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PubMed1977

A microbiologic disc assay for 5-methyltetrahydrofolate in the presence of methotrexate.

High-dose methotrexate-citrovorum factor rescue regimens are used extensively for the treatment of human malignancies. Since the rescue agent citrovorum factor is rapidly converted to 5-methyltetrahydrofolate, it is desirable to follow the distribution kinetics of both of these coenzymes to assess the selective therapeutic effectiveness of the regimen. The assay methods using methotrexate-resistant strains of Lactobacillus casei ATCC 7469 (LC/A), Streptococcus faecium var. durans ATCC 8043 (SF/A), and Pediococcus cerevisiae ATCC 8081 (PC/A) have been developed to determine the concentrations of 5-methyltetrahydrofolate and other folates in the presence of methotrexate. Since LC/A determines the total folates, SF/A deterimines the total folates except 5-methyltetrahydrofolate, and PC/A by our procedure determines only the most stable form of reduced folates, citrovorum factor, the quantitation of both 5-methyltetrahydrofolate and citrovorum factor can very easily be achieved by differential assays. This report describes the determination of as low as 10 ng/ml of 5-methyltetrahydrofolate in the presence of as high as 500 microgram/ml of methotrexate. The assay method described here is currently used in studying the pharmacokinetics of 5-methyltetrahydrofolate in patients with meningeal neoplasms.

Biological Assay↗
PubMed1995

Biodiversity of lactic acid bacteria from food-related ecosystems.

The diversity of lactic acid bacteria within a variety of food-related ecosystems was assessed and the strain as well as environment specific characteristics were investigated. The strains (108 in toto) were isolated from plant material, traditional fermented foods, dried marine algae, sea food, fungi as well as spoiled foods and beverages, and all were characterized to genus level. Lactobacillus strains dominated all ecosystems and contributed to 65% of the isolates while a further 13% consisted of Lactococcus strains, 12% of Leuconostoc strains, 5% of Pediococcus strains, and 5% of unidentified strains. Plant material was the most diverse ecosystem containing representatives from each of the genera as well as the majority of dextran and tyramine producers. Those strains able to grow at 45 degrees C as well as acid tolerant strains were predominantly isolated from traditional fermented beverages while halotolerant strains occurred mainly in sea food. Two bacteriocin producers but no histamine producers were isolated.

Bacteria↗
PubMed1993

[Utilization of lactic bacteria in the control of pathogenic microorganisms in food].

The lactic acid bacteria have the potential to inhibit the growth of pathogenic and spoilage bacteria and the possibility exists of using them to improve the hygienic quality and to extend the shelf-life of different foods. Among the many inhibitory substances produced by the lactic acid bacteria, the bacteriocins are of particular interest. It has been the objective of this work to review the bacteriocins produced by lactic acid bacteria from the genera Lactococcus, Lactobacillus and Pediococcus, as well as Leuconostoc and Carnobacterium to understand their relevant biochemical, immunological and genetic characteristics. The lactic acid bacteria may also express foreign genes codifying metabolites with antimicrobial activities against foodborne pathogens of interest, and this will also permit hypothesize about theoretical and experimental models of microbial antagonism mediated by the lactic acid bacteria.

Bacteriocins↗
PubMed1993

Role of flavin in acetoin production by two bacterial pyruvate oxidases.

Escherichia coli pyruvate oxidase (POXEC) requires FAD both for the oxidative decarboxylation of pyruvate to acetate and CO2 and for the formation of acetoin from pyruvate and acetaldehyde. Prior work has shown that the catalytic activity (kcat/Km) for POXEC in the oxidative reaction is stimulated approximately 450-fold by amphiphilic activators. This paper shows that the acetoin reaction does not respond to activation. The FAD requirement for acetoin formation can be replaced by 5-deaza-FAD and 6-hydroxy-FAD, FAD analogs which form kinetically stable oxidized and reduced enzyme species, respectively. As would be expected, the 5-deaza- and 6-hydroxy-FAD enzymes are not active in the oxidative reaction. A second flavin pyruvate oxidase from Pediococcus pseudomonas (POXPP), which catalyzes the oxidative decarboxylation of pyruvate to CO2 and acetyl phosphate, also requires FAD for acetoin formation. POXPP has an oxidative rate comparable to that of POXEC, but in comparison to POXEC, POXPP catalyzes acetoin formation at a much reduced rate. Again, as was found with the POXEC, an FAD analog incapable of undergoing facile oxidation-reduction reactions also could replace the FAD requirement in the POXPP acetoin reaction. The results indicate that the role for FAD in acetoin formation with both enzymes is based on a structural requirement and that FAD does not participate in a redox function in the acetoin reaction.

