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Structural characterization of oleogels from whey protein aerogel particles
Journal Pre-proofs Structural characterization of oleogels from whey protein aerogel particles S. Plazzotta, S. Calligaris, L. Manzocco PII: DOI: Reference:
S0963-9969(20)30124-1 https://doi.org/10.1016/j.foodres.2020.109099 FRIN 109099
To appear in:
Food Research International
Received Date: Revised Date: Accepted Date:
24 September 2019 21 December 2019 11 February 2020
Please cite this article as: Plazzotta, S., Calligaris, S., Manzocco, L., Structural characterization of oleogels from whey protein aerogel particles, Food Research International (2020), doi: https://doi.org/10.1016/j.foodres. 2020.109099
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Structural characterization of oleogels from whey protein aerogel particles Plazzotta, S.*, Calligaris, S., Manzocco, L. Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Via Sondrio 2/A, 33100 Udine, Italy *corresponding author: [email protected]; Tel: +39 0432-558571 [email protected]; [email protected] Abstract Oleogels intended as fat substitutes were prepared by oil dispersion of aerogel particles obtained through freeze-drying (FD) or supercritical-CO2-drying (SCD) of whey protein isolate (WPI) hydrogels (20 g/100 g). SEM revealed that freeze-dried particles presented larger dimensions than supercritical-dried ones. The latter also showed higher oil dispersibility, forming aggregates with lower dimension (300 nm) than those formed by freeze-dried particles (700 nm). Both particles presented oil structuring capability. Freeze-dried particles gave a weak oleogel, while supercritical-dried ones gave a strong (G’=3.1×105 Pa) and plastic (critical stress=723.2 Pa) oleogel, with rheological features comparable to those of traditional fats. These results can be explained based on the lower aggregation induced by SCD and on the higher capacity of supercritical-dried particles to form a network in oil through hydrophilic interactions, as suggested by FTIR. Therefore, WPI aerogel particles show the potentiality to be used as food ingredients to prepare oleogels with tailor-made physical properties. Keywords: lipid structuring; fat mimetics; dried template; indirect oleogelation; supercritical drying; freeze drying
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1
Introduction
Proteins are widely used in the food sector for their peculiar functional properties, including their ability to form aqueous gels (hydrogels) under defined physicochemical conditions. Gelation of proteins in water is often induced by heating above denaturation temperature. Heating favours protein unfolding and increases the exposure of hydrophobic groups originally buried inside the molecules. Aggregation is then obtained through surface hydrophobic interactions, leading to the formation of a three-dimensional network entrapping water (Loveday, 2019). Other approaches exploit the change of the aqueous phase pH to values close to the isoelectric point or the increase of ionic force. In these cases, the protein network is formed due to the prevalence of attractive van der Waals forces over electrostatic repulsions. The structure of the resulting hydrogels can be widely modulated by steering formulation and processing parameters (Sağlam, Venema, van der Linden, & de Vries, 2014). Conversely, the use of proteins to obtain oleogels is still a challenge (Scholten, 2019). Oleogels are defined as solid-like materials entrapping oil in a three-dimensional network of structuring molecules (Co & Marangoni, 2012). In recent years, oleogels and relevant oleogelation procedures have attracted large interest in different research areas. In the food sector, oleogels have been mainly proposed as fat substitutes to obtain healthier foods with reduced content of saturated/trans fatty acids (Patel & Dewettinck, 2016). More recently, they have been suggested as delivery systems of bioactive lipophilic molecules and lipolysis modulators during digestion (O’Sullivan, Barbut, & Marangoni, 2016; Tan, Wan-Yi Peh, Marangoni, & Henry, 2017). Although many oil gelators have been identified so far, most of them are liposoluble molecules (e.g. wax and wax esters, ethylcellulose, phytosterols, fatty acid derivatives) whose use in food is often subjected to compulsory limitations (Martins, Vicente, Cunha, & Cerqueira, 2018). The possible use of proteins as oil gelators could thus open new opportunities to enlarge oleogel applications, being proteins cheap, easily available and already widely used in foods (Scholten, 2019). Moreover, this could be an attractive approach meeting the requirements of circular economy. 2
Proteins can be actually derived from waste streams of a number of food production processes, contributing to increasing the sustainability of the overall food supply chain (Pojić, Mišan, & Tiwari, 2018). However, due to their predominant hydrophilic nature, proteins are not able to directly network in oil (Patel, 2018). To overcome this issue and include proteins into a hydrophobic environment, different approaches have been proposed. Romoscanu and Mezzenga (2006) suggested an emulsiontemplate approach. In this case, water is removed from a protein-stabilized emulsion, leading to the formation of a semi-solid high internal phase emulsion (HIPE), containing over 90% of oil. However, these systems do not properly fall under the oleogel definition, since they still contain water (Scholten, 2019). The complete removal of the water phase from oil-in-water emulsions stabilized by a polysaccharideprotein matrix was similarly reported to promote the physical trapping of oil, resulting in oleogels containing over 97% oil (Patel et al., 2015; Wijaya, Van Der Meeren, Dewettinck, & Patel, 2018). Another approach was proposed by de Vries and coworkers (de Vries, Gomez, van der Linden, & Scholten, 2017; de Vries, Hendriks, van der Linden, & Scholten, 2015; de Vries, Jansen, van der Linden, & Scholten, 2018; de Vries, Lopez Gomez, Jansen, van der Linden, & Scholten, 2017). These authors applied a solvent exchange method to generate semi-solid oleogels structured by a network of whey protein aggregates. In this procedure, proteins are firstly denaturated in water to obtain a hydrogel. Water is then progressively replaced by an intermediate solvent (i.e. acetone or THF), which is then substituted with oil. Although the resulting oleogels have tunable physical properties, their preparation requires the use of non-food-grade solvents. Finally, oleogelation by means of dried templates has been also proposed. The basic idea is to remove water from a bio-polymeric hydrogel to obtain a dried template, able to absorb oil. Oil absorption requires the availability of a highly porous material, undergoing limited structural collapse during drying (Manzocco et al., 2017; Patel, Schatteman, Lesaffer, & Dewettinck, 2013). 3
Aerogels are highly-porous ultra-light materials which are prepared by drying of hydrogels, via a suitable technique including air-drying, freeze-drying and supercritical-CO2-drying (García-González, Alnaief, & Smirnova, 2011). Freeze-drying (FD) and supercritical-CO2-drying (SCD) are particularly effective in preventing structural collapse. FD actually prevents capillary tensions and provides structural rigidity (Betz, García-González, Subrahmanyam, Smirnova, & Kulozik, 2012). Alternatively, in the case of SCD, hydrogel water is replaced by ethanol and the latter is then removed using a continuous flow of CO 2 in the supercritical state. This produces minimal capillary forces in the system, preventing structural collapse (Betz et al., 2012; Manzocco et al., 2017). Given their physical features, aerogels have been proposed for different applications, such as drug delivery, microencapsulation of bioactive compounds and aromas, sound insulation, absorption, and catalysis (García-González et al., 2011; García-González, Jin, Gerth, Alvarez-Lorenzo, & Smirnova, 2015; García-González & Smirnova, 2013; Parikka et al., 2017; Selmer, Kleemann, Kulozik, Heinrich, & Smirnova, 2015; Ubeyitogullari & Ciftci, 2017). It is noteworthy that the use of aerogels is currently considered an interesting approach to tackle two of the current main European challenges: active ageing and circular economy (AERoGELS Cost Action, ref. CA18125). In this context, aerogels have been suggested as elective candidates for oleogel preparation. For instance, bio-polymeric monolithic aerogels prepared via FD and SCD showed a good oil absorption capability, leading to oleogels presenting up to 95% oil (Ahmadi, Madadlou, & Saboury, 2016; Manzocco et al., 2017; Plazzotta, Calligaris, & Manzocco, 2019). However, the derived oleogels had a defined shape and a non-plastic structure, which would limit their use as fat substitutes (Plazzotta et al., 2019). These issues could be overcome by the production of aerogel particles able to absorb oil and form a network upon dispersion in oil. To this regard, freeze-dried whey protein particles have been shown to interact in oil by hydrophilic particle-particle interactions, showing a good potentiality as building blocks for oil gelation (de Vries et al., 2015; de Vries, Lopez Gomez, et al., 2017).
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Based on these considerations, the aim of the present paper was to investigate the possibility of using whey protein aerogel particles obtained by FD or SCD to structure liquid oil. The aerogel particles were analyzed for morphology and dispersibility in water and oil. The oleogels were characterized by microscopy, rheological and FTIR analyses. 2 2.1
Materials and Methods Materials
Whey protein isolate (WPI, 94.7% protein content; 74.6% β-lactoglobulin, 23.8% α-lactalbumin, 1.6% bovine serum albumin) was purchased from Davisco Food International Inc. (Le Sueur, MN, USA) Sigma-Aldrich (Milan, Italy); hydrochloric acid (HCl) was purchased from Carlo Erba Reagents (Milan, Italy); absolute ethanol was purchased from J.T. Baker (Griesheim, Germany); phosphorus pentoxide (P2O5) was purchased from Chem-Lab NV (Zedelgem, Belgium); liquid carbon dioxide (CO2) (purity 99.995%) was purchased from Sapio (Monza, Italy); sunflower oil was purchased in a local market (Giglio Oro, Carapelli, Firenze, Italy), commercial palm oil shortenings and margarines were selected among those typically used for the preparation of bakery goods and were furnished by local food companies; Fast Green and Nile Red dyes were purchased from Sigma Aldrich (Milan, Italy). All solutions were prepared using milli-Q water. 2.2
Preparation of hydrogel
The hydrogel was prepared according to the procedure described by de Vries et al. (2015) with some modifications. In particular, an amount of 0.20 g/g WPI was suspended in water, under continuous stirring at room temperature for 2 h and stored overnight at 4 °C to assure complete protein hydration. The pH of the WPI suspension was then adjusted to 5.7 using a 1 M HCl, which allows the preparation of a structurally stable gel (Betz et al., 2012). Aliquots of 30 mL were then introduced in 50 mL plastic tubes with screwcaps and heated at 85 °C for 15 min using a temperature-controlled water bath to induce 5
protein denaturation. The obtained protein gel was cooled in ice water and homogenized using a highspeed mixer at 13,000 rpm for 3 min (Polytron PT-MR3000, Kinematica AG, Littau, Switzerland). 2.3
Preparation of oleogels via oil dispersion of freeze-dried particles
The hydrogel was frozen at -80 °C for 24 h and freeze-dried for 72 h at 4,053 Pa by using the pilot plant model Mini Fast 1700 (Edwards Alto Vuoto, Milan, Italy), obtaining the aerogel particles. The latter were ground for 1 min using a domestic grinder (MC3001, Moulinex, Milan, Italy) and dispersed into sunflower oil (0.1 g/mL), homogenized by using a high-speed mixer at 13,000 rpm for 3 min (Polytron PT-MR3000, Kinematica AG, Littau, Switzerland) and collected by centrifugation at 13,000 × g for 5 min (Avanti J-25, Beckman, Palo Alto, CA, USA). This procedure was repeated twice, obtaining the oleogel. 2.4
Preparation of oleogels via oil dispersion of supercritical-CO2-dried particles
