Role of ovomucoid in the gelation of a [beta]-lactoglobulin-ovomucoid mixture

Abstract

The effect of ovomucoid on gelation of [beta]-lactoglobulin--as induced by heating and subsequent cooling--was investigated using a mixture of 5 % (w/v) ovomucoid/5 % (w/v) [beta]-lactoglobulin and pure [beta]-lactoglobulin solutions (5 and 10 % (w/v)) with subsequent analysis by rheological measurement, ultrasonic spectroscopy, scanning electron microscopy, and sodium dodecyl sulfate polyacrylamide electrophoresis. For the three systems, the dynamic modulus of the mixed-protein sample was smaller than that of either of the two pure [beta]-lactoglobulin samples. Although ultrasonic-relative velocity temperature sweeps for all samples showed that the relative velocities decreased with increasing temperature, the gradient values differed. Namely, the decrease for the mixed-protein sample (12 m/s) was intermediate between those of the pure [beta]-lactoglobulin systems. Ultrasonic attenuations of all samples increased with increasing temperature, and the absolute attenuation value of the mixed-protein sample was also intermediate between those of the two pure [beta]-lactoglobulin samples. Electrophoresis performed with or without 2-mercaptoethanol suggested that ovomucoid forms an aggregate with [beta]-lactoglobulin via intermolecular disulfide bonds. Together, these results suggest that ovomucoid has a synergistic effect on [beta]-lactoglobulin gelation despite the great heat stability.

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Role of ovomucoid in the gelationof a -lactoglobulin-ovomucoid mixture

Naoko Yuno-Ohta1,2 & Tsubasa Kato1 & Shiho Ashizawa2 & Yoko Kimura2 &

Nami Maruyama2 & Takahisa Nishizu3

Received: 10 December 2015 /Revised: 22 March 2016 /Accepted: 23 March 2016 /Published online: 8 April 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract The effect of ovomucoid on gelation of -lactoglobulinas induced by heating and subsequent coolingwas investigated using a mixture of 5 % (w/v) ovomucoid/5 % (w/v) -lactoglobulin and pure -lactoglobulin solutions (5 and 10 % (w/v)) with subsequent analysis by rheological measurement, ultrasonic spectroscopy, scanning electron microscopy, and sodium dodecyl sulfate polyacrylamide electrophoresis. For the three systems, the dynamic modulus of the mixed-protein sample was smaller than that of either of the two pure -lactoglobulin samples. Although ultrasonic-relative velocity temperature sweeps for all samples showed that the relative velocities decreased with increasing temperature, the gradient values differed. Namely, the decrease for the mixed-protein sample (12 m/s) was intermediate between those of the pure -lactoglobulin systems. Ultrasonic attenuations of all samples increased with increasing temperature, and the absolute attenuation value of the mixed-protein sample was also intermediate between those of the two pure -lactoglobulin samples. Electrophoresis performed with or without 2-mercaptoethanol suggested that ovomucoid forms an aggregate with -lactoglobulin via intermolecular disulfide bonds. Together, these results suggest that ovomucoid has a synergistic effect on -lactoglobulin gelation despite the great heat stability.

Keywords -lactoglobulin . Ovomucoid . Gelation . Ultrasonic spectroscopy . SDS-PAGE . Scanning electron microscopy

Introduction

Protein-protein interaction in heat-induced gel formation is important for food processing, in particular, for processing of albuminous food. -lactoglobulin (-LG) exists as a dimer in native state, and the gelation stepwise proceeds [1]. First, dimeric -LG easily dissociates to form monomers in a process controlled by various factors, e.g., mild heat treatment, pH change, and/or addition of salt, reductant, or detergent. Then, the monomers interact via intermolecular disulfide formation involving solvent-accessible cysteines and results in the formulation of the gel architecture stabilized by noncovalent intermolecular interactions. Conversely, egg-white ovomucoid (OM) is known as a heat-stable protein because it does not self-aggregate due to its large carbohydrate content. In addition, it has no free cysteines, only intramolecular disulfides [2]. We hypothesized, given the different properties of -LG and OM that if they were allowed to interact, the properties of the resultant material would be different from those of the starting proteins. Early on, Matsuda et al. [3] investigated the interaction between OM and lysozyme and found that the two native proteins interacted via electro-static forces. When those proteins were heat denatured, they aggregated via hydrophobic interactions, hydrogen bond, and disulfide bond formation if the pH of the solution was neutral to basic.

