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Feb 22, 2017 - Controlling Agglomeration of Protein Aggregates for Structure Formation in Liquid Oil: A Sticky Business. Auke de Vries†‡ , Yuly Lo...
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Controlling agglomeration of protein aggregates for structure formation in liquid oil: a sticky business Auke de Vries, Yuly Lopez Gomez, Bas Jansen, Erik van der Linden, and Elke Scholten ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00443 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on March 5, 2017

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Controlling Agglomeration of Protein Aggregates for Structure Formation in

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Liquid Oil: a Sticky Business

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Auke de Vries1,2, Yuly Lopez Gomez2, Bas Jansen2, Erik van der Linden2, Elke Scholten1,2*

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Top Institute Food and Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands.

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Laboratory of Physics and Physical Chemistry of Foods, Wageningen University, P.O. Box 17,

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6700 AA Wageningen, The Netherlands.

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*E-mail: [email protected]

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Abstract

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Proteins are known to be effective building blocks when it comes to structure formation in

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aqueous environments. Recently, we have shown that submicron colloidal protein particles can

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also be used to provide structure to liquid oil and form so-called oleogels.1 In order to prevent

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particle agglomeration, a solvent exchange procedure was used to transfer the aggregates from

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water to the oil phase. The aim of the current paper was to elucidate on the enhanced stability

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against agglomeration of heat-set whey protein isolate (WPI) aggregates to develop an alternative 1

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for the solvent exchange procedure. Protein aggregates were transferred from water to several

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solvents differing in polarity to investigate the effect on agglomeration and changes in protein

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composition. We show that after drying protein aggregates by evaporation from solvents with a

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low polarity (eg. hexane), the protein powder shows good dispersibility in liquid oil compared to

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powders dried from solvents with a high polarity. This difference in dispersibility could not be

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related to changes in protein composition or conformation but was instead related to the reduction

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of attractive capillary forces between the protein aggregates during drying. Following another

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route, agglomeration was also prevented by applying high freezing rates prior to freeze-drying.

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The rheological properties of the oleogels prepared with such freeze-dried protein aggregates

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were shown to be similar to that of oleogels prepared using a solvent exchange procedure. This

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paper provides valuable insights in how to tune the drying process to control protein

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agglomeration to allow for subsequent structure formation of proteins in liquid oil.

1. Introduction

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It has long been recognized that a diet rich in saturated and trans fats is associated with an

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increase in the amount of LDL cholesterol (Low Density Lipoprotein) at an expense of HDL

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cholesterol (High Density Lipoprotein),2 which is related to a higher risk of developing coronary

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artery disease. On the other hand, a diet rich in cis-unsaturated fatty acids decreases these risks. 3-

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technological benefits such as providing texture and oxidative stability to food products. One

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interesting alternative, which has gained much attention over the recent years, is the use of so-

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called ‘oleogels’.5-8 The purpose of designing edible oleogels is to be able to provide a solid-like

However, food reformulation is not straightforward as the use of saturated and trans fats has

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structure to liquid oil at room temperature other than by the conventional use of saturated and

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trans fatty acids.

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The most common gelling agents of organic liquids, called in a general term ‘organogelators’, are

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found within the class of low molecular weight organogelators (LMOG). Their network

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formation relies on the complex self-assembly of the components by noncovalent bonds such as

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dipolar interactions, π-stacking, and intermolecular hydrogen bonds into crystals, tubules or

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fibrillar structures responsible for the solid-like behaviour.9-11 These organogels have been

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studied for different applications, such as oil spills,12-13 electronics,14 and drug delivery.15-17 For

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food purposes, many different edible oil soluble LMOG’s have been studied for their gelling

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properties, such as lecithins,18-19 monoacylglycerides,20-21 fatty acids or alcohols,22 sterols,23-24 or

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waxes.25-27

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In contrast to the large diversity of low molecular weight compounds, only a limited amount of

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biopolymers have been studied to structure oil. Due to their predominant hydrophilic nature,

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these compounds usually do not dissolve in oil. A well-studied exception is the cellulose

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derivative ethyl cellulose (EC).28 EC dissolves in liquid oil at high temperature and upon cooling,

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the polymer chains interact to form solid structures. The resulting mechanical properties of the

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gels, depending on polymer-polymer interactions, can be tailored by changing the molecular

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weight of the polymer chain or by adding surfactants.28-29 Another example of a biopolymer

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studied for its oil structuring ability is the polysaccharide chitin. Modifying crude chitin into

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“nanocrystals” 30 or hydrophobic “whiskers” 31 was necessary to efficiently provide a structure to

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liquid oil. To overcome the problem of low dispersibility of biopolymers, other researchers have

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adopted a foam- or emulsion-template approach. In a first step, hydrophilic polymers like

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hydroxyl propyl methylcellulose, gelatin and xanthan are used to stabilize an oil-water or air-

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water interface. Subsequently, the water is removed and by shearing the dried product into the

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liquid oil, gelled structures are obtained.32-33 However, there is limited control over the network

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formation as the particle size of the building blocks might be difficult to tune, and shear forces

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may disrupt the network. A better control over the network formation of biopolymers is desirable

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to tune specific rheological characteristics of such gelled oils.

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Recently, we reported a new method to create oleogels using whey proteins.1, 34 Whey proteins

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are extensively used as ingredients in foods due to their high nutritional value and functional

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properties, and are therefore an ideal candidate as a structuring agent for liquid oil. First, we

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developed a method in which protein oleogels were prepared in a more direct way.34 To create

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oleogels, we prepared heat-set whey protein hydrogels, and a solvent exchange procedure was

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applied to include the oil in the interstitial areas of the protein network. In this process, the water

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is first replaced by an oil-miscible solvent, acetone, which is then replaced by liquid oil. To

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enhance the control over the network formation, we then prepared whey protein aggregates of

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colloidal size (~ 150 nm) as initial building blocks.1 In this paper, we showed that the solvent

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exchange procedure was able to prevent agglomeration of pre-formed heat-set aggregates and

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therefore enabled gel formation due to sufficient protein-protein interactions. Alternatively, when

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the protein aggregates were freeze-dried to remove the water before they were dispersed in oil,

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irreversible agglomeration of the initial particles was obtained. This agglomeration led to

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increased particle size in oil, which was related to an observed poor stability against

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sedimentation and poor gel formation. However, it is currently not precisely known how the

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solvent exchange procedure prevents such agglomeration.

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The aim of the current paper is to elucidate on the enhanced stability against agglomeration of

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whey protein aggregates present in oil after applying a solvent exchange procedure. To this end,

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we formulate two possible mechanisms:

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1) Prevention of stresses such as capillary pressure resulting from drying processes. By

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keeping the particles ‘solvated’ by the different solvents, starting with water, followed by

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acetone and finally liquid oil, the wet conditions may avoid strong forces arising from the

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formation of a solvent-air interface.

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2) Conformational changes of the proteins as a function of the properties of the surrounding

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solvent. Solvents with electronegative atoms, such as oxygen, are able to interfere with

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intramolecular hydrogen bonding,

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fraction of hydrophobic groups to the solvent. Potentially, this could result in more

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favourable protein-solvent interactions and enhanced stability against agglomeration.

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which could lead to the exposure of a larger

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To gain insight in the effect of the different solvents on possible agglomeration effects and

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conformational changes, we have used several solvents of different polarity in the solvent

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exchange procedure. In addition to acetone, we have included propanol as a solvent with the

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ability to form hydrogen bonds, and as an alternative for a hydrophobic liquid oil, we have used

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volatile apolar solvents such as hexane, decane and heptane. To analyse the protein composition

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and conformation, the protein material was dried from the solvent by evaporation, and

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agglomeration effects were tested by examining the dispersibility of the dried material in oil. The

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results were compared to aggregates freeze-dried from water. Understanding the mechanisms

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involved to prevent irreversible agglomeration could provide insights on how to design a route to

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obtain a dry protein material that is dispersible in liquid oil and allows for direct network

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formation without the need for a solvent exchange. This could be beneficial from a product

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development point of view, since the large amount of solvent needed for a solvent exchange

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procedure limits practical applicability.

