Stability of curcumin encapsulated in solid lipid microparticles incorporated in cold-set emulsion filled gels of soy protein isolate and xanthan gum

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Stability of curcumin encapsulated in solid lipid microparticles incorporated in cold-set emulsion filled gels of soy protein isolate and xanthan gum Thais C. Brito-Oliveiraa, Marina Bispoa, Izabel C.F. Moraesa, Osvaldo H. Campanellab,c, Samantha C. Pinhoa,⁎ a b c

Department of Food Engineering, School of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga, SP, Brazil Agricultural and Biological Engineering, Purdue University, West Lafayette, IN, United States Whistler Carbohydrate Research Center, Purdue University, West Lafayette, IN, United States

A R T I C L E I N F O

A B S T R A C T

Keywords: Curcumin Emulsion filled gels Cold-set gelation Soy protein isolate Xanthan gum Solid lipid microparticles

The objective of this study was to investigate the feasibility of producing cold-set emulsion filled gels (EFG), using soy protein isolate (SPI) and xanthan gum (XG) and incorporating curcumin-loaded solid lipid microparticles (SLM). For this purpose, the formulation GXG (15%, w/v SPI, 0.1%, w/v XG and 5 mM CaCl2) was selected for the production of EFG. A comparative study on the rheological and microstructural properties of non-filled gels and EFG revealed that SLM stabilized with Tween 80-Span 80 behaved as active fillers in the gel matrix, increasing the Young's modulus from 1.1 to 2.3 kPa, and also increasing the values of storage and loss moduli. The incorporation of SLM also affected the microstructural organization of the systems. Whereas unfilled gels presented a microstructural organization similar to that of interpenetrated networks, EFG exhibited a microstructure with clear phase separation. The stability of encapsulated curcumin in EFG was monitored using a colorimetric test and it was confirmed that the bioactive component showed a high stability for 15 days. After that period, the color started to change, indicating a decrease in curcumin concentration. The instability of curcumin was probably related to structural alterations of the EFG, which led to decreases of hardness after 7 days of storage at 10 °C, and to the collapse of the structures after 30 days. Although formulation improvements are required, the results indicate that the encapsulation of curcumin in SLM incorporated in EFG is a potential alternative for the replacement of yellow artificial dyes in gelled food products.

1. Introduction Curcumin is a hydrophobic polyphenol obtained from the rhizomes of Curcuma longa, that presents a strong yellowish color and a wide range of beneficial biological activities such as anti-inflammatory, anticancer, anti-microbial and neuroprotective properties (Anand, Kunnumakkara, Newman, & Aggarwal, 2007). Due to these properties, and also to its low toxicity, curcumin is a valuable ingredient to be used by the food industry, as a natural yellow pigment to replace allergenic artificial dyes (Anand et al., 2007; Basnet & Skalko-Basnet, 2011). Despite these characteristics, the application of this compound has been limited due to its high hydrophobicity, poor absorption, low bioavailability and spicy flavor, which can affect the sensory properties of food products (Anand et al., 2007). Among the techniques used to overcome these functional disadvantages, it is the encapsulation of curcumin in lipid carriers, such as the solid lipid particles (SLP). SLP are colloidal systems, similar to oil-

in-water emulsions, in which the oil phase is replaced by solid lipids (La Torre & Pinho, 2015). Besides its capacity to encapsulate, protect and deliver lipophilic functional components, the SLP present many advantages including the possibility of promoting controlled release of bioactive compounds in the absence of organic solvents, and also the possibility of large scale production at a relatively low cost (La Torre & Pinho, 2015; Mehnert & Mäder, 2001). Although the application of SLP may appear appealing to the food industry, special attention is required regarding the sensory characteristic of products containing SLP dispersions (Chojnicka-Paszun, Doussinault, & de Jongh, 2014). In products such as yogurts, dairy desserts, and starch puddings, for example, the incorporation of SLP dispersions may cause undesirable textural changes and decrease their acceptability. In such cases, the development of emulsion filled gels (EFG), which consist of dispersed lipid droplets/particles entrapped in a gelled matrix, could provide a viable alternative to overcome the textural problems (Liu, Stieger, van der Linden, & van de Velde, 2015;

⁎ Corresponding author at: Department of Food Engineering, School of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Av. Duque de Caxias Norte 225, Jd. Elite, Pirassununga 13635-900, SP, Brazil. E-mail address: [email protected] (S.C. Pinho).

http://dx.doi.org/10.1016/j.foodres.2017.09.071 Received 7 July 2017; Received in revised form 22 September 2017; Accepted 25 September 2017 0963-9969/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Brito-Oliveira, T.C., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.09.071

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curcumin (0.03%, w/w) was added to the lipid phase, and the aqueous phase, containing tween 80 (1.8%, w/w) dispersed in deionized water, was poured in the lipid phase. The resulting mixture was mixed using a rotor-stator device (T25, IKA, Staufen, Germany) at 18,000 rpm for 5 min and 80 °C. Sodium benzoate (0.02% m/m) was added to the samples to avoid microbiological spoilage. Immediately after preparation, the samples were subjected to centrifugation at 95g for 5 min at 25 °C (centrifuge Z-216 MK, Hermle, Wehingen, Germany) to remove non-encapsulated curcumin. All samples were prepared in triplicate and stored at a controlled temperature of 10 °C. The average particle size was obtained by a laser diffraction technique (SALD-201 V, Shimadzu, Kyoto, Japan).

