PILL-SOON SONG and C. 0. CHICHESTER Department of Food Science and Technology, University of California, Davis, California 95616

Kinetic Behavior and Mechanism of Inhibition in the Maillard Reaction.

III. Kinetic Behavior of the Inhibition in the Reaction

Between D-Glucose and Glycine

SUMMARY-Sodium bisulfite and various other inhibitors were studied as to efficiency and kinetic behavior in the Maillard reaction. One group of compounds (bisulfites and mercap- tans) inhibited the reaction step prior to the steady-phase browning step, whereas another group (benzoyl peroxide and disulfides) affected the steady-phase browning step. It is suggested that the inhibitory mechanism is due to attack on intermediates of the Maillard reaction by a reactive form of an inhibitor such as a free radical rather than by the original inhibitor molecule itself. Existing theories on the mechanism of inhibition by sulfites are critically reviewed, and their inade- quacy is pointed out.

INTRODUCTION A number of compounds with inhibitory effect on the

formation of melanoidins have been studied in connection with industrial applications. Information is scarce, how- ever, on the mechanism of the inhibition as compared to the mechanism of the reaction in the absence of an inhibitor such as bisulfite. Ellis (1959) proposed that ready com- bination of sulfur dioxide with furfurals in the Maillarcl reaction mixture may be significant in the inhibition, basing this on the assumption that the Maillard reaction proceeds via the formation of furfurals as intermediates. Our pre- vious papers have questioned the importance of furfurals as major intermediates in the reaction.

Ingram and Bas (1950) suggested that the sequence of reactions leading to the brown color development is interrupted at the stage where formaldehyde from the Strecker degradation of glycine reacts with the inhibitor sulfur dioxide. However, evidence contradictory to that proposal can be found elsewhere (Haas et al., 1948; Jos- lyn, 1941; Nomura, 1955).

The mechanism of inhibition by various known reagents other than sulfites has not been explored, except for a pro- posal that inhibition may possibly be caused by some carbonyl reagents which may reduce the concentrations of the reactant aldoses and free aldehyde intermediates in the reaction mixture (Ellis, 1959). Because understanding is poor of the Maillard reaction itself, no detailed inhibition studies have been made, either kinetically or mechanisti- cally, except for some practical aspects of the inhibition of the reaction.

It was hoped that inhibition studies would provide valu- able information on the mechanism of the Maillard reac- tion as well as on the mode of inhibition. Results are reported here on the kinetic behavior of inhibition. Sev- eral existing theories on the mechanism of inhibition by bisulfite are discussed critically.

Materials

EXPERIMENTAL

The materials used were sodium bisulfite,’ sodium meta- bisulfite, sodium dithionite, sulfur (Ss), bromine, hydro- gen iodide, and bromide from J. T. Baker Chemical Co.; Z-mercaptoethanol, mercaptoacetic acid, propanethiol, eth- anethiol, toluenethiol, thiophenol, benzyl disulfide, ,&ner- capto-DL-isoleucine, dimethyl sulfide, 3-mercaptopropionic acid, furfurylmercaptan, 0-mercaptobenzoic acid, 4-mer- captobutyric acid, and benzoyl peroxide from Eastman Organic Chemicals ; isoamylnitrite from Matheson Cole- man & Bell Co. ; formaldehyde from Allied Chemical & Dye CO. ; and D-glucose bisulfite synthesized by a modified method of Braverman and Kopelman (1961).

Preparation of reaction mixtures and kinetic runs

Parts I and II described preparation of the reaction mix- tures and kinetic measurements of the reaction, as well as other experimental details, such as autoradiography.

Identification and determination of gluconic acid in the sulfited reaction mixture

The following procedure was followed to isolate and identify gluconic acid, which is reported to be an oxidation product of D-glucose by sulfites.

