[advances in food research] advances in food research volume 3 volume 3 || enzyme-catalyzed...

Enzyme-Catalyzed Oxidative Browning of Fruit Products

BY M . A . JOSLYN AND J . D . PONTING

Division of Food Technology. College of Agriculture. Universi ty of California. and

Wes tern Regional Research Laboratory. Bureau o f Agricultural and Industr ial Chemistry. 0 . S . Department of Agricul ture

CONTENTS Page

I . Introduction . . . . . . . . . . . . . . . . . . . . . . 1 1 . G e n e r a l . . . . . . . . . . . . . . . . . . . . . . 1 2 . Theories of Enzymic Browning . . . . . . . . . . . . . 2

7 1 . Polyphenol Oxidase . . . . . . . . . . . . . . . . . 7 2 . Peroxidase . . . . . . . . . . . . . . . . . . . . . 8 3 . Flavoprotein Enzymes . . . . . . . . . . . . . . . . 9 4 . Cytochrome Oxidase . . . . . . . . . . . . . . . . . 9

9 11 14 18 18 20 22

V I I . Activation and Inactivation of Phcnolases . . . . . . . . . . . 24 1 . Effect of pH . . . . . . . . . . . . . . . . . . . . 24

3 . Effect of Temperature arid pH on Rate of Inactivation . . . . . 27 4 . Effect of Salts and Inhibitors . . . . . . . . . . . . . . 30

a . Commonly Used or Unpatcntcd Clicmicals . . . . . . . . 33 b . Patented Chemical Inhibitors . . . . . . . . . . . . 33

5 . Effect of Sugars . . . . . . . . . . . . . . . . . . 34 6 . Inactivation by Electrical Energy . . . . . . . . . . . . 35

References . . . . . . . . . . . . . . . . . . . . . . . 37

I1 . Nature and Properties of the Enzyme Systcms Involved . . . . . .

I11 . Location and Distribution of Polyphenolases . . . . . . . . . . I V . Nature of Substrates . . . . . . . . . . . . . . . . . . . V . Relation of Oxidizing Enzymes to Respiration . . . . . . . . . .

V I . Mechanism of Oxidation of Substrate . . . . . . . . . . . . . 1 . Initial Stage of Oxidation . . . . . . . . . . . . . . . 2 . Second Stage of Oxidation . . . . . . . . . . . . . . . 3 . Gcneral Aspects of Pigment Formation

2 . Effect of Temperature . . . . . . . . . . . . . . . . 26

. . . . . . . . . .

V I I I . Control of Enzymic Browning . . . . . . . . . . . . . . . 36

I . INTRODUCTION

1 . General

Many fruits undergo rapid changes in color following mechanical or physiological injury during harvesting and storage . Such color change in fruit products is accentuated during preparation for processing by canning, dehydration or freezing. and continues during freezing storage

1

2 M. A. JOSLYN AND J. D. PONTINQ

and subsequent defrosting of frozen fruits. The natural color of the product may be destroyed or masked by formation of dark brown or reddish pigments which cause the product to become unattractive in color. Undesirable changes in flavor, odor and nutritive value usually accompany this “browning. )’ Marked decreases occur, for example, in ascorbic acid content, (or even its complete loss), as well as decreases in other oxidizable nutrients, such as carotene.

Enzyme-catalyzed oxidative browning has long been recognized, according to Kastle (1910). Lindet (1895) concluded that the changes in color occurring in fresh cider are due to oxidation of tannin by a laccase-like enzyme contained in the tissues of the apple. The oxidizing enzymes of plants have been studied by plant biochemists and plant physiologists to determine their role in plant respiration (Onslow, 1931 ; Szent-Gyorgyi, 1937 ; Oppenheimer and Stern, 1939 ; Green, 1940 ; and Lardy, 1949), but investigations of the oxidizing enzymes of fruit have been very limited (Lindet, 1895; Martinand, 1895, 1897; Bassett and Thompson, 1911 ; Onslow, 1920 ; Overholser and Cruess, 1923 ; Cruess and Fong, 1926, 1929a, 1929b ; Ezell and Gerhardt, 1938a, 193810 ; Joslyn, 1941 ; Ponting and Joslyn, 1948) ; and Guadagni, Sorber and Wilbur, 1949.

Most of the research on enzymic oxidation of fruit products has been directed to qualitative characterization of the oxidizing enzymes present and to methods of inactivation o r control of the enzymes involved in browning, but without developing the chemistry of the enzyme systems involved, the nature of the substrates acted upon, or the mechanism of the transformations leading to the formation of the brown pigments.

Quantitative methods in studying plant oxidases were applied in the United States in the early extensive investigation of Bunzell (1912a, 1912b, 1916a, 1916b, 1916~) . Nelson and his collaborators a t Columbia University investigated, extensively, the tyrosinase of the common edible and the wild mushroom during the period of 1938-1941 and made many contributions to quantitative techniques (see review by Nelson and Daw- son, 1944). In investigations of enzymic oxidation of fruit products, however, quantitative methods have been applied only recently (Samisch, 1935a, 1937 ; Hussein and Cruess, 1940 ; Jimenez, 1947 ; Cruess and Sngi- hara, 1948 ; Ponting and Joslyn, 1948 ; El-Tabey and Cruess, 1949). The present knowledge of the role of oxidizing enzymes in the oxidative discoloration of fruit and fruit products has been reviewed briefly by Joslyn (1941), Cruess (1948), and Taubef (1949).

2. Theories of Enzymic Browning

Several theories have been proposed fo r the nature and course of enzymic browning, differing considerably in nomenclature as well as in

ENZYME-CATALYZED OXIDATIVE BROWXINQ OF FRUIT PRODUCTS 3

mechanism. This difference in nomenclature has been carried over even into the recent technological literature and has been the cause of some confusion. The early developments in this field have been discussed from the historical point of view by Kastle (1910), Oppenheimer (1926, 1939), Chodat (1936), Raper (1932, 1938), Onslow (1931), Sutter (1936), Oppenheimer and Stern (1939), Franke (1934, 1940).

Bach and Chodat (1904) (see Kastle, 1910, and Chodat, 1936) proposed the theory that plant tissues which darken on injury contain a substance termed “oxygenase” which in the presence of air undergoes autooxidation, yielding a peroxide. This peroxide, activated by the enzyme peroxidase present in most plants, then brings about the oxidation of the natural phenolic substances. Kastle and Loevenhart (1901) and Overhoher and Cruess (1923) ascribed discoloration to such a system.

During the period 1919-1924, Onslow (1931) who had been interested in the darkening shown by many plant tissues, especially when injured, reported that these plants contain phenolic compounds with free o-dihydroxy benzene groups such as catechol, protocatechuic acid, and caffeic acid. The catechol compound was separated by extraction with hot alcohol, removal of alcohol by distillation, precipitation of the compound in the residue with lead acetate, and deleading with sulfuric acid. The resulting solution was tested with ferric chloride to determine presence of catechol compound. It was observed that the darkening of the catechol compounds in plant tissue was faster than in aqueous solution, but did not occur when the plant tissue was boiled, and this fact led her to postulate the existence of an enzyme which catalyzed the autooxidation of the o-dihydroxy phenol derivatives in which both hydroxyl groups were functional. The presence of oxygenase was demonstrated by addition of enzyme extract from the same plant to the neutralized catechol compound solution and observing for oxidation with subsequent bluing of added guaiacum. This author modified the Bach and Chodat concept of oxygenase by considering i t as an enzyme which, in the presence of air, catalyzed the oxidation of o-dihydric phenols yielding as one of the oxidation products a peroxide or hydrogen peroxide. The latter, in the presence of peroxidase, was activated and oxidized a suitable chromogen. She believed that this secondary oxidation involved mono-, di- and poly- hydroxyphenolic compounds, including tannins, which after oxidation were transformed into the characteristic colored pigments.

Onslow systematically investigated the oxidizing enzymes present in higher plants and segregated them into two groups : those which contain oxygenase and catechol compound; and those in which oxygenase and catechol compound were absent, the peroxidase plants. The first group of plants discolor rapidly on injury and include, according to Onslow,


the following fruits : apple, apricot, banana, cherry, fig, grape, peach, pear, and strawberry. The second group of plants which do not discolor on injury include citrus fruit (lemon, orange, lime, and grapefruit), red currants, melon, pineapple, and tomato.

Szent-Gyorgyi (1925) ,* succeeded in showing that darkening of plant tissues could take place in the absence of peroxidase ; about this time Raper and coworkers ( loc. c i t . ) were engaged in unraveling the intermediate reactions involved in the conversion of tyrosine into melanin by tyrosinase without the intervention of peroxidase. During this period and subsequently, the earlier unreliable qualitative colorimetric methods of enzyme assay were displaced by manometric methods, and thus the ease of separation and purification of the enzyme was greatly enhanced. Sub- sequently, existence of copper-containing oxidases catalyzing the oxidation of phenols by molecular oxygen, as distinct from the iron porphyrin peroxidases, catalyzing the oxidation of phenols by hydrogen peroxide, was established.

Three groups of phenolases were recognized : tyrosinase (monophenol oxidase or cresolase) , catecholase (polyphenol oxidase) , and laccase (Craubard, 1939).

There is still some disagreement on nomenclature of the polyphenol oxidases. Boswell and Whiting (1938) prefer the term ‘ ‘polyphenol oxidase, ” or ‘ ‘ catechol oxidase ” to ‘ ‘ tyrosinase l 1 for potatoes and fruit, while Nelson and Dawson (1944) prefer “ tyrosinase. ” In the older literature and by some recent writers, e .g . , Cruess and Sugihara (1948), “ ~ x i d a s e ~ ~ is used for the mixture of polyphenol oxidases and peroxidases in fruit extracts or crude enzyme preparations from fruit tissues. We prefer “ polyphenol oxidase, ” abbreviated to polyphenolase or phenolase ; both terms are used interchangeably in this review. The phenolases catalyze the oxidation of a phenolic substrate by molecular oxygen t o some intermediary product, usually a quinone, which can then oxidize other constituents such as ascorbic acid, o r other phenols. The final pigmentation may be due to the oxidation and subsequent transformation of the original phenolic substrate o r to the induced oxidation of others.

Spitzer (1931), on the basis of the color change observed with ferric chloride, reported the occurrence of o-dihydroxyphenylalanine (dopa) in the tissues of apples, pears and other fruits. A dopa oxidase preparation from meal worm was found to oxidize the chromogen extracted with methyl alcohol from apple tissue, and he concluded that a specific phenolase, dopa oxidase, and a specific substrate, dopa, were involved in the browning of fruit. Neither dopa, as such, nor dopa oxidase was isolated from the fruit tissues, and in view of the limitations of the ferric

ENZYME-CATALYZED OXIDATIVE BROWNING OF FRUIT PRODUCTS 5

chloride test used and of the color changes in aqueous extracts of apple tissue (the formation of an initial yellow color on addition of Naz COB to apple extracts followed by reddish brown surface discoloration which extended into the solution so that the entire sohtion turned reddish brown) which are not sufficient for identification, Spitzer 's conclusions can not be accepted.