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Materials and Methods

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2.1 Materials

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Whey protein isolate (WPI, BiPro) was obtained from Davisco Foods International (Le Sueur,

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MN, USA). The protein concentration was 93.2% (N x 6.38) and was used as received. Acetone

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and n-hexane were supplied by Actu-All Chemicals (Oss, the Netherlands). Hydrogen chloride,

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sodium hydroxide, sodium dodecyl sulfate (SDS), and n-decane were purchased from Sigma-

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Aldrich (Steinheim, Germany). 1-propanol and n-heptane were purchased form Merck

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(Damstadt, Germany). N,N-Dimethyl-6-propionyl-2-naphthylamine (PRODAN) was obtained

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from Sigma Aldrich. Refined sunflower oil (Vandermoortele NV, Breda, the Netherlands) was

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bought at a local supermarket and was used without further purification. All chemicals used were

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of analytical grade. Demineralized water was used throughout the experiments.

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2.2 Methods

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2.2.1

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To prepare a protein stock solution, WPI powder (4% w/w) was dissolved in demineralized water

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under continuous stirring at room temperature for 2 h. Afterwards, the stock solution was stored

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overnight at 4 °C to assure complete protein hydration. The next day, the pH of the stock solution

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was adjusted to 5.7 using a 1 M HCl solution. The resulting solution was heated in 50 mL plastic

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tubes with screwcaps at 85 °C for 15 minutes using a temperature controlled water bath to induce

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protein denaturation. After cooling in ice water, a weak protein gel was obtained. This weak gel

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was easily broken into the smaller aggregates by hand shaking and vortexing. The resulting WPI

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aggregate dispersion was homogenized using a rotor stator homogenizer (Ultra Turrax, T25, IKA

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Werke, Germany) at 13.000 rpm for 3 min. The protein aggregates were then collected as a pellet

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by centrifugation at 3904 g (Hermle Z383K, Hermle Labortechnik GmbH, Wehingen, Germany)

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for 20 minutes at 20 °C. After collection, the pellet was re-dispersed and centrifuged twice with

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demineralized water to remove remaining soluble protein material. Afterwards, the sample was

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homogenized using a laboratory scale homogenizer (Labhoscope, Delta Instruments, Drachten,

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the Netherlands) at 200 bar (3 passes). The final pH of the WPI aggregate suspension was 8.0.

Preparation of WPI aggregates

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2.2.2

Preparation of the protein oleogels using a solvent exchange

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To prepare the protein oleogels, the WPI aggregates were transferred to the oil phase using a

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solvent exchange procedure, which was based on a method described in detail previously.1 In this

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procedure, the polarity of the solvent was changed gradually to remove the surrounding water

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from the WPI aggregates and replace the continuous phase for oil. In short, 15 g of aqueous

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pellet, containing the WPI aggregates, was re-dispersed in 150 mL acetone, and mixed 7

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thoroughly using rotor stator homogenization. Afterwards, the sample was centrifuged at 3904 g

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for 20 min at 20 °C. Excess acetone was removed by decanting and the pellet, containing the

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protein aggregates, was collected. The procedure of re-dispersing and centrifugation was repeated

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once more using acetone to assure water removal. The pellet was then re-dispersed twice in liquid

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oil. The obtained pellet of WPI aggregates in oil was diluted in a ratio of 1:10 with sunflower oil

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and left overnight under continuous stirring to allow for evaporation of the remaining acetone.

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The next day, the suspension was centrifuged at 4000 g for 20 min at 20 °C to increase the

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concentration of the protein aggregates and induce gel formation.

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2.2.3

Preparation of dried WPI aggregates

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Evaporation: To determine various properties of the WPI aggregates during the solvent exchange

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procedure, we dry the aggregates by evaporation from acetone, 1-propanol, hexane, heptane and

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decane. To produce protein aggregate suspensions in the different solvents, the water was

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exchanged for acetone or 1-propanol, as described above. The suspensions in hexane, heptane

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and decane were prepared using acetone as an intermediate solvent during the solvent exchange.

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In order to easily collect the protein material from its solvent, the protein suspensions were

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centrifuged at 3900 g for 20 min and the resulting pellet was placed in an aluminium tray (ø = 5

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cm) and dried in a fume hood for 16 h at room temperature. After drying, the powder was

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collected and grinded using a pestle and mortar until no further reduction in particle size was

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observed. To investigate the effect of the drying conditions, instead of drying from a concentrated

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pellet, a 1 wt% protein aggregate suspension was dried from water, acetone or hexane by

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evaporation. The solvent was evaporated in the same way as described above for the drying

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method using the concentrated pellets. 8

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Freeze drying: After the aqueous suspension was washed twice with demineralized water, the

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resulting suspension was homogenized using a lab scale homogenizer (Labhoscope) at 200 bar (3

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passes) followed by centrifugation. The pellet was frozen at -20 °C in a freezer for 16 h.

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Thereafter, the sample was freeze dried (Christ alpha 2-4 LD plus, Martin Christ

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Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) for 48 h to remove all water. In

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another experiment, the liquid suspension of WPI aggregates (1 wt%) was added dropwise into

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liquid nitrogen (-195 °C) to rapidly freeze the material. Thereafter, the frozen sample was freeze-

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dried as described above.

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2.2.4

Composition

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Protein content: The nitrogen content was determined using Dumas (Dumas Flash EA 1112

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Series, N Analyser, Thermo Scientific). After weighing, the samples were dried overnight in an

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oven at 60 ˚C before analysis. To calculate the protein content, a nitrogen conversion factor of

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6.38 was used.

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Water content: The water content in the oleogel was determined by dry matter determination.

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Aluminium cups (ø = 5 cm) were first heated to 105 °C in an oven (Venticell, BMT Medical

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Technology, Brno, Czech Republic) to remove any water contamination. Afterwards,

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approximately 1 g of oleogel sample was added to the cup, and its weight was recorded before

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and after drying for 4 h at 105 °C. Water content in the dried powders was determined by Karl

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Fisher titration. Measurements were performed in duplicate.

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2.2.5

Chemical stability

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In order to assess the internal bonds involved in the stabilizing mechanism of the WPI

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aggregates, several denaturants were added to a 1 wt% of WPI aggregate dispersion. The

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denaturants used were 10M Urea to examine disruption of hydrogen bonds, 140 mM Sodium

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Dodecyl Sulfate (SDS) for hydrophobic interactions and 50 mM dithiothreitol (DTT) for

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disulphide interactions.37 Several combinations of these denaturing agents were tested and

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whenever DTT was used, heat treatment was applied at 70 ˚C for 15 min.

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2.2.6

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)

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The protein composition of the freeze-dried aggregates, the acetone-dried aggregates, the hexane-

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dried aggregates, as well as the protein composition in the supernatant after centrifugation was

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analysed under reducing conditions by SDS-PAGE using the Novex NuPAGE gel system

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(Invitrogen, Thermo Fischer Scientific). Samples were prepared by the addition of NuPAGE LDS

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sample buffer (4x) and NuPAGE Reducing agent (10x) to a final protein concentration of 2

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mg/ml. Thereafter, samples were vortexed and heated at 75 ˚C for 10 min in a water bath. After

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cooling, samples were loaded into the wells of a NuPage 4-12% Bis-Tris gel. As a running buffer,

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NuPAGE MES SDS (20x) was used, with antioxidant in the cathode chamber. Electrophoresis

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was performed by applying a constant voltage of 200 V for 40 min. Afterwards, gels were stained

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using coomassie blue (SimplyBlue™). The apparent molecular weight of the proteins present in

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each sample was determined by comparing the position of the bands to a reference sample with

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proteins of various molecular weights (Mark12™ unstained standard, Invitrogen). The gels were

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scanned in a densitometer (Gelscanner GS-900, Bio-Rad, Hercules, CA, USA) with Image Lab

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software, which allows for the identification of the proteins present in each sample.

