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The effect of some pulsed electric field (PEF) processing parameters (electric field strength, pulse frequency and treatment time), on a mixture of microorganisms ( Kloeckera apiculata , Saccharomyces cerevisiae , Lactobacillus plantarum , Lactobacillus hilgardii and Gluconobacter oxydans ) typically present in grape juice and wine were evaluated. An experimental design based on response surface methodology (RSM) was used and results were also compared with those of a factorially designed experiment. The relationship between the levels of inactivation of microorganisms and the energy applied to the grape juice was analysed. Yeast and bacteria were inactivated by the PEF treatments, with reductions that ranged from 2.24 to 3.94 log units. All PEF parameters affected microbial inactivation. Optimal inactivation of the mixture of spoilage microorganisms was predicted by the RSM models at 35.0 kV cm − 1 with 303 Hz pulse width for 1 ms. Inactivation was greater for yeasts than for bacteria, as was predicted by the RSM. The maximum efficacy of the PEF treatment for inactivation of microorganisms in grape juice was observed around 1500 MJ L − 1 for all the microorganisms investigated. The RSM could be used in the fruit juice industry to optimise the inactivation of spoilage microorganisms by PEF. more...

Flow cytometry (FC) has been introduced to characterize and to assess the physiological states of microorganisms in conjunction with the classical plate-counting method. To show the applicability of the technique, in particular for the development of kinetic models, pure culture fermentation experiments were followed over time, using both prokaryotic ( Lactobacillus hilgardii ) and eukaryotic ( Saccharomyces cerevisiae ) microorganisms growing in standard culture media (MRS and YPD). The differences observed between the active and viable cells determined by FC and CFU, respectively, allowed us to determine that a large number of cells were in a viable but nonculturable (VBNC) state, which resulted in a subpopulation much larger than the damaged-cell (double-stained) subpopulation. Finally, the determination of the evolution of viable, the VBNC, and the dead cells allowed us to develop a segregated kinetic model to describe the yeast and the bacteria population dynamics and glucose consumption in batch cultures. This model, more complete than that which is traditionally used, based only on viable cell measurements, describes better the behavior and the functionality of the cultures, giving a deeper knowledge in real time about the status and the course of the bioprocesses. more...

A flow injection analysis (FIA) system was developed for the determination of phosphate efflux from wine lactic acid bacteria (Oenococus oeni and Lactobacillus hilgardii) as an indication of cell membrane damage. The system allowed the direct injection of the cell suspension, avoiding the filtration step, with minimum sample treatment and minimized reagent consumption. The developed system is characterized by a linear concentration zone between 3.23 × 10-5 and 4.84 × 10-4 mol L-1 PO4 3- and repeatability better than 2.9%. Bacterial suspensions were exposed to a chemical stress with phenolic acids and injected in the FIA system at regular intervals. The extracellular concentration of phosphate was measured spectrophotometrically. The experimental results obtained indicate that hydroxycinnamic acids (p-coumaric, ferulic and caffeic) induced faster phosphate leakage rates than hydroxybenzoic acids (vanillic and p-hydroxybenzoic) in both strains tested, which could be related to their higher lipophilic character. more...

Conventional phenotypic methods sometimes lead to misidentification of some heterofermentative wine lactobacilli such as Lactobacillus hilgardii, Lactobacillus buchneri, and Lactobacillus brevis. We establish the specificity of 16S rDNA sequencing in the differentiation of these species and in the rejection of the Lactobacillus vermiforme species name. Moreover, we succeeded in differentiating these heterofermentative species by means of recA gene sequence comparison. Short homologous regions were amplified by PCR with degenerate consensus primers, sequenced, and 280 bp were analysed and considered for the inference of phylogenetic trees. The phylogram obtained was coherent and clearly separated the three species. The recA gene sequence was a reliable and useful method that allowed a good discrimination among closely related species. The validity of the recA gene sequence, restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR), and random amplified polymorphic DNA (RAPD) to study the L. hilgardii intraspecies heterogeneity was tested in five strains isolated from stuck wine fermentations at the same winery in the same vintage. The results indicated that L. hilgardii is a heterogeneous species. Since L. hilgardii is a malolactic species that can influence the final quality of the wine, the presence of oenological relevant genes, such as those involved in ethyl carbamate or biogenic amine production, was investigated, This work was supported by grants AGL2005–00470 (CICYT), RM03–002 (INIA), and S-0505/AGR/000153 (CAM). We thank A. V. Carrascosa and R. González for critically reading the manuscript. The technical assistance of M.V. Santamaría and A. Gómez is greatly appreciated. H. Rodríguez is a recipient of an I3P predoctoral fellowship from the CSIC. more...

