Bacteriocin-producing Strains Research Articles

Antimicrobial effect of probiotic bacteriocins on Streptococcus mutans biofilm in a dynamic oral flow chamber model – an in vitro study

ABSTRACT Aim To determine the antimicrobial activity of the bacteriocin-producing probiotic strains Streptococcus salivarius K12 and Streptococcus salivarius M18 alone or in combination against caries-associated Streptococcus mutans. Methods Antimicrobial activity of S. salivarius K12 and/or S. salivarius M18 against S. mutans ATCC 25175 growth and biofilm formation on hydroxyapatite (HA) discs was determined in a flow chamber model by recording the colony forming units (CFU/ml) after 48 h of co-cultivation. The biofilm was analyzed by scanning electron microscopy (SEM) and by confocal laser scanning microscopy (CLSM). Additionally, the simultaneous antagonism assay was used to assess the inhibitory effect of S. salivarius K12 and/or S. salivarius M18 against S. mutans ATCC 25175 and 21 clinical isolates of S. mutans. Results Co-cultivation of S. mutans and S. salivarius K12 and/or S. salivarius M18 led to the inhibition of S. mutans viability, thereby, preventing its biofilm formation on HA discs. Furthermore, S. salivarius K12 and S. salivarius M18 exhibited antimicrobial activity against most clinical isolates of S. mutans. Conclusion The in vitro flow chamber system used in this study allows the simulation of time-dependent administration of S. salivarius probiotic strains, either alone or in combination, to investigate the prevention of S. mutans biofilm formation in a standardized model.

Studies on the mechanisms of action and development of resistance to class II bacteriocins of Gram-positive bacteria

Bacteriocins are peptides or proteins produced by bacteria to kill or inhibit the growth of other bacteria inhabiting the same ecological niche. The growing interest in bacteriocins reflects their potential use in food preservation and treatment of infections caused by antibiotic-resistant pathogenic bacteria, among other applications. The number of published studies on the identification of new bacteriocin-producing strains is constantly increasing. At the same time, there is a noticeable lack of research describing the mechanisms of action of most newly identified bacteriocins, as well as the mechanisms leading to the development of resistance to these bacteriocins and cross-resistance to antibiotics. Detailed understanding of these issues will allow to develop guidelines ensuring the most effective, safe and long-term use of bacteriocins without the risk of resistance development. This work describes the main assumptions of the doctoral dissertation of Aleksandra Tymoszewska, which objectives were to characterize the mechanisms of action and of resistance to class II bacteriocins of Gram-positive bacteria. Using the model bacterium Lactococcus lactis, two groups of bacteriocins were examined: (i) garvicins Q, A, B and C, and BacSJ; and (ii) aureocin A53 (AurA53)- and enterocin L50 (EntL50)-like bacteriocins. Bacteriocins of group (i) have been shown to recognize susceptible cells and form pores in the cell membrane using a specific receptor, the mannose-specific phosphotransferase system (Man-PTS), and sensitive bacteria have been shown to acquire resistance to the these bacteriocins by modifying the structure of Man-PTS. On the other hand, the acquisition of resistance to group (ii) membrane-targeting and receptor-independent bacteriocins occurs through changes in the structure of the bacterial cell wall and membrane, which are induced by changes in the expression of proteins involved in lipid metabolism or components of the YsaCB-KinG-LlrG regulatory system. The results shed new light on previous views on the mechanisms of action of bacteriocins and open up opportunities for their further study.

Enhanced production of bacteriocin by Bacillus subtilis ZY05.

