| With the development of the economy and society and the improvement of living standards,people pay more and more attention to life safety and health quality,which presents the characteristics of diversification and differentiation.The 14th Five-Year Plan clearly points out that "accelerating the development of TCMs(traditional Chinese medicine)" is one of the ways to effectively cope with various health challenges and better meet the health needs of people,and puts forward the goal of improving the high-quality development policies and systems of TCMs.High-quality development of TCMs needs to ensure their quality and safety,especially some "medicinal and edible" products,such as lotus seed(Nelumbinis semen)and malt.Fungi exist widely in nature and can be found as communities in a wide variety of substrates.Mycotoxins,as the secondary metabolites of toxigenic fungi,are easily introduced in the processes of planting,harvesting,processing,transportation,and storage of TCMs.Mycotoxigenic fungi and their secondary metabolites,mycotoxins,are considered as the key factors affecting the quality and safety of TCMs,which can produce irreversible acute and chronic cumulative toxic effects on the human body.Therefore,on the one hand,the community composition and diversity of fungi in TCMs were analyzed,especially those harmful and toxic fungi,so as to provide early warning for mycotoxigenic fungi and mycotoxin pollution,ensuring the quality and safety of TCMs and related products.On the other hand,the current methods(such as chromatography and spectroscopy techniques)for the detection of mycotoxin usually rely on heavy and expensive instruments and professional operators and are mostly confined to the laboratory,which is not suitable for the field analysis in some economically backward and remote areas.Therefore,it is urgent to develop a reliable and practical method for the rapid and on-site detection of mycotoxins.Among them,the lateral flow test strip is a low-cost,portable,stable,and rapid detection method,which can better meet the above requirements for the mycotoxin detection.In this study,from the two aspects of "mycobiome" and "mycotoxin",the fungal community structure and composition of medicinal and edible TCMs like lotus seed and malt,were revealed by the second-and third-generation sequencing technologies,respectively.Moreover,multiwall carbon nanotubes(MWCNTs)-based lateral flow immunochromatography test strip was developed for the low-cost,sensitive,and rapid detection of AFB1 in malt in order to ensure the quality and safety of these TCMs.The main studies were listed as follows:1.Fungal communities in lotus seed characterized by the second-generation highthroughput sequencing technologyIn this study,Illumina-MiSeq-based high throughput sequencing(HTS)technology was introduced to sequence the amplicons of fungi from normal and moldy lotus seed,and then to evaluate the diversity,community structure,composition,and differences of fungi in lotus seed.First,two different primers in internal transcription spacer 2(ITS2)were introduced to compare the performance of the target gene amplification.Then,the primers producing more reads were selected for the analysis.The results showed that 2 phyla,6 orders,8 families,9 genera,and 4 species of fungi were detected in 9 samples of lotus seed from normal and moldy groups.At the genus level,a total of 9 genera were identified,among which Aspergillus(0.04%-72.93%)and Rhizopus(0.002%-48.12%)accounted for the highest proportion.Alpha diversity analysis showed that each sample exhibited certain species diversity and abundance.Moreover,the sequencing had a high fungal coverage,which could represent the real situation of the fungal composition in the sample.Beta diversity analysis indicated some differences in species composition among all the samples.ANOISM analysis showed that there were no significant differences between normal and moldy lotus seed(p>0.05).Therefore,the HTS technology could detect the diversity and composition of fungal communities in complex lotus seed samples providing early warning for toxigenic fungi and mycotoxins(such as aflatoxins)pollution to ensure the quality and safety of TCMs.2.Mycobiome diversity in malt explored by the third-generation by single-molecule real-time sequencing technologyHerein,the third-generation single-molecule real-time(SMRT)sequencing technology based on PacBio was applied for the full-length amplicons of the ITS,to analyze the fungal composition of raw and roasted