Genomic, metabolomic, and functional characterisation of beneficial properties of Pediococcus pentosaceus ST58, isolated from human oral cavity.

Bacteriocins produced by lactic acid bacteria are proteinaceous antibacterial metabolites that normally exhibit bactericidal or bacteriostatic activity against genetically closely related bacteria. In this work, the bacteriocinogenic potential of Pediococcus pentosaceus strain ST58, isolated from oral cavity of a healthy volunteer was evaluated. To better understand the biological role of this strain, its technological and safety traits were deeply investigated through a combined approach considering physiological, metabolomic and genomic properties. Three out of 14 colonies generating inhibition zones were confirmed to be bacteriocin producers and, according to repPCR and RAPD-PCR, differentiation assays, and 16S rRNA sequencing it was confirmed to be replicates of the same strain, identified as P. pentosaceus, named ST58. Based on multiple isolation of the same strain (P. pentosaceus ST58) over the 26 weeks in screening process for the potential bacteriocinogenic strains from the oral cavity of the same volunteer, strain ST58 can be considered a persistent component of oral cavity microbiota. Genomic analysis of P. pentosaceus ST58 revealed the presence of operons encoding for bacteriocins pediocin PA-1 and penocin A. The produced bacteriocin(s) inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp. used to determine the activity spectrum. The highest levels of production (6400 AU/ml) were recorded against L. monocytogenes strains after 24 h of incubation and the antimicrobial activity was inhibited after treatment of the cell-free supernatants with proteolytic enzymes. Noteworthy, P. pentosaceus ST58 also presented antifungal activity and key metabolites potentially involved in these properties were identified. Overall, this strain can be of great biotechnological interest towards the development of effective bio-preservation cultures as well as potential health promoting microbes.

Lactobacillus probiotics restore vaginal and gut microbiota of pregnant women with vaginal candidiasis.

The development of probiotics has now included the areas along the gut-vaginal axis. We thus aimed to investigate the effects of lactobacilli probiotic to modulate and restore vaginal and gut microbiota of pregnant women with vaginal candidiasis (VC). A randomised, double-blind and placebo-controlled study was performed in 78 pregnant women with VC. Patients were randomised to either the probiotic (SynForU-HerCare) or placebo which were administered at baseline and continued for 8-weeks (two capsules/day of 9.5 log cfu/capsule). Microbiota profiles were assessed at time points of weeks-0, 4 and 8 for high vaginal swab and faecal samples. Shannon diversity index showed that after 8-weeks amid VC, a shift in microbial community compositional changes occurred in the high vaginal region at both genus (P=0.025) and species (P=0.044) levels, where the administration of probiotic prevented such a shift. These changes were mainly attributed to a decreased in abundance of Lactobacillus (P=0.042) accompanied by increased abundance of Prevotella (P=0.002) and Atopobium (P=0.002) in the placebo group while the probiotic group remained unchanged over time. The administration of probiotics also prevented a reduced abundance of faecal phylum Firmicutes after 8-weeks as seen in the placebo group (P<0.0001), which also showed reduction at subsequent taxonomic levels of class, family, genera and species. VC has not only altered the microbiota of vagina regions but also gut microbiota profiles, causing lessening of gut microbiota that are crucial for gut nutrient availability, protection and immunity. The administration of lactobacilli probiotics has prevented such a shift, leading to better modulated gut and vaginal microenvironment amid VC. The study was registered at ClinicalTrials.gov: identifier number NCT03940612.

Role of gastrointestinal microbial populations, a terra incognita of the human body in the management of intestinal bowel disease and metabolic disorders.

