Assessment of the Spoilage Microbiota during Refrigerated (4 °C) Vacuum-Packed Storage of Fresh Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P-Containing Extract

Although fresh whey cheeses are prone to rapid deterioration, mainly by psychrotrophic Gram-negative bacteria and lactic acid bacteria (LAB), data on the specific spoilage species in traditional Greek whey cheeses are scarce. Therefore, this study quantified growth and characterized the primary spoilage bacteria in fresh Anthotyros whey cheeses stored at 4 °C in a vacuum for 40 days, without or with an added 5% (v/w) of an enterocin A-B-P crude extract (CEntE). Psychrotrophic Pseudomonas spp., Aeromonas spp., Hafnia spp. and Serratia spp. grew faster than LAB during early storage. However, LAB outgrew the Gram-negative bacteria and prevailed by mid to late storage in all cheese batches, causing a strong or milder batch-dependent natural acidification. Two major non-slime-producing and two minor biotypes of Leuconostoc-like bacteria, all identified as Leuconostoc mesenteroides by 16S rRNA sequencing, dominated the LAB association (76.7%), which also included four subdominant Carnobacterium maltaromaticum biotypes (10.9%), one Leuconostoc lactis biotype (3.3%) and few Lactococcus (1.6%), mesophilic Lactobacillus (0.8%) and Enterococcus (0.8%). Growth and distribution of LAB and Gram-negative species were strongly batch-dependent and plant-dependent. The CEntE neither retarded growth nor altered the whey cheese spoilage association but enhanced LAB growth and the declines of Gram-negative bacteria by late storage.

Evaluation of Apoptotic Activity of Protein Fusion Enterocin A- Pyocin R-Lactocin, Bacteriocins and Specific Ligand of Gastric Cancer Cell Line (AGS)

Conventional antimicrobial and anti-cancer treatments, including chemotherapy, surgery, radiation therapy, etc., have all been associated with antimicrobial resistance (AMR) and side effects on healthy cell damage. Recently, bacteriocins have emerged as a promising option in antimicrobial and anticancer therapies.In this study, EntA-PynR-Lac gene sequence was obtained using bioinformatics software. The designed fusion gene was synthesized and cloned into the pET22b expression vector and transferred to the engineered E. coli BL21 bacterium for expression of the recombinant protein. The three-dimensional structures and stability of the designed recombinant protein were evaluated. Confirmation and purification of the recombinant protein was performed by Western blotting and nickel column chromatography and determination of cell lethality of different concentrations of the recombinant protein against AGS cell line by MTT technique was conducted. Treatment of cells with different concentrations of protein to evaluate the apoptosis of AGS cells was performed by flow cytometry. The results showed that the percentage of cells treated with recombinant protein at a concentration of 80µg/ml with total apoptosis was 46.91% and increased by 36.25% compared to untreated cells. Due to the anti-apoptotic properties of fusion protein, it can be used to inhibit cancer cells as a therapeutic supplement and prevention.

Manganese privation induced transcriptional upregulation of the class IIa bacteriocin plantaricin 423 in Lactobacillus plantarum 423

Plantaricin 423 is produced by Lactobacillus plantarum 423 using the pla biosynthetic operon located on the 8188 bp plasmid, pPLA4. As with many class IIa bacteriocin operons, the pla operon encodes biosynthetic genes ( plaA : precursor peptide, plaB : immunity, plaC : accessory and plaD : ABC transporter) but does not encode local regulatory genes. Little is known about the regulatory mechanisms involved in the expression of the apparently regulationless class IIa bacteriocins such as plantaricin 423. In this study, phylogenetic analysis of class IIa immunity proteins indicated that at least three distinct clades exist, which were then used to subgroup the class IIa operons. It became evident that the absence of classical quorum sensing genes on mobile bacteriocin encoding elements is a predisposition of the subgroup which includes plantaricin 423, pediocin AcH/PA-1, divercin V41, enterocin A, leucocin-A and -B, mesentericin Y105 and sakacin G. Further analysis of the subgroup suggested that the regulation of these class IIa operons may be linked to transition metal homeostasis in the host. By using a fluorescent promoter-reporter system in Lactobacillus plantarum 423, transcriptional regulation of plantaricin 423 was shown to be upregulated in response to manganese privation. IMPORTANCE Lactic acid bacteria hold huge industrial application and economic value, especially bacteriocinogenic strains which further aids in the exclusion of specific foodborne pathogens. Since bacteriocinogenic strains are sought after it is equally important to understand the mechanism of bacteriocin regulation. This is currently an understudied aspect of class IIa operons. Our research suggests the existence of a previously undescribed mode of class IIa bacteriocin regulation, whereby bacteriocin expression is linked to management of the producer’s transition metal homeostasis. This delocalized metalloregulatory model may fundamentally affect the selection of culture conditions for bacteriocin expression and change our understanding of class IIa bacteriocin gene transfer dynamics in a given microbiome.

Susceptibility to Enterocins and Lantibiotic Bacteriocins of Biofilm-Forming Enterococci Isolated from Slovak Fermented Meat Products Available on the Market

This study investigated eight types of Slovak dry fermented meat products (salami and sausages) that are available on the market and were produced by three different producers in different regions of Slovakia. The total counts of enterococci in these products ranged from 2.0 up to 6.0 cfu/g (log10). Three species were identified among the 15 selected enterococcal strains; Enterococcus faecium (8 strains), Enterococcus faecalis (3) and Enterococcus hirae (4). They were hemolysis-negative (γ-hemolysis) with a biofilm-forming ability, which was evaluated as low-grade biofilm formation, susceptible to conventional antibiotics and mainly susceptible to lantibiotic bacteriocins, namely, gallidermin and nisin; they even showed a higher susceptibility to gallidermin than to nisin. They were also susceptible to enterocin–durancin, but most strains showed resistance to enterocin A/P. This study indicated that bacteriocins can play a key role in preventing and/or protecting from undesirable bacterial multiplication or contamination in the food industry and that they have great potential for further experimental applications.

