Kinetics of Formation and Degradation of Morpholino-1-deoxy-D

Chapter 2

Kinetics of Formation and Degradation of Morpholino-1-deoxy-D-fructose 1

Alexis Huyghues-Despointes and Varoujan A. Yaylayan

Downloaded by PURDUE UNIV on August 23, 2016 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1995-0610.ch002

Department of Food Science and Agricultural Chemistry, McGill University, Quebec H9X 1C0, Canada

The kinetics of the reaction of glucose with morpholine to produce morpholino-1 -deoxy-D-fructose (Amadori product) was studied under experimental conditions that minimize side reactions and maximize Amadori product formation (pH 3.0, T=100°C, 90% methanol-water, 6 hr). Glucose (0.1 M) was reacted in sealed vials with a series of morpholine solutions (0.1 - 0.3 M) and similarly morpholine (0.1 M) was reacted with different concentrations of glucose (0.05 - 0.2 M). At specific time intervals, the samples were analyzed for the presence of reactants and Amadori product by a multidetector HPLC system (diode array and pulsed amperometric detector). Under the experimental conditions, morpholine alone (in the presence of glucose equivalent of sorbitol) and glucose alone (in the presence of morpholine equivalent of triethylamine) did not degrade. Similarly, the degradation of Amadori product (0.02 M) was also studied in the presence of excess glucose. The data obtained were used to calculate the rate constants for the formation and degradation of Amadori product.

Kinetics of the early Maillard reaction (Scheme 1) can be divided for convenience into two phases; one, generating the Amadori rearrangement product (ARP) (rate of formation) and the other, reactions leading to its destruction (rate of loss). However, the measured concentration of ARP at any time is the concentration of accumulated Amadori product. The factors that complicate the kinetic analysis are the side reactions of amino acid and sugar, and the regeneration of amino acid from the Amadori product. Thus, the rate of loss of either sugar or amino acid does not necessarily indicate the rate of formation of ARP. General kinetic models have been proposed for the Maillard reaction by Debrauwer et al., (7), Vernin et al., (2), Baisier and Labuza (3) and Yaylayan and Forage (4). Most of these 1

Corresponding author 0097-6156/95/0610-0020$12.00/0 © 1995 American Chemical Society Ho et al.; Flavor Technology ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

2.

HUYGHUES-DESPOINTES & YAYLAYAN

Morpholino-1-deoxy-D-fructose 21


models agree with a general scheme in which the reducing sugar (S) reacts with the amine (A) to produce a Schiff base which undergoes the Amadori rearrangement to produce the Amadori product (ARP), which itself degrades to a set of reactive compounds and free amines that recycle. In addition, the sugar may react with the Amadori product to produce diglycated Amadori products. These reactive intermediates then further react to producefluorescentcompounds and brown polymers. The second order rate equation for the formation of ARP can be written as shown below:

However, the observed or measured rate of the formation of ARP (which will be referred to as the rate of accumulation) is given by the following rate law:

Rate of accumulation =

ARP

^ ^ dt

= k{S][A] - k [ARP] - k [ARP] - • • • U2

n

where, lq, k and k are the rate constants for different decomposition reactions of Amadori product. Generally, the rate constants of reactions that follow second order kinetics of the type shown below, depend on the initial concentrations of the reactants and the first order rate loss of each reactant will increase when the concentration of the second reactant is increased. 2

n

^

= k[A)[B]

This has been observed at 37°C in the reaction of glycine with glucose (5). However, at 60 °C, Vernine et al (2) using computer simulated kinetic analysis of data obtainedfromthe reaction of glucose with three different amino acids have observed that irrespective of the initial ratio of the reactants the observed rate of glucose disappearance was always faster than the amino acids (except proline), if these results can be trusted it may mean glucose is undergoing other side reactions (such as forming diglycated products and amino acid catalyzed decompositions) more extensively than amino acids and that the rate of amino acid release from Amadori product is not negligible. In addition, the rate of sugar loss was also depended on the type of amino acid. Glucose disappeared ten time slower in presence of proline than in presence of valine or methionine, however, this rate was approximately the same as the rate of loss of proline at various sugar to

Ho et al.; Flavor Technology ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

FLAVOR TECHNOLOGY


22

proline ratios between 50 and 65°C (which can be explained by the fact that proline being a secondary amine its ARP cannot react with glucose). However, the thermal degradation of the Amadori product of methionine and proline was much slower (10,000 time slower) compared to valine (7,2). Glucose was reacted in sealed vials with a series of morpholine solutions and similarly morpholine was reacted with different concentrations of glucose. At specific time intervals, the samples were analyzed for the presence of reactants and Amadori product by a multidetector HPLC system (diode array and pulsed amperometric detector). The preliminary analysis of the kinetic data of the reaction between glucose and morpholine to produce morpholino-l-cfeoxv-D-fructose (Amadori product) is presented here. Statistical analysis will be reported elsewhere.

