Preparation Of Sheep Bone Polypeptide And Its Antioxidant Ability

Sheep bone has high nutritional value as a by-product.But its bioactive substances have not been studied in depth.In this study,antioxidant peptide was prepared from sheep bone through enzymolysis technology.The antioxidant ability,stability and secondary structure of the antioxidant peptides were analyzed.The peptides were purified,and the amino acid sequences were identified.Potential functional peptides were screened by functional annotation and bio-informatics prediction.Chemical synthesis of the selected peptides to verify the antioxidant activity.The results of this topic are as follows:The total content of amino acids in sheep bone is 15.75 g/100g,hydrophobic amino acids and essential amino acids accounted for 28.25%and20.44%,respectively.Sheep bone is an ideal raw material for the extraction of antioxidant peptides.The results of response surface methodology were as follows:alkaline protease,p H was 8.9,the amount of enzyme added was 6400U/g,hydrolysis time was 3.25 h,substance mass fraction was 8.8%and the hydrolysis temperature was 50?.Under these conditions,the degree of hydrolysis was(20.78±0.83)%,The IC₅₀values of·OH scavenging activity,DPPH·scavenging activity and ABTS⁺free radical scavenging capacity of antioxidant peptides from sheep bone were 6.07 mg/m L,23.45 mg/m L and1.18 mg/m L,respectively.When the concentration of hydrolysate was 2mg/m L,the reducing power was 0.15±0.003.The stability of sheep bone antioxidant peptide(SBAP)was studied.The results showed that SBAP was not tolerant to high temperature,acid and alkali.With the increase of Na Cl content,the reducing power of SBAP improved.Reducing sugar can keep the original activity of SBAP antioxidant peptide,or slightly increase the activity while metal ions decreased the antioxidant activity of SBAP.The activity of SBAP is stable to sodium benzoate and potassium sorbate.The simulated digestion in vitro shows that SBAP is inert to artificial gastrointestinal fluid.The structure characterization showed that SBAP retained the characteristic functional groups of the source protein,the micro morphology of SBAP was irregular macroporous spongy structure with loose pores.The decreased content of?-helices and random,increased content of?-sheets and?-turns were observed.Three components with different molecular weight were obtained:P-I(M_W<3 k Da),P-II(3 k Da<M_W<10 k Da)and P-III(M_W>10 k Da).The results of amino acids of ultrafiltration components showed that except(Gln/Glu),the content of other amino acids in SBAP was higher than that in sheep bone,and the ratio of essential amino acids to total amino acids(EAA/TAA)was 27.03%,and the hydrophobic amino acids were 39.96%.P-I had the highest proportion of hydrophobic amino acids 43.13%,including hydrophobic amino acids(Pro,Phe,Met,etc.)and non-polar aliphatic amino acids(Val).Under the same concentration,P-I had the highest activity of·OH scavenging activity,DPPH·scavenging activity,ABTS⁺scavenging activity and reducing power.It can be seen directly from the electron microscope that component P-I is more loose than other peptides,and its structure is like flocculent network.Simulated digestion in vitro shows that component P-I has higher activity.The results of LC-MS/MS showed that there were 17 peptides with peptide ranker score greater than 0.85 in P-I.GO and KEGG protein function annotation showed that the peptides prepared by enzymatic hydrolysis were involved in many biological processes,such as biological system,immune system,digestive system and so on.According to the literature reports and the function prediction website,the identified peptides were screened to synthesize VYPFPGPIPN,LGFPL and SLVYPFPGPIPN.The three peptides have antioxidant activity,but there are differences in antioxidant activity.At the same time,there are both positive and negative synergistic effects among synthetic peptides.