Analysis Of Microflora In Bee Bread And Screening,Identification And Application Of Lactic Acid Bacteria And Yeast

Bee pollen is a pollen grain collected by bees from the anthers of nectar-mining plants and plus the bees' own secretions.It is carried by the pollen basket on the hind limbs of the bees.Bee bread is a mixture of bee pollen,honey,and bee secretions,and fermented by specific microorganisms,the substances stored in the nest for the bees to eat.The purpose of this study was to analyze the bacterial community and identify the dominant bacteria in the bee bread.The lactic acid bacteria and yeast with excellent fermentation performance were screened from the bee grain.Besides,these microorganisms were applied to the fermentation of bee pollen to improve the taste as well as the digestion and absorption of bee pollen by human body or bees.First,the bee bread of Apis mellifera carnica(fermented for 0.5 d,4 d,7 d and 12 d),Apis mellifera carpatica Foti(fermented for 4 d)and Apis mellifera caucasica Gorb(fermented for 4 d)was sampled,and microbial 16 S rDNA of different samples were sequenced by high-throughput sequencing.Microbial diversity and composition of these samples were comparatively analyzed.As for the bee bread of different fermentation time,totally 27 phyla,112 genera of bacteria were identified in 4samples.Besides,there were 25 genera shared by the 4 samples.Moreover,the sequencing depth of this study effectively covered the microbial species,along with the fermentation,microbial richness and diversity increased first and then decreased.And Oxyphotobacteria and Proteobacteria were dominant on the phylum level.On the genus level,the dominant microorganism in bee bread is unidentified Oxyphotobacteria.Additionally,the differences in the microbial communities of Apis mellifera Linnaeus with different species are not significant.Next,the lactic acid bacteria and the yeast were screened from the bee bread using the MRS medium and the YPD medium.As a result,17 strains of lactic acid bacteria and 5 strains of yeasts were isolated.After comparison,6 strains of lactic acid bacteria and one strain of yeast with high fermentation performance were screened out.6 strains of lactic acid bacteria and one strain of yeast were respectively identified as Lactobacillus kunkeei and Saccharomyces cerevisiae by 16 S rRNA and ITS sequencing.The optimum growth temperature,pH,growth curve and pH curve of the two strains were determined.Finally,the screened lactic acid bacteria and yeasts were applied to the fermentation of bee pollen.The liquid fermentation and solid-state fermentation with single bacteria and mixed bacteria are respectively carried out.Results showed that:(1)After solid-state fermentation,the glucose content of bee pollen decreased,and the total phenolic and total flavonoids content increased.The total phenolic and total flavonoids content of the sterilized pollen inoculated with mixed bacteria increased the most,which were 25.3% and 7.2%,respectively.The content of total phenol and total flavonoids was the highest after inoculation of unsterilized pollen with mixed bacteria for five weeks,which was 11.65mg/g and 23.24 mg/g,respectively.(2)After liquid fermentation of bee pollen at a ratio of solid to liquid of 1:2,the glucose content generally showed a downward trend,except for a slight increase at thebeginning of the fermentation.This may be the result of microbial metabolic activity destroying the pollen cell wall and causing the dissolution of cellular contents at the beginning.At the same time,the total phenolic and total flavonoids content increased significantly.