Ovalbumin as an Outstanding Pickering Nanostabilizer for High

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Ovalbumin as an Outstanding Pickering Nanostabilizer for High Internal Phase Emulsions Yan-Teng Xu, Chuan-He Tang, Tong Xun Liu, and Rui Hai Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02183 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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Journal of Agricultural and Food Chemistry

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Ovalbumin as an Outstanding Pickering Nano-stabilizer for High Internal Phase

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Emulsions

4 †‡§ † ¶ Yan-Teng Xu†, Chuan-He Tang *, , , , Tong-Xun Liu , & Ruihai Liu

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†

Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, P. R. ‡

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China. State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou

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510640, People’s Republic of China.

§

Overseas Expertise Introduction Center for Discipline Innovation of Food ¶

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Nutrition and Human Health (111 Center), Guangzhou, China.

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New York, USA.

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Correspondence and requests for materials should be addressed to C. H. T. (email: [email protected]).

Department of Food Science, Cornell University,

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ACS Paragon Plus Environment


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Abstract: There is still a debate about the effectiveness of native globular proteins to perform as

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Pickering-like stabilizers for oil-in-water high internal phase emulsions (HIPEs). In the work, we

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report one native globular protein (ovalbumin) with strong structural integridity and high refolding

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ability, exhibit an outstanding Pickering stabilization for HIPEs. Ultrastable gel-like HIPEs can be

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formed through a facile one-pot homogenization, even at a concentration as low as 0.2 wt%. The

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HIPEs formed in the protein-poor regime are a kind of self-supporting and remoldable hydrogels

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consisting of bridging droplets. And the formed HIPEs also exhibit other unique characteristics, such

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as extraordinary coalescence stability (against prolonged storage or heating), susceptibility to

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freeze-thawing, enhanced oxidation stability (to encapsulated bioactives), and inhibited vaporization

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of volatile oils. The findings would be of importance for extending the HIPEs to be applied in food,

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cosmetic and petroleum industries.

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Keywords: Ovalbumin; High internal phase emulsions (HIPEs); Pickering stabilization; HIPE

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Hydrogels; Thermal stability

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INTRODUCTION

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High internal phase emulsions (HIPEs), also called as highly concentrated emulsions or gel

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emulsions, have recently received tremendous attention, in particular from the fields of food,

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cosmetic, tissue engineering, pharmaceutical and petroleum industries 1-5. The HIPEs are usually

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characterized by a minimal internal phase volume ratio (φ) of 0.74 for close hexagonal packing 6,7, or

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0.64 for random close packing 8. In addition to conventional surfactants, a broad range of solid

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colloidal particles of inorganic and organic origins, including silica 1, 9-11 or graphene oxide 12

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nanoparticles, microgels 13, cellulose or chitin nanocrystals 14, 15, assembled block copolymer 4, and

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more recently, protein-based colloidal particles 16-19, can be applied to act as effective Pickering

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stabilizers for HIPEs (w/o, or o/w). The need for biocompatible HIPEs that effectively protect

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oxidation of oils, inhibit vaporization of essential oils, and act as delivery systems for lipid-soluble

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nutraceuticals, or as hydrogel templates for tissue engineering arises. In this aspect, many

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protein-based colloidal particles to perform as effective HIPE stabilizers exhibit many advantages

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over other particles, thanks to their good hydrophilicity/hydrophobicity balance, high tendency to

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fabricate, and available at low cost or environment-friendly. However, only a limited number of

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works are available in the literature addressing the fabrication of Pickering HIPEs (mainly of o/w

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type) stabilized by protein-based colloidal particles (100~200 nm) 2, 16, 18, 19, though these particles to

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perform as Pickering stabilizers have been well documented 20-27.

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In general, the effectiveness of particles to perform as Pickering stabilizers is determined by their

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structural integrity (especially when adsorbed at the oil-water interface). For protein-based particles,

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the structural integrity can be strengthened by intra-particle covalent crosslinking 2, 23, 27. Without

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crosslinking, whey protein isolate (~153 nm; at pH 3.5) could not result in stable HIPEs (φ = 0.8) 19. 3



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This is consistent with the general viewpoint that many proteinaceous colloidal particles with a loose

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structural integrity (e.g., casein micelles) cannot be considered to be as effective Pickering stabilizers

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ovalbumin (OVA), β-lactoglobulin (β-LG) and lysozyme, and subunits of legume 7S or 11S

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globulins, in their native state, exhibit a high structural integrity with strong intramolecular

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interactions. Although it is well documented that many native globular proteins can be applied to

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stabilize highly concentrated emulsions (or HIPEs) 29-31, the coalescence stability of these emulsions

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is always the subject of concerns in the field. In most of cases using native globular proteins as

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stabilizers for HIPEs, a high protein concentration in the aqueous phase (c) is often applied, e.g. 10

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wt% for the stable and gel-like HIPE (at φ = 0.8) stabilized by native BSA 16. To the best knowledge

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of ours, no work is available addressing the HIPEs stabilized by these compact globular proteins at

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extremely low c values (e.g., < 0.5 wt%). On the other hand, we should always keep in mind that the

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structural characteristics and properties of globular proteins considerably vary. This leads us to

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wonder that there would be some native globular proteins of small dimension (e.g. with sizes of

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several nanometers) exhibiting a good emulsification efficiency, as well as an excellent Pickering

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stabilization for stable HIPEs. To differentiate from conventional small-molecular-weight surfactants

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and large-size solid or colloidal Pickering particles, we call these globular proteins with sizes from

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several to dozens of nanometers as ‘Pickering molecular stabilizers’. It therefore remains a

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considerable challenge to unravel what common structural characteristics these Pickering globular

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proteins share.

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. In fact, many globular proteins of several nanometers in size, e.g., bovine serum albumin (BSA),

Herein, we for the first time report that native OVA is such an outstanding Pickering nano-stabilizer for HIPEs. Stable and gel-like HIPEs stabilized solely by OVA can be fabricated 4


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using a facile one-pot homogenization, even at c values as low as 0.2 wt%. The rheological behavior

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and microstructure of these HIPEs can be delicately modulated by changing the c from 0.2 to 3.0

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wt%. Surprisingly, the HIPEs formed in the protein-poor regime are a kind of HIPE hydrogels with a

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self-supporting and remoldable gel network consisting of bridging droplets. The fabricated HIPEs or

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HIPE hydrogels exhibit some unique characteristics, such as extraordinary coalescence stability

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(against prolonged storage or heating), tempeature-responsiveness, enhanced oxidation stability (to

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encapsulated bioactives), and inhibited vaporization of volatile oils.

