Influence of different culture media on the antimicrobial activity of Pediococcus pentosaceus ST65ACC against Listeria monocytogenes.

Pediococcus pentosaceus ST65ACC is a bacteriocinogenic lactic acid bacteria (LAB) isolated from Brazilian artisanal cheese that is capable of inhibiting different food pathogens, mainly Listeria monocytogenes. The production of bacteriocins can be influenced by several growth conditions, such as temperature, pH, and medium composition. This study aimed to evaluate the effect of different culture media on the production of bacteriocins and antimicrobial activity of P. pentosaceus ST65ACC on L. monocytogenes Scott A. The strains were inoculated alone and in coculture in four different media: BHI broth, MRS broth, meat broth, and reconstituted skim milk (RSM) 10% (w/v). The culture media were then incubated at 37 °C for 96 h, and count analysis, pH measurement, and bacteriocin production were performed at 0, 24, 48, 72 and 96 h. L. monocytogenes was inhibited to nondetectable levels in coculture with P. pentosaceus ST65ACC in MRS broth within 96 h, consistent with the high production of bacteriocin throughout the analysis period (3,200-12,800 AU/mL). However, lower inhibitory activities of P. pentosaceus ST65ACC on L. monocytogenes Scott A were recorded in BHI, RSM, and meat broth, with low or no production of bacteriocins at the analyzed times. The composition of these culture media may have repressed the production and activity of bacteriocins and, consequently, the antagonist activity of P. pentosaceus ST65ACC on L. monocytogenes Scott A. The results showed that the antimicrobial activity was more effective in MRS broth, presenting greater production of bacteriocins and less variability when compared to the other media analyzed.

Influence of Emulsifying Salts on the Growth of Bacillus thuringiensis CFBP 3476 and Clostridium perfringens ATCC 13124 in Processed Cheese.

Processed cheese is a dairy product with multiple end-use applications, where emulsifying salts play a fundamental role in physicochemical changes during production. Moreover, some of these salts may be a strategy to control spoilage and pathogenic microorganisms, contributing to safety and shelf life extension. This study aimed to evaluate the in vitro inhibitory activity of two emulsifying salts (ESSP = short polyP and BSLP = long polyP) against Bacillus thuringiensis CFBP 3476 and Clostridium perfringens ATCC 13124, and to compare the in situ effects of two emulsifying salts treatments (T1 = 1.5% ESSP and T2 = 1.0% ESSP + 0.5% BSLP) in processed cheeses obtained by two different methods (laboratory- and pilot-scales), during 45-day storage at 6 °C. C. perfringens ATCC 13124 growth was not affected in vitro or in situ (p > 0.05), but both of the treatments reduced B. thuringiensis CFBP 4376 counts in the tested condition. Counts of the treatments with B. thuringiensis CFBP 3476 presented a higher and faster reduction in cheeses produced by the laboratory-scale method (1.6 log cfu/g) when compared to the pilot-scale method (1.8 log cfu/g) (p < 0.05). For the first time, the inhibitory effect of emulsifying salts in processed cheeses obtained by two different methods was confirmed, and changes promoted by laboratory-scale equipment influenced important interactions between the processed cheese matrix and emulsifying salts, resulting in B. thuringiensis CFBP 4376 growth reduction.

Physiological and molecular insights of bacteriocin production by Enterococcus hirae ST57ACC from Brazilian artisanal cheese.

