Studies on production,purification,properties of milk-clotting enzyme produced by Amylomyces rouxii on rice substrate and its application.

• Main Authors: Yu, Pei-Jing, 余佩璟
• Other Authors: Cheng-Chun Chou
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/75125588785779087618

博士 === 國立臺灣大學 === 食品科技研究所 === 85 === Amylomyces rouxii是甜酒釀菌元- "酒藥"中之主要黴菌之一，它在米中會產生風味 物質並產生凝乳酵素引起牛乳之凝乳，而使"光奶"，一種東方牛奶發酵產品除特具風味外 並形成凝膠質地。在本研究中吾人乃就此黴菌菌株於米基質中凝乳生產之情形，其所產 生之凝乳酵素之分離、純化與一些特性進行探討。此外並比較此Amy. rouxii與其他來源 之凝乳酵素對酪蛋白降解之情形，以及進行"光奶"之初步製備與品質之了解。 結果顯示 此黴菌在所試之五種米基質中，以在糯米為基質時凝乳酵素之產量最高。固態培養時糯米 基質含水量為50%時，於30℃培養5天後所得凝乳酵素之產量最高，達7.4 unit/g dry sub strate。液態靜置培養時則以含20%米基質，並於培養3天後所得凝乳酵素之活性最高。當 此黴菌與Endomycopsis capsularis或Saccharomyces cerevisiae同時培養會使其凝乳酵 素產量降低，添加Zn2+、Mg2+、Cu2+、Fe3+或Al3+ 則能提高其凝乳酵素產量。 Amy. rouxii粗凝乳酵素液以硫銨沈澱並經Sephadex G-75、DEAESephadex A-50膠體層析後可 純化7.73倍，回收率為65.38%。由SDS-PAGE及Native-PAGE電泳圖測得此酵素分子量為47. 5 kDa。此酵素是一種醣蛋白，含醣量為4.04%，其等電點為pI 3.9，凝乳活性與蛋白質分 解活性之比值為6.49，此外，此酵素於pH 4.0-6.5及30-50℃下穩定性較佳，由示差熱掃 瞄分析儀(DSC)之分析顯示74.6℃為其變性溫度，此酵素在pH 5.5及45℃時其凝乳活性最 高，Fe2+、Ca2+離子可提高此酵素之凝乳活性但Zn2+離子、KMnO4則會降低其活性。 SDS-PAGE 及膠體層析結果顯示，α-、β-casein 的殘餘量隨著與 Amy. rouxii 凝乳酵 素作用時間之增加而漸減，但無明顯之蛋白質小碎片出現，κ-casein可被 Amy. rouxii 凝乳降解為 11.1 kDa 及 6.0 kDa 之蛋白質碎片。穿透式電子顯微鏡圖顯示α-、β- 、κ- casein 及牛奶與Amy. rouxii 凝乳酵素反應後之產物皆為不規則塊狀。 利用 Amy. rouxii單獨或再配合不同酵母菌進行光奶之製備，發現光奶製品之品質深受釀製時 所添加糯米培養物汁液所影響，此外在製備過程中其成份亦會發生變化。在所製備之光奶 製品中以 Amy. rouxii 和 End. capsularis 共同培養所得汁液製成者較受歡迎。製備前 後之光奶產品 pH 值與酸度分別有小幅下降及增加之情形，含酵母菌者之pH值下降與酸度 增加之幅度大於未添加酵母菌者。光奶產品之有機酸以 tartaric acid 含量最多，且含 有酵母菌者之有機酸總量高於未添加酵母菌者。甲醛態氮含量以Amy. rouxii單獨培養之 萃出液製備者較高。游離脂肪酸及酒精含量則以含有 Sac. cerevisiae 者為最高。光奶 產品製備過程中，果糖與葡萄糖含量有顯著增加，蔗糖含量有顯著減少之情形。破裂強度 及硬度均以 Amy. rouxii 與 End. capsularis 共同培養者為最高，於 4℃放置 2 天後 ，其值無明顯差異。 Amylomyces rouxii is one of the main fungi exists in Chinese yeast, the s tarter of lao-chao. In addition to provide pleasant flavor, Amy. rouxii produc es milk-clotting enzyme which contribute to the formation of gelationous struc ture in gua-nai, an oriential style fermented milk product. These studies were first conducted to examine the production of milk-clotting enzyme in glutinou s rice substrate by Amy rouxii. Besides, purification and characterization of Amy. rouxii milk-clotting enzyme were conducted. Finally, quality of gua-nai p roducts prepared with Amy. rouxii were examined. Rresults show ed that the mold produced maximum amout of milk-clotting enzyme of 7.4 unit/g dry substrate was noted in 5-day solid culture of test organism in glutinous r ice substrate containing 50% moisture and at 30℃.The highest milk-clotting en zyme activity was noted in 3-day liquid culture of test organism in medium con taining 20% rice Inoculation of Endomycopsis capsularis or Saccharomyces cerev isiae in rice substrate resulted in the decreased yield of milk-clotting enzym e by Amy. rouxii. However, addition of Zn2+、Mg2+、Cu2+、Fe3+ or Al3+ increase d the production of milk-clotting enzyme in both solid and liquid culture cond itions. The crude solution of Amy. rouxii milk-clotting enzyme extracte d from rice culture was purified 7.73 fold by ammonium sulfate fractionation, Sephadex G-75 gel filtration, and DEAE Sephadex A-50 ion exchange column chrom atography. The recovery was 65.38%. Molecular weight of the enzyme is about 47 .5 kDa infered from SDS-PAGE and Native-PAGE, 48.6 kDa inferred from gel filtr ation. The enzyme is stable within pH 4.0-6.5 and 30-50℃, 74.6℃ is its denat ure temperature by DSC analysis. The enzyme has an optimum pH around 5.5, opti mum temperature 45℃. Fe3+ or Ca2+ ion can elevate milk-clotting enzyme activi ty, but Zn2+ or KMnO4 shows adverse result. SDS-PAGE and gel filtration r esults show that the amounts of α- andβ-casein were reduced gradually as rea ction time increased when incubated with Amy. rouxii milk-clotting enzyme, but no large hydrolysates appeared. κ-casein was broken down to a 11.1 kDa and a 6.0 kDa fragments by the enzyme. When α-、β-、κ- casein or milk incubated with Amy. rouxii milk-clotting enzyme results in irregular lump as observed wi th transmitting electron micrographys. Hadonic test showed that gua-nai product made with the juice of lao-chao prepared with Amy rouxii and End. caps ularis is more favorable for panels. Small magnitude of pH drop and increase i n acidity were noted during the gua-nai preparation process. Tartaric acid was the domain organic acid of gua-nai products, and the total amount of organic acids are higher when gua-nai containing yeasts. The contents of free fatty ac ids and ethanol are the highest when gua-nai product containing Sac. cerevisia e. Contents of fructose and glucose increased, but sucrose decreased significa ntly during the preparation process.