Safety and Beneficial Properties of Bacteriocinogenic Lactococcus lactis and Pediococcus pentosaceus Strains, and Their Effect Versus Oral Cavity Related and Antibiotic-Resistant Pathogens.

Pediococcus pentosaceus 732, Lactococcus lactis subsp. lactis 431, and Lactococcus lactis 808, bacteriocinogenic strains previously isolated from kimchi and banana, were investigated for their safety, beneficial properties and in vitro inhibition of pathogens such as Listeria monocytogenes ATCC 15313 and Staphylococcus simulans KACC 13241 and Staphylococcus auricularis KACC 13252. The results of performed physiological, biochemical, and biomolecular tests suggest that these strains can be deemed safe, as no virulence genes were detected in their DNA. Notably, only the gad gene associated with GABA production was identified in the DNA isolated of Lc. lactis 808 and Lc. lactis subsp. lactis 431 strains. All tested LAB strains exhibited γ-hemolysins and were non-producers of gelatinase and biogenic amines, which suggested their safety potential. Additionally, they were relatively susceptible to antibiotics except for streptomycin, tobramycin, and vancomycin for Pd. pentosaceus 732. The growth of Pd. pentosaceus 732, Lc. lactis subsp. lactis 431, and Lc. lactis 808 and their survival were minimally affected by up to 3% ox bile and low pH (except pH 2.0 and 4.0). Moreover, these LAB strains were not inhibited by various commercial extracts as well as most of the tested medications tested in the study. They did not produce proteolytic enzymes but exhibited production of D/L-lactic acid and ß-galactosidase. They were also hydrophilic. Furthermore, their survival in artificial saliva, gastric simulation, and enteric passage was measured followed by a challenge test to assess their ability to inhibit the selected oral pathogens in an oral saliva model conditions.

Identification of safe putative probiotics from various food products.

The objective of this study was to isolate, identify, and assess the safety and functionality in vitro of putative probiotic bacterial strains. Isolation procedures were based on standard methods using elective and selective media. The isolates were identified by comparative 16S rRNA sequencing analysis while their safety was determined according to the safety tests recommended by the FAO/WHO such as antibiotic resistance, hemolysin, and biogenic amine production. Most of the isolates did not pass the in vitro safety tests; therefore, only Lactiplantibacillus plantarum (from ant intestine and cheese), Lacticaseibacillus paracasei (from goat milk and kimchi), Enterococcus faecium (from chili doenjang and vegetables with kimchi ingredients), Limosilactobacillus fermentum (from saliva), and Companilactobacillus alimentarius (from kimchi) were identified and selected for further studies. The isolates were further differentiated by rep-PCR and identified to the strain level by genotypic (16S rRNA) and phenotypic (Gen III) approaches. Subsequently, the strain tolerance to acid and bile was evaluated resulting in good viability after simulated gastrointestinal tract passage. Adhesion to mucin in vitro and the presence of mub, mapA, and ef-tu genes confirmed the adhesive potential of the strains and the results of features associated with adhesion such as hydrophobicity and zeta potential extended the insights. This study reflects the importance of fermented and non-fermented food products as a promising source of lactic acid bacteria with potential probiotic properties. Additionally, it aims to highlight the challenges associated with the selection of safe strains, which often fail in the in vitro tests, thus hindering the possibilities of "uncovering" novel and safe probiotic strains.

Antimicrobial Peptides (Bacteriocins) Produced by Lactococcus lactis and Pediococcus pentosaceus Strains with Activity Against Clinical and Food-Borne Pathogens.

