Effects of Ohmic Heating on Denaturation of Whey Proteins Solutions: Influence on Whey-Derived Products

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1 Effects of Ohmic Heating on Denaturation of Whey Proteins Solutions: Influence on Whey-Derived Products Ricardo N. Pereira, António A. Vicente, José A. Teixeira IBB-Institute for Biotechnology and Bioengineering, Centre for Biological Engineering, University of Minho, Braga, Portugal ABSTRACT Ohmic heating is one of the earliest applications of electricity in thermal processing of food and is receiving increased attention because of its uniform heating of liquids with faster heating rates, which presumably enables obtaining products of a superior quality to those processed by conventional heating technologies. Given the biochemical structure of proteins, it is expectable that ohmic heating and its moderate electric fields will influence their properties and hence their behavior. The aim of this work was to evaluate the effects of moderate electric fields on denaturation and aggregation of whey proteins and its subsequent effects in whey-derived products. Results presented here show that ohmic heating led to lower whey protein denaturation at the early stages of heating, kinetically traduced by lower values of n and k, when compared to those from conventional heating (p < 0.05) under equivalent heating rate and holding times. Furthermore, whey protein aggregation (measured by dynamic light scattering in terms of aggregate size) was found to decrease with the presence of moderate electric fields applied during ohmic heating at 85 ºC, up to 30 min. Edible films prepared from ohmic heated film forming solutions present a decrease of water vapor permeability of 10%, when compared with films produced through conventional heating. Keywords: ohmic heating; whey proteins; denaturation, aggregation, edible films INTRODUCTION Thermal processing is believed to induce disruption of the native conformation of whey proteins, thus affecting their structure and functional properties of its derived products, such as whey protein isolate (WPI) and whey protein concentrate (WPC). Heating of WPI results in the exposure to the solvent of free sulphydryl groups (SH), normally occluded within β-lactoglobulin (β-lg) and bovine serum albumin (BSA), and hydrophobic amino acid side-chains [1]. SH may rapidly interchange with existing disulfide bonds to generate new inter- and intramolecular disulfide bonds [2]. During heating, the role of sulphydryl/disulfide interchange may promote intermolecular disulfide bond formation, which together with intermolecular protein protein interactions (such as entropic forces, dipolar and electrostatic interactions) between unfolded reactive proteins, determines protein aggregation. Whey protein aggregation can produce a number of undesired effects such as formation of deposits on heat exchangers [3, 4] and gelling during the production of concentrated milks [5]. On the other hand, for the production of edible whey protein films the formation of aggregates in the film-forming solutions (before drying) is required to form stiff and stretchable films.[6]. Therefore, the knowledge of proteins behavior during heating is essential for the control of their properties and characteristics during the recovery and application of whey derived products [7]. Ohmic heating (OH) is receiving increased attention due to its uniform heating of liquids with faster heating rates, which enables obtaining products of a superior quality to those processed by conventional heating technologies [8-10]. This heating method involves the passage of an alternating electrical current through a food, which will heat due to internal energy dissipation according to Joule s law. This technology offers great potential for use in a wide variety of food processing operations involving heat and mass transfer, such as blanching, evaporation, dehydration, fermentation [11], continuous cooking and sterilization of viscous and liquid food [12]. Given its volumetric heating, rapid heat penetration rates and also due to the presence of moderate electric fields, OH possibly influences denaturation and aggregation mechanisms of whey proteins, thus affecting WPI properties. However no experimental work has been reported to confirm this until the present moment. Therefore, the main objective of this study was to determine the levels of denaturation of whey proteins dispersions during ohmic heating and compare them with those obtained using a conventional heating

