STUDY ON PROTEIN FRACTIONATION AND IN VITRO DIGESTIBILITY OF SOME SELECTED LEGUMES Ranjani Amarakoon, Franti ek Bunka, Stanislav Kracmar, Franti ek Kocar, Ignác Hoza Abstract: The objective is to study on protein fractionation and in vitro digestibility of some legumes namely Pisum sativum (Terno, Xantos, Svit, Achat), Glycine max, Lupinus albus (Amiga), Pisum sativum var. arvense (Arkta), Faba vulgaris (Piestansky). Further, the changes of in vitro digestibility of protein and dry matter in cotyledons and radicles (shoot) separately of those legumes after germinating for 48 hours in comparison to the respective raw seeds were investigated with a view to assess the nutritional value. The ranges of the albumin, globulin, prolamin, glutelin and non-protein compound (residue) in the legumes tested were 40.3-48.5%, 38.6-42.0%, 3.5-5.3%, 3.4-6.4% and 2.8-7.2 % of the total extractable protein, respectively. Albumin was the main protein fraction while prolamin and glutelin were found as minor fractions in most of all legumes. In vitro digestibility of proteins in all legumes were improved significantly after germinating 48 hours in their cotyledons and radicles when compared with the raw seeds of the respective legumes. Key words: albumin, globulin glutelin, prolamin, in vitro digestibility, germinating INTRODUCTION Many studies on proteins of legumes explain different reasons for limited digestibility of the seeds. One of the main factors affecting their limited digestibility is the type of proteins present in the legume and its limited susceptibility to hydrolysis by digestive proteases due to its structural characteristics (Melito & Tovar, 1995; Tavano & Neves, 2008). Proteins in legumes are usually classified into two major fractions, namely, globulins and albumins. Further, it is noted that fractions of prolamin and glutamine have very low amounts (Adebowale et al., 2007). Albumins were much less digestible than globulins, mainly because of the primary structure, native conformation, involvement of disulphide bonds in the formation of complexes and those complexes are poorly susceptible to proteolysis. The low digestibility of globulins has been related to their compact structure and intracellular location that hinder the susceptibility to proteolysis (Clemente et al., 2000). Though many studies have been carried out on legumes, information regarding protein fractionation for the legumes considered and in vitro digestibility of cotyledons and radicles separately after germinating is not available. The main purpose of the present investigation was to study on fractionation of protein in selected legumes and In vitro digestibility of cotyledons and radicles separately after germinating 48 hours (h) and comparing with the respective raw seeds with the view to assessing nutritional quality of legumes. MATERIALS AND METHODS The eight legumes namely Pisum sativum (Terno, Xantos, Svit, Achat), Pisum sativum var. arvense (Arkta), Lupinus albus (Amiga), Glycine max, Faba vulgaris (Piestansky) were ground and part of seeds were germinated according to the method of Khalil et al.,
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2007 with a few modifications. Methods of AOAC were followed to determine dry matter (DM) and mineralization and micro Kjeldhal method for crude protein (CP). Fractionation of protein was carried out on the basis of solubility in different solvents, distilled water, 5% NaCl, 70% ethanol and 0.1 M NaOH into albumins, globulins, prolamins and glutelins as described by Chavan et al., 2001. In vitro digestibility of dry matter and crude protein for ground raw seeds and germinated legumes were determined by using pepsin (3g/1.5 l of 0.1 mol/l HCl) in the DaisyII Incubator (Ankom Technology, New York, USA) for digestion at 39±1°C 24 h and then jars were kept at 80±1°C for 30 minutes. Samples were dried in oven at 105±1°C for 24 h after washing and DM was recorded and undigested protein was measured by the micro Kjeldahl method. RESULTS AND DISCUSSION According to the results of protein fractionation presented in Table 1 albumin was found to be the main protein fraction in most of all legumes is in agree with some cultivars of