Sunflower oil organogels and organogel-in-water emulsions (part I): Microstructure and mechanical properties

Accepted Manuscript Sunflower oil organogels and organogel-in-water emulsions (part I): microstructure and mechanical properties T. Moschakis, E. Panagiotopoulou, E. Katsanidis PII:

S0023-6438(16)30141-4

DOI:

10.1016/j.lwt.2016.03.004

Reference:

YFSTL 5344

To appear in:

LWT - Food Science and Technology

Received Date: 12 November 2015 Revised Date:

24 February 2016

Accepted Date: 4 March 2016

Please cite this article as: Moschakis, T., Panagiotopoulou, E., Katsanidis, E, Sunflower oil organogels and organogel-in-water emulsions (part I): microstructure and mechanical properties, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.03.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Sunflower oil organogels and organogel-in-water emulsions (part I): microstructure and

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mechanical properties

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Moschakis T., Panagiotopoulou, E. and Katsanidis, E*

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Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of

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Thessaloniki, P.O. Box 235, Thessaloniki, Greece

* Corresponding author at: Department of Food Science and Technology, Faculty of Agriculture,

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Aristotle University of Thessaloniki, P.O. Box 235, Thessaloniki, Greece. Tel.: +30 2310991640; fax:

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+30 2310991632.

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E-mail address: [email protected] (E. Katsanidis).

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Abstract

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Currently, there is a growing interest for foods with not only sensorial quality, safety and

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shelf stability but also providing health benefits through certain added ingredients. The aim of

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the present study was to explore the potential use of vegetable oils structured with γ-oryzanol

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and phytosterols as an alternative to animal fat with regard to mechanical properties.

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Polarised microscopy and bulk rheological measurements have been employed to study the

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microstructure and the mechanical properties of organogels and organogel-in-water

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emulsions. Sterol ratio, total sterol content, storage duration and temperature were found to

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substantially affect the properties of the oil structures, both in organogels and organogels-in-

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water emulsions. However, the behaviour of the organogels structures does not always seem

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to match with the organogel-in-water emulsions response. Modulating the sterol ratio and

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total sterol content, the microstructure and mechanical properties of the vegetable oil

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organogels and organogels-in-water emulsions could be modified to mimic the mechanical

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properties of animal fat, without adversely affecting the textural quality and shelf-life of the

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food products.

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Highlights 

Mechanical properties of oil structures with γ-oryzanol & phytosterols were studied

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Sterol ratio and sterol content affect the properties of the oil structures

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Equimolar sterol structurant ratio resulted in stronger organogels

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Excess phytosterols crystallise and enhance the stability of organogel emulsions

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Keywords: organogels; organogel-based emulsions; phytosterols; γ-oryzanol; rheology

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1. Introduction

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Organogels are gels where the liquid (continuous) phase is comprised of oil (M. A. Rogers,

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2009) that is immobilized within a three-dimensional, cross-linked network. Mixtures of γ-

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oryzanol and β-sitosterol, among others, are used as gelators to form three-dimensional

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networked structures (Calligaris, Mirolo, Da Pieve, Arrighetti, & Nicoli, 2014; Nukit,

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Setwipattanachai, Chaiseri, & Hongsprabhas, 2014; Sawalha et al., 2015). The oil structuring

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ability of γ-οryzanol and different phytosterols is attributed to the formation of hollow

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tubules of 7 nm diameter and just under 1 nm wall thickness (Bot, den Adel, Roijers, &

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and γ-oryzanol, because individually neither one is capable of entrapping the oil phase (Bot

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& Agterof, 2006), while the sterol molecules are dynamically transferring from tubules to

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solution and vice versa (Sawalha et al., 2015). The oil phase is present both inside and

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outside of the tubule (Bot, den Adel, et al., 2009).

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The visual and mechanical properties of the organogels, e.g. firmness and transparency,

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depend on the oryzanol-sitosterol ratio (Bot & Agterof, 2006; Bot, den Adel, & Roijers,

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2008; Bot, Veldhuizen, den Adel, & Roijers, 2009). The turbidity of a sunflower oil gel

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decreased with increasing γ-oryzanol concentration and the firmest transparent gel is usually

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obtained at the ratio of 60:40 w/w oryzanol-sitosterol which corresponds to 1:1 molar ratio

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(Bot & Agterof, 2006; Bot et al., 2008; Bot, Veldhuizen, et al., 2009). Modifications to the

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ratio of phytosterols to γ-oryzanol affect not only the opacity of gels but also the aggregation

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into tubules and the activation energy of nucleation (M. A. Rogers, Bot, Lam, Pedersen, &

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May, 2010; Sawalha, Venema, Bot, Floter, & van der Linden, 2011).

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Moreover, γ-oryzanol and phytosterols are capable of structuring o/w and w/o emulsions.

