1株马乳酒样乳杆菌的分离鉴定及益生特性研究

doi:10.16431/j.cnki.1671-7236.2023.07.037

摘要/Abstract

摘要： 【目的】从新疆克州地区传统发酵马奶酒中分离既有抗菌活性又有抗氧化活性的乳酸菌，继而获得能用于微生态制剂开发的优良菌株。【方法】采用MRS固体培养基进行乳酸菌分离培养，筛选优势菌株，观察菌体形态学特征，并通过16S rRNA基因序列分析进行分子鉴定。研究菌株的生长曲线、产酸能力、耐酸能力、耐胆盐能力和抑菌能力，通过检测菌株耐H₂O₂能力及菌株发酵液、无细胞提取物、菌体混悬液的DPPH自由基清除率、超氧阴离子清除能力、羟自由基清除能力等相关指标来评价菌株体外抗氧化功能，并通过药敏试验和小鼠饲喂试验评估菌株的安全性。【结果】试验筛选出1株菌落形态圆形、白色半透明、边缘不整齐的革兰氏阳性菌，将其命名为JK-24，经16S rRNA克隆测序鉴定该菌株为马乳酒样乳杆菌（Lactobacillus kefiranofaciens）。在6~18 h，D_{600 nm}值增长速度较快，进入对数生长期，JK-24发酵液pH下降明显；JK-24在pH 2.5~6.5范围内均能生长，能耐受0.3%胆盐；发酵液对大肠杆菌和金黄色葡萄球菌的抑菌圈直径分别为15和20 mm，具有良好抑菌活性；能耐受浓度为3.0 mmol/L的H₂O₂，且在H₂O₂浓度为1.0和2.0 mmol/L时对菌株生长有促进作用；JK-24发酵液和无细胞提取物的DPPH自由基清除率、超氧阴离子清除能力和羟自由基清除能力均显著高于MRS培养基（P<0.05）；对青霉素等19种抗生素敏感；灌胃JK-24对小鼠生长性能和脏器系数均无显著影响（P>0.05），血液学参数均处于正常范围内，体内脏器无眼观病理变化。【结论】本试验分离到的马乳酒样乳杆菌JK-24安全、无致病性，具有良好的抑菌和抗氧化等益生特性，可作为生物饲料添加剂的菌种来源。

关键词: 马乳酒样乳杆菌; 鉴定; 益生特性; 安全性

Abstract: 【Objective】 The aim of this study was to isolate lactic acid bacteria with both antibacterial and antioxidant activities from traditional fermented horse milk wine in Kezhou, Xinjiang, and then obtain excellent strains that could be used for the development of micro-ecological agents.【Method】 MRS plate was used to isolate and culture lactic acid bacteria.Dominant strains were screened, morphological characteristics of bacteria were observed, and molecular identification was conducted by 16S rRNA gene sequence analysis.Then the growth curve, acid production ability, acid resistance, bile salt tolerance and bacteriostatic ability of the strain were studied. In vitro antioxidant function of the strain was evaluated by detecting the hydrogen peroxide resistance, the DPPH free radical scavenging ability, superoxide anion scavenging ability and hydroxyl radical scavenging ability of the strain's fermentation broth, cell-free extract and cell suspension.And the safety of the strain was evaluated by drug sensitivity test and feeding test in mice.【Result】 The results showed that a Gram-positive bacterium with round colony shape, white translucent and irregular edges was screened out and named JK-24.The strain was identified as Lactobacillus kefiranofaciens by 16S rRNA cloning and sequencing.From 6 to 18 h, D₆₀₀_nm value increased rapidly and entered the logarithmic growth phase, and the pH of JK-24 fermentation broth decreased obviously.JK-24 could grow in pH 2.5 to 6.5 and tolerate 0.3% bile salt.The diameters of bacteriostatic rings of fermentation broth against Escherichia coli and Staphylococcus aureus were 15 and 20 mm respectively.It could tolerate 3.0 mmol/L H₂O₂ and promote the growth of the strain at the concentration of 1.0 and 2.0 mmol/L.The DPPH free radical scavenging ability, superoxide anion scavenging ability and hydroxyl radical scavenging ability of the strain's fermentation broth and cell-free extract were significantly higher than those of MRS medium (P<0.05).It was sensitive to 19 antibiotics such as penicillin.Intragastric administration of JK-24 had no significant effect on growth performance and organ index of mice(P>0.05).The hematological parameters were in the normal range, and there were no pathological changes in organs.【Conclusion】 The Lactobacillus kefiranofaciens JK-24 isolated from horse milk wine was safe, non-pathogenic, and had good bacteriostatic and antioxidant properties, which could be used as a strain source of biological feed additives.

Key words: Lactobacillus kefiranofaciens; identification; probiotic properties; safety

中图分类号:

S816.79

周闯, 宋俊雅, 叶菁, 荀佳琦, 高海萍, 柳雅欣, 萨吉代姆·麦麦提, 林家琪, 张华. 1株马乳酒样乳杆菌的分离鉴定及益生特性研究[J]. 中国畜牧兽医, 2023, 50(7): 2974-2986.

ZHOU Chuang, SONG Junya, YE Jing, XUN Jiaqi, GAO Haiping, LIU Yaxin, SAJIDAIMU·Maimaiti, LIN Jiaqi, ZHANG Hua. Isolation, Identification and Probiotic Properties of a Strain of Lactobacillus kefiranofaciens[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2974-2986.

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