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PubMed1993

Carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various gram-positive bacteria including Listeria sp.

This paper describes a simple purification method for the purification of carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola UI49. The protocol was also applicable for the isolation of nisin Z, which is a biopreservative produced by Lactococcus lactis SIK-83. The protocol consists of only two purification steps, XAD chromatography and cation exchange chromatography. It is quick, easy, and can be used for large scale purification of these lantibiotics. The bactericidal activity of carnocin UI49 against carnobacteria, lactococci and Listeria was compared with that of nisin Z. The carnobacteria showed similar sensitivity towards carnocin UI49 and nisin. The nisin producing L. lactis strains were very sensitive towards carnocin UI49, while the non-producing L. lactis strains were more sensitive to nisin. The Listeria strains were weakly sensitive to carnocin UI49, lower concentrations of nisin were needed to inhibit growth.

Animals↗
PubMed1995

Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes.

A review is presented on the present status of biological preservation of foods. Recent developments are discussed with respect to underlying mechanisms of inhibition by 'protective' cultures, and special reference is made to lactic acid bacteria (LAB) and their 'food-grade' safety. The role of bacteriocins, their limitations and potentiating role in biological systems, is also addressed. The use of enzymes (e.g. lysozyme) for food preservation is mainly restricted by economic factors, their inactivation by endogenous food components and their limited activity spectrum. Practical applications of protective cultures refer to particular food commodities that either constitute novel systems with respect to packaging and/or composition, or represent special hygienic risks. It is concluded that biological preservation cannot substitute GMP; it, however, offers an additional (and acceptable) processing parameter for improving the safety and assuring the quality of a given food.

Antibiosis↗
PubMed1994

Characterisation of lactic acid bacteria isolated from naturally fermented Greek dry salami.

A total of 348 lactic acid bacteria isolated from five batches of naturally fermented dry salami at various stages of ripening were characterised. The majority of the strains were assigned to two main phylogenetic groups of species: (i) the psychrotrophic, formerly called atypical, meat streptobacteria (169 strains) and (ii) a new genus Weissella (120), which was recently proposed (Collins et al., 1993) to include Leuconostoc paramesenteroides and some other closely related species. Meat streptobacteria were identified as Lactobacillus curvatus (88 strains) and L. sake (76), whereas 5 strains were indistinguishable and, thus designated L. sake/curvatus. Non-psychrotrophic streptobacteria were also isolated and identified as L. plantarum (34 strains), L. farciminis (10), L. coryniformis (1) and L. casei subsp. pseudoplantarum (1). The majority of the Weissella strains (86) were leuconostoc-like bacteria; four of them were identified as W. viridescens, 11 belonged to the newly described W. hellenica (Collins et al., 1993), another 11 resembled W. paramesenteroides, whereas 60 isolates were not classified to any species. The latter group comprised strains that produced D(L)-lactate. The remaining Weissella were gas-forming, arginine-positive rods assigned to W. minor (31) and W. halotolerans (3). Other species identified were Enterococcus faecium (10), Leuconostoc mesenteroides (1), L. brevis (1) and Pediococcus sp. (1). The main criteria used to distinguish between above species as well as their distribution on the five salami batches in relation to their succession with time and suitability as starters were discussed.

Animals↗
PubMed1994

Evidence that dissipation of proton motive force is a common mechanism of action for bacteriocins and other antimicrobial proteins.

While bacteriocins from lactic acid bacteria (LAB) have generated tremendous interest among food microbiologists, they are not unique. The biosphere is awash with antimicrobial proteins such as colicins, defensins, cecropins, and magainins. These proteins share many characteristics. They are low molecular weight, cationic, amphiphilic, tend to aggregate and are benign to the producing organism. In cases where the mode of action has been investigated, the cell membrane appears to be the site of action. There is increasing evidence that bacteriocins from many bacterial genera also share these characteristics. After a brief introduction on the significance of LAB bacteriocins, this review provides some background on proton motive force. Current studies of mechanisms for various bacteriocins are reviewed. Evidence is then introduced that bacteriocins produced by lactic acid bacteria act by the common mechanism of depleting proton motive force. The role and importance of energized membranes in this process is examined. These observations are linked to literature which demonstrates that many other classes of antimicrobial proteins act by the same mechanism. Questions regarding the role of receptor proteins and the physical mechanism by which PMF is depleted remain unresolved.