P. sativum study by Mart nez-Villaluenga et al., (2008). Globulin was slightly higher than albumin fraction in P. sativum (Xantos) and Lupinus albus (Amiga). Prolamine and glutelin were the minor protein fractions in all legumes and similar results observed for many legumes in past literature (Adebowale et al., 2007). The ranges of content of albumins, globulins, prolamins, glutelins and residue in the legumes tested were 40.3-48.5%, 38.642.0%, 3.5-5.3%, 3.4-6.4%, and 2.8-7.2% of the total extractable protein, respectively. Prolamin and glutelin content were highest in Lupinus albus (Amiga) i.e. 5.3% and 6.4% respectively. Differences among cultivars were evident in all the legumes studied. According to Table 2, it is clearly seen that In vitro protein digestibility (IVPD) and In vitro digestibility of DM of cotyledons and radicles were increased after germinating for 48 h in comparison with the results of the IVPD of raw seeds and it has increased significantly in radicles of all legumes in this study. It ranged from 86.7 to 93.4% which is higher than both cotyledons and raw seeds. Since the radicles can be eaten even without further process, the high digestibility is very important from the point of view of health. The IVPD of cotyledon of legumes ranged from 79.1% to 86.4% and this appreciable increase at early stage of germination is in agreement with raw Indian bean (Ramakrishna et al., 2008). Significant variations of IVPD in different legume species and greatly improved with germination were noted (Khalil et al., 2007). Significant increase in IVPD after germinating 48 hours confirms the alterations in protein structure of different fractions. Low IVPD in raw seeds (54.0 - 75.0%) when compared to cotyledons and radicles may be due to more closed structure of polysachcharides in the seeds which does not facilitate to penetrate digestive proteolitic enzymes to digest proteins. CONCLUSION The legumes studied are good sources of protein and rich with albumin which seems to be one of the factors affecting low digestibility of raw seeds. A significant increase of CP and IVPD in radicles of all legumes may be due to structural changes of protein. In general the quality of proteins all legumes were improved significantly after germinating 48h in their cotyledons and radicles when compared with the raw seeds of the respective legumes.
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ACKNOWLEDGMENT This work was kindly supported by a project of Ministry of Education, Youth and Sports of the Czech Republic (Grant no. MSM 7088352101). REFERENCES 1. ADEBOWALE, Y. A., ADEYEMI, I., OSHODI, A. A., NIRANJAN. K., 2007. Isolation, fractionation and characterisation of proteins from Mucuna bean. Food Chemistry, 2007, 104, 287-299. 2. CHAVAN. U. D., MCKENZIE, D. B., SHAHIDI, F., 2001. Protein classification of beach pea (Lathyrus maritimus L.). Food Chemistry, 2001, 75, 145-153. 3. CLEMENTE, A., VIOQUE, J., SANCHEZ-VIOQUE, R., PEDROCHE, J., BAUTISTA, J., MILLAN, F., 2000. Factors affecting the in vitro protein digestibility of chickpea albumins. Journal of Science of Food Agriculture, 2000, 80, 79-84. 4. KHALIL, A. W., ZEB, A., MAHMOOD, F., TARIQ, S., KHATTAK, A. B., SHAH, H., 2007. Comparison of sprout quality characteristics of desi and kabuli type chickpea cultivars (Cicer arietinum L.). LWT - Food Science & Technology, 2007, 40, 937-945. 5. MELITO, C., TOVAR, J.,1995. Cell wall limit in vitro protein digestibility in processed legume seeds. Food Chemistry, 1995, 53, 305-307. 6. RAMAKRISHNA,V., RANI, P. J., RAO, P. R., 2008. Nutritional quality of storage proteins during germination of Indian bean (Dolichos lablab. var. lignosus) seeds. International Journal of Food Science & Technology, 2008, 43, 944-949. 7. SULIEMAN, M. A., HASSAN, A.B., OSMAN, G. A., EL TYEB, M. M., EL KHALIL, E. A. I., EL TINAY, E. H., BABIKER, E. E., 2008. Changes in total protein digestibility, fractions content and structure during cooking of lentil cultivars. Pakistan Journal of Nutrition, 2008, 7, 801-805. 8. TAVANO, O. L., NEVES. V. A., 2008. Isolation, solubility and in vitro hydrolysis of chickpea vicilin-like protein. LWT - Food Science and Technology, 2008, 41, 1244-1251.
