Natural oil emulsions stabilized by β-glucan gel

Accepted Manuscript Title: Natural oil emulsions stabilized by ␤-glucan gel Authors: Miroslav Veverka, Tibor Dubaj, Eva Veverkov´a, ˇ Peter Simon PII: DOI: Reference:

S0927-7757(17)30942-1 https://doi.org/10.1016/j.colsurfa.2017.10.043 COLSUA 22001

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

21-7-2017 18-10-2017 19-10-2017

Please cite this article as: Miroslav Veverka, Tibor Dubaj, Eva ˇ Veverkov´a, Peter Simon, Natural oil emulsions stabilized by ␤glucan gel, Colloids and Surfaces A: Physicochemical and Engineering Aspects https://doi.org/10.1016/j.colsurfa.2017.10.043 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.

Natural oil emulsions stabilized by β-glucan gel Miroslav Veverkaa, Tibor Dubajb,*, Eva Veverkovác, and Peter Šimonb a

Eurofins BEL/NOVAMANN Ltd., 940 02 Nové Zámky, Slovakia, e-mail: [email protected]

b

Department of Physical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia, tel.: +421-2-59325530, fax: +421-252493198, e-mail: [email protected]

c

Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia.

Graphical abstract

ABSTRACT Many foods and cosmetic products are applied in gel form that offers convenience to the consumers. In this work, we demonstrate a novel use of β-glucan (BG) isolated from Pleurotus ostreatus as a functional ingredient in food, cosmetics, and as a food additive. Stable emulsion gels were prepared by homogenization of BG hydrogel containing from 0.10 to 2.0 wt. % BG with natural oil without a cosolvent or surfactant addition. The oils, chosen as representative commercial beneficial fats, were conjugated linoleic acid (CLA), coconut oil, olive oil, and cocoa butter. Formulations containing from 10 to 50 wt. % of oil in BG gel exhibited stability for more than 12 months. Moreover, stable Pickering-type emulsions were prepared by adding micronized BG particles to 0.05 % BG hydrogel. The emulsion gels were characterized visually, SEM, cryo-electron microscopy, and viscosity measurements. The oil droplets are trapped and held in place by viscous BG hydrogel network structure created through interacting chain segment association and aggregated junction zones. Presence of crystalline cocoa butter in emulsion gels was confirmed by DSC and XRPD. The 1

study of the water mobility within the xerogels using the dynamic vapor sorption technique showed that cocoa butter loading of 69 % resulted in a reduction of equilibrium moisture content and hysteresis. The emulsion gels prepared show promising functional stability for emulsion-type cosmetics and as suitable carriers for dermal application. KEYWORDS: emulsion; microstructure; viscosity; xerogel; dietary fat

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1. INTRODUCTION Emulsion stability is an important factor for ensuring its quality and practical application. Addition of hydrocolloids to aqueous phase stabilizes emulsions via increased viscosity of the aqueous phase or by forming a film around the oil droplets (Leal-Calderon et al., 2007). In addition to hydrocolloids, solid particles are also efficient interface stabilizers (Leal-Calderon & Schmitt, 2008). A monolayer of particles (micro- or nanoparticles, granules) forms a rigid shell around the droplets and prevents the droplets from touching one another, thus hindering coagulation. Owing to their pH and thermoresponsive properties, soft particles like microgels are suitable emulsifiers of this type (Dickinson, 2010; Dickinson, 2012; Koenig et al., 2002; Brugger et al., 2008; Destribats et al., 2011; Ngai et al., 2006). In microgel-stabilized emulsions, the microgel particles are adsorbed at the oil–water interface. Simultaneously, the emulsion stability is enhanced when excess microgel particles form gel in the continuous phase which entraps the oil droplets (Li et al., 2009). Emulsion gels for controlled release applications consisting from oil-in-water emulsion stabilized by a highly viscous or gelled polymer matrix were proposed by Dickinson (2012), Sagiri et al. (2014) and Chen et al. (2007). In the case of food emulsion systems, polysaccharides from nanoscale to microscopic dimensions, such as cellulose (Dickinson, 1995; Wege et al., 2008) and starch (Timgren et al., 2013; Li et al., 2013) are used to improve the emulsion stability and textural properties. Incorporation of β-glucan (BG) into various products (baking products, pasta, noodles, muesli cereals, dairy products, soups, salad dressings, beverages, and meat products) showed that the important attributes, such as breadmaking performance, water binding and emulsion stabilizing capacity, thickening ability, texture and appearance, are related to the concentration, molecular weight, and structure of the polysaccharide (Havrlentová et al., 2011; Ramprasath et al., 2015; Zhu et al., 2015; Bird et al., 2008; Cleary et al., 2007; Omana et al., 2012). BG possesses several beneficial properties, for example, the ability to eliminate free radicals (Kofuji et al., 2012; Ahmad et al., 2014). There are glucan-enriched cereal products with 20–90 % glucan content in the form of highly viscous solutions or gels (Stevenson & Inglett, 2009) and formation of BG gels upon cooling was observed long time ago (Morgan & Ofman, 1998; Vaikousi et al., 2004). It was also found that the gel-forming ability of BG depends on its molecular weight (Lazaridou et al., 2003; Lazaridou & Biliaderis, 2007). Preferentially, cereal BGs are applied as hydrocolloids with modified rheological characteristics, i.e. their gelling capacity and ability to increase the viscosity of aqueous solutions (Burkus & Temelli, 1999, 2000). Kontogiorgos et al. (2004) and Santipanichwong & Suphantharika (2009) studied the influence of different BGs, i.e. curdlan, barley, oat, and yeast on the physical and rheological properties of oil-in-water emulsions. BG complexes with nutraceuticals were prepared by Veverka et al. (2014, 2016) and by Xiong et al. (2006); stabilizing effect on CLA was also observed (Veverka et al., 2017) Properties of polysaccharide-based dried porous gels (xerogels) indicate their great potential in the food and cosmetics, e.g., their high water-absorption capacity, safety, edibility, renewability, 3