Bacteriocin-producing strains were isolated from the soil of the rice field, screened out using an agar-well diffusion assay against six indicator bacterial strains, and the highest among them was selected for further investigation. The study focuses on how different growing conditions affect bacteriocin production. One-parameter-at-a-time (OPAT) and a central composite design of response surface methodology (RSM) were used to perform the optimization in two steps. In the OPAT trials, bacteriocin synthesis was elevated by 29%, 45%, and 34%, by employing sucrose as a carbon source and changing the NaCl concentration and pH at 7, respectively. To determine the linear, squared, and interaction correlations among the process variables to predict the ideal conditions for production, a four-factor central composite design (CCD) of RSM was used. It was determined that the analysis of variance (ANOVA), which produces a recognized model using RSM, is sufficient to describe bacteriocin production regarding activity (R2 = 0.9606). The ideal conditions for increased production were 1.51% sucrose concentration, 1.59% NaCl concentration, pH 6.35, and 28.66 (about 29) hours of incubation. The value predicted by RSM (4051.55 AU/mL) was approximately two times greater than the value of a non-optimized medium. The experimental value of 4403.85 AU/mL was closer to the expected value. According to the data, increasing bacteriocin activity required employing the ideal sucrose concentration, NaCl concentration, and incubation time. The partially purified bacteriocin was found stable at temperatures between 24 and 50°C and at pH 5-8. The molecular weight purified bacteriocin was determined to be between 13 and 35kDa.

Impact of bacteriocin-producing strains on bacterial community composition in a simplified human intestinal microbiota.

Bacteriocins are antimicrobial peptides that have been studied for decades as food bio-preservatives or as alternatives to antibiotics. They also have potential as modulators of the gut microbiome, which has been linked to human health. However, it is difficult to predict a priori how bacteriocins will impact complex microbial communities through direct and indirect effects. Here we assess the effect of different bacteriocin-producing strains on a Simplified Human Intestinal Microbiota (SIHUMI) model, using a set of bacteriocin-producing strains (Bac+) and otherwise isogenic non-producers (Bac-). Bacteriocins from different classes and with different activity spectra were selected, including lantibiotics such as lacticin 3147 and nisin A, and pediocin-like bacteriocins such as pediocin PA-1 among other peptides. SIHUMI is a bacterial consortium of seven diverse human gut species that assembles to a predictable final composition in a particular growth medium. Each member can be individually tracked by qPCR. Bac+ and Bac- strains were superimposed on the SIHUMI system, and samples were taken at intervals up to 48 h. The genome copy number of each SIHUMI member was evaluated using specific primers. We establish that the composition of the community changes in response to the presence of either broad- or narrow-spectrum bacteriocin producers and confirm that there are significant off-target effects. These effects were analyzed considering antagonistic inter-species interactions within the SIHUMI community, providing a comprehensive insight into the possible mechanisms by which complex communities can be shaped by bacteriocins.

Production and characterization of plantaricin Y11 from <i>Lactiplantibacillus Plantarum</i> strain y11 (MW642245.1) isolated from Nigerian food samples and their applications in preserving milk

Bacteriocin from lactic acid bacterium (LAB) was characterized and its biopreservation assay carried out using conventional methods and integrative approaches. About 66.7% (n=180) of the bacterial strains isolated from twelve different fermented/non-fermented food products were potential LAB strains by biochemical characterization. The highest bacteriocin producing strain was selected and confirmed with 16S rRNA sequencing and submitted to GenBank of National Center for Biotechnology Information as Lactiplantibacillus plantarum strain Y11 (Accession number: MW642245.1). Polymerase Chain Reaction (PCR) revealed the presence of virulence genes (cytotoxin K and haemolysin C) in L. plantarum Y11. The bacteriocin produced was labelled as plantaricin Y11 with molecular weight of 1540 Da, amino acid sequence was NSHGTADYCVMWLIXK, and possessed the merits of wide pH stability (2–10), high thermal stability and sensitivity to protease. The bacteriocin showed wide spectrum of activity against bacterial reference strains with bacteriocin activity ranging from 3200Au/mL to 6400Au/mL. Preservative potentials of the bacteriocin compared favourably with sodium benzoate in milk, inhibiting bacterial and fungal load compared to the control. There was log reduction of aerobic mesophilic bacterial count of 8.02±0.15 and 8.43±0.21 Log 10 CFU/mL in milk treated with plantaricin Y11 and sodium benzoate, respectively. The log reductions of fungal count in plantaricin Y11 and sodium benzoate treated milk ≥4.41±0.11 and ≥3.1±0.2 Log 10 CFU/mL respectively. The plantaricin Y11 was then classified as class IIa in bacteriocin classification schemes and it sustained some organoleptic conditions of milk making it a promising candidate for safe bio-preservatives.

