Effects of Hydrophobic and Ionic Interactions on Glycation of Casein

Article pubs.acs.org/JAFC

Effects of Hydrophobic and Ionic Interactions on Glycation of Casein during Maillard Reaction H. Gül Akıllıoğlu and Vural Gökmen* Department of Food Engineering, Hacettepe University, Beytepe Campus, 06800 Ankara, Turkey ABSTRACT: This study aimed to investigate the effects of hydrophobic and ionic interactions on glycation of native and highshear treated casein during heating. Casein−epicatechin and casein−calcium complexes were formed and glycated with glucose at different temperatures ranging from 70 to 150 °C in solution and dry states. Furosine, acid derivative of N-ε-fructoselysine (FL), and N-ε-carboxymethyl lysine (CML) were measured as indicators of early and advanced glycation, respectively. CML concentrations of casein−epicatechin and casein−calcium complexes heated in solution were significantly lower as compared to the control (p < 0.05). For instance, 182 ± 9.78 μg/g of CML formed in the control, while CML concentrations were 136 ± 10.7 and 101 ± 7.37 μg/g in casein−epicatechin and casein−calcium complexes, respectively, heated at 150 °C in the solution state. Treatment by high shear microfluidization further decreased the CML formed during heating at 70 °C in dry state. The results suggest that interactions with epicatechin molecule and calcium ion could be a useful strategy to limit advanced glycation of casein under certain conditions. KEYWORDS: casein, glycation, interaction, epicatechin, calcium ion

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INTRODUCTION The Maillard reaction, or nonenzymatic browning, is a series of complex reactions including condensation, elimination, and degradation mechanisms. It starts with the condensation between the amino group of an amino acid free or within a protein molecule and a carbonyl group. The so-called Schiff base then goes to an arrangement to form the Amadori product, which is the first stable product of the reaction. During prolonged heating or storage, the Amadori products undergo oxidative modifications (glycoxidations) to form dicarbonyl products. 1,2 Following these, ε-amino group of lysine, guanidino group of arginine, thiol group of cysteine, or any N-terminal amino group of amino acids react with these dicarbonyls to form stable end products. Glycation is the addition of a sugar moiety into a protein molecule, and occurs during Maillard reactions. It is known that glycation takes place both endogenously and exogenously. Endogenous glycation and its end products, advanced glycation end products (AGEs), lead to several consequences in the body. AGEs may contribute to the decline in tissue and organ function with age and are related chronic and degenerative diseases, such as diabetes, renal failure,3 atherosclerosis,4 Alzheimer’s, and Parkinson’s diseases.5 Exogenous glycation occurs during food processing, and severe heat treatment causes advance glycation in food products. Glycation during food processing leads to impairment of the nutritional quality of food, generation of toxic compounds such as HMF, acrylamide, and formation of AGEs. Dietary intake of exogenous AGEs may cause their accumulation in the bloodstream and in tissue proteins,6 then undergoing further reactions in the body.7 Thus, it is important to limit glycation of proteins during processing to reduce dietary AGEs. So far, several glycation products such as N-ε-fructoselysine (FL), pyrraline, pentosidine, N-ε-carboxymethyl-lysine (CML), N-ε-carboxyethyl-lysine (CEL), S-carboxymethyl cysteine, © 2014 American Chemical Society

glyoxal lysine dimer (GOLD), methylglyoxal lysine dimer (MOLD), and 3-deoxyglucosone lysine dimer (DOLD) have been identified in processed foods.2,8−10 Because of the ease of their analysis, some of these products are considered as indicators of glycation. CML, which is a marker of advanced glycation, can be formed through various pathways, such as by condensation of glucose with the ε-amino group of lysine, where the Amadori product FL is produced as an unstable intermediate and subsequently undergoes oxidation to form CML9 (Figure 1). Another pathway is the reaction of glyoxal directly with the ε-amino group in lysine.11 Furosine, which is a derivative of N-ε-fructoselysine, is widely used to assess the extent of early Maillard reaction, especially in dairy products.2 Casein is the major protein in milk, almost accounting for 80% of total milk protein. It is composed of four subunits, αs1casein, αs2-casein, β-casein, and κ-casein. αs1-, αs2-, β-caseins are highly phosphorylated, and together with κ-casein and calcium phosphate they form colloidal protein particles, called casein micelles.12 Because of its nutritional and functional features, casein is widely used as an ingredient in food products.13 Because it is rich in lysine, casein is glycated easily upon heating even at low temperatures.13,14 Few mechanisms have been proposed to inhibit glycation of proteins. These include blocking sugar attachment to amine groups of proteins, attenuating glycoxidation or oxidative stress through trapping or scavenging the intermediates such as dicarbonyls or free radicals, or breaking down formed AGE cross-links.15,16 This study aimed to investigate the effects of hydrophobic and ionic interactions of casein to limit its glycation during heating. Casein, native or high-shear treated, Received: Revised: Accepted: Published: 11289

