戊糖乳杆菌31-1菌株产细菌素研究
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摘要
作者从传统发酵肉制品云南宣威火腿中分离筛选出一株具有抑菌活性的乳酸菌31-1菌株，对多株乳杆菌具有抑菌作用。经排除干扰因素后确定其抑菌物质为蛋白类物质细菌素，菌株经鉴定后为戊糖乳杆菌(Lactobacillus pentosus)。
对戊糖乳杆菌31-1菌株产细菌素的条件进行优化，确定其有最大产量时的培养时间、培养温度、培养起始pH值，即30℃下培养24小时且初始pH为6.5；选取碳源、氮源、刺激因子等不同因素不同水平采用正交试验设计进行优化，结果得到细菌素产量是初始培养基培养的8倍，达到640AU／ml，最优培养基配方为乳糖3％、胰胨1.5％、豆胨2％、牛肉膏3％、蛋白胨2％、吐温80 0.1％、磷酸氢二钾0.2％、乙酸钠0.5％、柠檬酸铵0.2％、硫酸镁0.058％、硫酸锰0.025％，蒸馏水1000ml。
采用SP Sepharose Fast Flow阳离子交换树脂纯化细菌素，用含0.1M NaCl的0.02M醋酸盐缓冲液即可洗脱下细菌素峰，比活力达到1259.84(AU／mg)，是原发酵液的17倍。采用Tricine-SDS-PAGE法估测出细菌素分子量大小在14.2kDa以下。
31-1菌株所产细菌素在80℃热处理15min后活性基本不变，在121℃15min仍保持一定活性；在pH2-8范围内保持稳定，当pH值大于9时，活性逐渐降低。细菌素可被胃蛋白酶、胰蛋白酶、蛋白酶K、木瓜蛋白酶、中性蛋白酶完全失活，可被α-淀粉酶部分失活，酸性蛋白酶、溶菌酶不能使细菌素失活。31-1菌株所产的细菌素的抑菌谱较广，不仅可抑制实验所采用乳杆菌属、链球菌属、片球菌属中的大部分菌株，还可抑制金黄色葡萄球菌、大肠杆菌、芽孢杆菌中的部分菌株。31-1所产细菌素的抑菌作用效果明显，在3-5小时内可降低敏感菌活细胞2-3个数量级。该细菌素具有较好的热、酸稳定性及较高的安全性，具有作为食品防腐剂应用的良好前景。
31-1菌株作为发酵剂在河南双汇集团进行了实验室规模试制发酵香肠，结果表明香肠产酸迅速，风味优良，且在发酵过程中可明显降低肠杆菌的数量，作为肉类发酵剂使用具有较好的前景。
Lactobacillus pentosus 31-1 isolated from Xuan Wei Ham produced antimicrobial substance which was inhibitory to several Lactobacillus plantarum strains. After eliminating organic acid, oxid catalase, and so on, we have confirmed the antimicrobial role is because of bacteriocin.
To optimize bacteriocin production researching was done on the respect of incubation condition and the media components. And the optimum incubation time was 24h, the optimum temperature was 30℃, the optimum broth initial pH was 6.5. The optimum media component was: lactose 3%, trypone 1.5%, soybean peptone 2%,meat extract 3%, peptonethe 2%,Tween 80 0.1%, K2HPO40.2%, NaAc 0.5%, Tri-Ammonium citrate 0.2%, MgSO4 0.058%, MnSO4 0.025%, H2O 1000ml. Production of bactericoin under the optimum broth was 640AU/ml, 8 times than MRS media.
The bacteriocin was purified by ammonium sulphate precipitation, followed by SP-Sepharose FF cation exchange chromatography. And the bacteriocin was washed with 0.1M NaCl(in acetate buffer,pH5.5), at 1.2ml/min. And the specific activity was 1259.84AU/mg. The molecular size of bacteriocin was estimated to be under 14.2kDa, according to tricine-SDS PAGE.
Bacteriocin was inactivated when treated with pepsin, proteinase K, trypsin, papain, neutral proteinase, and part-inactivated by α-amylase.But remained active after incubation at pH 2-8 for 4h at 37℃ and when heated 15min at 80℃. The mechanism of activity of bacteriocin is bactericidal,as shown by a obvious decrease about 2-3 orders of magnitude in the viable cell numbers of L.plantarum over a period of 3-4h. The bacteriocin had a broad spectrum, including several strains from Lactobacillus, Streptococcus, Pediococcus, especially including Staphylococcus aureus, E.coli, Bacillus cereus, B.subtilis, B.megaterium.