The hydrogel was firstly converted into alcoholgel as described by de Vries et al. (2015), with some modifications. In particular, ethanol was used instead of acetone as intermediate-polarity solvent. The hydrogel was dispersed into ethanol (0.1 g/mL), homogenized by using a high-speed mixer at 13,000 rpm for 3 min (Polytron PT-MR3000, Kinematica AG, Littau, Switzerland) and collected by centrifugation at 13,000 × g for 5 min (Avanti J-25, Beckman, Palo Alto, CA, USA). This procedure was repeated twice to completely remove water, obtaining the alcoholgel. The latter was dried using the supercritical-CO2-drying plant developed at the Department of Agricultural, Food, Environmental and Animal Sciences (University of Udine) and previously described elsewhere (Manzocco et al., 2017). Alcohol removal was performed at 11 ± 1 MPa and 45 °C with an outlet flow of 6.0 NL/min for 8 h. Decompression from 11 MPa to atmospheric pressure was carried out at 6.0 NL/min in 30 min, obtaining the aerogel particles. The latter were ground and converted into oleogel by following the same procedure applied to the aerogel particles obtained upon freeze-drying (paragraph 2.3). 6
2.5
Analytical determinations
2.5.1
Image acquisition
Sample images were acquired using an image acquisition cabinet (Immagini & Computer, Bareggio, Italy) equipped with a digital camera (EOS 550D, Canon, Milano, Italy). The digital camera was placed on an adjustable stand positioned 45 cm above a black or white cardboard base where the samples were placed. The light was provided by 4 100 W frosted photographic floodlights, in a position allowing minimum shadow and glare. 2.5.2
Optical microscopy
Hydrogel and oleogel samples were gently placed on a glass slide, covered with a cover slide and observed using a Leica DM 2000 optical microscope (Leica Microsystems, Heerbrugg, Switzerland). The images were taken at 200× magnification using a Leica EC3 digital camera and elaborated with the Leica Suite Las EZ software (Leica Microsystems, Heerbrugg, Switzerland). 2.5.3
Scanning electron microscopy
For scanning electron microscopy (SEM), aerogel samples were mounted on aluminium sample holders and sputter-coated with 10 nm of gold using a Sputter Coater 108 auto (Cressington Scientific Instruments, Watford, United Kingdom). The aluminium holder was transferred to the SEM unit (EVO 40XVP, Carl Zeiss, Milan, Italy), which was at ambient temperature and under vacuum. Samples were imaged using an acceleration voltage of 20 kV and SmartSEM v. 5.09 (Carl Zeiss, Milan, Italy) application software was used to capture images of the samples at magnification from 100× to 25,000×. 2.5.4
Confocal microscopy
A 0.2% aqueous solution of Fast Green and Nile Red was used to stain respectively the proteins and the oil of the oleogel samples. After staining, the samples were gently mixed by hand, placed on the 7
microscope slide, covered with a cover slide and observed using a confocal laser scanning microscope at 100× magnification (Leica TCS SP8 X confocal system, Leica Microsystems, Wetzlar, Germany). Images were elaborated using the software LasX 3.5.5 (Leica Microsystems, Wetzlar, Germany). 2.5.5
Apparent density
The apparent density (g/cm3) of the aerogel particles was estimated by weighing 1 mL of dried material in a graded cylinder. 2.5.6
Particle size distribution
Aerogel particles were dissolved (1 mg/mL) in water or oil and sonicated 10 min into an ultrasonic bath (Ultrasonic Cleaner, VWR, Lovanio, Belgium). The mean particle dimension of the obtained dispersions was measured by using the dynamic light scattering instrument Zetasizer Nano ZS (Malvern, Milan, Italy). The angle of observation was 173°. Solution refractive index and viscosity were set at 1.333 and 0.001 Pa ∙ s, and 1.473 e 0.036 Pa ∙ s, corresponding to the values of pure water and sunflower oil at 25 °C, respectively. Particle mean diameter corresponding to intensity distribution was calculated by Distribution Analysis fitting. 2.5.7
Water and oil holding capacity
About 1 g of aerogel particles (P1) was dispersed into distilled water or sunflower oil (1 mg/mL), stirred using a vortex (Vortex 1, Ika, Milan, Italy) three times for 1 min and collected by centrifugation at 13,000 × g for 5 min at 20 °C (Avanti J-25, Beckman, Palo Alto, CA, USA). The supernatant was eliminated and the pellet was centrifuged again to remove the residual non-held solvent. The sediment obtained after centrifugation was weighted (P2). Water and oil holding capacities were calculated as g of water or oil held by 1 g of aerogel particles, following eq. 1: 𝑊𝐻𝐶 (
𝑔𝑤𝑎𝑡𝑒𝑟 𝑔𝑎𝑒𝑟𝑜𝑔𝑒𝑙 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠
) 𝑜𝑟 𝑂𝐻𝐶 (
𝑔𝑜𝑖𝑙 𝑔𝑎𝑒𝑟𝑜𝑔𝑒𝑙 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠
)=
𝑃2 −𝑃1 𝑃1
(eq. 1).
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2.5.8
Oil content
The oil content of oleogels was estimated based on oil holding capacity (paragraph 2.5.7) and defined as the percentage ratio between the amount of held oil and the overall weight of the sediment (i.e. the oleogel) obtained after centrifugation. The difference between oleogel weight and oil content was used to calculate the whey protein particle content in the gel. 2.5.9
Rheological analysis
The viscoelastic properties (moduli G’, G’’ and Tan ) of the oleogels were tested using an RS6000 Rheometer (Thermo Scientific RheoStress, Haake, Germany), equipped with a Peltier system for temperature control. Measures were performed using a parallel plate geometry at 20 °C with a gap of 2.0 mm. Oscillatory sweep tests to identify the linear viscoelastic region (LVR) were performed increasing stress from 1.0×10-3 to 1.0×103 Pa at 1 Hz frequency. Critical stress (Pa) was identified as the strain value corresponding to a 10% drop in G’ value. Frequency sweep tests were then performed increasing frequency from 0.1 to 120 Hz at stress values selected in the viscoelastic region. 2.5.10 FTIR measurement Spectra were recorded at 25 ± 1 °C by using an FTIR instrument, equipped with an ATR accessory and a Zn-Se crystal that allows the collection of FTIR spectra directly on a sample without any special preparation (Alpha-P, Bruker Optics, Milan, Italy). The “pressure arm” of the instrument was used to apply constant pressure to the samples positioned on the top of the Zn-Se crystal, to ensure good contact between the sample and the incident IR beam. Background scan of the clean Zn-Se crystal was acquired prior to sample scanning. All FTIR spectra were collected in the range from 4000 to 400 cm−1, at a spectrum resolution of 4 cm−1 and with 32 co-added scans. The second derivative of the spectra in the range 1600-1700 cm−1 was obtained by using the OPUS software (version 7.0 for Microsoft Windows, Bruker Optics). 9
2.5.11 Data analysis Determinations of apparent density, particle size distribution, water and oil holding capacity, oil content, and rheological properties were expressed as the mean ± standard error of at least three repeated measurements from two experiment replicates. Statistical analysis was performed by using R v. 3.0.2 (The R Foundation for Statistical Computing). Student’s t-test was used to determine statistically significant differences between means (p<0.05). At least 5 FTIR spectra were acquired for each sample and reported as mean spectra. Microscopy analyses were performed on at least 5 different sample portions to guarantee the representativeness of the presented images. 3 3.1
Results and discussion Characterization of the aerogel particles
A soft hydrogel with self-standing properties (Figure 1A) was obtained by heating and grinding of whey protein solutions (Figure 1A). This structure is due to the formation of disulfide bonds and interactions of hydrophobic residues, allowing the formation of a homogeneous network, well-visible in the microscopic image (Figure 1B) (de Vries, Wesseling, van der Linden, & Scholten, 2017). The hydrogel was subjected to FD or SCD and the resulting dried material was ground to unpack aggregates formed during drying. Obtained particles were analyzed by scanning electron microscopy (SEM) to determine their morphology. Figure 2 suggests that FD led to particles presenting much larger dimensions than those obtained via SCD. This result can be attributed to the ballooning effect of ice crystal growth during FD, which leads to dramatic contraction of the protein backbone and to the maximization of the interactions among protein particles in large aggregates (Tang, Wei, & Guo, 2014). By contrast, during the SCD procedure, proteins were always in a wet condition and evenly dispersed throughout the suspending medium, whose polarity slowly changed from hydrophilic to hydrophobic before solvent removal. These conditions probably favoured particle-solvent interactions, preventing 10
agglomeration of the protein particles (Ganesan et al., 2018). Such observations are supported by literature results showing that whey protein dried particles obtained through evaporation of solvents with a low polarity presented lower size than those obtained upon FD (de Vries, Lopez Gomez, et al., 2017; de Vries, Wesseling, et al., 2017). Higher SEM magnification highlighted that particles obtained upon both FD and SCD were characterized by a continuous matrix with evenly distributed pores (Figure 2). However, particles obtained through SCD presented an apparently finer porous surface as compared to that of freeze-dried particles (Figure 2). The porous nature of the particles was also supported by the apparent density calculation, which was of 0.070 and 0.021 g/cm3 for freeze-dried and supercritical-dried particles, respectively, confirming the higher porosity obtained upon SCD. Such density values are within the density range of aerogel materials (< 0.5 g/cm3) (Fricke & Tillotson, 1997). To understand if the morphological differences among aerogel particles obtained through FD and SCD could be explained based on the effect of the production process on protein conformation, FTIR spectra of the aerogel particles were acquired and compared to that of untreated whey proteins (Figure 3A). In addition, alcoholgel particles were analyzed to assess the effect of ethanol mass substitution on the conformation of protein aerogels obtained via SCD. Both aerogel particles exhibited the typical FTIR spectral bands of proteins, showing a broad tense peak at around 3280 cm −1, which corresponds to the N–H stretching vibration and IR bands at 1648 and 1530 cm−1, corresponding to the amides I and II, respectively (Parris, Purcell, & Ptashkin, 1991). As expected, the alcoholgel particles also showed the typical signals of ethanol, including intense peaks at 2973, 2972, 2881, 1087, 1045 and 880 cm-1 (Debebe, Redi-Abshiro, & Chandravanshi, 2017). The second derivative of the amide I region (1600-1700 cm−1) of the FTIR spectra was computed to better enlighten the changes in the secondary structure of whey proteins (Figure 3B). The untreated WPI showed specific bands in the range 1618-1640 and 1648-1657 cm-1 that have been attributed to β-sheets and α-helix structures of WPI proteins (Haque, Aldred, Chen, Barrow, & Adhikari, 2014; O’Loughlin, Kelly, Murray, Fitzgerald, & Brodkorb, 2015). As compared to 11
the untreated WPI, the spectra of both aerogel particles showed noticeable changes in the bands associated with the original WPI structures, as a consequence of heat-set denaturation of whey proteins. Denaturation actually decreased the occurrence of originally ordered structures in favour of disordered structures and intermolecular interactions. In particular, a decrease of original β-sheet structures was evidenced by the reduction of the peak at 1618 cm-1 and the broadening of the peak at 1628 cm-1. These changes were compensated by a new peak at about 1680 cm-1, associated with intermolecular β-sheets. The latter have been reported to be indicative of aggregation (Allain, Paquin, & Subirade, 1999; de Vries, Lopez Gomez, et al., 2017) and resulted more intense in the freeze-dried particles, confirming the role of FD in inducing intense particle aggregation, which was instead more limited upon SCD. The aerogel particle production process also affected the WPI α-helix structures. In particular, the bands at 1648 and 1657 cm-1 were reduced by the SCD process, while FD caused a considerable band shift, suggesting a reduction in these structures and a change in the hydrogen bonding pattern, respectively (Sharma & Kalonia, 2004). Moreover, the spectrum of freeze-dried particles also showed a new band at 1670 cm-1, which can be related to the presence of unordered random-coil structures. Since the latter result in a gain in conformational entropy, driving irreversible aggregation, their presence further confirms the intense aggregation induced by FD (O’Loughlin et al., 2015). It is noteworthy that the second derivative of the alcoholgel spectra in the Amide I region (Figure 3B) resulted comparable to that of the aerogel, demonstrating that mass substitution was the more important step in determining protein structure. Based on the solubility of ethanol in dense phase carbon dioxide, subsequent SCD allowed limiting further changes in protein conformation. In turn, this possibly accounted for the reduced particle aggregation and the high porosity of the supercritical-CO2-dried particles observed in Figure 2. 3.2