Balls and Hale (1935) reported that pigment formation in injured apple tissue was accelerated by the addition of horseradish peroxidase and inhibited by addition of direct inhibitors of peroxidase (cysteine, glutathione, and other sulfhydryl compounds) or by those substances which accelerate the inhibition of peroxidase by hydrogen peroxide. They concluded that the darkening of freshly cut surfaces of apples is a reaction catalyzed by peroxidase, and that the formation of hydrogen peroxide from molecular oxygen by a respiratory enzyme is a necessary preliminary step. This was not confirmed by Ponting and Joslyn (1948).

Cytochrome oxidase in the presence of cytochrome is also known to react with certain phenols in the presence of oxygen and to convert them into pigmented compounds similar to those observed in the phenolase or peroxidase oxidations. Although its occurrence in plants is limited, it is possible that under certain conditions it also may be involved in darkening.

The mechanism of the discoloration following hechanical or physiological injury is not established. It is known that discoloration does not occur in the intact cells of the fruit susceptible to enzymic oxidation. When the protoplasm of the cells is altered mechanically by cutting, bruising, or freezing, or by pathological damage, browning occurs rapidly. Heat damaged cells, or cells damaged by chemicals such as ether, will also brown, provided that the treatment kills the cells but does not inactivate the oxidizing enzymes concerned in browning. This discoloration may occur a t surfaces exposed to air, or in the interior of the fruit where sufficient oxygen for oxidation is present.

The internal browning of f rui t such as the Yellow Newtown apples caused by physiological injury to the large isodiametric cells of the fruit (Winkler, 1923 ; Overholser, Winkler, and Jacob, 1923), and the surface discoloration observed in apple scald are similar. Accumulation of respiratory products such as acetaldehyde, alcohol, etc., influence such discoloration by damaging the cells and increasing their permeability to oxygen. Storage at concentrations of oxygen low enough to result in the accumulation of respiratory intermediates in sufficient concentration to result in physiological injury or death of cells also will induce discoloration. Bennett and Bartholomew (1924), f o r example, found that the occurrence of black

Physiological injury to cells will also cause browning.


heart in potatoes resulted when anaerobic conditions were brought about in the tissues following exhaustion of most of the free oxygen in the surrounding atmosphere.

Lindet (1895) first explained browning following injury by assuming that the oxidase of fruits such as apples that brown on injury, and the tannin or chromogen upon which it acts, are stored in different cells, and that hence it is only when these are brought together by actual rupture of the cells that discoloration occurs. Since the occurrence of oxidase and chromogen in separate cells was recognized to be untenable, it was assumed later that either the enzyme or the chromogen existed in inactive condition prior to injury and became activated following injury and exposure to air. Palladin (1908) early proposed a more tenable theory. He showed that discoloration was connected with the enzymic oxidation of polyphenols. The oxidized polyphenol in intact cells, or under anaerobic conditions, becomes reduced again to the uncolored compound. If, however, the cells are damaged, this reduction does not take place and the oxidized phenol appears as a “respiratory pigment.”

It is well recognized today that the protoplasm, even in actively respiring cells, is under reducing conditions because the cellular oxidation- reduction potential is low enough to prevent the accumulation of the oxidized phenols, even if the polyphenol oxidase acts as a respiratory enzyme, which is not generally true. Szent-Gyorgyi (1937) pointed out that the lack of coloration in intact cells must be due either to reduction of oxidized phenols a t a rate equal to that of their oxidation or to the fact that the phenolase does not serve as a terminal respiratory enzyme in “oxidase” plants. I n damaged tissues, discoloration appears a t once indicating that the phenol is either oxidized faster or is reduced more slowly than in the intact plant tissue.

Szent-Gyorgyi and his collaborators actually found that macerated potato tissue (in the presence of ascorbic acid) takes up at least fif- teen times as much oxygen for the oxidation of the phenol as it uses in normal respiration, and concluded that in the intact tissue the oxidase does not act and that damage of the cells releases the phenol oxidase. Subsequently he demonstrated that addition of ascorbic acid to poly- phenoloxidase plants prevented pigment formation and suggested that inactivation of the dehydrogenases which would normally reduce dehy- droascorbic acid was responsible for pigmentation on injury. There is but little available information on plant dehydrogenases ; and the dehydrogenases of fruit tissue have not been investigated. The type of dehydrogenases present and their susceptibility to injury are not known.

ENZYME-CATALYZED OXIDATIVE BROWNINQ OF FRUIT PRODUCTS 7

11. NATURE AND PROPERTIES OF ENZYME SYSTEMS INVOLVED

Present knowledge of the enzyme systems which may play a part in browning of fruits is far from complete. Some of the oxidizing enzymes are known to be important while others are of doubtful importance, and some which could be involved have not yet been investigated from this standpoint. The enzyme systems which may be involved in oxidative browning of higher plants with some of their properties are briefly discussed.

1. Polyphenot Oxidase

There are evidently many different enzymes having the ability to catalyze oxidation of phenols by molecular oxygen. Nelson and coworkers (for references, see Nelson and Dawson, 1944) have found that the phenolase preparations they obtained from mushrooms always had the ability to catalyze oxidation of monophenols in addition to catalyzing oxidation of polyphenols. The ratio of rate of monophenol oxidation to that of polyphenol oxidation, however, varied considerably in preparations from different mushroom sources. Keilin and Mann (1938) purified polyplienol oxidase from mushrooms and found it did not oxidize monophenols appreciably. Kubowitz (1937) and others have found that the phenolase from potatoes oxidizes both monophenols and polyphenols. Eiger and Dawson (1949) found that purified sweet potato phenolase oxidizes only polyphenols with the o-dihydroxy grouping, although sweet potato slices will oxidize p-cresol also. I n almost all the above cases the activity of the enzyme was much greater toward catechol or other polyphenols with functional o-dihydroxy grouping than toward monophenols.

The phenolase in an apricot extract prepared by extraction of frozen ground tissue by Samisch and Cruess (1934) was able to catalyze only the oxidation of catechol and pyrogallol and not that of phenol, resorcinol, quinol, phloroglucinol or tyrosine. Samisch (1937) also reported that the extracts of fruit of apricot (var. Royal) and avocado (var. Spinx) oxidized catechol rapidly and pyrogallol only slowly. The avocado extract oxidized phloroglucinol at an exceedingly slow rate, while apricot extract caused no observable effect. The avocado extract, like the apricot extract, did not oxidize phenol, resorcinol, quinol, or tyrosine. El-Tabey and Cruess (1949), using cold acetone to extract the acetone-soluble pigments and other constituents, obtained a white colored enzyme preparation from apricots containing both phenolase and peroxidase. This apricot enzyme preparation catalyzed the oxidation of catechol, protocatechuic acid, caffcic acid, digallic acid, benzidine, gum guaiac, and o- and pphenylenediamine.

Jimenez (1947) found that guava extract catalyzed the oxidation of


catechol, pyrogallol and p-phenylenediamine, but not that of monohydric phenols, resorcinol or phloroglucinol. Cruess and Sugihara (1948) , using cold acetone-extracted olive tissue, found that it catalyzed the oxidation only of o-dihydroxy phenols. Ponting and Joslyn (1948) prepared a purified peroxidase-free polyphenolase from apple which was found to catalyze the oxidation of catechol and pyrogallol, but not that of hydroquinone, resorcinol, or monohydric phenols. The main reaction catalyzed by polyphenolase in fruits therefore appears to be oxidation of o-dihy- droxybenzene groups. This does not imply, however, that either free catechol or pyrogallol is oxidized in fruits. As pointed out in another section, the only natural substrate purified from fruits so far has a caffeic acid nucleus.

It is inactivated when it catalyzes the oxidation of catechol, etc., although whether this is due to reaction products or to the reaction process itself is not yet determined. Polyphenolase prepared from apples is very unstable, losing its activity rapidly at temperatures as low as -34°C. (-30°F.) (Ponting and Joslyn, 1948). It appears to be fairly stable when stored in a “dry- ice” box a t a temperature in the neighborhood of -60’ to -75°C. (-76” to -101.2’F.). Drying a solution from the frozen state dena- tures much or all of the enzyme. On account of its instability, polyphenolase has not been highly purified from fruits as yet.

Polyphenolase itself is a rather unusual enzyme.

2. Peroxidase

This enzyme catalyzes the oxidation of certain phenolic or aromatic amine compounds by H202 to form dark colored polymers. Balls and Hale (1934) reported that peroxidase requires for a substrate a di- (or more) substituted benzene ring, the substituents being ortho or para ; and one must be -OH or both must be -NH2.

Early work by Balls and Hale (1935) indicated that peroxidase is responsible for darkening of injured apple tissue. They showed that peroxidase and its substrate were present by the fact that apple tissue would darken in the absence of air if H202 were added. Hydrogen peroxide was also believed to be present naturally because guaiacum tincture or potassium iodide-starch mixture was oxidized (formed a blue color) in the presence of fresh apple tissue. They considered H202 formation by a direct oxidase to be a necessary preliminary step to darkening by peroxidase.

In the light of more recent knowledge, however, the reactions for “peroxide” can be explained as due to quinones formed by polyphenolase-catalyzed oxidation rather than to peroxides. It is true that peroxidase and a substrate are present, but the formation of H202 by a direct

ENZYME-CATALYZED OXIDATIVE BROWNING‘ OF FRUIT PRODUCTS 9

oxidase in apples is unproved. It has been shown by Dawson and Nelson (1938) and Dawson and Ludwig (1938) that H202 is not formed when catechol is oxidized by mushroom phenolase. Pugh (1930) found that banana skins would oxidize tyrosine and p-cresol, but not guaiacol unless H20z was added; therefore H202 was not formed in the skin.

There still remains the possibility that another enzyme system can produce some HzO2 from molecular oxygen ; flavoprotein enzymes are known to do this. Ponting and Joslyn (1948), however, demonstrated that peroxidase-catalyzed darkening of apples can be responsible for only a small portion, if any, of the total enzymic darkening; peroxidase is not required because darkening occurred when purified apple polyphenolase was added to boiled apple juice in which the peroxidase had been destroyed.

3. Flavoprotein Enzymes

Certain flavoprotein o r “yellow ” enzymes can cause oxidation of their specific substrates by molecular oxygen with formation of H202. These are usually considered as animal enzymes, but glucose oxidase is obtained from molds and others occur in yeast and bacteria. It is possible that enzymes of this group could produce some Hz02 which could be used by peroxidase to cause browning. The contribution of this group of enzymes cannot be large, however, as pointed out above.

