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Protein cross-linking in food: mechanisms, consequences, applications
International Congress Series 1245 (2002) 211 – 215
Protein cross-linking in food: mechanisms, consequences, applications Juliet A. Gerrard *, Paula K. Brown Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand
Abstract The Maillard reaction influences not only the colour and flavour of foods, but also their texture. One of the mechanisms by which this occurs is via protein cross-linking. The capacities of three molecules—formaldehyde, glyceraldehyde and glutaraldehyde—to cross-link wheat proteins were compared in vitro and in situ. All three molecules cross-linked wheat proteins in vitro, but only glutaraldehyde cross-linked the proteins when added to wheat flour dough. The effect of glutaraldehyde on the dough properties was marked. Upon baking, addition of glutaraldehyde was shown to alter crumb strength and texture of bread, but had no perceivable effect on croissants. A comparison to previous results employing enzymatic cross-linking reveals that cross-linking of specific wheat proteins can be correlated to particular properties of cereal foods. This suggests that the Maillard reaction may be harnessed by food processors to manipulate the texture of foods. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Protein cross-linking; Maillard; Glycation; Cereal foods; Food texture
1. Introduction In his closing address at the Sixth International Symposium on the Maillard Reaction, Mlotkiewicz [1] pointed out that ‘‘in exploiting the Maillard reaction, the key target for industry is to understand and harness the reaction pathways enabling improvement of existing products and the development of new products.’’ Three key properties of food to which consumers respond—colour, flavour and texture—are all influenced by the Maillard reaction, but the last of these, texture—has received the least attention [2]. We are engaged in a study of how the Maillard reaction can be harnessed to manipulate the texture of
*
Corresponding author. Tel.: +64-33667001; fax: +64-33642083. E-mail address: [email protected] (J.A. Gerrard).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 9 1 0 - X
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foods, particularly cereal foods. Our particular interest is in protein cross-linking, a known consequence of protein glycation, likely to occur during food processing [3]. Previous work has established unequivocally that protein cross-linking can profoundly affect the texture of food, specifically bread and croissants [4 –6]. In this paper, we explore the potential of three molecules known to cross-link proteins—formaldehyde, glyceraldehyde and glutaraldehyde—to alter the texture of cereal foods.
2. Materials and methods Unless otherwise stated, all materials were obtained from Sigma (St. Louis, MO, USA). Ribonuclease was Type XII-A from bovine pancreas. Wheat proteins were fractionated as previously described [4]. Protein cross-linking was monitored by previously published electrophoretic methods [3,7]. Lysine availability was monitored by a modification of the o-phthaldialdehyde method of Bertrand-Harb et al. [8]. Dough, bread and croissant properties were assessed using Crop and Food Research in-house methods [5,6].
3. Results Model studies using ribonuclease as a model protein confirmed the well-established fact that glutaraldehyde cross-links proteins almost instantaneously [9]. The cross-linking was accompanied by a dramatic loss in the availability of all measurable lysine residues in the protein, confirming that the cross-linking mechanism involves reaction of these amino groups. In contrast, both glyceraldehyde and formaldehyde cross-linked at a lower rate, and removed fewer lysine residues. Incubation of ribonuclease with formaldehyde or glyceraldehyde at 37 jC removed only 40% of the available lysines over 5 h, and resulted in substantially fewer cross-links. Wheat proteins were extracted from flour in four fractions, according to their solubility: the albumins and globulins, the gliadins, the soluble glutenins and the insoluble glutenins. Each of these fractions was incubated with each of the cross-linking molecules and the results compared to the model system. Comprehensive experiments were undertaken at a range of concentrations of each cross-linking reagent. A typical cross-linking pattern, for glutaraldehyde cross-linking the albumins and globulins, is shown in Fig. 1. Typical lysine availability results are shown in Fig. 2, for the gliadin fraction cross-linked with a lower concentration of glutaraldehyde. The results corroborated those of the model study, in that glutaraldehyde cross-linked very quickly, with glyceraldehyde and formaldehyde crosslinking more slowly. To establish whether the in vitro results were relevant to actual food systems, the three cross-linking reagents were each added to bread and croissant dough. Only glutaraldehyde, at concentrations of 200 mM and above, was found to have a marked effect in situ. Dough properties were influenced, confirming early reports, [10,11] and the crumb strength of loaves containing glutaraldehyde increased. No effect on croissant properties was observed. Extraction of the proteins from the treated doughs showed that
J.A. Gerrard, P.K. Brown / International Congress Series 1245 (2002) 211–215
213
Fig. 1. Typical SDS-PAGE results for the albumin and globulin wheat protein fraction incubated with 10 mM glutaraldehyde at 37 jC. From left to right: the frozen control (cf), which was immediately frozen, and the incubated control (ci), which was incubated for 96 h, do not contain glutaraldehyde. The large pore sizes of the stacking gel allow the free migration of all but very large proteins.
