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Changes in Organic Acid Composition of Citrus Juices During the 2000-2001 Season

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Changes in Organic Acid Composition of Citrus Juices During the 2000-2001 Season biocides both as alternatives to water chlorination and in al- Literature Cited ternate capacities to augment water chlorination. In all tests, Bartz, J. A., C. G. Eayre, M. J. Mahovic, D. E. Concelmo, J. K. Brecht, and S. alternate biocide efficacy, practicality and safety, as compared A. Sargent. 2001. Chlorine Concentration and the Inoculation of Tomato to that of chlorine, are major considerations in producing Fruit in Packinghouse Dump Tanks. Plant Disease 85:885-889. recommendations for or against their use in recirculated wa- White, C. 1999. Handbook of Chlorination and Alternative Disinfectants. ter systems. John Wiley & Sons, Inc., New York, NY. Proc. Fla. State Hort. Soc. 115:37-41. 2002. CHANGES IN ORGANIC ACID COMPOSITION OF CITRUS JUICES DURING THE 2000-2001 SEASON PAUL F. CANCALON1 AND YEHONG XU indicator of possible adulteration. If the level of isocitric acid Florida Department of Citrus in a juice is too low, it may indicate that non-citrus compo- 700 Experiment Station Road nents have been added to the juice. However, these values ap- Lake Alfred, FL 33850 pear too high for authentic Florida juices. To clarify the problem, the level of organic acids have been followed in au- Additional index words. capillary electrophoresis, citrate, isoci- thentic Florida orange and grapefruit juices collected during trate, citrus, organic acids the 2000-2001 season. Capillary electrophoresis (CE) has been used to examine Abstract. Both orange and grapefruit juice contain significant various food components (for review see Cancalon, 1997a, amounts of organic acids. Two, malic and citric acid, are 2000). The application of CE for the analysis of organic acids present in large quantity. The third one, isocitric acid, is in food samples was recently reviewed by Klampf et al. (2000). present in the juice in very small amounts but is particularly In the past, indirect UV was the main mode of detection for important in determining the authenticity of citrus juices. In the organic acids. With conductivity detection becoming com- present study, samples of orange and grapefruit juices were mercially available for CE (Jones 1996a, b), we have devel- collected between October 2000 and June 2001 and analyzed oped a conductivity method for the analysis of organic acids for organic acids by capillary electrophoresis with conductivi- in citrus juices (Cancalon, 1997b). Recently, however, Saave- ty detection with a method developed previously. Changes in organic acid levels were compared to the variations in Brix/ dra et al. (2001) developed a simple method to analyze organ- acid ratio and acidity. For isocitric acid, the same pattern was ic acids by direct UV detection at 200 nm with a found for each of the three orange varieties: the ratio varies in polyacrylamide coated capillary, that we adapted for bare sili- opposite manner as compared with isocitric acid. High acid ca capillary (Cancalon and Xu, 2001). levels and low ratios are found in the early fruit and the oppo- Several groups have reviewed the composition of many site become true for mature oranges. In the case of grapefruit juices including citrus (RSK, 1987; Dillon, 1995). The values juice, the same inverse relation between isocitric acid levels reported from several studies varied greatly. For orange juice, and ratio was also seen. However, since there are no varieties the ranges were 6-16g·L-1 for citric acid, 0.6 to 4 g·L-1 for malic maturing successively, the high level of acid recorded early in acid and 44 to 200 mg·L-1 for isocitric acid. The reported val- the season fell rapidly. ues for grapefruit juice were 9 to 20 g·L-1, 0.1 to 1.2 g·L-1 and 50 to 350 mg·L-1 for citric, malic, and isocitric respectively. With the development of the European Community, a European Code of Practice for the Evaluation of Fruit and Vegetable Juices (COP) was published (1990) by the Associa- Materials and Methods tion of the Industry of Juices and Nectars from Fruits and Veg- etables of the European Community (AIJN). Reference Apparatus. Conductivity detection analyses were per- chemical values were published for many juices, but mini- formed with a Thermo Bio Analysis Crystal 310 CE apparatus mum or maximum concentrations were also set by the COP equipped with a Crystal 1000 conductivity detector (Thermo for some components. For isocitric acid, the minimum con- Bio Analysis, Santa Fe, NM). Separations were performed centrations are 65 mg·L-1 for orange juice and 140 mg·L-1 for with uncoated fused silica capillaries 70 cm × 50 µm (Thermo grapefruit juice. Since this chemical is too expensive to be Bio Analysis). added back to a product, it has been used extensively has an Sample preparation. ‘Ambersweet.’ ‘Hamlin’ and ‘Valencia’ were collected successively, as they mature, during the entire 2000-2001 season from October to June. Grapefruit mature si- 1Corresponding author. multaneously during the season and white and red grape- Proc. Fla. State Hort. Soc. 115: 2002. 37 fruits were collected during the entire period. Juice samples were extracted with an FMC state house extractor at the FDOC pilot plant. The free form of isocitric acid was released from citrus juices following a procedure similar to that used for the enzy- matic analysis of isocitric acid (IFU, 1985). Ten mL of juice were centrifuged for 5 min at 10,000 rpm. Two and a half mL of 4 N NaOH were them added to 10 mL of supernatant. After 10 min at room temperature, the solution was neutralized with 2.5 mL of HCl. The juice was finally diluted to a total of 4 or 16 fold by mixing the neutralized solution with HPLC grade water. Running conditions. The conductivity detection was modi- fied for citrus juices from Jones (1996b), the buffer was made of 130 mM histidine/2-[N-morpholino]ethanesulfonic acid (MES), 0.7 mM tetradecyl-trimethyl-ammonium bromide (TTAB), 0.03% Triton X100, 10 ppm ethylenediamine tet- raacetic acid (EDTA), and 15% acetonitrile. After a 16-fold dilution, the samples were run at -18 Kv and 21 °C for 12 min and 25 mBar for 0.24 min, on a 70 cm × 50 um capillary col- Fig. 1. Electropherogram of orange juice organic acids with conductivity umn. An example of electropherogram of orange juice or- detection (uS = micro siemens). ganic acids is shown in Fig. 1. Fig. 2. Variations in isocitric acid concentration in grapefruit juice as com- Fig. 3. Changes in the citric/isocitric ratio in grapefruit juice (GJ)and in pared with the Brix/acid ratio (A) and % acid (B). orange juice (OJ). 38 Proc. Fla. State Hort. Soc. 115: 2002. Data analysis. Changes in chemical concentrations during lower than the 140 mg·L-1 set by AIJN (Fig. 2B). Under those the season were analyzed by curvilinear regression (Devore, conditions, 49% of the samples had an isocitric acid level low- 1991) using Excel (Microsoft) statistical data analysis pack- er than the limit. age. Orange. The opposite changes of ratio and acid seen with grapefruit were also seen with orange juice, but they occurred with each orange variety. Brix/acid ratio increased early in Results and Discussion the season with the maturation of ‘Ambersweet’, it then went down with the arrival of early ‘Hamlin’, peaked and with the Isocitric Acid arrival of ‘Valencia’, decreased and peaked again. Isocitric levels varied in the opposite manner, with high value associat- Grapefruit juice. Several grapefruit varieties are grown in ed with early fruit and low values with mature fruit (Fig. 4A). Florida but they all mature simultaneously during the season Again no clear relationship was seen between isocitric levels from about October to June. Therefore changes in chemical and acidity (Fig. 4B). composition during maturation can be follow during the en- Because of the successive changes, the number of samples tire season. The study has shown that during the season the below the minimum of 65 mg·L-1 was only 29%. As for the cit- Brix/acid ratio and isocitric levels vary in opposite manner, ric/isocitric ratio, 49% were above the AIJN maximum of 130 high ratio corresponding to low acid level and vice-versa (Fig. (Fig. 3 OJ). 2A). The last parameter examined was the ratio citric/isoc- itric. The AIJN standards set limits for this ratio between 50 and 95 (Fig. 3 GJ) among the samples we analyzed 54% had a Citric and Malic Acids ratio outside the limits. Percent acidity of the juice decreases during the entire season, the level of isocitric acid decreases Although the study was mainly aimed at examining isoc- even more rapidly, and by Jan. reaches a plateau of 117 mg·L-1 itric acid variations, citric and malic acids were also followed. Fig. 4. Variations in isocitric acid concentration in orange juice as com- Fig. 5. Variations in malic acid concentration in grapefruit juice as com- pared with the Brix/acid ratio (A) and % acid (B). pared with the Brix/acid ratio (A) and % acid (B). Proc. Fla. State Hort. Soc. 115: 2002. 39 Grapefruit juice. Citric and malic acids are the main acids in citrus juices. In grapefruit juice the average concentration for malic acid was 592 mg·L-1 but it should be noted that val- ues ranged from less than 300 to more than a 1,000 mg/L (Fig. 5). The range for citric acid was less drastic, varying from 8 to 13 g·L-1 with an average of 10.436 g·L-1 (Fig. 6). The changes for both acids follow a similar pattern. The trend lines indicate that the ratio Brix/acid increases during this period while the organic acids decrease steadily (Figs.
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