Bacteriocın, Plantaricin and Pediocin Bıosynthesis in Lactic Acid Bacteria, Antimicrobial Mechanism and Applications as Food Preservatives: Review

Bacteriocins are antimicrobial peptide compounds derived from gram-positive and gram-negative bacteria. Bacteriocins can be cationic, anionic, and neutral. These compounds are synthesized in bacterial ribosomes and have a broad spectrum of activity against pathogenic bacteria. Some bacteriocins from lactic acid bacteria used in the food industry are nisin, diplococcin, acidochilin, bulgarican, helventicin, lactacin, and plantaricin. Bacteriocins produced by Pediococcus spp., which are known as pediocins, can be used as alternative preservatives in the food industry. Another type of bacteriocin is plantaricin, which is produced by a group of gram-positive bacteria, Lactobacillus plantarum, and can inhibit growth and kill a group of gram-negative bacteria that are pathogenic. Bacteriocins derived from lactic acid bacteria and used as biopreservatives have several advantages, namely a) they are not toxic and biodegrade easily because they are protein compounds; b) they do not harm the intestinal microflora because they are easily digested by enzymes in the digestive tract; c) they can reduce the use of chemical food preservatives; and d) they are highly versatile and can be utilized in various forms such as the form of bacteriocin-producing bacterial culture strains or purified or semi-purified bacteriocin compounds.

Protective Effects of Bacteriocin-Producing Lactiplantibacillus plantarum on Intestinal Barrier of Mice.

Bacteriocins are crucial metabolites of probiotics that display beneficial functions. The intestinal barrier is an important target on which probiotics exert their intestinal health activity. However, the impacts of bacteriocin-producing probiotics on the intestinal barrier are unclear. In this study, the effects of bacteriocin-producing Lactiplantibacillus plantarum Q7 and L. plantarum F3-2 on the intestinal barrier of mice were explored. It was shown that L. plantarum Q7 promoted the expression of mucin MUC2 to enhance the protection provided by the intestinal mucus layer. L. plantarum Q7 up-regulated the gene expression of intestinal tight junction proteins ZO-1 and JAM-1 significantly, and L. plantarum F3-2 up-regulated ZO-1 and Claudin-1 markedly, which exhibited tight junction intestinal barrier function. The two strains promoted the release of IgA and IgG at varying degrees. The antimicrobial peptide gene RegIIIγ was up-regulated markedly, and the gene expression of inflammatory cytokines appeared to exhibit an upward trend with L. plantarum Q7 treatment, so as to enhance intestinal immune regulation function. Furthermore, L. plantarum Q7 and L. plantarum F3-2 increased the abundance of the beneficial bacteria Muribaculaceae, inhibited the growth of the harmful bacteria Parabacteroides, and facilitated the synthesis of total short-chain fatty acids (SCFAs), which seemed to favor the prevention of metabolic diseases. Our results suggested that L. plantarum Q7 and L. plantarum F3-2 showed strain specificity in their protective effects on the intestinal chemical, physical, immunological and biological barriers of mice, which provided theoretical support for the selective utilization of bacteriocin-producing strains to regulate host health.

