DETERIORATION OF DRIED FRUITS Use of Ion Exchange Resins to Identify Types of Compounds Involved in Browning VICTORIA A. HAAS AND E. R. STADTMAN University of California, Berkeley, Calif. Ion exchange resins have been used to separate apricot concentrates into three fractions of distinctly different composition: a cation fraction containing 81% of the nitrogenous constituents and all of the inorganic cationa, a neutral fraction containing 98cJ, of the sugars, and an anion fraction containing 88yo of the acids other than amino acids and acidic proteins. The browning of apricot concentrate was compared with the browning of the individual fractions and with combinations of these fractions. These experiments indicate that browning is the result of at least four types of reactions : between the nitrogenous constituents and the sugars, between the nitrogenous constituents and organic acids, between sugars and organic acids, and reactions involving only organic acids.
THE
4 identification of the reactions involved in browning of food products is complicated by the fact that browningis but one of many forms of deterioration which occurs during storage. The discovery that browning of citrus juice concentrates is accompanied by decreases in amino nitrogen and reducing sugar (4)has given rise t o the speculation that browning is the result of a reaction in which the condensation of sugars and amino compounds is followed by polymerization and pigment formation. Contrary t o the results with citrus concentrates, no change in amino nitrogen could be detected during the browning of unconcentrated orange juice (7, 10). Bedford ( 1 )was unable t o demonstrate a significant change in any nitrogen fraction or in sugar content during the browning of apricot concentrates. Using apricot concentrates (30% solids) which had been stored (15 days at 56" C.) until precipitation of dark material began, the authors were unable to find any change in total nitrogen or sugar concentration. Experimental evidence of this kind does not constitute proof either for or against the hypothesis that sugars and amino acids are involved in browning of fruit products. The mere fact that losses in these substances are concurrent with browning does not in itself show that the two phenomena are interdependent. Changes in flavor, texture, and odor which can occur independently of browning might equally well be attributed to observed changes in sugar and amino nitrogen content. Conversely, the failure to demonstrate losses in amino nitrogen and sugar during browning does not exclude the possibility that these substances are involved. It is possible, for example, that only minute quantities of both substances react to form highly colored pigments, in which case the absolute changes in concentration of the materials might be so small as to escape detection by means of the analytical methods available. There is, in fact, considerable evidence in support of the idea that browning involves only very small quantity changes (14). A more satisfactory approach t o a problem of this kind, which avoids the above difficulties, would be t o remove selectively specific components suspected of participation in the browning reaction and thus determine their effect. The difficulty lies in finding methods whereby the desired component can be quantitatively removed from the complex mixture without producing marked changesin theothercQmponentswhicharepresent. Sugars can be more or less selectively removed by fermentation with yeast.
By means of this technique, Stewart et al. ( 1 7 ) were able to prove that glucose is an essential precursor to the browning of dried eggs. With apricot concentrates, fermentation decreases the rate of browning to 30 to 40% of the original rate ( 3 ) . On the other hand, the removal of sugars from orange juice b y fermentation results in no significant change in the rate of browning ( 7 ) . The fermentation technique has certain disadvantages: It is not entirely specific. Growth or autolysis of the yeast during the fermentation may produce a change in some of t h e nitrogenous constituents. By-products such as aldehydes and ketones may be formed during fermentation and thereby modify the results. And finally, the sugars cannot be recovered for analysis or control testing. It occurred to the authors that synthetic organic ion exchange resins might be used to separate fruit juices into more or less homogeneous groups of chemical substances which could subsequently be tested for their ability t o produce browning. This paper describes a method of fractionating apricot concentrates into three distinctly different classes of compounds and illustrates the use of these fractions in the study of the browning reaction. PREPARATION OF FRACTIONS