Oliver, Scholten, & van Aken, 2015; Sala, Wijk, van de Velde, & van Aken, 2008). These complex systems have been largely investigated, as they present rheological and breakdown properties strongly affected by the characteristics of the gel matrix, lipid fillers and the interactions among the components (Lorenzo, Zaritzky, & Califano, 2013; van Aken, Oliver, & Scholten, 2015). Although a wide range of ingredients may be used to produce EFG, these systems are typically prepared using proteins and polysaccharides (Lorenzo et al., 2013; Vilela, Cavallieri, & Cunha, 2011). In addition, the combined application of these biopolymers may increase the gelling capacity of protein ingredients as the polysaccharides are, under some conditions, able to stabilize protein structures (Lorenzo et al., 2013; Vilela et al., 2011). Among the protein ingredients extensively applied in food preparations is the soy protein isolate (SPI), which has a low cost, high nutritional value and good functional properties, such as the ability to form cold-set gels (Maltais, Remondetto, & Subirade, 2009). Cold-set gelation methods have been increasingly investigated because they allow the incorporation of thermal-sensitive compounds and promote the formation of gel structures in foods, without the need of heating the final product (Alting, de Jongh, Visschers, & Simons, 2002). Although some studies have applied cold-set methods to produce SPI gels (Maltais et al., 2009; Maltais, Remondetto, Gonzalez, & Subirade, 2005; Maltais, Remondetto, & Subirade, 2008), the addition of polysaccharides to these systems was not largely investigated (Chang, Li, Wang, Bi, & Adhikari, 2014; Vilela et al., 2011). It is known that polysaccharides, such as xanthan gum (XG), may be used for the production of cold-set mixed gels through its incorporation to the protein solution before the second step of the gelation process (Chang et al., 2014; Jong, Jan Klok, & Van De Velde, 2009). XG is an anionic microbial polysaccharide, with high molecular weight (average molecular weight exceeds 106 Da), capable of forming mixed gels with protein ingredients, originating systems with different microstructural and rheological properties, which certainly has the potential for the development of new food products (Bertrand & Turgeon, 2007; Bryant & McClements, 2000; Chang et al., 2014). In this context, the main objective of this study was to investigate the feasibility of encapsulating curcumin in solid lipid microparticles (SLM) incorporated in cold-set EFG, produced with SPI and XG. For this purpose, the capacity of SPI to form mixed gels with XG under cold-set conditions was evaluated and the study of the effect of SLM incorporation to the gels was carried out. In addition, the stability of curcumin encapsulated in SLM incorporated in the EFG was evaluated using instrumental colorimetry.

2.3. Production of cold-set mixed gels The production of cold-set mixed gels was performed according to protocol adapted from Maltais et al. (2008). The SPI was hydrated to obtain samples with concentrations 25% higher than the final concentrations desired. Next, the samples had the pH adjusted to 7, were preheated up to 80 °C for 30 min and cooled to room temperature. Then, a concentrated solution of the polysaccharide (0.6%, w/v) was added to the SPI dispersions, which were, subsequently, diluted in a concentrated CaCl2 solution to the final desired concentration of protein, salt and polysaccharide. For the production of the concentrated solution of XG, the polysaccharide was hydrated with deionized water and subjected to magnetic stirring for 2 h, according to protocol adapted from Chang et al. (2014). In order to select the best formulation for the production of the mixed gels, different concentrations of SPI (5–15%, w/v), CaCl2 (0–15 mM) and XG (0.1–0.3%, w/v) were tested and visual phase diagrams were constructed (Perrechil, Sato, & Cunha, 2011). The systems were classified according to their visual appearance as: low viscosity dispersions, viscous dispersions, high viscosity dispersions, non self-supported gels and self-supported gels. The formulation GXG (15%, w/v SPI, 0.1%, w/v XG and 5 mM CaCl2) was selected for the preparation of EFG. For this purpose, different volumetric percentages (50%, 75%, and 100%) of deionized water used to hydrate SPI were replaced by SLM dispersions. After preparation, the samples were stored at 10 °C for 15 h, and visual phase diagrams were constructed. The formulation GXG was also used for the preparation of nonfilled gels with free (non-encapsulated) curcumin and, for this purpose, the bioactive was added to the SPI dispersion before the preheating process.

2.4. Texture profile analyses (TPA)

2. Material and methods

TPA was performed using a texturometer (TA-XT.plus Texture Analyser, Godalming, Surrey, UK), with pretest speed of 3 mm/s, test and post-test speed of 1 mm/s, and 5 mm compression. Gels with 20 mm height and 20 mm diameter were compressed by an aluminum probe (20 mm diameter). Each formulation was analyzed in six replicates, and the parameters hardness, springiness, and cohesiveness were analyzed using the Exponent software incorporated in the equipment.

