屠宰前后红肉N-羟乙酰神经氨酸解离的方法与分子模拟研究

Immunogenic N-glycolylneuraminic acid (Neu5Gc), mainly accumulated through red meat consumption in human body, have exhibited the risk of carcinogenic effects. It is the key of removing concerns over red meat to master the safe way to get rid of Neu5Gc. The applicant intends to use computational chemistry methods to predict bond dissociation energy of Neu5Gc and simulate environment condition of red meat pre-slaughter and post-slaughter. Exploring the dominant influencing factors of Neu5Gc dissociation could also provide basic theoretical guidance for the screening of scientific and rational processing technology. Then, an in vitro experiments is implemented for obtaining the safe dissociation formula on Neu5Gc; In accordance with the process of the production and processing of red meat, to construct the pre-slaughter solution from the guidance of toxicology studies in mice by hypodermic injection of enzymes adding and a variety of biochemical reagents adding, the key here is get data of Neu5Gc dissociation in vivo, and then implement a verification test via Guizhou miniature Xiang-pig model; In the end, to collect Neu5Gc dynamic variation trend in red meat combining with chemical and physical control results in the simulation process, the work is targeting at interpreting the effective regulation and control mechanism of Neu5Gc dissociation in pre-slaughter and post-slaughter red meat, and exploring an effective method to reduce the intake of Neu5Gc in human body.

免疫原性的N-羟乙酰神经氨酸（Neu5Gc）属致癌风险物质，人体中Neu5Gc主要是通过红肉摄入而积累。要解除红肉忧患，开展屠宰前后红肉中Neu5Gc 的安全稳妥的去除方法势在必行。申请人拟采用计算化学方法准确预测Neu5Gc的键解离能，模拟屠宰前后红肉中的Neu5Gc存在环境，探讨影响Neu5Gc解离的因素，获得有效的基本理论指导；继而进行体外实验，获取促Neu5Gc解离的安全配方；结合红肉的生产加工过程，以小鼠毒理学研究为指导，建立宰前酶制剂及多种生物质解离方案，获得体内解离Neu5Gc的关键数据，并用贵州微小型香猪模型予以验证；结合模拟加工中化学与物理调控研究结果，获取红肉中Neu5Gc动力学变化信息。本研究旨在阐释屠宰前后有效调控Neu5Gc解离的机制，为减少Neu5Gc的摄入摸索一套行之有效的方法。

红肉中广泛存在的非人源性唾液酸N-羟乙酰神经氨酸(Neu5Gc)，是与炎症相关的二类致癌物质，但对其解离的策略不够充分。本项目以红肉宰前和宰后视角入手，探索加工和合成过程中有效降低Neu5Gc的方法。为此，首先基于计算预测Neu5Gc不同条件下（溶剂、电场、自由基）键解离能与结构变化，其次模拟关键生化合成酶并筛选潜在抑制剂。综合考察了红肉热加工（油炸、蒸煮）和非热加工（脉冲强光、高压、电场、乳酸发酵、生物酶）处理效果。经大鼠和香猪实验放大验证了几种唾液酸转移酶和羟化酶抑制剂（染料木黄酮、山奈酚、槲皮素等）在体内干预合成Neu5Gc的效果。理论和光谱计算表明：外电场下Neu5Gc脱氢解离能降低最明显。极性溶剂下Neu5Gc亲核活性增强，自由基易抽提非羟基氢原子。分子对接模拟表明：菊粉酶活性残基可与Neu5Gc发生氢键相互作用，抑制剂通过分子间作用力占据Neu5Gc合成酶的活性口袋。加工处理和检测表明，电刺激使红肉中Neu5Gc含量降低达74.24%，外切菊粉酶可降低55.33±5.1%，植物乳杆菌GDM1.919降低效果最佳为80.26%。宰前合成干预试验表明，当以200mg/kg·d的槲皮素大鼠灌胃30d时，对肌肉中Neu5Gc抑制率最佳为24.74±0.07%，以1mg/kg·d的5'-CMP在香猪6月龄时饲喂30天后的最佳抑制效果为58.18%，且对肉品质无不良影响。以上研究为精准控制红肉Neu5Gc提供了方法参考。