Nonenzymatic Browning of
Foodstuffs PRODUCTION OF CARBON DIOXIDE V. M. LEWIS, W. B. ESSELEN, JR., AND C. R. FELLERS University of Massachusetts, Amherst, Mass.
.
A study of the production of carbon dioxide by foodstuffs venience, it was desired to keep the quantities of test reducing sugars and has revealed that many foods generate carbon dioxide materials as small as possible. spontaneously on storage. The reaction between reducing amino acids has been regarded for some time as one of the sugars and amino acids has also been studied with regard It was therefore necessary major causes of the nonto carbon dioxide production) it has been found that deto develop a satisfactory carboxylation of the amino acid is an integral part of the technique for estimating the enzvmatic browning of foodrate of carbon dioxide producstuffs. Maillard 710) obreaction at 100' C. tion under these conditions. served that in the later stages Since many of the testa of the reaction between gluwere to be run over periods of a month or even longer, it seemed cose and glycine, carbon dioxide was evolved. Although there has impracticable to use any continuous method of carbon dioxide been much recent work on the Maillard reaction, the importance measurement such as those used in plant respiration studies. of gas production as part of the reaction seems to have been negThe method final$ worked out consisted of sealing the reaction lected. I n several instances spontaneous production of carbon mixture or foodstuff in a glass tube, and after the required time dioxide in processed foodstuffs has been a serious problem. interval, analyzing the headspace gases for carbon dioxide in a I n the storage of molasses, the so-called frothy fermentation has micro-type of gas analysis apparatus developed for this purpose been attributed partially to the carbon dioxide resulting from the Maillard reaction (8, 6, 8). I n the storage of fruit juice concen(9). Pyrex tubing of 1-cm. internal diameter was cut into 30-cm. trates, swells are often caused as a result of the production bf lengths, and these were drawn out at the center to form two test carbon dioxide (3). During the Pacific war, canned rations of tubes about 15 cm. long. The test tube was filled partially with various types were found to swell undei tropical storage conditions 2 to 3 ml. of reaction mixture, or approximately 3 grams of foodowing to the spontaneous formation of carbon dioxide. stuff, and was then heated about its center in the oxygen flame I n all of his work, Maillard used highly concentrated solutions. until the glass was soft enough for the tube t o be drawn out into I n 1921 Grunhut and Weber ( 7 ) reported the results of a study the form of a fine bore (about 0.5 mm.) capillary tube (Figure 1,l). of the Maillard reaction using dilute solutions (0.025 N ) of the The capillary part of the tube was then bent in the manner shown sugars and amino acids. I n these concentrations, no evolution in Figure 1, 2, and excess tubing was removed. If the headof carbon dioxide was observed. Ambler (1) studied the respace was to be filled with nitrogen, the tube was placed in a action using alanine and glucose. He found that carbon dioxide vacuum desiccator and a 29-inch vacuum was applied. The was produced only with the more concentrated solutions and vacuum was released with nitrogen from a cylinder by means of a regarded his work as confirmation of that of Grunhut and two-way stopcock. This procedure was carried out three times Weber. Bauminger and Lieben ( 5 ) found that if a mixture of to remove the last traces of oxygen and obtain a nitrogen headglycine and glucose was subjected to a stream of oxygen a t 70' C. space in the tube. The tube was then removed from the desand a t a pH of 8.0, carbon dioxide was evolved, and the p H of the iccator and the end of the capillary quickly sealed by fusion solution decreased owing to the formation of lactic acid. They (Figure 1, 3). found that only a relatively small proportion of the sugar changed Before analysis of the headspace gas, the reaction tube was to carbon dioxide. Barnes and Kennedy ( 4 ) studied the prodplaced in a boiling water bath for a minimal period of 10 minutes. ucts of the reaction between glucose and glycine. They conThis served the dual purpose