Acetoin↗
PubMed1995

The lysins of bacteriophages infecting lactic acid bacteria.

This short review highlights the complete absence of literature on lysins of bacteriophages infecting species like S. salivarius subsp. thermophilus, Pediococcus and Leuconostoc species, L. helveticus, L. acidophilus, L. plantarum and L. brevis, which are also widely used in the dairy industry. The lysins described share some similar biochemical characteristics: optimal pH and temperature, site of hydrolysis inside the peptidoglycan, and some activators and inhibitors. The cloning of the genes encoding these lysins only began in the last few years and four of them have been completely sequenced. In the future, these lysin genes could be interestingly compared to the host autolysin(s) gene(s). By contrast, the passage of phage lysins through the cytoplasmic membrane of the host cell in order to reach the peptidoglycan (via a signal sequence or the presence of a holin) seems not to be clearly resolved. The presence of a second open-reading frame upstream from the gene of the lysin, enabling a putative holin to be encoded, has already been suggested. No doubt our ever increasing knowledge about bacteriophage genome organization will help to elucidate this question. Meanwhile the obtention of a Lactococcus strain with an autolytic phenotype, using a bacteriophage lysin gene, as well as the successful use of purified PL1 lysin to obtain protoplasts of L. casei encourage us to continue to explore the field of bacteriophage lysins.

Amino Acid Sequence↗
PubMed1988

Numerical taxonomy of psychrotrophic lactic acid bacteria from prepacked meat and meat products.

Ninety-four strains of lactic acid bacteria isolated from refrigerated, prepacked meat and meat products were together with 59 reference strains of Brochothrix, Lactobacillus, Leuconostoc, Pediococcus and Streptococcus phenotypically classified, using 96 unit characters. Data were examined using Simple Matching (SSM) or Jaccard coefficient (SJ), and unweighted pair group algorithm with arithmetic averages. Twenty-three clusters with two or more members were defined at the 84% SSM-similarity level which corresponded to the SJ-similarity level of 61%. Based on SSM, most field strains were included in nine clusters, and with three unsignificant exceptions these contained no reference strains. The field clusters were designated Carnobacterium piscicola (cluster 1; 5% of field isolates), Carnobacterium divergens (cluster 2; 9% of field isolates), Leuconostoc (cluster 9; 18% of field isolates) and Lactobacillus (cluster 4, 10, 11, 12, 13 and 14; together 60% of field isolates). The Lactobacillus clusters had many features in common with cluster II of Shaw & Harding (1984). Phenotypical characteristics of major clusters are given. The SSM and SJ based classifications basically coincided for the field strains; the exception was cluster 4 which now were split in two parts. Fourteen clusters were made up of mainly reference strains (SSM). Most of them included more than one type strain on species level; exceptions were Brochothrix thermosphacta (cluster 3), Lactobacillus salivarius (cluster 17) and Leuconostoc mesenteroides (cluster 18). Several rearrangements were seen amongst the clusters of the reference strains when SJ, instead of SSM, was used for clustering.

Food Microbiology↗
PubMed1976

[On the metabolism of amino acids by lactic acid bacteria isolated from wine (author's transl)].