Despite the large number of papers dealing with bacterial proteomes, very few include information about proteins with alkaline isoelectric points, because of the limits inherent in 2DE technology. Nonetheless, analyses of in silico proteomes of many prokaryotes show a bimodal distribution of their proteins based on their pIs, the most crowded areas lying between pI 4-7 and pI 9-11. The aim of the present research was to set up a general, simple and standardizable 2DE protocol suitable for studying alkaline proteome of L. hilgardii, a Gram-positive bacillus isolated from wine. The method has been also tested on a Gram-negative bacterium able to degrade aromatic pollutants, A. radioresistens S13. Optimization of the method was mainly focused on improving protein extraction and IEF (pI 6-11) separation protocols. Concerning IEF, different methods for sample loading (in-gel rehydration and cup-loading), and different reducing agents (DTT and HED) were tested and compared. The proposed protocol was found to resolve alkaline proteins from both our Lactobacillus and Acinetobacter strains efficiently, in spite of their different external layers, thus enabling more ....................... more...

The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His6 tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, [beta] = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å3 Da-1, respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code 1dxh) as the search model, JMM thanks the Ministerio de Educación y Ciencia for a research grant (BFU2004-01554/BMC; DGCYT) and `Factoría de Cristalización' Consolider-Ingenio 2010 for support of his research and also Dr Juliana Sanz for her helpful discussions. This work was also supported by grants AGL2005-00470 (CICYT) and S-0505/AGR000153 (CAM). HR is a recipient of a I3P predoctoral fellowship from the CSIC. more...

Trabajo presentado al IX Congreso Nacional de Investigación Enológica (GIENOL) celebrado en Badajoz del 29 de mayo al 1 de junio de 2007., Los lactobacilos heterofermentativos ailados de vino pertenecen a las especies Lactobacillus brevis, Lactobacillus hilgardii y Lactobacillus buchneri. Sin embargo, la taxonomía de estas especies es confusa puesto que a menudo los métodos fenotípicos convencionales producen identificaciones erróneas. La secuenciación del 16S rDNA es un método útil para la diferenciación de las especies de lactobacilos heterofermentativos aislados de vino. Mediante la secuenciación de un fragmento del gen recA también se ha podido discriminar entre las distintas especies de lactobacilos heterofermentativos., A los proyectos RM03-002 (INIA), 08G/0035/2003 (CAM) y AGL2005-00470 (CICYT). more...

Aims: A contribution towards the elucidation of the mechanisms of tannins on bacteria growth inhibition, with particular focus on the interaction between tannins and bacterial proteins. Methods and results: The interaction between tannic acid (TA) and Lactobacillus hilgardii, a wine spoilage bacterium, was investigated by a combination of physiologic and proteomic approaches. Growing tests were performed on medium supplemented with TA at concentrations ranging from 100 to 1000 mg l−1 demonstrating the inhibitory effect of TA on the growth rate. Total proteins extracted from cells unexposed and exposed to TA were then analysed by 2D-electrophoresis and significant quantitative variations with a marked decrease of protein intensity upon TA exposure were observed. Most of the proteins, identified by ESI tandem Mass Spectrometry, were metabolic enzymes of different pathways, located in cytoplasm and membrane. Conclusions: The effects of TA on cells are deduced by the involvement of metabolic enzymes, and functional proteins on the tannin–protein interaction. These results might be related to the altered functions of the cell metabolism. Significance and impact of the study: The possible role of tannins in the inhibition of the bacterial survival and growth in a natural environment such as wine. A similar approach could be applied for evaluating the effects of tannins on food borne and pathogenic bacteria. more...

