- Email: [email protected]
Heat-induced deamidation, dephosphorylation and breakdown of caseinate
International Dairy Journal 9 (1999) 237}241
Heat-induced deamidation, dephosphorylation and breakdown of caseinate Martinus A.J.S. van Boekel* Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, Netherlands
Abstract The kinetics of deamidation, dephosphorylation and protein breakdown in heated caseinate solutions were studied. The extent of deamidation corresponded to the level of the amide present in asparagine. The order with respect to concentration was 1, the order with respect to time could not be established unequivocally. The temperature dependence was in accordance with a mechanism in which a succinimide intermediate was formed. The extent of protein breakdown, measured as formation of non-protein nitrogen (NPN), was also characterized by an order of 1 with respect to concentration. The temperature dependence was in accordance with peptide bond hydrolysis (the order with respect to time could not be established unequivocally). Formation of inorganic phosphate and dehydroalanine was determined in heated b-casein solutions. More phosphate than dehydroalanine was formed, and based on literature results for lysinoalanine formation from dehydroalanine, it was concluded that hydrolysis of phosphoserine occurred more extensively than b-elimination of phosphoserine. The order with respect to time for dephosphorylation seemed to increase with temperature, possibly due to a di!erent temperature sensitivity of hydrolysis and b-elimination. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Deamidation; Dephosphorylation; Heat-induced proteolysis; Kinetics; Casein
1. Introduction Heat has a large e!ect on proteins. Apart from denaturation, heat-induced chemical reactions involving amino acid residues can signi"cantly alter protein properties. Such heat-induced chemical changes are as yet quantitatively not very well characterized. Therefore the kinetics of deamidation, dephosphorylation and fragmentation of caseinate were studied. Heat-induced deamidation (of the amides asparagine, Asn, and glutamine, Gln) has been studied to some extent for soy protein, egg white lysozyme, casein and gliadin (Zhang, Lee & Ho, 1993a, b, c). Deamidation of non-food proteins and model peptides (used to study the mechanism of deamidation) has been reviewed by Wright (1991). At pH(4}5, general acid/base catalysed hydrolysis is believed to be the mechanism, but at higher pH another mechanism speci"c for protein/peptides takes place. At the sequences Asn}glycine, Asn}serine and
* Tel.: #31-317-484281; fax: #31-317-483669. E-mail address: [email protected] (M.A.J.S. van Boekel)
Asn}alanine, a b-aspartyl shift mechanism occurs leading to a succinimide intermediate, which easily breaks down to either the a- or b-isomeric aspartate products in the presence of water; the ratio of a- to b-isomer is 3 : 1 (Wright, 1991). Deamidation of Asn occurs more readily than that of Gln because the amide group of Asn is closer to adjacent amino acid residues which may act as catalysts (Wright, 1991). Gln deamidation occurs via a similar (six-membered) ring intermediate, glutarimide. The NPN (non-protein nitrogen) content of heated protein solutions increases because of formation of ammonia due to deamidation. However, degradation of protein resulting in formation of small peptides and amino acids also results in NPN increase. To distinguish between NPN increase due to ammonia formation and peptide bond cleavage, we decided to study both ammonia and NPN formation. Heat-induced dephosphorylation of casein (having a high phosphoserine content) has been studied by Belec and Jenness (1962). The mechanism of heat-induced dephosphorylation is either hydrolysis of phosphoserine leading to formation of phosphate, or b-elimination leading to formation of dehydroalanine (DHA) and
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 6 8 - 0
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phosphate (Walstra & Jenness, 1984). DHA is quite a reactive intermediate, reacting with lysine to lysinoalanine, with histidine to histidinoalanine, with cystein to lanthionine, with ornithine/arginine to ornithinoalanine and with ammonia to b-aminoalanine. We studied heatinduced formation of phosphate and DHA in b-casein solutions; b-casein does not contain cystein (another source for DHA) so that DHA could only be formed from phosphoserine.