The changes in the concentrations of amino acids in the culture medium of lactic acid bacteria were determined by ion exchange chromatography after growth of 105 strains that were mainly isolated from wine. After growth of lactic acid bacteria a small but not significant decrease in the concentration of most amino acids is observed. There is apparently no difference between essential or not essential amino acids. Certain bacterial strains decompose the amino acids arginine, glutamic acid, histidine, and tyrosine completely. Other amino acids (tryptophane, aspartic acid, threonine, isoleucine, and phenylalanine) are only partially metabolized. Among 28 strains of Pediococcus cerevisiae only one strain was able to decarboxylate histidine to histamine. This was the only strain found to have this ability. Several strains of Lactobacillus brevis were able to form 4-aminobutyric acid and ornithine from glutamic acid and arginine respectively.

Amino Acids↗
PubMed1995

The formation of biogenic amines by fermentation organisms.

A total of 523 strains representing 35 species related to food fermentation organisms of practical importance were investigated for their potential for formation of biogenic amines (BA). The investigation was performed with resting cells in phosphate buffer (pH 5.5) and the formation of the following BAs was followed: putrescine, cadaverine, histamine, tyramine and 2-phenylethylamine. No potential was observed in species of lactococcus, Leuconostoc, Pediococcus, Streptococcus and several Lactobacillus spp., such as L. Pentosus and L. sake. A remarkable potential to form BA was observed in strains of carnobacteria, Lactobacillus buchneri, L. curvatus, L. reuteri, Staphylococcus carnosus and, to a lesser extent, in L. alimentarius, L. brevis, L. bavaricus, L. delbrueckii ssp. lactis, Micrococcus spp. and S. piscifermentans. In well known species with a practical function in the fermentation of dairy products, wine or cabbage a potential was observed for few strains only. In view of their role as starters in food fermentation, or their potential use in protective cultures and as probiotics, BA formation by the organisms has to be taken into consideration by selecting appropriate strains.

Bacteria↗
PubMed1995

Influences on the antimicrobial activity of surface-adsorbed nisin.

The efficacy of the antimicrobial peptide nisin was examined after adsorption to silica surfaces. Three protocols were used to evaluate nisin's activity against adhered cells of Listeria monocytogenes: bioassay using Pediococcus pentosaceous FBB 61-2 as the sensitive indicator strain; visualization and enumeration of cells by microscopic image analysis; and viability of adhered cells as determined by lodonitrotetrazolium violet uptake and crystallization. The activity of adsorbed nisin was highly dependent upon conditions of adsorption. The highest antimicrobial activity of adsorbed nisin occurred with high concentrations of nisin (1.0 mg ml-1) and brief contact times (1 h) on surfaces of low hydrophobicity. Sequential adsorption of a second protein (beta-lactoglobulin or bovine serum albumin) onto surfaces consistently resulted in decreased nisin activity. These data provide direction for the development of applications to limit microbial attachment on food contact surfaces through the use of adsorbed antimicrobial peptides.

Adsorption↗
PubMed1993

Partial characterization of an rpoD-like gene of Lactococcus lactis subsp. lactis ML3 with a polymerase chain reaction-based approach.

With degenerated oligonucleotide primers for conserved regions of bacterial sigma factor proteins, a 117-bp internal DNA fragment of an rpoD-like gene of Lactococcus lactis subsp. lactis ML3 was amplified by the polymerase chain reaction (PCR). The DNA sequence of this PCR product was determined by cycle sequencing, and the deduced amino acid sequence of this internal fragment showed an extensive homology with the known sigma factor sequences from six other microorganisms and present a 13-amino acid region corresponding to the typical "RpoD box" of primary sigma factors. This PCR product was used as a probe to specifically detect sigma homologs in Pediococcus acidilactici, Leuconostoc lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Enterococcus faecalis, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris. These data are consistent with the existence of a high similarity between the primary sigma factors from diverse Gram-positive microorganisms.

Amino Acid Sequence↗
PubMed1993

Alcohol production in submerged cashew pomace.

