Utilization of amino acids and dipeptides by Lactobacillus plantarum from orange in nutritionally stressed conditions.

AIMS: To investigate amino acid and dipeptide utilization by Lactobacillus plantarum N4 isolated from orange peel, in a nutritionally depleted medium based on MRS (Mann, Rogosa, Sharpe). METHODS AND RESULTS: In MRS with 0.1 g l(-1) of meat extract and without peptone and yeast extract, growth increased when essential and stimulatory amino acids and nonessential amino acid were added to the medium. Replacement of the essential amino acid, leucine, and the nonessential amino acid, glycine, by leucyl-leucine (Leu-Leu) and/or glycyl-glycine (Gly-Gly) significantly enhanced growth. Essential amino acids were mainly consumed and the dipeptides were almost completely used at the end of growth. Leucine and glycine accumulated internally from the peptides were higher than from the free amino acids. Glucose utilization increased in the media containing dipeptides compared with the medium containing free amino acids. CONCLUSIONS: In a N-depleted medium, Leu-Leu and/or Gly-Gly were more effective than the respective amino acids in supporting growth of the micro-organism. The more efficient internal accumulation of glycine and especially leucine from dipeptides confirmed the ability of the strain to assimilate mainly complex nitrogen molecules rather than simple ones. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of Lact. plantarum N4 to efficiently use dipeptides could contribute to spoilage development in the natural medium of the organism, orange juice.

Factors affecting the production of putrescine from agmatine by Lactobacillus hilgardii XB isolated from wine.

AIMS: To elucidate and characterize the metabolic putrescine synthesis pathway from agmatine by Lactobacillus hilgardii X(1)B. METHODS AND RESULTS: The putrescine formation from agmatine by resting cells (the normal physiological state in wine) of lactic acid bacteria isolated from wine has been determined for the first time. Agmatine deiminase and N-carbamoylputrescine hydrolase enzymes, determined by HPLC and LC-Ion Trap Mass Spectrometry, carried out the putrescine synthesis from agmatine. The influence of pH, temperature, organic acids, amino acids, sugars and ethanol on the putrescine formation in wine was determined. CONCLUSIONS: Resting cells of Lact. hilgardii X(1)B produce putrescine in wine. The putrescine production was carried out from agmatine through the agmatine deiminase system. SIGNIFICANCE AND IMPACT OF THE STUDY: These results have significance from two points of view, wine quality and toxicological and microbiological aspects, taking account that putrescine, which origin is still controversial, is quantitatively the main biogenic amine found in wine.

Comparative survey of putrescine production from agmatine deamination in different bacteria.

This article aims to study putrescine production in Lactobacillus hilgardii strain X(1)B, an agmatine degrader isolated from wine, and to compare it with three other different species, previously reported as putrescine producers from agmatine: Pseudomonas aeruginosa PAO1, Enterococcus faecalis ATCC11700 and Bacillus cereus CECT 148(T). The effect of different biogenic amines, organic acids, cofactors, amino acids and sugars on putrescine production was evaluated. In some cases, a similar effect was found in all the strains studied but the magnitude differed. Arginine, glucose and fructose showed an inhibitory effect, whereas the presence of agmatine induced the production of putrescine in all microorganisms. In other cases, the effect differed between P. aeruginosa PAO1 and the other microorganisms. Histamine and tyramine poorly influenced the utilization of agmatine, although a small increase in putrescine production was observed in P. aeruginosa PAO1. Succinate, spermidine and spermine also led to an increase in putrescine production in P. aeruginosa PAO1, whereas the succinate had no effect in the other microorganisms. Spermine and spermidine always produced a diminution in agmatine deamination. In this work, we have also demonstrated that pyridoxal 5-phosphate, Mg(2+) and Mn(2+) had no effect on putrescine production from agmatine. Results presented in this paper indicate differences in regulation mechanisms of agmatine deiminase pathway among P. aeruginosa PAO1 and L. hilgardii X(1)B, E. faecalis ATCC11700 and B. cereus CECT 148(T). These results are significant from two points of view, first food quality, and second the toxicological and microbiological aspects. It should be taken into account that putrescine, whose origin is still controversial, is quantitatively the main biogenic amine found in food.

Influence of artificial sweeteners on the kinetic and metabolic behavior of Lactobacillus delbrueckii subsp. bulgaricus.

