Enzymatic browning reactionsi appleand

This article was downloaded by: [North Carolina State University] On: 17 December 2012, At: 17:29 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Critical Reviews in Food Science and Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bfsn20 Enzymatic browning reactions in apple and apple products Jacques J. Nicolas a , Florence C. Richard‐Forget b , Pascale M. Goupy b , Marie‐Josèphe Amiot b & Serge Y. Aubert b a Chaire de Biochimie Industrielle et Agro‐Alimentaire, Conservatoire National des Arts et Métiers, 292 Rue Saint‐Martin, PARIS Cedex 03, 75141, France b Laboratoire de Biochimie des Dégradations, Station de Technologie des Produits Végétaux, Institut National de la Recherche Agronomique, Domaine Saint‐Paul, B.P. 91, Montfavet Cedex, 84143, France Version of record first published: 29 Sep 2009. To cite this article: Jacques J. Nicolas , Florence C. Richard‐Forget , Pascale M. Goupy , Marie‐Josèphe Amiot & Serge Y. Aubert (1994): Enzymatic browning reactions in apple and apple products, Critical Reviews in Food Science and Nutrition, 34:2, 109-157 To link to this article: http://dx.doi.org/10.1080/10408399409527653 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Critical Reviews in Food Science and Nutrition, 34(2): 109-157 (1994) Enzymatic Browning Reactions in Apple and Apple Products Jacques J. Nicolas Chaire de Biochimie Industrielle et Agro-Alimentaire, Conservatoire National des Arts et Metiers, 292 Rue Saint-Martin, 75141 PARIS Cedex 03, France Florence C. Richard-Forget, Pascale M. Goupy, Marie-Josèphe Amiot, and Serge Y. Aubert Laboratoire de Biochimie des Degradations, Station de Technologie des Produits V6getaux, Institut National de la Recherche Agronomique, Domaine Saint-Paul, B.P. 91,84143 Montfavet Cedex, France ABSTRACT: This review examines the parameters of enzymatic browning in apple and apple products that is, phenolic compounds, polyphenoloxidases, and other factors (ascorbic acid and peroxidases), both qualitatively and quantitatively. Then the relationships between intensity of browning and the browning parameters are discussed, including a paragraph on the methods used for browning evaluation. Finally, the different methods for the control of browning are presented. KEY WORDS: apple, browning, polyphenols, enzymes, polyphenoloxidases I. INTRODUCTION Based on size of production, the apple is one of the main fruit crops in the world. In 1980, the world apple production exceeded 35 million t, corresponding to fourth place after grapes, citrus, and banana. 356 The four major producers are the U.S., France, Italy, and China. More than half of the apple crop is sold in the fresh produce market. Depending on the year, 40 to 60% of apple pro- duction is used by the industry. Juice, either as single-strength sweet juice or as fermented juice (cider), apple sauce, and slices, are the main prod- ucts of processed apples. According to Salunkhe et al., 357 one fourth of the fruit and vegetables harvested is never consumed because of spoilage during postharvest manipulations or processing. Concerning apples, the postharvest losses are considerably less, because it has been estimated at less than 10% for the fresh market by Sparks 408 and about 14% in developing countries by Steppe. 410 The main causes of losses are physical injuries, physiological disorders or diseases dur- ing storage, 96 and improper conditions for pro- 1040-8398/94/$.50 © 1994 by CRC Press, Inc. cessing. In most, if not all cases, the observed symptoms correspond to a discoloration of the apple or the apple products. Thus, bruising, which is the most common defect of apples 186 seen on the market, results in a flattened area on the side of the fruit with the flesh browned beneath. 352 - 356 Similarly, bitter pit, 161 superficial 171 and senes- cent scalds, internal (senescent or low-tempera- ture) breakdown, 15 - 166 watercore, 239 and core flush 223 all result in browning of either the skin or the internal tissue. Finally, juice, puree, and apple slices that are badly processed brown intensely and give the final products a bad appearance which is rejected by the consumer. 74287 - 317 These general discoloration phenomena are mainly related to enzymatic browning. However, browning can also originate from nonenzymatic reactions such as the Maillard reaction, 16 - 106217 which occurs mainly in heat-processed apple products. 