Snake-gut bacterial isolates may have distinctive functional potential, but their application requires consideration of both strain-level functional evaluation and safety risk screening. This study combined in vitro phenotypic data, long-read whole-genome sequencing, species-matched comparative genomics, and multi-database screening to comprehensively characterize the previously high-performing Enterococcus hirae SA0215 and Lactococcus lactis SA0220 from Trimerodytes annularis and Enterococcus faecalis TP0208 from Trimerodytes percarinatus. All three strains yielded negative results in the gelatinase and catalase tests, and none could effectively use mucin as a carbon source to support vigorous growth; E. hirae SA0215 and L. lactis SA0220 tested qualitatively positive for bile salt hydrolase, and L. lactis SA0220 also exhibited significant auto-aggregation and co-aggregation capacities. Genomic analysis suggested that E. hirae SA0215 possessed features related to nutrient utilization and cell-surface homeostasis, together with features potentially related to antibacterial activity, whereas L. lactis SA0220 showed relatively prominent acid-stress-related modules and carbon-source utilization strategies under nutrient fluctuations. Although E. faecalis TP0208 possessed diverse metabolism-related modules, it also carried risk signals related to adhesion, biofilm formation, extracellular proteolysis, and antimicrobial resistance. Based on the combined phenotypic and genomic evidence, E. hirae SA0215 and L. lactis SA0220 may be further evaluated for in vivo safety and efficacy, whereas E. faecalis TP0208, as a subject for safety risk characterization, requires further targeted safety assessment. This study provides a strain-level framework for functional evaluation and risk stratification of isolates from nontraditional hosts.

The escalating global incidence of ulcerative colitis (UC) underscores the demand for novel therapeutic strategies. This study investigated the fermentation of Periplaneta americana (PA) powder using two conventional dairy starter strains, Lactobacillus delbrueckii subsp. bulgaricus SN22 and Streptococcus thermophilus SN05, to enhance its functional properties, particularly anti-inflammatory activity, via microbial processing. Both strains demonstrated favourable safety and antimicrobial activity. Untargeted metabolomics revealed that fermentation significantly altered the metabolite profile of the PA supernatant, enriching compounds with potential bioactivities, notably anti-inflammatory (e.g., 3-anisic acid) and antioxidant (e.g., vitamin U) properties. In the DSS-induced mouse colitis model, treatment with the fermented supernatant alleviated intestinal inflammation compared to the unfermented group. This was demonstrated by significantly reduced levels of the pro-inflammatory cytokines IL-1β and TNF-α, along with improved maintenance of intestinal barrier integrity. Further in vitro assays showed that the fermented supernatant significantly suppressed proliferation and clonogenicity in human HT-29 colon cancer cells, while also inducing reactive oxygen species accumulation and apoptosis. Results demonstrate these strains are multifunctional starters possessing superior antimicrobial and anti-inflammatory efficacy. This study employed LAB fermentation of insect-derived matrices to derive bioactive components. The fermentation products exhibited anti-inflammatory potential, offering a potential microbial transformation strategy for developing functional products for adjunctive UC intervention.