4. Cytochrome Oxidase

This enzyme has not been considered t o be very widespread in the plant kingdom, but is the main terminal oxidase in animal respiration. It catalyzes oxidation of cytochrome, an iron-porphyrin compound, by molecular oxygen. I n view of the work of Hussein (1944) showing that cytochrome oxidase was involved in respiration of the orange, and the work of Schade et al. (1948, 1949) showing that potato respiration may be mediated mostly by cytochrome oxidase, the occurrence and role of cytochrome oxidase in fruits warrants more investigation. Although this enzyme system does not cause darkening directly, there is a possibility that the oxidized cytochrome can cause the oxidation of phenolic or other chromogenic compounds.

111. LOCATION AND DISTRIBUTION OF POLYPIIENOLASES

It is a matter of common experience that the discoloration of plant tissue is not general, particularly in its initial stages and when care is taken to prevent distribution of the contents of cut cells over the entire area. The vascular elements of a number of fruits will darken more rapidly when the fruits are cut and exposed to air. Bunzell (1916b)

10 M. A. JOSLYN AND 3. D. PONTINQ

showed that the relative oxidase activity of different parts of the potato, tulip-tree and sugar beet plants varied. Thus, for spinach the rate of oxygen uptake by the leaves was smaller than by the roots, and the leaf extracts catalyzed oxygen uptake by catechol, p-cresol, pyrogallol and hydroquinone at a ratio in the order listed. The root extracts catalyzed the uptake of oxygen by p-cresol, hydroquinone, tyrosine, catechol, m- cresol, phlorhizin, guaiacol and pyrogallol. I n sugar beets, the root extracts were more active than the leaf extracts, but the relative oxidase activity toward various reagents was approximately the same for both roots and leaves.

Samisch (1935b) observed that when slices of firm ripe apricots were immersed in catechol solutions, the peel and vascular bundles, particularly the large ventral and dorsal vascular bundles, colored the most rapidly. The remainder of the pulp, especially the innermost portion of the pericarp, was exceedingly slow in staining.

The peroxidase is also localized in areas of greatest physiological activity. Samisch (1935b) found that the epidermal cells of apricot showed a higher peroxidase activity than the remainder of the tissue. The guard cells of the stomata were particularly active in catalyzing the bluing of benzidine in the presence of hydrogen peroxide. Within the vascular bundles which showed a high peroxidase activity, no reaction was detected in the spiral vessels, and the peroxidase in the vascular bundles was found to be localized in the bundle sheath. No peroxidase reaction was obtained within the protoplasm, although the vacuolar sap gave a positive test.

Hussein and Cruess (1940) found by immersing cut grape tissue in slightly oxidized benzidine solution that the greatest " oxidase " activity was in the epidermal cells, although it was also strong in the vascular bundles and less active in the pericarp. Hussein, Mrak and Cruess (1942), found that the peroxidase activity of Thompson Seedless and Muscat grapes was greater in the skins than in the flesh; the ratio of peroxidase activity of the skin to flesh for the two varieties was 31.1:l and 7.75 :1, respectively.

Jimenez (1947), using p-phenylenediamine, observed, microscopically, the distribution of oxidase in slices of firm ripe guavas and found highest activity in the inner flesh and in the vascular bundles, especially the tissues surrounding the fibrovascular bundles.

Rahman (1948) determined the distribution of peroxidase and phenolase in prunes, using quantitative colorimetric techniques, and found that both peroxidase and phenolase were more active in the prune flesh closer to the pit, lowest in the middle layer of flesh between pit and skin, and then increased again towards the skin. The flesh at the blossom end

E N Z Y M E - C A T A L Y m OXIDATIVE BROWNINQ O F FRUIT PRODUCTS 11

was higher in both peroxidase and phenolase activity than a t the stem end.

I n contrast with Samisch’s (193513) failure to observe phenolase activity in the protoplasm by qualitative methods is the clear-cut demonstration by Arnon (1949) that polyphenol oxidase is localized principally, if not entirely, in the choroplasts of the spinach beet (chard), Beta vulgaris.

More information is needed on the location and distribution of the polyphenolases and peroxidases in fruit tissues, particularly as affected by maturity, growing conditions and varietal differences.

IV. NATURE OF SUBSTRATES

The natural phenolic substrates in fruits which undergo enzymic oxidation and polymerization to dark colored pigments have scarcely been investigated chemically.

Most of the investigations of the natural substrates have been qualitative in nature and largely based on the technique introduced by Onslow (1931) for the demonstration of the presence of catechol compounds. She extracted the tissues with hot alcohol, removed the alcohol by distillation and treated the residue with aqueous lead acetate to precipitate the catechol compound. The lead was then removed with sulphuric acid and the extract tested with ferric chloride. She considered the formation of a green color which turned pink, purple and blue on addition of very weak alkali as a reaction specific for the ortho-dihydroxy benzene grouping. Spitzer (1931), using color reaction of methyl alcohol extracts of apple tissue (green color which turned dark red violet on addition of Na2C03) as characteristic of dihydroxyphenylalanine, reported the occurrence of 0.1% dopa in apple tissue. A wide variety of o-dihydroxy compounds could be involved as substrates : protocatechuic acid, caffeic acid, caff e- tannic acid or the so-called catechol tannins, dihydroxycinnamic acid, aesculetin, dihydroxyphenylalanine, chlorogenic acid, hydro-urushiol, etc. Onslow (1931) lists these compounds and the plants in which they have been reported.

Flavones and flavonols have long been spspected of being involved in discoloration. As early as 1921, Nagai reported that certain anthocyanins were completely decolorized by the action of oxidizing enzymes which also caused certain flavones, flavonols and their glucosides to yield characteristic oxidation colors. Nagai showed that the color of aqueous or alcoholic extracts of numerous plant tissues rich in flavones changes to brown or reddish-brown when the extracts were treated with freshly prepared plant juices containing oxidizing enzymes, and he also showed that purified preparations of the pigments themselves, yielded brownish colors by the action of these enzyme preparations. Nagai (1921) showed that


quercetin and quercitrin, a monorhamnoside of quercetin, were oxidized by plant oxidases into deep red colored pigments which rapidly changed to brown. Sando (1924) isolated and identified quercetin from the peels of McIntosh apple and postulated that it may be the chromogen involved in storage scald.

The chromogens involved in darkening have been referred to in the literature as “tannins.” Caffetannins, considered by Russell (1935) to be closely related to or even identical with chlorogenic acid, which do not precipitate proteins and which yield on hydrolysis quinic acid, caffeic acid and an unknown residue have been implicated, usually on the basis of color reactions alone. According to Russell (1935), most of the natural tannins are phlobatannins, polyhydroxyflavpinacols derived from the corresponding 4-hydroxyflavans and are related to plant pigments of the benzopyran type. Szent-Gyorgyi (1937) suggested that flavonones such as hesperitin, which occurs in lemon peel, served as intermediates in the oxidation chain between peroxidase and ascorbic acid and may be involved in browning. A similar compound, rutin, isolated from the flowers of elder by Sando and Lloyd (1924) and now known to be widely distributed in nature, may be involved as substrate for some of the plant oxidases.

Samisch and Cruess (1934) prepared a “tannin” extract from the lead precipitate of the water-soluble portion of an acetone extract of apricots. This solution was an excellent substrate for apricot phenolase, although it gave only a weak color test with ferric salts. El-Tabey and Cruess (1949) repeated this separation by extracting fresh apricot tissue with acetone, filtering, evaporating to dryness a t 70°C. (158”F.), redissolving in acetone and redrying, and obtained a preparation which gave the usual ferric chloride test for catechol compounds.

Balls and Hale (1935) partially purified the chromogenic substrate of “peroxidase” from apples. They found this material to be white, amorphous, nitrogen-free, slightly acid, autooxidizable in alkaline solution and rapidly oxidizable in neutral or slightly acid solution by peroxidase and H202 to form a deep brown color. The properties of this preparation are strikingly similar to those of the olive substrate prepared by Cruess and Alsberg (1934). It is quite probable that the material would be oxidized by polyphenolase as well as by peroxidase and H202.

Probably the most extensive study of natural substrates was that made by Cruess and Alsberg (1934) on the bitter principle of the olive, oleuro- pein. This substance, which was later found by Cruess and Sugihara (1948) to be a substrate for polyphenolase, proved to be a glucoside of caffeic acid, the latter also being esterified with a phenol. The purified material was creamy white, amorphous and did not contain nitrogen.


Only the caffeic acid portion of this compound contains the o-dihydroxy grouping necessary for polyphenolase oxidation. The other phenoIic portion is not required by the enzyme. This natural substrate, therefore, should not be called a tannin, particularly since Cruess and Alsberg (1934) found that their purified substrate was not precipitated by gelatin or absorbed by hide powder, reactions typieal of tannins. Thus the statement commonly made that the darkening of fruits is caused by oxidations of “tannins” does not apply in the case of the only well-investigated frui t ; and it probably does not apply to any other fruits.

The investigations noted above comprise essentially all of our present knowledge of the naturally-occurring oxidizable substrates of fruit. Some additional information, however, is available from studies on other plant materials with similar enzyme systems. Rudlrin and Nelson (1947), be- lieving that the phenolase of sweet potatoes was the terminal oxidase in the respiration chain, isolated its substrate and found i t to bc a mixture of o-dihydric phenolic compounds which, when added to respiring sweet potato slices, increased the rates of both oxygen uptake and carbon dioxide evolution. One component of this mixture proved to be chlorogenic acid and the other component a mixture of three compounds with the main one being similar to chlorogcnic acid. It is interesting that chlorogenic acid is an ester of caffeic acid, as is the substrate isolated from olives (Cruess and Alsberg, 1934). Its structure is given below :

OH

OH OH

Free phenols of low molecular weight evidently do not exist widely in nature, although Robinson and Nelson (1944) isolated the substrate of tyrosinase from white potatoes and found i t to be I-tyrosine. They suggested that the tyrosine is oxidized by the enzyme to dopa and that the latter could act as a hydrogen carrier in the respiration chain. Lamb and Sreerangachar (1940a) found free gallic acid in tea leaves and stated that catechol was probably also present in traces.

From the meager information in the literature it is obvious that the field of natural substrate chemistry is virtually untouched, and from the standpoint of prevention of f rui t discoloration it should be a profitable one to explore further.