2. Materials and methods Sodium caseinate (DMV, Veghel, NL) was dissolved in concentrations ranging from 1}5% (w/w) in a synthetic milk salt solution (Jenness & Koops, 1962) at pH 6.5 (a few experiments were also done at pH 7.5). b-casein (95% pure, Eurial, FR) was used in 3% solutions (w/w) in water, pH 7.0. Deamidation and NPN formation of sodium caseinate solutions was studied using 3.1 ml samples in stoppered stainless steel tubes rotated while heated (heating-up time 1 min). Dephosphorylation of b-casein solutions was studied in 13 ml samples heated in stoppered glass tubes without rotation (heating up time 2 min). The content of NPN (non-protein nitrogen) and the concentration of ammonia was determined in the same sample by adding trichloroacetic acid (TCA) to a "nal concentration of 12%. The resulting protein precipitate was removed by "ltration over 589 "lter paper (Schleicher & Schuell, Germany). The nitrogen content of the "ltrate was determined by the Kjeldahl method (FIL IDF, 1962). The ammonia content was determined in the same "ltrate by an enzymatic method (Boehringer, DE, cat no. 542 946). (The pH of the "ltrate was raised to 6}7 by adding 10 M KOH, results were not corrected for this small dilution e!ect.) Dephosphorylation was studied by determining the inorganic phosphorus content, after wet digestion by a colorimetric method (FIL IDF, 1990) in samples deproteinized in 12% TCA. Dehydroalanine (DHA) formation was studied according to a method described by Kleyn and Klostermeyer (1980) in which DHA is transformed into pyruvic acid by acid hydrolysis, and pyruvate is subsequently determined enzymatically (Boehringer, DE, cat no. 128 147).
3. Results and discussion A typical example of deamidation of caseinate at pH 6.5 is given in Fig. 1. The amount of ammonia released was never more than was present as amide N in Asn residues in casein, which would be in line with Asn being more easily deamidated than Gln (Wright, 1991).
Fig. 1. Deamidation of caseinate solutions at 120 (open symbols) and 1403C (closed symbols), pH 6.5, at initial amide concentration 18.9 mmol\ (*, 䢇), 28.35 mmol\ (䊐, 䊏) and 36.0 mmol l\ (䉫, 䉬). Closed line is for a "rst-order model, dotted line for a second-order model.
Kinetics of deamidation were studied following the general rate law dc rate"! "kcL, dt
(1)
in which c is concentration, t time, k the reaction rate constant and n the order of the reaction. Integration of this equation yields the order with respect to time, n (Atkins, 1986). n was obviously'0 (no straight line could be "tted to the data in Fig. 1) but n could not be estimated exactly because any order between 0 and 3 "tted almost equally well, probably because the extent of deamidation was not very high. (A marginally better "t was obtained for n "2.) The order with respect to con centration (n ) was determined from initial rates at the various concentrations studied by taking the logarithm of Eq. (1): log (rate)"log k#n log c; the slope is then n (Fig. 2). The results clearly pointed in the direction of n "1, in line with a pseudo "rst-order reaction in the case of hydrolysis as well as in the case of the succinimide intermediate. n does not necessarily need to be the same as n because the reaction may be autocatalytic (n (n ) or inhibited (n 'n ) as the reaction proceeds. Since we could not determine n exactly, an autocatalytic or inhibiting e!ect is not clear from our results. Heating the caseinate solutions resulted in a pH decrease due to changes in the milk salt solution (Walstra & Jenness, 1984), possibly causing an inhibiting e!ect because deamidation increases with pH according to the
M.A.J.S. van Boekel / International Dairy Journal 9 (1999) 237}241
Fig. 2. Logarithm of the initial rate of deamidation versus logarithm of amide concentration for 1103C (䉬), 1203C (䊐), 1303C (*), 1403C (ⴙ) and 1453C (䉱). The numbers indicate the slope of the line, n .
