生產角蛋白酶之最適化培養基並應用角蛋白酶於動物飼糧之研究

Medium optimization for keratinase production by Bacillus cereus H10 and application of using keratinase in the animal feed

鍾之儀(Chih-I Chung)

指導教授 ： 陳明汝

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01119

摘要

本研究室從豬毛上篩選出一株具有產生高活性角蛋白酶能力之細菌Bacillus cereus H10，而本研究擬利用畜產廢棄物－豬毛－做為誘導角蛋白酶生產的基質，探討培養此株菌之最適化培養基，以大量生產角蛋白酶，並且應用於動物飼糧中。
試驗中以不同比例的豬毛做為 B. cereus H10 生產角蛋白酶的誘導物，再以不同比例之葡萄糖、蛋白腖和糖蜜酵母粉作為培養基中的碳源及氮源，使用反應曲面法（response surface methodology，RSM）和序列二次規劃法（sequential quadratic programming，SQP）尋求最佳酵素活性表現，經分析為添加 3.00 % 豬毛及 1.20 % 糖蜜酵母粉於培養基中能夠得到最高之菌數，而添加 1.06 % 豬毛及 0.59 % 葡萄糖於培養基中則能得到最高之角蛋白酶活性，而含有1.06 % 豬毛及0.58 % 葡萄糖之培養基能同時得到最高菌數及角蛋白酶活性，接著以 3.5 L發酵槽大量生產角蛋白酶。為使角蛋白酶更利於應用，將粗萃H10酵素液製成H10酵素粉；角蛋白酶耐熱性佳，不管是噴乾或凍乾皆能維持酵素活性，其中以含有3.00 % 豬毛及1.20 % 糖蜜酵母粉培養基所製成的酵素粉能維持較佳的蛋白酶及角蛋白酶活性。最後將兩種酵素粉分別置於 37、25、4、-20 及 -80℃進行180天的保存試驗。凍乾之H10酵素粉其蛋白酶及角蛋白酶活性皆無明顯下降；噴乾之H10酵素粉，以25 ℃保存的蛋白酶保有較高活性，而其他溫度之保存則無顯著差異。
在in vitro的試驗中，豬毛粉添加2 % 的H10酵素粉或粗萃H10酵素液，培養經5日後，其可消化蛋白質含量及in vitro蛋白質消化率顯著高於未經酵素處理的豬毛粉。若分析豬毛及豬毛粉的水解液成分，豬毛經H10酵素水解後，其胺基酸含量皆高於未經酵素處理的豬毛；豬毛粉經H10酵素水解後，部分胺基酸的含量高於未經酵素處理的豬毛粉。實際應用H10酵素粉於1 ~ 21日齡肉雞飼糧中，無法增加肉雞的體增重、採食量及飼料換肉率，但可提高肉雞對於高蛋白質飼糧的消化率。
總結以上之結果，利用最適化培養基生產H10酵素，顯示培養基中含有1.06 % 豬毛及0.58 % 葡萄糖之培養基能同時得到最高菌數及角蛋白酶活性；不管是凍乾或噴乾之方式，皆可用來生產H10酵素粉，而在室溫下儲存180天，仍可維持良好之角蛋白酶活性。雖然H10酵素粉之發酵來源所產生的不良氣味，使得在肉雞飼糧試驗之應用具有限制性；但H10酵素具有蛋白酶及角蛋白酶分解活性之能力，若能改善氣味及其適口性，未來仍具有發展空間。

關鍵字

角蛋白酶豬毛 ； 最適培養基

並列摘要

Keratin, which is increasingly accumulating in the environment mainly in the form of feathers and hair, is a part of solid waste management. However, keratin is difficult to degrade due to its highly rigid structure rendered by extensive disulfide bonds and cross-linkages.
In our previous study, we isolated a bacterium with high keratinolytic activity from hog hair and identified as Bacillus cereus H10. The H10 enzyme produced by B. cereus H10 showed high thermal stability and were very effective in hog hair degradation, presenting potential uses for biotechnological processes involving keratin hydrolysis. Thus, in this study, we optimized the culture medium for B. cereus H10 to maximize the H10 enzymes production using a combination of response surface methodology (RSM) and sequential quadratic programming (SQP). Results indicated that B. cereus H10 incubated in a medium with 3.00% hog hair and 1.20% sugar yeast could have the highest cell counts, whereas this strain fermented in the culture medium with 1.06% hog hair and 0.58% glucose could yield the highest keratinolytic activities. After maximization of enzyme production, we further dehydrated the H10 enzyme using spray drying or freeze drying. Both drying methods could remain keratinolytic activity of the H10 enzyme for 6 months at room temperature with no significant difference.
The hog hair meal co-cultured with H10 enzyme showed significantly higher percentages of digestible protein and in vitro digestibility than hog hair meal without the enzyme. However, in vivo study, addition of H10 enzyme did not improve the growth performance of broiler, but H10 enzyme could increased digestibility of high protein content diet of 1 ~ 21-day-old broilers.
In this study, we maximize the production of the H10 enzyme by addition of 1.06% hog hair and 0.58% glucose in the culture medium for B. cereus H10 and dehydration the enzyme using spray drying/freeze drying. The keratinolytic activity of the H10 enzyme was stable for 6 months at room temperature. The in vitro digestibility of poultry indicated that addition of H10 enzyme did not improve the growth performance of broiler, but H10 enzyme could increased digestibility of high protein content diet of 1 ~ 21-day-old broilers.

並列關鍵字

Keratinase ； Optimization of medium ； Hog hair

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