FUNCTIONAL PROPERTIES OF ENZYMETICALLY MODIFIED WHEAT GLUTEN

777  FUNCTIONAL PROPERTIES OF ENZYMETICALLY MODIFIED WHEAT GLUTEN A. S. Jasim J. M. Nasser Researcher Assist. Prof.  Dep. of Food Sci., Coll. Agric. Engin. Sci. , University of Baghdad, Baghdad, Iraq  dr.jassim6699@yahoo.com ABSTRACT This study was aimed to investigate the potentiality of gluten inclusion into functional foods. The effect of controlled enzymatic hydrolysis on the antioxidant properties of Pepsin , Trypsin and Papain-assisted wheat gluten hydrolysates have been studied. Lyophilized and dried gluten from durum wheat, commercial durum gluten and whey proteins were enzymatically hydrolyzed. Based on antioxidant activity of the obtained hydrolysates, papain hydrolysed gluten were selected for this study. Functional properties (water holding capacity, emulsifying capacity and stability, foam formation and stability, protein solubility, and oil binding capacity) were investigated for the selected samples. Results revealed that the enzymatic modification improved the functional properties of all selected proteins significantly (P<0.05), with the superiority of the lyophilized and dried wheat gluten in some functional properties especially in alkaline pH and pH 4. . Key word: antioxidant properties , pepsin , trypsin , papain, gluten hydrolysates. Part of M. Sc. Thesis of the 1 st author.


INTRODUCTION
Preparation of wheat gluten:The wet gluten was extracted from durum wheat (Triticum durum). Wheat grains were conditioned to 14 % moisture before milling. AACC method No. 10-38 (1) was used for gluten extraction and estimation from flour. Chemical analysis: Proximate compositions of all wheat and flours were studied using AOAC methods (3). Total carbohydrate was calculated by difference. Enzymatic treatment of wheat gluten 1. Papain treated wheat gluten: Gluten hydrolysates were prepared using papain, according to Bandyopadhyay and Ghosh (4) with some modifications. The gluten was mixed with distilled water in ratio of 1:20 and the pH was adjusted to 10 with NaOH (0.1M) and incubated at 50 ̊ C for 1 hour until the protein completely dissolved . The pH re-adjusted to 8 using hydrochloric acid (0.1M), and incubated for 15 minutes at 37 ̊ C. 2000 & 3000 units per 1g of gluten was added individually , and incubated at 50 ̊ C for 7hr. Aliquot of the hydrolysates were taken after (1,2,3,4,5,6,7 )hrs., and the reaction terminated by placing the samples in boiled water bath for 5 minutes, centerfugated at 5000xg for 15 minutes. The supernatant collected and stored at (-18 ̊ C) until use.

