[Advances in Food Research] Advances in Food Research Volume 17 Volume 17 || Oxidation Systems in Fruits and Vegetables– their Relation to the Quality of Preserved Products
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OXIDATION SYSTEMS IN FRUITS AND VEGETABLES- THEIR RELATION TO THE QUALITY OF PRESERVED PRODUCTS
D. R. HAISMAN~ F. AYLWARD* AND
Fruit and Vegetable Preservation Research Association, Chipping Campden, England
I. Introduction . . . . . . 11. Oxidizing Enzyme S
A. Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... 9
I. Clycolate Oxidase . . . . . . . . . . . . . . . . . . . . . . 14
. . . . . . . . . .20
. . . . . . . . . . . . . . . 22
E. Antioxidants . . . . . . . . . .
'Present address: Department of Food Science, The University, Reading, Berks. England. +Present address: Unilever Research Laboratory, Colworth House, Sharnbrook, Beds., England.
OXIDATION SYSTEMS IN FRUITS AND VEGETABLES- THEIR RELATION TO THE QUALITY OF PRESERVED PRODUCTS
D. R. HAISMAN~ F. AYLWARD* AND
Fruit and Vegetable Preservation Research Association, Chipping Campden, England
I. Introduction. . . . . . . . . . . . 11. Oxidizing Enzyme Systems
A. Peroxidase.. . . . . . . . . B. Pseudo-Peroxidases . . . C. Catalase . . . . . . . . . . D. Cytochrome Oxidase . E. o-Diphenol Oxidase . F. p-Diphenol Oxidase . G. Ascorbate Oxidase . . I H. Amine Oxidases . . . . I. Glycolate Oxidase . . I J . Oxidation Mechanisms . K. Correlation between Enzyme Activity and Food Deterioration
111. Respiratory and Other Enzymes A. Respiration . . B. Fermentation . . . . . . . . . . . . . . C. Respiratory Enzymes and Food Deterioration D. Pectic Enzymes. E. Chlorophyllase . . F. Enzymes of Amino Acid Metabolism
IV. Oxidative and Other Changes in Lipids . A. Degradation of Lipids . B. Lipoxygenase . C. Autoxidation of Lipids D. Decomposition of Hydroperoxides E. Antioxidants . F. Lipid Oxidation in Relation to Food Quality
2 7 8 9
10 10 11 12 13 13 13 14 18 19 19 20 20 21 22 22 23 24 26
'Present address: Department of Food Science, The University, Reading, Berks. England. +Present address: Unilever Research Laboratory, Colworth House, Sharnbrook, Beds., England.
2 F. AYLWARD AND D. R. HAISMAN
V. Thermal and Other Environmental Factors Modifying Enzyme Activity . . . . . 34 A. Thermal Inactivation -General Principles . . . . . . . . . . . . . . . . . . . . . . . . . .35 B. Thermal Inactivation of Oxidizing Enzymes-Experimental Data . . . . . . 39 C. Enzyme Action at Low Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47 D. Effects of pH and Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47 E. Effects of Water. . . . . . . . . . . : . . . . . . . . . . . . . . .................... .48 F. Multiple Molecular Forms of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . t.48 G. Adsorption of Enzymes on Natural Substrates . . . . . . . . . . . . . . . . . . . . . . . .50
VI. Regeneration of Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53 A. Test Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Heat Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54 C. Storage Conditions .......................... .56
H. Specific Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
VII. Research Needs . . . . . . . . . .
B. Biochemical ......................... .58 C. Enzyme Systems: Substrates-Primary and Secondary Reactions . . . . . . 58 D. Plant Lipids: Oxidation Mechanisms and Reaction Products . . . . . . . . . . . 59 E. Plant Components Inhibiting or Modifying Enzyme Activity. . . . . . . . . . 59
G. New Methods of Preservation ....................................... .60 References ......................................................... 61
F. Heat Resistance of Enzymes . . . . . . . . . . ........................ .60
The fruit and vegetable canning, quick-freezing, and dehydration industries can be considered from the economic standpoint in terms of a chain stretching from farm to consumer and including growers and processors and groups concerned with transport, storage, and distribution. The scientific and technological problems of the industry must be considered in the same context.
During the growth of the canning industry, and later of the quick- freezing and dehydration industries, the importance of careful control of postharvest and processing conditions became well recognized, and scientific and technological investigations sponsored by indus- trial and other groups were directed to the production of preserved foods of good quality.
Not so much attention was given to the changes in quality of food after leaving the factory, but it was gradually recognized that changes1 in quality could and did take place, especially with frozen and dehydrated foods. Realization of the extent of such changes has led to various investigations, notably those sponsored by the Western Utilization Research Laboratory of the United States Department of Agriculture.
PLANT-TISSUE OXIDATION SYSTEMS 3
Quality changes may result from three main types of reaction within the foodstuffs or between the food and its environment: (a) microbiological; (b) enzymic, and (c) chemical nonenzymic changes. This review is concerned primarily with the effects of enzymes.
It is well known that active enzyme systems can spoil fruits and vegetables even at subzero temperatures and low moisture levels. For this reason most vegetables and some fruits which are to be pre- served by canning, freezing, or dehydration are given a preliminary heat treatment (for instance, blanching in boiling water) to inactivate the enzyme systems in the tissues. The blanching operation reduces the level of infection by microorganisms, and may improve color and flavor by expelling volatile degradation products formed during the postharvest interval (Adam et al., 1942). Blanching is omitted with certain products, such as onions and peppers and other strongly flavored materials, in which enzymic deterioration either does not occur or has no noticeable effect on quality (Makower, 1960).
The literature on the effect of enzymic activity on the quality of foods has been comprehensively reviewed by several workers. Joslyn (1949) examined the evidence for the enzymic nature of flavor changes, the effectiveness of blanching, and the tests available for measuring its efficiency. He noted that the best indicator for blanch- ing efficiency was peroxidase activity, and believed that until more was known about the enzymic systems involved in off-flavor forma- tion, technological developments would be limited to improvements in techniques of peroxidase estimation. From a survey of the experi- mental evidence, he concluded that there was no doubt of the activity of enzymes in frozen foods. In a later publication Joslyn (1951) dealt with enzyme activity in the dried state and in concentrated solutions, drawing parallels with the situation in frozen tissues. In more recent reviews Joslyn (1961, 1966) has emphasized that procedures for the control of enzymic activity during the preparation and freezing of fruits and vegetables are largely empirical; further biochemical re- search is required to define postharvest changes and changes during processing and storage.
Leeson (1957) reviewed the inactivation of enzymes by heat, the effect of residual enzyme activity on the quality of fruits and vege- tables, and evidence for the regeneration of enzyme activity. He emphasized that in devising tests of blanching efficiency the possi- bility of enzyme regeneration should not be overlooked. McConnell (1956) tabulated data on the heat resistance of enzymes in fruits and vegetables, and showed that in high-temperature short-time process-
4 F. AYLWARD AND D. R. HAISMAN
ing, enzyme inactivation may take longer than the destruction of microorganisms. Board (1961) surveyed the effect of enzymes on the stability of canned foods; in describing the changes caused by en- zymes he gave examples of nonenzymic catalysis such as the effect of copper and hemochromogens on the destruction of ascorbic acid and concluded that the mechanisms of the deteriorative changes were still obscure, but that both enzymic and nonenzymic reactions may take place.
The investigations of the Western Regional Laboratory, to which reference has already been made, were initiated in 1948 and covered not only fruits and vegetables but also other frozen products. The research program covered the behavior of frozen foods within the extremes of time and temperature which might be encountered in commercial practice. The main aims of the investigation were to establish tolerable deviations from ideal conditions for different products, to identify and improve critical operations, to seek techno- logical improvements ensuring greater tolerances, and to establish tests of product quality. The results were published from 1957 onward, starting with a definition of the problem (van Arsdel, 1957); surveying the quality of frozen soft fruits (Guadagni et al., 1957a,b,c, 1958, 1960; Guadagni, 1957; Guadagni and Nimmo, 1957a,b, 1958; Guadagni and Kelly, 1958), poultry products (Hanson and Fletcher, 1958; Hanson et al., 1959; Klose et al., 1959), vegetables (Dietrich et al., 1957, 1959a,b, 1960, 1962; Boggs et al., 1960), and liquid products (Hanson et al., 1957; McColloch et al., 1957), and covering relations between temperature history and product quality (van Arsdel and Guadagni, 1959) and bacterial population (Michener et al., 1960).
In frozen fruits the degree of browning and percentage loss of ascorbic acid (based on content of ascorbic acid, dehydroascorbic acid, and diketogulonic acid) were found to be closely related to overall quality. In peaches, partial inactivation of the oxidizing enzymes, followed by vacuum-packing in sealed containers and freezing, was sufficient to retard browning for a reasonable time after opening and thawing; complete inactivation of the enzymes by con- ventional heat processing caused serious flavor changes and increased leaching losses (Guadagni and Nimmo, 1957a).
In frozen vegetables, the loss of ascorbic acid was also found to be a useful index to quality, but the percentage retention of chlorophyll (considering chlorophyll-pheophytin ratios) was found to give the closest correlation with overall quality (Dietrich et al., 1959a,b).
PLANT-TISSUE OXIDATION SYSTEMS 5
Chlorophyll losses were affected by the initial heat treatment given the product: the greater the loss during blanching, the lower the stability during storage. Walker (1964a,b) also found that the rate of change of chlorophyll during frozen storage increased when green beans were overblanched. Chlorophyll degradation in green snap beans was minimized by the use of high-temperature short-time blanching (Dietrich et al., 1959b). Water-blanching of Brussels sprouts was more effective than steam-blanching in that enzyme in- activation was faster and chlorophyll degradation less (Dietrich and Neumann, 1965).
The search for improved quality in preserved fruits and vegetables has led to proposals to reduce the period of heat processing to a minimum, and so retain to the maximum extent the natural char- acteristics of the food. It is extremely important to have precise estimates of the effectiveness of different types of heat treatments and process times on enzymes, as well as on microorganisms. Many authors have stressed that with high-temperature short-time processes enzymes, such as peroxidase, may be more difficult to destroy than microorganisms; enzyme inactivation may therefore be the deciding factor in assessing the efficiency of the process (e.g., McConnell, 1956; Leeson, 1957; Adams and Yawger, 1961; Yamomoto et al., 1962).
Deterioration of foods through enzyme action can lead to the development of off-flavors and also to marked changes in color and texture. Despite the efforts of many investigators over the past thirty years the enzymes responsible for quality deterioration have not been positively identified except in a few cases, mostly concerned with changes in texture. There is general agreement that where flavor is concerned several enzymic systems may be involved, working in sequence or simultaneously.
The problem is complicated by the fact that the substances respon- sible for off-flavors are also largely unknown. Many compounds which could be involved can be detected by taste at extremely low concen- trations, of the order of 1 part in lo9 (Lea and Swoboda, 1958), that is, at levels at which chemical isolation and identification are difficult. In some frozen vegetables a good correlation can be obtained between acetaldehyde content and off-flavor, so that acetaldehyde levels can be used as an index of quality deterioration (Gutterman et al., 1951; Lovejoy, 1952). Acetaldehyde is not the cause of the off-flavor, but appears as a reaction by-product, possibly from anaerobic glycolysis. Anaerobic conditions may exist in frozen tissues (Fuleki and David, 1963). Joslyn (1966) has noted that the odors and flavors formed during
6 F. AYLWARD AND D. R. HAISMAN
the storage of frozen unblanched vegetables resemble those of fresh vegetables held in oxygen-deficient atmospheres at room temperature, and that particular vegetables develop quite characteristic odors.
Progress has been made in identifying both the precursors and volatile products responsible for flavor and odor changes in certain products. Falconer et al. (1964) found that the violetlike off-flavor in dehydrated carrot was closely related to the oxidation of p-carotene, and was derived from p-ionone and other oxidation products. The formation of pyrollidone carboxylic acid from glutamine has been shown to cause bitter phenolic off-flavors in beet products (Shallen- berger and Moyer, 1958).
Gas-liquid chromatographic examination of the volatile compounds from stored frozen vegetables has yielded interesting results. Bengts- son and Bosund (1964) evaluated the volatile substances from stored frozen peas, and found that the compounds formed slowly during frozen storage resembled those found in rapid postharvest changes at ordinary temperatures. The main components were acetaldehyde, ethanol, and hexanal; the hexanal content showed promise as an indi- cator for off-flavor development. It was shown later (Bengtsson et al., 1967) that the hexanal concentration in the vapor over cooked frozen peas correlated well with the postharvest deterioration which had occurred. Off-flavor development during the first months of storage at -5C coincided with the formation of hexanal, but over longer periods the hexanal concentration decreased, suggesting that it may be un- reliable as a single quantitative indicator of quality.
Other workers have examined hexanal formation in different products. Thus, Whitfield and Shipton (1966), in their examination of the volatile carbonyls from frozen unblanched peas in storage, found that the major components were acetaldehyde (96 %) and hexa- nal (3.5%). Hexanal was found to be a major component in the vola- tiles produced during low-temperature oxidation of sunflower oil (Swoboda and Lea, 1965) and oxidative degradation of potato granules (Buttery et al., 1961). In later experiments-during storage of potato granules for up to four months -the hexanal content of the vapor over the potato, after reconstitution at 93"C, followed subjective flavor scores closely (Boggs et al., 1964).
The experiments on the production of carbonyl compounds pro- vide support for the theory that the unsaturated lipids, although present in only trace amounts in some types of vegetable material may serve as substrates for oxidative degradative changes during storage.
PLANT-TISSUE OXIDATION SYSTEMS 7
The present review summarizes information available that is relevant to fruits and vegetables on: (1) the enzymes and, in par- ticular, oxidizing systems which may be important in postharvest changes; (2) the possible relationship of some of these oxidizing sys- tems to lipid oxidation by enzymic and nonenzymic mechanisms; (3) thermal and other factors modifying or inactivating the oxidizing enzyme systems; and (4) the relationship between enzymic activity and quality.
I I . OXIDIZING ENZYME SYSTEMS
There are many enzymes in plant tissues which possess an oxi- dizing function, and it is quite conceivable that most play some part in deteriorative processes, albeit a definite link has been established in only a very few cases. The enzymes which have been studied in this connection are listed in Table I, together with some of their dis- tinguishing features. Their properties are discussed in more detail below. The catalytic activity of most of these enzymes depends on a prosthetic group containing copper or iron, and consequently the pos- sibility cannot be ignored of nonspecific metallic catalysis of various
TABLE I OXIDIZING ENZYMES IN PLANTS
Nature of pH optimum Enzyme prosthetic group(s) for activity Comments
Peroxidase Heme ~
7.0 Widespread, concentrated in root material
conjunction with cytochrome systems
Catalase Heme 5.3-8.0 In all plants, in
Cytochrome oxidase Heme - Photodissociable carbon
o-Diphenol oxidase Copper 5.5-7.0 Inactivated by
p-Diphenol oxidase Copper - Insensitive to carbon
Ascorbate oxidase Copper 5.6 Insensitive to carbon
Amine oxidase Copper 8.5 Isolated from pea seedlings Glycolate oxidase Flavin 8.3 Isolated from spinach Lipoxygenase None 5-5-10.0 -
and copper monoxide complex
8 F. AYLWARD A N D D. R. HAISMAN
reactions before or after inactivation of the enzymes. This factor may be of some importance in the breakdown of lipid hydroperoxides, and examples of this, together with a detailed consideration of the action of lipoxygenase, are reserved for a later section, devoted to lipid oxidation.