228 - 244328 - 462 Basically, enzymatic browning can be de- fined as an initial enzymatic oxidation of phenols into slightly colored quinones. 13 - 47 - 101 - 185 - 251 - 350 - 470 These quinones are then subjected to further reac- tions, enzymically catalyzed or not, leading to the 109 Downloaded by [North Carolina State University] at 17:29 17 December 2012 formation of pigments. The colors of the latter differ widely in hue and intensity, following the phenols from which they originate and the environmental factors of the oxidation reac- tion _50.113,184,243,298,308-311,325,351,395,463 Enzymatic browning is mainly associated with polyphenol oxidases, which are able to act on phenols in the presence of oxygen. 253 " 256 - 338 - 350 - 489 - 510 Two kinds of enzymes are classified under this trivial name. The first class, catechol oxidases (E.C. 1.10.3.1), catalyze two distinct reactions (Figure 1): the hydroxylation of monophenols in o-diphenols (reaction 1) and the oxidation of o-diphenols in o-quinones (reaction 2). These two enzymatic reactions consume oxygen and are referred to as monophenolase (or cresolase) activity and o-diphenolase (or catecholase) activity, respec- tively. The former activity is not always present, and when both activities are present, the ratio of cresolase/catecholase activities varies widely from 1 to 10 or even 40. 34 - 443 The second class, laccases (E.C.I. 10.3.2), oxidizes o-diphenols as well as p-diphenols (Fig- ure 2), forming their corresponding quinones. Besides other differences in properties, 256 - 475 the unique ability to oxidize p-diphenols can be used to distinguish laccase activity from that of the first class. The nomenclature of these enzymes 176 is some- what confusing because besides the two numbers E.C.I.10.3.1 and E.C.I. 10.3.2, a third one exists, E.C.I.14.18.1. It is referred to as monophenol monooxygenase (tyrosinase) and corresponds to OH CRESOLASE 1/2 0, OH CATECHOLASE ,0H V2 0 2 H 2 0 (2) FIGURE 1. Reactions catalyzed by polyphenoloxidases (E.C. 1.14.18.1 and E.C. 1.10.3.1). (1) Hydroxylation of monophenol to o-diphenol; (2) dehydrogena- tion of o-diphenol to o-quinone. FIGURE 2. Reactions catalyzed by laccases (E.C. 1.10.3.2). 110 Downloaded by [North Carolina State University] at 17:29 17 December 2012 the same enzymes as E.C.I.10.3.1, which always catalyze the hydroxylation of monophenols. The peroxidases (E.C.I. 11.1.7) can also be considered as participating in enzymatic brown- ing. These enzymes, whose primary function is to oxidize hydrogen donors at the expense of perox- ides, are highly specific for hydrogen peroxide. On the other hand, they accept a wide range of hydrogen donors, including polyphenols. 36 - 342 - 343 - 443 The primary products of oxidized phenols are probably quinones similar to those obtained with polyphenol oxidases. Although peroxidases are distributed widely, especially in plants, they gen- erally appear to be little involved in enzymatic browning of fruits and vegetables following a mechanical stress. The explanation could be that the peroxidase activity is limited by the internal level of hydrogen peroxide. However, their in- volvement in a slow process such as internal browning is possible. 423424 Nevertheless, the di- rect involvement of peroxidase in enzymatic browning of apple and apple products remains questionable, as does that of laccases. The latter enzymes are mainly present in fungi and in cer- tain higher plants 256 and are almost always absent in sound fruits and vegetables, with the exception of peaches 146 and apricots. 87 Therefore, the bulk of the discussion below concentrates on the two main factors of apple enzymatic browning, that is, phenolic substrates and polyphenoloxidase (E.C.I.10.3.1) activity, with a small part devoted to other factors such as peroxidase and ascorbic acid. The different ways to estimate browning, the correlations among the intensity of browning and its causal factors, and, finally, the control of browning are then examined successively. II. PARAMETERS OF ENZYMATIC BROWNING IN APPLE AND APPLE PRODUCTS A. Phenolic Compounds of Apple and Apple Products 1. Total and Individual Phenolics in Ripe Apple Fruit and Apple Juice A great number of works have been devoted to the study of phenolics in apple and apple prod- ucts. Among the several classes of plant pheno- lics, six classes are present in apple fruit: hydroxycinnamic derivatives, flavonols, antho- cyanins, dihydrochalcones, monomeric flavan-3- ols, and tannins. 