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PubMed2002

The effect of temperature on the growth and persistence of Salmonella in fermented liquid pig feed.

Two studies were conducted to investigate the effect of temperature on the fate of Salmonella enterica subsp. enterica serovar typhimurium DT104:30 in fermented liquid pig feed. These were (1) by co-inoculation of feed with S. typhimurium DT104:30 and Pediococcus pentosaceus, as the fermenting organism, and (2) by fermenting feed for 48, 72 or 96 h prior to inoculation with S. typhimurium DT104:30. In co-inoculated feed incubated at 20 degrees C, S. typhimurium DT104:30 persisted for at least 72 h. In contrast, in feed incubated at 30 degrees C, no S. typhimurium DT104:30 were detectable 48 h after inoculation. In prefermented feed, S. typhimurium DT104:30 died four to five times faster in feed maintained at 30 degrees C (D(value) 34-45 min) compared with feed maintained at 20 degrees C (D(value) 137-250 min). This was not entirely due to differences in lactic acid concentration as feed fermented for 72 or 96 h at 20 degrees C and feed fermented for 48 h at 30 degrees C contained similar concentrations of lactic acid (160-170 mM). Low numbers of S. typhimurium DT104:30 were still detectable in fermented feed 24 h after inoculation at 20 degrees C. In contrast, none were detectable 6-7 h after inoculation at 30 degrees C. The results of these studies indicate that it would be advisable for pig producers to control the temperature of liquid feed tanks to reduce the risk of Salmonella contamination.

Animal Feed↗
PubMed2003

Proteolytic activities in togwa, a Tanzanian fermented food.

Proteolytic activities were investigated in sorghum-based togwa prepared by natural fermentation and using starter cultures previously isolated from the native product, i.e., Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus fermentum, Lactobacillus plantarum, Pediococcus pentosaceus, and Issatchenkia orientalis in coculture with either L. brevis or L. plantarum. Both proteinase and aminopeptidase activities were substantially higher in naturally fermented togwa than in those with starters (14-30%, 12-70%, respectively). A variable but substantial part of the proteinase activity followed the particulate fraction of togwa; aminopeptidase activity was mainly in that fraction. The breakdown of relatively high molecular mass protein (64 kDa) in togwa was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); the products were mainly in the 14-30 kDa range. Reversed-phase fast-protein liquid chromatography (RP-FPLC)-protein/peptide patterns changed during fermentation with some variation between togwa of different cultures. Supplementation of gruel with malt increased the concentration of total protein [from 9.5% to 11.0% (w/w) on dry weight basis)] and of most of the free amino acids. Fermentation had no effect on total protein content; however, the concentration of most of the amino acids was reduced, except for the proline content that increased. Natural fermentation also increased the concentration of glutamic acid and ornithine. Fermentation by P. pentosaceus increased aspartic acid, while L. cellobiosus, L. fermentum, and L. brevis in coculture with I. orientalis increased the concentration of citrulline.

Aminopeptidases↗
PubMed2004

Observations on the succession dynamics of lactic acid bacteria populations in chill-stored vacuum-packaged beef.

Drip samples were collected at 4-week intervals from 10 vacuum-packaged beef striploins stored for 16 weeks at -1.5 degrees C and assayed for populations of lactic-acid bacteria (LAB), pH and spoilage-causing fermentation products. A total of 15 LAB species were identified using pulsed-field gel electrophoresis and biochemical analysis. A pattern of succession was observed during storage between strains of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. Acetic acid production was associated with increasing LAB populations generally and butyric acid production was associated with the development of a particular strain of Leuconostoc. Changes in pH is postulated as a driver of succession.

Animals↗
PubMed2004

A rapid turbidometric microplate bioassay for accurate quantification of lactic acid bacteria bacteriocins.

A 1 day turbidometric microplate bioassay (TMB) was developed for the rapid, accurate and precise quantification of lactic acid bacteria (LAB) bacteriocins (nisin Z and pediocin PA-1). Parameters such as the concentration of the indicator strains and the incubation time were optimized for each bacteriocin. A high correlation coefficient (r(2)=0.992+/-0.004) was obtained for the exponential regression in the nisin Z concentration range of 20-120 ng/ml with 1 x 10(7) CFU indicator strain (Pediococcus acidilactici UL5) and an incubation time of 3 h. Using these parameters, the detection limit was estimated at 80 ng/ml (3.2 IU/ml), compared to 300 ng/ml for the agar diffusion assay (ADA). High precision (<7%) and accuracy (10%) were obtained for all nisin Z concentrations tested. Similar results were obtained with pediocin PA-1 with r(2)=0.993+/-0.005, a precision (8.2%) and an accuracy lower than 15%.

