- Email: [email protected]
Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate
Ultrasonics Sonochemistry 18 (2011) 951–957
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultsonch
Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate Jayani Chandrapala a,b, Bogdan Zisu c, Martin Palmer c, Sandra Kentish a,b, Muthupandian Ashokkumar a,b,⇑ a
School of Chemistry, University of Melbourne, VIC 3010, Australia Department of Chemical and Biomolecular Engineering, University of Melbourne, VIC 3010, Australia c Dairy Innovation Australia, Sneydes Road, Werribee, VIC 3030, Australia b
a r t i c l e
i n f o
Article history: Received 19 October 2010 Received in revised form 24 December 2010 Accepted 27 December 2010 Available online 31 December 2010 Keywords: Ultrasound Differential scanning calorimetry Circular dichroism Thiol groups Whey protein concentrates
a b s t r a c t The sonication-induced changes in the structural and thermal properties of proteins in reconstituted whey protein concentrate (WPC) solutions were examined. Differential scanning calorimetry, UV–vis, fluorescence and circular dichroism spectroscopic techniques were used to determine the thermal properties of proteins, measure thiol groups and monitor changes to protein hydrophobicity and secondary structure, respectively. The enthalpy of denaturation decreased when WPC solutions were sonicated for up to 5 min. Prolonged sonication increased the enthalpy of denaturation due to protein aggregation. Sonication did not alter the thiol content but resulted in minor changes to the secondary structure and hydrophobicity of the protein. Overall, the sonication process had little effect on the structure of proteins in WPC solutions which is critical to preserving functional properties during the ultrasonic processing of whey protein based dairy products. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Cheese whey, a by-product of the dairy industry is a source of valuable nutrients with important functional and nutritional properties. Whey protein concentrates and whey protein isolates are also important ingredients in the manufacture of value added products [1]. The major proteins in such whey systems are the milk globular proteins, a-lactalbumin (LA) and b-lactoglobulin (LG), along with small amounts of serum albumin and immunoglobulins [2]. The heat induced aggregation of whey protein based dairy systems has been an ongoing problem in dairy processing. Exposure of whey proteins to temperatures greater than 70 °C denatures the proteins and exposes buried thiol groups. It is recognised that the thermal behaviour is governed mainly by b-LG [3]. The heat induced unfolded conformation of this protein exposes functional groups, which are otherwise buried in the native structure, thereby increasing their reactivity. Particularly important is the increased reactivity of the thiol groups of cysteine. In addition to disulfide interchange, the unfolded protein is more susceptible to protein– protein interaction via calcium bridging and hydrophobic bonding [4]. The denatured whey proteins form complexes among themselves as well as with casein micelles, leading to the formation of large aggregates. This results in excessive thickening or gelling ⇑ Corresponding author at: School of Chemistry, University of Melbourne, Melbourne VIC 3010, Australia. Tel.: +61 3 83447090; fax: +61 3 93475180. E-mail address: [email protected] (M. Ashokkumar). 1350-4177/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2010.12.016
during thermal processing. The endothermic denaturation of the proteins and exothermic aggregation can both be monitored by changes in the heat flows to and from protein samples as a function of temperature [5,6]. The development of nonthermal processing methods to modify food ingredients has generated interest in the food industry recently [7]. In particular, the use of high intensity ultrasound has attracted considerable attention due to its promise in the development of novel, gentle but targeted processes to improve the quality and safety of processed foods. Furthermore, the technology offers the potential to improve existing processes [7,8]. Ultrasound has been developed for several food processing applications such as degassing of liquids, extraction of enzymes and proteins, inactivation of microorganisms [9], ultrasound-induced product modification [7], extraction of gingerol from ginger [10], homogenisation of milk [9], and acceleration of ageing of wines [11]. Acoustic cavitation is the fundamental process responsible for the initiation of many of the sonochemical reactions in liquids. In an acoustic field, bubbles can undergo growth by rectified diffusion and by bubble–bubble coalescence [12]. The collapse of cavitation bubbles results in the generation of high temperatures within the bubbles and is accompanied by emission of light (sonoluminescence). Cavitation also results in other mechanical, physical and chemical effects such as shockwave formation, turbulent motion of the liquid and generation of radicals [13]. The large shear forces generated can break inter and intramolecular bonds, which in turn may lead to fragmentation of clusters and aggregates.
952
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
Most recently, it has been shown that a short burst of ultrasound can help to break apart heat-induced aggregates and prevents their reformation on subsequent heating, thereby avoiding further increases in viscosity [14]. The physical shear forces generated during acoustic cavitation are believed to be responsible for the breakdown of the heat-induced aggregates and subsequent viscosity reduction. This approach is advantageous in a food application as it appears that changes in chemical composition are not required to achieve the heat stability in this case. Heat-induced changes to –SH groups and S–S bonds of food proteins [15,16], heat-induced secondary and tertiary structural changes of proteins [17,18] and thermal properties of protein solutions [5,6,19] have been extensively reported. However, less work has been reported on changes to protein properties as a result of sonication. Paulsson and Dejmek [5] state that ultrasound treatment acts differently to temperature treatment as it does not disrupt covalent bonds. They find that the effects of ultrasound are minimal on the secondary and tertiary structures of proteins. However, other researchers have found that sonication does result in some structural changes of individual proteins [20]. For example, Cavalieri et al. [21] have observed sonochemical cross-linking between the thiol groups of protein molecules under specific experimental conditions. Taylor and Richardson [16] found that the total thiol groups in skim milk decreased upon prolonged sonication under uncontrolled temperature conditions. In this case, it is possible that the heat generated during sonication reduced the thiol group content. In a separate study by Gulserren et al. [20], the amount of reactive thiol groups in BSA decreased after 90 min of sonication. The authors suggested that the decreased number of thiol groups may be attributed to the formation of protein aggregates. Further, a secondary structural analysis of native and ultrasonicated BSA showed a small increase in ordered structure upon ultrasonication which differed distinctly from any thermal treatment where ordered structure is lost. Stathpulos et al. [22] also observed aggregate formation following sonication of proteins. They used circular dichroism to show that these aggregates have high b-sheet content, and proteins containing significant native a-helical structure showed an increase in the b-sheet structure in the aggregates. Marchioni et al. [23] state that the behaviour of proteins under sonication depends mainly on the dominant secondary structure type (a-helix or b-sheets) and on the grade of the ordered structure. Their results suggest that free radicals produced by water sonolysis have an important role in causing the structural changes. Our previous work has shown a clear change in the physical and functional behaviour of sonicated whey protein concentrate (WPC) [24,25]. Sonication at 20 kHz for short times reduced the viscosity of WPC solutions, but an increase in viscosity was observed if sonication times were extended. When heated, the gel strength of sonicated solutions increased, while gelation time and gel syneresis were reduced. The microstructure of sonicated whey protein gels showed a densely packed protein network. Although highly reproducible, the precise mechanism or molecular basis for these effects is not known. The aim of the present study is to identify thermal and structural changes to the protein in whey protein concentrate solutions induced by sonication.
2. Materials and methods 2.1. Materials Commercial WPC powder was obtained from Warrnambool Cheese and Butter Factory (Allansford, Victoria, Australia). The ‘‘WPC 80’’ powder was derived from cheddar cheese whey and
contained 81.5% protein, 10.4% lactose and 4.4% fat. Ultra pure (MilliQ) water was used in all experiments. 2.2. Preparation of reconstituted WPC solutions WPC powder was reconstituted in MilliQ water to obtain 50 g/ kg (w/w) solutions. The solutions were continuously stirred for one hour at room temperature (25 ± 3 °C) to ensure complete mixing and stored overnight at 4 °C. On the following day, the solutions were equilibrated at 25 °C for 1 h prior to further analysis. 2.3. Sonication 60 mL solutions were sonicated in a glass vessel equipped with a cooling jacket using a 20 kHz, 450 W Ultrasonic horn (19 mm diameter, Branson Sonifier 450, Danbury, CT) at an amplitude of 50% for 1, 5, 10, 20, 30 and 60 min. The actual power delivered to the solution was 31 W as determined by calorimetry. This power was used in order to correlate the results with our previous study [14]. During sonication, chilled water was continuously circulated through the cooling jacket to maintain the sample temperature at 6 ± 4 °C. 2.4. DSC measurements Thermal analysis of whey proteins was carried out on a Perkin Elmer dynamic scanning calorimeter (DSC 2 Perkin Elmer Corporation, Winter Street, Waltham Massachusetts, 02451 USA) equipped with Pyris Manager, v.5.0002 software. The instrument was calibrated for temperature (T) and change in enthalpy (DH) using indium (Tpeak = 155.87 °C, DH = 28.234 J/g). Approximately 12– 15 mg of 12% (w/w) WPC solutions were accurately weighed into aluminium pans and hermetically sealed. An empty pan of equal weight was used as a reference. The scanning temperature was raised from 25 to 100 °C at 10 °C/min. The study was arranged as a randomised block design with time as the main factor. All experiments were replicated thrice with subsequent sub sampling. The pans were weighed before and after the DSC scan to ensure the effectiveness of sealing. Endothermic heat flow (endotherms) as shown by the instrument was represented as peaks in the downward direction (negative maxima). Temperature at maximum heat flow of endothermic transition and the area underneath the peak from the thermal curve which represents the enthalpy (J/g) was calculated based on the protein mass within the solution. 2.5. Surface hydrophobicity measurements For the fluorometric assay, the stock protein solutions were diluted with 0.1 M pH 7 phosphate buffer solutions to typical concentration ranges of 0.005–0.025% (w/w) using 1-anilinonaphthalene-8-sulfonate (ANS). A stock solution of 8 103 M ANS was prepared in 0.1 M pH 7 phosphate buffers. ANS stock solution was wrapped in aluminium foil to prevent light exposure and stored at room temperature. For hydrophobicity determination using ANS, the excitation and emission slits and wave lengths were set at 5/5 and 390/470 nm, respectively. 20 lL of ANS solution was added to 4 mL of diluted protein solution, vortexed and kept in the dark for 15 min. The Relative Fluorescence Intensity (RFI) of each solution was measured starting from buffer blank and then the lowest to highest protein concentration. The RFI of each dilution blank was subtracted from that of the corresponding protein solution with ANS to obtain the net RFI. Surface hydrophobicity was expressed as the initial slope of the plot of standardised net RFI values vs. % protein concentration computed using least squares linear regression analysis (Microsoft excel 8.0).