236 Since the first modern monograph published by Harborne 142 - 144 in 1964, considerable information has been compiled on the classes of these phenolics, 135 - 169 - 238 - 321 - 411 - 454 as well as on methodologies for their purification, isolation, quantitation, and structure elucida- tion i 27 . 143 . 162 ^™^. 455 The quantitation of total phenolic compounds is not accurate because of the extraction method, which often cannot guarantee a total solubiliza- tion of all phenolics, and to the assay method, which is unavoidably a compromise on the reac- tivity or absorption characteristics of the different classes of phenols. 27 - 28 - 81 - 234 - 396 - 398 - 413 Nevertheless, these measurements still are used for rough comparisons among samples. Indeed, a wide variability is observed in the total phenolic contents of ripe fruits and apple juices (Table 1). Obviously, this variation can be partly attributed to the different methods used by the authors for the quantitation of phenols. How- ever, even when the same method is used, consid- erable variation in results is still evident. 69150 - 448 As in other fruits, the levels of phenolic com- pounds in apple are highly dependent on many factors, such as variety, stage of maturity, and environmental factors. 236 - 491 Besides quantitative variations among the classes of phenolic com- pounds, large variations are also apparent in the qualitative distribution, which are caused by ge- netic and external factors. Modern methods, par- ticularly high-performance liquid chromatography, considerable progress in both the separa- tion and quantitation of individual phenolics in apple fruit. The main phenolics identified in apple fruit and apple products are given in Table 2, and the contents of the different classes are given in Table 3 for the ripe fruits. The tables show that: 1. In the cortex, three phenolics accounted for more than 90% of the total phenolic con- tent for most cultivars: one caffeoyl quinic acid (chlorogenic acid) and two flavan-3- ols ((-)-epicatechin and procyanidin B2). 2. In the peel, the hydroxycinnamic acid derivatives are not as high as in the cortex, 111 Downloaded by [North Carolina State University] at 17:29 17 December 2012 TABLE 1 Total Phenolic Compounds in Ripe Apple Fruit and Apple Juice Samples 7 varieties 8 10 varieties' 1 400 varieties' 1 7 varieties'* 2 varieties'" 8 varieties 8 3 varieties" Various varieties 6 8 varieties 6 8 varieties' 12 varieties^ 5 varieties 6 6 varieties 0 3 varieties (x 2 technologies)' 5 varieties 0 5 varieties' 22 varieties 3 10 varieties' 4 varieties' 2 varieties (x 6 technologies)* Industrial concentrate (72° Brix) e Ripe Fruit Peel Pulp 8.7-19.2 1.5-3.8 0.9-2.1 0.6-1.2 0.2-1.9 0.5-0.7 Apple Juice Total 0.7-2.5 0.6-2.0 0.5-17 0.15-0.21 0.6-0.8 1.0-1.9 0.49-0.84 0.5-11 0.16-0.44 0.15-0.58 0.24-0.62 0.14-0.37 0.21-15.2 0.37-0.62 0.02-0.14 0.3-2.7 1.49 Ref. 69 448 157 516 449 381 288 450 323 193 150 172 464 302 407 407 63 64 74 126 14 Note: Values are given in grams per kilogram (FW ripe fruit) or in grams per liter (juice). a Chlorogenic acid equivalent. b o-Diphenols (chlorogenic acid equivalent). c Gatechin equivalent. d Tannins. 8 Not given. ' Gallic acid equivalent. a o-Diphenols (catechol equivalent). h Tannic acid equivalent. whereas the flavan-3-ol and flavonol derivatives constitute the major part of the phenolic compounds. 3. In the hydroxycinnamic derivatives, quinic acid is the only hydroxyacid that forms esters with caffeic 403 and /7-coumaric 41 - 492 acids. Although not detected in the original fruit, 5'-feruloyl quinic acid has been reported in cell suspensions from apple fruit. 195 In the latter case, it accumulates in the latter stage of cell culture growth after the exponential growth phase. Similar behavior was shown for the sinapoyl glucose ester, which was observed only in in vitro cell suspension cultures from apple parenchyma. 