Labyrinthopeptin A2 disrupts raft domains.

Labyrinthopeptins constitute a class of ribosomal synthesized peptides belonging to the type III family of lantibiotics. They exist in different variants and display broad antiviral activities as well as show antiallodynic activity. Although their mechanism of action is not understood, it has been described that Labyrinthopeptins interact with membrane phospholipids modulating its biophysical properties and point out to membrane destabilization as its main point of action. We have used all-atom molecular dynamics to study the location of labyrinthopeptin A2 in a complex membrane as well as the existence of specific interactions with membrane lipids. Our results indicate that labyrinthopeptin A2, maintaining its globular structure, tends to be placed at the membrane interface, mainly between the phosphate atoms of the phospholipids and the oxygen atom of cholesterol modulating the biophysical properties of the membrane lipids. Outstandingly, we have found that labyrinthopeptin A2 tends to be preferentially surrounded by sphingomyelin while excluding cholesterol. The bioactive properties of labyrinthopeptin A2 could be attributed to the specific disorganization of raft domains in the membrane and the concomitant disruption of the overall membrane organization. These results support the improvement of Labyrinthopeptins as therapeutic molecules, opening up new opportunities for future medical advances.

Microencapsulação de enterocina e oleo de oregano em leitelho / Microencapsulation of the enterocin and essential oil oregano in buttermilk / Microencapsulación de enterocina y aceite esencial de orégano en suero de leche

A pesquisa busca técnicas alternativas para expansão da vida de prateleira dos alimentos, isto tem impulsionado estudos sobre a utilização de conservantes naturais, tais como as bacteriocinas e óleos essenciais, que são considerados agentes antimicrobianos naturais. No entanto estes antimicrobianos naturais, não são adicionados diretamente em produtos alimentícios, devido a alterações sensoriais e em suas características físico e química. Com avanço tecnológico da microencapsulação, tem sido um potencial em fornecer sistemas que garantem estabilidade para os antimicrobianos naturais desta forma podendo compor a matriz de alimentos. Portanto, o objetivo desse trabalho foi microencapsular a enterocina produzida por Enterococcus durans MF5 e óleo de orégano usando leitelho. Para a microencapsulação, foram realizados três tratamentos: T1 controle leitelho, T2 leitelho/enterocina (LE), e T3 leitelho/enterocina/óleo (LEO). O material foi submetido ao processo de spray dryer e foram realizados ensaios para determinar a atividade antimicrobiana do material encapsulado contra as bactérias Listeria monocytogenes, Listeria innocua e Listeria ivanovi. O rendimento da microencapsulação foi de 13,01% e 11,63% para LE e LEO, respectivamente. Os resultados apresentados nos microencapsulados LE e LEO mostraram inibição contra todas as bactérias teste, foi constatado que a microencapsulação de enterocina e óleo de orégano mantiveram seu poder antimicrobiano. A efetividade da microencapsulação foi realizada por (FTIR), onde picos de intensidade entre as amostras na região 1000 a 930 cm-¹ e 1800 a 1500 cm-¹ foram observadas. Os resultados apontam para mudança no perfil químico das amostras encapsuladas, corroborando com a hipótese que o leitelho apresentou papel encapsulante da bactericiona e óleo de orégano.Portanto a microencapsulação aumenta a eficácia antimicrobiana dos antimicrobianos.

The research seeks alternative techniques for expanding the shelf life of foods, this has driven studies on the use of natural preservatives, such as bacteriocins and essential oils, which are considered natural antimicrobial agents. However, these natural antimicrobials are not directly added to food products due to sensory changes and their physical and chemical characteristics. With technological advancement of microencap- sulation, it has been a potential to provide systems that ensure stability for natural anti- microbials in this way can compose the food matrix. Therefore, this study has an objective microencapsulated the interocin and essencial oil, used buttermilk as a encapsulating ma- terial where, T1 Buttermilk Control, T2 buttermilk/enterocin (LE), e T3 Buttermilk/en- terocin/oil (LEO). The product has been submitted to spray drier process, were conducted trials to determine antimicrobial activity. Was observed with mass yield 13,01% e 11,63% para LE e LEO. These results the microencapsulate indicate then LE e LEO there was inihibiton against bacteria tests. Was observed that the microencapsulated between enter- ocin and essential oil oregano maintained antimicrobial power. The effectiveness of the microencapsulated was performed by Fourier transform infrared (FTIR) analysis, where a sample in the region 1000 to 930 cm-¹ and 1800 to 1500 cm-¹ was observed. Therefore microencapsulation increases antimicrobial efficacy of antimicrobials.

