Table Of ContentSTUDIES ON THE MAILLAitD REACTION;
APPLICATION OF RADIOCHEMICAL TECHNIQUES
DISSERTATION
Presented in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy in the
Graduate School of The Ohio State
Univers ity
By
RAYMOND CHARLES SCHLICHT, B.S., M.S.
The Ohio State University
1952
Approved by:
Adviser
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
i
TABLE OF CONTENTS
Pa ge
I. INTRODUCTION AND STATEMENT OF THE PROBLEM........ I
II. HISTORICAL BACKGROUND................................ 4
A. Summary of Previously Reviewed Literature
on the Maillard Reaction............ 4
B. Recent Contributions tot he Literature........ 25
III. DISCUSSION OF RESULTS................................ 43
A. Radiochemical Tracer Reaction Series......... 43
1. Hexose-Glycine System...................... 45
2. Pentose-Glycine Systems................... 47
3. Comparison of the Hexose-Glycine
and Pentose-Glycine Systems....... 49
4. Ion-Exchange Separation of Tracer
Reaction Products.......................... 64
5. The Effect of Air on the Maillard
Reaction.................... 66
B. Degradation of a D-Xylose-Glycine Polymer.... 71
1. Alkaline Permanganate Oxidation.......... 71
2. Chromic Acid Oxidation.................... 72
C. Methylation of a D-Xylose-Glycine Polymer,... 72
D. Solubility in Organic Solvents of a
D-Xylose-Glycine Polymer....................... 75
E. Partial Decolorization of D-Xylose-
Glycine Polymers...... 76
F. Reactions of Aldoses and a-Aminoiso-
butyric Acid.......... 77
G. Recapitulation of the Definitive
Tracer Results.................... 81
H. Speculation Concerning the Possible
Means of Formation and the Nature of
the Polymer Repeating Unit..................... 83
SZS786
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE OF CONTENTSj (cont.)
Pa ge
IV. EXPERIMENTAL........................................ 87
A. Materials....................................... 87
B. Radiochemical Tracer Reaction Series........ 88
1. General "Browning" Reaction
Procedure .. .................. 88
2. Preparation and Treatment of
Radiochemicals. .......................... 89
(a) Ion-Exchange Recovery of
Radio Glycine . . 89
(b) Preparation of D-xylose-l-Cl4-. , 91
(c) Dilution of Radiochemicals.......... 93
3. Counting of Radioactivity and
Methods of Calculation................... 94-
(a) Dilution Factors in theP olymers.... 93
(b) Percentage of Carbon Dioxide
from the Sugar........................ 96
(c) Percentage of the Total Sugar
Number-One Carbons in the
Isolated Polymer , . 96
(d) Percentage of Carbon Dioxide
from Glycine-1- and -2-C^4-.......... 97
C. A D-Xylose-Glycine Polymer Prepared
Under Air....................................... 98
1. Preparation................................ 98
2. .Alkaline Permanganate Degradation....... 98
3. Chromic Acid Oxidation......... ......... 100
4.. Methylation of the Polymer............... 101
5. Solubility of the Polymer in
Organic Solvents...................... 102
D. Partial Decolonization of D-Xylose-
Glycine Polymers................ 103
E. Reactions of Aldoses and a-Aminoiso-
butyric Acid................................... IO4.
V. SUMMARY.............................................. 117
VI. SUGGESTIONS FOR FUTURE WORK............. .. .......120
VII. BIBLIOGRAPHY................................... 122
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE OF CONTENTS, (cont.)
Pa
rq
VIII. ACKNOWLEDGMENT.................................... 127
IX. A UTOBIOGkAFHI..................................... 12g
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
iv
LIST OF TABLES AMD FIGURES
Pa ge
TABLE I ............................................. 109
TABLE II ........................................ 110
TABLE III ........................................... 110
TABLE IV ............................................ Ill
TABLE V ............................................. 112
TABLE VI ............................................ 113
TABLE V I I .................................... 114.
Figure 1 ............................................ 115
Figure 2 ............................... 116
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
STUDIES Ql\i THE MAILLARD REACTION:
APPLICATION OF RADIOCHEMICAL TECHNIQUES
INTRODUCTION AND STATEMENT OF THE PROBLEM
The discoloration of foods has long been the
subject of research on its causes and on means for
its prevention or eradication. The discoloration,
commonly denoted browning, has been found to be due to
well-defined general reaction types which may occur
independently and simultaneously, depending upon the
nature of the material and its treatment. The three
main reaction types are: (a) high temperature decompo
sition of some of the components of the material,
(b) low temperature browning as a result of complex
enzymic reactions, and (c) the interaction of reducing
sugars, or other reactive carbonyl compounds with
nitrogenous compounds, usually proteins or free amino
acids. Reaction type (a) is easily prevented by not
allowing the temperature to approach that critical for
the particular material. Reaction type (b) is thwarted
by a "blanching" process, in which the enzymes are de
stroyed or inactivated by raising the temperature,
usually to 100°C., by steaming for a short time.