Application of Enterococcus faecium KE82, an enterocin-A-B-P-producing strain, as an adjunct culture enhances inactivation of Listeria monocytogenes during traditional PDO Galotyri cheese processing

The ability of the enterocin-A-B-P-producing Enterococcus faecium KE82 adjunct strain to inactivate Listeria monocytogenes during Galotyri PDO cheese processing was evaluated. Three artisan cheese trials from traditionally ‘boiled’ (85oC) ewe’s milk were processed. The milk cooled at 42oC was divided in two parts: A1 was inoculated with Streptococcus thermophilus ST1 and Lactococcus lactis subsp. cremoris M78, and A2 with the basic starter ST1+M78 plus the KE82 adjunct (step 1). All milks were fermented at 20-22oC for 24 h (step 2); the curds were drained at 12oC for 72 h (step 3) and then salted with 1.5-1.8% salt to obtain the fresh Galotyri cheeses (step 4), which were ripened at 4oC for 30 days (step 5). Because an artificial listerial contamination in the dairy plant was prohibited, A1 and A2 cheese milk (200-mL) or curd (200-g) portions were taken after steps 1 to 5, inoculated (3-4 log CFU/mL or g) with L. monocytogenes no.10, incubated at 37, 22, 12, and 4oC for predefined periods, and analyzed microbiologically and for pH. L. monocytogenes declined without growth in all cheese curd portions contaminated after steps 2 to 5 (pH 4.36 to 4.84), when stored at 4 or 12oC for 15 days. The final net reductions of Listeria populations were by 2.00, 1.07, 0.54 and 0.61 log units higher in the A2 than A1 curd portions after steps 2, 3, 4 and 5, respectively. As regards step 1 conducted in simulation of the whole cheese milk fermentation process, L. monocytogenes declined by 1.47 log units more in the A2 than A1 milk portions after 72 h at 22oC; however, a slight (0.6-log) growth was preceded during the first 6 h at 37oC. In conclusion, E. faecium KE82 showed growth compatibility with the starter and enhanced inactivation of L. monocytogenes across Galotyri cheese processing. Combined acid-enterocin antilisterial effects were the weakest in the fermenting milks, turned to the strongest in the unsalted fermented curds, and reduced in the salted fresh cheeses.

Molecular screening of the entA gene of Enterococcus faecium isolated from Food and clinical sources

Background: The microbial production of substances that have the potency to suppress the growth of other microorganisms is probably one of the prevalent defense strategy developed in nature, microorganisms produce a variable bunch of microbial defense systems, which include antibiotics, metabolic by-products, lytic agents, bacteriocins and others. Objective: The purpose of the present study was to isolate and identify Enterococcus faecium isolates then detecting its ability of carrying the gene responsible for enterocin production in this species. Materials and methods: Out of 50 samples from different sources (food and clinical sources) were collected for the Enterococcus faecium isolation, and the isolated bacteria Enterococcus faecium (37) isolates were detected for their harboring of Enterocin A gene (entA), using conventional PCR technique. Results: The identification revealed that 37(74%) isolates were considered as Enterococcus faecium, 20 isolates (54.05%) out of food samples (10 samples were collected from dairies, 7 from vegetables and 3 from fish samples), and 17 isolates 45.9% out of clinical samples (11 from stool and 6 from urine source). Genotypic Detection done by the amplification of the enterocin coding gene (ent A), and the results revealed that all the isolates were harboring that gene despite of the phonotypical differences, that they amplified entA gene and the PCR product size (362 bp) was detected using agarose gel electrophoresis. Conclusions: This study indicates the presence of Enterococcus spp. in food and clinical sources and the ability of these bacteria to produce antibacterial substances which is active against closely related clinical isolates.

Enterococci Isolated from Trout in the Bukovec Water Reservoir and Čierny Váh River in Slovakia and Their Safety Aspect

The aim of this study was to investigate enterococci as lactic acid bacteria and as part of Firmicutes phylum. We focused on the virulence factor, biofilm formation, and antibiotic resistance and also on lactic acid production and enterocin gene detection. Intestinal samples were taken from 50 healthy trout (3 Salmo trutta and 47 Salmo gairdneri) collected in April 2007, 2010, and 2015 from different locations at the Bukovec water reservoir and the Čierny Váh River in Slovakia. Twenty pure colonies were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification system based on protein fingerprints, and then seven identified strains were also phenotyped. Based on the identification methods used, the identified enterococci (7) belong taxonomically to four different enterococcal species: Enterococcus durans, E. faecium, E. mundtii, and E. thailandicus. They were hemolysis, DNase, and gelatinase negative with acceptable enzymatic activity. They did not form biofilm and were mostly susceptible to antibiotics. All strains produced lactic acid amounting to 1.78 ± 0.33 mmol/l on average and possessed the gene for enterocin A production. This is the first study reporting more detailed properties of enterococci from trout in Slovakian wild water sources, and it produces new possibilities for studying microbiota in trout.