Comparison of Different Mechanical Methods for the ... - Springer Link

Food Bioprocess Technol (2016) 9:1210–1218 DOI 10.1007/s11947-016-1711-4

ORIGINAL PAPER

Comparison of Different Mechanical Methods for the Modification of the Egg White Protein Ovomucin, Part B: Molecular Aspects Janina Brand 1 & Ulrich Kulozik 1,2

Received: 3 August 2015 / Accepted: 29 February 2016 / Published online: 7 March 2016 # Springer Science+Business Media New York 2016

Abstract It is indispensable to modify the physical properties of egg white prior to a fractionation of the included biofunctional proteins. It was already demonstrated that this can be realized with mechanical devices. However, until now, it was not clear by which kind of molecular changes this is accompanied. Thus, this study reports on the molecular changes in egg white proteins induced by various mechanical treatments (high-pressure homogenizer, colloid mill, toothed disc dispersing machine). Evaluation criteria were the particle size of the long-chain protein ovomucin, the content of thiol groups, and disulfide bridges in egg white as well as the amount of free lysozyme. In general, it was shown that these treatments led to changes in the molecular structure and that the obtained modifications were more pronounced the higher the applied energy was. In detail, it was found that the applied mechanical forces in the experimental range of this study were able to disrupt strong covalent bonds in the fibrillar protein ovomucin. Additionally, the bio-functional protein lysozyme that is partly entrapped in the natural egg white structure was released by the applied forces. Summing up, this study generates comprehensive knowledge concerning the underlying mechanisms that enable the release of lysozyme as well as the use of egg white for fractionation processes.

* Janina Brand [email protected]

1

Chair for Food Process Engineering and Dairy Technology, Technische Universität München, Weihenstephaner Berg 1, 85354 Freising, Germany

2

ZIEL Institute for Food and Health, Technische Universität München, Weihenstephaner Berg 1, 85354 Freising, Germany

Keywords Egg white . Fibrillar protein ovomucin . Mechanical devices . Disrupture of protein bonds

Introduction Based on its high protein content of around 90 % (w w−1) of the dry matter, egg white is a unique raw material that is often applied in food industry. Some of its proteins, like lysozyme and ovotransferrin, have bio-functional properties, which makes them a target for separation processes. However, these fractionations, especially the large scale applications, are limited because of the physical properties of egg white. In particular, the highly viscous, gel-like structure and the inhomogeneous distribution of different viscosities within the egg white that are both caused by the fibrillar protein ovomucin. Ovomucin is a filamentous biopolymer that is cross-linked through disulfide bridges, whereby it builds a network structure (Donovan et al. 1970; Rabouille et al. 1990; Ternes et al. 1994; Strixner and Kulozik 2011). At neutral pH or in the absence of denaturing agents, ovomucin is highly insoluble (Omana et al. 2010b). The protein itself consists of two subunits α-ovomucin and β-ovomucin that differ in their physical characteristics (e.g., molecular weight, amino acid composition etc.) (Itoh et al. 1987; Hayakawa and Sato 1978; Robinson and Monsey 1971; Hiidenhovi 2007). Ovomucin is also responsible for the low availability of free lysozyme, which limits the fractionation of this functional protein. This is on the one hand due to the ovomucin network structure in combination with the small size of the protein lysozyme (14.3 kDa). The network structure leads to water embeddings, in which this small molecule is entrapped (Brand et al. 2014). On the other hand, lysozyme has a high isoelectric point of 10.7,