The bacteriocinogenic Enterococcus hirae ST57ACC recently isolated from a Brazilian artisanal cheese was subjected here to additional analyses in order to evaluate its bacteriocin production and the potential influence of ABC transporter system in its expression. Besides these physiological and molecular aspects, the bacteriocin was evaluated for its cytotoxicity against HT-29. Differences in the inoculum size had no impact on the growth of E. hirae ST57ACC; however, the bacteriocin was only produced after 9 h of growth when the strain was inoculated at 5% or 10% (v/v), with similar levels of bacteriocin production obtained by both conventional growth and batch fermentation. Furthermore, potential expression of ABC transporters corresponding to the bacteriocin transport and sugar metabolism was identified. In terms of adverse effects, when a semi-purified fraction of the bacteriocin and the cell-free supernatant were tested against HT-29, total cell viability was similar to observed on untreated cells, indicating the absence of cytotoxic effect. Based on the obtained results, E. hirae ST57ACC can produce its bacteriocin at industrial level by using bioreactors, its bacteriocin expression is potentially influenced by the ABC transporter system, and no cytotoxic effects were observed on HT-29 cells, indicating its potential use as a bio-preservative.

Potential Control of Listeria monocytogenes by Bacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC Strains Isolated From Artisanal Cheese.

Bacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC strains, previously isolated from artisanal cheese, were evaluated for their safety with the aim to determine whether they could be used as beneficial strains, especially in the control of Listeria monocytogenes. Both isolates survived simulated gastrointestinal conditions and showed high levels of auto- and co-aggregation with L. monocytogenes, although the hydrophobicity of cells varied. Using the agar-spot test with 33 commercial drugs from different groups, only anti-inflammatory drugs and drugs containing loratadine and propranolol hydrochloride were able to affect the growth of the tested strains. Both strains were resistant to 3 out of 11 antibiotics tested by the disc diffusion method, and low frequencies of antibiotic resistance-encoding genes were observed by PCR analysis. Tested strains neither presented biogenic amine-related genes nor produced these substances. Aside from some antibiotic resistance characteristics, the tested strains were considered safe as they lack other virulence-related genes. E. hirae ST57ACC and P. pentosaceus ST65ACC both presented beneficial properties, particularly their ability to survive gastrointestinal conditions and to aggregate with L. monocytogenes, which can facilitate the elimination of this pathogen. Further studies should be conducted to better understand these interactions.

Inhibition of herpes simplex virus 1 (HSV-1) and poliovirus (PV-1) by bacteriocins from lactococcus lactis subsp. lactis and enterococcus durans strains isolated from goat milk.

Bacteriocins have unusual inhibitory activity, including antiviral properties, and this can be exploited to give alternative applications. Semi-purified bacteriocins of six lactic acid bacteria (LAB) strains isolated from goat milk (two Lactococcus lactis: GLc03 and GLc05, and four Enterococcus durans: GEn09, GEn12, GEn14 and GEn17) were tested for cytotoxicity in Vero cells (50% Cytotoxicity Concentration: CC50), and for their antiviral activities against herpes simplex virus 1 (HVS-1) and poliovirus (PV-1). Semi-purified bacteriocins presented low cytotoxicity, with CC50 varying from 256.2 µg/mL (GLc05) to 1084.5 µg/mL (GEn14). CC10 was determined for all isolates (GLc03: 36.9 µg/mL; GLc05: 51.2 µg/mL; GEn09: 88.1 µg/mL; GEn12: 99.9 µg/mL; GEn14: 275 µg/mL; and GEn17: 62.2 µg/mL) and considered for antiviral activity assays. Antiviral activity before virus adsorption was recorded against PV-1 for GLc05 (4.9%), GEn09 (3.4%), GEn12 (24.7%) and GEn17 (23.5%), and against HSV-1 for GEn12 (27.9%), GEn14 (58.7%) and GEn17 (39.2%). Antiviral activity after virus adsorption was identified against PV-1 for GLc05 (32.7%), GEn09 (91.0%), GEn12 (93.7%) and GEn17 (57.2%), and against HSV-1 for GEn17 (71.6%). The results obtained indicate the potential of some bacteriocins, particularly those produced by E. durans strains investigated in the present study, in viral inhibition and their application as new antiviral agents.

Molecular tracking of Salmonella spp. in chicken meat chain: from slaughterhouse reception to end cuts.

