Antibacterial activity of lactic acid bacteria isolated from vacuum-packaged meats

Lactic acid bacteria isolated from vacuum-packaged fresh meat stored at 4 degrees C were shown to produce antagonistic substances active against closely related bacteria. Growth medium, pH and growth temperature all affected the production of the inhibitory substances. Ten strains including aciduric Lactobacillus-type organisms, Carnobacterium spp. and Leuconostoc spp. were selected that produced protein-aceous substances that caused inhibition of indicator strains. These were considered to be bacteriocins or bacteriocin-like compounds based on their inactivation with protease, generally narrow spectra of antibacterial activity and bactericidal or bacteriostatic modes of action. Activity was not lost from supernatant fluids as a result of heat treatment at 62 degrees C for 30 min, except for the Leuconostoc strains. Inhibitory spectra of some strains included Enterococcus spp. and Listeria monocytogenes. Some strains were of interest because their inhibitory substances were detected in the supernatant fluid early in the growth cycle. The inhibitory substances differed in characteristics between strains and there is evidence that more than one bacteriocin-like substance may be produced by some strains.     

Inhibition of Vibrio parahaemolyticus by a bacteriocin-like inhibitory substance (BLIS) produced by Vibrio mediterranei 1

AIMS: The aim of this research was to identify and partially purify new bacteriocin-like substances from strains of halophilic 'non-cholera' vibrios isolated from food sources. METHODS AND RESULTS: Forty-five halophilic Vibrio spp. strains were screened for antimicrobial production. Vibrio mediterranei 1, a nonpathogenic strain, showed antimicrobial activity towards Vibrio parahaemolyticus spp. and related species. The bacteriocin-like inhibitory substance (BLIS), released by the bacteria into growth media, was concentrated by ultrafiltration and characterized. BLIS was sensitive to proteinase K, was stable in the pH range 5-9, was resistant to organic solvents and was heat stable up to 75 degrees C. Initial purification of BLIS by size exclusion chromatography showed an apparent molecular mass of 63-65 kDa. CONCLUSIONS: This study reports the ability of V. mediterranei 1 to produce a bacteriocin-like substance inhibiting growth of V. parahaemolyticus spp. and other closely related bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The strong activity of BLIS towards the human and fish pathogen V. parahaemolyticus and the persistence of antimicrobial properties under a variety of different conditions suggest its potential application in food microbiology.     

In vitro study on bacteriocin production of Enterococci associated with chickens

In recent years, the approach of using innovative strategies such as probiotics or bacteriocins for the prevention or treatment of bacterial infections has come into focus. The present study was undertaken to check in vitro ability of Enterococci-isolated from the gastrointestinal tract of chickens-to produce a bacteriocin-like substance and to describe some further probiotic properties in five selected Enterococcus faecium strains. All strains (n=17) were found to produce bacteriocin-like substances against 14 out of 20 indicator bacteria of animal, food or environmental origin. Selected E. faecium strains expressed sufficient survival by pH 3.0 after 3h, in the presence of 1% bile after 24h and they were sensitive to most of antimicrobials tested. All tested strains adhere to the human, canine and porcine intestinal mucus (between 1.5% and 9.2%). However, better adhesion ability was observed for the canine mucus. PCR detection of enterocin structural genes determined presence of enterocins A and P genes in all selected strains. Characterization of bacteriocin substance in detail was performed in E. faecium EF55. The EF55 strain produced a bacteriocin-like substance (during the late logarithmic and early stationary growth phase) with inhibitory activity mostly against Gram-positive bacteria (100-51,200 AU/mL) including Listeria monocytogenes. Proteinaceous character of the bacteriocin substance was confirmed (its inhibitory activity was lost after its treatment with proteases), it was found to be stable after heating (100 degrees C 10 min) and during 12 months storage at -20 degrees C. The highest inhibitory activity of bacteriocin produced by EF55 strain (growing in MRS) broth was achieved between pH 7.0 and 9.0.     

Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T

AIMS: To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins. METHODS AND RESULTS: Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase. The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. CONCLUSION: Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent. faecium JCM 5804T among LAB type and reference strains.     

