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JFS: Food Chemistry and Toxicology Maillard Browning in Ethanolic Solution ABSTRACT: Maillard browning in ethanolic solutions of a 0.2 M glucose–0.2 M glycine (G-G) system in a heat treatment becomes more pronounced with an increase in ethanol concentration (0% to 50%, v/v). Extents of browning, as measured by absorbance at 420 nm, of a fructose-glycine system in aqueous and 50% ethanolic solutions buffered at pH 4.3 are very close after the treatment, whereas that of the G-G system in 50% ethanolic solution is much enhanced. A higher hydroxymethylfurfural (HMF) content is present in the ethanolic G-G system, suggesting the involvement of ethanol in the G-G reaction to form HMF. Evidence also shows that ethanol inhibits the formation of HMF in an ethanolic solution containing glucose alone. Much more HMF is formed in the pH 4.3 buffered G-G system in an ethanolic solution at a water activity (aw) above 0.81 or at an ethanol concentration below 30% than in a glycerolic solution at the same aw. We propose that the reduction of aw is not the sole major mechanism for ethanol to accelerate HMF formation and Maillard browning in G-G solution. Keywords: Maillard reaction, browning, ethanol, hydroxymethylfurfural, water activity Introduction T he Maillard reaction is a class of nonenzymatic browning that involves the interaction of reducing sugars and free amino acids or a free amino group of an amino acid that is a part of the protein chain (BeMiller and Whistler 1996). The factors that influence the rate of the Maillard reaction in the processing and storage of food include the composition of food, the time-temperature condition, pH, water activity (aw), oxygen tension, the presence of promoters, and inhibitors, and so forth (Ames 1990, 1998). Conventionally, research on the Maillard reaction has used water as the solvent. Studies in nonaqueous systems are rare. In aqueous systems, the key chemical components that directly influence the progress of the Maillard reaction are sugars and amino acids. Del Pilar Buera and others (1987) compared the activities of various monosaccharides and disaccharides below pH 6 and found that they are xylose, fructose, glucose, lactose, maltose, and sucrose in descending order. Brands and others (2000) investigated the Maillard reaction in sugar-casein system and found that those sugars having a higher proportion of acyclic forms in the solution brown more rapidly. They also found that aldoses brown slower than their corresponding ketoses and that nonreducing disaccharides brown even slower because they need to be hydrolyzed to reducing sugars first. Ashoor and Zent (1984) compared the rate of Maillard browning among common amino acids and grouped them into high, intermediate, and low browning producing groups. Higher temperature and longer time result in more serious browning (Del Pilar Buera and others 1987; Lee and Nagy 1988). In alkaline condition, both of the browning rates in fructose-glycine and glucose-glycine systems reach maximum at pH 10 (Ashoor and Zent 1984). In the pH range between 3.4 and 7.7, the browning in starchglucose-lysine mixture increases with the increase in pH (Bates and others 1994). Renn and Sathe (1997) showed that the browning rate between L-leucine and D-glucose is positively correlated with pH. MS 20030383 Submitted 7/6/03, Revised 9/24/03, Accepted 1/29/04. Authors Shen and Wu are with the Inst. of Food Science and Technology, Natl. Taiwan Univ., P.O. Box 23-14, Taipei 106, Taiwan. Direct inquiries to author Wu (Email: [email protected]). © 2004 Institute of Food Technologists Further reproduction without permission is prohibited Research in Maillard browning regarding the effect of aw (Toribio and Lozano 1984; Cuzzoni and others 1988), organic acids (Akhavan and Wrolstad 1980), metal ions (Petriella and others 1988), and high pressure (Hill and others 1996) have also been reported. These reviewed studies were all done in aqueous systems to stand for