Livestock Research for Rural Development 18 (7) 2006 Guidelines to authors LRRD News

Citation of this paper

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

Amino acids were determined according to Hofmann et al (1997, 2003). Derivatization was done by esterification with trifluoroacetylation (Brand et al 1994). The amino acids are expressed as g/100 g of protein. The essential amino acid (EAA) score was determined by employing following formula:

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 = [Ni - (NF1 - NF2)] ÷ [Ni] × 100

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

where, Ni, 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.


Table 1. Characteristics features of tender pods of Canavalia cathartica compared with Canavalia maritima of coastal sand dunes (n=20; meanSD)

Pod feature

Canavalia+- cathartica

Canavalia maritima*

Fresh weight, g/pod

8.71.51a

6.32.65b

Dry weight, g/pod

2.20.27 a

1.50.61b

Length, cm

8.31.76 a

6.71.41b

Width, cm

2.60.38 a

1.70.21b

Thickness, cm

2.30.35 a

1.20.28b

*Bhagya (2006);  Figures across the column with different letters are significantly different (p<0.05, paired t-test)


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.


Table 2.  Proximate composition of tender pods and ripened beans of Canavalia cathartica on dry weight basis (n=5; meanSD)

Component

Tender pods

Ripened beans*

Raw

Cooked

Raw

Cooked

Moisture, %

11.20.45a

5.80.72bd

8.920.85c

6.440.48d

Crude protein, g/100g

21.73.12a

18.50.73bd

33.43.47c

30.11e

Crude lipid, g/100 g

1.080.08a

0.980.08ad

1.740.11c

1.560.15e

Crude fiber, g /100 g

17.30.14a

15.60.43bd

10.30.46c

10.40.83e

Ash, g/100 g

3.820.08a

3.180.08bd

3.340.27c

3.020.08e

Crude carbohydrates, g/100 g

56.13.21a

61.60.93bd

51.23.26c

54.91.04e

Energy value, kJ/100 g

13431.96a

13807.76bd

14827.15c

1483 15.04e

Vitamin C, mg/100 g

0.4330.02a

0.280.05bd

0.230.03c

0.080.01e

*Bhagya et al (2006)
Figures across the column with different letters are significantly different (p<0.05, paired t-test)


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.


Table 3.   Mineral compositions of tender pods and ripened beans of Canavalia cathartica on dry weight basis (mg/100 g) (n=5; meanSD)

Minerals

Tender pods

Ripened beans*

NRC/NAS pattern

for infants**

Raw

Cooked

Raw

Cooked

Sodium

50.612.3a

31.72.85bd

60.835.03c

32.14.89d

120-200

Potassium

1039136a

76433bd

132772.27c

74555.54d

500-700

Calcium

1473.8a

78.94.95bd

1407.76c

1012.68e

600

Phosphorus

1333.88a

1172.54bd

21414.07c

1774.12e

500

Magnesium

1056.23a

71.41.89bd

98.72.84c

88.52.44e

60

Iron

2.330.39a

1.170.15bd

1.210.07c

0.290.48e

10

Copper

0.380.02a

0.280.008bd

0.340.05a

0.240.02e

0.6-0.7

Zinc

11.71.22a

5.70.33bd

10.70.72a

2.820.36e

5

Manganese

2.250.08a

0.70.04bd

2.020.11c

0.260.04e

0.3-1

Selenium

10.20.27a

9.650.15bd

51.71.04c

47.00.46e

-

*Bhagya et al (2006)

 **NRC/NAS (1989)

Figures across the column with different letters are significantly different (p<0.05, paired t-test)


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).


Table 4.  Protein and carbohydrate fractions (g/100 g) of tender pods and ripened beans of Canavalia cathartica on dry weight basis (n=5; meanSD) (percent in parenthesis)

