Abstract

Background and Aim: Industrial-scale probiotic production requires economically viable media formulations that do not compromise strain functionality. This study aimed to develop a cost-effective medium for cultivating Lactiplantibacillus plantarum 22F (L22F), a probiotic candidate isolated from swine feces, while evaluating its industrial viability and functional metabolic profile.

Materials and Methods: Carbon (glucose, sucrose, and dextrose) and nitrogen (yeast extract, soy protein isolate, and whey protein concentrate) sources were screened using one-variable-at-a-time and Plackett–Burman design, followed by Response Surface Methodology for optimization. Fermentation was scaled from a flask to 50 L fermenters at 37 °C and pH 6.50 ± 0.05. Cell viability, pH, and residual sugar were monitored. Functional assessments included stress tolerance assays (heat, acid, bile, and oxidative stress) and untargeted metabolomic profiling using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Results: The optimal medium comprised 9 g/L glucose, 14.1 g/L soy protein isolate, and 14.1 g/L yeast extract, supplemented with minerals. In 50 L fermentation, L22F achieved 9.20 log colony-forming units/mL at 12 h, with residual sugar at 1.50 g/L and pH 3.99. Compared to de Man, Rogosa, and Sharpe, the modified medium reduced production cost by 70%–88%, improved fermentation efficiency, and supported enhanced stress resilience. Metabolomic analysis revealed an elevated production of bioactive metabolites, particularly 1,4-dihydroxy-2-naphthoic acid and indolelactic acid, which are known to support gut homeostasis, anti-inflammatory responses, and probiotic efficacy.

Conclusion: This study presents a cost-effective and scalable fermentation medium specifically designed for high-density L22F production. Beyond economic advantages, the medium enhanced the functional properties of L22F, supporting its application as a sustainable probiotic feed additive for swine. These findings establish a foundation for further industrial application and in vivo validation.

Keywords: cost-effective production, fermentation optimization, Lactiplantibacillus plantarum 22F, metabolomics, modified medium, stress tolerance, swine probiotic.