Intestinal bowel disease (IBD) is a chronic immune-mediated clinical condition that affects the gastrointestinal tract and is mediated by an inflammatory response. Although it has been extensively studied, the multifactorial aetiology of this disorder makes it difficult to fully understand all the involved mechanisms in its development and therefore its treatment. In recent years, the fundamental role played by the human microbiota in the pathogenesis of IBD has been emphasised. Microbial imbalances in the gut bacterial communities and a lower species diversity in patients suffering from inflammatory gastrointestinal disorders compared to healthy individuals have been reported as principal factors in the development of IBD. These served to support scientific arguments for the use of probiotic microorganisms in alternative approaches for the prevention and treatment of IBD. In a homeostatic environment, the presence of bacteria (including probiotics) on the intestinal epithelial surface activates a cascade of processes by which immune responses inhibited and thereby commensal organisms maintained. At the same time these processes may support activities against specific pathogenic bacteria. In dysbiosis, these underlying mechanisms will serve to provoke a proinflammatory response, that, in combination with the use of antibiotics and the genetic predisposition of the host, will culminate in the development of IBD. In this review, we summarised the main causes of IBD, the physiological mechanisms involved and the related bacterial groups most frequently associated with these processes. The intention was to enable a better understanding of the interaction between the intestinal microbiota and the host, and to suggest possibilities by which this knowledge can be useful for the development of new therapeutic treatments.

Safety and beneficial properties of bacteriocinogenic Pediococcus acidilactici and Pediococcus pentosaceus isolated from silage.

The control of Listeria monocytogenes is a relevant goal for dairy products, a process that begins from the supply of feed and management of animals' health. In the present study, we evaluated the safety of two bacteriocinogenic Pediococcus strains and show that both can be considered as safe, based on their haemolytic activity, biogenic amine production and antibiotic resistance, all evaluated through phenotypical and biomolecular approaches. Both strains have shown potential as a producer of γ-aminobutiric acid (GABA) and carry an incomplete set of genes related to folate biosynthesis; both strains were able to adhere to Caco-2 cell lines with adhesion rates of 6·59% ± 3·73 and 0·84% ± 0·48. Laboratory prepared clover silage, inoculated with each bacteriocinogenic Pediococcus strain and contaminated with L. monocytogenes, proved the hypothesis for bioprotective effect of the tested strains, with the tested pathogen eliminated in the first 24 h of the experiment. These results indicate that evaluated strains can be potential beneficial candidates for application in silage production.

Bacteriocin production by Leuconostoc citreum ST110LD isolated from organic farm soil, a promising biopreservative.

AIMS: The objective of this study was to isolate a bacteriocin-producing strain and to characterize the expressed bacteriocin for the control of Listeria monocytogenes with aim of biopreservation application. METHODS AND RESULTS: Soil samples from a Korean organic farm were subjected to microbiological analysis for isolation of potential bacteriocinogenic LAB, based on a three-level approach, using L. monocytogenes ATCC 15313 as an indicator test micro-organism. From a total of 17 isolates with inhibitory potential, seven were confirmed to be bacteriocin producers. The selected isolates were differentiated based on their morphology, catalase reaction, sugar fermentation profile obtained by API50CHL and by RAPD-PCR generating two unique profiles. One of the isolates, ST110LD, a specific strong producer of anti-Listeria bacteriocins (12 800 AU ml-1 ) was identified as Leuconostoc citreum. The proteinaceous nature of the inhibitory compound produced by Leuc. citreum ST110LD was confirmed through treatment with pepsin and α-chymotrypsin. Bacteriocin activity was observed to be not affected by the presence of milk, NaCl, SDS, Tween 80 or glycerol. Bacteriocin ST110LD effectively inhibited the growth of exponentially growing L. monocytogenes ATCC 15313 during a 10-h incubation period in BHI at 37°C. In addition, this bacteriocin showed specific inhibition of only Listeria spp., but did not inhibit the growth of beneficial cultures included in the microbial test panel for assessment of the spectrum of activity. CONCLUSIONS: Leuconostoc citreum ST110LD was evaluated as safe bacterium strain, producing bacteriocin with high specificity against listerial and enterococcal species. Specificity of producer strain and expressed bacteriocin can be explored in biopreservation of different fermented food products or applied in biotherapy of antibiotic resistant listerial or enterococcal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of bacteriocin produced by Leuc. citreum strain with highly specific antimicrobial activity against Listeria sp. and Enterococcus sp.