August 12, 2014 October 27, 2014 November 3, 2014 November 3, 2014 dx.doi.org/10.1021/jf5038954 | J. Agric. Food Chem. 2014, 62, 11289−11295

Journal of Agricultural and Food Chemistry

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Figure 1. Pathways of CML formation (adapted from Han et al.30). bath and heated at 70, 90, 110, 130, and 150 °C for 10 min. A set of samples was also heated at 70 °C for 6 h. To see the effect of protein conformation on glycation potential of casein, high shear microfluidization was applied to cause physical modification of the casein micelles. The casein suspension (5%) was treated under 30 000 psi for 5 min using a high-shear microfluidizer device (M110P, Microfluidics, Newton, MA). High-shear treated casein was dried by freeze-drying and kept at −20 °C. It was used to prepare the mixtures of high-shear treated casein−glucose−calcium (hsCN-G-Ca) and high-shear treated casein−glucose−epicatechin (hsCN-G-EC) complexes as described above. In this case, the control (hsCN-G) was a mixture of 5% high-shear treated casein and 5% glucose. The mixtures were glycated in dry state after freeze-drying by heating at 70 °C for 6 h. The concentrations of FL and CML were determined as early and advanced glycation products in the heated mixtures. To see the effect of storage on the concentrations of FL and CML concentrations, the samples were incubated at 37 °C for 7 days after heat treatment. Analysis of Furosine by HPLC. Analysis of furosine was performed according to Gökmen et al.17 30 mg of sample was weighed into a glass tube, and 10 mL of hydrochloric acid (8 N) was added. The tubes were flushed with nitrogen and tightly closed. They were hydrolyzed for 23 h at 110 °C. The hydrolyzates were filtered through filter paper, and then 100 μL was put into a glass vial. It was evaporated to dryness under nitrogen gas. The content then was washed with 1 mL of deionized water and passed through OASIS HLB cartridges. Ten microliters was injected into the HPLC system. Chromatographic separation was performed on an Atlantis HILIC column (250 × 4.6 mm, 5 μm) at 40 °C with a mobile phase of 1% formic acid at a flow rate of 1 mL/min. Data acquisition was performed acquiring chromatograms at the detection wavelength of 280 nm. Quantification was based on the calibration curve prepared using calibration solutions of furosine (1−10 mg/L), and was expressed as mg/g protein. Analysis of CML by LC−MS/MS. Analysis of CML was performed according to Palermo et al.18 with some modifications. A total of 20 mg of sample was mixed with 100 μL of deionized water in a glass tube. 450 μL of sodium borate buffer (0.2 M, pH 9.2) and 500

was interacted with calcium chloride and epicatechin to form casein−calcium and casein−epicatechin complexes. These complexes were heated in the presence of glucose at temperatures ranging from 70 to 150 °C, covering food processing conditions. FL and CML were determined in the heated mixtures as early and advanced glycation products, respectively.