Protein and

carbohydrate fraction

Tender pods

Ripened beans*

Raw

Cooked

Raw

Cooked

True protein

17.40.58 (100)a

10.70.19 (100)bd

26.3 (100)c

24.3 (100)e

Albumins

6.060.24 (34.9)a

2.260.11 (21.1)bd

8.240.56 (31.32)c

5.930.61 (24.38)e

Globulins 

6.590.19 (37.9)a

5.570.14 (52.1)bd

14.50.48 (55.23)c

16.01.15 (65.87)e

Prolamins  

1.850.07 (10.6)a

0.630.04 (5.89)bd

0.810.14 (3.08)c

0.630.06 (2.59)d

Glutelins

2.880.42 (16.8)a

2.240.06 (20.6)bd

2.80.42 (10.64)a

2.260.15 (9.29)d

Non-protein nitrogen

0.680.09a

1.250.04bd

1.130.4a

0.840.13e 

Starch

33.21.27a

49.20.67bd

43.61.04c

47.640.37e

Total sugars

7.360.17 (100)a

3.50.22 (100)bd

5.870.14 (100)c

3.560.14b (100)d

Reducing sugars

5.170.18 (70.2)a

2.260.1 (64.6)bd

0.210.01 (3.58)c

0.10.02b (2.81)e

Non-reducing sugars

2.190.3 (29.8)a

1.240.22 (35.5)bd

5.670.14 (96.4)c

3.460.12b (97.2)e

*Bhagya et al (2006)
Figures across the column with different letters are significantly different (p<0.05, paired t-test)


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).


Table 5.  Amino acid composition of tender pods and ripened beans of Canavalia cathartica (g/100 g protein)

 Amino acid

Tender pods

Ripened beansa

FAO/WHO/UNU

pattern for adultsb

Raw

Cooked

Raw

Cooked

Glutamic acid

16.6

0.39

10.4

0.62

 

Aspartic acid

18.7

7.84

10.5

4.35

 

Serine

2.03

1.24

1.77

1.36

 

Threonine

1.25

1.06

1.36

0.82

0.9

Proline

1.80

1.41

1.95

1.02

 

Alanine

2.77

1.72

2.07

1.33

 

Glycine

1.11

0.81

1.54

0.91

 

Valine

3.02

1.23

1.82

1.60

1.3

Cystine

ND

ND

ND

ND

 

1.7c

Methionine

0.87

0.39

1.36

0.32

Isoleucine

2.83

0.84

1.51

1.40

1.3

Leucine

2.78

0.18

1.63

0.26

1.9

Tyrosine

1.32

0.12

1.50

0.91

1.9d

Phenylalanine

3.88

2.86

3.90

1.12

Tryptophan

ND

ND

ND

ND

0.5

Lysine

3.50

2.69

4.12

2.03

1.6

Histidine

ND

ND

ND

ND

1.6

Arginine

ND

ND

ND

ND

 

aBhagya et al (2006)

bEssential amino acids for adults (FAO/WHO/UNU 1985) 

cMethionine+Cystine

dPhenylalanine+Tyrosine

ND, Not detectable


The elevated non-protein nitrogen in cooked pods (0.7 vs. 1.3%) unlike ripened beans (1.1 vs. 0.8%) can be attributed to partial hydrolysis of proteins during thermal processing. Starch was the dominant carbohydrate fraction of cooked pods, serving as a good source of carbohydrates for livestock, which has been elevated in cooked pods like ripened beans and cooked pods will be a good source of starch for livestock. Apart from calorific value, starch contributes to the texture and as a result to organoleptic properties of food (Tharanathan and Mahadevamma 2003). Starch-protein interaction in legumes contributes to decreased glycemic responses and is beneficial in the diet of diabetics and hyperlipidemia patients (Geervani and Theophilus 1981). Starch, total sugars, reducing sugar and non-reducing sugars also decreased in pods on cooking as seen in ripened beans.

Amino acids and fatty acids

Amino acid profile (mg/100 mg protein) of raw and cooked pods has been compared with ripened beans in Table 5. Cooking drained amino acids like the ripened beans. Glutamic acid and aspartic acid were high in pods as seen in ripened beans. As in common legumes, raw as well as cooked pods of Canavalia cathartica consist of adequate lysine and were deficient in sulfur-amino acids and this trend was similar in ripened beans too. Threonine, valine, isoleucine, leucine and tyrosine + phenylalanine of raw pods and threonine, valine, tyrosine + phenylalanine and lysine of cooked pods meet the FAO/WHO/UNU (1985) requirements. The isoleucine of raw pods was on par with FAO/WHO (1991) pattern (2.83 vs. 2.8 mg/100 g protein). The thermal treatment of the current study (household pressure-cooking) resulted in heavy removal of isoleucine (70.3%) and leucine (93.5%). Ecological conditions markedly influence the total nitrogen of seeds and thus affect the relative proportion of EAA (mainly cystine, methionine and lysine) (Baudoin and Maquet 1999). Possibly the pods of Canavalia spp. of different environmental conditions may possess different amino acid composition and need to be tested for domestication.

The FAMEs of pods have been compared with ripened beans in Table 6.