The protein population secreted from three wine-contaminant microorganisms was studied by two-dimensional electrophoresis and screened for proteases. Proteolytic enzymes were electrophoretically purified and their activity, optimum pH and temperature determined. The protease of Acetobacter aceti maintained its activity over the range of pHs 3.0-5.0, thus being of potential biotechnological interest. more...

Aims: To quantify the ability of 136 lactic acid bacteria (LAB), isolated from wine, to produce histamine and to identify the bacteria responsible for histamine production in wine. Methods and Results: A qualitative method based on pH changes in a plate assay was used to detect wine strains capable of producing high levels of histamine. Two quantitative, highly sensitive methods were used, an enzymatic method and HPLC, to quantify the histamine produced by LAB. Finally, an improved PCR test was carried out to detect the presence of histidine decarboxylase gene in these bacteria. The species exhibiting the highest frequency of histamine production is Oenococcus oeni. However, the concentration of histamine produced by this species is lower than that produced by strains belonging to species of Lactobacillus and Pediococcus. A correlation of 100% between presence of histidine decarboxylase gene and histamine production was observed. Wines containing histamine were analysed to isolate and characterize the LAB responsible for spoilage. Conclusions: Oenococcus was able to synthesize low concentrations of histamine in wines, while Pediococcus parvulus and Lactobacillus hilgardii have been detected as spoilage, high histamine-producing bacteria in wines. Significance and Impact of the Study: Information regarding histamine-producing LAB isolated from wines can contribute to prevent histamine formation during winemaking and storage. more...

Phenol-derived compounds are known to have membrane-active properties against microorganisms causing leakage of cell constituents including proteins, nucleic acids, ATP, and inorganic ions (such as potassium or phosphate). Phenolic (hydroxybenzoic and hydroxycinnamic) acids are components of the highly complex phenolic composition of wines. In this work, several wine-occurring phenolic acids, that were previously found to affect the growth and the survival of certain wine lactic acid bacteria, were tested for their effects on the cell membrane of these same strains. Selected strains of Lactobacillus hilgardii and Oenococcus oeni were cultivated to late exponential phase and then centrifuged, washed, and resuspended in buffer solution. Phenolic acids were added to the cell suspensions to induce chemical stress. Potassium leakage from cells was measured by determining the extracellular potassium concentration of the medium using a potassium ion-sensitive electrode. A combined glass electrode was used to monitor pH changes. Phosphate efflux was determined using a flow injection analysis (FIA) system with spectrophotometric detection. Samples were collected at regular intervals, microfiltered, and injected in the FIA system. Results indicate that p-coumaric acid had the strongest effect of all phenolic acids, causing rapid potassium and phosphate leakages from cells after exposure to this compound. Generally, hydroxycinnamic acids induced faster leakage rates than hydroxybenzoic acids, which could be related to their higher lypophilic character. These results agree with previous growth and survival experiments. The differences of phenolic acids toxicity toward lactic acid bacteria seem to be related to their effects on membrane permeability. more...

The ability of wine lactic acid bacteria to produce tyramine and phenylethylamine was investigated by biochemical and genetic methods. An easy and accurate plate medium was developed to detect tyramine-producer strains, and a specific PCR assay that detects the presence of tdc gene was employed. All strains possessing the tdc gene were shown to produce tyramine and phenylethylamine. Wines containing high quantities of tyramine and phenylethylamine were found to contain Lactobacillus brevis or Lactobacillus hilgardii. The main tyramine producer was L. brevis. The ability to produce tyramine was absent or infrequent in the rest of the analysed wine species. more...

The ability of Lactobacillus hilgardii 5w to metabolize gallic acid and catechin was evaluated. It was grown in a complex medium containing gallic acid or catechin. The metabolites were analyzed by high-performance liquid chromatography and identified by comparing the retention times and spectral data with the standards of a database. In gallic acid-grown cultures, gallic acid, pyrogallol, catechol, protocatechuic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, and p-hydroxybenzyl alcohol were detected. In catechin-grown cultures, catechin, gallic acid, pyrogallol, catechol, p-hydroxybenzoic acid, acetovanillone, and homovanillic acid were detected. This work presents evidence of gallic acid and catechin degradation by L. hilgardii from wine., This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Consejo de Investigaciones de la Universidad Nacional de Tucuman (CIUNT), Argentina and Programa de Reforma de la Educacion Superior (FOMEC). more...