Expression of coagulin A with low cytotoxic activity by Pediococcus pentosaceus ST65ACC isolated from raw milk cheese.

AIMS: We aimed to evaluate some specific conditions for growth of Pediococcus pentosaceus ST65ACC and its bacteriocin expression through ABC transporters; to purify the bacteriocin and determine its sequence; and to evaluate the cytotoxicity potential of the purified bacteriocin(s). METHODS AND RESULTS: The results presented for growth behaviour of P. pentosaceus ST65ACC showed that the bacterial growth was slightly influenced when cultured in MRS broth with different amounts of inoculum: 1, 2, 5 and 10%. The bacteriocin activity increased when 5 and 10% inocula were used. The carbon source (glucose) used in different amounts (1, 2, 3 or 4%) had no significant effect on growth and bacteriocin production. The studied strain P. pentosaceus ST65ACC was able to metabolize xylooligosaccharide (XOS) as the sole carbon source, resulting in the production of an antimicrobial peptide. The genes involved in the ABC transport system and sugar metabolism of P. pentosaceus ST65ACC were expressed at different levels. The bacteriocin produced by P. pentosaceus ST65ACC was partially purified by precipitation with ammonium sulphate (40% saturation), followed by reversed-phase liquid chromatography, resulting in the identification of an active bacteriocin. Tandem mass spectrometry was used to identify the partial sequence KYYGNGVTCGKHSCSVDWGK sharing high similarity to coagulin A. The semi-purified bacteriocin had low cytotoxicity based on estimated values for maximal nontoxic concentration (MNC) and cytotoxicity concentration (CC50 ). CONCLUSIONS: The bacteriocin produced by P. pentosaceus ST65ACC is similar to coagulin, with low cytotoxicity, strong antimicrobial activity and possible additional metabolite routes in the producer cell. In addition to MRS broth, bacteriocin was produced also in medium containing XOS (as the single carbon source). SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of evaluation of the role of ABC transporters in the expression of bacteriocin by P. pentosaceus, cultured in MRS and XOS.

Molecular screening of beneficial and safety determinants from bacteriocinogenic lactic acid bacteria isolated from Brazilian artisanal calabresa.

Despite of the beneficial relevance of several lactic acid bacteria (LAB) in the food industry, micro-organisms belonging to this group can determine spoilage in food products and carry a number of virulence and antibiotic resistance-related genes. This study aimed on the characterization of beneficial and safety aspects of five bacteriocinogenic LAB strains (Lactobacillus curvatus 12-named L. curvatus UFV-NPAC1), L. curvatus 36, Weissela viridescens 23, W. viridescens 31 and Lactococcus garvieae 36) isolated from an artisanal Brazilian calabresa, a traditional meat sausage. Regarding their beneficial aspects, all tested isolates were positive for mub, while EF226-cbp, EF1249-fbp and EF2380-maz were detected in at least one tested strain; none of the isolates presented map, EFTu or prgB. However, evaluated strains presented a variable pattern of virulence-related genes, but none of the strains presented gelE, cylA, efsA, cpd, int-Tn or sprE. Moreover, other virulence-related genes evaluated in this study were detected at different frequencies. L. curvatus 12 was generated positive results for ace, ccf, int, ermC, tetL, aac(6')-Ie-aph(2″)-Ia, aph(2″)-Ib, aph(2″)-Ic, bcrB, vanB and vanC2; L. curvatus 36: hyl, asa1, esp, int, ermC, tetK, aph(3')-IIIa, aph(2'')-Ic and vanC2; L. garvieae 32: asa1, ant(4')-Ia, aph(2'')-Ib, catA, vanA and vanC1; W. viridescens 23: esp, cob, ermB, aph(3')-IIIa, aph(2'')-Ic, vanA, vanB and vanC2; W. viridescens 31: hyl, esp, ermC, aph(3')-IIIa, aph(2'')-Ib, aph(2'')-Ic, catA, vanA and vanB. Despite presenting some beneficial aspects, the presence of virulence and antibiotic resistance genes jeopardize their utilization as starter or biopreservatives cultures in food products. Considering the inhibitory potential of these strains, an alternative would be the use of their bacteriocins as semi-purified or pure technological preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: The food industry has a particular interest in using bacteriocinogenic lactic acid bacteria (LAB) as starter, probiotics and/or biopreservatives in different food products. Characterization of additional beneficial features is important to identify new, multifunctional potential probiotic strains. However, these strains can only be applied in food products only after being properly characterized according their potential negative aspects, such as virulence and antibiotic resistance genes. A wide characterization of beneficial and safety aspects of bacteriocinogenic LAB is determinant to guide the proper utilization of these strains, or their purified bacteriocins, by the food industry.

