Sesame protein hydrolysate was prepared from defatted sesame flour after screening different proteases. Alcalase® 2.4L produced the highest value (96.68%) of protein recovery at 60°C and pH 8 followed by Flavourzyme (69.76%). However, at pH 7 and 50°C, Flavourzyme showed the highest protein yield value (79.28%) followed by Alcalase 2.4L (77.62%). Hydrolysis conditions (Temperature (T), pH, Enzyme/Substrate (E/?), time (t)) were investigated to optimize the degree of hydrolysis (DH) by response surface methodology (RSM). The DH ranged from 1.19 to 18.8 %. Solubility of the defatted sesame protein isolate (DSPI) increased as the pH increased. The SPH was observed to be a good emulsifier, foam, water and oil capacities were significantly higher than DSF, but the stability of the foam over a period of time was however not good. Sesame protein hydrolysates produced through the use of Alcalase was observed to have good functional attributes.

Key Words: Enzymes, Functional properties, Protein hydrolysate, Response surface methodology, Sesame

Introduction

Sesame belongs to the genus Sesamum, one of the sixteen genera in the family Pedaliaceae. Its protein has a high nutritional value compared with other proteins because of their relative high content of essential amino acids (Kanu et al., 2007; Radha et al., 2007). Sesame seeds have many nutrients needed for human health (Coulman et al., 2005). The chemical composition of sesame shows that the seeds are an important source of protein (Chen et al., 1998; Bandyopadhyay and Ghosh, 2002). Because of the increasing costs and limited supply of animal protein, plant protein has been investigated for possible incorporation into formulated foods particularly for its functional properties imparted in the food system (Bernard-Don et al., 1991; Chiang et al 1999; Chove et al., 2002).

Functional properties of food protein are important in food processing and food product formulation. Some of these properties are solubility, water holding capacity, oil holding capacity, emulsification, foaming properties, bulk density and viscosity (Bandyopadhyay and Ghosh; 2002 Jung et al., 2005). Nonetheless, some of these properties are affected by intrinsic factors of proteins such as molecular structure and size, and some environmental factors, including the method of protein isolation from the seed (Fuhrmeister and Meuser, 2003). Attention on plant protein isolates has been focused mainly on cotton seed, peanut, rapeseed, soyabean and sunflower seed and in some areas commercial preparations are even available for those oil seeds (Tsumura et al., 2005; Chove et al., 2002). In contrast the functionality of sesame protein has received little attention particularly when different production parameters, that is, more than two are combined to isolate the protein from sesame that could be used in food formulations. Few studies have reported mainly on the properties of the defatted sesame flour or meal, sesame oil, the antioxidant properties of sesame, and functional properties of sesame protein as influenced by pH only (Khalida et al., 2003; Shahidi et al., 2006; Aly et al., 2000) and the extraction of protein through the use of water (Kanu et al., 2007) . Very limited information is available for the functional properties of sesame protein isolate as influenced by other factors during its protein extraction from dehulled sesame seeds with the use of selected proteases as that has been speculated to give good functional properties by Nilo-Rivas et al., (1981). That speculation necessitated the present study. And enzymatic hydrolysis of food proteins generally results in profound changes in the functional properties of the protein treated like improved solubility, emulsification, water and oil holding capacities (Bernard-Don et al., 1991; Hrckova, et al., 2002; Ferreira et al., 2007).

Preliminary investigation using four kinds of microbial proteases (Flavourzyme, Protamex, Neutrase and Alcalase® 2.4L) was done for the hydrolysis of sesame seed protein. The critical parameters for better control of enzymatic hydrolysis are temperature, time of hydrolysis, pH, the nature of substrate, type of enzyme used, the enzyme/substrate ratio, the concentration of substrate and the degree of hydrolysis (Diniz and Martin, 1996; Tsumura et al., 2005).

When many factors and interactions affect a desired response, response surface methodology (RSM) is an effective tool for optimizing the process. The main advantage of RSM is the reduced number of experimental trials needed to evaluate multiple parameters and their interactions. Therefore, it is less laborious and less time-consuming than other approaches required optimizing a process (Cheison et al., 2007).

There is little information on the effect of enzyme treatment on the various functional properties of sesame seed protein. Therefore the objectives of this study were to screen different proteases, apply RSM to optimize the DH with the proteases that gives the highest protein recovery and study the effect on the functional properties of the resulting hydrolysates.

Materials and methods

Dehulled white sesame seeds of Sesamum indicum variety were obtained from a local supermarket in Wuxi, People’s Republic of China. The sesame seeds were ground and defatted according to Kanu et al., (2007) and kept in a freezer (Haier- BC/BD-275SB, Shanghai, People’s Republic of China) at -10oC till when needed for the experiments. Prior to the hydrolysis process, a portion of the defatted sesame flour was properly mixed.