Trypsin treated wheat gluten
Gluten protein hydrolysis was carried out using trypsin enzyme, according to Liu and Chiang (19) with some modifications. The gluten was mixed with distilled water in ratio of 1:20 and the pH was adjusted to 8 with NaOH (0.1M). The mixture was incubated at 50 ̊ C for 1hour until the protein completely dissolved, then incubated at 37 ̊ C for 15 minutes .The enzyme was added at different concentrations (4000 & 5000 units per 1g of gluten) and samples were taken after ( 1, 2, 3, 5, 6, 7)hrs, placed in boiling water bath for 5 minutes for enzyme inactivation and centrifuged at 5,000x g for 15 min. The supernatant collected and kept at (-18 ̊ C) until use 3.Pepsin treated wheat gluten Gluten hydrolysis was conducted, using pepsin, according to Chatterjee et al. (7) with some modifications. The gluten was mixed with distilled water in ratio of 1:20, pH was adjusted to 2 by HCl (0.1M). The mixture was incubated at 50 ̊ C for 1hour until the protein completely dissolved and was incubated at 37 ̊ C for 15 minutes. Different concentrations (4000 & 5000 units per 1g of gluten) of pepsin was used for gluten hydrolysis after ( 1, 2, 3, 5, 6, 7) hrs samples of hydrolysates were taken and placed in a boiling water bath for 5 minutes for enzyme inactivation then centrifuged at 5,000x g for 15 min. The supernatant was collected and kept at (-18 ̊ C) until use . Determination the Degree of Hydrolysis (DH): The degree of hydrolysis was tested according to Liu & Chiang (19). The standard solution of L-Lucien (55Mm) was prepared by dissolving 0.361g L-Lucien in small amount of distilled water and the volume was completed to 50 ml. The required concentrations were prepared as show in Table 1. Procedure To 0. 250 ml of each of the above solutions , 2 ml of SDS(1%) and 2 ml sodium phosphate (0.2125 M) at pH 8.2 and 2ml of TNBS solution (0.1%) were added. The mixture was incubated at 50 ̊ C for 1hour at dark place . The reaction was stopped by adding 4 ml of HCl solution (1M) . The samples were kept at room temperature for 30 minutes and the absorbency was read at 340 nm. The standard curve was plotted as the relation between the concentration of the L-Lucien and the absorbance reading at 340 nm.  0  0  1000  950  50  5  1000  850  150  15  1000  750  250  25  1000  650  350  35  1000  550  450  45  1000  450  550  55 The studied samples (0.250 ml of each) were transferred to a test tube and subjected to the above steps. NH 3 groups were calculated using the standard Lucien amino acid curve and the degree of degradation was calculated according to the following equation (14): DH =[ ( L t -L 0 ) / ( L max -L 0 )]*100 L t = concentration of α-NH 3 released in the time t .L 0 =α-NH 3 found in the original protein sample . L max = total α-NH3 in the undigested sample , which can be obtained after acidification using HCL (6 M) at 120̊ C for 24h Determination of antioxidant activity: DPPH Radical-Scavenging Activity (RSA) The RSA was measured according to Laohakungit et al.,(18) with some modulations. One ml of the sample (4 mg / ml) was mixed with 1 ml of DPPH solution (0.1 M). The mixture kept at dark place at room temperature for 30 minutes , and then centrifuged at 10,000x g for 5 min. The absorbency was measured at 517 nm , and the percentage of the scavenging activity was calculated according to the following equation :=

Radical Scavenging activity A = [C -(B-A )/ C] x100
A= Spectrophotometer reading of the tested sample at517 nm wavelength. B = the absorption reading of the control sample at 517 nm (prepared by mixing 1 ml of ethyl alcohol with 1 ml of the sample under study). C = reading of the positive control at 517 nm (obtained from mixing 1 ml of DPPH with 1 ml of distilled water). Functional properties 1. Solubility determination: Solubility of the protein was determined according to the method suggested by Catterjee et. al., (7) . A sample of gluten (50 mg ) was dissolved in 20 ml of distilled water and the pH adjust to different values ( 1,2,3,4,5,6,7,8,9,10,11,12 ), and left for 15 minutes under controlled pH, then centrifuged at 10000 * g for 15 minutes. The supernatant was collected and the total nitrogen content was estimated . The percentages of solubility were calculated as follows : Solubility % = protein content in the supernatant/ protein content in the sample x 100 .

Water holding capacity determination
Onsaard et al. (21) method was followed with some modification, 0.5 g of the experimental sample was mixed with 10ml distilled water, vortexed for 5 minutes . The pH was adjusted to (4,7,12) and left at room temperature for 15 minutes, and was centrifuged at 10,000 g for 10 minutes . Water holding capacity was calculated using the following equation : W.H.C = W2-W1/ W0 W2 = Tube weight + weight of the precipitate after water removal . W1 = Tube weight + Sample before water addition W0 = weight of Sample 3.Oil binding capacity determination Onsaard et al. (21) method was followed with some modification, 0.5 g of the sample was mixed with 10ml sun flower oil placed on the vortex for 15 minutes , then the pH adjusted to (4,7,12) and left at room temperature for 15 minutes , then centrifuged at 10,000 g for 10 minutes . Oil binding Capacity was calculated by the following equation : Oil Binding Determination ( gm oil /gm sample ) = F2-F1 / F0 F0 = Weight of the sample . F1 = Tube weight + sample weight before adding oil F2= Weight of the tube + weight of the deposit after removing the oil 4. Estimation the foam formation capacity and stability Cano -Medina et al., (23) method was adopted with some modification. One gram of the experimental samples was mixed with small amount of distilled water for one minute, and the volume completed to 100 ml , the pH of the experimental samples were adjusted to (4 , 7 , 12), 50 ml of each sample were placed in 150 ml flasks ,then vortexed for one minute at maximum speed and then transferred to a 100 ml graduated cylinder . The volume was measured before and after whipping. The ratio of foam capacity and stability was calculated as follow: Foam capacity % = volume after whiskingvolume before whipping / size before whisking*100 Foam stability % = foam size after a certain time/foam time zero x 100 5.Emulsification and emulsion stability Sharm et al., (23) method was adapted with some modification, 5 ml of the samples (1% ) at three different pH values (4,7,12) were mixed with 5 ml sunflower oil. The mixture was homogenized, (10,000 cycles / minute). Centrifuged at (3500*g) for 5 minutes and the emulsions layer volume was measured by the included cylinder. The percentage of emulsification capacity was calculated using the following equation: Emulsification capacity % = Emulsion layer size / Total size * 100 The stability of emulsion was measured by placing the emulsion in a water bath at 85 C for 30 min and then centrifugation (3500*g) for (5) minute and the emulsion layer volume was measured using the inserted cylinder. Emulsion stability was calculated using the following equation