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2.2.7

Surface hydrophobicity

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The surface hydrophobicity of the aggregates obtained through the solvent exchange method and

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freeze-drying was measured against the native WPI by means of the fluorescent probe method,

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with N,N-Dimethyl-6-propionyl-2-naphthylamine (PRODAN) as the binding probe. The

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procedure to determine the surface hydrophobicity was similar as reported by Haskard et al..38

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Five different concentrations of protein were prepared ranging from 0.04 to 0.2 mg/ml and

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analysed in duplicate. PRODAN was dissolved in acetone at a concentration of 0.0041 M and

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stored in a freezer (-20 ˚C) protected from light and evaporation. Ten µL of PRODAN solution

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was added to each 4 mL sample and vortexed well. The relative fluorescence intensity (RFI) was

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measured using a fluorimeter (Perkin Elmer luminescence spectrometer LS50B) after 15 min of

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reaction of the PRODAN with the proteins in the dark at room temperature, using disposable

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acrylic cuvettes (Sarstedt, Nümbrecht-Rommelsdorf, Germany). The measurement settings were

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set to an excitation wavelength of 365 nm, emission and excitation slit widths of 5 nm, emission

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scan from 300 to 600 nm, and a scan speed of 200 nm/min. The net RFI values of each sample

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(protein with PRODAN) was obtained by subtraction of the protein blank sample from the

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measured RFI value. The slope of the net RFI values taken from the maximum emission spectra

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of the bounded protein-PRODAN were plotted versus the protein concentration. The resulting

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slope of this linearization is used as a measure for the protein surface hydrophobicity.

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2.2.8

Attenuated Total Reflectance Fourier Transform Infrared Resonance (ATR-FTIR)

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To directly analyse protein conformation of the dried protein aggregates, samples were analysed

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using infrared spectroscopy. Dried protein material was placed directly on the crystal using an

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ATR-FTIR spectrometer (Platinum Tensor, Bruker Optics, Coventry, UK). The IR spectrum was

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recorded from 4000-600 cm-1 and for each sample, 64 scans with a resolution of 4 cm-1 were

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averaged. After averaging the scans, the spectrum was cut to obtain the amide I and II region at

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1400 – 1750 cm-1, baseline corrected, and vector normalized using the OPUS software to analyse

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the Amide I, II and III region. In order to assess the protein conformation, the second derivative

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of the amide I region (1600 – 1700 cm-1) was taken and smoothened using the OPUS software.

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All samples were measured in duplicate.

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2.2.9

Particle Size Analysis

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The particle size distribution of WPI aggregates was determined by static light scattering

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(Mastersizer 2000, Malvern Instruments, Worcestershire, UK) with either sunflower oil or

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demineralized water as the continuous phase. The refractive index of water was set to 1.33 and

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for sunflower oil to 1.469. The refractive index of the protein aggregates was set to 1.45 for

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aqueous protein samples and 1.54 for protein samples in sunflower oil to correct for the change in

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refractive index upon dehydration. The particle size distribution was determined as an average of

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three measurements.

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2.2.10 Scanning Electron Microscopy (SEM)

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A scanning electron microscope (Phenom G2 Pro, Phenom-World BV, Eindhoven, and The

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Netherlands) was used to visualize the structure of the different protein powders and used to

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analyse any structural differences between the different samples. To this end, a small sample was

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taken and fixated using carbon tabs on aluminium stubs (SPI Supplies/Structure Probe Inc., West

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Chester, USA). Conveniently, due to the low voltage used (5kV), sample pre-treatment was not

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necessary and the appearance of the powders could be visualized directly.

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2.2.11 Rheology

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Oscillatory rheology was performed on the oleogel made with WPA aggregates obtained via

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either the solvent exchange procedure or via freeze drying. Both samples were standardized to 10

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wt% protein and 1.2 wt% water to allow for comparison between the samples. Before the

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measurement, the samples were homogenized using rotor-stator homogenization (13.5000 x rpm)

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for 180 s. Afterwards, samples were degassed using a vacuum pump for 30 min and loaded into a

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stress-controlled rheometer (AP 502, Anton Paar GmbH, Graz, Austria) between sandblasted

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parallel plates (ø = 49.978 mm) to prevent slip phenomena. The temperature was controlled for

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all measurements at 20°C. Before any measurements were performed, the samples were allowed

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to equilibrate for 60 min at a frequency of 1 Hz and a strain (γ) of 0.01% (which was within the

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linear viscoelastic region). Frequency sweeps were performed by increasing the frequency

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logarithmically from 0.01 to 50 Hz at γ = 0.01%. Amplitude sweeps were performed by

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increasing the strain logarithmically from 0.001 to 100% at 1 Hz. All measurements were

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performed at in triplicate.

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3 Results and Discussion

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3.1 Characteristics of whey protein aggregates

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To understand possible changes in the protein aggregates during the solvent exchange procedure,

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we first examined the characteristics of the protein aggregates prepared in aqueous conditions.

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Figure 1A shows the particle size distribution of the whey protein isolate (WPI) aggregates after

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heat treatment. The major fraction of the WPI aggregates had a particle size around 150 nm,

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which is of comparable size to what has been reported in other studies.39 Upon heating a protein

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solution above the protein denaturation temperature, heat-set aggregates are formed that are

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stabilized through physical and chemical interactions. The strength of these interactions

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determine the stability of the protein aggregates against external forces such as those resulting

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from shear and drying processes. To assess which interactions are involved that lead to the

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stability of the protein aggregates, several denaturants were added to an aqueous 1% WPI

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aggregate suspension. After adding SDS, urea or a combination of both, we noticed that the

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suspensions remained turbid, suggesting that hydrogen bonds and hydrophobic interactions were

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not the only interactions responsible for the stabilization. However, when DTT was added, the

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suspension turned completely transparent as a result of disintegration of the aggregates. This

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suggests that the structure of the WPI aggregates is partially stabilized by internal covalent

300

disulphide bonds and is in agreement with other studies using heat-set WPI aggregates prepared

301

at similar conditions.40

302 303

After freeze-drying the WPI aggregates, the resulting powder was re-dispersed in demineralized

304

water. The resulting particle size distribution was comparable before freeze drying, as is

305

presented in Figure 1A. As already discussed in our previous research,1 the freeze-dried

306

aggregates do not readily disperse in oil and particles of more than 100 microns were obtained, as

307

is depicted in Figure 1B. Alternatively, when a solvent exchange procedure was used to disperse

308

the aggregates in the oil, a particle size of 150 nm was found (open circles in Figure 1B).

309

Although these results show that the solvent exchange procedure prevents agglomeration of the

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protein aggregates, the underlying mechanism is not yet understood. To gain more insight in the

311

effect of the solvents during the solvent exchange process, hexane was used as an alternative

312

nonpolar solvent in the following sections to easily isolate the aggregates during the solvent

313

exchange process by evaporation. Hereafter, we studied the properties of the dried aggregates in

314

terms of composition, conformation, and dispersibility in liquid oil.

