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Objective To explore the effects of ultrasonic synergistic enzymatic hydrolysis on the antioxidant activity and structure of donkey milk casein, optimize the process parameters of composite treatment, and analyze the promotion mechanism of ultrasonic pretreatment on the enzymatic hydrolysis process. Methods Using donkey milk casein as raw material, the ultrasonic parameters (200 W, 20 min, 6 s/6 s intermittency ratio) were optimized by single factor experiment, the neutral protease and trypsin (the mass ratio was 2:1) were screened, and the enzymatic hydrolysis conditions were optimized by response surface method (enzyme addition amount 4.86%, 55.28 °C, pH 7.76). The changes of protein molecular weight, secondary structure and functional groups were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared spectroscopy. The antioxidant indexes such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging rate were determined. Results Ultrasonic synergistic enzymatic hydrolysis resulted in antioxidant ability of 69.77% and a 65.3% increase in Fe3⁺ reducing capacity, which was significantly better than that of the single treatment. The structure showed that the α-helix decreased to 26.70%, the random coil increased to 28.35%, the aromatic amino acids were exposed, the sulfhydryl groups were oxidized to disulfide bonds, and the molecular weight was reduced. The predicted value of the response surface model was in good agreement with the measured value (R2=0.9937). Conclusion The results of ultrasonic pretreatment significantly improves the antioxidant activity by destroying the structure of protein micelles and releasing antioxidant short peptides in synergy with enzymes, which provides a reference process parameter for the production of high value-added donkey milk antioxidant products, which is helpful to promote the development of donkey milk industry and improve the economic value of donkey milk.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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