195 - 307 In apple fruit, besides quinic acid, hydroxycinnamic acids form esters with sugars and, more precisely, with glucose. Thus, Macheix 231 - 232 reported that 1-0-p-coumaroyl glucose was one of the major p-coumaric derivatives in apple (var. CalviUe blanc) together with several p-coumaroyl 112 Downloaded by [North Carolina State University] at 17:29 17 December 2012 TABLE 2 Main Phenolics Identified in Apple Fruit and Apple Products (According to Reference 236, with Modifications) Phenolic Acids 4 ' 95 ' 107 - 112 - 165 ' 229 ' 232 ' 235 ' 266 ' 279 ' 306 ' 336 ' 403 ' 487 ' 490 ' 492 ' 497 5'-caffeoylquinic, a 4'-caffeoylquinic, 3'-caffeoylquinic, 5'-p-coumaroylquinic, 4'-p-coumaroylquinic, 3'-p-coumaroylquinic, p-coumaroylglucose, caffeoylglucose, feruloylglucose Flavan-S-Ols 4 ' 107 - 112 - 119 ' 175 ' 203 ' 211 ' 229 ' 266 ' 279 ' 283 ' 336 ' 380 ' 385 - 392 - 429 - 453 ' 480 ' 481 ' 497 (-)-epicatechin, (+)-catechin, B1, B2, B5, C1, higher polymeric forms Flavonols 85 ' 86 - 294 ' 306 - 391 > 425 Quercetin-3-O-p-D-galactopyranoside (hyperin)," Quercetin-3-O-p-o-glucopyranoside (isoquercitrin), Querce- tin-3-O-p-D-xyloside (reynoutrin), Quercetin-3-O-a-L-rhamnopyranoside (quercitrin), Quercetin-3-O-oc-L-arabino- furanoside (avicularin), Quercetin-3-O-rutinoside (rutin) Dihydrochalcones 85 - 86 - 95 - 266 - 294 ' 497 Phloretin-2'-O-glucoside (phlorizin), Phloretin-2'-xyloglucoside AnthOCyaninS c 94,152,163,260,412,426,427,428 Cyanidin-3-galactoside (ideain), Cyanidin-3-glucoside (kuromarin), Cyanidin-3-xyIoside, Cyanidin-3-arabino- side; acylated derivatives of Cyanidin-3-galactoside, Cyanidin-3-arabinoside, Cyanidin-3-glucoside, and Cyanidin-3-xyloside Note: The major compounds in each class are written in boldfaced type. a The IUPAC recommendations 177 were applied for the nomenclature of the hydroxycinnamic quinic esters. Thus, 5'-caffeoylquinic is chlorogenic acid, 4'-caffeoylquinic acid is cryptochlorogenic acid, and 3'-caffeoylquinic acid is neochlorogenic acid. b A structure of quercetin-3-O-a-galactoside was proposed by Teuber and Herrmann. 425 0 Present in apple cultivars with red-colored skins. 4. quinic esters. Similarly, in their compilation, Risch and Herrmann 336 indicated that the glucose esters in apple were mainly p-coumaroyl and feruloyl glucose esters. Glucoside derivatives in which the phenolic group is engaged in bonding with glucose are not present in apple fruit, although they are often encountered in plants. 159 Finally, according to Macheix et al., 237 the overall balance of the three hydroxycinnamic acids (either as quinic esters or as glucose esters) ranges between 75 and 94% for caffeic acid, 5 and 20% forp-coumaric acid, and 1 to 5% for ferulic acid. In the flavan-3-ol derivatives, procyanidin B2 (dimer of two (—)-epicatechin units linked in a bond between C4, the "upper unit", and C8, the "lower unit") and (-)-epicatechin are the most abundant. 35 - 306 The (+)-catechin isomer is also always present in apple fruit, although sometimes only as traces. 336 According to Mosel and Herrmann, 283 the mean content of (-)-epicatechin in apples is five times higher than that of (+)-catechin. The latter authors indicated that (+)-gallocatechin and (—)-epigallocatechin, which are normally absent in apples, have been found in some cultivars 283 - 284 in relation to certain climatic factors and growing conditions. The data concerning the polymeric forms of flavan- 3-ols ("tannins") are scarce, probably owing to the difficulties encountered in their total extraction and precise quantitation. Almost 113 Downloaded by [North Carolina State University] at 17:29 17 December 2012 TABLE 3 Content in Chlorogenic Acid (CG), Other Hydroxycinnamic Derivatives (HCd), Flavan-3-Ols (F3OI), Flavonols (FVOd), and Dihydrochalcones (DHC) of Ripe Apple Fruit and Apple Juice Samples Compilation 7 var. 5 var. 3 var. 16 var. 8 var. 11 var.* 7 var. (pulp) 5 var. (pulp) 8 var. (pulp) 3 var. (pulp) 11 var. (pulp)* 12 var. (pulp)* 7 var. (peel) 5 var. (peel) 8 var. (peel) 3 var. (peel) 16 var. (peel)* 11 var. (peel)* 12 var. (peel)* 4 Com. samples 8 var. 5 var. 5 var. CG 5-51 2.6-13.6 15-33 6-51 4.3-19 a 18-173 19-55 4.2-32 9-^3 4-6 18-171 18-171 0.1-20 2-15 3-6 5-150 3-89 0.25-0.91 6.6-23.2 0.73-25 0.3-5.9 HCd F3OI Ripe Fruit 0.5-11.2 — — 25-65 — 4.4-10.6 0.7-5.2 1.9-2.6" 11-51 0-5.5 0.35-^.1 15-50 12-56 0.96-4.9 4-33 0.2-12 3.5-16 90-261 33-124 3-21.5 13-30 5-29 98-214 98-214 230-650 19.4-81.5 45.5-143 25-127 100-960 50-450 120-575 Apple Juice 0.23-0.45 tr-0.16 3.2-20 0.4-3.3 0.3-46.5 0.1-2.5 FVOd 0-2.9 6-26 4-15 tr-3.8 0.02-O.24 16-52 15.8-142 154-285 78-107 120-550 81-353 0.23-0.73 0-0.6 DHC 0.4-1.9 9-29 0.3-1.6 1.1-2.5 2-4 0.6-65 8.7-33 18-33 8-220 7-211 Ref. 236 69 172 158 336 283 5,7 322 305, 306 266 35 4 12 322 305, 306 85, 266 35 4 436 12 1.4-2.7 108 — 33 0.48-3.5 80 0.1-1.6 407 Note: All results are given in milligrams per 100 g FW basis or in milligrams per 100 ml (juice) except those marked by an asterisk (*), which are given in milligrams per 100 g DM basis. a expressed as caffeic acid equivalent (contains all the caffeoyl esters). b expressed as p-coumaric and ferulic acids (contains all the coumaroyl and feruloyl esters). all the data are concerned with apple juices and ciders, owing to the interest in the sensory and technological properties of these products. 