Amino Acid Sequence↗
PubMed1996

Influence of unsliced delicatessen meat freshness upon bacterial growth in subsequently prepared vacuum packed slices.

Unsliced beef pastrami, reformulated ham and bologna held at 6 degrees C were sliced 21, 17, 12 or 7 days before or at the assigned manufacturer's best before date and vacuum packaged. Packages of sliced meats were held at 6 degrees C for another 7, 12, 17 or 21 days, opened and analyses made for total bacteria, lactic acid bacteria, Enterobacteriaceae and Brochothrix thermosphacta. The maximum storage interval was 42 days; half this period unsliced, the remainder as repacked slices. Numbers of bacteria on pastrami were significantly greater than on ham and bologna (pastrami > ham > bologna) with the lactic acid bacteria dominating in all products. As unsliced meats approached their best before date, insignificant increases were generally noted for numbers of lactic bacteria, Enterobacteriaceae and B. thermosphacta. During subsequent storage of slices under vacuum, numbers of total and lactic bacteria increased exponentially at the same rate while B. thermosphacta growth was significantly slower. Numbers of Enterobacteriaceae remained low and were essentially unchanged during sliced meat storage. Within the context of study storage parameters, shelf-life appeared to be determined by length of time after slicing and repackaging rather than by best before date of the unsliced meat. Packages of sliced meat prepared from wholesale unsliced meats with 21 days left until their best before date or from unsliced meats with 12 days left until the best before date showed similar bacterial levels 21 days later. It was probable that the localization of bacterial growth at the meat surface-packaging film interface of the unsliced meats yielded slices with initially lowered bacterial content when repackaged and sampled from the uppermost slice. When Enterobacteriaceae and B. thermosphacta were absent from unsliced meats, extension of sliced meat package shelf-life beyond the best before date of the parent meat was possible. However, these bacterial groups were not always undetected.

Enterobacteriaceae↗
PubMed2005

Combined cold, acid, ethanol shocks in Oenococcus oeni: effects on membrane fluidity and cell viability.

The effects of combined cold, acid and ethanol on the membrane physical state and on the survival of Oenococcus oeni were investigated. Membrane fluidity was monitored on intact whole O. oeni cells subjected to single and combined cold, acid and ethanol shocks by using fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Results showed that cold shocks (14 and 8 degrees C) strongly rigidified plasma membrane but did not affect cell survival. In contrast, ethanol shocks (10-14% v/v) induced instantaneous membrane fluidisation followed by rigidification and resulted in low viability. Acid shocks (pH 4.0 and pH 3.0) exerted a rigidifying effect on membrane without affecting cell viability. Whatever the shock orders, combined cold (14 degrees C) and ethanol (14% v/v) shocks resulted in strong membrane rigidification. Interestingly, O. oeni survived combined cold and ethanol shocks more efficiently than single ethanol shock. Membrane rigidification was induced by ethanol-and-acid (10% v/v - pH 3.5) shock and correlated with total cell death. In contrast, O. oeni recovered its viability when subjected to cold (8 degrees C)-then-ethanol-and-acid shock which strongly rigidified the membrane. Our results suggested a positive short-term effect of combined cold, acid and ethanol shocks on membrane fluidity and viability of O. oeni.

Acids↗
PubMed2004

Identification and characterization of bacterial-binding property in the type III repeat domain of fibronectin.

To characterize fibronectin binding with Granulicatella adiacens, a causative agent of infective endocarditis, monoclonal antibodies were generated against human fibronectin and selected for their capacity to inhibit the fibronectin binding of the organism. Thermolysin and lysyl-endopeptidase digests of fibronectin were characterized by Western blot. The epitope of inhibitory monoclonal antibody was found in the central portion of fibronectin known as the cell-binding domain, and not in the N-terminal portion known to be the binding region of most microbial species, e.g., Staphylococcus aureus and Streptococcus pyogenes. While these two species could bind to both the N-terminal and central portion, Escherichia coli and G. adiacens bind only to the latter. Excess amounts of free fibronectin in the solution inhibited the bacterial adherence to the N-terminal fibronectin fragment, but not to the central region, thereby suggesting the central region plays a significant role for in vivo bacterial colonization in the presence of high concentrations of soluble fibronectin.

Animals↗
PubMed2005

Effects of integrated use of organic and inorganic nutrient sources with effective microorganisms (EM) on seed cotton yield in Pakistan.