We have reported on the contributions of the heat-stable food proteins, milk casein [4] as like as OM during gel formation of -LG, ovalbumin with fatty acid salts [5]. Milk casein easily self-assembles but does not form a gel when heat

* Naoko [email protected]; [email protected]

1 Junior College at Mishima, Nihon University, 2-31-145 Bunkyou-cho, Mishima, Shizuoka 411-8555, Japan

2 Advanced Course of Food and Nutrition, Junior College at Mishima, Nihon University, Shizuoka, Japan

3 Food Process Engineering Laboratory Course of Applied Life Science, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan

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treated. Fatty acid salts, acting as detergents in solution, improve the dispersibility of solutes as they act as ionic surfactants. However, because of their detergent-like properties, certain countries, including Japan, have limited the use of fatty acid salts during food processing. We expect generation of mixed-protein gels with available food materials such as OM may improve functional properties of major food proteins.

High-resolution ultrasonic spectroscopy can elucidate the structural changes and aggregation behavior of a protein undergoing gelation inducing during a heating-cooling cycle without damaging the sample [6]. Furthermore, ultrasound spectroscopy is very sensitive and, therefore, can be used to help us understand the phase transitions such as sol-gel transition of proteins [6]. However, ultrasound spectroscopy may not be a suitable technique to determine the differences in interaction strengths between different aggregates [7].

In this study, given the disparate physical properties of OM and -LG, we examined the effect of 5 % (w/v) OM on the gelation of 5 % (w/v) -LG using some complementary approaches (rheological measurements, ultrasound spectroscopy, scanning electron microscopy (SEM), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)). The gelation properties of 5 and 10 % (w/v) -LG samples were also examined to determine if OM improved the gelation properties of -LG.

Materials and methods

Materials

-LG was purchased from Sigma Chemical Co. (St. Louis, MO, USA; product number L3908). OM was purchased from Worthington Co. (Vassar, NJ, USA; product number 3087). All other reagents were obtained from Wako Pure Chemicals (Osaka, Japan).

Rheological measurements

Prior to the rheological measurements, solutions of 5 % (w/v) and 10 % (w/v) -LG, and 5 % (w/v) OM/5 % (w/v) -LG (mixed protein) were prepared in distilled water containing0.2 M NaCl. For example, in the case of preparing the mixed-protein solution 1 ml, each 50 mg freeze dried chemicals were measured into the separate test tubes, respectively. First, -LG protein was dissolved by adding 700 l distilled water and then the solubilized solution was poured into the other test tube containing OM. One M NaCl 200 l and 2.5 % NaN3 10 l were added to the mixed-protein solution, and the solution was mixed well and the pH (7.17.3) was checked with a pen-type pH meter (Compact pH meter twin pH B-212 type, HORIBA, Japan). Finally, the solution

was filled up to 1 ml. Rheological measurements were performed between 25 and 80 C using a dynamic strain-controlled rheometer (model RFS-III; TA Instruments, New Castle, DE, USA) in the oscillatory mode that was equipped with 25-mm-diameter parallel plates. Several kinds of rheological measurements (strain sweep, frequency sweep, and temperature ramp test) were performed to decide the changes in dynamic modulus (G and G) of the samples. The gap between the plates was 1.0 mm, and the sample volume was ~600 l. Measurements were performed at6.28 rad/s and used a maximum applied strain (10 %) which is the maximum strain in the linear region and a maximum allowed torque (3.0 g) in the optional auto strain mode, which make response to the change in the linear region of stress and strain for the sample [8]. The ramp rate in the temperature sweep test was 0.4 C/min to 80 from 25 C.

