Enzyme Activities Affecting End Product Distribution by Lactobacillus plantarum in Response to Changes in pH and O2

Lactobacillus plantarum catabolic end products changed in response to environmental conditions. While lactate was always the major end product, acetate was produced in alkaline and aerobic environments. Acetoin levels decreased under alkaline conditions. Changes in acetoin dehydrogenase, acetate kinase, NADH oxidase, pyruvate oxidase, and acetate kinase activities correlated with changes in end product distribution.     

Physiological Response of Lactobacillus plantarum to Salt and Nonelectrolyte Stress

In this report, we compared the effects on the growth of Lactobacillus plantarum of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K⁺ (or Na⁺) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.     

Changes in Photorespiratory Enzyme Activity in Response to Limiting CO(2) in Chlamydomonas reinhardtii

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO₂-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO₂. Adaptation to air levels of CO₂ by Chlamydomonas involves induction of a CO₂-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO₂-enriched cells. Unlike WT, the high-CO₂-requiring mutant, cia-5, does not respond to limiting CO₂ conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO₂ which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.     

益生乳酸菌Lactobacillus casei Zhang和Lactobacillus plantarum P8对全价饲料pH及微生物类群变化的研究

研究了益生乳酸菌干酪乳杆菌Zhang(Lactobacillus casei Zhang)和植物乳杆菌P8(Lactobacillus planta-rum P8)对全价饲料pH及微生物类群变化的影响。分别将L.casei Zhang、L.plantarum P8单一菌种及复合菌种(11)以6.30 lg cfu/g的接种总量发酵全价饲料,测定25℃10 d发酵期间全价饲料pH和微生物类群的变化,应用选择培养基测定发酵饲料中的乳酸菌及杂菌(酵母菌、霉菌、大肠菌群、芽胞杆菌和梭状芽胞杆菌)的动态变化,应用RT-PCR技术测定试验组中的L.casei Zhang和L.plantarum P8的动态变化。结果显示,试验组pH下降显著,发酵10 d时,L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料的pH分别为4.23、4.24和4.22,显著低于对照组(P0.05);L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料中的L.casei Zhang、L.plantarum P8活菌数分别为8.91、8.89、6.58和8.69 lg cfu/g。发酵期间,试验组中酵母菌、霉菌、大肠菌群、芽胞杆菌及梭状芽胞杆菌活菌数显著低于对照组(P0.05),其中L.plantarum P8单一菌种发酵和复合菌种发酵对杂菌抑制效果显著优于L.casei Zhang单一菌种发酵(P0.05)。结果表明,全价饲料经L.casei Zhang、L.plantarum P8发酵可以显著降低其pH,抑制其中杂菌的生长,同时L.casei Zhang、L.plantarum P8在饲料中具有良好的稳定性。     

Changes of ginsenosides in Korean red ginseng (Panax ginseng) fermented by Lactobacillus plantarum M1

To obtain microorganisms for the microbial conversion of ginsenosides in red ginseng powder (RGP), Lactobacillus species (M1–M4 and P1–P4) were isolated from commercial ginseng products. Strain M1 was determined to be L. plantarum by 16S rRNA sequencing. Red ginseng powder (RGP) fermented by L. plantarum M1 had a high total content of ginsenosides (142.4 mg/g) as compared to the control (121.8 mg/g). In particular, the ginsenoside metabolites Rg3, Rg5, Rk1, Compound K (CK), Rh1, and Rg2 showed a high level in the fermented RGP (65.5 mg/g) compared to the control (32.7 mg/g). During fermentation for 7 days, total sugar content decreased from 8.55 mg/g to 4 mg/g, uronic acid content reached its maximum (53.43 μg/g) at 3 days, and total ginsenoside content increased to 176.8 mg/g at 4 days. In addition, ginsenoside metabolites increased from 38.0 mg/g to 83.4 mg/g at 4 days of fermentation. Using everted instestinal sacs of rats, the fermented red ginseng showed a high transport level (10.3 mg of polyphenols/g sac) compared to non-fermented red ginseng (6.67 mg of polyphenols/g sac) after 1 h. These results confirm that fermentation with L. plantarum M1 is very useful for preparing minor ginsenoside metabolites while being safe for foods.     