Bacteriocins of Gram-positive bacteria having activity spectra extending beyond closely-related species.

Bacteriocins are bacterially-produced antimicrobial peptides that have killing activity principally against other relatively closely-related bacteria. Some bacteriocins of the lactic acid bacteria (LAB) have for many years been extensively applied in food biopreservation. However, especially during the last decade, a number of reports have appeared about unanticipated extensions to the generally rather narrow anti-bacterial activity spectrum of some of the LAB bacteriocins and novel applications have been proposed for bacteriocins ranging from controlling the growth of an increasingly-heterogeneous variety of pathogens, including Gram-negative multidrug resistant bacteria, viruses, yeasts, and in particular, difficult to control Mycobacterium spp., to their potential application as anticancer agents. How best can we assess this now rapidly-accumulating stream of reports on potential future applications of bacteriocins? Where is the line between realistic, science-based proposals and highly-speculative fiction and what are the 'critical points' that might help us to draw this line? In this review, we have attempted to analyse a selection of the presently-available data concerning relatively 'unorthodox' (i.e. beyond food preservation) applications of bacteriocins, and, by utilising our set of 'critical points', we endeavour to identify essential or/and missing information that appear crucial for success of the proposed applications.

Beneficial and Safety Properties of a Corynebacterium vitaeruminis Strain Isolated from the Cow Rumen.

Corynebacterium vitaeruminis MRU4 was isolated from the cow rumen and was differentiated from other isolates by rep-PCR and RAPD and identified by 16S rRNA sequencing. This strain presented higher survival rates for low pH and bile salts treatments, and it was able to survive and multiply in simulated gastric and intestinal environments. C. vitaeruminis MRU4 had a 53.2% auto-aggregation rate, 42.4% co-aggregation rate with Listeria monocytogenes Scott A, 41.6% co-aggregation rate with Enterococcus faecalis ATCC 19443, 10.0% co-aggregation rate with Lactobacillus sakei ATCC 15521, and 98.2% cell surface hydrophobicity rate. PCR analysis showed the presence of EFTu and map genes. The strain possessed positive results for deconjugation of bile salts (taurocholic acid, taurodeoxycholic acid, glycocholic acid, and glycodeoxycholic acid) and positive results for ß-galactosidase activity and lactose assimilation activity (glucose of 8.15 ± 0.01 CFU/ml and lactose of 9.24 ± 0.02 CFU/ml). No virulence was observed by phenotypical tests. C. vitaeruminis MRU4 was resistant to oxacillin, gentamicin, erythromycin, clindamycin, sulfa/trimethoprim, and rifampicin by the disc diffusion method and showed resistance just for vancomycin by the Etest® strips test. The strain was negative for 50 tested virulence and resistance genes based on performed PCR. Based on our knowledge, this is the first report regarding the beneficial potential of one C. vitaeruminis strain.

Novel bacteriocinogenic Enterococcus hirae and Pediococcus pentosaceus strains with antilisterial activity isolated from Brazilian artisanal cheese.

