Bacteriocin-like Inhibitory Substance Research Articles

Natural Antimicrobial Monoterpenes as Potential Therapeutic Agents in Vaginal Infections: A Review

The recurrence and relapse of vaginal infections in women is a major issue and a challenging pathway to identify and develop new approaches to treatment. In the case of antibiotic therapy, contraceptives, and dietary changes, the recurrence of vaginitis is more common these days. Anaerobic bacteria, Candida spp., and trichomonas in the vaginal microflora cause both symptomatic and asymptomatic vaginitis, which includes vaginal inflammation. It changes the vaginal microbiota and decreases Lactobacilli spp. growth, which is maintaining the vaginal pH (3.5-4.5) through lactic acid production, antimicrobial peptides, bacteriocin, and bacteriocin-like inhibitory substances. The remarkable antimicrobial activity of plant’s producing metabolites like alkaloids, tannins, phenolic compounds, flavonoids and terpenoids for several vaginal infections have been reported in previous studies. Presented review focuses on the pivotal role of monoterpenes, providing a detailed description of thymol, geraniol, limonene, eugenol, eucalyptol, and α-terpineol as antimicrobial molecules in the treatment of vaginal infections. These monoterpenes are very good at killing E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus niger, Trichomonas vaginalis and Candida albicans which are the main microbes that cause vaginitis. Future research could explore the latent combinations of such monoterpenes as synergistic antimicrobial agents to treat bacterial and fungal vaginal infections, trichomoniasis, among other conditions.

Production of bacteriocin-like inhibitory substance (BLIS) by Staphylococcus spp. isolates from dogs.

In the present study, 39 canine isolates of Staphylococcus spp. were tested for antimicrobial substance (AMS) production. Seven AMS producers were identified, whose products exhibited a non-acidic character and a proteinaceous nature, therefore being considered bacteriocin-like inhibitory substances (BLIS). The producer strains of BLIS P1, P16 and I3 showed a broad spectrum of antimicrobial activity. Human, veterinary and plant pathogens, such as Listeria monocytogenes, Klebsiella pneumoniae, Staphylococcus spp. and Clavibacter michiganensis, were among the inhibited micro-organisms, suggesting the potential biotechnological application of these peptides. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified the producer strains of BLIS P1, P16 and I3 as Staphylococcus pseudintermedius P1, Staphylococcus schleiferi P16 and Staphylococcus pseudintermedius I3. The plasmid profile of these strains suggests that the BLIS production is linked to biosynthetic genes located on plasmids. PCR analyses revealed that BLIS P1, P16 and I3 are different from 11 staphylococcins already described in the literature and that their genomic DNAs do not carry the most prevalent staphylococcal enterotoxin genes. The highest levels of BLIS production were achieved after 18-24h of growth of the producer strains in TSB medium. Moreover, BLIS P1 and I3 exhibited high resistance to temperature and pH variations, and BLIS P16 maintained 100% of its activity in almost all conditions tested. The characteristics associated with BLIS P1, P16 and I3 described in this work encourage further investigation of these substances, in addition to this study being the first report of BLIS production by a strain of S. schleiferi.

Efficient metabolic pathway modification in various strains of lactic acid bacteria using CRISPR/Cas9 system for elevated synthesis of antimicrobial compounds

Lactic acid bacteria (LAB) are known to exhibit various beneficial roles in fermentation, serving as probiotics, and producing a plethora of valuable compounds including antimicrobial activity such as bacteriocin-like inhibitory substance (BLIS) that can be used as biopreservative to improve food safety and quality. However, the yield of BLIS is often limited, which poses a challenge to be commercially competitive with the current preservation practice. Therefore, the present work aimed to establish an optimised two-plasmid CRISPR/Cas9 system to redirect the carbon flux away from lactate towards compounds with antimicrobial activity by disrupting lactate dehydrogenase gene (ldh) on various strains of LAB. The lactic acid-deficient (ldhΔ) strains caused a metabolic shift resulting in increased inhibitory activity against selected foodborne pathogens up to 78 % than the wild-type (WT) strain. The most significant effect was depicted by Enterococcus faecalis-ldh∆ which displayed prominent bactericidal effects against all foodborne pathogens as compared to the WT that showed no antimicrobial activity. The present work provided a framework model for economically important LAB and other beneficial bacteria to synthesise and increase the yield of valuable food and industrial compounds. The present work reported for the first time that the metabolism of selected LAB can be manipulated by modifying ldh to attain metabolites with higher antimicrobial activity.

A Novel C-Terminal Truncated Bacteriocin Found by Comparison between Leuconostoc mesenteroides 406 and 213M0 Isolated from Mongolian Traditional Fermented Milk, Airag.

Bacteriocins produced by lactic acid bacteria are known to be useful tools for food biopreservation and fermentation control. Leuconostoc mesenteroides subsp. mesenteroides 406 and 213M0 isolated from different samples of Mongolian traditional fermented milk, airag, had been reported to produce listericidal bacteriocin-like inhibitory substances with similar but slightly different properties. In this study, the antibacterial properties and the related gene sequences of both strains were compared, and then their bacteriocins were purified and identified. Strain 406 was superior to strain 213M0 in cell growth and antibacterial activity against many strains. However, the activity of 213M0 was stronger than that of 406 against a few strains. DNA sequencing revealed two and three plasmids in 406 and 213M0, respectively, and each one of them harbored an almost identical mesentericin Y105-B105 gene cluster. Removal of these plasmids resulted in a complete loss of activity, indicating that the antibacterial activity of both strains was generated by bacteriocins encoded on the plasmids. Mesentericins Y105 and B105 were purified from both cultures, and another novel bacteriocin, named mesentericin M, was identified from the 213M0 culture only. Its structural gene was coded on a 213M0 plasmid and, surprisingly, its C-terminal three amino acid residues were post-translationally cleaved. To our knowledge, this is the first report of a C-terminal truncated bacteriocin. In conclusion, the novel bacteriocin should be mainly responsible for the difference in antibacterial properties between the two strains.