Ultrasonic spectroscopy

Ultrasonic spectroscopy was performed using an HR-US 102 instrument (Ultrasonic Scientific, Dublin, Ireland) provided by ST Japan (Tokyo, Japan) [9]. The data were corrected with the manufacturers software (version 5.43). The spectrometer generates transverse sound waves that are passed through the sample and reference cells (1 ml each) and measures both the velocity and attenuation of the transmitted sound wave. Solutions were prepared as described above in BRheological measurements.^ The velocities of the sample and reference were recorded separately. Both solutions were degassed and equilibrated at 25 C. After loading 1 ml each of the sample and reference solutions into their respective cells, the instrument was tuned to 2.5, 5, and 8 MHz, (the frequency was calibrated with degassed water at 25 C before the experiments). The velocity and attenuation changes of the sound waves at the selective frequency were continuously measured in the sample and reference cells as the temperature increased, then held constant, and finally decreased. Temperature sweep was as follows. Temperature was increased up to 80 from 25 C at 0.3 C/min and then kept at 80 C for 30 min, followed by decreasing to 25 C at0.3 C/min. The internal temperature of the solution was controlled via a programmable circulation system Haake Phoenix II with bath (Haake C25P) (Thermo, Newington, NH, USA). The temperature of the spectrometer cell cavity was recorded using a thermocouple. For each sample, at least three independent experiments were executed. Data were analyzed and plotted using Microsoft Excel. Velocity gradient changes were calculated by separately summing five points before and five points after a given point, determining the difference and dividing the results by the temperature gradient in the same data range.

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SEM

Gels were sliced into small pieces (1 mm 1 mm 3 mm) with a razor blade. To prevent solubilization of the gels in the fixative, each sample was treated with a diluted fixative solution. That is, chemical fixation was performed two times for one cycle described below. First, preliminary conductive fixation and staining was performed using a solution containing 0.5 % (w/v) glutaraldehyde in 0.05 M sodium phosphate (pH 7.1) for 24 h at room temperature, followed by a solution of 0.5 % (w/v) glutaraldehyde-0.5 % (w/v) filtered tannic acid in 0.05 M sodium phosphate (pH 7.1) for 1 h at 4 C, and finally a solution of 0.5 % (w/v) glutaraldehyde-1.0 % (w/v) filtrated tannic acid in0.05 M sodium phosphate (pH 7.1) for 1 h at 4 C to increase the electrical conductivity of the sample. The samples were then washed with 0.05 M sodium phosphate (pH 7.1), fixed twice with 0.5 % (w/v) osmic acid solution in 0.05 M sodium phosphate (pH 7.1) for 24 h at 4 C, and finally fixed with 1.0 % (w/v) osmic acid solution in0.05 M sodium phosphate (pH 7.1) for 1 h at 4 C. After fixation, gels were dehydrated by soaking them in ethanol solutions of increasing concentration (50, 70, 80, 90, 95, and 100 % and again with 100 % (v/v) ethanol). Following dehydration, ethanol was completely replaced with tert-butyl alcohol by soaking them in tert-butyl alcohol solutions of increasing concentration (20, 40, 70, and 100 % and again in 100 % (v/v) tert-butyl alcohol). Samples were then dried using a tert-butyl-alcohol dryer (SINKU DEVISE Co. Ltd., VFD-21S, Ibaraki, Japan), glued to Au (150- thickness) on a quick coater SC-701 (SANYU DENSHI Co. Ltd.). Microscopy was performed using a JEOL Auto Analytical Scanning Microscope (JSM-6010LA, Japan).

SDS-PAGE

The mixed-protein gel and 5 % -LG gels were very fragile and prone to syneresis; therefore, we performed SDS-PAGE under reducing and nonreducing conditions (with or without 0.2 M 2-mercaptoethanol) to identify any protein compositional and physical changes in the gels and in the syneresis liquid (~50 to 25 l) from the gels (100 l each). The remained gel and syneresis, which was suctioned using a micropipette, were subjected to the Lowry method as modified by Peterson [10] to determine the amount of protein in each gel and the syneresis liquid. Treated samples and untreated -LG and OM (3 g protein each/well) were subjected to SDS-PAGE, and standard molecular weight markers (Bio-Rad, Hercules, CA, USA) were included in the electrophoresis run.