common foods. However, some foods, such as beer, sake, rice wine, many fortified wines, and liqueurs are types of ethanolic solutions that contain noticeable amounts of Maillard reaction substrates. The investigation of Maillard browning in ethanolic solution shall be worthwhile. Materials and Methods Reagents The sugar species tested in the present study covered pentose (xylose), hexoses (glucose and fructose), monosaccharides (xylose, glucose, fructose, and galactose), disaccharides (sucrose, lactose, and lactulose), aldoses (glucose and lactose), and ketoses (fructose and lactulose). All the sugars, amino acids, and hydroxylmethylfurfural (HMF) are the products of Sigma Co. (St. Louis, Mo., U.S.A.). Ethanol (95% v/v) is purchased from Taiwan Tobacco & Liquor Co. (Taipei, Taiwan). Preparation of reaction mixtures Among all the model systems for Maillard reaction, hexose- and pentose-glycine systems are the most frequently used (Wedzicha and Kaputo 1992). The present study uses glucose (0.2 M)-glycine (0.2 M) (G-G) model solutions to investigate the influence of ethanol concentration and pH on browning. The G-G solutions were prepared by dissolving glucose and glycine in 0.05 M succinic acid–sodium hydroxide buffered 0% to 50% ethanolic solutions. The pH value was adjusted to 4.3 to represent sake or to 5.5 to represent a liqueur made from spirit and a common low acid food (Leong and Wedzicha 2000). Aliquots (4 mL) of the model solutions were transferred to 4.5-mL glass vials, hermetically sealed, and heated in boiling water for 0 to 6 h as an accelerated storage test to simulate the storage at room temperature for approximately 8 mo. Glucose and glycine were also dissolved in ethanolic solutions without buffering for comparison. Vol. 69, Nr. 4, 2004—JOURNAL OF FOOD SCIENCE FCT273 Published on Web 4/15/2004 Food Chemistry and Toxicology S.-C. SHEN AND J.S.-B. WU Maillard browning in ethanolic solution . . . Food Chemistry and Toxicology Xylose, fructose, galactose, sucrose, lactose, or lactulose was then substituted for glucose in the preparation of solutions containing 0%, 15%, and 50% ethanol to investigate the browning potential of different sugars in an ethanolic system. Similarly, lysine, threonine, cysteine, serine, or alanine was substituted for glycine in the preparation of ethanolic solutions for investigating the effect of amino acid species on Maillard browning. The 0%, 15%, and 50% ethanol concentrations were chosen to represent the ethanol concentrations in aqueous solution, sake, and liqueur, respectively. Water used in the present study was obtained from a Milli-Q system (Millipore, Osaka, Japan). In comparison, glycerolic solutions were prepared by dissolving either 0.2 M glucose only or 0.2 M glucose-0.2 M glycine in aqueous solutions of glycerol (0% to 54.3%, w/w) buffered with 0.05 M succinic acid-sodium hydroxide at pH 4.3. Evaluation of aw The aw of all the ethanolic and glycerolic solutions were evaluated by the method described by Ferro Fontan and Chirife (1981) and Miracco and others (1981), with minor modifications. The freezing point of each solution was measured using a differential scanning calorimeter (DSC) (Model DSC 822e, Mettler-Toledo GmbH, MatChar, Switzerland). Purified nitrogen (99.999%) flowed at 80 mL/min and 200 mL/min were the purge gas and the dry gas, respectively. Samples of 14 to 18 mg were weighed into aluminum pans, and covers were hermetically sealed into place. An empty, hermetically sealed aluminum pan was used as the reference. Before the analysis of samples, the baseline was obtained with the reference pan. After both the sample pan and the reference pan were placed in the chamber at 25 °C, the cell block of the DSC was cooled to –50 °C at 10 °C/min and held there for 2 min, followed by heating at a rate of 5 °C/min to 25 °C. The heating thermograms of samples showed a single melting peak. The peak temperature and the enthalpy was obtained by analyzing the thermogram with Mettler Toledo Stare Software V6, 1.0. The