利用Bacillus sp.發酵豆渣與醬油粕混合物之研究

近年來隨著人口上升導致經濟作物需求增加，農業廢棄物產量亦隨之大增，其多伴隨難消化蛋白質及高纖維，而Bacillus菌屬所產生之蛋白酶、纖維素酶及澱粉酶等胞外酵素，可於發酵過程中將其降解為易吸收之小分子，以提高利用率。本研究自實驗室挑選15株Bacillus sp.分別進行蛋白酶及纖維素酶定性測試，將菌株振盪培養 (35℃、150 rpm) 48小時後取200 μL菌液，分別加入於已打洞 (10 mm) 之牛奶培養基及纖維素培養基中，經24小時後觀察培養基是否可產生分解環。結果顯示，15株Bacillus sp.中有12株皆可產生蛋白酶及纖維素酶之分解透明環，因此後續再將菌株進行蛋白酶活性及纖維素分解能力試驗。結果指出，Bacillus velezensis S3及Bacillus subtilis subsp. N1有較佳之蛋白酶活性，其分別為2.69 U及2.58 U。另外亦將菌株培養於含羧甲基纖維素 (CMC) 之TSB培養液中，測試菌株分解纖維素能力。結果顯示，Bacillus subtilis E20及Bacillus megaterium BM有較佳之纖維素分解能力，其分解CMC後可產生較高之葡萄糖含量，分別為5.84 μg/mL及5.61μg/mL。其結果得知Bacillus velezensis S3具有較佳之蛋白酶活性，而B. subtilis E20則有較佳之纖維素分解能力。接著將B. velezensis S3與B. subtilis E20進行菌株特性探討，結果顯示兩株菌株皆可以於10%氯化鈉添加之TSB培養基，但是其無法於pH 4培養基生長。接者將S3與E20分別添加1%於不同比例 (2:1、3:1、4:1、5:1及6:1) 之豆渣與醬油粕混合物 (Mix of okara and soya pomace, MOS) 共發酵，測定其菌數與總胺基酸增加率。結果顯示，發酵72小時後菌數皆可以維持在8 Log CFU/g，而總胺基酸增加率則是以6:1比例混合之MOS最好，與發酵0小時相比增加142.05±12.44%。接續探討6:1比例混合之MOS最適化之發酵條件。以一次一因子法探討葡萄糖添加量、氯化鈣添加量及調整發酵溫度，結果顯示添加0.75%葡萄糖及1.5%氯化鈣至6:1 MOS中再於45℃下可以產生最高含量之總游離胺基酸，再利用Box-Behnken設計方法來探討這三個變因對於產生總游離胺基酸之間關係。結果顯示，添加0.76%葡萄糖與1.49%氯化鈣至6:1比例混合之MOS經B. velezensis S3與B. subtilis E20於45.25℃下共發酵可獲得最高含量之游離胺基酸，為93.47±4.57 mg/g。最後再分析最適條件發酵後纖維與異黃酮之變化，結果顯示，半纖維素與纖維素含量相比，發酵後可分別降低6.64%及0.3%，表示B. velezensis S3與B. subtilis E20共發酵可降解MOS中纖維。另外，經發酵MOS其苷元型異黃酮Daidzein、Genistein分別下降30.56 ppm及37.78 ppm。綜合上述結果可以得知，B. velezensis S3與B. subtilis E20共發酵以6:1比例混合之MOS可以增加總游離胺基酸含量及降解纖維，使MOS自身之應用限制降低進而增加未來再利用性。

關鍵字

農業廢棄物； Bacillus sp. ；蛋白酶；纖維素分解能力；總游離胺基酸

並列摘要

According to the increase in population, the demand for cash crops has increased, and the output of agricultural waste has also increased significantly. Most of agricultural waste are accompanied by indigestible protein and high fiber. While extracellular protease, cellulase, and amylase produced by Bacillus sp., and the enzymes can degrad them into small molecules that are easily absorbed during the fermentation process to improve utilization. In this study, 15 strains of Bacillus sp. were selected from the laboratory for qualitative tests of protease and cellulase, respectively. The strains were shaken cultured (35℃, 150 rpm) for 48 hours, and take 200 μL of bacterial liquid and add to the holes (10 mm) in the milk medium and cellulose medium, after 24 hours, observe whether the medium could produce a decomposition ring. The results showed that 12 of the 15 strains of Bacillus sp. could produce protease and cellulase-decomposing transparent rings, so the strains were then tested for protease activity and cellulose decomposition ability. The results indicated that Bacillus velezensis S3 and Bacillus subtilis subsp. N1 had better protease activities, which were 2.69 U and 2.58 U, respectively. In addition, the strain was cultured in a TSB medium containing carboxymethyl cellulose (CMC) to test the ability of the strain to decompose cellulose. The results showed that Bacillus subtilis E20 and Bacillus megaterium BM had the better cellulose decomposition ability, and could produce higher glucose content after decomposing CMC, which were 5.84 μg/mL and 5.61 μg/mL, respectively. The results show that B. velezensis S3 has better protease activity, and B. subtilis E20 has the better cellulolytic ability. Then, the characteristics of B. velezensis S3 and B. subtilis E20 were investigated. The results showed that both strains could grow in a TSB medium supplemented with 10% sodium chloride, but they could not grow in a pH 4 medium. The receiver added 1% of S3 and E20 respectively in different ratios (2:1, 3:1, 4:1, 5:1, and 6:1) of the mixture of okara and soya pomace (MOS) After co-fermentation, the bacterial count and the increase rate of total amino acids were determined. The results show that the bacterial count can be maintained at 8 Log CFU/g after 72 hours of fermentation, and the increase rate of total amino acids is the best MOS mixed with a ratio of 6:1, which increases by 142.05±12.44% compared with 0 hour of fermentation. Continue to discuss the optimal fermentation conditions of MOS mixed in a ratio of 6:1. The addition of glucose, calcium chloride, and adjustment of fermentation temperature were investigated by one-factor method at a time. The results showed that adding 0.75% glucose and 1.5% calcium chloride to 6:1 MOS at 45℃ could produce the highest content of total free amino acids and the Box-Behnken design method were used to explore the relationship between these three variables for the production of total free amino acids. The results showed that the highest content of free amino acids was obtained by co-fermentation of B. velezensis S3 and B. subtilis E20 at 45.25℃ with MOS added with 0.76% glucose and 1.49% calcium chloride to a ratio of 6:1, which was 93.47± 4.57 mg/g. Finally, the changes in fiber and isoflavones after fermentation under optimum conditions were analyzed. The results showed that the content of hemicellulose and cellulose could be reduced by 6.64% and 0.3% respectively after fermentation, indicating that B. velezensis S3 and B. subtilis E20 co-fermented degradable MOS fiber. In addition, the aglycone isoflavones Daidzein and Genistein decreased by 30.56 ppm and 37.78 ppm respectively after fermented MOS. Based on the above results, it can be known that the co-fermentation of B. velezensis S3 and B. subtilis E20 with MOS mixed at a ratio of 6:1 can increase the content of total free amino acids and degrade fibers, to reduce the application limitation of MOS itself and increase future reusability.