Browning of Orange Juice Survey of the Factors Involved M. A. JOSLYNAND G. L. MARSH,University of California, Berkeley, Calif. The browning of orange juices involves oxidation as a primary step. The primary products of oxidation then apparently undergo condensation reactions in which secondary reactions, probably amino acid-sugar reactions, occur. Interference wiih the formation qf the primary products of oxidation by removal of oxygen or addition of reducing substances prevents browning. Sulfites and stannous salts are found to be of value in this regard. The addition of small quantities of sulJites or other antioxidants to pasteurized or benzoated juices or sirups preserves the color of such products. The
reducing action of stannous salts, as well as the absence of oxygen in the canned orange juice, accounts for the fact that browning of canned juice does not occur either in plain tin or citrus enamel cans when stored at high or low temperatures. A large number of experimentally packed as well as commercially packed canned orange juices have been examined in the last four years without any browning having been found. Von Loesecke (6) reports similar findings. Storage of unheated benzoated juices and sirups in cans is being used to some extent commercially.
M
OST preserved orange juice products readily lose their characteristic orange-yellow color after a short period, become increasingly brown, ftnally reach the color of black tea, and acquire an offensively acrid taste. Oxidation of, and chemical reactions between certain constituents of the extracted juice apparently proceed with the formation of dark pigments. The browning of orange juice and especially orange juice concentrate has been studied by several investigators with somewhat conflicting results. McDermott (7) found that the darkening of the juice was caused chiefly by oxidation. This was confirmed by Matthew (9) and by the evidence to be presented here. Wilson and his associates (3, 16) have stressed the role of amino acid-sugar reactions of the Maillard or melanoidin type, and have obtained data indicatipg marked reduction in amino nitrogen and slight reduction in reducing sugar during browning of orange juice concentrate. Joslyn, Marsh, and Morgan (6) have shown that decrease in reducing value and ascorbic acid content of the juice accompanies browning. The evolution of carbon dioxide in sterile concentrate noted by Wilson and Hall (3, 16) has been ascribed to decomposition of ascorbic acid (6, 11). A summary of the investigations in this field is presented here, together with an outline of experiments made to confirm the role of certain factors and to smooth out contradictions in previous work. The investigations discussed were carried out to obtain some information concerning the nature of reactions involved in browning from the catalytic or inhibitive effects of various substances.
EFFECT OB SUSPBNDBD PARTICLES The juice extracted by burring consists of fibrous partitions of pulp vesicles, the chromatophores contained in them, particles of albedo and carpellary membranes, and soluble pectin and other gums suspended in the juice proper. The chromatophores and other solid particles can be readily removed by centrifugalizing, leaving a light straw-colored fluid (4, 13). The orange color and most of the flavor is removed along with the chromatophores. Most of the fibrous particles can be removed by straining. The solid matter in the strained juice, if allowed to stand for some time, gradually settles to the bottom of the container, leaving a t first a cloudy supernatant liquid.
On hydrolysis of the pectin, which is chiefly responsible for the cloud, this liquid becomes water-clear and a white fluffy precipitate of pectic acid settles on top of the pulp. Matthew found that during darkening the clear liquid turns a decided brown color while the chromatophores lose their orange color, become muddy, and ultimately turn the same hue as the darkened juice. The writers found this to be true in general although a t times the browning of the pulp was more noticeable and occurred before the liquid browned. As a rule the pulp in contact with the supernatant liquid becomes a dirty gray to muddy brown in color while the lower pulp retains more of its original color. Matlack (8) has shown that the chromoplasts contained in the juice sacs bleach readily on exposure to air. This was also found to be true by Matthew and the present writers. The darkening apparently occurs only in the juice proper, and the intensity of color change is proportional to the amount of juice present (cf. 9). Browning was least in the washed pulp separated by centrifugalizing and greatest in the centrifugalized or filtered juice.
