泡菜中产细菌素植物乳杆菌的筛选及其所产细菌素生物学特性的研究
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摘要
细菌素是由某些细菌产生的一类可以杀灭或抑制其它微生物的蛋白类物质。本项研究旨在从泡菜中筛选得到具有天然食品防腐剂前景的产细菌素植物乳杆菌。
从发酵2周后的泡菜中共筛选得到乳酸菌119株,其中球菌53株,杆菌66株,对所有菌株进行两两相交拮抗,选定乳杆菌C6为指示菌菌株,乳杆菌C8、C10、C18和C25为待定的产细菌素菌株。对4株待定产细菌素菌株进行紫外诱变处理,并对诱变株进行有机酸排除、过氧化氢排除以及盐析产物抑菌试验表明,突变菌株C8-1为产细菌素菌株。基于细胞形态学、生理生化及16S rDNA测序数据,菌株C8-1与C6被鉴定为植物乳杆菌(Lactobacillus plantarum),菌株C8-1与植物乳杆菌WCFS1的同源性达99%。
对植物乳杆菌C8-1发酵条件优化结果表明,产细菌素的适宜培养温度为30°C,培养基pH为6.5,培养时间为28h;蔗糖为最佳碳源,大豆蛋白胨为最佳氮源,两者的最适添加量分别为2%和1.5%;培养基中牛肉膏与Tween80的最适添加量分别为1.5%和3‰。当以柠檬酸三铵为唯一氮源时,菌株C8-1不能合成细菌素。发酵条件优化后,植物乳杆菌C8-1所产细菌素的效价达到278.24 IU/mL,是优化前的10.7倍。对植物乳杆菌C8-1的发酵上清液按70%硫酸铵饱和度盐析或40%～70%硫酸铵饱和度的分段盐析,盐析产物分别经透析、蔗糖浓缩后同时进行Tricine-SDS-PAGE检测,确定该细菌素分子量约为16,500Da。
植物乳杆菌C8-1所产细菌素具有较宽的抑菌谱,不仅能够抑制革兰氏阳性的金黄色葡萄球菌和试验采用的大部分乳酸菌菌株,而且对大肠杆菌、假单胞菌、鞘氨醇杆菌、黄杆菌等革兰氏阴性菌也具有极强的抑制效果。该细菌素温度稳定性高,4°C冷藏5d、?20°C冷冻5d以及60°C、80°C、100°C和121°C分别加热15min,活性损失均不超过6%。该细菌素活性随pH增大逐步降低,当pH≥6时不显示抑菌活性。用胃蛋白酶、木瓜蛋白酶和胰蛋白酶处理该细菌素时,抑菌活性均有降低。显然,植物乳杆菌C8-1产生的细菌素具备进一步作为天然食品防腐剂开发的潜力。
Bacteriocins are proteinaceous antibacterial compounds produced by bacterias. This paper is aimed to screen the bacteriocin-producing Lactobacillus plantarum which prossess the prospect as natural food preservative.
There were 119 lactic acid bacteria strains including 53 lactococcus stains and 66 lactobacillus strains isolated from the pickle in fermentation anaphase. Lactobacillus C6 was determined the indicative strain and Lactobacillus C8、C10、C18 and C25 were determined the possible inhibitory strains by antibacterial test between any two strains. Some mutants were obtained by UV mutation treatment on four possible inhibitory strains. A mutant strain, C8-1, was determined the bacteriocin-producing strain after testing the inhibitory effect of protein salting-out product and eliminating orgnic acid and hydrogen peroxide. Inhibitory strain C8-1 and indicative strain C6 belong to Lactobacillus plantarum according to the indification of morphology, physiology and biochemistry and 16S rDNA sequence. The 16S rDNA homology of C8-1 shared 99% with Lactobacillus plantarum WCFS1.
Fermentation conditions for Lactobacillus plantarum C8-1 were optimizated and the results showed that the optimal temperature was 30°C, the optimal broth initial pH was 6.5 and the optimal incubation time was 28h; Sucrose was the optimum carbon source and soya peptone was the optimum nitrogen source, each addition is 2% and 1.5%; The optimal additions of beef extrat and Tween80 were 1.5% and 3‰. There was no synthesis of bacteriocin using triammonium citrate as the unique nitrogen source. The titer of this bactericin was 278.24 IU/mL after optimizing fermentation conditions which was 10.7 times higher than the original.
Using the method of 70% or 40%~70% saturated ammonium sulphate salting out separated the proteins in the supernatant of Lactobacillus plantarum C8-1. Then the protein concentrates were obtained respectively through treating the products of salting-out with dialysis and concentration. The result of Tricine-SDS-PAGE showed that there was a target protein band with Mr 16,500.
The bacteriocin of Lactobacillus plantarum C8-1 had a broad antimicrobial spectrum which not only include gram-positive bacteria for example Staphylococcus aureus and most of LABS used in this experiment but also include gram-negative bacteria such as E.coli, Pseudomonas, Sphingobacterium,Flavobacterium,etc. This bacteriocin had a good temperature stability in ?20°C~121°C. The antimicrobial activity loss was less than 6% when temperature treatment at 4°C for 5d, ?20°C for 5d and 60°C, 80°C, 100°C, 121°C for 15 minutes. With the increase of pH, the antimicrobial activity was gradually decreased and could not be showed at pH≥6. This bacteriocin was partly inactivated when treated with pepsin, papain and trypsin. Based on the above conclusions, the bacteriocin of Lactobacillus plantarum C8-1 had the conditions and prospect as the natural food preservative.
