藏猪源乳酸菌的分离鉴定及益生特性测定

Isolation, identification, and probiotic property evaluation of lactic acid bacteria from Xizang pigs

摘要:

乳酸菌(lactic acid bacteria, LAB)是益生菌的主要来源。我国珍贵的地方猪种藏猪的肠道乳酸菌资源仍有待挖掘。目的 筛选优质藏猪源乳酸杆菌。方法 采用平板划线法分离并通过16S rRNA基因测序鉴定乳酸菌，进一步选择2株乳酸产量有显著差异的藏猪源罗伊氏黏液乳杆菌和1株实验室此前保存杜×长×大(duroc×landrace×yorkshire, DLY)猪源的罗伊氏黏液乳杆菌，共3株菌进行耐酸耐胆盐、抗氧化试验、抑菌能力以及耐药性等生物学特性分析和比较。结果 从藏猪中分离鉴定到21株乳酸菌，分别为10株非解乳糖链球菌(Streptococcus alactolyticus)、6株罗伊氏黏液乳杆菌(Limosilactobacillus reuteri) [原名为罗伊氏乳杆菌(Lactobacillus reuteri)]、3株食淀粉乳杆菌(Lactobacillus amylovorus)、1株肠膜状明串珠菌(Leuconostoc mesenteroides)和1株阴道黏液乳杆菌(Limosilactobacillus vaginalis)。在挑选出的3株菌中，L. reuteri T-B5L2的乳酸产量最高，该菌在pH 3.0和0.1%胆盐的培养基中均表现出较强的生存能力，其抑制肠致病性大肠杆菌和猪霍乱沙门菌生长的能力最强。3株菌在抗氧化能力上无显著差异。3株菌均对青霉素类和头孢菌素类抗生素较敏感，对四环素类、氨基糖苷类和糖肽类抗生素有抵抗力。结论 本研究从藏猪粪便中分离到21株乳酸菌，其中L. reuteri T-B5L2的产酸能力最强。进一步的体外生化特性测定结果表明，L. reuteri T-B5L2生长性能良好，具有耐酸、耐胆盐、抗氧化和抑制病原菌生长的能力，是一株具有潜力的益生菌。

Abstract:

Lactic acid bacteria (LAB) are the predominant probiotics with significant health-promoting potential. Xizang pigs, an invaluable indigenous breed in China, harbor a unique and largely unexplored reservoir of intestinal LAB.Objective To screen high-quality LAB isolated from Xizang pigs.Methods LAB were isolated by the streak plate method and identified by 16S rRNA gene sequencing. Two Limosilactobacillus reuteri strains with significantly different lactic acid production capacities were selected from Xizang pigs. The two strains, together with one L. reuteri strain previously preserved in our laboratory, which originated from duroc×landrace×yorkshire pigs, were assessed in terms of acid and bile salt tolerance, antioxidant activity, antibacterial properties, and antibiotic resistance.Results A total of 21 LAB strains were isolated and identified from the intestinal microbiota of Xizang pigs, including ten Streptococcus alactolyticus strains, six L. reuteri strains, three Lactobacillus amylovorus strains, one Leuconostoc mesenteroides strain, and one Limosilactobacillus vaginalis strain. Among the three strains screened out, L. reuteri T-B5L2 exhibited the highest lactic acid production. Moreover, this strain demonstrated strong survival under pH 3.0 and 0.1% bile salts. L. reuteri T-B5L2 exhibited the strongest inhibitory activity against enteropathogenic Escherichia coli and Salmonella Choleraesuis. There were no significant differences in antioxidant activity among the three strains. All the three strains exhibited high sensitivity to penicillins and cephalosporins but displayed resistance to tetracyclines, aminoglycosides, and glycopeptides.Conclusion In this study, 21 LAB strains were isolated from the feces of Xizang pigs, among which L. reuteri T-B5L2 exhibited the highest acid production capacity. Further in vitro biochemical characterization demonstrated that this strain exhibited good growth performance, acid and bile salt tolerance, antioxidant activity, and the ability to inhibit pathogenic bacteria. These findings suggest that L. reuteri T-B5L2 is a promising probiotic candidate with potential applications in improving intestinal health and mitigating pathogenic infections.