基于MLST的婴幼儿配方乳粉中双歧杆菌溯源及功能相关性研究

Infant formula milk (IFM) powder is among the food products that are under most strict govermental regulation. Addition of bifidobacterial probiotics is a new trend in IFM powder industry. Since the probiotic effect is highly strain-specific, improper usage may cause harm to the health of infants. Therefore, precise tracing of the origin of strains is particularly important for probiotic function and product quality. Traditional biochemical and molecular typing techniques' resolving power is limited to the species and subspecies level. This project aims at a solution based on bifidobacterium multilocus sequence typing (MLST) data. Genes representing the evolutionary diversity of bifidobacteria will be identified using a combination of bioinformatics and experimental methods. High resolution SNP sites and their combinations in the genes will be predicted and be used to establish a Minim Typing model of bifidobacteria. This model will be used as the theoretical basis of stain-level accurately tracing to assist the development of rapid genotyping approaches based high resolution melting (HRM) techniques, which in turn will further enable the tracing of consistency and variability of bifidobacterium strains in IFM powder products collected from the market, the establishment of molecular traceability database, and the exploration of the effects of mutations on the biochemical and probiotic function of these strains. This study will provide theoretical basis for the establishment of the relevant testing standards, as well as scientific basis for risk detection, monitoring and safety supervision for the infant food.

婴幼儿配方乳粉是目前政府监管最严格的食品之一，添加以双歧杆菌为主的益生菌是其发展趋势。益生菌作用具有菌株特异性，使用不当会对婴幼儿健康产生危害，菌株的精确溯源对于益生菌发挥功能、确保产品品质尤为重要。但传统生化和普通分子分型技术仅能鉴定到种和亚种水平。本课题拟在双歧杆菌多位点序列分型（MLST）数据基础上，综合运用生物信息学和实验手段，寻找代表双歧杆菌进化多样性的基因；预测高分辨力单核苷酸多态性位点及组合，建立基于MLST的分型模型，作为双歧杆菌菌株水平精确溯源的理论基础；进一步拓展高分辨融解曲线技术进行快速分子分型检测；对市场婴幼儿配方乳粉来源菌株进行溯源，了解其与卫生部规定使用菌株的相符性及变异性，建立双歧杆菌分子溯源数据库；深入探索基因变异对益生菌生理生化和功能的影响。该研究将为建立相关检测标准提供理论基础，为婴幼儿食品的风险发现、监测和安全监管提供科学依据。

婴幼儿配方乳粉是目前政府监管最严格的食品之一，添加以双歧杆菌为主的益生菌是其发展趋势。益生菌作用具有菌株特异性，使用不当会对婴幼儿健康产生危害，菌株的精确溯源对于益生菌发挥功能、确保产品品质尤为重要。但传统生化和普通分子分型技术仅能鉴定到种和亚种水平。.本项目筛选获得代表双歧杆菌进化多样性的候选管家基因共7个，分析获得高分辨力SNP位点9个，D值均大于0.95，6种组合方式，建立双歧杆菌微小分型（Minim Typing）模型。开发建立针对模型中SNP组合的高分辨熔解曲线分析（HRM）检测技术，其中gyrB、rpo位点HRM方法能将长双歧杆菌、短双歧杆菌、两歧双歧杆菌、青春双歧杆菌、动物双歧杆菌进行种间区分。对于常用的动物双歧杆菌乳双歧亚种商业菌株，通过深入的全基因组比对，选择4个SNP位点用于乳双歧亚种菌株的精确鉴定，可将国际常用乳双歧杆菌商业菌株进行区分。 .本研究对国内流通领域主要益生菌婴幼儿配方乳粉品牌进行调研分析。共收集国内外益生菌配方乳粉共34个品牌，47个样品，分离菌株62个。综合多种方法，结合建立的HRM方法，将配方乳粉中常添加的3种商业菌株Bb-12，Bi-07，HN019精确区分，发现分离菌株与标签不相符的违规情况。同时建立了我国益生菌配方乳粉中分离菌株溯源数据库。API ZYM、API50CH、耐受人工胃液实验、药敏试验、抑菌实验结果显示样品菌株反应特征与双歧杆菌典型反应基本相符。个别从产品中分离商业菌株耐酸能力有所降低；不同菌株抗菌性差异较大。.本研究还使用多位点分析方法对7个管家基因进行研究，从系统发育的角度讨论利用多基因串联的方式对双歧杆菌属建立稳定可靠的分类方法的可行性。系统研究益生菌配方乳粉开封后保存条件对存活率的影响。发现储存14天和28天，37°C放置的存活率显著低于储存在4°C和25°C。益生菌配方乳粉中活菌存活率与储存温度和时间呈负相关，品牌和配方对存活率影响较小。为企业科学合理制定货架期、规范贮存条件及提示消费者正确消费行为提供科学借鉴。