Reaction mixture J

cation-exchange resin (Dowex-3) J displace with

eluate

I I

O.OlN HCl

anion-exchange resin (Duolite A-4)

“cation fraction”

displace with & O.OZN NaOH and eluate O.lN NHaOH for sulfites

I __ I “anion fraction” “neutral fraction”

Using the solvent n-butanol-formic acid-water (4 :l :5 V/V), the anion fraction was chromatographed on What- man No. 4 paper, and gluconic acid with R, 0.11 was iden- tified by comparison with authentic gluconic acid. The hydroxamate derivative of gluconic acid from the anion fraction was rechromatographed, and the Rr value was in accordance with the reported value of 0.63 in n-butanol- ethanol-water (4 :1 :5 v/v) (Abdel-Akher and Smith, 195 1). Since our concern was only gluconic acid, the other

MAILLARD REACTION-KINETIC BEHAVIOR-99

compounds possibly present in the anionic fraction were appreciably but do not affect k,, significantly. kst is defined excluded from the present experiment. as in Parts I and II.

Gluconic acid was further identified by gradient elution column chromatography using a Dowex-l-X8 ( loo-ZOO- mesh) by the method of Palmer (1955). The eluant was aqueous acetic acid with an initial concentration of 0.875N. Fig. 11 shows the chromatogram of the eluate plotted as the titration value versus fraction number.

Gluconic acid formed in the sulfited reaction mixture was determined as gluconic acid hydroxamate (Lien, 1959 ; Hilf and Castano, 1958). Its concentration in the anionic fraction from the sulfited reaction mixture was estimated from a standard calibration curve. The kinetics of gluconic acid formation were followed by this method (Hilf and Castano, 1958).

Type II : inhibitors that affect 17 tSl more significantly than the induction period of the reaction, or affect both Iz,~ and induction period moderately.

Actually, no accurate kinetic comparison is possible, be- cause different concentrations of inhibitor were used and inhibitors differ in solubility.

Values of 7 and 7in in Table 1 were obtained as described in Part I. kin was evaluated from the slope of the plot of the amount of production formation versus time in the presence of an inhibitor which was measured as the change in optical density.

RESULTS AND DISCUSSION Table 1 shows inhibitory kinetic data obtained with

various inhibitors, some of which have been used for the first time in the present work. For convenience, the inhibi- tors were arbitrarily classified as follows :

Type I : inhibitors that lengthen the induction period

Fig. 1 shows the effect of sodium bisulfite on the rate of the reaction, and Figs. 2, 3, and 4 show the effects of various inhibitors (Type I). Table 1 and Figs. l-4 show that the value of Y (defined in Table 1) is approxiunity except at higher concentrations of an inhibitor. It is pos- sible to evaluate the inhibitor efficiency from the relation- ship between 7in and concentration of an inhibitor, in view of the fact that the Type I inhibitor acts mainly during the induction period.

Table 1. Inhibition of Maillard reaction at 55°C at pH 5.5-5.6, except where otherwise indicated. TC,, and K4, are respectively induction period (minutes) and rate (ml-l mine*) in the nresence of an inhibitor. r is relative retardation constant Ck,t/K<,).

Inhibitor (G), ml-* (g), ml-l

Type 1 Sodium bisulfite 1

1

Sodium nz-bisulfite 1 1

Sodium dithionite 1

Sulfur ( SS) 1

Bromine 1

Furfuryl mercaptan 1 1

Z-Mercaptoacetic acid 1 3-Mercaptopropionic acid 1

1

4-Mercaptobutyric acid 1 1

I-Propanethiol 1 1

Ethanethiol 1 1

2-Mercaptoethanol 1 1 1 1 1

iso-Amylnitrite 1 1 1 1

0.25 0.1 9.14 3.35 5.94 1.20 0.50 0.1 9.14 2.93 12.50 1.09

0.50 0.1 5.25 1.87 13.40 1.02 0.50 0.19 10.0 3.74 9.75 1.41

0.50 0.1 5.75 1.76 12.10 1.13

0.25 0.1 3.90 0.39 6.27 1.13

0.50 0.062 3.92 3.84 3.12 4.40

0.50 0.23 19.8 0.50 10.10 1.35 0.50 1.15 98.0 1.71 11.00 1.25

0.50 0.57 0.62 5.03 10.20 1.09” 0.50 0.26 25.5 3.02 7.90 1.41 a 0.50 1.31 128 6.51 7.79 1.43”