literature (Zhang et al., 1993a, b). To investigate pH dependency, we carried out a few experiments at pH 7.5 for a casein solution containing 28.35 mmol\ amide (3% casein). The trend was that deamidation was somewhat stronger at pH 7.5 compared to pH 6.5. However, the di!erences, if any, were not very large. The temperature dependence of deamidation (at pH 6.5) was derived from the Eyring equation (Van Boekel & Walstra, 1995) and resulted in activation enthalpy *HS"92.0$13.6 kJ mol\ ($95% con"dence interval) and activation entropy *SS"!69.9$ 13.8 J mol\ K\ ($95% con"dence interval). The value for *HS is of the order of magnitude expected for a chemical reaction, while the negative *SS indicates a bimolecular (rate limiting) reaction, probably the formation of the succinimide intermediate. The parameters agree well with those for deamidation of a model hexapeptide (Patel & Borchardt, 1990), for which the mechanism of deamidation was shown to be exclusively via the succinimide intermediate. Zhang et al. (1993b) found an activation energy of 104 kJ mol\ at pH 7 for deamidation of soy protein. In addition to ammonia, heat-induced formation of NPN in caseinate solutions was determined. The NPN fraction contains low molecular weight nitrogenous compounds soluble in 12% TCA, including ammonia. Comparison of the formation of NPN with that of ammonia formed by deamidation may give some idea about heatinduced protein fragmentation besides deamidation. The quantity of NPN components formed was much larger than the ammonia content (Fig. 3 gives an example). The ammonia content was on average only 10}15% of the NPN content. The proportion of NPN to total nitrogen
239
Fig. 3. Formation of NPN (*, 䢇) and ammonia (䊐, 䊏) from 3% caseinate solutions heated at 120 (open symbols) and 1403C (closed symbols).
(TN) was quite high, at 1403C up to 20%, indicating that a considerable portion of protein was degraded into small fragments. If the results of Meisel and Schlimme (1995) for milk, i.e. no signi"cant formation of free amino acids due to heating, are also valid for casein solutions, it would mean that the NPN increase must be due to formation of small peptides. As with deamidation, NPN formation was slightly higher at pH 7.5. The order with respect to time for NPN formation gave a slightly better "t with n "2 but the di!erences were small for 0(n (3. Belec and Jenness (1962), Saidi and Wart hesen (1993) and Metwalli, Metwalli and Van Boekel (1996) found zero-order kinetics for NPN formation in heated milk or caseinate solutions. However, deviation from zero-order kinetics becomes obvious only at higher degree of conversion, as in the present study. The order with respect to concentration pointed to n "1. *HS for NPN formation was 107.0$27.6 kJ mol\ ($95% con"dence interval) and *SS was !37.4$13.7 J mol K\ ($95% con"dence interval), again pointing to a bimolecular rate-limiting step, presumably hydrolysis of peptide bonds. Dephosphorylation and formation of DHA was studied with b-casein solutions in water, pH 7 (Fig. 4). The pH change was negligible during heating. The total amount of organic phosphate in the b-casein solutions was 6 mmol l\. DHA formation was consistently lower than that of inorganic phosphate, but DHA may have reacted further into other reaction products. We did not determine lysinoalanine (LAL) but results of Meyer, Klostermeyer and Kleyn (1981) indicate that the amounts of LAL would be between 0.1 mmol l\ (60 min at 1103C) and 0.5 mmol l\ (60 min at 1403C). Allowing
240
M.A.J.S. van Boekel / International Dairy Journal 9 (1999) 237}241
Fig. 4. Formation of inorganic phosphate (*) and dehydroalanine (䢇) from 3% b-casein solutions (heating-up time of 2 min excluded). Solid lines represent kinetic models for n "0 at 1103C, n "0.7 at 1203C, n "1.3 at 1303C, n "1.8 at 1403C, dotted line represents a "rst-order model at each temperature.
for formation of the same amount of histidinoalanine (Henle, Walter and Klostermeyer, 1993), the amount of DHA having reacted further under the conditions used here would be between 0.4 mmol l\ at 1103C and 1 mmol l\ at 1403C at the most. This implies that more phosphate was formed via hydrolysis than via b-elimination, especially at the higher temperatures. Belec and Jenness (1962) concluded that dephosphorylation of casein was a "rst-order reaction. However, our results suggested that n increased with temperature from about zero at 1103C to about 2 at 1403C (as illustrated in Fig. 4). It may be an indication that the mechanism of dephosphorylation changed with increasing temperature, possibly due to a di!erent temperature sensitivity of the hydrolysis and b-elimination reaction. More research is needed to con"rm this. (The order with respect to concentration was unfortunately not determined.) The change in n with temperature made
it di$cult to estimate the temperature dependence of the reaction; in order to get an impression, n "1 was as sumed over the temperature range studied (see also Fig. 4). This yielded an apparent *HS of 97.2$4.2 kJ mol\ ($95% con"dence interval) and an apparent *SS of !41.1$ 3.2 J mol K\ ($95% con"dence interval). The negative (apparent) *SS indicates a bimolecular rate limiting reaction (in this case, probably a mixture of hydrolysis and b-elimination). Belec and Jenness (1962) found for b-casein *HS"117 kJ mol\ and *SS"!21 J mol K\.
Acknowledgements The author would like to thank Dr. A. Metwalli and Mr. H. Stempher for performing the deamidation and NPN experiments and Ms. K. Jukes for performing the dephosphorylation experiments.
M.A.J.S. van Boekel / International Dairy Journal 9 (1999) 237}241
References Atkins, P. W. (1986). Physical chemistry (3rd ed). Oxford: Oxford University Press. Belec, J., & Jenness, R. (1962). Dephosphorization of caseins by heat treatment. I. In caseinate solutions. Journal of Dairy Science, 45, 12}19. Henle, T., Walter, A. W., & Klostermeyer, H. (1993). Detection and identi"cation of the crosslinking amino acids NO- and NL-(2 -amino2 -carboxyethyl)-L-histidine (&histidinoalanine', HAL) in heated milk products. Zeitschrift fu( r Lebensmittel*Untersuchung und Forschung, 197, 114}117. FIL.IDF. (1962). Determination of nitrogen content. FIL.IDF Standard 20. FIL.IDF. (1990). Determination of total phosphorus content. FIL.IDF Standard 42B. Jenness, R., & Koops, J. (1962). Preparation and properties of a salt solution which simulates milk ultra"ltrate. Netherlands Milk and Dairy Journal, 16, 153}164. Kleyn, D. H., & Klostermeyer, H. (1980). Dehydroalanin als Reaktionsprodukt bei der Erhitzung von b-casein. Zeitschrift fu( r LebensmittelUntersuchung und- Forschung, 170, 11}13. Meisel, H., & Schlimme, E. (1995). Casein-gebundener Phosphor und der Gehalt an freien AminosaK uren in unterschiedlich waK rmebehandelter Milch. Kieler Milchwirtschaftliche Forschungsberichte, 47, 289}295. Metwalli, A. A. M., Metwalli, N. H., & Van Boekel, M. A. J. S. (1996). E!ect of urea on heat-induced changes in milk. Netherlands Milk Dairy Journal, 50, 427}457.
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Meyer, M., Klostermeyer, H., & Kleyn, D. H. (1981). Verminderte Bildung von Lysinoalanin bei enzymatisch dephosphoryliertem Casein. Zeitschrift fu( r Lebensmittel- Untersuchung und- Forschung, 172, 446}448. Patel, K., & Borchardt, R. T. (1990). Chemical pathways of peptide degradation. II. Kinetics of deamidation of an asparaginyl residue in a model hexapeptide. Pharmaceutical Research, 7, 703}711. Saidi, B., & Warthesen, J. J. (1993). Heat and fermentation e!ects on total nonprotein nitrogen and urea in milk. Journal of Food Science, 58, 548}551. Van Boekel, M. A. J. S., & Walstra, P. (1995). Use of kinetics in studying heat-induced changes in foods. In P. F. Fox, Heat-induced changes in milk (pp. 22}50). Brussels: International Dairy Federation. Walstra, P., & Jenness, R. (1984). Dairy Chemistry & Physics. New York: Wiley. Wright, H. T. (1991). Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins. Critical Reviews in Biochemistry and Molecular Biology, 26, 1}52. Zhang, J., Lee, T. C., & Ho, Ch-T. (1993a). Comparative study on kinetics of nonenzymatic deamidation of soy protein and egg white lysozyme. Journal of Agricultural and Food Chemistry, 41, 2286}2290. Zhang, J., Lee, T. C., & Ho, Ch-T. (1993b). Kinetics and mechanism of nonenzymatic deamidation of soy protein. Journal of Food Processing and Preservation, 17, 259}268. Zhang, J., Lee, T. C., & Ho, Ch-T. (1993c). Thermal deamidation of proteins in a restricted water environment. Journal of Agricultural and Food Chemistry, 41, 1840}1843.