sustainability, and low cost of polysaccharides (Chen et al., 1999; Coviello et al., 2005). BG aerogels are also hypothesized as delivery vehicles for nutraceuticals (Comin et al., 2012). Stabilization of emulsions using Pleorotus ostreatus BG at lower concentrations in the presence or without any stabilizer has not been reported yet. In this work, several model emulsion gel systems resembling real food or cosmetics oils were prepared with CLA, coconut oil, olive oil, and cocoa butter. The physicochemical nature of cocoa butter and coconut oil is different from that of CLA and olive oil as both cocoa butter and coconut oil are solids at room temperature and they melt at body temperature. These dissimilar types of oils were tested as they represent different breakdown and viscoelastic behavior. Further objective of this study was to evaluate microstructure, creaming stability, emulsion viscosity and xerogel formation of the oil emulsions stabilized by Pleorotus ostreatus BG gel. A model BG xerogel with cocoa butter was prepared and characterized; its isothermal swelling kinetics in water was investigated. 2. MATERIALS AND METHODS 2.1 Raw Materials and Emulsion Preparations BG as micronized particles with a mean diameter of 4.5 µm (from Pleorotus ostreatus, purity 93 %, Mw = 450 kDa) and BG hydrogel (2.0 wt. % BG) were both obtained from Natures Ltd. (Trnava, Slovakia). Conjugated linoleic acid (CLA; HBL 7, 82 % of the total CLA isomers from safflower oil) was purchased from BASF (Monheim, Germany), olive oil (HBL 7), cocoa butter (theobroma oil, HBL 6) and coconut oil (HBL 8) were purchased from a local supermarket and used without further purification. In all experiments, double distilled water was used; other chemicals of analytical grade were purchased locally and were used as received. Emulsion gels were initially prepared by a direct cold gelation of BG oil-in-water emulsions. A suspension of micronized BG was homogenized at 95 °C with an oil-in-water emulsion and yielded soft gels after cooling. However, for these systems, instability against creaming and low viscosity was observed; the failure of this approach indicates that the order of gelation/emulsification steps is important. Therefore, the emulsion gels were prepared from the stock BG gel to which an adequate amount of the oil phase was added slowly in two parts. After the first oil addition, the emulsion was stirred using homogenizer (Ultra Turrax T18, IKA, Germany) for 1 min at 4000 rpm; the second part of the oil was added and stirred at 1600 rpm for additional 4 min. During homogenization, the viscous mixture was immersed in a water bath to avoid excessive heating of the sample. The final product was stored in a 50-mL multi-dose container with airless pump and snap-on cap. For the control oil-in-water CLA emulsion, the emulsifying agents Tween 80 and Span 80 were used at the required HLB value of 7. To prevent microbial growth for stability testing samples, sodium azide (0.02 %, w/v) was added. Immediately after homogenization, the emulsion type was determined by a conductivity measurement and the drop test. In the latter, a drop of emulsion is added to a sample of water or oil. If it disperses in one of them, then that phase is the continuous phase of the emulsion. The morphology of emulsion 4

