Co-fermented whey protein by Lactobacillus plantarum HM1 and Lactobacillus rhamnosus HN001: Study on antibacterial, mechanisms, and bread preservation performance

Filamentous fungal contamination poses significant risks to bakery product safety, driving the demand for natural alternatives to synthetic preservatives within the “clean label” trend. We developed fermented whey powder (FWP) by co-fermenting whey protein concentrate with Lactobacillus plantarum HM1 and Lactobacillus rhamnosus HN001. Co-fermentation significantly enhanced the cell-free supernatant's antimicrobial activity against Staphylococcus aureus (86.99%), Escherichia coli (81.33%), Penicillium spp. (82.76%), and Aspergillus niger (83.09%). Metabolomics revealed upregulated antimicrobial metabolites and enrichment of the KEGG pathway “Biosynthesis of antibiotics”. When incorporated into bread at 3% w/w, FWP increased specific volume (3.84 cm 3 /g vs. 3.47 cm 3 /g) and pore count (2623 vs. 2249), enhancing crumb structure. FWP attenuated staling, reduced moisture migration, and extended shelf-life through suppressing microbial growth. Crucially, FWP (3–5%) extended the mold-free period by 3 days at 25°C in inoculated bread, matching or exceeding conventional synthetic preservatives. FWP is a multifunctional, clean-label bio-preservative from valorized dairy byproducts. • L. plantarum & L. rhamnosus co-fermentation boosts whey powder's antimicrobial effect. • Co-fermented CFS metabolomics: more antimicrobials & enriched antibiotic pathways. • Fermented whey powder (FWP) enhances freshly baked bread quality & sensory traits. • FWP: a clean-label multifunctional bio-preservative for bread.