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Improvement of functional properties of chickpea proteins by hydrolysis with immobilised Alcalase
Food Chemistry 122 (2010) 1212–1217
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Improvement of functional properties of chickpea proteins by hydrolysis with immobilised Alcalase María del Mar Yust *, Justo Pedroche, María del Carmen Millán-Linares, Juan María Alcaide-Hidalgo, Francisco Millán Instituto de la Grasa-CSIC, Av. Padre García Tejero, 4, 41012 Sevilla, Spain
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Article history: Received 21 December 2009 Received in revised form 18 February 2010 Accepted 29 March 2010
Keywords: Immobilised Alcalase Functional properties Chickpea hydrolysates
a b s t r a c t Limited chickpea protein hydrolysates ranging from 1% to 10% degree of hydrolysis were produced from chickpea protein isolate (CPI) using Alcalase immobilised on glyoxyl-agarose gels. Alcalase-glyoxyl derivative produced after 24 h of immobilisation at room temperature was 24 times more stable than soluble enzyme and presented approximately 51% of the activity of Alcalase. The chemical composition of chickpea hydrolysates were very close to that of CPI. Solubility, oil absorption, emulsifying activity and stability, and foaming capacity and stability were determined. All protein hydrolysates showed higher solubility than intact proteins, especially at pHs near isoelectric point of native chickpea proteins. Moreover, all hydrolysates had better functional properties, except emulsifying activity, than the original protein isolate. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Functional properties of proteins are those physicochemical properties that govern their performance and behaviour in food systems during their preparation, processing, storage, and consumption (Kinsella & Whitehead, 1989). A number of strategies have been suggested to improve the functional properties of proteins, including chemical and enzymatic modifications. Enzymatic hydrolysis may be preferable to chemical treatments because of milder process conditions, higher specificity, and minimal formation of by-products (Mannheim & Cheryan, 1992). To improve functional properties is generally admitted that a limited hydrolysis, between 1% and 10%, is needed (Vioque, Sánchez-Vioque, Clemente, Pedroche, & Millán, 2000). Commercially available crude proteinase preparations are used extensively in the food industry to prepare protein hydrolysates. Alcalase, produced by Novozymes, is a nonspecific serine-type protease from Bacillus licheniformis. Its optimum pH for catalysis ranges from 6.5 to 8.5. Alcalase has been widely used to produce protein hydrolysates with better nutritional or functional properties than intact protein (Doucet, Gauthier, Otter, & Foegeding,
Abbreviations: Boc-L-ala-ONp, tert-butoxycarbonyl-L-alanine-4-nitrophenilester; CPI, chickpea protein isolate; CPH, chickpea protein hydrolysate; DH, degree of hydrolysis. * Corresponding author. Tel.: +34 954611550; fax: +34 954616790. E-mail address: [email protected] (M.M. Yust). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.03.121
2003; Klompong, Benjakul, Kantachote, Hayes, & Shahidi, 2008; Pizones Ruiz-Henestrosa et al., 2007). Up to now, most studies on enzymatic hydrolysis to improve functional properties of proteins report the use of soluble enzymes. However, in order for enzymes to be viable from an industrial point of view, they must be recycled and reused. A second limiting factor for the implementation of enzymes as industrial biocatalysts is stability (Haki & Rakshit, 2003). Although several procedures have been employed to achieve stabilization of enzymes, coupling immobilisation to stabilization, has the advantage of facilitating both recycling and stabilization requirements. In this case, it has been reported that glyoxyl-agarose supports constitute a good substrate for the immobilisation/stabilization of proteins via multipoint covalent attachment (Mateo et al., 2006). The active glyoxyl groups found in these supports are aldehydes moderately separated from the support surface that form reversible and relatively weak Schiff bases with amine groups in enzymes (Guisán, 1988; Mateo et al., 2006). The Schiff bases can be later stabilized by reduction to amine bonds. Many enzymes have been stabilized using this technique, including trypsin, chymotrypsin, carboxypeptidase A, esterase, thermolysin, catalases, and lipases from different sources (Mateo et al., 2006; Pedroche et al., 2002). In addition to stabilization, the use of immobilised enzymes onto glyoxyl supports implies that inactivation of enzyme is not needed because the biocatalyst can be easily removed from the reaction medium. This is especially important in limited hydrolysis, where the time needed to reach inactivation conditions may accelerate the reaction rate, making difficult the control of desired DH.
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In the present paper we describe: (1) immobilisation/stabilization of Alcalase on glyoxyl-agarose gels, (2) the production of limited chickpea protein hydrolysates using Alcalase-glyoxyl derivatives and (3) studies on composition and functional properties of these hydrolysates. 2. Materials and methods 2.1. Materials Chickpea seeds were provided by Koipesol Semillas S.A. (Sevilla, Spain). Agarose cross-linked 4% (w/w) beads were purchased from Iberagar S.A. (Coina, Portugal). Alcalase 2.4 L was provided by Novozymes (Bagsvaerd, Denmark). Trinitrobenzenesulfonic acid (TNBS), and sodium borohydride were purchased from Sigma Chemical Co. (St. Louis, MO). Boc-L-alanine-4-nitrophenilester (boc-L-ala-ONp) was purchased from Bachem S.A. (Budendorf, Switzerland). All other chemicals were of analytical grade. 2.2. Methods 2.2.1. Preparation of CPI CPI was prepared according to Sánchez-Vioque, Clemente, Vioque, Bautista, and Millán (1999) with slight modifications. Chickpea defatted flour (100 g) was suspended in 1 l of 0.25% Na2SO3 (w/v) at pH 10.5. The suspension was extracted by stirring for 1 h at room temperature and pH was kept constant by adding 1 M NaOH. After centrifuging at 10,500g for 15 min, an additional extraction was carried out with half of the volume. The supernatants were pooled, and pH was adjusted to isoelectric point (4.3) of chickpea proteins. The precipitate formed, was recovered by centrifuging, washed with distiled water adjusted to pH 4.3, and spray-dried. 2.2.2. Activation of agarose gels Glyoxyl-agarose cross-linked 4% beads were prepared as described elsewhere (Guisán, 1988).
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conditions. The catalytic activity of commercial Alcalase was 2980 NPA units ml1. 2.2.5. Thermal stability Samples of soluble and immobilised enzyme were suspended in 0.1 M sodium phosphate, pH 8, and incubated at 50 °C. Aliquots were withdrawn periodically, and enzyme activities were measured at pH 7 and 25 °C as described above. Pseudo half-life times, pt1/2 (time necessary to reach 50% of residual activity), were taken directly from experimental time courses of inactivation for each sample. Stabilization factor of derivatives (gels with immobilised enzyme) was calculated as the ratio between pt1/2 of each derivative and pt1/2 of the soluble enzyme. 2.2.6. Hydrolysis of CPI Hydrolysis of CPI catalysed by immobilised Alcalase was performed batchwise in a reactor with stirring and controlled pH and temperature. Chickpea isolate was resuspended in distiled water (5% w/v) and pH was adjusted to 8 and temperature to 50 °C. Then, the 24 h Alcalase-glyoxyl derivative was suspended in a relation E/S = 1.14 mg immobilised enzyme/g chickpea protein. During the course of the reaction, pH was kept constant by titration of the released protons with a NaOH solution in appropriate concentration. Samples were withdrawn at different times to measure the degree of hydrolysis during the process and the biocatalyst was removed by filtration. 2.2.7. Determination of the degree of hydrolysis (DH) The DH, defined as the percentage of peptide bonds cleaved, was measured by the TNBS method according to Adler-Nissen (1979). The total number of amino groups was determined in a sample that had been 100% hydrolysed at 110 °C for 24 h in 6 N HCl. 2.2.8. Analytical methods Protein and peptide amounts were determined by elemental analysis as % nitrogen content 6.25, using a LECO CHNS-932 analyzer (St. Joseph, MI). Moisture and ash was determined using AOAC (1990) approved methods.
2.2.3. Preparation of Alcalase-glyoxyl derivatives Immobilisation of Alcalase was carried out according to Tardioli, Pedroche, Giordano, Fernández-Lafuente, and Guisán (2003) with slight modifications. One hundred millilitres of 100 mM sodium bicarbonate were mixed with 0.2 ml of Alcalase and then added to 15 g of glyoxyl-agarose. The reaction mixture was gently stirred at room temperature. Aliquots of the supernatant and whole suspension were withdrawn at different times and their catalytic activities were measured. After 1, 3, 6 or 24 h of contact between the enzyme and the support, derivatives were reduced with sodium borohydride, as described by Blanco and Guisán (1989). The reduced derivatives were washed successively with 100 mM sodium phosphate buffer at pH 7 and abundant distiled water.
2.2.9. Analyses of amino acid composition by HPLC The quantification of amino acids was done according to Alaiz, Navarro, Giron, and Vioque (1992). Tryptophan content was analysed according to the method of Yust et al. (2004).
2.2.4. Enzymatic activity assay Enzymatic activity in the soluble and immobilised Alcalase preparations was measured by following the increase of absorbance at 405 nm that accompanies hydrolysis of the synthetic substrate boc-L-ala-ONp (to 1.98 ml of 50 mM sodium phosphate, pH 7, containing 20% ethanol, were added 20 ll of soluble or suspended enzyme and 20 ll of 0.1 M boc-L-ala-ONp in acetonitrile). Assays were performed in spectrophotometric glass cuvettes at 25 °C with magnetic stirring. The gels did not produce interferences in spectrophotometric measurements. One nitrophenyl activity (NPA) unit was defined as the amount of enzyme that hydrolyses 1 lmol of boc-L-ala-ONp per min under the described
2.2.10.2. Oil adsorption. For determination of oil adsorption the method of Lin, Humbert, and Sosulski (1974) was used. Oil adsorption capacity was expressed as the number of grams of oil retained by 100 g of material.
2.2.10. Functional properties 2.2.10.1. Solubility. Samples were suspended in water (5% w/v) and pH was kept at different values between 2 and 10 using 1 N NaOH or 1 N HCl while stirring at room temperature for 1 h. The samples were then centrifuged at 10,000g for 15 min and nitrogen content was determined in the supernatants. Solubility was expressed as the percentage of total nitrogen of the original sample present in the soluble fraction.
2.2.10.3. Emulsifying activity and stability. Emulsifying activity and stability were determined according to Bejosano and Corke (1999), with slight modifications. Samples (3.5 g) were homogenised for 30 s in 50 ml of water using a model Omnimixer homogenizer (Sorvall Instruments, Wilmington, DE, USA) at approximately 10,000 rpm. Corn oil (25 ml) was added, and the mixture was homogenised again for 30 s. Other 25 ml of corn oil
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were added, and the mixture was homogenised for 90 s. The emulsion was centrifuged at 1100g for 5 min. Emulsifying activity was calculated by dividing the volume of the emulsified layer by the volume of emulsion before centrifugation. The emulsion stability was determined using similar samples as for measurement of emulsifying activity. They were heated for 15 min at 85 °C and after temperature came back to room temperature they were centrifuged at 1100g for 5 min. The emulsion stability was expressed as the percentage of emulsifying activity remaining after heating.
solvents, denaturating agents, etc. (Mateo et al., 2006; Pedroche et al., 2007). From this time on (3 h), the residual activity of the immobilised Alcalase (obtained as the difference between the activity of the suspension and that of the supernatant) maintained over 50% of the initial activity. This result is in agreement with those reported for other enzymes by Pedroche et al. (2002).
3.2. Thermal stability of Alcalase-glyoxyl derivatives
Alcalase is a commercial, not highly purified, enzymatic preparation and we were mostly concerned with its activity. For this reason, the progress of immobilisation was followed by measuring the activity (and not protein amount) of the supernatant, of the total suspension, and of the soluble enzyme. Several enzymatic loads were tested in the range 5–80 NPA units/g gel. After that, 38 NPA units/g gel was chosen as optimal because higher loads produced derivatives that preserved less than 40% of initial activity of Alcalase (data not shown). The time course of immobilisation of Alcalase with optimal enzymatic load is shown in Fig. 1. The immobilisation of the enzyme is reached at 3 h. However, a longer exposure time is required to obtain a higher number of bonds between the enzyme and the support, which may result in a higher resistance to small conformational changes caused by heat, organic
One extremely valuable advantage of conducting biotechnological processes at elevated temperatures is reducing the risk of contamination by common mesophiles. For that reason, the thermal stability of enzymes is an important feature for the application of the biocatalyst from a commercial point of view. Commercial Alcalase contains stabilizers that are lost during the washing of glyoxyl-agarose derivatives. Nevertheless, the stability of the derivatives was compared with that of the soluble enzyme, containing stabilizers, in order to study enzymatic preparations as they would actually be used in an industrial setting. The stabilization achieved through immobilisation is shown in Fig. 2. The 1 h derivative was about nine times more stable than soluble Alcalase. Moreover, stabilization of derivatives greatly increased as the contact time between enzyme and support did, which is a general result when an enzyme is immobilised onto glyoxyl-agarose supports (Pedroche et al., 2002, 2007; Tardioli et al., 2003; Yust et al., 2007). Our results indicated that the optimum time to obtain maximum stability is 24 h. Longer exposure times slightly increased the thermal stability but decreased enzyme activity as a consequence of higher distortion of enzyme structure. These stabilization factors are lower than those found in the immobilisation of other enzymes on glyoxyl-agarose gels (Pedroche et al., 2002, 2007; Tardioli et al., 2003), but we attributed this to two factors. Firstly, we used commercial soluble Alcalase, without removal of stabilizers, to calculate stabilization factors. Secondly, glyoxyl-agarose 10 BCL beads were used in articles cited above whereas we used glyoxyl-agarose 4 BCL beads, with a higher pore size, to avoid diffusion limitations in the hydrolysis of CPI, where 360 kDa proteins are predominant. It has been reported that 10 BCL beads, which have more active groups than 4 BCL beads, allow higher stabilization due to a higher number of interactions between enzyme and support (Pedroche et al., 2007). However, stabilization of Alcalase-glyoxyl derivative was higher than that obtained with other supports such as silica derivatives (Ferreira, Ramos, Dordick, & Gil, 2003).
Fig. 1. Time-course of the immobilisation of Alcalase onto glyoxyl-agarose bead, at 25 °C, pH 10.05. (}) Activity of soluble enzyme; (h) activity of whole suspension (supernatant plus agarose bead); and (s) activity of supernatant.
Fig. 2. Stabilization factor of Alcalase-glyoxyl derivatives, expressed as ratio between the half-life time of the immobilised enzyme and half-life of the soluble enzyme, obtained at different times of immobilisation.
