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Functional properties of native and chemically modified protein concentrates from bambarra groundnut
Food Research International 40 (2007) 1003–1011 www.elsevier.com/locate/foodres
Functional properties of native and chemically modified protein concentrates from bambarra groundnut O.S. Lawal a
a,*
, K.O. Adebowale b, Y.A. Adebowale
c
Department of Chemical Sciences, Olabisi Onabanjo University, Ago-Iwoye, P.M.B 2002, Ago-Iwoye, Ogun State, Nigeria b Department of Chemistry, University of Ibadan, Ibadan, Nigeria c Department of Food Technology, University of Ibadan, Ibadan, Nigeria Received 12 January 2007; accepted 6 May 2007
Abstract Protein concentrate (BNP) was prepared from bambarra groundnut (Voandzeia subterranean) and it was derivatised with succinic (BSP) and acetic anhydride (BAP). Chemical composition revealed significant (P < 0.05) increases in the moisture and ash contents following modifications, while the crude fat was reduced. Protein solubility reduced with increase in pH of the solution until it reached isoelectric points of 3, 3.5 and 4 for BSP, BAP and BNP, respectively, following which steady increases were observed for the proteins as the pH increased. The results indicate an initial increase in emulsifying activity (EA) with increase in protein concentration. Both acetylation and succinylation improved the emulsifying stability of the native protein. Foaming capacity of both native and modified proteins increased with increase in protein concentration. Except at pH 2, where BNP recorded higher foam capacity than the modified proteins, at all other pHs studied, both succinylation and acetylation improved foam capacity. Gelation studies revealed that initial increase in ionic strength enhanced gelation properties, while gelation capacities reduced at higher salt concentrations. Ó 2007 Published by Elsevier Ltd. Keywords: Bambarra groundnut; Protein concentrate; Acetylation; Succinylation
1. Introduction Recent developments in human nutrition concerns sourcing for cheap and abundant protein foods. This becomes necessary because animal protein is more expensive and is getting beyond the reach of many people in developing countries. Abundant proteins in legumes are cheaper sources of proteins that would serve the purpose. In previous publications, we have reported studies on functional properties and possible food applications of some legumes’ protein concentrates, particularly underutilized legumes (Adebowale & Lawal, 2003, 2004; Lawal, 2005, 2004; Lawal & Adebowale, 2006, 2004). In addition,
*
Corresponding author. E-mail address: [email protected] (O.S. Lawal).
0963-9969/$ - see front matter Ó 2007 Published by Elsevier Ltd. doi:10.1016/j.foodres.2007.05.011
several authors have also reported on functionality of other legumes that are lesser known but are potential sources of cheap protein in developing countries (Chavan, Mc Kenzie, & Shahidi, 2001; Chel-Guerrero, Perez-Flores, BentacurAncona, & Davila-Ortiz, 2002; Dzudie & Hardy, 1996). Bambarra groundnut seed is an underutilized legume (Adebowale, Afolabi, & Lawal, 2002). Numerous studies have been carried out on the chemical composition and nutritional properties of its seeds (Barimalaa & Auoghalu, 1997; Enwere & Hung, 1996). It is widely cultivated throughout tropical Africa, India, Sri Lanka, Indonesia and Malaysia (Duke, 1981). Of the total annual production of around 300,000 ton, approximately half is produced in West Africa (Doku & Karikari, 1971). The seed is consumed in various forms for food in different parts of the world. Fresh seed may be consumed raw, boiled, grilled or the dry seeds made into a powdery form to make cakes (Karikari & Lavoe, 1977).
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The effective utilization of proteins entails matching a wide variety of functional and nutritional characteristics to the complex needs of manufactured food products. This is often difficult because many native proteins posses limited functionality, therefore, there is technical need for development of methodology to manipulate plant proteins and endow them with desirable functional characteristics. Chemical derivatization through acylation of amino acid residues with acetic and succinic anhydride has been used to improve functional properties of many plant proteins (Dua, Mahajan, & Mahajan, 1996). Succinylation and acetylation involve chemical derivatization of groups such as e-amino group of lysine in proteins with succinic anhydride and acetic anhydride (see Scheme 1). Reaction of proteins with acetic anhydride results in elimination of the positive charges of lysyl residues and a corresponding increase in electronegativity. Acylation with succinic or other dicarboxylic anhydrides results in replacement of positive charge with a negative charge at lysyl residues. It has been noted that acylation of proteins in some cases causes an increase in the electrostatic repulsion forces in the protein, resulting in an expansion of the molecule (Johnson & Brekke, 1983). Acylated proteins are generally more soluble than native proteins. This suggests that solubility of less soluble proteins can be increased by acylation with succinic or acetic anhydride. These modifications have been applied to many plant proteins including mucuna bean (Lawal & Adebowale, 2004), Jack bean (Lawal & Adebowale, 2006), lablab bean (Lawal, 2005), Soybean (Franzen & Kinsella, 1976), peanut (Beuchat, 1977), sunflower (Kabirrulah & Wills, 1982), pea (Johnson & Brekke, 1983), winged bean (Narayana & Narasinga Rao, 1984), rapeseed (Dua et al., 1996), cotton seed (Rahma & Narasinga Rao, 1983). However, no report is available on functionality of modified bambarra groundnut proteins. The present study therefore concerns an investigation on improvement of the functional properties of a protein concentrate isolated from bambarra ground-
nut (Voandzeia subterranean) following acetylation and succinylation. It is hoped that the result from the present investigation could provide an important information about the protein structure–function properties of bambarra groundnut protein concentrate and its modified derivatives for diverse industrial applications.
2. Materials and methods 2.1. Materials Bambarra groundnut seeds were obtained from Bodija Market, Ibadan Nigeria. All chemicals used in the experiments were of analytical grade. 2.2. Preparation of flours The seeds were screened to eliminate the defective ones. Water was added to the samples (0.5 kg/l) and it was soaked for 12 h. The seeds were manually dehulled, dried for 48 h at 30 ± 2 °C, and then dry-milled to fine powders before sieving using 75 lm sieve. The flour was stored in polythene bags and kept in a refrigerator at 4 °C prior use. 2.3. Preparation of protein concentrates Flour (1 kg) was dispersed in distilled water (10 L) and the pH was adjusted to 8.0 with 1 mol/L NaOH, to facilitate protein solubilisation. It was stirred for 4 h at 30 ± 2 °C. The pH of the supernatant obtained after centrifuging at 4000g for 30 min was adjusted to 4.0 with 0.5 mol/L HCl to precipitate the protein concentrates, which was recovered by centrifugation at 5000g for 30 min. The protein concentrates were dried by freeze drying.
O
. H2N
NH2
+
pH9
H2N
NH O
O O
O
O O Succinyl lysine
Succinic anhydride
L-lysine
O
O
. H2N
NH2
O CH3
+
O L-lysine
H2N
NH
O CH3
O
pH9
Acetic anhydride
+
H3C
OH
O O
H3C Acetyl lysine
Scheme 1. The Acylation reactions of the lysine amino acid component of the protein concentrate.