The addition of artificial sweeteners to a LAPT (yeast extract, peptone, and tryptone) medium without supplemented sugar increased the growth rate and final biomass of Lactobacillus delbrueckii subsp. bulgaricus YOP 12 isolated from commercial yogurt. Saccharin and cyclamate were consumed during microorganism growth, while the uptake of aspartame began once the medium was glucose depleted. The pH of the media increased as a consequence of the ammonia released into the media supplemented with the sweeteners. The L. delbrueckii subsp. bulgaricus strain was able to grow in the presence of saccharin, cyclamate, or aspartame, and at low sweetener concentrations, the microorganism could utilize cyclamate and aspartame as an energy and carbon source.

Arginine dihydrolase pathway in Lactobacillus buchneri: a review.

The arginine dihydrolase system was studied in homo- and hetero-fermentative lactic acid bacteria. This system is widely distributed in Betabacteria lactobacilli subgroup (group II in Bergey's Manual). It is generally absent in the Thermobacterium lactobacilli subgroup (group IA in Bergey's Manual) and also in the Streptobacterium subgroup (group IB in Bergey's Manual). It is present in some species of the genus Streptococcus (groups II, III and IV in Bergey's Manual). In Lactobacillus buchneri NCDO110 the 3 enzymes of the arginine dihydrolase pathway, arginine deiminase, ornithine transcarbamylase and carbamate kinase, were purified and characterized. Arginine deiminase was partially purified (68-fold); ornithine transcarbamylase was also partially purified (14-fold), while carbamate kinase was purified to homogeneity. The apparent molecular weight of the enzymes was 199,000, 162,000 and 97,000 for arginine deiminase, ornithine transcarbamylase and carbamate kinase respectively. For arginine deiminase, maximum enzymatic activity was observed at 50 degrees C and pH 6; for ornithine transcarbamylase it was observed at 35 degrees C and pH 8.5, and for carbamate kinase at 30 degrees C and pH 5.4. The activation energy of the reactions was determined. For arginine deiminase, delta G* values were: 8,700 cal mol-1 below 50 degrees C and 380 cal mol-1 above 50 degrees C; for ornithine transcarbamylase, the values were: 9,100 cal mol-1 below 35 degrees C and 4,300 cal mol-1 above 35 degrees C; for carbamate kinase, the activation energy was: 4,078 cal mol-1 for the reaction with Mn2+ and 3,059 cal mol-1 for the reaction with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and partial characterization of Oenococcus oeni exoprotease.

The exoprotease from Oenococcus oeni produced in stress conditions was purified to homogeneity in two steps, a 14-fold increase of specific activity and a 44% recovery of proteinase activity. The molecular mass was estimated to be 33.1 kDa by gel filtration and 17 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggest that the enzyme is a dimer consisting of two identical subunits. Optimal conditions for activity on grape juice were 25 degrees C and a pH of 4.5. Incubation at 70 degrees C, 15 min, destroyed proteolytic activity. The SDS-PAGE profile shows that the enzyme was able to degrade the grape juice proteins at a significantly high rate. The activity at low pH and pepstatin A inhibition indicate that this enzyme is an aspartic protease. The protease activity increases at acidic pH suggesting that it could be involved in the wine elaboration.

Polysaccharide production by Pediococcus pentosaceus from wine.

Two polymer-forming strains of Pediococcus pentosaceus from Argentinian wines were grown under different conditions. Polysaccharides were produced independently of carbon source at the early logarithmic phase of growth. With the presence of ethanol and SO2, the specific polymers production was greater. The polysaccharides comprised of glucose, fructose and galactose, with predominant linkages of alpha-1,4- and alpha-1,6-glucosidic with a ratio 1:1. They contained approximately 5500 hexose molecules.

Arginine dihydrolase pathway in Lactobacillus plantarum from orange.

Lactobacillus plantarum N8 and N4 strains isolated from orange degraded L-arginine to citrulline, ornithine and ammonia. Citrulline and ornithine were consumed. Lactobacillus plantarum N4 utilized arginine and ornithine to a higher extent than Lactobacillus plantarum N8. Urea was not detected during arginine degradation, indicating that the amino acid degradation was carried out only by the arginine dihydrolase pathway. Citrulline increased the growth of the two strains, arginine only increased the growth of Lactobacillus plantarum N4. Ornithine did not modify the growth of the strains studied. With different behavior, Lactobacillus plantarum N8 and N4 strains were able to derive energy and ammonia from arginine or citrulline catabolism. This is interesting for microorganisms developing in a stressful environment.