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According to Duffy et al. (2009), oryzanol and sitosterol, under certain conditions, are

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capable of forming fibril structures also in aqueous phase. The formation of tubular

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microstructure in the oil phase of the emulsion can be promoted by reducing the water

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activity and/or by using oil of low polarity (Sawalha et al., 2012). It has also been reported

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that a mixture of oryzanol and sitosterol (16% in total, ratio 60:40) forms crystals in water

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phase that can be related to the crystals of the pure compounds (Bot et al., 2011; den Adel,

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Heussen, & Bot, 2010; Sawalha et al., 2012). Crystallization of the phytosterols in the

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aqueous continuous phase takes place during and after the emulsification process (Duffy et

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al., 2009). Phytosterols have surface activity, which would allow them to migrate to the oil–

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water interface of the emulsion droplets (Cercaci, Rodriguez-Estrada, Lercker, & Decker,

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that the presence of water interferes with the capacity of the phytosterols to structure the

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emulsion (Bot et al., 2011; Bot, Veldhuizen, et al., 2009; den Adel et al., 2010; Duffy et al.,

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2009). However, in oil gels a completely different structure is formed which is not related to

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the structures that are formed by both individual compounds, and which can be identified as a

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self-assembled tubular structure involving both molecules of sitosterol and oryzanol (den

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Adel et al., 2010).

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γ-Oryzanol is extracted from rice bran oil and is derived from a fraction containing ferulate

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(4-hydroxy-3-methoxycinnamic acid) esters of triterpene alcohols and plant sterols (E. J.

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Rogers et al., 1993). It is considered to be an antioxidant compound which is associated with

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decreasing plasma and serum cholesterol levels, cholesterol absorption and platelet

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aggregation (Patel & Naik, 2004). β-Sitosterol is a phytosterol widespread in plants (Moreau,

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Whitaker, & Hicks, 2002). Phytosterols are non-caloric compounds that occur naturally in

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vegetable products (such as fruits and nuts) as well as oils (Brufau, Canela, & Rafecas, 2008).

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Phytosterols appear to have bioactive properties and moderate dietary doses might have a

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positive impact on human health, by decreasing cholesterol absorption (Sanclemente et al.,

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2012). Dietary phytosterol supplementation of as little as 2 g per day has been reported to

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cause, on average, a 13% LDL-cholesterol reduction, and a 10% total cholesterol reduction

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(Romer & Garti, 2006).

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Oleogels and their emulsions could be used as trans- or saturated (animal) fat replacements

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and therefore, a better understanding of the factors affecting the mechanical properties and

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their stability is needed. The aim of this study (Part I) was to investigate the microstructure

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and the rheological properties of sunflower oil organogels and organogel-in-water emulsions

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in an attempt to characterise the mechanical properties of these systems for their potential to

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replace trans- or saturated aminal fat in structured food products.

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2. Materials & Methods

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2.1 Materials

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Sunflower oil and olive oil (Minerva SA, Metamorphosi, Greece) were obtained from the

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local market. γ-Oryzanol was purchased from Jan Dekker Nederland B.V. (Wormerveer, the

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Netherlands). Tetradecane was purchased from TCI Europe NV (Antwerp, Belgium) and

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phytosterols (Vegapure 867G) were kindly provided by BASF Group (Ludwigshafen,

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Germany). Tween® 20 and xanthan gum were obtained from Sigma-Aldrich (Hannover,

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Germany). All aqueous solutions were prepared with double distilled water. In preliminary

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experiments, sunflower lecithin (Solec™ SF-10, Solae Europe, S.A., Geneva, Switzerland)

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was added to sunflower oil prior to heating. All other chemicals used were of analytical

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grade.

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2.2 Preparation of organogels and organogel-based emulsions

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Organogels. Two different γ-oryzanol:phytosterol weight ratios, 30:70 and 60:40, were used

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in the preparation of sunflower oil solutions with total sterol content of 5%, 10%, 15% and

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20% w/w. The 60:40 w/w oryzanol-phytosterol ratio gives the firmest transparent gel which

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corresponds to 1:1 molar ratio (Bot & Agterof, 2006; Bot et al., 2008; Bot, Veldhuizen, et al.,

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2009). On the contrary, in the 30:70 ratio, the phytosterols are in excess and thus the effect of

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crystallization on the physicochemical properties of the organogels was investigated. A given

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amount of γ-oryzanol and phytosterols was added in oil at ambient temperature. The solutions

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were heated and maintained at the temperature range of 90-95 οC for 30 min, under constant

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stirring. Subsequently, the samples were cooled and stored at 4 οC or 25 οC, depending on the

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experimental procedure.

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ratios (10% and 20% w/w total sterol content in the oil phase, 30:70 and 60:40 γ-oryzanol to

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phytosterol weight ratio) were also prepared by mixing 50% w/w sunflower oil and 50% w/w

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aqueous phase. The aqueous phase of the emulsions containing Tween 20 (1.7% w/w) and

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xanthan gum (0.15% w/w) was heated up to 70 οC under stirring. The emulsions were

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prepared by homogenising the oil containing the structurants (γ-oryzanol and phytosterol)

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and the water phase using a high shear disperser (Ultra Turrax IKA T18 basic, IKA Works

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Inc., Wilmington, USA) for 1 min at 14.000 rpm at temperature > 70 οC. The produced

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emulsions were cooled immediately at ambient temperature and stored at 4 οC until further

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examination.