Characterization of bacteriocin produced by Brevibacillus laterosporus Tk3 isolated from ‘tatwakhar’ – a flour prepared from seeds of Aesculus indica in remote areas of Himachal Pradesh India

Bacteriocin producing strain Brevibacillus laterosporus TK3, was isolated from ‘Tatwakhar’- a flour prepared from seeds of Indian Horse Chestnut (Aesculus indica). Bacterial strain Brevibacillus laterosporus TK3 identified by morphological, biochemical techniques followed by 16S rRNA gene sequencing. The 16S rRNA sequence of bacteriocin producer was deposited in NCBI GenBank under accession no. KP861913.1. Bacteriocin of Brevibacillus laterosporus TK3 showed strong antagonistic activity against food spoiling/pathogenic bacteria viz. Listeria monocytogenes, Staphylococcus aureus and Clostridium perfringens. Bacteriocin production by Brevibacillus laterosporus TK3 was enhanced by optimizing production time, pH of the medium, inoculum size and incubation temperature. Maximum bacteriocin activity (6000 AU/ml) was recorded/obtained in basal salt medium of pH 5.5 with an inoculum size of 1.5 OD at 10% and incubation period of 24h at 35 °C. The bacteriocin was purified by single step gel exclusion chromatography. Molecular weight of active bacteriocin from Brevibacillus laterosporous TK3 was found to be 6 kDa according to SDS PAGE. The molecular mass of purified bacteriocin was confirmed as 5953.89 Da by MALDI TOF analysis. The purified bacteriocin was found desirable/suitable for food preservation as it showed wide spectrum of antimicrobial activity, resistance to high temperature, wide pH range and sensitivity to proteolytic enzymes thus, making it safe for human consumption.

Evaluation of probiotic and anti-inflammatory properties of bacteriocinogenic Pediococcus acidilactici HW01 and Leuconostoc citreum HW02 from malted barley

BackgroundBacteriocins are peptides with antibacterial properties that are released by lactic acid bacteria and affect genetically closely related bacteria. Settled bacteriocin-producing probiotics in the GI tract may contribute to bacteriocin formation, which can lead to pathogen inhibition and immune system regulation. The purpose of this study is to evaluate the probiotic capabilities of bacteriocin-producing LAB isolated from malted barley, Pediococcus acidilactici HW01, and Leuconostoc citreum HW02.ResultsBoth strains could survive in harsh GI conditions such as low pH, bile salt, pepsin, and lysozyme. Furthermore, exhibited non-haemolytic activity and tetracycline and erythromycin susceptibilities. In vitro adhesion of HW01 and HW02 strains to HT-29 cells was 76.28 and 75.99%, respectively. Auto-aggregation ability of HW01 and HW02 was about 14%, but their ability to aggregate with pathogens (Escherichia coli, Salmonella enterica ser. Typhimurium, Staphylococcus aureus and Listeria monocytogenes) ranged from 32.0% to 43.5%. Regarding the anti-inflammatory effect, heat-killed bacteriocin-producing strains suppressed the mRNA expression levels of cytokines and chemokines in RAW 264.7 macrophages induced by LPS (P < 0.005).ConclusionsBacteriocin-producing P. acidilactici HW01 and Leu. citreum HW02 may be good probiotic candidates.Graphical

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.

Bacteriocin production and technological properties of Enterococcus mundtii and Enterococcus faecium strains isolated from sheep and goat colostrum.

Enterococci are lactic acid bacteria (LAB) that play a role in the aroma formation, maturation, and sensory development of fermented foods such as meat and dairy products. They also contribute to the improvement of the extended shelf life of fermented foods by producing bacteriocin. The aim of this study was to isolate bacteriocin-producing LAB from sheep and goat colostrum, to characterize the bacteriocin-producing strains, and determine the technological properties of the strains. A total of 13 bacteriocin-producing LAB was isolated and identified as 11 Enterococcus mundtii and two Enterococcus faecium. The strains were found to be genetically different from each other by phylogenetic analysis of 16S rRNA gene sequences and random amplified polymorphic-DNA (RAPD-PCR). It has been determined that bacteriocins show activity in a wide pH range and are resistant to heat, lose their activity with proteolytic enzymes and α-amylase, but are resistant to detergents. While the presence of the munKS gene was detected in all of the strains, it was determined that E. faecium HC121.4, HC161.1, E. mundtii HC147.1, HC166.5, and HC166.8 strains contained multiple enterocin genes. Trisin-SDS-PAGE analysis revealed two active protein bands of approximately 5.1 and 5.5kDa in E. faecium HC121.4 and one active protein band with a weight of approximately 4.96kDa in other strains. E. mundtii strains and E. faecium HC161.1 were identified as mundticin KS producers, and E. faecium HC121.4 was defined as an enterocin A and B producer. Except for E. mundtii HC166.8, acid production of strains was found to be slow at 6h and moderate at 24h. None of them showed extracellular proteolytic and lipolytic activities. It was found that the strains had esterase, esterase lipase, leucine arylamidase, acid phosphatase, and naphthol-AS-Bl-phosphohydrolase activities, while protease activities were low and peptidase activities were high. In conclusion, bacteriocin producer 13 Enterococcus strains isolated from sheep and goat colostrum were found to have the potential to be included in starter culture combinations.

