論文引用生資中心保存菌株

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National Center for Biotechnology Information PubMed database 

共 541 筆
Record ID 第1筆 System ID 092TTU00106019
BCRC ID BCRC 10459, Public Year 92
Paper Name 利用Pseudomonas putida進行生物性鉻酸還原之研究 MICROBIAL REMOVAL OF CHROMATE BY PSEUDOMONAS PUTIDA
Student Name 周廷彥
Teacher Name 林銘澤
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 鉻是常見的工業污染物,常用於金屬加工、電鍍、皮革製作或色素相關應用。鉻在自然界中,經常以Cr(Ⅵ)和Cr(Ⅲ)的形式存在,六價鉻的溶解度大,較容易被生物體吸收累積,進而造成毒性。在自然環境中,常會形成鉻酸鹽化合物(CrO42-或是Cr2O72-);而三價鉻的溶解度低,常會行成氧化物、氫氧化物、或是硫化物。故關於鉻的處理都是將六價鉻轉變成三價鉻的形式來降低其毒性。 �Pseudomonas sp.相關的菌株大多具有在鉻酸環境下生長的能力,經分析Pseudomonas sp.發現,Pseudomonas putida BCRC 10459具有明顯的抗鉻能力,1000�嵱鉻酸處理5小時可還原223�嵱鉻酸;同時在50�嵱鉻酸處理2小時的情況下,可達到最大酵素比活性為每mg 粗蛋白質106.7 units Pseudomonas putida BCRC 10459之鉻酸還原酶經選殖後,建構表現質體後將之送入E. coli DH5α中,測試抗鉻能力發現,轉殖株明顯提升E. coli的抗鉻能力。測試不同濃度IPTG誘導發現,1mM IPTG誘導3小時,可達到最大酵素比活性為每mg粗蛋白質有39 units活性。以此誘導條件再處理鉻酸發現,1000�嵱鉻酸處理3小時,可達到152�嵱的最大還原量。經Ni-NTA resin純化後發現,可得到比活性提升1.6倍的純化酵素。 Chromium is common industrial pollution element. Cr is used in chemical industrial processes, mainly leather tanning, pigments and electroplating. The most stable and common forms are the trivalent Cr(Ⅲ) and hexavalent Cr(IV) species. Cr(Ⅵ), considered the most toxic form of Cr, is usually associated with oxygen as chromate (CrO42-) or dichromate (Cr2O72-) ions. In contrast, Cr(Ⅲ) in the form of oxides, hydroxides or sulfates, is much less mobile and exists mostly bound to organic matter in soil and aquatic environments. About the waste chromium, it usually reduce Cr(Ⅵ) to Cr(Ⅲ) is a common policy to detoxify Cr pollution. Pseudomonas species can survive or grow in chromium-related oxides. After biochemical analysis, the Pseudomonas putida BCRC 10459 have the best chromate resistance。 P. putida BCRC 10459 treated Cr(Ⅵ) with 5 hours can reduce 1000�嵱 Cr(Ⅵ) to 777�嵱。 P. putida BCRC10459 treated with 50�嵱 Cr(Ⅵ) for 2hours, it reached highest specific activity: 106。7 (units per mg of crude extract). Consequently, the chromate reductase was cloned from Pseudomonas putida CCRC10459。 An expression plasmid, pQE30, was ligated with it and then transferred to host E. coli DH5α. The chromium resistance of transgenic E. coli was also promoted. The highest specific activity was 39 units per mg of crude extract obtained from LB cultures for 3 hours after induction with 1mM IPTG. According this induction condition, the recombinant E. coli treated with 1000�嵱 Cr(Ⅵ) for 3 hours could remove or reduce 152mM chromate. After Ni-NTA method purification, the specific activity of purified chromate reductase was improved to 1.6 times of crude extract.

Record ID 第2筆 System ID 095TTU00106001
BCRC ID BCRC 12838, BCRC 16016, Public Year 94
Paper Name 利用QUANTITATIVE PCR定量硝酸廢水處理污泥中之脫硝菌 QUANTIFICATION OF DENITRIFYING BACTERIA IN SLUDGE FOR THE TREATMENT OF WASTEWATER WITH NITRATE BY QUANTITATIVE PCR
Student Name 薛伃君
Teacher Name 林銘澤
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 在氮循環過程中包含自營性的硝化及異營性的脫硝兩個過程,最後變成氮氣排放至大氣中。由於硝酸廢水處理所牽涉的菌種及化學反應過於廣泛,為維持大量且穩定均衡的菌相,隨時線上檢測為硝酸廢水處理中重要的一環。 於本系統以批次培養與連續式饋料培養脫硝菌株進行研究,並且應用分子生物檢測方法進行線上即時監測脫硝菌株之變化。藉由脫硝反應 (Denitrification) 中的重要酵素nitrite reductase之兩型基因 (nirK及nirS) 分別設計引子,分別對Alcaligenes xylosoxidans subsp. denitrificans BCRC 12838及Pseudomonas fluorescens Migula BCRC 16016標準菌株,與硝酸廢水活性污泥中之脫硝菌株進行聚合酶連鎖反應,其中nirK4f-nirK5r引子對與nirS2f-nirS3r引子對具專一反應,故選用此兩組引子利用定量競爭型聚合酶連鎖反應 (quantitative competitive polymerase chain reaction,QC-PCR),於模擬脫硝廢水處理過程,其污泥中脫硝菌株所含的兩型nitrite reductase基因數變化進行檢測及定量。 將已馴化之脫硝廢水污泥,用以處理含500 ppm硝酸鹽之模擬脫硝處理的批次培養反應槽中,分別於20℃、25℃、30℃與35℃下反應6小時,其脫硝率分別可達到28%、57%、86%及89%,取樣反應槽中之活性污泥,利用QC-PCR定量得於此6小時內,於25℃和30℃槽中,其nirK與nirS明顯增加,而低溫會減少污泥中脫硝菌,所以溫度會影響脫硝菌之生長。連續式脫硝反應槽以HRT 100ml/hr速率下,可連續操作150小時,其硝酸鹽脫硝率可達90%並維持80%以上,而處理過程中,溫度亦會影響脫硝菌之生長,於30℃可促進脫硝菌生長,但20℃時則會降低脫硝菌數。 The nitrogen cycle contains autotrophic nitrification and heterotrophic denitrification. The bacteria population and the chemical reaction were very complex to dispose the waste water with nitrate. We must to setup an on-line detectable system to quantify the bacteria population in the nitrate waste water for the evaluation of waste water treatment. The batch reaction (BR) and continue stirred tank reaction (CSTR) were setup to simulate the treatment of waste water with nitrate. Therefore, the molecular biologic assay was developed to detect the denitrifying bacteria on-line. There are many enzymes involving denitrification of nitrogen cycle, the nirK and nirS were selected to design the primer pairs for polymerase chain reaction. These primer pairs with the genomic DNAs of Alcaligenes xylosoxidans BCRC 12838 (nirK type), Pseudomonas fluorescens BCRC 16016 (nirS type), and denitrifying bacteria in wastewater carried out the polymerase chain reaction。 The primer sets of nirK4f-5r and nirS2f-3r were specific to the respective standard denitrifying bacterial strains, so these two primer sets were selected for quantitative competitive polymerase chain reaction (QC-PCR) to rapidly quantify the two type of denitrifying bacteria in active sludge of denitrifying bioreactor. The denitrification yield of simulated batch-type denitrifying bioreactor with 500 ppm nitrate were 28%, 57%, 86%, and 89% in 6 hr at 20�aC, 25�aC, 30�aC, and 35�aC, respectively. The copies of nirk and nirS in the bioreactors at 25�aC and 30�aC within 6 hr were obvious increase quantified by QC-PCR. However, treated at low temperature decreased the denitrifying bacteria in active sludge. It was evident that temperature effects the growth of denitrifying bacteria. In continuous stirred tank with HRT 100ml/hr, the denitrification yield was up to 90% and maintained over 80% under the continuous operation for 150 hr. The temperature was also effect the growth of denitrifying bacteria that elevated at 30 �aC and suppressed at 20 �aC.

Record ID 第3筆 System ID 092TTU01106011
BCRC ID BCRC 10451, BCRC 10067, BCRC 12450, BCRC 11634, BCRC 20511, Public Year 92
Paper Name 二氧化鈦塗料的殺菌作用 Disinfection Ability of TiO2-Containing Coating
Student Name 徐嘉玲
Teacher Name 許垤棊
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 奈米二氧化鈦是一種可淨化環境的光觸媒,可噴灑或塗佈在牆壁或器具上,經過光照射後可分解有機物,進而殺死細菌。二氧化鈦受波長385nm以下的光照射,在表面會產生氫氧自由基和超氧自由基。任何對於環境或人體有害之有機物質,包括各種能產生臭味之有機物質和各種病菌都會被光觸媒產生之氫氧自由基和超氧自由基破壞,因而達到光觸媒的消毒、除臭、自淨、抗污等功能。在室外,陽光含有很多紫外線;在室內,一般的日光燈也含有少量波長385nm以下的光線,這些光波的存在都可導致長期的光觸媒作用。 本研究先將含有二氧化鈦的塗料噴灑在玻璃表面,形成薄膜後,將數種病原菌分別分散於甘油溶液中,再塗佈在薄膜上,用日光燈照射。實驗組與對照組比較,發現含有奈米二氧化鈦的光觸媒塗料,對於金黃葡萄球菌、大腸桿菌、綠膿桿菌、白色念珠菌、糞腸球菌等,均有相當的殺菌效果。其中金黃葡萄球菌和腸球菌於照光過程中,對於熱、脫水和光觸媒作用具有適當的抵抗力,因此是用來檢測TiO2在日光燈下所產生的光觸媒消毒作用的最佳菌株。 Nano TiO2 is a kind of photocatalyst, which can be mixed with binder and then coated on the surfaces of construction matters or other objects, causing the light induced decomposition of organic compounds and even the death of microorganisms. When TiO2 crystals are irradiated with photons less than 385 nm, photocatalytic reactions occur, producing hydroxyl radicals and superoxide ions those have strong potential to oxidize organic compounds. This type of oxidation can serve as deodorant and allow surfaces to be self-cleaned and even self-disinfected. Outdoors sunlight has plenty of ultraviolet light to give considerable amount of photocatalysis. Indoors fluorescent lamps have a small fraction of lights with wavelengths shorter than 385 nm, which might result in rather weak disinfection ability. In the present study, pathogenic bacteria including Staphylococcus aureus CCRC 10451, Enterococcus faecium CCRC 10067, Pseudomonas aeruginosa CCRC 12450, E coli CCRC 11634 and Candida albicans CCRC 20511 were used to assay the photocatalytic disinfection ability of TiO2 induced by fluorescent light。 A smear of bacterial culture on the surface of the slide, which has been coated with TiO2-containing coating was irradiated by a fluorescent lamp in a short distance. Among the above-mentioned strains, S. aureus and E. faecium had moderate resistance to heat, dehydration and photocatalytic effect, being ideal germs for evaluation of photocatalytic disinfection ability of a TiO2-containing surface illuminated by fluorescent light.

Record ID 第4筆 System ID 092TTU05106007
BCRC ID BCRC 31535, BCRC 12325, Public Year 92
Paper Name 米醋與紅麴醋釀造過程中之生化變化及香氣成分分析 Biochemical changes and the flavor components analysis during the fermentation of rice vinegar and red yeast rice vinegar
Student Name 劉旭鈞
Teacher Name 李綉鈴
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 摘要 本研究探討米醋及米醋釀造過程中之製麴、酒精發酵、醋酸發酵及熟成等各階段之生化變化並對米醋及紅麴米醋之香氣成分加以分析。 結果顯示,Monascus ruber BCRC 31535接種於在米飯上,培養10天可獲得紅麴,其monacolin k含量為45.3 mg/kg dry weight。製麴過程中,添加紅麴均會降低米麴之α-amylase酵素活性,於酒精發酵過程中,米酒醪及紅麴酒醪中還原糖含量隨發酵時間增加而減少並伴隨酒精之逐漸生成,於5天後二者之酒精濃度均約達到10 %,紅麴酒中紅色素含量亦隨發酵時間增加而提高,米酒醪中之色素含量則是很低。紅麴酒醪中則無monacolin k之測得。米醋及紅麴醋醋酸於發酵過程中,Acetobacter aceti BCRC12325會消耗酒精產生醋酸,二者最終醋酸含量均約4.7 %。紅麴醋之色素含量則呈現些許下降,米醋與紅麴醋之香氣成分組成相似,共鑑定出種24香氣化合物,包括醇類化合物7種;酯類化合物7種;酸類化合物6種;醛類化合物2種;酮類化合物2種。 米醋與紅麴醋之香氣成分分析發現2,3-butanediol之含量最高,2-phenylethanol次之。於醋酸發酵終了 (48 hr),多數的香氣成分之含量多有增減,尤以2,3-butanediol含量會顯著增加,而於醋熟成2個月後,大多數之香氣成分含量均呈下降。 Abstract In this study, rice vinegars and red yeast rice vinegars were prepared. The biochemical changes of rice vinegars and red yeast rice vinegars during koji-making, alcohol fermentation, acetic acid fermentation and aging periods were under investigation. Meanwhile, aromatic volatiles of rice vinegars and red yeast rice vinegars were also identified and quantified. The results revealed that red yeast rice could be obtained after culturing Monascus ruber BCRC31535 on rice for 10 d。 The content of monacolin k increased to 45.3 mg/kg dry wt. Addition of red yeast rice to rice koji decreased the α-amylase activity of rice koji. During alcohol fermentation of rice moromi and red yeast rice moromi, the amount of reducing sugar decreased and the amount of alcohol increased with increasing time. The concentration of ethanol in rice moromi and red yeast rice moromi increased to 10% after 5 d of alcohol fermentation. The content of red pigment in red yeast rice moromi also increased. However, the content of red pigment content in rice moromi was much lower. The concentration of monacolin k in red yeast rice moromi was below detection limit. During acetic acid fermentation of rice wine by Acetobacter aceti BCRC 12325, the concentration of ethanol decreased and acetic acid formed。 The final concentration of acetic acid of rice vinegar and red yeast rice vinegar was 4.7% and the content of red pigment in red yeast rice vinegar slightly decreased at the end of fermentation. The aroma compounds identified in rice vinegar and red yeast rice vinegar were similar. Twenty-four compounds, including 7 alcohols, 7 esters, 6 acids, 2 aldehydes and 2 ketones, were identified. The amount of 2,3-butanediol was highest in rice vinegar and red yeast rice vinegar, followed by 2-phenylethanol. After acetic acid fermentation for 48h, the amount of 2,3-butanediol significantly increased. However, the amounts of most of the aroma compounds decreased after aging for 2 months.

Record ID 第5筆 System ID 092TTU01106006
BCRC ID BCRC 30428, BCRC 22292, Public Year 92
Paper Name 燒酎香味成分之探討 Studying of Flavor Ingredients for Shochu
Student Name 李水課
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 燒酎是日本傳統蒸餾酒。本研究利用本土不同種類原料米、紅、黃甘藷及沖繩黑糖、麥汁,小量製造燒酎。使用BCRC 30428 麴菌及BCRC 22292 酵母菌製米麴及酒母。酒母是以米麴、水、濃縮酵母菌及蒸米混合一週製成。將定量各種原料和水加入酒母進行酒精並行複式燒酎酒醪醱酵7~14 天後,以濾布濾除固態酒醪,取澄清醪液,以兩段式不同酒精度擷取酒液,一段取酒精度40%以上,一段取酒精度(19~40%)製造燒酎。並以玻璃瓶密封在常溫陰暗下貯存四個月後開封,以氣相層析儀分析酒液香味成分,並以市售五種不同燒酎(紅鶴酎(大麥)、樽藏紅鶴酎(大麥)、HONKAKU(大麥)燒酎、iichiko 大麥燒酎JOUGO 燒酎(黑糖)) 之香味成分作比較分析。經分析後獲得以下結果: 各種類燒酎之主體香味成份有羰基化合物、酯類、高級醇類、有機酸類、芳香族化合物構成,而以高級醇類佔其香味成份總量比最大,其次為有機酸類、羰基化合物、酯類。 自製各種燒酎(常壓蒸餾)香味成份較市售各種燒酎豐富,自製各種燒酎有11~16 種成份,市售各種燒酎僅有7~9 種成份。 市售燒酎之樽藏紅鶴酎(大麥)、HONKAKU(大麥)燒酎「無」有機酸類。自製燒酎有機酸含量高於市售燒酎,且自製同原料種類低酒精度(19~40 %,v/v)燒酎有機酸含量遠高於高酒精度(40%以上,v/v)燒酎。 各種燒酎以自製燒酎酯類及iichiko 大麥燒酎羰基化合物含量最少。 低酒精度米燒酎之芳香族化合物2-phenyl ethanol(β-苯乙醇) 佔總量比,遠高於各種類燒酎。 Shochu is a tranditional Japanese spirit. The present studies employed local agricultural products such as rice, sweet potato (red and yellow), brown sugar, and malt to make shochu in small scale. We used Aspergillus oryzae (BCRC 30428) as strain for koji rice and Saccharomyces cerevisiae (BCRC 22292) for Shubo (yeast starter) in our study。 The Shubo is made by mixing fresh koji rice, concentrated yeast slurry, water, and steamed rice for about one week. Shubo was then mixed with various raw materials and water for further fermentation. After 7 ~ 14 days, the solid part of mash was filtrated by cloth, and the liquid mash was distilled. Distillate was collected for alcohol content above 40 % (v/v) as part one, and alcohol content 19~40 % (v/v) as part two. The distillates (shochu) were stored in room temperature, dark place for four month. The flavor compounds of shochu in this study were analyzed by gas chromatography. Flavor compounds of shochu in this study were compared with that from five commercial shochus (Flamingo, Jar Flamingo, Honkaku, iichiko, and Jougo). The flavor compounds of shochu from gas chromatography can be classified as carboxylic compounds, higher alcohols (fusel oil), organic acids, and aromatics. The higher alcohols composed the major flavor compounds, followed by organic acid compounds, carboxylic compounds, and esters. The flavors of shochu in this study are between 11 to 16 compounds. That from commercial shochu is between 7 to 9 compounds. Organic acids of shochu in this study are more than commercial shochus. There are no organic acid compounds in Jar Flamingo shochu and Honkaku shochu. Organic acids of low alcohol content (19 ~ 40 %, v/v) were lower than that of high alcohol content (more than 40 %). Concentration of ester was very low for the shochus of this study. Concentrations of the carboxylic compounds were the lowest from iichiko shochu. The shochu made from rice (low alcohol content) had the highest aromatics (2-phenyl ethanol).

Record ID 第6筆 System ID 094TTU01106027
BCRC ID BCRC 30428, BCRC 22292, Public Year 94
Paper Name 利用台灣農產原料釀製日式燒酎之研究 Brewing Japanese Shochu Using local Crops
Student Name 簡茂生
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 摘要 本研究利用台灣農產原料米,紅甘薯,黃甘薯,黑糖,以及進口的濃縮麥芽精為原料,試驗製造日本燒酎。利用黃麴菌(BCRC 30428)製造麴米,以提供分解澱粉質原料所需要的糖化酵素,酒精發酵則採用一株日本燒酎用的酵母菌(BCRC 22292)。先以麴米,酵母菌製造酒母之後(一次醪),再加入蒸煮過的原料與麴米進行發酵(二次醪)。發酵完成的燒酎則以常壓蒸餾,分別擷取酒精度19∼40度及40∼55度兩段酒液裝瓶,在常溫密封貯存三個月後,取出以氣相層析儀做香味成分分析。 各種類燒酎之主體香味成分有羰基化合物、酯類、高級醇類、芳香族化合物構成,而其中以高級醇類所佔香味成分的總百分比最大(13~83%)。主要高級醇類為n-propanol、i-butanol、2-methyl-butanol(2-甲基丁醇)、3-methyl- butanol (異戊醇),其中以異戊醇含量最高。至於酯類香氣占總體香味成分的 1~5% 不等,乙酸乙酯以及己酸乙酯皆因濃度太稀,用我們現有的儀器測不出來,反而是高沸點的酯類如,乳酸乙酯則有出現。 研究結果顯示高酒精度的燒酎收集的香氣成分較佳,若要勾兌成低酒精度的燒酎商品,也應該利用高酒精度的燒酎為原料較好。 Abstract Shochu is a tranditional Japanese spirit. The present study employed local agricultural products such as rice, sweet potato (red and yellow), brown sugar, and malt extract to make shochu in small scale. We used Aspergillus oryzae (BCRC 30428) as strain for koji rice and Saccharomyces cerevisiae (BCRC 22292) for Shubo (yeast starter) in our study。 The Shubo mixed with steamed raw materials (rice and sweet potato), or brown sugar, malt extract for further fermentation for 2 weeks. The broth was distilled at 1 atmosphere. Distillate was collected for alcohol content above 40 % (v/v) as high alcohol shochu, and 19~40 % (v/v) as low alcohol shocho. The shochu was stored in room temperature for three month. The flavor compounds of shochu were analyzed by gas chromatography. The flavor compounds of shochu from gas chromatography can be clssified as carboxylic compounds, esters, higher alcohols, and aromatics. The higher alcohol shochu composed the major flavors (13 ~ 83 %). The major flavors are n-propanol, i-butanol, 2-methyl butanol, 3-methyl butanol. Among them, 3-methyl butanol is the most flavor compound. The esters are relatively low in concentration. Ethyl lactate cound be found in all of the shochu samples. The flavor compounds for high alcohol shochu are better than those for lower alcohol shochu. It is recommended to dilute high alcohol shochu to make low alcohol shochu product.

Record ID 第7筆 System ID 092TTU00106009
BCRC ID CCRC 14634, CCRC 14615, CCRC 11846, CCRC 14607, CCRC 15476, CCRC 14602, CCRC 14609, CCRC 11844, Public Year 92
Paper Name 比菲德氏菌對於高純度果寡糖的醱酵生長與代謝 Growth and Metabolism of Bifidobacteria in the Presence of High-Content Fructooligosaccharides
Student Name 何金柱
Teacher Name 許垤棊
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 比菲德氏菌存在於人體的大腸和結腸中,可促進人體的健康。消化性糖諸如:葡萄糖、蔗糖、麥芽糖等,均無法到達大腸。寡糖成分具有非消化性,可到達大腸,資化比菲德氏菌。利用不含消化性糖的高純度果寡糖於人體外資化比菲德氏菌,由於沒有葡萄糖和蔗糖的干擾,這樣的研究比前人利用含有大量消化性糖的寡糖的研究更具有意義。 本實驗的高純度果寡糖利用雙酵素系統製造,以蔗糖為原料,經黴菌Aspergillus japonicus的果糖轉移酵素催化,生成果寡糖和葡萄糖;同時,葡萄糖被葡萄糖氧化酵素催化,變成葡萄糖酸,葡萄糖酸和碳酸鈣形成鈣鹽沉澱。反應過程使用95 %氧氣,可得到95 %乾種以上的高純度果寡糖。 在五公升的醱酵槽中,有二公升的培養基,含5 % (w/v)的高純度果寡糖,在厭氧的條件下緩慢攪拌,觀察比菲德氏菌的生長。醱酵過程中,控制pH值為6.0,測定混濁度、生菌數、氧化還原電位(ORP)、醋酸對乳酸的比例,以及用HPLC分析二糖、三糖、四糖等諸寡糖成分的變化。在72小時醱酵中,本研究使用的八株比菲德氏菌是:Bifidobacterium longum (CCRC 14634 and 14602)、B. bifidum (CCRC 14615 and 11844)、B. breve (CCRC 11846)、B. adolescentis (CCRC 14607 and 14609)、B. pseudocatenulatum (CCRC 15476),其中以B. adolescentis CCRC 14607在16小時內消耗完1-Kestose和B. longum CCRC 14602在20小時內消耗完Nystose為最快產生最多乳酸的是B. adolescentis CCRC 14609,醋酸則是B. adolescentis CCRC 14607產生最多但B. bifidum CCRC 11844和B. breve CCRC 11846則在高純度果寡糖裡生長緩慢,因此寡糖的代謝較其他的菌株為差。氧化還原電位在菌體快速生長和消耗寡糖的過程中,急遽下降。生長對數期之後,氧化還原電位緩慢的上升。ORP的變化可反映出比菲德氏菌的生長和寡糖的代謝。 Bifidobacteria can promote human health. Bifidobacteria are regularly found in human large intestine and colon, where digestible sugars such as glucose, sucrose and maltose, are not attainable. The investigation of enhancing the in vitro growth of bifidobacteria with such oligosaccharides, which is free of the metabolic interference of glucose and sucrose must be more significant than past research with adding commercial oligosaccharides, in that a large amount of digestible sugars are present. High-content fructooligosaccharides are produced by a mixed-enzyme system with β-fructofuranosidase from Aspergillus japonicus and a commercial glucose oxidase as biocatalysts. Sucrose is catalyzed by β-fructofuranosidase, forming fructooligosaccharides and glucose, and this glucose is concurrently converted to gluconic acid which is then precipitated to calcium gluconate in solution through the catalysis of glucose oxidase. In such way and under an extreme aerobic condition made by aeration with 95 % (v/v) oxygen, up to 95 %, on a dry weight basis, of high-content fructooligosaccharides can be produced. Five percent (w/v) of such high-content oligosaccharides are added to a 2-L bifidus culture in a 5-L jar fermenter. The fermentation is carried out under an anaerobic condition with gentle stirring. The optical density and the viable count of the fermentation medium are monitored. Furthermore, the utilization of each sugar component such as disaccharide, trisaccharide or tetrasaccharide is analyzed by HPLC throughout the fermentation process. Other parameters such as ORP, pH and the acetate to lactate ratio are also determined. During 72 h of fermentation, eight bifidobacteria including Bifidobacterium longum CCRC 14634, B longum CCRC 14602, B bifidum CCRC 14615, B bifidum CCRC 11844, B breve CCRC 11846, B adolescentis CCRC 14607, B adolescentis CCRC 14609 and B pseudocatenulatum CCRC 15476 were investigated。 B. adolescentis CCRC 14607 depleted 1-kestose completely at h 16 and B longum CCRC 14602 consumed nystose completely at h 20, being the best strains among eight bifidobacteria。 B. adolescentis CCRC 14609 produced the maximum lactic acid of 40。88 g/L and B. adolescentis CCRC 14607 produced the maximum acetic acid of 27。82 g/L. But B. bifidum CCRC 11844 and B breve CCRC 11846 did not grow well and consumed fructooligosaccharides poorly。 As FOS was being consumed, the ORP of the culture broth declined, until most of the FOS was depleted, and then it would increase very slowly. The decreasing of ORP reflected the consuming of FOS and the growth of bifidobacteria.

Record ID 第8筆 System ID 092TTU05106004
BCRC ID BCRC 31494, Public Year 92
Paper Name 黑麴菌β-葡萄醣苷酶II之結構基因分析 STRUCTRURE GENE ANALYSIS OF ASPERGILLUS NIGER β-GLUCOSIDASE II
Student Name 周積孚
Teacher Name 顏聰榮
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract β-葡萄糖苷酶為催化醣基與氧親核基間反應屬於醣苷酶家族之酵素,由於作用機制為保留式反應,因此可能因其他非水配基作用釋放被酵素所包圍之醣基而達成糖轉移之效果,先前研究中,自黑麴菌Aspergillus niger BCRC 31494中純化之β-葡萄糖苷酶II,除水解活性外,亦具有特定之糖轉移活性,以纖維二醣為基質可合成纖維寡糖,若添加醇類為基質,則可合成相對應之烷基糖苷化合物,產物可成為機能性食品或利用於製藥原料之發展為達到利用遺傳工程技術將其基因選殖並改良其調控系統以提高酵素表現功能之目的,本研究利用已知β-葡萄糖苷酶II核苷酸序列設計適當引子,利用聚合酶連鎖反應技術,成功對A. niger BCRC 31494 β-葡萄糖苷酶II基因體DNA與RNA反轉譯之cDNA進行定序,基因體序列開放讀架區域核苷酸序列長度為2,948 bp,包括7個外顯子與6個內含子,cDNA核苷酸序列長度為2,583 bp,可轉譯成具860個胺基酸之酵素,根據轉譯之蛋白質序列,酵素屬於醣苷酶家族3,並且為β-葡萄糖苷酶II亞科B之成員。 β-Glucosidase is a member of glycosidase family that catalyzes the transfer of glycosyl group between oxygen nucleophiles. Because of its retaining mechanism, transglycosylation may occurs when the retained glucose released by aglycone other than water. In the previous study, β-glucosidase II was isolated from Aspergillus niger. Besides the typical hydrolysis activity, it was found that processes transglycosylation activity toward specific substrates. Cello-oligosaccharide was synthesized from cellobiose, and alkyl-glucosides were synthesized when supplied with alcohol. The products may apply to functional food and pharmaceutical industrial. For the purpose of enzyme cloning, properties enhancement, and regulation comprehension; we designed primers from known b-glucosidase sequences and succeed in obtaining the DNA and RNA-reversed-transcribed cDNA sequence of A. niger β-glucosidase II. The cloned genomic sequence revealed a 2,948 bp open reading frame with 6 introns and 7 exons. The cDNA showed a 2,583 sequence encoding an 860-amono-acid protein that belongs to glycosyl hydrolase family 3 as well as identified as a member of β-glucosidase subfamily B.

Record ID 第9筆 System ID 092TTU01106008
BCRC ID CCRC 21731, CCRC 30428, Public Year 92
Paper Name 小量清酒試釀及其香味之分析研究 SMALL SCALE OF SAKE BREWING AND ANALYSIS OF SAKE FLAVORS
Student Name 高珮琪
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 日本清酒之製法與我國之紹興酒及黃酒有頗多相似之處,屬米類釀造酒,其色澤清淡金黃,味道溫和且帶有果香,常作為餐前開胃酒或搭配日本菜飲用增加菜餚風味。我國近年來進口日本清酒數量,每年有快速增加的趨勢。清酒釀造所涉及之發酵技術層次比米酒或水果酒更複雜,另外,亦需特殊的低溫發酵環境,所以過去只有菸酒公賣局才有生產,民間釀酒業者似乎尚未見。台灣加入WTO後對國產稻米有相當大的衝擊,加強稻米製品加工技術,提升國產米附加價值,是因應稻米生產過量防止穀賤傷農的對策之一。 本研究利用台東縣池上鄉生產的有機米(高雄139號,台梗9號)為原料,進行小量清酒的釀造試驗。有機米利用小型碾米機精白數次,得到精米度75%的原料米。然後經過製麴、培養酒母、蒸米與麴米經過初添、中添、末添三個步驟,發酵約21天之後,進行壓榨酒醪得到的清酒,進行氣相(GC)與液相(HPLC)色層分析,得到主要的香味成份為:Ethyl acetate、Butyl acetate、Ethyl lactate、Ethyl Caprylate 、Isobutyric acetate、Isoamyl acetate、Ethyl alcohol、Propyl aIcohol、Isobutyl alcohol、Isoamyl alcohol、Phenylethyl alcohol、citric acid、malic acid、succinic acid、lactic acid、acetic acid 等,經比較日本高級清酒發現主要的香味成份皆類似,只是含量彼此之間有一些差異。試釀收得清酒的酒精度可達17%以上。本研究使用的酵母為日本協會6號,使用的麴菌為CCRC30428,經比較試釀清酒與日本清酒的風味發現,試釀的清酒果香較濃郁(Phenylethyl alcohol),而日本清酒的吟釀香(主要為Isoamyl acetate)較明顯。 Sake is a traditional Japanese alcohol beverage, which is similar to Shaohsing wine of China. Both the sake and Shaohing are made from rice. Sake is pale yellow in color with fruity flavor. Sake is very popular with Japanese cuisine in the world. Taiwan imported increasing amount of sake in the last few years. Sake is made with highly polished rice with very low fat and protein in content. The brewing processes include rice steaming, koji making, yeast starter (shubo), the mash (moromi), pressing. Sake is brewed in low temperature with very high rice concentration. In Taiwan, only Taiwan Tobacco and Liquor Corporation has this kind of product. The present study employed rice by organic agriculture techniques from Chih Shang, Taitung County for experimental brewing test of sake. The particular species of rice used are Kaoshing 139 and Tai-geng 9. Saccharomyces cerevisiae CCRC 21731 and Aspergillus oryzae CCRC 30428 were used in this study。 The rice was polished many times using a small scale rice polishing machine. A polished rice with 75 % of residual weight from brown rice was obtained. The polished rice was used to make sake by the standard brewing procedures as described above. The sake obtained in this study was analysed by GC and HPLC for the flavor components. The major flavor components include: ethyl acetate、butyl acetate、ethyl lactate、ethyl caprylate 、isobutyric acetate、isoamyl acetate、ethyl alcohol、propyl alcohol、isobutyl alcohol、isoamyl alcohol、phenylethyl alcohol、citric acid、malic acid、succinic acid、lactic acid、acetic acid. We found very similar flavor pattern in this study as comparing with those from several commercial Japanese sake. The alcohol concentration was 17 % (vol) in this study. The sake we brewed is very intensive fruity flavor (phenylethyl alcohol), while that for Japanese sakes have apparent isoamyl acetate flavor which is a particular flavor of ginjo-shu.

Record ID 第10筆 System ID 094TTU05106013
BCRC ID BCRC 30428, BCRC 22292, Public Year 94
Paper Name 蒸餾方法與酒精濃度對燒酎風味的影響 Effects of the distillation method and alcohol content on the flavor of Shochu
Student Name 陳淑清
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 本研究利用台灣農產原料米、黑糖、以及進口的濃縮麥芽精為原料,試驗製造日本燒酎。利用黃麴菌(BCRC 30428)製造麴米,以提供分解澱粉質原料所需要的糖化酵素,酒精醱酵則採用一株日本燒酎用的酵母菌(BCRC 22292)。先以麴米,酵母菌製造酒母之後(一次醪),再加入蒸煮過的原料與麴米進行醱酵(二次醪)。醱酵完成的燒酎則以常壓及減壓蒸餾,分別擷取酒精度19∼40度及40∼55度兩段酒液裝瓶,在常溫密封貯存三個月後,取出以氣相層析儀做香味成分分析。 各種類燒酎之主體香味成分有酯類、高級醇類、芳香族化合物構成,而其中以高級醇類所佔香味成分的總百分比最大(88~100%)。主要高級醇類為n-propyl Alcohol、i-butyl Alcohol、3-methyl-1-butanol (異戊醇)、Benzyl Alcohol(苯甲醇)中,其中以異戊醇含量最高。至於酯類香氣占總體香味成分的 0~12% 不等,只有出現乙酸乙酯。研究結果顯示高酒精度的燒酎收集的香氣成分較佳,若要勾兌成低酒精度的燒酎商品,也應該利用高酒精度的燒酎為原料較好。 Shochu is a traditional Japanese spirit. The present study employed local agricultural products such as rice, brown sugar, and malt extract to make shochu in small scale. We used Aspergillus oryzae (BCRC 30428) as strain for koji rice and Saccharomyces cerevisiae (BCRC 22292) for Shubo (yeast starter) in our study。 Shubo mixed with steamed raw materials (rice), or brown sugar, malt extract for further fermentation for 2 weeks. The broth was distilled at the condition of either atmospheric pressure or reduced pressure. The distillate was collected by following the concentrations of alcohol. High concentration of alcohol that was above 40 % (v/v) will be classified as high alcohol Shochu, and low concentration of alcohol that was between 19~40 % (v/v) will be classified as low alcohol Shochu. The shochu was stored in room temperature for three month. The flavor compounds of Shochu were analyzed by gas chromatography. The flavor compounds of Shochu analyzed by gas chromatography can be classified as carboxylic compounds, esters, higher alcohols, and aromatics. The higher alcohol in Shochu is composed the major flavors (88 ~ 100%). The major flavors are n-propanol, i-butanol, 2-methyl butanol, 3-methyl butanol. Among them, 3-methyl butanol is the most flavor compound. The esters are relatively low at concentration. Ethyl lactate could be found in all of the Shochu samples. The flavor compounds for high alcohol Shochu are better than those for lower alcohol shochu. It is recommended to dilute high alcohol Shochu to make low alcohol Shochu product.

Record ID 第11筆 System ID 094TTU00106016
BCRC ID BCRC 31535, Public Year 94
Paper Name 紅麴菌於黃豆基質上之生長及monacolin k 之產生 Growth of Monascus sp. and production of monacolin k by solid state fermentation of soybeans
Student Name 田甜
Teacher Name 李綉鈴
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 紅麴菌(Monascus sp.)可產生降血脂之monacolin k,黃豆中之異黃酮則可降低乳癌及前列腺癌之發生,本研究擬將紅麴菌培養於黃豆上,以開發新型保健食品—紅麴黃豆。於所測試之七株紅麴菌中,M . ruber BCRC 31535於黃豆上固態發酵培養14天後,所產生的monacolin k (62.4 mg/kg)含量最高;若額外於黃豆基質上添加1%或2%果糖,則可顯著促進M. ruber BCRC 31535之生長,而以添加1% 果糖時monacolin k之含量最高,達93.4mg/ kg。添加1%果糖之黃豆,再添加yeast extrate、monosodium glutamate或peptone等氮源時,雖可提高紅麴黃豆之橘色及紅色色素含量,但monacolin k之含量均顯著下降。將黃豆基質的初始水含量調整為64.4%時,色素及monacolin k含量均較其他初始水含量者高,以所得之最適培養條件,將M . ruber BCRC 31535在30℃下固態培養於黃豆基質,並於第六天及第七天分別補水2 ml,於 14天發酵過程中,紅麴黃豆之水分含量隨培養時間之延長而逐漸下降,M. ruber BCRC 31535 之生長與monacolin k含量則隨培養時間之增加而逐漸增加,培養14天後,M. ruber BCRC 31535之生長較控制組增加1.3倍,而 monacolin k含量則為144.5 mg/kg,較控制組提高2.2倍。 Monascus sp. can produce blood cholesterol lowering agent—monacolin k. The isoflavones of soybeans are reported to have a preventive effect on breast and prostate cancers. A novel functional food, monascal soybeans was prepared on soybeans fermented with Monascus sp. M. ruber BCRC 31535 showed the highest production of monacolin K (62。4 mg/kg) after 14 d of cultivation. Supplementation of 1% or 2% fructose to the soybeans enhanced the growth of M. ruber BCRC 31535。 The highest monacolin K production (93.4 mg/ kg) was found when 1% fructose was added to soybeans. Further supplementation of 1% of nitrogen sources includind yeast extract, monosodium glutamate or peptone to the fructose added soybeans increased the amounts of yellow and red pigment, but significantly reduced the monacolin K production by test organism. The highest amounts of pigment and monacolin K were found in monascal soybeans with an initial water content of 64.6 % . The content of water, glucosamine and monacolin k were determined during cultivation of monascal soybeans supplemented with 2 ml water each after fermentation for 6 d and 7 d under optimal fermentation condition at 30℃. The moisture content gradually decreased conteute of glucosamine and monacolin k increased as increased cultivation time. The contents of glucosamine and monacolin k were 1.3 and 2.2 times that of control, respectively. The highest content of monacolin k ,144.5 mg/ kg, was roted after cultivation of monascal soybeans for 14 d.

Record ID 第12筆 System ID 094TTU05106006
BCRC ID BCRC 31615, Public Year 94
Paper Name 探討培養基與培養條件對固態培養紅麴菌生產 Monacolin K、Citrinin 及色素的影響 Effect of Medium and Cultivation Condition on Monacolin K, Citrinin and Pigment Production During Solid State Fermentation of Monascus purpureus
Student Name 林秦瑋
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 本實驗以Monascus purpureus BCRC 31615 紅麴菌株,分別以白米、糙米為基質在 30℃ 下進行固態培養紅麴菌,並分析 monacolin K、citrinin 及色素產量。再藉由培養環境的改變,找出其最適的培養條件,以提高 monacolin K 產量。實驗結果發現,以白米培養紅麴時色素產量較高,而以糙米培養紅麴時 monacolin K 產量較高;在糙米的部份,接菌後第九天添加 60 ml 無菌水,monacolin K 產量由原本的 3813 ppm 增加到 5098 ppm,增加了 33.7%。而 monacolin K、citrinin 與色素的生成量皆有相似的趨勢。本研究成果是目前在文獻上所能得到的最佳成果。 In this study, the influence of different rice and cultivation condition on the production of monacolin K, citrinin and pigments by solid state fermentation of Monascus purpureus BCRC 31615 was studied。 Monascus was cultured at 30℃ on polished indica rice and de-hulled indica rice, separately. The results showed that the pigment is relatively high when cultivated on polished indica rice while monacolin K content is relatively high when cultivated on de-hulled indica rice. During solid state fermentation of de-hulled indica rice, adding 60 ml of water at day 9 improved 33.7% of the productivity of monacolin K from 3813 ppm to 5098 ppm. A similar tendency was observed in the formation amount of monacolin K, citrinin and pigments. The monacolin K production of this study was the most in the literatures ever published.

Record ID 第13筆 System ID 092TTU00106002
BCRC ID BCRC 36614, Public Year 92
Paper Name 深層培養松茸(Tricholoma matsutake) 之最適化培養液組成 Optimization of the medium components for Tricholoma matsutake submerged culture
Student Name 張志豐
Teacher Name 李綉鈴
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 松茸 (Tricholoma matsutake)又名松口蘑,是一種珍貴的食用菌,具有抗腫瘤及提高免疫力等生理活性。本研究乃在探討,於液態培養T. matsutake BCRC 36614中之培養液,包括碳源種類、胺基酸及銨態氮、有機氮peptone及yeast extract之添加,對松茸菌絲體及胞內多醣生產之影響,再利用反應曲面法找出各組成分之最適濃度結果顯示,glucose、maltose、mannose、lactose及malt extract不僅促進T. matsutake BCRC 36614之生長,亦能促進胞內多醣之累積,其中以含有2 % glucose之基礎培養液培養T. matsutake BCRC 36614至四十天後,可獲得最大菌絲乾重,約為8.4 g/L,而胞內多醣含量亦最高,約為3.6 mg/100 mg CDW,產量約為30.1 mg/100 mlL-arginine或L-glutamic acid之添加可促進T. matsutake BCRC 36614之生長及胞內多醣之累積;而ammonium tartrate或ammonium nitrate兩種銨態氮則有抑制效果添加0.2 % L-arginine於培養液中,T. matsutake BCRC 36614之生長狀況最佳,培養至第四十天時,可獲得最大菌絲體乾重,約為9.9 g/L,為不添加者之1.2倍;而胞內多醣含量亦最高,約為7.4 mg/100 mg CDW,為不添加者之2倍,產量約為73.4 mg/100 mlPeptone或yeast extract之添加,對T. matsutake BCRC 36614之生長及胞內多醣含量皆有顯著影響,以0.2 %為最適添加濃度經由反應曲面實驗設計法得知,當培養基組成為2.25 % glucose、0.2 % peptone、0.22 % yeast extract、0.2 % L-arginine、0.05 % KH2PO4、0.05 % MgSO4•7H2O時,為培養T. matsutake BCRC 36614獲得最大胞內多醣含量之最適組成,約為7.3 mg/ 100 mg CDW。 Tricholoma matsutake, also named matsutake, is a precious edible fungus which shows many pharmacological activities including anti-tumor effect and immunology enhancement. In this study, we investigated the effects of adding different carbon, amino acid, ammonium nitrogen and organic nitrogen (peptone and yeast extract) in T. matsutake BCRC 36614 submerged cultures on the growth and endopolysaccharide formation。 Finally, we used response surface methodology to find the optimal concentration of each medium component. Our results showed that glucose, maltose, mannose, lactose and malt extract promoted not only the growth of T. matsutake BCRC 36614, but also the formation of endopolysaccharide。 Among them, T. matsutake BCRC 36614 cultured in 2 % glucose-added basal medium after 40 days could result in maximal cell dry weight (8。4 g/l), endopolysaccharide content (3.6 mg/ 100mg CDW), and maximal endopolysaccharide production (30.1 mg/ 100 ml). Addition of L-arginine or L-glutamic acid promoted not only the growth of T. matsutake BCRC 36614, but also the formation of endopolysaccharide。 However, two ammonium nitrogen, ammonium tartrate and ammonium nitrate, showed suppressive effects. The best growth of T. matsutake BCRC 36614 was obtained in 0。2 % L-arginine-added medium. In the 40-day culture, we obtained maximal cell dry weight (9.9 g/l) which was 1.2 times compared with the control group, maximal endopolysaccharide content (7.4 mg/ 100mg CDW) which was 2 times compared with its control group, and maximal production (73.4 mg/ 100 ml). Addition of peptone or yeast extract could significantly affect the growth and endopolysaccharide formation of T. matsutake BCRC 36614。 The optimal concentrations of peptone and yeast extract were both 0.2 %. Using response surface methodology, we found the optimal medium components formula: 2.25 % glucose, 0.2 % peptone, 0.22 % yeast extract, 0.2 % L-arginine, 0.05 % KH2PO4, and 0.05 % MgSO4•7H2O. Cultured in the optimal medium, T. matsutake BCRC 36614 produced maximal endopolysaccharide content (7。3 mg/100 mg CDW).

Record ID 第14筆 System ID 094TTU05106003
BCRC ID BCRC 9363, Public Year 94
Paper Name 用米根黴的菌絲團生產L型乳酸 Production of L-Lactic Acid Using Mycelia Flocs of Rhizopus oryzae.
Student Name 吳靜怡
Teacher Name 許垤棊
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 在米根黴( Rhizopus oryzae BCRC 9363 )的傳統培養液中,菌絲會凝聚成菌塊,我們發現是因為培養液中的氮源耗盡的因素。因此在液態培養的過程定時補充氮源,可防止菌塊的產生,而形成棉絮狀的菌絲。因此,L型乳酸產量大大地提高。當在5公升攪拌式生物反應器中進行醱酵,用碳酸鈣乳液控制pH值在 4.3-4.5 之間,且每隔8小時補充少量的硫銨,以維持氨氮的濃度在每公升0.3-0.5公克之間。在此條件下,以每公升120克的葡萄糖為受質,L型乳酸產量為105公克,產率為0.88,生產速率為每小時每公升2.63公克。若沒有補充氮源,菌塊會纏繞在生物反應器內的裝置上面,而且L型乳酸產量顯著的下降。本研究展示一個簡單的方法使米根黴產生棉絮狀菌絲,而且L型乳酸的生產量達到最適化。 The formation of mycelial clumps in conventional cultures of Rhizopus oryzae resulted mainly from the depletion of nitrogen source in the cultural medium. Based on this behavior, L(+)-lactic acid production could be enhanced in the submerged culture using mycelial flocs induced by replenishing ammonium-nitrogen in the cultural medium, thereby the formation of mycelial clumps or pellets which often occurred in conventional cultures of R. oryzae was prevented. When the fermentation was performed in a 5-L stirred tank bioreactor with the pH being controlled in the range of 4.3 – 4.5 by adding calcium carbonate slurry, and small amounts of ammonium sulfate was added at 8-h intervals to sustain ammonium level of the culture in the range of 0.3 – 0.5 g/L, lactic acid production was optimized. The lactic acid concentration produced was 105 g/L, with the yield of 0.88 and the productivity of 2.63 g/L-h, using 120 g/L of glucose as substrate. Without replenish of ammonium-nitrogen, mycelial clumps attached everywhere in the bioreactor and lactic acid production dramatically declined. This article demonstrates a simple way to obtain mycelial flocs of R. oryzae thereby an optimized lactic acid production can be achieved.

Record ID 第15筆 System ID 093TTU00106004
BCRC ID BCRC 9363, Public Year 93
Paper Name 用米根黴的菌絲團生產L型乳酸 Production of L-Lactic Acid Using Mycelia Flocs of Rhizopus oryzae.
Student Name 吳靜怡
Teacher Name 許垤棊
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 在米根黴( Rhizopus oryzae BCRC 9363 )的傳統培養液中,菌絲會凝聚成菌塊,我們發現是因為培養液中的氮源耗盡的因素。因此在液態培養的過程定時補充氮源,可防止菌塊的產生,而形成棉絮狀的菌絲。因此,L型乳酸產量大大地提高。當在5公升攪拌式生物反應器中進行醱酵,用碳酸鈣乳液控制pH值在 4.3-4.5 之間,且每隔8小時補充少量的硫銨,以維持氨氮的濃度在每公升0.3-0.5公克之間。在此條件下,以每公升120克的葡萄糖為受質,L型乳酸產量為105公克,產率為0.88,生產速率為每小時每公升2.63公克。若沒有補充氮源,菌塊會纏繞在生物反應器內的裝置上面,而且L型乳酸產量顯著的下降。本研究展示一個簡單的方法使米根黴產生棉絮狀菌絲,而且L型乳酸的生產量達到最適化。 The formation of mycelial clumps in conventional cultures of Rhizopus oryzae resulted mainly from the depletion of nitrogen source in the cultural medium. Based on this behavior, L(+)-lactic acid production could be enhanced in the submerged culture using mycelial flocs induced by replenishing ammonium-nitrogen in the cultural medium, thereby the formation of mycelial clumps or pellets which often occurred in conventional cultures of R. oryzae was prevented. When the fermentation was performed in a 5-L stirred tank bioreactor with the pH being controlled in the range of 4.3 – 4.5 by adding calcium carbonate slurry, and small amounts of ammonium sulfate was added at 8-h intervals to sustain ammonium level of the culture in the range of 0.3 – 0.5 g/L, lactic acid production was optimized. The lactic acid concentration produced was 105 g/L, with the yield of 0.88 and the productivity of 2.63 g/L-h, using 120 g/L of glucose as substrate. Without replenish of ammonium-nitrogen, mycelial clumps attached everywhere in the bioreactor and lactic acid production dramatically declined. This article demonstrates a simple way to obtain mycelial flocs of R. oryzae thereby an optimized lactic acid production can be achieved.

Record ID 第16筆 System ID 091TTU00063002
BCRC ID CCRC 31494, Public Year 91
Paper Name 黑麴菌beta-葡萄糖苷酵酵素在強酸性陰離子交換樹脂Q-Sepharose之離子交換平衡 Adsorption Equilibrium of beta-Glucosidase from Aspergillus niger onto Strong Anion Exchange Resin Q-Sepharose
Student Name 韓安倫
Teacher Name 陳嘉明
School Department Name 大同大學 化學工程研究所 Academic Degree 碩士
Abstract 具熱敏感性的生物分子的分離常應用吸附及離子交換分離程序。為了設計及最適化整個吸附及離子交換程序,等溫吸附曲線十分重要。本研究有系統地探討從黑麴菌分泌的beta-葡萄糖苷酵素在Q-Sepharose陰離子交換樹脂上的離子交換吸附平衡。分別探討溫度(10-30deg.C)、氯化鈉濃度(0-0.5 M)、pH(3-6.2)對吸附平衡的影響。實驗結果顯示beta-葡萄糖苷酵素的吸附量隨溫度及pH增加而提高,但是隨鹽的濃度增加而減少。雖然在多種不同實驗條件下,吸附平衡實驗數據都與Langmuir或 Freundlich模式大致吻合,但本研究仍提出一個更為通用的模式,以方便用來評估溫度、氯化鈉濃度、pH對吸附平衡的影響。 Adsorption and ion exchange processes are widely used to separate heat-sensitive bio-products. In order to design and optimize the separation processes by adsorption or ion exchange, the adsorption isotherm is very crucial. The adsorption equilibrium of beta-glucosidase (EC 3.2.1.21) from the culture broth of Aspergillus niger CCRC 31494 onto Q-Sepharose ion- exchange resin was systematically studied。 The effects of temperature (10-30deg.C), concentration of sodium chloride (0-0.5 M) and pH (3-6.2) on the adsorption isotherm of beta-glucosidase were measured experimentally. The results showed that amounts of beta-glucosidase adsorption increased with increasing pH and temperature, but decreased with increasing concentration of sodium chloride. Although the adsorption isotherms under various operating conditions could be adequately fitted by the Langmuir and Freundlich models, a more general adsorption isotherm model was proposed to assess the effects of solution pH, temperature and concentration of sodium chloride.

Record ID 第17筆 System ID 094TTU00106001
BCRC ID BCRC 22637, Public Year 93
Paper Name 用酵母菌Pichia holstii醱酵生產胞外多醣的最適化 Optimization of Exopolysaccharide Production by Submerged Culture of Pichia holstii
Student Name 呂世淵
Teacher Name 許垤棊
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 硫化寡醣在臨床上具有抗凝血、抗病毒以及抗腫瘤的潛力。酵母菌的細胞外多醣可以當作硫化寡醣的原料,因而受到格外的重視。本研究主要是利用酵母菌Pichia holstii BCRC 22637,於五公升醱酵槽中,在不同培養基的條件下,探討產生細胞外多醣的最適化。醱酵條件包括:不同葡萄糖濃度、磷酸鉀濃度與不同氮源的濃度,以得到生產細胞外多醣的最大產量。醱酵槽操作條件為轉速450 rpm,通氣量2 L min-1,溫度維持在25℃,pH控制在5.0 ± 0.3之間。經過醱酵120小時後,發現培養基中葡萄糖的濃度為200 g L�{1 ;磷酸鉀濃度為15 g L�{1以及corn steep liquor 4 g L-1 和yeast extract 4 g L-1時,細胞外多醣的乾重高達72.4 g L�{1,而菌體量乾重達20.7 g L�{1。 Yeast exopolysaccharide (EPS) has been becoming attractive, because it can serve as raw material for the manufacture of sulfated oligosaccharide, which was currently under clinical evaluation as anticoagulant, anti-virus and anti-tumor agent. In the present study, batch cultures of Pichia holstii BCRC 22637 for EPS production under different conditions at various concentrations of glucose and potassium dihydrogen phosphate with various concentrations of different nitrogen sources were investigated。 The fermentation medium consisted of the following ingredients in gram per liter: MgSO4.7H2O, 0.2; MnSO4.H2O, 0.01; FeSO4.7H2O, 0.01 and NaCl, 0.01. Fermentation was carried out under the following conditions: initial working volume 2 L, agitation 450 rpm, aeration 2 vvm, temperature 25℃ and pH controlled at 5.0 ± 0.3. For the optimal production of EPS the medium consisted of the following ingredients in gram per liter: glucose 200, corn steep liquor 4, yeast extract 4 and KH2PO4 15. After an 120-h fermentation, up to 72.4 g L-1 EPS and 20.7g L-1 cells, on a dry weight basis, were obtained.

Record ID 第18筆 System ID 092TTU00106004
BCRC ID CCRC 9363為, CCRC 9363, Public Year 92
Paper Name 以米根黴生產L型乳酸 Production of L (+)-lactic acid by Rhizopus oryzae
Student Name 林怡君
Teacher Name 許垤棊
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract L型乳酸,分子式C3H6O3,為含有氫氧基的有機酸,是生物的代謝中間產物。L型乳酸可以在動物和人類細胞中新陳代謝因為這些細胞含有L型乳酸去氫酵素。L型乳酸還可用於生産L型乳酸聚合物。L型乳酸聚合物屬於無毒的高分子化合物,具有生物相容性,可用於製造生物可分解的塑膠、纖維以及生醫材料等。本研究以黴菌Rhizopus oryzae CCRC 9363為L型乳酸生產菌,針對生物反應器的種類與最適操作條件來進行探討。最後由實驗得知以改良型的生物反應器為最適生產L型乳酸的反應器。 L-lactic acid, C3H6O3, an organic acid with hydroxy group is a metabolic intermediate in living organisms. Only the L-form of lactic acid is metabolized in animal and human cells. This is due to the fact that only L-lactate dehydrogenase is synthesised by these cells. L-lactic acid can be polymerized to form Poly-lactic acid (PLA). L-form PLA is a non-virulent macromolecular compound with biocompatibility, which used in the manufacture of new biodegradable plastics, fiber, and biomedical material. This experiment used fungus, Rhizopus oryzae CCRC 9363 to produce L-lactic acid and discussed the types of bioreactor and best conditions。 Final, it is discovered that the modified bioreactor is the best bioreactor to produce L-lactic acid.

Record ID 第19筆 System ID 092TTU00106008
BCRC ID CCRC 14609, CCRC 11844, CCRC 11846, Public Year 92
Paper Name 高純度異麥芽寡糖對比菲德氏菌增值的影響 EFFECT OF HIGH-CONTENT ISOMALTOOLIGOSACCHARIDES ON THE GROWTH OF BIFIDOBACTERIA
Student Name 徐雅亭
Teacher Name 許垤
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 比菲德氏菌存在於人體的大腸和結腸中,消化性糖無法到達此處。然而,有一些寡糖成分,具有非消化性, 可到達大腸和結腸,進而資化比菲德氏菌。異麥芽寡糖,是以麥芽糖為基質,利用glucosyltransferase 催化反應生成,為機能性寡糖,是一種益生源,對於比菲德氏菌的生長具有很好的促進作用。利用酵母菌去除市售異麥芽寡糖中的消化性糖,亦即麥芽糖及葡萄糖,使寡糖的純度由58%提高至99%以上。在2公升醱酵槽中,用1.3公升的培養基,添加5%的高純度異麥芽寡糖,在礦油覆蓋下的厭氧條件、於37°C、攪拌速度100rpm,觀察比菲德氏菌的生長。醱酵過程中,監測培養液的混濁度、生菌數、氧化還原電位、pH值、醋酸與乳酸濃度,以及用HPLC分析寡糖成分的變化。 在72小時醱酵中,比菲德氏菌主要代謝panose, tertesaccharides 和isomaltose,因而產生glucose和maltose。B. adolescentis CCRC 14609利用異麥芽寡糖的速度為最快速且乳酸的產量37.41 g/L相較於其他菌株為最大而B. bifidum CCRC 11844和B. breve CCRC 11846則在高純度異麥芽寡糖裡生長緩慢,因此寡糖的代謝較其他的菌株為差。由於此寡糖不含消化性糖,所以這樣的研究比前人利用含有大量消化性糖的寡糖的研究更具有意義。因此可以了解比菲德氏菌對於異麥芽寡糖中不同成分的利用情形,也可以瞭解不同的比菲德氏菌利用寡糖的差異。 Bifidobacteria can promote human health. Bifidobacteria are regularly found in human large intestine and colon, where digestible sugars such as glucose, sucrose and maltose, are not attainable. Isomaltooligosaccharides (IMO), a function sugar, was made from maltose via a reaction catalyzed by glucosyltransferase. IMO is preprobiotic, being able to enhance the growth of bifidobacteria. High-content IMO can be produced, by treating commercial IMO syrup with yeast, thereby digestible sugar including maltose and glucose are depleted. In this way, the content of IMO increases from 58% to 99% on a dry weight basis. In a 2-L jar fermenter, bifidobacteria were cultured in 1.3 L broth in the presence of 5% (w/v) high-content IMO. The fermentation conditions were as following: anaerobic culture, overlaid with a layer of liquid paraffin; stir rate, 100rpm; temperature, 37°C. During fermentation, turbidity, viable count, ORP, pH, acetate, lactate and sugar components were determined periodically. During 72 h of fermentation, most of the bifidobacteria used in this study metabolize primarily panose, tertesaccharides and isomaltose and thus produced G and IG2. B. adolescentis CCRC 14609 consumed IMO much faster than other strains did and produced the maximum lactic acid of 37。41 g/L. But B. bifidum CCRC 11844 and B breve CCRC 11846 did not grow well and consumed isomaltooligosaccharides poorly。 The present investigation using such oligosaccharides which was free of digestible sugars was much more significant than past research, in which oligosaccharides accompanied with large amounts of digestible sugars were used. The utilization of various IMO components by eight bifidobacteria was clearly understood.

Record ID 第20筆 System ID 092TTU00106013
BCRC ID BCRC 31494, Public Year 92
Paper Name 黑麴菌β-葡萄醣苷酶II之結構基因分析 Structure Gene Analysis of Aspergillus niger β-Glucosidase II
Student Name 周積孚
Teacher Name 顏聰榮
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract β-葡萄糖苷酶為催化醣基與氧親核基間反應屬於醣苷酶家族之酵素,由於作用機制為保留式反應,因此可能因其他非水配基作用釋放被酵素所包圍之醣基而達成糖轉移之效果,先前研究中,自黑麴菌Aspergillus niger BCRC 31494中純化之β-葡萄糖苷酶II,除水解活性外,亦具有特定之糖轉移活性,以纖維二醣為基質可合成纖維寡糖,若添加醇類為基質,則可合成相對應之烷基糖苷化合物,產物可成為機能性食品或利用於製藥原料之發展為達到利用遺傳工程技術將其基因選殖並改良其調控系統以提高酵素表現功能之目的,本研究利用已知β-葡萄糖苷酶II核苷酸序列設計適當引子,利用聚合酶連鎖反應技術,成功對A. niger BCRC 31494 β-葡萄糖苷酶II基因體DNA與RNA反轉譯之cDNA進行定序,基因體序列開放讀架區域核苷酸序列長度為2,948 bp,包括7個外顯子與6個內含子,cDNA核苷酸序列長度為2,583 bp,可轉譯成具860個胺基酸之酵素,根據轉譯之蛋白質序列,酵素屬於醣苷酶家族3,並且為β-葡萄糖苷酶II亞科B之成員。 β-Glucosidase is a member of glycosidase family that catalyzes the transfer of glycosyl group between oxygen nucleophiles. Because of its retaining mechanism, transglycosylation may occurs when the retained glucose released by aglycone other than water. In the previous study, β-glucosidase II was isolated from Aspergillus niger. Besides the typical hydrolysis activity, it was found that processes transglycosylation activity toward specific substrates. Cello-oligosaccharide was synthesized from cellobiose, and alkyl-glucosides were synthesized when supplied with alcohol. The products may apply to functional food and pharmaceutical industrial. For the purpose of enzyme cloning, properties enhancement, and regulation comprehension; we designed primers from known β-glucosidase sequences and succeed in obtaining the DNA and RNA-reversed-transcribed cDNA sequence of A. niger β-glucosidase II. The cloned genomic sequence revealed a 2,948 bp open reading frame with 6 introns and 7 exons. The cDNA showed a 2,583 sequence encoding an 860-amono-acid protein that belongs to glycosyl hydrolase family 3 as well as identified as a member of β-glucosidase subfamily B.

Record ID 第21筆 System ID 092TTU00106018
BCRC ID BCRC 31535, BCRC 12325, Public Year 92
Paper Name 米醋及紅麴醋釀造過程中之生化變化及香氣成分分析 Biochemical changes and the flavor components analysis during the fermentation of rice vinegar and red yeast rice vinegar
Student Name 劉旭鈞
Teacher Name 李綉鈴
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 摘要 本研究探討米醋及米醋釀造過程中之製麴、酒精發酵、醋酸發酵及熟成等各階段之生化變化並對米醋及紅麴米醋之香氣成分加以分析。 結果顯示,Monascus ruber BCRC 31535接種於在米飯上,培養10天可獲得紅麴,其monacolin k含量為45.3 mg/kg dry weight。製麴過程中,添加紅麴均會降低米麴之α-amylase酵素活性,於酒精發酵過程中,米酒醪及紅麴酒醪中還原糖含量隨發酵時間增加而減少並伴隨酒精之逐漸生成,於5天後二者之酒精濃度均約達到10 %,紅麴酒中紅色素含量亦隨發酵時間增加而提高,米酒醪中之色素含量則是很低。紅麴酒醪中則無monacolin k之測得。米醋及紅麴醋醋酸於發酵過程中,Acetobacter aceti BCRC12325會消耗酒精產生醋酸,二者最終醋酸含量均約4.7 %。紅麴醋之色素含量則呈現些許下降,米醋與紅麴醋之香氣成分組成相似,共鑑定出種24香氣化合物,包括醇類化合物7種;酯類化合物7種;酸類化合物6種;醛類化合物2種;酮類化合物2種。 米醋與紅麴醋之香氣成分分析發現2,3-butanediol之含量最高,2-phenylethanol次之。於醋酸發酵終了 (48 hr),多數的香氣成分之含量多有增減,尤以2,3-butanediol含量會顯著增加,而於醋熟成2個月後,大多數之香氣成分含量均呈下降。 Abstract In this study, rice vinegars and red yeast rice vinegars were prepared. The biochemical changes of rice vinegars and red yeast rice vinegars during koji-making, alcohol fermentation, acetic acid fermentation and aging periods were under investigation. Meanwhile, aromatic volatiles of rice vinegars and red yeast rice vinegars were also identified and quantified. The results revealed that red yeast rice could be obtained after culturing Monascus ruber BCRC31535 on rice for 10 d。 The content of monacolin k increased to 45.3 mg/kg dry wt. Addition of red yeast rice to rice koji decreased the α-amylase activity of rice koji. During alcohol fermentation of rice moromi and red yeast rice moromi, the amount of reducing sugar decreased and the amount of alcohol increased with increasing time. The concentration of ethanol in rice moromi and red yeast rice moromi increased to 10% after 5 d of alcohol fermentation. The content of red pigment in red yeast rice moromi also increased. However, the content of red pigment content in rice moromi was much lower. The concentration of monacolin k in red yeast rice moromi was below detection limit. During acetic acid fermentation of rice wine by Acetobacter aceti BCRC 12325, the concentration of ethanol decreased and acetic acid formed。 The final concentration of acetic acid of rice vinegar and red yeast rice vinegar was 4.7% and the content of red pigment in red yeast rice vinegar slightly decreased at the end of fermentation. The aroma compounds identified in rice vinegar and red yeast rice vinegar were similar. Twenty-four compounds, including 7 alcohols, 7 esters, 6 acids, 2 aldehydes and 2 ketones, were identified. The amount of 2,3-butanediol was highest in rice vinegar and red yeast rice vinegar, followed by 2-phenylethanol. After acetic acid fermentation for 48h, the amount of 2,3-butanediol significantly increased. However, the amounts of most of the aroma compounds decreased after aging for 2 months.

Record ID 第22筆 System ID 093TTU05106006
BCRC ID BCRC 11844, BCRC 14607, Public Year 93
Paper Name 用比菲德氏菌醱酵黃豆豆漿 Fermentation of soybean milk by Bifidobacteria
Student Name 林雋軒
Teacher Name 許垤棊
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 豆漿的內容物中含有大量的蛋白質及多醣類,以及其他的醣包括:水蘇糖、棉籽糖、果糖、葡萄糖及蔗糖。本研究於豆漿醱酵過程中,觀察比菲德氏菌的生長及各種醣類的消耗,以及醋酸與乳酸產量的變化。利用下列四株比菲德氏菌:B.bifidum BCRC 11844 and 14615, B. adolescentis BCRC 14607 and 14609 對豆漿與牛奶於醱酵槽中進行72小時厭氧醱酵。醱酵過程中,監測豆漿的寡糖成分的變化、觀察比菲德氏菌的生長以及pH值。此外在牛奶與豆漿的72小時厭氧醱酵後,置於4℃、14天,監測生菌數以及pH值的變化。 Soybean milk contains large amounts of proteins and polysaccharides, and other sugar components, including stachyose, raffinose, sucrose, glucose and fructose. When Soybean milk was fermented by bifidobacteria, these sugar components were consumed, accompanying with the production of acetic acid and lactic acid. In this study, four bifidobacteria including: bifidobacterium bifidum BCRC 11844 and 14615, B adolescentis BCRC 14607 and 14609 were inoculated in soybean milk and milk and the 72-h anaerobic fermentations were carried out in jar fermenters。 During fermentation the consumption of sugar component, the growth of Bifidobacteria, the decrease of pH were monitored. Furthermore, the fermented milks were stored at chilling temperature for 14 days and the qualities were evaluated by monitoring the viability and the pH of the fermented milks.

Record ID 第23筆 System ID 091TTU00106002
BCRC ID CCRC 20306, CCRC 21509, Public Year 91
Paper Name D型胺基酸氧化酵素之蛋白質工程 Protein engineering of the D-amino acid oxidase
Student Name 吳東嶺
Teacher Name 官宜靜
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 將yeast Rhodosporidium toruloides (CCRC 20306) DAO cDNA基因置於E. coli strain BL-21(DE3)進行異源表現。於不同溫度下以IPTG誘導DAO表現後發現,較低溫度下DAO的活性表現較佳。於30℃下誘導5小時後,可測得最高胞內DAO活性約為5000 U/l culture。經檢測pH值及溫度對於DAO活性和穩定性之影響後發現,異源表現之R. toruloides和Trigonopsis variabilis (CCRC 21509) DAO有相同的最適反應pH (8.5~9),但前者能忍受較高之pH。T. variabilis DAO則有較高的最適反應溫度,且熱穩定性較佳。而His˙tag的有無與位置的變換並未顯著影響DAO的活性。此外,D-alanine的存在能明顯提高R. toruloides及T. variabilis DAO的pH及熱穩定性。另一方面,亦嘗試以R. toruloides及T. variabilis DAO cDNA基因進行DNA shuffling產生重組基因,以期產生高活性之DAO。經篩選後,僅獲得4個菌落產生具有DAO活性且其活性和原始之DAO相近。 Yeast Rhodosporidium toruloides (CCRC 20306) DAO cDNA gene was overexpressed in the heterologous host E。 coli strain BL-21(DE3). The DAO expression induced by IPTG at lower temperature was better than those induced at 37℃. The highest intracellular DAO activity obtained was ~5000 U/l from cultures induced 5 hours at 30℃. The effects of pH and temperature on the activity and stability of heterologous expressed R. toruloides and Trigonopsis variabilis (CCRC 21509) DAO were also examined。 The optimal pH for catalysis was 8.5~9 for both DAO, but the former had better pH stability. The optimal catalytic temperatures for R. toruloides and T. variabilis were 40~45℃and 45~50℃, respectively. The latter also had better thermostability. The presence of D-alanine could dramatically increase their pH stability and thermostability. The presence and position of His˙tag in DAOs showed little influence on their enzymatic characteristics. R. toruloides and T. variabilis DAO cDNA genes were together subject to DNA shuffling to generate mutant proteins. Only 4 active E. coli clones with DAO activity were obtained after extensive screening and showed similar DAO activity to the original proteins.

Record ID 第24筆 System ID 091TTU00106003
BCRC ID BCRC 12838, BCRC 16016, Public Year 91
Paper Name 含氮廢水中脫硝菌之檢測與定量 DETECTION AND QUANTIFICATION OF DENITRIFYING BACTERIA IN WASTEWATER WITH NITROGEN
Student Name 林冠妤
Teacher Name 林銘澤
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 由家庭生活污水及畜牧業與食品廠、ABS塑膠廠、石化工業、半導體等工廠廢水大量排放含銨氮的廢水,主要是以混合菌株培養處理。銨氮廢水處理的活性汙泥裡的微生物族群複雜,因此相關細菌的實際生長與分布研究困難。由於分子生物技術的進步,現在已經不須先經過會改變微生物組成與漏失部份訊息且耗時的集菌法,而可以直接鑑定菌種種類與定量。在氮循環之脫硝反應中包含多種酵素,選用nirK及nirS基因分別設計引子並以PCR之技術分別對標準菌Alcaligenes xylosoxidans BCRC 12838(nirK型)、Pseudomonas fluorescens BCRC 16016(nirS型)及含氮廢水中的脫硝菌進行定性及定量。 由實驗結果知9組nirK引子對中nirK3f-nirK5r此對引子與A. x 12838及含氮廢水中的脫硝菌皆有專一且正確之反應,而在16組nirS引子對中則是nirS2f-nirS3r此對引子能與P. f 16016及含氮廢水中的脫硝菌有專一且正確之反應。故選用此兩組引子對設計以競爭型PCR(competitive PCR)定量時所需之內標(internal control)之引子對,並於內標cnirK及cnirS製備完成後進行競爭型PCR。由結果可知在Tm值為60oC的條件下,含氮廢水中活性污泥裡脫硝菌之量為3.342108/g。 For health and environmental protection, municipal and industrial wastewaters have to be treated. Wastewater with ammonia-nitrogen, generally treated with mixed microbial populations, is produced by domestic wastewater, and wastewater from livestock and manufacture about food, ABS plastic, petroleum, and semiconductor at a large amount. The directly determination of the growths and distributions for relative bacteria are less or unsuccessful because of its complexity of populations in activated sludge. Due to the improvement of technology for molecular biology, the amounts and identify of the species can be directly determinated. There are many enzymes involved in denitrification of nitrogen cycle, we choose nirK and nirS these two enzymes to compare the genes sequences to design the primer pairs. These primer pairs with the genomis DNAs of Alcaligenes xylosoxidans BCRC 12838 (nirK type), Pseudomonas fluorescens BCRC 16016 (nirS type), and denitrifying bacteria in wastewater carry out the polymerase chain reaction for the rapidly determination and quantification of denitrifying bacteria From the results, we can see that there is only one primer set, nirK3f-nirK5r, can react properly with Alcaligenes xylosoxidans BCRC 12838 and denitrifying bacteria in wastewater among nine sets of primer pairs And there is also only one primer set, nirS2f-nirS3r, can react properly with Pseudomonas fluorescens BCRC 16016 and denitrifying bacteria in wastewater among sixteen set of primer pairs。 According to the sequences of nirK and nirS between the two specific primer sets, designed new primers to construct and clone the internal controls, cnirK and cnirS, for competitive PCR. There were 3.342108 denitrifying bacteria with nirS, determinated by competitive PCR, per gram of activated sludge in nitrogen compound-treated tank.

Record ID 第25筆 System ID 091TTU00106004
BCRC ID BCRC 20464, Public Year 91
Paper Name Candida boidinii D型胺基酸氧化酵素在大腸桿菌中的異源表現研究 HETEROLOGOUS EXPRESSION OF A D-AMINO ACID OXIDASE GENE FROM CANDIDA BOIDINII IN ESCHERICHIA COLI
Student Name 駱彥甫
Teacher Name 官宜靜
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract D-amino acid oxidase (DAO)為可以轉化D型胺基酸成α-酮酸並同時產生氨氣和過氧化氫的一種flavoprotein。Candida boidinii DAO genomic gene因不具intron,故被直接用來與表現載體pET-23am接合後,送入E. coli strain BL-21(DE3)進行大量異源表現。以TB medium進行培養時,於37℃下以IPTG誘導30小時後,可測得最高胞內DAO活性約為38024 U/l culture;但當誘導溫度下降至30℃時,DAO活性表現相近但活性高峰延遲至誘導後39小時。以LB medium進行培養時,在較低誘導溫度下,DAO的活性表現反而較佳,但表現高峰亦會向後延遲。於20℃下誘導12小時後,可測得最高胞內DAO活性約為4830 U/l culture。異源表現之C. boidinii DAO最適反應溫度及pH分別為55℃和pH 9。受質D-alanine的存在能明顯提高C. boidinii DAO的熱穩定度Tm由36℃增至60℃,但對其pH穩定度則幫助不大,僅在pH 6有較明顯的改善。另外,嘗試以C. boidinii、R. toruloides及T. variabilis DAO cDNA基因進行Family DNA shuffling,未能獲得full length之重組片段(1.1 kb),僅得500~800 bp之片段,故反應條件仍有待修正。 D-amino acid oxidase (DAO) is a flavoprotein which can catalyze the oxidative deamination of D-amino acids to α-keto acids, along with the generation of ammonia and hydrogen peroxide. Candida boidinii (BCRC 20464) DAO genomic gene containing no intron was directly inserted into the expression vector pET-23am for the heterologous expression in E。 coli strain BL-21(DE3). The highest activity expressed was 38024 U/l obtained from TB cultures 30 hours after induction with IPTG at 37℃. When the induction temperature was lowered to 30℃, the DAO expression was similar but peaked 39 hours after induction. In LB medium, the highest DAO activity expressed was 4830 U/l obtained 12 hours after induction at 20℃. The optimal temperature and pH for catalysis of the heterologously expressed C. boidinii DAO were 55℃ and pH 9, respectively. In the presence of D-alanine, the thermal stability of C. boidinii was dramatically improved with a Tm increasing from 36 to 60℃. However, the pH stability was not improved except at pH 6.0. Family DNA shuffling for in vitro enzyme evolution using C. boidinii, R. toruloides and T. variabilis DAO cDNA genes was performed. Only DNA fragments of 500~800 bp rather than the expected 1.1 kb full length were obtained. Possible causes and future adjustments of experimental conditions are discussed.

Record ID 第26筆 System ID 093TTU05106007
BCRC ID BCRC 11844, BCRC 14607, Public Year 93
Paper Name 用比菲德氏菌醱酵黑豆豆漿 Fermentation of black bean milk by Bifidobacteria
Student Name 林正安
Teacher Name 許垤棊
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 黑豆漿的內容物中含有大量的蛋白質及多醣類,以及其他的醣包括:水蘇糖、棉籽糖、果糖、葡萄糖及蔗糖。本研究於黑豆漿醱酵過程中,觀察比菲德氏菌的生長及各種醣類的消耗,以及醋酸與乳酸產量的變化。利用下列四株比菲德氏菌:Bifidum BCRC 11844 and 14615, B. adolescentis BCRC 14607 and 14609 對黑豆漿與牛奶於醱酵槽中進行72小時厭氧醱酵。醱酵過程中,監測黑豆漿的寡糖成分的變化、觀察比菲德氏菌的生長以及pH值。此外在牛奶與黑豆漿的72小時厭氧醱酵後,置於4℃、14天,監測生菌數以及pH值的變化。 Black bean milk contains large amounts of proteins and polysaccharides, and other sugar components, including stachyose, raffinose, sucrose, glucose and fructose. When black bean milk was fermented by bifidobacteria, these sugar components were consumed, accompanying with the production of acetic acid and lactic acid. In this study, four bifidobacteria including: Bifidobacterium bifidum BCRC 11844 and 14615, B adolescentis BCRC 14607 and 14609 were inoculated in black bean milk and milk and the 72-h anaerobic fermentations were carried out in jar fermenters。 During fermentation the consumption of sugar component, the growth of bifidobacteria, the decrease of pH were monitored. Furthermore, the fermented milks were stored at chilling temperature for 14 days and the qualities were evaluated by monitoring the viability and the pH of the fermented milks.