droplets was observed with an optical microscope. The emulsion viscosity was characterized by an Anton Parr viscometer; all measurements were performed within 2 h after emulsion gel preparation. The viscosity, pH, droplet size, and conductivity of emulsion gel are reported in Table 1. 2.2 Creaming index (CI) CI was determined according to Perrechil & Cunha (2010) as the ratio between the height of the lower layer and the initial height of the emulsion. A freshly prepared sample was transferred to a glass test tube (internal diameter 22 mm, height 65 mm, 0.05-ml graduations) sealed with a plastic cap and stored at 25 °C in dark. Subsequently, liquefaction, phase separation, and color characteristics of the emulsion were observed for 6 months. 2.3 Viscosity measurements Viscosity was measured using rotational viscometer (Anton Paar DV-1P, Berlin, Germany) equipped with tempering bath. The measurements were carried out at 25.0 ± 0.5 °C; the apparent viscosity was expressed in Pa∙s. 2.4 Optical micrographs Microstructure of the samples was observed using a polarizing microscope (Nikon Eclipse LV 100POL, Tokyo, Japan) and analyzed using NIS-Elements software. For the particle size and particle size distribution determination, a thin layer of the sample was spread on the microscope slide and covered with a cover clip. The diameters of approx. 100 drops were measured per sample using a magnification of 100; the average diameter was expressed as a simple arithmetic mean. 2.5 Scanning electron (SEM) and cryo-electron microscopy (cryo-EM) Structure of sample surface and structure of a cut through the sample was observed by scanning electron microscope JEOL 7500 F (JEOL, Japan) with field emission gun. For specimen preparation, aluminum target and carbon both-sided adhesive tapes were used. The samples were mounted using tape onto target (diameter of 2.5 cm) which is also used to transfer the specimen into the microscope. Before the sample was transferred, coating with gold/platinum plasma for 60 s using BALZERS coater was done. Coated samples were transferred into the SEM chamber and observed using SEI detector (secondary electron image) at the acceleration voltage of 10 kV; the working distance was 5–10 mm. For the cryo-EM, the same scanning electron microscope equipped with cryo-mode system (Quorum Technologies Ltd., UK) was pumped to high vacuum. Once the system reached high vacuum, it was cooled down to set temperatures. Set temperature of SEM and preparation chamber anticontaminator was −175 °C and temperature of SEM and preparation chamber stage was −140 °C; rivets were used for gel/oil specimen preparation. A drop of the sample was placed onto the top of a 3mm copper rivet fixed on a sample holder and inserted into liquid nitrogen. The sample was then transferred under vacuum to the preparation chamber using cryo-transfer device. Then, the sample was mounted on the precooled stage where the frozen sample was fractured by means of a front-mounted

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scalpel-bladed probe that was actively cooled by a copper braid connected to the cold shield in the preparation chamber. After sample fracturing, sublimation was done. The sublimation process removed fine vitrified water slivers generated during the fracture. The sublimation temperature was −90 °C; the sublimation time was 10 min. The samples were coated with platinum/palladium plasma for 60 s. Coated samples were transferred into the SEM chamber and they were mounted on the SEM cold stage. The prepared specimens were observed using SEI detector (secondary electron image) at acceleration voltage of 5 kV; working distance was 10 mm. 2.6 pH measurement A digital pH meter (Mettler Toledo, Columbus, OH, USA) was used to measure the pH values at 25.0 ± 0.5 °C of emulsion samples immediately after preparation. 2.7 Conductivity measurement An oil-BG emulsion gel was prepared and tempered at 25 ± 0.5 °C. The conductivity of emulsions was measured in situ by a conductometer Mettler Toledo, S230 (Switzerland) and electrode 731 ISM, directly in a reaction glass beaker. 2.8 Dynamic vapor sorption (DVS) Dynamic water sorption isotherms were obtained using automatic sorption analyzer Aquadyne DVS-2 (Quantachrome Ltd., UK). In all measurements, a sample of 20–60 mg was used, one cycle consisted of an initial drying phase, controlled temperature 25 °C, nitrogen flow 200 mL min−1, two complete cycles 0–95 % RH and backwards in 11 steps. First, each sample was equilibrated to 0 % RH in the DVS apparatus for 5 h. Moisture sorption isotherms were determined from the equilibrium moisture contents at each RH step. Equilibrium was considered reached when the weight change per minute was less than 0.002 %. 2.9 Gel drying A sample of 13.5 g of CB:BG (2.0 wt. %) 1:9 gel was transferred onto a plastic cup and left in open area at ambient temperature (28–30 °C, 40–45 % RH) until the equilibrium mass was reached (two weeks); solid xerogel was obtained as an irregular spheroid. Further drying was done via alcogel: samples of 13.5 g of CB:BG (2.0 wt. %) 1:9 gel were placed into 50-mL baths containing increasing ethanol concentration (20, 40, 60, 80, and 99.8 vol. %). Conversion of hydrogels to alcogels took place at 5 °C with a residence time of 2 h for each bath. After the remaining ethanol was carefully decanted, the resulting alcogel was dried at ambient conditions. In a second step, two different drying processes of alcogel to xerogel were used. In the first one, approx. 0.5 g of alcogel was carefully transferred into a 300-mL freeze-drying flask (ilShin Biobase, Korea) and frozen in an ultra-low-temperature freezer at −25 °C overnight. Subsequently, the flask was connected to a freeze-dryer (Coolsafe 55-4, Denmark) and freeze-dried at −55 °C under vacuum for 12 h to sublimate the solvent. The final desorption temperature was 20–22 °C after 24