La investigación busca técnicas alternativas para ampliar la vida útil de los alimentos, esto ha impulsado estudios sobre el uso de conservantes naturales, como las bacteriocinas y los aceites esenciales, que se consideran agentes antimicrobianos naturales. Sin embargo, estos antimicrobianos naturales no se añaden directamente a los productos alimentarios debido a los cambios sensoriales y a sus características físicas y químicas. Con el avance tecnológico de la microencapsulación, ha sido un potencial para proporcionar sistemas que garanticen la estabilidad de los antimicrobianos naturales de esta manera puede componer la matriz alimentaria. Por lo tanto, este estudio tiene como objetivo microencapsular la interocina y el aceite esencial, utilizando suero de leche como material encapsulante donde, T1 Suero de leche Control, T2 Suero de leche/enterocina (LE), e T3 Suero de leche/enterocina/aceite (LEO). El producto ha sido sometido al proceso de secado por pulverización, se realizaron ensayos para determinar la actividad antimicrobiana. Se observó con rendimiento de masa 13,01% e 11,63% para LE e LEO. Estos resultados indican que el microencapsulado LE e LEO fue inhibido contra las pruebas bacterianas. Se observó que el microencapsulado entre enterocina y aceite esencial de orégano mantuvo el poder antimicrobiano. La eficacia del microencapsulado fue realizada por análisis de infrarrojo transformado de Fourier (FTIR), donde fue observada una muestra en la región de 1000 a 930 cm-¹ y de 1800 a 1500 cm-¹. Por lo tanto, la microencapsulación aumenta la eficacia antimicrobiana de los antimicrobianos. PALABRAS CLAVE: Bacteriocina; Enterococcus durans; Suero de Leche; Origanum vulgare; Spray Dryer.

Polydiacetylene vesicles acting as colorimetric sensor for the detection of plantaricin LD1.

The interaction of antimicrobial peptides with membrane lipids plays a major role in numerous physiological processes. In this study, polydiacetylene (PDA) vesicles were synthesized using 10, 12-tricosadiynoic acid (TRCDA) and 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These vesicles were applied as artificial membrane biosensor for the detection of plantaricin LD1 purified from Lactobacillus plantarum LD1. Plantaricin LD1 (200 µg/mL) was able to interact with PDA vesicles by changing the color from blue to red with colorimetric response 30.26 ± 0.59. Nisin (200 µg/mL), used as control, also changed the color of the vesicles with CR% 50.56 ± 0.98 validating the assay. The vesicles treated with nisin and plantaricin LD1 showed increased infrared absorbance at 1411.46 and 1000-1150 cm-1 indicated the interaction of bacteriocins with phospholipids and fatty acids, respectively suggesting membrane-acting nature of these bacteriocins. Further, microscopic observation of bacteriocin-treated vesicles showed several damages indicating the interaction of bacteriocins. These findings suggest that the PDA vesicles may be used as bio-mimetic sensor for the detection of bacteriocins produced by several probiotics in food and therapeutic applications.

A plant endophyte Staphylococcus hominis strain MBL_AB63 produces a novel lantibiotic, homicorcin and a position one variant.

Here we report a jute endophyte Staphylococcus hominis strain MBL_AB63 isolated from jute seeds which showed promising antimicrobial activity against Staphylococcus aureus SG511 when screening for antimicrobial substances. The whole genome sequence of this strain, annotated using BAGEL4 and antiSMASH 5.0 to predict the gene clusters for antimicrobial substances identified a novel antimicrobial peptide cluster that belongs to the class I lantibiotic group. The predicted lantibiotic (homicorcin) was found to be 82% similar to a reported peptide epicidin 280 having a difference of seven amino acids at several positions of the core peptide. Two distinct peaks obtained at close retention times from a RP-HPLC purified fraction have comparable antimicrobial activities and LC-MS revealed the molecular mass of these peaks to be 3046.5 and 3043.2 Da. The presence of an oxidoreductase (homO) similar to that of epicidin 280- associated eciO or epilancin 15X- associated elxO in the homicorcin gene cluster is predicted to be responsible for the reduction of the first dehydrated residue dehydroalanine (Dha) to 2-hydroxypropionate that causes an increase of 3 Da mass of homicorcin 1. Trypsin digestion of the core peptide and its variant followed by ESI-MS analysis suggests the presence of three ring structures, one in the N-terminal and other two interlocking rings at the C-terminal region that remain undigested. Homicorcin exerts bactericidal activity against susceptible cells by disrupting the integrity of the cytoplasmic membrane through pore formation as observed under FE-SEM.

