EFFECT OF HYDROXYLAMINE, p-AMINOBENZOIC ACID AND p-AMINOSALICYLIC ACID ON THE OXIDATION OF O-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

The effect of hydroxylamine (NH₂OH), p-aminobenzoic acid (PABA) and p-aminosalicylic acid (PASA) on the spectrum of the final product (s) formed when o-dihydroxy- and trihydroxyphenols were oxidized by tyrosinase was examined. New pigmented product(s), probably oximes, were formed by the interaction of NH₂OH with the o-quinones of 4-methyl catechol, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylpropionic acid (3,4-DPPA) but not with the o-quinones of catechol or protocatechuic acid. Interaction of PABA or PASA with the o-quinones of catechol, 4-methyl catechol, protocatechuic acid, DOPAC and 3,4-DPPA also yielded pigmented oximes. The interaction of the o-quinones of trihydroxyphenols with NH₂OH, PABA or PASA had little effect on the spectrum of the final product (s), suggesting that oximes are not formed in these reactions.     

p-HYDROXYPHENYLACETIC ACID AND-3,4-DIHYDROXYPHENYLACETIC ACID AS SUBSTRATES FOR MUSHROOM TYROSINASE

The hydroxylation of p-hydroxyphenylacetic acid (PHPAC) and the oxidation of 3,4-dihydroxyphenylacetic acid (DOPAC) by mushroom tyrosinase are illustrated. DOPAC quinone (4-carboxy-methyl-o-benzoquinone (λ_max= 400±10 nm) is the initial pigmented product formed when DOPAC is oxidized by the enzyme. DOPAC quinone is very unstable, and, once formed, is converted rapidly to further oxidation product(s).
The relationships between the rate of p-dihydroxyphenylacetic acid hydroxylation and 3,4-dihydroxyphenylacetic acid oxidation as a function of various concentrations of each substrate and of mushroom tyrosinase, are described.
The effect of the addition of various chemicals that can potentially conjugate otherwise affect DOPAC quinone was studied The Km value of 3,4-dihydroxyphenylacetic acid for mushroom tyrosinase was estimated to be 4.0 mM.     

EFFECT OF KOJIC ACID ON THE OXIDATION OF TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE1

Kojic acid [5-hydroxy-2-(hydroxymethyl)-4-pyrone] inhibited effectively the rate of pigment formation during the oxidation of pyrogallol, 2, 3,4-THAP (2, 3,4-trihydroxyacetophenone) and 2, 4,5-THBP (2, 4,5-trihydroxybutyrophenone) by tyrosinase. On the other hand, kojic acid had a synergistic effect on the rate of methyl gallate and n-propyl gallate oxidation to pigmented product(s) (λ_max= 360 nm and λ_max= 380 nm, respectively). However, kojic acid inhibited effectively the rate of oxygen uptake when each of the above trihydroxyphenols was oxidized by tyrosinase. These results suggest that kojic acid inhibits tyrosinase per se (probably due to its ability to bind copper at the active site of the enzyme) and that it exerts only an apparent stimulatory effect during the formation of pigmented product (s) from methyl gallate and n-propyl gallate. Proof for the latter was obtained by a time-course experiment of kojic acid addition and examination of the spectra of pigmented product(s) formed in the absence versus presence of kojic acid, which suggested that the o-quinone of n-propyl gallate and the o-quinone of methyl gallate can each convert kojic acid to a yellow product(s) absorbing at the 360–380 nm region.     

EFFECT OF ACETOHYDROXAMIC ACID (AHA) AND SALICYLHYDROXAMIC ACID (SHAM) ON THE OXIDATION OF o-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

Acetohydroxamic acid (AHA) and salicylhydroxamic acid (SHAM) each inhibited the rate of oxidation of different o-dihydroxy- and trihydroxyphenols by tyrosinase when assayed spectrophotometrically or polarographically. SHAM was a much more effective inhibitor than AHA. Spectral changes occurring during the oxidation of different o-dihydroxyphenols by tyrosinase in the presence of AHA or SHAM were different than the spectral changes occurring in their absence. AHA and SHAM also had an effect on the spectrum of the final product(s) formed when different o-dihydroxyphenols were oxidized by the enzyme, suggesting that AHA and SHAM conjugate with the o-quinones formed. A lack of an effect of AHA and SHAM on the spectrum of product(s) formed when trihydroxyphenols were oxidized by tyrosinase suggest that AHA and SHAM do not conjugate with the o-quinones derived from trihydroxyphenols.     

