基于微纳流控液滴技术高通量激活QS调控IIa类乳酸菌细菌素Enterocin Y31合成的研究

Class IIa lactic acid bacteria bacteriocins have strong antibacterial activity against Listeria monocytogenes, which is a common pathogen in food. This class of bacteriocins has potential superiority as natural biopreservatives because of their unique characteristics of stable performance, safety and high efficiency. In our previous work, Enterococcus faecium Y31 producing a class IIa bacteriocin Enterocin Y31 with a broad spectrum antibacterial activity had been obtained from Chinese traditional fermentation paocai, but we found that the biosynthesis amount of Enterocin Y31 was very low so that it couldn’t reach the requirement of the industrial production. We had confirmed that biosynthesis of Enterocin Y31 was regulated by QS path. Traditional QS activition method need to cultivate a large number of strains producing bacteriocin in order to achieve the suitable threshold density, QS isn't able to be high throughput activated . In the present study, biosynthesis and regulation of Enterocin Y31 will be proceeded using confinement induced QS path, the model of the droplet -nanofluidic confinement induced QS path will be established, the hypothesis of confinement induced QS path will be verified, then biosynthesis of Enterocin Y31 will be regulated by establishing microfluidic and nanofluidic droplet cultivation model. The project will provide the theoretical guidance and technical support for biosythesising and regulating bacteriocin by confinement induced QS path hypothesis , and establish the basic for dissolving the food industrial bottleneck using microfluidic and nanofluidic technology.

IIa类乳酸菌细菌素对食品中常见的致病菌如单增李斯特氏菌有强烈的抑菌活性，性能稳定、安全高效，是潜在优良的天然防腐剂。前期申请人已在中国传统发酵酸菜中挖掘出一株产广谱抑菌活性IIa类细菌素Enterocin Y31的Enterococcus faecium Y31，研究过程中发现Enterocin Y31生物合成量极低，难以进行大规模工业化生产。前期实验确定Enterocin Y31的生物合成受QS通路调控，传统激活QS通路策略需培养大量的产细菌素菌株使其达到相应阈值密度，无法高通量激活QS。本项目拟通过限制诱导QS假说调控Enterocin Y31的生物合成，通过构建液滴-纳流控限制诱导QS通路模型，验证限制诱导QS通路假说，建立微纳流控液滴培养模型调控Enterocin Y31的合成。本项目将为限制诱导QS调控细菌素合成提供理论指导及技术支持，为微纳流控在食品领域的应用奠定基础。

IIa类乳酸菌细菌素对食品中常见的致病菌如单增李斯特氏菌有强烈的抑菌活性，性能稳定、安全高效，是潜在优良的天然防腐剂。前期申请人已在中国传统发酵酸菜中挖掘出一株产广谱抑菌活性IIa类细菌素Enterocin Y31的Enterococcus faecium Y31，研究过程中发现Enterocin Y31生物合成量极低，难以进行大规模工业化生产。前期实验确定Enterocin Y31的生物合成受QS通路调控，传统激活QS通路策略需培养大量的产细菌素菌株使其达到相应阈值密度，无法高通量激活QS。本项目拟通过限制诱导QS假说调控Enterocin Y31的生物合成，通过构建液滴-纳流控限制诱导QS通路模型，验证限制诱导QS通路假说。取得了以下成果：建立微纳流控液滴培养模型调控Enterocin Y31的合成。通过将包含 pAmilux 的 luxABCD 盒插入 1765bp NotI 酶切的 pJBA25 碎片中，成功构建了 pMH391 翻译融合载体；然后采用两步克隆法成功构建 entA 的 N 端部分和不稳定 luxABCD 基因突变株的翻译融合，将含有 pMHLAS 监控质粒转入到含有 QS 表达系统的 E.faecium Y31等步骤，成功建立自诱导肽合成基因簇entF基础的QS监控系统；最后将含有 QS 表达系统和监控质粒的 E.faecium Y31 与自诱导因子 IP-Y31 一起培养，通过检测荧光密度的大小确定合适的诱导因子 IP-Y31 添加浓度激活 QS，并利用琼脂扩散法和考马斯亮蓝法检测 Enterocin Y31 的生物合成量，同时利用常规方法验证此系统的专一性和敏感性；完成了NBD绿色光学标签的脂质体，通过制备二氧化硅原液，并加入荧光标签（NBD) liquid, 制备NBD绿色光学标签的脂质体。经过荧光显微镜检测，此脂质体发出的荧光密度符合作为标签的标准，达到了试验要求。本项目将为限制诱导QS调控细菌素合成提供理论指导及技术支持，为微纳流控在食品领域的应用奠定基础。