Development of safe food additive having antiviral and antimicrobial effects by polyglycosylation of food protein

本研究通过化学和遗传的方法将乳铁蛋白和胱抑素多糖基化，开发具有抗病毒和抗菌作用的安全食品添加剂。分别采用65℃、79% RH条件下的自然美拉德反应和毕赤酵母表达体系进行修饰。两种多糖基化均能显著改善食品蛋白的分子稳定性和表面功能特性。与天然形式相比，多糖基化胱抑素显示出较强的预防鼠伤寒沙门氏菌感染CV-1细胞的能力。多糖基化也提高了对轮状病毒的抗病毒功能。在鲱鱼鱼糜中添加了一种重组糖基化胱抑素，以防止烹饪过程中由于自溶而导致凝胶变弱。在40℃预孵育30 min后，90℃蒸煮20 min，可有效抑制鱼糜中肌球蛋白重链的蛋白水解。胱抑素的糖基化提高了鱼糜凝胶的耐热稳定性和组织蛋白酶d的蛋白水解能力，显著改善了鱼糜凝胶的质地，凝胶强度是未糖基化的对照鱼抑素的2.5倍。此外，通过细菌诱变试验和大鼠动物给药实验，证实了多糖基化蛋白的食品安全性。由此可见，多糖基化蛋白作为食品和医药助剂具有巨大的应用潜力。该研究表明，蛋白质的多糖基化是提高分子稳定性和功能特性的新途径。

This study deals with the development of safe food additive having antiviral and antimicrobial effects by chemical and genetic polyglycosylation of lactoferrin and cystatin. Naturally occurring Maillard reaction at 65℃ and 79% RH and a yeast expression system using Pichia pastoris were used as the modifications, respectively. Molecular stability and surface functional property of the food protiens were significantly improved by the both polyglycosylations. Polyglycosylated cystatins showed a strong ability to prevent infection of Salmonella typhimurium to CV-1 cells, compared to the native form. Antiviral function against rotavirus was also improved by the polyglycosylations. A recombinant glycosylated cystatin with a polymannosyl chain was added to roeherring surimi for preventing gel weakening due to autolysis during cooking. Proteolysis of myosin heavy chain in the surimi was effectively suppressed, while cooking at 90℃ for 20 min after preincubation at 40℃ for 30 min. The glycosylation of cystatin improved the stability against heating as well as proteolysis by cathepsin D.This process markedly improved the texture of the cooked surimi gel with gel strength 2.5 times greater than that of unglycosylated control cystatin. In addition, food safety of the polyglycosylated proteins was confirmed by bacterial mutagenesis tests and animal dose experiment using rats. Thus, it is revealed that polyglycosylated proteins gain great potential of the application as food and pharmaceutical aids. This study shows that polyglycosylation of protein is a new approach to enhance the molecular stability and functional properties.