315 316

3.2 Protein composition

317

Since WPI is a mixture of several proteins, the composition of the proteins present in the

318

aggregates might be different as a result of exposure to different solvents. To exclude these

319

differences, the freeze-dried, acetone-dried and hexane-dried WPA samples were analysed using

320

SDS-PAGE. After the heating step in aqueous conditions, only 80% of the protein material was

321

found to be included in the WPI aggregates (i.e. in the pellet), and 20% was still present in the

322

supernatant. For this reason, also the supernatant was analysed for its composition. Native WPI

323

was analysed for comparison. The SDS-PAGE electrophoretograms are shown in Figure 2. Lane

324

1 shows the electrophoretogram of native WPI and the major whey protein fractions can easily be

325

recognized. Major bands found around 66, 18 and 14 kDa correspond to Bovine Serum Albumin

326

(BSA), β-lactoglobulin (β-lac), and α-lactalbumin (α-lac), respectively. The major protein

327

fraction, as indicated by the higher band intensity, is β-lac. Comparable to the native sample, the

328

freeze-dried WPI aggregates (lane 2) showed bands at all major protein fractions. However, the

329

intensity of the band corresponding to α-lac (14 kDa) seems to be lower, as will be discussed in

330

more detail below. The acetone- and hexane-dried WPI aggregates (lane 3 and 4) showed a high

331

similarity with the freeze-dried sample. The protein composition of the supernatant, i.e. soluble

332

protein material after heat treatment and centrifugation, showed a distinctly different

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333

electrophoretogram (lane 5). The band corresponding to BSA is not detected, the band intensity

334

of β-lac is less intensive and the band intensity of α-lac has increased compared to the samples

335

containing the aggregates.

336 337

To estimate the relative content of α-lac and β-lac in each sample, we have taken the band

338

intensity of β-lac as an internal standard per electrophoretogram, and determined the ratio α-

339

lac/β-lac for the different samples (Figure 3). The α-lac/β-lac ratio of the aggregates was found to

340

be lower compared to the native WPI, whereas for the supernatant, this ratio was much higher.

341

Our result suggest that the disulphide crosslinked aggregates contain a higher amount of BSA and

342

β-lac compared to its native protein composition, and that α-lac is to a lesser extent incorporated

343

into the aggregates. Taking the gelation mechanism of the different proteins into account, the

344

results can be explained since both BSA and β-lac have a higher gelation rate and have a free

345

thiol group available to from disulphide bonds.41-42

346 347

From these results, we can conclude that changing the aqueous solvent to acetone or hexane had

348

no apparent influence on the internal protein composition of the aggregates. This indicates that

349

the protein aggregates were stable during the solvent exchange procedure, regardless of the

350

drying method or solvents used. Therefore, changes in oil dipsersibility as a result of the solvent

351

exchange, is not caused by a change in protein composition of the aggregates.

352

353

3.3 Particle Morphology

354

The obtained powders, dried by either freeze drying from water or evaporation from organic

355

solvents, were analysed by scanning electron microscopy (SEM) to determine the particle 16

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morphology. As can be seen in Figure 4, the freeze-dried powder (Figure 4A1 and A2) contained

357

large particle agglomerates (~ 50 – 100 µm), but appear to have an open, porous structure. The

358

morphology of the powder when evaporated from acetone (Figure 4B1 and B2) had a similar

359

appearance as the freeze-dried sample, i.e. large particle agglomerates with similar porosity. In

360

contrast, the hexane-dried protein powder is absent of any large agglomerates (Figure 4C1 and

361

C2), and the powder consists of smaller, porous agglomerates (10 – 20 µm). It shows that the

362

nature of the solvent from which the aggregates are dried had a large influence on the

363

agglomeration and powder morphology. The packing density of the powders was estimated by

364

weighing 1 mL of dried material in a graded cylinder. The packing densities were found to be

365

approximately 0.26 g/cm³ for the freeze-dried powder, 0.14 g/cm³ for the acetone-evaporated

366

powder, and 0.07 g/cm³ for the hexane-evaporated powder. Clearly, when WPI aggregates are

367

dried from solvents with low polarity, the powder morphology changed from large agglomerates

368

to agglomerates of smaller size consisting of more loosely packed particles.

369 370

The effects of different drying conditions are dependent on the hydrophobicity of the solvent,

371

which seem to change the interactions between the protein aggregates. By removal of the solvent

372

by evaporation, initially the solvent evaporates from the bulk, but will eventually form a liquid

373

bridge between two particles, as schematically depicted in Figure 5. This causes a capillary force

374

(Fc) across two particles as a result of a curved interface governed by the interfacial tension, γ.

375

When the distance between two particles approaches zero, Fc becomes:43

376 377

378

(1)

‫ܨ‬௖ = 2ߨܴߛ cos ߠ

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379 380

where R is the particle radius and θ is the contact angle between the liquid and the solid, which in

381

our case is the protein aggregate (Figure 5). When the capillary force is sufficiently high, this

382

may result in large attractive forces between the particles. Therefore, in the production of

383

nanometre sized ZrO2, Al2O3, or TiO2 particles, solvents with a low surface tension were used

384

during drying to reduce their agglomeration and increase their specific surface area.44-45 Similarly,

385

in the case of aerogels, it has been shown that large capillary forces leads to a collapse of the

386

initial microstructure. In order to prevent this undesired effect, often alternative solvents and/or

387

supercritical drying methods are used to change the wetting angle and interfacial tension.46-47 In

388

our case, the solvent evaporates from the interstitial spaces between and from the surface of the

389

aggregates, which may lead to increased capillary forces between the aggregates. When the

390

solvent has a high surface tension and a small contact angle, capillary pressure facilitates particle

391

agglomeration. The contact angle is related to the difference in polarity between the particle and

392

the solvent.48 In the case of water, a high surface tension of 73 mN/m and an estimated low

393

contact angle (θ < 90°), the resulting high capillary force would lead to a large degree of

394

agglomeration. However, in the case of hexane, the lower interfacial tension (19 mN/m) and a

395

higher contact angle, given the low polarity of hexane, leads to much lower values of the

396

capillary force and therefore a low degree of agglomeration, in accordance with our experiments.

397

In the case when θ > 90°, the resulting force could even lead to and effective repulsion between

398

two protein aggregates.

399 400

To visualize the effect of the drying conditions more clearly, we dried a liquid suspension of WPI

401

aggregates from the solvents water, acetone, or hexane and observed the appearance and

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microstructure of the film formed. Figure 6 shows the difference in the appearance of the dried

403

protein material as well as the microstructure of the films at a smaller length scale. When the

404

drying medium was water, the resulting film was hard and almost translucent. The SEM

405

micrograph shows a tight packing of the protein aggregates. Drying from acetone resulted in a

406

more brittle film, which was more difficult to handle than the water-dried protein film. The

407

microstructure shows more cracks or gaps between the protein aggregates. When the protein

408

aggregate suspension was dried from hexane, film formation was inhibited and the sample was

409

very brittle, resulting from limited interactions between the protein aggregates. In addition, the

410

resulting material was opaque (Figure 6C). The limited interactions can be seen more clearly in

411

Figure 6D, where a very porous structure can be observed with larger distances between the

412

individual aggregates. This is consistent with the observed powder morphology in Figure 4,

413

where drying from hexane prevents agglomeration. Prevention of strong capillary forces during

414

drying thus seems to reduce agglomeration between the particles.

415 416

3.4 Dispersibility of dried whey protein aggregates in liquid oil

417

The dried protein aggregates obtained from different drying methods were tested for their

418

dispersibility in liquid oil. To this end, a 1% w/w dispersion was prepared by adding the dried

419

powders to sunflower oil, and the resulting particle size was measured after homogenization. As

420

can be seen in Figure 7A, the acetone-dried sample showed poor dispersibility. A large increase

421

in particle sizes (10 – 300 µm) compared to the sizes observed in the original aqueous dispersion

422

is indicative of irreversible particle agglomeration during drying. Even though during drying the

423

surface tension and wettability of acetone is lower than that of water, apparently the capillary

424

forces are not yet decreased to such extent that agglomeration is prevented. In contrast, good

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425

dispersibility was obtained when the drying medium was hexane (Figure 7B), since a large

426

amount of small (< 1 µm) particles was obtained. Although agglomeration was not fully

427

prevented as shown by the presence of a smaller peak at 10 – 100 µm, the major fraction of the

428

protein aggregates retained their small initial size, shown by the large peak at ~150 nm. Since the

429

difference in particle agglomeration was observed by SEM, it seems that drying-induced

430

agglomeration was largely irreversible. Mixing the agglomerated powders into the oil by shear

431

hardly seems to break any formed agglomerates and leads to poor dispersibility in oil.