In mixed culture of Lactobacillus hilgardii X1B and Leuconostoc oenos X2L, isolated from Argentinian wines, an amensalistic growth response was observed: Leuconostoc oenos did not grow, and after 24 h of incubation at 30°C no viable cells were detected. In pure and mixed cultures, Lactobacillus hilgardii produced hydrogen peroxide early in the growth cycle, reaching the maximum at 24 h. The values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the action of hydrogen peroxide on the growth of Leuconostoc oenos were: 4.08μg ml-1 and 17.00 μg ml-1 respectively. more...

Aims: To determine the effect of several wine-associated, phenolic acids on the growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Methods and Results: Growth was monitored in ethanol-containing medium supplemented with varying concentrations of hydroxybenzoic acids (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acids) and hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids). Progressive inactivation was monitored in ethanolcontaining phosphate buffer supplemented in a similar manner to the growth experiments. Hydroxycinnamic acids proved to be more inhibitory to the growth of O. oeni than hydroxybenzoic acids. On the other hand, some acids showed a beneficial effect on growth of Lact. hilgardii. p-Coumaric acid showed the strongest inhibitory effect on growth and survival of both bacteria. Conclusions: Most phenolic acids had a negative effect on growth of O. oeni, for Lact. hilgardii this effect was only noted for p-coumaric acid. Generally, O. oeni was more sensitive to phenolic acid inactivation than Lact. hilgardii. Significance and Impact of the Study: Eight wine-derived, phenolic acids were compared for their effects on wine lactic acid bacteria. Results indicate that phenolic acids have the capacity to influence growth and survival parameters. The differences found between phenolic compounds could be related to their different chemical structures. more...

The genes implicated in the catabolism of the amino acid arginine by Lactobacillus hilgardiiX1Bwere investigated to assess the potential for formation of ethyl carbamate precursors in wine. L. hilgardii X1B can use arginine via the arginine deiminase pathway. The complete nucleotide sequence of the arc genes involved in this pathway has been determined. They are clustered in an operon-like structure in the order arcABC. No evidence was found for the presence of a homologue of the arcD gene, coding for the arginine/ornithine antiporter. The arc genes have been expressed in Escherichia coli resulting in arginine deiminase (ArcA), ornithine carbamoyltransferase (ArcB) and carbamate kinase (ArcC) activities. The results indicate the need for caution in the selection of lactic acid bacteria for conducting malolactic fermentation in wine since arginine degradation could result in high amounts of ethyl carbamate., This work was supported by grant VIN00-016 from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT). M.E. Arena was a recipient of a Contrato UNT-Proyecto FOMEC 1214 from the Programa de Reforma de la Educación Superior. The nucleotide sequence of the arcABC gene cluster has been deposited in the EMBL/GenBank/DDBJ databases under accession number AJ421514. more...

Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin. more...

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37°C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7°C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds. more...

The degradation of lactic acid under anoxic conditions was studied in several strains of Lactobacillus buchneri and in close relatives such as Lactobacillus parabuchneri , Lactobacillus kefir , and Lactobacillus hilgardii . Of these lactobacilli, L. buchneri and L. parabuchneri were able to degrade lactic acid under anoxic conditions, without requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels of NAD-linked 1,2-propanediol-dependent oxidoreductase (530 to 790 nmol min −1 mg of protein −1 ), a novel pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by L. buchneri does not support cell growth and is pH dependent. Acidic conditions are needed to induce the lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8 hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by L. buchneri is not certain, but some results indicate that it plays a role in maintaining cell viability. more...