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
Surface hydrophobicity was determined by taking the average of three analyses. In all cases, R2 values of >0.95 were noted for the linear regression analyses used to calculate the surface hydrophobicity. 2.6. Thiol group determination A method developed by Patrick and Swaisgood [26] for the determination of reactive and total thiol groups was adopted in the present study. The method uses Ellman’s reagent (5,50 Dithio-bis (2-nitrobenzoic acid (DTNB)) to react specifically with the thiol groups of the solutions to form a yellow coloured complex, which can be measured by absorbance at 412 nm. An aliquot (5 mL) of the WPC solution was diluted to 8 mL with 0.1 M phosphate buffer at pH 8. The pH of the solution was re-adjusted to pH 8 with 1 M NaOH. To this solution mixture, 1 mL of 2 mM DTNB (prepared using 0.1 M phosphate buffer at pH 7) was added. Colour development was allowed to proceed for 30 min. Separate experiments were performed in order to optimise the incubation time. A 30 min incubation time showed maximum absorbance which started to fade with longer exposure times. The solution was centrifuged at 35000g for 15 min (25 ± 2 °C) after adjusting the volume to 11 mL. The supernatant liquid was removed by syringe and filtered through a 0.45 lm filter. An aliquot (1 mL) of WPC solution was added to 1.44 g of urea to expose thiol groups that were buried inside the globular protein structure. 3 mL of 0.1 M phosphate buffer at pH 8 was added to the solution mixture. To this solution mixture, 1 mL of 2 mM DTNB (prepared using 0.1 M phosphate buffer at pH 7) was added. The volume of the solution mixture was adjusted to 7 mL. Colour was allowed to develop for 30 min and the solution mixture was filtered through a 0.45 lm filter. The absorbance of the thionitrobenzoate released by the reaction with thiols in the filtrates was read at 412 nm. The net absorbance was calculated as the difference between absorbance of samples with DTNB and that without DTNB:
DAbs412 ¼ Absðwith DTNBÞ Absðwithout DTNBÞ
ð1Þ
The concentration of thiol groups was then calculated according to the following formula [27]:
lmol SH=g ¼ ð73:53 DAbs412 DÞ=C
953
tained using a Jasco J-600 Spectropolarimeter (JASCO UK Ltd., Great Dunmow, UK) at 25 °C with a 0.1 cm path length quartz cell, a 10 nm/min scan rate and a 0.2 nm band width. Spectra were an average of 3 scans from 190 to 260 nm. The spectrum of 100 mM phosphate buffer was used as a blank and subtracted from the average three spectra to obtain a corrected spectrum for each sample. Spectra were determined in duplicate. Spectra were deconvoluted using the deconvolution software CONTIN to obtain information about the secondary structure. CONTIN, developed by Provencher and Glöckner (DICHROWEB website), fits the CD of unknown proteins by a linear combination of the spectra using a large data base of proteins with known conformations. An advantage of this method is the reliable estimate of b-turns in a protein. 2.9. Statistical analysis When necessary, one-way ANOVA with a 95% confidence interval was used. The ANOVA data with p < 0.05 were considered statistically significant. 3. Results and discussion 3.1. Differential scanning calorimetry Heat induced changes to whey protein structure are accompanied by changes in thermal properties. Fig. 1 represents the DSC thermograms of native and sonicated WPC solutions heated from 25 to 100 °C. All WPC samples showed a single broad endothermic peak at approximately 77 °C. This peak relates to the denaturation temperature of b-LG, the major whey protein and agrees with the work by others [5,29–31]. A shoulder peak also appears around 65 °C due to the denaturation of a-LA as confirmed by literature. Patel et al. [29] reported a shoulder between 62 and 72 °C resulting from the overlapping of two endothermic peaks and suspect that this may be due to the denaturation of a-LA. Fitzsimons et al. [32] also found a dominant endotherm centered at 75 °C and a shoulder at 62 °C. They assigned those peaks as the denaturation of b-LG and a-LA, respectively. Denaturation involves dissociation of intramolecular bonds (non-covalent and in some cases, covalent (disulfide) bonds) and
ð2Þ
where; DAbs412 is the net absorbance measured at 412 nm by the thiol groups with/without DTNB, D is the dilution factor (if any), C is the total solids content in milk expressed in mg/mL. 2.7. Reversed phase high performance liquid chromatography Reversed-phase (RP) HPLC was performed using a Shimadzu LC 20 HPLC system equipped with a Jupiter 5l C18 300A column (250 4.6 mm I.D.) and a UV–vis detector. Solvent A was a mixture of acetonitrile–water–trifluoroacetic acid (100:900:1 v/v/v) and solvent B was the same mixture with the proportions 900:100:0.7 (v/v/v). The solvent gradient reported by Visser et al. [28] was used for the elution. All whey protein samples were filtered through a 0.45 lm syringe filter before injection. The elution profiles were monitored at 280 nm. The total run time was 55 min. b-LG fractions were collected using a Shimadzu fraction collector. Fractions were freeze dried before further analysis by circular dichroism. 2.8. Circular dichroism spectroscopy Protein solutions were prepared at final concentrations of 0.5 mg/mL in 100 mM phosphate buffer at pH 7.0. Data were ob-
Fig. 1. DSC thermograms of reconstituted WPC solutions as a function of sonication time.
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
is therefore an endothermic process. Aggregation of the denatured molecules involves formation of new intermolecular bonds, and would therefore be expected to give rise to an exothermic peak in DSC measurements [33]. However, DSC studies have indicated that, when occurring concurrently, these two reactions can give rise to a single endothermic peak [34]. The DSC data in Fig. 1 reveal that in this case, endothermic denaturation reactions dominated within the temperature range studied, even though both denaturation and aggregation would be expected under these conditions [35]. Aouzelleg and Bull [30] stated that the reason for the broadness of the typical single DSC peak (Fig. 1) is due to the increasing number of proteins denaturing in response to continued heating. Furthermore, an unpredictable exothermic aggregation might also contribute to the broadness of thermograms [30]. In contrast, Choi et al. [33] concluded that the broadening of the thermogram is due to intermediate formations. In a given protein solution, although all molecules are in the same physicochemical conditions, a distribution of conformations is observed. The majority of proteins would be expected to be in a conformation state of minimal free energy but a number of molecules would be present in slightly higher energetic states. The proteins in higher energy states are partially or incorrectly folded conformers that have similarities with the native structure but contain non-native regions [30]. The variation in the depth of DSC profiles (Fig. 1) suggests that sonication does alter the thermal behaviour of whey proteins. These results are consistent with our previous work showing changes in the thermal gelling behaviour of sonicated WPC solutions [14,24,25]. However, the denaturation temperatures (75– 77 °C) of native and sonicated WPC solutions, as a function of sonication time, were not significantly different (Fig. 2). Fig. 3 represents the enthalpy changes of reconstituted WPC solutions as determined from these profiles. The enthalpy of native WPC was 0.19 J/g and agrees with data obtained by others [5,29– 31]. Dissanayake and Vasiljevic [31] suggest that these enthalpy values correlate with the content of ordered secondary structure and could be used to monitor the proportion of undenatured proteins. Their study shows a decrease in enthalpy upon high pressure shearing (micro fluidisation) and this was correlated to the destruction of hydrophobic interactions caused by the high pressure treatment. A slight increase in enthalpy following more extended micro fluidisation was correlated to a possible reformation of intra and intermolecular hydrophobic bonds from the resulting disordered protein molecules. These trends are consistent with our data. Sonication for up to 5 min, showed a decrease in enthalpy highlighting the
0.300 0.280
Enthalpy of Denaturation (J/g)
954
0.260 0.240 0.220 0.200 0.180 0.160 0.140 0.120 0.100
0
1
5
10
20
30
60
Sonication time (min) Fig. 3. Denaturation enthalpy changes of reconstituted WPC solutions as a function of sonication time.