0.50 0.13 10.8 2.29 11.30 1.21 0.50 0.26 21.6 4.45 11.20 1.22

0.50 0.17 0.22 2.08 9.15 1.49 0.50 0.42 0.56 5.13 4.03 3.40

0.50 0.084 0.14 1.73 11.10 1.24 0.50 0.168 0.27 3.18 6.34 2.16

0.50 0.112 0.141 1.25 12.00 1.14 0.50 0.224 0.282 3.63 10.90 1.26 0.50 0.448 0.564 4.54 10.40 1.32 0.50 0.672 0.846 7.30 8.50 1.61 0.50 0.224 0.282 1.90 49.60 2.5Zb

0.50 0.087 7.45 0.33 13.10 1.04 0.50 0.174 14.90 0.62 12.90 1.06 0.50 0.348 29.80 0.73 12.70 1.08 0.50 0.522 44.70 0.86 12.70 1.08

Inhibitor

763 ml-’ x 103

7ia (p-11)

7 Ktn x 10”

B At pH 4.7. The approximate value is estimated using k,t = 1.11 x lo-’ M ” min-1 at pH 4.5. b At pH 8.9.

(Table continued on next page)

+

lOO-JOURNAL OF FOOD SCIENCE-Volume 32 (1967)

Inhibitor

Table 1. (Concluded from preceding page). .

Inhibitor ~<n

(G), ml-l (g), ml-l 96, ml-’ x 103 (y-1)

Kbt x 104 r

Type II p-Mercapto-DL-

isoleucine L-Cystine

L-Cysteine.HCl

L-Cysteine (basic) Benzoyl peroxide

Hydrogen bromide Hydrogen iodide Benzoyl disulfide

Dimethyl disulfide Diethyl disulfide

Benzyl disulfide Thiophenol #-Toluenethiol 0-Mercapto benzoic

acid

Diphenyldisulfide

1 0.50

1 0.50 1 0.50

1 0.25 1 0.25 1 0.25 1 0.25

1 0.25 1 0.25 1 0.25 1 0.50

1 0.50

1 0.50 1 0.50

1 0.50 1 0.50 1 0.50 1 0.50 1 0.50

1 0.50

0.20 0.123 0.10 4.17 0.20 8.34 0.006 0.20 0.30 10.0 0.60 20.0

0.06 5.0

0.0048 0.2 0.0484 2.0 0.484 20.0

0.10 3.65 0.20 7.30 0.40 42.60 0.24 19.60 1.20 98.00 0.20 8.15 0.20 18.2 0.20 16.1

0.50 0.10 6.5 0.50 0.20 13.0 0.50 0.40 18.4

0.42 11.70 0.025 293 0.038 174 0.042 6.52 0.088 2.47 0.24 0.70 0.026 21.70 0.28 7.06 0.30 6.40 0.36 5.08 0.44 7.21 0.47 12.60 0.19 13.70 0.35 13.50 0.08 13.70 0.19 13.60 0.35 13.30 0.08 13.70 0.252 13.6

? 13.1

0.04 11.3 0.31 10.8

? 13.7

1.17 1.36" 2.29' 1.09 2.88

10.20 2.04d 1.01 1.11 1.40 1.54" 0.88' 1.00 1.02 1 .oo 1.01 1.03 1.00 1.01 1.01

1.21 1.27 1.00

’ At 94°C to dissolve the inhibitor. d At 80.5”C e At pH 4.5. r At pH 4.8. Approximate value estimated using the value of k,t at pH 4.5.

