VARIABILITY IN THE NUTRITIVE VALUE OF THREE NEW LIMA BEAN (PHASEOLUS LUNATUS L. WALP.) BREEDING LINES

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1 Legume Res., 29 () : 25-3, 2006 VARIABILITY IN THE NUTRITIVE VALUE OF THREE NEW LIMA BEAN (PHASEOLUS LUNATUS L. WALP.) BREEDING LINES Sunday Y. Giami Department of Food Science and Technology, Rivers State University of Science and Technology, Port Harcourt, Nigeria ABSTRACT Chemical composition and protein quality of raw and heat-processed (boiled and autoclaved) samples of selected advanced breeding lines of lima beans (Tpl B, Tpl 7 A and Tpl 75 A ), part of a larger collection developed by the International Institute for Tropical Agriculture (IITA), and evaluated in agronomic field trials in Nigeria, were studied. Protein quality was evaluated using weanling albino rats fed diets which we formulated to supply 0% protein using lima bean samples with casein as a control. Raw seeds contained % crude protein, mg iron per 00g, mg polyphenol per 00g and mg phytic acid per 00g seed flour. Tpl B had higher crude protein and mineral (calcium, iron and sodium) contents, and lower amounts of polyphenol and phytic acid compared with Tpl 7 A and Tpl 75 A. Boiling (moist heat) was more effective than autoclaving (dry heat) for reducing the levels of antinutrients and improving the protein quality of the beans, as shown by the higher values for weight gain, protein efficiency ratio (PER), net protein ratio (NPR) and true digestibility (TD) of boiled samples. There were no significant (p > 0.05) differences between the values obtained for PER, NPR, and TD of diets formulated with boiled samples of Tpl B and casein, indicating the superiority of the protein quality of this lima bean line compared with Tpl 7 A and Tpl 75 A. INTRODUCTION Lima beans (Phaseolus lunatus L. Walp.) are an important grain legume and source of protein and other nutrients in many developing countries. In Nigeria, lima beans are reported to provide from 25 to 30% of the protein in the diet and are second only to maize and cowpea as a staple food (Luse, 979). Efforts aimed at breeding and improvement of lima bean cultivars for specific characteristics, such as high yield, early maturity, improved nutritional properties, desired seed quality and resistance to insects and diseases have been carried out by the International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria. Advanced lines of lima beans, with good agronomic performance (high yield, early maturity and resistance to diseases) have been developed and released for cultivation by the IITA and they include Tpl B Tpl 7 A and Tpl 75 A. The presence of antinutrients, such as polyphenol and phytic acid has been identified as one major factor limiting the use of lima beans as food (Ologhobo, 986). Genetic manipulation in breeding new lima bean cultivars may produce wide variation in the contents of these antinutrients, their chemical composition and protein quality. The chemical composition and protein quality of lima beans and other legumes have been shown to vary according to cultivar (Apata and Ologhobo, 986; Singh et al., 99; Giami et al., 200a; Giami 2002). Removal or reduction of antinutrients and evaluation of the protein quality of newly developed lima bean cultivars is necessary for their effective utilization in human nutrition. Heat treatment has been shown to improve the nutritive value of legumes, oil seeds and other edible seeds by decreasing the levels of antinutrients and increasing protein digestibility (Kadam et al., 987; Giami and Wachukwu 997; Giami et al., 200b; Giami, 2002). Characterization of these improved lines of lima beans (Tpl B, Tpl 7 A and Tpl 75 A ) for physico-chemical properties and cooking quality has been reported (Giami,