Antimicrobial spectrum of bacteriocin-like substances produced by rumen staphylococci

Five strains of rumen coagulase-negative adherent and ureolytic staphylococci were obtained as bacteria producting bacteriocin-like substances or, lantibiotics. All examined staphylococci produced inhibitory agents which showed a wide range of inhibition against Gram-positive and Gram-negative indicator organisms from different sources. Clear zones of inhibition (diameter 1–6 mm) dominated. Most bacteriocin-like substances produced by the strains were stable and sensitive to trypsin, susceptible to chloroform vapours and heat-sensitive.     

Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4

A protease-sensitive antibacterial substance produced by Bacillus coagulans I₄ strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus , Leuconostoc , Oenococcus , Listeria and Pediococcus . Coagulin was stable at 60 °C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by α-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3–4 kDa by SDS-PAGE. The B. coagulans I₄ strain harbours a plasmid, pI₄, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI₄,whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for coagulin production.     

Antagonistic activity of Lactobacillus bacteria strains against anaerobic gastrointestinal tract pathogens (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile)]

Antagonistic activity of Lactobacillus strains has been known for some time. This property is connected with production of many active substances by lactobacilli e.g., organic acids and bacteriocin-like substances which interfere with other indigenous microorganisms inhabiting the same ecological niche, including also anaerobic gastrointestinal tract pathogens. Growing interest of clinical medicine in finding new approaches to treatment and prevention of common inflammatory infections of the digestive tract resulted in studies on a possible usage of lactic acid bacteria. Last years, several in vitro and in vivo experiments on antagonism of different Lactobacillus strains against Helicobacter pylori and Clostridium difficile were performed. These observations had been done on already established, well known probiotic Lactobacillus strains. We tested antibacterial activities of Lactobacillus strains isolated from human digestive tract. As indicator bacteria, four species known as anaerobic bacterial etiologic agents of gastroenteric infections: Helicobacter pylori, Campylobacter jejuni, C. coli and Clostridium difficile were used. Some of them were obtained from international collections, others were clinical isolates from specimens taken from patients with different defined gastrointestinal infections. We used a slab method of testing inhibitory activity described in details previously. Following conclusions were drawn from our study: All tested human Lactobacillus strains were able to inhibit the growth of all strains of anaerobic human gastrointestinal pathogens used in this study. Inhibitory activities of tested Lactobacillus strains against Helicobacter pylori, Campylobacter spp., and Clostridium difficile as measured by comparing mean diameters of the inhibition zones were similar. Differences in susceptibility of individual indicator strains of Campylobacter spp. and Clostridium difficile to inhibitory activity of Lactobacillus strains were small. A similar mechanism of inhibition of anaerobic bacteria by lactobacilli is postulated.     

Bacteria competing with the adhesion and biofilm formation by <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The activity of antagonistic substances produced by Pseudomonas aeruginosa and Lactobacillus acidophilus against the planktonic and sessile populations of Staphylococcus aureus strains was demonstrated. The strongest effects were caused by probiotic L. acidophilus strain — bacteriocin-like inhibitory substances (BLIS) positive. However, the S. aureus A3 growth, adhesion and biofilm formation was also limited by cell-free supernatant of L. acidophilus H-1 (BLIS negative). Moreover, competitive direct interactions were observed between staphylococci and the above bacteria, which influenced the formation of dualspecies aggregates on the surface.     

Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas hydrophila

The biochemical properties, virulence for mice and trout, and the extracellular virulence factors at 28° and 37°C of 11 environmental and nine human strains of Aeromonas hydrophila were compared. All the environmental isolates and four of the human group were virulent for trout at 3 x 10⁷ cfu, but only human strains were able to cause death or lesions in mice by the intramuscular route. Extracellular virulence factors such as haemolysins, cytotoxins and proteases were also investigated in supernatant fluids of cultures grown at 28°C and 37°C. The production of haemolysins, caseinases, elastases and growth yields of environmental strains decreased sharply during cultivation at 37°C but cytotoxins were produced to the same extent, or slightly less, than at 28°C. The human strains differed from the environmental strains in response to growth temperatures: protease activity decreased at 37°C, although growth yield was not affected, but more haemolysins and cytotoxins were produced by the virulent strains at this temperature than at 28°C. Sodium caseinate SDS-PAGE of culture supernatant fluids of selected human strains revealed that temperature selectively inhibited the production of certain proteases.     

