Chemometric methods for simultaneous quantification of lactic,malic and fumaric acids

Lactic, fumaric and malic acids are commonly used in food and pharmaceutical industries. During microbial production of these compounds, it is important to determine their concentrations in the fermentation broth with a rapid and sensitive method. Spectrophotometry is commonly used. However, UV‐spectral overlap between these organic acids makes it difficult to determine each of them individually from the mixture. In order to overcome this problem, statistical methods, namely principal component regression (PCR) and partial least squares‐1 methods, were tested and compared with conventional HPLC techniques. The absorbance data matrix was obtained by measuring the absorbances of 21 ternary mixtures of lactic, fumaric and malic acids in a wavelength range of 210–260 nm. Calibration and validation were performed by using the data obtained in a mixture of these organic acids. The prediction abilities of the methods were tested by applying them to fermentation broths. The precision of the PCR method was better than that of the partial least squares‐1 method. In the PCR method, the correlation coefficients between actual and predicted concentrations of the organic acids were calculated as 0.970 for lactic acid and 0.996 for fumaric acid in fermentation broths. The concentration of malic acid was not detected due to its low concentration in samples. These results show that the PCR method can be applied for simultaneous determination of lactic, fumaric and malic acids in fermentation broths.     

THE EFFECT OF DARKENING ON THE SUSCEPTIBILITY OF PLANTS TO INFECTION WITH VIRUSES

The susceptibility of French bean plants to infection by the Rothamsted strain of tobacco necrosis virus as measured by the local-lesion method is increased by a rise in temperature and usually by darkening the plant before inoculation. If part only of a leaf is darkened, that part becomes more susceptible. Plants in full light also become more susceptible if carbon dioxide is removed from the air, whereas the susceptibility of plants in the dark is not altered.
Darkening leaves decreases their content of malic, fumaric, succinic and glycolic acids and increases the content of citric acid; the content of oxalic and malonic acids remains constant. These changes occurred in winter and summer and whether or not darkening increased susceptibility.
The effect on susceptibility of individual acids infiltrated into the leaf was measured in leaves kept in the light or in the dark before inoculation. None of the acids used produced any large change in susceptibility.     

The Effect of CO2-Concentration on the Occurrence of a Number of Acids from the Citric Acid Cycle in Tomato Leaves

Examination of the effect of CO₂-concentration and time of day on the content of malic acid, citric acid, aconitic acid, isocitric acid, succinic acid and fumaric acid in tomato leaves, revealed that the total content of these acids will rise with the CO₂-concentration up to 0.10 vol% CO₂. In the morning up to 0.22 vol% CO₂ was needed for optimal effect. Samples of leaves picked at 1 a.m. showed the lowest content of these acids. At 9 a.m. the content had increased, and at 4 p.m. the increase was considerable. The content of malic and citric acid constituted 36 and 34% of the total acid content. In the afternoon and the night the aconitic acid represented 14% and in the morning 20% of the total acid content. Isocitric acid, fumaric acid and succinic acid occurred only in relatively small concentrations.     

Non-oxidative and oxidative alkaline degradation of pectic acid

As alkaline degradation products of pectic acid, 6 hydroxymonocarboxylic, 16 dicarboxylic, and 2 tricarboxylic acids were identified by g.l.c.-m.s. as their trimethylsilyl derivatives. In the absence of oxygen, the most abundant degradation products are 3-deoxy-2-C-(hydroxymethyl)pentaric, 2,3-dideoxypentaric, 2-deoxy-3-C-methyltetraric, malic, and 2¹,4-anhydro-3-deoxy-2-C-(hydroxymethyl)pentaric acids, whereas, in the presence of oxygen, glycolic, oxalic, malic, 3-deoxypentaric, and 2-C-carboxy-3-deoxypentaric acids preponderate. The routes of formation of these acids show many similarities with those encountered in the alkaline degradation of cellulose.     

Non-oxidative and oxidative degradation of d-galacturonic acid with alkali

Degradation products from non-oxidative and oxidative treatment of d-galacturonic acid with alkali have been analysed by g.l.c.-m.s.; 13 hydroxy monocarboxylic acids and 26 dicarboxylic acids were identified in addition to several isomerisation products. In the absence of oxygen, the main degradation products were 3-deoxy-lyxo-hexaric, 3-deoxy-xylo-hexaric, malic, tartonic, C-methyltartronic, lactic, and 3-deoxytetronic acids. In the presence of oxygen, arabinaric, threaric, malic, tartronic, and glycolic acids were formed in large proportions. The routes of formation for most of the compounds are outlined.     