Bacteriocins are ribosomal-synthesized peptides with antimicrobial activity, produced by different groups of bacteria, including lactic acid bacteria (LAB). Most of the produced by LAB bacteriocins can be described with rather broad spectra of inhibition and they offer suggested applications in food preservation and pharmaceutical sector. Different LAB were isolated from fermented food products and fruits, obtained from the region of Pohang, Korea, and identified based on physiological, biochemical, and molecular methods. The promising isolates, Pediococcus pentosaceus 732, Lactococcus lactis 808, and Lactococcus lactis subsp. lactis 431, were identified based on biochemical, physiological, and biomolecular approaches, including 16S rRNA partial sequencing, and were evaluated for production of bacteriocin, including stability in presence of enzymes, chemicals, pH, and temperatures. Adherence properties for the expressed bacteriocins by P. pentosaceus 732, Lc. lactis 808, and Lc. lactis subsp. lactis 431 were evaluated at presence of selected chemicals, pH, and temperatures. The presence of bacteriocin genes in the strains was investigated and analyzed. The bacterial effect of bacteriocin produced by studied strains on Listeria spp. and Staphylococcus spp. has been shown for actively growing and stationary cells. Similar growth and bacteriocin production were observed when studied strains were cultured in MRS at 30 °C or 37 °C. The presence of nisin operon with some point mutations on the genomic DNA was recorded based on the performed PCR reactions targeting different genes associated with nisin expression for both lactococcal strains. Pediocin PA-1 operon was evaluated in a similar manner for P. pentosaceus 732.

Bacteriocin and Antioxidant Production, a Beneficial Properties of Lactic Acid Bacteria Isolated from Fermented Vegetables of Northwest Bulgaria.

Lactiplantibacillus plantarum ST01BG, ST07BG, ST10BG, and ST15BG; Latilactobacillus curvatus ST02BG; Lacticaseibacillus paracasei ST04BG; Pediococcus pentosaceus ST05BG; Leuconostoc mesenteroides ST06BG; and Enterococcus faecium ST11BG were isolated from home-made fermented vegetables from Northwest Bulgaria and identified by biochemical, physiological, and biomolecular analyses, including partial 16S rRNA sequencing. The strains were designated as bacteriocin producers and the expressed antimicrobials partially characterized with a focus on their proteinaceous nature, stability to different pH and temperatures. The bacteriocins were effective in inhibiting different strains of Listeria spp., Enterococcus spp. (including vancomycin resistant enterococci) and Staphylococcus spp. These strains can be considered safe, based on the evaluation of hemolytic activity, production of biogenic amines, mucin degradation, antibiotic susceptibility/resistance, and gelatinase enzyme production. Moreover, the strains can be considered potentially beneficial based on their stability and survival under simulated gastrointestinal tract conditions (stomach and duodenum), the production of diacetyl, and specific levels of hydrophobicity. Special attention was given to antioxidant properties (DPPH radical, hydroxyl radical, superoxide anion radical scavenging activity, Fe+2 ion chelating activity, and anti-lipid peroxidation) of the strains. Antioxidant properties were found to be strain specific. The beneficial attributes (antimicrobial and antioxidant) of these cultures to fermented food products may enable the reduction of chemical additives in line with consumers' demand for more natural and chemical-free food commodities.

Dual Role of Probiotic Lactic Acid Bacteria Cultures for Fermentation and Control Pathogenic Bacteria in Fruit-Enriched Fermented Milk.

The food industry has been developing new products with health benefits, extended shelf life, and without chemical preservation. Bacteriocin-producing lactic acid bacteria (LAB) strains have been evaluated for food fermentation to prevent contamination and increase shelf life. In this study, potentially probiotic LAB strains, Lactiplantibacillus (Lb.) plantarum ST8Sh, Lacticaseibacillus (Lb.) casei SJRP38, and commercial starter Streptococcus (St.) thermophilus ST080, were evaluated for their production of antimicrobial compounds, lactic acid and enzyme production, carbohydrate assimilation, and susceptibility to antibiotics. The characterization of antimicrobial compounds, the proteolytic activity, and its inhibitory property against Listeria (List.) monocytogenes and Staphylococcus (Staph.) spp. was evaluated in buriti and passion fruit-supplemented fermented milk formulations (FMF) produced with LAB strains. Lb. plantarum ST8Sh was found to inhibit List. monocytogenes through bacteriocin production and produced both L(+) and D(-) lactic acid isomers, while Lb. casei SJRP38 mainly produced L(+) lactic acid. The carbohydrate assimilation profiles were compatible with those usually found in LAB. The potentially probiotic strains were susceptible to streptomycin and tobramycin, while Lb. plantarum ST8Sh was also susceptible to ciprofloxacin. All FMF produced high amounts of L(+) lactic acid and the viability of total lactobacilli remained higher than 8.5 log CFU/mL during monitored storage period. Staph. aureus ATCC 43300 in fermented milk with passion fruit pulp (FMFP) and fermented milk with buriti pulp (FMB), and Staph. epidermidis KACC 13234 in all formulations were completely inhibited after 14 days of storage. The combination of Lb. plantarum ST8Sh and Lb. casei SJRP38 and fruit pulps can provide increased safety and shelf-life for fermented products, and natural food preservation meets the trends of the food market.