freezing point is defined as the intersection of baseline and the tangent line of melting peak. The aw of a sample was calculated using the following equation: –ln aw = 9.6934 × 10–3 × F + 4.761 × 10–6 × F2 where F (freezing point depression) = To – TF; To = freezing point of pure water; TF = freezing point of sample solution. system to identify the corresponding retention time and elution profile in the chromatogram of the browning sample solutions. The content of HMF was analyzed adapting the method reported by Lee and Nagy (1988) by the constructed calibration curve of HMF (0.05 to 10 mg/mL) (y = 42849x + 1510.8; R2 = 0.999). Triplicate samples were prepared, and each sample was injected 3 times. Each experiment was repeated 4 times. The comparison among the values of optical absorbance or HMF content in different treatments was done by analysis of variance and Duncan’s multiple range test (P ⱕ 0.05). Results and Discussion Effect of ethanol concentration on pH and browning in nonbuffered G-G solutions The effect of ethanol concentration on the pH of nonbuffered GG solutions is shown in Figure 1. In the range of 0% to 50% ethanol content, a higher ethanol concentration corresponds to a higher initial pH value, in a well-adjusted quadratic relation (R2 = 0.985). Gutiérrez (2003) reported similar results in reconstituted wines containing 0% to 22% ethanol. The pH value in G-G solutions decreases with an increase in heating time (Figure 2a), similar to the findings in aqueous glucose-glycine solutions reported by Morales and Jimenez-Perez (2001). Among all the samples heated for the same duration, the one containing no ethanol shows the lowest pH value. The decrease in pH observed during the Maillard reaction can be attributed to the degradation of sugar into acid (Beck and others 1990) or the condensation between the free amino group of amino acid and the carbonyl group of glucose (Trifiro and others 1990). The effect of ethanol concentration on browning (A420nm) of the nonbuffered G-G solutions during incubation is shown in Figure 2b. It is noteworthy that browning is more severe in G-G ethanolic solutions containing a higher level of ethanol, revealing the involvement of ethanol in the glucose-glycine browning reaction, which has never been reported before. Effect of pH on browning in ethanolic solutions The effect of ethanol concentration on the absorbance at 420 nm in G-G ethanolic solutions buffered at pH 4.3 and pH 5.5 by 0.05 M succinic acid–sodium hydroxide is shown in Figure 3. At pH 4.3, the extent of browning increases with the increase in ethanol concentration. After heating in boiling water for 6 h, the absorbance at 420 Absorbance spectra of the solutions The absorbance spectra of solutions were read using a UV/VIS 8500 double-beam spectrophotometer (Lab Alliance, State College, Pa., U.S.A.). The absorbance at 420 nm was taken as a measure of browning (Baxter 1995; Ajandauz and Puigserver 1999). Analysis for HMF content The solutions were heated in boiling water for 6 h, followed by filtration through a Millex-HN nylon clarification kit with a 0.45-m pore size (Millipore, Bedford, Mass., U.S.A.). The filtrate was injected into a high-performance liquid chromatographic (HPLC) system that consisted of a Luna 5 C18 (2) Column (Phenomenex, Torrance, Calif., U.S.A.), a Series III Pump (Lab Alliance), a UV-970 Intelligent UV/VIS Detector (Jasco Co., Tokyo, Japan), and a Chem Lab Data Station Integrator (Scientific Information Service Co., Taipei, Taiwan). The quantity of injection was 20 L. The mobile phase was acetonitrile-water-acetic acid in a 10:89.5:0.5 volume ratio at a flow rate of 1.0 mL/min. The detected wavelength was 280 nm. Standard HMF (Sigma) was injected into the chromatographic FCT274 JOURNAL OF FOOD SCIENCE—Vol. 69, Nr. 4, 2004 Figure 1—Plot of pH compared with ethanol content for glucose (0.2 M)/glycine (0.2 M) solutions URLs and E-mail addresses are active links at www.ift.org Maillard browning in ethanolic solution . . . Table 