Rapid Identification Of Fungal Contaminated Fungi From Eupolyphaga Sinensis Walker And Aflatoxin Biodegradation

Eupolyphaga sinensis Walker is a valuable traditional Chinese animal medicine first recorded in Shennong Bencao.Previous research has shown that E.sinensis is easily contaminated by aflatoxins?AFs?,which are highly toxic mycotoxins,during harvest,storage,and transport,thereby posing a considerable threat to consumer health.Most often,these AFs are produced by Aspergillus species.Establishing a rapid identification method for contaminated Chinese medicines can detect potential toxin-producing fungi earlier,ensure the effective production and safety of Chinese medicines.Therefore,the main research contents of this study are as follows: 1.Determination of aflatoxin in 56 batches of E.sinensisDetermination of aflatoxin B₁?AFB₁?,aflatoxin B₂?AFB₂?,aflatoxin G₁?AFG₁?and aflatoxin G₂?AFG₂?in 56 samples from different medicinal material markets in five provinces by HPLC.The results showed that: AFB1 was detected in 38 batches,AFB2 was detected in 24 batches,AFG₁ was detected in 31 batches,AFG₂ was detected in 20 batches,25 %?14 batches?were detected.The AFB1 content exceeded the standard,the AFs content in 30.4 %?17 batches?of the sample exceeded the standard.2.Rapid detection identification of contaminated fungi from E.sinensis in Chinese medicinal materialsThis study contrasts the traditional culture-based dilution plating method to the high-throughput sequencing?HTS?technology for fungal identification in TCM E.sinensis.Both of the methods used internal transcribed spacer 1?ITS1?and calmodulin?CaM?sequencing for fungal molecular identification.The new CaM primer we designed in the study is suitable for Mi Seq PE300 sequencing used for identification of Aspergillus species in community DNA samples.More fungal species were found in the E.sinensis samples based on HTS than those found using the culture-based dilution plating method.Overall,combining the sequencing power of ITS1 and CaM is an effective method for the detection and monitoring of potential toxigenic Aspergillus species in E.sinensis.In conclusion,HTS can be used to obtain a large amounts of sequencing data about fungi contaminating animal medicine.3.Cloning of aflatoxin candidate enzyme genesUsing the cDNA of two edible fungi as templates,12 candidate aflatoxin candidate enzyme genes were cloned and encoded.Similarity with the NCBI database is 99 %.