76 - 203 - 208 - 209 - 212 - 280 - 282 - 302 - 407 - 451 - 452 A considerable range existed in the total tannin content of apple juices. 80 - 207 - 210 Thus, large differences were found in the phenolics of juices obtained from a dessert apple (var. Bramley) and a cider apple (var. Dabinett). 208 However, the largest difference was associated with the tannin fraction as the Dabinett juice contained twofold more phenolic acids, fivefold more phloridzin, and tenfold more epicatechin, but 20-fold more procyanidin B2 than the Bramley juice. 208 5. In the flavonol derivatives, the only characteristic feature is the presence of quercetin as aglycone. Methods have been proposed for the certification of apple juices based on their flavonol contents. 92 - 93 Although kaempferol derivatives have been reported by Van Buren 450 and Herrmann, 158 their presence has never been confirmed since then. Similarly, the presence of a 114 Downloaded by [North Carolina State University] at 17:29 17 December 2012 6. diglucoside quercetin found only by Fisher 1 •' seems unlikely. In apple fruit, the diversity is at the sugar level, because six different quercetin-3-glycosides have been characterized fully. They are all of a pyranose form with the exception of arabinose, which has a furanose form. The amount of each giycoside derivative (galactose, xylose, glucose, arabinose, rhamnose, and rutinose) and their relative balance are highly dependent on variety (Table 4). According to Teuber and Herrmann 425 and Oleszek et al., 294 hyperin (the galactoside derivative) is the most abundant. In the dihydrochalcone derivatives, two compounds have been characterized in apple and apple products. For a long time, it was stated that phloridzin (phloretin-2'-0- glucoside), a characteristic compound of the genusMalus (Rosaceae), 42 - 468 - 494 was present in leaves, stems, and seeds but absent in fruits. 493 However, a first description of phloridzin was reported in the ethyl acetate 7. extract of the core tissue of Mclntosh apples 95 and the methanol extract of peel of Democrat apples. 111 Then, using HPLC, this compound was fully characterized in the different parts of apple fruit 85 - 305 and in apple juice. 497 In the same extracts, another phloretin giycoside was also characterized corresponding to the xyloglucoside 294 as well as in Golden Delicious apple juices and jams. 431 - 432 The phloridzin content varied from 87 to 331 fig/g of fresh peel in eight cultivars grown in Canada 266 and from 21 to 105 |Xg/g for five cultivars grown in Spain. 306 In the cortex, levels were not as high as in the peel, as they ranged from 0.1 to 0.25 |Llg/g for the latter cultivars 306 on a fresh-weight basis and from 10 to 290 (ig/g for 11 French cultivars on a dry-weight basis. 7 In the anthocyanin derivatives, cyanidin was the only aglycone found in apple cultivars with red-colored skin. There is a general agreement to indicate that ideain (the cyanidin-3-galactoside) is the major pig- ment, 412 - 468 because it represented more than TABLE 4 Quercetin Giycoside Concentration in Apple Peel (tng.kg- 1 FW Basis) for Three Cultivars Grown in U.S., Eight Cultivars Grown in Canada, 266 Five Cultivars Grown in Spain, 306 and One in Germany 425 Cultivar Golden Delicious Empire Rl Greening Red Delicious Spartan Cortland Jerseyman Mclntosh Golden Delicious Gravenstein Northern Spy Starking Red Reinette Golden Delicious Verde Doncella Granny Smith Golden Delicious Rhamnoside 230 200 220 104 71 80 128 361 369 440 661 69.7 57.9 124 131 347 73 Rutinoside — — 117 57 159 185 117 110 139 169 0.5 1.1 3.3 15 19.2 3 Xyloside 100 110 150 210 244 192 119 254 195 145 297 — — — — — 34 Galactoside + glucoside 360 330 . 500 604 783 735 554 696 869 594 924 91 78 428 613 954 220 a Arabinosfde 130 140 200 546 662 589 556 777 539 508 801 21.1 21.3 29.1 47.7 105 75 Ref 35 35 35 266 266 266 266 266 266 266 266 306 306 306 306 306 425 a Only the galactoside derivative was assayed. 