THE INFLUENCE OF WATER AND pH ON THE REACTION BETWEEN AMINO COMPOUNDS AND CARBOHYDRATES*

BY LAWRENCE J. SCHROEDER, MICHAEL IACOBELLIS, AND

ARTHUR H. SMITH

(From the Department of Physiological Chemistry, Wayne University College of Medicine, Detroit, Michigan)

(Received for publication, May 3, 1954)

The mechanisms involved in browning and in the Maillard reaction (1)) together with their effect on the nutritive value of proteins, have been investigated extensively, yet much of the evidence is of a contradictory nature. These experimental results are to be expected if one considers the variety of conditions of temperature, pH, and reactant concentrations employed by the various investigators. In most cases the studies pub- lished have been concerned with the secondary changes induced in either the amino compounds or the reducing sugars by heat processing. This has led investigators to view the reaction of amino compounds and reduc- ing sugars (Maillard reaction) largely in its relation to browning, with the consequent tendency to attribute browning to the Maillard reaction.

In 1953, Schroeder, Iacobellis, Lees, and Smith (a), continuing a previ- ous investigation (3) on the effect of heat on milk products, presented data secured by the nitrogen balance method on intact animals and by diges- tion experiments with crystalline enzymes in vitro, showing that the de- crease in nutritive value suffered by the contained proteins when dried skim milk is autoclaved was prevented by prior reconstitution with water to protein levels as high as 24.5 per cent. It appeared that water pre- vented the deleterious effect of heat upon proteins. On the basis of these observations, it was postulated that browning and the Maillard reaction might not bear a causative relation to each other, but rather might be viewed as two separate and independent reactions. The purpose of the present investigation was to determine the influence of both water and pH on the interaction of amino compounds and reducing sugars.

* Preliminary reports of this work were presented before the Michigan Academy of Science, Arts and Letters at Detroit, April 17,1953, and before the American Institute of Nutrition at Chicago, April, 1953.

The data presented in this paper are taken from the dissertation submitted by Michael Iacobellis for the degree of Doctor of Philosophy, Wayne Universit,y, Febru- ary, 1954.

973

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974 AMINO COMPOUNDS AND CARBOHYDRATES

EXPERIMENTAL

Synthesis of Peptides

The peptides used in this investigation were synthesized by the phthalyl method (4, 5).

Phthalylglycyl-L-leucine-A solution of 13.41 gm. (0.06 mole) of phthalyl- glycyl chloride in 75 ml. of anhydrous chloroform was added dropwise during 30 minutes to a stirred, cold (0”) suspension of 7.87 gm. (0.06 mole) of n-leucinel and 15 gm. (0.18 mole) of sodium bicarbonate in 125 ml. of water. After stirring for an additional 30 minutes at room temperature, the solution was acidified (pH 5.0) with glacial acetic acid and then con- centrated under reduced pressure. The crude material was recrystallized from methyl alcohol, giving a total yield of 15.8 gm. of phthalylglycyl-L- leucine.

Glycyl-L-leucine-6.36 gm. (0.02 mole) of phthalylglycyl-n-leucine in 200 ml. of ethyl alcohol containing 6.0 ml. (0.12 mole) of hydrazine hydrate were refluxed for 2 hours. Water (100 ml.) was added to insure continued solubility of the hydrazine salt of phthalylhydrazide. The solution was then concentrated under reduced pressure. The dry solid residue was treated with 100 ml. of water and the pH brought to 5.0 with glacial acetic acid. After heating for 1 hour on a steam cone, the solution was cooled and filtered, and the precipitate (phthalylhydrazide) washed with 100 ml. of water. The combined filtrate and washings were concentrated under reduced pressure. The dry residue was recrystallized from ethyl alcohol and water. The yield was 3.5 gm.

Other peptides, as well as their phthalyl derivatives, were prepared in an analogous manner. Analytical data on these compounds appear in Table I.

