- Email: [email protected]
Corn gluten hydrolysis by Alcalase: Calibration of pH-stat
food and bioproducts processing 8 9 ( 2 0 1 1 ) 500–503
Contents lists available at ScienceDirect
Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp
Short communication
Corn gluten hydrolysis by Alcalase: Calibration of pH-stat Dilek Kilic¸ Apar, Belma Özbek ∗ Yıldız Technical University, Department of Chemical Engineering, Davutpas¸a Campus, 34210 Esenler/Istanbul, Turkey
a b s t r a c t In the present study, pH-stat method was calibrated by TNBS and OPA reactions for control of hydrolysis of corn gluten by Alcalase. Hydrolysis experiments were conducted in a batch reactor at the temperature of 50 ◦ C for various pH values, and at pH 7 for 40 and 60 ◦ C. The calibration was characterized by good linear correlations for both methods. The mean pK values of corn gluten hydrolysates were obtained as 6.83, 6.59 and 6.36 for 40, 50 and 60 ◦ C, respectively. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Corn gluten; Protein; Hydrolysis; Alcalase; pH-stat; pK
1.
Introduction
Plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries (Friedman, 1996). Hydrolytic modification of proteins is carried out for improving nutritional value, retarding deterioration, imparting texture, changing solubility, adding foaming or coagulation properties, adding emulsifying capacity, preventing undesired interactions, removing off-flavours or odours and removing toxic or inhibitory ingredients (Lahl and Braun, 1994). In protein hydrolysis, the key parameter for monitoring the reaction is the degree of hydrolysis. Several methods of monitoring the degree of hydrolysis have been described in the literature, for example pH-stat, osmometry, soluble nitrogen content, trinitro-benzene-sulfonic acid (TNBS) and ophthaldialdehyde (OPA) (Adler-Nissen, 1979, 1986; Nielsen et al., 2001). Determination of the degree of hydrolysis by colorimetric methods is time consuming. The pH-stat is by far the most convenient method and measures the rate of hydrolysis by quantifying the release of protons by titration. By measuring the amount of base added during the hydrolysis, it is possible to measure the rate of hydrolysis. However, to establish the relationship between the base consumption and degree of hydrolysis, it is essential to find out the mean pK value of the amino groups released during the hydrolysis process.
∗
The aim of the present work was to calibrate the pHstat by determining the mean pK values of the corn gluten hydrolysates at different temperatures and pH values for control of the hydrolysis reaction by Alcalase.
2.
Materials and methods
Corn gluten used in this research was obtained from Cargill (Istanbul, Turkey). The enzyme used in this work was Alcalase 2.4 L, a bacterial endo-peptidase produced by Bacillus licheniformis, obtained from Novozymes (Istanbul, Turkey). Hydrolysis experiments were carried out in a 200 ml jacked reactor with magnetic stirring and pH and temperature control. During the hydrolysis the pH was maintained by the addition of 0.2 N KOH. In this study, all experiments were carried out at least in duplicate and the deviation between trials was within ±5%. For determination of the free ␣-amino groups both TNBS (Adler-Nissen, 1979) and OPA (Nielsen et al., 2001) methods were used. For each sample, the assays were carried out in triplicate and their averages were taken.
2.1.
Theory of calibration
The equation which relates DH to base consumption is: HD = B × Nb ×
1 1 1 × × 100% × ˛ PM htot.
