Methodology. Indian Journal of Traditional Knowledge Vol. 12 (4), October 2013, pp

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1 Indian Journal of Traditional Knowledge Vol. 12 (4), October 2013, pp Improving nutritional value of velvet bean, Mucuna pruriens (L.) DC. var. utilis (Wall.ex.Wight) L. H. Bailey, an underutilized pulse, using microwave technology Gurumoorthi P 1 *, Janardhanan K 2 & Kalavathy G 3 1 Food and Process Engineering, School of Bioengineering, SRM University, Kattankulathur, , T N, India; 2 Department of Biotechnology, Prathyusha Institute of Technology and Management, Aranvoyal Kuppam, Thiruvallur, T N, India; 3 Department of Biotechnology, Dr MGR Educational and Research Institute, Chennai, , T N, India p_gurumoorthi@yahoo.com Received , revised The velvet bean is an underutilized legume species grown predominantly in Asia, Africa and in parts of America. A hill tribe by name Kanikkars inhabiting Western Ghats bordering Tamil Nadu and Kerala traditionally consume the seeds as source of protein. However, the seeds also are known to possess several undesirable antinutritional factors which elicit adverse effects in the consuming people. In the present study, an attempt has been made to study the effect of soaking followed by microwave cooking on the elimination or reduction of antinutritional factors (ANF s) and the overall improvement in nutritional quality of two germplasm seed samples collected from Western Ghats of Velvet bean. In both the germplasm, it has been detected and demonstrated that all the investigated ANF s except the pharmacologically important LDopa are significantly eliminated. The reduction in contents of total free phenolics, tannins and phytic acid due to microwave cooking is significant (87.5%, %, and %), respectively. The reduction in levels of LDopa due to microwave cooking is insignificant ( %) where as TIA and CIA are found to be significant (TIA: %; CIA: %), respectively. Among the three erythrocytes, the of lectin activity in respect of O blood in Kailasa Nadu germplasm is low compared to other values (76%). At the same time the seed protein quality of both the germplasm is not adversely affected. Keywords: Velvet bean, Germplasm, Microwave cooking, ANF s, Essential amino acid score IPC Int. Cl. 8 : A61K 36/00, A23, A23N, A23J, A47J 44/00, H01S 1/00, A61N 5/02 Velvet bean [Mucuna pruriens (L.) DC. var. utilis (Wall.ex.Wight) L. H. Bailey] is known to contain comparable level of nutrients (proteins, lipids, several minerals, and dietary fibers) on par with soybean 1. In recent years much research work has been focused on this underutilized pulse to explore whether it will serve as a source of food and feed to the evergrowing human population and livestock 2. The nutritional profile of velvet bean has been well documented by Prof Janardhanan and his team of researchers at Bharathiar University, Coimbatore 3,4,5,6. However, the nutrients of legumes including Velvet bean are associated with high concentration and more diverse array of toxic substances like the pharmaceutically active non protein amino acid, LDopa (L3, 4 dihydroxyphenylalanine), oligosaccharides, phytic acid, phenols, tannins and saponins, alkaloids, protease inhibitors, amylase inhibitors, lectins, etc. Several investigators have attempted to eliminate ANF s by both simple processing techniques as well as *Corresponding author advanced technological processing methods and to improve the protein digestibility of the velvet bean 7. However, the effect of microwave cooking on the elimination or reduction of antinutritional factors and the protein quality in Velvet bean has not been attempted. Studies on the impact of microwave heating on the levels of trypsin inhibitors and lectins in black gram, chickpea, soybean, green gram, faber gram and lentils have been undertaken. The studies have reviewed the reduction in the contents of harmful effects of ANF s without affecting protein efficiency ratio (PER) 8, 9. In view of the above said background two accessions (germplasm) of velvet bean were subjected to microwave cooking to evaluate the overall nutritional quality and results are discussed. Methodology Plant materials Two accessions of Velvet bean (white colored seed coat and black colored seed coat), Mucuna pruriens