Bacillus Strains Isolated from Korean Fermented Food Products with Antimicrobial Activity Against Staphylococci, an Alternative for Control of Antibiotic-Resistant Pathogens.

Multidrug resistance in foodborne and clinical pathogens is a worldwide health problem. The urgent need for new alternatives to the existing antibiotics is emerging. Bacteriocin-like inhibitory substances can be considered part of the new generation of antimicrobials, which can be potentially applied in the food industry and health care practices. This study aimed to select Bacillus strains with antimicrobial activity against Staphylococcus spp. with future application in the formulation of pharmaceutical antimicrobial preparations. Putative antimicrobial agent-producing strains, previously isolated and preidentified as Bacillus spp. were profiled by repetitive element sequence-based polymerase chain reaction (rep-PCR) and 16s rRNA sequencing identified the strains as Bacillus tequilensis ST1962CD with 99.47% identity confidence and as Bacillus subtilis subsp. stercoris ST2056CD with 98.45% identity confidence. Both the selected Bacillus strains were evaluated via biomolecular and physiological approaches related to their safety and virulence, beneficial properties, enzyme production profile, and presence of corresponding genes for the production of antimicrobials and virulence. Both strains were confirmed to harbor srfa and sbo genes and be free of hemolysin binding component (B) and two lytic components (L1 and L2) [BL] and nonhemolytic enterotoxin-associated genes. Produced antimicrobial agents by strains ST1962CD and ST2056CD were partially purified through the combination of ammonium sulfate precipitation and hydrophobic-based chromatography on SepPakC18 and evaluated regarding their cytotoxicity. The dynamics of bacterial growth, pH change, accumulation of produced antimicrobials, and the mode of action were evaluated. Obtained results were pointing to the potential application of safe B. tequilensis ST1962CD and B. subtilis subsp. stercoris ST2056CD strains as functional beneficial microbial cultures that are putative producers of surfactin and/or subtilosin, as potent antimicrobials, for the treatment of some staphylococcal-associated infections. Expressed antimicrobials were shown to be not cytotoxic, and appropriate biotechnological approaches need to be developed for cost-effective production, isolation, and purification of expressed antimicrobials by studied strains.

Beneficial, safety, and antioxidant properties of lactic acid bacteria: A next step in their evaluation as potential probiotics.

The role of lactic acid bacteria (LAB) as probiotics as health promoting factors for human or veterinary practice has gained increasing interest during the last three decades. This is reflected in screening approaches of LAB strains in line with minimal requirements for a "probiotic" with regard to safety and functionality. The latter might also include natural antioxidant properties, thereby constituting an additional benefit in substituting synthetic antioxidants. The in vitro antioxidant assays conducted in this study included the scavenging of the 2,2-diphenyl-1-picrylhydrazil (DPPH) free radical, metal (Fe+2) ion chelation, determining the scavenging properties of the hydroxyl and superoxide radicals, and anti-lipid peroxidation. Analysis of DPPH free radical scavenging property for the microorganisms included in current study, showed Streptococcus salivarius ST59HK to exhibit the highest activity at a level of 85.24%. The greatest Fe+2 chelation activity with 98.2% was recorded for Str. salivarius ST62HK while the lowest was recorded for Str. salivarius ST48HK at 71.5%. The greatest and minimal hydroxyl radical scavenging levels were detected for Str. salivarius ST59HK (98.6%) and Lactiplantibacillus plantarum ST63HK (35.60%), respectively. Superoxide anion radical scavenging activity was highly exhibited by Str. salivarius ST61HK (54.62%) and the least exhibited by Enterococcus faecium ST651ea (18.7%). Lastly, the strains Lactobacillus gasseri ST16HK and E. faecium ST7319ea showed the highest and lowest anti-lipid peroxidation levels with 69.43% and 26.15%, respectively. Anti-oxidative properties appear to be strain specific and thus some of these strains could be potentially applied as natural antioxidants in fermented food products.

Special Issue: Beneficial Properties and Safety of Lactic Acid Bacteria.