The activity of peroxidase with reference to deteriorative changes in vegetable tissues has been studied more extensively than any other enzyme system. Because of its relatively high resistance to thermal inactivation, and its extensive distribution, peroxidase has been widely used as an index of enzyme activity in plant tissues. It has been generally accepted that if peroxidase is destroyed by a given heat treatment it is unlikely that any other enzyme system will have sur- vived. As a practical test this has worked very well, its main disad- vantage being lack of agreement between different techniques for de- tecting the active enzyme. Methods of estimating peroxidase activity have recently been reviewed (Wood and Lopez, 1963), and levels of activity in various vegetables have been determined (Bottcher, 1961,1962).
A full account of the biochemistry of this enzyme has been given by Saunders et al. (1964). Various types of peroxidase exist, and the properties of the enzyme depend to some extent on its source. True peroxidases are hemoproteins, and have in common the prosthetic group protohematin IX in the proportion of one hematin residue per molecule of enzyme. Different peroxidases can be distinguished by differences in their absorption spectrum (Mehler, 1957) and in their behavior toward different reducing agents. Thus, milk peroxidase will oxidizes resorcinol but not nitrite (Elliott, 1932a,b). Even peroxidases isolated from the same plant source may exhibit different properties, and there is little doubt that multiple forms of the enzyme occur.
Horseradish peroxidase, the best characterized of the peroxidases, consists of a colorless protein reversibly bound to protohematin IX (Maehly, 1955). The iron atom in protohematin has six coordination positions, four of which are taken up by porphyrin nitrogens and the fifth by a protein attachment. The sixth position can be occupied by water, cyanide, or another radical, and the enzyme appears to operate by the exchange of groups at this position. The protohematin can be easily and reversibly detached by acetone and hydrochloric acid below WC, a property not shared by other peroxidases.
PLANT-TISSUE OXIDATION SYSTEMS 9
Peroxidases are quite specific in their primary reaction with peroxides, but, by means of coupled reactions whereby the primary oxidation products react with secondary substrates, they can promote a variety of consequential reactions. They appear to be most stable at pH 7.0, where they also exhibit maximum catalytic activity. Maehly (1955) found horseradish peroxidase to be stable between pH 3.5 and pH 12.0 in the absence of inhibitors, and Wilder (1962) confirmed this. Lenhoff and Kaplan (1955) found that at pH 7.0 cytochrome c peroxi- dase was most active and also stable. Axelrod and Jagendorf (1951) found peroxidase (and phosphatase and invertase) to be stable in auto- lyzing tobacco leaves. Even though the leaves lost 45% of the cyto- plasmic protein nitrogen during storage, the levels of enzyme activity were unaltered.
The enzyme is severely inhibited by azide, cyanide, fluorides, and other halides in acid solutions (Maehly, 1955; Lenhoff and Kaplan, 1955).
There are many organic and inorganic substances which can catalyze certain typical peroxidase reactions involving peroxides (Saunders et al., 1964). These include hematin compounds, chelated iron salts, amorphous heavy-metal hydroxides, aldehydes, granite, charcoal, platinum, and palladium. More specialized cases are acetyl choline, which can catalyze the oxidation of benzidine and vitamin A, and the carotenes, which can catalyze the oxidation of potassium indigosulfonate. Generally speaking, these substances are much less effective catalysts than peroxidase itself, as shown by the comparison between horseradish peroxidase and hematin compounds in Table 11.
TABLE I1 THE CATALYTIC ACTIVITY OF HEMATIN COMPOUNDS
Relative catalytic activity
Horseradish peroxidase 1,000,000 Pyridine hemochrome 4.5 Hemoglobin Denatured globin hemochrome Hematin
"Bancroft and Elliott, 1934.
10 F. AYLWARD AND D. R. HAISMAN
Catalase and peroxidase activities are often grouped together. Both are hemoproteins, use hydrogen peroxide as a substrate, and occur in several modifications according to source. Pure crystalline catalase can be obtained from blood, is red at a neutral pH, and contains 1.1 % protohemin and 0.09% iron, which is equivalent to four hematin residues per molecule of enzyme (Bonnichsen, 1955). Plant catalase has been isolated from spinach, and contains 0.049% iron, approxi- mately half the value for a pure four-hematin enzyme (Galston, 1955).
Preparations of the plant enzyme are stable indefinitely at 1C between pH 5.3 and 8.9, and the enzyme activity is greatest between pH 5.3 and 8.0, falling off quickly at more acid values, and slowly at more alkaline values (Galston, 1955). Sapers and Nickerson (1962a) prepared spinach catalase and found it to be quite stable below 36F. Otherwise, its stability was greatly influenced by storage tempera- ture and pH. At 80F and pH 7.0 it became inactivated rapidly. In- activation was more rapid in acid solutions. The enzyme was also susceptible to attack by microorganisms.
D. CYTOCHROME OXIDASE
The characterization of cytochrome oxidase is still the subject of experiment, but the available evidence suggests that it is constituted of heme, copper, lipid, and protein (Wainio, 1961). In a recent review, Beinert (1966) tentatively concludes that the enzyme is a 1: 1 combina- tion of cytochrome a and cytochrome a,. It is thought that the cyto- chrome a, reacts directly with oxygen (Smith and Conrad, 1961). The enzyme is readily distinguished by its absorption spectrum and by the spectral shift during the formation of its photo-dissociable carbon monoxide complex. It is specific to cytochrome c, which it converts to the oxidized form in the presence of molecular oxygen. The coupled system can oxidize many other substrates, such as phenols and amines, and sustains other oxidizing enzymes such as succinate de- hydrogenase, a particularly important system in many microorgan- isms. The estimation of the enzyme may be difficult in the presence of phenol oxidases. Hare1 and Mayer (1963), working with lettuce seeds, found that the presence of oxidized phenolic compounds de- pressed cytochrome oxidase activity. The activity was restored when phenolase inhibitors were added.
The final stages of respiration in potato tubers appear to be shared by cytochrome oxidase and o-diphenol oxidase, the relative impor-
PLANT-TISSUE OXIDATION SYSTEMS 11
tance of each depending on the maturity of the tuber (Mondy et al., 1960). Mapson and Burton (1962) found that 70% of the respiration of potato tubers passes over the cytochrome system. Other authors have found that cytochrome oxidase activity is high enough to account for the whole of the respiration of potato tubers (Goddard and Holden, 1950; Schade et al., 1949; Thimann et al., 1954).
The substrate of cytochrome oxidase, cytochrome c, is known as a stable hemoprotein. Its chemistry has recently been reviewed by Margoliash and Schejter (1966). It is notable for its remarkable stability in conditions commonly deleterious to proteins, and its re- sistance to dilute acid and alkali and to boiling (although a small proportion may be denatured). It is easily reduced by molecules such as cysteine and ascorbic acid, and reoxidized by cytochrome oxidase, and also by peroxidase. Cytochromes found in plant tissues have been surveyed by Bonner (1961), and may be slightly different from those from other sources. The purification of cytochrome c from wheat germ has been described (Wasserman et al., 1963), and this compound was found to be very unstable in solutions of low ionic strength at pH 7.0.
E. O-DIPHENOL OXIDASE
o-Diphenol oxidase is widespread in occurrence. Bonner (1957) re- viewed the function of the enzyme in plant tissues, and Brooks and Dawson (1966) surveyed aspects of its chemistry. It is a copper- containing enzyme, easily inactivated by carbon monoxide. o-Di- phenol oxidase displays activity toward a great range of substrates, as is indicated by the various names by which it is commonly known: catecholase, tyrosinase, cresolase, polyphenoloxidase, phenolase, etc. The enzyme apparently occurs in various forms, which can be classi- fied into two broad groups, both oxidizing o-dihydric phenols but not ascorbic acid, one of which possesses the added ability to catalyze the o-hydroxylation of monophenols (Robb et al., 1965). The ratio of the activities toward mono- and o-dihydric-phenols in an enzyme preparation varies according to the source and methods used in its isolation.
Although catechol and tyrosine or cresol are commonly used for estimation of the activity, the natural substrates are probably more complex phenolic compounds, such as chlorogenic acid. Alberghina (1964) found that o-diphenol oxidase from potato tubers had an affinity much higher for chlorogenic acid and methyl catechol than for catechol or dihydroxyphenylalanine. One enzyme from eggplant oxidized chlorogenic acid much faster than any other substrate tried,
12 F. AYLWARD A N D D . R. HAISMAN
while one from avocado showed greatest affinity for nordihydro- guaiaretic acid when compared with catechol and catechin (Knapp, 1965). Another eggplant phenol oxidase was active toward antho- cyanins (Sakamura et aZ., 1966). Tate et aZ. (1964) characterized an o-diphenol oxidase from Bartlett pears, and found it was active only toward o-dihydric phenols. Other phenols were not attacked. Walker (1964c,d) isolated the enzyme from apples and pears, and found that it was not only very active toward o-dihydric phenols but over longer times catalyzed the o-hydroxylation of p-coumaric acid to cafFeic acid. The conversion of p-coumaric acid to cafeic acid is efficiently catalyzed by mushroom o-diphenol oxidase in the presence of ascor- bic acid, but the reaction can also be brought about nonenzymatically by dihydroxymaleate or an iron-ascorbate system (Embs and Mar- kakis, 1966).
The optimum pH for maximum activity of the enzyme appears to vary between preparations and according to substrate from about pH 4.0 to pH 7.0. It is difficult to measure the activity of the enzyme directly, owing to the large number of secondary reactions which follow the initial enzymic oxidation. Assay methods have been com- pared critically (Mayer et al., 1966); polarographic measurement of the initial oxygen uptake appears to give the most sensitive estimate of the enzyme activity. The level of o-diphenol oxidase activity and the concentration of various substrates in different fruits and vege- tables have been determined by Herrmann (1957, 1958). The cellular location of the o-diphenol oxidase responsible for the darkening of cut red beet has been examined by Boscan et al. (1962). The inter- relationship between cytochrome oxidase and o-diphenol oxidase activities in potato tubers, particularly during storage at different temperatures, has been examined by Mondy et al. (1966a,b).
F. ~ D I P H E N O L OXIDASE
Commonly known as laccase, p-diphenol oxidase, another wide- spread copper-containing enzyme, has been reviewed by Bonner (1957) and Levine (1966). The pure enzyme is deep blue and is not appreciably inactivated by carbon monoxide. Alleged to be prin- cipally an extracellular enzyme, it catalyzes the oxidation of a large number of aryl dihydric phenols and diamines, where the functional groups have an o- or a p- relationship. Like o-diphenol oxidase, it in- directly oxidizes ascorbic acid through coupled oxidations with phenolic or amine substrates.
PLANT-TISSUE OXIDATION SYSTEMS 13
G. ASCORBATE OXIDASE
Ascorbic acid is a powerful reducing agent, and its oxidation is catalyzed by metals as well as by several enzyme systems. Methods have been devised (Butt and Hallaway, 1958) for distinguishing be- tween true ascorbate oxidase activity and the action of other less specific catalysts. Ascorbate oxidase is another copper-containing enzyme widespread in plants and microorganisms. Its chemistry has been reviewed by Bonner (1957) and Dawson (1966). The pure enzyme is blue and insensitive to carbon monoxide. Its natural sub- strate is assumed to be ascorbic acid, but it is also active toward ring analogs with a dienol grouping adjacent to a carbonyl group, and sub- stituted polyhydric and amino phenols, including 2,6-dichloroindo- phenol, which is oxidized to a blue quinoid dye.
The activity of the enzyme has been determined in a number of different fruits and vegetables (Huelin and Stephens, 1948; Mc- Combs, 1957).
H. AMINE OXIDASES
The characterization of these enzymes is still the subject of in- tensive investigation. Although a tentative classification into mono- amine oxidases and diamine oxidases has been proposed, Nara and Yasunobu (1966) suggest in a recent review that a rigid classifica- tion is no longer tenable. The enzyme is of interest since it catalyzes the conversion of amines to aldehydes; hydrogen peroxide and ammonia are the other primary reaction products. The production of hydrogen peroxide leads to various secondary reactions, including in- activation of the enzyme. Most work has been carried out on the amine oxidase system in pea seedlings (Kenten and Mann, 1955; Mann, 1961; Hill and Mann, 1962, 1964). The enzyme contains copper, and is inhibited by various chelating agents. It is unusual in that solu- tions of purified enzyme are pink. The preparation from pea seedlings catalyzes the oxidation of aliphatic monoamines, diamines, phenyl- alkylamines, histamines, spermidine, agmatine, lysine, and ornithine.
I. GLYCOLATE OXIDASE
Glycolate oxidase, a flavoprotein, catalyzes the oxidation of a- hydroxy acids by oxygen to the corresponding 0x0 acids, with the concomitant production of hydrogen peroxide. The most important
14 F. AYLWARD AND D . R. HAISMAN
substrates have been found to be glycolic and L-lactic acid (Zelitch and Ochoa, 1953). Kolesnikov (1948a,b; 1949) noted that glycolic acid had a catalytic effect on the degradation of chlorophyll and ascorbic acid in barley leaf macerates. In the presence of glycolic acid, oxygen absorption by the macerate increased up to 15 times over the amount needed to oxidize the glycolic acid. The rate of chlorophyll degrada- tion was dependent on the level of glycolic acid present. Tolbert and Burris (1950) also found that the oxidation of glycolic acid in green leaves was accompanied by bleaching of the chlorophyll. No11 and Burris (1954) detected glycolate oxidase activity in 17 species of plants. Recent work has shown that the enzyme also catalyzes the oxi- dation of glyoxylic and a-hydroxybutyric acids (Richardson and Tol- bert, 1961) and aromatic a-hydroxy acids (Gamborg et d., 1962).
J. OXIDATION MECHANISMS
Although quite distinct in their primary reactions, the oxidative enzymes are alike in their ability to utilize a wide range of secondary substrates. The main features of the oxidative reactions are shown in Table 111.
The combination of peroxidase with substrate can be followed spectrophotometrically, which facilitates study of the oxidation mechanism (Chance, 1949a,b). The cyclic reaction illustrated in Fig. 1 has been well established (Saunders et aZ., 1964). Methyl or
Oxidized donor Peroxidase
Hor e donor
Compounds If1 and N (enzymically inactive)
FIG. 1. The peroxidase oxidation cycle.