V. RELATION OF OXIDIZINQ ENZYMES TO RESPIRATION

Several hypotheses have been presented as to the function of polyphenolase in plants but none has been proven. Szent-Gyorgyi and Vietorisz (1931) suggested that the enzyme (of potatoes) is not concerned in respiration, but operates only in wound healing. When the tissue is crushed the quinone, formed by enzymic oxidation, could act as a germicide and could also precipitate protein to give mechanical protection. This view has not generally been accepted, probably because it is based mainly on the observation that the rate of oxidation in crushed tissue is about 15 times that in the intact potato. These workers rea- soned that if this large increase in oxidation rate by the enzyme could occur on in jury, only one-fifteenth of the potential enzyme activity would be used in normal respiration. They considered this as evidence against the enzyme being involved in respiration. However, this increase in rate of oxidation is separate from respiration. The rate of COz evolution does not increase on injury and the R.Q. decreases. Sreerangachar (1941 ) showed that the R.Q. (Respiration Quotient) in crushed tea leaves dropped from more than 1.0 to about 0.3.

A much more widely held view of the function of polyphenolase in plants is that it functions as the terminal oxidase in respiration. This was proposed by Boswell and Whiting (1938,1940). They added catechol to respiring potato slices (also apples and some vegetables) and found a sharp increase in rate of oxygen uptake, followed by a gradual decrease to 33% of that with potato slices alone. Further addition of catechol caused no response, indicating that the polyphenolase was inactive. They concluded that polyphenolase is the terminal oxidase for at least two- thirds of the respiration of potatoes, while some other system is responsible for the other one-third.

Baker and Nelson (1943) repeated these experiments using 4-tertiary butyl-catechol, which caused much less enzyme inactivation than catechol, and found that the decrease in respiration rate was as great as with catechol, although the enzyme remained active. These cuthors also added protocatechuic acid instead of catechol and found that the rate of oxygen uptake rose and was maintained for several hours. The rate of COZ evolution also increased and the R.Q. was near unity. The protocatechuic acid increased the respiration rate without causing an accumulation of quinone (or colored products), indicating that the quinone was reduced by the preceding part of the respiration chain. This finding, together with the inhibition of respiration to the extent of 85% by KCN and 4- nitrocatechol, which are also inhibitors of tyrosinme, led these authors to

ENZYME-CATALYZED OXIDATIVE EROWNINQ OF FRUIT PRODUCTS 15

conclude that a t least 85% and probably all the respiration of potatoes is dependent on tyrosinase.

Recently, Schade et d. (1949) have brought forth evidence that the rise and fall in respiration rate caused by addition of catechol to potato slices is due to the catechol acting as a cell poison rather than as a substrate for the tyrosinase. As little as 0.02 mg. of catechol added to a Warburg flask containing respiring potato slices caused a measurable inhibition of the normal respiration path, which shows a gradual increase in rate with time. These authors found that after treatment with sufficient catechol to evoke the maximum response in oxygen uptake the potato cells were no longer viable and showed no endogenous respiration. The so-called residual respiration of the slices after catechol treatment may represent enzymic oxidation of the catechol by the nonviable tissue slices, according to these workers. They suggest that the response of potato slices to addition of catechol is merely an expression of their catechol oxidase capacity without necessary implication of this enzyme in the endogenous respiration. In a later paper Schade and Levy (1949) presented evidence that two oxidases, polyphenolase and cytochrome oxidase, participate in the respiration (oxygen uptake) of potato slices. With prolonged washing times such as used by Boswell and Whiting (1938) and Baker and Nelson (1943) the participation of cytochrome oxidase decreases while that of polyphenolase increases. The polyphenolase also is much more sensitive to low oxygen pressure than is cytochrome oxidase. These authors concluded that in the intact potato tuber, cytochrome oxidase, and not tyrosinase, is the primary mediator of respiration.

Bonner and Wildman (1946) concluded that the terminal oxidase in spinach leaves is polyphenolase, mainly on the basis of experiments indicating that inhibitors of this enzyme inhibit both oxygen uptake and CO, evolution. They could find no cytochrome oxidase or ascorbic acid oxidase in spinach.

Bonner (1948), however, found that the terminal oxidase in the respiration of the Avena coleoptile was cytochrome oxidase, this plant material being devoid of polyphenolase.

Walter and Nelson (1945) found that when p-cresol and some other monophenols were added to respiring sweet potato slices the rate of oxygen uptake and COZ evolution were increased. They concluded that tyrosinase has the role of a terminal oxidase in sweet potato respiration.

Rothchild and Macvicar (1949) recently investigated the distribution of polyphenol oxidase and ascorbic acid oxidase in the leaf blades of several higher plants. They found that rapid oxidation of both catechol and dl-dopa occurred in presence of leaf tissue homogenates of several


species of “dicotyledonacae.” The tomato and sweet potato plants were found to contain the most highly active enzymes. Polyphenol oxidase could not be demonstrated in wheat leaf blade homogenates. Ascorbic acid oxidase activity was highest in sweet potato plants, lower in tomato plants, and lowest in Swiss chard and wheat plants. Sodium diethyl dithiocarbamate, in concentrations of 0.001 M and 0.01 M, was not found to inhibit oxygen consumption by leaf tissue homogenates in presence of added dopa, but ascorbic acid oxidation was marlredly inhibited at both concentrations. Copper-deficient tomato plants were markedly less active in polyphenol oxidase and ascorbic acid oxidase than normal plants ; but the addition of copper ions restored oxidative activity. Their data indicate that the copper-containing oxidases are not the principal terminal oxidases in leaf blade homogenates, particularly since they found that the endogenous rate of oxygen consumption was not significantly affected by the carbamate.

I n the case of fruit respiration, little work has been done on the elucidation of the role of polyphenolase or on the terminal oxidase of respiration. Hussein (1944) studied systems for oxygen uptake in the orange. He found no polyphenolase, but both cytochrome and 0- and p-phenylenediamine were oxidized, indicating the presence of cytochrome oxidase.

Ascorbic acid oxidase has been shown to be absent in oranges by Hus- sein (1944) and in apples by Ponting and Joslyn (1948), although Hackney (1946) claims to have found i t in Australian apples. Most likely i t is not concerned in fruit respiration, although it does act as a terminal oxidase in barley respiration, according to James and Cragg (1943).

Lamb and Sreerangaehar (1940b) made an interesting observation concerning the possible role of polyphenolase in tea leaf respiration. These workers noted that a particular tea bush had leaves which did not undergo the typical polyphenolase-catalyzed “fermentation” when crushed. Investigation showed that this was due to the absence of polyphenolase ; fermentation proceeded when an enzyme preparation from other tea leaves was added. Thus, respiration is not necessarily related to polyphenolase in tea leaves. Kertesz (1933) similarly found that Sunbeam peach tissue did not darken when crushed in air. I n this case, however, the absent factor was not polyphenolase but its substrate. When a substrate extract from a darkening variety of peach was added to Sunbeam tissue, darkening proceeded. Kertesz presented only qualitative data on phenolase activity and did not show that, quantitatively, the phenolase content of the Sunbeam peach was similar to that of other varieties. The content of permanganate-reducing matter adsorbable on charcoal, however, was markedly lower.


Strawberries are similar to oranges in that they do not contain polyphenolase (Hussein, 1944). They respire a t a higher rate than many other fruits (Haller, Rose and Harding, 1941). Whether cytochrome oxidase is present or not has not been determined.

I n view of the uncertainty as to the role of polyphenolase in potato respiration and the dearth of data on its role in fruit respiration, it can only be stated that the role of polyphenolase in plants remains unknown at present.

The role of flavoprotein enzymes, peroxidase, and catalase in the respiration of fruits is even more obscure than that of polyphenolase. The flavoprotein enzymes would logically be expected to play a role in the respiration systems having cytochrome oxidase as a terminal oxidase, if and when they occur together. However, the occurrence and function of flavoprotein enzymes in fruits has not been studied. Kuhn e t al. (1934) found considerable quantities of flavines in some fruits, but whether these are part of flavoprotein enzyme systems is not known. Peroxidase has generally not been considered a respiratory enzyme and has not been investigated from that standpoint in fruits. Keilin and Hartree (1945) have shown that catalase plus a flavoprotein oxidase can oxidize ethyl alcohol to acetaldehyde. Such a system might play a part in fruit respiration.

Data on the rate of respiration in fruit is limited and in only a few instances have attempts been made to correlate respiration with enzyme systems responsible for oxygen uptake. The relationship of carbon dioxide evolution and fruit ripening has been investigated for apples, pears, bananas and avocados as well as for citrus fruits (Biale, 1941; Cane, 1937 ; Kidd and West, 1938 ; Magness and Ballard, 1926 ; and Krotkov, 1941). With avocados, bananas, apples, and pears certain manifestations of maturity were found t o be accompanied by a greatly accelerated rate of respiration. Detailed data on respiratory metabolism of apples during ontogony is given by Krotkov (1941). The effect of temperature of storage and of storage conditions on respiratory rates of fruits has been investigated widely for some of the deciduous fruits. The early work in this field has been summarized by Gore (1911), who also measured the heat of respiration. More accurate data on the heat of respiration of apples, oranges, and strawberries a t 18.3"C. (65°F.) and 7.2"C. (45°F.) is given by Green, Hukill and Rose (1941). Some information is available on the relation of respiration to storage diseases such as soft scald of Jonathan apples; for example, see Miller and Xchomer (1940). More intensive investigation of the effects of maturity, harvesting and storage practices on respiration rates of fruits is needed.

Several general surveys have been made of the relation of "oxidase"

18 M. A. JOSLYN A N D J. D. PONTINQ

and catalase activity to intensity of respiration of fruit and to maturity and storage conditions. Ezell and Gerhardt (1938a), for example, investigated the “oxidase” (phenolase) activity of apple and pear fruits under various conditions in relation to rate of respiration of the whole fruit. They found a positive correlation between these from the time fruit was small and immature until it reached commercial maturity. Oxidase and catalase activity were not directly correlated with rate of respiration or with each other in fruit subjected to various storage temperatures or to various chemical respiratory stimulants or depressants. They found, however, that the decrease in oxidase activity in Bartlett pears as the fruit approaches maturity was in agreement with the observed behavior of pears used for canning. High oxidase activity in the immature pears was correlated with undesirable color (pale orange yellow) and low oxidase activity with desirable color (clear cream yellow) (Ezell and Gerhardt, 1938b). More investigations of this type are desirable to obtain a more objective grading of fruit for processing and to control discoloration in preparation.

VI. MECHANISM OF OXIDATION OF SUBSTRATE

1. Initial Xtage of Oxidation

The original mechanism for the oxidation of catechol to o-benzoquinone as proposed by Onslow and Robinson (1928) was a simple dehydrogena- tion with the formation of hydrogen peroxide :

OH 0

This mechanism was accepted by many workers untiI it was demonstrated by Dawson and Nelson (1938) and Dawson and Ludwig (1938) that hydrogen peroxide was not a product of the reaction.

