基于生物质谱和氧-18标记技术定量食品体系中食用明胶的方法学研究

At present, methodological study on traceability of edible gelatin becomes a research hotspot at home and abroad. However, few reports have been undertaken towards quantitative methodology of edible gelatin in food system. Previous research showed that biomass spectrometry and oxygen-18 labeling were able to quantify pure bovine and porcine gelatins. Whether this method is proper to quantify edible gelatin in complex food system, deserves to be explored further. In this project, bovine-hide, porcine-hide, and donkey-hide gelatins will be used as the materials. The difference of amino acid sequences will be analyzed by the sequence alignment software. And then, the database of theoretical characteristic peptides in gelatins will be established. Using single system (pure gelatin) and mixed system (gelatin mixture) as study objects, the rules of similar peptides disturbance and concentration dilution affecting the gelatin identification will be especially researched. The effects of system complexity and non-targeted peptides on gelatin quantification will be explored seriously. Under Selected Ion Monitoring (SIM) mode, the relationships between m/z setting range and key assessment indexes for quantitative results including detection limit will be established. Further using simulative system and food system (donkey-hide gelatin product) as the models, the effects of food matrixes including rock candy on gelatin quantitative results will be studied. Feasibility analysis will be done to examine the quantitative method, and then proper correction will be executed. Finally, based on biomass spectrometry and oxygen-18 labeling, a new method will be established to accurately quantify edible gelatin in food system. This project will innovate the gelatin traceability from qualitative diagnosis to quantitative determination, and enrich the methods and theories of edible gelatin traceability.

食用明胶的溯源方法研究是目前国内外的研究热点，然而，食品体系中食用明胶的定量方法学研究却鲜有报道。前期研究表明，联合生物质谱和氧-18标记技术能定量纯牛/猪皮明胶，但是否适用于复杂的食品体系？本项目拟选取牛皮、猪皮和驴皮明胶等为研究对象，利用蛋白质多序列比对软件分析氨基酸序列差异，建立明胶的理论特征性多肽数据库；以单一体系（纯明胶）和混合体系（明胶混合物）为对象，研究明胶鉴定过程中的相似多肽干扰效应和浓度稀释理论，探明体系复杂度和非目标多肽对明胶定量效果的影响，建立SIM模式下质荷比采谱区间与检出限等定量关键评价指标的相关性；进一步以模拟体系和食品体系（阿胶片）为模型，探究食品基质（冰糖等）对明胶定量效果的影响，检验定量方法的可行性，并对其进行修正，建立一种基于生物质谱和氧-18标记技术的准确定量食品体系中食用明胶的新方法。以实现明胶溯源从定性向定量的突破，完善食用明胶的溯源方法和理论。

建立食用明胶的溯源方法是目前该领域基础研究的难点和热点。在明胶定量方面，国内外仍处于起步阶段，亟需探寻一种准确定量食品体系中食用明胶的方法。前期研究表明，联合生物质谱和氧-18标记技术能定量纯牛/猪皮明胶，但是否能用于复杂体系中食用明胶的定量还有待探究。因此，本项目以牛皮、猪皮和驴皮明胶为研究对象，利用蛋白质多序列比对软件分析氨基酸序列差异，建立明胶的理论特征性多肽数据库；以纯明胶、明胶混合物等为对象，研究明胶鉴定过程中的相似多肽干扰效应和浓度稀释理论，建立一种基于生物质谱和氧-18标记技术的准确定量食品体系中食用明胶的新方法。此外，本项目进一步研究了无标记状态下生物质谱定量食用明胶的方法，开展了混合体系中明胶种类的溯源性研究。.研究结果表明，阿胶、牛皮明胶和猪皮明胶的理论特征性多肽数分别为55，49和50，虽然胶原蛋白α1链的氨基酸残基数比α2链多，但是三种明胶的α2链氨基酸序列差异性比α1链大；在纯明胶鉴定中，阿胶、牛皮明胶和猪皮明胶中可检测特征性多肽数目分别为14、28和27；在三种明胶的混合物中，当目标明胶含量低于10%，阿胶、牛皮明胶和猪皮明胶可检测特征性多肽数目分别为5、11和15；联合质谱和氧-18标记技术建立了牛明胶、猪明胶和阿胶的定量方法，准确度高且具有良好的特异性、灵敏度和重现性，动态范围为0.2-20（氧-18/氧-16浓度比）；当阿胶与牛皮明胶以1:10、1:1和10:1混合时，定量检测结果与实际值十分接近，表明该定量法可用于测定明胶体系中目标明胶的添加量；无标记状态下生物质谱定量食用明胶的方法具有良好的线性、特异性和重现性，可用于定量食品体系中的明胶；在阿胶块、阿胶口服液等体系中，鉴定了阿胶、牛明胶和猪明胶的溯源特征性多肽。.本项目的研究工作可以完善食用明胶的溯源基础理论，实现明胶溯源从定性向定量的突破，对促进食用明胶的规范应用具有重要的意义。