Contact address: Ranjani Amarakoon, Department of Biochemistry and Food Analysis, Faculty of Technology, Tomas Bata University in Zlin, Nam. T. G. Masaryka 275 762 72 Zlin, Czech Republic. E-mail:
[email protected]
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Table 1 Content (%) of protein fractions in some selected legumes Legume Pisum sativum
Cultivar
Albumin
Globulin
Prolamin
Glutamin
Residue
Terno
44.1
± 0.96
40.5 ± 0.09
4.5
± 0.13
5.8
± 0.01
4.3
± 0.01
Xantos
41.3
± 1.50
42.0 ± 2.28
4.5
± 0.07
6.2
± 0.11
4.7
± 0.21
Svit
44.1
± 0.48
40.3 ± 0.83
5.1
± 0.01
5.1
± 0.13
4.6
± 0.01
Achat
45.0
± 0.35
41.6 ± 0.24
4.5
± 0.11
4.1
± 0.11
3.6
± 0.06
48.5
± 0.25
38.6 ± 0.65
3.6
± 0.11
5.8
± 0.07
2.8
± 0.14
Glycine max Lupinus albus
Amiga
40.3
± 0.84
40.1 ± 1.23
5.3
± 0.17
6.4
± 0.10
7.2
± 0.09
P.sativum var. arvenes
Arkta
45.9
± 0.29
40.4 ± 0.58
4.3
± 0.11
3.4
± 0.09
5.4
± 0.19
Faba vulgaris
Piestansky
45.3
± 0.31
41.9 ± 0.36
3.5
± 0.16
4.0
± 0.14
3.6
± 0.02
Data shown are mean ± SD; n =10
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Table 2 Values and digestibility of dry matter and crude protein of raw seeds, cotyledons and radicles of legumes after germinating 48 hours Legume
Cultivar
Pisum sativum Terno
raw seeds cotyledons radicles Xantos raw seeds cotyledons radicles Svit raw seeds cotyledons radicles Achat raw seeds cotyledons radicles raw seeds Glycine max cotyledons radicles raw seeds Lupinus albus Amiga cotyledons radicles raw seeds Pisum sativum Arkta var. arvense cotyledons radicles Faba vulgaris Piestansky raw seeds cotyledons radicles
Dry matter (DM) %w/w 90.7 ± 0.01 41.9 ± 0.71 12.3 ± 1.49 91.5 ± 0.08 38.6 ± 0.50 12.1 ± 0.05 91.3 ± 0.09 39.6 ± 0.49 11.3 ± 0.63 91.5 ± 0.10 40.1 ± 0.55 10.4 ± 0.66 93.5 ± 0.11 45.8 ± 0.73 20.3 ± 1.07 92.1 ± 0.07 38.4 ± 2.84 26.8 ± 1.05 90.4 ± 0.09 49.8 ± 1.36 13.0 ± 0.06 91.7 ± 0.16 44.0 ± 2.45 20.3 ± 0.96
Digestibility of DM% 58.3 ± 2.51 66.2 ± 2.22 75.3 ± 1.88 60.8 ± 1.81 70.6 ± 2.40 74.9 ± 1.11 69.5 ± 4.71 79.2 ± 5.36 74.7 ± 1.65 69.5 ± 3.71 79.3 ± 4.14 71.1 ± 1.37 71.5 ± 2.49 76.7 ± 2.49 82.9 ± 1.98 69.6 ± 0.47 77.6 ± 4.36 85.2 ± 2.49 51.1 ± 2.24 56.5 ± 2.84 74.0 ± 5.08 67.8 ± 3.88 70.8 ± 3.04 76.7 ± 4.10
Data shown are mean ± SD; n =10
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Crude protein (CP) g/100g of DM 24.2 ± 0.51 25.5 ± 0.54 44.7 ± 0.61 21.9 ± 0.47 23.4 ± 0.48 47.4 ± 0.52 23.1 ± 0.35 28.8 ± 0.38 62.5 ± 0.39 22.4 ± 0.50 23.1 ± 0.52 41.6 ± 0.57 34.9 ± 0.40 36.2 ± 0.41 32.9 ± 0.46 33.9 ± 0.52 48.0 ± 0.60 46.6 ± 0.56 21.5 ± 0.39 19.0 ± 0.35 20.2 ± 0.40 29.0 ± 0.26 34.7 ± 0.30 64.9 ± 0.38
Digestibility of CP % 64.9 ± 4.41 83.7 ± 4.18 91.7 ± 1.84 62.6 ± 4.40 79.1 ± 4.56 88.2 ± 2.81 75.0 ± 3.55 82.9 ± 0.81 91.4 ± 1.71 73.7 ± 2.21 83.1 ± 1.39 88.3 ± 3.08 74.9 ± 3.95 86.4 ± 0.48 93.4 ± 4.43 66.0 ± 1.16 80.3 ± 1.04 86.7 ± 0.42 54.1 ± 3.84 64.5 ± 3.13 90.3 ± 2.19 65.6 ± 1.86 82.4 ± 4.74 92.6 ± 3.90