115 Downloaded by [North Carolina State University] at 17:29 17 December 2012 88% of the anthocyanins in 11 cultivars grown in England 427 and approximately 40% in the cultivar Scugog grown in Canada. 260 The presence of a cyanidin-7-arabinoside was postulated by Sun and Francis. 412 How- ever, later on, Timberlake and Bridle 427 raised some doubt as to the presence of derivatives with sugars linked in the 7 position of cyanidin. They indicated that all the deriva- tives are esterified by sugars in the 3 posi- tion. Moreover, the same authors 427 have found minor pigments corresponding to acylated forms of cyanidin-3-monoglyco- sides, but the nature of the acids involved in acylation was not determined. As already stated, several factors can induce large variations in both the quantitative and quali- tative content of phenolic compounds in apple. 2. Phenolic Variations at the Subcellular Level At the subcellular level, the phenolics are located mainly in the vacuoles. Yamaki 505 indi- cated that 97% of the total phenolics present in apple cells accumulate in vacuoles, while 3% are in free space and none in cytoplasm. According to the same author, 505 the calculated concentration of phenols is higher than 0.1 M in vacuoles of immature fruit flesh compared with the 1 to 10 mAf usually found in mature apples. 450 3. Phenolic Variations at the Tissue Level Although the subcellular distribution of soluble phenolics appears to be homogenous, the situation is different at the tissue level. Thus, it can be seen in Tables 1 and 3 that the epidermal and subepidermal layers (peel) have a higher con- tent of phenolics than the internal tissue (cortex). In different cultivars, the peel/cortex phenolic content ratio ranged from 3 to lO. 4 . 35 . 167 - 266 . 305 . 306 - 322 In a more precise study on the Calville Blanc variety, Macheix 232 - 234 divided apple fruit into four zones, from the outer to the inner parts of the fruit, corresponding to (1) the peel, (2) the outer part of the cortex (the major edible portion of apple), (3) the circular zone surrounding the car- pels, and (4) the central core, respectively. Zone 1 was the richest in chlorogenic acid, catechins, and flavonols. The flavonol content was less than 40 mg/kg (FW basis) in zones 2 to 4 compared with the 178 mg/kg in zone 1. The chlorogenic acid content was the lowest in zone 2 and in- creased slightly in zones 3 and 4. Although less important than in the peel, the catechin content was higher in zone 3 than in zones 2 and 4. Concerning chlorogenic acid content in the peel, this last result was slightly contradictory to the general comments given in Section 2.1.1. for Tables 2 and 3. This probably comes from differ- ences in the repartitions between peel and cortex used by the authors. Risch and Herrmann 336 also reported a greater amount of chlorogenic acid in the core than in the outer part of the cortex for the Jonathan cultivar. Similarly, Harel et al. 150 found a nonuniform concentration of o-diphenols in the flesh of the Grand Alexander cultivar. They re- ported that the amount of phenolics was highest in the peel, lowest in the outer part of cortex, and gradually increased toward the core. This uneven spatial distribution of phenolic compounds in apple fruit flesh is important, as it can induce different tissue sensitivities to enzymatic browning. 4. Phenolic Variations at the Cultivar Level A considerable variation was observed in the content of both total and individual phenolics among different cultivars of apple. Thus, in their compilations, Van Buren 450 and Herrmann 157 in- dicated ranges of 1 to 11 and 1 to 34, respectively, in the total phenolics content among varieties. Similarly, concerning the main individual phenolics, a variation of 1 to 10 was given for chlorogenic acid by Macheix et al. 236 and for (—)-epicatechin by Risch and Herrmann. 336 Simi- lar variations (1 to 9) were also found for fla- vonols in apple peel. 305 Apple cultivars with green- and yellow-colored skin are pigmented by chloro- phylls, carotenoids, 23 and quercetin derivatives 503 and are obviously devoid of anthocyanins. Never- 116 Downloaded by [North Carolina State University] at 17:29 17 December 2012 theless, a large variation (1 to 3) was also ob- served for ten Delicious apple strains with red- colored skin grown in the U.S. 394 Interestingly enough, the latter authors 394 found a correlation between the peel luminance L*, measured by a portable tristimulus colorimeter, and the antho- cyanin level of deep-red-colored fruits. 