destruction of bonds between proteins. However, the enthalpy increased on prolonged sonication beyond 5 min suggesting potential re-aggregation. Indeed, Choi et al. [33] confirmed the formation of network aggregates during prolonged sonication of soy protein isolate. These denaturation enthalpy changes (Fig. 3) also reflect the viscosity data obtained previously. Sonication for up to 5 min reduced the viscosity of protein solutions but increased when sonicating for longer periods (>5 min) [14]. The heating of globular proteins disrupts some of the forces responsible for the stability of tertiary and/or secondary protein structures. These forces include hydrogen interactions between the polar groups and interactions of non-polar groups (hydrophobic interactions) through the surrounding water molecules which form cages around hydrophobic groups. Electrostatic bonds and Vander Waals interactions are also involved in this heat denaturation process, although to a lesser extent [36,37]. However, on prolonged heat treatment, the process is reversed and heat enhances protein aggregation. Sonication may also result in disruption of intramolecular forces of proteins due to shear forces and lead to the formation of aggregates on prolonged sonication as is the case for heating.
300
250
Surface Hydrophobicity
Denaturation Temperature (C)
80.00
78.00
76.00
74.00
200
150
100
50 72.00 0 70.00
0
1
5
10
20
30
60
Sonication time (min) Fig. 2. Denaturation temperatures of reconstituted WPC solutions as a function of sonication time.
Samples
Native 10 min sonication 60 min sonication
1 min sonication 20 min sonication
5 min sonication 30 min sonication
Fig. 4. Surface hydrophobicities of reconstituted WPC solutions as a function of sonication time.
955
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
The importance of hydrophobic interactions for the stability, conformation, and function of proteins is well recognised. Due to the macromolecular structure of proteins, the surface hydrophobicity has more influence on functionality than the total hydrophobicity. The surface hydrophobicity of reconstituted WPC solutions as a function of sonication time is represented in Fig. 4. The surface hydrophobicity of the proteins increased for up to 5 min of sonication presumably due to the unfolding of the proteins resulting from minor structural changes. However, the surface hydrophobicities decreased after sonication for more than 5 min, which is a sign of protein aggregation which in turn protects the hydrophobic regions of the proteins. The partially denatured proteins with increased surface hydrophobicity might cause more extensive bonding, reducing the surface hydrophobicities on prolonged sonication. These results also support the trends observed in the enthalpy data (Fig. 3) and the viscosity data of previous studies [14]. Although the trends observed are
a
100.0
Absorbance
3.2. Surface hydrophobicity
0
5
10
15
20
25
Amount of Reactive Thiol Groups (umol)
5 min Sonication 10 min Sonication 20 min Sonication 30 min Sonication 60 min Sonication 50.0
25.0
0.0
Samples 400.0
Native 1 min Sonication
Amount of total Thiol Groups (umol)
350.0
5 min Sonication 10 min Sonication
300.0
20 min Sonication 30 min Sonication
250.0
40
45
50
Fig. 6. RP-HPLC profiles of reconstituted WPC as a function of sonication time (Control, 1, 5, 10, 20, 30 & 60 min from bottom to top, respectively).
1 min Sonication
b
35
Time/min
Native
75.0
30
60 min Sonication
200.0
150.0
100.0
50.0
0.0
Samples Fig. 5. The amount of reactive and total thiol group content of 5% w/w WPC subjected to sonication.
956
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957 30
20
10
0 200
210
220
230
240
250
260
270
-10
-20
Wavelength (nm) Fig. 7. Far UV CD spectrum of b-LG fractions obtained through HPLC of native (solid line) and 60 min sonicated (dashed line) WPC.
small, the changes in hydrophobicity as a function of sonication time are significant (p < 0.05). 3.3. Thiol content Fig. 5(a) and (b) represents the reactive- and total-thiol content of reconstituted WPC as a function of sonication time, respectively. Neither the reactive- nor total-thiol groups changed as a result of sonication. This is contrary to the results of other authors. Gulseren et al. [20] and Taylor and Richardson [16] observed a reduction in the free thiol content of proteins as a function of sonication time. This difference may arise due to the differences in the intensity and duration of the applied ultrasound. In particular, the lack of temperature control in the work of Taylor and Richardson [16] may explain their contradicting results. Another possibility is the complexity of the WPC solution, which contains a mixture of proteins rather than the pure BSA used by Gulseren et al. [20]. The intra-molecular location of the free thiol groups in the b-LG and a-LA proteins examined here may also make them less susceptible to degradation by ultrasound. 3.4. RP-HPLC Fig. 6 represents the RP-HPLC profiles of reconstituted WPC solutions sonicated for different time periods up to 60 min. The chromatographic profile for native WPC was similar to those reported previously [1,38,39]. The earliest eluting peaks (5– 10 min) are unidentified but Ferreira and Oliveira [39] suggest they may correspond to proteose-peptone derived from degradation of b-casein in milk and recovered in whey. The two large peaks eluting at 20 and 25 min represent a-LA and b-LG, respectively, as confirmed by standards. The b-LG peak was resolved in two parts highlighting the existence of b-LG’s A and B variants. Interestingly, the elution peaks did not show major changes after sonication for up to 60 min. HPLC analysis and UV-detection at this resolution failed to identify any minor protein structural changes associated with sonication. 3.5. CD measurements To identify sensitive structural changes beyond the resolution of HPLC, the sonicated and unsonicated b-LG HPLC fractions were collected, freeze dried and further analysed by CD. Fig. 7 shows the CD measurements of b-LG fractions collected by RP-HPLC of na-
tive WPC solutions and those sonicated for 60 min. When circularly polarised light passes through an absorbing optically active medium, the speeds between right and left polarisation differ as well as their wavelengths and the extent to which they are absorbed. One of the two polarised light beams is absorbed more than the other and this wavelength dependant difference in absorption is measured yielding the CD spectrum. A circular dichroism signal can be positive and negative depending on whether left-circularly polarised light absorbed to a greater extent than right or to a lesser extent. a-helix, b-sheet, and random coil structures each give rise to a CD spectrum of characteristic shape and magnitude. The secondary structure of b-LG comprises nine b-strands, a short a-helix segment and three helicoidal turns. The presence of a peak minimum at 218 nm confirms that both sonicated and unsonicated samples exhibit predominantly b-strand behaviour [20,37,40]. Approximately 38.2% b-strand was identified through CONTIN algorithm at 25 °C. This value is close to the 39% obtained by Lozano et al. [40] at ambient temperature. In contrast, De Jong et al. [41] identified 54% b-strands at the lower temperature of 15 °C. Sonication for 60 min resulted in minor changes in the b-LG spectra. Neither the shape nor the amplitude was affected to a significant extent but a slight shift towards the lower wavelengths was observed. Sonication resulted in a 10% increase in the a-helix component and a 6–9% decrease in the b-sheet and turn components. These changes are, however, small in comparison to those resulting from pressure and thermal treatments of b-LG [30,41]. Pressure induced spectral changes were reportedly indicative of an increase in the proportion of a-helix in the protein consistent with the present results. Considering that the secondary structure at a given point in a protein not only depends on the local sequence of amino acids but also on the interactions between different parts of the molecule, it is possible that these interactions were disrupted by sonication. Without these interactions, many parts of the b-LG protein that form b-sheets in the native form would assume a preferred a-helical secondary structure [30]. Stathpulos et al. [22] reported contradictory findings which showed that sonication-induced aggregates have high b-content, and proteins with significant native a-helical structure show increased b-structure with a concomitant decrease in a-helix structure in the aggregates. This difference might be due to sonication of different proteins in the two studies and the behavioural complexity of a WPC protein mixture in comparison with a pure protein solution. 4. Conclusions Sonication of reconstituted WPC solutions resulted in small changes to the thermal behaviour of proteins but did not change denaturation temperatures. Changes in the thiol group content were not observed following sonication. The enthalpy of denaturation decreased when sonicating WPC solutions for up to 5 min but increased thereafter. It is possible that prolonged sonication resulted in protein re-aggregation. Only minor changes to the protein secondary structural and surface hydrophobicity were observed. Although these minor changes cannot be ruled out completely, sonication in general does not appear to change the protein structure of WPC solutions to a significant degree, which may otherwise influence the functional properties of dairy systems during processing. Acknowledgement This study was supported by an Australian Research Council – Linkage Project and Dairy Innovation, Australia Ltd.
