纳豆菌液体发酵条件优化及抗菌物质分离纯化研究
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摘要
本论文对纳豆菌的分离鉴定、液体发酵条件及抗菌物质的分离纯化进行了研究,研究内容主要包括三个方面:
1纳豆菌的分离鉴定
本论文从日本纳豆中分离的到了5株有较强抗菌作用的菌株,对其中抗菌作用最强的1株菌BN5进行了菌落形态、菌体形态及生理生化特性鉴定,鉴定为枯草芽孢杆菌属纳豆菌亚种纳豆菌(Bacillus subtilis subsp.natto),简称纳豆菌BN5(Bacillus natto BN5)。
2纳豆菌液体发酵条件的研究
通过单因素和响应面试验,对纳豆菌发酵条件进行了试验研究。使用Minitab 15软件进行数据分析和作图,研究了菌种生长因素和发酵条件因素对纳豆菌BN5生长量的影响。试验结果表明:BN5的最优培养条件为,温度34.4℃、发酵液pH6.8、装瓶量56.8 mL/200 mL、接种量2%、摇床转速180 r/min。
3纳豆菌发酵液中抗菌物质的分离纯化
用饱和硫酸铵沉淀纳豆菌BN5发酵液中的抗菌物质,其中硫酸铵饱和度为30%-70%时提取的粗蛋白具有较强的抑菌活性,当饱和度达到70%时提取的抗菌物质抑菌活性最强。采用了层析技术分离和纯化了纳豆菌BN5发酵液的抗菌粗蛋白,利用DEAE-52阴离子交换层析后得到3个蛋白质吸收峰,其中Ⅱ峰、Ⅲ峰对大肠杆菌和金黄色葡萄球菌具有抑菌活性。将Ⅱ峰收集液过Sephadex G-50凝胶柱层析分离后得到2个吸收峰,其中Ⅳ峰、Ⅴ峰对肠杆菌和金黄色葡萄球菌具有抑菌活性。研究表明:Ⅴ峰对肠杆菌和金黄色葡萄球菌具有最强抑菌活性。
Studied on the isolating and identificating of the strain Bacillus natto BN5 from Natto,optimiziing the liqual fermentation conditions of Bcillus Natto BNS and purification antimicrobial substance from the liqual fermentation of Bcillus NattoBN5.
1 Isolating and identifing of the Bacillus Natto from Natto
Five strains of Bacillus natto with high antimicrobial activity were isolated from Natto.The strain Bacillus natto BN5 with the highest antimicrobial activity was identified as Bacillus subtilis subsp. natto accordin to morphological, physiologicl and biochemical characters. It was short of Bacillus natto.
2 Study on optimizing the liquid fermentation conditions of Bacillus Natto BN5
Through single factor experiment and orthogonal experiment,the conditions of liquid fermentation of Bacllius natto were experimented. The growth factors and conditions of liquid fermentation were studied according to analysis and construction of Minitab 15. The results indicated that optimal conditions of liquid fermentation of Bacillus natto BN5 were fermentation temperature 34.4℃, fermentation pH 6.86, volumes of fementation 56.8 mL /200 mL, inoculum levels 2%, shaking speeds 180r/min.
3 Study on purification antimicrobial substance from the liquid fermetation of Bacillus natto BN5
The filtrated cultured liquid of Bacillus natto strain BN5 was precipitated with the ammonium sulfate to draw the antimicrobial proteins. We studied character of crude proteins drawn with ammonium sulfate at 30%-70% saturation respectively,which showed a strong antimicrobial activity at 70%.Bacillus natto strain BNS secreted the antimicrobial protein,but litter was known about the antimicrobial protein. In this study,the cruded proteins were purified by Amersham AKTA Explore system. The crude protein were eluted through the DEAE-52 culumn and three peaks were found;the II and the III fraction had antimicrobial activity to Staphylococcus aureus and Escherichia coli..After the second fraction was pooled and eluted in the Sephadex G-50 culumn,there were alse two peaks fraction,which the IV and V fraction ,which had antimicrobial activity to Staphylococcus aureus and Escherichia coli. The study showed that the V fraction had the highest antimicrobial activity accoding to antimicrobial experiment to Staphylococcus aureus and Escherichia coli.