Involvement of Blueberry Peroxidase in the Mechanisms of Anthocyanin Degradation in Blueberry Juice

ABSTRACT: Addition of hydrogen peroxide (H₂ O₂) to blueberry juice changed the red coloration toward brown. Addition of ascorbic acid prevented the formation of brown polymers. An extract of peroxidase (POD) prepared from blueberry fruits was able to oxidize CG into the corresponding o-quinone but only in the presence of H₂ O₂. The chlorogenoquinone plays a dominant role in anthocyanin degradation. We demonstrated that peroxidase extract in the absence of CG showed a weak degradation activity toward blueberry anthocyanins, cyanidin 3-glucoside, and pelargonidin 3-glucoside. Nevertheless, addition of CG increased anthocyanin degradation, leading to formation of brown polymers. Therefore, blueberry POD could participate in the development of browning during blueberry-juice storage.     

The Flavonoid Eriodictyol as Substrate of Peach Polyphenol Oxidase

ABSTRACT: Spectral changes produced in the oxidation of eriodictyol by peach polyphenol oxidase were followed over time. A product with λ_max= 390 nm was seen to appear before another with λ_max= 475. The product absorbing at 390 nm must correspond to the o -quinone derived from eriodictyol. The compound absorbing at 475 nm must be derived from this eriodictyol-o-quinone. Progress curves at this wavelength revealed a lag, the length of which varied with enzyme and substrate concentrations. This lag must have been caused by chemical reactions taking place after the enzymatic reaction. When eriodictyol oxidation was studied in the presence of 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH), a potent nucleophilic reagent that reacts with the eriodictyol-o-quinone to form a dark pink product absorbing at 508 nm, the lag disappeared. When the kinetic parameter was evaluated in the presence of MBTH (K_m= 0.6 m M ), the results was similar to those obtained without MBTH. Eriodictyol oxidation was inhibited by tropolone, which behaved as a classic competitive inhibitor (K_I= 15 μ M ). The inhibition results reported show that eriodictyol oxidation was strictly dependent on the presence of polyphenol oxidase. In addition, other oxidase activities, such as laccase and H₂O₂ independent phenol oxidase, were not detected in the enzyme extract.     

2,3,4-TRIHYDROXYACETOPHENONE (2,3,4-THAP) AS A SUBSTRATE FOR MUSHROOM TYROSINASE1

2,3,4-Trihydroxyacetophenone (2,3,4-THAP) can serve as a substrate for mushroom tyrosinase with a K_m value of 1.2 mM. The product(s) formed is yellow, characterized by a peak at 420–430 nm. A lag period in 2,3,4-THAP oxidation to the yellow product(s) in the presence of ascorbate indicates that the initial product(s) is an o-quinone of 2,3,4-THAP. An oxime, characterized by a broad peak at 510–650 nm, is the likely product formed between o-quinone of 2,3,4-THAP and NH₂OH. The ?_m of the o-quinone of 2,3,4-THAP was estimated to be 1.6 × 10⁴ M^?1 cm^?1 at 425 nm. During relatively long incubation periods, the peak of the yellow product(s) shifts from 420–425 nm to 430–440 nm; the solution remains yellow and transparent for at least a week and no precipitate is formed. The final yellow product(s) is probably a low molecular weight polymer of THAP-o-quinone (dimer, tetramer, etc).     

