486 FLORIDA STATE HORTICULTURAL SOCIETY, 1975

6. Dougherty, R. H. and Koburger, J. A. 1972. Preparation 9. Krishnamurthy, S., Patwardham, M. V. and Subra- and storage of pasteurized-refrigerated mango fruit. Proc. manyam, H. 1971. Biochemical changes during ripening of Fla. State Hort. Soc. 85:190. the mango fruit. Phytochemistry 10(11) :2577. 7. Goldweber, S. 1967. Thoughts on the Florida mango 10. Mustard, M. J. 1948. Preservation of mangos by industry. Proc. Fla. State Hort. Soc. 80:384. freezing. Fla. Mango Forum Proc. 1948:27. 8. Harris, H. 1963. Pasteurized refrigerated peach prod 11. Thomas, P. 1975. Effectof post-harvest temperature ucts. Alabama Agric. Exp. Sta. Highlights of Agri. Res. on quality, carotenoids and ascorbic acid content of Alphonso 10(2) :11. mangos on ripening. J. Food Set. 40:704.

LEAVES FOR FOOD: AND CONTENTS OF FROM 23 TROPICAL AND SUBTROPICAL PLANTS

Nancy T. Hall, Steven Nagy could provide the advantages of exploiting the and Robert E. Berry year-round availability of high amounts of sun light and high yield of leaves due to multiple U.S. Citrus and Subtropical Products Laboratory1 cropping. Winter Haven Kohler and Bickoff (18), contributors to the Third International Congress of Food Science and Abstract Leaves are a potential source of low Technology (1970), recognized the research of cost protein. By dry weight, leaves of 23 plant Osborne and Wakeman (25) as one of the earliest types contained protein from 6 to 41%, of which in this field. Other early protein investigations 14 contained 20% or more. Notable were castor have been covered in a review by Tilley and Ray bean (Ricinus communis) 41%, balsam pear mond (30) and more recent work by Pirie (26,27), (Momordica charantia) 33%, cowpea (Vigna Akeson and Stahmann (1), and Oelshlegel et al. sinensis) 32%, and (Manihot esculenta) (24) have contributed considerably to knowledge 32%. The leaves had large quantities of the es in this emerging field. The nutritional values of sential amino acids lysine, leucine and isoleucine, leaf proteins have been investigated by Water- moderate amounts of valine, threonine and phen- low (32), Duckworth and Woodham (11), Gerloff ylalanine, and minor amounts of methionine and et al. (15) and Subba Rau et al. (29) while leaf tryptophan. Many nonessential amino acids were amino acid compositions have been researched by found in moderate quantities. Tyrosine and his- Chibnall et al. (10), Gerloff et al. (15) and Byers tidine were low, and cysteine and cystine were (9). detected at levels that were less than 1% of the Through extensive research leaf protein con total amino acids recovered. Leaves were es centrates (LPC) have become a reality. They are sentially similar in their amino acid compositions, currently used as an animal feed supplement and although several cultivars showed notable varia being tested for human consumption. Amino acid tions in methionine. compositions of many LPC's have been found to be as beneficial as soybean meal, and as digestible Because of increasing world population and and nutritious as milk. Waterlow (32) found that food scarcity the development of new protein when leaf proteins were combined with milk pro sources has been a high priority research goal teins in the diets of children suffering from pro for the past decade. Coordinated by N. W. Pirie tein malnutrition, weight gains were equivalent of Rothamsted, England, under the International to those of similarly afflicted children on milk at Biological Program (28), the development and use equal protein levels. Other investigations con of leaf protein have been actively pursued. Protein firmed that leaf proteins could provide essential from leaves that can be grown in tropical areas amino acids to supplement those already in the normal diet. LPC's have been prepared from lOne of the laboratories of the Southern Region, U. S. Department of Agriculture, Agricultural Research Service. (19,29), soybean, cowpea and peanut (5), water - The authors thank Dr. Franklin W. Martin and staff, of the Mayaguez Institute of Tropical Agriculture, Mayaguez, hyacinth (23), chenopodium, marrow, corn, nas Puerto Rico, for the samples ofdried leaves used in this turtium, red clover, rye grass, sanfoin, turnip and study, o Mention of brand names is for identification only and does wheat (15), ramie, swamp cabbage and brassica not imply endorsement by the U. S. Department of Agricul (8,14). Martin et al. (21) initiated a study screen- ture. HALL ET AL: LEAVES FOR FOOD 487 ing leaf species with characteristics ideal for use as protein sources in the tropics. The current study was made to determine the protein and amino acid contents of 23 types of tropical and subtropical leaves which were in r ' J J h 3 - .165 cluded in the study by Martin et al. (21). Leaves <" 32 5 - 2 107