2 678 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 12, No. 4, OCTOBER 2013 (L.) DC. var. utilis, were collected as mature parts from the natural stands of Kailasa Nadu, Idukki district, Kerala, Western Ghats. After drying thoroughly in sunlight for 23 days, the parts were thrashed to remove mature seeds; the seeds after thorough cleaning were stored in plastic containers at room temperature (25 C) until further use. Post harvest processing: (Microwave cooking) The collected seeds were soaked in distilled water (1:10 w/v) for 12 hrs. After that the excess water was drained off and exposed to microwave in a microwave oven (Megatron model IFB Industries Ltd., New Delhi, India) with a frequency of 2450 MHz and with an output of 750 watts and 230 voltage for 10 min. Preparation of seed flour About 50 gm of both processed and unprocessed (raw seeds) seed samples were powdered in a Wiley Mill to 60mesh size. Care was taken to cleave the Wiley Mill thoroughly after powdering a particular sample and before starting to powder a new sample to avoid mixing up of samples. The fine seed powder, so obtained will be redefined, hereafter as processed or unprocessed seed flour. The powder samples were stored in screwcap bottles until further use. Analysis of antinutritional factors Extraction and estimation of total free phenolics and tannins, LDopa and phytic acid The estimation of total free phenolics was carried out following a modified method of Maxon & Rooney 10 and estimated following the method of Bray & Thorne 11. Tannins were estimated based on Foiln Dens method of Schanderl 12. Following the method of Myhrman, LDopa was extracted from the samples and estimated by HPLC method (PerkinElmer model 200) 13. While, Phytic acid was extracted and estimated by following the method of Wheeler and Ferrel 14. Protease inhibitors Assays of trypsin iinhibitor activity (TIA) and chymotrypsin inhibitor activity (CIA) Trypsin inhibitor activity was determined according to the method of Smith et al. 15 Chymotrypsin inhibitor activity was determined according to the method of Norioka et al. 16. Assay of heamagglutinating activity Human blood (Blood A, B, and O) was procured from regional blood bank KG Hospital, Coimbatore. Blood erythrocyte suspension was prepared by washing blood samples separately with phosphate buffered saline and centrifuged for 3 min at low speed, supernatants were removed with pasture pipette. The washing procedure was repeated 3times. The washed cells were diluted by drops of cells with 24 drops of phosphate buffered saline. Phytoheamagglutinating activity was determined by the method of Tan et al. 17 Automated amino acid analysis Twenty five mg of both raw and processed seed meals were defatted by hydrolysis and then refluxing in 6 ml of 6 M HCl at 100 C for 24 hrs in Vacuo. The contents were centrifuged; the clear supernatant was dried in a rotatory evaporator to remove acid. After washing with glass distilled water and dried 3 times, the residue was dissolved in a known volume of KCl HCl buffer, ph 2.2. Aliquotes were analyzed in a Hitachi Perkin Elmer (Model KLA3B) automated amino acid analyzer. The estimation of tryptophan in seed proteins was carried out colorimetrically following a method of Spies and Chambers 18. Sulphur containing amino acids were oxidized using performic acid before the acid hydrolysis. The contents of different amino acids recovered were presented as gm 100 gm 1 of proteins and were compared with FAO/WHO 19. Stastical analysis All the values were estimated in triplicate determinations except phytoheamagglutinins (lectins) samples. The statistical values, mean, standard error and standard deviation were calculated using the software package, Genres Agres D square/ Path D square analysis. Significant differences between mean values were calculated by analysis of variance (ANOVA) using Ducan s Multiple Range Test at p<0.05. Results and discussion The data on the effect of soaking followed by microwave cooking on the levels of antinutritional factors, total free phenolics, tannins and phytic acid are shown in Table 1. The levels of LDopa, TIA, and CIA due to microwave cooking are depicted in Table 2. Similarly, the impact of microwave cooking on phytoheamagglutinating activity is shown in Table 3. Amino acid profiles of total proteins of raw and microwave cooked flours of Kailasa Nadu and Vala Nadu accessions are depicted in Tables 4 & 5, respectively.

3 GURUMOORTHI et al.: NUTRITIONAL VALUE OF VELVET BEAN, AN UNDER UTILIZED PULSE 679 Table 1 Effect of soaking in distilled water followed by microwave cooking on the levels of total free phenolics, tannins and phytic acid in Velvet bean accession (gm 100 gm 1 DM) a ACCESSIONS Treatment Total free phenolics % Tannins % Phytic acid % KAILASANADU Raw Seeds 5.74± ± ± 0.03 (WHITE COLORED) Microwave cooked 0.72 ± ± ± Raw Seeds 5.93 ± ± ± 0.01 (BLACK COLORED) Microwave cooked 0.74 ± ± ± A ALL VALUES ARE MEAN OF TRIPLICATE DETERMINATIONS. ± STANDARD ERROR Table 2 Effect of soaking in distilled water followed by microwave cooking on the levels Ldopa *, trypsin inhibitor activity (TIA) ** and chymotrypsin inhibitor activity(cia) *** in Velvet bean accessions. ACCESSIONS Treatment LDopa % TIA % CIA % KAILASANADU Raw Seeds 5.55 ± (WHITE COLORED) Microwave cooked seeds 5.19 ± Raw Seeds 4.60 ± (BLACK COLORED) Microwave cooked seeds 4.47 ± *g 100 gm1 DM; ** mg of pure trypsin inhibited g1 of sample; *** mg of pure Chymotrypsin inhibited g1 of sample; ±Standard Error; All values are mean triplicate determinations Table 3 Effect of soaking in distilled water followed by microwave cooking on phytohaemagglutinating activity in two different accesssions of Velvet bean a ACCESSIONS Treatment A Blood B Blood O Blood KAILASANADU Raw Seeds (WHITE COLORED) Microwave cooked seed Raw Seeds (BLACK COLORED) Microwave cooked seeds ND a Erythrocytes from human blood and haemaggulitinating activity expressed in units (HU mg 1 protein) The reduction in contents of total free phenolics, tannins and phytic acid due to microwave cooking is significant (87.5%, %, and %), respectively. The reduction in levels of LDopa due to microwave cooking is insignificant ( %) where as TIA and CIA are found to be significant (TIA: %; CIA: %), respectively. Similarly marked reduction in levels of phytoheamagglutinating activity in respect of human erythrocytes (A, B and O) in both the germplasm has taken place. Among the three erythrocytes, the of lectin activity in respect of O blood in Kailasa Nadu germplasm is low compared to other values (76%). Amino acid profiles of total proteins of raw seeds of both the investigated germplasm exhibit variations in levels of different amino acid. When subjected to microwave cooking the amino acid profiles of total proteins shows slightly lower values in respect of the essential amino acids isoleucine, leucine, lysine and tryptophan. Microwave cooking differs from conventional cooking because it is accomplished by means of electromagnetic waves, which penetrate deeply and heat up rapidly the material (food item) 20,21. Microwaves have been applied in cooking legume seeds like soybean to eliminate unpleasant flavour and destroy TIA. An increase in protein efficiency ratio (PER) in soybean compared with raw seeds also has been reported 22, 23. The results of the present study reveal that the microwave cooking has substantially eliminated almost all the antinutritional factors except LDopa. Moreover, microwave cooking caused minimum effect on the profiles of essential amino acids of the seed proteins in both the germplasm.

