Impact of starch gelatinization on the kinetics of Maillard reaction in ...

<strong>Impact</strong> <strong>of</strong> <strong>starch</strong> <strong>gelatinization</strong> on the kinetics <strong>of</strong> Maillard reaction in potato
dehydrated systems
Nuria C. Acevedo a , Carolina Schebor a,b , Pilar Buera a,b
a Departamentos de Industrias y de Química Orgánica. Facultad de Ciencias Exactas y
Naturales. Universidad de Buenos Aires. Ciudad Universitaria (1428). Ciudad de Buenos
Aires. ARGENTINA.e-mais: acevedonuria@yahoo.com.ar, cschebor@di.fcen.uba.ar,
pilar@di.fcen.uba.ar
b Members <strong>of</strong> CONICET, Argentina.
ABSTRACT
The non-enzymatic browning (NEB) reaction plays an essential role in food acceptance and quality. There
has been increased interest in revealing the role that water plays in native and gelatinized <strong>starch</strong>. However
there is a lack <strong>of</strong> information on the relationship between the water dynamics <strong>of</strong> water–<strong>starch</strong> interactions and
the kinetics <strong>of</strong> NEB reaction in native and gelatinized <strong>starch</strong> systems. The objective <strong>of</strong> the present work was
to analyze the changes in water-distribution and water-solids interactions that take place after <strong>starch</strong>
<strong>gelatinization</strong> and to elucidate their influence on the kinetics <strong>of</strong> the Maillard reaction in low moisture potato
<strong>starch</strong> systems. Freeze-dried native (NS) and gelatinized (GS) potato <strong>starch</strong> systems containing glycine and
glucose were prepared. 1 H NMR relaxation times (T 2 ), thermal transitions and water sorption isotherms were
analyzed. Maillard reaction was studied at 70ºC. In NS Maillard rate was inversely dependent on RH. In GS
the rate increased up to RHs between 75 and 84 % and then decreased at higher RHs. In the NS matrix,
which is almost inert towards Maillard reaction, reactants are concentrated in the inter-granule spaces. NS
has also lower tendency to retain water than GS, and the water formed during Maillard reaction is not
retained by the matrix, being available to act as inhibitor. This explains the high Maillard rate at low RHs and
the continuous inhibiting effect <strong>of</strong> water observed in NS. GS presents a more homogeneous distribution <strong>of</strong>
Maillard reagents within the matrix, which renders a more dilute system regarding browning reagents, and
also less water availability for the reaction. The “dilution” <strong>of</strong> the reagents makes this system more diffusion
dependent. This can explain the low Maillard rate at the lower RH values in the GS matrix, which increases
above the glass transition temperature (T g ) value and decreases when solvent water appears.
Keywords: potato <strong>starch</strong>; <strong>gelatinization</strong>; molecular mobility; Maillard reaction; glass transition.
INTRODUCTION
Maillard reaction plays an essential role in food acceptance and quality. It produces desirable flavors and
colors [1,2], but it may also cause undesirable loss <strong>of</strong> nutrients and brown pigment formation during
processing and storage [2]. Therefore, much attention has been given to control <strong>of</strong> the reaction rate. It is
known that Maillard reaction kinetics is affected by several physico-chemical factors such as concentration,
ratio and chemical nature <strong>of</strong> the reactants (type <strong>of</strong> amine and carbonyl groups involved), pH, relative
humidity, temperature, and time <strong>of</strong> heating [3]. One <strong>of</strong> the most familiar features <strong>of</strong> Maillard reaction is the
bell-shaped curve which relates the rate <strong>of</strong> the reaction to water activity (aw) [4]. Maximum browning rate
has been observed in most cases at water activities between 0.3 and 0.8 [5-9]. This is a consequence <strong>of</strong> the
low reaction rates due to mobility limitations <strong>of</strong> reactants at low water contents [10] and inhibition by
product/dilution <strong>of</strong> reactants at high water contents [11]. However, in liquids or systems where a low water
activity is not associated with a strong increase in viscosity, a maximum does not appear and the reaction rate
continuously decreases from low to high aw [12].
