Screening And Evaluation Of Bioactive Peptides From Acaudina Molpadioides

Objective:In this paper,two aqueous phase solvent systems based on eutectic solvents and medium-pressure preparative liquid phase combined technology are used to efficiently separate the active peptides in the polypeptides of Acaudina molpadioides,and the antihypertensive,antioxidant and hypoglycemic activities of each isolated component were studied by the chemical and biological evaluation methods.The above research will provide a basis for the research and development of Acaudina molpadioides active peptide functional food,special medical food and new drugs.Methods:1.The preparation process of Acaudina molpadioides polypeptide?1?The optimum enzymatic hydrolysis conditions was determined by orthogonal experiment with the angiotensin converting enzyme?ACE?inhibition rate as the evaluation index.?2?The main nutrients of Acaudina molpadioides and the amino acid content of Acaudina molpadioides polypeptide was determined by the national standard method.The amino acid score,chemical score,and essential amino acid index of Acaudina molpadioides and its polypeptide were calculated by the amino acid pattern and the whole egg protein model respectively,and the nutritional value was evaluated.Based on the above results,Acaudina molpadioides and its polypeptide nutritional value was evaluated2.Separation and purification of biologically active peptides from Acaudina molpadioides?1?The optimum separation and enrichment conditions of DES-based ATPS to Acaudina molpadioides active peptides was determined by a single factor experiment with the ACE inhibition rate as the evaluation index.?2?The middle-pressure preparative liquid chromatography was used to further separate and purify the enriched parts of the active peptides of Acaudina molpadioides with the best chromatographic separation conditions.3.Activity study on the Acaudina molpadioides active peptidesTo evaluate the ACE inhibitory ability of different fractionations of bioactive peptides from Acaudina molpadioides.The antioxidant capacity of different components of the peptide components was evaluated systematically by radical scavenging ability and total antioxidant capacity.Based on the glucose tolerance test in mice,the analysis of?-amylase inhibitory activity and the analysis of?-glucosidase inhibitory activity,the hypoglycemic ability of different fractionations of bioactive peptides from Acaudina molpadioides was evaluated systematically.Results:1.The preparation process of Acaudina molpadioides polypeptide?1?The optimal hydrolysis conditions:flavourzyme 1.5%?w/w?,substrate concentration 1/8?w/v?,hydrolysis time 4 h,pH 6.5.The IC₅₀ of enzymatic hydrolysis products is 4.0 mg/mL.?2?The protein content of Acaudina molpadioides is 44.2 g/100 g,the polysaccharide content is 2.2 g/100 g,and it is a fat-free food.The essential amino acids of Acaudina molpadioides and its peptides are complete and abundant.Acaudina molpadioides and its peptides were complete and rich in essential amino acids.Its first restricted amino acid were tryptophan,and the content of glycine and glutamic acid were higher than others.2.Separation and purification of biologically active peptides from Acaudina molpadioides?1?The optimal ATPS:CHCl/1,4-butanediol?12 g,1:2?:K₂HPO₄?0.95 g/mL?:H₂O.?2?The optimal conditions of middle-pressure preparation liquid chromatographic:ODS-BP C₁₈ preparation column?20.0×250 mm,5?m?;Flow rate8.0 mL/min;Detection wavelength 210 nm;Injection volume 1 mL;Column temperature 25?;The mobile phases consisted of 0.1%?v/v?trifluoroacetic acid water?A?and acetonitrile with 0.1%?v/v?trifluoroacetic acid?B?using a gradient elution of 5%B at 0-10 min,5-20%B at 10-40 min,20-40%B at 40-60min.3.Activity study on the Acaudina molpadioides active peptides?1?The ACE inhibitory ability of different fractionations of bio-peptides from Acaudina molpadioides is Hc>Hb>Ha.The Hc has the highest ACE inhibitory ability,wich is 74.78%at a concentration of 1 mg/mL.?2?The DPPH·scavenging ability of different fractionations of peptides from Acaudina molpadioides is Hc>Ha>Hb.The Hc had the highest the DPPH·scavenging ability,which is 76.12%at a concentration of 8 mg/mL.The⁺·scavenging ability is Hc>Hb>Hc.Hc has the highest⁺·scavenging ability,which was94.92%at a concentration of 8 mg/mL.The O₂^-·scavenging ability is Hc>Hb>Ha.The Hc had the highest O₂^-·scavenging ability,which is 94.33%at a concentration of 4 mg/mL.The total antioxidant capacity is Hc>Hb>Ha.The Hc has the highest total antioxidant capacity,which is 0.89 at a concentration of 8 mg/mL.?3?The glucose tolerance test in mice showed that 0.08 g/kg Acaudina molpadioides active peptide can significantly inhibit the increase of blood sugar caused by gavage starch?p<0.01?.The blood glucose level and the area under the blood glucose curve?AUC?were significantly lower than those of the control group after administration of starch for 2 hours?p<0.05?.The?-glucosidase inhibitory ability of fractionations of Acaudina molpadioides active peptide is Hc>Ha>Hb.The?-amylase inhibitory ability is Ha>Hc>Hb.Conclusion:The different fractionations of Acaudina molpadioides have certain antihypertensive,antioxidant and hypoglycemic activities,and the Hc fractionation has the highest activity.The combined method of ATPS based on DES enrichment and the middle-pressure preparative chromatography purification was established in this article,which provides a new separation mode for rapid separation and purification of Acaudina molpadioide active peptides.