Malic acid, detection

The reaction mixture for a coupled assay includes the substrates for the initial or test enzyme and also the additional enzymes and reagents necessary to convert the product of the first reaction into a detectable product of the final reaction. The enzyme aspartate aminotransferase (EC 2.6.1.1), for instance, results in the formation of oxaloacetate, which can be converted to malic acid by the enzyme malate dehydrogenase (EC 1.1.1.37) with the simultaneous conversion of NADH to NAD+, a reaction which can be followed spectropho-tometrically at 340 nm ... 

Figure 4.7 Anion exchange separation of carboxylic acids in red wine. Column, Shodex C811, 100 cm x 7.6 mm i.d. eluent, 3 mM perchloric acid flow rate, 0.9 ml min-1 temperature, 60 °C detection, reaction detection using chloro-phenol red at 430 nm. Peaks 1, citric acid 2, tartaric acid 3, malic acid 4, succinic acid 5, lactic acid 6, formic acid and 1, acetic acid.

FIGURE I I Separation of (I) trimethylamine, (2) morpholine, (3) isopropylamine, (4) tertbutylamine, and (5) cyclohexylamine with 20 mM aminopyridine, l7mM malic acid, l7mM l8-crown-6 as BGE. The capillary was first rinsed with a polyanion solution, followed by a polycation solution to coat the capillary. Indirect UV detection at 200 nm (filter). 

Detection of Malo-Lactic Fermentation. It is imperative that the winemaker, to control malo-lactic fermentation, has a satisfactory method for its detection. Disappearance of malic acid is the indication of the fermentation, but the formation of lactic acid is not sufficient evidence since it might also be formed by yeast and by bacteria from other carbohydrate sources. The rate of conversion of malic acid is expected to reflect bacterial metabolism and growth. In New York State wines, Rice and Mattick (41) showed bacterial growth (as measured by viable count) to be more or less exponential to 106-107 cells/ml, preceding disappearance of malic acid. The rate of loss of malic acid is probably also exponential. Malic acid seems to disappear so slowly that its loss is not detected until a bacterial population of about 106-107 cells/ml is reached then it seems to disappear so rapidly that its complete loss is detected within a few days (41). Rice and Mattick (41) also showed a slight increase in bacterial population for a few days following this. 

For faster results, thin layer chromatography has been used (66), but we are not confident that lower levels of malic acid (0.06% ) (cf. Ref. 11), sometimes found in California wine before malo-lactic fermentation, are easily detected by this means. Malic acid can be more precisely measured by using the quantitative enzymatic method (67). Only the l isomer, the natural form present in grapes and wine, is detected by this method. 

The NADH-forming activity described here is different from the classical malic enzyme activity found by London et al. (95) in Lacto-badUus casei. In their system, NADH is a major end product and detectable by spectrophotometry while lactic acid is only a minor product. L. casei uses malic acid as an energy source with carbon dioxide, acetate, and ethanol as the main fermentation products. The optimal pH... 

Fig. 3. IEC/CEC separation of anions and cations. Column TSK-Gel OA-Pak A (300 X 7.8 mm, 5 jam) eluent 5 mM malic acid-methanol (95 5) flow rate 1.2 ml/min sample volume 25 jal detection conductivity. Peaks (1) sulfate, (2) chloride, (3) nitrate, (4) fluoride, (5) sodium, (6) ammonium, (7) potassium, (8) magnesium, (9) calcium. Reprinted with permission from [19].

Fumaric acid has been found to be a good marker to detect the addition of D,L-malic acid to apple juice (Junge Spandinger, 1982). With the advent of the enzymic assay procedure for D-malic acid the method fell out of use. However, in 1995, a number of samples of apple juice in Germany were found to contain elevated levels of fumaric acid, which was attributed to the addition of L-malic acid. 

Sometimes it is even important to look at the internal isotope ratios seen within a molecule such as malic acid in apple juice. Two groups, Isolab in Germany and Eurofms in France, found it was useful to look at the carbon isotope ratios at the Q and C4 positions of malic acid extracted from apple juice. This allowed them to detect the addition of synthetic L-malic acid to apple juice at much lower levels than would be possible by other means (Jamin el al., 2000). 

Jamin, E., Lees, M. Fuchs, G. and Martin, G.G. (2000) Detection of added L-malic acid in apple and cherry juice—site specific 13C-IRMS method . Fruit Processing 11, 434-6. 