Rather than water affecting chemical reactions via water activity or by plasticizing amorphous systems, and
considering the inhibitory effect <strong>of</strong> water as a reaction product <strong>of</strong> Maillard reaction condensations, water
mobility itself may have a direct impact on chemical reactivity in low and intermediate moisture systems.
Solid state NMR is a useful tool to investigate the water dynamics in semisolid and solid states, such as
<strong>starch</strong> granules. The slowing <strong>of</strong> water motion in low-moisture samples reflects strong water-solid interactions

through hydrogen bonding [13] which corresponds to water molecules that are strongly influenced by their
proximity to the solids components.
In recent years, there has been increased interest in revealing the important role that water plays in native and
gelatinized <strong>starch</strong> [14-16]. However there is a lack <strong>of</strong> published studies on the relationship between the water
dynamics <strong>of</strong> water–<strong>starch</strong> interactions and the kinetics <strong>of</strong> Maillard reaction in native and gelatinized <strong>starch</strong>
systems.
The objective <strong>of</strong> the present work was to contribute to the understanding <strong>of</strong> the changes in water-distribution
and water-solids interactions that take place after <strong>starch</strong> <strong>gelatinization</strong> and to elucidate their influence on the
kinetics <strong>of</strong> Maillard reaction in low moisture potato <strong>starch</strong> systems.
MATERIALS & METHODS
Starch systems: Raw potatoes were washed, peeled and diced. A commercially available juice extractor was
employed to obtain a <strong>starch</strong>-rich extract. The extract was diluted with distilled water, centrifuged, and the
precipitate was recovered and re-suspended in water. This procedure was done many times in order to wash
the soluble components. The <strong>starch</strong>-rich aqueous suspensions were freeze-dried to obtain the <strong>starch</strong> powder.
This <strong>starch</strong> powder was considered “native” as it was not gelatinized. A portion <strong>of</strong> the extracted <strong>starch</strong> was
gelatinized by heating a 5 % aqueous suspension for 30 min at 80 °C. Aqueous suspensions containing 15 %
(w/w) <strong>of</strong> freeze-dried native or gelatinized <strong>starch</strong> powder, 0.5 % (w/w) glycine and glucose in phosphate
buffer pH 6, 0.175 M were prepared.
Freeze-drying: The freeze-drying process lasted 48 hours. An Alpha 1-4 LD / 2-4 LD-2, freeze drier (Martin
Christ, Gefriertrocknungsanlagen GmbH) was used; it was operated at -84 °C, at a chamber pressure <strong>of</strong> 0.04
mbar. The dehydrated <strong>starch</strong> powders were transferred into evacuated desiccators and kept for 14 days at 20
°C over saturated salt solutions that provided constant relative humidities in a range between 11 and 92 %
[17]. Part <strong>of</strong> the humidified powders was distributed into vials for the determination <strong>of</strong> water content, thermal
transitions and molecular mobility. The remaining material was used to determine Maillard reaction.
Water content: The water content was determined gravimetrically by vacuum drying at 97 °C for 48 h.
Heat treatment: After equilibration, potato <strong>starch</strong> powder systems were placed inside rubber o-rings which in
turn were sandwiched between two glass plates held hermetically with metal clamps to avoid water loss as
previously described [8]. The glass sample holder was then placed in an air-convection oven operated at 70 ±
1 ºC. At suitable intervals, samples were removed from the oven; color was measured and the samples were
placed back in the oven to continue with the heat treatment.
Thermal transitions: Glass transition temperatures (T g ) were determined by differential scanning calorimetry
(DSC; onset values) using a DSC 822e Mettler Toledo calorimeter (Schwerzenbach, Switzerland). All
measurements were performed at a heating rate <strong>of</strong> 10 °C/min. Hermetically sealed 40 l medium pressure
pans were used (an empty pan served as reference). Thermograms were evaluated using Mettler Star e
program. Average values <strong>of</strong> at least two replicates and standard deviations were reported.