We isolated and characterized bacteriocin producers Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC from raw milk artisanal cheeses. Their bacteriocins were tolerant to temperatures from 4°C to 100°C and under sterilization conditions (121°C for 15 min). Additionally, the tested bacteriocins remained active after being exposed to pH 2.0 to 10.0 for 2 h. The activity of the bacteriocins was affected by proteolytic enzymes but remained stable after treatment with EDTA, sodium dodecyl sulfate, NaCl, skim milk, and Tween 80. Cell-free supernatants were capable of inhibiting Listeria innocua and several strains of Listeria monocytogenes obtained from different sources and belonging to different serotypes. When L. monocytogenes 211 and L. monocytogenes 422 were treated with bacteriocins, growth was completely inhibited over 12 h. Cocultures of bacteriocinogenic strains and L. monocytogenes 422 in skim milk showed that E. hirae ST57ACC could control the growth of the pathogen in the matrix after 48 h. None of the selected isolates presented positive results on a screening panel for 25 bacteriocin-related genes, however, indicating that both strains might express novel bacteriocins.

Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins.

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and ß-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami.

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.

In vitro study of beneficial properties and safety of lactic acid bacteria isolated from Portuguese fermented meat products.

Many lactic acid bacteria produce bacteriocins with a rather broad spectrum of inhibition, which could offer potential applications in food preservation. Bacteriocin production by starter cultures may bring advantage to these strains in competitive interactions with pathogenic bacteria from the food matrix. The objective of this study was to determine the safety of beneficial strains (Lactobacillus plantarum ST202Ch and ST216Ch, Enterococcus faecium ST211Ch, and Lactobacillus sakei ST22Ch, ST153Ch and ST154Ch) previously isolated from fermented meat products and characterised as bacteriocin producers. Auto-aggregation was strain-specific, and values of 28.97, 27.86 and 28.56% were recorded for L. sakei ST22Ch, ST153Ch and ST154Ch, respectively, 16.95 and 14.58% for L. plantarum ST202Ch and ST216Ch, respectively, and 12.77% for E. faecium ST211Ch. Various degrees of co-aggregation between 28.85 and 44.76% for Listeria monocytogenes 211 and 409, and between 23.60 to 34.96% for E. faecium ATCC 19443 were observed. According to the results of the diffusion method, the studied strains demonstrated susceptibility to penicillin G, ampicillin, amoxicillin, amoxicillin/clavulonic acid, imipenem, linezolid, and tetracycline. In addition, the susceptibility of the six strains to various non-antibiotic commercial drugs was examined. Production of ß-galactosidase by L. sakei ST22Ch, ST153Ch and ST154Ch, L. plantarum ST202Ch and ST216Ch, and E. faecium ST211Ch was confirmed by employing sterile filter paper discs impregnated with o-nitrophenyl-ß-D-galactopyranose. A statistically significant (P<0.001) inhibition of Mycobacterium tuberculosis growth by bacteriocins produced by L. plantarum ST202Ch (38.3%) and ST216Ch (48.6%), L. sakei ST153Ch (16.2%) and ST154Ch (16.1%), and E. faecium ST211Ch (21.7%) was observed. As determined by the polymerase chain reaction, the tested strains showed a low virulence gene profile.

Technological properties of bacteriocin-producing lactic acid bacteria isolated from Pico cheese an artisanal cow's milk cheese.

AIM: Evaluate technologically relevant properties from bacteriocin-producing strains to use as starter/adjunct cultures in cheese making. METHODS AND RESULTS: Eight isolates obtained from Pico cheese produced in Azores (Portugal) were found to produce bacteriocins against Listeria monocytogenes and three isolates against Clostridium perfringens. They were identified as Lactococcus lactis and Enterococcus faecalis and submitted to technological tests: growth at different conditions of temperature and salt, acid production, proteolysis, lipolysis, coexistence, enzymatic profile and autolytic capacity. Safety evaluation was performed by evaluating haemolytic, gelatinase and DNase activity, resistance to antibiotics and the presence of virulence genes. Some isolates presented good technological features such as high autolytic activity, acid and diacetyl production. Lactococcus lactis was negative for all virulence genes tested and inhibit the growth of all Lactic acid bacteria (LAB) isolates. Enterococci were positive for the presence of some virulence genes, but none of the isolates were classified as resistant to important antibiotics. CONCLUSIONS: The bacteriocin-producing Lc. lactis present good potential for application in food as adjunct culture in cheese production. The study also reveals good technological features for some Enterococcus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocin-producing strains presented important technological properties to be exploited as new adjunct culture for the dairy industry, influencing flavour development and improve safety.

