Research Of Preparation Of Flounder(Paralichthys Olivaceus) Skin Collagen Peptide And ACE Activity

In recent years,China's fish processing industry has developed rapidly,produced a large number of processing by-products of fish skin.The fish skin is rich in collagen and is a good raw material for the preparation of collagen peptides.Since the use of fish skin is not fully utilized,resulting in waste of resources,this study investigated the ACE inhibitory activity of fish skin collagen peptides,providing a theoretical reference for the efficient and high-value utilization of fish skin.Taking the flounder as a research object,a research on the comprehensive utilization of fish processing by-product.The study consists of four parts:firstly,extraction of acid soluble collagen from the skin of flounder fish.With the black skin of the back of the flounder as the raw material,with 0.008 mol/L phosphoric acid,material-to-liquid ratio 1:20 g/mL,soaked for 1 h at 4?,the black skin was removed with a knife,and obtained the flounder dermis.Flounder dermis and non-pretreated flounder skin were extracted collagen using the traditional acetic acid?low temperature?method.UV scans have absorption peaks at 236 nm.After FTIR scanning,five characteristic absorption peaks of amide A,amide B,amide I,amide II and amide III were obtained.SDS-PAGE showed that both collagens contained three characteristic bands of?,?,and?,and the collagen,?,?,and?contents of pre-extracted collagen were higher than those of pretreated collagen.Thermal stability shows that the thermal shrinkage temperature is 110.6?and 118.1?,and the thermal denaturation temperature is22.2?and 17.7?.The experimental result showed that both of extracted collagens are the typical type I collagens,and the yields reached 41.1%and 22.2%,and the unique triple helix structure was retained.Secondly,preparation of flounder skin collagen peptide.With the black skin of the back of the flounder as the raw material,collagen peptide was prepared using the enzyme hydrolysis method.First establishment of an in vitro ACE inhibitory activity evaluation system.Choose protein enzyme,select five proteases:acid protease,alkaline protease,neutral protease,papain and pepsin.The optimum reaction temperature and pH of each protease were selected,and the same amount of enzyme was added at 250 U/g.After enzymolysis for 48 h,the ACE inhibitory rate of the hydrolyzate was determined at different time points.The curve was drawn and compared.The results showed that the ACE inhibitory activity of the neutral protease was 21.88%,and was the highest at 12 h,and the neutral protease was determined to be the optimal protease.What's more,the effects of enzymolysis time,enzyme dosage,and material-to-liquid ratio on the ACE inhibitory rate of collagen peptide powder after enzymatic hydrolysis were investigated by single factor experiments.Finally,the optimal conditions for enzymatic hydrolysis were determined by orthogonal test:enzymolysis time was 9 h,enzyme dosage was 1000 U/g,material-to-liquid ratio was1:32.5 g/ml,and the inhibition rate of ACE was 52.48%.Thirdly,separation,purification and structure identification of ACE inhibitory peptides.The collagen peptides were enzymatically digested under optimal conditions,separated and purified by membranes,separated and collected according to different molecular weight fractions,and concentrated and lyophilized to determine the ACE inhibitory activity.The results showed that the ACE inhibitory rate of the oligopeptide component with a molecular weight of 300¹000 Da was the highest,which was72.67%,and the IC₅₀ value was 1.72 mg/ml.Further,this component was further separated and purified by Sephadex LH-20 and RP-HPLC to obtain two peaks having ACE inhibitory activity of F6-14 and F7-17.Mass spectrometry indicated that the possible dipeptide structure of F6-14 was Ser-Trp,and the possible dipeptide structure of F7-17 was Gly-Trp.Fourth,the spontaneously hypertensive rats?SHR?was used as the subject,and SHR were fed with 300-1000 Da flounder skin collagen peptide and fed 200 mg/kg twice a day for four weeks,observed.After 15 days,SHR had blood pressure lowering effects.Blood samples were collected 36 hours after stop gavage,and ACE,Ang II,ATG,and ALD were measured.The results showed that the flounder skin collagen peptide had blood pressure lowering effect.