Dispersibility of aerogel particles in water and oil
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The size distribution of the aerogel particles in both water and oil was assessed (Figure 4). As a reference, the particle size distribution of untreated WPI was also determined. Both particles obtained by FD and SCD showed a mean dimension in water around 1300 nm, which resulted much higher than that of the untreated WPI (350 nm). This indicates a lower water dispersibility of the aerogel particles, which is probably due to the heat treatment applied for inducing WPI gelation. The latter is known to induce the exposure of hydrophobic groups, normally buried within the native folded structure of proteins and to induce the formation of large protein aggregates, confirming the observations relevant to Figure 2 and 3 (Fitzsimons, Mulvihill, & Morris, 2007). It must be noted that the freeze-dried particles resulted less dispersible in water than supercritical-dried ones, as indicated by the broader distribution and the presence of a second peak at larger size dimensions (about 5500 nm) (Figure 4A), confirming that FD provoked a more intense aggregation than SCD, as observed in the SEM images (Figure 2). By contrast, both aerogel particles showed a higher oil dispersibility than WPI. Indeed, the latter presented a largesize peak in oil at about 3000 nm and a second peak at about 120 nm (Figure 4B), while supercriticaldried particles in oil showed a homogeneous monomodal distribution, with particle dimensions around 300 nm. The latter resulted much smaller than those of the freeze-dried ones (700 nm). These results can be attributed to the specific effects of the drying technique on the ability of protein particles to interact with oil. In particular, SCD promoted the formation of small particles with a higher oil dispersibility than the large agglomerates generated during FD (Figure 2 and 3). The different ability of the particles to interact with solvents was also confirmed by the analysis of water and oil binding capacity (WHC and OHC). In fact, the supercritical-dried particles showed WHC and OHC values (4.51 ± 0.10 g water and 5.56 ± 0.09 g oil per g of aerogel particles) significantly higher than those of the freeze-dried one (3.14 ± 0.04 g water and 2.26 ± 0.05 g oil per g of aerogel particles), which is attributable to the lower particle dimension and higher dispersibility in both solvents (Figure 4). 3.3
Oleogels obtained from oil dispersion of the aerogel particles 13
The aerogel particles were dispersed in oil and collected by centrifugation, resulting in the materials shown in Figure 5 and having the composition reported in Table 1. Both aerogel particles presented an oil structuring ability, which can be attributed to their capacity of absorbing oil in the pores (Figure 2) by capillary forces and interacting with oil at the surface by particleoil interactions. In addition, oleogel structuring could further increase upon the formation of a network entrapping oil in the interstitial space among particles. To this regard, it has been shown that an increase of particle-particle interactions through hydrogen bonds in oil could contribute to gel strengthening (de Vries, Gomez, et al., 2017). The particle production process affected the physical properties of the oleogels (Figure 5). When SCD was applied, oleogels presented an oil content considerably higher than that of oleogels obtained by oil dispersion of freeze-dried particles (Table 1). In agreement with previous literature data (de Vries, Wesseling, et al., 2017), oil dispersion of the freeze-dried particles led to a system showing large aggregates coarsely dispersed in oil, as well-visible in the optical microscopy image and further evidenced by the application of confocal microscopy (Figure 5). By contrast, supercritical-dried aerogel particles were turned into an oleogel presenting an interesting plastic and deformable semi-solid texture (Figure 5). Optical microscopy evidenced the presence of a homogeneous network where proteins acted as building blocks (Figure 5). Confocal microscopy showed that this network was formed by highly porous protein particles able to embed the oil within pores as well as to hold it in the interparticle space (Figure 5). This suggests that supercritical-dried particles are able to form a more plastic and interconnected network in oil as compared to freeze-dried particles. To confirm this hypothesis, rheological analyses were performed (Table 1 and Figure 6). At low stress, both systems showed gel-like behaviour (G’ > G’’). With the increase of stress, the critical value was reached, determining the drop of elastic response (Figure 6A). The oleogel obtained from the supercritical-dried aerogel particles showed a linear viscoelastic region (LVR) limit significantly higher than that of the sample obtained from the 14
freeze-dried particles, thus showing a higher resistance to yielding and structure breakdown (Table 1). Figure 6B shows G’ and G’’ of oleogels in the frequency range of 0.01–100 Hz. A weak gel was obtained by oil dispersion of the freeze-dried aerogel particles, as suggested by the dependence of G’ and G’’ on the frequency. By contrast, the oleogel containing supercritical-dried particles was a strong gel, with G’ and G’’ patterns independent on frequency. Moreover, supercritical-dried aerogel particles gave an oleogel much more elastic than that obtained using freeze-dried particles, as shown by the higher G’ and the lower loss tangent (tan = G’’/G’), which measures the relative importance of elasticity in the system (Table 1). The values of G’ and critical stress have been previously indicated as key parameters for comparing oleogel rheological properties with those of traditional solid fats (Blake & Marangoni, 2015). Three commercial laminate fats were also subjected to rheological analyses, giving G’ and critical stress values in the range 8.2-26.7 ×105 Pa and 263.7-995.4 Pa, respectively. It must be noted that the oleogel obtained by the supercritical-dried aerogel particles (Table 1, Figure 6) gave rheological values in the same magnitude order of these commercial solid fats. Such rheological features have been previously obtained in solid monoglyceride emulsions containing 15% carnauba and sunflower waxes (Blake & Marangoni, 2015) or in 10%-ethylcellulose oleogels added with lecithin (Aguilar-Zárate, Macias-Rodriguez, ToroVazquez, & Marangoni, 2019). However, the first systems showed unpleasant waxy taste, not compatible with food, while the latter requires heating the oil at temperatures higher than 150 °C, possibly triggering oxidation (Aguilar-Zárate et al., 2019; Gravelle, Barbut, & Marangoni, 2012). Moreover, the use of these additives in food products is not desirable with respect to green labelling. By contrast, whey proteins used to prepare oleogels in the present paper are widely accepted by consumers and are not associated with negative food labelling impact (Scholten, 2019). The higher yielding resistance and elasticity of the oleogel containing supercritical-dried particles as compared to the one containing freeze-dried particles are much likely attributable to the different surface 15
properties of the dried particles. The lower aggregation (Figure 2) of supercritical-dried particles would guarantee a higher surface available for oil absorption, as well as higher availability of surface hydrophilic groups, which would drive particle-particle interactions in oil, leading to the formation of an interconnected network. By contrast, the intense particle agglomeration induced by FD (Figure 2) would reduce oil absorptive surface as well as the availability of hydrophilic surface groups required for interparticle interactions in oil (de Vries, Gomez, et al., 2017). To confirm these hypotheses, the structural features of the oleogels were further characterized using FTIR analysis, as shown in Figure 3C. Similar to aerogel particles, both oleogel samples exhibited the typical FTIR spectral bands of proteins (3279 cm−1, 1648 and 1530 cm−1, respectively). As expected, oleogel spectra also showed the typical bands of triglycerides, which are the main components of the sunflower oil used for oleogel production. In particular, the band at 3007 cm−1 is attributed to the stretching vibration of =C–H. Strong band absorptions were observed in the region of 3000–2800 cm−1, corresponding to C–H stretching vibrations of methylene (–CH2–) and methyl (–CH3) groups at 2922 and 2853 cm−1, respectively. The large peak around 1740 cm−1 is due to C=O double bond stretching vibration. Deformation and bending of C–H and stretching vibration of C–O result in peaks in the 1250– 1000 cm−1 region (Gurdeniz, Tokatli, & Ozen, 2007; Rohman & Che Man, 2012). It should be noted that there were no noticeable qualitative changes in FTIR profiles between the two oleogel samples produced by oil dispersion of freeze-dried and supercritical-dried aerogel particles (Figure 3C), indicating that the basic structural characteristics of protein and oil fractions in the oleogel samples were nearly unaffected by the production process. Nevertheless, the production process affected the intensity of the peaks at 1648 and 3279 cm−1. The intensity of the first band, which is influenced by hydrogen bonding pattern (Haque et al., 2014), resulted lower in the freeze-dried aerogel particles than in the supercritical-dried ones and was not affected by oil addition. This difference is possibly the result of the effect of drying technique on whey protein structures (Figure 3B), leading to different protein-protein hydrogen bonds. 16
Despite the lower oil content (and thus higher whey protein particle content) of the oleogel obtained using the freeze-dried particles (Table 1), the 3279 cm-1 peak resulted lower as compared to the oleogel containing supercritical-dried particles. Being these bands sensitive to hydrogen bonding strength (Haque et al., 2014), it can be inferred that the particles obtained via FD presented a lower capability of interacting through hydrogen bonding in oil as compared to those obtained via SCD. As a consequence, the freeze-dried particles would show a lower tendency to particle-particle interactions, while the supercritical-dried ones would form in oil a network mainly stabilized by such inter-particle interactions (de Vries, Gomez, et al., 2017). This supports the critical effect of particle networking by surface hydrophilic interactions on the oil structuring ability of aerogel particles. 4
Conclusions
Whey protein aerogel particles present interesting oil structuring functionality. By their simple addition into oil, oleogels with different physical properties, from those of a soft gel to those of a plastic solid fat, can be obtained. These differences can be attributed to the extent of structural collapse and agglomeration of particles during their preparation. The occurrence of these phenomena actually controls the aerogel particle size, porosity, dispersibility in oil, and capability of particle-particle interactions through hydrogen bonding in oil. These features not only tune the oil absorption ability of the particles but also their capacity of forming a network entrapping oil in the interstitial spaces. Whey protein aerogel particles show thus the potentiality to be used as innovative food ingredients to prepare oleogels with tailor-made physical properties. Further research is required to assess the performances of whey protein aerogel particles and the derived oleogels in real food matrices. Moreover, the real applicability of the production process should be carefully assessed. In this regard, the disadvantages of the proposed oleogel production process, which requires long times and dedicated
17
