发酵酸化技术对乳饼蛋白质降解及蛋白肽生物活性的影响

通过发酵酸化技术研制发酵型乳饼，以传统的直接酸化技术为对照，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE）、液相色谱-质谱测定乳饼蛋白质降解情况和游离氨基酸含量，通过用液相色谱-串联质谱鉴定乳饼蛋白肽。并通过测定体外抗氧化活性、α-葡萄糖苷酶抑制率和血管紧张素转换酶（angiotensin-converting enzyme，ACE）抑制率表征乳饼蛋白肽的生物活性。SDS-PAGE结果表明，两种酸化技术均能促进乳饼蛋白质的降解，发酵酸化技术对乳清蛋白的降解程度更高，能促进游离氨基酸的释放。从发酵酸化乳饼中鉴定出潜在的ACE抑制肽23?条、抗氧化肽6?条和降糖肽5?条，而传统的直接酸化乳饼中分别为13、4?条和1?条。发酵酸化技术能显著提升乳饼蛋白肽的α-葡萄糖苷酶抑制活性和ACE抑制活性（P＜0.05），其中分子质量＜3?kDa超滤组分对二者抑制率的IC50分别为1.250?mg/mL和0.416?mg/mL。发酵酸化技术能促进乳饼蛋白质降解，释放生物活性多肽，同时产生游离氨基酸，提升乳饼的生物学价值。

Effect of Lactic Acid Bacterial Fermentation on Protein Degradation and Biological Activity of Peptides in Rubing Cheese

Rubing cheese was manufactured by lactic acid bacterial fermentation or traditional direct acidification. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatography-mass spectrometry (LC-MS) were used to determine protein degradation and free amino acid contents in rubing cheese, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptides in rubing cheese. The biological activity of the hydrolysate of rubing cheese was characterized by in vitro antioxidant activity, α-glucosidase-inhibiting activity and angiotensin-converting enzyme (ACE)-inhibiting activity. According to the results of SDS-PAGE, both lactic acid bacterial fermentation and direct acidification could promote the degradation of milk proteins. Lactic acid bacterial fermentation caused a higher degree of degradation of whey protein, promoting the release of free amino acids. From the fermented rubing cheese and the directly acidified one, 23 and 13 potential ACE inhibitory peptides, six and four antioxidant peptides and five and one antiglycemic peptides were identified, respectively. Furthermore, lactic acid bacterial fermentation significantly improved the α-glucosidase-inhibiting activity and ACE-inhibiting activity of rubing cheese peptides (P < 0.05). The half-maximum inhibitory concentration (IC50) values of the fraction separated by ultrafiltration with a molecular mass of less than 3 kDa for α-glucosidase and ACE inhibitory activity were 1.250 and 0.416 mg/mL, respectively. In conclusion, lactic acid bacterial fermentation promotes protein degradation to release biologically active peptides and produce free amino acids, enhancing the biological value of rubing cheese.