Maillard反应修饰黄鲫蛋白抗菌液及其抑菌机制研究

Abuse of antibiotics has led to emerging phenomena of antibiotics resistance for some pathogenic bacteria. Chemicals added in food to control microbes has raised public concern. The seeking for novel natural antibacterial agents with high safety has been one of the most crucial topics in the arena of international food science research. By substituting chemical preservatives with natural antibacterial agents in foods would assure nowadays consumers for the health concern. Our previous study indicated that the hydrolysates of half-fin anchovy with pepsin (named as HAHp) demonstrated strong antibacterial effect on some common food pathogens. However, its addition in foods would be limited due to the taste flavoring of fishy smell and bitterness. In this project, HAHp is used as the starting substrate. Maillard reaction, one of the safest reactions in food processing, is used to enhance the antibacterial activity of HAHp. In addition, the Maillard reaction products will bring out good flavoring and food functional properties, which can contribute to the potential application of HAHp in foods. The main contents of the study contain: designing the rational model of Maillard reaction, elucidating the chemical structures of the newly formed antibacterial fractions, exploring the antibacterial mechanism, discovering the relationship between chemical structure and activity of the antibacterial fraction. The results of the project will provide a theoretical basis for modification of HAHp, as well as provide technical support for the antibacterial mechanism of Maillard reaction products. In a long run, this project would point out a new potential pathway for the high-added value application of the underused marine fish of half-fin anchovy.

由于抗生素的滥用导致耐药菌株不断产生，食物中添加化学药剂控制微生物的做法也引起公众的关注。寻找天然新型、且安全性高的抗菌剂是目前国际上食品科学研究的热点议题之一，使用天然高效抗菌剂替代合成防腐剂将有助于保障消费者的健康。本课题组前期研究发现，胃蛋白酶水解黄鲫得到的酶解液（HAHp）对食品中常见致病菌有较强抑制作用，但该抗菌物质有一定腥味和苦味，限制了在食品中应用范围。所以，本课题以HAHp为底物，利用食品加工中安全性高的美拉德反应进一步提高其抑菌性，同时赋予产物良好的风味性和功能性，为HAHp可持续性应用奠定基础。拟开展的主要研究有：建立黄鲫蛋白抗菌液的美拉德修饰反应模型，表征新获得的有效抑菌组分化学结构，研究其抑菌机制，探讨化学结构与抑菌活性关系。课题研究结果将为HAHp的修饰改性提供理论依据，也为美拉德反应产物的抑菌机制研究提供技术支持，最终为海产小杂鱼黄鲫的高值化利用提供一条新途径。

本项目以具有较高生物活性的黄鲫蛋白抗菌肽液（HAHp）为底物，通过美拉德反应修饰提高其抑菌活性，建立黄鲫蛋白抗菌液的美拉德修饰反应数学模型，表征有效抑菌组分的化学结构，研究美拉德产物的抑菌机制。研究表明HAHp与葡萄糖发生美拉德反应能有效提高产物的抑菌活性，经Box-Behnken响应面分析优化得出葡萄糖添加量3%，反应初始pH值9.0，加热温度120℃，加热时间100 min下生成的HAHp-葡萄糖美拉德产物抑菌性最强。HAHp-葡萄糖美拉德产物经离子交换、高效液相色谱（HPLC）法分离得到抑菌效果强组分P3-2，采用液质联用技术从P3-2中分离鉴定出9条肽，分子量在768~1763 Da。根据抗菌肽结构特性选择5条肽（WLPVVR、FFTQATDLLSR、VLLLWR、VLLVLLR、VLLALWR）进行化学合成，除了肽VLLVLLR外，其余4条合成肽（初始浓度20 mg/mL）对大肠杆菌均具有强抑菌作用。以大肠杆菌为模型菌，研究HAHp-葡萄糖美拉德产物的抑菌机制，结果表明HAHp-葡萄糖美拉德产物与大肠杆菌与作用3 h可造成大肠杆菌发生明显膜损伤，胞外K+离子和5’-腺苷三磷酸（ATP）量显著增加（P < 0.05），且胞内酶乳酸脱氢酶（LDH）释放量也增加显著（P < 0.05）。HAHp-葡萄糖美拉德产物与DAPI荧光标记大肠杆菌在室温下作用1 h、3 h，荧光显微镜观察发现大肠杆菌经HAHp-葡萄糖美拉德产物作用后荧光强度降低明显，其中3 h样品处理组大肠杆菌的DNA含量较对照组降低显著（P < 0.05）。以分离鉴定出的一条抑菌肽WLPVVR为出发肽，化学合成3条类似肽WLPVVH，WLPVVE和LPVVR，进一步分析抗菌肽N端和C端氨基酸残基对其抗菌性影响。结果表明抗菌肽WLPVVR的N端W（色氨酸）残基对其抗菌性起着重要作用，而C端R（精氨酸）替换成H（组氨酸）和E（谷氨酸）后对大肠杆菌仍然保持强抑菌性。此外，研究还发现WLPVVR和葡萄糖经过美拉德反应所得的美拉德产物抑菌性显著高于WLPVVR。综上，本项目研究结果说明适宜的美拉德反应能有效提高黄鲫蛋白抗菌液/抗菌肽的抑菌活性，所得美拉德产物可以通过膜损伤和抑制胞内DNA生成方式实现抑菌作用。