Corresponding author. Tel.: +90 212 383 4747; fax: +90 212 383 4725. E-mail address: [email protected] (B. Özbek). Received 27 April 2010; Received in revised form 27 July 2010; Accepted 25 August 2010 0960-3085/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2010.08.011
(1)
501
food and bioproducts processing 8 9 ( 2 0 1 1 ) 500–503
Fig. 1 – Calibration of pH-stat with TNBS reaction (a) at various pH values, T = 50 ◦ C ( pH 6.5, pH 7, pH 7.5, Ж pH 8, — models); (b) at various temperatures, pH = 7.5 ( 40 ◦ C, 50 ◦ C, 60 ◦ C, — models). B: base consumption in ml (or L), Nb : normality of the base, ˛: average degree of dissociation of the ␣-NH groups, PM : mass of protein in g (or kg), htot. : total number of peptide bonds in meqv/g protein, and HD : hydrolysis degree %. ˛−1 is the calibration factor for the pH-stat, and it is the reciprocal of the degree of dissociation;
˛=
10pH−pK 1 + 10pH−pK
(2)
For the calibration of the pH-stat; the amount of base consumed during hydrolysis was recorded. The samples of hydrolysates were taken at timed intervals and l-leucine and l-serine equivalents were determined by TNBS and OPA reactions, respectively. After correlation of the base consumption with l-leucine and l-serine equivalents, slopes (b) of the straight lines were obtained. Then, the pK values were calculated from the following equations given by Adler-Nissen (1986): pK = pH2 + log(b1 − b2 ) − log(10pH2 −pH1 × b2 − b1 )
Table 1 – Estimated pK values (by using Eq. (3)) for the reactions performed at different pH values, T = 50 ◦ C. pH 1
pH 2
Slope (b1 )
Data for the calibration by TNBS reaction 6.5 7 1.8479 7 7.5 1.2169 7.5 8 0.9696 6.5 7.5 1.8479 7 8 1.2169 6.5 8 1.8479 Data for the calibration by OPA reaction 6.5 7 2.0131 7 7.5 1.2782 7.5 8 1.0471 6.5 7.5 2.0131 7 8 1.2782 6.5 8 2.0131
Slope (b2 )
pK
1.2169 0.9696 0.8808 0.9696 0.8808 0.8808 Mean value
6.50 6.62 6.68 6.55 6.65 6.57 6.60
1.2782 1.0471 0.9615 1.0471 0.9615 0.9615 Mean value
6.56 6.56 6.63 6.56 6.57 6.57 6.58
(3)
Fig. 2 – Calibration of pH-stat with OPA reaction (a) at various pH values, T = 50 ◦ C ( pH 6.5, pH 7, pH 7.5, Ж pH 8, — models); (b) at various temperatures, pH = 7.5 ( 40 ◦ C, 50 ◦ C, 60 ◦ C, — models).
502
food and bioproducts processing 8 9 ( 2 0 1 1 ) 500–503
Table 2 – Estimated pK values (by using Eq. (4)) for the reactions performed at different temperatures, pH = 7.5. Temperature
Data for the calibration by TNBS reaction Slope (b)
40 ◦ C 50 ◦ Ca 60 ◦ C a
pK
1.0370 0.9696 0.9265
6.81 6.60 6.37
Slope (b)
pK
1.1348 1.0471 0.9996
6.84 6.58 6.34
Mean pK
6.83 6.59 6.36
Standard temperature.
pH2 > pH1 , b1 and b2 are the slopes which correspond to pH1 and pH2 . pK = pH + log
b b0
× (1 + 10pK0 −pH ) − 1
(4)
b and b0 measured at same pH.
3.
Data for the calibration by OPA reaction
Results and discussion
The mean pK value of the amino groups released during the hydrolysis process was first determined by Adler-Nissen (1986) by comparing the base consumption with the amino groups released. Since then, most researcher in this field have used these results for any substrates (Marquez and Vazquez, 1999; Spellman et al., 2003; Zhu et al., 2006; Kong et al., 2007; Klompong et al., 2008). In the present study, pH-stat method was calibrated by TNBS and OPA reactions for control of hydrolysis of corn gluten by Alcalase. The hydrolysis reactions were carried out for 60 min at 3% (w/v) protein concentration with addition of 0.15% (v/v) enzyme at 50 ◦ C for pH values of 6.5, 7, 7.5, 8 and at the pH value of 7.5 for the temperatures of 40 and 60 ◦ C. The relationship between consumption of base and content of amino groups determined both by TNBS and OPA reactions were given in Figs. 1a and b and 2a and b. As it can be seen from these figures, the calibration of pH-stat with TNBS and OPA reactions were characterized by good linear correlations that gave values for the coefficient of determination of greater than 0.9583 and 0.9877; and standard errors lower than 0.0672 and 0.0442, respectively. Similar high coefficient of determination values were reported in the literature for the calibration of pHstat with TNBS reaction for hydrolysis of pea protein isolate (Karamac et al., 1998); and hydrolysis of casein and