Enzyme Reactions and Acceptability of Plant Foods James K. Palmer Department of Food Science and Technology Virginia Polytechnic Institute and State University. Blacksburg, VA 24061 Plant foods contain an astonishing array of organic comDounds. most believed to be svnthesized via enzyme-catalyzed ieactiok. The nature and amount of certain of these com~ o u n d define s the very nature and character of the particular foods and also determine thrir acceptahility to the consumer. Elucidation of the enzyme reactions rcsponsihle fnr desirable and undesirable reactions is the basisfor controlling these reactions and enhancing acceptability. This report provides an overview of enzymt! reactions rontrihuting to churacter and acceptability and then discusses polgphenoloxidase in some detail, as mixample of an enzyme which can markedly affect character and acceptahility of plant foods. Organic Compounds in Foods and Contribution to Sensory Properties The table shows examples of organic compounds which occur in fruits and vegetahles. The compounds included are representative of a large number of compounds (from a wide variety of chemical ctasses) which directly affect character or acceptability by contributing to key sensory properties: texture, taste. color. or flavor. The contribution of each compound and eiamplks of its occurrence are indicated in the table and discussed brieflv below. @-caroteneis one of some 300 known carotenoids, many of which contribute to the vellow, orange, or red colors of foods (1,2).Lycopene, the pigment r e s p ~ ~ s i bfor l e the red color of tomatoes, is another example of this class. Cyanidin-3-glucoside is one of some 600 known flavonoids. Compounds in this class are responsihle for most of the red, purple, and blue colors of foods (1,2). There are also flavonoids which contribute yellow colors. Chlorophylls are virtually the only green pigments found in plant foods. Degradation products of chlorophyll can also contrihute green-brown or brown color (1,2). Glucose represents the sugars which are responsihle for the sweet taste of nlant foods. Sucrose and fructose are other important examples. Chemical components are also responsible for tastes such as bitter or sour. and for other tmical .. food characteristics such as nstringcnry. For example, certain ilavonoids contribute to bitterness in citrus (31. . . . and ohenoiics contribute to astringency (3,4). The ~ e c t i n sr e ~ r e s e n tthe structural ~olvsaccharidesof plant tksues. TheAhemicellulosesand celiuloie are other important structural polysaccharides. These polysaccharides interact to form extended structures, which ultimately determine the texturd characteristics of plant foods such as softness, dryness, or crispness. Recent evidence indicates these polysaccharide structures to be extraordinarily complex 1.5),. \-
Citral (actually a mixture of the isomers geranial and neral), 2-hexenal, isoamyl butyrate, and 3-methyl-2-isobutylpyrazine are all examples of compounds contributing to food aroma and flavor. Hundreds of such compounds have been identified in plant foods, representing a wide range of chemical classes and a great variety of aromas (6,7). Dopamine represents the phenolic constituents of plant foods. Many of the flavonoids (e.g., cyanidin-3-glucoside described above) are also phenols (4). Ortho-diphenols such as dopamine contrihute to desirable and undesirable browning reactions of foods, as described in more detail below. 284
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Chemlstry and Acceptability Acceptability is defined here in the broadest sense, in terns of certain characteristic features which are due to the presence of a particular array of organic compounds that make a fruit or vegetable generally acceptable. For example, a banana is expected to he appropriately soft, sweet, and flavorful from the time the peel is fully yellow until the peel becomes heavily flecked with brown. All of these desirable properties result f r o n ~the presence of certnin organic compounds, such as t he volatile esters responsihle for banana flavor, $carotene rontributine to vellow nee1 color. and elucose contributine to A sinhlar analisis (akhough there wouli be sweetness many gaps in identifying the particular compounds involved) could he made for all fruits and vegetables. Although some of the compounds responsible for sensory characteristics are also nutrients (e.g., sugars, pectins), the discussions here will deal only with sensory acceptability. Also, the discussions will concern only properties resulting from chemical reactions of the constituents of the foods themselves, and not the deterioration resulting from microbial attack.
(ST.