The third type of browning is the one of greatest
concern to food processors. The reaction of carbonyl
compounds and nitrogenous materials occurs with rapidity
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
at high temperatures, and, given sufficient time, will
take place to an injurious extent at relatively low
temperatures. Because of the importance to the armed
forces of preparing foods in dehydrated or concentrated
forms without appreciable loss in palatability and
nutrition, the Food and Container Research Institute
for the Armed Forces has carried out or supported a large
volume of research on reaction type (c). This reaction
has come to be named the Maillard reaction after L.-C.
Maillard, who found that amino acids and reducing sugars
react to give dark brown products akin to the coloring
matter formed when food3 and other natural products
disc olor.
Although considerable work of an empirical nature
has been and is being carried out with regard to the
more immediate practical aspects of- the browning of food
products, the Food and Container Research Institute
for the Armed Forces has recognized that, as long as
the fundamental nature of the reaction and the structure
of the products are unknown, or are clouded by the com
plexity of natural systems, it is probable that present
methods of inhibition of the reaction will not be used
with maximum effectiveness, and that new methods of in
hibition would only be discovered by haphazard trials in
the use of additives. Therefore, the Institute has
sponsored a project at this university, among others,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3
- -
to investigate the fundamental nature of the Maillard
reaction, in the hope that, eventually, effective means
of controlling or of inhibiting the browning reaction
may be contrived.
The work which is reported upon here is concerned
chiefly with the nature of the high molecular weight
pigments resulting from the reaction of simple sugars
and amino acids, free from the extraneous conditions
and materials which exist during the processing of food
products. A study of the nature of the "browning
polymer" is undertaken in order to ascertain the manner
in which the reactants combine as well as to shed more
light upon the precise structure of the polymer.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4
- -
II* HISTORICAL BACKGROUND
A . Summary of Previously Reviewed Literature
on the Maillard Reaction
The chemical literature contains numerous articles
on the Maillard reaction, but many of these concern the
browning of the complex mixtures found in foodstuffs
and other natural products, or in their processed forms.
Stadtman (1) has prepared an excellent review on the
(l) E. R. Stadtman, Advances in Food Research,
Vol. I, Academic Press, Inc., New York, II, Y., 1948,
p. 325.
nonenzymic browning of fruit products. However, this
writer is properly concerned more with the studies made
on relatively simple reaction systems. Langer (2) in
(2) A. W, Langer, Jr., Ph. D. Dissertation, The
Ohio State University, 1951.
his Ph. D. dissertation has reviewed most of those reports,
up to 1951, which were concerned with the fundamental
studies on model systems of amino acids and simple sugars
or carbonyl compounds. Danehy and Pigxnan (3) have pre-
(3) J. P. Danehy and W, W. Pigman, Advances in
Food Research, Vol. Ill, Academic Press, Inc., New York,
N. Y., 1951, p. 241.
sented a review which covers this limited field in a
somewhat more detailed fashion. Because of the existence
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
5
~ ~
of these quite extensive reviews, the aut- hor will give
a less detailed summary of the more impor tant works
which are included in the two reviews on the reactions
in relatively simple systems.
Maillard (4) made the discovery that- simple sugars
(4) L.-C. Maillard, Compt. rend., 1 54. 66 (1912);
Ann. chim., 9__/ 258 (1916).
and amino acids reacted quite readily to .give dark
brown pigments similar to those arising i jci the browning
of foods. He found that quite concentre t-ed solutions
D-glucose and glycine (5.55 M and 3.3 3 M, respective
ly) upon heating to temperatures ranging jfrom 34° to
150°C. gradually darkened with the evolutrion of carbon
dioxide. The nature of the atmosphere ha *d little effect,
since, in the presence of oxygen, the amo~unt of carbon
dioxide formed well exceeded the amount orf oxygen con
sumed. He drew the conclusion, now known to be false
(2), that oxygen had no role in the reactrion. Maillard
showed that extensive dehydration of the ssugar must have
occurred.
Maillard also found by visual comparrison that the
order of decreasing reactivity of various amino acids
with D-glucose at 100° was: alanine, valise, glycine,
glutamic acid, leucine, sarcosine, and tyrosine.
Similarly he showed that the order of decreasing re
activity of sugars with glycine at 100° was: pentose,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
