Ascorbic acid enzymatic oxidation

Enzymes known as polyphenol oxidases cause enzymatic browning. Other names of the enzyme include phenolases and tyrosinases. The enzymes catalyze the conversion of monophenols and diphenols to quinones. The quinones can undergo a series of non-enzymatic reactions to produce brown, gray and black colored pigments, collectively known as melanins (11). Maillard reactions, caramelizations and ascorbic acid oxidations can produce similar types of colored compounds (12). For some food processing... 

Although the inhibition of ascorbic acid oxidation in citrus juices (22,23) and other foods by EDTA was reported by several researchers (22-24), the possibility that EDTA could inhibit non-enzymatic browning in grapefruit or other citrus juices has not been explored. 

The oxidation of ascorbic acid in certain reactions has given evidence of an intermediate with the properties of a free radical which could be formed by one-electron oxidation. Thus, the rate-limiting step of ascorbic acid oxidation by Fe + and H2O2 was this one-electron oxidation (G12). Such a radical has now been identified in hydrogen peroxide-ascorbic acid solutions at pH 4.8 by electron paramagnetic resonance spectroscopy. The free radical, commonly referred to as monodehydroascorbic acid, decayed in about 15 minutes at this acid pH. It was also formed during the enzymatic oxidation of ascorbic acid by peroxidase (Yl). The existence of the monodehydroascorbic acid radical makes possible very... 

The specific enzymatic catalysis of ascorbic acid oxidation is known in plants but not in animal tissues. 

FIGURE 5. Ascorbic acid as an antioxidant. The free radical form of ascorbic acid, A , is the primary product of ascorbic acid oxidation, observed during catalytic, enzymatic, photooxidative, and free radical oxidation. It is relatively stable and, thus, can detected by electron spin resonance in ischemic reperfusion of the heart and iron overloaded blood plasma. Adapted from Bendich et al, (1986). 

Sulfur dioxide (bisulfites) also reacts with thiamine yielding inactive compounds (see Section 5.6.6), forms adducts with riboflavin, nicotinamide, vitamin K, inhibits ascorbic acid oxidation (see Section 5.14.6.1.6) and reacts with ascorbic acid degradation products, reduces o-quinones produced in enzymatic browning reactions back to 1,2-diphenols (see Section 9.12.4), causes decolourisation of fruit anthocyanins (see Section 9.4.1.5.7) and reacts with synthetic azo dyes to form coloured or colourless products (see Section 11.4.1.3.2). Sulfur dioxide also reacts with pyrimidine bases in vitro, specifically with cytosine and 5-methylcytosine. Important reactions of sulfur dioxide are shown in Figure 11.4. 

Chromatographic methods, notably hplc, are available for the simultaneous deterrnination of ascorbic acid as weU as dehydroascorbic acid. Some of these methods result in the separation of ascorbic acid from its isomers, eg, erythorbic acid and oxidation products such as diketogulonic acid. Detection has been by fluorescence, uv absorption, or electrochemical methods (83—85). Polarographic methods have been used because of their accuracy and their ease of operation. Ion exclusion (86) and ion suppression (87) chromatography methods have recently been reported. Other methods for ascorbic acid deterrnination include enzymatic, spectroscopic, paper, thin layer, and gas chromatographic methods. ExceUent reviews of these methods have been pubHshed (73,88,89). 

Drugs can also Interfere with laboratory results by negating certain nonspecific oxidation and reduction reactions essential for the chemical assay. Penicillin, streptomycin and ascorbic acid are known to react with cupric Ion thus, false positive results for glucose may occur If a copper reduction method Is used. If the specific enzymatic glucose-oxidase method Is employed, ascorbic acid can cause a false negative result by preventing the oxidation of a specific chromogen In the reaction. 

Needless to say, there is a definite possibility that, if reactions as just described participate in the transformations of polyuronide polysaccharides, enzymatic control of these may exist by systems leading to the oxidation of ascorbic acid or producing peroxide-type intermediaries. 

A combination of chemical and enzymatic reactions is used to eliminate two of the original chiral centers (C-4 and 5 in D-glucose) by changing the oxidation level. 60,000 tons of ascorbic acid are produced per year by this method in an overall yield of 60 % from D-glucose. 

It should be noted that ascorbic acid is more stable at pH 4-5 than at pH 7, at which the folacin vitamers are more stable. Additional protection from oxidation can be achieved by degassing the extraction solution with an inert gas, such as helium. Homogenization is followed immediately by protein precipitation and release of bound folacin vitamers. This can be accomplished by mild acidification, heating, addition of organic compounds such as trichloroacetic acid, and/or enzymatic (e.g., papain) hydrolysis. The specific conditions used for homogenization and protein precipitation are dictated by the food matrix and the expected profile of folacin vitamers. 

Most ultramicrobiosensors use differential measurement to overcome the problems of interferences and electrode fouling. The practical use of these biosensors for direct measurement is limited by interferents, such as ascorbic acid, acetaminophen (paracetamol), uric acid, etc., which are present in complex matricies such as serum. The specificity of the biochemical system is compromised by the partial selectivity of the electrode. The electrode not only oxidizes the desired product (e.g., H2O2 formed in the enzymatic oxidation of glucose by glucose oxidase), but also any other species oxidizable at the working potential. This produces a larger current response and a positive error. 

During the crushing process, ascorbic acid can be added to protect against oxidation. This prevents non-enzymatic browning until the juice is pasteurized. 

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