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Together with the facile fabrication, these unique characteristics impart these novel HIPEs or HIPE

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hydrogels a great potential to be applied in the food, cosmetic and even petroleum fields.

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MATERIALS AND METHODS Matrerials. OVA (≥ 90%) from chicken egg was purchased from Sigma-Aldrich Co. LLC.

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(USA). Dodecane (99%) was bought from Adamas Reagent, Ltd. (China). 30% β-carotene

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(Lucarotin® 30 sun) was purchased from BASF Corporation (Germany). Tween 20 (polyoxyethylene

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glycol sorbitan monolaurate; also named Polisorbate 20; purity = 100%) and Tween 80 (polyethylene

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glycol sorbitan monooleate; also named Polisorbate 80; purity = 100%) were purchased form

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Aladdin Industrial Corporation (Shanghai, China). All of other reagents are analytical reagents.

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Emulsion preparation. Prior to emulsification, native OVA was solubilized in 5 mM phosphate

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buffer (pH 7.0). The protein solution containing 0.02 wt% sodium azide (for the inhibition of

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microbial growth) was stored overnight to achieve complete hydration of OVA. The physicochemical

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and structural characteristics, including hydrodyanmic size, ζ-potential and surface hydrophobicity,

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of all OVA were determined according to the methods as described in Supporting Information and 5



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summarized Supplementary Table S1. Oil-in-water (o/w) emulsions with different φ values of 0.1-0.92, or HIPEs at φ = 0.8, were directly

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prepared by homogenizing the mixtures of the continueous phase (protein solutions; 0.2-3.0 wt%)

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and the dispersed phase (dodecane) at the corresponding ratios, at 5000 rpm for 2 min, using a

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XHF-DY high-speed dispersing unit with a 10 mm head (Ningbo Scientz Biotechnology Co., China).

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For the HIPEs loaded with β-carotene (0.003 or 1.0 wt%; relative to oil phase), β-carotene was added

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into the dispersed phase prior to the emulsification. The HIPEs stabilized by low-molecular weight

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surfactants Tween 20 or 80 at φ = 0.8 and c = 5.0 wt% were prepared according to the same process

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as described above.

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Microstructure of emulsions or HIPEs. The microstructure of emulsions or HIPEs stabilized

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by OVA was evaluated by optical microscopy (Olympus BX51 with an Olympus DP70 camera)

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and/or confocal laser scanning microscopy (CLSM) using a Leica DMRE-7 (SDK) upright

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microscope (Leica Microsystems Inc., Heidelberg, Germany) with a Leica TCS SP5 confocal laser

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scanning head. Surface-average droplet size (d3,2 = Σnidi3/Σnidi2) of these emulsions was determined

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using a microscopic image analysis software (Nano Measurer 1.2, Fudan University, China), on their

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optical microscopic images. For each sample, three different captured images were selected

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randomly, and all the droplets (150 ~ 1500) in every image were measured to obtain their d3, 2. For

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CLSM observations, all the samples were dyed with Nile Blue (Sigma Aldrich; for proteins). Each

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HIPE or HIPE hydrogel (5 mL) was mixed with 15 µL propylene glycol solution containing 0.1%

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(w/v) Nile Blue. The tested samples were then put on concave confocal microscope slides (Sail,

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Sailing Medical-Lab Industries Co. Ltd., Suzhou, China) covered by glycerol-coated coverslips, and

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observed using a 40 × magnification lens with a He-Ne laser excitation wavelength at 633 nm. 6


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Rheological behavior test. The dynamic viscoelastic behavior of HIPEs or HIPE hydrogels was

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characterized using a HAAKE RS600 Rheometer (HAAKE Co., Germany) with parallel plates (d =

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27.83 mm) at 25 °C. The gap between two plates was set to 1.0 mm. For the strain sweep mode,

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elastic (G’) and loss moduli (G’’) were recorded with strain (γ) changing from 0.002 to 1.0 at a fixed

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frequency of 1.0 Hz. And for the frequency sweep mode, the frequency was oscillated from 0.1 to 10

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Hz and all measurements performed at 0.5% strain which was within the identified linear viscoelastic

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region.

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Surface coverage of OVA at o/w interface. Suppose that all the droplets in the HIPEs (φ = 0.8)

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are stabilized by OVA in a closely packed monolayer way, and OVA molecules are hard spheres

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without deformation occurring when adsorbed at the interface, the theoretical full surface

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concentration, Γfull, can be obtained as the following equation 47:

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Γfull =

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Taking the density of OVA molecules ρova = 1.426 g cm-3 and the radius of OVA molecules a = 2.6

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nm as measured by DLS (Supplementary Table S1), we can calculate Γfull = 9.76 mg m-2. The

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densest packing of hard spheres is reached with a hexagonal packing array (hpa): Γhpa=

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0.907 Γfull = 8.85 mg m-2.

=

=

√

Γfull =

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The non-adsorbed OVA in the different HIPEs was removed by mixing the HIPE with 5 mM

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phosphate buffer at a weight ratio of 1:20 for more than 12 h, followed by a dispersion using a vortex

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for 10 s and centrifugation at 2500 g for 15 min. The adsorbed amount of OVA at the interface was

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calculated by substracting the amount of OVA in the above supernatants from the total protein

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amount. The surface concentration for the HIPEs was then determined by dividing the adsorbed OV

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amount against total interface area (A = π (d3,2)2, where d3,2 is the surface-average droplet size of the 7



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HIPEs). For instance, the surface concentration for the HIPE at c = 0.2 wt% was determined to be 8.0

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mg m-2, which corresponds to a percentage of surface coverage (SC%) of approximately 90% (=

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×100%).

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. .

Dispersion test of HIPEs. The dispersion behavior of the HIPEs or HIPE hydrogels stabilized by

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OVA, at c values of 0.2 (0.4) wt% and 2.0 wt%, was evaluated by mixing them (0.2-0.25 g) with

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water or 6.0 M urea at a weight ratio of 1:9. The mixtures were quiescently incubated at room

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temperature for 12 h, followed by a vortexing for 10 s. The appearance and microstructure of the

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resultant dispersions were evaluated using visual observation and optical microscopy.