E$ect of Water and Heat on Amino Acid- and Peptide-Glucose Mixtures

Glycyl-L-leucine and glycyl-nn-valine, as well as their constituent amino acids, were autoclaved at 15 pounds pressure for 30 minutes with and without glucose in the dry state and in the presence of water. All samples were weighed in calibrated non-protein nitrogen tubes and the ratio of amino compounds to glucose was 1: 1 on a molar basis. The amount of water added to the samples autoclaved in aqueous medium was 25 ml. All samples after autoclaving were brought to the 50 ml. mark and aliquots taken for the determination of total nitrogen and amino nitrogen and for paper chromatography. The total nitrogen was determined by the micro- Kjeldahl method, while the amino nitrogen was estimated by the method of Pope and Stevens (S), as modified by Schroeder, Kay, and Mills (9).

1 The amino acids were kindly supplied by Merck and Company, Inc.

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L. J. SCHROEDER, M. IACOBELLIS, AND A. H. SMITH 975

In these investigations a modification of the chromatographic technique of Williams and Kirby (10-13) was used. RF values were calculated in the usual manner.

TABLE I

Analytical Data on Synthetic Peptides and Their Phthalyl Derivatives*

Compound

Phthalylglycyl-n- leucine

Glycyl-n-leucinet Phthalylglycyi-n-

glutamic acid Giycyi-n-giutamic

acid1 Phthalylglycyi-on-

valine Giycyi-nn-valine Phthalyl-nn-phenyl

alanylglycines Phenylalanylglycint Phthaiyigiycyi-nL-

phenylalanylgly- tine

Glycyl-nn-phenyl- alanylglycine

-

-

! 1

Nitrogen

Total Amino

CZkll- lated

>er cent

8.8

P

14.9 14.8 7.45 8.38 8.30

11.76 11.68 5.88

9.21 9.3

16.07 16.10 8.04 7.95 7.85

12.6 12.3 6.3 10.27 10.25

15.0 15.2 5.0

‘ound

:r cent

7.2

5.84

8.0

6.45

Neutralization equivalent

318

304.3

352

Found

317.3

301.5

350

* For properties of the free peptide see Fruton (6). t M.p. 242” (decomposition); [ol]z -35.2” (water); Fischer and Steingroever (7),

[a]$ -36.1”. $ [CL]; +6.9” (water). $ Phthalylphenylalanyl chloride, m.p. 130-131”. Sheehan and Frank (4), m.p.

121-124” (124-126”).

Effect of pH and Heat on Amino Acid- and Peptide-Glucose Mixtures

Series of amino acids-glucose and peptides-glucose mixtures were weighed in calibrated 50 ml. non-protein nitrogen Pyrex tubes as described before. The relative concentrations of both glucose and the amino compounds1 were varied. Several series of the same mixtures were prepared: one was dissolved in water, brought to volume, and its pH checked, while the other was dissolved in buffers of different pH values. Aliquots were taken at this time for total nitrogen, amino nitrogen, and paper chromatography de-

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976 AMINO COMPOUSDS jLK:D CARBOHYDIL~TES

terminations. The remainder of the solution was autoclaved at 15 pounds pressure for 30 minutes. Samples of the amino compound alone and of the glucose alone under the identical conditions served as controls and these were compared to other samples similarly treated but unautoclaved. Color development was evaluated grossly by direct visual inspection. Total nitrogen and amino nitrogen determinations, as well as ascending paper chromatography, were carried out as previously described. All pH determinations were made with the Beckman titration pH meter (model H) standardized at pH 4.0 and 7.0.

Results

Effect of Water

The average results of the amino nitrogen determination for the peptides and amino acids after being autoclaved with and without mater appear in Fig. 1. Only in those samples of amino compounds which had been autoclaved with glucose in the dry state were there appreciable decreases in amino nitrogen. These samples were accompanied by browning. On the other hand, whereas the amino compounds which had been reconsti- tuted with water before autoclaving showed browning, no decrease in amino nitrogen was detected. The total nitrogen in these samples auto- claved with water, and in those autoclaved in the dry state with and with- out glucose, was the same as that of the control unautoclaved samples. All samples which had been autoclaved with glucose in the presence of water gave spots with ninhydrin having the same intensity and RF values shown by the control unautoclaved amino acids and peptides. The spots from the samples of amino compound-glucose mixtures which had been autoclaved in the dry state were by comparison less intense, but of the same RF value as those of the control samples. Two exceptions were observed : each of the glycine-glucose and L-leucine-glucose mixtures autoclaved in the dry state showed two comparatively weak spots, one corresponding to the amino acid and the other having an RF value of 0.22 for glycine-glucose and 0.43 for L-leucine-glucose. These two extra RF values are quite different from those of the corresponding amino acids which in the case of glycine is 0.4 and of L-leucine 0.82. They apparently represent the reaction compounds formed between amino compounds and glucose.

