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This study isolated and characterized bioactive peptides with dipeptidyl peptidase-IV (DPP-IV) inhibitory activity from walnut protein hydrolysates. The hydrolysis process was optimized using response surface methodology with alkaline protease, yielding optimal conditions as follows: Substrate concentration 2.23% (w/v), enzyme dose 9100 U/g, and hydrolysis time 180 min. Under these conditions, the degree of hydrolysis reached 25.47%, and the DPP-IV inhibition rate was 77.85%. The hydrolysate was further separated, purified, and characterized. The fraction with molecular weight <3 kDa (U1) demonstrated the strongest DPP-IV inhibitory activity (IC50=2.81 mg/mL). Subsequent purification of U1 by Sephadex C-25 column chromatography yielded two sub-fractions (F1 and F2), with F2 exhibiting superior activity. A total of 6557 peptides were identified, and five potential hypoglycemic peptides were screened via in silico approaches. Among these, the peptide KFPF showed the highest DPP-IV inhibitory activity (IC50=0.26 mg/mL). Molecular docking simulations revealed that KFPF binds to DPP-IV via six hydrogen bonds and ten hydrophobic interactions. In vitro assays indicated that KFPF acts as a competitive inhibitor. Furthermore, secondary structure analysis by infrared spectroscopy suggested a high content of β-turn (82.03%) and α-helix (15.18%), implying a possible correlation between these structural features and DPP-IV inhibition. These findings demonstrated that KFPF derived from walnut protein has potential as a functional dietary component for blood glucose management.
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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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