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

本研究旨在通过对乳铁蛋白和半胱氨酸蛋白酶抑制剂进行化学和遗传的多糖化修饰，开发具有抗病毒和抗菌作用的安全食品添加剂。在65℃和79%RH下自然发生的美拉德反应和使用毕赤酵母的酵母表达系统分别用作修饰。这两种多糖化方法均能显著提高蛋白质的分子稳定性和表面功能特性。与天然形式相比，聚糖基化半胱氨酸蛋白酶抑制剂显示出较强的防止鼠伤寒沙门氏菌感染CV-1细胞的能力。多糖基化还提高了抗轮状病毒的功能。将一种含有多聚甘露糖链的重组糖基化半胱氨酸蛋白酶抑制剂添加到鱼糜中，以防止鱼糜在蒸煮过程中由于自溶而导致凝胶弱化。在40℃预孵育30 min后，再在90℃蒸煮20 min，可有效抑制鱼糜中肌球蛋白重链的蛋白水解。半胱氨酸蛋白酶抑制剂的糖基化可提高鱼糜的热稳定性和组织蛋白酶D对蛋白质的水解作用，从而显著改善鱼糜凝胶的质构，其凝胶强度是未糖基化半胱氨酸蛋白酶抑制剂的2.5倍。此外，通过细菌致突变试验和大鼠动物剂量试验证实了多糖化蛋白的食品安全性。因此，它揭示了多糖蛋白质作为食品和药物助剂的应用潜力巨大。本研究表明，蛋白质的多糖基化是提高蛋白质分子稳定性和功能特性的一条新途径。

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.