The application of LAB in various sectors, including in the biotechnical and food industry, in human and veterinary practice, and in health-promoting practices and cosmetics, has been the subject of intensive research across the globe, with a range of traditional and innovative methods currently being explored [...].

Diversity of the bacteriocins, their classification and potential applications in combat of antibiotic resistant and clinically relevant pathogens.

There is almost a century since discovery of penicillin by Alexander Fleming, a century of enthusiasm, abuse, facing development of antibiotic-resistance and clear conclusion that the modern medicine needs a new type of antimicrobials. Bacteriocins produced by Gram-positive and Gram-negative bacteria, Archaea and Eukaryotes were widely explored as potential antimicrobials with several applications in food industry. In last two decades bacteriocins showed their potential as promising alternative therapeutic for the treatment of antibiotic-resistant pathogens. Bacteriocins can be characterised as highly selective antimicrobials and therapeutics with low cytotoxicity. Most probably in order to solve the problems associated with the increasing number of antibiotic-resistant bacteria, the application of natural or bioengineered bacteriocins in addition to synergistically acting preparations of bacteriocins and conventional antibiotics, can be the next step in combat versus drug-resistant pathogens. In this overview we focussed on diversity of specific lactic acid bacteria and their bacteriocins. Moreover, some additional examples of bacteriocins from non-lactic acid, Gram-positive and Gram-negative bacteria, Archaea and eukaryotic organisms are presented and discussed. Therapeutic properties of bacteriocins, their bioengineering and combined applications, together with conventional antibiotics, were evaluated with the scope of application in human and veterinary medicine for combating (multi-)drug-resistant pathogens.

Evaluation of Antifungal Metabolites Produced by Lactic Acid Bacteria.

This study aimed to select and characterize lactic acid bacteria (LAB) with potential antifungal activities against the filamentous fungi Alternaria alternata ATCC MYA-4642, Aspergillus flavus KACC 45470, Aspergillus niger KACC 42589, Cladosporium sphaerospermum ATCC MYA-4645, Penicillium chrysogenum ATCC MYA-4644, and Penicillium expansum KACC 40815. Initial screening of the antifungal activity has identified six LAB strains belonging to the genera Enterococcus and Leuconostoc, selected by their antagonistic activities against at least three of the filamentous fungi in the test panel. Preliminary prediction of bioactive compounds was carried out to narrow down the possible identity of the antagonistic metabolites produced by the studied LAB. Furthermore, metabolic profiles were assessed and used as a basis for the identification of key metabolites based on VIP scores and PCA plot scores. Key metabolites were identified to be ß-phenyllactic acid, ⍺-hydroxyisobutyric acid, 1,3-butanediol, phenethylamine, and benzoic acid. Individual assessment of each metabolic compound against the test panel showed specificity inhibitory patterns; yet, combinations between them only showed additive, but not synergetic effects. The pH neutralization significantly reduced the antifungal activity of the cell-free supernatant (CFS), but no bioactive compounds were found to be stable in high temperatures and pressure. This study will be beneficial as an additional building block on the existing knowledge and future antifungal application of LAB produced metabolites. Furthermore, this study also provides a new bio-preservative perspective on unexplored antifungal metabolites produced by LAB as biocontrol agents.

The Evaluation of Different Bacteriocinogenic Bacillus spp. with Activity Against Staphylococcus spp. and Their Beneficial and/or Hazardous Properties.

The aim of this project was to screen for bacteriocinogenic Bacillus strains with activity versus Staphylococcus spp. with future application in formulation of pharmaceutical antimicrobial preparations. Putative bacteriocinogenic strains, isolated and pre-identified as Bacillus spp. were selected for future study and differentiated based on repPCR and identified as Bacillus subtilis for strains ST826CD and ST829CD, Bacillus subtilis subsp. stercoris for strain ST794CD, Bacillus subtilis subsp. spizizenii for strain ST824CD, Bacillus velezensis for strain ST796CD, and Bacillus tequilensis for strain ST790CD. Selected strains were evaluated regarding their safety/virulence, beneficial properties, and potential production of antimicrobials based on biomolecular and physiological approves. Expressed bacteriocins were characterized regarding their proteinaceous nature, stability at different levels of pH, temperatures, and the presence of common chemicals applied in bacterial cultivation and bacteriocin purification. Dynamic of bacterial growth, acidification, and cumulation of produced bacteriocins and some aspects of the bacteriocins mode of action were evaluated. Based on obtained results, isolation and application of expressed antimicrobials can be realistic scenario for treatment of some staphylococcal associated infections. Appropriate biotechnological approaches need to be developed for cost effective production, isolation, and purification of expressed antimicrobials by studied Bacillus strains.