2,3-DIHYDROXYBENZOIC ACID AS A SUBSTRATE FOR MUSHROOM TYROSINASE1,2

When 2, 3-dihydroxybenzoic acid (2, 3-DBA) is acted upon by mushroom tyrosinase, a yellow intermediate, 2, 3-DBA-o-quinone, characterized by a peak at 415 nm, is the first product detected. 2, 3-DBA-o-quinone gives rise to a “final blue product” (λmax = 230, 410, 620 nm), and to “soluble oxidation product(s)” (λmax = 275–280, 350–360 nm). Kinetic data (assayed spectrophotometrically and polarographically) obtained when different concentrations of 2, 3-DBA were oxidized by a fixed amount of mushroom tyrosinase, deviated from classic Michaelis-Menten kinetics. Reduction of the “final blue product” with ascorbate resulted in the loss of the blue chromophore at 620 nm and the concomitant appearance of a “yellowish reduced final product.” The “yellowish reduced final product” could be reoxidized with either mushroom tyrosinase or with NaIO4 to the “final blue product,” indicating that the latter has carbonylic quinonoid groups in ortho position to each other.     

KOJIC ACID CONVERSION TO A YELLOW PRODUCT(S) BY THE HEMOGLOBLIN/H2O2 SYSTEM1

Kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one; also named 5-hydroxy-2-(hydroxymethyl)-γ-pyrone) in the presence of hydrogen peroxide, but not in its absence, can be oxidized by hemoglobin (Hb) to a yellow product(s). The yellow product(s) formed is characterized by a peak at 370–380 nm and is fluorescent. The relationship between the rate of oxidation of kojic acid and various concentrations of hemoglobin and of H₂O₂ is described. The changes with time in the spectrum of product(s) obtained when kojic acid is acted upon by the Hb/H₂O₂ system and the relationship between the various concentrations of hemoglobin, of kojic acid and of H₂O₂ on the spectrum of the “final yellow product(s)” are shown.     

EFFECT OF KOJIC ACID ON THE HYDROXYLATION OF L-TYROSINE AND TYRAMINE BY MUSHROOM TYROSINASE1

Kojic acid inhibits effectively the rate of L-tyrosine and tyramine hydroxylation by mushroom tyrosinase. It also affects the spectrum of product (s) formed, this being due to the ability of dopaquinone and dopamine-o-quinone to oxidize kojic acid to a yellow product(s). Kojic acid prevents the conversion of these o-quinones to their corresponding melanins. An insoluble yellow product(s) is formed when tyramine, but not L-tyrosine, is incubated with tyrosinase for 20 h in the presence of excess kojic acid.     

OXIDATION OF INDOLEACETIC ACID BY AN APPARENTLY HOMOGENEOUS PEROXIDASE FROM THE FLAVEDO OF WASHINGTON NAVEL ORANGES (CITRUS SINENSIS)

Orange flavedo peroxidase was purified by ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. The anionic fraction, representing 51% of the total peroxidase activity, was an apparently homogeneous isoenzyme with an pI of 4.0. The cationic fraction was composed of two isoenzymes with pI of 10.0 and 11.5.
The anionic peroxidase oxidized indoleacetic acid in the presence of H₂O₂ and 2,4-dichlorophenol. This reaction was inhibited by Mn²⁺ and enzyme activity decreased at increasing pH in the range of 4.0 to 5.5. This isoenzyme also oxidized indoleacetic acid in the reaction mediated by 2,4-dichlorophenol, Mn²⁺ and phosphate. In this case the optimum pH was 4.5 and the reaction was inhibited by H₂O₂.
Study of the characteristics of both reactions indicates that, even though they could share some common steps and have similar end products, they probably differ in their reaction mechanism. The possible physiological significance of the H₂O₂ mediated oxidation of indoleacetic acid is briefly discussed.     

Reaction Between Malvidin 3-Glucoside and (+)-Catechin in Model Solutions Containing Different Aldehydes

ABSTRACT: The contribution of the aldehyde composition of wine spirit to the color changes in Port red wine was studied in model solutions. Malvidin 3-glucoside was shown to be very reactive towards catechin in the presence of different aldehydes: acetaldehyde, isovaleraldehyde, benzaldehyde, propionaldehyde, isobutyraldehyde, formaldehyde, and 2-methylbutyraldehyde. LC/MS data confirmed the formation of oligomeric pigments resulting from the reaction between the anthocyanin and the flavanol (colored products) and between 2 flavanol units (colorless products) mediated by each aldehyde assayed. The UV-visible spectra of the colored pigments showed a λ_max bathochromically shifted relatively to the λ_max of original anthocyanins. All samples revealed a "blueing" and "darkening" color effects using the CIELAB system.     

