Although high-protein diets are widespread, the fate of dietary protein, its fermentation by gut microbiota, and the resulting effects on intestinal health are not yet fully understood. This article provides a comprehensive overview of microbial protein fermentation and its impact on intestinal health. We focus on the bacterial anabolic and catabolic pathways involved in microbial protein metabolism and the generation of metabolites such as ammonia, biogenic amines (BAs), and short-chain fatty acids (SCFAs). This review also examines how excessive dietary protein affects intestinal health. Elevated protein levels may disrupt microbial homeostasis, promoting the proliferation of pathogenic bacteria while reducing beneficial microbiota. Furthermore, enhanced bacterial metabolic activity can lead to greater production of harmful compounds such as BAs. These alterations are associated with impaired intestinal barrier function, immune dysregulation, and elevated inflammatory responses. Further research is necessary to clarify the metabolism of high-protein diets and their consequences for intestinal health.

The ban on antibiotic growth promoters in livestock production has created an urgent demand for safe and effective probiotic alternatives derived from indigenous animal microbiota. Here, we isolated Enterococcus hirae HI3 from Hu sheep jejunum and confirmed its species assignment via whole-genome sequencing, with ANI analysis showing 98.92% identity to the E. hirae reference genome. HI3 showed moderate acid tolerance, with survival rates of 19.31% (95% CI: 15.83–22.78%) at pH 2 and 18.40% (95% CI: 17.87–18.94%) at pH 3 after 4 h, and maintained > 50% survival in 2% bile salts. It was susceptible to penicillin, ampicillin, erythromycin, and chloramphenicol. No hemolytic activity was observed, and major enterococcal virulence genes (gelE, cyl, esp, hyl) were absent from the HI3 genome. The HI3 genome (3.06 Mb) consists of one circular chromosome and three circular plasmids. Functional annotation identified 229 carbohydrate metabolism genes (including 65 glycoside hydrolases) and 43 probiotic-related genes involved in acid tolerance (atpA–G, nhaC), bile salt tolerance (cbh), stress responses (clp family, groEL, dnaK, cspA, sod2), and adhesion (ltaS, srtA, eno, epsA). Multiple bacteriocin biosynthetic gene clusters (enterolysin A, class II lanthipeptide, etc.) were identified. Plasmid-borne tetracycline resistance genes tet(M) and tet(L) were detected; however, their functional transferability requires experimental validation. No other known transferable resistance determinants were identified in the genomic analysis. These in vitro and genomic findings represent an initial characterization of E. hirae HI3 and support its potential as a ruminant probiotic candidate. However, in vivo studies are required to validate its colonization capacity, safety, and efficacy in the target ruminant species.