Combined Action of Antibiotics and Bacteriocins against Vancomycin-Resistant Enterococci.

Antibiotics have been one of the most important discoveries in the area of applied medical microbiology; however, as a result of various factors, we are currently facing a dramatic and relatively dangerous increase in the number of cases of antibiotic resistance, and the need for new types of antimicrobials continues to grow. New approaches are needed to combat antibiotic-resistant pathogens. Bacteriocins, as part of the group of antimicrobial peptides, can be considered as alternatives and/or complements to known antibiotics. Their narrow spectra of activity can be explored for the control of various pathogens, such as vancomycin-resistant enterococci (VRE), as single therapies or in combination with known antibiotics. In the present study, we isolated bacteriocins from different lactic acid bacteria (LAB) strains, including Enterococcus and Pediococcus, and explored the possible synergistic inhibition of growth by bacteriocins and vancomycin. It was observed in the growth dynamics with previously selected VRE strains that the bacteriocins had a high specificity and a promising inhibitory effect against the VRE strains, and these results were validated by a propidium iodide viability test using flow cytometry. The data obtained indicate that the selected bacteriocins can be used to control VRE in the food industry or even as an alternative treatment to combat infections with antibiotic-resistant bacteria.

Selection of Beneficial Bacterial Strains With Potential as Oral Probiotic Candidates.

This study aimed to select beneficial strains from the oral cavity of healthy volunteers and to evaluate these as potential oral probiotic candidates. The selection process was based on the isolation, differentiation, identification, and safety assessment of LAB strains, followed by a series of experiments for the selection of appropriate candidates with beneficial properties. In the screening procedure, 8 isolates from the oral cavity of a Caucasian volunteers were identified as Streptococcus (Str.) salivarius ST48HK, ST59HK, ST61HK, and ST62HK; Lactiplantibacillus plantarum (Lb.) (Lactobacillus plantarum) ST63HK and ST66HK; Latilactobacillus sakei (Lb.) (Lactobacillus sakei) ST69HK; and Lactobacillus (Lb.) gasseri ST16HK based on 16S rRNA sequencing. Physiological and phenotypic tests did not show hemolytic, proteinase, or gelatinase activities, as well as production of biogenic amines. In addition, screening for the presence of efaA, cyt, IS16, esp, asa1, and hyl virulence genes and vancomycin-resistant genes confirmed safety of the studied strains. Moreover, cell-to-cell antagonism indicated that the strains were able to inhibit the growth of tested representatives from the genera Bacillus, Enterococcus, Streptococcus, and Staphylococcus in a strain-specific manner. Various beneficial genes were detected including gad gene, which codes for GABA production. Furthermore, cell surface hydrophobicity levels ranging between 1.58% and 85% were determined. The studied strains have also demonstrated high survivability in a broad range of pH (4.0-8.0). The interaction of the 8 putative probiotic candidates with drugs from different groups and oral hygiene products were evaluated for their MICs. This is to determine if the application of these drugs and hygiene products can negatively affect the oral probiotic candidates. Overall, antagonistic properties, safety assessment, and high rates of survival in the presence of these commonly used drugs and oral hygiene products indicate Str. salivarius ST48HK, ST59HK, ST61HK, and ST62HK; Lb. plantarum ST63HK and ST66HK; Lb. sakei ST69HK; and Lb. gasseri ST16HK as promising oral cavity probiotic candidates.

Antimicrobial properties of Pediococcus acidilactici and Pediococcus pentosaceus isolated from silage.

