Hydrolysis of chickpea proteins with Flavourzyme immobilized on glyoxyl-agarose gels improves functional properties

Abstract

Chickpea protein isolate was hydrolyzed using Flavourzyme immobilized on glyoxyl-agarose beads by multipoint covalent attachment. This Flavourzyme-glyoxyl derivative, produced after 1 h of immobilization at 4 °C followed by 5.5 h at room temperature, presented approximately 51% of the endoprotease activity of Flavourzyme but was around 700 times more stable than soluble enzyme. Chickpea protein hydrolysates ranging from 1% to 10% degree of hydrolysis were produced and their chemical composition was very close to that of protein isolate used as starting material. 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.

Keywords

Chickpea proteins enzymatic hydrolysis Flavourzyme functional properties immobilized enzyme

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 and 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 and Cheryan, 1992). To improve functional properties is generally admitted that a limited hydrolysis, between 1% and 10%, is needed (Vioque et al., 2000).

The products of enzymatic proteolysis frequently present a bitter taste due to the presence of hydrophobic amino acids (Kukman et al., 1995). Flavourzyme, produced by Novozymes, is a nonspecific protease obtained by fermentation of a selected strain of Aspergillus oryzae and contains both endoprotease and aminopeptidase activities. Using Flavourzyme, protein hydrolysates without bitter taste can be obtained. The optimal pH for the enzyme complex is in the range of 5.0–7.0 and the optimal temperature is around 50 °C.

Up to now, most studies on enzymatic hydrolysis to improve functional properties of proteins report the use of soluble enzymes. The main drawback of using soluble enzymes from an industrial point of view is that they cannot be reused. This can be overcome by employing membrane reactors, special solvent systems, or immobilization techniques (Giorno and Drioli, 2000; Okahata and Mori, 1997). So far most efforts have focused on immobilization, of which many approaches are known including adsorption on appropriate supports or covalent attachment to such materials as well as encapsulation in polymers or in sol–gel materials (Mateo et al., 2007). A second limiting factor for the implementation of enzymes as industrial biocatalysts is stability (Haki and Rakshit, 2003). Although several procedures have been employed to achieve stabilization of enzymes, coupling immobilization to stabilization has the advantage of facilitating both recycling and stabilization requirements. In this sense, it has been reported that glyoxyl-agarose beads constitute a good substrate for the immobilization/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 bead 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 (Guisán, 1988; Mateo et al., 2006; Pedroche et al., 2002).

In addition to stabilization, the use of immobilized enzymes onto glyoxyl-agarose beads 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 degree of hydrolysis (DH).

In the present paper, we describe the chemical composition and some functional properties of limited chickpea protein hydrolysates (CPHs) obtained hydrolysing chickpea protein isolate (CPI) by means of Flavourzyme immobilized on glyoxyl-agarose beads.

Materials and methods

Materials

Glyoxyl-agarose cross-linked 4% beads were prepared as described elsewhere (Guisán, 1988). CPI was prepared according to Yust et al. (2010). Trinitrobenzenesulfonic acid (TNBS), L-leucine-p-nitroanilide, and sodium borohydride were purchased from the Sigma Chemical Co. (St. Louis, MO). Boc-L-alanine-4-nitrophenyl ester was from Bachem S.A. (Budendorf, Switzerland). All other chemicals were of analytical grade.

Preparation of Flavourzyme-glyoxyl derivative

Immobilization of Flavourzyme was carried out according to Yust et al. (2007) with slight modifications. One hundred millilitres of 100 mM sodium bicarbonate were mixed with 0.4 mL of Flavourzyme and then added to 18 g of glyoxyl-agarose gel. The reaction mixture was gently stirred at 4 °C for 1 h followed by 5.5 h of incubation at room temperature and reduced with sodium borohydride, as described by Blanco and Guisán (1989). The reduced derivative was washed successively with 100 mM sodium phosphate buffer at pH 7 and abundant distilled water.