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Stability evaluation. The heat stability of the HIPEs was evaluated by treating the sealed samples

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in boiling water for 15 min, immediately followed by cooling in an ice bath to room temperature. For

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the freeze-thawing stabiity, the HIPEs were frozen at -20 °C for 24 h and thawed at 25 °C for 4 h. If

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necessary, the heating-cooling or freeze-thawing treatment was repeated 3 times or more. The

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appearance, microstructure and rheological behavior of the heated or freeze-thawed HIPEs were

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characterized by visual observation, optical microscopy and dynamic oscillatory measurements, as

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descibed above.

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Structural integrity evaluation of adsorbed proteins. The adsorbed proteins of the unheated or

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heated (100 °C, 15 min) HIPEs formed at the cm value of OVA (0.2 w%) were obtained by a

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freeze-thawing treatment, followed by a centrifugation at 5000 g for 15 min and subsequently,

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resolubilization in 5 mM phosphate buffer (pH 7.0), or additionally containing 6 M urea. The

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structural characteristics (e.g., hydrodynamic size, tertinary conformation, and/or secondary structure)

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of these adsorbed globular proteins were evaluated using multi-spectroscopic techniques and

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compared with native globular proteins. The particle size distribution and hydrodyanmic size of these 8


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proteins were determined by dynamic light scattering (DLS) technique using a Zetasizer Nano-ZS

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instrument (Malvern Instruments Ltd., Malvern, Worcestershire, UK) equipped with a 4 mW He-Ne

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laser (633 nm wavelength) at 25 °C. The tertinary conformation of the proteins was evaluated by

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intrinsic fluorescence technique using a F-7000 fluorescence spectrophotometer (Hitachi Co. Ltd,

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Japan) at 25 °C. A quartz cuvette with an optical path of 1 cm was used. The excitation wavelength

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was set as 295 nm (Trp), 275 nm (Tyr) and 258 nm (Phe), respectively. And the emission spectra

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ranged from 300 to 450 nm. The excitation and emission slit widths were 5 nm, the scanning speed

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was 240 nm/min, and the voltage was fixed at 550 V. The secondary structure of native or adsorbed

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proteins was assessed by Far-UV circular dichroism (CD) spectrascopy, using a MOS-450

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spectrometer (BioLogic Science Instrument, France). The far-UV CD spectrum (190 ~ 250 nm) were

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obtained at fixed protein concentration of 0.02 wt%. And the secondary structures (α-helix, β-sheet,

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β-turn, and random coil) of proteins were calculated applying the CONTINLL program in CDPro

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software 48. All the above tested protein solutions were filtered through PVDF Millipore membrane

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filters (0.22 µm), prior to the determinations.

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Oxidation stability test. β-Carotene (0.003 wt%; relative to dodecane) was chosen as a

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representative lipid-soluble bioactive to be loaded in the HIPEs for oxidation stability test (under

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sealed and dark conditions). A heating at 95 °C for 5 h was applied to accelerate the oxidation of

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β-carotene, in free dodecane or HIPEs (φ = 0.8) stabilized by OVA at various c values of 0.2-3.0 wt%.

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The HIPEs stabilized by Tween 20 or 80 at c = 5.0 wt% were tested as the controls. After the heating,

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all samples were immediately cooled with ice bath, then frozen at -20 °C for 24 h and thawed at

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25 °C for 4 h. Thanks to the de-emulsification by the freeze-thawing, a certain volume of oil phase

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was readily taken and diluted using dodecane. The concentration of remaining β-carotene in the oil 9



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phase was determined spectroscopically at 450 nm using a UV-Visible spectrophotometer (Shanghai

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Precision & Scientific Instrument Co., China). The retention ratio (%) of β-carotene was obtained by

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dividing the concentration of remaining β-carotene by the corresponding initial concentration.

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Volatility test. Hexane was chosen as the representative volatile oil for the volatility test of free oil

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and the HIPEs (as the above). All the samples were placed in bottles with a sectional diameter of 2

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cm, and placed in a fume cupboard without lids at 25 ºC for 2 h. After volatilization, all the samples

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were sealed with plastic wraps and lids, and subjected to a freeze-thawing treatment (-20 °C, 24 h

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→25 °C, 4 h). The retention ratio (%) of hexane was estimated by calculating the percentage of the

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height difference between the catactrophic emulsion system and aqueous phase.

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Statistics. An analysis of variance (ANOVA) of the data was performed using Origin Pro 2017

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(OriginLab Corporation, Northampton, MA, USA), and a least significant difference (LSD) with a

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confidence interval of 95% was applied to compare the means of duplicate or triplicate

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measurements on separate samples (n = 2 or 3).

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RESULTS AND DISCUSSION

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Fabrication of o/w HIPEs stabilized by OVA at different c values: Importance of bridging

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monolayer to the hydrogel network formation. Using a one-pot homogenization process, we first

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evaluated the formation and microstructure of OVA-stabilized emulsions (with dodacane as the

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dispersed phase) at c = 1.0 wt% with ø increasing from 0.1 to 0.92. As expected, the emulsions at ø

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values less than 0.64 (a critical value of random close packing 8) underwent a creaming upon

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elongated storage over 30 min after preparation, with the theight of creamed layer progressively

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increasing with the ø (Fig. 1 a, top row). When the ø was in the range 0.7-0.91, homogenous and 10


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gel-like emulsions that could adhere to the glass vials were formed; the emulsions became very

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unstable at ø > 0.91 (Fig. 1 a, top row). All the creamed emulsions or HIPEs exhibited a similar

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translucent appearance and a gel-like behavior. The optical microscopic observations of creamed

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emulsions (Fig. 1 a, bottom row) indicated that the creamed layers of the emulsions at ø < 0.7 were a

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kind of highly concentrated emulsions with undeformed droplets more packed at higher ø values,

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while in the gel-like emulsions (ø > 0.7), all the droplets were interconneted with adhesive patches

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between neighoring droplets. Interestingly, the gel-like emulsions at ø values of 0.8-0.91 exhibited a

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good self-supporting network, and could be even well remoldable (Fig. 1 b, c). Despite of the

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applied ø, all the formed emulsions (or creamed layers of emulsions) did not suffer a noticeable

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change in appearance upon storage up to 3 months (data not shown), indicating an extraordinary

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stability against creaming and coalescence. If β-carotene (a lipid-soluble bioactive) was encapsulated

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in the dispersed phase of the emulsion at ø = 0.8, no penetrating of the color occurred when the

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colored HIPE loaded with β-carotene was contacted with another uncolored HIPE, even after storage

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up to 3 months (Fig. 1 c), reflecting that these gel-like HIPEs were a kind of hydrogels in essence.