equipment, can be counter-balanced by the high added-value and remarkable performances of the final product. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. References Allain, A. F., Paquin, P., & Subirade, M. (1999). Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy. International Journal of Biological Macromolecules, 26, 337–344. Aguilar-Zárate, A., Macias-Rodriguez, B. A., Toro-Vazquez, J. F., & Marangoni, A. G. (2019). Engineering rheological properties of edible oleogels with ethylcellulose and lecithin. Carbohydrate Polymers, 205, 98–105. Ahmadi, M., Madadlou, A., & Saboury, A. A. (2016). Whey protein aerogel as blended with cellulose crystalline particles or loaded with fish oil. Food Chemistry, 196, 1016–1022. Betz, M., García-González, C. A., Subrahmanyam, R. P., Smirnova, I., & Kulozik, U. (2012). Preparation of novel whey protein-based aerogels as drug carriers for life science applications. Journal of Supercritical Fluids, 72, 111–119. Blake, A. I., & Marangoni, A. G. (2015). Factors affecting the rheological properties of a structured cellular solid used as a fat mimetic. Food Research International, 74, 284–293. Co, E. D., & Marangoni, A. G. (2012). Organogels: An alternative edible oil-structuring method. JAOCS, Journal of the American Oil Chemists’ Society, 89, 749–780. COST-Action "Advanced Engineering of Aerogels for Environment and Life Sciences" (AERoGELS, ref. CA18125) funded by the European Commission. https://cost-aerogels.eu/. Last accessed 18
19/12/2019 Debebe, A., Redi-Abshiro, M., & Chandravanshi, B. S. (2017). Non-destructive determination of ethanol levels in fermented alcoholic beverages using Fourier transform mid-infrared spectroscopy. Chemistry Central Journal, 11, 1–8. de Vries, A., Gomez, Y. L., van der Linden, E., & Scholten, E. (2017). The effect of oil type on network formation by protein aggregates into oleogels. RSC Advances, 7, 11803–11812. de Vries, A., Hendriks, J., van der Linden, E., & Scholten, E. (2015). Protein oleogels from protein hydrogels via a stepwise solvent exchange route. Langmuir, 31, 13850–13859. de Vries, A., Jansen, D., van der Linden, E., & Scholten, E. (2018). Tuning the rheological properties of protein-based oleogels by water addition and heat treatment. Food Hydrocolloids, 79, 100–109. de Vries, A., Lopez Gomez, Y., Jansen, B., van der Linden, E., & Scholten, E. (2017). Controlling agglomeration of protein aggregates for structure formation in liquid oil: A sticky business. ACS Applied Materials and Interfaces, 9, 10136–10147. de Vries, A., Wesseling, A., van der Linden, E., & Scholten, E. (2017). Protein oleogels from heat-set whey protein aggregates. Journal of Colloid and Interface Science, 486, 75–83. Fitzsimons, S. M., Mulvihill, D. M., & Morris, E. R. (2007). Denaturation and aggregation processes in thermal gelation of whey proteins resolved by differential scanning calorimetry. Food Hydrocolloids, 21, 638–644. Fricke, J., & Tillotson, T. (1997). Aerogels: production, characterization, and applications. Thin Solid Films, 297, 212–223. Ganesan, K., Budtova, T., Ratke, L., Gurikov, P., Baudron, V., Preibisch, I., … Milow, B. (2018). Review on the production of polysaccharide aerogel particles. Materials, 11, 2144. García-González, C. A., Alnaief, M., & Smirnova, I. (2011). Polysaccharide-based aerogels - Promising biodegradable carriers for drug delivery systems. Carbohydrate Polymers, 86, 1425–1438. 19
García-González, C. A., Jin, M., Gerth, J., Alvarez-Lorenzo, C., & Smirnova, I. (2015). Polysaccharidebased aerogel microspheres for oral drug delivery. Carbohydrate Polymers, 117, 797–806. García-González, C. A., & Smirnova, I. (2013). Use of supercritical fluid technology for the production of tailor-made aerogel particles for delivery systems. Journal of Supercritical Fluids, 79, 152–158. Gravelle, A. J., Barbut, S., & Marangoni, A. G. (2012). Ethylcellulose oleogels: Manufacturing considerations and effects of oil oxidation. Food Research International, 48, 578–583. Gurdeniz, G., Tokatli, F., & Ozen, B. (2007). Differentiation of mixtures of monovarietal olive oils by mid-infrared spectroscopy and chemometrics. European Journal of Lipid Science and Technology, 109, 1194–1202. Haque, M. A., Aldred, P., Chen, J., Barrow, C., & Adhikari, B. (2014). Drying and denaturation characteristics of α-lactalbumin, β-lactoglobulin, and bovine serum albumin in a convective drying process. Journal of Agricultural and Food Chemistry, 62, 4695–4706. Loveday, S. M. (2019). Food proteins: Technological, nutritional, and sustainability attributes of traditional and emerging proteins. Annual Review of Food Science and Technology, 8, 13–29. Manzocco, L., Valoppi, F., Calligaris, S., Andreatta, F., Spilimbergo, S., & Nicoli, M. C. (2017). Exploitation of κ-carrageenan aerogels as template for edible oleogel preparation. Food Hydrocolloids, 71, 68–75. Martins, A. J., Vicente, A. A., Cunha, R. L., & Cerqueira, M. A. (2018). Edible oleogels: An opportunity for fat replacement in foods. Food and Function, 9, 758–773. O’Loughlin, I. B., Kelly, P. M., Murray, B. A., Fitzgerald, R. J., & Brodkorb, A. (2015). Concentrated whey protein ingredients: A Fourier transformed infrared spectroscopy investigation of thermally induced denaturation. International Journal of Dairy Technology, 68, 349–356. O’Sullivan, C. M., Barbut, S., & Marangoni, A. G. (2016). Edible oleogels for the oral delivery of lipid soluble molecules: Composition and structural design considerations. Trends in Food Science and 20
Technology, 57, 59–73. Parikka, K., Nikkilä, I., Pitkänen, L., Ghafar, A., Sontag-Strohm, T., & Tenkanen, M. (2017). Laccase/TEMPO oxidation in the production of mechanically strong arabinoxylan and glucomannan aerogels. Carbohydrate Polymers, 175, 377–386. Parris, N., Purcell, J. M., & Ptashkin, S. M. (1991). Thermal denaturation of whey proteins in skim milk. Journal of Agricultural and Food Chemistry, 39, 2167–2170. Patel, A. R. (2018). Structuring edible oils with hydrocolloids: Where do we stand? Food Biophysics, 13, 113–115. Patel, A. R., & Dewettinck, K. (2016). Edible oil structuring: An overview and recent updates. Food and Function, 7, 20–29. Patel, A. R., Rajarethinem, P. S., Cludts, N., Lewille, B., De Vos, W. H., Lesaffer, A., & Dewettinck, K. (2015). Biopolymer-based structuring of liquid oil into soft solids and oleogels using watercontinuous emulsions as templates. Langmuir, 31, 2065–2073. Patel, A. R., Schatteman, D., Lesaffer, A., & Dewettinck, K. (2013). A foam-templated approach for fabricating organogels using a water-soluble polymer. RSC Advances, 3, 22900–22903. Plazzotta, S., Calligaris, S., & Manzocco, L. (2019). Structure of oleogels from κ-carrageenan templates as affected by supercritical-CO2-drying, freeze-drying and lettuce-filler addition. Food Hydrocolloids, 96, 1–10. Pojić, M., Mišan, A., & Tiwari, B. (2018). Eco-innovative technologies for extraction of proteins for human consumption from renewable protein sources of plant origin. Trends in Food Science and Technology, 75, 93–104. Rohman, A., & Che Man, Y. B. (2012). Quantification and classification of corn and sunflower oils as adulterants in olive oil using chemometrics and FTIR spectra. The Scientific World Journal, 2012, 1–6. 21
Romoscanu, A. I., & Mezzenga, R. (2006). Emulsion-templated fully reversible protein-in-oil gels. Langmuir, 22, 7812–7818. Sağlam, D., Venema, P., van der Linden, E., & de Vries, R. (2014). Design, properties, and applications of protein micro- and nanoparticles. Current Opinion in Colloid and Interface Science, 19, 428– 437. Scholten, E. (2019). Edible oleogels: how suitable are proteins as a structurant? Current Opinion in Food Science, 27, 36–42. Selmer, I., Kleemann, C., Kulozik, U., Heinrich, S., & Smirnova, I. (2015). Development of egg white protein aerogels as new matrix material for microencapsulation in food. Journal of Supercritical Fluids, 106, 42–49. Sharma, V. K., & Kalonia, D. S. (2004). Effect of vacuum drying on protein-mannitol interactions: The physical state of mannitol and protein structure in the dried state. AAPS PharmSciTech, 5, 58–69. Tan, S. Y., Wan-Yi Peh, E., Marangoni, A. G., & Henry, C. J. (2017). Effects of liquid oil vs. oleogel co-ingested with a carbohydrate-rich meal on human blood triglycerides, glucose, insulin and appetite. Food and Function, 8, 241–249. Tang, Z., Wei, Q., & Guo, B. (2014). A generic solvent exchange method to disperse MoS 2 in organic solvents to ease the solution. Chemical Communications, 50, 3934–3937. Ubeyitogullari, A., & Ciftci, O. N. (2017). Generating phytosterol nanoparticles in nanoporous bioaerogels via supercritical carbon dioxide impregnation: Effect of impregnation conditions. Journal of Food Engineering, 207, 99–107. Wijaya, W., Van Der Meeren, P., Dewettinck, K., & Patel, A. R. (2018). High internal phase emulsion (HIPE)-templated biopolymeric oleofilms containing an ultra-high concentration of edible liquid oil. Food and Function, 9, 1993–1997.
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Table 1. Whey protein particle and oil content, G’, G’’, Tan (at 1 Hz) and critical stress (Pa), of the oleogels produced by oil dispersion of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Procedure for aerogel production
Whey protein particle content (g/100 g)
Oil content (g/100 g)
Critical stress (Pa)
G’ (×105 Pa)
G’’ (×105 Pa)
Tan
Freeze-drying
30.7 ± 0.2
69.3 ± 0.1
0.0756 ± 0.0003
0.011 ± 0.004
0.0048 ± 0.0009
0.455 ± 0.085
Supercritical-CO2drying
15.2 ± 0.4
84.8 ± 0.6
723.2 ± 38.4
3.1 ± 0.1
0.16 ± 0.02
0.066 ± 0.008
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Figure captions Figure 1. Appearance (A) and optical microscopy image (B) of whey protein hydrogel. Figure 2. Appearance (1) and microscopic images at different magnitude (2 and 3) of aerogels obtained by freeze-drying (FD) and supercritical-CO2-drying (SCD). Figure 3. FTIR spectra (A) and second derivative of Amide I region (B) of whey protein isolate, alcoholgel particles, aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). FTIR spectra of the oleogels containing aerogel particles obtained applying FD and SCD are also reported (C). Figure 4. Size distribution in water (A) and oil (B) of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Untreated whey protein isolate was added as reference. Figure 5. Appearance (1) and microscopic structure assessed by optical (2) and confocal (3) microscopy, of oleogels produced by oil dispersion of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Green = oil; Red = proteins. Figure 6. Amplitude sweep (A) and frequency sweep (B) test of oleogels produced by oil dispersion of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Error bars of triplicate measurements were typically not larger than the symbols and were left out for clarity.
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Figure
Figure 1. Appearance (A) and optical microscopy image (B) of whey protein hydrogel.
Figure
Figure 2. Appearance (1) and microscopic images at different magnitude (2 and 3) of aerogels obtained by freeze-drying (FD) and supercritical-CO2-drying (SCD).
Figure
Figure 3. FTIR spectra (A) and second derivative of Amide I region (B) of whey protein isolate, alcoholgel particles, aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2drying (SCD). FTIR spectra of the oleogels containing aerogel particles obtained applying FD and SCD are also reported (C).
Figure
40
Intensity (%)
Reference
A
FD SCD
30 20 10 0 10
100
10000
1000
10000
B
40
Intensity (%)
1000
30 20 10 0 10
100
Figure 4. Size distribution in water (A) and oil (B) of aerogel particles obtained applying freezedrying (FD) and supercritical-CO2-drying (SCD). Untreated whey protein isolate was added as reference.
Figure
Figure 5. Appearance (1) and microscopic structure assessed by optical (2) and confocal (3) microscopy, of oleogels produced by oil dispersion of aerogel particles obtained applying freezedrying (FD) and supercritical-CO2-drying (SCD). Green = oil; Red = proteins.
Figure
1.0E+06 1.0 x 106
A 1.0E+05 1.0 x 105
G', G'' (Pa)
1.0E+04 1.0 x 104 1.0E+03 1.0 x 103 1.0E+02 1.0 x 102 1.0 x 101 1.0E+01 1.0E+00 0.001
G' FD G' SCD 0.01
0.1
1 10 Stress (Pa)
100
1000
G'' FD G'' SCD 10000
1.0E+06 1.0 x 106
B 1.0E+05 1.0 x 105
G', G'' (Pa)
1.0E+04 1.0 x 104 1.0 x 103 1.0E+03 1.0 x 102 1.0E+02
1.0E+01 1.0 x 101 1.0E+00 0.01
0.1
1 10 Frequency (Hz)
100
1000
Figure 6. Amplitude sweep (A) and frequency sweep (B) test of oleogels produced by oil dispersion of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Error bars of triplicate measurements were typically not larger than the symbols and were left out for clarity.
*Graphical Abstract
WPI Aerogel
WPI Hydrogel
WPI Oleogel Weak gel
Freeze-drying
Large aggregates
Dispersion in oil
Supercritical-CO2-drying Small particles Plastic gel
*Credit Author Statement
Author contribution Stella Plazzotta: Investigation, Formal analysis, Writing - Original Draft, Review & Editing Sonia Calligaris: Writing - Review & Editing, Supervision. Lara Manzocco: Conceptualization, Writing - Review & Editing, Supervision
*Conflict of Interest form
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Whey protein aerogel particles show oleogelation ability Hydrogel drying technique affects aerogel and oleogel structure Oil structuring is controlled by surface absorption and particle-particle interactions Aerogel particles form a network in oil thanks to hydrophilic interactions Oleogels from supercritical-dried particles show rheological features of common fats
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GA
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S0963-9969(20)30124-1 https://doi.org/10.1016/j.foodres.2020.109099 FRIN 109099
To appear in:
Food Research International
Received Date: Revised Date: Accepted Date:
24 September 2019 21 December 2019 11 February 2020
Please cite this article as: Plazzotta, S., Calligaris, S., Manzocco, L., Structural characterization of oleogels from whey protein aerogel particles, Food Research International (2020), doi: https://doi.org/10.1016/j.foodres. 2020.109099
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Structural characterization of oleogels from whey protein aerogel particles Plazzotta, S.*, Calligaris, S., Manzocco, L. Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Via Sondrio 2/A, 33100 Udine, Italy *corresponding author: [email protected]; Tel: +39 0432-558571 [email protected]; [email protected] Abstract Oleogels intended as fat substitutes were prepared by oil dispersion of aerogel particles obtained through freeze-drying (FD) or supercritical-CO2-drying (SCD) of whey protein isolate (WPI) hydrogels (20 g/100 g). SEM revealed that freeze-dried particles presented larger dimensions than supercritical-dried ones. The latter also showed higher oil dispersibility, forming aggregates with lower dimension (300 nm) than those formed by freeze-dried particles (700 nm). Both particles presented oil structuring capability. Freeze-dried particles gave a weak oleogel, while supercritical-dried ones gave a strong (G’=3.1×105 Pa) and plastic (critical stress=723.2 Pa) oleogel, with rheological features comparable to those of traditional fats. These results can be explained based on the lower aggregation induced by SCD and on the higher capacity of supercritical-dried particles to form a network in oil through hydrophilic interactions, as suggested by FTIR. Therefore, WPI aerogel particles show the potentiality to be used as food ingredients to prepare oleogels with tailor-made physical properties. Keywords: lipid structuring; fat mimetics; dried template; indirect oleogelation; supercritical drying; freeze drying
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1
Introduction
Proteins are widely used in the food sector for their peculiar functional properties, including their ability to form aqueous gels (hydrogels) under defined physicochemical conditions. Gelation of proteins in water is often induced by heating above denaturation temperature. Heating favours protein unfolding and increases the exposure of hydrophobic groups originally buried inside the molecules. Aggregation is then obtained through surface hydrophobic interactions, leading to the formation of a three-dimensional network entrapping water (Loveday, 2019). Other approaches exploit the change of the aqueous phase pH to values close to the isoelectric point or the increase of ionic force. In these cases, the protein network is formed due to the prevalence of attractive van der Waals forces over electrostatic repulsions. The structure of the resulting hydrogels can be widely modulated by steering formulation and processing parameters (Sağlam, Venema, van der Linden, & de Vries, 2014). Conversely, the use of proteins to obtain oleogels is still a challenge (Scholten, 2019). Oleogels are defined as solid-like materials entrapping oil in a three-dimensional network of structuring molecules (Co & Marangoni, 2012). In recent years, oleogels and relevant oleogelation procedures have attracted large interest in different research areas. In the food sector, oleogels have been mainly proposed as fat substitutes to obtain healthier foods with reduced content of saturated/trans fatty acids (Patel & Dewettinck, 2016). More recently, they have been suggested as delivery systems of bioactive lipophilic molecules and lipolysis modulators during digestion (O’Sullivan, Barbut, & Marangoni, 2016; Tan, Wan-Yi Peh, Marangoni, & Henry, 2017). Although many oil gelators have been identified so far, most of them are liposoluble molecules (e.g. wax and wax esters, ethylcellulose, phytosterols, fatty acid derivatives) whose use in food is often subjected to compulsory limitations (Martins, Vicente, Cunha, & Cerqueira, 2018). The possible use of proteins as oil gelators could thus open new opportunities to enlarge oleogel applications, being proteins cheap, easily available and already widely used in foods (Scholten, 2019). Moreover, this could be an attractive approach meeting the requirements of circular economy. 2
Proteins can be actually derived from waste streams of a number of food production processes, contributing to increasing the sustainability of the overall food supply chain (Pojić, Mišan, & Tiwari, 2018). However, due to their predominant hydrophilic nature, proteins are not able to directly network in oil (Patel, 2018). To overcome this issue and include proteins into a hydrophobic environment, different approaches have been proposed. Romoscanu and Mezzenga (2006) suggested an emulsiontemplate approach. In this case, water is removed from a protein-stabilized emulsion, leading to the formation of a semi-solid high internal phase emulsion (HIPE), containing over 90% of oil. However, these systems do not properly fall under the oleogel definition, since they still contain water (Scholten, 2019). The complete removal of the water phase from oil-in-water emulsions stabilized by a polysaccharideprotein matrix was similarly reported to promote the physical trapping of oil, resulting in oleogels containing over 97% oil (Patel et al., 2015; Wijaya, Van Der Meeren, Dewettinck, & Patel, 2018). Another approach was proposed by de Vries and coworkers (de Vries, Gomez, van der Linden, & Scholten, 2017; de Vries, Hendriks, van der Linden, & Scholten, 2015; de Vries, Jansen, van der Linden, & Scholten, 2018; de Vries, Lopez Gomez, Jansen, van der Linden, & Scholten, 2017). These authors applied a solvent exchange method to generate semi-solid oleogels structured by a network of whey protein aggregates. In this procedure, proteins are firstly denaturated in water to obtain a hydrogel. Water is then progressively replaced by an intermediate solvent (i.e. acetone or THF), which is then substituted with oil. Although the resulting oleogels have tunable physical properties, their preparation requires the use of non-food-grade solvents. Finally, oleogelation by means of dried templates has been also proposed. The basic idea is to remove water from a bio-polymeric hydrogel to obtain a dried template, able to absorb oil. Oil absorption requires the availability of a highly porous material, undergoing limited structural collapse during drying (Manzocco et al., 2017; Patel, Schatteman, Lesaffer, & Dewettinck, 2013). 3
Aerogels are highly-porous ultra-light materials which are prepared by drying of hydrogels, via a suitable technique including air-drying, freeze-drying and supercritical-CO2-drying (García-González, Alnaief, & Smirnova, 2011). Freeze-drying (FD) and supercritical-CO2-drying (SCD) are particularly effective in preventing structural collapse. FD actually prevents capillary tensions and provides structural rigidity (Betz, García-González, Subrahmanyam, Smirnova, & Kulozik, 2012). Alternatively, in the case of SCD, hydrogel water is replaced by ethanol and the latter is then removed using a continuous flow of CO 2 in the supercritical state. This produces minimal capillary forces in the system, preventing structural collapse (Betz et al., 2012; Manzocco et al., 2017). Given their physical features, aerogels have been proposed for different applications, such as drug delivery, microencapsulation of bioactive compounds and aromas, sound insulation, absorption, and catalysis (García-González et al., 2011; García-González, Jin, Gerth, Alvarez-Lorenzo, & Smirnova, 2015; García-González & Smirnova, 2013; Parikka et al., 2017; Selmer, Kleemann, Kulozik, Heinrich, & Smirnova, 2015; Ubeyitogullari & Ciftci, 2017). It is noteworthy that the use of aerogels is currently considered an interesting approach to tackle two of the current main European challenges: active ageing and circular economy (AERoGELS Cost Action, ref. CA18125). In this context, aerogels have been suggested as elective candidates for oleogel preparation. For instance, bio-polymeric monolithic aerogels prepared via FD and SCD showed a good oil absorption capability, leading to oleogels presenting up to 95% oil (Ahmadi, Madadlou, & Saboury, 2016; Manzocco et al., 2017; Plazzotta, Calligaris, & Manzocco, 2019). However, the derived oleogels had a defined shape and a non-plastic structure, which would limit their use as fat substitutes (Plazzotta et al., 2019). These issues could be overcome by the production of aerogel particles able to absorb oil and form a network upon dispersion in oil. To this regard, freeze-dried whey protein particles have been shown to interact in oil by hydrophilic particle-particle interactions, showing a good potentiality as building blocks for oil gelation (de Vries et al., 2015; de Vries, Lopez Gomez, et al., 2017).
4
Based on these considerations, the aim of the present paper was to investigate the possibility of using whey protein aerogel particles obtained by FD or SCD to structure liquid oil. The aerogel particles were analyzed for morphology and dispersibility in water and oil. The oleogels were characterized by microscopy, rheological and FTIR analyses. 2 2.1
Materials and Methods Materials
Whey protein isolate (WPI, 94.7% protein content; 74.6% β-lactoglobulin, 23.8% α-lactalbumin, 1.6% bovine serum albumin) was purchased from Davisco Food International Inc. (Le Sueur, MN, USA) Sigma-Aldrich (Milan, Italy); hydrochloric acid (HCl) was purchased from Carlo Erba Reagents (Milan, Italy); absolute ethanol was purchased from J.T. Baker (Griesheim, Germany); phosphorus pentoxide (P2O5) was purchased from Chem-Lab NV (Zedelgem, Belgium); liquid carbon dioxide (CO2) (purity 99.995%) was purchased from Sapio (Monza, Italy); sunflower oil was purchased in a local market (Giglio Oro, Carapelli, Firenze, Italy), commercial palm oil shortenings and margarines were selected among those typically used for the preparation of bakery goods and were furnished by local food companies; Fast Green and Nile Red dyes were purchased from Sigma Aldrich (Milan, Italy). All solutions were prepared using milli-Q water. 2.2
Preparation of hydrogel
The hydrogel was prepared according to the procedure described by de Vries et al. (2015) with some modifications. In particular, an amount of 0.20 g/g WPI was suspended in water, under continuous stirring at room temperature for 2 h and stored overnight at 4 °C to assure complete protein hydration. The pH of the WPI suspension was then adjusted to 5.7 using a 1 M HCl, which allows the preparation of a structurally stable gel (Betz et al., 2012). Aliquots of 30 mL were then introduced in 50 mL plastic tubes with screwcaps and heated at 85 °C for 15 min using a temperature-controlled water bath to induce 5
protein denaturation. The obtained protein gel was cooled in ice water and homogenized using a highspeed mixer at 13,000 rpm for 3 min (Polytron PT-MR3000, Kinematica AG, Littau, Switzerland). 2.3
Preparation of oleogels via oil dispersion of freeze-dried particles
The hydrogel was frozen at -80 °C for 24 h and freeze-dried for 72 h at 4,053 Pa by using the pilot plant model Mini Fast 1700 (Edwards Alto Vuoto, Milan, Italy), obtaining the aerogel particles. The latter were ground for 1 min using a domestic grinder (MC3001, Moulinex, Milan, Italy) and dispersed into sunflower oil (0.1 g/mL), homogenized by using a high-speed mixer at 13,000 rpm for 3 min (Polytron PT-MR3000, Kinematica AG, Littau, Switzerland) and collected by centrifugation at 13,000 × g for 5 min (Avanti J-25, Beckman, Palo Alto, CA, USA). This procedure was repeated twice, obtaining the oleogel. 2.4
Preparation of oleogels via oil dispersion of supercritical-CO2-dried particles