In chromatograms developed with aniline hydrogen phthalate, samples autoclaved in the presence of water showed spots with RF values similar to those of the control unautoclaved glucose solution (0.39), but smaller in size. On the other hand, the mixtures autoclaved in the dry state failed to give any spot under identical conditions.

It would appear, therefore, from both the analytical and chromato-

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L. J. SCHROEDER, M. LkCOBELLIS, AND A. H. SMITH 977

graphic methods employed that the reaction between amino compounds and glucose in the dry state results in a decrease in amino nitrogen, a decrease in the size of the spot on the paper chromatograms developed with ninhydrin (except in the cases of glycine and L-leucine which gave an

PLUS GLUCOSE

Unautoclaved

Amino nitrogen (mg.)

21=FFY

Autoclaved with water

Autoclaved dry

Unautoclaved

Autocloved with water

Amino nitrogen (mg .)

21=E-F

GLYCYL-L-LEUCINE GLYCYL-DL-VALINE

FIG. 1. Effect of water on the amino nitrogen content of glycine, L-leucine, and glycyl-L-leucine and of glycine, nL-valine, and glycyl-DL-valine autoclaved (15 pounds pressure for 30 minutes) with and without glucose.

extra spot), and a failure to demonstrate any spot whatever with aniline- phthalic acid. On the other hand, in an aqueous medium, one observes only a decrease in the aldehyde group concentration, as evidenced by the weak spot observed with aniline-phthalic acid.

E$ect of pH

It was seen that a pH depression is caused by autoclaving, particularly evident in those glucose-containing solutions autoclaved in an initial

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978 liMIN0 COMPOUNDS BND CllRBOHYDRaTES

alkaline medium. Inasmuch as the spots on the aniline hydrogen phthal- ate chromatograms decrease in size as the pH increases, it appears that the browning of glucose at higher pH levels is characterized by a partial conversion of glucose to acidic intermediates which no longer give the typical aldehydic reaction. The intensity of the color of the autoclaved glucose solutions is somewhat dependent on the initial concentration of glucose present in solution.

TABLE II Effect of Autoclaving (15 Pounds Pressure for SO Minutes) on Glucose, Glycine,

and Glucose-Glycine Mixtures in Phosphate Buffer at Various pH Levels

Glucose-glyci

~lllt&S

50:o

0:50

10:50*

ne -

-_

PH

Before autoclaving

3.2 6.0 9.7

6.0 (Un- buffered)

3.2 6.0 9.7

5.9 (Un- buffered)

3.2 6.0 9.7

6.2 (Un- buffered)

After utoclavinj

2.9

5.0 6.0 5.3

3.2 6.0 9.7

5.9

3.2 5.2 7.9

5.5

Total nitrogen

697.5

697.5

: a After

utoclavine

mz.

0

After .utoclaving

nzg.

0

697.0 697.6 697.6 697.3

697.3 697.7

697.2 697.8

697.6

697.2

697.1 697.3

697.8

697.6

520.3 697.2

a-Amino nitrogen

25.4

COlOi-

-

++

+++++

-

-

-

-

-

-

+++

++++

++

* Comparable results were obtained on lO:lO, 30:10, 10:30, and lo:50 mixtures.

The amino acid-glucose solutions were next studied. Glycine, L-lysine, and D-glutamic acid were first investigated; these amino acids were chosen because they are representative of neutral, basic, and acidic amino acids. A series of glycine-glucose mixtures was autoclaved in a buffer at pH 6.0 corresponding to the isoelectric point of glycine, another in a buffer at pH 9.7 corresponding to the isoelectric point of lysine, a third at pH 3.2 corres- ponding to the isoelectric point of glutamic acid, and finally a series in aqueous medium without addition of any buffer. The lysine series was autoclaved at the same pH levels, as well as in an unbuffered solution, and at pH 8.0, representing an alkaline medium but still acid to the isoelectric point, and at pH 11.0, on the basic side of the isoelectric point of lysine.