432 433

To test which physical properties of the solvent have an effect on particle agglomeration and

434

resulting dispersibility in oil, 1-propanol, heptane and decane were also used as the suspending

435

solvents during drying. These solvents differ in surface tension and dielectric constant as can be

436

seen in Table 1. Here, we use the dielectric permittivity as a measure for the polarity. As can be

437

seen in Figure 7, drying from 1-propanol led to a similar size distribution as acetone, whereas

438

decane and heptane led to a similar particle size distribution as hexane. Interestingly, although the

439

surface tension of decane and 1-propanol is similar, the resulting dried aggregates showed a very

440

different size distribution when dispersed in oil. A low surface tension does not seem to be the

441

most important prerequisite to prevent irreversible agglomeration. Particle agglomeration seems

442

to be better related to the dielectric permittivity i.e. the polarity of the solvent. The low polarity

443

causes a low wettability with the mainly hydrophilic proteins and subsequently a large contact

444

angle. In turn, this results in a lower capillary pressure across two protein aggregates during

445

solvent evaporation, which leads to less irreversible agglomeration and better dispersibility of the

446

aggregates into oil.

447

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448

3.5 Protein conformation

449

The previous results show that prevention of agglomeration by choosing a solvent with a low

450

polarity is an important factor for the increased dispersibility of protein aggregates. However,

451

changes within the proteins aggregates may also add to this effect. Since proteins are subjective

452

to structural reorientation as solvent conditions are changed, it is possible that the protein

453

conformation is altered upon contact with solvents like acetone or hexane. Possibly, this

454

contributes to increased protein-solvent interactions by structural reorientations. To determine if

455

the protein conformation differed amongst WPI aggregates as a result of the type of solvent it

456

was dried from, we analysed the different powders by Attenuated Total Reflectance Fourier

457

Transform Infrared Resonance (ATR-FTIR). This technique is capable to determine the protein

458

conformation (i.e. secondary structure) as well as the hydration level.49 The amide I region (1600

459

– 1680 cm-1) is mostly due to C=O stretching and is closely related to changes in protein

460

conformation. The amide II region (1480 – 1560 cm-1), caused by NH bending and CN stretching

461

is closely related to protein hydration and less sensitive to conformational changes.50

462

Conveniently, we can probe the conformation as well as the hydration level of the obtained dried

463

samples directly. Figure 8A shows the amide I and II region of different WPI aggregate samples

464

as well as the native WPI sample. We display the results for aggregates dried from hexane (HD)

465

and acetone (AD), as these solvents gave a large difference in morphology and particle size, and

466

compared these samples to aggregates dried via freeze-drying (FD). Looking at the amide I

467

region, no clear changes can be observed. directly. Changes in the shape of the amide II region

468

are more extensive between the samples than changes in the amide I region. This may indicate a

469

change in hydration level of the proteins, which can be probed by comparing the intensity ratio of

470

the peak at 1535 cm-1 and 1541 cm-1 to the peak at 1515 cm-1. The vibration at 1515 cm-1 is

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471

related to tryptophan and is insensitive to hydration,51 which makes this a valuable internal

472

standard. These intensity ratios were used by other researchers to assess the hydration level

473

during film drying of β-lactoglobulin films.52 Figure 8B shows that the I1535/I1515 and I1541/I1515

474

ratios are higher for all dried aggregates compared to the native WPI powder. Aggregates

475

obtained from freeze drying showed lower hydration levels than the aggregates dried from both

476

acetone and hexane, but no differences were seen between acetone or hexane dried aggregates.

477

The lower hydration levels were confirmed by Karl Fisher titration, where the measured water

478

content was 6.3 % (± 0.1) for native WPI, 7.0 (± 0.1) for the freeze-dried aggregates, 7.9 (± 0.1)

479

for acetone-dried aggregates and 7.9 (± 0.3) for hexane-dried aggregates. Most likely, the amount

480

of water present in the solvent-dried samples (either acetone or hexane) was slightly higher

481

because of the hygroscopic nature of the protein powder, attracting moisture from the air. This

482

effect can be enhanced for powders with low density and large contact area. However, the

483

increased dispersibility and the small particle size of the hexane-dried aggregates compared to

484

acetone-dried protein aggregates in oil is not related to these differences in water content.

485 486

To determine the protein conformation, the second derivative of the amide I region was obtained

487

for native WPI and the WPI aggregates and displayed in Figure 8C-E. Since the solvent exchange

488

did not affect protein composition of the sample, this allows for direct comparison between the

489

aggregates. In all graphs, the second derivative of native WPI was added as a reference (dotted

490

line). Compared to the signal from the native WPI, the aggregates did not show major

491

differences. Only a broadening of the major band is noticeable at 1630 cm-1, assigned to an

492

increase of intermolecular β-sheet formation, and is indicative of aggregation.53-54 Between the

493

WPI aggregates, however, regardless of the drying method, no obvious differences were seen.

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494

The only noticeable difference is the slight increase in the intensity around 1640 cm-1 for the

495

samples dried from acetone and hexane compared to the freeze-dried sample, which might be

496

related to the small difference in hydration. Although small changes are observed between the

497

aggregates dried from the different solvents, we suspect that these differences are not large

498

enough to account for significant changes in the structure of the proteins. Note that also the

499

spectra of the second derivative of WPI aggregates dried from 1-propanol, heptane, and decane

500

did not differ from those shown in Figure 8. Moreover, we observed no changes in the second

501

derivative of the amide I region when WPI aggregates were suspended in oil using a solvent

502

exchange procedure (data not shown). The increased dispersibility of the aggregates as a result of

503

the solvent exchange procedure thus seems to be unrelated to any changes in protein

504

conformation since the results from the FTIR measurements for these samples show a high level

505

of similarity.

506

507

3.6 Surface hydrophobicity

508

Though FTIR was unable to detect differences in conformation, we checked the surface

509

hydrophobicity of the freeze-dried and the hexane-dried WPI aggregates. Using PRODAN, a

510

fluorescent probe, the relative fluorescence intensity (RFI) was measured as a function of protein

511

concentration and the results are displayed in Figure 9. The slope of RFI versus protein

512

concentration, is used as a measure of hydrophobicity. We have added the results of native WPI

513

as a comparison. As can be seen, the affinity for PRODAN increased as a result of applying a

514

heat treatment, as a higher slope was measured for the aggregates than for the native proteins.

515

This increase in hydrophobicity is expected as the heating process leads to exposure of

516

hydrophobic groups normally buried within the native folded structure of proteins.55 Between the 23

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517

two WPI aggregate samples, however, there was no clear difference between the freeze-dried and

518

hexane-dried sample, as the slope shows a high similarity. From this, we conclude that the

519

hydrophobicity does not change resulting from drying from different solvents, consistent with the

520

results discussed before. However, these results have to be taken with care, since these

521

measurements were performed in aqueous environments and therefore only irreversible changes

522

as affected by the different drying method can be measured.