of reducing as far as possible the cluded that the mechanism of the reaction was a direct condensasolubility of gases in the liquid in the tube and of developing a tion of the glycine with the glucose, followed or accompanied by positive pressure in the tube, thereby allowing easy removal of a rearrangement, but did not consider that any decarboxylation sample of the gases. occurred in the formation of the brown pigments which they After removal of the reaction tube from the water bath, 8, isolated. scratch was made on the glass of the capillary with a tungsten The purpose of this study was to determine to what extent carbide glass knife about 0.5 to 0.75 inch from its end. The end carbon dioxide production occurred in foodstuffs and to investiof the capillary was then placed inside a mercury-filled gas reagate the mechanism of the production of this gas. ervoir, and by pressing it against the wall of the reservoir, the tip was broken off (Figure 1, 4) and a gas sample obtained from EXPERIMENTAL the tube. Part of the sample was transferred to the gas analysis A number of studies were made on the production of carbon chamber of the microanalysis apparatus, and the percentage of dioxide both in foodstuffs and in synthetic reaction mixtures. carbon dioxide in the headspace was determined. In most instances the rate of production of carbon dioxide The capillary portion of the reaction tube was next removed w w extremely slow. For reasons of both economy and con(Figure 1, 5), and the volume of the headspace was determined by filIing the tube with water from a buret equipped with a 1 Present address, Southern Can Company, Fty. Ltd., Gsalong Road, W. finely drawn out delivery point and measuring the quantity Footscrsy, W 11, Viotory, Austrdie.
T
HE reaction between
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Vol. 41, No. 11
added, and the tube was sealed in the usual manner (Figure 2 ) . After sealing, the tube was inverted to allow the contents of the inner and outer glass tubes t o react, and the carbon dioxide produced as a result of the reaction was determined. The results obtained are presented in Table I, which shows that there was good recovery over a wide range of headspace gas compositions.
TABLEI. RECOVERY O F CARBON DIOXIDE FROM REACTIOS MIXTURESOF SODIUM CARBONATE AND SULFURIC &ID Expected Yield
Actual Yield of coz, Mg.
11.00 11.00 2.20 2.20 2.20 2.20 2.20 2.20 2.20 2.20 2.20 2.20 0.11 0.11
10.50 10.33
of
\
4
u
1
Figure 1. Stages in Preparation of Reaction Tubes Used for Measuring Carbon Dioxide Production
required. The volume of the capillary was ignored. Knowing the volume of the headspace and the percentage of carbon dioxide in the gas, i t was possible to calculate the weight of carbon dioxide that had been produced. Sources of error in this method of estimating carbon dioxide production are: 1. An error due to ignoring the volume of the capillary part of the reaction tube; this volume was very small compared with t h e total volume of the tube. 2. An error due to the probable presence of some residual dissolved gas in the liquid in the tube; to minimize this, the tube was heated to 100 O C. before a sample was taken. 3. I n making the calculations, i t u-as assumed that the tube had been sealed under uniform conditions of temperature and pressure (20" C. and 760 mm.). No effort was made t o standardize sealing conditions rigidly, but the tube was always allowed to come to room temperature before sealing. Errors due to variation in room temperature and atmospheric pressure a r e comparatively small.
Although most sources of error are relatively small, the gross accuracy of the determination of carbon dioxide production cannot be considered high. However, the method appeared to be the only one that could be conveniently used under the experimental conditions. I n all determinations of carbon dioxide production, duplicate tubes were used, and the figures given represent mean values obtained. Recovery tests to check the accuracy of the method for determining carbon dioxide production were made in the following manner : One ml. of sodium carbonate solution of the required concentration was placed in the bottom of a reaction tube. An inner glass tube containing a slight excess of sulfuric acid was then
cog, hlg.
