戊糖乳杆菌C50-6细菌素的纯化和特性研究
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摘要
为了获取广谱、高效的乳酸菌细菌素产生菌,本文对分离自四川传统发酵肉制品(香肠、腊肉)中的91株乳酸菌,以大肠杆菌(Escherichia coli)ATCC 25922、金黄色葡萄球菌(Staphylococcus aureus)ATCC 25923、藤黄微球菌(Micrococcusluteus)10209、铜绿假单胞杆菌(Pseudomonas aeruginosa)ATCC 27853、枯草芽孢杆菌(Bacillus subtilis)为指示菌,采用打孔法初筛,进一步通过排除有机酸、过氧化氢干扰及发酵液的蛋白酶降解实验复筛,得到1株产广谱(能抑制G~+菌和G~-菌)细菌素的菌株C50-6,经表型特征和16S rDNA系统发育特征鉴定为戊糖乳杆菌(Lactobacillus pentosus)。
采用单因素实验,对戊糖乳杆菌C50-6产细菌素的培养时间、温度、起始pH条件进行优化,结果表明:将菌株C50-6种子液按2%接种量接入起始pH6.0的MRS肉汤,经30℃培养36h能获得较高的细菌素产量,其发酵液效价可达1151IU/ml,是原培养条件细菌素产量的3.6倍。
分别采用硫酸铵盐析、有机溶剂沉淀、正丙醇提取、pH吸附几种方法对戊糖乳杆菌C50-6细菌素进行粗分级分离,综合分析后选取效果最好的硫酸铵盐析法。该细菌素的纯化步骤依次为:首先取发酵液离心得去菌体细胞的上清液,再向其中加入硫酸铵固体粉末至饱和度70%,静置过夜,离心取沉淀溶于pH4.5的柠檬酸缓冲液得到细菌素粗提液,用相同的缓冲液进行过夜透析脱盐,再经过SP-Toyopeal-650M阳离子交换色谱纯化,采用含0.1mol/L NaCl的pH4.4的柠檬酸缓冲液洗脱可得细菌素的活性峰,最终细菌素比活提高了39倍,得率为21.8%。经Tricine-SDS-PAGE检测该细菌素的分子量约为2500Da,抑菌实验表明该蛋白条带具有很强的抑菌活性,证实该电泳条带即是细菌素。
对细菌素的发酵液和粗提液的特性进行对比研究,发现发酵液在pH2.0-5.0、粗提液在pH2.0-6.0时显示活性;发酵液100℃耐受20min,粗提液具有更高的热稳定性,121℃加热20min后活性无损失;发酵液和粗提液都被木瓜蛋白酶和胰蛋白酶失活,被蛋白酶K部分失活,不被胃蛋白酶失活;发酵液和粗提液具有较一致的抑菌谱,二者均能有效抑制金黄色葡萄球菌和大肠杆菌等G~+和G~-细菌、能抑制戊糖乳杆菌(Lactobacillus pentosus) G21-10和植物乳杆菌(Lactobacillusplantarum)C21-6等近源乳酸菌、对红酵母(Rhodotorula sp.)和青霉(Penicilliumsp.)也有微弱的抑制作用,细菌素粗提液的效价明显提高,经过排除有机酸干扰后粗提液对G~+菌抑制作用更为显著。总之,戊糖乳杆菌C50-6所产细菌素在酸性和中性环境下稳定、对热稳定、能被部分蛋白酶失活、抑菌谱较广,具有作为食品生物防腐剂的潜在应用价值。
Ninety-one strains of lactic acid bacteria (LAB) isolated from traditional Sichuan fermented sausage and cured meat were tested for antagonistic activities by punch method. Cell-free supernatants from seven of these strains were shown to inhibit the growth of four indicators (Escherichia coli ATCC 25922、Staphylococcus aureus ATCC 25923、Micrococcus luteus 10209、Pseudomonas aeruginosa ATCC 27853、Bacillus subtilis), which contained both gram-positive bacteria and gram-negative bacteria, after eliminating the interferences of organic acid and H_2O_2. Strain C50-6 provided the most effective of inhibitory activity and was selected for further study. The inhibitory activity of cell-free supernatant from strain C50-6 decreased sharply after the treatment with trypsin and pepsin. It confirmed that this inhibitory material was a kind of protein and C50-6 was a broad-spectrum bacteriocin-producing strain. Its phenotype characteristics and 16S rDNA sequence analysis showed highly homology to Lactobacillus pentosus.
