Biochemical and protein quality evaluation of tender pods of wild legume Canavalia cathartica of coastal sand dunes

B Bhagya, K R Sridhar and S Seena

Microbiology and Biotechnology, Department of Biosciences, Mangalore University,
Mangalagangotri, Mangalore 574 199, Karnataka, India
sirikr@yahoo.com

Abstract

The wild legume, Canavalia cathartica is widely distributed on the coastal sand dunes of southwest coast of India. It is one of the major nitrogen fixing sand-binding creeper with high nutritional value. Raw and pressure-cooked tender pods of Canavalia cathartica were evaluated for biochemical composition and protein qualities in comparison with raw and pressure-cooked ripened beans. The pods are rich in protein (18.6-21.7%) and fiber (15.7-17.3%). Potassium, magnesium, zinc and manganese meet the recommended pattern of NRC/NAS for infants. Globulins were the major protein fractions (5.6-6.6%), while starch among the carbohydrates (33.2-49.2%). Sulphur-amino acids were limiting among the essential amino acids. Threonine, valine, isoleucine, leucine, tyrosine + phenylalanine and lysine of pods fulfill the FAO/WHO/UNU requirements for adults. Pods consist of all essential fatty acids (linoleic, linolenic and arachidonic acid).

Trypsin inhibitor activity was absent and pressure-cooking decreased the total phenolics, orthodihydric phenols, tannins and phytohemagglutination activity. On feeding rats with raw and cooked pod diets, the latter showed an increase in protein efficiency ratio (PER), net protein retention (NPR) and protein retention efficiency (PRE), true digestibility (TD), biological value (BV) and net protein utilization (NPU). In vitro starch digestibility doubled on pressure-cooking. This is the first detailed investigation on the nutritional, antinutritional and protein quality evaluation of Canavalia cathartica tender pods of coastal sand dunes. The pods of Canavalia cathartica may meet the protein and energy requirement of rural population and livestock on judicious application of methods to overcome the toxic features.

Key words: amino acids, antinutritional factors, Canavalia cathartica, coastal sand dunes, nutrition, protein quality, tender pods, wild legume

Introduction

Under explored tropical wild legumes are of immense value in human and livestock nutrition. As a large number of farmers are dependent on livestock for their livelihood, animal husbandry plays an important role in the rural economy of developing countries like India. Nutrition remains by far the most critical constraint to increased animal productivity and more efficient performance across the developing countries (ILRI 1995). There is a perpetual gap between the demand and supply of digestible crude protein and total digestible nutrients (Singh et al 1997). One of the means of elevating livestock production is to increase the quality of legume-based pastures. Supplementation of feeds with legumes increased more roughage intake and digestion in sheep (Adu et al 1992). Due to shortage of feed during dry season, animals lose weight, exhibit low fertility and susceptible to diseases and death. Feed supplementation that provides additional protein, minerals and energy during dry season can be achieved inexpensively using native wild legumes. Most of the unconventional legumes are resistant to drought, competing effectively with other species and quickly cover the ground. Germplasm of underutilized legumes with high nutritional potential is a valuable source for the improvement of feed quality and quantity.

One of the potential wild legumes of edible value is Canavalia cathartica Thouars, which is drought-tolerant, grow and widely distributed in coastal sand dunes of the southwest coast of India (Arun et al 1999, 2003, Bhagya et al 2005). It is a wild ancestral form of Canavalia gladiata and distributed throughout the tropical Asia and Africa (Purseglove 1974). In the vicinity of mangroves and coastal sand dunes, naturally grown or cultivated Canavalia cathartica serve as green manure and mulch in agricultural practices (Seena and Sridhar 2006) This legume pasture serves partly as a cattle fodder and the tender pods are consumed by coastal dwellers as famine food (Arun et al 1999, Bhat et al 2005, Seena and Sridhar 2006). The whole twines with pods serves as rabbit-feed in coastal area. Although the dry seeds of Canavalia cathartica possess high proteins, carbohydrates, energy and essential amino acids, their antinutritional factors limits its edibility (Seena and Sridhar 2004, 2006; Seena et al 2006). Pressure-cooked ripened beans of Canavalia cathartica possess better nutritional qualities including improved protein and starch digestibility with relatively less antinutritional features than dry seeds (Bhagya 2006, Bhagya et al 2006). It has larger and heavier pods than Canavalia maritima, another coastal sand dune legume. The tender pods of Canavalia cathartica during post-monsoon season (September) on the sand dunes of southwest coast of India are smooth, green, elongated and less fibrous turning yellow on ripening. There is no information on the nutritional qualities of tender pods of Canavalia cathartica. Thus, the current investigation attempts to evaluate the nutritional features, antinutritional factors, protein quality and starch digestibility of raw and pressure-cooked tender pods of Canavalia cathartica of coastal sand dunes of southwest coast of India in view of its importance to human and livestock.