As will be shown later, the Type I inhibitor (1%) may function by trapping reaction intermediates (Z) , which must accumulate to a steady state concentration to initiate the formation of the melanoidins. Thus, assuming a steady- state approximation for the following mechanism,

kl G+g---+I

k3 I+g-B

kia I +I,-+1 -In

the following relation can be derived :

(40 1 = ka (1%) Tin -- s--

(4 kz+ Jz3 (9)

1 [II 7

where (I), and (I) are concentrations of intermediates respectively in the absence and presence of an inhibitor. The last similarity in Eq. 1 is defined only as a semiquanti- tative proportionality relation. Figs. 5, 6, and 7 show plots of (&T) - 1 against concentration of inhibitor. The slope of such a plot gives the apparent inhibition constant E = Iza[ ka + ha(g) ] see constants in Table 2).

Fig. 8 shows typical kinetic behavior of the reaction in the presence of inhibitors which do not affect T but retard Kat significantly (Type II). The relative retardation con-

Fig. 1. Effect of sodium bisulfite on the Maillard reaction (1M D-glucose DIUS 0.25M glvcine: 55°C and DH 5.3:

j at

A: no inhibitor added, B : 3.08 x 10m3 Ml-’ NaHS03, C: 6.15 x lOa Ml-‘, D: 9.03 x lOa Ml-l, E: 1.52 x lo-’ Ml-‘. F: 3.04 x‘ lo-’ 1MI”.

MAILLARD REACTION-KINETIC BEHAVIOR-101

Table 2. Apparent inhibition constants (E) of inhibitors in the Maillard reaction.

Inhibitor E x 1O-2 E/EN.R~

Sodium bisulfite 2.42 1.00 Sodium m-bisulfite 3.68 1.52 iso-Amylnitrite 0.043 a 1.82 x 10” Z-Mercaptoethanol 0.86 0.36 4-Mercaptobutyric acid 2.02 0.84 Ethanethiol 1.18 0.49 1-Propanethiol 0.913 0.38 Furfurylmercaptan 0.186 7.7 x 1o-z

’ Calculated from the initial slope of Fig. 6.

t. d.p

Fig. 2. Effect of inhibitors on the Maillard reac- tion (l&f D-glucose plus 0.25M glycine for Curves A and B, or 0.5M glycine for C, D, E, and F) at 55°C and pH 5.5 : A & C: no inhibitor added. B ~~3.9 x 10” M/L Ss, ’ D: 5.75 x lo9 M/L Na&O+ E: 1.36 x lo-’ M/L CH,CH,SH, F: 2.71 x 10” M/L CHXH,SH.

10

A B C D E

0.0

j 0.4 <

0.b

Fig. 3. Effect of 2-mercaptoethanol on the Mail- lard reaction (1M D-glucose plus 0.5M glycine) at 55°C and pH 5.5 : A : no inhibitor added, Fig. 5. Plot of inhibition according to Eq. 1: B: 1.41 x lo-* M/L HSCHKHzOH, A: plot of (~1. - T) vs. (NaHSOa), C: 2.82 x lWM/L, B : plot of (T,~/T) - 1 vs. ( NaHS03), D : 5.64 x lo-’ M/L, and C: plot of ~6~7) - 1 vs. (HSCHKHSOH), and E: 8.46 x lo-* M/L. D : plot of (T,,, - T) vs. (HSCHzCHzOH).

stants (r) for Type I inhibitors in Table 1 are seen to be close to unity at relatively low concentration of an inhibitor except bromine, although s’s are affected greatly. In con- trast, Type II inhibitor (“retarder”) appears to show more deviation from unity, although a fractional induction period is not remarkable. Some of Type I inhibitors also affect r at increased concentration of inhibitor, as can be seen from Fig. 9 and Table 3.

The following reactions (evidence to be presented here- inafter and in Part IV) with inhibition are now assumed

Fig. 4. Effect of isoamylnitrite on the Maillard reaction (1M D-glucose plus 0.5M glycine) at 55°C and DH 5.5: A: no inhjbitor added, B: 8.72 x 10.’ M/L (CH,),CHCH20NO, c: 0.174 M/L, D : 0.348 M/L, and E : 0.522 M/L.