2, 4, 5-TRIHYDROXYBUTYROPHENONE (2, 4, 5-THBP)a AS A SUBSTRATE FOR MUSHROOM TYROSINASEb

2, 4, 5-THBP (2, 4, 5-trihydroxybutyrophenone) is a substrate for mushroom tyrosinase with a K_m value of 1.00 mM. The final product(s) formed is red (λ._max 480–490 nm) and it is rather stable with time. An intermediate red product(s), having a maximum at λ 505–515 nm, was detected and the time course of its conversion to the final red product(s) was studied. The data suggest that the intermediate red product(s) is 2, 4, 5-THBP-quinone which is converted to colorless polymerized 2, 4, 5-THBP-OH, which is then oxidized to a red polymerized 2, 4, 5-THBP-quinone(s), which is the final red product(s).     

EFFECT OF KOJIC ACID ON THE OXIDATION OF o-DIHYDROXYPHENOLS BY MUSHROOM TYROSINASE1

Kojic acid is a very effective inhibitor of mushroom tyrosinase as judged by its effect on the rate of pigmented products formation and on the rate of oxygen uptake when different o-dihydroxyphenols are oxidized by the enzyme. In addition to the ability of kojic acid to inhibit the enzyme per se, the data show that kojic acid can change the spectrum of some pigmented products formed in its absence, probably due to the ability of some o-quinones formed enzymatically to oxidize kojic acid to a yellow product(s). These findings call for caution in the use of kojic acid as an inhibitor of enzymatic browning in tissues and processed foods that are not yellow originally.     

SIMULTANEOUS DIFFUSION of CITRIC ACID and ASCORBIC ACID IN PREPEELED POTATOES

The study of the simultaneous diffusion of chemical preservatives in vegetable tissues permits the determination of the time required to complete this process and the concentration distributions of the preservatives.
The individual or simultaneous diffusion of citric and ascorbic acids in pre-peeled potatoes was analyzed; the effect of pH decrease on the diffusive flux of ascorbic acid and the interaction between both acids was considered as the multicomponent diffusion problem.
Potato spheres of different radii were immersed in individual solutions or mixtures of citric and ascorbic acids in concentration ranging from 0.5% to 2% W/V for different immersion times and agitation conditions. the residual concentration of citric acid was determined by titrable acidity (22058 AOAC method) and that of ascorbic acid by 2–6 dichlorophenol-indophenol method.
Experimental data were fitted to the mathematical models and the effective diffusion coefficients were determined for citric (D_e= 4.3 ± 0.2 × 10⁻¹⁰ m²/s) and ascorbic acids (D_e= 5.45 ± 0.4 × 10⁻¹⁰ m²/s) diffusing individually. When mixtures of two acids were used, multicomponent analysis was adopted and interaction coefficients were evaluated (D₁₂= 6.67 ± 0.8 × 10⁻¹¹ m²/s and D₂₁= 8.33 ± 0.8 × 10⁻¹¹ m²/s); they were an order of magnitude lower than binary diffusion values.
The pH effect on the diffusive flux of ascorbic acid was decoupled from the interaction of both acids during simultaneous diffusion by studying the diffusion of the first acid in potatoes preacidified with the second acid.     

Calcium Chloride Added to Irrigation Water of Mushrooms (Agaricus Bisporus) Reduces Postharvest Browning

The influence of 0.3% CaCl₂ added to irrigation water on mushroom tyrosinase activity and postharvest browning was examined. Calcium concentration of mushrooms was significantly increased with the addition CaCl₂ to irrigation water but had no effect on inherent tyrosinase activity but reduced postharvest browning. The CaCl₂ had even more pronounced improvement in shelf life as indicated by reduced browning following a standard bruising treatment. Transmission electron micrographs indicate that increased levels of calcium in mushrooms irrigated with CaCl₂may have decreased browning by increasing vacuolar membrane integrity; thereby reducing the opportunity for tyrosinase to react with its substrates.     