Tannin Degradation by a Novel Tannase Enzyme Present in Some Lactobacillus plantarum Strains

Lactobacillus plantarum is frequently isolated from the fermentation of plant material where tannins are abundant. L. plantarum strains possess tannase activity to degrade plant tannins. An L. plantarum tannase (TanB_Lp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanA_Lp). While all 29 L. plantarum strains analyzed in the study possess the tanB_Lp gene, the gene tanA_Lp was present in only four strains. Upon methyl gallate exposure, the expression of tanB_Lp was induced, whereas tanA_Lp expression was not affected. TanA_Lp showed only 27% sequence identity to TanB_Lp, but the residues involved in tannase activity are conserved. Optimum activity for TanA_Lp was observed at 30°C and pH 6 in the presence of Ca²⁺ ions. TanA_Lp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanA_Lp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanA_Lp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments.     

Enzymatic regulation of glucose catabolism by Lactobacillus plantarum in response to pH shifts in a chemostat

Summary The influence of medium pH on the regulation of glucose catabolism by Lactobacillus plantarum 8014 was examined in anaerobic chemostat cultures. When L. plantarum was grown in a chemostat at pH 5.5, and the pH shifted to pH 7.5, acetate was produced in addition to lactate and acetoin. After the shift, acetate kinase and NAD-dependent lactate dehydrogenase activities increased while the acetoin dehydrogenase and alpha-acetolactate synthase activities decreased. The high acetate kinase activity together with low acetoin dehydrogenase and alpha-acetolactate synthase activities may explain why L. plantarum made more acetate at the expense of acetoin in response to alkaline conditions.Offprint requests to: T.J. Montville     

Generation of H2O2 by Human Neutrophils and Changes of Cytosolic Ca2+ and pH of Rat Thymocytes in Response to Galactoside-Binding Proteins (Lectins or Immunoglobulins)

In contrast to plant agglutinins, biological activities of animal/human lectins are not well defined yet. Testing a panel of seven mammalian carbohydrate-binding proteins we have found that the dimeric lectin from chicken liver (CL-16) was a stimulator of H₂O₂ release from human neutrophils as well as effector for induction of cytosolic Ca²⁺ and pH increase in rat thymocytes. Activity of this lectin was comparable to potent galactoside-specific plant lectins such as Viscum album L. agglutinin. The activities of the tested plant lectins depended significantly on their nominal carbohydrate specificity as well as on the source. The results indicate that endogenous lectins may be involved in the regulation of neutrophil and lymphocyte functions by elicitation of selective biosignaling reactions.     

Conversion of Pyruvate to Acetoin Helps To Maintain pH Homeostasis in Lactobacillus plantarum

Pyruvate is the substrate for diacetyl and acetoin synthesis by lactobacilli. Exogenous pyruvate stimulates acetoin production when glucose is present as an energy source. In Lactobacillus plantarum ATCC 8014, the energy derived from glucose via glycolysis generated a constant proton motive force of about -120 mV. At a low external pH, energized cells rapidly transported and accumulated pyruvate but did not do so when they were deenergized by nigericin. When large amounts of pyruvate were transported and subsequently accumulated internally, the cotransported protons rapidly lowered the internal pH. The conversion of pyruvate to acetoin instead of acidic end products contributed to the maintenance of pH homeostasis. This is the first report showing that the conversion of pyruvate to acetoin serves as a mechanism of pH homeostasis.     

