免疫磁性微球偶联特异细菌素捕获-杀灭果汁中脂环酸芽孢杆菌理论方法

Alicyclobacillus spp. have become one of the major "green technical barriers" set by developed countries to Chinese juice products along with the international trade. To establish the rapid and accurate theory and methods for the identification, capture, inactivation and inhibition of Alicyclobacillus spp. in apple juice products has become a focus of investigation in beverage safety. On the basis of previous successful preparation of immuno-magnetic microspheres and the discrimination, discovery and acquisition of bacterocins capable of effective inactivation of Alicyclobacillus spp, the molecular mechanism underlying the function will be further revealed. In this program, it will be established by a novel low temperature approach to capture-inactivate Alicyclobacillus spp in apple juice by coupling specific bacterocin with immune-magnetic microspheres. The systematical revelation of action site, binding type and its molecular mechanism are assisted by structural analysis of specific antibodies and bacteriocins, as well as chemical composition analysis of functional magnetic microspheres. The validity, specificity and safety of immunocapture-control system for Alicyclobacillus spp. are comfirmed, and the most suitable function range and methods are optimized. Finally, the theoretical basis and methodology of systems for rapid recognition and control of Alicyclobacillus spp. in fruit juice samples will be established. This work is believed to be of both academic interest and practical importance for monitoring and controlling Alicyclobacillus spp. in fruit juice and other beverage industry.

脂环酸芽孢杆菌已成为发达国家对中国果汁产品设置“绿色安全壁垒”的主要指标之一。建立一种快速有效的识别、捕获、杀灭、控制理论与方法成为产业急需。本项目在已获得脂环酸芽孢杆菌免疫磁性微球基础上，甄别、发现、获得有效抑制杀灭脂环酸芽孢杆菌的特异性细菌素，揭示抑制杀灭脂环酸芽孢杆菌的分子机制；通过甄别发现特异性细菌素偶联免疫磁性微球，获得可以捕获、杀灭脂环酸芽孢杆菌的免疫磁性微球-特异性细菌素偶联体，建立果汁中脂环酸芽孢杆菌的低温免疫捕获-杀灭新理论方法；通过脂环酸芽孢杆菌多克隆抗体、特异性细菌素结构分析及功能化磁性材料化学构成解析与表征，系统揭示免疫磁性微球与特异性细菌素耦合体对脂环酸芽孢杆菌捕获-杀灭作用部位、化学位点、结合方式，建立免疫捕获-杀灭分子机制；确证与评价免疫捕获-抑制杀灭的有效性、特异性、安全性及最佳作用方式，系统构建免疫捕获-抑制杀灭果汁中脂环酸芽孢杆理论方法。

脂环酸芽孢杆菌作为果汁生产加工中关键的质量安全指标，已严重制约了我国水果生产及果汁加工产业的快速、可持续国际化发展，也成为发达国家为果汁产品设置“绿色安全壁垒”的主要依据之一。本项目通过甄别筛选获得了有效抑制杀灭脂环酸芽孢杆菌的乳酸菌细菌素；将细菌素偶联免疫磁性微球，获得可以捕获-杀灭脂环酸芽孢杆菌的免疫磁性微球-特异性细菌素耦合体；通过多种表征方法，系统揭示耦合体对脂环酸芽孢杆菌捕获-杀灭作用部位、化学位点、结合方式；通过转录组分析揭示了耦合体对脂环酸芽孢杆菌的具体杀灭机制；确证与评价了免疫捕获-抑制杀灭的有效性、特异性、安全性及最佳作用方式，系统构建免疫捕获-抑制杀灭果汁中脂环酸芽孢杆菌理论方法。主要取得以下成果：.1、从20株实验室保藏的乳酸菌中筛选出三株具有抗嗜酸耐热菌活性菌株L. paracasei CICC 20241、L. rhamnosu CICC 20975和 B. animalis subsp. animalis CICC 6165，从三株活性乳酸菌发酵液中分离获得了三种能有效抑杀嗜酸耐热菌的细菌素bificin c6165、paracin C和bacteriocin RC20975。.2、成功制备出IONPs @ pDA - nisin磁性纳米偶联体材料，对脂环酸芽孢杆菌DSM 3922具有很好的抑菌效果，试验优化了IONPs @ pDA - nisin磁性纳米材料的制备条件：nisin : IONPs @ pDA1 : 1，反应体系的pH在4.0-11.0，反应时间30 min。.3、通过转录组测序分析，耦合体的抑杀机制是通过细胞膜，导致转录调节和能量代谢发生改变，细胞膜表面开始产生芽孢，细胞壁生物合成被抑制，细胞膜转运功能紊乱，细胞的运动性和芽孢萌发被抑制，最终细胞内容物泄漏，导致菌体细胞死亡。.4、测定了IONPs @ pDA - nisin耦合体的最小杀菌浓度，系统建立了免疫捕获-抑制杀灭果汁中脂环酸芽孢杆理论方法。