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
References [1] M.E. Lucena, S. Alvarez, C. Menendez, R.A. Riera, R. Alvarez, D-Lactoglobulin removal from whey protein concentrates production of milk derivatives as a base for infant formulas, Separation and Purification Technology 52 (2006) 310–316. [2] E.E.G. Rojas, J.S.R. Coimbra, L.A. Minim, A.D.G. Zuniga, S.H. Saraiva, V.P.R. Minim, Size-exclusion chromatography applied to the purification of whey proteins from the polymeric and saline phases of aqueous two-phase systems, Process Biochemistry 39 (2004) 1751–1759. [3] V. Bernal, P. Jelen, Thermal stability of whey proteins – a calorimetric study, Journal of Dairy Science 68 (1985) 2847–2852. [4] A. Kato, Y. Osako, N. Matsudomi, K. Kobayashi, Changes in the emulsifying and foaming properties of proteins during heat denaturation, Agricultural Biological Chemistry 47 (1983) 33–37. [5] M. Paulsson, P. Dejmek, Thermal denaturation of whey proteins in mixtures with caseins studied by differential scanning calorimetry, Journal of Dairy Science 73 (1990) 590–600. [6] P. Relkin, L. Eynard, B. Launay, Thermodynamic parameters of b-lactoglobulin and a-Lactalbumin. A DSC study of denaturation by heating, Thermochimica Acta 204 (1992) 11–121. [7] M. Ashokkumar, D. Sunartio, S. Kentish, R. Mawson, L. Simons, K. Vilkhu, C. Versteeg, Modification of food ingredients by US to improve functionality: a preliminary study on a model system, Innovative Food Science and Emerging Technologies 9 (2008) 155–160. [8] D. Knorr, M. Zenker, V. Heinz, D. Lee, Application and potential of ultrasonics in food processing, Trends in Food Science & Technology 15 (2004) 261–266. [9] M. Villamiel, P. De Jong, Influence of high intensity US and heat treatment in continuous flow in fat, proteins and native enzymes of milk, Journal of Agricultural and Food Chemistry 48 (2000) 472–478. [10] S. Balachandran, S. Kentish, R. Mawson, M. Ashokkumar, Ultrasonic enhancement of the supercritical extraction from ginger, Ultrasonics Sonochemistry 13 (2006) 471–479. [11] R. Bhaskaracharya, S. Kentish, M. Ashokkumar, Selected applications of ultrasonics in food processing, Food Engineering Review 1 (2009) 31–49. [12] E. Ciawi, J. Rae, M. Ashokkumar, F. Grieser, Determination of temperatures within acoustically generated bubbles in aqueous solutions at different US frequencies, Journal of Physical Chemistry 110 (2006) 13656–13660. [13] M. Ashokkumar, J. Lee, S. Kentish, F. Grieser, Bubbles in an acoustic field: An overview, Ultrasonics Sonochemistry 14 (2004) 470–475. [14] M. Ashokkumar, J. Lee, B. Zisu, R. Bhaskarcharya, M. Palmer, S. Kentish, Sonication increases the heat stability of whey proteins, Journal of Dairy Science 92 (2009) 5353–5356. [15] M.J. Taylor, T. Richardson, Antioxidant activity of skim milk: effect of heat and resultant sulfhydryl groups, Journal of Dairy Science 63 (1980) 1783–1795. [16] M.J. Taylor, T. Richardson, Antioxidant activity of skim milk: effect of sonication, Journal of Dairy Science 63 (1980) 1938–1942. [17] L.A. Tedford, C.J. Schaschke, Induced structural change to b-lactoglobulin by combined pressure and temperature, Biochemical Engineering Journal 5 (2000) 73–76. [18] Y. Hong, L.K. Creamer, Changed protein structures of bovine b-lactoglobulin B and a-lactalbumin as a consequence of heat treatment, International Dairy Journal 12 (2002) 345–359. [19] T.X. Liu, B.P. Relkin, B. Launay, Thermal denaturation and heat-induced gelation properties of b-lactoglobulin. Effects of some chemical parameters, Thermochimica Acta 246 (1994) 387–403. [20] I. Gulserren, D. Guzey, D. Bruce, J. Weiss, Structural and functional changes in ultrasonicated bovine serum albumin solutions, Ultrasonics Sonochemistry 14 (2007) 173–183.
957
[21] F. Cavalieri, M. Ashokkumar, F. Grieser, F. Caruso, Ultrasonic synthesis of stable, functional Lysozyme microbubbles, Langmuir 24 (2008) 10078–10083. [22] P.B. Stathpulos, G.A. Scholz, Y. Hwang, J.A.O. Rumfeldt, J.R. Lepock, E.M. Meiering, Sonication of proteins causes formation of aggregates that resemble amyloid, Protein Science 13 (2004) 3017–3027. [23] C.E. Marchioni et al., Structural changes induced in proteins by therapeutic ultrasounds, Ultrasonics 49 (2009) 569–576. [24] M. Ashokkumar, S. Kentish, J. Lee, B. Zisu, M. Palmer, M.A. Augustin, Processing of dairy ingredients by ultra-sonication, PCT Int. Appl. 2009, WO2009/079691 A1. [25] B. Zisu, R. Bhaskaracharya, S. Kentish, M. Ashokkumar, Ultrasonic processing of dairy systems in large scale reactors, Ultrasonics Sonochemistry 17 (2010) 1075–1081. [26] P.S. Patrick, H.E. Swaisgood, Sulfhydryl and disulfide groups in skim milk as affected by direct ultra high temperature heating and subsequent storage, Journal of Dairy Science 59 (1976) 594–600. [27] S. Ou, K.C. Kwok, Y. Wang, H. Bao, An improved method to determine SH and S–S group content in soy milk protein, Food Chemistry 88 (2004) 317–320. [28] S. Visser, C.J. Slangen, H.S. Rollema, Phenotyping of bovine milk proteins by reversed-phase-high performance liquid chromatography, Journal of Chromatography 548 (1991) 361–370. [29] M. Patel, A. Kilara, L.M. Huffman, S.A. Hewitt, A.V. Houlihan, Studies on whey protein concentrates 1.Compositional and thermal properties, Journal of Dairy Science 73 (1990) 1439–1449. [30] A. Aouzelleg, L. Bull, DSC study of pressure/temperature processed b-LG: the effect of dextran sulphate, Food Research International 37 (2004) 933–940. [31] M. Dissanayake, T. Vasiljevic, Functional properties of whey proteins affected by heat treatment and hydrodynamic high pressure shearing, Journal of Dairy Science 92 (2009) 1387–1397. [32] S.M. Fitzsimons, D.M. Mulvihill, E.R. Morris, Denaturation and aggregation processes in thermal gelation of whey proteins resolved by DSC, Food Hydrocolloids 21 (2007) 638–644. [33] S.M. Choi, S.T. Kwok, C.Y. Ma, C.Y. Conformational study of SPI treated with ultrasound, (2009) Poster presentation. [34] S.M. Fitzsimons, E.R. Morris, D.M. Mulvihill, Resolution of the denaturation and aggregation processes during whey protein gelation by differential scanning calorimetery, Irish Journal of Agricultural and Food Research 43 (2004) 109– 146. [35] A. Aouzelleg, L. Bull, N.C. Price, S.M. Kelly, Molecular studies of pressure/ temperature induced structural changes in bovine b-lactoglobulin, Journal of Food and Agriculture 84 (2004) 398–404. [36] P. Relkin, Differential scanning calorimetry: a useful tool for studying protein denaturation, Thermochimica Acta 246 (1994) 371–386. [37] L. Nygren-Babol, K.L. Karonen, The effect of different folate forms on denaturation of bovine folate binding protein, International Dairy Journal 19 (2009) 437–442. [38] C. Holt, C. Apparent, Apparent chemical composition of nine commercial or semi-commercial whey protein concentrates, isolates and fractions, International Journal of Food Science & Technology 34 (1999) 543–556. [39] I.M.P.L.V.O. Ferreira, M.B.P.P. Oliveira, Determination of Casein macropeptide by an RP-HPLC method and monitoring of the addition of Rennet Whey to powdered milk, Journal of Liquid Chromatography & Related Technologies 26 (2003) 99–107. [40] J.M. Lozano, G.I. Giraldo, C.M. Romero, An improved method for isolation of blactoglobulin, International Dairy Journal 18 (2008) 55–63. [41] H.H.J. de Jong, T. Groneveld, J. de Groot, Mild Isolation Procedure Discloses New Protein Structural Properties of b-Lactoglobulin, Journal of Dairy Science 84 (2001) 562–571.