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The gel network formation in these emulsions was verified by the dynamic oscillatory measurements.

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The emulsions at ø = 0.8-0.91 exhibited a viscoelastic property dominated by elasticity, with a weak

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dependence of the moduli over the test frequency range of 0.1-10 Hz (Fig. 1 d), indicating a good

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tolerence to strain (e.g., less than 1.0) under the investigated conditions. Chen et al. 32 successfully

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fabricated a kind of moldable HIPE hydrogels stabilized by polymer nanogel, by means of

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non-covalently crosslinking through hydrogen bond interactions. Zhang et al.4 reported a similar

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HIPE hydrogel stabilized by assembled block copolymer at a comparable solid concentration (1.0

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wt%), but the HIPE had to be obtained through a complex emulsification process. In contrast, the 11



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fabrication of the HIPE hydrogel in the current work is simple (with a one-pot homogenization) with

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OVA as the sole emulsifer and stabilizer, and biocompatible with food or cosmetic formulations. This

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is the first observation for the facile fabrication of a kind of o/w HIPE hydrogels stabilized solely by

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native globular proteins at low c values.

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The hydrogel network formation in these HIPEs stabilized by OVA is expected to depend on the

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applied c. To verify this, we characterized the formation, microstructure and rheology of the HIPEs

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or HIPE hydrogels at ø = 0.8 with c values changing in the range 0.1-2.0 wt%. Surprisingly,

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homogeneous and gel-like HIPEs could be formed at a c value as low as 0.2 wt% (Fig. 2 a, top row).

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This minimal c value (cm) for the formation of a gel-like HIPE is within the range 0.05-0.5 wt%, as

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previously reported for a number of Pickering HIPEs stabilized by microgel particles 13, cellulose

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nanocrystals 14, gelatin particles 2 or hydrophobically modified silica particles 9, suggesting that OVA

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was an effective globular protein stabilizer for HIPEs with a comparable stabilization to these

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Pickering particles. As expected, the droplet sizes (d3,2) of these HIPEs dramatically decreased with

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increasing the c in the range 0.2-1.0 wt%, and after that, the rate of decrease gradually slowed down

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[Fig. 2 a (bottom row), b (top)], which is basically characteristic of proteins 31, 33 or Pickering

240

particles 9, 34 stabilized emulsions.

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On the other hand, dynamic oscillatory measurements showed that the elastic modulus (Gʹ; at a

242

specific frequency of 1.0 Hz) of these gel-like HIPEs progressively increased to a maximal value

243

with the c increasing up to 1.6 wt%, and then dramatically decreased with the c further increasing to

244

1.9 wt%; as the c increased from 1.9 to 2.0 wt%, the Gʹ slightly increased (Fig. 2 b, bottom row).

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The progressive increase in elasticity of these HIPEs with the c increasing up to 1.6 wt% has been

246

similarly observed for Pickering HIPEs stabilized by gelatin particles (from 0.5 to 1.5 wt%) 2, and 12


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supramolecular cellulose nanocrystals 5. For Pickering particles, in some cases, the use of a high

248

concentration (e.g., > 2.0 wt%) of particles might impair the formation of stable HIPEs, due to

249

particle agglomeration 2, 10. In general, protein-stabilized emulsions at low electrolyte concentrations

250

are considered to be stabilized by means of repulsive (electrostatic and steric) stabilization

251

mechanism 8, 29. To reflect this, an effective volume fraction (øeff = ø (1 + 3h/(2R); where h is the

252

thickness of the films between droplets, and R the mean droplet radius), is often introduced 8, 29.

253

Considering the fact that 1) at low electrolyte concentrations globular proteins adsorb to the interface

254

in a monolayer 33; 2) their h in emulsions is of the order of several nanometers 8, 29 but the droplet

255

sizes of the HIPEs in the current work were of the order of 20-50 µm (Fig. 2 b, top row), the

256

enhanced jamming packing due to the increased øeff (as a result of reduced droplet sizes) would be

257

negligible. Instead, Horozov & Binks 35 interestingly proposed that besides the steric stabilization,

258

solid particles can also contribute to the emulsion stabilization by means of a bridging monolayer,

259

especially when the droplets are sparsely covered. Thus, all the evidences suggest that besides the

260

repulsive stabilization, OVA might play a Pickering particle-like role in the stabilization of its HIPEs,

261

despite the fact that the dimension of monomeric OVA (2.9 nm × 3.5 nm × 7.2 nm) 36 is considerably

262

smaller than that of reported solid or colloidal Pickering particles (usually of 200-1000 nm in size) 1,

263

2, 5

.

264

Suppose that all the droplets in the HIPEs are stabilized by OVA in a closely packed interfacial

265

monolayer, and OVA molecules do not suffer a deformation when adsorbed at the interface, we can

266

calculate their maximal percentage of surface coverage (SC%), as displayed in Fig. 2 b. The SC%

267

for the HIPE at c = 0.2 wt% was approximately 90%, which is well consistent with its CLSM

268

observation (Fig. 2 c), indicating that a bridging with two droplets sharing a same protein monolayer 13



269

occurred in this HIPE. We can interestingly see that the SC% underwent a progressive decrease,

270

followed by a gradual recover, and subsequently, a dramatic increase, as the c increased from 0.2 to

271

2.0 wt% (Fig. 2 b). Given this c dependent profile of SC%, we classified three types of HIPEs

272

formed in three c regimes: protein-poor regime (I; 0.2-1.2 wt%), protein-saturated regime (II; 1.2-1.6

273

wt%), and protein-rich regime (III; > 1.6 wt%). The progressive decrease in SC% in the regime I

274

reflected the enhanced formation of bridging emulsions, while the contrary increase in SC% in the

275

regime II was clearly associated with the lessening of this bridging network (in the presence of

276

saturated protein). In contrast, the remarkable increase in SC% in the regime III (143% vs 94 % for

277

full coverage of droplets) might be largely due to the fact that in the protein-rich regime, all the OVA

278

molecules would suffer a orintation and conformation arrangment at the interface to form a more

279

packed protein monolayer. The high extent of overlapping of the Debye layer, as well as strong

280

interdroplet electrostatic repulsion of adsorbed OVA molecules, led to droplet deformation for the

281

HIPE in the regime III, as confirmed by the CLSM observation (Fig. 2 c) that almost all the droplets

282

of the HIPE at c = 2.0 wt% (with much smaller droplet sizes than those at c = 0.2 wt%) were

283

deformed, and packed in a separate polyhedral array (surrounded by the continuous phase of OVA).