Single factor level test was adopted to optimize the incubation condition of this bacteriocin. As a result, the highest production would be obtained at adding 2% of seeds into MRS broth (pH6.0), cultivating 36h at 30℃. The potency of cell-free supernatant from L. pentosus C50-6 under the optimal incubation condition was 1151IU/ml, resulted in 3.6-fold increase to the primal incubation condition.
Contrasting the results of rough fractionation by ammonium sulfate precipitation, organic solvents precipitation, n-propanol extraction and pH adsorption, ammonium sulfate precipitation was finally chosen for rough fractionation of the bacteriocin. The bacteriocin was purified sequentially from culture supernatant, first precipitated with 70% saturation of solid ammonium sulfate, then purified by SP-Toyopeal-650M cation exchange chromatography, using gradient NaCl concentrtion stepwise elution with citrate buffer (pH4.4). The entire purifation protocol led to a 39-fold increase in the specific activity of the bacteriocin, and yield of 21.8%. The purified bacteriocin was identified as a ca. 2500Da peptide by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), and the protein band had strongly antibacterial activity.
The culture supernatant and crude extract were characterized. The former showed activity at pH 2-5, and the latter showed activity at pH 2-6. The former maintained the most activity when heated 20min at 100℃, and the latter was stable when heated 20min at 121℃. Both of them were inactivated by papain and trypsin, and part-inactivated by proteinase K, but remained activity after treating by pepsin. The two had almost the same inhibition spectrum. Both of them could effectively inhibit gram-positive bacteria and gram-negative bacteria, could inhibit the growth of Lactobacillus pentosus G21-10 and Lactobacillus plantarum C21-6, and weakly inhibit the growth of Rhodotorula sp. and Penicillium sp.. The potency of crude extract increased remarkably, and it had more effectively inhibition to gram-positive bacteria than the culture supernatant. In a word, the bacteriocin produced by L. pentosus C50-6 is heat stable, acid and neutral stable, proteinaceous, and had a broad spectrum, seen to have the potential application value as a kind of food biological preservative.
采用单因素实验,对戊糖乳杆菌C50-6产细菌素的培养时间、温度、起始pH条件进行优化,结果表明:将菌株C50-6种子液按2%接种量接入起始pH6.0的MRS肉汤,经30℃培养36h能获得较高的细菌素产量,其发酵液效价可达1151IU/ml,是原培养条件细菌素产量的3.6倍。
分别采用硫酸铵盐析、有机溶剂沉淀、正丙醇提取、pH吸附几种方法对戊糖乳杆菌C50-6细菌素进行粗分级分离,综合分析后选取效果最好的硫酸铵盐析法。该细菌素的纯化步骤依次为:首先取发酵液离心得去菌体细胞的上清液,再向其中加入硫酸铵固体粉末至饱和度70%,静置过夜,离心取沉淀溶于pH4.5的柠檬酸缓冲液得到细菌素粗提液,用相同的缓冲液进行过夜透析脱盐,再经过SP-Toyopeal-650M阳离子交换色谱纯化,采用含0.1mol/L NaCl的pH4.4的柠檬酸缓冲液洗脱可得细菌素的活性峰,最终细菌素比活提高了39倍,得率为21.8%。经Tricine-SDS-PAGE检测该细菌素的分子量约为2500Da,抑菌实验表明该蛋白条带具有很强的抑菌活性,证实该电泳条带即是细菌素。
对细菌素的发酵液和粗提液的特性进行对比研究,发现发酵液在pH2.0-5.0、粗提液在pH2.0-6.0时显示活性;发酵液100℃耐受20min,粗提液具有更高的热稳定性,121℃加热20min后活性无损失;发酵液和粗提液都被木瓜蛋白酶和胰蛋白酶失活,被蛋白酶K部分失活,不被胃蛋白酶失活;发酵液和粗提液具有较一致的抑菌谱,二者均能有效抑制金黄色葡萄球菌和大肠杆菌等G~+和G~-细菌、能抑制戊糖乳杆菌(Lactobacillus pentosus) G21-10和植物乳杆菌(Lactobacillusplantarum)C21-6等近源乳酸菌、对红酵母(Rhodotorula sp.)和青霉(Penicilliumsp.)也有微弱的抑制作用,细菌素粗提液的效价明显提高,经过排除有机酸干扰后粗提液对G~+菌抑制作用更为显著。总之,戊糖乳杆菌C50-6所产细菌素在酸性和中性环境下稳定、对热稳定、能被部分蛋白酶失活、抑菌谱较广,具有作为食品生物防腐剂的潜在应用价值。
