一株植物乳酸杆菌LABR-X产细菌素的研究
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摘要
细菌素(bacteriocin)是某些细菌产生的抑菌肽或蛋白,这些物质可以杀灭和抑制与之相同或相似生境的其他微生物。虽然许多细菌都能产生细菌素,但乳酸菌产生的细菌素更适合在食品工业中应用。现在只有几种商品化的细菌素,如nisin,nisin作为食品添加剂可以非常有效的抑制许多食品中的腐败菌,并延长其货价期。但由于其不能抑制革兰氏阴性菌和抑菌活性的不稳定限制了它在食品工业与饲料工业中的发展,所以我们要继续大力开发其他乳酸菌细菌素。
本文从自然的酸菜发酵液中挑选分离出形态良好的8株菌落菌株进行初筛,通过抑菌实验,选取抑菌最大的菌株作为诱变出发菌株LABR,经微生物常规鉴定方法初步鉴定LABR为植物乳杆菌(Lactobacillus plantarum)。利用紫外线与硫酸二乙酯对LABR多轮复合诱变筛选出抑菌活性最强的菌株LABR-X,在排除有机酸和过氧化氢的干扰后,离心浓缩后的发酵液对指示菌有强烈的抑制作用。
优化菌株LABR-X的培养条件,通过正交实验确定了最佳培养条件:培养温度为37℃,MRS培养基的初始pH值为6,接种量为5%(V/V),装液量为60%。在该培养条件下,LABR-X的生长稳定期为18～26小时。LABR-X发酵液经蛋白酶稳定性实验发现,发酵液中的抑菌物质对胰蛋白酶和蛋白酶K敏感,同时结合水合茚三酮反应和双缩脲反应得到的阳性结果,充分证明LABR-X的发酵液中的抑菌物质含有多肽类物质成分,即乳杆菌素。
按照蛋白质物质提取纯化原理,LABR-X发酵产物经硫酸铵盐析法粗提、弱酸性阳离子交换树脂层析纯化、Tricine-SDS-PAGE电泳分离等方法提取精制乳杆菌素,通过透析袋处理得到的抑菌活性最大的乳杆菌素组分分子量约20.1kDa,其效价为nisin的2倍,经氨基酸分析仪检测乳杆菌素中含有13种氨基酸和5种未知寡肽。
利用光学显微镜与扫描电镜菌体形态观察、碱性磷酸酶活性测定和β-半乳糖苷酶活性测定等方法,对乳杆菌素抑菌机理进行了初步研究与探讨,实验结果显示乳杆菌素能够破坏指示菌的细胞壁和细胞膜,从而达到抑菌的作用。
Bacteriocins produced by certain bacteria are antimicrobial peptides or proteins, in sufficient amounts, these peptides can kill or inhibit bacteria competing for the same ecological niche or the same nutrient pool. Although bacteriocins are produced by many Gram-positive and Gram-negative species, those produced by the LAB are of particular interest to the food industry. To date, the only commercially produced bacteriocins is nisin. While nisin has been found to be extremely effective as an additive to prevent spoilage and increase shelf-life in a number of foods. But its effectiveness has been more variable with respect to other applications. So the new bacteriocins should be attractive to explore.
In the paper, 8 clonies of good shape were isolated from sauerkraut fermentation broth, the starting strain mutagenesis LABR of the largest inhibitory strains was selected by the antibacterial test. The LABR was identified as Lactobacillus plantarum by the conventional microbiological identification. The LABR-X of the best antibacterial activity was compound selected by UV and DNS from LABR,the fermentation supernatant by centrifugation and concentrated can inhibit indicator strains strongly excluding inhibitive effect of hydrogen peroxide and organic acid.
To optimize mutagenesis strains LABR-X culture conditions, the best culture conditions were determined by orthogonal experiments: the temperature 37℃, the initial pH of culture media 6,the culture starter is 5%(v/v),the volume of liquid is 60%. The period of stable growth of LABR-X from 18 to 26 hours under the culture conditions. The inhibitive activity decreased sharply after treatment with proteinase K and Trypsin, with Ninhydrin and biuret reaction response to positive. The results confirmed that these inhibitory materials were protein, could be classed as bacteriocins.
Through the principle of protein extraction and purification, the fermentation product of LABR-X were precipitated by ammonium sulphate, followed by weak acid cation exchange chromatography and Tricine-SDS-PAGE to purification. The molecular weight of elution protein of the best inhibition was 20.1 kDa, the inhibition titer double than nisin. The bacteriocins contain 13 kinds of amino acids and 5 kinds of unknown oligopeptide.