Effect of Inoculating Two Bacteriocin-Producing Lactiplantibacillus plantarum Strains at Ensiling on In Vitro Rumen Fermentation and Methane Emissions of Alfalfa Silage with Two Dry Matter Contents.

The objective of this study was to investigate the effects of inoculating two bacteriocin-producing strains, Lactiplantibacillus plantarum ATCC14917 and LP1-4, at ensiling on the in vitro ruminal fermentation characteristics and methane production of alfalfa silage with two dry matter (DM)contents. Before ensiling, fresh alfalfa was wilted to a moderate DM content (355 g/kg) and a high DM content (428 g/kg). The wilted alfalfa was treated with (1) distilled water (control), (2) commercial strain L. plantarum MTD/1 (MTD/1), (3) bacteriocin-producing L. plantarum ATCC14917 (ATCC14917), and (4) a bacteriocin-like substance producing L. plantarum LP1-4 (LP1-4) at 1 × 105 colony forming units (CFU)/g fresh weight. After 90 d of ensiling, the silages were used for in vitro rumen fermentation. Inoculation with the two bacteriocin-producing strains at ensiling remarkably reduced (p < 0.05) in vitro ruminal CH4 production and enhanced DM digestibility compared with the control group regardless of DM content. For silages with high DM content, inoculation with the bacteriocin-producing strains even increased (p < 0.05) in vitro ruminal total volatile fatty acid production. Therefore, the bacteriocin-producing inoculants have a great potential to mitigate ruminal methane emission but without an adverse effect on rumen fermentation of the inoculated alfalfa silage.

A novel bacteriocin isolated from Lactobacillus plantarum W3-2 and its biological characteristics.

In this study, screening bacteriocin-producing strains from 2,000 plant-derived strains by agar well diffusion method was conducted. The corresponding produced bacteriocin was purified and identified by Sephadex gel chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Meanwhile, the biological characteristics of bacteriocin were investigated. The targeted strain W3-2 was obtained and identified as Lactobacillus plantarum by morphological observation and 16S rRNA gene sequence analysis. Correspondingly, a novel bacteriocin (named plantaricin W3-2) produced by L. plantarum W3-2 with a molecular weight of 618.26 Da, and an amino acid sequence of AVEEE was separated, purified by Sephadex gel chromatography and RP-HPLC, and identified by LC-MS/MS. Further characteristics analysis displayed that plantaricin W3-2 had good thermal, pH stability, and broad-spectrum antimicrobial ability. In conclusion, plantaricin W3-2 can be used as a new food preservative.