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MATERIALS AND METHODS

Chemicals. Casein from bovine milk, glucose, (−)-epicatechin, sodium borohydride (Fluka), and CML standard were purchased from Sigma-Aldrich Chemie Gmbh (Germany), whereas calcium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, and boric acid were from Merck (Germany). Furosine standard was purchased from Neosystem Laboratoire (Strasbourg, France). Hydrochloric acid and formic acid were also purchased from Sigma-Aldrich (Germany) and were of HPLC grade. Consumables. Syringe filters (nylon, 0.45 μm), Oasis HLB (1 mL, 30 mg) solid-phase extraction cartridges, Acquity UPLC HSS T3 column (4.6 × 150 mm, 3 μm), and Atlantis dC18 column (250 × 4.6 mm, 5 μm) were supplied by Waters (Millford, MA). Preparation of Casein Complexes. Casein was suspended in 50 mM phosphate buffer (pH 6.8) at a concentration of 5%. The suspension was continuously stirred for 20 min using a magnetic stirrer. Next, 0.1% of calcium chloride was added to promote ionic interaction between casein molecules and calcium ions, or 0.1% of epicatechin was added to promote hydrophobic interaction between casein and epicatechin molecules. The contents were continuously stirred for 20 min to maintain the casein−calcium or casein− epicatechin complexes. Afterward, 5% of glucose was added onto these complexes and stirred for further 20 min to obtain final mixtures of casein−glucose−calcium (CN-G-Ca) and casein−glucose−epicatechin (CN-G-EC). The control (CN-G) was a mixture of 5% casein and 5% glucose. Finally, the mixtures were glycated in solution state or in dry state after freeze-drying. The glycation reactions were performed in tightly closed test tubes containing the mixtures (in solution or dry states) prepared as described above. The tubes were placed in an oil 11290

dx.doi.org/10.1021/jf5038954 | J. Agric. Food Chem. 2014, 62, 11289−11295


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μL of sodium borohydride (1 M solution prepared in 0.1 M NaOH) were added. They were incubated at room temperature for 4 h to reduce fructoselysine into hexitol lysine. Afterward, 2 mL of hydrochloric acid (8 N) was added, and the tubes were closed under nitrogen flush. They were hydrolyzed for 24 h at 110 °C. Twenty microliters of hydrolyzate was evaporated with nitrogen. After the content was washed with 1 mL of deionized water, it was passed through a preconditioned Oasis HLB cartridge for cleanup. The first 8 drops of the eluent were discarded, and the rest was collected and diluted properly prior to LC−MS/MS analysis. The sample was injected into an Acquity UPLC HSS T3 column (4.6 × 150 mm, 3 μm) at 40 °C coupled to a Waters LC−MS/MS system operated in positive ionization mode using the following interface parameters: source temperature of 120 °C, desolvation temperature of 450 °C, collision gas flow of 0.20 mL/min, desolvation gas flow of 900 L/min, capillary voltage of 3 kV, cone voltage of 25 V, and extractor voltage of 2 V. Chromatographic separation was performed by using a mobile phase consisting of 0.1% formic acid in water:0.1% formic acid in acetonitrile (90:10, v/v) at a flow rate of 0.50 mL/min. Acquisition was performed by monitoring m/z ratio of 205.10 for CML, 84.10 and 130.10 for its product ions. Quantification was performed by means of a matrix-matched calibration curve. Hydrolyzate of unheated protein was used as blank matrix. Calibration solutions of CML were prepared in the blank matrix at concentrations of 0, 1, 2.5, 5.0, and 10 μg/mL. They then were subjected to the lengthy extraction procedure that was used for actual samples, and analyzed as described above. Statistical Analysis. The samples were analyzed at least in triplicate. Statistical analyses were performed by using IBM SPSS Statistics 19. Differences between mean values of samples were evaluated by analysis of variance (ANOVA), and the Duncan test was applied to determine significant differences at p < 0.05 level. Determination of significant differences between samples before and after incubation was performed with independent samples t test.

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Figure 2. Effects of complex formation on furosine concentration of samples heated at different temperatures in (a) solution state, and (b) dry state. Bars with different letters indicate significant difference for control (CN-G) at p < 0.05 level. Bars with an asterisk indicate significant differences of complexes from control (CN-G) within the same temperature (p < 0.05).

RESULTS AND DISCUSSION Effects of Complex Formation on Glycation of Casein. Exogenous glycation of proteins that takes place during food processing may occur in a wide range of temperatures. In this study, glycation behavior of casein was investigated by forming hydrophobic or ionic interactions prior to heating. In the presence of glucose, casein−calcium and casein−epicatechin complexes were heated at temperatures of 70, 90, 110, 130, and 150 °C for 10 min to monitor glycation under aqueous and dry conditions. Furosine is a good marker of early glycation especially for monitoring modification of proteins rich in lysine like casein. Figure 2 shows furosine concentrations of casein complexes heated at different temperatures in solution and dry state conditions. In solution state, furosine concentrations of all samples gradually increased as the temperature increased from 70 to 130 °C during heating. Further increase of temperature from 130 to 150 °C did not cause a remarkable increase in furosine concentrations. There were statistically significant differences (p < 0.05) in furosine concentrations of the samples heated at 70, 90, 110, and 130 °C. In comparison to the control, there were no significant differences (p > 0.05) in furosine levels for casein complexes formed with calcium and epicatechin at all temperatures studied except 130 and 150 °C. The effect of temperature on furosine concentrations was different for the samples heated in dry state than in solution state. Furosine levels increased when the temperature was raised from 70 to 110 °C. With an exception for 150 °C, furosine concentrations of the samples glycated in dry state were higher than those of the samples glycated in solution state at all temperatures studied. This indicates a higher glycation potential of casein in dry conditions. It is known that the first