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2.3 Optical Microscopy

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Micrographs of the organogels and the organogel-in-water emulsions were captured by using

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an Olympus BX51 (Olympus Optical Co Ltd, Tokyo, Japan) polarising microscope equipped

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with a microscope digital camera (Olympus DP 70, Japan). The freshly prepared organogels

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and organogel-based emulsions were placed onto a microscope slide with a cover glass and

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were observed after 24h at ambient temperature without any further preparation.

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2.4 Large deformation measurements: Penetration Test

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Penetration tests were performed in organogel samples on a TA-XT2i Texture Analyser

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(Godalming, Surrey, UK) using a stainless steel cylindrical probe with a diameter of 6 mm.

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Hot solutions of sunflower oil with 5%, 10%, 15% and 20% w/w sterols (30:70 and 60:40 γ-

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oryzanol to phytosterol weight ratio) were poured into plastic cylindrical containers and

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stored at 4 οC or 25 οC. After a period of approximately 24 h, in which gelation had taken

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place, the organogels were removed and cut to self-sustained cylinders of 10 mm height and

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19 mm diameter. Penetration experiments were performed at a penetration speed of 0.5 mm s-

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distance of 5 mm, corresponding to the 50% of the height of the samples. The parameters

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extracted were hardness (N) as the maximum force that occurred during penetration and total

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work for penetration defined as the gel strength (mJ) represented by the surface under the

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force deformation curve (Vancamp & Huyghebaert, 1995; Verbeken, Bael, Thas, &

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Dewettinck, 2006). At least five measurements were performed at ambient temperature for

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each sample and the data were collected by using the software Texture Expert Version 1.22

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of Stable Micro Systems (Godalming, Surrey, UK).

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(probe speed before and after penetration 1.0 mm s-1 and 5.0 mm s-1, respectively) over a

2.5 Rheological Measurements

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The rheological properties of the organogel-based emulsions were studied by a rotational

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Physica MCR 300 rheometer (Physica Messtechnic GmbH, Stuttgart, Germany) using a

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parallel plate geometry (50 mm diameter and 1 mm gap); the temperature was regulated by a

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Paar Physica circulating bath and a controlled peltier system (TEZ 150 P/MCR) with an

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accuracy of ± 0.1 οC. The data of the rheological measurements were analysed with a

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supporting rheometer software US200 V2.21. Two recordings were made per sample and

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each measurement was carried out on two separate prepared samples (total four

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measurements per sample). All tests were performed at 25 οC.

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Initially, strain sweep tests were performed at 1 Hz for all the samples in order to determine

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the linear viscoelastic region (LVR). Deviation from the linear viscoelastic region occurs

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when the sample starts to permanently deform, implying the destruction of the transient

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network structure. The apparent yield stress τy of the samples was determined as the stress at

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remains constant.

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A target strain of 0.5% was used in the subsequent experiments, which was within the LVR

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for each sample examined. Small deformation oscillatory measurements for evaluation of the

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viscoelastic properties, G' (storage modulus), G'' (loss modulus), and phase angle δ (defined

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by tanδ= G''/G') were performed over the frequency range 0.1 to 100 Hz at 25 οC. In addition,

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the flow behaviour of the samples was assessed by measuring steady shear viscosity η (Pa·s)

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over a range of shear rates 0.001–100 s-1 at 25 οC.

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2.2.7 Statistical Analysis

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All data collected during measurements were analysed by using the General Linear Model.

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Means were compared using Tukey’s multiple range test. All analyses were performed using

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the Minitab (Minitab Inc., USA) statistical software.

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3. Results & Discussion

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3.1 Organogels

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3.1.1 Polarized microscopy

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Figure 1 illustrates the microstructure of sunflower oil organogels with 10% and 20% w/w

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structurants at different sterol weight ratios, 60:40 and 30:70 (γ-oryzanol:phytosterol), after

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24h at ambient temperature. In the 30:70 samples (Figure 1 a & b), crystal formation of

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sterols is favoured. The microstructure was studied under polarised light, thus the liquid oil

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phase, optically isotropic, appears dark, while crystals appear as bright white features. The

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crystals resemble typical phytosterol crystals observed in phytosterol-saturated oil systems

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(Vaikousi, Lazaridou, Biliaderis, & Zawistowski, 2007). As can be seen from Figure 1a & b,

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w/w sterol content sample (Figure 1 b), the size of the phytosterol crystals appears to be

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larger and the shape seems to be more blade-shaped in comparison with needle-shaped

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crystals at 10% w/w sterol content. On the other hand, the 60:40 sterol weight ratio, i.e. 1:1

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molar ratio, has been characterised as ideal for creating sterol structured organogels (Pernetti,

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van Malssen, Floter, & Bot, 2007). Indeed, in this ratio, no crystals were observed (Figure 1 c

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& d), only some feather-like formations. These formations are likely to be the result of the

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sterol fibrillar network, described by other researchers (Bot et al., 2008).

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3.1.2 Mechanical properties

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Effect of sterol content: Visual observations of olive and sunflower oil organogels at

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concentrations of 5%, 10% and 20% w/w sterols and 60:40 weight ratio are shown in Figure

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The mixture of γ-oryzanol and plant sterols is capable of structuring a liquid fat of vegetable

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origin (Pernetti et al., 2007). During preliminary experiments, it was found that dispersions of

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6% w/w γ-oryzanol and 4% w/w phytosterols also exhibited significant mechanical properties

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by using tetradecane oil as the liquid lipid phase (results not shown) implying that the

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structuring effect occurs in non-vegetable origin oils. That is, at the equimolar oryzanol and

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phytosterol ratio a network can be established in other apolar liquid solvents apart from the

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polyunsaturated triglyceride oils. However, in this study only sunflower oil was selected as

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the continuous phase of organogels to be further examined.

