# Biofilm-Forming Lactic Acid Bacteria in Sausages: Isolation, Characterization, and Inhibition Using Eisenia bicyclis-Based Nanoparticles

## TL;DR

This study explores biofilm-forming bacteria in sausages and tests natural compounds and nanoparticles to inhibit their growth, aiming to improve meat product safety and shelf life.

## Contribution

The study introduces nano-formulated phloroglucinol from Eisenia bicyclis as a novel natural antibiofilm agent for food preservation.

## Key findings

- PG-AuNPs showed strong antibiofilm activity, especially against Pseudomonas aeruginosa and L. sakei S10.
- PG and PG-AuNPs reduced biofilm biomass more effectively in vitro than in situ on sausage surfaces.
- PG-AuNPs had a spherical shape, nanoscale size, and good colloidal stability.

## Abstract

Background/Objectives: Lactic acid bacteria produce biofilms in meat products that contribute to the products’ deterioration, reduction in quality, and shortened shelf life. Although LAB are generally considered benign, certain strains create slime and cause significant drops in pH. The study’s goal was to identify and characterize LAB strains from sausage products that are capable of biofilm formation, and to evaluate the inhibitory effects of E. bicyclis methanol extract, its ethyl acetate fraction, and phloroglucinol, as well as to synthesize AuNPs, and assess their efficacy in controlling biofilm formation. Methods: Slime or biofilm-producing LAB bacteria were isolated from commercial sausages and identified using 16S rRNA gene sequencing. Lactobacillus sakei S10, which can tolerate high salt concentrations and cold temperatures, was chosen as a representative strain. The isolates were subsequently tested for hemolytic activity, salt and temperature tolerance, and carbohydrate consumption patterns. To evaluate antibiofilm potential, marine-derived compounds from Eisenia bicyclis, such as phloroglucinol (PG), crude methanolic extracts, ethyl acetate fractions, and gold nanoparticle (AuNP) formulations, were tested in situ on sausage surfaces against L. sakei S10 and common pathogens (Pseudomonas aeruginosa and Staphylococcus aureus). The biofilm-inhibitory effects of the extracts, PG, and PG-AuNPs were estimated using the colony-counting method. Results: The PG-AuNPs had an average particle size of 98.74 nm and a zeta potential of −29.82 mV, indicating nanoscale dimensions and considerable colloidal stability. Structural analysis confirmed their spherical form and crystalline structure, as well as the presence of phenolic groups in both reduction and stabilization processes. Among the studied treatments, the PG and PG-AuNPs had the strongest antibiofilm activities, dramatically lowering biofilm biomass, particularly for P. aeruginosa and L. sakei S10. However, the inhibitory effects were less prominent in in situ conditions than in in vitro testing, highlighting the complexity of real food matrices. Conclusions: The results of this study indicate that polyphenolic compounds obtained from marine sources, particularly in nano-formulated forms, have a great deal of potential as natural antibiofilm products. Enhancing the microbiological safety of processed meat products and extending their shelf life could be accomplished through the application of these polyphenolic compounds in food packaging or surface treatments.