Anti-Listerial Activity of Bacteriocin-like Inhibitory Substance Produced by Enterococcus lactis LBM BT2 Using Alternative Medium with Sugarcane Molasses

Abstract

Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, thus reaching final products by cross-contamination. With the growing demand for clean-label products, the search for natural antimicrobials as biopreservants, such as bacteriocins, has shown promising potential. In this context, this study aimed to evaluate the anti-listerial action of bacteriocins produced by Enterococcus lactis LBM BT2 in an alternative medium containing sugarcane molasses (SCM). Molecular analyses were carried out to characterize the strain, including the presence of bacteriocin-related genes. In the kinetic study on SCM medium E. lactis, LBM BT2 showed biomass and bacteriocin productions similar to those observed on a sucrose-based medium (control), highlighting the potential of the sugarcane molasses as a low-cost substrate. Stability tests revealed that the molecule remained active in wide ranges of pH (4-10) and temperature (60-100 °C). Furthermore, the proteolytic treatment reduced the biomolecule's antimicrobial activity, highlighting its proteinaceous nature. After primary purification by salting out and tangential flow filtration, the bacteriocin-like inhibitory substance (BLIS) showed bacteriostatic activity on suspended L. monocytogenes cells and against biofilm formation at a concentration of 0.625 mg/mL. These results demonstrate the potential of the produced BLIS as a biopreservative in the food industry.

Keywords: Enterococcus lactis; Listeria monocytogenes; anti-listerial activity; bacteriocin-like inhibitory substance; sugarcane molasses.

Figure 1

Agarose gel electrophoresis of genomic DNA (DNAg) extraction from strain E. lactis LBM BT2 (A), 16S rRNA amplification using DNAg from LBM BT2 (B), PCR products using gluP primer set (gluP1 or gluP2) (C) or entP oligonucleotides (entP1 or entP2) (D). As PCR template, it employed DNAg strains from collection of the Microbial Biomolecules Laboratory (LBM), e.g., E. lactis LBM BT2 (BT2), E. lactis LBM M3D (M3D), E. faecalis LBM 3148 or Lactococcus lactis LBM B1 (B1). DNA Ladder: 100 bp DNA Ladder RTU (Kasvi, São José dos Pinhais, PR, Brazil).

Figure 2

Enterococcus lactis LBM BT2 growth (optical density, OD) in MRS-basal medium with different carbon sources. Data are presented as mean ± standard deviation.

Figure 3

Shake flask cultivation of E. lactis LBM BT2 in molasses (A) and sucrose-based medium (B); cell dry concentration (X (g/L), O); pH variation (Δ); total sugars (S, (g/L), ◊); BLIS activity (AU/mL, □).

Figure 4

Batch fermentation of E. lactis LBM BT2 growth and bacteriocin production in a stirred tank 2 L bioreactor; cell dry concentration (X (g/L), O); total sugars (S (g/L), ◊); BLIS activity (AU/mL, □).

Figure 5

Tricine-SDS-PAGE of partially purified BLIS produced by E. lactis LBM BT2 after staining with Coomassie Brilliant Blue G250. Proteins from salted-out (from 10 to 60% w/v), CFS and semi-purified BLIS samples were separated in 16% of acrylamide gel in the presence of 6 M urea. MW: molecular weight: Precision plus protein dual Xtra Standards (Bio-Rad, Hercules, CA, USA).

Figure 6

Listeria monocytogenes growth (optical density, OD) in BHI medium with different concentrations of the semi-purified BLIS produced by Enterococcus lactis LBM BT2. Data are presented as mean ± standard deviation.

Figure 7

Inhibitory effect of semi-purified BLIS from Enterococcus lactis BT2 against Listeria monocytogenes biofilm formation. Average values with different superscripts indicate significant differences (p < 0.05).