Oxygen metabolism of catalase-negative and catalase-positive strains of Lactobacillus plantarum.

Two catalase-negative strains of Lactobacillus plantarum and a strain producing the atypical, nonheme catalase were studied to determine if the ability to produce the atypical catalase conferred any growth advantage upon the producing strain. Both catalase-negative strains grew more rapidly than the catalase-positive strain under aerobic or anaerobic conditions in a glucose-containing, complex medium. Upon exhaustion of glucose from the medium, all three strains continued growth under aerobic but not under anaerobic conditions. The continued aerobic growth was accompanied by production of acetic acid in addition to the lactic acid produced during growth on glucose. Oxygen was taken up by exponential phase-cell suspensions grown on glucose when glucose or glycerol were used as substrates. Cells harvested from glucose-exhausted medium oxidized glucose, glycerol, and pyruvate. Oxygen utilization by a catalase-negative strain increased as did the specific activity of reduced nicotinamide adenine dinucleotide peroxidase during late growth in the glucose-exhausted medium. The catalase-positive strain and the catalase-negative strain tested both possessed low but readily detectable levels of superoxide dismutase throughout growth. The growth responses are discussed in terms of the presence of enzymes which would allow the cells to remove potentially damaging reduction products of O2.     

Anaerobic l-lactate degradation by Lactobacillus plantarum

Abstract Lactobacillus plantarum strains used as silage inoculants were investigated for their ability to metabolize lactic acid anaerobically after prolonged incubation (7–30 days) when glucose was absent from the medium. When citrate was present in the medium together with glucose during the initial fermentation, the lactic acid produced was degraded. Citrate was concomitantly degraded, resulting in accumulation of formic, acetic and succinic acids along with CO₂. The anaerobic degradation was confirmed by the use of l ¹⁴C(U) labelled lactate. The existence of pyruvate formate lyase in L. plantarum was indicated by using ¹⁴C-labelled pyruvate and HPLC identification of end-products. The 1-¹⁴C-carboxylic acid group of pyruvate was converted to formic acid, and the 3-¹⁴C was found in acetic acid. The key enzyme(s) in this metabolic pathway appears to require anaerobic conditions and induction by citrate.     

Involvement of pyruvate oxidase activity and acetate production in the survival of Lactobacillus plantarum during the stationary phase of aerobic growth

In addition to the previously characterized pyruvate oxidase PoxB, the Lactobacillus plantarum genome encodes four predicted pyruvate oxidases (PoxC, PoxD, PoxE, and PoxF). Each pyruvate oxidase gene was individually inactivated, and only the knockout of poxF resulted in a decrease in pyruvate oxidase activity under the tested conditions. We show here that L. plantarum has two major pyruvate oxidases: PoxB and PoxF. Both are involved in lactate-to-acetate conversion in the early stationary phase of aerobic growth and are regulated by carbon catabolite repression. A strain devoid of pyruvate oxidase activity was constructed by knocking out the poxB and poxF genes. In this mutant, acetate production was strongly affected, with lactate remaining the major end product of either glucose or maltose fermentation. Notably, survival during the stationary phase appeared to be dramatically improved in the poxB poxF double mutant.     

Amplifying the cellular reduction potential of Streptococcus zooepidemicus

The valuable pharmaceutical polymer, hyaluronic acid, is produced industrially using the gram-positive bacterium Streptococcus zooepidemicus. Synthesis of this polymer is a significant energetic burden upon the microorganism hence the native NADH oxidase gene was cloned and overexpressed to increase the energy yield of catabolism during aerobic cultivation on glucose. Elevated NADH oxidase levels led to a decline in lactic acid generation and prevented ethanol formation, leaving acetate as the main fermentation product. Biomass yield increased due to the energy gained from the formation of acetate. Evaluation of the acetate flux control coefficient over a range of NADH oxidase expression levels revealed that acetate production was sensitive to the NADH oxidase level. However, at high NADH oxidase levels, the acetate flux was mainly influenced by another factor. The concomitant excretion of pyruvate at high NADH oxidase levels suggested that the flux through the pyruvate dehydrogenase enzyme complex was limiting the conversion of pyruvate to acetate.     

Enzyme Activities Affecting End Product Distribution by Lactobacillus plantarum in Response to Changes in pH and O(2)

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.     