Materials and methods

Characteristics of pods and processing

Tender pods of Canavalia cathartica were harvested during post-monsoon season (September 2003) from the coastal sand dunes of Someshwara (12º 47¢ N, 74º 52¢ E), Mangalore, southwest coast of India. Fresh and dry weights of tender pods were determined gravimetrically. The dimensions of pods were recorded. Each pod was cut into four pieces and divided into two sets. First set was sun dried and the second set was pressure-cooked in household pressure-cooker (Prestige, ttk product, Bangalore, India) with freshwater (1:3 v/v) and sun dried. Dried raw and cooked pods were ground (Wiley Mill, 30 mesh) and stored in air-tight containers and refrigerated prior to use.

Proximate composition and minerals

Moisture of the pod flours was determined by gravimetric method on drying (100°C) until attaining constant weight. The total nitrogen and crude protein content (N × 6.25) were determined by mciro-Kjeldahl method (Humphries 1956). The crude lipid was estimated on extraction with a Soxhlet extractor using diethyl ether, crude fiber by acid and alkaline digestion method and ash was determined gravimetrically on incineration in a muffle (550°C) (AOAC 1990). The crude carbohydrate content was calculated by difference (Müller and Tobin 1980):

Crude carbohydrates (%) = 100 - [crude protein (%) + crude lipid (%) + crude fiber (%) + ash (%)]

The gross energy estimation was based on Osborne and Voogt (1978):

Gross energy kJ/100g DM = [crude protein (%) × 4] + [crude lipid (%) × 9] + [crude carbohydrates (%) × 4]

Vitamin C was determined according to Roe (1954).

Sodium, potassium and calcium were determined by flame photometry (Systronics, Mediflame 127 Sr. No. 2083, India) (AOAC 1990). Magnesium, iron, copper, zinc, manganese and selenium were estimated by atomic absorption spectrophotometer (GBC 904AA, Germany) (AOAC 1990). The total phosphorus was determined as orthophosphate by ascorbic acid method (APHA 1995).

Protein and carbohydrate fractions

True protein of raw and cooked pod flours was extracted according to Basha et al (1976) with a slight modification. The protein fractions were extracted (1:10 w/v) with different solvents (distilled water; 0.25M NaCl; 70% ethanol and 0.05N NaOH). Extracted proteins were precipitated with 10% trichloroacetic acid (TCA) and estimated protein by micro-Kjeldahl method (nitrogen × 6.25) (Humphries 1956). The non-protein nitrogen was determined by precipitating protein in the flour using TCA (10%) (Sadasivam and Manickam 1992) and the supernatant was estimated for nitrogen by micro-Kjeldahl method (Humphries 1956).

Starch of pod flours was analyzed according to Clegg (1956). Total sugars was determined according to Dubois et al (1951). Reducing sugars were determined based on method outlined by Nelson (1944). Non-reducing sugars was calculated by subtracting reducing sugar from total sugars.

Amino acids and fatty acids

EAA score = [EAA in 100 g test protein (g)] ¸ [EAA in 100 g FAO/WHO/UNU (1985) reference pattern (g)] × 100

Fatty acid methyl esters (FAMEs) were determined according to Garces and Mancha (1993). Polyunsaturated and saturated fatty acid ratio was calculated on dividing sum of saturated fatty acids by sum of polyunsaturated fatty acids.

Antinutritional factors

Total phenols of pod flours were estimated following procedure by Rosset et al (1982) using tannic acid (Merck) as standard. Orthodihydric phenols were determined by the method of Mahadevan (1966) with caeffic acid (Sigma) as standard. Tannins were detected by Vanillin-HCl method (Burns 1971) using catechin as standard. The activity of trypsin was analyzed using the method described by Kakade et al (1974). Hemagglutination activity was analyzed for rabbit erythrocytes as described by Hankins et al (1980).

