Malic Acid Utilization Research Articles

Metabolomic analysis of the effects of a mixed culture of Saccharomyces cerevisiae and Lactiplantibacillus plantarum on the physicochemical and quality characteristics of apple cider vinegar.

This study compared differences in physicochemical characteristics of the vinegar made by a mixed culture (MC) of Saccharomyces cerevisiae and Lactiplantibacillus plantarum and a pure culture (PC) of Saccharomyces cerevisiae. The fermentation process was monitored, and metabolomics analysis by Liquid Chromagraphy-Mass Spectrometry (LC-MS) was applied to the compositional differences between PC and MC vinegars, combined with quantification of organic acids, amino acids and B vitamins. A total of 71 differential metabolites including amino acids, organic acids and carbohydrates, and six possible key metabolic pathways were identified. MC enhanced the malic acid utilization and pyruvate acid metabolism during fermentation, increasing substrate-level phosphorylation, and supplying more energy for cellular metabolism. Higher acidity at the beginning of acetic acid fermentation, resulting from lactic acid production by Lactiplantibacillus plantarum in MC, suppressed the cellular metabolism and growth of Acetobacter pasteurianus, but enhanced its alcohol metabolism and acetic acid production in MC. MC vinegar contained more vitamin B, total flavonoids, total organic acids, amino acids and had a higher antioxidant capacity. MC enhanced the volatile substances, particularly ethyl lactate, ethyl caprate and ethyl caproate, which contributed to a stronger fruity aroma. These results indicated the mixed culture in alcoholic fermentation can effectively enhance the flavor and quality of apple cider vinegar.

Ideonella alba sp. nov. and Ideonella aquatica sp. nov. isolated from an aquaculture farm.

The three novel bacterial strains designated as 3Y2T, 4Y16 and 4Y11T were isolated from an aquaculture farm and characterized using a polyphasic taxonomic approach. These strains were determined to be catalase- and oxidase-positive and to hydrolyze gelatin and aesculin. The results of 16S rRNA gene-based phylogenetic analysis indicated that the three strains were related to members of the genus Ideonella. The phylogenomic results further indicated that the three strains formed two independent branches distinct from reference type strains within this genus. The digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) and average amino acid identity (AAI) values between the three strains and their relatives were far below the thresholds of 70 % dDDH, 95-96 % ANI and 95 % AAI for species definition, respectively, indicating that the three strains represent two novel genospecies. The results of chemotaxonomic characterization indicated that the major cellular fatty acids of the three strains were summed feature 3 (C16 : 1ω6c and/or C16 : 1 ω7c) and C16 : 0; the common main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol; the respiratory quinone was ubiquinone-8. The genomic DNA G+C contents of the three strains were 70.2, 70.1 and 69.7%, respectively. On the basis of the different genotypes and distinctive phenotypes such as the phosphatidylcholine and glycolipid only in 3Y2T and the utilization of malic acid and trisodium citrate only in 4Y11T, strains 3Y2T and 4Y11T are concluded to represent two novel species of the genus Ideonella, for which the names Ideonella alba sp. nov. (type strain 3Y2T = GDMCC 1.2584T = KCTC 82813T) and Ideonella aquatica sp. nov. (type strain 4Y11T = GDMCC 1.1935T = JCM 34285T) are proposed.

Influence of Altered Microbes on Soil Organic Carbon Availability in Karst Agricultural Soils Contaminated by Pb-Zn Tailings.

Soil organic carbon (SOC) availability is determined via a complex bio-mediated process, and Pb-Zn tailings are toxic to the soil microbes that are involved in this process. Here, Pb-Zn-tailings- contaminated karst soils with different levels (paddy field > corn field > citrus field > control group) were collected to explore the intrinsic relationship between Pb-Zn tailings and microbes due to the limited microbial abundance in these soils. The SOC concentration in the paddy fields is the highest. However, based on the soil microbial diversity and sole-carbon-source utilization profiles, the rate of SOC availability, McIntosh index, Shannon-Wiener diversity index, Simpson’s diversity index and species richness are the lowest in the rice paddy soils. According to the results of Illumina sequencing of the 16S rRNA gene, Acidobacteria and Proteobacteria are the dominant phyla in all samples, accounting for more than 70% of the reads, while the majority of the remaining reads belong to the phyla Verrucomicrobia, Chloroflexi, Actinobacteria, Bacteroidetes, and Nitrospirae. We also observed that their class, order, family, genus and operational taxonomic units (OTUs) were dependent on SOC availability. Pearson correlation analysis reveals that L-asparagine utilization profiles show significant positive correlation with OTUs 24, 75, and 109 (r = 0.383, 0.350, and 0.292, respectively), and malic acid utilization profiles show significant positive correlation with OTUs 4, 5, 19, 27 (Bradyrhizobium), 32 (Burkholderia), 75 and 109 (r = 0.286, 0.361, 0.387, 0.384, 0.363, 0.285, and 0.301, respectively), as also evidenced by the redundancy analysis (RDA) biplot and heat map. These results indicate that the most abundant groups of bacteria, especially the uncultured facultative Deltaproteobacteria GR-WP33-30 (OTU 24), after long-term acclimation in heavy metal-contaminated soil, are associated with the variance of labile carbon source such as L-asparagine and may have considerable control over the stability of the vast SOC pool in karst surface soils with different agricultural land-use practices. These findings can expand our understanding of global soil-carbon sequestration and storage via changes in microbial community structure of the most abundant species.