AIMS: The objective of this study was to isolate multifunctional bacteriocin-producing strains; to characterize the expressed bacteriocin for the control of Listeria monocytogenes and vancomycin-resistant Enterococcus; to evaluate the safety of studied strains; and to explore their antifungal activity. METHODS AND RESULTS: Two Pediococcus strains were isolated from silage samples obtained from an organic farm in Belogradchik, Bulgaria. The strains were identified by 16S rRNA sequencing analysis and characterized as bacteriocins producers. Strong antimicrobial activity was detected against more than 74 different strains of Listeria monocytogenes, 27 different vancomycin-resistant Enterococcus strains. In addition, studied strains were able to inhibit the growth of strains of Alternaria alternate, Aspergillus flavus, Aspergillus niger, Cladosporium sphaerospermum, Penicillium chrysogenum and Penicillium expansum. Some aspects of the antimicrobial mode of action were evaluated, including killing curves and aggregation properties. Both strains generated positive PCR results for the presence of pediocin PA-1, but not for other bacteriocins evaluated in this screening process. Metabolomic analysis of the cell-free supernatants from both strains was performed in order to explain the observed antifungal activity against different moulds. According to PCA and PLS-DA score plot, P. acidilactici ST3522BG and P. pentosaceus ST3633BG were clearly clustered from control (MRS). Increases in the production of benzoic acid, 2-hydroxyisocaproic acid, ß-phenyl-lactic acid, α-hydroxybutyric acid and 1,3-butanediol were recorded, these metabolites were previously described as antifungal. CONCLUSIONS: Pediococcus acidilactici ST3522BG and P. pentosaceus ST3633BG were evaluated as producing bacteriocin strains with high specificity against Listeria and vancomycin-resistant Enterococcus species. In addition, both investigated Pediococcus strains were evaluated as producer of effective antifungal metabolites with potential for the inhibition of mycotoxin-producing moulds. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this report is a pioneer in the evaluation of Pediococcus strains isolated from silage with highly specific bacteriocinogenic antimicrobial activity against Listeria spp. and vancomycin-resistant Enterococcus spp., and antifungal activity against mycotoxin-producing moulds.

Characterization and safety evaluation of two beneficial, enterocin-producing Enterococcus faecium strains isolated from kimchi, a Korean fermented cabbage.

Enterococcus faecium ST20Kc and ST41Kc were isolated from kimchi, a traditional Korean fermented cabbage. Bacteriocins produced by both strains exhibited strong activity against Listeria monocytogenes and various Enterococcus spp., including 30 vancomycin-resistant enterococcal strains, but not against other lactic acid bacteria (LAB) on the evaluated test panel. The antimicrobials produced by the strains were found to be proteinaceous and stable even after exposure to varying pH, temperature, and chemicals used in the industry and laboratory processes. Antimicrobial activity of both strains was evaluated as bactericidal against exponentially growing cultures of L. monocytogenes ATCC® 15313™ and Enterococcus faecalis 200A. Based on tricine-SDS-PAGE, the molecular weights of the bacteriocins produced by the strains were between 4 and 6 kDa. Additionally, both strains were susceptible to antibiotics, including vancomycin, kanamycin, gentamycin, ampicillin, streptomycin, tylosin, chloramphenicol, clindamycin, and tetracycline. Adhesion genes, map, mub, and EF-Tu, were also detected in the genomes of both strains. With gastrointestinal stress induction, both strains showed high individual survival rates, and capability to reduce viable counts of L. monocytogenes ATCC® 15313™ and Enterococcus faecalis 200A in mixed cultures. Based on the metabolomics analysis, both strains were found to produce additional antimicrobial compounds, particularly, lactic acid, phenyllactic acid, and phenethylamine, which can be potentially involved in the antimicrobial interaction with pathogenic microorganisms.

Probiotic potential and safety assessment of bacteriocinogenic Enterococcus faecium strains with antibacterial activity against Listeria and vancomycin-resistant enterococci.

Enterococcus spp., known for their wide ecological distribution, have been associated with various fermented food products of plant and animal origin. The strains used in this study, bacteriocinogenic Enterococcus faecium previously isolated from artisanal soybean paste, have shown strong activity against Listeria spp. and vancomycin-resistant enterococci. Although their antimicrobial activity is considered beneficial, the potential application of enterococci is still under debate due to concerns about their safety for human and other animal consumption. Therefore, this study not only focuses on the screening of potential virulence factors, but also the auxiliary beneficial properties of the strains Ent. faecium ST651ea, ST7119ea, and ST7319ea. Phenotypic screening for gelatinase, hemolysin, and biogenic amine production showed that the strains were all safe. Furthermore, the antibiogram profiling showed that all the strains were susceptible to the panel of antibiotics used in the assessment except for erythromycin. Yet, Ent. faecium ST7319ea was found to carry some of the virulence genes used in the molecular screening for safety including hyl, esp, and IS16. The probiotic potential and other beneficial properties of the strains were also studied, demonstrating high aggregation and co-aggregation levels compared to previously characterized strains, in addition to high survivability under simulated gastrointestinal conditions, and production of numerous desirable enzymes as evaluated by APIZym, indicating diverse possible biotechnological applications of these strains. Additionally, the strains were found to carry genes coding for γ-aminobutyric acid (GABA) production, an auxiliary characteristic for their probiotic potential. Although these tests showed relatively favorable characteristics, it should be considered that these assays were carried out in vitro and should therefore also be assessed under in vivo conditions.