Heat-induced deamidation, dephosphorylation and breakdown of caseinate Martinus A.J.S. van Boekel* Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, Netherlands
Abstract The kinetics of deamidation, dephosphorylation and protein breakdown in heated caseinate solutions were studied. The extent of deamidation corresponded to the level of the amide present in asparagine. The order with respect to concentration was 1, the order with respect to time could not be established unequivocally. The temperature dependence was in accordance with a mechanism in which a succinimide intermediate was formed. The extent of protein breakdown, measured as formation of non-protein nitrogen (NPN), was also characterized by an order of 1 with respect to concentration. The temperature dependence was in accordance with peptide bond hydrolysis (the order with respect to time could not be established unequivocally). Formation of inorganic phosphate and dehydroalanine was determined in heated b-casein solutions. More phosphate than dehydroalanine was formed, and based on literature results for lysinoalanine formation from dehydroalanine, it was concluded that hydrolysis of phosphoserine occurred more extensively than b-elimination of phosphoserine. The order with respect to time for dephosphorylation seemed to increase with temperature, possibly due to a di!erent temperature sensitivity of hydrolysis and b-elimination. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Deamidation; Dephosphorylation; Heat-induced proteolysis; Kinetics; Casein
1. Introduction Heat has a large e!ect on proteins. Apart from denaturation, heat-induced chemical reactions involving amino acid residues can signi"cantly alter protein properties. Such heat-induced chemical changes are as yet quantitatively not very well characterized. Therefore the kinetics of deamidation, dephosphorylation and fragmentation of caseinate were studied. Heat-induced deamidation (of the amides asparagine, Asn, and glutamine, Gln) has been studied to some extent for soy protein, egg white lysozyme, casein and gliadin (Zhang, Lee & Ho, 1993a, b, c). Deamidation of non-food proteins and model peptides (used to study the mechanism of deamidation) has been reviewed by Wright (1991). At pH(4}5, general acid/base catalysed hydrolysis is believed to be the mechanism, but at higher pH another mechanism speci"c for protein/peptides takes place. At the sequences Asn}glycine, Asn}serine and
* Tel.: #31-317-484281; fax: #31-317-483669. E-mail address: [email protected] (M.A.J.S. van Boekel)
Asn}alanine, a b-aspartyl shift mechanism occurs leading to a succinimide intermediate, which easily breaks down to either the a- or b-isomeric aspartate products in the presence of water; the ratio of a- to b-isomer is 3 : 1 (Wright, 1991). Deamidation of Asn occurs more readily than that of Gln because the amide group of Asn is closer to adjacent amino acid residues which may act as catalysts (Wright, 1991). Gln deamidation occurs via a similar (six-membered) ring intermediate, glutarimide. The NPN (non-protein nitrogen) content of heated protein solutions increases because of formation of ammonia due to deamidation. However, degradation of protein resulting in formation of small peptides and amino acids also results in NPN increase. To distinguish between NPN increase due to ammonia formation and peptide bond cleavage, we decided to study both ammonia and NPN formation. Heat-induced dephosphorylation of casein (having a high phosphoserine content) has been studied by Belec and Jenness (1962). The mechanism of heat-induced dephosphorylation is either hydrolysis of phosphoserine leading to formation of phosphate, or b-elimination leading to formation of dehydroalanine (DHA) and
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 6 8 - 0
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M.A.J.S. van Boekel / International Dairy Journal 9 (1999) 237}241
phosphate (Walstra & Jenness, 1984). DHA is quite a reactive intermediate, reacting with lysine to lysinoalanine, with histidine to histidinoalanine, with cystein to lanthionine, with ornithine/arginine to ornithinoalanine and with ammonia to b-aminoalanine. We studied heatinduced formation of phosphate and DHA in b-casein solutions; b-casein does not contain cystein (another source for DHA) so that DHA could only be formed from phosphoserine.