Stability of emulsion = Emulsion layer after heating / Total volume before heating *100 Statistical analysis
Statistical Analysis System (SAS) (22) was used for the analysis of data, to study the effect of different treatments in the studied traits in full randomized design (CRD). The differences between mean were compared with the least significant difference (LSD). Table (2) shows the percentages of moisture, protein, fat, ash, fiber and carbohydrates of durum wheat and wheat flour(72-76% extracted), dried Lyophilized durum wheat gluten and commercial gluten. The percentages of moisture were (6.61, 9.6, 4.49, 3.72, 5%) respectively. Moisture content has a significant impact on the quality of wheat storage and is also an important factor in determining the quality of the resulting flour and its water absorption. Due to the wheat conditioning, moisture percentage has increased in the flour. Protein content of wheat and flour were 17.5% and 13%, respectively. Protein is of great importance in determining product quality. The same table also showed that the fat percentage were (2.64 , 1.91%) respectively. Many studies confirm the importance of flour fat bread manufacturing and the rheological properties of the dough, despite their small quantity. The percentage of ash was 3.07 and 1.9 %, respectively. Ash content is an important measure related to the quality of milling process and also it is a strong indicator of flour color and purity. It is noted that the ratio of fibers does not correspond to the ratios indicated by Zain Elabideen, (29), who pointed out that the percentage of fiber in Iraqi wheat varieties ranged between 2 -2.7%. Iuliana et al., (16) reported that the percentage of carbohydrates for wheat varieties ranged from 65-75%, and this is similar to our finding in the Iraqi wheat strain in this study.

Enzymatic treatment of wheat gluten
The effect of enzymes concentrations (pepsin, papain and trypsin) on the hydrolysis of dried, lyophilized and commercial gluten and antioxidant properties were studied individually.

1-Pepsin treated wheat gluten and whey protein
Tables 3 show the degrees of hydrolysis(DH) of dried, lyophilized and commercial gluten and whey proteins treated with pepsin (3000, 4000 units / g protein). As it is obvious the degree of hydrolysis increased, as the enzyme concentrations increased with the hydrolysis time. The bitter taste appeared in the hydrolysates after (3) hours of enzymatic hydrolysis when (3000units / g protein) was used, and after (1 ) hours when (4000units / g protein) was used. Elmalimadi, (11) studied the effect of heat pretreatment for wheat gluten on the enzymatic hydrolysis induced by alcalase. The results indicated that the heat treatment significantly improved susceptibility of WGPs to alcalase and the DH (%) varied from 2 to 30 % over 195 min of hydrolysis.  Table 4 indicates the degrees of hydrolysis (DH) of dried, lyophilized and commercial gluten and whey proteins treated with trypsin (4000, 5000 units / g protein). The bitter taste was observed after 6 hours of enzymatic hydrolysis of gluten samples under study. It was noted that the trypsin was less effective in gluten hydrolysis as compared to pepsin, in contrast it was more effective in hydrolysis of whey proteins. ECabrera-Chávez et al., ( 12 ) found " that the DH of hydrolysis of trypsin treated durum, bred wheat and gluten fractions were 1.16-1.40%. The influence of hydrolysis on the isoelectric point was more evident in durum wheat gluten.