2.12 2.18 2.18 2.18 2.12 2.08 2.08 2.08 2.16 2.18 0.12 0.13
Because previous work had indicated that carbon dioxide was produced as a result of the reaction of amino acids with reducing sugars, i t was believed that all foodstuffs might produce a certain amount of carbon dioxide on storage, since all contain amino acids and reducing sugars. Further, it was thought that carbon dioxide production might be used as an index of the extent of the browning reaction in foods. A number of foodstuffs, therefore, were selected to cover a wide range of types of product. Three grams of each of these foods were placed in the reaction tubes described above, and the tubes were sealed in an atmosphere of nitrogen. Although Maillard (IO)had pointed out that the production of dark pigments and carbon dioxide was independent of oxygen, it was deemed advisable to use B nitrogen atmosphere in these studies because i t was thought that if oxygen were present i t might react with other constituents of the food and thereby tend to mask results. The tubes were incubated at 100" C. and were analyzed for carbon dioxide production at the end of 3- and 7-day intervals. The results of these analyses are shown in Table 11. All of the foods tested showed some production of carbon dioxide, but with some foods it was much higher than with others. TVith prunes, the rate of gas evolution was exceptionally high; this was attributed to the high amount of soluble solids. However, orange juice and string beans, neither of which are high in soluble solids, also gave fairly high figures for carbon dioxide production. To check whether the formation of carbon dioxide occurred only a t higher temperatures, several of the foods were incubated in the same manner a t temperatures of 50" C. Because microbiological spoilage could possibly have occurred at this temperature, the tubes were first processed for 20 minutes a t 115" C. Figure 2. Reacbefore incubation. Analyses were tion Tube Used made for carbon dioxide production in Determination after the initial processing and again of Carbon Dioxide Recovery after a storage period of 2 months. A Sulfuric acid The difference between these figures B Sodium oarbongave the amount of carbon dioxide ate solution .
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INDUSTRIAL AND ENGINEERING CHEMISTRY
that had been produced during the 2-month period a t 50" C. Results are shown in Table 111. Although carbon dioxide production was much less rapid a t this temperature than at 100' C., it can readily be seen that gas evolution does occur a t the lower temperature. It therefore seemed apparent that some chemical reaction was occurring during the storage of foodstuffs which gave rise to the production of carbon dioxide. It was not possible t o say a t this stage whether the carbon dioxide produced was the result of the reaction between amino acids and reducing sugars in the food. The best approach to the problem seemed to be to study carbon dioxide and color production in a model system and to determine in this manner how the two are related. The system chosen for most of the work was a glucose-glycine reaction mixture. When such a mixture was boiled under reflux, after about 0.5 hour the solution became pale yellow and on continued boiling darkened to an orange color, thence t o a reddish brown, and after about 20 hours the solution appeared almost black. Between 20 and 30 hours dark insoluble matter began to appear in the solution, and this increased in amount progressively until after about a week there was no soluble coloring matter remaining in the flask. The dark, insoluble matter can be filtered off from the mixture and washed with hot water until free from extractives. A crude preparation of the pigments was obtained by boiling under reflux for 24 hours a mixture of 10 grams of glycine and 24 grams of glucose in 150 ml. of water. The solution, after refluxing, was filtered and then concentrated under vacuum t o a volume of 60 ml. The sirupy black liquid obtained was then slowly poured into ten times its volume of acetone and the mixture agitated violently. Although neither glucose nor glycine is soluble to any great extent in this concentration of acetone, a considerable amount of the residual glucose can be removed from the sirup by this means since it tends to remain in suspension in the acetone and can be decanted off along with it. A further washing with acetone removed additional glucose in suspension. It was found necessary to remove as much of the sugar as possible before chromatography in order t o reduce the viscosity of the solution sufficiently to allow it to be run satisfactorily through a column of adsorbent. About 30 ml. of water were then added to the remaining pigments, and the solution was placed on the water bath for a short time to boil off remaining acetone. I n this way, a crude concentrated solution of the coloring matter from the original mixture was obtained. The pigments in this mixture were next fractionated by a chromatographic procedure. The only satisfactory adsorbent thus far tested was ignited C.P. alumina. When used alone,