Biosynthesis: Enzymes and Reactions The next auestion concerns the origin of these many organic c o m p o u n d s . ~ o s are t believed to he synthesized aid/& degraded in enzvme reactions. Before looking.a t particular en. iyme reactions, a few words on the properties of enzymes. Briefly, enzymes are proteins which are able to catalyze various reactions and are especially notahle for their efficiency and specificity. Efficiency means that many reactions which normally proceed at slow or negligible rates and/or require high temperatures for initiation will proceed rapidly a t room temperature in the presence of trace amounts of the appropriate enzyme. The actual rate of an enzyme reaction depends on many factors, including enzyme concentration, reactant (substrate) concentration, pH, and temperature. Since enzvmes are rotei ins. thev are suscentible to denaturation bv G a t . ~ x ~ d s u to r etemperatures abov? 80°C for only a few minutes will cause severe loss in catalvtic activitv of most enzymes. Specificity means that a given enzyme will catalyze e reaction. onlv one articular reaction or t v ~ of here h e two basic experimental approaches to identifying enzvmes and elucidatine the reactions thev catalvze. To ilB is a flavor lustrate these approaches, assume t h ~compound t com~oundfound in a frnit. Recause of similarities in chemical structure, B is proposed to be synthesized from amino acid A. The first approach would involve making an extract of the plant tissue, mixing the extract with a solution of amino acid A and measuring the rate of formation of flavor compound B a t room temperature. Formation of B at a measurable rate in this "test tube" (in oitro) system, under conditions (such as pH) approximating those in the original tissue, is evidence that A is probably the precursor of B in the intact tissue. confirmation that conv&sion of A to B is an enzyme reaction is obtained by repeating the experiment with enzyme extract which has heen boiled ~ r i oto r incubation with A. The reaction -~~~~~~~~~~ is confirmed to he enzymatic if there is little or no conversion of A to B in the "boiled control." In the second so-called tracer approach, compound A would he "labelled" by synthetically introducing radioactke carbun ~~~
~~
~
Organic Compounds In Frults and Vegetables Chemical Name
Class
Structural Formula
Function & Occurrence
ca~otenoid (terpenoid)
Yeilow-arange pigment in carrots, bananas
anthocyanin (flavonoid)
red pigment in
porphyrin
green pigment in many fruits and vegetables.
glucose
sugar (carbohydrate)
sweetener in many fruitsand vegetables.
pectins
poiysaccharide (carbohydrate)
SlruCt~raImaterial which imparts texture. Degradation results in sdlening In fruits and vegetables.
blackberries. cabbage, peaches, raspberries.
Pectin Fragment
monotwpene (terpenaid)
"lemon" flavor in cihus fruits.
i-hexenal
aldehyde
"green" flavor in fruits and YegetabieJ.
isoarnyi butyrate
estw
''huitV flavorin several fruits.
3methoxy2 isobuiyl pyrazine
pyrazine
green pepper flavor: also Contributes to flavor of peas.
dopamine
polyphen01
substrata tor enzymatic brownina in bananas.
("C) or hydrogen (tritium. 3H) into the molecule. Extremely small amounts of A could then be detected hy techniques for measuring radioactivity. A small amount nf compound A would be introdured intocells of the tissue of interest. After several hours of incubation, compound B would be isolated and the amount ol' radioactivity in B determined. The presence of significant radioactivi& in B is taken as strong evidence for the A to B conversion. Demonstration that the accumulation of radioactivity in B is prevented hy prior heat treatment of the tisue provides evidence for emit catalysis of the -~~~~~ reaction..and the rate of accumulation in B ~rovidesan .indication of the enzyme activity. ~~~~
~
Only the simplest forms of the two approaches for studying enzyme reactions have bean described. Biorhemists have developed many ingenious variations of these approaches, so that it is now possihle in most cases to provide strong support for the existence of enzyme r e ~ r t i o n and s to charocterirr the enzyme reactions in termsof specificity, pH effecte,etr. Utilizing these techniques, biochemists have identified and characterized thousands of enzymes, in terms of their occurrence, specificitv and efficiency. Enzymes play a central role in biological processes hy making possihle the essential series of reactions involved in synthesis and metabolism of "~rimarv metabo1ites"such as proteins, amino acids, lipids, &sugars. Volume 61
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HYDROXYMETHYL GLUTARYL CoA M U N U l bKPfiNUIUb
MEVALONIC ACID
Ha)_/"
t
PYROPHOSPHATE
COA
I
CAROTENOIDS
I
FARNESYL PYROPHOSPHATE
LIPIDS CH,
I
NH,
I
TRANSAMIWAJE
CHCH&HCOOH
I
.