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2.4. Acetylation Protein concentrates were made into 25% w/v slurry with distilled water in 250 mL beaker and the temperature was maintained at 30 °C in a water bath (HAAKE W19, Gallenkamp, Germany) and the pH was adjusted to 7.5– 8.5 with 1 mol/L NaOH using a pH meter (MP 220 Mettler Toledo, GmbH, Switzerland). Acetic anhydride was added to the suspension over 30–90 min at the level of 0.5 g/g of protein. The slurry was dialysed with dialysis tubing (Spectra/por 1, MWCO 6000–8000) against distilled deionised water for 16 h at 30 ± 2 °C and the water was changed four times at 4 h intervals before freeze drying. 2.5. Succinylation The same procedure as for acetylation was used for succinylation. Succinic anhydride was used in place of acetic anhydride at 0.5 g/g of protein (Groninger, 1973). 2.6. Extent of modification The trinitrobenzene sulphonic acid (TNBS) method of Habeeb (1966) was used to determine the extent of acylation of proteins. The procedure involved the addition of 1 mL of a 0.1% w/v TNBS solution to 1 mL protein suspension (0.5 mg/mL). The samples were heated in a 60 °C water bath (HAAKE W19, Gallenkamp, Germany) for 2 h and then cooled to room temperature. Sodium dodecyl sulphate (1 mL, 10%) and HCl (0.5 mL, 1 mol/L) were added to the protein solutions. The absorbance of solution was read at 335 nm in a spectrophotometer against a reagent blank. The absorbance of the control protein concentrate was set equal to 100% free amino groups and the degree of acylation of the modified samples was calculated based on the decrease in absorbance because fewer amino groups were able to react with the TNBS reagent. 2.7. Chemical composition Standard Association of Official Analytical Chemistry methods, AOAC (1996) were adopted for estimating moisture, ash, crude fibre, protein crude fat and carbohydrate contents. The pH was determined with a pH meter (MP 220 Mettler Toledo, GmbH, Switzerland). Protein solutions (2% w/v) were prepared and the pH recorded at room temperature (30 ± 2 °C). 2.8. pH-solubility profile Samples (125 mg) were dispersed in distilled water (25 mL) and the solution was adjusted to pH 2–10 using either 0.5 mol/L NaOH or 0.5 mol/L HCl. The slurries were mixed for 1 h at 30 °C using magnetic bar before centrifuging at 1200g (Type GLC-1 Ivan Sovall Inc., USA) for 20 min at 4 °C. Protein content in the supernatant was determined by kjeldahl method (AOAC, 1996). Triplicate
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determinations were carried out and the solubility profile was obtained by plotting averages of protein solubility (%) against pH Solubility ð%Þ ¼
Amount of protein in the supernatant : Amount of protein in the sample
2.9. Emulsifying activity and stability Emulsifying activity and stability were determined using the method of Neto, Narain, Silva, and Bora (2001). Five milliliter portions of protein solution were homogenised with 5 mL canola oil. The emulsions were centrifuged at 1100g for 5 min. The height of emulsified layer and that of the total contents in the tube was measured. The emulsifying activity (EA) was calculated as EA ð%Þ ¼
Height of emulsified layer in the tube 100 : Height of the total contents in the tube
Emulsion stability was determined by heating the emulsion at 80 °C for 30 min before centrifuging at 1100g for 5 min. ES ð%Þ ¼
Height of emulsified layer after heating 100 : Height of emulsified layer before heating
Influence of pH was investigated by preparing protein solutions of various pHs ranging from 2 to 10. Effect of ionic strength was studied at 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mol/L KCl solutions. All studies were conducted in triplicates. 2.10. Foaming capacity and stability The foaming capacity and stability were studied according to the method of Coffman and Garcia (1977). Weighed amount of protein concentrate (2–10 g) was dispersed in 100 mL-distilled water. The resulting solution was whipped vigorously for 2 min in a Phillips Kitchen blender set at speed 2. Volumes were recorded before and after whipping. The percentage volume increase was calculated according to the following equation: % Volume increase ¼ ðV 2 V 1 Þ=ðV 1 Þ 100; V2 = volume of protein solution after whipping; V1 = volume of solution before whipping. Foam stability was determined as the volume of foam that remained after 8 h (30 ± 2 °C) expressed as a percentage of the initial foam volume. The effect of pH on foaming properties was carried out by adjusting 2%w/v dispersion to the desired pH range from 2.0 to 10.0, using either 1 mol/L HCl or 1 mol/L NaOH, followed by vigorous whipping as described above. The final volume resulting after addition of HCl or NaOH for pH adjustment was used as V1 in the calculation of % volume increase. Influence of ionic strength was evaluated by dispersing 2 g of protein concentrate in 100 mL KCl solution of
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known ionic strength. Studies were conducted in solutions with ionic strength (l) of 0.0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0, mol/L after which they were whipped vigorously. 2.11. Gelation properties Gelation properties were investigated using the method described by Coffman and Garcia (1977). Sample suspensions of 2–20% were prepared in distilled water. Each of the prepared dispersions (10 mL) was transferred into a test tube. It was heated in a boiling water bath for 1 h, followed by rapid cooling in a bath of cold water. The test tubes were further cooled at 4 °C for 2 h. The least gelation concentration was determined as the concentration when the sample from the inverted test tube did not slip or fall. Studies on the effect of pH were conducted on the sample at various concentrations by adjusting the pH to desired value from 2.0 to 10.0, prior heating, using either 0.5 mol/L HCl or 0.5 mol/L NaOH. Calculation of the concentrations was based on the final volume after pH adjustment. Least gelation concentration was determined as described above. Effect of ionic strength was investigated by preparing sample suspension (2–20%w/v) at various concentrations in KCl solution of known ionic strength (l). The pH was adjusted to 7.0 in each case. Studies were conducted at ionic strength (l) of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mol/L. 3. Statistical analysis Triplicate collection data were used for the analysis. Analysis of variance was performed to calculate significant differences in treatment means, and LSD (P < 0:05) was used to separate means (SAS, 1988). 4. Results and discussion 4.1. Chemical composition The chemical composition of modified and native bambarra groundnut protein concentrate is presented in Table 1. BAP had the highest moisture content and the lowest was recorded in native protein. Significant (P < 0.05) changes were observed in the moisture contents following modification. Ash content increased significantly
after acetylation and succinylation. Increase in ash content is attributed to reagents used for the chemical modification. Fat composition ranged between 0.2% and 0.3%. These results compare favourably with data presented by ChelGuerrero et al. (2002) on flours and protein isolate of Phaseolus lunatus. No Significant changes were observed in the crude fat following modifications. Percent protein ranged between 78.1% and 79.43% and it reduced significantly after modification. In the literature, yield of 67.9– 77.3% total protein content has been presented for beach pea protein isolated from NaOH and sodium hexametaphosphate solutions (Chavan et al., 2001). Crude fibre was below detection limit and this observation agrees with the previous studies (Lawal & Adebowale, 2006; Tomoskozi, Lasztity, Sule, Gaugecz, & Varga, 1998). The result indicates that 28.42 g-protein concentrate was recovered from 100 g of flour. Previously, Sathe, Deshpande, and Salunkhe (1982) presented similar result on flours and protein concentrates of winged bean. 4.2. Protein solubility Protein solubility in native and modified derivatives responded to changes in pH of the solution as indicated in Fig. 1. Native protein and the modified derivatives recorded highest solubility at the pH of 12. Protein solubility reduced with increase in pH of the solution until it reached isoelectric points of 3, 3.5 and 4 for BSP, BAP and BNP, respectively, following which steady increases were observed for the proteins as the pH increased. These observations have been reported previously for lablab proteins (Lawal, 2005), African locust bean (Lawal, 2004) and soy protein (Achouri, Zhang, & Shying, 1998). The result shows that acylation improved the solubility of BNP at its isoelectric point. At pH range 4–12, acylated derivatives had higher solubility compared with native protein. It is due to combination of intra and intermolecular charge repulsion that promoted protein unfolding and produced fewer protein–protein interactions and more protein–water interactions after derivatization. Similar observations had also been reported for sunflower protein (Kabirrulah & Wills, 1982) and canola proteins (Paulson & Tung, 1987). In addition, the result indicates that BSP has higher solubility than BAP at pH 4–12. Improved solubility following succinylation could be explained based on the structure of
Table 1 Chemical composition of native (BNP), acetylated (BAP), and succinylated (BSP) protein concentrates of bambarra groundnut Sample BNP BAPd BSPe
Moisture (%) a
7.24 ± 0.04 7.53 ± 0.02b 7.42 ± 0.04c
Ash (%)
Crude fibre (%) a
2.06 ± 0.01 3.05 ± 0.01b 3.03 ± 0.01b
ND ND ND
Crude fat (%) a
0.30 ± 0.01 0.20 ± 0.01a 0.20 ± 0.01a
All Values are means of triplicate determinations ± standard deviation. Means within columns with different letter (a, b or c) are significantly different (P < 0.05) ND: Not detected. d Degree of acetylation: 78%; percentage yield calculated on native protein basis. e Degree of succinylation: 72%; percentage yield calculated on native protein basis.
Protein (%) a
79.43 ± 2.34 78.5 ± 5.22b 78.1 ± 4.61b
Carbohydrate (%)
pH
Yield (%)
12.97 13.72 14.25
5.6 5.4 4.8
28.42 96.74 91.41
O.S. Lawal et al. / Food Research International 40 (2007) 1003–1011
80
60
40
BNP BAP BSP
20
0 2
4
6
pH
8
10
12
Fig. 1. pH dependent protein solubility profile of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
succinylated proteins. Succinylation involves the replacement of short-range attractive forces in the native molecule with short-range repulsive ones and this results in subsequent unfolding of polypeptide chains. Electrostatic attractions between neighbouring ammonium and carboxyl, which reduced protein solubility by protein–protein interactions in native bambarra protein concentrate have been limited in succinylated protein derivatives and this lead to better solubility.
4.3. Emulsifying activity and stability Studies conducted on the effect of concentration of protein solution on emulsifying activity of native and derivatised bambarra protein indicate an initial increase in emulsifying activity with increase in concentration. When protein concentration is low, protein adsorption at the oil–water interface is diffusion controlled. However, at high protein concentration, activation energy barrier does not allow protein migration to take place in a diffusion dependent manner. Initial increase in protein concentration facilitated enhanced interaction between the oil phase and the aqueous phase. However, as the concentration increased, a point was reached where further increase in protein concentration led to accumulation of proteins in the aqueous phase, this development resulted in decrease in emulsifying activity. The results also indicate that acylation improved the emulsifying activity of the native protein at all concentrations. This observation is consistent with the report on mung bean protein isolate (EL-Adawy, 2000). A similar pattern was also observed for emulsion stability. Initial increases were observed with increase in concentration up to 3, 4 and 5% w/v for BNP, BAP and BSP, respectively. In addition, both acetylation and succinylation improved the emulsifying stability of the native protein. Acylation brings about unfolding of protein molecules and subse-
quently, increases the number of active binding sites, which facilitates emulsion stability. Both EA and ES were pH dependent as indicated in Figs. 2 and 3, respectively. Generally, the observed trend of increase and decrease is in agreement with the pH dependent solubility of the respective proteins. pH has pronounced effect on emulsifying activity because emulsifying activity of soluble proteins depend upon the hydrophilic–lipophilic balance (Sosulski & Fleming, 1977), which is affected by pH. At the oil–water interface, the protein orients lipophilic residues to the oil phase and hydrophilic residues to the aqueous phase, thus reducing surface tension at the interface. Acylation enhanced exposure of lipophilic and hydrophilic residues and this facilitated improved EA. ES of acylated proteins were higher than those of native proteins in the range of pH 4–10 but lower when the pH was 2. The increase in ES with increasing pH after isoelectric point has been attributed to formation of charged layers around fat globules, which caused mutual repulsion and or by forming a hydrated layer around the interfacial material, which lower interfacial energy and retarded droplet coalescence. Addition of succinic and acetic anhydride increased the electrical potential of the ionised layer of the interfacial film around oil droplets, and this enhanced the emulsion stability. Studies conducted on the effect of ionic strength on EA and ES revealed that emulsifying activity and stability of BNP increased progressively with increase in ionic strength of the medium. Similarly, progressive increases in EA and ES were also observed in BAP and BSP until the ionic strength of the solution reached 0.6 mol/L, following which decreases were observed with further increases in ionic strength.
BNP BAP BSP
80 70
Emulsifying activity (%)
Protein Solubility (%)
100
1007
60 50 40 30 20 10 0 2
4
6
7
8
10
pH
Fig. 2. Effect of pH on emulsifying activity of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
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BNP BAP BSP
BNP BAP BSP
240 220
100
200 180
Foam capacity (ml)
Emulsion stability (%)
90 80 70 60 50 40
160 140 120 100 80 60
30
40
20
20
10
0 2
4
6
7
8
10
pH
0 2
4
6
7
8
10
pH
Fig. 3. Effect of pH on emulsion stability of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
Fig. 4. Effect of pH on foam capacity of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
100
BNP BAP BSP
90 80
Foam stability (%)
In a previous work, effect of ionic strength on emulsifying properties of cowpea protein had earlier been reported by, Aluko and Yada (1995). Similarly, Chavan et al. (2001) also reported ionic strength dependent emulsifying properties for beach pea. Higher emulsion stability of protein at low ionic strength has been attributed to dissociation of Oligomeric structure of 11S-glycinin and subsequent improvement of surface behaviour (Wagner & Gueguen, 1995).
70 60 50 40 30 20 10
4.4. Foaming capacity and stability
0 2
Protein concentration has pronounced effect on foaming capacity and stability. Foaming capacity of both native and modified proteins increased with increase in protein concentration. Increase in foam capacity as observed in this work agrees with those reported for mung bean protein isolate (EL-Adawy, 2000) and succinylated soy protein (Franzen & Kinsella, 1976). Acylation can cause unfolding of the protein, which increases protein–water interaction. Also, the increased net negative charge of succinylated proteins would especially promote protein–water interaction, which facilitates improved foaming capacity. Decrease in foam stability following modifications is a result of increased charge density of succinylated proteins, since it inhibits the protein–protein interactions (Townsend & Nakai, 1983). Effect of pH on foam capacity and stability is presented in Figs. 4 and 5. The result indicates that both native and modified proteins respond to changes in pH of the solution. Except at pH 2, where BNP recorded higher foam capacity than the modified proteins, at all other pHs studied, both succinylation and acetylation improved foam capacity. Maxi-
4
6
pH
7
8
10
Fig. 5. Effect of pH on foam stability of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
mal foam capacities were observed at pH 10 in all the cases. Contrarily, succinylation and acetylation reduced foam stability at all other pHs except pH2. This result suggests that solubility of protein determines the foaming properties. In a previous study, Lin, Humbert, and Sosulski (1974) have reported a pH dependent foaming properties for sunflower meal products. In addition, pH dependent foaming properties have also been reported for P. lunatus and Canavalia ensiformis (Chel-Guerrero et al., 2002). The high foaming capacity at pH 2 and pH 12 may be due to an increase in the net charge of the protein molecules, which weakens hydrophobic interactions and increases protein flexibility. This allowed them to spread to the air water interface more quickly thus encapsulating air particles and increasing foam formation.
