Bacteriocin-like Inhibitory Substance Research Articles

Lactic Acid Bacteria and Bacillus subtilis as Potential Protective Cultures for Biopreservation in the Food Industry

The use of bacteria and/or their compounds is an alternative to the use of positive-list additives that the food industry is using as a tool to meet consumer demands for more natural, long-shelf-life, and healthy products, in short, to offer clean label foods. The aim of this study is to investigate the suitability of cell-free supernatants (CFSs) from Qualified Presumption of Safety strains as bioprotective cultures. Out of an initial screening panel of about 200 isolates, strains Pediococcus acidilactici CNTA 1059, Lactiplantibacillus plantarum CNTA 600, Levilactobacillus brevis CNTA 1374, and Bacillus subtilis CNTA 517 demonstrated strong antimicrobial activity against, especially, Gram-positive bacteria. The CFSs of these four strains showed minimum inhibitory concentration values between 0.15% and 5% against Listeria monocytogenes and Lentilactobacillus parabuchneri. None of the four selected strains exhibited acquired resistance to target antibiotics, and the non-toxigenicity of all the CFSs was demonstrated. In the case of the three lactic acid bacteria, the presence of bacteriocin-like inhibitory substances was confirmed following the decline in antimicrobial activity due to treatment with proteases. Regarding B. subtilis, biosynthetic gene clusters for different bacteriocin-like substances, including protease-resistant lipoproteins, were found via whole-genome sequencing. In addition, all of the CFSs exhibited stable antimicrobial activity at a wide range of temperatures (70–121 °C) used for the pasteurization and sterilization of food products, with a loss of antimicrobial activity ranging from 3% to 28%. These results point to the possibility that CFSs from these strains could be used in the food industry as a biocontrol tool to develop new products.

Selection of starter lactic acid bacteria capable of forming biofilms on wooden vat prototypes for their future application in traditional Sicilian goat's milk cheese making

In this study, 327 presumptive lactic acid bacteria (LAB) were isolated from goats' milk acid curds produced at a Sicilian dairy farm with the aim to identify potential starter cultures for traditional cheeses. All isolates were first processed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. This approach identified 63 distinct strains which were evaluated for their acidifying capacity. Only 15 strains specifically stood out for their acidification capacity and were identified through 16S rRNA gene sequencing as Lactococcus lactis (11 strains) Enterococcus faecalis (three strains), and Ligilactobacillus animalis (one strain). Notably, all 15 LAB isolates produced bacteriocin-like inhibitory substances and anti-biofilm compounds, against both planktonic and biofilm forms of Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, and Staphylococcus aureus, albeit at varying levels. Among these 15 LAB, En. faecalis RGM25 and Lc. lactis RGM55, susceptible to five antibiotics tested, were put in contact with wooden vat prototypes, because all equipment used in traditional cheese production in Sicily are made of wood. Scanning electron microscopy and bacterial plate counts of the wooden vat prototypes showed the development of biofilms at levels of approximately 6.0 log CFU/cm2. Overall, this study contributes to establishing a custom-made LAB starter cultures with bio-preservatives properties for Sicilian cheese productions.

Characterization of the broad-spectrum antibacterial activity of bacteriocin-like inhibitory substance-producing probiotics isolated from fermented foods

Antimicrobial peptides, such as bacteriocin, produced by probiotics have become a promising novel class of therapeutic agents for treating infectious diseases. Selected lactic acid bacteria (LAB) isolated from fermented foods with probiotic potential were evaluated for various tests, including exopolysaccharide production, antibiotic susceptibility, acid and bile tolerance, antibacterial activity, and cell adhesion and cytotoxicity to gastric cell lines. Six selected LAB strains maintained their high viability under gastrointestinal conditions, produced high exopolysaccharides, showed no or less cytotoxicity, and adhered successfully to gastric cells. Furthermore, three strains, Weissella confusa CYLB30, Lactiplantibacillus plantarum CYLB47, and Limosilactobacillus fermentum CYLB55, demonstrated a strong antibacterial effect against drug-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica serovar Choleraesuis, Enterococcus faecium, and Staphylococcus aureus. Whole genome sequencing was performed on these three strains using the Nanopore platform; then, the results showed that all three strains did not harbor genes related to toxins, superantigens, and acquired antimicrobial resistance, in their genome. The bacteriocin gene cluster was found in CYLB47 genome, but not in CYLB30 and CYLB55 genomes. In SDS-PAGE, the extract of CYLB30 and CYLB47 bacteriocin-like inhibitory substance (BLIS) yielded a single band with a size of less than 10 kDa. These BLIS inhibited the growth and biofilm formation of drug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA), causing membrane disruption and inhibiting adhesion ability to human skin HaCaT cells. Moreover, CYLB30 and CYLB47 BLIS rescued the larvae after being infected with P. aeruginosa and MRSA infections. In conclusion, CYLB30 and CYLB47 BLIS may be potential alternative treatment for multidrug-resistant bacteria infections.