Nelson and his coworkers (see Nelson and Dawson, 1944) proposed an oxidation reaction having water and o-benzoquinone as products, based on the observation by Wagreich and Nelson (1938) that the initial product of oxidation as determined by iodometric titration corresponded exactly to an uptake of one atom of oxygen:

OH 0


This equation for the overall reaction of the initial stage of the oxidation of catechol is generally accepted at present. This reaction, however, is only the sum of several other intermediate reactions. LuValle and God- dard (1948) proposed the following simplified mechanism for the enzymic oxidation of a substrate such as catechol to o-quinone and water, based on the free radical or semiquinone theory of oxidation developed by Hantzsch (1921)) Barnett e t al. (1923), Michaelis (1935, 1938), Lu Valle and Weissberger (1947a, 1947b, 1947c), and L u Valle (1948). These workers assume, among other things, that a trimolecular complex is formed between enzyme, substrate and oxidizing agent ; also that electron transfer in univalent steps constitutes the oxidation, and hydrogen transfer occurs t,hrough the solvent by ionic association and dissociation. I n this scheme RH2 = substrate, R = oxidized substrate and E = enzyme:

It is interesting to note that in this mechanism the well-known inactivation of the enzyme is due to formation of an enzyme peroxide (eq. 8) in a reaction competing with catechol oxidation (eq. 6 ) , and not to the products of catechol oxidation. This is in agreement with the findings of Ludwig and Nelson (1939) that o-benzoquinone does not inactivate the enzyme. According to the above mechanism, however, increasing the catechol concentration should shift the equilibrium in equation 6 to the right, thereby leaving less of the free radical IIO;*E. to form inactive enzyme peroxide. Thus the degree of enzyme inactivation should depend on catechol concentration, which according to the data of Ludwig and Nelson (1939) it does not.

Whether o-benzoquinone does or does not inactivate the enzyme is still a controversial point. Dawson and Nelson (1938) suggested that o-quinone might be the cause of enzyme inactivation, but Ludwig and Nelson (1939) concluded that it was ineffective since inactivation of their mushroom phenolase persisted in the presence of sodium benzene-sul- finate, which combines with o-benzoquinone. On the other hand, Richter (1934) had found previously tha t addition of aniline to combine with o-quinones prevented inactivation of potato phenolase during oxidation of catechol derivatives (although aniline does not combine with the


o-quinone group itself) ; also oxidation of the monophenols, tyrosine and p-cresol, was completely inhibited for 5 hours by addition of o-phenylenediamine, sodium bisulfite, potassium iodide or aniline to combine with o-quinone. As Richter pointed out, however, phenolases from different sources show marked differences in inhibition characteristics.

Preliminary experiments by one of the authors (J.D.P.) have indicated that polyphenolase of apples is inhibited by products of the oxidation of catechol. I f the reaction in the presence of excess catechol is allowed to proceed until color formation stops, further addition of catechol or enzyme produces no increase in color. Furthermore, addition of a small amount (ca. 10%) of a previously reacted mixture which has ceased to darken to a fresh mixture of enzyme and catechol will completely prevent oxidation of the latter. Thus apple polyphenolase appears to show inactivation characteristics almost opposite to those described for the mushroom enzyme by Ludwig and Nelson. If fur ther investigation with oxygen uptake experiments, etc., should confirm the above preliminary observations on apple polyphenolase the mechanism proposed by LuVallc and Goddard (1948) would need revision as to the effect of the oxidation product on inactivation of the enzyme.

2. Becond Stage of Oxidation

The discussion of the oxidation mechanism so fa r has concerned only the oxidation of catechol to o-benzoquinone, which requires only one atom of oxygen per molecule of catechol. It is well known, however, that the oxidation does not cease at this point. Nelson and his group (Wagreich and Nelson, 1938 ; Dawson and Nelson, 1938 ; Ludwig and Nelson, 1939 ; and Cushing, 1948), found that, with their enzyme preparations, two atoms of oxygen are taken up per molecule of catechol oxidized, as was also found by Robinson and McCance (1925), Kubowitz (1937) and Jack- son (1939). Wagreich and Nelson (1938) proposed a mechanism for the uptake of the second atom of oxygen involving a reaction of o-quinone with water to form hydroxyquinone, which then polymerizes to a dark colored pigment of unspecified structure, evidently without further uptake of oxygen:

O H


There are several reasons why this mechanism is not acceptable. First, Wright and Mason (1946) found that, by systematically varying the conditions of pH, enzyme concentration and catechol concentration, the number of atoms of oxygen taken up per molecule of catechol oxidized varied between 2.34 and 3.35. Second, Mason e t al. (1945) followed the oxidation of catechol spectrophotometrically by scanning the wavelength range of 220 to 400 mp a t intervals of 4 minutes during the reaction, and could not detect any hydroxy-p-quinone ; they could detect only o-quinone and suggested that this compound polymerized to a phenolic chain susceptible to further enzymatic oxidation. Third, Jackson (1939) showed that 1,2,4, trihydroxybenzene was not an intermediate in the oxidation of catechol, and Cushing (1948) found tha t substitution of -CH3, -C1, etc., in the 4 position of catechol did not change the oxygen uptake, Fur- thermore, as Nelson and Dawson (1944) pointed out, other reactions can occur such as the reaction of o-quinone with catechol. LuValle and Weissberger (1947a) listed several possible reactions that may occur and derived rate laws for them. The rates and equilibria involved determine the pathway of the oxidation. F o r the overall reaction, R + 02* T + 05, which is a n autooxidation reaction but applies, except for the peroxide product, to enzymic oxidation, these reactions are as follows : (R = reduced compound, S = semiquinone, T = oxidized compound, D = dimer, X = compound reacting irreversibly with solvent, forming Y ; T-S = addition complex. Symbols stand for neutral molecules and ion or ions in equilibrium with them.)

22 M. A. JOSLYN A N D J. D. PONTING

T + X e Y T + S T * S T*S + S --+ 2T + R 2T.S + 3T + R

According to these reactions, o-quinone would react with catechol to form a dimer (eq. 15), as suggested by Dawson and Nelson (1938), or it could be formed from the semiquinone (eq. 16) . This dimer could then be further oxidized enzymically as suggested by Mason e t ul. (1945), or nonenzymically, t o form a higher polymer with its oxygen content dependent upon the conditions and extent of oxidation. The experimental determination of the structure of such a polymer has not been made as yet, nor has the elucidation of the extent of oxidation which is catalyzed directly by the enzyme.

3. General Aspects of Pigment Formation

Although the mechanism of pigment formation from phenolic substrates is not known in detail, the general course of pigmentation is known to involve enzymic oxidation, nonenzymic oxidation, nonoxidative transformations and polymerizations. Only in one case, the formation of purpurogallin from pyrogallol, has the final product been identified and the course of oxidation established to some degree of certainty.

Purpurogallin, the pigment formed by the polyphenolase-catalyzed oxidation of pyrogallol or by the oxidation of pyrogallol by peroxidase and hydrogen peroxide, has been the subject of a series of investigations from 1869 to 1923 and more recently. Nierenstein (1934) reviewed the earlier investigations which led to the establishment of the empirical formula CllH805. Willstatter and Heiss (1923), as a result of a series of investigations, indicated that the first phase of the oxidation is the conversion of pyrogallol to its ortho-quinone, 3-hydroxy-o-quinone. The quinone then condenses with pyrogallol into a semi-quinone, half quinoid -half hydroquinoid diphenyl derivative of the quinhydrone type which is darkly colored. This semi-quinone on further oxidation then forms the ortho-qninone bipyrogallol. This adds water and loses one molecule of carbon dioxide by a type of benzilic acid transformation. The intermediate so obtained is then further oxidized and after oxidation undergoes a second benzilic acid transformation, without loss of COZ, into purpurogallin :

OH C0,H


Willstatter and Heiss (1923) thus account for the 3 atoms of oxygen required to form 1 mol of purpurogallin, for its empirical formula, CllH806, and the presence of 3 hydroxyl groups and 1 carboxyl group ; the latter, however, is not detectable by the usual reagents.

This mechanism has been widely accepted, but it was questioned recently by Barltrop and Nicholson (1948) who, largely on the basis of hydrogenation experiments with pure purpurogallin and its methylated derivatives, proposed a new structure containing a seven-membered tropolone ring :

HO O--H

Melanin formation by the tyrosinase-induced oxidation of tyrosine also has been widely investigated but so far only the general course of oxidation has been established. The chemistry of the polymerized products of oxidized tyrosine, the melanins, is not known, nor is the constitution of the naturally-occurring melanins (See Mason, 1947, 1948 ; and Furth and Thallrnayer, 1937). The intermediate reactions involved in the conversion of tyrosine into melanin have been largely worked out by Raper and coworkers. They showed the first visible change in the oxidation of tyrosine by tyrosinase is the production of a red pigment. This red pigment is very unstable and changes spontaneously to a colorless substance which in turn undergoes oxidation in air to produce melanin. Raper and his coworkers demonstrated that tyrosine is first converted into dihydroxyphenylalanine which is then oxidized to the corresponding ortho-quinone. The ortho-quinone, on intramolecular rearrangement, is converted into 5,6-dihydroxy indole- 2-carboxylic acid which on further oxidation is converted into the red 5,6-quinone indole carboxylic acid. From this, by subsequent changes, varying according to conditions, the black melanins arise. Raper (1932) summarized these reactions as follows :

(See review by Raper, 1932).

Tyrosine __+ red substance (oxidative, enzymatic)

Red substance + colorless substance (nonoxidative, nonenzymatic)

Colorless substance --f melanin (oxidative, nonenzymatic)

Denny (1935) suggested that the browning of apple juice by oxidation involves two different reactions and two different chromogens, as follows :

24 M. A. JOSLYN AND J. D. PONTING

reversible, rapid

reduced to relatively colorless compounds by addition of both thiourea and pineapple juice.

not reversible, slow

thiourea inhibits but pineapple juice does not.

Chromogen A t b dark brown oxidation products

Chromogen B t --k light brown oxidation products

Although the chemistry of pigment formation is still largely unknown, the evidence summarized above indicates that pigmentation involves a series of consecutive reactions. It is possible to regenerate the original phenol from an intermediate by reduction provided that oxidation and subsequent transformation has not gone too far. The determination of the readiness with which the intermediate pigments can be reduced would be applicable in practice in control of discoloration. Apparently, in later stages the browning is no longer reversible.

VII. ACTIVATION AND INACTIVATION OF PHENOLASES

The activity of phcnolases, as of all enzymes, is influenced by concentration of substrate, of enzyme, temperature, pH, salts, etc. Phenolases are inactivated by the ions of heavy metals, cyanides, azides, high temperatures, ultrasonic and high frequency radio waves, etc. The more important factors limiting phenolase activity, particularly those that can be used for control, are briefly discussed in the following sections.