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultsonch
Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate Jayani Chandrapala a,b, Bogdan Zisu c, Martin Palmer c, Sandra Kentish a,b, Muthupandian Ashokkumar a,b,⇑ a
School of Chemistry, University of Melbourne, VIC 3010, Australia Department of Chemical and Biomolecular Engineering, University of Melbourne, VIC 3010, Australia c Dairy Innovation Australia, Sneydes Road, Werribee, VIC 3030, Australia b
a r t i c l e
i n f o
Article history: Received 19 October 2010 Received in revised form 24 December 2010 Accepted 27 December 2010 Available online 31 December 2010 Keywords: Ultrasound Differential scanning calorimetry Circular dichroism Thiol groups Whey protein concentrates
a b s t r a c t The sonication-induced changes in the structural and thermal properties of proteins in reconstituted whey protein concentrate (WPC) solutions were examined. Differential scanning calorimetry, UV–vis, fluorescence and circular dichroism spectroscopic techniques were used to determine the thermal properties of proteins, measure thiol groups and monitor changes to protein hydrophobicity and secondary structure, respectively. The enthalpy of denaturation decreased when WPC solutions were sonicated for up to 5 min. Prolonged sonication increased the enthalpy of denaturation due to protein aggregation. Sonication did not alter the thiol content but resulted in minor changes to the secondary structure and hydrophobicity of the protein. Overall, the sonication process had little effect on the structure of proteins in WPC solutions which is critical to preserving functional properties during the ultrasonic processing of whey protein based dairy products. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Cheese whey, a by-product of the dairy industry is a source of valuable nutrients with important functional and nutritional properties. Whey protein concentrates and whey protein isolates are also important ingredients in the manufacture of value added products [1]. The major proteins in such whey systems are the milk globular proteins, a-lactalbumin (LA) and b-lactoglobulin (LG), along with small amounts of serum albumin and immunoglobulins [2]. The heat induced aggregation of whey protein based dairy systems has been an ongoing problem in dairy processing. Exposure of whey proteins to temperatures greater than 70 °C denatures the proteins and exposes buried thiol groups. It is recognised that the thermal behaviour is governed mainly by b-LG [3]. The heat induced unfolded conformation of this protein exposes functional groups, which are otherwise buried in the native structure, thereby increasing their reactivity. Particularly important is the increased reactivity of the thiol groups of cysteine. In addition to disulfide interchange, the unfolded protein is more susceptible to protein– protein interaction via calcium bridging and hydrophobic bonding [4]. The denatured whey proteins form complexes among themselves as well as with casein micelles, leading to the formation of large aggregates. This results in excessive thickening or gelling ⇑ Corresponding author at: School of Chemistry, University of Melbourne, Melbourne VIC 3010, Australia. Tel.: +61 3 83447090; fax: +61 3 93475180. E-mail address: [email protected] (M. Ashokkumar). 1350-4177/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2010.12.016
during thermal processing. The endothermic denaturation of the proteins and exothermic aggregation can both be monitored by changes in the heat flows to and from protein samples as a function of temperature [5,6]. The development of nonthermal processing methods to modify food ingredients has generated interest in the food industry recently [7]. In particular, the use of high intensity ultrasound has attracted considerable attention due to its promise in the development of novel, gentle but targeted processes to improve the quality and safety of processed foods. Furthermore, the technology offers the potential to improve existing processes [7,8]. Ultrasound has been developed for several food processing applications such as degassing of liquids, extraction of enzymes and proteins, inactivation of microorganisms [9], ultrasound-induced product modification [7], extraction of gingerol from ginger [10], homogenisation of milk [9], and acceleration of ageing of wines [11]. Acoustic cavitation is the fundamental process responsible for the initiation of many of the sonochemical reactions in liquids. In an acoustic field, bubbles can undergo growth by rectified diffusion and by bubble–bubble coalescence [12]. The collapse of cavitation bubbles results in the generation of high temperatures within the bubbles and is accompanied by emission of light (sonoluminescence). Cavitation also results in other mechanical, physical and chemical effects such as shockwave formation, turbulent motion of the liquid and generation of radicals [13]. The large shear forces generated can break inter and intramolecular bonds, which in turn may lead to fragmentation of clusters and aggregates.
952
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
Most recently, it has been shown that a short burst of ultrasound can help to break apart heat-induced aggregates and prevents their reformation on subsequent heating, thereby avoiding further increases in viscosity [14]. The physical shear forces generated during acoustic cavitation are believed to be responsible for the breakdown of the heat-induced aggregates and subsequent viscosity reduction. This approach is advantageous in a food application as it appears that changes in chemical composition are not required to achieve the heat stability in this case. Heat-induced changes to –SH groups and S–S bonds of food proteins [15,16], heat-induced secondary and tertiary structural changes of proteins [17,18] and thermal properties of protein solutions [5,6,19] have been extensively reported. However, less work has been reported on changes to protein properties as a result of sonication. Paulsson and Dejmek [5] state that ultrasound treatment acts differently to temperature treatment as it does not disrupt covalent bonds. They find that the effects of ultrasound are minimal on the secondary and tertiary structures of proteins. However, other researchers have found that sonication does result in some structural changes of individual proteins [20]. For example, Cavalieri et al. [21] have observed sonochemical cross-linking between the thiol groups of protein molecules under specific experimental conditions. Taylor and Richardson [16] found that the total thiol groups in skim milk decreased upon prolonged sonication under uncontrolled temperature conditions. In this case, it is possible that the heat generated during sonication reduced the thiol group content. In a separate study by Gulserren et al. [20], the amount of reactive thiol groups in BSA decreased after 90 min of sonication. The authors suggested that the decreased number of thiol groups may be attributed to the formation of protein aggregates. Further, a secondary structural analysis of native and ultrasonicated BSA showed a small increase in ordered structure upon ultrasonication which differed distinctly from any thermal treatment where ordered structure is lost. Stathpulos et al. [22] also observed aggregate formation following sonication of proteins. They used circular dichroism to show that these aggregates have high b-sheet content, and proteins containing significant native a-helical structure showed an increase in the b-sheet structure in the aggregates. Marchioni et al. [23] state that the behaviour of proteins under sonication depends mainly on the dominant secondary structure type (a-helix or b-sheets) and on the grade of the ordered structure. Their results suggest that free radicals produced by water sonolysis have an important role in causing the structural changes. Our previous work has shown a clear change in the physical and functional behaviour of sonicated whey protein concentrate (WPC) [24,25]. Sonication at 20 kHz for short times reduced the viscosity of WPC solutions, but an increase in viscosity was observed if sonication times were extended. When heated, the gel strength of sonicated solutions increased, while gelation time and gel syneresis were reduced. The microstructure of sonicated whey protein gels showed a densely packed protein network. Although highly reproducible, the precise mechanism or molecular basis for these effects is not known. The aim of the present study is to identify thermal and structural changes to the protein in whey protein concentrate solutions induced by sonication.
2. Materials and methods 2.1. Materials Commercial WPC powder was obtained from Warrnambool Cheese and Butter Factory (Allansford, Victoria, Australia). The ‘‘WPC 80’’ powder was derived from cheddar cheese whey and
contained 81.5% protein, 10.4% lactose and 4.4% fat. Ultra pure (MilliQ) water was used in all experiments. 2.2. Preparation of reconstituted WPC solutions WPC powder was reconstituted in MilliQ water to obtain 50 g/ kg (w/w) solutions. The solutions were continuously stirred for one hour at room temperature (25 ± 3 °C) to ensure complete mixing and stored overnight at 4 °C. On the following day, the solutions were equilibrated at 25 °C for 1 h prior to further analysis. 2.3. Sonication 60 mL solutions were sonicated in a glass vessel equipped with a cooling jacket using a 20 kHz, 450 W Ultrasonic horn (19 mm diameter, Branson Sonifier 450, Danbury, CT) at an amplitude of 50% for 1, 5, 10, 20, 30 and 60 min. The actual power delivered to the solution was 31 W as determined by calorimetry. This power was used in order to correlate the results with our previous study [14]. During sonication, chilled water was continuously circulated through the cooling jacket to maintain the sample temperature at 6 ± 4 °C. 2.4. DSC measurements Thermal analysis of whey proteins was carried out on a Perkin Elmer dynamic scanning calorimeter (DSC 2 Perkin Elmer Corporation, Winter Street, Waltham Massachusetts, 02451 USA) equipped with Pyris Manager, v.5.0002 software. The instrument was calibrated for temperature (T) and change in enthalpy (DH) using indium (Tpeak = 155.87 °C, DH = 28.234 J/g). Approximately 12– 15 mg of 12% (w/w) WPC solutions were accurately weighed into aluminium pans and hermetically sealed. An empty pan of equal weight was used as a reference. The scanning temperature was raised from 25 to 100 °C at 10 °C/min. The study was arranged as a randomised block design with time as the main factor. All experiments were replicated thrice with subsequent sub sampling. The pans were weighed before and after the DSC scan to ensure the effectiveness of sealing. Endothermic heat flow (endotherms) as shown by the instrument was represented as peaks in the downward direction (negative maxima). Temperature at maximum heat flow of endothermic transition and the area underneath the peak from the thermal curve which represents the enthalpy (J/g) was calculated based on the protein mass within the solution. 2.5. Surface hydrophobicity measurements For the fluorometric assay, the stock protein solutions were diluted with 0.1 M pH 7 phosphate buffer solutions to typical concentration ranges of 0.005–0.025% (w/w) using 1-anilinonaphthalene-8-sulfonate (ANS). A stock solution of 8 103 M ANS was prepared in 0.1 M pH 7 phosphate buffers. ANS stock solution was wrapped in aluminium foil to prevent light exposure and stored at room temperature. For hydrophobicity determination using ANS, the excitation and emission slits and wave lengths were set at 5/5 and 390/470 nm, respectively. 20 lL of ANS solution was added to 4 mL of diluted protein solution, vortexed and kept in the dark for 15 min. The Relative Fluorescence Intensity (RFI) of each solution was measured starting from buffer blank and then the lowest to highest protein concentration. The RFI of each dilution blank was subtracted from that of the corresponding protein solution with ANS to obtain the net RFI. Surface hydrophobicity was expressed as the initial slope of the plot of standardised net RFI values vs. % protein concentration computed using least squares linear regression analysis (Microsoft excel 8.0).