2.2.10.4. Foaming capacity and stability. The activity and the stability of foam were determined by the method of Fuhrmeister and Meuser (2003) with modifications. The samples (50 ml of 3% w/v dispersion in distiled water) were homogenised using a model Omnimixer homogenizer at 10,000 rpm. The foaming capacity was expressed as the percentage of volume increase. Foam stability was expressed as the percentage of foam volume remaining after 60 min at room temperature. 2.2.11. Statistical analysis Statistical analysis was performed with the STATGRAPHICS Plus 5.0 program. Data were analysed with One-Way ANOVA Tests and pairwise multiple comparisons were conducted with Fisher’s least significant difference (LSD) procedure. Differences between the means were considered to be significant when P 6 0.05. 3. Results and discussion 3.1. Immobilisation of Alcalase
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3.3. Production and characterisation of chickpea protein hydrolysates CPI is a good substrate, with approximately 90% protein on a dry basis, to be used as starting material for the enzymatic hydrolysis (Table 1). To obtain chickpea protein hydrolysates (CPH) in the range 1–10% DH, a very low proportion E/S ratio should be used to get a very slow hydrolysis rate. In that way, several hydrolysates with successively increasing DH can be produced in the same assay. Thus, after several trials, an E/S ratio of 1.14 mg immobilised Alcalase per g of CPI protein was chosen. At different times, samples were withdrawn and the catalyst was removed by filtration. The variation of DH versus time is shown in Fig. 3. The hydrolysates obtained after 10, 50, 150 and 300 min had DH = 1%, 2.9%, 4.9% and 10%, respectively, and were selected to study their functional properties. The chemical composition of the hydrolysates was similar to chickpea isolate (Table 1). In fact, the main difference was ash content, which was higher in the hydrolysates due to the addition of alkali to keep the pH constant during hydrolysis. The amino acid composition of CPI and CPHs is presented in Table 2. CPI had an amino acid composition that meets the nutritional requirements proposed by FAO/WHO, except in sulphur amino acids, which is common in legume proteins (Singh, Rao, Singh, & Jambunathan, 1988). The amino acid composition of hydrolysates did not show significant differences with respect to isolate, which demonstrates that enzymatic hydrolysis, in opposition to acid or basic hydrolysis, do not alter the nutritional value of original proteins. 3.4. Functional properties of CPI and CPHs 3.4.1. Solubility A high solubility is necessary to use a product in a lot of manufactured foods. It is also important to improve other functional
Table 1 Composition of chickpea protein isolate and hydrolysates obtained after treatment with immobilised Alcalase.
CPI CPH CPH CPH CPH a
1% 2.9% 4.9% 10%
Protein
Moisture
Ash
Othersa
89.3 ± 0.98 89.6 ± 0.47 87.1 ± 0.81 87.7 ± 0.44 86.1 ± 0.56
3.41 ± 0.09 5.32 ± 0.16 8.11 ± 0.27 6.54 ± 0.23 6.23 ± 0.22
2.32 ± 0.01 3.17 ± 0.20 4.71 ± 0.08 5.14 ± 0.09 7.40 ± 0.26
4.97 1.90 0.09 0.65 0.27
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properties, which depend on an adequate initial solubilisation of proteins. Fig. 4 shows the protein solubility profiles of CPI and CPHs as a function of pH. Intact CPI had very low solubility between pHs 4 and 6, with a minimum at pH 4.3 (isoelectric point), where approximately 95% of the proteins precipitated. Similar solubility patterns were reported in other legume protein isolate such as cowpea (Ragab, Babiker, & Eltinay, 2004), bean (Makri & Doxastakis, 2006), and soy (Fuhrmeister & Meuser, 2003). Hydrolysis increased the solubility at all pH values in this study, except for DH = 1%. Moreover, the main differences between solubility of CPI and the hydrolysates were observed around isoelectric point. When the degree of hydrolysis increased, a rapid increase of the solubility at this pH could be observed. This effect has been described in the partial hydrolysis of other legume proteins such as pea (Periago et al., 1998) and soy (Molina Ortiz & Wagner, 2002). The enhanced solubility of the hydrolysates may be attributed to their smaller molecular size and the newly exposed ionisable amino and carboxyl groups, which increase the hydrolysates’ hydrophilicity. However, the decrease in size of CPH 1% in relation to CPI was too small to get higher solubility. 3.4.2. Oil absorption Oil absorption capacity represents the ability of proteins to interact with lipid materials, which is important in food formulation and processing since many properties of foods involve the interaction of proteins and lipids, e.g., fat entrapment and flavour absorption. Oil absorption of CPI (Table 3) was higher than that observed in other plant protein isolates such as rapeseed (Mansour, Peredi, & Dworschak, 1992) or soy protein isolate (Wang & Johnson, 2001), which showed oil absorption of 225 and 204 g oil/ 100 g, respectively. Furthermore, all hydrolysates studied showed higher oil-holding capacity than the original isolate. Similar results were obtained by Periago et al. (1998) when pea flour was hydrolysed and by Vioque et al. (2000) in the hydrolysis of rapeseed protein isolate with Alcalase. This may be due to the fact that the hydrolysis of proteins exposes non-polar side chains that bind hydrocarbon moieties of oil, contributing to increase oil absorption. The hydrolysate with DH = 4.9% presented the highest oil absorption capacity (628). If the DH increases further the oil absorption capacity decreases, what may be attributed to the major exposure of ionic groups after hydrolysis. From these results, we can conclude that chickpea hydrolysates with DH around 5% can be used in food industry as ingredients in products like meat replacers and extenders, doughnuts, baked goods and soups, where fat retention is important.
Calculated as 100-protein-moisture-ash.
Fig. 3. Time course of the hydrolysis of CPI with Alcalase immobilised onto glyoxylagarose at pH 8 and 50 °C.
3.4.3. Emulsifying activity and emulsion stability The emulsifying properties determined in this study were the emulsifying activity and emulsion stability. The former is related to the ability of a protein to form an emulsion, i.e., to assist in the dispersion of oil into small globules that are homogeneously distributed throughout the continuous aqueous phase. The emulsion stability is related to the ability of a protein to preserve homogeneity of the oil-in-water emulsion on storage. The literature is ambiguous about the improvement of emulsifying properties with limited hydrolysis. Some studies reported that the emulsifying properties of proteins are improved by limited hydrolysis, due to the exposure of hydrophobic amino acid residues that may interact with the oil whereas the hydrophilic residues interact with water (Vioque et al., 2000). However, it is speculated that peptides, of smaller size than intact protein, generated in the hydrolysis cannot form the same molecular interactions as proteins of higher molecular weight (Panyam & Kilara, 1996; Singh & Dalgleish, 1998). Limited proteolysis with the Alcalase-glyoxyl derivative had none or little effect on emulsifying properties of CPI (Table 3). Only the hydrolysate with DH = 2.9% showed higher emulsifying activity
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Table 2 Amino acid composition of chickpea protein isolate and hydrolysates obtained with immobilised Alcalase.
Asp + Asn Glu + Gln Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Trp Leu Phe Lys
CPI
CPH 1%
CPH 2.9%
CPH 4.9%
CPH 10%
13.2 ± 0.53 17.1 ± 0.65 5.99 ± 0.19 2.82 ± 0.10 3.91 ± 0.09 4.55 ± 0.10 9.18 ± 0.08 4.86 ± 0.20 4.82 ± 0.63 2.40 ± 0.00 4.26 ± 0.21 0.24 ± 0.02 0.93 ± 0.09 3.39 ± 0.12 0.63 ± 0.03 9.00 ± 0.27 6.40 ± 0.26 6.70 ± 0.02
13.6 ± 0.24 18.3 ± 0.08 6.38 ± 0.01 2.64 ± 0.08 3.88 ± 0.01 4.31 ± 0.07 8.87 ± 0.07 4.79 ± 0.03 4.15 ± 0.33 2.25 ± 0.02 4.20 ± 0.18 0.33 ± 0.02 0.91 ± 0.05 3.20 ± 0.05 0.65 ± 0.03 8.87 ± 0.10 6.31 ± 0.12 6.75 ± 0.06
14.3 ± 0.20 17.4 ± 0.85 6.85 ± 0.09 2.70 ± 0.13 4.09 ± 0.15 3.89 ± 0.13 9.22 ± 0.08 4.64 ± 0.05 3.61 ± 0.02 2.65 ± 0.13 3.76 ± 0.01 0.23 ± 0.03 0.98 ± 0.05 3.34 ± 0.08 0.78 ± 0.02 8.88 ± 0.20 6.35 ± 0.21 6.75 ± 0.10
14.5 ± 0.66 19.8 ± 0.46 6.86 ± 0.11 2.43 ± 0.12 3.94 ± 0.03 3.74 ± 0.17 9.50 ± 0.17 4.22 ± 0.02 4.31 ± 0.20 2.21 ± 0.11 3.71 ± 0.14 0.21 ± 0.03 0.94 ± 0.06 3.17 ± 0.11 0.61 ± 0.03 8.28 ± 0.24 5.82 ± 0.09 6.92 ± 0.23
14.2 ± 0.11 18.7 ± 0.82 7.01 ± 0.07 2.40 ± 0.12 3.86 ± 0.18 4.16 ± 0.15 9.22 ± 0.23 4.31 ± 0.17 3.82 ± 0.18 2.13 ± 0.09 4.17 ± 0.14 0.17 ± 0.04 0.80 ± 0.06 3.34 ± 0.14 0.55 ± 0.06 8.50 ± 0.15 5.83 ± 0.05 7.11 ± 0.19
FAOa
1.9 3.4
3.5 2.5b 2.8 6.6 6.3c 5.8
Data expressed as g/100 g are the mean ± standard deviation of three determinations. a FAO/WHO/ONU. Energy and protein requirements, 1985. b Methionine + cysteine. c Phenylalanine + tyrosine.
soy (Tsumura et al., 2005), and whey (van der Ven, Gruppen, de Bont, & Voragen, 2001) amongst others.
Fig. 4. Nitrogen solubility as a function of pH of CPI and CPHs obtained with Alcalase-glyoxyl derivative. (—) isolate; (h) CPH 1%; (s) CPH 2.9%; () CPH 4.9%; and (}) CPH 10%.
than CPI, but emulsion stability decreased. In many cases, a direct relationship between nitrogen solubility and emulsifying properties of proteins has been demonstrated (Narayana & Narasinga Rao, 1984). In our research, although CPHs had excellent nitrogen solubility, they showed poor emulsification properties; therefore, emulsifying properties ultimately depend upon a suitable balance between the hydrophiles and lipophiles and do not necessarily increase as a protein becomes more soluble. In fact, reduction of emulsion-forming ability after hydrolysis has been reported for
3.4.4. Foaming capacity and foam stability Foams are biphasic colloidal systems with a continuous liquid or aqueous phase and a dispersed gas or air phase. Food proteins are capable of forming good foams, and ingredients such as egg white have long been an integral part of recipes that require whipping or foaming. It has also been established that whey proteins possess good foaming properties (Panyam & Kilara, 1996). Partial hydrolysis of CPI with immobilised Alcalase improved foaming capacity to a great extent (Fig. 5). In fact, CPI did not show foaming capacity whereas all CPH had good foaming properties. This could be attributed to the production of amphiphilic peptides after hydrolysis. Their reduced molecular weight will make them more flexible, forming a stable interfacial layer and increasing the rate of diffusion to the interface, improving the foamability properties (Liceaga-Gesualdo & Li-Chan, 1999). This improvement in foaming capacity for enzymatically hydrolysed food proteins was reported by several authors (Pizones Ruiz-Henestrosa et al., 2007; Tsumura et al., 2005). The best result was for DH = 4.9%; this hydrolysate had a value of 158% of foam capacity. Moreover, this hydrolysate showed the highest foam stability, preserving approximately 60% of foam after 60 min at room temperature. In conclusion, the partial hydrolysis of chickpea protein isolate with immobilised Alcalase is a helpful strategy to improve some functional properties of intact proteins, such as solubility, oil absorption capacity, and foaming capacity and stability. Specifically, the hydrolysis of 5% peptidic bonds of chickpea proteins seems to be the most appropriate to be considered as an alternative for other protein ingredient sources that are being used in the food industry.
Table 3 Functional properties of chickpea protein isolate and hydrolysates with different DH.
Oil adsorption Emulsifying activity Emulsion stability
Isolate
CPH 1%
CPH 2.9%
CPH 4.9%
CPH 10%
308 ± 6.6a 44.7 ± 1.2a 76.5 ± 0.9a
542 ± 9.0b 39.1 ± 0.8b 100 ± 0.0b
618 ± 5.1c 54.0 ± 0.6c 51.8 ± 0.7c
628 ± 5.2c 37.8 ± 0.6b 16.8 ± 0.4d
443 ± 7.1d 35.1 ± 0.4d 17.5 ± 1.2d
Data represent the mean ± standard deviation of three experiments. Different letters within the same row indicate significant differences (P 6 0.05).
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Fig. 5. Foaming capacity (open bars) and foam stability (closed bars) of CPI and CPHs obtained with Alcalase-glyoxyl derivative. Values marked by the same letter are not significantly different (P 6 0.05).
Acknowledgement This research work was supported by a fellowship of Spanish Ministry of Education and Science (M.M.Y.).
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Makri, E. A., & Doxastakis, G. I. (2006). Emulsifying and foaming properties of Phaseolus vulgaris and coccineus proteins. Food Chemistry, 98, 558– 568. Mannheim, A., & Cheryan, M. (1992). Enzyme-modified proteins from corn gluten meal: Preparation and functional properties. Journal of the American Oil Chemists Society, 69(12), 1163–1169. Mansour, E. H., Peredi, J. M., & Dworschak, E. (1992). Preparation and functional properties of rapeseed protein products. Acta Alimentaria, 21, 293–305. Mateo, C., Palomo, J. M., Fuentes, M., Betancor, L., Grazu, V., López-Gallego, F., et al. (2006). Glyoxyl-agarose: A fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzyme and Microbial Technology, 39, 274–280. Molina Ortiz, S. E., & Wagner, J. R. (2002). Hydrolysates of native and modified soy protein isolates: Structural characteristics, solubility and foaming properties. Food Research International, 35, 511–518. Narayana, K., & Narasinga Rao, M. S. (1984). Effect of acetylation and succinylation on the functional properties of winged bean (Psophorcarpus tetragonolobus) flour. Journal of Food Science, 49, 547–550. Panyam, D., & Kilara, A. (1996). Enhancing the functionality of food proteins by enzymatic modification. Trends in Food Science and Technology, 7, 120–125. Pedroche, J., Yust, M. M., Girón-Calle, J., Vioque, J., Alaiz, M., Mateo, C., et al. (2002). Stabilization–immobilization of carboxipeptidase A to aldehyde-agarose gels. A practical example in the hydrolysis of casein. Enzyme and Microbial Technology, 31, 711–718. Pedroche, J., Yust, M. M., Mateo, C., Fernandez-Lafuente, R., Girón-Calle, J., Alaiz, M., et al. (2007). Effect of the support and experimental conditions in the intensity of the multipoint covalent attachment of proteins on glyoxyl-agarose supports: Correlation between enzyme-support linkages and thermal stability. Enzyme and Microbial Technology, 40, 1160–1166. Periago, M. J., Vidal, M. L., Ros, G., Rincón, F., Martínez, C., López, G., et al. (1998). Influence of enzymatic treatment on the nutritional and functional properties of pea flour. Food Chemistry, 63, 71–78. Pizones Ruiz-Henestrosa, V., Carrera Sánchez, C., Yust, M. M., Pedroche, J., Millán, F., & Rodríguez-Patino, J. M. (2007). Limited enzymatic hydrolysis can improve the interfacial and foaming characteristics of b-conglycin. Journal of Agricultural and Food Chemistry, 55, 1536–1545. Ragab, D. D. M., Babiker, E. E., & Eltinay, A. H. (2004). Fractionation, solubility and functional properties of cowpea (Vigna unguiculata) proteins by pH and/or salt concentration. Food Chemistry, 84, 207–212. Sánchez-Vioque, R., Clemente, A., Vioque, J., Bautista, J., & Millán, F. (1999). Protein isolates from chickpea (Cicer arietinum L.): Chemical composition, functional properties and protein characterization. Food Chemistry, 64, 237–243. Singh, A. M., & Dalgleish, D. G. (1998). The emulsifying properties of hydrolysates of whey proteins. Journal of Dairy Science, 81, 918–924. Singh, D. K., Rao, A. S., Singh, R., & Jambunathan, R. (1988). Amino acid composition of storage proteins of a promising chickpea (Cicer arietinum L.) cultivar. Journal of the Science of Food and Agriculture, 43, 373–379. Tardioli, P. W., Pedroche, J., Giordano, R. L. C., Fernández-Lafuente, R., & Guisán, J. M. (2003). Hydrolysis of proteins by immobilized–stabilized alcalase-glyoxyl agarose. Biotechnology Progress, 19, 352–360. Tsumura, K., Saito, T., Tsuge, K., Ashida, H., Kugimiya, W., & Inouye, K. (2005). Functional properties of soy protein hydrolysates obtained by selective proteolysis. LWT, 38, 255–261. van der Ven, C., Gruppen, H., de Bont, D. B. A., & Voragen, A. G. J. (2001). Emulsion properties of casein and whey protein hydrolysates and the relation with other hydrolysate characteristics. Journal of Agricultural and Food Chemistry, 49, 5005–5012. Vioque, J., Sánchez-Vioque, R., Clemente, A., Pedroche, J., & Millán, F. (2000). Partially hydrolyzed rapeseed protein isolates with improved functional properties. Journal of the American Oil Chemists Society, 77(4), 447– 450. Wang, C., & Johnson, L. A. (2001). Functional properties of hydrothermally cooked soy protein products. Journal of the American Oil Chemists Society, 78(2), 189–195. Yust, M. M., Pedroche, J., Alaiz, M., Girón-Calle, J., Vioque, J., Mateo, C., et al. (2007). Partial purification and immobilization/stabilization on highly activated glyoxyl-agarose supports of different proteases from flavourzyme. Journal of Agricultural and Food Chemistry, 55, 6503–6508. Yust, M. M., Pedroche, J., Girón-Calle, J., Vioque, J., Millán, F., & Alaiz, M. (2004). Determination of tryptophan by high-performance liquid chromatography of alkaline hydrolysates with spectrophotometric detection. Food Chemistry, 85, 317–320.
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Improvement of functional properties of chickpea proteins by hydrolysis with immobilised Alcalase María del Mar Yust *, Justo Pedroche, María del Carmen Millán-Linares, Juan María Alcaide-Hidalgo, Francisco Millán Instituto de la Grasa-CSIC, Av. Padre García Tejero, 4, 41012 Sevilla, Spain
a r t i c l e
i n f o
Article history: Received 21 December 2009 Received in revised form 18 February 2010 Accepted 29 March 2010
Keywords: Immobilised Alcalase Functional properties Chickpea hydrolysates
a b s t r a c t Limited chickpea protein hydrolysates ranging from 1% to 10% degree of hydrolysis were produced from chickpea protein isolate (CPI) using Alcalase immobilised on glyoxyl-agarose gels. Alcalase-glyoxyl derivative produced after 24 h of immobilisation at room temperature was 24 times more stable than soluble enzyme and presented approximately 51% of the activity of Alcalase. The chemical composition of chickpea hydrolysates were very close to that of CPI. Solubility, oil absorption, emulsifying activity and stability, and foaming capacity and stability were determined. All protein hydrolysates showed higher solubility than intact proteins, especially at pHs near isoelectric point of native chickpea proteins. Moreover, all hydrolysates had better functional properties, except emulsifying activity, than the original protein isolate. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Functional properties of proteins are those physicochemical properties that govern their performance and behaviour in food systems during their preparation, processing, storage, and consumption (Kinsella & Whitehead, 1989). A number of strategies have been suggested to improve the functional properties of proteins, including chemical and enzymatic modifications. Enzymatic hydrolysis may be preferable to chemical treatments because of milder process conditions, higher specificity, and minimal formation of by-products (Mannheim & Cheryan, 1992). To improve functional properties is generally admitted that a limited hydrolysis, between 1% and 10%, is needed (Vioque, Sánchez-Vioque, Clemente, Pedroche, & Millán, 2000). Commercially available crude proteinase preparations are used extensively in the food industry to prepare protein hydrolysates. Alcalase, produced by Novozymes, is a nonspecific serine-type protease from Bacillus licheniformis. Its optimum pH for catalysis ranges from 6.5 to 8.5. Alcalase has been widely used to produce protein hydrolysates with better nutritional or functional properties than intact protein (Doucet, Gauthier, Otter, & Foegeding,
Abbreviations: Boc-L-ala-ONp, tert-butoxycarbonyl-L-alanine-4-nitrophenilester; CPI, chickpea protein isolate; CPH, chickpea protein hydrolysate; DH, degree of hydrolysis. * Corresponding author. Tel.: +34 954611550; fax: +34 954616790. E-mail address: [email protected] (M.M. Yust). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.03.121
2003; Klompong, Benjakul, Kantachote, Hayes, & Shahidi, 2008; Pizones Ruiz-Henestrosa et al., 2007). Up to now, most studies on enzymatic hydrolysis to improve functional properties of proteins report the use of soluble enzymes. However, in order for enzymes to be viable from an industrial point of view, they must be recycled and reused. A second limiting factor for the implementation of enzymes as industrial biocatalysts is stability (Haki & Rakshit, 2003). Although several procedures have been employed to achieve stabilization of enzymes, coupling immobilisation to stabilization, has the advantage of facilitating both recycling and stabilization requirements. In this case, it has been reported that glyoxyl-agarose supports constitute a good substrate for the immobilisation/stabilization of proteins via multipoint covalent attachment (Mateo et al., 2006). The active glyoxyl groups found in these supports are aldehydes moderately separated from the support surface that form reversible and relatively weak Schiff bases with amine groups in enzymes (Guisán, 1988; Mateo et al., 2006). The Schiff bases can be later stabilized by reduction to amine bonds. Many enzymes have been stabilized using this technique, including trypsin, chymotrypsin, carboxypeptidase A, esterase, thermolysin, catalases, and lipases from different sources (Mateo et al., 2006; Pedroche et al., 2002). In addition to stabilization, the use of immobilised enzymes onto glyoxyl supports implies that inactivation of enzyme is not needed because the biocatalyst can be easily removed from the reaction medium. This is especially important in limited hydrolysis, where the time needed to reach inactivation conditions may accelerate the reaction rate, making difficult the control of desired DH.
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In the present paper we describe: (1) immobilisation/stabilization of Alcalase on glyoxyl-agarose gels, (2) the production of limited chickpea protein hydrolysates using Alcalase-glyoxyl derivatives and (3) studies on composition and functional properties of these hydrolysates. 2. Materials and methods 2.1. Materials Chickpea seeds were provided by Koipesol Semillas S.A. (Sevilla, Spain). Agarose cross-linked 4% (w/w) beads were purchased from Iberagar S.A. (Coina, Portugal). Alcalase 2.4 L was provided by Novozymes (Bagsvaerd, Denmark). Trinitrobenzenesulfonic acid (TNBS), and sodium borohydride were purchased from Sigma Chemical Co. (St. Louis, MO). Boc-L-alanine-4-nitrophenilester (boc-L-ala-ONp) was purchased from Bachem S.A. (Budendorf, Switzerland). All other chemicals were of analytical grade. 2.2. Methods 2.2.1. Preparation of CPI CPI was prepared according to Sánchez-Vioque, Clemente, Vioque, Bautista, and Millán (1999) with slight modifications. Chickpea defatted flour (100 g) was suspended in 1 l of 0.25% Na2SO3 (w/v) at pH 10.5. The suspension was extracted by stirring for 1 h at room temperature and pH was kept constant by adding 1 M NaOH. After centrifuging at 10,500g for 15 min, an additional extraction was carried out with half of the volume. The supernatants were pooled, and pH was adjusted to isoelectric point (4.3) of chickpea proteins. The precipitate formed, was recovered by centrifuging, washed with distiled water adjusted to pH 4.3, and spray-dried. 2.2.2. Activation of agarose gels Glyoxyl-agarose cross-linked 4% beads were prepared as described elsewhere (Guisán, 1988).
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conditions. The catalytic activity of commercial Alcalase was 2980 NPA units ml1. 2.2.5. Thermal stability Samples of soluble and immobilised enzyme were suspended in 0.1 M sodium phosphate, pH 8, and incubated at 50 °C. Aliquots were withdrawn periodically, and enzyme activities were measured at pH 7 and 25 °C as described above. Pseudo half-life times, pt1/2 (time necessary to reach 50% of residual activity), were taken directly from experimental time courses of inactivation for each sample. Stabilization factor of derivatives (gels with immobilised enzyme) was calculated as the ratio between pt1/2 of each derivative and pt1/2 of the soluble enzyme. 2.2.6. Hydrolysis of CPI Hydrolysis of CPI catalysed by immobilised Alcalase was performed batchwise in a reactor with stirring and controlled pH and temperature. Chickpea isolate was resuspended in distiled water (5% w/v) and pH was adjusted to 8 and temperature to 50 °C. Then, the 24 h Alcalase-glyoxyl derivative was suspended in a relation E/S = 1.14 mg immobilised enzyme/g chickpea protein. During the course of the reaction, pH was kept constant by titration of the released protons with a NaOH solution in appropriate concentration. Samples were withdrawn at different times to measure the degree of hydrolysis during the process and the biocatalyst was removed by filtration. 2.2.7. Determination of the degree of hydrolysis (DH) The DH, defined as the percentage of peptide bonds cleaved, was measured by the TNBS method according to Adler-Nissen (1979). The total number of amino groups was determined in a sample that had been 100% hydrolysed at 110 °C for 24 h in 6 N HCl. 2.2.8. Analytical methods Protein and peptide amounts were determined by elemental analysis as % nitrogen content 6.25, using a LECO CHNS-932 analyzer (St. Joseph, MI). Moisture and ash was determined using AOAC (1990) approved methods.
2.2.3. Preparation of Alcalase-glyoxyl derivatives Immobilisation of Alcalase was carried out according to Tardioli, Pedroche, Giordano, Fernández-Lafuente, and Guisán (2003) with slight modifications. One hundred millilitres of 100 mM sodium bicarbonate were mixed with 0.2 ml of Alcalase and then added to 15 g of glyoxyl-agarose. The reaction mixture was gently stirred at room temperature. Aliquots of the supernatant and whole suspension were withdrawn at different times and their catalytic activities were measured. After 1, 3, 6 or 24 h of contact between the enzyme and the support, derivatives were reduced with sodium borohydride, as described by Blanco and Guisán (1989). The reduced derivatives were washed successively with 100 mM sodium phosphate buffer at pH 7 and abundant distiled water.
2.2.9. Analyses of amino acid composition by HPLC The quantification of amino acids was done according to Alaiz, Navarro, Giron, and Vioque (1992). Tryptophan content was analysed according to the method of Yust et al. (2004).
2.2.4. Enzymatic activity assay Enzymatic activity in the soluble and immobilised Alcalase preparations was measured by following the increase of absorbance at 405 nm that accompanies hydrolysis of the synthetic substrate boc-L-ala-ONp (to 1.98 ml of 50 mM sodium phosphate, pH 7, containing 20% ethanol, were added 20 ll of soluble or suspended enzyme and 20 ll of 0.1 M boc-L-ala-ONp in acetonitrile). Assays were performed in spectrophotometric glass cuvettes at 25 °C with magnetic stirring. The gels did not produce interferences in spectrophotometric measurements. One nitrophenyl activity (NPA) unit was defined as the amount of enzyme that hydrolyses 1 lmol of boc-L-ala-ONp per min under the described
2.2.10.2. Oil adsorption. For determination of oil adsorption the method of Lin, Humbert, and Sosulski (1974) was used. Oil adsorption capacity was expressed as the number of grams of oil retained by 100 g of material.
2.2.10. Functional properties 2.2.10.1. Solubility. Samples were suspended in water (5% w/v) and pH was kept at different values between 2 and 10 using 1 N NaOH or 1 N HCl while stirring at room temperature for 1 h. The samples were then centrifuged at 10,000g for 15 min and nitrogen content was determined in the supernatants. Solubility was expressed as the percentage of total nitrogen of the original sample present in the soluble fraction.
2.2.10.3. Emulsifying activity and stability. Emulsifying activity and stability were determined according to Bejosano and Corke (1999), with slight modifications. Samples (3.5 g) were homogenised for 30 s in 50 ml of water using a model Omnimixer homogenizer (Sorvall Instruments, Wilmington, DE, USA) at approximately 10,000 rpm. Corn oil (25 ml) was added, and the mixture was homogenised again for 30 s. Other 25 ml of corn oil
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were added, and the mixture was homogenised for 90 s. The emulsion was centrifuged at 1100g for 5 min. Emulsifying activity was calculated by dividing the volume of the emulsified layer by the volume of emulsion before centrifugation. The emulsion stability was determined using similar samples as for measurement of emulsifying activity. They were heated for 15 min at 85 °C and after temperature came back to room temperature they were centrifuged at 1100g for 5 min. The emulsion stability was expressed as the percentage of emulsifying activity remaining after heating.
solvents, denaturating agents, etc. (Mateo et al., 2006; Pedroche et al., 2007). From this time on (3 h), the residual activity of the immobilised Alcalase (obtained as the difference between the activity of the suspension and that of the supernatant) maintained over 50% of the initial activity. This result is in agreement with those reported for other enzymes by Pedroche et al. (2002).
3.2. Thermal stability of Alcalase-glyoxyl derivatives
Alcalase is a commercial, not highly purified, enzymatic preparation and we were mostly concerned with its activity. For this reason, the progress of immobilisation was followed by measuring the activity (and not protein amount) of the supernatant, of the total suspension, and of the soluble enzyme. Several enzymatic loads were tested in the range 5–80 NPA units/g gel. After that, 38 NPA units/g gel was chosen as optimal because higher loads produced derivatives that preserved less than 40% of initial activity of Alcalase (data not shown). The time course of immobilisation of Alcalase with optimal enzymatic load is shown in Fig. 1. The immobilisation of the enzyme is reached at 3 h. However, a longer exposure time is required to obtain a higher number of bonds between the enzyme and the support, which may result in a higher resistance to small conformational changes caused by heat, organic
One extremely valuable advantage of conducting biotechnological processes at elevated temperatures is reducing the risk of contamination by common mesophiles. For that reason, the thermal stability of enzymes is an important feature for the application of the biocatalyst from a commercial point of view. Commercial Alcalase contains stabilizers that are lost during the washing of glyoxyl-agarose derivatives. Nevertheless, the stability of the derivatives was compared with that of the soluble enzyme, containing stabilizers, in order to study enzymatic preparations as they would actually be used in an industrial setting. The stabilization achieved through immobilisation is shown in Fig. 2. The 1 h derivative was about nine times more stable than soluble Alcalase. Moreover, stabilization of derivatives greatly increased as the contact time between enzyme and support did, which is a general result when an enzyme is immobilised onto glyoxyl-agarose supports (Pedroche et al., 2002, 2007; Tardioli et al., 2003; Yust et al., 2007). Our results indicated that the optimum time to obtain maximum stability is 24 h. Longer exposure times slightly increased the thermal stability but decreased enzyme activity as a consequence of higher distortion of enzyme structure. These stabilization factors are lower than those found in the immobilisation of other enzymes on glyoxyl-agarose gels (Pedroche et al., 2002, 2007; Tardioli et al., 2003), but we attributed this to two factors. Firstly, we used commercial soluble Alcalase, without removal of stabilizers, to calculate stabilization factors. Secondly, glyoxyl-agarose 10 BCL beads were used in articles cited above whereas we used glyoxyl-agarose 4 BCL beads, with a higher pore size, to avoid diffusion limitations in the hydrolysis of CPI, where 360 kDa proteins are predominant. It has been reported that 10 BCL beads, which have more active groups than 4 BCL beads, allow higher stabilization due to a higher number of interactions between enzyme and support (Pedroche et al., 2007). However, stabilization of Alcalase-glyoxyl derivative was higher than that obtained with other supports such as silica derivatives (Ferreira, Ramos, Dordick, & Gil, 2003).
Fig. 1. Time-course of the immobilisation of Alcalase onto glyoxyl-agarose bead, at 25 °C, pH 10.05. (}) Activity of soluble enzyme; (h) activity of whole suspension (supernatant plus agarose bead); and (s) activity of supernatant.
Fig. 2. Stabilization factor of Alcalase-glyoxyl derivatives, expressed as ratio between the half-life time of the immobilised enzyme and half-life of the soluble enzyme, obtained at different times of immobilisation.
2.2.10.4. Foaming capacity and stability. The activity and the stability of foam were determined by the method of Fuhrmeister and Meuser (2003) with modifications. The samples (50 ml of 3% w/v dispersion in distiled water) were homogenised using a model Omnimixer homogenizer at 10,000 rpm. The foaming capacity was expressed as the percentage of volume increase. Foam stability was expressed as the percentage of foam volume remaining after 60 min at room temperature. 2.2.11. Statistical analysis Statistical analysis was performed with the STATGRAPHICS Plus 5.0 program. Data were analysed with One-Way ANOVA Tests and pairwise multiple comparisons were conducted with Fisher’s least significant difference (LSD) procedure. Differences between the means were considered to be significant when P 6 0.05. 3. Results and discussion 3.1. Immobilisation of Alcalase
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3.3. Production and characterisation of chickpea protein hydrolysates CPI is a good substrate, with approximately 90% protein on a dry basis, to be used as starting material for the enzymatic hydrolysis (Table 1). To obtain chickpea protein hydrolysates (CPH) in the range 1–10% DH, a very low proportion E/S ratio should be used to get a very slow hydrolysis rate. In that way, several hydrolysates with successively increasing DH can be produced in the same assay. Thus, after several trials, an E/S ratio of 1.14 mg immobilised Alcalase per g of CPI protein was chosen. At different times, samples were withdrawn and the catalyst was removed by filtration. The variation of DH versus time is shown in Fig. 3. The hydrolysates obtained after 10, 50, 150 and 300 min had DH = 1%, 2.9%, 4.9% and 10%, respectively, and were selected to study their functional properties. The chemical composition of the hydrolysates was similar to chickpea isolate (Table 1). In fact, the main difference was ash content, which was higher in the hydrolysates due to the addition of alkali to keep the pH constant during hydrolysis. The amino acid composition of CPI and CPHs is presented in Table 2. CPI had an amino acid composition that meets the nutritional requirements proposed by FAO/WHO, except in sulphur amino acids, which is common in legume proteins (Singh, Rao, Singh, & Jambunathan, 1988). The amino acid composition of hydrolysates did not show significant differences with respect to isolate, which demonstrates that enzymatic hydrolysis, in opposition to acid or basic hydrolysis, do not alter the nutritional value of original proteins. 3.4. Functional properties of CPI and CPHs 3.4.1. Solubility A high solubility is necessary to use a product in a lot of manufactured foods. It is also important to improve other functional
Table 1 Composition of chickpea protein isolate and hydrolysates obtained after treatment with immobilised Alcalase.
CPI CPH CPH CPH CPH a
1% 2.9% 4.9% 10%
Protein
Moisture
Ash
Othersa
89.3 ± 0.98 89.6 ± 0.47 87.1 ± 0.81 87.7 ± 0.44 86.1 ± 0.56
3.41 ± 0.09 5.32 ± 0.16 8.11 ± 0.27 6.54 ± 0.23 6.23 ± 0.22
2.32 ± 0.01 3.17 ± 0.20 4.71 ± 0.08 5.14 ± 0.09 7.40 ± 0.26
4.97 1.90 0.09 0.65 0.27
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properties, which depend on an adequate initial solubilisation of proteins. Fig. 4 shows the protein solubility profiles of CPI and CPHs as a function of pH. Intact CPI had very low solubility between pHs 4 and 6, with a minimum at pH 4.3 (isoelectric point), where approximately 95% of the proteins precipitated. Similar solubility patterns were reported in other legume protein isolate such as cowpea (Ragab, Babiker, & Eltinay, 2004), bean (Makri & Doxastakis, 2006), and soy (Fuhrmeister & Meuser, 2003). Hydrolysis increased the solubility at all pH values in this study, except for DH = 1%. Moreover, the main differences between solubility of CPI and the hydrolysates were observed around isoelectric point. When the degree of hydrolysis increased, a rapid increase of the solubility at this pH could be observed. This effect has been described in the partial hydrolysis of other legume proteins such as pea (Periago et al., 1998) and soy (Molina Ortiz & Wagner, 2002). The enhanced solubility of the hydrolysates may be attributed to their smaller molecular size and the newly exposed ionisable amino and carboxyl groups, which increase the hydrolysates’ hydrophilicity. However, the decrease in size of CPH 1% in relation to CPI was too small to get higher solubility. 3.4.2. Oil absorption Oil absorption capacity represents the ability of proteins to interact with lipid materials, which is important in food formulation and processing since many properties of foods involve the interaction of proteins and lipids, e.g., fat entrapment and flavour absorption. Oil absorption of CPI (Table 3) was higher than that observed in other plant protein isolates such as rapeseed (Mansour, Peredi, & Dworschak, 1992) or soy protein isolate (Wang & Johnson, 2001), which showed oil absorption of 225 and 204 g oil/ 100 g, respectively. Furthermore, all hydrolysates studied showed higher oil-holding capacity than the original isolate. Similar results were obtained by Periago et al. (1998) when pea flour was hydrolysed and by Vioque et al. (2000) in the hydrolysis of rapeseed protein isolate with Alcalase. This may be due to the fact that the hydrolysis of proteins exposes non-polar side chains that bind hydrocarbon moieties of oil, contributing to increase oil absorption. The hydrolysate with DH = 4.9% presented the highest oil absorption capacity (628). If the DH increases further the oil absorption capacity decreases, what may be attributed to the major exposure of ionic groups after hydrolysis. From these results, we can conclude that chickpea hydrolysates with DH around 5% can be used in food industry as ingredients in products like meat replacers and extenders, doughnuts, baked goods and soups, where fat retention is important.
Calculated as 100-protein-moisture-ash.
Fig. 3. Time course of the hydrolysis of CPI with Alcalase immobilised onto glyoxylagarose at pH 8 and 50 °C.
3.4.3. Emulsifying activity and emulsion stability The emulsifying properties determined in this study were the emulsifying activity and emulsion stability. The former is related to the ability of a protein to form an emulsion, i.e., to assist in the dispersion of oil into small globules that are homogeneously distributed throughout the continuous aqueous phase. The emulsion stability is related to the ability of a protein to preserve homogeneity of the oil-in-water emulsion on storage. The literature is ambiguous about the improvement of emulsifying properties with limited hydrolysis. Some studies reported that the emulsifying properties of proteins are improved by limited hydrolysis, due to the exposure of hydrophobic amino acid residues that may interact with the oil whereas the hydrophilic residues interact with water (Vioque et al., 2000). However, it is speculated that peptides, of smaller size than intact protein, generated in the hydrolysis cannot form the same molecular interactions as proteins of higher molecular weight (Panyam & Kilara, 1996; Singh & Dalgleish, 1998). Limited proteolysis with the Alcalase-glyoxyl derivative had none or little effect on emulsifying properties of CPI (Table 3). Only the hydrolysate with DH = 2.9% showed higher emulsifying activity
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Table 2 Amino acid composition of chickpea protein isolate and hydrolysates obtained with immobilised Alcalase.
Asp + Asn Glu + Gln Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Trp Leu Phe Lys
CPI
CPH 1%
CPH 2.9%
CPH 4.9%
CPH 10%
13.2 ± 0.53 17.1 ± 0.65 5.99 ± 0.19 2.82 ± 0.10 3.91 ± 0.09 4.55 ± 0.10 9.18 ± 0.08 4.86 ± 0.20 4.82 ± 0.63 2.40 ± 0.00 4.26 ± 0.21 0.24 ± 0.02 0.93 ± 0.09 3.39 ± 0.12 0.63 ± 0.03 9.00 ± 0.27 6.40 ± 0.26 6.70 ± 0.02
13.6 ± 0.24 18.3 ± 0.08 6.38 ± 0.01 2.64 ± 0.08 3.88 ± 0.01 4.31 ± 0.07 8.87 ± 0.07 4.79 ± 0.03 4.15 ± 0.33 2.25 ± 0.02 4.20 ± 0.18 0.33 ± 0.02 0.91 ± 0.05 3.20 ± 0.05 0.65 ± 0.03 8.87 ± 0.10 6.31 ± 0.12 6.75 ± 0.06
14.3 ± 0.20 17.4 ± 0.85 6.85 ± 0.09 2.70 ± 0.13 4.09 ± 0.15 3.89 ± 0.13 9.22 ± 0.08 4.64 ± 0.05 3.61 ± 0.02 2.65 ± 0.13 3.76 ± 0.01 0.23 ± 0.03 0.98 ± 0.05 3.34 ± 0.08 0.78 ± 0.02 8.88 ± 0.20 6.35 ± 0.21 6.75 ± 0.10
14.5 ± 0.66 19.8 ± 0.46 6.86 ± 0.11 2.43 ± 0.12 3.94 ± 0.03 3.74 ± 0.17 9.50 ± 0.17 4.22 ± 0.02 4.31 ± 0.20 2.21 ± 0.11 3.71 ± 0.14 0.21 ± 0.03 0.94 ± 0.06 3.17 ± 0.11 0.61 ± 0.03 8.28 ± 0.24 5.82 ± 0.09 6.92 ± 0.23
14.2 ± 0.11 18.7 ± 0.82 7.01 ± 0.07 2.40 ± 0.12 3.86 ± 0.18 4.16 ± 0.15 9.22 ± 0.23 4.31 ± 0.17 3.82 ± 0.18 2.13 ± 0.09 4.17 ± 0.14 0.17 ± 0.04 0.80 ± 0.06 3.34 ± 0.14 0.55 ± 0.06 8.50 ± 0.15 5.83 ± 0.05 7.11 ± 0.19
FAOa
1.9 3.4
3.5 2.5b 2.8 6.6 6.3c 5.8
Data expressed as g/100 g are the mean ± standard deviation of three determinations. a FAO/WHO/ONU. Energy and protein requirements, 1985. b Methionine + cysteine. c Phenylalanine + tyrosine.
soy (Tsumura et al., 2005), and whey (van der Ven, Gruppen, de Bont, & Voragen, 2001) amongst others.
Fig. 4. Nitrogen solubility as a function of pH of CPI and CPHs obtained with Alcalase-glyoxyl derivative. (—) isolate; (h) CPH 1%; (s) CPH 2.9%; () CPH 4.9%; and (}) CPH 10%.
than CPI, but emulsion stability decreased. In many cases, a direct relationship between nitrogen solubility and emulsifying properties of proteins has been demonstrated (Narayana & Narasinga Rao, 1984). In our research, although CPHs had excellent nitrogen solubility, they showed poor emulsification properties; therefore, emulsifying properties ultimately depend upon a suitable balance between the hydrophiles and lipophiles and do not necessarily increase as a protein becomes more soluble. In fact, reduction of emulsion-forming ability after hydrolysis has been reported for
3.4.4. Foaming capacity and foam stability Foams are biphasic colloidal systems with a continuous liquid or aqueous phase and a dispersed gas or air phase. Food proteins are capable of forming good foams, and ingredients such as egg white have long been an integral part of recipes that require whipping or foaming. It has also been established that whey proteins possess good foaming properties (Panyam & Kilara, 1996). Partial hydrolysis of CPI with immobilised Alcalase improved foaming capacity to a great extent (Fig. 5). In fact, CPI did not show foaming capacity whereas all CPH had good foaming properties. This could be attributed to the production of amphiphilic peptides after hydrolysis. Their reduced molecular weight will make them more flexible, forming a stable interfacial layer and increasing the rate of diffusion to the interface, improving the foamability properties (Liceaga-Gesualdo & Li-Chan, 1999). This improvement in foaming capacity for enzymatically hydrolysed food proteins was reported by several authors (Pizones Ruiz-Henestrosa et al., 2007; Tsumura et al., 2005). The best result was for DH = 4.9%; this hydrolysate had a value of 158% of foam capacity. Moreover, this hydrolysate showed the highest foam stability, preserving approximately 60% of foam after 60 min at room temperature. In conclusion, the partial hydrolysis of chickpea protein isolate with immobilised Alcalase is a helpful strategy to improve some functional properties of intact proteins, such as solubility, oil absorption capacity, and foaming capacity and stability. Specifically, the hydrolysis of 5% peptidic bonds of chickpea proteins seems to be the most appropriate to be considered as an alternative for other protein ingredient sources that are being used in the food industry.
Table 3 Functional properties of chickpea protein isolate and hydrolysates with different DH.
Oil adsorption Emulsifying activity Emulsion stability
Isolate
CPH 1%
CPH 2.9%
CPH 4.9%
CPH 10%
308 ± 6.6a 44.7 ± 1.2a 76.5 ± 0.9a
542 ± 9.0b 39.1 ± 0.8b 100 ± 0.0b
618 ± 5.1c 54.0 ± 0.6c 51.8 ± 0.7c
628 ± 5.2c 37.8 ± 0.6b 16.8 ± 0.4d
443 ± 7.1d 35.1 ± 0.4d 17.5 ± 1.2d
Data represent the mean ± standard deviation of three experiments. Different letters within the same row indicate significant differences (P 6 0.05).
María del Mar Yust et al. / Food Chemistry 122 (2010) 1212–1217
Fig. 5. Foaming capacity (open bars) and foam stability (closed bars) of CPI and CPHs obtained with Alcalase-glyoxyl derivative. Values marked by the same letter are not significantly different (P 6 0.05).
Acknowledgement This research work was supported by a fellowship of Spanish Ministry of Education and Science (M.M.Y.).
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