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.

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.

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.

Probiotics for Aquaculture: Hope, Truth, and Reality.

The use of microorganisms as beneficial crops for human and animal health has been studied for decades, and these microorganisms have been in practical use for quite some time. Nowadays, in addition to well-known examples of beneficial properties of lactic acid bacteria, bifidobacteria, selected Bacillus spp., and yeasts, there are several other bacteria considered next-generation probiotics that have been proposed to improve host health. Aquaculture is a rapidly growing area that provides sustainable proteins for consumption by humans and other animals. Thus, there is a need to develop new technologies for the production practices associated with cleaner and environment-friendly approaches. It is a well-known fact that proper selection of the optimal probiotics for use in aquaculture is an essential step to ensure effectiveness and safety. In this critical review, we discuss the evaluation of host-specific probiotics in aquaculture, challenges in using probiotics in aquaculture, methods to improve the survival of probiotics under different environmental conditions, technological approach to improving storage, and delivery along with possible negative consequences of using probiotics in aquaculture. A critical analysis of the identified challenges for the use of beneficial microbes in aquaculture will help in sustainable aquafarming, leading to improved agricultural practices with a clear aim to increase protein production.

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.

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.

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.

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.

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.

Exploring Beneficial Properties of the Bacteriocinogenic Enterococcus faecium ST10Bz Strain Isolated from Boza, a Bulgarian Cereal-Based Beverage.

The bacteriocin-producing strain Enterococcus faecium ST10Bz, isolated from boza, a Bulgarian cereal-based beverage, exhibited strong activity against Listeria strains, vancomycin-resistant and other Enterococcus strains, but not against most of the other lactic acid bacteria (LAB) strains included in the test panel. Bacteriocin ST10Bz was proven as a stable antimicrobial, even after exposure to various environmental conditions, including varying pH values, temperatures, and commonly used chemicals in industry and laboratory practice. Bacteriocin activity against L. monocytogenes ATCC®15313™ was recorded at 25,600 AU/mL when the producer strain was cultured in MRS broth at 25 °C and 30 °C, and 19,200 AU/mL, when cultured at 37 °C. Additionally, bacteriocin ST10Bz exhibited bactericidal mode of action when added to actively growing cultures of L. monocytogenes ATCC®15313™ and Enterococcus faecalis 200A. E. faecium ST10Bz was susceptible to the antibiotics kanamycin, gentamycin, ampicillin, streptomycin, tylosin, chloramphenicol, clindamycin, tetracycline, and vancomycin; with no evidence for vanA, B, C, D, E, or G genes. PCR analysis of DNA from strain ST10Bz generated positive results for presence of some bacterial adhesion genes, including map, mub and ef-tu, as well as the gamma aminobutyric acid (GABA) production-related gene, gad. Under simulated gastrointestinal conditions in single and co-culture with L. monocytogenes ATCC®15313™ and E. faecalis 200A, E. faecium ST10Bz showed a high survival rate and the ability to reduce the viable numbers of the two test strains.