Two organic exchange resins (manufactured by Chemical Process Company San Francisco, Calif.), Duolite C-3, a cation exchange resin, a n 4 Duolite A-3, a n anion exchange resin, were used. The Duolite A-3 resin was chosen because it is reported to be less reactive with aldehydic sugars than other resins (a). Columns (5 X 50 cm.) of the type described by McCready and Hassid ( 1 1 ) were used. The general method of operation was t h e same as previously described (2). The Duolite C-3 column was pretreated with 4 volumes of 2 N hydrochloric acid and washed with distilled water until a negative test for chloride was obtained with silver nitrate. The Duolite A-3 column was pretreated Rith 4y0sodium hydroxide and washed until the p H of the wash water fell below 8.5. To produce the various fractions, 100 ml. of apricot concentrate. (30.4% solids), diluted t o 500 ml., were passed over the Duolite C-3 (cation) resin and washed with three bed-volumes of distilled water which was added to the effluent material. After washing with 40 t o 60 bed-volumes of distilled water the Duolite C-3 resin was eluted with 1 liter of 2 N hydrochloric acid. This eluate was called the cation fraction. The effluent from the Duolite C-3 resin was then passed over the Duolite A-3 (anion) resin and after washing as described above, the resin was eluted with 250 ml. of 1 N sodium hydroxide. This eluate was immediately passed over a freshly regenerated Duolite C-3 (cation) column to replace the sodium with hydrogen ions. This fraction is called the anion or acid fraction. Those substances not removed by either the Duolite A-3 or C-3 resin make up the neutral fraction. Each fraction was concentrated in vacuo (2 to 5 mm. of mercury, T < 35 ' C.) to the original volume of 100 ml. During this vacuum concentration the greater portion of excess hydrochloric acid present in the cation fraction volatilized. COMPOSITION OF FRACTIONS
The Kjeldahl nitrogen and reducing values of each fraction were determined. The results (Table I) show that clear-cut separations of groups of compounds had been made. T h e cation fraction contained 95y0 of the recovered nitrogen, 55% of which is a-amino nitrogen (formol titration), and should also contain the inorganic cations. The neutral fraction after acid
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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hydrolysis contained 08% of the reducing value of the original concentrate (after acid hydrolysis) and therefore contains at least 98% of the original sugars. The anion fraction contained negligible amounts of nitrogenous and reducing substances. This fraction should contain all the fruit acids other than the amino acids and acidic proteins, which would be found in the cation fraction.
I n addition t o studying the influence of the gross fract,ioiis otjtained by ion exchange resins it is also possible t o evaluate ttic effect of specific compounds 011 firom-riing. The conccntratiori of a specific compound in a fraction may be determined and t,lw effect, of this concentration of the pure compound on browning may be estimated by adding this component, rather tlian 1,l~i~ fraction itself to a mixture of the other fractions. This t.echniquc: is illustrated in the following experiments. INFLUENCE OF GLUCOSE ON BRQWNINC,
TABLE I. ANALYSIS OF APRICOT SIRUPAXD FRACTIONS (All values expressed on d r y weight basis) Total Reducing Valuea Reducing Valueb, hlg. G l ~ c o ~ e / G . hlg. G1ucose;G. 740 243
Kjeldahl, 3Ig. N / G 13.: 0.l 10.6
Original sirup "Inion fraction Ij.2 Cation fraction ... 1.6 Neutral fraction 699 238 0.3 a Determined by Hassid's Ce(SO4)n method (6)after inversion with invertase. b Determined by IIassid's Ce(SOd? method (0)before inversion.
...