Bioinformatics analysis of purified and identified novel bacteriocin‐like inhibitory substances produced by Streptomyces sp.

SummaryBacteriocins are antimicrobial peptides synthesised ribosomally by bacteria that can be used as natural preservatives. One potential bacteriocin‐producing bacteria that has yet to be widely studied is Streptomyces sp. Bioinformatics analysis has been conducted on the purified and identified bacteriocin‐like inhibitory substances (BLIS) produced by Streptomyces sp. The BLIS fraction, purified by reversed‐phase high‐performance liquid chromatography, showed significantly increased specific activities against E. coli, L. monocytogenes, S. aureus and S. typhimurium (1.2–189.4 folds). Identification of four purified BLIS fractions using HR‐LC‐ESI‐MS/MS revealed molecular mass from 1287.51 to 1812.85 Da with the sequences of TAEAEAAEGAEEAAP (fraction AS531), TQGGINANKTGSQGYG (fraction AS822), SGGYGGMGAGGIGGQRSGPQ (fraction AS857) and PGGAKGGFGGGGRPGGQGMPGG (fraction AS1121). These peptide sequences have a minimum of 2 hydrophobic amino acids, a net charge of −4 to +2, an isoelectric point of 2.68–11.71 and a hydrophobicity of 20.44–27.46 kcal mol−1. The results of molecular docking analysis between purified BLIS and the penicillin‐binding protein 3 (PBP‐3) receptor by methicillin‐resistant Staphylococcus aureus (MRSA) also showed that four fractions had low binding energy (−7.5 to −8.1 kcal mol−1) with 17–25 hydrogen bonds and distances <4 Å. Furthermore, in vitro characterisation revealed that the four purified‐BLIS have a low to moderate LC50 (68.81–200.35 μgmL−1) against A. salina and the ability to maintain 50% antibacterial activity against E. coli up to 100 °C for 30 min and in the pH range of 2.0–8.0, especially fractions AS531 and AS822. Based on the antibacterial spectrum, physicochemical characteristics and peptide sequence, BLIS produced by Streptomyces sp., is a novel bacteriocin with promising applications as a food preservative.

Anti-Listerial Activity of Bacteriocin-like Inhibitory Substance Produced by Enterococcus lactis LBM BT2 Using Alternative Medium with Sugarcane Molasses.

Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, thus reaching final products by cross-contamination. With the growing demand for clean-label products, the search for natural antimicrobials as biopreservants, such as bacteriocins, has shown promising potential. In this context, this study aimed to evaluate the anti-listerial action of bacteriocins produced by Enterococcus lactis LBM BT2 in an alternative medium containing sugarcane molasses (SCM). Molecular analyses were carried out to characterize the strain, including the presence of bacteriocin-related genes. In the kinetic study on SCM medium E. lactis, LBM BT2 showed biomass and bacteriocin productions similar to those observed on a sucrose-based medium (control), highlighting the potential of the sugarcane molasses as a low-cost substrate. Stability tests revealed that the molecule remained active in wide ranges of pH (4-10) and temperature (60-100 °C). Furthermore, the proteolytic treatment reduced the biomolecule's antimicrobial activity, highlighting its proteinaceous nature. After primary purification by salting out and tangential flow filtration, the bacteriocin-like inhibitory substance (BLIS) showed bacteriostatic activity on suspended L. monocytogenes cells and against biofilm formation at a concentration of 0.625 mg/mL. These results demonstrate the potential of the produced BLIS as a biopreservative in the food industry.

Isolation and characterization of lactic acid bacteria producing a potent anti-listerial bacteriocin-like inhibitory substance (BLIS) from chhurpi, a fermented milk product.

