微生物发酵替代肉制品中亚硝酸盐的呈色和抑菌作用及机理研究

Nitrite is a widely used ingredient in meat products. However, its use is limited due to its potential carcinogenicity. The objective of this project is to evaluate microbial fermentation as an alternative to nitrite addition during manufacturing of fermented meat products, and also, to discuss its mechanism. The metmyoglobin, a pigment protein extrated from fresh meat, is added into culture medium to formulate a meat model system, where the "reddening" microorganisms are selected. The red pigment, as a derivative of myoglobin, is traced during conversion process and identified by four kinds of spectra analysis. The determination of enzyme activity and 2D-PAGE (two direction-polyacrylamide gel electrophoresis) of the bacteria proteome are used as the tools to illuminate the mechanism of the conversion in a view of metabolism and comparative proteomics. Also, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is employed to investigate the antimicrobiologic characteristic of the "reddening" bacteria, as well as the other strains commercially used in meat industry. The strains chosen will be inoculated into meat to evaluate the antioxidant activity and the contribution to flavor, compared with the nitrite-cured meat. The strains chosen are combined as a mixed starter culture, with which fermented dried sausage would be produced without nitrite addition. This would be a potential way for nitrite substitute and play theoretical basis for novel fermented meat products.

亚硝酸盐是肉类腌制的关键组分，但由于其潜在的致癌性，其应用受到限制。本项目应用微生物发酵法替代肉体系中亚硝酸盐的多重作用，并对其机理进行阐述。通过向培养基中添加肌肉色素，模拟肉基质环境，筛选出具有"呈色"功能的微生物菌株，并利用四种光谱技术对所得红色衍生物进行鉴定与转化过程跟踪；采用酶活测定和双向电泳手段，从菌体代谢和比较蛋白质组学角度阐明其转化机理。同时利用聚合酶链式反应-变性梯度凝胶电泳（PCR-DGGE）技术，进一步研究考察"呈色"菌株和其它菌株作为肉品生物保护菌的可能性和对某些腐败菌和特定致病菌的抑制作用，阐明其抑菌机理。对所得菌株接种发酵过程中肉制品的蛋白和脂肪氧化指标和风味物质成分进行分析测定，探讨其抗氧化能力和风味形成情况；最终对筛选所得功能菌株进行复配，制成复合发酵剂，开发无硝或低硝发酵肉制品。该研究成果将丰富亚硝酸盐替代途径，为发酵肉制品工艺改进提供理论依据。

亚硝酸盐在肉类腌制中具有呈色和抑菌等重要作用，但由于其潜在的致癌性，应用受到限制。本项目采用微生物发酵替代肉中亚硝酸盐的呈色和抑菌作用，并对其机理进行了阐述。通过向培养基中添加肌肉色素-高铁肌红蛋白（Met-Mb）模拟肉基质环境，筛选出4株具有“呈色”功能的微生物菌株：弯曲乳杆菌、发酵乳杆菌、植物乳杆菌和木糖葡萄球菌。通过紫外/可见光谱和电子自选共振光谱对肌红蛋白红色衍生物进行扫描分析，表明木糖葡萄球菌和发酵乳杆菌可转化Met-Mb形成了六配位的亚硝基肌红蛋白，而弯曲乳杆菌和植物乳杆菌生成了氧合肌红蛋白。对细菌转化Met-Mb过程追踪发现，该转化速度与原始接种细菌数和生长速度密切相关，并且菌株的一氧化氮合酶促进了肉中亚硝基肌红蛋白的形成。在模拟体系中考察受试菌株对产气荚膜梭菌的抑制效果，表明不同乳酸菌对产气荚膜梭菌及芽孢均有一定抑制作用，其中植物乳杆菌作用最显著，且具有杀死活细胞的作用。发酵乳杆菌、植物乳杆菌和戊糖片球菌培养48h后形成的条件培养基，可以抑制其芽孢形成。乳酸菌发酵降低pH值和植物乳杆菌产生的耐热细菌素是其抑制产气荚膜梭菌的主要原因。在肉糜体系中考察了筛选菌株的呈色和抑菌效果，表明木糖葡萄球菌可在肉体系中转化Met-Mb生成亚硝基肌红蛋白，适度增加木糖葡萄球菌接种量，可提高肉糜红度值。同时筛选得到两株细菌，清酒乳杆菌和木糖葡萄球菌，可作为“生物保护菌”应用到肉制品中，抑制其中腐败菌的生长。最后将木糖葡萄球菌、清酒乳杆菌和植物乳杆菌应用到发酵风干肠中，评价其作为发酵剂对风干肠颜色、质构、理化特性以及安全性的影响，结果表明接种含有木糖葡萄球菌的风干肠红度值显著高于对照组，且与亚硝酸钠腌制组无显著差异，接种发酵抑制了产气荚膜梭菌的生长，加速了优势菌群的形成，对风干肠品质特性有改善作用。该研究成果表明微生物发酵可以部分替代亚硝酸盐在肉制品中的发色和抑菌作用，为生产低硝肉制品及发酵肉制品工艺改进提了供理论依据。