p 31.8 i having greater than an arbitrarily selected level vine 1.8 .182 of 20% protein were subjected to acidic and basic p chrub 31.6 1 .5 .107 hydrolyses and their amino acid compositions were A herb 27-5 - .3 .2U3 determined by semiquantitative thin-layer chroma- P vine 2«i.l» - .0 .162 tree 23-9 - .3 .128 tography (TLC). This method of amino acid P vine 23.3 - .2 .152 analysis was chosen because it is fast, inexpensive P shrub 23.3 - .081 and requires minimal equipment. This information P shrub 22.U i .7 .091 on protein content and amino acid composition A herb 21.6 i .3 .109 should be helpful in the nutritional assessment of P herbaceous 20.9 - .6 .133 these leaf cultivars as future sources of food. P tree 20.li - .1 .12U

P shrub 19.5 - .2 .076 Materials and Methods P shrub 17.5 i .2 N.D.'

Protein Determinations P herb 16.3 i .It N.D.

P tree 15.5 i .2 N.D. Leaves, picked at maturity stages known to be P tree 1U.5 - .3 N.D. safe for animal and human consumption, were P herb 13.0 i .2 N.D. dried at 58°C. Within 24 hr of sampling for micro P tree 11. b - .2 N.D. Kjeldahl nitrogen (2) and total free amino acids P tree 10.8 - .2 N.D. determinations (4), the leaves were redried in P tree 7.5 - .!» N.D. vacuo at 95-100°C for 5 hr. The catalyst in the P tree 5.9 i .2 N.D. micro Kjeldahl total nitrogen method was modified by using copper sulfate instead of mercuric sul- fate (6). The mean crude protein value was ob tained from 2 to 5 Kjeldahl nitrogen determina > acid per gin of dry lei tions on 100 mg samples: mg nitrogen/100 mg leaves times 6.0 (21). Amino acid mixtures (standards and leaf Amino Acid Determinations hydrolysates) were separated by two-dimensional TLC on precoated 250 fi silica gel GF plates For free amino acid determination by formal (Analtech, Inc., Wilmington, Del.). Separation titration, three 10-ml aliquots were taken from a was effected in the first dimension with solvent I 200 ml aqueous leaf homogenate prepared by dis (CHCl3-Me0H-17% NH40H, 2:2:1, v/v/v); the integrating 5 g of dried leaves in a Waring plates were dried for ca. 1 hr in a forced air-draft blendor. Total free amino acids were reported hood, turned 90°, and separated in the second di (Table 1) as milliequivalents per gram dry leaves. mension with solvent II (phenol -H2O, 75:25, Three to 6 samples of the 14 species having w/w). The plates were dried for an hour and 20% or more protein were subjected to both acid sprayed with ninhydrin cupric nitrate indicator. (6N HC1, 22 hr, 110°) and basic [250 mg (Ba Amino acids were identified by their relative Rf (OH)2-8H2O) dissolved in 5 ml H2O, 24 hr, values and by their specific reaction to the poly 128°] " hydrolysis (7,20). Following ~ hydrolysis chromatic ninhydrin indicator (22). The analyses amino acids were extracted with H2O, CHC13 and of the amino acids was semiquantitative. A stand CH3OH (7:8.7:17.3 v/v/v). The CH3OH-H2O ard curve was prepared for each amino acid re layer was passed through an Amberlite CG-120 lating area of chromatographic spot with weight cation exchange column (Rohm and Haas, Phila of the amino acid. Aliquots (0.5, 1, 2 and 4 fil) of delphia, Pa.) which retained the amino acids (16). standard amino acid mixtures were spotted on 4 These were eluted with IN NH40H, and the efflu plates and the area of each amino acid spot was ent concentrated to dryness on a rotoevaporator. determined from photocopies of the plates by The dried amino acid sample was dissolved in 0.1 planimeter tracing. Weights were determined by N HC1 and refrigerated until analyzed by TLC. the formula (31): VA = m log wt + C. Aliquots 488 FLORIDA STATE HORTICULTURAL SOCIETY, 1975

