动物双歧杆菌乳亚种YYS-J9的筛选、鉴定及体外功效评价

Screening, identification, and in vitro functional evaluation of Bifidobacterium animalis subsp. lactis YYS-J9

中文关键词:  动物双歧杆菌乳亚种  低聚糖  短链脂肪酸  生物膜  人工胃液  人工胰液  疏水性  白假丝酵母

英文关键词:Bifidobacterium animalis subsp. lactis  oligosaccharides  short-chain fatty acids  biofilm  simulated gastric juice  simulated pancreatic juice  hydrophobicity  Candida albicans

中文摘要:

      目的 挖掘出可利用低聚糖为唯一碳源生长、能吸附重金属铅的乳酸菌, 并评估其重金属吸附能力、短链脂肪酸生产水平、胃肠耐受力、黏附力、生物膜形成、疏水性能和抑菌水平。方法 采用革兰氏染色和扫描电镜观察菌形态特征, 生理生化和分子生物学鉴定筛选的YYS-J9菌株, 电感耦合等离子体质谱法(inductively coupled plasma mass spectrometry, ICP-MS)进行铅/镉吸附水平测试, 气相色谱-质谱法(gas chromatography-mass spectrometry GC-MS)分析其短链脂肪酸水平, 主成分分析(principal component analysis PCA)法综合评价YYS-J9利用低聚糖生长和产短链脂肪酸水平, 流式细胞仪检测分析YYS-J9人工模拟胃液、胰液耐受力, 分光光度法检测YYS-J9的生物量、生物膜形成水平、黏附聚集水平和疏水作用力, 牛津杯法检测抑菌水平。结果 从自然发酵乳中分离出一株能以多种低聚糖为唯一碳源的可高效吸附重金属铅/镉的菌株YYS-J9, 经菌落形态观察、生理生化、16S rDNA和GroEL基因鉴定, 确定为动物双歧杆菌乳亚种(Bifidobacterium animalis subsp. lactis) YYS-J9对铅和镉的吸附率高达95.46%和84.62%; 可利用海藻糖、水苏糖、菊粉、低聚半乳糖、低聚异麦芽糖和低聚果糖为唯一碳源进行生长, 可产较高的乙酸、丙酸、丁酸、异丁酸和戊酸, PCA分析获得低聚半乳糖、海藻糖和水苏糖是YYS-J9最适生长和产短链脂肪酸的低聚糖, 并可利用各种低聚糖形成生物膜, 其中以菊粉和低聚半乳糖为碳源时生物膜形成能力最佳, 分别是以葡萄糖为碳源的2.853倍和3.211倍; YYS-J9可在pH为2.0~3.0的人工模拟胃液中处理1~5 h, 存活率不低于83.67%, 并可高达95.81%, 在pH为7.5~8.0人工模拟胰液中处理3~6 h, 存活率范围在75.46%~86.02%之间, 具有良好的胃液胰液耐受力; 其疏水率高达78.424%, 对白假丝酵母具有一定的抑菌作用和黏附能力。结论 从发酵乳中筛选出一株可利用多种低聚糖为唯一碳源生长并产短链脂肪酸的动物双歧杆菌乳亚种YYS-J9, 该菌同时具有显著的铅/镉吸附、生物膜形成、良好的胃液胰液耐受力、疏水性和对白假丝酵母的抑制和黏附特征, 这为便秘、重金属排毒和抑菌产品的开发提供了新的益生菌源。

英文摘要:

      Objective To identify lactic acid bacteria capable of utilizing oligosaccharides as the sole carbon source for growth and adsorbing heavy metal lead, and to evaluate their metal adsorption capacity, short-chain fatty acid production levels, gastrointestinal tolerance, adhesion, biofilm formation, hydrophobicity, and antibacterial activity. Methods Morphological characteristics of the selected strain YYS-J9 were observed using Gram staining and scanning electron microscopy. Physiological, biochemical, and molecular biological methods were used for identification. Inductively coupled plasma mass spectrometry (ICP-MS) was employed to test the lead/cadmium adsorption capacity. Gas chromatography-mass spectrometry (GC-MS) was used to analyze short-chain fatty acid levels. Principal component analysis (PCA) method was conducted to comprehensively evaluate YYS-J9’s ability to utilize oligosaccharides for growth and short-chain fatty acid production. Flow cytometry was used to analyze the tolerance of YYS-J9 to simulated gastric and pancreatic juices. Spectrophotometry was applied to detect biomass, biofilm formation, adhesion and aggregation levels, and hydrophobic interaction of YYS-J9. The oxford cup method was used to detect antibacterial activity. Results A strain, YYS-J9, capable of efficiently adsorbing heavy metals lead and cadmium using various oligosaccharides as the sole carbon source was isolated from naturally fermented milk. Based on colony morphology, physiological and biochemical tests, 16Sr DNA, and GroEL gene sequencing, it was identified as Bifidobacterium animalis subsp. lactis. YYS-J9 exhibited adsorption rates of 95.46% and 84.62% for lead and cadmium, respectively. It could utilize trehalose, stachyose, inulin, galactooligosaccharides, isomaltooligosaccharides, and fructooligosaccharides as the sole carbon source for growth and produced high levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid. PCA analysis revealed that galactooligosaccharides, trehalose, and stachyose were the optimal oligosaccharides for YYS-J9 growth and short-chain fatty acid production, respectively. It could form biofilms using various oligosaccharides, with the best biofilm formation ability observed when using inulin and galactooligosaccharides as carbon sources, which were 2.853 and 3.211 times higher, respectively, than when using glucose as the carbon source. YYS-J9 exhibited good tolerance in simulated gastric juice at pH 2.0–3.0 for 1–5 h, with survival rates not lower than 83.67% and up to 95.81%, and in simulated pancreatic juice at pH 7.5–8.0 for 3–6 h, with survival rates ranging from 75.46% to 86.02%. It had a high hydrophobicity rate of 78.424% and demonstrated antibacterial activity and adhesion capacity against Candida albicans. Conclusion A strain of Bifidobacterium animalis subsp. lactis YYS-J9, capable of utilizing various oligosaccharides as the sole carbon source for growth and producing short-chain fatty acids, is screened from fermented milk. This strain also exhibites significant lead/cadmium adsorption, biofilm formation, good tolerance to simulated gastric and pancreatic juices, hydrophobicity, and inhibitory and adhesive properties against Candida albicans. This provides a new probiotic source for the development of products for constipation relief, heavy metal detoxification, and antibacterial applications.