The hydrogel was firstly converted into alcoholgel as described by de Vries et al. (2015), with some modifications. In particular, ethanol was used instead of acetone as intermediate-polarity solvent. The hydrogel was dispersed into ethanol (0.1 g/mL), homogenized by using a high-speed mixer at 13,000 rpm for 3 min (Polytron PT-MR3000, Kinematica AG, Littau, Switzerland) and collected by centrifugation at 13,000 × g for 5 min (Avanti J-25, Beckman, Palo Alto, CA, USA). This procedure was repeated twice to completely remove water, obtaining the alcoholgel. The latter was dried using the supercritical-CO2-drying plant developed at the Department of Agricultural, Food, Environmental and Animal Sciences (University of Udine) and previously described elsewhere (Manzocco et al., 2017). Alcohol removal was performed at 11 ± 1 MPa and 45 °C with an outlet flow of 6.0 NL/min for 8 h. Decompression from 11 MPa to atmospheric pressure was carried out at 6.0 NL/min in 30 min, obtaining the aerogel particles. The latter were ground and converted into oleogel by following the same procedure applied to the aerogel particles obtained upon freeze-drying (paragraph 2.3). 6
2.5
Analytical determinations
2.5.1
Image acquisition
Sample images were acquired using an image acquisition cabinet (Immagini & Computer, Bareggio, Italy) equipped with a digital camera (EOS 550D, Canon, Milano, Italy). The digital camera was placed on an adjustable stand positioned 45 cm above a black or white cardboard base where the samples were placed. The light was provided by 4 100 W frosted photographic floodlights, in a position allowing minimum shadow and glare. 2.5.2
Optical microscopy
Hydrogel and oleogel samples were gently placed on a glass slide, covered with a cover slide and observed using a Leica DM 2000 optical microscope (Leica Microsystems, Heerbrugg, Switzerland). The images were taken at 200× magnification using a Leica EC3 digital camera and elaborated with the Leica Suite Las EZ software (Leica Microsystems, Heerbrugg, Switzerland). 2.5.3
Scanning electron microscopy
For scanning electron microscopy (SEM), aerogel samples were mounted on aluminium sample holders and sputter-coated with 10 nm of gold using a Sputter Coater 108 auto (Cressington Scientific Instruments, Watford, United Kingdom). The aluminium holder was transferred to the SEM unit (EVO 40XVP, Carl Zeiss, Milan, Italy), which was at ambient temperature and under vacuum. Samples were imaged using an acceleration voltage of 20 kV and SmartSEM v. 5.09 (Carl Zeiss, Milan, Italy) application software was used to capture images of the samples at magnification from 100× to 25,000×. 2.5.4
Confocal microscopy
A 0.2% aqueous solution of Fast Green and Nile Red was used to stain respectively the proteins and the oil of the oleogel samples. After staining, the samples were gently mixed by hand, placed on the 7
microscope slide, covered with a cover slide and observed using a confocal laser scanning microscope at 100× magnification (Leica TCS SP8 X confocal system, Leica Microsystems, Wetzlar, Germany). Images were elaborated using the software LasX 3.5.5 (Leica Microsystems, Wetzlar, Germany). 2.5.5
Apparent density
The apparent density (g/cm3) of the aerogel particles was estimated by weighing 1 mL of dried material in a graded cylinder. 2.5.6
Particle size distribution
Aerogel particles were dissolved (1 mg/mL) in water or oil and sonicated 10 min into an ultrasonic bath (Ultrasonic Cleaner, VWR, Lovanio, Belgium). The mean particle dimension of the obtained dispersions was measured by using the dynamic light scattering instrument Zetasizer Nano ZS (Malvern, Milan, Italy). The angle of observation was 173°. Solution refractive index and viscosity were set at 1.333 and 0.001 Pa ∙ s, and 1.473 e 0.036 Pa ∙ s, corresponding to the values of pure water and sunflower oil at 25 °C, respectively. Particle mean diameter corresponding to intensity distribution was calculated by Distribution Analysis fitting. 2.5.7
Water and oil holding capacity
About 1 g of aerogel particles (P1) was dispersed into distilled water or sunflower oil (1 mg/mL), stirred using a vortex (Vortex 1, Ika, Milan, Italy) three times for 1 min and collected by centrifugation at 13,000 × g for 5 min at 20 °C (Avanti J-25, Beckman, Palo Alto, CA, USA). The supernatant was eliminated and the pellet was centrifuged again to remove the residual non-held solvent. The sediment obtained after centrifugation was weighted (P2). Water and oil holding capacities were calculated as g of water or oil held by 1 g of aerogel particles, following eq. 1: 𝑊𝐻𝐶 (
𝑔𝑤𝑎𝑡𝑒𝑟 𝑔𝑎𝑒𝑟𝑜𝑔𝑒𝑙 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠
) 𝑜𝑟 𝑂𝐻𝐶 (
𝑔𝑜𝑖𝑙 𝑔𝑎𝑒𝑟𝑜𝑔𝑒𝑙 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠
)=
𝑃2 −𝑃1 𝑃1
(eq. 1).
8
2.5.8
Oil content
The oil content of oleogels was estimated based on oil holding capacity (paragraph 2.5.7) and defined as the percentage ratio between the amount of held oil and the overall weight of the sediment (i.e. the oleogel) obtained after centrifugation. The difference between oleogel weight and oil content was used to calculate the whey protein particle content in the gel. 2.5.9
Rheological analysis
The viscoelastic properties (moduli G’, G’’ and Tan ) of the oleogels were tested using an RS6000 Rheometer (Thermo Scientific RheoStress, Haake, Germany), equipped with a Peltier system for temperature control. Measures were performed using a parallel plate geometry at 20 °C with a gap of 2.0 mm. Oscillatory sweep tests to identify the linear viscoelastic region (LVR) were performed increasing stress from 1.0×10-3 to 1.0×103 Pa at 1 Hz frequency. Critical stress (Pa) was identified as the strain value corresponding to a 10% drop in G’ value. Frequency sweep tests were then performed increasing frequency from 0.1 to 120 Hz at stress values selected in the viscoelastic region. 2.5.10 FTIR measurement Spectra were recorded at 25 ± 1 °C by using an FTIR instrument, equipped with an ATR accessory and a Zn-Se crystal that allows the collection of FTIR spectra directly on a sample without any special preparation (Alpha-P, Bruker Optics, Milan, Italy). The “pressure arm” of the instrument was used to apply constant pressure to the samples positioned on the top of the Zn-Se crystal, to ensure good contact between the sample and the incident IR beam. Background scan of the clean Zn-Se crystal was acquired prior to sample scanning. All FTIR spectra were collected in the range from 4000 to 400 cm−1, at a spectrum resolution of 4 cm−1 and with 32 co-added scans. The second derivative of the spectra in the range 1600-1700 cm−1 was obtained by using the OPUS software (version 7.0 for Microsoft Windows, Bruker Optics). 9
2.5.11 Data analysis Determinations of apparent density, particle size distribution, water and oil holding capacity, oil content, and rheological properties were expressed as the mean ± standard error of at least three repeated measurements from two experiment replicates. Statistical analysis was performed by using R v. 3.0.2 (The R Foundation for Statistical Computing). Student’s t-test was used to determine statistically significant differences between means (p<0.05). At least 5 FTIR spectra were acquired for each sample and reported as mean spectra. Microscopy analyses were performed on at least 5 different sample portions to guarantee the representativeness of the presented images. 3 3.1
Results and discussion Characterization of the aerogel particles
A soft hydrogel with self-standing properties (Figure 1A) was obtained by heating and grinding of whey protein solutions (Figure 1A). This structure is due to the formation of disulfide bonds and interactions of hydrophobic residues, allowing the formation of a homogeneous network, well-visible in the microscopic image (Figure 1B) (de Vries, Wesseling, van der Linden, & Scholten, 2017). The hydrogel was subjected to FD or SCD and the resulting dried material was ground to unpack aggregates formed during drying. Obtained particles were analyzed by scanning electron microscopy (SEM) to determine their morphology. Figure 2 suggests that FD led to particles presenting much larger dimensions than those obtained via SCD. This result can be attributed to the ballooning effect of ice crystal growth during FD, which leads to dramatic contraction of the protein backbone and to the maximization of the interactions among protein particles in large aggregates (Tang, Wei, & Guo, 2014). By contrast, during the SCD procedure, proteins were always in a wet condition and evenly dispersed throughout the suspending medium, whose polarity slowly changed from hydrophilic to hydrophobic before solvent removal. These conditions probably favoured particle-solvent interactions, preventing 10
agglomeration of the protein particles (Ganesan et al., 2018). Such observations are supported by literature results showing that whey protein dried particles obtained through evaporation of solvents with a low polarity presented lower size than those obtained upon FD (de Vries, Lopez Gomez, et al., 2017; de Vries, Wesseling, et al., 2017). Higher SEM magnification highlighted that particles obtained upon both FD and SCD were characterized by a continuous matrix with evenly distributed pores (Figure 2). However, particles obtained through SCD presented an apparently finer porous surface as compared to that of freeze-dried particles (Figure 2). The porous nature of the particles was also supported by the apparent density calculation, which was of 0.070 and 0.021 g/cm3 for freeze-dried and supercritical-dried particles, respectively, confirming the higher porosity obtained upon SCD. Such density values are within the density range of aerogel materials (< 0.5 g/cm3) (Fricke & Tillotson, 1997). To understand if the morphological differences among aerogel particles obtained through FD and SCD could be explained based on the effect of the production process on protein conformation, FTIR spectra of the aerogel particles were acquired and compared to that of untreated whey proteins (Figure 3A). In addition, alcoholgel particles were analyzed to assess the effect of ethanol mass substitution on the conformation of protein aerogels obtained via SCD. Both aerogel particles exhibited the typical FTIR spectral bands of proteins, showing a broad tense peak at around 3280 cm −1, which corresponds to the N–H stretching vibration and IR bands at 1648 and 1530 cm−1, corresponding to the amides I and II, respectively (Parris, Purcell, & Ptashkin, 1991). As expected, the alcoholgel particles also showed the typical signals of ethanol, including intense peaks at 2973, 2972, 2881, 1087, 1045 and 880 cm-1 (Debebe, Redi-Abshiro, & Chandravanshi, 2017). The second derivative of the amide I region (1600-1700 cm−1) of the FTIR spectra was computed to better enlighten the changes in the secondary structure of whey proteins (Figure 3B). The untreated WPI showed specific bands in the range 1618-1640 and 1648-1657 cm-1 that have been attributed to β-sheets and α-helix structures of WPI proteins (Haque, Aldred, Chen, Barrow, & Adhikari, 2014; O’Loughlin, Kelly, Murray, Fitzgerald, & Brodkorb, 2015). As compared to 11
the untreated WPI, the spectra of both aerogel particles showed noticeable changes in the bands associated with the original WPI structures, as a consequence of heat-set denaturation of whey proteins. Denaturation actually decreased the occurrence of originally ordered structures in favour of disordered structures and intermolecular interactions. In particular, a decrease of original β-sheet structures was evidenced by the reduction of the peak at 1618 cm-1 and the broadening of the peak at 1628 cm-1. These changes were compensated by a new peak at about 1680 cm-1, associated with intermolecular β-sheets. The latter have been reported to be indicative of aggregation (Allain, Paquin, & Subirade, 1999; de Vries, Lopez Gomez, et al., 2017) and resulted more intense in the freeze-dried particles, confirming the role of FD in inducing intense particle aggregation, which was instead more limited upon SCD. The aerogel particle production process also affected the WPI α-helix structures. In particular, the bands at 1648 and 1657 cm-1 were reduced by the SCD process, while FD caused a considerable band shift, suggesting a reduction in these structures and a change in the hydrogen bonding pattern, respectively (Sharma & Kalonia, 2004). Moreover, the spectrum of freeze-dried particles also showed a new band at 1670 cm-1, which can be related to the presence of unordered random-coil structures. Since the latter result in a gain in conformational entropy, driving irreversible aggregation, their presence further confirms the intense aggregation induced by FD (O’Loughlin et al., 2015). It is noteworthy that the second derivative of the alcoholgel spectra in the Amide I region (Figure 3B) resulted comparable to that of the aerogel, demonstrating that mass substitution was the more important step in determining protein structure. Based on the solubility of ethanol in dense phase carbon dioxide, subsequent SCD allowed limiting further changes in protein conformation. In turn, this possibly accounted for the reduced particle aggregation and the high porosity of the supercritical-CO2-dried particles observed in Figure 2. 3.2
