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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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1
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] 14
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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
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disulphide bonds and is in agreement with other studies using heat-set WPI aggregates prepared
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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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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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Acknowledgements
628
This research was funded by the Top Institute Food and Nutrition, Wageningen, The Netherlands.
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Graphical abstract
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References
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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.
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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
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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%
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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
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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
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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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