Early Stages of Maillard Reaction in Dehydrated Orange Juice M. Dolores del Castillo, Nieves Corzo,* and Agustı ´n Olano Instituto de Fermentaciones Industriales (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain The formation of furoylmethyl derivatives of amino acids as indicators of the early stages of Maillard reaction in dehydrated orange juices and model systems was studied. In stored dehydrated orange juices, the presence of furoylmethyl derivatives of arginine, asparagine, proline, alanine, glutamic acid, and GABA was detected. Their formation increased with temperature of storage. After 2 weeks at 30 °C and a w ) 0.44, the reconstituted orange juice contained 94 mg/L furoylmethyl derivatives, whereas up to 1215 mg/L was detected in samples stored at 50 °C. Keywords: Maillard reaction; storage; orange juice INTRODUCTION The Maillard reaction is one of the most complex reactions that occur during the processing and storage of foods. The reaction is initiated by a condensation between the free amino group of an amino acid, peptide, or protein and the carbonyl group of a reducing sugar, leading to the formation of Amadori compounds. The activity of water, pH, time, and temperature govern the extent of the reaction. In the case of foods containing proteins and glucose, Amadori compounds are largely represented by protein-bound ǫ-fructosyl-lysine (Olano and Martı ´nez-Castro, 1996). The determination of the level of Amadori compounds present in foods allows one to detect the onset of the reaction before detrimental changes occur (del Castillo et al., 1998) as well as to retrospectively assess the heat treatment or storage conditions to which a product has been submitted. In the case of foods containing free amino acids, free Amadori compounds can be present. Although Amadori compounds can be analyzed by HPLC and detected by differential refractometry (Mo ¨ll and Gross 1981), the sensitivity of the method does not allow the detection of the low amounts present in foods. To improve sensitivity, HPLC methods involving derivatization have been proposed (Walton and McPherson 1987; Reutter and Eichner 1989). A sensitive method for evaluating Amadori com- pounds involves an acid hydrolysis that releases protein- bound ǫ-fructosyl-lysine and generates furosine (ǫ-N-2- furoylmethyl-lysine) (Erbersdobler et al., 1995), which can be directly measured by HPLC (Resmini et al., 1990). In foods containing free Amadori compounds, acid hydrolysis would give rise to the formation of the corresponding 2-furoylmethyl derivatives, which could be easily determined by UV detection. The purpose of this paper was to study the formation of furoylmethyl derivatives from the Amadori com- pounds of major amino acids present in orange juice and further study their presence in the hydrolysates of processed orange juices. MATERIALS AND METHODS Model Systems. Mixtures of D-glucose and the correspond- ing L-amino acid [alanine, γ-aminobutyric acid (GABA), argi- nine, asparagine, aspartic acid, glutamic acid, proline, and serine] in molar ratios of 6:1, in water (5 mL), were lyophilized and equilibrated to aw ) 0.44 in a desiccator over saturated K2CO3 solution using the method of Labuza and Saltmarch (1981) and then stored at 50 °C. Dehydrated Orange Juice Samples. Freshly squeezed orange juice was prepared in the laboratory from 2 kg of oranges (navel variety) using a domestic juicer Braun Citro- matic MPZ2 (Braun Espan ˜ ola, S.A., Spain). The extracted juice was filtered through a double layer of gauze to remove seeds and albedo fragments and to reduce pulp content. pH was measured (pH-meter MicropH2001 Crison Instruments, S.A., Barcelona, Spain) in aliquots (5 mL), which were then lyo- philized, equilibrated to aw ) 0.44, and stored at the desired temperature (30 or 50 °C) for 14 days. Samples were taken at 4, 7, and 14 days of storage. Before analysis, samples were reconstituted to initial volume. Synthesis of N-(1-Deoxy-D-frutosyl)-γ-aminobutyric Acid. N-(1-Deoxy-D-frutosyl)-γ-aminobutyric acid was obtained according to the procedure of Reutter and Eichner (1989). A mixture of glucose (3.25 g), γ-aminobutyric acid (0.5 g), microcrystalline cellulose (16.41 g), and water (100 mL) was lyophilized and stored for 14 days at 40 °C and aw ) 0.35 in a desiccator over saturated CaCl2 solution. Following the stor- age, the mixture was treated with water (3 × 80 mL) at vacuum and the aqueous solution lyophilized. The reaction mixture was subjected to a purification by ion-exchange chromatography (Finot and Mauron, 1969) on Dowex 50 WX4 in pyridinium form. The characterization of N-(1-deoxy-D- frutosyl)-γ-aminobutyric acid was made by 13 C NMR spectros- copy (del Castillo et al., 1998). HPLC Analysis of Furoylmethyl Derivatives. An ion- pair RP-HPLC method (Resmini et al., 1990) was used to analyze furoylmethyl derivatives using a C8 column (250 mm × 4.6 mm i.d.) (Alltech Furosine-dedicated) with a linear binary gradient. A Dionex chromatograph (DX-300) and vari- able wavelength detector (LDC Analytical, SM 4000) were used. Acquisition and processing of data were achieved in an HPChem Station (Hewlett-Packard). Before HPLC analysis, 1.3 mL of sample was hydrolyzed with 3 mL of 11.4 M HCl at 110 °C for 24 h in a Pyrex screw- cap vial with a PTFE-faced septum. High-purity helium gas was bubbled through the solution for 2 min. The hydrolysate was filtered with a medium-grade paper filter. A 0.5 mL portion of the filtrate was applied to a Sep-Pak C 18 cartridge (Millipore, Madrid, Spain) prewetted with 5 mL of methanol * Author to whom correspondence should be addressed [telephone (34-91) 562 29 00, ext. 307; fax (34-91) 564 48 53; e-mail ifics19@ifi.csic.es]. 4388 J. Agric. Food Chem. 1999, 47, 4388-4390 10.1021/jf990150x CCC: $18.00 © 1999 American Chemical Society Published on Web 09/23/1999