Record ID 第27筆 System ID 091TTU00106014
BCRC ID CCRC 22637, Public Year 91
Paper Name 利用酵母菌Pichia holstii醱酵生產胞外甘露多糖 Production of phosphomannan by Pichia holstii
Student Name 朱朝麟
Teacher Name 段國仁
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 中文摘要 酵母菌Pichia holstii的細胞外多糖含有大量的磷酸甘露聚糖,經過部分水解、純化、硫化之後,形成硫化寡糖,可以作為一種抗凝血藥物。利用酵母菌Pichia holstii生產磷酸甘露聚糖具有發展的潛力。本研究主要利用酵母菌Pichia holstii CCRC 22637,在氮源(Yeast extract)濃度改變下,探討磷酸甘露聚糖的生產 酵母菌Pichia holstii CCRC 22637於5公升醱酵槽醱酵的最適條件:碳源,葡萄糖120 g/L;氮源,Yeast extract 8g/L;轉速維持在450 rpm,通氣量為2L/min,溫度維持於25℃,pH控制在4.7~5.3,藉由批次醱酵培養,經過88小時菌體可達27 g/L,細胞外多糖可達54.7 g/L。以Yeast extract濃度8g/L進行饋料批次方式培養,經過314小時,多糖可達到106 g/L,菌體量可達到31.63 g/L;以Yeast extract濃度12 g/L進行饋料批次培養,菌體量可達到60 g/L,多糖可達到40g/L;以Yeast extract濃度16 g/L進行饋料批次培養,菌體量可達到47 g/L,多糖可達到40 g/L。培養基中含有較多量的yeast extract,雖然使菌體量增加,但是多糖並未增加。 利用凱氏氮法分析醱酵液中總氮含量,可以得知當菌體停止生長,此時凱氏氮的含量最少。在饋料批次實驗中有機氮會轉換氨氮,在醱酵培養結束之後,氨氮離子依然會殘存於醱酵液中。整個醱酵的過程中,Pichia holstii CCRC 22637便開始生產的磷酸甘露聚糖,而不是在氨氮耗盡時才生產所以,Pichia holstii CCRC 22637生產的磷酸甘露聚糖不同於 Agrobacterium sp.生產的curdlan 或是從Alcaligene entrophus 生產的PHB,因為後者的多糖都是在氨氮耗盡之後才開始生產多糖。 ABSTRACT Extra-cellular phosphomannan can be produced by Pichia holstii. The phosphomannan was partially hydrolyzed to phosphomannan oligosacchrides. The sulfated phosphomannan oligosacchrides can be a candidate of anticoagulant drug. The present study employed Pichia holstii CCRC 22637 to study the effect of nitrogen concentrations to the production of phosphomannan。 The optimum culture conditions in a fermenter are as follows: carbon source, glucose 120 g/L; nitrogen source, yeast extract 8 g/L; agitation speed, 450 rpm; aeration rate, 2 L/min; temperature at 25 °C; pH was controlled at 4.7~5.3.We applied a batch fermentation to achieve 27 g-DCW/L and 54.7 g/L phosphomannan after 88 h. The fed-batch fermentation was applied by using yeast extract 8 g/L, and achieved 106-g/L phosphomannan, 31.6 g-DCW/L after 314 h. The same fed-batch fermentation was performed by using 12 g/L yeast extract, the maximum dry cell weight and phosphomannan were 60 g-DCW/L, 40 g/L respectively. In addition, 16 g/L of yeast extract was applied for the same fed-batch fermentation; the maximum dry cell weight and phosphomannan were 47 g-DCW/L, 40 g/L respectively. More yeast extract in the medium resulted in more cell mass; however, less oligosaccharide was produced. Analysis of TKN indicates that cell growth stopped upon TKN decreased to minimum. Organic nitrogen source converted to ammonium during fed-batch fermentation, the ammonium ion remained in the broth at the end of fermentation. Phosphomannan was produced substantially throughout the period of fermentation. Phosphomannan production from Pichia holstii was not the same as curdlan from Agrobacterium sp. or PHB from Alcaligene entrophus that polymer was produced upon exhaustion of ammonium.

Record ID 第28筆 System ID 091TTU00106015
BCRC ID CCRC 21498, Public Year 91
Paper Name 利用複合酵素系統生產高純度的半乳寡醣 Studies on the Production of High-Content Galactooligosaccarides from Lactose by the Complex Enzyme System
Student Name 鄭昭君
Teacher Name 許垤棊
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 半乳寡醣(galactooligosaccharides,GOS)可促進腸道內的比菲德氏菌的增殖,藉此促進人體的健康。一般半乳寡醣商品是利用β-半乳糖苷酶的半乳糖苷轉移活性催化乳糖反應生成寡醣,然而產品中尚含有大量的葡萄糖和乳糖;而研究中所謂的「高純度半乳寡醣」的主要成分則是寡醣。高純度半乳寡醣不會引起蛀牙,而且熱量很低,可供糖尿病患者食用。本研究是利用一複合酵素系統用來生產高純度半乳寡醣。此系統是利用乳糖當作原料,而且含有來自環泰公司的β-半乳糖苷酶和一種葡萄糖氧化酶,商品名是Gluzyme。在五公升的醱酵槽中反應,通入95﹪的氧氣,用碳酸鈣懸浮液控制反應液的酸鹼度。乳糖經由β-半乳糖苷酶的半乳糖苷轉移活性催化反應生成寡醣,而副產物葡萄糖是此反應的抑制劑。反應中,葡萄糖經由葡萄糖氧化酶的催化作用生成葡萄糖酸,葡萄糖酸繼續和碳酸鈣作用,形成葡萄糖酸鈣沉澱物。改變基質的濃度、酵素劑量、溫度以及酸鹼度,做不同反應條件的探討。最適的反應條件:乳糖濃度是45% (重量/體積)、對每公克乳糖添加3.5單位的β-半乳糖苷酶和28單位的葡萄糖氧化酶、溫度是55℃、酸鹼度是pH 5.0。結果得到最高的半乳寡醣純度是60% (以乾重計算),而殘餘成分是33%的乳糖和7% 的半乳糖。此外,利用一株具有β-半乳糖苷酶酵素活性的酵母菌(Kluyveromyces marxianus CCRC 21498)可移除半乳寡醣糖漿中所殘存的乳糖以及副產物半乳糖。經由此菌的醱酵,半乳寡醣含量則可達到100% (以乾重計算)。 關鍵詞:半乳寡醣、複合酵素系統、β-半乳糖苷酶、葡萄糖氧化酶。 Galctooligosaccharides (GOS) can strongly stimulate the proliferation of colonic bifidobacteria and thereby improves human health. Commercial GOS produced via the enzymatic transgalactosylation of β-galactosidase on raw material lactose contains large amounts of glucose and lactose, while the so-called “high-content GOS” in this study, composed mainly of oligosaccharide ingredients. High-content GOS is non-cariogenic, low in calorie and might be ingested by diabetic patients. In this study, the complex enzyme system for the production of high-content GOS was investigated. The complex enzyme system contained a b-galactosidase obtained from Huan-Tai Company and a commercial glucose oxidase, Gluzyme, using lactose as raw material. The complex enzyme system was carried out in a 5-liter jar fermenter and was aerated with 95% oxygen. Calcium carbonate slurry was added to the reaction mixture to control pH at a predetermined value. GOS was formed via the transgalactosylation of b-galactosidase on lactose and the byproduct glucose, a strong inhibitor for GOS forming reaction, was converted to gluconic acid through the oxidation catalyzed by glucose oxidase. The gluconic acid was reacted with calcium carbonate, forming a calcium gluconate precipitate. The reactions under various conditions at different substrate concentration, enzyme dose, temperature and pH were investigated. For the production of high-content GOS by this complex enzyme system, the optimum conditions were:lactose concentration, 45% (w/v);enzyme doses per gram lactose, 3.5 units of b-galactosidase and 28 units of glucose oxidase;temperature, 55 ℃;pH, 5.0. A maximum GOS content of 60%, on a dry weight basis, was achieved, the remainder being 33% lactose and 7% galactose. Furthermore, a β-galactosidase-inherited yeast─Kluyveromyces marxianus CCRC 21498─is used to deplete both lactose and galactose present in GOS syrups produced by the complex enzyme system。 GOS content up to 100% on a dry weight basis can be achieved. Keywords:galctooligosaccharides, complex enzyme system, β-galactosidase, glucose oxidase.

Record ID 第29筆 System ID 093TTU00106006
BCRC ID BCRC 10094, Public Year 93
Paper Name 利用嗜鹼性芽胞桿菌Bacillus circulans No. 38-2 (BCRC 10094)進行醱酵生產環狀糊精葡萄糖苷轉移酶之研究 Production of cyclodextrin glucanotransferase (CGTase) by Bacillus circulans No. 38-2 (BCRC 10094)
Student Name 林清安
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 本研究利用嗜鹼性芽胞桿菌(Bacillus circulans No. 38-2,BCRC 10094)進行醱酵生產CGTase的研究。分別使用玉米澱粉和可溶性澱粉為碳源,能夠有效的誘導生產CGTase。而澱粉同時扮演碳源與誘導劑的功能。在33 �aC下,以基礎培養基與澱粉20 (g/L)為碳源的批式醱酵能生產約17319 Unit/ml的酵素活性。在pH-stat的饋料批式醱酵培養中,以澱粉的培養基,能夠得到約63165 Unit/ml的酵素活性。若先以葡萄糖為碳源將細胞的濃度提高,然後再饋入澱粉來誘導酵素,則能得到約39500 Unit/ml的酵素活性。在連續式醱酵培養的操作條件下,以稀釋率0.15倍進行CGTase的生產,結果能得到 31722 Unit/ml的酵素活性,以稀釋率0.3倍進行 CGTase的生產,則能得到約14320 Unit/ml的酵素活性。本結果顯示,利用pH-stat的饋料批式醱酵培養,在以澱粉為碳源的條件下,能夠得到最大量的酵素活性。若以單位體積產率(volumetric productivity)看則以連續式醱酵在稀釋率為0.3倍時,得到的酵素活性最高(約2864 Unit/h/L),其次為連續式醱酵在稀釋率為0.15倍(約2379 Unit/h/L),以pH-stat 饋料批式醱酵及批式醱酵以澱粉為碳源的體積生產速率分別為 (638, 444 Unit/h/L)。 Production of cyclodextrin glucanotransferase (CGTase) by Bacillus circulans No. 38-2 (BCRC 10094) in alkaline medium under various conditions was investigated。 Corn starch and soluble starch were employed as the carbon source for culturing the strain and performing as an inducer of CGTase, The basal medium (2 % yeast extract, 1 % Na2CO3, 0.1 % KH2PO4, 0.1 % K2HPO4, 0.02 % CaCl2, 0.02 % MgSO4, 0.02 % Mn(NO3)2 ) and 2 % starch as carbon source could produce the CGTase activity of 17319 unit/ml in batch culture at 33 �aC. The CGTase activity could be achieved 63165 unit/ml using soluble starch as carbon source in pH-state fed-batch fermentation. Another fermentation strategy was performed by culturing the cell to high density using glucose as carbon source in pH-stat strategy, and then CGTase was induced by soluble starch to achieve 39500 unit/ml activity. In continuous culture, 31722 unit/ml and 14320 unit/ml were achieved at respective diluting rate of 0.15 and 0.3. The results indicated that the most volumetric activity (2864 unit/h/L) was achieved for continuous fermentation at a dilution rate of 0.3, and then at a dilution rate of 0.15 (2379 unit/h/L). The volumetric activities of 638 and 444 unit/h/L were achieved for fed-batch and batch fermentation respectively.

Record ID 第30筆 System ID 093TTU00106011
BCRC ID BCRC 36348, Public Year 93
Paper Name 羊肚菌與蜜環菌之抗氧化性質分析 Analysis of antioxidant properties of Morchella esculenta and Armellaria mellea
Student Name 梁靖宗
Teacher Name 官宜靜
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 本研究主要在評估以YM或modified Bisakowski’s formula (MBF) broth培養的Morchella esculenta BCRC 36348與Armillaria mellea ATCC 11113之多醣生產與抗氧化性質。當M. esculenta培養於MBF broth 有最大胞內外多醣,分別為166 mg/g DW與0.275 mg/ml,而A. mellea亦以培養於MBF broth者有較多之胞內外多醣,分別為82.6 mg/g DW與0.731 mg/ml。 抗氧化性質的分析上,則分別量測各樣品的抗氧化力(共軛雙烯)、捕捉DPPH能力、還原力(普魯士藍)和螯合亞鐵離子能力並作比較。抗氧化力方面,熱水萃取物中,以萃自M. esculenta YM培養者最佳,濃度在10 mg/ml時,抗氧化效率可達78.74%;甲醇萃取物中,以萃自A. mellea YM (pH 5)培養者較佳,濃度在10 mg/ml時,其抗氧化效率達91.6%;在胞外發酵液中,以取自A. mellea YM (pH 5)培養者較佳,在濃度5 mg/ml時,抗氧化力達86.5%。捕捉DPPH能力方面,在熱水萃取物中,取自A. mellea兩種培養者皆呈現較高效率,在濃度2 mg/ml時,皆達76 %以上;甲醇萃取物與胞外發酵液中,皆以萃自A. mellea YM (pH 5)培養者較佳,在濃度為5 mg/ml時,分別達95.2%與93.9%。還原力方面,熱水和甲醇萃取物以及胞外發酵液中,皆以萃自A. mellea YM (pH 5)者較佳,還原力(OD700)分別為0.56 (在濃度1 mg/ml時)、0.59 (在濃度1 mg/ml時)和0.81 (在濃度5 mg/ml時)。螯合亞鐵離子能力方面,熱水萃取物中,以萃自A. mellea MBF培養者較佳,在濃度20 mg/ml時,達90%;在甲醇萃取物中,以萃取自M. esculenta MBF培養者較佳,在5 mg/ml時,達91.59%;在胞外發酵液中,以取自A. mellea YM培養者較佳,在濃度5 mg/ml時,達89.84%。四種抗氧化性質中,僅有A. mellea之還原力和多酚量有較明顯的正相關性。 The production of endopolysaccharide and exopolysaccharide as well as antioxidant properties of Morchella esculenta BCRC 36348 and Armillaria mellea ATCC 11113 grown in YM or modified Bisakowski’s formula (MBF) broth were investigated。 The amount of endopolysaccharide and exopolysaccharide produced by M. esculenta in MBF broth were 166 mg/g DW and 0.28 mg/ml, respectively. The amount of endopolysaccharide and exopolysaccharide produced by A. mellea in MBF broth were 82.6 mg/g DW and 0.73 mg/ml, respectively. Both M. esculenta and A. mellea grown in MBF broth produced more endopolysaccharide and exopolysaccharide than those in YM broth. To evaluate the antioxidant properties, we examined the antioxidant activity by conjugated diene method, the scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals, the reducing power and the chelating effects on ferrous ions of hot water extract, methanol extract and extracellular fluid. The higher antioxidant activities were obtained in hot water extract from M. esculenta in YM broth, 78.7% at 10 mg/ml, methanol extract from A. mellea in YM (pH 5) broth, 91.6% at 5 mg/ml and extracellular fluid from A. mellea in YM (pH 5) broth, 86.5% at 5 mg/ml. The better scavenging effects were observed in hot water extract from A. mellea both in YM and MBF broth, ~76% at 2 mg/ml, methanol extract and extracellular fluid from A. mellea in YM (pH 5) broth, 95.2 and 93.9% at 5 mg/ml, respectively. The higher reducing powers were obtained in hot water extract, methanol extract and extracellular fluid from A. mellea in YM (pH 5) broth, 0.56 at 1 mg/ml,0.6 at 1 mg/ml, and 0.81 at 5 mg/ml, respctively. The better chelating effects on ferrous ions were observed in hot water extract from A. mellea in MBF broth, 90% at 20 mg/ml, methanol extract from M. esculenta in MBF broth, 91.6% at 5 mg/ml, extracellular fluid from A. mellea in YM (pH 5) broth, 89.8% at 5 mg/ml. The reducing powers of hot water extract, methanol extract and extracellular fluid from A. mellea showed positive correlation with their total phenol amount.

Record ID 第31筆 System ID 090TTU00106004
BCRC ID CCRC 10459, Public Year 90
Paper Name 以分子生物方法處理鉻酸鹽之研究 Studies on The Treatment of Chromate by Molecular Biology
Student Name 林隆誌
Teacher Name 林銘澤
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract CrO42-多來自於工業廢水,其中以金屬和皮革製造業所佔比例最大。而CrO42-容易造成腸胃方面的疾病,近幾年更證明與胃癌有相和肝癌有相當直接的關係,所以一直是公共衛生上的嚴重問題。本實驗室由過去的研究中,了解Pseudomaons putida CCRC10459可對鉻酸還原,主要是藉由chromate reductase進行鉻酸的還原作用。所以本實驗室選殖chromate reductase的基因,建構一表現質體並送入大腸桿菌進行表現。將已先誘導表現兩小時的轉形株於LB培養基中添加不同鉻酸鹽處理6小時,發現添加量於50μM至1000μM範圍內,鉻酸有顯著地被還原消耗。而在defined medium培養下,重組大腸桿菌於含鉻酸環境下,生長較LB培養差,但是以低濃度鉻酸處理的殘量,則比以LB培養時低。另外,不論轉型菌株或是Pseudomonas putida野生株對於鉻酸的消耗,隨著時間增長而增加,但是在靜置下,卻只在第一小時內鉻酸有明顯的被消耗。 Chromates are released into environment from wastewater and ash of manufactures for pigments, alloy, and leather tanning. Chromate usually causes the sickness in gastrointestinal tract, and is directly relative to the cancers of stomach and liver reported in nearly years. Pseudomonas putida CCRC 10459 was studied for the resistant and reduction of chromate in our laboratory for several years。 In order to resolve the problem, the chromate reductase gene (chrR) was cloned from its genome and expressed in E. coli BL-21. The transformant, induced by IPTG for 2 hours, was cultured in LB medium with different concentrations of chromate ion. After 6 hours, all the concentrations of chromate are obviously reduced from the cultures with 50 to 1000M of chromate. The induced recombinant E. coli grows more poorly in the defined medium than in LB medium, but reduces more chromate at low concentration. On the other hand, the induced recombinant and Pseudomonas putida CCRC 10459 can reduce chromate continuously in aerobic culture, but only at the first hours in anaerobic culture

Record ID 第32筆 System ID 093TTU05106003
BCRC ID CCRC 14609, CCRC 11844, CCRC 11846, Public Year 93
Paper Name 高純度異麥芽寡糖對比菲德氏菌增值的影響 EFFECT OF HIGH-CONTENT ISOMALTOOLIGOSACCHARIDES ON THE GROWTH OF BIFIDOBACTERIA
Student Name 徐雅亭
Teacher Name 許垤棊
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 比菲德氏菌存在於人體的大腸和結腸中,消化性糖無法到達此處。然而,有一些寡糖成分,具有非消化性, 可到達大腸和結腸,進而資化比菲德氏菌。異麥芽寡糖,是以麥芽糖為基質,利用glucosyltransferase 催化反應生成,為機能性寡糖,是一種益生源,對於比菲德氏菌的生長具有很好的促進作用。利用酵母菌去除市售異麥芽寡糖中的消化性糖,亦即麥芽糖及葡萄糖,使寡糖的純度由58%提高至99%以上。在2公升醱酵槽中,用1.3公升的培養基,添加5%的高純度異麥芽寡糖,在礦油覆蓋下的厭氧條件、於37°C、攪拌速度100rpm,觀察比菲德氏菌的生長。醱酵過程中,監測培養液的混濁度、生菌數、氧化還原電位、pH值、醋酸與乳酸濃度,以及用HPLC分析寡糖成分的變化。 在72小時醱酵中,比菲德氏菌主要代謝panose, tertesaccharides 和isomaltose,因而產生glucose和maltose。B. adolescentis CCRC 14609利用異麥芽寡糖的速度為最快速且乳酸的產量37.41 g/L相較於其他菌株為最大而B. bifidum CCRC 11844和B. breve CCRC 11846則在高純度異麥芽寡糖裡生長緩慢,因此寡糖的代謝較其他的菌株為差。由於此寡糖不含消化性糖,所以這樣的研究比前人利用含有大量消化性糖的寡糖的研究更具有意義。因此可以了解比菲德氏菌對於異麥芽寡糖中不同成分的利用情形,也可以瞭解不同的比菲德氏菌利用寡糖的差異。 Bifidobacteria can promote human health. Bifidobacteria are regularly found in human large intestine and colon, where digestible sugars such as glucose, sucrose and maltose, are not attainable. Isomaltooligosaccharides (IMO), a function sugar, was made from maltose via a reaction catalyzed by glucosyltransferase. IMO is preprobiotic, being able to enhance the growth of bifidobacteria. High-content IMO can be produced, by treating commercial IMO syrup with yeast, thereby digestible sugar including maltose and glucose are depleted. In this way, the content of IMO increases from 58% to 99% on a dry weight basis. In a 2-L jar fermenter, bifidobacteria were cultured in 1.3 L broth in the presence of 5% (w/v) high-content IMO. The fermentation conditions were as following: anaerobic culture, overlaid with a layer of liquid paraffin; stir rate, 100rpm; temperature, 37°C. During fermentation, turbidity, viable count, ORP, pH, acetate, lactate and sugar components were determined periodically. During 72 h of fermentation, most of the bifidobacteria used in this study metabolize primarily panose, tertesaccharides and isomaltose and thus produced G and IG2. B. adolescentis CCRC 14609 consumed IMO much faster than other strains did and produced the maximum lactic acid of 37。41 g/L. But B. bifidum CCRC 11844 and B breve CCRC 11846 did not grow well and consumed isomaltooligosaccharides poorly。 The present investigation using such oligosaccharides which was free of digestible sugars was much more significant than past research, in which oligosaccharides accompanied with large amounts of digestible sugars were used. The utilization of various IMO components by eight bifidobacteria was clearly understood.

Record ID 第33筆 System ID 090TTU00106006
BCRC ID CCRC 21436, CCRC 21326, CCRC 21854, Public Year 90
Paper Name 利用ORP-stat控制策略培養酵母菌生產木糖醇 Fermentation of Xylose into Xylitol by Yeasts using ORP-stat Feeding Strategy
Student Name 周宣如
Teacher Name 許垤綦
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 木糖醇是五個碳的醣醇,木糖醇具有甜味及蛀牙特性,且木糖醇廣泛的被添加於食品和飲料中,當作甜味劑。木糖醇的生產可以利用酵母醱酵法,將木醣還原成木糖醇。本實驗使用Candida tropicalis CCRC 21436, Candida guilliermondii CCRC 21326和 Debaryomyces hansenii CCRC 21854當作饋料批式醱酵的種菌。在木醣的醱酵過程中添加葡萄糖,並且控制在低溶氧狀態下,可以得到較高的木糖醇產量。在低溶氧狀態下,溶氧會接近於零,造成溶氧值難被偵測,因此以氧化還原電位來代替溶氧的偵測。在木醣的醱酵過程中添加葡萄糖,會造成醱酵培養液中氧化還原電位的下降,但是當醱酵培養液中的葡萄糖被消耗完畢,氧化還原電位則會上升,因此利用上述特性,木醣醱酵產生木糖醇的過程中,則可利用ORP-stat來控制葡萄糖的添加。比較Candida tropicalis CCRC 21436, Candida guilliermondii CCRC 21326和 Debaryomyces hansenii CCRC 21854利用ORP-stat醱酵木醣產生木糖醇,實驗結果發現Candida tropicalis CCRC 21436,在ORP為-180mv,通氣量為0.2 L/min時可得最高的木糖醇產量。木糖醇的產量會受到醱酵液、饋料策略、攪拌速率、通氣量等影響。比較Candida tropicalis CCRC 21436利用ORP-stat醱酵木醣產生木糖醇,醱酵液及溶氧對木糖醇生產之影響實驗結果發現Candida tropicalis CCRC 21436培養在2升的醱酵液中,其醱酵液成分為40 g/L glucose; 100 g/L D-xylose, 20 g/L yeast extract, 5 g/L KH2PO4及0.2 g/L MgSO4。醱酵溫度控制在30°C,攪拌速率控制在300 rpm,通氣量控制在2.0 L/min,pH控制在5.0。在醱酵8小時,當葡萄糖消耗完畢時,添加500 g/L的饋料液,饋料速度為4 mL/min。在醱酵12小時,當酵母菌量達到最高值時,將通氣量由2.0 L/min 降低到0.2 L/min並且將ORP控制在-180 mv。在醱酵24小時,可得到最多的木糖醇累積量(91 g/L),最佳的木糖醇轉化率(100 %),最佳的木糖消耗率(95 %)及最多的木糖醇比生長速率(0.6 g/g/h)。基於上述可以得知,在木糖醱酵產生木糖醇的過程中,可利用ORP-stat來控制葡萄糖之添加,以克服溶氧電極於低溶氧時,不易偵測控制的困難。 Xylitol is a naturally occurring sugar alcohol. Its high sweetening power, anti-cariogenic properties and possibilities for use in diabetic food products makes xylitol an attractive sucrose substitute in a wide variety of foods and beverages. Xylitol can be produced by biological reduction of xylose, a five-carbon sugar, using microorganisms. The fed-batch fermentation processes were carried out by using three yeasts-Candida tropicalis CCRC 21436, Candida guilliermondii CCRC 21326 and Debaryomyces hansenii CCRC 21854。 In the process of xylose fermentation into xylitol, feeding glucose could enhance xylitol production. The xylose fermentation was carried out under oxygen-limited conditions. Under such condition DO is too low to be precisely detected, therefore ORP can be used as a control index of oxygen supply during micro-aerobic fermentation. Intermittent glucose-feeding caused the fermentation medium to get decreased ORP. After glucose in medium was used up ORP fluctuated. According to this property, ORP-stat glucose-feeding fermentation processes were carried out to optimize the production of xylitol from xylose. The processes of ORP-stat glucose-feeding fermentation using strain Candida tropicalis CCRC 21436, Candida guilliermondii CCRC 21326 and Debaryomyces hansenii CCRC 21854 were compared Candida tropicalis CCRC 21436 had the optimum production of xylitol at ORP of -180 mv and aeration rate of 0。2 L/min. Xylitol yield in the xylose fermentation processes were influenced by the factors, such as composition of fermentation medium, feeding strategy, stir rate and aeration rate. Comparison in the influence of medium composition and oxygen transfer on the ORP-stat fermentation by Candida tropicalis CCRC 21436 was investigated The optimum results were obtained by Candida tropicalis CCRC 21436 Candida tropicalis CCRC 21436 was cultivated in the medium consisting of the following ingredients per liter: glucose, 40 g; D-xylose, 100 g; yeast extract, 20 g; KH2PO4, 5 g; MgSO4, 0。2 g. The fermentation was carried out in a 5-liter jar fermenter under the following conditions: initial working volume of fermentation medium, 2 liters; temperature, 30°C; stir rate, 300 rpm; aeration rate, 2.0 L/min; automatically controlled pH, at 5.0. After glucose was used up, at hr 8, an autoclaved sugar solution consisting of 500 g/L glucose was fed at a rate of 4 mL/min. At hr 12, glucose was fed by ORP-stat at -180 mv and the aeration rate was switched from 2.0 to 0.2 L/min. At hr 24 in the ORP-stat fermentation, 91 g/L accumulated xylitol, 100 % xylitol conversion from xylose, 95 % xylose consumption and 0.6 g/g/h maximun specific productivity of xylitol, were obtained respectively. The fact that the ORP-stat glucose-feeding fermentation for the production of xylitol was successfully carried out manifested the advantage of monitoring of ORP in the fermentation process. In the case of xylose fermentation into xylitol, ORP-stat glucose feeding was a novel feeding strategy in fermentation process.

Record ID 第34筆 System ID 093TTU00106007
BCRC ID BCRC 10094, Public Year 93
Paper Name 利用不同澱粉與環狀糊精葡萄糖苷轉移酶生產生產環狀糊精之研究 Production of cyclodextrin using cyclodextrin glycosyltransferase and different starch
Student Name 陳鈞浩
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 環狀糊精( cyclodextrins , CDs)是由6~8個葡萄糖單元,以α-1,4-糖苷鍵結形成的環狀化寡糖。廣泛應用於許多領域,例如醫藥、化妝品、食品工業等。環狀糊精是由是由利用葡萄糖苷轉移酵素催化澱粉而生成。本研究以Bacillus sp. BCRC10094為CGTase 的生產菌株進行搖瓶振盪培養,探討碳源對發酵活性的影響,比較玉米澱粉、木薯澱粉、可溶性澱粉(關東化學株式會社)、麥芽糖、以及 DE10, DE20 的可溶性澱粉(環泰果糖股份有限公司)作為震盪培養的碳源,結果發現使用2 ﹪(w/v)玉米澱粉、木薯澱粉、可溶性澱粉為基質,在 33 ℃、160 rpm下得到 1400 unit/ml 的活性,但使用麥芽糖與DE10, DE20的可溶性澱粉作為碳源, 得到較低的活性。將本研究的酵素與NOVO的商業酵素進行環狀糊精的合成實驗,配製 15% (w/v) 的各種澱粉,以活性250 unit/g-starch 的 Novo 酵素與玉米澱粉在 75 ℃進行澱粉轉化實驗反應 24 小時,結果發現玉米澱粉可得到 33 % 的β - CD。若使用自己發酵的CGTase則需要 1000~2000 unit/g-starch的酵素,反應24小時,可以得到 18 ~23 % 的轉化率,顯示NOVO酵素有很好的熱穩定性。 Cyclodextrins (CDs) are cyclic oligosaccharides composed of 6-8 glucose with α-1, 4-linkages. CDs have been used in food, pharmaceutics, cosmetics industries. CDs are produced by enzymatic catalyzation of cyclodextrin glucanotransferase (CGTase) from starch. In this study, CGTase is produced using starch or starch hydrolyzates as carbon sources from a Bacillus sp. BCRC 10094。 The maximum CGTase activity was about 1400 unit/ml in a shaking culture using 2%(v/w)corn starch, cassava starch, soluble starch (Kanto Chemical Co. Ltd) at 33℃and 160 rpm. The carbon sources using DE10, DE20 soluble starch (Taiwan Fructose Co.,Ltd) and maltose in the medium produced less CGTase activity. The CGTase of this study and commercial CGTase (NOVO) were employed to produce CDs from 15 % (w/v) starch. CDs were produced from corn starch catalyzed by CGTase (NOVO, 250 unit/g-starch) at 75 C for 24 hrs. The yield of CDs from starch was 33 %. However, it was 1000 ~ 2000 unit needed per gram of starch for the CGTase cultured from Bacillus sp. BCRC 10094 in this study。 The yield of CDs from starch was 18 ~23 % using our enzyme. The results indicated that CGTase from NOVO had better thermal stability.

Record ID 第35筆 System ID 090TTU00106008
BCRC ID CCRC 16016, Public Year 90
Paper Name 以PCR檢測硝化及脫硝菌方法之研究 STUDY FOR THE DETECTION METHOD OF NITRIFYING AND DENITRIFYING BACTERIA BY PCR
Student Name 黃惠貞
Teacher Name 林銘澤
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 由家庭生活污水及畜牧業與食品廠、ABS塑膠廠、石化工業、半導體等工廠廢水大量排放含銨氮的廢水,主要是以混合菌株培養處理。銨氮廢水處理的活性汙泥裡的微生物族群複雜,因此相關細菌的實際生長與分布研究困難。由於分子生物技術的進步,而可以直接鑑定菌種種類與定量。在氮循環之脫硝反應中包含多種酵素,分別比對其基因序列,並設計出nosZ,norB,nirS與nirK基因之引子,配合作為正反應控制組的16S rDNA基因序列的通用引子,而與Alcaligenes xylosoxidans、Bacillus cereus、E. coli、Paracoccus denitrificans、Pseudomonas aeruginosa、Pseudomonas fluorescens、Pseudomonas stutzeri、Rhodobacter sphaeroides等細菌的DNA,進行聚合酵素連鎖反應(PCR),以進行脫硝菌的定性、定量快速檢測。結果Pseudomonas fluorescens CCRC 16016可以和多數的nirS引子反應,Alcaligenes xylosoxidans CCRC12838則和nirK引子能獲得預期的PCR產物,而能被準確的偵測。而norB引子和多數的菌沒有反應,nosZ引子則只和Alcaligenes faecalis會有專一的反應,因此在此實驗中,此二基因不適合作為檢測之用。 抽取含氮廢水處理槽中活性汙泥裡的細菌DNA,分別和nirK與nirS引子進行PCR反應,結果汙泥菌中的DNA可和nirS1f-nirS3r,nirS1f-nirS7r,nirS2f-nirS7r,nirS4f-nirS6R引子對,經PCR產生正確DNA片段;而nirK引子則僅和nirK1f-nirK5r引子對有反應,因此這些引子對可用於含氮廢水處理槽脫硝菌之檢測。 另外並設計硝化作用中AMO基因之各種amoA引子對,直接和硝化槽中之硝化菌進行檢測,但反應並不理想。 For health and environmental protection, municipal and industrial wastewaters have to be treated. Wastewater with ammonia-nitrogen, generally treated with mixed microbial populations, is produced by domestic wastewater, and wastewater from livestock and manufacture about food, ABS plastic, petroleum, and semiconductor at a large amount. The directly determination of the growths and distributions of relative bacteria are less or unsuccessful because of its complexity of populations in activated sludge. Due to the improvement of technology for molecular biology, the amounts and identify of the species can be directly determinated. There are many enzymes involved in nitrification of nitrogen cycle. Compare the genes sequences to design the primer pairs for nosZ, norB, nirS, and nirK. These primer pairs and the universal primer of 16S rDNA with the genomic DNAs of Alcaligenes xylosoxidans, Bacillus cereus, E. col, Paracoccus denitrificans, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas stutzer, Rhodobacter sphaeroides carry out the polymerase chain reaction for the rapidly determination of denitrifying bacteria. Pseudomonas fluorescens CCRC 16016 can react properly with almost nirS primer pairs, and Alcaligenes xylosoxidans CCRC12838 with nirK pimer pairs can produce the expect PCR product。 However, norB primers can’t react with bacterial genomic DNA, and nosZ primers are specific to Alcaligenes faecalis DNA. So these primer pairs of the two genes are unsuitable for the detection of denitrifying bacteria. Isolate the bacterial genomic DNA from the activated sludge in treatment tank of wastewater with nitrogen compounds to do PCR with nirK and nirS primers. nirS1f-nirS3r, nirS1f-nirS7r, nirS2f-nirS7r, and nirS4f-nirS6R primer pairs can produce correct DNA fragments, and the nirK primer pairs are only the nirK1f-nirK5r primer pair with positive reaction. Thus, these primer pairs can be used for the determination of nitrifying bacteria in treatment-tank of wastewater. Additionally, used the designed amoA primer pairs, according to the AMO gene, to determinate the nitrifying bacteria in nitrifying tank, but they were inappropriate.

Record ID 第36筆 System ID 093TTU05106002
BCRC ID CCRC 9363為, CCRC 9363, Public Year 93
Paper Name 以米根黴生產L型乳酸 Production of L (+)�{lactic acid by Rhizopus oryzae
Student Name 林怡君
Teacher Name 許垤棊
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract L型乳酸,分子式C3H6O3,為含有氫氧基的有機酸,是生物的代謝中間產物。L型乳酸可以在動物和人類細胞中新陳代謝因為這些細胞含有L型乳酸去氫酵素。L型乳酸還可用於生産L型乳酸聚合物。L型乳酸聚合物屬於無毒的高分子化合物,具有生物相容性,可用於製造生物可分解的塑膠、纖維以及生醫材料等。本研究以黴菌Rhizopus oryzae CCRC 9363為L型乳酸生產菌,針對生物反應器的種類與最適操作條件來進行探討。最後由實驗得知以改良型的生物反應器為最適生產L型乳酸的反應器。 L-lactic acid, C3H6O3, an organic acid with hydroxy group is a metabolic intermediate in living organisms. Only the L-form of lactic acid is metabolized in animal and human cells. This is due to the fact that only L-lactate dehydrogenase is synthesised by these cells. L-lactic acid can be polymerized to form Poly�{lactic acid (PLA). L-form PLA is a non�{virulent macromolecular compound with biocompatibility, which used in the manufacture of new biodegradable plastics, fiber, and biomedical material. This experiment used fungus, Rhizopus oryzae CCRC 9363 to produce L-lactic acid and discussed the types of bioreactor and best conditions。 Final, it is discovered that the modified bioreactor is the best bioreactor to produce L-lactic acid.

Record ID 第37筆 System ID 090TTU00106012
BCRC ID CCRC 31494, Public Year 90
Paper Name 黑麴菌α-半乳糖苷酵素純化與性質研究 Purification and characterization of α-galactosidase from Aspergillus niger CCRC31494
Student Name 郭建平
Teacher Name 顏聰榮
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 我們從Aspergillus niger CCRC 31494的發酵液中純化出具有高活性的α-半乳糖苷酶。在SDS PAGE電泳片上我們可以得知此純化出來的酵素的次單元體分子量是64KDa,從膠體層析中我們可以得到245 Kda分子量的酵素,這兩者之間的差距,可進一步推測α-半乳糖苷酶是由四個次單元體所構成。以Endoglycosidase H來切除α-半乳糖苷酶外以N鍵結的寡糖,在SDS PAGE的電泳片上可看到一個55KDa拖曳狀的色帶,這個分子量差距的結果,我們認為α-半乳糖苷酶含有high mannose與hybride這兩種型式的寡醣,且佔總分子量的14%。α-半乳糖苷酶對pNP-α-Gal最適反應條件是在pH4.0,60℃的環境下;在pH 3~4之間的酸性水溶液有較佳的活性表現;在溫度40℃以下的熱環境可穩定存在長達4個小時。α-半乳糖苷酶對mNP-α-Gal,oNP-α-Gal及pNP-α-Gal的酵素動力學參數Km值分別是5.01 mM,Km=1.79 mM和Km=1.405 mM。此外,我們實驗証實了α-半乳糖苷酶可分解天然基質melibiose、raffinos、stachyose,尤其是它對melibiose有很高的水解效率,適合使用在食品加工業與製糖工業上來去除這些干擾物質。在酵素活性抑制的研究發現加入銀離子,汞離子, galactose, melibiose對α-半乳糖苷酶有競爭型抑制的情況。 An extracellular enzyme with high α-galactosidase activity was purified from Aspergillus niger CCRC31494 culture filtrate。 The molecular mass of this enzyme was estimated to be 254 kDa on Sephacryl S-300HR gel filtration, and 64 kDa by SDS-PAGE. Therefore, purified α-galactosidase was considered to be a tetramer composed of four 64 kDa subunits. After treating the purified enzyme with Endoglycosidase H, we could observe a 55 kDa dragged band in SDS-PAGE, so we supposed that purified α-galactosidase has 14 % N-linked oligosaccharides. α-Galactosidase, optimally active at pH 4.0 and 60oC, was measured by p-nitrophenyl α-D-galactopyranoside. In pH 3.0~4.0, it could retain most of its activity. At the optimum temperature, this enzyme was unstable, but it was stable at 40oC up to 4 hours. Kinetic parameters of α-galactosidase toward mNP-α-Gal, oNP-α-Gal, and pNP-α-Gal were Km=5.01 mM, Km=1.79 mM, and Km=1.405 mM, respectively. Some natural substrates such as melibiose, raffinose, and stachyose could be hydrolyzed by α-galactosidase. This enzyme hydrolyzed melibiose more efficiently than that of raffinose and stachyose in 6 hours. So it can be used in sugar-making and food industry to remove these interfering substrates. In addition, we investigated that the activity of α-galactosidase was strongly inhibited by 1mM metal ions─Ag+, Hg2+,and 25mM saccharides─ galactose, melibiose.

Record ID 第38筆 System ID 090TTU00106014
BCRC ID CCRC 22637, Public Year 90
Paper Name 利用批式和饋料批式醱酵生產酵母菌之胞外多醣 Production of Extracellular Polysaccharide from Yeasts by Batch Fed-batch Fermentation
Student Name 黃世偉
Teacher Name 許垤棋
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 酵母菌的細胞外多醣可以當作硫化寡糖(一種抗凝血藥物)的原料,因而受到格外的重視。酵母菌的細胞外多醣含有大量的磷酸甘露聚醣,經過部分水解、管柱分離、純化以及硫化 (加入硫酸根後),形成硫化寡糖,是一種口服型的抗凝血藥物,可防止血拴、心肌梗塞、增生性視網膜剝離、關節惡化等疾病以及有些病毒的感染。本研究主要是利用數株酵母菌,於五公升醱酵槽中,在各種條件下,探討細胞外多醣的最大產量。研究的醱酵條件包括:不同的溫度、pH值、葡萄糖濃度、通氣量、攪拌速度、饋料方式等。批式、饋料批式、重複地饋料批式以及連續式的生產都將納入研究範圍。利用”Genie”軟體和個人電腦控制生物反應槽的溶氧偵測器、pH偵測器、氧化還原偵測器、葡萄糖分析儀、饋料蠕動幫浦、空氣閥等週邊設備,以得到醱酵槽生產細胞外多醣的最適條件。實驗發現,Pichia holstii CCRC 22637.在轉速為450 rpm與通氣量為2 L/min,溫度維持在25℃,pH控制在4.7~5.3之間,經由長時間醱酵培養120小時後,菌體量可達10.9 g L-1,細胞外多醣產量為53.4 g L-1。若將培養基中葡萄糖的濃度增加為兩倍(120 g L-1)時,細胞外多醣的產量可提升至73.2 g L-1,菌體量可達10.6 g L-1。以饋料批次方式培養,在實驗過程中補充消耗的葡萄糖與磷酸鹽,來增加細胞外多醣的產量。實驗發現,在醱酵培養336小時後,細胞外多醣的產量可達112 g L-1,菌體量可達12.3 g L-1。若將培養基中氮源的含量增加為兩倍時,菌體量可達21.6 g L-1,細胞外多醣的產量可達129 g L-1。 Recently, being able to serve as a raw material for the synthesis of sulfated oligosaccharide, a potential anticoagulant, anti-virus and anti-tumor agent, yeast extracellular polysaccharide was becoming attractive. Batch and fed-batch fermentation was carried out in a 5-liter jar fermenter for the production of extracellular polysaccharide from Pichia holstii CCRC 22637。 The fermentation medium consisted of the following ingredients per liter: glucose, 60; tryptone, 1; corn steep liquor, 1; KH2PO4, 10; MgSO4Ÿ7H2O, 0.2; MnSO4ŸH2O, 0.01; FeSO4Ÿ7H2O, 0.01 and NaCl, 0.01. Fermentation was carried out under the following conditions: initial working volume, 2 L; agitation, 450 rpm; aeration, 2 L min-1; temperature, 25°C and pH controlled at 4.7-5.3. After a 80-hr batch fermentation, 10.9 g L-1 dry weight of cells and 53.4 g L-1 dry weight of extracellular polysaccharide were obtained. When glucose and phosphate content in the medium was doubled, 10.6 g L-1 dry weight of cells and 73.2 g L-1 dry weight of extracellular polysaccharide was achieved at hr 124. In fed-batch fermentation, when half of initial glucose in the medium was exhausted, one tenth working volume of a solution containing 60 % (w/v) glucose and 5% (w/v) KH2PO4 was fed into fermenter and three repeated operations for such feeding were carried out throughout the fermentation process. After a 336-hr fed-batch fermentation, 12.3 g L-1 dry weight of cells and 112 g L-1 dry weight of extracellular polysaccharide were obtained. In a similar process of fed-batch fermentation, when nitrogen source was doubled in concentration, 21.6 g L-1 dry weight of cells and 129 g L-1 dry weight of extracellular polysaccharide were achieved at hr 364.

Record ID 第39筆 System ID 093TTU00106003
BCRC ID BCRC 930007(patent, Public Year 93
Paper Name 固定化酵素生產果寡糖與半乳寡糖 Production of fructooligosaccharides and galactooligosaccharides by immobilized enzymes
Student Name 鄭慈千
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 博士
Abstract 中文摘要 本研究以Tris(hydroxymethyl)phosphine (THP) 為交聯劑與幾丁聚醣共交聯分別與β–夫喃果糖苷酶和β–半乳糖苷酶固定生產果寡糖(FOS)與半乳寡糖(GOS)。結果顯示使用THP為交聯劑與幾丁聚醣共交聯固定酵素後,酵素之熱穩定性、重複使用性與產率良好。 從黴菌Aspergillus japonicus (BCRC 930007)純化得到β–夫喃果糖苷酶,分別以Tris(hydroxymethyl)phosphine (THP)與戊二醛為交聯劑與幾丁聚醣共交聯反應以固定β–夫喃果糖苷酶生產果寡糖。以2.5 mg/ml THP或5 % (w/v)戊二醛分別與0.1 g幾丁聚醣及4.2 μg/ml的酵素進行固定化反應,結果顯示自由酵素與固定化酵素的最適pH皆為5.5,最適溫度為60 oC。自由酵素保存於37 oC下,第二天只剩下60 %活性,但是若將THP-固定化酵素保存於37 oC下,11天後仍然有80 %活性,顯然使用THP為交聯劑固定後酵素的熱穩定性良好。THP-固定化酵素在重複使用實驗中,於37 oC下經過11天後仍然有75 %的活性。自由酵素,THP-固定化酵素與戊二醛-固定化酵素的酵素動力參數Km,分別為0.03 g/l (s.d.=0.03), 0.06 g/l (s.d.=0.05) 與0.05 g/l (s.d.=0.03) ,顯示固定化酵素在反應過程中有立體障礙與擴散阻力效應。以50 %蔗糖的基質溶液分別加入固定化酵素與自由酵素(1 unit/g),於pH 5.5,50 oC下反應,果寡糖產率分別為48 %與58 %。 利用Tris(hydroxymethyl) phosphine(THP)與幾丁聚醣共交聯反應以固定β–半乳糖苷酶生產半乳寡糖(GOS)。以2.5 mg /ml THP活化0.5 g幾丁顆粒及1 mg/ml的酵素進行固定化反應。固定化酵素與自由酵素最適反應溫度為55 oC,最適反應pH為5.0。固定化酵素在重複使用實驗中,於55 oC下經過13天後仍然有75 %的活性,顯示使用THP為交聯劑固定化酵素的熱穩定性良好。自由酵素在pH 5.0,55 oC,36 %乳糖溶液下反應,GOS的產率為43 %,而固定化酵素在相同條件下GOS的產率則為41 %兩者約略相同。 ABSTRACT We present the results of this study in the production of fructooligosaccharides (FOS) and galactooligosaccharides (GOS) by immobilized enzymes from sucrose and lactose solution. The β-frutofuranosidase and β-galactosidase were immobilized onto chitosan using tris (hydroxymethyl) phosphine (THP) as a coupling agent to produce fructooligosaccharides and galactooligosaccharides, respectively. Our results demonstrated the possible industrial application of the THP-immobilized β–frutofuranosidase and ��-galactosidase for continuous production of fructooligosaccharides and galactooligosaccharides at longer duration and higher temperature tolerance at 55 oC. Our study show the THP-immobilization has better thermal stability then the glutaraldehyde as a coupling agent. For the production of fructooligosaccharides, β–frutofuranosidase from Aspergillus japonicus (BCRC 930007) was immobilized onto chitosan using tris (hydroxymethyl) phosphine (THP) and glutaraldehyde as coupling agents to produce FOS。 The optimal pH was 5.5 and the optimal temperature was 60oC for both free and two immobilized enzymes. The THP-immobilized β–frutofuranosidase retained more than 75 % activity after 11 batches (or days) of FOS production at 37oC. The THP-immobilized enzyme had higher reusability than that immobilized by glutaraldehyde. The Km for the free, THP-immobilized and glutaraldehyde-immobilized enzyme were 0.03 g/l (s.d.= 0.03), 0.06 g/l (s.d.= 0.05) and 0.05 g/l (s.d.= 0.03), respectively. The yield of FOS with the free enzyme from the sucrose solution (50 %, w/v) at 50 oC was 58 % and the THP-immobilized enzyme (48 %) is less than the free enzyme. For the production of galactooligosaccharides, the β-galactosidase was immobilized on to chitosan using tris (hydroxymethyl) phosphine (THP) as a coupling agent to produce galactooligosaccharides from lactose solution. Both the THP-immobilized and the free enzymes were maximally achieved at pH 5.0 and the optimal temperature was 55 oC. The THP-immobilized enzyme still can remain 70 % residual activity from 36 % (w/v) of lactose after 13 batches (or days) at 55oC. This show the THP-immobilized has better thermal stability. The yield of GOS with the free enzyme from the lactose solution (36 %, w/v) at 55 oC is 43 %, which is about the same as that with the THP-immobilized counterpart with 41 % yield.

Record ID 第40筆 System ID 093TTU00106002
BCRC ID BCRC 36348, Public Year 93
Paper Name 深層培養羊肚菌(Morchella esculenta)之最適化培養液組成 Optimization of the medium components for Morchella esculenta submerged culture
Student Name 劉梅玉
Teacher Name 李繡鈴
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 本研究乃在探討液態培養M. esculenta BCRC 36348以獲得其多醣之最適培養液組成。當以glucose為碳源時,其菌絲體乾重、胞內多醣及總多醣產量最高,添加濃度為2 %時,可獲得最高之總多醣產量及產率。yeast extract為培養M. esculenta BCRC 36348 生產多醣之最適氮源,添加0.5 %yeast extract時,可獲得最高之總多醣產量及產率培養液中添加植物油雖會抑制M. esculenta BCRC 36348之生長,但會顯著提高其總多醣產量、含量及產率,以添加1 % 玉米油之效果最佳當以2 % glucose、0.5 % yeast extract、1 % corn oil、0.0068 % KH2PO4 及10 % mineral solution之培養液於25℃下進行M. esculenta BCRC 36348之液態培養6天後,可獲得最高總多醣之產量及含量,分別為2.18 g/L及335.64 mg/g DW經由反應曲面試驗設計得知,當培養液組成為3.31 % glucose、0.39 % yeast extract、 1 % corn oil、0.0068 % KH2PO4 及10 % mineral solution時,培養M. esculenta BCRC 36348 6天後,可獲得最大之胞內多醣產量,約為1.97 g/L。若培養液組成為3.34 % glucose、0.41 % yeast extract、1 % corn oil、0.0068 % KH2PO4 及10 % mineral solution時,則可獲得最大之總多醣產量,約為2.22 g/L。 In this study, the optimized medium composition for polysaccharide production in submerged cultures of M. esculenta BCRC 36348 were investigated。 When using 2% glucose as carbon source, maximal mycelium dry weight, formation of intracellular polysaccharide and total polysaccharide were obtained. Yeast extract is the optimal nitrogen source. When 0.5% yeast extract were added, maximal production and yield of total polysaccharide were found. Although addition of plant oils inhibit the growth of M. esculenta BCRC 36348, production, content and yield of total polysaccharide significantly increased。 Addition of 1% corn oil to the medium gave the best effect. After cultivation for 6 d, the maximum production and content of total polysaccharide, i.e. 2.18 g/L and 335.64 mg/g DW, respectively, by M. esculenta BCRC 36348 were found in the medium containing 2 % glucose, 0。5 % yeast extract, 1 % corn oil, 0.0068 % KH2PO4 and 10 % mineral solution. With response surface methodology, maximal production of intracellular polysaccharide (1.97 g/L) was obtained by M. esculenta BCRC 36348 after 6 d of cultivation when the medium contained 3。31 % glucose, 0.39 % yeast extract, 1 % corn oil, 0.0068 % KH2PO4 and 10 % mineral solution. While maximal production of total polysaccharide (2.22 g/L) was found when medium contained 3.34 % glucose, 0.41 % yeast extract, 1 % corn oil, 0.0068 % KH2PO4 and 10 % mineral solution.

Record ID 第41筆 System ID 093TTU01106002
BCRC ID BCRC 31615, BCRC 31499, Public Year 93
Paper Name 以米為基質固態培養紅麴菌生產色素、Monacolin K及Citrinin產量變化之研究 Solid fermentation of Monascus sp. using rice substrates in production of the pigment, monacolin K and citrinin
Student Name 鄧化瑜
Teacher Name 段國仁
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract 本研究選擇兩株紅麴菌株(BCRC 31615、BCRC 31499),分別以在來米(精白米),蓬萊米(糙米、精白米及發芽米)為基質於30℃培養,探討其色素及二次代謝產物Monacolin K之變化。   由市售購得之在來米及糙米(蓬萊米)作為原料,另將糙米分為三份,其中一份送至米廠脫去外層麩皮精白,另一份則浸水兩日使其發芽成為發芽米。米首先泡水蒸熟,接菌後以第六日至第十四日為採樣點,乾燥後保存,取1克麴米加入5毫升乙酸乙酯萃取後分析之。   在培養過程中觀察其外觀,發現BCRC31615的顏色比BCRC31499的顏色要深色素分析上分別以OD500nm及OD400nm偵測紅色色素及黃色色素之吸光值,BCRC31615的色素產量較高,尤其是發芽米蓬萊糙米及發芽米不利BCRC31499之色素的生成。   在Monacolin K的生成上與色素的生成有相似的趨勢。利用BCRC31615接種於在來米及三種不同形式蓬萊米(發芽米、糙米、精白米),以蓬萊發芽米培養第12日有最高產量381 mg/kg,在來米在第12日有下降的現象,隨後在第14日達最大值287 mg/kgBCRC31499則以在來米培養第十日有最高產量34 mg/kg,蓬萊糙米及發芽米則未超過10 mg/kgBCRC31615之Monacolin K生產量,無論是以何種基質培養,皆高於BCRC 31499 然而citrinin常伴隨著紅色色素生成,遂決定分析BCRC31615菌株citrinin之生成變化。在來米、去皮白米、糙米三種基質所產生的citrinin大約在80-120ppm之間,然而以發芽米培養所產生的citrinin最大值接近300ppm。以發芽米培養雖可得較高的monacolin K,但同時也有最高的citrinin產生,且其增加的幅度遠超過其他基質,所以發芽米確實存有特殊成分可以提高BCRC31615的二次代謝產物生成。 In this research, two Monascus sps. (BCRC 31615, BCRC 31499) were choosed for culturing on Japanica and Indica type of rices at 30 ℃ to understand the yields change of pigments and secondary metabolic product, Monacolin K。 The brown rice (Japanica type rice) purchased from local market was divided into three parts, the one was polished to remove the bran, the other part was immersing in water for two days to become germinated. The other kind of polished rice (Indica type rice) was also purchased from local market. Samples of the red rice were taken and dried from 6 ~14 th day after inoculated with Monascus sp. Then 1 gram of red rice was extracted with 5 ml ethyl acetate to obtain the pigment, monacolin K, and citrinin. We found that the color of red rice cultured by BCRC31615 was deeper than that from BCRC31499 during the culturing process。 The absorbance values of red and yellow pigments were determined with OD500nm and OD400nm. It was found that BCRC31615 has higher yields in pigments than BCRC31499 The brown rice and geminated rice were unfavorable for the synthesis of pigment by BCRC31499 The geminated rice has the highest yields of pigment by BCRC31615。 The production curves of monacolin K and pigment had similar trend with time for the brown rice and the germinated rice. 381 mg/kg of monacolin K was achieved for the germinated rice cultured by BCRC31615 at the 12th day of cultivation。 While that for the polished Indica rice was 287 mg/kg at the 14th day of cultivation. Monacolin K production from BCRC 31499 was only 34 mg/kg at the 10th day of cultivation for the polished Indica rice。 The brown rice and germinated rice were not good in the production of monacolin K. Yield of monacolin K was more by BCRC 31615 than that by BCRC 31499 no matter what kinds of rice were used。 Production curve of citrinin has similar trend as that of pigment. Concentrations of citrinin of red rice from BCRC 31615 were in the range of 80~120 ppm for the polished Indica rice and polished Japanica rice and brown rice The most citrinin (300 ppm) was obtained from the germinated rice which also achieved the most monacolin K from BCRC 31615。 It is suggested that some ingradients are in the germinated rice which are beneficial for the production of secondary metabolites.

Record ID 第42筆 System ID 089TTU00106009
BCRC ID CCRC 14634, Public Year 89
Paper Name 以氧化還原電極監控雙歧桿菌之醱酵研究 Monitor the fermentation process of Bifidobacterium longum by ORP
Student Name 何顯令
Teacher Name 段國仁
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 培養比菲德氏菌會產生一些代謝性有機酸如乳酸與醋酸,這些有機酸會抑制比菲德氏菌的生長。目前並無較可靠快速的方法來偵測培養比菲德氏菌過程中所產生的有機酸與乳糖的代謝情形。本研究探討培養比菲德氏菌longum CCRC14634過程中氧化還原電位的變化。我們利用二種不同的培養基以及批次、饋料批次、循環批次式、透析式和連續式等五種培養方式,來探討醱酵比菲德氏菌過程中細胞密度、培養基代謝(乳糖、葡萄糖)、代謝性有機酸濃度(乳酸、醋酸)與氧化還原電位的關係。 本研究結果發現偵測氧化還原電位可以作為判斷醱酵比菲德氏菌醱酵狀況的依據。隨著醱酵過程中主要碳源的消耗,氧化還原電位逐漸降低。以循環批次式醱酵培養可以氧化還原電位變化作為控制參數,當氧化還原電位開始持平或上升時,移除 3/4 體積的培養液,並饋入 3/4 體積的新鮮培養基以進行下一循環批次醱酵之用。在厭氧培養條件下,培養於 MRS 和 WY 培養基中,分別得到平均約 1.9×109 CFU/ml和3.42×109 CFU/ml的比菲德氏菌。另外,以 MRS 培養基進行批次饋料醱酵和透析式以去除代謝性有機酸醱酵,可得菌體量分別為 5.2×109 CFU/ml 和 1.8×1010 CFU/ml。以 WY 培養基進行連續式醱酵培養時可得菌體量為 3.1×109 CFU/ml。 The lactic acid and acetic acid are produced in the fermentation of Bifidobacteria. The metabolic organic acids inhibit cell growth during fermentation of Bifidobacteria. There are no reliable methods to detect the inhibitory organic acids and lactose uptake during fermentation. The present study monitored the ORP changes during fermentation of Bifidobacteria longum CCRC 14634。 The batch, fed-batch, cyclic batch, continuous and dialysed fermentation were performed to culture Bifidobacteria. Cell densities, uptake of carbon sources (glucose, lactose), metabolic organic acids, and ORP were detected during fermentation. It was found that ORP was very close related to cell growth of Bifidobacteria. ORP decreased continuously during fermentation until exhausting of carbon source. We have performed six cycles of batch fermentation using ORP as control parameter. When ORP remained constant or increased, three fourth of the medium was removed, and the same volume of fresh medium was fed to the fermentor for each cycle. We have achieved 1.9×109 CFU/ml and 3.4×109 CFU/ml in average for cyclic batch fermentation in MRS and WY medium respectively. Furthermore, cell densities of 5.2×109 CFU/ml and 1.8×1010 CFU/ml were achieved by fed-batch and dialysed fermentation respectively in MRS medium. Continuous fermentation was performed using WY medium to achieve 3.1×109 CFU/m.

Record ID 第43筆 System ID 089TTU00106010
BCRC ID CCRC 21436, Public Year 89
Paper Name 用酵母菌Candida tropicalis醱酵生產木糖醇之研究 Studies on the Fermentative Production of Xylitol by Candida tropicalis
Student Name 陳盈州
Teacher Name 許垤棋
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 木糖醇(xylitol)為五碳的糖醇,具有抗蛀牙、與蔗糖有相同的甜度,可當做糖尿病患者的代糖,在歐洲,70%的口香糖添加木糖醇。利用半纖維素的水解物,經化學合成法可將其中的木糖還原成木糖醇,此法需耗費較高的分離純化成本,來移除葡萄糖。而利用酵母菌將木糖醱酵轉化成木糖醇,可同時將葡萄糖消耗殆盡。利用酵母菌Candida tropicalis CCRC 21436以及它的NTG突變菌M40,進行饋料醱酵生產木糖醇的研究。醱酵過程中饋入葡萄糖,可提高木糖醇的產量。每次加入葡萄糖時,醱酵液的pH和氧化還原電位(ORP)下降,而醱酵液的葡萄糖被酵母菌消耗殆盡後,pH和氧化還原電位就逐漸上升,依此特性可進行pH-stat和ORP-stat的葡萄糖饋料醱酵以生產木糖醇。 比較pH-stat和ORP-stat的葡萄糖饋料。pH-stat控制於pH 5經30小時醱酵,可從196克的木糖原料得到83克的木糖醇,木糖醇轉化率(xylitol conversion yield)有74%、木糖消耗率為57%以及比產率(specific productivity)為0.065 克(木糖)/克(細胞乾重)/小時。而ORP-stat將ORP值控制於-200mV經27小時醱酵,可從193克的木糖原料得到105克的木糖醇,木糖醇轉化率為86%、木糖消耗率為63%以及比產率為0.091 克(木糖)/克(細胞乾重)/小時。ORP-stat 優於pH-stat。 不同ORP(-100,-150, -200 mv)和通氣量(0.2,2,4 l/min)的條件互相比較時,Candida tropicalis CCRC 21436的最適條件是ORP控制於-150 mv,通氣量0.2 l/min。經28小時培養可從203克的木糖得到148克的木糖醇,木糖醇轉化率為75%、木糖消耗率為98%以及比產率為0.095 克(木糖)/克(細胞乾重)/小時。突變菌M40的最適條件是ORP控制於-150 mv,通氣量2 l/min。經28小時培養可從199克的木糖得到144克的木糖醇,木糖醇轉化率為74%、木糖消耗率為98%以及比產率為0.105 克(木糖)/克(細胞乾重)/小時。 連續式醱酵生產木糖醇時,連續饋入葡萄糖(50 g/l)和木糖(150 g/l),稀釋倍率0.022 hr-1,第12到第72小時之間的木糖消耗率、木糖醇轉化率及木糖醇產率,分別穩定維持於81~93%、82~87%及6.2 ~7.1 g/h。這個結果優於稀釋倍率為0.044 hr-1的結果。 Xylitol, a five-carbon sugar alcohol, was an anti-cariogenic substance and has the same sweetness of sucrose by weight. It could serve as a diabetic sweetener. About 70% chewing gums in Europe are xylitol-added. Fed-batch and continuous fermentation processes by Candida tropicalis CCRC 21436 and its mutant, M40, for the production of xylitol were investigated。 Feeding glucose in fermentation process could enhance xylitol production. Intermittent glucose-feeding caused the decreasing in pH and ORP of the fermentation medium and after glucose in medium was used up, pH and ORP fluctuated. According to this property, pH-stat and ORP-stat glucose-feeding fermentation processes were carried out for the optimum production of xylitol from xylose. The processes of glucose-feeding fermentation using strain M40 via pH-stat at pH 5 and ORP-stat at -200 mv were compared. At hr 30 in the pH-stat fermentation at pH 5, 83 g xylitol was produced from 196 g xylose and xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption, specific productivity were 74%, 57%, 0.065 g (xylitol)/g (cell dry weight)/hr, respectively. Whereas at hr 27 in ORP-stat fermentation at -200 mv, 105 g xylitol was produced from 196 g xylose and xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption, specific productivity were 86%, 63%, 0.091 g (xylitol)/g (cell dry weight)/hr, respectively. The processes of ORP-stat fermentationat at ORP values of -100, -150, or -200 mv and at aeration rates of 0.2, 2, or 4 l/min were compared. The optimum production by Candida tropicalis CCRC 21436 was obtained at ORP of -150 mv and aeration rate of 0。2 l/min. At hr 28, 148 g xylitol was produced from 203 g xylose and xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption, specific productivity were 75%, 98%, 0.095 g (xylitol)/g (cell dry weight)/hr, respectively. By the mutant M40, the optimum production was achieved under a condition with ORP of -150 mv and aeration rate of 2 l/min. At hr 28, 144 g xylitol was produced from 199 g xylose and xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption, specific productivity were 74%, 98%, 0.105 g (xylitol)/g (cell dry weight)/hr, respectively. Both strains had the similar optimum productivities of xylitol via the ORP-stat fermentation under different conditions. In continuous process of fermentation, at a dilution rate of 0.022 hr-1, from hr 12 to hr 72, xylitol conversion yield (xylitol produced/xylose consumed), xylose consumption and xylitol productivity were in the ranges of 81-93%, 82-87% and 6.2-7.1 g/h, respectively. These results were better than those were obtained at the dilution rate of 0.044 hr-1.

Record ID 第44筆 System ID 088TTU00106006
BCRC ID CCRC 21945, Public Year 88
Paper Name 以酵母菌生產木糖醇之研究 THE STUDIES ON THE PRODUCTION OF XYLITOL BY YEAST
Student Name 許力中
Teacher Name 許垤
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 木糖醇(Xylitol)是一種糖醇類,由木糖(Xylose)還原而來,目前共有數種技術可用來生產木糖醇:固相-液相萃取法、化學合成法與生物技術法,其中,又以生物技術法生產木糖醇較為經濟。來自於自然界中的35株酵母菌中,23號酵母菌為一 Saccharomyces sp.,具有最高的木糖醇轉換產率 (xylitol conversion yield),Y p/s = 0.68 (g/g),另一方面,由菌種中心 (CCRC) 購得的酵母菌,除了CCRC 21945外,其木糖消耗率 (xylose consumption) 介於 19﹪~ 80﹪,累積木糖醇的能力也不高在振盪培養實驗中,添加甲醇或乙醇作為替代葡萄糖的能量來源,對於CCRC 21945有助於木糖醇的生成,然而,對於23號酵母菌卻沒有助益。 在攪拌式生物反應器中,23號酵母菌醱酵生產木糖醇的最適條件為:溫度,30℃;攪拌速度,300 min-1;通氣量,0.02 vvm (KLa = 8.64 h-1);pH控制在5.0以上;饋料葡萄糖與木糖比例為 1 : 3,在此條件下,木糖醇最大產量 (maximum xylitol production)、木糖醇轉換產率 (xylitol conversion yield) 與體積產量 (volumetric productivity) 分別為:26.7 (g/l)、0.863 (g/g)以及0.477 (g/liter h)。若提高饋料的總糖濃度,可以增加醱酵液中木糖醇濃度,但卻會造成醱酵時間的增加導致木糖無法完全被利用而殘留於醱酵液中。於是,有必要發展出雙或序列醱酵槽複合系統 (Dual- or sequencing-bioreactor system) 用來解決醱酵時間過長與木糖殘留於醱酵液中之缺點。 Xylitol (C5H12O5) is a sugar alcohol derived from the reduction of xylose (C5H10O5). There are several technologies available for xylitol production, which were solid-liquid extraction, chemical synthesis, and biotechnological methods. However, production of xylitol by microorganisms is more economic than others. Among 35 yeast strains isolated from natural sources, Yeast NO. 23, a Saccharomyces sp., was found to have the highest xylitol conversion yield, Y p/s = 0.68 (g/g). On the other hand, except CCRC 21945, the strains purchased from the Culture Collection and Research Center, with D-xylose consumption varied from 19 % to 80 %, accumulated low xylitol The addition of methanol or ethanol substituting for glucose as energy source in the shaking culture of CCRC 21945 enhanced xylitol production。 Whereas, the culture of Yeast NO. 23 was unaffected by the addition of methanol or ethanol. The optimum conditions of Yeast NO. 23 for fermentation of xylitol in stirred tank bioreactor were as follows: temperature, 30℃; stirring rate, 300 min-1; aeration rate, 0.02 vvm (KLa = 8.64 h-1); pH controlled above 5.0; the ratio of supplementary substrate, glucose : xylose = 1 : 3. Under these conditions, maximum xylitol production, xylitol conversion yield and volumetric productivity were 26.7 (g/l), 0.863 (g/g), and 0.477 (g/ liter h), respectively. Raising the concentration of total sugar in supplementary substrate was able to increase xylitol concentration in culture broth. But the more the concentration of total sugar the more fermentation time was required resulting in considerable amount of xylose remained in final culture broth. Therefore, it is necessary to develop a dual- or sequencing-bioreactor system to solve these problems.

Record ID 第45筆 System ID 094TTU00106005
BCRC ID BCRC 21731, Public Year 94
Paper Name 利用Pichia pastoris異源表現Saccharomyces cerevisiae之alcohol acetyltransferase I基因以生產isoamyl acetate Heterologous expression of the Saccharomyces cerevisiae alcohol acetyltransferase I gene in Pichia pastoris for production of isoamyl acetate
Student Name 陳姿伶
Teacher Name 李綉鈴
School Department Name 大同大學 生物工程學系(所) Academic Degree 碩士
Abstract Isoamyl acetate為發酵性酒精飲品之重要香味化合物,其由Saccharomyces cerevisiae之alcohol acetyltransferase (AATase)催化isoamyl alcohol與acetyl-CoA所生成。本研究乃將S. cerevisiae之ATF1 架構於一持續表現載體(pGAPZA),轉殖進入P. pastoris GS115,以大量表現ATF1來生產天然芳香化合物isoamyl acetate,並探討表現ATF 1,生產isoamyl acetate之最適培養條件,冀能提高芳香化合物isoamyl acetate 之產量 吾人以不同的基質濃度isoamyl alchol探討其最適條件,發現當培養液額外添加20mM isoamyl alchol可獲得最高isoamyl acetate之含量,約為0.87 mM,而在碳源的試驗上則是以2 % glucose為最佳。Yeast extract之濃度試驗則發現,當yeast extract添加濃度為1~3 %時,其isoamyl acetate之含量則無顯著性差異。而在peptone之濃度試驗則發現,培養液之isoamyl acetate含量並不受到peptone濃度之影響。因此,根據本研究結果得到,當轉型酵母菌P. pastoris培養於含2% glucose、1 % yeast extrace與20 mM isoamyl alcohol時,可獲得最佳isoamyl acetate之產量,約為0.87 mM。 Isoamyl acetate is an important flavor compound in wine and beverage. It is formed from isoamyl alcohol and acetyl-CoA catalyzed by alcohol acetyltransferase (AATase) in Saccharomyces cerevisiae. In this study, the gene ATF1 coded for AATase from S. cerevisiae BCRC 21731 was cloned and constructed into a constitutive expression vector pGAPZA。 A recombinant Pichia pastoris strain was obtained express high level natural flavor to produce isoamyl acetate. The optimal culture condition to enhance the production of isoamyl acetate by recombinant P. pastoris was also investigated. The results showed that recombinant strain GS115/GAPZA-ATF1 produced twice as much isoamyl acetate as GS115/GAPZA (control strain). When glucose, other than glycerol or methanol, was used as carbon source, recombinant strain produced the highest amount of isoamyl acetate. The optimal medium composition for isoamyl acetate production by recombinant strain was 2% glucose, 1% yeast extract and 20 mM isoamyl alcohol. The highest production of isoamyl acetate, is 0.87 mM, was obtained when the recombinant strain GS115/GAPZA-ATF1 was cultivated in the optimal medium at 30℃ for 4 days.

Record ID 第46筆 System ID 094TTU00106007
BCRC ID BCRC 31615, Public Year 94
Paper Name 探討培養基與培養條件對固態培養紅麴菌生產 Monacolin K、Citrinin 及色素的影響 Effect of Medium and Cultivation Condition on Monacolin K, Citrinin and Pigment Production During Solid State Fermentation of Monascus purpureus
Student Name 林秦瑋  
Teacher Name 段國仁
School Department Name 大同大學 生物工程研究所 Academic Degree 碩士
Abstract 本實驗以Monascus purpureus BCRC 31615 紅麴菌株,分別以白米、糙米為基質在 30℃ 下進行固態培養紅麴菌,並分析 monacolin K、citrinin 及色素產量。再藉由培養環境的改變,找出其最適的培養條件,以提高 monacolin K 產量。實驗結果發現,以白米培養紅麴時色素產量較高,而以糙米培養紅麴時 monacolin K 產量較高;在糙米的部份,接菌後第九天添加 60 ml 無菌水,monacolin K 產量由原本的 3813 ppm 增加到 5098 ppm,增加了 33.7%。而 monacolin K、citrinin 與色素的生成量皆有相似的趨勢。本研究成果是目前在文獻上所能得到的最佳成果。 In this study, the influence of different rice and cultivation condition on the production of monacolin K, citrinin and pigments by solid state fermentation of Monascus purpureus BCRC 31615 was studied。 Monascus was cultured at 30℃ on polished indica rice and de-hulled indica rice, separately. The results showed that the pigment is relatively high when cultivated on polished indica rice while monacolin K content is relatively high when cultivated on de-hulled indica rice. During solid state fermentation of de-hulled indica rice, adding 60 ml of water at day 9 improved 33.7% of the productivity of monacolin K from 3813 ppm to 5098 ppm. A similar tendency was observed in the formation amount of monacolin K, citrinin and pigments. The monacolin K production of this study was the most in the literatures ever published.

Record ID 第47筆 System ID 086TTIT0063029
BCRC ID CCRC 14104, Public Year 86
Paper Name 由Gluconobacter oxydans CCRC 14104生產煙草醯胺腺嘌呤二核酸磷酸依賴型葡萄糖去氫之純化與性質 PURIFICATION AND CHARACTERIZATION OF THE NADP-DEPENDENT GLUCOSE DEHYDROGENASE FROM Gluconobacter oxydans CCRC 14104
Student Name 吳國怡
Teacher Name 許垤
School Department Name 大同工學院 化學工程研究所 Academic Degree 碩士
Abstract 我們從Gluconobacter oxydans CCRC 14104菌株中純化出一煙草醯胺腺嘌呤二核酸磷酸依賴型葡萄糖去氫(NADP-dependent glucose dehydrogenase). 純化的過程中,使用了Blue Sepharose Fast Flow affinity column chromatography, Red Sepharose CL-6B affinity column chromatography, Sephacryl S-300 gel filtration column chromatography, DEAE Sepharose ion-exchange column chromatography以及Hydroxyapatite column. 經過純化的酵素其酵素活性比酵素粗抽液提高約117倍, 純化所得的酵素產率約5.20%.純化得到的酵素其pI值約介於pH4.1至4.2之間. 此酵素的分子量經過分析約為140,000左右, 若依據SDS-PAGE所得到的結果, 此酵素應具有四個分子量相等之次單體, 其分子量約為35,000daltons. 酵素在pH8.0至9.0之間具有最佳之酵素活性, 而且在pH9.0左右酵素活性最穩定. 酵素在攝氏40到45度具有最佳的催化能力. 至於金屬離子對酵素的影響, 本論文中所使用之金屬離子包括: 鋅離子、鐵離子、鈣離子、鎂離子、銅離子、汞離子、錳離子、鉛離子、鈷離子等均對酵素之活性有所抑制, 其中又以鋅離子對酵素活性具有最強的抑制效果. 此酵素對葡萄糖的Km值和Vmax值分別為0.34mM和37.7mM/min.。 NADP-dependent glucose dehydrogenase(EC 1.1.1.47) was purified homogeneity from the cell of Gluconobacter oxydans CCRC 14104。 The enzyme was solublizedfrom the cell sonicate and purified through Blue Sepharose Fast Flow affinity column chromatography, Red Sepharose CL-6B affinity column chromatography, Sephacryl S-300 gel filtration column chromatography, DEAE Sepharose ion-exchange column chromatography, and hydroxyapatite column. The specific activity of the purified enzyme was 117-fold of specific activity of the crude enzyme, and the final yield of the purified enzyme was about 5.20%.The enzyme was purified as a single molecular species with no evidence form of the enzyme. The Gluconobacter oxydans CCRC 14104 NADP-dependent glucose dehydrogenase had an apparent isoelectric point of pH4。1~4.2 and an Mr=140,000 and was comprised of four subunits of wach molecular weight about 35,000 daltons. Its substrate specificity of glucose was close to the substrate specificity of the similar enzyme from other microorganisms such asBacillus subtilis, Bacillus megaterium, and Pseudomonas spp.. The enzyme was most active from pH8.0 to pH9.0, and is quite stable at pH9.0. The optimum temperature is around 40`C to 45`C. The metal ions, such as Zn2+, Fe2+, Ca2+, Mg2+, Cu2+, Hg2+, Mn2+, Pb2+, Co2+, and Zn2+ ions showed significant inhibition on the NADP-dependent glucose dehydrogenase, whereas Zn2+ ion showed the strongest inhibition on the enzyme. The Km and Vmax value were0.34mM and 37.7mM/min for glucose as the substrate.

Record ID 第48筆 System ID 086TTIT0063032
BCRC ID CCRC 31494, Public Year 86
Paper Name 藍藒葸廇lpha-葡萄醣甘轉移酵素生成分枝麥芽寡醣之研究 synthesis of isomaltooligosaccharides with Aspergilus niger alpha-glucosidase
Student Name 邱仁德
Teacher Name 顏聰榮
School Department Name 大同工學院 化學工程研究所 Academic Degree 碩士
Abstract 由Aspergillus niger CCRC 31494所純化出來的transglucosidase (TGase)具有轉移生成isomaltooligosaccharides的活性,可將maltose之非還原端的葡萄糖殘基轉移至D-glucose及maltose的C6-OH上,形成isomaltose和panose,亦可轉移至maltose的C4-OH上,形成maltotriose.酵素之耐熱性,於40 C保溫9小時,TGase仍有殘存活性85%;有機溶劑ethanol,1-propanol, 2-propanol和hexane,可有提高水解活性約120%;另外,glucose和mannose有競爭性抑制作用,其Ki分別為12.9mM及75.9mM,而fructose和galactose則無抑制作用.以maltose為基質,濃度分別為20, 30, 40, 60及80 mg/ml,加入適當的酵素量反應,以HPLC分析產物,確認在30~60 mg/ml,其分枝麥芽寡醣之生成率約為33~40%,依據實驗數據,分析轉移產物生成之變化,探討生成IMOS之反應機制,並進一步建立TGase轉移反應途徑之動力學模式.A transglucosidase (TGase) of Aspergillus niger CCRC 31494 can catalyse bothnydrolytic and transfer reactions on incubation with isomalto-oligosaccharides。TGase can transfer the non-reducing D-glucosyl residue to water (hydrolysis), or to the non-reducing residue producing isomaltose from glucose or panose(6-O-a-D-glucosylmaltose) from maltose. It can also transfer the non-reducing D-glucosyl residue to C4-OH position of maltose and produce maltotriose. The enzyne is stable in storage at 40 C at least 9 hours. In organic solvent, ethanol,1-propanol, 2-propanol, and hexane can enhance hydrolysis about 120%. The activityis competitively inhibited by glucose and mannose, but is not ingibited by galactose and fructose. The Kis of glucose and mannose are 12.9 mM and 75.9 mM,respectively.Using maltose as substrate, the transglucosylation products were analyzed withHPLC. Based on product analysis and the kinetic properties of TGase. A suitablekinetic modle for illustrating the synthesis of isomaltooligosaccharides was deduced. The practical data were coincident to the deduced result.