Arginine, citrulline and ornithine metabolism by lactic acid bacteria from wine.

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.

Pediocin N5p from Pediococcus pentosaceus: adsorption on bacterial strains.

Pediocin N5p is a bacteriocin produced by Pediococcus pentosaceus isolated from wine. It is absorbed on both sensitive and resistant Gram-positive and Gram-negative bacteria in non specific (non lethal) sites and higher values, up to 30% are observed in sensitive strains suggesting the presence of particular (lethal) receptors. Cell death without lysis is produced by the action of pediocin on sensitive strains. The cations Mg2+ and Mn2+ increase the pediocin binding by 80 and 100%, respectively. Treatment of sensitive cells with proteolytic enzymes and 1% SDS increases the subsequent binding of bacteriocin by 100 and 25%, respectively. As a lipid moiety of pediocin N5p is critical to its activity, probably a hydrophobic interaction with the peptidic receptor, stabilized by Mn2+ or Mg2+ might be established. Pediocin N5p adsorption is not affected by ethanol and SO2, two factors involved in vinification.

Effect of gallic acid and catechin on Lactobacillus hilgardii 5w growth and metabolism of organic compounds.

The effects of different concentrations of (+)-catechin and gallic acid on the growth and metabolism of Lactobacillus hilgardii in different media were evaluated. These phenolic compounds at concentrations normally present in wine not only stimulated the growth rate but also resulted in greater cell densities during the stationary phase of growth in both media. During the first hours of growth both phenolic compounds activated the rate of glucose and fructose utilization and only catechin increased the malic acid consumption rate. Gallic acid and catechin were consumed from the beginning of L. hilgardii growth. All cited effects were increased when the cells were precultivated in the presence of phenolic compounds, especially in the FT80 medium. As stimulating agents of L. hilgardii 5w growth, gallic acid and catechin could increase the risk of spoilage lactic acid bacteria in wine.

Aspartate aminotransferase of Lactobacillus murinus.

Aspartate aminotransferase from Lactobacillus murinus is thermostable, its activity being not changed for two months at temperatures between 4 and -70 degrees C. Maximum activity was observed at 40 degrees C and pH 7.3 in phosphate buffer (30 mmol/L). delta G* Value of 26.3 kJ/mol was calculated from the Arrhenius plot. The Km values for L-aspartate and 2-oxoglutarate at pH 7.3 were 25 and 100 mmol/L, respectively. Sodium maleate and glutamate acted as inhibitors of the enzyme activity. The Ki values for sodium maleate with L-aspartate of 2-oxoglutarate as variable substrates were 1.1 and 0.5 mmol/L, respectively. The Ki values for glutamate with L-aspartate or 2-oxoglutarate were 8.0 and 4.0 mmol/L, respectively. An inhibitory effect was observed with 1 mM Hg2+ ions (1 mmol/L). The activity of the enzyme was diminished by only 12% in the absence of pyridoxal 5'-phosphate.

Effect of cultivation conditions on proteinase production by Lactobacillus murinus.

Proteinase production by Lactobacillus murinus was influenced by temperature, glucose concentration, initial pH and nitrogen sources. Maximum proteinase production occurred at 45 degrees C, pH 6.6 and with 0.5% (W/V) glucose. Tryptone, peptone and gelatin inhibited it.

beta-D-galactosidase of Lactobacillus species.

The activity of beta-D-galactosidase was studied in 13 strains of lactobacilli (groups Streptobacterium, Thermobacterium and Betabacterium). Using 2-nitrophenyl galactopyranoside as substrate, the enzyme activity varied with the strain. The values found in the Thermobacterium group were superior to those in the Streptobacterium group. The optimum pH for the species belonging to the Thermobacterium group was uniform, in contrast to the pH for those from the Streptobacterium which varied according to the species. The optimum temperature was quite uniform within each group and higher in the Streptobacterium. Lactose acted as a competitive inhibitor. MgCl2 protected the enzyme from thermal denaturation. The calcium ions inhibited the activity in all cases. The behaviour of the protectors of the SH groups varied according to the strain. 6-Phospho-beta-D-galactosidase activity was also determined, levels lower than beta-D-galactosidase were found, except in Lactobacillus plantarum ATCC 8014 and 14917.

Characterization of a Lactobacillus plantarum strain able to produce tyramine and partial cloning of a putative tyrosine decarboxylase gene.

