| Objective:In recent years,the clinical efficacy and medicinal value of traditional Chinese medicine have received more and more attention.As the demand for traditional Chinese medicine continues to increase,the safety of traditional Chinese medicine materials has gradually attracted widespread attention.Traditional Chinese medicine will introduce exogenous harmful substances in the collection,transportation,storage,etc.,among which aflatoxin is one of the exogenous harmful substances.Aflatoxin is a serious threat to people's health due to its high toxicity and strong carcinogenicity.Among them,aflatoxin B1 is the most harmful,which makes the safety of Chinese medicine not guaranteed and hinders the modernization of Chinese medicine.Therefore,Chinese medicine was found the method of removing aflatoxin is particularly important.This study tried to find a strain with high degradation rate,safe and pollution-free to degrade aflatoxin B1,in order to achieve a green detoxification effect;secondly,the screened aflatoxin B1 degrading bacteria were identified and their strains were clarified.And make a preliminary study on the degradation characteristics.This research aims to discover an efficient and environmentally friendly biological method to degrade aflatoxin B1 in Chinese medicinal materials,so as to realize the green conservation of Chinese medicinal materials and ensure the safety of clinical medication.Methods:In the first chapter of this article,12 batches of Polygalae Radix samples from different origins were collected as the source of strains.The culture medium with coumarin as the sole carbon source was used for preliminary screening,and then the grown bacteria were streaked and purified to obtain the preliminary screening.Then,the primary screened bacteria were co-cultured with aflatoxin B1,and the residual amount of aflatoxin B1 was determined by ultra performance liquid chromatography triple quadrupole tandem mass spectrometry(UPLC-MS/MS),and rescreening bacteria with high degradation rate.The second chapter of this article identified and screened the aflatoxin B1 degrading bacteria,using morphological,microscopic and 16S rDNA molecular identification methods to identify,and then used the third-generation sequencing technology to sequence the whole genome of the bacteria,using the NR database and ANI analysis comprehensively identifiesd bacterial species,and characterized the genomic information of the strains through GO,COG and KEGG databases to discover possible aflatoxin B1 degrading enzymes.The third chapter of this article firstly divided the components of the fermentation broth of aflatoxin B1 degrading bacteria to clarify the active site and then studied the degradation mode;secondly,by measuring the OD600 and free protein content to explore the dynamics of the flora and determine the best strain cultivation time;finally,the single-factor control variable method was used to investigate the influence of time,temperature,and initial pH on the degradation process.Results:1.There were coumarin-utilizing strains on the surface of 11 batches of Polygalae Radix samples from different origins,a total of 55 strains.After the UPLC-MS/MS re-screening results,there were five strains with a higher degradation rate,respectively,50.08%,81.34%,40.00%,44.13%,38.49%,and they all came from the samples of Polygalae Radix from Wenxi County,Shanxi Province.2.The morphological identification result of aflatoxin B1 degrading bacteria showed that the colony was round,milky white,the edges were neat and thin,and the center of the bacteria was slightly raised and mound-shaped,The microscopic identification results showed that the cell morphology was rod-shaped and Gram stained Positive.Combined with the 16S rDNA molecular identification results,the strain was of the genus Bacillus.The whole genome sequencing results showed that the size of the aflatoxin B1 degrading bacteria was 5.54 Mb and the GC content was 37.1%.The strain genome contained 8633 genes,7059 coding genes,147 non-coding RNAs,including 127 tRNAs,13 rRNAs(11 5s rRNA,1 16s rRNA,1 23s rRNA),and 7 sRNAs.Both NR database annotation and ANI analysis indicated that the aflatoxin B1 degrading bacterium was Bacillus megaterium.Annotated and analyzed the GO,COG,and KEGG databases,an aflatoxin-degrading enzyme was preliminarily located as laccase,and the functional gene encoding laccase was yfiH.3.The results of the active components of aflatoxin B1 degrading bacteria showed that the degradation rate of the supernatant was 97.45%,the bacterial suspension was 57.19%,and the intracellular fluid was 39.63%.The degradation rates of supernatant after protease K and heating treatment were 25.17%and 18.72%,respectively.The study of flora dynamics showed that 12 h was the best culture time for aflatoxin B1 degrading bacteria.At this time,the OD600 was 1.46 and the protein content was 198.11 ?g·mL-1.The experimental results of the influence of environmental factors on the degradation process showed that the best degradation conditions were 72 h,57?,and the initial pH value was 8.0.Conclusion:There were beneficial bacteria on the surface of Polygalae Radix that could degrade aflatoxin B1,and the degradation rate could reach 81.34%.The strain was Bacillus megaterium.The detoxification mechanism of this strain was biodegradation.The extracellular enzymes in the supernatant were working.The best culture time of the strain was 12 h,and the best degradation conditions were 72 h,57?,and the initial pH value was 8.0.This study provided theoretical support for the application of the biodegradation of aflatoxin B1 to the green conservation of Chinese medicine. |
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