Bacteriocins as promising antimicrobial peptides, definition, classification, and their potential applications in cheeses

There is increased importance to finding alternative solutions to antibiotic resistance which require more research, bacteriocins are promising antimicrobial peptides with inhibitory and bactericidal activities that might be one of these solutions. Bacteriocins are small antimicrobial peptides synthesized by bacterial ribosomes, active against the bacterial pathogen, multidrug-resistant bacteria, and cancer therapy. Lactic acid bacteria (LAB) are one of the most used bacteria to produce bacteriocins and dairy products (i.e. cheeses) consider rich sources of LAB isolates. Enterococcus faecalis, Lactobacillus fermentum, L. plantarum, L. helveticus, L. pentosus, L. paracasei subsp. paracasei, L. rhamnosus I, and L. delbrueckii subsp. lactis are strong strains in bacteriocins production. Several applications were applied to control bacterial pathogens spread in cheeses, one of them is using bacteriocins and bacteriocins-like inhibitory substances (BLIS). To reduce foodborne pathogens and spoilage bacteria in cheese, bacteriocins can be applied in several means such as inoculating cheese with bacteriocin-producer strain and adding purified or semi-purified bacteriocin as a food additive. This review is focused on bacteriocins and BLIS classification, mechanism, and applications in dairy products i.e. cheeses.

Phenotypic and genomic analyses of bacteriocin-producing probiotic Enterococcus faecium EFEL8600 isolated from Korean soy-meju.

Enterococcus faecium is a prevalent species found in fermented soybean products, known for its contributions to flavor development and inhibition of pathogenic microorganisms during fermentation. This study aims to provide comprehensive phenotypic and genomic evidence supporting the probiotic characteristics of E. faecium EFEL8600, a bacteriocin-producing strain isolated from Korean soy-meju. Phenotypic analysis revealed that EFEL8600 produced a peptide with inhibitory activity against Listeria monocytogenes, estimated to be 4.6 kDa, corresponding to the size of enterocins P or Q. Furthermore, EFEL8600 exhibited probiotic traits, such as resilience in gastrointestinal conditions, antioxidant and anti-inflammatory activities, and protection of the intestinal barrier. Safety assessments demonstrated no hemolytic and bile salt deconjugation activities. Genomic analysis revealed the presence of several genes associated with probiotic characteristics and bacteriocin production, while few deleterious genes with a low likelihood of expression or transferring were detected. Overall, this study highlights E. faecium EFEL8600 as a potent anti-listeria probiotic strain suitable for use as a starter culture in soymilk fermentation, providing potential health benefits to consumers.

Effects of Bacteriocin-Producing Lactiplantibacillus plantarum on Fermentation, Dynamics of Bacterial Community, and Their Functional Shifts of Alfalfa Silage with Different Dry Matters

This study investigated the effects of two bacteriocin-producing Lactiplantibacillus plantarum strains on fermentation, bacterial communities, and their functions of alfalfa silage with two dry matter (DM) contents of 355 (moderate DM) and 428 (high DM) g/kg fresh weight. Before ensiling, alfalfa was treated with (1) distilled water (control), (2) the commercial strain L. plantarum MTD/1, (3) bacteriocin-producing L. plantarum ATCC14917, and (4) bacteriocin-producing L. plantarum LP1-4, and ensiled for 3 d, 7 d, 14 d, 60 d, and 90 d, respectively. Application of ATCC14917 promoted lactic acid production in the moderate DM silage at the early fermentation stage (3 d). Silages treated with ATCC14917 and LP1-4 showed lower DM losses and non-protein nitrogen concentrations versus the control or MDT/1-treated silage (p &lt; 0.05). During fermentation, a high proportion of Weissella cibaria was observed in the silages with high DM content from 3 to 60 d of ensiling, and the functions of carbohydrate and amino acid metabolisms of silage bacterial community were decreased by ATCC14917 before 60 d of ensiling. In addition, ATCC14917 also inhibited the growth of Aerococcus and Enterobacter in silage. Therefore, the bacteriocin-producing L. plantarum ATCC14917 has a great potential to improve alfalfa silage quality, nutritive value, and safety as well.

Isolation, Characterization, and Effect on Biofilm Formation of Bacteriocin Produced by Lactococcus lactis F01 Isolated from Cyprinus carpio and Application for Biopreservation of Fish Sausage.