The variations in the total titratable acidity, hydrogen ion concentration, reducing sugar, soluble solids and alcohol contents of the supernatant of the submerged cashew pomace in a non-aseptic condition were investigated. The crude fibre content of the pomace was also determined during fermentation. At intervals, the changes in microbial load in both the pomace and its supernatant were examined during the 14-day study. Fifty four percent of the total soluble solids (TSS) was utilised within 7 days of degradation while an increase of about 91% in the total titratable acidity occurred in the cashew pomace within the same period bringing its pH to 3.24. Acid hydrolysis of the pomace accounted for the relative high value of 2.15% TSS on the 8th day. Isolates of the genera Leuconostoc, Lactobacillus, Pediococcus, Aspergillus, Rhizopus and some yeast strains were obtained from the fresh pomace. The fungal colonies constituted about 76% of the population in the cashew pomace. The 86% drop in microbial population of the 8th day biodegraded pomace could be attributed to a decrease in nutrients of the substrate and the inhibitory effect of the organic acids produced during fermentation.

Alcohols↗
PubMed2003

Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of gram-positive bacteria.

Lactobacillus plantarum NC8 was shown to produce plantaricin NC8 (PLNC8), a recently purified and genetically characterized inducible class IIb bacteriocin, only when it was co-cultured with other gram-positive bacteria. Among 82 strains belonging to the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Staphylococcus, and Streptococcus, 41 were shown to induce PLNC8 production in L. plantarum NC8. There was apparently no relationship between the sensitivity of the strains and their ability to induce the bacteriocin, indicating that the inducer and sensitive phenotypes may not be linked. In some instances, induction was promoted by both living and heat-killed cells of the inducing bacteria. However, no PLNC8-inducing activity was found in the respective cell-free, pure culture supernatants. Inducer strains also promoted the production of a PLNC8-autoinducing activity by L. plantarum NC8, which was found only in the cell-free co-culture supernatants showing inhibitory activity. This PLNC8-autoinducing activity was diffusible, heat resistant, and of a proteinaceous nature, and was different from the bacteriocin itself. Taken together, the results suggest that the presence of specific gram-positive bacteria acts as an environmental stimulus activating both PLNC8 production by L. plantarum NC8 and a PLNC8-autoinducing activity, which in turn triggers or maintains bacteriocin production in the absence of inducing cells.

Antibiosis↗
PubMed2006

Modification of the data-processing method for the turbidimetric bioassay of nisin.

The data processing method of the turbidimetric bioassay of nisin was modified to facilitate its industrial application. The influence of the initial indicator concentration was minimized by a redefined specific dose of the bacteriocin as the quotient between the titer of the added bacteriocin and the initial population density of the indicator in the suspension. It was found that dc = 0.125 microg ml(-1) was the critical dose of nisin that can cause a complete inhibition of the indicator, Pediococcus acidilactici UL5, with an initial OD of 0.135. To eliminate the interference of the cell debris, an equation, epsilonI = ODi(1-di)/OD0 = epsilonA(1-di/dc), exploiting dc, was formulated to obtain the intrinsic survival proportion. The use of the specific dose of the bacteriocin and the intrinsic survival proportion as parameters of the dose/response curve greatly enhanced its repeatability and feasibility. A dual-dosage approach was developed to further simplify the conventional standard dose/response curve method.

Anti-Bacterial Agents↗
PubMed2001

Flocculation and coflocculation of bacteria by yeasts.

Biotransformations in natural environments frequently involve interactions between microorganisms. Although there are many reports on the interactions between bacteria, interactions between yeasts and bacteria have not been extensively studied. Previously we reported on the flocculation and coflocculation of Pediococcus damnosus by Saccharomyces cerevisiae. Now we report that several other yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, and Schizosaccharomyces pombe, induce flocculation with several industrially or medically relevant bacteria, including Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Candida utilis was one of the best flocculation inducers. The results are discussed with respect to interactions between yeasts and bacteria and their applications in industry and medicine.