1—Absorbance at 420 nm of the ethanolic solutions of glycine (0.2 M)/sugar (0.2 M) buffered at pH 4.3 with 0.05 M succinic acid–sodium hydroxide and heated in boiling water for 6 ha,b Ethanol conc. (v/v) 0% 15% 50% Glucose Fructose Lactose Lactulose Sucrose Galactose Xylose 0.26 ± 0.01ey 0.25 ± 0.01dy 1.29 ± 0.02bx 1.14 ± 0.01bx 0.86 ± 0.01by 1.14 ± 0.01bx 0.23 ± 0.01ex 0.17 ± 0.01dy —c 0.56 ± 0.01dx 0.47 ± 0.01cy 0.49 ± 0.02cy 0.25 ± 0.03ey 0.20 ± 0.01dz 0.43 ± 0.02cx 0.75 ± 0.02cy 0.77 ± 0.01by 0.97 ± 0.07bx 6.28 ± 0.08az 10.94 ± 0.28ay 33.51 ± 2.04ax a Values shown are the average of 3 replicates. b Different letters (a–e) within the same row are significantly different ( P < 0.05) in Duncan’s multiple range test. Different letters (x–z) within the same column nm of G-G system in 50% ethanolic solution reaches 1.03, approximately 3.8 to 3.6 times the absorbance value of 0.27 to 0.29 in the 0% to 20% ethanolic solutions. For samples with the same ethanol level, more severe browning occurs at pH 5.5 than at pH 4.3. This suggests that a high pH is favorable for the browning reaction in ethanolic solution, as has been reported in aqueous solution (Ames 1998). Table 1 presents the absorbance at 420 nm in 0%, 15%, and 50% ethanolic solutions of 0.2 M glycine-0.2 M sugar buffered at pH 4.3 with 0.05 M succinic acid–sodium hydroxide after the heat treatment. Regardless of the species of sugar, the extent of browning is more pronounced in ethanolic solution than in aqueous solution. The data for lactose are not available because of the low solubility of this sugar in the solution. Xylose, the most potent browning-inducing sugar species in the glycine-sugar aqueous system (Del Pilar Buera and others 1987), is also proven to be most active among the sugars tested in 50% ethanolic solution with an absorbance reading at 420 nm of 33.5, about 5 times of that in aqueous solution (Table 1). This shows that ethanol enhances browning, as also indicated by Figure 2 and 3. Interestingly, the readings of absorbance at 420 nm for glucose in aqueous and 50% ethanolic solution are about 0.26 and 1.29, respectively, whereas those of fructose in both systems had the same value of 1.14 after heating (Table 1). Restated, glucose as an aldose browns less severely than its ketose isomer, fructose, in aqueous solution and in 15% ethanol, but more severely in 50% ethanol. It appears that the concentration of ethanol may influence the reactivity of some sugar species in the formation of Maillard reaction intermediates. Figure 2—Effect of ethanol concentration on (a) the pH and (b) the absorbance at 420 nm in nonbuffered glucose (0.2 M)/glycine (0.2 M) solutions during heating in boiling water Figure 3—Effect of ethanol concentration on the absorbance at 420 nm in glucose (0.2 M)/glycine (0.2 M) solutions buffered at pH 4.3 and 5.5 by 0.05 M succinic acid– sodium hydroxide during heating in boiling water Effect of sugar species on browning in ethanolic solutions URLs and E-mail addresses are active links at www.ift.org Vol. 69, Nr. 4, 2004—JOURNAL OF FOOD SCIENCE FCT275 Food Chemistry and Toxicology are significantly different ( P < 0.05) in Duncan’s multiple range test. c Insoluble in 50% (v/v) ethanol. Maillard browning in ethanolic solution . . . Table 2—Absorbance at 420 nm of the ethanolic solutions of glucose (0.2 M)/amino acid (0.2 M) buffered at pH 4.3 with 0.05 M succinic acid–sodium hydroxide and heated in boiling water for 6 ha,b Ethanol conc. (v/v) 0% 15% 50% Alanine Cysteine 0.10 ± 0.01cy 0.07 ± 0.01cy 0.32 ± 0.04cx 0.08 ± 0.01cz 0.15 ± 0.02cy 0.59 ± 0.05cx Glycine Serine 0.24 ± 0.02cy 0.22 ± 0.02bcy 1.00 ± 0.02cx 0.28 ± 0.04cy 0.27 ± 0.01bcy 1.03 ± 0.05cx Threonine Lysine 0.64 ± 0.02bz 0.79 ± 0.02by 2.52 ± 0.09bx 3.23 ± 0.40az 5.73 ± 0.79ay 15.94 ± 0.42ax a Values shown are the average of 3 replicates. b Different letters (a–e) within the same row are significantly different ( P < 0.05) in Duncan’s multiple range test. Different letters (x–z) within the same column are significantly