MATERIALS AND METHODS

Dehulled white sesame seeds of Sesamum indicum variety were obtained from a local supermarket in Wuxi, Jiangsu Province, People’s Republic of China. The sesame seeds were ground and defatted using the method of John Kanu et al. (2007) and kept in a freezer (Haier-BC/BD-275SB, Shanghai, People’s Republic of China) at -10°C until required for further experiments. Prior to the hydrolysis process, a portion of the defatted sesame flour was mixed properly for the purpose of homogeneousness.

Alcalase^® (in solution form with declared activity of 2.4 Activity Units (AU) kg^-1 and density of 1.18 g mL^-1) a bacterial endoproteinase from a strain of Bacillus licheniformis was provided by Novo Nordisk’s Enzyme Business in Wuxi, Jiangsu Province, PR China and stored at 5°C until required for the hydrolysis experiments. All chemicals and reagents used in the experiment were of analytical or food grade standard. This research was conducted in the State Key Lab of Food Science and Technology between the periods of 25th July 2008 to 30 June 2009.

Protease Selection
Protamex, a Bacillus protease complex, Neutrase from Bacillus subtulis strain, Alcalase^® 2.4 L from a strain of Bacillus licheniformis and Flavourzyme from Aspergillus oryzae were obtained from Novo Nordisk’s Enzyme Business in Wuxi, Jiangsu Province, People’s Republic of China. The four proteases were evaluated for their ability to hydrolyze sesame seed protein. The four bacterial enzymes were screened using the pH Stat method as described by Adler-Nissen (1986), for the purpose of identifying those with better capabilities of hydrolyzing the defatted sesame seed protein. The DH is intricately related to the properties of the hydrolysates (Mahmoud et al., 1992) and was used as a factor to determine the effectiveness of the enzymes in hydrolyzing the protein.

Enzymatic Hydrolysis
Experiments to study the effect of hydrolysis variables in the range given in Table 1 were carried out in accordance with the experimental design depicted in Table 2. All reactions were carried out in triplicates in a 1 L polyethylene-jacketed glass vessel in a thermostatically controlled water bath (NUOHAI-XMTD-204, Tokyo, Japan) with constant stirring (700 rpm). The experimental vessel was covered with a tight fitting lid with bore holes to accommodate 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 pouring required reagents during the course of the hydrolysis process. In the reaction vessel, 50 g of defatted sesame flour was mixed in 500 mL of distilled water and placed in a water bath at the required temperature. The pH of the reaction content was maintained at desired values by adding required amounts of 0.2 mol L^-1 NaOH.

Table 1:	Hydrolysis variables and experimental design levels for response surface analysis
T: Temperature, pH, E/S: Enzyme/Substrate ratio (% v/w of Defatted Sesame flour), t: Time (min)

Table 2:	Protein yield of enzymatically hydrolyzed defatted sesame flour using different proteases

Homogenization was carried out for 5 min in order to adjust the pH (through addition of 0.2 mol L^-1 NaOH) and temperature to the desired values. After equilibrium was reached, the enzyme (Alcalase^®, which gave the highest protein recovery from the preliminary tests) 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 reaction was terminated by immersing the reaction vessel into hot water at 95°C for 15 min with continuous stirring to ensure enzyme inactivity. The temperature of the reaction mixture at the end of the inactivation was 90 to 95°C. The resulting slurry was cooled to room temperature (23-25°C) and then centrifuged using a Beckman Coulter Centrifuge (Avanti J-26XPI, Beckman Co., USA) at 2800x g for 20 min 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 (1986). Degree of hydrolysis is the ratio (expressed in percent) of the number of peptide bonds cleaved (h) and the total number of peptide bonds in the substrate studied (h_tot). The degree of hydrolysis was determined (Eq. 1) based on the quantity of base required to maintain the pH during the hydrolysis process in the batch assay.

(1)

where, h_tot is the total number of peptide bonds in the protein substrate in mol g^-1_protein, h is the number of hydrolyzed peptide bonds, B is the base consumed in mL, N_b is base normality and 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 Eq. 2.

(2)

where, pK is the average value of the α-amino groups liberated during the hydrolysis which varies significantly with temperature but relatively independent of the substrate. The pK at different temperatures (T in °C) was calculated according to Eq. 3.