Production, Purification and Characterization of a Novel Bacteriocin Produced by Bacillus subtilis VS Isolated from Mango (Mangifera indica L)

Abstract Bacteriocin has been identified as an excellent alternative to chemical preservatives due to its astonishing antimicrobial activity against food spoiling and food-borne pathogens. So there is a need to identify the newer and potent sources of bacteriocin producers. This study aims the isolation of potent bacteriocin producing microorganism from fresh fruits and vegetables, its production, purification, and characterization. Firstly, 43 isolates were analysed for its antimicrobial potential, out of which7 were found to inhibit the growth of various pathogens. Considering the results of antimicrobial activity; the microorganism isolated from mango was regarded as the most potent one; which was identified as Bacillus subtilis VS.70% ammonium sulphate precipitated and dialysed bacteriocin was purified using DEAE cellulose and sephadex G75 chromatography. Bacteriocin was purified by 24.64 fold with 8.65% recovery and its molecular weight was found to be 31.2kDa. The Purified bacteriocin was found to be stable at broad pH and temperature. It was found to be degraded by various proteases studied confirming its proteinaceous nature. Considering all these attributes; the purified bacteriocin isolated from Bacillus subtilis VS can be exploited by various food industries.

Identification and characterization of Pseudomonas aeruginosa derived bacteriocin for industrial applications.

Drug resistance has become a major threat due to the frequent use of commercial antibiotics and there is an urgent need to combat this problem. Having this in mind, the present research was aimed at developing a novel P. aeruginosa puBac bacteriocin molecule. The bacteriocin was purified by ammonium sulfate precipitation followed by Sepharose FF and Sephadex G15 column purification and the purified bacteriocin has been reported to have the molar mass of 43 kDa. The findings of the optimization showed that 3500 AU/mL of bacteriocin was obtained at 37 °C, 3410 AU/mL of bacteriocin at 6.5 pH and 3780 AU/mL of bacteriocin at 48 h of incubation time. In addition, 3863 AU/mL of bacteriocin activity was obtained with Tween-80 followed by 3789 AU/mL with a concentration of 2% NaCl and 4200 AU/mL for Fe2+. PuBac bacteriocin has been shown to have a significant effect on test pathogens. For example, E. coli was found to have 3.6 µM of MIC, followed by Staphylococcus sp. with 6.15 µM of MIC and Bacillus sp. with a 7.5 µM of MIC. The remarkable properties of bacteriocin suggest that it could be used in various pharmaceutical and food industries.

Metagenomics-based approach for studying and selecting bioprotective strains from the bacterial community of artisanal cheeses.

A metagenome-based approach was used to assess the taxonomic affiliation and functional potential for bacteriocin production of the bacterial community in cow's milk artisanal cheeses from Northwestern Argentina. Three different samples were analyzed by high-throughput sequencing of the V4 region of the 16S rRNA gene and shotgun metagenomics. Taxonomic analysis showed that cheese A and C were quite similar whereas cheese B displayed a rather different bacterial composition. Overall, two families, Streptococceae and Enterococceae, dominated the artisanal cheese microbiota, being the former family prevalent in cheese B and the later family the most important in samples A and C. Besides the usual species associated to cheeses, a number of bacterial taxa that have not been previously found in Argentinean artisanal cheeses were reported in the present work such as Macrococcus caseolyticus and Streptococcus macedonicus Functional metagenomics analysis using the bacteriocin mining software BAGEL3, identified 2 ORFs encoding antimicrobial peptides in cheese B and 42 different peptides in sample C. The bacteriocin genes found showed good correlation with taxonomy. Based on the microbial diversity and functional features found through shotgun metagenomic sequencing, a culture-dependent approach was applied aiming to isolate bacteriocin-producing bacteria able to inhibit the growth of the foodborne pathogen Listeria monocytogenes. From 151 bacterial colonies derived from the cheese samples, 10 were associated to high anti-Listeria activity. Based on partial 16S rRNA gene sequencing and RAPD-PCR analysis, all bacteriocinogenic isolates were identified as Enterococcus faecium. Finally, we carried out a pilot experiment with L. monocytogenes-contaminated cheese using one of the enterococcal isolates as a bioprotective adjunct culture. The use of E. faecium CRL1879 during artisanal cheese manufacturing did not alter the main organoleptic properties of the cheese and ensured an efficient control of the foodborne pathogen up to 30 days. This finding supports the use of E. faecium CRL1879 as an adjunct culture in the cheese-making process with a combination of both safety and minimal processing.

Characterization of robust Lactobacillus plantarum and Lactobacillus pentosus starter cultures for environmentally friendly low-salt cucumber fermentations.

