Identification Of Fungi On The Surface Of Six Kinds Of Chinese Herbal Medicines Such As Dried Tangerine Peel And Their Effects On The Growth Of Aspergillus Flavu

1 Objection1.1To study which fungi exist on the surface of six kinds of TCM,such as Citrus reticulate Blanco.Which are susceptible fungi.Is there a harmful fungus,Aspergillus flavus.1.2 To study whether there are differences in Aspergillus flavus between six kinds of TCM and mildew,and to explore the causes of aflatoxin contamination.1.3 To study the relationship between the water extract and the growth of Aspergillus flavus.2 Research content and research methods2.1 samples collectionIn March 2017,Citrus reticulata Blanco,Hordeum vulgare L.,Coix lacryma-jobi L.var.mayuen(Roman.)Stapf,batryticated silkworm,Chinemys reevesii(Gray),Pheretima aspergillum(E.Perrier)were collected from different sources in Beijing,among which were collected in 8 different retail drugstores.The samples was collected in sterile bags,sealed,stored at 4℃.2.2 Culture and Identification of Fungi on six kinds of TCMThe fungi on the Chinese medicine slices were washed with aseptic water,and the fungi were cultured by plate method.The colony,microscopic characteristic and DNA bar code were used to identify the fungi on Traditional Chinese medicine.In order to find out the main contamination fungi and harmful fungi(Aspergillus flavus),the number of fungi on the surface of traditional Chinese medicine and the species of fungi on the surface of Chinese medicine were counted.2.3 Analysis of fungal Diversity on traditional Chinese Medicine based on Illumina HiSeq sequencing platform2.3.1 Extraction of DNA samplesDNA samples of fungi were extracted from TCM,primers were designed,amplified by PCR,then,the products were purified and quantified.2.3.2 Construction of libraries for sequencingThe qualified PCR products were checked,the library was constructed and sequenced by Illumina Hiseq 2500.2.3.3 Data analysisThe original data obtained by high-throughput sequencing are recognized by base base to form the original sequence,and stored in FASTQ format,including sequence information and quality information of sequencing.Sequencing results of PE reads were jointed and got Raw Tags.The effective number of sequences was obtained by filtering the original sequences and removing chimerism.2.3.4 Species annotationThe OUT is divided by the Effective Tags and the Species is annotated.The fungal communities at the taxonomic level of phylum,class,order,family,genus,specie were obtained.Phylogenetic tree and taxonomic tree at taxonomic level were obtained.To analyze the diversity of fungi on TCM.2.4 Pollution ofAspergillus flavus on TCM2.4.1 Investigation on mildew of Chinese Herbal MedicineEach kind of Traditional Chinese Medicine was weighed 10 g,and 6 kinds of Traditional Chinese Medicine were put into paper bag and self-sealing bag respectively.At a temperature of 30℃ and a relative humidity of 85%.to make the TCM mildew.Record the time required for mildew and observe the mildew of Chinese medicine under different packaging.The occurrence of visible plaque,that is,mildew success.2.4.2 Analysis on the contamination of Aspergillus flavus in TCMMildew was taken out and fungi on the sample were eluted and diluted to 10-1,10-2,10-3,10-4,10-5.The eluant of 10-1,10-3,10-5 concentration gradient was cultured.The contamination rate of Aspergillus flavus on TCM was analyzed.2.5 Effects of Water extracts from six kinds of TCM on the growth of Aspergillus flavusThe water extract of TCM was prepared and diluted into different concentration gradient with salsa glucose liquid medium.Aspergillus flavus spores were added to culture medium which was containing water extract of TCM.5 days later,the weight of the hypha was weighed.The relationship between the water extract and the growth of Aspergillus flavus at different concentrations was analyzed by statistical analysis.3 Results3.1 Analysis of fungal contamination on six kinds of TCMA total of 62 strains of 7 kind fungi were isolated from the Citrus reticulata Blanco.Penicillium sp.and Aspergillus sp.were the main contaminated fungi.A total of 33 strains of 7 kind fungi were isolated from Coix lacryma-jobi L.var.mayuen(Roman.)Stapf.Penicillium sp.and Aspergillus sp.were the main contaminated fungi.Three kinds of fungi were identified on Hordeum vulgare L..Rhizopus oryzae and penicillium sp.were the main contaminated fungi.A total of 126strains of 10 fungi were isolated from Bombyx bassiana.Penicillium and Aspergillus were the main contaminating fungi.A total of 287 strains of 9 fungi were isolated from Pheretima aspergillum(E.Perrier).Penicillium sp.and Aspergillus sp.were the main contaminated fungi.A total of 42 strains 10 species of fungi from Chinemys reevesii(Gray),Penicillium sp.Aspergillus sp.were the main pollution of fungus.3.2 Analysis of fungal Diversity on TCM based on Illumina HiSeq sequencing platform3.2.1 Sequencing quality assessmentThe Reads numbers obtained from the samples of Citrus reticulata Blanco,Hordeum vulgare L.,Coix lacryma-jobi L.var.mayuen(Roman.)Stapf,batryticated silkworm,Chinemys reevesii(Gray),Pheretima aspergillum(E.Perrier)were as follows:79570,80057,79757,79750,79979,79967.The