产广谱细菌素植物乳杆菌的初步研究及其在泡菜中的应用
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摘要
从四川传统发酵食品中筛选出一株安全的具有广谱抗菌活性(对G~+菌和G~-菌均有较强抑制作用)的产细菌素植物乳杆菌，通过对其产细菌素发酵条件的优化、生物学性质及其在泡菜中的应用研究，为微生物生物防腐剂的开发及其在食品中的应用提供了理论依据。
1．采用打孔法研究分离自四川传统发酵食品的267株乳酸菌对大肠杆菌(Escherichia coli)ATCC25922、金黄色葡萄球菌(Staphylococcus aureus)ATCC25923、藤黄微球菌(Micrococcus luteus)10209、铜绿假单胞杆菌(Pseudomonas aeruginosa)ATCC27853、枯草芽孢杆菌(Bacillus subtilis)的抑菌作用，初筛出64株具有广谱抗菌活性的乳酸菌：再采用牛津杯法研究初筛出的乳酸菌菌株做对藤黄微球菌、铜绿假单胞杆菌的抑菌作用，排除酸性产物、H_2O_2的干扰，并进行蛋白酶酶解，复筛出一株具有产广谱细菌素的乳酸菌菌株P158，分离自醪糟。根据其形态学和生理生化特征，以及16S rDNA分子遗传学分析(该菌株的16S rDNA序列在Genbank上的登录号为EF114393)，鉴定其为植物乳杆菌(Lactobacillus plantarum)。
2．优化了植物乳杆菌P158产细菌素发酵条件，具体探讨了不同培养基、培养温度、培养时间、培养方式、接种量、起始培养pH值，培养基碳源、氮源、金属离子、吐温-80等因素对细菌素产量的影响。优化出最佳培养条件和培养基成分为：34℃静置发酵42h、接种量为3％、起始pH为6.0、葡萄糖2％、大豆蛋白胨1.5％、酵母膏2％、乙酸钠0.5％、K_2HPO_40.2％、MgSO_4 0.58％、MnSO_4 0.25％、FeSO_4 0.04％、吐温-80 0.08％，在此条件下培养，P158菌株细菌素相对效价可达到1145IU／mL，与起始培养基相比细菌素产量提高了216％。
3．研究了植物乳杆菌P158发酵上清液的生物学性质，结果表明：①P158在培养36h生物量达到最大，在40h对藤黄微球菌、铜绿假单胞杆菌的抑菌圈直径都达到最大值，而此时发酵上清液的pH值也降至最低，乳酸菌处于稳定期内。②P158发酵上清液具有较好的酸稳定性，较高的热稳定性；对蛋白酶敏感；对抗生素普遍敏感；③具有较广泛的抑菌谱，对大肠杆菌等G。菌、藤黄微球菌等G~+菌有较强的抑制作用，对一些真菌有一定的抑制作用，而对近源的乳酸菌几乎没有抑制作用；④经对P158菌株质粒提取与消除实验研究，抑菌活性基因没有存在于质粒上。
4．对植物乳杆菌P158纯种半固态发酵什锦泡菜工艺参数进行了优化，提高了泡菜品质，最佳工艺参数为：盐加量4％，糖加量3％，接种量0.1％，发酵温度34℃。将纯种半固态发酵与纯种液体发酵、传统自然发酵方式制作泡菜进行比较，分析三种泡菜产品在发酵和低温贮藏过程中感官品质、pH值、总酸、盐度、亚硝酸盐含量和微生物指标的变化，结果表明，P158菌株纯种半固态发酵的什锦泡菜品质与纯种液体发酵的什锦泡菜品质相差不明显，但纯种发酵泡菜明显优于传统自然发酵泡菜；与纯种液体发酵和自然发酵相比，半固态发酵能明显提高泡菜总酸含量、降低盐度、降低亚硝酸盐含量，保持较高乳酸菌数量，并且细菌、霉菌、酵母菌、MPN数量均较低。
A LAB strain, producing a novel broad-spectrum bacteriocin, was screened from the traditional fermented food in Sichuan province. By studying the strain fermentation conditions, biological property and it's apply on pickled vegetable in order to afford theoretic data for the exploiture and application of microbial bio-preservative in food industry.
