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Physico-chemical and foaming properties of nanofibrillated egg white protein and its functionality in meringue batter
Journal Pre-proof Physico-chemical and foaming properties of nanofibrillated egg white protein and its functionality in meringue batter Farhad Alavi, Zahra Emam-Djomeh, Mehdi Mohammadian, Maryam Salami, Ali Akbar Moosavi-Movahedi PII:
S0268-005X(19)31566-8
DOI:
https://doi.org/10.1016/j.foodhyd.2019.105554
Reference:
FOOHYD 105554
To appear in:
Food Hydrocolloids
Received Date: 19 July 2019 Revised Date:
26 October 2019
Accepted Date: 27 November 2019
Please cite this article as: Alavi, F., Emam-Djomeh, Z., Mohammadian, M., Salami, M., MoosaviMovahedi, A.A., Physico-chemical and foaming properties of nanofibrillated egg white protein and its functionality in meringue batter, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/ j.foodhyd.2019.105554. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Physico-chemical and foaming properties of nanofibrillated egg white protein and its
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functionality in meringue batter
3 4 5
Farhad Alavia, Zahra Emam-Djomeh*abd, Mehdi Mohammadiana, Maryam Salamia, Ali Akbar Moosavi-Movahedicd
6 7 8 9 10 11 12 13 14 15
a
Department of Food Science, Engineering and Technology, College of Agriculture & Natural Resources, University of Tehran, Karaj Campus, Karaj, Iran b Transfer Phenomena Laboratory (TPL), Controlled Release Center, Department of Food Science, Engineering and Technology, College of Agriculture & Natural Resources, University of Tehran, Karaj Campus, Karaj, Iran c Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran d Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran
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*Corresponding Author:
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Transfer Phenomena Laboratory (TPL), Department of Food Science, Technology and
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Engineering Faculty of Agricultural Engineering and Technology, Agricultural Campus of the
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University of Tehran, 31587-11167 Karadj, Iran, Tel. and Fax:+98 263224 8804.
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[email protected]
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Physico-chemical and foaming properties of nanofibrillated egg white protein and its
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functionality in meringue batter
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Abstract
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In the current study, the foaming properties of native egg white proteins (EWP) were compared
27
to those of fibrillated EWP formed by the heating of EWP solution at acidic condition.
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Transmission electron microscopy (TEM) revealed that different fibrillated morphologies were
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formed dependent on heating duration. In general, foams produced from fibrillated EWP,
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particularly the fibrillated EWP prepared from incubation time of 48 h, showed greater stability
31
when compared with those from native EWP at a pH range of 3 to 9. As a proof-of-concept for
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the functionality of these fibrillated EWP in a real food system, overrun and rheological
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properties of meringue batters produced using the fibrillated EWP compared to those made by
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native EWP. While meringue batters prepared from native EWP showed a runny consistency and
35
could not maintain their shape after shaping, meringue batters made by fibrillated EWP have
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more solid consistency and overrun and were able to maintain their shape. The rheological data
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showed batters prepared from fibrillated EWP had higher zero-shear viscosity, yield stress,
38
elastic module, and greater shear thinning behaviour over those made from native EWP,
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attributing to the presence of fibril chain entanglements in former batters. This study suggested
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that fibrillated EWP could be used as versatile thickening and texturizing agents in aerated
41
confectionery products that will offer food manufacturers greater control over the texture and
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consistency of formulated foods.
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Keywords: Egg white protein, Fibrillation, Foam, Rheology
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1. Introduction
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A variety of unique textures can be formed by foam formation at many foods including ice
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cream, cake, bread, and confectionery products (Gharbi & Labbafi, 2019). The foam may be a
47
product in itself, such as whipped topping, or be produced as a step in the product processing,
48
such as angel food cake, cookies, marshmallow and chocolate mousses, and must experience
49
further processing before the product is complete (Mardani et al., 2019; Yang & Foegeding,
50
2010). Thus, foam stability is the main parameter for the final quality of these products, and the
51
stability must be maintained when subjected to a variety of processes (Gharbi & Labbafi, 2019).
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Egg white protein (EWP) is known as the most common commercial foaming ingredients and
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exploited to provide structure and texture in aerated food products due to its high foaming
54
properties (Gharbi & Labbafi, 2019). However, foams are thermodynamically unstable colloidal
55
systems and have a considerably shorter lifetime over other colloidal systems such as emulsions
56
(Lazidis et al., 2016). The main destabilisation mechanisms of foams are drainage of the thin
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film between bubbles, disproportionation and coalescence (Yang & Foegeding, 2010). In
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general, an increase in the continuous phase viscosity can decrease drainage rate and increase
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foam stability through slowing down the movement of liquid through the network of thin films
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and plateau borders (Lau & Dickinson, 2005; Yang & Foegeding, 2010). To increase the
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continuous phase viscosity, sugars in high concentration are used to obtain stable confectionery
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foams. In fact, besides adding sweetness, sugar is required to stabilize the structure of aerated
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confectionery systems. When sugar is beaten into an egg-white foam, it dissolves in the protein
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film on the surface of the air bubbles and improves stability of aerated confectionery foods
65
through decreasing the drainage rate as a result of viscosity increase in the continuous liquid
66
phase (Lau & Dickinson, 2005; Raikos, Campbell, & Euston, 2007; Yang & Foegeding, 2010).
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Also, sugar used to increase product bulk or weight, giving body or mouthfeel to the products.
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On the other hand, hydrocolloid gums are used in admixture to EWP to enhance the stability of
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foam systems (Sadahira, Rodrigues, Akhtar, Murray, & Netto, 2018; Dabestani & Yeganehzad,
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2018). The increased stability of egg white foams in the presence of hydrocolloid gums would be
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related to the increased bulk viscosity and the formation of a strong elastic film at the interface
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(Sadahira et al., 2018).
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An alternative strategy to improve the foam stability lies in the ability of proteins to produce
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tailored aggregates with more effective volume fraction than native proteins, increasing bulk
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viscosity of the dispersions (Alavi et al., 2019). The aggregates may provide more foam stability
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through adsorbing on the interface and forming rigid films or creating a viscoelastic network in
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the continuous phase (Lazidis et al., 2016; Alavi et al., 2019). In this context, it was observed
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that the ultraviolet-induced aggregates of EWP produced foams with higher stability (Manzocco,
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Panozzo, & Nicoli, 2012). Likewise, Alavi et al. (2019) showed that the free radical-induced
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aggregation of EWP could increase its foam stability and produce ultra-stable foams.
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The aggregates also can be achieved through the heat-induced denaturation of EWP (Van der
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Plancken, Van Loey, & Hendrickx, 2007). The morphology of heat-induced protein aggregates
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depends on the processing condition and ionic strength and may create different structures such
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as flexible strands, fibrils, and nano and microparticles (Mohammadian & Madadluo, 2018).
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Heat treatment of proteins above their denaturation temperature for prolonged durations at pH
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values far from the isoelectric point (pI) (commonly at pH ≈ 2.0) and at low ionic strength,
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aggregates proteins into fibrils of micron length (1–10 µm), nanometric diameter (1–10 nm) and
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multi-stranded twisted like structure (Mohammadian & Madadlou, 2018; Jansens et al., 2019).
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At a currently more accepted model, it is suggested that during heating in acidic condition
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proteins at first are hydrolyzed to generate the fibril-forming peptides. Pre-hydrolyzed proteins
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rather than intact proteins have therefore been utilized for fabrication of the fibrils. The
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unidirectional growth of protein aggregates rather than being randomly packed dictated by the
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domination of electrostatic repulsive forces over attractive interactions among the fibril-forming
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units. The major driving force of fibrillation and stabilization mechanism of fibrils is generally
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believed to be hydrophobic interactions (Akkermans et al., 2008; Jansens et al., 2019;
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Mohammadian & Madadlou, 2018).
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Previous studies have indicated that the fibrillar aggregates of whey protein and soy protein can
98
form more stable foams over their non-fibrillated counterparts (Loveday, Su, Rao, Anema, &
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Singh, 2011; Oboroceanu, Wang, Magner, & Auty, 2014; Wan, Yang, & Sagis, 2016; Peng,
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Yang, Li, Tang, & Li, 2017). Some studies have investigated the formation of nanofibrillar
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structures from pure main fractions of egg proteins such as ovalbumin (Humblet-Hua, Scheltens,
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Van Der Linden, & Sagis, 2011; Lara, Gourdin-Bertin, Adamcik, Bolisetty, & Mezzenga, 2012;
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Lassé et al., 2016; Jansens, Brijs, Delcour, & Scanlon, 2016) and ovotransferrin (Wei & Huang,
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2019; Wei, Cheng, & Huang, 2019), and lysozyme (Song, Shimanovich, Michaels, Ma,
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Knowles, & Shum, 2016). However, none of these studies focused on the production and
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characterization of nanofibrillar structures from the whole egg white protein. Besides, to the best
107
of our knowledge, no studies so far have been carried out on investigating the foaming properties
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of fibrillated EWP, either pure fractions of EWP or whole EWP. Besides, the potential of the
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fibrillated proteins as functional ingredients in high sugar aerated systems and their impacts on
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the mechanical properties of the systems has not been fully investigated. An example of a high
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sugar aerated food system is that of meringues which are prepared by whipping of EWP while
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gradually adding sugar. This batter can be shaped in a range of forms and is then baked. Thus,
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the batters must retain their stability and shape when subjected to heat-solidifying and drying
114
processes in the oven. Furthermore, the meringue batter may also serve as a culinary ‘scaffold’,
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for example, in the production of angel food cakes, or they can use as soft, cloudlike toppings for
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pies and tarts (Wouters, Rombouts, Fierens, Brijs, & Delcour, 2018). Therefore, due to their
117
simple composition, meringue batters represent a model system to study the behaviour of
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proteins in high sugar aerated systems, which are the basis for other, more complex, bakery and
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confectionery goods.
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It has been reported that the heat-induced conversion of protein monomers into fibrils at acidic
121
pH dependence on heating time, where the conversion yield increases with heating time (Lara et
122
al., 2012; Bolder, Vasbinder, Sagis, & van der Linden, 2007). So, firstly, we investigated
123
physicochemical and foaming properties of fibrillated EWP prepared from various heating
124
duration (6-48 h). Secondly, we prepared a high sugar aerated model system (meringue) from
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native and fibrillated EWP suspensions and compared the physical and mechanical properties of
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the resulting systems.
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2. Materials and methods
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2.1. Materials
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Egg white protein powder with 88.5 protein content based on a dry matter (containing 80.7% ±
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1.1% protein, 8.8% ± 0.4% moisture, 6.2% ± 0.2% ash content based on wet matter) was gifted
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from Pulviver Company (EAP-R™, Bastogne, Belgium). Sugar powder was purchased from the
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local market. 8-Anilino-1-naphthalene sulfonic acid (ANS), 5-5 -Dithio-bis (2-nitrobenzoic acid)
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(DTNB), sodium azide, sodium hydroxide (NaOH), and hydrochloric acid (HCl) were purchased
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from Sigma-Aldrich (Sa. Louis, MO, USA). All of the chemicals that used for the running of gel
135
electrophoresis were also purchased from Merck and Sigma-Aldrich.
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2.2. EWP fibril formation
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EWP solutions (at a protein concentration of 3.0%, w/v) were prepared with deionized water and
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stirred (1500 rpm) for 2 h at room temperature. Sodium azide (0.02%, w/v) was added to avoid
139
microbial growth during storage. Then the solutions were kept at 4 ℃ overnight to complete
140
hydration. Fibrillated EWP dispersions were produced by heating of EWP dispersions (at a
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protein concentration of 3.0%, w/v) at pH 2.0 and 85 °C for 6, 24, and 48 h. This pH value was
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selected according to previous studies that reported heating of pure solutions of ovalbumin,
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ovotransferrin, and lysozyme (as main fractions of egg white protein) at pH 2.0 caused forming
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nanofibrillar structures (Humblet-Hua et al., 2011; Wei & Huang, 2019; Wei et al., 2019; Song et
145
al., 2016). The pH was adjusted using 8 N HCl. To end the fibrillation process, dispersions were
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cooled down rapidly to 20 ℃ with cold tap water followed by storing at 4 ℃. Based on
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preliminary studies to obtain nanofibrillar structures with different length, the three heating time
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were chosen to form EWP nanofibrils. Furthermore, the concentration of 3% EWP (w/v) was
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chosen to secure high conversion rates of the monomers into fibrils and to avoid gelation (that
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occurred for protein concentrations higher than 3 % (w/v)). The following coding for the samples
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will be used throughout the text: N sample corresponded with native EWP, and F-6, F-24, and F-
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48 samples were corresponding with dispersions of EWP heated for 6, 24, and 48 h, respectively.
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It should be noted that since some unconverted peptides and intact monomers existed in addition
154
to fibrillar structures in the final solution, we address it as “fibrillated EWP” solution throughout
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the paper. 2.3. Transmission Electron Microscopy (TEM).
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For TEM, fibrillated EWP dispersions were diluted to 0.03 wt % protein with distilled water (pH
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2). A drop of the diluted sample was set down onto a 5 nm thick carbon support film on a copper
158
grid (400 mesh). A droplet of staining solution (2% uranyl acetate at pH 3.8) was then added for
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negative staining, and the excess was removed after 15 s with filter paper. Electron micrographs
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were made using an LEO 906 TEM (LEO, Oberkochen, Germany), operating at 100 kV.
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2.4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE)
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SDS-PAGE under reducing condition at a constant voltage of 150 V was employed to study the
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changes in the molecular weight profiles of EWP dispersions heated for different durations (0, 6,
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24, and 48 h). The EWP dispersions were diluted with distilled water (pH 2) to the protein
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concentration of 4.0 mg mL-1 and were run by the method of Laemmli (1970) via 15%
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acrylamide gels.
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2.5. Circular dichroism (CD) spectroscopy
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The modifications in the secondary structures of EWP upon fibrillation were studied by CD
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spectroscopy with a spectropolarimeter (Jasco J-810, Jasco Corporation, Japan) in far-UV (190-
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260 nm) region. Different samples were diluted with distilled water (pH 2) to the final protein
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concentration of 0.4 mg mL-1. Estimates of secondary structure contents of CD spectra data for
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different
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(http://bioinformatik.biochemtech.uni-halle.de/cdnn/). CDNN program is a free software which
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quantitatively analysis the protein far UV circular dichroism spectra to estimate the protein
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conformation by neural networks (Böhm, Muhr, & Jaenicke, 1992). For each sample, three CD
176
spectra were recorded and used to estimate the secondary structure by CDNN software.
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2.6. Free -SH content
samples
were
performed
by
the
CDNN
software
(version
2.1)
178
0.2 mL of the sample solutions (protein concentration of 30 mg/mL) was added to 1.8 mL of
179
Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, 0.004 M EDTA, pH 8.0) in a 2 mL
180
microcentrifuge tubes, to reach a final protein concentration of 3 mg mL-1. Also, the same buffer
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having 2.0% SDS was used to measure the total free -SH groups. After the addition of 20 μL of
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DTNB solution (Ellman's reagent, 8 mg mL-1) to the tubes, samples were incubated for 15 min at
183
room temperature. The samples were then centrifuged at 10,000 × g at room temperature ℃ for 5
184
min to remove possible insoluble turbid particles (Van der Plancken, Van Loey, & Hendrickx,
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2005; Mishyna, M., Martinez, Chen, Davidovich-Pinhas, & Benjamin, 2019). Clear, yellow
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supernatant was obtained over a white precipitate, indicating no noticeable absorption of DTNB
187
to the pellet (Van der Plancken, Van Loey, & Hendrickx, 2005). The supernatant fractions were
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analyzed for free –SH content at 412 nm using a CecilCE2502 UV-Vis spectrophotometer (Cecil
189
Ins., Cambridge, UK).
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The free -SH content (mmol SH g-1) was calculated as follows:
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(Eq. 1)
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Where C is the protein content in reaction mixture (4 mg/mL), A412 = the absorbance at 412 nm;
193
D = the dilution factor (2.02 2.00), and the factor 73.53 is from 104/(1.36 × 104); 1.36 × 104 =
194
the molar absorptivity constant (M-1 cm-1). All measurements were repeated 3 times.
195
2.7. ζ -Potential measurements
196
The ζ-potential of samples was determined by a Brookhaven's NanoBrook 90Plus PALS
197
instrument (Brookhaven, USA). The sample solutions were diluted 300 times and their pH was
198
adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. All measurements were carried out at room
199
temperature and repeated 3 times.
/ = (75.53 × × )/
200
2.8. Surface tension
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The surface tension of native and fibrillated EWP solutions was measured by a tensiometer
202
(Krüss K100, Germany) using the Du Noüy ring method at room temperature. To do this, the pH
203
of native and fibrillated dispersions was firstly adjusted to 3, 7, and 9 using 6 N NaOH and then
204
the dispersions were diluted to a concentration of 10.0 mg mL-1. The surface tension plot versus
205
time was then produced for 360 s. All measurements were repeated 3 times.