Record ID 第49筆 System ID 087TTIT0063035
BCRC ID CCRC 31494, Public Year 87
Paper Name 黑黴菌屬beta-葡萄糖甘酵素II基因之選殖及其序列分析 Cloning and characterization of -glucosidase II gene from Aspergillus niger
Student Name 李振漢
Teacher Name 顏聰榮 博士
School Department Name 大同工學院 化學工程研究所 Academic Degree 碩士
Abstract 中文摘要 Aspergillus菌屬在工業上的用途相當廣泛,能生產各種工業用酵素,為高經濟利益菌屬,而且可分泌高活性之β-葡萄糖酵素,我們從Aspergillus niger CCRC 31494菌體外培養基中發現兩種β-葡萄糖甘酵素(β-Glc-I及β-Glc-II),除具有水解活性外,亦有高轉移活性。而β-Glu-II之水解及轉移活性又比β-Glu-I強,可用以合成纖維寡醣及烷基醣甘化合物,在工業上利用性高。在本研究中希望經由基因選殖及序列分析來瞭解β-Glu-II之生理功能。先從GCG基因庫中搜尋Aspergillus菌屬之β-葡萄糖甘酵素基因,結果發現在A. aculeatus及A. kawachii菌株中分別有β-葡萄糖甘酵素基因被發表,DNA序列也被確定,兩酵素基因之搜尋序號分別為D64088及AB003470。依同源性高區域設計7個引子,其中B1、B2、B3及B4分別為forward primers,而B5、B6及B7為reverse primers。以B3 X B7及B4 X B7可經PCR合成1.3 kb之DNA片段。經核酸限制酵素及nested PCR之雙重確認,可確認其應為β-葡萄糖甘酵素之基因片段,因此經DIG labeling使可為探針來搜尋A. niger之genomic DNA及cDNA libraries之正反應菌落。將與探針有正反應菌落之insert DNA進行次選殖及定序,將所得到之序列與A. aculeatus No. F-50及A. kawachii IFO4308之β-葡萄糖酵素DNA序列進行比對其DNA序列的相似性為74.5%及81.9%;amino acid 序列的相似性為79%及90%。以ScanProsite分析amino acid序列上的結構功能區域,發現其上有十個N-glycosylation位置及一個glycosyl hydrolase family 3 活性位置。 Abstract Aspergillus species are widely used in fermentation food, and pharmaceutical industry. They produce many enzymes which can be applied to industry. β-glucosidase is one of the well-known enzymes produced by Aspergillus niger. Two β-glucosidases (β-Glu-I and β-Glu-II) have been isolated and purified from Aspergillus niger CCRC 31494。 Both of these two β-glucosidases have high hydrolysis and transglucosylation activities. The β-Glu-II has higher enzyme activity than that of β-Glu-I, and is better for synthesis of cellooligosaccharides and alkyl glucosides. In this study, we try to clone the β-Glu-II gene from A. niger CCRC 31494 and to determine its DNA sequence in attempt to predict the possible physiological functions of the β-Glu-II。 There are two fungal β-glucosidase genes were found after searching the genetic database of the GCG genebank. The two β-glucosidase genes were isolated from A. aculeatus No. F-50 and A. kawachii IFO4308. By others based on DNA sequence of the highly conserved region, seven different primers were designed. Among which B1、B2、B3 and B4 were used as the forward primers and B5、B6 and B7 were used as the reverse primers. The designed primer pairs were used to perform the normal PCR experiments with both of the genomic DNA and cDNA of A. niger CCRC 31494。 An expected 1.3 kb fragment was obtained using primer pair of B3 X B7 and B4 X B7. Furthermore, the restriction enzyme mapping of DNA fragment also confirmed that this 1.3 kb was one part of the β-glucosidase gene. This 1.3 kb was then randomly labeled with the DIG-labeling kit as a non-isotopic labeling probe, BG-P-1. The BG-P-1 was used to screening the genomic DNA and cDNA libraries of A. niger CCRC 31494。 From the obtained positive colonies, the cloned DNA fragments were recovered 、purified and sequenced. About 3032 kb nucleotide sequence was determined. The DNA sequence was found to be 74.5% and 81.9% homology respectively to that of A. aculeatus No. F-50 and A. kawachii IFO4308. The amino acid sequence was 79% and 90% homology to that of A. aculeatus No. F-50 and A. kawachii IFO4308 respectively. The analysis of its amino acid sequence using ScanProsite program showed ten N-glycosylation sites and one glycosyl hydrolase family 3 active site.

Record ID 第50筆 System ID 084TTIT0063009
BCRC ID CCRC 30414, CCRC 30210, Public Year 84
Paper Name 以固定化-葡萄糖生產異麥芽寡糖之研究 STUDIES ON THE PRODUCTION OF ISOMALTOOLIGOSACCHARIDES BY IMMOBILIZED -GLUCOSIDASE
Student Name 黃金義
Teacher Name 段國仁
School Department Name 大同工學院 化學工程學系 Academic Degree 碩士
Abstract 本研究是利用由 Aspergillus carbonarious CCRC 30414 和 Aspergillus niger CCRC 30210 這兩株黴菌所產生的胞內型-葡萄糖  (-glucosidase) 以麥芽糖為基質進行催化轉糖基反應來生產異麥 芽寡糖。在過去研究中發現這兩株菌都有具有胞內型及胞外型兩種酵素, 其中胞內型酵素具有葡萄糖轉移活性,胞外型沒有葡萄糖轉移活性,而只 有水解活性,為了避免胞外型 -葡萄糖的水解作用提高酵素的穩定 性和濃度,以及連續式生產,提高寡糖的產率,在本研究中利用兩種不同 型態的固定化酵素的方法來達到此效果,以利將來工業化生產異麥芽寡糖 之參考: 一、固定化部份純化酵素: 將部份純化所得之-葡萄糖 以不同的擔體固定之,包括幾丁質擔體 (chitopearl beads) 和幾丁 質 (chitosan)。 二、固定化細胞: 將培養之菌絲直接冷凍乾燥,再 分別用聚乙烯亞胺和戊二醛處理將-葡萄糖 固定在菌體內。 結 果發現,來自 Asp. carbonarious 的酵素, 經幾丁質擔體固定之酵素和 沒有經過處理的菌絲生產異麥芽寡糖的產率分別可以達到60﹪和46﹪,在 操作穩定性方面, 經幾丁質擔體固定之酵素和聚乙烯亞胺、戊二醛處理 後的菌絲, 連續六天的反應中仍可維持分別80﹪和70﹪的酵素活性。 菌 絲在聚乙烯亞胺、 戊二醛處理期間酵素活性沒有明顯的下降。 另一方面 , 來自 Asp. niger 的酵素, 經幾丁質固定之酵素和沒有經過處理的 菌絲生產異麥芽糖寡糖的產率分別可以達到52﹪和43﹪,在操作穩定性方 面,經幾丁質固定之酵素和聚乙烯亞胺、戊二醛處理後的菌絲,連續六天 的反應中沒有明顯的酵素活性下降。 但是菌絲在聚乙烯亞胺、戊二醛處 理期間酵素活性下降了80﹪。

Record ID 第51筆 System ID 084TTIT0063021
BCRC ID CCRC 10791, Public Year 84
Paper Name 乳酸球菌Lactococcus lactis subsp. lactis CCRC10791質體pL2複製元之研究 Study on the replicon of plasmid pL2 isolated from Lactococcus lactis subsp. lactis CCRC10791
Student Name 高炳煌
Teacher Name 顏聰榮
School Department Name 大同工學院 化學工程學系 Academic Degree 碩士
Abstract 經比較三種質體微量抽取法後,選擇以Anderson和McKay的方法進 行28株乳酸菌質體之篩檢。結果有一半菌株含1至6個質體,分子量介 於2.0至83.5 kb,並定出其中三個分子量較小的質體pL1、pL2、p10357-1 之限制圖譜。根據不同複製型態質體的複製起始區及複製蛋白質同源性 較高之區域,設計了TA1至TA4四條探針。TA1及TA2分別為Rolling-circle 之複製起始區及複製蛋白質探針,TA3及TA4則屬theta-type。結果只有 TA4與兩株乳酸球菌Lactococcus lactis subsp. lactis CCRC 10791 (ATCC11454)及CCRC 14016 (IFO12007)的質體有反應。有雜交反應中分子 量最小的一個質體為pL2 (5.3 kb),因為分子量小較易送入宿主,因此選 擇pL2作為複製元研究之對象。利用限制將pL2之1.8 kbHpaII片段插入 pUC19的AccI位置,得到pUH1。pUH1插入DNA的5?端序列和其他乳酸球菌 質體之最小複製元的DNA序列具有很高的同源性,但是缺少起始區上游部 份AT-rich的一段序列。因此改以將pL2之3.3 kb EcoRI片段及完整質體長 度插入pUC19的EcoRI位置,得到pUL6及pUL4。利用pUL6當template,定出 pL2的1475個核酸序列。根據序列比對,pUL6具有典型的乳酸球菌 theta-type質體複製元的特徵,在複製起始區及複製蛋白質的位置相同性 非常高,因此推論pL2 的複製型態應屬theta-type,具有用來建構一穩定 複製穿梭載體的價值性。

Record ID 第52筆 System ID 084TTIT0063024
BCRC ID CCRC 14018, Public Year 84
Paper Name 抑菌素 Fermentcin B 的純化及其性質之探討 Studies on Fermentcin B, a Bacteriocin Produced by Lactobacillus fermentum
Student Name 李宗憲
Teacher Name 顏聰榮
School Department Name 大同工學院 化學工程學系 Academic Degree 碩士
Abstract 利用 disc diffusion method 從乳酸菌屬中篩選具有抑菌能力的菌 株, 發現 Lactobacillus fermentum Beijerinck CCRC 14018 在 pH 5.0 時具有較高之活性. 菌體培養液經離心除菌後再以硫銨沉澱, Q Sepharose Fast Flow 及 CM Sepharose 結合的管柱層析後得到部份純化 的抑菌素 - Fermentcin B. 純化倍率約為 270 倍, 回收率約為 6%. 此 抑菌素只對某些革蘭式陽性菌有抑制性, 顯現出較窄的抑制範圍. 其抑菌 機制為 bactericidal-type. 離心後之菌體再分別以 0.1% CaCl2 或 2% ethanol 處理可使 Fermentcin B 的回收產率增加為 1.5 及 1.25 倍. 純化的抑菌素以 proteolytic 及 glyclytic 的酵素作用後,無法測得活 性; 這顯示其可能為醣蛋白. 其在 100C 下加熱 30 分鐘後仍保其活性, 在pH 3.0 -pH 8.0 的範圍中皆具有活性且相當穩定. 由 ultrafiltration 實驗發現, Fermentcin B 的分子量介於 1000 到 3000 Da 之間. 由以上的結果雖可將 Fermentcin B 歸類為 class II 抑菌素, 但其卻含有醣分子. 故 Fermentcin B 可能為一介於 class II 和 class IV 之間的醣蛋白抑菌素.

Record ID 第53筆 System ID 094DYU00515006
BCRC ID CCRC 12826, Public Year 94
Paper Name 以化學法及微生物法生產γ-聚麩胺酸 之衍生物及其應用 Chemically and Microbiologically Synthesized Poly-γ-Glutamic Acid Derivatives and Their Applications
Student Name 吳嘉興
Teacher Name 施英隆
School Department Name 大葉大學 環境工程學系碩士班 Academic Degree 碩士
Abstract 聚麩胺酸是經由微生物發酵生產的天然生物性材料,其具有水溶性、生物可分解與可食等特性,且聚麩胺酸本身及其分解物對人體與環境無毒害,目前聚麩胺酸已知可應用於食品、化妝品、醫藥材料、環境保護等領域,因此工業化大量生產聚麩胺酸此一對環境友善之生物性材料,對於環境及環境保護將有相當的貢獻。 本研究以十升發酵槽培養Bacillus subtilis C1並探討pH、曝氣量、葉片轉速對菌株生長、聚麩胺酸產量的影響,當Bacillus subtilis C1培養於T1培養基中(Glycerin:17 ﹪、Citric acid:2.2 ﹪、NH4Cl:0.7 ﹪、K2HPO4:0.05 ﹪、MgSO4‧7H2O:0.05 ﹪、CaCl2‧2H2O:0.015 ﹪、FeCl3‧6H2O:0.004 ﹪、MnSO4‧4~6H2O:0.0104 ﹪),發現發酵槽條件為pH=6.00、曝氣量為5、葉片轉速為150rpm、37℃下培養84小時,聚麩胺酸最高產量為8.25g/L,約為搖瓶培養生產聚麩胺酸的1倍(平均產量4.36g/L),Bacillus subtilis C1所生產之產物經結構鑑定證實為聚麩胺酸與甘油之共聚物,其光譜圖中化學位移1.8-2.1(m,β,2H)、2.3-2.4(b,γ,2H) 、4.1-4.2(b,α,1H)代表γ-聚麩胺酸各波峯之位移,而化學位移3.50-3.54(dd,2H)、3.60-3.62(dd,2H)、3.72-3.77(m,1H)相當於甘油各波峯之化學位移。此產物之分子量為2,929,844。 本研究亦探討聚麩胺酸衍生物之吸水性應用,使用Bacillus licheniformis CCRC 12826菌株所生產聚麩胺酸,並利用化學方法偶合聚麩胺酸與環氧樹脂以交聯形成結構堅固之凝膠,並測試其吸水性,實驗發現2.5 ﹪聚麩胺酸溶液於pH值為5.33與150μl環氧樹脂進行反應4天,所得之凝膠可達到吸水重為乾凝膠重的50~60倍,吸水性較其它條件製備之凝膠優越。 不同吸水材料之吸水實驗中,發現Bacillus subtilis C1所生產之聚麩胺酸-甘油複合物於30分鐘吸水實驗中,其吸水重為乾凝膠重之26倍,然而較長時間之吸水,此材料即完全溶解。 poly-γ-glutamic acid (PGA) is a naturally occurring bio-material produced by microbial fermentation. It is water soluble, biodegradable, edible and nontoxic toward humans and the environment. PGA has potential applications in the field of food, cosmetics, medicine and environmental. Development of this material is both environmentally and economically valuable. In this study, we investigated the effects of pH, aeration and agitation on γ-polyglutamic acid (γ-PGA) production of Bacillus subtilis C1 in a 10-L fermenter. When the bacteria were cultivated in medium T1 composed of Glycerin ( 17 ﹪), Citric acid ( 2.2﹪), NH4Cl ( 0.7 ﹪), K2HPO4 ( 0.05 ﹪), MgSO4‧7H2O ( 0.05 ﹪), CaCl2‧2H2O ( 0.015﹪), FeCl3‧6H2O ( 0.004 ﹪), MnSO4‧4~6H2O ( 0.0104 ﹪), the optical PGA production was pH6.00, aeration rate 5L/min, agitation speed 150rpm, temperature 37℃;the highest yield was 8.25g/L after 84 hr cultivation. The yield increased 89﹪from 4.36g/L in shake flask culture to 8.25g/L in a 10-L fermemter. The chemical shifts in ppm, 1.8-2.1(m,β,2H), 2.3-2.4(b,γ,2H)and 4.1-4.2(b,α,1H), correspond to the peak positions of the authentic γ-PGA previously synthesized ;in addition, the chemical shifts in ppm, 3.50-3.54(dd,2H), 3.60-3.62(dd,2H), 3.72-3.77(m,1H) corresponds to the peak positions of glycerol. The number-average molecular weight (Mw) determined by gel permeation chromatography was 2,929,844. This product is a γ-PGA-glycerol conjugate. We also investigated the water-absorption properties of γ-PGA derivatives. A crosslinked product produced by coupling of 2.5 wt ﹪γ-PGA of Bacillus licheniformis CCRC 12826 and 150μl Diglycidyl ether of bisphenol A (DGEBA) at pH5。33, for 4 days, displayed strong water-absorption activity;the specific water content (wt of water / wt of polymer) was approximately 50-60. It was also found that the specific water content (wt of water / wt of polymer) was approximately 26 in 30min water-absorption period for the γ-PGA-glycerol conjugate produced by Bacillus subtilis C1;however, this material totally dissolved after 30min in water.

Record ID 第54筆 System ID 089DYU00250007
BCRC ID CCRC 31535, Public Year 89
Paper Name 利用回應曲面法尋求紅麴菌生產膽固醇合成抑制劑之培養基最適化 Optimization of Medium Composition for Monacolin K Production by Monascus ruber Using Response Surface Methodology
Student Name 黃壬章
Teacher Name 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究以Monascus ruber CCRC 31535 為生產紅麴菌株,先進行甘油碳源濃度之探討,再利用回應曲面法以尋求白米粉-甘油複合培養基中四種組成分(白米粉、peptone、甘油、葡萄糖)等最適化濃度探討,以期得到最高產量的膽固醇合成抑制劑(monacolin K)。甘油碳源濃度探討之實驗結果顯示,在培養溫度為25℃,白米-甘油複合培養基之起始酸鹼值為pH 5.0,培養體積為25 mL 等培養條件下,甘油濃度為26.4 mL/L時,monacolin K的平均產量可達到0.131 mg/mL。 本研究亦利用回應曲面法來尋求白米粉-甘油複合式培養基中四種組成分(白米粉、peptone、甘油、葡萄糖)之最適化濃度條件,並分析其對於monacolin K 產量之影響,結果發現四種組成分之平方項對monacolin K 產量達到顯著之影響(p<0.05)。由回應曲面法尋得最適培養基組成為34.4 g/L白米粉、10.8 g/L peptone、26.4 mL/L甘油、129.2 g/L葡萄糖、2 g/L KNO3、1 g/L MgSO4.7H2O。在此組成下,經過十天培養後,可得到本研究monacolin K平均最高產量為0.131 mg/mL,而二階模式預測值則為0.130 mg/mL。此外在回應曲面模式適切性之統計檢驗上,R2為0.876,表示此一回應模式能適切地作為描述實驗數據參考用。 ABSTRACT In this study, monacolin K was produced by Monascus ruber CCRC 31535 in flask culture。 In preliminary study, the suitable concentration of the glycerin carbon source was investigated. The optimum concentration of glycerin was found at 26.4 mL/L in the culture of 25℃, pH 5.0 and 25 mL of the rice-glycerin complex medium, and 150 rpm in the shaker. The average yield of monacolin K was 0.131 mg/mL. In addition, response surface methodology was used to optimize the concentrations of the rice powder-glycerin complex compositions and to evaluate the effects of the composition concentrations on monacolin K productivity. The analysis of variance indicated that the quadratic terms of four compositions (rice powder, peptone, glycerin, glucose) in the quadratic model were significant. The optimum composition for monacolin K production was found to be 34.4 g/L rice powder, 10.8 g/L peptone, 26.4 mL/L glycerin, 129.2 g/L glucose, 2 g/L KNO3 and 1 g/L MgSO4.7H2O. With these compounds, the average monacolin K production was 0.131 mg/mL after 10 days of cultivation, while the predicted maximum production was 0.130 mg/mL. For this kind of complex medium, it would be good for monacolin K production by M. ruber CCRC 31535

Record ID 第55筆 System ID 089DYU00250020
BCRC ID CCRC 35515, Public Year 89
Paper Name 綠殭菌發酵最適化條件之探討 Optimization of fermentation process for Nomuraea rileyi
Student Name 薛一祥
Teacher Name 謝建元
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究以回應曲面法分別進行綠殭菌(Nomuraea rileyi CCRC 35515)液態與固態發酵,最適化生產菌絲體和分生孢子之探討。在液態發酵方面,以葡萄糖、果糖、蔗糖、麥芽糖與糖蜜為碳源,其中以葡萄糖為最佳;另一方面,在酵母萃出物、neopeptone與玉米浸粉的氮源試驗中,則以玉米浸粉為最佳。於葡萄糖及玉米浸粉培養基中添加綜合蔬果汁(V8 juice)具有促進菌體生長之功效,而搖瓶培養的其它物理因子:培養液起始酸鹼值以pH 6 為最佳,菌體絲乾重亦隨搖瓶轉速上升而提高。以回應曲面法所得之最適化培養基組成為葡萄糖3.2﹪、V8 juice 29﹪及玉米浸粉0.15﹪,可得到最高菌絲體乾重為12.1 g/L。將此最適化培養基條件應用於5 L液態發酵槽試驗中,在1 vvm通氣量條件下,較高的發酵槽攪拌速率可得到較佳的菌絲體產量,且排出氣體的含氧量較低;但是當通氣量為2 vvm時,不同攪拌速率(250、350及450 rpm)對菌絲體生長並無明顯影響,而菌µ²體乾重可高達16.4 g/L。 在固態發酵方面,RSM同樣應用在最適化分生孢子的生產,糖蜜、玉米浸粉、酵母粉、魚漿及高梁仁等培養基對綠殭菌生長最佳,且產孢量可達4.79×109 conidia/g-dry weight。在固態發酵量產方面,將平板及太空包之結果轉移至22-L固態發酵槽,以2800 Lumens/m2且光照週期為L/D=12/12,其產孢量最佳可達6.8×109 conidia/g-dry weight。 固態發酵槽所生產之分生孢子,其對四齡甜菜夜蛾的致死率為56.05±5.43﹪,與SMAY斜面培養基比較其活性並無明顯差異。 The purpose of this research was studying the optima production of mycelia and conidia spore of Nomuraea rileyi by using the response surface method in submerged cultural fermentation and solid-stat fermentation, respectively. In submerged cultural fermentation, the glucose was found to be the best carbon source for the growth of mycelia among glucose, fructose, sucrose, maltose, and molasses. On the other hand, the corn steep powder was the better nitrogen in the selected testing nitrogen source such as yeast extract, peptone, and corn steep powder. The V8 juice as the additive was found the promoting mycelia growth effect in the media with glucose and corn steep powder. Other physical conditions in the shake flask study, the pH6 as initial growth pH and higher shaker rotation speed were also found favor the mycelia growth. The optima growth compositions from the RSM was 3.2% glucose, 29% V8 juice, and 15% corn steep powder resulted 12.1 g-cell dry weight per liter. This result was applied to scale up in the 5-L fermentor. The higher agitation speed of the fermentor has higher cell production and has lower oxygen contain in the output gas with 1 vvm aeration. However, at higher aeration of 2 vvm the speed of mixing was not affected significant at 250 rpm, 350 rpm, and 450 rpm. Finally, the best cell production was found as high as 16.4 g-cell dry weight per liter. In solid-stat fermentation, the RSM was also applied in the optima spore production. The molasses, corn steep powder, yeast powder, fishmeal, and sorghum were found to be the best medium for the growth and conidia spore production with 4.76×109 conidia/g-dry weight. The scale up study of solid-stat fermentation was carrying out from the plate, plastic bag, and all the way to 22-L bioreactor. In 22-L bioreactor the higher spore production 6.8×109 conidia/g-dry weight was found at light intensity 2800-Lumens/m2 and light period of L/D=12/12. Against 4-th star larva of Spodoptera exigua the conidia spore from the bioreactor has 56.05±5.43% mortality. This result was closes to the conidia spore from the SMAY slant.

Record ID 第56筆 System ID 089DYU00250029
BCRC ID CCRC 31535, Public Year 89
Paper Name 白米複合培養基對紅麴菌膽固醇合成抑制劑產量影響之研究 Effect of Rice Complex Medium on Monacolin K Production of Monascus ruber
Student Name 李志誠
Teacher Name 涂瑞澤 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 中文摘要 本研究的目的在於探討以Monascus ruber CCRC 31535 為紅麴生產菌株,利用食品級白米-酵母粉或白米-蔬菜油等複合培養基,進行其影響紅麴膽固醇合成抑制劑產量之研究。結果顯示白米-蔬菜油複合培養基為本研究最佳培養基,而其最適培養溫度為25℃,培養基體積在250 mL三角錐瓶中為50 mL;另外,最佳培養基起始酸鹼值為pH 5-6,但最佳培養基起始酸鹼值將隨白米含量而有所改變,當0.06 g/mL白米含量時,其起始酸鹼值為 pH 5,相對地,當0.20 g/mL白米含量時,則起始酸鹼值為pH 6。一般對紅麴膽固醇合成抑制劑產量而言,白米粒形態較白米粉形態為佳。 最後,當白米粒-蔬菜油複合培養基起始酸鹼值為pH 5,其培養體積為 50 mL,且白米粒濃度為 0.10 g/mL,蔬菜油為0.036 mL/mL,蔗糖為0.13 g/mL,酵母萃取物為0.01 g/mL,可得到本研究最高紅麴膽固醇合成抑制劑產量為0.058 mg/mL;若以此最佳白米粒-蔬菜油複合培養基條件進行五公升小型發酵槽培養條件探討,當槽內攪拌速率為400 rpm時,發酵至第九天時,可得到最高monacolin K產量,約為0.023 mg/mL,無法預期達到搖瓶培養方式之最佳結果,主要由於紅麴菌的生長極易受培養條件的不同而有很大的變化,因此食品級白米複合培養基中組成分及其濃度最適化,仍待利用回應曲面法探討,而且五公升小型發酵槽培養條件之最適化,亦待進一步探討。 ABSTRACT In this study, monacolin K was produced by Monascus ruber CCRC 31535 in flask culture。 The objective of this study was to develop the effects of the food-grade rice-yeast powder or rice-vegatable oil complex media on monacolin K production by M. ruber. Several different strategies of manipulating variables, such as culture temperature, initial pH and volume of the complex medium, were investigated. It was found that the optimum culture medium was the rice-vegatable oil complex medium. The temperature and medium volume in 250-mL flask culture were at 25℃and 50 mL, respectively. The optimum initial pH of the rice complex medium was to be 5.0-6.0. The optimum initial pH for 0.06 g/mL rice particle in the complex medium was to be 5.0, while that for 0.20 g/mL rice particle was to be 6.0. In general, the rice particle, rather than the rice powder, was considered to be a suitable composition form for improving yield of monacolin K. Finally, the result indicated that the addition of vegatable oil in the rice complex medium was found to increase the monacolin K production. The maximum yield was 0.058 mg/mL when 0.10 g/mL rice particle, 0.036 mL/mL vegatable oil, 0.13 g/mL sucrose, 0.01 g/mL yeast extract was used in the complex medium. By using the rice-vegatable oil complex medium describle above, the maximum monacolin K yield, 0.023 g/L, was obtained at the 9th day under controlling 400 rpm stirred speed in a 5-L stirred tank fermentor. This research demonstrated the food-grade rice complex medium was worth improving the yield of monacolin K by using response surface methodology to optimze the composition concentration and to study the fermentation conditions by using a 5-L stirred tank fermentor.

Record ID 第57筆 System ID 089DYU00250031
BCRC ID CCRC 10695, CCRC 10699, Public Year 89
Paper Name 以紅麴發酵蝦蟹殼粉生產抗菌幾丁質酶之研究 The Studies on the Production of Antimicrobial Chitinase by Monascus sp. Using Shrimp and Crab shell as a Carbon source
Student Name 蕭惟仁
Teacher Name 王三郎
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究主要係以Monascus sp.發酵蝦蟹殼水產廢棄物,生產符合具有安全、有效及無污染等特點的環保生物制劑為目的,並探討其較適生產條件、生化性質及抑制機制等等相關之研究。 實驗結果,選擇出最具抑制活性並最節省成本培養之菌株,即Monascus 31499真菌抑制劑之較適生產條件培養基中含有0.l% K2HPO4 、0.05% MgS04 .7H20 、0.001﹪FeSO4.7H2O 、0.3﹪NaNO3 、0.05﹪KCl 、0.l% 酵母萃出物、0.l% 聚蛋白、1% SCSP;培養條件為pH 7、25℃、4 days、100 ml/250ml三角錐瓶,針對抑制Fusarium oxysporum,其活性為0.35U/ml;對各種微生物之抑制測定結果Lactobacillus acidophilus CCRC 10695、Lactobacillus delbrueckii subsp. lactis CCRC 10699 、M1001和W113則具有較明顯的抑制情形;應用於田間試驗部分,可知粗酵素液不但可抑制植物病源真菌Fusarium oxysporum生長,亦可促進植物重量的增加。而且其pH值安定性為6~8,卻不具有100℃熱安定性。然而針對抑制Fusarium oxysporum來進行顯微鏡觀察,其抑制作用機制包括了溶解菌絲之細胞壁,改變病原菌細胞表面通透性,因而造成其菌絲末端的膨大現象,且其最適反應溫度為40℃,最適反應pH為7。 以Monascus 31499所得粗酵素液,經硫酸銨沈澱、濃縮透析並於DEAE Sepharose CL-6B中,分離出分子量約為81kDa(SDS-PAGE),等電點為5.4,且其性質為之最適反應溫度為40℃,最適反應pH值為pH7,並具有幾丁質酶及蛋白質酶活性;且其幾丁質酶及蛋白質酶活性pH值穩定性為pH 6~9,仍不具有100℃熱安定性;其胺基酸成分經推算約佔樣品總量64﹪,其中以asparagine含量最高,其次為glutamine、alanine、glycine、leucine。針對幾丁質酶活性來說Fe2+可促進其活性,而Zn2+卻抑制其活性較強,而對於Hg2+及Acetone幾乎受到完全抑制;Hg2+幾乎抑制蛋白質酶活性,EDTA、Methanol ,Ethanol對兩者均具有高抑制性。而分離純化後之真菌抑制劑,可發現在稀釋五倍的濃度下,其孢子幾乎不具有發芽狀,故以此可知此真菌抑制劑具有抑制孢子生長之機制。 This thesis is a study of the utilization of shrimp shell wastes by Monascus spp. to produce antifungal substance. The purification and characterization of fungicides were described. By resulting, we choose Monascus 31499 as the fungicide producer. That inhibitory activity(0.35U/ml)for Fusarium oxysporum was obtained when the strain was grown aerobically in a medium consisting of 1% shrimp shell wastes,0.l% K2HPO4 、0.05% MgS04 .7H20 、0.001﹪FeSO4.7H2O 、0.3﹪NaNO3 、0.05﹪KCl 、0.l% yeast extract and 0.l% poly-peptone in 100ml medium at 25℃(pH 7)for 4 days. Besides Fusarium oxysporum ,it can also to fight the Lactobacillus acidophilus CCRC 10695、Lactobacillus delbrueckii subsp lactis CCRC 10699 、Pseudomonas aeruginosa M1001 and Bacillus subtilus W113 。The fungicide was stable at pH from 6 to 8,but was not stable at 100℃. The culture supernatant was tested for hyphal growth, it caused abnormal hyphal swelling on the tip of Fusarium oxysporum. However, the optimum pH was 7 and optimum temperature was 40 degree C. The fungicide was purified from the culture supernatant of Monascus 31499 by ammonium sulfate fractionation and DEAE Sepharose CL-6B column chromatography. The purified enzyme was estimated to be 81kDa by SDS-PAGE have molecular weight.It showed chitinase activity and antifungal activity. It’s found to be an acidic protein with pI at 5.4. The optimum pH for enzymatic activity was approx.7 and the optimum temperature was 40 degree C. The enzyme was stabile at pH from 6 to 9 and 100℃ thermal stability for inhibition times less than 3 min. The activity of chtinase and protease was activated by Fe2+,but strongly inhibited by Hg2+and Acetone.

Record ID 第58筆 System ID 089DYU00250032
BCRC ID CCRC 31527, CCRC 31499, CCRC 31530, CCRC 31535, CCRC 32966, Public Year 89
Paper Name 紅麴發酵茶葉渣生產除臭劑之研究 Fermentation of Tea Powder Waste by Monascus to Produce Deodorant.
Student Name 徐士喬
Teacher Name 王三郎 顏裕鴻
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究為利用加工及農業廢棄物為原料,藉由微生物之發酵加工及農業廢棄物生產有效的除臭物質。 研究中用以生產除臭劑之五株紅麴菌,Monascus purpureus CCRC 31527、Monascus purpureus CCRC 31499、Monascus purpureus CCRC 31530、Monascus rubber CCRC 31535及Monascus purpureus CCRC 32966係購自新竹之食品工業發展研究所菌種保存中心(Culture Collection and Research Center;CCRC),其餘之Lactic acid bacteria、 Streptococcus actuosus A151、Pseudomonas fluorescens K-188及Bacillus subtilis V656係本研究室自行篩得之菌株 本研究除臭力之初步篩選結果,篩選出具除臭潛力之菌株為Monascus ruber CCRC 31535,在掃描式電子顯微鏡下顯示於TPW表面生長情況最為良好。因此,在本研究中利用此株菌,進一步探討生產除臭劑之最適條件,探討結果為:培養基水分含量於60﹪(w/v),利用TPW作為培養基質,基質粒徑大小不拘,培養時間9天,培養基質不需經過熱處理及鹼處理,除臭劑利用烘箱以100 ℃進行乾燥,如此可得到除臭效果較佳之除臭劑。 在除臭劑之特性方面,本除臭劑適合於室溫環境下進行脫臭,在冷藏溫度之下其除臭效果較弱;以Monascus rubber CCRC 31535生產之除臭劑其除臭能力在48小時達到飽和,而以Lactic acid bacteria生產之除臭劑其除臭能力則在12小時達到飽和;在本研究中除臭能力的比較結果市售除臭劑除臭率為之除臭劑效果遠不及本研究室自製之除臭劑。除臭劑之應用結果,以利用TPW為基質者的到整體上最佳之抗菌效果。 The purpose of this research is to find the best method to ferment industrial waste and agricultural waste to produce deodorants. Nine microorganism were studied in this research. Five of them are Monascus obtained from Culture Collection and Research Center (CCRC) They are Monascus purpureus CCRC 31527, Monascus purpureus CCRC 31499, Monascus purpureus CCRC 31530, Monascus rubber CCRC 31535 and Monascus purpureus CCRC 32966。 The other four microorganism, Lactic acid Bacteria, Streptococcus actuosus A151, Pseudomonas fluorescens K188 and Bacillus Subtilis V656, were the microorganism incubated in the lab. After testing the nine microorganism, Monascus rubber CCRC 31535 is proved to be the microbe that meets the need of the project best The best condition for Monascus rubber CCRC 31535 to grow was therefore investigated The result showed that Monascus rubber CCRC 31535 grows best when the water content of the medium is 60% (w/v) The best medium for Monascus rubber CCRC 31535 to grow is TPW。 The size of the medium is proved to have no effect on deodorization. The days for incubating the Monascus rubber CCRC 31535 for the best result are nine days。 The medium does not need to be heat treatment and NaOH treatment. The deodorant is dried with 100 °C in the oven for the best result. The characters of the deodorants produced in this research are as follows: The deodorant produced using Monascus rubber CCRC 31535 works best in room temperature; when the temperature drops below 4 °C, the effect of deodorization of the deodorant decreases The deodorant produced using Monascus rubber CCRC 31535 is saturated in 48 hours。 The deoroizant produced using Lactic acid bacteria is saturated in 12 hours. Comparing with the deorizants sold in the market, the deodorants produced in this research are better in deodorization.

Record ID 第59筆 System ID 089DYU00250033
BCRC ID CCRC 21761, Public Year 89
Paper Name 巨峰葡萄酒釀製過程品質及風味變化之探討 Changes in Quality and Flavor during Brewing of Ge-Hon wine
Student Name 賴映汝
Teacher Name 游銅錫 陳齊聖
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 中文摘要 巨峰葡萄為本省夏、冬季盛產之水果之一,主要產於中部之大村鄉、東勢鎮等地。由於近年來國人對葡萄酒之喜好性增加,且因應我國即將加入世界貿易組織,所造成對葡萄果農及酒類工業的衝擊。因此,本論文特別選擇具有地方特色之大村巨峰葡萄來釀製葡萄酒。 本研究中分為二個部份,第一部份主要探討葡萄破碎後利用外加酵母菌來釀造巨峰葡萄酒之最適發酵條件。於此部份之結果顯示添加Saccharomyces cerevisiae CCRC 21761菌株發酵,控制糖度30。Brix,偏亞硫氫鉀用量為75ppm,主酵控溫度在25℃ 15天,後醱酵控溫在18℃15天,而後在4℃熟成6個月之巨峰葡萄酒最受喜愛。 本研究之第二部份為探討傳統民間製法及以第一部份所品評之最佳條件釀製之巨峰葡萄酒,經由上部空隙吹除捕捉法配合氣相層析質譜儀鑑定其香氣成分。由研究結果得知巨峰葡萄酒的香氣成分約有39種。可分為aldehydes類、alcohols類、esters類、ketones類、hydrocarbons類及miscellaneous類。 經由官能品評之結果顯示以添加Saccharomyces cerevisiae CCRC 21761菌株釀製之巨峰葡萄酒較傳統釀製之巨峰葡萄受消費者喜愛,可能原因為esters類及alcohols類組成間之百分比例才是決定巨峰葡萄酒之風味好壞的重要因子。 關鍵字:巨峰葡萄、啤酒酵母、偏亞硫氫鉀、葡萄酒、揮發性香氣成份。 ABSTRACT Ge-Hon grape, harvested twice a year (summer and winter), is a major domestic agriculture product of western Taiwan, which is distributed in the rural areas of Da-Tsuen and Don-She. The market of grape wine has been steadily increasing in recent years. In order to confront the approaching impact, due to joining WTO, on local farmers and the wine industry, this research studied the manufacture of grape wine using Da-Tsuen’s Ge-Hon grape. This research was divided into two phases. In the first phase, the optimum fermentation conditions, while adding various strains of brewing yeast, were studied. Results showed that the following brewing condition was optimum: 30 。Brix (initial sugar concentration), 75 ppm potassium bisulfite, fermentation at 25°C for 15 days, followed by 18°C for another 15 days, and maturation at 4°C for 6 months. In the second phase of this study, the volatile flavor compounds collected by aeration and adsorption were identified. The analysis of volatile flavor compounds came up with 39 different compounds. These flavor compounds were categorized as aldehydes, alcohols, esters, hydrocarbons and miscellaneous. Key word: Ge-Hon grape, potassium bisulfite, Saccharomyces cerevisiae, volatile flavor vompounds, wine.

Record ID 第60筆 System ID 093DYU00111007
BCRC ID CCRC 12826, Public Year 93
Paper Name 利用二水準部分因子實驗設計法尋求苔蘚桿菌生產聚麩胺酸之最適化饋料溶液濃度與饋料時間 Optimization of Feeding Solution Concentration and Feeding Time for Poly(glutamic acid) Production by Bacillus licheniformis with Two-Level Fractional Factorial Design
Student Name 陳宥瑾
Teacher Name 張耀南 吳建一
School Department Name 大葉大學 生物產業科技學系 Academic Degree 碩士
Abstract 本研究以苔蘚桿菌Bacillus licheniformis CCRC 12826為生產菌株,利用二水準部分因子實驗設計法探討苔蘚桿菌生產聚麩胺酸(poly-γ-glutamic acid, PGA)之最適化饋料溶液濃度與饋料時間。實驗結果得知目前饋料溶液中主要組成分的最適化濃度分別為40 g/L麩胺酸、42 g/L檸檬酸、158 g/L甘油、1 g/L氯化銨,於其最適饋料時間20h時,將其最適饋加體積25 mL之饋料溶液添加原修飾培養基後繼續培養至120 h,可得到PGA產量為27.4 g/L,略低於未添加此饋料溶液之產量(28.3 g/L);若將25 mL饋料溶液的添加體積濃縮為5 mL,即饋料溶液中主要組成分濃度分別為200 g/L麩胺酸、210 g/L檸檬酸、790 g/L甘油、5 g/L氯化銨,於相同饋料時間,將5 mL饋料溶液添加原修飾培養基後繼續培養至120 h,PGA產量為31.2 g/L,較未添加此饋料溶液之產量增加10.21%,若繼續培養至144 h時,PGA產量可高達37.7 g/L,較未添加此饋料溶液之產量增加33.25%。由此可知饋料添加程序雖然使得苔蘚桿菌生產高產量PGA所需時間延長,但卻能提高PGA產量,故此饋料溶液的添加程序策略仍然值得深入探討。 In this study, the optimization of feeding solution concentration and feeding time for poly-γ-glutamic acid (γ-PGA) by Bacillus licheniformis CCRC 12826 was investigated by using two-level factional factorial design。 It was found that the optimal volume of the suitable feeding solution comprising 40.0g/L glutamic acid, 42.0g/L citric acid, 158.0g/L glycerol, 1.0g/L NH4Cl, was 25mL and the optimal feeding time was at 20h of cultivation. When 25mL of the suitable feeding solution was added to the originally designed medium at 20h of cultivation, the yield of γ-PGA production was 27.4g/L at 120h of cultivation. It was little less than the yield (28.3g/L) without any feeding solution. When the volume of the feeding solution was reduced from 25mL to 5mL, the component concentrations were calculated to be 200.0g/L glutamic acid, 210.0g/L citric acid, 790.0g/L glycerol, 5.0g/L NH4Cl. When this concentrated feeding solution was fed at the optimal feeding time described above, the yield of γ-PGA production was 31.2g/L at 120h of cultivation. The γ-PGA production was increased by 10.21% from 28.3 to 31.2g/L, while the yield was 37.7g/L after 144h of cultivation and it was increased significantly by 33.25%. With these feeding processes, the culture time for the highest yield of γ-PGA production was delayed. However, this research demonstrated that the feeding processes with the two-level factional factorial design were worth using to improve the yield of γ-PGA production by B. licheniformis CCRC 12826

Record ID 第61筆 System ID 089DYU00250046
BCRC ID CCRC 12511, CCRC 12509, Public Year 89
Paper Name 發酵培養生產色胺酸之研究 Studies on Production of L-Tryptophan by Fermentative Cultivation
Student Name 黃淑貞
Teacher Name 涂瑞澤 洪淑嫻
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 色胺酸在自然界中的含量並不多,其主要存在於肉類、蛋、奶等動物性食品中;植物性食品除馬鈴薯外,在禾榖類如稻米、小麥等主要糧食及飼料作物中含量均低,其製備法可分為下列四種,分別為:蛋白質水解法、化學合成法、酵素轉換法及發酵法,而本研究中所的用的方法為發酵法。影響L-tryptophan產率的因素主要有培養基組成與培養條件。培養基組成包括碳源種類、天然氮源種類和無機氮源種類,而培養條件包括 pH對生產L-tryptophan的影響,以及利用非連續饋料培養對生產L-tryptophan之影響。本研究先以Corynebacterium glutamicum 21334 (CCRC 12511)為生產菌株,但是此菌株在培養過程中並無L-tryptophan的生產,因此決定以人工突變方式改良菌株。在突變改良過程中,先以化學突變劑NTG處理,再配合適當之篩選策略篩選出可能具生產力之菌株,然而經過一連串試驗後發現無法達成改良菌種之目的,亦即,變異株仍無顯著的跡象能生產L-tryptophan。 因此,決定更換菌株,改用Brevibacterium flavum ATCC 21427(CCRC 12509),經生產培養基測試後該菌株確定有L-tryptophan的產生,因此,以此菌株進行培養基的配方與培養條件的探討,而培養基配方包括碳源的種類(葡萄糖、果糖及蔗糖)、天然氮源的種類(酵母萃出物與蛋白胴)以及無機氮源的種類(硫酸銨與氯化銨),培養條件包括 pH對生產L-tryptophan的影響,以及利用非連續饋料培養對生產L-tryptophan之影響。培養基配方實驗結果顯示,碳源與天然氮源對菌體與L-tryptophan之生長均有顯著影響。使用葡萄糖與酵母萃出物時可得最佳L-tryptophan之產量;而使用蔗糖時,菌體量雖可達最高,但是L-tryptophan偏低,而無機氮源對菌體生長與L-tryptophan之產量則無顯著差異。利用30 mL 之培養基裝液量,於30 ℃, 150 rpm 下振盪培養96 h 後,其發酵液中累積0.01 g/L之L-色胺酸。 此外,本研究並探討pH與生產L-tryptophan之關係,分別將pH控制在6.0、7.0和8.0進行培養,結果顯示,當pH 值控制在7.0時為最佳。 以3 L 之發酵槽進行非連續批式培養法進行L-色胺酸生產試驗,其操作條件為:接種量2﹪,培養溫度 30 ℃,攪拌速率250 rpm,pH 7.0,裝液量2 L,總糖濃度約為4﹪,在此條件培養約 35 h,L-色胺酸產量可達0.02 g/L。因此,以葡萄糖為追加液時,可使碳源濃度維持固定濃度,使菌體在碳源充足且不至於引起抑制效果的狀態下生長,因此利用非連續饋料批式培養法,可得較高之L-tryptophan產量。 關鍵詞:L-tryptophan、變異、發酵、非連續饋料批式培養。 Abstract L-tryptophan is not abundant in the natural world. It mainly exists in the food of animal type such as meat, egg, and milk. For the food of plant type, except potatoes, the content of L-tryptophan in grains like rice and wheat is scare. Four methods to produce L-tryptophan include hydrolysis of protein, chemical synthesis, enzymatic transformation and fermentation. In this study, fermentation is used to produce L-tryptophan. The yield of L-tryptophan may be affected by many factors such as the composition of media, carbon sources, organic and inorganic nitrogen sources, operating conditions such as temperature and pH, and batch or continuous. Corynebacterium glutamicum 21334 (CCRC 12511) was used to begin。 Unfortunately, this strain produced no L-tryptophan. Mutant of this strain was then considered to improve the L-tryptophan yield. NTG (N-Methyl-N’-nitro-N-nitrosoguani -dine) was then used to treat the original strain, and several potential strains were selected. Through a series of screening experiments, the mutant strains still showed no evidence to produce L-tryptophan. In other words, mutant didn’t succeed in selecting a potential candidate for L-tryptophan production. Therefore, we decided to replace the strain with Brevibacterium flavum ATCC 21427 (CCRC 12509)。 The strain showed evidence to produce L-tryptophan after preliminary tests, so we used it to study optimal medium composition and culture conditions. The following three major components, carbon sources (glucose, fructose, and sucrose), organic nitrogen sources (yeast extract and peptone), and inorganic nitrogen sources ((NH4)2SO4 and NH4Cl), were considered. The pH value during batch fermentation was also explored. Experimental results showed that carbon and organic nitrogen sources had significant effect to the yield of L-tryptophan, and inorganic nitrogen sources had not. The combination of glucose and yeast extract could have the highest yield of L-tryptophan. Under a medium volume of 30 mL (flask volume 250 mL) and 30 ℃, the concentration of L-tryptophan could reach 0.01 g/L after a cultivation of 96 h in a shaker of 150 rpm. In addition, this experiment also studied the effect of pH on the production of L-tryptophan. Three pH’s ( 6.0, 7.0 and 8.0 ) were selected for testing. The results showed that the microbial growth and the L-tryptophan production were the best when the pH was set at 7.0. Furthermore, a fed-batch cultivation of Brevibacterium flavum ATCC 21427 was run with the following conditions:seed 2﹪, temperature 30 ℃, stirring rate 250 rpm, pH 7.0, working volume 2 L, and mass fraction of glucose around 4﹪. The concentration of L-tryptophan could reach 0.02 g/L under the above condition after a cultivation of 35 h. The concentration of glucose could almost remain constant if a proper control was imposed. Consequently, the microbial growth could sustain longer because a proper concentration of carbon source could maintain. Therefore, fed-batch cultivation may be a better way than a batch fermentation to produce L-tryptophan. Key words:L-tryptophan, mutant, fermentation, fed-batch cultivation.

Record ID 第62筆 System ID 092DYU00111041
BCRC ID BCRC 36355, Public Year 92
Paper Name 蓮花菌胞內及胞外多醣體的發酵及分離純化程序之研究 Studies on the Production and Purification of Intracellular and Extracellular Polysaccharides from Grifola frondosa by Submerged Fermentation
Student Name 游清源
Teacher Name 徐泰浩 張德明
School Department Name 大葉大學 生物產業科技學系碩士班 Academic Degree 碩士
Abstract 蓮花菌(Grifola frondosa)可以用來治療小便不利、水腫、腳氣、肝硬化及腹水、高血壓和肥胖症等疾病。而蓮花菌所產的多醣含有β-葡聚醣,與豬苓多醣相似,具有明顯的抗腫瘤效果,是故蓮花菌的大量培養有其經濟價值。由於液態培養具有週期短、效益高及易於規模放大的優點。而菇類的種類與來源的多樣化,最適化的培養條件差異頗大,必須個別進行系統化的試驗。故這方面的研究需藉由測試培養條件對菌絲體及菌液多醣體之影響,進而找出蓮花菌發酵生長之優化條件。藉由連續式發酵與回應曲面法在蓮花菌液態發酵系統中搜尋最佳比生長速率(μ)及胞外多醣單位體積產量(EPS)。其萃取出的多醣體為粗多醣體,所以將所得之胞多外粗多醣做一個純化之步驟,以得到較精緻且純之多醣體。在探討蓮花菌 BCRC 36355 於不同培養條件下對發酵產程之影響之前期實驗,結果發現,果糖1.5%、起始pH 4、25℃為較佳之培養條件。以此作為原點,以回應曲面法(RSM)探討各因子間對菌體生長速率交互作用。將陡升路徑實驗設計逼近比生長速率的極值範圍。將部分因子實驗及中心混成補充實驗所得之μ及EPS產量與發酵條件回歸而得的公式計算,比生長速率的最適條件為pH 4.01、DO 71.93 % 、果糖1.65 %、25.3℃。蓮花菌於不同發酵方式下比較在相同發酵體積下的平均每日產量,菌絲體生物質量是以5L連續式發酵最高,而EPS則是以5L批式發酵最高。純化過程中所分離各波峰皆含有(1→3)β-D-glucan,含量與波峰大小成正比。以胞外粗多醣溶液測定(1→3)β-D-glucan含量後發現其含量約31.68 %。 Grifola frondosa have been reported in many research articles include cure of edema, beriberi, ascites, hypertension and adiposis. Grifola frondosa’s polysaccharide is a β-(1-3)-linked glucan with branches of β-(1-6)-D-linked glucan showing the main of antitumor activity. Submerged fermentation of great advantage to cycle brevity, productive pregnant. Because of medicine fungus breed is biodiversity, then have different optimize cultured control factor, need respective to proceed systematized experimental. This study on the mycelial biomass growth and extracellular Polysaccharides from Grifola frondosa by continuous stirred tank reactor and response surface methodology. Production pure polysaccharides by purification of extracellular polysaccharides. The study shows that, in different cultured control experimental, best cultured control is achieved under the condition of 1.5% fructose, medium pH 4, cluture temp for 25℃; in steepest ascent path experimental, the highest mycelial biomass growth is achieved under the condition of 1.65% fructose, medium pH 4.01, cluture temp for 25.3℃, diffluence oxygen 71.93%; in different fermentation culture experimental, the highest mycelial biomass growth by continuous stirred tank reactor in 5L fermenter; the highest extracellular polysaccharides by 5L batch fermenter. All wave crest to have β-D-glucan in extracellular polysaccharides purification by ion exchange chromatography, all wave crest and β-D-glucan concentration is directly_proportional; coarse extracellular polysaccharides’s β-D-glucan concentration for 31.68 %.

Record ID 第63筆 System ID 090DYU00250011
BCRC ID CCRC 36355, CCRC 36434, Public Year 90
Paper Name 化學合成與天然浸液培養基及培養溫度對蓮花菌菌株間發酵產程菌絲體及多醣之影響 Effect of chemical defined and natural infusion media and cultural temperature on mycelium and polysaccharide from Maitake Grifola frondosa strains during fermentation
Student Name 張雅雯
Teacher Name 徐泰浩
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 蓮花菌(Grifola frondosa)又稱舞菇、千佛菌、灰樹花,是一種藥用和高價值的真菌,具有抗腫瘤、增加免疫力、抗糖尿病、抗愛滋病之多種生理活性,是中國及日本近年熱門之保健食品。應用醱酵技術生產菌絲體及生物活性多醣,其醱酵菌絲體具有生長期短、品質穩定,易於控制成本較低及可調控生理活性物質之優點。本研究使用醱酵技術培養蓮花菌,以便了解蓮花菌之發酵產程中之變化。本研究探討之變化包括:(一)蓮花菌之形態發生(二)使用不同之培養基對搖瓶培養菌絲體生長之影響(三)不同複合培養基時對搖瓶培養菌絲體生長之影響(四)使用發酵槽進行大量培養時對菌絲體生長之影響(五)不同溫度下胞內多醣和胞外多醣的變化(六)使用GPC 及HPLC對胞內多醣和胞外多醣進行分析。 研究結果顯示: 固態培養之下,以碳源為葡萄糖、氮源為氯化氨、碳氮比為5:1、微量元素為1%時菌落生長速率較佳,而在液態培養之下,以起始pH值為5.5、碳源為葡萄糖、氮源為硫酸氨、碳氮比為20:1、微量元素為1%、葡萄糖濃度為2%時,菌絲體生物質量較高。選擇六種不同的複合培養基,比較對菌絲體生物質量、發酵液還原糖及最終pH值之影響。其結果顯示10% 糙米粉對於蓮花菌生長為培養基時,可得到最高的菌絲體生物質量。5公升發酵槽試驗中於168小時發酵產程探討菌絲體產量。研究結果顯示於N1培養基下菌絲體生物質量最高。選用六株蓮花菌,評估於不同溫度下,其胞內多醣、胞外多醣、黏度及菌絲體生物質量之變化。結果顯示於25℃時,可得到最大胞內多醣及胞外多醣量,而 CCRC 36355 的胞內多醣生成量最多, CCRC 36434 的胞外多醣生成量最多。以GPC及HPLC來分析多醣成份,胞內多醣及胞外多醣分子量分別為103,900及70,200,且分析多醣成份中具有葡萄糖和木糖。 Grifola frondosa is a kind of fungi which can be used in medicine, therefore it has high economic value. Plenty of researches on the medical effects of Grifola frondosa have pointed out that it has positive effects on health in terms of antitumor, immunological enhancement, antidiabetic and anti-HIV….etc. It is now a popular health-food product in China and Japan. According to the related studies, employing mycelium of submerged culture to grow fungi has the advantages of minimizing growing time, stablizing the quality, and lowering cost. In addition, it also makes the biological activity easy to control. Thus, submerged culture of mycelium and polysaccharide have been adopted in this study to grow Grifola frondos. This study investigates the process of the growth of Grifola frondosa in terms of the following issues:(1) The difference of the morphological development of Grifola frondosa (2) the different growing effects caused by different elements of media in the shaking bottles (3) the different growing effects caused by different complex media in the shaking bottles (4) the growing effects of using fermentor tank to grow Grifola frondosa. (5) the variation of viscosity, endopolysaccharide and expolysaccharide in varied temperatures (6) the variances of the components and the molecular weight of polysaccharide. The study shows that, in solid culture, the best growing speed of the colony is achieved under the condition of glucose as carbon source, ammonium cholride as nitrogen source, the ratio of carbon and nitrogen as five to one, and trace element as one percent. In liquid culture, the highest mycelium biomass is achieved under the condition of glucose as carbon source, ammonium sulfate as nitrogen source, the ratio of carbon and nitrogen as twenty to one and trace element as one percent, the initial pH as 5.5, and glucose concentration as two percent. Regarding the different growing effects caused by different complex media in the shaking bottles, in this study, among the culture conditions of the six complex medium, the 10% brown rice powder achieved the highest mycelium biomass. The study also shows that using 5L fermentor tank to grow Grifola frondosa, N1 medium proves to be the best medium for mycelia production. It is also found that the optimun temperature for producing polysaccharide is 25℃. The subject that has the highest contents of endopolysaccharide is Grifola frondosa 36355. The subject that has the highest contents of expolysaccharide is Grifola frondosa 36434. The molecular weight of Endopolysaccharide is 103,900, and the molecular weight of expolysaccharide is 70,200. The analysis of the polysaccharide shows that it includes glucose and xylose.

Record ID 第64筆 System ID 091DYU00250022
BCRC ID CCRC 12826, Public Year 91
Paper Name 利用苔蘚桿菌生產聚麩胺酸之搖瓶 饋料批式培養探討
Student Name 邱紫與
Teacher Name 張耀南 洪淑嫻
School Department Name 大葉大學 食品工程學系碩士班 Academic Degree 碩士
Abstract 本研究目的在探討以苔蘚桿菌Bacillus licheniformis CCRC 12826 為生產菌株,探討以液態搖瓶培養方式生產γ-聚麩胺酸之不同饋料基質組成濃度、饋料時間與饋料體積之研究。苔蘚桿菌培養於起始酸鹼值pH 6.5之50 mL修飾培養基中,於37℃溫度下,以150 rpm振盪培養120h時,可產生聚麩胺酸產量為34.5g/L。當饋料基質組成分為40 g/L麩胺酸、42 g/L檸檬酸、158 g/L甘油、1 g/L氯化銨,且在第20h培養時間添加25 mL於修飾培養基後繼續培養至120h,可得到聚麩胺酸產量為24.5 g/L;若將饋料添加體積由25mL濃縮為5mL,即是饋料組成分濃度為200 g/L麩胺酸、210 g/L檸檬酸、790 g/L甘油、5 g/L氯化銨,如上述饋料與培養條件繼續培養120h後,γ-聚麩胺酸產量即可達到33.5 g/L,在144h培養後,γ-聚麩胺酸產量卻能高達45.3 g/L。雖然饋料程序使高產量γ-聚麩胺酸所需時間延長,但卻能提高產量,故饋料策略值得探討研究。 The object of this study is to study γ-poly(glutamic acid) (γ-PGA or PGA) production by Bacillus licheniformis CCRC 12826 in fed batch flask culture, to investigate the component concentrations if feeding medium and the feeding time for the high γ-PGA production。 The γ-PGA production was 34.5 g/L when B. licheniformis was cultured in the 50mL modified medium in 250mL flask with the initial pH6.5 at 37℃ shaker (150rpm) for 120h of cultivation. When the 25mL feeding medium containing glutamic acid (40 g/L), citric acid (42 g/L), glycerol (158 g/L), NH4Cl (1 g/L) was fed in the culture medium after 20h of cultivation, the γ-PGA yield was only 24.5 g/L. If the volume of the feeding medium was reduced from 25mL to 5mL, the component concentrations should become 200 g/L glutamic acid, 210 g/L citric acid, 790 g/L glycerol, 5 g/L NH4Cl. When this concentrated feeding medium was fed as described above, the γ-PGA yield became 33.5 g/L for 120h of cultivation. The overall γ-PGA product isolated after in cultivation of 144h was 45.3 g/L. The time for the highest γ-PGA production was delayed, but the feeding process was worth using to improve the γ-PGA yield.

Record ID 第65筆 System ID 091DYU00250029
BCRC ID CCRC 36434, CCRC 36355, CCRC 36357, Public Year 91
Paper Name 食藥用真菌-蓮花菌菌絲體及多醣體發酵產程之研究 Production of Mycelium and Polysaccharide from the Edible and Medicinal Fungus Grifola frondosa by Submerged Fermentation
Student Name 王懿丞
Teacher Name 徐泰浩
School Department Name 大葉大學 食品工程學系碩士班 Academic Degree 碩士
Abstract 摘 要 蓮花菌(Grifola frondosa)又稱灰樹花、舞茸等,屬於非摺菌目、多孔菌科、豬苓屬,是木生腐敗菌種的好氣性真菌,其多醣體具有抗腫瘤、降血壓、降血脂、治療肝炎、減肥、糖尿病等功效,以具有β-1,6支鏈之β-1,3葡聚糖為其生物活性主要成份,在其子實體及菌絲體均有此結構的多醣體。一般人工栽培由接種到生成子實體需要3個月以上的時間,應用發酵技術生產菌絲體及生物多糖體,具有發酵時間短、品質穩定等優點。本研究主要探討:(一)、篩選不同品系之最適產醣蛋白質之菌株,並分析其游離胺基酸、總胺基酸、主要元素(C, N, O)含量及酵素群活性;(二)、探討蓮花菌在搖瓶與靜置培養時,在不同培養條件下對生物質量、胞內與胞外粗多醣蛋白質複合物之影響;(三)、比較合成、半合成培養基培養前後之粗多醣蛋白質複合物與游離胺基酸差異;(四)、以搖瓶培養探討產程變化,並再擴展到5L、20L發酵槽階段。 研究結果顯示:不同品系蓮花菌株比較,在PDA培養時以CCRC 36434生長直徑對大,以PDB與基礎培養基培養以CCRC 36355胞內多糖體產量最高,以PDB培養胞外多糖體以CCRC 36357最高;以游離胺基酸含量,顯示CCRC 36355含量最高總胺基酸以CCRC 36434含量最高;酵素群譜分析,以胞內酵素群酵素含量最高,不同品系蓮花菌株酵素含量和種類不相同。振盪培養時,胞內多糖體以碳源為4%葡萄糖、氮源為0.1%草酸胺、其它添加物為0.15%磷酸氫鉀能有最高產量;游離胺基酸含量在碳源為3%蔗糖、氮源為0.2%草酸銨、其它添加物為0.45%磷酸氫鉀最佳。半化學合成培養基氮源含量,以胞內多糖體以0.2%蛋白胨、胞外多糖體以0.4%酵母萃出物、菌絲體生物質量以0.8%胰蛋白最佳。靜置培養時,胞內多糖體以碳源為3%果糖與氮源為0.2%硝酸銨最佳;游離胺基酸含量在碳源為4%甘露糖醇、氮源為0.4%草酸銨最高。探討產程,以搖瓶培養EPS在第11天最高,以5公升、 20公升發酵槽皆在第5天達最高量。在5公升發酵槽培養之游離胺基酸含量以第2天最高,隨時間延長,胺基酸含量減少。胞外多糖體與胞內多糖體在經陰離子交換樹脂可得正電荷之多糖體蛋白質複合物;胞內多糖體有少許負電荷之多糖體蛋白質複合物。 ABSTRACT Grifola frondosa in Japan as maitake (dancing mushroom), in China as gray tree flower, it is a Basidiomycete fungus belonging to the order Aphyllopherales, and family Polyporaceae, as a white-rot and acreobe fungus. It is polysaccharides have been reported in many research articles include antitumor, immunological enhanc- ement, antidiabetic and anti-HIV, etc. A β-(1-3)-linked glucan with branches of β-(1-6)-D-glucose showing the main of pharma- cological activity has been isolated from fruit bodies and mycelium. By synthetic-log cultivation, when the young mycelium grown to fruit body need of three months.according to the related studies, employing mycelium of the submerged culture to grow the fungus has the advantages of the shorter growth time, better product quality, and lower cost. This study investigates the process of the growth of Grifola frondosa in terms of the following issues:(1)to screen different strains producing polysaccharides and analysis of free amino acid、total amino acid、main element (C、N及O)、enzyme activity;(2)studies biomass、extracellular polysaccharide and intracellular polysaccharide under shaking and static bottles;(3)to compare of the free amino acid and polysaccharides by the chemical and semi-chemical medium;(4) effect of submerged fermentation in shaking bottles, and to expand of 5 and 20L fermentor. The study shows that, in PDA culture, the CCRC 36434 have the best growing speed of the colony, in PDB and base medium culture, the CCRC 36355 have best yields of intracellular poly- saccharides, CCRC 36357 which yields the highest content of extracellular polysaccharide;in content of free amino acid, shows the higher of CCRC 36355;in content of total amino acid by mycelium, the higher of CCRC 36434;in api-ZYM system, intracellular enzyme have higher activity;in shaking culture by chemical medium , the highest intracellular polysaccharides is achieved under the condition of 4﹪glucose, 0。1﹪ammonium oxalate, 0.15﹪potassium phosphate, the highest the free amino acid is achieved under the condition of 3﹪sucrose, 0.2﹪ammonium oxalate, 0.45﹪potassium phosphate;in shaking culture by semi- chemical medium , the highest intracellular polysaccharides is achieved under the condition of 0.2﹪peptone, the highest extra- cellular polysaccharides is 0.4﹪yeast extract, the highest the mycelium biomass is 0.8﹪tryptone;in static culture, the highest intracellular polysaccharides is achieved under the condition of 3﹪fructose, 0.2﹪ammonium nitrate, the highest the free amino acid is achieved under the condition of 4﹪mannose, 0.4﹪ammonium oxalate;studies submerged fermentation, the higher extracellular polysaccharides on day 11 by shaking culture and 5、20L fermentor on day 5;in 5L fermentor, free amino acid have best yield on day 2, the free amino acid follow time to decreased. extracellular polysaccharides and intracellular polysaccharides contain acidic glucan by ion exchange chromatography and intracellular polysaccharides contain less basic glucan.

Record ID 第66筆 System ID 091DYU00250033
BCRC ID CCRC 35515, Public Year 91
Paper Name 二階段醱酵時不同生長條件對綠殭菌產孢之影響 Effects of growth condition to conidial production of Nomuraea rileyi by two-stage fermentation
Student Name 林明申
Teacher Name 謝建元
School Department Name 大葉大學 食品工程學系碩士班 Academic Degree 碩士
Abstract 蟲生真菌綠殭菌能感染30多種鱗翅目害蟲,特別對夜蛾科幼蟲致病力強,其分生孢子對害蟲具感染性,是極具開發潛力的殺蟲微生物。本研究以Nomuraea rileyi CCRC 35515為試驗菌株,探討二階段醱酵時不同生長條件對綠殭菌產孢之影響。於醱酵中途加入營養源試驗中,液態醱酵第五天時加入20 mL氮源(80% V8 juice及0.6%玉米浸粉),液態醱酵至第六天接種至固態基質其產孢量可達6.97×109 conidia/g-dry material。 此外在添加界面活性劑於液態培養基試驗中,以添加全透力於液態培養基,菌體生長至第五天時菌體濃度最高可達0.0453 g/mL。在產量方面,亦是以添加全透力產孢量最高達4.89×109 conidia/g-dry material。研究結果發現,在搖瓶試驗中,使用棉花塞(瓶塞32 mm、棉花塞重量約4.8 g)為封瓶者,菌體濃度較使用橡皮塞(瓶塞中心之通氣孔洞,直徑約4 mm,孔隙中填充0.25 g棉花)佳,可達0.03 g/mL,璀¢量亦是以棉花塞者最佳達1.11×1010 conidia/g-dry material。此外在添加幾丁聚醣試驗中,以添加1%幾丁聚醣於固態基質中最佳,產孢量達5.69×109 conidia/g-dry material。生物檢定中對三齡期之甜菜夜蛾死亡率並無助益,死亡率為45%±3.1%。 Nomuraea rileyi has been reported that it is able to infect more than 30 species of lepidopterous pests and it makes the larva of several noctuid pests cause high mortality. Thus, it has great potential to be a microbial control agent. The purpose of this research was studying the effects of growth condition on conidia production of Nomuraea rileyi by using two-stage fermentation. 20 mL nitrogen source (80% V8 juice and 0.6% C.S.P.) was added to liquid culture at day 5, and then liquid culture was transfered to solid substrate at day 6. For another ten days’ fermentation, the production of conidia was 6.97 ×109 conidia/g-dry material. Effect of addition of surfactant, the total wet addition was found to have the best production on both mycelia cell concentration and conidia concentration with 0.0453 g/mL and 4.89×109 conidia/g-dry material, respectively. In this study, we found that flask with cotton plug (using 4.8g cotton as the plug in flask bottle neck with I.D. 32mm) had better fungus growth and conidia production than flask with rubber plug (with a hole of I.D. 4mm with 0.25g cotton), and the best production on both mycelia cell concentration and conidia concentration with 0.03 g/mL and 1.11×1010 conidia/g-dry material, respectively. Furthermore, addition of 1% chitosan to product of conidia (5.69×109 conidia/g-dry material) was better than others, but the is high production was not favorite the mortality (45%±3.1%) of S. exigua.

Record ID 第67筆 System ID 086DYU00250001
BCRC ID CCRC 31499, Public Year 86
Paper Name 利用紅麴菌釀造水果酒之研究 Production of Fruit Red Wine Made by Monascus anka CCRC 31499
Student Name 林東穎
Teacher Name 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 紅麴菌的一級代謝產物與多種水解酵素利用於水果紅酒的釀造。最適紅麴菌釀造之水果種類為蘋果汁所釀造水果酒品質最佳, 酒精度可達 4.1 %,且消耗單位糖度轉換酒精值高達至 0.347 %,表示糖分轉換酒精利用率最佳, 另在官能試驗(色澤與外觀),蘋果紅酒亦有最佳之接受度,故選定蘋果汁作為釀造之對象。利用紅麴液態培養與酵母發酵蘋果酒,兩者於不同天數混合培養至第十天進行上述分析測試,結果顯示在第四天混合時酒精度為 6.12 %,消耗單位糖度轉換酒精值高達至 0.313 %, 由此可知,將紅麴菌與酵母菌於不同天數混合培養之方法比單獨培養後再混合佳。 單獨以 Monascus anka CCRC 31499 為紅麴釀酒菌株, 進行培養(釀酒)溫度(25-37 ℃)、糖類碳源及培養天數等影響因素的探討,結果顯示最適釀酒溫度為 25 ℃; 在 25 ℃培養下蘋果紅酒具有 3.12 %酒精度, 其最佳消耗單位糖度轉換酒精度為0.62 %,培養溫度愈高,蘋果紅酒愈呈現鹼性狀態。另外,在色澤方面,△ a 值僅有0.1039/ 消耗糖度,紅色色素呈現不明顯。利用紅麴菌釀造蘋果紅酒的最適天數為二十天,且葡萄糖及果糖為其較佳醣類碳源,對果糖而言,酒液的酒精度可高達 4.98 %,然而△ a 值卻下降為 -0.12/ 消耗糖度,另外麩胺酸鈉添加有助於提高酒精度, 而且縮短 釀造天數,對葡萄糖及果糖而言,釀酒十天後, 即可達到最高產量,濃度分別為5.75 %與 5.80 %, 但在色澤轉變方面略為不足,由此可知,利用紅麴菌釀造蘋果紅酒未來仍值得探討研究。 In this study, fruit red wine was made from fruit juice by Monascus ankaCCRC 31499 and brewer`s yeast。 Apple juice was one of the best source foryield and quality improvement in wine making. The red wine was mixed by the 1:1 ratio of the apple wines made by M.anka and yeast after ten-day fermentation. The ethanol yield of the applered wine was 4.1% and 0.347% /0Brix.The color of the wine was moreacceptable than those of the wines made from grape and orange juice. Theother apple red wine was made by both M. anka and yeast from four-day toten-day mixture fermentation. The ethanol yield of the wine was 6.12% and0.313%/0Brix.It was found that the ethanol yield of the wine made by mixturefermentation, while the ethanol%/0Brix yield was decreased. The apple red wine only made by Monascus anka CCRC 31499 was alsostudied。 Three different strategies of operation variables, such as winemaking temperature, time and sugar-type carbon source, were investigated.The optimal wine making temperature was at 25 ℃ for 10 days The ethanolproduction was 3.12% and 6.12%/0Brix.The pH value of the wine becomeincrease as winemaking temperature increased. The color of the wine made at25 ℃ become more red when the △ a increase was 0.1039/0Brix. ℃.Fructoseand Glucos e were the optimal sugar-type carbon sources for M. anka winemaking. The ethanol yield for fructose was up to 4.98% but the △ a increasewas -0.12/ 0Brix. In addition, monosodium glutamate(MSG) addition was usedto improve the increase of ethanol production and color of winemaking. Forglucose and fructose the ethanol yields of red wines were 5.75% and 5.80%,respectively, while the colors were not changed. This research demonstratedthat M. anka CCRC 31499 was worth using to improve the ethanol yield an dcolor quality improvements in apple red winemaking

Record ID 第68筆 System ID 086DYU00250012
BCRC ID CCRC 31527, Public Year 86
Paper Name 利用紅麴菌以固-液態培養方式產生膽固醇合成抑制劑- Production of Hypocholesterolemic Agent by Monascus pilosus in Solid-Liquid Culture
Student Name 謝鳳龍
Teacher Name 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究以 Monascus pilosus CCRC 31527 為主要紅麴生產菌株, 進行最適搖瓶培養條件及培養基碳、氮源等因素之探討。 結果顯示最適培養溫度介於 25-30 ℃之間,而最佳培養基起始酸鹼值及其體積含量亦隨溫度而有所改變。在 25 ℃培養下,培養基的最佳起始酸鹼值為 pH 8,最適體積含量為 25 ml,可達產量 1.54 × 10-3 mg/ml;而在 30 ℃培養時,培養基最佳起始酸鹼值為 pH 5,體積含量為 125 ml 最適合。 另外,最佳澱粉類或醣類碳源為白米澱粉,有機氮源會隨培養溫度而有所不同,在 25 ℃培養下,酵母萃取物為最佳者;而 30 ℃下,則以 peptone 為佳。 在 25 ℃溫度下培養 ,以白米為澱粉類碳源時,在 25 ml 的培養基體積中, 使其起始酸鹼值為 pH 8.0的固 - 液態培養條件下, 可使本研究 monacolin K 產量達到最高, 約為 7.178 ×10-3 mg/ml。 可知,以固 - 液態培養培養方式提高膽固醇合成抑制劑產量是值得探討的。 另外,利用回應曲面法尋求紅麴菌生產膽固醇合成抑制劑之最適化培養條件時,發現酵母萃取物的濃度設計,應偏向於愈低濃度的設計,中心點設為 0.5 g/L;甘油則要偏向高濃度設計, 中心點設為 120 ml/L,其他如白米澱粉、葡萄糖等的濃度則不作改變,如此才可能獲得較好之結果。 In this study, monacolin K was produced by Monascus pilosus CCRC 31527in solid-liquid culture。 Several different strategies of manipulatingvariables, such as culture temperature, initial pH and volume of medium,carbon and nitrogen source, were investigated. It was found that the optimumculture temperature was between 25 oC and 30 oC. The optimum media in theculture of 25 oC were found to be pH 8.0 and 25 ml; while those in theculture of 30 oC were to be pH 5.0 and 125 ml. Furthermore, rice powder wa sconsidered to be the best carbon source in giving the maximum yield ofmonacolin K. The maximum yield was 7.178(10-3 mg/ml in the culture of 25 oC.The appropriate organic nitrogen source was changed with the culturetemperature. Among the nitrogen sources tested, yeast extract and peptonewere found to be suitable for monacolin K production in the culture of 25 oCand 30 oC, respectively. This research demonstrated that the solid-liquidculture was worth improving the yield of monacolin K. Response surface methodology was used to optimite monacolin K productionby M. pilosus CCRC 31527 in flask culture。 Analysis of variance indicatedthat the interaction between operation variables (yeast extract andglycerin) in the quadratic model was only significant. The concentration ofyeast extract at the design center point was recommended to be at 0.5 g/L,while the concentration of glycerin was at 120 ml/L and the concentrationsof both rice starch and glucose were 30 g/L. For this kind of complexmedium, it would be good for monacolin K production by M. pilosus CCRC31527

Record ID 第69筆 System ID 091DYU00515004
BCRC ID CCRC 12826, Public Year 91
Paper Name 以批次醱酵槽生產聚麩胺酸及其抗凍性之研究 Investigation of γ-poly(glutamic acid) production by batch reactor and antifreeze activity by Differential Scanning Calorimeter
Student Name 邵奕遠
Teacher Name 施英隆
School Department Name 大葉大學 環境工程學系碩士班 Academic Degree 碩士
Abstract 本研究係以醱酵槽探討B.licheniformis CCRC 12826生產聚麩胺酸最佳條件,採用逐步固定法之方式,探討曝氣量、攪拌速度、pH值對聚麩胺酸產量影響,並於培養期間探討碳源消耗,溶氧,黏度,細胞生長及聚麩胺酸產量變化,以找出聚麩胺酸之最適生產條件。 結果顯示在pH=6.0時最適合菌的生長,而在pH=6.5時則較適合聚麩胺酸之生合成。於攪拌速度之探討中發現在100rpm時,由於基質質傳速率慢且菌量生長緩慢,導致聚麩胺酸的產量不高,而在300rpm時,雖然其基質消耗快速,但是可能因轉速快而造成聚合酉每不易產生或是易被破壞,因此產量相對較低,200rpm最適合聚麩胺酸的聚合。於曝氣量的變化中,以曝氣量(3L/min)的產量最高,其產量較曝氣量(2L/min)多了33%。總之,本研究發現當B.licheniformis CCRC 12826培養於培養基F(NH4Cl 7.0(g/L) , K2HPO4 0.5(g/L) , MgSO4․7H2O 0.5(g/L) , FeCl3 ․6H2O 0.04(g/L) , CaCl2․2H2O 0.15(g/L) , MnSO4․4~6H2O 0.104(g/L), Citric acid 22(g/L), Glutamic acid 65(g/L), Glycerol 170(g/L)),於pH=6.5,攪拌速率200rpm,曝氣量(3L/min)之10L醱酵槽中,所得之最佳產率為25.93g/L,較搖瓶培養時增加了23%。 除運用醱酵槽生產聚麩胺酸之外,所得之聚麩胺酸則進行抗凍性之研究。我們製備不同的光學異構,不同分子量及各種金屬鹽(鈉、鉀、鎂、鈣)之聚麩胺酸並探討其分子結構與抗凍活性之關係。 結果顯示不同光學異構組成之聚麩胺酸之抗凍活性是相似的,顯然光學異構不影響聚麩胺酸抗凍活性。在不同分子量之聚麩胺酸方面,分子量越小者其抗凍活性越高,而在分子量為15,151時,其抗凍活性(AF)達到了5.57。在不同金屬鹽之聚麩胺酸中,其抗凍活性為Mg >Na ≈Ca >K,這趨勢與無機金屬鹽之抗凍性相似,即高離子電荷具高抗凍性,然而如何解釋分子結構與抗凍性之反應機構則尚待研究。 In this study we investigated the effects of pH, aeration and agitation on γ-polyglutamic acid(γ-PGA) productivity in a 10-L fermenter . In addition, the changes of carbon source ( citric acid, glutamic acid, glycerol), dissolved oxygen, cell density, andγ-PGA production were monitored during fermentation process. The results showed that the cell growth is most suitable at pH=6.0, but the highest yield of γ-PGA is at pH=6.5. Because of low mass-transfer rate and low cell growth at 100rpm , theγ-PGA yield is low. Although mass-transfer rate is high at 300rpm, the γ-PGA yield at 300rpm is very low as well. Good yield ofγ-PGA was obtained when the agitation was at 200rpm. In addition, it was found that theγ-PGA yield is highest at aeration rate of 3L/min. In conclusion, when B.licheniformis CCRC 12826 was cultivated in medium F (NH4Cl 7。0(g/L) , K2HPO4 0.5(g/L) , MgSO4․7H2O 0.5(g/L) , FeCl3 ․6H2O 0.04(g/L) , CaCl2․2H2O 0.15(g/L) , MnSO4․4~6H2O 0.104(g/L), Citric acid 22(g/L), Glutamic acid 65(g/L), Glycerol 170(g/L)), pH 6.5, 200rpm, 3L/min, the highest yield of γ-PGA was obtained ;it was 25.93g/L. The yield increased 23% from 21g/L in shake flask culture to 25.93g/L in 10-L fermenter. In addition to investigation of γ-PGA production in fermenter, we also investigated the antifreeze activity of γ-PGA. Various enatiomeric isomers, metals salts and molecular weights of γ-PGA, produced by B.licheniformis CCRC 12826 were prepared and their antifreeze activities were studied by differential scanning calorimetry(DSC)。 The antifreeze activity of γ-PGA was significant;it increased as its molecular weight decreased. However, the antifreeze activity ofγ-PGA was indifferent to its enatiomeric content. The antifreeze activity was cation dependent;it decreased in the order Mg salt > Ca salt ≈ Na salt >K salt. This trend agrees with that for inorganic chlorides;that is high ionic charge leads to high antifreeze activity. The mechanism by which the cryoprotective effects of γ-PGA can be explained is still yet to be determined.