1. 267 LAB strains were isolated from Sichuan traditional fermented food. Using agar plate proliferation, 64 LAB strains which can inhibit indicator strains (Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Micrococcus luteus 10209, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis) were screened from those isolated LABs. The LAB P158 strain could inhibit indicator strains (Micrococcus luteus 10209 and Pseudomonas aeruginosa ATCC27853) strongly excluding the disturbances of organic acid and H_2O_2 by using double-layer agar plate proliferation, but the inhibition decreased sharply after the treatment with trypsin and pepsin. The results confirmed that this inhibitory material was a kind of protein and it could inhibit both G~+ and G~-strains, so it could be classed as a broad-spectrun bacteriocin. According to morphological, physiological properties and 16S rDNA sequence (Accession No. EF114393) analysis, P158 strain was identified as Lactobacillus plantarum.
2. Optimized the condition of bacteriocin production, research was done on five types of medias, the incubation condition and the media components. The optimum incubation time was 42h without shaking, the optimum temperature was 34℃, the optimum broth initial pH was 6.0. The optimum inoculating amount was 3%. And the optimum media's component was: glucose 2%, soybean peptone 1.5%, yeast extract 2%, K_2HPO_4 0.2%, NaAc 0.5%, MGSO40.58%, MnSO4 0.25%,FESO40.04%, Tween 80 0.08%. With the fermentation condition, the relative active of P158 was 1145IU/mL, raise 216%.
3. The biological property of bacteriocin of L. plantarum P158 indicated that P158 cultured best on 36h, but have the best active against Micrococcus luteus and Pseudomonas aeruginosa on 40h, and have the lowest pH at the same time. The bacteriocin of P158 was steady in acid conditions and it had good thermal stability. It was sensitive to proteinase. It was sensitive to antibiotics. It had broad antimicrobial spectrum, was active against G~-strains(such as E. coli), G~+ strains(such as Micrococeus luteus) and could inhibit some fungi, but almost have no active against LABs. The experiment of plasrnid elimination confirmed that the gene was not located on the plasmid.
4. The technology parameters to produce the semisolid pickled vegetables inoculated P158 were researched and optimized, and the quality of the products were improved. The traditional submerged pickled vegetables which were inoculated P158 and the traditional natural fermented pickled vegetables were compared with the semisolid one and the organoleptic, the chemical and the microbiological quality were studied. The results indicated that there were few discrepancies between the inoculated submerged and the inoculated semisolid one, but they both were evidently better than the traditional natural fermented pickled vegetables.
1．采用打孔法研究分离自四川传统发酵食品的267株乳酸菌对大肠杆菌(Escherichia coli)ATCC25922、金黄色葡萄球菌(Staphylococcus aureus)ATCC25923、藤黄微球菌(Micrococcus luteus)10209、铜绿假单胞杆菌(Pseudomonas aeruginosa)ATCC27853、枯草芽孢杆菌(Bacillus subtilis)的抑菌作用，初筛出64株具有广谱抗菌活性的乳酸菌：再采用牛津杯法研究初筛出的乳酸菌菌株做对藤黄微球菌、铜绿假单胞杆菌的抑菌作用，排除酸性产物、H_2O_2的干扰，并进行蛋白酶酶解，复筛出一株具有产广谱细菌素的乳酸菌菌株P158，分离自醪糟。根据其形态学和生理生化特征，以及16S rDNA分子遗传学分析(该菌株的16S rDNA序列在Genbank上的登录号为EF114393)，鉴定其为植物乳杆菌(Lactobacillus plantarum)。
2．优化了植物乳杆菌P158产细菌素发酵条件，具体探讨了不同培养基、培养温度、培养时间、培养方式、接种量、起始培养pH值，培养基碳源、氮源、金属离子、吐温-80等因素对细菌素产量的影响。优化出最佳培养条件和培养基成分为：34℃静置发酵42h、接种量为3％、起始pH为6.0、葡萄糖2％、大豆蛋白胨1.5％、酵母膏2％、乙酸钠0.5％、K_2HPO_40.2％、MgSO_4 0.58％、MnSO_4 0.25％、FeSO_4 0.04％、吐温-80 0.08％，在此条件下培养，P158菌株细菌素相对效价可达到1145IU／mL，与起始培养基相比细菌素产量提高了216％。