TABLE I11 OXIDATION MECHANISMS
Enzyme S ubstrate( s) Primary product Secondary reaction
Peroxidase HzOz Oxidized peroxidase Electron transfer to
(and some other peroxides)
hydrogen donor such as m i n e or phenol
2 HZO + 0 2 Cytochrome oxidase Cytochrome c and oxygen Oxidized cytochrome c Coupled oxidation of
0- and p-Dihydric phenols
Quinone phenols, mines, etc.
Quinone formation may lead to further coupled oxidations
o-Dihydric phenol Oxidation of dihydric
Quinones May lead to further
Ascorbate oxidase Ascorbic acid Dehydroascorbic acid Spontaneous delactonization to diketogulonic acid -
At low concentrations of hydrogen peroxide, catalase can act as a peroxidase, using aliphatic alcohols and other compounds as hydrogen donors.
16 F. AYLWARD AND D. R. HAISMAN
ethyl hydroperoxides, peracids, or hypochlorite can be substituted to some extent for hydrogen peroxide in the first stage of the reac- tion. The commonest hydrogen donors are amines (such as aniline, p-toluidine, mesidine, etc.) or phenols. Side reactions are very com- mon, and methyl, methoxy groups, or halogen atoms may be elimi- nated from the ring during the oxidation of aromatic amines.
An alternative oxidative pathway involving peroxidase was dis- covered when Kenten and Mann (1950) showed that MnZf was oxi- dized by peroxidase systems. Since then, systems containing peroxi- dase, manganese, and mono- or dihydric phenols have been found to oxidize a variety of substrates including dicarboxylic acids (Kenten and Mann, 1953), phenylacetaldehyde (Kenten, 1953), NAD, and NADP (Akazawa and Conn, 1958). Mudd and Burris (1959) observed that plant peroxidases have broad substrate specificities, acting peroxidatively in the presence of hydrogen peroxide, or oxidatively in the presence of phenols and manganous or cerous ions. The peroxi- dase-manganese-phenol system also oxidizes indoleacetic acid and, although the point is still controversial, may account for activity for- merly attributed to a specific enzyme, indoleacetic acid oxidase (Hare, 1964). This oxidation is inhibited by certain quercetin deriva- tives (Furuya et al., 1962).
The oxidation of crocin in sugar-beet leaves may be mediated by a similar system. Dicks and Friend (1966a,b) found two enzyme systems associated with mitochondria from sugar-beet leaves which could accomplish crocin oxidation. One was attributed to a coupled oxida- tion involving lipoxygenase and unsaturated lipids, and the other, which was stimulated by 8-hydroxyquinoline and other phenols, probably involved peroxidase or another hemoprotein.
The presence of free radicals during peroxidase oxidations was demonstrated by Yamazaki et al. (1960) with peroxidase from turnips on substrates of hydroquinone and ascorbic and dihydroxyfumaric acid.
The characteristic reaction of catalase is the catalytic decomposition of hydrogen peroxide to water and oxygen. This is a two-stage re- action and can be followed spectrophotometrically.
Catalase + 2 H,O, - Compound I - Catalase + 2 H,O + 0, Keilin and Hartree (1955) discovered that, in the presence of the
very low concentrations of peroxide generated by oxidase systems in
PLANT-TISSUE OXIDATION SYSTEMS 17
oitro (e.g., xanthine oxidase, glucose oxidase, etc.), catalase could bring about the coupled oxidation of alcohols and other donors, such as nitrites, carboxylates, and aldehydes. Compared with the decom- position of peroxide, the coupled oxidation is a slow reaction.
3. Diphenol Oxidases
Characteristically, the phenol oxidases catalyze the aerial oxidation of dihydric phenols. Mayer studied the effect of various inhibitors on the activity of the o-diphenol oxidase (Mayer, 1962; Mayer et al., 1964) and also the effect of nuclear substituents on the rate of enzymic oxi- dation. It appears that the oxidation takes place by electrophilic attack. The inactivation of o-diphenol oxidase during the oxidative reactions which it catalyzes is well known and was investigated by Ingraham (1954). He concluded that no extensive damage occurred to the pro- tein in this inactivation, which seems most likely to be due to an inter- action between the enzyme and the quinone produced by the oxida- tion (Brooks and Dawson, 1966).
4 . Ascorbate Oxidase
The first product of oxidation of ascorbic acid is dehydroascorbic acid. Opening of the lactone ring, with the formation of diketogulonic acid, can follow spontaneously or by enzymic catalysis. Roe et al. (1948) devised a method of estimating all three compounds in the presence of one another which has been much used in subsequent in- vestigations. The mechanisms of the destruction of ascorbic acid in cauliflower, bitter gourd, and tapioca leaves have been investigated in detail by Tewari and Krishnan (1960, 1961). They found that both steps from ascorbic acid to diketogulonic acid were enzymically catalyzed, and that tapioca leaves contained a further enzyme system which degraded diketogulonic acid. Tapioca leaves also contained a natural inhibitor of ascorbate oxidase which stabilized the natural ascorbic acid.
Like o-diphenol oxidase it is reaction-inactivated, but in this case the inactivation is thought to be due to hydrogen peroxide produced nonenzymically by traces of free copper associated with the enzyme (Dawson, 1966). Lillehoj and Smith (1966) found that an ascorbate oxidase from Myrothecium uerucaria took up more than 0.5 mole of oxygen per mole of ascorbate oxidized, and that 10% of the oxidized product disappeared. They thought that either the ascorbate oxidase had peroxidative capacity for a reductant other than ascorbic acid, or free radicals were produced during the oxidation.
18 F. AYLWARD A N D D . R. HAISMAN
K. CORRELATION BETWEEN ENZYME ACTIVITY AND FOOD DETERIORATION
Wagenknecht and Lee (1958) added various enzymes to blanched peas and found a good correlation between added peroxidase and off- flavor production, but emphasized that the flavor changes were only minor. In a similar experiment Zoueil and Esselen (1959) added peroxidase to sterile packs of green beans and turnips and found that off-flavors and off-odors developed and the acetaldehyde content of the pack increased up to fivefold. Joslyn and Neumann (1963) used the decrease in ascorbic acid content in frozen vegetables as an index of peroxidase activity. Pinsent (1962) noted that when peroxidase was not completely inactivated during the blanching of green peas, off- flavors developed during storage of the frozen product.
Grommick and Markakis (1964) found that anthocyanin pigments could be discolorized by peroxidase.
Wagenknecht and Lee (1958) found that additions of catalase to blanched peas resulted in a mild off-flavor when the peas were stored frozen. A later experiment, with added endogenous catalase, produced a disagreeable off-flavor over 18 months of frozen storage.
Although the function of the oxidases in the metabolic processes of plants is still obscure, their ability to catalyze direct oxidation by molecular oxygen makes them potential agents in quality deterioration.
The browning of plant tissues, particularly after injury, due to the oxidation of polyphenolic constituents is a familiar problem. Joslyn and Ponting (1951) reviewed the enzymes responsible for the brown- ing of fruit and pointed out that, although the phenol oxidases are the primary browning agents, other enzymes, such as cytochrome oxidase, which participate in coupled oxidations, may easily be involved. This applies to both oxidative and reductive changes. Makower (1964a,b) showed that adenosine triphosphate (ATP) inhibited the browning of potato slices although it was not itself a reducing agent. It appeared that reduced nicotinamide adenine dinucleotide (reduced NAD) was the effective reducing agent, and that the function of the ATP was to maintain the supply of reduced NAD.
PLANT-TISSUE OXIDATION SYSTEMS 19
Investigations into the effect of oxidase activity on quality have concentrated on color changes and oxidation of vitamin C. Phenolic oxidation products may also contribute to changes in flavor (Mapson and Swain, 1961), however, and the possibility should not be over- looked of apparently unrelated effects due to secondary reactions of the oxidation products.
I l l . R E S P I R A T O R Y AND O T H E R E N Z Y M E S
Although peroxidase and catalase may be involved in the respiratory process, their exact role is not clearly understood. There are, however, many enzyme systems operating along the respiratory pathways whose function is defined, and most of them have been detected at one time or another in the higher plants (e.g., Bonner and Varner, 1965).
In essence, respiration is controlled oxidation of organic material to carbon dioxide and water, producing energy in a form which can be utilized in other cellular processes. It is carried out by a sequence of enzyme systems that transfer electrons from successive degradation products to molecular oxygen by a stepwise process. The energy pro- duced is transported to other systems by means of the reduced forms of coenzymes such as nicotinamide-adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), and energy-rich organic phosphates such as adenosine triphosphate
(ATP). The process can be divided into two stages. In the first instance,
hexoses are oxidized to pyruvate ions under the action of phosphoryl- ating, isomerizing, chain-splitting, and dehydrogenating enzymes. Next, the pyruvate is oxidized to carbon dioxide and water through operation of the Krebs cycle, again involving a variety of phosphoryl- ating, isomerizing, dehydrogenating, and decarboxylating enzymes.
Most of the oxidative reactions in the respiratory chain are coupled, often using common intermediates such as NAD or NADP, and the pathways of electron transport from the original substrates are ob- viously complex and probably involve cytochrome systems and flavo- proteins. Although various oxidases have been proposed as terminal oxidases (Lee, catalyzing the final step in the transfer of electrons to oxygen) most have a comparatively low affinity for oxygen and are more likely to operate through some other acceptor (Bonner, 1957).
20 F. AYLWARD AND D. R. HAISMAN
Cytochrome oxidase has a high affinity for oxygen, and almost cer- tainly catalyzes at least part of the oxygen uptake of all plant tissues.
Under anaerobic conditions the terminal respiratory pathways are blocked. The breakdown of hexose continues, but the pyruvate pro- duced is no longer oxidized via the Krebs cycle. Instead, it is either reduced to lactate through the agency of lactate dehydrogenase and reduced NAD (the predominant mechanism in muscle tissue) or decarboxylated under the action of pyruvate decarboxylase to acetal- dehyde and carbon dioxide. The acetaldehyde can then be reduced to alcohol by alcohol dehydrogenase in the presence of reduced NAD. The latter mechanism predominates in plant tissues. Hatch and Turner (1958) showed that pea extracts could quantitatively convert starch, hexoses, and hexose phosphates to carbon dioxide and ethyl alcohol when cofactor levels of ATP, NAD, and magnesium were supplied.
c. RESPIRATORY ENZYMES AND FOOD DETERIORATION
Delay between vining and processing peas brings about rapid deterioration in quality, and the conditions are often such that respira- tory by-products might be expected to accumulate. Off-flavors can develop in less than two hours (Talburt and Legault, 1950) and, even when ice-cooling is used, are noticeable after four hours (Lynch et al., 1959).
The enzymes of the respiratory pathways have been suspected as factors in the development of off-flavors mainly because acetaldehyde and alcohol, typical products of anaerobic glycolysis, have been found in relatively large amounts in some deteriorated products (Gutterman et d., 1951; Joslyn and David, 1952). More extensive investigations with peas (David and Joslyn, 1953) and broccoli (Buck and Joslyn, 1953) showed that, in addition to acetaldehyde and alcohol, smaller amounts of acetoin and diacetyl were produced. In the presence of thiamine pyrophosphate, pyruvate decarboxylase is known to catalyze acyloin formation as well as a-keto acid decarboxylation (Singer, 1955):
RCOCOOH + RCHO + CO, RCOCOOH + RICH0 -+ RCHOHCOR' + CO,
RCHO + RICH0 + RCHOHCOR'
PLANT-TISSUE OXIDATION SYSTEMS 21
and the results indicated that a pyruvate decarboxylase system was still active in underblanched peas.
David and Joslyn (1953) demonstrated that pyruvate decarboxylase was active in green peas but was inactivated within 2 min at 60C. Buck and Joslyn (1956) followed the activity of the enzyme in broccoli, but found that the amounts of acetaldehyde, acetoin, and diacetyl produced could not be correlated with the intensity of undesirable flavors. Ralls (1959) reported that levels of acetoin are appreciable (up to 300 ppm) in many canned vegetables and can be induced in frozen peas through the nonenzymic thiamine-catalyzed conversion of pyruvic acid. Although acetoin is probably a flavor component, it cannot be classed as an off-flavor.
Fuleki and David (1963) examined the production of alcohol, acetaldehyde, and off-flavors in frozen snap-beans, and concluded that neither acetaldehyde nor alcohol level gave an objective measure of off-flavor development. Controlled blanching experiments showed that the fermenting enzymes were more easily inactivated than the off-flavor-producing enzymes, and that most of the acetaldehyde and alcohol were produced under anaerobic conditions before and during freezing. In a study of immediate postharvest changes, Wager (1964) measured the respiration of peas in and out of pods. Although shelled peas deteriorated fairly rapidly there was little change in their respiration pattern. On the other hand, the respiration of the pod changed markedly after removal of the peas. Wager postulated that a translocation of hormone from pod to peas delayed senescence.
Experimental evidence thus far indicates that the occurrence of acetaldehyde and related substances in deteriorated products may be coincidental and unrelated to the more objectionable flavor changes. Conditions which favor the development of off-flavors may also en- courage fermentation, though not necessarily. Generally, the products of fermentative processes occur at quite high levels. The amounts of acetaldehyde reported in the literature are commonly of the order of 1 mmole/kg, and thus are far in excess of the putative off-flavor components, such as hexanal and TBA-reactive substances, reported at levels of between 0.01 and 0.05 mmole/kg.
D. PECTIC ENZYMES
Among other plant enzymes, the pectic enzymes have been studied extensively because of their influence on the texture and appearance of plant products. The occurrence, effects, and applications of pecto- lytic activity, particularly in fruits and fruit juices, have been discussed
22 F. AYLWARD AND D. R. HAISMAN
in many reviews (e.g., Charley, 1961; Reid, 1950; Demain and PhafF, 1957). Pectinesterase, which catalyzes the deesterification of poly- gacturonates, brings about the gelation of soluble pectins and in- creases the potential cross-linking of structural pectins through divalent ions such as calcium and magnesium, leading to a firmer texture. The enzyme is structure-bound and insoluble in its natural state. It can be solubilized in the presence of salts in slightly alkaline solutions. The bound enzyme is active over a smaller pH range than its soluble form, but both show maximum activity at pH 7.5 (Jansen et al., 1960). The optimum pH for the associated enzyme, polygalac- turonase, is 2.5 to 4.5, depending on the substrate (Pate1 and Phaff, 1960a,b).
The innate pectinesterase activity of tomatoes has been used to improve the texture of the canned product; there is some evidence that short heat treatments (such as a 30-second blanch) activate the enzyme in situ (Hsu et al., 1965).
Pectic enzymes are not inhibited by sulfur dioxide, and can show considerable activity at temperatures as low as - 12C (Doesburg, 1951).