The degradation of lactic acid under anoxic conditions was studied in several strains of Lactobacillus buchneri and in close relatives such as Lactobacillus parabuchneri, Lactobacillus kefir, and Lactobacillus hilgardii. Of these lactobacilli, L. buchneri and L. parabuchneri were able to degrade lactic acid under anoxic conditions, without requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels of NAD-linked 1,2-propanediol-dependent oxidoreductase (530 to 790 nmol min-1 mg of protein-1), a novel pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by L. buchneri does not support cell growth and is pH dependent. Acidic conditions are needed to induce the lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8 hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by L. buchneri is not certain, but some results indicate that it plays a role in maintaining cell viability. Chemicals/CAS: acetic acid, 127-08-2, 127-09-3, 64-19-7, 71-50-1; alcohol, 64-17-5; lactic acid, 113-21-3, 50-21-5; nicotinamide adenine dinucleotide, 53-84-9; oxidoreductase, 9035-73-8, 9035-82-9, 9037-80-3, 9055-15-6; propylene glycol, 57-55-6; Acetic Acid, 64-19-7; Culture Media; Lactic Acid, 50-21-5; NADH Dehydrogenase, EC 1.6.99.3; Propylene Glycol, 57-55-6 more...

The catabolism of arginine, an amino acid found in grape juice and wine, citrulline and ornithine was investigated in four lactic acid bacteria. Only Lactobacillus hilgardii X1B catabolized arginine and excreted citrulline into the medium. The recovery of arginine as ornithine was lower than the expected theoretical value. The arginase-urease pathway was not detected indicating that the amino acid degradation was carried out only by the arginine dihydrolase pathway. Oenococcus oeni m, a strain not able to utilize arginine, degraded citrulline that was completely recovered as ornithine, ammonia and CO2. Lactobacillus hilgardii X1B catabolized citrulline but it was only 44% recovered as ornithine. The citrulline utilization by Oenococcus oeni m may be important for two reasons: it can gain extra energy for growth from citrulline metabolism, and the amino-acid diminution could avoid the possibility of ethyl carbamate formation from the citrulline naturally present in wine. more...

Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacillus hilgardii and Lactobacillus brevis. Random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP-PCR) techniques were developed for a molecular study of these two species. The taxonomic relationships were confirmed by analysis of the ribosomal operon. Amplified DNA fragments were chosen to isolate L. hilgardii-specific probes. In addition to rapid molecular methods for identification of L. hilgardii, these results convincingly proved that some strains first identified as L. brevis must be reclassified as L. hilgardii. The data clearly showed that these molecular methods are more efficient than phenotypic or biochemical studies for bacterial identification at the species level. more...

Fifteen heterofermentative lactobacilli belonging to the Lactobacillus hilardii and Lactobacillus brevis were studied. In a DNA/DNA hybridization test most of them give the expected answer since their DNA hybridized with the reference strain DNA probe from the same species. However, strains (of L. brevis) strongly hybridized with L. hilgardii ATCC 8290. This raised doubts on their taxonomical position because they had previously been identified as L. brevis. Restriction enzymes (EcoRI, BamHI, HindIII, PstI) were used in an RFLP (restriction fragment length polymorphism) study of these strains. 16S + 23S rRNA from Escherichia coli constituted the probe. Statistical analysis of all the patterns (i.e. four enzymes and 15 strains) led to the construction of a dendrogram. Finally, two main branches corresponded well with the results of conventional identification, because they associated L. hilgardii strains on one side and L. brevis on the other side. In addition, each branch was divided into subgroups. This method proved adapted to ribotype all the 15 strains except two, using these four enzymes. more...

A variant ofLactobacillus hilgardii was immobilized by its own production of dextran gel, forming grains. The best rate of weight increase of the gel in continuous fermentation was 16.3±3.3%/h, at pH 4.8±0.1 and with a dilution rate of 0.22 to 0.26/h. Observation by scanning electron microscopy located most of the bacteria as microcolonies on the surface. A similar arrangement appeared in calcium alginate beads. The best population density (10(10) cells/g) was obtained in grains at pH 5.8, after 30h. At a similar pH value, 4.8, the growth rate was higher in alginate beads than in dextran gel but the final population density was approximately the same. Acidification rate increased faster with mixed gel at pH 5.2 than with dextran at pH 5.8. more...