2 26 LEGUME RESEARCH 200), but, information on their composition and protein quality, as affected by moist and dry heat treatments, is lacking. The aim of this study, therefore was to evaluate the chemical composition and protein quality of these lima bean breeding lines and the effects of boiling (moist heat) and autoclaving (dry heat) of the beans on these properties. MATERIAL AND METHODS Lima bean samples Seeds of three advanced breeding lines (Tpl B, Tpl 7 A and Tpl 75 A ) of lima beans (Phaseolus lunatus L. Walp.) were obtained from International Institute for Tropical Agriculture (IITA), Ibadan (Onne Station), Nigeria. Preparation of samples Lima bean seeds were divided into three groups. Samples (50± g) from one group were boiled in distilled water (2000 ml) in a covered aluminum pot for 30 min. Samples (50 ± g) from the second group were first ground in raw form, then autoclaved (2 0 C,.05 kg cm -2 ) for 20 min. Samples from the third group received no heat treatment and served as the (raw) control. Raw and heat processed samples were oven dried (50 0 C, 24h) in a hot-air fan oven (Model QUB 30500G, Gallenkamp, UK), ground using a laboratory mill (Numex Pep Grinding Mill, India) and screened through a 0.25mm British standard sieve (Model BS 40, Endecotts Ltd UK). Flours from raw and heat-processed samples to be used in test diet formulations for rat studies were defatted by solvent extraction in soxhlet apparatus (Tecator Inc., Co, USA) for 8h using n-hexane. All samples were stored in screw capped plastic bottles at 4 0 C until used. Chemical analyses Crude protein (method 2.057) and ether extract (method were determined according to AOAC (984) procedures. The factor 6.25 was used for conversion of nitrogen to crude protein. Polyphenol was determined using the vanillin/h 2 SO 4 assay described by Wilson and Blunden (983), with reference to a standard graph based on phloroglucinol. The results were expressed as mg phloroglucinol equivalents per 00g seed flour. In-vitro protein digestibility was assessed using pepsin and pancreatin (Ali et al., 2003). For the determination of phytic acid, a combination of two methods was used. The extraction and precipitation of phytic acid were performed according to the method of Wheeler and Ferrel (97); iron in the precipitate was measured using method 4.03 of the AOAC (984). A 4:6 Fe/P, molecular ratio was used to calculate the phytic acid content. Pepsin and pancreatin were manufactured by Sigma Chemical Co (St. Louis, MO, USA). All reagents used were of analytical grade (BDH Chemicals, Poole UK). Biological assay Protein quality of diets containing raw, autoclaved and boiled samples of selected advanced lines of lima bean was evaluated using a rat bioassay. Ten per cent protein diets containing casein (control) and flour samples from raw and heat-processed lima bean breeding lines were prepared based on the formulation of the AOAC (984) as described in a previous paper (Giami, 2002). The composition is shown in Table. Weanling male albino rats of the Wistar strain, 28 days old and weighing between 34 and 37g were grouped by randomized block design into eleven groups on the basis of weight, such that mean initial weights were similar. Each group consisted of ten rats and was housed in individual wire-bottom cages that allowed for easy faecal collection and the measurement of food intake. One group was fed a nitrogenfree diet, which consisted entirely of the basal diet. Another group was fed casein (control)