stage of the glycation reaction is a condensation between the amino group of casein and the carbonyl group of glucose, yielding one molecule of water. Hence, the presence of water in the medium might be suppressing the reaction. The peak levels of furosine were observed for the samples heated at 110 °C for 10 min in dry state that were significantly higher (p < 0.05) than the levels observed for the samples heated in solution state. Increasing the temperature in the dry state from 110 to 130 and to 150 °C caused a gradual decrease in furosine levels. It is a fact that furosine, an acid derivative of FL, is the indicator of early glycation. As an intermediate compound, FL can be converted faster to advanced glycation products at higher temperatures. So, the furosine concentration decreases as glycation progresses. FL is considered as the main precursor of CML.19 It was shown that decomposition of FL leads to formation of CML.20 Similar to the results observed in solution state, there were no significant differences (p > 0.05) in furosine levels for casein complexes formed with calcium and epicatechin. Figure 3 shows CML concentrations of casein complexes heated at different temperatures in solution and dry state conditions. In solution state, there was a gradual increase in CML concentrations of the samples as the temperature increased from 70 to 150 °C (Figure 3a). For the control (CN-G), the CML level increased by a factor of 114 when the temperature was increased from 70 to 150 °C during heating. However, this increase was limited to 39 and 60 times for 11291



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therefore have inhibited advanced glycation of casein. In our experiments, epicatechin did not cause a significant reduction in early glycation as stated earlier; however, the CML level significantly decreased (p < 0.05). There is another potential mechanism for epicatechin to limit glycation; Yin et al.25 reported that epicatechin inhibited formation of intermediary radicals during the Maillard reaction. It was explained that autoxidation of epicatechin yielded formation of quinones that reacted with lysine through either a Michael-type addition or a Strecker-like reaction. There is additional evidence that supports such a reaction happening at 110 °C in semi dry mixtures; the reaction of glycine and the oxidized form of catechin leads to the formation of single- and double-addition products.26 However, that kind of mechanism is favorable at alkaline conditions in which polyphenols may readily oxidize to respective quinones.27 Therefore, this mechanism should occur to a limited extent at the conditions applied in this study. In dry state, there was a remarkable increase in the amounts of CML formed in the samples when the temperature was increased from 70 to 90 °C (Figure 3b). In general, the CML levels were comparable to each other for all samples heated at 90, 110, 130, and 150 °C. In detail, the casein−epicatechin complex had significantly lower amounts of CML during heating at 90, 110, and 130 °C (p < 0.05). The casein−calcium complex had a lower amount of CML during heating only at 130 °C (p < 0.05). It is thought that the thermal impact suppressed molecular interactions because the treatment was harsh during heating casein complexes in dry state. Calcium ion accelerates dehydration of sugar molecules as do all mono-, di-, and trivalent cations.28 Dehydration of glucose causes ian ncrease in the formation of CML through the glyoxal pathway, as explained in Figure 1. This is the most probable reason for higher amounts of CML found in heated casein−calcium complexes in dry state. Effects of Protein Conformation on Glycation of Casein. In the second part, the complexes were formed using high-shear treated casein to understand the effects of physical modifications on the glycation tendency of casein. High-shear application was performed by treatment of casein suspension using a microfluidizer at 30 000 psi for 5 min. As mentioned before, formation of casein complexes with calcium and epicatechin before heat treatment did not cause a change in the formation of early glycation products measured as furosine in the samples (Figure 4). However, heating the complexes prepared from high-shear treated casein formed lower amounts of furosine than the complexes prepared from native casein (p < 0.05). This indicated that physical modification has an effect on complex formation, and therefore on protein glycation. Among others, high-shear treated casein− epicatechin complex (hsCN-G-EC) had the lowest amount of furosine (p < 0.05). It is thought that more hydrophobic and ionic interaction sites might become available for epicatechin and calcium to bind when casein micelle was dissociated. This consequence could have yielded a decrease in glycation reaction. Altuner et al.29 showed that high hydrostatic pressure affected noncovalent interactions and especially hydrophobic bonds in milk. It was reported that as the pressure applied increased, the casein micelles decomposed into submicelles and the embedded hydrophobic areas inside the micelles repositioned in such a way that they readily interfered with the fluorescent marker, 1,8-naphthaleresulfonic acid (ANS).