The original apricot concentrate contained 49 rnilliequivalents of titratable acids (phenolthalein end point). After passage of the concentrate over the Duolite C-3 (cation) resin the effluent contained 93 niilliequivalents of titratable acids. Thus 47 % of the acids were originally present as salts. The anion fraction contained 82 milliequivalents or 55% of the acids present in thf. effluent from the Duolite C-3 (anion) resin. APPLICATIOSS TO STUDIES ON BROWNING
With this separat,ion of apricot concentrate into three fractions, each containing dist,inct groups of substances, it became possible t o det,ermine the influence of each group on the over-all browning reaction. T o determine the browning produced by any fraction or combination of fractions, the fractions to be tested were combined and the mixture was adjusted to p H 3.6 (the p H of original coneent,rate) with 1 N sodium hydroxide or 1 N hydrochloric acid. T h e samples were then coiiceiit,rated in vacuo a t room temperature to the same volume concentration as the original concentrate. The samples were stored in air at 56 ' C. and the browning was measured colorimetrically ( 3 ) . The relative importance of various types of browning reactions might be different at 56" C. than at room temperature, but the same reactions probably occur a t both temperatures. Experiments with dried apricots (16, 16), showed t h a t oxygen has a, relatively great'er effect' on deterioration at 50" C. than at lower temperatures, but in other respects the deterioration appeared to be qualitatively the same between 22 a and 50 O C. When the three fractions, anion, neutral, and cation, were combined and stored, the mixture darkened a t the same rate as the original unfractionated concentrate (Table 11); this indicated that most' of the constituents involved in browning were rccovered in the three fractions. Examination of Table I1 shows that when stored separately, little or no browning occurred in the neutral or cation fractions and browning wa,s relatively slight in the anion fraction. When any two fractions were combined, appreciable browning occurred, but at a considerably slower rate than in a mixture of all three fractions. Moreover, the summation of the browning of the three possible combinations (anion-neutral, anion-cation, and cation-neutral) is less than that observed in a mixture of all three fractions. However, in the anion-cation mixture precipitation of the dark material occurred. Thus the browning values reported for this mixture are too low. Because there was no simple way of evaluating the browning represented by this precipitate, it was not possible t o determine lvhether t h e discrepancy between the browning of the anion-cation-neutral nlixture and t h e summation of the browning of the three binary mixtures was due t o the precipitate in t h e anion-cation mixture, or t o secondary reactions possible only when all three fractions were present.
Vol. 41, No. 5
T o determine the effect of glucose on browning, the rleutr,ill fract'ion was analyzed for total reducing value after inversioii and glucose equivalent t o this value was added to a mixt>ureo f the other two fractions. The followiiig mixtures Jvere storeil: anion-cation, anion-cation-neutral, and union-cation-glucose. The addition of glucose gave a rntje of browning 60% of that of the, neutral fraction. This shows that glucose is involved in browning but that glucose does not coinpletely substitute for the neutral fraction. Therefore, other substances present in the neutra I fraction must also be involved in browning. Analysis of tho neutral fraction showed that 82% of the total solids were sugam [52% sucrose (6, 61,17% glucose (9),and 13% fructose]. I n n + much as fructose is a morc reactive sugar than glucose ( I S ) t h ~ substitution of glucose for fructose in the above experinleiit may account for the low rate of browning as compared with t l i i z neutral fraction. An experiment in which a 52% sucros(' 13% fructose --17% glucose mixture is substitute3 For tllr neutral fraction would show whether only these sugars contributcb to the browning of the neutral I'ractiori or tho 18% notisiig:ir' solids of the neutral fract,ion also contribute to brox-ning. IYFLUESCE O F FRI;I'C ACIDS
I t was of int,eresl t)o know whet'her phosphate might be i i i ~ important agent in browning, as it has been shown to contribut (' t o the brownirig of milk powder ( 8 ) . Any pliosplialc present ~ I I the original apricot' concentrate should bc found in the a,riiorl fraction. Analysis of t,he anion fraction shon.cd 12.4 ing. o f potassium dihydrogen phosphatk per grain of conceiitratt:. on a dry weight basis; therefore, the follon~ingmixtures w ( > i ' ~ prepared t o determine the cffcct of phosphate on browning: neutral-cation, neutral-cation-anion, arid iieutral-cation-pliorphate where phosphate wa? added in the same conccntratiori as was present in the anion fraction. Table I1 shows that phospkmtt: present in this concent,ration had little or no effcc(' on the rnti: OF darkening.
