PROXIMATE COMPOSITIONS, MINERAL CONTENTS AND DETERMINATION OF PROTEASE ACTIVITY FROM GREEN GRAM (Vigna radiata l. Wilczek)

BANGLADESH RESEARCH PUBLICATIONS JOURNAL ISSN: 1998-2003, Volume: 5, Issue: 3, Page: 207-213, May -June, 2011 PROXIMATE COMPOSITIONS, MINERAL CONTENT...
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BANGLADESH RESEARCH PUBLICATIONS JOURNAL ISSN: 1998-2003, Volume: 5, Issue: 3, Page: 207-213, May -June, 2011

PROXIMATE COMPOSITIONS, MINERAL CONTENTS AND DETERMINATION OF PROTEASE ACTIVITY FROM GREEN GRAM (Vigna radiata l. Wilczek) Tapash Paul1, N.H.M. Rubel Mozumder 2*, M. A. Sayed3 and M. Akhtaruzzaman2 Tapash Paul, N.H.M. Rubel Mozumder, M. A. Sayed and M. Akhtaruzzaman (2011). Proximate Compositions, Mineral Contents and Determination of Protease Activity from Green Gram (Vigna radiata L. Wilczek). Bangladesh Res. Pub. J. 5(3): 207-213. Retrieve from

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Abstract The present study was conducted to analyze the proximate compositions and mineral contents of green gram/mungbean (Vigna radiata L. Wilczek) collected from local markets of Dhaka city as well as determination of protease enzyme activity isolated from germinated seeds. The results showed that the green gram contained 21.57g/100g protein, 1.53g/100g crude fat, 0.63g/100g crude fiber, 12.07 g/100g moisture, 3.85g/100g total ash and 60.35g/100g carbohydrate, respectively. The mineral contents of green gram were found to be with (k> P> Mg) increasing order; potassium (1145.88mg/100g sample) was the most abundant mineral followed by phosphorus (315.30mg/100g sample) and magnesium (132.69 mg/100g). The iron (5.04mg/100g sample), zinc (2.83mg/100g sample), sodium (32.92mg/100g sample) and calcium (72.89 mg/100g sample) were found in moderate level. The procedures for protease determination involved a combination of extraction, ammonium sulfate precipitation, protein determination and finally assay of activity on buffer solution using hemoglobin as a substrate. Green gram bean showed protease activity (0.0026 Units/mg) optimum at pH 4 and at 37o temperature using spectrophotometer at 600nm absorbance.

Key Words: Proximate compositions, mineral contents, protease activity and green gram.

Introduction Lack of protein is one of the major world food problems in the diet of many people and the evidence from nutritional surveys in Bangladesh and others developing countries confirm that nearly 80% of proteins consumed by humans are supplied by plants or plant products specially pulses (Osho S.M, 1988; Singh U & Jambunathan, 1991; Jahan & Hossain, 1998). The estimated total production of pulses in Bangladesh is about 0.55 million tons. Though green gram contributes only 38% pulses (second largest) of total production in compare to lentil which contributes 58% of total production (BBS, 2002), it is relatively free from antinutritional factors (low Lectins or haemagglutins content) compared with lentil and other pulses (Chau, Cheung, & Wong, 1997; Wang, Lewis, Brennan, & Westby, Corresponding Author’s email: [email protected] 1 Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000; 2 Department of Food Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200; 3 Department of Biochemistry and Molecular Biology, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200.

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1997; Vijayakumari, Siddhuraju, Pugalenthi, & Janardhanan, 1998). Green gram is an excellent source of protein (25%), high in dietary fiber, rich sources of vitamin and minerals and its essential amino acid composition has been compared favorably with that FAO/WHO reference protein (El-Adawy, 1996; Fan & Sosulski,1974; Thompson, Hung, Wang, Rapser, & Gade, 1976). Its low glycemic index and high folate content reduce blood glucose level and neural tube defects in newborn babies (GRDC, 1994; USDA Nutrient Database for Standard Reference, 2001). A longitudinal study, “The Food Habit in Later Life” conducted in Japan showed that a higher pulse intake (63- 86g/d) is the most protective dietary predictor of survival amongst the elderly (I Darmad et al, 2004). Proteases, a group of hydrolytic enzymes mostly exist in microorganism. Ashton, F. (1976) and Ichihima (1964) reported proteases which are widely distributed in plant especially leguminous seeds and found to be mainly as neutral and alkaline but few acidic with pH optimum at 2-3. The activity of proteases during germination in green gram has been reported by several studies (Lin & Yao, 1995; Rahman et al., 2007). This plant protease plays various important physiological roles in the metabolism of seeds protein by inducing the formation of enzymes that eliminate or reduce the ant-nutritional and indigestible factors in legumes during germination (Bau,Villanme, Nicolos,& Mejean, 1997). Besides theses, proteases can be used in pharmaceutical industries, leather industries and various foods processing industries (Fox PF and Morrissey PA., 1980). Therefore, the present study conducted to determine the proximate compositions and mineral contents of green gram for the best possible utilization for the poor people of Bngaldesh as well as isolation and determination of protease to provide data for future work as an excellent source of protease for industrial purposes.

