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Literature Cited 9. Lu, P. and J. E. Kinsella. 1972. Extractability and properties of 1. Boyd, C. E. Leaf protein from aquatic plants. Leaf Protein IBP Handbook #20. Ed. by N. W. Pirie, Blackwell Scientific Publica 10. Morrison, Frank B. 1951. Feeds and Feeding, Morrison Publishing tions, Oxford, p. 9. 2. Bruemmer, J. H. 1979. Solubility of proteins in DMSO. (in prepara tion). 3. and B. Roe. 1979. Protein extraction from aquatic plants. (In preparation). 4. Delory, G. E. and E. J. King. 1945. Sodium carbonate-bicarbonate buffer for alkaline phosphatases. Biochem. J. 39:245-247. 5. Edie, H. H. and B. W. C. Ho. 1969. Ipomoea aquatica as a veget able in Hong Kong. Econ. Bot. 23(l):32-36. 6. Edwards, R. H., Miller, R. E., deFremery, D., Knuckles, B. E., Bickoff, E. M. and G. O. Kohler. 1975. Pilot plant production of an edible white fraction leaf protein concentrate from alfalfa. /. Agric. Food Chem. 23:620-626. 7. Gornall, A. G., Bardawill, C. S. and M. M. David. 1949. Determina tion of serum proteins. J. Biol. Chem. 177:751-766. 8. Kohler, G. O. and E. M. Bickoff. 1971. Commercial production from alfalfa in USA. Leaf protein IBP Handbook No. 20. Ed. by N. W. Pirie Blackwell Scientific Publications, Oxford, p 69. protein from alfalfa leaf meal. /. Food Sci. 37:94-99. Company, Ithaca, New York, 21st Ed., p 1086. 11. Morton, J. F. and G. H. Snyder. 1976. Aquatic crops vs organic soil subsidence. Proc. Fla. State Hortic. Soc. 89:125-129. 12. Morton, R. K. 1955. Extraction of enzymes from animal tissues. Methods in Enzymology, Ed. by S. P. Colowick and N. O. Kaplan. Vol. 1, p 25. 13. Potty, V. H. 1969. Determination of proteins in the presence of phenols and pectins. Anal. Biochem. 29:535-539. 14. Singer, S. J., Eggman, L., Campbell, J. M. and S. G. Wildman. 1952. The proteins of green leaves. IV. A high molecular weight protein comprising a large part of the cytoplasmic proteins. /. Biol. Chem. 197:233-239. F 15. Spies, J. R. and D. C. Chambers. 1951. Determination of tryptophan using photochemical development of color. /. Biol. Chem. 191:787- 789. 16. Snyder, G. H. 1977. Personal communication. 17. Takeda, K. Y. 1975. Personal communication. 18. Watt, B. K. and A. L. Merrill. 1963. Composition of Foods, Agri cultural Handbook No. 8, USDA, Washington, D.C. p 61. Proc. Fla. State Hort. Soc. 92:143-145. 1979. PROLINE CONTENT IN FLORIDA FROZEN CONCENTRATED ORANGE JUICE AND CANNED GRAPEFRUIT JUICE1 S. V. Ting and R. L. Rouseff Florida Department of Citrus, AREC, P. O. Box 1088, Lake Alfred, FL 33850 juices, its use as an index has been suggested by several Abstract. Proline is the most abundant amino acid in citrus juices, and has been suggested as one of the indices of orange juice purity by some European drink manufacturers. Using a modified photometric method of acidic ninhydrin re action on proline developed by Chinard, the proline concn of Florida frozen concnd orange juice (FCOJ) and canned sinale strength grapefruit juice were determined. The proline content in reconstituted FCOJ varied as much as two and half fold from a low of around 60 mg/100 ml to as high as 150 mg/100 ml. Canned grapefruit juice was much lower in its proline content than orange juice, varying from about 20 mg-60 mg/100 ml. With the exception of ornithine, other amino acids found in citrus juices did not interfere in this column chromatography. However, the same reagent pro analysis. Proline, 2, pyrollidine carboxylic acid, is the most abundant of all amino acids in citrus juices (2, 14, 16). Vandercook and Price (14) found an average of 169 mg of proline in 100 ml of California Valencia orange juice but only 94 mg in the same amount of Florida orange juice. Ting and Deszyck (13) using the procedure of Moore and Stein (7) reported a range of about 45 to 75 mg proline in 100 ml frozen concentrated orange juice (FCOJ) reconsti tuted to 11.8° Brix. Niedermann (8) and Koch (6) both found that proline values in orange juice might vary from 2.5 to 3-fold among the samples. In grapefruit juice, Brenoe (2) reported values varying from 35 to 292 mg per 100 ml. The use of concentrations of various chemical con stituents of fruit juices as indices of juice authenticity or as a measure of juice content in