102-JOURNAL OF FOOD SCIENCE-Volume 32 (1967)

Table 3. Approximate estimates of apparent re- tardation constants, E, (calculated from slope of the plot in Fig. 9 and from the data in Table 1).

Inhibitor Er x 10”

~(Ct,,12CHCH2CH20NO). ML-’ X IQ2

Fig. 6. Plot of inhibition by isoamylnitrite (data obtained from Fig. 4).

Type II L-Cysteine . HCl L-Cysteine’ (basic) Benzoyl peroxide

Type 1 Sodium bisulfite 2-Mercaptoethanol

1.90 1.45 0.21

0.147 0.073

1

Fig. 7. Plot of inhibition by various inhibitors (data from Table 1) : A : sodium bisulfite B : 4-mercaptoethanol, C : ethanethiol, D : 1-propanethiol, and E : furfuryl mercaptan.

I- /AL. J 1

Fig. 8. Effects of retarders on the Maillard tion (l&f D-glucose plus 0.25M glycine) at and pH 5.5: A : no retarder added, B : 2 x lO”M/L benzoylperoxide, c : 2 x 1o-3 M/L, D : 0.02 M/L, E: 2 x lO+ M/L L-cysteine, F: 0.01 M/L, and G : 0.02 M/L.

reac- 55°C

for discussing the kinetic behavior, of the reaction in the presence of an inhibitor :

k3 I+g-B

ka I + In-I-In

and (or) via a free radical mechanism,

k In OIn * . . . . . . . . . . . . . . . . . ...(b)

I ko

+ In- I-In * (c)

I-In * + In kc

+ I - In + In . ..,,......(d)

or

I-In . + SH kO I - In + s (d’)

In . + In . ka

+ In-In

where In and In* are respectively the inhibitor and its free radical, and SH is a third body in the system which sup- plies an electron.

It can be predicted that, in the reactions considered above, strong inhibition will occur under the following conditions :

(I) cc (In* ), or (I) < (1%).

Under certain conditions, after a sufficient reaction time

Fig. 9. Plot of fractional retardation against the initial concentrations of inhibitors (data from Table 1 and Figs. 1 and 3) :

-o- :NaHS03 and -0- :HSCH,CH20H.

MAILLARD REACTION-KINETIC BEHAVIOR-103

elapses, the following steady-state approximation can be written :

d(I)/dt = PI [h&q + &(41(g) - kL(4 (In) - kb(O (1%. > E

d(ln.)/dt = [31 [koof k,(I-In.)] - k,(I)(In.) - ~,(IYz.)~ e

Integrating eqs. 2 and 3 and solving for (In . ), we have :

(In.) = [41

[

[ko + k,(l-In.) + k.(l)](h) - [kl(G) - k%(l)] (9) I” kg 1

Also,

(0 = k,(G) (g)/[ka(g) + ka(ln) + ka(Zn.11 [51

From Eq. 4 it is evident that the reactive intermediate (1% . ) is proportional to In but inversely proportional to to kd, which determines the efficiency of an inhibitor. Eq. 5 indicates that I in the presence of an inhibitor is strongly dependent on whether or not a given inhibitor is efficient.

It is significant to note that an inhibitor (for example, sodium m-bisulfite) inhibits the reaction strongly if added initially, but if added during the later stage in the steady- phase reaction retards the reaction only slightly or merely bleaches the products (Burton et al., 1962).

It is likely that the condition, instead of being (I) < (In), must be either (I) < (In . ) or (I) < (In*), where In* is an active form of an inhibitor other than free radical, since, if the first condition was applied, the concentration of 1n (approximately 2.1 X lOm”M-l of sodium ++-bisulfite) would have been much higher than the steady-state concen- tration of the intermediates. This argument is partially substantiated in Part IV. Since it is unlikely that the concentration of intermediates is higher than the concen- tration of In used (see Part IV, in which only a small fraction of D-glucose was shown to react), the second or third condition represents the correct situation. The validity of such a condition involving free radicals or other reactive species is suggested in Eqs. 4 and 5 as mentioned previously.

