Nutritional Value and Food Safety Assessment of Single-Cell Protein Derived from <i>Ralstonia eutropha</i> for Food Applications

The growing global protein demand and environmental concerns from conventional animal agriculture have driven the exploration of sustainable alternative protein sources. Single-cell proteins (SCPs) from microbial fermentation offer a promising solution. This study comprehensively evaluated the nutritional value and safety profile of SCP produced from <i>Ralstonia eutropha</i> H16 through integrated <i>in vitro</i> and <i>in vivo</i> assessments. Nutritional analyses revealed a high crude protein content of 71.87 ± 5.05 g/100 g dry weight, with total amino acids of 53.67 ± 1.05 g/100 g. The essential amino acid content was 24.38 ± 0.51 g/100 g, accounting for 45% of the total amino acids. An essential amino acid index (EAAI) of 1.46 ± 0.04 and an amino acid score (AAS) of 0.83 ± 0.06 confirmed its classification as a high-quality protein source according to FAO/WHO standards. <i>In vivo</i> rat feeding trials demonstrated an adjusted protein efficiency ratio (PER) of 1.81, exceeding common plant proteins such as wheat (0.8-1.1). True digestibility (TD) reached 85.73%, with a biological value (BV) of 49.37%, net protein utilization (NPU) of 42.33%, and protein digestibility-corrected amino acid score (PDCAAS) of 0.71. Comprehensive safety assessments included chemical contaminant screening, acute oral toxicity studies in rats and mice, <i>in vitro</i> chromosome aberration tests, and erythrocyte micronucleus tests. Heavy metals and aflatoxin B₁ levels were below regulatory limits. Acute oral toxicity studies established LD₅₀ values exceeding 10,000 mg/kg body weight in both rodent species, classifying this protein source as practically non-toxic. The 28-day sub-acute toxicity study showed no significant adverse effects at low doses (6.25% protein replacement). Both genotoxicity assays (mammalian cell chromosome aberration assay and mammalian erythrocyte micronucleus test) returned negative results. These findings establish <i>R. eutropha</i> H16-derived SCP as a safe, nutritious, and sustainable protein source with considerable potential for feed and food applications, contributing to global food security and environmental sustainability.