206
2.9. Flow behaviour
207
The apparent viscosity of native and fibrillated EWP solutions was measured by a rotational
208
viscometer (Model LV-DV3T, Brookfield Engineering Inc., Middleborough, MA, USA)
209
equipped with small spindle sampler adaptor with spindle SC4-34 at room temperature. For this
210
purpose, the pH of native and fibrillated dispersions was firstly adjusted to 3, 7, and 9 using 6 N
211
NaOH. Before viscosity measurement, a pre-shear rate of 30 s-1 for 1 min was applied on
212
samples to obtain a homogenous suspension. The obtained data were fitted to the Power-law
213
model Eq. (2) with Curve Expert Professional software version 2.6.4.
214
(Eq. 2)
215
Where η is the apparent viscosity (mPa. s), K is the consistency coefficient (mPa. sn), γ is the
216
shear rate (s-1) and n is the flow behaviour index (dimensionless). All measurements were
217
repeated 3 times.
218
2.10. Foam characteristics
219
2.10.1. Foam capacity
η = Kγ"#
220
Foam characteristics of native and fibrillated EWP suspensions were investigated at pH values of
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3, 7, and 9. The foam was prepared at room temperature by whipping 40 mL of corresponding
222
suspensions in a graduated 250 mL-beaker using an Electric-Hand-Mixer (Philips- HR1459-
223
300W) at a constant speed setting of 5 during 4 min. For calculation of foam capacity (overrun),
224
volumes were determined before and after whipping and the percentage of increase in foam
225
volume (overrun) was measured.
226
2.10.2. Foam stability
227
Foam stability was determined by measuring foam drainage. To determine liquid drainage, foam
228
samples were whipped according to above method in 250 mL beaker and stored at 4 °C. The
229
beakers were sealed with the aluminium foil to avoid further contact with air. The drainage liquid
230
was taken with a pipette from the bottom of the beaker and weighed. Liquid drainage was
231
followed for 5 h by comparing the weight of drained liquid to the initial weight of suspensions
232
before whipping. All tests were performed in triplicates and means from the results were
233
reported.
234
2.10.3. Foam density
235
Foam density was determined by weighing a fixed foam volume. The foam was scooped out
236
using a spoon, and transferred to a cylindrical container (13.8 mL volume), carefully avoiding
237
the trapping of air pockets. The top of the container was levelled with a metal spatula to obtain a
238
uniform and plane surface. Measurements were performed in four repetitions at room
239
temperature °C.
240
2.10.4. Foams micromorphology
241
The size and morphology of the air bubbles were evaluated using an OMAX M8333Z-
242
PHIPeC180U3 light microscope (OMAX, Gyeonggi-do, South Korea) equipped with an 18.0
243
MP digital USB microscope camera with a ×4 objective lens. A small part of the foam was
244
deposited on a concave glass (with well diameter 6 mm and well depth about 1.8–2 mm) and
245
covered with a cover glass and stored at 4 °C for further observation. Observations were
246
monitored at intervals of 0, 1, 2, 3, and 5 h.
247
All foaming experiments described in the above paragraphs were replicated at least three times
248
for statistical purpose and means from the results of foam properties were reported.
249
2.11. Meringue batter preparation
250
The meringue batters were made based on Wouters et al. (2018), with some modification. The
251
meringue recipe used here contained 40.0 mL of 3% dispersions of native or fibrillated EWP and
252
100.0 g powder sugar. A reference meringue recipe was also made comprising of 40.0 mL of
253
10% dispersions of native EWP and 100.0 g powder sugar. In all cases, the pH of the protein
254
dispersions was adjusted to 7.0 and the dispersions were transferred to a beaker with a volume of
255
250 mL. These dispersions were whipped for 2 min using an Electric-Hand-Mixer at a constant
256
speed setting of 5 at room temperature, after which the sugar was gradually added over an
257
additional 4 min of whipping using the same mixer at the same setting. Once all sugar had been
258
added, the batter was whipped for an additional 14 min under the same conditions.
259
The following coding for the batters used throughout the text: N-3% batter was corresponding
260
with batter prepared from native EWP suspension with 3% protein concentration, N-10% batter
261
corresponded with batter prepared from native EWP suspension with 10% protein, and F-48-3%
262
batter corresponded with batter prepared from F-48 fibrillated EWP suspension with 3% protein
263
concentration.
264
2.12. Meringue batter properties
265
2.12.1. Density
266
Meringue batter samples were carefully filled into cylindrical containers (13.8 mL) to avoid the
267
trapping of air pockets. To take constant volume the top of the container was levelled with a
268
metal spatula to obtain a uniform and plane surface. Measurements were performed in four
269
repetitions at room temperature. The foam weight was recorded and then the foam density was
270
determined according to the equation (3):
271
(Eq. 3)
272
Measurements were performed in four repetitions at room temperature.
273
2.12.2. Rheological properties
274
A Physica MCR 301 rheometer (Anton Paar GmbH, Graz, Austria) equipped with parallel-plate
275
geometry (25 mm flat plate) was used to measure the rheological properties of meringue batters
276
at room temperature. The apparent viscosity of meringue batters was measured as a function of
277
shear rate (0.001–100 s-1), using a 3 mm gap. Data were fitted to the Cross model (4) and Ellis
278
model (5) equations by Curve Expert Professional software version 2.6.4 to describe their flow
279
behaviours:
280
(Eq. 4)
%&''() *(+,-'. (//) =
η = CD +
0122 34 567 8729:5;<= >15578 ?3:907 34 @A:;
(FG # FH ) I(JK L)M
η = CD +
(FG # FH)
281
(Eq. 5)
282
Where η is apparent viscosity, CD is infinite-shear rate viscosity, CO is zero-shear rate viscosity,
283
P@ is Cross time constant (or sometimes the consistency) and has dimensions of time, γ is shear
284
rate, m is dimensionless exponent index, σ is shear stress, and R@ is critical stress or yield stress
285
and could be defined as the stress above which the structure of the system is broken down.
286
Because CD value of food polymer dispersions in practical concentrations are very low, CD is
287
neglected during the fitting process (Rao, 2014).
288
The dynamic viscoelastic moduli (elastic modulus G’, viscous modulus G”) of the foams were
289
determined at a gap of 3 mm, which was selected to avoid crushing or destroying of the gas
290
bubbles (Sadahira et al., 2018). To determine the linear viscoelastic region, shear stress sweep
291
tests were carried out at 1 Hz. It allowed defining the value of strain amplitude 0.1%, common
292
for all systems, as a point within the LVR which was used in the frequency sweep tests. Samples
293
were then subjected to a frequency sweep from 0.1 to 10 Hz at a constant strain amplitude
294
(0.1%).
295
All rheological tests were performed in three repetitions and means of the results were reported.
296
To each repetition, a fresh meringue batter was prepared.
297
2.13. Statistical analysis
298
Data were analyzed by one-way ANOVA with SPSS software version 23 (IBM software, NY,
299
USA) by using Duncan's test at 0.05 level of p for the examination of differences among mean
300
values.
301
3. Results and discussion
I(N / NK )M
302
3.1. Molecular characterization of fibrillated EWP
303
Fig. 1 illustrates TEM images of the fibrillated structures formed at different incubation times.
304
As shown in Fig. 1A, EWP appeared like points with an average diameter below 10 nm before
305
heating. When the protein solution of EWP at pH 2 was heated at 85 °C, the visual appearance of
306
fibrils was observed (Fig. 1B). As the heating duration increased from 6 to 24, the end to end
307
length and the density of the curly fibrils increased (Fig. 1C). These curly fibrils possess a
308
similar shape to the ovalbumin fibrils and bovine serum albumin fibrils as reported previously
309
(Humblet-Hua et al., 2011; Usov, Adamcik, & Mezzenga, 2013). Previous studies have been
310
revealed that whey protein isolate (WPI) and β-lactoglobulin (β-Lg) formed long and straight
311
fibrils with an end to end length of above 5 µm (Loveday, Anema, & Singh, 2017;
312
Mohammadian et al., 2019). It suggested that EWP fibrils are different in term of morphology
313
and length than WPI and β-Lg fibrils. It was noteworthy that the some long and straight EWP
314
fibrils were first appeared after heating for 48 h, and coexist with the curly fibrils. The
315
coexistence of curly and straight fibrils has been reported so far for ovalbumin and bovine serum
316
albumin fibrillation upon extending the heating duration (Lara et al., 2012; Usov et al., 2013).
317
It is suggested the combination of low pH and heat initially hydrolyze proteins to generate the
318
fibril-forming peptides. The peptides have then been utilized for fabrication of the fibrils
319
(Mohammadian & Madadluo, 2018; Jansens et al., 2019). This hypothesis is in accordance with
320
our SDS-PAGE results (Fig. 2A), where the band intensity of three major fractions of egg white
321
protein, namely, ovalbumin (45 kDa), ovotransferrin (78 kDa), and lysozyme (14.3 kDa)
322
decreased upon heating at pH 2; with the longer the heating time, these bands became the less
323
intense and peptides with lower molecular weight formed (Fig. 2A, lanes 3 and 4). Because TEM
324
images revealed that more and longer EWP fibrils formed at 24 and 48 h than 6 h heating
325
duration, it may be assumed that peptide fragments with smaller molecular weights that formed
326
at the longer heating time are more beneficial to EWP fibrillation (Wei & Huang, 2019).
327
Previous studies have shown that pure form of ovalbumin, ovotransferrin, and lysozyme (as main
328
fractions of EWP) are able to form nanofibrillated structures by heating at acidic condition (Lara
329
et al., 2012; Wei & Huang, 2019; Knowles, Oppenheim, Buell, Chirgadze, & Welland, 2010).
330
Given that SDS-PAGE showed that band intensity of these major fractions of egg white protein
331
(i.e. ovalbumin, ovotransferrin, and lysozyme) decreased upon heating at pH 2 and peptides with
332
low molecular weight formed, however, it is not clear that these nanofibrillated EWP structures
333
observed in TEM images are formed by combination of peptides derived from different protein
334
fractions of EWP or by combination of those peptides derived from a single protein fraction of
335
EWP.
336
Fibrillated EWP samples (lanes 2, 3, and 4) did not show bonds corresponding to high molecular
337
weight fractions and only peptides were observed. It was in agreement with previous studies
338
(Oboroceanu, Wang, Brodkorb, Magner, & Auty, 2010; Mohammadian & Madadlou, 2016;
339
Mohammadian et al., 2019), confirming fibrils were derived from assembling of the peptide
340
fragments formed during heating. These assemblies were broken down by sample buffer so any
341
high molecular bonds corresponding to these fibrillated structures were not observed. Likewise,
342
Mantovani, Fattori, Michelon, and Cunha (2016) reported that only bands and smears
343
corresponding to the low molecular peptides appeared in both reducing and non-reducing SDS-
344
PAGE gels of whey protein fibrils. Given that Tris-glycine and SDS in the non-reducing sample
345
buffer, as well as β-mercaptoethanol in the reducing sample buffer, only dissociate hydrogen
346
bonds, hydrophobic interactions, and disulfide links, respectively, it can therefore be tentatively
347
concluded that EWP components (i.e. peptides) within the nanofibrils were associated via non-
348
covalent interactions (Oboroceanu et al., 2010; Mantovani et al., 2016).
349
The average contents of total and surface free -SH groups of native EWP were 30.13 ± 1.68 and
350
2.05 ± 0.09 µmol/g protein, respectively (Fig. 2B). It indicates that in native EWP the free -SH
351
groups mostly existed in the interior of protein molecules and were not available to react with
352
DNTB, but they were exposed in the presence of 2.0% SDS (a denaturant agent). The surface
353
free -SH groups of EWP significantly increased after 6 h fibrillation suggesting most of buried
354
free -SH groups in native EWP have become exposed by the heating at the acidic condition as a
355
result of heat-induced denaturation and hydrolysis. Beyond 6 h heating, surface free –SH groups
356
tent to decreased, which might be explained by the fact that some of the surface –SH groups
357
reburied within the fibrillated structures resulting from more self-assembling of EWP toward
358
longer fibril structures by increasing the heating time. In this situation, the free –SH groups are
359
located within the fibrillated aggregates, and DNTB cannot interact with them. However, the
360
amount of total -SH groups of EWP did not show any significant changes after fibrillation, where
361
the total free –SH groups of fibrillated EWP prepared from incubation time of 6 h (F-6), 24 h (F-
362
24), and 48 h (F-48) was similar to that of native EWP. The lack of change in total free –SH
363
groups during fibrillation indicate that the newly exposed surface free –SH groups do not
364
participate in–SH/SH oxidation and disulfide bonds formation during fibrillation reactions. It is
365
known that the reactivity of SH groups and in turn the oxidation of -SH groups into S-S bonds
366
are progressively inhibited under acidic conditions because the –SH groups are predominantly
367
protonated at this condition (Hoffmann & van Mil, 1997). Our results are in agreement with Gao,
368
Xu, Ju, & Zhao (2013) who concluded the non-covalent interactions such as hydrophobic
369
interactions, hydrogen bonding, van der Waals’ forces, and the ionic bond play important roles in
370
stabilizing of the fibrils, whereas the disulfide bonding between protein molecules does not occur
371
to any significant extent because cysteine residues are predominantly protonated at very acidic
372
pH (2.0) used for fibrillation process.
373
Fig. 2C shows the far-UV CD spectra of the fibrillated EWP samples compared to native EWP.
374
The far-UV CD spectrum of native EWP showed double minima, at 208 and 222 nm, indicative
375
of a predominant α-helical structure. Presence of peaks at 210–220 nm in the CD spectra is
376
generally ascribed to the β-sheet structures (Mohammadian & Madadlou, 2016). The spectra
377
indicate that the fibrillation process had a great impact on the secondary structure of EWP. To
378
obtain a quantitative vision of these changes, the secondary structure of the different samples
379
was estimated by CDNN software and the results were summarized in Table 1. Upon fibrillation,
380
there was a loss of random coil structures, indicating the formation of more ordered structures.
381
After 6 h incubation time, the proportion of β-sheet structures significantly increased (p< 0.05),
382
suggesting that internal structures of EWP fibrils stacked as β-sheet. It is believed that the β-
383
sheets usually are a structural characteristic of amyloid fibrils and amyloid-like fibrils and the β-
384
sheet rich structures are important for amyloid stabilization and formation (Mohammadian &
385
Madadlou, 2018; Jansens et al., 2019). The proportion increase of β-sheet structure was
386
intensified with extending of heating duration from 6 h to 24 h and 48 h, indicating more
387
extensive fibrillation happened by increasing the heating time. The increase of β-sheets in
388
circular dichroism spectra upon amyloid fibrillation previously reported for WPI and
389
ovotransferrin (Mohammadian & Madadlou, 2016; Wei & Huang, 2019).
390
The pH dependence of the electric charge of EWP before and after heat-induced fibrillation for
391
6, 24, and 48 h are shown in Fig. 2D. The both native and fibrillated EWP showed similar trends
392
in their ζ-potential-pH profile, where ζ-potential changed from positive to negative as the pH
393
increased from 3 to 9. As compared to the native EWP, fibrillation of EWP raised both the ζ-
394
potential magnitude and pI, especially in the pH range of 3-6. As mentioned above, the
395
combination of low pH and heat during the fibrillation process initially hydrolyze the peptide
396
bonds in proteins to generate the fibril-forming peptides. During the hydrolysis of the peptide
397
bonds, the new terminal carboxyl (with pKa ~ 3.6) and amine groups (with pKa ~8.0) are
398
released (Hass & Mulder, 2015). At acidic pH below and far from their pKa (8.0), -NH3+ groups
399
govern the charge of protein molecules resulting from the protonation of the amino groups. Due
400
to the fact that a large part of the native egg white proteins are converted to peptide fractions
401
with more NH3+ terminal groups during the fibrillation process, one can expect that these
402
fibrillated EWP samples have a higher ζ-potential magnitude at acidic pH condition (e.g. 3-6)
403
compared to the native EWP as a result of protonation of the terminal amino groups. Our results
404
were in agreement with Liu and Zhong (2013) and Mantovani et al. (2016) who reported that the
405
magnitude of absolute ζ-potential and pI of whey proteins increased after fibrillation.
406
3.2. Flow behaviour and surface tension of fibrillated EWP
407
The rheological properties of protein solutions are an important factor contributing to their
408
capacity to develop stable foams. Flow behaviour curves of native and fibrillated EWP solutions
409
at pH of 3, 7 and 9 as a function of heating duration are presented in Fig. 3. Furthermore, Table 2
410
shows the parameters obtained by fitting the flow curve data with Power law model. At pH 3,
411
while the native EWP solution had relatively low viscosity, the fibrillated protein samples
412
showed a significantly higher apparent viscosity (stated as consistency coefficient, K value).