3 Table. Composition of experimental, casein and protein-free diets Diet/lima bean line Ingredients (g per 00) Sample Corn oil Salt Vitamin Cellulose Cassava Casein mixture mixture 2 starch Tpl B Raw Boiled Autoclaved Tpl 7 A Raw Boiled Autoclaved Tpl 75 A Raw Boiled Autoclaved Casein Protein-free Salt mixture (composition per 00g): calcium (0.6g); chloride (0.5g); copper (.0mg); iodine (0.02mg); iron (0mg); magnesium (0.2g); manganese (7.5mg), phosphorus (0.5g); potassium (0.5g); sodium (0.5g); zinc (.8mg). 2 Vitamin mixture (composition per 00g): vitamin A (700 i.u.); vitamin D (30 i.u); Vitamin E (6 i.u.); vitamin K (0.29mg); thiamine hydrochloride (40mg); riboflavin (0.5mg); pyridoxine hydrochloride (0.6mg); niacin (.0mg); pantothenic acid (.2mg); cyanocobalamin, B 2 (0.5mg). diet, while the remaining nine groups were fed the experimental diets. The rats had free access to the diets and water for 28 days, the duration of the experiment for the protein efficiency ratio (PER) study. Digestibility study was started on the 4 th day of the PER study and lasted for 7 days, while the net protein ratio (NPR) Vol. 29, No., determination was done on the 0 th day of the PER study. Daily records on weight gain or loss, food and protein intakes and faecal output by the rats were taken and used in calculating the PER, NPR and true digestibility (TD) using Standard recommended equations (Pellet and Young, 980) as: PER (Unadjusted) = Gain (or loss) in body weight (g) Protein consumed (g) NPR = Weight gain of test group + weight loss of protein - free group Protein consumed by test group Where, = Protein intake of rats fed test diet; F = Protein excreted in faeces of rats fed test diet; F K = Protein excreted in faeces of rats fed protein-free diet. - (F - F K ) TD (%) = x 00 Statistical design and analyses The study was conducted in two parts. In the first part, in which the aim was to investigate the effects of heat treatment on the chemical composition of the beans, the experimental design was a complete

4 28 LEGUME RESEARCH randomized 3 x 3 x 3 factorial with three lima bean lines (Tpl B, Tpl 7 A and Tpl 75 A ), three processing operations (raw as control, boiled and autoclaved) and three replications (for each constituents and anitnutritional factors.) The models included all main effects and interaction terms. In the second part, investigating the nutritional values of the beans, a 3x3 factorial design with two replicates was used. Mean values were calculated and the data subjected to analysis of variance for treatments arranged factorially by a completely randomized statistical model (Wahua, 999): Y ijk = μ + A i + B j + (AB) ij + E (ij) K Where Y ijk represents each observable measurement; μ represents mean of each measurement; A i represents breeding line fixed effect (i =, 2, 3 breeding lines); B j represents processing fixed effect (j =, 2, 3 processing methods); (AB) ij represents interaction effect of breeding lines and processing methods; E (ij) K represents residual random term. If a significant F-test was noted, means were separated using Duncan s multiple range test (Duncan, 995). Significances was accepted at P < RESULTS AND DISCUSSION Data on the chemical composition of raw and heat-processed lima bean breeding lines are presented in Table 2. The crude protein contents of Tpl B and Tpl 75 A were %, higher than the values ( %) reported for other lima bean cultivars (Apata and Ologhobo, 994) and some commonly consumed Nigerian legumes, such as, pigeon peas and African yam bean with values ranging from 2.. to 22.5% (Ene- Obong and Carnovale, 992). The crude protein content of Tpl B was significantly higher than those of Tpl 7 A and Tpl 75 A. The levels of polyphenol (55.7mg/00g) and phytic acid (8.8 mg/00g) in raw samples of Tpl B