Nowadays, the bacteriocin industries have seen significant growth, supplanting chemical preservatives in its ability to improve the shelf-life and safety of food. The increasing customer desire to use natural preservatives has fueled advancing bacteriocin research. The objective of this study was to identify lactic acid bacteria (LAB) that produce bacteriocin-like inhibitory substance (BLIS) and have strong anti-listerial activity. We have identified and analyzed a LAB obtained from chhurpi samples, a popular milk-derived product in the Himalayan regions of India and Nepal. The strain was studied and identified based on its morphological, biochemical, and physiological characteristics. Furthermore, the molecular 16s-rDNA analysis suggests that the strain was Lactococcus sp. RGUAM1 (98.2% similar to Lactococcus lactis subsp. hordniae NBRC 100931T). The isolated strain can produce a potent BLIS, which has shown efficacy against three gram-positive bacteria responsible for food spoilage, such as Listeria monocytogenes (MTCC 657), Staphylococcus aureus subsp. aureus (MTCC 87), Lactobacillus plantarum (MTCC 1407), Lactobacillus paraplantarum (MTCC 12904). The scanning electron microscope (SEM) image illustrates that the crude cell-free supernatant (CFS) disrupts the cell envelope, leading to the release of cellular contents and the clustering of cells. In addition, this BLIS can easily withstand a wide range of pH (2-12), temperature (up to 100°C for 15min), bile salt (0.3% W/V), salinity (4% W/V), and enzyme activity of 1600AU/ml against Listeria monocytogenes. Our research offers a robust framework and valuable insights into bio-preservation and its potential applications in diverse food products.

Enterococcus faecium BACTERIOCIN EFFLUX PUMP MexA GENE AND PROMOTE SKIN WOUND HEALING IN MICE

The process involved isolating E. faecium from the gut of honeybees, screening the bacterium for bacteriocin-like inhibitory substance (BLIS), evaluating its impact on the expression of the mexA gene in multidrug-resistant (MDR) P. aeruginosa, and determining the role of bacteriocin in treating infected wounds in mice through histopathological examination. After evaluating the best circumstances for producing BLIS, it was discovered that glucose was a superior carbon source and yeast extract was the best source of nitrogen. The pH was found to be 5, the ideal incubation time was 72 hours, and ammonium sulfate salt was used for partial purification at 80% saturation. The identification of MDR P. aeruginosa isolates from pus infections was a further focus of the study. The VITEK 2 system was used to perform the identification. The results of antibiotic susceptibility tests revealed that the greatest resistance rates were found against Meropenem (83.3%) and Gentamicin (73.3%), followed by beta-lactam antibiotics (Ticarcillin, Ticarcillin/Clavulanic Acid, Piperacillin, and Aztreonam), which showed resistance in about 66.6 and 36.6% of the study isolates, respectively. Followed by Imipenem (63.3%), Ceftazidime (36.6%), and Cefepime (36.6%). The mexA gene was detected in all nine strains. The study also investigated the impact of the bacteriocin of the chosen strain on the expression of the mexA gene. An in vivo study revealed that wound healing was enhanced by treating infected wounds with E. faecium bacteriocin. Conclusion: Down-regulation and up-regulation in the expression of the genes following exposure to Bacteriocin indicate the potential of E. faecium as an effective antimicrobial agent against MDR P. aeruginosa infections.

The composition of Tibetan kefir grain TKG-Y and the antibacterial potential and milk fermentation ability of S. warneri KYS-164 screened from TKG-Y.

This study utilized high-throughput sequencing and SEM observation to elucidate the microbial composition of a Tibetan herder's homemade kefir grain named TKG-Y. Subsequently, S. warneri KYS-164 was isolated from TKG-Y, which can produce mixed protein substances with antibacterial activity, namely bacteriocin-like inhibitory substances (BLIS). BLIS can significantly reduce the growth rate of Escherichia coli 366-a, Staphylococcus aureus CICC 10384 and mixed strains at low concentrations (1 × MIC). The presence of the warnericin-centered gene cluster in KYS-164 may explain the antibacterial properties of the BLIS. Pepsin and an acidic environment can reduce the number of colonies of KYS-164 by 2.5 Log10 CFU mL-1 within 1 h, and reduce the antibacterial activity of BLIS by 21.48%. S. warneri KYS-164 showed no antibiotic resistance and biological toxicity after 80 subcultures, while BLIS produced by 40 generations of the strain retained their inhibitory efficacy against pathogenic bacteria. After 48-hour fermentation of milk with KYS-164, volatile compounds such as aldehydes, phenols, esters, and alcohols, giving it a floral, fruity, milky, oily, and nutty aroma, were released, enriching the sensory characteristics of dairy products. This study not only revealed the bacterial colony composition information of home-made kefir grain TKG-Y but also discovered and proved that S. warneri KYS-164 has the potential to inhibit bacteria and ferment dairy products. This will provide a basis for subsequent applied research on KYS-164.