Enzymatic activity assays

Endoprotease and aminopeptidase activities of Flavourzyme-glyoxyl derivative was measured following the hydrolysis of synthetic substrates Boc-L-alanine-4-nitrophenyl ester and L-leucine-p-nitroanilide, respectively, as described by Yust et al. (2007). Residual activity of Flavourzyme-glyoxyl derivative was expressed as percentage of activity of commercial Flavourzyme.

Thermal stability

Samples of soluble and immobilized Flavourzyme were suspended in 0.1 M sodium phosphate, pH 7, and incubated at 50 °C. Aliquots were withdrawn periodically and enzyme activities were measured as described above. Pseudo-half-life time (pt_1/2), time necessary to reach 50% of residual activity, was taken directly from experimental time course of inactivation for each sample. Stability was calculated as pt_1/2 immobilized Flavourzyme/ pt_1/2 soluble enzyme.

Hydrolysis of chickpea protein isolate

CPI was hydrolyzed using Flavourzyme 1000 L immobilized onto glyoxyl-agarose supports. CPI was resuspended in distilled water (50 g/L) and pH was adjusted to 7 and temperature to 50 °C. Then the enzyme was added in a relation E/S = 1.38 mg immobilized protein/g protein in CPI. Samples were withdrawn at different times and the biocatalyst was removed by filtration.

Determination of the DH

The DH, defined as the percentage of peptides bonds cleaved, was calculated by the TNBS method according to Adler-Nissen (1979). The total number of amino groups was determined in a sample that had been 100% hydrolyzed at 110 °C for 24 h in 6 M HCl.

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 was calculated as the loss in weight after drying at 105 °C for 24 h. Ash content was determined by the direct ignition method (550 °C for 36 h).

Analyses of amino acid composition by high-performance liquid chromatography

The quantification of amino acids was done according to Alaiz et al. (1992). The determination of tryptophan was made following the method described by Yust et al. (2004).

Functional properties

Solubility

Samples were suspended in water (50 g/L) 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 10 000 g 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.

Oil absorption

For determination of oil absorption, the method of Lin et al. (1974) was used. Oil absorption capacity was expressed as the number of grams of oil retained by 100 g of material.

Emulsifying activity and stability

Emulsifying activity and stability were determined according to Bejosano and Corke (1999), with slight modifications described by Yust et al. (2010).

Foaming capacity and stability

The activity and the stability of foam were determined by the method of Fuhrmeister and Meuser (2003). The foaming capacity was expressed as the percentage of volume increase. Foam stability was expressed as foam volume remaining after 60 min at room temperature.

Statistical analysis

Data were expressed as the mean ± standard deviation of three independent determinations. Statistical analysis was performed with the STATGRAPHICS Plus 5.0 program (Statistical Graphic, Rockville, MD, USA). Data were analyzed with one-way analysis of variance (ANOVA) tests and multiple range tests were conducted with Fisher’s least significant difference procedure. Differences between the means were considered to be significant when p < 0.05.

Results and discussion

Obtaining of Flavourzyme immobilized on glyoxyl-agarose gel

The immobilization method chosen was the multipoint covalent attachment of Flavourzyme (through amino groups) to glyoxyl-agarose beads. This method has been successfully used to achieve immobilization/stabilization of several proteases (Mateo et al., 2006). In a previous study (Yust et al., 2007), we have optimized the process concluding that the best derivative was obtained with an enzymatic load of 22 µL commercial Flavourzyme/g glyoxyl-agarose gel and after 1 h of incubation at 4 °C followed by 5.5 h at room temperature. The endoprotease and aminopeptidase activities of this biocatalyst are shown in Table 1. Due to immobilization process, both activities decreased, which is a usual result when enzymes are immobilized by multipoint covalent attachment to glyoxyl-agarose beads (Pedroche et al., 2002). This drop of enzymatic activity was especially high for aminopeptidase whereas endoprotease activity was 50% of the commercial Flavourzyme, a typical value in protease immobilization to glyoxyl supports (Mateo et al., 2006; Pedroche et al., 2002).

Table 1.