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hours. The second method consisted from drying in a vacuum oven at 30–32 °C and 1.3 kPa. The same procedures were also applied to neat BG gel. Moisture content in the xerogels was determined by Karl Fischer titration; the experiments were done in duplicate. 2.10 CB recovery from xerogel A sample of xerogel (approx. 0.1 g) was crushed and gently stirred with 20 ml of petrol ether for 20 min and sonicated for 5 min. The resulting mixture was centrifuged and the supernatant liquid containing CB was collected. The centrifugation procedure was repeated 3 times; all extracts were combined. The extracts were filtered and concentrated under vacuum, the CB residue was weighted. The conversion of the hydrogels and recovery of oil in the terms of weight percentage is summarized in Table 2. The CB percentage was also determined as the xerogel melting enthalpy divided by that of the neat CB. 2.11 Swelling experiments Swelling characteristics of xerogels were carried out using a simple gravimetric procedure (Dragan & Apopei, 2011). The neat BG and CB:BG xerogels (0.1 g) were swollen to equilibrium at regular time intervals. Samples dried in open area were used. Since the xerogel was fragile, it was placed in a preweighed sintered glass a mesh size of 1 mm, carefully wiped off the excess water with a filter paper and the sample was weighed. This handling allowed placement of the material in water and weighing of the sample without any significant disturbance. Each xerogel with initial mass m0 was immersed in distilled water for a specified time at 25 °C; the hydrogel was then separated and dried as before and weighed at regular time intervals to obtain m(t). The swelling ratio (SR) was calculated as SR(t ) 

m(t )  m0 . m0

(1)

The percentage of swelling was expressed as %SR(t ) 

S (t ) 100%, Seq

(2)

where Seq is the equilibrium swelling ratio. The measurements were performed in duplicate. 3. RESULTS AND DISCUSSION 3.1 Model CLA:BG gel emulsions and general remarks Preliminary tests were carried out with CLA as a model oil to determine feasibility of emulsion stabilization using BG hydrogel. If not stated otherwise, the BG concentration in the hydrogel was 2.0 wt. %. CLA emulsification was performed in a homogenizer; the mean CLA droplet size ranged from 1.0 to 2.9 µm; appearance of milky-white color without any phase separation was an indication of successful emulsification. The formulations which failed to flow under gravity were considered emulsion gels. Prepared emulsion gels showed sufficiently small droplet size with narrow monomodal distribution without any flocculation or droplet aggregation (see the optical micrographs in Figure 1). 7

Microstructure analysis was carried out to determine the distribution of CLA droplets in the gel matrix. Table 1 shows a negligible effect of CLA concentration on the droplet size. The results show that 2.0 wt. % BG gel can readily accommodate 50 wt. % CLA while homogeneous distribution of droplets is still maintained. However, CLA droplet aggregation was observed under microscope by changing the temperature from 25 °C to 46–48 °C. This implicates that the decreasing temperature increases viscosity and prevents droplet aggregation and phase separation. Therefore, during the homogenization the samples were immersed in a water bath to avoid excessive heating. Optical microscopy images of all emulsion gels showed regular spatial distribution of individual droplets; however, the method does not allow observation of gel network structure. Hence, there is lack of the direct observation of the microstructure of emulsions combined with gels (Chen et al., 2007). Evaluation of cryo-EM images (Figure 2) provides some insight into the structural organization of the oil–gel network. In general, the droplets are spherical as expected for a typical emulsion; the only exception are cocoa butter emulsions. Considering the BG hydrogel as a continuous network with dispersed oil phase, some droplets are entrapped in the free volume of the gel matrix. Thus, the emulsions are partially stabilized via immobilization of the oil droplets in the gel matrix as suggested by Gao et al. (2009) and Liang et al. (2011). Among this steric effect caused by increased viscosity, electrostatic hindrance can also contribute to the emulsion stability as it presents a significant barrier for coalescence. This effect was also hypothesized in case of chitosan–paraffin emulsions (Payet & Terentjev, 2008). Prolongation of the homogenization time above 5 min did not significantly affect the mean diameter of oil droplets. Similarly, the mean droplet size remains practically constant with respect to concentration of BG in gel matrix and no significant difference was observed between the droplet size of the control CLA–water–Tween 80/Span 80 emulsion (mean droplet size 1.26 µm) and other emulsions. However, the mean droplet size decreased under 1 µm in emulsions containing phospholipids as emulsifier. Emulsion of CLA in BG gel (2.0 wt. %) 1:1 exhibited phase separation just after two days when stored at 28 °C. Addition of emulsifiers (Tween80/Span80) resulted in slightly more stable emulsions with phase separation observed after five days. However, these formulations also have lower viscosity and exhibit coalescence. Quite surprisingly, CLA:BG gel (0.50 wt. %) 1:1 emulsion with addition of emulsifiers exhibited stability for 6 months with only sporadic occurrence of oil globules. When phospholipid (Phospholipon 90G) was added, the same behavior was observed; however, the mean droplet size decreased with only negligible increase in emulsion viscosity (see Figure S1 in the electronic supplementary material). Therefore, a formation of interfacial complexes between oil and gel matrix is hypothesized and different stabilization mechanisms are anticipated in this case (Dickinson & Yamamoto, 1996). Gentle shaking of all phase-separated emulsions over the tested period restored homogenous emulsion with a better result for 0.50 wt. % BG emulsion. (Table 1). When the BG concentration was increased from 0.10 wt. % to 2.0 wt. %, the emulsion gel changed its 8