a fruit drink has been widely accepted. For citrus juice, formol numbers, poly phenols, some individual amino acids, and several minerals have been proposed either singly or in combinations for this purpose (10). Since proline is a major amino acid of citrus iFlorida Agricultural Experiment Stations Journal Series No. 1995. Proc. Fla. State Hort. Soc. 92: 1979. authors (1, 5, 6, 8, 15). Proline produces a yellow color with ninhydrin buffered at pH 5, and this color reaction was used by Moore and Stein (7) in the determination of proline separated by duces a blue or bluish red color with other amino acids. Ting and Deszyck (13) using a mixed standard of several major amino acids of orange juice determined proline along with the other amino acids. Chinard (3) developed a photo metric procedure for the estimation of proline using nin hydrin reagent with concentrated formic acid. Under the acidic condition with ninhydrin reagent proline first forms a yellow color, followed by a red color having an absorption maximum at 515 nm. Onlv orinthine and hydroxyproline interfere in this reaction. None of the other amino acids tested by Chinard produced any significant color with this method. Ouch (9) used this procedure to analyze proline in grape juice and wine. He found no significant interference from other grace amino acids in amounts normally present in that fruit. He also demonstrated that the red color de veloped need not be extracted with a water immiscible solvent as previously proposed by Chinard (3), but instead that the color may be diluted with isopropanol and measured directly. Wallrauch (15) found that the use of ethyl acetate to extract the color produced by the acidic ninhydrin reaction was necessary for highly colored juices with low proline content. The purpose of this paper is to report the use of this acidic ninhydrin method with modification and simplifica tion to study the range of proline content in Florida FCOJ and canned grapefruit juice as produced commercially. Variations in proline content of orange juice due to fruit cultivars and maturity were also studied. The simplified method could be easily used in most quality control labora tories equipped with an inexpensive colorimeter. Materials and Methods Samples One hundred samples of FCOJ and 80 samples of canned 143 single-strength grapefruit juice used in this study were from various processing plants in Florida. The juice samples used to study variations of proline content with fruit maturity in different orange cultivars were from another experiment. These juices were extracted using an FMC Model 091 InLine Juice Extractor from fruits harvested at different times of the season, and were pasteurized and sealed in cans. All juice samples were stored frozen until ready for analysis. Analytical procedure A. Reagents. Only two reagents are needed in this simplified method for the determination of proline. 1. Ninhydrin reagent. The ninhydrin reagent is prepared by dissolving 3 g of reagent grade nin hydrin (Nutritional Biochemical Co., Cleveland, OH) in 100 ml of diethyleneglycol (Eastman Organic Chemicals, Rochester, NY). The mix ture is stirred on a magnetic stirrer until dis solved and is stable for at least 2 weeks when 2. B. stored in a refrigerator. Formic acid. The concentrated formic acid (Fisher reagent grade 90%) is used without dilution. Procedure. Reconstituted FCOJ or the singlestrength canned grapefruit juice was centrifuged, and the clarified juice diluted with water. For orange juice a dilution of 1 to 100 is necessary in order that the proline content in the test solution falls within the range of linear response of the colorimetric analysis. For grapefruit juice the dilution is made 1 to 50. To 1 ml of the diluted juice in a 13 x 100 mm screw cap culture tube (Corning #9826) is added 1 ml of concn formic acid. After thorough mixing, 2 ml of the ninhydrin reagent are added. The screw caped tube is immersed in a boiling water bath for 15 min and cooled in tap water for 10 min. The absorbance of the sample is determined at 515 nm on a colorimeter or spectrophotometer and the concn calculated from the standard curve of proline. Stock proline standards of 100 mg per liter of water was diluted to a series of working standards containing 