Arabinose fermentation by Lactobacillus plantarum in sourdough with added pentosans and alphaalpha-L-arabinofuranosidase: a tool to increase the production of acetic acid

Sixty-five strains of obligately and facultatively heterofermentative sourdough lactic acid bacteria were screened for their capacity to grow optimally in the presence of arabinose, ribose and xylose as carbon sources. Lactobacillus alimentarius 15F, Lact. brevis 10A, Lact. fermentum 1F and Lact. plantarum 20B showed higher growth rate, cell yield, acidification rate and production of acetic acid when some pentoses instead of maltose were added to the SDB medium. Lactobacillus plantarum 20B used arabinose also in a synthetic medium where complex growth factors such as yeast extract were omitted. Other Lact. plantarum strains did not show the same property. Pentosan extract was treated with alpha-L-arabinofuranosidase from Aspergillus niger or endo-xylanase from Bacillus subtilis to produce hydrolysates containing mainly arabinose and xylose, respectively. In particular, the hydrolysate containing arabinose substantiated the growth and the production of lactic acid and, especially, of acetic acid by Lact. plantarum 20B. Sourdough fermentation by Lact. plantarum 20B with addition of pentosan extract and alpha-L-arabinofuranosidase increased the acidification rate, titratable acidity and acetic acid content compared with traditional sourdough. A facultatively heterofermentative strain, Lact. plantarum 20B, also produced a sourdough with an optimal fermentation quotient.     

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.     

Effect of oxygenation and sulfate concentrations on pyruvate and lactate formation inKlebsiella oxytoca ZS growing in chemostat cultures

Klebsiella oxytoca ZS fermented glucose to ethanol and lactic, formic, and acetic acids, but, in contrast to many strains, accumulates pyruvic and acetic acids as the principal end products in aerobic growth conditions. This strain was grown in sulfate-limited chemostat at a fixed low dilution rate (D=0.033 h^–1) with glucose present in excess. When oxygen was supplied at a high level, pyruvate and acetate were produced, and the ratio NADH/NAD⁺ was low (0.04) while the internal pyruvate concentration increased to 100 mol (g dry wt)^–1. A shortage of oxygen supply was accompanied by lactate production, an increase of the ratio NADH/NAD⁺ (0.53), and an undetectable level in internal pyruvate concentration. The observed changes in LDH activity found in vitro in extracts of the cells are not strictly related to those found in vivo. In fact, the specific activity of LDH was essentially stable at 30% of dissolved oxygen tension (d.o.t.) and decreased slightly at 60% of d.o.t., whereas specific lactic acid production decreased rapidly. The in vitro LDH activity was strongly affected by the NADH/NAD⁺ ratio.     

Metabolism of pyruvate and citrate in lactobacilli

Lactobacillus acidophilus, L. bulgaricus, L. casei, L. delbrueckii , L. lactis and L. plantarum contained a pyruvate oxidase for the oxidation of pyruvate to acetyl phosphate and acetate. The presence of an acetate kinase converted the acetyl phosphate to acetate. L. casei and L. plantarum produced lactate and acetoin, in addition to acetate, under the conditions used while L. casei also produced diacetyl. L. casei and L. plantarum were the only species to utilize citrate. L. helveticus and L. helveticus subsp. jugurti did not utilize pyruvate under the conditions used.     

Glucose degradation, molar growth yields, and evidence for oxidative phosphorylation in Streptococcus agalactiae

In a complex medium with the energy source as the limiting nutrient factor and under anaerobic growth conditions, Streptococcus agalactiae fermented 75% of the glucose to lactic acid and the remainder to acetic and formic acids and ethanol. By using the adenosine triphosphate (ATP) yield constant of 10.5, the molar growth yield suggested 2 moles of ATP per mole of glucose from substrate level phosphorylation. Under similar growth conditions, pyruvate was fermented 25% to lactic acid, and the remainder was fermented to acetic and formic acids. The molar growth yield suggested 0.75 mole of ATP per mole of pyruvate from substrate level phosphorylation. Under aerobic growth conditions about 1 mole of oxygen was consumed per mole of glucose; about one-third of the glucose was converted to lactic acid and the remainder to acetic acid, acetoin, and carbon dioxide. Molar growth yields indicated 5 moles of ATP per mole of glucose. Estimates based on products of glucose degradation suggested that about one-half of the ATP was derived from substrate level phosphorylation and one-half from oxidative phosphorylation. Addition of 0.5 m 2,4-dinitrophenol reduced the growth yield to that occurring in the absence of oxygen. Aerobic pyruvate degradation resulted in 30% of the substrate becoming reduced to lactic acid and the remainder being converted to acetic acid and carbon dioxide, with small amounts of formic acid and acetoin. The molar growth yields and products found suggested that 0.70 mole of ATP per mole of pyruvate resulted from substrate level phosphorylation and 0.4 mole per mole of pyruvate resulted from oxidative phosphorylation.     

Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters

AIMS: The characterization of global aerobic metabolism of Lactobacillus plantarum LP652 under different aeration levels, in order to optimize acetate production kinetics and to suppress H2O2 toxicity. METHODS AND RESULTS: Cultures of L. plantarum were grown on different aeration conditions. After sugar exhaustion and in the presence of oxygen, lactate was converted to acetate, H2O2 and carbon dioxide with concomitant ATP production. Physiological assays were performed at selected intervals in order to assess enzyme activity and vitality of the strain during lactic acid conversion. The maximal aerated condition led to fast lactate-to-acetate conversion kinetics between 8 and 12 h, but H2O2 immediately accumulated, thus affecting cell metabolism. Pyruvate oxidase activity was highly enhanced by oxygen tension and was responsible for H2O2 production after 12 h of culture, whereas lactate oxidase and NADH-dependent lactate dehydrogenase activities were not correlated to metabolite production. Limited NADH oxidase (NOX) and NADH peroxidase (NPR) activities were probably responsible for toxic H2O2 levels in over-aerated cultures. CONCLUSION: Modulating initial airflow led to the maximal specific activity of NOX and NPR observed after 24 h of culture, thus promoting H2O2 destruction and strain vitality at the end of the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimal aeration conditions were determined to minimize H2O2 concentration level during growth on lactate.     

Pyruvate fermentation by Oenococcus oeni and Leuconostoc mesenteroides and role of pyruvate dehydrogenase in anaerobic fermentation

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate --> 1 lactate + 1 acetate + 1 CO(2)). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.     

Induced lactic acid fermentation during the preservation stage of ripe olives from Hojiblanca cultivar

The influence of initial sodium chloride concentration (6 and 0%, w/v), acetic acid concentration (0.6, 0.3 and 0.0%, v/v), type of process (natural and inoculated), and storage system (anaerobic and aerobic) on the inducement of a lactic fermentation for the preservation stage of Hojiblanca cultivar ripe olives was investigated. The addition of 6% NaCl prevented colonization by lactic acid bacteria in all cases. A high level of acetic acid (0.6%) was effective in preserving olives for 2 months, although yeast growth was not inhibited for longer periods of storage. Natural growth of Lactobacillus plantarum did not occur. Inoculation with this micro-organism was effective only in the two treatments with tap water (with no NaCl) as the initial covering solution, although survival was reduced to a half of the added organisms when the initial pH was corrected with 0.3% acetic acid. In these two treatments pH quickly reached appropriate values (<4.0) for olive stabilization. Aerobic conditions led to low concentrations of carbon dioxide, without disturbing growth of lactic acid bacteria. Thus, the aerobic lactic acid fermentation, with tap water initially, was the most adequate preservation procedure for the storage of ripe olives prior to their oxidation treatment. Results of trials conducted on an industrial scale showed the same pattern and confirmed the viability of the new procedure.     

Aerobic Metabolism of Streptococcus agalactiae

Streptococcus agalactiae cultures possess an aerobic pathway for glucose oxidation that is strongly inhibited by cyanide. The products of glucose oxidation by aerobically grown cells of S. agalactiae 50 are lactic and acetic acids, acetylmethylcarbinol, and carbon dioxide. Glucose degradation products by aerobically grown cells, as percentage of glucose carbon, were 52 to 61% lactic acid, 20 to 23% acetic acid, 5.5 to 6.5% acetylmethylcarbinol, and 14 to 16% carbon dioxide. There was no evidence for a pentose cycle or a tricarboxylic acid cycle. Crude cell-free extracts of S. agalactiae 50 possessed a strong reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase that is also cyanide-sensitive. Dialysis or ultrafiltration of the crude, cell-free extract resulted in loss of NADH(2) oxidase activity. Oxidase activity was restored to the inactive extract by addition of the ultrafiltrate or by addition of menadione or K(3)Fe(CN)(6). Noncytochrome iron-containing pigments were present in cell-free extracts of S. agalactiae. The possible participation of these pigments in the respiration of S. agalactiae is presently being studied.     

Characteristics of some lactic Acid bacteria used as starter cultures in dry sausage production

The fermentation characteristics of two commercial (Duploferment 66 and Saga II) and five Norwegian lactic acid bacteria used in dry sausage production were compared with those of Lactobacillus plantarum ATCC 8014. The Norwegian strains lacked the ability to ferment mannitol, sorbitol, lactose, and d-(+)-raffinose and grew at 8 but not at 42 degrees C, in contrast to the ATCC culture and the two commercial strains. The lactate dehydrogenase activity of the Norwegian strains was not stimulated by pyruvate. All strains were capable of peroxide destruction when grown in the presence of myoglobin.     