Further evidence in favor of the inhibition mechanism by free radicals of sulfites will be presented in a forthcom- ing paper (Part IV). At present it is sufficient to point out that benzoyl peroxide, which produces a free radical, readily inhibits the Maillard reaction, as shown in Fig. 8 and Table 3, indicating that the free radical may be such a reactive species for the inhibition.

The ratio of the rate of the inhibited reaction and the reaction in the absence of an inhibitor can be written as follows :

-d(ln)/di --- -d(In)/dt _ d(B)/dt - -d(I)/dt -

[k, + k,(I) + kc(I - In.)](In)

[kz + ksk7>1 (4 d(In)/d(I) = (k’,/kst) (In)/(l)

where k’, = k, + k,(I) + k, (1-m * ) , with the assumption that (I) = (I),t and (I-In*) = (l-ln.),t are constant.

If ionic mechanisms are unimportant in the inhibition and if free radicals of original inhibitors are the only reac- tive species, then the first assumption is unnecessary, and we have

d(In)/d(I) = k, + k&I - In.) (In)

k at 1 (0 and, rearranging and integrating, we have

1p)t [

k,+k, (I-In*)

(In>o- k,t 1 ln(Qt/(~), =

k, + k, (I - In.) k 1 ln(B)T/(B)t t61 st

Then, r - 1 reflects the ratio

[ (k. + k,[I - In])/k,, 1-l

In the presence of an inhibitor, -d(I)/dt can be rewrit- ten, and we have

d(In)/d(l) = PI [k, + k,(I) -t k,(l - In.)] (In)

[kz + b(g) + ka(In) + kb(In.)] (I - B) = II&‘/(&t + k~)] (In)/(l - B)

where kob = .Iz, (In) + kb (In. ) , in which In and 1%. at steady state are assumed to be constant. The first assump- tion seems valid at least for the inhibition by sodium bisul- fite, a large fraction of which remained unconsumed at the end of inhibition, as shown by autoradiographic data (Part IV). It is also likely that 1%. is constant throughout the inhibitory action. Eq. 7 then yields an expression similar to Eq. 6.

We now critically review hypotheses proposed for the mechanism of inhibition of the Maillard reaction by sulfur dioxide and sulfite. Other inhibitors have not been studied in detail.

Inhibition at the initial step

Bisulfite addition comzpound tlzeory (Stadtman, 1948; Ingram and Bas, 1950; Ingles, 1959a,b; Cantor et al., 1945 ; Gehman and Osman, 1954; Braverman, 1963). Some workers have stressed the importance of bisulfite addition compounds of carbonyl compounds, including D-glucose, with the association constant K. In order to examine this theory critically, consider the following reac- tions at moderate pH :

G +g%I _.._..., [II - km

G+HSOs+ G-SO,H (D-glucose- k, bisulfite) (f) .

Rate of reaction is given by

-d(G)/dt = h(G) (g)/( 1 + K(HS03)) [S]

where K = km/k,. Integration of Eq. 8 between limits yields

ln[(G),, - (G),] = ln(G)O - kt 191 where k = k, (c~)/[l + K(HSOs)].

104-JOURNAL OF FOOD SCIENCE-Volume 32 (1967)

It is apparent that the slope of k in Eq. 9 (In(G) vs. t) should be smaller in the presence of the inhibitor, whether its concentration is high or moderately low, than in the absence of the inhibitor. In fact, it will be shown by auto- radiographic studies (Part IV) that the above is not the case, since the decrease in radioactive D-glucose is about the same in both the absence and presence of the inhibitor.

The same treatment can be applied to the following reac- tion (for example, Adams and Lipscomb, 1949 ; Adams and Garber, 1949) :

g + HSOS- = complex ._.. (g) .

To examine the significance of these mechanisms as part of the inhibition, we must consider further the following kinetic conditions which will govern the behavior of the inhibitor sulfite.