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Increase in the foam stability at region of isoelectric point is due to the formation of stable molecular layers in the air–water interface of the foams. Protein adsorption and viscoelasticity at an air–water interface is maximal near or at isoelectric pH because protein is not strongly repelled. In addition, the protein possesses low net charge near isoelectric pH, which may contribute to the formation of stable molecular layers in the air–water interface, a development that improves foam stability. Studies revealed that foaming capacity and stability of native and modified proteins increased with increase in ionic strength of the media. Except in the control solution, modifications improved foam capacity and stability at all other ionic strengths. This development is attributed to increased solubility, thus leading to enhanced whippability and formation of stable cohesive films around the air vacuoles following modifications. 4.5. Gelation properties Effect of ionic strength on gelation capacities is presented in Table 2. Taking the least gelation concentration (LGC) as the index of gelation capacity, lower LGC means better gelation capacity. The result shows that for all the proteins, initial increase in ionic strength enhanced gelation properties, while gelation capacities reduced at higher salt concentrations. In previous reports, Otte, Schumacher, Ipsen, Ju, and Qvist (1999) reported a reduction of gel firmness of whey proteins, when the NaCl content of the mixture was increased. Initial increase in ionic strength facilitated a shielding effect on the surface charges; this development reduced the repulsive forces acting among the protein molecules, creating an identical situation as for isoelectric region, whereas further increase in ionic strength influenced the gel forming process negatively by decreasing the protein unfolding. Similar observation has been reported for whey protein (Boye, Alli, Ismail, Gibbs, & Konishi, 1995). Gelation capacities of the proteins were also pH dependent as presented in Table 3. The result indicate that gelation of the modified derivatives were best at pH 2, while the BNP recorded the highest gelation at pH 4 corresponding to the regions of isoelectric point. Protein gelation is vital in the preparation and acceptability of many foods, including vegetable and other products. Gelation mechanism and gel appearance are fundamentally controlled, by the bal-
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Table 3 Effect of pH on gelation properties of native, acetylated and succinylated bambarra protein concentrate Sample
pH 2
pH 4
pH 7
pH 8
pH 10
BNP BAP BSP
14 10 8
10 12 10
14 12 12
14 16 14
14 16 14
Values are least gelation concentrations expressed as % w/v. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
ance between attractive hydrophobic interactions and repulsive electrostatic interactions (Egelandsal, 1980). The repulsive forces are due to surface charges and the attractive forces are due to various functional groups exposed by the thermal unfolding of the protein (Kojima & Nakamura, 1985). The pH of protein dispersions has profound effect on gelation reactions by influencing the balance of polar and non-polar residues. At pH values in the region of isoelectric pH, protein–protein interactions are generally favoured because the net surface charge is close to zero, which significantly reduces the repulsive interactions between protein molecules thus enhancing gelation, while at pH far removed from isoelectric points, the surface charge on the protein is large and significant repulsive forces prevent protein–protein interaction. 5. Conclusion The effects of acetylation and succinylation on the solubility profile, gelation, foaming and emulsifying properties of bambarra bean protein concentrate have been studied in this work. Acylation of bambarra bean protein concentrate produced pronounced changes in its functional properties. The study also revealed that solubility of protein concentrate improved following acetylation and succinylation and this improved both emulsifying and foaming properties of the native protein concentrate. The study provides fundamental information about the changes in protein function – properties of a new source of protein after acylation. The strength of the investigation is that an under utilized legume is used for the study and these fundamental information could be useful in providing insights into diverse applications of the bambarra groundnut protein,
Table 2 Effect of ionic strength on gelation properties of native, acetylated and succinylated bambarra protein concentrate Sample
Control
0.1 mol/L
0.2 mol/L
0.4 mol/L
0.6 mol/L
0.8 mol/L
1.0 mol/L
BNP BAP BSP
14 12 12
8 10 10
10 8 10
14 8 14
14 14 14
14 14 14
18 16 16
Values are least gelation concentrations expressed as % w/v. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
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particularly in the developing nations in the tropics where this plant is readily available. Acknowledgement The corresponding author is grateful to Alexander von Humboldt foundation, Germany for the award of postdoctoral fellowship and the Abdus salam International Centre for theoretical physics, Trieste, Italy for the award of associate of biophysics of living state. References Achouri, A., Zhang, W., & Shying, X. U. (1998). Enzymatic hydrolysis of soy protein isolate and effect of succinylation on functional properties of resulting protein hydrolysates. Food Research International, 31, 617–623. Adebowale, K. O., Afolabi, T. A., & Lawal, O. S. (2002). Isolation, chemical modification and physicochemical characterisation of bambarra groundnut (Voandzeia subterranean) starch and flour. Food Chemistry, 78, 305–311. Adebowale, K. O., & Lawal, O. S. (2003). Foaming, gelation and electrophoretic characteristics of mucuna bean (Mucuna pruriens) protein concentrates. Food Chemistry, 83, 237–246. Adebowale, K. O., & Lawal, O. S. (2004). A Comparative study of the functional characteristics of some underutilized African Legume flours: Bambarra groundnut (Voandzeia subterranean), Jack bean (Canavalia ensiformis) and Mucuna bean (Mucuna pruriens). Food Research International, 37, 335–365. Aluko, R. E., & Yada, R. Y. (1995). Structure–function relationships of cowpea (Vigna unguiculata) globulin isolate. Influence of pH and NaCl on physicochemical and functional properties. Food Chemistry, 53, 259–265. AOAC (1996). Official methods of analysis. Airlington: Association of official Analytical chemistry. Barimalaa, I. S., & Auoghalu, S. C. (1997). Effect of processing on certain antinutrients in Bambara groundnut cotyledon. Journal of the Science of Food and Agriculture, 73(2), 186–188. Beuchat, L. R. (1977). Functional and Electrophoretic characteristics of succinylated peanut flour protein. Journal of Agricultural and Food Chemistry, 25, 258–261. Boye, J. I., Alli, I., Ismail, A. A., Gibbs, B. F., & Konishi, Y. (1995). Factors affecting molecular characteristics of whey protein gelation. International Dairy Journal, 5, 337–353. Chavan, U. D., Mc Kenzie, D. B., & Shahidi, F. (2001). Functional properties of protein isolates from beach pea (Lathyrus maritimus L). Food Chemistry, 74, 177–187. Chel-Guerrero, L., Perez-Flores, V., Bentacur-Ancona, D., & DavilaOrtiz, G. (2002). Functional properties of flours and protein isolates from Phaseolus lunatus and Canavalia ensiformis seeds. Journal of Agricultural and Food Chemistry, 50, 584–591. Coffman, C. W., & Garcia, V. V. (1977). Functional properties and amino acid content of a protein isolate from Mung bean flour. Journal of Food Technology, 12, 473–484. Doku, E. V., & Karikari, S. K. (1971). Bambarra groundnut. Economic Botany, 25, 255–262. Dua, S., Mahajan, A., & Mahajan, A. (1996). Improvement in functional properties of rapeseed (Brassica Campestris var. Toria) preparations by chemical modification. Journal of Agricultural and Food Chemistry, 44, 706–710. Duke, J. A. (1981). Hand book of legumes of world economic importance. New York: Plenum press [pp. 307–310]. Dzudie, T., & Hardy, J. (1996). Physicochemical and functional properties of flours prepared from common beans and Green Mung beans. Journal of Agricultural and Food Chemistry, 44, 3029–3032.
Egelandsal, B. (1980). Heat induced gelling in solutions of ovalbumin. Journal of Food Science, 45, 570–573. EL-Adawy, T. A. (2000). Functional properties and nutritional quality of acetylated and succinylated mung bean protein isolate. Food Chemistry, 70, 83–91. Enwere, N. J., & Hung, Y. C. (1996). Some chemical and physical properties of bambara groundnut seeds and products. International Journal of Food Sciences and Nutrition, 47(6), 469–475. Franzen, K. L., & Kinsella, J. E. (1976). Functional properties of succinylated and acetylated leaf protein. Journal of Agricultural and Food Chemistry, 24, 914–919. Groninger, H. S. (1973). Preparation and properties of succinylated fish myofibrillar Protein. Journal of Agricultural and Food Chemistry, 21, 978–981. Habeeb, A. F. (1966). Determination of free amino groups in proteins by trinitrobenzenesulphonic acid. Analytical Biochemistry, 14, 328– 336. Johnson, E. A., & Brekke, C. J. (1983). Functional properties of acylated pea protein isolates. Journal of Food Science, 48, 722–725. Kabirrulah, M., & Wills, R. B. H. (1982). Functional properties of acetylated and succinylated sunflower protein isolate. Journal of Food Technology, 17, 235. Karikari, S. K., & Lavoe, S. K. (1977). Preliminary evaluation and utilization of fourteen cultivars of bambarra groundnut (Voandzeia subterranean Thours). Acta Horticulture, 53, 195–199. Kojima, E., & Nakamura, R. (1985). Heat gelling properties of hen’s egg yolk low-density lipoprotein (LDL) in the presence of other proteins. Journal of Food Science, 50, 63–66. Lawal, O. S. (2004). Functionality of African locust bean (Parkia biglobossa) protein isolate: Effect of pH, ionic strength and various protein concentrations. Food Chemistry, 86, 345–355. Lawal, O. S. (2005). Functionality of native and succinylated Lablab bean (Lablab purpureus) protein concentrate. Food Hydrocolloids, 19, 63–72. Lawal, O. S., & Adebowale, K. O. (2004). Effect of Acetylation and succinylation on solubility profile, water absorption capacity, oil absorption capacity and emulsifying properties of Mucuna bean (Mucuna pruriens) protein concentrate. Nahrung/Food, 48, 129– 136. Lawal, O. S., & Adebowale, K. O. (2006). The acylated protein derivatives of Canavalia ensiformis (jack bean): A study of functional characteristics. LWT/ Food Technology, 39, 918–929. Lin, M. J. Y., Humbert, E. S., & Sosulski, F. (1974). Certain functional properties of sunflower meal products. Journal of Food Science, 39, 368–372. Narayana, K., & Narasinga Rao, M. S. (1984). Effect of acetylation and succinylation on the functional properties of winged bean (Psophocarpus tetragonolobus) flour. Journal of Food Science, 49, 547– 550. Neto, V. Q., Narain, N., Silva, J. B., & Bora, P. S. (2001). Functional properties of raw and heat processed cashew nut (Anarcarduim occidentale, L.) kernel protein isolate. Nahrung/Food, 45, 258– 262. Otte, J., Schumacher, E., Ipsen, R., Ju, Z. Y., & Qvist, K. B. (1999). Protease induced gelation of unheated and heated whey proteins. Effects of pH, temperature and concentration of proteins, enzyme and salts. International Dairy Journal, 9, 801–812. Paulson, A. T., & Tung, M. A. (1987). Solubility, hydrophobicity and net charge of succinylated canola protein isolate. Journal of Food Science, 52, 1557–1561. Rahma, E. H., & Narasinga Rao, M. S. (1983). Effect of acylation and succinylation of cottonseed flour on its functional properties. Journal of Agricultural and Food Chemistry, 31, 351–355. SAS (1988). SAS SAS/STAT users guide, release 6.03. Carey, NC: SAS Institute Inc. Sathe, S. K., Deshpande, S. S., & Salunkhe, D. K. (1982). Functional properties of winged bean (Psophocarpus tetragonolobus, L.) proteins. Journal of Food Science, 47, 503.
O.S. Lawal et al. / Food Research International 40 (2007) 1003–1011 Sosulski, F. W., & Fleming, S. E. (1977). Chemical functional and nutritional properties of sunflower protein products. Journal of American Oil Chemist Society, 54, 100. Tomoskozi, S., Lasztity, R., Sule, E., Gaugecz, J., & Varga, J. (1998). Functional properties of native and modified wheat germ protein isolates and fractions. Nahrung/Food, 42, 245–247.
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Townsend, A., & Nakai, S. (1983). relationships between hydrophobicity and foaming characteristics of food proteins. Journal of Food Science, 48, 588–594. Wagner, J. R., & Gueguen, J. J. (1995). Effect of dissociation, deamination and reducing treatment on structural and surface active properties of soy glycinin. Journal of the Agricultural and Food Chemistry, 43, 1993–2000.
Functional properties of native and chemically modified protein concentrates from bambarra groundnut O.S. Lawal a
a,*
, K.O. Adebowale b, Y.A. Adebowale
c
Department of Chemical Sciences, Olabisi Onabanjo University, Ago-Iwoye, P.M.B 2002, Ago-Iwoye, Ogun State, Nigeria b Department of Chemistry, University of Ibadan, Ibadan, Nigeria c Department of Food Technology, University of Ibadan, Ibadan, Nigeria Received 12 January 2007; accepted 6 May 2007
Abstract Protein concentrate (BNP) was prepared from bambarra groundnut (Voandzeia subterranean) and it was derivatised with succinic (BSP) and acetic anhydride (BAP). Chemical composition revealed significant (P < 0.05) increases in the moisture and ash contents following modifications, while the crude fat was reduced. Protein solubility reduced with increase in pH of the solution until it reached isoelectric points of 3, 3.5 and 4 for BSP, BAP and BNP, respectively, following which steady increases were observed for the proteins as the pH increased. The results indicate an initial increase in emulsifying activity (EA) with increase in protein concentration. Both acetylation and succinylation improved the emulsifying stability of the native protein. Foaming capacity of both native and modified proteins increased with increase in protein concentration. Except at pH 2, where BNP recorded higher foam capacity than the modified proteins, at all other pHs studied, both succinylation and acetylation improved foam capacity. Gelation studies revealed that initial increase in ionic strength enhanced gelation properties, while gelation capacities reduced at higher salt concentrations. Ó 2007 Published by Elsevier Ltd. Keywords: Bambarra groundnut; Protein concentrate; Acetylation; Succinylation
1. Introduction Recent developments in human nutrition concerns sourcing for cheap and abundant protein foods. This becomes necessary because animal protein is more expensive and is getting beyond the reach of many people in developing countries. Abundant proteins in legumes are cheaper sources of proteins that would serve the purpose. In previous publications, we have reported studies on functional properties and possible food applications of some legumes’ protein concentrates, particularly underutilized legumes (Adebowale & Lawal, 2003, 2004; Lawal, 2005, 2004; Lawal & Adebowale, 2006, 2004). In addition,
*
Corresponding author. E-mail address: [email protected] (O.S. Lawal).
0963-9969/$ - see front matter Ó 2007 Published by Elsevier Ltd. doi:10.1016/j.foodres.2007.05.011
several authors have also reported on functionality of other legumes that are lesser known but are potential sources of cheap protein in developing countries (Chavan, Mc Kenzie, & Shahidi, 2001; Chel-Guerrero, Perez-Flores, BentacurAncona, & Davila-Ortiz, 2002; Dzudie & Hardy, 1996). Bambarra groundnut seed is an underutilized legume (Adebowale, Afolabi, & Lawal, 2002). Numerous studies have been carried out on the chemical composition and nutritional properties of its seeds (Barimalaa & Auoghalu, 1997; Enwere & Hung, 1996). It is widely cultivated throughout tropical Africa, India, Sri Lanka, Indonesia and Malaysia (Duke, 1981). Of the total annual production of around 300,000 ton, approximately half is produced in West Africa (Doku & Karikari, 1971). The seed is consumed in various forms for food in different parts of the world. Fresh seed may be consumed raw, boiled, grilled or the dry seeds made into a powdery form to make cakes (Karikari & Lavoe, 1977).
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The effective utilization of proteins entails matching a wide variety of functional and nutritional characteristics to the complex needs of manufactured food products. This is often difficult because many native proteins posses limited functionality, therefore, there is technical need for development of methodology to manipulate plant proteins and endow them with desirable functional characteristics. Chemical derivatization through acylation of amino acid residues with acetic and succinic anhydride has been used to improve functional properties of many plant proteins (Dua, Mahajan, & Mahajan, 1996). Succinylation and acetylation involve chemical derivatization of groups such as e-amino group of lysine in proteins with succinic anhydride and acetic anhydride (see Scheme 1). Reaction of proteins with acetic anhydride results in elimination of the positive charges of lysyl residues and a corresponding increase in electronegativity. Acylation with succinic or other dicarboxylic anhydrides results in replacement of positive charge with a negative charge at lysyl residues. It has been noted that acylation of proteins in some cases causes an increase in the electrostatic repulsion forces in the protein, resulting in an expansion of the molecule (Johnson & Brekke, 1983). Acylated proteins are generally more soluble than native proteins. This suggests that solubility of less soluble proteins can be increased by acylation with succinic or acetic anhydride. These modifications have been applied to many plant proteins including mucuna bean (Lawal & Adebowale, 2004), Jack bean (Lawal & Adebowale, 2006), lablab bean (Lawal, 2005), Soybean (Franzen & Kinsella, 1976), peanut (Beuchat, 1977), sunflower (Kabirrulah & Wills, 1982), pea (Johnson & Brekke, 1983), winged bean (Narayana & Narasinga Rao, 1984), rapeseed (Dua et al., 1996), cotton seed (Rahma & Narasinga Rao, 1983). However, no report is available on functionality of modified bambarra groundnut proteins. The present study therefore concerns an investigation on improvement of the functional properties of a protein concentrate isolated from bambarra ground-
nut (Voandzeia subterranean) following acetylation and succinylation. It is hoped that the result from the present investigation could provide an important information about the protein structure–function properties of bambarra groundnut protein concentrate and its modified derivatives for diverse industrial applications.
2. Materials and methods 2.1. Materials Bambarra groundnut seeds were obtained from Bodija Market, Ibadan Nigeria. All chemicals used in the experiments were of analytical grade. 2.2. Preparation of flours The seeds were screened to eliminate the defective ones. Water was added to the samples (0.5 kg/l) and it was soaked for 12 h. The seeds were manually dehulled, dried for 48 h at 30 ± 2 °C, and then dry-milled to fine powders before sieving using 75 lm sieve. The flour was stored in polythene bags and kept in a refrigerator at 4 °C prior use. 2.3. Preparation of protein concentrates Flour (1 kg) was dispersed in distilled water (10 L) and the pH was adjusted to 8.0 with 1 mol/L NaOH, to facilitate protein solubilisation. It was stirred for 4 h at 30 ± 2 °C. The pH of the supernatant obtained after centrifuging at 4000g for 30 min was adjusted to 4.0 with 0.5 mol/L HCl to precipitate the protein concentrates, which was recovered by centrifugation at 5000g for 30 min. The protein concentrates were dried by freeze drying.
O
. H2N
NH2
+
pH9
H2N
NH O
O O
O
O O Succinyl lysine
Succinic anhydride
L-lysine
O
O
. H2N
NH2
O CH3
+
O L-lysine
H2N
NH
O CH3
O
pH9
Acetic anhydride
+
H3C
OH
O O
H3C Acetyl lysine
Scheme 1. The Acylation reactions of the lysine amino acid component of the protein concentrate.
O.S. Lawal et al. / Food Research International 40 (2007) 1003–1011
2.4. Acetylation Protein concentrates were made into 25% w/v slurry with distilled water in 250 mL beaker and the temperature was maintained at 30 °C in a water bath (HAAKE W19, Gallenkamp, Germany) and the pH was adjusted to 7.5– 8.5 with 1 mol/L NaOH using a pH meter (MP 220 Mettler Toledo, GmbH, Switzerland). Acetic anhydride was added to the suspension over 30–90 min at the level of 0.5 g/g of protein. The slurry was dialysed with dialysis tubing (Spectra/por 1, MWCO 6000–8000) against distilled deionised water for 16 h at 30 ± 2 °C and the water was changed four times at 4 h intervals before freeze drying. 2.5. Succinylation The same procedure as for acetylation was used for succinylation. Succinic anhydride was used in place of acetic anhydride at 0.5 g/g of protein (Groninger, 1973). 2.6. Extent of modification The trinitrobenzene sulphonic acid (TNBS) method of Habeeb (1966) was used to determine the extent of acylation of proteins. The procedure involved the addition of 1 mL of a 0.1% w/v TNBS solution to 1 mL protein suspension (0.5 mg/mL). The samples were heated in a 60 °C water bath (HAAKE W19, Gallenkamp, Germany) for 2 h and then cooled to room temperature. Sodium dodecyl sulphate (1 mL, 10%) and HCl (0.5 mL, 1 mol/L) were added to the protein solutions. The absorbance of solution was read at 335 nm in a spectrophotometer against a reagent blank. The absorbance of the control protein concentrate was set equal to 100% free amino groups and the degree of acylation of the modified samples was calculated based on the decrease in absorbance because fewer amino groups were able to react with the TNBS reagent. 2.7. Chemical composition Standard Association of Official Analytical Chemistry methods, AOAC (1996) were adopted for estimating moisture, ash, crude fibre, protein crude fat and carbohydrate contents. The pH was determined with a pH meter (MP 220 Mettler Toledo, GmbH, Switzerland). Protein solutions (2% w/v) were prepared and the pH recorded at room temperature (30 ± 2 °C). 2.8. pH-solubility profile Samples (125 mg) were dispersed in distilled water (25 mL) and the solution was adjusted to pH 2–10 using either 0.5 mol/L NaOH or 0.5 mol/L HCl. The slurries were mixed for 1 h at 30 °C using magnetic bar before centrifuging at 1200g (Type GLC-1 Ivan Sovall Inc., USA) for 20 min at 4 °C. Protein content in the supernatant was determined by kjeldahl method (AOAC, 1996). Triplicate
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determinations were carried out and the solubility profile was obtained by plotting averages of protein solubility (%) against pH Solubility ð%Þ ¼
Amount of protein in the supernatant : Amount of protein in the sample
2.9. Emulsifying activity and stability Emulsifying activity and stability were determined using the method of Neto, Narain, Silva, and Bora (2001). Five milliliter portions of protein solution were homogenised with 5 mL canola oil. The emulsions were centrifuged at 1100g for 5 min. The height of emulsified layer and that of the total contents in the tube was measured. The emulsifying activity (EA) was calculated as EA ð%Þ ¼
Height of emulsified layer in the tube 100 : Height of the total contents in the tube
Emulsion stability was determined by heating the emulsion at 80 °C for 30 min before centrifuging at 1100g for 5 min. ES ð%Þ ¼
Height of emulsified layer after heating 100 : Height of emulsified layer before heating
Influence of pH was investigated by preparing protein solutions of various pHs ranging from 2 to 10. Effect of ionic strength was studied at 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mol/L KCl solutions. All studies were conducted in triplicates. 2.10. Foaming capacity and stability The foaming capacity and stability were studied according to the method of Coffman and Garcia (1977). Weighed amount of protein concentrate (2–10 g) was dispersed in 100 mL-distilled water. The resulting solution was whipped vigorously for 2 min in a Phillips Kitchen blender set at speed 2. Volumes were recorded before and after whipping. The percentage volume increase was calculated according to the following equation: % Volume increase ¼ ðV 2 V 1 Þ=ðV 1 Þ 100; V2 = volume of protein solution after whipping; V1 = volume of solution before whipping. Foam stability was determined as the volume of foam that remained after 8 h (30 ± 2 °C) expressed as a percentage of the initial foam volume. The effect of pH on foaming properties was carried out by adjusting 2%w/v dispersion to the desired pH range from 2.0 to 10.0, using either 1 mol/L HCl or 1 mol/L NaOH, followed by vigorous whipping as described above. The final volume resulting after addition of HCl or NaOH for pH adjustment was used as V1 in the calculation of % volume increase. Influence of ionic strength was evaluated by dispersing 2 g of protein concentrate in 100 mL KCl solution of
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known ionic strength. Studies were conducted in solutions with ionic strength (l) of 0.0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0, mol/L after which they were whipped vigorously. 2.11. Gelation properties Gelation properties were investigated using the method described by Coffman and Garcia (1977). Sample suspensions of 2–20% were prepared in distilled water. Each of the prepared dispersions (10 mL) was transferred into a test tube. It was heated in a boiling water bath for 1 h, followed by rapid cooling in a bath of cold water. The test tubes were further cooled at 4 °C for 2 h. The least gelation concentration was determined as the concentration when the sample from the inverted test tube did not slip or fall. Studies on the effect of pH were conducted on the sample at various concentrations by adjusting the pH to desired value from 2.0 to 10.0, prior heating, using either 0.5 mol/L HCl or 0.5 mol/L NaOH. Calculation of the concentrations was based on the final volume after pH adjustment. Least gelation concentration was determined as described above. Effect of ionic strength was investigated by preparing sample suspension (2–20%w/v) at various concentrations in KCl solution of known ionic strength (l). The pH was adjusted to 7.0 in each case. Studies were conducted at ionic strength (l) of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mol/L. 3. Statistical analysis Triplicate collection data were used for the analysis. Analysis of variance was performed to calculate significant differences in treatment means, and LSD (P < 0:05) was used to separate means (SAS, 1988). 4. Results and discussion 4.1. Chemical composition The chemical composition of modified and native bambarra groundnut protein concentrate is presented in Table 1. BAP had the highest moisture content and the lowest was recorded in native protein. Significant (P < 0.05) changes were observed in the moisture contents following modification. Ash content increased significantly
after acetylation and succinylation. Increase in ash content is attributed to reagents used for the chemical modification. Fat composition ranged between 0.2% and 0.3%. These results compare favourably with data presented by ChelGuerrero et al. (2002) on flours and protein isolate of Phaseolus lunatus. No Significant changes were observed in the crude fat following modifications. Percent protein ranged between 78.1% and 79.43% and it reduced significantly after modification. In the literature, yield of 67.9– 77.3% total protein content has been presented for beach pea protein isolated from NaOH and sodium hexametaphosphate solutions (Chavan et al., 2001). Crude fibre was below detection limit and this observation agrees with the previous studies (Lawal & Adebowale, 2006; Tomoskozi, Lasztity, Sule, Gaugecz, & Varga, 1998). The result indicates that 28.42 g-protein concentrate was recovered from 100 g of flour. Previously, Sathe, Deshpande, and Salunkhe (1982) presented similar result on flours and protein concentrates of winged bean. 4.2. Protein solubility Protein solubility in native and modified derivatives responded to changes in pH of the solution as indicated in Fig. 1. Native protein and the modified derivatives recorded highest solubility at the pH of 12. Protein solubility reduced with increase in pH of the solution until it reached isoelectric points of 3, 3.5 and 4 for BSP, BAP and BNP, respectively, following which steady increases were observed for the proteins as the pH increased. These observations have been reported previously for lablab proteins (Lawal, 2005), African locust bean (Lawal, 2004) and soy protein (Achouri, Zhang, & Shying, 1998). The result shows that acylation improved the solubility of BNP at its isoelectric point. At pH range 4–12, acylated derivatives had higher solubility compared with native protein. It is due to combination of intra and intermolecular charge repulsion that promoted protein unfolding and produced fewer protein–protein interactions and more protein–water interactions after derivatization. Similar observations had also been reported for sunflower protein (Kabirrulah & Wills, 1982) and canola proteins (Paulson & Tung, 1987). In addition, the result indicates that BSP has higher solubility than BAP at pH 4–12. Improved solubility following succinylation could be explained based on the structure of
Table 1 Chemical composition of native (BNP), acetylated (BAP), and succinylated (BSP) protein concentrates of bambarra groundnut Sample BNP BAPd BSPe
Moisture (%) a
7.24 ± 0.04 7.53 ± 0.02b 7.42 ± 0.04c
Ash (%)
Crude fibre (%) a
2.06 ± 0.01 3.05 ± 0.01b 3.03 ± 0.01b
ND ND ND
Crude fat (%) a
0.30 ± 0.01 0.20 ± 0.01a 0.20 ± 0.01a
All Values are means of triplicate determinations ± standard deviation. Means within columns with different letter (a, b or c) are significantly different (P < 0.05) ND: Not detected. d Degree of acetylation: 78%; percentage yield calculated on native protein basis. e Degree of succinylation: 72%; percentage yield calculated on native protein basis.
Protein (%) a
79.43 ± 2.34 78.5 ± 5.22b 78.1 ± 4.61b
Carbohydrate (%)
pH
Yield (%)
12.97 13.72 14.25
5.6 5.4 4.8
28.42 96.74 91.41
O.S. Lawal et al. / Food Research International 40 (2007) 1003–1011
80
60
40
BNP BAP BSP
20
0 2
4
6
pH
8
10
12
Fig. 1. pH dependent protein solubility profile of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
succinylated proteins. Succinylation involves the replacement of short-range attractive forces in the native molecule with short-range repulsive ones and this results in subsequent unfolding of polypeptide chains. Electrostatic attractions between neighbouring ammonium and carboxyl, which reduced protein solubility by protein–protein interactions in native bambarra protein concentrate have been limited in succinylated protein derivatives and this lead to better solubility.
4.3. Emulsifying activity and stability Studies conducted on the effect of concentration of protein solution on emulsifying activity of native and derivatised bambarra protein indicate an initial increase in emulsifying activity with increase in concentration. When protein concentration is low, protein adsorption at the oil–water interface is diffusion controlled. However, at high protein concentration, activation energy barrier does not allow protein migration to take place in a diffusion dependent manner. Initial increase in protein concentration facilitated enhanced interaction between the oil phase and the aqueous phase. However, as the concentration increased, a point was reached where further increase in protein concentration led to accumulation of proteins in the aqueous phase, this development resulted in decrease in emulsifying activity. The results also indicate that acylation improved the emulsifying activity of the native protein at all concentrations. This observation is consistent with the report on mung bean protein isolate (EL-Adawy, 2000). A similar pattern was also observed for emulsion stability. Initial increases were observed with increase in concentration up to 3, 4 and 5% w/v for BNP, BAP and BSP, respectively. In addition, both acetylation and succinylation improved the emulsifying stability of the native protein. Acylation brings about unfolding of protein molecules and subse-
quently, increases the number of active binding sites, which facilitates emulsion stability. Both EA and ES were pH dependent as indicated in Figs. 2 and 3, respectively. Generally, the observed trend of increase and decrease is in agreement with the pH dependent solubility of the respective proteins. pH has pronounced effect on emulsifying activity because emulsifying activity of soluble proteins depend upon the hydrophilic–lipophilic balance (Sosulski & Fleming, 1977), which is affected by pH. At the oil–water interface, the protein orients lipophilic residues to the oil phase and hydrophilic residues to the aqueous phase, thus reducing surface tension at the interface. Acylation enhanced exposure of lipophilic and hydrophilic residues and this facilitated improved EA. ES of acylated proteins were higher than those of native proteins in the range of pH 4–10 but lower when the pH was 2. The increase in ES with increasing pH after isoelectric point has been attributed to formation of charged layers around fat globules, which caused mutual repulsion and or by forming a hydrated layer around the interfacial material, which lower interfacial energy and retarded droplet coalescence. Addition of succinic and acetic anhydride increased the electrical potential of the ionised layer of the interfacial film around oil droplets, and this enhanced the emulsion stability. Studies conducted on the effect of ionic strength on EA and ES revealed that emulsifying activity and stability of BNP increased progressively with increase in ionic strength of the medium. Similarly, progressive increases in EA and ES were also observed in BAP and BSP until the ionic strength of the solution reached 0.6 mol/L, following which decreases were observed with further increases in ionic strength.
