JFS: Food Chemistry and Toxicology Effects of pH on Caramelization and Maillard Reaction Kinetics in Fructose-Lysine Model Systems E.H. AJANDOUZ, L.S. TCHIAKPE, F. DALLE ORE, A. BENAJIBA, AND A. PUIGSERVER ABSTRACT: The nonenzymatic browning reactions of fructose and fructose-lysine aqueous model systems were Food Chemistry and Toxicology investigated at 100 8C between pH 4.0 and pH 12.0 by measuring the loss of reactants and monitoring the pattern of UV-absorbance and brown color development. At all the pH values tested, the loss of fructose was lower in the presence than in the absence of lysine. And, in lysine-containing fructose solution, the sugar disappeared more rapidly than the amino acid. Lysine was moderately lost below pH 8.0. Caramelization of fructose, which accounted for more than 40% of total UV-absorbance and 10 to 36% of brown color development, may therefore lead to overestimating the Maillard reaction in foods. Keywords: Maillard reaction, caramelization, lysine, fructose Introduction (Ellingson and others 1954; Bobbio and The aim of this study was, there- HE MAILLARD REACTION, WHICH others 1981; Baxter 1995). It has also fore, to describe the kinetics of the Tlinks the carbonyl group of reduc- been reported that the browning of nonenzymatic browning reaction in ing carbohydrates and the amino group fructose solutions was either more or fructose solutions heated either alone of free amino acids as well as of lysyl less extensive than that of glucose, de- or in the presence of lysine at 100 8C at residues in proteins, may have either pending on the heating conditions initial pH values ranging from 4.0 to beneficial or detrimental effects. At ear- (Kato and others 1969; Buera and oth- 12.0. The extent of both the caramel- ly stages of the reaction, an improve- ers 1987; Wijewickreme and others ization and the Maillard reactions was ment of the functional properties of 1997). On the other hand, the influence compared at the initial, intermediate, proteins was generally observed (Handa of pH on the Maillard reaction of amino and final stages. The kinetic behavior and Kuroda 1999), and afterwards anti- acid-containing glucose or fructose of the nonenzymatic browning reac- oxidant compounds are formed (Monti model systems has been studied at dif- tions of fructose was also compared to and others 1999) as well as highly ap- ferent pH ranges and conditions of that of glucose under the same experi- preciated browned flavors (Ho 1996). temperature and concentration of reac- mental conditions (Ajandouz and However, loss of lysine and decrease in tants, namely pH 4-6 (Buera and others Puigserver 1999). protein digestibility may also occur 1987), pH 5-7 (Petriella and others (Friedman 1996), together with some 1985), pH 5.5-7.5 (Baxter 1995) and pH Materials and Methods antinutritive (Oste and others 1987; 6-12 (Ashoor and Zent 1984). It is worth O’Brien and others 1994), toxic (O’Brien mentioning here that only the Maillard Materials and Morrissey 1989) or mutagenic browning intensity was measured in L-lysine, D-fructose, L-a-amino-n- (Wang and others 1999) effects. The most of these studies. butyric acid and triethylamine (TEA) caramelization of sugars, which takes Very little attention has been paid so were purchased from Sigma Chemical place at the same time, also contributes far to the contribution of carameliza- Co. (St. Louis, Mo., U.S.A.). Phenyl- to nonenzymatic browning reactions. tion to the nonenzymatic browning re- isothiocyanate (PITC) and the standard In both the Maillard and caramelization actions of glucose or fructose, although mixture of amino acids were supplied reactions, highly UV-absorbing and col- studying the chemical reactions in- by Pierce Chemical Co. (St. Louis, Mo., orless compounds are formed at inter- volved in caramelization is a prerequi- U.S.A.). All the other chemicals used mediate stages, whereas the brown site for understanding the Maillard re- were of the purest commercially avail- polymers are formed at final stages action (Mauron 1981). It has, however, able grade. (Hodge 1953; Mauron 1981). been clearly established that the frag- The nonenzymatic browning reac- mentation of sugars occurs to a signifi- Heating procedure tion of fructose has not been as thor- cant extent at pH values below neutrali- An equimolar (0.05 M) mixture of oughly investigated as that of glucose, ty (O’Beirne 1986; Buera and others fructose and lysine (3 mL) was heated and it has usually been compared to the 1987) and increases considerably at in a 15-ml screw-sealed tubes for vari- latter. In several early studies (Maillard high pH values and temperatures, yield- ous periods of time in boiling water at 1912; Hodge 