Heat-induced changes in some technological properties of whey proteins concentrate

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1 Available online at Journal of Agroalimentary Processes and Technologies 2010, 16 (2), Journal of Agroalimentary Processes and Technologies Heat-induced changes in some technological properties of whey proteins concentrate Loredana Dumitraşcu *, Nicoleta Stănciuc *, Alina Ardelean, Silvius Stanciu, Gabriela Râpeanu Dunărea de Jos University of Galaţi, Faculty of Food Science and Engineering, 111 Domneasca Street, , Galati, Romania Received: 10 May 2010; Accepted: 15 June 2010 Abstract Whey protein concentrate (WPC) is used as food ingredients due to their commercially important functional properties. The effects of heat treatment on the components of milk are very important for the final product character, since they undergo modifications that affect sensory and nutritional quality of milk. As foodstuffs they are applied not only because of their functional properties, but also because of their high nutritive value. Improvements in functional properties may be achieved by modifying the protein structure by chemical, enzymatic or physical treatments. Functional properties of whey proteins such as solubility, viscosity, water-holding capacity, gelation, adhesion, emulsification and foaming are affected by their structure and mainly reflect the functionality of β-lactoglobulin as the most abundant protein. The objective of this work was to evaluate the effect of heat treatment on dispersions of whey proteins concentrate on some technological properties. The heat induced changes were monitored by measurement of thiol availability, protein solubility and turbidity at neutral ph (7.5). Keywords: Whey proteins, heat-treatment, technological properties 1. Introduction Whey protein concentrate (WPC) are used as food ingredients due to their commercially important functional properties such as solubility, viscosity, water-holding capacity, gelation, adhesion, emulsification and foaming [7]. As ingredients in food, whey proteins are used also for of their high nutritive value, reasonable cost and GRAS status. Generally, the functional properties of food proteins may be classified into three main groups: (a) hydration properties, dependent upon proteinwater interactions which have an important bearing on wetability, swelling, adhesion, dispersibility, solubility, viscosity, water absorption and water holding; (b) interfacial properties including surface tension, emulsification and foaming characteristics; and (c) aggregation and gelation properties, which are related to protein-protein interactions [9]. Heat-treatment of can cause the unfolding and denaturation of the whey proteins, followed by aggregation through hydrophobic interaction and disulphide-thiol interchanges to form heat-induced aggregates. These heat-induced changes in proteins structure can affect the technological properties of whey proteins such as emulsification, foaming and gelation as they are mainly determined by the structure of β-lactoglobulin. The objective of this work was to evaluate the effect of heat treatment on dispersions of whey proteins at a concentration which is close to but insufficient for gelation, on the some technological properties. Corresponding author: nsava@ugal.ro; loredana.dumitrascu@ugal.ro