Protein quality assessment

The experimental protocol has been followed as approved by the ethics committee (Ministry of Social Justice and Empowerment, Government of India no. 25/1/99 - AWD). Weaned male Wistar 21-day-old albino rats with an average weight (30±5 g) were selected for experimental trials. The rats were sorted into four groups each of 5 rats, kept individually in polypropylene metabolic cages receiving water and feed ad libitum. Suitable room temperature (22±1°C), and humidity (50%) were maintained with a fixed light-dark cycle (12 hr).

Standard diet was prepared using casein as reference protein and control diet devoid of protein. The test diets were formulated with raw and cooked pod flours (10% crude protein on dry weight basis). All the experimental diet were prepared, labeled and stored in air-tight containers prior to use. Protein efficiency ratio (PER) and net protein ratio (NPR) were carried out according to the method outlined by Pellet and Young (1980) and performed over 28-day period. Food consumption and body weight of rats were observed at weekly and 10-day interval. The PER, corrected PER, food efficiency ratio (FER) (4 weeks) and NPR (10 days) was calculated:

PER = [Weight gain of the test animal (g)] ÷ [Protein consumed (g)]

Corrected PER = (PER ´ 2.5) ÷ (Determined PER for reference casein)

where, 2.5 as standard value for casein

FER = [Weight gain of the test animal (g)] ÷ [Food consumed (g)]

NPR = [Weight gain of the test animal (g)] + [Weight loss of the protein free test animal (g)] ÷ [Weight of test protein consumed (g)]

The protein retention efficiency (PRE) was calculated according to Bender and Doel (1957):

PRE = NPR ´ 16

Nitrogen balance studies were carried out according to Chick et al (1935). Twenty adult male albino rats (60-68 g) were distributed into four groups in polypropylene metabolic cages. One group of rat was fed a protein-free diet another group with casein diet and the rest with diet containing raw and cooked pod flour diet respectively. Food and water were provided ad libitum. The experiment was carried out for 14 days, nine days for acclimatization and remaining five days as sampling period. On each day, urine and faeces were sampled and pooled separately. The nitrogen of urine and faeces were estimated by micro-Kjeldahl method (AOAC 1990). True digestibility (TD) and biological value (BV) were calculated:

TD = [N_i - (NF₁ - NF₂)] ÷ [N_i] × 100

BV = [N_i - (NF₁ - NF₂)] - [(NU₁ - NU₂)] ÷ [N_i - (NF₁ - NF₂)] × 100

where, N_i, Nitrogen intake of animal fed test diet; NF₁, Nitrogen excreted in faeces of animals fed test diet; NF₂, Nitrogen excreted in faeces of animal fed protein-free diet; NU₁, Nitrogen excreted in urine of animals fed test diet; NU₂, Nitrogen excreted in urine of animals fed protein-free diet.

Net protein utilization (NPU) was calculated according to Platt et al (1961):

NPU = [BV × TD] ÷ 100

The protein digestibility-corrected amino acid score (PDCAAS) of essential amino acids was calculated based on amino acid requirements for adults (FAO/WHO/UNU 1985):

PDCAAS (%) = [EAA in 100 g test protein (g)] ¸ [EAA in 100 g FAO/WHO/UNU (1985) reference pattern (g)] × D

where, D is the in vivo protein digestibility (%)

Starch digestibility

The in vitro starch digestibility was estimated based on Beutler (1984). Defatted test flour (100 mg) was incubated (37ºC, 3 hr) with diastase (1300 α-amylase units/g) (Hi-Media, Mumbai, India) (2 mg/12.5 ml 0.02 M potassium phosphate buffer, pH 7.0) followed by inactivation with NaOH (0.5 N, 1 ml). Zero-time control was maintained by inactivating the enzyme before addition of substrate. The inactivated reaction mixtures were centrifuged and supernatants were made up to 10 ml with distilled water. Reducing sugar liberated by the enzyme was estimated by Nelson's (1944) method using maltose (0-100 µg) as standard.

Statistical analysis

The difference in pod features of Canavalia cathartica and Canavalia maritima, raw vs. cooked pod or raw bean, cooked pod vs. cooked bean for proximate composition, minerals, protein and carbohydrate fractions, total phenols, tannins and orthodihydric phenols was assessed by paired t-test (Stat Soft Inc 1995). The paired t-test was also employed to ascertain the difference between casein vs. raw or cooked pod diet and between raw and cooked pod.