Seven candidates for starter cultures for cucumber fermentations belonging to the Lactobacillus pentosus and Lactobacillus plantarum species were characterized based on physiological features desired for pickling. The isolates presented variable carbohydrate utilization profile on API® 50CHL test strips. The L. pentosus strains were unable to utilize d-xylose in MRS broth or the M medium. The lactobacilli were unable to produce histamine, tyramine, putrescine, and cadaverine in biogenic amine broth containing the necessary precursors. Production of d-lactic acid by the lactobacilli, detected enzymatically, was stimulated by growth in MRS broth as compared to cucumber juice medium (CJM). The lactobacilli utilized malic acid in the malate decarboxylase medium. Exopolyssacharide biosynthesis related genes were amplified from the lactobacilli. A sugar type-dependent-ropy phenotype was apparent for all the cultures tested in MRS and CJM. The genes associated with bacteriocin production were detected in the lactobacilli, but not the respective phenotypes. The antibiotic susceptibility profile of the lactobacilli mimics that of other L. plantarum starter cultures. It is concluded that the lactobacilli strains studied here are suitable starter cultures for cucumber fermentation. PRACTICAL APPLICATION: The availability of such starter cultures enables the implementation of low salt cucumber fermentations that can generate products with consistent biochemistry and microbiological profile.

The Evolution of Human Probiotics: Challenges and Prospects.

In recent years, the intestinal microbiota has been found to greatly influence a number of biological processes important for human health and longevity. Microbial composition changes easily in response to external factors, such as an unbalanced diet, lack of physical activity, and smoking. Probiotics are a key factor in maintaining the optimal composition of the intestinal microbiota. However, a number of important questions related to probiotics, such as indication for prescription, comparative efficacy of monostrain and multistrain probiotics, methods of delivery, and shelf life, remain unresolved. The aim of this review is to highlight existing issues regarding probiotic production and their prescription. The review presents the most recent findings regarding advantages and efficacy of monostrain and multistrain probiotics, preservation of probiotic strains in capsules and microcapsules, production of probiotics in the form of biofilms for improved efficacy and survival, and results of clinical studies evaluating the benefits of probiotics against different pathologies. We believe that this work will be of interest to physicians and researchers alike and will promote the development of new probiotics and ensuing regimens aimed at the treatment of various diseases.

Identification and characterisation of capidermicin, a novel bacteriocin produced by Staphylococcus capitis.

One hundred human-derived coagulase negative staphylococci (CoNS) were screened for antimicrobial activity using agar-based deferred antagonism assays with a range of indicator bacteria. Based on the findings of the screen and subsequent well assays with cell free supernatants and whole cell extracts, one strain, designated CIT060, was selected for further investigation. It was identified as Staphylococcus capitis and herein we describe the purification and characterisation of the novel bacteriocin that the strain produces. This bacteriocin which we have named capidermicin was extracted from the cell-free supernatant of S. capitis CIT060 and purified to homogeneity using reversed-phase high performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric (MS) analysis revealed that the capidermicin peptide has a mass of 5,464 Da. Minimal inhibitory concentration (MIC) experiments showed that capidermicin was active in the micro-molar range against all the Gram-positive bacteria that were tested. Antimicrobial activity was retained over a range of pHs (2-11) and temperatures (10-121°C x 15 mins). The draft genome sequence of S. capitis CIT060 was determined and the genes predicted to be involved in the biosynthesis of capidermicin were identified. These genes included the predicted capidermicin precursor gene, and genes that are predicted to encode a membrane transporter, an immunity protein and a transcriptional regulator. Homology searches suggest that capidermicin is a novel member of the family of class II leaderless bacteriocins.

Bacteriocin Occurrence and Activity in Escherichia coli Isolated from Bovines and Wastewater.

The increasing prevalence of antimicrobial resistant (AMR) E. coli and related Enterobacteriaceae is a serious problem necessitating new mitigation strategies and antimicrobial agents. Bacteriocins, functionally diverse toxins produced by most microbes, have long been studied for their antimicrobial potential. Bacteriocins have once again received attention for their role as probiotic traits that could mitigate pathogen burden and AMR bacteria in livestock. Here, bacteriocins were identified by activity screening and whole-genome sequencing of bacteriocin-producers capable of inhibiting bovine and wastewater E. coli isolates enriched for resistance to cephalosporins. Producers were tested for activity against shiga toxin-producing E. coli (STEC), AMR E. coli, and related enteric pathogens. Multiple bacteriocins were found in 14 out of 90 E. coli isolates tested. Based on alignment within BACTIBASE, colicins M, B, R, Ia, Ib, S4, E1, E2, and microcins V, J25, and H47, encoded by identical, variant, or truncated genes were identified. Although some bacteriocin-producers exhibited activity against AMR and STEC E. coli in agar-based assays, most did not. Despite this idiosyncrasy, liquid co-cultures of all bacteriocinogenic isolates with luciferase-expressing generic (K12) or STEC E. coli (EDL933) resulted in inhibited growth or reduced viability. These abundant toxins may have real potential as next-generation control strategies in livestock production systems but separating the bacteriocin from its immunity gene may be necessary for such a strategy to be effective.