Anti-Bacterial Agents↗
PubMed1998

Comparative study of lactic acid bacteria house flora isolated in different varieties of 'chorizo'.

A total of 516 strains of lactic acid bacteria isolated from 'chorizo' (a Spanish dry fermented sausage) were identified. The 'chorizo' was from three zones of Castilla and León in Spain: Burgos, Segovia and Salamanca. Two factories were chosen in each zone and the samples were taken at three stages of ripening. L. sake was the most predominant species present (68.8%) followed by L. curvatus (16.47%) and Pediococcus sp. (8.52%). Different strains that do not belong to the above species were grouped as Lactobacillus sp. Group S1 comprising maltose and lactose negative L. sake was the main group present in all factories except in a factory in Segovia where group S3 comprising lactose positive L. sake and pediococci were the predominant ones. Group S1 increased during the ripening process in all six factories and it dominated in the ripened 'chorizo' except in the mentioned factory in Segovia. In general strains of L. sake and L. curvatus which fermented maltose but not lactose were more dominant at the beginning and in the middle of the process, whereas, L. sake and L. curvatus which could ferment lactose, or lactose and maltose occurred in higher numbers in semi-ripened 'chorizo' and in the final product. This indicates that strains which could ferment lactose were more competitive towards the end of the process. Strains from group S1 were the microorganisms responsible for the pH drop in most of the factories, giving the correct texture. As a result it would appear that a strain from this group would be most suitable for use as starter culture.

Animals↗
PubMed2003

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria.

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.

Animals↗
PubMed2000

Isolation and identification of tetracycline resistant lactic acid bacteria from pre-packed sliced meat products.

In recent years, the food chain has been recognised as one of the main routes for transmission of antibiotic resistant bacteria between the animal and human population. In this regard, the current study aimed to investigate if tetracycline resistant (tetR) lactic acid bacteria (LAB) are present in ready-to-eat modified atmosphere packed (MAP) sliced meat products including fermented dry sausage, cooked chicken breast meat and cooked ham. From population graphs based on doubling tetracycline concentrations between 0 and 256 microg ml(-1), only fermented dry sausage was shown to contain a high-level retR LAB population (5.10(1) - 2,23.10(4) CFU/g), and this in four out of ten examined sausages. From these four positive sausages, a total of 100 strains were isolated on de Man, Rogosa and Sharpe-sorbic acid (MRS-S) agar without tetracycline (n = 45) and on MRS-S agar supplemented with a tetracycline breakpoint concentration of 64 microg ml(-1) (n = 55). Using resistance histograms derived from the disc diffusion method, all these strains were grouped as sensitive to rifampicin, erythromycin and ampicillin. All strains from the tetracycline-containing MRS-S plates were resistant to tetracycline. Identification with whole-cell protein profiling revealed that the total strain set represented four different species: Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sakei subsp. carnosus and Lactobacillus curvatus. All species are commonly associated with fermented dry sausage, either as starter culture or as natural contaminants. The latter three species were found to comprise all tetracycline resistant strains. To our knowledge, this is the first report providing evidence for the presence of tetR LAB in final ready-to-eat pre-packed fermented dry sausages.

Food Microbiology↗
PubMed2000

Molecular diversity of lactic acid bacteria from cassava sour starch (Colombia).

Lactic acid bacteria and more particularly lactobacilli and Leuconostoc, are widely found in a wide variety of traditional fermented foods of tropical countries, made with cereals, tubers, meat or fish. These products represent a source of bacterial diversity that cannot be accurately analysed using classical phenotypic and biochemical tests. In the present work, the identification and the molecular diversity of lactic acid bacteria isolated from cassava sour starch fermentation were assessed by using a combination of complementary molecular methods: Randomly Amplified Polymorphic DNA fingerprinting (RAPD), plasmid profiling, hybridization using rRNA phylogenetic probes and partial 16S rDNA sequencing. The results revealed a large diversity of bacterial species (Lb. manihotivorans, Lb. plantarum, Lb. casei, Lb. hilgardii, Lb. buchneri, Lb. fermentum, Ln. mesenteroides and Pediococcus sp.). However, the most frequently isolated species were Lb. plantarum and Lb. manihotivorans. The RAPD analysis revealed a large molecular diversity between Lb. manihotivorans or Lb. plantarum strains. These results, observed on a rather limited number of samples, reveal that significant bacterial diversity is generated in traditional cassava sour starch fermentations. We propose that the presence of the amylolytic Lb. manihotivorans strains could have a role in sour starch processing.

