Research On The Quality Of Tortoise Shell And Its Processed Products Based On Label Free And High-throughput Sequencing Technology

1 ObjectionTortoise shells are the carapace and belly of the Chinese Pond Turtle Chinemys reevesii(Gray),As one of the animal medicines,it has a long history and is widely used.Tortoise shell is mostly used as medicine in the form of decoction pieces of tortoise shell and vinegar tortoise shell,which is in great demand.The external visual characteristics and the internal chemical composition of animal medicinal materials will change greatly in the process of processing.The difficulty of research will also increase,and the related reports are less,so the modern research needs to be strengthened urgently.The protein or amino acid component is the main medicinal substance of tortoise shell.The 2015 edition of the Pharmacopoeia of the People's Republic of China only specifies the traits of tortoise shell and thin layer chromatography(TLC),which is far from the purpose of controlling its quality.In order to improve the quality control system of tortoise shell,pre-column derivatization AQC high performance liquid chromatography(HPLC)and Label Free(Label Free)were used to study the amino acids,peptide segments and proteins of tortoise shell and its processed products.Amino acids in tortoise shell and its processed products were determined by pre-column derivatization AQC-HPLC,and the contents and types of amino acids before and after processing were compared.Using Label Free technology,the protein of tortoiseshell and its processed products was studied by protein extraction,reductive alkylation and enzymatic hydrolysis,peptide quantification,mass spectrometry detection,and database search.According to the changes of amino acid-peptide-protein before and after tortoise shell processing,the quality of tortoise shell was comprehensively evaluated,which provided a reference for the subsequent development of turtle nail quality standards.At the same time,this experiment also focused on the microbial contamination of traditional Chinese medicines,and used morphological observation,microscopic identification and DNA barcode technology to collect high-throughput sequencing methods to compare the differences of surface fungi before and after processing of tortoise shell,to explore the diversity of surface fungi of tortoise shell and its processed products,so as to provide a basis for better quality control and evaluation of tortoise shell.2 Research content and research methods2.1 Sample CollectionSeventeen batches of raw tortoise shells and seventeen batches of vinegar tortoise shells were collected from drug markets in Anguo,Anhui,Bozhou,Sichuan and Chengdu,respectively,and major pharmacies in Beijing.Three batches of vinegar tortoise shells were made according to the processing method of tortoise shells in Chinese Pharmacopoeia 2015 edition.2.2 Precolumn Derivatization ACQ High Performance Liquid Chromatography for the Determination of Amino Acids in Tortoise Carapace and Its Processed ProductsThe content of amino acids in tortoise shell and its processed products was determined by pre-column derivatization AQC high performance liquid chromatography,including glutamic acid,glycine,aspartic acid,etc.The variety and content of amino acids in tortoise shell before and after processing were analyzed.2.3 Protein research of tortoise shell and its processed products based on Label Free TechnologyLabel Free method was used to study the proteomics of tortoise shell and its processed products.The differential proteins before and after processing were identified.The functional annotation of tortoise shell whole protein was made.The GO enrichment and Pathway enrichment of the differential proteins were analyzed.2.4 Culture and identification of surface fungi on tortoise shell and its processed productsThe surface fungi of tortoise shell and its processed products were eluted by sterile water and cultured by plate method.Morphological observation,microscopic identification and DNA barcode were used to identify the fungi on tortoise shell pieces.2.5 Fungal diversity of tortoise shell and its processed products based on high-throughput sequencingHigh throughput sequencing technology based on Illumina HiSeq sequencing platform was used to study the diversity of fungi on the surface of tortoise shell and its processed products.Species of fungi on tortoise shell surface were classified.3 Results3.1 According to the analysis of the kinds and contents of amino acids before and after processing,the total amount of amino acids in raw tortoise shell is significantly different from that in processed tortoise shell,and the total amount of amino acids in raw tortoise shell is higher than that in processed tortoise shell.The total content of amino acids in raw tortoise shell products ranged from 24.38%to 35.33%,with an average content of 28.77%.The total content of amino acids in processed tortoise shell products ranged from 19.51%to 25.67%,with an average content of 22.92%.There are 15 kinds of amino acids in raw tortoise shell and processed products.Compared with