Activity and stability (at pH 7 and 50 °C) of Flavourzyme immobilized on glyoxyl-agarose beads

Endoprotease	Aminopeptidase
Residual activity	51.0 ± 0.9	8.4 ± 0.4
Stability	676.2 ± 5.8	73.2 ± 2.5

Stability of Flavourzyme-glyoxyl derivative was studied at pH 7 and 50 °C because these were the conditions to be used in the hydrolysis of CPI. The stabilization achieved through immobilization was very high, especially in endoprotease activity whose pt_1/2 was 676 times higher than that of soluble Flavouryzme. This improvement may be attributed to several factors including prevention of autolysis and protection of enzymes from structural rearrangements due to multipoint attachment to the support (Ferreira et al., 2003). The high stability achieved offsets the loss of activities, making this derivative an appropriate catalyst to be used in protein hydrolysis on an industrial scale.

Production and characterisation of chickpea protein hydrolysates

Protein hydrolysates were obtained from CPI using Flavourzyme immobilized onto glyoxyl-agarose as described in Materials and Methods. At different times, aliquots were withdrawn, removing the biocatalyst by filtration. The variation of DH versus time is shown in Figure 1. The hydrolysates obtained after 20, 75, 120 and 300 min had DH 1, 3, 5, and 10%, respectively, and were selected to study their functional properties.

Figure 1.

Time course of the hydrolysis of chickpea protein isolate (CPI) with Flavourzyme immobilized on glyoxyl-agarose gel at pH 7 and 50 °C.

The chemical composition of the hydrolysates was very similar to chickpea isolate (Table 2). 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.

Table 2.

Composition of chickpea protein isolate and hydrolysates obtained after treatment with immobilized Flavourzyme

Protein	Moisture	Ash	Others^a
CPI	89.30 ± 0.98 a	3.41 ± 0.09 a	2.32 ± 0.01 a	4.97
CPH 1%	88.21 ± 0.42 ab	6.65 ± 0.10 b	3.27 ± 0.01 b	1.87
CPH 3%	87.02 ± 0.67 bc	7.04 ± 0.08 c	5.12 ± 0.09 c	0.82
CPH 5%	86.04 ± 0.63 c	6.38 ± 0.16 d	6.76 ± 0.07 d	0.82
CPH 10%	84.77 ± 0.40 d	7.16 ± 0.03 c	6.93 ± 0.02 e	1.14

Data, expressed as g/100 g of sample, represent the mean ± standard deviation of three determinations. Different letters within the same column indicate significant differences (p < 0.05).

Calculated as 100-protein-moisture-ash.

The amino acid compositions of CPI and CPHs are shown in Table 3. The main amino acids in CPI are glutamic and aspartic acids, followed by arginine and leucine. Overall, CPI had an amino acid composition that meets the nutritional requirements proposed by FAO/WHO, except in sulphur amino acids, which is a common profile in legume proteins (Singh et al., 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.

Table 3.

Amino acid composition of chickpea protein isolate and hydrolysates obtained with immobilized Flavourzyme