DNA, Ribosomal↗
PubMed1999

Gene expression systems for lactic acid bacteria.

Considerable advances have been made in the genetics and molecular biology of lactic acid bacteria, including Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Streptococcus spp. These have resulted in the construction of constitutive gene expression cassettes, inducible gene expression systems, and specific protein targeting systems for these bacteria. These developments are important in the food industry where lactic acid bacteria can be exploited as food-grade cell factories.

Gene Expression Regulation, Bacterial↗
PubMed1995

UDP-N-acetylmuramyl-L-alanine functions as an activator in the regulation of the Escherichia coli glutamate racemase activity.

D-Glutamate is an essential component of the bacterial peptidoglycan. In Escherichia coli, the biosynthesis of D-glutamate is catalyzed by a glutamate racemase (encoded by the dga gene) and is regulated by UDP-N-acetylmuramyl-L-alanine [Doublet et al. (1994) Biochemistry 33, 5285], a bacterial peptidoglycan subunit precursor. Investigation was conducted to elucidate the interaction between the enzyme and its regulator. Whole and N-terminal truncated enzymes, encoded by individual constructs containing either a full-length or an N-terminal truncated dga gene, were evaluated. In the absence of the regulator, the purified whole enzyme showed a low-level basal racemase activity for which a Km value of 18.9 mM and a Vmax of 0.4 mumol/(min.mg) were determined, using D-glutamate as the substrate. Using the same substrate, in the presence of 6.5 microM UDP-N-acetylmuramyl-L-alanine, a Km value of 4.2 mM and a Vmax of 34 mumol/(min.mg) were measured. Similar kinetic parameters for the activated enzyme were obtained using L-glutamate as the substrate. The N-terminal truncated E. coli enzyme, with a 21 amino acid region removed, is similar in size to the Pediococcus pentosaceus glutamate racemase. Effects of the regulator on the full-length and the N-terminal truncated enzyme in the dialyzed cell lysate were compared. A host cell line, E. coli WM335 delta recA, containing a nonfunctional chromosomal dga gene was used to minimize the background interference. With 6.5 microM regulator added, the N-terminal truncated enzyme displayed a loss of more than 80% of the activity compared to the full-length enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗
PubMed2004

Resistance screening essay of wine lactic acid bacteria on lysozyme: efficacy of lysozyme in unclarified grape musts.

In wine making, the bacteriolytic activity of lysozyme has primarily been used to control the malolactic fermentation in wines. The use of lysozyme in musts before settling and the beginning of the alcoholic fermentation to inhibit the growth of lactic acid bacteria could be very beneficial. In a resistance test carried out in MT/b broth, lysozyme had greater antimicrobial activity toward Oenococcus oeni than Lactobacillus species. Several strains of wine bacteria belonging to Oenococcus proved sensitive to the bacteriolytic activity of lysozyme at low concentrations in both synthetic medium (MT/b) (50 mg/L), white must, or red must made with or without the skins (100 mg/L). Lactobacillus and Pediococcus strains survived at lysozyme concentrations of 200-500 and 500 mg/L, respectively, in MT/b and musts. Suspended solids in unclarified musts may strongly bind to lysozyme thereby causing its removal by filtration or centrifugation. One hour after lysozyme was added to musts, it was quantified by HPLC and found after centrifugation to be 40-50% and only 10% in musts made with or without the skins, respectively. Although appreciable amounts of lysozyme were bound to wine components, this did not appear to be a serious hindrance to lysozyme activity.

Enzyme Stability↗
PubMed2006

Glucan and fructan production by sourdough Weissella cibaria and Lactobacillus plantarum.

After a large screening on sourdough lactic acid bacteria, exopolysaccharide (EPS)-forming strains of Weissella cibaria, Lactobacillus plantarum, and Pediococcus pentosaceus were selected. After 6 days of incubation at 30 degrees C, the synthesis of EPS in MRS-based broth ranged from 5.54 to 7.88 mg mL-1. EPS had an apparent molecular mass of ca. 104 Da. As shown by carbohydrate consumption, the synthesis of EPS was found from sucrose only. Two types of homopolysaccharides were synthesized: glucans simultaneously with growth and fructans after 1 day of incubation. Two protein bands of ca. 180-200 kDa were in situ detected on SDS-PAGE gels incubated with sucrose. PCR products of ca. 220 bp were found for L. plantarum PL9 (100% of identity to putative priming glycosyltransferase of L. plantarum WCFS1) and W. cibaria WC4 (80% of identity to putative glycosyltransferase, epsD, of Bacillus cereus G9241) by using hybrid primers for the priming gtf genes. Degenerated primers DexreuR and DexreuV showed a unique PCR product, and the predicted amino acid sequences were identical for W. cibaria WC4 and L. plantarum PL9. The sequence had similarity with polysaccharide biosynthesis glycosyltransferases. W. cibaria WC4 or L. plantarum LP9 synthesized ca. 2.5 g kg-1 EPS during sourdough fermentation with sucrose added. Compared to the sourdough started with an EPS-negative strain, the sourdough started with W. cibaria WC4 or L. plantarum LP9 increased the viscosity, and the resulting bread had higher specific volume and lower firmness. The synthesis of EPS by selected sourdough lactic acid bacteria could be considered as a useful tool to replace the additives for improving the textural properties of baked goods.