Figure 3. Effects of complex formation on CML concentration of samples heated at different temperatures in (a) solution state, and (b) dry state. Bars with different letters indicate significant difference for control (CN-G) at p < 0.05 level. Bars with an asterisk indicate significant differences of complexes from control (CN-G) within the same temperature (p < 0.05).

casein complexes formed with calcium and epicatechin, respectively. This revealed that formation of casein−calcium and casein−epicatechin complexes before heat treatment has a potential to limit the formation of advanced glycation end products during heating under certain conditions. At 130 and 150 °C, the amounts of CML formed during heating in the samples of casein complexes (CN-G-Ca and CN-G-EC) were found significantly lower (p < 0.05) than those formed in the control (CN-G). It is known that calcium ions act as a crosslinker forming bridges between casein micelles.21 Formation of the casein−calcium complex through ionic interaction makes it difficult for glucose to bind amine groups of casein. On the other hand, there are different possible mechanisms for epicatechin to limit glycation. Protein−phenol interactions are known so far by taking place between hydrophobic residues of protein and polyphenols, mainly flavonoids. Noncovalent interaction between protein molecules and polyphenols results in bridges that make protein molecules hold together.22 Hence, the first mechanism of epicatechin is to form a complex with casein, which is sterically hindered for glucose molecules. Because polyphenols are known to have carbonyl-trapping activity,23,24 the second mechanism of epicatechin might be trapping glucose derived dicarbonyl compounds like glyoxal. CML may be formed from FL or from the reaction between glyoxal and lysine residues of protein (Figure 1). Hence, the decreased CML level besides the undiminished FL level indicates that epicatechin may have trapped glyoxal, and 11292



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conflicting reports about epicatechin; some reported an antiglycation effect,23−26,31 while others reported an increased glycation.32,33 Epicatechin was found to be an effective agent for the inhibition of thermally developed Maillard reaction products and reduced the cooked flavor in UHT milk.31 Some reported antiglycation effect of epicatechin by carbonyl trapping,23,24 while others explained the mechanism through quinone formation reacting with amines25,26 as mentioned above. Also, Zhang et al.34 reported that polyphenols reduce glyoxal generation by inhibiting lipid peroxidation. However, in a recent review, it was stated that epicatechin could both inhibit and enhance glycation.33 It was explained by Fujiwara et al.32 that catechol group containing polyphenols generates hydrogen peroxide during autoxidation of catechol structure, then hydrogen peroxide may generate hydroxyl radicals, and finally CML is generated by the reaction of hydroxyl radicals and Amadori products. So, the increased CML level of the complex prepared from the native casein in this study could be explained by the oxidation triggering effect of epicatechin. However, the CML level could be decreased if the complex was prepared using high-shear treated casein (Figure 5). It is thought that epicatechin can bind to casein structure more easily when it is treated using high-shear microfluidizer because the hidden hydrophobic sites within intact protein are exposed to interaction. Epicatechin has the ability to make weak hydrophobic interactions through hydrophobic amino acids of protein such as proline, tryptophan, tyrosine, and phenylalanine.35 So far, several methods have been proposed to inhibit protein glycation during food processing. The main thought is the blockage of carbonyl attachment to proteins, preventing oxidative stress or breaking down formed AGE cross-links. As exemplified for casein, this study proposes ionic and weak hydrophobic interactions as a strategy to limit advanced glycation of proteins during heating in a wide range of temperatures in both solution and dry states. It is certain that epicatechin or calcium ions do not block free amine groups of proteins, but they form steric hindrance that makes it difficult for carbonyl compounds to bind to amine groups on protein molecule. Casein micelle has a hydrophobic core and a hydrophilic surface. High shear treatment of casein allows more interaction sites, causing exposure of embedded hydrophobic regions within the intact molecule. So if the hydrophobic complex is to be formed to reduce glycation, physical modification of protein prior to complexation seems advantageous. Under the stated conditions, antiglycation effects of epicatechin molecules and calcium ions as hydrophobic and ionic interaction agents are remarkable in the advanced state. However, these interactions do not make significant differences in the early stage of glycation. In conclusion, calcium ions and epicatechin molecules may inhibit the glycation of casein during heating under aqueous and dry conditions as follows: • In aqueous solution state, calcium ions act as crosslinking agent forming bridges between casein micelles that make it difficult for carbonyl compounds to bind to glycation sites on protein. • In dry state, calcium ions accelerate dehydration of glucose that may enhance advanced glycation of casein through the formation of glyoxal at high temperatures. • Epicatechin forms a complex with casein through a wellknown protein−phenolic interaction phenomenon that

