Preparation Of ACE Inhibitory Peptides From Tilapia Scale By Hydrothermal Pretreament And Enzymatic Hydrolysis

Tilapia(Oreochromis niloticus)is one of the major farmed species in freshwater aquaculture,with annual production accounting for about half of the world's total production.Large quantities of by-products(such as fish skin,fish scales and fish bones)are inevitably produced during processing and are mostly disposed as solid wastes.Fish scales are rich in protein and can be used to produce collagen or peptides,but the natural bone layer barrier prevents the extraction and utilization of fish scale protein.How to develop green and efficient pretreatment methods to break the natural resistance barrier and improve the enzymatic efficiency is one of the key scientific problems that need to be solved urgently.To this end,this study used tilapia scales as raw materials,The effects of different pretreatment methods on the recovery rate of fish scale protein were compared.The hydrothermal pretreatment process was determined.The optimal protease species for the preparation of antihypertensive peptides were screened and the enzymatic hydrolysis process parameters were optimized.The physicochemical properties and activity stability of fish scale ACE inhibitory peptide were further analyzed,and the fish scale ACE inhibitory peptide was further isolated and purified.The research results can provide a theoretical basis for the development of tilapia fish scale antihypertensive peptides.The main findings are as follows:The effects of pretreatment methods such as smash,decalcification and hydrothermal pretreatment on the recovery rate of fish scale protein were compared.The structure,enzymatic properties and amino acid composition of pretreated fish scale protein were characterized.It was found that hydrothermal treatment can significantly improve the protein recovery and enzymatic efficiency of fish scale.The optimal hydrothermal pretreatment conditions for fish scales are:treatment temperature at 135 ? for 90 min,substrate concentration 8%.Under these conditions,the recovery rate of fish scale protein was 84.81%,The degree of hydrolysis(DH)and ACE inhibition of enzymatic hydrolysate were 12.88%and 82.77%,respectively,which were significantly higher than the control group.Scanning electron microscopy and infrared spectroscopy were used to investigate the effects of hydrothermal pretreatment on the morphology and chemical structure of fish scales.The results show that the hydrothermal treatment can basically separate the fish scale protein from the hydroxyapatite skeleton.The results of SDS-PAGE showed that the molecular weight of the fish scale protein after hydrothermal pretreatment was mainly concentrated below 20 kDa.Using enzymatic hydrolyzate degree and ACE inhibition rate as evaluation indicators,based on single factor test,the process parameters of hydrothermal pretreatment and enzymatic preparation of fish scale ACE inhibitory peptide were optimized by response surface methodology.The results showed that alkaline protease was the optimal enzyme,and the optimal enzymatic hydrolysis parameters were as follows:substrate concentration 8%,enzymatic time 1.5 h,enzymatic temperature 55.37 ?pH value 7.99 and enzyme-substrate ratio 0.59%.The basic composition,physicochemical properties of fish scale peptide and the effects of various factors in vitro on its blood pressure lowering activity were analyzed.The peptides has a crude protein content of 97.01%and the relative molecular mass is mainly distributed in 200?1000 Da.The fish scale peptides is rich in glycine,proline,glutamic acid and alanine.The peptides had a solubility of about 85%at different pH values.The foaming,foam stability,emulsion stability and oxidation resistance are relatively good.Compared with the oven-dried peptide samples,The freeze-dried peptide sample is whiter in color,larger in size and looser in irregularity,and has better ACE inhibitory activity than the oven-dried sample,the IC50 was 730 ?g/mL;Fish scale peptides have good heat resistance and acid and alkali resistance,and can maintain its activity well after gastrointestinal digestion.After the fish scale peptide solution having separated with an ultrafiltration membrane with a molecular weight cut off of 3 kDa,the IC50 value was 423.30 ?g/mL;After further separation by gel filtration chromatography,the IC50 was 167.30 ?g/mL,and the purification ratio was 4.36 times.