Chlorophyllase hydrolyzes chlorophyll to a chlorophyllide and phytol, converting it from a fat-soluble to a water-soluble pigment. Its influence on retention of the natural color of green vegetables during processing has not been investigated. The enzyme is localized in the chloroplasts, and is easily solubilized and activated by treat- ment with trypsin (Boger, 1965). Holden (1961) obtained a soluble preparation from sugar-beet leaves. She found it was most active in sugar beet, peas, beans, wheat, and barley. Brussels sprouts contained only a low level of activity.
F. ENZYMES OF AMINO ACID METABOLISM
Eriksson and von Sydow (1964) examined the effect of postharvest treatments on the levels of glutamic, y-aminobutyric, and aspartic acid in green peas. When peas were damaged during harvesting, y-aminobutyric acid was formed through the action of glutamate decarboxylase. The concentration of this acid increased with the time the peas were held after vining, indicating its possible value as an index of quality. The glutamic acid concentration in the peas was maintained by proteolytic enzymes and aspartate aminotransferase
PLANT-TISSUE OXIDATION SYSTEMS 23
acting on aspartic and a-ketoglutaric acid. Enzyme activity was highest in the skins of the peas; it was postulated that, in damaged peas, changes in permeability brought enzymes and substrates into proximity.
IV. OXIDATIVE AND OTHER CHANGES IN LIPIDS
There is now substantial evidence that changes in the lipids of food components may play an important, sometimes dominant, role in deterioration in the quality of foodstuffs. Our knowledge of lipid chemistry and biochemistry has advanced rapidly in recent years, both because of more workers in this field and because of the avail- ability of new analytical techniques - in particular, chromatographic methods.
Various standard textbooks and monographs (e.g., Witcoff, 1951; Eckey, 1954; Lovern, 1955; Deuel, 1951, 1955, 1957; Hilditch and Williams, 1964) give authoritative reviews of lipids in terms of their types, composition, and distribution in plants, animals, and micro- organisms.
It has long been recognized that two major types of lipids are widely distributed in plant and animal cells, namely the mono-, di-, and tri- glycerides, and the phospholipids (such as phosphatidyl choline), containing glycerol, fatty acids, phosphoric acids, and a nitrogenous base. Work over the past twenty years has established the existence of several other types of lipids of varying degrees of complexity. Authors frequently group within the term lipids the sterols and fat-soluble pigments and vitamins, which are often associated with glycerides and phospholipids in tissues, and are extracted from tis- sues by similar solvents. These sterols and pigments (including polyene pigments) of varying degrees of unsaturation may participate in oxidation processes in foodstuffs.
The glycerides and phospholipids contain a range of fatty acids which differ in the number of carbon atoms and in the number of unsaturated links. In plant tissues the lipid content varies greatly from one type of material to another. Large quantities are present in oilseeds, and substantial amounts in cereals. Vegetables and fruits may contain only small amounts (perhaps less than 1% of the wet weight). The fact that the lipids are only minor components of the common fruits and vegetables in no way detracts from their potential importance in quality deterioration.
There has been an increasing interest in the location and function
24 F. AYLWARD AND D. R. HAISMAN
of lipids in the plant cell. In this connection the protein and carbo- hydrate complexes of lipids are important, both in the living plant and in plant foodstuffs.
Much of the earlier work on lipids (cf. Hilditch and Williams, 1964) was concerned with the nature and distribution of fatty acid compo- nents. A second stage of investigations covered the isolation of individual lipids and their characterization in terms of both fatty acids and other components. Much work has been carried out on oil- seeds (texts already cited, and Markley, 1950; Aylward and Nichols, 1961, 1962). An increasing amount of effort has been devoted to cereals (Aylward and Showler, 1962a,b; Fisher, 1962; Fisher et al., 1964). Relatively little systematic work has been carried out on the common fruits and vegetables, although mention should be made of investigations by Wagenknecht (1957a,b) into the lipids of peas.
Reviews of the literature on plant phospholipids (e.g., Aylward, 1956) show that proper characterization of lipids is difficult and that reliable information is scarce for most plant materials, although many gaps in knowledge are being filled by the application of newer tech- niques (James, 1960). In the absence of detailed information about lipids in many plant foodstuffs, the role of lipids in deteriorative processes in such foodstuffs is necessarily obscure, although much can be learned by analogy with studies on animal products (such as milk and fish).
A. DEGRADATION OF LIPIDS
Changes in lipids can be brought about by different methods (see Fig. 2), which can be summarized as follows: (1) Partial or complete hydrolysis of the lipid to fatty acids and other components, followed by oxidation of unsaturated fatty acids; and (2) direct oxidation of the unsaturated acids (or other unsaturated components) in the intact lipids, followed by hydrolytic degradations.
The hydrolytic processes are brought about by enzymes normally classified as lipases; the oxidation processes may be catalyzed by enzymes (in particular the enzyme lipoxygenase) or by metals and their salts or organic complexes.
Among the degradation products likely to be of special importance in relation to food quality are (1) short-chain (< 12C) volatile fatty acids present in some intact lipids and liberated by hydrolysis; (2) other volatile substances (such as acids and aldehydes) formed by oxidation of fatty acids or polyene components; and (3) nitrogenous or other nonfatty components (e.g., from phospholipids).
PLANT-TISSUE OXIDATION SYSTEMS 25
ted and saturated
Action of lipases- - stepwise hydrolysis T Y - - . . L
.. . . \
\ \ J t Mono- and Phosphatidic Nitrogenous , Fatty acids diglycerides acids bases Chain length Chain length 1,4-Pentadiene
reduced by reduced by structure attacked one carbon two carbon 1,3-Diene-5-hydro- atom atoms peroxide produced
FIG. 2. T h e degradation of lipids.
Most of the work on lipases has been in connection with animals rather than vegetables. Studies on vegetable lipases include those of Hanahan and Chaikoff (1947a,b, 1948) on the degradation of phospho- lipids from carrots and cabbage, and of Long et al. (1962) on phospho- lipase D from cabbage.
It is probable that several lipase systems exist, some of which are selective, preferentially attacking either the a or p fatty acid chains of triglycerides. There is some evidence that the enzymes in animal tissue are activated by freezing and can operate at moisture levels down to 1.5% (Lea, 1961a).
The general outlines of the biological oxidation of lipid materials have been well defined (e.g., Mahler, 1964). Probably the most important degradative pathway is the @-oxidation spiral, which in- volves coenzyme A, NAD+, and magnesium ions and shortens the fatty acid chain by two carbon units in successive cycles to produce acetyl coenzyme A. The p-oxidative enzymes have been shown to be present in both the mitochondria and the soluble proteins of plant cell homogenates, and the acetyl coenzyme A which is produced is subsequently consumed in the tricarboxylic acid and glyoxylate cycles.
The longer chain fatty acids (C13-C18) can also be degraded by a-oxidation through a two-enzyme sequence, reducing the chain length by one carbon atom per cycle. Both saturated and unsaturated acids are susceptible to this attack (Martin and Stumpf, 1959; Hitch-
26 F. AYLWARD A N D D. R. HAISMAN
cock and James, 1963). The reaction requires continuous generation of hydrogen peroxide, such as might be provided by the action of glycolate oxidase (widespread in plant tissues). The degradation proceeds through the action of a specific fatty acid peroxidase fol- lowed by a long-chain aldehyde dehydrogenase in the presence of NAD+.
The enzymic oxidative degradation which has received most attention in relation to deterioration of the quality of foods is that due to the enzyme lipoxygenase.
Bergstrom and Holman (1948) reviewed the chemistry and proper- ties of lipoxygenase, an enzyme which is distributed widely, par- ticularly in legumes, potatoes, tomatoes, and various herbs. According to the earlier literature it displays maximum activity at pH 9.0. No known prosthetic group or cofactor is involved in lipoxygenase catalysis, but there have been reports of an activating agent in seeds, which may in fact be an emulsifier assisting close approach of enzyme and substrate.
The enzyme is specific in attacking only 1,Cpentadiene structures, e.g., it attacks linoleic, linolenic, and arachidonic acids, but not oleic acid (Dillard et al., 1961). The product of the action of lipoxy- genase is an optically active cis-trans-conjugated hydroperoxide (Privet? et al., 1955).
At one time it was thought that the enzyme attacked free acids only, but now it is accepted that it will act also on triglycerides and other esters. Koch et al. (1958) isolated two groups of lipoxygenases from soybeans, one of which was active toward triglycerides and the other toward free acids. Dillard et al. (1960) showed both triglyceride and fatty acid types of activity in beans, peanuts, and peas, and found that the optimum pH varied with the substrate. Siddiqi and Tappel (1956) identified a lipoxygenase in peas which had maximum activity at pH 6.9 at temperatures between 0" and 15C. Surrey (1964) introduced an improved spectrophotometric method for the estimation of lipoxy- genase activity, and found pH optima for maximum activity ranging from pH 5.5 to 7.0, depending on the substrate. The discrepancies in the reported effects of pH on the activity of the enzyme are probably due to variations in the ionic strength of the reaction medium, Ames and King (1966) found that the pH profile of lipoxygenase activity varied between pH 5.5 and 10.0, depending on the type of medium used.
PLANT-TISSUE OXIDATION SYSTEMS 27
The enzymic reaction is inhibited by antioxidants such as a-tocoph- erol and dibutylhydroxytoluene and to some extent by surface-active agents such as Triton X-100 (Dillard et al., 1961). Blain and Shearer (1965) found that long-chain polyacetylenic acids were potent com- petitive inhibitors, in contrast to nordihydroguaiaretic acid, which they found prolonged the induction period. A natural inhibitor of lipoxygenase activity, found in peanut testa, has also been reported (Narayanan et al., 1963).
c. AUTOXIDATION OF LIPIDS
As anyone with experience in lipid studies will testify, constant and elaborate precautions are necessary to guard against autoxida- tion. The oxidation of unsaturated fats in the presence of molecular oxygen proceeds through free-radical chains and is thus autocatalytic (Lundberg, 1962). It is accelerated by heat and light and catalyzed by heavy metals and their salts and organometallic compounds; by all oxidative enzymes; by hemoglobin and all hematin compounds; and by photochemical pigments. Autoxidation reactions are readily dis- tinguished from lipoxygenase activity because: (a) they are quite unspecific; (b) they are autocatalytic; and (c) they yield oxidation products which are not optically active.
The catalytic action of metals and their compounds has been re- viewed by Ingold (1962). Examples of simple hematin compounds acting as oxidative catalysts are quoted by Lemberg and Legge (1949); the hematin compound is destroyed, indicating that this is a peroxida- tive reaction. Tappel (1962) states that all naturally occurring hematin compounds catalyze the oxidation of unsaturated lipids and other olefins.
The hematin compounds hemoglobin, myoglobin, hemin, and cytochrome c all have similar catalytic activities (Tappel, 1955). Hematin catalysis appears to be unspecific, requiring only an un- saturated compound which forms a peroxide. The rate of oxidation is proportional to the square root of the catalyst concentration. Hematin-catalyzed linoleate oxidation has a relatively low activation energy of 3 to 5 Kcal/mole, of the same order as lipoxygenase catalysis. The occurrence of cytochrome systems in vegetable tissue, similar to the cytochromes a, b, and c of animal tissue, was demonstrated by Lundegardh (1954), and Butler and Baker (1965) have found an active ferri-hematin compound in peanuts. Peroxidase and catalase, both containing hematin prosthetic groups, are widespread in vegetable tissue, so that this oxidative pathway could be of considerable im-
28 F. AYLWARD AND D. R. HAISMAN
portance. Blain and Styles (1961) attempted to elucidate the function of hematin compounds in soya extracts in p-carotene destruction. They concluded that carotene was destroyed by a coupled oxidation using linoleate hydroperoxide and catalyzed by hematin compounds. In the absence of p-carotene, cytochrome c promoted the formation of conjugated diene hydroperoxides. The relative concentrations of catalyst and fatty acid may be critical in this process. Lewis and Wills (1963) reported the inhibition of autoxidation by high concentrations of hematin proteins, which catalyzed peroxidation within a certain concentration range.
D. DECOMPOSITION OF HYDROPEROXIDES
As indicated above, hydroperoxides can be formed either through the action of lipoxygenase or by autoxidation. Lea (1962) outlined decomposition routes of hydroperoxides (Fig. 3), and Keeney (1962) showed how aldehydes, alcohols, ketones, epoxides, and esters are produced by free-radical chain reactions. Free radicals have been detected during the action of soybean lipoxygenase (Walker, 1963), and in lyophilized lipid-rich foods in the presence of oxygen or after heating to 100C (Munday et al., 1962). The production of dec-1-yne, a compound with a potent off-flavor, together with dec-1-ene and n-decanol during the autoxidation of soybean and cottonseed oils has
oxidation I Hydroperoxide
further oxidation, further oxidation, polymerization,
or fission / coupled oxidations Aldehydes, acids, ketones, Destruction of vitamins,
pigments, and other hydroxy- and epoxy- compounds, polymers constituents
various secondary reactions
causing "Off" odors and flavors and
FIG. 3. Formation and decomposition of hydroperoxides. Formation: catalyzed by lipoxygenase, oxidative enzymes, hemes, pigments, and metals and their salts. De- composition: catalyzed by metals, hemes, and oxidative enzymes.
PLANT-TISSUE OXIDATION SYSTEMS 29
also been reported (Smouse et al., 1965). The decomposition can be accelerated by heat. Lea and Hobson-Frohock (1965), using autoxi- dized sunflower and linseed oils, showed that Iinolenate peroxides decomposed more readily than linoleate peroxides under the action of heat and produced a higher proportion of volatile carbonyl compounds.
Apart from the fatty acids, many other substances, such as pigments, vitamins, and proteins, are susceptible to free-radical action once this is initiated. Thus oxygen-labile compounds such as vitamin A and p-carotene are readily cooxidized, and amino acids such as histidine, serine, cystine, and methionine can be destroyed. Glycosides and aglycones have been found to break the chain reaction in the heme- catalyzed oxidation of lipids (Pratt and Watts, 1964; Pratt, 1965). Mapson and Moustafa (1955) demonstrated the coupled oxidation of glutathione in the presence of linoleic acid by a lipoxygenase from pea seeds.
The decomposition of hydroperoxides into free radicals is catalyzed by many substances, and, once more, heavy metals and hematin com- pounds constitute an important catalytic group. Mikhlin and Brono- vitskaya (1948) observed that cytochrome c peroxidase could utilize the hydroperoxides produced by the action of lipoxygenase on lino- leic acid. Maier and Tappel (1959a) measured the comparative catalytic activity of hematins and other metal catalysts in decomposing linoleate hydroperoxide, with results shown in TabIe IV. They also (1959b) examined the products of the heme-catalyzed oxidation of unsaturated fatty acids, and found the concentrations of catalyst and substrate to be critical.