Food Bioprocess Technol (2016) 9:1210–1218

which leads to electrostatic interactions between the positively charged lysyl-ε-amino group of lysozyme and the negative charges of the sialic acid of the protein ovomucin. These interactions are described to be stronger for β-ovomucin than for the α-subunit (Kato et al. 1971). Due to these primarily electrostatically bound lysozymeovomucin complexes the availability of lysozyme is further reduced. The processing restrictions by viscosity are hitherto solved with an isoelectric precipitation step of ovomucin that leads to a low viscous and homogeneous structure (Croguennec et al. 2001; Guérin-Dubiard et al. 2005; Omana et al. 2010a; Wang and Wu 2014). However, the limitations concerning the availability of lysozyme for fractionation processes are not solved thereby, but even worsened. This is due to the unavoidable loss of co-precipitating proteins. Depending on the method, this can be up to 22.5 % in the case of lysozyme (Brand et al. 2014). As already described in the part of the study dealing with the macroscopic effects (Brand et al. 2015), mechanical devices like the high-pressure homogenizer (HPH), the colloid mill (CM) as well as the toothed disc dispersing machine (TDDM) are able to modify the physical properties of egg white targeted. It was shown that the viscosity can be decreased and that the egg white structure becomes homogenous by the destruction of the ovomucin network. This diminution was assessed by the determination of the fibril sizes. Based on the effects observed, it can be suspected that changes occur also on the molecular level. Panozzo et al. (2014) found an increase of free thiol groups resulting from a high-pressure homogenization treatment of egg white. However, other authors like Arzeni et al. (2012) did not find any changed while treating egg white with ultrasound. Thus, until now, it is not clear, which kind of molecular changes occur due to treating egg white mechanically. Further, there are different forces, power densities, and residence times in the shear zone of each device used in the study, which presumably results in different effects. The HPH combines a high power density with a short residence time in the shear zone (Karbstein 1994; Schuchmann 2005; Jafari et al. 2008; Schultz et al. 2004). The diminution forces that occur in the HPH are based on laminar and turbulent flows as well as on cavitation (Karbstein and Schubert 1995; Stang et al. 2001; Dumay et al. 2013). In the CM, there is a laminar shear combined with disrupting Taylorvortexes and turbulences (Urban et al. 2006; Köhler 2012). The power density in the CM is much lower compared to the HPH, whereas the residence time is clearly higher (Karbstein 1994; Schuchmann 2005; Jafari et al. 2008; Schultz et al. 2004). The forces in the TDDM are mainly based on turbulences that are in a medium power density area (Schuchmann 2005; Köhler 2012). So, the

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question arises whether these differences in the devices will lead to variations on the molecular level of egg white. Consequently, the aim of the study was to understand the structural egg white changes induced by different mechanical treatments on a molecular level. For a better understanding of the destruction mechanism, the content of free sulfhydryl groups and disulfide bridges was analyzed. These results were correlated to the fibrils sizes, which gave information referring to the cleavage of peptide bonds. Furthermore, concerning the downstream fractionation process, the release of inaccessible lysozyme from the network structure was determined, which is also based on the residual structure of egg white entrapping lysozyme.

Materials and Methods Raw Material Eggs used in this study were collected from the university’s research farm (Thalhausen, breed: BLohmann Tradition^) and used within 24 h after collection. The egg white was separated manually from the egg yolk, and the chalaza was removed. Pre-treatment Methods High-Pressure Homogenization The high-pressure homogenizer (HPH) used in this study was the APV 1000 (SPX Flow Technology Rosista, Charlotte, USA; initially APV Products, Albertslund, Denmark). Variations were based on the pressures (1, 5, 10, 25, 50, 75, and 100 MPa) as well as on the number of passes (one; three discontinuously). Colloid Mill The colloid mill (CM) applied was the Labor Pilot 2000/4 (IKA®, Staufen, Germany). The rotor as well as the stator possesses a toothed surface, whereby turbulent forces occur. The gap in between rotor and stator was adjusted to 1.040 mm. During the operation, the rotational speeds (3170– 7524 min−1) as well as the number of passes (one; three discontinuously) were varied. Toothed Disc Dispersing Machine The toothed disc dispersing machine (TDDM) that was used was the Ultra-Turrax® T25 digital (IKA®, Staufen, Germany) with the geometry S 25 N-18 G. The treatment was carried out at rotational speeds between 3000 and 25,000 min−1 for 1 or 3 min. Further details are reported in part A of this study (Brand et al. 2015).

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Analyses-Measurements

Sulfhydryl Group and Disulfide Bridge Content

Effective Viscosity

Free thiols (SH) as well as disulfide bridges (SS) were quantified using the method of Hansen et al. (2007) with slight modifications. In short, the protein molecules were unfolded with guanidine-HCl (Sigma-Aldrich, Steinheim, Germany) to expose all SH. Afterwards, the SH react with 4-DPS, whereby it can be detected by HPLC. For the determination of the SS, they were reduced to SH by sodium borohydride (SigmaAldrich, Steinheim, Germany), whereby the resulted SH can be analyzed with the same method afterwards. Thereby, the total amount of SH (SHtot) available in the sample was determined. The amount of SS was calculated by the difference of the SHtot after SS cleavage and the amount of SH before cleavage. For a normalization of the diversity existing between eggs, the determined SH as well as the amount of SS were referred to SHtot. These values were used to calculate the SH/SS ratio (Eq. 1).

The effective viscosities of the samples were measured using the Advanced Rheometer AR 1000 (TA Instruments, New Castle, USA) controlled by AR Instrument Control software (MathWorks, Natick, Massachusetts, USA) with concentric cylinder geometry (gap 500 μm). According to the method described by Brand et al. (2015), the shear profile consisted of an increasing ramp (0.1–120 s−1, 3 min), followed by a holding step (30 s) and a decreasing shear ramp (120– 0.1 s−1, 3 min). Afterwards, the data of the increasing ramp were modelled using Herschel-Bulkley, and the effective viscosity at a shear rate of 20 s−1 was taken.