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this was far too impervious to give a satisfactory column, but a good column was obtained by mixing 2 parts by weight of alumina with 1 part of Dicalite. It was found that if the acidity of the crude preparation of pigments, which when prepared had a pH value of 4.0, was adjusted to p H 7.0 with sodium hydroxide solution, the coloring matter could be separated into several components by passage through the column. Three milliliters of the crude preparation were run onto the top of a column, 1 inch in diameter and 8 inches in height, and the dark material eluted initially with water. A large part of the pigment was retained near the top of the column but one fraction was not adsorbed a t all and, along with the residual glycine and glucose, could be completely eluted from the column with water. This nonadsorbable part of the pigment has been designated as fraction A. PURIFICATION OF FRACTION A
Fraction A, as obtained by elution of the alumina column with water, still retained residual glycine and glucose as impurities, as well as the sodium added to adjust the p H of the crude pigment, and probably other by-products of the reaction. A method of purification was worked out based on the fact that the pigment forms an insoluble silver salt, which glycine does not. The aqueous eluate containing fraction A was vacuum concentrated to as small a volume as was practicable without crystallization of the glycine, and an equal volume of 30% silver nitrate solution was added. To obtain good precipitation a t this stage, the pH value of the pigment concentrate should be close to 7.0. This condition will be met if the procedure outlined above has been followed. Precipitation occurred immediately but a greater yield was obtained if the mixture was allowed to stand overnight. The precipitate, which was black in color, was removed by centrifugation and washed repeatedly until the wash waters were free from any trace of silver. When this stage had been reached, the precipitate was also free from glucose and glycine. Trouble was experienced after the last trace of silver had been removed, because in the absence of soluble electrolytes, the precipitate tended t o disperse and could not be thrown out of suspension by ordinary centrifugal forces. This was avoided by adding a trace of acetic acid to the wash water. The washed precipitate was next decomposed into fraction A by the addition of 6 N hydrochloric acid. A few ml. of the acid were added to the centrifuge tube containing the precipitate, the mixture waa stirred with a glass rod, and the silver chloride formed was removed by further centrifuging. The silver chloride formed was reasonably white, indicating that reduction of the silver nitrate had not occurred. The acid solution of the pigment was then immediately poured off into 15 volumes of acetone, where it was quantitatively precipitated. The acetone was removed by centrifugation, end the pigment was then washed several times t o TABLE 11. PRODUCTION OF CARBON DIOXIDEBY FOODSTUFFS remove residual acid. The pigment was then dissolved in a small INCUBATED AT 100" C. IN ABSENCE OF OXYGEN volume of water, centrifuged to remove residual silver chloride, Carbon Dioxide Produced, Mg./G. and, if necessary, boiled under a high vacuum to remove residual Foodstuff 3 Days 7 Days acetone. The pigment should be kept as an aqueous solution Oranze iuice 1.39 2.86 0.50 0.80 in a refrigerator, for when the pure solution is dried, it cannot 0.48 1 .oo 2.46 3.46 readily be brought into solution again. In drying samples for 0.89 2.00 analysis, heat should not be used, but the pigment may be dried 8.20 12.41 Black currant jam 3.64 5.40 by freeze drying or under vacuum after precipitation with aceCheddar cheese 1.14 1.80 Reconstituted dry peas 1.24 ... tone. The absorption spectrum OY fraction A was studied between 400 and 650 mp, and maximum absorption was shown a t 400 mw TABLE111. PRODUCTION OF CARBON DIOXIDE BY FOODSTUFFS with the percentage absorption decreasing smoothly to the red INCUBATED AT 50" C. IN ABSENCEOF OXYGEN end of the spectrum. The color of a concentrated solution of the Carbon Dioxide Produced, Mg./G. pigment was reddish brown, but dilute aqueous solutions were. After yellowish; the dried pigment was black. The color of the solu2 months' By After Foodstuff sterilization storage difference tion was not affected by changes in pH value. When measured! Beef muscle 0.04 0.07 0.03 a t 400 mw the percentage transmission of a 0.0085% solution of Apple tissue 0.07 0.1, 0.04 the pigment was equivalent to that of a 0.025% solution of potasPrunes (18% moisture) 0.66 1.09 0.43 sium dichromate.
