Leaves for Food: Protein and Amino Acid Contents of Leaves from 23 Tropical and Subtropical Plants

Leaves are a potential source of low cost protein. By dry weight, leaves of 23 plant types contained protein from 6 to 41%, of which 14 contained 20% or more. Notable were castor bean (Ricinus communis) 41%, balsam pear (Momordica charantia) 33%, cowpea (Vigna sinensis) 32%, and cassava (Manihot esculenta) 32%. The leaves had large quantities of the es sential amino acids lysine, leucine and isoleucine, moderate amounts of valine, threonine and phenylalanine, and minor amounts of methionine and tryptophan. Many nonessential amino acids were found in moderate quantities. Tyrosine and histidine were low, and cysteine and cystine were detected at levels that were less than 1% of the total amino acids recovered. Leaves were es sentially similar in their amino acid compositions, although several cultivars showed notable varia tions in methionine. Because of increasing world population and food scarcity the development of new protein sources has been a high priority research goal for the past decade. Coordinated by N. W. Pirie of Rothamsted, England, under the International Biological Program (28), the development and use of leaf protein have been actively pursued. Protein from leaves that can be grown in tropical areas lOne of the laboratories of the Southern Region, U. S. Department of Agriculture, Agricultural Research Service. The authors thank Dr. Franklin W. Martin and staff, of the Mayaguez Institute of Tropical Agriculture, Mayaguez, Puerto Rico, for the samples of dried leaves used in this study, o Mention of brand names is for identification only and does not imply endorsement by the U. S. Department of Agricul ture. could provide the advantages of exploiting the year-round availability of high amounts of sun light and high yield of leaves due to multiple cropping. Kohler and Bickoff (18), contributors to the Third International Congress of Food Science and Technology (1970), recognized the research of Osborne and Wakeman (25) as one of the earliest in this field. Other early leaf protein investigations have been covered in a review by Tilley and Ray mond (30) and more recent work by Pirie (26,27), Akeson and Stahmann (1), and Oelshlegel et al. (24) have contributed considerably to knowledge in this emerging field. The nutritional values of leaf proteins have been investigated by Waterlow (32), Duckworth and Woodham (11), Gerloff et al. (15) and Subba Rau et al. (29) while leaf amino acid compositions have been researched by Chibnall et al. (10), Gerloff et al. (15) and Byers (9). Through extensive research leaf protein con centrates (LPC) have become a reality. They are currently used as an animal feed supplement and being tested for human consumption. Amino acid compositions of many LPC's have been found to be as beneficial as soybean meal, and as digestible and nutritious as milk. Waterlow (32) found that when leaf proteins were combined with milk pro teins in the diets of children suffering from pro tein malnutrition, weight gains were equivalent to those of similarly afflicted children on milk at equal protein levels. Other investigations con firmed that leaf proteins could provide essential amino acids to supplement those already in the normal diet. LPC's have been prepared from alfalfa (19,29), soybean, cowpea and peanut (5), water hyacinth (23), chenopodium, marrow, corn, nas turtium, red clover, rye grass, sanfoin, turnip and wheat (15), ramie, swamp cabbage and brassica (8,14). Martin et al. (21) initiated a study screenHALL 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 cluded in the study by Martin et al. (21). Leaves having greater than an arbitrarily selected level of 20% protein were subjected to acidic and basic hydrolyses and their amino acid compositions were determined by semiquantitative thin-layer chromatography (TLC). This method of amino acid analysis was chosen because it is fast, inexpensive and requires minimal equipment. This information on protein content and amino acid composition should be helpful in the nutritional assessment of these leaf cultivars as future sources of food. Materials and Methods Protein Determinations Leaves, picked at maturity stages known to be safe for animal and human consumption, were dried at 58°C. Within 24 hr of sampling for micro Kjeldahl nitrogen (2) and total free amino acids determinations (4), the leaves were redried in vacuo at 95-100°C for 5 hr. The catalyst in the micro Kjeldahl total nitrogen method was modified by using copper sulfate instead of mercuric sulfate (6). The mean crude protein value was ob tained from 2 to 5 Kjeldahl nitrogen determina tions on 100 mg samples: mg nitrogen/100 mg leaves times 6.0 (21). Amino Acid Determinations For free amino acid determination by formal titration, three 10-ml aliquots were taken from a 200 ml aqueous leaf homogenate prepared by dis integrating 5 g of dried leaves in a Waring blendor. Total free amino acids were reported (Table 1) as milliequivalents per gram dry leaves. Three to 6 samples of the 14 species having 20% or more protein were subjected to both acid (6N HC1, 22 hr, 110°) and basic [250 mg (Ba (OH)2-8H2O) dissolved in 5 ml H2O, 24 hr, 128°] " hydrolysis (7,20). Following ~ hydrolysis amino acids were extracted with H2O, CHC13 and CH3OH (7:8.7:17.3 v/v/v). The CH3OH-H2O layer was passed through an Amberlite CG-120 cation exchange column (Rohm and Haas, Phila delphia, Pa.) which retained the amino acids (16). These were eluted with IN NH40H, and the efflu ent concentrated to dryness on a rotoevaporator. The dried amino acid sample was dissolved in 0.1 N HC1 and refrigerated until analyzed by TLC. r