Junge, C. and Spandinger, C. (1982) Detection of the addition of L and D, L malic acids in apple and pear juice by quantitative determination of fiimaric acid . Flussiges Obst 49(2), 57-62. 

The formol number is checked for authenticity by means of its ratios with proline, ammonia, alpha-amino nitrogen, and amino acid analyses. The malic acid is checked by enzyme and chemical assays. Based on their data, the Subgroup considers the system to be accurate enough to detect a 10% difference between the measured and stated juice concentration. Their data indicate good agreement between six cooperating laboratories. 

More recently, the carbon stable isotope ratio test (SIRA) has become an easy method to detect adulteration with cane and corn syrup (Carro et al, 1980). Because maple trees are C3 plants with a somewhat different photosynthetic pathway for carbon fixation, the ratio of 13C/12C in the sugar produced is different than cane or com. Maple has a 813C of approximately —24.5, whereas com and cane are closer to a 813C of —8 to —12. Thus, even a small addition of cane or corn syrup is readily detectable. Because beets are also C3 plants, the SIRA test is not able to detect adulteration with beet sugar. Improvement of the SIRA method is possible using malic acid as an internal standard (Tremblay and Paquin, 2007). 

Tremblay, P. and Paquin, R. (2007). Improved detection of sugar addition to maple syrup using malic acid as internal standard and in 13C isotope ratio mass spectrometry (IRMS).. Agric. Food Chem. 55,197-203. 

Fig. 5 Separation of the organic acid on CIM disk monolithic column. Conditions—mobile phase 130 mM NaCl in 20 mM phosphate buffer, pH 8.0 separation unit CIM disk monolithic column comprising of four CIM QA disks flow rate 5 mL/ min sample (1) 0.03 g/L pyruvic acid, (2) 0.5 g/L malic acid, (3) 0.2 g/L a-ketoglutaric acid, (4) 0.007 g/L fumaric acid, (5) 2 g/L citric acid, and (6) 2 g/L isocitric acid injection volume 20 pL detection UV at 210 nm. (From Ref. [17].)...

Many plants contain a variety of free acids such as acetic acid, citric acid, fumaric acid, malic acid, succinic acid, oxalic acid, glycohc acid, etc. They are components of citric cycle, whereas the others are intermediates in the pathway from carbohydrates to aromatic com-pounds. Following extraction, organic acids can be separated and detected with a variety of techniques. Thin layer chromatographic methods have been also employed to separate certain organic acids,as presented in Table 3. 

Figure 1.7 HPLC chromatogram of organic acids analysis of Prosecco grape must sample. 1. tartaric acid, 2. malic acid, 3. citric acid, 4. shikimic acid. Analytical conditions Lichrospher 100 RP-18 (250 x4mm, 5p,m) column (Merck, Darmstadt, Germany) at room temperature, detection at wavelength 210 nm, sample volume injected 20/xL solvent H3P04 5 x 10 3 M with isocratic elution at flow rate 0.6 mL/min...

Figure 1.11 Analysis of an organic acids standard solution. 1. citric acid, 2. tartaric acid, 3. malic acid, 4. succinic acid, 5. lactic acid, 6. acetic acid. Analytical conditions column Aminex HPX-87H (300 x 7.8mm, 9(jim) at 65 °C (Bio-Rad Laboratories, Richmond, CA) detection at wavelength 210 nm sample volume injected 10p,L solvent H2S04 0.026 N with isocratic elution at flow rate 0.8mL/min...

Fig. 4-2. Separation of organic acids on PRP-X300. - Eluent 0.0005 mol/L H2S04 flow rate 1 mL/min detection direct conductivity injection volume 100 pL solute concentrations 4 ppm tartaric acid, 7.5 ppm malic acid and citric acid, 10 ppm lactic acid, 25 ppm acetic acid, and 40 ppm succinic acid.

Fig. 4-10. Separation of organic acids on IonPac ICE-AS5. - Eluent 0.0016 mol/L perfluorobu-tyric acid flow rate 0.3 mL/min detection suppressed conductivity injection volume 50 pL solute concentrations fully dissociated compounds (1), 10 ppm oxalic acid (2), 25 ppm pyruvic acid (3), and tartaric acid (4), 30 ppm malonic acid (5), lactic acid (6), malic acid (7), and acetic acid (8), 20 ppm isodtric acid (9), 30 ppm citric acid (10), 40 ppm / -hydroxybutyric acid (11), succinic acid (12), and propionic acid (13).

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