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The production of carbon dioxide and color was studied in reaction tubes containing 1 ml. of 0.25 M glucose, 1 ml. of 0.25 M glycine, and 0.5 ml. of water. T h e tubes were nitrogen filled before settling and were incubated at 100.5' C. for different time intervals. The initial pN of the reaction mixture was 6.2. Parallel determinations were made on the carbon dioxide produced and on the amount of color developed. The color readings were made directly in terms of the glucose-glycine melanoidin pigment, fraction A, Fraction A was determined by measuring the percentage transmission of the unknown solution at 400 mp and referring to a n analytical curve which had previously been constructed using known concentrations of the pure, isolated pigment. These results are given in Table IV and show that color production and gas production proceed at the same time. The velocity constants at the different periods of time were calculated from the figures for carbon dioxide production using the formula for a second-order reaction. The values obtained for k were: Velocity constant ( k ) , Set.-' a t 100.5O C. 1 . 9 5 x 101.91 X 1 0 - 6 1.64 X 10-6
Time, Hr 20.75 46.00 72.50
The extent of the agreement obtained a t the different time intervals indicates that the reaction is of the simple second-order type-that is, the rate of reaction is proportional to the concentrations of the two reacting substances. It is thought that the decrease in value of the velocity constant with time is due to the progressive decrease in the p H value of the solution. The production of carbon dioxide was also studied with several amino acids in different concentrations to find the quantitative relation between carbon dioxide produced and the amino acid destroyed. Preliminary work was undertaken to find what length of incubation at 100 C. was necessary for carbon dioxide evolution to be complete. Using molar Concentrations of glycine and glucose, i t was found that carbon dioxide production was virtually complete after 10 days. Since the reaction rate with lower concentrations of amino acids would be slower than this, the mixtures mere incubated for 1 month before examination for gas production. I n the experiment reported here, 1 ml. of the amino acid solution of the particular concentration used was sealed in the reaction tube with 1 ml. of 20% glucose solution in an atmosphere of nitrogen. A tube was also carried containing a mixture of glucose with 0.125 M propionic acid. Buffer solutions were not used in any instance. The results are shown in Table V, O
OF CARBOKDIOXIDE AND COLORIN TABLE IV. PRODUCTION
GLUCOSE-GLYCINE REACTION MIXTURE
(Mixture, 0.25M, incubated a t 100.5O C. i n absence of oxygen, initial o H 6.21 Carbon Color, as Dioxide Fraction A, per Tube, per Tube, Time, Hours Mg. Mg. 0.147 20.75 0.314 1:iS 46 2.30 0.421 72.5 IN REACTION TABLE V. CARBONDIOXIDEPRODUCTION MIXTURESOF 20% GLUCOSE AND VARIOUS AMINOACIDS (Incubation 1 moAth st looo C.)
Amino Acid U8ed Glycine Glycine Glycine Aspartic acid Aspsrtic acid Cystine As aragine g&ninobenzoic acid ropionic acid
Concontration, 144 1/1 1/8 1/1a
'/a 1/19
l/r 1/18 1/10 1/8
Carbon Dioxide Found, Jlg./Tube 9.65 4.63 2.93 11.50 6.20 22.17 1.85 2.33 0.05
Carbon Dioxide Expected, Mp./Tube 11.00 5.50 2.75 5.50 2.76 22.00 2.75 0.00 0.00
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which includes the expected yield of carbon dioxide if complete dccarboxylation of the carboxyl group adjacent to the -amino group were to occur. I n most instances the actual results agree fairly closely with the figures in the expected yield column; in two concentrations of aspartic acid approximately twice the expected yield was obtained; and with p-aminobenzoic acid, decarboxylation also occurred. The results demonstrate that complete decarboxylation is possible as a result of the Maillard reaction. However, there was practically no decarboxylation in the case of the propionic acid. With asparagine, i t is uncertain as to whether only the free carboxyl was decarboxylated, since the reaction was evidently far from completion even after a month a t 100" C. CARBON DIOXIDE AND COLOR PRODUCTION IN GLUCOSEGLYCINE REACTION MIXTURES
When a mixture of glycine and glucose reacts in solution, there is a progressive increase in the intensity of the color produced, and a t the same time, continued production of carbon dioxide occurs. There has been no reported attempt to relate the production of carbon dioxide with the development of color. Originally it was believed that gas production occurred only toward the final stages of the reaction, and currently the general concensus is that the gas production is a separate reaction from that involved in color formation.
