Method Development Of Collagen Detection And Its Application In Food

As an important animal protein,collagen is widely used in food,beauty,tissue engineering,biological materials and other fields.Due to collagen and its degradation products of different types and different animal origins have no significant differences in amino acid composition,isoelectric point,molecular weight,hydrophobicity,immunogenicity,etc.,this makes collagen quality control very difficult.Traditional detection methods(such as SDS-PAGE,capillary electrophoresis,colorimetry,high performance liquid chromatography,immunological detection,etc.)for collagen detection have certain limitations in terms of quantitative and qualitative research.There are significant differences in the amino acid sequence of collagen of different types and different animal origins.These sequence differences can be used as the basis for type identification or animal traceability.Detection of the different sequences by appropriate methods provides a theoretical basis for the quality control of collagen.This study is based on high performance liquid chromatography-mass spectrometry,and aims to establish a collagen detection and analysis method.High performance liquid chromatography-mass spectrometry combines the advantages of high resolution of chromatography for complex samples and high selectivity,high sensitivity of mass spectrometry,and the ability to provide relative molecular mass and structural information.This method can separate and quantify the marker peptides of different types of collagen in a large number of peptides after collagen hydrolysis.The main research results obtained in this paper are as follows:(1)The feasibility of collagen type recognition and quantitative detection based on marker peptides was studied by model proteins.Bovine type I collagen was used as a model protein,and marker peptides were compared by sequence comparison.Qualitative analysis of collagen degradation products was performed by high performance liquid chromatography-ion trap mass spectrometry,and bovine type I collagen marker peptide GFSGLDGAK was screened.The results show that the marker peptide has a strong signal in the mass spectrometry data,and the peptide fragment of the secondary mass spectrum is clear.The selected peptide can be used as the marker peptide of the corresponding animal collagen.This method can be used to screen marker peptides of other species or other types of collagen.The experiment further studied the feasibility of the method for type I,III collagen and rat I,III collagen type recognition,and selected marker peptides for type recognition,including porcine type I collagen marker peptide GETGPAGPAGPVGPVGAR,porcine type III Collagen marker peptide GPP*GAVGPSGPR,rat type I collagen marker peptide GPAGPSGPIGK,rat type III collagen marker peptide GPVGPHGPPGK.(2)Screening of sample processing methods and optimization of analysis conditions.Using bovine type I collagen as the actual sample,the pretreatment conditions were optimized,and the results showed that when the sample to trypsin ratio was 50:1,the best results were obtained by enzymolysis at 37? for 16 h.In the pre-treatment step,the sample is subjected to two heat treatments without damaging the marker polypeptide and affecting the accuracy of quantification.In mass spectrometry,ion secondary mass spectrometry can improve the stability and repeatability of the method.Under optimized conditions,the signal remains stable.The same sample was injected three times,and the relative standard deviation of the measured peak area values was 0.38% to 1.45%.(3)A quantitative detection method for collagen based on marker peptides was established.After the qualitative analysis screened the marker peptides and optimized the pretreatment conditions,the marker peptide,GFSGLDGAK was chemically synthesized.Quantitative analysis of 90 bovine type I collagen samples proved the feasibility of quantitative analysis based on marker peptides.The methodological research of quantitative analysis was completed,and the stability,uniformity and fixed value of collagen products were analyzed by this method,and the bovine type I collagen content in this product was determined to be 0.926±0.014mg/mg,which proved the accuracy of the method.(4)Application of marker peptide-based detection method in the quality control method of collagen degradation products.The established method was used to detect collagen-based products-Ejiao and raw materials-skin,which proved the feasibility of this method for the detection of collagen-based products and raw materials.Donkey skin collagen peptides(MW 1077.6Da)were detected in the Ejiao samples.Although collagen in Ejiao products has been degraded,the greater the proportion of protein peptides with molecular weights above 1077.6 Da,the higher the content of marker peptides in the sample.The content of collagen in pig skin and rat skin was measured.The results showed that the collagen content in the skin first increased with age,and the collagen content gradually decreased after adulthood.The experiment accurately determined the type I and type III collagen content in pig skin and rat skin at different ages.The content of type I collagen in pig skin at 3 weeks of age was 0.256mg/mg,and the content of type III collagen was 0.104mg/mg.Subsequently,the content of type I collagen continued to increase and the content of type III collagen continued to decrease.In 12-week-old pig skin,the type I collagen content was 0.497mg/mg,and the type III collagen content was 0.079mg/mg.At the age of 7 weeks,the collagen content in the rat skin was the highest at 0.7738mg/mg,and the type I collagen content was 0.7329mg/mg.The determination of skin collagen content at different ages provides a basis for related research such as anti-aging,or for the production of collagen using skin as the raw material,and provides a basis for selecting suitable skin raw materials.Collagen detection method based on marker peptides has broad application prospects in collagen-containing foods,medicines,collagen-based medical materials,etc.