CHI
HYDROLASE
CH,CH&H=CHCHICH=CHCH~CH=CH(CH~)FOOH LINOLENIC ACID
L-LEUCINE CHI
I
CHCHSOCOOH
DBCARBOXYLASE
I
*
CH.CHICH=CHCHICHCH=CHCH=CH(CH~)FOOH I OOH
CHa e-KETOISOCAPROIC ACID
IALDEWDB
13-HYDROPEROXY ACID
LYASE
(HYDROPEROXIDE LYASE)
ISOVALERALDEHYDE
TRANS-2-HEXENAL
FH"
12-OXO-TRANS-10DODECENOIC ACID
STARCH ( C 6 H ~ ~ O 5 ) , (POLYSACCHARIDE)
1
&VYMs8S
CHI
I
~~~H,OOCCH~CH,CH, ISOAMYL BUTYRATE
MALTOSE C,~H.IOII
(DISACCHARIDE)
1 GLUCOSE
(MONOSACCHARIDE)
CrHtrO. D
I
Figure I. Blosynthesis and degradation of selected componems in plant fwd A. carotenoids and monoterpenes [adapted from Robinson (lo)] 8. isoamyl butyrate
286
Journal of Chemical Education
C. 2-hsxenal D. glucose from starch
synthesis or degradation and markedly reduce acceptahility. But they also catalyze production of characteristic "secondary Softening in ripening fruits is a prime example. A certain metabolites," such as the flavors and pigments of the tahle. degree of softening is characteristic and essential for acceptFigure 1outlines the reactions and enzymes involved in the ability, whereas excessive softening is perhaps the greatest hiosynthesis andlor degradation of components selected from single cause of fruit losses in the post harvest period. the tahle. Monoterpenes, such as geranial, and carotenoids, such as p-carotene, are hiosynthesized from acetate via the Polyphenoloxidase and Acceptability: Bananas and Tea central metabolite mevalonic acid (Fig. 1A) (9.10). As indicated in Figure lA, geranyl pyrophosphate is another key The discussions to this point give an over-view of the wide intermediate which occupies the branch point between morange of organic compouids a i d enzyme reactions which noterpenes and higher terpenes. Cyclization of the geranyl contribute to the character and acceptability of fruits and pyrophosphate and subsequent reductions, oxidations, rearvegetahles. The remaining discussion will look in more depth rangements, etc. of the dephosphorylated products result in a t oolwhenoloxidase (catechol oxidase), as an example of an . .. formation of the many different monoterpenes. Alternatively, enzyme which can have profound effectson the character and one molecule of geranyl pyrophosphate and one of isopentenyl acceptahility of plant foods. A look a t the properties and role pyrophosphate can react to form farnesyl pyrophosphate (the of polyphenoloxidase in hananas and tea will illustrate the precursor of sesquiterpenes), and the farnesyl pyrophosphate, kinds of information the food scientist obtains from research by similar reaction with an additional isopentenyl pyroon food enzymes and how this information can be used to phosphate, can form geranyl-geranyl pyrophosphate. Conmaintain and/or enhance acceptahility. densation of two molecules of farnesyl pyrophosphate forms Figure 2 shows the reactions which lead to hrowning in the the C30 precursor of the steroids, and condensation of two hanana fruit (17). Research has established that polyphenolmolecules of geranyl-geranyl pyrophosphate forms the Cia oxidase catalyzes only the first reaction, the conversion of precursor of &carotene and other carotenoids. Only a few of dopamine to dopamine quinone. The suhsequent series of the individual enzymes involved in terpenoid synthesis have reactions are nonenzymatic, leading finally to the formation been isolated. The pathways have been worked out for the of the brown melanins. The detailed structure of the melanin most part by radioactive tracer methods. However, in recent pigments is still somewhat of a mystery, hut they are known studies (11) enzymes that catalyze key steps in terpenoid to he insoluble polymers of high molecular weight formed by synthesis, such as cyclization of geranyl pyrophosphate, have condensation of various products of dopamine oxidation, hut been isolated and characterized for the first time. also containing varying amounts of peptides and amino acids A pathway for