Materials and Methods Seeds of Green gram (Vigna radiata L. Wilczek) were purchased from six local markets of Dhaka city. For the preparation of composite sample, the seeds were then grinded in a mechanical grinder, mixed, and finally kept in an airtight glass-container. All the chemicals used in this experiment were analytical grade. Proximate composition Proximate composition such as moisture, ash, crude fat (solvent extraction), crude protein (Kjeldahl N × 6.25), and crude fiber were determined in at least triplicate using the methods as described by AOAC, 1984). The nitrogen content was estimated by the micro- Kjeldahl method and the crude protein content was calculated (N x 6.25). Crude lipid content was determined using Soxhlet apparatus. The Total carbohydrate content was determined by difference and energy value was calculated following the method as described by Osborne and voogt (1987). Mineral content Sodium and potassium were determined by using a flame photometer (AA-6800) and phosphorus by using spectrophotometer (JASCO V-630) followed by the method as described by Pearson 1976. All determinations were done in triplicate. The concentration of phosphorus was determined through the measurement of the yellow phosphor vanado-molybdate complex using Cecil Carating Digital Spectrophotometer Series. All other minerals were determined using atomic absorption spectrophotometer. Isolation and preparation of protease (crude) enzyme Crude enzyme was extracted by following the method given by Dahot MU et al., 1985. Fifty grams of Green gram (Vigna radiata) seeds were washed and soaked in distilled water for over night at room temperature for germination. Then seeds http://www.bdresearchpublications.com/journal/

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were grinded by electric homogenizer using cold acetone to remove the fat and filtered by suction through double rings filter papers. Then the homogenates were air dried for overnight. After that, seeds were powdered finely under ice-cold condition in a mortar using pestle and finally suspended in 10mM Tris-HCl buffers at pH 8 containing two molar (2M) NaCl for four hrs. The extracted mixture was then filtered through gauge in a cold environment. The filtrate was centrifuged at 10000 rpm for 8 minutes below 4oC. The supernatant was designated as the crude extract for further investigations. Determination of water-soluble protein Protein concentration was determined by the method of Lowry et al., 1951 using bovine serum albumin as a standard and the absorbance was measured at 660nm by spectrophotometer. The amount of the soluble protein was calculated from the standard curve as mg of protein per ml of test samples. Assay of protease activities Protease assay using hemoglobin as a substrate was done according to the method outlined by Anson (1938). The reaction mixture consisting of 250 µl of enzyme solution and 1 ml of substrate (2% hemoglobin for acidic pH 4 in glycineHCl buffer ) was incubated at 37oC for 30 minutes after which the reaction was terminated by the addition of 500µl of 10 % TCA. For the control, the enzymes were used after addition of 500µl 10% TCA. The precipitated material was removed by centrifugation (10000 rpm for 10 minutes) and collected 0.3ml supernatant was mixed with 2.5ml Cu-alkaline solution using vortex machine and allowed to stand for 15 minutes. Thereafter, 0.2ml FCR (double times diluted) reagent was added to mixture and allowed to stand for another 20 minutes. Then the reading was taken in spectrophotometer at 660 nm. The protease activity was expressed as difference of absorbance between the control sample and the test sample and specific activity of the protease is calculated by the activity of protease per milligram protein per minute.

Results and Discussion Proximate composition The proximate compositions of green gram are given in Table 1. The moisture content was observed in green gram is 12.07 g/100g. The data from different workers (Table 1) showed that moisture in green gram ranged from 8.25 to 9.75 g/100g that is much lower than those our findings (Bhatty et al, 2000; Mubarak A.E., 2005 and U.A, Agugo et al., 2009). However, much higher moisture content has also been reported (Siddique M, 1989). Green gram was found to contain highest ash content (3.85g/100g) in this study which is good agreement with the value (3.45g/100g sample) reported by several studies (Savage and Deo, 2000; U.A, Agugo et al., 2009). However, Bhatty et al., 2000 reported the higher ash content (4.63 g/100g) of Green gram. Ash content in these pulses indicates that the pulse seed provides essential minerals. The protein content of green gram was 21.57 g/100g sample that is lower than the values reported by three previous studies shown in Table 1. From the literature, protein content of green gram was reported to range from 20.3 to 29.0 percent (Augustine and Klein, 1989 and Calloway et al., 1994). The differences may be due to location and variety effect. Crude Fat content of green gram have been reported to 1.53 percent. While Bhatty et al., 2000 showed a higher crude fat content (2.83%) for Green gram but Savage and Deo (2000); A.E. Mubarak, 2005 and Agugo. U.A, et al., 2009 reported quite similar value of fat content for Green gram. All these variations may be due to variety differences. The fiber content of green gram was found to be 0.63 percent per 100 g sample that is a quite inconsistent value reported by http://www.bdresearchpublications.com/journal/