2 method, under identical temperature history conditions. The effects of ohmic heating technology on aggregation patterns of WPI solutions and physical properties of whey protein edible films were also assessed. MATERIALS & METHODS Whey protein dispersions: Aqueous solution of WPI (3% w/w, protein), kindly supplied by Arla Foods Ingredients (Viby, Denmark), was prepared by dispersing WPI powder in a 0.05 M phosphate buffer. Heating treatments: WPI dispersions were heated through conventional (indirect) heating () and OH at temperatures ranging from 75 to 90 C, for various time periods. During the OH experiments, the nominal electric field was varied through a rheostat to adjust the supplied voltage and simulate the thermal history of samples observed during conventional heating experiments. During heating phase and holding treatment the nominal electrical field applied varied from 15 to 22 V cm -1 and 4 to 8 V cm -1, respectively. A close coincidence of the temperature profiles during the sample in both conventional and ohmic treatments is always a necessary condition to evaluate the non-thermal effects of ohmic heating. Determination of free sulphydryls (SH): Unfolding and exposure of SH groups initially buried inside the protein macromolecules was measured through reactivity of free thiol groups using Ellman's 5,5'-dithiobis- (2-nitrobenzoic acid) or DTNB method [13]. Determination of levels of denaturation of whey proteins: Whey protein denaturation was followed by means of soluble tryptophan (Trp) fluorescence value (F Trp ) at excitation/emission of 290/340 nm on the ph 4.6- soluble fraction of the heated protein solutions. Analysis of the kinetics of whey protein denaturation was carried out by non linear regression (NLR) The denaturation process WPI solutions was described by the general-rate equation (1): = (1) where (dc/dt) represents the rate of protein denaturation, k the rate constant, C the protein concentration and n the reaction order. For reactions of order n 0, equation 1 has as general solution of: = (2) where t is the time (s), C 0 the initial protein, C t the concentration (g/l) of native protein at each holding time. In Eq. (2) the product gives an apparent rate constant k with units of (s 1 ), which is the basis of comparison between different kinetic models, circumventing the fact that k n has different units for different reaction orders [14]. The concentration and time data were fitted directly into Eq. 2 and C 0, n and k determined simultaneously by NLR [15, 16]. The overall fit of the model to the experimental data was given by the r 2. For the experimentally obtained kinetic parameters to be valid over a wide temperature range, degrees of denaturation up to >90% have to be achieved [17]. Particle size analysis: The average size of WPI aggregates formed during heating treatments at 85 ºC was measured by dynamic light scattering (DLS), using a Zetasizer Nano (ZEN 3600, Malvern Instruments Ltd., Malvern, U.K.). Average diffusion coefficients were determined by the method of cumulants fit and were translated into average particle diameters (Z-value) using Stokes-Einstein relationship [18]. Heating conditions (85 C up to 30 min), were selected to avoid coagulation or gel formation during heating. Production of WPI films: Glycerol (Gly, Panreac, Barcelona, Spain) was added (as a plasticizer) to WPI solutions subjected to conventional and ohmic heating, in a 1:3 ratio of Gly:WPI. WPI film-forming solutions were cast onto 8 cm diameter glass plates in order to maintain film thickness. Films were then dried in a convection oven at 35 ºC during 12 h. After this dehydration step, films were peeled from the plate and conditioned, at 20 C during 48 h, in desiccators at a controlled RH atmosphere of 50%, for further property testing. For each property, a minimum of three replicates for each heating treatment were prepared.

3 Characterization of WPI films: WPI edible films were characterized in terms of thickness, water vapour permeability (WVP), solubility and moisture content. The measurement of WVP was performed gravimetrically based on ASTM E96-92 method [19-22]. The weight loss of the film sample was determined, from which the moisture content was calculated. Film solubility in water was determined according to the method reported by Gontard et al. and Cuq et al.[23, 24], and it was defined by the content of dry matter solubilised after 24 h immersion in water. RESULTS & DISCUSSION At the end of heating treatments, concentrations of free SH groups in WPI solution were 10-fold higher than those observed in unheated solutions. WPI samples treated by heating exhibited more reactive free SH than samples treated by OH at the end of 30 minutes of heating (Figure 1a), being this difference statistically relevant (p < 0.05). The results obtained are still in good agreement with published data, indicating unmasking and activation of SH groups in whey proteins upon extended heat treatment at temperatures above 70 ºC [25-27]. For each heating treatment, the F Trp value of the acid (ph 4.6) protein solutions, was monitored as a function of heating time. Due to the very low concentration of lactose (< 0.5%) and temperature used (75 to 90 ºC), the F Trp value was strictly correlated to the protein concentration during thermal denaturation [28]. For equivalent holding times, the susceptibility of the whey proteins to denaturation was higher under than OH treatment (p < 0.05), particularly during the early stages of heating (first 2 minutes) for treatments at temperatures above 75 ºC (Figure 1b). Figure 1. Example of conventional () and ohmic (OH) treatment at 85 ºC: (a) unfolding of whey proteins