different (P < 0.05) in Duncan’s multiple range test. Food Chemistry and Toxicology Effect of amino acid species on browning in ethanolic solutions The amino acids tested beside glycine were lysine, alanine, serine, threonine, and cysteine. Table 2 shows the browning in ethanolic solutions of 0.2 M glucose and 0.2 M amino acid buffered at pH 4.3. Among all the samples, the most severe browning occurred in glucose-lysine solutions. In 15% and 50% ethanolic solutions of glucose-lysine, the readings of absorbance at 420 nm are 5.73 and 15.94, respectively, which are about 1.8 and 4.9 times the value of 3.23 in aqueous solution. Similar browning-enhancing effects are observed when other amino acids react with glycine in ethanolic solutions at 50% ethanol concentration. This phenomenon is consistent with the tendency of discoloration in liqueur. It is well recognized that the carbonyl group of glucose condenses most easily with the ⑀-amino group of lysine in aqueous solutions to form Nsubstituted glycosylamine in the initiation stage of Maillard browning (Van Martins and others 2001). The same thing would be true in ethanolic solutions, with more pronounced browning reaction at high ethanol concentrations (Table 1 and 2). Mechanisms are not clear. However, fructose browns faster than glucose in aqueous system because of the higher proportion of the acyclic form (Davies and others 1998). It is possible that ethanol increases the concentration of the acyclic form of glucose. Reduction of aw by ethanol and its effect on Maillard browning Ethanol, a highly volatile solvent, would interfere with any direct instrumental measurement for aw. Therefore, we measured the freezing point depression and calculated aw. Eichner and Karel HMF content in ethanolic solutions Figure 4 provides evidence for the involvement of ethanol in the browning of ethanolic solutions containing either 0.2 M glucose alone or G-G system. An absorption peak occurs at 280 to 290 nm. The height of the absorption peak in the heated and diluted G-G solutions decreases with the decreasing ethanol concentration, from about 2.42 at 288 nm in 50% ethanol to about 0.62 in deionized water (Figure 4a). In comparison, the height of the absorption peak at 286 nm in the solution containing 0.2 M glucose alone decreases with the increasing ethanol concentration, from about 0.57 in aqueous solution to 0.27 in 50% ethanolic solution (Figure 4b). In the system containing glucose alone, ethanol suppresses the absorbance at 280 to 290 nm (Figure 4b). However, in G-G ethanolic solutions, higher absorbance readings correspond to higher ethanol concentrations (Figure 4a). At pH 4.3, hexoses may react with amino acids to form Amadori rearrangement products, which then undergo 1,2-enolisation to form HMF as a key intermediate in the Maillard browning (Tress and others 1995). The maximum absorbance in the spectrum of HMF occurs at approximately 280 nm (Lee and Nagy 1988). Accordingly, we recognize the occurrence of a dominant absorption peak at 280 to 290 nm in HPLC analysis as evidence for HMF formation (Figure 5). HMF is the dominant peak in the chromatogram for all the samples tested (Figure 5), revealing this compound as the main intermediate in ethanolic solutions in the present browning reaction. An increase in ethanol concentration favors the formation of HMF in the G-G system more than that in the fructose-glycine system. The content of HMF in the G-G system starts to surpass that in the fructose-glycine system before the ethanol concentration reaches 50% (Figure 6). Changes in browning with increasing level of ethanol as shown in Figure 3a and Table 1 are consistent with these findings. FCT276 JOURNAL OF FOOD SCIENCE—Vol. 69, Nr. 4, 2004 Figure 4—Absorbance spectra of (a) glucose (0.2 M)/glycine (0.2 M) and (b) glucose (0.2 M) solutions buffered at pH 4.3 with 0.05 M succinic acid–sodium hydroxide, heated in boiling water for 6 h and then diluted 8-fold URLs and E-mail addresses are active links at www.ift.org Maillard