新疆维吾尔自治区呼图壁县驼乳源乳酸菌分离鉴定及其对金黄色葡萄球菌的抑制作用

doi:10.12160/j.issn.1672-5190.2024.02.001

摘要/Abstract

摘要： [目的]分离、鉴定新疆维吾尔自治区呼图壁县驼乳源乳酸菌,评价其对金黄色葡萄球菌的抑制作用。[方法]从呼图壁县某驼场采集健康驼乳样本45份,使用MRS培养基分离乳酸菌,采用细菌16S rDNA序列PCR扩增及测序方法进行分子生物学鉴定。利用点种法、琼脂斑点法和牛津杯双层琼脂扩散法筛选对金黄色葡萄球菌ATCC 29213具有抑制作用的乳酸菌。将MRS培养基的pH值调节至1~10,测定乳酸菌的耐酸能力;在MRS培养基中添加0~0.30%的牛胆盐,测定乳酸菌的耐胆盐能力。应用微量肉汤倍比稀释法测定乳酸菌无细胞上清液（cell-free culture supernatant,CFCS）对金黄色葡萄球菌ATCC 29213的最低抑菌浓度（minimum inhibitory concentration,MIC）和亚抑菌浓度（subinhibitory concentration,SIC）;考查CFCS在不同pH值条件下及经酶处理后对金黄色葡萄球菌ATCC 29213的抑制作用变化,初步分析CFCS的抑菌活性物质;利用牛津杯双层琼脂扩散法测定CFCS对驼乳源和奶牛乳房炎源金黄色葡萄球菌野生株的抑制作用。[结果]从45份驼乳中分离得到5种20株乳酸菌,分别为粪肠球菌（Enterococcus faecalis,n=13）、鼠李糖乳杆菌（Lacticaseibcillus rhamnsus,n=1）、发酵黏液乳杆菌（Limosilactobacillus fermentum,n=3）、副干酪乳杆菌（Lactobacillus paracasei,n=1）和融合魏斯氏菌（Weissella confusa,n=2）。经3种方法筛选,鼠李糖乳杆菌KC4株对金黄色葡萄球菌ATCC 29213的抑菌作用最强,且具有良好的耐酸和耐胆盐特性,在pH值为4~7和牛胆盐浓度为0~0.20%的条件下均可正常生长。KC4株的CFCS对金黄色葡萄球菌ATCC 29213的MIC和SIC分别为62.5 mg/mL和7.812 5 mg/mL;在pH值为2~5时仍具有抑菌效果,经过胃蛋白酶、过氧化物酶、胰蛋白酶、肽酶、蛋白酶K、过氧化氢酶和木瓜蛋白酶处理后抑菌效果极显著（P<0.01）降低;对驼源和牛源金黄色葡萄球菌野生株均具有良好的抑菌效果。[结论]在呼图壁县驼乳中筛选出1株对金黄色葡萄球菌标准菌株以及驼源和牛源金黄色葡萄球菌野生菌株均具有较强抑制作用的鼠李糖乳杆菌,具有较强的耐酸和耐胆盐能力,其抑菌活性物质可能包括酸、过氧化物和蛋白类物质等。研究结果为驼乳源乳酸菌的开发利用提供了潜在菌种资源。

关键词: 驼乳, 乳酸菌, 金黄色葡萄球菌, 抑菌活性

Abstract: [Objective] The aims of the present study were to isolate and identify lactic acid bacteria from camel milk in Hutubi County of Xinjiang Uygur Autonomous Region, and to evaluate their inhibitory effects on Staphylococcus aureus （S. aureus）. [Method] A total of 45 healthy camel milk samples were collected from a camel farm in Hutubi County. Lactic acid bacteria were isolated using MRS medium, and molecular biological identification was carried out using PCR amplification and sequencing targeting 16S rDNA sequence. Point inoculate method, agar spot method and Oxford cup double-layer agar diffusion method were used to screen the lactic acid bacteria with inhibitory effects on S. aureus ATCC 29213. The acid tolerance of the lactic acid bacteria was measured by adjusting pH value of MRS medium to 1-10. The bile salt tolerance of the lactic acid bacteria was assessed by adding 0-0.30% ox bile salt to MRS medium. The minimum inhibitory concentration （MIC） and subinhibitory concentration （SIC） of cell-free culture supernatant （CFCS） of the lactic acid bacteria against S. aureus ATCC 29213 were determined using micro-broth dilution method. Changes in inhibitory effect of CFCS on S. aureus ATCC 29213 under different pH value conditions and after enzymatic treatments were examined to preliminarily analyze the antibacterial active substances of CFCS. The inhibitory effect of CFCS on wild strains of S. aureus isolated from camel milk and dairy cow with mastitis were evaluated by Oxford cup double-layer agar diffusion method. [Result] A total of 20 lactic acid bacterial strains belonging to 5 species were isolated from the 45 camel milk samples, including Enterococcus faecalis （n=13）, Lacticaseibcillus rhamnsus （n=1）, Limosilactobacillus fermentum （n=3）, Lactobacillus paracasei （n=1） and Weissella confusa （n=2）. After screening with the above mentioned three methods, a strain of Lacticaseibcillus rhamnsus （L. rhamnsus, designated as KC4） exhibiting the strongest antibacterial effect on S. aureus ATCC 29213 was obtained. KC4 strain had good acid and bile salt tolerance, and showed normal growth in MRS medium with pH values adjusting to 4-7 and addition of ox bile salt at concentrations of 0-0.20%. The MIC and SIC of KC4 strain CFCS against S. aureus ATCC 29213 were 62.5 mg/mL and 7.812 5 mg/mL, respectively. Its antibacterial activity was still effective at pH values of 2-5, but reduced extremely significantly （P<0.01） after enzymatic treatments with pepsin, peroxidase, trypsin, peptidase, protease K, catalase and papain. It had good antibacterial effect on wild strains of camel and dairy cow derived S. aureus. [Conclusion] One strain of L. rhamnsus was obtained from the camel milk in Hutubi County, which had strong inhibitory effect on standard strain of S. aureus and wild strains of S. aureus isolated from camel milk and dairy cow with mastitis. It also exhibited high level tolerance to acid and bile salt, and the possible antibacterial active substances included acids, peroxides and proteins. The results obtained in this study provided potential bacterial resources for the development and utilization of lactic acid bacteria derived from camel milk.

Key words: camel milk, lactic acid bacteria, Staphylococcus aureus, antibacterial activity

中图分类号:

S852.6
Q93-3

苏帆帆, 毛相清, 赵耀, 邢国锋, 吴自豪, 吴静. 新疆维吾尔自治区呼图壁县驼乳源乳酸菌分离鉴定及其对金黄色葡萄球菌的抑制作用[J]. 畜牧与饲料科学, 2024, 45(2): 1-8.

SU Fanfan, MAO Xiangqing, ZHAO Yao, XING Guofeng, WU Zihao, WU Jing. Isolation and Identification of Lactic Acid Bacteria from Camel Milk in Hutubi County of Xinjiang Uygur Autonomous Region and Their Inhibitory Effects on Staphylococcus aureus[J]. Animal Husbandry and Feed Science, 2024, 45(2): 1-8.

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