traduced by activation of free sulphydryls (SH) groups; (b) contents of soluble native whey proteins in heated solutions; and (c) increase of average hydrodynamic diameter (Z value) of protein aggregates upon heating. The kinetic parameters derived from NLR analysis are shown in Table 1. In all plots, the kinetic parameters obtained by NLR analysis were highly significant (p < 0.001) and their fit to the experimental data was satisfactory once it gave adjusted r 2 values > The k and n values obtained in this work were well comparable with published literature for the denaturation of β-lg [29], the most abundant protein in WPI. Heating at 75 ºC did not promote significant differences (p > 0.05) on kinetic parameters for denaturation of whey protein. Otherwise, at temperatures above 75 ºC, OH treatments presented in general lower values of n and k (p < 0.05), when compared to those from heating, under identical thermal profiles. Through Eq. 2 and kinetic parameters obtained from NLR analysis was possible to determine D and t 1/2 values, which were the times needed for 90% and 50% of denaturation, respectively, at constant temperature (Table 1). In range of temperatures studied no significant differences (p > 0.05) were found on D values, between the several treatments. Concerning the t 1/2 value, at temperatures above 75 ºC significant differences were noticed (p < 0.05) among the treatments. Particularly, at temperatures of 80 and 85 ºC, OH treatment presented higher t 1/2 values than the ones obtained for the heating. Concerning particle size analysis, when WPI solutions were heated (85 ºC, up to 30 min), the average hydrodynamic diameter of the whey protein aggregates was found to increase considerably in both and OH treatments. However, OH produced

4 smaller changes (p < 0.05) in whey protein aggregates size when compared with heating (Figure 1c). WPI solutions treated by heating presented a Z-value of 86.0 ± 0.5 nm, while ohmic heating determined a value of 76.6 ± 0.5, at the end of 30 minutes of heating at 85 ºC. Table 1. Kinetic parameters derived from non linear regression (NLR) for the denaturation of WPI solutions subjected to conventional () and ohmic heating (OH) treatments. Treatment T(ºC) n k (s ) t 1/2 (min) D (min) r ± 0.1 a 3.8 ± 1.0 a 4.0 ± 0.9 a 25.0 ± 3.0 a OH 1.4 ± 0.3 a 2.3 ± 0.2 a 5.9 ± 0.1 a 28.8 ± 6.6 a ± 0.1 a 13.1 ± 1.2 a 1.3 ± 0.1 a 11.0 ± 2.0 a OH 1.4 ± 0.1 b 6.1 ± 0.8 b 2.2 ± 0.2 b 10.5 ± 0.7 a ± 0.2 a 39.7 ± 4.8 a 0.46 ± 0.02 a 5.4 ± 1.2 a OH 1.7 ± 0.1 b 17.4 ± 1.6 b 0.87 ± 0.06 b 5.4 ± 0.4 a ± 0.1 a ± 10.2 a 0.17 ± 0.01 a 1.9 ± 0.2 a OH 2.1 ± 0.0 a 71.8 ± 7.4 b 0.24 ± 0.02 a 2.5 ± 0.1 a, a b For each temperature and kinetic parameter, means in the same column with different superscripts are significantly different (p < 0.05). The levels of aggregation of whey proteins observed were comparable with other literature reports [30]. Protein edible films were produced from heated WPI solutions and characterized in terms of their permeability properties. Table 2 shows the results for characterization of WPI intact films. The thickness of films prepared through heating ranged from to mm. These values were slightly higher than those reported by other authors for whey protein films [31, 32], essentially due to differences in film-forming solutions formulations and in the procedures used. WPI films prepared from OH presented a mean thickness of mm, which was significantly lower (p < 0.05) than that of films produced by (0.202 mm). OH seems to have affected the properties of WPI film-forming solutions, which determined thinner films after the drying step. Concerning the permeability properties, despite of the ohmic film being thinner than the conventional one, the same amount of water vapour passed through both films (equivalent permeance). Since WVP is the product of film permeance and thickness, ohmic films presented lower permeability to water vapour than conventional films (p < 0.05), due to the lower thickness of the former. WVP values for conventional and ohmic films, 0.60 and 0.66 g.mm.kpa -1.h -1.m -2, respectively, are in agreement with published data for WPI films, which can range from 0.2 to 6.4 g.mm.kpa -1.h -1.m -2 [33]. Ohmic and conventional films presented almost 20% of moisture content and were partially soluble in water, both showing >50% of soluble matter. The type of heating treatment ( or OH) had no significant effect (p > 0.05) on films solubility and moisture content. The insolubility in water of whey protein films has been attributed to the presence of high concentrations of intermolecular disulfide bonds. In the present work, the higher values of solubility may be explained by the lower content of WPI in the film-forming solutions ( 3%), which led to a lower occurrence of intermolecular disulfide bonds. Table 2. Characterization of conventional () and ohmic (OH) WPI edible films OH Thickness (mm) 0.20 ± 0.01 a 0.17 ± 0.01 b Permeance (g. kpa -1. h -1.m -2 ) 3.27 ± 0.08 a 3.44 ± 0.08 a WVP (g.mm.kpa -1. h -1.m -2 ) 0.66 ± 0.02 a 0.60 ± 0.01 b Solubility (%) 58.0 ± 0.8 a 57.1 ± 0.5 a Moisture (%) 