whey proteins (Dzwolak and Ziajka, 1999). However poor correlations were reported by Spellman et al. (2003) between the methods of pH-stat, TNBS and OPA for Debitrase HYW20 whey protein hydrolysates. The extent of the hydrolysis reaction increased with the increase on temperature and pH as the base consumption and amino group concentration increased with the temperature (Figs. 1b and 2b) and pH (Figs. 1a and 2a). At the end of 60 min of processing time; at pH 8 (T = 50 ◦ C), 1.63 meqv/g protein base consumption was obtained with respect to 1.71 meqv LeusineNH2 /g protein and 1.87 meqv Serine-NH2 /g protein; and at 60 ◦ C (pH 7.5), 1.55 meqv/g protein base consumption was obtained with respect to 1.75 meqv Leusine-NH2 /g protein and 1.89 meqv Serine-NH2 /g protein. The slopes obtained from the linear correlations of base consumption and concentration of free amino groups; and calculated pK values for corn gluten hydrolysates were given in Tables 1 and 2. As it can be seen from these tables, the pK values obtained as a result of the calibration performed by TNBS
and OPA reactions were found almost same. This result was not surprised as the calibration of pH-stat with both methods was characterized by good linear correlations. Nielsen et al. (2001) has also reported high correlation (r = 0.999) between the TNBS and OPA method for soy protein hydrolysates. Hence, considering all the results, the mean pK values of corn gluten hydrolysates were obtained as 6.83, 6.59 and 6.36 for 40, 50 and 60 ◦ C, respectively. The variation on pK with temperature could be calculated from Gibss–Helmholtz equation. The ionisation enthalpy of the amino group was reported as 45 kJ/mol by Steinhardt and Beychok (1964). By inserting this value in Gibss–Helmholtz equation, it was found that for a change of 10 ◦ C, pK will change about 0.23 pH units. In the present study, the difference between the pK values obtained for 40 and 50 ◦ C is 0.24 pH units, and the difference between the pK values obtained for 50 and 60 ◦ C is 0.23 pH units. These results confirm the reliability of the study.
Acknowledgements Writers gratefully acknowledges to Novoyzmes and Cargill for their supports.
References Adler-Nissen, J., 1979. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry 27, 1256–1262. Adler-Nissen, J., 1986. Enzymic Hydrolysis of Food Proteins. Elsevier Applied Science Publishers, New York, USA, pp. 132–142. Dzwolak, W., Ziajka, S., 1999. Enzymatic hydrolysis of milk proteins under alkaline and acidic conditions. Journal of Food Science 64, 393–395. Friedman, M., 1996. Nutritional value of proteins from different food sources. Journal of Agricultural and Food Chemistry 44, 6–29. Karamac, M., Amarowicz, R., Kostyra, H., Sijtsma, L., 1998. Hydrolysis of pea protein isolate piyasane by Trypsin. Nahrung 42, 219. Klompong, V., Benjakul, S., Kantachote, D., Hayes, K.D., Shahidi, F., 2008. Comparative study on antioxidative activity of yellow stripe trevally protein hydrolysate produced from Alcalase and Flavourzyme. International Journal of Food Science and Technology 43, 1019–1026. Kong, X., Zhou, H., Qian, H., 2007. Enzymatic preparation and functional properties of wheat gluten hydrolysates. Food Chemistry 101, 615–620. Lahl, W.J., Braun, S.D., 1994. Enzymatic production of protein hydrolysates for food use. Food Technology 48, 68–71. Marquez, M.C., Vazquez, M.A., 1999. Modelling of enzymatic protein hydrolysis. Process Biochemistry 35, 111–117. Nielsen, P.M., Petersem, D., Dambmann, C., 2001. Improved method for determining food protein degree of hydrolysis. Journal of Food Science 66, 642–646.
food and bioproducts processing 8 9 ( 2 0 1 1 ) 500–503
Spellman, D., McEvoy, E., O’Cuinn, G., FitzGerald, R.J., 2003. Proteinase and exopeptidase hydrolysis of whey protein: comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis. International Dairy Journal 13, 447–453. Steinhardt, J., Beychok, S., 1964. Interactions of proteins with hydrogen ions and other small ions and molecules. In:
503
Neurath, H. (Ed.), The Proteins, vol. II. Academic Press, New York, USA, pp. 139–304. Zhu, K., Zhou, H., Qian, H., 2006. Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with Alcalase. Process Biochemistry 41, 1296–1302.