Table 6.  Fatty acid composition of tender pods and ripened beans of Canavalia cathartica (mg/g lipid; n=3, mean)

Fatty acid

Tender pods

Ripened beans*

Raw

Cooked

Raw

Cooked

Saturated fatty acids

Lauric acid (C12:0)

-

-

0.19

-

Tridecanoic acid (C13:0)

0.013

0.343

-

6.6

Myristic acid (C14:0)

7.805

1.618

-

0.68

Pentadecanoic acid (C15:0)

0.422

0.058

-

0.27

Palmitic acid (C16:0)

-

6.896

0.66

0.8

Heptadeconoic acid (C17:0)

-

0.029

0.35

10.67

Stearic acid (C18:0)

6.631

-

11.01

-

Nonadeconoic acid (C19:0)

-

-

0.28

-

Arachidic acid (C20:0)

2.66

-

-

-

Heneicosanoic acid (C21:0)

-

10.345

4.33

-

Behenic acid (C22:0)

-

-

4.83

1.28

Tricosanoic acid (C23:0)

-

-

0.88

-

Lignoceric acid (C24:0)

0.179

-

0.6

0.003

Polyunsaturated fatty acids

Myristoleic acid (C14:1)

1.337

-

0.27

-

Palmitoleic acid (C16:1)

-

0.925

0.5

0.73

Elaidic acid (C18:1)

-

-

0.95

3.37

Oleic acid (C18:1)

0.058

25.617

-

-

Linoleic acid (C18:2)

-

9.097

-

2.94

Linolenic acid (C18:3)

0.922

0.158

0.59

8.49

Eicosenoic acid (C20:1)

-

24.656

-

13.66

Eicosadienoic acid (C20:2)

6.095

-

-

-

Arachidonic acid (C20:4)

2.941

-

5.1

0.82

Docosahexaenoic acid (C20:6)

-

5.342

-

5.34

Nervonic acid (C24:1)

0.032

-

0.03

-

Sum of essential fatty acids

3.86

9.26

5.69

12.25

Sum of saturated fatty acids

17.7

19.3

23.3

20.3

Sum of polyunsaturated fatty acids

10.7

65.8

7.44

35.35

P/S ratioa

0.604

3.41

0.32

1.74

*Bhagya et al (2006)

 -, Not detectable

aRatio of polyunsaturated/saturated fatty acids


Sum of polyunsaturated fatty acids of cooked pods was higher than raw pods as well as raw and cooked beans. Myristic acid among saturated fatty acid and eicosadienoic acid among unsaturated fatty acid was highest in raw pods, while heneicosanoic acid and oleic acid in cooked pods. Sum of essential fatty acids was higher in cooked pods than raw pods. Raw pods consist of linolenic acid and arachidonic, while in cooked pods linoleic and linolenic acid. The polyunsaturated/saturated fatty acid (P/S) ratio elevated over five times between raw and cooked pods. Oleic acid in cooked pods was high (25.6 mg/g lipid), which was very low in raw pods and not detected in ripened beans. Surprisingly, oleic acid was very low (trace-0.08 mg/g lipid) in roasted as well pressure-cooked dry seeds (Seena et al 2006). Similarly, eicosenoic acid was high in cooked pods (24.7 mg/g lipid), while it was low in roasted and pressure-cooked dry seeds (7.12-7.44 mg/g lipid) (Seena et al 2006). Thus, the essential fatty acids of cooked tender pods have beneficial effect on consumption as human food or livestock feed.

Antinutritional factors

Legumes commonly consist of toxic factors, which decrease the digestibility and prevent bioavailability of nutrients. Among the antinutritional factors evaluated in our study, the raw pods were devoid of trypsin inhibitor activity (Table 7).


Table 7.   Antinutritional components of tender pods and ripened beans of Canavalia cathartica (g/100g) (n=5; mean SD)

Component

Tender pods

Ripened beans*

Raw

Cooked

Raw

Cooked

Total phenolics

3.560.2a

2.840.06bd

50.1c

2.420.15e

Orthodihydric phenols

14.70.33a

2.420.27b

NP

NP

Tannins

0.280.02a

0.190.03bd

0.0430.01c

0.0190.01e

Trypsin inhibitor activity

NP

NP

NP

NP

Phytohemagglutination activity**

14

4

26

14

*Bhagya et al (2006)

**, Titre: maximum dilution where agglutination was observed

NP, Not present

Figures across the column with different letters are significantly different (p<0.05, paired t-test)


Total phenolics of pods was less than ripened beans, while tannins were high in pods and decreased on cooking. Orthodihydric phenols are known to confer resistance against pests and considerably decreased in pods on cooking, while was absent in ripened beans. The phytohemagglutination activity was also drastically decreased (up to 70%) in pods on cooking unlike ripened beans. Thermal treatment is known to improve the nutritive value of legumes through inactivation of hemagglutinins (Swaminathan 1974). As phenolics and tannins are water-soluble, they may be eliminated by decortication soaking or cooking (Reddy et al 1985). Considerable decrease of antinutritional factors in cooked Canavalia cathartica pods qualifies for human or livestock consumption.