Amino acid +

OH C - N cleavage

C - N rem ains intact

Am ino acid D eoxy glucosones Furan and pyran derivatives FLAVOR AND

D ecarboxy lation D ehydrations Retro-AIdol rxs

COLOR

Scheme 1. General scheme of Maillard reaction


2. HUYGHUES-DESPOINTES & YAYLAYAN

Morpholim-l-deoxy-D-jructose

23

MATERIALS AND METHODS Reagents and Chemicals. All reagents were purchasedfromAldrich Chemical Company (Milwaukee, WI). HPLC solvents werefilteredand degassed (ultrasound/vacuum) prior usage. The Amadori compound of morpholine was purchasedfromSigma Chemical Company (St. Louis, MO).


Kinetic Runs D-glucose (0.05 M - 0 . 2 M ,finalconcentrations) and morpholine (0.1 M - 0.3 M , final concentrations) solutions (pH = 3, adjusted with phosphoric acid) in 90 % methanol/water (see Table I for relative ratios) were incubated in sealed tubes at 100°C for 6 hr. The progress of the reaction was monitored at specific time intervals. Glucose degradation alone was performed in the presence of triethylamine and morpholine degradation alone was performed in the presence of sorbitol (both solutions were at pH = 3, adjusted with phosphoric acid). The results represent the average of doublicate analysis.

T A B L E I. Final concentrations (M) of reactants in different kinetic runs

Experiment product

Sugar

Amine

Amadori

a b c d e f

0.1 0.1 0.1 0.2 0.05 0.1 0.1* 0

0.1 0.2 0.3 0.1 0.1 0 0 0.1**

0 0 0 0 0 0.02 0 0

g h

* in the presence of triethylamine, **in the presence of sorbitol HPLC Analysis Concentrations were determined by a multidetector HPLC system (5) which consisted of a programmable solvent module (Beckman, system GOLD, module 126), a diode array detector (Beckman, system GOLD, module 168), an analog interface (Beckman, system GOLD, module 406), and a programmable


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electrochemical detector (Hewlett Packard, model 1049A). Glucose was measured by pulsed amperometric detector (PAD) at a gold electrode (E2 = +0.650 V, t2 = 50 ms; E3 = -1.050 V, t3 = 50 ms; E i = +0.10 V, ti = 200 ms; vs. Ag/AgCl reference electrode with internal electrolytes). Morpholine and Amadori morpholine were measured by diode array detector, set to monitor the 190 nm to 460 nm region. The carbohydrate dedicated column of Waters (10 mM.; 4.6 mm x 30 cm) was used. The mobile phase consisted of 70 % acetonitrile and 30 % phosphate buffer (0.04 M, pH 2.9). Theflowrate was set at 1 mL/min.


RESULTS AND DISCUSSION The mechanism of the reaction of reducing sugars with amino acids and subsequent decomposition of the Amadori product is a complex process which depends primarily on the reaction conditions such as temperature, time, water content and the relative ratio of the reactants and their concentrations. In the context of the kinetic analysis certain assumptions should be made regarding the mechanism in order to simplify the mathematical calculations. The assumption that sugar and amino acid alone do not undergo decomposition in our model system consisting of glucose and morpholine holds true under the experimental conditions as verified by heating these reactants separately (see Table I, runs g and h). The general kinetic model for the reaction of glucose with morpholine as proposed in Scheme 2, stipulates formation of the Amadori product with a rate constant of ICQ and subsequent decomposition by two pathways one initiated by CN bond cleavage to regenerate the amino group with a rate constant of kj and the other decompositions without C-N bond cleavage with a rate constant of k . 2

Glucose - morpholine reaction kinetics In order to calculate the rate constants k , kj and k , it was assumed that at any time t = tj, x moles of sugar react with x moles of amine to produce x moles of Amadori product (SA). A certainfractionof SA (rx moles) decomposes to regenerate the amine, and r*x moles decompose through other pathways (0 < r and f < 1) therefore: 0

at t = ti

S = (S - x); 0

2

A = (Ao - x + rx);

SA = (x - rx - r'x)

where S, A and SA are the measured concentrations of sugar, amine and the Amadori product respectively. By solving simultaneously the above three equations with three unknowns, the following values for x, r and r' can be obtained with either known or experimentally measured values: Ao - A + S - So , , SA x = S -S, r= — , r'=l-rS - So So - S 0

n

0

:



2.


MorpholinO'l-deoxy-D-Jructose 25

Kinetic Model

Scheme 2. General kinetic model for the reaction of glucose with morpholine

where S and A Q are the initial concentrations of sugar and amine respectively. Knowledge of thefractionof Amadori product undergoing C-N cleavage (rx) and otherfragmentations(r'x) under specific conditions, allows the evaluation of their relative importance and contribution to the overall aroma profile. Such analysis can eventually lead to controlled production of Maillard reaction mixtures, enriched with specific types of products forflavorapplication purposes. Under the experimental conditions, the absolute value of the rate of loss of sugar is equal to the rate of formation of Amadori compound, and the differential rate law for the second order reaction is given by the following equation: 0

=

-ko[So-x][Ao-x]

Solving the above equation will generate the rate law for second order reactions as shown below:


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1/S VSt

R

2

= 0.9996

•

13

A ,A

,..4

1/S A

A

A

A

A

A

A

f

•

1/S

10

Linear (1/S)