413
Fibrillation process significantly increased the end to end length and subsequently the volume
414
ratio of primary globular monomeric proteins (Mcclements, 2015), so entanglement of the long
415
nanofibrillated structures with each other increase energy dissipation during untangling the
416
chains and raise viscosity of the nanofibrillated EWP solutions compared with the native EWP
417
counterpart (Mohammadian & Madadlou, 2018; Jansens et al., 2019).
418
Whereas for short heating times (6 h), the apparent viscosity at pH 3 only showed a moderate
419
increase, the apparent viscosity tended to a huge increase with heating and maximum viscosity
420
was attained within 48 h. The viscosity of fibril dispersions depends on the effective volume
421
fraction of fibrils (Loveday, Rao, Anema, & Singh, 2012). The viscosity of biopolymers
422
increases dramatically when the effective volume of biopolymer molecules start to increase. The
423
effective volume of a biopolymer is directly dependent on its volume ratio, wherewith increasing
424
end to end length of the biopolymer molecule its volume ratio increases (Mcclements, 2015). So,
425
poor viscosity enhancement in F-6 sample is probably related to the short length of its fibrils
426
compared those fibrils produced by 24 and 48 h heating (see Fig. 1). Fitting of the flow
427
behaviour of fibrillated EWP solutions with Power Law model showed that the solutions
428
behaved as a shear-thinning fluid in the shear rate range of 1 to 100 s-1 (i.e., n < 1; Table 2). The
429
shear-depending behaviour of fibrillated solutions is attributed to the entanglement and
430
entanglement of fibrils at low and high shear rates (Loveday et al., 2012; Mohammadian &
431
Madadlou, 2016). The shear-thinning behaviour was more prominent in F-24 and F-48 samples
432
over F-6 one, probably due to the presence of longer fibrils and higher fibrillation yield in the
433
former samples, as confirmed by TEM, SDS-PAGE and CD data. The longer fibrils in F-24 and
434
F-48 samples strengthen entanglement and disentanglement behaviours at low and high shear
435
rates, respectively, thereby exhibiting more considerable shear-thinning character than the F-6
436
sample.
437
At the same shear rate range, the consistency coefficient (K) increased as the pH increased from
438
3 to 7. As shown in Fig. 2, fibrillated EWP solutions tend to have a significantly lower absolute
439
ζ-potential value at pH 7 (between −15.26 to -13.91 mV) as compared to those of pH 3 (between
440
31.85 to 38.85 mV). The reduced electrostatic repulsion is not strong enough to prevent the
441
aggregation of EWP nanofibrils at pH 7.0, so viscosity tends to increase as a result of higher
442
entanglement of the aggregated fibrils (Peng et al., 2017). At a high shear rate, the aggregated
443
fibrils progressively detangled and oriented toward the shear field direction. This can also
444
explain why the fibrillated solutions at pH 7 showed a more intensive shear-thinning character
445
(lower n values) as compared to those at pH 3. When the pH further raised from 7 to 9 the net
446
charges of fibrils slightly but significantly increased, resulting in increased electrostatic repulsion
447
between fibrils. With the improved electrostatic repulsion, the entanglement of the nanofibrils
448
decrease, thus a lower viscosity was observed for nanofibril solutions in pH 9 over pH 7.
449
The surface tension of different samples as a function of pH is presented in Fig. 3. All of the
450
samples including the native EWP and fibrillated EWP samples at all studied pH values could
451
reduce the surface tension at the air/water interface as compared with distilled water. As can be
452
seen in Fig. 3, the F6, F24 and F48 samples showed the lowest surface tension (the most surface
453
activity) at pH 3, 7 and 9, respectively. Based on ζ-potential data, at pH 3, 7 and 9, the F-6, F-24,
454
and F-48 samples have the lowest absolute surface charges, respectively. So, electrostatic
455
repulsion between F-6, F-24, and F-48 fibril molecules at the liquid-air interface was presumed
456
the weakest at pH 3, 7, and 9, respectively, resulting in easier and faster aligning to form a film
457
in the interface, thus more surface activity. It should be noted that the differences in surface
458
tension of fibrillated EWP solutions in various pH were small (less than 2 units), so surface
459
tension data confirmed that all fibrillated EWP samples had a good affinity towards the liquid-air
460
interfaces at pH values 3, 7, and 9.
461
Protein fibrils are large-sized assemblies. Therefore one should expect that the fibrillated
462
structures will not have a proper surface activity, because they should diffuse more slowly to the
463
air-water interface. However, the fibril suspensions consist of a mixture of fibril structures and
464
hydrolyzed proteins (peptides) that unconverted to fibril. Peng et al. (2017) stated that the
465
adsorption process of the protein fibril systems at the air-water interface was mainly dominated
466
by the high proportion of unconverted peptides, since, compared to large fibrils, these small
467
peptide materials (free peptides and/or peptide aggregates) have faster mass transport rate toward
468
the interface, decreasing the surface tension more rapidly.
469
3.3. Foam properties
470
Foaming properties of native and fibrillated EWP dispersions at pH values of 3, 7 and 9 were
471
studied (Fig. 4). At pH 3, the native EWP showed very low foam capacity (65% overrun) and the
472
foam was also very unstable and collapsed quickly. It should be noted that, at this pH (3), when
473
protein concentration increased to 10%, foam capacity dramatically increased and a relatively
474
stable foam was formed (data not shown). This may be due to the fact that the adsorption amount
475
of egg white proteins at the air/water interface will increase when the concentration of protein
476
increase from 3% to 10%. This can improve the interaction of protein and film thickness to form
477
a stable foam with good overrun.
478
Unlike the native EWP, fibrillated EWP suspensions showed satisfactory foam capacity at pH 3
479
(Fig. 4A) even at 3% protein concentration, suggesting the fibrillation process improved the
480
foaming capacity of the EWP at this condition. At pH 7 and 9, native EWP samples showed high
481
foaming capacity (Fig. 4B and C), suggesting that at neutral and alkaline pH values the native
482
EWP suspensions are capable of forming the high-overrun. At pH 7 and 9, the main protein
483
fractions of EWP (i.e. ovalbumin and ovotransferrin) are negatively charged, while lysozyme is
484
are positively charged. At the conditions, the basic protein lysozyme (pI 10.7) can interact
485
electrostatically with negatively charged proteins, and develop a cohesive viscoelastic film at
486
air/water interface even at low protein concentration (Mine, 1995). Furthermore, at pH 7 and 9,
487
foam capacity of F-24 and F-48 samples did not much differ from their capacity at pH 3 (Fig. 4B
488
and C), indicating that these fibrillated EWP show a good foaming capacity in a wide range of
489
pH from acidic to alkaline conditions. It should be noted that, at these pH values (7 and 9),
490
fibrillated EWP suspensions showed inferior foaming capacity than native EWP ones. It is
491
credited to the higher viscosity of the fibrillated EWP suspensions (see flow behaviour section)
492
that did not allow sufficient incorporation of air during aeration. It is in accordance with those of
493
Mohammadian and Madadlou (2016) and Wan et al. (2016) who reported that fibrillated WPI
494
and soy protein isolate (SPI) yielded lower overrun values than corresponding non-fibrillated
495
proteins.
496
Foam stability was quantitatively estimated by weighing the drained liquid over time. To observe
497
the changes in the fibrillated EWP foam stability, a graph of EWP dispersions foam drainage (%)
498
as a function of time was plotted (Fig. 4D-F). At pH 3, whereas foam prepared from native EWP
499
was very unstable and completely collapsed just after preparation, fibrillated EWP samples were
500
able to produce more stable foams, where the most stable foams were formed by dispersions of
501
the F-48 sample since they exhibit the longest drainage time (Fig. 4D). Furthermore, even though
502
foams prepared from native EWP at pH values 7 and 9 were more stable compared to those
503
prepared at pH 3, their stability was still significantly lower than those prepared by fibrillated
504
EWP suspensions at the same pH values. Similar observations were also reported by Oboroceanu
505
et al. (2014) who found that WPI fibrils at both pH values of 2 and 7 had better foaming stability
506
than native WPI. Our study confirmed that even at alkaline pH 9, EWP fibrils have a
507
significantly higher capacity to produce stable foams compared to native EWP. As mentioned
508
above, fibrillated proteins form a high elastic layer with solid-like behaviour at the air-water
509
interface, preventing coalescence, drainage, and coarsening of foams (Wan et al., 2016; Peng et
510
al., 2017). Furthermore, foam stability is influenced by the viscosity of the continuous fluid
511
phase, where a high viscosity of continuous phase delay the movement of liquid through the
512
network of thin films and plateau borders, thereby slowing the drainage rate (Gharbi & Labbafi,
513
2019). Foams under gravity-induced drainages typically experienced a shear rate of about 8.5 1/s
514
(Yang & Foegeding, 2010). The solution apparent viscosity of native EWP and fibrillated EWP
515
F-6, F-24, and F-48 samples at pH 3.0 and shear rate 8.5 1/s were 2.2 ± 0.1, 16.9 ± 1.6, 103.6 ±
516
8.3, and 164.2 ± 11.7 mPa s. Therefore, a negative relationship observed between foam drainage
517
and solution apparent viscosity at a shear rate of 8.5 1/s, explaining the longer drainage time
518
observed by fibrillated EWP suspensions as compared with native ones. It also explains why,
519
among fibrillated EWP suspensions, the F-48 sample showed the longest foam drainage time at
520
pH 3 (because this suspension has the highest apparent viscosity at a shear rate of 8.5 1/s).
521
In accordance with increased apparent viscosity of fibrillated EWP suspensions at pH 7
522
compared to pH 3 (see Fig. 3A-C), a significant increase in foam stability of these fibrillated
523
systems was observed at pH 7 in comparison with pH 3 (Fig. 4E). As seen in Fig. 4E, at pH 7 a
524
lag in drainage onset (up to 30 min) was observed in foams produced by fibrillated EWP
525
samples, and only 15-27% drainage occurred at these foams after 300 min, while at native EWP
526
foam mostly 50% drainage was observed in the first 1 h of foam formation. In addition to the
527
increased apparent viscosity at pH 7 compared to pH 3, fibrils had a high negative surface charge
528
at pH 3.0 (see Fig. 2D), thus the surface of bubbles was not densely packed by fibrils, owing to
529
the strong electrostatic repulsion among fibrils at the interface. Hence, at pH 3, the foams had the
530
lowest foam stability. However, as evidenced by the zeta-potential data (Fig. 2D), fibrils at pH 7
531
had lower net charges as compared to pH 3 so that at this pH fibrils are able to form a thicker
532
protective layer around bubbles by overlapping and entanglement of the fibrils leading to the
533
higher foam stability (Peng et al., 2017).
534
Foams produced by fibrillated EWP suspensions at pH 9 showed an even longer lag phase in
535
drainage onset compared to pH 7. While the drainage onset in foam produced by F-6 suspension
536
detected after 180 min of foam formation, at foams produced by F-24 and F-48 suspensions the
537
lag phase of drainage onset even was longer and any drainage was not detected after 300 min.
538
Nevertheless, the consistency coefficient of the fibrillated EWP suspensions at pH 7 was higher
539
than pH 9 (Table 2). On the other hand, due to increased electrostatic repulsion among fibrils at
540
pH 9 than pH 7, it should expect to a drop in packing intensity of fibrils at the interfacial layer
541
leading to the lower foam stability at pH 9 than pH 7. A rational reason for the unexpected
542
greater foaming stability observed at pH 9 than pH 7 at fibrillated EWP samples maybe this fact
543
that disulphide bonds could be formed between fibrils at interface or continuous phase causing
544
the formation of clusters in the interface or continuous phase. As seen in Fig. 2B, fibrillated
545
EWP samples have a very high surface free –SH groups compared native EWP counterpart. The
546
reactivity of the surface free –SH groups in alkaline pH (e.g. 9.0) is very high and significant
547
SH-SH oxidation to S-S occurred even at room temperature (Hoffmann & van Mil, 1997). In this
548
regard, Bolder et al. (2007) observed that the fibrillated WPI samples which were stored at pH 7
549
showed increased viscosity after overnight storage at cold temperature, and the samples that were
550
stored at pH 10 gelled overnight. Using gel electrophoresis, the authors confirmed that storage of
551
the fibrils at higher pH conditions induces the formation of larger structures due to the formation
552
of disulphide bonds in the sample (Bolder et al., 2007). Similarly, in foam systems, one can
553
expect that the formation of the disulfide bonds induces developing a gel-like structure with the
554
high elastic module at interface layer or continues phase, thus the ultra-high stable foams can be
555
produced by fibrils at alkaline pH.
556
Changes of bubbles size of foams formed from native and fibrillated EWP at pH values of 3, 7
557
and 9 with the extension of time are presented in Figure 5. At pH 3, as mentioned above, foams
558
prepared by native EWP were unstable and collapsed quickly. So, the foams were not evaluated
559
for changes in bubbles size. As can be seen from Figure 5A, the bubbles formed from F-6 sample
560
showed a larger initial bubble size as compared to those prepared from F-24 and F-48 samples,
561
which suggests more liquid may already drain during foam creation from F-6 samples.
562
Moreover, for all systems, it can be observed that with increasing time the bubble size gradually
563
increased due to the destabilization processes, such as liquid drainage, coarsening, and
564
coalescence (van der Plancken et al., 2007). However, in accordance with foam stability data, by
565
comparing the images of the bubbles with time (Fig. 5A), the rate of bubble size increase of the
566
F-48 foam system was significantly lower than those of F-24 and F-6 ones, confirming the foam
567
stability of the F-48 system appeared to be better than later ones.
568
At pH 7, the bubbles formed from the fibrillated EWP systems showed comparable initial bubble
569
size (at 0 min), which were much smaller than that observed in native EWP (Fig. 5B).
570
Additionally, a foam prepared from fibrillated EWP samples shows a lower rate of increase in
571
the bubble size over time compared to the native EWP. Same with pH 7, the foams made from
572
all fibrillated EWP samples at pH 9 showed much smaller bubbles size as compared to foams
573
formed from native EWP (Fig. 5C). Furthermore, whereas the initial bubbles of the fibrillated
574
EWP systems remained their spherical shape even after 300 min, the bubbles from native EWP
575
tend to be polyhedral only after 60 min, implying the liquid drainage in foams formed from
576
native EWP is much faster than in the fibrillated EWP systems. Additionally, as it is inferred by
577
comparing the microscopic images of the bubbles with time, the size of bubbles stabilized by
578
fibrils at pH 9 displayed the slowest rate of increase over pH values of 3 and 7. It is in
579
accordance with foam stability data that presented foams formed from fibrillated EWP
580
suspensions at pH 9 showed a longer lag phase in drainage onset compared to pHs of 7 and 3.
581
The data obtained from the analysis of foam density showed that foams produced by fibrillated
582
EWP samples had significantly higher foam density than that of native EWP (Fig. 6). At pH 3, as
583
said above, foams prepared by native EWP were unstable and collapsed quickly. So, the foams
584
were not evaluated for foam density. As expected, the density of the foams had an inverse
585
relationship with their overrun. The higher density of foams prepared from fibrillated EWP
586
suspensions resulted in a moist and creamy appearance, contrasting with the crispy and dry
587
appearance of the foams prepared from native EWP suspensions. The lower liquid volume
588
fractions of the native EWP foams at pH 7 and 9 (corresponding to their higher overrun) results
589
in that these foams behaviour like dry foams. However, the high liquid volume fraction of
590
fibrillated EWP foams (corresponding to their lower overrun) probably modulate the properties
591
of these foams correspond to wet foams or “bubbly liquid” systems (Nicorescu et al., 2011;
592
Furuta, Oikawa, & Kurita, 2016). This result was very similar to van der Plancken et al. (2007),
593
which found that heat-treated EWP produced moist and sticky foam.
594
3.4. Meringue batter characterisation
595
Meringues (sweetened egg white foams) are the main part of a wide range of culinary recipes
596
such as soufflés, macarons, tiramisu, mousses, and angel food cake, where they provide most of
597
the structural support (Vega & Sanghvi, 2012). Therefore, due to their simple composition, we
598
investigated the rheological characteristics of meringue batters as a model system to study the
599
behaviour of fibrillated EWP in aerated systems containing a high level of sugar. Because in our
600
preliminary investigation on foaming capacity and foam stability of fibrillated EWP suspension
601
(see above), F-48 suspension showed the promising results, this fibrillated EWP system was
602
selected to prepare meringue batter.