3-Papain treated wheat gluten and whey protein
Tables 5 shows degrees of hydrolysis (DH) of dried, lyophilized and commercial gluten and whey proteins treated with papain (2000, 3000 units / g protein). It has been noticed from tables (3,4,5) that papain was more effective in the hydrolysis of all protein samples under study as compared with pepsin and trypsin. The DH of papain treated samples increased rapidly in the first four hours, then began to slow down. It nate worthily that the whey protein hydrolysates showed no bitter taste through the entire hydrolysis time.  Table 6 shows the Radical-scavenging activity (using DPPH) of pepsin, trypsin and papain treated proteins (dried, lyophilized, commercial and whey proteins). It was observed that radical-scavenging activity of all hydrolysates increased as hydrolysis time increased. The difference in the radicalscavenging activity of the treated proteins can be attributed to the differences in the degrees of enzymatic degradation, the type, molecular weight of product, size and configuration of peptide produced, as well as the type and sequence of amino acids ( 13 ; 18 ; 25). Gluten hydrolysates (induced by pepsin enzyme) had a higher radical scavenging activity compared with other hydrolysates, however, these hydrolysates were excluded because the bitter taste was appeared at the first hour of the hydrolysis. The hydrolysates which obtained after four hours papain induced hydrolysis was free of bitter taste and gave higher RSA as compared to trypsin induced hydrolysates. Therefore, this group was selected to complete this study.  Table 7 shows the solubility (%) of the experimental proteins before and after enzymatic treatment at different pH values. The solubility of modified whey proteins were ( 0.52, 0 .56, 0.68, 0.81, 0.75, 0.61, 0.60, 0,78, 1.03, 1.34, 1.48, 1.41) at pH (1,2,3,4,5,6,7,8,9,10,11,12) (1,2,3,4,5,6,7,8,9,10,11,12 ) respectively. Solubility of Papain treated proteins were the best in alkaline pH especially at PH 8 and 10 (Table  5).These results are similar to that of Bomara et al. (5) who recorded that the enzymatic treatment improved solubility, and close to results of Olanca and Ozay (20) who noticed a significant increase in gluten solubility at neutral and alkaline pH especially at pH 7, 8, and 10. The results reveal a significant improvement (P<0.05)in the other functional properties of the tested proteins treated enzymatically with papain (3000 units/ g protein) ( Table 8): the statistical analysis indicate that water holding capacity (WHC) of wheat gluten was increased significantly for all tested proteins especially at a pH (12) with insignificant improvement at the other pH values. These results are in agreement with Bomara et el. (5) who found a significant improvement in WHC of wheat gluten. Additionally, Deng et al., (10) found slight increase in water holding capacity of enzymatically modified wheat gluten and an improvement in emulsifying and stability of gluten hydrolysates (using wheatbug protease )at neutral and alkaline pH , and our results were close to their results except at pH 4. Table 9 indicates that the emulsifying capacity of dried, lyophilized and commercial gluten was improved at pH (4, 7 and 12). Meanwhile the emulsion stability was also significantly improved at pH 12 in all tested proteins except the dried sample. This result was similar to that of several researchers findings (17; 28) who noticed an improvement in capacity with no change in emulsifying stability of wheat gluten modified ( using acid protease from  Bombara et al. (5) found the same increasing in emulsifying capacity of wheat flours modified using protease.mTable 10 illustrates the foaming ability of the experimental proteins. It has been noticed that the best improvement in this property achieved after 60 minutes especially in lyophilized gluten. The same improvement also seen in foam stability, but the best effect was after 30 minutes. Table 9. Effect of enzymatic treatment of wheat gluten (Triticum durum) with Papain (3000 units/g protein) on emulsion ability (%) and emulsion stability(%) at different pH values  This result was similar to that of researchers finding (9 , 4) who reported a significant increase in oil holding capacity of wheat gluten. But we can say that there is no difference in those results when we view to the type of the tested wheat that they use which was the soft wheat.