EFFECT OF OXIDASE The browning of injured oxidase plants and their products has been ascribed to the activity of phenolases ( l a ) , but, although enzyme activity is responsible for part of the flavor changes observed, there is no evidence that it is involved in the browning of orange juice. A complete oxidizing enzyme system was not found in the orange by Onslow ( l a ) , McDermott (7), Willimott and Wokes (16),Matthew (9), and others; the peroxidase which was found occurs only in the flavedo, albedo, and carpellary membranes. Matthew (9)found that juice pasteurized a t 175' F. (79.5' C.) did not differin behavior from benzoated juice. The writers found no difference in the rate and intensity of browning in juices either benzoated, pasteurized a t 175' F., pasteurized a t 175' F. and then benzoated, or pasteurized at 212' F. (100' C.). Removal of suspended pulp by centrifugalizing, filtering brilliantly clear with Filter-Cel, or decolorizing with charcoal had no effect on the rate of browning. Furthermore, browning was found to continue and become more rapid a t higher temperatures. McDermott (7) reported that samples which were pasteurized a t higher temperatures darkened even more than those which had received less heat; and Matthew
186
February, 1935
INDUSTRIAL AND ENGINEERING CHEMISTRY
(9) found that heated samples darkened more than unheated. However, unless the heating is prolonged, and if care is taken to maintain other conditions alike, no appreciable differences are found between the pasteurized and benzoated samples. However, storage a t elevated temperatures does increase the rate of browning. The data available are too meager to make possible the calculation of the temperature coefficient for the reaction.
EFFECT
EFFECTOF EXTENTOF >RAPIDOXIDATION WITH OXYGEN O N IMMEDIATE COLOR C6LORUNITB
TIMESHAKEN IODINE TITRATION Red
~
Yellow
Hours 23.3 17.3 12.7 6.9 4.2 1.3
1.2
Apparently primary products of oxidation, probably peroxides, are involved in browning. The reducing substances present in the juice may act as inhibitors or antioxidants. McDermott also found that the browning of juice in the presence of oxygen was but little different from that in air alone. ilpparently oxygen is necessary only t o destroy certain inhibitors or form certain primary products, and only enough oxygen is necessary to produce these initial changes. Additional oxygen is apparently unnecessary.
OF OXYGEN
McDermott (7) thought that the darkening of orange juice was due to a simple process of autoxidation and showed that it could be prevented by removing the oxygen dissolved in the juice and that in contact with it. Matthew found that oxygenation increased darkening, and that very small quantities of oxygen can bring about considerable changes in color when the storage temperature is sufficiently high. Hall ( 3 ) found that long standing of dilute juices in the presence of oxygen causes darkening due to oxidation, and that bottled concentrate develops a dark layer a t the top in contact with the air in the neck of the bottle. The writers have found that in the absence of oxygen practically no browning of the juice occurs. Increasing the amount of air in contact with the juice to a certain extent increases the extent of browning and in some cases the rate of browning. It was found that browning of juice in the presence of air was not apparent until the iodine reducing value of the juice decreased to approximately half its initial value. After this point was reached, browning became more marked and increased in intensity a t an apparently faster rate than the decrease in iodine reducing value. However, complete oxidation of the iodine reducing substances with iodine, hydrogen peroxide, or oxygen did not result in any immediate color change. It was found that the iodine reducing value could be rapidly reduced by shaking the juice with oxygen without having any immediate effect on color. The following data are typical of the results obtained. The volume of 0.01 N iodine solution required to oxidize 50-cc. portions of filtered Valencia juice shaken with about 200 cc. of oxygen a t 25' C. is shown in Table I. The color of the samples was determined in a color comparator, using Lovibond color slides. TABLEI.
187
1.3 1.3 1.3 1.3 1.3 1.3 1.3
12 12 12 12 12 12 12
To determine the length of time necessary for color to develop in oxidized juice in comparison with unoxidized juice, a sample of the above juice was shaken with oxygen for 15 hours until its iodine reducing value was about 1.0. One thousand cubic centimeters of the same juice were exposed to air in a cotton-stoppered, 4-liter bottle. Color readings and, on the latter lot of juice, iodine titrations were made a t periodic intervals. The results shown in Table I1 indicate that the browning proceeded at practically the same rate in both cases. This was confirmed by other tests. It was found that browning occurred in oxygenated orange juice even in the absence of air. Samples of juice shaken with oxygen for 15 hours were slightly boiled under vacuum and sealed in an atmosphere of carbon dioxide. Browning occurred to the same extent as in samples exposed to air.