Results and discussion

Tender pods of Canavalia cathartica possess significantly (P<0.05) higher fresh weight, dry weight, length, width and thickness than Canavalia maritima (Table 1) and qualify its suitability as green vegetable or livestock food.

Proximate features and mineral constituents

Proximate composition of raw and cooked tender pods has been compared with ripened beans of Canavalia cathartica in Table 2.

Moisture of raw pods was higher than raw beans (11.2 vs. 8.9%), while it was reverse in cooked pods and beans (5.8 vs. 6.4%). The crude protein of pods was lower than ripened beans (18.6-21.7 vs. 30.1-33.4%) as protein concentrate more on seed maturity. Protein of raw and cooked pods of Canavalia cathartica surpassed or was within the range of seeds of many wild legumes (Atylosia scarbaeoides, 17.3%; Erythrina indica, 21.5%; Neonotonia wightii, 15.1%; Rhynchosia filipes, 16.9%; Tamarindus indica, 14%) (Arinathan et al 2003) and edible legumes (Cajanus cajan, 19.4%; Cicer arietinum, 20.7%; Vigna trilobata, 20.2%, and V. unguiculata, 15.9%) (Arinathan et al 2003, Jambunathan and Singh 1980, Nwokolo 1987) qualify its use as protein food. Generally, legumes are low in fat except for soybean and groundnut. Crude lipid of pods was lower than ripened beans (0.98-1.08 vs. 1.6-1.7%), so also seeds of many edible as well as wild legumes including Canavalia spp. (Seena et al 2006). Crude fiber is quite high in pods (15.7-17.3%) than ripened beans (10.3-10.4%) and dry seeds of Canavalia spp. (2.4-12.8%) (Seena et al 2005, 2006) and on par with dry seeds of Canavalia maritima of Central America (17.3%) (Bressani et al 1987). Crude fiber in diet is known to enhance the digestibility, low levels traps less proteins and carbohydrates (Balogun and Fetuga 1986), but high levels cause low digestibility and decrease nutrient utilization (Oyengu and Fetuga 1975). The high fiber in Canavalia cathartica pods can be brought down to the recommended levels on addition of fiber-poor flours (e.g. corn, wheat, rice). The ash of the pod was higher than ripened beans (3.2-3.8 vs. 3-3.3%), but on par or less than dry seeds of other Canavalia spp. (Seena et al 2005, 2006). Low ash in pods than ripened beans is due to low minerals (see Table 4). The crude carbohydrates were higher in cooked pods than raw pods (56.1-61.6%), so also ripened beans (51.5-55%) and dry seeds of Canavalia spp. (Seena et al 2005) qualify for animal feed. The calorific value of pods was lower than ripened beans (1343-1380 vs. 1482-1483 kJ/100g), so also to dry seeds of other Canavalia spp. (Seena et al 2005, 2006). The vitamin C was significantly higher than ripened beans (0.28-0.43 vs. 0.08-0.23 mg/100g).

Table 3 shows the mineral constituents of Canavalia cathartica pods in comparison with ripened beans.

All the minerals tested drained on cooking in pods as seen in ripened beans. Sodium, calcium, phosphorus and selenium were lower in pods than ripened beans, while it was vice-versa in iron, copper, zinc and manganese. Potassium was higher in raw beans than raw pods, while it was opposite in cooked beans and cooked pods. However, the overall mineral constituents of pods are on par with or surpassed the dry seeds of Canavalia gladiata (Seena et al 2005) and mangrove Canavalia cathartica (Seena et al 2006). The low sodium (32-51 mg/100g) in pods helps to elevate blood pressure of hypertensive patients. Magnesium was higher in raw pods than beans, while it was reverse between cooked pods and beans. Iron, selenium, zinc and manganese are antioxidants (Talwar et al 1989), which strengthens the immune system. Potassium, magnesium, zinc and manganese met the recommended pattern of NRC/NAS (1989) for infants.

Protein and carbohydrate fractions

Globulins and albumins constitute the major fractions of true protein in pods as seen in ripened beans, but in lower concentration than ripened beans (Table 4) and dry seeds of other Canavalia spp. (Seena et al 2005, 2006).

However, the true protein in raw beans exceeded winged bean (15.2%) Albumins, globulins and prolamins are lower than dry seeds of Canavalia maritima (Seena et al 2005). Albumins are known to be rich in sulphur-amino acids and other EAA (Baudoin and Maquet 1999). As albumin concentration is less in pods, it resulted in low sulphur-amino acids (see Table 5).