Differences in Amino Acid Composition in Commercial Orange Juices M. Dolores del Castillo, Guillermo Santa-Marı ´a, Encarnacio ´n Pueyo, Nieves Corzo,* and Agustin Olano Instituto de Fermentaciones Industriales (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain The amino acid composition of two sets of commercial orange juice was determined, and the mean value and range of values for each amino acid were tabulated. Single-strength juices from concentrates showed lower content of amino acids than straight-processed juices. Statistical comparison by ANOVA revealed statistical significance (p e 0.001) for aspartic, arginine, asparagine, glutamine, serine, threonine, and isoleucine, between straight-processed orange juices, and those single-strength juices from concentrates. The combination of glutamine, asparagine, and alanine allowed a 100% correct classification. Keywords: Commercial orange juices; amino acids; discriminant analysis INTRODUCTION During the last years, orange juice demand has been expanding at a rapid rate, in part due to the constant modernization of the technologies which have had the effect of improving the quality of commercial juices. Since there is a shift in consumer preferences from pasteurized orange juice to totally fresh juice, one of the major efforts underway is to minimize changes during the orange juice processes. Even under mild processing conditions, changes in the volatile constituents of orange juice take place, and quantitative data have been used to correlate changes in components with processing conditions. Velez et al. (1993) classified orange juice samples which had been stored at different temperatures, based on analysis of volatile constituents, and Shaw et al. (1993, 1994) were able to classify commercial orange juice samples into four types, based on the type of processing. Although the mechanism responsible for the deterio- ration of citrus juice during processing and storage is not well understood, nonenzymatic browning reactions between carbohydrates and amino acids are generally believed to be involved (Kimball, 1991; Del Castillo et al., 1998). Since prolonged storage of juice concentrates may cause changes in the amino acid composition, this study was undertaken to determine whether the quan- titative determination of individual amino acids would allow the distinction between two types of commercial orange juices: straight-processed and single-strength juices from concentrates. MATERIALS AND METHODS Samples. Seven samples of straight-processed orange juices and 17 samples of single-strength orange juices from concentrates were purchased at different local markets. Determination of Amino Acids. Aliquots (10 mL) of orange juice were centrifuged at 6940 g for 20 min at 20 °C, and free amino acids were determined in the supernatant diluted (1:25) with borate buffer 0.4 M, pH ) 10, and were filtered through a 0.22-μm membrane filter (Millipore Ibe ´rica, Madrid, Spain). Analysis, in duplicate, was carried out by HPLC using a Waters (Milford, MA) liquid chromatograph controlled by a Maxima 820 chromatography workstation (Waters). Samples were submitted to an automatic precolumn derivatization with o-phthaldialdehyde (OPA) (Gonza ´lez de Llano et al., 1991) to determine primary amino acids. The quantification of proline was performed by automatic precol- umn double derivatization with OPA and 9-fluorenylmethyl- chloroformate (FMOC) (Fluka Quimica, Madrid, Spain) (Einarsson, 1985). The separation of amino acids was per- formed on a Novapak C-18 60-Å 4-μm column (3.9 cm × 150 mm) (Waters). Detection was by fluorescence using the wavelengths of excitation and emission at 340 and 425 nm, respectively, for OPA derivatives. For FMOC derivatives the excitation and emission wavelengths were 250 and 335 nm, respectively. All reagents used were HPLC grade. Statistical Analysis. The BMDP package (Dixon, 1988) was used for variance (BMDP7D program) and discriminant analysis (BMDP7M). RESULTS AND DISCUSSION Table 1 shows the mean values, ranges, and relative standard deviations of the amino acids of orange juices marketed as straight-processed and as single-strength from concentrates. In the two sets of samples, γ-ami- nobutyric acid, arginine, asparagine, aspartic acid, glutamic acid, proline, and serine were present in larger amounts than the rest of amino acids and they ac- counted for some 89% of total amino acids. In general, single-strength juices from concentrates showed lower mean content of amino acids (2740 mg/L) than straight- processed juices (3835 mg/L). Considerable dispersion in amino acid content between samples of the same set was observed. Except for glutamic acid, histidine, leucine, ornithine, proline, tyrosine, and methionine, higher dispersion was found in single-strength juices from concentrates than in straight-processed juices, mainly for glutamine which showed a relative standard deviation of 65.7% in single-strength juices from con- centrates. Apart from variations due to maturity, cultural practices, and variety of the fruit (Aristoy et al., 1989), * To whom correspondence should be addressed [telephone (34-1) 562 29 00, ext. 307; fax (34-1) 564 48 53; e-mail ifics19@fresno.csic.es]. 2329 J. Agric. Food Chem. 1998, 46, 2329-2331 S0021-8561(97)00900-X CCC: $15.00 © 1998 American Chemical Society Published on Web 05/13/1998