Isolation and identification of bacteriocinogenic strain of Lactobacillus plantarum with potential beneficial properties from donkey milk.

AIMS: The goal of this study was to isolate and characterize a lactic acid bacteria (LAB) from donkey milk with potential beneficial properties. METHODS AND RESULTS: Lactic acid bacteria were isolated from donkey milk and identified based on physiological, biochemical and molecular methods. The isolate that presented highest bacteriocin potential (Lactobacillus plantarum LP08AD) was evaluated for the production of bacteriocin, including stability in the presence of various enzymes, surfactants, salts, pH and temperatures. Bactericidal effect of bacteriocin LP08AD on Listeria monocytogenes, Enterococcus faecium and Lactobacillus curvatus was shown for actively growing and stationary cells. Similar growth and bacteriocin production were observed when strain LP08AD was cultured in MRS broth at 30°C or 37°C. Bacteriocin LP08AD adhered at low levels on the producer cells (200 AU ml(-1) ). The presence of plantaricin W gene on the genomic DNA was recorded based on PCR. Good growth for strain LP08AD was recorded in MRS broth with pH from 5·0 to 9·0 and LP08AD grew well in the absence of oxbile or concentration below 0·8%. Lact. plantarum LP08AD was applied to the small intestinal epithelial polarized monolayers of H4, PSIc1 and CLAB and demonstrated low attachment ability on all cell lines studied, with values with a similar behaviour for cells from human and pig origin. CONCLUSIONS: Bacteriocin-producing Lact. plantarum LP08AD might be useful in the design of novel functional foods with potential probiotic or biopreservation properties. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report on detection and characterization of bacteriocinogenic Lact. plantarum from donkey milk. The strain LP08AD shows to have potential beneficial properties, as demonstrated by the use of noncancerogenic cell lines.

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui, a Brazilian fermented, salted and dried meat product.

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0-10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production.

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese.

AIMS: To provide molecular and phenotypical characterization of Enterococcus isolates obtained from raw milk and cheese, regarding their bacteriocinogenic and virulence activity. METHODS AND RESULTS: Forty-three bacteriocinogenic enterococci isolates were identified by 16s rDNA, fingerprinted by RAPD-PCR analysis and tested by PCR for the presence of genes for lantibiotics (lanM, lanB and lanC) and enterocins (entA, entB, entP, entL50AB and entAS48) and by phenotypical methods for bacteriocin production and inhibitory spectrum. Also, the virulence of the isolates was evaluated by PCR for genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for gelatinase, lipase, DNAse and α- and ß-haemolysis. Most isolates (93·0%) harboured at least one lantibiotic or enterocin gene and were positive for several tested virulence genes, mainly asa1 (100%), gelE (93·0%) and efaA (83.7%). 53.5% of the isolates presented ß-haemolysis [corrected]. CONCLUSIONS: Enterococcus spp. isolates presented an interesting potential application for food preservation because of bacteriocin production; however, virulence-related genes were identified in all RAPD profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrated the contradictory characteristics of the tested Enterococcus isolates: they presented a good potential for application in food biopreservation but contained several virulence factors.

Potential beneficial properties of bacteriocin-producing lactic acid bacteria isolated from smoked salmon.

AIMS: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. METHODS AND RESULTS: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell-free supernatants containing bacteriocins, added to 3-h-old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto-aggregation was strain-specific, and values ranged from 7·2% for ET35 to 12·1% for ET05. Various degrees of co-aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco-2 cells was within the range reported for Lactobacillus rhamnosus GG, a well-known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61·9­64·6%), Lact. fermentum (78·9%), Lact. delbrueckii (43·7%) and Ped. acidilactici (51·3%), which are higher than the one recorded for Lact. rhamnosus GG (53·3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain-dependent manner. CONCLUSIONS: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco-2 cells was within the range reported for Lact. rhamnosus GG, a well-known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics.