# Bactericidal and antibiofilm activity of lactic acid bacteria-derived cell free extracts against dairy-associated spoilage and pathogenic bacteria

## TL;DR

This study shows that extracts from lactic acid bacteria can effectively kill and prevent biofilms of spoilage and harmful bacteria in dairy products.

## Contribution

The study identifies specific lactic acid bacteria strains and their extracts with strong antimicrobial and antibiofilm properties for use in dairy systems.

## Key findings

- Non-neutralized cell-free supernatants showed strong bactericidal activity against spoilage and pathogenic bacteria.
- Lactiplantibacillus plantarum Q4C3 and other strains were identified as the most effective biocontrol agents.
- SEM analysis revealed membrane disruption in treated cells, confirming the efficacy of the extracts.

## Abstract

This study aimed to evaluate the antimicrobial and antibiofilm potential of 15 LAB strains using five types of LAB-derived preparations: cell-free supernatants (CFS), sonicated-inactivated cells (IC), their combination (ICS), and their neutralized variants (CFS N, ICS N) to identify the most effective strain-extract combinations for potential application as natural biocontrol agents in dairy systems. Antimicrobial activity was assessed through agar diffusion assays, growth and biofilm inhibition, and determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations. The extracts were further characterized by pH and organic acid profiles using high-performance liquid chromatography (HPLC), and their mechanisms of action were investigated through cellular leakage assays, time–kill kinetics, and scanning electron microscopy (SEM). Several LAB-derived extracts exhibited strong antagonistic and antibiofilm activity against both spoilage and pathogenic bacteria. Non-neutralized CFS and ICS showed pronounced bactericidal activity, confirming the central role of organic acids in microbial inhibition. Z-score ranking and leakage assays identified Lactiplantibacillus plantarum Q4C3, Lactococcus lactis subsp. lactis biovar diacetylactis SBR4, Weissella cibaria W21, and Weissella viridescens W23 as the most effective strains. Time–kill assays demonstrated rapid microbial reductions (>3 log CFU/mL) within 4 h by CFS, whereas ICS required longer exposure. SEM analysis revealed severe membrane disruption in CFS-treated cells and the presence of LAB-derived debris surrounding ICS-exposed cells. These findings demonstrate that acidic LAB-derived extracts, particularly CFS, efficiently disrupt microbial cells and support their use as safe and effective natural biocontrol agents for improving the microbial safety and quality of dairy products.