Study And Application Of Proteomics Technology In Adulteration Identification Of Donkey-hide Gelatin Pastry And Gelatin Drink

In recent years,due to the shortage of donkey skin resources,the price of ejiao has risen sharply,and a large number of fake ejiao made from horses,mules,pigs,cows and sheepskins have appeared in the market.Recently,it has been reported that camel skin and deerskin have been adulterated.At present,fluorescence quantitative PCR,liquid chromatography-tandem mass spectrometry and other methods are used for the determination of ingredients from gelatin skin.Ejiao is both a food and a medicine.As a medicine,ejiao has been included in the 2020 edition of the Chinese Pharmacopoeia,which stipulates the identification of ingredients from donkey skin.The State Food and Drug Administration issued supplementary inspection methods for ingredients from cow skin in2012 and pig skin in 2019.At present,there is no national standard for the source detection of ejiao skin in food.The local standard of Shandong Province DB37/T 4009-2020 is the identification method for the source ingredients of ejiao skin and animal skin in food.It only detects the source ingredients of donkey,horse,ox and pig skin,but does not cover common large-scale livestock such as donkey,horse,mule,ox,pig and sheep.In addition,the adulteration of camel skin and deerskin has also been found in the flight inspection of ejiao production enterprises in recent years.The current quality standard of donkey-hide gelatin products has no specific detection method for adulteration,and the identification method of donkey-hide gelatin pharmacopoeia is liquid mass spectrometry for the detection of characteristic peptide,which is not the specific peptide of donkey,and can also be detected in horses with similar genetic relationship with donkey,so its specificity is not strong.At present,there are only supplementary testing methods for the possible adulterated skin ingredients of cattle and pigs,but there are no testing standards for the skin ingredients of horses,mules,sheep,camels,deer,etc.,so it is not good to distinguish whether there is counterfeit or adulterated.At present,quality standards can not control product quality effectively,and it is necessary to further improve the existing quality standards.Aiming at these problems,in order to better improve the quality of donkey-hide gelatin containing food to reduce security risks exist,this study of donkey-hide gelatin cake and gelatin drink(glue mucilage,donkey-hide gelatin,oral liquid,glue paste)explores the original ingredients adulterated,liquid ms method is adopted to establish the donkeys,horses,cattle,pigs,sheep source composition detection method,can be used to glue pudding and gelatin drink daily risk regulation.In this study,proteomics technology was used to carry out the following work:(1)Blast multi-sequence comparison was performed on type Ⅰ α1 and α2collagen sequences of donkey,horse,cattle,pig and sheep using bioinformatics analysis technology,and the different sequences of collagen of different species were determined,which further simulated trypsin digestion of collagen to obtain theoretical characteristic peptides.(2)Donkey-hide gelatin,horseskin glue,mule skin glue,yellow gelatin,pig skin glue,sheep skin glue and other different samples were prepared,digested by trypsin and analyzed by high resolution mass spectrometry.The data were systematically analyzed by Thermo Proteome Discovery 2.0 software using the Proteome of donkey,horse,pig,cattle and sheep as the database.The theoretical characteristic peptides analyzed and studied were verified in the above gum samples.(3)Donkey skin characteristic peptide 1-5,horse skin characteristic peptide,cow skin characteristic peptide and pig skin characteristic peptide were synthesized.Liquid chromatography-triple four-pole mass spectrometry was used to establish the identification method of donkey skin origin ingredients in donkey-hide gelatin cake and donkey-hide gelatin drink and the detection method of horse,cow,pig and sheep skin origin ingredients in donkey-hide gelatin cake and donkey-hide gelatin drink.(4)The homemade gelatin samples,market donkey-hide gelatin cakes and donkeyhide gelatin drinks were tested.It is worth noting that horseskin characteristic peptide can be detected in both horseskin glue and mule skin glue.The reason is that mule is the product of donkey and horse cross,and mule contains two sets of genetic information of horse and donkey,so horseskin characteristic peptide is also of screening significance for donkey and horse skin with the same illegal feeding.