2. Materials and methods Sodium caseinate (DMV, Veghel, NL) was dissolved in concentrations ranging from 1}5% (w/w) in a synthetic milk salt solution (Jenness & Koops, 1962) at pH 6.5 (a few experiments were also done at pH 7.5). b-casein (95% pure, Eurial, FR) was used in 3% solutions (w/w) in water, pH 7.0. Deamidation and NPN formation of sodium caseinate solutions was studied using 3.1 ml samples in stoppered stainless steel tubes rotated while heated (heating-up time 1 min). Dephosphorylation of b-casein solutions was studied in 13 ml samples heated in stoppered glass tubes without rotation (heating up time 2 min). The content of NPN (non-protein nitrogen) and the concentration of ammonia was determined in the same sample by adding trichloroacetic acid (TCA) to a "nal concentration of 12%. The resulting protein precipitate was removed by "ltration over 589 "lter paper (Schleicher & Schuell, Germany). The nitrogen content of the "ltrate was determined by the Kjeldahl method (FIL IDF, 1962). The ammonia content was determined in the same "ltrate by an enzymatic method (Boehringer, DE, cat no. 542 946). (The pH of the "ltrate was raised to 6}7 by adding 10 M KOH, results were not corrected for this small dilution e!ect.) Dephosphorylation was studied by determining the inorganic phosphorus content, after wet digestion by a colorimetric method (FIL IDF, 1990) in samples deproteinized in 12% TCA. Dehydroalanine (DHA) formation was studied according to a method described by Kleyn and Klostermeyer (1980) in which DHA is transformed into pyruvic acid by acid hydrolysis, and pyruvate is subsequently determined enzymatically (Boehringer, DE, cat no. 128 147).
3. Results and discussion A typical example of deamidation of caseinate at pH 6.5 is given in Fig. 1. The amount of ammonia released was never more than was present as amide N in Asn residues in casein, which would be in line with Asn being more easily deamidated than Gln (Wright, 1991).
Fig. 1. Deamidation of caseinate solutions at 120 (open symbols) and 1403C (closed symbols), pH 6.5, at initial amide concentration 18.9 mmol\ (*, 䢇), 28.35 mmol\ (䊐, 䊏) and 36.0 mmol l\ (䉫, 䉬). Closed line is for a "rst-order model, dotted line for a second-order model.
Kinetics of deamidation were studied following the general rate law dc rate"! "kcL, dt
(1)
in which c is concentration, t time, k the reaction rate constant and n the order of the reaction. Integration of this equation yields the order with respect to time, n (Atkins, 1986). n was obviously'0 (no straight line could be "tted to the data in Fig. 1) but n could not be estimated exactly because any order between 0 and 3 "tted almost equally well, probably because the extent of deamidation was not very high. (A marginally better "t was obtained for n "2.) The order with respect to con centration (n ) was determined from initial rates at the various concentrations studied by taking the logarithm of Eq. (1): log (rate)"log k#n log c; the slope is then n (Fig. 2). The results clearly pointed in the direction of n "1, in line with a pseudo "rst-order reaction in the case of hydrolysis as well as in the case of the succinimide intermediate. n does not necessarily need to be the same as n because the reaction may be autocatalytic (n (n ) or inhibited (n 'n ) as the reaction proceeds. Since we could not determine n exactly, an autocatalytic or inhibiting e!ect is not clear from our results. Heating the caseinate solutions resulted in a pH decrease due to changes in the milk salt solution (Walstra & Jenness, 1984), possibly causing an inhibiting e!ect because deamidation increases with pH according to the
M.A.J.S. van Boekel / International Dairy Journal 9 (1999) 237}241
Fig. 2. Logarithm of the initial rate of deamidation versus logarithm of amide concentration for 1103C (䉬), 1203C (䊐), 1303C (*), 1403C (ⴙ) and 1453C (䉱). The numbers indicate the slope of the line, n .
literature (Zhang et al., 1993a, b). To investigate pH dependency, we carried out a few experiments at pH 7.5 for a casein solution containing 28.35 mmol\ amide (3% casein). The trend was that deamidation was somewhat stronger at pH 7.5 compared to pH 6.5. However, the di!erences, if any, were not very large. The temperature dependence of deamidation (at pH 6.5) was derived from the Eyring equation (Van Boekel & Walstra, 1995) and resulted in activation enthalpy *HS"92.0$13.6 kJ mol\ ($95% con"dence interval) and activation entropy *SS"!69.9$ 13.8 J mol\ K\ ($95% con"dence interval). The value for *HS is of the order of magnitude expected for a chemical reaction, while the negative *SS indicates a bimolecular (rate limiting) reaction, probably the formation of the succinimide intermediate. The parameters agree well with those for deamidation of a model hexapeptide (Patel & Borchardt, 1990), for which the mechanism of deamidation was shown to be exclusively via the succinimide intermediate. Zhang et al. (1993b) found an activation energy of 104 kJ mol\ at pH 7 for deamidation of soy protein. In addition to ammonia, heat-induced formation of NPN in caseinate solutions was determined. The NPN fraction contains low molecular weight nitrogenous compounds soluble in 12% TCA, including ammonia. Comparison of the formation of NPN with that of ammonia formed by deamidation may give some idea about heatinduced protein fragmentation besides deamidation. The quantity of NPN components formed was much larger than the ammonia content (Fig. 3 gives an example). The ammonia content was on average only 10}15% of the NPN content. The proportion of NPN to total nitrogen
239
Fig. 3. Formation of NPN (*, 䢇) and ammonia (䊐, 䊏) from 3% caseinate solutions heated at 120 (open symbols) and 1403C (closed symbols).