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Organogels produced with 5% w/w sterol content were not free-standing, and thus they could

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not be subjected to penetration tests even after being stored at ambient temperature for 10

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days (Figure 2 a&b). On the other hand, the organogels of 10%, 15% and 20% w/w of sterols

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were strong enough to be subjected to penetration tests 24 h after preparation. These results

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dimensional network at a very short time period at ambient temperature. Obviously, 5%

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structurant concentration exhibited a time dependent gelation process, since it did not flow

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upon container’s inversion 21 days after preparation (Figure 2 c&d). Similar results were

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obtained in organogels produced with 30:70 weight γ-oryzanol:phytosterol ratio.

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Hardness and gel strength of the organogels increased statistically significantly with

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increasing total sterol content after 24 h storage at 25 oC (Figure 3 A).

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Effect of sterol mass ratio: As previous research has shown, the sterol mass ratio affects in a

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large extent the appearance and mechanical properties of organogels (Bot et al., 2008).

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Modifying the oryzanol and phytosterol amount in the system, results in drastic changes to

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the kinetics of the tubule self-assembly, as well as the final physical properties of the material

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(M. A. Rogers et al., 2010). Indeed, the 60:40 ratio resulted in transparent gels while in the

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case of 30:70 ratio more turbid gels were formed (results not shown). This implies that the

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gel structuring blocks at 60:40 ratio are considerably smaller than the wavelength of visible

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light (Pernetti et al., 2007). However, the turbidity at 30:70 gels can be explained by the

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crystallization of excess phytosterols to large-scale structures, much larger than the fiber

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structures of γ-oryzanol and phytosterol network (Bot et al., 2008). Moreover, according to

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macroscopic observation, the 30:70 gels had an oily-greasy surface and were much softer

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than those of 60:40 ratio at all total sterol contents tested. At high sterol levels, such as 20%

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w/w, the 30:70 ratio exhibited a waxy-like texture and brittleness, opposed to the elastic

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behaviour of 60:40 ratio. Indeed, the penetration test confirmed the increased (twofold)

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hardness and gel strength of 60:40 sterol ratio gels in comparison to 30:70 ones in both sterol

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contents examined (Figure 3 A). Assuming that both sterols are involved in the fibrillar

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network at 60:40 ratio which corresponds to 1:1 molar ratio, then in 30:70 gels, the

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30:70 ratio there is not enough γ-oryzanol to associate with all the available phytosterols and

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form the aforementioned fibrillar network. Thus, phytosterols in excess tend to crystallize. In

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this case, a weaker fibrillar network is formed resulting in lower mechanical responses of the

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30:70 organogels; i.e., the network formed by oryzanol and phytosterol seems to play the

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important role in the overall mechanical properties.

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Effect of ageing: Hardness increased statistically significantly (p < 0.05) with time for both

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total sterol concentrations and ratios (Figure 3 B). This implies that the gel network

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undergoes reorganization, rearrangement and restructuring resulting in firmer gel networks.

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This becomes more pronounced at longer times (see Figure 2 and total sterol content 5%

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w/w). Nevertheless, although the absolute values of gel strength increased with time, no

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statistically significant differences were detected (Figure 3 B).

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Effect of storage temperature: The effect of two temperatures, 4 οC and 25 οC for 48h

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storage, on hardness and gel strength was studied in 30:70 ratio organogels with 10% and

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20% w/w total sterol content. Samples showed no differences in the macroscopic

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examination. However, the penetration test (all the measurements were conducted at 25 oC

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for uniformity reasons) indicated that by reducing the storage temperature, the hardness of

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organogels increased statistically significantly for both samples (Figure 3 C). Undoubtedly, at

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low temperatures (4 οC) crystal growth occurred more extensively and rapidly and thus

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crystal-structured gels of 30:70 ratio became more resilient. Gel strength increased at 4 οC,

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and statistically significant differences were detected only for the 20% w/w sample. These

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results show that apart from its time dependence, gelation process is additionally temperature

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dependent due to the enhanced interactions between the organogel components at higher

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temperatures. Consequently, different sterol concentrations provide different levels of

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structural organization. An increase in gel stability at low temperatures (5 οC) has been also

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observed in rapeseed oil organogels with 12-hydroxy-stearic acid (M. A. Rogers, Wright, &

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Marangoni, 2008).

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3.2 Organogel-based emulsions

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3.2.1 Polarized microscopy

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Sterol-structured organogels emulsified in water in the presence of Tween 20 and xanthan

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gum led to organogel-in-water emulsions. The microstructure was studied under polarised

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light (Figure 4), thus the liquid oil phase, optically isotropic, appears dark, while crystals

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appear as bright white spots.