Fig. 2. Typical lysine availability results for the gliadin fraction incubated with 1 mM glutaraldehyde. Each point represents the mean of triplicate measurements. Error bars represent the standard error of the mean.
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J.A. Gerrard, P.K. Brown / International Congress Series 1245 (2002) 211–215
the albumin and globulin fraction had been cross-linked, whilst the other fractions were not affected.
4. Discussion The order of cross-linking activity is not surprising, since glutaraldehyde is a dicarbonyl compound with two reactive moieties, whereas direct cross-linking by formaldehyde or glyceraldehyde, with only one carbonyl group per molecule, is less likely. This study has shown that although wheat proteins are amenable to Maillard cross-linking, only the most reactive of the cross-linkers is able to act within this food system—dough— on a timescale sufficient to have an effect on the final product. Glutaraldehyde, although not an appropriate ingredient for food use, provides a useful model for molecules that can be generated during the breakdown of sugars and cross-link proteins via Maillard chemistry. Glutaraldehyde specifically cross-linked only one fraction of wheat proteins—the albumins and globulins. Interestingly, this is in contrast to our earlier work on enzymatic cross-linking, where the enzyme transglutaminase was shown to cross-link two fractions—the albumins and globulins and the high molecular weight glutenins. Glutaraldehyde changed the dough properties and improved the crumb strength of baked loaves, but had no effect on baked croissants. Transglutaminase, on the other hand, changed dough properties, improved crumb strength and dramatically increased croissant volume [3]. These results suggest that the albumins and globulins are specifically responsible for dough properties and crumb strength, whilst croissant volume is influenced by the cross-linking of high molecular weight glutenins. The fact that different crosslinking reagents cause specific changes in food properties augurs well for future research into the controlled manipulation of food texture via protein cross-linking. The Maillard reaction offers a viable method of achieving this.
Acknowledgements This work was funded by the Crop and Food Research as part of contract C02X0001 for the New Economy Research Fund, Foundation for Research Science and Technology, New Zealand. We thank Dr. Siaˆn Fayle for her input into this project and Jackie P. Healy for invaluable technical expertise.
References [1] J.A. Mlotkiewicz, The role of the Maillard reaction in the food industry, in: J. O’Brien, M.J.C. Crabbe, J.M. Ames (Eds.), Maillard Reaction in Foods and Medicine, vol. 223, Royal Society of Chemistry Special Publication, Cambridge, 1998, pp. 19 – 27. [2] S. Hill, A.M. Easa, in: J. O’Brien, M.J.C. Crabbe, J.M. Ames (Eds.), Maillard Reaction in Foods and Medicine, vol. 223, Royal Society of Chemistry Special Publication, Cambridge, 1998, pp. 133 – 138. [3] S.E. Fayle, J.A. Gerrard, L. Simmons, et al., Crosslinkage of proteins by dehydroascorbic acid and its degradation products, Food Chem. 70 (2) (2000) 193 – 198.
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[4] J.A. Gerrard, S.E. Fayle, P.K. Brown, et al., Effects of microbial transglutaminase on the wheat proteins of bread and croissant dough, J. Food Sci. 66 (6) (2001) 782 – 786. [5] J.A. Gerrard, S.E. Fayle, A.J. Wilson, et al., Dough properties and crumb strength of white pan bread as affected by microbial transglutaminase, J. Food Sci. 63 (3) (1998) 472 – 475. [6] J.A. Gerrard, M.P. Newberry, M. Ross, et al., Pastry lift and croissant volume as affected by microbial transglutaminase, J. Food Sci. 65 (2) (2000) 312 – 314. [7] S.E. Fayle, J.P. Healy, P.A. Brown, et al., Novel approaches to the analysis of the Maillard reaction of proteins, Electrophoresis 22 (8) (2001) 1518 – 1525. [8] C. Bertrand-Harb, M.-G. Nicolas, M. Dalgalarrondo, J.-M. Chobert, Determination of alkylation degree by three colorimetric methods and amino acid analysis. A comparative study, Sci. Aliments 13 (3) (1993) 577 – 584. [9] G.T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996. [10] D.H. Simmonds, C.W. Wrigley, P.W. Gras. Treatment of flour to control physical properties of doughs made therefrom, Aust. Patent Appl. PA7902, 1972. [11] C.W. Wrigley, L.W. Lee, P.W. Gras, The mechanical properties of dough in relation to the molecular weight distribution of gluten proteins, Royal Australian Chemical Institute Cereal Chemistry Group Conference (1972) 25 – 32.
