Study On The Extraction Technology And Properties Of Sika Deer Antler Protein Peptides

Purpose:Based on the kjeldahl method and the Folin-Ciocalteu method,the protein polypeptide of pilose antler of sika deer was extracted by enzymatic hydrolysis.Through optimizing the enzymatic hydrolysis process,a large amount of pilose antler protein polypeptide can be obtained,and the maximum value of the protein polypeptide in pilose antler can be obtained.Then,the membrane separation technique was used to isolate and purify pilose antler protein polypeptide.The molecular weight distribution of protein polypeptides was studied by Tricine-SDS-PAGE analysis and HPGFC analysis.And the kinds and contents of amino acids in protein polypeptides were determined by precolumn derivation.The above experiments provide experimental basis for the study of the properties of pilose antler polypeptide.And it is expected to lay a theoretical foundation for the study of quality standard of pilose antler protein polypeptide.Materials and methods:This experiment is to study the pilose antler of sika deer.At first,the protein content in pilose antler powder of sika deer was determined by automatic kai-type nitrometer.Then,the trichloroacetic acid solution?TCA?precipitates macromolecules of protein.The applicability of the method for determination of protein polypeptide in enzymatic hydrolysis was investigated by using the Folin-Ciocalteu method.Then Alcalase AF 2.4L alkaline protease was used to treat pilose antler powder.Orthogonal design of L₉?3⁴?was carried out with hydrolysis degree and polypeptide content as evaluation indexes.The content of hydrolysis and protein polypeptide was determined by the formol titration method and the Folin-Ciocalteu method.The effects of four factors including enzymolysis temperature,PH,enzyme dosage and substrate concentration on the enzymatic hydrolysis process were investigated.To optimize the extraction technology of velvet antler protein polypeptide.It provides material basis for later experiments.The ultrafiltration membrane of5KDa was used to separate and purify the enzymatic hydrolysate.Then,the Tricine-SDS-PAGE method was used to determine the optimum electrophoresis conditions by investigating the concentration of different gel separators.The molecular weight distribution of purified protein polypeptides was detected.At the same time,the relationship between the relative molecular mass logarithm and the retention time of five standard samples of bovine serum albumin?five components?,ovalbumin,cytochrome C,insulin and bacitide was determined by high performance gel filtration chromatography?HPGFC?of COSMOSIL5DioL-300-II gel filtration column.Drawing the standard curve and calculating the molecular weight to determine the distribution of molecular weight of pilose antler protein polypeptide.Finally,pre column derivatization was used to separate and purify protein peptide samples.And high performance liquid chromatography?HPLC?was used to determine the types and contents of hydrolyzed and free amino acids in protein polypeptides by amino acid ODS column.Results:1.The total protein content of Cervus nippon antler was 56.48%by the automatic kjeldahl method.The correlation coefficient of the standard curve for the determination of pilose antler protein polypeptide by Folin-Phenol method was 0.9990 after macromolecular protein was precipitated by TCA solution.The RSD values of stability,precision and repeatability tests were less than 2.0%,the average recovery was between 95%and 97%,and the RSD values were less than 2.0%.2.Alcalase AF 2.4L alkaline protease enzymatic extraction of velvet antler protein polypeptides was the best technology:at 55?,PH7.5 conditions,substrate concentration of2%?m/v?,adding 8%?v/m?enzyme reaction for 2 hours.A clear and transparent protein polypeptide solution was obtained by batch extraction and 5KDa ultrafiltration membrane.3.The molecular weight distribution of velvet antler protein polypeptides analyzed by Tricine-SDS-PAGE method showed that the molecular weight distribution of velvet antler protein polypeptides after enzymatic hydrolysis ranged from 2kDa to 50kDa,mainly below15kDa.A band of 50kDa was missing and the molecular weight distribution of protein polypeptides ranged from 2kDa to 37kDa.4.The molecular weight of protein polypeptides in the enzymatic hydrolysate of velvet antler determined by HPGFC was mostly about 11.9kDa.The Protein Polypeptides Purified by5kDa ultrafiltration membrane were concentrated about 11.6kDa,and the purity was higher than that of enzymatic hydrolysate.5.There were 17 peaks in the chromatogram of purified protein peptides?except solvent peaks?,of which 16 were known amino acid peaks and 1 was unknown amino acid peaks.In this method,the R of each amino acid standard was more than 0.9985,RSD of precision,stability and repeatability test was less than 3.0%,average recovery was more than 96.4%,RSD was less than 2.5%.Conclusion:1.The content of protein in antler of sika deer is 56.48%,which indicates that the main substance in antler of sika deer is protein,which provides material basis for the following experimental study.The protein peptide content was determined by the Folin-Ciocalteu method after TCA solution precipitated large protein.This method is stable,reasonable and feasible.It is suitable for the determination of protein polypeptide in enzymatic hydrolysate of velvet antler.2.The Alcalase AF 2.4L alkaline protease was used to hydrolyze velvet antler.The degree of hydrolysis was 9.22%and the content of protein polypeptide was 36.28%.The extraction rate of velvet antler protein polypeptide was greatly improved.The ultrafiltration membrane separation technology has the advantages of easy operation,low cost,batch treatment and protection of heat sensitive substances.3.The Tricine-SDS-PAGE method was used to investigate the distribution of protein polypeptides.The results showed that the molecular weight distribution could be seen directly,and the separation rate and accuracy were high.4.The HPGFC method is convenient,fast,time-saving and labor-saving.It can provide a simple,fast and reliable method for the determination of protein polypeptide molecular weight in pilose antler.5.The pre-column derivatization was used to treat the samples.The method was stable,reliable and accurate for the determination of amino acids in pilose antler by HPLC.