Effect of Fermentation Time and Heat Treatment on Yield and In Vitro Antioxidant and Antimicrobial Activity of Postbiotics from Mixed Bifidobacterium and Lactobacillus Co-Fermentations

Abstract

This study evaluated the effects of fermentation time (24, 48, and 72 h), and post-fermentation heat treatment (PFHT) on yield, physicochemical properties, antioxidant activity, and antimicrobial activity of postbiotics produced from the co-fermentation of Lactobacillus rhamnosus GG, Lactobacillus plantarum, Bifidobacterium longum, and Bifidobacterium breve. Cross-streak assays confirmed strain compatibility under anaerobic conditions. Net postbiotic yield (dry mass corrected against an uninoculated control) was significantly influenced by fermentation time and PFHT (P < 0.05), with the highest yield observed at 48 h under PFHT (70.97 ± 0.36 g/L). Fermentation was characterized by progressive acidification (pH 6.37-3.81) and biomass accumulation (OD₆₀₀: 0.15-1.66). The solubility of freeze-dried postbiotics was 95-98% and was not significantly affected by fermentation time or PFHT (P > 0.05). Antioxidant activity increased with fermentation time and was significantly higher in PFHT samples, with DPPH (62.09-83.43%) and ABTS⁺ (79.82-95.62%) scavenging activities, reaching a plateau at 48 h. Antimicrobial activity was greater in PFHT samples, with the largest inhibition zones of 14.33 ± 0.58 mm were observed at 48 h. The correlation and principal component analyses indicated associations between acidification and antioxidant activity, although causality cannot be inferred. Within the limitations of this study, including a single heat treatment condition, absence of minimum inhibitory concentration data, lack of metabolomic characterization, and exclusive in vitro evaluation, the findings suggest that 48 h fermentation combined with PFHT provides a favorable balance of postbiotic yield and bioactivity. Further studies are required to support optimization and application.