(0.5, 1, 2, 4 ittl) of the acid hydrolyzates of each of the 14 samples were spotted and the correspond ing weights of each amino acid were obtained v Solvent I from the standard curves. Weights for all the amino acids were totaled and the relative weight percent calculated, e.g., wt specific amino acid/ 12 total amino acid wt x 100. Values were tabulated (Table 2) and represent the mean of 3 to 6 de terminations. The coefficient of variation (CV) was determined for several mean ranges (MR) with the following results: MR 1-3, CV 25-65%; MR 3-8, CV 10-40%; MR 8-17, CV 5-20%. 3o 6 x°y

Results and Discussion

The percentages of protein calculated for the ,O2O 23 leaf samples ranged from 6 to 41% (Table 1). Solvent U Protein content, based on Kjeldahl values, include Ori gin protein nitrogen, free amino acid nitrogen, and nonproteinaceous nitrogen. The free amino acid contents were noticeably low and ranged from .076 Fig. 1. Thin-layer chromatographic separation of amino to .243 meq/g dry leaves. The highest protein acids of coffee leaves after acid and basic (dotted areas) hydrolysis: (1) lysine, (2) arginine, (3) aspartic acid, (4) content was found in Ricinus communis, the com serine, (5) glycine, (6) glutamic acid, (7) proline, (8) histi- mon castor bean. Of the remaining 13 species whose dine, (9) threonine, (10) alanine, (11) valine, (12) methio- nine, (13) tyrosine, (14) leucine and isoleucine, (15) phen leaves contained 20% or more protein, 6 of these, ylalanine, (16) tryptophan, (17) oxidative breakdown prod ucts of cysteine, (x) ?-aminobutryic acid and (y) tentatively viz., cowpea, cassava, chayote, pigeon pea, banana identified as hydroxyproline. and coffee, are currently grown for other food uses. Thus, processing their leaves for protein could leaves examined. Methionine values ranged from provide an additional product or by-product. 1% in castor bean, Ceylon spinach and pigeon Fig 1 shows a typical TLC amino acid profile pea to 4% in cassava. This amino acid has often resulting from hydrolysis of coffee leaves. Acid been noted as a limiting factor in many proteins hydrolysis resulted in nearly complete decomposi of different origin. tion of tryptophan. Basic hydrolysis, however, In comparing the essential leaf amino acids to liberated tryptophan intact but caused partial de other amino acid sources, the percentages of lysine, composition of serine, threonine, arginine, and threonine, valine, phenylalanine and leucine- cystine. Both acid and basic hydrolyses caused isoleucine equaled or exceeded those in the FAO some oxidative degradation of cystine and cysteine. reference protein (13). Methionine content was For semiquantitation of leaf amino acids, only lower than the FAO percentage in only 3 of the acid-hydrolyzed mixtures were used. To confirm leaves; Ceylon spinach, pigeon pea and castor the presence of tryptophan and to approximate bean. Tryptophan was not definitively quantitated its percentage, basic hydrolysis was employed. but probably is at least equal to the 1.4% in the Table 2 lists the proximate weight percentage FAO standard. With the exception of banana and distribution of essential and nonessential amino coffee leaves, the nonessential amino acids, histi- acids in the 14 types of leaves with highest pro dine, glycine, alanine, serine, proline and arginine tein content. Although tryptophan could not be were similar to the average reported for these quantitated in the acid hydrolyzed mixture, rela amino acids in leaf protein concentrates (15). In tive area comparisons of valine and phenylalanine approximately one-third of the leaves, percentages to tryptophan from a basic hydrolyzed mixture of aspartic and glutamic acids were ca. 1 to 2 per resulted in an estimate of 1-3% for tryptophan. centage points lower than values reported by Ger- Valine and phenylalanine TLC standard curves loff et al. (15) for LPC. These lower values may were essentially similar to the standard curve of be due to greater variation in the TLC technique tryptophan. Leucine and isoleucine were unresolved than in the ion-exchange method of Gerloff et al. by TLC and were reported as an entity. Lysine and Banana leaves showed the lowest combined total threonine values were similar for all types of of aspartic and glutamic acids. For cowpea, cassa- HALL ET AL: LEAVES FOR FOOD 489