0, 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 ^g per ml, which were analyzed in the same manner as described above. A linear absorbance response with concn was obtained within the concn range. The amount of proline in a sample was cal culated from the standard curve. A set of standards was run each time with the samples. Results and Discussion Proline in reconstituted FCOJ. The mean proline content of 100 samples of reconstituted FCOJ (12.8° Brix) was 107.8 mg/100 ml with a range from 60 to 153 mg per 100 ml. A distribution of these samples is shown in Fig. 1. A large number of samples had proline contents above 100 mg/100 ml with a second peak in the distribution centered around 130 mg/100 ml. Variations of proline in orange juice due to maturity and cultivar. An investigation was carried out to determine the seasonal variations of proline in juice of the main cultivars of Florida oranges. The results are shown in Fig. 2. The juices of early and mid-season oranges are low in proline when the fruit is immature or just begins to reach the Florida standards (11). Proline increased rapidly with season. However, when the fruit reach their maturity, the proline content was low compared to late season fruit. 'Hamlin', the early maturing orange cultivar, was found to 144 PROLINE CONTENT mg/ 100ml Fig. 1. Frequency distribution of proline content in 12.8° Brix reconstituted FCOJ. Mean = 107.8 mg/100 ml; standard deviation = 21.5 mg/100 ml; n = 100. be extremely low in proline even after the fruit had attained maturity but continued to increase in proline as the season advanced. 'Pineapple' oranges ripen during January and February and its proline content was considerably higher than 'Hamlin'. With 'Valencia' oranges the proline content fluctuates although it is always high as compared to the earlier maturing cultivars. The fluctuation of proline in the late season fruit probably is caused by the climate during that time of the year when the weather favors vegetative growth of the tree (12, 16). \CKJ \ 100 \ VALENCIA -80 PINEAPPLE I n^* * o o ^.60 £ / 40 / HAMLIN 20 OCT NOV DEC JAN FEB MONTH MAR APR MAY JUN Fig. 2. Seasonal variations of proline content in juices of 3 Florida orange cultivars. (1973-74 season). The mean and range of proline content of Florida canned grapefruit juice. The average proline content of 80 samples of canned grapefruit juice was 39 mg/100 ml, about one-third of that of reconstituted FCOJ, and the coefficient of variation was 37% as compared to about 20% for orange juice. A distribution of the proline values of these samples is shown in Fig. 3. The lowest value for grapefruit juice was 16 mg/100 ml and the highest was 92 mg/100 ml. It is not known whether the seasonal and varietal variation in proline content as found in oranges also occur in grape fruit. Conclusion Due to its wide variations, the use of proline concn of citrus juices as an index of juice content suffers from the Proc. Fla. State Hort. Soc. 92: 1979. most citrus quality control laboratories for the Davis test The ninhydrin reagent will also produce a red color with hydroxyproline and orinthine. No hydroxyproline has ever been reported to occur in citrus, and orinthine is only about 0.5-1.5% of proline as determined in orange juice (2. 6). Literature Cited 15 25 35 45 55 PROLINE CONTENT 65 95 75 mg / 100ml Fig. 3. Frequency distribution of proline content in Florida canned grapefruit juice. Mean = 39.1 mg/100 ml; standard deviation = 14.6 mg/100 ml; n « 80. same shortcomings of many other juice characteristics used for this purpose. However, in the 100 samples of orange juice analyzed, the mean was 107.8 mg/100 ml with a standard deviation of 21.5 mg/100 ml. A confidence at the level of 95% could be expected that the juice should not have a value below the 2 standard deviations of the mean, or 63.8 mg/100 ml for a 12.8° Brix orange juice. The amount must be adjusted for juices reconstituted to lower Brix values. The use of proline content as an index for grapefruit juice will be even less meaningful because of its low values and high standard deviation (14.6 mg/100 ml). Products containing significant amount of juices derived from less mature early and mid-season Florida oranges may have low proline content. With proportioned amount of the late season orange juice added to blend for Brix to acid ratios and for color, the resulting product may reach the minimum values for this amino acid as proposed by Bielig et al. (1) and Koch (5, 6). The simplified method of determining proline could give the quality control laboratories an additional tool for monitoring the characteristics of the juice. The reagents are easy to prepare and stable. The diethyleneglycol used to prepare the ninhydrin reagent is generally available in 1. Bielig, H. J., W. Feathe, J. Koch, S. Wallrauch and K. Wucherpfennig. 