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L. J. SCHROEDER, M. IACOBELLIS, AND A. H. SMITH 979

In the case of glutamic acid, a similar series was autoclaved, except that the solution at pH 11.0 was replaced by one at pH 2.0 (below the isoelectric point of glutamic acid). The average results of these investigations appear in Tables II, III, and IV.

TABLE III

Effect of Autoclaving (15 Pounds Pressure for SO Minutes) on Glucose, Lysine, and Glucose-Lysine Mixtures in Phosphate Buffer at Various pH Levels

Glucose-lysine monohydro-

chloride

mnaoles

50:o

0:50

10: 50t

-

PH

Before autoclaving

3.2 6.0 8.0 9.7

11.0 6.0 (Un-

buffered 3.2 6.0 8.0 9.7

11.0 5.7 (Un-

buffered 3.2 6.0 8.0 9.7

11.0 5.6 (Un-

buffered)

3.0 5.2 5.9 6.2 6.5 5.3

3.2 6.0 8.0 9.7

11.0 5.6

3.2 5.5 7.5 8.0 7.8 5.3

Total nitrogen

a1cu1atet

WY.

0

1396.9

1396.9

.fter auto- claving

m.s.

0

1321.5 1313.0 1313.5 1312.8 1314.2 1313.8

1319.1 1313.1 1312.5 1314.5 1315.1 1314.7

-

A

-

-

a-Amino nitrogen*

.fter autc- claving

w.

0

698.7 698.0 698.5 698.1 698.5 698.5

698.3 698.8 502.9 497.3 494.5 697.9

-

.- LOSS

per cent

28.0 28.8 29.2

Color

- ++

++++ ++++ ++++

+

- - - - -

- +++

++++ ++++ ++++

++

* a-Amino nitrogen is one-half of the total nitrogen. t Comparable results were obtained on lO:lO, 30:10, 10:30, and lo:50 mixtures.

From the experiments in which the amino compound-glucose mixtures are autoclaved in unbuffered aqueous medium or at acid reaction, it ap- pears that browning may be due to the effect of the pH of the solution on the carbohydrate alone. The color development occurs at the same rate and to the same extent in the autoclaved solutions of glucose alone as in those of glucose heated with amino compounds, provided they are at the same pH. Under these non-alkaline conditions, the amino nitrogen of the autoclaved mixtures containing amino compounds is the same as that of

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980 AMINO COMPOUNDS AND CARBOHYDRATES

the unautoclaved samples. Total nitrogen values likewise are unchanged. Furthermore, paper chromatograms when developed with ninhydrin show only one spot having the same RF value and intensity as that of the un- heated control, indicating that the amino compound remains intact. In these experiments the color development was found to be accompanied by a pH depression, as already pointed out. With aniline-phthalic acid the

TABLE IV Effect of Autoclaving (15 Pounds Pressure for 30 Minutes) on Glucose, Glutamic

Acid. and Glucose-Glutamic Acid Mixtures in Phosphate Buffer at Various pH Levels

GlUCOSf? glutamic acid

___- ?ltlltoles

50:o

0:50

10: 50*

PH

Before autoclaving

2.0 3.2 6.0 9.7 6.0 (Un-

buffered 2.0 3.2 6.7 9.7 4.8 (Un-

buffered) 2.0 3.2 6.0 9.7 4.8 (Un-

buffered)

a.1 After

utoclavin@

2.0 3.0 5.2 6.0 5.3

2.0 3.2 6.0 9.7 4.8

2.0 3.1 5.6 7.9 4.4

.-

Total nitrogen

%%-

mg.

0

698.2

698.2

A

-

fter autc- claving

mg.

0

698.0 698.0 698.2 698.4 699.0 698.2 698.9 698.4 698.5 698.6

698.8 698.2 698.9 697.9 698.9 698.0 698.5 535.5 699.0 698.1

a-Amino nitrogen

LOSS

per cent

23.3

-- C&r

- - +

++++ +

- - - - -

- +

++ ++++

+

* Comparable results were obtained on 10: 10, 30: 10, 10: 30, and 10: 50 mixtures.

autoclaved mixtures gave one spot which had an RF value corresponding to that of the unautoolaved glucose but much smaller in size.