?varf

1 c 15 15 15 I.yr.iiie 7 7 7 C 7 7 ft 7 7 0 7 7 Valino M (' 8 C 6 7 o 7 7 8 6 8 Thvoonine € n C 7 j, 6 6 £ Thenyl-a-nine r 6 6 6 5 5 5 Methionine ",, 2 ■5. p 3 d'- t/" D- D1" E* DW

3 3 3 Olutamic acid 10 11 10 ^ 11 10 10 7 12 10 6 JJyeine " 7 7 ? 5 6 ' G 7 8 7 7 Aspartic acid 8 10 10 9 9 1 * 10 8 9 9 9 11 9 Alrarlnv: f, r. 5 7 b 6 7 7 7 6 7 oeri »■;•.'.• ('• r. 6 6 f. fi 5 c )i 5 It 6 PvnliiiO r> 6 (•" ? 5 , c 5 )i 5 6 •Vrrlr.irie " 7 7 c' 7 7 7 7 6 5 iyvosinc <■■ 2 2 2 3 2 • 2 4 3 k 3 3 Cy3tei:ie?ra:id

cyst i no <__ <1 <1 <1 <"- <1

"A.'\ i:."> aoias ex? r-.-cscd as percent ty veipht of tot-:! n::.ir.o aMir: >••?_• eh G-'r.ir.C! acid pcrccr.t.i^o vni ue represer.4:: the i:.'.:-ar. f-r* '—' '":•:-•:•:•••!:.!!".

"Lourtoe and iroleucino uin-.^lY-a ry TI.C; expressed as cor.l.?r.-:-d 4 o' • WTryrihni!}i:.«i detected by ha^ic hydrolysis mvI ert U-ini-.cd as 1-3^.

