籽鹅肠道中非解乳糖链球菌的分离鉴定及益生特性研究

doi:10.16431/j.cnki.1671-7236.2024.10.038

摘要/Abstract

摘要： 【目的】从籽鹅肠道中分离筛选用于改善籽鹅健康状况的潜在益生菌株，并研究其益生特性。【方法】采集健康籽鹅不同肠段内容物，梯度稀释后涂布于含碳酸钙的MRS(CaCO₃-MRS)固体培养基上，经厌氧培养后，通过形态学、生化试验、16S rRNA基因PCR扩增对分离菌株进行鉴定，采用耐酸试验和抑菌试验对菌株益生特性进行筛选，评价其抑菌能力、抗生素敏感性、耐酸和耐胆盐能力、胃肠液耐受能力，并测定其生长曲线和产酸曲线。【结果】从健康籽鹅肠道中分离得到4株疑似乳酸菌，命名为RS-1、RS-2、RS-3和RS-4，其中RS-3菌株具有良好的益生特性。RS-3菌株在CaCO₃-MRS培养基上的菌落形态为表面光滑、淡黄色、产生明显溶钙圈的圆形菌落，为革兰阳性杆菌。生化试验结果显示，分离菌株均可发酵麦芽糖、纤维二糖、蔗糖、棉子糖、菊糖和乳糖，也可发酵甘露醇、山梨醇和水杨苷，且七叶苷试验和1%马尿酸钠试验均为阳性，初步判定分离菌株均为乳酸菌。经16S rRNA基因序列分析鉴定，4株分离菌均为非解乳糖链球菌(Streptococcus alactolyticus)，其中RS-3菌株耐受性优于其他3株分离菌。RS-3菌株发酵液对金黄色葡萄球菌有较好的抑制效果，抑菌圈直径达10 mm以上，对卡那霉素、阿米卡星、罗红霉素、磺胺嘧啶、林可霉素、多黏菌素B和诺氟沙星耐药。RS-3菌株具有酸、胆盐及人工胃液和肠液的耐受性，灌胃小鼠后其生长状况良好。【结论】试验分离得到4株籽鹅肠源型非解乳糖链球菌，其中RS-3菌株具有较好的抑菌、耐酸、耐胆盐益生特性，可作为后续开发鹅用益生菌制剂的一种可靠的菌株来源。

关键词: 籽鹅; 非解乳糖链球菌; 肠道; 益生菌; 微生态

Abstract: 【Objective】 The aim of this study was to isolate and screen potential probiotic strain from the intestinal tract of Zi geese to improve their health status，and analyze its probiotic properties. 【Method】 The contents of different intestinal tract of healthy Zi geese were collected，diluted in gradient and coated on (CaCO₃-MRS) solid medium containing calcium carbonate. After anaerobiccultivation，the isolates were identified through morphology，biochemical tests，16S rRNA gene PCR amplification，respectively. The probiotic properties of the isolates were screened by acid resistance and antibacterial tests. The antibacterial ability，antibiotic sensitivity，acid and bile salt tolerance，gastric and intestinal fluid tolerance were evaluated，and the growth curve and acid production curve were measured. 【Result】 4 suspected lactic acid bacteria strains were isolated from the intestinal tract of healthy Zi geese，named RS-1，RS-2，RS-3 and RS-4，of which RS-3 had good probiotic properties. The colony morphology of RS-3 in CaCO₃-MRS medium was smooth，light yellow and round colony with obvious calcium-soluble ring，Gram-positive Bacillus. The results of biochemical tests showed that the isolates could ferment maltose，cellobiose，sucrose，raffinose，inulin and lactose，as well as mannitol，sorbitol and salicin. The results of esculin test and 1% sodium hippurate test were positive，and the isolates were initially determined to be lactic acid bacteria. 16S rRNA gene sequence analysis showed that 4 isolates were all Streptococcus alactolyticus，and the tolerance of RS-3 was better than that of the other three strains. The fermentation broth of RS-3 had a good inhibitory effect on Staphylococcus aureus. The diameter of inhibitory zone was more than 10 mm，and it was resistant to kanamycin，amicacin，roxithromycin，sulfadiazine，lincomycin，polycolistin B and norfloxacin. Moreover，RS-3 had tolerance to acid，bile salts artificial gastric and intestinal fluids. The mice grew well after administration of RS-3. 【Conclusion】 4 isolates enterogenic Streptococcus alactolyticus of Zi geese were isolated in this experiment，of which RS-3 had good antibacterial and probiotic properties with acid and bile salt tolerance，and could be used as a reliable strain source for the subsequent development of probiotic preparations for geese.

Key words: Zi geese; Streptococcus alactolyticus; intestinal tract; probiotics; microecology

中图分类号:

S852.61⁺1

张红, 田秋丰, 邹跃, 尹珺伊, 白长胜, 黄宇翔, 王志强, 马志刚, 霍明东, 董佳强, 杨昊天, 杨坤, 苗艳, 陈志峰. 籽鹅肠道中非解乳糖链球菌的分离鉴定及益生特性研究[J]. 中国畜牧兽医, 2024, 51(10): 4578-4586.

ZHANG Hong, TIAN Qiufeng, ZOU Yue, YIN Junyi, BAI Changsheng, HUANG Yuxiang, WANG Zhiqiang, MA Zhigang, HUO Mingdong, DONGJiaqiang, YANG Haotian, YANG Kun, MIAO Yan, CHEN Zhifeng. Isolation，Identification and Probiotic Characteristics of Streptococcus alactolyticus from Intestinal Tract of Zi Geese[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4578-4586.

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