5. Influence of Maturity and Postharvest Storage Although, as already mentioned, the assay methods for total phenol content are not accu- rate, there is general agreement that the con- centrations of phenolic compounds are very high in young fruits and then rapidly decrease during fruit development. 236 Numerous studies carried out on the phenolic content of developing apples reflect this general trend. 69 - 150 - 157 - 229 - 466 - 488 - 516 The phenolic concentrations decline sharply 1 month after the petal drop, reaching a low level 10 weeks later, and remain approximately constant thereafter. 150 - 488 On a fruit basis, the change in total phenols is less pronounced dur- ing the 4- to 14-week period after the petal drop. Because in that period the number of cells is fixed, the decrease in the concentration of total phenols is primarily the result of a dilution of phenolic compounds in the vacuole. 236 After harvest, the concentration of total phenols remains essentially constant or decreases slightly. 35 - 69 - 150 - 172 - 229 - 445 - 448 The changes in content of the different classes of phenols and of some individual phenols have also been followed during apple fruit develop- ment and its subsequent storage after harvest. Thus, during apple development and 1 month after the petal drop, a general decrease in the hydroxycinnamic (caffeic, p-coumaric, and feru- lic) acid derivatives was observed. 284 An example is shown in Figure 3A concerning changes in the levels of /j-coumaroylquinic acid, p-coumaroyl- glucose, and chlorogenic acid (by far the most important). 233 For the latter phenolic compound, similar results were given by several au- thors. 35 - 69 - 230 - 466 During cold storage, the variations in the content of hydroxycinnamic acid deriva- tives were less important and, depending on the cultivars, the authors indicated a decrease, 283 - 284 a constant level, 69 or fluctuations. 172 - 445 On a fruit basis (Figure 3B), after the initial and rapid rise in chlorogenic acid (the same was observed for p-coumaroylquinic acid and p-coumaroyl- glucose), this phenol steadily accumulated, and it was only after 10 weeks that its amount decreased slightly. 233 Depending on the cultivars, this last decline was not observed 283 - 284 or only during cold storage. 69 - 172 The variations in catechins were simi- lar to those obtained with hydroxycinnamic de- rivatives. The concentrations in catechins rose sharply during the 30 to 40 d after flowering and then decreased rapidly to stabilize at a low level in mature fruit 230 - 283 - 284 and throughout the storage period. 35 On a fruit basis, a later peak was ob- served and the decrease was delayed to the end of the maturation period. 283 Moreover, in two apple varieties, an increase in the (-)-epicatechin-to- (+)-catechin ratio was observed during the pro- gressive growth of the fruit. 284 Only a few works have been devoted to the variations of quercetin and phloretin glycosides. The quercetin glyco- sides per gram of fresh weight remained fairly constant during the 2 months preceding the com- mercial harvest (var. Golden Delicious and Grimes Golden) but, because of fruit growth, increased on a per fruit basis. 503 Moreover, inside the com- mercial harvest, the same author 503 indicated that the flavonol content of green fruit was only half of that of yellow fruit (var. Golden Delicious). In that case, no change was observed in the total flavonol content during storage. However, for Mclntosh apples, a two- to threefold increase in the levels of quercetin glycosides was found during the first 2 months of cold storage. 85 A similar trend was found for phloridzin. 85 Finally, although the total quercetin glycosides remained relatively constant until the climacteric, considerable varia- tions were observed among the individual glycosides. 85 6. Influence of External and Internal Factors during Cultivation and Storage As in other fruits, the regulation of phenolic metabolism in apple depends greatly on both ex- ternal and internal factors (light, temperature, ethylene, growth regulators, nutrients, pesticides, 117 Downloaded by [North Carolina State University] at 17:29 17 December 2012 [...]