1. Effect of p H

The effect of p H and type of buffer on phenolase activity have been widely investigated, but most of the data has been obtained with crude preparations, a t first with fruit tissue extracts; later with acetone or alcohol extracted residues. Qualitative colorimetric tests, manometric measurements and more recently quantitative colorimetric tests were used in measuring enzyme activity. Jeffrey and Cruess (1933), using a gasometric method of estimating LLoxidase” activity, reported that reducing the p H value of apple juice from its normal pI-1 4.0 to 3.7 greatly retarded oxygen absorption in presence of added catechol and a t pH 2.25-3.0 absorption of oxygen practically ceased, being approximately the same as in boiled juice. Increasing the pH to 5.4 and 6.75 greatly increased the absorption of oxygen.

Samisch (1935a), using a manometric method, found that with the same substrate, catechol, extracts from apricots and peaches exhibited a sharp


optimum rate of oxygen absorption a t p H 4.9, falling off on either side of the optimum. With the same substrate, the rate of oxygen uptake by apple tissue did not exhibit any optimum between pH 3.7 and 6.4, but increased slowly with increase in pH. The oxygen absorbed in 3 hours (mg. x for apricot extracts was 370 at pH 3.6, 445 a t pH 3.9 and 360 at pH 6.6; for apples it varied from about 400 a t p H 3.7 to 420 a t pH 6.6. Samisch found that the optimum p H for the activity of the enzyme preparation from any one source varied with different substrates.

Ponting and Joslyn (1948) found that purified peroxidase-free preparations of apple polyphenolase catalyzed the oxidation of catechol a t a rate which increased with pH to an optimum at pH 7.0. The pH-activity curve of the rate of oxidation of ascorbic acid by the apple polyphenolase in presence of catechol in oxalate-phosphate buffer increased rapidly with p H from pH 4.5 to about 6 and then slowly up to p H 9.5 ; in citrate buffer i t increased slowly at first in the region of p H 3-3.7, then rapidly from 3.7 to 4.0, and remained essentially constant over the range 4 to 8. El-Tabey and Cruess (1949) found the phenolase activity of acetone-treated apricot tissue preparations (using catechol as substrate) to increase from 0 at p H 2.2 to a maximum at pH 7. Cruess and Sugi- hara (1948) reported the optimum p H for olive oxidase with catechol as substrate a t pH 7.5.

Most of the data available in the literature on the effect of p H on the activity of fruit “oxidase” is for fruit peroxidase in the presence of variable amounts of phenolase and other accompanying enzymes. Cruess and Sugihara (1948) reported that for purified olive oxidase preparations (using guaiacol and H202) maximum color formation occurred at p H 5, decreasing to 56% of the maximum activity a t pH 3 and to 20% a t pH 8. For guava peroxidase (in acetone-treated extracts) Jimenez (1947) found highest activity a t pII 5 to 6 with an optimum at pH 5.5; the activity decreased rapidly on either side of the optimum, reaching minimum values a t pH 2 and p H 9. Hussein and Cruess (1940) reported for Tokay grape peroxidase an optimum activity at pH 5, falling off sharply to minimal values at pH 2.5 and 8.

Rahman (1948) observed the activity of mixed prune peroxidase and phenolase in McIlvaine’s buffers in the pII range 2.5 to 8.0 and found that prune phenolase was most active to catechol a t pH 7.0, its activity at pH 2.5 and 3.0 being 0 and 1276, respectively, of that a t pH 7.0; its activity at pH 8 was also but 12% of that at p H 7. The peroxidase activity to guaiacol showed an optimum a t p H 5.6, decreasing to about 5% of optimal activity a t pH 2.5 and 8.0. The polyphenolase activity in acetate, phosphate-citrate, phosphate and oxalate buffers at p H 5 was about the same, but it was noticeably lower in borate buffers. At pH 6

26 M. A. J O S L Y N A N D J. D. PONTING

the polyphenolase activity was about the same in phosphate and phosphate-citrate buffers, lower in acetate and oxalate buffers and least in borate buffers. Similar effects were observed for peroxidase activity which was greatest at both p H 5 and 6 in phosphate-citrate buffer, next active in acetate and phosphate and least active in oxalate buffers.

The optimum p H for the activity of an enzyme preparation from any one source usually varies with different substrates and is characteristic of the substrate as well as the enzyme preparation used (Samisch, 1935a). It is evident, however, that decreasing the pH by the addition of acids will assist in controlling enzymic discoloration by reducing the activity of the enzymes concerned. The type of acid as well as the pH will influence the results obtained. This property has been used to advantage in checking the browning of lye-peeled peaches during handling. Im- mersing the fruit in a 1% solution of citric acid after washing was found beneficial by Woodroof (1930). Cruess, Quin, and Mrak (1932) reported that the pH of the outer surface of lye-peeled clingstone canning peaches varied from 4.2 to 6.2 after washing; the normal p H of untreated peaches being 3.9 to 4.0. Since the rate of enzymic darkening increases above pH 4.5 and is very rapid a t p H 5 to 7, they suggested dipping the thoroughly washed lye-peeled peaches in dilute acid to reduce the p H and decrease the browning of canning peaches during preparation. These workers reported that the fruit acids such as citric and tartaric were not as effective as dilute hydrochloric acid.

The use of a combination of citric and ascorbic acid has been suggested for preventing the browning of frozen cut fruits (Luther and Cragwall, 1946). Joslyn and Hohl (1948) reported that sirups containing 0.5% citric acid and 0.03% ascorbic acid were as satisfactory for color retention in apricots, peaches and nectarines during freezing storage as one containing 0.170 ascorbic acid. The fruit frozen with citric and ascorbic acids, however, does not retain its full fruit flavor as well as fruit frozen with ascorbic acid alone. Upon storage at room temperature after defrosting, the citric-acid-ascorbic-acid-treated fruit, particularly apricots, darkened more rapidly than that treated with ascorbic acid alone (Joslyn, 1949; Joslyn et al., 1949). Strachan and Moyls (1949) reported that citric acid had no significant value in preventing oxidation or in reducing the quantity of ascorbic acid required.

2. Effect of Temperature

The effect of temperature on the activity of phenolases has not been investigated intensively. As with enzymes in general, phenolase activity increases with increase in temperature until temperatures high enough o r exposures for periods of time long enough to inactivate the enzymes


are reached. The point of balance between the accelerating effect of temperature upon the enzyme-catalyzed reaction and the effect of destruction of the enzyme by heat is known as the temperature optimum. Tammann (1895) very early pointed out that increase of temperature increases the rate of enzyme destruction more than it does enzyme action so that the optimum temperature of enzyme action is not definite, but varies with time, shifting to lower temperatures with increasing time. Quantitative data on the effect of temperature on fruit phenolase and fruit peroxidase activity are meager. The earlier, largely qualitative data are briefly discussed by Kastle (1910), Cruess and Fong (1926) and Fong and Cruess (1929). The earlier data of Cruess and his coworkers was concerned largely with determination of inactivation temperatures.

Hussein and Cruess (1940) found that activity of purified grape peroxidase in the range of 0"-40°C. (32"-104"F.), reached an optimum a t 36.3"C. (97.4"F.). Jimenez (1947) found that the activity of guava peroxidase preparations increased with temperature from 5" to 50°C. (41"-122"F.), reaching a sharp optimum at 50°C. (122°F.). Ponting and Joslyn (1948) studied the effect of temperature on the rate of oxidation of ascorbic acid by purified apple phenolase in presence of catechol in the range of 5" to 65°C. (41°-149"F.) and found that the rate of oxidation increased with t,emperature, reaching an optimum at 40°C. (104°F.).

Cruess and Sugihara (1948) observed the intensity of pigment formation with guaiacol, HzOz and purified olive "oxidase" in the range of 5" to 4OOC. (41"-104"F.). Maximum peroxidase activity occurred at 315°C. (88.6"F.) and was considerably less at 375°C. (99.4"F.) and 40°C. (104°F.) j a t 5°C. (41°F.) it was only about one-fourth as rapid as a t 31.5"C. (88.6"F.).

3. Effect of Temperature and p H on Rate of Inactivation

The effect of temperature, time of heating, and pH on inactivation of fruit enzymes has been studied by few investigators and only in one case was polyphenolase studied specifically. Bouffard (1895) (see Kastle, 1910) found that the "oenoxidase" of grapes could be destroyed by heating below the boiling point. Overholser and Cruess (1923) heated slices of Newtown apples for 5 minutes in a water bath at various temperatures and found the benzidine test for oxidase to be negative at 735°C. (164.2"F.) and weakly positive at 71°C. (159.8"F.), the benzidine plus H202 test for peroxidase to be negative at 100°C. (212°F.) and positive a t 90°C. (194"F.), and the starch-iodide test for organic peroxide to be negative a t 735°C. (164.2"F.) and doubtful at 71°C. (159.8"F.). Cruess

28 M. A. JOSLYN AND J. D. PONTINB

and Fong (1926) and Fong and Cruess (1929) found that the temperature required to inactivate oxidizing enzymes of several fruits varied with pH. They carried out tests with freshly extracted fruit juices and with suspensions of fruit “oxidase” precipitated with alcohol and purified by redissolving in water and reprecipitation with alcohol or acetone. Cruess and Fong (1926) reported that the inactivation temperature of the organic peroxide of several fruits was considerably below that of the peroxidase. When fruit juices were brought to various pH values and heated in 10 ml. portions in small test tubes immersed in a water bath f o r 10 minutes, it was found that the inactivation temperature of the peroxidase, as observed qualitatively by cooling and testing the samples by addition of H202 and benzidine or guaiac, was markedly affected by pH. The resistance to heat increased rapidly at first with increase in pH up to 4.5, and slowly from 4.5 to 7.0, and then decreased from pH 8 to 12. Samisch and Cruess (1934) reported that preparations of the “oxidase” of apricots were completely inactivated by 1-hr. exposure at 71°C. (159.8”F.) and 6 hrs. a t 60°C. (140°F.).

Hussein and Cruess (1940) reported, on the basis of quantitative de- terminations, that grape peroxidase was destroyed in 5 minutes a t 85°C. (185°F.) in grape juice at p H 4, a t 85°C. (185°F.) in wine at pH 3.1, and at 90°C. (194°F.) in buffer suspensions of purified enzyme preparations at pH 5.2. Additional data on the effect of various pretreatments, including heat treatment and drying, on activity of grape peroxidase were reported by Hussein, Mrak and Cruess (1942). Immersion of grapes for an appreciable period of time in hot dipping solutions reduced peroxidase activity, whereas dipping for a very short time increased it. Heating grape peroxidase preparations at 82.2”C. (179.6”F.) for 0 to 30 seconds resulted in a progressive decrease in activity reaching 91% at 30 seconds.

Cruess and Sugihara (1948) reported that the peroxidase in olive tissue preparations was destroyed a t pH 6.7 by heating for 5 minutes a t 80°C. (1’76°F.).