Anti-Listeria monocytogenes Bacteriocin-Like Inhibitory Substances From Enterococcus faecium UQ31 Isolated from Artisan Mexican-Style Cheese

Artisan fresh Mexican-style cheeses are commonly made from raw milk that provides not only rich flavors, but also a diversity of associated lactic acid bacteria (LAB) strains. Enterococcus faecium UQ31 was isolated from panela cheese and produced bacteriocin-like inhibitory substances (BLIS) with a strong anti-Listeria activity. A modified pH–mediated adsorption-desorption purification process resulted in (after SDS-PAGE) two bands showing antimicrobial activities, where most of the activity corresponded to the band with an estimated molecular weight of 7.5 kDa. The BLIS produced by E. faecium UQ31 were heat resistant, stable at ambient storage conditions, and active in the pH range 5–9. The BLIS antimicrobial activities were detected during logarithmic growth phase and remained constant until the end of incubation time (19 h). These BLIS showed a wide anti-Listeria monocytogenes spectra. The E. faecium UQ31 strain or their BLIS represent a promising potential as antimicrobial food preservatives.     

Production of histamine and tyramine by lactic acid bacteria isolated from vacuum-packed sugar-salted fish

The incidence of histamine- or tyramine-producing lactic acid bacteria was examined in several products of vacuum-packed sugar-salted fish (salmon, halibut, mackerel). No histamine-producing isolates were observed, whereas the majority of tyramine-producing isolates were identified as Carnobacterium spp. These organisms were shown to be important members of the microbial flora during storage of vacuum-packed sugar-salted salmon at 5°C. The amount of tyramine produced was reduced by lowering the temperature from 9°C to 4°C for all of five strains of carnobacteria or lactobacilli. The majority of tyramine was produced during the exponential growth phase for Carnobacterium piscicola N 5 and Lactobacillus viridescens N 69. The ability of these bacteria to produce tyramine may be used as an index of microbial quality/acceptability of stored vacuum-packed sugar-salted fish.     

In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk

AIMS: Food-borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions. METHODS AND RESULTS: The growth of Escherichia coli O157:H7, Salmonella serotype Typhimurium, Staphylococcus aureus, Listeria innocua, Enterococcus faecium and Enterococcus faecalis was evaluated for 48 h at 37 degrees C. In the presence of the lactic acid bacteria (LAB), an increase of the generation time was observed for all the gram-positive bacteria evaluated. Staphylococcus aureus was the most sensitive strain showing an increase of the generation time by 210%. However, for all the gram-negative bacteria evaluated, no inhibition occurred after 8 h of fermentation. The soluble portion of Lact. acidophilus- and Lact. casei-fermented milk was recuperated and tested for its antimicrobial activity. Listeria innocua and Staph. aureus were the most sensitive to the presence of fermented milk supernatant showing an inhibition of 85.9% and 84.7%, respectively. This soluble fraction was neutralized to eliminate the antimicrobial effect of the organic acids produced; the most sensitive strains were L. innocua and E. coli O157:H7 showing an inhibition of 65.9% and 61.9%, respectively. Finally, the soluble fraction was neutralized and irradiated at 45 kGy using a (60)Co source to eliminate the possible antimicrobial effect of both organic acids and bacteriocin-like substances. Enterococcus faecalis, E. coli O157:H7 and Staph. aureus were the most affected bacteria by this fraction, showing 39.1, 32 and 31.2% inhibition, respectively. CONCLUSIONS: The results obtained in this study suggest the implication of both organic acids and bacteriocin-like inhibitory substances in the antimicrobial activity observed in the soluble fraction of the probiotic preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the antimicrobial mechanisms of action of Lact. acidophilus- and Lact. casei-fermented milk used to prevent antibiotic-associated diarrhoea.     

