Preparation And Physicochemical Properties Of ACE-inhibitory Peptides From Silkworm Pupae Protein

Silkworm pupae is a by-product of silk reeling industry. The protein source has poor utility for the applications of food and medicine industry due to its bad color and taste. The objective of this study was to prepare angiotensin-converting enzyme (ACE) inhibitory peptides from silkworm pupae protein by enzymatic hydrolysis. The physicochemical properties of silkworm pupae protein and its fractions were investigated by the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid analysis and differential scanning calorimetry analysis (DSC). According to ACE-inhibitory activity of hydrolysates, the enzymatic hydrolysis technology of ACE-inhibitory peptides from silkworm pupae protein was also optimized by response surface methodology. ACE-inhibitory peptides of silkworm pupae were isolated and purificated from hydrolysates of silkworm pupae protein by ultrafiltration, DEAE-52 ion-exchange chromatography, and Sephadex G-50 gel chromatography. Furthermore, the interaction mechanism of the ACE-inhibitory peptides and ACE was investigated.The content of silkworm pupae protein in silkworm pupae (Dry base) was about 57.61 %. The water-soluble protein and alkali-soluble protein were the main protein fractions in silkworm pupae protein, the two protein fractions had complete kinds of amino acids and were well-balanced. The water-soluble protein and alkali-soluble protein had higher ratio of essential to total amino acids (41.24 % and 39.16 %, respectively) than the pattern recommended by WHO (at least 36 %). Two endothermic denaturation transitions were observed in the DSC thermograms of silkworm pupae water-soluble protein. The denaturation temperature (Td) was observed at 45.98°C and 88.87°C. The alkali-soluble protein of silkworm pupae had a endothermic denaturation transition, and its Td was 180.37°C. The alcalase hydrolysates for the water-soluble protein and alkali-soluble protein of silkworm pupae had strong ACE-inhibitory activities (Their IC50 values were 0.121 and 0.113 mg/mL, respectively).Taking ACE inhibitory activity as index, alcalase was chosen from three commercial proteases (alcalase,α-chymotrypsin and trypsin), which was used to prepare ACE-inhibitory peptides from silkworm pupae protein. The hydrolysis process of alcalase was optimized by response surface methodology. The results showed that the optimal hydrolysis conditions were pH 9.0, temperature of enzymatic hydrolysis 50.8°C and enzyme/substrate (E/S) 3500 U/g. Under the optimal conditions, the IC50 value of alcalase hydrolysates of silkworm pupae protein was 0.102 mg/mL.The hydrolysates fractions of molecular weight (MW) <5 kDa, 5~10 kDa, and MW>10 kDa were obtained from hydrolysates of silkworm pupae protein by ultrafication. Their ACE-inhibitory activities decreased in the following order: MW <5 kDa, 5~10 kDa, and MW>10 kDa. The ACE inhibition pattern of MW <5 kDa fractions isolated from hydrolysates of silkworm pupae protein was investigated using Lineweaver–Burk plots, and found to be competitive. In addition, ultraviolet spectra revealed that the MW <5 kDa fractions could cause a change on molecular structure of ACE. The performance tests showed that ACE-inhibitory peptides obtained from silkworm pupae protein had good acidic, alkaline, and thermal stability and ability of anti-intestinal digestion.The activities of ACE-inhibitory peptides from silkworm pupae protein, which were purificated by DEAE-52 ion-exchange chromatography and Sephadex G-50 gel chromatography, were markedly increased. Their IC50 value was 0.072 mg/mL. The purificated ACE-inhibitory peptides were composed of dipeptide to octapeptide, and their molecular weights were 226.34 ~ 983.61 Da. Among them, the peptides of 474.63 Da were the main activity fractions.