603
Fig. 7A shows density values of meringue batters produced from native and fibrillated EWP
604
samples. In the meringue batters prepared from 3.0% and 10.0 % native EWP (called N-3% and
605
N-10% meringue batters, respectively), batter density values were 0.61 and 0.58 mg mL-1,
606
respectively, while this value for the meringue made from 3.0% F-48 fibrillated EWP (called F-
607
48-3% meringue batter) was significantly lower (0.49 g mL-1). These results interestingly
608
indicated that the F-48 fibrillated EWP incorporated higher air to its meringue batter than that of
609
native EWP at 3.0% and even 10.0% of protein concentrations, even though the latter contained
610
three times more protein than the F-48 sample. It suggested that although the native EWP
611
solutions showed greater overrun (see Fig. 4B) than fibrillated F-48 solutions at pH 7 (as pH that
612
meringue batters produced), the air volume incorporated into F-48 meringue batter was superior
613
to N-3% and N-10% meringue batters. Thus, while foam capacity (indicated as overrun) is
614
considered an important parameter determining the foaming properties of protein solutions, it
615
does not seem to be of particular relevance in the meringue system studied here. This is probably
616
relevant to the very high sugar content of meringues. Sugar has two roles here. First, the
617
presence of sucrose in protein solution increased solution viscosity and slow down diffusion rate
618
of the protein molecules toward the interface, so less air incorporated into the foam in a given
619
period (Raikos et al., 2007). As previously stated, the adsorption process of the protein fibril
620
systems at the air-water interface was mainly dominated by unconverted peptides (hydrolyzed
621
proteins that unconverted to fibril). In the condition that the continuous phase viscosity of both
622
meringue batters prepared from native and fibrillated EWP increases due to high content of
623
sugar, peptides in protein fibril system (i.e. F-48 sample) may have a faster and more efficient
624
absorption toward the air/water interface in the viscous continuous phase when compared to
625
native protein molecules. Second, sucrose competes for available water molecules, where the
626
direct contact between protein and water is considered thermodynamically unfavourable in the
627
presence of a high concentration of sugars and this can be correlated directly with an
628
enhancement of hydrophobic interactions between protein molecules (Dickinson & Merino,
629
2002). Given that the free –SH groups and CD spectroscopy data showed that fibrillation process
630
caused profound changes in the molecular structure of egg white proteins, it can be expected that
631
the sugars have very different effects on these two proteins in terms of sugar-induced
632
hydrophobic interactions and thus affect the amount of air that these two different protein
633
structures can incorporate in a high sugar aerated system.
634
To observe the visual flow behaviour of the resulting meringue batters, about 6 g of meringue
635
batters were shaped as cones of about 4.5 cm diameter on a plane surface using a manual pastry
636
bag with a tip diameter of 7 mm. As is evidenced in Fig. 7B, when N-3% batter shaped on the
637
surface, they spread quickly. With the N-10% batter, although the rate of spreading was a bit less
638
than N-3% batter, they also spread significantly after a while (about 2 min). However, the F-48
639
batter retained their shape significantly better than those containing native EWP (Fig. 7B). In
640
accordance with our results, Wouters et al. (2018), that used the same sugar concentration to
641
prepare their meringue batters, observed that batters containing EWP at both concentrations of
642
5% and 10% were runny and did not retain their shape when shaped on baking paper.
643
Steady-state flow curves of all meringue batter samples matched to a structured fluid with a clear
644
shear-thinning behaviour over a wide range of shear rates (10-3-102 1/s). Furthermore, there are
645
two well-defined regions in the flow curves of all samples; at low shear rates, viscosity reaches a
646
limiting value, namely zero-shear viscosity (CO ); as the shear rate rises, a sudden power-law
647
decay in the viscosity was detected (Fig. 8A). Thus, the flow behaviour of the meringue batters
648
was satisfactorily fitted to the Cross and Ellis model (R2 > 0.99). These models successfully
649
applied to other systems containing CO (Rao, 2014). Table 3 shows the parameters obtained with
650
both Cross and Ellis models for various meringue batters.
651
Although there were some differences in values of the rheological parameters in Cross and Ellis
652
models, it can be seen that the meringue batters produced from fibrillated F-48 EWP (F-48
653
batter) showed the highest zero-shear rate viscosity (CO ) in both fitted models. N-3% batters had
654
the lowest CO between studied meringue batters. Furthermore, the dynamic yield stress
655
(determined by fitting the data using the Ellis model) of F-48 meringue batters were significantly
656
higher than those meringue batters produced from native EWP solutions at both protein
657
concentrations of 3% and 10% (Table 3). Similar to the observed trend in CO , the lowest dynamic
658
yield stress corresponded to N-3% meringue batters. The reciprocal 1/P@ gives us a critical shear
659
rate as an indicator of the onset shear rate for shear-thinning region (Yang & Luo, 2013). So,
660
high Cross time constant (P@ ) in F-48 meringue batter implied that onset of the shear-thinning
661
region in this batter occurs at a lower shear rate as compared to batters prepared from native
662
EWP. The parameter “m” is considered as a measure of the shear rate dependence of viscosity in
663
the shear-thinning region and a higher “m” indicated stronger shear-thinning properties. It can be
664
seen from Table 3 that the F-48 meringue batters showed significantly higher “m” when
665
compared with those prepared from native EWP, suggesting the former batters had a stronger
666
shear-thinning behaviour than latter ones.
667
The higher zero-shear viscosity and yield stress in meringue batters produced from fibrillated F-
668
48 EWP when compared to those prepared by native EWP could explain that why former batters
669
retain shape significantly superior to latter ones (see Fig. 7B). The higher zero-shear viscosity
670
and yield stress of F-48 meringue batter were attributed to the presence of fibril chain
671
entanglements; the fibrillated EWP system has a curly shape, and compared to spherical-shape
672
native EWP are more easily tangled. Thus, at zero-shear viscosity regions (unsheared state), the
673
F-48 meringue batters had a very high viscosity (CO = 9.08 × 103 Pa s) due to these
674
entanglements. However, after sufficient shear, almost all of the entanglement was disentangled
675
and quick align in the direction of flow as shear rate increases and therefore physical interactions
676
between adjacent fibril chains decrease. Therefore, F-48 meringue batter showed a stronger and
677
earlier shear-thinning behaviour (higher m and P@ values, respectively) as compared to meringue
678
batters containing native EWP. The strong shear-thinning behavior of fibrillated EWP containing
679
meringue batters (F-48 batters) allows the batters to be pumped easily. Furthermore, these batters
680
with higher shear-thinning behaviour and zero-shear rate viscosity are beneficial for extruding
681
and 3D printing of confectionery goods, as they can be easily extruded out from the nozzle with
682
the application of shear force and solidify rapidly again after leaving the nozzle (Liu, Zhang,
683
Bhandari, & Wang, 2017). On the other hand, given that the applied shear rate upon a whipping
684
process usually is sufficiently high (more than 102 1/s) (Yang & Foegeding, 2010), the extensive
685
shear-thinning behaviour of fibrillated EWP in the presence of high sugar content (e.g. in
686
meringue batter recipe) may be another beneficial characteristic; the shear-thinning behaviour
687
allows that air easily incorporated in batter system upon whipping and then the air bubbles be
688
stabilized at a viscoelastic matrix after stopping the whipping process (zero-shear rate condition).
689
So, the superior air incorporation of F-48 batters compared to native EWP batters can be
690
explained by the more and earlier shear-thinning behavior of former batters than latter ones as
691
well as the existence of peptides in protein fibril system (i.e. F-48 batter) having a faster and
692
more efficient absorption toward the air/water interface when compared to native protein
693
molecules.
694
To analysis the oscillatory response of the meringue batters, frequency sweep measurements
695
were applied. The frequency sweeps plots of different meringue batter formulations are depicted
696
in Fig. 8B. The frequency (ω) dependence of G’ and G” can indicate the type of structure present
697
in the batters. N-3% meringue batter exhibited a strong dependence of G’ with ω, and one can
698
see an overlapping of the G’ and G” curves indicating a viscose-like behaviour (Zhang, Arrighi,
699
Campbell, Lonchamp, & Euston, 2018). With F-48 meringue batter, G’ was always greater than
700
G'' and its G’ only had a weak dependence on ω values, suggesting the formation of a solid-like
701
structure. Between these two batters was the N-10% meringue batter that showed a moderate
702
frequency dependence of G’ on ω and a G'/G” crossover in high ω. To quantitative analyse the
703
degree of frequency dependence of the storage modulus (G′), a Power-law model (] ^ = &_< )
704
was fitted to the results from Fig. 8B and the fitted Power-law parameters are shown in Table 4.
705
The coefficients “a” and “n” represent the magnitude of the intercepts at frequency 1 Hz and the
706
slopes of G′ as a function of frequency (ω), respectively. The “a” value is related to the strength
707
(elastic structure) of a sample. The “n” value close to zero is characteristic of a truly solid-like
708
material. For “n’ value = 1 the system behaves as a viscous material (Sadahira et al., 2018). The
709
higher “a” value and lower “n” value at F-48 meringue batter than N-10% and N-3% meringue
710
batters show that former batter is more solid than the latter ones.
711
The Cox-Merz rule is an empirical relationship that enables comparison of steady shear viscosity
712
to complex oscillatory viscosity in equal shear rates and frequencies range. When a system is a
713
liquid (viscose), it will obey the Cox-Merz rule i.e. the steady shear viscosity curve completely
714
overlap on complex oscillatory viscosity curve, while those that are elastic will not. Thus,
715
adherence to the Cox-Merz rule can be used to assess further the structure in a system (Young,
716
2014). As shown in Fig. 8C, plots of complex oscillatory viscosity and shear viscosity for the F-
717
48 meringue batter indicate a strong deviation from the Cox-Merz rule. Such behaviour suggests
718
that the solid-like (elastic) structure is formed in these batters (Zhang et al., 2018), probably due
719
to the occurrence of strong inter-fibrillated bonds (such as disulfide bonds, as previously
720
discussed) and formation of strong entanglements of the curly protein structures in these batters
721
prepared at pH 7. The complex viscosity of N-3% meringue batter (Fig. 8C) was found to be
722
close to the shear viscosity, indicating that this batter complied with the Cox-Merz rule and thus
723
had a predominantly viscose weak structure. Between these two batters was the N-10% meringue
724
batter where moderate deviation from Cox-Merz behaviour was seen. Generally, the rheological
725
data showed that the meringue batters containing fibrillated EWP exhibit a stronger rheological
726
behaviour with more elastic elements over meringue batters made of native EWP.
727
4. Conclusions
728
This study showed that whole EWP can be fibrillated by heating at acidic condition (pH 2).
729
Fibrillated EWP has curly shape, although some of the long straight fibrils appeared with
730
increasing of heating time to 48 h. Fibrillation increased β-sheet elements, surface free –SH
731
groups, and consistency. Furthermore, foams produced from fibrillated EWP showed significant
732
stability when compared with native EWP at all of the studied pH values. By assessment of the
733
functionality of these fibrillated EWP in a standard meringue batter, we also conclude that the
734
improved viscosity and foam properties of fibrillated EWP had great relevance for using in high
735
sugar aerated systems. Thus, these results suggested that fibrillated EWP systems could be used
736
as versatile thickening and texturizing agents in high sugar aerated confectionery products that
737
will offer food manufacturers greater control over the texture and consistency of formulated
738
foods.
739
Acknowledgement:
740
The authors thank the Iran National Science Foundation (INSF) for financial support of the
741
project.
742
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743
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Table captions Table 1. Estimates of secondary structures content for native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48). Table 2. Parameters from Power law model fitting for native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48). K, n, and R2 are consistency coefficient (mPa sn), flow behaviour index (dimensionless) and confidence of fit, respectively. Table 3. Rheological parameters from Cross and Ellis models fitting of the flow behaviour of meringue batters prepared from F-48 fibrillar EWP (F-48), native EWP with a protein concentration of 3% (N-3%), and native EWP with protein concentration of 10% (N-10%). ߟ is zero-shear rate viscosity, ߙ is Cross time constant, m is dimensionless exponent index, and ߪ is critical stress or yield stress. Table 4. Power law parameters for storage modulus (G’) of meringue batters prepared from F-48 fibrillar EWP (F-48), native EWP with protein concentration of 3% (N-3%), and native EWP with protein concentration of 10% (N-10%).
Figure captions Fig. 1. TEM images of native (A) and fibrillar EWP prepared from incubation time of 6 h (B), 24 h (C), and 48 h (D) at ×27800 magnification. Fig. 2. (A) SDS-PAGE of native EWP (lane 1), fibrillar EWP prepared from incubation time 6 h (lane 2), 24 h (lane 3), 48 h (lane 4), and molecular-weight size marker (lane M). (B) surface and total free –SH groups, (C) Circular dichroism spectrum, (D) pH dependence of the ζ-potential of the native of EWP (N), and fibrillar EWP prepared from incubation time 6 h (F-6), 24 h (F-24), and 48 h (F-48). Fig. 3. Flow curve of native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48) at pH 3 (A), 7 (B), and 9 (C). Surface tension as a function of time for native EWP (N), and fibrillar EWP prepared from incubation time 6 h (F-6), 24 h (F24), and 48 h (F-48) at pH 3 (D), 7 (E), and 9 (F). Fig. 4. Overrun of native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F6), 24 h (F-24), and 48 h (F-48) at pH 3 (A), 7 (B), and 9 (C). Foam drainage (%) as a function of time for native EWP (N), and fibrillar EWP prepared from incubation time 6 h (F-6), 24 h (F24), and 48 h (F-48) at pH 3 (D), 7 (E), and 9 (F). Fig. 5. Changes of bubbles size of foams formed from native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48) at pH 3 (A), 7 (B), and 9 (C). Fig. 6. Density of foams formed from native EWP and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48) at pH 3, 7, and 9. Because the foams prepared by
native EWP at pH 3 were unstable and collapsed quickly, the foams were not evaluated for foam density.
Fig. 7. Density (A) and representative images (B) of meringue batters prepared from F-48 fibrillar EWP (F-48), native EWP with protein concentration of 3% (N-3%), and native EWP with protein concentration of 10% (N-10%). Fig. 8. Flow curve (A) as a function of shear rate, (B) Storage (G′, solid symbols) and loss modulus (G″, open symbols) as a function of frequency, and Cox-Merz plots (C) of meringue batters prepared from F-48 fibrillar EWP (F-48), native EWP with protein concentration of 3% (N-3%), and native EWP with protein concentration of 10% (N-10%).
Table 1. Samples N
F-6
F-24
F-48
α-helix
24.8 ± 0.2a
21.5 ± 0.3b
20.0 ± 0.2c
19.1 ± 0.4d
β-sheets
23.7 ± 0.1d
36.2 ± 0.5c
39.1 ± 0.4b
40.0 ± 0.4a
β-turn
15.6 ± 0.2b
16.2 ± 0.2a
15.6 ± 0.3b
15.0 ± 0.4b
Random coil
35.9 ± 0.2a
26.1 ± 0.3b
25.2 ± 0.3c
25.9 ± 0.4b
Different superscripts in each column represent a significant difference (p<0.05). Data are means ± SD.
Table 2. Sample
K
n
R2
pH 3 N
2.8 ± 0.2i
0.895 ± 0.02a
0.948
F-6
25.5 ± 2.2g
0.823 ± 0.03b
0.978
F-24
217.7 ± 11.1f
0.647 ± 0.03c
0.997
F-48
349.6 ± 14.3e
0.63 ± 0.03c
0.994
pH 7 N
3.3 ± 0.1h
0.870 ± 0.03a
0.941
F-6
632.8 ± 24.1c
0.287 ± 0.04f
0.993
F-24
919.8 ± 41.1b
0.113 ± 0.03g
0.992
F-48
1190.3 ± 31.8a
0.019 ± 0.04h
0.993
pH 9 N
3.0 ± 0.2j
0.913 ± 0.01a
0.921
F-6
462.4 ± 18.4d
0.439 ± 0.03e
0.992
F-48
246.6 ± 17.1f
0.546 ± 0.04d
0.991
F48-9
1082.9 ± 90.2a
0.248 ± 0.02
0.991
Different superscripts in each column represent a significant difference (p<0.05). Data are means ± SD.
Table 3. Batter type
Cross model
Ellis model
ߟ (Pa s)
ߙ (s)
m
R2
ߟ (Pa s)
ߪ (Pa)
m
R2
N-3%
1.58 × 102
7.59
0.61
0.993
1.49 × 102
10.8
0.99
0.994
N-10%
3.99 × 103
79.3
0.79
0.999
3.38 × 103
30.2
1.90
0.999
F-48-3%
9.08 × 103
147.8
1.11
0.999
7.23 × 103
34.3
5.73
0.994
Table 4. Sample
a
n
R2
N-3%
131.1 ± 8.7c
0.59 ± 0.02a
0.988
N-10%
502.3 ± 24.3b
0.31 ± 0.01b
0.994
F-48
1722.5 ± 54.1a
0.22 ± 0.01c
0.996
Different superscripts in each column represent a significant difference (p<0.05). Data are means ± SD.
Fig. 1.
Fig. 2
Fig. 3
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.
Highlights •
Egg white proteins (EWP) were fibrillated via heating at pH 2.0.
•
Foams made by fibrillar EWP showed greater stability over those formed by native EWP.
•
Fibril-based meringue batter had higher yield stress over that from native EWP.
•
Meringue batters based on native EWP behaving as a predominantly viscose system.
•
Batters prepared form fibrillar EWP had a solid-like structure.
The authors declare that there is no conflict of interest regarding the publication of this article except with our former colleague Dr. Ashkan Madadlou ([email protected]) who left our department three years ago.