Condition I: (In) <c(G), (g). Under this condition, reaction 1 and the over-all Maillard reaction in the presence of an inhibitor cannot be inhibited significantly. In con- trast, data in Fig. 1 suggest that the inhibition is appre- ciable under the conditions specified, thus ruling out reac- tion f as a possible mechanism of inhibition by bisulfite.

Condition II : (In) <c(g)% (G). The reaction, again, can- not be inhibited under this condition if mechanism g is op- erative. Fig. 10 gives (Q/T) - 1 = 5.82, and r = 1.1. These results, again, rule out mechanism g. When 2M glycine was used, inhibition was still appreciable [ (T~~/T) - 1 = 5.25, and r = 1.011.

The kinetic analyses presented above therefore lead to the conclusion that neither mechanism f nor g is likely to be the initial mechanism of inhibition. Furthermore, for inhibition to be effective, the dissociation constant of the

I

Fig. 10. Effect of sodium bisulfite on the Mail- lard reaction (O.lM D-glucose plus 144 glycine) at 55°C and pH 5.6. A : no inhibitor added. B : 9.61 x lo4 M/L NaHSOs.

D-glucose-bisulfite addition compound must be much smaller than unity. In fact, the experimental values for Kdissoc. for that compound have been found to be 0.4 at 33°C (Jatkar and Dangre, 1954) and 0.64 at room tem- perature (Burroughs and Whiting, 1960)) compared with 1.5 x 1O-6 for acetaldehyde-bisulfite (Burroughs and Whiting, 1960). We have prepared D-glucose-bisulfite and added it to the reaction mixture. The reaction was equally well inhibited, indicating that dissociation of the bisulfite was appreciable.

Thus, any proposals involving reactions of the inhibitor with initial reactants can be ruled out as significant mecha- nism for the inhibition. For example, it is kinetically improbable that the conformational stabilization of D-glu- cose by sulfur dioxide (Burton and McWeeny, 1963 ; Burton et al., 1963) or the formation of sugar sulfates (Ingles, 1960) plays a major role in the inhibition. It has been found that, when D-glucose-6-sulfate was added to the Maillard reaction mixture (minus a corresponding amount of D-glucose), the reaction was not inhibited (McWeeny and Burton, 1963).

Oxidation of D-glucose by birulfite

Oxidation theory (Ingles, 1959a,c). This proposal is based on the fact that under some conditions bisulfite has been shown to oxidize reducing sugars, including D-glu- cose, which was oxidized to gluconic acid with liberation of elementary sulfur. The analyses presented in the last section make it immediately obvious that this theory also suffers from the kinetic difficulty that the inhibition depends upon reaction between inhibitor and initial reactant. In fact, Fig. 11 confirms that gluconic acid is formed in the sulfited reaction mixture, but adding gluconic acid in an amount corresponding to that formed in the reaction mix- ture (i.e., minus the concentration of D-glucose corre- sponding to the amount of the gluconic acid added) none- theless accelerated the reaction rather than inhibiting it. Again, this suggests that such a trapping of a reactant by oxidation cannot be a possible inhibition mechanism in the reaction.

Gluconic acid in the sulfited reaction mixture increased

: , i

FRACTION NUMBER

Fig. 11. Gradient elution chromatogram of anion fraction from the sulfited reaction mixture : - : sample, and : commercial gluconic acid.

MAILLARD REACTION-KINETIC BEHAVIOR-105

initially, and at a later stage decreased, indicating that it is incorporated into the reaction system.