(3)

Determination of Free Amino Groups
The number of free amino groups was determined by using Trinitrobenzene sulfonate (TNBS) as described by Mutilangi et al. (1995) with slight modifications. TNBS (1 mL of 1% solution) was added to 1 mL of protein solution (0.15 mg mL^-1) containing 1% Sodium Dodecyl Sulfate (SDS) and 4% NaHCO₃, pH 9.5. After rapid mixing, the mixture was kept at 40°C for 2 h in a water bath and after which, the reaction was stopped by adding 0.5 mL of 1 mol HCl and 1 mL of 10% SDS. The absorbance of the sample was read at 335 nm 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. The 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 with DH considered the dependent variable. To predict the optimal point, experimental data were fitted into a second order polynomial parameter model, according to Eq. 4. The regression model between dependent variable (Y) and independent variable (X) was in accordance with Eq. 4.

(4)

where, Y is the measured response variable, β_o, β_i, β_ii and β_ij are the constant, linear, quadratic and cross product regression coefficients of the model respectively, X_i and X_j represent the independent variables (hydrolysis parameters), e is the random error.

Regression coefficients of the model were obtained and tested by analysis of variance (ANOVA), performed by SAS method according to SAS (2002). The R² value, residual error, pure error and lack of fit were calculated by the model.

Proximate Composition
Moisture content was determined by placing 2 g of SPH sample into a preweighed aluminum dish. The sample was then dried in a forced-air convection oven at 105°C until a constant weight was reached. Ash content was estimated by charring in a crucible at 600°C until the ash had a white appearance. The total crude protein (N x 6.25) content of the samples was determined using the Kjeldahl method. The extraction and determination of fat from the samples were performed using the soxhlet method described by James (1995).

Nitrogen Solubility Index
The Nitrogen Solubility Index (NSI) procedure was used to measure the solubility of the SPH according to Hrckova et al. (2002) with slight modifications. SPH (10 g) were dispersed in water (100 mL) and the solution’s pH was adjusted to the desired values (3.0, 5.0, 7.0 and 9.0) with either 0.5 N HCl or 0.5 N NaOH while continually stirring for 45 min. At the end of this period, the mixture was centrifuged at 2800x g for 30 min.

A 15 mL aliquot of the supernatant was analyzed for nitrogen content (N) by the Kjeldahl method according to AOAC and NSI calculated using Eq. 5.

(5)

Foaming Capacity and Stability
Foaming capacity and stability were evaluated according to the method of Darwicz et al. (2000). A 0.5 g of the SPH was dissolved in a 30 mL distilled water; the aqueous dispersion was mixed thoroughly using an Ultra-Turrax T18 homogenizer (Shanghai, People’s Republic of China) at 3000 rpm for 3 min in a 250 mL graduated cylinder. The same was done for the DSF. Foaming capacity was calculated as percentage increase in volume upon mixing. Foam stability was estimated as the percent of foam remaining after 5, 10, 20, 40 and 60 min.

Emulsification Capacity
Emulsification Capacity (EC) was measured by an oil titration method described by Chove et al. (2002) with slight modification. 0.5 g of freeze-dried SPI and 30 mL of soybean oil were added to 60 mL of 0.5 N NaCl solution and mixed using an Ultra-Turrax T18 homogenizer (Shanghai, People’s Republic of China) at 3000 rpm for 10 min. Thereafter, another 20 mL of soybean oil were added and mixed for 5 min again. The mixture was transferred to centrifuge tubes, held in a water-bath at 85°C for 15 min and then centrifuged at 2800x g for 30 min. Emulsification capacity was calculated as given in Eq. 6.

(6)

where, O_A is the volume of oil added to form an emulsion, O_R is the volume of oil released after centrifugation and W_S is the weight of the SPH. The same was done for the DSF.

Water-Holding Capacity
Water-Holding Capacity (WHC) was determined using the centrifugation method as described by Jung et al. (2005). Triplicate of the SPH (0.5 g each) was dissolved in 20 mL of water in centrifuge tubes and dispersed with a vortex mixer for 10 min. The dispersion was allowed to stand at room temperature for 2 h and it was then centrifuged at 2800x g for 30 min. The supernatant was filtered with Whatman No. 1 filter paper and the volume recovered was accurately measured. The difference between the initial volume of distilled water added to the protein sample and the volume of the supernatant was determined and the results were reported as mL of water absorbed per gram of protein sample.

Oil-Holding Capacity
Oil-Holding Capacity (OHC) was measured as the volume of edible oil held by 0.5 g of the SPH and DSF as described by Xiangzhen et al. (2007). About 0.5 g of the SPI was added to 10 mL of soybean oil in a centrifuge tube and mixed for 10 min in a vortex mixer in triplicates. The oil dispersion was centrifuged at 2800x g for 30 min.

The volume of oil separated from the hydrolysate was measured and OHC was calculated as mL of oil absorbed per gram of protein sample.