Homo- and heterofermentative species of Lactobacillus have been isolated from sugary kefir grains. Most of the homofermentative strains fermented tagatose and aldonitol and presented 48-54% of homology with Lactobacillus paracasei ssp. paracasei NCDO 151 (ex Lactobacillus casei). The two variants of a hetero-fermentative species, although fermenting arabinose, were related to Lactobacillus hilgardii NCDO 264 (type strain) with 88% of homology. One of them produced polysaccharide from sucrose at pH 4.8 and 30 degrees C; the best glucose conversion into polysaccharide was obtained from 3% of sucrose (81.8%), and the maximum production occurred about 35 hours after the end of the log phase of growth, in MRS sucrose broth. Polysaccharide formation did not occur above 40 degrees C, a temperature at which no growth was observed. The two variants were forming minicells by abnormal divisions. more...

The bacterium Lactobacillus hilgardii (previously named L. brevis ), isolated from sugary kefir grains, has been found to produce a gelling extracellular polysaccharide. Methylation analysis and 1 H- and 13 C-NMR analysis revealed a dextran-like structure for the polysaccharide. A high proportion of α-(1,6)-Glc and small proportions of α-(1,3,6)-Glc, α-(1,4,6)-Glc and terminal α-Glc were detected. The L. hilgardii polysaccharide differed from normal dextrans in containing a small proportion of α-(1,3)-Glc. The structure of the polysaccharide was found to be independent of harvesting time and unaffected by changes in the growth medium. The development of the gel structure has been followed by transmission electron microscopy. more...

In a mixed culture of Lactobacillus hilgardii and Pediococcus pentosaceus on commerical grape juice, growth of the latter was inhibited until 24 h; after 24 h no viable cells were detected. During the early stages of growth, sugars and malic acid were consumed and production of M- and L-lactic acids was greater in the mixed culture than in either of the pure cultures. more...

The continuous decolorization of molasses waste water (MWW) by immobilized cells of Lactobacillus hilgardii W-NS was studied. The immobilized cells showed the maximal decolorization yield in the presence of 1% glucose with a medium pH of 5.0 at 45°C. On successive decolorization of MWW, with recycling of the immobilized cells, more than 90% of the maximal decolorization yield was maintained for one month when 0.05% peptone was added to MWW. In contrast, on continuous decolorization in a column type reactor, a sufficient decolorization yield could not be maintained, the decolorization yield dropping to half the maximal level during operation for 5 days. But the feeding of MWW adjusted to the pH of 7.3, compared with the maximal pH value (5.0), slowed down the decrease in the decolorization yield. more...

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum. more...

The microflora of sugary kefir grains was principally mesophilic and consisted chiefly of lactic acid bacteria [Lactobacillus casei, Lactobacillus hilgardii (=brevis),Leuconostoc mesenteroides ssp.dextranicum, Streptococcus lactis] and a small proportion of yeasts (Zygosaccharomyces florentinus, Torulospora pretoriensis, Kloeckera apiculata, Candida lambica andC. valida). Few coliforms and faecal streptococci were observed. Observation by scanning electron microscopy revealed that the filamentous yeasts adhered to the bacteria on the periphery of the grain.Lactobacillus hilgardii, the single microorganism isolated which was able to produce a gelling polysaccharide, was important in the biosynthesis of the grain. Pieces of gel produced by this strain, and transferred in a yeast extract-sucrose solution, grew and resembled the household kefir grains. This represents a new, cheap way of producing immobilized cells by self-embedding in a neutral polysaccharide. more...