1 H Relaxation molecular mobility: A Bruker Minispec mq 20 pulsed nuclear magnetic resonance (NMR)
instrument, with a 0.47 T magnetic field operating at resonance frequency <strong>of</strong> 20 MHz, was used for
measurements. The spin-spin relaxation time (T 2 ) associated to the fast relaxing protons (related to the solid
matrix and to water interacting tightly with solids) was measured using a free induction decay analysis (FID)
after a single 90º pulse. The decay envelopes were fitted to mono-exponential behavior. T 2 associated to slow
relaxing protons (related to the populations <strong>of</strong> water molecules displaying less interactions with solids) were
measured using the Carr−Purcell-Meiboom−Gill pulse sequence (CPMG). The decay envelopes were fitted
to bi-exponential. All determinations were performed in duplicate.
Degree <strong>of</strong> Maillard reaction: The degree <strong>of</strong> Maillard reaction was determined by reflectance measurements <strong>of</strong>
the color attribute luminosity (L*) with a white background <strong>of</strong> reflectance. A handheld tristimulus reflectance
spectrocolorimeter with an integrating sphere (Minolta CM-508-d, Minolta Corp., Ramsey, NJ, USA) was
employed. Color functions were calculated for illuminant D65 at 2° standard observer and in the CIELAB
uniform color space. The measurements were performed excluding the specular component, to avoid the
contribution <strong>of</strong> reflection from the glass plate. The color function L*, (Lo*- L*) was found to be an
adequate parameter to evaluate the non enzymatic browning reactions in opaque samples [18] being Lo* and
L* the sample color attribute before and after heat treatment respectively. Six replicates were analyzed for
each storage time and an average value was reported. The standard error was lower than 2 % (P < 0.05) for
all the analyzed samples.

RESULTS & DISCUSSION
Figure 1 shows the Maillard reaction rate coefficient (K) versus RH for native (NS) and gelatinized (GS)
potato <strong>starch</strong> systems stored at 70 °C. The rate <strong>of</strong> Maillard reaction for each system was calculated from a
pseudo first order kinetic model from the plot <strong>of</strong> L* versus storage time at 70 ºC. It can be seen that for the
NS system the rate coefficients were relatively high at very low RH values and significantly decreased as RH
increased. The GS system, however, did not show important differences in browning rate up to 52 % RH, and
at higher RH values it showed an increase and a maximum rate coefficient between 75 and 84 % RH. The GS
system behavior resembles that observed for most <strong>of</strong> model and food systems, showing a bell-shaped curve
with a maximum rate at intermediate/high RH values [4]. In this case, the disruption <strong>of</strong> the granule upon
<strong>gelatinization</strong> caused a more homogeneous distribution <strong>of</strong> the reactants within the <strong>starch</strong> matrix. Thus, the
diffusion effects and the availability <strong>of</strong> water greatly affected the Maillard reaction rate, giving kinetic
coefficients up to 4 times lower than those observed for NS systems. Acevedo et al. [7] studied the Maillard
reaction kinetics at 70 ºC on dehydrated potato systems and found that the RH value for the maximum
Maillard reaction rate was 75 %. Hendel et al. [19] studied dehydrated potato systems at temperatures in a
range from 40 to 99.5 °C and reported a maximum Maillard reaction rate corresponding to 70–75 % RH. The
NS system consists <strong>of</strong> the intact <strong>starch</strong> granules and the Maillard reactants (glycine and glucose). Upon
freeze drying it is likely that glucose and glycine remain adsorbed on the granules surface, and do not
penetrate the compact structure <strong>of</strong> the granules. Thus, the Maillard reaction would only take place in the
inter-granule space, in which reactants would be highly concentrated. Therefore, at low RH values, there
would be enough reactant concentration and water adsorbed to allow a high browning development. The NS
systems presented a behavior similar to that observed in liquid systems for which only an inhibitory effect <strong>of</strong>
water is manifested [5]. The confinement <strong>of</strong> water and reactants in the inter-granule spaces can support the
fact that a possible effect <strong>of</strong> dilution <strong>of</strong> reactants, besides an inhibitory effect <strong>of</strong> water, could influence
Maillard reaction kinetics even at very low RH values [20, 21].
