Fermentation and Production of Bacillus amyloliquefaciens as Microbial Additives

• Main Authors: Chung-Pao Lin, 林崇寶
• Other Authors: 胡淼琳
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/86496979876240373383

碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 102 === We made use of Bacillus amyloliquefaciens (BPD1), to conduct development of microbial feed addictives. At the beginning, we analyzed the characteristics of BPD1, engaging in heat tolerance test with 0.5% molasses, 1% lactose, and 2% soybean peptone at 60°C, 70°C, 80°C, 90°C and 100°C water bath separately for ten minutes to detect influences on BPD1. Data reveal that the survival rate of BPD1 only slightly decreased at 100°C water bath. For bile salt resistance test, through test of LB medium with added 0.1% to 0.3% bile salt, it showed that BPD1 couldn’t grow but was with 40% survival rate under the density of 0.3%. For acid and alkali resistance analysis, we cultured BPD1 on LB medium, from pH2 to pH10, and found that part of bacteria decreased under environment of pH2, pH3 and pH10 while there were no obvious effects on growth of BPD1 from pH5 to pH9. For salt tolerance test, we showed that BPD1 could grow by testing LB medium with added 1% to 3% NaCl. LB with 2% and 3% NaCl were even better for the growth of strains. For shake flask culture, we used 2% soybean peptone as fixed nitrogen source and tested with different carbon sources such as sugar, lactose, brown sugar, and molasses. The result shows that BPD1 cultured by 0.5% molasses and 1% lactose reached 5.1±1.4× 10 9 CFU/ml. For nitrogen source test, we used 0.5% molasses and 1% lactose as fixed carbon source, testing with nitrogen sources such as soybean flour, yeast powder, soybean peptone, and corn steep liquor. Results show that yeast powder and soybean peptone reaching > 1.1× 10 10 CFU/ml. Other fermentation conditions such as pH value and temperature showed no obvious effects on BPD1. BPD1 grew well from pH5 to pH9, with no differences under 30℃ or 25℃ . For 10-liter fermentation tank, we took 0.5% molasses, 1% lactose, and 2% soybean peptone to detect effects on growth of BPD1 caused by stirring speed and ventilation. The increase of stirring speed and ventilation slightly boosted bacteria number; however, the increase of stirring speed leaded to high foam at the same time. Trough 10-liter test, we can make sure that BPD1 reached> 4.5±2.1× 10 9 CFU/ml under conditions of stirring speed 200 to 300rpm, 30℃ , no adjustment of pH value and culture for five days. For 1-ton fermentation tank, BPD1 reached 6.0±0.5× 10 8 CFU/ml by taking 0.5% molasses, 1% lactose and 2% soybean peptone as culture medium for five days under conditions 30℃ , stirring speed 160rpm and ventilation 0.5vvm. BPD1 could reach to 1.2±0.7× 10 9 CFU/ml if increasing the stirring speed. When concentrating fermentation liquid from 1000 ml to 500 ml, either 28℃ or 32℃, came out bacteria number increased by one times, which is 2.4±1.0× 109CFU/ml. For enzyme activity test, we cultured four formulae in flask and got the result that these formulae effectively fermented BPD1 > 1.2× 10 10 CFU/ml. But for formula with added yeast powder, the enzyme activity of protease is higher, which is 501.3 U/ml; on the other hand, for formula with added 1% NaCl, the enzyme of amylase was higher, reaching 63.8 U/ml. For 1-ton fermentation tank, we took 3% molasses, 1% soybean peptone, and 0.045% FeSO 4 to ferment and detect enzyme activity, finding that protease activity of fermentation liquid reached the highest the next day, which was 1398 U/ml; amylase and cellulase activity reached the highest on the fourth day, which were 14.6 U/ml and 55.7 U/ml; lipase activity reached the highest on the sixth day, which was13.1 U/ml.