Keywords: potato starch; gelatinization; molecular mobility; Maillard reaction; glass transition.
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1 Impact of starch gelatinization on the kinetics of 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 of 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 starch. However there is a lack of information on the relationship between the water dynamics of water starch interactions and the kinetics of NEB reaction in native and gelatinized starch systems. The objective of the present work was to analyze the changes in water-distribution and water-solids interactions that take place after starch gelatinization and to elucidate their influence on the kinetics of the Maillard reaction in low moisture potato starch systems. Freeze-dried native (NS) and gelatinized (GS) potato starch 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 of water observed in NS. GS presents a more homogeneous distribution of Maillard reagents within the matrix, which renders a more dilute system regarding browning reagents, and also less water availability for the reaction. The dilution of 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 starch; gelatinization; 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 of nutrients and brown pigment formation during processing and storage [2]. Therefore, much attention has been given to control of the reaction rate. It is known that Maillard reaction kinetics is affected by several physico-chemical factors such as concentration, ratio and chemical nature of the reactants (type of amine and carbonyl groups involved), ph, relative humidity, temperature, and time of heating [3]. One of the most familiar features of Maillard reaction is the bell-shaped curve which relates the rate of 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 of the low reaction rates due to mobility limitations of reactants at low water contents [10] and inhibition by product/dilution of 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 of water as a reaction product of 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 starch granules. The slowing of water motion in low-moisture samples reflects strong water-solid interactions
2 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 starch [14-16]. However there is a lack of published studies on the relationship between the water dynamics of water starch interactions and the kinetics of Maillard reaction in native and gelatinized starch systems. The objective of the present work was to contribute to the understanding of the changes in water-distribution and water-solids interactions that take place after starch gelatinization and to elucidate their influence on the kinetics of Maillard reaction in low moisture potato starch systems. MATERIALS & METHODS Starch systems: Raw potatoes were washed, peeled and diced. A commercially available juice extractor was employed to obtain a starch-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 starch-rich aqueous suspensions were freeze-dried to obtain the starch powder. This starch powder was considered native as it was not gelatinized. A portion of the extracted starch was gelatinized by heating a 5 % aqueous suspension for 30 min at 80 C. Aqueous suspensions containing 15 % (w/w) of freeze-dried native or gelatinized starch powder, 0.5 % (w/w) glycine and glucose in phosphate buffer ph 6, 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 of 0.04 mbar. The dehydrated starch 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 of the humidified powders was distributed into vials for the determination of 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 starch 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 of 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 of 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 of 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 of 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 of Maillard reaction: The degree of Maillard reaction was determined by reflectance measurements of the color attribute luminosity (L*) with a white background of 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 of 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.
3 RESULTS & DISCUSSION Figure 1 shows the Maillard reaction rate coefficient (K) versus RH for native (NS) and gelatinized (GS) potato starch systems stored at 70 C. The rate of Maillard reaction for each system was calculated from a pseudo first order kinetic model from the plot of 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 of model and food systems, showing a bell-shaped curve with a maximum rate at intermediate/high RH values [4]. In this case, the disruption of the granule upon gelatinization caused a more homogeneous distribution of the reactants within the starch matrix. Thus, the diffusion effects and the availability of 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 % RH. The NS system consists of the intact starch 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 of 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 of water is manifested [5]. The confinement of water and reactants in the inter-granule spaces can support the fact that a possible effect of dilution of reactants, besides an inhibitory effect of water, could influence Maillard reaction kinetics even at very low RH values [20, 21] K 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 of the average K value. Figure 2 shows the experimental points of the water sorption isotherm at 20 ºC for NS and GS systems together with the results of the fitting with the GAB model [22], and the glass transition temperatures. The GAB fitting gave monolayer water contents (mo) of 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 starch at similar temperatures [23, 24], and also with data for starch from different sources [15, 23, 25-27]. Water adsorption on starch occurs through hydrogen-bonding of water molecules to the available hydroxyl groups of the substrate [28]. The starch granule is chemically micro-heterogeneous: it consists of 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 of 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 starch up to 75 % RH. The sorption isotherms were determined at 20 C. The slope
4 change in the sorption isotherm at high RH levels has been attributed to the differences in the number of starch hydroxyl groups available [28], and to the glass transition region, where the amorphous parts of starch 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 starch [29-31]. Water content, %d.b NS GS Water activity 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 of 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 of the isotherm) were in the supercooled region, and this RH region coincides with the decrease of 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 of 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 of 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 starch 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 of the more mobile protons (which account mainly for water protons) was studied through the analysis of 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 starch 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 of NS reveal the less affinity of the native starch by water. The RMN findings obtained for NS and GS systems (Fig. 3) agree with the interpretation of the Maillard reaction rate (Fig. 1), and of 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 of starch within the gelatinization temperature range, suggesting that the increase in starch 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 of water-starch molecules interactions, than the observed for NS system. Moreover, the sorption isotherm shape indicates the higher tendency of the gelatinized matrix to adsorb water. The GS system presents also a more homogeneous distribution of Maillard reagents within the matrix, which renders a more dilute system regarding browning reagents, and also less water availability for the reaction. The
5 dilution of 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 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 of the average value. CONCLUSION The relationships among browning rate, sorption behavior, molecular mobility and physical state have been confirmed in starch matrices. Since the general water sorption behavior does not take into account the heterogeneities in the water distribution within the matrix, a general relationship of the sorption phenomenon with the browning rate is difficult to establish. In the case of NS systems, the starch 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 of water. In native starch systems the Maillard reaction rate is thus only inversely dependent on the water content. On the other hand, due to a more homogeneous distribution of water and reactants within the gelatinized starch matrix, in these systems the Maillard reaction rate can be related to the physical properties of 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. REFERENCES [1] Carabasa-Giribet M. & Ibarz-Ribas A Kinetics of Colour Development in Aqueous Glucose Systems at High Temperatures. Journal of Food Engineering, 44, [2] Martins S.I.F.S., Jongen W.M.F. & van Boekel M.A.J.S A Review of Maillard Reaction in Food and Implications to Kinetic Modelling. Trends in Food Science and Technology, 11, [3] Buera M.P., Chirife J., Resnik S.L. & Wetzler G Nonenzymatic Browning in Liquid Systems of High Water Activity: Kinetics of Color Changes due to Maillard Reaction's Between Different Single Sugars and Glycine and Comparison with Caramelization Browning. Journal of Food Science, 52, [4] Labuza T.P., Tannenbaum S.R. & Karel M Water Content and Stability of Low Moisture and Intermediate- Moisture Foods. Food Technology, 24, [5] Eichner K. & Karel M The Influence of Water Content and Water Activity on the Sugar-Amino Browning Reaction in Model Systems Under Various Conditions. Journal of Agricultural and Food Chemistry, 20, [6] Labuza T. & Saltmarch M Kinetics of Browning and Protein Quality Loss in Whey Powders During Steady State and Nonsteady State Storage Conditions. Journal of Food Science, 41(1), [7] Acevedo N.C., Schebor C. & Buera M.P Water solids Interactions, Matrix Structural Properties and the Rate of Non-Enzymatic Browning. Journal of Food Engineering, 77,
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