va, mulberry and pigeon pea, the percentage of to foods have been extraction, palatability, unde tyrosine (2%) was ca. 1 percentage point lower sirable color due to chlorophyll, biological un than the average reported by Gerloff et al. availability of amino acids, and the occasional Any leaf crop with high protein and significant presence of toxic substances. Many of these prob amounts of most essential amino acids should be lems have been partially solved by new processes strongly considered as a source of LPC or as a such as the Pro-Xan method (17), separation of supplementary food, or both, especially if the crop LPC into chloroplastic and cytoplasmic (white or can be widely grown in tropical and subtropical colorless) fractions, and heat destruction of regions. The leguminous plants, cowpea and pigeon poisonous cyanides (12). Through improved pro pea, already widely grown for their high protein cessing techniques, increased research efforts, and seeds, should be considered as a source of supple continued studies on plant hybridization, many of mentary amino acids because of their high leaf the problems that currently prevent the wide protein content and high lysine, which is common spread use of leaf proteins may be overcome. ly limiting in diets based on cereals such as rice, Solution of these problems may be the next decade's maize and wheat (5). The nutritional quality of greatest contribution to the world's starving and cassava leaves, which are high in lysine and malnourished peoples. The study of leaf amino methionine (6 and 4%, respectively) would en acid balance and protein content is a beginning. hance a diet of cassava root (3), a staple food in the tropics of America, Africa, and Asia. Literature Cited Purslane and Ceylon spinach leaves are currently 1. Akeson, W. R. and M. A. Stahmann. 1966. Leaf pro tein concentrates: a comparison of protein production per used as greens in some areas. These crops should acre of forage with that from seed and animal crops. Econ. be encouraged to expand because of their high Bot. 20:244-250. 2. American Public Health Association. 1965. Standard leaf-protein levels. Methods for the Examination of Water and Waste Water. New York. 402-404 p. The castor bean, grown in India for castor oil 3. Anon. 1972. Amino Acid Content of Foods. Food and production, has a high leaf-protein content but Agriculture Organization of the United Nations. Rome, Italy. 46-47 p. has not been processed because, although the im 4. Association of Official Agricultural Chemists. 1965. Of ficial Methods of Analysis. W. Horowitz (ed.), Washington, mature leaves are safe to consume, a poisonous D. C. 324 p. alkaloid combined with the protein, ricin, forms in 5. Betscart, A. A. and J. E. Kinsella. 1974. Influence of storage on composition, amino acid content and solubility of the mature leaf (21). Research to solve this prob soybean leaf protein concentrate. J. Agric. Food Chem 22* lem through improved processing techniques or 116-122. 6. Blanded, W. J. and V. W. McLoche (eds.). 1963. Ele plant hybridization could provide access to a new mentary Quantitative Analysis—Theory and Practice. Harper and Row, New York. 379 p. and potentially valuable protein source. 7. Brenner, M., A. Niederwieser and G. Pataki. 1969. Other problems encountered in processing leaves Amino acids and derivatives. Thin-Layer Chromatography E. Stahl (ed.). Springer-Verlag, New York. 730-785 p. 490 FLORIDA STATE HORTICULTURAL SOCIETY, 1975