Biological properties and production of bacteriocins-like-inhibitory substances by Lactobacillus sp. strains from human vagina.

AIMS: The aim of this study was to characterize Lactobacillus strains for their biological properties and amensalistic activities against genital and nongenital pathogens. METHODS AND RESULTS: For the purpose, some special characteristics (H2 O2 , biofilm and antimicrobial substances production) as well as safety properties of 112 lactobacilli were evaluated. All the strains had good amensalistic characteristics, in particular cell-free supernatants of 10 strains showed antibacterial activity against bacteria, as well as Candida sp. Moreover, these 10 strains were excellent biofilm producers. CONCLUSIONS: These results provide evidence for the possible use as probiotics for vaginal co-therapy in case of dysbiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Recently, the problem of antibiotic resistance is constantly increasing, even though resources and energy are invested in order to increase knowledge on the mechanisms of action. Bacteriocins have a rapid mechanism of action, act at extremely low concentrations, are generally sensitive to proteases and they usually have a narrow killing spectrum; these characteristics reduce the possibility of the bacterium to develop resistance. This study is focused on the feasibility of a high production of antimicrobial substances and their characterization in order to be exploited as a therapeutic alterative or in co-therapy with antibiotics in case of vaginal dysbiosis.

Papel da interação entre bactérias láticas isoladas de alimentos na produção de bacteriocinas / Role of interactions among lactic acid bacteria isolated from foods on production of bacteriocins

Bacteriocinas produzidas por bactérias láticas (BAL) apresentam um importante potencial de aplicação na bioconservação de alimentos, por sua ação antimicrobiana contra algumas espécies de microrganismos patogênicos de relevância, como Listeria monocytogenes. Este estudo analisou o efeito da interação entre cepas selecionadas de BAL produtoras de bacteriocinas com outras BAL viáveis ou não viáveis (bacteriocinogênicas ou não) na indução da produção de bacteriocinas. O efeito dos metabólitos produzidos por estas cepas na indução da bacteriocinogênese também foi avaliado. As cepas produtoras de bacteriocinas selecionadas para o estudo foram Lactobacillus sakei MBSa1, produtora de sakacina A e Pediococcus acidilactici ET34, produtora de pediocina, isoladas de salame e salmão defumado, respectivamente. A produção de pediocina por P. acidilactici ET34 foi avaliada também em leite em pó desnatado reconstituído, além de meio de cultura (caldo MRS). Os resultados indicaram que, quando em co-cultura com Enterococcus faecalis ATCC12755, Lactobacillus sakei ATCC15521 ou Listeria monocytogenes (cepas 104, 711 e 637), ou na presença do sobrenadante livre de células (SLC) dessas culturas, nenhuma das duas cepas testadas produziu maior quantidade de bacteriocina do que a produzida quando em monocultura ou na ausência do SLC. A bacteriocina produzida por P. acidilactici ET34 apresentou um efeito bacteriostático contra L. monocytogenes 104 no leite em pó desnatado reconstituído nas 12 h analisadas, com extensão da fase lag, de forma dose-dependente. Os resultados indicaram, também, que P. acidilactici ET34 não foi capaz de produzir pediocina no leite em pó desnatado reconstituído quando em monocultura ou em co-cultura, ao contrário do observado para o caldo MRS. Mais investigação é necessária para esclarecer os efeitos de possíveis interações entre as BAL presentes em um alimento, bem como o efeito dos componentes dos alimentos na produção das bacteriocinas pelas BAL bacteriocinogênicas