Ninety-one strains of lactic acid bacteria (LAB) isolated from traditional Sichuan fermented sausage and cured meat were tested for antagonistic activities by punch method. Cell-free supernatants from seven of these strains were shown to inhibit the growth of four indicators (Escherichia coli ATCC 25922、Staphylococcus aureus ATCC 25923、Micrococcus luteus 10209、Pseudomonas aeruginosa ATCC 27853、Bacillus subtilis), which contained both gram-positive bacteria and gram-negative bacteria, after eliminating the interferences of organic acid and H_2O_2. Strain C50-6 provided the most effective of inhibitory activity and was selected for further study. The inhibitory activity of cell-free supernatant from strain C50-6 decreased sharply after the treatment with trypsin and pepsin. It confirmed that this inhibitory material was a kind of protein and C50-6 was a broad-spectrum bacteriocin-producing strain. Its phenotype characteristics and 16S rDNA sequence analysis showed highly homology to Lactobacillus pentosus.
Single factor level test was adopted to optimize the incubation condition of this bacteriocin. As a result, the highest production would be obtained at adding 2% of seeds into MRS broth (pH6.0), cultivating 36h at 30℃. The potency of cell-free supernatant from L. pentosus C50-6 under the optimal incubation condition was 1151IU/ml, resulted in 3.6-fold increase to the primal incubation condition.
Contrasting the results of rough fractionation by ammonium sulfate precipitation, organic solvents precipitation, n-propanol extraction and pH adsorption, ammonium sulfate precipitation was finally chosen for rough fractionation of the bacteriocin. The bacteriocin was purified sequentially from culture supernatant, first precipitated with 70% saturation of solid ammonium sulfate, then purified by SP-Toyopeal-650M cation exchange chromatography, using gradient NaCl concentrtion stepwise elution with citrate buffer (pH4.4). The entire purifation protocol led to a 39-fold increase in the specific activity of the bacteriocin, and yield of 21.8%. The purified bacteriocin was identified as a ca. 2500Da peptide by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), and the protein band had strongly antibacterial activity.
The culture supernatant and crude extract were characterized. The former showed activity at pH 2-5, and the latter showed activity at pH 2-6. The former maintained the most activity when heated 20min at 100℃, and the latter was stable when heated 20min at 121℃. Both of them were inactivated by papain and trypsin, and part-inactivated by proteinase K, but remained activity after treating by pepsin. The two had almost the same inhibition spectrum. Both of them could effectively inhibit gram-positive bacteria and gram-negative bacteria, could inhibit the growth of Lactobacillus pentosus G21-10 and Lactobacillus plantarum C21-6, and weakly inhibit the growth of Rhodotorula sp. and Penicillium sp.. The potency of crude extract increased remarkably, and it had more effectively inhibition to gram-positive bacteria than the culture supernatant. In a word, the bacteriocin produced by L. pentosus C50-6 is heat stable, acid and neutral stable, proteinaceous, and had a broad spectrum, seen to have the potential application value as a kind of food biological preservative.
引文
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