家蝇蛹多肽的制备及促伤口愈合活性评价Preparation and evaluation of wound healing-promoting activity of peptides from housefly pupa

为了确定酶解法制备家蝇蛹多肽（PHP）的工艺条件，并初步评价PHP的促伤口愈合活性，以家蝇（Musca domestica L.）蛹为原料，采用蛋白酶酶解法制备PHP，通过单因素实验优化酶解法制备PHP的工艺条件，并在细胞水平上评价PHP的促伤口愈合活性。结果表明，10种蛋白酶酶解法制备的PHP中，胃蛋白酶酶解法制备的PHP得率最高，其次是风味蛋白酶酶解法制备的PHP（PHF）。另外，PHF的多肽含量及水解度均最高。单因素实验结果表明，制备PHF的最佳条件为酶解温度60 ℃、加酶量8 000 U/g、酶解时间4 h，在此条件下制得的PHF得率最高、水解最完全。细胞实验结果显示，不同蛋白酶酶解法制备的PHP具有不同程度促进HaCaT细胞增殖及迁移的作用。其中，PHF（分子质量主要分布在500~1 300 Da之间）促伤口愈合活性最佳。同时，PHF可显著促进RAW264.7细胞转化生长因子TGF-β1及炎性细胞因子TNF-α、IL-6的表达。综上，风味蛋白酶酶解法是一种制备高得率、高多肽含量及高水解度的PHP的方法，PHF具有促进伤口愈合的潜力。In order to determine the process conditions for preparing peptides from housefly pupa (PHP) using enzymolysis method and evaluate the wound healing-promoting activity of PHP, PHP were prepared with enzymolysis methods using pupa of Musca domestica L. as the raw material. The single factor experiment was designed to optimize the preparation process of PHP and the wound healing-promoting activity of PHP was studied at the cellular level. The results showed that among the PHP prepared with ten proteases, the yield of PHP prepared with pepsin was the highest, followed by flavourzyme hydrolysis (the peptide was referred to as PHF). In addition, PHF showed the highest peptides content and degree of hydrolysis. The results of single factor experiment displayed that PHF presented the highest yield and degree of hydrolysis under the conditions of temperature 60 ℃, enzyme dosage 8 000 U/g, and time 4 h. Besides, PHP prepared with different proteases had different degrees of promoting proliferation and migration of HaCaT cells, among them, PHF (molecular weight mainly distributed between 500 Da and 1 300 Da) exhibited the best wound healing-promoting activity. Meantime, PHF significantly promoted the expression of transforming growth factor TGF-β1 and inflammatory cytokines TNF-α, IL-6 in RAW264.7 cells. In conclusion, the enzymolysis method with flavourzyme contributes to prepare PHP with high-yield, high-peptides content, and high-degree of hydrolysis, and PHF has the potential to promote wound healing.