INTRODUCTION:

The genus Mucuna is one of the lesser known underutilized legume, belongs to the family fabaceae (leguminosae), include up to 150 species. They are annual or perennial legumes and used as a minor food crop by certain tribal and ethnic groups of Asia and Africa. Many parts of the plants are useful such as immature pods and leaves serve as vegetables, while seeds as condiment and main dish by ethnic groups in Nigeria¹. More than 15 varieties/accessions are available in southern India and seeds are relatively good source of protein and have a relatively favorable amino acid composition. Mucuna seeds are a promising source of protein. However, Protein digestibility is affected by the presence of protease inhibitors which inhibits the proteolytic enzymes. A high level of protease inhibitors in the diet stimulates pancreatic juice secretion and causes pancreatic hypertrophy and poor growth performance in animals². Soaking and germination slightly reduces the levels of protease inhibitory activity.

Germination is an economical and effective technology for improving the quality of legumes by enhancing their digestibility³, and reducing the content of antinutritional factors ⁴.

In the present study, variation and stability of storage proteins and chymotrypsin inhibitors from Mucuna seeds during germination have been studied.

Materials and Methods:

The seeds of Mucuna hirsute, Mucuna cochinensis, Mucuna cochinensis MP9, Mucuna pruriens MP7, Mucuna species NRC, Mucuna species IIHR MP5, Mucuna species IC 2199 and Mucuna utilis IC25333 were collected from different parts of Karnataka, Tamilnadu, Kerala and IIHR, Bangalore, Karnataka, India. Bovine pancreatic trypsin, α – chymotrypsin, Casein, N – acetyl – DL – phenylalanine – β – naphthyl ester (APNE), acrylamide, N,N methylene bis acrylamide were obtained from Sigma Chemical Co. All other chemicals were of technical grade.

The dry seeds of eight varieties of Mucuna were soaked in distilled water for 8 - 12 hours and germinated for six days under standard conditions. The acetone powder (10 %) was prepared according to the method of Wetter⁵. Seeds (10 g) were blended in a blender for 5 min using chilled acetone, then filtered using suction pump under vacuum and dried at 37^o C. The crude extract from the acetone powder of dry, soaked and germinated seeds of eight varieties of Mucuna were prepared using 0.05 M phosphate buffer, pH 7.0 by stirring over a magnetic stirrer for 1 1/2 hr at 4^o C. It was then centrifuged at 10,000 rpm for 15 min at 4^o C. The supernatants were collected and used for qualitative and quantitative analysis of total proteins and chymotrypsin inhibitory activity. The total protein content was determined according to the method of Lowry et al⁶. The chymotrypsin activity was determined using casein as the substrate according to the method of Kakade et al⁷. Twenty four µg of chymotrypsin was taken in 2.0 ml of sodium phosphate buffer, pH 7.6 containing 0.15 M NaCl. The reaction was initiated by the addition of 2.0 ml of 2% casein at 37^oC. The reaction was stopped after 20 minutes by the addition of 6% trichloroacetic acid (6.0 ml) and after standing for 1 hr, the suspension was filtered through whatman no. 1 filter paper. Absorbance of the filtrate was measured at 275 nm using spectrophotometer. One chymotrypsin unit is arbitrarily defined as an increase in absorbance by 0.01 at 275 nm under conditions of assay. The chymotrypsin inhibitor activity was determined using casein as the substrate according to the method of Kakade et al⁸. 24 µg of chymotrypsin was preincubated with known aliquots of the inhibitor extract in a total volume of 2 ml at 37^oC for 10 min in 0.01 M sodium phosphate buffer, pH 7.6, containing 0.15 M NaCl. The residual enzyme activity was determined as described above. Chymotrypsin inhibitory unit is defined as the number of chymotrypsin units inhibited under the assay conditions.

Polyacrylamide gel electrophoresis: the native polyacrylamide gel electrophoresis was carried out according to the method of Davis and Ornstein⁹. A discontinuous gel system consisting of 8% separating gel and 4% spacer gel was used. The electrophoresis was carried out in cold applying a current of 20 – 25 mA for 4 hours using tris–glycine (pH 8.3) as electrode buffer and bromophenol blue as marker dye. After the electrophoresis, the proteins were stained with coomassie brilliant blue R – 250 for 1 hour and destained using 7 % acetic acid. Visualization of chymotrypsin inhibitor in polyacrylamide gel was performed according to Filho and Moriera¹⁰. The gel was stained for chymotrypsin inhibitory activity by incubating the gel in 100 µg chymotrypsin / ml in 0.1 M phosphate buffer, pH 7.6 for 20 min at 37^o C and then stained using 0.1 M phosphate buffer pH 7.6 containing 0.8 mM N – acetyl – DL – phenylalanine – β – naphthyl ester (APNE) and 0.5 mg Diazo Blue B / ml for 1 hr. The gels were stored in 7% acetic acid.

