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研究生:陳小玲
研究生(外文):Hsiao-Ling Chen
論文名稱:酒釀菌元篩選與其凝乳特性
論文名稱(外文):Screening of starters of lao-chao and characterization of milk-clotting enzyme
指導教授:林 慶 文
指導教授(外文):Chin-Wen Lin
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:畜產學研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:162
中文關鍵詞:酒釀菌元篩選凝乳酵母菌
外文關鍵詞:lao-chaoscreening startermilk-clotting enzymefungiyeast
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本研究旨在利用酒釀萃中凝乳與風味成分,開發饒富東方口味的低酸酒釀萃凝乳。由於傳統製法的酒釀菌元(酒藥)係自然發酵而成,菌相複雜,控制不易。為求製品的均一化,必須篩選適合的純菌元,以生產具有凝乳活性及良好風味的酒釀萃。
選擇Rhizopus javanicus、Aspergillus oryzae、Rhi. oryzae、Rhi. chinensis、Rhi. oligosporus、以及Mucor racemosus 等6株黴菌菌株進行篩選。結果顯示,所有菌株皆能液化蒸煮糯米產生酒釀萃。除Asp. oryzae 之外,其餘5株黴菌發酵的酒釀萃具有凝乳活性與蛋白質分解活性。試驗組中,Rhi. javanicus 產生多量具凝乳活性的酒釀萃,所製得的酒釀萃凝乳質地細緻且風味合宜,官能品評接受度僅次於酒藥組。然而,其製品凝乳堅實度較低,此缺陷雖可利用混合接種Muc. racemosus 來改善,卻會使風味接受度略為降低。
在酵母菌菌元篩選方面,選定Rhi. javanicus為黴菌菌元配合接種Candida magnoliae、Endomyces fibuligera、Saccharomyces bayanus、 Sac. cerevisiae、Sac. diastaticus、 Sac. formosensis、以及 Pichia anomala 等酵母菌,並以酒藥為對照組。結果顯示,單獨接種Rhi. javanicus 及將其分別配合7株酵母菌之試驗組中,無論酒釀萃產量、pH值、凝乳活性、以及製品凝乳堅實度等均無顯著性差異(P>0.05)。經4天發酵後,接種Rhi. javanicus 配合Sac. cerevisiae 所得之酒釀萃,相較於其他試驗組,具有最低的還原糖以及最高的乙醇濃度,而香氣成分最近似酒藥組。因此,就酒釀萃凝乳開發而言,液化與糖化米基質,以及產生凝乳,端賴Rhi. javanicus;酵母菌則可依個別菌株之風味特性作選擇性的搭配。
為掌握酒釀萃凝乳之製程,必須瞭解擔任凝乳與風味劑雙重角色的酒釀萃特性。Rhi. javanicus發酵蒸米所得之酒釀萃經超過濾系統進行除鹽、緩衝液替換、以及濃縮成粗凝乳後供試。Rhi. javanicus粗凝乳表現最強蛋白質分解活性的溫度及pH值,分別為40℃ 及pH 3。而於pH 3-7以及30-50℃ 具有較好的安定性 。Mg2+、Ca2+、Fe2+等離子對凝乳活性有促進效果,對蛋白質分解活性則無顯著作用。蔗糖會造成凝乳的延遲與製品堅實度的降低,此缺陷可利用Ca2+離子之添加來改善。
在Rhi. javanicus 凝乳的純化方面,以超過濾取代一般使用的硫酸銨沈澱濃縮程序,配合快速蛋白質液相層析系統,先後經Hitrap Desalting、Hitrap Q、以及Hiprep Sephcryl S-100 等管柱純化酒釀萃中凝乳。並由SDS-PAGE 及IEF 推論其分子量與等電點分別為35 kDa、5.45。然而,Rhi. javanicus 凝乳之凝乳活性與蛋白質分解活性比值為0.525,低於其他的市售凝乳。SDS-PAGE的結果更顯示其對α-,β-,κ- 酪蛋白均表現較強的分解力。如此的高蛋白質分解力及低專一性可能會導致貯存期間的苦味與乳清分離,而成為商品化的困難點。因此,本研究室已著手開發結合凝乳與酒釀風味的家庭用製劑,以研製即食酒釀萃凝乳來解決。
This study was conducted to develop an oriental-style dairy product coagulated with culture filtrates from lao-chao, a fermented rice product well known in China, for meeting consumer preferences for a low-acid or non-sour tasting yogurt-like product. However, it is well known that the commercial starter of lao-chao, chiu-yao, is often prepared under relatively poor microbiological conditions. Therefore, the properties of the fermented products from such starters cannot be made uniform. It is necessary to screen pure cultures of microorganism for those which exhibit milk-clotting activity and produce a pleasant aroma in rice fermentation.