Analysis of organic acids of tricarboxylic acid cycle in plants using GC-MS, and system modeling

BackgroundLeaves of 15 plant species were collected from the catchment areas of the river Beas, Punjab, India, and analyzed for organic acids of tricarboxylic acid cycle, viz., citric acid (CA), succinic acid (SA), fumaric acid (FmA), and malic acid (MA).MethodsGas chromatography-mass spectrometry (GC-MS) was used to determine the content of organic acids in the leaves of plant species. Two microliters of plant sample was injected into the GC-MS, and the concentration of organic acids was quantified using standard curve.ResultsAverage concentrations of these acids in the leaves of plants studied were 4.79, 0.98, 0.54, and 8.36 mg/g dw, respectively. The maximum contents of these acids were found in the leaves of Chenopodium album (CA = 6.42 mg/g dw), Argemone mexicana (SA = 1.27 and FmA = 0.73 mg/g dw), and Rumex dentatus (MA = 18.0 mg/g dw). Factor analysis revealed mainly two underlying factors for organic acids: Factor-1 having maximum loadings on SA and FmA and Factor-2 had maximum loadings on CA and MA. Multiple linear regression analysis of MA on other acids showed that CA and SA have positive regressions, whereas FmA has a negative regression on MA. In artificial neural network analysis, correlation between the target and output values of MA was found to be highly significant. System transfer coefficients were calculated from simulation graphs fitted to the mean values of different organic acids by using difference equations.ConclusionsFrom the present study, it was found that citric acid has a maximum direct effect on the malic acid as compared to succinic and fumaric acids as revealed by path analysis. System modeling revealed that the rate of utilization of malic acid is about 9%. The present study describes a new system simulation technique in which a pathway comprising of linear transformation of biochemical constituents may be characterized in terms of its rate transfer coefficients.

Pseudomonas lini Strain ZBG1 Revealed Carboxylic Acid Utilization and Copper Resistance Features Required for Adaptation to Vineyard Soil Environment: A Draft Genome Analysis.

Pseudomonas lini strain ZBG1 was isolated from the soil of vineyard in Zellenberg, France and the draft genome was reported in this study. Bioinformatics analyses of the genome revealed presence of genes encoding tartaric and malic acid utilization as well as copper resistance that correspond to the adaptation this strain in vineyard soil environment.

Use of flow cytometry with fluorescent antibodies in real-time monitoring of simultaneously inoculated alcoholic-malolactic fermentation of Chardonnay

To monitor in real-time the changes in microbial populations and chemistry of grape juice simultaneously inoculated with Saccharomyces cerevisiae and Oenococcus oeni. Viable populations of S. cerevisiae and O. oeni in Chardonnay fermentations were identified and quantified using fluorescent dyes and fluorescently labelled antibodies in a flow cytometric assay. Fermentation chemistry was monitored using Fourier transform infrared (FTIR) spectroscopy, except for malic acid which was measured enzymatically. Malic acid utilization by O. oeni was greatest in the presence of the yeast Cepage. Growth of O. oeni was substantially slower in the presence of the yeast VL1. The three yeasts had similar fermentation rates in the presence and absence of O. oeni. Viable and nonviable yeast and bacterial populations can be rapidly discriminated in simultaneous malolactic-alcoholic wine fermentations using antibodies, fluorescent dyes and flow cytometry. This is the first study using fluorescently labelled antibodies to discriminate and monitor yeast and bacterial populations in wine fermentations and offers a new approach to investigating microbial interactions in wine fermentations.

Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose

The mutant IGC 40 × 1001 of the yeast Pichia anomala IGC 4380, which displays inverse diauxic growth in a medium with glucose and malic acid, was studied to elucidate the biochemical mechanisms underlying that behavior. Time course changes of enzyme activities during growth of the mutant in that mixture of substrates indicated that the gluconeogenic enzymes remained active during the first phase of diauxic growth, while glycolytic enzyme activities were significantly reduced. This reduction was essentially due to an alteration in the maximum velocity and not in substrate affinity. Malate, citrate, and adenosine triphosphate did not affect significantly the activities of the glucose phosphorylating enzymes in cell extracts of either the mutant or the wild strain. In P. anomala, unlike Saccharomyces cerevisiae, the fructose/glucose phosphorylating ratio was not associated with repression/derepression conditions.

Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose.

The mutant IGC 40 x 1001 of the yeast Pichia anomala IGC 4380, which displays inverse diauxic growth in a medium with glucose and malic acid, was studied to elucidate the biochemical mechanisms underlying that behavior. Time course changes of enzyme activities during growth of the mutant in that mixture of substrates indicated that the gluconeogenic enzymes remained active during the first phase of diauxic growth, while glycolytic enzyme activities were significantly reduced. This reduction was essentially due to an alteration in the maximum velocity and not in substrate affinity. Malate, citrate, and adenosine triphosphate did not affect significantly the activities of the glucose phosphorylating enzymes in cell extracts of either the mutant or the wild strain. In P. anomala, unlike Saccharomyces cerevisiae, the fructose/glucose phosphorylating ratio was not associated with repression/derepression conditions.

Influence of Pre-culture Conditions on the Ability ofLeuconostoc oenosto Conduct Malolactic Fermentation in Wine

Most industrially freeze-dried malolactic starters must undergo a pre-culture or reactivation process. This step permit a good implantation into wine. Variables of pre-culture conditions studied were the duration of the reactivation process and initial reactivated cell population. Reactivation in half-strength wine followed by growth in a medium with 75% wine allowed a gradual readjustment of bacteria to counterbalance the effect of wine components. The inoculum of the pre-culture media has to be, at most, 10⁷ cfu/mL so that malic acid degradation follows cellular proliferation. The utilization of malic acid in the pre-culture medium determines the time to collect bacteria for their inoculation into wine.

Sugar and malic acid utilization and acetic acid formation byLeuconostoc oenos

Leuconostoc oenos ST8, isolated from an Argentinian red wine, utilized glucose, fructose and malic acid and produced acetic acid. Fructose was preferred to glucose as a source of carbohydrate. Malic acid was almost completely degraded in 3 days at 25°C. Acetic acid formation correlated with fructose utilization.

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.

Malo‐alcoholic fermentation: the influence of operating conditions on the kinetics of deacidification

Abstract Vinho Verde wines are typical of Portuguese wines, and have a high level of malic acid. Sometimes, the grape musts of such wines have too high a malate level, and this paper studies the possibility of using a strain of Schizosaccharomyces pombe to remove this excess of malate. The influence of malic acid concentration, ethanol concentration and aeration rate on the kinetics of malic acid utilization by a strain of S. pombe is studied. A linear model is developed that allows for the time control of final malic acid concentration. The maximum specific malic acid consumption rate (qm,max) is shown to increase in a linear relationship with initial malic acid concentration. For ethanol concentrations below 40 g l‐1 no significant change in the specific malate consumption rate (qm.max) is detected. Above this value, a drastic decrease is observed. Aeration also plays an important role in malo‐alcoholic fermentation. When no air is sparged in the fermentation medium, ethanol and qm,max are maximal. The ...

Isolation and characterization of Schizosaccharomyces pombe mutants with defective NAD-dependent malic enzyme

To obtain NAD-dependent malic enzyme mutants of Schizosaccharomyces pombe, a colony color indicator screening system was developed. Mutants defective for malic acid utilization (mau mutants) are yellow, while wild-type colonies are blue on the defined bromcresol green based indicator medium. NAD-dependent malic enzyme mutants were distinguished from other mau mutants by subsequent, starch gel electrophoresis, spectrophotometry, complementation tests, and intermediate pool analysis with cell-free extracts.