Protein cross-linking in food: mechanisms, consequences, applications Juliet A. Gerrard *, Paula K. Brown Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand
Abstract The Maillard reaction influences not only the colour and flavour of foods, but also their texture. One of the mechanisms by which this occurs is via protein cross-linking. The capacities of three molecules—formaldehyde, glyceraldehyde and glutaraldehyde—to cross-link wheat proteins were compared in vitro and in situ. All three molecules cross-linked wheat proteins in vitro, but only glutaraldehyde cross-linked the proteins when added to wheat flour dough. The effect of glutaraldehyde on the dough properties was marked. Upon baking, addition of glutaraldehyde was shown to alter crumb strength and texture of bread, but had no perceivable effect on croissants. A comparison to previous results employing enzymatic cross-linking reveals that cross-linking of specific wheat proteins can be correlated to particular properties of cereal foods. This suggests that the Maillard reaction may be harnessed by food processors to manipulate the texture of foods. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Protein cross-linking; Maillard; Glycation; Cereal foods; Food texture
1. Introduction In his closing address at the Sixth International Symposium on the Maillard Reaction, Mlotkiewicz [1] pointed out that ‘‘in exploiting the Maillard reaction, the key target for industry is to understand and harness the reaction pathways enabling improvement of existing products and the development of new products.’’ Three key properties of food to which consumers respond—colour, flavour and texture—are all influenced by the Maillard reaction, but the last of these, texture—has received the least attention [2]. We are engaged in a study of how the Maillard reaction can be harnessed to manipulate the texture of
*
Corresponding author. Tel.: +64-33667001; fax: +64-33642083. E-mail address: [email protected] (J.A. Gerrard).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 9 1 0 - X
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J.A. Gerrard, P.K. Brown / International Congress Series 1245 (2002) 211–215
foods, particularly cereal foods. Our particular interest is in protein cross-linking, a known consequence of protein glycation, likely to occur during food processing [3]. Previous work has established unequivocally that protein cross-linking can profoundly affect the texture of food, specifically bread and croissants [4 –6]. In this paper, we explore the potential of three molecules known to cross-link proteins—formaldehyde, glyceraldehyde and glutaraldehyde—to alter the texture of cereal foods.
2. Materials and methods Unless otherwise stated, all materials were obtained from Sigma (St. Louis, MO, USA). Ribonuclease was Type XII-A from bovine pancreas. Wheat proteins were fractionated as previously described [4]. Protein cross-linking was monitored by previously published electrophoretic methods [3,7]. Lysine availability was monitored by a modification of the o-phthaldialdehyde method of Bertrand-Harb et al. [8]. Dough, bread and croissant properties were assessed using Crop and Food Research in-house methods [5,6].
3. Results Model studies using ribonuclease as a model protein confirmed the well-established fact that glutaraldehyde cross-links proteins almost instantaneously [9]. The cross-linking was accompanied by a dramatic loss in the availability of all measurable lysine residues in the protein, confirming that the cross-linking mechanism involves reaction of these amino groups. In contrast, both glyceraldehyde and formaldehyde cross-linked at a lower rate, and removed fewer lysine residues. Incubation of ribonuclease with formaldehyde or glyceraldehyde at 37 jC removed only 40% of the available lysines over 5 h, and resulted in substantially fewer cross-links. Wheat proteins were extracted from flour in four fractions, according to their solubility: the albumins and globulins, the gliadins, the soluble glutenins and the insoluble glutenins. Each of these fractions was incubated with each of the cross-linking molecules and the results compared to the model system. Comprehensive experiments were undertaken at a range of concentrations of each cross-linking reagent. A typical cross-linking pattern, for glutaraldehyde cross-linking the albumins and globulins, is shown in Fig. 1. Typical lysine availability results are shown in Fig. 2, for the gliadin fraction cross-linked with a lower concentration of glutaraldehyde. The results corroborated those of the model study, in that glutaraldehyde cross-linked very quickly, with glyceraldehyde and formaldehyde crosslinking more slowly. To establish whether the in vitro results were relevant to actual food systems, the three cross-linking reagents were each added to bread and croissant dough. Only glutaraldehyde, at concentrations of 200 mM and above, was found to have a marked effect in situ. Dough properties were influenced, confirming early reports, [10,11] and the crumb strength of loaves containing glutaraldehyde increased. No effect on croissant properties was observed. Extraction of the proteins from the treated doughs showed that
J.A. Gerrard, P.K. Brown / International Congress Series 1245 (2002) 211–215
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Fig. 1. Typical SDS-PAGE results for the albumin and globulin wheat protein fraction incubated with 10 mM glutaraldehyde at 37 jC. From left to right: the frozen control (cf), which was immediately frozen, and the incubated control (ci), which was incubated for 96 h, do not contain glutaraldehyde. The large pore sizes of the stacking gel allow the free migration of all but very large proteins.