TABLE11. BROWNING OF OXIDIZED AND UNOXIDIZED JUICE -UNITS
OF JUICE EXPOBED
IODINETITRATION OF TO A I R PERIODJUICEEXPOSED TO AIR Yellow Red
STORAQE
Days 0 2 3 5 6
23.3 11.6 6.0 0.7
..
10 12 19
12 12 12 15 17 20 25 25
COLOROXIDIZED JUICE
Yellow
Red
12
1.3 1.3 1.8 2.5 3.1 4.8 5.0 5.5
1.3 1.3 15
12 12 15 20 20 25 25
2.6 2.5
4.5 5.0 5.5
EFFECT OF CERTAIN REDUCING OR OXIDIZING SUBSTANCES The addition of 0.05 milliequivalent of aluminum, barium, copper, iron, magnesium, manganese, nickel, or zinc ions did not influence the behavior of juices tested by Matthew (9). However, the writers found that the addition of ferrous salts markedly increased the rate and extent of browning in concentrations as low as 25 p. p. m. The browning increased with increase in concentration of ferrous salt. Ferric salts increased the browning to an appreciable extent but not as markedly as ferrous salts. Nickel, copper, or stannic salts were without effect. Stannous salts and sulfites protected the juice exposed to air from browning until they were oxidized to an extent sufficient for the inception of the primary oxidation processes involved in browning. A concentration of 1000 p. p. m. of sulfur dioxide will check browning for a period of 60 days in juice exposed to air in cotton-stoppered containers; smaller amounts of sulfur dioxide are adequate to prevent browning in sealed containers. Matthew (9) found that samples of pasteurized juice containing 90 p. p. m. of added sulfur dioxide kept well in color for over a year a t 60" or 90" F. (15.6" or 32.2' C.) when sealed with about 25 cc. headspace in 120-cc. bottles. The effect of sulfur dioxide on the iodine titration of orange juice exposed to air is shown in Table 111. To one lot of juice containing 0.2 per cent sodium benzoate was added 2.2 grams of XatS03.7HzO per 100 grams (about 500 p. p. m. of sulfur dioxide) and 1000-cc. portions of the sulfited and untreated juice were exposed in cotton-plugged, 4-liter bottles. TABLE111. EFFECTO F SULFITE ON R l T E O F REDUCTION IN IODINEREDUCIKG VALUE STORAQE
PERIOD
UNTREATED SULFITED STORAGE UNTREATED SULFITED JUICE JUICE PERIOD JUICE JUICE
Days
0
2 6 10
29.7 26.9
17.8 9.6
79.5 71.9 61.6 55.6
Days 14 20 25 35
2.2 1.0 1.0
1.0
48.9 40.4 33.1 15.6
The untreated juice began to brown after 10 days and was very dark after 20 days; the sulfited juice began to darken after 30 days. It is thought that the effect of reducing substances such as sulfites, phosphites, arsenites, or stannous salts may be due t o their being more readily oxidized than the naturally occurring reducing substances. There is a little evidence that in sulfited juice the sulfur dioxide is oxidized before ascorbic acid, etc. However, the mechanism of the protective influence of such reducing substances is under investigation.