Result and Discussion:

The total protein content of eight varieties of Mucuna seeds ranged between 15 – 26% (Table 1). The protein content of Mucuna pruriens and Mucuna utilis IC25333 was higher than that of commonly consumed legumes. The protein content of the Mucuna seeds (Mucuna pruriens and Mucuna utilis IC25333) compared well with that of cowpea (Vigna unguiculata) at 29.3% and mung bean (Phaseolus aureus) at 26.5% ^{11& 12}. It was reported that the protein content of Mucuna bean grown in different locations of the tropics and subtropics is between 21.0 - 30.3% and this variation may be due to interaction between genetic makeup and the environment¹³.

Analysis of protein profile of raw and germinated seeds of different varieties of Mucuna revealed that there is no significant change in the protein content up to 48 hrs of germination (Table 2). After 72 hrs of germination, among eight varieties, M. utilis, M. hirsute and M. pruriens showed increase in the protein content by 8.7%, 9.4% and 12.2% respectively, where as M. cochinensis, M. sps.NRC and M.cochinensis MP9 showed increase in the protein content by 19.8%, 23% and 24.5% respectively. A remarkable increase in the protein content was observed in M. pruriens MP7 (34%) and M sps IIHR MP5 (39%). During 92 hrs of germination, M. hirsute, M. utilis IC25333, M. pruriens MP7, M. pruriens MP9, M. sps.NRC and M. pruriens showed decrease in the protein content, however, increase in the protein content was observed in M.cochinensis (21.4%) and M.sps. IIHR MP5 (45%). The seeds of all the varieties/species of Mucuna showed decrease and increase in the protein content after 120 and 144 hrs of germination. The observed changes in soluble protein content indicated that these were intimately involved in the process of germination.

The major drawback which limits the nutritional and food qualities of the legumes is the presence of anti-nutritional factors. The presence of protease inhibitors in the diet inhibits proteolytic enzymes leads to the formation of the irreversible protease-protease inhibitor complex, resulting a decrease in protein digestibility, leading to slower animal growth². Seeds of different varieties of Mucuna contain chymotrypsin inhibitor activity (Table 1) and ranged from 3.23 – 7.0 CIU / mg of protein which less than in red kidney bean extract (12.62 U/mg proteins). This result was in agreement with Ramirez et al¹⁴ who reported the presence of chymotrypsin inhibitors in soybean and red kidney beans. The variation in the amount of chymotrypsin inhibitors between the present result and other reports is due to the different cultivars, climate, variety or other factors, which can influence the production of inhibitors in legume seeds¹⁵. The chymotrypsin inhibitor profile of dry, soaked and germinated seeds of M. pruriens is shown in Table 3. There is no significant decrease in CIA level was noticed up to 48 hrs of germination and gradual reduction followed by significant reduction with the increase of germination time. After 144 hrs of germination, CIA was significantly reduced (CIA was decreased by 82%). The results obtained in this study agree with those reported for kidney bean ¹⁶.

Electrophoretic analysis of chymotrypsin inhibitors revealed the presence of five to seven isoinhibitors in the seeds of different varieties of Mucuna (Fig. 1a). The Mucuna hirsute and Mucuna pruriens contained a maximum of seven chymotrypsin isoinhibitors. The seeds of M. pruriens and M. utilis IC25333 showed higher amount of chymotrypsin inhibitory activity. M. pruriens is one of the major legume seed commonly available and it was chosen for further studies to analyze the effect of germination on proteins and chymotrypsin inhibitory activity. Electrophoretic analysis of chymotrypsin inhibitors during germination of Mucuna pruriens are shown in Fig. 1b. It is observed the gradual decrease in the intensity of chymotrypsin isoinhibitor bands up to 4^th day of germination and steep decrease in the intensity of the bands thereafter.