raw tortoise shell,processed products reduce two kinds of amino acids:Cys and Tyr.Among the total 15 amino acids,glycine,proline and glutamic acid were the three most abundant amino acids.The average contents of three amino acids in raw tortoise shell products were 6.04%,3.04%and 2.80%,respectively.The average contents in processed tortoise shell products were 5.40%,2.65%and 2.42%.The contents of amino acids in raw tortoise shell and processed tortoise shell are basically the same.3.2 The proteomics of tortoise shell and its processed products was studied based on Label Free technique.A total of 179 peptide segments and 55 proteins were retrieved.The protein molecular weight of tortoise shell and its processed products is mostly below 80 KDa.The correlation coefficient ranged from 0.17 to 0.48 between raw and processed tortoise shells,which indicated that the similarity of protein expression between raw and processed tortoise shells was different.There are 30 proteins in crude and processed turtle shell products,of which 16 proteins are up-regulated and 14 proteins are not significantly different.There are 23 specific proteins in crude turtle shell products,so there are 39 differential proteins between crude turtle shell products and processed turtle shell products.Functional annotation of tortoise-shell protein showed that tortoise-shell protein was mainly related to cellular and biological metabolic processes by GO analysis.GO enrichment analysis of differentially expressed proteins in tortoise shell and its processed products showed that the specific enrichment of GO involved many cellular metabolic processes,such as redox,monomer catabolism,monocarboxylic acid metabolism,organic matter decomposition,phosphoriboside metabolism,phosphoribose metabolism,etc.KEGG Pathway analysis showed that tortoise beetle protein mainly involved in human diseases,biological systems and other metabolic pathways.Pathway enrichment analysis of differential proteins in tortoise shell and its processed products showed that KEGG Pathway was involved in many metabolic pathways,such as HIF-1 signal,calcium signal,cell apoptosis,Alzheimer's disease,measles and systemic lupus erythematosus.3.3 Through the cultivation and identification of the fungi on the surface of tortoise shell,it was found that there were some differences between the raw and processed products of tortoise shell.The number of fungi on the surface of raw tortoise shell is more than that of vinegar tortoise shell,but the species are not more than that of vinegar tortoise shell.The surface of raw tortoise shell is mostly Aspergillus fungi,Aspergillus on the surface of vinegar tortoise shell and Penicillium fungi.Aspergillus flavus was not detected on the surface of raw tortoise shell and vinegar tortoise shell.3.4 Based on high-throughput sequencing technology,the diversity of fungi on the surface of tortoise shell and its processed products was studied.By evaluating the sequence number,sequence length,GC content,Q20 and Q30 quality values,Effective and other parameters of samples in each stage,the results show that the quality of sequencing data is excellent and the accuracy is high.At the same time,the ACE,Chao1,Simpson and Shannon indices of each sample were comprehensively measured,which proved that the sequencing results truly reflected the diversity of fungi on the surface of raw and processed tortoise shell products.Fungi with high abundance on tortoise shell surface include Aspergillus,Sporofilament,Candida,Pichia,Arthrobasidium,Cladosporium,Cordyceps,Verticillium and Penicillium.The composition of fungi on the surface of raw tortoise shell and processed tortoise shell is different.Aspergillus,Sporisorium and Candida were the most abundant fungi on the surface of crude products,followed by Sporisorium and Candida.The surface of processed products is mainly composed of Sporisorium and Aspergillus.4 Conclusion4.1 Precolumn derivatization AQC high performance liquid chromatography can qualitatively and quantitatively determine amino acids in tortoise shell and its processed products,which provides a basis for the subsequent formulation of quality standards for tortoise shell.4.2 Using Label Free technology to study the proteomics of tortoise shell and its processed products,we can get the information of different proteins and protein functions,which can lay a foundation for further exploring the molecular functions and pharmacological activities of tortoise shell.4.3 Morphological observation,microscopic identification and DNA barcode technology combined with high throughput sequencing can comprehensively study the fungal diversity on the surface of tortoise shell and its processed products.It can be used for better quality control and quality evaluation of tortoise shell medicinal materials.5 InnovationFor the first time,Label Free technique was used to study the proteomics of tortoise shell and its processed products.For the first time,morphological observation,microscopic identification,DNA barcode and high throughput sequencing techniques were used to evaluate fungal diversity on tortoise shell surface.