| Bee bread is the main source of protein for the development of honey bee larvae and an essential ingredient for maintaining a healthy colony.One of the main differences between bee bread and bee pollen is that bee bread is a bee feed with higher biological value,faster digestibility and better chemical composition than bee pollen,formed by bees using fresh pollen as raw material and fermented by microbial action under certain humidity conditions,and is also a health product that can be consumed directly by humans.A.cerana and A.mellifera are the two most important bee species kept in China,and they both feed on nectar and pollen.However,due to the difference in collection habits,there is a lack of systematic research on the nutritional formation and microbial diversity in the fermentation process of bee bread of the two species.Therefore,this study was conducted to identify and analyze the diversity of bacteria and fungi in the bee bread of A.cerana and A.mellifera,the most widely used in Chinese farming,in all seasons,and to determine their nutritional composition,revealing the seasonal dynamic changes in the microbial composition and differences in the main nutrients in the bee bread of the two bee species.The results of the study are as follows.1: Structural diversity of bacterial flora in different seasons of bee bread of two honey bee speciesBased on the sequencing results of bee bread samples from A.cerana and A.mellifera,the16S rRNA sequences of bacteria from different bee bread species in different seasons were analyzed to compare the species composition and abundance information of the flora.The combination of NMDS,PCo A analysis and differences in diversity indices indicated significant differences in bacterial community composition between A.cerana and A.mellifera grains.At the phylum level,the eastern and western honey bee bee bread samples from four seasons were mainly composed of five phylum: Cyanobacteria,Proteobacteria,Firmicutes,Actinobacteriota,and Bacteroidota.The highest abundance of Cyanobacteria in the A.cerana bee bread samples was above 75.4%,reaching 90.0% in autumn,while the A.mellifera bee bread samples differed in that the higher abundance of Proteobacteria,31.0%-56.7%,and Cyanobacteria,22.7%,in the four seasons.Cyanobacteria accounted for 22.5%-48.1%,and interestingly,the percentages of the two phyla were relatively close.At the genus level,the top five in order of relative abundance were norank＿f＿＿norank＿o＿＿Chloroplast,Acinetobacter,norank＿f＿＿Mitochondria,Lactobacillus,and Sphingomonas.A.cerana bee bread samples in all seasons In the A.mellifera bee bread samples,norank＿f＿＿norank＿o＿＿Chloroplast and Acinetobacter were the dominant genera,with abundance percentages ranging from 75.4% to 90.0%;in the A.mellifera bee bread samples,norank＿f＿＿norank＿o＿＿Chloroplast and Acinetobacter were the dominant genera,with abundance percentages exceeding 20% in all four seasons.The abundance of Acinetobacter spp.was more than 20% in all four seasons.The abundance of the top five colonies was significantly different between the A.cerana and A.mellifera bee bread groups in different seasons:norank＿f＿＿norank＿o＿＿Chloroplast(P=0.00027),Acinetobacter(P=0.00026),norank＿f＿＿Mitochondria(P= 0.015),Lactobacillus(P=0.0002),Sphingomonas(P=0.0335).2: Structural diversity of fungal flora in different seasons of bee bread of two honey bee speciesThe 16S rRNA sequences of the fungal flora of different honey bee species in different seasons were analyzed to compare the species composition and abundance information of the flora based on the sequencing results of the bee bread samples of A.cerana and A.mellifera.At the phylum level,the top five relative abundance rankings of A.cerana and A.mellifera bee bee bread samples in the four seasons were Ascomycota,unclassified＿k＿＿Fungi,Basidiomycota,Chytridiomycota,and Mortierellomycota in that order.The highest relative abundance of Ascomycota was found in both A.cerana and A.mellifera bee bread samples in all four seasons,accounting for more than 55.8%,and in the summer time,the percentage of Ascomycota in A.mellifera bee bread reached 98.5%.In the spring,the second highest ranking A.cerana bee bread was unclassified＿k＿＿Fungi with 35.4% and the A.mellifera bee bread was Basidiomycota with21.1%;in the summer,the second highest ranking A.cerana bee bread was Basidiomycota with4.9% and the A.mellifera bee bread was Basidiomycota,accounting for 1%;in autumn,the second highest ranking A.cerana diet was Basidiomycota,accounting for 34.4%,and the Western honey bee diet was unclassified＿k＿＿Fungi,accounting for 30.9%;in winter,the second highest ranking Eastern honey bee diet was Basidiomycota,accounting for 8.1%,and the Apis mellifera diet was unclassified＿k＿＿Fungi,accounting for 30.9%;in winter,the second highest ranking A.cerana diet was Basidiomycota,accounting for 8.1%,and The western honey bee bread was unclassified＿k＿＿Fungi with 29.5%.From the NMDS and PCo A analyses,the fungal diversity of A.cerana and A.mellifera grains was similar in terms of colony composition,with differences between seasons and closer affinities between the seasons.Significant differences in fungal composition were found between A.cerana and A.mellifera bee bread in each season for Ascomycota(P=0.0017),unclassified＿k＿＿Fungi(P=0.0084),and Basidiomycota(P=0.0042),respectively.At the genus level,there were 41 genera with relative abundance exceeding 1%,and the top five were the genera Sawadaea,unclassified＿k＿＿Fungi,Cladosporium,Metschnikowia,and unclassified＿o＿＿Saccharomycetales,respectively.Significant differences in fungal composition were found between A.cerana and A.mellifera grains for each season for unclassified＿k＿＿Fungi(P=0.0085),Cladosporium(P=0.00032),Epicoccum(P=0.01499),and Nigrospora(P=0.02311),respectively.3: Comparison of nutrient contents of two honey bee diets in different seasonsThe results of statistical analysis showed that the nutrient contents of the bee bread of A.cerana and A.mellifera differed in different seasons,and the pH values were different in each period;there were also slight differences between different honey bee bread in the same season.pH values varied in the same pattern spring > summer > autumn > winter,with the highest pH in the spring and the lowest pH in the winter;the total sugar content varied less throughout the year,with a significant difference between winter and the other three seasons;soluble protein content and total flavonoid content varied in temperature throughout the year and were not significantly different;free amino acid content and total flavonoid content were not significantly different from the other three seasons;free amino acid content and total flavonoid content varied in temperature throughout the year and were not significantly different from the other three seasons.There was a significant difference between winter and the other three seasons;soluble protein content and total flavonoid content varied at the same temperature throughout the year with no significant difference;free amino acid content and water content of bee bread were the highest values in summer. |
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