Rehydration before Application Improves Functional Properties of Lyophilized Lactiplantibacillus plantarum HAC03.

Preservation of probiotics by lyophilization is considered a method of choice for developing stable products. However, both direct consumption and reconstitution of dehydrated probiotic preparations before application "compromise" the survival and functional characteristics of the microorganisms under the stress of the upper gastro-intestinal tract. We evaluated the impact of different food additives on the viability, mucin adhesion, and zeta potential of a freeze-dried putative probiotic, Lactiplantibacillus (Lp.) plantarum HAC03. HAC03-compatible ingredients for the formulation of ten rehydration mixtures could be selected. Elevated efficacy was achieved by the B-active formulation, a mixture of non-protein nitrogen compounds, sugars, and salts. The survival of Lp. plantarum HAC03 increased by 36.36% compared rehydration with distilled water (4.92%) after passing simulated gastro-intestinal stress conditions. Cell viability determined by plate counting was confirmed by flow cytometry. B-active formulation also influenced Lp. plantarum HAC03 functionality by increasing its adherence to a Caco-2 cell-line and by changing the bacterial surface charge, measured as zeta potential.Hydrophobicity, mucin adhesion and immunomodulatory properties of Lp. plantarum HAC03 were not affected by the B-active formulation. The rehydration medium also effectively protected Lp. plantarum ATCC14917, Lp. plantarum 299v, Latilactobacillus sakei (Lt.) HAC11, Lacticaseibacillus (Lc.) paracasei 532, Enterococcus faecium 200, and Lc. rhamnosus BFE5263.

Characterization of Partially Purified Bacteriocins Produced by Enterococcus faecium Strains Isolated from Soybean Paste Active Against Listeria spp. and Vancomycin-Resistant Enterococci.

Three out of one hundred eighty putative LAB isolates from Korean traditional fermented soybean paste were identified to be unique and bacteriocinogenic strains. Based on phenotypic and 16S rRNA sequencing analysis, selected strains were identified as Enterococcus faecium ST651ea, E. faecium ST7119ea and E. faecium ST7319ea. The bacteriocinogenic properties of the studied strains were evaluated against Listeria monocytogenes ATCC15313, Listeria innocua ATCC33090 and vancomycin-resistant E. faecium VRE19 of clinical origin. The strains E. faecium ST651ea, ST7119ea and ST7319ea expressed bacteriocins with an activity of 12,800 AU/mL, 25,600 AU/mL and 25,600 AU/mL, respectively, recorded against L. monocytogenes ATCC15131. According to the PCR-based screening of bacteriocin-related genes, which was further confirmed through amplicon sequencing, showed that strain E. faecium ST651ea carries entB and entP genes, whereas both E. faecium ST7119ea and ST7319ea strains harbor entA and entB genes. The molecular size of expressed bacteriocins was estimated by tricine-SDS-PAGE showing an approximative protein size of 4.5 kDa. The assessment of the spectrum of activity of bacteriocins ST651ea, ST7119ea and ST7319ea showed strong activity against most of clinical VRE isolates, majority of other Enterococcus spp. and Listeria spp. Bacteriocins ST651ea, ST7119ea and ST7319ea were partially purified by combination of 60% ammonium sulfate precipitation and hydrophobic chromatography on the SepPakC18 column. Challenge test with semi-purified (60% 2-propanol fraction) bacteriocins resulted in a significant reduction of viable cells for all test organisms. Thus, indicating that all the bacteriocins evaluated can be used as potential biocontrol in food and feed industries as well as an alternative treatment for VRE-related infections in both veterinary and clinical settings.