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three studies (Bhatty et al, 2000; Mubarak A.E., 2005 and Agugo. U.A, et al., 2009). However, Augustine and Klein (1989) showed the fiber content of green gram ranged from 1.6 to 3.3 percent. Moreover high fiber content makes green gram a good digestive food. Green gram was found to contain 60.35 percent carbohydrate. This finding slightly corresponds with of the value reported by Agugo. U.A, et al. (2009) but lower than those reported by Bhatty et al., 2000. The variations observed may be due to different processing methods. However, Savage and Deo, (2000) and Mullar, (1988) repoted as low as 35 and 40 percent carbohydrate. Mineral composition The minerals content of Green gram are depicted in Table 2 under present study with increasing order (K > P > Mg): potassium was found to be the most abundant mineral that is slightly lower than the value (1246mg/100g) reported by USDA Nutrient Database for Standard Reference (2001) but larger than the values reported by Olaofe.O.et al., 2009 for Chinese bottle. Phosphorous was found to be the next highest mineral component of 315.30±10.79 mg/100g sample that was lower than the value (367 mg/100g) reported by USDA Nutrient Database for Standard Reference (2001). However, Aremu et al., 2006 reported lower Phosphorous content (196.4mg/100g sample) in P. Africana. The iron content in green gram was 5.04±0.24mg/100g sample. Nevertheless, Agugo U.A, et al., 2009 found iron content in green gram was to be 4.23mg and 6.45mg respectively. The bean was richer in magnesium (132.69±0.68 mg/100g), calcium (72.89±0.73) and sodium (32.92±0.07 mg/100g) but the values of copper and zinc were low which was1.66±0.09 mg/100g and 2.83±0.03mg/100g sample respectively. These values are in good agreement with those values reported by Gopalan et al., 1993. Assay of Activity of protease enzyme The activity of protease enzyme of green gram bean compared with several values reported by previous studies is presented in Table 3. In present study, the protease activity was found to be a value of 0.0026 units per mg protein per minute in crude extracts isolated from germinated green gram when estimated with haemoglobin as substrates. From the table, Rahman et al., 2007 showed the range of protease activity was 3.05-7.78Unit/g of protein for locally available green gram during different germination period which is not consistent with our value. Similarly, Lin and Yao (1996) and Vidyavati et al.,(1983) reported higher protease activity. These fluctuations may be due to the germination period and effect of several inhibitory factors of green gram as reported by several studies (Dahut MU, 1992; Krishna Chaitanya, Keshavkant and Naithani, 2000; ElMorsi, 2001 and Shaoyun Wanga et al., 2006).

Conclusion The proximate compositions, mineral contents and protease activity were determined from green gram. The results of proximate analysis and mineral values indicated Green gram might be excellent source of protein that supplements our diets especially for protein. The protease activity revealed that it would be a good source of enzyme for industrial purposes.

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Table 1: Proximate composition (g/100g sample) a of green gram compared with previous studies Parameters Moisture Ash Crude Protein Crude Fat Crude fiber Carbohydrate Caloric value(Kcal)

Nutrients (Present study) 12.07±0.25 3.85±0.05 21.57±0.18 1.53±0.05 0.63±0.01 60.35

Reference By A.E. Mubarak, 2005 9.75 3.76 27.5 1.85 4.63 62.3

Reference By Bhatty et al. 2000 8.25 4.63 25.00 2.83 1.68 65.86

Reference By U.A, Agugo et al. 2009 NG 3.34 22.90 1.43 8.95 61.47

342.71

NG

NG

NG

Table 2: Minerals content of green gram (Green gram) Nutrients(Mean±SD) (mg/100 g sample)a 5.04±0.24 72.89±0.73 2.83±0.03 1.66±0.09 132.69±0.68 315.30±10.79 32.92±0.07 1145.88±8.78

Parameters Fe(mg) Ca(mg) Zn(mg) Cu(mg) Mg(mg) P(mg) Na(mg) K(mg)

Table 3: Protease activity (Unit/mg) of green gram compared with previous studies Source of Protease

Green gram Mungbean Mungbean Cotton seed Mungbean

Protein conc. (mg/ml)

Protease activity (Specific activity) (Unit/mg)

Reference Values

3.75 NG NG 2.30 NG

0.0026 a 3.05-7.78 b 0.62 c 3.0 d 1.0-3.5 e

Present study Rahman et al., 2007 Lin and Yao, 1996 Dahot M. Umar, 1992 Vidyavati et al. 1983

(a) † Value is the average of triplicate samples ± standard deviation (SD) NG: protein concentration not given in study report a: specific activity determined as acidic protease using hemoglobin substrate b: specific activity determined as alkaline protease using casein substrate c: specific activity determined as acidic protease using hemoglobin substrate d: specific activity determined as acidic protease using hemoglobin substrate

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