LINOLEATE HYDROPEROXIDE CATALYSIS OF DECOMPOSITION OF
Catalvst Rate constant
Catalase 36 Hemoglobin 29 Hematin 4.8 Cytochrome c 1.7 Peroxidase 0.22 Ferric triethylenc tetramine 0.45 Manganic protoporphyrin 0.17
30 F. AYLWARD AND D . R. HAISMAN
Banks et al. (1961) studied inhibition of the decomposition of methyl linoleate hydroperoxide by different concentrations of cyto- chrome c. OBrien (1966, 1967) examined the kinetics of the reaction between linoleic acid hydroperoxide and cytochrome c and other heme catalysts at various pH values, and postulated that the hemo- protein coordination bonds are loosened to expose the hematin ring.
The mechanism of destruction of carotenoids is still obscure. It has been attributed to the action of free radicals produced during the chain oxidation of fats, but the oxidative bleaching of carotene in sugar-beet chloroplasts appears to be independent of the peroxidation of the unsaturated fats present (Lea, 1961b). Dahle (1965) suggested that lipoxygenase activity was a major factor in carotene destruction in the milling of semolina, but found that the destruction was de- pendent on the concentration of free fatty acids, which are the prin- cipal substrate for the lipoxygenase. Friend and Nakayama (1959) measured the destruction of carotenoids in the chloroplasts of dif- ferent leaves and postulated the existence of two differently located enzyme systems. Friend and Mayer (1959) found that the action of chloroplasts on crocin (the glycoside of crocetin, a polyene dicar- boxylic acid) was due to aerobic oxidation catalyzed by a metallo- protein. Reviewing the oxidation of carotenoids in green plant tissue, Friend (1961) noted the loss of p-carotene from frozen un- blanched spinach and examined degradation mechanisms. The coupled oxidation of &carotene with linoleate was catalyzed by lipoxygenase and ferrous phthalocyanin; epoxides, hranoxides, and conjugated polyene aldehydes were produced. Friend concluded that two different carotenoid-destroying enzyme systems were operating, with pH optima of 4.8 and 7.5. Since one system was inhibited by cyanide, it probably included a hemoprotein.
There is some evidence that hydroperoxides can be enzymically decomposed (e.g., Mikhlin and Bronovitskaya, 1948). Gini and Koch (1961) found that soya-flour extracts contained a heat-labile factor which accelerated the breakdown of lipoxygenase-formed hydro- peroxides and had many of the properties of a peroxidase. It was less heat-stable than lipoxygenase, had an optimum pH of 8 to 9, and was activated by storage at 4C.
Frankel (1962) reviewed the properties of various hydroperoxides, including their isolation by molecular distillation, for example, and their characterization. Fat hydroperoxides can be relatively stable substances that, when formed, can persist for relatively long storage periods. This observation may provide an explanation of delayed changes in food quality during storage (see Section IV,F).
PLANT-TISSUE OXIDATION SYSTEMS 31
E . ANTIOXIDANTS
Extensive studies have been made of the use of antioxidants in preserving edible oils and fats (Tappel, 1961), but their effect on de- teriorative changes in preserved vegetables has not been fully ex- plored. Mention has already been made of various inhibitors of lipoxygenase activity; similar compounds effectively inhibit the heme-catalyzed oxidation of unsaturated fatty acids (Tappel, 1954). The effect of the naturally occurring inhibitors, particularly cy-toco- pherol, on oxidative changes during the storage of plant materials may have considerable significance, particularly since the level of vitamin E in different organs of the plant is known to fluctuate during growth and according to season (Sironval and El Tannir-Lomba, 1960).
Rhee and Watts (1966b) investigated the effect of various additives on lipoxygenase activity in different systems and found that propyl gallate, turnip-green extract, and sodium tripolyphosphate retarded the oxidation of pea lipids. They also found evidence (1966~) of a certain amount of natural antioxidant activity in plant systems. Pratt (1965) attributed significant antioxidant activity to the flavone glyco- sides and cinnamic acid derivatives in onions, peppers, and potato skins. The presence of water inhibits oxidation in freeze-dried ma- terial, and this has been demonstrated in the autoxidation of methyl linoleate with and without metal catalysis. Amino acids were also effective in prolonging the induction period and reducing the rate of oxidation (Maloney et al., 1966; Labuza et al., 1966; Karel et al., 1966).
I?. LIPID OXIDATION IN RELATION TO FOOD QUALITY
Some instances of food deterioration during storage can be ex- plained by assuming that hydroperoxides are formed before or during processing and then decompose by a free-radical path, gradually producing rancid odors and flavors. As already mentioned, some peroxides have been shown to be relatively stable compounds and may not decompose for some weeks or months (depending, perhaps, on the diffusion of a particular catalyst). Consequently, if this theory is correct, eventual deterioration of quality may be long delayed.
Koch (1962) showed that flavor deterioration in precooked dehy- drated beans was due to lipoxygenase activity while the beans were soaking, prior to cooking. It has been postulated that peroxy free radicals, either produced by lipoxygenase or generated by heat processing, propagate chain reactions that degrade chlorophyll and
32 F. AYLWARD A N D D. R. HAISMAN
pheophytin (C.S.I.R.O., 1962- 1964). This theory accounts for the deleterious effect of both over- and underblanching. The same re- port noted, however, that free fatty acid, peroxide, or diene content could not be correlated with off-flavor development. In fuller publi- cations, Walker (1964a,b) showed that the oxidative degradation of chlorophyll and pheophytin in frozen French beans occurred only after 12 months storage and was coincident with an increase in fat peroxide. He thought that the time lag could be attributed to a natural antioxidant which slowly became exhausted.
Wagenknecht and Lee (1958) have examined the part played by lipids in the deterioration of frozen peas. They showed that the addi- tion of lipase and lipoxygenase preparations to blanched peas caused off-flavors and chlorophyll breakdown. Lipids extracted from frozen- stored unblanched peas contained a high proportion of unsaturated carbonyl compounds not present in fresh or cold-stored blanched peas (Lee, 1958). They were able to isolate from peas a lipase which ef- fectively degraded chlorophyll to pheophytin (Wagenknecht et al., 1958). Using enzymes isolated from fresh peas, they showed that the addition of these enzymes to blanched peas led to the degradation of chlorophyll and produced some flavor changes during storage (Lee and Wagenknecht, 1958). They concluded that the mechanism of off- flavor production during frozen-storage was complex and that several enzymes were involved, including lipase and lipoxygenase, acting in sequence.
In investigations of underblanched corn on the cob, Wagenknecht (1959) detected residual lipoxygenase activity which appeared to be partly responsible for off-flavor production. Lee and Mattick (1961) showed a considerable breakdown of phospholipids and losses of unsaturated fatty acids from the triglycerides in unblanched peas stored one year at -17.8"C. Pendlington (1962) presented evidence for lipid breakdown, with a corresponding increase in choline and phosphate, in unblanched peas, but concluded that the mechanism of off-flavor production remained obscure.
In further studies of frozen unblanched peas, Bengtsson and Bosund (1966) found that, between -20" and -5"C, the temperature co- efficients remained about the same for the rates of production of off- flavors and formation of free fatty acids, and that both were much lower than would be expected if the degradative changes were nonenzymic. Holden (1964) reported that the chlorophyll-bleaching activity of legume seed extracts is correlated with the action of lipoxy- genase and (1965) appears to arise from a chain reaction involving the
PLANT-TISSUE OXIDATION SYSTEMS 33
peroxidation of long-chain fatty acids which are subsequently de- composed by a heat-labile factor.
Lea and Parr (1961) described the off-flavors which arise during oxidative deterioration of the lipids in crude leaf protein, dividing them into two categories: polyunsaturated acids of galactoglycerides, or phospholipids which give fishy flavors; and carotenoids and chlorophylls, which produce violet or haylike odors. Some aspects of quality deterioration attributed to lipids may be non- enzymic in origin. Thus, Burton and McWeeny (1963) implicated phosphatides, such as lecithin, in nonenzymic browning reactions.
In contrast, experiments using the 2-thiobarbituric acid (TBA) test as a measure of lipid oxidation in frozen peas appeared to es- tablish that rancidity was not a primary cause of flavor deteriora- tion (Rhee and Watts, 1966~). The TBA test depends on the formation of a red pigment when thiobarbituric acid reacts with oxidized fat. In many cases the intensity of the pigment has been found to be propor- tional to the oxidative degradation which has taken place. The re- active material is malonaldehyde, which is produced from the hydro- peroxides of unsaturated fatty acids containing three or more methyl- ene-interrupted double bonds (Dahle et al., 1962). Oleic and linoleic acid do not react, although it is reported that malonaldehyde may be a secondary oxidation product in other lipid oxidations (Lillard and Day, 1964). It is unlikely that malonaldehyde exists in the free state in oxidized products; it is probably combined with other food com- ponents, particularly some proteins (Crawford et al., 1967).
Most workers have agreed on the necessity of using acid extraction of the food material to obtain maximum color development. Like other reactive intermediates, malonaldehyde is itself destroyed during the later stages of oxidation, and, in view of this and the various side reactions which can occur (Tarladgis et al., 1962), the results obtained with the test must be treated cautiously.
Rhee and Watts (1966a) used the TBA test on plant materials and suggested that a comparison of the TBA values of tissues blended with and without acid (to inactivate lipoxygenase) might provide a useful measure of the lipid oxidation potential. As already men- tioned, when the test was applied to stored frozen blackeye peas, whether raw, blanched, or cooked, the TBA numbers were all less than the threshold of 1.0 established for rancid odors in animal products, even though the raw peas were judged unpalatable by sen- sory methods (Rhee and Watts, 1966~). Rancid peas were found to have a TBA number of 7.8.
34 F. AYLWARD AND D. R. HAISMAN
V. THERMAL AND OTHER ENVIRONMENTAL FACTORS MODIFYING ENZYME ACTIVITY
The activity of an enzyme, whether considered in its natural en- vironment in the living cell or in isolated tissues or extracts, is de- pendent on many factors, and much work has been carried out on activating and inhibiting agents in various enzyme systems, as well as on the effects of changes of pH, temperature, and other conditions.
Some of the factors that have been studied are summarized in Table V. I t is well known that many enzymes consist of two linked com- ponents, the protein moiety (or apoenzyme) and a prosthetic group (coenzyme). Destruction or modification of activity can be brought about by dissociation of this complex or by changes in either com- ponent. On this basis it is to be expected that enzyme activity will be lost through conditions that lead to the destruction of proteins. The prosthetic groups are all molecules of relatively low molecular weight; many of the oxidizing enzymes embody metallic (e.g., Fe or Cu) compounds in the prosthetic group, and in such cases reagents which will react with these metals would be expected to reduce activity.
In view of the importance of thermal processes in food preserva- tion, the effects of heating and cooling on plant enzymes will be con- sidered in some detail before discussing the other factors outlined in Table V.
TABLE V hfODIFICATION OF ENZYME ACTIVITY
1. Methods of inactiuation (a) Destruction or dissociation of protein-prosthetic group
(apoenzyme/coenzyme) complex leaving complex
(c) Changes in prosthetic group (coenzyme) 1 intact (b) Changes in protein (apoenzyme) (a) Temperature
(c) Water (d) Sugar and related compounds (e) Multiple forms (0
2. Factors modifying actiuity
Adsorbents of different types (inert substrates, active substrates)
(g) Action of specific inhibitors, e.g., agents affecting the prosthetic groups (coenzyme)
PLANT-TISSUE OXIDATION SYSTEMS 35
A. THERMAL INACTIVATION - GENERAL PRINCIPLES
1 . Effect of Temperature on Enzyme Activity
In any enzyme system, two independent processes are simul- taneously accelerated by an increase in temperature: the catalyzed reaction; and thermal inactivation of the enzyme. The rate of the catalyzed reaction normally follows the general pattern of other chemical reactions; the temperature coefficient, Qlo, defined as the factor by which the velocity is increased on raising the temperature by 10"C, is normally between 1 and 2 (Dixon and Webb, 1958). The temperature coefficient for enzyme inactivation is usually very much higher (see Section V,B,7).
As a consequence of these two different temperature coefficients, an optimum temperature for the reaction is observed. Below the op- timum, changes in temperature have the greater effect on the cata- lyzed reaction, whereas above it the inactivation of the enzyme becomes the predominant factor.
Both coefficients are highly dependent on the environment of the system (e.g., the ionic strength and pH). The optimum temperature for most enzyme reactions usually lies between 30" and 50C.
2. Temperature Coefficients of Reactions
As noted above, the temperature coefficients for enzyme reactions usually lie between 1 and 2, but exact measurements have been made on comparatively few systems. Sizer and Josephson (1942) measured the kinetics of three enzyme systems over the range -70" to +50"C, and found that the rates conformed to the Arrhenius equation:
d In k' - E dT R P
(where k' is the specific rate constant, T is the temperature in degrees Kelvin, R is the gas constant, and E is the energy of activation) except for a discontinuity at the melting point of each system. Abnormal temperature coefficients were obtained below the melting point; lipase had a Q," of 1.5 in the range 0" to 50"C, changing to 26 in the range -70" to 0C. Maier et al. (1955) studied phosphatase- and peroxidase-catalyzed reactions at subzero temperatures, and also found pronounced deviations from the Arrhenius equation. This point is taken up later (Section V,C).
36 F. AYLWARD AND D. R. HAISMAN
3. Znactivation of Enzymes by Heat
Like all proteins, enzymes have a definite three-dimensional structure, maintained by a multitude of secondary bonds (hydrogen bonds and London dispersion forces), which is easily disrupted by thermal or chemical attack. The processes of denaturation of proteins and inactivation of enzymes appear to comprise a structural break- down of this sort. Breaking bonds, even weak secondary bonds, needs energy, and only those molecules possessing energy much above the average can rearrange to the denatured or inactive conformation. At a fixed temperature, the Maxwell-Boltzmann distribution law shows that, for any random assembly of molecules, the number, ni, having an energy in excess of a certain value is proportional to the total number of molecules,
(2) !!! = e-EIRT n
Hence, the rate of denaturation or inactivation is proportional to the concentration of the unaltered protein or enzyme. Let this be c ; then
-dc -- - kc d t (3)
Integrating between times t , and tz
In 5 = k( t2 - t , ) (4) C2
If x is the fraction of activity remaining after heating for t min at temperature T
2.3 log x = kt (5 ) and k , the specific rate constant, can be obtained from a plot of log x against t .
4 . Practical Units
The thermal inactivation of enzymes is in many ways analogous to the thermal destruction of bacterial spores, and, when estimating degrees of enzyme inactivation, food technologists have found it convenient to adopt the conventions and mathematical treatments introduced by Ball (1923) for the evaluation of sterilizing processes. Ball (1943) applied these methods in a study of phosphatase in- activation during the pasteurization of milk.