1纳豆菌的分离鉴定
本论文从日本纳豆中分离的到了5株有较强抗菌作用的菌株,对其中抗菌作用最强的1株菌BN5进行了菌落形态、菌体形态及生理生化特性鉴定,鉴定为枯草芽孢杆菌属纳豆菌亚种纳豆菌(Bacillus subtilis subsp.natto),简称纳豆菌BN5(Bacillus natto BN5)。
2纳豆菌液体发酵条件的研究
通过单因素和响应面试验,对纳豆菌发酵条件进行了试验研究。使用Minitab 15软件进行数据分析和作图,研究了菌种生长因素和发酵条件因素对纳豆菌BN5生长量的影响。试验结果表明:BN5的最优培养条件为,温度34.4℃、发酵液pH6.8、装瓶量56.8 mL/200 mL、接种量2%、摇床转速180 r/min。
3纳豆菌发酵液中抗菌物质的分离纯化
用饱和硫酸铵沉淀纳豆菌BN5发酵液中的抗菌物质,其中硫酸铵饱和度为30%-70%时提取的粗蛋白具有较强的抑菌活性,当饱和度达到70%时提取的抗菌物质抑菌活性最强。采用了层析技术分离和纯化了纳豆菌BN5发酵液的抗菌粗蛋白,利用DEAE-52阴离子交换层析后得到3个蛋白质吸收峰,其中Ⅱ峰、Ⅲ峰对大肠杆菌和金黄色葡萄球菌具有抑菌活性。将Ⅱ峰收集液过Sephadex G-50凝胶柱层析分离后得到2个吸收峰,其中Ⅳ峰、Ⅴ峰对肠杆菌和金黄色葡萄球菌具有抑菌活性。研究表明:Ⅴ峰对肠杆菌和金黄色葡萄球菌具有最强抑菌活性。
Studied on the isolating and identificating of the strain Bacillus natto BN5 from Natto,optimiziing the liqual fermentation conditions of Bcillus Natto BNS and purification antimicrobial substance from the liqual fermentation of Bcillus NattoBN5.
1 Isolating and identifing of the Bacillus Natto from Natto
Five strains of Bacillus natto with high antimicrobial activity were isolated from Natto.The strain Bacillus natto BN5 with the highest antimicrobial activity was identified as Bacillus subtilis subsp. natto accordin to morphological, physiologicl and biochemical characters. It was short of Bacillus natto.
2 Study on optimizing the liquid fermentation conditions of Bacillus Natto BN5
Through single factor experiment and orthogonal experiment,the conditions of liquid fermentation of Bacllius natto were experimented. The growth factors and conditions of liquid fermentation were studied according to analysis and construction of Minitab 15. The results indicated that optimal conditions of liquid fermentation of Bacillus natto BN5 were fermentation temperature 34.4℃, fermentation pH 6.86, volumes of fementation 56.8 mL /200 mL, inoculum levels 2%, shaking speeds 180r/min.
3 Study on purification antimicrobial substance from the liquid fermetation of Bacillus natto BN5
The filtrated cultured liquid of Bacillus natto strain BN5 was precipitated with the ammonium sulfate to draw the antimicrobial proteins. We studied character of crude proteins drawn with ammonium sulfate at 30%-70% saturation respectively,which showed a strong antimicrobial activity at 70%.Bacillus natto strain BNS secreted the antimicrobial protein,but litter was known about the antimicrobial protein. In this study,the cruded proteins were purified by Amersham AKTA Explore system. The crude protein were eluted through the DEAE-52 culumn and three peaks were found;the II and the III fraction had antimicrobial activity to Staphylococcus aureus and Escherichia coli..After the second fraction was pooled and eluted in the Sephadex G-50 culumn,there were alse two peaks fraction,which the IV and V fraction ,which had antimicrobial activity to Staphylococcus aureus and Escherichia coli. The study showed that the V fraction had the highest antimicrobial activity accoding to antimicrobial experiment to Staphylococcus aureus and Escherichia coli.
引文
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