browning in ethanolic solution . . . Figure 5—High-performance liquid chromatographic patterns of glucose (0.2 M)/glycine (0.2 M) and fructose (0.2 M)/glycine (0.2 M) in pH 4.3, 0.05 M succinic acid–sodium hydroxide buffered solutions after heating in boiling water for 6 h URLs and E-mail addresses are active links at www.ift.org Table 3—Water activity (aw)a values of pH 4.3, 0.05 M succinic acid–sodium hydroxide buffered ethanolic or glycerolic solutions of glucose (0.2 M) alone or glucose (0.2 M)/glycine (0.2 M) Ethanol conc. glucose (v/v) only 0% 10% 15% 20% 30% 40% 50% 0.98 0.96 0.93 0.90 0.86 0.83 0.76 aw glucoseglycine 0.97 0.91 0.89 0.87 0.81 0.77 0.70 aw Glycerol conc. (w/w) glucose only glucose glycine 0% 13.9% 20.9% 27.9% 41.3% 54.3% 0.98 0.91 0.87 0.83 0.76 0.73 0.97 0.89 0.86 0.82 0.73 0.67 a Calculated from Ferro Fontan and Chirife (1981) equation. in both the glycerolic solutions of glucose alone (Figure 7a) and GG (Figure 7b). However, a lower aw value may correspond to either a lower HMF content in the ethanolic solution of glucose alone (Figure 7a) or a higher HMF content in the ethanolic solution of G-G (Figure 7b). Restated, ethanol has a net inhibitory effect on HMF formation in the system containing glucose alone. The same phenomenon is also shown in Figure 4b. The mechanism of inhibition may be as follows: the hydroxyl group of an ethanol molecule reacts with the carbonyl group of a glucose molecule, loses 1 molecule of water, and forms 1 chemically stable molecule of ethyl -D-glucopyranoside (BeMiller and Whistler 1996), thus avoiding the 3-deoxyglucosone pathway and blocking the formation of HMF. The trends in the formation of HMF in both glycerolic solution and ethanolic solution of the G-G system are presented in Figure 7b. The HMF content in glycerol solution changes from 4.38 mg/100 mL at aw 0.82 to 8.95 mg/100 mL at aw 0.67, having an increment of 104%. Interestingly, in a similar change of aw, from 0.81 in 30% ethanol to 0.70 in 50% ethanol, the HMF content shifts from 6.05 mg/ 100 mL to 19.41 mg/100 mL, having an increment of 221%. At an aw of approximately 0.70, the HMF content in the ethanolic solution is more than double that in the glycerolic solution. It is evident that Figure 6—The relationship between ethanol concentration and HMF content in pH 4.3, 0.05 M succinic acid–sodium hydroxide buffered ethanolic solutions of fructose (0.2 M)/ glycine (0.2 M) and glucose (0.2 M)/glycine (0.2 M) after heating in boiling water for 6 h Vol. 69, Nr. 4, 2004—JOURNAL OF FOOD SCIENCE FCT277 Food Chemistry and Toxicology (1972) added glycerol to an aqueous solution to investigate the effect of aw reduction on Maillard browning. We substituted ethanol with glycerol in the solutions containing either 0.2 M glucose alone or the G-G system and then evaluated the aw for the same reason. The aw values of aqueous, ethanolic, and glycerolic solutions are shown in Table 3. It appears that the presence of ethanol reduces aw noticeably. For example, the aw values of aqueous solutions of 0.2 M glucose alone and G-G are 0.98 and 0.97, respectively, whereas those of 50% ethanolic solutions containing the same solutes are reduced to 0.76 and 0.70. The ranges of aw values in the glycerolic solutions containing 0.2 M glucose alone and G-G are 0.91 to 0.76 and 0.89 to 0.67, respectively. Each covers the range of aw values of the ethanolic solution. Figure 7 shows the relationship between aw and HMF content in pH 4.3, 0.05 M succinic acid–sodium hydroxide buffered ethanolic and glycerolic solutions of 0.2 M glucose alone and the G-G system after the heat treatment. An increase in the concentration of ethanol causes a reduction of aw value in the ethanolic model solutions. As expected, a lower aw value corresponds to a higher HMF content Maillard browning in ethanolic solution . . . Food Chemistry and Toxicology the reduction of aw is not the sole major mechanism responsible for the acceleration in the Maillard reaction by ethanol. This may also explain the marked