S0268-005X(19)31566-8
DOI:
https://doi.org/10.1016/j.foodhyd.2019.105554
Reference:
FOOHYD 105554
To appear in:
Food Hydrocolloids
Received Date: 19 July 2019 Revised Date:
26 October 2019
Accepted Date: 27 November 2019
Please cite this article as: Alavi, F., Emam-Djomeh, Z., Mohammadian, M., Salami, M., MoosaviMovahedi, A.A., Physico-chemical and foaming properties of nanofibrillated egg white protein and its functionality in meringue batter, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/ j.foodhyd.2019.105554. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Physico-chemical and foaming properties of nanofibrillated egg white protein and its
2
functionality in meringue batter
3 4 5
Farhad Alavia, Zahra Emam-Djomeh*abd, Mehdi Mohammadiana, Maryam Salamia, Ali Akbar Moosavi-Movahedicd
6 7 8 9 10 11 12 13 14 15
a
Department of Food Science, Engineering and Technology, College of Agriculture & Natural Resources, University of Tehran, Karaj Campus, Karaj, Iran b Transfer Phenomena Laboratory (TPL), Controlled Release Center, Department of Food Science, Engineering and Technology, College of Agriculture & Natural Resources, University of Tehran, Karaj Campus, Karaj, Iran c Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran d Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran
16
*Corresponding Author:
17
Transfer Phenomena Laboratory (TPL), Department of Food Science, Technology and
18
Engineering Faculty of Agricultural Engineering and Technology, Agricultural Campus of the
19
University of Tehran, 31587-11167 Karadj, Iran, Tel. and Fax:+98 263224 8804.
20
[email protected]
21
22
23
Physico-chemical and foaming properties of nanofibrillated egg white protein and its
24
functionality in meringue batter
25
Abstract
26
In the current study, the foaming properties of native egg white proteins (EWP) were compared
27
to those of fibrillated EWP formed by the heating of EWP solution at acidic condition.
28
Transmission electron microscopy (TEM) revealed that different fibrillated morphologies were
29
formed dependent on heating duration. In general, foams produced from fibrillated EWP,
30
particularly the fibrillated EWP prepared from incubation time of 48 h, showed greater stability
31
when compared with those from native EWP at a pH range of 3 to 9. As a proof-of-concept for
32
the functionality of these fibrillated EWP in a real food system, overrun and rheological
33
properties of meringue batters produced using the fibrillated EWP compared to those made by
34
native EWP. While meringue batters prepared from native EWP showed a runny consistency and
35
could not maintain their shape after shaping, meringue batters made by fibrillated EWP have
36
more solid consistency and overrun and were able to maintain their shape. The rheological data
37
showed batters prepared from fibrillated EWP had higher zero-shear viscosity, yield stress,
38
elastic module, and greater shear thinning behaviour over those made from native EWP,
39
attributing to the presence of fibril chain entanglements in former batters. This study suggested
40
that fibrillated EWP could be used as versatile thickening and texturizing agents in aerated
41
confectionery products that will offer food manufacturers greater control over the texture and
42
consistency of formulated foods.
43
Keywords: Egg white protein, Fibrillation, Foam, Rheology
44
1. Introduction
45
A variety of unique textures can be formed by foam formation at many foods including ice
46
cream, cake, bread, and confectionery products (Gharbi & Labbafi, 2019). The foam may be a
47
product in itself, such as whipped topping, or be produced as a step in the product processing,
48
such as angel food cake, cookies, marshmallow and chocolate mousses, and must experience
49
further processing before the product is complete (Mardani et al., 2019; Yang & Foegeding,
50
2010). Thus, foam stability is the main parameter for the final quality of these products, and the
51
stability must be maintained when subjected to a variety of processes (Gharbi & Labbafi, 2019).
52
Egg white protein (EWP) is known as the most common commercial foaming ingredients and
53
exploited to provide structure and texture in aerated food products due to its high foaming
54
properties (Gharbi & Labbafi, 2019). However, foams are thermodynamically unstable colloidal
55
systems and have a considerably shorter lifetime over other colloidal systems such as emulsions
56
(Lazidis et al., 2016). The main destabilisation mechanisms of foams are drainage of the thin
57
film between bubbles, disproportionation and coalescence (Yang & Foegeding, 2010). In
58
general, an increase in the continuous phase viscosity can decrease drainage rate and increase
59
foam stability through slowing down the movement of liquid through the network of thin films
60
and plateau borders (Lau & Dickinson, 2005; Yang & Foegeding, 2010). To increase the
61
continuous phase viscosity, sugars in high concentration are used to obtain stable confectionery
62
foams. In fact, besides adding sweetness, sugar is required to stabilize the structure of aerated
63
confectionery systems. When sugar is beaten into an egg-white foam, it dissolves in the protein
64
film on the surface of the air bubbles and improves stability of aerated confectionery foods
65
through decreasing the drainage rate as a result of viscosity increase in the continuous liquid
66
phase (Lau & Dickinson, 2005; Raikos, Campbell, & Euston, 2007; Yang & Foegeding, 2010).
67
Also, sugar used to increase product bulk or weight, giving body or mouthfeel to the products.
68
On the other hand, hydrocolloid gums are used in admixture to EWP to enhance the stability of
69
foam systems (Sadahira, Rodrigues, Akhtar, Murray, & Netto, 2018; Dabestani & Yeganehzad,
70
2018). The increased stability of egg white foams in the presence of hydrocolloid gums would be
71
related to the increased bulk viscosity and the formation of a strong elastic film at the interface
72
(Sadahira et al., 2018).
73
An alternative strategy to improve the foam stability lies in the ability of proteins to produce
74
tailored aggregates with more effective volume fraction than native proteins, increasing bulk
75
viscosity of the dispersions (Alavi et al., 2019). The aggregates may provide more foam stability
76
through adsorbing on the interface and forming rigid films or creating a viscoelastic network in
77
the continuous phase (Lazidis et al., 2016; Alavi et al., 2019). In this context, it was observed
78
that the ultraviolet-induced aggregates of EWP produced foams with higher stability (Manzocco,
79
Panozzo, & Nicoli, 2012). Likewise, Alavi et al. (2019) showed that the free radical-induced
80
aggregation of EWP could increase its foam stability and produce ultra-stable foams.
81
The aggregates also can be achieved through the heat-induced denaturation of EWP (Van der
82
Plancken, Van Loey, & Hendrickx, 2007). The morphology of heat-induced protein aggregates
83
depends on the processing condition and ionic strength and may create different structures such
84
as flexible strands, fibrils, and nano and microparticles (Mohammadian & Madadluo, 2018).
85
Heat treatment of proteins above their denaturation temperature for prolonged durations at pH
86
values far from the isoelectric point (pI) (commonly at pH ≈ 2.0) and at low ionic strength,
87
aggregates proteins into fibrils of micron length (1–10 µm), nanometric diameter (1–10 nm) and
88
multi-stranded twisted like structure (Mohammadian & Madadlou, 2018; Jansens et al., 2019).
89
At a currently more accepted model, it is suggested that during heating in acidic condition
90
proteins at first are hydrolyzed to generate the fibril-forming peptides. Pre-hydrolyzed proteins
91
rather than intact proteins have therefore been utilized for fabrication of the fibrils. The
92
unidirectional growth of protein aggregates rather than being randomly packed dictated by the
93
domination of electrostatic repulsive forces over attractive interactions among the fibril-forming
94
units. The major driving force of fibrillation and stabilization mechanism of fibrils is generally
95
believed to be hydrophobic interactions (Akkermans et al., 2008; Jansens et al., 2019;
96
Mohammadian & Madadlou, 2018).
97
Previous studies have indicated that the fibrillar aggregates of whey protein and soy protein can
98
form more stable foams over their non-fibrillated counterparts (Loveday, Su, Rao, Anema, &
99
Singh, 2011; Oboroceanu, Wang, Magner, & Auty, 2014; Wan, Yang, & Sagis, 2016; Peng,
100
Yang, Li, Tang, & Li, 2017). Some studies have investigated the formation of nanofibrillar
101
structures from pure main fractions of egg proteins such as ovalbumin (Humblet-Hua, Scheltens,
102
Van Der Linden, & Sagis, 2011; Lara, Gourdin-Bertin, Adamcik, Bolisetty, & Mezzenga, 2012;
103
Lassé et al., 2016; Jansens, Brijs, Delcour, & Scanlon, 2016) and ovotransferrin (Wei & Huang,
104
2019; Wei, Cheng, & Huang, 2019), and lysozyme (Song, Shimanovich, Michaels, Ma,
105
Knowles, & Shum, 2016). However, none of these studies focused on the production and
106
characterization of nanofibrillar structures from the whole egg white protein. Besides, to the best
107
of our knowledge, no studies so far have been carried out on investigating the foaming properties
108
of fibrillated EWP, either pure fractions of EWP or whole EWP. Besides, the potential of the
109
fibrillated proteins as functional ingredients in high sugar aerated systems and their impacts on
110
the mechanical properties of the systems has not been fully investigated. An example of a high
111
sugar aerated food system is that of meringues which are prepared by whipping of EWP while
112
gradually adding sugar. This batter can be shaped in a range of forms and is then baked. Thus,
113
the batters must retain their stability and shape when subjected to heat-solidifying and drying
114
processes in the oven. Furthermore, the meringue batter may also serve as a culinary ‘scaffold’,
115
for example, in the production of angel food cakes, or they can use as soft, cloudlike toppings for
116
pies and tarts (Wouters, Rombouts, Fierens, Brijs, & Delcour, 2018). Therefore, due to their
117
simple composition, meringue batters represent a model system to study the behaviour of
118
proteins in high sugar aerated systems, which are the basis for other, more complex, bakery and
119
confectionery goods.
120
It has been reported that the heat-induced conversion of protein monomers into fibrils at acidic
121
pH dependence on heating time, where the conversion yield increases with heating time (Lara et
122
al., 2012; Bolder, Vasbinder, Sagis, & van der Linden, 2007). So, firstly, we investigated
123
physicochemical and foaming properties of fibrillated EWP prepared from various heating
124
duration (6-48 h). Secondly, we prepared a high sugar aerated model system (meringue) from
125
native and fibrillated EWP suspensions and compared the physical and mechanical properties of
126
the resulting systems.
127
2. Materials and methods
128
2.1. Materials
129
Egg white protein powder with 88.5 protein content based on a dry matter (containing 80.7% ±
130
1.1% protein, 8.8% ± 0.4% moisture, 6.2% ± 0.2% ash content based on wet matter) was gifted
131
from Pulviver Company (EAP-R™, Bastogne, Belgium). Sugar powder was purchased from the
132
local market. 8-Anilino-1-naphthalene sulfonic acid (ANS), 5-5 -Dithio-bis (2-nitrobenzoic acid)
133
(DTNB), sodium azide, sodium hydroxide (NaOH), and hydrochloric acid (HCl) were purchased
134
from Sigma-Aldrich (Sa. Louis, MO, USA). All of the chemicals that used for the running of gel
135
electrophoresis were also purchased from Merck and Sigma-Aldrich.
136
2.2. EWP fibril formation
137
EWP solutions (at a protein concentration of 3.0%, w/v) were prepared with deionized water and
138
stirred (1500 rpm) for 2 h at room temperature. Sodium azide (0.02%, w/v) was added to avoid
139
microbial growth during storage. Then the solutions were kept at 4 ℃ overnight to complete
140
hydration. Fibrillated EWP dispersions were produced by heating of EWP dispersions (at a
141
protein concentration of 3.0%, w/v) at pH 2.0 and 85 °C for 6, 24, and 48 h. This pH value was
142
selected according to previous studies that reported heating of pure solutions of ovalbumin,
143
ovotransferrin, and lysozyme (as main fractions of egg white protein) at pH 2.0 caused forming
144
nanofibrillar structures (Humblet-Hua et al., 2011; Wei & Huang, 2019; Wei et al., 2019; Song et
145
al., 2016). The pH was adjusted using 8 N HCl. To end the fibrillation process, dispersions were
146
cooled down rapidly to 20 ℃ with cold tap water followed by storing at 4 ℃. Based on
147
preliminary studies to obtain nanofibrillar structures with different length, the three heating time
148
were chosen to form EWP nanofibrils. Furthermore, the concentration of 3% EWP (w/v) was
149
chosen to secure high conversion rates of the monomers into fibrils and to avoid gelation (that
150
occurred for protein concentrations higher than 3 % (w/v)). The following coding for the samples
151
will be used throughout the text: N sample corresponded with native EWP, and F-6, F-24, and F-
152
48 samples were corresponding with dispersions of EWP heated for 6, 24, and 48 h, respectively.
153
It should be noted that since some unconverted peptides and intact monomers existed in addition
154
to fibrillar structures in the final solution, we address it as “fibrillated EWP” solution throughout
155
the paper. 2.3. Transmission Electron Microscopy (TEM).
156
For TEM, fibrillated EWP dispersions were diluted to 0.03 wt % protein with distilled water (pH
157
2). A drop of the diluted sample was set down onto a 5 nm thick carbon support film on a copper
158
grid (400 mesh). A droplet of staining solution (2% uranyl acetate at pH 3.8) was then added for
159
negative staining, and the excess was removed after 15 s with filter paper. Electron micrographs
160
were made using an LEO 906 TEM (LEO, Oberkochen, Germany), operating at 100 kV.
161
2.4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE)
162
SDS-PAGE under reducing condition at a constant voltage of 150 V was employed to study the
163
changes in the molecular weight profiles of EWP dispersions heated for different durations (0, 6,
164
24, and 48 h). The EWP dispersions were diluted with distilled water (pH 2) to the protein
165
concentration of 4.0 mg mL-1 and were run by the method of Laemmli (1970) via 15%
166
acrylamide gels.
167
2.5. Circular dichroism (CD) spectroscopy
168
The modifications in the secondary structures of EWP upon fibrillation were studied by CD
169
spectroscopy with a spectropolarimeter (Jasco J-810, Jasco Corporation, Japan) in far-UV (190-
170
260 nm) region. Different samples were diluted with distilled water (pH 2) to the final protein
171
concentration of 0.4 mg mL-1. Estimates of secondary structure contents of CD spectra data for
172
different
173
(http://bioinformatik.biochemtech.uni-halle.de/cdnn/). CDNN program is a free software which
174
quantitatively analysis the protein far UV circular dichroism spectra to estimate the protein
175
conformation by neural networks (Böhm, Muhr, & Jaenicke, 1992). For each sample, three CD
176
spectra were recorded and used to estimate the secondary structure by CDNN software.
177
2.6. Free -SH content
samples
were
performed
by
the
CDNN
software
(version
2.1)
178
0.2 mL of the sample solutions (protein concentration of 30 mg/mL) was added to 1.8 mL of
179
Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, 0.004 M EDTA, pH 8.0) in a 2 mL
180
microcentrifuge tubes, to reach a final protein concentration of 3 mg mL-1. Also, the same buffer
181
having 2.0% SDS was used to measure the total free -SH groups. After the addition of 20 μL of
182
DTNB solution (Ellman's reagent, 8 mg mL-1) to the tubes, samples were incubated for 15 min at
183
room temperature. The samples were then centrifuged at 10,000 × g at room temperature ℃ for 5
184
min to remove possible insoluble turbid particles (Van der Plancken, Van Loey, & Hendrickx,
185
2005; Mishyna, M., Martinez, Chen, Davidovich-Pinhas, & Benjamin, 2019). Clear, yellow
186
supernatant was obtained over a white precipitate, indicating no noticeable absorption of DTNB
187
to the pellet (Van der Plancken, Van Loey, & Hendrickx, 2005). The supernatant fractions were
188
analyzed for free –SH content at 412 nm using a CecilCE2502 UV-Vis spectrophotometer (Cecil
189
Ins., Cambridge, UK).
190
The free -SH content (mmol SH g-1) was calculated as follows:
191
(Eq. 1)
192
Where C is the protein content in reaction mixture (4 mg/mL), A412 = the absorbance at 412 nm;
193
D = the dilution factor (2.02 2.00), and the factor 73.53 is from 104/(1.36 × 104); 1.36 × 104 =
194
the molar absorptivity constant (M-1 cm-1). All measurements were repeated 3 times.
195
2.7. ζ -Potential measurements
196
The ζ-potential of samples was determined by a Brookhaven's NanoBrook 90Plus PALS
197
instrument (Brookhaven, USA). The sample solutions were diluted 300 times and their pH was
198
adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. All measurements were carried out at room
199
temperature and repeated 3 times.
/ = (75.53 × × )/
200
2.8. Surface tension
201
The surface tension of native and fibrillated EWP solutions was measured by a tensiometer
202
(Krüss K100, Germany) using the Du Noüy ring method at room temperature. To do this, the pH
203
of native and fibrillated dispersions was firstly adjusted to 3, 7, and 9 using 6 N NaOH and then
204
the dispersions were diluted to a concentration of 10.0 mg mL-1. The surface tension plot versus
205
time was then produced for 360 s. All measurements were repeated 3 times.