Bacillus subtilis↗
PubMed1996

The effect of cations on the hydrolysis of lactose and the transferase reactions catalysed by beta-galactosidase from six strains of lactic acid bacteria.

beta-Galactosidases from Lactobacillus delbruekii subsp. bulgaricus 20056, Lb. casei 20094, Lactococcus lactis subsp. lactis 7962, Streptococcus thermophilus TS2, Pediococcus pentosaceus PE39 and Bifidobacterium bifidum 1901 were partially purified. The rate of hydrolysis of lactose given by the predominant beta-galactosidase activity from each of the bacteria studied was in all cases enhanced by Mg2+, while the effect of K+ and Na+ differed from strain to strain. The beta-galactosidases from all strains also catalysed trans-galactosylation reactions. The types of oligosaccharides produced appeared to be very similar in each case, but the rates of their production differed. All the beta-galactosidases were also capable of hydrolysing galactosyl-lactose although, unlike the other bacteria studied, Lb. delbruekii subsp. bulgaricus 20056 and Lc. lactis subsp. lactis 7962 were unable to utilise galactosyl-lactose as a carbon source for growth.

Cations↗
PubMed1998

Construction of a food-grade multiple-copy integration system for Lactococcus lactis.

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

Blotting, Southern↗
PubMed2006

Benefits of a synbiotic formula (Synbiotic 2000Forte) in critically Ill trauma patients: early results of a randomized controlled trial.

BACKGROUND: Since probiotics are considered to exert beneficial health effects by enhancing the host's immune response, we investigated the benefits of a synbiotics treatment on the rate of infections, systemic inflammatory response syndrome (SIRS), severe sepsis, and mortality in critically ill, mechanically ventilated, multiple trauma patients. Length of stay in the intensive care unit (ICU) and number of days under mechanical ventilation were also evaluated. METHOD: Sixty-five patients were randomized to receive once daily for 15 days a synbiotic formula (Synbiotic 2000Forte, Medipharm, Sweden) or maltodextrin as placebo. The synbiotic preparation consisted of a combination of four probiotics (10(11) CFU each): Pediococcus pentosaceus 5-33:3, Leuconostoc mesenteroides 32-77:1, L. paracasei ssp. paracasei 19; and L. plantarum 2,362; and inulin, oat bran, pectin, and resistant starch as prebiotics. Infections, septic complications, mortality, days under ventilatory support, and days of stay in ICU were recorded. RESULTS: Synbiotic-treated patients exhibited a significantly reduced rate of infections (P = 0.01), SIRS, severe sepsis (P = 0.02), and mortality. Days of stay in the ICU (P = 0.01) and days under mechanical ventilation were also significantly reduced in relation to placebo (P = 0.001). CONCLUSION: The administration of this synbiotic formula in critically ill, mechanically ventilated, multiple trauma patients seems to exert beneficial effects in respect to infection and sepsis rates and to improve the patient's response, thus reducing the duration of ventilatory support and intensive care treatment.

Critical Illness↗
PubMed2002

Cloning of the L-lactate dehydrogenase gene from the ruminal bacterium Selenomonas ruminantium HD4.

A clone from a Selenomonas ruminantium HD4 Lambda ZAP II genomic library was isolated by its ability to complement the anaerobic growth deficiency of an Escherichia coli (pfl, ldh) double mutant. The 1.0-kb insert from the clone was sequenced and revealed a single open reading frame (ORF, 957-bp) which was preceded by a putative Shine-Dalgarno (SD) sequence (AGGGGG). The potential SD sequence corresponded to 3' 16S rRNA sequences of various Selenomonas strains. The ORF was predicted to encode a protein of 318 amino acids with a calculated molecular mass of 34,975 Da and an isoelectric point of 5.54. In addition, the ORF contained 51 mol % G + C and this is consistent with the average G + C content (54%) of the S. ruminantium chromosome. The cloned S. ruminantium gene exhibited 59% nucleotide identity and 61% deduced amino acid similarity with L-lactate dehydrogenases (L-LDH) of Pediococcus acidilactici and Bacillus megaterium, respectively. Incorporation of the cloned S. ruminantium gene into E. coli DC1368 (pfl, ldh) restored anaerobic growth on glucose and L-LDH activity was detected in cell extracts. Because lactate accumulation within the rumen can be detrimental to animal performance, characterizing the gene(s) involved in lactate production by predominant ruminal bacteria will lead to a better understanding of lactate metabolism within the rumen.

Amino Acid Sequence↗