A new rapid method of glycolate test by diethyl ether extraction,which is applicable to a small amount of bacterial cells of less than one milligram

The glycolate test is a method to discriminate N-acyl groups of muramyl residue in peptidoglycan of bacterial cell walls by color reaction without purification of the cell walls. The glycolyl residue presents red purple color by heating with 0.02% 2,7-dihydroxynaphthalene (DON) dissolved in concentrated H(2)SO(4). Instead of the previous column methods for quantitative analysis, a qualitative method by solvent works was developed to simplify and to miniaturize the analysis. In this method, solvents played two roles, removal of interfering materials and extraction of glycolic acid from the cell hydrolysates. Of several solvent systems tested, diethyl ether was studied in detail on such properties as the efficiency of glycolic acid extraction under several conditions, the ability of removing various interfering compounds, and the advantage on evaporation procedure of the solvent from extracts. DON reaction of the second diethyl ether extract from cell hydrolysate of "Micromonospora nigra" JCM 3328 showed a clear red purple color of a strong absorbance at 530 nm, which is the same as that of authentic glycolic acid. The solvent method was applied to 20 strains of typical actinomycete species whose acyl types have already been known (Uchida and Seino, 1997). All glycolate test positive strains showed the clear red purple color mentioned above, whereas acetyl type strains revealed no apparent color by the same procedures. Additional experiments indicated that the glycolate test could be determined with less than 1 mg of actinomycete cells by using a smaller amount of DON reagent and ordinary polypropylene tubes. The new method was discussed for advantages in the identification of actinomycetes and for possible applications to other fields.     

Catabolism of aromatic acids in Trichosporon cutaneum.

Trichosporon cutaneum readily metabolized protocatechuate, homoprotocatechuate, and gentisate, but lacked ring fission dioxygenases for these compounds. Benzoic, salicylic, 2,3-dihydroxybenzoic, and gentisic acids were converted into beta-ketoadipic acid before entry into the Krebs cycle. Benzoic acid gave rise successively to 4-hydroxybenzoic acid, protocatechuic acid, and hydroxyquinol (1,3,4-trihydroxybenzene), which underwent ring fission to maleylacetic acid. Salicylate and 2,3-dihydroxybenzoate were both initially metabolized to give catechol. 2,3-Dihydroxybenzoate was the substrate for a specific nonoxidative decarboxylase induced by salicylate, although 2,3-dihydroxybenzoate was not a catabolite of salicylate. Gentisate was metabolized to maleylacetic acid and was also readily attacked by salicylate hydroxylase at each stage of a partial purification procedure. Phenylacetic acid was degraded through 3-hydroxyphenylacetic, homogentisic, and maleylacetoacetic acids to acetoacetic and fumaric acids. All the reactions of these catabolic sequences were catalyzed by cell extracts, supplemented with reduced pyridine nucleotide coenzymes where necessary, except for the hydroxylations of benzoic and phenylacetic acids which were demonstrated with cell suspensions and isotopically labeled substrates.     

Effect of organic acids on the biosynthesis of carotenes by an Actinomyces chrysomallus strain

Synthesis of carotenes by Actinomyces chrysomallus var. carotenoides was stimulated by citric, acetic, oxalacetic, fumaric, succinic, malic, alpha-ketoglutaric, tartaric, pyruvic, and propionic acids. Acetic acid acts as a precursor of carotene synthesis and also has another stimulating mechanism of action on carotenogenesis of the actinomycete. Acetic, furmaric, malic, succinic, and alpha-ketoglutaric acids stimulate cyclization of lycopene yielding beta-carotene.     

Carboxylic Acids as Biomarkers of Biomphalaria alexandrina Snails Infected with Schistosoma mansoni

Biomphalaria alexandrina snails play an indispensable role in transmission of schistosomiasis. Infection rates in field populations of snails are routinely determined by cercarial shedding neglecting prepatent snail infections, because of lack of a suitable method for diagnosis. The present study aimed at separation and quantification of oxalic, malic, acetic, pyruvic, and fumaric acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to test the potentiality of these acids to be used as diagnostic and therapeutic biomarkers. The assay was done in both hemolymph and digestive gland-gonad complex (DGG) samples in a total of 300 B. alexandrina snails. All of the studied acids in both the hemolymph and tissue samples except for the fumaric acid in hemolymph appeared to be good diagnostic biomarkers as they provide not only a good discrimination between the infected snails from the control but also between the studied stages of infection from each other. The most sensitive discriminating acid was malic acid in hemolymph samples as it showed the highest F-ratio. Using the Z-score, malic acid was found to be a good potential therapeutic biomarker in the prepatency stage, oxalic acid and acetic acid in the stage of patency, and malic acid and acetic acid at 2 weeks after patency. Quantification of carboxylic acids, using HPLC strategy, was fast, easy, and accurate in prediction of infected and uninfected snails and possibly to detect the stage of infection. It seems also useful for detection of the most suitable acids to be used as drug targets.     