browning in ethanolic solutions at low aw, such as a liqueur, during storage. Figure 8 shows the relationship between aw and the extent of browning in the 0.2 M glucose solution and the G-G solution. The readings of absorbance at 420 nm of the samples containing 0.2 M glucose alone are very low (Figure 8a). The contribution to browning from caramelization should be negligible in the present study. Generally, severe heating and extreme pH are necessary for the caramelization of sugar in aqueous solutions (Morales and Van Boekel 1998). The same thing could also be true in ethanolic solutions. Both ethanol and glycerol accelerate the Maillard browning in GG solution through the formation of more HMF (Figure 7b and 8b). Hofmann (1998) heated D-xylose, alanine, and furane-2-carboxaldehyde mixture in aqueous and methanolic solutions and reported the formation of 3 additional color-active compounds in the presence of methanol. He concluded that methanol shifted the pathway for Maillard browning. Similar mechanism may present in ethanolic solutions as well. Conclusions T he present study is concerned with the nonenzymatic browning in ethanolic solution. Maillard browning and HMF in etha- Figure 7—The relationship between aw and hydroxymethylfurfural (HMF) content in pH 4.3, 0.05 M succinic acid–sodium hydroxide buffered ethanolic and glycerolic solutions of (a) glucose (0.2 M) alone and (b) glucose (0.2 M)/glycine (0.2 M) after heating in boiling water for 6 h. Different letters (a–j) within the same graph indicate significant difference (P < 0.05) in HMF content in Duncan’s multiple range test. FCT278 JOURNAL OF FOOD SCIENCE—Vol. 69, Nr. 4, 2004 nolic solutions of the G-G system are found to increase with an increase in ethanol concentration between 0% to 50%, suggesting the enhancement of ethanol on the browning reaction. The results also indicate that the mechanisms of the Maillard reaction in ethanolic solution and aqueous solution are not the same. Interestingly, HMF content decreases with the increasing ethanol level in a system containing glucose alone. Experiments using glycerol as a control concluded that the reduction of aw is not the sole major mechanism for ethanol to accelerate HMF formation and Maillard browning in the G-G solution. The mechanism of the Maillard reaction in ethanolic solutions is not known in detail. Further studies including the purification and identification of the browning products are being done in our laboratory for a better understanding of this subject. References Akhavan I, Wrolstad RE. 1980. Variation of sugars and acids during ripening of pears and in the production and storage of pear concentrate. J Food Sci 45:499– 501. Ajandouz EH, Puigserver A. 1999. Nonenzymatic browning reaction of essential amino acids: Effect of pH on caramelization and Malliard reaction kinetics. J Agric Food Chem 47:1786-93. Ames JM. 1990. Control of the Maillard reaction in food systems. Trends Food Sci Technol 1:150–4. Ames JM. 1998. Applications of the Maillard reaction in the food industry. Food Chem 62:431–9. Ashoor SH, Zent JB. 1984. Maillard browning of common amino acids and sugars. J Food Sci 49:1206–7. Baxter JH. 1995. Free amino acid stability in reducing sugar systems. J Food Sci Figure 8—The relationship between aw and absorbance at 420 nm in pH 4.3, 0.05 M succinic acid–sodium hydroxide buffered ethanolic and glycerolic solutions of (a) glucose (0.2 M) alone and (b) glucose (0.2 M)/glycine (0.2 M) after heating in boiling water for 6 h. Different letters (a–j) within the same graph indicate significant difference (P < 0.05) in absorbance at 420 nm in Duncan’s multiple range test. URLs and E-mail addresses are active links at www.ift.org 60:405–8. Beck J, Ledl F, Sengl M, Severin T. 1990. Formation of acids, lactones and esters through the Maillard reaction. Z Lebensm U Forsch 190:212–6. BeMiller JN, Whistler RL. 1996. Carbohydrates. In: Fennema OR, editor. Food chemistry. 