were significantly (p < 0.05) lower than the levels of these anitnutrients found in raw samples of Tpl 7 A and Tpl 75 A. The variation in the content of these antinutrients show the possibility of breeding lines which have little or none of these antinutrients, thus improving the quality of lima beans. The level of phytic acid (8.8mg/00g) in the raw sample of Tpl B was lower than the levels reported for various legumes, particularly those of bambara groundnut (294mg/00g), winged bean (65mg/00g,) moth bean ( mg/ 00g) and various cowpea cultivars ( mg/00g) (Ologhodo and Fetuga, 984); Khokhar and Chauhan, 986; Kadam et al., 987; Igbedioh et al., 994). The lower level of phytic acid in Tpl B, compared with the various legumes cited above, seem to indicate that the nutritive value of this lima bean line would be impaired to a comparatively lesser extent, leading to an overall better nutritional quality of this legume line. Besides, lowering the bioavailability of minerals and inhibiting the digestibility of proteins (Lopez et al., 2002), phytic acid is also implicated in the hard-tocook phenomenon of legumes (Stanley and Aguilera, 985). The cooking time of Tpl B was found in a previous study (Giami, 200) to be shorter than those of Tpl 7A and Tpl 75 A. There were significant (P < 0.05) differences between the values obtained for invitro protein digestibility, polyphenol and phytic acid contents of raw and heat-processed samples (Table 2). Autoclaving was noted to be less effective in improving protein digestibility and lowering polyphenol and phytic acid contents of the samples compared with boiling. Similar observations have been made during heat processing of soybeans (Giami, 2002), winged bean (Kadam et al., 987), African breadfruit (Giami et al., 200b) and groundnuts (Singh et al., 99). Boiling has

5 Vol. 29, No., Table 2. Chemical composition of raw and heat-processed samples of lima bean breeding lines Sample Crude protein (%) Ether extract In-vitro protein Polyphenol Phytic acid N x 6.25 (%) digestibility (%) (mg per 00g) (mg per 00g) Tpl B Raw 27.8±0.2 a 2.2±0.2 a 56.7±0.3 c 55.7±0.3 b 8.8±0.8 b Boiled 27.4± 0. a.6±0. b 77.8±0.4 a 6.6±0.2 d 6.4±0.3 c Autoclaved 27.3±0. a.8±0. b 65.9±0.4 b 33.5±0.2 c 6.8±0.6 b Tpl 7 A Raw 24.0±0.2 b.9±0.2 b 5.8±0.3 c 77.8±0.7 a 222.0±0.8 a Boiled 23.7±0. b 0.9±0. c 66.7±0.2 b 8.4±0.3 d 6.2±0.3 b Autoclaved 23.6±0.3 b.0±0. c 65.3±0.2 b 37.4±0.4 c 29.5±0.5 a Tpl 75 A Raw 24.5±0. b.6±0.2 b 52.8±0.2 c 79.0±0.8 a 225.9±0.7 a Boiled 24.3±0.2 b 0.8±0. c 68.4±0.4 b 39.6±0.4 c 8.6±0.4 b Autoclaved 24.2±0.3 b.0±0. c 64.±0.3 b 56.6±0.6 b 28.5±0.4 a Mean±standard error of the mean of triplicate determinations; a, b, c, d means with the same superscript within a column do not differ significantly; (P > 0.05) by the Duncan s multiple range test. Table 3. Mineral contents (mg per 00g) of raw and heat-processed samples of lima bean breeding lines Sample Calcium Phosphorus Sodium Iron Tpl B Raw 78.5±0.6 a 360.8±0.7 b 7.9±0.2 a 7.7±0. a Boiled 64.8±0.2 b 269.4±0.8 c 4.4±0. b 6.8±0.2 b Autoclaved 77.5±0.4 a 360.5± 0.6 b 7.7±0. a 7.4±0. a Tpl 7 A Raw 65.7±0.5 b 407.4±0.5 a 4.9±0. b 6.9±0.2 b Boiled 49.9±.06 c 368.0±0.3 b 2.3±0. c 4.9±0.c Autoclaved 64.4±0.4 b 40.6±0.4 a 4.7±0.2 b 6.8±0.2b Tpl 75 A Raw 66.5±0.4 b 374.8±0.3 b 5.0±0. b 6.8±0.2 b Boiled 50.5±0.3 c 27.6±0.2 c 2.5±0. c 4.6±0. c Autoclaved 64.9±0.2 b 370.3±0.8 b 4.7±0. b 6.7±0. b Mean ± standard error of the mean of triplicate determination; a, b, c Means with the same superscript within a column do not differ significant (P > 0.05) by the Duncan s multiple range test. been reported to improve protein digestibility of legumes, most likely by destroying heat-liable