Fig. 2. Typical lysine availability results for the gliadin fraction incubated with 1 mM glutaraldehyde. Each point represents the mean of triplicate measurements. Error bars represent the standard error of the mean.
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J.A. Gerrard, P.K. Brown / International Congress Series 1245 (2002) 211–215
the albumin and globulin fraction had been cross-linked, whilst the other fractions were not affected.
4. Discussion The order of cross-linking activity is not surprising, since glutaraldehyde is a dicarbonyl compound with two reactive moieties, whereas direct cross-linking by formaldehyde or glyceraldehyde, with only one carbonyl group per molecule, is less likely. This study has shown that although wheat proteins are amenable to Maillard cross-linking, only the most reactive of the cross-linkers is able to act within this food system—dough— on a timescale sufficient to have an effect on the final product. Glutaraldehyde, although not an appropriate ingredient for food use, provides a useful model for molecules that can be generated during the breakdown of sugars and cross-link proteins via Maillard chemistry. Glutaraldehyde specifically cross-linked only one fraction of wheat proteins—the albumins and globulins. Interestingly, this is in contrast to our earlier work on enzymatic cross-linking, where the enzyme transglutaminase was shown to cross-link two fractions—the albumins and globulins and the high molecular weight glutenins. Glutaraldehyde changed the dough properties and improved the crumb strength of baked loaves, but had no effect on baked croissants. Transglutaminase, on the other hand, changed dough properties, improved crumb strength and dramatically increased croissant volume [3]. These results suggest that the albumins and globulins are specifically responsible for dough properties and crumb strength, whilst croissant volume is influenced by the cross-linking of high molecular weight glutenins. The fact that different crosslinking reagents cause specific changes in food properties augurs well for future research into the controlled manipulation of food texture via protein cross-linking. The Maillard reaction offers a viable method of achieving this.
Acknowledgements This work was funded by the Crop and Food Research as part of contract C02X0001 for the New Economy Research Fund, Foundation for Research Science and Technology, New Zealand. We thank Dr. Siaˆn Fayle for her input into this project and Jackie P. Healy for invaluable technical expertise.
References [1] J.A. Mlotkiewicz, The role of the Maillard reaction in the food industry, in: J. O’Brien, M.J.C. Crabbe, J.M. Ames (Eds.), Maillard Reaction in Foods and Medicine, vol. 223, Royal Society of Chemistry Special Publication, Cambridge, 1998, pp. 19 – 27. [2] S. Hill, A.M. Easa, in: J. O’Brien, M.J.C. Crabbe, J.M. Ames (Eds.), Maillard Reaction in Foods and Medicine, vol. 223, Royal Society of Chemistry Special Publication, Cambridge, 1998, pp. 133 – 138. [3] S.E. Fayle, J.A. Gerrard, L. Simmons, et al., Crosslinkage of proteins by dehydroascorbic acid and its degradation products, Food Chem. 70 (2) (2000) 193 – 198.
J.A. Gerrard, P.K. Brown / International Congress Series 1245 (2002) 211–215
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[4] J.A. Gerrard, S.E. Fayle, P.K. Brown, et al., Effects of microbial transglutaminase on the wheat proteins of bread and croissant dough, J. Food Sci. 66 (6) (2001) 782 – 786. [5] J.A. Gerrard, S.E. Fayle, A.J. Wilson, et al., Dough properties and crumb strength of white pan bread as affected by microbial transglutaminase, J. Food Sci. 63 (3) (1998) 472 – 475. [6] J.A. Gerrard, M.P. Newberry, M. Ross, et al., Pastry lift and croissant volume as affected by microbial transglutaminase, J. Food Sci. 65 (2) (2000) 312 – 314. [7] S.E. Fayle, J.P. Healy, P.A. Brown, et al., Novel approaches to the analysis of the Maillard reaction of proteins, Electrophoresis 22 (8) (2001) 1518 – 1525. [8] C. Bertrand-Harb, M.-G. Nicolas, M. Dalgalarrondo, J.-M. Chobert, Determination of alkylation degree by three colorimetric methods and amino acid analysis. A comparative study, Sci. Aliments 13 (3) (1993) 577 – 584. [9] G.T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996. [10] D.H. Simmonds, C.W. Wrigley, P.W. Gras. Treatment of flour to control physical properties of doughs made therefrom, Aust. Patent Appl. PA7902, 1972. [11] C.W. Wrigley, L.W. Lee, P.W. Gras, The mechanical properties of dough in relation to the molecular weight distribution of gluten proteins, Royal Australian Chemical Institute Cereal Chemistry Group Conference (1972) 25 – 32.