Statistical Analysis
The results were subjected to statistical Analysis of Variance (ANOVA), using a Statistical Analysis Software (SAS, 2002). The significant difference between means was determined by Duncan’s Multiple Rang Test (DMRT), where p<0.05 was considered for significant difference.

RESULTS AND DISCUSSION

Protease Selection
The results for DH are presented in Fig. 1a and b. Among all proteinases, Alcalase 2.4 L at pH 7 and 8 determined at 50 and 60°C, respectively, showed the highest values DH. A rapid increase in DH with time was observed during the initial stages of hydrolysis as more

Fig. 1:

Enzymatic hydrolysis of defatted sesame flour by different proteases (a) at 55°C and pH 7, (b) at 60°C and pH 8

NaOH was required to maintain the pH during this period. This is because the enzymes were largely saturated within the substrate resulting to the cleavage of large numbers of peptide bonds. The hydrolysis rate increased in an exponential manner with time until a stationary phase was attained with lesser number of peptide bonds to be broken in the substrate. The screening results for the four proteases are shown in Table 2. Alcalase 2.4 L produced the highest value (96.68%) of protein recovery at 60°C with 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 2.4 L (77.62%). This observation could be attributed to the fact that different enzymes require specific reaction conditions for optimal activity. The conditions for optimal activity for Alcalase 2.4 L, 50°C and pH 7 (for defatted sesame flour hydrolysis) differ from those reported by Adler-Nissen (1986) but the results nonetheless corroborates the fact that the enzyme works more effectively above pH 7. The difference in protein recovery between Flourzyme and Alcalase was not significant at p<0.05. At a temperature of 60°C with pH-8, Alcalase 2.4 L gave a 96.68% protein recovery while Flourzyme, Neutrase and Protamex gave a protein recovery of 69.76, 50.12 and 45.18%, respectively. These results reinforces observations made by Hrckova et al. (2002), in their investigation of enzymatic hydrolysis of defatted soy flour by three different proteases and their effect on the functional properties of the resulting protein hydrolysates.

Determination of Free Amino Groups
Free amino acid groups increased through the course of hydrolysis and the increase was related to increase in DH. The number of generated free amino acid groups increases as the DH increases for all the enzymes except Alcalase which produced the lowest free amino acid groups throughout the experiments as shown in Fig. 2. Enzyme specificity is a key factor controlling the generation of free amino groups (Mutilangi et al., 1995) which may be attributed to the above observation. Alcalase produced little free amino groups compared to Flavouzyme, Protamex and Neutrase as observed in this study.

Alcalase 2.4 L was selected for the current study because of its high protein yield under optimum conditions, low free amino groups generated, ready availability, less expensive and ease of handling.

Fig. 2:

Free amino groups for the four enzymes

Table 3:	Box-Behnken design matrix and the response of the dependent variable degree of hydrolysis (DH) for defatted Sesame flour hydrolysis by Alcalase 2.4 L®

Optimization of Hydrolysis Conditions
The effect of the independent variables on the dependent variable for the hydrolysis of defatted sesame flour protein using Alcalase^® is shown in Table 3. The DH ranged from 1.19 to 18.8% at design points 5 and 16, respectively.

The ANOVA of the coefficients of the response surface model as provided by Eq. 4 were evaluated. Statistical analysis indicated that all the four hydrolysis factors (temperature, pH, enzyme/substrate ratio and time) had significant influence on DH. Similar observations have been reported in previous studies that show the combined effect of pH, temperature and enzyme/substrate ratio on the hydrolysis of some oil seeds (Taha and Ibrahim, 2002) and soy protein (Adler-Nissen, 1986) and also having markedly influence on peptide bond cleavage in the protein substrate. The same effect was observed for whey protein concentrates (Mota et al., 2004).

Regression coefficients in their linear form (T, pH, E/S and t) as well as one quadratic term (t²) were significant at p< 0.05. One crossproduct interaction (T^.t) was not significant at p<0.05.

The final response model equation to estimate the enzymatic hydrolysis in terms of DH of the defatted sesame flour is shown in Eq. 7.

where, DH is the response factor degree of hydrolysis (%). T, pH, E/S and t are the values of the independent factors, reaction temperature (°C), reaction pH, enzyme/substrate ratio (% v/w of defatted sesame flour) and reaction time (min), respectively.

The equation, in terms of coded factors, was generated using regression coefficients with statistical significance of up to p<0.05. The equation indicates that the variables had almost equal effect on the DH of defatted sesame flour protein, since they had similar slope values.

