Preparation And Functional Properties Of Watermelon Seed Peptide And Calcium Chelate

Watermelon seed is the seed of watermelon,a Cucurbitaceae plant.It has high protein content,balanced distribution of amino acids,rich in arginine,glutamic acid,aspartic acid,leucine and other essential amino acids.It is a high-quality protein source with great development potential.At present,the research on watermelon seed protein at home and abroad mainly focuses on the extraction and separation of protein,structural characterization and the preparation of antioxidant peptides.In recent years,peptide calcium chelates have attracted much attention because of their good stability,high calcium bioavailability,and the dual nutrition of active peptides and calcium.Our research group has also confirmed that watermelon seed peptide has antioxidant activity and digestive stability.The peptide contains aspartic acid(Asp),glutamic acid(Glu)and cysteine(Cys).These amino acids are typical amino acids that can chelate with calcium ions.Therefore,watermelon seed peptide is likely to be a high-quality peptide source for peptide calcium chelation.However,there is no report on the preparation of peptide chelated calcium from watermelon seed protein so far.Based on this,the peptide fragments with different molecular weights were obtained by ultrasonic assisted enzymatic hydrolysis and fractionation of watermelon seed protein,and the peptide calcium chelate was prepared,and its structure was characterized.On this basis,the stability,digestion and absorption characteristics of peptide calcium chelate were studied,which laid a theoretical and scientific foundation for the efficient preparation of watermelon seed peptide calcium chelate and the development of calcium supplements.The main contents and results of this paper are as follows:1.Ultrasonic assisted enzymatic hydrolysis of watermelon seed proteinTaking the degree of hydrolysis as the index,the optimal technological parameters for ultrasonic assisted enzymatic hydrolysis of watermelon seed protein were obtained through single factor step-by-step Optimization: substrate protein concentration 2%,ultrasonic frequency combination 40/60 k Hz,power density 120W/L,ultrasonic pretreatment 45 min,p H 9.0,alkaline protease addition 8000 U/g,enzymatic hydrolysis temperature 50 ℃,enzymatic hydrolysis time 4 h.Under these conditions,the degree of hydrolysis of watermelon seed protein can reach 23.37%,which is 7.25% higher than that of traditional enzymatic hydrolysis.2.Study on the preparation of watermelon seed protein peptide calcium chelateThrough single factor and response surface optimization,the preparation parameters of watermelon seed protease hydrolysate protein peptide chelating with calcium were as follows: the mass concentration of protein peptide mixture was 3%,the mass ratio of watermelon seed protein peptide to calcium was 6:1,the solution p H was 8.2,the temperature was 40 ℃,the chelating time was 90 min,and the volume ratio of absolute ethanol to chelating solution was 8:1.Under these conditions,the calcium binding capacity of watermelon seed protein peptide chelated calcium reached 57.44 mg/g,indicating that watermelon seed protein peptide is a potential resource for the preparation of calcium chelates.3.Preparation and structural characterization of watermelon seed peptide calcium chelate(1)Four peptide fragments,W1(Mw < 1 k Da),W2(Mw 1 ～ 3 k Da),W3(Mw 3～ 5 k Da)and W4(Mw > 5 k Da),were obtained from watermelon seed protease hydrolysate by ultrafiltration.The calcium binding amounts of the four peptide calcium chelates were 72.32 mg/g,73.23 mg/g,64.16 mg/g and 51.79 mg/g,respectively.Except that the calcium binding capacity of W4-Ca was lower than that of non ultrafiltration,the calcium binding capacity of the other three peptide fragments was higher than that of non ultrafiltration hydrolysates.It showed that watermelon seed peptide with Mw < 5 k Da was more likely to chelate with calcium ion.(2)The structure of watermelon seed peptide calcium chelate was characterized by UV and FTIR.The results showed that the N-H,C-N,C=O,-COOH and-OH groups in the peptide fragments participated in the chelation reaction of calcium ions;The amino acids involved in the chelation reaction mainly include aspartic acid(Asp),glutamic acid(Glu)and cysteine(Cys),and the main binding sites are the carboxyl group on the side chain of Asp and Glu and the sulfhydryl group on the side chain of Cys.4.Study on the stability,digestion and absorption characteristics of watermelon seed peptide calcium chelate(1)Watermelon seed peptide calcium chelate has good thermal stability and acid-base stability.Under the conditions of 60～90 ℃ and p H 5-9,the calcium retention rate can reach 80%～90%.The watermelon seed peptide calcium chelate has good anti digestibility.After the combined action of pepsin and trypsin,the calcium retention rates of W1-Ca,W2-Ca and W3-Ca all reach more than 90%,indicating that the watermelon seed peptide calcium chelate has good stability.(2)The results of Caco-2 cell transport test showed that when the concentration of chelate calcium was in the range of 0.5～1.5 mmol/l,it showed concentration dependence,and the bioavailability of calcium was higher than that of calcium chloride.When the calcium ion concentration of chelate was 1.5 mmol/l,the bioavailability of W1-Ca,W2-Ca and W3-Ca were 42.03%,41.55% and 38.94%respectively,significantly higher than that of calcium chloride(27.51%,all p < 0.05),indicating that W1-Ca,W2-Ca and W3-Ca can effectively improve the bioavailability of calcium.