284

Furthermore, we observed that the HIPE hydrogel at c = 0.2 wt% was extremely resistant to the

285

dilution with distilled water, under vortexed conditions, and could not be completely disrupted by 6

286

M urea; in contrast, the HIPE at c = 2.0 wt% could be easily disrupted by dilution with distilled water,

287

and completely dispersed by 6 M urea (Fig. 2 d). The observations clearly confirmed that the HIPEs

288

formed in the protein-poor regime (I) were a kind of Pickering-type emulsion hydrogels with a

289

network consisting of bridging droplets, while in the protein-rich regime (III) , the HIPEs were

290

mainly a kind of concentrated Pickering-type emulsions, as illustrated in Fig. 2 e. The importance of 14


Page 14 of 34

Page 15 of 34


291

a percolating network consisting of bridged droplets to the gel-like behavior of emulsions has also

292

been confirmed for w/o Pickering emulsions stabilized by silica microspheres (with a diameter of

293

975 nm) 37.

294

Stability of the HIPEs stabilized by OVA. All the fabricated HIPEs or HIPE hydrogels

295

stabilized by OVA at c values of 0.2-2.0 wt% were extraordinarily stable against creaming and

296

coalescence upon prolonged quiescent storage at 25 ºC. For example, the visual appearance and

297

microstructure of the HIPE hydrogel at c = 0.2 wt% did not undergo noticable changes upon storage

298

up to 60 days (Fig. 3 a). However, the dynamic oscillatory data (Fig. 3 b) indicated that the storage

299

led to a distinct strengthening of the hydrogel network, suggesting that adsorbed OVA at the interface

300

would suffer a structural unfolding and arrangement to form a more viscoelastic interfacial film, after

301

a prolonged storage. All the HIPEs were extremely resistant to a heating (100 ºC, 15 min)-cooling

302

treatment, with no distinct droplet coalescence occurring after the heating (Fig. 3 c). The

303

heating-cooling treatment could be repeated up to 5 times, without affecting the appearance and

304

microstructure of these HIPEs (data not shown). However, it can be still observed that all the

305

droplets in the HIPEs at c values of 0.8 wt% or above seemed to be more interconneted (Fig. 3 c),

306

which is clearly as a result of enhanced protein-protein interactions between adsorbed proteins, or

307

between adsorbed and unadsorbed proteins, due to heating-induced denaturation of the protein.

308

Accordingly, the elasticity of these HIPEs was significantly enhanced by the heating, with greater

309

extent of improvement observed at higher c values (Fig. 3 d).

310

On the other hand, we can see that these HIPEs or HIPE hydrogels at c values of 0.2-2.0 wt%

311

were very prone to freeze (at -20 ºC)-thaw treatment; the heating could alleviate the susceptibility of

312

these HIPEs to the freeze-thawing, but to a limited extent (Fig. 3 c). The high susceptibility of these 15



313

HIPEs to freeze-thawing might be associated with the desorption of the OVA molecules from the

314

interface of droplets, as a result of solidication of aqueous and dispersed phases during freezing.

315

Structural integrity of OVA adsorbed at interface: Evidence for Pickering particle nature of

316

OVA. A strong structural integrity is one of the basic requirements for particles to perform as

317

effective Pickering stabilizers. This is consistent with the general consensus in the field that many

318

food proteins or proteinaceous colloidal particles (e.g., casein micelles) cannot be considered to be a

319

kind of effective Pickering stabilizers 38, since once the colloidal particles of weak integrity become

320

adsorbed, they start to break down into smaller fragments. It has also been well accepted that once

321

adsorbed at the interface, many globular proteins would partially unfold and interact to form a

322

cohesive viscoelastic interfacial protein layer 39, 40. Furthermore, globular proteins, unadsorbed or

323

adsorbed, would undergo a structural unfolding and denaturation, when subjected to a heat treatment

324

at temperatures far above their denaturation temperatures.

325

By means of freeze-thawing (Fig. 3 b), we obtained the adsorbed OVA of the unheated or heated

326

(at 100 ºC for 15 min) HIPE at c = 0.2 wt% (in the protein-poor regime) by centrifugation, and

327

resolubilized it in water. The structural characteristics (including protein-protein association, tertiary

328

conformation and secondary structure) of the adsorbed OVA (unheated or heated) were evaluated and

329

compared with native OVA at a comparable c value. Dynamic laser scattering (DLS) results (Fig. 4 a)

330

showed that OVA did not suffer a noticeable change in particle size distribution profile, as well as

331

hydrodynamic diameter (Dh), when subjected to an emulsification and subsequent freeze-thawing.

332

Surprisingly, the hydrodynamic size of the adsorbed OVA was slightly but insignificantly increased

333

by an additional heating at 100 ºC (much higher than the thermal denaturation temperatures of 77.7

334

ºC for native OVA 41) (Fig. 4 a). Intrinsic fluorescence (excited at 258, 275 and 295 nm 16


Page 16 of 34

Page 17 of 34


335

corresponding to the Phe, Tyr and Trp fluophores, respectively) and far-UV CD spectra (Fig. 4 b, c)

336

further indicated that, there were no distinct variations in intrinsic fluorescence characteristics

337

(reflecting tertiary conformation) and secondary structure between the adsorbed OVA and its native

338

counterpart. By comparison, the additional heating led to a significant decrease in magnitude and a

339

blue-shift (about 3-6 nm) of the maximal fluorescence peak (Fig. 4 b), and accordingly, the relative

340

ratio of β-sheet slightly increased at the expense of α-helix (Fig.4 c, inset). The DLS and

341

spectroscopic observations indicated that the adsorbed OVA suffered a structural unfolding and

342

rearrangement to form a more compact conformation upon heating, but it still kept structural

343

integridity at the interface, which behaves like the thermalostable form of OVA (S-OVA) 41.

