Study On Binding Mechanism And Properties Of Calcium-Binding Soy Peptides

Calcium is an indispensable inorganic mineral element for human growth and maintenance.Calcium deficiency may lead to a series of diseases such as chondropathy and rickets.Traditional calcium supplements often have defects such as easy sedimentation,low absorption and utilization,and toxic side effects.Calcium binding peptides can effectively solve these problems.Soy protein is rich in source,low in price,and the preparation process of soy peptides is simple,which is a good choice for preparing calcium binding peptides.It is not clear about the binding mode of calcium-binding peptides.Based on the study of calcium-binding soy peptides,the paper studied the interaction between Ca²⁺and characteristic amino acids and peptides in order to understand the interaction between calcium and peptides.Provided a theoretical basis for the preparation and application of calcium-binding peptides.Firstly,the effect of the composition and amino acid composition of the peptides on calcium-binding ability was examined.The calcium-binding abilities of four peptides were compared,including soy peptides,pea peptides,corn peptides and fish collagen peptides?>80%of peptides with molecular weight less than 1000 Da?.It was found that the calcium-binding abilities of soy peptides and pea peptides were found up to 60.34 mg/g and 61.26 mg/g,respectively.Amino acid analysis showed that the proportions of Glx+Asx in soy peptides and pea peptides were 34.33%and 35.03%,respectively,which may be the important amino acids involved in the binding reaction.Soy peptides was prepared from the soybean meal by enzymatic hydrolysis,centrifugation and membrane separation.It was found that the calcium binding ability can up to 64.91 mg/g after the pH 7.0 centrifugation and membrane separation.The amino acid analysis of calcium-binding soy peptides showed that the contents of Glx and Asx were 35.69% and 14.14%,respectively.And the Cys content was 3.20%,which was doubled compared with peptides before binding.The F1 component peptides had a great calcium-binding ability which was isolated by RP-HPLC,and its calcium-binding ability can be up to 158.63 mg/g.Through peptide sequence identification,it showed that the F1components were mainly Glu,Gln and Asp,which accounted for more than 60%.Secondly,the binding mode of the calcium-binding peptides was studied.The results of Zeta potential analysis and X-ray diffraction showed that a new compound was produced after the soy peptide was bound with Ca²⁺.The ¹H NMR results showed that the characteristic peak of the carbonyl group migrated from 3.55 ppm to 2.60 ppm after binding.The integrated results showed that the integral values of the carbonyl peak and the amine peak increased,indicating that the carboxyl group and amine group might be participating in the reaction.FTIR results further showed that the-COO^-stretching vibration moved from 1400 cm^-1 to 1408 cm^-1,C=O stretching vibration moved from 1658 cm^-1 to 1650 cm^-1,N-H stretching vibration peak moved from 3338 cm^-1 to 3359 cm^-1,C-O stretching vibration moved from 1114 cm^-1 to 1110 cm^-1,while the N-H bending vibration at 1558 cm^-1 did not change,indicating that carboxyl oxygen and primary amide amino nitrogen were the binding sites of Ca²⁺.The FTIR analysis of calcium-binding showed that in addition to carboxyl oxygen and amino nitrogen,the side chain thiol also was involved in the binding with Ca²⁺.The FTIR of Glu and Asp indicated that the binding sites of two amino acids were consistent with soy peptides.Further,by LC-MS analysis,a possible chemical structure of calcium-binding GSH and calcium binding with two amino acid compounds was obtained.These provided a powerful basis for us to understand the combination of calcium-binding soy peptides.On this basis,the thermodynamic study of the interaction between peptide and Ca²⁺by isothermal titration calorimetry showed that the main forces binding to Ca²⁺at pH 7.8 and 40°C were van der Waals forces and hydrogen bonds,but when sulfhydryl groups participated in the reaction,the main force was ionic interaction.Finally,the stability of the calcium-binding soy peptides was investigated.The calcium-binding soy peptides had a certain stability in phosphate buffer,and the calcium retention rate of the calcium-binding soy peptides could reach 62.17%in 20 mmol/L phosphate buffer.The stability of calcium-binding soy peptides under different pH conditions showed that the calcium-binding soy peptides did not dissociate significantly in the range of pH 2.0⁸.0,and had good pH stability.DSC and TGA results showed that the calcium-binding soy peptides had better thermal stability than the soy peptides.The results of in vitro simulated digestion showed that the calcium dissociation rate of calcium-binding soy peptides was 14.58%when digested in simulated gastric juice for 30 min,and the calcium dissociation rate increased to 43.51%at120 min.While simulating the duodenum and small intestine digestion,soy peptides had a good stability during digestion and the calcium dissociation rate was always below 10%.