Research On The Preparation Of ACE Inhibitory Peptides From Skim Milk By The Multi-enzymatic Hydrolysis

Hypertension is one of the main diseases for human health.The study found that food antihypertensive peptides,compared with the traditional antihypertensive drugs,had high safety and non-toxic side effects.Milk and dairy products not only contain the various nutrients needed by the human body,but also have a variety of bioactive peptides.Skim milk was hydrolyzed by multi-enzymatic to prepare ACE inhibitory peptides with the regulation of hypertension.Which contributes to the development of functional dairy products,increases the added value of dairy products,and provides therapeutic ways for the prevention of hypertension.Skim cow and goat milk were used as raw materials in present study,and eight kinds of proteases were used to hydrolyze skim milk respectively.The optimum enzyme for preparing ACE inhibitory peptides was screened.Mixture experiment design determined the optimum enzyme ratio;single factor and response surface method optimized the enzymatic hydrolysis process.A single component of ACE inhibitory peptides was obtained by the combination of ultrafiltration,macroporous resin DA201-C,gel chromatography and HPLC separation technologies.Finally,components of enzymatic hydrolysate that have been purified were determined amino acid composition and the molecular weight of the polypeptide.And compared the differences of peptides production and recovery rate,ACE inhibitory activity between skim cow and goat milk.The results are follows:(1)Among the eight kinds of proteases,ACE inhibitory activity of enzymatic hydrolysate for alkaline protease,bacillus licheniformis protease and Alcalase were significantly higher than those of the other 5 species both in skim cow and goat milk.The optimum protease ratio in skim cow milk was: EAlcalase:Ebacillus licheniformis protease=0.566: 0.434,the degree of hydrolysis and ACE inhibitory activity can reach at 60.29±0.28%,84.86±0.33%,respectively.While for skim goat milk,the optimum protease ratio was: EAlcalase: Ebacillus licheniformis protease : Ealkalineprotease= 0.388: 0.406: 0.206,the degree of hydrolysis and ACE inhibitory activity can reach at 64.56±0.21% and 82.54±0.18%,respectively.(2)Optimization of multi-enzymatic hydrolysis skim milk process by single factor and response surface methodology.The optimum conditions for enzymatic hydrolysis skim cow milk were: pH 9.01,E/S ratio 6.50%,temperature 61.80? and hydrolysis time 50 min,under this optimized condition,the degree of hydrolysis and ACE inhibitory activity of enzymatic hydrolysate were 61.12±0.38% and 85.02±0.22%,respectively.As for skim goat milk,the optimum conditions for enzymatic hydrolysis were: pH 8.49,E/S ratio 8.04%,temperature 61.54? and hydrolysis time 90 min,the degree of hydrolysis and ACE inhibitory activity were 65.39±0.01% and 84.65±0.03% respectively under its optimized conditions.(3)Enzymatic hydrolysate were separated and purified by ultrafiltration and macroporous resin DA201-C,ACE inhibitory activity for skim cow and goat milk were 91.90±0.36% and 92.07±0.06%,respectively.And IC50 were0.258±0.007 mg/mL and 0.253±0.011 mg/mL,respectively.(4)Sephadex G-25 was used to purify the above resin eluates,and three components were obtained for both skim cow and goat milk.Among them,IC50 of component G2 was 0.175±0.003 mg/mL,which was the smallest of the three components in the skim cow milk hydrolyzate,and its ACE inhibitory activity was 92.23±0.13%;IC50 of component F2 was 0.154±0.006 mg/mL,which was the smallest of the three components in the skim goat milk hydrolyzate,and its ACE inhibitory activity was 93.88±0.33%.Sephadex G-15 continued to separate and purify the components G2 and F2,respectively,components G2-1,G2-2;F2-1,F2-2 were obtained.The ACE inhibitory activity of G2-2 and F2-2 was relatively high,were 92.54±0.22% and 93.97±0.15%,respectively.IC50 were0.167±0.007 mg/mL and 0.121±0.004 mg/mL,respectively.(5)The content of hydrophobic amino acid,aliphatic amino acid and aromatic amino acid for component G2-2 were 76.59%,40.05% and 31.86%,respectively.While for component F2-2,the corresponding amino acid content were 73.17%,33.86% and 33.72%,respectively.Hydrophobic amino acids and aromatic amino acids are the main active components of ACE inhibitory peptides.Hydrophobic amino acid and aromatic amino acid for G2-2 and F2-2 wererelatively higher than G2-1,F2-1,respectively.The skim goat milk F2-2 had a higher ACE inhibitory activity than skim cow milk G2-2,IC50 of F2-2 was0.121±0.004 mg/mL,which was 0.046 mg/mL less than G2-2.The components G2-2 and F2-2 were further separated by reverse phase high performance liquid chromatography(HPLC),and identified by Matrix assisted laser desorption ionization time-of-flight mass spectrometry.The G2-1 and G2-2 of multienzymatic hydrolyzate from skim cow milk were identified 10,8 peptides,respectively,and peptides were mainly derived from ?-casein and ?S1-casein;The F2-2 of multi-enzymatic hydrolyzate from skim goat milk were identified 11 peptides,and they were mainly derived from ?-casein,?S1-casein and ?S2-casein.In this study,the high active ACE inhibitory peptides were prepared by multi-enzymatic hydrolysis skim milk.The differences in the composition and activity of ACE inhibitory peptides for skim cow and goat milk were compared.Which provided theoretical basis and technical reference for future development of functional dairy products with regulation of hypertension.