state and became a strong gel. When BG concentration drops from 2.0 wt. % to 0.10 wt. % at a fixed CLA:BG gel mass ratio of 1:1, a significant increase in droplet size and viscosity decrease was visible with some free CLA floating on the emulsion surface. Figure S2 in the electronic supplementary material shows the emulsions stabilized by BG hydrogel with different BG concentrations. High viscosity increasing from 6.2 Pa∙s to 1507 Pa∙s stabilizes emulsions at 2.0 wt. % of BG in gel. It was possible to prepare stable emulsion with the CLA mass ratio 1:2 in gel containing only 0.50 wt. % of BG; however, emulsion stabilization using 0.10 wt. % BG gel was ineffective and extensive coalescence was observed for 6 months’ period (see Figure S2). The viscosity of CLA:BG (0.10 wt. %) 1:2 system is slightly lower than that of CLA:BG (0.50 wt. %) 1:2 emulsion. Thus, it can be concluded that only emulsions containing more than 0.50 wt. % of BG in gel are stable and exhibit negligible creaming index. The emulsions with 2.0 wt. % BG gel form much more viscous systems where the high BG content decreases mobility and results in increased emulsion stability. This demonstrates that the increase in viscosity was a primary factor in the emulsion stabilization. Table 2 shows that the apparent viscosity gradually increased with BG concentration from 0 to 0.50 wt. % and sharply increased when BG concentration reached 2.0 wt. %. Note that emulsion CLA:BG 2:1 with both 0.10 wt. % and 0.050 wt. % BG gel was stable for almost 6 months. As shown in Table 1, initial pH of the emulsions does not depend on the oil. Addition of sodium hydroxide solution (5 wt. %) to CLA:BG (2.0 wt. %) (1:9) emulsion to pH 7.8 leads to a more viscous emulsion (7854 mP s) with somewhat smaller droplet size (1.16 µm). For particle-stabilized (Pickering) emulsions, the particle concentration is an important parameter affecting the stability against coalescence (Dickinson, 2010; Destribats et al., 2011; Ngai et al., 2006). Introducing solid BG particles that are unconnected in the pre-existing BG gel network can adsorb on the CLA–BG interface and behave as an additional barrier against droplet coalescence. This assumption was confirmed by addition of micronized BG particles (4.5 µm) into 0.050 wt. % BG gel to yield a total concentration 0.10 wt. %. The resulting gel stabilized the emulsion with mass ratio CLA:BG 1:2 while a particle-free BG gel with the same total BG content was ineffective at this CLA concentration. A Pickering formulation prepared by a stepwise addition of CLA from 5.0 wt. % to 25 wt. % was stable, however, at 30 wt. % flocculation of emulsion was observed after 24 h. During this stepwise CLA additions, the system viscosity increased about tenfold, from 31 mPa∙s (for 5.0 wt. % CLA) to 293 mPa∙s (for 30 wt. % CLA) and, simultaneously, the droplet size increased from 1.85 µm to 3.40 µm. If an additional amount of BG particles was added to this mixture (to reach the final BG concentration of 3.5 wt. %), the flocculation of 30 wt. % emulsion disappeared with negligible increase in viscosity and the droplet size decreased to 1.73 µm with narrow distribution (SD 0.62 µm). Moreover, even heating the sample at 55 °C for 1 h did not lead to phase separation These results suggest that in this case the emulsion stability is mainly driven by the BG particle concentration rather than by the elevated viscosity.