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Irrespective of sterol weight ratio, 10% w/w organogels (Figure 4 a & c) produced stable

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emulsions with small-sized oil droplets dispersed in the aqueous phase, where no crystal

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formation was detected 24 h after storage at 4 οC. However, in 20% w/w organogel-based

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emulsions of 30:70 ratio (Figure 4 b), numerous crystalline structures were observed. Crystals

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were larger in the aqueous phase and limited due to the droplet size in the oil phase. Some of

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the crystals (Fig. 4b) revealed in the micrographs – captured using polarised optical

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microscopy – were larger than the emulsion droplets with irregular crystal morphology and

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located in the water phase. In addition, flocculated and crystallised oil droplets that exhibit

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birefringence were displayed in the micrograph. The presence of a non-adsorbing biopolymer

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like xanthan leads to the formation of a transient but potentially long-lasting network from

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emulsion droplets due to attractive depletion flocculation interactions (Thomas Moschakis,

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2013; T. Moschakis, Murray, & Dickinson, 2006). This flocculation may have resulted in

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partially crystalline oil droplets (partial coalescence) with marked increase in viscosity (see

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section 3.2.2). Further experiments are needed to clarify this point.

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(Figure 4 d). At 30:70 ratio (Figure 4 a & b), only at high sterol content used, i.e. 20% w/w

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(Figure 4 b), the crystal formation was extensive. Thus, the γ-oryzanol:phytosterols weight

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ratio seems to also play an important role in crystal formation in the produced emulsions.

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Moreover, it has been found that structurants (γ-oryzanol and phytosterols), in the presence of

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water, crystallise into forms similar to those that each sterol would individually form alone as

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it has also been reported in other studies (Bot et al., 2011; den Adel et al., 2010; Duffy et al.,

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2009). Basically, the water appears to inhibit or destabilize the tubular structures of sterols

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that occur in organogels. The absence of crystalline features in 60:40 weight ratio is probably

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due to the 1:1 molar ratio of sterols.

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In general, the crystallization of a structurant spreads rapidly in the continuous phase once

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initiated (Bot & Agterof, 2006; Bot, Veldhuizen, et al., 2009). However, in the case of an oil-

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in-water emulsion, the oil phase is dispersed into the aqueous phase and therefore

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crystallization or extensive crystal structure formation takes place more slowly since it has to

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be initiated independently in each oil droplet (Bot & Agterof, 2006; Bot, Veldhuizen, et al.,

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2009). Of course, the absence of large crystals at the time of the microscopic observation of

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the emulsions, i.e., 24 h after preparation, does not preclude their creation in the future.

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However, evolution of the emulsion microstructure over time was not further investigated in

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the present study.

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Lecithin has been reported as a phytosterol-crystallization inhibitor (Engel & Schubert,

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2005). During preliminary experiments, sunflower lecithin was added before emulsification

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to 30:70 organogel (lipid) phase of the emulsion along with sterols prior to heating, with the

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aim to inhibit crystallization of plant sterols in excess. Surprisingly, in such an organogel-

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based emulsion, lecithin not only did not inhibit the crystallization of phytosterols in excess

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in the system, but on the contrary, numerous crystallization nuclei formed even in 10% w/w

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According to Han et al. (2014) the addition of lecithin modifies the crystal morphology of

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phytosterols. The addition of lecithin in the organogel phase probably interferes in the

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crystallisation of phytosterols and led to more unstable emulsions and larger crystal structures

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formation. Therefore, the morphology of phytosterol crystals seems to be related with the

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stability of the organogel-based emulsions.

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3.2.2 Rheological measurements

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Amplitude sweep. The shear-stress amplitude sweeps for organogel-based emulsions are

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shown in Figure 6A. It is observed that the 30:70 samples exhibited longer linear viscoelastic

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region (LVR) for both total sterol contents (10% and 20%). That is, the storage modulus

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remained relatively constant over a broader range of shear stress applied in the emulsions

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produced with 30:70 ratio. In addition, the 30:70 emulsion samples exhibited much higher G'

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values at the LVR than the respective 60:40 samples.

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The shear stress where a noticeable deviation from the linear viscoelastic region during the

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strain sweep test occurs could be a rough estimate of the sample’s yield stress (Figure 6B).

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Deviation from the LVR occurs when the sample starts to permanently deform, implying the

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destruction of the structure. In this study, the tolerated deviation was defined as 10%.

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Although the existence of yield stress has been questioned in the literature (Barnes, 1999), it

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is a good indication of a gel network strength.

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As can be seen from Figure 6, the 20%-30:70 emulsion gel exhibited the highest level of

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internal structure against all other samples tested. Consequently, in contrast to organogels, the

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crystallization of the excess phytosterols resulted in an overall stronger structure. This

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reinforcement was not observed in organogels (without emulsion droplets) where the

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equimolar sterol ratio plays the key role in mechanical properties. In emulsions, the increase

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phytosterol) did not augment substantially the overall viscosity, whereas an enhancement of

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the mechanical properties in the continuous phase and/or in the interconnected aggregated

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crystallised (or partially crystallised) oil droplets phase, predominantly determines the

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rheological behaviour of the overall bulk mechanical properties.

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Frequency sweep. Frequency sweep tests show that the elastic behaviour dominates in all the

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organogel-in-water emulsions (Figure 7) since the G' was always higher than G'' over the

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entire frequency range explored. However, a weak frequency dependence of the phase angle

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was observed which is more pronounced for the samples containing 20% structurants

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indicating a weak gel behaviour.

