Carbon–nitrogen interaction controls postbiotic short-chain fatty acid spectrum in a bacterial–yeast consortium: a central composite design approach

Background and Aim: Postbiotics, particularly short-chain fatty acids (SCFAs), play critical roles in gut health, immune modulation, and animal productivity. However, nutrient-driven metabolic regulation of SCFA production in mixed microbial systems under rumen-simulated conditions remains poorly understood. This study aimed to optimize SCFA production and to evaluate how carbon (C) and nitrogen (N) concentrations, and incubation time, interact to control metabolic outputs in a bacterial–yeast consortium during in vitro rumen fermentation. Materials and Methods: A co-culture of Schleiferilactobacillus harbinensis LH991 and Pichia kudriavzevii B-5P was incubated anaerobically with goat rumen fluid using a response surface methodology–central composite design. Three variables were tested: glucose (0.1–0.3 g/L), yeast extract (5–15 g/L), and incubation time (24–72 h). Individual SCFAs (acetate, propionate, butyrate, iso-butyrate, valerate, and iso-valerate) were quantified by gas chromatography, and quadratic polynomial models were used to determine optimal conditions and interaction effects. Results: Model adequacy was confirmed with R² values ranging from 0.82 to 0.94 and non-significant lack-of-fit tests (p > 0.05). Optimal acetate production occurred at moderate C (0.2 g/L), N (10 g/L), and 48 h incubation. In contrast, propionate, butyrate, iso-butyrate, and iso-valerate production were maximized under low C (0.1 g/L), high N (15 g/L), and extended incubation (72 h). Valerate production showed dual optima depending on incubation duration and substrate balance. Response surface plots demonstrated clear nutrient-dependent metabolic shifts, indicating that N enrichment combined with C limitation redirected metabolic flux toward branched-chain and energy-dense SCFAs. Conclusion: This study demonstrates a previously unreported nutrient-dependent metabolic switching mechanism in a bacterial–yeast consortium under rumen-simulated conditions. Precise manipulation of C, N, and incubation time enables targeted modulation of SCFA profiles, providing a scalable strategy for cost-effective postbiotic production. These findings support the development of optimized microbial fermentation systems for animal nutrition, functional feeds, and industrial postbiotic applications. Keywords: bacterial–yeast consortium, central composite design, in vitro rumen fermentation, nitrogen source optimization, postbiotic production, response surface methodology, short-chain fatty acids, Schleiferilactobacillus harbinensis.