Isolation And Identification Of Lactic Acid Bacteria From Sour Honey And Their Application In Millet Fermentation

Stingless bee（Hymenoptera,Apidae,Trigona） honey is also known as sour honey due to its low pH and distinct sour taste.Sour honey has been shown to be highly valuable in the treatment of cataracts,gastrointestinal conditioning,and wound healing.However,there is very little research on the microorganisms in sour honey in Yunnan,China.Honey bees are purely natural microbial collectors who spread microorganisms through nectar collection and pollination.Stingless bees being one of them,the sour honey they produce may be a good resource for novel lactic acid bacteria.Therefore,in this study,Xishuangbanna native sour honey from four different production areas in Yunnan,China was selected for microbial community analysis after which several strains of Lactobacillus strains were isolated,identified and applied to foxtail millet fermentation.The main research results are as follows:（1）High-throughput sequencing was used to analyze the microbial diversity and bacterial community structure in four Yunnan native sour honey samples（Y1–4）.The results showed that the main bacterial genus in sour honey were Carnimonas（33%～74%）,Clostridium（8%～27%）,Staphylococcus（1%～10%）,Lactobacillus（3%～16%）,and Bacillus（4%～15%）;the main fungal species were Wickerhamiella（26%～50%）and Monascus（1%～5%）.The bacterial diversity of the four sour honeys was ranked Y3>Y1>Y2>Y4 and the fungal diversity was ranked Y3>Y2>Y4>Y1.（2）The species composition did not differ significantly between the original sour honey samples,but the microbial community structure differed significantly from that of the sour honey samples（X1–4） after domestication with de Man,Rogosa and Sharpe（MRS）medium.The relative abundance of Carnimonas in all of the domesticated sour honey samples decreased substantially to less than 0.5%.In contrast,the relative abundance of Lactobacillus reached 98.03%,which was five times higher than that before domestication,and became the dominant group.Seven Lactobacillus strains（S1–7）were isolated from the sour honey after multiple rounds of anaerobic enrichment culture（domestication culture）.The results of comprehensive sequence comparison and phylogenetic analysis as well as morphological and physiological and biochemical tests showed that all strains were phylogenetically located in the Lactobacillus pentosus subgroup.（3）Seven Lactobacillus pentosus（S1–7） strains isolated from sour honey were applied to foxtail millet fermentation,and the concentrations of most free amino acids increased with fermentation time compared to the characteristics of the initial foxtail millet fermentation broth.The increase of threonine among the essential amino acids was the most pronounced,with the concentration after 10 days of fermentation by Lactobacillus pentosus S1–6 being on average 3.19 times higher than the value in the initial foxtail millet fermentation broth.The essential amino acid fraction increased the most in the millet fermentation broth of Lactobacillus pentosus S2,with the total content of eight essential amino acids at the end of fermentation being 293.41 mg·L^-1,accounting for 30%of the total free amino acids.In addition,the changes in taste-presenting amino acids varied.After comprehensive consideration,it was found that 6 days of fermentation was the best time for foxtail millet fermentation,when the fermentation broth of almost all isolates had the lowest content of bitter amino acids and the highest content of fresh and sweet amino acids.（4）During the fermentation of foxtail millet by seven Lactobacillus pentosus（S1–7） strains isolated from sour honey,a potential tryptophan accumulating isolate,Lactobacillus pentosus S4,was obtained,which could reach a maximum tryptophan content of 238.43mg·L^-1 in the fermentation broth,1.80 times the initial tryptophan content in the fermentation broth.The whole genome sequencing analysis was performed and the strain was named Lactobacillus pentosus SYBC M1.This strain has strong acid tolerance,salt tolerance and strong fermentation acid production ability,and its ability to degrade nitrite reached more than99%,and has good probiotic potential.