Results

Appearance of the protein solutions after the heating and cooling treatment

Figure 1 shows the appearance of the protein solutions after heating them in petri dishes at 80 C for 30 min using a water bath and then keeping them in ice water for 30 min. A 10 % OM sample (pH 6.9; dish d) was a transparent solution; the mixed-protein sample (pH 7.3; dish b) formed a somewhat darker gel than did the 5 and 10 % -LG samples (pH 7.3; dishes a and c, respectively). Namely, the color of mixed-protein sample reflected the black background. Furthermore, although not visible, the 5 % -LG and mixed-protein gels underwent syneresis.

Rheology

Plots of the changes in the storage (G) and loss (G) moduli and the temperature vs. time for the 5 % -LG, mixed-protein, and 10 % -LG samples are shown in Fig. 2ac, respectively.

Although the G of starting values of each samples is larger than those of G like an ovalbumin in aqueous solution etc. [11], the rapid increase of the modulus for the three solutions (namely, the onset temperature for the increases in G and G) occurred between 64 and 68 C. Just for the record, we also characterized the phase transition of OM by differential scanning calorimetry and found that it appeared as a very broad endothermic between ~60 and 90 C by temperature sweep at 1 C per min (data no shown). Generally, the difference between G and G is ~tenfold for each of the samples suggesting that each formed a homogeneous gel [12]. From these reorganizations, although the mixed protein is also a homogeneous gel state as so -LG pure gel, the result in the mixed-protein sample showed an OM had an inhibitory effect on the storage modulus of -LG during the heating and cooling process because of the relatively small storage modulus. OM may interact with the heat-denatured -LG molecules by disrupting the hydrophobic interactions between -LG molecules.

Ultrasonic velocity measurements

Ultrasonic velocity is defined as the distance an ultrasonic wave moves through a sample per unit time [13]. The ultrasonic velocity was measured at 5 MHz. The solid lines in Fig. 3 show changes in ultrasonic velocity over temperature up to 80 from 25 C for sample solutions in 0.2 M NaCl for 5 % -LG (a), 5 % -LG + 5 % OM (b), and 10 % -LG (c), respectively. During heating, the velocity increased and then decreased with increasing temperature in both the -LG solutions (solid lines) and the reference salt solutions (thin lines). To determine the effect of heat on the interactions

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(a) (b) (c) (d)

Fig. 1 Appearances of the protein solutions after the heating and cooling treatment. a 5 % -LG, pH 7.3; b 5 % OM/5 % -LG, pH 7.3; c 10 % -LG, pH 7.3; d 10 % OM, pH 6.9. All solutions contained 0.2 M NaCl.

Aliquots (900 l) of the protein solution in a glass petri dish were covered with heat seal and heated at 80 C for 30 min in a water bath and cooled at 0 C for 30 min in an ice water

between the proteins alone, the velocity measured in the reference cell was subtracted from the ultrasound velocity

measured in the protein solution. Namely, the relative velocity(d), (e), and (f) is the difference between the velocity measured

Fig. 2 Development of the dynamic storage (G) and loss (G ) moduli during heating and cooling of the -LG and mixed-protein solutions. a 5 % -LG, pH 7.3; b 5 % OM/5 % -LG, pH 7.3, c 10 % -LG, pH 7.3. All solutions contained 0.2 M NaCl. Thick line, G; thin line, G. The curve that is shaped like a flattened pyramid represents the temperature change. Each arrow indicates the temperature at which the values of the moduli began to increase

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Fig. 3 Temperature dependencies of the ultrasonic velocity, relative velocities, and velocity gradients for the 5 w/v % -LG, mixed-protein, and 10 w/v % -LG samples at 5 MHz. The ultrasonic velocity of protein in 0.2 M NaCl (thick line) and the reference solution (0.2 M NaCl) (thin line) during 25 to 80 C (0.3 C/min) shown in Fig. 3 a 5 % (w/v) -LG; b 5 % (w/v) OM/5 % (w/v) -LG; c 10 % (w/v) -LG. The relative velocity is the difference between the velocity measured in the sample cell (protein in 0.2 M NaCl) and that measured in the reference cell (0.2 M

