食品源特异性抗冻多肽的结构基础与低温保护作用的分子机制

Antifreeze proteins，because of its characteristics of inhibition of ice crystal growth, reducing cell damage and maintaining the quality of products in low temperature storage chain，have been becoming the popular research topic. The present study focused on the purification of antifreeze proteins from cold organisms. However, the quantity limitation of antifreeze protein from cold organisms and the negative fact of low antifreeze efficiency caused by the uncertain structural domains, have restricted its spread application as major bottle-neck problem. The research on efficient food-based antifreeze peptides and elucidating the mechanism of action is the key breakthrough to the bottle-neck problem. This project, based on achieved controllable preparation and biochemical isolation of food-based antifreeze peptide in the preliminary research, aims to the research on structural specificity and molecular mechanisms of action. The specific antifreeze peptide is to be separated and identified, the structure and domain specificity of specific antifreeze peptide is to determined through the determination of amino acid full sequence, circular dichroism chromatography and X - ray diffraction, the relationship between the activity of ice crystal growth inhibition and the specific structure will be determined. Through thermal differential method, ice-etching technique, nuclear magnetic resonance and scanning electron microscopic analysis, combination with the molecular dynamics simulations and quantum chemical calculations, site-directed mutagenesis and chemical synthesis, the ice-binding sites, the ice-action model, the process dynamics of thermal hysteresis effect are to be determined, and the molecular mechanisms of action on ice crystal growth inhibition and recrystallization inhibition is to be elucidated. This research will provide the theoretical foundation for the the active molecular design and potential application of food-based specific antifreeze peptides in the areas of food system.

抗冻蛋白因为具有抑制冰晶生长、减少细胞损伤而保持低温冷链产品品质的特点而成为研究热点。目前研究大多集中从耐寒生物体中纯化获取，而其数量的局限性以及因冰晶抑制结构域不明确导致抗冻效率不理想的现实成为制约其广泛应用的瓶颈，研究具有高效结构域和确切冰晶抑制位点的特异性抗冻多肽是关键的突破口。本项目在已实现可控制备、生化分离食品源抗冻多肽的基础上，旨在阐明特异性抗冻多肽的结构基础与低温保护作用的分子机制。通过全序列测定、圆二色谱和X-衍射确定其结构特征和冰晶抑制结构域，解析特定肽链长度、亲/疏水序列和特异结构域与其抑制冰晶生长的构效关系；利用热力示差、冰蚀刻、核磁共振和低温扫描电镜技术，结合分子动力学和量子化学计算、定点突变和化学合成，确定冰晶结合位点、晶格匹配和作用模式，解析过程动力学，阐明其抑制冰晶生长和重结晶的低温保护分子机制。本研究将为食品源特异性抗冻多肽的分子设计及开发应用提供理论依据。

本项目以食品加工副产物为原料，通过生物可控酶解技术制备抗冻多肽，获得具有特定结构域、肽链长度的抗冻多肽，实现抗冻多肽可控、高效的制备；通过分子质量大小、氨基酸组成、表面亲疏水荧光探针等，解析特定肽链长度、亲/疏水序列和特异结构域与其抑制冰晶生长的构效关系；利用热力示差、低温显微镜、低场核磁共振和低温扫描电镜技术等，解析抗冻多肽抑制冰晶生长和重结晶的低温保护分子机制；通过构建乳酸菌低温胁迫模型，阐明抗冻多肽对冷冻制品低温品质劣变干预及其干预的分子机制。本研究为食品源特异性抗冻多肽的开发、精准筛选及应用奠定理论基础。