On the other hand, in the experiments carried out in alkaline buffers, pH depression, color formation, decrease in amino nitrogen, and appear- ance of an extra spot on the paper chromatograms developed with nin- hydrin were observed, indicating that both browning and the Maillard reaction had occurred. Moreover, when the solutions of glucose alone were compared with those of glucose and amino compounds, both solu- tions having been autoclaved in an alkaline medium at the same pH, it

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L. J. SCHROE-DER, iM.. I.K!OBELLIS, AND .i. H. SMITH 981

was found that the extent of pH depression and of color formation was the same in both cases, changes that would hardly be observed if browning mere the result of the Maillard reaction. These observations, together with those obtained with the mixtures autoclaved in unbuffered and acid solutions, suggest that the underlying mechanism of color development is the same at all pH values; it is completely independent of the concomitant interaction of amino compounds with glucose which takes place only in an alkaline medium.

TABLE V Chronzatographic Analysis* of Amino Compound-Glucose Mixtures Autoclaved

in Alkaline Buffers

Glycine .................... L-Lysine ................... n-Glutamic acid. .......... L-Arginine ................. L-Leucine .................. nn-Valine .................. Glycyl-r-leucine .......... Glgcyl-nn-valine. ..........

I- Before

auto&wing

RF

0.40 0.30 0.29 0.54 0.82 0.79 0.72 0.80

After autoclaving (2 spots)

1 Extra

RF RF

0.40 0.22 0.30 0.55

Longer spot with 2 humps 0.54 0.91 0.82 0.40 0.79 (Smaller in size) 0.72 L‘ tc ‘6

0.80 “ “ “

* Paper chromatography (phenol-water; ninhydrin) for amino compound-glucose mixtures. In all autoclaved solutions treated with aniline hydrogen phthalate a spot was obtained Fhich had an RF value of 0.39, corresponding to that of the unau- toclaved glucose solution, but smaller in size. The size of the spot decreased with increasing pH levels.

As stated earlier, the experiments were set up to determine the possible part played by the electronic state of the amino compounds in the Maillard reaction. From the results obtained with lysine autoclaved at pH 8.0 and 11.0, it may be seen that a positive Maillard reaction can be demon- strated in all cases. This signifies that the electronic state of the amino acid has little effect on the interaction between amino compounds and reducing sugars. The same conclusions were drawn on the basis of other amino compounds studied, namely, with L-arginine, L-leucine, m-valine, glycyl-L-leucine, and glycyl-nn-valine. In these latter experiments again, browning only was observed in the samples autoclaved in neutral and acidic medium, whereas in alkaline medium both browning and the Mail- lard reaction occurred. That a definite reaction did occur between amino compounds and glucose was evidenced also by the decrease in amino

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982 AMINO COMPOUNDS AND CARBOHYDRATES

nitrogen and by the appearance of an extra spot on the chromatograms developed with ninhydrin. The RF values of this extra spot, together with those of the spot corresponding to the control amino acids, appear in Table V.

DISCUSSION

After numerous studies on proteins and allied compounds heated with carbohydrates, the over-all view-point persists that browning is the result of the interaction between amino compounds and reducing sugars as postulated for the first time by Maillard (1). The present study with model amino acid- and peptide-glucose mixtures further substantiates the view advanced previously (2, 3) that water has a definite inhibitory effect on this apparent interaction. However, it is now suggested that browning and the Maillard reaction occur independently of one another, the extent of each being determined by the conditions, most particularly by the degree of heating and the pH of the solution.

Whereas Frankel and Katchalsky (14-16) had found a parallelism between the pH depression, browning, and the amino compounds-sugar interaction, our trial experiments showed a pH depression and browning but no amino compounds-sugar interaction, as evidenced by the amino nitrogen values as well as by chromatographic analyses. However, Englis and Dykins (17) failed to observe any decrease in a-amino nitrogen even in solutions in which marked sugar rotation changes were found. Kass and Palmer (18), in their study on the effect of buffers on lactose at autoclave temperatures, concluded that color development is related to the loss of optical activity and is accompanied by the development of acidity. However, Friedman and Kline (19) report that color formation per se is an inadequate criterion for evaluating this reaction.