8. Brown, H. E., E. R. Stein and G. Saldana. 1975. Evalu and nonprotein amino acids in citrus leaves as affected by ation of Brassica carinata as a source of plant protein. J. sample preparation and species differences. Am. Soc. Hortic. Agric. Food Chem. 23:545-547. Sci. 96:514-518. 9. Byers, M. 1971. Amino acid composition and in vitro 21. Martin, F. W., L. Telek and R. Ruberte'. 1975. Some digestibility of some protein fractions from three species of tropical leaves as feasible sources of protein. In review. leaves of various ages. J. Sci. Food Agric. 22:242-251. 22. Moffat, E. D. and R. I. Lytle. 1959. Polychromatic 10. Chibnall, A. C, M. W. Rees and J. W. H. Lugg. 1963 techniques for the identification of amino acids on paper The amino acid composition of leaf proteins. J. Sci. Food chromatograms. Anal. Chem. 31:926-928. 23. Niyogy, S- C. and J. J. Ghosh. 1975. Biochemical and ^U.* Duckworth, J. and A. A. Woodham. 1961. Leaf protein nutritional studies on leaf proteins. Final Tech. Report., concentrates I. Effect of source of raw material and methods Dept. of Applied Science, Calcutta, University, Calcutta, In of drying on protein value for chicks and rats. J. Sci. Food dia. 24. Oelshlegel, F. J., Jr., J. R. Schroeder and M. A. Stah Agric. 12:5-15. 12. Eggum, B. O. 1970. The protein quality of cassava mann. 1969. Potential for protein concentrates from alfalfa leaves. Br. J. Nutr. 24:761. and waste green plant material. J. Agric. Food Chem. 17: 13. Food and Agriculture Organization. 1965. Nutrition 791-795. Meetings Report, Series No. 37. Food and Agriculture Organi 25. Osborne, T. B. and A. J. Wakeman. 1920. The pro zation of the United Nations. Rome, Italy. teins of green leaves. J. Biol. Chem. 42:1-26. 14. Garcha, J. S., B. L. Kawatra and D. S. Wagle. 1970. 26. Pirie, N. W. 1969. The production and use of leaf Evaluation of different leaf protein concentrates for some protein. Proc. Nutr. Soc. 28:85-91. essential amino acids Curr. Sci. 39(12) :269-270. 27. . 1969. The present position of research on 15. Gerloff, E. D., I. H. Lima and M. A. Stahmann. 1965. the use of leaf protein as a human food. PL Fds. Hum. Nutr. Amino acid composition of leaf protein concentrates. Agric. 1:237-246. 28. . 1971. Leaf Protein: Its Agronomy, Prep 16 Kaiser, F. E., C. W. Gehrke, R. W. Zumwalt and K. C. aration and Use. International Biological Program Handbook Kuo (eds.). 1974. Amino Acid Analysis. Analytical Bio 20, Blackwell Scientific Publications, Oxford, England. 192 p. chemistry Laboratories, Inc. Columbia, Missouri. 48 p. 29. Subba Rau, B. H., S. Mahadeviah and N. Singh. 1969. 17. Kohler, G. O. 1974. Wet processing of alfalfa. 12th Nutritional studies on whole extract coagulated leaf protein Tech. Alfalfa Conference, Proc, Overland Park, Kansas. and fractionated chloroplastic and cytoplasmic proteins from lucerne (Medicago sativa). J. Sci. Food Agric. 20:355. 65-66 p. 18. , and E. M. Bickoff. 1971. Leaf Protein. 30. Tilley, J. M. A. and W. F. Raymond. 1957. The ex Proc SOS/70 Third International Congress Food Science and traction and utilization of leaf protein. Herb. Abst. 27:235- Technology. Institute of Food Technology, Washington, D. C. 245. 31. Truter, E. V. 1963. Thin Film Chromatography. John 290-295 p. 19. ., and D. deFremery. 1974. Green Wiley and Sons, Inc., New York. 112-118 p. leaves—a potential new source of protein for human nutri 32. Waterlow, J. C. 1962. The absorption and retention tion. Protein Symposium at University of California. Davis, of nitrogen from leaf protein by infants recovering from malnutrition. Br. J. Nutr. 16:531. 20. Labanauskas, C. K. and M. F. Handy. 1971. Protein

SUSCEPTIBILITY OF WEST INDIAN AVOCADOS TO CHILLING INJURY AS RELATED TO RAPID COOLING WITH LOW TEMPERATURE AIR OR WATER1

J. J. GAFFNEY subject to chilling injury when stored at tempera tures lower than 55 °F. The use of low tempera USD A, Agricultural Research Service tures for the cooling medium can greatly reduce Gainesville the time required for cooling. Such practice, how ever, raises the question of whether the fruit C. D. Baird might suffer from chilling injury when cooled in IF AS, Agricultural Engineering Department this way. Studies were conducted, over a period Gainesville of three seasons, on the rapid cooling of 12 West Indian varieties of avocados to determine whether Abstract Removal of field heat from avocados this manner of cooling would cause chilling in before shipment reduces decay and retards the jury to the fruit. Air temps were as low as ripening process, thus extending market life. Some 17 °F, and water temp used were as low as 33°F. avocados, expecially West Indian varieties, are Fruit were cooled to mass-average temps that gen erally ranged from 40° to 50 °F but was as low as

lFlorida Agricultural Experiment Stations Journal Series 33 °F in one test, and were then allowed to ripen No 7079. Appreciation is expressed to Dr. Charles Barmore, at 70°F. No definite evidence of chilling injury AREC, Lake Alfred, Dr. James Soule, Department of Fruit Crops, University of Florida, and to Dr. Donald Spalding, was found in any of the samples tested. ARS USDA, Miami, Florida, for assisting in the chilling injury evaluations involved in this investigation. Grateful acknowledgement is given to J. R. Brooks and Son, Inc., Homestead, Florida for providing fruit samples and to the Removal of field heat from avocades before Florida Avocado Administrative Committee, Homestead, for shipment is desirable in order to lower the respira- providing financial assistance.