synthesis of isoamyl hutyrate from the amino as Dart of the structure. Since it is impossible to remove these acid leucine (Fig. 1B) has been proposed by Tressl et al. (12) insoluble polymers once formed, the research emphasis has and Tressl and Drawert (13). They utilized radioactive tracer been on preventing formation of the melanin pigments. methods, measuring radioactivity in isoamyl hutyrate after Briefly, hanana polyphenoloxidase has been found to he a introducing 14Cleucine into banana slices. copper-containing enzyme which requires oxygen for activity, Biosynthesis of 2-hexenal has been studied also, as sumto have a pH optimum of 7 and little activity below pH 4, and marized in the review of Tressl and Drawert (13). These aut o he relatively sensitive to heat denaturation (17). The prithors and their co-workers reported the enzymatic formation mary substrate in bananas is dopamine (see tahle, Fig. 2). The of 2-hexenal from linolenic and linoleic acid in hanana, apple, dopamine is compartmentalized in special cells and hrowning pear, plum, and grape homogenates in 1966. In later experioccurs only when tissue damage releases the dopamine and ments, isolation and identification of labelled products after allows i t to come in contact with the enzyme. incubation of hanana homogenates with 14C-lahelledlinolenic Based on this information, techniques for inhibiting acid led them to propose the pathway shown in Figure 1C. hrowning in sliced hananas or other banana products were Tressl and Drawert were unable to isolate any of the individual developed (18, 19). Examples are treatment with reducing enzymes proposed in Figure 1C. Hatanaka and co-workers agents such as ascorbic acid (to reduce dopamine quinone (14) have studied the biosynthesis of 2-hexenal in tea. They hack to dopamine); treatment with acids such as citric acid proposed a pathway similar to Figure lC, except that cis-3(to keep pH below 4); treatment with chelating agents such hexenal was the initial product formed by the action of hyas ethvlene diamine tetra acetic acid (to complex copper ions droperoxide lyase. The cis-3-hexenal was then converted to ind &ke them unavailahle~and blanching (heat in&tivation trans-2-hexenal, possihly catalyzed by an isomerase. These of thr enzvmer. Similar studies oi ~olvnhenoloxidasein other workers isolated a hydroperoxide lyase which was specific for fruits and vegetables have resulted'& an array of specific the 13-hydroperoxy acid. technioues to reduce enzvmatic hrowning durinz slicina, hoTurning to hydrolytic or degradative reactions, it is well operati& (20). mogenikng, or other estahlished that the increase in sweetness during ripening of there is no effective chemical treatment for preventing the fruits or during storage or cooking of sweet potatoes results from enzymic hydrolysis of starch to glucose (Fig. ID). See (15) for discussion of enzvme reactions involved in sweetening of sweet potatoes. pI* There is considerable evidence that CH, +to, the degradation of pectin by polygalacturonases is a key reaction in DOPAMINE DOPAMINE 2 3-DIHYDROINDOLE QUINONE softening of plant tissues (16). -6.6 MAX. QU =I300 N O Nrn@ E (RE? LO r = 3,95 The examples given in this section 470 rn@: LOG E = 3.40emphasize the desirable aspects of food enzymes. In fact, the enzymes in foods are "good news and bad news." On the one hand, they catalyze the MELANIN production of the characteristic and ABSORPTION) +!O. deliahtful flavors, colors, and .. texGres of fruits and vegetahles. On 5.6 = DIHYDROXYINDOLE INDOLE-5 6 QUINONE (PU~PLE) the other hand, if the fruit or vegeMAX = 540 rmr table is not carefullv handled, the Figure 2. Reaction mechanism lor oxidation of dopamine by banana polyphenoioxidase. From Palmer ( 1 7 ) . enzymes can quickly cause excessive
o ow ever,
I
1
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Figure 3. Substrates for pOlyphenoloxidaSe in tea. From Sanderson (21).