344

OVA is a phosphoglycoprotein with more than 50% of hydrophobic amino acids, exhibiting a

345

highly hydrophilic nature 42, 43. Taking into account the fact that in the protein-poor regime, an

346

interfacial protein monolayer that may be shared by two droplets is formed in OVA-stabilized

347

emulsions 43, it can be reasonably hypothesized that all the OVA molecules with a relatively thick

348

Debye layer (largely due to the presence of a carbohydrate chain 44, 45) would array in a close packing

349

way (with the Debye layer of two molecules overlapping with each other), as illustrated in Fig. 4 d.

350

Although the OVA molecules at the interface might undergo a structural unfolding upon heating, the

351

enhanced steric repulsion due to increased overlapping of the Debye layer seems to be still enough to

352

keep them in a separate state at the interface. Once cooled, the strong refolding ability of denatured

353

OVA 41 ensures it rapidly refold into a stable conformation (similar to that of native OVA). Thus, all

354

the observations confirmed that OVA is a highly efficient Pickering molecular stabilizer for HIPEs

355

with a strong structural integrity that could resist the disruption from both interfacial Laplace

356

pressure (tangential to the interface) and repulsive force between two droplets pushed against each 17



357

other (perpendicular to the interface) 30.

358

Enhanced heat stability of encapsulated lipid-soluble bioactives and vaporization inhibition

359

of volatile oils. If β-carotene (0.003 wt%) was loaded in the dispesed phase of these HIPEs stabilized

360

by OVA at c values of 0.2-2.0 wt%, we can see that, almost no degradation occurred for β-carotene in

361

these HIPEs, after an extensive heating at 95 °C for 5 h, while about 15% of β-carotene in free oil

362

degraded (Fig. 5 a). In the HIPEs stabilized by a concentration of 5 wt% of Tween 20 and Tween 80,

363

the loss of β-carotene reached about 80% and 25%, respectively (Fig. 5 a). The observations

364

indicated that these HIPEs imparted an extraordinary heat stability to the encapsulated bioactives.

365

The extraordinary chemical stability would be largely ascribed to the antioxdiative nature of applied

366

proteins. The high heat stability for the HIPEs by OVA might be due to the high free sulfhydryl (SH)

367

content per molecule of OVA. Even in the aqueous phase, OVA could produce an antioxidative

368

protection on lipid oxidation in the emulsions stabilized by Tween 20 4. Heat-induced exposure of

369

SH groups, as well as improvement of interfacial protein layer interconnectivity might also

370

contribute to the strong oxidative stability of encapsulated β-carotene. In contrast, the incorporation

371

of β-carotene in Tween 20-stabilized HIPEs remarkably accelerated its oxidation, which is related to

372

the increased interfacial area in the system.

373

The formation of o/w HIPE hydrogels has been suggested as one promising strategy for

374

enhancing safety of a number of liquid fuels and organic solvents 46. We evaluated the volatility of

375

the dispersed phase in these HIPEs stabilized by OVA with hexane as a representative volatile oil.

376

The results (Fig. 5 b) showed that all the HIPEs stabilized by OVA even at c = 0.2 wt% exhibited a

377

considerably higher retention ratio of hexane than unemulsified control (95-98% vs 56%), after

378

storage of 2 h at room temperature. The vaporization inhibition in the HIPEs stabilized by OVA was 18


Page 18 of 34

Page 19 of 34


379

also significantly greater than that by Tween 20 or 80 (at a much higher c of 5.0 wt%) (Fig. 5 b).

380

Since the HIPEs stabilized by OVA are very prone to freeze-thawing, the oils trapped in them can be

381

readily recovered. Thus, the fabricated HIPEs stabilized by such Pickering molecular stabilizer, OVA,

382

exhibits a great potential to be applied for preservation and transportation of a number of volatile

383

essential oils and even fuels and organic solvents. In conclusion, we, for the first time, demonstrate that native OVA exhibit an excellent potential to

384 385

perform as an outstanding Pickering molecular stabilizer for oil-in-water HIPEs. This Pickering

386

molecular stabilizer exhibits strong intermolecular structural integridity, high refolding ability of

387

unfolded molecules. The HIPEs or HIPE hydrogels can be obtained with a facile one-pot

388

homogenization, with the rheological behavior and microstructure delicately modulated by changing

389

the c. A kind of novel HIPE hydrogels showing a good gel self-supporting and remoldable ability can

390

be easily produced at c values as low as 0.2 wt%. All the fabricated HIPEs exhibit an extraordinary

391

stability upon storage, or against heating. The HIPE hydrogels formed in the protein-poor regime are

392

temperature-reponsive, with the gel network easily broken down when subjected to a freeze-thawing

393

treatment. This unique behavior imparts these HIPE systems a great potential to be applied in the

394

fields of essential oils and even fuels and highly volatile organic solvents for storage, delivery and

395

safety considerations. The OVA-stabilized HIPEs also exhibit a great potential to act as

396

encapsulation systems for improved oxidation stability of lipids or oil-soluble bioactives.

397 398

399

Supporting Information.

400

The Supporting Information is available free of charge on the ACS Publications website at DOI:

ASSOCIATED CONTENT

19



401

XX.XXXX/JAFC.XXXXXXX.

402

Figures S1 and Table S1.

403 404

405

Corresponding author

406

* E-mail: [email protected] (C.H.T.). Tel: +86 20 87111707.

AUTHOR INFORMATION

407 408

Funding

409

This work is supported by The National Key Research and Development Program of China

410

(2017YFD0400200), the NNSF of China (serial number: 31471695), GDHVPS (2017), and the 111

411

Project (B17018). We are very thankful for the critical review about our manuscript by Professor

412

Qixin Zhong, University of Tennessee Knoxville, TN 37996-4539, USA.

413 414

Notes

415

The authors declare no competing financial interest.