2 Functionality was limited to solubility, turbidity and thiol availability. We suppose that this research could encourage attempts to use these ingredients in a wide range of formulated food products to fully replace traditional additives such as milk powder or skim milk. 2. Materials and methods WPC was purchased from KUK Romania. The chemicals 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB), sodium dodecylsulfate (SDS) were obtained from Sigma Chemicals (St. Louis, MO, USA). All solvents and chemical reagents were of analytical grade. Isothermal Treatment of WPC solutions. WPC solutions (10 ml of 5 mg/ml in distillated water, ph 7.5) were heated in glass tubes in a thermostatically controlled water bath at constant temperatures between 70 and 85 C for 0 to 40 min. After heat treatment, samples were immediately transferred to ice-cold water to prevent further denaturation. The samples were stored at 4 0 C over night. The samples of (un)treated WPC solutions were centrifuged for 15 min (Eppendorf centrifuge, Eppendorf AG) at g for 20 min after lowering the ph to 4.6 ± 0.1 using 0.1 and 1 N of HCl. Turbidity. Turbidity was determined spectrophotometrically after diluting the samples (100 µl to 2.5 ml 0.02 M Tris-HCl buffer, ph 6.6) at a wavelength of 600 nm and 20 C. One hundred percent turbidity was defined as 0% transmission of light. Solubility. The soluble protein (SP) content was determined after isoelectric precipitation of denatured /aggregated whey protein [10]. The SP content in the supernatants was determined by measuring absorption at 280 nm after appropriate dilution in a dissociating buffer (EDTA 50 mm, urea 8 M, ph 10) using a Cintra spectrophotometer (GBC Scientific Equipment). Insoluble protein content of suspensions at ph 4.6 was defined as the difference between TP (total protein) and SP contents and was used to estimate the extent of denaturation/aggregation of whey proteins. The percentage of DP content was calculated with the following equation 1: TP SP DP = 100(%) (1) TP Thiol availability. The exposed, total and slow reacting SH groups were determined as described by Sava et al. (2005) [12]. The (un) heated samples (50 µl) were diluted with appropriate buffer solution (standard solution at ph 8.0 for exposed SH groups, standard solution with 8 M uree, ph 8.0 for total SH groups and standard solution with 8 M urea and 0,5% SDS for slow reacting groups). DTNB reagent (10 µl) was added to the (un)treated samples. The absorbance at 412 nm was measured against a reagent blank after 2 min (total SH groups) or 15 min (surface SH groups) at 20 C. The SH groups (µmol/g protein) were calculated with the equation 2: ( ) D µmol SH/g protein = A1 A0 (2) C13600 where A 1 is the absorbance of (un)heated sample at 412 nm, A 0 is the absorbance of the martor, d the dilution factor, the molar extinction coefficient, and c the protein concentration (mg/ml). For the slow reacting SH groups, the absorbance at 412 nm was recorded during 5 minutes of reaction at room temperature. The amount of free SH groups was calculated at each time. The slow reacting SH groups can be obtained using the following pseudofirst-order equation (equation 3): ln [ SHt SH r ] = kobs t + ln[ SH s ] (3) where SH t is the total SH groups content obtained from the maximum absorbance value, SH r is the content of SH groups having reacted at time t, k obs is the reaction rate constant for the reaction between DTNB and SH 121, and SH s is the content in slow reacting SH groups [13]. 3. Results and discussion Solubility is an important characteristic for functional application of proteins in food systems, not only per se but also as a prerequisite for derived functional properties like emulsification, foaming, and gelation [4]. The commonly used heat treatments such as pasteurization and sterilization always affect the structure and properties of whey proteins, either reversibly or irreversibly. 131

3 The solubility at control and heat-treated samples was determined by measuring the protein concentration in the supernatant after centrifugation, and it should be considered a reliable predictor of functionality. The main components of the whey protein fraction of bovine milk are β-lactoglobulin, α-lactalbumin and bovine serum albumin (BSA). During heat treatment, the denaturation of whey protein concentrate is mainly determined by the parameters as temperature and holding time. Figure 1 shows the denaturation degree of the protein at neutral ph. Treatment of whey proteins concentrate at temperature over 80 0 C leads to a progressive protein denaturation, estimated by the loss of solubility at ph 4.6. The maximum extent of denaturation was reached at 85 0 C after 40 minutes, when the proteins aggregate and the solubility decreased with almost 77%. In the temperature range of C, the denaturation degree varied from 5 to 30%, with a minimal increase in turbidity (Figure 2). Figure 1. The denaturated protein percent content as a function of heating time and temperature Figure 2. The increase in turbidity during heat treatments of WPC solutions at neutral ph The denaturation curves measured by the changes in the exposure degree of SH groups are given in Figure 3. Heat treatment induced significant changes in the extent of denaturation, with a decrease between 30% at 70 0 C to 100% at 80 0 C after 40 minutes of holding. Figure 3. Denaturation curves of whey protein concentrate expressed as degree of SH groups exposure The first order kinetic model was used to define the heat-induced changes on the degree of SH exposure. The temperature dependence of the rate constant, k (min -1 ) was described by the Arrhenius equation 4 (figure 4): k = k ref E a 1 1 exp (4) R T Tref with T and T ref the absolute temperature (K) and the reference temperature (K), respectively; k ref the rate constant at T ref, E a the activation energy (kjmol -1 ), R the universal gas constant (8.314 Jmol -1 K -1 ). Kinetic parameters were estimated by nonlinear regression analysis. The activation energy had been the important and widely used parameters for predicting the effect of temperature on any specific reaction. By monitoring the kinetics of SH group s exposure in protein solutions, it was observed that the rate constant had different values in the temperature range of C. The rate constants k for this model are given in table 1. The temperature dependence of k between 70 and 85 C could accurately be described by the Arrhenius equation (r 2 = 0.99), resulting in an activation energy of 111.3±2.7 kj.mol

Table 1. Kinetic parameters k and E a of the first order model describing heat-induced changes in exposed SH groups Temperature k (min -1 ) ( C) 70 0.0333±0.012 a 75 0.0617±0.016 Figure 4.