NaCl). d 5 % (w/v) -LG; e 5 % (w/v) OM/5 % (w/v) -LG; f 10 % (w/v) -LG. The final pH of the mixed-protein solution was between 7.3 and7.7, depending on the sample. Three independent experiments were carried out, and typical results are presented. The gradient velocity calculation is described in the BMaterials and methods.^ Panels g, h, and i present the gradient velocities as functions of temperature for panels d, e, and f, respectively

in the sample cell and that measured in the reference cell (containing the 0.2 M NaCl without protein) which is corresponding to the gap between two curves for the velocity of samples and reference in (a), (b), and (c). All the solutions showed the relative ultrasonic velocity decreased with the increment of temperature (d), (e), and (f). Gekko & Noguchi [14] investigated compressibility of several kinds of protein solutions and indicated that the velocity has inverse correlation with density and compressibility of the sample system. Namely, a decrease in the ultrasonic velocity of a sample indicates that the compressibility of a system increases, and, for protein gelation process, this increase is due to a consequence of a high local concentration of nonpolar groups [14]. In other words, an increase in compressibility means the formation of a gel network by a denatured protein that has many voids. Therefore, these protein solutions in Fig. 3 proceeded to their gelation. However, the gradients are different from each other. Namely, the amount of decrease of 5 % -LG is 7 m/s, the mixed protein is 12 m/s, and 10 % -LG is 14 m/s during heat treatment from 25 to 80 C, respectively. Ten percent -LG solution (f) showed the largest gradient; it suggests that 10 % -LG solution (f) formed the most compressible gel (hydrophobic gel) than the 5 % -LG solution (d) or the mixed-protein solution (e) or during heat treatment. Furthermore, these graphs showed small inflection points at ~70 and 67 C. To clarify each inflection point in

Fig. 3df, the velocity gradients of the curves in Fig. 3gi were obtained as the inflection points situated at the trough minima (see the BMaterial and methods^ section). The temperature negative peaks are 67.8, 69.7, and 66.9 C, respectively. Furthermore, in our previous study, we found that the temperature at which the gradient velocity curves of -LG first decreased was approximately the same as the initial temperature increase for the dynamic viscoelastic modulus [15]. In this study, although the peak flexion temperature or onset temperature for the gradient velocity and the initial temperature increase for the corresponding viscoelastic modulus were similar, but not identical, the temperature studies suggested that OM is weakly involved in -LG gelation given the magnitude of decrease in the relative velocity.

Ultrasonic attenuation measurements

Figure 4a shows the ultrasonic attenuation dependencies of the samples on temperature. Ultrasonic attenuation caused by scattering is a measure of the reduction in the amplitude of the ultrasonic wave as it propagates through a material, and can be attributed to a number of factors including the heterogeneity of the dispersed system [16, 17]. The thin arrows and the numerical value in each sub panel of Fig. 4a indicate the direction of the temperature sweep and the magnitude of the difference of attenuation between the attenuation at the start of

1070 Colloid Polym Sci (2016) 294:10651073

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Fig. 4 Temperature dependence of the ultrasonic attenuation for the 5 % -LG, mixed-protein, and 10 % -LG samples at 8 MHz. a (i) 5 % -LG; (ii) 5 % OM/5 % -LG; (iii) 10 % -LG. The final pH of the mixed-protein solution was between 7.3 and 7.7, depending on the assay. The arrows and the numbers indicate the directions of the temperature sweeps and the magnitudes of the attenuation increases at 80 C, respectively. b Frequency dependencies of the ultrasonic attenuations at 25 C after gel formation. Dashed line, 5 % -LG; thick line, 5 % OM/ 5 % -LG; thin line, 10 % -LG

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the increase and that after heat treatment from 25 to 80 C for 30 min, respectively. The attenuation value for the mixed-protein sample is intermediate between those of the 5 and 10 % -LG samples. In addition, the increase in attenuation (11 m1) of the 10 % -LG sample is ~1.5 times larger than that of the mixed-protein sample (8 m1), and the attenuation of the 10 % -LG sample during the cooling treatment increased to a greater extent than did those of the other two samples. In general, several types of intermolecular forces promote protein gelation, including hydrophobic interactions among denatured protein molecules during heating, and hydrogen bond formation among the molecules during cooling [18]. Therefore, both types of forces substantially contributed to the gelation of the 10 % -LG sample.