CPI	CPH 1%	CPH 3%	CPH 5%	CPH 10%	FAO^a
Asp + Asn	13.19 ± 0.53	13.52 ± 0.51	13.54 ± 0.36	13.13 ± 0.05	12.95 ± 0.13
Glu + Gln	17.11 ± 0.65	17.58 ± 0.18	17.38 ± 0.01	17.58 ± 0.06	17.19 ± 0.51
Ser	5.99 ± 0.19	6.23 ± 0.06	6.11 ± 0.23	6.07 ± 0.07	6.25 ± 0.01
His	2.82 ± 0.10	2.75 ± 0.08	2.69 ± 0.09	2.82 ± 0.05	2.91 ± 0.00	1.9
Gly	3.91 ± 0.09	3.76 ± 0.02	3.77 ± 0.17	3.65 ± 0.05	3.68 ± 0.04
Thr	4.55 ± 0.10	4.31 ± 0.28	4.37 ± 0.07	4.62 ± 0.05	4.66 ± 0.00	3.4
Arg	9.18 ± 0.08	9.28 ± 0.05	9.36 ± 0.21	9.34 ± 0.13	9.34 ± 0.07
Ala	4.86 ± 0.20	4.53 ± 0.01	4.65 ± 0.13	4.54 ± 0.04	4.42 ± 0.01
Pro	4.82 ± 0.63	4.62 ± 0.24	4.62 ± 0.31	4.65 ± 0.92	4.62 ± 1.07
Tyr	2.40 ± 0.00	2.23 ± 0.25	2.40 ± 0.22	2.53 ± 0.01	2.64 ± 0.00
Val	4.26 ± 0.21	4.01 ± 0.57	3.94 ± 0.04	4.11 ± 0.04	3.98 ± 0.03	3.5
Met	0.24 ± 0.02	0.28 ± 0.02	0.30 ± 0.06	0.23 ± 0.04	0.31 ± 0.09	2.5 ^b
Cys	0.93 ± 0.09	1.11 ± 0.18	1.03 ± 0.13	1.13 ± 0.25	0.96 ± 0.05
Ile	3.39 ± 0.12	3.62 ± 0.04	3.41 ± 0.04	3.52 ± 0.03	3.62 ± 0.04	2.8
Leu	9.00 ± 0.27	8.51 ± 0.10	8.76 ± 0.01	8.71 ± 0.04	8.80 ± 0.08	6.6
Phe	6.40 ± 0.26	6.33 ± 0.13	6.24 ± 0.02	6.21 ± 0.01	6.47 ± 0.07	6.3 ^c
Lys	6.70 ± 0.02	6.61 ± 0.09	6.78 ± 0.02	6.56 ± 0.01	6.61 ± 0.05	5.8
Trp	0.63 ± 0.03	0.59 ± 0.12	0.65 ± 0.02	0.63 ± 0.04	0.59 ± 0.01

Data, expressed as g/100 g of protein, are the mean ± standard deviation of three determinations.

FAO/WHO/ONU. Energy and protein requirements, 1985.

Methionine + cysteine.

Phenylalanine + tyrosine.

Functional properties of CPI and CPHs

Solubility

Solubility is one of the most important characteristics of proteins because it is not only important by itself but it also influences other functional properties. The protein solubility profiles of CPI and CPHs as a function of pH are shown in Figure 2. Intact CPI had very low solubility between pHs 4 and 6, with a minimum at pH 4.3 (isoelectric point of chickpea proteins), where approximately 95% of the proteins precipitated. Similar solubility patterns were reported in other legume protein isolates such as soy (Fuhrmeister and Meuser, 2003), cowpea (Ragab et al., 2004), and bean (Makri and Doxastakis, 2006). Hydrolysis increased the solubility at all pH values in this study, except for DH = 1%. The main differences between solubility of CPI and the hydrolysates were observed around isoelectric point. When DH increased, a quick rise of the solubility around this pH could be observed, especially from DH = 3% to 5%. These results are similar to that obtained after hydrolysis of CPI with Alcalase (Yust et al., 2010), although CPH 3% obtained with Flavourzyme had lower solubility at acidic pHs than CPH 3% obtained with Alcalase. Therefore, the increase in solubility not only depends on DH but also on specificity of proteases.

Figure 2.

Nitrogen solubility as a function of pH of chickpea protein isolate (CPI) and chickpea protein hydrolysates (CPHs) obtained with Flavourzyme-glyoxyl derivative. (—) CPI; (□) CPH 1%; (○) CPH 3%; ( * ) CPH 5%; (⋄) CPH 10%.

Oil absorption

Oil absorption of CPI (Table 4) was higher than the one corresponding to some commercial products such as sodium caseinate or dry egg white, with range between 150 and 200 g oil/100 g sample (Ahmedna et al., 1999). Moreover, the absorption capacity of CPI was higher than that observed in other plant protein isolates such as rapeseed (Mansour et al., 1992) or soy protein isolate (Wang and Johnson, 2001), which showed oil absorption of 225 and 204 g oil/100 g, respectively.

Table 4.