188
INDUSTRIAL AND ENGINEERING
The addition of catechol and tannin to orange juice increases browning, whereas the addition of maleic acid, an oxygen acceptor, decreases the rate and extent of browning. EFFECTOF AMINOACIDS The amino acids shown to be present in orange juice by Hall (3), Smith (IS), and Nelson (11) may form condensation products of several kinds. These may be similar to those occurring in neutral or slightly alkaline solutions, known as the “Maillard” or “melanoidin” type, which have been referred to by Wilson ( I @ , Hall (S), Browne ( 2 ) , and Nelson (11). They may be similar to those responsible for humin formation in acid hydrolysate of protein (10); or they may be similar to the condensation of formaldehyde and other aldehydes with aromatic amines in neutral, alkaline, or acid media (14). Various nitrogenous substances (aniline, diphenyl amine, n-butyl amine, di-n-butyl amine, alanine, glycine, glutamic acid, aspartic acid, leucine, tyrosine, cystine, tryptophan, arginine, histidine, asparagine, and ammonia) were added in amounts to give 0.1 per cent nitrogen to orange juice and to acid solutions of invert sugar to determine their effect on browning. In 10 per cent invert sugar solutions in the presence of 1 per cent citric acid, the addition of aniline and tryptophan caused an immediate browning; butyl amine caused a slight yellowing. The reaction of aniline with an acid solution of dextrose or invert sugar was very rapid; change in color occurred almost immediately. Evacuation and closure under vacuum did not have any effect on the reactions induced by these substances. Gas evolution was noted in certain cases during evacuation. The substitution of filtered navel orange juice for Valencia juice changed the extent and type of effect. Thus, in Valencia juice after storage for 6 months in closed containers, the order of increasing darkening was as follows (parentheses enclose those closely similar in color): (blanks), (glutamic acid, cystine, leucine, ammonia, asparagine, glycine, butyl amine), (di-nbutyl amine, tyrosine, tryptophan, aspartic acid, alanine, diphenyl amine), aniline. In navel juice the order was: di-+butyl amine, (checks), n-butyl amine, (alanine, ammonia, tyrosine), aspartic acid, (glycine, diphenyl amine), cystine, (asparagine, glutamic acid, leucine), aniline. In general the addition of asparagine, aspartic acid, or histidine did not appreciably increase the browning although these substances were reported present in Florida Valencia juice (11). The addition of aniline or tryptophan markedly increased browning. The amino acid reaction which may occur in orange juice is thus probably a condensation between sugars and aromatic amines-(ld). These results explain Matthew’s findings (9) that the addition of small amounts of asparagine, urea, or casein to the juice did not increase the rate of browning; that the same substances in water solutions of citric acid and glucose did not form a brown soluble substance; and that no carbon dioxide formation was found in the juice during browning. Changes in amino nitrogen during browning were followed in both Valencia and navel juice preserved with sodium benzoate by both titration in the presence of formaldehyde and by the Van Slyke method. The results obtained for one 1000-cc. lot of Valencia juice stored in a 4-liter, cottonstoppered bottle and for a 3000-cc. lot of navel juice so stored is shown in Table IV. Practically no change in amino nitrogen accompanied darkening. Similar results were obtained when the changes in amino nitrogen were followed by formaldehyde titration. Although the juice exposed to air browned and rapidly decreased in iodine reducing value, practically no change in formaldehyde titration was found.
CHEMISTRY
Vol. 21, No. 2
It is possible that hydrolysis of a soluble polypeptide might maintain a fairly constant amino nitrogen content even though the latter substances were involved in the reaction. Proteins were demonstrated to be present in the chromatophores by Smith (IS). However, the present results support those previously obtained by Smith and subsequent results by Nelson (11) on the negligible decrease in amino nitrogen content of orange juice on browning. Nelson (11) reported no change in the nature of the bases present in Florida Valencia orange juice on browning. TABLE IV. CHANGES IN IODINE REDUCING VALUEAND AMINO NITROGEN CONTENTDURING THE BROWNING OF NAVEL AND VALENCIA ORANGEJUICE STORAQE IODINE PERIOD TITRATION AMINON Days Mg./lOO cc.
COLOR
NAVEL JUICE
0 7
27.0 18.2
33.7 34.0
Light straw-yellow Very light amber, incipient brown-
35 71
12.5 7.5
33.4 33.4
Very brown Dark brown
0
18.8 15.9 11.5 6.3 3.0 2.5 2.5 2.5
iw
VALIONCIA JUICE
1 3
5 7 9 17 126
80.4
*...