Mechanism of passive malic-acid efflux from vacuoles of the CAM plantKalanchoë daigremontiana

An analysis was carried out of the mechanism of malic-acid efflux from vacuoles of mesophyll cells of the crassulacean acid metabolism (CAM) plantKalanchoe daigremontiana. Following its accumulation in the vacuole as a result of nocturnal CO2 fixation, the malic acid is passively transported back across the tonoplast in the subsequent light period and is decarboxylated in the cytoplasm. Malic-acid efflux was studied using leaf slices in solution or by following malic-acid utilization (deacidification) in leaves of intact plants. Samples of leaf-cell sap were taken at different times during the day-night rhythm to establish the relation between cell-sap pH and malate content. From the empirically determined pK values for malic acid in the cell sap, it was then possible to calculate the proportion of malate existing as the undissociated acid (H2mal0) and in the anionic forms (Hmal1− and mal2−) for all times during the CAM rhythm. In leaf-slice experiments it has been found that the rate of malic-acid efflux increases exponentially with the malic-acid content of the tissue. This is shown to be related to the increasing amounts of H2mal0 present at high malic-acid contents. At low malic-acid contents (<65 mol m−3), when H2mal0 is not present in significant amounts, efflux must be in the form of Hmal−1 and/or mal2−. At high malic-acid contents it is suggested that efflux occurs predominantly in the form of passive, noncatalyzed diffusion of H2mal0 across the tonoplast by a ‘lipid-solution’ mechanism. This is supported by the fact that the slope of the curve relating efflux to H2mal0 concentration, when corrected for the presumed contributions from Hmal1− and mal2− transport and plotted on a log-log basis, approaches 1.0 at the highest malic-acid contents. Moreover, the permeability coefficient required to be consistent with such a mechanism\((P_{H_2 mal^0 } = 1.0to2.0 \times 10^{ - 8} m\sec ^{ - 1} )\) is similar to that estimated from a Collander plot, using the partition coefficient of malic acid between ether and water. We suggest that\(P_{H_2 mal^0 } \) may be important in determining the maximum amounts of malic acid that can be accumulated during the CAM rhythm.

Effect of Sulfur Dioxide on the Activity of Schizosaccharomyces Pombe

<i>Schizosaccharomyces pombe</i> was more resistant to sulfur dioxide than <i>Saccharomyces cerevisiae</i> in wine fermentation. <i>Sch. pombe</i> was as active in the presence of 300 ppm added SO₂ as <i>Sac. cerevisiae</i> was in 100 ppm added SO₂. Utilization of malic acid by <i>Sch. pombe</i> was rapid and complete, and was not affected by 300 ppm added SO₂. The only effect noted was the slower starts with the higher SO₂ concentrations. In a mixed culture of <i>Sch. pombe</i> and <i>Sac. cerevisiae</i>, the <i>Sch.</i> yeast was not overgrown by the <i>Sac.</i> yeast when 150 ppm or more of SO₂ was added.

Effect of Sulfur Dioxide on the Activity of Schizosaccharomyces Pombe

<i>Schizosaccharomyces pombe</i> was more resistant to sulfur dioxide than <i>Saccharomyces cerevisiae</i> in wine fermentation. <i>Sch. pombe</i> was as active in the presence of 300 ppm added SO₂ as <i>Sac. cerevisiae</i> was in 100 ppm added SO₂. Utilization of malic acid by <i>Sch. pombe</i> was rapid and complete, and was not affected by 300 ppm added SO₂. The only effect noted was the slower starts with the higher SO₂ concentrations. In a mixed culture of <i>Sch. pombe</i> and <i>Sac. cerevisiae</i>, the <i>Sch.</i> yeast was not overgrown by the <i>Sac.</i> yeast when 150 ppm or more of SO₂ was added.

Proton and malate fluxes in cells of Bryophyllum daigremontianum leaf slices in relation to potential osmotic pressure of the medium

Summary Leaf slices obtained from the CAM plant Bryophyllum daigremontianum at the end of the dark phase were used to study proton und malate fluxes. Increasing potential osmotic pressure (π*) of the medium decreases loss of H + and malate from the leaf cells to the medium and increases the amounts of H+ and malate retained in the cells. As compared to the initial H + and malic acid levels, after the efflux experiments a certain amount of H+ and malate is not accounted for by efflux and retainment in the tissue. This amount is thought to be due to metabolic utilization of malic acid, it is smaller in the dark than in the light, and it is not clearly correlated to π * of the medium. Changes in potential osmotic pressure of extracts (Δπ * ) of the leaf slices are highly correlated to changes in malate content of the extracts. Malic acid accounts for about 55% of Δπ * of the extracts. It is suggested that net efflux of malic acid from the vacuoles of B. daigremontianum depends on turgor of the cells and that this mechanism plays a role in regulation of crassulacean acid metabolism (CAM) in vivo .