Particle Size Distribution The particle size distribution of the samples indicates the extent of bond cleavage of the ovomucin network. It was analyzed using the Malvern Mastersizer 2000 with a Malvern Mastersizer Hydro 2000S measurement unit (Malvern Instruments, Herrenberg, Germany) according to the method described by Brand et al. (2014) as well as by Brand et al. (2015). For the measurements, a diffraction index of 1.41 for protein and 1.33 for water was used.

Protein Content Using reversed-phase high-performance liquid chromatography (RP-HPLC), the concentration of the main egg white proteins ovalbumin, ovotransferrin, lysozyme, and ovomucoid was analyzed. Especially the concentration of lysozyme was of interest, because, as already explained in Brand et al. (2014), this was used to determine the extent of lysozyme release from the ovomucin network due to the inserted mechanical forces. For preparation, the samples were diluted in deionized water (Milli-Q Integral 3, Merck KGaA) depending on their protein concentration (target concentration approx. 2.5 mg mL −1 ), filtered through syringe filters (RC-45/25 Chromafil® Xtra ϕ = 0.45 μm, Macherey-Nagel GmbH & Co. KG, Düren, Germany), and placed into HPLC vials (Macherey-Nagel GmbH & Co. KG, Düren, Germany). The separation of the egg white proteins was performed on an Agilent 1100 series (Santa Clara, CA, USA) with a Jupiter 5 μ C4 300 A column (Phenomenex, Aschaffenburg) (main column 250 × 4.6 mm, 2 × pre-columns 4 × 3.0 mm). The elution gradient was run at 40 °C with two different eluents (A and B) and at a flow rate of 1 mL min−1. Further details are reported in Brand et al. (2014).

SH ¼ SS

=S H tot =S H tot

SH SS

ð1Þ

Statistical Analysis All experiments were performed in triplicate. As the following analyses were done in duplicate, a sixfold determination was carried out. Data were analyzed SigmaPlot 12.3 (Systat Software, USA). Energy Density The energy density Ev [J m−3] of the used devices was calculated based on the following equations (Jafari et al. 2008; Schultz et al. 2004; Karbstein and Schubert 1995; Brand et al. 2015; Stang et al. 2001): &

High-pressure homogenizer, Eq. (2). EV ¼ Δp

ð2Þ

where Δp [Pa] is the difference between homogenization pressure and atmospheric pressure. & Colloid mill, Eq. (3). EV ¼

&

P : V

ð3Þ

with P [W] as power intake of the sample and V : [m3 s−1] as the volumetric flow. Tooth disc dispersing machine, Eq. (4) EV ¼

E V

ð4Þ


where E [J] is the energy input and V [m3] is the dispersing volume.

Results and Discussion Impact of Mechanical Energy Input on Covalent Bonds As already shown in the part A of the study dealing with the macroscopic effects of the treatment (Brand et al. 2015), the highly viscous egg white structure can be destroyed by the described mechanical devices (Fig. 7, part A). This destruction is based on the diminution of the ovomucin network, and afterwards of the single ovomucin fibrils. This was determined by particle size measurements. Resulting from this diminution, there must be a cleavage of bonds included in the fibrillar, long-chain protein ovomucin. It was shown that the proportion of particles that are smaller than 0.45 μm, as this particle size was defined to be the threshold for the required fibril diminution, increased for each device with higher energy input. The lowest amounts of small particles were produced with the TDDM and the highest amount with the HPH. This extent of fibril diminution was not only influenced by the energy input, but also by the kind of energy that depends on the specific destruction mechanism of the respective device. The results indicate that the covalent peptide bonds of ovomucin can be cleaved by the used devices. Thereby, a higher Ev leads to more cleavage, and hence to a higher amount of small fibril particles. It is generally known that the forces in the HPH are intensive enough to disrupt weak interactions like hydrophobic bonds. According to literature, these forces are not known to be able to affect covalent bonds (Subirade et al. 1998). However, it was shown that this seems to be different treating long-chain fibrillar structures. Effects of mechanical energy input on long-chain molecules can also be found in literature. Floury et al. (2002, 2003), for example, worked with methylcellulose, which was treated with a HPH. Although methylcellulose is not a protein, it is nevertheless comparable to ovomucin in terms of its long-chain structure and its similar size of 300 kDa, which results in highly viscous, gel-like solutions. By treating methylcellulose with HPH, the authors achieved a significant viscosity decrease, as well as a decrease in molecular weight of the macromolecules. They concluded that the HPH provides sufficient energy to disrupt covalent bonds in long-chain structures. Additionally, they found that small molecules were not affected by this treatment. The covalent bonds cleaved in the case of methylcellulose were between C and O or between C and C. The binding energy of these bonds is 335 and 347 kJ mol−1, respectively (Mortimer and Müller 2010). As shown in our work, the ovomucin fibrils could also be destructed by the forces resulting from the different mechanical devices.