Simple Purification and Antimicrobial Properties of Bacteriocin-like Inhibitory Substance from Bacillus Species for the Biopreservation of Cheese.

Bacteriocins may be used as natural preservatives and antibiotic substitutes in various foods. However, the multistep purification process of bacteriocins results in high production costs, which is an obstacle to their commercial use and consumer accessibility. In this study, a bacteriocin-like inhibitory substance (BLIS) from Bacillus spp. isolated from Korean fermented foods was partially purified using the aqueous two-phase system (ATPS). The maximum activity of the BLIS was achieved for ATPS composed of PEG 1000 (15% [w/w])/ammonium sulfate (20% [w/w])/sodium chloride (2% [w/w]), which caused BLIS activity to increase by 3 times with a 99% recovery rate. In particular, B. amyloliquefaciens Y138-6 BLIS exhibited broad antibacterial activity, high resistance to acid-base stress, and excellent thermal stability. This antibacterial substance inhibited the growth of aerobic bacteria and fungi on the walls of cheese and ripening rooms. These antibacterial properties have been shown to increase food safety and have the potential for use as biopreservatives. Moreover, considering that the execution of the ATPS requires only salts and PEG, it is a simple, environmentally friendly, and cost-effective process and may have industrial applications in the recovery of BLIS from fermentation broth.

The Bacteriocin-like Inhibitory Substance Producing Lacticaseibacillus paracasei LPa 12/1 from Raw Goat Milk, a Potential Additive in Dairy Products

Goat milk is gradually becoming the preferred milk by consumers worldwide, including Slovakia. It is also demanded as a functional and/or nutraceutical drink as it is rich in bioactive components. However, another new development is the research into the beneficial autochthonous strains used to enrich goat dairy products. Among individual species, representatives in raw goat milk are also involved in lacticaseibacilli. Bacteriocin-like inhibitory substance-producing Lacticaseibacillus paracasei LPa 12/1 was isolated from raw goat milk. This study focuses on its potential as a new additive in dairy products. No mortality was found when checking the safety of the LPa 12/1 strain using Balb/c mice. The strain reached higher counts in ewe–goat milk yoghurt (up to 6.1 cfu/g log 10) than in cow milk yoghurt (almost 5.0 cfu/g log 10). Cow milk yoghurts remained in more stable consistency after LPa 12/1 supplementation compared with ewe–goat milk yoghurts, although specific organoleptic tests were not performed. However, LPa 12/1 has survived sufficiently in both types of yoghurts. This bacteriocin-like inhibitory substance-producing strain LPa 12/1 in its encapsulated form applied in yoghurts seems suitable to supplement dairy products.

Under conditions closely mimicking vaginal fluid, Lactobacillus jensenii strain 62B produces a bacteriocin-like inhibitory substance that targets and eliminates Gardnerella species.

Within the vaginal ecosystem, lactobacilli and Gardnerella spp. likely interact and influence each other's growth, yet the details of this interaction are not clearly defined. Using medium simulating vaginal fluid and a two-chamber co-culturing system to prevent cell-to-cell contact between the bacteria, we examined the possibility that Lactobacillus jensenii 62B (Lj 62B) and/or G. piotii (Gp) JCP8151B produce extracellular factors through which they influence each other's viability. By 24 h post-inoculation (hpi) in the co-culture system and under conditions similar to the vaginal environment - pH 5.0, 37 °C, and 5% CO2, Lj 62B viability was not affected but Gp JCP8151B had been eliminated. Cell-free supernatant harvested from Lj 62B cultures (Lj-CFS) at 20 hpi, but not 16 hpi, also eliminated Gp JCP8151B growth. Neither lactic acid nor H2O2 production by Lj 62B was responsible for this effect. The Lj-CFS did not affect viability of three species of lactobacilli or eight species of Gram-positive and Gram-negative uropathogens but eliminated viability of eight different strains of Gardnerella spp. Activity of the inhibitory factor within Lj-CFS was abolished by protease treatment and reduced by heat treatment suggesting it is most likely a bacteriocin-like protein; fractionation revealed that the factor has a molecular weight within the 10-30 kDa range. These results suggest that, in medium mimicking vaginal fluid and growth conditions similar to the vaginal environment, Lj 62B produces a potential bacteriocin-like inhibitory substance (Lj-BLIS) that clearly targets Gardnerella spp. strains. Once fully characterized, Lj-BLIS may be a potential treatment for Gardnerella-related BV that does not alter the vaginal microflora.

A new bacteriocin- like-inhibitory substance from Egyptian dairy products isolated from Enterococcus faecium with anti-SARS-CoV-2 activity

Bacteriocins generated by lactic acid bacteria have increased in popularity due to their safety and versatility of application. Dairy products are rich sources of lactic acid bacteria. This work screened 31 Egyptian dairy product isolates for antimicrobial activity. Two bacteriocinogenic isolates, Enterococcus faecium CH019 and Enterococcus faecium CM019, were identified using molecular techniques. The production conditions of Bacteriocin-like inhibitory substances (BLIS) were optimized. The physical and chemical properties of the Enterococcus faecium CM019 bacteriocin-like compound were analyzed. Cytotoxic and antiviral properties against SARS-CoV-2 were evaluated. Enterococcus faecium CM019 produces bacteriocin-like substances most effectively in MRS broth with a pH of 6.5 and incubation temperature of 35 °C for 24 h. In the presence of surfactant, solvent, and high temperature, the bacteriocin-like substance activity was persistent, but it was eliminated by proteinase K, trypsin, and lipase. The BLIS proved effective against Listeria monocytogenes and Staphylococcus aureus. The CC50 of the bacteriocin-like compound was 108.3 µg/ml, while the IC50 against SARS-CoV-2 was 22.9 µg/ml. With an acceptable toxicity limit, BLIS generated by Enterococcus faecium CM019 isolated from Egyptian dairy products has a broad antimicrobial spectrum and can be employed as an antibacterial agent against Gram-positive bacteria and anti-SARS-CoV-2.

Enhancing food preservation with postbiotic metabolites γ-aminobutyric acid (GABA) and bacteriocin-like inhibitory substances (BLIS) produced by Lactobacillus brevis C23 co-cultures in plant-based medium

Lactic acid bacteria (LAB) can produce γ-aminobutyric acid (GABA) with antioxidant properties and sedative effects when it binds to the GABA receptor in the human brain. LAB can also produce bacteriocin-like inhibitory substances (BLIS) with antimicrobial capabilities during carbohydrate fermentation. GABA and BLIS are natural compounds with potential health benefits and food preservation properties. Lactobacillus brevis C23 was co-cultured with three different LABs as inducers, which produced the highest GABA content and BLIS activity. They were cultured in various plant-based media to obtain an edible and better-tasting final product over commercially available media like MRS broth. A coconut-based medium with additives was optimized using response surface methodology (RSM) to increase GABA and BLIS production. The optimized medium for maximum GABA production (3.22 ± 0.01 mg/mL) and BLIS activity (84.40 ± 0.44%) was a 5.5% coconut medium containing 0.23% glucose, 1.44% Tween 20, 0.48% L-glutamic acid, and 0.02% pyridoxine. Due to the presence of GABA, the cell-free supernatant (CFS) as a postbiotic showed higher antioxidant activity than other food preservatives like nisin and potassium sorbate. Finally, microbiological tests on food samples showed that the postbiotic was more effective than other preservatives at combating the growth of LAB, molds and coliform bacteria, making it a possible food preservative.

Antimicrobial activity of lactiplantibacillus plantarum against shiga toxin-producing escherichia coli.

The aim of the present work was to characterize Lactiplantibacillus sp. LP5 strain, isolated from pork production, and identify bacteriocin-like inhibitory substances produced by this strain. In this study, LP5 was identified by specie-specific PCR and 16S rDNA sequencing. Additionally, bacterial growth kinetics, antimicrobial activity, detection of the genes related to plantaricins production and genetic expression of plantaricins, were determined. Lactiplantibacillus sp. LP5 was identified as Lactiplantibacillus plantarum. The well-diffusion test using cell-free supernatants (CFS), neutralized CFS, CFS treated with catalase and CFS treated with proteinase K showed that inhibitory effects on a Shiga toxin-producing E. coli (STEC) strain were produced by bacteriocins. The PCR technique allowed the detection of genes encoding E/F plantaricins, as well as J/K and whole genome sequencing and bacteriocin mining analysis allowed us to confirm the presence of these plantaricins. We can conclude that the inhibitory effect of L. plantarum LP5 isolated from pigs against STEC EDL933 strain could be associated with the bacteriocins production and represents a potential use as a probiotic strain.