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The emulsions' stability was observed at temperature 25 °C by their visual inspection at regular time intervals (0.5, 1, 2, 3, 4, 5 and 6 months). Creaming and the resulting destabilization was observed for CLA:BG (0.10 wt. %) 1:1 emulsion after 24 h. After 1 month, only negligible creaming and drop in viscosity was observed for CLA:BG (0.50 wt. %) 1:1 emulsion and stability after 12 months was confirmed. Paradoxically, instability was observed after 3 months for the same emulsion formulated with additional emulsifiers (Span 80, Tween 80). Emulsions formulated as CLA:BG (2.0 wt. %) 1:9 and 1:5 were stable during 12 months, however, the 1:2 emulsion broke down after 5 months. Thus, the results indicate that only hydrogel with 2.0 wt. % of BG guarantees emulsion stability for CLA:BG gel mass ratios ranging from 1:9 to 1:2. 3.2 Cocoa butter, coconut oil, and olive oil emulsion gels After developing a set of suitable model gels with CLA, we investigated the effect of the BG gel matrix on other oils with relevance for food or cosmetics products. Obviously, the presence of different dispersed oils in the gel affects the system viscosity. For the same oil:gel mass ratio of 1:9 in gel containing 2.0 wt. % BG, the highest viscosity is observed for CLA followed by CB and coconut oil gel and olive oil gel. Comparing the intrinsic viscosities of the emulsion gels with that of pure oils (CB 76.5 mPa∙s at 60 °C, olive oil 76.2 mPa∙s at 20 °C, virgin coconut oil 54.9 mPa∙s at 27 °C and CLA 53.6 mPa∙s at 20 °C) reveals that the more viscous oils do not necessarily give the more viscous emulsion gels. Similarly, the type of oil did not influence the droplet parameters such as diameter and organization. The emulsion gels were prepared from CB, coconut oil, olive oil using 2.0 wt. % BG gel at oil:gel ratios of 1:4, 1:9, 1:18. For all these gels the stability against phase separation was observed after 12 months. When the emulsions gels were stored at 25 °C in closed flasks, only a small increase in the mean diameter after storage period was observed and the microscopy showed a homogeneous distribution of the emulsion droplets over the whole sample column. Moreover, the CB afforded stable emulsion gel even at 1:1 mass ratio. Two systems under study are composed from oils whose melting temperatures are slightly above the room temperature (coconut oil and cocoa butter). Saturated fatty acids of cocoa butter behave as solid at the oil–gel interface and form a spatial pattern which prevents droplet coalescence (Giermanska et al., 2007; Ghosh & Rousseau, 2011; Thivilliers et al., 2006). The cryo-EM micrograph of the cocoa butter emulsion gel (Figure 2c) indicated the presence of crystalline particles. In addition, examination of light-polarized microscopy images for CB:BG (1:9) and CB:BG (1:4) formulations reveals bright spots appearing in each droplet. Sagiri et al. (2014) suggested the presence of crystalline CB in its gelatin emulsion gel by XRPD and DSC. Indeed, the XRPD pattern of directly prepared CB:BG (1:9) emulsion gel confirmed the presence of stable polymorph (β) of CB in the emulsion gels (data not shown). Comparison of the emulsion gel melting enthalpy measured by DSC with that of bulk CB allows to determine the portion of CB that is dispersed as a solid. The DSC measurements

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depicted in Figure 3 revealed that the ratio of sample melting enthalpy to that of bulk CB agrees well with the total CB content. Thus, practically the whole oil phase is present as a solid. 3.3 Xerogel formation and swelling kinetics Comin et al. (2012) reported that upon hydration the BG xerogel forms a cohesive gel with flax oil which can be applied as an ointment base in cosmetics. Thus, the ability of BG emulsion gels with various natural oils to form a xerogel was tested. Xerogels are solid structures formed from gels by their drying accompanied with shrinkage (Walter, 1998) and are suitable as materials for delivery and stabilization of bioactive compounds. Literature on BG-based xerogels is quite scarce (see, for example, Hromádková et al., 2003; Comin et al., 2012; and Mikkonen et al., 2013). Removal of water by solvent exchange and lyophilization enables the formation of larger dispersed particles and porosity with a sheet-like microstructure. Emulsified CLA, CB, coconut oil and olive oil gels initially containing 10 wt. % of the respective oil were successfully reconstituted from emulsion gels which were previously freely dried at 25 °C for 4 weeks and then again rehydrated (swelled) in water for 3– 4 h at 25 °C (see Figure 4). During the drying process, the gels lost viscoelastic properties, color and reduced their volume (shrinkage) without blooming of the oil phase on the xerogel surface. Surprisingly, no oil droplet aggregation was found on the surface of the dried gel and the overall shape of the original matter was conserved. This suggests that the dispersed oil droplets are regularly and deeply captured in the BG gel network. The CB:BG 1:9 emulsion gel has been further studied as a model for preparing a low-density xerogel. In our study, CB was emulsified with the BG hydrogel and the ethanol washing and free (open area) drying were applied to yield a xerogel (Table 2). The simple ethanol washing and freeze-drying methods resulted in the maximum CB loading with 62.8 % and 69.0 % respectively of the final xerogel mass; slightly more than reported by Comin et al. (2012). However, the suitability of washing method can be questioned since CB is partially soluble in ethanol and also some of the gel/alcogel is lost in ethanol bath. The samples of these emulsion gels were dried in open area and via alcogel drying at 35 °C and 1.3 kPa in oven with final water content (determined by K. F. titration) 8.7 % and 6.2 %, respectively. The CB:BG xerogel dried via alcogel by freeze drying procedure (water content 2.1 %) is brittle, porous and can be easily pulverized (Figure S3 in the electronic supplementary material) while the sample dried freely in open area is a plastic material. As discussed previously in the case of hydrogels, DSC of CB:BG xerogel (Figure 3) revealed that crystalline CB is present. Crystallization of CB may occur during the drying of the sample; however, crystallization primarily takes place in the emulsification step as confirmed by XRPD in hydrogels emulsions. Figure 5 shows the scanning electron micrographs of neat BG and CB:BG xerogels. The CB:BG xerogel has many closed pores without interconnecting capillary channels in its inner surface. Comparison of corresponding SEMs of CB:BG gel and xerogel indicates an incorporation of the oil to gel matrix which results into filled pores and increased water repulsion. 11