206
2.9. Flow behaviour
207
The apparent viscosity of native and fibrillated EWP solutions was measured by a rotational
208
viscometer (Model LV-DV3T, Brookfield Engineering Inc., Middleborough, MA, USA)
209
equipped with small spindle sampler adaptor with spindle SC4-34 at room temperature. For this
210
purpose, the pH of native and fibrillated dispersions was firstly adjusted to 3, 7, and 9 using 6 N
211
NaOH. Before viscosity measurement, a pre-shear rate of 30 s-1 for 1 min was applied on
212
samples to obtain a homogenous suspension. The obtained data were fitted to the Power-law
213
model Eq. (2) with Curve Expert Professional software version 2.6.4.
214
(Eq. 2)
215
Where η is the apparent viscosity (mPa. s), K is the consistency coefficient (mPa. sn), γ is the
216
shear rate (s-1) and n is the flow behaviour index (dimensionless). All measurements were
217
repeated 3 times.
218
2.10. Foam characteristics
219
2.10.1. Foam capacity
η = Kγ"#
220
Foam characteristics of native and fibrillated EWP suspensions were investigated at pH values of
221
3, 7, and 9. The foam was prepared at room temperature by whipping 40 mL of corresponding
222
suspensions in a graduated 250 mL-beaker using an Electric-Hand-Mixer (Philips- HR1459-
223
300W) at a constant speed setting of 5 during 4 min. For calculation of foam capacity (overrun),
224
volumes were determined before and after whipping and the percentage of increase in foam
225
volume (overrun) was measured.
226
2.10.2. Foam stability
227
Foam stability was determined by measuring foam drainage. To determine liquid drainage, foam
228
samples were whipped according to above method in 250 mL beaker and stored at 4 °C. The
229
beakers were sealed with the aluminium foil to avoid further contact with air. The drainage liquid
230
was taken with a pipette from the bottom of the beaker and weighed. Liquid drainage was
231
followed for 5 h by comparing the weight of drained liquid to the initial weight of suspensions
232
before whipping. All tests were performed in triplicates and means from the results were
233
reported.
234
2.10.3. Foam density
235
Foam density was determined by weighing a fixed foam volume. The foam was scooped out
236
using a spoon, and transferred to a cylindrical container (13.8 mL volume), carefully avoiding
237
the trapping of air pockets. The top of the container was levelled with a metal spatula to obtain a
238
uniform and plane surface. Measurements were performed in four repetitions at room
239
temperature °C.
240
2.10.4. Foams micromorphology
241
The size and morphology of the air bubbles were evaluated using an OMAX M8333Z-
242
PHIPeC180U3 light microscope (OMAX, Gyeonggi-do, South Korea) equipped with an 18.0
243
MP digital USB microscope camera with a ×4 objective lens. A small part of the foam was
244
deposited on a concave glass (with well diameter 6 mm and well depth about 1.8–2 mm) and
245
covered with a cover glass and stored at 4 °C for further observation. Observations were
246
monitored at intervals of 0, 1, 2, 3, and 5 h.
247
All foaming experiments described in the above paragraphs were replicated at least three times
248
for statistical purpose and means from the results of foam properties were reported.
249
2.11. Meringue batter preparation
250
The meringue batters were made based on Wouters et al. (2018), with some modification. The
251
meringue recipe used here contained 40.0 mL of 3% dispersions of native or fibrillated EWP and
252
100.0 g powder sugar. A reference meringue recipe was also made comprising of 40.0 mL of
253
10% dispersions of native EWP and 100.0 g powder sugar. In all cases, the pH of the protein
254
dispersions was adjusted to 7.0 and the dispersions were transferred to a beaker with a volume of
255
250 mL. These dispersions were whipped for 2 min using an Electric-Hand-Mixer at a constant
256
speed setting of 5 at room temperature, after which the sugar was gradually added over an
257
additional 4 min of whipping using the same mixer at the same setting. Once all sugar had been
258
added, the batter was whipped for an additional 14 min under the same conditions.
259
The following coding for the batters used throughout the text: N-3% batter was corresponding
260
with batter prepared from native EWP suspension with 3% protein concentration, N-10% batter
261
corresponded with batter prepared from native EWP suspension with 10% protein, and F-48-3%
262
batter corresponded with batter prepared from F-48 fibrillated EWP suspension with 3% protein
263
concentration.
264
2.12. Meringue batter properties
265
2.12.1. Density
266
Meringue batter samples were carefully filled into cylindrical containers (13.8 mL) to avoid the
267
trapping of air pockets. To take constant volume the top of the container was levelled with a
268
metal spatula to obtain a uniform and plane surface. Measurements were performed in four
269
repetitions at room temperature. The foam weight was recorded and then the foam density was
270
determined according to the equation (3):
271
(Eq. 3)
272
Measurements were performed in four repetitions at room temperature.
273
2.12.2. Rheological properties
274
A Physica MCR 301 rheometer (Anton Paar GmbH, Graz, Austria) equipped with parallel-plate
275
geometry (25 mm flat plate) was used to measure the rheological properties of meringue batters
276
at room temperature. The apparent viscosity of meringue batters was measured as a function of
277
shear rate (0.001–100 s-1), using a 3 mm gap. Data were fitted to the Cross model (4) and Ellis
278
model (5) equations by Curve Expert Professional software version 2.6.4 to describe their flow
279
behaviours:
280
(Eq. 4)
%&''() *(+,-'. (//) =
η = CD +
0122 34 567 8729:5;<= >15578 ?3:907 34 @A:;
(FG # FH ) I(JK L)M
η = CD +
(FG # FH)
281
(Eq. 5)
282
Where η is apparent viscosity, CD is infinite-shear rate viscosity, CO is zero-shear rate viscosity,
283
P@ is Cross time constant (or sometimes the consistency) and has dimensions of time, γ is shear
284
rate, m is dimensionless exponent index, σ is shear stress, and R@ is critical stress or yield stress
285
and could be defined as the stress above which the structure of the system is broken down.
286
Because CD value of food polymer dispersions in practical concentrations are very low, CD is
287
neglected during the fitting process (Rao, 2014).
288
The dynamic viscoelastic moduli (elastic modulus G’, viscous modulus G”) of the foams were
289
determined at a gap of 3 mm, which was selected to avoid crushing or destroying of the gas
290
bubbles (Sadahira et al., 2018). To determine the linear viscoelastic region, shear stress sweep
291
tests were carried out at 1 Hz. It allowed defining the value of strain amplitude 0.1%, common
292
for all systems, as a point within the LVR which was used in the frequency sweep tests. Samples
293
were then subjected to a frequency sweep from 0.1 to 10 Hz at a constant strain amplitude
294
(0.1%).
295
All rheological tests were performed in three repetitions and means of the results were reported.
296
To each repetition, a fresh meringue batter was prepared.
297
2.13. Statistical analysis
298
Data were analyzed by one-way ANOVA with SPSS software version 23 (IBM software, NY,
299
USA) by using Duncan's test at 0.05 level of p for the examination of differences among mean
300
values.
301
3. Results and discussion
I(N / NK )M
302
3.1. Molecular characterization of fibrillated EWP
303
Fig. 1 illustrates TEM images of the fibrillated structures formed at different incubation times.
304
As shown in Fig. 1A, EWP appeared like points with an average diameter below 10 nm before
305
heating. When the protein solution of EWP at pH 2 was heated at 85 °C, the visual appearance of
306
fibrils was observed (Fig. 1B). As the heating duration increased from 6 to 24, the end to end
307
length and the density of the curly fibrils increased (Fig. 1C). These curly fibrils possess a
308
similar shape to the ovalbumin fibrils and bovine serum albumin fibrils as reported previously
309
(Humblet-Hua et al., 2011; Usov, Adamcik, & Mezzenga, 2013). Previous studies have been
310
revealed that whey protein isolate (WPI) and β-lactoglobulin (β-Lg) formed long and straight
311
fibrils with an end to end length of above 5 µm (Loveday, Anema, & Singh, 2017;
312
Mohammadian et al., 2019). It suggested that EWP fibrils are different in term of morphology
313
and length than WPI and β-Lg fibrils. It was noteworthy that the some long and straight EWP
314
fibrils were first appeared after heating for 48 h, and coexist with the curly fibrils. The
315
coexistence of curly and straight fibrils has been reported so far for ovalbumin and bovine serum
316
albumin fibrillation upon extending the heating duration (Lara et al., 2012; Usov et al., 2013).
317
It is suggested the combination of low pH and heat initially hydrolyze proteins to generate the
318
fibril-forming peptides. The peptides have then been utilized for fabrication of the fibrils
319
(Mohammadian & Madadluo, 2018; Jansens et al., 2019). This hypothesis is in accordance with
320
our SDS-PAGE results (Fig. 2A), where the band intensity of three major fractions of egg white
321
protein, namely, ovalbumin (45 kDa), ovotransferrin (78 kDa), and lysozyme (14.3 kDa)
322
decreased upon heating at pH 2; with the longer the heating time, these bands became the less
323
intense and peptides with lower molecular weight formed (Fig. 2A, lanes 3 and 4). Because TEM
324
images revealed that more and longer EWP fibrils formed at 24 and 48 h than 6 h heating
325
duration, it may be assumed that peptide fragments with smaller molecular weights that formed
326
at the longer heating time are more beneficial to EWP fibrillation (Wei & Huang, 2019).
327
Previous studies have shown that pure form of ovalbumin, ovotransferrin, and lysozyme (as main
328
fractions of EWP) are able to form nanofibrillated structures by heating at acidic condition (Lara
329
et al., 2012; Wei & Huang, 2019; Knowles, Oppenheim, Buell, Chirgadze, & Welland, 2010).
330
Given that SDS-PAGE showed that band intensity of these major fractions of egg white protein
331
(i.e. ovalbumin, ovotransferrin, and lysozyme) decreased upon heating at pH 2 and peptides with
332
low molecular weight formed, however, it is not clear that these nanofibrillated EWP structures
333
observed in TEM images are formed by combination of peptides derived from different protein
334
fractions of EWP or by combination of those peptides derived from a single protein fraction of
335
EWP.
336
Fibrillated EWP samples (lanes 2, 3, and 4) did not show bonds corresponding to high molecular
337
weight fractions and only peptides were observed. It was in agreement with previous studies
338
(Oboroceanu, Wang, Brodkorb, Magner, & Auty, 2010; Mohammadian & Madadlou, 2016;
339
Mohammadian et al., 2019), confirming fibrils were derived from assembling of the peptide
340
fragments formed during heating. These assemblies were broken down by sample buffer so any
341
high molecular bonds corresponding to these fibrillated structures were not observed. Likewise,
342
Mantovani, Fattori, Michelon, and Cunha (2016) reported that only bands and smears
343
corresponding to the low molecular peptides appeared in both reducing and non-reducing SDS-
344
PAGE gels of whey protein fibrils. Given that Tris-glycine and SDS in the non-reducing sample
345
buffer, as well as β-mercaptoethanol in the reducing sample buffer, only dissociate hydrogen
346
bonds, hydrophobic interactions, and disulfide links, respectively, it can therefore be tentatively
347
concluded that EWP components (i.e. peptides) within the nanofibrils were associated via non-
348
covalent interactions (Oboroceanu et al., 2010; Mantovani et al., 2016).
349
The average contents of total and surface free -SH groups of native EWP were 30.13 ± 1.68 and
350
2.05 ± 0.09 µmol/g protein, respectively (Fig. 2B). It indicates that in native EWP the free -SH
351
groups mostly existed in the interior of protein molecules and were not available to react with
352
DNTB, but they were exposed in the presence of 2.0% SDS (a denaturant agent). The surface
353
free -SH groups of EWP significantly increased after 6 h fibrillation suggesting most of buried
354
free -SH groups in native EWP have become exposed by the heating at the acidic condition as a
355
result of heat-induced denaturation and hydrolysis. Beyond 6 h heating, surface free –SH groups
356
tent to decreased, which might be explained by the fact that some of the surface –SH groups
357
reburied within the fibrillated structures resulting from more self-assembling of EWP toward
358
longer fibril structures by increasing the heating time. In this situation, the free –SH groups are
359
located within the fibrillated aggregates, and DNTB cannot interact with them. However, the
360
amount of total -SH groups of EWP did not show any significant changes after fibrillation, where
361
the total free –SH groups of fibrillated EWP prepared from incubation time of 6 h (F-6), 24 h (F-
362
24), and 48 h (F-48) was similar to that of native EWP. The lack of change in total free –SH
363
groups during fibrillation indicate that the newly exposed surface free –SH groups do not
364
participate in–SH/SH oxidation and disulfide bonds formation during fibrillation reactions. It is
365
known that the reactivity of SH groups and in turn the oxidation of -SH groups into S-S bonds
366
are progressively inhibited under acidic conditions because the –SH groups are predominantly
367
protonated at this condition (Hoffmann & van Mil, 1997). Our results are in agreement with Gao,
368
Xu, Ju, & Zhao (2013) who concluded the non-covalent interactions such as hydrophobic
369
interactions, hydrogen bonding, van der Waals’ forces, and the ionic bond play important roles in
370
stabilizing of the fibrils, whereas the disulfide bonding between protein molecules does not occur
371
to any significant extent because cysteine residues are predominantly protonated at very acidic
372
pH (2.0) used for fibrillation process.
373
Fig. 2C shows the far-UV CD spectra of the fibrillated EWP samples compared to native EWP.
374
The far-UV CD spectrum of native EWP showed double minima, at 208 and 222 nm, indicative
375
of a predominant α-helical structure. Presence of peaks at 210–220 nm in the CD spectra is
376
generally ascribed to the β-sheet structures (Mohammadian & Madadlou, 2016). The spectra
377
indicate that the fibrillation process had a great impact on the secondary structure of EWP. To
378
obtain a quantitative vision of these changes, the secondary structure of the different samples
379
was estimated by CDNN software and the results were summarized in Table 1. Upon fibrillation,
380
there was a loss of random coil structures, indicating the formation of more ordered structures.
381
After 6 h incubation time, the proportion of β-sheet structures significantly increased (p< 0.05),
382
suggesting that internal structures of EWP fibrils stacked as β-sheet. It is believed that the β-
383
sheets usually are a structural characteristic of amyloid fibrils and amyloid-like fibrils and the β-
384
sheet rich structures are important for amyloid stabilization and formation (Mohammadian &
385
Madadlou, 2018; Jansens et al., 2019). The proportion increase of β-sheet structure was
386
intensified with extending of heating duration from 6 h to 24 h and 48 h, indicating more
387
extensive fibrillation happened by increasing the heating time. The increase of β-sheets in
388
circular dichroism spectra upon amyloid fibrillation previously reported for WPI and
389
ovotransferrin (Mohammadian & Madadlou, 2016; Wei & Huang, 2019).
390
The pH dependence of the electric charge of EWP before and after heat-induced fibrillation for
391
6, 24, and 48 h are shown in Fig. 2D. The both native and fibrillated EWP showed similar trends
392
in their ζ-potential-pH profile, where ζ-potential changed from positive to negative as the pH
393
increased from 3 to 9. As compared to the native EWP, fibrillation of EWP raised both the ζ-
394
potential magnitude and pI, especially in the pH range of 3-6. As mentioned above, the
395
combination of low pH and heat during the fibrillation process initially hydrolyze the peptide
396
bonds in proteins to generate the fibril-forming peptides. During the hydrolysis of the peptide
397
bonds, the new terminal carboxyl (with pKa ~ 3.6) and amine groups (with pKa ~8.0) are
398
released (Hass & Mulder, 2015). At acidic pH below and far from their pKa (8.0), -NH3+ groups
399
govern the charge of protein molecules resulting from the protonation of the amino groups. Due
400
to the fact that a large part of the native egg white proteins are converted to peptide fractions
401
with more NH3+ terminal groups during the fibrillation process, one can expect that these
402
fibrillated EWP samples have a higher ζ-potential magnitude at acidic pH condition (e.g. 3-6)
403
compared to the native EWP as a result of protonation of the terminal amino groups. Our results
404
were in agreement with Liu and Zhong (2013) and Mantovani et al. (2016) who reported that the
405
magnitude of absolute ζ-potential and pI of whey proteins increased after fibrillation.
406
3.2. Flow behaviour and surface tension of fibrillated EWP
407
The rheological properties of protein solutions are an important factor contributing to their
408
capacity to develop stable foams. Flow behaviour curves of native and fibrillated EWP solutions
409
at pH of 3, 7 and 9 as a function of heating duration are presented in Fig. 3. Furthermore, Table 2
410
shows the parameters obtained by fitting the flow curve data with Power law model. At pH 3,
411
while the native EWP solution had relatively low viscosity, the fibrillated protein samples
412
showed a significantly higher apparent viscosity (stated as consistency coefficient, K value).
413
Fibrillation process significantly increased the end to end length and subsequently the volume
414
ratio of primary globular monomeric proteins (Mcclements, 2015), so entanglement of the long
415
nanofibrillated structures with each other increase energy dissipation during untangling the
416
chains and raise viscosity of the nanofibrillated EWP solutions compared with the native EWP
417
counterpart (Mohammadian & Madadlou, 2018; Jansens et al., 2019).