4 Table 1. Kinetic parameters k and E a of the first order model describing heat-induced changes in exposed SH groups Temperature k (min -1 ) ( C) ±0.012 a ±0.016 Figure 4. The temperature dependence of the rate constant (k, min -1 ) describing changes in SH ex at ph 7.5 Dannenberg and Kessler (1988) determined, for the heat-induced denaturation of β-lactoglobulin A and B in milk, an activation energy of about 270 kj mol -1 in the temperature range C and of about 50 kj mol -1 above 90 0 C [3]. According to the well-known model of thermal denaturation of β- lactoglobulin in milk and solutions of WPC, denaturation reactions are limited by unfolding in the lower temperature range and by aggregation in the upper temperature range. The estimated activation energies for WPC denaturation shown in Table 1 lead to the conclusion that aggregation is the limiting step for the denaturation of whey proteins in simple medium in the temperature range C. Heating treatment weakens the structure of protein molecules by weakening stabilizing non-covalent bonds; this facilitates the first step of the denaturation reaction, unfolding of whey proteins which promote the aggregation. It can be observed from table 1 that the rate constant is 5 times higher at 85 0 C when compared with 70 0 C. De Witt (2009) suggested that the unfolding of β- lactoglobulin is considered to be a first-order reaction, which may involve a number of consecutive conformational changes in the molecule [5]. The denaturation temperature varies with the ph and is about 70 0 C at ph 7.0. The present value for Ea is consistent with the Ea range for β-lactoglobulin denaturation (148.2 kj/mol) in the temperature range of 67.5 to 82.5 C as reported by Sava et al., (2005), but it is much lower than the Ea value reported by Galani and Apenten (1997) for thermal denaturation of β-lactoglobulin in Tris-HCl buffer in the temperature range of 70 to 95 0 C ( kj/mol) [6, 12]. The calculated value for Ea is characteristic for chemical reactions in complex media, as reported by Walstra and Jenness (1984) [14]. Anema and McKenna (1996) suggest that in an aggregation process in which a few intermolecular bonds are formed and the state of order of the system is increased, Ea will be lower [15]. The total SH groups content for native WPC was ± 0.7 µmol/g of proteins. The heat-induced changes in total SH groups are depicted in Figure 5. The total SH groups content varied significantly during thermal treatment. After 40 minutes of heating at 85 0 C, the total SH group content was 0,52 ± 0.5 µmol/g, representing 2.3% of the total SH groups for untreated protein. Figure 5. Denaturation curves of whey protein concentrate expressed as changes in total SH groups With increasing temperature, whey protein molecules undergo a sequence of conformational changes due to the balance of stabilizing interactions which are altered. After or/and during heat treatment new intermolecular interactions are formed and the proteins may be newly structured. In figure 6 is given the increase in absorbance for (un) treated samples at different temperature for 40 minutes. The initial slow-reacting SH group content was found to be ± 0.37 µmol/g protein, representing 87.3% of the initial total SH groups. 133