Bacteriocinogenic Potential of Bacillus amyloliquefaciens Isolated from Kimchi, a Traditional Korean Fermented Cabbage.

Bacteriocin production is considered a favorable property for various beneficial cultures. In addition to their potential as biopreservatives, bacteriocins are also promising alternatives for the control of multidrug-resistant pathogens and the inhibition of some viruses and cancer cells. The objective of this study was to screen and characterize a bacteriocin-producing strain with the aim of its future application for control of Listeria monocytogenes, an important food-borne pathogen. A total of 22 potentially bacteriocinogenic strains active against L. monocytogenes ATCC15313 were isolated from locally produced kimchi through a three-level approach. Pure cultures were obtained according to good microbiological practices and differentiated through RAPD-PCR using the primers OPL01, OPL09, and OPL11. Altogether, 5 strains were selected for further study. Specific focus was given to strain ST05DL based on its specific inhibitory activity against L. monocytogenes ATCC15313, while not affecting different strains belonging to the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella, most of which are beneficial microorganisms. The strain ST05DL was identified as Bacillus amyloliquefaciens based on its sugar fermentation profile obtained through API50CHB analysis and 16S rRNA partial sequencing. The antimicrobial compound produced by B. amyloliquefaciens ST05DL was found to be sensitive to pepsin and α-chymotrypsin, evidence of its proteinaceous nature. The presence of skim milk, NaCl, Tween 80, glycerol, and SDS did not affect the antimicrobial activity. The addition of 20% cell-free supernatant (CFS) obtained from a 24-h culture of B. amyloliquefaciens ST05DL to an exponentially growing culture of L. monocytogenes ATCC15313 successfully inhibited the test microorganisms during the monitored 10-h incubation. Optimal bacteriocin production by B. amyloliquefaciens ST05DL was observed during the stationary phase at 12 h (800 AU/mL) and remained stable for the next 15 h. The ratio between live and dead cells during this period was 74.37% and 25.66%, respectively, as determined by flow cytometry. The presence of the virulence genes hblA, hblB, hblC, nheA, nheB, and nheC was not detected in the total DNA of B. amyloliquefaciens ST05DL, and the strain was resistant only to ampicillin out of 10 tested antibiotics. Future evaluation of expressed bacteriocin/s by B. amyloliquefaciens ST05DL (amino acid sequence, molecular mass, cytotoxicity, detailed mode of action, etc.), will be the next step in the characterization and its potential application as biopreservative and/or pharmaceutical product.

Modulation of the Gut Microbiome and Obesity Biomarkers by Lactobacillus Plantarum KC28 in a Diet-Induced Obesity Murine Model.

Lactobacillus plantarum KC28 showed a beneficial (anti-obesity) effect in a diet-induced obese (DIO) C57BL/6 murine model receiving an intermediate high-fat diet (IF). This diet was selected for probiotic studies by prior comparisons of different combinations of basic (carbohydrate, protein and fat) components for optimized induction of dietary obesity in a murine model. Prior selection of Lact. plantarum strain KC28 was based on different physiological tests for safety and functionality including cell line adhesion and anti-adipogenic activity. The strain was administered at 5.0 × 109 CFU/mouse/day to the DIO mice (control mice received a normal diet). The anti-obesity effect of KC28 and the well-known probiotic strains Lact. rhamnosus GG (LGG) and Lact. plantarum 299v was assessed over 12 weeks. Xenical served as anti-obesity control. The high-fat diet groups receiving strains KC28 and LGG and the control Xenical group showed significant weight loss and notable changes in some obesity-related biomarkers in the liver (significant up-regulation of PGC1-α and CPT1-α only by KC28; p < 0.05) and mesenteric adipose tissue (significant down-regulation of ACOX-1, PPAR-γ, and FAS; KC28 p < 0.001 for PPAR-γ and FAS), compared with the IF control. Favourable changes in the studied biomarkers suggest a similar beneficial influence of Lact. plantarum KC28 on the alleviation of obesity comparable with that of the two well-studied probiotic strains, LGG and 299v. This probably resulted from a modulation in the cecal microbiota of the IF group by either probiotic strain, yet in a different manner, showing a highly significant increase in the families Desulfovibrionaceae and Lactobacillaceae only in the group receiving Lact. plantarum KC28.