Record ID 第70筆 System ID 088DYU00250001
BCRC ID CCRC 11847, Public Year 88
Paper Name 製備Bifidobacterium longum 保健性菌種之噴霧乾燥方法之開發 Development of spray drying method for the preparation of probiotic bacteria Bifidobacterium longum
Student Name 江捷峰
Teacher Name 陳鴻章 李範亞
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究主要利用噴霧乾燥方式對人體腸胃系統有益之促生菌-雙歧桿菌(Bifidobacterium longum , CCRC 11847)進行加工處理,期能獲得良好的保存方法以供商業應用。首先以一次只改變一個變因之方式探討菌齡、進料速度、出口溫度、乳化劑種類、乳化劑濃度、抗氧化劑種類和抗氧化劑濃度等因子對菌體存活率之影響,而後將各因子之最適化條件予以組合進行噴霧乾燥,以期求得最佳之菌體存活率。 將菌體接種至 MRS broth 中,並在 37 ℃ 厭氧狀態下培養不同時間後進行噴霧乾燥測試以探討最佳菌齡,結果發現培養36 小時後之菌數可達 3.0*108 CFU/mL,菌種之生長狀態為最佳。 而進行噴霧乾燥時出、入口溫度分別設定為 90 ℃ 及 37 ℃ 所達到的效果為最佳。 以不同種類之乳化劑如glycerol monostearate、sorbitan monostearate 及 stearic acid 作為菌種保護劑進行噴霧乾燥,發現 glycerol monostearate 對 Bifidobacterium longum 的保護效果最好,存活菌數可達 9*105 CFU/g。而在乳化劑濃度方面,使用1%、2%、3% 三種不同濃度,其保存效果隨著乳化劑的濃度的提高而有增加的趨勢。 不同種類抗氧化劑如 butyl hydroxyanisol (BHA)、dibutyl hydroxy toluene (BHT) 及抗壞血酸進行噴霧乾燥後,發現,BHA 對菌體的保護效果最佳。當改變抗氧化劑濃度為 1,000 ppm、2,000 ppm、3,000 ppm 進行菌體存活率試驗時,發現高濃度的抗氧化劑保護效果略佳於低濃度的抗氧化劑。 將以上之最適條件因子組合後進行噴霧乾燥,結果發現 Bifidobacterium longum 此菌株於噴霧乾燥後及室溫下保存 30天後之菌體存活率均高於使用基本條件之菌體存活率。當初始菌數為 3.0*108 CFU/g 時,以基本條件進行噴霧乾燥後之存活菌數 420 CFU/g,貯存30天後菌株之存活率為零。若以最適條件進行噴霧乾燥,則存活之菌數為 9.0*105 CFU/g,於室溫下保存 30 天後菌體之存活數為 4.4*105 CFU/g,其存活率可達到 49% 左右。 In order to obtain a better preservation method for commercial purpose, this research investigated the feasibility of preserving Bifidobacterium longum CCRC 11847, a probiotic bacterium in human gastrointestinal tract, with spray-drying process。 Initially, the effect of each factor, such as time course, outlet temperature of spray-drying, feed velocity and types and concentration of emulsifiers and antioxidants, on the survival ratio of the bacteriumwere was investigaed by changing one factor at a time. Then the most suitable conditions of each factor were combined for spray-drying process to obtain the best survival ratio of the bacteria. The bacterium was cultured anaerobically at 37 ℃in the MRS broth and spray-dried at different time interval to obtain the best time course for preservation. After 36 hours , the total viable plate count was 3*108 CFU/mL and the growth of the bacteria was optimum. The effect was the best when the outlet and inlet temperature of the spray dryer were set at 90 ℃ and 37 ℃, respectively. When different kinds of emulsifers such as glycerol monostearate, sorbitan monostearate, and stearic acid were used as the protective agent in the spray-drying process, it was found that glycerol monostearate had the best protective effect on Bifidobacterium longum. The total viable plate count after spray-drying was 9*105 CFU/g. As for the concentration of the emulsifers, three different concentrations (1%, 2%, 3%) were used and the results indicated that the protective effect increased with increasing concentration of the emulsifers. When different kinds of antioxidants, such as butyl hydroxyanisol (BHA), dibutyl hydoxytoluene (BHT), and ascorbic acid (Vitamin C) were used as the protective agent in the spray-drying process, it was found that BHA had the best protective effect on the bacterium among the three. When the survival ratio test of the bacteria was conducted at antioxidant concentrations of 1000 ppm, 2000 ppm, and 3000 ppm, it was found that higher antioxidant concentrations had better protective effect than lower concentrations. When the optimal conditions of each factor in the spray-drying process were combined, the survival ratios of Bifidobacterium longum right after spray-drying and after 30 days storage at room temperature were higher than those of basal set. With an initial cell concentration of 3*108 CFU/mL, the survival count right after spray-drying with basal set conditions was 420 CFU/g, and none survived after 30 days of storage. However, the survival count right after spray-drying reached 9*105 CFU/g when the optimal set of conditions was employed, and the survival count of Bifidobacterium longum after 30 days storage at room temperature was 4.4*105 CFU/g , representing a survival ratio of 49%.

Record ID 第71筆 System ID 088DYU00250005
BCRC ID CCRC 31535, Public Year 88
Paper Name 紅麴菌二級代謝物中紅麴色素與膽固醇合成抑制劑共存性之探討 Studies on Coexistence of Monascus Secondary Metabolites-Red Pigments and Monacolin K
Student Name 黃育輝
Teacher Name 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究利用回應曲面法(RSM),探討紅麴菌Monascus ruber CCRC 31535最適培養基(白米粉、peptone、甘油與葡萄糖)之濃度影響,以了解紅麴菌產生孢內與孢外紅色色素與膽固醇合成抑制劑之共存性,其結果經由RSM試驗顯示,發現peptone與甘油添加對孢內monacolin K產量有顯著影響;此外,白米粉、peptone 與葡萄糖添加濃度加倍分別對其產量亦有顯著影響;相對地,白米粉、peptone與葡萄糖添加對孢外monacolin K產量則有顯著影響;而且白米粉-葡萄糖交互作用對其產量有顯著影響;對孢內紅色色素產量而言,白米粉、peptone與甘油添加對其產量有顯著影響,並發現白米粉-甘油及peptone-甘油交互作用明顯影響其產量,另外,甘油添加濃度加倍對孢內紅色色素有顯著影響;對孢外紅色素產量而言,白米粉、peptone與甘油添加對其產量顯著影響,並發現白米粉-peptone 交互作用有明顯影響其產量。 本研究發現紅麴菌二級代謝物(monacolin K與紅色色素)孢內產量較孢外產量為高,並由回應曲面法尋求較合適培養基組成分為34.4 g/L白米粉、10.8 g/L peptone、36.4 mL/L 甘油、129.2 g/L 葡萄糖、2 g/L KNO3 及1 g/L MgSO4.7H2O。以此培養基,經過十天搖瓶培養後,得到目前本研究最高孢內monacolin K 產量約為0.157 mg/mL、而孢外 monacolin K 產量為 0.33 μg/mL;孢內紅色色素產量則為 3.488 mg/mL,而孢外紅色色素產量僅有0.113 mg/mL濃度。 In this study, response surface methodology (RSM) was used to optimize the concentrations of the compositions (rice powder, peptone, glycerin and glucose) of the rice-glycerin complex medium for Monascus ruber CCRC 31535。 The coexistence of Monascus intracellular and extracellular secondary metabolites (red pigments and monacolin K) was also studied. For the intracellular monacolin K production, the effects of peptone and glycerin on monacolin K production were only significant. In addition, analysis of variance indicated that the quadratic terms of rice powder, peptone and glucose in the quadratic model were significant. For the extracellular monacolin K production, the effects of rice powder, peptone and glucose were significant. The interaction red pigments, analysis of variance indicate that the effects of rice powder, peptone and glycerin on the intracellular and extracellular pigment production were only significant. It also indicated the interaction terms of rice powder-glycerin and peptone-glycerin were significant. In addition, the quadratic term of glycerin in the quadratic model was significant. For the extracellular red pigments, the interaction term of rice powder-peptone in the quadratic model was significant. In this study, the productivity of the intracellular secondary metabolites ( monacolin K and red pigments ) was higher than that of the extracellular ones. At present, the most suitable composition for the intracellular monacolin K production was found to be 34.4 g/L rice powder, 10.8 g/L peptone, 36.4 mL/L glycerin, 129.2 g/L glucose, 2 g/L KNO3 and 1 g/L MgSO4.7H2O. With these compounds, the production was 0.157 mg/mL after 10 days of cultivation, while the extracellular monacolin K production was 0.33 μg/mL. The intracellular red pigment production was 3.488 mg/mL, while the extracellular one was 0.113 mg/mL. Whith the compounds described above.

Record ID 第72筆 System ID 090DYU00250005
BCRC ID CCRC 12511, CCRC 11631, Public Year 90
Paper Name 利用變異技術提昇Corynebacterium glutamicum生產色胺酸之研究 Promotion of L-Tryptophan Production by Mutation of Corynebacterium glutamicum
Student Name 李明貞
Teacher Name 涂瑞澤 洪淑嫻
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 摘 要 色胺酸在自然界中的含量並不多,其主要存在於肉類、蛋、奶等動物性食品中;植物性食品除馬鈴薯外,在禾榖類如稻米、小麥等主要糧食與飼料作物中含量均低,其製備法有下列四種,分別為:蛋白質水解法、化學合成法、酵素轉換法及發酵法,其中發酵法是利用廉價之碳源與氮源等物質,直接進行發酵反應,符合低生產成本,故本研究中所的用的方法為發酵法。本實驗先以Corynebacterium glutamicum CCRC 12511,進行搖瓶培養,但並無生產L-色胺酸之跡象,因此決定以人工突變方式改良菌株再配合適當之篩選策略篩選出可能具生產力之菌株,然而 Corynebacterium glutamicum CCRC 12511經過一連串試驗後發現無法達成改良菌種之目的,即變異株仍無顯著的跡象能生產L-色胺酸因而改用Corynebacterium glutamicum CCRC11631,進行搖瓶培養,但其無生產L-色胺酸之跡象,故亦對Corynebacte- rium glutamicum CCRC1163進行突變,再經篩選出可能具生產力之變異株No.195 以Corynebacterium glutamicum CCRC 11631所篩選出可能具有生產L-色胺酸潛力之變異株No.195,利用實驗設計法進行探討此菌株在不同培養條件對菌體濃度與L-色胺酸濃度的影響,培養條件包括:葡萄糖初始濃度、有機氮源種類及培養時間。實驗結果經變異數分析 (Analysis of Variance, ANOVA)得知,葡萄糖初始濃度、有機氮源種類及培養時間對菌體濃度皆有影響。葡萄糖初始濃度與有機氮源種類對L-色胺酸濃度有影響,而培養時間對L-色胺酸濃度並無影響。 根據實驗結果所得之最適培養條件─葡萄糖初始濃度為40 g/L、有機氮源種類為玉米浸泡液及培養時間為72 h。再以變異株No.195於30 ℃,150 rpm振盪培養72 h,其發酵液中L-色胺酸濃度累積達8.374 mg/L。再以3 L發酵槽進行L-色胺酸生產發酵培養試驗,其操作條件為:接種量2﹪,培養溫度 30 ℃,攪拌速率250 rpm,pH 7.0,裝液量2 L,總糖濃度為4﹪,L-色胺酸產量可達13.5 mg/L。 關鍵詞:L-色胺酸、變異、發酵、Corynebacterium glutamicum。 Abstract L-Tryptophan is not abundant in the natural world. It mainly exists in the food of animal type such as meat, egg, and milk. For the food of plant type, except potatoes, the content of L-tryptophan is very scarce especially in grains like rice and wheat. Four methods, including hydrolysis of protein, chemical synthesis, enzymatic transformation, and fermentation, are used to produce L-tryptophan. Among them, fermentation is most frequently used because it could use low-priced carbon and nitrogen sources. Therefore, fermentation is the method used in this study to produce L-tryptophan. Corynebacterium glutamicum CCRC 12511 was the first strain to begin。 Unfortunately, this strain appeared to produce no L-tryptophan. Therefore, mutation of this strain was then considered to improve the L-tryptophan yield. NTG (N-Methyl-N’- nitro-N-nitrosoguanidine) was then used as the mutant reagent to treat the original strain. Through a series of screening experiments, several potential strains were selected. Unfortunately, the mutated strains still showed no evidence to produce L-tryptophan. Therefore, the strain of Corynebacterium glutamicum CCRC 11631 was then selected to replace CCRC 12511 However, the strain of CCRC 11631 did not produce L-tryptophan either Then, the strain of CCRC 11631 was mutated One of the mutated strains from CCRC 11631, namely No。195, was selected as a potential candidate for producing L-tryptophan. An experimental design method was used to study the optimal cultivating condition for mutated strain No.195. The three major factors, including the concentration of glucose, the type of nitrogen sources and the cultivation time, were considered. Through the analysis of ANOVA (analysis of variance), experimental results showed that the initial glucose concentration, the organic nitrogen type and the cultivation time all had a significant effect to the yield of biomass. The initial glucose concentration and the cultivation time had a significant effect to the yield of L-tryptophan production, and the cultivation time had not. Experimental results showed that the optimal cultivating condition is as follows: the initial glucose concentration 40 g/L, the organic nitrogen type corn steep liquor and the cultivation time 72 h. The concentration of L-tryptophan could reach 8.347 mg/L after 72 h of cultivation of strain No. 195 in a shaker with a speed of 150 rpm and 30 ℃. Later, a jar fermentor of 3 L was used to cultivate strain No. 195 under 30 ℃ and pH 7.0. The concentration of L-tryptophan could reach 13.5 mg/L after 72 h cultivation in the fermentor with a working volume of 2 L and a stirring speed of 250 rpm. Keywords: L-tryptophan, mutant, fermentation, Corynebacterium glutamicum.

Record ID 第73筆 System ID 090DYU00250015
BCRC ID CCRC 31535, Public Year 90
Paper Name 紅麴菌生產膽固醇合成抑制劑之食品級培養基最適化探討 Optimization of Food-Grade Medium Composition for Monacolin K Production by Monascus ruber
Student Name 穆春菊
Teacher Name 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究的目的在於探討以Monascus ruber CCRC 31535 為紅麴生產菌株,先進行不同食用油最適化之探討,再利用回應曲面法以尋求食品級白米粒-蔬菜油複合培養基中四種組成分 ( 白米粒、peptone、蔬菜油、葡萄糖 ) 等對紅麴膽固醇合成抑制劑產量影響之探討。不同食用油最適化探討之實驗結果顯示,在培養養溫度25℃,白米-不同食用油複合培養基之起始酸鹼值為 pH 5.0,培養基體積在250 mL三角錐瓶中為25mL等相同培養條件下,與不同的食用油 (甘油、蔬菜油、橄欖油、葵花油、芥花油、沙拉油) 進行搖瓶培養,當以白米粒-蔬菜油複合培養基培養時,所生產的膽固醇合成抑制劑產量可達0.1 mg/mL。 本研究亦利用回應曲面法來尋求白米粒-蔬菜油複合培養基中四種組成分 ( 白米粒、peptone、蔬菜油、葡萄糖 ) 之最適化濃度條件,由實驗結果得知,最適培養基組成為37 g/L白米粒、5 g/L peptone、43 ml/L 蔬菜油、7.6 g/L 葡萄糖,在此組成分培養下,經過十天培養後,可得到本研究最高膽固醇合成抑制劑產量為0.141 mg/mL。此外,在回應曲面法模式適切性之統計檢驗上,R2為0.86,表示此一回應模式能適切地作為描述實驗數據參考用。 In this study, monacolin K was produced by Monascus ruber CCRC 31535 in flask culture。 In preliminary study, the suitable effect of the different cooking oil in rice complex medium was investigated. The optimum cooking oil of the rice complex medium was vegetable oil. The initial pH and volume of the complex medium were set be 5.0 and 25mL, respectively. The culture condition 25℃ and 150 rpm in the shaker. The average yield of monacolin K was 0.100 mg/mL. In addition, response surface methodology was used to optimize the concentrations of the rice-vegetable oil complex compositions and to evaluate the effects of the composition concentrations on monacolin K productivity. The optimum composition for monacolin K production was found to be 37 g/L rice-particle, 5 g/L peptone, 43 mL/L g vegetable oil, 7.6 g/L glucose. With these compounds, the highest monacolin K production was 0.141 mg/mL after 10 days of cultivation. For this kind of complex medium, it would be good for monacolin K production by M. ruber CCRC 31535

Record ID 第74筆 System ID 090DYU00250016
BCRC ID CCRC 31535, Public Year 90
Paper Name 甲硫胺酸與乙酸鈉對紅麴生產膽固醇合成抑制劑產量影響之研究 Study on Influence of Sodium Acetate and Methionine on Monacolin K production by Monascus ruber
Student Name 鄭顏昆
Teacher Name 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究以Monascus ruber CCRC 31535 為生產紅麴菌株,進行甲硫胺酸與乙酸鈉添加比例之探討,尋求甲硫胺酸與乙酸鈉之最適添加比例,以期得到最高產量之膽固醇合成抑制劑(monacolin K)。實驗結果顯示,於第二或三天培養時間添加任何比例之甲硫胺酸及乙酸鈉於白米-甘油複合培養基中,對紅麴膽固醇合成抑制劑生化合成有明顯抑制作用,而且隨著甲硫胺酸及乙酸鈉添加時間的延後,則對紅麴膽固醇合成抑制劑之抑制影響愈不顯著。對任何添加比例之甲硫胺酸及乙酸鈉而言,於第二天及第十一天培養時間添加,則紅麴膽固醇合成抑制劑產量分別降至0.0 mg/L及88.0 mg/L,而未添加對照組之產量為137.0 mg/L。 本研究同時於第3、4、5、6、7天單獨添加甲硫胺酸或乙酸鈉,並分析其對於monacolin K 產量之影響,結果發現單加甲硫胺酸或乙酸鈉皆對紅麴菌生合成膽固醇合成抑制劑有抑制效果。甲硫胺酸方面,於第三天添加後,繼續培養七天,膽固醇合成抑制劑產量降為6.3 mg/L,而未添加之對照組產量為98 mg/L。在乙酸鈉方面,於第三天添加後,繼續培養七天,膽固醇合成抑制劑產量為0 mg,而未添加之對照組產量為98 mg/L。故甲硫胺酸與乙酸鈉對紅麴菌生合成膽固醇合成抑制劑有抑制之效果,此抑制現象被假設為甲硫胺酸及乙酸鈉之添加濃度過高。 Monacolin K produced by Monascus ruber CCRC 31535 was studied in solid-liquid flask culture。 Several different strategies of manipulating the addition of methionine and sodium acetate in rice-glycerin complex medium, such as addition time and ratio, were investigated. It was found that the production of Monascus monacolin K was inhibited by the addition of methionine and sodium acetate in complex medium at the first two or three days for any kind of addition ratio. The inhibition of monacolin K production was decreased as the addition time of methionine and sodium acetate increased. The monacolin K productions with the addition of methionine and sodium acetate at the 2nd and 11th day became close to 0 mg/L and 88 mg/L, respectively, while that without any addition was 137 mg/L. The inhibition phenomena of monacolin K production by the individual addition of methionine or sodium acetate were similar to those described above. At the first three days, the inhibition effect of sodium acetate was better than that of methionine. The monacolin K production with the addition of sodium acetate at the 3rd culture day became 0 mg/L, while that for methionine addition was 6.3 mg/L. This research demonstrated that the monacolin K production of M. ruber CCRC 31535 was inhibited by the addition of methionine and sodium acetate presumably due to the high addition concentration

Record ID 第75筆 System ID 088DYU00250022
BCRC ID CCRC 31619, Public Year 88
Paper Name 以發酵槽高密度培養Penicillium chrysogenum 生產penicillin V之研究 High Cell Density Cultivation of Penicillium chrysogenum:Production of Penicillin V in a Fermentor
Student Name 陳惠婷
Teacher Name 瑞澤 洪淑嫻
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 青黴素屬於微生物的二次代謝產物,具有抑菌的作用,依所結合側鏈的不同分為天然型青黴素、半合成青黴素及生合成青黴素,各有不同的構造與特性。生合成青黴素中的青黴素V具有對酸安定的特性,適用於口服藥劑之調製。本實驗以Penicillium chrysogenum (CCRC 31619 or ATCC 28089)為菌株生產青黴素V,首先以振盪培養探討起始pH值與不同碳源對培養過程之影響,之後將振盪培養最適的起始pH值與碳源用於發酵槽批次培養,於發酵槽培養中所改變的變因有二:(1)酵母萃取物的添加,(2)發酵槽的攪拌速率。 振盪培養結果顯示:孢子濃度為7.3*106 spores/mL時,培養基起始pH值為6.0、6.5及7.0之下,菌體濃度以起始pH 6.5時為最多,而青黴素的產量也最高,可達0.15 g/L。葡萄糖與硫酸銨消耗情形也以起始pH值為6.5時最為顯著。其次,將起始pH值固定在6.5,探討不同碳源培養之關係時發現:葡萄糖做為碳源時,青黴素V產量最多;而蔗糖做為碳源時的青黴素V最終產量與葡萄糖為碳源時相差不多;以乳糖做為碳源時,會因為乳糖的消耗速率較慢而使青黴素V產量較差。所以由實驗結果得知,振盪培養P. chrysogenum生產青黴素V的最適起始pH值為6.5,最佳碳源為葡萄糖。 發酵槽批次培養中,添加酵母萃取物做為氮源,結果發現,可能是因菌體細胞發生自溶而使菌體濃度與青黴素V產量稍微下降,但因為酵母萃取物成分複雜,可能對之後的研究條件有所影響,所以探討發酵槽攪拌速率時仍以硫酸銨做為唯一的氮源。 在發酵槽的攪拌速率對培養之影響方面,當攪拌速率為350 rpm時,菌體濃度最高,達到8.7 g/L,此時青黴素V的產量也最高,為0.40 g/L。溶氧量在攪拌速率為350 rpm時最高,可提供較充足的氧氣使菌體生長。雖然攪拌速率愈高產生的剪應力會愈大,理應對菌體的生長有所影響,但在此可能是因為發酵槽攪拌速率在150 ~ 350 rpm時之間,剪應力對於菌體細胞的影響並不顯著之因素所致。 由於P. chrysogenum屬於好氧菌,培養時應維持較高的溶氧量。以發酵槽培養時,因為能不斷通入空氣提高發酵液之溶氧量,所以青黴素V產量比以振盪培養的產量高出許多,其中最大的因素應該是,振盪培養時,氧氣供應不足,以致菌體生長受到限制,而使得青黴素V產量比以發酵槽培養時為低。 Penicillin is a kind of the secondary metabolites produced by Penicllium sp., and it possesses antimicrobial capability. Penicillin can be classified into three categories as natural, biosynthetic and semi-biosynthetic penicillins according to their binding side-chains. Penicillin V belongs to the category of biosynthetic penicillins, and it is quite stable in acidic environment, and can be used as an oral medicine. In this study, the strain of Penicillium chrysogenum (CCRC 31619 or ATCC 28089) was used to produce penicillin V。 First of all, the effect of initial pH values and different carbon sources on the penicillin V production was explored. Once the optimun initial pH value and carbon sources have been determined, they would be used for later experiment. Secondly, the experiment was carried out in a batch fermentor by using media with and without yeast extract (YE). The rotational speed of impeller in the fermentor was another factor being taken into consideration. The spore concentration was 7.3*106 spores/mL for the flask culture under various pH values (6.0, 6.5 and 7.0). The culture had highest penicillin V production, reaching 0.15 g/L under an initial pH value of 6.5. The consumption rates of glucose and (NH4)2SO4 were also the highest for the case of pH 6.5. Then, pH 6.5 was fixed when different carbon sources including glucose, sucrose and lactose, were under consideration. Experimental results show that glucose is better than sucrose or lactose as a carbon source for producing biomass and penicillin V. Therefore, pH 6.5 and glucose were the best combined condition for the flask culture. For the batch culture in a fermentor, the addition of YE as a nitrogen source could lead to higher production of biomass and penicillin V. However, the existence of YE also leads to the decrease of biomass and penicillin V toward to the end of cultivation. This phenomenon might be due to the cellular autolysis in the culture. Because the composition of YE is not well defined and might affect the experimental results in some ways, (NH4)2SO4 was used as the only nitrogen source for later cultivation of Penicillium chrysogenum in a fermentor. The D.O. (dissolved oxygen) value increased as the agitation rate increased. Three rates, say 150, 200 and 350 rpm, were selected for the experiment. The highest biomass and penicillin V concentrations were 8.7 g/L and 0.4 g/L, respectively, for the agitation rate of 350 rpm. However, the increase of the agitation rate will increase the shear stress which is harmful to the microbial growth. In this study, the influence of shear stress seems not significant, this may be due to the rates of 350 rpm is still high enough to produce harmful shear stress to microorganism. Penicillium chrysogenum is an aerobic fungal, and must be cultivated in an environment with high dissolved oxygen. Aerated air can enhance the dissolved oxygen in the medium if cultivated in a fermentor, and hence the penicillin V production is higher than that of penicillin V produced in a shaker flask culture. Note that the lack of dissolved oxygen may limit microbial growth and penicillin V production if cultivated in a shaker flask.

Record ID 第76筆 System ID 088DYU00250027
BCRC ID CCRC 10674, CCRC 10746, CCRC 12652, Public Year 88
Paper Name 含硫率對磺酸幾丁聚醣與磺酸苯幾丁聚醣抑菌作用及水溶性之影響 The effects of sulfur content on antimicrobial activity and water solubility of sulfonated chitosan and sulfobenzoyl chitosan
Student Name 蘇俊旗
Teacher Name 陳齊聖
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 利用來自蝦殼的幾丁聚醣產品,以化學法製造出不同去乙醯度(DD)的幾丁聚醣,分別在 pyridine 及 methanol 相中,進行化學修飾,得磺酸幾丁聚醣(SC)和磺酸苯幾丁聚醣(SBC)。本研究選擇不同含硫率(1∼5%)及不同去乙醯度的 SC 及 SBC 對 Escherichia coli (CCRC 10674)、Salmonella typhimurium (CCRC 10746)及 Staphylococcus aureus (CCRC 12652)做最低抑菌濃度(MIC)的測試,更進一步探討含硫率對 SC 及 SBC 水溶性之影響。研究結果顯示,溶解度隨著含硫率的增加而增加,然而含硫率的增加並不會有較好的抑菌效果,另一方面,不同的菌株對含硫率的敏感度不一,所以各種菌株對含硫率的要求也不盡相同。而去乙醯度也會影響 SC 及 SBC 的性質。SC 及 SBC 均比幾丁聚醣有較顯著的抑菌效果,且 SBC 富水溶之特性,可增加未來應用之潛力。 Shrimp shell derived chitosan was chemically modified to obtain sulfonated chitosan ( SC ) and sulfobenzoyl chitosan ( SBC ) in pyridine and methanol solvent system respectively. Antimicrobial activity and aqueous solubility of these two chitosan derivatives as functions of sulfur content ( ranged from 1 to 5% ) were studied. Test microorganism included Escherichia coli(CCRC 10674), Salmonella typhimurium(CCRC 10746) and Staphylococcus aureus(CCRC 12652)。 Generally speaking, solubility increased with increasing sulfur content. However, increased sulfur content did not result in higher antimicrobial activity. It was also found that there existed optimal sulfur content in terms of antimicrobial activity and the sensitivities toward sulfur content were strain dependent. Degree of deacetylation also affected these two properties of SC,SBC and underivatised chitosan﹒ Similar conclusion that the influence was strain dependent was obtained. Relatively speaking, SC and SBC demonstrated superior antimicrobial activity and aqueous solubility than underivatised chitosan..

Record ID 第77筆 System ID 083DYU00250002
BCRC ID CCRC 31840, Public Year 83
Paper Name γ-次亞麻油酸之發酵量產研究 The study of large-scale production of gamma-linolenic acid
Student Name 劉哲明
Teacher Name 陳鴻章
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本實驗使用 Cunninghamella echinulata CCRC 31840 為菌種,並以於三 角錐瓶中生產γ-次亞麻油酸之最佳培養基,做為發酵槽基本發酵液,來 探討量產γ-次亞麻油酸之最佳發酵槽條件。發現培養溫度為 22℃、pH 值為 7.0、溶氧值 7.5 ppm 、轉速 600 rpm 及螺旋槳式攪拌翼是其量產 γ-次亞麻油酸的個別最佳發酵槽條件。而組合這些最適條件其γ-次亞麻 油酸產量達 1,720 mg/l,是前人研究中於三角錐瓶培養相同菌株產量的 1.8 倍。於三角瓶中分別改變無機氮源 (硝酸銨) 與碳源 (可溶性澱粉) 之濃度,以不同起始碳氮比之培養基培養,分析碳氮比發現當比值為 40 : 1-80 : 1 之間時,可大量累積油脂。在五天之饋料批式發酵實驗中, 發現固定間隔 24 小時饋入無機氮源硝酸銨,其饋料總量為 2.2 g/l 時 ,有最高γ-次亞麻油酸產量 ,達 1,156 mg/l。

Record ID 第78筆 System ID 087DYU00250007
BCRC ID CCRC 31535, Public Year 87
Paper Name 紅麴菌生產膽固醇合成抑制劑搖瓶培養條件之探討 Studies on Production of Monacolin K by Monascus ruber in Flask Culture
Student Name 林怡昌
Teacher Name 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 紅麴菌的二級代謝物中具有的monacolin K是降體人體內膽固醇過高的一種優良藥劑,故本研究以M. ruber CCRC 31535為主要生產菌株,以固-液態培養方式來進行紅麴菌生產膽固醇合成抑制劑的最適培養環境探討以及基質中澱粉類碳源及有機性氮源合適性之篩選。結果顯示以脫脂黃粉-甘油複合培養基作為培養基質時,在最適培養溫度25℃下,當培養基的起始酸鹼值為pH 5.0,而體積含量為25 ml時,其產量可達到1.37510-2 mg/ml;若將白米澱粉替換原先培養基中的澱粉類碳源-脫脂黃豆粉時,則其產量可高達到4.16510-2 mg/ml;此外在醣類碳源及有機性氮源上的篩選上,發現以脫脂黃豆粉-甘油複合培養基為基質時,葡萄糖及Tryptone為較佳之選擇,其產量濃度為2.03510-3 mg/ml。 另外利用回應取面法尋求紅麴菌生膽固醇合成抑制劑之最適培養基組成份及其最適化濃度之條件時,在二階模式中發現當以白米澱粉為34.4 g/L、Peptone為10.8 g/L、甘油為36.4 ml/mL、葡萄糖為129.2 g/L時及KNO3為8g/L、MgSO4.7H.2O為4 g/L時,可找到本研究monacolin K最高產量濃度為0.157 mg/mL。 In this study, monacolin K was produced by Monascus ruber CCRC 31535 in solid-liquid culture。 Several different strategies of manipulating variables, such as culture conditions (temperature, initial pH and volume of medium) and complex medium compounds (carbon and nitrogen source), were investigated. It was found that the optimum culture temperature was 25 oC .The optimum soy flour-glycerol media in culture of 25 oC were found to be pH 5.0 and 25 ml; while the soy powder-glycerol media in the culture were to be pH 6.0 and 25 ml. Furthermore, rice powder was considered to be the best carbon source in giving the maximum yield of monacolin K. The maximum yield was 0.042 mg/ml in the culture of 25 oC. Among the sugar-type carbon and nitrogen sources tested, glucose, tryptone, respectively, were found to be suitable for monacolin K production in the culture of 25 oC. The yield was 0.0203 mg/ml. This research demonstrated that the solid-liquid culture was worth improving the yield of monacolin K. Response surface methodology was used to optimite monacolin K production by M. ruber CCRC 31535 in flask culture。 Analysis of variance indicated that the interaction between operation variables (yeast extract and glycerin) in the quadratic model was only significant. The concentration of yeast extract at the design center point was recommended to be at 0.5 g/L, while the concentration of glycerin was at 120 ml/L and the concentration of both rice starch and glucose were 30 g/L. For this kind of complex medium, it would be good for monacolin K production by M. ruber CCRC 31535。 (Key words:Monascus ruber, monacolin K, response surface methodology).

Record ID 第79筆 System ID 087DYU00250019
BCRC ID CCRC 32512, CCRC 32513, Public Year 87
Paper Name 利用 Gibberella 屬絲狀真菌生產激博素 Production of Gibberellin by Fungal Strains of Gibberella sp.
Student Name 李延卿
Teacher Name 陳鴻章
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究以生產激博素之絲狀真菌Gibberella fujikuroi CCRC 32512及CCRC 32513,先利用一次只改變培養條件中一個因子之方式探討培養環境如菌絲密度及均質時間、接菌量、發酵容積、振盪轉速、生長溫度等對生產激博素之影響就菌絲密度而言,G. fujikuroi CCRC 32512及CCRC 32513 兩菌株以刮入0.6 g菌絲施以240 sec均質處理可得最高量之激博素,產量分別為130.31 mg/L 及 140.31 mg/L。在接菌量方面,則兩菌株皆以接入4% (v/v) 懸浮菌絲量於 50 mL 培養液中進行發酵時有最大激博素產量。就發酵容積而言,發酵容積為50 mL 時兩菌株有較高之激博素產量,分別為76 mg/L 和 98 mg/L。振盪轉速對於激博素之生產亦有顯著的影響。例如,G. fujikuroi CCRC 32512 及 CCRC 32513 在250 rpm下,其激博素產量分別為 96 mg/L 和145 mg/L,係 100 rpm 時激博素產量的 1.7倍及 2.0 倍。就生長溫度而言,在 31 ℃ 的溫度環境下,兩菌株有較高之激博素產量,分別為 74 mg/L 和118 mg/L。 由以上結果另外得知G. fujikuroi CCRC 32513 無論在發酵容積、振盪轉速及生長溫度方面的整體表現均優於G. fujikuroi CCRC 32512,因此選擇前者繼而探討培養基組成如碳、氮源種類及濃度、緩衝劑添加量等對生產激博素之影響。就緩衝劑添加量而言,以添加 0.5 g/L 磷酸氫鈉和1.0 g/L 磷酸氫二鉀之用量時有最大之激博素產量。就碳源而言,Gibberella fujikuroi CCRC 32513 以蔗糖為最佳之碳源種類,而含量以添加 40 g/L 為最佳。就氮源而言,此菌株以蛋白為較佳氮源,而含量以添加 12 g/L 為最佳。而若以最適化條件組合培養 Gibberella fujikuroi CCRC 32513七天後其最大激博素產量達 289 mg/L,是基本培養條件培養相同菌株最大產量的 3.1 倍 此外,本研究亦利用L16(215) 直交表探討培養 Gibberella fujikuroi CCRC 32513之培養環境和培養基組成之因子主效應和因子間交互作用關係。先由第一次直交表設計及變異數分析後得知影響激博素生產之因子主效應有碳源濃度、氮源濃度及培養時間等,其餘因子對產物產量並無顯著之影響。再將碳源濃度、氮源濃度及培養時間等因子提高水準範圍配置於第二次直交表,得知 G. fujikuroi CCRC 32513 於 40 g/L 蔗糖、12 g/L 蛋白、培養 4 天後所得激博素產量較 60 g/L 蔗糖、18 g/L 蛋白、培養 8 天之結果為高。最後再將上述因子朝向低水準方向配置於第三次直交表後得知,生產激博素之最適碳、氮源濃度範圍分別為30 至 40 g/L、9 至 12 g/L、培養時間為 4 天,其餘因子為一次一因子所得之最適化條件,經此組合試驗可得一較高產量為 348 mg/L,且因子主效應及其交互作用對產量有著顯著之影響。 The research studied the production of gibberellin by filamentous fungal strains : Gibberella fujikuroi CCRC 32512 and CCRC 32513。 By changing only one variable on each trial, it examined the effect on the production of gibberellin in relation to variables such as mycelium density, homogenization time, inoculum ratio, fermentation volume, shaker velocity and growth temperature, etc. In relation to mycelium density, the greatest production of 130.31 mg/L and 140.31 mg/L respectively occurs when 0.6 g each of Gibberella fujikuroi CCRC 32512 and CCRC 32513 were scraped and put into homogenization for 240 sec。 As to the inoculum ratio, the gibberellin grew the most when 4% (v/v) of suspending filamentous fungal strain were cultivated in the 50 mL medium for fermentation. The production of gibberellin by both Gibberella fujikuroi CCRC 32512 and CCRC 32513 reached the respective optimum of 76 mg/L and 98 mg/L in the fermentation volume of 50 mL。 Shaker velocity has a significance on the production of gibberellin too. For example, under 250 rpm, Gibberella fujikuroi CCRC 32512 and CCRC 32513 each produces 96 mg/L and 145 mg/L of gibberellin, 1。7 and 2.0 times of that under 100 rpm. In terms of growth temperature, the production of gibberellin was the most desirable under the temperature of 31 ℃ for both strains with respective yields of 74 mg/L and 118 mg/L. Through the result of the above experiments, we found G. fujikuroi CCRC 32513 performed more outstandingly than G fujikuroi CCRC 32512 in respect of fermentation volume, shaker velocity and growth temperature。 Thus, G. fujikuroi CCRC 32513 was selected to continue the studies of the effect on the production of gibberellin in relation to variables such as source and concentration of carbon and nitrogen and the amount of buffering agent, etc。 The relevance between the production of gibberellin and buffering agents showed the addition of 0.5 g/L sodium dihydrophosphate and 1.0 g/L di-potassium hydrophosphate promoted the most the production of gibberellin, while sucrose served as the best carbon source at the amount of 40 g/L. In relation to nitrogen source, G. fujikuroi CCRC 32513 was able to generate the most gibberellin with peptone being its best nitrogen source at the amount of 12 g/L。 In the optimal combination of culture media, the production of gibberellin by G. fujikuroi CCRC 32513 reached as high as 289 mg/L 7 days later, 3。1 times of that in the basal medium. By means of orthogonal array design L16(215), the research also examined the major effect of each factor and the interaction effect among the factors on the production of gibberellin by G. fujikuroi CCRC 32513。 The first L16(215) matrix showed the major factors that affected the production of gibberellin included the concentration of carbon source, nitrogen source and the cultural time, Other factors did not have a significant influence on the production. The factors such as concentration of carbon source, nitrogen source and the cultural time in high levels were further set in the second L16(215) design, and the analysis showed more gibberellin was generated when G. fujikuroi CCRC 32513 was cultured in the media of 40 g/L sucrose, 12 g/L peptone for 4 days than in the media of 60 g/L sucrose and 18 g/L peptone for 8 days。 At last, with the rest factors fixed at the optimal condition obtained from the tests in which only one factor was changed each time, the setting of the above mentioned factors in low levels in the third L16(215) matrix showed that the most favorable range of carbon and nitrogen concentration were 30 — 40 g/L and 9 —12 g/L respectively, and the optimal cultural time was 4 days. The experiment showed the combination led to a higher production of 348 mg/L, and the major effect of each factor and the interaction among factors have a significant influence on the production of gibberellin. Key words : filamentous fungal strains, gibberellins, orthogonal array design.

Record ID 第80筆 System ID 087DYU00250026
BCRC ID CCRC 31499, Public Year 87
Paper Name 以梅胚醃漬液為基質利用紅麴菌釀造梅子紅酒 Plum Red Wine Made by Monascus anka
Student Name 丁正芳
Teacher Name 張耀南
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 紅麴菌的一級代謝產物與多種水解酵素可利用於梅子紅酒的釀造。本研究是以紅麴菌 Monascus anka CCRC 31499 為釀酒菌株,以梅胚醃漬液(脫鹽及未脫鹽醃漬液)為基質,探討不同培養(釀酒)條件對梅子紅酒品質之影響。結果顯示當基質的起始酸鹼值為pH 6.0、起始糖度20。Brix時,以最適培養溫度為25℃經培養第十五天結果為最佳,此時脫鹽與未脫鹽醃漬液為基質時分別含有2.67%與3.36%酒精度,而每消耗單位糖度增加△a值分別為2.68/。Brix與1.82。Brix。此外基質添加MSG不僅有助於提升酒精度,在紅色色度也有顯著性的改變,並且將培養的天數縮短成為十天。 當基質起始糖度始糖度為20°Brix,基質起始酸鹼值為 pH 7.0 時,經培養第十天酒精度方面每消耗單位糖度轉換酒精度為最高,色澤方面脫鹽與未脫鹽醃漬液分別在第十天與第十五天為最佳;此外當麥芽糖作為脫鹽醃漬液醣類碳源,葡萄糖作為未脫鹽醃漬液醣類碳源時,經培養第 10 天時每消耗單位糖度轉換酒精度的為最高;菌體外的紅色素方面,以果糖作為脫鹽醃漬液的醣類碳源時,在培養天數為第 20 天時,可獲得到較高的菌體外的紅色素,而以葡萄糖作為未脫鹽醃漬液,則是在培養天數為第 15 天時為最高。未來利用紅麴菌釀造梅子紅酒仍值得繼續探討研究。 In this study, fruit red wine was made from mei juice by Monascus anka CCRC 31499 and brewer`s yeast。 Apple juice was one of the best source for yield and quality improvement in wine making. The red wine was mixed by the 1:1 ratio of the apple wines made by M. anka and yeast after ten-day fermentation. The ethanol yield of the apple red wine was 4.1% and 0.347% /0Brix.The color of the wine was more acceptable than those of the wines made from grape and orange juice. The other apple red wine was made by both M. anka and yeast from four-day to ten-day mixture fermentation. The ethanol yield of the wine was 6.12% and 0.313%/0Brix.It was found that the ethanol yield of the wine made by mixture fermentation, while the ethanol%/0Brix yield was decreased. The apple red wine only made by Monascus anka CCRC 31499 was also studied。 Three different strategies of operation variables, such as wine making temperature, time and sugar-type carbon source, were investigated. The optimal wine making temperature was at 25℃for 10 days The ethanol production was 3.12% and 6.12%/0Brix.The pH value of the wine become increase as winemaking temperature increased. The color of the wine made at 25℃become more red when the △a increase was 0.1039/0Brix.℃.Fructose and Glucose were the optimal sugar-type carbon sources for M. anka wine making. The ethanol yield for fructose was up to 4.98% but the △a increase was -0.12/ 0Brix. In addition, monosodium glutamate(MSG) addition was used to improve the increase of ethanol production and color of winemaking. For glucose and fructose the ethanol yields of red wines were 5.75% and 5.80%, respectively, while the colors were not changed. This research demonstrated that M. anka CCRC 31499 was worth using to improve the ethanol yield and color quality improvements in apple red winemaking

Record ID 第81筆 System ID 082DYU00250011
BCRC ID CCRC 31840, Public Year 82
Paper Name 以微生物生產具生理活性之多元不飽和脂肪酸 Microbial production of bioactive polyunsaturated fatty acids
Student Name 張志嘉
Teacher Name 陳鴻章
School Department Name 大葉大學 食品工程研究所 Academic Degree 碩士
Abstract 本研究先自菌種中心蒐購文獻中能生產具生理活性之多元不飽和脂肪酸之 菌株或類似菌共二十株,以基本培養基於不同溫度篩選具量產潛力之菌株 ,發現 Cunninghamella echinulata CCRC 31840 含有最高產量之 gama- 次亞麻油酸,但僅 Mortierella alpina CBS 210.32 一株有較明 顯之花生四烯酸含量 (7.6-10.2 %) 。同時探討培養條件對此二菌生產 γ-次亞麻油酸及花生四烯酸之影響,發現在最適培養基中培養五天後 gama-次亞 麻油酸及花生四烯酸產量分別達 964 及 965 mg/l,分別為以 基本培養基培養時之9.6倍及55倍。續自各地泥土中篩選多元不飽和脂肪 酸之生產菌株,其中以篩選株 H4 能產生504 mg/l 之花生四烯酸且其含 量佔油脂總量之 42 % 最為突出。在確定最佳碳源為可溶性澱粉,最佳氮 源為尿素,最適培養溫度為 24 ℃後,其花生四烯酸產量於五天後已提升 為 1,817 mg/l 。經反應曲面實驗法求篩選株 H4生產花生四烯酸之各因 子之最佳濃度及探討各因子之交互影響。可得到花生四烯酸平均產量為 2,169 mg/l, 為自泥土中剛篩出時之產量之 4.3 倍。

Record ID 第82筆 System ID 091DYU00250021
BCRC ID CCRC 12826, Public Year 91
Paper Name 利用回應曲面法尋求苔蘚桿菌生產聚麩胺酸之培養基最適化 Use of Response Surface Methodology to Optimize Culture Medium for Production of Poly(γ-glutamic acid) by Bacillus licheniformis
Student Name 呂文凱
Teacher Name 張耀南 洪淑嫻
School Department Name 大葉大學 食品工程學系碩士班 Academic Degree 碩士
Abstract 本研究以苔蘚桿菌Bacillus licheniformis CCRC 12826 為培養菌株,並以搖瓶培養方式,進行回應曲面法實驗設計探討培養基主要組成分 (麩胺酸、甘油、檸檬酸及氯化銨) 對苔蘚桿菌生合成聚麩胺酸 (γ-PGA) 產量之影響。由實驗結果得知,當培養基組成分濃度為34 g/L麩胺酸、26 g/L檸檬酸、146 g/L甘油、11 g/L氯化銨時,可得到本研究目前最高γ-PGA產量為35.33 g/L。若由回應曲面法實驗設計,分析得知,當培養基組成分濃度為34.70 g/L麩胺酸、26.97 g/L檸檬酸、145.45 g/L甘油、10.49 g/L氯化銨時,可得到理論最高γ-PGA產量為35.51 g/L。 In this study, optimization of medium E composition for γ-PGA (poly-γ-glutamic acid) production by Bacillus licheniformis CCRC 12826 with response surface methodology (RSM)。 The factors chosen for optimization were glutamic acid, glycerol, citric acid and NH4OH. The maximal yield of γ-PGA (35.33 g/L, average of four repeats) appeared at the region where the respective concentrations of glutamic acid, citric acid, glycerin and NH4Cl were around 34 g/L, 26 g/L, 146 g/L and 11 g/L, respectively. The predicted optimal composition derived from RSM regression was 34.07 g/L glutamic acid, 27.00 g/L citric acid, 145.45 glycerin and 10.49 g/L NH4Cl. With this composition, the predicted maximum γ-PGA production was 35.51 g/L.

Record ID 第83筆 System ID 094DYU00061009
BCRC ID CCRC 12827, Public Year 94
Paper Name L-aminoacylase 基因選殖與酵素活性分析 Gene Cloning and Enzyme Characterization of L-aminoacylase from Deinococcus radiodurans CCRC12827
Student Name 郭怡君
Teacher Name 簡宏堅
School Department Name 大葉大學 分子生物科技學系碩士班 Academic Degree 碩士
Abstract 中文摘要 根據 Deinococcus radiodurans R1之基因體中找出 兩段基因序列(DR1711和DR0339),它們與L-aminoacylase的相同度很高,以此作設計為引子之依據,並以抗輻射細菌 D. radiodurans CCRC 12827 的染色體DNA當模板,利用PCR分別放大laa1和laa2之不同DNA序列片段。將laa1或laa2基因選殖入Escherichia coli 菌體內,可表現L-aminoacylase 的活性。laa1的open reading frame(ORF)全長1,167 bp,laa2的ORF長度則是1,179 bp,各自均可轉譯出LAA1蛋白分子量41,443 Da與LAA2蛋白分子量42,607 Da,兩者都是胞內酵素。LAA1經胺基酸殘基序列比對,結果與 D. radiodurans R1 N-acyl-L-amino acid amidohydrolase(DR1711)的胺基酸相同度98%,LAA2也作胺基酸殘基序列比對,和D. radiodurans R1 probable N-acyl-L-amino acid amidohydrolase (DR0339)相同度高達 99%。將D. radiodurans之 L-aminoacylase ORF選殖入pQE30載體,轉形至E. coli Nove Blue,再用Co-NTA管柱回收此酵素。經過酵素活性分析後,發現LAA1最適反應溫度為45℃,而LAA2最適反應溫度是35℃。在最適pH的實驗中得知LAA1是在pH 8.0有最佳活性,而LAA2是在pH 7.0有最好的活性。在無金屬離子條件下,LAA1與LAA2不具任何催化能力,而Mn2+及Co2+等離子均能提昇酵素活性,結果顯示其可能是metalloenzyme。此外,用N-CBZ-Gly-Ala 為基質時,LAA1與LAA2的carboxypeptidase 比活性最大,而N-acetyl-L-His當基質時,LAA1的L-aminoacylase 比活性最佳,在LAA2 L-aminoacylase 比活性的測試,是在以N-chloroacetyl-L-Phe作基質時有最佳的比活性,用L-ornithine-L-ala為基質時,僅有LAA2具dipeptidase活性。此結果顯示LAA2為carboxypeptidase、L-aminoacylase與dipeptidase參功能酵素,而LAA1是carboxypeptidase及L-aminoacylase的雙功能酵素。 ABSTRACT Two genes(DR1711 and DR0339) on Deinococcus radiodurans R1 genome had high similarity with L-aminoacylase (laa)genes from different spieces. So both genes were used as template to desigin primer pairs. Chromosome DNA of the radioactivity resistant bacterium D. radiodurans CCRC 12827 was used as template to amplify laa1 and laa2 genes by PCR, respectively。 Cloning of laa1 or laa2 genes into Escherichia coli and expression of L-aminoacylase activity were performed. The length of laa1 open reading frame(ORF) was 1,167 bp, however, the laa2 ORF was 1,179 bp. The molecular weight of the translation product LAA1 and LAA2 were 41,443 Da and 42,607 Da, respectively. Both LAA1 and LAA2 were intracellular enzymes. Sequence alignment between LAA1 and D. radiodurans R1 N-acyl-L-amino acid amidohydrolase shows 98% identity, while comparison of LAA2 and D. radiodurans R1 probable N-acyl-L-amino acid amidohydrolase shows 99% identity. The D. radiodurans L-aminoacylase gene was cloned into pQE30 expression vecter and transformed to E. coli Nove Blue. Finally, we used Co-NTA column to purify the enzymes. The optimal temperatures of enzymes were measured as 45℃and 35℃ for LAA1 and LAA2, respectively. The optimal pH for LAA1 and LAA2 were pH 8.0 and pH 7.0, respectively. Without metal ion, LAA1 and LAA2 were inactivated. Mn2+and Co2+ ions promote enzyme activity, because both enzymes could be metalloenzymes. Besides, using N-CBZ-Gly-Ala as substrate, LAA1and LAA2 showed maximal carboxypeptidase specific activity. When the substrate switched to N-acetyl-L-His, LAA1 showed the best L-aminoacylase activity. Using N-chloroacetyl-L-Phe as substrate, LAA2 had maximal L-aminoacylase specific activity. In addition, LAA2 had dipeptidase activity of hydrolyzing the dipeptide L-ornithine-L-alanine. These results indicate that LAA1 is a bifunctional enzyme and LAA2 is a trifunctional enzyme.

Record ID 第84筆 System ID 094DYU00111023
BCRC ID BCRC 13036, Public Year 94
Paper Name 限磷條件下添加丙酸鈉/戊酸鈉對連續式生產PHBV之影響 Effect of Addition of Sodium Propionate or Sodium Valerate on Continuous Production of Poly(hydroxybutyrate-co-hydroxyvalerate) under a Phosphorus-Limiting Condition
Student Name 李志韋
Teacher Name 凃瑞澤 余世宗
School Department Name 大葉大學 生物產業科技學系 Academic Degree 碩士
Abstract 聚羥基烷酯類(polyhydroxyalkanoates, PHAs)為一種微生物生合成的可分解塑膠,其性質與傳統塑膠材料聚丙烯相似,其中以聚羥基丁酯(polyhydroxybutyrate, PHB)與聚羥基丁酯和戊酯的共聚物(poly(hydroxybutyrate-co-hydroxyvalerate), PHBV)最受到注意。 Ralstonia eutropha在簡單碳源,以葡萄糖為單一碳源基質可生合成累積PHB;若以有機酸鹽(如:丙酸鈉或戊酸鈉)作為第二碳源,即可生產含HV的聚合物。有機酸鹽的含量不宜過高,否則會影響菌體的生長。 本研究於限磷條件下,以微生物進行連續式發酵生產PHB(V)。使用菌株為R. eutropha(ATCC 17699;BCRC 13036),以葡萄糖為第一碳源(20 g/L),硫酸銨為氮源(10 g/L),磷酸氫二鈉與磷酸二氫鉀為磷源(分別為0.1與0.2 g/L),於限磷條件下,進行連續式發酵,溫度維持30℃,可得最大PHB含量,約2.20 g/L,佔總菌體33.0% ,隨著時間增加,碳源使用殆盡,可看出生質量仍在增加,PHB含量卻在減少。 添加有機酸鈉(丙酸鈉或戊酸鈉)於饋料溶液中,添加丙酸鈉5 g/L,可得到較多PHBV含量,約2.1 g/L,佔總菌體的31.8% (生質量6.79 g/L)。添加戊酸鈉5 g/L,可得HV含量較高的PHBV,約1.61 g/L,佔總菌體的25.9% (生質量5.94 g/L)。比較兩種添加有機酸鹽生合成之PHBV,以添加戊酸鈉可累積較多的HV。 Polyhydroxyalkanoate, a kind of biodegradable plastics, can be biosynthesized by various microorganisms, and possesses physical properties similar to conventional plastics such as polypropylene. Especially, polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) are two polymers frequently brought much attention. Ralstonia eutropha can produce PHB with a simple carbon source. For instance, glucose can be used as a sole carbon source to produce PHB. If organic acidic substrates, such as sodium propionate and valerate, are used to be the second carbon source, R. eutropha can produce PHBV. However, if the concentration of sodium propionate or valerate is too high, the microbial growth of R. eutropha may be refrained. In this study, PHB(V) was produced by R. eutropha (ATCC 17699; BCRC 13036) that was cultivated in a continuous fermenter in a phosphorus-limiting condition。 The major carbon source was glucose (20 g/L), the nitrogen source was (NH4)2SO4 (10 g/L) and the phos-phorous sources included Na2HPO4 (0.1 g/L) and KH2PO4 (0.2 g/L). At 30 ℃, PHB was biosynthesized by R. eutropha with a concentration of 2.19 g/L which was about 33% of the total biomass. As the carbon substrate was depleting, the biomass increased a little, but the PHB was decreasing. In the other two experiments, the second carbon source (sodium propionate or valerate) was added in the feed in order to produce PHBV. When the medium containing 5 g/L sodium propionate, PHBV (13.2% HV and 86.6% HB) was bio- synthesized with a concentration of 2.1 g/L, and was about 31.8% of the biomass (6.79 g/L). When the medium containing 5 g/L sodium valerate, PHBV (containing 47% HV and 53% HB) was biosynthesized with a concentration of 1.61 g/L, and was about 25.9% of the biomass (5.94 g/L). Comparison of these two cases revealed that adding sodium valerate in the feed would raise the ratio of HV in the PHBV.

Record ID 第85筆 System ID 094DYU01111007
BCRC ID BCRC 16064, Public Year 94
Paper Name 省產紅葡萄酒降酸之研究 Studies on the Deacidification of Red Wine in Taiwan
Student Name 林頌樺
Teacher Name 陳鴻章
School Department Name 大葉大學 生物產業科技學系碩士在職專班 Academic Degree 碩士
Abstract 省產黑后葡萄是本省栽種的唯一釀酒用紅葡萄品種,但因品種、栽種環境等因素影響,酸度偏高,釀製成酒後其口感刺激而降低酒質。本研究將黑后葡萄酒應用不同化學降酸劑 (碳酸鈣、碳酸氫鉀、酒石酸鉀)及蘋果酸乳酸發酵進行降酸,以釀製出品質高、酸度低、口感柔醇的紅葡萄酒。 於紅葡萄酒發酵的不同皆段,直接添加碳酸鈣、碳酸氫鉀或酒石酸鉀等化學降酸劑與葡萄酒中的有機酸中和,可形成降酸作用。使用化學降酸劑中,以碳酸鈣2.01 g/L或碳酸氫鉀2.55 g/L降酸效果較佳,由品評結果得知添加碳酸氫鉀2.55 g/L的紅葡萄酒最受到大眾所喜好。 在蘋果酸乳酸發酵中發現並無法將紅葡萄酒中所有蘋果酸轉換成乳酸,實際上只能轉換大約1/3的量;其最佳發酵條件為5%接菌量、發酵溫度20℃及營養素0.2 g/L,而菌種以商業菌種Biolact Acclimatée®降酸效果較單一菌種Oenococcus Oeni BCRC 16064佳。 在化學降酸劑配合蘋果酸乳酸發酵之試驗中,兩株不同的蘋果酸乳酸菌商業菌種Biolact Acclimatée®及Lalvin X-3®,之降酸結果相似,而紅葡萄酒中可滴定酸度均可降低至0.85 g/100mL,且品評結果在嘗味及整體喜好的評分為最高,具有顯著的差異。採用化學降酸劑配合蘋果酸乳酸發酵對紅葡萄酒降酸的效果最佳且最受到大眾所喜好。經熟陳三個月後的紅葡萄酒更能受到大眾所喜好。 Black Queen is the only grape variety for making red wine in Taiwan, but its high acidity, influenced by factors such as variety and planting environment, has resulted in the production of wine with biting mouthfeel and inferior quality. Different chemicals (calcium carbonate, potassium hydrogen carbonate, potassium bitartrate) and malolactic fermentation bacteria were used to deacidify the wine into a product with high quality, low acidity and mellow mouthfeel. Direct addition of calcium carbonate, potassium hydrogen carbonate and potassium bitartrate to the various stages of winemaking to neutralize the organic acids in the wine can achieve the deacidification effect. A dosage of 2.01 g/L of calcium carbonate or 2.55 g/L of potassium hydrogen carbonate gave the best deacidification effect when chemical deacidificants were exploited. The red wine deacidified with 2.55 g/L of potassium hydrogen carbonate was most acceptable in the sensory test. The malic acid in the red wine couldn’t be completely converted to lactic acid during malolactic bacteria fermentation. In practice, only one-third of malic acid was converted. The optimum conditions for malolactic fermentation included an inoculum of 5%, fermentation temperature of 20℃, and addition of 0.2 g/L of malolactic nutrients. The commercial multiple strain product, Biolact Acclimatée® was a batter source of malolactic bacteria than single strain Oenococcus oeni BCRC 16064 in deacidifying red wine。 In the experiments combining the chemical deacidificants and malolactic bacteria to deacidify red wine, two commercial malolactic bacteria products, Biolact Acclimatée® and Lalvin X-3® have similar effect. Both deacidified red wine to a total acidity content of 0.85 g/100mL, and had highest scores of taste and overall preference with significant difference. In conclusion, treatment with chemical deacidificants combined with malolactic bacteria have the best deacidifiction effect on red wine and resulted in improved acceptability of the wine. The deacidified red wine was even more acceptable after 3 months of storage.

Record ID 第86筆 System ID 084DYU00250004
BCRC ID CCRC 10695, CCRC 12580, CCRC 10069, Public Year 84
Paper Name 低鹽小黃瓜醃漬物製造技術之研究 Studies of the pickling technology of low-salt pickled cucumbers
Student Name 張志豪
Teacher Name 陳鴻章, 顏裕鴻
School Department Name 大葉工學院 食品工程研究所 Academic Degree 碩士
Abstract 本研究由小黃瓜之前處理、醃漬及儲藏等方面,探討利用低鹽醃漬 技術來製作高品質且 具開胃、健康等訴求之低鹽醃漬產品之可行性。前 處理方法包括不同程度之熱水殺菁、熱鹽 水及 / 或熱氨水殺菁、不同 程度之鹽搓及鹽浸。 醃漬條件包括於 2% 之鹽漬液中接種不同 之乳酸 菌及添加 0 - 300 ppm 之亞硫酸氫鈉、0.1 - 0.3 M 檸檬酸 / 0.2 - 0.6 M 磷酸氫 二鈉緩衝劑、添加 1% 葡萄糖及 / 或 0.5% 酵母萃取物 , 並於室溫、15 ℃或 4 ℃下行低 鹽發酵;而其品質固定方式包括真 空包裝、充氮裝填或不抽空氣直接包裝後以不同條件殺菌 並於 4 ℃或 室溫下儲藏。 於前處理、醃漬及儲藏期間分析其酸鹼值、總菌體量、多 元酚氧 化酵素活性、表皮色差值、硬度、有機酸含量、可滴定酸、總生 菌數、總乳酸菌數、大腸桿 菌群或黴菌與酵母菌數等。 結果顯示,只 用熱水殺菁對小黃瓜綠色之保存並無明顯之功效 。而使用熱鹽水殺菁對 小黃瓜綠色之保存稍有助益,這可能是因為這些不同程度之熱鹽水殺 菁 皆足比單用熱水殺菁更能有效地使多元酚氧化酵素失活;然而不同程度之 熱鹽水殺菁對小 黃瓜顏色保存之效果差異不大。 另外,添加 50、100 或 300ppm 之亞硫酸氫鈉雖然仍使漢 特色澤?巫雂亅穻 V 紅色, 但顯然可減少 a 值之上升,即可減少綠色之消退;且輕微 之鹽搓與鹽 漬前處理配合亞硫酸氫鈉之添加對小黃瓜綠色保存有更明顯之效果。然而 ,醃漬 期間之酸鹼值若未加以控制皆會迅速降至 3.7 以下, 導致葉綠素發生脫鎂反應( pheophytinization ),中心鎂離子被氫取 代而形成暗褐色之脫鎂葉綠素 (pheophytin) 或 脫鎂葉綠酸; 若以不 同濃度及比例之檸檬酸 / 磷酸氫二鈉緩衝液為漬液,即可控制漬液之 酸鹼度, 而由小黃瓜表皮色差值可發現其?珙鬼 t 值,可見酸鹼度 之控制對於小黃瓜 表皮綠色保存之影響最大。且鹼液殺菁亦具有保存綠 色色澤之效果。綜上所述,原料小黃瓜 經 1) 鹽搓 1 min 及鹽浸 30 min, 2 ) 以 70 ℃之 0.05% 氨水 / 5% 鹽水殺菁 1 min; 3) 於醃漬 期間加入 50 ppm 亞硫酸氫鈉, 及 4) 使用 0.1 M 檸檬酸 / 0.2 M 磷 酸氫二鈉 緩衝液控制酸鹼度等多重處理時, 再以 Lactobacillus acidophilus CCRC 10695 及 Leuconostoc mesenteroides subsp. mesenteroides CCRC 12580 進行發酵,確可保持醃漬 三天期間小黃瓜 之質地及表皮之完整與綠色 以上述最適前處理與醃漬條件製作低鹽小 黃 瓜醃漬物並接種篩選出之低溫乳酸菌- Lactobacillus plantarum CCRC 10069,於 4 ℃下 發酵時,雖可避免大腸桿菌群及黴菌與酵母菌 之生長及保存良好綠色色澤;但乳酸菌之生長 與產酸速率緩慢, 即使 添加 1% 葡萄糖及 / 或 0.5% 酵母萃取物亦無法促進乳酸菌生長以 產 生良好風味。 而不添加基質於 15 ℃下醃漬 2 天後之小黃瓜,不但可控 制菌相且在表皮 顏色、瓜肉完整度、香氣、脆度、酸度及整體接受性上 的表現亦較好。 若用耐龍 / 聚丙 烯材質之積層膜真空包裝不添加基 質於 15 ℃下醃漬 2 天後之小黃瓜,並以 70 ℃,10 分 鐘之熱處理殺 菌後於 4 ℃下儲存,能保持醃漬小黃瓜之綠色達十二週, 而儲存期間之 酸鹼 值約在 4.1 - 4.5, 可滴定酸度約 0.06 - 0.12%, 且可控制 總生菌數在 103 CFU / ml 以下。經較高溫之熱處理及儲藏或以空氣、 氮氣包裝之醃漬小黃瓜其綠色皆迅速消退。 然 而,上述低鹽發酵法於 大量生產時可能面臨設備及冷藏等成本之增加、醃漬工廠衛生環境必 需 改善與無法於短時間內處理大量原料等問題;因此,本研究最後階段實驗 係在小黃瓜汁中 接種 Lactobacillus plantarum CCRC 10069 於室溫下 發酵三天,以此發酵液為漬液添加於 經最適前處理過之切片小黃瓜中, 探討製造輕度加工小黃瓜醃漬物之可行性。結果發現,以 添加或不加入 防腐劑並調整酸鹼值至 4.5 後再滅菌之醱酵漬液處理, 並以前述最適條 件包 裝、殺菌及儲藏之小黃瓜輕度加工產品之顏色、質地在四週後仍保 存良好;但無論防腐劑添 加與否,醱酵漬液若未調整酸鹼值、滅菌或包 裝後殺菌條件不足,皆會導致小黃瓜輕度加工 產品之顏色、質地及整體 接受性快速劣變。 The pretreatments, pickling conditions and storage methods were examined for the production of high quality, appetizing and healthy pickles from cucumber by low-salt pickling technology. Pretreatments included hot water blanching, hot brine and/or hot NH3 solution blanching, and salt scrubbing and salt soaking. Pretreated cucumber was then pickled in a 2% brine solution at 4 ℃, 15 ℃ or room temperature with the inoculation of different lactic acid bacteria and/or addition of 0-300 ppm of NaHSO3, 0.1-0.3 M citric acid/0.2-0.6 M Na2HPO4 buffers, 0-0.1% glucose and/or 0-0.5% yeast extract. The storage methods include different packaging/pasteurization condition and storage temperature. The pH value, microbial growth (OD660), polyphenol oxidase (PPO) activity, color values, hardness, organic acid concentration, titratable acidity, total count, total lactics, yeast and mold count, coliform count were analyzed during the pretreatment, pickling and storage periods. The result indicated that hot water blanching alone could not preserve the green color of cucumbers. The green color was better preserved by hot brine blanching due to the inactivation of PPO and thus the inhibition of enzymatic browning by salt, but different levels of hot brine blanching appeared to have similar results. Besides, the addition of 50, 100, or 300 ppm of NaHSO3 could slow down the increasing in color value "a" and delay the disappearing of green color. The effect was more obvious when salt scrubbing and salt soaking and the additi on of NaHSO3 were combined. However, the pH without control rapidly decreased to 3.7 or lower during pickling. Consequently, the Mg++ of chlorophyll was substituted by H+ in pheophytinization to form pheophytin or pheophorbide and the color of cucumber turned dark brown. When pH was buffered with citric acid/Na2HPO4, the color value "a" of cucumbers was kept negative. Controlling pH was most effective in preserving the green color of pickling cucumbers. Overall, the multitreatment described as follow can pr eserve both the texture and green color of cucumber after 3-days light pickling with 1: 1 Lactobacillus acidophilus CCRC 10695 and Leuconostoc mesenteroides CCRC 12580: the cucumber was 1) salt scrubbing for 1 min and then salt soaking for 30 min; 2) blanched at 70 ℃ in 0。05% ammonia / 5% brine for 1 min; and 3) pickled in a solution containing 50 ppm of NaHSO3 with its pH buffered with 0.1 M Citric acid / 0.2 M Na2HPO4. When the cucumber was pretreated with the above optimal methods, inoculated with Lactobacillus plantarum CCRC 10069 and fermented at 4 ℃, the yeast and mold and coliforms did not grow。 However, the growth of lactics and production of acid at 4 ℃ was slow even if 1% glucose and/or 0.5% yeast extract were added. Without substrate addition, the pickling of optimally pretreated cucumbers at 15 ℃ for 2 days could not only control the microflora, but also yield a better pickle product in terms of the quality of the green color, pulp perfectness, flavor, brittleness and acidity. The green color of pickling cucumbers can be preserved after 12-weeks storage, if the cucumber was pickled at 15 ℃ for 2 days, pasteurized at 70 ℃ for 10 min after vacuum packaging with the O-Ny/CPP laminated film and stored at 4 ℃. The pH of the pickle products was 4.1-4.5, titratable acidity was 0.06%-0.12%, total counts was lower than 1,000 CFU/ml during storage. The green color disappeared rapidly if the pickle products were pasteurized or stored at higher temperatures and packed under nitrogen or atmosphere. The low-salt pickling process described above, however, could encounter the problems such as the increasing cost of equipments or refrigeration, the necessity in improving pickling sanitation and the impossibility of pickling at once a great quantity of raw materials. Thus, the feasibility of using minimal processing to produce low-salt cucumber sliced pickles to overcome these problems was investigated. Pickling brine was prepared from cucumber juice inoculated with Lactobacillus plantarum CCRC 10069 at room temperature for 3days。 The texture and color of the cucumber slices were well preserved 4 weeks after minimal processing if the slices were pretreated, pickled in fermented, steriled and pH-adjusted (4.5) cucumber juice with/without preservatives, then pasteurized, packed and stored under the previously- observed optimal conditions. On the other hand, the texture, color and overall acceptance of cucumber slices deteriorated rapidlly if the slices were pickled in fermented brine without pH adjustment or without sterilization, or were inadequately pasteurized after packaging.

Record ID 第87筆 System ID 093PCCU0115006
BCRC ID BCRC 14085, BCRC 10357, BCRC 10695, Public Year 93
Paper Name 乳酸菌酛固定化技術對於共軛亞麻油酸生成之研究 Study of Immobilization of Lactic acid bacteria on Conjugated Linoleic Acid Production
Student Name 張甘霖
Teacher Name 林棟雍
School Department Name 中國文化大學 生活應用科學研究所 Academic Degree 碩士
Abstract 中文摘要 共軛亞麻油酸(CLA)具多種健康功能文獻已有詳載,而以乳酸菌生產CLA極具潛力,然而,乳酸菌生產CLA之產量仍低,如何有效提升CLA產量為當務之急,因此本研究以褐藻酸鈉將乳酸菌酛固定之,以探討其對促進乳酸菌生成CLA之影響。 經活化之五株乳酸菌酛:Lactoacillus helveticus (BCRC14030)、Streptococcus thermophilus (BCRC 14085)、L. plantarum (BCRC 10357)、Strep. thermophilus (BCRC12257)、與L. thermophilus (BCRC 10695)分別以2%褐藻酸鈉固定,再取20 g與0.065 g bovine serum albumin及0.1mL 亞麻油酸於25、37、50℃下反應0、24、48、72、96、120、144、與168 hr,以生產CLA後,進行CLA之定性與定量 結果發現:以褐藻酸鈉固定化此五株乳酸菌酛之CLA生成量皆比未經固定化之菌酛顯著為高;經固定化後之L. helveticus (BCRC14030)與Strep. thermophilus (BCRC14085)及L. plantarum (BCRC 10357)處理組之最適CLA反應條件與最大總CLA產量分別為50℃、96 hr之988.01 μg,37℃、120 hr之1293.37 μg與50℃、120 hr之717.91 μg;經固定化後之Strep. thermophilus (BCRC12257)及L. thermophilus (BCRC 10695)處理組之三個反應溫度對總CLA產量之影響則不顯著,而其最適反應時間與最大總CLA產量分別介於72~120 hr與105.11~448.89 μg之間 本研究顯示以褐藻酸鈉固定化乳酸菌確能促進CLA生成,而此五株乳酸菌中,以L. helveticus (BCRC14030)經固定化後在37℃經120 hr反應之總CLA產量最高。 關鍵詞:乳酸菌、共軛亞麻油酸、褐藻酸鈉、固定化。 Abstract Conjugated linoleic acid (CLA) had been reported to be responsible for many biological activities that relate to health. Although lactic acid bacteria were observed to be capable of converting linoleic acid (LA) into CLA, the yield was low. The immobilization technique has been used on many microorganisms to improve their enzyme activities and the product yields. However, the technique has not yet been applied on lactic acid bacteria for CLA production. The study, therefore, was to determine the effect of alginate immobilized lactic acid bacteria on the production of CLA. Five lactic acid bacteria: Lactoacillus helveticus (BCRC14030), Streptococcus thermophilus (BCRC 14085), Strep thermophilus (BCRC12257), L plantarum (BCRC 10357), and L acidophilus (BCRC 10695) were immobilized by 2% sodium alginate and reacted with 0。1 mL linoleic acid for 0, 24, 48, 72, 96, 120, 144, and 168 hr at 25, 37, and 50oC for CLA production. The results showed that CLA yields were higher in the treatments of alginate immobilized lactic acid bacteria compared with the control. The optimal reaction conditions of alginate immobilized Lactoacillus helveticus (BCRC14030), Strep thermophilus (BCRC 14085), and L plantarum (BCRC 10357)were 37oC, 120 hr, 50oC, 96 hr, and 50oC, 120 hr, respectively, and CLA yields were 1293。37, 988.01, and 717.91 μg, respectively. Whereas, the reaction temperature did not affect CLA yield in the treatments of alginate immobilized Strep. thermophilus (BCRC12257) and L acidophilus (BCRC 10695), and the optimal reaction times and the yields of those two treatments were 72~120 hr and 05。11~448.89 μg, respectively. The results demonstrated that lactic acid bacteria immobilized by sodium alginate could enhance CLA production. Alginate immobilized L. helveticus (BCRC14030) produced highest CLA yield among five lactic acid bacteria tested and was suggested for CLA production。 Key word: Lactic acid bacteria, Conjugated linoleic acid, Sodium alginate,Immobilization.

Record ID 第88筆 System ID 093PCCU0115002
BCRC ID CCRC 1403、, CCRC 14076, CCRC 14085, CCRC 14030, Public Year 93
Paper Name 不同乳酸菌元對胞外多醣生成之研究 Studies of Exopolysaccharides Production by Different Lactic Acid Bacteria
Student Name 張簡玫芳
Teacher Name 林棟雍 博士
School Department Name 中國文化大學 生活應用科學研究所 Academic Degree 碩士
Abstract 摘要 本研究旨在探討不同乳酸菌元生成胞外多醣之黏絲性、產量、單醣組成、含量比例及不同分子量胞外多醣之關係,以篩選出具黏絲性高產量之產胞外多醣乳酸菌元及其最適發酵時間。 利用三株購自新竹食品工業發展研究所菌種保存及研究中心(Culture Collection and Research Center, CCRC)之乳酸菌元:Lactobacillus helveticus CCRC 1403、Lactobacillus helveticus CCRC 14076與Streptococcus thermophilus CCRC 14085,分別以5L脫脂乳(skim milk, 10%, w/v)於37℃、pH 5之5-L發酵槽培養84 hr,分別於0, 12, 16, 20, 24, 32, 36, 48, 60, 72及84 hr定時取樣,進行胞外多醣黏絲性、產量、分子量及單醣組成之分析 在黏絲性方面,L. helveticus CCRC 14030在32~84 hr發酵時間之胞外多醣黏稠物皆具有黏絲性,32 hr至48 hr之黏絲性介於1.13 ~1.33 cm,差異不顯著(p>0.05),黏絲性於60 hr達最高值,為2.1 cm,並在發酵72與 84 hr後黏絲性分別下降至0.6和0.56 cm,而L. helveticus CCRC 14076與Strep. thermophilus CCRC 14085均不具有黏絲性;產量方面,L. helveticus CCRC 14030在60 hr發酵時間所生成胞外多醣產量顯著高於32、36、48、72與84 hr所生成之胞外多醣產量(p<0.05)(0.73 g/L vs. 0.25, 0.25, 0.48, 0.54, 0.53 g/L),另L. helveticus CCRC 14076 與Strep. thermophilus CCRC 14085之胞外多醣產量在32~84 hr六個發酵時間下分別為0.19- 0.28 g及0.22-0.28 g,皆無明顯差異(p>0.05);分析三株乳酸菌元在不同發酵時間生成之分子量最大之胞外多醣,L. helveticus CCRC 14030在32~60 hr與72~84 hr生成之分子量最大之胞外多醣分別為196300 kDa及24640 kDa,明顯大於12~24 hr生成之分子量最大之胞外多醣(47 kDa),且L. helveticus CCRC 14030於32~84 hr生成之最大分子量胞外多醣含量佔總胞外多醣含量比例介於19~52%,另外,L. helveticus CCRC 14076與Strep. thermophilus CCRC 14085在12~84 hr十個發酵時間下生成之分子量最大之胞外多醣分別為589 kDa與264-480 kDa。 三株乳酸菌元生成之胞外多醣在各發酵時間皆會分離出二個或以上之不同分子量胞外多醣,不因菌元或發酵時間不同有所差異,且各發酵時間之胞外多醣分子量大小與同一發酵時間生成之不同分子量之胞外多醣含量比例兩者間並無相關性;分析三株乳酸菌元生成胞外多醣之單醣組成與其含量比例,結果顯示,從同一菌株因不同發酵時間,可分離出兩個或以上不同單醣組成及含量比例之胞外多醣,且三株乳酸菌元在不同發酵時間,生成之胞外多醣皆含有葡萄糖、半乳糖、鼠李糖及乙醯葡萄糖胺。 綜上所述,三株測試之乳酸菌元以Lactobacillus helveticus CCRC 14030具產黏絲性,而其能生成具黏絲性胞外多醣之發酵時間為32~60 hr,其中又以60 hr之胞外多醣產量及黏絲性程度最高,故欲得到具黏絲性高產量之胞外多醣,建議以此株菌元培養於含脫脂乳之發酵槽,在37℃與pH 5之發酵條件下經60 hr發酵最為合適。 關鍵詞:胞外多醣、乳酸菌。 Abstract The objective of this study was to determine thread forming properties, yields, monosaccharide compositions, and content proportion of different molar mass of exopolysaccharides produced by three lactic acid bacteria in order to find the lactic acid bacteria with the highest thread forming property and yields and the optimal fermentation time. The three lactic acid bacteria strains:Lactobacillus helveticus CCRC 14030、Lactobacillus helveticus CCRC 14076 and Streptococcus thermophilus CCRC 14085 were obtained from the Culture Collection and Research Center。 Each of the three strains was inoculated in a 5-L fermentor filled with 10% skim milk and inoculated at 37℃ and pH 5 for 0~84 hr. Fermented samples were withdrawn at 0, 12, 16, 20, 24, 32, 36, 48, 60, 72 and 84 hr, and thread forming properties yields, molar mass and monosaccharide compositions were determined. L. helveticus CCRC 14030 was the only ropy strain observed among the three strains tested。 Exopolysaccharides with ropiness was produced at 32~84 hr of fermentation and the thread forming property was between 1.13 and 1.33 cm. The highest thread forming property (2.1 cm) was observed at 60 hr. It decreased at 72 and 84 hr (0.6 and 0.56 cm). Neither L. helveticus CCRC 14076 nor Strep thermophilus CCRC 14085 was ropy strain。 The highest yield of exopolysaccharides was produced at 60 hr by L. helveticus CCRC 14030 and the yield was 0。73 g/L which was much higher than 0.25, 0.25, 0.48, 0.54, 0.53 g/L produced at 32, 36, 48, 72 and 84 hr. Exopolysaccharides yields produced by L. helveticus CCRC 14076 and Strep thermophilus CCRC 14085 at 32~84 hr were 0。19-0.28 g and 0.22-0.28 g, respectively. Comparing the largest molar mass of exopoly- saccharides produced by the three lactic acid bacteria at different fermentation times, exopolysaccharides with the largest molar mass were at 32~60 hr and 72~84 hr produced by L. helveticus CCRC 14030, and the molar mass was between 196300 kDa and 24640 kDa, much greater than 47 kDa produced at 12~24 hr by this strain。 The proportion of content of exopoly- saccharides with the largest molar mass to that of total exopolysaccharides ranged from 19 to 52% at 32~84 hr of fermentation in this ropy strain treatment. Largest molar mass of exopolysaccharides produced by L. helveticus CCRC 14076 and Strep thermophilus CCRC 14085 at 12~84 hr were 589 kDa and 264-480 kDa, respectively。 Exopolysaccharides with more than two different molar mass were produced by the three lactic acid bacteria at each ferment- ation time, regardless of culture strain and fermentation time. In addition, no corelation between molar mass and the content of each molar mass of exopolysaccharides was found at different fermentation times. Exopolysaccharides with various monomers and the content proportion were observed at different ferment- ation times for each strain, and exopolysaccharides produced by those strains contained all four monomers tested. In conclusion, L. helveticus CCRC 14030 was the only ropy strain found among those three strains。 Exopolysaccharides with thread forming property was produced at 32~60 hr, the highest yield and thread forming property were observed at 60 hr of fermentation time. L. helveticus CCRC 14030 inoculated in skim milk and incubated at 37℃ for 60 hr, therefore, was suggested for producing exopolysaccharides with highest yield and thread forming property。 Key words:Exopolysaccharides, Lactic acid bacteria.