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
Surface hydrophobicity was determined by taking the average of three analyses. In all cases, R2 values of >0.95 were noted for the linear regression analyses used to calculate the surface hydrophobicity. 2.6. Thiol group determination A method developed by Patrick and Swaisgood [26] for the determination of reactive and total thiol groups was adopted in the present study. The method uses Ellman’s reagent (5,50 Dithio-bis (2-nitrobenzoic acid (DTNB)) to react specifically with the thiol groups of the solutions to form a yellow coloured complex, which can be measured by absorbance at 412 nm. An aliquot (5 mL) of the WPC solution was diluted to 8 mL with 0.1 M phosphate buffer at pH 8. The pH of the solution was re-adjusted to pH 8 with 1 M NaOH. To this solution mixture, 1 mL of 2 mM DTNB (prepared using 0.1 M phosphate buffer at pH 7) was added. Colour development was allowed to proceed for 30 min. Separate experiments were performed in order to optimise the incubation time. A 30 min incubation time showed maximum absorbance which started to fade with longer exposure times. The solution was centrifuged at 35000g for 15 min (25 ± 2 °C) after adjusting the volume to 11 mL. The supernatant liquid was removed by syringe and filtered through a 0.45 lm filter. An aliquot (1 mL) of WPC solution was added to 1.44 g of urea to expose thiol groups that were buried inside the globular protein structure. 3 mL of 0.1 M phosphate buffer at pH 8 was added to the solution mixture. To this solution mixture, 1 mL of 2 mM DTNB (prepared using 0.1 M phosphate buffer at pH 7) was added. The volume of the solution mixture was adjusted to 7 mL. Colour was allowed to develop for 30 min and the solution mixture was filtered through a 0.45 lm filter. The absorbance of the thionitrobenzoate released by the reaction with thiols in the filtrates was read at 412 nm. The net absorbance was calculated as the difference between absorbance of samples with DTNB and that without DTNB:
DAbs412 ¼ Absðwith DTNBÞ Absðwithout DTNBÞ
ð1Þ
The concentration of thiol groups was then calculated according to the following formula [27]:
lmol SH=g ¼ ð73:53 DAbs412 DÞ=C
953
tained using a Jasco J-600 Spectropolarimeter (JASCO UK Ltd., Great Dunmow, UK) at 25 °C with a 0.1 cm path length quartz cell, a 10 nm/min scan rate and a 0.2 nm band width. Spectra were an average of 3 scans from 190 to 260 nm. The spectrum of 100 mM phosphate buffer was used as a blank and subtracted from the average three spectra to obtain a corrected spectrum for each sample. Spectra were determined in duplicate. Spectra were deconvoluted using the deconvolution software CONTIN to obtain information about the secondary structure. CONTIN, developed by Provencher and Glöckner (DICHROWEB website), fits the CD of unknown proteins by a linear combination of the spectra using a large data base of proteins with known conformations. An advantage of this method is the reliable estimate of b-turns in a protein. 2.9. Statistical analysis When necessary, one-way ANOVA with a 95% confidence interval was used. The ANOVA data with p < 0.05 were considered statistically significant. 3. Results and discussion 3.1. Differential scanning calorimetry Heat induced changes to whey protein structure are accompanied by changes in thermal properties. Fig. 1 represents the DSC thermograms of native and sonicated WPC solutions heated from 25 to 100 °C. All WPC samples showed a single broad endothermic peak at approximately 77 °C. This peak relates to the denaturation temperature of b-LG, the major whey protein and agrees with the work by others [5,29–31]. A shoulder peak also appears around 65 °C due to the denaturation of a-LA as confirmed by literature. Patel et al. [29] reported a shoulder between 62 and 72 °C resulting from the overlapping of two endothermic peaks and suspect that this may be due to the denaturation of a-LA. Fitzsimons et al. [32] also found a dominant endotherm centered at 75 °C and a shoulder at 62 °C. They assigned those peaks as the denaturation of b-LG and a-LA, respectively. Denaturation involves dissociation of intramolecular bonds (non-covalent and in some cases, covalent (disulfide) bonds) and
ð2Þ
where; DAbs412 is the net absorbance measured at 412 nm by the thiol groups with/without DTNB, D is the dilution factor (if any), C is the total solids content in milk expressed in mg/mL. 2.7. Reversed phase high performance liquid chromatography Reversed-phase (RP) HPLC was performed using a Shimadzu LC 20 HPLC system equipped with a Jupiter 5l C18 300A column (250 4.6 mm I.D.) and a UV–vis detector. Solvent A was a mixture of acetonitrile–water–trifluoroacetic acid (100:900:1 v/v/v) and solvent B was the same mixture with the proportions 900:100:0.7 (v/v/v). The solvent gradient reported by Visser et al. [28] was used for the elution. All whey protein samples were filtered through a 0.45 lm syringe filter before injection. The elution profiles were monitored at 280 nm. The total run time was 55 min. b-LG fractions were collected using a Shimadzu fraction collector. Fractions were freeze dried before further analysis by circular dichroism. 2.8. Circular dichroism spectroscopy Protein solutions were prepared at final concentrations of 0.5 mg/mL in 100 mM phosphate buffer at pH 7.0. Data were ob-
Fig. 1. DSC thermograms of reconstituted WPC solutions as a function of sonication time.
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
is therefore an endothermic process. Aggregation of the denatured molecules involves formation of new intermolecular bonds, and would therefore be expected to give rise to an exothermic peak in DSC measurements [33]. However, DSC studies have indicated that, when occurring concurrently, these two reactions can give rise to a single endothermic peak [34]. The DSC data in Fig. 1 reveal that in this case, endothermic denaturation reactions dominated within the temperature range studied, even though both denaturation and aggregation would be expected under these conditions [35]. Aouzelleg and Bull [30] stated that the reason for the broadness of the typical single DSC peak (Fig. 1) is due to the increasing number of proteins denaturing in response to continued heating. Furthermore, an unpredictable exothermic aggregation might also contribute to the broadness of thermograms [30]. In contrast, Choi et al. [33] concluded that the broadening of the thermogram is due to intermediate formations. In a given protein solution, although all molecules are in the same physicochemical conditions, a distribution of conformations is observed. The majority of proteins would be expected to be in a conformation state of minimal free energy but a number of molecules would be present in slightly higher energetic states. The proteins in higher energy states are partially or incorrectly folded conformers that have similarities with the native structure but contain non-native regions [30]. The variation in the depth of DSC profiles (Fig. 1) suggests that sonication does alter the thermal behaviour of whey proteins. These results are consistent with our previous work showing changes in the thermal gelling behaviour of sonicated WPC solutions [14,24,25]. However, the denaturation temperatures (75– 77 °C) of native and sonicated WPC solutions, as a function of sonication time, were not significantly different (Fig. 2). Fig. 3 represents the enthalpy changes of reconstituted WPC solutions as determined from these profiles. The enthalpy of native WPC was 0.19 J/g and agrees with data obtained by others [5,29– 31]. Dissanayake and Vasiljevic [31] suggest that these enthalpy values correlate with the content of ordered secondary structure and could be used to monitor the proportion of undenatured proteins. Their study shows a decrease in enthalpy upon high pressure shearing (micro fluidisation) and this was correlated to the destruction of hydrophobic interactions caused by the high pressure treatment. A slight increase in enthalpy following more extended micro fluidisation was correlated to a possible reformation of intra and intermolecular hydrophobic bonds from the resulting disordered protein molecules. These trends are consistent with our data. Sonication for up to 5 min, showed a decrease in enthalpy highlighting the
0.300 0.280
Enthalpy of Denaturation (J/g)
954
0.260 0.240 0.220 0.200 0.180 0.160 0.140 0.120 0.100
0
1
5
10
20
30
60
Sonication time (min) Fig. 3. Denaturation enthalpy changes of reconstituted WPC solutions as a function of sonication time.
