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

**Background** Stingless bee honey (sour honey) has excellent medicinal value and a low pH, suggesting it may contain acid-resistant lactic acid bacteria (LAB). However, limited studies have isolated and characterized such bacteria from stingless bee honey in Yunnan, China. This study aimed to assess bacterial diversity in sour honey, isolate acid-resistant Lactobacillus strains, and evaluate their probiotic potential and ability to accumulate L-tryptophan during millet fermentation.

**Methods** Four sour honey samples (Y1–4) were collected from Xishuangbanna, Yunnan. Bacterial diversity was analyzed by high-throughput sequencing of the 16S rRNA V3–V4 region after enrichment in MRS medium. LAB were isolated using MRS agar with CaCO3 and identified by 16S rRNA sequencing. Strain S4 (Lactobacillus pentosus SYBC-MI) was selected for whole-genome sequencing (Illumina NovaSeq/Oxford Nanopore ONT). Probiotic properties were assessed in vitro: acid and bile tolerance (pH 2.0–4.0, 0.2–1.0% bile salts), simulated gastric (pH 2.5, pepsin 3 g/L) and intestinal juice (pH 8.0, trypsin 1 g/L) survival, cell surface hydrophobicity (xylene), auto-aggregation, antagonistic activity against Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, and Salmonella typhimurium ATCC14028, antibiotic susceptibility (7 antibiotics), hemolysis (sheep blood agar), and nitrite degradation (125 μg/mL nitrite). Millet fermentation was performed with millet flour and grains at 30°C for 10 days; free amino acids (including tryptophan) were quantified by HPLC.

**Key Results** High-throughput sequencing generated 318,346 high-quality sequences (average 79,587 per sample). Dominant phyla were Firmicutes and Proteobacteria; at genus level, Carnimonas (33.36–75.29%), Clostridium (up to 27.60%), Lactobacillus (2.58–16.90%), Bacillus (4.41–15.33%), and Staphylococcus (1.31–10.53%) were prevalent. After MRS enrichment, Firmicutes exceeded 98.5% relative abundance. Seven LAB strains (S1–S7) were isolated from sample Y1; all showed 99.26–99.89% 16S rRNA similarity to Lactobacillus pentosus. Strain S4 (L. pentosus SYBC-MI) had a complete genome of 3,287,615 bp (44.77% GC), 3,129 protein-coding genes, 16 rRNAs, 74 tRNAs, and 6 prophages. It grew at pH 2.5 (the only strain), at 6% NaCl, and optimally at 30°C. Lactic acid yield after 24 h was 17.62 g/L (highest among isolates). Survival rates in simulated gastric juice (8 h) and intestinal juice (12 h) were 78% ± 3.66% and 70% ± 4.49%, respectively. Cell surface hydrophobicity was 28.05% ± 1.32%; auto-aggregation was 25.06% ± 1.41%. S4 showed antagonistic activity against E. coli (12.69 mm inhibition zone) and S. aureus (10.7 mm), but not Salmonella. It was sensitive to gentamicin, erythromycin, chloramphenicol, ampicillin, and vancomycin; resistant to streptomycin and kanamycin. Hemolysis was γ (non-hemolytic). Nitrite degradation exceeded 99.5% (final pH 4.04). During millet flour fermentation, S4 produced the highest L-tryptophan content of 238.43 mg/L at day 8 (1.80 times initial), and the total essential amino acids in S2 fermentation reached 293.41 mg/L (30% of total free amino acids).

**Clinical Implications** This study identifies stingless bee honey as a novel source of acid-resistant Lactobacillus pentosus with strong probiotic potential, including high acid and bile tolerance, antimicrobial activity, and nitrite degradation. The strain's ability to accumulate L-tryptophan during millet fermentation suggests applications in functional foods that may support sleep and mental health. Whole-genome analysis confirms safety (no transferable antibiotic resistance genes, γ-hemolysis) and provides a basis for future in vivo studies. These findings could lead to development of probiotic-enriched honey or fermented millet products with enhanced health benefits.