Record ID 第89筆 System ID 092PCCU0111012
BCRC ID BCRC 12586, Public Year 92
Paper Name 家禽乳酸菌之食用型疫苗應用與遺傳組分型覈實 Poultry Lactic Acid Bacteria for Edible Vaccine Applications with Genotyping Verification
Student Name 姚佩君
Teacher Name 張春梵
School Department Name 中國文化大學 生物科技研究所 Academic Degree 碩士
Abstract 本項論文研究目標為發展特定功效雞隻乳酸菌種原之基因重組轉殖與基因分型確證技術,初步開發雞隻疫苗基因選殖乳酸菌食用疫苗的重組核酸,以及進行基因分型確認乳酸菌種原相關菌株的遺傳品質正確性。大規模雞隻飼養的強調高度經濟效益須倚賴全力防治疾病的最小感染損失,但需耗費大量人力施打疫苗及投與藥物,造成雞群緊迫型育成率下降的經濟效益嚴重損失。初步開發重組病原基因核酸片段轉形乳酸球菌(Lactococcus)的食用疫苗菌株,將可投與雞隻經腸道相關淋巴組織(GALT)誘發雞隻免疫反應,以利減低雞隻育成損失與節省人工物力成本。 食用疫苗應用方面,利用乳酸球菌屬乳酸菌種Lactococcus lactis subsp. Cremoris (BCRC 12586)為疫苗主體,於質體pRSET選殖冠病毒屬雞隻傳染性支氣管炎病毒(IBV)突觸蛋白(spike protein)基因的互補核酸片段,賦予大腸桿菌和乳酸球菌等菌體間穿梭表現能力,經定序確認重組基因序列為符合譯讀框(in-frame),製備菌株蛋白質萃取物經免疫沈降與膠板電泳確認表現蛋白質分子量約58 kDa,後續將可製備具抗原性且無病原性之食用疫苗菌株。遺傳分型確證方面,應用高度效能屬內專一性(genus-specific)擴增引子16S rRNA進行乳酸菌核苷酸序列遺傳分型之快速鑑定,擴增引子Blb11-Blb12對Lactobacillus擴增250 bp條帶,簡易區分不同動物種系乳酸菌之動物腸道益生效果與優良實驗材料菌種效益,可以補足傳統分型技術無法依據顯性特質進行乳酸菌種分型的不足部分。 The purpose of this thesis study is to develop both recombinant DNA construct of pathogen gene fragment within edible lactococcus strains and the genotyping verification system on genetic quality. Preliminary achievements include recombinant construction for transforming edible poultry lactococci vaccine and as well genotyping verification of lactococcus genetic quality from possibly contaiminated bacteria strains. The poultry farms suffer great profit loss represented by reduced batch production percentage ironically due to repetitive stress caused by costy vaccine application and labor in order to build maximal disease control system for minimizing loss. The solution herein may be the edible lactococcus vaccine with transformed recombinant pathogen gene construct in order to maximize disease control by enteral immunity induction of gut associated lymphoid tissues (GALT). In the aspect of edible vaccine, the host system of Lactococcus lactis subsp. Cremoris (BCRC 12586) is transformed with recombinant pRSET cloned with spike cDNA insert of infectious bronchitis virus (IBV) of poultry coronavirus。 With shuffle protein expression ability of pRSET vector, the pRSET-ibv-spike clone is verified with in-frame inserted cDNA sequence and as well with 58 kDa protein by both immunoprecipitation with anti-spike antibody and SDS-PAGE separation of propagated protein extracts from pRSET-ibv-spike transformed strains. In the aspect of genotyping verification, high-efficient genus-specific PCR primers with 16S rRNA sequence are adopted in the fast differential genotyping of related strains. The Blb11-Blb12 primer set with 250 bp PCR yield on Lactobacillus simplifies lactobacteria differentiation of different animal host species. The genotyping verification completes the shortage of conventional phenotyping system due to the lacking of phenotypic criteria in special cases of probiotic and experimental strains of enteral bacteria. Keyword: Lactobacteria, edible vaccine, genotyping, coronavirus, infections bronchitis virus, spike protein.

Record ID 第90筆 System ID 089PCCU0111003
BCRC ID CCRC 35396, CCRC 35398, Public Year 89
Paper Name 利用細胞核傳送方法進行牛樟芝的擬有性雜交 Parasexual Crosses of Antrodia Camphorata By nuclear transfer method
Student Name 王威閔
Teacher Name 吳瑞鈺 博士
School Department Name 中國文化大學 生物科技研究所 Academic Degree 碩士
Abstract 牛樟芝Antrodia camphorata(Zang & Su)被喻為台灣最珍貴真菌,有台灣紅寶石之稱,為台灣特有的真菌之一。在傳統療法上牛樟芝常被利用為解毒劑(antidote),用以治療癌症(cancer)、高血壓(hypertension)及肝癌 (liver cancer), 目前已有報告指出牛樟芝子實體可被分離出固醇酸(steroid acid) 等成分。牛樟芝目前仍無法以人工栽培的方式生產子實體,但隨著生物科技的發展牛樟芝目前正發展菌絲體培養法進行發酵,大量生產菌絲體及有效成分,因此企望能經由選種或育種,找出具有生長快速、有效成分含量高的優良菌種,以進行工業化生產。 傳統真菌育種大多是以原生質體融合的方式來進行育種,本實驗開發傳送 核 入 原 生 質 體的方法,以二株不同的牛樟菌株 CCRC 35396 (C96)與 CCRC 353989 (C98)當親本,分別將C96的核傳送入C98原生質體(68傳送組)及 C98的核傳送入 C96原生質體(86傳送組)來進行雜交育種。首先以酵素Novozyme 234 分別製備原生質體然後純化出細胞核,再分別進行傳送核入原生質體,而所獲得的68傳送組與86傳送組的雜交體共有10群,這些雜交體經過多次繼代培養與單株化後共獲得13株穩定的雜交株系這些雜交株系包括S68A、S68B、S68C、S8D S68E、S68F、S68G、S68H、S68I、S68J、與S86A、S86B、S86C等。雜交株系的外觀表現經分析後發現13個雜交株系在19.5、27.5、35.5℃三種溫度與 BM、MEA、 PDA三種培養基培養,其菌落型態與顏色皆呈現不同的表現型。利用AFLP DNA指紋分析技術,由64組引子對中篩選出可以擴增標誌片段並區別親本的E1M3、E2M4、E4M6、E7M3四組引子,再利用這四組引子對進行牛樟菌親本與雜交株系的 AFLP DNA圖譜分析,用以鑑別其基因型,經由4組引子的DNA標誌片段的比對分析,S68D的基因型與 C98相同,而其他12株系皆呈現不同基因型。比對牛樟菌二親本 (CCRC35396、CCRC35398)與13個雜交株系的外表型(phenotype)與基因型(genotype),發現13個雜交株系的外表型態與基因型有一致性,而且可以加以區別,顯示13個雜交株系為不同於C98與C96牛樟菌的新菌株。 本實驗建立牛樟芝以傳送核入原生質體的擬有性生殖育種方法、輔以菌種外表型鑑別法與AFLP DNA指紋分析技術鑑別牛樟菌菌種之雜交株系,藉由這些方法建立,更育出13種不同的牛樟芝新株系。 Antrodia camphorata , is considered the most precious and unique fungus in Taiwan. It was also nicknamed as “Ruby of Taiwan”. It has been utilized as an antidote in the treatment for cancer, hypertension, and hepatocellular carcinoma in Taiwan folk medicine. Studies indicated that steroid acid and other active components could be isolated from the fruiting bodies of A. comphorata. But it was still unsuccessful to reproduce the fruiting body through mycelium cultivation. However, the rapid progress in biotechnology has made the mass production of these active components possible, by scale-up fermentation of the mycelium of A. comphorata. The conventional methods for fungi breeding mainly depends on the successful protoplast fusion technology. In our studies, a nuclei transfer method was developed and used for A. comphorata breeding. Two parental stocks of A. comphorata ( C96 and C98 ) were purchased from Culture Collection and Research Center(CCRC)。 The nuclei were isolated and purified from protoplast of mycelium treated by Novozyme 234. The isolated nuclei from C96 and C98 were transferred into C98 and C96 protoplasts, respectively, to regenerate new A. comphorata hybrids. In the beginning, 10 groups of hybrids with a differential characteristic in colony were obtained after the first subculture. Finally, 13 stable hybrid strains (designated as S68A S68B S68C S68D S68E S68F S68G S69H S68I S68J and S86A S86B S86C) were generated through a serial of subculture and clonization process. Different culture conditions, including 3 temperature of 19.5 27.5 35.5 C and 3 media of BM MEA PDA, had been tested to differentiate these 13 hybrid strains. The morphology and pigment production of fungal colonies were compared and distinct from each other under some culture conditions. The AFLP DNA fingerprinting assay with 64 pairs of primers was further used to study the genotype of hybrid strains. Results showed that E1M3, E2M4, E4M6, and E7M3, 4 out of the 64 primer pairs, were able to distinguish the PCR-amplification products of C96 from those of C98. Using these 4 primer pairs, we had completed the genotype identification of 13 hybrid strains and compared these data with those of their parental stocks, C96 and C98. The results demonstrated that these 13 hybrid strains were distinct in both genotype and phenotype and they should be identified as new strains of A. comphorata. In our studies, we demonstrated the success of parasexual reproduction of A. comphorata by transferring nuclei into protoplast. In addition to the morphological classification method, the AFLP DNA fingerprinting method could be utilized for identification of hybrid strains and their parental stocks. In this report, 13 new strains of A. comphorata were obtained and identified.

Record ID 第91筆 System ID 086PCCU1344006
BCRC ID CCRC 13032, CCRC no, Public Year 86
Paper Name 乳酸菌醱酵及凍乾保存條件之探討 Study on Fermention and Freeze Dry of Lactic Acid Bacteria
Student Name 張惠雯
Teacher Name 向明, 張玲玲
School Department Name 中國文化大學 造紙印刷研究所 Academic Degree 碩士
Abstract 本研究主要探討乳酸菌醱酵之最適條件,並探討乳酸菌醱酵液進 行冷凍乾燥時,添加物對乳酸菌株存活之影響。 自食品工業發展研究所菌種中心(CCRC.)購得的八株乳酸菌,經 MRS及Skim milk培養基初步篩選後,以Enterococcus faecalis (CCRC 13032)菌株的生長情形較好,產酸適中且具香氣,故選定E. faecalis 進行醱酵培養。將菌株分別培養於37℃、42℃、45℃之結果中測得E. faecalis在37℃時培養的效果最好。 經培養基探討結果顯示,glucose 2%、yeast extract 0.4%、peptone 0. 5%、MgSO4×7H2O 0.02%、K2HPO4 0.1%為培養基時,在醱酵24~26 小時之間,可獲得約6.5×109/ml的最高活菌數。若醱酵時以不同濃度 之Glucose、Yeast extract作為饋料,對於E. faecalis 的醱酵與未饋料 的效果相當,無法達提昇活菌數的效果。改變醱酵槽轉速發現,轉速 增高至250或300rpm,對醱酵液的pH值和酸度並沒有明顯的影響, 但是活菌數則有下降的趨勢。故仍採用原先之50rpm進行醱酵試驗。 冷凍乾燥過程,將醱酵液離心,菌體以蒸餾水水洗後,添加吃 trehalose及glycerol再進行冷凍乾燥程序,所獲得之效果最好,活菌數 約可達2.66×109/g。完全不添加添加物時,所獲得之活菌數最多達2.42 ×105/g。 凍乾後之乳酸菌於室溫下儲存,結果顯示活菌數慢慢下降,至第 12週測試時,活菌數平均只剩下2.0~3.0×105/g,顯示E. faecalis其保 存的效果不佳。 The culture method of lactic acid bacteria fermentation for this study. To use the lactic acid bacteria broth for additive of freeze dry and cell existing after freeze dry. In this experiement, the eight strains of lactic acid bacteria is from Culture Collection & Research Center (CCRC) After the first selection of cultivation from MRS broth and skim milk, therefore, choose CCRC no。 13032 to be the fermentation. The lactic acid bacteria was cultured respectively at temperature of 37℃, 42℃ and 45℃, and it was found that the optimal temperature for CCRC no。13032 was 37℃. From the result of fermentation finds that when the mixture of glucose(2%), yeast extract(0.4%), peptone(0.5%), MgSO4×7H2O(0.2g/L), K2 HPO4(1.0g/L) to be the medium, it can get the highest cell density of 6.5×109/ml during the fermentation period of 24~26 hours. While fermentation increases the different mixture concentration feeding of glucose and yeast extract, the effect will be the same compared to the CCRC no。 13032 and without increasing the feeding. Therefore, we can`t approach the effect of cell count. To change the speeding of jar-fermentor, while the speed up to 250 or 300rpm, the pH value and acidity of fermentation will not have the obvious influence, but the cell density will tend to decrease. To get the best result of freeze drying, the fermentation broth needs to be centrifugalized and water washed. Then add in trehalose and glycerol in order to process the freeze dry procedure, the cell density might reach to 2.66×109/g. The second best result will be add in mannitol and glycerol, the cell density might reach to 8.41×108/g, the effect will be third best. Without adding in any additive, the cell density will only reach to 2.42×105/g. After freeze dry, the lactic acid bacteria stores at the room temperature, the result reveals that cell count decrease gradually. After 12 weeks, the cell count is only left 2.0~3.0 ×105/g in average, and the result of existing cell is not well.

Record ID 第92筆 System ID 080PCCU2262008
BCRC ID CCRC 10355, Public Year 80
Paper Name 利用細菌基因體限制�t圖譜與全蛋白質圖譜作為菌種鑒定之可行性研究 Application of bacterial genomic restriction fingerprints and whole protein fingerprints for bacterial identification -- a feasibility study
Student Name 陳毓玲
Teacher Name 歐樂君
School Department Name 中國文化大學 家政學研究所 Academic Degree 碩士
Abstract 微生物在農業、工業、食品工業及醫藥上都有其應用及研究的價值,而菌種鑑定是 最基礎而又重要的。傳統的鑑定方法是依據菌落菌體外觀、對氧氣、養分的需求性 、在特殊培養基中生長、產生代謝物的情形、噬菌體分析、血清抗原抗體反應等, 十分費時費事,有時結果亦不明確,一般而言,對於大多數菌種之鑑定極限,只能 鑑定至〝屬〞或〝種〞之差異性,種以下〝株〞之差異性則無法區分。 近年來由於分子生物學的進步,利用分子生物學的方法應用於細菌鑑定確實有一定 價值。本論文擬有系統地建立一套結合菌株基因體限制圖譜及全蛋白圖譜的分析 方法以補強目前傳統以外觀、生長需求、生物特徵作鑒定可能發生的疏漏,另一方 面由此方法得到的資訊或許可縮簡原來檢定的流程。 本實驗首先將 15 株不同屬或不同種的 G(-) 菌株以 TYBN 培養液培養至定常期 ( stationary phase) ,抽取其高分子基因體以洋菜膠電泳,以確定所得DNA 之品質 ,並檢視該菌株是否有質體DNA (plasmid) 之存在,質體之存在與否必然影響原菌 株之基因體限制圖譜及全蛋白質圖譜乃至菌株特性。其次基因體DNA 分別以限制  Dra I(辨識 TTT ↓ AAA )及 Eag I(辨識 C↓ GGCCG)水解之,所得DNA 片 AAA ↑ TTT G CCGGC 段以Hoechst dye 33258 螢光比色法定量後,取約2 μg DNA 以 0.6﹪洋菜膠電泳 展開,染色後顯示其圖譜。 另一方面菌株以TYBN培養至對數期 Mid-Log Phase,菌體以 0.5﹪ NaCl 洗去培養 液後以Biuret Method 定蛋白質含量,取含0.05 mg 蛋白質之菌體加入 SDS/β- mercaptoethanol 溶液,經煮沸後以 SDS/PAGE 展開並以 Coomassie Brilliant Blue染色顯現其圖譜。 實驗結果顯示: 1. 以本實驗方法抽取的高分子DNA 品質良好 2. 菌株 Alcaligenes faecalis ATCC 25094, Pseudomonas putida ATCC 17484, ▔▔▔▔▔▔▔▔▔▔ ▔▔▔▔▔▔▔▔▔ Serratia grimesii ATCC 14460, Serratia marcescens ATCCc 8100 有等質 ▔▔▔▔▔▔▔▔▔ ▔▔▔▔▔▔▔▔▔▔ 體DNA 存在。 3. 以Eag I 及Dra I 水解基因體DNA,圖譜可明顯反應該菌株基因體G+C Content 。如G+C content 高者以Eag I 切割所得的DNA 片段平均較短,反之以Dra I 切割者則多較長。 4. 以15株菌株(除E coli外)有其各別之基因體限制圖譜,尤以 Alcaligenes ▔▔▔ ▔▔▔▔▔▔ faecalis ATCC 8750, Alcaligenes faecalis CCRC 10355 , Alcaligenes ▔▔▔▔ ▔▔▔▔▔▔ ▔▔▔▔ ▔▔▔▔▔▔ faecalis ATCC 25094 雖為同屬同種(僅不同株)其差異性仍大。 ▔▔▔▔ 5. 全蛋白圖譜 Salmonella Sp,Pseudomonas Sp,Serratia Sp,Ecoli species ▔▔▔▔▔▔▔ ▔▔▔▔▔▔▔ ▔▔▔▔▔▔ ▔▔▔▔▔▔▔ 等同屬、同種間有相似性。但 Alcaligenes Sp 同種間差異極大而Alcaligens ▔▔▔▔▔▔▔ ▔▔▔▔▔ faecalis 10355、Alcaligenes denitrficansubsp denitrifican 15137 ,雖 ▔▔▔▔ ▔▔▔▔▔▔▔▔▔▔▔▔▔▔▔▔▔▔▔▔▔ 不同種圖形卻極為相似,與傳統覽定結果不同,似乎意味這兩隻菌株在遺傳上 很親近。 6. 由於基因體限制圖譜各別菌株間特異性很大,故可用來鑑定二同種菌株是否 來自同株,而全蛋白圖譜由於同屬、同種或同株間有相似性,以可作為菌種鑑 定的一項參考。

Record ID 第93筆 System ID 095PCCU0115002
BCRC ID BCRC 14030, Public Year 95
Paper Name 糖類之添加對乳酸菌胞外多醣生成之研究 Effect of sugar addition on exopolysaccharides production by lactic acid bacteria
Student Name 林佩璇
Teacher Name 林棟雍 博士
School Department Name 中國文化大學 生活應用科學研究所 Academic Degree 碩士
Abstract 摘要   本研究在探討乳酸菌酛與單醣對EPS(exopolysaccharides)生成之影響。接種Yogurt bacteria(Lactobacillus delbrueckii subsp. bulgaricus與 S.thermophilus)與L. helveticus BCRC 14030於添加1、5%葡萄糖、半乳糖及果糖之含脫脂乳培養基之發酵槽中在,25 ℃、pH 6培養96小時後,進行發酵液黏度、乳清黏度、發酵槽菌數、黏絲性、EPS產量、單醣組成及分子量之分析,以篩選最適乳酸菌與單醣,以生產高產量具黏性之EPS   發酵液黏度在Yogurt bacteria與L. helveticus BCRC 14030各處理組間差異皆不顯著(p > 0.05),但隨著滴定量上升,發酵液黏度有下降的趨勢。添加1%的葡萄糖不能促進Yogurt bacteria菌體與酪蛋白結合,造成發酵液黏度(3.09 cp)上昇,去除酪蛋白後之黏度(1.50 cp)隨之降低,顯示:酪蛋白為發酵液黏度主要影響因素。由於菌體對酪蛋白的利用率不高,EPS無法與酪蛋白結合,造成EPS產量(0.32 g/L)下降。   Yogurt bacteria與L. helveticus BCRC 14030之黏絲性分別以添加1%半乳糖(1.35 cm)與5%半乳糖(2.43 cm)處理組較佳比較Yogurt bacteria與L. helveticus BCRC 14030處理組之黏絲性,以添加1%葡萄糖(0.86 vs.1.83 cm)、5%半乳糖(0.86 vs.2.43 cm)與5%果糖(0.32 vs.1.86 cm)都以接種L. helveticus BCRC 14030處理組顯著為高   Yogurt bacteria 之EPS產量,以添加1%半乳糖(2.12 g/L) 與5%半乳糖(1.72 g/L)處理組較高;不同單醣、濃度之L. helveticus BCRC 14030 EPS處理組產量介於1.44~3.42 g/L之間,差異並不顯著(p > 0.05)比較Yogurt bacteria(YC-380)與L. helveticus BCRC 14030間之EPS產量,以L. helveticus BCRC 14030之未添加單醣、1%葡萄糖及5% 葡萄糖三個處理組之EPS產量(2.72、2.98、3.42 g/L) 比Yogurt bacteria (0.91、0.32、1.40 g/L)顯著為高(p < 0.05)。   綜上所述,Yogurt bacteria於添加1%半乳糖處理組之黏絲性最佳,EPS產量亦最高。在L. helveticus BCRC 14030處理組中,則以添加5%半乳糖之黏絲性最佳;添加1%、5%葡萄糖、半乳糖及果糖無法提升L. helveticus BCRC 14030之EPS產量;而L. helveticus BCRC 14030生成之EPS產量與黏絲性較Yogurt bacteria為佳。 Abstract The objective of the study was to investigate the effects of lactic acid bacteria and monosaccharide addition on EPS (exopolysaccharides) production. Yogurt bacteria (Lactobacillus delbrueckii subsp. Bulgaricus and S.thermophilus) or Lactobacillus helveticus BCRC 14030 was inoculated into a fermentor containing skim milk medium with 1% or 5 % glucose, galactose, or fructose added, and incubated at 25 ℃, pH 6 for 96 hr。 The viscosity of fermented medium, viable count, thread forming property of EPS, EPS yield, the composition of monosaccharide, and molecular weight of EPS were analyzed, in order to find an optimal lactic acid bacterium and monosaccharide for producing EPS with high yield and good thread forming property. While the viscosity of fermented medium decreased with increase in titration level, the viscosity of fermented medium produced between the treatments of yogurt bacteria and L. helveticus BCRC 14030 was not significantly different (p > 0。05). The addition of 1 % glucose did not improve the binding between yogurt bacteria and casein, which resulted in the decrease in the viscosity of the fermented medium (1.50 cp). The finding indicated the importance of casein on the viscosity. In addition, the inability of binding between lactic acid bacteria and casein could also result in the decrease in EPS yield (0.32 g/L). Higher thread forming properties were observed in 1 and 5 % galactose (1.35 and 2.43 cm) additions of both yogurt bacteria and L. helveticus BCRC 14030 treatments。 Comparing the treatments of yogurt bacteria with L. helveticus BCRC 14030, 1 % glucose (0。86 vs.1.83 cm), 5 % galactose (0.86 vs.2.43 cm), and 5 % fructose (0.32 vs.1.86 cm) of L. helveticus BCRC 14030 treatments were found to be higher in thread forming property。 Higher EPS yields were observed in 1 and 5 % galactose (2.12 and 1.72 g/L)additions of yogurt bacteria treatment. The yields of EPS were not significant different (1.44-3.42 g/L) (p > 0.05)in the treatments of L. helveticus BCRC 14030 with the addition of monosaccharide of different concentrations。 Furthermore, EPS yields were significantly higher in the control and the treatments of 1 and 5 % glucose (2.72、2.98、3.42 g/L) additions than those of yogurt bacteria (0.91、0.32、1.40 g/L) treatments. In summary, the best thread forming property was found in 1 % galactose of yogurt bacteria treatment. Whereas 5 % galactose was the best in the L. helveticus BCRC 14030 treatment。 Although the addition of 1 and 5 % glucose, galactose and fructose could not improve EPS yield of L. helveticus BCRC 14030 treatment, inoculation of this culture strain produced higher EPS yield and thread forming property, compared to yogurt bacteria

Record ID 第94筆 System ID 094PCCU0115007
BCRC ID BCRC 14030, Public Year 94
Paper Name 固定化凝膠對乳酸菌與發酵乳之共軛亞麻油酸生成之研究 Study of Immobilization of Lactic acid bacteria and Yogurt bacteria on Conjugated Linoleic Acid Production
Student Name 田巧怡
Teacher Name 林棟雍
School Department Name 中國文化大學 生活應用科學研究所 Academic Degree 碩士
Abstract 共軛亞麻油酸(CLA)具多種健康功能,而乳酸菌能促進亞麻油酸轉變成CLA,不過以乳酸菌生產CLA之產量低,因此如何提升CLA產量為當前研究趨勢。本研究以褐藻酸鈉、聚丙烯醯胺、幾丁聚醣與鹿角菜膠固定化Lactobacillus helveticus (BCRC 14030),在pH 5、6、7與8下進行CLA生成反應,以探討不同凝膠與反應pH值對促進此一菌酛生成CLA之影響,及其應用於生產富含CLA發酵乳之可行性 經活化之L. helveticus分別以褐藻酸鈉、聚丙烯醯胺、幾丁聚醣與鹿角菜膠固定後,取20 g與0.065 g BSA及0.1ml亞麻油酸(LA)於37 ℃下反應120小時,以生產CLA;再以不同比例褐藻酸鈉與鹿角菜膠固定化發酵乳菌酛(YC380)與不同比例褐藻酸鈉與鹿角菜膠固定化發酵乳菌酛添加2 %L. helveticus(BCRC 14030)各置入43 ℃培養至pH值4.3後生成發酵乳,其後以HPLC定量CLA,並取經10 %鹿角菜膠、10 %褐藻酸鈉固定化、5 %未固定化發酵乳菌酛生產之發酵乳與市售發酵乳做Hunter L,a,b值測定,就外觀、氣味、口味、濃稠度與總接受度,進行官能品評試驗。 結果發現:褐藻酸鈉固定化L. helveticus在pH 7下之總CLA生成量為584.80 μg,顯著地(p<0.05)比其它pH處理組及未固定化菌酛組為高;聚丙烯醯胺固定化L. helveticus在pH 6下之總CLA生成量為382.66 μg,顯著地(p<0.05)比pH 5與8處理組為高;幾丁聚醣固定化L. helveticus在pH 8下之總CLA生成量為23.70 μg,顯著地(p<0.05)比其它pH處理組為高;鹿角菜膠固定化L. helveticus在pH 6下之總CLA生成量為71.56 μg,顯著地(p<0.05)比pH5與8處理組及未固定化菌酛組為高。褐藻酸鈉與鹿角菜膠固定化發酵乳菌酛,與褐藻酸鈉與鹿角菜膠固定化發酵乳菌酛添加2 %L. helveticus(BCRC 14030)下之總CLA生成量皆顯著(p<0.05)比未固定化菌酛為高;Hunter測定中L值與a值皆以市售發酵乳顯著(p<0.05)比其他處理組為高、b值顯著(p<0.05)比其他處理組為低;而官能品評試驗中所有處理組之整體接受度大致相似。 本研究顯示經固定化之菌酛確能促進CLA生成,而四種不同凝膠固定化後,褐藻酸鈉固定化L. helveticus在pH 7下之總CLA生成量最高為584.80 μg,Hunter L,a,b值測定結果以市售發酵乳之L值與a值最高,b值最低(p<0.05),而官能品評試驗中所有處理組之整體接受度大致相似。 Conjugated linoleic acid (CLA) has many bioactivities related to the health, and linoleic acid can be promoted to convert into CLA by lactic acid bacteria. Lactic acid bacteria were observed to be capable of converting linoleic acid (LA) into CLA. However the yield was low. It is important to study how to improve the yield of CLA. Therefore, the research was performed using alginate, polyacrylamide, chitosan and carrageenan immobilized L. helveticus (BCRC14030) to convert LA into CLA at pH 5, 6, 7 and 8 in order to evaluate the effect of different gels and pH on CLA yield and the feasibility to produce CLA-rich fermented milk。 We used alginate, polyacrylamide and chitosan to immobilize L. helveticus after activation, and mixed 20 g of immobilized cell, 0.065 g of BSA and 0.01 ml of linoleic acid (LA) together for 120 hours at 37 ℃ to produce CLA; then use different ratios of alginate and carrageenan immobilized yogurt bacteria (YC380) with 2 % of L. helveticus (BCRC 14030) to make fermented milk。 Then we measured CLA content by HPLC and Hunter L, a, b value then sensory evaluation with appearance, smell, taste, thickness and total acceptability ratings were also determined. The results showed that when pH of alginate L. helveticus is 7, total CLA yield (584.80 μg) was significantly (p>0.05) higher than other pH and free cell groups. When pH of polyacrylamide immobilized is 6, total CLA yield (382.66 μg) was significantly (p>0.05) higher than other groups that pH are 5 and 8; when pH of chitosan immobilized L. helveticus is 8, total CLA yield (23.70 μg) was significantly (p>0.05) higher than other pH groups; when pH of carrageenan immobilized L. helveticus is 6, total CLA yield (71.56 μg) was significantly (p>0.05) higher than other pH groups that pH5, 8 and groups of free cell. When alginate and carrageenan immobilized yogurt bacteria and 2 % L. helveticus (BCRC 14030), total CLA yield was significantly (p>0。05) higher than other free cell; in Hunter L, a value, commercial yogurt products were significantly (p>0.05) higher than other groups, b value were significantly (p>0.05) lower than other groups, the acceptability ratings in sensory evaluation are almost the same. The results showed that immobilization can improve the yield of CLA, and cells immobilized by 4 kinds of gel, and when pH of alginate immobilized L. helveticus is 7, total CLA yield is 584.80 μg. In HUNTER L, a and b values of commercial yogurt products, a value was the highest, b value was the lowest (p>0.05), and in sensory evaluation, the acceptability ratings of all groups are almost the same.

Record ID 第95筆 System ID 094CMCH5513005
BCRC ID BCRC 14647, BCRC 10744, BCRC 10695, BCRC 14009, BCRC 14615, BCRC 11847, Public Year 94
Paper Name 菊糖有孢子乳桿菌潛在益生功效的研究 Studies on the Potential Probiotic Effects of Sporolactobacillus inulinus BCRC 14647
Student Name 陳佩玉
Teacher Name 曾政鴻
School Department Name 中國醫藥大學 營養學系碩士班 Academic Degree 碩士
Abstract 本研究目的在探討產孢性乳酸菌-菊糖有孢子乳桿菌Sporolactobacillus inulinus BCRC 14647對模擬人體腸道中不良環境因子之抵抗性以及其在腸道中定殖與拮抗病原菌腸炎沙門氏菌Salmonella enteritidis BCRC 10744之能力,同時探討該菌株對人體腸道細胞之安全性,並與Lactobacillus acidophilus、L. bulgaricus、Bifidobacterium bifidum、B. longum等市售益生菌產品常添加之乳酸菌就上述試驗性質進行比較。酸與膽鹽耐受性,分別是以pH 2∼4之酸液及0.1∼0.4%之膽鹽溶液試驗之,而模擬腸道吸附性則是利用Caco-2 cells作為吸附試驗模式,另外抑菌試驗方面包含了乳酸菌培養基上清液抑制S. enteritidis生長之抑菌圈試驗以及上清液或其菌體拮抗S. enteritidis對模擬腸道細胞之吸附試驗。結果顯示在耐酸性方面,孢子形態的S. inulinus存活率顯著高於營養細胞形態的S. inulinus及Bifidobacterium spp. (p < 0.05),而其中以Lactobacillus spp.的存活率最佳(p < 0.05),此情形於耐膽鹽試驗中亦可看到類似的結果。而在模擬腸道細胞吸附試驗,則發現營養細胞形態的S. inulinus (65.1%)、L. bulgaricus (78.7%)和Bifidobacterium spp. (74.9%, 52.7%)皆具有良好的吸附性,其中以L. acidophilus對Caco-2 cells呈現出強力的吸附性(92.3%),而孢子形態的S. inulinus則是低吸附性的(10.5%)。另外在抑菌圈之抑菌試驗方面,發現S. inulinus的培養基上清液可顯著地抑制S. enteritidis的生長,且其營養細胞或上清液亦具有干擾S. enteritidis吸附於模擬腸道細胞的能力,同時預先以S. inulinus吸附於模擬腸道細胞亦可預防S. enteritidis的吸附。此外,侵入性試驗也顯示了S. inulinus具有高度的安全性。本研究顯示,營養細胞形態之S. inulinus因能夠吸附於模擬人類腸道細胞並拮抗腸炎沙門氏菌而具有潛在之益生功效。 The basic characteristics of the spore-forming lactic acid bacterium (SFLAB), Sporolactobacillus inulinus BCRC 14647, associated with its potential probiotic effects were evaluated in vitro Assessments including acid and bile salt tolerance, adhesiveness, and antagonistic effects on pathogenic Salmonella enteritidis BCRC 10744, and an invasion assay were conducted using lactic acid bacteria (LAB) Lactobacillus spp (Lactobacillus acidophilus BCRC 10695, L bulgaricus BCRC 14009) and Bifidobacterium spp (Bifidobacterium bifidum BCRC 14615, B longum BCRC 11847) as a reference。 For the acid and bile tolerance test, the samples were treated with pH 2~4 acid solutions and 0.1~0.4% oxgall bile solutions, respectively. The adhesion test was conducted using Caco-2 cells as the assay model. For the antagonistic activity of LAB toward pathogen S. enteritidis, the well diffusion assay method was used, the inhibition effect of LAB cells or their spent culture supernatants (SCS) on the binding of the pathogen to the Caco-2 cells was explored. In the results, S. inulinus spores presented significant higher survival rates than the vegetative cell form in acidic conditions as well as than the reference Bifidobacterium spp. However, Lactobacillus spp. showed the highest viability among all tested strains. Similar results were found in the bile tolerance test. Compared to the reference strains, the vegetative cell form of S. inulinus possessed a proper adhesive characteristic (65.1% for S. inulinus, 78.7% for L. bulgaricus and 74.9% and 52.7% for B. bifidum and B. longum, respectively). In the adhesion assay, the spore form of S. inulinus displayed weak adhesive traits (10.5%). On the other hand, L. acidophilus showed a strong adhesive property (92.3%). The vegetative cells of S. inulinus and its SCS both dramatically reduced the adhesion of S. enteritidis to Caco-2 cells; meanwhile, the SCS of S. inulinus vegetative cells inhibited the growth of S. enteritidis in the inhibition zone test. From the results of the invasion assay, S. inulinus showed high safety properties. Based on the results of in vitro tests, S. inulinus shows the potential probiotic effects via the adherence to simulated human intestinal epithelial cells and the antagonistic activity against S. enteritidis. The vegetative cells of S. inulinus may be considered a candidate for the probiotic use.

Record ID 第96筆 System ID 095CHPI0015015
BCRC ID BCRC 17059, Public Year 95
Paper Name 以固定化菌株處理甲苯及乙酸乙酯
Student Name 虞佩瑾
Teacher Name 黃思蓴
School Department Name 中華大學 土木與工程資訊學系碩士班 Academic Degree 碩士
Abstract 就噴塗業來說,其製程中所產生之甲苯(T)和乙酸乙酯(E)皆為高生物分解性之揮發性有機化合物,理當適合採用生物方法來處理。然而,本實驗室先前的研究結果顯示,增加乙酸乙酯的添加量,甲苯的處理效率會受到抑制。因此,為了更確切地了解菌種間相互抑制的作用,本實驗將Rhodococcus sp. AC6(BCRC 17224)的衍生菌株A3(簡稱A3)、Rhodococcus sp. B5(BCRC 17223)的衍生菌株R3(簡稱R3)及Pseudomonas putida F1 (BCRC 17059)的衍生菌株G2(簡稱G2)等三種菌株,利用分開包埋與共同包埋等不同的包埋方法,來探討菌種間種種作用機制。其中分開包埋可以約束其菌量於一定比例,而共同包埋可任由共同包埋的菌株特性而增減。經由實驗結果發現,(1)基質適應性抑制,發生在反應的初期,R3和G2菌株對於甲苯的降解能力有限,但可以藉由重複批次培養來改善。之後的實驗,仍會有基質適應性的抑制。在甲苯/乙酸乙酯比(T/E比)<1的條件下,而甲苯的添加量相對於乙酸乙酯較低時,甲苯和乙酸乙酯兩者的降解速率都因為有機負荷增加會變慢。在T/E比>1的條件,由於甲苯的添加量相對於乙酸乙酯較高,此時基質適應性抑制的效應僅次於基質競爭抑制,因此對甲苯的降解效率不會因為進料形式的不同而有太大的差異。(2)基質競爭性抑制,發生在T/E比<1的條件下,尤其是在甲苯添加量相對較高時更加明顯。反之,在T/E比>1的條件下,乙酸乙酯的添加量雖然相較於甲苯較低,但仍然造成基質競爭性的抑制,在共同進料的條件下,對甲苯的降解速率會變慢。(3)有關族群間的協調與抑制,混合分開包埋A3+R3、A3+G2及A3+R3+G2菌株顆粒,分別以序列處理的方式進行代謝,所獲得的甲苯處理效率最佳。反之,進行平行處理的分開包埋R3 +G2菌株顆粒,因為共同競爭乙酸乙酯而抑制甲苯,所以甲苯的處理效率不佳。共同包埋之A3R3、A3G2及A3R3G2菌株顆粒比分開包埋時好,是因為基質競爭和取得食物的機率等二種效應來幫助各族群在序列處理污染物時獲得空間上的最有效利用。在基質競爭時,A3菌株會先降解乙酸乙酯,然後再換R3或G2等其它族群;或者在取得食物來源上,因為只有A3菌株會挑食,所以在族群上所佔的比例較小,使可以代謝甲苯的其它菌株變成顆粒中之優勢菌種,而不像分開包埋空間始終受限於1/2或1/3。

Record ID 第97筆 System ID 093HWAI7111009
BCRC ID CCRC 32068, Public Year 93
Paper Name 利用土麴菌移除水中重金屬最適化條件探討 Development of the treatment for the heavy metal wastewater removal by Aspergillus terreus
Student Name 廖宜亭
Teacher Name 孫逸民
School Department Name 中華醫事學院 生物科技研究所 Academic Degree 碩士
Abstract 目前利用傳統處理廢水的方法來移除水中之重金屬,仍然有許多缺點存在,然而在近代環境生物技術領域的發展下,處理水中重金屬方法則逐漸轉向尋找微生物來當做生物吸附材料用以移除水中的重金屬。本研究所採用之菌株是由食品工業發展研究所菌種保存中心所購得之18株Aspergillus terreus,首先由此18株菌中篩選出對金屬離子吸附能力較佳之菌株,接著進一步的研究其水中重金屬移除之最適化與探討複合金屬存在下之影響,最後進行再生研究以了解其重複使用之可行性。 利用Aspergillus terreus吸附各重金屬離子(Ni2+、Cr3+、Pb2+)試驗,均利用搖瓶震盪培養來進行。對於此18株菌中,我們篩選出其中的Aspergillus terreus CCRC 32068為本研究之菌株;對移除水中重金屬最佳操作條件實驗部分,針對Ni2+、Cr3+、Pb2+金屬離子之最適化條件(pH值、菌量、溫度、金屬濃度)探討,我們得到其最佳pH值分別為7、3、5,以不同菌體濃度吸附水中重金屬之最佳吸附效率皆為0.01g,最佳操作溫度皆為28℃,最佳金屬濃度吸附效率操作值皆為150 ppm;而就此操作條件下所進行之各金屬離子(Ni2+、Cr3+、Pb2+)之吸附效率分別為33.54、33.05、97.09(mg of metal/g of biomass)。此外於複合金屬影響試驗部分,我們發現其吸附能力為Pb2+>Ni2+、Cr3+。 利用Aspergillus terreus CCRC 32068菌株於重複使用的實驗上,我們發現本菌株經過5次的循環利用,其吸附效率仍可維持50%以上綜合上述結果,本研究顯示利用Aspergillus terreus CCRC 32068來移除水中酀金屬具有可行性,且值得進行後續之研究。 Conventional techniques commonly applied to remove heavy metals from wastewater, but these methods have several disadvantages. Recent developments in the field of environmental biotechnology include the search for microorganisms as sorbents for heavy metals. The Aspergillus terreus 18 strains used in this study was obtained from the Food Industry Research and Development Institute. From the Food Industry Research and Development Institute, we sift through one of the strains, which has better capacities to bind metal ions, and then go step further, development of the optimum to treatment for the heavy metals, in addition to evaluate of the metal ions mixture, and reuse of regenerated biomass in biosorption. Biosorption of each of the ions Ni2+、Cr3+、Pb2+ on Aspergillus terreus in a batch stirred system was investigated. From the Aspergillus terreus 18 strains. We selected the Aspergillus terreus CCRC 32068 strain in our study。 The study of the best operating condition, the influence of the solution pH, biomass dosage, temperature, metal concentration were studied. The optimum pH for biosorption of Ni2+、Cr3+、Pb2+ were found to be 7、3、5, respectively. Optimum biomass dosage was observed as 0.01g. The adsorption capacity of temperature, the optimum was determined as 28℃, and the optimum of metal concentration was 150ppm. In the operating condition, the Aspergillus terreus CCRC 32068 strain adsorbed metal ions in the order of Ni2+、Cr3+ and Pb2+, with the biosorption capability of 33。54、33.05、97.09(mg of metal/g of biomass). Experiments were also conducted to evaluate the interactive biosorption of nickel、chromium and lead ions, and the Aspergillus terreus biomass biosorbed the heavy metals in the following order:lead > nickel、chromium. Aspergillus terreus could be used for five cycles of biosorption-elution of biosorbed ion-regeneration for biomass. This research showed that Aspergillus terreus CCRC 32068 biosorption had a potential to be used in the removal of heavy metal ions from water

Record ID 第98筆 System ID 094YZU05159034
BCRC ID BCRC 31996, BCRC 31499, Public Year 94
Paper Name 以微生物生產葡萄糖胺之動力學研究 Production Kinetic of Glucosamine by Microorganism
Student Name 謝睿文
Teacher Name 吳和生 王孝憲
School Department Name 元智大學 化學工程與材料科學學系 Academic Degree 碩士
Abstract 葡萄糖胺(glucosamine)為一種單胺醣類物質,是構成幾丁質(chitin)和幾丁聚醣(chitosan)之單糖;為人類關節間潤滑結締組織之主要成分,具修復、補充、滋潤、再造受損的軟骨,可治療退化性關節炎。本研究主要是利用微生物醱酵生產葡萄糖胺,討論製備葡萄糖胺的可行性以及分離純化葡萄糖胺。使用四種菌株: Rhizopus oligosorus BCRC 31996; Monascus purpures BCRC 31499; Monascus pilosus BCRC31527; Aspergillus sp. BCRC31742,實驗主要分成三部份討論:(1)分析純化,分析方式為利用1-naphthyl isothiocyanate為衍生試劑,衍生反應在50°C下反應1小時,以吡啶為反應溶劑,衍生物利用逆向液相層析分離管柱分離,以230 nm 紫外光檢出器偵測生產之葡萄糖胺含量,其精密度可達1.69%、準確度可達2.73%; (2)以搖瓶培養探討培養條件,如菌種種類、培養基、酸鹼度、碳氮比等等,菌種產量最高為Aspergillus sp. BCRC31742培養於葡萄糖胺(glucose)和蛋白質(peptone)複合培養基(3430 mg/L),在培養條件下以酸鹼度影響最大;(3)以4公升醱酵槽進行培養,探討變因有酸鹼度、轉速、碳氮比,實驗中使用Aspergillus sp. BCRC31742培養於葡萄糖胺和蛋白質複合培養基中,可獲得葡萄糖胺濃度為2310 mg/L、生物量為10.1g/L、含量為229mg/g biomass、產率為92.4mg/g carbon source、生產力13.8mg/L×h;(4)純化方式使用Aspergillus sp. BCRC31742醱酵所得之菌塊經6N HCl在100oC下反應24小時,以NaOH中和至pH 7,使用活性碳除色,再由減壓濃縮機濃縮成葡萄糖胺鹽酸鹽濃縮液,經烘箱烘乾後,其純度由原來2.29 %提升至13.4 %。 Glucosamine is an amino-monosaccharide and one of the basic constituents of Chitin and Chitosan. Glucosamine is the major component of human inter-articular lubricant connective tissue. It can repair and rebuild the damaged cartilage and it also can cure the disease of osteoarthritis. The objective of this work is to study the parameters affecting the production of glucosamine hydrochloride by microbial fermentation and to obtain the method of separation and purification of glucosamine hydrochloride.This study uses four funguses Rhizopus oligosorus BCRC 31996, Monascus purpures BCRC 31499, Monascus pilosus BCRC31527 and Aspergillus sp BCRC31742 to product glucosamine hydrochloride by using fermentation。Three parts were discussed in this study.First is the kinetic and strategy by flask culture which conditions include kinds of fungus, mediums, pH value, and carbon and nitrogen source.The experimental result shows that the glucosamine concentration had an optimum value and was 3428mg/L by using Aspergillus sp. BCRC31742 culture in GP medium, the pH controlled an important rate in culture。 Second, the kinetics and strategoy by fermenter culture that the factors were pH value, incubation time and carbon and nitrogen source in the 4L fermentor fermentation.The result shown the glucosamine concentration was 2311mg/L; biomass, 10.1g/L; content, 229(mg/g biomass); yield, 92.4mg/g of carbon source; productivity, 13.8mg/L×h-1 that Aspergillus sp. BCRC31742 was incubated in GP medium。 Third is the part of analysis and purification of glucosamine. The glucosamine was analyzed with 1-naphthyl isothiocyanate (NITC) as derivatizing agent. The reaction was carried out in pyridine at 50oC for 1 h. The derivative was analyzed of High Performance Liquid Chromatography. The precision of glucosamine is below to 1.69% and the accuracy is to 2.73%. Purify method is the dry cell reacting with 6N HCl at 100oC for 24 h, then neutralization with NaOH to pH 7 to obtain glucosamine hydrochloride aqueous solution. The purity of glucosamine is from 2.29% to 13.4% after the purification process containing depigmentation and condensing by Rotary Evaporator.

Record ID 第99筆 System ID 093YZU00063057
BCRC ID CCRC 14365, Public Year 93
Paper Name 高濃度酚在微孔性中空纖維模組中生物降解之程序與動力學 Biodegradation Process and Kinetics of High-Strength Phenol in Microporous Hollow Fiber Modules
Student Name 鍾翠萍
Teacher Name 莊 瑞 鑫
School Department Name 元智大學 化學工程學系 Academic Degree 博士
Abstract 實驗細菌Pseudomonas putida CCRC 14365經培養後,自五個不同的生長時期(遲滯期、對數期、後指數期、靜止期及死亡期)收集作實驗,量測各生長期細菌之比生長速率及酚之比降解速率。結果發現細胞經由100 mg/L的酚活化誘導後,能在15至26小時內完全降解100 mg/L的酚,若未經酚化合物的誘導,則需23至33小時才能完全降解。再者,從後指數期收集的細胞,不論是否經過誘導適應過程,均能最有效率的完全降解酚化合物。與未受誘導的細胞相較之下,經過誘導的細胞雖然比生長速率較大,其比降解速率卻較小。也就是說,實驗前經過受質誘導的細菌能長出更高密度的細菌生物量,從而加強分解效率,儘管比降解速率並不高。 有關P. putida CCRC 14365在懸浮態系統中降解酚化合物及細胞生長動力學的情況,由於存在基質本身的毒性抑制效應,懸浮態細胞完全降解酚濃度大約到400 mg/L,最高能忍受約600 mg/L,但無法完全降解。在動力學方面,懸浮態細胞降解酚有基質抑制的現象,在酚濃度25-600 mg/L下,求得Haldane model之動力參數值。由於使用傳統的Haldane model預測批次反應中的細胞生長量及酚降解程序,其準確度不理想。於是進一步推演出Two-phase model,它植基於Haldane model,以細胞生長期中的兩個時期,包含遲滯期及對數期的代謝生長模式。它的有效性經由實驗數據驗證,結果顯示本研究所開發的Two-phase model可以更準確的預測批次反應中的細胞生長量及酚降解動態,特別是亦涵蓋了遲滯期轉變到對數期之間的過渡狀態,解決了使用傳統Haldane model的最大困擾。 比較P. putida CCRC 14365在懸浮態系統及藻酸鈉固定化系統中降解酚化合物的情況,發現兩個系統在酸鹼值(pH)及溫度效應的趨勢相似。由於基質的毒性抑制效應,藻酸鈉固定化系統雖然降解速率較低,但可忍受酚的濃度高達1000 mg/L。固定化細胞在85-400 mg/L酚濃度範圍,能看到降解過程之中間產物(鄰苯二酚),但是在懸浮態及較低與較高濃度之固定化實驗下均不明顯。推論酚傳輸進入藻酸鈣固定化材質時,受到擴散延遲的影響,這可能有助於中間產物的探求。 建立微孔性中空纖維膜生物反應器模組 (HFMBR) 以探討P. putida對酚之生物降解,模組中的聚丙烯纖維先經過乙醇潤濕為親水性膜。主要探討流速、不同酚濃度及分散劑(四鈉焦磷酸鹽)等對降解速率及細胞生長之影響。結果顯示,當細胞被中空纖維膜固定化及與廢水端分開時,實驗一開始約10%的酚就被中空纖維吸附,P. putida細胞能在92小時內完全降解3000 mg/L的酚。四鈉焦磷酸鹽分散劑經酚降解及電子顯微鏡檢實驗證實,其對生物反應器中殼側細菌端控制薄膜上生物積垢有明顯的效果,減少生物膜上的積垢可以加速酚的降解效率。在中空纖維生物反應器中降解的過程發展,將依不同濃度範圍分階段充分討論。在高初始酚濃度的降解反應中,酚通過薄膜的質傳並非限制步驟,伴隨細胞生長的降解反應才是其限制步驟。為瞭解中空纖維膜生物反應器模組中酚的傳送,算出從管側穿過薄膜至殼側酚的總質傳係數的理論值及實驗值。建立中空纖維生物反應器的生物膜質傳模式,以零次方平板模式(zero-order flat sheet model) 預測反應器的去除率其修正係數 R2 可達0.94。對這個零次方平板模式各個重要參數作敏感度測試,發現去除率與生物膜的相關參數有很強的函數關係,尤其是當生物膜的生物量密度及擴散係數下降時,去除率會呈現線性下降的趨勢。 當以懸浮態、藻珠固定化及中空纖維固定化三個反應系統作比較時,在較低的酚濃度(< 400 mg/L),基質抑制不嚴重的情況下,以懸浮態系統處理含酚廢液較有利。然而,只有固定化系統能忍受高於600 mg/L的酚濃度,特別是在高於1000 mg/L 酚濃度時,這套中空纖維生物反應器中的固定化細胞,能先將酚降解至可忍受的基質濃度,接著伴隨著反應器中快速生長的懸浮態細胞,可以更快速地將酚完全降解。中空纖維生物反應器系統同時具有基質分配控制能力及含有提高耐受力的生物膜,這兩種功能使HFMBR有效率的降解高濃度的酚有機污染物。 The specific growth rates of Pseudomonas putida CCRC 14365 abstracted from five different phases (the lag, log, late-exponential, stationary, and death), along with its specific rates for phenol degradation, were determined。 The cells harvested from the late-exponential phase were the most effective for complete consumption of phenol. Phenol degradation by P. putida CCRC 14365 and cell growth kinetics were detected in the free suspension systems。 Due to the substrate inhibitory effect, the free cells could completely degrade phenol only up to about 400 mg/L within 43 h. However, free cells have poor degradation efficiency when initial concentration up to 600 mg/L. The growth kinetics of free cells for degradation of phenol in the concentration range 25~600 mg/L was described by the Haldane model. A simple two-phase model, originated from the Haldane model, was presented to predict the behavior of batch culture operations. The model was based on the two regions of metabolic activity: the lag phase and the log phase. In contrast to the one-phase Haldane model, it was demonstrated that the proposed two-phase Haldane model much better predicted the dynamics of biomass growth including the transient region from the lag to the log phases. Phenol degradation by P. putida CCRC 14365 were compared between the free and Ca-alginate gel-immobilized systems。 It was shown that the trends of the effects of pH and temperature on phenol degradation were similar for both free and immobilized cells. Due to the substrate inhibition effect, the free cells could degrade phenol only up to about 600 mg/L. The immobilized cells could tolerate a higher level up to 1000 mg/L, though the degradation rate was slower. Unlike the case of free cells, an intermediate catechol was detected using the immobilized cells at a phenol level of 85~400 mg/L. This implied that the occurrence of medium diffusion resistance in the immobilized systems, which retarded the degradation reaction, might be useful for detection of the intermediates. The degradation of phenol (100-3000 mg/L) by the cells of P. putida CCRC 14365 in a microporous hollow fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were pre-wetted with ethanol。 The effects of flow velocity, pH, the concentrations of phenol and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were focused. It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process. The cells of P. putida fully degraded 3000 mg/L of phenol within 92 h when the cells were immobilized and separated by the fibers. SEM studies showed that the biofilm become thinner on addition of TSP. The effect of thinner biofilm company with more free cells resulting from TSP addition is more advantageous for biodegradation. The process development in HFMBR system was discussed. Judging from the high residual phenol concentration in shell side of HFMBR, it obvious that the mass transfer of phenol across the membrane was sufficient to supply the carbon source to the microorganism and that the bacterial growth was the limiting-step for phenol biodegradation. The mass transfer of phenol across the membranes was estimated by theoretical and experimental study. Biofilm model of the HFMBR were created. The zero-order flat sheet model fit the data well, correlation coefficient R2 = 0.94. Sensitivity analysis of the zero-order model indicated that removal was a strong function of the biofilm phase. It is especially for biomass density and also of the biofilm diffusion coefficient, with both values downward resulting in linear decreased removal rates. When comparing with free and Ca-alginate gel-immobilized systems, it was more advantageous to treat the solution in a suspended system at relatively low phenol levels (< 400 mg/L), where substrate inhibition was not severe. However, only immobilized cells can tolerate higher phenol levels (> 600 mg/L). Especially higher than 1000 mg/L phenol levels, HFMBR system, which combined the functions of substrate partition control and the tolerance enhancement by biofilm, could be applied to degrade phenol down to a tolerable concentration with weak substrate-inhibition, and the followed degradation alternately accompanied by a suspended culture would result in larger degradation rate.

Record ID 第100筆 System ID 091YZU00063044
BCRC ID CCRC 17015, Public Year 91
Paper Name 利用脂肪酵素進行甘油三丁酸酯之水解反應 Hydrolysis of Tributyrin using Lipase
Student Name 蔡明儒
Teacher Name 吳和生
School Department Name 元智大學 化學工程學系 Academic Degree 碩士
Abstract 脂肪酶(Lipase,EC 3.1.1.3)主要是水解三甘油脂成甘油及脂肪酸。廣泛地應用於脂肪、油品、食品及清潔劑工業。大多數的脂肪酶為適中溫性的酵素,無法水解在常溫下為固體的受質及對化學變性劑較無抗性,因此在工業應用上有所限制。但因其催化作用通常伴隨著高特定選擇和高鏡像選擇性,所以在生物技術上反而擁有巨大發展潛力。因此本實驗利用磁石攪拌器乳化油-水反應液以研究從 Pseudomonas fluorescenes CCRC 17015所的抽取的粗製脂肪酶,在批式反應器內催化甘油三丁酸酯的水解反應速率,並探討基質濃度、溫度、pH值、轉速等因素對水解速率之影響。而為了增加酵素的實用性,本實驗以多孔隙性的甲基丙烯醯胺共聚物為載體,此載體經由乙二胺改質、戊二醛活化後,做為酵素固定化的材質,並利用共價結合的方法,將鍵酵素結以形成固定化酵素。對此,固定化酵素以溫度、pH、轉速等不同的操作條件加以探討,並且計算出Km、Vmax。實驗結果顯示,脂肪酶固定化後擁有良好的熱穩定性與耐鹼性。 The primary purposes of lipase is use to hydrolyze triacylglyceol becomes the glycerol and the fatty acid. This enzyme has been widely used in fat, Food ingredients and Detergents industry. Most of lipases are mesophilic enzyme, which can’t hydrolyze substrate that exists in solid form at room temperature and are unstable when chemical solvents are present. This has severely restricted the application of lipase in industries. But in this catalysis reaction, it came with substrate specificity and enantioselectivity. Therefore, the the lipase is one of the most potential in biotechnological. The purpose of this study is research the hydrolysis rate of tributyrin in batch reactor use crude lipase from Pseudomonas fluorescenes CCRC-17015 and using magnetic stir reactor to emulsify oil-water phase。 In order to increase the usability of enzyme, the study use lipase immobilization onto porous polymethyacrylamide by covalent fixation. The carrier modified with ethylenediamine and activated by glutaraldehyde. The primary purposes of this thesis are to study the effect of hydrolysis rate at different substrate concentrations, temperature, pH and stirring speed. Final, to determine the operating conditions of free enzyme and immobilized enzyme then we can get kinetic parameters Km and Vmax values. The experimental results showed lipase after immobilization own good thermal stability and greater stability at higher pH value.

Record ID 第101筆 System ID 083TMC03534002
BCRC ID Public Year 83
Paper Name 靈芝屬三帖類成分之分佈模式與對肝功能及HL-60細胞之影響 Distribution and pattern of Triterpenoids in the Genus Ganoderma and the Physiological Activities on Hepato-protection and HL-60 Cell Line
Student Name 楊依珍
Teacher Name 蘇慶華
School Department Name 台北醫學大學 醫學研究所 Academic Degree 碩士
Abstract 靈芝屬內已命名者超過50種,其形態類似,不易以簡單方法鑑別之。本論文嘗試以簡便快速之HPLC及TLC方法分析源自於CCRC及農試所等不同國家之靈芝標本約64個;其學名分別為:G. neo-japonicum, G. formosanum, G. australe, G. calidophilum, G. mastopolvm, G. webenanum, G. pfeifferi, G. resinaceum, G. lucidum, G. subamboiaefise var. laevisponim, G. boniense, G. tropicum, G. fornicatum, G. tsugae, G. curtisli, G. lobatum, G. mirabile, G. oerstedii由其酒精萃取物中,以靈芝酸B及C2 為標準品,歸納成為18種類型,此18種類型與臺灣省農試所值病系之形態及雜交可孕性試驗吻合。同時由於靈芝酸B,C2為已知具肝臟保護作用之三帖類純化成分,利用HPLC分析而得知各種類型靈芝中靈芝酸B,C2含量情形;並以四氯化碳誘發小白鼠急性肝障礙之模式,以GOT,GPT及組織切片評估各種類型靈芝三帖類排除急性肝障礙之效果;結果顯示,相同劑量(30mg/kg老鼠體重)之含三帖類萃取物投與小白鼠3劑後,以G. tropicum排除急性肝障礙效果最佳。另外,以人類急性前骨髓白血病細胞株(HL-60 Cell)為體外治療血癌之模式,了解各種類型靈芝三帖類對於HL-60細胞分化作用及分裂作用之影響;在分化方面,進行血球功能特性包括吞噬作用,NBT還原作用之評估,細胞表面抗原之偵測,及細胞形態之觀察;在分裂方面,測試靈芝三帖類對於細胞數,細胞存活率,增殖能力之影響,同時了解靈芝三帖類對於HHL-60細胞之細胞週期(Cell Cycle)之影響。首先利用流動細胞分析儀(Flow Cytometer)篩檢以18種類型靈芝酒精萃取物50μg/ml,0.5μg/ml,0.005μg/ml處理5天後之細胞吞噬能力,同時以NBT還原作用確定各種類型靈芝酒精萃取物50μg/ml,5μg/ml,0.5μg/ml,0.5μg/ml處理5天後細胞分化情形;發現 G. weberianum (F, 50 μg/ml),G. lucidum (I,50 μg/ml ), G. lucidum (K, 50 μg/ml), G. tsugae (O, 50μg/ml),G.tsugae(P, 50μg/ml), G. lobatum( Q, 5μg/ml, 0.5 μg/ml),G. mirabile(R, 5μg/ml,0.5μg/ml)與未處理之細胞比較有顯著差異;再利用流動細胞分析儀偵測細胞表面CD標記CD11b,CD14百分比,以得知分化成熟之細胞百分比,結果顯示G. weberianum(F, 50μg/ml)具有輕微促進HL-60細胞分化為顆粒性白血球之效果(p<0.05):而G. lucidum(K,50μg/ml),G. tsugae(0,50μg/ml), G. lobatum (Q, 5μg/ml, 0.5μg/ml)具有輕微促進 HL-60細胞分化為單核球之效果(p<0.05)。此外,G.resinaceum (G, 50μg/ml), G. mirabile (R, 50μg/ml)顯著降低細胞存活率、增殖能力及細胞數目。探討 G. resinaceum(G, 50μg/ml), G. lucidum (K, 50μg/ml), G. tropicum (M, 50μg/ml)對於HL-60細胞週期之影響,亦發現某些靈芝含三帖類酒精萃取物似乎其有細胞毒性 (Cytotoxic activity)。 To distinguish among the known over 50 recorded species in the genus Ganodenna by morphological characters of either mycelia or fruiting bodies usually leads to an ambigous result. In the present study, 64 strains of Ganoderma from Culture Collection and Research Center (CCRC) and the fruiting bodies cultivated by Taiwan Agricultural Research Institute (TARI) were used for analysis by HPLC and TLC。 The taxon of the strains include: G. neo-japonicum, G. formosanum, G. australe, G. calidophilum, G. mastoporum, G. weberianum, G. pfeifferi, G. resinaceum, G. lucidum, G. subamboinense var. laevisporum, G. boniense, G. tropicum, G. fornicatum, G. tsugae, G. curtisii, G. lobatum, G. mirabile, and G. oerstedii. Ethanol extracts of these fruiting bodies were used for the analysis and ganoderic acid B and ganoderic acid C2, both known as hepato-protective triterpenoids, were employed as external standards for the analysis. From the patterns of HPLC and TLC, 18 groups (A to R) were classified with the contents of ganoderic acids B and C2 determined in each group and the result was in good agreement to that from inter-fertility test carried out by TARI. Carbon tetrachloride (CCl4) induced liver damage in mice was used as an animal model to evaluate hepato-protective effect of the three doses of the 18 ethanolic extracts (total 30 mg/kg of mouse) orally administrated with an interval of 4 hours. It indicated that the extract of M group (G. tropicum ) manifested the strongest effect by lowering SGOT and SGPT values and also showed prominent repair effect to the hepatocytes around central veins. HL-60 cell line, an acute leukemic cell culture was employed as an in vitro model for the effect of the triterpenoid containing extracts on cell differentiation and proliferation. The cell behaviours, including phagocytosis, NBT reduction, surface marker, change in morphology, cell growth curve, survival rate, cell proliferation and cell cycle of HL-60 , with retinoic add as positive control, were observed or analyzed by microscopy or flow cytometry. It revealed that in the treatment of G. weberianum (F, 50 μg/ml), G. lucidum (I, 50 κg/ml) G. tsugae (O, 50 κg/ml), G. tsugae (P, 50κg/ml), G. lobatum (Q, 0.5 &: 5 κg/ml) and G. mirabile (R, 0.5 & 5 κg/ml) showed significant enhancement of phagocytosis and NBT reduction. Further study on expression of myeloid-spedfic differentiate antigens (CD11b and CD14 ) indicated the extract of G. weberianum (F, 50 κg/ml) slightly induced HL-60 cells to differentiate to mature granulocyte and G. lucidum (K, 50 μg/ml), G. tsugae (O, 50 μg/ml), G. lobatum (Q, 0.5 & 5 μg/ml) also mildly (p<0.05) induced HL-60 cell to differentiate to monocytes. In addition, the extracts of G. resinaceum (G, 50 μg/ml) and G. mirabile (R, 50 μg/ml) strongly suppressed the survival rate and proliferation. The extracts of G. resinaceum (G, 50 μg/ml), G. lucidum (K, 50 μg/ml) and G. tropicum (M, 50 μg/ml) severely changed cell behaviour during the stages of cell cycle. Certain compounds in the crude extracts are proposed to be responsible for cytotoxicity to the cell line.

Record ID 第102筆 System ID 086TMC00339008
BCRC ID CCRC 32219, CCRC 32220, CCRC 36421, CCRC 32221, CCRC 31900, CCRC 31775, CCRC 36124, CCRC 36159, Public Year 86
Paper Name 利用核醣體DNA的朮轉錄區鑑定蟲草屬真菌 Identification of fungi in the genus Cordyceps by ribosomal DNA internal transcribed spacers (ITS)
Student Name 趙書慶
Teacher Name 蘇慶華
School Department Name 台北醫學院 細胞及分子生物研究所 Academic Degree 碩士
Abstract 近年來國內由於藥膳與食療風氣漸開,珍貴之中藥-冬蟲夏草(Cordyceps sinensis) 其市場需求亦逐漸增大,目前冬蟲夏草之新種仍陸續被發現,其中已有記載者達282種 ; 基本上蟲草屬(Cordyceps)之鑑定乃是依菌種之形態來分類的,然而菌種在一般培養基 時不易產生子座等可作為鑑定依據的特徵,且亦常有健康食品以乾燥的醱酵菌絲經研磨加 工後之型式販售,因此鑑定此些菌種時會受到許多限制。因而本研究基於上述之原因,期 能以rDNA之片段進行蟲草屬中各種(Species)之鑑定。 於本研究中已成功地建立了由蟲草屬真菌抽得高純度、高產量Genomic DNA的方法,且可 順利地利用聚合脢連鎖反應法及兩套普遍性引子(ITS1 & ITS2、ITS3 & ITS4) 增幅得 到C. memorabilis CCRC(Culture Collection and Research Center)32218、C. milit aris CCRC 32219、C. ophioglossoides CCRC 32220、C. sinensis CCRC 36421、C. sp. CCRC 32221、C. sinensis(西藏野外採集)、C. nutans(日月潭野外採集)、C. myrme cophila(日月潭野外採集)、傳統中藥店之冬蟲夏草藥材、Phytocordyceps ninchukisp ora CCRC 31900、Claviceps purpurea CCRC 31775、Ganoderma lucidum CCRC 36124、 Ganoderma pfeifferi CCRC 36159等菌種的兩段以核醣體DNA之內轉錄區(Internal Tran scribed Spacer,簡稱ITS)為主的DNA片段(ITSI & ITSII),之後我們從所得到的DNA 片段之大小來進行分析,發現到各菌種間的差異並不具有分類學上的意義,故以聚合脢連 鎖反應產物之大小來作為分類標準的方式並不可行;接著乃是將此些菌種的ITSI及ITSII 之核酸序列互相比對,首先與發表於GenBank的G. lucidum及G. pfeifferi之ITSI及ITSII 的核酸序列比對後,證實了經由本研究所建立的系統得到的菌種之核酸序列的可信賴度, 且發現到屬於C. sinensis的同種菌株間其ITSI及ITS片段之核酸序列的相似性高達99.5 % 、99.7 %;且同時根據種源關係樹與自西藏4000公尺以上的草原中採集所得的C. sinensi s比對後,我們發現食品工業發展研究所之菌種保存及研究中心的C. sinensis有可能是與 C. capitata於種源關係上較近的菌種,而非其所聲稱的C.s sinensis;同時我們亦利用 此種方法發現到,自野外採集所得的Wild specimen-3可能即是C. militaris的無性世代 ,而我們也成功地將此套系統實際應用於了解如市售商品中所加入的菌絲成份其品種及真 偽等方面。 Fresh specimens of Cordyceps sinensis collected from Tibet ( Wild specimen-4 ) were compared with the special nuclear ribosomal DNA ( rDNA ) fragments to t hat of other fungi including C. memorabilis CCRC ( Culture Collection and Rese arch Center ) 32218、C militaris CCRC 32219、C ophioglossoides CCRC 32220、C sinensis CCRC 36421、C。 sp. CCRC 32221、C。 nutans ( collected in the field; Wild specimen-2 )、C. myrmecophila ( collected in the field ; Wild specimen -1 )、dried specimen of C. sinensis from Chine herb store ( Dongchongxiachao-1、 Dongchongxiachao-2 )、Phytocordyceps ninchukispora CCRC 31900、Claviceps purpu rea CCRC 31775、Ganoderma lucidum CCRC 36124、Ganoderma pfeifferi CCRC 36159。. ....etc. The method of DNA extraction from cultured mycelia or dried specimens of thefungi was firstly studied to establish a stable procedure for high quan tity and purity of genomic DNA. It was found that a modified rapidpreparation method was the most efficient one. Then , the ITSI and ITSII DNAregions were f urther amplified by polymerase chain reaction ( PCR ) with twopairs of fungal universal primers ( ITS1 and ITS2 , ITS3 and ITS4 ). Sequence analysis of the amplified DNA were followed and the data from G.lucidum ( CCRC 36124 ) and G pfeifferi ( CCRC 36159 ) were confirmed as ITSIand ITSII of them in 99。5 - 100 % match by the published data of GenBank. Thepercentages of sequence similari ty analyzed by DNASTAR revealed that ITSIand ITSII of C. sinensis derived from fresh specimens and the dried specimensfrom chinese herb store were 99.5 % an d 99.7 % , respectively. In addition , the results also indicated that the C. sinensis ( CCRC 36421 ) could be morerelated to C。 capitata ( ITSI→81.5 % , I TSII→95.8 % ) rather than C. sinensis( Wild specimen-4 ) ( ITSI→71 % , ITSII →81.5 % ) and the specimen ( Wild specimen-3 ) could be the anamorph of C. mi litaris. According to the similarity of the ITSI and ITSII sequence , a prelim inary phylogenetic tree was proposed to explain the possible distance of evolu tion in the genus of Cordyceps when the key of Kobayasi ( 1982 ) was compared. The methods and the results of present study can be useful in determining the Cordyceps species in anamorphic state or the commercial Cordyceps product in pulverized mycelial form , since two commercial products ( CP-1 , CP-2 ) were found to be C. sinensis ( CCRC 36421 ) and C militaris ( CCRC 32219 ) , respe ctively

Record ID 第103筆 System ID 087TMC00339009
BCRC ID CCRC 36147, Public Year 87
Paper Name 靈芝三類成分對蛋白質水解酵素抑制機轉及抑制HeLa細胞生長之研究 Triterpenoids from Ganoderma resinaceum used as proteinase inhibitors and a growth inhibitor of HeLa cells
Student Name 謝翊翎
Teacher Name 蘇慶華
School Department Name 台北醫學院 細胞及分子生物研究所 Academic Degree 碩士
Abstract 由以前實驗證明Ganoderma resinaceum ( CCRC 36147 )在65種靈芝中,具有最豐富之三類類型,並於細胞實驗中證實對HL-60細胞具有Apoptosis作用。本研究延續此一結果針對G. resinaceum的三類酒精萃取物,進行蛋白質水解酵素的抑制物之篩選。其三類酒精萃取物,分別做Collagenase inhibitor assay 、Chymotrypsin抑制活性測試,並以HeLa cell line做細胞實驗,篩選出有效成份。每一成分別經由MPLC及TLC將其粗分離,再經由PLC純化,找出最後最有效的單一成分。實驗結果可知由G. resinaceum的酒精萃取物由MPLC及TLC粗分離成4個部分,最有效成分為T組,再由PLC純化成8個成分,最有效為T-2組,再由T-2 組純化出T-2-1為有效成分及再次純化出T-2-1-1為T組中單一有效成分。T-2-1-1對細胞存活率有明顯的抑制其存活,在流式細胞儀實驗中,看出加入T-2-1-1其細胞碎片增加至73.59%,且細胞型態變得比較不規則。在in vitro assay中,T-2-1-1對Collagenase抑制其鈄率為-1.9E-6 ( Negative contorl 為0.86E-6 )明顯有抑制效果;在Chymotrypsin抑制活性測式方面,T-2-1-1其水解圈為0.4cm ( Negative control為0.9cm )有明顯的抑制效果。最後推測T-2-1-1的結構為一個27個碳之三類物質。經由本研究結果證實;此一三類能當蛋白質水解酵素的抑制物,將大量收集,做為腫瘤細胞之抑制物質之研究。 Previous study on survey of 65 ganoderma fruiting bodies demonstrated that alcoholic extract of G. resinaceum (CCRC-26147) enhanced apoptosis of HL-60 cell lines。 The present study has focused on the proteinase inhibition activity against collagenase and chymotrypsin of this species as well as a growth inhibitor of HeLa cell. Fruiting body ( 10 kg ) of G. resinaceum was extracted and fractionation into four groups ( C, B, M, and T ) by MPLC system, in which only T fraction showed inhibitory on the assays. Then, T fraction was further separated into T-2, T-2-1, and T-2-1-1 by preparative TLC. All these fractions demonstrated strong inhibitory activity on collagenase, chymotrypsin and cell growth of HeLa cell line. NMR spectrum suggested T-2-1-1 was C27 triterpenoid related to lucidenic acid in literature. The result indicated that triterpenoids in G. resinaceum could be a potential drug for the treatment of carcinoma.

Record ID 第104筆 System ID 094SCU05381007
BCRC ID BCRC 36319, Public Year 94
Paper Name 探討白腐真菌之錳離子過氧化酵素於液態和半固態環境下的最適生產條件 Optimization of Manganese Peroxidase Production by White Rot Fungi During Liquid and Semi-Solid State
Student Name 游凱翔
Teacher Name 趙維良
School Department Name 東吳大學 微生物學系 Academic Degree 碩士
Abstract 白腐真菌能產生三種主要的胞外木質素分解酵素:漆氧化酵素,木質素過氧化酵素以及錳離子過氧化酵素。錳離子過氧化酵素為具有高氧化還原電位,是一種帶有亞鐵離子的的血紅素醣蛋白分子,能應用於許多頑強污染物的氧化與降解,例如多環芳香碳氫化合物、偶氮染料。 本篇研究從菌種中心、林業試驗所取得和本實驗室篩得一共八株白腐真菌 (BCRC 36319、TFRI 20、TFRI 31、TFRI 45、TFRI 554、TFRI 691、TFRI707、F5)。針對此八株菌的錳離子過氧化酵素 (MnP) 進行初步的酵素產量分析,以低氮源 (1.2 mM)的無機鹽培養基培養篩選出TFRI 20、TFRI 554及TFRI 691此三株具較高錳離子過氧化酵素之菌株。在高/低氮源的培養實驗中,高氮源培養基 (含有3 g tryptone peptone)對於錳離子過氧化酵素具有抑制的效果。另外針對在不同時間點各菌株的MnP活性進行測試,最高活性出現在培養後第4至6天,TFRI 20的 MnP活性為13631 U/L,TFRI 554為21931 U/L,TFRI 691為37619 U/L。TFRI 554與TFRI 691在後續不同氮源種類培養的實驗中,TFRI 554的MnP活性受到氯化銨以及硝酸鉀的抑制,而TFRI 691的MnP在酒石酸銨及硝酸鉀的環境下都有高量的活性表現(約為6,0000 U/L),但仍會受到氯化銨些微的影響。更換不同pH值的酵素分析試劑實驗中TFRI 554及TFRI 691兩者的MnP活性皆在pH 6下有最佳的反應活性,隨著反應環境越趨酸性,酵素活性則逐漸減弱。而在不同溫度測試中TFRI 691的MnP在40℃及45℃下加熱一小時後酵素活性有些微的提高,但兩株菌的MnP對於50℃的高溫並沒有明顯的耐受性,處理三小時後僅剩餘40%~50%的活性。不論添加少量的碎稻桿(0.5% W/V)或veratryl alcohol (1mM、2mM),這些誘導物都能顯著的提升TFRI 554及TFRI 691的MnP產量,但過量的veratryl alcohol (>2 mM)則反而會抑制MnP的生成。TFRI 554與TFRI 691兩株菌在半固體培養實驗中均有很好的MnP產量,甚至比一般的液體震盪培養還要來的高(74700 U/L; 45553 U/L),而通氣培養則能維持MnP的活性穩定,持續到培養後第18天。TFRI 554與691的MnP經由SDS-PAGE及Native PAGE分析後發現具有不同的酵素圖譜。TFRI 554的錳離子過氧化酵素具有蛋白質條帶,大小分別為65 kDa、40 kDa、35 kDa;TFRI 691的錳離子過氧化酵素三個蛋白質條帶,大小分別為40kDa、38 kDa、35 kDa。 White-rot fungi is the most common degrader in lignin degradation. They produce three major extra cellular lignolytic enzymes-laccase, lignin peroxidase and manganese peroxidase. Manganese peroxidase (MnP) is a heme containing glycoprotein which has a high redox potential, could be applied in oxidizing and degrading many recalcitrant pollutants such as PAHs (polycyclic aromatic hydrocarbons), azo dyes. There are eight isolates were screened for the production of MnP. TFRI 20, TFRI 554 and TFRU 691 cultivated in low nitrogen phosphate (1.2 mM) buffer medium has the highest MnP activities (13631 U/L, 21931 U/L, 37619 U/L, respectively). In the later experiment different nitrogen sources were applied in cultivation with TFRI 554 and TFRI 691. The MnP activity of TFRI 554 was suppressed by ammonium chloride and potassium nitrate; TFRI 691 under ammonium tartrate and potassium nitrate exhibited high amount of MnP activity (around 6,0000 U/L), but the activity was also reduced slightly while cultivating under ammonium chloride. In the analysis of MnP activities, different pH of analysis buffer were applied, the MnP of TFRI 554 and TFRI 691 both exhibited highest activity under pH 6, but the MnP activities decreased as the pH of analysis buffer decreased. When study the effect of temperature on MnP stability, MnP activity of TFRI 691 raised slightly after one hour treatment under 40℃ even 45℃. MnP of both TFRI 554 and TFRI 691 did not exhibit the stability to high temperature. Whether the addition of milled rice straw (0.5% W/V) or veratryl alcohol (1, 2 mM), these inducers could be suitable for inducing higher MnP production of TFRI 554 and TFRI 691, but an excess addition of veratryl alcohol (4 mM) could conversely inhibit the MnP production. In the semi-solid state study, both TFRI 554 and TFRI 691 produce high amounts of MnP (74700 U/L; 45553 U/L, respectively), even higher than which in liquid cultivation. MnP activity of TFRI 554 maintained stable amount in air pumping experiment until the eighteenth day of cultivation, much longer than that of non-air pumping cultivation. According the SDS-PAGE analysis, the MnP zymogram of TFRI 554 and TFRI 691 are different. There were three bands of MnP found in TFRI 554 (65 kDa, 40 kDa, 35 kDa respectively); and there were three bands found in TFRI 691 (40 kDa, 38 kDa, 35 kDa respectively).

