Bacterial diversity of stingless bee honey in Yunnan, China: isolation and genome sequencing of a novel acid-resistant () with probiotic and L. tryptophan producing potential via millet fermentation.

Stingless bee (, and ) honey is a remarkable "miracle liquid" with a wide range of medical benefits for conditions including gastroenteritis, cataracts, and wound healing. Our study aimed to isolate, identify, and characterize acid-resistant spp. from sour honey distributed in Yunnan, China. To assess the safety of an entirely novel strain, S4 (OM618128), based on probiotic property evaluation and whole-genome sequencing analysis. A 16S rRNA gene high-throughput sequencing analysis showed that was abundant at the genus level in sour honey. Seven strains (viz. S1-7) were isolated from sour honey using a multiple-anaerobic culture enrichment method. One potential acid-resistant isolate, sp. S4, was obtained after screening the seven isolates, and it had the highest lactic acid production (17.62 g/L), followed by sp. S3 (17.07 g/L). Phylogenetic and comparative analyses of conserved sequence regions have shown that all seven strains are phylogenetically located in the sub-cluster. In , there is a circular chromosome (3288615 bps) and 11,466 bps plasmids. GC content is 44.03%. The number of predicted genes is 3,129, with 16 rRNAs and 74 tRNAs present. During the fermentation of foxtail millet by seven (S1-7) strains isolated from sour honey, a potential tryptophan accumulating isolate, S4, was obtained, which could reach a maximum tryptophan content of 238.43 mgL that is 1.80 times the initial tryptophan content in the fermentation broth. This strain has strong acid tolerance, salt tolerance, and fermentation acid production abilities. This strain degrades nitrite at a rate of over 99%, and it has high probiotic potential as well. This project has established a solid foundation for further exploring the excellent lactic acid bacteria in sour honey. It is also investigating the key taxa and their role in the environment. According to the results of our studies, these LAB isolates provide a lot of potential for use in the future, as a source of probiotics for human, animals, and starter cultures for food applications.