Lantibiotics biosynthesis genes and bacteriocinogenic activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. isolated from raw milk and cheese

Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by 16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or 48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.     

Utilization of glucose, fructose and malic acid by malolactic bacteria: effect of ethanol and formation of mannitol and volatile acids

Five lactic acid bacteria capable of carrying out the malolactic fermentation were studied at 15°C and 25°C. Results indicated that at 15°C hardly any glucose, fructose and l -malic acid was utilized over a 9d period. After 9d at 25°C very little glucose and fructose and most (if not all) of the l -malic acid was degraded. The addition of ethanol markedly affected the l -malic acid utilization by the four Leuconostoc oenos strains at 25°C. Large differences in their ability to convert l -malic acid were found amongst the four strains in media containing 5% and 10%(v/v) ethanol.     

The combination effect of pH, SO2, ethanol and temperature on the growth of Leuconostoc oenos

A factorial design was used to determine the main effects and interactions of pH (3.6, 3.8 and 4.0), SO₂ (25 and 50 mg/l), ethanol (7%, 10% and 13%, v/v) and temperature (15°C and 25°C) on the growth of 54 Leuconostoc oenos strains. These differed greatly in their ability to survive adverse conditions. Under the test conditions, temperature had a profound effect on growth. The power pH values of 3.6 and 3.8 had a marked inhibitory effect on growth, as did at the lower temperature of 15°C. The higher concentrations of SO₂ (50 mg/l) and ethanol (13%) had the greatest inhibitory effect on growth, and with a synergistic effect when both SO₂ and ethanol were added to the test medium. Based on these results, five strains were selected that were best capable of surviving the adverse conditions studied. These strains will be used for further study on the induction of malolactic fermentation in wine.     

Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832

Pediocin PD-1, produced by Pediococcus damnosus NCFB 1832, is inhibitory to several food spoilage bacteria and food-borne pathogens. However, pediocin PD-1 is not active against other Pediococcus spp. and differs in this respect to other pediocins produced by Pediococcus acidilactici and Pediococcus pentosaceus. Production of pediocin PD-1 starts during early growth and reaches a plateau at the end of exponential growth. Pediocin PD-1 was partially purified and its size was determined by tricine-SDS-PAGE as ≈ 3·5 kDa. The isoelectric point (pI) of pediocin PD-1 is ≈ 3·5, as determined with the Rotofor electrofocusing cell (BioRad). Pediocin PD-1 is heat-resistant (10 min at 121°C) and remains active after 30 min of incubation at pH 2–10. Pediocin PD-1 is resistant to treatment with pepsin, papain, α-chemotrypsin and trypsin, but not Proteinase K. Pediocin PD-1 is bactericidal against sensitive cells of Oenococcus oeni (previously Leuconostoc oenos ).     

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an α-(1→6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30°C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives.     

Antibacterial activity and lantibiotic post-translational modification genes in Streptococcus spp. isolated from ruminal fluid

The production of bacteriocins is frequently described in high microbial diversity environments. The aims of this study were to screen Streptococcus spp. isolated from rumen for their antibacterial potential and to determine the presence of post-translational modification genes for lantibiotic class of bacteriocins. The isolates were tested for production of antibacterial compounds by the spot-on-lawn assay. Presence of interfering factors and the sensitivity to proteinase K were evaluated. The ruminal bacteria were identified by 16S rRNA gene sequencing and the subspecific discrimination of the isolates belonging to the same specie was performed by PFGE. The presence of lantibiotic post-translational modification genes (lanB, lanC, and lanM) into bacterial genomes was performed by PCR. The bacteriocin-like inhibitory substances showed broad inhibitory activity and the producer cells were identified as S. equinus, S. lutetiensis, and S. gallolyticus. According to PFGE, the isolates identified as S. equinus belong to different strains. Three ruminal isolates showed at least one of the lantibiotic post-translational modification genes, and lanC was more frequently detected (75%). The production of broad-spectrum bacteriocin-like inhibitory substances by rumen strains suggests that antimicrobial peptides may play an important role in competition in the complex ruminal ecosystem.