Bacteriocin-like inhibitory substances as green bio-preservatives; nanoliposomal encapsulation and evaluation of their in vitro/in situ anti-Listerial activity

In this study, predominant lactic acid bacteria (LAB) isolated from a traditional pickle (Persian pickled Litteh) were screened based on their ability to produce bacteriocin-like inhibitory substances (BLIS) with inhibitory effects on foodborne bacteria tested. The isolated BLIS was also characterized using liquid chromatography-mass spectrometry (LC-MS), and loaded into nanoliposomes; then, it's in vitro and in situ inhibitory effects were evaluated. Sequencing results of the PCR products led to the identification of Lactococcus lactis LI04 as the selected BLIS-producing LAB. The highest inhibitory activity of the BLIS was observed against Listeria monocytogenes PTCC 1298, which was significantly (P < 0.05) higher than the other foodborne bacteria studied. According to LC-MS analysis, presence of two peptides with retention times of 1.45 and 1.76 min corresponding to mass to charge ratio of 456.45 and 194.79 was approved in the BLIS. Produced empty and BLIS-loaded nanoliposomes had diameters of 92.99 and 139.10 nm, respectively. Results of Fourier transform infrared analysis showed that BLIS loading induced high degree of hydration in nanoliposomes. The increased Tm of loaded nanoliposomes (verified by differential scanning calorimetry) implied that the rigidity of liposomal membrane was enhanced. The loaded nanoliposomes were also able to maintain their inhibitory activity after treatment in simulated gastrointestinal conditions and commercial sterilization; whereas, the free (non-encapsulated) BLIS lost its effect. Furthermore, anti-Listerial activity of nanoliposomal BLIS after pasteurization was higher than the free BLIS. The highest release of the BLIS from nanoliposomes at 4 °C was observed after 16 days' storage. After this period, anti-Listerial activity of nanoliposomal BLIS was significantly higher than the free BLIS in the produced homemade beef steak.

Antimicrobial Activity of Peptides Produced by Lactococcus lactis subsp. lactis on Swine Pathogens.

Swine production is of great importance worldwide and has huge economic and commercial impact. Due to problems with bacterial infection, the use of antimicrobials has increased in the last decades, particularly in Latin America and Asia. This has led to concerns about antimicrobial resistance, which poses risks to human health and the environment. The use of probiotic organisms has been proposed as an alternative to this use, as these beneficial bacteria can produce antimicrobial peptides, such as bacteriocins, which allow the induction of inhibitory effects against pathogenic microorganisms. Among probiotics, some bacteria stand out with the inhibition of animal pathogens. The bacteriocin-like inhibitory substances (BLISs) of Lactococcus lactis subsp. lactis strain L2, present in its cell-free supernatant, were tested against pathogenic strains isolated from pig samples, such as Escherichia coli, Salmonella enterica, Streptococcus suis, Streptococcus dysgalactiae, Staphylococcus hyicus, and Enterococcus faecalis. Compounds secreted by L. lactis L2 have been shown to inhibit the growth of some pathogenic species, particularly Gram-positive bacteria, with S. suis being the most prominent. Antimicrobial peptides with a molecular size of 500-1160 Daltons were isolated from BLISs. The results highlight the potential of L. lactis BLISs and its peptides as natural antimicrobials for use in the food industry and to reduce the use of growth promoters in animal production.

Partial characterization of a bacteriocin-like inhibitory substance produced by Streptococcus equinus C6I9, a bovine rumen isolate.

The present study aimed to evaluate the production and the factors that influence the antimicrobial activity of a bacteriocin-like inhibitory substance (BLIS) produced by Streptococcus equinus C6I9, a bovine rumen isolate. The antagonist activity was evaluated by the spot-on-lawn assay. Antimicrobial activity of the crude extracts obtained using NaCl acidic solution (pH 2, 100 mM) was evaluated along the bacterial growth and in growth medium containing different carbon and nitrogen sources, by agar diffusion method. Crude extract was evaluated regarding temperature stability, storage conditions, and pH sensitivity. S. equinus C619 inhibited both Gram-positive and Gram-negative strains. The crude extract was sensitive to proteinase K, and showed great stability to different temperatures and pH (2 to 12). The presence of meat extract (1.5 g/L) and sucrose (8 g/L) resulted in higher production of BLIS by S. equinus C6I9. The BLIS molecular mass was determined as approximately 3.5 kDa. The results obtained in this study evidenced the production of a broad spectrum activity, thermo- and pH stable BLIS by the ruminal isolate S. equinus C6I9, important features for its biotechnological application.