BNP BAP BSP
80 70
Emulsifying activity (%)
Protein Solubility (%)
100
1007
60 50 40 30 20 10 0 2
4
6
7
8
10
pH
Fig. 2. Effect of pH on emulsifying activity of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
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BNP BAP BSP
BNP BAP BSP
240 220
100
200 180
Foam capacity (ml)
Emulsion stability (%)
90 80 70 60 50 40
160 140 120 100 80 60
30
40
20
20
10
0 2
4
6
7
8
10
pH
0 2
4
6
7
8
10
pH
Fig. 3. Effect of pH on emulsion stability of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
Fig. 4. Effect of pH on foam capacity of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
100
BNP BAP BSP
90 80
Foam stability (%)
In a previous work, effect of ionic strength on emulsifying properties of cowpea protein had earlier been reported by, Aluko and Yada (1995). Similarly, Chavan et al. (2001) also reported ionic strength dependent emulsifying properties for beach pea. Higher emulsion stability of protein at low ionic strength has been attributed to dissociation of Oligomeric structure of 11S-glycinin and subsequent improvement of surface behaviour (Wagner & Gueguen, 1995).
70 60 50 40 30 20 10
4.4. Foaming capacity and stability
0 2
Protein concentration has pronounced effect on foaming capacity and stability. Foaming capacity of both native and modified proteins increased with increase in protein concentration. Increase in foam capacity as observed in this work agrees with those reported for mung bean protein isolate (EL-Adawy, 2000) and succinylated soy protein (Franzen & Kinsella, 1976). Acylation can cause unfolding of the protein, which increases protein–water interaction. Also, the increased net negative charge of succinylated proteins would especially promote protein–water interaction, which facilitates improved foaming capacity. Decrease in foam stability following modifications is a result of increased charge density of succinylated proteins, since it inhibits the protein–protein interactions (Townsend & Nakai, 1983). Effect of pH on foam capacity and stability is presented in Figs. 4 and 5. The result indicates that both native and modified proteins respond to changes in pH of the solution. Except at pH 2, where BNP recorded higher foam capacity than the modified proteins, at all other pHs studied, both succinylation and acetylation improved foam capacity. Maxi-
4
6
pH
7
8
10
Fig. 5. Effect of pH on foam stability of native, acetylated and succinylated protein concentrate of bambarra groundnut. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
mal foam capacities were observed at pH 10 in all the cases. Contrarily, succinylation and acetylation reduced foam stability at all other pHs except pH2. This result suggests that solubility of protein determines the foaming properties. In a previous study, Lin, Humbert, and Sosulski (1974) have reported a pH dependent foaming properties for sunflower meal products. In addition, pH dependent foaming properties have also been reported for P. lunatus and Canavalia ensiformis (Chel-Guerrero et al., 2002). The high foaming capacity at pH 2 and pH 12 may be due to an increase in the net charge of the protein molecules, which weakens hydrophobic interactions and increases protein flexibility. This allowed them to spread to the air water interface more quickly thus encapsulating air particles and increasing foam formation.
O.S. Lawal et al. / Food Research International 40 (2007) 1003–1011
Increase in the foam stability at region of isoelectric point is due to the formation of stable molecular layers in the air–water interface of the foams. Protein adsorption and viscoelasticity at an air–water interface is maximal near or at isoelectric pH because protein is not strongly repelled. In addition, the protein possesses low net charge near isoelectric pH, which may contribute to the formation of stable molecular layers in the air–water interface, a development that improves foam stability. Studies revealed that foaming capacity and stability of native and modified proteins increased with increase in ionic strength of the media. Except in the control solution, modifications improved foam capacity and stability at all other ionic strengths. This development is attributed to increased solubility, thus leading to enhanced whippability and formation of stable cohesive films around the air vacuoles following modifications. 4.5. Gelation properties Effect of ionic strength on gelation capacities is presented in Table 2. Taking the least gelation concentration (LGC) as the index of gelation capacity, lower LGC means better gelation capacity. The result shows that for all the proteins, initial increase in ionic strength enhanced gelation properties, while gelation capacities reduced at higher salt concentrations. In previous reports, Otte, Schumacher, Ipsen, Ju, and Qvist (1999) reported a reduction of gel firmness of whey proteins, when the NaCl content of the mixture was increased. Initial increase in ionic strength facilitated a shielding effect on the surface charges; this development reduced the repulsive forces acting among the protein molecules, creating an identical situation as for isoelectric region, whereas further increase in ionic strength influenced the gel forming process negatively by decreasing the protein unfolding. Similar observation has been reported for whey protein (Boye, Alli, Ismail, Gibbs, & Konishi, 1995). Gelation capacities of the proteins were also pH dependent as presented in Table 3. The result indicate that gelation of the modified derivatives were best at pH 2, while the BNP recorded the highest gelation at pH 4 corresponding to the regions of isoelectric point. Protein gelation is vital in the preparation and acceptability of many foods, including vegetable and other products. Gelation mechanism and gel appearance are fundamentally controlled, by the bal-
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Table 3 Effect of pH on gelation properties of native, acetylated and succinylated bambarra protein concentrate Sample
pH 2
pH 4
pH 7
pH 8
pH 10
BNP BAP BSP
14 10 8
10 12 10
14 12 12
14 16 14
14 16 14
Values are least gelation concentrations expressed as % w/v. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
ance between attractive hydrophobic interactions and repulsive electrostatic interactions (Egelandsal, 1980). The repulsive forces are due to surface charges and the attractive forces are due to various functional groups exposed by the thermal unfolding of the protein (Kojima & Nakamura, 1985). The pH of protein dispersions has profound effect on gelation reactions by influencing the balance of polar and non-polar residues. At pH values in the region of isoelectric pH, protein–protein interactions are generally favoured because the net surface charge is close to zero, which significantly reduces the repulsive interactions between protein molecules thus enhancing gelation, while at pH far removed from isoelectric points, the surface charge on the protein is large and significant repulsive forces prevent protein–protein interaction. 5. Conclusion The effects of acetylation and succinylation on the solubility profile, gelation, foaming and emulsifying properties of bambarra bean protein concentrate have been studied in this work. Acylation of bambarra bean protein concentrate produced pronounced changes in its functional properties. The study also revealed that solubility of protein concentrate improved following acetylation and succinylation and this improved both emulsifying and foaming properties of the native protein concentrate. The study provides fundamental information about the changes in protein function – properties of a new source of protein after acylation. The strength of the investigation is that an under utilized legume is used for the study and these fundamental information could be useful in providing insights into diverse applications of the bambarra groundnut protein,
Table 2 Effect of ionic strength on gelation properties of native, acetylated and succinylated bambarra protein concentrate Sample
Control
0.1 mol/L
0.2 mol/L
0.4 mol/L
0.6 mol/L
0.8 mol/L
1.0 mol/L
BNP BAP BSP
14 12 12
8 10 10
10 8 10
14 8 14
14 14 14
14 14 14
18 16 16
Values are least gelation concentrations expressed as % w/v. Degree of acetylation: 78%; percentage yield calculated on native protein basis. Degree of succinylation: 72%; percentage yield calculated on native protein basis.
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