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Genomic and functional characterization of bacteriocinogenic lactic acid bacteria isolated from Boza, a traditional cereal-based beverage.

ABSTRACT: Boza is a traditional low-alcohol fermented beverage from the Balkan Peninsula, frequently explored as a functional food product. The product is rich in Lactic Acid Bacteria (LAB) and some of them can produce bacteriocins. In this study, a sample of Boza from Belogratchik, Bulgaria, was analyzed for the presence of bacteriocinogenic LAB, and after analyses by RAPD-PCR, three representative isolates were characterized by genomic analyses, using whole genome sequencing. Isolates identified as Pediococcus pentosaceus ST75BZ and Pediococcus pentosaceus ST87BZ contained operons encoding for bacteriocins pediocin PA-1 and penocin A, while isolate identified as Pediococcus acidilactici ST31BZ contained only the operon for pediocin PA-1 and a CRISPR/Cas system for protection against bacteriophage infection. The antimicrobial activity of bacteriocins produced by the three isolates was inhibited by treatment of the cell-free supernatants with proteolytic enzymes. The produced bacteriocins inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp., among other tested species. The levels of bacteriocin production varied from 3200 to 12,800 AU/ml recorded against L. monocytogenes 104, 637 and 711, measured at 24 h of incubation at 37 °C. All bacteriocins remained active after incubation at pH 2.0-10.0. The activity mode of the studied bacteriocins was bactericidal, as determined against L. monocytogenes 104, 637 and 711. In addition, bactericidal activity was demonstrated using a cell leakage β-galactosidase assay, indicating a pore formation mechanism as a mode of action. The present study highlights the importance of combining genomic analyses and traditional microbiological approaches as way of characterizing microbial interactions in fermented foods.

SUBMITTER: Queiroz LL

PROVIDER: S-EPMC8795150 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The multifunctional properties of autochthonous lactic acid bacteria can be of use for enhancing the sensorial properties of food, as well as in food preservation. An initial screening for antimicrobial, proteolytic, and lipolytic capacities was done in 214 presumptive lactic acid bacteria isolates obtained from Chihuahua cheese manufacturing and during a ripening period of nine months. The antimicrobial screening was done by spot-on-the-lawn tests, using Listeria monocytogenes and Escherichia coli as indicator microorganisms; proteolysis was tested in casein-peptone agar and lipolysis in Mann-Rogosa-Sharpe (MRS)-tributyrin agar. More than 90% of the isolates hydrolyzed the casein, but only 30% hydrolyzed tributyrin; the inhibition of L. monocytogenes in the spot-on-the-lawn assay was used to select 39 isolates that had a bigger inhibition zone (>11.15 mm ± 0.3) than the control (Nisin producer Lactococcus lactis BS-10 Chr Hansen). The selected isolates were grown in MRS to obtain the neutralized cell-free supernatants and verify their antimicrobial activity by agar diffusion and the percentage of growth inhibition techniques. The selected isolates were also growth in casein peptone broth, and the cell-free supernatants were used for the determination of antioxidant activity by the radical scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) techniques. The results were analyzed to identify similarities by cluster analysis, based on their antimicrobial and antioxidant capacities. The isolates were arranged into six clusters; one cluster that included 12 isolates demonstrated L. monocytogenes (784-2811 mm2/mL AU by agar diffusion assay) and E. coli (41%-47% growth inhibition) antimicrobial activity. The isolates clustered in these groups also showed competitive inhibition of both radicals (11%-19% of DPPH and 50%-60% of ABTS). The isolates from cluster one were also identified by 16S rDNA amplification and were identified as Enterococcus faecium. Traditional products such as Chihuahua cheese can be a source or lactic acid bacteria with metabolic properties that can be used in food preparation and preservation.

Project description:BackgroundLactic acid bacteria (LAB) are part of the gut microbiota and produce ribosomally synthesized antimicrobial peptides or bacteriocins with interest as natural food preservatives and therapeutic agents. Bacteriocin-producing LAB are also attractive as probiotics. Griffon vultures (Gyps fulvus subspecies fulvus) are scavenger birds that feed almost exclusively on carrion without suffering apparent ill effects. Therefore, griffon vultures might be considered a reservoir of bacteriocin-producing lactic acid bacteria (LAB) with potential biotechnological applications.ResultsGriffon vulture feces were screened for LAB with antimicrobial activity, genes encoding bacteriocins, potential virulence determinants, susceptibility to antibiotics, genotyping and characterization of bacteriocins. In this study, from 924 LAB evaluated 332 isolates (36 %) showed direct antimicrobial activity against Gram-positive bacteria only. The molecular identification of the most antagonistic 95 isolates showed that enterococci was the largest LAB group with antimicrobial activity (91 %) and E. faecium (40 %) the most identified antagonistic species. The evaluation of the presence of bacteriocin structural genes in 28 LAB isolates with the highest bacteriocinogenic activity in their supernatants determined that most enterococcal isolates (75 %) encoded multiple bacteriocins, being enterocin A (EntA) the largest identified (46 %) bacteriocin. Most enterococci (88 %) were resistant to multiple antibiotics. ERIC-PCR and MLST techniques permitted genotyping and recognition of the potential safety of the bacteriocinogenic enterococci. A multiple-step chromatographic procedure, determination of the N-terminal amino acid sequence of purified bacteriocins by Edman degradation and a MALDI TOF/TOF tandem MS procedure permitted characterization of bacteriocins present in supernatants of producer cells.ConclusionsEnterococci was the largest LAB group with bacteriocinogenic activity isolated from griffon vulture feces. Among the isolates, E. faecium M3K31 has been identified as producer of enterocin HF (EntHF), a bacteriocin with remarkable antimicrobial activity against most evaluated Listeria spp. and of elevated interest as a natural food preservative. E. faecium M3K31 would be also considered a safe probiotic strain for use in animal nutrition.

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Genomic and functional characterization of bacteriocinogenic lactic acid bacteria isolated from Boza, a traditional cereal-based beverage.

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