The effect of temperature and pH on the activity of purified polyphenol oxidase in solution was studied by Ponting and Joslyn (1948), but the inactivation conditions were not determined. The optimum temperature (for maximum activity) was 43°C. (109.4”F.). The optimum p H for catechol oxidation was 7.0, but for ascorbic acid oxidation (with oatechol as catalyst) the optimum pH seemed t o depend on the buffer used ; in citrate-phosphate buffer the optimum pH was 7.7-7.8, but in oxalate-phosphate buffer there was no detectable optimum up to pH 9.5.

El-Tabey and Cruess (1949) reported data on the activity of apricot phenolase and apricot peroxidase in tissue preparations after heating at various temperatures a t p H 4, 5 and 6. After one hour, at 55°C. (131°F.),


the activity of the phenolase decreased from 100% at 10°C. to 30% at pII 4,51% at p H 5, and 17% at pH 6. After one hour, a t 55°C. (131°F.), the activity of the peroxidase decreased from 100% at 10°C. to 59% at pII 4, 44% at pH 5, and 48% a t pH 6. Data for activity in the range of 10" to 55°C. (50°-1310F.) for exposures of 1 and 24 hours, indicate that phenolase was more easily inactivated than peroxidase. A t higher temperatures at p H 5 and a 5-minute heating period, apricot peroxidase activity in purified "oxidase" preparations dropped to 2% of the initial value at 90°C. (192.2"F.) and to 0% a t 100°C. (212°F.) ; apricot phenolase activity dropped to 2% at 85°C. (185°F.) and 0% at 90°C. (194°F.).

Rahman (1948) reported that for a heating period of 5 minutes the activity of prune phenolase in prune tissue preparations dropped to 2% of its initial value at 90°C. (194°F.) and that of prune peroxidase to 5% of its initial value at 90°C. (194°F.) ; the polyphenolase activity dropped with temperature more rapidly than did the peroxidase activity. Chari e t ul. (1948), using qualitative tests, found that their prune enzyme preparation was inactivated by heating at 81.5"C. (178.6"F.) for 2 minutes a t p H 5.0.

An investigation of the time-temperature conditions for the inactivation of polyphenol oxidase in purees of several fruits, as well as the effect of pH, was made by Dimick and Ponting (1949). They used a continuous flow system for heating, holding and cooling the purees in a few seconds. They found that all the fruits tested gave similar curves of activity ws. temperature. The activity decreased only slightly with increase in temperature until a critical temperature was reached, after which the rate of inactivation increased very sharply. With a holding time of 7 seconds, the temperature for approximately 99.9% inactivation varied from 90"-91°C. (194"-195.8"F.) for Royal apricots to 78"-79°C. (172.4O-174.2"F.) for Gravenstein apples. The estimated temperature coefficient of the inactivation (QI0) varied from 15.7 for Concord grapes to 120 for Elberta peaches, over the range 70"-80°C. (15S0-176"F.). I n all cases the coefficient is much higher than for an ordinary chemical reaction, but similar to the coefficients found for heat denaturation of other proteins.

The pH of maximum heat stability of polyphenol oxidase was 3.9 for apricots, 4.5 for grapes, 6.0 for pears, and 6.2 for apples, while the natural p H of the purees were respectively 4.0, 3.3, 3.9 and 3.1. Apricots appear somewhat anomalous in being the only fruit tested having a p H of maximum stability lower than the natural pH. All of the fruits had a sharp pH optimum.

I n the application of data on the thermal rate of inactivation of phenolase to the control of darkening in fruits during preparation for


canning, dehydration, or freezing, heat transfer determinants as well as physiological factors have to be considered. Cruess, Quin and Mrak (1932) called attention to the importance of completely heating the fruit tissue to inactivate enzymes in the interior as well as a t the surface. Un- less this is done, the halved or sliced fruit will show a brown crescent in the flesh a t the line of demarcation between that tissue in which enzymes were inactivated and that which was not heated to a high enough temperature. In fruit that is scalded for freezing, browning will occur in the unheated or partially heated areas (Joslyn and Hohl, 1948). Control tests to determine the extent of heat penetration and extent of enzyme inactivation during scalding were suggested by Ponting (1944). The fruit, after scalding and cooling, is cut across and the cut surface is covered with a 1% solution of catechol. Areas which have not been heated sufficiently will turn dark brown in color; the rest will remain light.

4. Effect of Salts and Inhibitors

It has long been known that various ions, hydrocyanic acid, and hydrogen sulfide inhibit oxidase activity. Kastle and Loevenhart (1901) observed early that the oxidizing power of aqueous extracts of potatoes was destroyed by hydrocyanic acid, hydroxylamine, sodium thiosulfate, phenyl hydrazine, acids such as HC1, HBr, oxalic, salicylic, etc. Bouffard as early as 1894 (see Kastle, 1910), pointed out that sulfurous acid prevented the action of oenoxidase (grape oxidase) ; Lindet (1895) called attention to its use in controlling the darkening of apple products. One of the earliest systematic investigations of the effect of neutral salts, alkalies, sulfurous acid and other acids on the darkening of apple tissue was made by Overholser and Cruess (1923). They found that chlorides prevented the browning of juice, sulfates, acetates and tartrates had no effect, oxalates inhibited browning, while nitrates increased it. Sulfurous acid, sodium sulfite and sodium sulfite plus HCl markedly inhibited browning.

Samisch and Cruess (1934) reported that only the anions of neutral alkali or alkaline earth salts inhibited the phenolase of apricots, and that cations had no effect. Of the halides tested, NaF was most effective and NaI least. Hussein and Cruess (1940) reported that grape peroxidase was completely inhibited a t 1 x M NaF it had only 47% of its normal activity. Jimenez (1947) reported that guava peroxidase was completely inactivated in 7 x M NaF ; 84% of its activity was destroyed by 5.5 x 10ks M KCN. Cruess and Sugihara (1948) found the purified olive oxidase to be extremely sensitive to NaCN, being inactivated at a concentration of 22c 10W4 M. Fluoride ion was less

M NaF, and with 4 x


toxic to olive oxidase than cyanide ; a t 5 x 10 -3 M K F the oxidase activity was only 5% of that in absence of KF, and a t 5 x 10P4 M K F it was 56.8%. They found that NaCl reduced oxidase activity to about 78% of the initial at a concentration of 0.1 N but apparently had a minimum effect at 0.2 N. El-Tabey and Cruess (1949) studied the comparative effects of various concentrations of KCN, NaN, and NaF on apricot peroxidase and phenolase. Both were sensitive to KCN, but the‘ peroxidase was somewhat more so than the phenolase. The phenolase was also slightly more resistant than the peroxidase to the inhibiting action of sodium azide. The apricot peroxidase was more resistant than the phenolase to NaCl and NaF. Similar effects were observed for prune phenolase and peroxidase by Rahman (1948).

Ascorbic acid, both d- and 1- forms, and related compounds have been introduced for the control of enzymic and autooxidative discoloration. I-ascorbic acid is widely used in preventing the discoloration of fruit products during freezing storage (Joslyn and Hohl, 1948 ; Hohl, 1946 j Bauernfeind et al., 1946 ; Bauernfeind and Siemers, 1946 ; Esselen e t al., 1949; and DuBois, 1949). It acts primarily by reducing the oxygen present in or surrounding the fruit tissues and in maintaining a reducing condition in the fruit tissues.

The relative efficiency of I-ascorbic acid, reductic acid, reductone and dihydroxymaleic acid in retarding enzymic browning of defrosted apples, apricots, peaches and pears were investigated by Tarr and Cooke (1949). These carbonyl enediols were found to be similar in their action. Strachan and Moyls (1949) , however, reported that I-ascorbic acid was superior to dihydroxymaleic acid in inhibiting browning and loss in flavor during freezing storage.

Sulfurous acid and its salts (bisulfites, sulfites and metabisulfites) have long been known as efficient inhibitors of enzymic as well as nonenzymic browning, but little is known concerning their mode of action (Joslyn and Mrak, 1930, 1933). Overholser and Cruess (1923) believed that sulfurous acid and sulfites prevented browning by destroying the “organic peroxide.” Cruess and Fong (1926) exposed pears, peaches, cher- ries, and apricots t o fumes of burning sulfur and then tested them for peroxidase, “organic peroxide” and catalase. Only the peroxidase test was positive, but after the “free” SO2 was leached out of the fruit in running water, positive tests for “organic peroxide l f were obtained with all samples, indicating that the SOz had acted upon the peroxide in a temporary manner only. The concentration of SO2 required to inactivate the “organic peroxide” was very small, about 60 p.p.m. When higher concentrations of SO2 were used, the peroxidase was also destroyed, but this required 1600 p.p.m. of SO2. When these experiments were repeated


by adding various amounts of SOz to fruit juices (Cruess and Fong, 1929a), positive tests were obtained for peroxidase when the excess acidity was neutralized by NaHC03, and hydrogen peroxide sufficient to oxidize the sulfurous acid present was added prior to addition of benzidine.

Pouting and Johnson (1945) reported that the sulfur dioxide ,in freshly sulfured and in frozen sulfured fruit is rapidly oxidized by blending the fruit containing active oxidizing enzymes in a Waring blendor. This enzyme-catalyzed oxidation of sulfur dioxide has not been reported here- tofore, and further investigation of this phenomenon may help to clarify the role of sulfur dioxide in inhibiting discoloration.

Denny (1935) reported that the darkening of apple juice could be inhibited by thiourea and related thioamides ; and later extended this treatment to control of browning in frozen fruits (Denny, 1942). Wink- ler (1949) observed incomplete recoveries of known amounts of thiourea added to frozen fresh peaches, although good recoveries were obtained if the frozen peaches with added thiourea were immersed immediately in boiling water or in a solution of sodium sulfite. These findings led this author to the conclusion that the oxidizing enzymes of the peach destroy the added thiourea. The amounts of thiourea destroyed at room temperatures varied with samples of frozen peaches used ; many samples did not destroy thiourea in amounts greater than 25 or 30 p.p.m., others oxidized 80-100 p.p.m., while a few were capable of readily destroying 130-140 p.p.m. Winkler studied the reactions of thiourea with the oxidizing systems of frozen peaches, particularly to determine whether the course of oxidation was similar to that which occurs with chemical oxidizing agents. The oxidation of thiourea with the latter is known to occur in a t least four stages, depending on the agents and conditions of oxidation. The following successive oxidation products have been identified : formamide disulfide salts, formamidine sulfinic acid (dioxide of thiourea), formamidine sulfonic acid (trioxide of thiourea) and urea and sulfuric acid. No evidence was obtained of the presence of formamidine disulfide, dioxide of thiourea or of significant amounts of urea in mixtures of frozen peaches blended with thiourea solutions. Strong tests were obtained for trioxide of thiourea, however, and it was concluded that the reaction of thiourea with the oxidizing systems of peaches pro- ceeds rapidly to the stage where the trioxide of thiourea is formed and that this is the chief end product.