从发酵2周后的泡菜中共筛选得到乳酸菌119株,其中球菌53株,杆菌66株,对所有菌株进行两两相交拮抗,选定乳杆菌C6为指示菌菌株,乳杆菌C8、C10、C18和C25为待定的产细菌素菌株。对4株待定产细菌素菌株进行紫外诱变处理,并对诱变株进行有机酸排除、过氧化氢排除以及盐析产物抑菌试验表明,突变菌株C8-1为产细菌素菌株。基于细胞形态学、生理生化及16S rDNA测序数据,菌株C8-1与C6被鉴定为植物乳杆菌(Lactobacillus plantarum),菌株C8-1与植物乳杆菌WCFS1的同源性达99%。
对植物乳杆菌C8-1发酵条件优化结果表明,产细菌素的适宜培养温度为30°C,培养基pH为6.5,培养时间为28h;蔗糖为最佳碳源,大豆蛋白胨为最佳氮源,两者的最适添加量分别为2%和1.5%;培养基中牛肉膏与Tween80的最适添加量分别为1.5%和3‰。当以柠檬酸三铵为唯一氮源时,菌株C8-1不能合成细菌素。发酵条件优化后,植物乳杆菌C8-1所产细菌素的效价达到278.24 IU/mL,是优化前的10.7倍。对植物乳杆菌C8-1的发酵上清液按70%硫酸铵饱和度盐析或40%～70%硫酸铵饱和度的分段盐析,盐析产物分别经透析、蔗糖浓缩后同时进行Tricine-SDS-PAGE检测,确定该细菌素分子量约为16,500Da。
植物乳杆菌C8-1所产细菌素具有较宽的抑菌谱,不仅能够抑制革兰氏阳性的金黄色葡萄球菌和试验采用的大部分乳酸菌菌株,而且对大肠杆菌、假单胞菌、鞘氨醇杆菌、黄杆菌等革兰氏阴性菌也具有极强的抑制效果。该细菌素温度稳定性高,4°C冷藏5d、?20°C冷冻5d以及60°C、80°C、100°C和121°C分别加热15min,活性损失均不超过6%。该细菌素活性随pH增大逐步降低,当pH≥6时不显示抑菌活性。用胃蛋白酶、木瓜蛋白酶和胰蛋白酶处理该细菌素时,抑菌活性均有降低。显然,植物乳杆菌C8-1产生的细菌素具备进一步作为天然食品防腐剂开发的潜力。
Bacteriocins are proteinaceous antibacterial compounds produced by bacterias. This paper is aimed to screen the bacteriocin-producing Lactobacillus plantarum which prossess the prospect as natural food preservative.
There were 119 lactic acid bacteria strains including 53 lactococcus stains and 66 lactobacillus strains isolated from the pickle in fermentation anaphase. Lactobacillus C6 was determined the indicative strain and Lactobacillus C8、C10、C18 and C25 were determined the possible inhibitory strains by antibacterial test between any two strains. Some mutants were obtained by UV mutation treatment on four possible inhibitory strains. A mutant strain, C8-1, was determined the bacteriocin-producing strain after testing the inhibitory effect of protein salting-out product and eliminating orgnic acid and hydrogen peroxide. Inhibitory strain C8-1 and indicative strain C6 belong to Lactobacillus plantarum according to the indification of morphology, physiology and biochemistry and 16S rDNA sequence. The 16S rDNA homology of C8-1 shared 99% with Lactobacillus plantarum WCFS1.
Fermentation conditions for Lactobacillus plantarum C8-1 were optimizated and the results showed that the optimal temperature was 30°C, the optimal broth initial pH was 6.5 and the optimal incubation time was 28h; Sucrose was the optimum carbon source and soya peptone was the optimum nitrogen source, each addition is 2% and 1.5%; The optimal additions of beef extrat and Tween80 were 1.5% and 3‰. There was no synthesis of bacteriocin using triammonium citrate as the unique nitrogen source. The titer of this bactericin was 278.24 IU/mL after optimizing fermentation conditions which was 10.7 times higher than the original.
Using the method of 70% or 40%~70% saturated ammonium sulphate salting out separated the proteins in the supernatant of Lactobacillus plantarum C8-1. Then the protein concentrates were obtained respectively through treating the products of salting-out with dialysis and concentration. The result of Tricine-SDS-PAGE showed that there was a target protein band with Mr 16,500.
The bacteriocin of Lactobacillus plantarum C8-1 had a broad antimicrobial spectrum which not only include gram-positive bacteria for example Staphylococcus aureus and most of LABS used in this experiment but also include gram-negative bacteria such as E.coli, Pseudomonas, Sphingobacterium,Flavobacterium,etc. This bacteriocin had a good temperature stability in ?20°C~121°C. The antimicrobial activity loss was less than 6% when temperature treatment at 4°C for 5d, ?20°C for 5d and 60°C, 80°C, 100°C, 121°C for 15 minutes. With the increase of pH, the antimicrobial activity was gradually decreased and could not be showed at pH≥6. This bacteriocin was partly inactivated when treated with pepsin, papain and trypsin. Based on the above conclusions, the bacteriocin of Lactobacillus plantarum C8-1 had the conditions and prospect as the natural food preservative.
引文
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