418
Whereas for short heating times (6 h), the apparent viscosity at pH 3 only showed a moderate
419
increase, the apparent viscosity tended to a huge increase with heating and maximum viscosity
420
was attained within 48 h. The viscosity of fibril dispersions depends on the effective volume
421
fraction of fibrils (Loveday, Rao, Anema, & Singh, 2012). The viscosity of biopolymers
422
increases dramatically when the effective volume of biopolymer molecules start to increase. The
423
effective volume of a biopolymer is directly dependent on its volume ratio, wherewith increasing
424
end to end length of the biopolymer molecule its volume ratio increases (Mcclements, 2015). So,
425
poor viscosity enhancement in F-6 sample is probably related to the short length of its fibrils
426
compared those fibrils produced by 24 and 48 h heating (see Fig. 1). Fitting of the flow
427
behaviour of fibrillated EWP solutions with Power Law model showed that the solutions
428
behaved as a shear-thinning fluid in the shear rate range of 1 to 100 s-1 (i.e., n < 1; Table 2). The
429
shear-depending behaviour of fibrillated solutions is attributed to the entanglement and
430
entanglement of fibrils at low and high shear rates (Loveday et al., 2012; Mohammadian &
431
Madadlou, 2016). The shear-thinning behaviour was more prominent in F-24 and F-48 samples
432
over F-6 one, probably due to the presence of longer fibrils and higher fibrillation yield in the
433
former samples, as confirmed by TEM, SDS-PAGE and CD data. The longer fibrils in F-24 and
434
F-48 samples strengthen entanglement and disentanglement behaviours at low and high shear
435
rates, respectively, thereby exhibiting more considerable shear-thinning character than the F-6
436
sample.
437
At the same shear rate range, the consistency coefficient (K) increased as the pH increased from
438
3 to 7. As shown in Fig. 2, fibrillated EWP solutions tend to have a significantly lower absolute
439
ζ-potential value at pH 7 (between −15.26 to -13.91 mV) as compared to those of pH 3 (between
440
31.85 to 38.85 mV). The reduced electrostatic repulsion is not strong enough to prevent the
441
aggregation of EWP nanofibrils at pH 7.0, so viscosity tends to increase as a result of higher
442
entanglement of the aggregated fibrils (Peng et al., 2017). At a high shear rate, the aggregated
443
fibrils progressively detangled and oriented toward the shear field direction. This can also
444
explain why the fibrillated solutions at pH 7 showed a more intensive shear-thinning character
445
(lower n values) as compared to those at pH 3. When the pH further raised from 7 to 9 the net
446
charges of fibrils slightly but significantly increased, resulting in increased electrostatic repulsion
447
between fibrils. With the improved electrostatic repulsion, the entanglement of the nanofibrils
448
decrease, thus a lower viscosity was observed for nanofibril solutions in pH 9 over pH 7.
449
The surface tension of different samples as a function of pH is presented in Fig. 3. All of the
450
samples including the native EWP and fibrillated EWP samples at all studied pH values could
451
reduce the surface tension at the air/water interface as compared with distilled water. As can be
452
seen in Fig. 3, the F6, F24 and F48 samples showed the lowest surface tension (the most surface
453
activity) at pH 3, 7 and 9, respectively. Based on ζ-potential data, at pH 3, 7 and 9, the F-6, F-24,
454
and F-48 samples have the lowest absolute surface charges, respectively. So, electrostatic
455
repulsion between F-6, F-24, and F-48 fibril molecules at the liquid-air interface was presumed
456
the weakest at pH 3, 7, and 9, respectively, resulting in easier and faster aligning to form a film
457
in the interface, thus more surface activity. It should be noted that the differences in surface
458
tension of fibrillated EWP solutions in various pH were small (less than 2 units), so surface
459
tension data confirmed that all fibrillated EWP samples had a good affinity towards the liquid-air
460
interfaces at pH values 3, 7, and 9.
461
Protein fibrils are large-sized assemblies. Therefore one should expect that the fibrillated
462
structures will not have a proper surface activity, because they should diffuse more slowly to the
463
air-water interface. However, the fibril suspensions consist of a mixture of fibril structures and
464
hydrolyzed proteins (peptides) that unconverted to fibril. Peng et al. (2017) stated that the
465
adsorption process of the protein fibril systems at the air-water interface was mainly dominated
466
by the high proportion of unconverted peptides, since, compared to large fibrils, these small
467
peptide materials (free peptides and/or peptide aggregates) have faster mass transport rate toward
468
the interface, decreasing the surface tension more rapidly.
469
3.3. Foam properties
470
Foaming properties of native and fibrillated EWP dispersions at pH values of 3, 7 and 9 were
471
studied (Fig. 4). At pH 3, the native EWP showed very low foam capacity (65% overrun) and the
472
foam was also very unstable and collapsed quickly. It should be noted that, at this pH (3), when
473
protein concentration increased to 10%, foam capacity dramatically increased and a relatively
474
stable foam was formed (data not shown). This may be due to the fact that the adsorption amount
475
of egg white proteins at the air/water interface will increase when the concentration of protein
476
increase from 3% to 10%. This can improve the interaction of protein and film thickness to form
477
a stable foam with good overrun.
478
Unlike the native EWP, fibrillated EWP suspensions showed satisfactory foam capacity at pH 3
479
(Fig. 4A) even at 3% protein concentration, suggesting the fibrillation process improved the
480
foaming capacity of the EWP at this condition. At pH 7 and 9, native EWP samples showed high
481
foaming capacity (Fig. 4B and C), suggesting that at neutral and alkaline pH values the native
482
EWP suspensions are capable of forming the high-overrun. At pH 7 and 9, the main protein
483
fractions of EWP (i.e. ovalbumin and ovotransferrin) are negatively charged, while lysozyme is
484
are positively charged. At the conditions, the basic protein lysozyme (pI 10.7) can interact
485
electrostatically with negatively charged proteins, and develop a cohesive viscoelastic film at
486
air/water interface even at low protein concentration (Mine, 1995). Furthermore, at pH 7 and 9,
487
foam capacity of F-24 and F-48 samples did not much differ from their capacity at pH 3 (Fig. 4B
488
and C), indicating that these fibrillated EWP show a good foaming capacity in a wide range of
489
pH from acidic to alkaline conditions. It should be noted that, at these pH values (7 and 9),
490
fibrillated EWP suspensions showed inferior foaming capacity than native EWP ones. It is
491
credited to the higher viscosity of the fibrillated EWP suspensions (see flow behaviour section)
492
that did not allow sufficient incorporation of air during aeration. It is in accordance with those of
493
Mohammadian and Madadlou (2016) and Wan et al. (2016) who reported that fibrillated WPI
494
and soy protein isolate (SPI) yielded lower overrun values than corresponding non-fibrillated
495
proteins.
496
Foam stability was quantitatively estimated by weighing the drained liquid over time. To observe
497
the changes in the fibrillated EWP foam stability, a graph of EWP dispersions foam drainage (%)
498
as a function of time was plotted (Fig. 4D-F). At pH 3, whereas foam prepared from native EWP
499
was very unstable and completely collapsed just after preparation, fibrillated EWP samples were
500
able to produce more stable foams, where the most stable foams were formed by dispersions of
501
the F-48 sample since they exhibit the longest drainage time (Fig. 4D). Furthermore, even though
502
foams prepared from native EWP at pH values 7 and 9 were more stable compared to those
503
prepared at pH 3, their stability was still significantly lower than those prepared by fibrillated
504
EWP suspensions at the same pH values. Similar observations were also reported by Oboroceanu
505
et al. (2014) who found that WPI fibrils at both pH values of 2 and 7 had better foaming stability
506
than native WPI. Our study confirmed that even at alkaline pH 9, EWP fibrils have a
507
significantly higher capacity to produce stable foams compared to native EWP. As mentioned
508
above, fibrillated proteins form a high elastic layer with solid-like behaviour at the air-water
509
interface, preventing coalescence, drainage, and coarsening of foams (Wan et al., 2016; Peng et
510
al., 2017). Furthermore, foam stability is influenced by the viscosity of the continuous fluid
511
phase, where a high viscosity of continuous phase delay the movement of liquid through the
512
network of thin films and plateau borders, thereby slowing the drainage rate (Gharbi & Labbafi,
513
2019). Foams under gravity-induced drainages typically experienced a shear rate of about 8.5 1/s
514
(Yang & Foegeding, 2010). The solution apparent viscosity of native EWP and fibrillated EWP
515
F-6, F-24, and F-48 samples at pH 3.0 and shear rate 8.5 1/s were 2.2 ± 0.1, 16.9 ± 1.6, 103.6 ±
516
8.3, and 164.2 ± 11.7 mPa s. Therefore, a negative relationship observed between foam drainage
517
and solution apparent viscosity at a shear rate of 8.5 1/s, explaining the longer drainage time
518
observed by fibrillated EWP suspensions as compared with native ones. It also explains why,
519
among fibrillated EWP suspensions, the F-48 sample showed the longest foam drainage time at
520
pH 3 (because this suspension has the highest apparent viscosity at a shear rate of 8.5 1/s).
521
In accordance with increased apparent viscosity of fibrillated EWP suspensions at pH 7
522
compared to pH 3 (see Fig. 3A-C), a significant increase in foam stability of these fibrillated
523
systems was observed at pH 7 in comparison with pH 3 (Fig. 4E). As seen in Fig. 4E, at pH 7 a
524
lag in drainage onset (up to 30 min) was observed in foams produced by fibrillated EWP
525
samples, and only 15-27% drainage occurred at these foams after 300 min, while at native EWP
526
foam mostly 50% drainage was observed in the first 1 h of foam formation. In addition to the
527
increased apparent viscosity at pH 7 compared to pH 3, fibrils had a high negative surface charge
528
at pH 3.0 (see Fig. 2D), thus the surface of bubbles was not densely packed by fibrils, owing to
529
the strong electrostatic repulsion among fibrils at the interface. Hence, at pH 3, the foams had the
530
lowest foam stability. However, as evidenced by the zeta-potential data (Fig. 2D), fibrils at pH 7
531
had lower net charges as compared to pH 3 so that at this pH fibrils are able to form a thicker
532
protective layer around bubbles by overlapping and entanglement of the fibrils leading to the
533
higher foam stability (Peng et al., 2017).
534
Foams produced by fibrillated EWP suspensions at pH 9 showed an even longer lag phase in
535
drainage onset compared to pH 7. While the drainage onset in foam produced by F-6 suspension
536
detected after 180 min of foam formation, at foams produced by F-24 and F-48 suspensions the
537
lag phase of drainage onset even was longer and any drainage was not detected after 300 min.
538
Nevertheless, the consistency coefficient of the fibrillated EWP suspensions at pH 7 was higher
539
than pH 9 (Table 2). On the other hand, due to increased electrostatic repulsion among fibrils at
540
pH 9 than pH 7, it should expect to a drop in packing intensity of fibrils at the interfacial layer
541
leading to the lower foam stability at pH 9 than pH 7. A rational reason for the unexpected
542
greater foaming stability observed at pH 9 than pH 7 at fibrillated EWP samples maybe this fact
543
that disulphide bonds could be formed between fibrils at interface or continuous phase causing
544
the formation of clusters in the interface or continuous phase. As seen in Fig. 2B, fibrillated
545
EWP samples have a very high surface free –SH groups compared native EWP counterpart. The
546
reactivity of the surface free –SH groups in alkaline pH (e.g. 9.0) is very high and significant
547
SH-SH oxidation to S-S occurred even at room temperature (Hoffmann & van Mil, 1997). In this
548
regard, Bolder et al. (2007) observed that the fibrillated WPI samples which were stored at pH 7
549
showed increased viscosity after overnight storage at cold temperature, and the samples that were
550
stored at pH 10 gelled overnight. Using gel electrophoresis, the authors confirmed that storage of
551
the fibrils at higher pH conditions induces the formation of larger structures due to the formation
552
of disulphide bonds in the sample (Bolder et al., 2007). Similarly, in foam systems, one can
553
expect that the formation of the disulfide bonds induces developing a gel-like structure with the
554
high elastic module at interface layer or continues phase, thus the ultra-high stable foams can be
555
produced by fibrils at alkaline pH.
556
Changes of bubbles size of foams formed from native and fibrillated EWP at pH values of 3, 7
557
and 9 with the extension of time are presented in Figure 5. At pH 3, as mentioned above, foams
558
prepared by native EWP were unstable and collapsed quickly. So, the foams were not evaluated
559
for changes in bubbles size. As can be seen from Figure 5A, the bubbles formed from F-6 sample
560
showed a larger initial bubble size as compared to those prepared from F-24 and F-48 samples,
561
which suggests more liquid may already drain during foam creation from F-6 samples.
562
Moreover, for all systems, it can be observed that with increasing time the bubble size gradually
563
increased due to the destabilization processes, such as liquid drainage, coarsening, and
564
coalescence (van der Plancken et al., 2007). However, in accordance with foam stability data, by
565
comparing the images of the bubbles with time (Fig. 5A), the rate of bubble size increase of the
566
F-48 foam system was significantly lower than those of F-24 and F-6 ones, confirming the foam
567
stability of the F-48 system appeared to be better than later ones.
568
At pH 7, the bubbles formed from the fibrillated EWP systems showed comparable initial bubble
569
size (at 0 min), which were much smaller than that observed in native EWP (Fig. 5B).
570
Additionally, a foam prepared from fibrillated EWP samples shows a lower rate of increase in
571
the bubble size over time compared to the native EWP. Same with pH 7, the foams made from
572
all fibrillated EWP samples at pH 9 showed much smaller bubbles size as compared to foams
573
formed from native EWP (Fig. 5C). Furthermore, whereas the initial bubbles of the fibrillated
574
EWP systems remained their spherical shape even after 300 min, the bubbles from native EWP
575
tend to be polyhedral only after 60 min, implying the liquid drainage in foams formed from
576
native EWP is much faster than in the fibrillated EWP systems. Additionally, as it is inferred by
577
comparing the microscopic images of the bubbles with time, the size of bubbles stabilized by
578
fibrils at pH 9 displayed the slowest rate of increase over pH values of 3 and 7. It is in
579
accordance with foam stability data that presented foams formed from fibrillated EWP
580
suspensions at pH 9 showed a longer lag phase in drainage onset compared to pHs of 7 and 3.
581
The data obtained from the analysis of foam density showed that foams produced by fibrillated
582
EWP samples had significantly higher foam density than that of native EWP (Fig. 6). At pH 3, as
583
said above, foams prepared by native EWP were unstable and collapsed quickly. So, the foams
584
were not evaluated for foam density. As expected, the density of the foams had an inverse
585
relationship with their overrun. The higher density of foams prepared from fibrillated EWP
586
suspensions resulted in a moist and creamy appearance, contrasting with the crispy and dry
587
appearance of the foams prepared from native EWP suspensions. The lower liquid volume
588
fractions of the native EWP foams at pH 7 and 9 (corresponding to their higher overrun) results
589
in that these foams behaviour like dry foams. However, the high liquid volume fraction of
590
fibrillated EWP foams (corresponding to their lower overrun) probably modulate the properties
591
of these foams correspond to wet foams or “bubbly liquid” systems (Nicorescu et al., 2011;
592
Furuta, Oikawa, & Kurita, 2016). This result was very similar to van der Plancken et al. (2007),
593
which found that heat-treated EWP produced moist and sticky foam.
594
3.4. Meringue batter characterisation
595
Meringues (sweetened egg white foams) are the main part of a wide range of culinary recipes
596
such as soufflés, macarons, tiramisu, mousses, and angel food cake, where they provide most of
597
the structural support (Vega & Sanghvi, 2012). Therefore, due to their simple composition, we
598
investigated the rheological characteristics of meringue batters as a model system to study the
599
behaviour of fibrillated EWP in aerated systems containing a high level of sugar. Because in our
600
preliminary investigation on foaming capacity and foam stability of fibrillated EWP suspension
601
(see above), F-48 suspension showed the promising results, this fibrillated EWP system was
602
selected to prepare meringue batter.