416 417

418

(1) Kim, K.; Kim, S.; Ryu, J.; Jeon, J.; Jang, S. G.; Kim, H.; Gweon, D.-G.; Im, W. B.; Han, Y.; Kim H.;

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Choi, S. Q. Processable High Internal Phase Pickering Emulsions Using Depletion Attraction. Nature

420

Comm. 2016, 8, 14305.

421 422

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Stabilized Solely by Whey Protein Isolate-Low Methoxyl Pectin Complexes: Effect of pH and

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Polymer Concentration. Food Funct. 2017, 8, 584-594.

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Whey Protein Microgel Particles: towards Food-Grade Pickering Emulsions. Soft Matter 2014, 22


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Characteristics, Emulsifying Properties, and Interfacial Adsorption/Arrangement at Interface.

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Food Hydrocolloids 2016, 60, 606-619.

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Emulsification and Interfacial Adsorption by Electrostatic Screening. Food Hydrocolloids 2016,

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Nano-Particles under in vitro Digestion Conditions. Food Hydrocolloids 2013, 33, 264-272.

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Food Emulsions. Trends Food Sci. Technol. 2012, 24, 4-12. (29) Dimitrova, T. D.; Leal-Calderon, F. Rheological Properties of Highly Concentrated

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from Non-Covalently Crosslinked Poly(N-Isopropylacrylamide) Nanogel Dispersions. Chem.

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Containing Globular Milk Proteins. Adv. Colloid Interface Sci. 2006, 123-126, 259-293. (34) Chevalier, Y.; Bolzinger, M. A. Emulsions Stabilized with Solid Nanoparticles: Pickering

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Ovalbumin at 1.95 Å Resolution. J. Mol. Biol. 1991, 221, 941-959. (37) Lee, M. N.; Chan, H. K.; Mohraz, A. Characteristics of Pickering Emulsion Gels Formed by

Droplet Bridging. Langumir 2012, 28, 3085-3091. (38) Dickinson, E. Use of Nanoparticles and Microparticles in the Formation and Stabilization of

Food Emulsions. Trends Food Sci. Technol. 2012, 24, 4-12. (39) Dickinson, E. Exploring the Frontiers of Colloidal Behavior Where Polymers and Particles Meet.

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(42) Mine, Y. Recent Advances in the Understanding of Egg White Protein Functionality. Trends

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(43) Delahaije, R. J. B. M.; Wierenga, P. A.; van Nieuwenhuijzen, N. H.;Giuseppin, M. L. F.;

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Gruppen, H. Protein Concentration and Potein-Exposed Hydrophobicity as Dominent

515

Parameters Determining the Flocculation of Protein-Stabilized Oil-in-Water Emulsions.

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Langmuir 2013, 29, 11567-11574.

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(44) Enomoto, H.; Ishimaru, T.; Li, C. P.; Hayashi, Y.; Matsudomi, N.; Aoki, T. Phosphorylation of

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Ovalbumin by Dry-Heating in the Presence of Pyrophosphate: Effect of Carbohydrate Chain on

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the Phosphorylation Level and Heat Stability. Food Chem. 2010, 122, 526-532.

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(45) Huntington, J. A.; Stein, P. E. Structure and Properties of Ovalbumin. J. Chromatog. B 2001, 756,

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526

(48) Sreerama, N.; Woody, R. W. Estimation of Protein Secondary Structure from Circular Dichroism

527

Spectra: Comparison of CONTIN, SELCON and CDSSTR Methods with an Expanded

528

Reference Set. Anal. Biochem. 2000, 287, 252-260.

529 530

25



531 532

Figure captions. Figure 1. . Formation and remoldablity of HIPE hydrogels. (a) Visual appearance (top row) and optical

533

microscopy images (bottom row) of OVA-stabilized emulsions with a ø increasing from 0.1 to 0.92. Scale bars, 100

534

µm. (b) Visual observation for gel-like self-supporting emulsions stabilized by OVA at ø values in the range

535

0.8-0.91. (c) Moldable HIPE hydrogels stabilized by OVA at ø = 0.8, with the dispersed oil phase uncolored or

536

colored with 1.0 wt% β-carotene. (d) Representative profiles of elastic (G′, filled) or loss (G″, hollow) moduli

537

against frequency (in the range 0.1-10 Hz) for the HIPE hydrogels at ø values of 0.8, 0.9 and 0.91. The inset figure

538

represents a typical strain sweep of the HIPE hydrogel at ø = 0.8. All the emulsions were obtained at a constant

539

protein concentration in the aqueous phase (c) of 1.0 wt%.

540

Figure 2. . Microstructure and properties of HIPEs/HIPE hydrogels. (a) Visual observations and optical

541

micrographs of the OVA-stabilized HIPEs (or HIPE hydrogels; oil fraction, ø = 0.8) at selected protein

542

concentrations (c) of 0.2, 0.5, 1.6, 1.9 and 2.0 wt%, respectively. Scale bars, 100 µm. (b) Evolution of

543

surface-average droplet size (d3,2), percentage of surface coverage (SC%) and elastic modulus (G′) of the HIPEs (or

544

HIPE hydrogels), as a function of c in the range 0.1-2.0 wt%. The d3,2 data were obtained using microscopic image

545

analysis software (Nano Measurer 1.2, Fudan University, China) over 100 droplets per optical microscopic image.

546

Based on the c-dependent profile of SC%, we classified the HIPEs into three regimes: (I) protein-poor (0.2-1.2

547

wt%), (II) protein-saturated (1.2-1.6 wt%), and (III) protein-rich (> 1.6 wt%). The data for d3,2 and G′ are the means

548

and standard deviations of the size of at least 100 droplets, and of three measurements, respectively. (c)

549

Representative confocal laser scanning microscopy (CLSM) images of the HIPE hydrogels in the protein-poor (0.2

550

wt%) or protein-rich (2.0 wt%) regmines. The protein was stained with Nile Blue, as excited at 633 nm. The white

551

and blue bars represent 50 and 10 µm in scale, respectively. (d) Visual and optical microscopic observations of the

552

HIPEs formed at c values of 0.2 and 2.0 wt%, diluted with deionized water or 6.0 M urea, respectively. The 26


Page 26 of 34

Page 27 of 34


553

mixtures of the HIPEs and dispersing solvents at a weight ratio of 1:9 were incubated quiescently for 12 h, and then

554

vortexed for 10 s at room temperature. Scale bars, 100 µm. (e) Scheme illustration for the microstructure of the

555

HIPEs stabilized by OVA: bridging hydrogel network (regime I), closely packed (without particle deformation;

556

regime II) and closely packed (with particle deformation; regime III).