destruction of bonds between proteins. However, the enthalpy increased on prolonged sonication beyond 5 min suggesting potential re-aggregation. Indeed, Choi et al. [33] confirmed the formation of network aggregates during prolonged sonication of soy protein isolate. These denaturation enthalpy changes (Fig. 3) also reflect the viscosity data obtained previously. Sonication for up to 5 min reduced the viscosity of protein solutions but increased when sonicating for longer periods (>5 min) [14]. The heating of globular proteins disrupts some of the forces responsible for the stability of tertiary and/or secondary protein structures. These forces include hydrogen interactions between the polar groups and interactions of non-polar groups (hydrophobic interactions) through the surrounding water molecules which form cages around hydrophobic groups. Electrostatic bonds and Vander Waals interactions are also involved in this heat denaturation process, although to a lesser extent [36,37]. However, on prolonged heat treatment, the process is reversed and heat enhances protein aggregation. Sonication may also result in disruption of intramolecular forces of proteins due to shear forces and lead to the formation of aggregates on prolonged sonication as is the case for heating.
300
250
Surface Hydrophobicity
Denaturation Temperature (C)
80.00
78.00
76.00
74.00
200
150
100
50 72.00 0 70.00
0
1
5
10
20
30
60
Sonication time (min) Fig. 2. Denaturation temperatures of reconstituted WPC solutions as a function of sonication time.
Samples
Native 10 min sonication 60 min sonication
1 min sonication 20 min sonication
5 min sonication 30 min sonication
Fig. 4. Surface hydrophobicities of reconstituted WPC solutions as a function of sonication time.
955
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
The importance of hydrophobic interactions for the stability, conformation, and function of proteins is well recognised. Due to the macromolecular structure of proteins, the surface hydrophobicity has more influence on functionality than the total hydrophobicity. The surface hydrophobicity of reconstituted WPC solutions as a function of sonication time is represented in Fig. 4. The surface hydrophobicity of the proteins increased for up to 5 min of sonication presumably due to the unfolding of the proteins resulting from minor structural changes. However, the surface hydrophobicities decreased after sonication for more than 5 min, which is a sign of protein aggregation which in turn protects the hydrophobic regions of the proteins. The partially denatured proteins with increased surface hydrophobicity might cause more extensive bonding, reducing the surface hydrophobicities on prolonged sonication. These results also support the trends observed in the enthalpy data (Fig. 3) and the viscosity data of previous studies [14]. Although the trends observed are
a
100.0
Absorbance
3.2. Surface hydrophobicity
0
5
10
15
20
25
Amount of Reactive Thiol Groups (umol)
5 min Sonication 10 min Sonication 20 min Sonication 30 min Sonication 60 min Sonication 50.0
25.0
0.0
Samples 400.0
Native 1 min Sonication
Amount of total Thiol Groups (umol)
350.0
5 min Sonication 10 min Sonication
300.0
20 min Sonication 30 min Sonication
250.0
40
45
50
Fig. 6. RP-HPLC profiles of reconstituted WPC as a function of sonication time (Control, 1, 5, 10, 20, 30 & 60 min from bottom to top, respectively).
1 min Sonication
b
35
Time/min
Native
75.0
30
60 min Sonication
200.0
150.0
100.0
50.0
0.0
Samples Fig. 5. The amount of reactive and total thiol group content of 5% w/w WPC subjected to sonication.
956
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957 30
20
10
0 200
210
220
230
240
250
260
270
-10
-20
Wavelength (nm) Fig. 7. Far UV CD spectrum of b-LG fractions obtained through HPLC of native (solid line) and 60 min sonicated (dashed line) WPC.
small, the changes in hydrophobicity as a function of sonication time are significant (p < 0.05). 3.3. Thiol content Fig. 5(a) and (b) represents the reactive- and total-thiol content of reconstituted WPC as a function of sonication time, respectively. Neither the reactive- nor total-thiol groups changed as a result of sonication. This is contrary to the results of other authors. Gulseren et al. [20] and Taylor and Richardson [16] observed a reduction in the free thiol content of proteins as a function of sonication time. This difference may arise due to the differences in the intensity and duration of the applied ultrasound. In particular, the lack of temperature control in the work of Taylor and Richardson [16] may explain their contradicting results. Another possibility is the complexity of the WPC solution, which contains a mixture of proteins rather than the pure BSA used by Gulseren et al. [20]. The intra-molecular location of the free thiol groups in the b-LG and a-LA proteins examined here may also make them less susceptible to degradation by ultrasound. 3.4. RP-HPLC Fig. 6 represents the RP-HPLC profiles of reconstituted WPC solutions sonicated for different time periods up to 60 min. The chromatographic profile for native WPC was similar to those reported previously [1,38,39]. The earliest eluting peaks (5– 10 min) are unidentified but Ferreira and Oliveira [39] suggest they may correspond to proteose-peptone derived from degradation of b-casein in milk and recovered in whey. The two large peaks eluting at 20 and 25 min represent a-LA and b-LG, respectively, as confirmed by standards. The b-LG peak was resolved in two parts highlighting the existence of b-LG’s A and B variants. Interestingly, the elution peaks did not show major changes after sonication for up to 60 min. HPLC analysis and UV-detection at this resolution failed to identify any minor protein structural changes associated with sonication. 3.5. CD measurements To identify sensitive structural changes beyond the resolution of HPLC, the sonicated and unsonicated b-LG HPLC fractions were collected, freeze dried and further analysed by CD. Fig. 7 shows the CD measurements of b-LG fractions collected by RP-HPLC of na-
tive WPC solutions and those sonicated for 60 min. When circularly polarised light passes through an absorbing optically active medium, the speeds between right and left polarisation differ as well as their wavelengths and the extent to which they are absorbed. One of the two polarised light beams is absorbed more than the other and this wavelength dependant difference in absorption is measured yielding the CD spectrum. A circular dichroism signal can be positive and negative depending on whether left-circularly polarised light absorbed to a greater extent than right or to a lesser extent. a-helix, b-sheet, and random coil structures each give rise to a CD spectrum of characteristic shape and magnitude. The secondary structure of b-LG comprises nine b-strands, a short a-helix segment and three helicoidal turns. The presence of a peak minimum at 218 nm confirms that both sonicated and unsonicated samples exhibit predominantly b-strand behaviour [20,37,40]. Approximately 38.2% b-strand was identified through CONTIN algorithm at 25 °C. This value is close to the 39% obtained by Lozano et al. [40] at ambient temperature. In contrast, De Jong et al. [41] identified 54% b-strands at the lower temperature of 15 °C. Sonication for 60 min resulted in minor changes in the b-LG spectra. Neither the shape nor the amplitude was affected to a significant extent but a slight shift towards the lower wavelengths was observed. Sonication resulted in a 10% increase in the a-helix component and a 6–9% decrease in the b-sheet and turn components. These changes are, however, small in comparison to those resulting from pressure and thermal treatments of b-LG [30,41]. Pressure induced spectral changes were reportedly indicative of an increase in the proportion of a-helix in the protein consistent with the present results. Considering that the secondary structure at a given point in a protein not only depends on the local sequence of amino acids but also on the interactions between different parts of the molecule, it is possible that these interactions were disrupted by sonication. Without these interactions, many parts of the b-LG protein that form b-sheets in the native form would assume a preferred a-helical secondary structure [30]. Stathpulos et al. [22] reported contradictory findings which showed that sonication-induced aggregates have high b-content, and proteins with significant native a-helical structure show increased b-structure with a concomitant decrease in a-helix structure in the aggregates. This difference might be due to sonication of different proteins in the two studies and the behavioural complexity of a WPC protein mixture in comparison with a pure protein solution. 4. Conclusions Sonication of reconstituted WPC solutions resulted in small changes to the thermal behaviour of proteins but did not change denaturation temperatures. Changes in the thiol group content were not observed following sonication. The enthalpy of denaturation decreased when sonicating WPC solutions for up to 5 min but increased thereafter. It is possible that prolonged sonication resulted in protein re-aggregation. Only minor changes to the protein secondary structural and surface hydrophobicity were observed. Although these minor changes cannot be ruled out completely, sonication in general does not appear to change the protein structure of WPC solutions to a significant degree, which may otherwise influence the functional properties of dairy systems during processing. Acknowledgement This study was supported by an Australian Research Council – Linkage Project and Dairy Innovation, Australia Ltd.