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.

Harnessing the probiotic properties and immunomodulatory effects of fermented food-derived Limosilactobacillus fermentum strains: implications for environmental enteropathy.

Environmental enteropathy (EE), a chronic small intestine disease characterized by gut inflammation, is widely prevalent in low-income countries and is hypothesized to be caused by continuous exposure to fecal contamination. Targeted nutritional interventions using potential probiotic strains from fermented foods can be an effective strategy to inhibit enteric pathogens and prevent chronic gut inflammation. We isolated potential strains from fermented rice water and lemon pickle and investigated their cell surface properties, antagonistic properties, adhesion to HT-29 cells, and inhibition of pathogen adherence to HT-29 cells. Bacteriocin-like inhibitory substances (BLIS) were purified, and in vivo, survival studies in Caenorhabditis elegans infected with Salmonella enterica MW116733 were performed. We further checked the expression pattern of pro and anti-inflammatory cytokines (IL-6, IL8, and IL-10) in HT-29 cells supplemented with strains. The strains isolated from rice water (RS) and lemon pickle (T1) were identified as Limosilactobacillus fermentum MN410703 and MN410702, respectively. Strains showed probiotic properties like tolerance to low pH (pH 3.0), bile salts up to 0.5%, simulated gastric juice at low pH, and binding to extracellular matrix molecules. Auto-aggregation of T1 was in the range of 85% and significantly co-aggregated with Klebsiella pneumoniae, S. enterica, and Escherichia coli at 48, 79, and 65%, respectively. Both strains had a higher binding affinity to gelatin and heparin compared to Bacillus clausii. Susceptibility to most aminoglycoside, cephalosporin, and macrolide classes of antibiotics was also observed. RS showed BLIS activity against K. pneumoniae, S. aureus, and S. enterica at 60, 48, and 30%, respectively, and the protective effects of BLIS from RS in the C. elegans infection model demonstrated a 70% survival rate of the worms infected with S. enterica. RS and T1 demonstrated binding efficiency to HT-29 cell lines in the 38-46% range, and both strains inhibited the adhesion of E. coli MDR and S. enterica. Upregulation of IL-6 and IL-10 and the downregulation of IL-8 were observed when HT-29 cells were treated with RS, indicating the immunomodulatory effects of the strain. The potential strains identified could effectively inhibit enteric pathogens and prevent environmental enteropathy.

Screening of bacteriocin-producing dairy Enterococcus strains using low-cost culture media.

This study was carried out to select the bacteriocinogenic strains among Enterococcus strains isolated from Ukrainian traditional dairy products using a low-cost media for screening, that containing molasses and steep corn liquor. A total of 475 Enterococcus spp. strains were screened for antagonistic activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes indicator strains. The initial screening revealed that 34 Enterococcus strains during growth in low-cost medium containing corn steep liquor, peptone, yeast extract, and sucrose produced metabolites with inhibition activity against at least of the indicator strains used. Enterocin genes entA, entP, and entB were detected in 5 Enterococcus strains by PCR assay. Genes of enterocins A and P were found in E. faecalis 58 and Enterococcus sp. 226 strains, enterocins B and P - in Enterococcus sp. 423, enterocin A - in E. faecalis 888 and E. durans 248 strains. Bacteriocin-like inhibitory substances (BLIS) produced by these Enterococcus strains were thermostable and sensitive to proteolytic enzymes. To our knowledge, this is the first report on the isolation of enterocin-producing wild Enterococcus strains from traditional Ukrainian dairy products using a low-cost media for screening bacteriocinogenic strains. Strains E. faecalis 58, Enterococcus sp. 423, and Enterococcus sp. 226 are promising candidates for practical use as producers of bacteriocins with inhibitory activity against L. monocytogenes using molasses and steep corn liquor as cheap sources of carbon and nitrogen, that can significantly reduce the cost of industrial bacteriocin production. Further studies will be required to determine the dynamic of bacteriocin production, its structure, and mechanisms of antibacterial action.