Bacteria↗
PubMed1979

Synthesis of aza homologues of folic acid.

Folic acid analogues containing an additional nitrogen atom between the phenyl ring and the carbonyl group of the side chain were synthesized. None of the compounds showed significant inhibitory activity against human lymphoblastic leukemia cells (CCRF-CEM) in culture or against Lactobacillus casei (ATCC 7469) growth. Against L1210 leukemia in mice, the aza homologue of folic acid, 4, and the aspartic acid analogue, 14, showed no increase in life span over control animals. These compounds were more toxic in vivo than the corresponding methotrexate analogues. Compound 4 supported the growth of Streptococcus faecium (ATCC 8043), and its tetrahydro derivative supported the growth of Pediococcus cerevisiae (ATCC 8081). These results strongly suggest that 4 can substitute for folate derivatives as cofactors for serine transhydroxymethylase, thymidylate synthetase, and dihydrofolate reductase.

Animals↗
PubMed1977

Diastereoisomers of 5,10-methylene-5,6,7,8-tetrahydropteroyl-D-glutamic acid.

The diastereoisomers of 5,10-methylene 5,6,7,8-tetrahydropteroyl-D-glutamate were resolved and tested as substrates and inhibitors of Lactobacillus casei thymidylate synthetase. No activity was observed. The compounds were neither growth factors nor inhibitors for Lactobacillus casei, Streptococcus faecium, or Pediococcus cerevisiae. 7,8-Dihydropteroyl-D-glutamate is 50% as active as 7,8-dihydropteroyl-L-glutamate (dihydrofolate) as a substrate for L. casei dihydrofolate reductase.

Lacticaseibacillus casei↗
PubMed1971

Pteroylglutamate derivatives in Pisum sativum L. Biosynthesis of cotyledonary tetrahydropteroylglutamates during germination.

1. The concentrations of pteroylglutamate derivatives in the cotyledons of pea (Pisum sativum L.) seedlings were determined by microbiological assay by using Lactobacillus casei (A.T.C.C. 7469), Streptococcus faecalis (A.T.C.C. 8043) and Pediococcus cerevisiae (A.T.C.C. 8081). During germination the pteroylglutamate content of the cotyledons increased rapidly from 0.2mug/g dry wt. to 4.0mug/g dry wt., the maximum values being reached approx. 120h after imbibition. 2. Approx. 50% of the pteroylglutamate pool of 3-day-old seedlings was accounted for by highly conjugated derivatives. The concentrations of such derivatives were greatest when precautions were taken to inactivate endogenous enzymes before extraction of the tissues. 3. Individual derivatives present in the tissue extracts were separated by chromatography on DEAE-cellulose. Synthesis of the major derivative, 5-methyltetrahydropteroylmonoglutamate, was inhibited by administration of 0.1mm-aminopterin and -amethopterin solutions during imbibition. Under these conditions pteroylglutamic acid accumulated in the tissues. 4. Feeding experiments employing [2-(14)C]pteroylglutamic acid and 5[(14)C]-methyltetrahydro-pteroylmonoglutamic acid revealed that both compounds were incorporated into conjugated and unconjugated derivatives of the pteroylglutamate pool.

Aminopterin↗
PubMed1972

Localization and interconversion of tetrahydropteroylglutamates in isolated pea mitochondria.