The aim of this work was the screening of bacteriocin-producing LABs isolated from fish, the selection of promising/prominent strain(s), the characterization of the bacteriocin produced, and the evaluation of its potential to be used as biopreservative(s). Amplification and sequencing of the 16S rRNA gene of the bacteriocin-producing strain was performed. Then a partial purification of the produced bacteriocin, using a combination of ammonium sulfate and chloroform-methanol precipitation, was done. Its molecular weight was determined by SDS-PAGE. In addition, the action spectrum, the hemolysis test, and its ability to inhibit biofilm formation were analyzed. A total of 88 isolates of lactic acid bacteria (LAB) including one bacteriocin producer, which was identified as Lactococcus lactis F01, were collected. The bacteriocin was partially purified with an estimated yield of 40%. Regarding the SDS-PAGE profile, the secreted bacteriocin has molecular weight of about 3.5 kDa and was identified as class I bacteriocin. The antimicrobial test showed that the bacteriocin inhibits pathogenic and/or spoilage bacteria, 10 Gram-positive and 16 Gram-negative bacterial species. Moreover, it can inhibit biofilm formation from 1.3% (Escherichia coli) to 63.92% (Pseudomonas aeruginosa ATCC15692) depending on the strain. The hemolytic activity of novel bacteriocin was observed at the concentration of 10 μg/ml of bacteriocin crude extract, which was 0.7 ± 0.0029%. In addition, it exhibited good thermal and pH stability with retained antibacterial activity of 85.25% after treatment at 121°C for 20 min, as well as at a pH range between 2.0 and 10.0. Moreover, this bacteriocin showed the ability to inhibit the growth of bacterial culture load in fish sausage stored at 8°C for 28 days. Considering the results obtained, bacteriocin could be potentially exploited as an alternative to chemical preservatives or as a substitute for antibiotics.

Rapid Proteomic Characterization of Bacteriocin-Producing Enterococcus faecium Strains from Foodstuffs.

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (&gt;1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry.

Characterization of novel bacteriocin produced by bacteriocinogenic Carnobacterium maltaromaticum isolated from raw milk

This study was designed to assess newly isolated bacteriocin-producing strain as potential food preservative. A bacteriocin producing lactic acid bacterium, named Carnobacterium maltaromatium KCA018, was screened from raw milk using deferred and spot-on-the-lawn assays. The crude cell free supernatant (CFS) was purified to obtain proteinaceous bacteriocin by ammonium sulfate precipitation (assigned as bacteriocin KCA) and tested for bacteriocin production, physical stability, antimicrobial activity, and bacteriocin-encoding gene detection. The growth curves of C. maltaromatium KCA018 reached late exponential phase after 15 h of incubation at 25 °C and 30 °C (Fig. 2). The maximum production of bacteriocin KCA was reached after 12 h of incubation at 25 °C, showing the antimicrobial activity of more than 3000 AU/ml against Listeria monocytogenes. The purified bacteriocin KCA was stable up to 67 °C for 30 min of exposure and between pH 4 and 7, showing more than 6000 AU/ml. The antibacterial activity of bacteriocin KCA was lost in the presence of pronase, proteinase K, and trypsin. Purified bacteriocin KCA showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. The CFS and purified bacteriocin KCA effectively inhibited the growth of L. monocytogenes ATCC 1911, E. faecalis ATCC 19433, and E. feacium ATCC 11576. The molecular weight of purified bacteriocin KCA was estimated at approximately 5 kDa. The positive amplification was observed for pisA and cbnBM1 with approximately between 100 and 200 bp. The newly identified bacteriocin can be a promising preservative for application in food.

Draft Genome Sequences of Two Bacteriocin-Producing Enterococcus faecium Strains Isolated from Nonfermented Animal Foods in Spain.

Here, we report the draft genome sequences of two bacteriocin-producing Enterococcus faecium strains isolated from nonfermented animal foods in Spain. The genomes of the strains contain at least three different regions encoding bacteriocins, and the strains comply with the European Food Safety Authority guidance for use in animal nutrition.