Bacteriocins produced by lactic acid bacteria (LAB) present an important application potential in food biopreservation, by their antimicrobial activity against some species of pathogenic microorganisms of relevance, such as Listeria monocytogenes. This study analyzed the effect of the interaction between selected strains of bacteriocin-producing LAB with other viable or non-viable LAB (bacteriocinogenic or not) in the induction of bacteriocin production. The effect of the metabolites produced by these strains on the induction of bacteriocinogenesis was also evaluated. The bacteriocin-producing strains selected for the study were Lactobacillus sakei MBSa1, producer of sakacin A and Pediococcus acidilactici ET34, producer of pediocin, isolated from salami and smoked salmon, respectively. The production of pediocin by P. acidilactici ET34 was also evaluated in reconstituted skimmed milk powder as well as culture medium (MRS broth). The results indicated that when co-cultivated with Enterococcus faecalis ATCC12755, Lactobacillus sakei ATCC15521 or Listeria monocytogenes (strains 104, 711 and 637), or in the presence of the cell free supernatant (SLC) of these cultures, neither of the two strains tested produced greater amount of bacteriocin than that produced in monoculture or in the absence of SLC. The bacteriocin produced by P. acidilactici ET34 presented a bacteriostatic effect against L. monocytogenes 104 in skimmed milk powder reconstituted in 12h, with extension of lag phase, in a dose-dependent manner. The results also indicated that P. acidilactici ET34 was not able to produce pediocin in the reconstituted skimmed milk powder when in monoculture or in co-culture, unlike that observed for the MRS broth. More research is needed to clarify the effects of possible interactions between BAL present in a food and the effect of food components on bacteriocin production by bacteriocinogenic BAL

Exploring bioactive peptides from bacterial secretomes using PepSAVI-MS: identification and characterization of Bac-21 from Enterococcus faecalis pPD1.

As current methods for antibiotic drug discovery are being outpaced by the rise of antimicrobial resistance, new methods and innovative technologies are necessary to replenish our dwindling arsenal of antimicrobial agents. To this end, we developed the PepSAVI-MS pipeline to expedite the search for natural product bioactive peptides. Herein we demonstrate expansion of PepSAVI-MS for the discovery of bacterial-sourced bioactive peptides through identification of the bacteriocin Bac-21 from Enterococcus faecalis pPD1. Minor pipeline modifications including implementation of bacteria-infused agar diffusion assays and optional digestion of peptide libraries highlight the versatility and wide adaptability of the PepSAVI-MS pipeline. Additionally, we have experimentally validated the primary protein sequence of the active, mature Bac-21 peptide for the first time and have confirmed its identity with respect to primary sequence and post-translational processing. Successful application of PepSAVI-MS to bacterial secretomes as demonstrated herein establishes proof-of-principle for use in novel microbial bioactive peptide discovery.

Strategies for screening, purification and characterization of bacteriocins.

Bacteriocins are bioactive antimicrobial peptides synthesized in the ribosome of numerous bacteria and released extracellularly. Bacteriocins have the ability to kill or inhibit the growth of prokaryotes and could potentially be useful against pathogens and antibiotic-resistant strains of bacteria. The antimicrobial mechanisms and relatively narrow killing spectrums of bacteriocins distinguish them from traditional broad-spectrum antibiotics, making them possible candidates to replace antibiotics in the future. In recent years, researchers have discovered many new bacteriocins from different sources. However, the discovery of new bacteriocins involves complicated screening, identification, purification, and characterization processes. This review describes the strategies currently employed for screening, identification, purification, and characterization of bacteriocins. Approximately 40 novel bacteriocins are reviewed in this paper. Our conclusions about the research process will guide the development of novel methods and allow for the faster, easier, and more efficient discovery of novel bacteriocins.

Estimation of the bacteriocin ColE7 conjugation-based "kill" - "anti-kill" antimicrobial system by real-time PCR, fluorescence staining and bioluminescence assays.

The efficiency of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system, was assessed using real-time PCR, flow cytometry and bioluminescence. The ColE7 antimicrobial system consists of the genetically modified Escherichia coli strain Nissle 1917 harbouring a conjugative plasmid (derivative of the F-plasmid) encoding the "kill" gene (ColE7 activity gene) and a chromosomally encoded "anti-kill" gene (ColE7 immunity gene). On the basis of traJ gene expression in the killer donor cells, our results showed that the efficiency of the here studied antimicrobial system against target E. coli was higher at 4 than at 24 h. Flow cytometry was used to indirectly estimate DNase activity of the antimicrobial system, as lysis of target E. coli cells in the conjugative mixture with the killer donor strain led to reduction in cell cytosol fluorescence. According to a lux assay, E. coli TG1 (pXen lux+ Apr ) with constitutive luminescence were killed already after 2 h of treatment. Target sensor E. coli C600 with DNA damage SOS-inducible luminescence showed significantly lower SOS induction 6 and 24 h following treatment with the killer donor strain. Our results thus showed that bioluminescent techniques are quick and suitable for estimation of the ColE7 bacterial conjugation-based antimicrobial system antibacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial antimicrobial resistance is worldwide rising and causing deaths of thousands of patients infected with multi-drug resistant bacterial strains. In addition, there is a lack of efficient alternative antimicrobial agents. The significance of our research is the use of a number of methods (real-time PCR, flow cytometry and bioluminescence-based technique) to assess the antibacterial activity of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system. Bioluminescent techniques proved to be rapid and suitable for estimation of antibacterial activity of ColE7 bacterial conjugation-based antimicrobial system and possibly other related systems.