Considering the xerogel application, its absorption kinetics is indicative to give an idea how quickly the water would be absorbed to form a hydrogel. Figure 6 depicts the swelling ratio (SR) as a function of time for the freeze-dried BG and CB:BG xerogels. As it can be seen, at the beginning of swelling, CB:BG xerogel absorbs water more slowly compared with the neat BG xerogel which is a clear consequence of the water-repulsive nature of CB:BG xerogel. For both xerogels, the swelling curve follows roughly the same two-stage profile. During the first 100 min, the hydrogels attain about 50 % of their equilibrium water content. Subsequently the neat BG and CB:BG xerogels undergo a slow absorption of water until saturation is attained, the time for this slower stage is about 3 h and 4 h respectively. Obviously, the swelling dramatically altered the mechanical properties of xerogel. The BG xerogels were very brittle and difficult to handle without breaking which led to irreproducible results. Therefore, a more appropriate non-invasive DVS technique was applied to get insight into the hydration process. The moisture sorption isotherms of the xerogels were determined isothermally at 25 °C using a DVS apparatus. The DVS shows the water sorption isotherms for the freeze-dried BG and CB:BG xerogels as a function of RH from 0 to 95 %. According to the classification of BET sorption isotherms, group III shape characteristic for sorption isotherm of carbohydrates was observed. (Hoobin et al., 2013; Despond et al., 2005). In the RH range from 10 to 80 % the water content measured in the BG xerogel was higher (based on the percentage of dry mass) than that in the CB:BG xerogel. The results confirmed that the water content of the BG xerogel (26 % at 90 % RH) agrees with that of water-soluble barley BG films (28 % at 90 % RH). In this part of the isotherm, water sorption is mainly driven by water condensation and reflects the availability of the BG matrix for binding water to specific sorption sites. Above 80 % RH, water sorption in BG xerogel increases exponentially. The highest hysteresis (8.5 %) of CB:BG xerogel was reached at 70 % RH, and clearly demonstrates changes of properties such as shape of the capillaries or differences in mobility of the matrix connected with the composition of the matrix (Aguirre-Álvarez et al., 2013; Lu & Pignatello, 2002). It is assumed that the hydroxyl groups of BG can form hydrogen bonds with water molecules, the DVS cycles involving drying period induced dehydration of samples, indicated that water molecules are held in these materials through weak bonding. Nevertheless, atmosphere with ambient relative humidity of about 40 % is adequate for material storage at 20 °C. Absence of physical changes upon exposure to water vapor was inspected by the microscopic images collected during the sorption where the DVS exposed samples did not show any sign of swelling. From comparison between DVS results depicted in Figure 7 and swelling behavior it can be inferred that a layer of hydrogel on the surface of xerogel is formed during the first stage of monomolecular sorption. Thus, the diffusion of water vapor into the saccharide network prior to swelling is thereby hindered. On the other hand, the swelling in liquid water induces an osmotic effect in the network and facilitates much more rapid uptake of water.

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CONCLUSIONS We have demonstrated the use of Pleorotus ostreatus BG hydrogel for formulation of stable emulsion gels with natural oils such as CLA, coconut oil, olive oil, and cocoa butter. It was found that oil droplets could be trapped and held in place by viscous BG hydrogel network structure created through interacting chain segment association and aggregated junction zones. The hydrogel formed with only 0.50 wt. % of BG was able to stabilize CLA in 2:1 mass ratio for 6 months against coalescence. Increasing the BG concentration in hydrogel from 0.10 to 2.0 wt. % results in more viscous systems which can readily accommodate up to 50 wt. % of the oil phase. Furthermore, the addition micronized solid BG particles to the CLA:BG emulsion gels was found to enhance the emulsification via phenomenon known as the Pickering stabilization. Versatility of emulsion stabilization using BG hydrogel was demonstrated by cocoa butter xerogel formation; loading of 69 wt. % was confirmed by DSC and gravimetrically. The combination of natural oil phase with BG hydrogel matrix into an emulsion gel along with the possibility of balancing the final composition can be exploited for transdermal delivery of both hydrophobic and hydrophilic substances. Moreover, BG may replace surfactants necessary to ensure sufficient stability of food and cosmetics.

ACKNOWLEDGEMENTS Financial support from the Scientific Grant Agency of the Slovak Republic (VEGA 1/0592/15) is gratefully acknowledged (T. D., P. Š.). This work was also supported by the Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic for the Structural Funds of EU, OP R&D of ERDF by realization of the project “Evaluation of natural substances and their selection for prevention and treatment of lifestyle diseases”, ITMS 26240220040 (M. V.).