植物乳杆菌LpT1和LpT2体外降胆固醇机制

【目的】初步探讨植物乳杆菌LpT1和LpT2的体外降胆固醇机制。【方法】以MRS、MRS+CH、MRS+CH+S和MRS+CH+N四种培养基为基础,接种植物乳杆菌LpT1和LpT2进行培养,通过分析比较培养基上清液、菌体沉淀和菌体细胞内部胆固醇含量以及接种和未接种两种情况上清液、沉淀和细胞内胆固醇总量变化,推测植物乳杆菌体外降胆固醇机制。【结果】乳酸菌体外降胆固醇存在非代谢和代谢降解两条途径,非代谢途径与共沉淀作用和菌体吸收有关。代谢降解是由于植物乳杆菌在生长过程中产生了特殊的酶系,从而将胆固醇降解成其他物质,导致其含量降低。【结论】研究结果为进一步研究植物乳杆菌体外降胆固醇的机制奠定了良好基础。     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Characterization of wine Lactobacillus plantarum by PCR-DGGE and RAPD-PCR analysis and identification of Lactobacillus plantarum strains able to degrade arginine

Summary Screening of strains isolated from red wine undergoing malolactic fermentation allowed the identification of lactic acid bacteria able to degrade arginine. A denaturing gradient gel electrophoresis approach, using the rpoB gene as the molecular target, was developed in order to characterize the isolated strains. Several strains were identified as Lactobacillus plantarum and were typed by RAPD-PCR with several randomly designed primers. Almost all of the␣L. plantarum strains identified were able to produce citrulline and ammonia, suggesting that the ability of␣L.␣plantarum to degrade arginine is a common feature in wine. During the characterization of the newly identified L.␣plantarum strains, the presence of genes coding for the arginine deiminase (ADI) pathway was observed in the strains able to produce citrulline, while the lack of this genes was observed in strain unable to produce citrulline. These results suggest that the degradation of arginine in L. plantarum is probably strain-dependent.     

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.     

植物乳杆菌LpT1和LpT2大鼠体内降胆固醇特性

【目的】探讨植物乳杆菌LpT1和LpT2大鼠体内降胆固醇特性。【方法】将高脂血症的大鼠随机分成4组,分别进行灌胃。A、B、C和D组分别灌胃菌株LpT1、菌株LpT2、洛伐他汀和蒸馏水。灌胃28d后,断尾采血,分离血清,分别测定总胆固醇、总甘油三酯、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇的含量并进行肝脏组织切片的制作与电镜观察。【结果】饲喂高脂饲料7d后,成功构建出高脂血症大鼠模型。植物乳杆菌菌株LpT1和阳性对照洛伐他汀降胆固醇效果极其显著(p<0.01),菌株LpT2次之(p<0.05),而阴性对照水几乎无降胆固醇效果。从电镜扫描结果看,植物乳杆菌LpT1和LpT2在大鼠肠道中定植后,能很好的调节肝脏代谢脂类物质朝着正常化趋势发展。【结论】研究结果为进一步明确植物乳杆菌体内降胆固醇机制奠定了良好基础。     

Bioenergetic consequences of catabolic shifts by Lactobacillus plantarum in response to shifts in environmental oxygen and pH in chemostat cultures.

Proton motive force (PMF), intracellular end product concentrations, and ATP levels were determined when a steady-state Lactobacillus plantarum 8014 anaerobic chemostat culture was shifted to an aerobic condition or was shifted from pH 5.5 to 7.5. The PMF and intracellular ATP levels increased immediately after the culture was shifted from anaerobic to aerobic conditions. The concentrations of intracellular lactate and acetate, which exported protons that contributed to the proton gradient, changed in the same fashion. The H+/lactate stoichiometry, n, varied from 0.8 to 1.2, and the H+/acetate n value changed from 0.8 to 1.6 at 2 h after the shift to aerobic conditions. The n value for acetate excretion remained elevated at aerobic steady state. When the anaerobic culture was shifted from pH 5.5 to 7.5, intracellular ATP increased 20% immediately even though the PMF decreased 50% as a result of the depletion of the transmembrane proton gradient. The H+/lactate n value changed from 0.7 to 1.8, and n for H+/acetate increased from 0.9 to 1.9 at pH 7.5 steady state. In addition, the H+/acetate stoichiometry was always higher than the n value for H+/lactate; both were higher in alkaline than aerobic conditions, demonstrating that L. plantarum 8014 coexcreted more protons with end products to maintain intracellular pH homeostasis and generate proton gradients under aerobic and alkaline conditions. During the transient to pH 7.5, the n value for H+/acetate approached 3, which would spare one ATP.     