Alcalase® (in solution form with declared activity of 2.4 Activity units(AU)/kg and a density of 1.18g/mL) a bacterial endoproteinase from a strain of Bacillus licheniformis was provided by Novo Nordisk’s Enzyme Business in Wuxi, PR China and stored at 5°C until it was used for hydrolysis experiments. The chemicals and reagents used in the experiment were of analytical or food grade quality.

Protease selection

Protamex, a Bacillus protease complex, Neutrase from Bacillus subtulis strain, Alcalase® 2.4L from a strain of Bacillus licheniformis and Flavourzyme from Aspergillus oryzae were obtained from Novo Nordisk’s Enzyme Business in Wuxi, People’s Republic of China. The four proteases were evaluated for ability to hydrolyze sesame seed protein. The four bacterial enzymes were screened using a pH Stat method according to Adler-Nissen, (1977) in order to select one with the best properties to be used for the defatted sesame seed protein hydrolysis. The DH to which an enzyme can hydrolyze a protein is a popular parameter for protein hydrolysis experiments according to the manufacturers of such proteases. DH is intricately related to the properties of the hydrolysates (Mahmoud et al., 1992).

Enzymatic hydrolysis

Experiments to study the effects of hydrolysis variables in the range given in Table 1 were done in accordance with the experimental design depicted in Table 2. All reactions were done in triplicates in a 1L polyethylene-jacketed glass vessel in a thermostatically controlled water bath (NUOHAI- XMTD-204, Tokyo, Japan) with constant stirring (700 rpm). The vessel was covered with a close fitting lid which was given an opening for an automatic temperature compensator (ATC) probe, a pH electrode (Hanna Precision pH meter (Model pH 212, SIGMA, USA ), an over head mixer shaft (KIKA- WERKE KMO2, KIKA Co. Tokyo, Japan) and for the addition of acid or base. During the reactions, pH was maintained at a desired value by the addition of 0.2 mol/L NaOH. The reaction vessel with 50g of defatted sesame flour was mixed with 500mL of distilled water and placed in a previously heated water bath.

Homogenization was carried out for 5 minutes in order to adjust the pH (through addition of 0.2 mol/L NaOH) and temperature to the desired values. After equilibrium was reached, the enzyme (Alcalase®, which gave the highest protein recovery in this study) was added and the reaction was allowed to proceed. The amount of alkali added to keep the pH constant during the hydrolysis was recorded and used to calculate the DH. The reactions were terminated by immersing the reaction vessel into hot water at 95 °C for 15 minutes with continuous stirring to ensure the inactivation of the enzyme. The temperature of the reaction mixture at the end of the inactivation was 90 to 95°C. The resultant slurry was cooled at room temperature (23-25 oC) and then centrifuged in a Beckman Coulter Centrifuge (Avanti J-26XPI, Beckman Co. USA) at 2800 x g for 20 minutes at room temperature. The supernatant was collected and freeze dried.

Determination of degree of hydrolysis

The hydrolysis was carried out using the pH-stat method as described by Adler-Nissen, (1977). Degree of hydrolysis is the percentage ratio between the number of peptide bonds cleaved (h) and the total number of peptide bonds in the substrate studied (htot). The degree of hydrolysis was determined based on the consumption of the base necessary for controlling the pH during the batch assay as depicted in equation 1.

DH%= Eq. (1)

Where htot is the total number of peptide bonds in the protein substrate, in mol/gprotein; h is the number of hydrolyzed peptide bonds; B the base consumed in mL; Nb is base normality; α is the average degree of dissociation of the α-NH groups and MP is the mass of protein in g (N x 6.25).

The degree of dissociation was calculated as in equation 2.

α = Eq. (2)

Where pK is the average value of the α-amino groups liberated during the hydrolysis and varies significantly with temperature but is relatively independent of the substrate as such. The pK at different temperatures (T in oC) was calculated according to equation 3.

pK = Eq. (3)

Determination of free amino groups

The number of free amino groups was determined by using TNBS following the method of Mutilangi et al. (1995) with some modifications. TNBS (1 mL of 1% solution) was added to 1mL of protein solution (0.15 mg/mL) containing 1% sodium dodecyl sulfate (SDS) and 4% NaHCO3, pH 9.5. After rapid mixing, the mixture was held at 40 °C for 2hr in a water bath and the reaction was stopped by adding 0.5 mL of 1mol HCL and 1 mL of 10% SDS. The absorbance of the sample was read at 335nM against a blank and the readings were converted to free amino acids by preparing a standard curve.

Experimental design

To establish optimal conditions for hydrolysis of defatted sesame flour, RSM was used. Processing variables investigated were temperature (T), pH, enzymes/substrate (E/S) and time (t). A Box-Behnken factorial design with four factors and three levels was applied according to Box and Behnken, (1960) as shown in Table 1. Three levels were adopted and coded as -1, 0 and +1. DH was the dependent variable. To predict the optimal point, experimental data were fitted to a second order polynomial parameter of model, according to equation (4). The regression model between dependent variable (Y) and independent variables was according to equation 4.

Υ =

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