1953; Reynolds 1965), the ing colored N-free polymers (Myers various pH values. After 5, 15, 30, 60, 90, browning of fructose aqueous solutions and Howell 1992; Clarke and others and 120 min, the tubes were removed in the presence of amino acids in model 1997). The Maillard reaction in foods and immediately cooled in ice. Part of systems was found to take place more may then be overestimated and the em- the heated solution was used directly rapidly than that of glucose, although phasis placed on its detrimental rather for UV-absorbance, browning, and final the contrary was also reported to occur than its beneficial effects. pH measurements, while the rest was 926 JOURNAL OF FOOD SCIENCE—Vol. 66, No. 7, 2001 © 2001 Institute of Food Technologists Maillard and Caramelization Reaction Kinetics . stored at –20 8C for fructose and lysine intensity of the aqueous solutions con- well as in glucose solutions containing loss determinations. Fructose and taining fructose or fructose and lysine either lysine, or methionine or threo- lysine were separately heated under the were measured at room temperature at nine when heated to 100 8C at different same experimental conditions. The fol- 294 nm and 420 nm, respectively, using pH values (Ajandouz and Puigserver lowing buffers were used: 0.05 M sodi- a Beckman model DU 640 spectropho- 1999). In a number of studies in which um acetate adjusted to pH 4.0 with 1 M tometer (Beckman Instruments, Irwin, proteins were heated in the presence of acetic acid, 0.05 M sodium phosphate Calif., U.S.A.). When necessary, appro- reducing sugars at an intermediate adjusted to pH 6.0, pH 7.0 and pH 8.0 priate dilutions were made in order to moisture content and a wide range of using either monobasic or dibasic sodi- obtain an optical density of less than temperatures, a no-loss period was um phosphate, 0.05 M Tris-carbonate 1.5. found to occur when 50 to 75% of the adjusted to pH 9.0 with 1 M hydrochlo- All the experiments were carried out lysine was destroyed (Wolf and Thomp- ric acid, 0.05 M sodium carbonate-bi- in triplicate and the mean values (over- son 1977; Labuza and Saltmarch 1981). carbonate adjusted to pH 10.0 and pH all less than 10% standard deviation) The no-loss period might be due to 11.0 using either sodium carbonate or were used to draw up the kinetic plots. some limitation in the reactants or to sodium bicarbonate, and finally 0.05 M the release of amino groups at ad- sodium bicarbonate adjusted to pH Results and Discussion vanced stages of the Maillard reaction. 12.0 with 1 M sodium hydroxide. The decrease in pH throughout the Early stages in the browning heat treatment may also contribute to Fructose loss reactions the progressive lowering of the rates of The remaining nondegraded fruc- The initial stages in the nonenzymat- lysine loss. Under our experimental tose was monitored on high-perfor- ic browning reaction of the heated fruc- conditions, a pH drop of 0.25-3.8 units mance anion exchange-pulsed ampero- tose solutions, with or without lysine, and of 0.5 to 1.8 units occurred in fruc- metric detection equipment. Fructose was studied by measuring the extent of tose and fructose-lysine model systems Food Chemistry and Toxicology was eluted under isocratic conditions fructose and lysine degradation over after a 2-h heating period at starting pH from the CarboPac PA-100 (Dionex time. As expected, an increase in pH of values of 6.0 to 12.0, respectively. A Corp., Sunnyvale, Calif., U.S.A.) analyti- the heated solutions led to an increase more severe pH decrease has been re- cal anion exchange column (250 3 4 in the initial rate of degradation of both ported in unbuffered solutions of gly- mm) equipped with an IonPac AG4A- fructose and lysine (Figure 1). Lysine cine and glucose (Nicoli and others SC (Dionex Corp., Sunnyvale, Calif., degradation was already reduced at pH 1993). More attention should, therefore, U.S.A.) guard column (25 3 4 mm) con- 8.0, since it did not exceed 22% of the be paid to the pH decrease during non- nected to a gold working electrode cell. amino acid after 2 h of boiling, and only enzymatic browning reactions, espe- The eluent, a 5 mM sodium acetate so- 5% after 15 min as compared to higher cially at intermediate moisture content lution containing 0.1 M NaOH, was de- pH values (13 to 34% loss after 15 min and pH values below neutrality. livered at a rate of 1 mL.min–1 by a GP of heating at pH 9.0 to 12.0). Moreover, Studies on the Maillard reaction 40 Dionex gradient pump (Dionex lysine loss resulted almost completely, if placing emphasis on the degradation of Corp.,). The injection volume was deliv- not exclusively, from Maillard reaction, reducing sugars are not so numerous.