乳蛋白遗传多态性及翻译后修饰水平对牛乳凝胶特性影响机制研究

Dairy enterprises often encounter the phenomenon of “non-antibiotic residue” of raw milk noncoagulation or poor coagulation, which causing the rejection of dairy farmers' raw milk. If used in fermented milk production, it will lead to poor water-binding capacity of yogurt, serious separate out of whey, and lower yield of cheese. Large losses have been caused to enterprises. Milk coagulation properties(MCP) is highly correlated with milk protein genetic polymorphisms and posttranslational modification. However, this scientific problems are still lack of enough study and the mechanism has not been elucidated clearly. The objectives of this study will be to use proteomics techniques to detect genetic polymorphism of milk protein in raw milk with different MCP. Identification of significant differences milk protein variants, and establishment of a relationship between milk protein variants and the characteristics of milk coagulation. To establishment of screening markers of protein genotypes by high-throughput sequencing for screening out of noncoagulation or poor coagulation dairy cows. To study the types and sites of milk protein phosphorylation and glycosylation, and to reveal the mechanism of milk protein posttranslational modification on the MCP. Furthermore, rheological methods and confocal laser spectroscopy will be used to analysis the differences in rheological properties and microstructure of different milk proteins gels in order to ascertain the relationship between the milk coagulation properties and gel microstructures. The research results would be helpful and meaningful for dairy enterprises to make use of raw milk rationally and improve the quality of fermented milk based on MCP.

乳品企业在原料奶验收中经常会遇到“非抗生素残留”的不凝乳或弱凝乳现象，导致奶农原料乳被拒收，若用于生产会导致酸奶保水力差、乳清析出严重、干酪得率降低，给企业造成较大损失。据报道凝乳不良可能与乳蛋白遗传多态性及其翻译后修饰水平等因素相关，但尚缺乏系统深入研究，机制尚不清楚。本项目拟在前期对凝乳特性调查基础上，以凝乳差异显著的原料乳为研究对象，采用蛋白组学技术开展乳蛋白质遗传多态性对凝乳特性的影响规律，鉴定出差异显著的乳蛋白变异体，建立乳蛋白变异体与凝乳特性之间的关联关系；利用高通量测序建立不凝乳蛋白基因型筛选标记，用于不凝乳奶牛筛查；考察乳蛋白磷酸化及糖基化种类及位点，揭示乳蛋白翻译后修饰对凝乳品质的影响机制；采用流变学及激光共聚焦显微镜等方法探明乳蛋白凝胶流变学特性及微观结构差异，解析凝胶特性与其微观结构的内在联系。该研究对乳品企业合理利用原料乳，改善发酵乳品质，具有重要的理论和应用价值。

乳品企业在原料奶验收中经常会遇到“非抗生素残留”的不凝乳或弱凝乳现象，导致奶农原料乳被拒收，若用于生产会导致酸奶保水力差、乳清析出严重、干酪得率降低，给企业造成较大损失。本项目主要研究了不同基因型乳蛋白对牛乳凝乳特性的影响规律。采集了1071头荷斯坦奶牛血样，重点了分析κ-酪蛋白（κ-CN）和β-乳球蛋白（β-LG）的基因型，在明确基因型的基础上，采集样品开展牛乳凝乳能力评价。在初步筛选的基础上，选择凝乳好、差和不凝乳样品开展凝乳流变学特性、蛋白多态性及矿物离子分布分析。通过分析牛乳的凝乳时间，胶体钙、镁、磷含量差异，探明不同基因型导致蛋白多态性及含量对牛乳凝乳能力的影响。结果表明，凝固性好的牛奶具有更短的凝乳酶凝固时间和更高的凝胶硬度，在形成的凝胶内连接更紧密。较低的牛奶pH值和酪蛋白磷酸化度，以及较高的蛋白质、酪蛋白和钙含量可能都是良好凝固性的原因。与其他国家的荷斯坦奶牛报告的频率相比，中国荷斯坦奶牛的κ-CN AA和AB变体以及β-LG AB和AA变体的频率更高。在所有奶牛组中，β-LG的AB基因型（占比48.48%）最常见，但AA型基因（30.97%）的原料乳凝乳效果较好；κ-CN的BB基因型（12.00%）凝乳效果较好，较AA、AB等其凝乳时间更短和凝胶强度更强。凝乳性能好的样品中CN含量及胶体钙含量较高，凝乳时间与κ-CN含量呈反比，酪蛋白和乳清蛋白组成和基因频率的变化会影响牛乳凝乳性能的变化。通过蛋白质组学研究了κ-CN变异体对牛奶成分、牛奶蛋白组成和牛奶蛋白磷酸化程度的影响。结果表明一些κ-CN变异体与总酪蛋白中αS2-CN比例明显降低和κ-CN比例升高有关。此外，在具有κ-CN BE变体的牛奶中观察到κ-CN和α-CN的高磷酸化程度。该研究对乳品企业合理利用原料乳，改善发酵乳品质，具有重要的理论和应用价值。