Due to the importance of Salmonella spp. in poultry products, this study aimed to track its main contamination routes since slaughtering reception to processing of chicken end cuts. Samples from different steps of slaughtering and processing (n = 277) were collected from two chicken slaughterhouses (Sl1 and Sl2) located in Minas Gerais state, Brazil, and subjected to Salmonella spp. detection. The obtained isolates were subjected to serological identification and tested by PCR for specific Salmonella spp. genes (ompC and sifB). Also, Salmonella spp. isolates were subjected to XbaI macrorestriction and pulsed-field gel electrophoresis (PFGE). Sixty-eight samples were positive for Salmonella spp. and 172 isolates were obtained. Sl1 and Sl2 presented similar frequencies of Salmonella spp. positive samples during reception, slaughtering and processing (p > 0.05), except for higher frequencies in Sl1 for chicken carcasses after de-feathering and evisceration (p < 0.05). PFGE allowed the identification of cross contamination and persistence of Salmonella spp. strains in Sl1. The results highlighted the relevance of the initial steps of chicken slaughtering for Salmonella spp. contamination, and the pre-chilling of carcasses as an important controlling tool. In addition, the presence of Salmonella spp. in chicken end cuts samples represents a public health concern.

Serotypes and Pulsotypes diversity of Listeria monocytogenes in a beef-processing environment.

Utensils and equipment from meat-processing facilities are considered relevant cross-contamination points of Listeria monocytogenes to foods, demanding tracking studies to identify their specific origins, and predict proper control. The present study aimed to detect L. monocytogenes in a beef-processing facility, investigating the diversity of serotypes and pulsotypes in order to identify the possible contamination routes. Surface samples from knives (n=26), tables (n=78), and employees hands (n=74) were collected before and during the procedures from a beef-processing facility, in addition to surface samples of end cuts: round (n=32), loin (n=30), and chuck (n=32). All samples were subjected to L. monocytogenes screening according ISO 11.290-1, and the obtained isolates were subjected to serotyping and pulsed-field gel electrophoresis. Listeria spp. were identified in all processing steps, in 61 samples, and L. monocytogenes was detected in 17 samples, not being found only in knives. Eighty-five isolates were identified as L. monocytogenes, from serotypes 1/2c (n=65), 4b (n=13), and 1/2b (n=7), being grouped in 19 pulsotypes. Considering these results, cross-contamination among hands, tables, and beef cuts could be identified. The obtained data indicated the relevance of cross-contamination in the beef-processing facility, and the occurrence of serotypes 1/2b and 4b in beef cuts distributed for retail sale is a public health concern.

Genetic diversity and some aspects of antimicrobial activity of lactic acid bacteria isolated from goat milk.

Lactic acid bacteria (LAB, n = 57) were previously obtained from raw goat milk, identified as Lactococcus spp. (n = 24) and Enterococcus spp. (n = 33), and characterized as bacteriocinogenic. Fingerprinting by pulsed field gel electrophoresis (PFGE) demonstrated high genetic diversity, and 30 strains were selected and exhibited strong antimicrobial activity against 46 target strains (LAB, spoilage, and foodborne pathogens). Six strains (Lactococcus lactis: GLc03 and GLc05; and Enterococcus durans: GEn09, GEn12, GEn14, and GEn17) were selected to characterize their bacteriocinogenic features, using Listeria monocytogenes ATCC 7644 as the target. The six strains produced bacteriocins at higher titer when incubated in MRS at 37 °C up to 12 h, when compared to growth at 25 and 30 °C. The produced bacteriocins kept their antimicrobial activity after exposure to 100 °C for 2 h and 121 °C for 20 min; the antimicrobial activity was also observed after treatment at pH 2.0 to 10.0, except for GLc03. L. monocytogenes populations were reduced approximately two logs after treatment with cell-free supernatants from the selected strains. These data show that goat milk can contain a diverse microbiota able to inhibit L. monocytogenes, a common pathogen found in dairy products, and can be potentially employed in biopreservation of food produced under different processing conditions.