523 524

In summary, from the results obtained by SDS-PAGE, FTIR and hydrophobicity measurements,

525

we can conclude that no differences in protein composition, conformation or hydrophobicity

526

occurred as affected by the presence of different types of solvents. This shows that structural

527

changes on a molecular level are therefore most likely not responsible for the enhanced

528

dispersibility of WPI aggregates in oil. A slight difference in water content was found as result of

529

the different drying methods, but this does not seem to explain the difference in dispersibility

530

between the acetone- and hexane-dried aggregates. Instead, other factors than water content or

531

conformational changes seem to dominate the dispersibility. We propose that prevention of

532

capillary forces between the aggregates during drying is most likely the cause for the increased

533

dispersibility of the aggregates in oil. It suggests that an optimized drying process to avoid

534

particle agglomeration could be an alternative for the solvent exchange procedure. Since a

535

solvent exchange requires a large amount of solvent, an alternative method would provide many

536

advantages from a practical point of view. Therefore, we examined the process of freeze-drying

537

more closely by considering different rates of freezing.

538

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539

3.7 Effect of freeze drying conditions

540

During the process of freeze-drying, water is removed from the sample by sublimation of ice at

541

low pressure. Although freeze-drying is considered a mild drying technique, freezing effects can

542

play a significant role in the agglomeration of particles. Ice formation in a colloidal suspension,

543

such as our suspension of aggregates, typically expels the particles from the frozen areas,

544

effectively increasing the particle concentration locally. The formed ice crystals pack the particles

545

close to one another with high forces that can overcome repulsive forces and thus induce

546

(irreversible) particle agglomeration. When conditions are carefully chosen, in a process called

547

‘freeze casting’, directional ice formation can even occur, leading to the formation of a layered

548

pattern.56 When the freezing rate is increased, however, the formed ice crystals are small, and

549

tight packing of the particles is prevented to some extent and instead, the particles remain more

550

evenly distributed.57 Therefore, as an alternative to the slow freezing at -20 °C, we investigated

551

the effect of a higher freezing rate by dripping a 1 wt% aqueous WPI aggregate suspension

552

directly into liquid nitrogen. The temperature difference, and subsequently the freezing rate, was

553

thus roughly increased by a factor 5. After the material was freeze-dried, the dried powder was

554

dispersed directly into oil by homogenization. The resulting particle size distribution was

555

measured and as can be seen in Figure 10, the sample contains two main size populations as is

556

noticeable by the appearance of two distinct peaks. One population having a size of

557

approximately 150 nm, the other a broad range of larger particle sizes (~ 10 – 500 µm). The

558

resulting size distribution was different from the sample frozen at -20 °C (Figure 1B), where only

559

large agglomerates were observed. The peak at 150 nm shows that irreversible agglomeration of

560

the aggregates was prevented to some extent by the process of fast freezing and subsequent

561

freeze-drying. Even though the average particle size was reduced, we found that agglomeration

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562

into larger agglomerates was still unavoidable. These larger aggregates (>100 µm) were removed

563

by centrifugation at low speeds (500 g). The particle size distribution of the supernatant (Figure

564

10, open squares) shows that the peak at smaller particle sizes became more prominent. Even

565

though a bimodal distribution can be seen, the major fraction of the particles was now below 1

566

µm. Comparing the size as a surface weighted diameter, d3,2, we found an average of 140 nm for

567

the aggregates obtained with the solvent exchange procedure, and 220 nm for the WPI aggregates

568

in the supernatant obtained with the freeze-drying method using rapid freezing. This shows that a

569

high freezing rate can prevent particle size agglomeration to a large extent.

570

571

3.8 Rheology of WPI oleogels prepared via solvent exchange and freeze drying

572

From a practical point of view, freeze-drying as an alternative for a solvent exchange is desirable

573

since much less solvent is needed to transfer the protein aggregates to the oil phase and the dried

574

protein powder can be added to the oil directly. To assess the capability of the freeze-dried WPI

575

aggregates to form a network in liquid oil, the supernatant was centrifuged at higher speeds (4000

576

g, 40 min) to collect the protein aggregates as a dense pellet. The rheological properties of the

577

protein oleogel prepared from freeze-dried aggregates were compared to those of the oleogel

578

prepared using the solvent exchange procedure. To allow for comparison between the different

579

oleogels, the composition of the samples was standardized for protein (10 wt%) and water

580

content (1.2 wt%) as determined by Dumas and dry matter analysis, respectively. Both samples

581

were paste-like gels and the results of the frequency dependence can be seen in Figure 11A. Both

582

oleogels show a high degree of similarity as G’ was roughly an order of magnitude higher than

583

G” for both samples, indicating an elastic network had formed. Since G’ was only slightly

584

dependent on frequency and the complex viscosity decreased linearly with the applied frequency, 26

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585

it shows that for both gels, the viscoelastic response was not significantly affected by the rate of

586

deformation. Figure 11B displays for both oleogels the rheological response to an increased strain

587

amplitude of deformation. It can be seen that both samples had a similar linear viscoelastic

588

region, as the G’ deviated from linearity at roughly the same strain value. Furthermore, the

589

overshoot in G” can be noticed in both samples, which indicated fast rearrangements in the

590

network structure during deformation.58 Even though the rheological response is highly similar,

591

the magnitude of G’ was somewhat lower for the sample with freeze-dried WPI aggregates. This

592

small difference can be explained by the larger particles present in the freeze-dried sample, which

593

were not observed by applying a solvent exchange procedure. Since larger particles are less

594

effective in creating a network structure, given the lower surface area available, this leads to a

595

less efficient network formation and lower values for the moduli. Nonetheless, by preventing

596

severe particle agglomeration due to the high freezing rate, the increased surface area available

597

for protein-protein interactions resulted in effective gel formation. Given that the results are

598

similar for both type of oleogels, we can conclude that the solvent exchange and the freeze-

599

drying method both lead to effective network formation of the protein aggregates in liquid oil.

600

Therefore, tuning the conditions during drying, such as a fast freezing process, could be an

601

effective strategy to produce a protein powder which is well dispersible in oil and directly

602

capable of forming a gelled structure.

603

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604

Conclusions

605

The aim of the current paper was to elucidate on the enhanced stability against agglomeration of

606

whey protein isolate (WPI) aggregates in oil after applying a solvent exchange procedure. To this

607

end, heat-set WPI aggregates were transferred from water to several solvents differing in polarity.

608

We have shown that drying protein aggregates by evaporation from solvents with a low polarity

609

(eg. hexane) resulted in a low density powder, which showed good dispersibility of the

610

aggregates into liquid oil. When the aggregates were dried from more hydrophilic solvents, such

611

as acetone, 1-propanol or water, the drying process resulted in agglomeration of the protein

612

aggregates, and poor dispersibility in oil. No change in protein composition, protein

613

conformation, or surface hydrophobicity was observed as a result of the solvent exchange

614

procedure. Therefore, we concluded that reduced agglomeration is dominated by a reduction of

615

attractive capillary forces between the protein aggregates during drying. Nonpolar solvents such

616

as hexane, having a low surface tension and low wettability, prevent agglomeration by avoiding a

617

capillary pressure build up during drying. This result suggests that the drying conditions can be

618

tuned to minimize the degree of irreversible agglomeration of the protein aggregates. Indeed, we

619

were able to show that by increasing the freezing rate prior to freeze-drying the water,

620

irreversible agglomeration was prevented to a large extent. The resulting particle size distribution

621

of the freeze-dried WPI aggregates after fast freezing showed to be close to that of the solvent

622

exchange sample. For both methods, the small aggregates were effective in forming a gel

623

network where G’ > G”. This research has shown that by carefully designing a drying process,

624

irreversible agglomeration of WPI aggregates can be prevented to obtain a dried protein material

625

that can be used directly for structure formation in liquid oil.

626

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627

Acknowledgements

628

This research was funded by the Top Institute Food and Nutrition, Wageningen, The Netherlands.