Amino Acid Catabolism and Malic Enzyme in Differentiating Dictyostelium discoideum

Amino acids produced from protein degradation are the major energy source for differentiation and aging in Dictyostelium discoideum. Considering the reactions involved in the conversion of amino acids from an average protein into tricarboxylic acid cycle intermediates, a route from a cycle intermediate (probably malate) to acetyl coenzyme A is required for the complete utilization of amino acids. Citrate was isolated from cells pulse-labeled with (14)C-labeled amino acids and was cleaved with citrate lyase. When cells were pulse-labeled with [U-(14)C]-glutamate the specific radioactivity of the acetate and oxaloacetate portions of citrate were consistent with the conclusion that one-third of the carbon flowing through the tricarboxylic acid cycle is removed for the synthesis of acetyl coenzyme A. The data were also consistent with the patterns of carbon flux required to maintain steady-state levels of cycle intermediates in cells catabolizing amino acids. It is suggested that the malic enzyme (EC 1.1.1.40) catalyzes the synthesis of acetyl coenzyme A from malate and is responsible for the observed citrate labeling pattern. In cell extracts the activity of this enzyme increased markedly with the onset of differentiation. The properties of partially purified (40-fold) malic enzyme isolated at culmination indicated that the enzyme was allosteric and was positively affected by aspartate and glutamate. Thus, amino acid production from protein degradation would stimulate a reaction essential for the efficient utilization of these amino acids for energy.     

Nicotinamide adenine dinucleotide phosphate-malic enzyme of rat liver. Purification, properties, and immunochemical studies.

Rat liver malic enzyme (EC 1.1.1.40) was purified from livers of rats fasted and refed a high sucrose diet containing 1% desiccated thyroid powder. The purification was accomplished by a six-step procedure. The specific activity of the purified enzyme was increased 181-fold above that of the initial high speed supernatant of liver extracts. Slight additional purification of malic enzyme was achieved with preparative disc electrophoresis. The specific activities of the purified rat liver malic enzyme from the least two steps were between 28.0 and 30.5 units per mg of protein. Homogeneity of the purified enzyme was determined by disc and starch gel electrophoresis as well as sedimentation velocity and sedimentation equilibrium studies. The molecular weight and S20, w values of rat liver malic enzyme are 268,000 and 10.2, respectively. Amino acid analysis based on milligram of protein hydrolyzed yielded higher amounts of leucine and glutamic acid but lower quantities of alanine and voline per subunit than the corresponding Escherichia coli enzyme...     

Enzymatic synthesis of L-malic acid from fumaric acid using immobilized Escherichia coli cells

Optimal conditions were chosen for cultivation of Escherichia coli 85 cells with a rather high fumarate-hydratase activity on a cheap medium containing no edible raw material. An active biocatalyst for the synthesis of L-malic acid from fumaric acid was obtained based on E. coli 85 cells immobilized in carrageenan. The enzymatic synthesis of L-malic acid from potassium fumarate was kinetically studied and optimized. Some thermodynamic parameters of fumaric acid hydration into malic acid were determined. A technique for assaying the reaction mixture was developed that involved high performance liquid chromatography.     

Detection of Glycollic Acid in Etiolated Barley Shoots

A modified method for carboxylic acid analysis has been developedin order to study the metabolism of glycollic acid in barleysap. Glycollic, malic, citric, malonic, succinic, and fumaric acidshave been detected in alcoholic extracts from etiolated barleyshoots, and the amounts present roughly estimated.     

Influence of pH,malic acid and glucose concentrations on malic acid consumption by Saccharomyces cerevisiae

Malic acid consumption by Saccharomyces cerevisiae was studied in a synthetic medium. The extent of malic acid degradation is affected by its initial concentration, the extent and the rate of deacidification increased with initial malate concentration up to 10 g/l. For malic acid consumption, an optimal pH range of 3–3.5 was found, confirming that non-dissociated organic acids enter S. cerevisiae cells by simple diffusion. A full factorial design has been employed to describe a statistical model of the effect of sugar and malic acid on the quantity of malate degraded (g/l) by a given amount of biomass (g/l). The results indicated that the initial malic acid concentration is very important for the ratio of malate consumption to quantity of biomass. The yeast was found to be most efficient at higher levels of malate.     

The degradation of malic acid by high density cell suspensions of Leuconostoc oenos

Washed cell suspensions of Leuconostoc oenos catalysed the degradation of L-malic acid to L-lactic acid. Cell suspensions of 10₁₀ cfu ml_-1 degraded 90–95% of the malic acid in a buffer assay system and in wine within 30 min. A reaction time of 6 h was needed to obtain the same extent of degradation with suspensions of 10₉ cfu ml_-1. With a reaction period of 6 h and an initial malic acid concentration of 3 g 1_-1, reaction variables of pH 2.5-4.0, temperature 10–30°C, ethanol up to 15%, and L-lactic acid up to 4 g 1_-1 did not decrease the degradation of malic acid to below 90–95%. Total SO₂ at 100 mg 1_-1 decreased the degradation of malic acid to 80%. The degradation (%) of malic acid was decreased when the concentration of malic acid was decreased below 2 g 1_-1. The results indicate the prospect of using high densities of Leuc. oenos cells in membrane bioreactor systems for the rapid, continuous, deacidification of wine.     