Record ID 第105筆 System ID 080SCU02381001
BCRC ID CCRC 36200, Public Year 80
Paper Name 影響白腐菌分解偶氮染料因子的探討 Biodegradation of azo dyes by white rot fungi
Student Name 李淑蘭
Teacher Name 趙維良
School Department Name 東吳大學 微生物研究所 Academic Degree 碩士
Abstract 針對五種偶氮染料,利用選擇性培養基從環境中篩選可使染料褪色的白腐菌(white rot fungi),共得到120 株篩選株,其中15個篩選株可使染料Orange G褪色。篩選 株S1、S2與從菌種中心購得的Phanerochaete chrysosporium CCRC 36200、36201 均可在洋菜固體培養基及液體培養基中使偶氮染料Orange G、Amaranth (acid-red 27)、Tropaeolin O 及異環染料Azure B 褪色,其中篩選株S1在固體及液體均可使 Congo red褪色。而Phanerochaete chrysosporium 31891在固體培養基中可使染料 褪色但在液體培養基卻失去褪色能力。在不同氮源的培養液中,測試菌株使Orange G 褪色情形,發現以 1﹪ urea 、ammonium tartract 、glycine 及 peptone、 NHCl作為氮源時,除篩選株S1外,其餘菌株均無法表現褪色能力,篩選株 S1 於 1﹪ NHCl作為氮源時,仍可使染料Orange G褪色。若氮源為12mM urea、glycine 、peptone 條件下,會均抑制或延遲了受試菌株使Orange G的褪色現象;反之,培 養液若含 12mM NHCl、ammonium tartrate 時,受試的菌株除了Phanerochaete chrysosporium 36201 外,其餘菌株均能表現褪色現象。當氮源為低濃度的urea、 glycine (1.2mM)、peptone (0.05﹪) 受試菌株使染料 Orange G 褪色快速、若以 NHCl取代時,則延遲退色現象。大致上,以低濃度的有機氮為氮源時,菌株均能 表現其褪色能力。因菌株的差異和培養狀況的不同,veratric acid 會促進或抑制 菌株使Orange G褪色的能力。而Phanerochaete chrysosporium 36201 之褪色在各 種處理中均不明顯。若以cellulose、cellobiose、starch、dextrin取代glucose 作為碳源配合不同濃度的NHCl (1﹪, 12mM, 1.2mM) ,結果篩選株S1均能表現其 褪色現象,其中以在cellulose 為碳源含1.2mM NHCl的培養液中褪色趨勢最快。 改以 starch 為碳源則 veratric acid 在低氮源 (1.2mM) 下,可促進受試菌株使 Orange G褪色。至於 cellobiose、dextrin 和 glucose 作為碳源時大致相近,但 Phanerochaete chrysosporium 36201 則在dextrin 為碳源低NHCl (1.2mM)的培 養液中,其褪色驅勢最快。所以菌株褪色能力的表現會因菌株的差異及不同氮源濃 度及添加中間物而受影響。以Phanerochaete chrysosporium 36200 及篩選株S1的 胞外濃縮液進行褪色實驗,發現胞外濃縮液可改變染料的吸收波形及最大吸收波長 的吸收值。於蛋白質的圖譜分析中,兩株菌在SDS-PAGE的電泳片上於40kd至70kd之 間有蛋白質條紋,在活性染色的電泳片上,Phanerochaete chrysosporium 36200 於分子量50kd左右及篩選株S1則於40kd左右的蛋白質具有過氧化氫的活性。由實 驗結果顯示白腐菌的胞外過氧化氫在偶氮染料褪色過程中扮演重要的角色。

Record ID 第106筆 System ID 094THU00289001
BCRC ID CCRC 14146, CCRC 14667, CCRC 10695, CCRC 12257, CCRC 11844, CCRC 11847, Public Year 94
Paper Name 益生菌之耐酸和耐膽鹽能力及其於酸酪乳之應用 The acid and bile resistant ability of probiotics and its application in yoghurt
Student Name 游淑惠
Teacher Name 洪連欉
School Department Name 東海大學 畜產與生物科技學系 Academic Degree 碩士
Abstract 本研究目的為探討益生菌之耐酸及耐膽鹽能力,及其於酸酪乳之應用,針對耐酸、耐膽鹽能力較佳之菌株直接製作益生菌酸酪乳;耐酸、耐膽鹽能力較差的菌株則給予包埋,並測試包埋的效果及其於酸酪乳中的應用。 試驗菌株選用東海大學畜產與生物科技學系應用微生物研究室貯存之雙叉桿菌及乳酸桿菌…等益生菌以及一般乳酸菌,首先進行益生菌之耐酸、耐膽鹽、模擬胃腸道消化測試及對抗性澱粉及異麥芽寡醣的利用性。選取耐酸、耐膽鹽較佳的菌株,於牛乳中培養觀察益生菌的生長,並測試抗性澱粉與異麥芽寡醣對益生菌增殖之影響,選取適合在牛乳中生長的菌株,與傳統酸酪乳菌酛配合製作益生菌酸酪乳,並探討其菌數、理化學性質及感官品評。結果顯示耐酸且耐膽鹽的菌株為Bifidobacterium bifidum CCRC 14146、B. catenulatum CCRC 14667和Lactobacillus acidophilus CCRC 10695,且牛乳中添加抗性澱粉、異麥芽寡醣及葡萄糖可縮短益生菌達pH 4.6所需時間(P<0.05)於牛乳中以37及43℃下培養比較,以B. bifidum CCRC 14146的生長最佳,達到pH 4.6的時間最短分別為21及12小時,且菌數可達到108 cfu/mL。抗性澱粉添加並不影響益生菌酸酪乳貯存期間之活菌數(P>0.05),然經35天貯存,菌數仍可維持約107 cfu/mL。此外利用選取的益生菌B. bifidum CCRC 14146(B)與傳統酸酪乳菌酛Streptococcus thermophilus CCRC 12257(ST)以1:1混合菌株,於43℃培養製作益生菌酸酪乳(STB),結果顯示其菌數、理化學性質及感官品評相較於一般傳統酸酪乳皆無顯著差異(P>0.05),且乳酸菌與益生菌數分別為1.2×107 cfu/mL與2.5×108 cfu/mL。 對酸與膽鹽耐受性較差的菌株,分別以未煮與預煮的抗性澱粉(resistant starch)依1:1比例混合進行包埋,然後模擬胃腸道消化測試其耐受性;並將包埋菌體添加於滅菌酸酪乳中進行貯存試驗。顯示對酸與膽鹽耐受性較差的菌株分別為B. bifidum CCRC 11844和B. longum CCRC 11847;利用預煮的抗性澱粉包埋此兩株菌,其耐酸測試之存活菌數較未包埋者高1 log cfu/mL,且耐膽鹽測試之存活菌數較未包埋者高4 log cfu/mL;而在模擬胃腸道消化測試之存活菌數,預煮的抗性澱粉包埋較未包埋者高3 log cfu/mL以上;將此益生菌包埋體添加於滅菌酸酪乳於4℃貯存35天,B. longum CCRC 11847存活菌數較未包埋者高1 log cfu/mL。 The healthy benefits of probiotics have been demonstrated in recent years, but most probiotics are low acid- and bile-resistant. The survival of probiotics passing through the digestive tract was very low, and these probiotics couldn’t grow well in milk. Therefore the cultures for probiotic yoghurt need to be selected or microencapsulated appropriately to process healthy probiotic products. The purpose of this study was to investigate the acid and bile resistant ability of probiotics and its application in yoghurt. The acid and bile resistant probiotics were selected under pH 1.0, 2.0 and 0.3% bile potassium chloride buffer. The ability to hydrolyze resistant starch(RS)by these probiotics which grew on broth containing RS as the sole carbon source was measured. In addition, the growth ability of the acid and bile resistant probiotic culture in milk at 37 and 43℃ was tested. Finally, the yoghurt was manufactured by using the strains that showed the best survival under gastric fluid tests and growth rate in milk in combination with yoghurt starter. The physicochemical characteristics and sensory evaluation were investigated. The results demonstrated that these probiotics utilized RS with different ability and three probiotics(Bifidobacterium bifidum CCRC 14146, B catenulatum CCRC 14667, and Lactobacillus acidophilus CCRC 10695)had the highest survival under gastric fluid tests。 Further study showed that the B. bifidum CCRC 14146 grew well in milk at 37 and 43℃ and reached the pH 4。6 after 21 and 12 hours, respectively, with a viable count of 108 cfu/mL. The viable counts of B. bifidum CCRC 14146 could’t enhance in probiotic yoghurt containing resistant starch during 35d storage at 4℃。 There were no significant differences of the sensory evaluation scores between probiotic yoghurt and traditional yoghurt(P>0.05). The viable counts of yoghurt starter and probiotics were 107 cfu/mL and 108 cfu/mL, respectively. According to this study, B. bifidum CCRC 14146 might be a promising new yoghurt culture for probiotic yoghurt。 The low acid- and bile-resistant probiotics were microencapsulated by mixing with equal volume of the uncooked or precooked RS. Then, the viable counts of free and microencapsulated probiotics in the simulated digestive tract and yoghurt during storage were measured respectively. This study aimed to test how microencapsulation with RS affected the acid- and bile-resistant properties of probiotic culture. The results demonstrated that low acid- and bile-resistant probiotics were B. bifidum CCRC 11844 and B longum CCRC 11847。 Microencapsulation with precooked RS improved the acid- and bile-resistant by 1 and 4 log cfu/mL respectively compared with those of the free cultures. Furthermore, the counts of precooked RS-microencapsulated cultures were 3 log cfu/mL higher than free cultures in the simulated digestive tract test. The counts of precooked RS-microencapsulated B. longum CCRC 11847 were enhanced by 1 log cfu/mL in yoghurt stored at 4℃ for 35 days。 Therefore the low acid- and bile-resistant probiotics could be microencapsulated by mixing with precooked resistant starch and application in probotic yoghurt.

Record ID 第107筆 System ID 093THU00063007
BCRC ID BCRC 21727, Public Year 93
Paper Name 改良酵母菌種及培養基組成以增進γ-GC (γ-Glutamylcysteine)產量之研究 Studies of γ-Glutamylcysteine production by improving yeast strains and medium compositions
Student Name 林欣儀
Teacher Name 楊芳鏘 博士 黃進發 博士
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 本研究主要目的為改良酵母菌BCRC 21727,藉由篩選MG (MethylGlyoxal)之敏感性變異株,提高酵母菌體胞內麩胺半胱胺酸(γ-glutamylcysteine ;γ-GC )之生產能力,並分析其特性與培養基組成之影響等 在實驗項目方面首先以EMS進行酵母菌BCRC 21727突變,篩選γ-GC產能較高之菌株,並比較原始菌株與突變菌株之GSH I及GSH II酵素活性,H2O2、不同培養基組成、與胺基酸之添加等對菌體及γ-GC產能之影響,最後於5L發酵槽進行初步批式與饋料批式發酵試驗,並與三角瓶進行比較 實驗結果顯示,以EMS進行酵母菌BCRC 21727突變,篩得MG敏感性變異株中,以編號SY-35679其胞內γ-GC含量顯著提高達4.77 ± 0.17 mg/g DCW約為原始菌株(0.59 ± 0.06 mg/g DCW)之8倍,γ-GC/GSH比值為0.53約為原始菌株(0.09 mg/g DCW)之6倍。此γ-GC高產量變異株SY-35679之酵素GSH I比活性提高,而對酵素GSH II則沒有明顯影響。另一方面,突變株單位菌體對H2O2之抗性有提升之現象。 在培養基方面,突變菌株SY-35679,在代號為 TM的培養基中,γ-GC含量為6.85 ± 0.36 mg/g DCW約為YPD培養基的1.4倍。在不同組成份濃度之試驗中,當葡萄糖濃度為20 g/L時結果較好,菌體內GSH含量為8.95 ± 0.33 mg/g DCW,γ-GC含量為4.77 ± 0.16 mg/g DCW。而氮源Peptone濃度在6 g/L時,各項分析指標顯示較高。於YPD培養基中添加適量L-Cysteine有助於細胞生長及增加胞內含硫物質,在12 hr添加5 mM L-Cysteine,可獲得較高之GSH與γ-GC含量。 以三角搖瓶或5L發酵槽初步培養比較,結果顯示突變菌株SY-35679胞內GSH與γ-GC含量均明顯較原始菌株高。而批式發酵槽所生產的菌體胞內γ-GC及GSH含量均較以三角搖瓶培養者高。然而,在5L發酵槽以非連續饋料批式發酵方式培養結果末期菌體量顯著增加,但是胞內γ-GC與GSH含量明顯降低,此結果顯示不同培養環境或條件會顯著影響菌體生長及菌體胞內含硫物質之生產。 The purpose of this study is to improve the γ-glutamylcysteine(γ-GC) production of a yeast strain BCRC 21727 by mutating and screening the MethylGlyoxal-sensitive mutants。 Besides, we also analysis the mutant’s characteristics and effects of media on the cell growth and the intracellularγ-GC content. In experimental, first, the parent yeast strain, BCRC 21727, was mutated by EMS in order to isolate higherγ-GC-producing strains Then, this isolated mutant with improved γ-GC production was compared with the parent strain, BCRC 21727, for their GSH I and GSH II enzyme activities, as well as, the effects of H2O2, medium compositions, or amino acid additions on the cell growth and the γ- GC productivity。 Finally, we also do a preliminary test by batch or discontinuous fed-batch fermentation in 5L fermentors in order to provide data information for comparing with those obtained from the shake flasks. The results show that one significantly high γ-GC-producing mutant, SY-35679, was isolated from the MG-sensitive mutants derived from yeast BCRC 21727 through the EMS treatment。 The intracellular γ- GC content(4.77 ± 0.17 mg/g DCW)in strain SY-35679 is around eight times compared to that (0.59 ± 0.06mg/g DCW) of the parent strain, meanwhile, the γ-GC/GSH ratio (0.53) of the mutant is about six times higher than that of the parent strain (0.09). Besides, the GSH I specific activity of strain SY-35679 was higher than the parent strain, however, no significant increase in GSH II activity was observed. On the other hand, cell growth against hydrogen peroxide was slightly inhibited, however, the anti-H2O2 ability in mutant was better than the parent strain based on evaluating the equal amount of cell mass. In medium studies, the γ- GC content of strain SY-35679 in TM medium was 6.85 ±0.36 mg/g DCW, which was 1.4 time of that in YPD medium. From the different concentration of media used, 20 g/L of glucose can reach better results with 8.95 ±0.33 mg/g DCW of GSH and 4.77 ±0.16 mg/g DCW of γ- GC. In the meantime, nitrogen source with 6g/L of peptone could reach the best results. On the other hand, higher amounts of GSH andγ- GC content were obtained with adding 5 mM L-Cysteine to the YPD medium at 12hr. Comparison of the γ- GC fermentation in either shake flasks or 5L fermentors, the results showed that intracellular γ- GC and GSH contents in mutant SY-35679 were significantly higher than those in the parent strain. Besides, higher amounts and content of γ- GC were obtained at 30 h batch fermentation in a 5L fermentor when comparing with those from shake flasks. However, different profiles of GSH orγ- GC production were observed in a discontinuous fed-batch fermentation. These results indicate that incubation situations significantly affect the production of sulfur-containing compounds.

Record ID 第108筆 System ID 093THU00063019
BCRC ID BCRC 35396, Public Year 93
Paper Name 培養條件對樟芝菌絲固態培養之影響 The Effect of Cultural Conditions on the Solid-State Culture of Antrodia camphorate
Student Name 薛姿涓
Teacher Name 楊芳鏘
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 本研究的重點著重於利用固態培養的方式進行樟芝(Antrodia camphorate BCRC 35396)菌絲體的培養,藉由改變不同的培養條件(培養容器、培養時間、初始含水量、初始pH值及培養容器大小)及添加物與攪拌頻率對樟芝菌絲體生長及抗氧化能力(捕捉DPPH自由基能力、總酚含量及抗氧化能力)之影響。 由實驗結果得知以小薏仁當做固態培養基,初始含水量為55%,在25℃下培養14天,在菌體濃度方面添加木屑、較低比例之液態培養基、樟腦油有助於菌體之生長,其中以添加牛樟木屑0.3g、樟腦油0.4ml,菌體濃度可達0.90g(D.W.)/g substrate及0.98g(D.W.)/g substrate,約為未添加的1.40倍及1.53倍。在添加木屑萃出物方面,以添加少量的木屑水萃出液、木屑乙酸乙酯萃出液及丙酮萃出液有助於菌體生長。 在抗氧化能力方面,添加樟腦油及木屑其捕捉DPPH自由基能力約為未添加的3.5倍左右,添加初始液態培養基捕捉效果最好可達70.54%。添加木屑乙酸乙酯及甲醇萃出液有提高菌體捕捉DPPH自由基之能力的效果。攪拌頻率會破壞菌絲生長及其抗氧化之能力,且攪拌頻率越高影響越大。

Record ID 第109筆 System ID 091THU00063003
BCRC ID CCRC 36123, Public Year 91
Paper Name 固態培養生產靈芝菌絲體之研究 The Research on the production of mycelium of Ganoderma Lucidum by Solid-state cultures
Student Name 廖仁宏
Teacher Name 楊芳鏘
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 本研究主要目的在於利用固態基質進行靈芝菌絲體的培養,探討不同培養條件對固態培養靈芝菌絲體生長與生理活性成分生成之影響。 在實驗項目方面,以培養皿、三角瓶與發酵槽培養,在培養皿的試驗中,分別進行不同菌種、培養時間及不同培養基質之試驗,也對物理與化學培養因子進行探討,物理因子方面有培養溫度、初始pH值、初始含水量及接菌量等、而化學因子則為不同碳源、氮源和其他營養成分,如天然穀物及木屑等。三角瓶的試驗中,探討了攪拌與表面通氣量對菌絲生長的影響。發酵槽的試驗中,比較填充床與攪拌式反應器培養之差異,及不同發酵體積對菌絲固態培養的影響。 實驗結果發現以麥糠添加蒸餾水為基礎培養基, CCRC 36123 為菌株來做固態培養,培養溫度為25~35℃,初始含水量66~75﹪最佳。初始含水量66﹪,30℃下培養7天之菌體濃度可達1.72g dry weight/g substrate。以添加4﹪Fructose當碳源與添加8﹪Malt extract當氮源為最佳。添加穀物方面,以大薏仁為最佳,比起添加碳氮源對於菌體生長更有幫助。添加木屑對菌體生長雖沒有明顯的幫助,但可以誘導其分泌代謝產物,約3~4個星期左右,氣生菌絲表面就可形成類似子實體的紅棕色表面,也可能加速菌絲分化。 實驗中也對靈芝多醣與靈芝酸的生成,分別以GPC及HPLC來作分析。由GPC層析圖得知,與液態培養菌絲球相比,固態培養菌絲體分泌的多醣,分子量有較大的趨勢,對抗癌有很大的功效。而靈芝酸部分則因為固態培養的菌絲體其培養時間遠短於子實體,故固態菌絲之靈芝酸比子實體少。 本研究也對固態菌絲的形態構造以光學顯微鏡進行剖析,並與液態菌絲球之菌絲結構比較。 The main purpose of this research was to study the feasibility of solid-state culture of Ganoderma lucidum for the production of mycelia and physiology activity compositions. The culture tests were carried out in plates, flasks, and a fermentor to determine optimal cultivating conditions, including physical and chemical factors. The physical factors included temperature, initial pH, initial water content, and inoculum. The chemical factors included carbon sources, nitrogen sources, wood chip, and various types of grain. Based on the results obtained from plate cultures, the optimal temperature and initial water content were suggested to be around 25~35℃ and 66﹪~75﹪. Biomass concentration could reach to 1.72g dry weight/g substrate at initial water content of 66﹪and 30℃ in 7 days. The results also indicate that the additions of fructose (4%) and malt extract (8%) as the carbon source and nitrogen source would increase the biomass concentration. Some grain also found to be effective to the enhancement of biomass. In contrast, there was no obvious effect on biomass production by adding wood chip, but it might induce the secretion of metabolite and accelerated differentiation. The research also probed into the production of polysaccharides and Ganoderma acid by the analysis by GPC and HPLC. The molecular weight of polysaccharide secreted by mycelia in solid-state cultures was estimated to be higher than that in submerged culture. However, since it took less time in mycelial solid-state cultures, and the Ganoderma acid produced in mycelia by solid-state cultures had a less variety, compared with those in a fruit-body. The research also studied the change of mycelial morphology by microscope and compared structure difference of mycelia.

Record ID 第110筆 System ID 091THU00063031
BCRC ID CCRC 36123, Public Year 91
Paper Name 不同培養方式對靈芝菌絲體抗氧化活性成分與多醣體生成之影響 The effects of cultural methods on the antioxidant activity and polysaccharide of Ganoderma lucidum mycelia
Student Name 詹宏偉
Teacher Name 楊芳鏘
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 本研究主要目的在於利用固態基質進行靈芝菌絲體的培養,探討不同培養條件對固態培養靈芝菌絲體生長與生理活性成分生成之影響,並與液態培養做比較。 在實驗項目方面,以培養皿、三角瓶培養,在培養皿的試驗中,分別進行培養時間及不同培養基質之試驗,也對物理培養因子進行探討,物理因子方面有培養溫度、初始pH值、初始含水量及接菌量等,在添加物方面有添加木屑、木屑萃出液、液態培養基、麥糠等。三角瓶的試驗中,探討了攪拌與表面通氣量對菌絲生長的影響。 實驗結果發現以燕麥添加蒸餾水為基礎培養基, CCRC 36123 為菌株來做固態培養,培養溫度為25~33℃、初始含水量50﹪及pH2~6最佳。初始含水量50﹪,30℃下培養14天之菌體濃度可達0.42 g(D.W.)/g substrate。整體來說,固態培養的抗氧化能力比液態培養好,溫度是主要的影響關鍵,培養溫度在25~33℃下都能維持不錯的抗氧化能力,而DPPH自由基掃除活性固態培養和液態培養分別約為40%和10%,相差4倍左右。多醣分子量以固態培養方式有助於高分子量的產生,分子量分布與子實體較相似。 添加木屑、木屑萃出液、液態培養基和麥糠都有助於提升菌體濃度和DPPH。適當的通氣及攪拌有助於菌體生長,抗氧化方面,不通氣和適當的攪拌,對抗氧化能力有幫助,減少通氣和增加攪拌有助於DPPH自由基掃除活性的提高。 The main purpose of this research was to study the feasibility of solid-state culture of Ganoderma lucidum for the production of mycelia and physiology activity compositions. Various cultural systems were developed and evaluated. The culture tests were carried out in plates and flasks to determine optimal cultivating conditions. The physical factors included temperature, initial pH, initial water content, and inoculum. Based on the results obtained from plate cultures, the optimal temperature, initial water content and pH were suggested to be around 25~35℃, 50﹪, pH2~4, resceptively. Biomass concentration could reach to 0.42 g(D.W.)/g substrate at the conditions of initial water content of 50﹪and 30℃ in 14 days. However, the formation of antioxidant components in solid-state fermentation was greater than that of submerged culture, ranging from 20 to 80% and the culture temperature was found to be a key factor. DPPH radical scavenging activities in solid-state fermentation and submerged culture were around 40% and 10%, respectively. Moreover, solid-state fermentation facilitated the production of higher molecular weight of polysaccharide. The additions, which included wood chip, wood chip extract, liquid medium and wheat bran, could enhance biomass concentration and DPPH scavenging ability. Proper aeration and agitation were also proved to be beneficial to the cell growth. In contrast, the data showed that the higher level of aeration might have the negative effect on antioxidant activity and DPPH radical scavenging activity.

Record ID 第111筆 System ID 090THU00063013
BCRC ID CCRC 37200, Public Year 90
Paper Name 培養條件對靈芝菌絲體超氧歧化酶(SOD)生成之影響
Student Name 張德玉
Teacher Name 楊芳鏘
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 本研究主要目的在探討Ganoderma lucidum CCRC 37200菌株,利用三角瓶液態培養時,各種外在培養環境與培養基組成對靈芝菌絲體胞內SOD生成活性之影響,再以「一次一因子」方法為基礎,配合回應曲面法進行環境因子對SOD活性的探討,希望藉由培養環境因子的調整與控制提高靈芝菌體濃度及胞內SOD的活性。 結果顯示250毫升三角瓶批次基礎培養基中,靈芝菌絲生長的最適溫度為30℃,初始pH值為5,轉速為100rpm,在此條件下培養7天的菌體濃度能達到586.89mg/100ml,單位菌體胞內SOD的活性亦能達到255.46U/g d.w.。 在培養基組成方面,以蔗糖為碳源所得結果優於葡萄糖,氮源以Yeast extract和Peptone兩種氮源搭配為最佳,而在菌體生長期時,當添加適量的過氧化氫,雖然會對菌體濃度稍微抑制,但可刺激胞內SOD生成,使靈芝胞內SOD活性提高2~3倍。 利用回應曲面法(RSM)探討碳源濃度、兩種不同氮源濃度和初始pH值因子交錯影響,來決定最佳培養基的組成。最後決定蔗糖2.33﹪、Peptone 0.106﹪、Yeast extract 2.59﹪、初始pH6.05為最適的培養基條件,以此條件培養7天菌體SOD活性能達到1285.35U/g.d.w.,比原用基礎培養基所得菌體SOD活性,可提昇5倍以上。 The main objective of this research is to study the effects of cultural conditions on the formation of superoxide dismutase (SOD) in the submerged culture of Ganoderma lucidum. In addition, the response surface method (RSM) was employed to determine the interaction effects of factors, such as C-source, N-source and initial pH. The strain of Ganoderma lucidum CCRC 37200 was chosen and the experiments were carried out in shake flasks to determine the optimal cultural conditions。 The results indicated that the condition with the temperature of 30℃, was the initial pH of 5, the rotary speed of 100rpm, and the inoculums density of 8% was in favor of the SOD production. Under that condition, the mycelium concentration and intracellular SOD activity could reach 586.89mg/100ml and 255.46/g d.w., respectively, in a 7-day culture. A number of carbon sources were tested in order to determine their effects on the growth of mycelium and SOD production. The activity of SOD obtained with sucrose was higher than that with glucose. The results showed that the combination of yeast extract and peptone was the best choice for nitrogen source. The addition of H2O2 could induce the formation of SOD, although it might reduce the growth rate of mycelia. By examination different carbon and nitrogen sources, sucrose, peptone, and yeast extract were selected for the following test of RSM to study the interaction effects of C-source concentration, N-source concentration, and initial pH. With the optimal composition determined by RSM, which was consist of 2.33% of sucrose, 0.106% of peptone, 2.59% of yeast extract, and the initial pH of 6.05, the concentration of SOD could reach 1604mg/100ml on the 7th day and which was more 5-fold. The results demonstrated that the cultural conditions had a great influence on the formation of SOD. The optimal conditions determined by the RSM effectively increase the productivity of SOD in shake flask cultures of Ganoderma lucidum.

Record ID 第112筆 System ID 092THU00289007
BCRC ID CCRC 14009, CCRC 10940, CCRC 10697, CCRC 10695, CCRC 11052, CCRC 10791, CCRC 12268, Public Year 92
Paper Name 選殖乳酸菌暨發酵乳清生產乳酸之研究 The Studies for the Selection of Lactic Acid Bacteria and the Production of Lactic Acid from Whey Fermentation
Student Name 黃培鈞
Teacher Name 施宗雄 博士
School Department Name 東海大學 畜產學系 Academic Degree 碩士
Abstract 本試驗為選殖乳酸菌以乳清為發酵基質生產乳酸之研究,期能提高乳酸生產效率而達到降低生產成本之目的。本試驗選取Lb. rhamnosus THSH-1、Lb. delbrueckii subsp. bulgaricus CCRC 14009、Lb. casei subsp. rhamnosus CCRC 10940、Lb. casei subsp. casei CCRC 10697、Lb. acidophilus CCRC 10695、Lb. helveticus CCRC 11052、Lactococcus lactis subsp. lactis CCRC 10791及Streptococcus thermophilus CCRC 12268等8菌株為供試之乳酸菌株,期能選殖乳酸生成效率最佳的乳酸菌株及尋找最佳補充氮源並應用於乳清發酵生產乳酸。試驗結果顯示,在乳清滲流液與乳清中接種10﹪Lb. rhamnosus THSH-1菌量、調節pH值及42℃振盪培養的發酵條件下,添加3﹪酵母萃取物的營養補充物時發酵乳清滲流液及乳清生產乳酸的效果最佳。而添加泛酸亦可以促進Lb. rhamnosus THSH-1發酵補充豆奶的乳清滲流液生產乳酸。 This study aimed at selecting homofermentative lactic acid strains for lactic acid production from whey fermentation. According to this study results, Lb. rhamnosus THSH-1 performed the better results than other strains in lactic acid productivity and lactose utilization(P<0.05). Optimisation of temperature for lactic acid production by Lb. rhamnosus THSH-1 was 42℃. pH adjustment and agitation culture provided beneficial effects on lactic acid production during the fermentation process(P<0.05). Inoculum of 10﹪Lb. rhamnosus THSH-1 was the superior to 1﹪inoculum on production of lactic acid(P<0.05). The addition of nutrients presented a positive effect on the lactic acid production. Among the different nitrogen sources supplemented to whey, yeast extract performed the best lactic acid productivity(P<0.05). At 3﹪w/v yeast extract supplementation, the lactic acid productivity was the best(P<0.05). The addition of pantothenic acid in whey permeate supplemented with soymilk presented a significant beneficial affect with lactic acid production(P<0.05).

Record ID 第113筆 System ID 092THU00289008
BCRC ID CCRC 12264, CCRC 14117, CCRC 11198, CCRC 10940, CCRC 10696, Public Year 92
Paper Name 利用乳酸菌自體水解能力促進乾酪熟成 Acceleration of Cheese Ripening Using Autolytic Ability of Lactic Acid Bacteria
Student Name 劉哲君
Teacher Name 施宗雄 博士
School Department Name 東海大學 畜產學系 Academic Degree 碩士
Abstract 本實驗旨在篩選自體水解能力較佳之乳酸菌株應用在製作乾酪上以促進乾酪熟成為目標。實驗菌株選用購自食品工業發展研究所菌種中心而於本實驗室保存之乳酸球菌菌株、乳酸桿菌菌株及嗜熱乳鏈球菌菌株作為篩選試驗菌株,首先進行乳酸菌之蛋白分解能力與自體水解能力測試:乳酸球菌菌酛以Lactococcus lactis subsp. cremoris CCRC 12264具有最高之蛋白分解能力與自體水解能力,Lc. lactis subsp. lactis CCRC 14117次之,Lc. lactis subsp. lactis CCRC 11198最差;乳酸桿菌菌株之乳酸去氫酶(lactate dehydrogenas;LDH)活性以Lactobacillus casei subsp. rhamnosus CCRC 10940活性最高,Lb. bulgaricus CCRC 10696活性最差 其次依乳酸菌之蛋白分解能力與自體水解能力測試之結果,以混合菌酛方式接種於全脂殺菌乳製作乾酪,共四個處理組,分別為(1)由高自體水解能力及高蛋白分解力的Lc. lactis subsp. cremoris CCRC 12264與次高自體水解能力之Lc. lactis subsp. lactis CCRC 14117為混合菌酛;(2)以Lc. lactis subsp. cremoris CCRC 12264與Lc. lactis subsp. lactis CCRC 14117添加LDH酵素活性最佳之Lb. casei subsp. rhamnosus CCRC 10940作為輔助菌酛;(3) 以Lc. lactis subsp. cremoris CCRC 12264與Lc. lactis subsp. lactis CCRC 14117添加LDH酵素活性最差之Lb. bulgaricus CCRC 10696作為輔助菌酛及(4)以高自體水解能力及高蛋白分解力的Lc. lactis subsp. cremoris CCRC 12264與自體水解能力最差之Lc. lactis subsp. lactis CCRC 11198混合菌酛結果顯示,所製作的四組乾酪於熟成期間,水分皆呈逐漸降低之趨勢,蛋白質及脂肪則隨水分之減少而有逐漸上升的趨勢,pH值、NaCl含量及S/M值則有波動,乳酸菌數則以Lc. lactis subsp. cremoris CCRC 12264與Lc. lactis subsp. lactis CCRC 11198混合菌酛之Lc. lactis subsp. cremoris CCRC 12264存活率最低所製作之乾酪於熟成第84天時,四組乾酪之熟成度與游離胺基酸之產量,皆以添加LDH酵素活性最差之Lb. bulgaricus CCRC 10696作為輔助菌酛之組別有最高之熟成度與游離胺基酸之產量(p < 0.05),但pH值較低(p < 0.05)若以自體水解能力菌酛比較,則以使用Lc. lactis subsp. cremoris CCRC 12264與Lc. lactis subsp. lactis CCRC 14117為混合菌酛組有較高熟成率與游離胺基酸產量(p < 0.05),但以Lc. lactis subsp. cremoris CCRC 12264與Lc. lactis subsp. lactis CCRC 11198混合菌酛組有較高之自體水解能力。 The process of cheese making is an ancient craft that dated thousands of years ago. There is approximately 35% of the total milk supply in the world, which is converted into cheese. Ripening time of cheese varies from four weeks (soft cheeses) to three years (very hard cheeses), which depends on the varieties of cheese. If the ripening time of cheese was accelerated, it would provide both the economic and technical advantages such as reducing the refrigeration costs, weight loss and microbiological risks. The purpose of this experiment was to examine and select the best lactic acid bacteria in autolysis to accelerate cheese ripening. Seventeen strains of lactic acid bacteria were used as starters in this experiment, which included 4 lactococci, 11 lactobacilli, and 2 streptococci. According to the results, Lactococcus lactis subsp. cremoris CCRC 12264 performed the highest ability in proteolysis and autolysis and Lc。 lactis subsp. lactis CCRC 14117 presented the second high ability。 However, Lc. lactis subsp. lactis CCRC 11198 performed the lowest ability in proteolysis and autolysis。 In addition, the results for testing the LDH activity presented that Lactobacillus. casei subsp. rhamnosus CCRC 10940 was the most active strain and Lb bulgaricus CCRC 10696 is the less active one。 In order to identify the autolysis ability of Lc. lactis subsp. lactis CCRC 14117 and Lc。 lactis subsp. cremoris CCRC 12264, four mixed cultures were prepared to manufacture cheese samples, which included sample A (Lc。 lactis subsp. cremoris CCRC 12264 + Lc。 lactis subsp. lactis CCRC 14117 + Lb。 casei subsp. rhamnosus CCRC 10940 as adjunct), sample B (Lc。 lactis subsp. cremoris CCRC 12264+ Lc。 lactis subsp. lactis CCRC 14117+ Lb bulgaricus CCRC 10696 as adjunct), sample C as control (Lc。 lactis subsp. cremoris CCRC 12264+ Lc。 lactis subsp. lactis CCRC 14117), and sample D (Lc。 lactis subsp. cremoris CCRC 12264 + Lc。 lactis subsp. lactis CCRC 11198)。 After experiment, the results indicated that the components of sample cheeses were changed that included moisture content decreased along with ripening days, protein and fat contents increased and pH and S/M value were unstable. In addition, Lc. lactis subsp. cremoris CCRC 12264 in sample D presented the lowest viability (p<0。05). After 84 days of ripening, sample A performed the highest ripening ratio and the production of free amino acid (p<0.05), however sample A had low performance in pH value (p<0.05). Based on the results of comparing autolysis abilities within two samples, sample C presented the higher ripening ratio and the production of free amino acid (p<0.05) than the other sample. To sum up, using the highest autolytic starter and adjunct culture with the high LDH activity can accelerate proteolysis and cheese ripening.

Record ID 第114筆 System ID 091THU00253020
BCRC ID CCRC 10695, CCRC 10697, CCRC 14009, CCRC 12257, CCRC 11844, CCRC 11847, Public Year 91
Paper Name 利用單獨及混合乳酸菌試製黑豆奶酸凝酪之研究 A study on the manufacture of blackbean milk yogurt using single and mixed strains of lactic acid starters
Student Name 石湘翎
Teacher Name 閻立平
School Department Name 東海大學 食品科學系 Academic Degree 碩士
Abstract 摘要 本實驗選取Lactobacillus bulgaricus(CCRC 14009)、Streptococcus thermophilus(CCRC 12257)、Lactobacillus acidophilus (CCRC 10695) 、Lactobacillus casei (CCRC 10697)、Bifidobacterium bifidum(CCRC 11844)及Bifidobacterium longum(CCRC 11847) 等六株乳酸菌為黑豆奶酸凝酪(blackbean milk yogurt)之發酵菌,嘗試比較單獨及混合菌對酸凝酪菌數、成分及物理性質等之影響,以篩選較佳之發酵菌組並嘗試添加不同醣類於黑豆奶基質以改善黑豆奶酸凝酪之風味品質。 結果顯示:混合菌製成之酸凝酪較單獨菌者具較高之菌數、較強之產酸能力、較快之完成發酵(pH=4.6)時間及較大黏度等優點。此外,黑豆奶中之主要醣類(蔗糖、水蘇糖及果糖)之利用、總糖消耗率、乳酸產量及維生素B群(B1及B2)產量亦以混合菌組高於單獨菌組。因此,以混合菌組可得較高營養價值及較佳物性之酸凝酪。 五種不同混合菌製成之酸凝酪,其中以B. longum+S. thermophilus+L. bulgaricus組在4℃,28天之貯存期間,具有最高之殘存菌數(8.78 Log CFU / ml)、存活率(98.7﹪)、滴定酸度(0.495﹪)及黏度與最低之乳清析出率等較佳之微生物與物理性質。 以B. longum+S. thermophilus+L. bulgaricus混合菌試製酸凝酪之感官評估顯示,添加10﹪(w/v)蔗糖或6﹪(w/v)蔗糖+2﹪(w/v)乳糖+2﹪(w/v)葡萄糖於黑豆奶基質可改善酸凝酪風味品質及提高接受度。以經濟上考量,可考慮選擇前者。 ABSTRACT Six strains of lactic acid bacteria(Lactobacillus bulgaricus CCRC 14009, Streptococcus thermophilus CCRC 12257, Lactobacillus acidophilus CCRC 10695, Lactobacillus casei CCRC 10697, Bifidobacterium bifidum CCRC 11844, and Bifidobacterium longum CCRC 11847)were selected as the starter organisms in this research to study the effect of using single and mixed strains of lacic acid starters on the manufacture of blackbean milk yogurt。 Result showed that blackbean milk yogurt products made from mixed strains had higher viable count, higher titratable acidity, shorter fermentation time (pH=4.6) and higher viscosity as campared with those made from single strains. In addition, mixed strains showed higher sugar utilization (sucrose, stachyose and fructose) and higher production of lactate and vitamin B complex (B1&B2) than single strains. Therefore, using mixed-strain starters could produce blackbean milk yogurt with better nutritious value and physical property. After stored at 4℃ for 28 days, blackbean milk yogurt made from mixed strains of B. longum+S. thermophilus+L. bulgaricus had better microbiological and physical properties than those made from other 4 mixed-strain starters. This yogurt product showed the highest viable count (8.78 Log CFU/ml), survival rate (75.9%), titratable acidity (0.495%) and viscosity; and the lowest syneresis rate. Sensory evaluation of the yogurt product made from mixed strains of B. longum+S. thermophilus+L. bulgaricus indicated that adding 10% (w/v) sucrose or 6% (w/v) sucrose+2% (w/v) lactose+2% (w/v) glucose could improve the sensory property and raise the acceptability of this product. For economical concern, the former would be recommended.

Record ID 第115筆 System ID 091THU00253022
BCRC ID CCRC 31535, Public Year 91
Paper Name 利用不同發酵槽培養紅麴菌(Monascus rubber)生產monacolin K和紅麴色素之研究 A study on production of monacolin k and Monascus pigments from Monascus rubber using different types of fermentors
Student Name 廖玉萍
Teacher Name 顏文義博士
School Department Name 東海大學 食品科學系 Academic Degree 碩士
Abstract 摘要 紅麴是中國傳統用於釀酒的菌種,近年來被發現其二次代謝產物中的monacolin K,可做為降低人體內膽固醇的一種藥劑,而紅麴色素則是優良的天然食品色素。本研究的目的在於探討以Monascus ruber CCRC 31535 為紅麴生產菌株,進行monacolin K產量和紅麴色素生產之研究。利用攪拌式發酵槽(Stirred tank fermentor)以及氣舉式發酵槽(Airlift fermentor)做為培養方法的比較,並使用白米-甘油,和白米-蔬菜油兩種複合培養基為測試,而且是採用饋料式培養,也就是剛開始發酵時只放入培養基總米量的40%,分別在發酵的第四天和第七天再加入總米量的30%。 結果顯示,在5公升攪拌式發酵槽中,25℃,轉速為250rpm,通氣量為3.5vvm時起始pH值為5時M.rubber以白米-甘油培養基在發酵的第十天時達到monacolin K最高產量156μg/ml,白米-蔬菜油培養基,也在發酵第十天時monacolin K最高產量164μg/ml,而紅麴色素的最大產量在第八天,達到高峰為30.822mg/ml。而M.rubber在白米-蔬菜油培養基,未調起始pH值時,monacolin K的最大產量是也在發酵後第十天時的66μg/ml,而其紅麴色素的最大產量在第十天時達到18.705mg/ml。 M.rubber在七公升的氣舉式發酵槽中,發酵條件為通氣量3.5vvm在25℃發酵培養十四天,以白米-蔬菜油培養基起始pH值為5,到第五天時monacolin K達到最大產量為51μg/ml,紅麴色素在第十四天時達最大產量為12.480mg/ml。 由上述結果可知,以Monascus rubber CCRC31535為實驗菌株不論是要生產monacolin K或是以生產紅麴紅色素為重點,都是以攪拌式發酵槽且起始pH值為5的發酵產量最佳,氣舉式發酵槽的效果比較差,而且實驗中所用之培養基皆為食品級,所以將來可以應用在工業上來大量生產,做為保健食品。 Abstract Monacolin K is a secondary metabolite of Monascus spp.,which is a mold used in brewing by the Chinese. It can be used as a medicine to lower cholesterol in human body. The red color Monascus pigment is a good source of natural food colorant. The purpose of this study is using surbmerged culture of Monascus rubber CCRC 31535 for process optimization in production monacolin K and Monascus pigment。 Fed-batch fermentation using air lift fermentor and stirred tank equipped with marine type impeller (MST) fermentor, and using rice-glycerol (RG) or rice-vegetable oil (RV) complex medium was examed. The results showed that, for the 5 liter MST fermentor, at 25℃, agitation speed 250 rpm, aeration rate 0.85 vvm, utilizing RG medium at initial pH 5, the production of monacolin K had the maximum of 156 μg/ml after 10 day fermentation. Higher result was found in RV medium (164 μg/ml) on day 10, and Monascus pigment had the maximal productivity of 30.822 mg/ml on day 8. For RV medium but without adjusting initial pH, the maximal monacolin K and Monascus pigment production were 66μg/ml and 18.705 mg/ml, respectively, under the same fermentation conditions. In the 7 liter air lift fermentor, utilizing RV medium with initial pH 5,aeration rate 0.6 vvm, at 25 ℃, although the fermentation run for 14 days, the maximal productivity for monacolin K was 51μg/ml on day 5 , and Monascus pigment reaches its maximal production ,12.480 mg/ml on day 14. Results indicated when using M. rubber CCRC31535 for both monacolin K and Monascus pigment production at the same time, the best result was in using the MST fermentor and RV medium with initial pH 5。Air lift fermentor showed poor results. The medium used in the experiments are food grade. They may be adapted in the industry for health food production.

Record ID 第116筆 System ID 090THU00253013
BCRC ID CCRC 12257, CCRC 10695, CCRC 10697, CCRC 11844, CCRC 11847, Public Year 90
Paper Name 利用單獨及混合乳酸菌試製羊乳酸凝酪之研究 A study on the manufacture of goat milk yogurt using single and mixed strains of lactic acid starters
Student Name 蕭如吟
Teacher Name 閻立平
School Department Name 東海大學 食品科學系 Academic Degree 碩士
Abstract 本實驗目的在於利用Lactobacillus bulgaricus(CCRC 14009)、Streptococcus thermophilus (CCRC 12257)、Lactobacillus acidophilus (CCRC 10695)、Lactobacillus casei (CCRC 10697)、Bifidobacterium bifidum (CCRC 11844)及Bifidobacterium longum (CCRC 11847) 為菌,單獨或混合接種於羊乳中發酵製成酸凝酪。實驗共分成三部份:試驗一,接種3﹪單獨或混合菌於羊乳中,探討羊乳於乳酸菌發酵期間之菌數、pH值、滴定酸度及黏度之變化,以比較單獨與混合接種組對發酵羊乳含菌量及性狀之差異。試驗二,測定羊乳於乳酸菌發酵前後乳糖、半乳糖、有機酸、L(+)型乳酸及葉酸之含量,以比較單獨與混合接種組對發酵羊乳成分之差異。試驗三,探討以混合接種組之羊乳酸凝酪貯存期間菌數、滴定酸度、黏度及乳清析出率之變化,以篩選較佳之混合乳酸菌組。 試驗一結果:所有單獨接種組完成發酵後,其菌數均達1.0×108 CFU/ml(8.0 Log CFU/ml)以上;混合接種組則皆達1.0×109 CFU/ml(9.0 Log CFU/ml)以上;因此乳酸菌混合培養於羊乳中的生長狀況比單獨培養時好。單獨接種組產酸能力以L. bulgaricus組較強,完成發酵(pH=4.5)所需時間較短;混合接種組則以S. thermophilus+L. bulgaricus組最強,所需發酵時間最短;大部分混合接種組產酸速度皆較單獨接種組快。黏度方面,單獨接種組以L. bulgaricus組上升最快;混合接種組則以S. thermophilus+L. bulgaricus組的黏度上升最快亦最高,並且混合接種組可以提高單獨接種組之黏度。 試驗二結果:單獨接種組之乳糖殘留量,以L. bulgaricus及B. longum顯著最少;混合接種組則是S. thermophilus+L. bulgaricus組殘留量顯著最少;而在以上各接種組之半乳糖增加量皆為最高;混合接種組皆較單獨接種組有較佳的乳糖分解能力。乳酸及L(+)型乳酸產量,單獨接種組以L. bulgaricus組較高;混合培養組則以S. thermophilus+L. bulgaricus組生成量最多;混合接種組之產量亦多高於單獨接種組。在醋酸方面,使用混合接種組可降低單獨使用B. bifidum或B. longum組醋酸造成的不良風味。各單獨接種組發酵後,葉酸含量都有增加,以B. bifidum組產量最高;而混合接種組也都有顯著增加。 試驗三結果:接種七組混合菌製成羊乳酸凝酪,貯存28天後其菌數均仍有5.0 ×108 CFU/ml(8.7 Log CFU/ml)以上;在滴定酸度與乳清析出方面,皆隨著貯存時間增加而有增加之現象;但在黏度則有下降之趨勢。七接種組中以B. longum+ S. thermophilus+ L.acidophilus及B. bifidum+S. thermophilus+L. bulgaricus組貯存至28天其菌數、酸度皆符合CNS 3058之標準;彼等乳糖分解能力也較佳,所產生之L(+)型乳酸量較高、醋酸含量較低,在葉酸產量也顯著高於原料羊乳之含量。 綜合上述實驗結果可以選擇B. longum+ S. thermophilus+ L.acidophilus及B. bifidum+S. thermophilus+L. bulgaricus混合乳酸菌組作為較佳之羊乳酸凝酪菌,以得到較高營養價值之產品。 The purpose of this research is to study the manufacture of goat milk yogurt by using single or mixed strains of Lactobacillus bulgaricus(CCRC 14009), Streptococcus thermophilus (CCRC 12257), Lactobacillus acidophilus (CCRC 10695), Lactobacillus casei (CCRC 10697), Bifidobacterium bifidum (CCRC 11844) and Bifidobacterium longum (CCRC 11847) as the starter organisms。 The objective of the first part of this research - Experiment 1, was to compare the effect of using single-strain starters and mixed- strain starters on the viable count and characteristics(pH, titratable acidity and viscosity)of the goat milk yogurt during fermentation. The objective of the second part of this research - Experiment 2, was to compare the effect of using single-strain starters and mixed-strain starters on the nutrient contents(lactose, galactose, organic acid, L(+) lactate and folic acid)of the goat milk yogurt before and after fermentation . The objective of the third part of this research - Experiment 3, was to select the proper mixed-strain starters based on changes of viable count, titratable acidity, viscosity and syneresis during refrigerated storage of the yogurt. In Experiment 1, the yogurt inoculated with mixed-strain starters showed higher average viable count (>1.0×109 CFU/ml or 9.0 Log CFU/ml) than that (>1.0×108 CFU/ml or 8.0 Log CFU/ml) of the yogurt inoculated with single-strain starters. L. bulgaricus and S. thermophilus+ L. bulgaricus showed highest acid-producing ability, shortest fermentation time (pH=4.5) and highest viscosity among the single-strain starters and mixed-strain starters, respectively. Most mixed-strain starters had higher acid-producing ability and viscosity than single-strain starters. In Experiment 2, the residual lactose content was significantly least in yogurts inoculated with L. bulgaricus, B. longum and S. thermophilus+ L. bulgaricus, which expressed highest increase in galactose content consequently. Mixed-strain starters showed better lactose-hydrolyzing ability than single-strain starters. L. bulgaricus and S. thermophilus+ L. bulgaricus showed highest content of lactate and L(+) lactate among the single-strain starters and mixed-strain starters, respectively. Most mixed-strain starters had higher lactate and L(+) lactate-producing ability than single-strain starters. The acetate content, which may cause off-flavor in yogurt, was effectively reduced when using mixed-strain starters instead of single-strain starters of B. bifidum or B. longum. Folic acid content increased in yogurt inoculated with either single or mixed-strain starters. Among the single-strain starters, B. bifidum caused the highest increase of folic acid. In Experiment 3, viable counts of all yogurt samples inoculated with mixed-strain starters maintained above 5.0×108 CFU/ml (8.7 Log CFU/ml) at the end of 28-day storage. Titratable acidity and syneresis of yogurt increased with increased storage time. Viscosity of yogurt decreased with increased storage time. Among the 7 mixed-strain starters, B. longum+S. thermophilus+ L. acidophilus and B. bifidum+ S. thermophilus+ L. bulgaricus met the viable count and titratable acidity standards of CNS 3085 at the end of 28-day storage. The above-mentioned two mixed-strain starters also showed better ability in lactose hydrolysis, higher production of L(+) lactate, lower production of acetate and a significant increase of folic acid content after fermentation. Concluding from the above results, mixed-strain starters of B. longum +S. thermophilus+ L. acidophilus and B. bifidum+ S. thermophilus+ L. bulgaricus could be selected as the better starters to manufacture more nutritive products.

Record ID 第117筆 System ID 090THU00289008
BCRC ID CCRC 12257, CCRC 14009, Public Year 90
Paper Name 產胞外多醣體之乳酸菌對羊乳酸酪乳品質之影響 The effects of exopolysaccharide-producing lactic acid bacteria on the qualities of caprine yogurt
Student Name 盛兆盈
Teacher Name 洪連欉
School Department Name 東海大學 畜產學系 Academic Degree 碩士
Abstract 實驗旨在利用可產胞外多醣體之黏質乳酸菌以改善羊乳酸酪乳之品質。實驗菌株為本實驗室篩選出之黏質乳酸菌Streptococcus thermophilus A1、A2、Lactobacillus bulgaricus B2、E、與一般商用菌酛S. thermophilus CCRC 12257(S) 與L. bulgaricus CCRC 14009(L)。 分別接種球菌A1、A2與桿菌B2、E等四株黏質乳酸菌於10% TS之還原羊乳中,並於32℃與42℃發酵72小時。結果顯示,四株黏質乳酸菌於發酵期間之pH值隨培養時間而下降,菌數、黏絲性、黏度則呈現出二次元程式之曲線,產酸能力以42℃大於32℃,而32℃時以球菌A1、A2之產酸能力較高。最高乳酸菌數與最大黏度皆為42℃高於32℃者,且以球菌A1、A2高於桿菌B2、E。黏絲性與胞外多醣體產量皆為32℃高於42℃,且球菌A1、A2高於桿菌B2與E。而胞外多醣體產量則隨著培養時間而呈降低之趨勢。 分別以黏質乳酸菌與一般商用乳酸菌單獨使用、黏質乳酸菌之球菌與桿菌互相配對、黏質乳酸菌與一般商用乳酸菌之球菌與桿菌互相配對之方式,在27、32、37與42℃下發酵至pH值達4.5-4.6,製成羊乳酸酪乳。結果顯示,所有組別之酸酪乳其乳酸菌數皆能達8個對數值以上。在各培養溫度下之黏絲性與黏度,黏質酸酪乳皆比一般酸酪乳高(P<0.05),且以培養溫度32℃與37℃培養者高於27℃與42℃者(P<0.05),單株菌為球菌高於桿菌,混合菌則為黏質乳酸菌互相配對者高於黏質乳酸菌與一般商用乳酸菌配對者(P<0.05)。在各培養溫度下之乳清析出率,黏質酸酪乳皆比一般酸酪乳低(P<0.05),且32℃與37℃培養者低於27℃或42℃(P<0.05),單株菌之乳清析出率為球菌低於桿菌(P<0.05),混合菌之乳清析出率則以黏質乳酸菌互相配對者低於黏質乳酸菌與一般商用乳酸菌配對者。胞外多醣體之產量單株菌於27℃培養者為最低,而混和菌低於單株菌(P<0.05),胞外多醣體產量與酸酪乳黏度兩者間並無相關。物性方面,以培養溫度為主要影響因子,黏質酸酪乳之凝乳強度隨培養溫度之上昇而提高,在同樣培養溫度下比較,單株黏質乳酸菌之凝乳強度較單株商用菌者高,而混合菌在黏質酸酪乳與一般酸酪乳間並無明顯之差異。在貯存期間,黏質酸酪乳皆能保持較高之黏度與較低之乳清析出率(P<0.05)。官能品評之結果,黏質酸酪乳之外觀及口感較一般酸酪乳黏稠,且第零週之風味與總接受度亦較高,但經貯存兩週後則無明顯差異。 Ropy lactic acid bacteria were used to manufacture ropy caprine milk yogurt, in order to improve the qualities of caprine milk yogurt. Four ropy strains, Streptococcus thermophilus A1, A2, Lactobacillus bulgaricus B2, E and two commercial strains, Streptococcus thermophilus CCRC12257 (S) and Lactobacillus bulgaricus CCRC14009 (L) were used。 Ropy strains were incubated in 10% TS caprine milk at 32 and 42℃ for 72 hours. The pH value of samples decreased during the incubation. The bacterial counts, thread forming properties and viscosity displayed a quadratic curve. The exopolysaccharide (EPS) concentration had a trend toward reduction. The lactic acid production, maximal bacterial counts and viscosity of samples fermented at 42℃ greater than those were fermented at 32℃, while A1 and A2 sets were predominant. The thread forming properties and EPS concentration of samples fermented at 32℃ greater than those were fermented at 42℃, while A1 and A2 sets were predominant. Caprine yogurt were made at 27, 32, 37 and 42℃ by single ropy strains or mixed strains .The bacterial counts of all samples reached 108 cfu/ml. The thread forming properties and viscosity of ropy yogurt were higher than those of commercial yogurt at all incubation temperature. The incubation temperature of 32 and 37℃ resulted in the higher viscosity while the A1 and A2 sets were better than B2 and E sets. The mixed strains sets of ropy and ropy strains were better than the mixed strains sets of ropy and commercial strains. The syneresis of ropy yogurt were lower than commercial yogurt at all incubation temperature and the incubation temperature of 32 and 37℃ resulted in the lower syneresis while the A1 and A2 sets were lower than B2 and E sets. The mixed strains sets of ropy and ropy strains were lower than the mixed strains sets of ropy and commercial strains. Ropy yogurt had the least amounts of EPS at 27℃.The mixed strains resulted in less amounts of EPS than single strains, but did not affect the other characteristics. There’s no relationship between the viscosity and EPS concentration. The rheology of caprine yogurt was mainly affected by the incubation temperature. The gel strength of ropy yogurt increased with the increased incubation temperature. There’s no difference between ropy yogurt and commercial yogurt at the same incubation temperature. The ropy yogurt had the higher viscosity and the lower syneresis than commercial yogurt during the storage. The appearance and mouth-feel of ropy yogurt were higher than those of commercial yogurt. The flavor and overall acceptability of ropy yogurt were higher than those of commercial yogurt at the beginning of storage, but there’s no difference after 2 weeks.

Record ID 第118筆 System ID 089THU00063003
BCRC ID CCRC 35396, Public Year 89
Paper Name 樟芝菌絲體深層培養之研究
Student Name 黃惠琴
Teacher Name 楊芳鏘
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 本研究以Antrodia cinnamomea CCRC 35396為主要試驗菌株,利用三角瓶液態培養,探討不同物理及化學環境因子,對樟芝菌絲體生長之影響,希望藉由培養因子的調整與控制提高菌絲生長速率及菌體濃度。 主要實驗項目:以三角瓶批式培養,探討時間、pH、溫度、轉速、表面通氣、培養成分等因子對菌絲生長與多醣生成之影響,並利用回應曲面法提高菌絲體產量。 結果顯示:在250ml三角瓶批式培養中,樟芝菌絲生長的最適培養溫度為25℃,初始pH值為5,轉速為100rpm,在此條件下培養14天菌體濃度達486.8mg/100ml。過高的表面通氣效應及過快的振盪轉速皆不利於菌絲球生長,最適迴轉速率在100rpm左右。在碳、氮源種類方面:玉米粉及YM Broth對菌體生長有較佳表現。 利用回應曲面法(RSM)探討碳源濃度、氮源濃度和初始pH值因子交錯影響,決定最佳培養基組成的條件下,培養7天菌體濃度可達到2164.2mg/100ml,產量比原濃度提昇5倍以上。本研究結果證實利用三角瓶培養條件控制,配合回應曲面法培養組成決定,可以有效的縮短樟芝培養時間,並且能夠快速提高菌絲體產量。 In this study the influence of different physical and chemical factors on the production of biomass and polysaccharide by Antrodia cinnamomea CCRC 35396 was investigated in the submerged culture of using shake flasks。 Optimal cultural conditions were expected to decide to enhance the growth rate and the mycelium concentration. The main concerned cultural conditions include: time, pH, temperature, rotation speed, surface aeration and medium compositions, etc. In additions, Response Surface Methodology (RSM) was used to optimize the medium composition in order to increase the mycelium concentration. The results indicated that when the submerged culture in shake flasks was operated at 25℃, initial pH 5 and rotation speed 100rpm, the mycelium concentration reached to 486.8mg/100ml at 14th day. Both the high surface aeration rate and rotation speed were unfavorable to the growth of mycelium. The optimal rotation speed was shown to be around 100rpm. After the comparison of various sources of carbon and nitrogen, corn powder and YM broth were selected for the further tests of RSM. According to the RSM, the medium composition was modified and the mycelium concentration of Antrodia cinnamomea reached to 2164.2mg/100ml at the 7th day and had a 5-fold increase, compared to the growth in basal medium. The results proved that the production rate of mycelium of Antrodia cinnamomea could be highly enhanced by means of the control of cultural conditions and the modification of medium compositions based on the RSM.

Record ID 第119筆 System ID 085THU00289008
BCRC ID CCRC 10695, CCRC 14009, CCRC 12257, CCRC 11844, CCRC 11847, Public Year 85
Paper Name 不同乳酸菌應用於乾燥酸酪乳製造之研究 Studies of different lactic acid bacteria on the production of dried yogurt
Student Name 黃俊達
Teacher Name 施宗雄
School Department Name 東海大學 畜產學研究所 Academic Degree 碩士
Abstract 本實驗目的在探討於製造乾燥酸酪乳時,分別添加Lactobacillus bulgaricus(CCRC 14009)、Streptococcus thermophilus(CCRC 12257)、 L. acidophilus (CCRC 10695)、 Bifidobacterium bifidum(CCRC 11844)及B. longum(CCRC 11847) 為乳酸菌菌元,測定 其理化學性狀和 微生物學之變化,以供日後有關研究和利用之參考。試驗共分為兩個階段 ,第一階段為菌元的生長試驗,將混合菌元接種3%(V/V)於含固形物16%的 滅菌脫脂乳中,測定其發酵過程中的乳酸菌數、酸度、黏度、糖類及有機 酸之變化以作為基本的發酵條件;第二階段為乾燥酸酪乳之製備與貯存, 將混合菌元製成的酸酪乳依噴霧乾燥與冷凍乾燥的方法製成乾燥酸酪乳, 並於4℃及25℃下貯存五個月,每月採樣測其乳酸菌數、半乳糖  ((-galactosidase)活性、溶解度與滴定酸度之變化。 其結果顯示如下:1、混合菌元以L. bulgaricus與Str. thermophilus(1:1)混合,在42℃培養時的乳糖分解能力與產酸能力最高 。2、三組混合菌元之生長菌數,在第48小時 ,各組的菌數仍可維持 在108 CFU/ml 以上。3、以L. bulgaricus與Str. thermophilus (1:1)混合培養時,平均有較佳的黏度表現。4、L(+)型乳酸的含量會隨著 培養時間的增加而增加,以L. bulgaricus與Str. thermophilus(1:1)混 合培養時的生成量最高(p<0.05) ,在第48小時的生成量達1.14 g/dl。5 、噴霧乾燥過程中,在入風口溫度140℃,出風口 70℃的處理組,其水活 性含量有偏高的情形,平均達0.798。6、β-半乳糖 以冷凍 乾燥處 理有較高的活性,而在噴霧乾燥時,出風口溫度越高,其活性越低。7、 在4℃下貯存時,β-半乳糖 的活性較高。8、在貯存期間,以L. bulgaricus與Str. thermophilus (1:1)混合處理,保有較高的 β-半乳糖 活性;其次為L. acidophilus與B. longum(1:1)混合處理 。9、在溶解度方面,以L. bulgaricus與Str. thermophilus 製成的乾燥 酸酪乳,溶解度指數較低。10、在貯存期間,以 L. bulgaricus與Str. thermophilus混合製成的乾燥酸酪乳,有較高的滴定酸度表現。11、在4 ℃下貯存的乾燥 酸酪乳其乳酸菌比在25℃下貯存時要高p<0.05);在菌元 的選擇上,以L. acidophilus 與B. longum混合處理有較高的菌數表 現(p<0.05)。12、在乾燥條件上,以噴霧乾燥法的 第二組(入風口140℃ ,出風口80℃)處理有最佳表現(p<0.05)。 綜合以上結論,乾燥酸酪乳中的活性乳酸菌以L. acidophilus與B. longum (1:1)混 合培養,經噴霧乾燥(入風口140℃、出風口80℃)處理後 ,在4℃下貯存有最高的活性菌數值,五個月後仍可達108 CFU/g 以上。 The purpose of this experiment is to study that adding Lactobacillus bulgaricus (CCRC 14009), Streptococcus thermophilus (CCRC 12257), L acidophilus (CCRC 10695), Bifidobacterium bifidum (CCRC 11844) and B longum (CCRC 11847) as starters during production process of dried yogurt totest the characteristic of physico-chemical and the changes of microbiologicalfor the reference of later usage。 In exp.1,the grown-up test of starters wasperformed for testing of variation of the numbers of lactic acidbacteria, TA%, viscosity, carbohydrate and organic acid in the process offermentation as basic condition. In exp.2, yogurt fermented by mixedstarters was preparation of dried yogurt by the methods of spray drying and freeze drying and storage at 4 ℃ and 25 ℃ for 5 months. The number of lacticacid bactereia, lactase activity, solubility index and TA% were tested. The results were as follows:1. The starter of L. bulgaricus and Str. thermophilus fermented by the mix ratio of 1:1 at 42 ℃ has the greatest ability of hydrolyzing lactose and producing acid. 2. The numbers of bacteria fermented by mixed starters can keep above 108 CFU/ml at 48 hours. 3.The starter of L. bulgaricus and Str. thermophilus fermented by the mix ratio of 1:1 at 42 ℃ has the better performance of viscosity.4. The maximum production of L(+) lactic acid is the mixture of L. bulgaricus and Str. thermophilus at the mix ratio of 1:1, is 1.14 g/dl at 48 hours during the periods of fermentation (p<0.05).5. In the process of spraydrying, inlet temperature is 140 ℃ and outlet temp. is 80 ℃, water activity of sample is tend to be higher.6. The activity of lactase of freeze drying was the higher, and decreased with the increased outlet temp. duringthe process of spray drying.7. The activity of lactase under the storage temp. of 4 ℃ is higher.8. The mixture of L. bulgaricus and Str. thermophilus (1:1) has the highest lactase activity under storage, the mixture of L.acidophilus and B. longum (1:1) is next to the above. 9. The mixture of L. bulgaricus and Str. thermophilus (1: 1) has the lowest solubility index. 10. Dried yogurt manufactured from the mixture of L. bulgaricus and Str. thermophilus (1:1) has the higher titrable acidity.11.During the storage period within five months at 4 ℃ and 25 ℃, the numbers of lactic acid bacteria were large under 4 ℃ (p<0.05), the numbers of lactic acid bacteria were large with the mixture of L. acidophilus and B. longum (p<0.05). 12.In the conditions of drying,the process of spray drying (inlet temp.140 ℃ and outlet temp.80 ℃ ) has the bestperformance on the numbers lactic acid bacteria. In general, the lactic acid bacteria in dried yogurt manufactured from themixture of L. acidophilus and B.longum (1:1), by spray drying (inlet temp. 140 ℃ and outlet temp. 80 ℃ ) has the highest performance under 4 ℃ storage for five months.

Record ID 第120筆 System ID 085THU00289009
BCRC ID CCRC 12257, CCRC 10695, CCRC 11844, CCRC 11847, Public Year 85
Paper Name 不同乳酸菌應用於羊乳酸酪乳製造之研究 Studies of Different Lactic acid Bacteria on the Production of
Student Name 葉金桂
Teacher Name 施宗雄
School Department Name 東海大學 畜產學研究所 Academic Degree 碩士
Abstract 本研究旨在探討於羊乳中分別接種 Lactobacillus. bulgaricus(CCRC 14009)、Streptococcus. thermophilus (CCRC 12257)、 L. acidophilus (CCRC 10695)、 Bido-bacterium. bifidum( CCRC 11844)、B. longum(CCRC 11847) 為菌元調製成酸酪乳, 分別測定 其理化學性狀和生物學之變化並探討將來利用之可行性。試驗共分三階段 :第一階段為不同菌元之生長試驗, 採用單一或混合菌元接種 3%(V/V) 於滅菌羊乳中,測定發酵過程中之菌數、酸度、糖類及有機酸的變化以做 為其他階段之基本發酵條件;第二階段測定菌元發酵前後膽固醇、 脂肪 酸、 葉酸及游離胺基酸之變化; 第 T 階段為接種 (S.thermophilus + L. bulgaricus、L. acidophilus + B. bifidum、L. acidophilus + B.longum) 製成羊乳酸酪乳之貯存試驗。 第一階段試驗結果顯示:接種單株菌中以L. bulgaricus於第6小時最早達 到最高菌數,混合菌元則以S. thermophilus 與L. bulgaricus混合接種 組於第6小時最早達到最高菌數,所有菌元平穩期可維持14小時左右;菌 元分解乳糖與產酸能力,單株菌元以L. bulgaricus最強(p<0.05), 混合菌元則以S. thermophilus與L. bulgaricus組較強;L(+)型乳酸,單 株菌以L. bulgaricus 及S. thermophilus產生量最高(p<0.05),混合菌 元則 以S. thermophilus與L. bulgaricus混合組產生量顯著較高( p<0.05);黏度方面,混合菌元可改善單一菌元不佳之問題。 第二階段試驗結果: 脂肪酸組成方面, 所有菌元於發酵後飽和短 中鏈脂肪酸(C4~C10) 有下降的趨勢 (p>0.05); 膽固醇方面,發酵後皆 有上升情形; 葉酸含量,除L. bulgaricus 於發酵後有下降情形外, 其 餘各組皆有增加, 其中以 S. thermophilus增加 1.75 倍為最高 (p<0.05); 游離胺基酸的變化, S. thermophilus 及 B. bifidum於發 酵後,游離必需胺基酸皆有增加趨勢。 第三階段試驗結果: 三組接種不同混合菌元羊乳酸酪乳於貯存四週後其 菌數均尚達109 CFU/ml;貯存期間酸度有輕微上升 (p<0.05);三組中β - 半乳糖活性皆有阀¨貯存時間而下降之趨勢; 乳清析出,三組皆隨 貯存時間增加而輕微增加 (p<0.05);但是黏度則有些許下降之趨勢;在 貯存期間,其組成分 ( 蛋白質、脂肪、灰分等 ) 皆無顯著的改變 (p>0.05); 而在官能品評方面,則隨貯存時間之增加,其總接收性平均 皆有下降之趨勢,然而質地組織並無顯著的改變 (p>0.05);於貯存期間 ,以 L. acidophilus 與 B.bifidum 混合組平均外觀品評值最低。 綜合上述結論, 試製羊乳酸酪乳於貯存四週後,均可維持菌數在 109 CFU/ml,符合中國國家標準對一般發酵乳的規定 (107 CFU/ml); 而原本 羊乳中就含有較低的乳糖,製成酸酪乳後有部分被分解再加上含有大量的 乳糖分解存在,因此有利於乳糖不耐症者食用;然羊乳酸酪乳仍具有的 特殊羊騷味,是否能廣受大眾喜愛,如何改善並研發多種適當調合口味, 以符合產品多元化的市場需求,則有待將來進一步的研究探討。 Five strains of lactic acid bacteria Lactobacillus bulgaricus (CCRC14009), Streptococcus thermophilus (CCRC 12257), L acidophilus (CCRC 10695),B bifidum (CCRC 11844) and B longum (CCRC 11847) were used as startersculture on the manufacturing of goat(s yogurt。 In experiment Ⅰ the grown-uptest of single or mixed starters was performed for testing of variation of thenumbers of lactic acid bacteria, TA%, carbohydrate and organic acid in theprocess of fermentation as basic condition. In exp. Ⅱ, C ange on content ofcholesterol, free fatty acid, folic acid and free amino acid by exp. Ⅰstarter used after fermentation.In exp. Ⅲ, three goat(s yogurt ( L.bulgaricus + S. thermophilus、 L. acidophilus + B. bifidum and L. acidophilus +B. longum ) was examined during processing and storaging at 4 ℃ for 4 weeks. The results were as follows: Experiment Ⅰ. 1. L. bulgaricus was the starter first reached stationary phase within sixhours. Mixed starters of S. thermophilus and L. bulgaricus (1:1), were the early reached the stationary phase within six hours. The stationary phase can keep going for about 14 hours. 2. L. bulgaricus had the strongest ability in lactose hydrolysis and lacticacid forming ( p<0.05). Mixed starters of S. thermophilus and L. bulgaricus had the strongest ability in lactose hydrolysis and lactic acid forming(p<0.05). 3. The maximum production of L(+) lactic acid is the mixture L. bulgaricus andS. thermophilus (p<0.05) at the mix ratio of 1:1, the average is 1.42 (g/dl)during the periods of fermentation. In the single starter was L. bulgaicusproducing maximum L(+) lactic acid (p<0.05). 4. The L. bulgaricus has the better perfermance of viscosity in the singlestarter treatments, and the mixed starter can improve the viscosity fermentedwith single starter. Exp. Ⅱ. 5. After fermentation, the amont of saturated short chain fatty acid (C4-C10)had decreased (p>0.05) in all starters. 6. Cholesterol content was increased after fermentation in all starters. 7. Folic acid content, except L. bulgaricus was decreased after fermentation,others were increased. 8.After fermentation, free amino acids content of S. thermophilus and B.bifidum group increased, individually, which higher than other strains. Exp.Ⅲ 9. 4 weeks of storage, the lactic acid bacteria counts of three goat(s yogurttreatments were 109 CFU/ml, there were slightly increased during storage. 10.With the increasing of storage time, the activity of β -galactosidase ofthree goat milk yogurt treatments was decreased. 11. With the increasing of storage time, the syneresis increase slightly amongthe three treatments, and the viscosity decreased slightly. 12. During storage, three goat(s yogurt were not different significantly inyogurt composition (protein, fat and ash,etc) (p>0.05). 13. In respect of organoleptic test, with the increasing of storage time, themean total acceptability decrease, but there is not different significantly intexture (p>0.05). During the storage time of goat milk yogurt, both L.acidophilus and B. bifidum had the lowest mean appearance score. In general, the lactic acid bacteria in goat yogurt decreased duringstorage. But they can still maintain above 109 CFU/ml during four weeksstorage which is reaching the Chinese National Standard about the fermentedmilk ( 107 CFU/ ml). Not only goat milk was low lactose content than cow milk, but also a great deal of lactose were hydrolyzed in the goat,s yogurt whichshould be advantagous of lactase intolerance consume it. Concering goat milkordor, which applied to developing market, many kinds of fla ored productsshould be developed to satisfy the request of the multiplicities of products.

Record ID 第121筆 System ID 088THU00289003
BCRC ID CCRC 14098, CCRC 12268, Public Year 88
Paper Name 篩選產黏乳酸菌以改善酸酪乳品質 Selecting Slime-producing Lactic Acid Bacteria for Improving the Quality of Yogurt
Student Name 簡君祐
Teacher Name 洪連欉
School Department Name 東海大學 畜產學系 Academic Degree 碩士
Abstract 本研究旨在篩選產黏乳酸菌株試製黏質酸酪乳,期能取代傳統酸酪乳中添加之膠類,以節省生產成本和改善酸酪乳品質。 利用10株商用乳酸菌分別於MEPS-1, MEPS-2, MEPS-3, MRS plus broth及Elliker broth等五種培養基,以27, 32, 37, 42℃等溫度培養8, 16及24小時後,結果以MEPS-2培養基(9% SNF脫脂乳中添加0.35% 酵母萃取物、0.35% 蛋白 及5% 乳糖)於32℃經16小時培養者之黏絲性程度顯著大於其他組(P<0.05),故選定其作為產黏乳酸菌株之篩選條件。 自市面上採集12種醱酵乳製品,利用MEPS-2培養基於32℃經16小時培養以篩選產黏乳酸菌株,共得Ⅰ、Ⅱ、Ⅲ、Ⅳ及Ⅴ等5個具產黏特性之乳製品;經進行分離菌株並純化培養後,取得4株產黏乳酸菌,其代號分別為A1, A2, B2及E,經鑑定A1和A2為 Streptococcus thermophilus,而B2和E為Lactobacillus bulgaricus。 依桿菌與球菌混合培養方式將產黏菌株配對後,比較接種A1+B2及A2+E菌 於32℃或42℃製備之10% SNF酸酪乳與接種傳統菌 Lactobacillus bulgaricus CCRC 14098 (L) + Streptococcus thermophilus CCRC 12268 (S) 之10% SNF酸酪乳於4℃貯存四週期間之差異。結果顯示,不論是32℃或42℃,以產黏菌株A1+B2及A2+E組之黏絲性程度顯著大於L+S組(P<0.05)、且黏度顯著比L+S組者高(P<0.05)、而乳清析出率則顯著比L+S組者低(P<0.05)、乳酸菌數也高於L+S組,其中又以42℃者之乳酸菌數高於32℃者。官能評估方面,黏質酸酪乳A1+B2組和A2+E組與傳統酸酪乳L+S組比較,結果顯示,黏質酸酪乳之乳清析出明顯較低、黏度明顯較高、質地明顯較平滑及整體總接受性明顯較高。 傳統酸酪乳菌 Lactobacillus bulgaricus CCRC 14098 (L) 和Streptococcus thermophilus CCRC 12268 (S) 混合接種產黏菌株於12% SNF脫脂乳,並分別於27, 32, 37及42℃製備酸酪乳(L+S, L+S+A1, L+S+A2, L+S+B2, L+S+E),比較其品質之差異。結果顯示,L+S+A1, L+S+A2, L+S+B2, L+S+E組之黏絲性程度、黏度皆顯著大於L+S組(P<0.05),而乳清析出率則顯著低於L+S組(P<0.05),其中又以32℃製備者較其他組者佳。 黏質酸酪乳與添加果膠之傳統酸酪乳間產黏特性的比較。結果顯示,傳統酸酪乳之黏絲性程度、黏度隨著乳無脂固形物含量及果膠添加量的增加而顯著增加(P<0.05),而乳清析出率則隨著乳無脂固形物含量及果膠添加量的增加而降低(P<0.05);黏質酸酪乳之黏絲性程度、黏度仍顯著高於添加0.5% 果膠之傳統酸酪乳(P<0.05),而乳清析出率則顯著低於添加0.5% 果膠之傳統酸酪乳(P<0.05)。 The study was conducted to select slime-producing lactic acid bacteria for manufacturing ropy yogurt, in order to replace the addition of pectin in the traditional yogurt, and to save the cost of production and improve the quality of yogurt. Ten strains of commercial lactic acid bacteria, five modified media (MEPS-1, MEPS-2, MEPS-3, MRS plus broth and Elliker broth), four incubation temperatures (27, 32, 37 and 42℃) and three incubation times (8, 16 and 24 hours) were used in the experiments. The results showed that the thread forming property of MEPS-2 medium(9% SNF skimmilk containing 0.35% yeast extract, 0.35% peptone and 5% lactose)under 32℃ at 16 hours was significantly higher than other treatment combinations (P<0.05), so using it as the selective conditions. Twelve commercial fermented dairy products were collected and then the above selective conditions were used to select the slime-producing lactic acid bacteria. It was found that five products named Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ, had the thread forming property. After a series of isolation procedures, four slime-producing strains named A1, A2, B2 and E were purified. A1 and A2 were identified as Streptococcus thermophilus, and B2 and E were identified as Lactobacillus bulgaricus. According to the combination of rod and coccus to match A1, A2, B2 and E. The change of qualities during four-weeks storage of 10% SNF yogurt made with L+S, A1+B2 or A2+E culture incubated at 32℃ and 42℃ was determined. The results showed that, the thread forming property, viscosity , syneresis and lactic acid bacterial counts of A1+B2 and A2+E sets were significantly better than L+S set when incubating at 32℃ or 42℃ (P<0.05), and 32℃ group was better than 42℃ group. The sensory evaluation results of ropy and traditional yogurt showed that, A1+B2 and A2+E sets had lower syneresis, higher viscosity, smoother texture and higher overall aceeptiablity. Evaluating the qualities of 12% SNF yogurt made with Lactobacillus bulgaricus CCRC 14098 (L) + Streptococcus thermophilus CCRC 12268 (S), L+S+A1, L+S+A2, L+S+B2 and L+S+E culture incubated at 27, 32, 37 and 42℃。 The results showed that, the thread forming property, viscosity and syneresis of L+S+A1, L+S+A2, L+S+B2 and L+S+E sets were significantly better than L+S set (P<0.05), and 32℃ group was significantly better than others (P<0.05). Comparing the qualities of ropy yogurt and traditional yogurt containing pectin. It was found that, the thread forming property and viscosity of traditional yogurt were significantly increased with the amount of SNF and pectin increased (P<0.05), and the syneresis was significantly decreased with the amount of SNF and pectin increased (P<0.05). In addition, the thread forming property, viscosity and syneresis of ropy yogurt were still significantly better than the traditional yogurt containing 0.5% pectin (P<0.05).