Relatedness among strains of Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus hilgardii, Lactobacillus confusus, Lactobacillus fermentum, Lactobacillus cellobiosus, Lactobacillus reuteri, Lactobacillus collinoides, Lactobacillus fructivorans, Lactobacillus kandleri, and Lactobacillus viridescens was determined by computerized comparison of total soluble cell protein patterns. Two related subgroups of L. buchneri were obtained: the first comprised a tight cluster of strains ATCC 11579 and ATCC 9460 with neotype strain ATCC 4005, and the second contained strains ATCC 12936, ATCC 12935, and ATCC 11307. L. brevis ATCC 13648, ATCC 4006, ATCC 367, and ATCC 8287 appear to be related to the neotype strain of L. brevis (ATCC 14869), L. brevis ATCC 11577 to L. hilgardii, and L. confusus ATCC 27646 to L. brevis ATCC 367 and ATCC 8287. L. brevis ATCC 14434 and L. cellobiosus ATCC 11740 were originally misclassified. The protein patterns of these strains did not resemble those of any of the other named lactobacilli which were examined. Our findings support the reclassification of L. brevis ATCC 8007 as Lactobacillus kefir, L. cellobiosus ATCC 11739 as L. fermentum, and L. fermentum ATCC 23272 as L. reuteri. L. collinoides, L. fructivorans, L. hilgardii, L. kandleri, L. reuteri, and L. viridescens are distinct, separate species. more...

The screening of facultative anaerobes with melanoidin-decolorizing activity (MDA) was performed, and some strains were isolated from stored waste water of an alcohol fermentation involving molasses. One of them, strain W-NS, showed high and stable MDA, and was identical with Lactobacillus hilgardii. The decolorization yield of this strain under the optimal conditions was only 28%, however, it was improved to 40% by immobilization of cells within Ca-alginate gel. Unlike Basidiomycetes and Ascomycetes, this strain decolorized smaller molecular weight fractions of melanoidin, quickly. Also, the MDA of this strain toward various synthetic melanoidin was quite different from that of Basidiomycetes. more...

Nineteen atypical heterofermentative strains of Lactobacillus spp. isolated from South African red wines were identified with the API 50 CHL system and by computerized comparison of their total soluble cell protein patterns. Eleven strains were identified as Lactobacillus hilgardii and eight strains as L. brevis. Three strains of L. hilgardii and three strains of L. brevis produced small amounts of H2S in peptone water supplemented with 001%l-cysteine. All strains produced mannitol from fructose. Proteolytic activity was detected in all except two strains of L. hilgardii. Strains capable of digesting gelatine, hydrolized casein, but not vice versa. The excessive production of sulphur-containing compounds, mannitol and acetaldehyde by lactic acid bacteria could have serious quality implications for the wine industry. Three strains of L. hilgardii and all strains of L. brevis decarboxylated l-malic acid to l-lactic acid. more...

The influence of various yeasts on the growth of lactic acid bacteria in wine was tested by inoculating Lactobacillus hilgardii, L. brevis and two strains of Leuconostoc mesenteroides into experimental wines made with twelve different yeasts of the genus Saccharomyces. Wines made from juice which had been infected with several spoilage yeasts and then fermented with a wine yeast were also tested in this way. It was found that the yeasts differed considerably in their effects on bacterial growth. In some of the experimental wines bacterial growth was delayed or failed altogether. Generally, the unfavourable influence of any yeast on bacterial growth was much reduced if the wines were left in contact with the yeast cells for some weeks after the fermentation. The significance of these results in relation to the occurrence of malo-lactic fermentation in commercial wineries is discussed. more...

SUMMARY: An improved growth medium for lactobacilli is described. It supports good growth of lactobacilli generally and also is particularly useful for a number of fastidious strains which grow only poorly in other general media. In addition, tomato juice, a highly variable material, is not required. In a slightly modified form, it can also be used as a basal medium for fermentation tests. more...

Growth of two heterofermentative lactic acid bacteria (Lactobacillus hilgardii and Leuconostoc mesenteroides) isolated from wine, was inhibited in a medium to which sulphurous acid and an excess of acetaldehyde had been added. It was found that the bacteria rapidly attacked the aldehyde and liberated sufficient free sulphur dioxide to prevent further growth. A homofermentative bacterium (Lactobacillus arabinosus) consumed much less aldehyde than the two heterofermentative organisms, but nevertheless failed to grow in the presence of 100 p.p.m. of bound sulphur dioxide. The significance of these results in relation to the malo-lactic fermentation in wines is discussed. more...

International audience; Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1,3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pastertrianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GD1 and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider. more...