603
Fig. 7A shows density values of meringue batters produced from native and fibrillated EWP
604
samples. In the meringue batters prepared from 3.0% and 10.0 % native EWP (called N-3% and
605
N-10% meringue batters, respectively), batter density values were 0.61 and 0.58 mg mL-1,
606
respectively, while this value for the meringue made from 3.0% F-48 fibrillated EWP (called F-
607
48-3% meringue batter) was significantly lower (0.49 g mL-1). These results interestingly
608
indicated that the F-48 fibrillated EWP incorporated higher air to its meringue batter than that of
609
native EWP at 3.0% and even 10.0% of protein concentrations, even though the latter contained
610
three times more protein than the F-48 sample. It suggested that although the native EWP
611
solutions showed greater overrun (see Fig. 4B) than fibrillated F-48 solutions at pH 7 (as pH that
612
meringue batters produced), the air volume incorporated into F-48 meringue batter was superior
613
to N-3% and N-10% meringue batters. Thus, while foam capacity (indicated as overrun) is
614
considered an important parameter determining the foaming properties of protein solutions, it
615
does not seem to be of particular relevance in the meringue system studied here. This is probably
616
relevant to the very high sugar content of meringues. Sugar has two roles here. First, the
617
presence of sucrose in protein solution increased solution viscosity and slow down diffusion rate
618
of the protein molecules toward the interface, so less air incorporated into the foam in a given
619
period (Raikos et al., 2007). As previously stated, the adsorption process of the protein fibril
620
systems at the air-water interface was mainly dominated by unconverted peptides (hydrolyzed
621
proteins that unconverted to fibril). In the condition that the continuous phase viscosity of both
622
meringue batters prepared from native and fibrillated EWP increases due to high content of
623
sugar, peptides in protein fibril system (i.e. F-48 sample) may have a faster and more efficient
624
absorption toward the air/water interface in the viscous continuous phase when compared to
625
native protein molecules. Second, sucrose competes for available water molecules, where the
626
direct contact between protein and water is considered thermodynamically unfavourable in the
627
presence of a high concentration of sugars and this can be correlated directly with an
628
enhancement of hydrophobic interactions between protein molecules (Dickinson & Merino,
629
2002). Given that the free –SH groups and CD spectroscopy data showed that fibrillation process
630
caused profound changes in the molecular structure of egg white proteins, it can be expected that
631
the sugars have very different effects on these two proteins in terms of sugar-induced
632
hydrophobic interactions and thus affect the amount of air that these two different protein
633
structures can incorporate in a high sugar aerated system.
634
To observe the visual flow behaviour of the resulting meringue batters, about 6 g of meringue
635
batters were shaped as cones of about 4.5 cm diameter on a plane surface using a manual pastry
636
bag with a tip diameter of 7 mm. As is evidenced in Fig. 7B, when N-3% batter shaped on the
637
surface, they spread quickly. With the N-10% batter, although the rate of spreading was a bit less
638
than N-3% batter, they also spread significantly after a while (about 2 min). However, the F-48
639
batter retained their shape significantly better than those containing native EWP (Fig. 7B). In
640
accordance with our results, Wouters et al. (2018), that used the same sugar concentration to
641
prepare their meringue batters, observed that batters containing EWP at both concentrations of
642
5% and 10% were runny and did not retain their shape when shaped on baking paper.
643
Steady-state flow curves of all meringue batter samples matched to a structured fluid with a clear
644
shear-thinning behaviour over a wide range of shear rates (10-3-102 1/s). Furthermore, there are
645
two well-defined regions in the flow curves of all samples; at low shear rates, viscosity reaches a
646
limiting value, namely zero-shear viscosity (CO ); as the shear rate rises, a sudden power-law
647
decay in the viscosity was detected (Fig. 8A). Thus, the flow behaviour of the meringue batters
648
was satisfactorily fitted to the Cross and Ellis model (R2 > 0.99). These models successfully
649
applied to other systems containing CO (Rao, 2014). Table 3 shows the parameters obtained with
650
both Cross and Ellis models for various meringue batters.
651
Although there were some differences in values of the rheological parameters in Cross and Ellis
652
models, it can be seen that the meringue batters produced from fibrillated F-48 EWP (F-48
653
batter) showed the highest zero-shear rate viscosity (CO ) in both fitted models. N-3% batters had
654
the lowest CO between studied meringue batters. Furthermore, the dynamic yield stress
655
(determined by fitting the data using the Ellis model) of F-48 meringue batters were significantly
656
higher than those meringue batters produced from native EWP solutions at both protein
657
concentrations of 3% and 10% (Table 3). Similar to the observed trend in CO , the lowest dynamic
658
yield stress corresponded to N-3% meringue batters. The reciprocal 1/P@ gives us a critical shear
659
rate as an indicator of the onset shear rate for shear-thinning region (Yang & Luo, 2013). So,
660
high Cross time constant (P@ ) in F-48 meringue batter implied that onset of the shear-thinning
661
region in this batter occurs at a lower shear rate as compared to batters prepared from native
662
EWP. The parameter “m” is considered as a measure of the shear rate dependence of viscosity in
663
the shear-thinning region and a higher “m” indicated stronger shear-thinning properties. It can be
664
seen from Table 3 that the F-48 meringue batters showed significantly higher “m” when
665
compared with those prepared from native EWP, suggesting the former batters had a stronger
666
shear-thinning behaviour than latter ones.
667
The higher zero-shear viscosity and yield stress in meringue batters produced from fibrillated F-
668
48 EWP when compared to those prepared by native EWP could explain that why former batters
669
retain shape significantly superior to latter ones (see Fig. 7B). The higher zero-shear viscosity
670
and yield stress of F-48 meringue batter were attributed to the presence of fibril chain
671
entanglements; the fibrillated EWP system has a curly shape, and compared to spherical-shape
672
native EWP are more easily tangled. Thus, at zero-shear viscosity regions (unsheared state), the
673
F-48 meringue batters had a very high viscosity (CO = 9.08 × 103 Pa s) due to these
674
entanglements. However, after sufficient shear, almost all of the entanglement was disentangled
675
and quick align in the direction of flow as shear rate increases and therefore physical interactions
676
between adjacent fibril chains decrease. Therefore, F-48 meringue batter showed a stronger and
677
earlier shear-thinning behaviour (higher m and P@ values, respectively) as compared to meringue
678
batters containing native EWP. The strong shear-thinning behavior of fibrillated EWP containing
679
meringue batters (F-48 batters) allows the batters to be pumped easily. Furthermore, these batters
680
with higher shear-thinning behaviour and zero-shear rate viscosity are beneficial for extruding
681
and 3D printing of confectionery goods, as they can be easily extruded out from the nozzle with
682
the application of shear force and solidify rapidly again after leaving the nozzle (Liu, Zhang,
683
Bhandari, & Wang, 2017). On the other hand, given that the applied shear rate upon a whipping
684
process usually is sufficiently high (more than 102 1/s) (Yang & Foegeding, 2010), the extensive
685
shear-thinning behaviour of fibrillated EWP in the presence of high sugar content (e.g. in
686
meringue batter recipe) may be another beneficial characteristic; the shear-thinning behaviour
687
allows that air easily incorporated in batter system upon whipping and then the air bubbles be
688
stabilized at a viscoelastic matrix after stopping the whipping process (zero-shear rate condition).
689
So, the superior air incorporation of F-48 batters compared to native EWP batters can be
690
explained by the more and earlier shear-thinning behavior of former batters than latter ones as
691
well as the existence of peptides in protein fibril system (i.e. F-48 batter) having a faster and
692
more efficient absorption toward the air/water interface when compared to native protein
693
molecules.
694
To analysis the oscillatory response of the meringue batters, frequency sweep measurements
695
were applied. The frequency sweeps plots of different meringue batter formulations are depicted
696
in Fig. 8B. The frequency (ω) dependence of G’ and G” can indicate the type of structure present
697
in the batters. N-3% meringue batter exhibited a strong dependence of G’ with ω, and one can
698
see an overlapping of the G’ and G” curves indicating a viscose-like behaviour (Zhang, Arrighi,
699
Campbell, Lonchamp, & Euston, 2018). With F-48 meringue batter, G’ was always greater than
700
G'' and its G’ only had a weak dependence on ω values, suggesting the formation of a solid-like
701
structure. Between these two batters was the N-10% meringue batter that showed a moderate
702
frequency dependence of G’ on ω and a G'/G” crossover in high ω. To quantitative analyse the
703
degree of frequency dependence of the storage modulus (G′), a Power-law model (] ^ = &_< )
704
was fitted to the results from Fig. 8B and the fitted Power-law parameters are shown in Table 4.
705
The coefficients “a” and “n” represent the magnitude of the intercepts at frequency 1 Hz and the
706
slopes of G′ as a function of frequency (ω), respectively. The “a” value is related to the strength
707
(elastic structure) of a sample. The “n” value close to zero is characteristic of a truly solid-like
708
material. For “n’ value = 1 the system behaves as a viscous material (Sadahira et al., 2018). The
709
higher “a” value and lower “n” value at F-48 meringue batter than N-10% and N-3% meringue
710
batters show that former batter is more solid than the latter ones.
711
The Cox-Merz rule is an empirical relationship that enables comparison of steady shear viscosity
712
to complex oscillatory viscosity in equal shear rates and frequencies range. When a system is a
713
liquid (viscose), it will obey the Cox-Merz rule i.e. the steady shear viscosity curve completely
714
overlap on complex oscillatory viscosity curve, while those that are elastic will not. Thus,
715
adherence to the Cox-Merz rule can be used to assess further the structure in a system (Young,
716
2014). As shown in Fig. 8C, plots of complex oscillatory viscosity and shear viscosity for the F-
717
48 meringue batter indicate a strong deviation from the Cox-Merz rule. Such behaviour suggests
718
that the solid-like (elastic) structure is formed in these batters (Zhang et al., 2018), probably due
719
to the occurrence of strong inter-fibrillated bonds (such as disulfide bonds, as previously
720
discussed) and formation of strong entanglements of the curly protein structures in these batters
721
prepared at pH 7. The complex viscosity of N-3% meringue batter (Fig. 8C) was found to be
722
close to the shear viscosity, indicating that this batter complied with the Cox-Merz rule and thus
723
had a predominantly viscose weak structure. Between these two batters was the N-10% meringue
724
batter where moderate deviation from Cox-Merz behaviour was seen. Generally, the rheological
725
data showed that the meringue batters containing fibrillated EWP exhibit a stronger rheological
726
behaviour with more elastic elements over meringue batters made of native EWP.
727
4. Conclusions
728
This study showed that whole EWP can be fibrillated by heating at acidic condition (pH 2).
729
Fibrillated EWP has curly shape, although some of the long straight fibrils appeared with
730
increasing of heating time to 48 h. Fibrillation increased β-sheet elements, surface free –SH
731
groups, and consistency. Furthermore, foams produced from fibrillated EWP showed significant
732
stability when compared with native EWP at all of the studied pH values. By assessment of the
733
functionality of these fibrillated EWP in a standard meringue batter, we also conclude that the
734
improved viscosity and foam properties of fibrillated EWP had great relevance for using in high
735
sugar aerated systems. Thus, these results suggested that fibrillated EWP systems could be used
736
as versatile thickening and texturizing agents in high sugar aerated confectionery products that
737
will offer food manufacturers greater control over the texture and consistency of formulated
738
foods.
739
Acknowledgement:
740
The authors thank the Iran National Science Foundation (INSF) for financial support of the
741
project.
742
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743
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Table captions Table 1. Estimates of secondary structures content for native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48). Table 2. Parameters from Power law model fitting for native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48). K, n, and R2 are consistency coefficient (mPa sn), flow behaviour index (dimensionless) and confidence of fit, respectively. Table 3. Rheological parameters from Cross and Ellis models fitting of the flow behaviour of meringue batters prepared from F-48 fibrillar EWP (F-48), native EWP with a protein concentration of 3% (N-3%), and native EWP with protein concentration of 10% (N-10%). ߟ is zero-shear rate viscosity, ߙ is Cross time constant, m is dimensionless exponent index, and ߪ is critical stress or yield stress. Table 4. Power law parameters for storage modulus (G’) of meringue batters prepared from F-48 fibrillar EWP (F-48), native EWP with protein concentration of 3% (N-3%), and native EWP with protein concentration of 10% (N-10%).
Figure captions Fig. 1. TEM images of native (A) and fibrillar EWP prepared from incubation time of 6 h (B), 24 h (C), and 48 h (D) at ×27800 magnification. Fig. 2. (A) SDS-PAGE of native EWP (lane 1), fibrillar EWP prepared from incubation time 6 h (lane 2), 24 h (lane 3), 48 h (lane 4), and molecular-weight size marker (lane M). (B) surface and total free –SH groups, (C) Circular dichroism spectrum, (D) pH dependence of the ζ-potential of the native of EWP (N), and fibrillar EWP prepared from incubation time 6 h (F-6), 24 h (F-24), and 48 h (F-48). Fig. 3. Flow curve of native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48) at pH 3 (A), 7 (B), and 9 (C). Surface tension as a function of time for native EWP (N), and fibrillar EWP prepared from incubation time 6 h (F-6), 24 h (F24), and 48 h (F-48) at pH 3 (D), 7 (E), and 9 (F). Fig. 4. Overrun of native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F6), 24 h (F-24), and 48 h (F-48) at pH 3 (A), 7 (B), and 9 (C). Foam drainage (%) as a function of time for native EWP (N), and fibrillar EWP prepared from incubation time 6 h (F-6), 24 h (F24), and 48 h (F-48) at pH 3 (D), 7 (E), and 9 (F). Fig. 5. Changes of bubbles size of foams formed from native EWP (N), and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48) at pH 3 (A), 7 (B), and 9 (C). Fig. 6. Density of foams formed from native EWP and fibrillar EWP prepared from incubation time of 6 h (F-6), 24 h (F-24), and 48 h (F-48) at pH 3, 7, and 9. Because the foams prepared by
native EWP at pH 3 were unstable and collapsed quickly, the foams were not evaluated for foam density.
Fig. 7. Density (A) and representative images (B) of meringue batters prepared from F-48 fibrillar EWP (F-48), native EWP with protein concentration of 3% (N-3%), and native EWP with protein concentration of 10% (N-10%). Fig. 8. Flow curve (A) as a function of shear rate, (B) Storage (G′, solid symbols) and loss modulus (G″, open symbols) as a function of frequency, and Cox-Merz plots (C) of meringue batters prepared from F-48 fibrillar EWP (F-48), native EWP with protein concentration of 3% (N-3%), and native EWP with protein concentration of 10% (N-10%).
Table 1. Samples N
F-6
F-24
F-48
α-helix
24.8 ± 0.2a
21.5 ± 0.3b
20.0 ± 0.2c
19.1 ± 0.4d
β-sheets
23.7 ± 0.1d
36.2 ± 0.5c
39.1 ± 0.4b
40.0 ± 0.4a
β-turn
15.6 ± 0.2b
16.2 ± 0.2a
15.6 ± 0.3b
15.0 ± 0.4b
Random coil
35.9 ± 0.2a
26.1 ± 0.3b
25.2 ± 0.3c
25.9 ± 0.4b
Different superscripts in each column represent a significant difference (p<0.05). Data are means ± SD.
Table 2. Sample
K
n
R2
pH 3 N
2.8 ± 0.2i
0.895 ± 0.02a
0.948
F-6
25.5 ± 2.2g
0.823 ± 0.03b
0.978
F-24
217.7 ± 11.1f
0.647 ± 0.03c
0.997
F-48
349.6 ± 14.3e
0.63 ± 0.03c
0.994
pH 7 N
3.3 ± 0.1h
0.870 ± 0.03a
0.941
F-6
632.8 ± 24.1c
0.287 ± 0.04f
0.993
F-24
919.8 ± 41.1b
0.113 ± 0.03g
0.992
F-48
1190.3 ± 31.8a
0.019 ± 0.04h
0.993
pH 9 N
3.0 ± 0.2j
0.913 ± 0.01a
0.921
F-6
462.4 ± 18.4d
0.439 ± 0.03e
0.992
F-48
246.6 ± 17.1f
0.546 ± 0.04d
0.991
F48-9
1082.9 ± 90.2a
0.248 ± 0.02
0.991
Different superscripts in each column represent a significant difference (p<0.05). Data are means ± SD.
Table 3. Batter type
Cross model
Ellis model
ߟ (Pa s)
ߙ (s)
m
R2
ߟ (Pa s)
ߪ (Pa)
m
R2
N-3%
1.58 × 102
7.59
0.61
0.993
1.49 × 102
10.8
0.99
0.994
N-10%
3.99 × 103
79.3
0.79
0.999
3.38 × 103
30.2
1.90
0.999
F-48-3%
9.08 × 103
147.8
1.11
0.999
7.23 × 103
34.3
5.73
0.994
Table 4. Sample
a
n
R2
N-3%
131.1 ± 8.7c
0.59 ± 0.02a
0.988
N-10%
502.3 ± 24.3b
0.31 ± 0.01b
0.994
F-48
1722.5 ± 54.1a
0.22 ± 0.01c
0.996
Different superscripts in each column represent a significant difference (p<0.05). Data are means ± SD.
Fig. 1.
Fig. 2
Fig. 3
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.
Highlights •
Egg white proteins (EWP) were fibrillated via heating at pH 2.0.
•
Foams made by fibrillar EWP showed greater stability over those formed by native EWP.
•
Fibril-based meringue batter had higher yield stress over that from native EWP.
•
Meringue batters based on native EWP behaving as a predominantly viscose system.
•
Batters prepared form fibrillar EWP had a solid-like structure.
The authors declare that there is no conflict of interest regarding the publication of this article except with our former colleague Dr. Ashkan Madadlou ([email protected]) who left our department three years ago.