Dispersibility of aerogel particles in water and oil
12
The size distribution of the aerogel particles in both water and oil was assessed (Figure 4). As a reference, the particle size distribution of untreated WPI was also determined. Both particles obtained by FD and SCD showed a mean dimension in water around 1300 nm, which resulted much higher than that of the untreated WPI (350 nm). This indicates a lower water dispersibility of the aerogel particles, which is probably due to the heat treatment applied for inducing WPI gelation. The latter is known to induce the exposure of hydrophobic groups, normally buried within the native folded structure of proteins and to induce the formation of large protein aggregates, confirming the observations relevant to Figure 2 and 3 (Fitzsimons, Mulvihill, & Morris, 2007). It must be noted that the freeze-dried particles resulted less dispersible in water than supercritical-dried ones, as indicated by the broader distribution and the presence of a second peak at larger size dimensions (about 5500 nm) (Figure 4A), confirming that FD provoked a more intense aggregation than SCD, as observed in the SEM images (Figure 2). By contrast, both aerogel particles showed a higher oil dispersibility than WPI. Indeed, the latter presented a largesize peak in oil at about 3000 nm and a second peak at about 120 nm (Figure 4B), while supercriticaldried particles in oil showed a homogeneous monomodal distribution, with particle dimensions around 300 nm. The latter resulted much smaller than those of the freeze-dried ones (700 nm). These results can be attributed to the specific effects of the drying technique on the ability of protein particles to interact with oil. In particular, SCD promoted the formation of small particles with a higher oil dispersibility than the large agglomerates generated during FD (Figure 2 and 3). The different ability of the particles to interact with solvents was also confirmed by the analysis of water and oil binding capacity (WHC and OHC). In fact, the supercritical-dried particles showed WHC and OHC values (4.51 ± 0.10 g water and 5.56 ± 0.09 g oil per g of aerogel particles) significantly higher than those of the freeze-dried one (3.14 ± 0.04 g water and 2.26 ± 0.05 g oil per g of aerogel particles), which is attributable to the lower particle dimension and higher dispersibility in both solvents (Figure 4). 3.3
Oleogels obtained from oil dispersion of the aerogel particles 13
The aerogel particles were dispersed in oil and collected by centrifugation, resulting in the materials shown in Figure 5 and having the composition reported in Table 1. Both aerogel particles presented an oil structuring ability, which can be attributed to their capacity of absorbing oil in the pores (Figure 2) by capillary forces and interacting with oil at the surface by particleoil interactions. In addition, oleogel structuring could further increase upon the formation of a network entrapping oil in the interstitial space among particles. To this regard, it has been shown that an increase of particle-particle interactions through hydrogen bonds in oil could contribute to gel strengthening (de Vries, Gomez, et al., 2017). The particle production process affected the physical properties of the oleogels (Figure 5). When SCD was applied, oleogels presented an oil content considerably higher than that of oleogels obtained by oil dispersion of freeze-dried particles (Table 1). In agreement with previous literature data (de Vries, Wesseling, et al., 2017), oil dispersion of the freeze-dried particles led to a system showing large aggregates coarsely dispersed in oil, as well-visible in the optical microscopy image and further evidenced by the application of confocal microscopy (Figure 5). By contrast, supercritical-dried aerogel particles were turned into an oleogel presenting an interesting plastic and deformable semi-solid texture (Figure 5). Optical microscopy evidenced the presence of a homogeneous network where proteins acted as building blocks (Figure 5). Confocal microscopy showed that this network was formed by highly porous protein particles able to embed the oil within pores as well as to hold it in the interparticle space (Figure 5). This suggests that supercritical-dried particles are able to form a more plastic and interconnected network in oil as compared to freeze-dried particles. To confirm this hypothesis, rheological analyses were performed (Table 1 and Figure 6). At low stress, both systems showed gel-like behaviour (G’ > G’’). With the increase of stress, the critical value was reached, determining the drop of elastic response (Figure 6A). The oleogel obtained from the supercritical-dried aerogel particles showed a linear viscoelastic region (LVR) limit significantly higher than that of the sample obtained from the 14
freeze-dried particles, thus showing a higher resistance to yielding and structure breakdown (Table 1). Figure 6B shows G’ and G’’ of oleogels in the frequency range of 0.01–100 Hz. A weak gel was obtained by oil dispersion of the freeze-dried aerogel particles, as suggested by the dependence of G’ and G’’ on the frequency. By contrast, the oleogel containing supercritical-dried particles was a strong gel, with G’ and G’’ patterns independent on frequency. Moreover, supercritical-dried aerogel particles gave an oleogel much more elastic than that obtained using freeze-dried particles, as shown by the higher G’ and the lower loss tangent (tan = G’’/G’), which measures the relative importance of elasticity in the system (Table 1). The values of G’ and critical stress have been previously indicated as key parameters for comparing oleogel rheological properties with those of traditional solid fats (Blake & Marangoni, 2015). Three commercial laminate fats were also subjected to rheological analyses, giving G’ and critical stress values in the range 8.2-26.7 ×105 Pa and 263.7-995.4 Pa, respectively. It must be noted that the oleogel obtained by the supercritical-dried aerogel particles (Table 1, Figure 6) gave rheological values in the same magnitude order of these commercial solid fats. Such rheological features have been previously obtained in solid monoglyceride emulsions containing 15% carnauba and sunflower waxes (Blake & Marangoni, 2015) or in 10%-ethylcellulose oleogels added with lecithin (Aguilar-Zárate, Macias-Rodriguez, ToroVazquez, & Marangoni, 2019). However, the first systems showed unpleasant waxy taste, not compatible with food, while the latter requires heating the oil at temperatures higher than 150 °C, possibly triggering oxidation (Aguilar-Zárate et al., 2019; Gravelle, Barbut, & Marangoni, 2012). Moreover, the use of these additives in food products is not desirable with respect to green labelling. By contrast, whey proteins used to prepare oleogels in the present paper are widely accepted by consumers and are not associated with negative food labelling impact (Scholten, 2019). The higher yielding resistance and elasticity of the oleogel containing supercritical-dried particles as compared to the one containing freeze-dried particles are much likely attributable to the different surface 15
properties of the dried particles. The lower aggregation (Figure 2) of supercritical-dried particles would guarantee a higher surface available for oil absorption, as well as higher availability of surface hydrophilic groups, which would drive particle-particle interactions in oil, leading to the formation of an interconnected network. By contrast, the intense particle agglomeration induced by FD (Figure 2) would reduce oil absorptive surface as well as the availability of hydrophilic surface groups required for interparticle interactions in oil (de Vries, Gomez, et al., 2017). To confirm these hypotheses, the structural features of the oleogels were further characterized using FTIR analysis, as shown in Figure 3C. Similar to aerogel particles, both oleogel samples exhibited the typical FTIR spectral bands of proteins (3279 cm−1, 1648 and 1530 cm−1, respectively). As expected, oleogel spectra also showed the typical bands of triglycerides, which are the main components of the sunflower oil used for oleogel production. In particular, the band at 3007 cm−1 is attributed to the stretching vibration of =C–H. Strong band absorptions were observed in the region of 3000–2800 cm−1, corresponding to C–H stretching vibrations of methylene (–CH2–) and methyl (–CH3) groups at 2922 and 2853 cm−1, respectively. The large peak around 1740 cm−1 is due to C=O double bond stretching vibration. Deformation and bending of C–H and stretching vibration of C–O result in peaks in the 1250– 1000 cm−1 region (Gurdeniz, Tokatli, & Ozen, 2007; Rohman & Che Man, 2012). It should be noted that there were no noticeable qualitative changes in FTIR profiles between the two oleogel samples produced by oil dispersion of freeze-dried and supercritical-dried aerogel particles (Figure 3C), indicating that the basic structural characteristics of protein and oil fractions in the oleogel samples were nearly unaffected by the production process. Nevertheless, the production process affected the intensity of the peaks at 1648 and 3279 cm−1. The intensity of the first band, which is influenced by hydrogen bonding pattern (Haque et al., 2014), resulted lower in the freeze-dried aerogel particles than in the supercritical-dried ones and was not affected by oil addition. This difference is possibly the result of the effect of drying technique on whey protein structures (Figure 3B), leading to different protein-protein hydrogen bonds. 16
Despite the lower oil content (and thus higher whey protein particle content) of the oleogel obtained using the freeze-dried particles (Table 1), the 3279 cm-1 peak resulted lower as compared to the oleogel containing supercritical-dried particles. Being these bands sensitive to hydrogen bonding strength (Haque et al., 2014), it can be inferred that the particles obtained via FD presented a lower capability of interacting through hydrogen bonding in oil as compared to those obtained via SCD. As a consequence, the freeze-dried particles would show a lower tendency to particle-particle interactions, while the supercritical-dried ones would form in oil a network mainly stabilized by such inter-particle interactions (de Vries, Gomez, et al., 2017). This supports the critical effect of particle networking by surface hydrophilic interactions on the oil structuring ability of aerogel particles. 4
Conclusions
Whey protein aerogel particles present interesting oil structuring functionality. By their simple addition into oil, oleogels with different physical properties, from those of a soft gel to those of a plastic solid fat, can be obtained. These differences can be attributed to the extent of structural collapse and agglomeration of particles during their preparation. The occurrence of these phenomena actually controls the aerogel particle size, porosity, dispersibility in oil, and capability of particle-particle interactions through hydrogen bonding in oil. These features not only tune the oil absorption ability of the particles but also their capacity of forming a network entrapping oil in the interstitial spaces. Whey protein aerogel particles show thus the potentiality to be used as innovative food ingredients to prepare oleogels with tailor-made physical properties. Further research is required to assess the performances of whey protein aerogel particles and the derived oleogels in real food matrices. Moreover, the real applicability of the production process should be carefully assessed. In this regard, the disadvantages of the proposed oleogel production process, which requires long times and dedicated
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equipment, can be counter-balanced by the high added-value and remarkable performances of the final product. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. References Allain, A. F., Paquin, P., & Subirade, M. (1999). Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy. International Journal of Biological Macromolecules, 26, 337–344. Aguilar-Zárate, A., Macias-Rodriguez, B. A., Toro-Vazquez, J. F., & Marangoni, A. G. (2019). Engineering rheological properties of edible oleogels with ethylcellulose and lecithin. Carbohydrate Polymers, 205, 98–105. Ahmadi, M., Madadlou, A., & Saboury, A. A. (2016). Whey protein aerogel as blended with cellulose crystalline particles or loaded with fish oil. Food Chemistry, 196, 1016–1022. Betz, M., García-González, C. A., Subrahmanyam, R. P., Smirnova, I., & Kulozik, U. (2012). Preparation of novel whey protein-based aerogels as drug carriers for life science applications. Journal of Supercritical Fluids, 72, 111–119. Blake, A. I., & Marangoni, A. G. (2015). Factors affecting the rheological properties of a structured cellular solid used as a fat mimetic. Food Research International, 74, 284–293. Co, E. D., & Marangoni, A. G. (2012). Organogels: An alternative edible oil-structuring method. JAOCS, Journal of the American Oil Chemists’ Society, 89, 749–780. COST-Action "Advanced Engineering of Aerogels for Environment and Life Sciences" (AERoGELS, ref. CA18125) funded by the European Commission. https://cost-aerogels.eu/. Last accessed 18
