1株植物乳杆菌的分离鉴定、生物学特性和安全性研究

doi:10.16431/j.cnki.1671-7236.2025.07.009

摘要/Abstract

摘要： 【目的】本研究旨在分离筛选1株鸭源益生菌，探究其生物学特性，选育应用于肉鸭生产的益生菌株，为开发新型益生菌制剂提供参考依据。【方法】试验从鸭盲肠内容物中分离纯化了1株细菌，通过钙溶圈法、革兰染色镜检、生化试验和16S rDNA检测对该菌进行了分离鉴定，测定其生长特性、耐酸性和胆盐耐受性，游离氨基酸产量和有机酸产物含量，检测其对大肠杆菌、鸭疫里默氏杆菌、沙门菌、金黄色葡萄球菌、猪链球菌和副鸡嗜血杆菌的抑制率和凋亡率，电镜法观察抑菌形态，超滤法分离鸭源植物乳杆菌胞外蛋白，液相色谱串联质谱分析其中的抑菌成分，并评估对肉鸭使用的安全性。【结果】分离得到1株革兰阳性杆状的无芽孢杆菌，通过生化鉴定试验确定其为乳杆菌属，再经16S rDNA检测确定其为植物乳杆菌。该株鸭源植物乳杆菌与泡菜源植物乳杆菌相比，能够快速进入对数生长期和平缓期，具有较好的耐酸和耐胆盐特性；可产生较多游离氨基酸、乳酸和乙酸(P＜0.05)；在1∶1添加比例下对大肠杆菌和副鸡嗜血杆菌有显著抑制效果(P＜0.05)；在1∶4添加比例下对大肠杆菌、鸭疫里默氏杆菌和沙门菌的生长均有显著抑制作用(P＜0.05)，在1∶9添加比例下对大肠杆菌和鸭疫里默氏杆菌有显著抑制作用(P＜0.05)，1∶4添加比例下，大肠杆菌、鸭疫里默氏杆菌、猪链球菌和副鸡嗜血杆菌的凋亡率显著升高(P＜0.05)。经液相色谱串联质谱分析，共检测出10种蛋白，其中2种蛋白与抑菌作用相关，分别为肽聚糖结合蛋白和乙酸激酶；对肉鸭安全性试验表明，该菌对肉鸭安全、无毒副作用。【结论】本试验分离筛选出了1株鸭源植物乳杆菌，其具有良好的生长特性、耐酸性、胆盐耐受性、产游离氨基酸和有机酸特性以及较好的抑菌效果，其抗菌机制可能通过抑菌蛋白及有机酸协同发挥作用，破坏细胞壁和细胞膜导致菌体死亡，对肉鸭安全、无毒副作用，具备作为益生菌饲料添加剂的潜力。

关键词: 植物乳杆菌; 分离鉴定; 生物学特性; 安全性; 肉鸭

Abstract: 【Objective】 This study aimed to isolate and screen a duck-origin probiotic strain,investigate its biological characteristics,and select candidate strains for meat duck production,providing reference for developing novel probiotic preparations. 【Method】 Bacterial strain was isolated and purified from duck cecal contents.Identification was performed through calcium dissolution zone assay,Gram staining,biochemical tests,and 16S rDNA sequencing.Growth characteristics,acid tolerance,bile salt tolerance,free amino acid production,and organic acid content were determined.Antimicrobial efficacy against Escherichia coli,Riemerella anatipestifer,Salmonella spp.,Staphylococcus aureus,Streptococcus suis,and Avibacterium paragallinarum were evaluated through inhibition rate and apoptosis rate assays.Scanning electron microscopy characterized antibacterial morphology.Duck-derived Lactiplantibacillus plantarum extracellular proteins were isolated by ultrafiltration,with antibacterial components analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Safety assessment was conducted in meat ducks. 【Result】 A Gram-positive,non-spore-forming bacillus was isolated.and it was determined to be Lactobacillus genus through biochemical identification tests.Then,it was confirmed to be Lactiplantibacillus plantarum through 16S rDNA detection.Compared with Lactiplantibacillus plantarum from pickle (pickle-LP),this Lactiplantibacillus plantarum from duck (duck-LP) could quickly enter the logarithmic phase and plateau of growth,and had a better acid and bile salt resistance.It could produce more free amino acids,lactic acid,and acetic acid (P＜0.05).It had a significant inhibitory effect on Escherichia coli and Avibacterium paragallinarum at a 1∶1 addition ratio (P＜0.05).At a 1∶4 addition ratio,there was a significant inhibitory effect on the growth of Escherichia coli,Riemerella anatipestifer,and Salmonella spp.(P＜0.05).At a 1∶9 addition ratio,there was a significant inhibitory effect on Escherichia coli and Riemerella anatipestifer (P＜0.05).At a 1∶4 addition ratio,the apoptosis rates of Escherichia coli,Riemerella anatipestifer, Streptococcus suis,and Avibacterium paragallinarum were significantly increased (P＜0.05). Through LC-MS/MS,a total of 10 proteins were detected,including two antimicrobial components:Peptidoglycan-binding protein and acetate kinase.The safety test on meat ducks showed that the bacterium was safe and had no toxic side effects on meat ducks. 【Conclusion】 This experiment isolated and screened a strain of duck-LP,which had excellent growth characteristics,acid/bile tolerance,amino acid/organic acid production,and broad-spectrum antimicrobial activity.Its antibacterial mechanism might be through synergistic effects of antimicrobial proteins and organic acids,disrupting cell wall/membrane integrity.The strain showed potential as a safe probiotic feed additive for meat ducks.

Key words: Lactiplantibacillus plantarum; isolation and identification; biological characteristics; safety; meat ducks

中图分类号:

S852.61

代林, 怀志芳, 王宝维, 张名爱, 凡文磊, 张晶, 王秉翰, 齐国峰. 1株植物乳杆菌的分离鉴定、生物学特性和安全性研究[J]. 中国畜牧兽医, 2025, 52(7): 3070-3083.

DAI Lin, HUAI Zhifang, WANG Baowei, ZHANG Ming'ai, FAN Wenlei, ZHANG Jing, WANG Binghan, QI Guofeng. Isolation, Identification and Biological Properties and Safety of A Lactiplantibacillus plantarum Strain[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3070-3083.

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