5 Figure 6. The increase in absorbance of WPC solutions treated at different temperatures for 40minutes The decrease in the amount of slow-reacting SH groups is depicted in Figure 7, indicating SH/SS interchange reactions, as described by the polymerization mechanism [11]. Figure 7. Time-dependent structural changes in slowreacting SH groups In native β-lactoglobulin, the free thiol group is masked in the hydrophobic interior of the protein and does not normally participate in a disulfide linkage[2]. The reactivity of the free thiol group can be markedly increased by protein unfolding induced by, for example, thermal treatment [12]. Denatured whey proteins have an activated thiol group that can oxidize with another thiol group to form a disulfide bond, or that can exchange with a disulfide bond to rearrange the position of the disulfide bond. Aggregates formed in these polymerization reactions still have reactive thiol groups present [1]. Sava et al. (2005) suggested that in the first stage of heating, the exposed thiol group (Cys 121 ) may react with a solvent-exposed Cys 66 -Cys 160 bond of another β-lactoglobulin molecule, resulting in a relatively stable Cys Cys 66 bond and a free non-native thiol group (Cys 160 )[12]. These newly formed SS bonds play an important role in the heat-induced aggregation and gelation of β-lg [8]. 4. Conclusions The results presented in this paper demonstrate that heat-induced changes in WPC greatly influence its technological properties. Solubility decrease in combination with protein aggregation was related to the intensity of applied treatment time. Heat treatment of WPC in the temperature range of C and at neutral ph leads to the exposure of the free thiol groups. This process is accompanied by the aggregation of the molecules because of sulfhydryldisulfide interchange reactions, with consequences for protein solubility. Acknowledgment This work was supported by CNCSIS UEFISCSU, project number PNII IDEI 517/2008. References 1. Alting, A. C., Hamer, R. J., De Kruif, C. G., Visschers, R. W. Formation of disulfide bonds in acid-induced gels of preheated whey protein isolate. Journal of Agricultural and Food Chemistry, 2000, 48(10), , doi: /jf000474h 2. Bryant, C. M., and D. J. McClements. Molecular basis of protein functionality with special consideration of cold-set gels derived from heat-denatured whey. Trends in Food Science and Technology, 1998, 9(4), , doi: /s (98) Dannenberg, F., and H. G. Kessler. Thermodynamic approach to kinetics of β-lactoglobulin denaturation in heated milk and sweet whey. Milchwissenschaft 1988, 43(3), de Wit, J. N., and G. Klarenbeek, Effects of various heat treatments on structure and solubility of whey proteins, Journal of Dairy Science, 1984, 67(11), , doi: /jds.s (84) de Wit, J.N. Thermal behavior of bovine b- lactoglobulin at temperatures up to 150ºC. A Review, Trends in Food Science and Technology, 2009, 20(1), 27-34, doi: /j.tifs Galani, D., O. R. K. Apenten. The comparative heat stability of bovine β-lactoglobulin in buffer and complex media, Journal of Science and Food Agriculture, 1997, 74(1), 89 98, doi: / (SICI) (199705) 7. Harper W.J. Biological Properties of Whey Components: A Review, American Dairy Product Institute, Chicago IL, 1 67, 2000, People/HARPER/Functional-foods/2003%20%20 %20BIOLOGICAL%20PROPERTIES%20OF%20W HEY%20COMPONENTS%20-Final.doc 134

6 8. Hoffman, M. A. M., P. J. J. M. van Mill. Heatinduced aggregation of β-lactoglobulin: Role of the free thiol group and disulphide bonds, Journal of Agricultural and Food Chemistry, 1997, 45(8), , doi: /jf960789q 9. Kresic, G., Lelas, V, Herceg, Z., Rezerk, A. Effects of high pressure on functionality of whey protein concentrate and whey protein isolate, Lait, 2006, 86(4), , doi: /lait: Meza, B.E., Verdini,. R:A:, Rubiolo, A.C: Viscoelastic behavior of heat-treated whey protein concentrate suspensions, Food Hydrocolloids, 2009, 23(3), , doi: /j.foodhyd Roefs, S. P. F. M., and C. G. De Kruif. A model for the denaturation and aggregation of β- lactoglobulin, European Journal of Biochemistry, 1994, 226(3), , doi: /j x 12. N. Sava, I. Van der Plancken, Claeys, W., Hendrickx, M., The kinetics of heat-induced structural changes of β-lactoglobulin, Journal of Dairy Science, 2005, 88(5), , doi: /jds.s (05) Shimada, K. and Cheftel, J.C. Sulfhydryl group/disulphide bond interchange reactions during heat induced gelation of whey protein isolate, Journal of Agriculture and Food Chemistry, 1989, 37(1), , doi: /jf00085a Walstra, P. and R. Jenness, Dairy Chemistry and Physics. John Wiley and Sons, New York., Anema, S. G. and McKenna, A. B., Reaction kinetics of thermal denaturation ofwhey proteins in heated reconstituted whole milk. Journal Agriultural and Food Chemistry, 1996, 44(2),

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