19/12/2019 Debebe, A., Redi-Abshiro, M., & Chandravanshi, B. S. (2017). Non-destructive determination of ethanol levels in fermented alcoholic beverages using Fourier transform mid-infrared spectroscopy. Chemistry Central Journal, 11, 1–8. de Vries, A., Gomez, Y. L., van der Linden, E., & Scholten, E. (2017). The effect of oil type on network formation by protein aggregates into oleogels. RSC Advances, 7, 11803–11812. de Vries, A., Hendriks, J., van der Linden, E., & Scholten, E. (2015). Protein oleogels from protein hydrogels via a stepwise solvent exchange route. Langmuir, 31, 13850–13859. de Vries, A., Jansen, D., van der Linden, E., & Scholten, E. (2018). Tuning the rheological properties of protein-based oleogels by water addition and heat treatment. Food Hydrocolloids, 79, 100–109. de Vries, A., Lopez Gomez, Y., Jansen, B., van der Linden, E., & Scholten, E. (2017). Controlling agglomeration of protein aggregates for structure formation in liquid oil: A sticky business. ACS Applied Materials and Interfaces, 9, 10136–10147. de Vries, A., Wesseling, A., van der Linden, E., & Scholten, E. (2017). Protein oleogels from heat-set whey protein aggregates. Journal of Colloid and Interface Science, 486, 75–83. Fitzsimons, S. M., Mulvihill, D. M., & Morris, E. R. (2007). Denaturation and aggregation processes in thermal gelation of whey proteins resolved by differential scanning calorimetry. Food Hydrocolloids, 21, 638–644. Fricke, J., & Tillotson, T. (1997). Aerogels: production, characterization, and applications. Thin Solid Films, 297, 212–223. Ganesan, K., Budtova, T., Ratke, L., Gurikov, P., Baudron, V., Preibisch, I., … Milow, B. (2018). Review on the production of polysaccharide aerogel particles. Materials, 11, 2144. García-González, C. A., Alnaief, M., & Smirnova, I. (2011). Polysaccharide-based aerogels - Promising biodegradable carriers for drug delivery systems. Carbohydrate Polymers, 86, 1425–1438. 19
García-González, C. A., Jin, M., Gerth, J., Alvarez-Lorenzo, C., & Smirnova, I. (2015). Polysaccharidebased aerogel microspheres for oral drug delivery. Carbohydrate Polymers, 117, 797–806. García-González, C. A., & Smirnova, I. (2013). Use of supercritical fluid technology for the production of tailor-made aerogel particles for delivery systems. Journal of Supercritical Fluids, 79, 152–158. Gravelle, A. J., Barbut, S., & Marangoni, A. G. (2012). Ethylcellulose oleogels: Manufacturing considerations and effects of oil oxidation. Food Research International, 48, 578–583. Gurdeniz, G., Tokatli, F., & Ozen, B. (2007). Differentiation of mixtures of monovarietal olive oils by mid-infrared spectroscopy and chemometrics. European Journal of Lipid Science and Technology, 109, 1194–1202. Haque, M. A., Aldred, P., Chen, J., Barrow, C., & Adhikari, B. (2014). Drying and denaturation characteristics of α-lactalbumin, β-lactoglobulin, and bovine serum albumin in a convective drying process. Journal of Agricultural and Food Chemistry, 62, 4695–4706. Loveday, S. M. (2019). Food proteins: Technological, nutritional, and sustainability attributes of traditional and emerging proteins. Annual Review of Food Science and Technology, 8, 13–29. Manzocco, L., Valoppi, F., Calligaris, S., Andreatta, F., Spilimbergo, S., & Nicoli, M. C. (2017). Exploitation of κ-carrageenan aerogels as template for edible oleogel preparation. Food Hydrocolloids, 71, 68–75. Martins, A. J., Vicente, A. A., Cunha, R. L., & Cerqueira, M. A. (2018). Edible oleogels: An opportunity for fat replacement in foods. Food and Function, 9, 758–773. O’Loughlin, I. B., Kelly, P. M., Murray, B. A., Fitzgerald, R. J., & Brodkorb, A. (2015). Concentrated whey protein ingredients: A Fourier transformed infrared spectroscopy investigation of thermally induced denaturation. International Journal of Dairy Technology, 68, 349–356. O’Sullivan, C. M., Barbut, S., & Marangoni, A. G. (2016). Edible oleogels for the oral delivery of lipid soluble molecules: Composition and structural design considerations. Trends in Food Science and 20
Technology, 57, 59–73. Parikka, K., Nikkilä, I., Pitkänen, L., Ghafar, A., Sontag-Strohm, T., & Tenkanen, M. (2017). Laccase/TEMPO oxidation in the production of mechanically strong arabinoxylan and glucomannan aerogels. Carbohydrate Polymers, 175, 377–386. Parris, N., Purcell, J. M., & Ptashkin, S. M. (1991). Thermal denaturation of whey proteins in skim milk. Journal of Agricultural and Food Chemistry, 39, 2167–2170. Patel, A. R. (2018). Structuring edible oils with hydrocolloids: Where do we stand? Food Biophysics, 13, 113–115. Patel, A. R., & Dewettinck, K. (2016). Edible oil structuring: An overview and recent updates. Food and Function, 7, 20–29. Patel, A. R., Rajarethinem, P. S., Cludts, N., Lewille, B., De Vos, W. H., Lesaffer, A., & Dewettinck, K. (2015). Biopolymer-based structuring of liquid oil into soft solids and oleogels using watercontinuous emulsions as templates. Langmuir, 31, 2065–2073. Patel, A. R., Schatteman, D., Lesaffer, A., & Dewettinck, K. (2013). A foam-templated approach for fabricating organogels using a water-soluble polymer. RSC Advances, 3, 22900–22903. Plazzotta, S., Calligaris, S., & Manzocco, L. (2019). Structure of oleogels from κ-carrageenan templates as affected by supercritical-CO2-drying, freeze-drying and lettuce-filler addition. Food Hydrocolloids, 96, 1–10. Pojić, M., Mišan, A., & Tiwari, B. (2018). Eco-innovative technologies for extraction of proteins for human consumption from renewable protein sources of plant origin. Trends in Food Science and Technology, 75, 93–104. Rohman, A., & Che Man, Y. B. (2012). Quantification and classification of corn and sunflower oils as adulterants in olive oil using chemometrics and FTIR spectra. The Scientific World Journal, 2012, 1–6. 21
Romoscanu, A. I., & Mezzenga, R. (2006). Emulsion-templated fully reversible protein-in-oil gels. Langmuir, 22, 7812–7818. Sağlam, D., Venema, P., van der Linden, E., & de Vries, R. (2014). Design, properties, and applications of protein micro- and nanoparticles. Current Opinion in Colloid and Interface Science, 19, 428– 437. Scholten, E. (2019). Edible oleogels: how suitable are proteins as a structurant? Current Opinion in Food Science, 27, 36–42. Selmer, I., Kleemann, C., Kulozik, U., Heinrich, S., & Smirnova, I. (2015). Development of egg white protein aerogels as new matrix material for microencapsulation in food. Journal of Supercritical Fluids, 106, 42–49. Sharma, V. K., & Kalonia, D. S. (2004). Effect of vacuum drying on protein-mannitol interactions: The physical state of mannitol and protein structure in the dried state. AAPS PharmSciTech, 5, 58–69. Tan, S. Y., Wan-Yi Peh, E., Marangoni, A. G., & Henry, C. J. (2017). Effects of liquid oil vs. oleogel co-ingested with a carbohydrate-rich meal on human blood triglycerides, glucose, insulin and appetite. Food and Function, 8, 241–249. Tang, Z., Wei, Q., & Guo, B. (2014). A generic solvent exchange method to disperse MoS 2 in organic solvents to ease the solution. Chemical Communications, 50, 3934–3937. Ubeyitogullari, A., & Ciftci, O. N. (2017). Generating phytosterol nanoparticles in nanoporous bioaerogels via supercritical carbon dioxide impregnation: Effect of impregnation conditions. Journal of Food Engineering, 207, 99–107. Wijaya, W., Van Der Meeren, P., Dewettinck, K., & Patel, A. R. (2018). High internal phase emulsion (HIPE)-templated biopolymeric oleofilms containing an ultra-high concentration of edible liquid oil. Food and Function, 9, 1993–1997.
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Table 1. Whey protein particle and oil content, G’, G’’, Tan (at 1 Hz) and critical stress (Pa), of the oleogels produced by oil dispersion of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Procedure for aerogel production
Whey protein particle content (g/100 g)
Oil content (g/100 g)
Critical stress (Pa)
G’ (×105 Pa)
G’’ (×105 Pa)
Tan
Freeze-drying
30.7 ± 0.2
69.3 ± 0.1
0.0756 ± 0.0003
0.011 ± 0.004
0.0048 ± 0.0009
0.455 ± 0.085
Supercritical-CO2drying
15.2 ± 0.4
84.8 ± 0.6
723.2 ± 38.4
3.1 ± 0.1
0.16 ± 0.02
0.066 ± 0.008
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Figure captions Figure 1. Appearance (A) and optical microscopy image (B) of whey protein hydrogel. Figure 2. Appearance (1) and microscopic images at different magnitude (2 and 3) of aerogels obtained by freeze-drying (FD) and supercritical-CO2-drying (SCD). Figure 3. FTIR spectra (A) and second derivative of Amide I region (B) of whey protein isolate, alcoholgel particles, aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). FTIR spectra of the oleogels containing aerogel particles obtained applying FD and SCD are also reported (C). Figure 4. Size distribution in water (A) and oil (B) of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Untreated whey protein isolate was added as reference. Figure 5. Appearance (1) and microscopic structure assessed by optical (2) and confocal (3) microscopy, of oleogels produced by oil dispersion of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Green = oil; Red = proteins. Figure 6. Amplitude sweep (A) and frequency sweep (B) test of oleogels produced by oil dispersion of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Error bars of triplicate measurements were typically not larger than the symbols and were left out for clarity.
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Figure
Figure 1. Appearance (A) and optical microscopy image (B) of whey protein hydrogel.
Figure
Figure 2. Appearance (1) and microscopic images at different magnitude (2 and 3) of aerogels obtained by freeze-drying (FD) and supercritical-CO2-drying (SCD).
Figure
Figure 3. FTIR spectra (A) and second derivative of Amide I region (B) of whey protein isolate, alcoholgel particles, aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2drying (SCD). FTIR spectra of the oleogels containing aerogel particles obtained applying FD and SCD are also reported (C).
Figure
40
Intensity (%)
Reference
A
FD SCD
30 20 10 0 10
100
10000
1000
10000
B
40
Intensity (%)
1000
30 20 10 0 10
100
Figure 4. Size distribution in water (A) and oil (B) of aerogel particles obtained applying freezedrying (FD) and supercritical-CO2-drying (SCD). Untreated whey protein isolate was added as reference.
Figure
Figure 5. Appearance (1) and microscopic structure assessed by optical (2) and confocal (3) microscopy, of oleogels produced by oil dispersion of aerogel particles obtained applying freezedrying (FD) and supercritical-CO2-drying (SCD). Green = oil; Red = proteins.
Figure
1.0E+06 1.0 x 106
A 1.0E+05 1.0 x 105
G', G'' (Pa)
1.0E+04 1.0 x 104 1.0E+03 1.0 x 103 1.0E+02 1.0 x 102 1.0 x 101 1.0E+01 1.0E+00 0.001
G' FD G' SCD 0.01
0.1
1 10 Stress (Pa)
100
1000
G'' FD G'' SCD 10000
1.0E+06 1.0 x 106
B 1.0E+05 1.0 x 105
G', G'' (Pa)
1.0E+04 1.0 x 104 1.0 x 103 1.0E+03 1.0 x 102 1.0E+02
1.0E+01 1.0 x 101 1.0E+00 0.01
0.1
1 10 Frequency (Hz)
100
1000
Figure 6. Amplitude sweep (A) and frequency sweep (B) test of oleogels produced by oil dispersion of aerogel particles obtained applying freeze-drying (FD) and supercritical-CO2-drying (SCD). Error bars of triplicate measurements were typically not larger than the symbols and were left out for clarity.
*Graphical Abstract
WPI Aerogel
WPI Hydrogel
WPI Oleogel Weak gel
Freeze-drying
Large aggregates
Dispersion in oil
Supercritical-CO2-drying Small particles Plastic gel
*Credit Author Statement
Author contribution Stella Plazzotta: Investigation, Formal analysis, Writing - Original Draft, Review & Editing Sonia Calligaris: Writing - Review & Editing, Supervision. Lara Manzocco: Conceptualization, Writing - Review & Editing, Supervision
*Conflict of Interest form
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Whey protein aerogel particles show oleogelation ability Hydrogel drying technique affects aerogel and oleogel structure Oil structuring is controlled by surface absorption and particle-particle interactions Aerogel particles form a network in oil thanks to hydrophilic interactions Oleogels from supercritical-dried particles show rheological features of common fats
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GA
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