Figure 4b shows the frequency dependency of the attenuations, which reflects the oscillation of the interconnected proteins within a gel network. The slope of the 10 % -LG curve is ~twofold greater than those of the mixed protein and the 5 % -LG curves, indicating that the 10 % -LG gel caused a greater change in the acoustic energy than did the other two gels. These results suggest that, for the 10 % -LG gel, more protein molecules are interconnected and that OM may interact with -LG molecules because the slope for the mixed-protein gel is somewhat greater than that for the 5 % -LG gel.

Microstructures of the gels

Protein concentration and composition affect a gel network [19]. We examined the microstructures of the three gels by SEM (Fig. 5). Relatively small clusters of particles (~400 nm in diameter) were observed in the 5 % -LG gel (Fig. 5a, d), suggesting that concentration of denatured -LG was not sufficient to aggregate in that solution. In the mixed-protein system (Fig. 5b, e), it appears that the denatured proteins aggregated to form particles ~1.2 m in diameter. The largest aggregates

(~1.7 m in diameter) are observed for the 10 % -LG gel (Fig. 5c, f). The sizes of the ultrasonic attenuations for the gels may be related to the sizes of the aggregates (see Fig. 4a). Given that the particle size of the mixed-protein gel is intermediate between those of the 5 and 10 % -LG gels, these observations suggest that when 5 % OM is mixed with 5 % -LG, it positively affects the development of the network structure.

SDS-PAGE

As mentioned above, the 5 % -LG gel and the mixed-protein gel gradually released water. The amounts of water released from the 5 % -LG and mixed-protein gels were ~25 and ~50 % of the gel volumes, respectively. Conversely, the 10 % -LG gel did not release water. The syneresis of the 5 % -LG and mixed-protein gels may be a consequence of their inability to retain water. To examine the composition of the mixed protein gel and the syneresis, we characterized the protein composition of its extract from the gel retained and its syneresis liquid by performing SDS-PAGE in the presence or absence of 2-mercaptoethanol (Fig. 6). In the presence of 2-mercaptoethanol, at most five protein bands were found in the syneresis liquid from the mixed protein gel (bands a, b, c, d, and e; lane 8). Band a, a high molecular weight component, did not enter the gel for electrophoresis. Band b has a molecular weight of ~70,000. Although band b is not clear, it might be aggregates produced by the interaction of OM and -LG. Band c corresponds to a minor component in the OM starting material. Band d is the major form of OM, a heterogeneous mixture with a molecular weight range between 27,000 and 43,000. Band d shows the most heavily stained part at ~34,000. The molecular weight of band e (17,500) corresponds to that of -LG.

By comparing the proteins found in the extract of the 5 % -LG gel (lane 5) and its syneresis liquid (lane 6), we see that more monomeric -LG (band e) is found in lane 5 than in lane 6, indicating that the majority of -LG remained in the gel.

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Fig. 5 SEM of the gels (a, d), 5 % -LG; (b, e) 5 % OM/5 % -LG; (c, f), 10 % -LG gels. Micrographs shown in d, e, and f are higher magnification images of a, b, and c, respectively. (a, b, c) The bars represent 5 m; (d, e, f) the bars represent 2 m

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Lane No. 1 2 3 4 5 6 7 8 9 10 11 12

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Fig. 6 SDS-PAGE of the 5 % -LG and mixed-protein gel extracts and syneresis liquids in the presence or absence of 2-mercaptoethanol. Lane 1, standard molecular weight protein markers; lane 2, untreated -LG; lane 3, untreated OM; lane 4, untreated mixture of 5 % -LG and 5 % OM; lane 5, extract from the 5 % -LG gel; lane 6, syneresis liquid from the 5 % -LG gel; lane 7, extract from the mixed-protein gel; lane 8, syneresis liquid from the mixed-protein gel. Proteins in lanes 18 were subjected to reduction by 2-mercaptoethanol. Lanes 912 contain the same samples as lanes 58, respectively, but the samples were not reduced

Conversely, band a from the syneresis liquid is more intense than that found in the 5 % -LG gel extract, suggesting that this high molecular weight component was excluded from the gel matrix.