Contents lists available at ScienceDirect
Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp
Short communication
Corn gluten hydrolysis by Alcalase: Calibration of pH-stat Dilek Kilic¸ Apar, Belma Özbek ∗ Yıldız Technical University, Department of Chemical Engineering, Davutpas¸a Campus, 34210 Esenler/Istanbul, Turkey
a b s t r a c t In the present study, pH-stat method was calibrated by TNBS and OPA reactions for control of hydrolysis of corn gluten by Alcalase. Hydrolysis experiments were conducted in a batch reactor at the temperature of 50 ◦ C for various pH values, and at pH 7 for 40 and 60 ◦ C. The calibration was characterized by good linear correlations for both methods. The mean pK values of corn gluten hydrolysates were obtained as 6.83, 6.59 and 6.36 for 40, 50 and 60 ◦ C, respectively. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Corn gluten; Protein; Hydrolysis; Alcalase; pH-stat; pK
1.
Introduction
Plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries (Friedman, 1996). Hydrolytic modification of proteins is carried out for improving nutritional value, retarding deterioration, imparting texture, changing solubility, adding foaming or coagulation properties, adding emulsifying capacity, preventing undesired interactions, removing off-flavours or odours and removing toxic or inhibitory ingredients (Lahl and Braun, 1994). In protein hydrolysis, the key parameter for monitoring the reaction is the degree of hydrolysis. Several methods of monitoring the degree of hydrolysis have been described in the literature, for example pH-stat, osmometry, soluble nitrogen content, trinitro-benzene-sulfonic acid (TNBS) and ophthaldialdehyde (OPA) (Adler-Nissen, 1979, 1986; Nielsen et al., 2001). Determination of the degree of hydrolysis by colorimetric methods is time consuming. The pH-stat is by far the most convenient method and measures the rate of hydrolysis by quantifying the release of protons by titration. By measuring the amount of base added during the hydrolysis, it is possible to measure the rate of hydrolysis. However, to establish the relationship between the base consumption and degree of hydrolysis, it is essential to find out the mean pK value of the amino groups released during the hydrolysis process.
∗
The aim of the present work was to calibrate the pHstat by determining the mean pK values of the corn gluten hydrolysates at different temperatures and pH values for control of the hydrolysis reaction by Alcalase.
2.
Materials and methods
Corn gluten used in this research was obtained from Cargill (Istanbul, Turkey). The enzyme used in this work was Alcalase 2.4 L, a bacterial endo-peptidase produced by Bacillus licheniformis, obtained from Novozymes (Istanbul, Turkey). Hydrolysis experiments were carried out in a 200 ml jacked reactor with magnetic stirring and pH and temperature control. During the hydrolysis the pH was maintained by the addition of 0.2 N KOH. In this study, all experiments were carried out at least in duplicate and the deviation between trials was within ±5%. For determination of the free ␣-amino groups both TNBS (Adler-Nissen, 1979) and OPA (Nielsen et al., 2001) methods were used. For each sample, the assays were carried out in triplicate and their averages were taken.
2.1.
Theory of calibration
The equation which relates DH to base consumption is: HD = B × Nb ×
1 1 1 × × 100% × ˛ PM htot.