The protein banding pattern revealed the presence of total 11 bands on anionic polyacrylamide gel electrophoresis (Fig 2a). Mucuna cochinensis and Mucuna cochinensis MP9 were identical in both number of bands (total of 09 bands) and banding pattern. Mucuna species NRC and Mucuna species IC2199 were identical in both number of bands (total of 10 bands) and banding pattern. Mucuna species IIHR MP5, Mucuna utilis IC25333 and Mucuna pruriens were identical in both number of bands (total of 11 bands) and banding pattern. However, a change in the intensity of bands was observed among these samples. The electrophoretic protein band pattern during germination of Mucuna pruriens is shown in Fig. 2b. Electrophoretic analysis of proteins revealed the presence of total 11 bands in the soaked seeds of Mucuna. After 24 hrs of germination, there are total 12 protein bands and intensity of the band 9 was increasing and was maximum at 48 hrs of germination. Some of the bands were disappeared and new bands were appeared during germination.

Electrophoretic protein profiles of different accessions of the same subspecies showed identical or similar patterns, confirming the stability of seed storage proteins within these subspecies. However, considerable variation of protein patterns was observed among the seeds of different varieties of Mucuna pruriens, Mucuna cochinensis and Mucuna hirsute. This could be correlated to different geographical origins.

CONCLUSION:

The protein profile of Mucuna germplasms suggests that mature Mucuna bean seeds can be used as food source and that the bean merits wider use by tribals of Karnataka and other parts of India. The high protein content in the seeds of Mucuna indicated that seeds are good source of proteins, if the seeds are properly processed. The presence of chymotrypsin inhibitors as anti-nutritional factors identified in the current study should pose a problem in human consumption if the beans are not properly processed. Germination is one such seed processing method and this method can be used to process Mucuna beans before consumption.

ACKNOLDGEMENTS:

The author wish to thank UGC, SWRO, Bangalore, Karnataka, India for financial assistance, Prof. M. R. Doreswamy, Founder Chairman, PES Group of Institutions, Bangalore and Prof. D. Jawahar, CEO, PES Group of Institutions, Bangalore for providing research facility to carry out this research work. The author also wishes to thank IIHR, Bangalore, for providing few varieties of seeds of Mucuna and people who are directly or indirectly involved in the collection of Mucuna seeds from different parts of Karnataka as well as Tamilnadu and Kerala.

Table 1: Protein and chymotrypsin inhibitor profile of seeds of different varieties of Mucuna

NO	Samples	Total protein (mg / gm)	CIA (CIU/mg)
1	Mucuna hirsute	206	5.68
2	Mucuna cochinensis	188	5.20
3	Mucuna utilis IC25333	261	7,00
4	Mucuna sps. IIHR MP5	162	4.00
5	Mucuna pruriens MP7	211	4.10
6	Mucuna cochinensis MP9	161	3.76
7	Mucuna sps. NRC	154	3.23
8	Mucuna pruriens	251	7.00

Table 2: Protein profile of raw and germinated seeds of different varieties /species of Mucuna

Plant material	Dry seeds	Soaked seeds	24 hrs	48 hrs	72 hrs	96 hrs	120 hrs	144 hrs
Mucuna hirsute	203	206	206	213	222	219	158	179
Mucuna cochinensis	182	188	191	208	218	221	114	142
Mucuna utilis IC25333	254	261	253	272	276	181	108	121
Mucuna sps. IIHR MP5	151	162	162	178	210	219	144	179
Mucuna pruriens MP7	206	211	223	240	276	219	132	156
Mucuna cochinensis MP9	159	161	174	179	198	119	111	123
Mucuna sps. NRC	143	154	153	170	176	149	114	164
Mucuna pruriens	246	251	253	260	276	219	124	161

Table 3: Proteins and protease inhibitor profile of dry, soaked and germinated seeds of Mucuna pruriens

Germination Time	Total protein (mg/gm)	CIA (CIU/mg)
Dry Seeds	246	6.78
Soaked Seeds	251	7.00
24 hrs	253	6.41
48 hrs	260	7.25
72 hrs	276	7.76
96 hrs	219	5.86
120 hrs	124	3.35
144 hrs	161	1.24

CIA-Chymotrypsin inhibitor activity; CIU-Chymotrypsin inhibitor units

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Received on 20.10.2012 Modified on 05.11.2012

Asian J. Research Chem. 5(11): Nov., 2012; Page 1388-1391