A field experiment was conducted to determine the effects of integrated use of organic and inorganic nutrient sources with effective microorganisms on growth and yield of cotton. Treatments included: control; organic materials (OM); effective microorganisms (EM); OM+EM; mineral NPK (170:85:60 kg); 1/2 mineral NPK+EM; 1/2 mineral NPK+OM+EM and mineral NPK+OM+EM. OM and EM alone did not increase the yield and yield attributing components significantly but integrated use of both resulted in a 44% increase over control. Application of NPK in combination with OM and EM resulted in the highest seed cotton yield (2470 kg ha-1). Integrated use of OM+EM with 1/2 mineral NPK yielded 2091 kg ha-1, similar to the yield (2165 kg ha-1) obtained from full recommended NPK, indicating that this combination can substitute for 85 kg N ha-1. Combination of both N sources with EM also increased the concentrations of NPK in plants. Economic analysis suggested the use of 1/2 mineral NPK with EM+OM saves the mineral N fertilizer by almost 50% compared to a system with only mineral NPK application. This study indicated that application of EM increased the efficiency of both organic and mineral nutrient sources but alone was ineffective in increasing yield.

Agriculture↗
PubMed2006

Isolation of lactic acid bacteria from Malaysian foods and assessment of the isolates for industrial potential.

Two traditional fermented food 'tapai' (fermented tapioca) and 'tempoyak' (fermented durian flesh), chilli puree and fresh goat's milk were used as sources for the isolation of lactic acid bacteria (LAB). A total of 126 isolates were obtained and by sequential screening for catalase activity and Gram-staining, 55 were determined to be LAB out of which 16 were established to be homofermentative by the gel plug test. Seven isolates were identified by use of the API 50CHL kit and two lactobacilli strains and one lactococci strain were selected to study their growth and lactic acid production profiles in a time course experiment. The lactobacilli strains, both isolated from 'tapai', produced higher amounts of cells and lactic acid from glucose as compared to the lactococci strain isolated from fresh goat's milk.

Animals↗
PubMed2004

Prebiotic effects of chicory inulin in the simulator of the human intestinal microbial ecosystem.

The prebiotic potential of native chicory inulin was assessed in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) by monitoring microbial community from the colon compartments, its metabolic activity and community structure. Inulin addition selected for a higher short chain fatty acid production with shifts towards propionic and butyric acid. Conventional culture-based techniques and PCR-denaturing gradient gel electrophoresis analysis showed no remarkable changes in the overall microbial community from the colon compartments of the SHIME, whereas selective effects were seen for lactic acid bacteria. Quantitative PCR with bifidobacteria-specific primers revealed a significant increase with more than 1 log CFU ml(-1) from the proximal to distal colon, in contrast to culture-based techniques, which only showed a minor bifidogenic effect in the ascending colon. Our results indicate that inulin purports prebiotic effects from the proximal to distal colon and that real-time PCR is a more precise technique to detect differences in bifidobacterial populations whereas conventional culturing techniques are much more variable.

Bifidobacterium↗
PubMed2004

Purification, characterization and amino acid sequencing of divergicin M35: a novel class IIa bacteriocin produced by Carnobacterium divergens M35.

Carnobacterium divergens M35, isolated from a commercial sample of frozen smoked mussels, produces a new bacteriocin, divergicin M35, a class IIa bacteriocin. Divergicin M35 is sensitive to pronase-E, alpha-chymotrypsin and proteinase K, but not to trypsin and withstands thermal treatments up to 121 degrees C for 30 min. Divergicin M35 was extracted from the culture supernatant of C. divergens M35 using an SP-Sepharose cation-exchange column, desalted and purified on a C18 Sep-Pack column and further purified by reverse phase-high pressure liquid chromatography. This procedure allowed the recovery of 10% of the bacteriocin present in the culture supernatant with purity higher than 99%. Divergicin M35 had a molecular mass of 4518.75 Da as determined by mass spectrometry, a pI value of 8.3 and positive net charge (+3). The amino acid sequence of divergicin M35 was found to consist of 43 amino acid with four cysteine residues (Cys10, 15, 25, 43) and showed 80.5% homology with divercin V41 (80.5%) and 80.0% with bavaricin MN. Divergicin M35 showed powerful antilisterial activity, especially against Listeria monocytogenes and was also active against carnobacteria but not against strains of Lactococcus, Lactobacillus, Enterococcus, Bifidobacteria and Escherichia. Divergicin M35 production began in late exponential phase and reached a maximum activity of 65,000 AU/ml in early stationary phase. Initial broth pH, Tween 80 and acetate did not affect C. divergens M35 growth or divergicin production. This bacteriocin may be a potential tool for inhibiting L. monocytogenes in seafood products that do not usually undergo an adequate heat treatment.