The second order model showed a good fit, the adjusted coefficient of determination (R² adj), which can check the appropriateness of a model, was 0.9870. This implies that 98.70% of the variation of DH of DSF could be explained by Eq. 7 in terms of the independent variables, within the range of values studied. The data proved that the developed model could adequately represent the real relationship among the parameters chosen for study.

Canonical analysis showed a maximum predicted DH of 16.34% and had the following critical values for the hydrolysis factors: T = 58.79°C, pH = 8.32, E/S = 1.80% (% v/w of defatted sesame flour) and t = 82.85 min. The experimental DH was 15.78%.

Contour plots generated from the predicted data illustrated the effect of each pair of independent variables (T, pH, E/S and t) on DH as shown in Fig. 3a-f. As observed from the plots, an increase in DH during the hydrolysis of defatted sesame flour is achieved by increasing temperature, pH and enzyme/substrate ratio. Similar results have been reported by Cheison et al. (2007), when they optimized the hydrolysis of whey protein. An increase in DH was also achieved as the time increased up to a certain level beyond which DH slightly decreases. The decrease in percentage hydrolysis can be explained by the increasing denaturation of the protein molecules hence reducing its biological activity (Claver and Huiming, 2005). It could also result from the exhaustion of the substrate or reduction of substrate concentration if all other reaction conditions remained the same. Similar observations have been reported for hydrolytic response of food proteins using three different kind proteases (Hrckova et al., 2002).

The non-linear relation between time and DH as noticed in Fig. 3b, d and f implies that the hydrolytic reaction depends on the availability of susceptible peptide bonds on which the primary enzymatic attack is based and also the physical structure of the protein molecule.

The DH was optimized using the freeze dried hydrolysate following the critical values for maximizing DH for the hydrolysis factors. This result was not significantly different (p<0.05) compared to the optimal value predicted by the design model. It further confirms that the process could be used to optimize the DH on DSF protein hydrolysates using RSM. A high protein recovery by Alcalase and its low cost may provide an incentive for using it in commercial operations.

Fig. 3:

Surface plots to show the combined effect of (a) pH and temperature, (b) time and temperature, (c) enzyme/substrate ratio and temperature, (d) time and pH, (e) enzyme/substrate ratio and pH and (f) enzyme/substrate ratio and time the on hydrolysis of defatted sesame flour by Alcalase®

Composition and Functional Properties of Defatted Sesame Flour and its Hydrolysate
The composition of defatted sesame flour and its hydrolysate is shown in Table 4. The hydrolysates contained a higher percentage protein (96.68%) compared to the native protein estimated at 52.04% as reported by John Kanu et al. (2007) after defatting the sesame flour. The moisture and ash content of the hydrolysate were estimated at 2.87 and 6.87%, respectively with insignificant amounts of fat and carbohydrate. A Significant increase of protein was observed when the DSF was hydrolyzed and this observation may be attributed to the degrading process by the enzymes that would have resulted to the release of more protein molecules trapped within the flour. Ash content is normally high in defatted sesame protein hydrolysates (Bandyopadhyay and Ghosh, 2002). The insignificant amounts of fat and carbohydrate could be attributed to the fact that the two components most times become diminished during protein extraction. The protein content of defatted sesame flour hydrolysate ranging between 93 to 98% is similar to that previously reported by Bandyopadhyay and Ghosh (2002).

The solubility was observed to increase gradually from pH 2 to 7 with a slight decrease at pH 8 but maintained afterwards as shown in Fig. 4. The native protein was lower in solubility right through the experiment and was observed to decrease between pH 4-5. The solubility of the hydrolysate obtained in this work differs on the grounds that solubility always fall within the isoelectric points (pI) of most protein isolates (Khalida et al., 2003). We observed an increase in solubility as the pH increases. It has been suggested that an increase in solubility of protein hydrolysate is due to the reduction of its secondary structure and also due to the enzymatic release of smaller polypeptide units from the protein (Mahmoud, 1994). Related studies have demonstrated that enzymatic hydrolysis of soy protein (Bernard-Don et al., 1991; Tsumura et al., 2005; Jung et al., 2005) and whey protein (Sinha et al., 2007) produced good functional properties, particularly solubility, as nitrogen solubility of the hydrolysate was pH dependent over the range studied. Such behavior is explained by the fact that smaller, more hydrophilic and more solvated polypeptide units are produced as a consequence of enzymatic hydrolysis.