... limitation in browning because of the lack of substrate or soluble enzymatic activity.236 Another published approach predicted the browning potential of grape varieties from their phenolic contents.213 A browning index was established for each individual phenolic compound using the maximum absorbance obtained after enzymatic oxidation The browning potential was then calculated as the sum of the browning. .. peroxidase in the presence of hydrogen 132 A Methods for Evaluation of Browning Accurate methods are required for the measurement of browning in tissue slices and extracts This need is obvious when different cultivars are compared for susceptibility to browning or for evaluation of experimental treatments designed to control enzymatic browning. 12 Basically, two kinds of methods are available.236 The first... of the enzymatic reaction and the nonenzymatic ways of o-quinone degradation.333 Finally, besides chemical parameters, physical parameters such as those related to firmness or bruise resistance193 are obviously of importance by limiting mechanical damage and therefore the amount of bruised tissue (i.e., of enzyme-substrate contacts).345-346-484 IV CONTROL OF BROWNING The control of enzymatic browning. .. Vm (Table 7) Use of cinnamic acids to control enzymatic browning in fruit juices has been suggested.471 Cinnamic acid added to Granny Smith juice at concentrations greater than 0.5 mM resulted in an inhibition of browning for over 7 h.471 However, apple plugs dipped in 10 mAf sodium cinnamate were protected for several hours but then exhib128 ited a severe browning over extended storage times.369 It... the surface of the examined object.198 During browning, variations in tristimulus data are the result of both chemical (browning) and physical changes, the relative importance of the two processes being difficult to assess Most authors use the decrease in lightness oL (i.e., the difference in L* values before and after browning) to evaluate the extent of browning Some authors have proposed a more sophisticated... evaluation of the browning sensitivity of apple cultivars.7 After suspension in water, the browned tissues are centrifuged and simultaneous measurements of soluble (absorbance at 400 run of the supernatant) and insoluble (lightness of the precipitate) pigments are carried out The degree of browning is defined as the sum of the two values after normalization.7 B Browning Susceptibility and Browning Parameters... methods for browning evaluation, numerous authors have attempted to correlate their results with the phenolic content and/or enzymatic activity of a p p l e s 7,69,150,172,193,230,323,446.448,465,488 As already emphasized for many other fruit species,236 there was no clear-cut relationship between the extent of browning and the substrate and/or the enzyme activities in apples Thus, it was found that browning. .. 2012 flavan-3-ols Thus, the two main classes of apple phenolics contributed to browning Because browning susceptibility resulted from both soluble and insoluble pigments, it was proposed to express the degree of browning as the normalized sum of the two parameters ( A ^ and L*) after normalization.7 Thus, the degree of browning of the 11 cultivars appeared to be strongly correlated (r = 0.933) to the... activity in bruised tissues during browning. 449 Other phenols may also be of importance for the final color, positively through cooxidation reactions298 or negatively by inhibition of the enzymatic reaction.7 Although the degree of browning did not appear to be correlated to ascorbic acid content,230-465-488 this compound could play a minor role in the extent of browning The same holds for acidity,... single wavelength correlate poorly with visual evaluation of browning In order to obtain information on the relative importance of browning parameters, it has been Downloaded by [North Carolina State University] at 17:29 17 December 2012 proposed to evaluate tissue browning under different conditions.374-488 Thus, "actual"488 or "real"374 browning was measured after homogenization of ground tissue in . of browning and the browning parameters are discussed, including a paragraph on the methods used for browning evaluation. Finally, the different methods for the control of browning . These general discoloration phenomena are mainly related to enzymatic browning. However, browning can also originate from nonenzymatic reactions such as the Maillard reaction, 16 - 106217 . estimate browning, the correlations among the intensity of browning and its causal factors, and, finally, the control of browning are then examined successively. II. PARAMETERS OF ENZYMATIC BROWNING