J. Chandrapala et al. / Ultrasonics Sonochemistry 18 (2011) 951–957
References [1] M.E. Lucena, S. Alvarez, C. Menendez, R.A. Riera, R. Alvarez, D-Lactoglobulin removal from whey protein concentrates production of milk derivatives as a base for infant formulas, Separation and Purification Technology 52 (2006) 310–316. [2] E.E.G. Rojas, J.S.R. Coimbra, L.A. Minim, A.D.G. Zuniga, S.H. Saraiva, V.P.R. Minim, Size-exclusion chromatography applied to the purification of whey proteins from the polymeric and saline phases of aqueous two-phase systems, Process Biochemistry 39 (2004) 1751–1759. [3] V. Bernal, P. Jelen, Thermal stability of whey proteins – a calorimetric study, Journal of Dairy Science 68 (1985) 2847–2852. [4] A. Kato, Y. Osako, N. Matsudomi, K. Kobayashi, Changes in the emulsifying and foaming properties of proteins during heat denaturation, Agricultural Biological Chemistry 47 (1983) 33–37. [5] M. Paulsson, P. Dejmek, Thermal denaturation of whey proteins in mixtures with caseins studied by differential scanning calorimetry, Journal of Dairy Science 73 (1990) 590–600. [6] P. Relkin, L. Eynard, B. Launay, Thermodynamic parameters of b-lactoglobulin and a-Lactalbumin. A DSC study of denaturation by heating, Thermochimica Acta 204 (1992) 11–121. [7] M. Ashokkumar, D. Sunartio, S. Kentish, R. Mawson, L. Simons, K. Vilkhu, C. Versteeg, Modification of food ingredients by US to improve functionality: a preliminary study on a model system, Innovative Food Science and Emerging Technologies 9 (2008) 155–160. [8] D. Knorr, M. Zenker, V. Heinz, D. Lee, Application and potential of ultrasonics in food processing, Trends in Food Science & Technology 15 (2004) 261–266. [9] M. Villamiel, P. De Jong, Influence of high intensity US and heat treatment in continuous flow in fat, proteins and native enzymes of milk, Journal of Agricultural and Food Chemistry 48 (2000) 472–478. [10] S. Balachandran, S. Kentish, R. Mawson, M. Ashokkumar, Ultrasonic enhancement of the supercritical extraction from ginger, Ultrasonics Sonochemistry 13 (2006) 471–479. [11] R. Bhaskaracharya, S. Kentish, M. Ashokkumar, Selected applications of ultrasonics in food processing, Food Engineering Review 1 (2009) 31–49. [12] E. Ciawi, J. Rae, M. Ashokkumar, F. Grieser, Determination of temperatures within acoustically generated bubbles in aqueous solutions at different US frequencies, Journal of Physical Chemistry 110 (2006) 13656–13660. [13] M. Ashokkumar, J. Lee, S. Kentish, F. Grieser, Bubbles in an acoustic field: An overview, Ultrasonics Sonochemistry 14 (2004) 470–475. [14] M. Ashokkumar, J. Lee, B. Zisu, R. Bhaskarcharya, M. Palmer, S. Kentish, Sonication increases the heat stability of whey proteins, Journal of Dairy Science 92 (2009) 5353–5356. [15] M.J. Taylor, T. Richardson, Antioxidant activity of skim milk: effect of heat and resultant sulfhydryl groups, Journal of Dairy Science 63 (1980) 1783–1795. [16] M.J. Taylor, T. Richardson, Antioxidant activity of skim milk: effect of sonication, Journal of Dairy Science 63 (1980) 1938–1942. [17] L.A. Tedford, C.J. Schaschke, Induced structural change to b-lactoglobulin by combined pressure and temperature, Biochemical Engineering Journal 5 (2000) 73–76. [18] Y. Hong, L.K. Creamer, Changed protein structures of bovine b-lactoglobulin B and a-lactalbumin as a consequence of heat treatment, International Dairy Journal 12 (2002) 345–359. [19] T.X. Liu, B.P. Relkin, B. Launay, Thermal denaturation and heat-induced gelation properties of b-lactoglobulin. Effects of some chemical parameters, Thermochimica Acta 246 (1994) 387–403. [20] I. Gulserren, D. Guzey, D. Bruce, J. Weiss, Structural and functional changes in ultrasonicated bovine serum albumin solutions, Ultrasonics Sonochemistry 14 (2007) 173–183.
957
[21] F. Cavalieri, M. Ashokkumar, F. Grieser, F. Caruso, Ultrasonic synthesis of stable, functional Lysozyme microbubbles, Langmuir 24 (2008) 10078–10083. [22] P.B. Stathpulos, G.A. Scholz, Y. Hwang, J.A.O. Rumfeldt, J.R. Lepock, E.M. Meiering, Sonication of proteins causes formation of aggregates that resemble amyloid, Protein Science 13 (2004) 3017–3027. [23] C.E. Marchioni et al., Structural changes induced in proteins by therapeutic ultrasounds, Ultrasonics 49 (2009) 569–576. [24] M. Ashokkumar, S. Kentish, J. Lee, B. Zisu, M. Palmer, M.A. Augustin, Processing of dairy ingredients by ultra-sonication, PCT Int. Appl. 2009, WO2009/079691 A1. [25] B. Zisu, R. Bhaskaracharya, S. Kentish, M. Ashokkumar, Ultrasonic processing of dairy systems in large scale reactors, Ultrasonics Sonochemistry 17 (2010) 1075–1081. [26] P.S. Patrick, H.E. Swaisgood, Sulfhydryl and disulfide groups in skim milk as affected by direct ultra high temperature heating and subsequent storage, Journal of Dairy Science 59 (1976) 594–600. [27] S. Ou, K.C. Kwok, Y. Wang, H. Bao, An improved method to determine SH and S–S group content in soy milk protein, Food Chemistry 88 (2004) 317–320. [28] S. Visser, C.J. Slangen, H.S. Rollema, Phenotyping of bovine milk proteins by reversed-phase-high performance liquid chromatography, Journal of Chromatography 548 (1991) 361–370. [29] M. Patel, A. Kilara, L.M. Huffman, S.A. Hewitt, A.V. Houlihan, Studies on whey protein concentrates 1.Compositional and thermal properties, Journal of Dairy Science 73 (1990) 1439–1449. [30] A. Aouzelleg, L. Bull, DSC study of pressure/temperature processed b-LG: the effect of dextran sulphate, Food Research International 37 (2004) 933–940. [31] M. Dissanayake, T. Vasiljevic, Functional properties of whey proteins affected by heat treatment and hydrodynamic high pressure shearing, Journal of Dairy Science 92 (2009) 1387–1397. [32] S.M. Fitzsimons, D.M. Mulvihill, E.R. Morris, Denaturation and aggregation processes in thermal gelation of whey proteins resolved by DSC, Food Hydrocolloids 21 (2007) 638–644. [33] S.M. Choi, S.T. Kwok, C.Y. Ma, C.Y. Conformational study of SPI treated with ultrasound, (2009) Poster presentation. [34] S.M. Fitzsimons, E.R. Morris, D.M. Mulvihill, Resolution of the denaturation and aggregation processes during whey protein gelation by differential scanning calorimetery, Irish Journal of Agricultural and Food Research 43 (2004) 109– 146. [35] A. Aouzelleg, L. Bull, N.C. Price, S.M. Kelly, Molecular studies of pressure/ temperature induced structural changes in bovine b-lactoglobulin, Journal of Food and Agriculture 84 (2004) 398–404. [36] P. Relkin, Differential scanning calorimetry: a useful tool for studying protein denaturation, Thermochimica Acta 246 (1994) 371–386. [37] L. Nygren-Babol, K.L. Karonen, The effect of different folate forms on denaturation of bovine folate binding protein, International Dairy Journal 19 (2009) 437–442. [38] C. Holt, C. Apparent, Apparent chemical composition of nine commercial or semi-commercial whey protein concentrates, isolates and fractions, International Journal of Food Science & Technology 34 (1999) 543–556. [39] I.M.P.L.V.O. Ferreira, M.B.P.P. Oliveira, Determination of Casein macropeptide by an RP-HPLC method and monitoring of the addition of Rennet Whey to powdered milk, Journal of Liquid Chromatography & Related Technologies 26 (2003) 99–107. [40] J.M. Lozano, G.I. Giraldo, C.M. Romero, An improved method for isolation of blactoglobulin, International Dairy Journal 18 (2008) 55–63. [41] H.H.J. de Jong, T. Groneveld, J. de Groot, Mild Isolation Procedure Discloses New Protein Structural Properties of b-Lactoglobulin, Journal of Dairy Science 84 (2001) 562–571.