(TN) was quite high, at 1403C up to 20%, indicating that a considerable portion of protein was degraded into small fragments. If the results of Meisel and Schlimme (1995) for milk, i.e. no signi"cant formation of free amino acids due to heating, are also valid for casein solutions, it would mean that the NPN increase must be due to formation of small peptides. As with deamidation, NPN formation was slightly higher at pH 7.5. The order with respect to time for NPN formation gave a slightly better "t with n "2 but the di!erences were small for 0(n (3. Belec and Jenness (1962), Saidi and Wart hesen (1993) and Metwalli, Metwalli and Van Boekel (1996) found zero-order kinetics for NPN formation in heated milk or caseinate solutions. However, deviation from zero-order kinetics becomes obvious only at higher degree of conversion, as in the present study. The order with respect to concentration pointed to n "1. *HS for NPN formation was 107.0$27.6 kJ mol\ ($95% con"dence interval) and *SS was !37.4$13.7 J mol K\ ($95% con"dence interval), again pointing to a bimolecular rate-limiting step, presumably hydrolysis of peptide bonds. Dephosphorylation and formation of DHA was studied with b-casein solutions in water, pH 7 (Fig. 4). The pH change was negligible during heating. The total amount of organic phosphate in the b-casein solutions was 6 mmol l\. DHA formation was consistently lower than that of inorganic phosphate, but DHA may have reacted further into other reaction products. We did not determine lysinoalanine (LAL) but results of Meyer, Klostermeyer and Kleyn (1981) indicate that the amounts of LAL would be between 0.1 mmol l\ (60 min at 1103C) and 0.5 mmol l\ (60 min at 1403C). Allowing
240
M.A.J.S. van Boekel / International Dairy Journal 9 (1999) 237}241
Fig. 4. Formation of inorganic phosphate (*) and dehydroalanine (䢇) from 3% b-casein solutions (heating-up time of 2 min excluded). Solid lines represent kinetic models for n "0 at 1103C, n "0.7 at 1203C, n "1.3 at 1303C, n "1.8 at 1403C, dotted line represents a "rst-order model at each temperature.
for formation of the same amount of histidinoalanine (Henle, Walter and Klostermeyer, 1993), the amount of DHA having reacted further under the conditions used here would be between 0.4 mmol l\ at 1103C and 1 mmol l\ at 1403C at the most. This implies that more phosphate was formed via hydrolysis than via b-elimination, especially at the higher temperatures. Belec and Jenness (1962) concluded that dephosphorylation of casein was a "rst-order reaction. However, our results suggested that n increased with temperature from about zero at 1103C to about 2 at 1403C (as illustrated in Fig. 4). It may be an indication that the mechanism of dephosphorylation changed with increasing temperature, possibly due to a di!erent temperature sensitivity of the hydrolysis and b-elimination reaction. More research is needed to con"rm this. (The order with respect to concentration was unfortunately not determined.) The change in n with temperature made
it di$cult to estimate the temperature dependence of the reaction; in order to get an impression, n "1 was as sumed over the temperature range studied (see also Fig. 4). This yielded an apparent *HS of 97.2$4.2 kJ mol\ ($95% con"dence interval) and an apparent *SS of !41.1$ 3.2 J mol K\ ($95% con"dence interval). The negative (apparent) *SS indicates a bimolecular rate limiting reaction (in this case, probably a mixture of hydrolysis and b-elimination). Belec and Jenness (1962) found for b-casein *HS"117 kJ mol\ and *SS"!21 J mol K\.
Acknowledgements The author would like to thank Dr. A. Metwalli and Mr. H. Stempher for performing the deamidation and NPN experiments and Ms. K. Jukes for performing the dephosphorylation experiments.
M.A.J.S. van Boekel / International Dairy Journal 9 (1999) 237}241
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