Corresponding author. Tel.: +90 212 383 4747; fax: +90 212 383 4725. E-mail address: [email protected] (B. Özbek). Received 27 April 2010; Received in revised form 27 July 2010; Accepted 25 August 2010 0960-3085/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2010.08.011
(1)
501
food and bioproducts processing 8 9 ( 2 0 1 1 ) 500–503
Fig. 1 – Calibration of pH-stat with TNBS reaction (a) at various pH values, T = 50 ◦ C ( pH 6.5, pH 7, pH 7.5, Ж pH 8, — models); (b) at various temperatures, pH = 7.5 ( 40 ◦ C, 50 ◦ C, 60 ◦ C, — models). B: base consumption in ml (or L), Nb : normality of the base, ˛: average degree of dissociation of the ␣-NH groups, PM : mass of protein in g (or kg), htot. : total number of peptide bonds in meqv/g protein, and HD : hydrolysis degree %. ˛−1 is the calibration factor for the pH-stat, and it is the reciprocal of the degree of dissociation;
˛=
10pH−pK 1 + 10pH−pK
(2)
For the calibration of the pH-stat; the amount of base consumed during hydrolysis was recorded. The samples of hydrolysates were taken at timed intervals and l-leucine and l-serine equivalents were determined by TNBS and OPA reactions, respectively. After correlation of the base consumption with l-leucine and l-serine equivalents, slopes (b) of the straight lines were obtained. Then, the pK values were calculated from the following equations given by Adler-Nissen (1986): pK = pH2 + log(b1 − b2 ) − log(10pH2 −pH1 × b2 − b1 )
Table 1 – Estimated pK values (by using Eq. (3)) for the reactions performed at different pH values, T = 50 ◦ C. pH 1
pH 2
Slope (b1 )
Data for the calibration by TNBS reaction 6.5 7 1.8479 7 7.5 1.2169 7.5 8 0.9696 6.5 7.5 1.8479 7 8 1.2169 6.5 8 1.8479 Data for the calibration by OPA reaction 6.5 7 2.0131 7 7.5 1.2782 7.5 8 1.0471 6.5 7.5 2.0131 7 8 1.2782 6.5 8 2.0131
Slope (b2 )
pK
1.2169 0.9696 0.8808 0.9696 0.8808 0.8808 Mean value
6.50 6.62 6.68 6.55 6.65 6.57 6.60
1.2782 1.0471 0.9615 1.0471 0.9615 0.9615 Mean value
6.56 6.56 6.63 6.56 6.57 6.57 6.58
(3)
Fig. 2 – Calibration of pH-stat with OPA reaction (a) at various pH values, T = 50 ◦ C ( pH 6.5, pH 7, pH 7.5, Ж pH 8, — models); (b) at various temperatures, pH = 7.5 ( 40 ◦ C, 50 ◦ C, 60 ◦ C, — models).
502
food and bioproducts processing 8 9 ( 2 0 1 1 ) 500–503
Table 2 – Estimated pK values (by using Eq. (4)) for the reactions performed at different temperatures, pH = 7.5. Temperature
Data for the calibration by TNBS reaction Slope (b)
40 ◦ C 50 ◦ Ca 60 ◦ C a
pK
1.0370 0.9696 0.9265
6.81 6.60 6.37
Slope (b)
pK
1.1348 1.0471 0.9996
6.84 6.58 6.34
Mean pK
6.83 6.59 6.36
Standard temperature.
pH2 > pH1 , b1 and b2 are the slopes which correspond to pH1 and pH2 . pK = pH + log
b b0
× (1 + 10pK0 −pH ) − 1
(4)
b and b0 measured at same pH.
3.
Data for the calibration by OPA reaction
Results and discussion
The mean pK value of the amino groups released during the hydrolysis process was first determined by Adler-Nissen (1986) by comparing the base consumption with the amino groups released. Since then, most researcher in this field have used these results for any substrates (Marquez and Vazquez, 1999; Spellman et al., 2003; Zhu et al., 2006; Kong et al., 2007; Klompong et al., 2008). In the present study, pH-stat method was calibrated by TNBS and OPA reactions for control of hydrolysis of corn gluten by Alcalase. The hydrolysis reactions were carried out for 60 min at 3% (w/v) protein concentration with addition of 0.15% (v/v) enzyme at 50 ◦ C for pH values of 6.5, 7, 7.5, 8 and at the pH value of 7.5 for the temperatures of 40 and 60 ◦ C. The relationship between consumption of base and content of amino groups determined both by TNBS and OPA reactions were given in Figs. 1a and b and 2a and b. As it can be seen from these figures, the calibration of pH-stat with TNBS and OPA reactions were characterized by good linear correlations that gave values for the coefficient of determination of greater than 0.9583 and 0.9877; and standard errors lower than 0.0672 and 0.0442, respectively. Similar high coefficient of determination values were reported in the literature for the calibration of pHstat with TNBS reaction for hydrolysis of pea protein isolate (Karamac et al., 1998); and hydrolysis of casein and