Record ID 第122筆 System ID 087THU00289010
BCRC ID CCRC 14601, CCRC 14604, CCRC 14605, CCRC 11847, CCRC 14146, CCRC 11844, Public Year 87
Paper Name 冷凍乾燥對標準雙叉桿菌及其篩選菌株生理特性影響之研究 Study of the Effect of Freeze-Drying on the Physiological Characteristics of the Standard and the Selected Bifidobacteria
Student Name 張贊岩
Teacher Name 施宗雄
School Department Name 東海大學 畜產學系 Academic Degree 碩士
Abstract 本試驗先篩選乳酸菌中的雙叉桿菌(Bifidobacteria)之耐氧性菌株再與標準菌株進行冷凍乾燥處理,藉由其生理及生化特性之測定,以了解冷凍乾燥對雙叉桿菌及所篩選菌株之影響。本次試驗選用Bifidobacterium breve CCRC 14601、B. infantis CCRC 14604、B. longum CCRC 14605、B. longum CCRC 11847、B. bifidum CCRC 14146與B. bifidum CCRC 11844作為試驗標準菌株。 試驗一為採用純系選種(Pure culture selection)方式,以標準菌株為基礎,進行選育在有氧環境下能穩定生長的雙叉桿菌,選出後之菌株再與標準菌株比較其生理特性上的差異,包括乳凝固時間、菌數、滴定酸度、β-半乳糖甘脢(β-galactosidase)及α- 半乳糖甘脢(α-galactosidase)活性。試驗二乃將標準菌株與所篩選出之菌株共10株,在冷凍乾燥處理前後,進行各項生理生化試驗,以比較冷凍乾燥對雙叉桿菌的影響,期能篩選出耐氧之雙叉桿菌,以提供乳業界實際之應用。 試驗一結果︰六株標準雙叉桿菌在有氧狀態下經15次繼代培養並加以篩選後,發現除B. longum CCRC 14605與B. bifidum CCRC 11844之外其餘四株雙叉桿菌可明顯在選育後生長於有氧環境下。不過篩選菌株與標準菌株相互比較,發現乳凝固時間顯著比標準菌株長(p<0.05),且產生的凝乳並不完全呈半凝狀態,同時篩選菌株比標準菌株之菌數明顯減少了將近1∼2 log CFU/ml(p<0.05)。在酸度上亦顯著較標準菌株低(p<0.05),可能與各篩選菌株之β-galactosidase活性顯著比標準菌株低(p<0.05)有關。篩選菌株中除B. bifidum 14146的α-galactosidase活性顯著高於其標準菌株外(p<0.05),其餘皆顯著低於標準菌株的活性(p<0.05)。 試驗二結果︰冷凍乾燥後標準菌株之存活率以B. bifidum 14146降低最顯著(p<0.05),而篩選出之雙叉桿菌B. infantis 14604對冷凍乾燥的耐受性最高,其菌數與乾燥前無顯著差異(p>0.05),B. breve 14601與B. bifidum 14146之菌數則降至6 log10/ml以下。乾燥菌體在經溶菌酵素(lysozyme)、牛膽汁(oxgall)及氯化鈉(NaCl)處理後發現,不論標準雙叉桿菌或篩選菌株皆顯著增加對這些物質的敏感性(p<0.05)。另外,冷凍乾燥後的標準菌株及篩選菌株,經培養於含2%乳糖的MRS液態培養基後發現,B. breve 14601及B. infantis 14604在厭氧與有氧的狀態下,β-galactosidase及α-galactosidase活性有顯著增加的現象(p<0.05)。各培養菌液經離心後所得上清液部份,不論標準菌株或篩選菌株皆顯著測得乳糖酵素的存在(p<0.05),代表細胞有通透性改變的現象而造成酵素從菌體內洩漏至細胞外。細胞萃取物方面,從聚丙烯凝膠電泳的結果發現,冷凍乾燥之菌體不論是標準菌株及篩選菌株,經0.2% SDS處理後,明顯比未乾燥菌體多出了一分子量約30 Kd的蛋白質,這可能是菌體受冷凍乾燥的影響,使得在細胞壁上鍵結的蛋白質變得不穩定,故易被萃取出來。 The aims of this study were to select the oxygen-tolerant bifidobacteria first and then investigate the effect of freeze drying on the standard and the selected bifidobacteria which were subjected to this treatment. Six bifidobacteria including Bifidobacterium. breve CCRC 14601, B infantis CCRC 14604, B longum CCRC 14605, B longum CCRC 11847, B bifidum CCRC 14146, and B bifidum CCRC 11844 were studied。 In the first part of this study, pure culture selection was adopted to select the bifidobacteria that could survive normally under aerobic environment and the physiological characteristics which included milk coagulation time, total plate counts of bifidobacteria, titration acidity, activity of β-galactosidase and α-galactosidase for the standard and the selected bifidobacteria were compared. In the second part, the effect of freeze drying on ten standard and selected bifidobacteria in total was further investigated by conducting physiological and biochemical experiments before and after freeze drying. The results of the first part study indicated that four out of six bifidobacteria under aerobic condition, except B. longum CCRC 14605 and B longum CCRC 11847, could normally survive through 15 times transfer cultivation and selection。 It was found that milk coagulation time for the selected bifidobacteria was generally longer than that for the standards (p<0.05) and also the coagulated milk was not completed in the state of semi-coagulation. Moreover, the population of the selected bifidobacteria was obviously less than that of the standards by nearly 1~2 log CFU/ml (p<0.01). The selected bifidobacteria were also markedly lower than the standards in acidity (p <0.05). It was suggested that it might be related to the activity of β-galactosidase for the selected bifidobacteria which was significantly lower than the standards (p<0.01). The yield of lactic acid was indirectly affected and , the acidity was limited for ascending. The selected bifidobacteria were significantly weaker than the standards for the activity of α-galactosidase (p<0.01), except for B. bifidum CCRC 14146 that was much stronger than standards (p<0。01). The results of the second study were indicated that the survival percentage of the standard B. bifidum CCRC14146 significantly declined after freeze drying (p<0。01) and also the population for the two out of four selected bifidobacteria sharply dropped to less than 6 log10/ml. However the selected B. infantis 14604 showed the highest tolerance to the freeze drying, so its population was not obviously changed by freeze drying (p>0.05). Both the standard and the selected bifidobacteria which treated with lysozyme, oxygall and NaCl after freeze drying had significantly increased their sensitivity to these materials (p<0.01). It suggested that freeze drying would affect the stability of cell wall and the permeability of cell membrane for bifidobacteria. In addition, the standard, B. breve CCRC 14601, and the selected B infantis CCRC 14604 incubated in MRS broth with 2% lactose after freeze drying had apparently increased the activity of β-galactosidase and α-galactosidase under anaerobic and naerobic conditions。 The supernatant for the standard and the selected bifidobacteria were detected obviously to have the above two enzymes after centrifuging (p<0.01), which indicated that the permeability of bifidobacteria cell membrane were changed, therefore the enzymes could flow and leak in outside of the cell. The SDS-PAGE for the cell extract revealed that both the standard and the selected bifidobacteria treated with 0.2% SDS after freeze drying had apparently about 30Kd molecular weight of protein as compared to that without freeze drying. It suggested that freeze drying had made the linkage of protein on the cell wall of bifidobacteria unstable, so that the protein could be readily extracted.

Record ID 第123筆 System ID 083THU00286004
BCRC ID CCRC 14009, CCRC 12257, CCRC 14072, CCRC 10791, Public Year 83
Paper Name 不同乳酸菌應用於製造冷凍酸酪乳其生化特性之研究 Studies on the Biochemical Characteritic of Different Lactic Acid Bacteria of Frozen yogurt Processing
Student Name 顏成君
Teacher Name 施宗雄
School Department Name 東海大學 畜牧學系 Academic Degree 碩士
Abstract 於調製冷凍酸酪乳時分別添加保加利亞乳桿菌(~u2;Lactobacillus~u1; u1; CCRC 14009)、嗜熱乳鏈球菌(~u2;Streptococcus~u1;s~u1; CCRC 12257)、嗜酸乳桿菌 (~u2;Lactobacillus~u1;~u1; CCRC 14072)及乳鏈 球菌 (~u2;Lactococcus~u1; ~u2;lactis~u1; w其理化學性狀和生 物學影響以及將來利用之可行性。試驗共分為三個階階段為菌元之生長試 驗,採用單一或混合菌元接種3%(V/V)於滅菌脫脂乳中中孝葝①B酸度、糖 類及有機酸的變化以做為基本發酵之條件。過不同菌元發酵過後之脫脂酸 酪乳為主要材料再調配冰淇淋混料經均質混缶-31℃等三種冷凍溫度下, 然後每週測定其中乳酸菌數及乳糖分解▋之變士採用~u2;L~u1;.~u2; bulgaricus~u1; CCRC 14009 與~u2;Str~u1;.s~u1; CCRC?2℃下培養為 製做冷凍酸酪乳之基質,並調配成混合料並經正規操作過程A然後測定其 製程及成品保存五週其品質之變化。其結果顯示如下:1.上述鷎i後6-7小 時達到生長平穩期(stationary phase),並且可維持16小時左右。N凍狀 態下,在-5℃下乳酸菌數均有明顯下降之趨勢,且桿菌大於球菌,然下, 經儲存五週,各菌株之菌數並未造成明顯地減少(P>0.01)。s程中均質化 處理有使乳酸菌數增加的效果(P<0.01)。4.冷凍攪打的過程中a下降( P<0.01)。5.經冷凍攪打後乳糖▋活性有增高的趨勢(P<0.01)。6.冷18℃ 及-31℃儲存五週過程中,活性乳酸菌數以-31℃較高,而乳糖▋活性 ?01)。 |株試驗菌株在製作冷凍酸酪乳中的活性乳酸菌雖然在製程及冷 凍保存保存l傷或死亡,但在五週保存期間,均可維持在 7 LogX中國國家 標準對一般發酵乳的規定(7 Log中又有大量的乳糖▋,因此該產品應可被 接受。不過實際應用於市場開發元g過適當調合的口味,以符合產品多元化 的訴求,則有待將來進一步之研究。 Four strains of lactic acid bacteria ~u2;L~u1;.~u2; bulgaricus~u1;;Str~U1;.~U2;thermophilus~U1; CCRC 12257,cidopholus~U1; CCRC 14072, ~U2;Lac~U1;~U2;lactis~U1; CCRC 10791arter culture on the manufacturing of frozen yogurt。 Yogurtdifferent starters was combined with ice cream mix. Yogurt2;L~U1;.~U2;bulgaricus~U1; and ~U2;Str~U1;. ~U2; thermophilus~U1;as selected for manufacturing of frozen yogurt. The quality of the frozen yogurt was examinedng and storage for 5 weeks. The results were as follows: 1. Theteria reached the stationary phase within six or seven hours. Thee kept going for about 16 hours. 2. The bacteria numbers of fourains did not decrease under the temperature of -31℃ for fiveThe numbers of lactic acid bacteria reduced significantly under the range of reduction Lactobacillus are larger than. Homogenization had the effect of increasing the number ofteria (p<0.01). 4. The process of soft serving decreased theic acid bacteria significantly (p<0.01). 5. The activity ofed (p<0.01) after the process of soft serve treatment. 6. Duringiod within five weeks at -18℃ and -31℃, the numbers lactic acidarge under -31℃, but the activity of lactase was not significantly different (p>0.01) under the twogeneral, the lactic acid bacteria in frozen yogurt was damagedthe process and frozen storage. But they still maintained above 7ng five weeks storage which is reaching the Chinese Nationalthe fermented milk (7 Log CFU/ml). There was a great deal offrozen yogurt which should be acceptable.

Record ID 第124筆 System ID 081THU00253004
BCRC ID CCRC 30196, Public Year 81
Paper Name 米麴菌蛋白生產及應用之研究 Studies on production and application of Aspergillus oryzae proteases
Student Name 蔡慧霖
Teacher Name 李根永
School Department Name 東海大學 食品科學系 Academic Degree 碩士
Abstract 米麴菌(Aspergillus oryzae CCRC 30196)在米糠加磷酸緩衝液之固態 發酵培養基,31℃,pH10.5的條件下,培養3-5天,可得最佳蛋白之產量。 此培養基不需要添加任何蛋白質誘導米麴菌生成蛋白,若於培養基中添 加葡萄糖則會有catabolite repression 的現象發生。培養完成,加水於 米糠培養基中抽取酵素,酵素液再經由下列步驟純化:硫酸銨沈澱,丙酮 脫色,Sephadex G-150膠體過濾,以及DEAE-Sephadex A-50 離子交換層析 。結果純化之蛋白,比活性由1.4μmol/mg protein ,提高到17.14μ mol/mg protein 。而在 DEAE離子交換層析步驟中,可分離中性及鹼性 蛋白。  蛋白水解魚肉之研究中,蛋白與魚肉基質的體積比 為10:1 (蛋白活性單位為8.83μmol/ml),反應溫度為50℃。在魚肉水 解液胺基酸組成分析中,比較純化蛋白、市售米麴菌蛋白NP-10及硫 酸銨沈澱酵素三種水解液,發現本實驗純化蛋白處理之水解液中,以麩 胺酸(Glu)、苯丙胺酸(Phe)、白胺酸(Leu)及甲硫胺酸 (Met)有明 顯游離,可能與蛋白作用位置有關。  魚肉水解液有苦味產生,此苦 味胜經由Sephadex G-50及Cellulose GCL-25之管柱層析分離,可得單 一波峰。以Cellulose GCL-25膠體層析法測定此苦味胜分子量,經計算 估計約為1400道爾頓。 Asp. oryzae produced fair amount of proteases at 31℃ in rice bran media with phosphate buffer adjusting pH to 10.5. In this medium,adding protein to induce proteases producing was not necessary.However,affluent glucose in the medium would cause catabolite repression on enzyme producing. The adequate fermentation time for enzyme production was 3-5 days.After fermentation,the proteases were extracted with distilled water from the fermented media.The procedures of enzyme purification are:(NH4)2SO4 precipitation,acetone decoloring,gel filtration chromatography with Sephadex G-150, and ion exchange chromatography with DEAE-Sephadex A-50.As a result,the specific activity of purified proteases was improved from 1.4(μmol)/ (mg protein) to 17.14(μmol)/(mg protein), and the proteases could be separated into neutral protease and alkaline proteases by ion exchange chromatography. The amino acid composition of the fish hydrolysates tre- ated with purified proteases was analyzed, and compared with those of the hydrolysates treated with commercial protease NP-10 and the (NH4)2SO4;precipitation enzyme.It was shown that Glu、Phe、Leu and Met were released obviously from the hydro- lysate treated with purified proteases.It might mean those amino acid residues were related to the cleavage site of the proteases on the substrate proteins. A fraction of bitter peptides was separated with the chromatography of Sephadex G-50 from fish hydrolysates,and a bitter peptide was ioslated with the chromatography Cellulose GCL-25.The molecular weight of the bitter peptide is approximatly 1400 daltons.

Record ID 第125筆 System ID 081THU00253005
BCRC ID CCRC 10313, Public Year 81
Paper Name 高鹽濃度對麩胺酸生產菌胺基酸生合成的影響 The influence of NaCl at high concentration on the glutamate metabolism of Corynebacterium glutamicum
Student Name 吳佳怡
Teacher Name 李根永 博士
School Department Name 東海大學 食品科學系 Academic Degree 碩士
Abstract 培養基中生物素的含量對 Corynebacterium glutamicum CCRC10313在麩 胺酸的生產是極為重要的決定因素。於本實驗中依實驗結果決定種母培養 基及主醱酵培養基的生物素含量分別為 10μg/l、 1μg/l。鹽濃度愈高 對菌體的生長愈不利。由實驗得知 NaCl對此支菌株生長的最低抑制濃度 為 7% 。添加 3%NaCl的培養基,菌體的生長速率降低,但麩胺酸的產量 卻增加。而天門冬胺酸與息寧胺酸的產量亦增加,但胺基丙酸及離胺酸的 產量卻降低。這些胺基酸生成量的變化,可能是由於培養基中的NaCl使麩 胺酸池擴大所造成的。經3%NaCl處理的菌體轉移到不含NaCl的培養基中, 經NaCl誘導合成胺基酸的能力仍能維持一段時間。因滲透壓力誘導所產生 的麩胺酸,可排出菌體外,並且擴大的胺基酸池可能利於天門冬胺酸與息 寧胺酸的生合成。 It is well known that the amount of biotin in the medium is a crucial factor to the glutamate productivity of Corynebacterium glutamicum CCRC 10313。 In this research, the biotin concertration of seed medium and fermentation medium were 10μg/l and 1μg/l, respectively. High concentration of NaCl in the medium showed a negative effect on the microbial growth. It was detected that the minimal inhibition concentration of NaCl for the microorganism is approximately 7% (w/v) . In the medium containing 3% of NaCl, the growth rate of the microorganism declined , but the productivity of glutamate was increased obviously. The productivity of aspartate and threonine were also raised , but those of lysine and alanine were decreased. The variation of amino acid productivity in the miroorganism might be attributed to the enlarged glutamate pool induced by the NaCl in the medium. The induced capabitity of synthesizing amino acid were sustained for a period of time, when the miroorganisms were transfered from the media containing 3% of NaCl to the media whithout adding NaCl. It was also shown that the glutamate induced by osmotic stress would be secreted out of the cell, and the enlarged amino acids pool might be benefited the biosynthesis of aspartate and threonine .

Record ID 第126筆 System ID 094THU00063023
BCRC ID BCRC 36099, Public Year 94
Paper Name Coprinus cinereus 真菌菌絲體自溶現象之探討 Research on the autolysis of Corprinus cinereus Fungi mycelium
Student Name 陳啟倫
Teacher Name 楊芳鏘
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 鬼傘屬真菌主要特色是生長週期短,子實體成熟後菌蓋會自溶呈墨汁狀,本研究重點著重於探討不同條件下,鬼傘屬真菌菌絲體自溶進行程度之差異。首先利用液態培養方式挑選生長迅速之菌株,經七天液態搖瓶培養後,Coprnius cinereus BCRC 36099生長最為迅速,第七天菌絲體乾菌體量可達1.215(g/100ml 培養基)。 在不同溫度與緩衝溶液pH值對真菌自體溶解影響試驗中,當pH值=6、溫度=50℃、反應24小時,自體溶解率達66.92%。在時間因素對真菌自體溶解影響試驗中,在第1小時自體溶解率已達到60.16%,至第24小時,自體溶解率僅上升了6.76%。在添加不同酵素對真菌自體溶解影響試驗中,添加木瓜酵素自體溶解率為69.99%,添加鳳梨酵素自體溶解率為68.06%。 當pH值=6、溫度=50℃、反應24小時,鳳梨酵素對於幾丁質與蛋白質的分解成效最佳,反應後得葡萄糖氨與游離胺基酸濃度分別是85.7835μg/ml與1.1894mg/ml。另外,在探討發酵槽自體溶解試驗中,菌絲體濃度減少39.5%,葡萄糖氨與游離胺基酸的濃度分別是270.7550 (μg/ml)與5.3519(mg/ml)。 The character of fungi Coprinus is the short growing periods. The cap of the fruit body will autolysis and be ink-like after maturity. This research focuses on the effects of Corprnius mycelial autolysis degree under different reaction conditions. By using a liquid culture method, we select the strain in which growth is the most rapid。After seven days of flask liquid cultivation, the Coprnius cinereus BCRC 36099 exhibited the most rapid growth, and based on the dry weight, the mycelium mass can be up to 1。215(g/100ml substrate). The optimum condition for fungi autolysis was determined to be a temperature equal to 50℃. Initial pH=6 was controlled before reaction. The autolysis solubility can reach to 60.16% at 1st hour, and 66.92% after 24 hours of reaction in 0.1M sodium acetate buffer. In addition, adding 0.1g of the papain and bromelain can bring the autolysis solubility up to 69.99% and 68.06%. During the reaction in 24 hours, the addition of bromelain can obtain the best dissolution result for chitin and protein; the concentration of glucosamine and free amino acids after reaction are 85.7835(μg/mL) and 1.1894(mg/mL). Moreover, in the fermenter operation for autolysis reaction, mycelium concentration decreased 39.5%, glucosamine concentration and free amino acids concentration are 270.7550 (μg/mL) and 5.3519(mg/mL) individually.

Record ID 第127筆 System ID 094THU00063020
BCRC ID BCRC 35253, Public Year 94
Paper Name 培養條件對雲芝菌絲體生長與多醣生成之影響 Effect of culture conditions on the mycelia growth and the formation of polysaccharides of Coriolus versicolor
Student Name 林嘉隆
Teacher Name 楊芳鏘
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 摘 要 本研究主要目的在利用液態培養方式,藉由改變在不同的培養條件下雲芝(Coriolus versicolor BCRC 35253)菌絲體生長、多醣體生成及形態變化之影響,希望藉由控制培養因子來提高菌絲濃度與多醣體的產量。三角瓶培養實驗,主要探討培養基的成分、時間、pH值、不同高分子添加等環境因子對菌絲生長與多醣生成之影響。 結果顯示:雲芝在搖甁實驗的培養基中培養第七天時,菌絲濃度可達到11.63 (g/L),且多醣體的產量高達2.1 (g/L)。添加不同高分子對菌絲濃度與形態有明顯的差異,在低接種量下實驗,添加carboxymethyl cellulose sodium salt (0.2 %)菌絲濃度可達到10.27 (g/L),菌絲粒徑大小約4~8 mm,添加agar (0.2 %)菌絲濃度可達到16.23 (g/L),菌絲粒徑大小約4 mm。綠茶在最適添加量3 %時,有效增加雲芝多醣的產量,濃度可達到3.16 (g/L),比控制組多醣濃度高出許多。此外,發酵槽操作方面,在5 L的攪拌式發酵槽中,轉速500 rpm、 pH值6.0及25 ℃下菌絲能在短時間內達到高濃度,反之在轉速100 rpm下菌絲濃度低,而多醣體濃度較高。 關鍵字:雲芝、高分子添加、多醣生成、形態。 Abstract The main purpose of this research was to study the effects of different culture conditions on the mycelium growth, polysaccharides formation and morphology of Coriolus versicolor (BCRC 35253) using liquid state fermentation to determine the optimum conditions for the the production of biomass and polysaccharide。 The experiment items in shake-flasks cultures included the effects of medium compositions, time, pH value and polymer additions on mycelium growth and polysaccharides formation. The results show that the biomass concentration reached to 11.63 (g/L) at 7th day and had a high level of polysaccharide of 2.1 (g/L). The additions of polymers were found to have a great influence on biomass concentration and morphology. When carboxymethyl cellulose sodium salt of 0.2 % was added, the biomass concentration reached to 10.27 (g/L) and mycelium size was around 4 mm to 8 mm. In contrast, with the addition of 0.2 % agar, the biomass concentration rose to 16.23 (g/L). In additions, the extract of green tea 3 % was proved to have a beneficial effect on the production of polysaccharide, in which the level was about 3.16 (g/L) and higher than that of the control. Moreover, high agitation speed at 500 rpm was demonstrated to be beneficial to the growth of mycelium, but not the production of polysaccharide in the cultures using a fermentor. Key words: Coriolus versicolor; polymer addition; polysaccharide production; morphology.

Record ID 第128筆 System ID 094THU00289006
BCRC ID CCRC 12944, Public Year 94
Paper Name 添加不同乳酸菌對尼羅草青貯料品質之影響 The Effect of Silage Quality of Nilegrass Inoculated with Lactic Acid Bacteria
Student Name 蘇志宛
Teacher Name 施宗雄
School Department Name 東海大學 畜產與生物科技學系 Academic Degree 碩士
Abstract 本試驗探討以乳酸菌為添加劑對尼羅草青貯料發酵特性的影響。試驗使用的乳酸菌為Lactobacillus plantarum CCRC 12944菌液 ( LP ) , Lactobacillus rhamnosus THSH-1菌液 ( LR ), 以及L. plantarum 與 L. rhamnosus兩種乳酸菌混合的混合菌液 ( LPR ) ,其濃度皆為106 CFU/g (新鮮草料重),及未添加菌液者為對照組(C)。於青貯後2到4天,LPR組之pH值下降速率最快且最低(P < 0.05),分別為3.21與3.46。青貯60天時,各處理之品質,pH值方面,以LP組最低(P < 0.05);乾物質以LP組最高(29.5%),LPR組最低(25.2%)(P < 0.05);氨態氮以C組最低(73.6 g/kg DM),LR組次之(74 g/kg DM)(P < 0.05);水溶性碳水化合物、中洗纖維和酸洗纖維處理組間皆無顯著差異;酵母菌菌數在C組和LPR組皆小於2 log CFU/g,LP組和LR組分別為3.45 log CFU/g 和3.56 log CFU/g, 且四個處理組的黴菌菌數皆低於2 log CFU/g;有機酸方面,醋酸含量則是LR組最低,為16.23 g/kg DM ( P < 0.05 ),而丁酸含量為LP組最低,為0.95 g/kg DM ( P < 0.05 );乳酸含量以LP組最高( P < 0.05 ),達28.73 g/kg DM。Flieg氏青貯料脂肪酸組以LP組分數最高,為70分,品質為四組中最佳者。 This study was conducted to determine the fermentation characterics of nilegrass silage inoculated with lactic acid bacteria. The inoculants contained L. plantarum CCRC 12944 (LP) , L。 rhamnosus THSH-1 (LR) , and a mixture of L. plantarum and L. rhamnosus (LPR) (106 CFU/g FW) which were added to nilegrass, respectively, while the treatment without adding any inoculant was used as control (C) . After ensiling for 2 and 4 days, the pH values of LPR decreased faster than the other treatments ( P < 0.05 ), and these were 3.21 and 3.46. As to the quality of all silages after 60 days ensiling, LP had the lowest pH value ( P < 0.05 ) ; LP had the highest dry matter content ( 29.5% ), followed by LPR ( 25.2% ) ( P<0.05 );ammonium nitrogen contents of C was 73.6 g/kg DM, which was the highest one, followed by that of LR ( 74 g/kg DM ) ( P<0.05 ) ; no significant effects were observed among the treatments for water soluble carbohydrates, neutral detergent fiber, or acid detergent fiber; after 60 days, the yeast numbers of C and LPR were below 2 log CFU/g, and the yeast numbers of LP and LPR were 3.54 log CFU/g and 3.56 log CFU/g respectively, the mold numbers of four treatments were below 2 log CFU/g;for the fatty acids, the acetic acid content of LR was 16.23 g/kg DM ( P < 0.05 ), and the butyric acid content of LP was 0.95 g/kg DM ( P < 0.05 ) . These were both the lowest treatments; the lactic acid content of LP was 28.73 g/kg DM ( P < 0.05 ), it was the highest one. Flieg’s score of LP was 70, and higher than those of the other treatments. It was concluded that using LP could make the best quality of silage.

Record ID 第129筆 System ID 093THU00063016
BCRC ID BCRC 32219, Public Year 93
Paper Name 培養條件對北冬蟲夏草(Cordyceps militaris)菌絲生長之影響 Effect of cultural conditions on mycelium growth of Cordyceps militaris
Student Name 姚淑玲
Teacher Name 楊芳鏘
School Department Name 東海大學 化學工程學系 Academic Degree 碩士
Abstract 摘要 本研究主要目的是在於利用液態培養北冬蟲夏草(Cordyceps malitaris),探討不同培養條件以及添加物對於北冬蟲夏草菌絲體生長與抗氧化活性生成之影響,並測量抗氧化成份總酚類化合物含量。 結果以 BCRC 32219 為培養菌株,蔗糖、酵母萃出物和一些微量元素為基礎培養基,在25℃、100 rpm下培養15天,菌體濃度可達2.0368 g/100 ml,捕捉DPPH能力可達24.54%,總酚類化合物含量為18.634 mg/ml。添加沙拉油和消泡劑等都可增加菌體生成量,如添加5%沙拉油菌絲體濃度可達4.1596 g/100 ml,添加5%消泡劑可達3.2576 g/100 ml。過多的精油添加反而會抑制菌體的生成,添加2%精油菌絲體濃度只有0.0668 g/100 ml。於第7天時添加1%檸檬精油,捕捉DPPH能力(49.74%)比未添加精油(24.54%)時,高2倍;就總酚類化合物含量來說,以添加2%精油測出的總酚量化合物含量最高,可達46.022 mg/ml。 固態培養中間添加水份時,菌絲體濃度於第25天時達到0.7511 g/g substrate,相對於固態培養時(最大為0.6400 g/g substrate)還要高上0.1 g/g substrate。捕捉DPPH之能力到第25天可達39.83%的抗氧化活性,比固態培養(29.65%)多了10%以上。 關鍵字:北冬蟲夏草,深層培養,DPPH,總酚類化合物。 Abstract The main purpose of this research was to study effect of cultural condition onthe production of mycelia of Cordyceps militaris and physiology activity compositions, such as total polyphenols. Effects of the supplementation of various components were also investigated. Based on the results, the mycelium weight could reach 2.0368 g / 100 ml at 25℃, 100 rpm in the 15th day. Culture medium was made up of sucrose, yeast extract and some trace elements. The antioxidant activity and total polyphenols was around 24.54% and 18.634 mg/ml, respectively. The proper additions of some components could enhance mycelium concentration. For instance, 5% of salad oil caused the mycelium concentration increase to 4.1596 g/100 ml. However, the additions of essential oil had a negative effect on the mycelium growth and the mycelium concentration was only 0.0668 g/ 100 ml in the medium containing 2 % essential oil. When 1% lemon essential oil was supplemented at the 7th day, the effect of scavenging of DPPH reached 49.74%, which was two-folds higher than that of the control. For production of total polyphenols, the optimal supplementation amount of essential oil was 2% and the value could reach 46.022 mg/ml. In semi-solid state fermentation, the mycelium weight could reach 0.7511 g/g medium, which was 0.2 g more than that of solid state fermentation. In additions, effect of scavenging of DPPH of 39.83% at the 25th day was 10% higher than that of solid state fermentation. Key words: Cordyceps militaris, submerged culture, DPPH and total polyphenols.

Record ID 第130筆 System ID 093CGU00396002
BCRC ID CCRC used, CCRC shoul, CCRC (clus, Public Year 93
Paper Name 決策樹於連續性照護退休社區市場區隔之研究-以長庚養生文化村為例 Using Decision Tree for Exploring the Bases of Market Segmentation of CCRCs-Chang Gung Silver Village for Example
Student Name 賀千盈
Teacher Name 王日昌 陳亭羽
School Department Name 長庚大學 資訊管理研究所 Academic Degree 碩士
Abstract 台灣現有之長期照護機構不論社區式或機構式皆有其缺失,故連續性照護退休社區的發展提供了高齡者另一種退休安排選擇。國內目前針對連續性照護退休社區的市場區隔研究尚未涵蓋完整之服務設施項目,故本研究之目的除了要瞭解完整的連續性照護退休社區所應提供的服務類型,從資料中選取適當的研究變數,以決策樹演算法找出市場區隔及有效區分市場區隔之區隔基礎,使有意在台灣經營連續性照護退休社區之企業,有相關資訊及模式參考。 本研究以人口統計變數、連續性照護退休社區提供之五大類服務設施問項為自變數,利用因素分析、集群分析及其他各種統計檢定方法進行資料前置處理,選取三項不同的依變數,利用決策樹建立預測模型,得出市場區隔及市場區隔變數。為證實區隔結果之強健性,利用卡方檢定及Schiffman and Kanuk學者提出之市場區隔準則(鑑別性、足量性、穩定性和接近性)評定是否為有效的市場區隔。 實驗結果顯示,若以「入住意願」為目標類別標籤,則最佳之市場區隔變數為「影響入住選擇因素區隔」、「財務協助之工作抵費」、「不動產擁有狀況」及「每月可支配所得」,決策樹預測準確率為66.0%;若以「影響入住選擇因素區隔」作為目標類別標籤,則最佳之市場區隔變數為「入住意願」、「子女狀況」、「獨立自理能力」、「不動產擁有狀況」、「目前住處之房屋所有權」,決策樹預測準確率為64.5%。最後,將決策樹之區隔結果與其他方法作比較,決策樹不論在區隔變數的強健性及分析區隔結果的方便性上,皆較其他方法之結果佳。 In Taiwan, a new model, “Continuing Care Retirement Communities (CCRCs),” is proposed for the retired people In the previous research, there was no any variable about the services and facilities within a complete CCRC used for segmenting the CCRCs market in Taiwan Therefore, the goals of our research are not only to understand the types of services and facilities a complete CCRC should provide, but also to use decision tree in finding the valid bases of market segmentation which can effectively segment the CCRCs market in Taiwan We analyse the result and provide suggestions for the enterprises who want to operate the CCRCs business in Taiwan We take the demographic variables and variables about the five types of services and facilities a CCRC should provide as independent variables, and preprocess the variables with factor analysis, cluster analysis, and some statistical tests。 There are three cases for decision tree induction, for each case we use different dependent variable. After building the tree-like prediction models, the bases of market segmentation which are took out from the tree model must be testified by Chi-square test and evaluated by Schiffman and Kanuk’s criterions. The results are as the follows. If “the will to live in a CCRC” is the dependent variable, the best segmenting variables are “factors effect the will to live in a CCRC (clustered),” “financial support-working,” “owned realty,” and “monthly income。” In this case, the accuracy of prediction is 66.0%. If “factors effect the will to live in a CCRC (clustered)” is the dependent variable, the best segmenting variables are “the will to live in a CCRC,” “children,” “self-conducting,” “owned realty,” and “residence ownership。” In this case, the accuracy of prediction is 64.5%. At last, comparing the result of decision tree induction and other market segmenting method, our conclusion is that using decision tree to segment the CCRCs market in Taiwan is better than other methods

Record ID 第131筆 System ID 088CGU00457015
BCRC ID CCRC devel, Public Year 88
Paper Name 都會區中齡者對連貫性照顧退休社區態度之研究--以台北市為例 A Study of Middle Age People's Attitude toward Continuing Care Retirement Communitites in Urban Area : A Case Study of Taipei
Student Name 劉燕瑩
Teacher Name 王惠玄
School Department Name 長庚大學 管理學研究所 Academic Degree 碩士
Abstract 本研究探討都會區中齡者對於退休社區的態度認知以及對「連貫性照顧退休社區」的行動傾向,配合人口學統計變數、健康特性、資源特性與生活特質等變項,找出何為影響態度的重要因子,進而提出策略之建議,期提供政府與民間企業設計規劃老人安養型態之參考。 本研究以台北市40-64歲居民為母群體,以12個行政區為抽樣層級,於民國89年4月8日至5月7日間對舉辦社區活動的8個里進行便利取樣,共發出554份問卷,剔除資料不全者,有效問卷523份,實際有效回收率達94.4%。 本研究以態度認知量表、行動傾向量表與AIO(心理偏好變數)量表為主要材料。以30題AIO問句進行因素分析萃取6項生活型態構面,再根據這些構面進行集群分析,將受訪者分成4個集群(開放重品牌、保守傳統勤勉、重健康輕物質與樂天知命四型),再以複迴歸模型來驗證生活型態、人口學變項、健康特性與資源特性對於態度認知與行動傾向是否有顯著影響。研究結果顯示男性、非行政主管人員、基督教徒、傾向品牌取向、家庭取向、及時行樂、健康外向與適應力強等生活特質者對於退休社區的認知較良好;而勞保、具有家庭取向、及時行樂、健康外向與適應力強等生活型態、對於退休社區認知分數較高等變項是影響連貫性照顧退休社區行動傾向的顯著因素。 本研究對於連貫性照顧退休社區提出之行銷策略建議為:對「開放重品質」與「重健康輕物質」二型,應塑造專業、健康、精緻品牌形象為訴求的退休社區,並擬定行銷組合(4P)作為未來推出此類產品之行銷策略。 As the population in Taiwan is aging, difference approaches to caring for the increasing number of elderly are blooming. The objective of this study is to examine the attitude and action tendency of urban middle age people toward Continuing Care Retirement Communities (CCRCs) in Taipei city。 The impacts from demographic characteristics、perceived health、social resource and lifestyle factors were examined to offer strategies for government and enterprises in designing and planning the new elderly care model. Questionnaires on attitudes and action tendency towards CCRCs, as well as general consumption preference (AIO measures) were distributed to 554 adults aged between 40 to 64, who attended community activities in various regions of Taipei city during April 8 and May 7。 With the response rate of 94.4%, information from 523 subjects are analyzed with principal component method and cluster analyses. The results indicated that subjects bearing the following characteristic are more likely to have positive attitude towards CCRCs: male, non-managers, Christian, brand-name fan, family oriented, fun-seeking, out-going, and strong adaptation skills Those who are family oriented, outgoing; having labor insurance, fun-seeking lifestyle, strong adaptation skills, positive opinions towards CCRCs, are more likely to consider living in a CCRC Cluster analyses identified four groups of middle age people for whom corresponding marketing strategies are suggested for future reference by the CCRC developers

Record ID 第132筆 System ID 094STUT0111020
BCRC ID BCRC C, BCRC 32219, Public Year 94
Paper Name 以蛋白質體學技術研究蟲草屬分離菌株蛋白質圖譜 The investigation of the protein profiles of the isolates of Cordyceps spp. by proteomics
Student Name 高怡蜓
Teacher Name 吳定峰
School Department Name 南台科技大學 生物科技系 Academic Degree 碩士
Abstract 蟲草屬真菌被作為傳統中藥已有很長的一段時間了,雖然蟲草屬真菌種類很多但僅有少數種類具有藥理功效,因此需要一個精確的方法來分析其藥理功效,目前已有人利用5.8S rDNA、28S rDNA、5S rDNA、18S rDNA、ITS、IGS區域的序列來做為分類上的依據,但這僅只能看見DNA的層級,並不能夠完整了解其基因的表現,因為藥理功效與代謝路徑及蛋白質的表現有關,本論文是第一次利用蛋白質體學技術來看不同蟲草屬真菌之蛋白質表現情形,論文中主要利用二維電泳技術來看八種不同蟲草屬分離菌株蛋白質之表現,這八株蟲草屬分離菌株分別是Cordyceps sinensis(冬蟲夏草BCRC C.S)、Cordyceps militaries (蛹蟲草,BCRC 32219)、Paecilomyces tenuipes(細腳擬青黴菌 IUM077、IUM067)和四株Beauveria amorpha (球孢白疆菌ROKI36、ROKI2229、IUM0922、BCRC35502)每種菌株均以相同的培養環境包括溫度、pH、培養基轉速等,抽取蛋白質進行二維膠體電泳pH4-7 12.5%SDS-PAGE;接著以PDQuest軟體進行蛋白質圖譜的分析,結果顯示蛹蟲草和冬蟲夏草的圖譜幾乎完全不同,IUM67和IUM77有大約851個蛋白質點只有184個蛋白質點有相同的分子量大小(Mw)跟等電點(pI)、以ROKI36和ROKI2229、IUM0922、BCRC35502的圖譜比較分別有342、140、70個蛋白質點有相同的MW/pI;另外我們以統計學方式計算每對二維電泳圖譜的蛋白質點的相似度結果如下:BCRC32219和BCRC C.S相似度14%、IUM067和IUM077相似度12%、ROKI36和ROKI2229相似度24%、ROKI36和IUM0922相似度9%、ROKI36和BCRC 35502相似度4%,由於這些菌株有不同的宿主,所以儘管這些蟲草屬分離菌株核醣體DNA序列很相似,但因為宿主不同而使代謝路徑改變進而造成圖譜的差異,這也很可能是造成不同地區採集到的蟲草藥理活性不同的原因。 Cordyceps spp. have been used in the traditional Chinese medicine for a long time. Few medicinal Cordyceps spp. possess the curative effects. Therefore it is worth to identify the Cordyceps spp. with the pharmacological activities utilizing the accurate analytical method such as classification by 5.8S rDNA、28S rDNA、5S rDNA and 18S rDNA regions However, the rDNA identification can not distinguish the different pharmacological activities of Cordyceps spp.. In this study, we used two dimensional (2-D) gel electrophoreses to compare the proteomic profiles of 8 related isolates of the genus Cordyceps¾ Cordyceps sinensis BCRC C。S. 、Cordyceps militaries BCRC 32219、Paecilomyces tenuipes IUM077 、IUM067 、Beauveria amorpha ROKI2229、ROKI36、IUM0922 and BCRC35502。 The 2-D gels were compared by PDQuest software. The comparative results demonstrated that the proteome map of C.S. was almost totally different from that of BCRC 32219。 Among ~731 protein spots in IUM067 only 184 spots were found in IUM077 with the same pI and molecular weight. Compared to ROKI36, 342, 140 and 71 matched protein spots were found in ROKI2229, IUM0922 and BCRC35502 respectively。 In order to estimate the similarities between the Cordyceps spp. isolates protein profile similarities were calculated between pairs of isolates using a matching co-efficient. The matching analyses results indicated that BCRC32219 with BCRC CS similarity 14%, IUM067 with IUM077 similarity 12%, ROKI36 with ROKI2229 similarity 24%, ROKI36 with IUM922 similarity 9% and ROKI36 with BCRC35502 similarity 4%, suggesting that the isolates of a species collected from the different host might have the different protein expressions even though they are supposed to have the same genome

Record ID 第133筆 System ID 088NPUST541007
BCRC ID CCRC 12557, Public Year 88
Paper Name 溶血性大腸桿菌clyA基因分子選殖及表現之研究 Study on molecular cloning and expression of the clyA gene from hemolytic Escherichia coli
Student Name 徐麗卿
Teacher Name 張甘楠
School Department Name 屏東科技大學 獸醫學系 Academic Degree 碩士
Abstract 本試驗旨在研究溶血性大腸桿菌clyA基因(細胞溶解素A,簡稱為EclyA)並對此基因之產物加以定性。利用聚合鏈反應(plymerase chain reaction,PCR)選殖方法,設計特定的引子自溶血性大腸桿菌(CH9802)之菌體DNA,合成912 bp之DNA片段,並將此段DNA與pQE30載體接合後,轉殖至E. coli SG13009,經選殖後得到兩個clones,EclyA-543及EclyA-1011,並進行蛋白質表現(expression),經純化後,結果可得到表現之融合蛋白(fusion protein, 6  His)分子量約為34 kDa。本研究以所選殖的EclyA-543、EclyA-1011及未帶有EclyA基因之宿主細胞E. coli SG13009培養於血液培養基,含有EclyA基因之菌株產生溶血現象,而E. coli SG13009宿主細胞則未見溶血現象,上述表現之融合蛋白34 kDa對山羊紅血球具有溶血作用。由此證明clyA基因之產物具有溶血作用,而且對小鼠巨噬細胞株(J774-A1)及Vero細胞具有細胞毒性作用。本試驗自Salmonella choleraesuis亦選殖到clyA基因(簡稱SclyA基因)該SclyA基因具有19個stop codon,此與現場分離之S. choleraesuis不產生溶血有密切關係。另將EclyA基因定序,證明EclyA基因非屬於RTX family之成員,因其與hly基因DNA序列比對相似度僅有23 %。而與verotoxin中之SLT-I及SLT-II基因DNA序列之相似度甚低分別為24.5 %及25.0 %,由此可知EclyA與SLT是不同的。另者,將EclyA基因與Enterococcus faecalis之clyA基因比較其DNA序列相似度亦僅為22.4 %,顯示EclyA與Enterococcus faecalis之clyA基因亦不相同。但是將EclyA與非病原性E. colik-12之clyA基因DNA序列比較之結果,其相似度高達99.3 %,顯示兩者同源性極高,該E. coli K-12 (CCRC 12557,野外株)雖具有clyA及slyA基因,但是培養於血液培養基時不產生溶血現象。 The purpose of this study is to analyze and identify the clyA gene of hemolytic E. coli. By using polymerase chain reaction (PCR) and a pair of specific primers, we were able to amplify the 912 bp fragment from hemolytic E. coli (CH9802). The resulting PCR product was then used as insert DNA to ligate with vector PQE30 DNA and transformed to E. coli SG13009. After screening, we got two clones, termed EclyA-543 and EclyA-1011 respectively. Both expressed fusion proteins of these two clones have the same molecular weight of 34 kDa. The results showed that the host cells, E. coli SG13009, harbored the recombinant EclyA gene were able to cause -hemolysis on blood agar, but did not the host cells E. coli SG13009. Therefore, the EclyA gene (912 bp) was involved in the hemolytic activity. After purification with affinity column (Ni-NTA column), the purified fusion proteins not only caused goat RBCs hemolysis but also cytotoxicity to the mouse macrophage cell line (J774-A1) and Vero cells. Moreover, Salmonella clyA gene (termed SclyA gene) was also cloned from Salmonella choleraesuis in this study. After DNA sequencing the SclyA gene revealed 19 stop codons which could be tightly associated with non-hemolysis characteristics of S. choleraesuis being isolated from the field cases. The resulting data showed that EclyA gene does not belong to RTX family since the similarity of DNA sequences between EclyA and hly gene is only 23 %. The similarity of DNA sequences between EclyA gene and verotoxin genes (SLT-I and SLT-II) are as low as 24.5 % and 25.0 % respectively. Furthermore, the similarity of DNA sequence between EclyA and clyA of Enterococcus faecalis is only 22.4 % indicating low homology in betweens. However, it showed a very high similarity (99.3 %) of DNA sequences between the EclyA gene and the clyA gene of nonpathogenic E. coli K-12 (CCRC 12557, wild type) which does not exhibit any hemolysis on blood agar

Record ID 第134筆 System ID 091NPUST253014
BCRC ID CCRC 31499, Public Year 91
Paper Name 以大型製麴機培養紅麴之研究 An Investigation on Mass Cultivation of Monascus purpureus Using Commercial Scale Koji Making Equipment
Student Name 倪光輝
Teacher Name 古源光
School Department Name 屏東科技大學 食品科學系 Academic Degree 碩士
Abstract 摘 要 學號:n8936012 論文名稱:以大型製麴機培養紅麴之研究 總頁數:89 學校名稱:國立屏東科技大學食品科學研究所碩士班 畢業時間及摘要別:九十一學年度第一學期碩士學位論文摘要 研究生:倪光輝 指導教授:古源光 博士 論文摘要內容: 將紅麴菌接種在米飯所培養製成之紅麴,內含有高經濟價值的膽固醇抑制劑Monacolin K、降血壓物質γ-胺基丁酸(GABA)及色素等代謝產物。然而,由於傳統培製紅麴方法既費時且耗工,需寬廣的製造場所,製程亦相當繁雜,故產製單位成本相對偏高。因此,本研究選定能產生紅麴色素及膽固醇抑制劑Monacolin K之紅麴菌株 Monascus purpureus CCRC 31499,來製造高濃度液體麴,以取代傳統的麴種。研究結果顯示,利用120公升液體發酵槽,以20%在來米為碳源,並以耐高溫液化酵素取代乳酸,將可有效液化米粒,並降低液體麴之黏稠度,以利紅麴菌接種繁殖。製程溫度控制在34℃左右,以60 rpm的速率攪拌、無菌空氣通氣量保持在2vvm,經過五天的培養,即可製得含3.5g/100 mL以上紅麴菌絲體的液體麴,將之均勻拌合後,即可接著以2% v/w之液體麴接種於在來米所蒸成之米飯上,並投入直徑500公分製麴機內培製紅麴。以此種製麴機製造紅麴,入機米飯含水量為35%、品溫控制在37 — 38℃,濕度在RH90%以上,並以自動溫控之循環風散熱,必要時輔以攪拌,紅麴菌在16小時左右達到生長遲滯期 (Lag phase)。入料經過36小時 (含水量約30%) 需在麴層上加水,以補充麴菌代謝或高熱蒸發所需之水份。加水方式則以大量淋水法,可使麴粒含水量達50%以上效果最佳。製麴後段將品溫調降至34℃,以幫助紅麴色素之產生。依此製程,麴層厚度在20公分培製7天後,每批次製得紅麴約1200 kg,而製造時間只需傳統式的1/2。 Abstract Monascus spp. cultivated in the rice medium contains several high value substances, such as monacolin K, γ- aminobutyric acid (GABA), and natural red colorant. These substances are the secondary metabolites of fermentation, and are medicinally proved to possess anti-cholesterol activity. Traditional methods for cultivating Monascus spp. need large space, long fermentation time, and high labor cost, while with very low efficiency. In this investigation, Monascus purpureus CCRC 31499 was selected for its higher production capacity of monacolin K and the red pigment than traditional species。 The selected strain was first cultivated at 34℃, agitation of 60 rpm and under aeration (2 vvm) in a 120 L submerged type fermentor using 20﹪rice porridge as carbon source. To make it more feasible for inoculation, heat-stable liquefying enzyme was added instead of lactate for better liquefying efficiency and less fermentation viscosity. Five days later, high concentrated liquid red mold rice (above 3.5 g/mL) was ready to inoculate. The whole fermentation broth was well mixed with cooked rice to the final concentration of 2﹪v/w in a commercial scale koji making equipment (diameter: 500 cm), in which temperature was kept at 37-38℃and humility at 90﹪RH automatically. Air was circulated to remove fermentation heat while the rotary bed of 20 cm high and 500 cm diameter was slowly agitated. Lag phase was about 16 hours. Water was added by falls into bed at 36 th hour for keeping moisture of the cooked rice at about 50﹪or above. At the final stage of fermentation, temperature was adjusted to 34 ℃ to increase the production of red colorant. After 7 days solid-state fermentation, the depth of the layer of red mold rice was about 20 cm and the weight of red molded rice harvested each batch was about 1200 kg. The total process time was reduced to 1/2 compared with those made by traditional methods.

Record ID 第135筆 System ID 092NPUST253039
BCRC ID BCRC 20666, BCRC 21808, Public Year 92
Paper Name 低溫處理與酵母發酵對椰子酯類生物合成之影響 Effects of cold treatment and yeast fermentation on coconut esters biosynthesis
Student Name 王玉淳
Teacher Name 黃卓治 博士
School Department Name 屏東科技大學 食品科學系 Academic Degree 碩士
Abstract 本研究為探討低溫處理與酵母發酵對椰子酯類生物合成之影響。利用可可椰子(Cocos nucifera Linn.)果實為原料,將其在低溫逆境環境下貯藏,而後再利用加熱乾燥處理,取其椰肉及椰子內果皮的部份,以水蒸氣蒸餾法萃取其揮發性成分,再以GC及GC-MS進行分析與鑑定,結果顯示椰子精油中至少含有19種揮發性化合物,其中以椰子內果皮之揮發性化合物含量較高,包括2-nonanone、nonanal、octanoic acid ethyl ester、octanoic acid、2-undecanone、decanoic acid methyl ester、decanoic acid ethyl ester、n-decanoic acid、dodecanoic acid methyl ester、dodecanoic acid ethyl ester、dodecanoic acid、tetradecanoic acid ethyl ester、tetradecanoic acid、ethyl-9-hexadecenoate、n-hexadecanoic acid、hexadecanoic acid ethyl ester、10-octadecenoic acid methyl ester、linoleic acid ethyl ester、ethyl oleate等;就整體而言,揮發性化合物的總含量以50℃加熱乾燥的椰子內果皮中dodecanoic acid ethyl ester(24.6%)含量最多。而脂肪酶及酯酶之酵素活性隨低溫處理時間增加而略增可能是形成酯類的原因。 由椰子中分離出的酵母菌為Saccharomyces cerevisiae A/Tor. Pretorien、Saccharomyces boulardii、Candida tropicalis B及Candida fluviatilis,將其培養在含有1%椰子油的基礎培養液中進行發酵,再抽取培養液之香氣並經由GC及GC-MS分析鑑定後,其香氣成分主要為酯類,包括dodecanoic acid ethyl ester、pentanoic acid ethyl ester、tetradecanoic acid ethyl ester、hexadecanoic acid ethyl ester、11-hexadecenoic acid ester、9,12-octadecadienoic acid ethyl ester,而形成酯類的原因是酵母菌中的脂肪酶及酯酶所產生的。實驗又以四株Saccharomyces cerevisiae(BCRC 20666、BCRC 21808、Saccharomyces cerevisiae、Saccharomyces cerevisiae A/Tor. Pretorien)利用椰子水進行酒精發酵而得椰子酒,其香氣成分經GC及GC-MS分析鑑定包括了醇類、脂肪酸及酯類。 Effects of cold treatment and yeast fermentation on the formation of esters were studied. Separated coconut endocarp and flesh dried by hot air, and the volatile components were extracted by steam distillation. The extractant was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The results indicated at least 19 volatile components could be identified from coconut, while the coconut endocarp contained the most flavor and aroma components. These volatile components included 2-nonanone, nonanal, octanoic acid ethyl ester, octanoic acid, 2-undecanone, decanoic acid methyl ester, decanoic acid ethyl ester, n-decanoic acid, dodecanoic acid methyl ester, dodecanoic acid ethyl ester, dodecanoic acid, tetradecanoic acid ethyl ester, tetradecanoic acid, ethyl-9-hexadecenoate, n-hexadecanoic acid, hexadecanoic acid ethyl ester, 10-octadecenoic acid methyl ester, linoleic acid ethyl ester, ethyl oleate. Hot air drying at 50℃ following the cold treatment led to the highest amount of dodecanoic acid ethyl ester (24.6%) in the endocarp. Lipase and esterase may account for the formation of these esters. Saccharomyces cerevisiae A/Tor. Pretorien, Saccharomyces boulardii, Candida tropicalis B and Candida fluviatilis were isolated from coconut. Significant amount of dodecanoic acid ethyl ester, pentanoic acid ethyl ester, tetradecanoic acid ethyl ester, hexadecanoic acid ethyl ester, 11-hexadecenoic acid ester, 9,12-octadecadienoic acid ethyl ester were detected in the fermented basal medium enriched with 1% coconut oil. Lipase and esterase in these yeasts were proposed to be responsible for the esters formation. Four strains Saccharomyces cerevisiae (BCRC 20666, BCRC 21808, Saccharomyces cerevisiae and Saccharomyces cerevisiae A/Tor。 Pretorien) were fermented in coconut juice. Volatile flavor compounds include ethanol, fatty acid and ester were identified from coconut wine by GC and GC-MS.

Record ID 第136筆 System ID 092NPUST253049
BCRC ID CCRC 20581, CCRC 21673, Public Year 92
Paper Name 洛神葵酒製備及其抗氧化活性之研究 Studies on the preparation and antioxidant activity of Roselle wine
Student Name 孫丕忠
Teacher Name 古源光 博士
School Department Name 屏東科技大學 食品科學系 Academic Degree 碩士
Abstract 本研究以市售乾燥洛神葵(Hibiscus sabdariffa L.)為原料,原料與水採3:100之固液比例混合,於100℃下加熱萃取20分鐘,萃取液以55%果糖糖漿調整糖度至20oBrix後,分別植入三株單一酵母菌株(A:CCRC 20581 Saccharomyces ellipsoideus、B:CCRC 21673 Saccharomyces bayanus、C:自市售白麴中分離之菌株,編號為P13)及其四組混合菌株組(AB、AC、BC及ABC組),植入之酵母菌量為10%(v/v),置於25℃下進行前醱酵14天,再於10℃下後醱酵10天。研究中探討不同之試驗酵母菌種對洛神葵萃取液之醱酵情形,醱酵期間測定oBrix值、總酵母菌數、還原糖、可滴定酸、有機酸、pH值、總色素、色素褪變指數、酒精含量之變化,醱酵完成後之新鮮酒液進行抗氧化活性、香氣成分分析以及嗜好性感官品評。 實驗結果顯示,酒精產量以BC組最高(10.0%, v/v)、B組次之(9.5%, v/v)、C及AC組最低(5.0%, v/v),可滴定酸含量以C組最高(0.82%)、BC組最低(0.64 %),還原糖變化範圍介於0.44~0.56%之間,總色素含量(以花青素計)介於47.45~58.37 mg/L之間,七組實驗組之pH值,於醱酵期間並無顯著之變化,介於pH2.17∼2.36之間,含量最高之有機酸除C組為琥珀酸(succinic acid,2052.53 ppm)外,其餘各組均為乳酸(lactic acid),其濃度範圍為728.54~2259.77 ppm。洛神葵酒重要香氣成分中主要為醇類化合物及酯類化合物,醇類化合物以2,3-丁二醇(2,3-butanediol)、 苯乙醇(phenylethyl alcohol)及乙二醇(ethylene glycol)為主;而酯類化合物則以醋酸乙酯(ethyl acetate)及琥珀酸氫乙酯(ethyl hydrogen succinate)居多。醱酵完成後之洛神葵酒之抗氧化活性,以C及BC組較佳。醱酵完成後之新鮮酒液置於10℃下貯存一週後取出,以55%果糖調整糖度至16oBrix後進行嗜好性感官品評,結果顯示,以B組、AB組及BC組之整體接受性較佳。編號P13之酵母菌株,經以bioMérieux API鑑定試劑組鑑定為Saccharomyces cerevisiae。 This study utilized commercial dried Roselle (Hibiscus sabdariffa L.) as raw materials for Roselle wine. Roselle extract was prepared by mixing dried Roselle and water at a weight ratio of 3:100 and cooking at 100℃ for 20 minutes. The sugar content of the extract was adjusted to 20oBrix with 55% fructose syrup and fermented with three groups of single strain (A: CCRC 20581 Saccharomyces ellipsoideus, B: CCRC 21673 Saccharomyces bayanus, C: P13) and four groups of mixed strains (group AB, AC, BC, and ABC)。 The amount of yeast inoculated was 10%(v/v). The extract was pre-fermented at 25℃ for 14 days and then post-fermented at 10℃ for another 10 days. Each tested group during fermentation was analyzed for the following parameters: oBrix, pH value, degradation index (DI) value, total pigment, total yeast count, residual reducing sugar, total titrable acidity, organic acids, and alcohol content. The finished products were determined for antioxidant activity, flavor compounds, and sensory evaluation. The results showed that the highest alcohol content was found in group BC (10.0%, v/v), followed by group B (9.5%, v/v), and the lowest were in group C and AC (5.0%, v/v). Group C (0.82%) was determined to contain the highest total acidity while group BC (0.64%) was the lowest. Residual reducing sugar of each group was between 0.44% and 0.56%. Total pigment was between 47.45 mg/L and 58.37 mg/L expressed as anthocyanin. pH values of all tested groups were between 2.17 to 2.36, and there was no significant change during winemaking process. The highest organic acid content of each group was lactic acid (728.54~2259.77 ppm), except for group C in which succinic acid (2052.53 ppm) was highest. The major flavor compounds of the Roselle wine were 2,3-butanediol, phenylethyl alcohol, propylene glycol, ethyl hydrogen succinate, and ethyl acetate etc. The antioxidant activities of all Roselle wine were comparatively stronger in group C and BC. All of the freshly finished Roselle wine were stored at 10℃ for one week, then adjusted sugar content to 16oBrix by 55% fructose syrup prior to sensory evaluation. The results demonstrated that group B, AB, and BC were satisfactorily accepted. The yeast strain of P13 was identified as Saccharomyces cerevisiae with bioMérieux API Identification System.

Record ID 第137筆 System ID 092NPUST289018
BCRC ID CCRC 31499, Public Year 92
Paper Name 紅麴萃出液之抗氧化力及其對豬漢堡肉保存效果之評估 Evaluation of Antioxidative Activities of Monascus Extracts and It’s Preservation Potential on Pork Hamburger
Student Name 李惠平
Teacher Name 曾穎玉 博士
School Department Name 屏東科技大學 畜產系 Academic Degree 碩士
Abstract 本試驗目的在選取具較佳抗氧化活性之紅麴萃出液,並將之添加於豬漢堡肉中,測試其對生豬漢堡肉之保鮮效果。含瘦肉80 % 之豬漢堡肉,分成四個處理組,A組為對照組添加基礎配方,B、C及D組分別於配方中再添加紅麴萃出液3 %、5 % 及抗壞血酸鈉500 ppm。紅麴菌(Monascus purpureus) CCRC 31499、31746及31747接種於固態蒸米培養,再分別以乙酸乙酯、乙醇及去離子水三種溶劑萃取,比較不同菌株及萃出溶劑之抗氧化力。選擇最佳抗氧化力之紅麴萃出液,測定其對生豬漢堡肉保存安定性之影響,並進行加熱後漢堡之風味品評。 Monascus purpureus 31499之麴米,以乙酸乙酯為溶劑之萃出液其紅色度最高,萃出液之紅色度,於2℃貯存14日維持穩定,沒有顯著降低。菌株31746麴米之酒精萃出液,其清除DPPH自由基之能力及對氯化鐵之還原力最高,其次為31747麴米之酒精萃出液。選用31746酒精萃出液添加於豬漢堡肉。 漢堡肉添加紅麴萃出液3 % 或5 %,pH值於2~3℃貯存期間較為穩定,生鮮或加熱過之漢堡肉其紅色值明顯的高於對照組及D組(P<0.05)。添加紅麴萃出液3 % 及5 % 之處理組TBARS高於D組,但顯著的低於對照組(P<0.05)。漢堡肉添加紅麴萃出液不能防止VBN值的上升,貯存期間B、C兩組顯著高於D組(P<0.05)。添加紅麴萃出液之豬漢堡肉,貯存期間之總生菌數與低溫菌數顯著低於A、D兩組(P<0.05)。官能品評之結果顯示,加熱過之四組豬漢堡肉在多汁性、嫩度及色澤上均無顯著的差異性(P>0.05),但添加紅麴萃出液降低風味及可接受性。添加紅麴萃出液3 % 之豬漢堡肉色澤評分最高,添加5 % 最低,但四組間沒有顯著差異性(P>0.05)。綜合上述結果,豬漢堡肉添加紅麴萃出液對貯存期限具有正面的作用,漢堡肉風味及可接受性之降低,可能是因為酒的味道不能被品評人員所接受。 The objective of this study was to select the highest antioxidative activity of Monascus extracts the culture extracts were added in raw pork hamburger to investigate the effects on freshness preservation. Hamburger (80 % lean) were divided into four treatments, A (control) added with basal formula, treatments B, C, and D additional added with 3 % extracts, 5 % extracts and 500 ppm sodium ascorbate, respectively. Monascus purpureus CCRC 31499, 31746 and 31747 were cultured in solid rice and extract by 3 kinds solvent, ethyl acetate, ethanol and distill water。 The best antioxidative activities of solvent extracts were selected to examine the effect on storage stability of raw hamburger and sensory evaluation of cooked hamburger. Monascus purpureus CCRC 31499 extracts by ethyl acetate had the highest redness, the extracts stored at 2℃ for 14 day, redness maintained stability without significantly decreased (P>0。05); the 31746 ethanol extracts showed the highest in scavenging DPPH free radical and reducing power on FeCl3, the 31747 ethanol extracts was the second. The 31746 ethanol extracts was selected addition in pork hamburger. Hamburger added with 3 % or 5 % of Monascus extracts, the pH maintance stability during storage at 2~3℃, and the redness both in raw and cooked hamburger were higher than control and treatment D (P<0.05). The TBARS of treatments added with 3 % and 5 % Monascus extracts was higher than treatment D, but significantly lower than control (P<0.05). Hamburger treatment added with Monascus extracts could not prevent VBN value increasing during storage time, treatment B and C were significantly higher than treatment D (P<0.05). The total plate counts and psychrotrophic counts were significantly lower in hamburger added with Monascus extracts than A and D treatment. The panel test showed that the juiciness, tenderness and color in cooked hamburger without significantly different of four treatments (P>0.05), but hamburger addition with Monascus extracts, the flavor and acceptability were decreased. Hamburger added with 3 % extracts showed the highest color score, and the 5 % addition was the lowest, without significantly different in color evaluation within the four treatments (P>0.05). Comprehensive from these results, pork hamburger addition with Monascus ethanol extracts had positive effect on the storage life, the decreased flavor and acceptability of hamburger may be due to the wine flavor could not acceptable by panelist.

Record ID 第138筆 System ID 088NPUST253012
BCRC ID CCRC 12377, Public Year 88
Paper Name 葡萄子與皮抗氧化性之研究 Studies on antioxidant activity of grape seeds and skin
Student Name 王子慶
Teacher Name 楊季清
School Department Name 屏東科技大學 食品科學系 Academic Degree 碩士
Abstract 摘 要: 本研究主要是針對國產黑后葡萄子與皮甲醇萃取物,進行抗氧化性之探討,包括抗氧化特性、於不同脂質過氧化系統中之抗氧化性及抗氧化成分分析,並對其毒性和致突變性加以評估。 以甲醇萃取葡萄子與皮可得到較高的萃取率,且甲醇萃取物比其他溶劑萃取物者之抗氧化性高,對亞麻油酸之氧化抑制率略低於BHA,但優於α-tocopherol,故選取此葡萄子與皮甲醇萃取物作為研究材料。 在抗氧化特性探討方面,葡萄子與皮甲醇萃取物添加量為400ppm時,對亞麻油酸具有良好之過氧化抑制率分別可達到90.1%與75.3%。在對α,α-diphenyl picrylhydrazyl(DDPH)自由基清除方面,當葡萄子與皮甲醇萃取物添加劑量為100ppm時,清除效果分別達到94.5%與50.5%。在對超氧陰離子清除方面,當葡萄子與皮甲醇萃取物添加劑量為100ppm時,清除效果分別達到88.2%與75.7%。在對氫氧自由基清除方面,當葡萄子與皮甲醇萃取物添加劑量為1mg/mL時,清除效果分別達到25.3%與24.2%。在對過氧化氫清除方面,當葡萄子與皮甲醇萃取物添加劑量為1mg/mL時,清除效果分別達到40.0%與38.7%。此外,並發現葡萄子甲醇萃取物之還原力高於十倍濃度之葡萄皮甲醇萃取物者,而葡萄子甲醇萃取物之螯合亞鐵離子能力亦與二倍濃度之葡萄皮甲醇萃取物者相近。由以上結果顯示,葡萄子與皮甲醇萃取物可藉由不同之作用模式來終止或延緩脂質過氧化作用之進行,而且,葡萄子甲醇萃取物所具有之提供氫原子以終止脂質氧化連鎖反應的作用模式,較葡萄皮甲醇萃取物者為強。 在不同促氧化因子 (包括自氧化Fe+2 、Fe+3 、Fe+2/H2O2 及 Fe+3/H2O2/ascorbic acid)催化之亞麻油酸乳化系統中,以葡萄子與皮甲醇萃取物在自氧化系統中之抗氧化性最佳。於其他之催化系統中,則發現當葡萄子或皮甲醇萃取物添加劑量為400ppm時,其脂質過氧化抑制率均以Fe+3催化系統者最高,就葡萄子萃取物而言,最低者為Fe+2/H2O2催化系統者,而就葡萄皮萃取物而言,於Fe+2/H2O2 、Fe+3/H2O2/ascorbic acid 或Fe+2三催化系統中,其脂質過氧化抑制率無甚差異;而當葡萄子或皮甲醇萃取物添加劑量為1000ppm時,其脂質過氧化抑制率之大小,則依序均為Fe+2/H2O2 、Fe+3/H2O2/ascorbic acid 及Fe+2 催化系統者。此外,不論在何催化系統下,葡萄子甲醇萃取物脂質過氧化抑制率均高於葡萄皮甲醇萃取物者,唯在各系統中,二者均呈現隨濃度增加而其脂質過氧化抑制率亦增加之趨勢。 由成分分析知,葡萄子甲醇萃取物之抗壞血酸、類黃酮、總多酚類及β-胡蘿蔔素含量,分別為3.4ppm、67.4mg/100g、702.9mg/g及36.8ppm,而葡萄皮甲醇萃取物者,則分別為172.2ppm、353.0mg/100g、53.1mg/g及未檢出。在所含生育醇之形式與含量方面,葡萄子甲醇萃取物之-α、-β、-γ及-δ形式之tocopherol含量,分別為91.8、4.3、2.0ppm及未檢出,而葡萄皮甲醇萃取物者,則分別為11.3、3.3、15.8及3.2ppm。就生育醇總量而言,葡萄子甲醇萃取物者約為葡萄皮甲醇萃取物者之三倍,並推測葡萄子與皮甲醇萃取物之抗氧化性是多種抗氧化物質之綜合表現,而總多酚類物質應扮演相當重要之角色。 本研究採用Ames test來探討葡萄子與皮甲醇萃取物之毒性及致突變性,測試菌株為購自食品工業發展研究所之Salmonella choleraesuis subsp. Choleraesuis CCRC 12377 及 CCRC12378二隻菌株,結果顯示於所測試之濃度範圍(0.5-5.0 mg/plate)內,無論有無添加S-9 mix,均未對該二隻菌株造成毒性或致突變性,此表示葡萄子與皮甲醇萃取物可通過微生物基因毒性及致突變性試驗。 Abstract The objectives of this research were to investigate the antioxidative properties of methanolic extracts from grape (cv. Black Queen) seeds and skin in Taiwan. The study items including antioxidative characteristics, antioxidative activity in different lipid peroxidation systems, contents of some antioxidant, the estimation of their toxicity, and mutagenicity by Ames test. The methanolic extracts of grape seeds (MEGSE) and skin (MEGSK) exhibited higher extract yield and showed greater antioxidative activity than n-hexane and ethyl-acetate extracts. The antioxidation activity of MEGSE and MEGSK at 400 ppm was lower than butylate hydroxyanisole(BHA), however, stronger than α-tocopherol. The MEGSE and MEGSK were slected to be experimental materials. The antioxidative characteristics of MEGSE and MEGSK were investigated. MEGSE and MEGSK exhibited remarkable antioxidative activity inhibited 90.1% and 75.3% peroxidation of linoleic acid at 400 ppm, respectively. MEGSE and MEGSK showed 88.2% and 75.7% scavenging effect onα,α-diphenyl-β-picryhydrazyl(DPPH) at 100 ppm, respectively. The scavenging effects of MEGSE and MEGSK on superoxide anion were 88.2% and 75.7% at 100ppm, and on hydroxyl radical were 25.3% and 24.2% at 1mg/mL, and on hydrogen peroxide were 40.0% and 38.7% at 1mg/mL, respectively. In addition, the reducing power of MEGSE was higher than 10-fold concentration MEGSK. The chelating ability on Fe+2 of MEGSE was near to the 2-fold concentration MEGSK. Antioxidative activity of MEGSE and MEGSK were determined using different linoleic acid peroxidation systems that included by various prooxidants (including autoxidation, Fe+2, Fe+3, Fe+2/H2O2, and Fe+3/H2O2/ascorbic acid). MEGSE and MEGSK exhibited the high antioxidative activity in autoxidation system than in other induced systems. The inhibition of peroxidation in Fe+3 system of MEGSE and MEGSK were higher than those induced by Fe+2, Fe+2/H2O2, and Fe+3/H2O2/ascorbic acid at 400 ppm. When concentration was 1000 ppm, the inhibition of peroxidation of MEGSE and MEGSK was also in order of Fe+3 > Fe+2/H2O2 > Fe+3/H2O2/ascorbic acid > Fe+2. The inhibition of peroxidation(%) of MEGSE was better than MEGSK in any peroxidation system and in any concertration, but it were increased with the increasing of concentration in various peroxidation systems. The contents of ascorbic acid, flavonoid, total phenol and β-carotene in MEGSE were 3.4 ppm, 67.4 mg/100g, 702.9 mg/g and 36.8 ppm, and in MEGSK were 172.2 ppm, 353.0mg/100g, 53.1 mg/g and non-detectable, respectively. The contents of -α, -β, -γ, and -δ form tocopherol in MEGSE were 91.8, 4.3, 2.0 ppm, and non-detectable, and in MEGSK were 11.3, 3.3, 15.8, and 3.2 ppm, respectively. The content of total tocopherol in MEGSE was 3-fold MEGSK. No toxicity and mutagenicity were found in MEGSE and MEGSK toward Salmonella choleraesuis subsp. choleraesuis CCRC 12377 and CCRC12378 (ordered from food industry research and development institute) with or without S-9 mix at tested dosage (0。5-5.0 mg/plate) by Ames test. The results indicated that MEGSE and MEGSK can passed the microorganism gene toxicology and mutagenic test.

Record ID 第139筆 System ID 093NPUST253003
BCRC ID BCRC 17023, BCRC 17027, Public Year 93
Paper Name 抑制幽門桿菌發酵乳之研究 Study on the fermented milk for inhibit Helicobacter pylori
Student Name 陳義昌
Teacher Name 謝寶全
School Department Name 屏東科技大學 食品科學系 Academic Degree 碩士
Abstract   本研究收集市售商品及自然界樣品分離出生長快速之乳酸菌,並測試對幽門桿菌的抑制能力,挑選出兩株不同來源的乳酸菌,經菌種鑑定後,認定分離自市售發酵乳的2號菌株為Lactobacillus paracasei;分離自醃漬蔬菜的26號菌株為Lactobacillus plantarum,兩株乳酸菌發酵牛乳之後能在培養皿上抑制幽門桿菌BCRC 17023和BCRC 17027,產生17 - 22 mm的抑菌圈。   L. paracasei菌株和產生的乳酸為抑制幽門桿菌的主要原因,在發酵乳中能夠抑制幽門桿菌產生明顯的抑菌圈,而發酵乳上清液(FMS)在體外2個小時能抑制45%的幽門桿菌,並降低幽門桿菌所產生的尿素酶活性56 %;L. plantarum能分泌出某種抑菌物質,在中性FMS中2個小時能抑制33 - 37%的幽門桿菌,並降低40 - 60%的尿素酶活性,乳酸存在的低pH值的條件下,FMS可以在1個小時內殺死試管中所有的幽門桿菌(8.8 Log CFU/mL)。但是兩株乳酸菌的發酵乳經過100℃,30分鐘殺菌後則失去對幽門桿菌的抑制能力。動物試驗中,兩株乳酸菌的發酵乳讓小鼠連續飲用3週,能減少小鼠胃中之幽門桿菌數量約3個對數值,並相對抑制尿素酶活性反應。顯示本研究之乳酸菌可以預防幽門桿菌感染。在乳酸菌的安全性上,ICR、C57BL/6J兩個品系的小鼠飲用發酵乳6 - 8週後,都沒有發生生理上的明顯差異及病變。   在發酵乳的應用上,L. paracasei對脫脂乳的發酵性較好,可以直接發酵脫脂乳,但無法忍受消化道環境之低pH值(pH 3.0)和高濃度0.3%膽鹽;L. plantarum需要添加0.5%以上的yeast extract於脫脂乳中,才可以得到較好的發酵性,而此菌對消化道環境適應性良好,可以存活於腸胃中。兩株乳酸菌在共同生長時不會有互相抑制的情形,並在製成發酵乳後儲藏於4℃中60天後,仍可保有活菌數7.05 Log CFU/mL。   綜合上述,本研究所使用的兩株乳酸菌可以預防幽門桿菌感染,在食用上為安全無虞,可以用於製造發酵乳並具有良好的儲藏性。 Isolated strains of lactic acid bacteria (LAB) from commercial production and natural sources were tested for their efficiency in inhibiting Helicobacter pylori in this study. The No.2 strain of Lactobacillus paracasei was isolated from commercial fermented milk and the NO.26 strain of Lactobacillus plantarum was isolated from pickled vegetables. Two LAB strains could inhibited H. pylori BCRC 17023 and BCRC 17027 on the fermented milk plate and produced the inhibitory zones were 17 to 22 mm。 The main reasons of inhibition H. pylori were alive bacteria and produced lactic acid by L. paracasei that was incubated with the fermented milk could produce obvious inhibitory zone. In vitro test, fermented milk supernatant (FMS) of L. paracasei could kill 45% H. pylori and decreased 56% the urease activity in 2 hours. In addition, L. plantarum could excrete certain bacteriocins. In vitro test, neutral fermented milk supernatant (NFMS) of L. plantarum could kill 33 to 37% H. pylori and decreased 40 to 60% the urease activity in 2 hours. Using FMS would be kill the H. pylori (8.8 Log CFU/mL) for 100% in 1 hour. But two LAB strains of FMS after, 30 min sterilized 100℃ lost the ability to inhibit H. pylori. In vivo, mice drank fermented milk for four week, the results showed that the H. pylori decreased to approximately 3 log cfu/ml and inhibited urease activity reaction in stomach. On the security of the LAB, after the mice of ICR and C57BL/6J two strains drank fermented milk 6 to 8 weeks, there were no obvious difference in the occurrence of physiological and pathological change. Application of fermented milk, the ferment of L. paracasei was better in the skim milk and could directly fermented the skim milk, but unable to tolerate low pH value 3.0 and high concentration of 0.3% bile salts in the environmental of alimentary canal. When skim milk added more than 0.5% the yeast extract, L. plantarum were got better fermentation, survivability and adapted fine in the environmental of alimentary canal. While two LAB strains each other growth will be no inhibition condition and made fermented milk was 7.05 Log CFU/mL of alive LAB in store 60 days at 4℃. The results showed that fermented milk in this research can prevent the infecting from H. pylori and do not have any toxic for the security in eating, may be used for making fermented milk and possess good shelf life.

Record ID 第140筆 System ID 092NSYS5112034
BCRC ID CCRC 17200, Public Year 92
Paper Name 熱穩定性纖維素分解細菌分離株之特性探討與親緣關係的研究 Characterizations and Phylogeny of Thermostable Cellulolytic Bacterial Isolates
Student Name 戴上凱
Teacher Name 劉仲康 林畢修平
School Department Name 國立中山大學 生物科學系研究所 Academic Degree 博士
Abstract 本研究探討 52 株具纖維素分解能力之嗜高溫微生物的生理特性及演化分類。根據 16S rDNA 序列之分析,3 株來自生技中心和 4 株分離自南台灣的細菌分別近似於菌屬 Bacillus 和 Geobacillus。新屬 Geobacillus 的四株菌中,菌株 T4 為可移動、有氧生長且可形成孢子的革蘭氏陰性桿菌,能分泌熱穩定的內切型纖維素分解酵素。當菌株 T4 生長於含 carboxymethylcellulose 的液體培養基時,其在培養懸浮液中的纖維素分解酵素活性可耐受 70℃ 的高溫。根據 16S rDNA 序列、DNA G+C 含量、生理生化特徵以及 DNA-DNA 雜交反應的分析,菌株 T4 可被鑑定為 Geobacillus thermoleovorans。此菌種已分別存放德國與中華民國菌種中心,其編號分別為 DSM 14791 及 CCRC 17200。此外,也根據熱穩定纖維素分解酵素之氨基酸序列構築一個含 20 個相關微生物的親緣演化樹。本研究結果建議這些屬於醣苷水解酵素大家族中的熱穩定纖維素分解酵素可分成 4 個亞族:GH family 12 subgroup TC-1、bacterial subgroup TC-2、bacterial subgroup TC-3 以及 GH family 1 subgroup TC-4。再結合以 16S rDNA 序列將菌株 T4 和 10 株相關微生物進行的分析,菌株 T4 近似於亞族 subfamily TC-4 但與亞族 subfamily TC-1 在演化關係上較遠。 Fifty two cellulolytic thermophilic microorganisms were analyzed for their physiological characterization and phylogenetic systematics. Based on 16S rDNA sequence analysis, 3 strains from DCB and 4 novel isolates from southern Taiwan are close related to the genera of Bacillus and Geobacillus respectively. Among 4 new Geobacillus strains, strain T4, a Gram negative, motile, aerobically growing sporulating rod, can secrete thermostable endoglucanase. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70°C. Based on 16S rDNA sequence analysis, DNA G+C content, phenotypic and physiological characteristics, as well as DNA-DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200)。 Furthermore, a phylogenetic tree of 20 related microorganisms was also constructed based on their thermostable cellulase amino acid sequences. Our sequence analysis shows that cellulases belonging to the large family of glycoside hydrolases (GHs) can be divided into four subfamilies: TC-1 (GH family 12 group), TC-2 (bacterial group Ι in which fungal species Thermoascus aurantiacus fits), TC-3 (bacterial group ΙΙ), and TC-4 (GH family 1 group). Together with the 16S rDNA sequence analysis of strain T4 and 10 related microorganisms, strain T4 has a close phylogenetic relationship with subfamily TC-4 but far from subfamily TC-1.