18.8 ± 0.8 a 17.8 ± 0.1 a a b Means in the same row with different superscripts are significantly different (p < 0.05). The present study clearly shows that OH interferes with the mechanisms of unfolding and aggregation or, at least, in the interactions between whey proteins. OH have determined less activation of free SH groups, lower whey protein denaturation rates, both leading to less pronounced aggregation, when compared with conventional heating, under similar thermal profiles. This effect was particularly evident at the early stages of heating and supported by the higher values of t 1/2, obtained when OH was applied. During OH treatment, heat is generated directly within the food sample (volumetric or direct heating) and hence the problems associated with heat transfer surfaces are eliminated [34]. The results presented here also suggest that extent of WPI

5 protein aggregation seems to influence films properties. Despite of the importance of stabilizing forces, such as hydrogen bonding, on the functional properties of films made from WPI [2], heat-induced aggregates have been recently considered as the building blocks involved in the formation of protein films networks [6]. Films produced by OH presented particular physical and structural properties, and this in part may be explained by different patterns of denaturation, interaction or aggregation of whey proteins observed. Moreover, aggregation, and consequently the viscosity, of cast solutions may have played an important role in establishing the film thickness of ohmic films, once the less viscous a solution, the more it spreads during casting and drying [35]. Lower thickness values for WPI films may be an important goal from a sensory perspective, once it makes them less perceptible in a food system[36]. CONCLUSION The present study clearly shows that ohmic heating potentially reduces denaturation of whey proteins during heating. In general, under identical temperature history conditions the rate of loss of native whey proteins was lower when ohmic effect was present. This was kinetically traduced by lower k and n values for the ohmic heating denaturation reactions. The obtained results also suggest that the ohmic heating influences the mechanisms of association/aggregation of denaturated whey proteins bringing implications on quality and functional and technological properties of whey-derived products, such as edible films. Further studies are needed to determine the effects that moderate electric fields may have on the structure of whey proteins and its subsequent effects on the acid-induced gelation properties of milk. REFERENCES [1] Shimada, K. and J.C. Cheftel, Sulfhydryl-Group Disulfide Bond Interchange Reactions during Heat-Induced Gelation of Whey-Protein Isolate. J. Agric. Food Chem., (1): p [2] Fairley, P., et al., Mechanical properties and water vapor permeability of edible films from whey protein isolate and N-ethylmaleimide or cysteine. J. Agric. Food Chem., (12): p [3] Burton, H., Reviews of Progress of Dairy Science - Section G Depsosits from Whole Milk in Heat Treatmentplant- a Review and Discussion. Journal of Dairy Research, (2): p. 317-&. [4] Fryer, P.J., M.T. Belmar-Beiny, and P.J. Schreier, Fouling and cleaning in milk processing., in Heat-Induced Changes in Milk, P.F. Fox, Editor. 1995, International Dairy Federation: Brussels, Belgium. p [5] Singh, H. and L.K. Creamer, Heat stability of milk, in Advanced Dairy Chemistry - 1: Proteins, P.F. Fox, Editor. 1992, Elsevier Applied Science: London. p [6] Lefevre, T., M. Subirade, and M. Pezolet, Molecular description of the formation and structure of plasticized globular protein films. Biomacromolecules, (6): p [7] de la Fuente, M.A., H. Singh, and Y. Hemar, Recent advances in the characterisation of heat-induced aggregates and intermediates of whey proteins. Trends in Food Science & Technology, (8): p [8] Castro, I., et al., The influence of field strength, sugar and solid content on electrical conductivity of strawberry products. J. Food Process Eng., (1): p [9] Parrot, D., Use of ohmic heating for aseptic processing of food particulates. Food Technol. (Chicago, IL, U. S.), (12): p [10] Machado, L.F., et al., Moderate electric fields can inactivate Escherichia coli at room temperature. J. Food Eng., (4): p [11] Cho, H.Y., A.E. Yousef, and S.K. Sastry, Growth kinetics of Lactobacillus acidophilus under ohmic heating. Biotechnol. Bioeng., (3): p [12] Icier, F. and C. Ilicali, The effects of concentration on electrical conductivity of orange juice concentrates during ohmic heating. Eur. Food Res. Technol., (3-4): p [13] Ellman, G.L., Tissue sulfhydryl groups. Arch. Biochem. Biophys., (1): p [14] Zuniga, R.N., et al., Kinetics of Formation and Physicochemical Characterization of Thermally-Induced beta- Lactoglobulin Aggregates. Journal of Food Science, (5): p. E261-E268. [15] Oldfield, D.J., et al., Kinetics of denaturation and aggregation of whey proteins in skim milk heated in an ultrahigh temperature (UHT) pilot plant. International Dairy Journal, (4): p

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