Content And Bioactivities Of Micropeptides In Skim Milk Powder

Through the long-term research on dairy chemistry and nutrition, milk proteins are now recognized as precursors of many different bioactive peptides. These peptides, which are inactive within the sequence of the milk protein, can be released and activated by fermentation or ripening during physical, chemical and biological processing. These peptides consisted of 2-20 amino acid(aa) are defined as specific protein fragments that have a wide range of positive impact on body functions or conditions including digestive system, cardiovascular system, immune system and nervous system, and may ultimately influence health, such as antimicrobial, antihypertensive, antioxidant, immunomodulatory, etc.This study aimed to evaluate the content, the antioxidant and antihypertensive characteristics, and the amino acid composition of micropeptides in skim milk powder. The skim milk powder was firstly rehydrated and salted-out. After centrifugation, the supernatants were subjected to microfiltration and then separated with Superdex-30 column fractionation, producing three fractions (PF-I to PF-III). We choose Bacitracin from Bacillus Licheniformis with molecular weight of 1.4kDa as standard reference based on the peptide resources and molecular weight distribution, and found that the molecular weight of fraction PF-III was about 1.0 kDa, thus the PF-III fraction was collected and lyophilized. The peptide content was measured using the standard curve of concentration-integral peak area of Bacitracin from Bacillus Licheniformis constructed by the method of SEC, and the result was about 0.1313±0.0052 mg mL-1 in the sample. Based on this, we estimated the content of micropeptide with molecular weight less than 1.0 kDa in skim milk powder was about 1.09%. Then the bioactivities of the peptide fraction were evaluated. Fraction PF-III at 10 mg mL-1 exhibited a modest antioxidant activity with DPPH free radical scavenging rate of 36.69±0.92%, which was significantly different from deionized water(26.05±0.34%) as the negative control; and that of 0.44 mg mL-1 BHT (66.29±0.66%) and that of 0.44 mg mL-1 VE (97.20±0.53%) as positive control (P < 0.05). To test the angiotensin I-converting enzyme (ACE) activity inhibitory rate of the peptide fraction, we firstly set up the equation of integral eluted peak area and concentration of Hippuric acid (HA) (R2=0.9956), for HA released from HHL is directly related to the ACE activity. We got ACE crude extracts (with enzymatic activity of 4.887±0.192U/mL) from rabbit lung, and found the PF-III at concentration of 3 mg mL-1 showed weaker ACE inhibitory activity (20.12±7.59%) compared with that of the positive control 10μM VPP (37.62 6.68%) and 10μM IPP (56.97 2.40%) and PBS with no ACE inhibitory activity (P < 0.01). Moreover, the amino acid compositions of PF-III revealed it is rich in Glu, Gly, Asp and Leu, and the total hydrophobic amino acids content was as high as 50.53%, which might contribute to the antioxidant and ACE-inhibitory activities.