Amino Acid Sequence↗
PubMed2005

Analysis of kimchi microflora using denaturing gradient gel electrophoresis.

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to determine the microfloral composition during the fermentation of kimchi, a traditional Korean fermented vegetable food. The kimchi was fermented at 10 degrees C or 20 degrees C for 30 or 20 days, respectively. DGGE of the partially amplified 16S rDNA was performed and the most intense bands sequenced. The application of this culture-independent molecular technique determined that the lactic acid bacteria Weissella confusa, Leuconostoc citreum, Lactobacillus sakei, and Lactobacillus curvatus were the main microorganisms responsible for kimchi fermentation.

Brassica↗
PubMed2005

Effect of inoculation of Carnobacterium divergens V41, a bio-preservative strain against Listeria monocytogenes risk, on the microbiological, chemical and sensory quality of cold-smoked salmon.

The aim of this study was to develop a bio-preservation strategy for cold-smoked salmon (CSS) by the use of lactic acid bacteria previously selected for their capability to inhibit the growth of Listeria monocytogenes in the product. The spoiling potential of three Carnobacterium strains (Carnobacterium divergens V41, Carnobacterium piscicola V1 and SF668) was tested in sterile CSS blocks inoculated by 10(4-5) CFU g(-)(1) and stored under vacuum for 9 days at 4 degrees C followed by 19 days at 8 degrees C. C. divergens V41 grew a little faster than the other strains and none of the three carnobacteria showed any adverse effect on quality of the product, i.e. no off-odour detected by a trained panel, no total volatile basic nitrogen (TVBN) production, no acidification and no biogenic amine except a slight production of tyramine. An application on commercial CSS was tested by spraying C. divergens V41 (10(4-5) CFU g(-1)) on slices of four batches freshly processed in different smoke-houses. Microbial, chemical and sensory characteristics were weekly compared to a control during 4 weeks of vacuum storage. When the natural microflora was initially weak (two batches<20 CFU g(-1)), C. divergens V41 quickly reached 10(7-8) CFU g(-1) and a slight inhibition of endogenous Enterobacteriaceae, lactobacilli and yeasts was observed. The presence of C. divergens V41 was slightly detected (odour and flavour) but none of the sample was considered as spoiled by the sensory panel. When the natural microflora was initially high (2 batches>10(4-5) CFU g(-1)), no effect on the microflora, TVBN and biogenic amine production, nor on the sensory characteristics was observed in presence of C. divergens V41. In conclusion, bio-preservation of CSS using lactic acid bacteria such as C. divergens V41 is a promising way to inhibit the growth of pathogenic bacteria such as L. monocytogenes with low effect on the quality of the product.

Animals↗
PubMed2004

Evaluation of antiplaque activity of Azadirachta indica leaf extract gel--a 6-week clinical study.

Various chemical agents have been evaluated over the years with respect to their antimicrobial effects in the oral cavity; however, all are associated with side effects that prohibit regular long-term use. Therefore, the effectiveness of neem (Azadirachta indica A. Juss) leaf extract against plaque formation was assessed in males between the age group of 20-30 years over a period of 6 weeks. Present study includes formulation of mucoadhesive dental gel containing Azadirachta indica leaf extract (25 mg/g). A 6-week clinical study was conducted to evaluate the efficacy of neem extract dental gel with commercially available chlorhexidine gluconate (0.2% w/v) mouthwash as positive control. Microbial evaluation of Streptococcus mutans and Lactobacilli species was carried out to determine the total decrease in the salivary bacterial count over a period of treatment using a semi-quantitative four quadrant streaking method. The results of the study suggested that the dental gel containing neem extract has significantly (P<0.05) reduced the plaque index and bacterial count than that of the control group.

Adult↗
PubMed2004

Rapid screening method for the detection of antimicrobial substances.