The aim of this article was to analyze the ability of wine Lactobacillus plantarum strains to form tyramine. Preliminary identification of L. plantarum strains was performed by amplification of the recA gene. Primers pREV and PlanF, ParaF and PentF were used respectively as reverse and forward primers in the polymerase chain reaction tests as previously reported. Furthermore, the gene encoding for the tyrosine decarboxylase (TDC) was partially cloned from one strain identified as L. plantarum. The strain was further analyzed by 16S rDNA sequence and confirmed as belonging to L. plantarum species. The tyrosine decarboxylase activity was investigated and tyramine was determined by the high-performance liquid chromatography method. Moreover, a negative effect of sugars such as glucose and fructose and L: -malic acid on tyrosine decarboxylase activity was observed. The results suggest that, occasionally, L. plantarum is able to produce tyramine in wine and this ability is apparently confined only to L. plantarum strains harboring the tdc gene.

Interaction between Lactobacillus hilgardii and Pediococcus pentosaceus and their metabolism of sugars and organic acids.

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.

Growth and sugars utilization by mixed cultures ofLactobacillus delbrueckii subsp.bulgaricus andStreptococcus salivarius subsp.thermophilus isolated from Argentina yogurt.

Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss1 and Yop12) andStreptococcus salivarius subsp.thermophilus (Ss2 and Yop9) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop12+Ss2) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.

Transcription from complementary deoxyribonucleic acid strands in various sporogenic and asporogenic mutants of Bacillus subtilis. Hybridization-competition studies on ribonucleic acid synthesized in vivo by a thermosensitive sporulation mutant (ts-4).

The present paper describes an investigation, at the transcription level, in a thermosensitive sporulation mutant of Bacillus subtilis (ts-4) grown at the permissive (30 degrees C) or restrictive (42 degrees C) temperature where sporulation capacity is respectively expressed or arrested. These studies were carried out by analysing the ribonucleic acid from vegetative and stationary phase cells (t3 cells) grown under both conditions, by hybridization-competition experiments.

Biogenic amine production by Lactobacillus.

AIMS: The aim of this work was to demonstrate that strains of Lactobacillus may be able to produce putrescine and agmatine from one of the major amino acids present in fruit juices and wine, arginine, and from amino acid-derived ornithine. METHODS AND RESULTS: Biogenic amines were determined by HPLC. Their production in the culture medium was similar under both microaerophilic and anaerobic conditions. The presence of Mn2+ had a minimal influence on the results, whereas the addition of pyridoxal phosphate increased amine production 10-fold. Lactobacillus hilgardii X1B, isolated from wine, was able to degrade arginine by two pathways: arginine deiminase and arginine decarboxylase. The isolate was able to produce putrescine from ornithine and from agmatine. Lactobacillus plantarum strains N4 and N8, isolated from orange, utilized arginine via the arginine deiminase system. Only the N4 strain was able to produce putrescine from ornithine. CONCLUSION: It has been demonstrated that Lact. hilgardii X1B is able to produce the most important biogenic amine found in wine, putrescine, and also agmatine from arginine and ornithine, and that Lactobacillus plantarum, considered to be an innocuous spoilage micro-organism in fruit juices, is able to produce amines. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to food poisoning caused by beverages that have been contaminated with biogenic amines.

Flocculation and cell surface characterization of Kloeckera apiculata from wine.

AIMS: To characterize and analyze the flocculation phenomenon of Kloeckera apiculata mc1 from Argentinian wine to understand the cell-cell interaction pattern. METHODS AND RESULTS: Kloeckera apiculata mc1 possess intense cell-cell interactions in MYPG medium (0.5% malt extract, 1% yeast extract, 2% glucose, 2% peptone), pH 5.5 by shaking at 25 degrees C. Optimum flocculation is observed at pH 4.5 in the presence of 3 mmol l-1 Ca2+. The flocculation is induced by peptone and malt extract and not by yeast extract and is reversed by 50 mmol l-1 galactose or lactose. The flocculation is highly susceptible to pronase, chymotrypsine and proteases types IV and XXVII and is partially resistant to trypsin. The electronic microscopy shows that the cells are attached to each other along their sides by fine hair-like threads. CONCLUSIONS: The mechanism of flocculation of K. apiculata mc1 is mediated by protein-carbohydrate interaction, stabilized by Ca2+. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of selected pure yeast inocula of known ability is preferred to wine elaboration, therefore the indigenous flora must be avoided and the flocculation of K. apiculata could be an economic method to do it.