Preparation,Purification And Bioactive Of Shellfish Umami Peptide

Bay scallops(Argopecten iridians)are one of the important marine cultured shellfish in China.Over the past 10 years,their production has accounted for more than10 % of the national shellfish production.At present,the production and sales of scallops are mainly for fresh sale and processing of scallops,while there are few deeply processed products.Low level overcapacity and high value-added product shortages have become bottlenecks in the development of shellfish industry,especially scallop industry.Protease hydrolysis is widely used in the utilization of protein resources,but there are also problems such as low hydrolysis efficiency and significant bitterness in products.In this paper,with the goal of deep development of bay scallop resources,the fresh enzymatic hydrolysate was prepared by optimizing the two-step enzymatic hydrolysis of endonuclease and exonuclease,and the umami peptides were separated,purified and evaluated for biological activity through nanofiltration,ultrafiltration,gel filtration chromatography,reverse phase high performance liquid chromatography,in vitro tests and in vivo tests,providing a theoretical basis for the development of bay scallop flavor active peptides.The main research results are as follows:Firstly,using the degree of hydrolysis,protein recovery,molecular weight distribution of enzymatic hydrolysis products,and sensory evaluation as measurement indicators,the enzymatic hydrolysis effects of four endonucleases(neutral protease,AP-200 A alkaline protease,papain,and trypsin)on bay scallop were comprehensively investigated.Finally,AP-200 A alkaline protease was determined as the best endonuclease for enzymatic hydrolysis of bay scallop.Through single factor and orthogonal optimization experiments,the optimal enzymatic hydrolysis conditions for AP-200 A alkaline protease were obtained as follows: enzyme addition of 2000 U/g,enzymatic hydrolysis time of 6 hours,p H 9,enzymatic hydrolysis temperature of 35 ℃.Under these conditions,the degree of hydrolysis of bay scallop protein was 26.07 %.Secondly,on the basis of endonuclease hydrolysis,the enzymatic hydrolysis effects of two kinds of exoenzymes(FF106 flavor protease and MF105 complex protease)were evaluated,and finally,FF106 flavor protease was determined as the best exoenzyme for enzymatic hydrolysis of bay scallop.Through single factor and orthogonal optimization experiments,the optimal enzymatic hydrolysis conditions for FF106 flavor protease were obtained as follows: enzyme addition amount 0.75 %,enzymatic hydrolysis time 5 h,p H 7,enzymatic hydrolysis temperature 55 ℃.Under these conditions,the degree of hydrolysis of bay scallop protein was 37.12 %.Through sensory evaluation,free amino acid composition,and molecular weight distribution analysis of enzymatic hydrolysis products,it was determined that the reason for the better flavor of two-step enzymatic hydrolysis was that the proportion of fresh and sweet amino acids in the two-step enzymatic hydrolysis solution reached 54.56 %,and the proportion of small molecular peptides and free amino acids with molecular weight less than 1000 Da reached 92.53 %.Thirdly,the enzymolysis products obtained from the two steps of enzymolysis were separated and purified step by step by means of nanofiltration,ultrafiltration,gel filtration chromatography,reverse phase high performance liquid chromatography,and the purified components were screened with sensory evaluation and taste dilution value as indicators.The results showed that the components of 200-3000 Da obtained by ultrafiltration were the most delicious components,with 53.30 % of the fresh sweet amino acids and 40.79 % of the essential amino acids;Four separation peaks were obtained by gel filtration chromatography,of which F1 was the strongest for flavor;The analysis by reversed phase high performance liquid chromatography showed that the F1 component was a mixed flavor peptide component.If a single flavor peptide component was to be obtained,further separation and purification were still needed.Finally,in order to further explore the biological activity of the Bay Scallop Flavor Peptide,its antioxidant activity was evaluated using in vitro experiments.The results showed that the Bay Scallop Flavor Peptide had an antioxidant activity with a DPPH free radical scavenging rate of 57.30 %,a hydroxyl free radical scavenging rate of81.14 %,and a ferrous ion chelating rate of 65.42 %.Using this as a starting point,we evaluated the activation of delicious peptides on SOD and ADH through in vitro experiments,and found that their activation rates were up to 19.83% and 18.18%,respectively,with potential antialcohol and liver protection activities.After that,the acute alcohol toxicity experiment in mice was designed to evaluate its antialcohol and liver protection effects in vivo.The results showed that bay scallop umami peptide can effectively improve the oxidative stress state of the body caused by alcohol by increasing the activities of antioxidant enzymes such as GSH-Px,SOD,and CAT in the body,reducing the accumulation of lipid peroxidation products in the body;By activating the activities of ADH and ALDH in the liver,it can reduce liver cells from damage caused by alcohol,effectively prolong the time to enter the drunken state,reduce the length of sober up,and have good antialcohol and liver protection activities.