We researched on mechanism of antibacterial by optical microscope and scanning electron microscope observation of cell morphology and activity assay of alkaline Phosphatase andβ-galactosidase. The experimental results show the bacteriocins inhibit bacteria by destroying the cell wall and membrane of bacteria.
本文从自然的酸菜发酵液中挑选分离出形态良好的8株菌落菌株进行初筛,通过抑菌实验,选取抑菌最大的菌株作为诱变出发菌株LABR,经微生物常规鉴定方法初步鉴定LABR为植物乳杆菌(Lactobacillus plantarum)。利用紫外线与硫酸二乙酯对LABR多轮复合诱变筛选出抑菌活性最强的菌株LABR-X,在排除有机酸和过氧化氢的干扰后,离心浓缩后的发酵液对指示菌有强烈的抑制作用。
优化菌株LABR-X的培养条件,通过正交实验确定了最佳培养条件:培养温度为37℃,MRS培养基的初始pH值为6,接种量为5%(V/V),装液量为60%。在该培养条件下,LABR-X的生长稳定期为18～26小时。LABR-X发酵液经蛋白酶稳定性实验发现,发酵液中的抑菌物质对胰蛋白酶和蛋白酶K敏感,同时结合水合茚三酮反应和双缩脲反应得到的阳性结果,充分证明LABR-X的发酵液中的抑菌物质含有多肽类物质成分,即乳杆菌素。
按照蛋白质物质提取纯化原理,LABR-X发酵产物经硫酸铵盐析法粗提、弱酸性阳离子交换树脂层析纯化、Tricine-SDS-PAGE电泳分离等方法提取精制乳杆菌素,通过透析袋处理得到的抑菌活性最大的乳杆菌素组分分子量约20.1kDa,其效价为nisin的2倍,经氨基酸分析仪检测乳杆菌素中含有13种氨基酸和5种未知寡肽。
利用光学显微镜与扫描电镜菌体形态观察、碱性磷酸酶活性测定和β-半乳糖苷酶活性测定等方法,对乳杆菌素抑菌机理进行了初步研究与探讨,实验结果显示乳杆菌素能够破坏指示菌的细胞壁和细胞膜,从而达到抑菌的作用。
Bacteriocins produced by certain bacteria are antimicrobial peptides or proteins, in sufficient amounts, these peptides can kill or inhibit bacteria competing for the same ecological niche or the same nutrient pool. Although bacteriocins are produced by many Gram-positive and Gram-negative species, those produced by the LAB are of particular interest to the food industry. To date, the only commercially produced bacteriocins is nisin. While nisin has been found to be extremely effective as an additive to prevent spoilage and increase shelf-life in a number of foods. But its effectiveness has been more variable with respect to other applications. So the new bacteriocins should be attractive to explore.
In the paper, 8 clonies of good shape were isolated from sauerkraut fermentation broth, the starting strain mutagenesis LABR of the largest inhibitory strains was selected by the antibacterial test. The LABR was identified as Lactobacillus plantarum by the conventional microbiological identification. The LABR-X of the best antibacterial activity was compound selected by UV and DNS from LABR,the fermentation supernatant by centrifugation and concentrated can inhibit indicator strains strongly excluding inhibitive effect of hydrogen peroxide and organic acid.
To optimize mutagenesis strains LABR-X culture conditions, the best culture conditions were determined by orthogonal experiments: the temperature 37℃, the initial pH of culture media 6,the culture starter is 5%(v/v),the volume of liquid is 60%. The period of stable growth of LABR-X from 18 to 26 hours under the culture conditions. The inhibitive activity decreased sharply after treatment with proteinase K and Trypsin, with Ninhydrin and biuret reaction response to positive. The results confirmed that these inhibitory materials were protein, could be classed as bacteriocins.
Through the principle of protein extraction and purification, the fermentation product of LABR-X were precipitated by ammonium sulphate, followed by weak acid cation exchange chromatography and Tricine-SDS-PAGE to purification. The molecular weight of elution protein of the best inhibition was 20.1 kDa, the inhibition titer double than nisin. The bacteriocins contain 13 kinds of amino acids and 5 kinds of unknown oligopeptide.
We researched on mechanism of antibacterial by optical microscope and scanning electron microscope observation of cell morphology and activity assay of alkaline Phosphatase andβ-galactosidase. The experimental results show the bacteriocins inhibit bacteria by destroying the cell wall and membrane of bacteria.
引文
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