By comparing the proteins found in the extract of the mixed-protein gel (lane 7) and its syneresis liquid (lane 8), we see that somewhat more -LG is found in the gel extract than in the syneresis liquid, whereas more OM is found in the syneresis liquid. Furthermore, band Ba^ is found in greater amounts in the syneresis liquids from both gels (lane 6 and8) than in the gel extracts (lane 5 and lane 7), suggesting, again, that the components of band Ba^ was excluded from the gels.

Discussion

Buckin and Smith described the basic principles concerning ultrasonic velocity and attenuation for high-resolution ultrasonic measurements [9], and they stated that ultrasonic velocity is inversely related to the compressibility and density of the system. Furthermore, Gekko et al. [14, 20] reported that the increased compressibility of a protein after denaturation is a consequence of increased local concentrations of nonpolar groups in the denatured protein as they found a direct relationship between the adiabatic compressibilities and

hydrophobicities or polarities of proteins. Namely, in a native globular protein, its bulky hydrophobic groups must pack together with little or no solvent penetration into any remaining voids. In the denatured state, however, hydrophobic residue packing is imperfect and results in larger interior voids. In other words, voids should be formed in the interior of a globular protein where most hydrophobic residues are located. Voids are formed mainly by imperfect packing of interior hydrophobic residues, and the total void volume is a function of the hydrophobicity of a protein. The changes in densities of samples are much less than those in compressibility for the thermally denatured protein as reported by Tamura and Gekko [21].

In this paper, two ultrasound parameters, velocity and attenuation, were measured as a function of temperature. Ultrasonic velocity is defined as the distance an ultrasonic wave moves through a sample per unit time [13]. Therefore, comparison of the velocity/temperature gradients (Fig. 3gi) might shed light on the differences in the gelation processes. Ultrasonic attenuation can reflect different factors including thermal loss and intrinsic attenuation. The increased attenuation in the mixed-protein system should also be related to its heterogeneity. However, the frequency sweep of attenuation is an interesting analysis because more protein is interconnected within the network structure for a 10 % -LG gel. In summary, the rheological characteristics, ultrasonic velocity, and attenuation data including frequency dependence of the attenuation suggest that the mixed-protein gel has properties intermediate to those of the 5 % (w/v) and 10 % (w/v) -LG gels.

We also performed SDS-PAGE in the presence or absence of 2-mercaptoethanol to further investigate the interaction(s) between -LG and OM. Interestingly, the gel profiles strongly suggest that OM associates with -LG in the mixed-protein gel. Namely, under the nonreducing condition for the syneresis from mixed-protein gel (lane 12), bands d and e are absent, but a high molecular weight protein smear is present, suggesting that the smear is mostly composed of -LG and OM (Fig. 6, lanes 12 and 8). Furthermore, by comparing the protein found in the extract of the mixed-protein gel between reducing (lane 7) and nonreducing conditions (lane 11), we found that high molecular compound (band a) in lane 11 was composed from -LG and OM in lane 7 via disulfide bond. Previously, Matsuda et al. investigated the interaction between OM and lysozyme using heat-induced precipitation experiments. They presumed that OM and lysozyme molecules were brought close together by the electrostatic attractive forces and unfolded under heat treatment and then aggregated through intermolecular forces such as hydrophobic interaction, hydrogen bond, and disulfide bond [3]. Their report and ours suggested that OM would be able to interact with -LG via intermolecular forces. Together, these results suggest that OM has a synergistic effect on -LG gelation despite the great heat stability.

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Conclusions

We investigated how OM affects gelation of a 5 % -LG solution using rheological and other analytical methods. Addition of OM caused a 5 % -LG solution to form a softer gel. OM interacted with -LG via disulfide bonds, which resulted in aggregates intermediate in size between those of the 5 and 10 % -LG gels. Given our results, we expect that OM will be able to interact with other types of proteins, resulting in new textured materials, which depend on the properties of the starting protein composition.

