Protocols for the isolation and detection of lactic acid bacteria with bacteriocinogenic potential

Abstract

a b s t r a c tThe objective of this study was to evaluate culture media and methodologies for isolation and detection of lactic acid bacteria (LAB) capable to produce bacteriocin-like substances. Samples of milk and cheese were pour plated on de Mann-Rogosa-Sharpe agar (MRS) and Kang-Fung-Sol agar (KFS) (both at 35 C/48 h, under anaerobiosis), from which 389 and 256 LAB cultures were selected. The antagonistic activity of them was evaluated using the spot-on-the-lawn and two culture media: brain-heart infusion agar with catalase (BHI þ C) and M17 (both at 35 C/24 h). The proteinaceous nature of the antagonistic cultures was verified using: spot-on-the-lawn (MRS, 25 C/24 h, under anaerobiosis) and well-diffusion (cultures amplified on modified MRS broth at 25 C/24 h, and then neutralized using NaOH). Listeria monocytogenes ATCC 7644 was used as indicator. A larger number of antagonist cultures were isolated from MRS (83 by M17 and 65 by BHI þ C) in comparison to KFS (24 by M17 and 15 by BHI þ C). The spot-onthe-lawn identified a higher frequency of LAB capable of producing bacteriocin-like substances. MRS was considered to be the best culture media for the isolation of LAB capable to produce bacteriocin-like substances, activity that was better identified using the spot-on-the-lawn methodology.

Partial Characterization of Bacteriocin Produced by Halotolerant Pediococcus acidilactici Strain QC38 Isolated from Traditional Cotija Cheese

“…The isolates were evaluated by cell morphology and Gram's stain reaction. The isolates were screened for bacteriocin production using the spot on the lawn assay using MRS plates (Moraes et al, 2010). Five µl of each isolated strain was spotted on MRS plates and incubated at 28°C for 24 h. After incubation, the plates were overlaid with 10 ml of BHI soft agar and inoculated with 10 5 CFU/ml of every tested pathogenic indicator strain.…”

Molecular Characterization of Bacteriocinogenic, Antifungal and Probiotic Lactic Acid Bacteria Isolated from Chicken Gastrointestinal Tract

“…The bacteriocinogenic nature of antibacterial activity was determined by using 10-fold-concentrated CFS treated with acid protease (2 mg•ml −1 ) (Sigma, India) in 10 mM citrate buffer pH 3.0 and incubated at 37˚C for 3 h. Antibacterial activity was checked at pH 6.0 against Staphylococcus aureus and compared with control which was also processed in a similar way without enzyme [14].…”

Biopreservation potential ofEnterococcus faecalisisolated from Italian traditional raw milk cheeses

“…In fact, the lack of antimicrobial activity expression in phenotypical assay was previously reported. It was revealed as dependent on numerous factors, such as the interference of environmental conditions and the non-induction of the expression of genes encoding bacteriocins (Moraes et al, 2010). In particular, bacteriocins seem to be commonly regulated and subsequently produced only under appropriate growth conditions, such as incubation temperatures.…”

“…Some bacteriocins are also produced on solid growth media but not in liquid cultures (Strompfová, Lauková, Simonová, & Marciňáková, 2008). Moraes et al (2010) assessed that the performance of the antimicrobial activity assay of LAB varies in relation to culture media and protocols too. E. faecalis strains were subsequently subjected to PCR analysis.…”

“…In fact, bacteriocins may serve as an alternative to chemical preservatives in foods (Casaus et al, 1997): their production may represent an additional functional property of value for selected food applications (Cleveland, Montville, Nes, & Chikindas, 2001), above all as a consequence of the increased demand by consumers for products that are less processed and are free of preservatives. They are also an interesting option for the food industry as their addition does not modify the taste or smell of the final products (Moraes et al, 2010). Furthermore, the application of bacteriocin-producing strains as starter or adjunct cultures in the production process of fermented food provides an attractive and economic alternative to the addition of bacteriocins, whose purification is expensive if compared to cultivation of strains for inoculation purposes (O'Sullivan, Ross, & Hill, 2002).…”