A great many substances have been investigated as possible inhibitors of enzymic and auto-oxidative discoloration, and several patents have been obtained. Most of these substances are sulfur compounds or aro-


matic copper deactivators. A list of compounds which have been suggested for use in fruits or juices follows:

a. Cornmolzly Used or U n p t e n t e d Chemicals: Cysteine, cystine, glutathione, sulfonamides, sulfurous acid and its salts, sodium sulfide, sodium chloride, citric acid, hydrochloric acid, ascorbic acid.

b. Patented Chemical Inhibitors: (a) Sodium thiosulfate : Elion (1942) ; (b) thioamides such as thiocarbamide: Denny (1943) ; (c) sodium chloride, ascorbic acid, and sodium bisulfite : Johnson and Guadagni (1949).

Hydroquinone, toluhydroquine, hydroquinone + lecithin, hydroquinone + triethanolamine, diphenylamine, pyrocatechol, p-aminophenol or resorcylaldehyde : Johnston e t al. (1943).

o-hydroxy aromatic oximes free of strongly acidic groups : Downing et al. (1943).

Product of condensation of one mol. of an alkali primary amino ali- phatic carboxylate with at least one mol. of an o-hydroxy-substituted aromatic aldehyde. Preferred deactivators are salicylal derivatives of sodium glycinate, disodium glutamate, sodium tyrosinate and sodium cysteinate : Downing and Pedersen (1944).

Thiosemicarbazide and its derivatives : Clarkson (1946). Several natural products have been suggested fo r the control of fruit

discoloration. Balls and Hale (1935) proposed the use of pineapple juice because of its high content of naturally occurring sulfhydryl compounds. Woodmansee, Baker, and Gilligan (1948) have suggested the use of water extracts of rhubarb as a preventative of browning of apple slices in home-frozen storage. The high oxalic acid content of rhubarb is probably involved, although the nature of its antibrowning power is not known. Citrus juices, because of their high content of ascorbic acid, may be used also.

I n treating fruits with inhibitors such as sulfites, conditions of treatment (particularly concentration and time) must be so chosen as to obtain the maximum protection against oxidation. I n some cases protection against oxidation at exposed surfaces is all that is necessary; in other cases complete and thorough penetration of the inhibitor into all of the tissue treated is required. The structure of the fruit, the residual oxygen content of the interior cells, and the rate of diffusion of oxygen into the interior are limiting factors.

At low concentrations of sulfites or at short periods of exposure, the sulfite will penetrate through the surface cells only into part of the interior of the sliced or halved fruit, treated. Apple tissue so treated when stored at room temperature will show discoloration in the interior a t the borders between areas where the cells are killed and the enzymes inhibited and cells which are killed but whose phenolase activity iY not

34 M. A. JOSLYN AND J. D. PONTINO

inhibited (Joslyn and Mrak, 1933). When such fruit is frozen it will show a general browning in the interior. Ponting (1944) reported that storage of sulfited apple slices after treatment and before freezing would permit diffusion of the sulfite into the interior cells. Complete penetration of other inhibitors, e.g., ascorbic acid, and their use in concentrations sufficient to reduce all the oxygen present and maintain a reducing condition during freezing storage and subsequent defrosting is usually necessary for adequate control of enzymic oxidation. This is also true of fruit products such as purees and juices.

5. Efect of lrugars

Sugars and sugar solutions are used in the freezing preservation of fruit products for sweetening and to exclude direct contact of the fruit tissue with molecular oxygen (Joslyn and Hohl, 1947). The sugar solutions inhibit discoloration by their effect in reducing the concentration of dissolved oxygen and reducing the rate of diffusion of the oxygen of the air into the fruit tissues. Joslyn (1949) presented some data on the effect of type of sugar and concentration on the solubility of oxygen. Concentrated sugar solutions also exert an inhibiting effect on fruit oxidases. Quin (1929) found that a t the same concentration the retarding effect of sucrose was greater than that of glycerol or dextrose. He reported that the activity of peach oxidase (peroxidase) was completely inhibited in solutions containing 70% of sucrose or 60% of glycerol. El-Tabey and Cruess (1949) observed the effect of sucrose and glucose in the concentration range of 0, 10, 20, 30, 40, 50 and 60% for sucrose, and 0, 10, 20 and 30% for glucose, on the activity of their apricot phenolase-peroxidase preparation. Glucose was found to have no effect on peroxidase activity ; i t had an appreciable stimulating effect on phenolase at 10 and 20% concentrations and no effect a t 30%. Sucrose, however, had but little effect on peroxidase at 10% and slightly inhibited it in the range of 20-60% ; the peroxidase activity in the latter range was about 80% of that in the sugar-free preparation. Sucrose in the concentration range of 10 to 40% increased the phenolase activity to 110% of that in the sugar-free preparation; but phenolase activity dropped to 89% of the initial in 50% sucrose solution and to 77% of the initial in 60% sucrose solution.

Rahman (1948) found similar results for prune phenolase and peroxidase. At lower concentrations (10 and 20% ), he found that glucose had a slightly stimulating effect in both phenolase and peroxidase activity, while with maltose and sucrose there was a slight but definite inhibition. At 50% concentration, the polyphenolase activity was reduced to 80% of that in sugar-free solutions for glucose, to 50% for maltose and to 80%


f o r sucrose. The prune peroxidase activity in sugar solutions a t 50% by weight was 90% of the initial for glucose, 50% of the initial for maltose, 90% for “Cerelose,” 19% for “Puritose,” and 17% for “Sweetose.” Rahman (1948) found, however, that the time of measurement markedly affected the relative results. When the transmittancy of prune phenolase-catechol mixtures was taken over a period of 60 minutes, it decreased rapidly for the first 10 minutes and then more slowly. In the presence of 25% of added sucrose, there was little difference between the transmittancy of it and the control for the first 5 minutes, and then the optical density of the sugar solution increased less rapidly, becoming practically constant after 30 minutes. In the presence of 50% sucrose, the effect was even more pronounced so that when the effect of sugar is compared at later periods, the differences are more marked. Similar behavior was found with glucose. The effect of sugars on the course of enzymic oxidation of catechol and of natural substrates, therefore, must be studied more thoroughly, particularly a t both the initial and secondary stages.

Glycerol and the higher alcohols insofar as they have been investigated are similar to the sugars in their effect. Glycerol, particularly, may have a protective effect. Kastle (1910) reported that many “oxidases” are more stable in the presence of glycerol. The lower alcohols, however, are more toxic or inhibitive on a weight basis but not as high on a molar basis. Rahman (1948) found that both prune peroxidase and prune phenolase activity decreases with increase in concentration of ethyl alcohol. Their activity is approximately halved in 3.4 M alcohol and is only a fourth of that in the absence of alcohol a t 6.8 M ; the polyphenolase is somewhat more resistant to alcohol than the peroxidase. I n comparison, the prune peroxidase activity in 3.2 M glycerol is 58% of the initial value; and that of the phenolase is about 80%. On the same basis, the activity of peroxidase in 2.75 M solution is reduced to 9070 of its initial value in glucose solutions and to 80% in levulose ; that of the phenolase is reduced to 80% and 70%, respectively, for glucose and levulose solutions of this molarity.

6. Inactivation. hy Electrical Energy

During the past few years, numerous investigations have been made of the chemical and biological effects of high-frequency sound radiations, high-frequency radio waves, ultra-violet rays, X-rays, cathode rays, etc. (Moyer, 1946). There is some evidence that supersonic waves, electro- magnetic waves and electron beams have an inactivating effect on fruit enzymes. The extent of inactivation, the mechanism involved and the practicability of applying energy sources of this type still remain to be investigated. Christensen and Samisch (1934) found that the phenolase

36 M. A. JOSLYN AND J . D. PONTINQ

activity of extracts of apricots, peaches and avocados was markedly reduced by exposure to high frequency sound waves although at the €re- quency used, 450,000 cycles, complete inactivation was not obtained in treatments up to 12 hrs. They found that temperature rise in the medium, oxygen tension during treatmeni, and the amount of HzOz formed, had little effect on the rate of inactivation produced. They concluded that oxidation played a minor role in this destruction.

VIII. CONTROL OF ENZYMIC BROWNING

The control of enzymic browning in industry is based on the assump- tion that this browning is largely due to interaction of the enzyme, polyphenolase, with molecular oxygen and a suitable phenolic substrate. The enzymic browning is controlled by selection of varieties of fruit least susceptible to discoloration either because of the absence of the phenolic substrate [as in the case of the Sunbeam peach (Kertesz, 1933)], or the presence of the substrate or enzyme a t low concentration; selection of the fruit at the stage of maturity a t which discoloration is at a minimum (such evidence as is available indicates that the immature fruit is higher in the content of color base and in phenolase activity) ; removal of oxygen from the fruit tissues as well as from the atmosphere surrounding the frui t ; addition of acids to reduce the pH and so reduce phenolase activity ; addition of antioxidants or reducing substances which may act either by reducing free oxygen concentration or as phenolase inhibitors (ascorbic acid and sulfites are used most commonly) ; addition of or treatment with permissible inhibitors such as salt; and heat inactivation of the phenolase. At present the application of these control measures is based largely on empirical observations.

The available information on varietal and maturity factors was summarized by Hohl (1946) and Joslyn and Hohl (1948). Guadagni, Sorber, and Wilbur (1949) presented quantitative data indicating that the susceptibility of several varieties of peaches to browning could be correlated with the amounts of polyphenol oxidase and substrate present. Storage temperature and p H were found by them to have considerable effect on the rate of browning of frozen peaches. The effect of removal of oxygen by deaeration and deaeration-impregnation as well as vacuum closing has been discussed by Joslyn (1934). The removal of tissue gases by vacuum and replacement of the gases by solutions of sucrose with and without small amounts of antioxidants has been under investigation for some time. Recent data on this were presented by Joslyn e t al. (1949) for apricots, peaches, and nectarines, and by Guadagni (1949) €or apples. The latter author reported quantitative data on weight losses and solids changes occurring in sliced apples during treatment, and similar data on


apricots have been summarized by Joslyn (1949). For sliced apples in sirup, Guadagni found vacuum closing to be equivalent to deaeration- impregnation, although this was not the experience with other fruits, particularly whole berries (Joslyn, 1934).

The application of other treatments is discussed by Anon (1944) ; Joslyn (1934,1942) ; Joslyn and Hohl (1948) ; Joslyn e t al. (1949) ; Lee (1944) ; Sater et al. (1947) j Sorber et al. (1944) ; Strachan and Moyls (1949) ; Tarr and Cooke (1949) ; Tressler and DuBois (1944) j Wiegand (1946) ; and Woodroof (1946).

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