Preparation Of Sheep Bone Collagen Peptide-calcium Chelate Using Enzymolysis-fermentation Methodology And Its Structural Characterization And Stability Analysis

In this study,calcium chelate was prepared from sheep bone meal.With the free calcium content as the index,the two enzymes with the best enzymatic hydrolysis effect were screened by single factor test and response surface test optimization method,and the sequential enzymatic hydrolysis was conducted.The obtained enzymatic solution was fermented with lactic acid bacteria and the fermentation conditions were optimized.On this basis,the bone calcium and polypeptide of sheep bone were chelated with anhydrous ethanol to obtain collagen polypeptide-calcium chelate.The results are as follows:?1?Screening of optimal enzymes:the optimal conditions for the hydrolysis of three enzymes?papain,pepsin,trypsin?to extract soluble calcium from sheep bone were:enzymatic hydrolysis temperature 55?,40?,45?;The enzymatic hydrolysis time was 4 h,4 h and 5 h.Enzymatic hydrolysis at pH 6.0,2.5,7;Add enzyme amount 5000 U/g,5300 U/g,1500 U/g.The free calcium content reached 78.30 mg/100g,1008.68 mg/100g and 45.68 mg/100g,respectively.Therefore,papain and pepsin were selected for subsequent experiments.?2?The optimal enzymatic hydrolysis combination of the two proteases was as follows:under the conditions of the optimal enzymatic hydrolysis temperature,enzymatic hydrolysis pH and enzymatic amount of the two enzymes,papain was hydrolyzed for 3 h at first,followed by pepsin for 3 h,and the final free calcium content was the highest,reaching1839.14 mg/100g.?3?The optimal technological conditions for the fermentation of enzymatic hydrolysate:the glucose dosage was 3%,the inoculation dosage was 6%,the fermentation time was 24.5 h,the fermentation temperature was 37?and the initial ph was 6.5.The free calcium content reached 2212.58mg/100g.?4?The calcium chelation ability was 42.57±0.09%;The calcium content in the peptid-calcium chelate was 15.81%±0.06%.?5?Ultraviolet absorption spectrum analysis revealed the formation of new substances after the chelation of collagen peptide and calcium;The change of fluorescence spectrum indicates that the chelation reaction may be caused by the interaction between the side chain residues and Ca²⁺.Fourier transform infrared spectroscopy speculated that the main binding sites of collagen polypeptides were carboxyl oxygen,hydroxyl oxygen,amino nitrogen and oxygen on the peptide chain,and these groups cooperated with Ca²⁺to form peptide-calcium chelate.The emergence of new absorption peaks in DSC spectrum and new diffraction peaks in X-diffraction spectrum further proves the occurrence of chelation reaction.Amino acid analysis showed that through the residues of amino acids carboxyl group in the reaction process of calcium binding;SEM also indicated that the initial structure of the peptide was changed due to the interaction between the peptide and calcium ions during the chelation process.?6?Stability of the peptide-calcium chelate was studied:the peptide-calcium chelate was relatively stable at different temperatures;The peptide-calcium chelate was more stable at different pH values than in acidic,neutral and alkaline environments.?7?In vitro digestion of peptide-calcium chelates:after digestion with pepsin,trypsin,and gastric+trypsin,the calcium content of peptide-calcium chelates decreased?p<0.05?to87.3±2.3%,73.5±4.2%,and 57.6±3.8%,respectively,compared with the control group.The results showed that the peptid-calcium chelate was stable in the gastrointestinal environment and could promote calcium absorption.?8?The antioxidant activity of peptide-calcium chelate was preliminarily studied:compared with collagen peptide,the antioxidant activity of peptide-calcium chelate was improved.The purpose of this experiment is to lay a foundation for the high value utilization of sheep bone and the development of related products and explore its potential application value.