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Figure 8 shows the G' and tanδ at 1 Hz of the emulsion gels. A large increase in G' was

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obtained with increasing total sterol content while higher values were exhibited at 30:70

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emulsions. The sterol ratio seems to affect the rheological properties of the organogel-in-

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water emulsions. In addition, all emulsions had a dominant elastic character (tanδ < 1)

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(Thomas Moschakis, 2013), and no statistical differences were found; i.e., the emulsions

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exhibited the same elastic character independently of the sterol content and ratio (Figure 8).

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Large deformation steady-state viscometry. During large deformation steady-state

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viscometry (Figure 9), the curves show pronounced shear-thinning behaviour, irrespectively

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to sterol ratio and sterol content, and increasingly high viscosities at low-shear rates for

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increasing sterol content and percentage of phytosterols in the sterol ratio (γ-

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oryzanol:phytosterols ratio from 60:40 to 30:70) (Figure 9). This behaviour is typical of weak

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associative interactions and suggests the formation of a weak droplet network structure.

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Addition of xanthan in stable emulsions causes depletion flocculation, network formation,

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Moschakis, 2013; T. Moschakis, Murray, & Dickinson, 2005; T. Moschakis et al., 2006). In

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such systems, the rheological behaviour is dependent on the viscoelastic properties of the

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interconnected depletion-flocculated network and not on the rheology of the aqueous

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continuous phase (T. Moschakis et al., 2006).

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At high oil volume fractions (e.g. 50%) the emulsion droplets begin to interact with each

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other due to hydrodynamic interactions, apart from the colloidal interactions. As a result, the

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viscosity of the system is modified. The hydrodynamic interactions are the result of the

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relative motion of neighbouring particles (McClements, 2005), and are important in all types

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of concentrated emulsions (say oil volume fraction > 0.4) (T. Moschakis et al., 2006). In

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such systems, shear thinning occurs when the shear stress is large enough to overcome the

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rotational but mainly translational Brownian motion of the emulsion droplets (McClements,

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2005). As the shear stress increases, the hydrodynamic forces dominate, and thus

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rearrangement and reordering of the emulsion droplets along the shearing occurs which cause

386

a decrease in the overall viscosity (shear- thinning behaviour). At low oil volume fractions

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(20% wt % oil), enhanced shear thinning behaviour was exhibited only on systems that

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extensive crystallization of phytosterols in the continuous phase was observed (unpublished

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data).

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When the phytosterols were added at higher proportions than γ-oryzanol (ratio 30:70),

391

crystals of phytosterols were formed, especially at high sterol content and upon storage (see

392

Figure 4), which enhances the overall viscosity. The low shear rate viscosity measured in

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10%-30:70 emulsion was found to be ~90 Pa·s while for the 10%-60:40 emulsion was ~70

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Pa·s illustrating the higher structural organization level in the first sample. At 20% total sterol

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content, the low shear rate viscosity was an order of magnitude higher indicating that the

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enhancement in the organogel viscoelastic properties resulted in an substantial increase of the

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overall bulk viscosity.

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4. Conclusions

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Sterol ratio, total sterol content, storage duration and temperature was found to define the

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properties of the oil structures, both in organogels and organogels-in-water emulsions. In

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30:70 sterol ratio, phytosterols in excess crystallize and therefore enhance the stability of the

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system while the 60:40 ratio favours fibril formation. However, fibril formation resulted in

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stronger gels indicating a remarkable fibril structuring effect on pure oil phase. On the

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contrary, the 30:70 ratio, having excess amount of phytosterols, produced stronger emulsion

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gels compared to the 60:40 ratio due to the interconnected aggregated crystallised (or

407

possibly partially crystallised) oil droplets and/or the enhancement of the mechanical

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properties in the aqueous continuous phase containing phytosteol crystals. An increase in

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total sterol content reinforced and amplified the structuring potential of both sterol ratios in

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organogels and emulsions.

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Vegetable oils structured with γ-oryzanol and phytosterols, undoubtedly, constitute a

412

noteworthy alternative to trans- or saturated (animal) fat. Vegetable oil gels could be utilized

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in foods containing relatively high amounts of solid fat. Organogel-based emulsions, on the

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other hand, are more appropriate for foods with emulsion composition such as margarine,

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yoghurt, spread and processed cheese, mayonnaise and dressings.