黄曲霉发酵薯干粉水解液生产L－苹果酸

经选育和诱变得到一株产苹果酸的黄曲霉菌ＴＨ５００７,通过发酵条件的优化,以薯干粉的水解液为主要原料,发酵５ｄＬ－苹果酸可达到６％左右。在总酸中苹果酸含量平均达７２％以上,杂有机酸主要是柠檬酸,延胡索酸的含量在０．０２％以下。补糖发酵比分批发酵产酸性能更佳。     

Anaerobic biosynthesis of intermediates of reductive branch of tricarboxylic acids cycle by <Emphasis Type="Italic">Escherichia coli</Emphasis> strains with inactivated <Emphasis Type="Italic">frdAB</Emphasis> and <Emphasis Type="Italic">sdhAB</Emphasis> genes

The genes frdAB and sdhAB, which encode components of fumarate reductase and succinate dehydrogenase, have been deleted in a recombinant E. coli strain with the inactivated pathways of mixed-acid fermentation and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. Under anaerobic conditions, the parental strain efficiently converted glucose to succinic acid without synthesizing notable amounts of fumaric or malic acid. Upon individual deletion of the frdAB genes, the mutant strain fermented glucose to succinic acid less efficiently secreting notable amounts of malic and fumaric acids. Individual deletion of the sdhAB genes in the parental strain did not significantly affect the formation of the main fermentation end-product. The combined inactivation of fumarate reductase and succinate dehydrogenase in the constructed strain enhanced the anaerobic conversion of glucose to fumaric and malic acids with the activation of the glyoxylate bypass and decrease in the contribution of the reductive branch of the TCA cycle to the formation of the target products.     

Comparison of GC and HPLC for the quantification of organic acids in coffee

A GC and an HPLC method for the quantification of organic acids OAs in coffee have been compared. The GC procedure, employing trimethylsilyl derivatives, was found to be very tedious. The HPLC method, which employed an ion exchange column using a flow gradient of water containing 1% phosphoric acid and UV detection (210 nm), was found to be much simpler for the quantification of eight organic acids (oxalic, succinic, fumaric, malic, tartaric, citric, quinic and fumaric acids) in four representative coffee samples. The HPLC procedure was more convenient than that described in the literature since no pre-purification was required for quantification of the OAs.     

Metabolic engineering of Rhizopus oryzae: Effects of overexpressing fumR gene on cell growth and fumaric acid biosynthesis from glucose

Fumaric acid is a dicarboxylic acid used extensively in synthetic resins, food acidulants, and other applications, including oil field fluids and esters. The filamentous fungus Rhizopus oryzae is known for its ability to produce and accumulate high levels of fumaric acid under aerobic conditions. In this work, the overexpression of native fumarase encoded by fumR and its effect on fumaric acid production in R. oryzae were investigated. Three plasmids containing the endogenous fumR gene were constructed and used to transform R. oryzae, and all transformants showed significantly increased fumarase activity during both the seed culture (growth) and fermentation (fumaric acid production) stages. However, fumarase overexpression in R. oryzae yielded more malic acid, instead of fumaric acid, in the fermentation because the overexpressed fumarase also catalyzed the hydration of fumaric acid to malic acid. The results suggested that the overexpressed fumarase, encoded by fumR, by itself was not responsible for the over-production of fumaric acid in R. oryzae.     

Sensory evaluation of acids by free-choice profiling

The technique of free-choice profiling was applied in orderto characterize the sensory properties of some common organicand inorganic acids. Analysis of panelists' scores by generalizedProcrustes Analysis (GPA) provided information on the relationshipsamong samples and assessors for both the consensus and individualconfigurations. Results indicated that on a weight basis (w/vor v/v), acids differed in their flavor and taste dynamics.Acids were described differently by individual panelists. The GPA resulted in three important principal axes (PA). Thefirst PA had astringency/mouthfeel as the most important factor,while bitterness and sourness were the most important for thesecond and the third PAs, respectively. At 0.08% (w/v or v/v),the inorganic acids, hydrochloric and phosphoric, were moreastringent than sour. The bitterness of succinic (S) was intenseas was the sourness of fumaric, malic and the combinations offumaric:malic (FM), citric:malic (CM) and citric:fumaric (CF).The sensory characteristics of adipic and quinic were perceivedto be very weak at this concentration. The relationship betweenastringency and pH was more evident than was the relationshipbetween pH and sourness.