Novel approaches to purifying bacteriocin: A review.

Bacteriocin is a proteinaceous biomolecule produced by bacteria (both Gram-positive and Gram-negative) that exhibits antimicrobial activity against closely related species, and food-borne pathogens. It has recently gained importance and attracted the attention of several researchers looking to produce it from various substrates and bacterial strains. This ushers in a new era of food preservation where the use of bacteriocin in food products will be an alternative to chemical preservatives, and heat treatment which are understood to cause unwanted side effects, and reduce sensory and nutritional quality. However, this new market depends on the success of novel downstream separation schemes from various types of crude feedstocks which are both effective and economic. This review focuses on the downstream separation of bacteriocin from various sources using both conventional and novel techniques. Finally, recommendations for future interesting areas of research that need to be pursued are highlighted.

Importance of the agar-media in the evaluation of bacteriocin activity against the same test-microorganisms

Abstract Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria, which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. Successful application of techniques for quantitative or qualitative bacteriocin determination relies not only on the sensitivity of the test-microorganisms, but also on the agar-medium employed. Cell free supernatants are routinely used to preliminary screen for antimicrobial activity of bacteria by means of the agar well diffusion method, but the supernatant may also include other molecules (such as medium components and/or intracellular compounds) accidentally released during cell free supernatant preparation, which may interfere with the assay. Reproducibility of bacteriocin activity against the same test-microorganisms is an important factor to be considered. Unfortunately, no specific information about bioassays standardization to determine bacteriocin activity is available in the literature. In this work, growth inhibition by means of the agar well diffusion assays were carried out on different agar-media showing a strong dependence on the agar-medium used, indicating that the inhibitory effects could also depend on the diffusion of exudates that are included in the cell-free supernatant. The results presented in this communication show that selection of the agar-medium is crucial for the bioassay response.

Optimization of the yield of bacteriocin-like substance (BLIS) produced by Pediococcus pentosaceus and its application as food bioconservative / Otimização do rendimento de substância semelhante a bacteriocina (BLIS) produzido por Pediococcus pentosaceus e sua aplicação como bioconservante de alimentos

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria (LABs), which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. However, when this bacteriocin has not been completely characterized regarding its amino acid and the nucleotide sequences of the corresponding gene, the qualified term bacteriocin-like inhibitory substance (BLIS) is recommended. In order to increase the antimicrobial activity of bacteriocins, the ability of probiotics LABs, such as Pediococcus pentosaceus, to ferment different carbon and nitrogen sources has been studied. For the development of an improved culture medium, carbon and nitrogen sources must be considered as nutrients responsible for cell growth and bacteriocin production. The best condition, after 48 h of cultivation, for growth (3.420 g/L) and for BLIS production by Pediococcus pentosaceus ATCC 43200 was in Man, Rogosa and Sharp (MRS) culture medium supplemented with 1.5% peptone, initial pH 6.0 and under the following culture conditions: anaerobiosis, 30oC and agitation of 200 rpm. Compared with control (MRS without supplement), the growth of Pediococcus was significantly lower (1.995 g/L) as well as it reduced significantly its generation time from 2.05 h (control) to 1.28 h (MRS supplemented), a reduction of approximately 62.5%. Moreover, addiction of peptone to MRS medium promoted reduction of 4 h to the Pediococcus exponential phase onset. Regarding BLIS antimicrobial activity, addition of nitrogen source to MRS medium was also quite significant. Through the agar diffusion method, BLIS showed inhibition halos between 12.50 and 19.50 mm against LABs strains (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 and Carnobacterium piscicola CECT 4020). Against Listeria strains (Listeria innocua NCTC 111288 and Listeria seeligeri NCTC 11289), their antimicrobial activity was better detected in liquid medium assay, evaluating the minimal inhibitory concentration of 50%. BLIS was able to inhibit 60 and 100% of L. seeligeri and L. innocua, respectively, as well as, diluted 1x (v/v) in water was able to inhibit 100% growth of both Listeria. BLIS 17 showed also good results as food preservative when applied in ready-to-eat pork ham artificially contaminated with L. seeligeri in vacuum-package at 4oC during shelf life of 10 days. BLIS was able to maintain low Listeria multiplication, lower samples weight loss, low lipid peroxidation and good color parameters during samples storage. Results demonstrated the importance of optimizing the culture medium to increase microbial mass, to produce and to improve the activity of this antimicrobial molecule. Moreover, results also suggest the possible application of BLIS as a natural food preservative

