Identification Of Sources Of Bee Pollen,Microbial Species Of Bee Pollen And Yeasts In Honey

There are many kinds of bee pollen.Different bee pollen has different pharmacological effects and nutritional value.However,most of the identification methods for bee pollen varieties are traditional methods,such as microscopic observation,physical and chemical identification,and so on.At present,as a new analysis and identification technology,DNA barcoding technology has been applied to many fields,such as identification of Chinese medicinal materials,food safety,identification of exotic species,and so on.The DNA metabarcoding technology developed based on DNA barcoding technology and high-throughput sequencing technology is a new species identification technology that can analyze the composition of all species in a mixed sample.At present,the application of this type of technology in the field of bee products is still at a blank stage.This paper applied DNA barcodes to identify the origin of bee pollen and applied DNA metabarcoding to identify microorganisms on bee pollen and yeast in honey.This study applied DNA metabarcoding technology to identify the source of bee pollen.First,the source of bee pollen was identified using DNA barcoding technology based on ITS2 sequence and 18S sequence.After DNA extraction,target region PCR amplification,sequencing,NCBI sequence comparison and other steps,identification of bee pollen types.The results of the DNA barcoding technique were then verified by scanning electron microscopy.Finally,identification of a toxic honey using ITS2-based DNA metabarcoding technology and analyze the toxic sources of toxic honey.The results showed that among the 41 bee pollen samples sequenced by ITS2 sequence,the identification results of 24 samples were consistent with the sample information,15 samples were inconsistent with sample information,and 2 comparisons failed;21 bee pollen samples sequenced by 18S sequence Among the 13 samples,the identification results were consistent with the sample information,and the identification results of the 8 samples were inconsistent with the sample information.Partial identification of ITS2 and 18S showed differences in the identification of species levels.This is because in the market,in addition to common bee pollen such as.buckwheat,rape,and lotus,etc.;most of them are mixed pollen which contains two or more bee pollen.The results showed that the ITS2 sequence was more suitable for the identification of bee pollen.Scanning electron microscopy(SEM)was used to verify the results of DNA barcoding.The results were basically consistent with DNA barcoding.In this paper,DNA metabarcoding based on ITS and 18S sequences were used to identify fungi in bee pollen.After genomic DNA extracting,target region amplification,database constructing,high-throughput sequencing,database comparison and other steps,identification of fungi on bee pollen.Among them,58 species of fungi can be identified by the ITS region,mainly include Aspergillus.sp,Candida.sp,Hyphopichia burtonii,Rhizopus.sp,etc.;only 24 fungi were identified by the 18S region,mainly Aspergillus.sp,Candida.sp and Rhizopus.sp.Among them,Alternaria alternata,Aspergillus fumigatus,Candida albicans and Fusarium.sp may have human pathogenicity.In addition,the identification of the 18S sequence contained some fungi that were not present in the identification of the ITS sequence.This shows that the ITS region is more suitable for the identification of fungi on bee pollen,and the results of the 18S region can complement the results of ITS region.Finally,this paper applies the DNA metabarcoding technology based on ITS sequence to identifiy the yeast in honey.The yeast in honey was separated by honey-coating,and then shaking for 3 to 5 days,extracting DNA,PCR amplification,sequencing,and NCBI sequence comparison were extracted to identify the type of yeast.Through the method of picking a single colony from the coated plate,Zygosaccharomyces rouxii and Zygosaccharomyces siamensis were predominantly identified in honey and by DNA metabarcoding technology,Zygosaccharomyces mellis in honey was also identified.The results fully demonstrate that DNA metabarcoding can quickly,effectively,and comprehensively identify yeast in honey.In addition,the determination of the sugar content in honey yields the possibility of linking yeast to its total sugar content in honey:when the total sugar content in honey reaches a certain level,the hyperosmophilic yeast strain will hardly grow in honey.