1977. Standards and variations of the determined charac teristics for apple, grape and orange juices. Obst Gemuese-Verwert. Ind. 62:209-219, und Fluss. Obst 44:215-225. (In German). 2. Brenoe, C. 1971. Research studies of the distribution of free amino acids and other compounds in orange fruits picked in Greece during the period January to March 1968 and collected during the period January to March 1969. Husholdingsradets Tekniske Meddeleser 11:15-59. 3. Chinard, F. P. 1952. Photometric estimation of proline and orinthine. /. Biol. Chem. 199:91-95. 4. Davis, W. B. 1947. Determination of flavanone in citrus fruits. Anal. Chem. 19:476-478. 5. Koch, J. 1975. About the characteristics of manufactured orange juice as basis for a uniform estimation of the product by means of official food regulations. Fluss. Obst. 42:217-222. (In German). 6. . 1979. The free amino acids in commercial orange juice. Fluss. Obst. 46:212-216. (In German). 7. Moore, S. and W. H. Stein. 1948. Photometric ninhydrin method for use in the chromatography of amino acids. /. Biol. Cheim. 176:367-387. 8. Niedmann, P. D. 1976. A contribution to the quantitative deter mination of free amino acids and ammonia in orange juice. Dtsch. Lebensm. Rundsch. 72:119-126. (In German). 9. Ouch, C. S. 1969. Rapid determination of proline in grapes and wines. /. Food Sci. 34:228-230. 10. Royo Iranzo, J. 1974. Methods for the detection of adulteration in citrus juices. Proceeding International Conference on Quality and Detection of Adulteration in Citrus Juices. Instituto de Agroquimica y Technologia de Alimentos, Valencia, Spain. (In Spanish). 11. State of Florida, 1974 Citrus Fruit Laws. Editorially Revised. Florida Department of Citrus, Lakeland, Florida. 12. Ting, S. V. and J. A. Attaway. 1971. Citrus Fruits. In The Bio chemistry of Fruits and their Products, A. C. Hulme, Editor. Aca demic Press, New York and London. 13. and E. J. Deszyck. 1960. Total amino acid content of chilled orange juice and frozen concentrate. Fla. State Hort. Soc. 73:252-257. 14. Vandercook, C. and E. L. Price. 1972. The application of amino acid composition to the characterization of citrus juices. /. Food Sci. 37:384-386. 15. Wallrauch, S. 1976. Determination of proline in fruit juices, Sig nificance for the estimation. Fluss. Obst 45:430-437. 16. Wedding, W. T. and R. P. Horspool. 1955. Juice composition change during orange development. Calif. Citrog. 40:106-107. Proc. Fla. State Hort. Soc. 92:145-148. 1979. TANGERETIN CONTENT OF FLORIDA CITRUS PEEL AS DETERMINED BY HPLC R. L. ROUSEFF AND S. V. TlNG Florida Department of Citrus, Agriculture Research and Education Center, IFAS, P. a. Box 1088, Lake Alfred, FL 33850 Additional index words, albedo, flavedo, dietary fiber, fiavones. Abstract, Eleven varieties of Florida citrus have been analyzed for peel tangeretin contents using a new HPLC method. Highest tangeretin concentrations are found in the flavedo. Of the varieties tested, the average flavedo con- Proc. Fla. State Hort. Soc. 92: 1979. tributes to only 42.5% of the fresh peel weight, yet it contains 87.5% of the total amount of tangeretin in the peel. Mandarin peel had the greatest tangeretin concentration (183 ppm) while lemon peel had the least (.06 ppm). Orange peel contained, on the average, slightly greater amounts of tangeretin than grapefruit peel. The tangeretin content of any citrus peel based product can be increased or decreased dramatically depending on the variety and the portion of the peel used as a starting material. Tangeretin is one of several methoxylated fiavones found in citrus and has been found in all of the component parts of the Valencia orange (4). It has been reported by Robbins 145