Sufficient evidence to propose the theory that browning and the Maillard reaction occur independently of each other is at hand. The present experiments have shown that the interaction between amino compounds and reducing sugars is limited to alkaline solutions and to the dry state. From our data it is also evident that browning appears to be due largely to the effect of pH on carbohydrates and not to the interaction of carbo- hydrates and amino compounds. A definite interaction does occur be- tween amino compounds and reducing sugars in alkaline solutions, as evidenced by the appearance of an extra spot on the chromatograms when glycine, L-leucine, n-glutamic acid, L-lysine, and arginine were autoclaved with glucose. Similar results were obtained recently by Taufel and Iwainsky (20), who concluded that the formed complex is usually com- posed of carbohydrates and amino compounds. More recent studies (21) have produced strong evidence that the type of compound formed in the

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L. J. SCHROEDER, M. IACOBELLIS, AND A. H. SMITH 983

Maillard reaction is a glyconyl peptide. The assumption by many in- vestigators that browning was the result of the Maillard reaction was logical, if one considers that in alkaline solutions, in which the interaction between amino compounds and reducing sugars takes place, browning is at its maximum. However, the mechanism by which the Maillard reac- tion takes place in the dry state is the topic of continued investigation.

The authors wish to express their appreciation for a research grant from the American Dairy Association which has made the present investigation possible. They would like also to acknowledge the cooperation of Dr. H. L. Sipple of the Evaporated Milk Association.

SUMMARY

The effect of water and pH on browning and the Maillard reaction was studied by comparing model amino acids and synthetic peptides autoclaved with and without glucose in the dry state and in buffered solutions. It is evident that browning appears to be due to the effect of pH on carbohy- drates only and not to the interaction of carbohydrates with amino com- pounds, whereas this interaction, the so called Maillard reaction, is limited and takes place in alkaline solutions. These findings were substantiated by chromatographic analyses as well as by the more conventional total nitrogen and amino nitrogen determinations.

BIBLIOGRAPHY

1. Maillard, L. C., Compt. rend. Ad., 157, 850 (1913). 2. Schroeder, L. J., Iacobellis, M., Lees, H., and Smith, A. H., J. Nutr., 60, 351

(1953). 3. Schroeder, L. J., Iacobellis, M., and Smith, A. H., J. Nutr., 49, 549 (1953). 4. Sheehan, J. C., and Frank, V. S., J. Am. Chem. Sot., 71, 1856 (1949). 5. Grassmann, W., and Schulte-Uebbing, E., Ber. them. Ges., 83, 244 (1950). 6. Fruton, J. S., Advances in Protein Chem., 6, 1 (1949). 7. Fischer, E., and Steingroever, J., Ann. Chem., 365, 167 (1909). 8. Pope, C. G., and Stevens, M. F., Biochem. J., 33,107O (1939). 9. Schroeder, W. A., Kay, L. M., and Mills, R. S., Anal. Chem., 22, 760 (1950).

10. Williams, R. J., and Kirby, H., Science, 107,481 (1948). 11. Bull, H. B., Hahn, J. W., and Baptist, V. H., J. Am. Chem. Sot., 71,550 (1949). 12. Consden, R., and Gordon, A. H., Nature, 162, 180 (1948). 13. Partridge, S. M., Nature, 164, 443 (1949). 14. Frankel, M., and Katchalsky, A., Biochem. J., 31, 1595 (1937). 15. Frankel, M., and Katchalsky, A., Biochem. J., 35, 1028 (1941). 16. Katchalsky, A., Biochem. J., 35, 1024 (1941). 17. Englis, D. T., and Dykins, F. A., Ind. and Eng. Chem., AnuE. Ed., 3,17 (1931). 18. Kass, J. I’., and Palmer, L. S., Ind. and Eng. Chem., 32,136O (1940). 19. Friedman, L., and Kline, 0. L., J. Biol. Chem., 184, 599 (1950). 20. Taufel, K., and Iwainsky, H., Biochem. Z., 323, 299 (1952). 21. Iacobellis, M., Federation Proc., 13, 235 (1954).

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Arthur H. SmithLawrence J. Schroeder, Michael Iacobellis andCOMPOUNDS AND CARBOHYDRATESON THE REACTION BETWEEN AMINO THE INFLUENCE OF WATER AND pH

1955, 212:973-984.J. Biol. Chem. 

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