Lactobacillus pentosu 31-1 was successfully used as starter culture of fermented sausages in He Nan province Shuan hui meat company. It can made sausages acid-producing very fast, finally up to pH 5.2, especially inhibit Escherichia strains growing and finally dispear in product. This indicated that bacteriocin produced from Lactobacillus pentosus 31-1 have great potential application as preservatives in food, and the strain as starter culture of fermented sausages.
对戊糖乳杆菌31-1菌株产细菌素的条件进行优化，确定其有最大产量时的培养时间、培养温度、培养起始pH值，即30℃下培养24小时且初始pH为6.5；选取碳源、氮源、刺激因子等不同因素不同水平采用正交试验设计进行优化，结果得到细菌素产量是初始培养基培养的8倍，达到640AU／ml，最优培养基配方为乳糖3％、胰胨1.5％、豆胨2％、牛肉膏3％、蛋白胨2％、吐温80 0.1％、磷酸氢二钾0.2％、乙酸钠0.5％、柠檬酸铵0.2％、硫酸镁0.058％、硫酸锰0.025％，蒸馏水1000ml。
采用SP Sepharose Fast Flow阳离子交换树脂纯化细菌素，用含0.1M NaCl的0.02M醋酸盐缓冲液即可洗脱下细菌素峰，比活力达到1259.84(AU／mg)，是原发酵液的17倍。采用Tricine-SDS-PAGE法估测出细菌素分子量大小在14.2kDa以下。
31-1菌株所产细菌素在80℃热处理15min后活性基本不变，在121℃15min仍保持一定活性；在pH2-8范围内保持稳定，当pH值大于9时，活性逐渐降低。细菌素可被胃蛋白酶、胰蛋白酶、蛋白酶K、木瓜蛋白酶、中性蛋白酶完全失活，可被α-淀粉酶部分失活，酸性蛋白酶、溶菌酶不能使细菌素失活。31-1菌株所产的细菌素的抑菌谱较广，不仅可抑制实验所采用乳杆菌属、链球菌属、片球菌属中的大部分菌株，还可抑制金黄色葡萄球菌、大肠杆菌、芽孢杆菌中的部分菌株。31-1所产细菌素的抑菌作用效果明显，在3-5小时内可降低敏感菌活细胞2-3个数量级。该细菌素具有较好的热、酸稳定性及较高的安全性，具有作为食品防腐剂应用的良好前景。
31-1菌株作为发酵剂在河南双汇集团进行了实验室规模试制发酵香肠，结果表明香肠产酸迅速，风味优良，且在发酵过程中可明显降低肠杆菌的数量，作为肉类发酵剂使用具有较好的前景。
Lactobacillus pentosus 31-1 isolated from Xuan Wei Ham produced antimicrobial substance which was inhibitory to several Lactobacillus plantarum strains. After eliminating organic acid, oxid catalase, and so on, we have confirmed the antimicrobial role is because of bacteriocin.
To optimize bacteriocin production researching was done on the respect of incubation condition and the media components. And the optimum incubation time was 24h, the optimum temperature was 30℃, the optimum broth initial pH was 6.5. The optimum media component was: lactose 3%, trypone 1.5%, soybean peptone 2%,meat extract 3%, peptonethe 2%,Tween 80 0.1%, K2HPO40.2%, NaAc 0.5%, Tri-Ammonium citrate 0.2%, MgSO4 0.058%, MnSO4 0.025%, H2O 1000ml. Production of bactericoin under the optimum broth was 640AU/ml, 8 times than MRS media.
The bacteriocin was purified by ammonium sulphate precipitation, followed by SP-Sepharose FF cation exchange chromatography. And the bacteriocin was washed with 0.1M NaCl(in acetate buffer,pH5.5), at 1.2ml/min. And the specific activity was 1259.84AU/mg. The molecular size of bacteriocin was estimated to be under 14.2kDa, according to tricine-SDS PAGE.
Bacteriocin was inactivated when treated with pepsin, proteinase K, trypsin, papain, neutral proteinase, and part-inactivated by α-amylase.But remained active after incubation at pH 2-8 for 4h at 37℃ and when heated 15min at 80℃. The mechanism of activity of bacteriocin is bactericidal,as shown by a obvious decrease about 2-3 orders of magnitude in the viable cell numbers of L.plantarum over a period of 3-4h. The bacteriocin had a broad spectrum, including several strains from Lactobacillus, Streptococcus, Pediococcus, especially including Staphylococcus aureus, E.coli, Bacillus cereus, B.subtilis, B.megaterium.
Lactobacillus pentosu 31-1 was successfully used as starter culture of fermented sausages in He Nan province Shuan hui meat company. It can made sausages acid-producing very fast, finally up to pH 5.2, especially inhibit Escherichia strains growing and finally dispear in product. This indicated that bacteriocin produced from Lactobacillus pentosus 31-1 have great potential application as preservatives in food, and the strain as starter culture of fermented sausages.
引文
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