The proteins and trypsin inhibitors were isolated from the seeds of different varieties/accessions of an underutilized legume, Mucuna. The crude protein content of all the germplasms of Mucuna is varied from 15% - 26%, showed little variation and contain higher crude protein when compared with other Mucuna species reported earlier and the pulse crops commonly consumed in India. The seeds of all the varieties of Mucuna exhibited trypsin inhibitor activity. The trypsin inhibitor activity varied from 11 - 14 TIA/mg of protein. Not much variation was observed in trypsin inhibitory activities in soaked seeds compared to dry seeds. Germination of Mucuna pruriens has been carried out and the change in the protein content and trypsin inhibitors were monitored. The protein content of the endosperm increased up to 72 hrs of germination and then decreased. The trypsin inhibitory activity decreased with increase in germination time. The trypsin inhibitor activity was decreased from 14.81 TIA/mg to 2.62 TIA/mg (82% reduction in the trypsin inhibitor activity) after 144 hrs germination.
The seeds of the plants belonging to the family Leguminosae generally rich in proteins required for human consumption in developing countries. Although legumes are rich in proteins, they have limited utilization because of the presence of various anti-nutritional factors including enzyme inhibitors such as trypsin and chymotrypsin inhibitor. Trypsin inhibitors when ingested in large quantities may disrupts the digestive process and leads to undesirable physiological reactions. The suggested functions of protease inhibitors are including acting as storage proteins, regulation of endogenous proteinases, or acting as protective agents against insects or microbial predators. Germination is an inexpensive and effective technology for improving the quality of legumes by reducing the content of antinutritional factors such as protease inhibitors [
Mucuna is one of the lesser known under utilized legume, grown as a minor food crop by tribal and ethnic groups of Asia and Africa. The immature pods and leaves serve as vegetables, while seeds as condiment and main dish by ethnic groups in Nigeria [
In the present study, the variability of proteins and trypsin inhibitors of eight varieties of Mucuna seeds and their stability during germination have been studied.
The seeds of different verities/species of Mucuna (Mucuna hirsute, Mucuna cochinensis, Mucuna cochinensis MP9, Mucuna pruriens MP7, Mucuna species NRC, Mucuna species IIHR MP5, Mucuna species IC2199 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 analytical grade.
The seeds of eight varieties of Mucuna were soaked in distilled water for 12 hours and germinated for six days under standard conditions. The acetone powder (10%) of dry, soaked and germinated seeds of eight varieties of Mucuna were prepared according to the method of [
Protein was estimated according to the method of Lowry et al. (1951) [
The trypsin activity was determined using casein as the substrate [
An anionic disc gel electrophoresis was carried out essentially according to the method of Davis and Ornstein [
The protein content of eight varieties of Mucuna samples ranged between 15% - 26% (
Seeds of different varieties of Mucuna contain trypsin inhibitor activity (
Electrophoretic analysis of trypsin inhibitors revealed the presence of five to seven isoinhibitors in the seeds of different varieties of Mucuna (Figures 1(a) and (b)). The Mucuna hirsute and Mucuna pruriens contained a maximum of seven trypsin isoinhibitors. Analysis of proteins during germination of eight varieties of Mucuna seeds are shown in
TIA: trypsin inhibitor activity; TIU: trypsin inhibitor unit.
(a)
(b)
Figure1. (a) Electrophoretic pattern of trypsin inhibitors isolated from 1) Mucuna hirsute; 2) Mucuna cochinensis; 3) Mucuna utilis IC25333; 4) Mucuna sps. IIHR MP5; 5) Mucuna pruriens MP7; 6) Mucuna cochinensis MP9; 7) Mucuna sps. NRC; 8) Mucuna pruriens; (b) Electrophoretic pattern of trypsin inhibitors during germination of seeds of Mucuna pruriens: D: dry; S: soaked, 24 hrs, 48 hrs, 72 hrs, 96 hrs, 120 hrs and 144 hrs of germination.
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 electrophoretic protein band pattern obtained for different varieties of Mucuna are shown in
The electrophoretic protein band pattern during germination of Mucuna pruriens is shown in
hrs of germination and reappeared on 144 hrs of germination. The intensity of the minor bands increased on germination. In addition to band 6 new bands appeared after 72 hrs of germination and disappeared there onwards. The intensity of band 10 and 11 gradually decreased up to 48 hrs of germination and disappeared up to 120 hrs of germination. These bands were again expressed at 144 hrs of germination. The intensity of the bands 4 and 5 increased gradually throughout the 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.
The seeds of M. pruriens and M. utilis IC25333 showed higher amount of proteins, trypsin 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 trypsin inhibitory activity. The proteins and trypsin inhibitor profile of dry, soaked and germinated seeds of M. pruriens is shown in
Soluble proteins are the physiologically active fractions which constitute the major bulk of enzymes involved in plant metabolism. During germination and plant development, specific metabolic changes have been observed by many investigators. The analysis of zymogram of protein banding pattern revealed the variation in the protein pattern. This result suggests the requirement of these for the development of the plant. The protein content also increased at 72 hrs of germination and decreased thereafter. Analyses of protein banding pattern revealed the wide variation in banding pattern. The intensity of some of the major protein bands gradually decreased and few bands were disappeared followed by
appearance of small molecular weight new protein bands during the germination. The electrophoretic studies showed that high molecular weight polypeptide bands disappeared with the appearance of new low molecular weight polypeptide bands in the endosperm proteins of the germinating seeds.
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 trypsin 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. However, the presence pharmacologically active compound L-DOPA (L-3,4-dihdroxy phenylalanine) is potentially toxic if large amounts are ingested.
The author wish to thank UGC, SWRO, Bangalore, Karnataka, India for financial assistance, Prof. M. R. Doreswamy, Founder Chairman, PES Group of Institutions, Bangalore, Prof. D. Jawahar, CEO, PES Group of Institutions, Bangalore and Dr. K. N. B. Murthy, Principal, PES Institute of Technology, 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.