Figure 4. Furosine levels of native and high-shear treated casein complexes heated at 70 °C for 6 h. Bars with different letters indicate significant difference between complexes at p < 0.05 level. Bars with an asterisk indicate significant differences between samples before and after incubation (p < 0.05).

To see the progression of glycation during storage, the heated mixtures of casein complexes were incubated at 37 °C for 7 days. As shown in Figure 4, furosine concentrations decreased slightly during incubation. This is not surprising because FL being an intermediate compound formed in the Maillard reaction would decrease as glycation further developed under the stated conditions. Ahmed et al.20 reported that FL content decreased during 15 days of reaction at 37 °C. Also, Han et al.30 reported a decrease in FL and its simultaneous conversion to CML in the lysine−glucose−lipid model system during 4 h of incubation at 100 °C. Figure 5 shows the amounts of CML formed in the mixtures of native and high-shear treated casein complexes during

Figure 5. CML levels of native and high-shear treated casein complexes heated at 70 °C for 6 h. Bars with different letters indicate significant difference between complexes at p < 0.05 level. Bars with an asterisk indicate significant differences between samples before and after incubation (p < 0.05).

heating at 70 °C for 6 h. Interestingly, the presence of calcium ions showed a distinct antiglycation effect on native casein in terms of the formation of CML as the indicator of advanced glycation, whereas epicatechin caused an increase in CML level during heating. During incubation at 37 °C for 7 days, CML levels increased in all samples with an exception for the heated mixture of casein−epicatechin complex (CN-G-EC). It is a fact that calcium ions act as a cross-linking agent, forming bridges between casein micelles.21 The results of the present study bring evidence that forming casein−calcium complex makes it difficult for glucose to bind amine groups of casein. There are 11293