PLANT-TISSUE OXIDATION SYSTEMS 37
The decimal reduction time, D, defined as the time taken for a 90 % decrease in the activity of the enzyme, can be obtained from the relationship
D = 2.3/k'
D values provide a practical measure of the heat resistance of an enzyme system at a particular temperature. A related practical parameter that is used more widely is the FT value, the time required to reduce the activity to a desired level, again at a particular tempera- ture. The chosen level of activity usually corresponds to the detection limit of the enzyme system in question (say 1 % or 0.1 % of the original activity ).
A complete description of the heat resistance of an enzyme system requires that we know not only the rate of inactivation at one tempera- ture, but also the way in which this rate varies as the temperature is changed. Experimentally, for both enzymes and bacterial spores it is found that, over short temperature ranges, plots of the logarithms of the rates of destruction against either the temperature or the recip- rocal of the absolute temperature approximate to straight lines (Gillespy, 1948).
(7) log D = a - ( t F / z )
log D = (M/TK) - b (8)
where a, z, M , and b are constants. The former equation is very useful in practice, and defines the factor
z as the temperature change in degrees Fahrenheit necessary to pro- duce a tenfold change in the rate of inactivation. Thus, the parameters D or F , together with z, completely describe the heat inactivation of an enzyme system over a restricted range of temperature.
5. Thermodynamics of lnactivation
Although Eq. (7) has practical value, it cannot be justified theo- retically. On the other hand, Eq. (8), which is of the same form as the Arrhenius equation [Eq. (l)], can be derived from strict postulates and, properly treated, can yield thermodynamic constants which illumine the mechanism of inactivation. Unfortunately, in practice the full potential of this treatment is seldom realized, because of the difficulty of applying the necessary restrictions to the system.
The earliest relationship between rate of inactivation and tempera-
38 F. AYLWARD A N D D. R. HAISMAN
ture was derived from the collision theory applied to the conversion of normal molecules to the activated transition form, which preceded the final structural rearrangement:
where A came to be identified with the energy required to activate the molecule, and C was a constant embodying frequency and probability factors. In practice, both A and C were found to vary with tempera- ture, and various modifications have been suggested to produce a better fit between the theoretical equation and practical data. The advent of statistical mechanical treatment of the problem in terms of absolute reaction rates (Stearn, 1938; Eyring and Stearn, 1939), however, showed that the collision theory amounted to a special case of a more general equation:
k' = C e - A / R T
kT -F*IRT h
k ' = K - e
where K is a transmission coefficient (usually taken as unity), k is the Boltzmann constant, h is Planck's constant, F' is the Gibbs free energy of activation, K" is the equilibrium constant between acti- vated complex and reactant, and y" and yo are activity coefficients.
Thus, discrepancies between experimental data and theory could always be attributed to neglect of the correct activity coefficients.
Using the transformation
AF' = AH' - T AS' (11) (with H" and S' denoting enthalpy and entropy of activation respec- tively) Eq. (10) can be written
from which the usual Arrhenius equation (1) can be derived. The free energy, AF', the enthalpy, AH', and the entropy, AS', of activa- tion can be calculated from the activation energy, AE', and the rate constant, k'. The values obtained will depend on the standard states which are adopted. Provided the inactivation rates are measured with due regard to the activity coefficients of the reactants and the acti- vated complex, they will vary only with respect to temperature and pressure. As already mentioned, these requirements are most often
PLANT-TISSUE OXIDATION SYSTEMS 39
experimentally unrealizable, so that the thermodynamic constants refer to the unspecified standard states under which the measure- ments happen to be made.
6. Znterconversion of Parameters
In the literature, heat inactivation data have been expressed in a variety of forms. The following relationships have been used in comparing results from different sources:
D = 2.303Ik' (6) 10E"
2.303 X 1.987 X T 2 Qlo = antilog
where T is the average temperature in degrees Kelvin, and E" is in calories.
T2 - 8.237
18 z=-- log Qio E
B. THERMAL INACTIVATION OF OXIDIZING ENZYMES - EXPERIMENTAL DATA
1 . General Considerations
The earlier literature on the heat resistance of different enzymes has been surveyed by McConnell (1956) and Leeson (1957). Both workers concluded that many of the published observations were of limited value in that experiments were often confined to one tempera- ture or were only semiquantitative, and the modifying action of pH and other environmental conditions were usually not taken into account.
McConnell tabulated all the data from which thermal inactivation curves could be drawn, recalculating, where necessary, the constants in terms of F7. and z values to facilitate comparison. The enzymes studied included the pectic enzymes, peroxidase, o-diphenol oxidase, catalase, ascorbate oxidase, and phosphatase. In vegetables, peroxi- dase was found to be by far the most heat-resistant enzyme. In fruits, where conditions are more acid, peroxidase was less stable, but still the most difficult enzyme to inactivate; ascorbate oxidase displayed almost as great a resistance to heat under these conditions. Pectic enzymes required, on average, long periods for destruction at moder- ate temperatures, but the rate of inactivation increased rapidly as
40 F. AYLWARD A N D D. R. HAISMAN
the temperature was raised. Both McConnell and Leeson emphasized that at high temperatures, processes adequate for sterilization pur- poses may not wholly inactivate the more heat-resistant enzymes.
A selection of results from more recent experimental work on oxi- dizing enzymes is presented below. The information is presented first in terms of individual enzymes, and secondly for different systems. The empirical parameters D, F , and z have been chosen to express the results. Thermodynamic data, which must be regarded with greater caution (for the reasons already outlined), are presented in a later section.
Many workers have studied the thermal inactivation of peroxidase under conditions corresponding, on the one hand, to industrial processes and, on the other, to precise examinations of highly purified systems. Thus, Lopez et ul. (1959) investigated the effect of blanching time (at 100C) on peroxidase and catalase activity in peas, and found that 70 to 100% of the activity was destroyed in 1 to 3 minutes. Baker and Goldblith (1961) investigated the effect of both heat and ionizing radiations on peroxidase activity in green beans. Ionizing energy had a measurable effect on enzymic activity, but this was insignificant compared with the effect of heat. Kyzlink and Chytra (1959) deter- mined the thermal death rates of peroxidase in fourteen kinds of fruit and vegetables, and found large variations in the heat resistance of the enzyme systems, particularly among fruits. They emphasized the importance of the pH of the system, which may in fact be the only fac- tor determining the thermal resistance of enzymes during the ripening of fruits. They found that the heat resistance of the enzyme was greater when the enzyme concentration was high. Addition of salt often de- creased the heat resistance of the system.
Wilder (1962) also found that the ionic strength of the medium had a profound effect on the heat inactivation of purified peroxidase in buffered solutions. Joffe and Ball (1962) measured the thermal in- activation of pure horseradish peroxidase precisely, and summarized the results of previous workers in a comparative survey of heat- inactivation data. This information is presented in Table VI, together with some later work, they did not include. F values refer to the heating necessary to reduce the enzyme activity to below the limits of detection. The exceptional heat resistance of peroxidase is evident from the fact that nearly all the measurements have been made at temperatures above the boiling point of water. The great variations in
TABLE VI THE THERMAL INACTIVATION OF PEROXrDAsE
Highest experimental temperature z FZSO Fsoo Dzoo Dzm
Medium ( O F ) ( O F ) (min) (min) (min) (min) Investigators
HRP in PO, buffer HRP in PO, buffer Green beans, aqueous extract Turnips Yellow turnips White turnips Broccoli Green beans Peas Peas Sweet corn, whole-
kernel heat-labile fraction heat-stable fraction heat-stable fraction
Sweet maize, aqueous extract
Sweet maize, whole- kernel
302 302 250 270 300 300 300 300 300 260
200 200 290 270
49.8 64.3 25.7 47.0 3.0 46.0 11.3 72.5 3.1 57.5 2.1 63.0 1.4 86.0 0.6 48.0 6.0 52.0 7.7
87.0 54.0 65.0 40.0 1.78
310 86.0 0.26 270 7.0 310 2.1
7.5 ] Joffe and Ball (1962)
0.4 1.8 I Zoueil and Esselen (1959) Esselen (unpubl.) quoted by Joffe and Ball (1962)
Farkas et al. (1956) Guyer and Holmquist (1954)
0.2 Yamamoto et al. (1962) 11.0
Vetter et al. (1958) i
42 F. AYLWARD A N D D . R. HAISMAN
F and D values are almost certainly a consequence of differences in pH, ionic strength, cellular materials, and assay and heating tech- niques. The temperature coefficient, z, is affected less by such vicis- situdes, so there is closer agreement about its value, which on average is about 60F. An investigation by Yamamoto and co-workers (1962) is of great interest since they demonstrated the existence of a peroxi- dase fraction (about 5% of the total activity) with abnormal thermal stability. The rate of inactivation of this heat-resistant form was fifty times lower than that of the heat-labile fraction.
The thermal lability of catalase is well established, and there have been several semiquantitative determinations of its inactivation by different blanching treatments (e.g., Lopez et aZ., 1959), showing that its heat resistance is low. Basic studies of the inactivation process have been confined principally to catalases isolated from animal and microbiological sources, and there are indications that plant catalases may show some differences in behavior. Frazer and Kaplan (1955), studying yeast catalase, found marked differences in behavior be- tween the native (Lea, in situ) enzyme and crystalline preparations or aqueous extracts. Unaltered catalase in the yeast cell was less active but more heat-resistant than extracted or altered catalase. Deutsch (1951) found that the heat inactivation of crystalline horse erythrocyte catalase did not follow first-order kinetics, a fact he attributed to the presence of various species of catalase of differing thermal lability.
In a basic investigation of a plant catalase (from spinach), Sapers and Nickerson (1962b) found that the rate of inactivation by heat was not first-order. They postulated the presence of a heat-labile inhibitor. Kinetic constants derived from the data of Sapers and Nickerson are shown in Table VII. All investigators are agreed on the relative ease with which catalase is inactivated.
4 . Lipoxygenase
There has been only one basic investigation of the inactivation of lipoxygenase by heat (Farkas and Goldblith, 1962). The enzyme, a purified soybean lipoxygenase, showed a typical protein-inactivation response to heat treatment, varying with the pH of the system. The enzyme was most stable between pH 5 and pH 7. Heat resistance was also enhanced up to tenfold by the addition of 20% pea solids to the system. Under the same conditions the enzyme was quite sensitive to
PLANT-TISSUE OXIDATION SYSTEMS 43
ionizing radiation. Kinetic constants calculated from the heat in- activation data are shown in Table VIII.
5. o-Dipheno2 Oxidase
Leeson (1957) compiled miscellaneous data from numerous investi- gations on thermal inactivation of the phenol oxidases. Of particular interest were results of a comprehensive investigation by Dimick et uZ. (1951) on the effects of time and temperature on the activity of the enzyme in fruit purbes. A D value of 3.5 min at 75C, and a z value of about 10, were obtained with pear puree. The thermal stability of the enzyme was dependent on pH, but, surprisingly, the pH for maximum thermal stability varied widely between different fruits (apricots 3.9, grapes 4.5, pears 6.0, and apples 6.2). Studying the properties of the oxidase from apples, Walker (1964a) found that in solution at pH 5.0, where it displayed maximum activity, the enzyme had a half-life of 12 min at 70C. This corresponds to a decimal reduction time of 50 min, in reasonable agreement with previous work.
TABLE VII THE THERMAL INACTIVATION OF CATALASE
Highest experimental Temperature for temperature z D determination of D
Medium (OF) (OF) (min) (F)
Spinach catalase purified in buffer 140 - ca 2 140
Spinach extract 140 37 22-31 140 Spinach extract 149 15 ca 1 149
TABLE VIII THE THERMAL INACTIVATION OF LIPOXYGENASE
Highest experimental Temperature for temperature z D determination of D
Medium (F) (F) (min) (F)
McIlvaine buffer, pH 7.0 167 9.2 44 149
+ 20% pea solids 158 15.7 85 154 + 20% pea solids 171 6.1 12 163
44 F. AYLWARD AND D. R. HAISMAN
6. Other Systems
Some of the experimental work on nonoxidizing plant enzyme sys- tems will be noted for comparative purposes. The heat resistance of the pectolytic enzymes has been investigated extensively, particularly in connection with the firmness of fruits and gels and the manufacture of fruit juices. Quantitative data on heat resistance are limited, but the pectolytic enzymes, on the whole, appear to be labile and readily inactivated at temperatures over 70C. Some peculiarities in behavior have been reported, however. Kohn et al. (1953) examined the de- struction of pectic enzymes used for the clarification of apple juice, and found that polygalacturonase was rather more heat-stable than pectinesterase. The F values were 7.2 and 255 min at 65.5"C, and associated z values were 11.3 and 11.5. McColloch and Kertesz (1948) isolated a polygalacturonase from tomatoes which appeared unusually stable, retaining 5% of its original activity even after boiling for 1 hour. The polygalacturonase in papaya also appears to possess a higher degree of heat resistance (Aung and Ross, 1965). The decimal reduction time at 82.2"C was found to be 10 min (pH 4.2), and the z value 11. When heat processes based on these data were evaluated, however, residual activities were higher than expected, indicating the existence of a more heat-resistant form.
7. Comparative Thermal Stability of Diferent Enzymes
Comparative values for the heat-inactivation constants from the sources already quoted are shown in Table IX. D values have been extrapolated to 176F to facilitate comparison. Since z values are constant over only short temperature ranges, the estimates of D must be viewed with caution. The results substantiate data tabulated by McConnell (1956) and, once more, illustrate the exceptional position of peroxidase.
8. Thermodynamic Constants for Thermal Znactivation
As explained in the derivation of the thermodynamic properties relating to inactivation, proper restrictions are seldom applied to the experimental system. In consequence, although it is permissible to compare the values for any property taken from one experimental set (in which standard states, although undefined, are probably con- stant), a comparison of results obtained in different systems may be quite misleading. Nevertheless, inspection of data available in the
PLANT-TISSUE OXIDATION SYSTEMS 45
TABLE IX COMPARATIVE THERMAL STABILITY OF DIFFERENT ENZYMES
z D, 16 Enzyme ( O F ) (min)
Peroxidase (horseradish) 49.8 232 Peroxidase (green beans) 47.0 15
Peroxidase (sweet corn) 54.0 30 Peroxidase (turnips) 46.0 73
Catalase (spinach extract) 15.0 0.02 Lipoxygenase (+ pea solids) 15.7 0.09 o-Diphenoloxidase (pear) 10 0.82 Polygalacturonase (papaya) 11 23
literature does reveal certain salient features which are at least qualitatively significant.
The changes in total and free energy, heat content, and entropy which are usually calculated refer to the transition of the enzyme molecule from the normal to the activated state. Thus they apply only to the first part of the denaturing transformation. The whole process has been studied only in a reversible system (e.g., the de- naturation of trypsin), in which an equilibrium constant for the com- plete reaction can be calculated. A selection of calculated thermo- dynamic properties for the inactivation of various enzymes is shown in Table X.