4 680 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 12, No. 4, OCTOBER 2013 Table 4 Amino acid profiles of total proteins of raw, microwave cooked seed flours of Kailasa Nadu accession Amino acid Aspartic acid Glutamic acid Alanine Valine Glycine Arginine Serine Cystine Methionine Threonine Phenylalanine Tyrosine Isoleucine Leucine Histidine Lysine Tryptophan Proline Total Protein Raw seeds EAA score Microwave cooked ± ND ±0.13 EAA score essential amino acid score; ND not detectable EAA core Table 5 Amino acid profiles of total proteins of raw, microwave cooked seed flours of Vala Nadu accession. Amino acid Raw seeds EAA score Aspartic acid Glutamic acid Alanine Valine Glycine Arginine Serine Cystine Methionine Threonine Phenylalanine Tyrosine Isoleucine Leucine Histidine Lysine Tryptophan Proline Total Protein ± Microwave cooked ND ±0.91 EAA score essential amino acid score; ND not detectable EAA score Conclusion To sum up, the nutritional value of velvet bean can be improved by soaking the seeds followed by microwave cooking so as to reduce/eliminate most of the antinutritional factors without affecting the protein quality and digestibility of the legume proteins. Furthermore, after through processing this underutilized tribal pulse may be used to alleviate Protein Energy Malnutrition and it also be recommended for commercialization and functional food development. Acknowledgement The authors, KJ and GK are grateful to Shri P Raja Rao, Chairman Prathyusha s of companies T Nagar, Chennai. PG is grateful to Dr C Muthamizhchelvan, Director (E&T) and Management Authorities of SRM University. References 1 Janardhanan K & Gurumoorthi P, In: Velvet bean: as source of food, feed and pharmaceuticals, (Wisdom Publications, Chennai (In press). 2 Buckles D, Velvet bean: A new plant with a history, Econ Bot, 49 (1995) Janardhanan K & Lakshmanan KK, Studies on the pulse, Mucuna utilis: Chemical compopsition and antinutritional factors, J Food Sci Technol, 22 (1985) Janardhanan K, Gurumoorthi P & Pugalenthi M, Nutritional potential of five accessions of a South Indian tribal pulse, Mucuna pruriens var.utilis I. The effect of processing methods on the content of LDopa, phytic acid, and oligosaccharides, J Trop Subtrop Agroecosys, 1 (2003) Vadivel V & Janardhanan K, Nutritional and antinutritional composition of velve bean: An underutilized food legume in South India, Intl J Food Sci Nutr, 51 (2000) Mohan VR & Janardhanan K, Chemical analysis and nutritional assessment of the less know pulses of the genes, Mucuna, Food Chem, 52 (1995) Gurumoorthi P Janardhanan K & Myhrman VR, Effect of differential processing methods on LDOPA and protein quality in velvet bean, an underutilized pulse, LWT Food Sci Technol, 41(2008) Infante MH Sousa V Montalvo I & Tena E, Impact of microwave heating on heamagglutinins, trypsin inhibitors and protein quality of selected legume seeds, Plant Food Hum Nutr, 52 (1998) Sasikiran K & Padmaja G, Inactivation of trypsin inhibitors in sweet potatos and taro tuber during processing, Plant Food Hum Nutr, 58 (2003) Maxon ED & Rooney LW, Two methods of tannin analysis for Sorghum bicolor (L.). Moench, grain, Crop Sci, 12 (1972) Bray HG & Thorne WV, Analysis of phenolic compunds, Methods Biochem Anal, 1 (1954) Schanderl S, In: Method in Food Analysis, (Academic Press, New York), 1970, 709.

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