1. Mitochondria were extracted from 4-day-old pea cotyledons and purified on a sucrose density gradient. 2. Microbiological assay of the purified mitochondrial fraction with Lactobacillus casei (A.T.C.C. 7469), Streptococcus faecalis (A.T.C.C. 8043) and Pediococcus cerevisiae (A.T.C.C. 8081) revealed a discrete pool of conjugated and unconjugated derivatives of tetrahydropteroylglutamic acid. 3. Solubilization and chromatographic studies of the mitochondrial fraction demonstrated the presence of formylated and methylated derivatives, 10-formyltetrahydropteroylmonoglutamic acid, 5-formyltetrahydropteroylmonoglutamic acid and 5-formyltetrahydropteroyldiglutamic acid being the major derivatives present. 4. The principal mitochondrial pteroylglutamates were labelled when dry seeds were allowed to imbibe [2-(14)C]pteroylglutamic acid and 5-[methyl-(14)C]-methyltetrahydropteroylmonoglutamic acid. 5. The ability of isolated mitochondria to catalyse oxidation and reduction of tetrahydropteroylglutamic acid derivatives was demonstrated in feeding experiments in which [(14)C]formaldehyde, [3-(14)C]serine, sodium [(14)C]formate, 5-[methyl-(14)C]methyltetrahydropteroylmonoglutamic acid or [2-(14)C]-glycine served as C(1) donor. In addition, (14)C was incorporated into free amino acids related to C(1) metabolism.

Carbon Isotopes↗
PubMed1972

Regulation of C metabolism by L-methionine in Saccharomyces cerevisiae.

1. The concentrations of folate derivatives in aerobic cultures of Saccharomyces cerevisiae (A.T.C.C. 9763) were determined by microbiological assay employing Lactobacillus casei (A.T.C.C. 7469) and Pediococcus cerevisiae (A.T.C.C. 8081). Cells cultured in media lacking l-methionine contained higher concentrations of folate derivatives than cells grown in the same media supplemented with 2.5mumol of l-methionine/ml. The concentrations of highly conjugated derivatives were also decreased by supplementing the growth medium with l-methionine. 2. DEAE-cellulose column chromatography of extracts prepared from cells grown under these conditions revealed that the concentrations of methylated tetrahydrofolates were drastically decreased by the methionine supplement. Smaller decreases were also observed in the concentrations of formylated and unsubstituted derivatives. 3. The concentrations of four enzymes of C(1) metabolism were compared after 6h of growth in the presence and in the absence of l-methionine (2.5mumol/ml). The specific activities of formyltetrahydrofolate synthetase, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase were not altered by this treatment but that of 5-methyltetrahydrofolate-homocysteine methyltransferase was decreased by approx. 65% when l-methionine was supplied. The activities of 5-methyltetrahydrofolate-homocysteine methyltransferase, serine hydroxymethyltransferase and formyltetrahydrofolate synthetase were not appreciably altered by l-methionine in vitro. In contrast this amino acid was found to inhibit the activity of methylenetetrahydrofolate reductase. 4. Feeding experiments employing sodium [(14)C]formate indicated that cells grown in the presence of exogenous methionine, although having less ability to convert formate into methionine, readily incorporated (14)C into serine and the adenosyl moiety of S-adenosylmethionine. 5. It is suggested that exogenous l-methionine controls C(1) metabolism in Saccharomyces principally by regulation of methyl-group biogenesis within the folate pool.

Alcohol Oxidoreductases↗
PubMed1973

Identification and measurement of the folates in sheep liver.

1. Methods are described for the extraction, separation by ion-exchange chromatography and estimation by microbiological assay of the folates in sheep liver. 2. Injection of [2-(14)C]-pteroylglutamate into a sheep fed on a stock diet led to extensive labelling of chromatographically separable liver folates. About 12% of the label in the liver could not be extracted by the method used. 3. Liver folates were examined in five ewes fed on restricted amounts of a diet of wheaten hay-chaff and gluten and injected weekly with vitamin B(12). Chromatographic separation was followed by microbiological assay with Lactobacillus casei, Streptococcus faecalis R. and Pediococcus cerevisiae both before and after treatment of fractions with conjugase (gamma-glutamylcarboxypeptidase). Evidence was obtained that the folates present were predominantly polyglutamate forms of tetrahydropteroylglutamate, 5-methyltetrahydropteroylglutamate and 5- (and 10-) formyltetrahydropteroylglutamates. Differences in the responses of the assay organisms permitted quantitative distinction between these three main classes of folates. 4. Methyltetrahydrofolates were eluted in seven successive peaks that were separated by constant increments in the logarithm of eluant [P(i)]. A similar relationship existed for seven successive peaks of tetrahydrofolate and may also have existed for each of the two series of formyltetrahydrofolates. 5. Based on these and other observations it is proposed that sheep liver folates consist predominantly of the mono- to hepta-glutamates of each of the reduced pteroates identified. The methods employed allowed quantitative determinations to be made of most of the folates present. The predominant forms were hexaglutamates. 6. Four components active for L. casei were detected that could not be identified. Three of them were polyglutamates.

Animal Feed↗