whey proteins (Dzwolak and Ziajka, 1999). However poor correlations were reported by Spellman et al. (2003) between the methods of pH-stat, TNBS and OPA for Debitrase HYW20 whey protein hydrolysates. The extent of the hydrolysis reaction increased with the increase on temperature and pH as the base consumption and amino group concentration increased with the temperature (Figs. 1b and 2b) and pH (Figs. 1a and 2a). At the end of 60 min of processing time; at pH 8 (T = 50 ◦ C), 1.63 meqv/g protein base consumption was obtained with respect to 1.71 meqv LeusineNH2 /g protein and 1.87 meqv Serine-NH2 /g protein; and at 60 ◦ C (pH 7.5), 1.55 meqv/g protein base consumption was obtained with respect to 1.75 meqv Leusine-NH2 /g protein and 1.89 meqv Serine-NH2 /g protein. The slopes obtained from the linear correlations of base consumption and concentration of free amino groups; and calculated pK values for corn gluten hydrolysates were given in Tables 1 and 2. As it can be seen from these tables, the pK values obtained as a result of the calibration performed by TNBS
and OPA reactions were found almost same. This result was not surprised as the calibration of pH-stat with both methods was characterized by good linear correlations. Nielsen et al. (2001) has also reported high correlation (r = 0.999) between the TNBS and OPA method for soy protein hydrolysates. Hence, considering all the results, the mean pK values of corn gluten hydrolysates were obtained as 6.83, 6.59 and 6.36 for 40, 50 and 60 ◦ C, respectively. The variation on pK with temperature could be calculated from Gibss–Helmholtz equation. The ionisation enthalpy of the amino group was reported as 45 kJ/mol by Steinhardt and Beychok (1964). By inserting this value in Gibss–Helmholtz equation, it was found that for a change of 10 ◦ C, pK will change about 0.23 pH units. In the present study, the difference between the pK values obtained for 40 and 50 ◦ C is 0.24 pH units, and the difference between the pK values obtained for 50 and 60 ◦ C is 0.23 pH units. These results confirm the reliability of the study.
Acknowledgements Writers gratefully acknowledges to Novoyzmes and Cargill for their supports.
References Adler-Nissen, J., 1979. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry 27, 1256–1262. Adler-Nissen, J., 1986. Enzymic Hydrolysis of Food Proteins. Elsevier Applied Science Publishers, New York, USA, pp. 132–142. Dzwolak, W., Ziajka, S., 1999. Enzymatic hydrolysis of milk proteins under alkaline and acidic conditions. Journal of Food Science 64, 393–395. Friedman, M., 1996. Nutritional value of proteins from different food sources. Journal of Agricultural and Food Chemistry 44, 6–29. Karamac, M., Amarowicz, R., Kostyra, H., Sijtsma, L., 1998. Hydrolysis of pea protein isolate piyasane by Trypsin. Nahrung 42, 219. Klompong, V., Benjakul, S., Kantachote, D., Hayes, K.D., Shahidi, F., 2008. Comparative study on antioxidative activity of yellow stripe trevally protein hydrolysate produced from Alcalase and Flavourzyme. International Journal of Food Science and Technology 43, 1019–1026. Kong, X., Zhou, H., Qian, H., 2007. Enzymatic preparation and functional properties of wheat gluten hydrolysates. Food Chemistry 101, 615–620. Lahl, W.J., Braun, S.D., 1994. Enzymatic production of protein hydrolysates for food use. Food Technology 48, 68–71. Marquez, M.C., Vazquez, M.A., 1999. Modelling of enzymatic protein hydrolysis. Process Biochemistry 35, 111–117. Nielsen, P.M., Petersem, D., Dambmann, C., 2001. Improved method for determining food protein degree of hydrolysis. Journal of Food Science 66, 642–646.
food and bioproducts processing 8 9 ( 2 0 1 1 ) 500–503
Spellman, D., McEvoy, E., O’Cuinn, G., FitzGerald, R.J., 2003. Proteinase and exopeptidase hydrolysis of whey protein: comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis. International Dairy Journal 13, 447–453. Steinhardt, J., Beychok, S., 1964. Interactions of proteins with hydrogen ions and other small ions and molecules. In:
503
Neurath, H. (Ed.), The Proteins, vol. II. Academic Press, New York, USA, pp. 139–304. Zhu, K., Zhou, H., Qian, H., 2006. Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with Alcalase. Process Biochemistry 41, 1296–1302.