Methodological Study On The Quantitative Of Edible Gelatin By Label-free Biomass

As an important additive in the food industry,gelatin is widely used in the production of candy,ice cream and other products.Internationally,factors such as food safety,religious beliefs,and vegetarianism have made consumers pay close attention to the types of gelatin in the marketed products(especially pig gelatin and bovine gelatin with the largest market share).In China,Ejiao(donkey skin gelatin),as a traditional Chinese medicinal material,has been expanding its market scale.However,the lack of donkey skin resources and huge commercial interests have frequently exposed the problem of adulteration of Ejiao.The identification of the authenticity of Ejiao products is a common concern for the Ejiao industry and consumers.Based on the above considerations,if a simple,fast,and accurate method of gelatin identification and quantification can be established,it will help consumers eliminate their concerns about gelatin products,and will also help combat the illegal acts of Ejiao adulteration.Therefore,based on the identification of characteristic peptides of gelatin,this paper uses high-performance liquid chromatography-high-resolution mass spectrometry(HPLC-MS/MS)to establish label-free quantitative methods for bovine gelatin and donkey-hide gelatin,and applies them to complex systems to evaluate their accuracy.The main conclusions can be summarized as follows:(1)Use HPLC-MS/MS to detect and analyze the bovine gelatin after trypsin digestion by mass spectrometry and compare the obtained bovine gelatin peptide mass spectrometry results with the bovine gelatin theoretical characteristic peptide database and the bovine gelatin traceability database constructed by the research group in the early stage.After comparison,17 characteristic peptides with quantitative potential were finally found.Further analyze the peak area diagrams of 17 characteristic peptides at different concentrations,construct a standard curve with the sample concentration and the peak area corresponding to the characteristic peptide as the horizontal and vertical coordinates,and calculate the linear regression equation.In order to make the quantitative effect more accurate,the characteristic peptides with regression equation R²?0.99 were selected to screen out 7 characteristic peptides:bovine gelatin?₁ chain⁴⁵¹GEPG*PTGIQGP*PGPAGEEGK⁴⁷⁰,¹⁰³⁹GETGPAGP*PGA*PGAPGAPGPVGPAGK¹⁰⁶⁴,?₂chain³²⁸GI*PGPVGAAGATGAR³⁴²,³⁸²GSTGEIGPAGP*PGP*PGLR³⁹⁹,⁵⁷⁴GI*PGEFGL*PGPAGAR⁵⁸⁸,⁶²²GE*PGVVGA*PGTAGPSGPSGL*PGER⁶⁴⁵ and⁸³¹SGETGASGP*PGFVGEK⁸⁴⁶ have 7 characteristic peptides,which are used to draw quantitative equations.Finally,the specificity and reproducibility of these peptides are tested.They all meet the standards and can be used for bovine gelatin Quantitative testing.(2)Combine MS/MS to analyze the peptide sequence and amino acid modification of Ejiao enzymatic hydrolysis,compare the obtained peptide information with Ejiao's theoretical characteristic peptide database,screen out 5 characteristic peptides,which are further compared with the Ejiao traceability database established by the research group to determine that these five characteristic peptides all have quantitative potential.Then five sample concentrations were selected for quantitative analysis,the sample concentration and characteristic peptide peak area were used as the abscissas and ordinates to establish a standard curve and fit a linear regression equation.The regression equation R²?0.99 characteristic peptides was selected,and finally 4peptides that satisfy the quantitative requirements were obtained.The characteristic peptides are ⁴⁷²GE*PGPTGL*PGP*PGER⁴⁸⁶,¹⁰⁶⁶GEAGPAGPAGPIGPVGAR¹⁰⁸³,¹⁰⁶²SGDRGEAGPAGPAGPIGPVGAR¹⁰⁸³ of the?₁ chain of Ejiao and⁹⁷⁷GE*PGPVGSVGPVGAVGPR⁹⁹⁴ of the?₂ chain.Finally,the specificity and reproducibility of these 4 peptides are analyzed,and the results meet the quantitative conditions.Therefore,these four characteristic peptides can be used for the quantification of donkey-hide glue and can provide data support for combating illegal behaviors of donkey-hide glue adulteration.(3)In order to expand the application scenarios of the label-free quantification method of cattle gelatin and donkey-hide gelatin,apply it to the quantification of target gelatin in a complex system to test its accuracy.First,analyze the influence of food matrix on the detection of characteristic peptides of gelatin.The results show that food matrices such as sorbitol and glycerin have little influence on the detection of characteristic peptides.Secondly,the mass spectrometry analysis of the bovine gelatin-donkey-hide gelatin mixed gelatin system is carried out by HPLC-MS/MS.Through the mass spectrum peaks and peak areas of four quantitative characteristic peptides of Ejiao,the recovery rate and the reproducibility of characteristic peptides of Ejiao are analyzed.The results show that the characteristic peptide?₁:⁴⁷²GE*PGPTGL*PGP*PGER⁴⁸⁶ has a good quantitative effect and can be used for accurate quantification of donkey-hide gelatin in the mixed system;finally,bovine gelatin and porcine gelatin that are supplemented with sorbitol and glycerin are used as raw materials to prepare gelatin film,and the prepared gelatin film is analyzed by mass spectrometry using HPLC-MS/MS technology.Through the mass spectrum peak and peak area of the 7 quantitative characteristic peptides,the quantitative recovery and reproducibility were studied,and the results showed that the recovery and reproducibilityof?₂:³⁸²GSTGEIGPAGP*PGP*PGLR³⁹⁹and?₂:⁶²²GE*PGVVGA*PGTAGPSGPSGL*PG ER⁶⁴⁵ meet the quantitative conditions and can be regarded as complex Quantitative characteristic peptides of bovine gelatin in the system.