One of the most striking points about the inactivation of enzymes and the denaturation of proteins by heat is the very large activation energy involved. Chemical processes requiring the same apparent energy of activation proceed at an infinitesimal rate below 100C; for instance, the rate of depolymerization of dianthracene in phenetole, activation energy 39.4 kcal, is about 3 x lo-'* (sec-I) (Moelwyn- Hughes, 1933). The comparatively rapid rate of enzyme inactivation is explained by the large increase in entropy involved, which counter- balances the high activation energy. The entropy of activation is visualized as the result of the fission of many secondary bonds in the protein, so that the activated molecule has a greater freedom in its spatial configuration.
As might be expected, peroxidase proves an anomaly in this concept of thermal inactivation. Results from two independent investigations corroborate in showing that, for this molecule, the entropy of activa- tion is negative; other results (e.g., Zoueil and Esselen, 1959) indicate a low positive entropy when a negative value is not obtained. On balance, it appears that the peroxidase molecule is more rigid in the
TABLE X THERMODYNAMICS OF INACTIVATION
Temperature Inactivation rate A H* A Fo A So Enzyme ("C) (sec-' x 104) (cal/mole) (caVmole) (caVmole "K) Investigators
Peroxidase (horseradish) 85 2.69 24,300 27,000 -7.39 Joffe and Ball
Peroxidase (sweet corn) 85 34.9 15,700 24,900 - 26.0 Yamamoto et az. (1962)
Catalase (spinach) 60 14.5 60,900 23,900 + 111 Sapers and Nickerson (1962b)
Lipoxygenase (soybean) 65 8.71 101,000 22,900 + 242 Farkas and
o-Diphenol oxidase (pear) 80 59.1 100,000 24300 + 212 Dimick et nZ. (1951)
PLANT-TISSUE OXIDATION SYSTEMS 47
activated state, which would indicate an unusual mechanism of inactivation in this case.
c . ENZYME ACTION A T LOW TEMPERATURES
Although it is normally assumed that the rate of enzyme action diminishes rapidly on cooling, there have been several reports of the activation of tissue enzymes by freezing. Kiermeier (1947) found that the activities of catalase and lipase were increased after freezing and thawing in the presence of substrate, although repeating the opera- tion had an adverse effect. Lea (1961a) also mentions the activation of lipases by freezing. Peroxidase activity in kohlrabi was stimulated eightfold by freezing (Kiermeier, 1951). It has been suggested that the activation during freezing is due to the liberation of endoenzymes from associated inhibitory constituents (Kiermeier, 1951; Sukhorokov and Barkovskaya, 1953).
Some enzymic reactions appear to be accelerated by freezing. Thus, the transfer reaction between amino acid esters and hydroxylamine, catalyzed by trypsin, was twice as rapid at -23C as at +1"C (Grant and Alburn, 1966), although in one case the pathway of the reaction changed. Such behavior is exceptional, for enzyme activity in frozen media is generally drastically reduced, being comparable to that in very concentrated solutions or dried tissues (Joslyn, 1951; Tappel, 1966).
The important effect of freezing may be that the decrease in activity is not the same for all enzyme systems, so that interrelated reactions get out of balance, allowing particular intermediates to accumulate. Tappel and others (1953) found that lipoxygenase near the freezing point displayed relatively higher activity than did other systems, although the activity was sharply reduced (to 1 % of the liquid system) upon actual freezing.
Some enzymes are partially inactivated at low temperatures. The inactivation observed with phosphatase and peroxidase is reversible and is thought to be due to the formation of internal hydrogen bonds and associated conformational changes (Maier et al., 1955).
D. EFFECTS OF PH AND IONIC STRENGTH
In solution, the secondary structure of enzymes is highly dependent on pH and ionic strength, and examples of the effect of these variables on heat inactivation have already been quoted (Farkas and Goldblith, 1962; Sapers and Nickerson, 196213). Nakayama and Kono (1957)
48 F. AYLWARD AND D. R. HAISMAN
found that the rate of inactivation of sweet potato P-amylase increased as the buffer concentration was raised from 0.01 to 0.1 M and as the enzyme concentration was lowered over a fivefold range. The heat inactivation of glucose dehydrogenase is affected markedly by dimer- monomer conversion, which occurs in solutions of this enzyme. Since the equilibrium between dimer and monomer depends on pH and ionic strength, the stability of the enzyme can be increased by a factor of lo6 by adding sodium chloride and changing the pH from 7.5 to 6.5 (Sadoff et al., 1965).
E. EFFECTS OF WATER
Enzymic activity may persist in foods at quite low moisture levels (Acker, 1962; Blain, 1962). Peroxidase activity in solvent mixtures has been measured down to 12.5% water, and lipase activity in oats has been measurable at moisture levels between 6 and 12 %. Amylases were found to be active in the hydrolysis of glycogen in freeze-dried model systems containing meat enzymes at moisture levels of 3% or lower (Matheson, 1962).
It is established that enzymes, like other proteins, are much more stable (e.g., to heat treatment) when dry, but in practice much will depend on the amounts of residual moisture present.
The techniques of food preservation include not only dehydration by heat but methods (e.g., sugar and salt preservation) in which the effect is due in large part to reduction of the activity of water in the system.
Sugars and other substances (e.g., lyophilic colloids) have been shown to inhibit enzyme actions in some types of experiments, and to stabilize enzyme systems in others-for example, during heat treatment. Thus, Chang et al. (1965) found that sucrose inhibited pectin methylesterase activity in papaya, and Kiermeier and Kober- lein (1957) found that mono- and disaccharides could reduce the effect of heat treatment on the activity of catalase. In the presence of sugar, heating times had to be increased by factors up to 100, or temperatures raised 15"C, to inactivate the enzyme to the same degree. It seems likely that the action of sugar, at least in some cases, can be considered the equivalent of dehydration.
F. MULTIPLE MOLECULAR FORMS OF ENZYMES
The idea is not new that an enzyme may occur in different forms, and it has often been used to explain discrepancies in properties in
PLANT-TISSUE OXIDATION SYSTEMS 49
comparisons of enzyme preparations isolated from different sources. Interest in the concept was stimulated by the unequivocal demon- stration, in 1957, of different forms of lactate dehydrogenase, and this has led to the subsequent discovery of physically distinct forms of many of the common enzymes. The subject has been reviewed in a recent monograph (Wilkinson, 1965). The occurrence of multiple molecular forms is of both theoretical and practical importance. The different forms may display differences in substrate specificity and react differently to changes in pH or ionic strength and to heat.
The properties of the five isoenzymes of lactic dehydrogenase were studied in detail by Wilkinson (1965). He found differences not only in chemical composition and physical properties but also in thermal stability, substrate specificity, response to coenzyme analogs and susceptibility to inhibitors. Moreover, the relative proportions of the isoenzymes were found to vary between different tissues and between individuals.
The differences in lability between the isoenzymes have been used to devise a chemical test (Wroblewski and Gregory, 1961). Compar- isons of activities before and after heating to 57" and 65C provide approximate values for the relative proportions of labile, stable, and intermediate fractions.
Peroxidases have long been known to differ in properties according to source. Horseradish peroxidase, verdoperoxidase, lactoperoxidase, and cytochrome c peroxidase have all been obtained in crystalline form, and chromatographic and electrophoretic techniques have shown that many of the crystalline preparations comprise several peroxidases (Saunders et al., 1964). McCune (1961) obtained six active peroxidase fractions from corn. Siege1 and Galston (1966) separated iso-peroxidases in peas by starch gel electrophoresis, and found that they differed in their secondary reactions. Kon and whit- aker (1965) separated three peroxidases from fig latex, all more heat-stable than horseradish peroxidase.
The confusion surrounding the properties of various phenol oxi- dases can be attributed, in part at least, to the existence of many variant forms. Hare1 et al. (1965) isolated four distinct o-diphenol oxidases from the subcellular particles of apples, and postulated that each enzyme was bound to a specific site in the cell, where it fulfilled a particular physiological function. Heterogeneous forms of this enzyme have also been recently reported in broad beans (Robb et al., 1965), tea (Gregory and Bendall, 1966), and eggplant (Sakamura et al., 1966). There is some evidence for transmutation between different forms; Jolley and Mason (1965) observed changes in the
50 F. AYLWARD A N D D. R. HAISMAN
forms of phenol oxidase from mushrooms when the enzyme was incubated for a short time in dilute buffers within a pH range of 3.8 to 10.4.
Hultin and Levine (1963) isolated from bananas three forms of pectin methyl esterase, which differed in pH activity curves, response to surface-active agents, and differential temperature inactivation. One fraction could be completely inactivated by freezing to -30C for 3 days, but was comparatively stable at 0C and above. It appeared that the forms could be interconverted to some extent by treatment with sodium dodecyl sulfate (Hultin et al., 1966).
G. ADSORPTION OF ENZYMES ON NATURAL SUBSTRATES
Some of the variants in multiform enzymes may be cases where the enzyme is combined with another compound such as a protein, lipid, carbohydrate, or inorganic ion. Such a combination often greatly modifies the properties, especially the stability, of the enzyme.
Since enzyme inactivation is frequently due to disruption of the protein structure through the breaking and rearrangement of second- ary bonds, any compound or process which stabilizes the structure of the molecule will, to some extent, protect it against inactivation. Okunuki (1961), examining the relationship between the denaturation and inactivation of enzyme proteins, showed that inactivation, like denaturation, rendered enzymes more susceptible to attack by proteolytic enzymes. Taka-a-amylase was activated by calcium ions, which were bound to the enzyme molecule. Over a certain level, the bound calcium was found to protect the enzyme against heat inactiva- tion and proteinase attack.
London et al. (1958) related the potency of inhibitors of prostatic acid phosphatase to their structural affinities to the principal sub- strate, P-glycerophosphate. They found that inhibitors enjoying a multipoint attachment to the enzyme stabilized it against denatura- tion. Inhibitors such as chloride and sulfate, which made only single- point contact, had no effect on the stability of the enzyme.
Adsorption of enzymes also leads to increased stability. Lilly et al. (1965) observed that chymotrypsin, ficin, and ribonuclease had a greater heat resistance when chemically bound to modified celluloses. The stabilization of glucose oxidase-catalase preparations by mixing with methylcellulose has been patented (Scott, 1961). In its natural state, pectin methylesterase adsorbed on cell walls has been found to be very resistant to heat inactivation (McDonnell et al., 1945). In a like manner, the presence of excess substrate can protect an enzyme,
PLANT-TISSUE OXIDATION SYSTEMS 51
presumably because of the increased rigidity of the enzyme-substrate complex.
Taka-a-amylase was more heat resistant in the presence of starch and its hydrolysis products (Tonita and Kim, 1965), and the heat resistance of almond P-glucosidase was doubled in the presence of 0.2% or more of amygdalin, the primary substrate (Haisman and Knight, 1967). Enzymes are denatured when adsorbed at interfaces at low surface pressures (i.e., when the molecule is completely un- folded). At higher concentrations, allosteric effects may be observed, with the activity and stability depending on the conformation of the enzyme molecule (James and Augenstein, 1966). Frazer et al. (1955) found that catalase adsorbed at an oil-water interface was stabilized by lipids.
The general protective action of plant constituents associated with enzymes has often been demonstrated, although the responsible factors have not been identified. Examples have already been quoted of the protection of lipoxygenase by pea solids and of catalase by spinach extract. Frequent references have been made to the stabiliza- tion of pectin methylesterase by associated tissue components, e.g., in snap beans (van Buren et al., 1962) and in apple juice (Pollard and Kieser, 1951). Manolkides (1962) attributed the heat resistance of catalase and phosphatase in milk to their association with other milk proteins.
A striking example of the effect of associated constituents on the stability of enzymes has been observed with adenylate kinase, which controls the equilibrium between the adenosine phosphates:
ATP + AMP 2 ADP Colowick and Kalckar (1943) observed that this enzyme possessed an unusual stability and survived boiling with mineral acids and pre- cipitation with trichloroacetic acid. Bowen and Kenvin (1956), how- ever, found that purification of the enzyme drastically reduced its heat resistance.
The intrinsic stabilization of enzymes and metabolic systems against extremes of temperature is demonstrated in the vegetation of the tundra and the desert, and in thermophilic microorganisms. Langridge (1 963) reviewed the proposed biochemical mechanisms to account for the thermal stability of proteins in living organisms subject to extreme temperatures. Enzymes isolated from cacti have been found to be more heat-resistant than their analogs in temperate- zone plants. Theories involving adsorption on cellular surfaces and combination with other proteins and metal ions have also been ad-
52 F. AYLWARD A N D D. R. HAISMAN
vanced to account for cases of unusual stability. A special mechanism appears to operate in certain bacterial spores, where the heat resist- ance has been shown to depend on the presence of dipicolinic acid, a substance found in large quantities in resting spores of Bacillus species (Powell, 1953; Church and Halvorson, 1959). Dipicolinic acid also retards the denaturation of bovine serum albumin (Mishiro and Ochi, 1966).
There is indirect evidence for the existence of natural inhibitors, which may or may not have a stabilizing function, in association with many native enzymes. I t has often been observed that the enzymic activity in tissue extracts can be stimulated by the addition of surface- active or other agents, or by mild heat treatments. For example, the activity of NAD pyrophosphatase in trichloroacetic acid extracts of Proteus vulgaris was enormously enhanced by boiling the extract for two minutes in the presence of inorganic pyrophosphate (Swartz e t al., 1956). Similarly, o-diphenol oxidases, isolated from lower forms of animals or from the leaves of broad beans, have been activated by treatment with detergents, acetone, or urea or by heating to 60"-70"C (Swartz et al., 1956; Bailey, 1961; Kenten, 1958; Robb e t al., 1966). In other cases, oxidase activity has been stimulated by the action of unrelated enzymes; thus, pectic enzymes have been shown to induce increased phenol oxidase activity in injured or infected potato-tuber tissue (Tomiyama and Stahmann, 1964), and proteolytic enzymes produced a fourfold increase in the activity of o-diphenol oxidase from sugar-beet chloroplasts (Mayer, 1966). The activity of pectin- methylesterase in fruit has been found to increase after mild blanch- ing treatments (Hsu et al., 1965) or irradiation (Somogyi and Romani, 1964), and a thennolabile inhibitor of pectolytic enzymes has been reported in pear juices (Weurman, 1953). Lipoxygenase in pea seeds has been activated by the addition of certain alcohols (Mapson and Moustafa, 1955).
The explanation usually advanced for heat activation postulates some form of combination between a relatively stable enzyme and a labile inhibitor. The inhibitor has generally been assumed to be an associated protein. Swartz e t al. (1956) suggested that some enzymes may complex with RNA, since RNA breakdown products have been found in activated P-galactosidase systems. Proteins and pigments have been identified 'as phenoloxidase inhibitors in broad beans (Bailey, 1961) and mushrooms (Karkhanis and Frieden, 1961).