3rd ed. New York: Marcel Dekker. p 157–223. Brands CMJ, Alink GM, van Boekel MAJS, Jongen WMF. 2000. Mutagenicity of heated sugar-casein systems: effect of the Maillard reaction. J Agric Food Chem 48:2271–5. Cuzzoni MT, Stoppini G, Gazzani G, Mazza P. 1988. Influence of water activity and reaction temperature of ribose-lysine and glucose-lysine Maillard systems on mutagenicity, absorbance and content of furfurals. Food Chem Toxic 26:815– 22. Davies CGA, Kaanane A, Labuza TP, Moscowitz AJ, Guillaume F. 1998. Evaluation of the acyclic state and the effect of solvent type on mutarotion kinetics and on Maillard browning rate of glucose and fructose. In: O’Brien J, Nursten H, Crabbe MJ, Ames J, editors. The Maillard reaction in foods and medicine. London: Royal Society of Chemistry. p 166–71. Del Pilar Buera M, Chirife J, Resnik SL, Wetzler G. 1987. Nonenzymatic browning in liquid model systems of high water activity: kinetics of color changes due to Maillard’s reaction between different single sugars and glycine and comparison with caramelization browning. J Food Sci 52:1063–7. Eichner K, Karel M. 1972. The influence of water content and water activity on the sugar-amino browning reaction in model systems under various conditions. J Agric Food Chem 20:218–23. Ferro Fontan C, Chirife J. 1981. The evaluation of water activity in aqueous solutions from freezing point depression. J Food Technol 16:21–30. Gutiérrez IH. 2003. Influence of ethanol content on the extent of copigmentation in a Cencibel young red wine. J Agric Food Chem 51:4079–83. Hill VM, Ledward DA, Ames JM. 1996. Influence of high hydrostatic pressure and pH on the rate of Maillard browning in a glucose-lysine system. J Agric Food Chem 44:594–8. Hofmann T. 1998. Studies on the influence of the solvent on the contribution of single Maillard reaction products to the total color on browned pentose/ala- URLs and E-mail addresses are active links at www.ift.org nine solutions—a quantitative correlation using the color activity concept. J Agric Food Chem 46:3912–7. Lee HS, Nagy S. 1988. Quality changes and nonenzymic browning intermediates in grapefruit juice during storage. J Food Sci 53:168–72. Leong LP, Wedzicha BL. 2000. A critical appraisal of the kinetic model for the Maillard browning of glucose with glycine. Food Chem 68:21–8. Miracco JL, Alzamora M, Chirife J, Ferro Fontan C. 1981. On the water activity of lactose solutions. J Food Sci 46:1612–3. Morales FJ, Jimenez-Perez S. 2001. Free radical scavenging capacity of Maillard reaction products as related to color and fluorescence. Food Chem 72:119–25. Morales FJ, Van Boekel MAJS. 1998. A study on advanced Maillard reaction in heated casein/sugar solutions: color formation. Int Dairy J 8:907–15. Petriella C, Resnik SL, Lozano RD, Chirife J. 1985. Kinetics of deteriorative reactions in model food systems of high water activity: color changes due to nonenzymatic browning. J Food Sci 50:622–6. Renn PT, Sathe SK. 1997. Effects of pH, temperature, and reactant molar ratio on L -leucine and D-glucose Maillard browning reaction in an aqueous system. J Agric Food Chem 45:3782–7. Toribio JL, Lozano JE. 1984. Nonenzymatic browning in apple juice concentrate during storage. J Food Sci 49:889–92. Tress R, Nittka C, Kersten E, Rewicki D. 1995. Formation of isoleucine-specific Maillard products from[1-13C]- D-glucose and [1-13C]- D-fructose. J Agric Food Chem 43:1163–9. Trifiro A, Gherardi S, Belloli S, Saccani G, Aldini R. 1990. Effetti della tecnologia di lavorazione e delle condizioni di magazzinaggio sulle reazioni d’imbrunimento non enzimatico in derivati del pomodoro. Industr Conserv 65:210–5. Van Martins SIFS, Jongen WMF, Boejel MAJS. 2001. A review of Maillard reaction in food and implications to kinetic modeling. Trends Food Sci Technol 11:364– 73. Wedzicha BL, Kaputo MT. 1992. Melanoidins from glucose and glycine: composition, characteristics and reactivity toward sulfite ion. Food Chem 43:359–67. Vol. 69, Nr. 4, 2004—JOURNAL OF FOOD SCIENCE FCT279 Food Chemistry and Toxicology Maillard browning in ethanolic solution . . .