Figure 4. Proposed structure of thesflavin. From Sanderson (21)
a major role in determining the quality of brewed tea. Additional polymerization yields the so-called thearuhigens, chemically ill-defined hut brown in color and important in determinine the color of tea infusions, especiallv of the black teas. secondary oxidation of various &ino acid;, carotenoids, and lipids hv the enzvme-generated auinones leads to formation of c&nponen& whGh contrihke to the delicate tea aroma. For example, carotenes are oxidized by the quinones to yield key tea aroma compounds such as @-ionone,theaspirone, linalool (Fig. 5 ) , and a whole series of unidentified terpenoids. The central role of polyphenoloxidase (catechol oxidase) in generating color and flavor in tea is summarized in Figure 6. This information on the enzyme reactions involved in making tea has contributed to obtaining better control of tea production. These brief discussions of polyphenoloxidase in hananas and tea illustrate the central role enzyme reactions can play in determining the character and acceptahility of food products. Similar analyses could be made in relation to desirable and undesirable sensory characteristics of mans other plant foods, although again there would he considerahle gaps [n our knowledge of many of the systems. For example, recent research has generated considerahle information on the enzyme reactions involved in fruit softening (16) and further research will no douht ultimately lead to gaining better control of softening, and specifically to prolonging the period of optimum softness.
Summary The enzyme reactions which ultimately determine the character and acceptahility of plant foods are relatively complex. Nevertheless, food chemists have learned much about these reactions in recent years. This knowledge has been used in many cases to define acceptability in more objective terms and to enhance or maintain acceptabilitv via modification of post harvest handling and/or procedures, for example. Research continues in this interesting area and will no douht lead to further improvements.
hrowning of intact fresh fruits and vegetahles which results from senescence, bruising, etc. The only recourse is careful handling to prevent stress or damage and thus limit mixing of suhstrate and enzyme. In the future, i t may he possible to prevent hrowning by breeding varieties free of suhstrate and/or enzyme. Overall, although much progress has been made, enzymatic hrowning continues to be a serious prohlem during post harvest handling and processing of many fruits and vegetahles. Research directed toward solving this prohlem is continuing. Tea provides an interesting contrast to hananas. Research Figure 5. Aroma compounds in tea resulting from oxidation of carotenoids by has shown that many of the key characteristics of tea in fact enzymegeneratedquinanes. result from and depend on the action of polyphenoloxidase (catechol oxidase), as detailed by Sanderson (21). Tea manufacture consists of a series of steps which promote a controlled enzymatic oxidation of polyphenols. Freshly harvested leaves are partially desiccated (withered) and then macerated to promote extensive mixing of cell contents. The macerated leaves are incubated (fermented) Theaflavins and Binflavanols for 1-4 hr and finallv are heat treated (fired) \(transitory) I to stop the enzymicprocesses and to d;y thk product. Extensive research