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5. References

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Barnes, H. A. (1999). The yield stress - a review or 'pi alpha nu tau alpha rho epsilon iota' - everything flows? Journal of Non-Newtonian Fluid Mechanics, 81(1-2), 133-178. doi: 10.1016/s03770257(98)00094-9 Bot, A., & Agterof, W. G. M. (2006). Structuring of edible oils by mixtures of gamma-oryzanol with beta-sitosterol or related phytosterols. Journal of the American Oil Chemists Society, 83(6), 513-521. doi: 10.1007/s11746-006-1234-7 Bot, A., den Adel, R., Regkos, C., Sawalha, H., Venema, P., & Floter, E. (2011). Structuring in betasitosterol plus gamma-oryzanol-based emulsion gels during various stages of a temperature cycle. Food Hydrocolloids, 25(4), 639-646. doi: 10.1016/j.foodhyd.2010.07.026 Bot, A., den Adel, R., & Roijers, E. C. (2008). Fibrils of gamma-oryzanol plus beta-sitosterol in edible oil organogels. Journal of the American Oil Chemists Society, 85(12), 1127-1134. doi: 10.1007/s11746-008-1298-7 Bot, A., den Adel, R., Roijers, E. C., & Regkos, C. (2009). Effect of sterol type on structure of tubules in sterol plus gamma-oryzanol-based organogels. Food Biophysics, 4(4), 266-272. doi: 10.1007/s11483-009-9124-9 Bot, A., Veldhuizen, Y. S. J., den Adel, R., & Roijers, E. C. (2009). Non-TAG structuring of edible oils and emulsions. Food Hydrocolloids, 23(4), 1184-1189. doi: 10.1016/j.foodhyd.2008.06.009 Brufau, G., Canela, M. A., & Rafecas, M. (2008). Phytosterols: physiologic and metabolic aspects related to cholesterol-lowering properties. Nutrition Research, 28(4), 217-225. doi: 10.1016/j.nutres.2008.02.003 Calligaris, S., Mirolo, G., Da Pieve, S., Arrighetti, G., & Nicoli, M. C. (2014). Effect of Oil Type on Formation, Structure and Thermal Properties of gamma-oryzanol and beta-sitosterol-Based Organogels. Food Biophysics, 9(1), 69-75. doi: 10.1007/s11483-013-9318-z Cercaci, L., Rodriguez-Estrada, M. T., Lercker, G., & Decker, E. A. (2007). Phytosterol oxidation in oilin-water emulsions and bulk oil. Food Chemistry, 102(1), 161-167. doi: 10.1016/j.foodchem.2006.05.010 den Adel, R., Heussen, P. C. M., & Bot, A. (2010). Effect of water on self-assembled tubules in betasitosterol plus gamma-oryzanol-based organogels. Xiv International Conference on SmallAngle Scattering (Sas09), 247. doi: 10.1088/1742-6596/247/1/012025 Duffy, N., Blonk, H. C. G., Beindorff, C. M., Cazade, M., Bot, A., & Duchateau, G. S. M. J. E. (2009). Organogel-based emulsion systems, micro-structural features and impact on in vitro digestion. Journal of the American Oil Chemists Society, 86(8), 733-741. doi: 10.1007/s11746009-1405-4 Engel, R., & Schubert, H. (2005). Formulation of phytosterols in emulsions for increased dose response in functional foods. Innovative Food Science & Emerging Technologies, 6(2), 233237. doi: 10.1016/j.ifset.2005.01.004 Han, L., Li, L., Li, B., Zhao, L., Liu, G.-q., Liu, X., & Wang, X. (2014). Structure and Physical Properties of Organogels Developed by Sitosterol and Lecithin with Sunflower Oil. Journal of the American Oil Chemists Society, 91(10), 1783-1792. doi: 10.1007/s11746-014-2526-y McClements, D. J. (2005). Food Emulsions: Principles, Practice, and Techniques (2nd ed.). Florida: CRC Press. Moreau, R. A., Whitaker, B. D., & Hicks, K. B. (2002). Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses. Progress in Lipid Research, 41(6), 457-500. doi: 10.1016/s0163-7827(02)00006-1 Moschakis, T. (2013). Microrheology and particle tracking in food gels and emulsions. Current Opinion in Colloid & Interface Science, 18(4), 311-323. doi: 10.1016/j.cocis.2013.04.011 Moschakis, T., Murray, B. S., & Dickinson, E. (2005). Microstructural evolution of viscoelastic emulsions stabilised by sodium caseinate and xanthan gum. Journal Of Colloid And Interface Science, 284(2), 714-728.