3．研究了植物乳杆菌P158发酵上清液的生物学性质，结果表明：①P158在培养36h生物量达到最大，在40h对藤黄微球菌、铜绿假单胞杆菌的抑菌圈直径都达到最大值，而此时发酵上清液的pH值也降至最低，乳酸菌处于稳定期内。②P158发酵上清液具有较好的酸稳定性，较高的热稳定性；对蛋白酶敏感；对抗生素普遍敏感；③具有较广泛的抑菌谱，对大肠杆菌等G。菌、藤黄微球菌等G~+菌有较强的抑制作用，对一些真菌有一定的抑制作用，而对近源的乳酸菌几乎没有抑制作用；④经对P158菌株质粒提取与消除实验研究，抑菌活性基因没有存在于质粒上。
4．对植物乳杆菌P158纯种半固态发酵什锦泡菜工艺参数进行了优化，提高了泡菜品质，最佳工艺参数为：盐加量4％，糖加量3％，接种量0.1％，发酵温度34℃。将纯种半固态发酵与纯种液体发酵、传统自然发酵方式制作泡菜进行比较，分析三种泡菜产品在发酵和低温贮藏过程中感官品质、pH值、总酸、盐度、亚硝酸盐含量和微生物指标的变化，结果表明，P158菌株纯种半固态发酵的什锦泡菜品质与纯种液体发酵的什锦泡菜品质相差不明显，但纯种发酵泡菜明显优于传统自然发酵泡菜；与纯种液体发酵和自然发酵相比，半固态发酵能明显提高泡菜总酸含量、降低盐度、降低亚硝酸盐含量，保持较高乳酸菌数量，并且细菌、霉菌、酵母菌、MPN数量均较低。
A LAB strain, producing a novel broad-spectrum bacteriocin, was screened from the traditional fermented food in Sichuan province. By studying the strain fermentation conditions, biological property and it's apply on pickled vegetable in order to afford theoretic data for the exploiture and application of microbial bio-preservative in food industry.
1. 267 LAB strains were isolated from Sichuan traditional fermented food. Using agar plate proliferation, 64 LAB strains which can inhibit indicator strains (Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Micrococcus luteus 10209, Pseudomonas aeruginosa ATCC27853 and Bacillus subtilis) were screened from those isolated LABs. The LAB P158 strain could inhibit indicator strains (Micrococcus luteus 10209 and Pseudomonas aeruginosa ATCC27853) strongly excluding the disturbances of organic acid and H_2O_2 by using double-layer agar plate proliferation, but the inhibition decreased sharply after the treatment with trypsin and pepsin. The results confirmed that this inhibitory material was a kind of protein and it could inhibit both G~+ and G~-strains, so it could be classed as a broad-spectrun bacteriocin. According to morphological, physiological properties and 16S rDNA sequence (Accession No. EF114393) analysis, P158 strain was identified as Lactobacillus plantarum.
2. Optimized the condition of bacteriocin production, research was done on five types of medias, the incubation condition and the media components. The optimum incubation time was 42h without shaking, the optimum temperature was 34℃, the optimum broth initial pH was 6.0. The optimum inoculating amount was 3%. And the optimum media's component was: glucose 2%, soybean peptone 1.5%, yeast extract 2%, K_2HPO_4 0.2%, NaAc 0.5%, MGSO40.58%, MnSO4 0.25%,FESO40.04%, Tween 80 0.08%. With the fermentation condition, the relative active of P158 was 1145IU/mL, raise 216%.
3. The biological property of bacteriocin of L. plantarum P158 indicated that P158 cultured best on 36h, but have the best active against Micrococcus luteus and Pseudomonas aeruginosa on 40h, and have the lowest pH at the same time. The bacteriocin of P158 was steady in acid conditions and it had good thermal stability. It was sensitive to proteinase. It was sensitive to antibiotics. It had broad antimicrobial spectrum, was active against G~-strains(such as E. coli), G~+ strains(such as Micrococeus luteus) and could inhibit some fungi, but almost have no active against LABs. The experiment of plasrnid elimination confirmed that the gene was not located on the plasmid.
4. The technology parameters to produce the semisolid pickled vegetables inoculated P158 were researched and optimized, and the quality of the products were improved. The traditional submerged pickled vegetables which were inoculated P158 and the traditional natural fermented pickled vegetables were compared with the semisolid one and the organoleptic, the chemical and the microbiological quality were studied. The results indicated that there were few discrepancies between the inoculated submerged and the inoculated semisolid one, but they both were evidently better than the traditional natural fermented pickled vegetables.
引文
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