T&BIaJC TI.
DARKENING OIi' APRICOT COSCEKTRATE ~ T O R I G I ),\'I' 56" C .
Lnfractionated coni,entrate .Inion-neiitral-carion Anion-neutral-cation ash Anion-neutral Anion-cation Neutral-cation Anion Cation Neutral Neutral-phosphate Neutral-cation-phosphate a Precipitate of dark material
Change of Darkenink. iii,lex .. 4 days 11 days Is (I?,\... 17.1 62.5 203 20.0 9 1
63.3 17:6
5.8
13.8
7.0"
3.9 4.9" 0 0
0
4.6
247
11.8R
12.3
4.4" 0.4 0 0
14.2
The neut,ral fract,ion shomed no browning whcn st,orod n,lorlc., although the p H had been adjusted to 3.6 with hydrochloric acid. When the neutral and anion fractions were mixed, adjusted t o pH 3.6, and stored, considerable browning occurred. Therefore, the accelerated ratc of browning produced by the anion fractioii when added t o the neutral fraction is not a general effect nf hydrogen ions, but, must be caused by some specific acid. The iiifluerice of specific organic acids on the browning reactiori was
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TABLE 111. EFFECTOF ORGANIC ACIDSON DARKENING Neutral-cation-anion Neutral-cation-citratea Neutral-cation-malatea Neutral-cation-galacturonateO Neutral-cation
Change of Darkening Index 5 days 16 days 32.9 292.9 13.9 78.2 9.7 54.9 31.6 47.9 4.0 25.0
a Amount of acid added per 5 ml. of mixture = 232 mg. of citric acid, I12 mg. of malic acid, or 11.6 mg. of galacturonic acid.
then determined by storing the following series of samples: neutral-cation-anion, neutral-cation, and neutral-cation-specific organic acid. Galacturonic, malic, and citric acids were added in concentrations similar to that found in the anion fraction. [Galacturonic acid has been showed to ’be involved in some browning reactions (la).] The addition of each acid increased the rate of browning above that of the neutral-cation mixture. However, the sum of the effect of all three acids was not so great as that of the anion fraction, so that some untested substance must contribute to the browning caused by the anion fraction, When calculated on a n equal molecular basis galacturonic acid is the most effective as a browning agent of the acids tested, causing twelve times as much biowning as the same amount of citric acid and fourteen times as much as malic acid. The cation fraction contains both inorganic cations and amino acids and proteins. To determine the relative importance of each group of cations t o the browning reaction the cation fraction was ashed t o destroy all organic material and the following mixtures were stored: anion-neutral-cation, anion-neutral, and anion-neutral-cation ash. The inorganic cations were responsible for only 6% of the browning caused by the complete cation fraction and are therefore of relatively little importance to the browning reaction as compared with the organic cations. DISCUSSION
For many years the browning of fruit products has been ascribed to a sugar-amino acid reaction. Strong evidence against this hypothesis, as applied t o apricot concentrates, was obtained by Bedford ( I ) , who was unable t o detect changes in any nitrogen fraction or in reducing sugars during the browning of this product. However, with the fractionation of apricot concentrate by means of ion exchange resins the authors were able to demonstrate in the experiments described above that a sugar-amino acid reaction does contribute to browning. I n addition, it was shown that browning is produced by reactions between cations and anions, anions and neutral substances, and reactions involving the anions only. These represent only general types of reactions. Undoubtedly, each of the three groups of compounds contains several substances which are capable of participating in browning reactions. For example, in the study i t was shown not only t h a t the importance of the anion fraction was due to acidity but that specific organic acids contained in this fraction differed in their ability to produce browning. Thus browning is not the result of a simple process but is the manifestation of a large number of unrelated reactions of various kinds, each giving rise t o dark pigments. With the evidence that browning is produced by several mechanisms it is obvious that future research must be directed toward the oomplete characterization of the individual reactions capable of producing browning. In view of the fact that only small quantity changes are involved in these reactions, it is not possible to determine the identity of the reactions by correlating the change in color with change in concentration of suspected precursors. The inadequacy of this approach has been demonstrated with studies on apricot concentrates where it was impossible to detect any change in the concentration of sugars or of any nitrogen fraction