number of Clean tags produced by filtration by splicing the two-terminal Reads of each sample is as follows:73917,74550,75487,74537,74549,73177.The number of Clean tags were about 73000,Q20>98%,Q20>98%,Effective tags>91%.3.2.2 Species annotationA total of 155 genera and 176 species of fungi were found on the Citrus reticulata Blanco.A total of 89 genera and 96 species of fungi were found on the Coix lacryma-jobi L.var.mayuen(Roman.)Stapf.A total of 36 genera and 36 species of fungi were found on the Hordeum vulgare L..A total of 22genera and24 species of fungi were found on the Bombyx bassiana.A total of 127 genera and 121 species of fungi were found on the Pheretima aspergillum(E.Perrier).A total of 173genera and 201 species of fungi were found on the Chinemys reevesii(Gray).3.3 Contamination of Aspergillusflavus on TCMUnder the condition of temperature of 30℃ and relative humidity of 85%.the Chinese medicine in the paper bag packaging is easy to mould and Aspergillus flavus Increase.Aspergillus flavus was isolated from 6 batches of Citrus reticulata Blanco with a pollution rate of 72.Aspergillus flavus was isolated from 5 batches of Bombyx bassiana and Chinemys reevesii(Gray).Aspergillus flavus was isolated from 4 batches of Hordeum vulgare L.Coix lacryma-jobi L.var.mayuen(Roman.)Stapf.Aspergillus flavus was isolated from 2 batches of Pheretima aspergillum(E.Perrier).3.4 Effects of Water extracts from six kinds of TCM on the growth of Aspergillus flavus3.4.1 Effects of water extracts of different concentrations on the growth of Aspergillus flavusWhen the water concentration of lacryma-jobi L.var.mayuen(Roman.)Stapf was 3.125%,the dry weight of mycelium was 18.60 mg,and the promoting rate was 29.17%.when the concentration of water extract was 50%,the dry weight of hyphae was 21.50 mg,24.80 mg,42.20 mg,22.60 mg,and the promoting rate was 49.30%、72.22%、185.42%、56.94%,respectively..The growth of Aspergillus flavus and the concentration of water extract of Hordeum vulgare L.had no obvious regularity.When the concentration of water extract was 50%,the dry weight of mycelium was 14.00 mg,which showed that the growth of Aspergillusflavus was inhibited and the inhibition rate was-2.78 mg.3.4.2 Effects of High concentration Water extract on the growth of Aspergillus flavusThe results showed that the P value of dry weight of Hordeum vulgare L.mycelium was higher than 0.05,which indicated that there was no significant difference in dry weight of mycelium between water extract of Hordeum vulgare L.and Aspergillus flavus weight.What is noteworthy is that,P value of dry weight of lacryma-jobi L.var.mayuen(Roman.)Stapf,Citrus reticulata Blanco,Chinemys reevesii(Gray),Bombyx bassiana and Pheretima aspergillum(E.Perrier)L.mycelium was lower than 0.05.4 Conclusion4.1 Fungi could be isolated from the surfaces of six kinds of Chinese medicines.Penicillium and Aspergillus were the main contaminating fungi.The harmful Aspergillus flavus was isolated from the surface of batryticated silkworm and Pheretima aspergillum(E.Perrier).4.2 The results of high throughput sequencing showed that Aspergillus was one of the main fungi of Coix lacryma-jobi L.var.mayuen(Roman.)Stapf,batryticated silkworm,Pheretima aspergillum(E.Perrier),Chinemys reevesii(Gray)。The surface of the Citrus reticulata Blanco were mainly Penicillium.The surface of the Hordeum vulgare L.,were mainly Wickerhamomyces.The diversity of fungi on the surface of six kinds of traditional Chinese medicine was:Chinemys reevesii(Gray)>Citrus reticulata Blanco>Pheretima aspergillum(E.Perrier)>Coix lacryma-jobi L.var.mayuen(Roman.)Stapf>Hordeum vulgare L.>batryticated silkworm.4.3 In the environment of high temperature and high humidity,the Chinese medicine in the paper bag package is easy to mildew,and the self-sealed bag package is not easy to mildew.The results of aspergillus flavus in six kinds of traditional Chinese medicine without mildew treatment were different from those in mildew treatment.4.4 The effect of different concentrationwater extract on Aspergillus flavus was different,and the growth of Aspergillus flavus was more obvious in high concentration water extract.The effects of high concentration water extract of six kinds of traditional Chinese medicine on the growth of Aspergillus flavus were as follows:batryticated silkworm>Chinemys reevesii(Gray)>Pheretima aspergillum(E.Perrier)>Citrus reticulata Blanco>Coix lacryma-jobi L.var.mayuen(Roman.)Stapf>Hordeum vulgare L..5 Innovation5.1 Illumin Hiseq 2500 was used to sequence the fungi on 6 kinds of traditional Chinese medicine,such as Citrus reticulata Blanco,Coix lacryma-jobi L.var.mayuen(Roman.)Stapf;Hordeum vulgare L.,batryticated silkworm,Pheretima aspergillum(E.Perrier),Chinemys reevesii(Gray),to evaluate the diversity of fungi.5.2 The water extracts of 6 kinds of Chinese medicine pieces were extracted from Chinese medicine such as Citrus reticulata Blanco,Coix lacryma-jobi L.var.mayuen(Roman.)Stapf,Hordeum vulgare L.,batryticated silkworm,Pheretima aspergillum(E.Perrier),Chinemys reevesii(Gray).The effects of water extract of traditional Chinese medicine on the growth of Aspergillus flavus were studied by co-culture with Aspergillus flavus spores.