0.16
0.12
K
0.08
0.04
0.00
0 20 40 60 80 100
Relative Humidity (%)
Figure 1. Maillard reaction rate coefficients (K) versus RH for NS (■) and GS () systems stored at 70 °C. The error
bars represent the standard deviation <strong>of</strong> the average K value.
Figure 2 shows the experimental points <strong>of</strong> the water sorption isotherm at 20 ºC for NS and GS systems
together with the results <strong>of</strong> the fitting with the GAB model [22], and the glass transition temperatures. The
GAB fitting gave “monolayer” water contents (mo) <strong>of</strong> 6.1 % and 8.7 % d.b. (dry basis) for NS and GS
systems respectively. Both values correspond to RHs between 22 and 33 %. The mo values are in agreement
with previously reported “monolayer” water contents for potato <strong>starch</strong> at similar temperatures [23, 24], and
also with data for <strong>starch</strong> from different sources [15, 23, 25-27]. Water adsorption on <strong>starch</strong> occurs through
hydrogen-bonding <strong>of</strong> water molecules to the available hydroxyl groups <strong>of</strong> the substrate [28]. The <strong>starch</strong>
granule is chemically micro-heterogeneous: it consists <strong>of</strong> both crystalline and amorphous regions. The
crystalline regions typically exhibit resistance to solvent penetration. It can be observed that at low RH
values, up to 52 %, the GS system adsorbs approximately 2 % more water than the NS system. This could be
due to the presence <strong>of</strong> granules in the NS system that present higher crystallinity and less sites available to
interact with the water molecules. Charles et al. [15] reported the same behaviour when they compared native
and gelatinized wheat <strong>starch</strong> up to 75 % RH. The sorption isotherms were determined at 20 °C. The slope

change in the sorption isotherm at high RH levels has been attributed to the differences in the number <strong>of</strong>
<strong>starch</strong> hydroxyl groups available [28], and to the glass transition region, where the amorphous parts <strong>of</strong> <strong>starch</strong>
start to plasticize [22]. The glass transition temperature (T g ) values obtained for the studied samples are close
to those reported by several authors for native and gelatinized potato <strong>starch</strong> [29-31].
Water content, %d.b.
70
60
50
40
30
150
120
20
30
10
• NS
GS
0
0
0.0 0.2 0.4 0.6 0.8 1.0
Water activity
90
60
T g , °C
Figure 2. Glass transition temperatures (dashed lines) and water sorption isotherm at 20 °C: experimental points and the
corresponding GAB fitting (solid lines) versus aw. NS (■) and GS (□) systems.
All <strong>of</strong> the samples shown in the water sorption isotherm were in the glassy state at 20 °C (Fig. 2). At 70°C
the GS samples above 90 % RH (lying on the ascending branch <strong>of</strong> the isotherm) were in the supercooled
region, and this RH region coincides with the decrease <strong>of</strong> the Maillard rate coefficient (Fig. 1). Thus,
particularly for the GS systems, the rate coefficient seems to be influenced by the glass transition.
Above 90 % RH, frozen water was detected by DSC both in the GS and NS samples (not shown). As shown
in Fig. 2, in the GS samples the Maillard reaction rate coefficient decreased above 84 % RH. This could be
due, in part, to the dilution effect <strong>of</strong> water, since solvent water is available to dilute the reactants at high RH
values. In coincidence with the findings by Acevedo et al. [9], for the GS samples the Maillard reaction rate
decrease after the maximum is related to the appearance <strong>of</strong> mobile water.
As water and matrix molecular mobility can be related to the Maillard reaction rate, 1 H NMR relaxation
times were determined in the samples equilibrated at the different RHs analyzed (Figure 3). A free induction
decay (FID) analysis was performed at 20 ºC to analyze the molecular mobility corresponding to protons in
solids and tightly bound water. Both native and gelatinized <strong>starch</strong> showed a single T 2 -FID value in the whole
RH scale, which was in a range between 8 and 10 microseconds, and, for a given RH value, no differences in
T 2 -FID values between NS and GS samples were observed (p < 0.05).
