Study The Effect Of Pretreatments On The Release Of ACE Inhibitory Peptides From Collagen In The Hydrolysis Process

There are abundant ACE inhibitory peptides in collagen three spiral area, as the spiral area is remarkably stable and not easy to be degraded by non-matrix metalloproteinase, pretreatments before hydrolysis were usually required in order to expose more enzyme loci, promote the release of ACE inhibitory peptides. Currently, there are no reports about pretreatments promote the release of ACE inhibitory peptides from collagen. This study aims to explore a sample and efficient pretreatment to product ACE inhibitory peptides and further study the mechanism. The effect of five pretreatments (including acid, alkali, heat, ultrasonic, superhigh pressure) and ultrasonic synergy acid/alkali on collagen ACE inhibitory peptide release were studied, filtered the best pretreatment, and then the ACE inhibitory peptides obtained were purified and identified.With no treatment group for comparison, the degree of hydrolysis, ACE inhibition activity, SDS-PAGE, gel permeation liquid chromatography, DSC and FTIR results of five pretreatments were analyzed. These results showed that all pretreatments were conducive for Alcalase to hydrolyze collagen, however, the effects of ACE inhibitory activity varied from pretreatments. The ACE inhibition activity of alkali and ultrasonic treatment groups were highest and those peptide release rate were more rapidly, so alkali and ultrasonic treatment are better effectively pretreatments to produce ACE inhibitory peptides. Results of the molecular weight distribution, DSC and FIRT indicated that, alkali destroy the balance of collagen non-covalent bond, changed collagen conformation characteristics, exposed more enzyme loci from spiral area, so more ACE inhibitory peptides from spiral area were get, the inhibition activity were higher and increased more rapidly; ultrasonic could make collagen polypeptide chain extend and expose internal hydrophobic enzyme site which were advantageous for Alcalase to hydrolyze; Other pretreatments could destroy collagen structure to some extent and mainly damage telopeptide region.Base on previous studies, ultrasonic pretreatment combined with acid and alkali were studied, the results showed that ultrasonic synergy acid/alkali groups could release collagen ACE inhibitory peptides more rapidly than each individual pretreatment, after 1min hydrolysis, the ACE inhibition activity were above 88% which were higher than individual pretreatment group 4h hydrolysis level. Otherwise, ultrasonic synergy alkali treatment group could release collagen ACE inhibitory peptides in a short time compared with ultrasonic synergy acid treatment. Combined with the molecular weight distribution, DSC and FITR results, ultrasonic synergy acid destroy the collagen molecules covalent cross-linking, and ultrasonic synergy alkali had little effect on collagen covalent bond, it mainly damaged the hydrogen which hold collagen three helical structure, changed collagen sub-molecular conformation, its spiral area exposed more enzyme loci than ultrasonic synergy acid and thus more conductive to hydrolyze. According to the results of scan electron microscopy (SEM), alkali could damage collagen, but the damage had been restricted to collagen surface. After ultrasonic synergy alkali, forming small particles of collagen, increasing the specific surface area, exposed more enzyme loci, conducive to hydrolyze.The Sephadex G-15, semi-preparation of high performance liquid chromatography were used to separated and purified the sample of 30min hydrolysis of ultrasonic synergy alkali group, and the sequences were identified. The best separation condition was:sample concentration of 100mg/mL, injection volume 2mL, flow rate 2mL/min, distilled water act as eluent and chromatography column was 1m×10mm. Under the separation condition, hybrid peptides were divided into AP-Ⅰ, AP-Ⅱ two components, the IC50 value were 19.501mg/mL and 1.005mg/mL. The strong inhibitory activity of AP-Ⅱ was further separated by semi-high performance liquid chromatographic, the optimum separation conditions were column temperature 30℃, injection volume 43.5μL (sample concentration 10mg/mL), flow rate 4.35mL/min, with 10% acetonitrile(containing 0.05% TFA) eluted 40min. There are 8 peaks after semi-high performance liquid chromatographic separation, the peak 5 and peak 6 IC50 value was higher, and then peak 2. Considering IC50 value and the sample weight collected after semi-high performance liquid chromatographic separation, the peak 2 and peak 6 sequences were identified by LC-MS/MS, the results show that peak 2 peptide sequence was QGPPGPAGPR and peak 6 was AGPPGPPGPA, this two sequence respectively derive from526-535,730-739 of collagen al chain, the amino acids were accordance with the structure-activity characteristics of ACE inhibitory peptides, and the sequences were not reported by now.