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Moschakis, T., Murray, B. S., & Dickinson, E. (2006). Particle tracking using confocal microscopy to probe the microrheology in a phase-separating emulsion containing nonadsorbing polysaccharide. Langmuir, 22(10), 4710-4719. doi: 10.1021/la0533258 Nukit, N., Setwipattanachai, P., Chaiseri, S., & Hongsprabhas, P. (2014). Effects of Surfactants and Aging Time on Solidification of Rice Bran Oil at Room Temperature. Journal of Oleo Science, 63(11), 1099-1107. doi: 10.5650/jos.ess14099 Patel, M., & Naik, S. N. (2004). Gamma-oryzanol from rice bran oil - A review. Journal of Scientific & Industrial Research, 63(7), 569-578. Pernetti, M., van Malssen, K. F., Floter, E., & Bot, A. (2007). Structuring of edible oils by alternatives to crystalline fat. Current Opinion in Colloid & Interface Science, 12(4-5), 221-231. doi: 10.1016/j.cocis.2007.07.002 Rogers, E. J., Rice, S. M., Nicolosi, R. J., Carpenter, D. R., McClelland, C. A., & Romanczyk, L. J. (1993). Identification and quantitation of gamma-oryzanol components and simultaneous assessment of tocols in rice bran oil. Journal of the American Oil Chemists Society, 70(3), 301-307. doi: 10.1007/bf02545312 Rogers, M. A. (2009). Novel structuring strategies for unsaturated fats - Meeting the zero-trans, zerosaturated fat challenge: A review. Food Research International, 42(7), 747-753. doi: 10.1016/j.foodres.2009.02.024 Rogers, M. A., Bot, A., Lam, R. S. H., Pedersen, T., & May, T. (2010). Multicomponent hollow tubules formed using phytosterol and gamma-oryzanol-based compounds: an understanding of their molecular embrace. Journal of Physical Chemistry A, 114(32), 8278-8285. doi: 10.1021/jp104101k Rogers, M. A., Wright, A. J., & Marangoni, A. G. (2008). Crystalline stability of self-assembled fibrillar networks of 12-hydroxystearic acid in edible oils. Food Research International, 41(10), 10261034. doi: 10.1016/j.foodres.2008.07.012 Romer, S., & Garti, N. (2006). The activity and absorption relationship of cholesterol and phytosterols. Colloids and Surfaces a-Physicochemical and Engineering Aspects, 282, 435456. doi: 10.1016/j.colsurfa.2005.12.032 Sanclemente, T., Marques-Lopes, I., Fajo-Pascual, M., Cofan, M., Jarauta, E., Ros, E., . . . Garcia-Otin, A. L. (2012). Naturally-occurring phytosterols in the usual diet influence cholesterol metabolism in healthy subjects. Nutrition Metabolism and Cardiovascular Diseases, 22(10), 849-855. doi: 10.1016/j.numecd.2011.01.010 Sawalha, H., den Adel, R., Venema, P., Bot, A., Floter, E., & van der Linden, E. (2012). Organogelemulsions with mixtures of beta-sitosterol and gamma-oryzanol: influence of water activity and type of oil phase on gelling capability. Journal of Agricultural and Food Chemistry, 60(13), 3462-3470. doi: 10.1021/jf300313f Sawalha, H., Venema, P., Bot, A., Floter, E., den Adel, R., & van der Linden, E. (2015). The Phase Behavior of gamma-Oryzanol and beta-Sitosterol in Edible Oil. Journal of the American Oil Chemists Society, 92(11-12), 1651-1659. doi: 10.1007/s11746-015-2731-3 Sawalha, H., Venema, P., Bot, A., Floter, E., & van der Linden, E. (2011). The influence of concentration and temperature on the formation of gamma-oryzanol + i-2-sitosterol tubules in edible oil organogels. Food Biophysics, 6(1), 20-25. doi: 10.1007/s11483-010-9169-9 Vaikousi, H., Lazaridou, A., Biliaderis, C. G., & Zawistowski, J. (2007). Phase transitions, solubility, and crystallization kinetics of phytosterols and phytosterol-oil blends. Journal of Agricultural and Food Chemistry, 55(5), 1790-1798. doi: 10.1021/jf0624289 Vancamp, J., & Huyghebaert, A. (1995). High pressure-induced gel formation of a whey-protein and hemoglobin protein-concentrate. Food Science and Technology-Lebensmittel-Wissenschaft & Technologie, 28(1), 111-117. Verbeken, D., Bael, K., Thas, O., & Dewettinck, K. (2006). Interactions between kappa-carrageenan, milk proteins and modified starch in sterilized dairy desserts. International Dairy Journal, 16(5), 482-488. doi: 10.1016/j.idairyj.2005.06.006

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Figure 1. Polarized micrographs of organogels, after storage at ambient temperature for 24 h; (a) 10% w/w sterol content 30:70 weight γ-oryzanol:phytosterol ratio, (b) 20% w/w - 30:70 γ-oryzanol:phytosterol, (c) 10% w/w - 60:40 γ-oryzanol:phytosterol and (d) 20%

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Figure 2. Vegetable oil organogels of 60:40 weight γ-oryzanol:phytosterol ratio in various total concentrations at ambient temperature; after 10 days storage (a) olive oil and

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Figure 3. Mechanical properties, hardness (N) and gel strength (mJ), of sunflower organogels with different total sterol contents (10% and 20% w/w). A) Effect of oryzanol-phytosterol weight ratio, 30:70 and 60:40, after 24 h storage at 25 °C.

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C) Effect of temperature after 48h storage at 4 οC and 25 οC for the 30:70

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Figure 4. Polarized micrographs of emulsions, after storage at 4 οC for 24 h. The emulsions contain 50% w/w aqueous phase and 50% w/w organogel; (a) 10% w/w sterol content 30:70 weight ratio, (b) 20% w/w - 30:70, (c) 10% w/w - 60:40 and (d) 20% w/w -

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Figure 5. Polarized micrographs of 30:70 organogel-based emulsions, after storage at 4 ο

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Figure 6. Α) Strain sweep tests of organogel-based emulsions, after storage at 4 οC for 24 h. The emulsions contain 50% w/w aqueous phase and 50% w/w organogel

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Β) Yield stress τy (Pa) of organogel-based emulsions after storage at 4 οC for 24 h.

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Figure 8. Variation of G' and tanδ at 1 Hz among different organogel composition (30:70 and 60:40 γ-oryzanol:phytosterol weight ratio, 10% and 20% w/w total sterol content) of emulsion gels, after storage at 4 οC for 24 h.

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