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629

Graphical abstract

630

631

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References

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634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675

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676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719

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19. Pernetti, M.; van Malssen, K.; Kalnin, D.; Flöter, E., Structuring Edible Oil with Lecithin and Sorbitan Tri-Stearate. Food Hydrocoll. 2007, 21 (5-6), 855-861. 20. Da Pieve, S.; Calligaris, S.; Co, E.; Nicoli, M. C.; Marangoni, A. G., Shear Nanostructuring of Monoglyceride Organogels. Food Biophys. 2010, 5 (3), 211-217. 21. Ojijo, N. K. O.; Neeman, I.; Eger, S.; Shimoni, E., Effects of Monoglyceride Content, Cooling Rate and Shear on the Rheological Properties of Olive Oil/Monoglyceride Gel Networks. J. Sci. Food Agric. 2004, 84 (12), 1585-1593. 22. Schaink, H. M.; van Malssen, K. F.; Morgado-Alves, S.; Kalnin, D.; van der Linden, E., Crystal Network for Edible Oil Organogels: Possibilities and Limitations of the Fatty Acid and Fatty Alcohol Systems. Food Res. Int. 2007, 40 (9), 1185-1193. 23. Bot, A.; Agterof, W. G. M., Structuring of Edible Oils by Mixtures of Γ-Oryzanol with ΒSitosterol or Related Phytosterols. J. Am. Oil Chem. Soc. 2006, 83 (6), 513-521. 24. Sawalha, H.; Margry, G.; den Adel, R.; Venema, P.; Bot, A.; Flöter, E.; van der Linden, E., The Influence of the Type of Oil Phase on the Self-Assembly Process of Γ-Oryzanol + ΒSitosterol Tubules in Organogel Systems. Eur. J. Lipid Sci. Technol. 2013, 115 (3), 295-300. 25. Blake, A. I.; Co, E. D.; Marangoni, A. G., Structure and Physical Properties of Plant Wax Crystal Networks and Their Relationship to Oil Binding Capacity. J. Am. Oil Chem. Soc. 2014, 91 (6), 885-903. 26. Hwang, H. S.; Kim, S.; Singh, M.; Winkler-Moser, J. K.; Liu, S. X., Organogel Formation of Soybean Oil with Waxes. J. Am. Oil Chem. Soc. 2012, 89 (4), 639-647. 27. Toro-Vazquez, J. F.; Morales-Rueda, J. A.; Dibildox-Alvarado, E.; Charó-Alonso, M.; Alonzo-Macias, M.; González-Chávez, M. M., Thermal and Textural Properties of Organogels Developed by Candelilla Wax in Safflower Oil. J. Am. Oil Chem. Soc. 2007, 84 (11), 989-1000. 28. Davidovich-Pinhas, M.; Barbut, S.; Marangoni, A. G., The Gelation of Oil Using Ethyl Cellulose. Carbohydr. Polym. 2015, 117, 869-878. 29. Davidovich-Pinhas, M.; Barbut, S.; Marangoni, A. G., The Role of Surfactants on Ethylcellulose Oleogel Structure and Mechanical Properties. Carbohydr. Polym. 2015, 127, 355362. 30. Nikiforidis, C. V.; Scholten, E., Polymer Organogelation with Chitin and Chitin Nanocrystals. RSC Adv. 2015, 5 (47), 37789-37799. 31. Huang, Y.; He, M.; Lu, A.; Zhou, W.; Stoyanov, S. D.; Pelan, E. G.; Zhang, L., Hydrophobic Modification of Chitin Whisker and Its Potential Application in Structuring Oil. Langmuir 2015, 31 (5), 1641-1648. 32. Patel, A. R.; Rajarethinem, P. S.; Cludts, N.; Lewille, B.; De Vos, W. H.; Lesaffer, A.; Dewettinck, K., Biopolymer-Based Structuring of Liquid Oil into Soft Solids and Oleogels Using Water-Continuous Emulsions as Templates. Langmuir 2015, 31 (7), 2065-2073. 33. Patel, A. R.; Schatteman, D.; Lesaffer, A.; Dewettinck, K., A Foam-Templated Approach for Fabricating Organogels Using a Water-Soluble Polymer. RSC Adv. 2013, 3 (45), 2290022903. 34. de Vries, A.; Hendriks, J.; van der Linden, E.; Scholten, E., Protein Oleogels from Protein Hydrogels Via a Stepwise Solvent Exchange Route. Langmuir 2015, 31 (51), 13850-13859. 35. Barteri, M.; Gaudiano, M. C.; Mei, G.; Rosato, N., New Stable Folding of ΒLactoglobulin Induced by 2-Propanol. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1998, 1383 (2), 317-326.

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36. Yoshida, K.; Yamaguchi, T.; Osaka, N.; Endo, H.; Shibayama, M., A Study of AlcoholInduced Gelation of Β-Lactoglobulin with Small-Angle Neutron Scattering, Neutron Spin Echo, and Dynamic Light Scattering Measurements. PCCP 2010, 12 (13), 3260-3269. 37. Havea, P.; Carr, A. J.; Creamer, L. K., The Roles of Disulphide and Non-Covalent Bonding in the Functional Properties of Heat-Induced Whey Protein Gels. J. Dairy Res. 2004, 71 (3), 330-339. 38. Haskard, C. A.; Li-Chan, E. C. Y., Hydrophobicity of Bovine Serum Albumin and Ovalbumin Determined Using Uncharged (Prodan) and Anionic (Ans-) Fluorescent Probes. J. Agric. Food Chem. 1998, 46 (7), 2671-2677. 39. Schmitt, C.; Bovay, C.; Vuilliomenet, A. M.; Rouvet, M.; Bovetto, L.; Barbar, R.; Sanchez, C., Multiscale Characterization of Individualized Beta-Lactoglobulin Microgels Formed Upon Heat Treatment under Narrow Ph Range Conditions. Langmuir 2009, 25 (14), 7899-7909. 40. Schmitt, C.; Moitzi, C.; Bovay, C.; Rouvet, M.; Bovetto, L.; Donato, L.; Leser, M. E.; Schurtenberger, P.; Stradner, A., Internal Structure and Colloidal Behaviour of Covalent Whey Protein Microgels Obtained by Heat Treatment. Soft Matter 2010, 6 (19), 4876-4884. 41. Hines, M. E.; Foegeding, E. A., Interactions of Alpha-Lactalbumin and Bovine SerumAlbumin with Beta-Lactoglobulin in Thermally Induced Gelation. J. Agric. Food Chem. 1993, 41 (3), 341-346. 42. Edwards, P. B.; Creamer, L. K.; Jameson, G. B.; Boland, M.; Singh, H., Chapter 6 Structure and Stability of Whey Proteins A2 - Thompson, Abby. In Milk Proteins, Academic Press: San Diego, 2008, pp 163-203. 43. Strauch, S.; Herminghaus, S., Wet Granular Matter: A Truly Complex Fluid. Soft Matter 2012, 8 (32), 8271. 44. Shan, H.; Zhang, Z., Preparation of Nanometre-Sized Zro2/Al2o3 Powders by Heterogeneous Azeotropic Distillation. J. Eur. Ceram. Soc. 1997, 17 (5), 713-717. 45. Hu, Z. S.; Dong, J. X.; Chen, G. X., Replacing Solvent Drying Technique for Nanometer Particle Preparation. J. Colloid Interface Sci. 1998, 208 (2), 367-372. 46. Kraiwattanawong, K.; Tamon, H.; Praserthdam, P., Influence of Solvent Species Used in Solvent Exchange for Preparation of Mesoporous Carbon Xerogels from Resorcinol and Formaldehyde Via Subcritical Drying. Microporous Mesoporous Mater. 2011, 138 (1-3), 8-16. 47. Betz, M.; García-González, C. A.; Subrahmanyam, R. P.; Smirnova, I.; Kulozik, U., Preparation of Novel Whey Protein-Based Aerogels as Drug Carriers for Life Science Applications. J. Supercrit. Fluids 2012, 72, 111-119. 48. Giovambattista, N.; Debenedetti, P. G.; Rossky, P. J., Effect of Surface Polarity on Water Contact Angle and Interfacial Hydration Structure. J. Phys. Chem. B 2007, 111 (32), 9581-9587. 49. Barth, A., Infrared Spectroscopy of Proteins. Biochim. Biophys. Acta-Bioenerg. 2007, 1767 (9), 1073-1101. 50. Wellner, N.; Belton, P. S.; Tatham, A. S., Fourier Transform Ir Spectroscopic Study of Hydration-Induced Structure Changes in the Solid State of Ω-Gliadins. Biochem. J. 1996, 319 (3), 741-747. 51. Almutawah, A.; Barker, S. A.; Belton, P. S., Hydration of Gluten: A Dielectric, Calorimetric, and Fourier Transform Infrared Study. Biomacromolecules 2007, 8 (5), 1601-1606. 52. Bouman, J.; de Vries, R.; Venema, P.; Belton, P.; Baukh, V.; Huinink, H. P.; van der Linden, E., Coating Formation During Drying of Beta-Lactoglobulin: Gradual and Sudden Changes. Biomacromolecules 2015, 16 (1), 76-86.