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microscopy and fluorescent polyphenols. Food Hydrocolloids 2014, DOI: 10.1016/j.foodhyd.2014.03.022. (13) Jindal, S.; Naeem, A. Consequential secondary structure alterations and aggregation during prolonged casein glycation. J. Fluoresc. 2013, 23, 367−374. (14) Jing, H.; Kitts, D. D. Chemical and biochemical properties of casein−sugar Maillard reaction products. Food Chem. Toxicol. 2002, 40, 1007−1015. (15) Peng, X.; Ma, J.; Chen, F.; Wang, M. Naturally occurring inhibitors against the formation of advanced glycation end-products. Food Funct. 2011, 2, 289−301. (16) Mesías, M.; Navarro, M.; Gö kmen, V.; Morales, F. J. Antiglycative effect of fruit and vegetable seed extracts: inhibition of AGE formation and carbonyl-trapping abilities. J. Sci. Food Agric. 2013, 93, 2037−2044. (17) Gökmen, V.; Serpen, A.; Morales, F. J. Determination of furosine in thermally processed foods by hydrophilic interaction liquid chromatography. J. AOAC Int. 2009, 92, 1460−1463. (18) Palermo, M.; Fiore, A.; Fogliano, V. Okara promoted acrylamide and carboxymethyl-lysine formation in bakery products. J. Agric. Food Chem. 2012, 60, 10141−10146. (19) Lima, M.; Assar, S. H.; Ames, J. M. Formation of Nε(carboxymethyl)lysine and loss of lysine in casein glucose-fatty acid model systems. J. Agric. Food Chem. 2010, 58, 1954−1958. (20) Ahmed, M. U.; Thorpe, S. R.; Baynes, J. W. Identification of Nεcarboxymethyllysine as a degradation product of fructoselysine in glycated protein. J. Biol. Chem. 1986, 261, 4889−4894. (21) Nair, P. K. Colloidal behaviour of casein micelles with concentration. Ph.D. Thesis, Canada, 2012; pp 7−12; https:// atrium.lib.uoguelph.ca/xmlui/handle/10214/3997. (22) Siebert, K. J.; Troukhanova, N. V.; Lynn, P. Y. Nature of polyphenol-protein interactions. J. Agric. Food Chem. 1996, 44, 80−85. (23) Totlani, V. M.; Peterson, D. G. Influence of epicatechin reactions on the mechanisms of Maillard product formation in low moisture model systems. J. Agric. Food Chem. 2007, 55, 414−420. (24) Sang, S.; Shao, X.; Bai, N.; Lo, C. Y.; Yang, C. S.; Ho, C. T. Tea polyphenol (−)- epigallocatechin-3-gallate: A new trapping agent of reactive dicarbonyl species. Chem. Res. Toxicol. 2007, 20, 1862−1870. (25) Yin, J.; Hedegaard, R. V.; Skibsted, L. H.; Andersen, M. L. Epicatechin and epigallocatechin gallate inhibit formation of intermediary radicals during heating of lysine and glucose. Food Chem. 2014, 146, 48−55. (26) Guerra, P. V.; Yaylayan, V. A. Interaction of flavanols with amino acids: Postoxidative reactivity of the B-ring of catechin with glycine. J. Agric. Food Chem. 2014, 62, 3831−3836. (27) Rawel, H. M.; Kroll, J.; Rohn, S. Reactions of phenolic substances with lysozyme-physicochemical characterisation and proteolytic digestion of the derivatives. Food Chem. 2001, 72, 59−71. (28) Gökmen, V.; Şenyuva, H. Z. Effects of some cations on the formation of acrylamide and furfurals in glucose-asparagine model system. Eur. Food Res. Technol. 2007, 225, 815−820. (29) Altuner, E. M.; Alpas, H.; Erdem, Y. K.; Bozoglu, F. Effect of high hydrostatic pressure on physicochemical and biochemical properties of milk. Eur. Food Res. Technol. 2006, 222, 392−396. (30) Han, L.; Li, L.; Li, B.; Zhao, D.; Li, Y.; Xu, Z.; Liu, G. Hydroxyl radical induced by lipid in Maillard reaction model system promotes diet-derived Ne-carboxymethyllysine formation. Food Chem. Toxicol. 2013, 60, 536−541. (31) Colahan-Sederstrom, P. M.; Peterson, D. G. Inhibition of key aroma compound generated during ultrahigh-temperature processing of bovine milk via epicatechin addition. J. Agric. Food Chem. 2005, 53, 398−402. (32) Fujiwara, Y.; Kiyota, N.; Tsurushima, K.; Yoshitomi, M.; Mera, K.; Sakashita, N.; Takeya, M.; Ikeda, T.; Araki, T.; Nohara, T.; Nagai, R. Natural compounds containing a catechol group enhance the formation of Nε-(carboxymethyl)lysine of the Maillard reaction. Free Radical Biol. Med. 2011, 50, 883−891.

may cause reduction in the accessibility of glycation sites on protein. • Epicatechin may limit early glycation of protein through the formation of quinones that further react with amine residues on protein. However, this effect is more favorable under strong alkaline and oxidizing conditions. • Epicatechin may limit advanced glycation by trapping glyoxal formed from the degradation of glucose. The reduced CML concentrations despite undiminished furosine levels indicate that mechanism is dominating in our experimental conditions. The above-mentioned effects of calcium and epicatechin observed in the model system can be useful to prevent casein glycation in actual food systems such as pasteurized or sterilized milk and spray dried milk powder. Inhibition of Maillard reaction during milk processing not only improves the nutritional value of milk products, but also brings some sensorial advantages such as prevention of off-flavor. However, it should be kept in mind that the ingredient profile has substantial effects on advanced glycation.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +90-312-2977108. Fax: +90-312-2992123. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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Effects of Hydrophobic and Ionic Interactions on Glycation of Casein

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