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Table 1 Physico-chemical properties of BG gel emulsions determined 2 h after preparation. If not stated otherwise, gel containing 2.0 wt. % BG was used. Numerical values are arithmetic means from measurements done in triplicates. Oil

pH

Oil:gel (m:m)

Viscositya Conductivity (Pa∙s) (µS/cm)

Droplet sizeb (µm)

Appearance, emulsion typec

CLA

nc

1:1

unstable emulsion, w/o white gelled emulsion, w/o white gelled emulsion, w/o white gelled emulsion, w/o white gelled emulsion, w/o white gelled emulsion, w/o unstable emulsion, w/o white gelled emulsion, o/w white gelled emulsion, o/w yellowish gelled emulsion yellowish emulsion, w/o yellowish gelled emulsion white gelled emulsion, o/w white gelled emulsion, w/o brownish-white emulsion, o/w brownish-white emulsion, o/w brownish-white gelled emulsion, o/w

Olive oil

Coconut oil

CB

nc

2.80

nc

5.5

1:1

d

0.140

0.44

2.04

5.8

1:1e

3.370

0.075

<1.0

5.7

1:1d, f

0.910

0.99

1.59

5.8

1:2

6.215

125

1.98

5.8

1:2f

4.710

234

2.93

5.7

g

1:2

1.255

62.9

1.39

5.4

1:4

1080

2.80

1.72

5.5

1:9

1507

1370

1.54

5.0

1:9

216

1294

1.95

7.0

1:1

114

2.22

1.96

0.671

2.95

<1.0

e

7.0

1:1

5.8

1:9

232.8

1252

2.16

5.7

1:4

1626

715

2.45

5.9

1:9

1084

1275

1.94

7.0

1:9

1012

205

1.80

5.8

1:18

1948

1547

2.43

a

apparent value mean droplet size determined by statistical analysis of optical image c determined by conductivity measurement and drop test d emulsifiers (Tween80/Span80) were used e Phospholipon 90 G was used f 0.5 wt. % BG gel g 0.1 wt. % BG gel nc, test not conducted b

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Table 2 Properties of CB:BG and BG xerogels prepared using different drying techniques

a b c

Treatment

Residual water (wt. %)

Xerogel mass (wt. % of hydrogel)

Xerogel mass (% of alcogel mass)

CB recovery (%)

Freeze drying

2.1

7.6

14.5

62.8a, 69b

Oven drying

6.2

10.9

17.1

54.2a, nc

Open area

8.7

12.8

nc

58.8a, 44b

Open areac

11.2

8.8

nc

nc

gravimetric method DSC method neat BG xerogel

19

(a)

(b)

(c)

(d)

(e)

(f)

Figure 1 Optical micrographs (magnification 100×) of emulsion gels prepared from BG gel and (a) CLA (9:1), (b) cocoa butter (9:1), (c) coconut oil (3:1), (d) olive oil (9:1), (e) CLA (2:1, in 0.50 wt. % BG gel), and (f) control CLA-in-water emulsion (9:1) with Tween 80/Span 80. The continuous phase of mixtures consists of 2.0 wt. % BG gel

20

Figure 2 Representative cryo-EM micrographs of microstructures: neat BG 2.0 wt. % gel (a), coconut oil 9:1 (b), cocoa butter 9:1 (c), and olive oil 9:1 (d). The continuous phase of mixtures consists of 2.0 wt. % BG gel

21

Normalized heat flow (endotherms up)

0,5 W/g

5

10 15

20 25 30 35 40 45 Temperature (°C)

50 55

60 65

Figure 3 From top to bottom: DSC records (heating at 5 °C/min) of neat cocoa butter, CB:BG (2.0 wt. %) 1:9 emulsion, CB:BG (2.0 wt. %) 1:18 emulsion, and freeze-dried CB:BG xerogel prepared via alcogel

22

Figure 4 Appearance of the emulsion gels formed by BG 2.0 wt. % and oil at mass ratio 9:1; right to left: olive oil, coconut oil, cocoa butter, CLA. Top: emulsions after preparation; middle: drying for 14 days at 28–30 °C and 40–45 % RH; bottom: reconstitution after swelling.

23

Figure 5 Inner surface SEMs of the neat BG xerogel (left) and CB:BG xerogel (right) prepared by drying in open area

24

Percentage of swelling (% SR)

90 80 70 60 50 40 30 20

CB:BG xerogel BG xerogel

10 0

20

40

60

80 100 120 140 160 180 200 Time (min)

Figure 6 Swelling kinetics of xerogels in water at 25 °C

25

Figure 7 (a) Moisture sorption isotherms of neat BG xerogel (green) and freeze-dried CB:BG xerogel (red) at 25 °C. (b) Moisture sorption kinetics of neat BG xerogel (green) and freeze-dried CB:BG xerogel (red) at 25 °C with controlled relative humidity ranging from 0 to 90 % (blue).

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