The effect of sodium acetate on the growth yield,the production of L- and D-lactic acid,and the activity of some enzymes of the glycolytic pathway of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T)

The effect of sodium acetate was studied on the change of the growth yield, the production of L- and D-lactic acid, and the activity of lactate dehydrogenases (LDHs; L-lactate dehydrogenase [EC 1.1.1.27, L-LDH] plus D-lactate dehydrogenase [EC 1.1.1.28, D-LDH]), fructose-1, 6-bisphosphate aldolase [EC 4.1.2.13, FBP-aldolase], and phosphofructokinase [EC 2.7.1.11, PFK] of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T). The growth yield of L. sakei NRIC 1071(T) was increased 1.6 times in the presence of sodium acetate compared with its absence. The activity of LDHs in L. sakei NRIC 1071(T) and L. plantarum NRIC 1067(T) was retained longer under the addition of sodium acetate in the reaction mixture. As a result, these strains produced much more lactic acid in the presence of sodium acetate compared with its absence. Furthermore, the activity of L-LDH in L. sakei NRIC 1071(T) cultivated in the presence of sodium acetate increased three times or more compared with the activity of the cells cultivated in its absence. Consequently, the type of stereoisomers of lactic acid produced by L. sakei shifted from the DL-type to the L-type because the ratio of L-lactic acid to D-lactic acid produced became larger with the addition of sodium acetate to culture media. This phenomenon was not observed in L. plantarum NRIC 1067(T). Further, the participation of lactate racemase is discussed from the viewpoint of the production of D-lactic acid by L. sakei.     

Regulatory mechanisms of cellular respiration. III. Enzyme distribution in the cell. Its influence on the metabolism of pyruvic acid by bakers' yeast

The rate of the aerobic metabolism of pyruvic acid by bakers' yeast cells is determined mainly by the amount of undissociated acid present. As a consequence, the greatest rate of oxidation was observed at pH 2.8. Oxidation, at a slow rate, started at pH 1.08; at pH 9.4 there was no oxidation at all. The anaerobic metabolism, only a fraction of the aerobic, was observed only in acid solutions. There was none at pH values higher than 3. Pyruvic acid in the presence of oxygen was oxidized directly to acetic acid; in the absence of oxygen it was metabolized mainly by dismutation to lactic and acetic acids, and CO₂. Acetic acid formation was demonstrated on oxidation of pyruvic acid at pH 1.91, and on addition of fluoroacetic acid. Succinic acid formation was shown by addition of malonic acid. These metabolic pathways in a cell so rich in carboxylase may be explained by the arrangement of enzymes within the cell, so that carboxylase is at the center, while pyruvic acid oxidase is located at the periphery. Succinic and citric acids were oxidized only in acid solutions up to pH 4. Malic and α-ketoglutaric acids were not oxidized, undoubtedly because of lack of penetration.     

植物乳杆菌DY6主要抑菌代谢物的分析和鉴定

【背景】被广泛应用于食品和饲料等多个行业的乳酸菌已成为制作生物防腐剂的研究热点。【目的】探究抑菌性能良好的植物乳杆菌DY6的抑菌物质,为其进一步应用提供参考依据。【方法】对植物乳杆菌发酵液中抑菌物质的理化特性进行研究,采用GC-MS分析发酵上清液代谢物,通过多元统计学分析方法推测主要抑菌物质,抑菌物质通过半制备进行初步分离后用GC-MS鉴定。【结果】植物乳杆菌DY6对金黄色葡萄球菌、大肠杆菌、沙门氏菌都有较强的抑制作用。采用不同发酵时间的发酵液作为研究对象,测定发酵上清液的抑菌能力,发酵0-4 h上清液无抑菌能力,发酵至8 h抑菌能力逐步上升,发酵24-48 h发酵上清液抑菌能力趋于稳定,在48 h时抑菌能力最佳,抑菌直径为15.28mm。通过多元统计学分析乳酸菌发酵液差异标志物,发现主要差异物为有机酸(如乳酸、乙酸、丙酸等)和脂肪酸(如辛酸、癸酸等)。经过半制备液相分离发酵上清液得到的抑菌组分,主要有有机酸(如乳酸、乙酸、3-苯基乳酸、苯丙酸等)和脂肪酸(如癸酸、辛酸、壬酸等),另外还有少量的醛类和醇类物质。【结论】确定了植物乳杆菌DY6的抑菌物质主要为有机酸和脂肪酸,为其进一步防腐应用提供了理论基础。