7913 79:3 78.5
Verylight yellow No change Light amber Very brown Darkbrown Dark brown Dark brown Very dark brown
EFFECTOF FERMENTATION If reducing sugars are involved in the browning of orange juice, then their removal by fermentation might be expected to prevent browning. Hall found fermented juice and juice concentrate to be more stable. Furthermore, Bennett and Tarbert (1) report that the reducing power of orange juice toward dichlorophenolindophenol does not diminish much in storage in the absence of preservatives; this juice fermented. In order to determine the effect of fermentation, alcohol, and oxido-reductase enzymes secreted by yeast, 1300-cc. portions of the following lots of juice were stored in cotton-stoppered, 4-liter bottles a t room temperature: A = B = C = D = E = F = G = H = I =
unfermented juice plus 0.2 per cent sodium benzoate fermented juice fermented juice, dealcoholized by vacuum distillation fermented juice, dealcoholizedby distillation fermented juice, heated under reflux for 30 minutes benzoated juice, heated under reflux for 30 minutes fermented juice plus 0.2 per cent sodium fluoride benzoated juice plus 5 per cent alcohol benzoated juice plus yeast juice
Valencia orange juice was used in these tests; fermentation was carried out with Fleischmann’s compressed yeast. The analysis of the juice follows: BEFORIO FERMENTATION AFTIOR FBRYENTATION Before After BefoFe After filtration filtration filtration filtration 1.0020 1.0020 1.0533 1.0458 (23/20) (21/20) ‘2;w;’
y y
Sp. gr. Balling degree Acidity, gram citric/100 Alcohol, yoby vol. Iodine titration CoLor; Hed Yellow
CC.
0.928
0.800
... ...
19.3
... ...
2.1 15.0
...
... ...
5.0
... ... ...
... ...
4.6 11.0 2.1 20.0
The color and iodine reducing value of the samples were observed periodically with the results shown in Table V. Fermentation did not inhibit browning, and destruction of yeast enzymes by heat, their inhibition by fluoride, or the addition of yeast juice to orange juice did not have any marked effect on browning.
February, 1935
INDUSTRIAL AND ENGINEERING CHEMISTRY TABLEV.
SAMPLE A
O
189
EFFECTOF FERMENTATION, ALCOHOL, AND YEASTJUICE ON BROWNING 0
lodine titration Units red Units yellow B Iodine titration Units red Units yellow C Iodine titration Units red Units yellow D Iodine titration Units red Units yellow E Iodine titration Units red Units yellow F Iodine titration Unit6 red Units yellow G Iodine titration Units red Units yellow H Iodine titration Units red Units yellow Iodine titration I Units red Units yellow Number of days elapsed in parentheses.
16.6 2.2 15.0 11.0 2.2 20.0 9.8 2.2 20.0 8.2 5.5 20.0 9.6 6.3 20.0 14.9 11.0 20 11.0 2.2 16 16.2 2.2 16 13.5 2.2 15
2 12.0 2.3 15
4 8.1
6.7
6.3
2.6 20.0 6.6 2.6 20.0 3.2 5.3 20.0 2.7 5.0 20.0 10.3 11.0 20 6.2 2.5 20 9.7 1.8
EFFECTOF ACIDITY
If decomposition of ascorbic acid by a process similar to that occurring during heating of uronic acids with 12 per cent hydrochloric acid is involved in browning, then increasing the acidity should increase the rate of browning. Matthew (9) reported that increasing the concentration of hydrogen ions increased darkening, and decreasing it decreased browning. McDermott (7) found that increasing the concentration of citric acid had no effect on browning, neutralizing the acid prevented browning in some cases, but juice made alkaline darkened rapidly. I n the presence of sufficient air, increasing the concentration of hydrogen ions by addition of citric acid decreases the rate and extent of browning. In the presence of a limited amount of oxygen, increasing the acidity has little effect. NATUREOF BROWNPIGMENT A resinous brown precipitate gradually forms in juice exposed to air for several months. It was found that the brown pigment could be, in large part, precipitated with excess alcohol or acetone after concentration in vacuo. However, not all of the brown color could be so removed. The brown pigment is also precipitated by neutral lead acetate and by some of the other heavy metals. An amorphous brown pigment was separated from Valencia and navel juice and from old Valencia concentrate by precipitating with lead, deleading with dilute sulfuric acid, and concentrating and reprecipitating with alcohol. The material was found to be insoluble in ethyl ether, petroleum ether, benzene, toluene, and practically insoluble in strong ethanol, methanol, or ethyl acetate. Fusion with potassium hydroxide failed to yield a test for phenolic residues; tests for aromatic nuclei were negative. On drying in vacuo a t 70” C., some of the material sublimed. The ash content of the pigment obtained in this manner from benzoated browned Valencia orange juice, benzoated browned navel juice, and old Valencia concentrate, respectively, was as follows (in per cent) : 28.4, 18.1, and 8.0. On an ash-free basis the respective elementary compositions were as follows : carbon, 40.6, 41.8, 46.0; hydrogen, 5.0, 4.15, 4.8; nitrogen 2.44, 2.44, 2.22. It is surprising that the pigments from such varied sources were practically similar in composition. Their most striking property is their high nitrogen content. Apparently the substances formed in all cases were essentially the same.