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Peptide bonds have a binding energy of 330–400 kJ mol−1 (Messens et al. 1997), which is similar to the one of methylcellulose. Hence, it can be concluded that the cleavage of strong covalent bonds is possible if the structure is fibrillar instead of globular. This is due to the good accessibility of these bonds. The extent of cleavage depends on the applied forces. With the strong forces in the HPH, more covalent bonds can be cleaved compared to the relatively weaker forces in the CM or the TDDM. Impact of Mechanical Energy Input on Disulfide Bridges As mentioned above, the ovomucin network is cross-linked through disulfide bridges, which have a binding energy of 213 kJ mol−1 (Mortimer and Müller 2010). The question arises, whether these comparably weak bonds were also disrupted by the mechanical treatments. In the literature, reports about the effect of different treatments concerning oxidation of SH groups or cleavage of SS are quite controversial. Panozzo et al. (2014) found that the egg white treatment with HPH at 150 MPa led to an increase in free SH groups. They stated that this is based on the disruption of large protein aggregates. However, Arzeni et al. (2012) did not find any significant changes after treating egg white with highintensity ultrasound. Thus, for a better understanding of the disintegrating mechanisms and positions resulting from the shear devices in this study, the amount of free SH groups as well as of the intact SS bridges was measured. Figure 1 shows that the ratio between free thiol groups (SH) and disulfide bonds (SS) shifted towards more SS with higher energy input. This was in contrast to the assumption mentioned before. Pretreating egg white with one pass through the HPH led to a clear decrease of the SH/SS ratio with increasing pressure from 1.6 (1 MPa) down to 0.9 (100 MPa) (Fig. 1a). With three passes, the energy was so high that the ratio was consistently low. There were only marginal changes depending on the setting from 1.0 (1 MPa) to 0.9 (100 MPa). The treatment of egg white in the CM led to a SH/SS ratio in the medium range (1.3) that did not change above the applied range of rotational speed (Fig. 1b). Three passes through the CM led to a little lower ratio of 1.1, also independent of the used speed. Using the TDDM (Fig. 1c), there was also a decreasing trend with higher rotational speed. The effect of a prolonged residence time showed a further decreasing effect using the TDDM. The highest amount of SS occurred when the HPH was used. This could be due to the appearing cavitation eventually leading to the generation of hydrogen oxide, which may oxidize susceptible functional groups like SH. Contrarily to expectations, the decline of the ratio displays a formation of SS instead of a cleavage. This is presumably due to a division of the underlying reaction into an intermediate state and a final state (Fig. 2). Thereby, the intermediate

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Fig. 1 Ratio between thiol groups and disulfide bridges in EW in dependence of the applied shear treatment. a HPH. b CM. c TDDM. One pass (closed symbols) and three passes (open symbols)

state is the cleavage of SS into SH, combined with a cleavage of peptide bonds. Both factors leading to a reduction of fibril size, and therefore, a significant viscosity decrease. Resulting from this, the molecules can move and interact to a higher extent, which facilitates the reactive SH to build new SS. It is known that SH groups show an increased reactivity at pH values above 7, and fresh egg white naturally has a pH around 9. This is probably the reason why the amount of SS in the final state increases with intensity of the egg white treatment, although there is a cleavage of SS in the intermediate state. Hence, for further considerations, it is assumed that a high amount of SS corresponds to a high cleavage of the native SS combined with a subsequent oxidation of SH creating SS bonds between destructed particles. Thereby, a network of very small particles was partially reestablished. One of the questions of this study was whether the molecular changes primarily resulted from the inserted energy Ev or whether the impact of the individual devices with its respective characteristics (peak shear, kind of introduced forces, residence time) had a particular impact. Part A of this study (Brand, Silberbauer, and Kulozik 2015) already shows that the changes based on peptide bond cleavage were not alone

an effect of inserted energy, but that there was also a strong impact of the specific forces, which depend on the used device. To confirm this, the medium Ev range (E2 in Fig. 7, part A) is exemplarily regarded. Applying this medium level of Ev resulted in samples without any small fibril fragments (E2a) as well as in samples with 20-40 % particles