has demonstrated Thearubiginr that the primary and central reaction during (S,-S,.S,,+Insoluble) fermentation is enzymatic oxidation of tea catechins. The resulting quinones are suhsequently involved in a complex series of reacVolatile Epitheaflavic Acids tions which lead to formation of tea aroma and flavor Carotenoids constituento color. Figure 3 shows typical catechin (flavowhich contribute %tP;',"; flavor nol) polyphenols found in tea. Figure 4 shows to black preeu*sors tea aroma. a typical theaflavin, a product of the oxidation of two flavonols, (-)-epigallocatechin and (-)-epigallocatechin gallate. The theaflavins F'@xe 6. haposed mechanism in. formation of pigments and aroma canpounds in tea. From Sacderwrn have a bright reddish color in solution and play 121). 288
Journal of Chemical Education
Literature Cited (1) Clydesdale, F. M., and Francis. F. J., in "Principlps of Foad Science. Part 1. Food Chemistry,ll Fennema, 0. R. (Editor), Dekker, New York, 1976, pp. 385-426. (2) Alkema,J.,and Seager,S. L.,J.CHEM.EDUc.,59,183 (1982). (3) Van Buren, J.. in "The Biochemistry of Fruits and Their Products: Hulme, A. C. (Editor), Academic P~ess,New York, 1970, Vol 1, pp. 269-304. 11963). (4) Go1drtein.J. L.,snd Swain,T.S.,Phylorhamisfry,2,371 (5) Alberrheim, P., Scienlilic Amerimn, April 1975, pp. 81-99. (6) Nunten, H. E., in "The Binchemi8try of Fruits and Their Products," Hulme, A. C. (Edito~l,Academic Pleas, New York, 1970, Val. 1,pp. 23?-268. (7) Nursten, H. E., m "Progress in Flavour Research: Land, D. G.. and Nursten, H. E. (Editors), Applied Science Publishers Ltd.. Essex. England, 1979, pp. 837-355. (8) Palmer. J. K.. in "The Binchemistry of Fruits and Their Producfs: Hulme, A. C. iEdiior1, Academic Presa, New York. 1970, Vol. 2, pp. 65-105. (9) Hendrickson, J. B.;'TheMoloculeaofNsture~ W. A. Benjamin, Ina.,NewYork, 1965. PP.3&39. (10) Robinson, T., "The Organic Constituents of Higher Plants," 3rd ed.. Cordm Press. N. Amherat, MA, 1975,~.182. (11) Croteau. R., in "Biosynthesis of Monoterpenes: Porter. J. W.. and Spurgeon, S. L.
(Editors),John Wileyand Sons, New York. 1981,Vol. 1, pp. 225-282. (12) Tressl, R., Embever, R.. Drawert, F., and Heimann, W., Z Noturffrffh., 25b, 704 119701 ,. .. .,.
(13) Tresl. R., and Drawert, F., J. Agr Fuod Chem., 21,560 (1973). (14) Sekiya,J., Nums, S., Kajiwars, T.,and Hatsnake, A,, Agrir. B i d Chem., 40,185 11976): Hatsnske.A.,Ksjiwsra,T.,Sskiya,J.,and1nouye.S..Phylochem..21.13 (19821. (15) Palmer, J . K., in "Sweet Potato," Yilla~eal.R L., and Griggs, T. D. iEdilorsl. Asian Vegetable h a a r e h a n d Development Center,Shanhua.Tsinan,Tsiwan. 1982,pp. 135-140. (16) Pre~y.R.,in"EneymesinFoadmdBeverage Proceasing,"Ory,R. L.,and St. Angelo. A. J . (Editom), American Chemical Society, Washinaton, DC, 1977, PD.172-191. (18) Palmer. J. K.,in "Phenolicsin Normal and D i s d F r u i L s a n d Vegetshles,"Runwkles. V. C. (Editor), Plant Phenolics Group of N. America, Montreal, 1965. pp. 7-12. (19) Luh.N. and ~ a l m e r . ~ . ~ . , " D e h y d r a t~e adn a n a ~ r o d u n : u . S . ~ a t e n t 3 . 9 ~ , 3 0 1 (1576). (20) Mathew, A. G., and Ps1pia.A. B., Ad". Fond Re&. 19.75 (1971). (21) Sandenon, G. W., in "Swetursl and Functional Aspects of Phytmhemistry."Recent Adv. in Phytochem. Vol. 5, Runeckles, V. C., and Tso, T. C. (Editors), Academic Press, New York, 1972, pp. 247-316.
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