CONCENTRATION OF GAMMA LINOLENIC ACID (GLA) FROM BORAGE OIL BY UREA COMPLEXATION: OPTIMIZATION OF REACTION CONDITIONS

Production of gamma-linolenic acid (GLA, 18:3ω6) concentrates from borage oil (BO) was optimized. A 3-factor-3-level face-centered cube design was used to study the effect of reaction time (X₁), reaction temperature (X₂) and urea-to-fatty acid ratio (X₃) in order to maximize the GLA content(Y). A second-order polynomial model was employed to generate a surface response. Under optimum conditions, a maximum of 91% GLA was obtained from borage oil at a reaction time of 16 h, reaction temperature of -7C and a urea-to-fatty acid ratio(w/w) of 3.7.     

EFFECT OF BENZENESULFINIC ACID ON THE OXIDATION OF o-DIHYDROXY - AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

Benzenesulfinic acid inhibits the rate of oxidation of different o-dihydroxy-phenols and trihydroxyphenols by tyrosinase when assayed spectrophotometrical-ly but barely has an effect when assayed polarographically. Benzenesulfinic acid is a much more effective inhibitor of the rate of oxidation to pigmented product(s) by tyrosinase of o-dihydroxyphenols than of trihydroxyphenols. The spectrum of the final product(s) formed by the oxidation of most of the tested substrates was different in the absence versus presence of benzenesulfinic acid suggesting that the latter traps the o-quinones forming phenylsulfonyl derivatives in the reaction.     

LIPID CLASSES, FATTY ACID COMPOSITIONS AND TRIACYLGLYCEROL MOLECULAR SPECIES FROM ADZUKI BEANS (VIGNA ANGULARIS)

The lipids extracted from adzuki beans grown in Japan were classified by thin-layer chromatography (TLC) into eight fractions. Molecular species and fatty acid distributions of triacylglycerols (TAGs) isolated from the total lipids in the beans were determined from a combination of argentation-TLC and gas chromatography. The major lipid components were phospholipids (PL; 63.5%) and TAG (21.2%), while hydrocarbons (5.1%), steryl esters (7.5%), free fatty acids (0.9%), diacylglycerols (1.3%) and monoacylglycerols (0.5%) were also present in minor proportions. Both major samples had high amounts of total unsaturated fatty acids, representing 62.1% for TAG and 65.9% for PL. Seventeen different molecular species were detected. The major TAG components were SMD (5.0%), S₂T (19.3%), SD₂ (13.8%), SMT (9.3%), MD₂ (4.5%), SDT (7.0%), D₃ (8.8%) and ST₂ (15.9%), where S, M, D and T denote a saturated fatty acid, a monoene, a diene and a triene, respectively.

PRACTICAL APPLICATIONS

This article describes the characteristics of lipid components, fatty acid compositions as well as the profiles of triacylglycerol (TAG) molecular species of adzuki beans. α-Linolenic (18:3 n -3) acid was detected as 24.8, 21.2 and 15.2% in the TAG, total lipids and phospholipids, respectively. The oil from legumes, except the profitable fatty acid content, could be a potential source of tocopherols. The data obtained in this study would be useful to both consumers and producers for manufacturing traditional adzuki confectionaries ( wagashi ) in Japan and elsewhere.     

FATTY ACIDS FROM LARVAL SULCASCARIS SP. (NEMATODA) AS POSSIBLE INDICATORS OF INFECTION OF CALICO SCALLOPS (ARGOPECTEN GIBBUS)

Fatty acid profiles of larval nematodes (stage- 4 Sulcascaris sp.), of tissue from their intermediate host (calico scallops, Argopecten gibbus), and of the host capsule that surrounds the larvae were prepared in an attempt to identify infected scallops. Nematode tissue showed lower ratios of C_14:O/C_14:1, C_16:0/C_16:1 and C_18:0/C_18:1 than did scallop tissue. The nematodes contained relatively less C_16:0 and more C_18:2 than did scallops. Fatty acids shorter than C_14:0 were found in small amounts in both organisms. Fatty acid profiles of capsules differed little from those of normal scallop tissue.