OF
STORAQE 6 3.3 3.3
15
6.6 2.1
6.0 4.5 20.0 5.0 4.5 20.0 2.1 6.0
1.9
7.1 8.0 6.6
20
11.2~ 1.8 15
DAY^
7.OP)O 2.3
20.0
20.0 1.6 6.5 20.0 3.3 7.8 20 1.3 3.5 20 1.5 2.5 20 3.0P)‘ 4.3 20.0
7
15
27
5.8
16: 5 20
13:o 20
20.0
20.0
8 0.5
15 5.0 5.0 4.0 5.0
20.0
2.1 8.0 20.0 2.0 6.5 20.0
S:O 13:o
20.0
15:o
20.0
7:0
20.0
0.8
10.5 20 0.8
3.8
20.0 0.5 4.3 20.0
i.’s
20.0
*.
.... ..
.. *. .. .*
.. ... . .,
1215 20
13:o 20
4:5 20.0
i:2 20.0
7:5 20
8:3 20.0
0.8(“)0
9:3(1’)”
20: 0
..
Attempts were made to precipitate orange juice fractionally with lead acetate a t various pH levels in order to determine in which fraction the browning was concentrated. Both the lead precipitate and the filtrate were found, after deleading and bringing to the initial pH, to brown a t about the same rate. However, the addition of neutral lead acetate made the solutions sufficiently basic t o cause color change; this bleached on deleading with dilute sulfuric acid. Precipitation with lead, however, was found to be of value in partially purifying the brown pigment before precipitation with alcohol. ACKNOWLEDGMENT The authors wish to thank W. E. Baier, director of the Research Laboratories of the California Fruit Growers Exchange, for making available to them the reports of Hall and his associates on the darkening of orange concentrate.
LITERATURE CITED (1) Bennett, A. H., and Tarbert, D . J., Biochem. J., 27,1294 (1933).
(2) Browne, C. -4.. IND. ENG.CHEM.,21,600 (1929). (3) Hall, J. A., and Nedvidek, R. D., unpub. repts. to Research Lab., Calif. Fruit Growers Exchange, 1924-27. (4) J o s h M. A,, IND.ENG.CHEM.,24,665 (1932). (5) Joslyn, M. A,, Marsh, G. L., and Morgan, A. F., J. Biol. Chem., 105, 17 (1934). (6) Loesecke, H. W. von, Mottern, H. H., and Pulley, G. K., ISD. ENG.CHEM.,26,771 (1934). ( 7 ) McDermott, F. A., J. IND.ENG. CHEM., 8, 136 (1916); Fla. Agr. Expt. Sta., Bull. 135, 130 (1917). (8) Matlack, M. B., Am. J . Pharm., 100,243 (1928). (9) Matthew, rllexander, hl. S. thesis in agricultural technology, Univ. Calif., 1928. (10) Mitchel, H. H., and Hamilton, T . S., “Biochemistry of a m i n o Acids,” p. 119, New York, Chemical Catalog Co., 1929; Gortner, R. A,, “Outlines of Biochemistry,” pp. 332-3, New York, John Wiley & Sons, 1929. (11) Nelson, E. K., Mottern, H. H., and Eddy, C. W., Fruit Products J.,12,231 (1933). (12) Onslow, M. W., Biochem. J., 13, 1 (1919) : 14,535, 541 (1920); 20,1138 (1926). (13) Smith, A. H., J . B i d . Chem., 63, 71 (1924): unpub. repts. to Research Lab., Calif. Fruit Growers Exchange, 1924. (14) W a g n e r , E. C., J. Am. Chem. Sac., 55, 724 (1933). (15) Willimott, S. G., and Wokes, Frank, Bz’ochem. J., 20, 1008 (1926). (16) Wilson, c. P., I N D . ENG.CHEM., 20,1302 (1928). RECEIVBID September 25, 1934. Presented before the Division of Agricultural and Food Chemiatry a t the 88th Meeting of the American Chemical Society, Cleveland, Ohio, September 10 to 14, 1934.