557

Figure 3. . Stability of HIPEs or HIPE hydrogels. (a, b) Visual appearance, optical micrographs (a) and

558

rheology (b) of the OVA-stabilized HIPE hydrogel at c = 0.2 wt%, freshly prepared or after storage of 60 days. The

559

bars, 100 µm. (c) Visual appearance and/or optical micrographs of unheated and heated (at 100 ºC for 15 min)

560

HIPEs stabilized by OVA at selected c values of 0.2, 0.8, 1.4 and 2.0 wt%, before or after a freeze-thaw treatment,

561

respectively. (d) Elastic modulus (at a frequency of 1.0 Hz) of unheated and heated HIPEs stabilized by OVA at

562

different c values of 0.2-2.0 wt%. All the emulsions were obtained at φ = 0.8. Data are reported as means ± S.D. (n

563

=3). The symbols * and ** represent significant difference at p < 0.05 and p < 0.01 level, respectively.

564

Figure 4. . Structural integridity of adsorbed OVA. (a-c) The hydrodynamic size distribution (a), typical

565

intrinsic emission fluorescence (excited at 258, 275 and 295 nm) (b) and far-UV CD (c) spectra of native and

566

adsorbed OVA (c = 0.1 wt%; at pH 7.0) at 25 °C. Solid lines: native OVA; dash lines: adsorbed OVA (unheated);

567

dot lines: adsorbed OVA (heated). The adsorbed OVA from unheated and heated HIPE, formed at c = 0.2 wt%, was

568

obtained via emulsion breaking by freeze-thawing (-20 °C, 24 h→25 °C, 4 h), and then resolubilized in water.

569

Figure 4 (a) Inset: hydrodynamic diameter data (means ± S. D; n = 3) of native OVA, and adsorbed OVA (unheated

570

or heated), are included. (d) Schemic illustration showing orientation, structural unfolding and arrangement of

571

adsorbed OVA at oil-water interface. When adsorbed at the oil-water interface in initial periods, OVA molecules

572

suffer a speading, orintation and even limited structural unfolding. Once the interface is completely covered by

573

OVA molecules, the strong repulsive force between unadsorbed and adsorbed OVA molecules leads to formation of

574

a close ordered packing of adsorbed OVA at the interface. The presence of the Debye layer makes adsorbed OVA 27



575

molecules array in a separate state in the monolayer. When an additional heating is applied, the adsorbed OVA

576

molecules would undergo a distict structural unfolding and even denaturation. As a consequence, the Debye layer

577

of different adsorbed OVA molecules overlaps, thus strenghening the elasticity of the interfacial film. The strong

578

steric repulsion and high refolding ability of denatured OVA molecules ensure the structural integridity, once

579

cooled.

580

Figure 5. . Novel applications of HIPEs or HIPE hydrogels. (a) Heat stability of β-carotene (0.003 wt%) in

581

free oil phase (control), or loaded in the dispersed phase of HIPEs (φ = 0.8) stabilized by OVA at different

582

concentrations (c) of 0.2-2.0 wt%, or by Tween 20 or 80 at a conconcentration of 5.0 wt%. All the samples in sealed

583

containers were heated at heated at 95 °C for 5 h at dark to initiate oxidation, followed by a freeze-thawing

584

treatment. (b) Loss of volatile for hexane (as the representative volatile dispersed phase), free or in the HIPEs

585

stabilized by OVA (at c = 0.2-2.0 wt%) or Tween 20 or 80 (at c = 5.0 wt%), after storage at room temperature for 2

586

h. Data reported are the means and standard deviations (n = 3). Different letters (a-d) on the top of columns mean

587

significant difference at p < 0.05 level between different samples.

588

28


Page 28 of 34

Page 29 of 34

589


Figure 1. (Xu et al.)

590

591

d

100 0.1

592

Moduli (Pa)

Moduli (Pa)

1000 φ = 0.8 φ = 0.9 φ = 0.91

1000

G' G" 100 0.1

1

Strain (%)

1

Frenquency (Hz)

593

29


10


594


595

b

Regime

II

I

III

50

d3,2 (µm)

40 30 20

Storage moduli (Pa)

Surface Coverage (%)

180 150 120 90 60 1400 1200 1000 800

0.0

596 597

0.5

1.0 c (wt%)

1.5

2.0

598 599

30


Page 30 of 34

Page 31 of 34


d 5000

Moduli (Pa)

b 1000 0d 60 d 100 0.1

601

1

10

Frenquency (Hz)

602 603

31


Elastic modulus (Pa)

600


Unheated Heated

4000

**

**

1.4

2.0

3000 **

2000 1000 0

*

0.2

0.8

OVA concentration (wt%)



a

b

Volume (%)

40 30

336

λex = 295 nm

20 10 Dh

0 1

10

100

Size (nm)

c 2 [θ] (degcm / dmol)

6000 4000

Fluorescence Intensity (A.U)

604

Page 32 of 34

0

339

6000

334

200

210

220

230

240

250

λex = 275 nm

4000 338

2000 4000 2000

-10000 -20000

2000

333

λex = 258 nm

339

300 320 340 360 380 400 420 440

Wavelength (nm)

Wavelength (nm)

605

606 607

32


Page 33 of 34


a a

c

80

d e

10

b

90

c

80 70

d

60

33


80 -5

.0

.0 TW

20 -5

-2 .0 A V O

TW

-1 .4 A V

V

A O

ol C

on tr

C on tr ol O V A -0 .2 O V A -0 .8 O V A -1 .4 O V A -2 .0 TW 20 -5 .0 TW 80 -5 .0

-0 .8

50

0

609 610

a

ab

ab

-0 .2

20

ab

100

O

a

A

Retention rate (%)

90

a

V

b

100

Retention rate (%)

b

O

608



TOC Graphic

611

34


Page 34 of 34

Ovalbumin as an Outstanding Pickering Nanostabilizer for High

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