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53. O'Loughlin, I. B.; Kelly, P. M.; Murray, B. A.; Fitzgerald, R. J.; Brodkorb, A., Concentrated Whey Protein Ingredients: A Fourier Transformed Infrared Spectroscopy Investigation of Thermally Induced Denaturation. Int. J. Dairy Technol. 2015, 68 (3), 349-356. 54. Shivu, B.; Seshadri, S.; Li, J.; Oberg, K. A.; Uversky, V. N.; Fink, A. L., Distinct BetaSheet Structure in Protein Aggregates Determined by Atr-Ftir Spectroscopy. Biochemistry 2013, 52 (31), 5176-5183. 55. Stănciuc, N.; Aprodu, I.; Râpeanu, G.; Bahrim, G., Fluorescence Spectroscopy and Molecular Modeling Investigations on the Thermally Induced Structural Changes of Bovine ΒLactoglobulin. Innov. Food Sci. Emerg. Technol. 2012, 15, 50-56. 56. Deville, S.; Saiz, E.; Nalla, R. K.; Tomsia, A. P., Freezing as a Path to Build Complex Composites. Science 2006, 311 (5760), 515-518. 57. Lee, J.; Cheng, Y., Critical Freezing Rate in Freeze Drying Nanocrystal Dispersions. J. Controlled Release 2006, 111 (1-2), 185-192. 58. Hyun, K.; Kim, S. H.; Ahn, K. H.; Lee, S. J., Large Amplitude Oscillatory Shear as a Way to Classify the Complex Fluids. J. Non-Newtonian Fluid Mech. 2002, 107 (1-3), 51-65.

780 781 782

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783

Figures and Tables

784 785

Table 1. Properties of the solvents used for drying WPI aggregates. Dielectric permittivity, ε (298K)

Surface tension mN/m (293K)

Water

80.1

72.8

Acetone

21.4

25.2

1-Propanol

19.4

23.7

n-Hexane

1.8

18.4

n-Heptane

1.8

20.1

n-Decane

1.8

23.8

786

787 8

8

A

B

6

6

Volume%

Volume%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4

2

4

2

0

0 0.01

0.1

788

1 10 Size [μm]

100

1000

0.01

0.1

1 10 Size [μm]

100

1000

789

Figure 1. Particle size distribution of dispersions of WPI aggregates. A) Particle size in water

790

measured either directly after homogenization () or re-dispersed after freeze-drying (). B)

791

Particle size of aggregates in sunflower oil using freeze-drying () or a solvent exchange

792

procedure ().

793

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794 795

Figure 2. Scans of SDS-PAGE electrophoretograms of native whey protein isolate (1), freeze-

796

dried WPI aggregates (2), acetone-dried WPI aggregates (3), hexane-dried aggregates (4),

797

supernatant after centrifuging the whey protein aggregates (5). M: molecular weight markers.

798

2.0

Ratio α-lac / β-lac [-]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 41

1.5

1.0

0.5

0.0

799

Native

FD

AD

HD

Supernatant

800

Figure 3. Ratio α-lactalbumin/β-lactoglobulin in the different protein samples. FD, AD and HD

801

represent freeze-dried, acetone-dried and hexane-dried aggregates respectively. Supernatant was

802

taken after centrifuging the whey protein aggregates.

803

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804 805

Figure 4. SEM micrographs of dried WPI aggregates: (A) freeze-dried from water, (B)

806

evaporated from acetone, (C) evaporated from hexane. Numbers 1 and 2 refer to a different

807

magnification of the same sample.

808

Wet pellet

Solvent evaporation

Protein aggregate R

θ

809 810

Figure 5. Schematic drawing of a liquid bridge between two spherical particles during

811

evaporation of the solvent.

812

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813 814

Figure 6. Appearance of dried protein suspensions from water (A), acetone (B), or hexane (C) at

815

ambient conditions and corresponding SEM micrographs (D-F).

816 8

8

B

A 6 Volume%

6 Volume%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4

2

2

0

0 0.01

817

4

0.1

1

10 Size [μm]

100

1000

0.01

0.1

1

10 Size [μm]

100

1000

818

Figure 7. Particle size distribution of dispersed WPI aggregates in liquid oil. A) size distribution

819

of aggregates dried from 1-propanol () and acetone (). B) Particle size distribution of

820

aggregates dried from hexane (), heptane () or decane ().

821 822 823

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A

Amide II

Absorbance [-]

Absorbance [-]

Amide I

C

FD

1680

FD

1640

1600

AD 1560

1520

Wavenumber [cm-1]

1.1

B

D

HD

1535/1515

1480

Absorbance [-]

Native WPI

1541/1515

AD

1.0

E Absorbance [-]

I/I1515 [-]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

0.9

HD 0.8 Native

824

FD

Sample

AD

1700

HD

1680

1660 1640 1620 Wavenumber [cm-1]

1600

825

Figure 8. FTIR results for native WPI powder, freeze-dried aggregates (FD), acetone-dried

826

aggregates (AD) and hexane-dried aggregates (HD). A) Amide I and II IR-spectra, B) Band

827

intensity ratio of selected band/1515 cm-1 (I/I1515). White bars: 1535 cm-1 black bars: 1541 cm-1.

828

C-E) Second derivative of Amide I band of dried whey protein aggregates (solid lines), in each

829

figure, native WPI powder was added as a reference (dotted line).

830

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1000

RFI [-]

800 600 400 200 0 0

0.05

831

0.1 0.15 0.2 Protein Concentration [mg/mL]

0.25

832

Figure 9. Relative fluorescence intensity (RFI) as a function of protein concentration for native

833

WPI (), freeze-dried WPA (), and hexane-dried WPA ().

834 8

6 Volume%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 41

4

2

0 0.01

835

0.1

1 10 Size [μm]

100

1000

836

Figure 10. Particle size distribution of freeze dried aggregates in sunflower oil using a high

837

freezing rate (), and the supernatant of the same sample after centrifugation at 500 x g ().

838

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10000

100000

10000

B

A 10000 1000

100

G', G" [Pa]

1000

η* [Pa.s]

1000

G', G" [Pa]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100

100 10 10 G’ G” η*

G’ G”

10

1 0.1

1

1 0.001

10

0.01

Frequency [Hz]

0.1

1

10

100

Strain [%]

839 840

Figure 11. Frequency sweeps (A) and strain sweeps (B) of 10 wt% protein oleogels prepared by

841

solvent exchange () or by freeze-drying (). A) frequency sweep, B) strain sweep. Error bars

842

of triplicate measurements were typically not larger than the symbols and were left out for

843

clarity.

844

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