A Comprehensive Safety Assessment of Ralstonia eutropha H16 for Food Applications: Integrating Genomic, Phenotypic, and Toxicological Analyzes

Abstract

Ralstonia eutropha H16, a metabolically versatile bacterium, has gained prominence as a microbial platform for sustainable bioproduction. While its capabilities in synthesizing single-cell proteins and biodegradable materials are well documented, comprehensive strain-level safety evaluations remain insufficient for food-grade applications. This study systematically assessed the safety of R. eutropha H16 through genomic, phenotypic, and toxicological analyzes. Genomic analyzes revealed the absence or minimal presence of virulence factors and antibiotic resistance genes, aligning with microbiological safety standards. Phenotypic investigations demonstrated a limited gastric fluid tolerance (pH 2.5, survival rate 25.70% after 3 h) and intestinal fluid persistence (pH 8, 44.67% viability after 3 h), coupled with an exceptional bile salt tolerance (0.2% w/v). Antioxidant assays confirmed the fermentation broth specifically scavenges DPPH free radicals (14.60 ± 1.24 μg Trolox/mL), whereas bacterial suspensions and cell-free supernatants exhibited a strong hydroxyl radical scavenging (>90 U/mL) and superoxide anion inhibition (>100 U/L). Acute toxicity testing indicated no mortality or histopathological abnormalities, with an LD₅₀ value exceeding 1 × 10¹¹ CFU/kg. Subacute toxicity studies (28-day, 1 × 10⁸-1 × 10¹⁰ CFU/kg) revealed no significant effects on growth, hematology, or organ function. Minor alterations in serum biochemistry might be attributed to physiological adaptation. Subacute exposure induced transient serum ALT fluctuations without hepatorenal dysfunction, while maintaining hematological parameters within physiological ranges. Collectively, these results substantiate the safety of R. eutropha H16 for food-related applications while underscoring the necessity of strain-specific risk assessments for industrial microbial platforms.

Keywords: Ralstonia eutropha H16; acute and subacute toxicity; phenotypic analysis; safety assessment.

Figure 1

The genomic annotation and analysis of R.eutropha H16. (A) GO classification statistics. (B) KEGG classification statistics. (C) COG classification statistics. (D) The CAZymes classification of protein functions. (E) MirvDB classification statistics. (F) PHI-base classification statistics.

Figure 2

The physiological features analysis of R. eutropha H16. (A) Growth curves under different acidic conditions (pH = 2, 3, and 4) and different concentrations of bile salts (0.1%, 0.2%, and 0.3%). (B) R. eutropha H16 viable bacteria at 0 h and 3 h in simulated gastric fluids (SGF) and simulated intestinal fluids (SIF). (C) The oxidation resistance of different parts. ·OH, hydroxyl radical U/mL; O₂⁻, superoxide anion, U/mL; DPPH, 1,1-diphenyl-2-picrylhydrazyl, ug Trolox/mL. (D) Biofilm forming capacity. (E) Auto-aggregation. (F) The hydrophobicity of R. eutropha H16 towards xylene, hexadecane, and octane at different time points. “RH16” means R. eutropha H16.

Figure 3

Acute toxicity assessment results of R. eutropha H16. (A) Acute toxicity experiment design. (B) The body weight of male and female rats. (C) The food intake of male and female rats. (D) The organ coefficient of male rat liver and female rat kidney. (E) The MPV value of male rats. MPV mean platelet volume. (F) Histopathological results of organs with liver and kidney. a. Liver, male control group, 20×. b. Liver, male RH16 group, 20×. c. Kidney, female control group, 20×. d. Kidney, female control group, 20×. “ns”, no significant difference compared to the control group, p > 0.05. * Significantly different from the control group, p < 0.05.

Figure 4

Subacute toxicity assessment results of R. eutropha H16. (A) Subacute toxicity experiment design. (B) Body weight of male. (C) Body weight of female. (D) Food intake of male. (E) Food intake of female. RC control group; RL R. eutropha H16 low-dose, 10⁸ CFU/kg/day; RM R. eutropha H16 medium-dose, 10⁹ CFU/kg/day; RH R. eutropha H16 high-dose, 10¹⁰ CFU/kg/day. (F) The histopathological results of the control group and high-dose group. Heart, liver, spleen, kidney, pancreas, testis, ovary, 20×; thymus, 40×. RH16-H, R. eutropha H16 high-dose group.