Protein quality assessment

The diet formulation used for protein quality assessment in rats has been given in Table 8.


Table 8.  Diet composition

Ingredients, g/100 g

Basal diet

Casein diet

Test diets

Raw pod

Cooked pod

Corn starch

80

70

33.9

26.1

Corn oil

10

10

10

10

Non-nutrition cellulose

5

5

5

5

Casein

-

10

-

-

Raw pod flour

-

-

46.1

-

Cooked pod flour

-

-

-

53.9

Salt mixture*

4

4

4

4

Vitamin mixture**

1

1

1

1

*Salt mixture: CaCO3, 78.6 g; Ca3C12H10O144H2O, 308.3 g; CaHPO42H2O, 112.8 g;

K2HPO4, 218.8 g; KCl, 124.7 g; NaCl, 77.1 g; MgSO4, 38.3 g; MgCO3, 35.2 g;  Fe(C6H17N3O7), 15.3 g; MnSO4H2O, 0.201 g; CuSO45H2O, 0.078 g; KI, 0.041 g; AlNH4(SO4)212H2O, 0.507 g.

**Vitamin mixture: Vitamin A, 1000 IU; Vitamin D, 100 IU; Vitamin E, 10 IU; Vitamin K, 0.5 mg; Thiamine, 0.5 mg; riboflavin, 1 mg; Pyrodoxine, 0.4 mg; Pantothenic acid, 4 mg; Niacin, 4 mg; Choline, 200 mg; Inositol, 25 mg; Para-aminobenzoinc acid, 10 mg; Vitamin B12, 2 mg; Biotin, 0.02 mg; Folic acid, 0.2 mg; added cellulose to make up to 1 g


The biological trials revealed no considerable weight gain on feeding diet consisting of raw as well as cooked pod flour on comparison with casein diet (Table 9).


Table 9.   Food intake, protein intake, gain in body weight, food efficiency ratio (FER), net protein retention (NPR), protein retention efficiency (PRE), true digestibility (TD), biological value (BV) and net protein utilization  (NPU) of casein and tender pod flours of Canavalia cathartica fed to albino rats (n=5; meanSD)

Assay

Casein

Tender pods

Raw

Cooked

Growth studies

Food intake for 28 d, g

1432.39a

69.31.72bc

78.11.85bd

Protein intake for 28 d, g

14.30.23a

6.930.17bc

7.810.18bd

Gain in body weight for 28 d, g

33.00.42a

0.130.04bc

0.920.08bd

FER

0.230.01a

0.0020.001bc

0.010.001bd

PER

2.320.06a

0.020.007bc

0.120.011bd

Corrected PER*

2.5a

0.020.007bc

0.130.02bd

Gain in weight for 10 d, g

9.720.17a

0.020.005bc

0.330.007bd

Weight loss for 10 d, g

2.80.03

2.80.03

2.820.03

Protein consumed for 10 d, g

4.730.04a

2.490.12bc

2.60.06bc

NPR

2.650.02a

1.140.06bc

1.210.03bc

PRE

42.30.41a

18.211bc

19.240.54bc

Nitrogen balance studies

TD (%)

98.50.37a

38.13.27bc

47.12.15bd

BV (%)

87.00.24a

30.22.3bc

41.00.46bd

NPU (%)

85.80.47a

11.40.92bc

19.31.02bd

*Based on values of 2.5 as standard for casein

Figures across the column with different letters are significantly different (p<0.05, paired t-test)