TABLEVI. C a R B o X DIOXIDEAND COLOR PRODUCTION GLUCOSE-GLYCINE REACTION MIXTURES Conditions - of Incubation Concn. Time, Temp., ofants, react- Initial hr. 144 144 46 46 72 72 18 18
C. 90
90 100.5 100.5 100.5 100.5 110 110
iM
pH 7.2 6.6 6.2
6.1
6.2 6.1 7.2 6.6
IN
Calcd. Mg. CotFound, per Fraction A (Fraction TAu ger e), tube 0,336 0,260 0.314 0.271 0.421 0.394 0.363 0.325
per tube 1.51 1.24 1.58 1.25 2.30 1.95 1.61 1.35
Mg. 1.50 1.18 1.43 1.24 1.92 1.80 1.65 1.47
I n order to demonstrate the relation between carbon dioxide production and color formation, parallel determinations of carbon dioxide and color were made in a series of experiments, in which equimolecular proportions of glucose and glycine were incubated in a n atmosphere of nitrogen. The color readings were made directly in terms of the melanoidin pigment, fraction A, and all were made at comparatively early stages in the reaction, as it had been found that at the early stages the color formed is due practically entirely to the so-called fraction A. This was checked furthcr and found to be true by passing the mixtures from the reaction tubes, after incubation, through an alumina column, when quantitative recovery of pigment occurred. Table VI shows the conditions of incubation; the amount of carbon dioxide produced in each tube; the amount of fraction A produced in each tube; and the amount of pigment that would be expected in the tube, calculated from the observed figures for carbon dioxide, taking the molecular weight of the pigment, as determined, a t 200. The calculations were made on the basis of 1 molecule of pigment being produced for every molecule of carbon dioxide split from the glycine. There is excellent agreement between the observed values for pigment production and those calculated from the carbon dioxide production. Therefore, the data from this system indicate that the carbon dioxide production and pigment formation are both part of the same reaction, The results also show that one of the possibk sources of spontaneous carbon dioxide production in foods is the glucoseamino acid reaction.
November 1949
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I N D U S T R I A LA N D E N G I N E E R I N G C H E M I S T R Y
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SUMMARY
LITERATURE CITED
A method is described for studying the production of carbon dioxide, in small amounts over long periods of time, by foodstuffs and reaction mixtures by the use of a sealed-tube technique. All foodstuffs studied were shown to produce carbon dioxide spontaneously on storage a t 100' C., but the rate of carbon dioxide production varied considerably between the different foodstuffs. I n reaction mixtures of glucose with various amino acids, complete decarboxylation of tKe amino acid occurs on incubation for a sufficient length of time a t 100' C. I n glucoseglycine mixtures, the development of color is directly correlated with the production of carbon dioxide, and decarboxylation is an integral part of the reaction between these two molecules. One of the sources of carbon dioxide production by foodstuffs is, therefore, the reaction between reducing sugars and amino acids.
(1) Ambler, J. A., IND. ENG.CHEM.,21,47(1929). (2) Ambler, J. A., Intern. Sugar J . , 29,382(1927). (3) Anon., Food Packer, 26 (4),32 (1945). (4)Barnes, H.M., and Kennedy, C. A., presented as a part of the Symposium on Nonenzymatic Browning before the Division of Sugar ChemisOry and Technology at the 112th Meeting Of the AMERICAN CHEMICAL SOCIETY, New York, N. Y. (5) Bauminger, B., and Lieben, F., Biochem. Z., 292,92(1937). (6) Browne, C.A., IND. ENQ.CHEM.,21,600 (1929). (7) Grunhut, L.,and Weber, J., Biochem. Z., 121,109 (1921). (8) Hucker, G.J., and Brooks, R. F., Food Research, 7,481 (1942), (9) Lewis, V. M.,Anal. Chem., 21,635 (1949). (10) Maillard, L.C., Ann. Chim. 5, 258 (1916).
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RECEIVED July 22, 1949.