Bioluminescence is phenomenon where living organisms produce light and this production is directly dependent on metabolic activity of the organism. Genes encoding enzymes, luciferases, responsible for light production can be cloned into indicator strains, thus allowing sensitive detection of antimicrobial activity. This study utilized bacterial luciferase genes cloned into Staphylococcus aureus, Escherichia coli and Salmonella enterica serovar Typhimurium indicator strains and showed that the detection of antimicrobial activity can be obtained already in 2 h without laborious plate counting and overnight incubation. Indicator strains used in the study harboured luxAB genes responsible of producing light as well as luxCDE genes for synthesis of long-chain fatty aldehyde as substrate for light production. As a consequence, no exogenous aldehyde addition was needed allowing stable light production. Furthermore, the method was used for the detection of antimicrobial activity from lactic acid bacteria after the effect of organic acids was eliminated.

Anti-Bacterial Agents↗
PubMed2006

Use of unsupervised and supervised artificial neural networks for the identification of lactic acid bacteria on the basis of SDS-PAGE patterns of whole cell proteins.

Conventional multivariate statistical techniques (hierarchical cluster analysis, linear discriminant analysis) and unsupervised (Kohonen Self Organizing Map) and supervised (Bayesian network) artificial neural networks were compared for as tools for the classification and identification of 352 SDS-PAGE patterns of whole cell proteins of lactic acid bacteria belonging to 22 species of the genera Lactobacillus, Leuconostoc, Enterococcus, Lactococcus and Streptococcus including 47 reference strains. Electrophoretic data were pre-treated using the logistic weighting function described by Piraino et al. [Piraino, P., Ricciardi, A., Lanorte, M. T., Malkhazova, I., Parente, E., 2002. A new procedure for data reduction in electrophoretic fingerprints of whole-cell proteins. Biotechnol. Lett. 24, 1477-1482]. Hierarchical cluster analysis provided a satisfactory classification of the patterns but was unable to discriminate some species (Leuconostoc, Lb. sakei/Lb. curvatus, Lb. acidophilus/Lb. helveticus, Lb. plantarum/Lb. paraplantarum, Lc. lactis/Lc. raffinolactis). A 7x7 Kohonen self-organizing map (KSOM), trained with the patterns of the reference strains, provided a satisfactory classification of the patterns and was able to discriminate more species than hierarchical cluster analysis. The map was used in predictive mode to identify unknown strains and provided results which in 85.5% of cases matched the classification obtained by hierarchical cluster analysis. Two supervised tools, linear discriminant analysis and a 23:5:2 Bayesian network were proven to be highly effective in the discrimination of SDS-PAGE patterns of Lc. lactis from those of other species. We conclude that data reduction by logistic weighting coupled to traditional multivariate statistical analysis or artificial neural networks provide an effective tool for the classification and identification of lactic acid bacteria on the basis of SDS-PAGE patterns of whole cell proteins.

Bacterial Proteins↗
PubMed2005

Influence of ethanol and low pH on arginine and citrulline metabolism in lactic acid bacteria from wine.

The aim of this work was to study the effects of ethanol on cell growth and arginine and citrulline metabolism in two heterofermentative lactic acid bacteria from wine, and to determine their possible association with the formation of ethyl carbamate (EC), a carcinogenic compound. Lactobacillus hilgardii X1B is able to utilize arginine and citrulline, while Oenococcus oeni m can only use citrulline, a precursor of EC. Growth of both microorganisms was partially inhibited by 10 and 15% (v/v) ethanol. Specific arginine consumption by L. hilgardii increased when the pH value diminished from 6.5 to 3.8, but was not affected by an increasing ethanol concentration. However, the ethanol concentration affected the specific citrulline consumption of both microorganisms. Arginine metabolism by L. hilgardii X1B increased the amount of citrulline, thus allowing production of EC in the medium. Citrulline utilization by both microorganisms, at all pH values studied, indirectly inhibited the formation of EC; indeed, one of the precursors had practically disappeared after 48 h of incubation. Due to its ability to form precursors, L. hilgardii X1B has the potential to contribute to EC formation, whereas citrulline utilization by O. oeni m in the presence of ethanol may contribute to diminishing the formation of EC. Rapid degradation of citrulline in the presence of ethanol by O. oeni m is important from a toxicological point of view, because it is important to keep the EC levels as low as possible.

Arginine↗
PubMed2005

Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l.).

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod (Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.

Animals↗