Table 5:	Proximate analysis (%) of defatted sesame flour protein hydrolysates
Values are Mean±SEM (n = 3), different letter(s) in the same column are not significant at level (p<0.05) but significant at p<0.01, DSF: Defatted Sesame Flour, ND: Not Detected

Fig. 4:

Solubility of the hydrolysate and NP in water as affected by pH using Alcalase assisted hydrolysis process

Table 5:	Functional properties of defatted sesame flour protein non-hydrolyzed and hydrolyzed
Values are Mean±SEM (n = 3), different letter(s) in the same column are not significant at level (p<0.05) but significant at (p<0.01)

Hence, protein aggregates are usually formed even at isoelectric pH (Cheison et al., 2007). The surface of protein has a net charge that depends on the number and identities of the charged amino acids and on pH. There could have been different charges generated at the protein surface after the hydrolysis process. These charges make the hydrolysates more soluble as without a net charge, protein-protein interaction and precipitation are more likely to occur which could have been reflected in a decrease in the solubility of the protein hydrolysates. The results of other functional properties of defatted sesame flour hydrolysates prepared from Alcalase treatment are presented in Table 5. The hydrolysate produced was highly water-soluble with good water and oil holding capacities and an enhanced emulsifying property than the DSF. Foaming capacity was observed to be higher than the DSF but its stability over a period of time (5-60 min) was however not good. The high foaming capacity could be attributed to the protein in dispersion causing a decrease in surface tension at the water air interface thus leading to a higher foam forming tendency (Taha and Ibrahim, 2002). High foaming capacity is also determined by molecular flexibility and physicochemical properties (hydrophobicity, net charge distribution, hydrodynamic properties) of the proteins. To foam effectively (to develop high foamability), a protein needs to adsorb air rapidly during the transient stage of foam formation (Martin et al., 2002; Gonzalez-Perez et al., 2005) and it is envisaged that this phenomenum could have influenced the above result. Other results indicate an increase in surface activity, probably due to the initially greater number of polypeptide chains which may have been formed from the proteolytic activities, thus, allowing more air to be incorporated into the hydrolysates (Khalida et al., 2003). For the emulsifying capacity, it was observed that the hydrolysate was a good emulsifier than the DSF and according to the result in Table 5 the difference observed was significant. This might be because proteins are composed of charged amino acids, non-charged polar amino acids and nonpolar amino acids-the surfactant possessing both hydrophilic and hydrophobic properties that can interact with both water and oil in the food system, thus, making the protein a good emulsifier. (Martin et al., 2002). It is also possible that the protein molecules on the surface unfolded enough thus exposing the hydrophobic groups. This phenomenon could also enhance the emulsifying capability of the protein. Water holding capacity was also observed to be significantly higher in the hydrolysate compared to the native protein. The interaction of water and oil with protein is very important in food systems because of its effect on flavor and texture. Intrinsic factors affecting water holding capacity of food proteins include amino acid composition, protein conformation and surface polarity/hydrophilicity (Barbut, 1999). However, the method of protein extraction also had an important impact on the protein conformation and hydrophobicity/hydrophilicity which could lead to an enhanced water holding capacity of the protein. During hydrolysis the enzymes denaturated the protein molecules into smaller peptides, exposing more sites for water interaction with the protein molecules (Barbut, 1999). Oil holding capacity of the hydrolysate was also observed to be significantly higher compared to DSF. This could be attributed to the fact that the protease degraded the defatted sesame flour protein from its original protein structure to various peptides, thus exposing more hydrophobic groups to the oil interface resulting to an increased oil holding capacity (Barbut, 1999), since denaturation can improve the oil holding capacity of proteins due to increased hydrophobic surface and flexibility of the protein molecules.

CONCLUSION

Protein hydrolysates were produced from defatted sesame flour using different kinds of proteases to enhance higher protein recovery carried out by imposing engineered reaction conditions to optimize the degree of hydrolysis. The results show Alcalase 2.4 L to be a better enzyme in the preparation of protein hydrosylates from defatted sesame flour compared to the other proteases used in this work. Its effect produced the highest protein recovery and the hydrolysates obtained showed good functional properties. The study reveals a better alternative to protein enrichment procedures from plant materials and could serve as a blue print for the commercial production of protein supplements from plant materials. The findings prove the designed technique could be useful in the food industry especially in the realm of producing food formulations requiring high protein supplements. It could be applied in production of infant food formulations or poultry feeds etc with possible remarkable outcomes. The success of this technique further stresses the central role biotechnology plays in the food industry as efforts are stepped up to move away from conventional chemical food treatment procedures.

ACKNOWLEDGMENT

This study was financially supported by both the Government of Sierra Leone and Government of People’s Republic of China through the Sierra Leone-Sino relationship. The Authors are very grateful to the two governments.