Tannins and other polyphenolic compounds have been identified as potent enzyme inhibitors in many systems, including mitochondria1 preparations from apples and flowers (Hulme e t al., 1964), pectic
PLANT-TISSUE OXIDATION SYSTEMS 53
enzymes from grape leaves (Porter et al., 1961; Porter and Schwartz, 1962), and glucosidases (Goldstein and Swain, 1965).
H. SPECIFIC INHIBITORS
There is an extensive literature on enzyme inhibition (e.g., Dixon and Webb, 1958; Hochster and Quastel, 1!363), and it will be recog- nized that various groups of chemical substances can act as inhibitors for many different enzyme systems.
In some cases the chemical inhibitors may act through combination with, or destruction of, the protein part of the enzyme system. In other cases, the effect may be through the coenzyme.
As already noted, many oxidizing enzymes have a prosthetic group incorporating iron or copper, and reagents which can combine with, or alter, the valency states of these metals may be expected to produce inactivation (reversible or irreversible) of the enzyme. The suggestion has been made that such inhibitory mechanisms might have practical applications in food preservation, and, in fact, treatment with carbon monoxide gas has been used as a substitute for blanching in the pro- duction of dehydrated vegetables (Brooks, 1955). Carbon monoxide inactivates o-diphenol oxidase (and also, in the dark, cytochrome oxidase), and hence might be expected to reduce browning reactions. Workers in Louisiana have found that vegetables dried in a stream of air containing 0.3% carbon monoxide retain an acceptable color after dehydration (Anon., 1966).
VI. REGENERATION OF ENZYME ACTIVITY
The reversibility of the heat inactivation of certain enzymes is well established and is best illustrated by the completely reversible inactivation of trypsin, in which the equilibrium between native and denatured forms has been measured (Anson and Mirsky, 1934). The partial reversibility of the inactivation of peroxidase is also well authenticated. Leeson (1957) quoted many examples of the revival of peroxidase activity after apparently complete inactivation, and some features of the earlier work are worth recalling.
The regeneration of peroxidase activity has been found to depend on three main factors: the test used for detecting the activity; the severity of the heat treatment applied to the system; and the con- ditions under which the inactivated system has been kept.
54 F. AYLWARD A N D D . R. HAISMAN
A. TEST METHODS
Balls (1942) pointed out that some tests for peroxidase activity were unspecific, depending on the catalytic activity of the prosthetic groups alone; many instances of pseudo-peroxidase activity have been quoted in this review. Some inconsistencies in earlier reports of peroxidase regeneration may be attributed to this factor. Schwimmer (1944), studying the regeneration of peroxidase in turnip and cabbage juices, found that heating separated the enzyme system into soluble and insoluble components. Although the soluble fraction retained some catalytic activity, regeneration was observed only when both components were incubated together for some time. Some evidence was adduced that the precipitated protein characterized the specificity of the peroxidase while the prosthetic group remained in the supernatant liquid.
Reddi et al. (1950) used three different substrates - guaiacol, o- phenylenediamine, and pyrogallol- to detect the regeneration of peroxidase activity in an apple extract, and although all the results followed the same general pattern, the differences found between substrates showed that experiments limited to one aspect of per- oxidase activity could give misleading results. Nebesky et al. (1950) obtained similar results in tests on other products, and concluded that the guaiacol test gave the most accurate indication of peroxidase activity.
B. HEAT TREATMENT
It has been found consistently that the regeneration of peroxidase activity can be prevented by prolonged or rigorous heat treatment (Kaplan et al., 1949; Reddi et al., 1950; Farkas et al., 1956), and that regeneration is most likely to occur when the enzyme has not been wholly inactivated, or has been heated just to the elimination point as judged immediately after the heat treatment. Figure 4 illustrates some examples of regeneration, based on data obtained by Schwimmer (1944) and Farkas et al. (1956), in which the regeneration of peroxi- dase activity, during the day following the heat treatment, is shown for both the longest heating periods used and the heating period just sufficient to inactivate the enzyme at various temperatures. Where the enzyme was not completely inactivated, the residual activity is also depicted. It can be seen that the extent of regeneration was
PLANT-TISSUE OXIDATION SYSTEMS 55
::I, , ~ , ~ 10
0 70 80 90 100 110 I20
Temperature of inactivation ('C)
FIG. 4. The regeneration of peroxidase. Blank area of column, residual activity after heat treatment; striped area, regenerated activity 24 hr after heat treatment; solid area, regenerated activity 24 hr after maximum heat treatment. A: Data of Schwimmer, 1944. B: Data of Farkas et nl. . 19.56.
greater when there was residual activity after heat treatment or when no extra heating was given after inactivation appeared complete. Furthermore, the amount of regeneration varied with the temperature of the heat treatment given and was considerably greater after short- time high-temperature processes. Esselen and Anderson (1956) tested 17 different vegetables after processing at temperatures up to 300"F, and calculated the heat treatment required, first, to inactivate peroxi- dase, and, second, to prevent regeneration. The F values at 212F to prevent regeneration were about 2-4 times as great as those for in- activation based on testing immediately after heating, and the z values were lower.
56 F. AYLWARD AND D. R. HAISMAN
C. STORAGE CONDITIONS
Other factors which have been found to affect the recovery of peroxi- dase after inactivating treatments include the concentration of neutral salts, the concentration of the enzyme itself, and pH (Herrlinger and Kiermeier, 1944). As more and more cases of regenerated activity have been examined, however, it has become apparent that conditions of time and temperature during the storage of the "inactivated" products are critical factors determining whether or not regeneration will be observed.
Schwimmer (1944) found that the activity regenerated in an inacti- vated turnip peroxidase preparation stored for 20 hours was greater at 25C than at 6C. Studying canned sweet corn, Vetter et al. (1958) found that regeneration of peroxidase took place for two days after processing and was greater when the cans were stored at higher temperatures in the range of 2"-38"C. Zoueil and Esselen (1959) found that peroxidase activity in green beans and turnips stored at 22"-23"C regenerated within 24 hours of processing. Pinsent (1962) observed regeneration of peroxidase activity in peas within a few hours after blanching if the peas were kept at room temperature. At -18"C, regeneration took several months. Working with solutions of pure peroxidase, Wilder (1962) observed a gradual regeneration of activity over 9 days after heat inactivation, increasing most in the first day. In a comparison of three storage temperatures - lo, 22", and 38C -the middle temperature was found to be optimum for maximum regeneration.
Joffe and Ball (1962) investigated the regeneration of pure peroxi- dase in greater detail. In solutions stored at 30C after heat treatment, the enzyme activity was slowly regenerated over 2 to 10 days after an initial time lag of 20 hours. Thereafter, the activity slowly decreased to zero again. At higher temperatures, both regeneration and the sub- sequent decrease in activity were accelerated. The amount of enzyme activity regenerated varied from 4 to 24%. Thus, for any particular product the amount of regenerated peroxidase activity which can be detected will depend both on the time elapsed since heat treatment and on the ambient temperature. Neglect of these factors probably accounts for discrepancies in the earlier literature.
There are few examples of the regeneration of other enzymes con- sidered important in relation to food preservation, but mention should be made of the transitory reappearance of catalase activity after heat treatment, reported by Sapers and Nickerson (1962~). Significantly affected by pH, storage temperature, and heating conditions, up to 29% regeneration of the original activity was observed at pH 7 and
PLANT-TISSUE OXIDATION SYSTEMS 57
30C within 2 hours of inactivation. The reactivated enzyme was evidently unstable, and the regenerated activity decayed within 24 hours.
D. EFFECT ON QUALITY
It has been generally assumed that regenerated enzyme activity must have an undesirable effect on product quality, but, because the borderline between residual and regenerated activities is ill-defined and depends on the sensitivity of the test method used, unequivocal measurement of this effect is difficult. In one of the few published investigations of this aspect of regeneration, Guyer and Holmquist (1954) found that canned peas given a minimum process at 250" or 260F showed a very low level of regenerated peroxidase activity, and after eight months of storage possessed a definite off-flavor.
VII. RESEARCH NEEDS
This review has made many references to off-flavors and deteriora- tion in the color and texture of foods. Any discussion on food quality, however, must focus attention also on the remarkable progress made by the food industries in the past half century in providing an ever- widening range of products both for the domestic consumer and for catering and institutional use (Aylward, 1966, 1967). The impetus for advances in food technology comes not only from the food industries but also from increasing demands by the consumer for variety in foods and for rising standards of quality. The search for improved quality is therefore almost self-perpetuating.
A. THE SCOPE OF RESEARCH
In surveying research needs we must consider not only individual topics, such as the properties of some particular enzyme system, but also the place of this enzyme system in the chain of reactions from farm to consumer. Deterioration from enzyme action may result from changes which occur at different points in this chain.
a. After Harvest and before Processing. Undesirable enzymic re- actions may be stimulated by bruising and other injury, changes in temperature, and anaerobic or oxidative conditions, leading to prote- olysis, foreign flavors, and the possible production of reactive inter- mediate products which may induce further changes at a later stage.
b. During Processing. Enzymic reactions may be accelerated or
58 F. AYLWARD AND D. R. HAISMAN
activated by a rise in temperature; autoxidation and textural changes may be promoted.
c. During Storage. As a result of residual enzymic activity, off- flavors will become more pronounced as storage time is prolonged. Moreover, chemical and physical reactions may be stimulated by reactive intermediate compounds produced at an earlier stage in storage or processing. Such reactions by free-radical or other path- ways can cause the destruction of pigments, vitamins, proteins, and other compounds. Autoxidation in particular may lead to flavor and color changes, and phase separation and conformational changes may lead to changes in texture.
Poor quality may arise from the operation of any or all of these factors, and it is seldom possible to deduce the mechanism from a consideration of quality defects alone. Investigations carried out at the Western Regional Laboratory and elsewhere have shown that the quality of frozen foods may deteriorate even when the possibility of continuing enzymic activity is minimal. Although present-day processing procedures go far toward stabilizing the raw material, it is still potentially reactive, particularly if mistreated during storage. How far this potential is created or modified during processing treat- ments poses one of the more interesting problems requiring further investigation.
B . BIOCHEMICAL ENGINEERING
There will clearly be differences in individual processes such as canning, dehydration (by various methods), and quick freezing, and further work is still required on the effects of these processes on the plant constituents. Moreover, modifications of processes require in- vestigation so that a balance sheet of advantages and disadvantages can be obtained. This points to the need for collaboration between chemical and biochemical engineers, concerned with machinery design and process innovation, and the biochemist and microbi- ologist. There is ample scope for investigations in this field, which can be broadly described as biochemical engineering.
c. ENZYME SYSTEMS: SUBSTRATES - PRIMARY AND SECONDARY REACTIONS
In surveying the enzymes which may be involved in the deteriora- tion of fruit and vegetable products, it appears that only a few (for example, ascorbate oxidase and the pectolytic and proteolytic en-
PLANT-TISSUE OXIDATION SYSTEMS 59
zymes) affect quality directly. The undesirable effects of the majority of oxidative enzymes arise almost entirely from secondary reaction products and the reaction chains they initiate. It has been observed that the off-flavors and off-odors produced during the storage of under- blanched frozen vegetables are characteristic of the vegetable con- cerned, which suggests that although the initiation of degradation may be a common reaction, its development depends on secondary reactions involving indigenous components.
Considerable effort has been applied to identifying and deter- mining enzyme activity in various plant tissues; it is probable that there is now a greater need for more information about enzyme sub- strates and the nature of the secondary reactions. In this connection, work of recent years on reactions involving the unsaturated lipids of plant material is of great importance.
D. PLANT LIPIDS: OXIDATION MECHANISMS AND REACTION PRODUCTS
Unsaturated lipids can be oxidized under a wide range of con- ditions: enzymically by the action of lipoxygenase, and otherwise by heat, light, and a variety of metallic catalysts including hemo- proteins such as peroxidase (not necessarily active) and the cyto- chromes. The peroxides, once formed, may be long-lived; their eventual breakdown may be spontaneous or catalyzed by metals, hemoproteins, and the like, and can initiate chain reactions promoting more extensive degradation.
The oxidation and decomposition of lipids in foods has been followed through the assay of intermediate products such as perox- ides, malondialdehyde, and hexanal. These products represent phases in a complex sequence of reactions, and in isolation may not give an accurate picture of either the extent or course of degradation. An ex- tension of work on the degradation pathways in these systems would be of great value.
E. PLANT COMPONENTS INHIBITING OR MODIFYING ENZYME ACTIVITY
There are numerous examples of modification of the properties of an enzyme system by factors in its natural environment. In situ, enzymes usually show an enhancement in heat resistance, and various inhibitors may disguise the existence of certain enzymes altogether. Natural antioxidants, such as the E and K groups of vitamins and the
60 F. AYLWARD AND D. R. HAISMAN
ubiquinones, may delay or prevent the onset of oxidative changes; analysis of many of these components presents difficulties, but there are indications that some at least vary in concentration according to season and maturity. The effect of these and other components on the potential stability of vegetable products is worth investigation in greater detail.
F. HEAT RESISTANCE OF ENZYMES
Study of the heat resistance of enzymes has been confined to comparatively few systems and has centered on peroxidase because of its demonstrably superior stability and reversible inactivation. There is general agreement that peroxidase activity should be com- pletely suppressed to ensure that quality is maintained in long-term storage, but the actual effect of regenerated peroxidase activity has not been firmly established. More data are undoubtedly needed on the heat resistance of other enzyme systems, such as ascorbate and cytochrome oxidase, particularly in relation to subcellular environ- ment. At the same time it is worth recalling the point emphasized by Joslyn (1966) that critical evaluation of the lability of enzymes is useful as a criterion for the technical treatment of raw materials only when the processing conditions are constant and reproducible.
G. NEW METHODS OF PRESERVATION
There are two approaches to food preservation: (1) to seek meth- ods whereby the essential attributes of the fresh material are least changed; and (2) to produce new types of products which have their own special desirable characteristics as well as stability during storage.
If the first is the objective, then the ideal process would arrest all natural changes in perpetuity. In practice, no method has been found of controlling the complex biochemical balance maintained in natural organisms, although modern freezing techniques go some distance toward this goal.
The second objective is the basis of many traditional methods for preserving both plant and animal materials. Products such as cheese, kippers, and dried fruits are different in many respects from their fresh counterparts. They are accepted as foodstuffs in their own right (Aylward, 1967).
Despite the very considerable advances in knowledge of food science and technology, the majority of the methods used have their
PLANT-TISSUE OXIDATION SYSTEMS 61
origins in antiquity (Tilgner, 1965). The possibility of new approaches to the preservation of foods should not be ignored, and must increase as knowledge grows on the biochemical mechanisms underlying maturation, senescence, and dormancy. There is agreement in many countries on the role of biochemistry as the theoretical basis for food technology (Oparin, 1966).
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