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during the browning of this product; yet with fractionation of the apricot concentrate by means of ion exchange resins, it was possible t o prove that both groups of compounds were involved in browning. By further development of the ion exchange technique it should be possible to determine which fruit constituents are important in browning and to identify the reactions in which they are involved. The ion exchange technique should have wide application to problems where it is of interest to characterize a particular reaction in a complex mixture. SUMMARY
Ion exchange resins have been used to separate apricot concentrates into three fraction$ of distinctly different composition: a cation fraction containing 81yo of the nitrogenous constituents and all of the inorganic cations, a neutral fraction containing 98% of the sugars, and an anion fraction containing 88% of the acids other than amino acids and acidic proteins. Most of the constituents responsible for browning were recovered in these three fractions. Storage experiments show that the over-all browning is the result of at least four distinctly different types of reaction: reactions between the nitrogenous constituents and the sugar, reactions between the nitrogenous constituents and organic acids, reactions between sugars and organic acids, and reactions involving only organic acids. Glucose is one of the important constituents of the neutral fraction with respect t o browning. Not only the hydrogen ion concentration but also the nature of the anion is important in browning. Thus, when compared on an equimolar basis, galacturonic acid is twelve times more effective in producing browning than citric acid and fourteen times more effective than malic acid. Phosphate ion was found to be of no importance in the browning of apricot concentrate (3001, solids). The inorganic cations were found t o contribute to browning but were of secondary importance. LITERATURE CITED
Bedford, C. L., Food Eesearch, 1, 337 (1936). Chemical Process Co., San Francisco, Calif., “Properties and Uses of Duolite Ion Exchanger,” 1946. Haas, V. A., Stadtman, E. R., Stadtman, F. H., and MaoKinney, G., J.Am. Chem. SOC.,70, 3676 (1948). Hall, J. A . , Research Laboratory, California Fruit Growers Exchange,,LosAngeles, unpublished report, 1927. Hassid, W. Z., IND. ENG.CHEM.,ANAL.ED.,8, 138 (1936). Ibid., 9, 228 (1937). Joslyn, M. A., and Marsh, G. L., IND.ENG.CHEM.,27, 186 (1935).
Kass, J. P., and Palmer, L. S., Ibid., 32, 1360 (1940). Lathrop, R. E., and Holmes, R. L., IND.ENG.CHEM.,’ANAL. ED.,3 , 3 3 4 (1931).
Loeffler, H. J., IND.ENG.CHEW.,33, 1308 (1941). McCready, R. M., and Hassid, W. Z., J . Am. Chem. SOC.,66, 560 (1944).
Seaver, J. L., and Xertess, 2. I., I b i d , 68, 2178 (1946). Singh. B., Dean, G. R., and Cantor, 8. M., Ibid., 70, 517 (1948). Stadtman, E. R., in “Recent Advances in Food Research,” Vol. 1, New York, Academic Press, 1948. Stadtman, E. R., Barker, H. A . , Haas, V., and Mrak, E. M., IND. ENQ.CHEM.,38, 541 (1946). Stadtman, E. R., Barker, H A . , Haas, V., Mrak, E. M., and MacKjnney, G., I b i d , 38, 324 (1946). Stewart, G. F., Best, L. R., and Lowe, B., Proc. Inst. Food Technol., 1943, 77. RECBXVEDAugust 19, 1948. Undertaken in cooperation with the Committee on Food Research, Quartermaster Food and Container Institute for the Brmed Forces, under contract Wll-009-q.m.-70216with the University of California. The opinions or conclusions contained i n this report are those of the authors. They are not to be construed as neceksarily reflecting the views or the endorsement of the War Department.