Acknowledgments This study was partly supported by a Grant-in Aid for Scientific Research in Priority Areas (nr 25350116) of Ministry of Education, Science and Culture of Japan. We thank Professor H. Ohta of Tokyo Institute of Technology, Japan, for the helpful suggestion and discussion.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

References

1. Roefs SP, De Kruif KG (1994) A model for the denaturation and aggregation of -lactoglobulin. Eur J Biochem 226:883889

2. Kato I, Schrode J, Kohr WJ (1987) Chicken ovomucoid: determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains. Biochemistry 26:193201

3. Matsuda T, Watanabe K, Sato Y (1982) Interaction between ovomucoid and lysozyme. J Food Sci 47:637641

4. Yuno-Ohta N, Corredig M (2011) -casein aids in the formation of a sodium caprate-induced -lactoglobulin B gel. Colloids Surf B: Biointerfaces 84:442446. doi:http://dx.doi.org/10.1016/j.colsurfb.2011.01.039

Web End =10.1016/j.colsurfb.2011.01.039

5. Yuno-Ohta N, Endo M, Sawaki M, Kishikawa M (2014) Effects of a-casein and sodium caprate on the formation of heat-induced ov-albumin gels. Nippon Shokuhin Kagaku Kogaku Kaishi 61:183 191. doi:http://dx.doi.org/10.3136/nskkk.61.183

Web End =10.3136/nskkk.61.183

6. Buckin V, Kudryashov E (2001) Ultrasonic shear wave rheology of weak particle gels. Adv Colloid Interf Sci 89-90:401422

7. Corredig M, Verespej E, Dalgleish DG (2004) Heat-induced changes in the ultrasonic properties of whey proteins. J Agric Food Chem 52:44654471

8. Yuno-Ohta N (2006) Mechanism for formation of ovalbumin-fatty acid salt mixed gels. Food Hydrocoll 20:357360. doi: http://dx.doi.org/10.1016/j.foodhyd.2004.03.008

Web End =10. 1016/j. http://dx.doi.org/10.1016/j.foodhyd.2004.03.008

Web End =foodhyd.2004.03.008

9. Buckin V, Smith C (1999) High-resolution ultrasonic resonator measurements for analysis of liquids. Sem Food Anal 4:113130

10. Peterson GL (1979) Review of the folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem 100:201220

11. Ikeda S, Nishinari K (2001) Solid like mechanical behaviors of ovalbumin aqueous solution. Int J Biol Macromol 28:315320

12. Almdal K, Dyre J, Hvidt S, Kramer O (1993) Towards a phenomenological definition of the term gel. Polm Gels Netw 1:517

13. Ochenduszko A, Buckin V (2010) Real-time monitoring of heat-induced aggregation of -lactoglobulin in aqueous solutions using high-resolution ultrasonic spectroscopy. Int J Thermophys 31:113 130. doi: http://dx.doi.org/10.1007/s10765-010-0705-0

Web End =10. 1007/s 107650100705-0

14. Gekko K, Noguchi H (1979) Compressibility of globular proteins in water at 25 C. J Phys Chem 83:27062714

15. Yuno-Ohta N (2009) Use of ultrasound spectroscopy to examine the effect of cysteine on -lactoglobulin interactions. Colloid Polym Sci 287:14871491

16. Mc Clements DJ (2000) Ultrasonic measurements in particle size analysis. In: Meyers RA (ed) Encyclopedia of analytical chemistry. John Wiley & Sons, Ltd. Chichester, England, pp. 18

17. Dukhin AS, Goetz PJ, Wines TH, Somaundaran P (2000) Acoustic and electroacoustic spectroscopy. Colloids Surf A Physicochem Eng Asp 173:127158

18. Nagano T, Mori H, Nishinari K (1994) Effect of heating and cooling on the gelation kinetics of 7S globulin from soybean. J Agric Food Chem 42:14151419

19. Yuno-Ohta N, Toryu H, Higasa T, Maeda H, Okada M, Ohta H (1996) Gelation properties of ovalbumin as affected by fatty acid salts. J Food Sci 61:906910 & 920

20. Gekko K, Hasegawa Y (1989) Effect of temperature on the compressibility of native globular proteins. J Phys Chem 93:426429

21. Tamura Y, Gekko K (1995) Compactness of thermally and chemically denatured ribonuclease A revealed by volume and compressibility. Biochemistry 34:18781884

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Springer-Verlag Berlin Heidelberg 2016

Role of ovomucoid in the gelation of a [beta]-lactoglobulin-ovomucoid mixture

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