0 8

Time(hr)

1 2

Figure 1. Plot of 1/S vs time for model system of glucose : morpholine (1:1 molar ratio)


2. HUYGHUES-DESPOINTES & YAYLAYAN

Morpholino-1-deoxy-D-jructose 27

- = — + kot S So If the reaction is indeed second order then plotting 1/S vs. t will be a straight line with a slope of ICQ and intercept 1/S . Figure 1 shows the plot of 1/S vs. t for the sugar/amine ratio of 1:1. Table II lists the calculated initial rates for other model systems in which the ratio of reactants is not 1:1, using the following equation (6*):


0

where, r is the ratio of reactants, a and b are the initial concentrations of the reactants and x is the number of moles/liter of reactant which have reacted at time t. The data obtained indicate that the reaction of glucose with morpholine to form Amadori product follows second order kinetics and that highest amount of Amadori product was accumulated when the starting amine concentration was three times in excess to that of glucose. A detailed statistical analysis of the data will be published elsewhere.

T A B L E n . Calculated initial rate constants (0 - 2 h)

System

ko(M-ih-i)

3A: IS 1A:2S 1A: IS 1A:0.5S 2A: IS 5S: ISA

1.2 0.6 2.1 1.0 0.8

1

Mh" )

1

Mh- )

R

2

0.9987 0.9981 0.9996 0.9999 0.9927 0.35

0.32

S = glucose; A = morpholine; SA = Amadori product

Kinetics of decomposition of morpholine Amadori compound To calculate kj and k the rate constants for the different decomposition pathways of the Amadori product, the decomposition of the morpholino-D-fructose (final concentration of 0.02 M) was studied under the same reaction conditions in five 2


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Ln(SAo - y/SAo) and Ln(SAo - x/SAo) vs t —


0.8 T

0* 0

— I

0.2

1

1

1

1

1

1

1

1

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

1 2

Time (hr)

Figure 2. Plots of Ln(SA - y / SAo) and Ln(SA -x / SA ) vs time for morpholine Amadori: Glucose model system. 0

c

C


2.


Morpholino-1-deoxy-D-fructose 29

molar excess of glucose (final concentration of 0.1 M). Assuming that at any time t = ti, x moles of the Amadori product decomposes through C-N bond cleavage and y moles through other pathways then the values for x and y can be calculated as shown below.

A

SA

+ Pi

P y SAo SAo - x + x - y


2

So So-x

t= 0 t = t!

During the reaction of Amadori product in the presence of excess glucose, the concentration of the sugar diminished, indicating a reaction with the amine, since it has been established that sugar alone, under the experimental conditions, do not decompose and that the Amadori product being a tertiary amine cannot react with the sugar. However, the concentrations of the amine produced through C-N cleavage were below the detection limits of the system, indicating that in the presence of excess sugar the released amine reacted quickly with the sugar to form Amadori product. Hence the measured concentration of the sugar (S) and measured Amadori concentration (SA) are given by the following equations:

S = So - x ;

SA = SAo-x + x - y

Rearranging these equations will yield the values for x = So - S, and for y = SAo - SA, where So and SAo are the initial sugar and Amadori concentrations respectively. For a first order decomposition of Amadori product plotting SAo - x

Ln

vs. t should generate a straight line with a slope of ki similarly plotting SAo SAo - y

Ln

vs. t should generate a straight line with a slope of k . Figure 2 shows 2

SAo

the above plots during the initial two hour period. These plots deviate slightly from linearity at the later stages of the reaction due to the regeneration of the Amadori product. The calculated values for the decomposition rate constants (ki and k ) from these plots are listed in Table II. 2


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Conclusion The kinetic data indicate that the reaction of morpholine with glucose to form the Amadori product follows a second order kinetics and that the relative importance of the decomposition pathway of the resulting Amadori product depends on the sugar amine ratio in the reaction mixture. Acknowledgments


The author (V. Y.) acknowledges funding for this research by the Natural Sciences and Engineering Research Council (NSERC) of Canada. Literature Cited 1. Debrauwer, L.; Vernin, G.; Metzger, J.; Siouffi, A.M.,; Larice, J.L. Bull. Soc. Chim. Fr. 1991, 128, 244-254. 2. Vernin, G.; Debrauwer, L.; Vernin, G. M. F.; Zamkotsian, R-M.; Metzger, J.; Larice, J. L. and Parkanyi, C. In Off-flavors in foods and beverages, Charalambous, G., Ed.; Elsevier, 1992; pp. 567-623. 3. Baisier W.M.; Labuza, T.P. J. Agric. Food Chem., 1992, 40, 707-713. 4. Yaylayan, V.; Forage, N. Food Chem. 1992, 44, 201-208. 5. Huyghues-Despointes, A.; Yaylayan, V. Food Chem. 1994, 51, 109-117. 6. Castellan, G. W. In Physical Chemistry, Addison-Wesley, 1973; pp. 738-739. RECEIVED April 25, 1995


Kinetics of Formation and Degradation of Morpholino-1-deoxy-D

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