Production and SERS characterization of bacteriocin-like inhibitory substances by latilactobacillus sakei in whey permeate powder: exploring natural antibacterial potential.

Bacteriocins are antimicrobial compounds that have awakened interest across several industries due to their effectiveness. However, their large-scale production often becomes unfeasible on an industrial scale, primarily because of high process costs. Addressing this challenge, this work analyzes the potential of using low-cost whey permeate powder, without any supplementation, to produce bacteriocin-like inhibitory substances (BLIS) through the fermentation of Latilactobacillus sakei. For this purpose, different concentrations of whey permeate powder (55.15gL-1, 41.3gL-1 and 27.5gL-1) were used. The ability of L. sakei to produce BLIS was evaluated, as well as the potential of crude cell-free supernatant to act as a preservative. Raman spectroscopy and surface-enhanced Raman scattering (SERS) provided detailed insights into the composition and changes occurring during fermentation. SERS, in particular, enhanced peak definition significantly, allowing for the identification of key components, such as lactose, proteins, and phenylalanine, which are crucial in understanding the fermentation process and BLIS characteristics. The results revealed that the concentration of 55.15gL-1 of whey permeate powder, in flasks without agitation and a culture temperature of 32.5°C, presented the highest biological activity of BLIS, reaching 99% of inhibition of Escherichia coli and Staphylococcus aureus with minimum inhibitory concentration of 36-45%, respectively. BLIS production began within 60h of cultivation and was associated with class II bacteriocins. The results demonstrate a promising approach for producing BLIS in an economical and environmentally sustainable manner, with potential implications for various industries.

Effect of Enterococin – Zinc Oxide Nanoparticles on Gene Expression of rsbA Swarming Genes in Proteus mirabilis isolation Catheter urine.

Urinary tract infections linked to catheters are believed to be caused most frequently by Proteus mirabilis. It produces urease, which greatly increases the potency of catheter occlusion caused by swarming. Pathogenic bacteria use swarming as one of their main virulence mechanisms to evade antibiotics; as a result, there is an increasing need to develop novel antibiotic substitutes. Investigating the possible antibiofilm capabilities of artificial zinc oxide nanoparticles (ZnO NPs) made from E. Faecium was the aim of this study. By generating reductive enzymes, bacterial cells are able to catalyze the biosynthesis process. Bacteriocin-like inhibitory substance (BLIS) was used to create the nanoparticles. AFM, TEM, FESEM, and other analytical tools were used to characterize the synthesized zinc nanoparticles and determine the chemical and physical characteristics of the products. Weak swarming is shown by microorganisms that develop strong swarming. After incubation, the ZnO nanoparticles were incubated for 24 or 48 hours at 37°C at a sub-MIC of 32 µg/ml. After these isolates were treated with zinc nanoparticles, downregulation of rsbA expression was detected via real-time PCR compared to that in the untreated isolates. Zinc oxide nanoparticles can serve as antibacterial agents in a concentration-dependent manner, according to all of the study's findings. This was demonstrated by the notable downregulation of rsbA gene expression, which effectively inhibits the production of biofilms and swarming motility. This was demonstrated by their noteworthy downregulation of rsbA gene expression, which effectively promoted swarmed motility.

Antagonistic Activity of Bacteriocin-like Inhibitory Substances from Enterococcus lactis Isolated from the Surface of Jalapeno Pepper against Foodborne Pathogens

Lactic acid bacteria (LAB) can produce peptides known as bacteriocins with antagonistic activity against foodborne pathogens. The potential of LAB isolated from the surface of jalapeno peppers to produce bacteriocins with antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium was evaluated. Previously isolated LAB strains were reactivated, and their cell-free supernatants (CFSs) were evaluated. Out of 390 reactivated strains, 60 produced bacteriocin-like inhibitory substances (BLIS) since their antagonistic activity was lost after proteases addition. Subsequently, 16 BLIS showed heat resistance (HR-BLIS), retaining their bioactivity after heat treatment (121 °C for 15 min). By 16S rRNA gene sequencing and antibiotic susceptibility tests, LAB strains producing HR-BLIS were identified as Enterococcus lactis. Four HR-BLIS exhibited a minimum inhibitory concentration (MIC) of 80 mg/mL against L. monocytogenes. MIC and minimum bactericidal concentration (MBC) of HR-BLIS-67 for S. aureus (MIC = 80 mg/mL; MBC = 320 mg/mL), S. Typhimurium (MIC = 150 mg/mL; MBC = 250 mg/mL), and E. coli O157:H7 (MIC = 250 mg/mL; MBC = 400 mg/mL) were determined. LAB isolated from the surface of jalapeno pepper produced HR-BLIS (possibly enterocin) that exhibited broad-spectrum antagonistic activity against foodborne pathogens; therefore, they are a promising source of natural antimicrobials to ensure food safety.

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.

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.

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.