Plant-Based Protein Fermentation Medium Optimization for Antimicrobial Cell-Free Supernatant Production by Lactiplantibacillus plantarum K014 Against Cutibacterium acnes

Acne vulgaris is one of the most common dermatological conditions. Growing consumer demand for natural, vegan-friendly skincare has driven exploration of plant-based therapeutics. This study evaluates the antimicrobial potential of cell-free supernatants (CFS) from lactic acid bacteria (LAB) fermented in plant-based media against Cutibacterium acnes (C. acnes). Ten LAB strains were initially screened using agar-well diffusion, and six strains, namely, Lactiplantibacillus plantarum K014, Levilactobacillus brevis C23, Limosilactobacillus reuteri BM5, Lacticaseibacillus paracasei FD1 and FD2, and Lacticaseibacillus rhamnosus CM1, exhibited inhibition zones of 10.33 to 23.67 mm. Strain K014, which exhibited the highest activity, was further evaluated across five plant-based protein substrates. Brown rice yielded the strongest inhibition (16.00 mm). Optimization via response surface methodology (RSM) using central composite design (CCD) identified an optimal formulation of 35 g/L brown rice, 15 g/L yeast extract, and 30 g/L lactose, producing a 21.67-mm inhibition zone. The optimized CFS was freeze-dried (lyophilized CFS, LCFS) and characterized, revealing MIC₅₀ and MIC₉₀ values of 12.5 and 25 mg/mL, respectively. LCFS K014 demonstrated thermal stability between 60 and 121 °C and retained antimicrobial activity at pH 2-5, with reduced efficacy under alkaline conditions. Lactic acid and protein contents were quantified at 20% w/w and 0.15% w/w, respectively. These findings support LCFS K014 as a promising, stable, plant-based anti-acne agent and underscore lactic acid bacterial fermentation as a sustainable strategy for topical therapeutic development.