Bacteriocinas são peptídeos produzidos por várias espécies de bactérias, especialmente bactérias ácido-láticas (BALs) e apresentam um amplo espectro de ação contra bactérias deteriorantes e patógenos de origem alimentar. Entretanto, quando estas bacteriocinas não foram completamente caracterizadas quanto a sequência de seus nucleotídeos e do seu gene correspondente, é recomendada a denominação de substância semelhante a bacteriocina (BLIS). Para aumentar a atividade antimicrobiana de bacteriocinas, a habilidade de BALs probióticas, como Pediococcus pentosaceus, em fermentar diferentes fontes de carbono e nitrogênio tem sido estudado. Para o desenvolvimento de um meio de cultura melhorado, fontes de carbono e nitrogênio devem ser consideradas como nutrientes responsáveis pelo crescimento celular e pela produção de bacteriocina. A melhor condição, após 48 h de cultivo, para o crescimento (3,420 g/L) e para a produção de BLIS por P. pentosaceus ATCC 43200 foi em meio de cultivo Man, Rogosa e Sharp (MRS) suplementado com 1,5% de peptona, pH inicial 6,0 e sob as seguintes condições de cultivo: anaerobiose, 30oC e agitação de 200 rpm. Comparado ao controle (MRS sem suplementação), o crescimento de Pediococcus foi significativamente menor (1,995 g/L) assim, como também, reduziu significativamente o tempo de geração de 2,05 h (controle) para 1,28 h (MRS suplementado), uma redução de aproximadamente 62,5%. Além disso, a adição de peptona ao meio MRS promoveu redução de 4 h para o início da fase exponencial de Pediococcus. Quanto a atividade antimicrobiana de BLIS, a adição de fonte de nitrogênio ao meio MRS também foi bastante significativa. Através do método ágar difusão, BLIS apresentou halos de inibição entre 12,50 a 19,50 mm contra cepas de BALs (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 e Carnobacterium piscicola CECT 4020). Contra cepas de Listeria (Listeria innocua NCTC 11288 e Listeria seeligeri NCTC 11289), a sua atividade inibitória foi melhor detectada em meio líquido, através da determinação da concentração mínima inibitória de 50%. BLIS sem diluição foi capaz de inibir 60 e 100% de L. seeligeri e L. innocua, 15 respectivamente, assim como, diluído 1x (v/v) em água foi capaz de inibir 100% o crescimento de ambas Listeria. BLIS também apresentou bons resultados como conservante de alimento quando aplicado em presunto contaminado artificialmente com L. seeligeri e armazenado a 4oC a vácuo por 10 dias. BLIS foi capaz de manter baixa a multiplicação de Listeria, menor perda de peso das amostras, baixa peroxidação lipídica e bons parâmetros de cor durante o armazenamento das amostras. Os resultados demonstraram a importância de se otimizar meio de cultivo tanto para o aumento da massa microbiana como para a produção e melhoramento da atividade desta molécula antimicrobiana. Além disso, os resultados também sugerem a possível aplicação de BLIS como conservante natural de alimentos

Rapid detection of Lactococcuslactis isolates producing the lantibiotics nisin, lacticin 481 and lacticin 3147 using MALDI-TOF MS.

The aim of the study was to evaluate the potential use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for fast and reliable detection of strains producing the lantibiotics nisin, lacticin 481 and lacticin 3147 in a large collection of lactococci. A total of one hundred lactococcal isolates from traditional ewe's and goat's raw milk cheeses were identified to the species level as Lactococcuslactis by MALDI-TOF MS based on comparison with lactococcal entries in the BioTyper database. Mass spectra in the range 2000-4000Da of the identified isolates were compared to reference spectra of three lactococcal strains producing lacticin 481 (IFPL 330), lacticin 3147 (IFPL 105) and nisin (IFPL 503). Only eight isolates had mass spectra with peaks that could be unequivocally identified as lacticin 481 (2900.47Da) or nisin (3330.31Da). None of the assayed isolates matched the mass spectra corresponding to the two-peptide lacticin 3147 (2847.97 and 3306.29Da). The results obtained by MALDI-TOF MS were genetically validated by amplification of the corresponding structural gene coding for lacticin 481, nisin and lacticin 3147. MALDI-TOF MS can be used as a fast and reliable technique to screen a large number of lactococcal isolates for the ability to produce the lantibiotics nisin, lacticin 481 and lacticin 3147.