protease inhibitors (Tan et al., 984), or denaturing globulin, which are highly resistant to proteases in the native state (Walker and Kochhar, 982). Loss of polyphenol and phytic acid in boiled legumes have been attributed to leaching of these anitnutients into the boiling water (Kadam et al., 987). Tpl B had higher levels of calcium, sodium and iron, compared with Tpl 7 A and Tpl 75 A (Table 3). Variations in the content of minerals for different cultivars of the same legume have been reported by Apata and Ologhobo (994) and attributed to differences in origin, geographical location and soil fertility. Autoclaving had a negligible effect on mineral contents of the lima bean lines compared with boiling. Diets formulated with raw samples of lima bean lines resulted in poor protein quality indices, such as negative values for protein efficiency ratio (PER), low values for net protein ratio (NPR) and true digestibility (TD)

6 30 LEGUME RESEARCH Table 4. Protein quality of diets containing raw (r ), boiled (b) and autoclaved (a) samples of lima bean breeding lines Protein quality Diet/Sample 2 indices Tpl B Tpl 7 A Tpl 75 A Reference diets r b a r b a r b a Casein Proteinfree Weight gain/loss (g) d a 2.75 b d 2.0 b 5.62 c d.74 b 5.68 c 29.5 a e Protein intake (g) 4.84 c 0.4 a 8.56 b 4.33 c 7.57 b 5.87 c 4.36 c 7.03 b 5.94 c 2.37 a 0.00 PER d 2.35 a.49 b d.60 b 0.96 c 0.62 d.67 b 0.96 c 2.36 a 0.00 NPR c 4.2 a.80 b 0.83 c.84 b 0.87 c 0.84 c.82 b 0.79 c 4.50 a 0.00 TD 5 (%) c a b 47.5 d 73.4 b c d 7.88 b 60.2 c a 0.00 Mean of ten rats per group; a, b, c, d Means with the same superscript within a row do not differ significantly (P > 0.05) by the Duncan s multiple range test; 2 Samples were: r = raw; b = boiled and a = autoclaved; 3 Protein efficiency ratio (unadjusted); 4 Net protein ratio; 5 True digestibility. when fed to rats (Table 4). Rats fed diets formulated with raw samples lost weight. Feeding studies by other researchers (Fetuga et al., 973; Kadam et al., 987; Giami, 2003) showed that raw legumes and oilseeds did not support the growth of rats and attributed to toxic components such as growth inhibitors in the raw seeds. The weight gains of rats fed diets containing boiled beans were significantly higher than those of rats fed diets containing autoclaved beans. The values obtained for PER, NPR and TD of diets containing boiled samples of Tpl B were similar to those of the casein (control) diet, indicating that this lima bean line is nutritionally superior to Tpl 7 A and Tpl 75 A. In conclusion, this study has shown that Tpl B has desirable attributes such as high crude protein and iron contents, low levels of polyphenol and phytic acid and superior protein quality compared with Tpl 7 A and Tpl 75 A. REFERENCES Ali, M.A.M. et al. (2003). Food Chem., 80: AOAC (984). Official methods of Analysis, 4 th Edn. Association of Official Analytical Chemists, Washington DC. Apata, D.F. and Ologhobo, A.D. (994). Food Chem., 49: Duncan, D.B. (955). Biometrics, : -5. Ene-bong, H.N. and Carnvale, E. (992). Food Chem., 43: Fetuga, B.L. et al. (973). J. Sci. Food Agric., 24: Giami, S.Y. and Wachuku, O.C. (997). Plant Foods Hum. Nutr., 50: Giami, S.Y. (200). Plant Foods Hum. Nutr., 56: Giami, S.Y. et al. (200a). Plant Foods Hum. Nutr., 56: Giami S.Y. et al. (200b). Plant Foods Hum. Nutr., 56: Giami, S.Y. (2002). J. Sci. Food Agric., 82: Giami, S.Y. (2003). J. Dairying, Foods and Home Sci., 22: Igbedioh, S.O. et al. (994). Food Chem., 50: Kadam, S.S. et al. (987). J. Sci. Food Agric., 39: Khokhar, S. and Chauhan, B.M. (986). J. Food Sci., 5: Lopez, H.W. et al. (2002). Int. J. Food Sci. Technol., 37:

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