The relaxation behavior <strong>of</strong> the more mobile protons (which account mainly for water protons) was studied
through the analysis <strong>of</strong> the spin-spin relaxation times after the CPMG pulses sequence. Proton populations
with medium (T 2-1 , from 0.1 to 0.9 ms) and high mobility (T 2-2 , from 1.5 to 3 ms) were detected. Both T 2
values in NS and GS <strong>starch</strong> systems are shown in Fig. 4. It can be observed that NS system always showed
higher values for both T 2-1 and T 2-2 than those for GS system, particularly at high RH (p < 0.05). As a larger
T 2 value indicates a more mobile proton population, the larger T 2 values <strong>of</strong> NS reveal the less affinity <strong>of</strong> the
native <strong>starch</strong> by water.
The RMN findings obtained for NS and GS systems (Fig. 3) agree with the interpretation <strong>of</strong> the Maillard
reaction rate (Fig. 1), and <strong>of</strong> the water sorption properties (Fig. 2). The rapid decrease in the Maillard reaction
rate with increasing RH in NS samples, which could be caused by product inhibition, is also in accord with
the higher water molecular mobility (Fig. 4) detected by 1H NMR. Regarding the GS systems, several NMR
studies have revealed a drastic decrease in T 2 <strong>of</strong> <strong>starch</strong> within the <strong>gelatinization</strong> temperature range,
suggesting that the increase in <strong>starch</strong> polymers hydration diminishes the water molecules mobility [14, 32].
The results shown in Fig. 3 are in accordance with those findings. It can be proposed that water adsorbed in
the gelatinized matrix is more homogeneously distributed and renders less mobile protons due to the
increased number <strong>of</strong> water-<strong>starch</strong> molecules interactions, than the observed for NS system. Moreover, the
sorption isotherm shape indicates the higher tendency <strong>of</strong> the gelatinized matrix to adsorb water. The GS
system presents also a more homogeneous distribution <strong>of</strong> Maillard reagents within the matrix, which renders
a more dilute system regarding browning reagents, and also less water availability for the reaction. The

“dilution” <strong>of</strong> the reagents makes this system more diffusion dependent. This can explain the initial low
Maillard reaction rate, which increases upon the increase in RH and decreases when solvent water appears.
T 2 (millisecond)
3.0 • NS
2.5
GS
2.0
1.5
0.9
0.6
0.3
0.0
0 20 40 60 80 100
Relative Humidity (%)
Figure 3. Spin-spin relaxation times T 2-1 and T 2-2 (inset) at 20 °C obtained by CPMG sequence for NS (■) and GS (□)
systems versus RH. The bars represent the standard deviation <strong>of</strong> the average value.
CONCLUSION
The relationships among browning rate, sorption behavior, molecular mobility and physical state have been
confirmed in <strong>starch</strong> matrices.
Since the general water sorption behavior does not take into account the heterogeneities in the water
distribution within the matrix, a general relationship <strong>of</strong> the sorption phenomenon with the browning rate is
difficult to establish.
In the case <strong>of</strong> NS systems, the <strong>starch</strong> matrix is almost inert towards Maillard reaction. Thus, reactants are
very concentrated in the inter-granule spaces. Also, NS has lower tendency to retain water than GS, and the
water formed during Maillard reaction is not retained by the matrix, being available to act as inhibitor. These
characteristics account for the high Maillard reaction rate at low RH and for its continuous attenuation given
by the inhibiting effect <strong>of</strong> water. In native <strong>starch</strong> systems the Maillard reaction rate is thus only inversely
dependent on the water content.
On the other hand, due to a more homogeneous distribution <strong>of</strong> water and reactants within the gelatinized
<strong>starch</strong> matrix, in these systems the Maillard reaction rate can be related to the physical properties <strong>of</strong> the
matrix: Tg, water sorption and water mobility as determined by 1 H NMR T 2 relaxation times. These
properties can be relevant tools to predict the Mallard reaction rate dependence on water content.
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