Antioxidant theory (Stadtman, 1948 ; Hodge, 1953). This theory proposes that the inhibitor reduces the inter- mediates in the reaction mixture to inhibit the formation of melanoidins, while the inhibitor itself is being oxidized. This theory appears attractive, since sulfur dioxide or bisulfite is a reducing agent. However, the theory lacks the details of the mechanism. It is possible that the inhibi- tor may also reduce the reactant and other reducible sub- stances as well as products. Furthermore, sulfur dioxide or bisulfite can act as both an oxidizing and a reducing agent :

fl Lewis acid, : oxidizing agent o-s=0

E‘\, Lewis base : reducing agent

The following thermodynamic data indicate that the re- ducing power increases with an increase in pH of the solu- tion in the range where SOS-- species predominates (Cot- ton and Wilkinson, 1962) ;

so2 . at neutral pH

x Hz0 - \ SO,--- -t 4H+ ix - 2)HzO + Ze-;

E, = 0.17 volts

SOS-- + 2oH- at a1ka1ine PH \ . SO,-- + HZ0 + 2e-; E, = 0.93 volts.

Therefore, if the above theory is correct, the fractional inhi- bition would be expected to increase as the pH of the solu- tion rises. It is also well known that SOS-- is a better nucleophile than HS03-, attacking olefinic compounds more readily at high pH values (Hine, 1962).

It was found, however, as will be shown in Part IV, that the inhibition increases with pH, being maximum around pH 8, above which inhibition decreases, in spite of the fa- vorable standard E”. Some of the inhibitors listed in Table 1 are not reducing agents, e.g., benzoylperoxide. Further- more, good reducing agents such as thiophenol and p-tolu- enethiol are noninhibitory or only slightly retarding. A strong reducing agent, sodium borohydride, actually accel- erated the rate in our system, although it reduced the reductones in a model reaction mixture studied (Hodge and Rist, 1953).

Although this theory may account for the inhibition qualitatively, it is still insufficient to consider as estab- lished. This view becomes further obvious from the fact that a high concentration of the original bisulfite still re- mained after the inhibition was completed, as shown by Stadtman et al. (1946) and autoradiographic evidence obtained in the present work (see also Part IV).

Bleaching of the products (Stadtman, 1948). This pro- posal, which is based on the assumption that the inhibitor bleaches the colored products as soon as the latter are formed, is somewhat analogous to the antioxidant theory in that sulfites reduce olefinic bonds. However, it is still obscure whether the bleaching is due to reductive or oxi- dative action of the inhibitor, although the former appears more likely. It was, however, not possible to bleach the

products significantly with a low concentration of sodium bisulfite corresponding to the lowest concentration of the inhibitor added initially for the study of inhibition kinetics. A high concentration was required to bleach the products effectively. The large portion of bisulfite still unconsumed after completion of the inhibition, again, substantiates the conclusion that the bleaching of products cannot be a major inhibitory mechanism. Definite evidence against this theory has been obtained autoradiographically.

After inhibition was completed, aliyuots of both the control and inhibited reaction mixtures with either D-glu- case-Cl,4 or glycine-Cy were chromatographed and auto- autoradiographed (Figs. 12, 13). It is seen that the sta- tionary spots at the origin, which represent the melanoidins, are lower in concentration for the inhibited reaction mix- ture. A counting of the radioactivity on the stationary

x

A 6 Fig. 1’2. Autoradiograms of the reaction mixtures

containing D-glucose-C:. Solvent for the chroma- tography was isopropanol-water (4 : 1 v/v) : A : without sodium bisulfite. B : with sodium bisulfite.

106-JOURNAL OF FOOD SCIENCE-Volume 32 (1967)

B C 0 Fig. 13. Autoradiograms of the reaction mix-

tures containing glycine-Cy. Solvent used was iso- propanol-water (4 :l v/v) : B : B of Fig. 12. C : without sodium bisulfite. D : with sodium bisulfite.

spots revealed that D-glucose was incorporated into the products 0.27% and 0.06% in the absence and presence, respectively, of sodium bisulfite after the induction period (4 days). Since sulfite-S”” was incorporated into the sta- tionary spots as the inhibited reaction proceeded, it seetns justified to assume that the products in the presence of the inhibitor had not moved from the chromatographic origin after being bleached, if bleached products were actually present. Therefore, bleaching of products can be consid- ered to make only limited contribution to the inhibition mechanism.

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