However all parameters studied under growth studies was higher in cooked pod diet than raw pod diet. Even with low NPU (raw pod, 11.44%; cooked pod, 19.24%), the rats in our study were in positive nitrogen balance. Shermer and Perkins (1975) indicated that the decreased bioavailability of amino acids, especially methionine lowers net protein utilization. The TD and BV were higher in cooked pod diet than raw pod diet and close to 50% of casein diet. The antinutritional factor, mainly the lectin limits the use of raw pods of Canavalia cathartica. Other toxins like urease and canatoxin are not known to exert toxic effect on oral administration, thus not considered as antinutritional factors (Udedibie and Carlini 1998). Lectins are known to adversely affect protein digestibility (Singh 1984; Tan et al 1984, Knuckles et al 1985). Our study revealed drastic decrease of hemagglutinin activity of raw pods on pressure-cooking. The lectin, con A is known for its resistance to digestion in Canavalia ensiformis (Udedibie and Carlini 1998). It has been predicted that con A-like lectin in tender pods of Canavalia cathartica in our study is responsible for low growth and nitrogen balance in rats. Thus, domestic pressure-cooking of Canavalia cathartica tender pods is not very efficient in detoxification and necessary to adapt other thermal strategies.

In EAA score, sulphur- amino acids, cystine + methionine in both raw and cooked pods were limiting (Table 10).


Table 10.   Essential amino acid (EAA) score, protein digestibility corrected amino acid score (PDCAAS) of tender pods ripened beans of Canavalia cathartica

 

Tender pods

Ripened beans*

Raw

Cooked

Raw

Cooked

EAA score**

Threonine

139

118

151

91.1

Valine

232

94.6

140

123

Cystine + Methionine

51.2

22.9

80.0

18.8

Isoleucine

218

64.6

116

108

Leucine

146

9.47

85.8

13.7

Tyrosine + Phenylalanine

274

157

284

107

Tryptophan

ND

ND

ND

ND

Lysine

219

168

257

127

Histidine

ND

ND

ND

ND

PDCAAS, (%**

Threonine

52.9

55.5

63.4

64.2

Valine

88.4

44.6

58.7

86.7

Cystine + Methionine

19.5

10.8

33.6

13.3

Isoleucine

82.9

30.4

48.4

75.8

Leucine

55.7

4.46

36.0

9.64

Tyrosine + Phenylalanine

104

73.9

119

75.2

Tryptophan

ND

ND

ND

ND

Lysine

83.3

79.2

108

89.3

Histidine

ND

ND

ND

ND

*Bhagya (2006)

**Calculated based on FAO/WHO/UNU (1985) pattern for adults


In addition, valine, isoleucine and leucine were limited in cooked pods due to draining. Cystine + methionine and isoleucine were limited in both raw and cooked ripened beans of Canavalia cathartica. The percent PDCAAS of raw and cooked pods is similar to EAA score (Table 10). Appreciable amount of lysine was available in both raw and cooked pods as in ripened beans. This suggests that to uplift the utility of cooked tender pods of Canavalia cathartica, blending with suitable foodstuff consisting of sulphur-amino acids, valine, isoleucine and leucine is necessary.

Starch digestibility

The in vitro starch digestibility was significantly doubled in cooked pods (1.35 vs. 2.62 mg maltose/hr/100 g), however, it was not improved as in beans (1.34 vs. 6.73 mg maltose/hr/100 g) (Table 11).


Table 11.   In vitro starch digestibility of tender pods and ripened beans of Canavalia cathartica (n=5; meanSD)

Starch digestibility, mg maltose/hr/100 g

Raw

Cooked

Tender pods

1.350.3a

2.620.17bc

Ripened beans*

1.340.08a

6.730.08bd

*Bhagya et al (2006)

Figures across the column and rows with different letters are significantly different

(p<0.05, paired t-test)


Starch in raw tender pods is indigestible as it forms granules and less susceptible to hydrolytic enzymes (Colonna et al 1992). High ratios of amylose/amylopectin, high dietary fiber and and various antinutritional factors in raw pods reduce the starch bioavailability (Deshpande and Cheryan 1984). Intact tissue/cell structure enclosing starch granules are the other factors hindering the swelling and solubilization of starch (Tovar et al 1990). Enhancement of starch digestibility due to starch gelatinization and destruction of antinutrients have been reported (Mbofung et al 1999; Yu-Hui 1991). Cooking leads to hydrolysis of starch and gelatinization, which allow easy access to enzymatic attack (Bishnoi and Khetrapaul 1993). The improvement of starch digestibility in cooked tender pods in our study can be attributed to the hydrolysis of starch and partial elimination of antinutritional factors.


Conclusions


Acknowledgements

Authors are grateful to Mangalore University for granting permission to carry out this study at the department of Biosciences. We are indebted to editors and anonymous referees for comments to improve the manuscript.


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Received 27 April 2006; Accepted 7 May 2006; Published 18 July 2006

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