Functional properties of chickpea protein isolate and hydrolysates with different degree of hydrolysis (DH)

CPI	CPH 1%	CPH 3%	CPH 5%	CPH 10%
Oil absorption	308.1 ± 6.6 a	646.0 ± 4.4 b	660.1 ± 8.1 c	503.2 ± 4.4 d	489.8 ± 7.1 e
Emulsion activity	44.7 ± 1.5 a	53.1 ± 0.9 b	41.6 ± 0.3 cd	39.8 ± 0.6 d	42.9 ± 1.4 ac
Emulsion stability	76.5 ± 0.9 a	70.3 ± 0.8 b	83.9 ± 0.8 c	76.9 ± 0.7 a	77.1 ± 0.9 a

Data represent the mean ± standard deviation of three experiments. Different letters within the same row indicate significant differences (p < 0.05).

As a consequence of enzymatic hydrolysis, oil absorption capacity of chickpea proteins was doubled, which 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 = 3% presented the highest oil absorption capacity (660 g oil/100 g sample) and if the DH increases further, the oil absorption capacity decreases, what may be attributed to the major exposure of ionic groups after hydrolysis. This result is better than obtained in the hydrolysis of CPI with Alcalase (Yust et al., 2010) where the highest oil absorption was found in DH around 5% (628 g oil/100 g sample).

Emulsifying activity and emulsion stability

In principle, emulsifying properties of proteins should be improved by limited hydrolysis, due to the exposure of hydrophobic amino acid residues that may interact with the oil while the hydrophilic residues interact with water. But it has also been described that peptides, smaller than intact proteins, cannot form the same molecular interactions than larger proteins, resulting in a less-stable emulsion (Panyam and Kilara, 1996; Singh and Dalgleish, 1998). Limited proteolysis with the Flavourzyme-glyoxyl derivative had none or little effect on emulsifying properties of CPI (Table 4). Only CPH 1% showed higher emulsifying activity than CPI, but emulsion stability decreased.

In some cases, a direct relationship between nitrogen solubility and emulsifying properties of proteins has been demonstrated (Narayana and Narasinga Rao, 1984). But in our research, although CPHs had improved nitrogen solubility, they showed poor emulsifying properties; therefore, the ability of proteins to form stable emulsions may ultimately depend upon a suitable balance between the hydrophilic and lipophilic residues and do not necessarily increase as a protein becomes more soluble. In fact, reduction of emulsion-forming ability after hydrolysis has been reported for soy (Tsumura et al., 2005) and whey (Van der Ven et al., 2001) among others.

Foaming capacity and stability

Foam capacity of proteins depends on several factors such as the type of protein, degree of denaturation, pH, temperature, and the method used to produce the foam. Partial hydrolysis of CPI with immobilized Flavourzyme improved foaming capacity to a great extent (Figure 3). In fact, CPI did not show foaming capacity whereas hydrolysis of only 1% led to foam capacity of 126%. This property increased as long as hydrolysis progressed, reaching maximum at DH = 5%. The same pattern was observed in the partial hydrolysis of CPI by Alcalase immobilized on agarose beads (Yust et al., 2010) although foam capacities of CPHs obtained with were lower than the corresponding to CPHs obtained with Flavourzyme. This improvement in foaming capacity for enzymatically hydrolyzed food proteins has been reported for other pulses such as soy and rapeseed (Pizones Ruiz-Henestrosa et al., 2007; Tsumura et al., 2005; Vioque et al., 2000). With regard to foam stability of chickpea proteins, it was also increased after partial hydrolysis with immobilized Flavourzyme, and the highest value was shown by CPH 3%, which preserved approximately 60% of foam after 60 min at room temperature. Considering the two foaming properties studied, the best way to improve CPI characteristics is by hydrolysing proteins up to DH = 3%.

Figure 3.

Foaming capacity (closed bars) and foam stability (open bars) of chickpea protein isolate (CPI) and chickpea protein hydrolysates (CPHs) obtained with Flavourzyme-glyoxyl derivative. Values marked by the same letter are not significantly different (p < 0.05).

In conclusion, the partial hydrolysis of chickpea proteins with Flavourzyme immobilized on glyoxyl-agarose beads is a successful strategy to improve some functional properties of intact proteins, such as solubility (especially at acidic pHs), oil absorption, and foaming capacity and stability. Besides, the improvement of functional properties depends not only on degree of hydrolysis but also on specificity of the enzyme. The chickpea protein hydrolysates could be an alternative for other protein ingredient sources that are being used in the food industry.

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