Population Heterogeneity of Lactobacillus plantarum WCFS1 Microcolonies in Response to and Recovery from Acid Stress

Within an isogenic microbial population in a homogenous environment, individual bacteria can still exhibit differences in phenotype. Phenotypic heterogeneity can facilitate the survival of subpopulations under stress. As the gram-positive bacterium Lactobacillus plantarum grows, it acidifies the growth medium to a low pH. We have examined the growth of L. plantarum microcolonies after rapid pH downshift (pH 2 to 4), which prevents growth in liquid culture. This acidification was achieved by transferring cells from liquid broth onto a porous ceramic support, placed on a base of low-pH MRS medium solidified using Gelrite. We found a subpopulation of cells that displayed phenotypic heterogeneity and continued to grow at pH 3, which resulted in microcolonies dominated by viable but elongated (filamentous) cells lacking septation, as determined by scanning electron microscopy and staining cell membranes with the lipophilic dye FM4-64. Recovery of pH-stressed cells from these colonies was studied by inoculation onto MRS-Gelrite-covered slides at pH 6.5, and outgrowth was monitored by microscopy. The heterogeneity of the population, calculated from the microcolony areas, decreased with recovery from pH 3 over a period of a few hours. Filamentous cells did not have an advantage in outgrowth during recovery. Specific regions within single filamentous cells were more able to form rapidly dividing cells, i.e., there was heterogeneity even within single recovering cells.     

Changes in gene expression induced by H(2)O(2) in cardiac myocytes

Oxidative stress induces cardiac myocyte apoptosis. At least some effects are probably mediated through changes in gene expression. Using Affymetrix arrays, we examined the changes in gene expression induced by H(2)O(2) (0.04, 0.1, and 0.2mM; 2 and 4h) in rat neonatal ventricular myocytes. Changes in selected upregulated genes were confirmed by ratiometric RT-PCR. p21(Cip1/Waf1) was one of the only two genes upregulated in all conditions studied. Of the heat shock proteins, only Hsp70/70.1 was induced by H(2)O(2) with no change in the expression of Hsp25, Hsp60 or Hsp90. Heme oxygenase 1 was also potently upregulated, but not heme oxygenases 2 or 3. Of the intercellular adhesion proteins, syndecan-1 was significantly upregulated in response to H(2)O(2), with little change in the expression of other syndecans and no change in expression of any of the integrins studied. Thus, oxidative stress, exemplified by H(2)O(2), selectively promotes the expression of specific gene family members.     

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.     

Influence of CO(2)-HCO(3) Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO(2)-HCO(3) growth conditions and suggest that there exists a threshold level of CO(2) for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO(2)-HCO(3) growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K') was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO(2)-HCO(3) as a consequence of regulating electron sink metabolism.     

Effect of acidic pH and salt on acid end-products by Lactobacillus plantarum in aerated, glucose-limited continuous culture

In aerated, glucose-limited continuous culture, Lactobacillus plantarum altered its metabolic pathways of acid production in response to changes in environmental pH. Under acidic conditions, acetate and L-lactate production decreased with decreasing pH, whereas levels of D-lactate increased. In the presence of 6% NaCl, acetate and lactate production continued, but no acetate was detected at pH 4.5.