Contribution 873 of the Massachusetts Agricultural Experiment Station, Amherst, Mass.
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(Nonenzymatic Browning of Foodstuffs)
NITROGEN-FREE CARBOXYLIC ACIDS IN THE BROWNING REACTION V. M. LEWIS, W. B. ESSELEN, JR., AND C. R. FELLERS University of Massachusetts, Amherst, Mass. T h e reaction between nitrogen-free carboxylic acids and glucose may be a cause of browning in foodstuffs. This reaction may be similar in nature to the Maillard reaction, but because the nitrogen-free acids are more abundant in foodstuffs than the amino acids, their importance in the browning reaction should not be overlooked.
T
HE isolation and characterization of certain of the glycineglucose melanoidin pigments have been reported in a previous paper (f ). It was pointed out that the nonadsorbable pigment, fraction A, was probably the initial colored reaction product of glycine and glucose, and that this fraction could readily be oxidized, first to darker soluble pigments and later to insoluble dark material. It was also indicated that the reaction between amino acids and reducing sugars involved decarboxylation of the amino acids. I n the present paper, work on the role of the nitrogen-free carboxylic acids in the browning reaction is presented. ANALYTICAL METHODS
Carbon dfoxide measurements were made in the manner described previously (1). Color measurements are for the most r t expressed in terms of the isolated glycine-glucose pigment raction A ( 1 ) . The use of fraction A as a standard permitted ready comparison of the amount of color produced in various reaction mixtures. Color readings were made by measuring the percentage transmission of the unknown solution in the Evelyn colorimeter using the 400 mp filter, and reading the corresponding concentration of fraction A directly from an analytical curve prepared from the purified fraction A. REACTION BETWEEN GLUCOSE AND NITROGEN-FREE CARBOXYLIC ACIDS
I n order to determine the conditions necessary for decarboxylation of organic acids to occur in their reaction with glucose, experiments were carried out using carboxylic acids other than those containing an amino group. It was previously reported that in the incubation of propionic acid with glucose, there was virtually no carbon dioxide production, but in this experiment there w&s no attempt to control the pH value, which was found to be 2.8 before incubation. Since it is well known that the browning reaction
is inhibited greatly by high acidity, in the next series of experiments steps were taken to have the initial p H in the range in which the glucose-glycine reaction was known to proceed rapidly. The sodium salts of the various organic acids were consequently used, and these were prepared in 0.25 M concentrations, the pH of each solution being adjusted to 7.2 by the addition of either sodium hydroxide or hydrochloric acid before making up to final volume. For comparative purposes, a 0.25 M solution of glycine was prepared having the same pH value. Reaction tubes were prepared containing 1 ml. of the various acid solutions, and 1 ml. of 0.25 M glucose. The tubes were nitrogen-filled before sealing, and were incubated a t 100' C. for 70 hours. At the end of this period, analyses were made for carbon dioxide production, and a t the same time color readings were made on the contents of the tubes in the manner described above. When the tubes were removed from incubation, all of the reaction mixtures showed some browning. The results obtained are presented in Table I. The color values were obtained in the manner described, but for convenience in comparing the extent of browning caused by the different acids with that for the glucose-glycine reaction, the color value for glycine was set arbitrarily a t 100 and the other values adjusted accordingly. The results show that both carbon dioxide production and browning occurred with all the acids tested. Among the different acids, however, carbon dioxide production was not related to the extent of browning-for example, with lactate, 3.22 mg. of carbon dioxide were associated with only 55 units
TABLEI. PRODUCTION OF CARBON DIOXIDEAND COLORI N REACTIONMIXTURESOF ORGANICACIDSAND GLUCOSE (Initial pH of 7.2 after incubation in the absence of oxygen for 70 hours a t loOD C.) Carbon Dioxide Produced per Relative Darkness Acid Tube, Mg. a t 400 Mp Acetate 0.64 53.5 Oxslate 0.49 38.2 Fumarate 0.71 41.2 Citrate 1.32 112.3 Tartrate 1.05 41.9 Lactate 3.22 56.0 Glycine 0.89 100.0