The fermenting fungal cultures screened were Rhizopus javanicus, Aspergillus oryzae, Rhi. oryzae, Rhi. chinensis, Rhi. oligosporus, Mucor racemosus. All the inocula liquefied a steamed glutinous rice base and produced culture filtrates with both milk-clotting and proteolytic activities except Asp. oryzae. Of the fungal strains tested, Rhi. javanicus was the most successful, producing a good yield of culture filtrate with the desired milk-clotting activity, and the resultant yogurt-like product with a pleasant flavor and fine texture was more acceptable to consumers than any except that from chiu-yao. The firmness was less acceptable, but this could be improved by using mixed pure cultures of Rhi. javanicus and Muc. racemosus. The yeast cultures including Candida magnoliae, Endomyces fibuligera, Saccharomyces bayanus, Sac. cerevisiae, Sac. diastaticus, Sac. formosensis, and Pichia anomala were screened for combination with Rhi. javanicus. Treatment with chiu-yao was used as the control. In the treatments with Rhi. javanicus alone and combined with each the seven yeast cultures, there was no difference in volume of culture filtrates, pH value, milk-clotting activity or curd firmness of products. Of the nine treatments, Rhi. javanicus combined with Sac. cerevisiae produced the most ethanol and the least reducing sugars after 4 days fermentation. This treatment also exhibited the most similar components of aroma to that of chiu-yao. Therefore, Rhi. javanicus was important in liquefying and saccharifying the steamed glutinous rice and in producing the milk-clotting enzyme used to clot milk in the new dairy product. However, the seven yeast cultures could be inoculated for the purpose of contributing to the formation of various kinds of pleasant aroma according to the unique characteristics of various yeasts.
For the manufacture of the new dairy products, the characteristics of culture filtrates from lao-chao used as both milk-clotting and flavor agents were determined. The concentrates of culture filtrate from lao-chao with Rhi. javanicus after desalting and buffer exchange by ultrafiltration were obtained for further analysis. The highest proteolytic activity exhibited around 40℃ and pH 3. Rhi. javanicus rennet were stable at pH 3-7 and at 30-50℃. Ca2+, Mg2+, and Fe2+elevated milk-clotting activity but not proteolytic activity. Sucrose resulted in the retardation of milk-clotting. Ca2+could be used to increase curd firmness.
The ultrafiltration was used for replacing ammonium sulfate fractionation to concentrate protein. Fast protein liquid chromatography system with columns including Hitrap Desalting, Hitrap Q, and Hiprep Sephacryl S-100 was applied to purify rennin in culture filtrate from lao-chao with Rhi. javanicus. The molecular weight of Rhi. javanicus rennin was about 35 kDa inferred from SDS-PAGE and pI was about 5.45 inferred from IEF . The MCA/PA ratio of Rhi. javanicus rennin was 0.525 lower than all of the commercial rennets. SDS-PAGE patterns showed that commercial rennets including calf rennin, calf rennet, and Muc. miehei rennet had less proteolytic on α-, β- and κ- caseins than Rhi. javanicus rennin. The high proteolytic activity and low specificity of Rhi. javanicus rennin could result in the bitterness and whey-off of the products during storage, which became a problem for sale. Therefore, we still develop a agent combining the milk-clotting enzyme with aroma components of lao-chao to be used for the family manufacture of the new dairy products.
封面

壹、中文摘要
貳、緒言
參、文獻檢討
一、扣碗酪
二、酒釀
(一)簡介
(二)菌元
(三)發酵過程中微生物之作用
三、酵奇菌發酵風味之生成機制
(一)高級醇之生合成
(二)酯類化合物之生合成
(三)有機酸之生合成
四、凝乳
(一)凝乳之種類
(二)凝乳之作用機制
(三)影響微生物生產凝乳之因子
(四)影響凝乳凝乳作用之因子
五、基因工程技術於微生物凝乳醃之應用
(一)基因工程技術於原核細胞凝乳之發展
(二)基因工程技術於真菌細胞凝乳之發展
(三)基因重組凝乳於乾酪之應用
肆、實驗
第一章 酒釀之黴菌菌元篩選
一、前言
二、材料與方法
三、結果興討論
(一)接種不同黴菌菌株之酒釀萃產量
(二)酒釀萃於發酵期間pH值及滴定酸度之變化
(三)酒釀萃於發酵期間還原糖及乙醇之變化
(四)酒釀萃於發酵期間凝乳活性及蛋白質分解活性之變化
(五)酒釀萃凝乳與風味劑之綜合評估
第二章 酒釀之酵母菌菌元篩選
一、前言
二、材料與方法
三、結果興討論
(一)Rhi.javanicus 配合不同酵母菌株發酵酒釀之酒釀萃產量
(二)接種不同酵母菌酒釀萃之癡乳活性、蛋白質分解活性、以及凝乳堅實度
(三)接種不同酵母菌酒釀萃之pH值、滴定酸度、還原糖以及乙醇濃度
(四)酒釀萃之香氣成分
第三章 酒釀萃中Rhizopus javanicus粗凝乳之特性
一、前言
二、材料與方法
三、結果興討論
(一)Rhi. javanicus 粗凝乳之最適作用pH值及溫度
(二)Rhi. javanicus 粗凝乳之pH值安定性及熱安定性
(三)金屬離子對Rhi. javanicus 粗凝乳凝乳活性及蛋白質分解活性之影響
(四)原料乳熱處理及凝乳溫度對Rhi. javanicus 粗凝乳凝乳堅實度之影響
(五)添加蔗糖與鈣離子對RAi.@avanicus粗凝乳凝乳堅實度之影響
(六)Rhi. javanicus 粗凝乳凝乳之顯微構造
第四章 Rhizopus javanicus 凝乳之純化與特性
一、前言
二、材料與方法
三、結果興討論
(一)純化
(二)分子量及等電點
(三)凝乳活性及蛋白質分解活性
(四)酪蛋白分解特性
伍、結論
陸、參考文獻
柒、英文摘要
捌、小傳
玖、附錄
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