malts,for identifying the enriched fungal species.The results showed that a total of 1 kingdom,6 phyla,23 classes,56 orders,120 families,188 genera,333 species,and 780 operational taxa(OTUs)were detected,and the sequencing depth and sample size were qualified.Alpha diversity analysis displayed that all the samples had certain species diversity and richness,while Beta diversity analysis showed certain differences in the fungal community composition among different samples,as well as between the raw and roasted malt groups.Wickerhamomyces(56%),Cyberlindnera(15%),Dipodascus(12%),and Candida(6.1%)were the dominant genera in the raw malt,while,Aspergillus(35%),Dipodascus(21%),Wickerhamomyces(11%),and Candida(3.5%)in the roasted malt samples.Aspergillus proliferans,Aspergillus penicillioides,and Wickerhamomyces anomalus are the key species that lead to differences between the two groups.The correlation analysis of environmental factors showed that the Aw significantly affected the fungal community composition in malt.The field mildew tests confirmed that raw malt samples were more susceptible to mildew than the roasted malts,which indicated that roasting process of the raw malt could reduce the Aw,change the fungal community structure and composition,further reduce the incidence of toxigenic fungal contamination,and ensure the safety of the malt.Therefore,in the practical application of the malt,special attention should be paid to toxigenic fungi and pathogenic fungi to ensure the safety and quality of the malt and its final products.3.Rapid and on-site detection of AFB1 in malt by developing a MWCNTs-based lateral flow immunochromatographic test stripIn this study,a MWCNTs-based lateral flow immunochromatographic test strip was developed by combining visual recognition and gray value analysis to realize the rapid detection of AFB1 in malt.Through the carboxyl and amino reaction,carboxylated MWCNTs were coupled with AFB1 monoclonal antibody(mAb)to prepare the signal probe,which was coated on the binding pad of the test strip.The AFB1 complete antigen and goat anti-mouse secondary antibody were fixed on the test line(T line)and control line(C line),respectively,on the NC membrane of the test strip.Then,the sample pad,binding pad,NC membrane,absorbent paper,and bottom plate were assembled into the immunochromatographic test strips.The color(black)signal of MWCNTs was identified by the naked eye and the gray value analysis was carried out by smartphone for the qualitative and quantitative determination of AFB1.The results showed that the developed test strip could realize the detection of AFB1 in malt within 15 min under optimal conditions.The linear range was 0～5 ng/mL with a limit of detection(LOD)of 0.25 ng/mL.The average recoveries of low(0.5 ng/mL),medium(1.5 ng/mL),and high(4.5 ng/mL)were 116.75%,99.33%,and 110.1%,respectively,with relative standard deviations(RSDs)lower than 9.5%.The method was applied to testing 14 batches of actual samples.The results showed that one batch was positive and the others were negative.The positive detection rate of AFB1 was 7.14%in malts.Simultaneously,in order to avoid the"false positive" and "false negative" of the new method,both positive and negative samples were verified by the UFLC-MS/MS method.The verification results were consistent with the lateral flow test strip.The MWCNTs-based immunochromatographic strip is simple and portable,with low-cost,which can meet the requirements of rapid field detection of AFB1 in a large number of malt samples.In this study,from the aspect of "fungal community",the source of a mold in TCMs,the fungal diversity and community structure of fungi in lotus seed and malt were analyzed by the second-generation HTS(Illumina-based)and third-generation SMRT sequencing(PacBio-based)technologies,respectively,to explore the factors influencing the fungal community composition.All the results could provide early warning for the pollution of toxigenic fungi,pathogenic fungi,and mycotoxins,as well as important references and data support for the safe use of TCMs.A simple and low-cost MWCNTs-based immunochromatographic test strip could achieve the rapid and on-site detection of AFBi in malt from the perspective of "mycotoxin".The comprehensive analysis of the fungal community and mycotoxins can explain the causes and consequences of the moldy TCMs more comprehensively,which is more in line with the requirements for the safe application of TCMs. |
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