Record ID 第141筆 System ID 091NSYS5112037
BCRC ID Public Year 91
Paper Name 樟芝(Antrodia camphorata)菌絲體培養及粒線體rDNA序列分析之研究 Studies on the culture and mitochondrial rDNA sequence analysis of Antrodia camphorata mycelium
Student Name 薛文明
Teacher Name 卓忠隆
School Department Name 國立中山大學 生物科學系研究所 Academic Degree 碩士
Abstract 樟芝(Antrodia camphorata)為台灣特有真菌,僅生長於台灣牛樟樹(Cinnamomum kanehirae),具有許多的保建功效,目前是以菌絲體醱酵培養為主。 本研究第一部份在探討樟芝菌絲體(CCRC 35716 )液態培養中其胞外多醣體、三萜類、pH值及生長之變化,並藉由HPLC分析菌絲體三萜類差異。實驗結果顯示以PDB為液態培養基之液態醱酵14天,在第11天時胞外多醣體含量最高(0.23 �b 0.002 g/L),第8天時之乾菌重最高(6.19 �b 0.190 g/L),第12天三萜類含量達最高,第8天時其pH值最低(3.20 �b 0.032),碳源在第8-9天時達最低。起始pH值在3-6時均適合菌絲體生長,pH 2時生長受抑制。起始pH 7時三萜類含量最高,以PDA為培養基之固態培養,照光對三萜類代謝量有負面的影響。由三萜類之HPLC分析圖譜可以分辨樟芝、靈芝、雲芝、冬蟲夏草、蜜環菌、猴頭菇等菌絲體三萜類之差異,可藉此一特性來進行藥用真菌產品的相關檢驗分析。 第二部份在分析樟芝(Antrodia camphorata)粒線體rDNA序列,探討在演化上的親緣關係。利用粒線體DNA序列之ssu rDNA及lsu rDNA二段序列並以Neighbor Joining及UPGMA法產生的演化樹圖譜結果顯示,可用mtDNA ssu rDNA序列來確定牛樟芝屬內的演化地位,可作為分子生物學上鑑定的依據之一。但種內之親緣關係則無法提供有效的解析。 Antrodia camphorata is a Taiwan-unique fungus which grows on the walls inside the core of decayed Cinnamomum kanehirae. Antrodia camphorata provides many bioactive substance and it is cultivated mainly via mycelium nowadays. First section of this research is focused on Antrodia camphorata (CCRC-35716) in liquid cultivation medium and the changes within extracellular polysaccharides, trierpenoids, pH and growth。 It also uses HPLC to analyze the differences of mycelium triterpenoids. This experiment showed when Antrodia camphorata was cultivated in PDB liquid medium for 14 days; it had the highest extracellular polysaccharides (0.23 �b 0.002 g/L) in day 11, the highest dry mycelium (6.19 �b 0.190 g/L)in day 8 and the highest triterpenoids in day 12,the lowest pH (3.20 �b 0.032) in day 8, and the lowest carbon source in day 8 and day 9. pH of 3-6 in most favorable for the growth of mycelium and pH 2 inhibits its growth. When growing at pH 7, triterpenoids volume is at the highest. When cultivating with PDA soil medium, light has negative effects on triterpenoids metabolism. Based on the triterpenoids HPLC analysis chart, Antrodia camphorata, Ganoderma lucidum, Coriolus versicolor, Hericium erinaceum, Armillaria mellea, Cordyceps sinensis, etc. can be identified easily. Difference in Triterpenoids can also be used to analyze other fungi used in medicine. Second section of this research is to analyze the sequence of mitochondrial rDNA and discuss its phylogeny. By utilizing two mitochondrial DNA sequences, ssu rDNA and lsu rDNA, and Neighbor Jointing and UPGMA method, Phylogeny tree was developed which can be used to identify in molecular biology the Antrodia camphorata in the phylogeny. However, it can not offer effective analysis in identifying relationships among the same species .

Record ID 第142筆 System ID 085NSYS3277008
BCRC ID CCRC 10670, Public Year 85
Paper Name 噬菌體控制鰻魚(Anguilla Japonica)病原菌Aeromonas hydrophila, Edwardsiella tarda之研究
Student Name 羅仲邑
Teacher Name 徐基新
School Department Name 國立中山大學 海洋資源學系 Academic Degree 碩士
Abstract Aeromonas hydrophila及Edwardsiella tarda為鰻魚最主要之病原菌。目前在養殖界控制病源菌的方式仍以施用抗生素等藥劑為主,然而在細菌抗藥性不斷增強下,發展另類拮抗物質有其必要性。在自然界中,噬菌體是造成細菌死亡的重要因子,本研究的目的在於了解噬菌體於自然水相中溶裂宿主菌的能力,提供實際應用參考。以A.hydrophila ATCC 7966、E.tarda CCRC 10670及來自病鰻所分離之E.tarda為宿主,自南台灣水域中分離出5株強溶裂型噬菌體,分別定名為A1、A2、E1、E2及φE1。經初步鑑定,A2、E1、E2遺傳物質皆為雙股DNA且以限制酉每EcoR I切割時,可分別為酵素切割成至少6、20及22條片段。在實驗室中觀察顯示,25℃下,在感染倍數(multiplicity of infection, M.O.I.)約等於10時,皆可在2小時內使菌數降低3-5次方。在鑑別培養基評估方面,starch-ampicillin agar在計數及準確性上皆相當優越,可直接用來計數環境水相中的A.hydrophila菌數;salmonella-shigellaagar也同樣可直接用來計數環境水相中的E.tarda菌數。田間試驗裡,額外添加A.hydrophila至每毫升6 ×105菌數而噬菌體A1的感染倍數為0.234時,在8小時內即將A.hydrophila菌數降低245倍,同時產生高量的噬菌體。而E.tarda並不能在水體長存。在沒有添加噬菌體的情況下,即使額外添加菌數至每毫升四次方,終究會在48小時內菌數降低為原菌數之千分之一。 Aeromonas hydrophila and Edwardsiella tarda are the major pathogen of eels. At present, antibiotics are widely used in culture ponds in order to kill pathogenic bacteria, but the chemotherapeutic method is not successful for controlling the drug-resistant strains. It``s necessary to find another way for killing pathogenic bacteria. In the nature, bacteriophages are an important factor of bacterial mortality. The purpose of this studies was to understand the ability of bacteriophages lysis of bacterium in the natural water. In this study, five bacteriophages were isolated from southern Taiwan aquatic environment. The bacteriophages isolated on A. hydrophila ATCC 7966, E.tarda CCRC 10670 and the E。tarda strain from diseased eel were named as A1, A2, E1, E2 and ψE1. After characterization, bacteriophages A2, E1 and E2 contained double-stranded DNA. The bacteriophage genomes could be cut with restriction endonuclease EcoR I into at least 6, 20 and 22 fragments. Those five bacteriophages could lyse their hosts quickly. The total viable cells could be reduced to less than 10-3-10-5 of starting concentration by infection at an M.O.I.=10 in 2 hours. In selective media evaluation, starch-ampicillin agar permitted quantitative recovery and showed good accuracy of A. hydrophila for counting from natural water, the salmonella-shigella agar can also be using for counting E. tarda. In field experiment, the viable A. hydrophila concentration was reduced to 0.004 of original concentration 6 ×105/ml after adding bacteriophage A1 to M.O.I.=0.234 within 8 hours and relased large quantity bacteriophages. However, E.tarda could not be matained in nature water. Without adding bacteriophage, the viable E.tarda concentration was reduced to 0.001 of original concentration 104/ml in nature water during 48 hours.

Record ID 第143筆 System ID 093NCU05063052
BCRC ID BCRC 35396, Public Year 93
Paper Name 探討樟芝發酵生產Aflatoxin B1解毒酵素與純化 none
Student Name 林育成
Teacher Name 徐敬衡
School Department Name 國立中央大學 化學工程與材料工程研究所 Academic Degree 碩士
Abstract 在現今的食品當中,潛藏著許多對人體造成健康危害的毒素,這些毒素來源有:黴菌毒素、細菌類毒素、重金屬毒素、農藥和化學污染等等,對人體可是造成疾病的發生。其中黃麴毒素AFB1為黴菌毒素的一種,主要污染來源為豆類食品。在毒性上,相對於其它具有相似結構的黃麴毒素,被認為是毒性較強的黴菌毒素。從文獻上,也發現在真菌類當中,具有其降低AFB1毒性的解毒酵素,反應後發現在螢光下偵測其AFB1吸收值降低。因此在實驗研究中,也希望能從真菌類樟芝發酵中,找出具有可降低AFB1毒性的解毒酵素。 本研究以樟芝Antrodia camphorata BCRC 35396於氣舉式發酵槽培養下,找尋具有降低毒素毒性的解毒酵素,其反應上造成AFB1螢光降低,並將發酵液進行酵素分離純化。 解毒酵素的純化步驟是將發酵澄清液利用硫酸銨沉降、透析,再經過DEAE Sepharose陰離子交換管柱層析分離純化。最後由SDS-PAGE電泳分析,找出此解毒酵素分子量為54kDa。 在反應部份,則是將純化之酵素與AFB1反應,利用AFB1在螢光excitation 365nm偵測下,emission 420nm 會有吸收值,來作為判斷毒素反應變化情況。 none.

Record ID 第144筆 System ID 092NCU05063043
BCRC ID BCRC 35396, Public Year 92
Paper Name 探討通氣量對於樟芝發酵生產與純化脂解酵素之研究 The study of production and purification of lipase fermented from Antrodia camphorata by aeration rate
Student Name 林志成
Teacher Name 徐敬衡
School Department Name 國立中央大學 化學工程與材料工程研究所 Academic Degree 碩士
Abstract 本研究以樟芝Antrodia camphorata BCRC 35396於攪拌式發酵槽培養下,探討不同通氣量對於樟芝發酵生產脂解酵素的影響,並將最適條件之發酵液進行酵素的分離純化。由實驗結果顯示,0.3 vvm的最大脂解酵素活性為1.151 U/ml,而1.0 vvm的最大酵素活性為2.588 U/ml,大約提升了2.25倍,所以通氣量的增加有利於脂解酵素活性的提升。而YL/P(specific activity)顯示,0.8 vvm有較高的比活性(29.493 U/mg)。在蛋白質的生成方面,0.3 vvm的最大蛋白質含量為0.061 mg/ml,而1.0 vvm的最大蛋白質含量為0.120 mg/ml,大約提升1.97倍,所以提升通氣量對於蛋白質含量有提高的效果。 脂解酵素的純化步驟是將發酵澄清液利用冷凍乾燥再經由DEAE Sepharose陰離子交換管柱層析分離純化。經過冷凍乾燥處理的脂解酵素活性及比活性分別為341.6 U及29.19 U/mg。而通過陰離子交換管柱層析後,純化倍率為4.50倍,回收率為8.28%。最後由SDS-PAGE電泳分析,發現最後純化的脂解酵素分子量是60.0 kDa。 This work studied the production and purification of lipase fermented from Antrodia camphorata BCRC 35396 by aeration rate in the stired tank。 Shown by the experimental result, the more aeration rate can enhance the lipase activity. The biggest lipase activity of 1.0 vvm was 2.25 times higher than that of 0.3 vvm. The specific activity YL/P show the 0.8 vvm has the higher quality than other aeration rate. The biggest protein of 1.0 vvm was 1.97 times higher than one of 0.3 vvm. Lipase fermented from Antrodia camphorata BCRC 35396 was purified by lyophilization as concentration, follow by anion exchange chromatography(DEAE-Sepharose)。 The specific activity of the purified enzyme was 4.5 times higher than that of the crude broth after lyophilization. The lipase had a molecular weight of 60.0 kDa when examined by SDS-polyacrylamide gel electrophoresis.

Record ID 第145筆 System ID 092NCU05105001
BCRC ID CCRC 12422, Public Year 92
Paper Name Actinomycetes H12所產轉麩胺酸醯胺基酶之生產、特性及加工製程之探討 Production, characterization and procession of transglutaminase produced by Actinomycetes H12
Student Name 許寶月
Teacher Name 黃雪莉
School Department Name 國立中央大學 生命科學研究所 Academic Degree 碩士
Abstract 摘要 本研究由50多個土壤樣品中分離出15,000株放線菌,其中以Actinomycetes H12有最高之轉麩胺酸醯胺基酶酵素活性。Actinomycetes H12於最適化液態培養條件下,培養可得轉麩胺酸醯胺基酶產量2.7 U/ml,較原始培養(TSB)條件下之轉麩胺酸醯胺基酶產量提高1.4倍,較S. ladakanum CCRC 12422之轉麩胺酸醯胺基酶高1.8倍,而與日本生產轉麩胺酸醯胺基酶之菌株Streptoverticillium sp. S-8112 產量2.5 U/ml相似。 Actinomycetes H12 之轉麩胺酸醯胺基酶蛋白質特性分析:其最適作用溫度40℃,熱穩定性在50℃下加熱30分鐘仍保有50%活性,最適作用pH 6-8。此轉麩胺酸醯胺基酶活性會受到Cu2+、 Zn2+明顯之抑制,但一價離子、Fe3+與Ca2+則不影響其活性。 Actinomycetes H12發酵槽液態培養至40-48小時,此時其酵素分泌至胞外比率仍低,利用此特性將發酵液高速離心後,可直接回收帶有酵素之菌泥,不經純化下其收率為85%,經冷凍乾燥後其活性不變,但發現有Escherichia coli,因之再輔以Co 60-10kGy照射殺死病原菌及生產菌,然其活性下降至65%,即使照射時加入抗氧化劑(β-carotene, ascorbic acid)亦不能改善提高其殘留活性。 We isolated 15,000 Actinomycetes strains from over 50 soil samples, one isolated strain H12 have the highest TGase activity. The TGase activity (2.7U/ml) under the optimal conditions was about 1.4-fold than the TSB medium condition and 1.8-fold than the TGase from S. ladakanum CCRC 12422, but it is similar to the enzyme from Streptovertecillium sp。 S-8112 (2.5 U/ml). The crude TGase from Actinomycetes H12 had the optimum pH and temperature being pH 6-8 and 40℃, respectively. The stable pH range was 5-9 and thermal stability of the crude TGase remained 50% activity after treatment at 50℃ for 30 min. The metal ions, Cu2+ and Zn2+, inhibited the activity of TGase. The addition of monovalence, Fe3+ and Ca2+ did not affect the activity of TGase. When Actinomycetes H12 was cultured on fermentor after 40-48 hr, the extracellular TGase still low. So we centrifuged the culture fluid in a single step and with high yields (85%), then the TGase activity didn’t decrease after freeze-dry. There were contaminants in products, therefore we treatment with Co 60-10 kGy killing germs and Actinomycetes H12, but the enzyme activity is decreased (65%). It didn’t improve to raise the activity, even thought added β-carotene and ascorbic acid.

Record ID 第146筆 System ID 089NCU00063042
BCRC ID CCRC 36431, Public Year 89
Paper Name 探討不同培養方式對猴頭菇抗氧化與抗腫瘤性質的影響
Student Name 吳姿宜
Teacher Name 徐敬衡
School Department Name 國立中央大學 化學工程研究所 Academic Degree 碩士
Abstract 文獻指出人體老化、病變與體內氧化還原平衡失調有關,科學家證實自由基(free radical)與多種疾病的產生有密切的關係,他能引起動脈粥狀硬化、糖尿病、癌症、發炎反應、老化等疾病。近年來發現許多的菇類(如舞菇、猴頭菇、金針菇、香菇、雲芝和赤芝等)以中極性甲醇萃取評估其具有抗氧化性質的成分,菇類的抗氧化性更值得去探討。猴頭菇在中國是著名的藥用真菌之一,其生理活性成分豐富,據文獻記載,猴頭菇子實體中的抗氧化物質以酚類物質所佔的比率最高,加上有許多的研究指出酚類物質具有相當強的抗氧化力與抗腫瘤能力,以茶多酚為例,能抗低密度脂蛋白氧化作用、有抗衰老作用、對抗紫外線及放射線致癌作用、抗突變等作用。 本研究將探討不同萃取溶液(甲醇、乙醇、丙酮)、不同菌種(猴頭王、中特猴、 CCRC 36431 、 CCRC 36431 )、不同培養方式(固態培養、搖瓶培養、發酵槽培養)對猴頭菇子實體萃取酚類物質能力或菌絲體的菌體產率、總酚產率及其清除DPPH自由基的影響進行比較,以選出較佳的培養條件與分析方法。然後在以最適條件進行改變培養基實驗,在培養基中添加有利物質-綠茶多酚,探討添加綠茶多酚是否能增加猴頭菇抗氧化、及抗腫瘤性質。 實驗結果發現,猴頭菇的酚類物質萃取溶液以乙醇較為適合,菌種篩選部份則以猴頭王生長速率最快,萃取出來的酚類物質含量及清除自由基能力最強,品質最好,而培養方式則以2L bubble column發酵槽效率最好,菌絲體萃取液酚類物質產率(1.12mg/g.day)遠比子實體含量高,約為子實體的16.4倍,且其菌絲體清除DPPH自由基能力(86.8%)約為子實體的2.3倍。在培養基中添加不同量綠茶多酚部份,隨著綠茶多酚添加量的增加,猴頭菇的生長速率、菌體轉化率、總酚產率、水溶性酚類物質之抗腫瘤能力有增加的趨勢,以添加3.3g綠茶多酚為例,他的生長速率為0.0364 1/hr,約為control組的2倍,總酚產率為0.113 mg/g-hr為control組的2.3倍。探討水溶性酚類物質對不同癌細胞的抑制能力部份,發現菌絲體所得的腫瘤抑制率較子實體抑制能力高,隨著綠茶多酚添加量的增加,其抑制能力有增加的趨勢。以胃癌為例,添加3.3g綠茶多酚所得菌體水溶性酚類濃度為7.5ppm,胃癌細胞的存活率為35.8%較control組的存活率72.6%高。

Record ID 第147筆 System ID 088NCU00105003
BCRC ID CCRC 12567, CCRC 10069, Public Year 88
Paper Name 利用乳酸菌表達大腸桿菌之酸性磷酸與其在模擬腸胃道環境之定性探討
Student Name 曾季清
Teacher Name 董啟功
School Department Name 國立中央大學 生命科學研究所 Academic Degree 碩士
Abstract 本研究藉由基因重組技術,將植酸與乳酸菌二者相結合,欲促使乳酸菌群在動物體內多了一項代謝植酸的功能,希望藉此能幫助加強宿主動物營養份吸收,並間接達到減少以植酸的形式隨動物體的排遺排出體外之無機磷所造成的環境污染問題。 本研究首先利用聚合鏈鎖反應(PCR)的方式成功的篩選出大腸桿菌(Escherichia coli B strain CCRC 12567)之酸性磷酸基因,其長度為1237 bp;接著將該基因送入大腸桿菌之蛋白質表現系統(pET system),並證明所篩選出之基因的確能轉譯成具有能夠分解抗營養因子植酸之酵素 然後將酸性磷酸基因與穿梭載體pGIT032重新構築後,以電穿孔(electroporation)將此重組載體送入Lactobacillus plantarum CCRC 10069、Lactobacillus casei subsp. rhamnosus GG、Lactobacillus casei Shirota等三種不同乳酸菌。結果顯示轉殖後之菌種胞內酵素活性表現以Lactobacillus casei subsp. rhamnosus GG轉殖株之植酸表現最高,達未轉殖單一宿主菌株之62.4倍,而Lactobacillus casei Shirota之轉殖株之植酸表現亦分別為2倍,但Lactobacillus plantarum的活性幾乎與對照菌株相同。 最後將此三種轉殖菌株進行模擬體內消化道環境酸度、膽鹽耐受性試驗。在酸度耐受性試驗方面,三種轉殖株經pH 2.0之Rogosa and Sharpe(MRS)處理、培養後均不生長;但經pH 3.0處理後,任一轉殖菌均發現當酸度處理時間愈長,其菌液於培養皿內培養後之生成菌落數愈少。此外,三種菌轉殖株均不具0.3和0.5%Oxygall膽鹽之耐受性。 Abstract In this study, an acid phosphatase gene coding for phytase activity was obtained from Escherichia coli B strain and transferred to several species of Lactobacillus using recombinant technology. This process was expected to provide the increase of nutrient absorption and decrease of phytates excretion for the animals that harbor these genetically engineered bacteria carrying extra phytase activity. The acid phosphatase gene of Escherichia coli B strain was cloned using polymerase chain reaction (PCR) technique. Subsequently, the gene was transferred into pET expression system using BL21 cells as host. The recombinant Escherichia coli successfully expressed fused protein affirmatively carrying phytase activity. The same gene was set to transform the Lactobacillus using shuttle vector pGIT032. After the recombinant vector was constructed, it was transferred to Lactobacillus plantarum, Lactobacillus casei subsp. rhamnosus GG and Lactobacillus casei Shirita using electroporation technique. The results showed the intracellular phytase activity of the transformants of Lactobacillus casei subsp. rhamnosus GG increased 62.4 times and Lactobacillus casei Shirita increased 2 times while Lactobacillus plantarum remained unchanged. All three different transformed Lactobacillus were subjected to simulation tests for gastrointestinal tract in animals including acid tolerance and bile-salt tolerance. In pH 2.0, all three showed no tolerance. In pH 3.0, the tolerance decreased as the incubation time increased. Furthermore, no tolerance was observed in the treatments by bile salt of 0.3 and 0.5% of Oxygall.

Record ID 第148筆 System ID 090CCU00063010
BCRC ID CCRC 10809, Public Year 90
Paper Name 選殖有胞外蔗糖水解酵素基因之Zymomonas mobilis的乙醇醱酵與蛋白質分析研究 Ethanol fermentation and protein characterization of Zymomonas mobilis cloned with extracellular sucrase gene
Student Name 詹議翔
Teacher Name 李文乾
School Department Name 國立中正大學 化學工程研究所 Academic Degree 碩士
Abstract 革蘭氏陰性菌Zymomonas mobilis具有優異的乙醇生產力,唯一的缺點是其在厭氧情況下經由恩-杜氏途徑醱酵代謝醣類的能力只限於葡萄糖、果糖及蔗糖,其它的單醣或多醣則無法被吸收利用。以蔗糖為碳源時,Z. mobilis無法將蔗糖磷酸化而直接消化代謝,必須分泌酵素將蔗糖水解成葡萄糖與果糖才能利用,且會產生副產物果聚糖和山梨糖醇。 本研究所使用的菌種即為選殖有胞外蔗糖水解酵素SacC之Z. mobilis CCRC 10809,含有質體pZM-SacC,質體大小為7.7 Kb,具有抗氯黴素(chloramphenicol, Cm)基因標記 在本研究中,利用不同濃度蔗糖為碳源進行Z. mobilis CCRC 10809之乙醇醱酵。隨著蔗糖濃度的增加,重組菌的乙醇收率提高,並維持在0.334 ~ 0.357 g/g (理論值的73~78 ﹪)左右,野生菌的乙醇收率則明顯下降,從0.346 g/g降到0.285 g/g(理論值的75.7降到62.3﹪)。在相同蔗糖濃度比較下,重組菌的乙醇收率皆高於野生菌,尤其以200 g/L蔗糖濃度時,重組菌乙醇收率與野生菌的乙醇收率差距較大,約0.049 g/g。 比較蛋白質二維電泳圖發現重組菌之胞外蔗糖水解酵素蛋白質的分泌量沒有明顯的比野生菌高出許多,重組質體胞外蔗糖水解酵素分泌的表現似乎並不明顯,可能是造成重組菌乙醇收率沒有大幅提高的原因。 本實驗最後也推導Z. mobilis以蔗糖為碳源之醱酵計量平衡式與動力學模式。由計量平衡式計算之數據可顯示蔗糖之比水解速率 (αqS)大於果聚糖之比生成速率(βqS),可表示胞內與胞外蔗糖水解酵素的活性總和大於胞外蔗糖-6-果糖基轉移酵素的活性。若將野生菌與重組菌在蔗糖水解速率增加的時間範圍計算結果,重組菌αqS/βqS的比值皆大於野生菌,顯示經選殖胞外蔗糖水解酵素的重組菌具有較高的蔗糖水解活性,使蔗糖在胞外能較充分水解成葡萄糖和果糖,有助於Z. mobilis對蔗糖的攝食。動力學模式方面,其中蔗糖、菌體濃度和乙醇的實驗數值大致和模擬的數值曲線符合。此結果顯示,對於菌體生長、蔗糖的消耗與主要產物乙醇的生成能夠較準確的預測,將有助於未來Z. mobilis利用蔗糖大量生產乙醇的醱酵程序設計,以期獲得更佳的經濟產值。 Zymomonas mobilis is a Gram-negative bacterium that is known as an efficient ethanol producer. It uses an exclusively fermentative metabolism through the Entner-Doudoroff pathway under anaerobic conditions to metabolize glucose, fructose, or sucrose. On sucrose-based substrate, it may be postulated that sucrose is first hydrolyzed to form glucose and fructose, and then, these monosaccharides are uptaken by the cell. The ethanol yield from sucrose is lower due to the formation of by-products, mainly levan and sorbitol. In this study, we used the strain of Zymomonas mobilis CCRC 10809 cloned with extracellular sucrase gene (SacC gene)。 The recombinant plasmid was pZM-SacC, which is a 7.7 kb molecule and codes for the chloramphenicol resistance gene (Cmr) as a selectable marker gene. Compared with the batch ethanol fermentation of wild-type Zymomonas mobilis CCRC 10809 on different concentrations of sucrose, the recombinant Z。 mobile led to higher ethanol yields. The ethanol yield of recombinant strain could keep at 0.334 to 0.357 g/g (73.1 to 78.1﹪of the theoretical value). But the ethanol yield of wild-type strain was reduced from 0.346 to 0.285 g/g (75.7 to 62.3 ﹪of the theoretical value) by the increase of sucrose concentration from 150 to 200 g/L. At the same sucrose concentration, the ethanol yield of recombinant cell was always higher than that of wild-type cell; especially at the sucrose concentration of 200 g/L, the difference in ethanol yield was greater than 0.049 g/g. A comparison of results from the two-dimensional gel electrophoresis of extracellular proteins secreted by wild-type and recombinant strains indicates that the recombinant strain did not secret more amount of extracellular sucrase than the wild-type. This may be why the ethanol yield by the recombinant strain wasn’t significantly higher than that of the wild-type. In this study, the stoichiometric and kinetic models for Zymomonas mobilis fermentation using sucrose as the sole carbon source system were also developed. According to the calculation based on mass balance, the specific sucrose hydrolyze rate (αqS) was higher than the specific levan formation rate (βqS). That meant the total activity of extracellular and intracellular sucrase was higher than the activity of extracellular levansucrase. When the αqS/βqS values were calculated based on the time of the largest sucrose-hydrolyzing rate, the recombinant strain was significantly higher than the wild-type strain. It meant the recombinant strain had a highly extracellular sucrase activity, which was helpful for sucrose hydrolysis to form glucose and fructose, and then, for the cell to assimilate these sugars easily. In the model simulation, the experimental data of sucrose, biomass and ethanol concentrations were fitted to the kinetic model. Modeling work on the kinetics of ethanol fermentation by Zymomonas mobilis on sucrose will be helpful for scaling-up such ethanol-producing process.

Record ID 第149筆 System ID 094NCHU0253002
BCRC ID BCRC 12947, BCRC 14848, BCRC 12963, BCRC 14824, BCRC 13825, Public Year 94
Paper Name 食品病原菌與金黃色葡萄球菌腸毒素檢測用生物晶片之發展以及金黃色葡萄球菌之分子分類與即時聚合酶鏈反應 Development of Microarrays for Food Bacterial Pathogens and Staphylococcus aureus Enterotoxin Genes, as well as Molecular Typing and Real-Time PCR for Staphylococcus aureus
Student Name 蔣育錚
Teacher Name 曾浩洋
School Department Name 國立中興大學 食品科學系 Academic Degree 博士
Abstract 在食品或臨床樣品中快速檢測、鑑定其可能污染或感染的微生物菌屬(genus)或菌種 (species) 相當的重要。本研究利用微生物之16S rDNA發展出一套寡核苷酸生物晶片系統來鑑定微生物,可成功鑑定出的微生物包括Bacillus 屬、Escherichia coli、Salmonella 屬、 Staphylococcus 屬以及Vibrio屬,這些微生物常見於食品中毒案件裡以及一些偶發性的中毒案件中。本研究利用生物素(biotin)標定的共通引子組(universal primer)對檢測目標基因的變異區(variable region)進行PCR增幅,而標定後的PCR產物與寡核苷酸生物晶片進行雜交,於四小時的時間內可快速的鑑定微生物菌屬。本研究以尼龍膜型生物晶片上佈放有15條寡核苷酸探針,用以決定生物晶片雜交圖譜。依照圖譜,可成功區分出Escherichia coli、 Bacillus 屬、 Salmonella 屬、Staphylococcus 屬以及Vibrio 屬。Bacillus 屬、 Salmonella 屬、Staphylococcus 屬以及Vibrio 屬的生物晶片檢測僅限於屬的層次(genus level),沒有到種的層次(species level)。在182株隨機挑選的微生物菌株中,本寡核苷酸生物晶片的檢測正確率可高達98%以上。除了其中3株的微生物無法成功的進行PCR及雜交圖譜外,其他的179株可以正確的鑑定出該目標微生物,而且沒有交叉雜交的反應出現。這些179株的微生物可以區分為5種雜交圖譜。藉由其他特異性探針的增加,也許可以增加此型生物晶片對更多種微生物的檢測,而不用額外的增加成本及複雜度。 另一方面,本研究利用隨機引子擴增的方法,針對檢測微生物之DNA進行標定螢光Cy3-dUTP、Cy5-dUTP之工作,並利用文獻發表針對特定病原菌株之基因探針,如腸炎弧菌之 pR72H 片段,沙門氏菌屬(Salmonella spp.)之1.8 kb 片段之特異性探針,並自行設計多組PCR引子組,分別針對Salmonella Typhimurium、Staphylococcus aureus、E. coli O157:H7及 Listeria monocytogenes 等,以其PCR產物做為生物晶片探針,佈放於生物晶片上,並與上述細菌DNA雜交,其結果顯示,本生物晶片可成功的鑑定微生物至血清型以及種的層次(serotype and species level)如:Salmonella Typhimurium (BCRC 12947)、 Listeria monocytogenes (BCRC 14848)、 Vibrio parahaemolyticus (BCRC 12963)、 Escherichia coli O157:H7 (BCRC 14824)、 B. anthracis (ATCC14578, ATCC 8705)、Staphylococcus aureus (BCRC 13825)。另外在Salmonella Enteritidis (ATCC 13076)方面,則可以鑑定至屬的層次(genus level)。本研究成功的建立螢光型生物晶片系統,並發展出能同時檢測多種微生物之生物晶片。 在S. aureus 的腸毒素檢測方面,為了調查台灣地區金黃色葡萄球菌中毒案件中傳統及新型腸毒素之分佈情形,本研究設計了新型腸毒素基因sek、sel、sem、sen、seo和sep之PCR引子組。針對新型腸毒素所設計的引子組,除帶有sek、sel、sem、sen、seo和sep等腸毒素基因之金黃色葡萄球菌會有PCR產物之外,其餘之非目標菌,如葡萄球菌屬,皆無PCR產物的出現。本研究亦利用反轉錄聚合酶鏈鎖反應 ( RT-PCR ) 對帶有新型腸毒素基因sek、sel、sem、sen、seo及sep的金黃色葡萄球菌菌株,進行鑑定其是否有基因表現之可能,並利用傳統及新型腸毒素之PCR引子組(包括SEG, H, I, J),對臨床樣品及食品中毒案件分離之金黃色葡萄球菌進行腸毒素分佈調查。結果顯示,當新型腸毒素PCR引子組SEK1/SEK2, SEL1/SEL2, SEM1/SEM2, SEN1/SEN2, SEO1/SEO2 以及SEP1/SEP2應用於鮮乳及肉品樣品中之金黃色葡萄球菌腸毒素基因sek、sel、sem、sen、seo、sep檢測時,在經預培養10小時後,其靈敏度可達到N × 100 CFU/每毫升食品均質液。本研究中,隨機挑選帶有新型腸毒素基因之金黃色葡萄球菌菌株,以RT-PCR方法檢測時,發現其皆具有新型腸毒素基因RNA的表現。由傳統及新型腸毒素基因的分佈調查發現,百分之70以上的金黃色葡萄球菌,常帶一個或一個以上之腸毒素基因。 在進行臨床及食品中毒案件分離之金黃色葡萄球菌抗生素基因mecA及vat(A)的檢測方面,發現醫院來源之S. aureus 有相當高的MRSA(Methicillin-resistant S. aureus)分離比率,達到百分之60左右。於本研究中並未發現vancomycin 抗藥性S. aureus菌株之存在。在生物晶片檢測系統檢測腸毒素基因方面,本研究建立之螢光型生物晶片系統,可同時、快速且準確的鑑定出S. aureus 之腸毒素基因。本研究亦利用脈衝電泳(Pulsed field gel electrophoresis)分析收集至醫院臨床分離S. aureus菌株及食品中毒案件分離S. aureus菌株,結果顯示,於2003年分離至台中榮總分離之S. aureus可區分為20分型(C1~C20),而2001~2003年分離至疾病管制局第三分局之食品中毒人體來源S. aureus可區分為38型(Y1~Y38)。本研究所收集之MRSA菌株,經PFGE分析後,得到18種分型 (subtypes)。並由PFGE分型結果可知,醫院來源主要流行菌株與食品中毒來源之主要流行菌株不同。且2003年台中榮總之S. aureu分離株與1998年台中榮總之臨床分離株具有高度的同源相關性。在MRSA菌株與傳統腸毒素基因方面,醫院來源之MRSA菌株常帶有sea基因而食品中毒來源之MRSA菌株常帶有seb基因。醫院來源MRSA菌株之分離比率比食品中毒來源之MRSA菌株有較高的分離頻率。藉由PFGE圖譜發現,1998年至2003年台中榮總之MRSA菌株具有相當的同源相關性,且同源相關性高的MRSA菌株重複出現。在臨床來源S. aureu分離株與食品中毒來源之S. aureu分離株中,S. aureu菌株之間傳染的可能性似乎是低的。 最後,本研究嘗試以Real-Time PCR(RT-PCR)系統,在純菌及食品樣品中,直接檢測金黃色葡萄球菌及其SEN、SEO、SEP腸毒素基因。結果顯示,利用Sa1S/Sa1A、SEN1/SEN2、SEO1/SEO2及SEP1/SEP2引子組,在食品樣品中(如牛奶及肉品中)之檢測方面,在樣品不經預培養或經預培養10小時後之狀況下,其Real-Time PCR之檢測極限皆可達到N×100 CFU /每毫升食品均質液。RT-PCR在食品樣品的檢測靈敏度方面,在不經預培養的步驟下,其檢測靈敏度可達到100 CFU /每毫升食品均質液。結果顯示,RT-PCR比傳統PCR (conventional PCR)比較,Real-Time PCR 具有較高的靈敏度、快速等優點。 Rapid identification of the genus and species of bacteria in foods and clinical specimens is important. In this report, DNA sequences of bacterial 16S rDNA were used to develop the oligonucleotide array for the identification of bacterial strains of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. Most of these bacterial strains may cause food-borne outbreaks or sporadic cases. A rapid (<4 h) detection method that used universal PCR primers to amplify the variable regions of bacterial 16S rDNA, followed by reverse hybridization of the PCR products, which were biotin labeled, to the oligonucleotides arrayed on the chip was developed. Fifteen oligonucleotide probes were selected and spotted on the nylon strip to determine the array hybridization patterns. It was successful in discriminating Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. with identification, in general, to the genus level, not species level. As 182 randomly selected strains were assayed, the detection rate was found higher than 98%. Except for 3 strains, the remaining 179 strains were correctly identified and no cross reactions were observed. These 179 strains generated five hybridization patterns. Adding more oligonucleotide probes to the array may allow the detection of more bacterial genera and species without significant increasing the complexity or cost. On the other hand, random primering method was used to amplify the total genomic DNA isolated from target organism. Cy3-dUTP and Cy5-dUTP were used for DNA labeling. Using the published unique DNA sequences for target organisms, for example, those from Vibrio parahaemolyticus pR72H DNA fragment or those from 1.8kb DNA fragment for Salmonella spp., and the self-designed PCR primers for Salmonella Typhimurium, S. aureus, E. coli O157:H7, and Listeria monocytogenes, we prepared PCR products and spotted them on glass-slides for microarray design. By using such microarray, we were able to detect Salmonella Typhimurium (BCRC 12947), Listeria monocytogenes (BCRC 14848), Vibrio parahaemolyticus (BCRC 12963), Escherichia coli O157:H7 (BCRC 14824), B anthracis (ATCC14578, 8705), and Staphylococcus aureus (BCRC 13825) on the species and serotype level。 As for Salmonella Enteritidis (ATCC 13076), the microarray we designed allowed the detection of S. Enteritidis at genus level. Overall, we have established a fluorogenic type microarray system for the specific and simultaneous detection of multi pathogens on microarray. For S. aureus enterotoxins (SEs), with an attempt to survey the distribution of classical and new SEs in staphylococcal intoxication in Taiwan, we developed PCR primers for SEK, SEL, SEM, SEN, SEO, and SEP genes. Bacterial strains other than sek, sel, sem, sen, seo, and sep S. aureus including strains of other Staphylococcus species, would not generate any false positive results. The expression potential for these sek, sel, sem, sen, seo, and sep strains were also identified by reverse transcription (RT)-PCR. Together with the PCR primers specific for classical SEs and other new SEs, including SEG, H, I, J, we surveyed the SE genes in S. aureus strains isolated from food-poisoning cases and clinical samples. When SEK1/SEK2, SEL1/SEL2, SEM1/SEM2, SEN1/SEN2, SEO1/SEO2, and SEP1/SEP2 primers were used for monitoring of the sek, sel, sem, sen, seo, and sep S. aureus strains in food samples, such as whole milk and pork, a detection limit of N ×100 CFU/ml of food homogenate could be obtained if a 10h pre-culture step was performed prior to the PCR. In this study, the RNA transcripts from sek, sel, sem, sen, seo, and sep strains were assayed by RT-PCR. It was found that all the randomly selected S. aureus strains with sek, sel, sem, sen, seo, and sep genes were positive by RT-PCR. More than 70% of S. aureus were found positive for one or more SE genes. S. aureus strains from clinical samples and food-poisoning cases were also assayed for their methicillin and vancomycin resistance genes, mecA and vat(A). The ratio of MRSA strains in clinical isolates of S. aureus was near 60%. Vancomycin resistance strains were not observed in any of these isolates. For the microarray detection system for the staphylococcal enterotoxin genes, we established a fluorogenic type microarray system for the specific and simultaneous detection of classical and new enterotoxins on microarray. Pulse field gel electrophoresis (PFGE) patterns for the SmaI digested chromosomal DNA from S. aureus strains were also investigated. Twenty subtypes (C1~C20) were found in clinical samples isolated from Veternary Genernal Hospital, Taichung, in 2003, and 38 subtypes (Y1~Y38) of food poisoning cases isolated from Center for Disease Control, Taichung during 2001~2003 were observed. In MRSA strains, eighteen PFGE types were found for all these MRSA strains from hospitals and food-poisoning cases. However, the major PFGE subtypes for strains from hospitals were different from those of the food-poisoning isolates. The subtypes for clinical strains isolated in 2003 were clonally highly similar to those isolated in 1998. For classical enterotoxin genes, sea and seb are the major types in MRSA strains from hospitals and food-poisoning cases, respectively. MRSA strains were more frequently found in clinical isolates than in food-poisoning isolates. Major MRSA strains may circulate in hospitals during 1998~2003 as evidenced by PFGE patterns. Also, possibility for strain transmission between hospital isolates and food-poisoning isolates seems to be low. Finally, we also tried to use Real-Time PCR (RT-PCR) for the specific detection of S. aureus and their SEN, SEO, and SEP enterotoxin genes in pure culture and food samples. When Sa1S/Sa1A, SEN1/SEN2, SEO1/SEO2 and SEP1/SEP2 primers were used for monitoring of S. aureus and the sen, seo and sep S. aureus strains in food samples, such as whole milk and pork, a detection limit of 100 CFU/ml of food homogenate could be obtained with or without a 10 h pre-culture step. The RT-PCR detection sensitivity for food samples was 100 CFU/ml of food homogenate without a pre-culture step. Such data indicates that RT-PCR is more sensitive and rapid than the conventional PCR method.

Record ID 第150筆 System ID 083NCHU0366013
BCRC ID CCRC 21509, Public Year 83
Paper Name Trigonopsis variabilis D-型氨基酸氧化�@基因在 Escherichia coli的 表現及突變分析 D-型胺基酸氧化 活性相關之胺基酸殘基 Expression of Trigonopsis variabliis D-amino acid oxidase gene in Escherichia coli and mutational anaysis of amino acid residues involved in the catalytic acitivity of D-amino acid oxidase
Student Name 傅惠美
Teacher Name 許文輝博士 賴美津博士
School Department Name 國立中興大學 植物學系 Academic Degree 碩士
Abstract Trigonopsis variabilis 的 D-amino aicd oxidase(EC.1.4.3.3, 以下 簡稱 DAO)可用於生產頭孢菌素類抗生素的先驅物 7- amino- cephalosporanic acid 。此酵素是 flavoprotein,由兩個相同的次單元 (subunit)所組成,且需與 FAD 結合才具有活性。T.variabilis dao 基因長 1,107 bp,並包含一個內子(intron)位於第 25 到 60 bp之間 。將 dao cDNA 基因選殖至表現載體 pET-21b 再轉形入 Escherichia coli BL-21(DE3),以 T7 啟動子控制其表現,在 IPTG誘導之下,其比 活性為 0.3 U/mg,為 T.variabilis CCRC 21509 (基因供給者 ) DAO 活 性的九倍。且 dao 基因於 E。coli BL-21(DE3)表現時,轉形菌株的生 長狀況良好。然而,構築於 pKm-cDAO 上的 dao 基因,於 E.coli DH5α 中表現時,轉形菌株的生長不良。為解決此問題,設計了七種培養基,發 現 LA 培養基添加 L-glutamic aicd 或 D-glutamic acid 時,會大幅提 高pKm-cDAO 對 E.coli DH5α 的轉形效率 transformation efficiency )。然而,對轉形菌株的生長,仍然無明顯的改善。將 dao 基因 構築 於 pALTER-cDAO 上,利用定點突變法,將 DAO 上的 His-324 改變為 Leu-324,發現此種 DAO 的活性消失。顯示 His-324 和 DAO 的活性有關 。將具有不同 DAO活性的質體 pKm-cDAO ,pKm-cDAOm1, pKm-cDAOm2, pTrc-cDAO7 及 pTrc-cDAO28 上的 dao 基因進行核甘酸定序及氨基酸比 對,得到 5 個氨基酸殘基的變異點,這些變異點未來可分別進行定點突 變,以確定這些氨基酸殘基是否確實與 DAO 的活性有關。.

Record ID 第151筆 System ID 093NCHU0061006
BCRC ID BCRC 12444, BCRC 13677, Public Year 93
Paper Name Pseudonocardia autotrophica BCRC12444的cytochrome P450 monooxygenase基因以及脂肪酸代謝基因串 Cytochrome P450 monooxygenase gene and a gene cluster for the fatty acid catabolism from Pseudonocardia autotrophica BCRC12444
Student Name 陳昭賢
Teacher Name 許文輝
School Department Name 國立中興大學 分子生物學研究所 Academic Degree 博士
Abstract (一) Pravastatin是一種高脂血症治療劑,可降低血中膽固醇,商業化製造包括兩個步驟,首先利用微生物生產compactin,接著將compactin 氫氧化而產生pravastatin。 Compactin生成可由Penicillium citrinum大量生產而得,然而,compactin的氫氧化必需靠cytochrome P450的催化才會轉變成pravastatin。如果利用化學合成法將compactin轉變成pravastatin,其效果不佳,易將部份產物轉成其他異構物,而且所需的成本非常的高。若用微生物來轉換compactin,可有效的在6 b位置氫氧化而形成pravastatin。在本研究希望尋找具有高轉換能力的菌種,選殖其cytochrome P450 monooxygenas的基因,利用轉形菌株大量轉換compactin形成pravastatin,來替代化學合成法的傳統方式。利用HPLC方法分析,雖然許多細菌都具有轉換compactin的能力,但只有P. autotrophica BCRC 12444及Steptomyces griseolus BCRC 13677真正能將compactin轉成pravastatin,雖然S. griseolus BCRC 13677的轉換能力較P. autotrophica BCRC 12444強,但較不穩定,pravastatin易再轉成其他產物,故選擇P. autotrophica做為標的菌株。針對此菌設計cytochrome P450 monooxygenase 基因的退化性引子,利用PCR方法找到一屬於此基因的DNA片段,利用此片段做為探針,以plaque hybridization方法,由 P. autotrophica的基因庫篩選二個含cytochrome P450 monooxygenase 基因的轉殖株,經定序及上網分析比對,整合出一含有六個基因的DNA片段,六個基因分別為acyl-CoA ligase (fadA)、enoyl-CoA hydratase (fadB)、transcriptional regulator (fadR)、cytochrome P450 monooxygenase (fadC)及ferredoxin (fadD)及hypothetical protein基因。純化的cytochrome P450 monooxygenase及ferredoxin無法將將compactin轉換成pravastatin,以RNA即時定量 PCR方法測試fadC基因的表現,發現此基因的表現不會因為compactin的誘導而提升。 (二) 從Pseudonocardia autotrophyca BBRC12444篩選到一段參與脂肪酸代謝的基因串,以各種DNA序列分析方法及引子延伸反應分析,此基因串除了六個ORF外,還包含一個雙向的啟動子。此fad基因串上被確定的五個基因分別命名為fadA、fadB、fadR、fadC及 fadD,基於其胺基酸的相似度,各基因的產物分別為acyl-CoA ligase (FadA)、enoyl-CoA hydratase (FadB)、transcriptional regulator (FadR)、cytochrome P450 monooxygenase (FadC)及ferredoxin (FadD)。以膠體泳動遲滯技術法及DNase I footprinting分析法,證實調節蛋白FadR可結合在位於fadR及fadC之間的雙向的啟動子區域的操縱子序列上,顯示此蛋白可調控脂肪酸的代謝作用。以即時定量PCR的方法證實了長鏈脂肪酸可誘導fadA、fadB、fadR及fadC基因的表現,而葡萄糖則會抑制這四個基因的表現,所有的數據均顯示此fad基因串與脂肪酸的代謝有關。 (1) Cholesterol is synthesized from acetyl-coenzyme A (Co A) in a pathway that includes more than 20 enzymatic steps. The rate-limiting step of this pathway is catalyzed by 3-hydroxy-3-methygultaryl (HMG)-CoA reductase. Pravastatin is an effective and tissue-selective hypolipidemic drug that inhibits the enzyme in cholesterol synthesis and is commonly used as cholesterol lowering therapeutic medicine. Pravastatin is produced by a two-step fermentation process. The first step is production of compactin by microorganisms and the second is a hydroxylation step that converts compactin to provastatin. Penicillium citrinum can be used as a compactin-producing strain. The cytochome P450-dependent monooxygenase systems are found to participate in the hydroxylation mechanism. Although many microorganisms were found with potently convertible properties, P. autotrophica could potently convert compactin to pravastatin and was used to clone the p450 monooxygenase gene from it. Three degenerate primers were designed from the conserved region of p450 monooxygenase gene. A DNA fragment was amplified from P. autotrophica using nested polymerase chain reaction. DNA sequence analysis revealed that this DNA fragment is part of p450 monooxygenase gene and can be used as a probe to screen for the clones that harbored the cytochrome P450 monooxygenase gene from P. autotrophica genomic library. Two positive clones that contained the plasmid were obtained. Analysis of nucleotide sequence of the inserts revealed that contained six ORFs predicted as acyl-CoA ligase (fadA), enoyl-CoA hydratase (fadB), transcriptional regulator (fadR), cytochrome P450 monooxygenase (fadC), ferredoxin (fadD) and hypothetical protein genes. The cytochrome p450 monooxygenase and ferredoxin genes were cloned into pET32a expression vector and expressed in E. coli Nova Blue. After purification by affinity chromatography, we found that these two enzymes could not convert compactin to pravastatin. Real-time quantitative PCR assays also found that the expression of genes in the cluster could not be induced by compactin. (2) Genes involved in fatty acid degradation (fad) were isolated from Pseudonocardia autotrophyca BBRC12444. Six open reading frames (ORFs) and a bi-directional promoter region were identified by DNA sequence analyses and primer extension. The fad gene cluster included five ORFs, designated fadA, fadB, fadR, fadC and fadD. Base on their amino acid sequence identity, the gene products were identified as acyl-CoA ligase (FadA), enoyl-CoA hydratase (FadB), transcriptional regulator (FadR), cytochrome P450 monooxygenase (FadC) and ferredoxin (FadD). Regulatory protein, FadR, could bind to an operator sequence located in the divergent promoter region between fadR and fadC genes, implicating the control of fatty acids degradation. The real-time quantitative PCR assays revealed that the expression of the fadA, fadB, fadR, and fadC genes was induced by long chain fatty acids and repressed by glucose. All results demonstrated that the fad gene cluster participated in the pathway of the fatty acid catabolism.

Record ID 第152筆 System ID 093NCHU0185019
BCRC ID CCRC 15211, Public Year 93
Paper Name 白腹叢蚊抗菌蛋白cecropin基因表現之研究 Study on the expression of cecropin genes in the mosquito, Armigeres subalbatus
Student Name 劉威廷
Teacher Name 路光暉 杜武俊
School Department Name 國立中興大學 昆蟲學系 Academic Degree 碩士
Abstract 本研究利用表現序列標記(expressed sequence tags, EST)建構之資訊,選取七條白腹叢蚊(Armigeres subalbatus)抗菌蛋白cecropin序列進行基因歸群之比對,並分別設計專一性引子,對經以熱殺死之大腸桿菌(Escherichia coli O157:H7)免疫刺激之白腹叢蚊雌蟲蟲體之RNA進行PCR增輻反應,經選殖而獲得五條具開放讀架之cecropins序列,將之分別命名為Ar. subalbatus CecA1、Ar. subalbatus CecA2、Ar. subalbatus CecB1、Ar. subalbatus CecB2及Ar. subalbatus CecN。利用白腹叢蚊這五條cecropins核酸序列之cDNAs分別設計探針(probe),以北方墨點法探討此五基因之表現,結果顯示CecA1、CecA2、CecB1和CecN基因可經大腸桿菌誘導表現,且表現類型各異。至於CecB2基因則於正常無處理之蟲體呈持續表現現象,且不因大腸桿菌之誘導而增加其表現量。為進一步探討CecB2基因作用情形,再利用金黃葡萄球菌(Staphylococcus aureus CCRC 15211)與綠殭菌(Nomuraea rileyi SH1)等微生物進行白腹叢蚊雌蚊、幼蟲及蛹期之免疫刺激,結果顯示注射這些微生物的雌蟲與四齡幼蟲期皆無法誘導CecB2基因表現量產生增加作用。至於正常無處理之蛹,在化蛹後1小時Ar. subalbatus CecB2表現量產生減少現象,其表現量並隨時間呈遞減現象,至化蛹後48小時幾乎無法偵測到CecB2的表現,惟在化蛹72小時後CecB2表現又回復,其表現量與蛹期初期相若。若於蛹初期注射大腸桿菌,則可偵測得CecB2被誘導表現現象,此誘導表現量於處理18小時後呈降下現象,36小時後降到最低,至處理48小時後其表現量再上升並持續表現到羽化成成蟲,此結果相較於正常蟲體之CecB2有較多的mRNA表現量。除此之外,利用干擾RNA (RNAi)技術,注射雙股之dsCecB2至白腹叢蚊蛹體,令CecB2基因表現沉寂後,可明顯導致部分蛹體無法順利羽化,或令羽化的成蟲產生畸形足的出現。綜合以上結果,顯示CecB2可能與白腹叢蚊變態具相關性。 In this study, five cecropin peptides, i.e. Armigeres subalbatus CecA1, Ar. subalbatus CecA2, Ar. subalbatus CecB1, Ar. subalbatus CecB2 and Ar. subalbatus CecN, were isolated from the adult mosquito, Armigeres subalbatus, by injecting with heat-killed Escherichia coli O157:H7. The gene expression of CecA1, CecA2, CecB1 and CecN in adult mosquitoes were induced and performed different patterns by challenging with E. coli. An intriguing phenomenon was observed, that is, CecB2 was constitutively expressed in adult and larvae and the expression was not affected by challenging with E. coli, Staphylococcus aureus CCRC 15211 or Nomuraea rileyi SH1, respectively。 On the other hand, a novel gene expression profile of CecB2 was revealed that it was declined 1 h after pupariation, disappeared after 48 h and reappeared after 72 h; during this period, however, CecB2 was inducible and persistently expressed in the period of 18-h treatment with E. coli, and decreased after 36 h, and then increased again after 48 h until emergence. Furthermore, the gene function of CecB2 with dsRNA was revealed by using the RNAi technique. Silencing of CecB2 resulted in the failure of emergence or morphogenetic abnormality of emerged adult legs. In conclusion, the function of CecB2 might not only serve as antimicrobial peptide, but also is involved in the process of metamorphosis.

Record ID 第153筆 System ID 093NCHU0253002
BCRC ID BCRC 12936, Public Year 93
Paper Name 兒茶素於經Lactobacillus helveticus發酵黑豆奶對低密度脂蛋白氧化及免疫調節能力之探討 Effect of black bean soymilk fermented by Lactobacillus helveticus with tea catechins on LDL oxidation and immunomodulatory activity
Student Name 林佾傑
Teacher Name 方繼
School Department Name 國立中興大學 食品科學系 Academic Degree 碩士
Abstract 近年來從食物蛋白來源的機能性胜肽逐漸的受到食品科學家的重視。其中,有許多胜肽片段已被證實具有某些特定的活性,如從β-酪蛋白(β-casein)水解產生的類鴉片(opioid peptide)胜肽、以及降血壓胜肽等等。 本研究以市售的黑豆為原料,製成豆漿,利用乳酸菌Lactobacillus helveticus BCRC 12936及添加不同濃度的兒茶素進行發酵,分析發酵液中胜肽類物質對血管收縮素轉換酵素(Angiotensin Ⅰ converting enzyme, ACE)活性的影響,並以MTS分析法,分析發酵液對BALB/c老鼠脾臟細胞調節細胞免疫能力的影響。另外,使用細胞株 J774A.1分析發酵液對吞噬細胞吞噬能力的影響。最後,以敘利亞黃金倉鼠(Syrian golden hamster)為動物模式,探討此發酵液中胜肽類物質對血清低密度脂蛋白氧化(Serum LDL oxidation)之影響。 結果發現,接菌量2%(v/w)及添加200 ppm兒茶素進行48小時發酵後的上清液,能得到最高ACEi活性的胜肽物質,其ACE抑制活性達78.5%。發酵豆奶之上清液,會延長低密度脂蛋白(LDL)在銅離子誘導氧化過程中的氧化遲滯時間。發酵豆奶之上清液對BALB/c小鼠脾臟淋巴細胞增生能力的影響,在原發性的實驗中,發酵豆奶上清液在濃度70及140 μg/ml時會刺激脾臟淋巴細胞的增生;當以Con A作為裂殖素時,添加200 ppm兒茶素發酵的豆奶上清液,在濃度17.5、35及70 μg/ml時會刺激T淋巴細胞的增生;以LPS作為裂殖素時,發酵豆奶上清液在濃度35、70及140μg/ml時會刺激B淋巴細胞的增生。發酵豆奶上清液亦會增加吞噬細胞吞噬能力,FS、FSC2及FSC4組的吞噬細胞吞噬能力與對照組相較,分別增加84%、74%及57%。 In the recent years, peptides from hydrolysates of food proteins have received greater attention from food scientists than ever before. Many biological peptides, with health benefits such as opioid activity, antihypertensive activity etc. have been classified and identified from food proteins hydrolysates. In this study, we obtain the ACE inhibitory peptides from supernatant of soy milk fermented by Lactobacillus helveticus BCRC 12936 with different concentrations of catechins, and investigate the effects of fermented soy milk supernatant on macrophage phagocytosis and cell immunity。 We also test the ability of soy milk supernatant to influence serum LDL oxidation in Syrian golden hamster model. In order to characterize the ACE inhibitory ability of fermented soy milk supernatant, we added different concentrations of catechins in the fermented process. The results revealed that the supernatant of soy milk fermented by 2% BCRC 12936 with 200 ppm catechins have the highest ACE inhibitory activity (78。5%). In the serum LDL oxidation experiment, we found that fermented soy milk supernatant can delay time of cupper induced serum LDL oxidation. Furthermore, we used the BALB/c mice model to prove that 70 and 140 μg/ml fermented soy milk supernatant can stimulate spleen lymphocytes proliferation . When we used Con A as mitogen, 17.5、35、70 μg/ml supernatant of soy milk fermented with 200 ppm catechins can stimulate T-lymphocytes proliferation ; in condition of using LPS as mitogen, the amounts of 35、70、140 μg/ml supernatant can stimulate B-lymphocytes proliferation. In J774A.1 cell line, we also observed that supernatant of fermented soy milk (FS、FSC2、FSC4)compared with control have higher abilities of macrophage phagocytosis (84%、74%、57%).

Record ID 第154筆 System ID 093NCHU0253060
BCRC ID BCRC 10697, Public Year 93
Paper Name 轉殖CpG及AT寡核苷酸片段以提升乳酸菌之免疫調節能力 Cloning of CpG motifs and AT oligonucleotides to stimulate the immunomodulation of lactic acid bacteria
Student Name 陳文萱
Teacher Name 林美吟
School Department Name 國立中興大學 食品科學系 Academic Degree 碩士
Abstract 近年來研究指出,病原菌和酵母菌DNA中的CpG motifs 具有誘發B淋巴細胞產生分裂之作用,並可刺激IgM、IFN-、IL-6、IL-12、IL-18及TNF-之產生;進而提高T細胞及自然殺手細胞之活性以預防傳染性疾病。另有研究指出,乳酸菌之genomic DNA經內切限制酶水解後的DNA片段具有促B淋巴細胞分裂的能力,該片段中含有大量的A、T寡核苷酸。本研究將此兩種DNA片段接合於可在乳酸菌體內複製的質體上,構築出含有5個CpG及4個AT寡核苷酸的載體pHY-5CpG4AT,並轉形入益生菌Lactobacillus casei ( BCRC 10697 ) 中,以期透過腸道免疫反應,提昇乳酸菌免疫調節的功能。 將轉形成功之乳酸菌宿主及其胞內液進行免疫相關測試。測試的項目包括:刺激巨噬細胞株J774A.1的吞噬能力、激活細胞株分泌TNF-α 及IL-6的量等。結果顯示,含有質體pHY-5CpG4AT的L. casei活菌株可顯著刺激巨噬細胞分泌TNF-α;而其胞內液則可刺激巨噬細胞增加吞噬的能力。由此可知,含有質體pHY-5CpG4AT的L. casei可激活巨噬細胞,具有增強乳酸菌免疫反應之應用潛力。 It has been reported that pathogenic bacterial DNA and yeast DNA induce B-lymphocyte mitogenicity, and these mitogenic DNA sequences consist of CpG motifs. CpG motifs are reported to promote several cytokine productions, such as TNF-α, IFN-γ, IL-6, IL-12 and IL-18, and play a beneficial role against infectious diseases through the activation of T lymphocytes and NK cells. Moreover, the nucleotide sequences that consist of only A and T nucleotides ( AT oligonucleotides ) were characterized as B lymphocyte specific mitogens because they resulted in proliferation of B lymphocytes. In this experiment, CpG motifs and AT oligonucleotides were cloned into pHY300PLK and transformed to Lactobacillus casei ( BCRC 10697 ) to improve its immunomodulation。 Viable bacteria and bacteria extract were used to test the effect of immunomodulation. The testing items include phagocytosis, and the secretion of TNF-α and IL-6 of murine macrophage J774A.1 cell line. The objectives of this study were to reveal that the sequences cloned to lactic acid bacteria play a beneficial role in human health by immunomodulation. The result showed that co-culture with viable bacteria of L. casei which contained plasmid pHY-5CpG4AT with macrophage cell line significantly increased its production of TNF-α. The bacteria extract of L. casei which contain plasmid pHY-5CpG4AT could stimulate phagocytosis of macrophage cell line. Our results indicated that plasmid pHY-5CpG4AT in L. casei can stimulate the macrophage cell line and may improve immunomodulation of lactic acid bacteria.

Record ID 第155筆 System ID 093NCHU0515038
BCRC ID BCRC 16048, Public Year 93
Paper Name 柴油分解菌之生物降解與浮起特性 Biodegradation and floating characteristics of diesel-assimilating bacteria
Student Name 林大成
Teacher Name 楊秋忠 Young,Chiu-Chung
School Department Name 國立中興大學 環境工程學系 Academic Degree 博士
Abstract 本研究目的在開發具浮起特性之柴油降解優勢菌種,以篩選環保菌種裨益於產業應用,開創菌種之浮起特性以建立技術專利,擴展菌株之多功能特性以利生物復育,期能提高油污染水體之處理效果。本研究主要內容以菌種特性、浮起特性及生物降解柴油等試驗為三大探討之主軸。菌種特性試驗首先篩選本土性柴油分解優勢菌種,經16S rRNA基因序列鑑定為Acinetobacter sp. CC-CF5(CF5)、Alcaligenes sp. CC-ESB2(ESB2)、Comamonas testosteroni CC-CF3(CF3)、Gordonia alkanivorans CC-JG39(JG39)、Rhodococcus erythropolis CC-BC04(BC04)、Rhodococcus erythropolis CC-BC11(BC11)及Sphingomonas yanoikuyae CC-CG22(CG22)等菌株,同時以生物資源保存及研究中心(BCRC)之菌株Bacillus subtilis BCRC16048 (BS)作為實驗之對照,進行各項生化與生理試驗。研究結果在菌種特性方面,以菌株ESB2 與 JG39降低柴油BH溶液之表面張力最低約為47 mN/m,而菌株ESB2之乳化活性最高(E24 = 68%);在生長特性、耐鹽性、酸鹼性及溫度效應等之生理特性,菌株ESB2 與 CF3與照菌株BS功能相當;菌株BC04、BC11、CF3、CG22及JG39等在脂肪分解酵素反應與毒化物利用性之生化特性,皆優於對照菌株BS或功能與之相當。故菌株CF3、CG22、JG39 及ESB2等為適於油污染環境生存之環保功能優勢菌種,將具有產業應用之潛力與價值。在浮起特性試驗中,本研究首次發現菌株JG39、BC04、BC11及ESB2具有浮起特性;而菌株CF3、CF5、CG22及對照菌BS則不具有浮起特性,其中菌株JG39、BC04、BC11及對照菌BS皆屬格蘭氏陽性菌(G+);而菌株CF3、CF5、CG22及ESB2則屬格蘭氏陰性菌(G−)。研究顯示浮起特性可能與菌種親油性、細胞壁結構及細胞壁有機分子具有相關性;而與菌體比重、氧氣需求性及碳源分佈等無關。助浮介質試驗顯示,由菌株JG39所分泌之胞外多糖體具有助浮效果,而親油水兩性之環狀糊精則無助浮效應,故菌株BC04、BC11與JG39,以及助浮介質胞外多糖體,將有利於浮油污染生物復育之專利應用。在柴油之生物降解試驗顯示,菌株CF3、CG22及JG39等之降解效果皆優於對照菌株BS,其中菌株JG39分泌之生物界面活性劑對菌株CF3與CG22具有促進柴油降解之顯著作用(P <0.05),為多功能菌株,其最大比生長速率(µmax = 0.158 h-1) 與最大比降解速率(vmax = 3.59 mg/h/mg cell)之動力參數,在油污染生物復育之整治設計上極具參考。混合菌組試驗顯示對柴油之生物降解,浮起菌組比非浮起菌較具有顯著效果(P < 0.05),因此浮起菌組將具有應用於海洋溢油或地下水體浮油(smear zone)之生物復育潛力。故本研究篩選本土性具浮起特性之柴油分解菌種,對浮油污染之整治將具有相當之應用價值。 Potential diesel-degrading bacteria were isolated from different locations around Taiwan. Biodegradation, floating characteristics and physiological properties of isolates were investigated to promote the efficiency of bioremediation and to apply for industrial patent. Identification of isolates by 16S rDNA sequencing assigned these isolates as Acinetobacter sp. CC-CF5(CF5), Alcaligenes sp. CC-ESB2(ESB2), Comamonas testosteroni CC-CF3(CF3), Gordonia alkanivorans CC-JG39(JG39), Rhodococcus erythropolis CC-BC04(BC04), Rhodococcus erythropolis CC-BC11(BC11) and Sphingomonas yanoikuyae CC-CG22(CG22). A commercially available strain Bacillus subtilis BCRC 16048 (BS) obtained from Bioresource Collection and Research Center (BCRC) was used for comparison。 From the study results, it was evident that strains ESB2 and JG39 were able to decrease the surface tension of the BH medium amended with diesel to 47 mN/m. Strain ESB2 also showed the highest emulsification index (E24) of 68%. Physiologically, strains ESB2 and CF3 had similar growth patterns to that of the control strain Bacillus subtilis(BS), especially showing similar characteristics while growing at different temperatures, pH and salinity. Lipase analysis and utilization of oil additives (BTEX) by strains BC04, BC11, CF3, CG22, and JG39 were comparatively higher than strain BS. Therefore, strains CF3, CG22, JG39 and ESB2 can be recommended and used in bioremediation of petroleum hydrocarbon polluted environment. Further, the novel study on floating characteristics revealed that strains JG39, BC04, BC11 and ESB2 had floating activities, while strains CF3, CF5, CG22 and BS were unable to float and suspended uniformly in the liquid medium. Strains showed that strains JG39, BC04, BC11 and BS are Gram-positive, while strains CF3, CF5, CG22 and ESB2 were Gram-negative. Hence, it can be assumed that the floating activity of these strains might be attributed to the hydrophobicity which is presumably related to the nanoparticle present in structure of cell wall, but is independent of bacterial density, oxygen utilization, and carbon distribution in medium. In addition, an exopolysaccharide isolated from strain JG39 was shown to play a role in facilitating the floating activity of strains CF3 and CG22. However cyclodextrin did not show the same feature. Exopolysaccharide produced by strain JG39 was evidenced by the scanning electronic microphotography. Therefore, it is claimed that strains BC04, BC11 and JG39, as well as exopolysaccharide produced by strain JG39 have novelty for application in bioremediation of oil-polluted aquatic ecosystems. The results of diesel biodegradation indicate that strains CF3, CG22 and JG39 were more efficient than strain BS. The biosurfactant extracted from strain JG39 significantly promoted diesel biodegradation of strains CF3 and CG22 (P<0.05). The kinetics of specific growth rate (µmax=0.158 1/h) and specific degradation rate (vmax=3.59 mg/h/mg cell) of strain JG39 are beneficial for designing a bioreactor for bioremediation. Statistical analysis showed that a consortium of floating group was more significantly efficient than non-floating strains in diesel biodegradation (P<0.05). Therefore, a consortium of floating strains has potential application in bioremediation of oil spilled sea surface and smear zone of underground water. The diesel-degrading bacteria selected have unique characteristics that can promote bioremediation efficiently. Hence, this study recommends that strains JG39, BC04, BC11 and ESB2 are potential candidates for bioaugmention on site.

Record ID 第156筆 System ID 093NCHU0253012
BCRC ID BCRC 11569, Public Year 93
Paper Name 以反應曲面法探討蜂蜜酒糟醋發酵較適條件之研究 Optimization of Vinegar Fermentation from Honey Distiller’s Water with Response Surface Methodology
Student Name 曾貽湧
Teacher Name 柯文慶
School Department Name 國立中興大學 食品科學系 Academic Degree 碩士
Abstract 摘要 蒸餾為酒廠製造蒸餾酒之必要程序,蒸餾後常副生大量俗稱酒糟的蒸餾殘液,此液常供作飼料,但有些則當廢棄物處理。為避免酒廠排除蒸餾廢液造成環境污染,同時提高其利用性,本研究以蜂蜜為基質,利用商業菌種利用不完全蒸餾模式釀造蜂蜜酒,將其蒸餾後所得之酒糟進行醋酸發酵,用反應曲面實驗設計法求出最佳發酵條件之組合,結果如下: 1. 篩選之商業酵母菌中以最終酒精濃度最高者(可達 8.94%)進行較適酒精發酵條件之探討,所用之 3 個因子為溫度(18-26℃)、基質濃度(18-260Brix)、pH值(3-4),而探討發酵 15 天後之酒精含量變化,顯示基質濃度 260Brix、pH 值 4.0、發酵溫度 22℃ 下有最高酒精產量 10.63%。 2. 醋酸菌的篩選中,以 Acetobacter aceti BCRC 11569 產醋效率最高,在含 5% 酒精的 mannitol medium 中發酵 5 天產出 4.45% 的醋酸,以此菌進行最適醋酸發酵條件之探討。所用之 3 個因子為溫度(27-33℃)、基質濃度(5-7%)、接種量(5-15%),而探討發酵 8 天後醋酸含量變化,顯示基質濃度 6%、發酵溫度 27℃、接種量 5% 時有最高醋酸產量 6.33%。 3. 醋酸發酵之改進試驗顯示,添加生長促進因子時,碳源方面以添加 10% sucrose 可得 6.71% 醋酸,氮源方面以添加 0.1% peptone可得 6.24% 醋酸,添加有機酸方面以 1% 檸檬酸可得到最高的醋酸產量 7.35%;在促進因子之濃度方面,正交試驗法證明 5% sucrose、0.5% citric acid、0.15% peptone的添加可獲得高濃度的醋酸產量達 8.28%。 Abstract Distillation is a necessary process for factories that produce spirits. Besides offering as feed, the stillage produced after distillation is often discarded as wastes. For preventing from environmental contamination and increasing the availability of the stillage, honey was used as substrate for partial fermentation with commercial yeast. The stillage obtained was further used as substrate for acetic acid fermentation. The most appropriate fermentation condition was studied by means of response surface methodology (RSM). The derived results were discussed as follows: 1. A commercial yeast ultimately produced 8.94% ethanol was screened to investigate the conditions of ethanol fermentation. Temperature (18-26℃), sugar concentration (18-26˚Brix) and pH value (3-4) were three independent variables, while ethanol concentration produced after fermentation for 15 days was a dependent variable. The highest yield of ethanol up to 10.63% was obtained under the optimum fermentation conditions, sugar concentration 26˚Brix, pH 4.0 and 22℃. 2. Acetobacter aceti BCRC 11569 showed the highest yield among the screened acetic bacteria。 This strain produced up to 4.45% acetic acid in mannitol medium containing 5% ethanol after fermentation for 5 days. Thus we chose this strain to investigate the optimal condition of acetic acid fermentation. Three independent variables were temperature (27-33℃), ethanol concentration (5-7%), inoculation concentration (5-15%), while acetic acid concentration which was obtained after fermentation for 8 days was a dependent variable. The highest yield of acetic acid up to 6.63% was obtained under the optimum fermentation conditions, sugar concentration 6%, 27℃ and inoculation concentration 5%. 3. For improvement of acetic acid fermentation, various growth factors were added. As the result, 6.71%, 6.24%, and 7.35% acetic acid could be got by adding 10% sucrose as C-source, 0.1% peptone as nitrogen source, and 1% citric acid as organic acid source, respectively. Orthogonal Experimental Design verified that simultaneous addition of 5% sucrose, 0.5% citric acid and 0.15% peptone could obtain acetic acid up to 8.28%.

Record ID 第157筆 System ID 093NCHU0111020
BCRC ID BCRC 35290, Public Year 93
Paper Name 抗真菌胜肽於大腸菌之異源表達與其活性測試 Heterogeneous Expression of an antifungal peptide in Escherichia coli and its activity assay
Student Name 梁棟榮
Teacher Name 孟孟孝
School Department Name 國立中興大學 生物科技學研究所 Academic Degree 碩士
Abstract 真菌病原之感染常導致經濟作物產量的大幅減少。文獻上,有很多藉由植物抗真菌基因(如幾丁質水解酶基因),導入植物以增加植物抵抗真菌病原的例子。其成果變異很大。主要的限制之一是由單一植物抗真菌基因所獲得的抵抗力相當低,所以需要同時使用多種的基因。顯然,植物病原已經對源自植物的抗真菌物質演變出耐受性。本實驗,我們選擇一段已被發現具有抗真菌活性的胜肽-cicadin,它是源由中藥材料乾的「蟬幼蟲」中分離出來的蛋白質,將cicadin 基因以pTYB2為載體,構築後送入Escherichia coli Novablue (DE3)表現。純化後,做抗真菌活性分析,初步我們發現由大腸桿菌所表現的cicadin 胜肽,對豌豆鐮胞菌(Fusarium oxysporum f. sp. pisi; 只有些微的抗菌能力。期待將來藉由分析方法的修飾及多測試幾種真菌,我們能夠確認由大腸桿菌所表達的cicadin胜肽具有抗真菌的活性。 Yields of agricultural products are often reduced severely by fungal pathogens. Attempts to increase plant resistance to fungal pathogens through introduction of plant antifungal genes ( e.g. chitinase gene) into the plant have been reported repeatly. The outcome varies. One of the main limitations has been the relatively low level of resistance obtained with a single plant antifungal gene, which has resulted in the need to use gene combinations. Obviously, plant pathogens have already evolved tolerance to plant-derived antifungal compounds. In this experiment, we chose cicadin peptide whose antifungal activities have been reported. Cicadin is from dried juvenile cicadas which are used in traditional Chinese medicine for treatment of certain diseases. The synthetic cicadin gene was inserted into pTYB2 plasmid and introduced into Escherichia coli Novablue (DE3). After purification, the antifungal activity of the E. coli-expressed peptide was analyzed. Preliminary results show that the E. coli-expressed cicadin has some but not strong antifungal activity against Fusarium oxysporum f. sp. pisi; BCRC 35290。 Yet, the antifungal activity of the recombinant cicadin needs to be confirmed through modifications of our assay method and tests of the other kinds of fungus in the future.

Record ID 第158筆 System ID 093NCHU0289018
BCRC ID CCRC 31499, CCRC 31494, Public Year 93
Paper Name 添加不同量紅麴(Monascus purpureus)及煙燻對低硝鴨賞於4℃儲存期間其品質之影響 Effect of Various Levels of Monascus purpureus and usage of smoking on the Quality of Low-Nitrite Cured Ducks During Storage at 4℃
Student Name 李莉瑋
Teacher Name 劉登城
School Department Name 國立中興大學 畜產學系 Academic Degree 碩士
Abstract 本試驗係分別添加Monascus purpureus CCRC No. 31499製成之紅糟3%、5%及10%於鴨賞醃料中,再分以煙燻與否(S-煙燻處理;R-未煙燻處理)處理,探討紅麴添加及煙燻處理對低硝鴨賞於4℃儲存期間其產品品質及風味物質之影響。於第0天測定製品之一般化學組成、含鹽量及剪力值。製品經真空包裝後儲藏在4℃,於第0、7、21、35及49天測試其pH值、2-硫巴比妥酸(TBA)、揮發性鹽基態氮(VBN)、總生菌數、厭氣菌數、色澤和亞硝根殘留量之變化,並於第28天及第49天進行感官品評。另外分析紅麴(Monascus purpureus CCRC No. 31494)及鴨賞醃料中有無添加10%紅麴和煙燻與否之風味化合物質。 結果顯示:低硝鴨賞經煙燻處理之組別(S1:3%、S2:5%、S3:10%),其水分、粗蛋白及粗脂肪含量與對照組(C)間無顯著差異(p>0.05),但C、S1、S2及S3組其水分及粗脂肪含量皆顯著低於未煙燻處理組(R1:3%、R2:5%、R3:10%) (p<0.05);而添加紅麴及煙燻對鴨賞之灰份含量並無顯著差異(p>0.05)。對照組含鹽量為3.34%顯著高於其他處理組(2.26~2.28%) (p<0.05)。隨著儲藏天數增加,各組之微生物及VBN值皆顯著上升(p<0.05);總生菌數及厭氣菌數隨著紅麴添加量之增加及煙燻處理有減少之趨勢但無顯著差異(p>0.05);VBN值隨著紅麴添加量之增加而越高,且S1、S2及S3其VBN值於儲藏期間皆較R1、R2及R3為低。 各組pH值隨著儲藏天數及紅麴添加量增加而降低,而S1、S2、S3及C組之pH值有較R1、R2、R3高之趨勢。鴨賞添加不同量之Monascus purpureus及煙燻處理於4℃儲存期間,第0至7天時TBA值於各組皆顯著增加(p<0.05),且S1、S2、S3及C組其TBA值於儲藏期間皆顯著較R1、R2及R3為高(p<0.05)。另外紅麴的添加可減緩亞硝根殘留量隨儲藏天數的增加而減少之速率,但具有較高之TBA值。 隨著儲藏天數增加,各組L值皆有下降之趨勢而a值皆有上升之趨勢,且紅麴添加量越高a值則顯著增加(p<0.05),而紅麴添加量之多寡對L值無顯著差異。鴨賞隨著紅麴添加量之增加其剪力值有降低的趨勢,而經煙燻處理之組別其剪力值則有增加之趨勢。於儲藏至第28天及第49天時,紅麴處理各組之得分皆低於對照組,而以S2組有較佳之品評得分。 揮發性風味成分方面,萃取出的揮發性成分主要分為醇類、醛類、酸類、酯類、酚類及雜環化合物等共四十六種化合物,其中紅麴產生的揮發性成分主要為醇類、醛類、酸類及酯類等二十一種風味化合物,且隨著紅麴之添加,這些物質有增加之趨勢,而煙燻會產生較多之酚類化合物。 The purpose of this study was to investigate effects of adding Monascus purpureus 3%, 5%and 10% to curing agent and usage of smoking (S-smoking; R-without smoking) on the quality and flavor of low-nitrite cured ducks storage at 4℃ for 49 days. The pH value, TBA value, VBN value, total plate count, anaerobic bacteria count, color and residual nitrite content of the products with vacuum package were to determine at the 0th, 7th, 21th, 35th and 49th day during cold storage. The sensory panels of all cured ducks were also performed at the 28th and 49th day in this experiment. On the other hand, the shear force, the content of salt and the chemical composition of cured ducks were to determine at the 0th day. Since Monascus and smoking could produce some flavor compounds, the volatile compounds of Monascus purpureus culture and cured ducks with 10% Monascus purpureus and usage of smoking were extracted and analyzed by GC. The results showed that the control (C) and the low-nitrite cured ducks with Monascus purpureus and smoking (S1:3%, S2:5%, S3:10%) were