从牛奶和奶制品中分离的潜在益生菌乳酸菌的分离和鉴定

J. Vanniyasingam, R. Kapilan, S. Vasantharuba
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引用次数: 12

摘要

益生菌是指通过肠道微生物平衡对人体健康产生有益作用的活微生物。应用最广泛的益生菌是牛奶和奶制品中的乳酸菌群。本研究旨在研究从切达奶酪、酸奶和牛奶中分离的乳酸菌(LAB)菌株的益生菌特性。在牛奶、切达奶酪和酸奶中自然生长的细菌菌株采用MRS琼脂培养基分离,在37℃好氧和厌氧条件下分别培养48 h。在好氧条件下对8株菌株进行分离纯化,并通过革兰氏染色、过氧化氢酶试验和运动性试验等形态学和生化分析对其进行鉴定。8株分离株均为棒状或球菌状,革兰氏阳性,过氧化氢酶阴性,无运动和无孢子形成。这8株分离菌株经鉴定为乳酸菌。对8株分离菌株进行了对大肠杆菌、肺炎克雷伯菌、铜绿假单胞菌、沙门氏菌和金黄色葡萄球菌等5种人致病菌的抑菌活性筛选,其中3株(M6、C1和Y1)表现出广谱抑菌活性。通过酸耐受性、NaCl耐受性、胆汁耐受性、乳糖利用率、抗生素耐药性等试验,对3株菌株的益生菌特性进行了研究。3株菌株均表现出对胃pH (pH 3.0)的耐受性,但菌株C1对胃pH的耐受性明显高于其他菌株。虽然3株菌株在NaCl和0.3%胆汁的作用下生长良好,但菌株M6在NaCl和0.3%胆汁的作用下生长显著高于其他菌株。虽然3株菌株都有利用乳糖的能力,但在乳糖利用试验中,分离株M6比其他2株表现出明显的颜色变化。采用氨苄西林、链霉素、杆菌霉素、庆大霉素4种抗生素对分离实验室进行药敏评价,结果显示菌株M6对氨苄西林、杆菌霉素耐药较高,对链霉素、庆大霉素敏感。另外两株(C1和Y1)对所有4种抗生素均敏感。通过药敏试验和益生菌特性分析,确定菌株M6为潜在的益生乳酸菌。通过16s rDNA序列分析,鉴定该菌株为植物乳杆菌CIP 103151。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Isolation and characterization of potential probiotic lactic acid bacteria isolated from cow milk and milk products
Probiotics refer to the living microorganisms that exhibit beneficial effect on the health of human by the intestinal microbial balance. Most widely used probiotics are lactic acid bacterial group found in milk and milk products. This study was aimed to characterize the probiotic properties of lactic acid bacterial (LAB) strains isolated from cheddar cheese, yoghurt and cow milk. Bacterial strains naturally grown in milk, cheddar cheese and yoghurt were isolated using De Mann Rogosa Sharpe (MRS) agar medium and incubated at 37oC for 48 h separately under aerobic and anaerobic conditions. There were eight strains grown under aerobic conditions and they were isolated, purified and characterization was done based on the morphological and biochemical analysis such as gram staining, catalase test and motility test. All the eight isolates were either rod or cocci shaped, gram positive, catalase negative, non-motile and non-spore formers. These eight isolated strains were identified as lactic acid bacteria. When screening of the 8 isolates was done to determine their antimicrobial activities against five human pathogenic strains such as E.coli, Klebsiella pneumoniae, Pseudomonas eaeruginosa, Salmonella sp and Staphylococcus aureus, three isolates (M6, C1 and Y1) showed wide spectrum antimicrobial activity. To determine the probiotic properties of these three isolates, different tests such as tolerance to acid, NaCl and bile, lactose utilization and antibiotic resistance were done. Though all the three isolates showed resistance to stomach pH (pH 3.0), the strain C1 showed significantly higher tolerance to stomach pH than the other strains. Though the three isolates grew well in the presence of NaCl and 0.3% bile, the isolate M6 showed significantly higher growth with NaCl and 0.3% bile than the others. Even though all the three isolates had the capacity of utilizing lactose, the isolate M6 showed prominent colour change in the lactose utilization test than the other two strains. When antibiotic susceptibility of the isolated LABs were evaluated using four antibiotics such as Ampicillin, Streptomycin, Bacitracin and Gentamycin, the strain M6 showed significantly higher resistance to Ampicillin and Bacitracin but sensitive to Streptomycin and Gentamycin. Other two isolates (C1 and Y1) were sensitive to all the four antibiotics used. Based on the antibiotic sensitivity tests and the analysis of probiotic properties, the isolated strain M6 was confirmed as a potential probiotic lactic acid bacterium. This strain was identified as Lactobacillus plantarum strain CIP 103151 through16S rDNA sequence analysis.
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