2.1. Chemicals and reagents Soy protein isolate (SPI, Protimarti M-90, 84.3% protein) was obtained from Marsul (Montenegro, RS, Brazil), and Xanthan Gum (Grindsted Xanthan 80®) was donated by Danisco (Cotia, SP, Brazil). For the production of solid lipid microparticles, palm stearin (melting point = 50.1 °C) was donated by Agropalma (Belém, PA, Brazil), and Tween 80, Span 80 and curcumin (CAS 7727) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were reagent grade. Ultra-pure water (from a Direct Q3 system, Millipore, Billerica, MA, USA) was used throughout the experiments.

2.5. Water holding capacity (WHC) WHC analyses were performed according to Beuschel, Culbertson, Partridge, and Smith (1992). For this purpose, the gel samples were weighed on Whatman paper number 1, placed in a falcon tube type and centrifuged at 6g for 10 min at 6 °C (Hermle centrifuge Labortechnik GmbH, model Z-216 MK, Wehingen, Germany). Subsequently, the samples were removed from the filters and the masses of the papers were determined. WHC values were then calculated according to the Eq. (1).

2.2. Production of curcumin-loaded solid lipid microparticles (SLM) Curcumin-loaded SLM were produced using palm stearin as the lipid phase, and Tween 80 and Span 80 as hydrophilic and lipophilic surfactants, respectively. Initially, the lipid phase, consisting of palm stearin (4.5%, w/w) and span 80 (2.7%, w/w), was maintained at 80 °C for 30 min in order to eliminate the lipid thermal memory. Afterwards, 2

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WHC (%) = 100 −

mf − mi ms

2.9. Uniaxial compression tests (1) Uniaxial compression measurements were carried out according to the protocol adapted from Oliver et al. (2015) using a texturometer (TA-XT.plus Texture Analyser, Godalming, Surrey, UK). The gel samples were prepared in a cylindrical shape of 30 mm height and 20 mm diameter, and compressed to 80% of their original height by an aluminum probe lubricated with silicon oil to minimize friction, using a deformation speed of 1 mm/s. All formulations were analyzed in five replicates, and the values of Hencky stress (σH) and Hencky strain (εH) were obtained from the force-deformation data according to the Eqs. (4) and (5), respectively,

where mf is the mass of the wet filter paper (g), mi is the initial mass of the dry filter paper (g), and ms is the mass of the SPI sample (1–2 g). All systems were analyzed in triplicate. 2.6. Confocal laser scanning optical microscopy (CLSM) Confocal laser scanning microscopy (CLSM) (Inverted Microscope LSM 780 NLO-Zeiss, Zeiss, Germany) was carried out using simultaneous dual-channel imaging, and the protocol adapted from Abhyankar, Mulvihill, and Auty (2014). Rhodamine B and Fluorescein isothiocyanate (FITC) were used to dye the protein and the polysaccharide compounds, respectively. For this purpose, stock solutions of Rhodamine B (0.2%, w/v, in deionized water) and FITC (1 mg/mL deionized water) were prepared and added to the gels in the concentrations 10 μL Rhodamine B solution/mL of gel and 0.05 mL FITC solution/mL of gel. In order to dye the lipid phase, Nile Red solution (0.1 g/100 mL methanol) was prepared and added to the lipid phase (10 μL solution/g lipid) during the production of SLMs. The excitation wavelengths and the emission wavelength ranges applied were: 543 nm and 551–655 nm for Rhodamine B, 488 nm and 493–543 nm for FITC, 543 nm and 551–655 nm for Nile Red. This protocol was adapted from Abhyankar et al. (2014).

σH = F(t).

εH = ln

H (t ) H0. A0

H(t) H0

(4)

(5)

where F(t) is the force at time t, A0 is the initial area, H0 is the initial height, and H(t) is the height at time t. The rupture parameters were associated to the maximum point of the stress-strain curve and the values of apparent Young's modulus (Eg) were determined by the slope of the Hencky stress (σH) against Hencky strain (εH) curves. 2.10. Instrumental colorimetry Gels containing curcumin were monitored by colorimetry in order to evaluate the stability of the bioactive compound using a colorimeter (Miniscan XEm Hunterlab, Reston, VA, USA), with an illuminator D65 and observer at 10° (Geremias-Andrade, Souki, Moraes, & Pinho, 2017). The parameters of the tristimulus color system (L*a*b*) were obtained, and the values of total color difference (TCD), chroma (C*ab) and Hue angle (h°) parameters were calculated using Eqs. (6), (7) and (8), respectively (Pathare, Opara, & Al-Said, 2013). All measurements were performed in triplicate.

2.7. Cryo scanning electronic microscopy (Cryo-SEM) Cryo-SEM tests were performed, according to Spotti, Tarhan, Schaffter, Corvalan, and Campanella (2016), using a FEI Nova Nano630 SEM (Hillsboro, Oregon, USA), with a spot size of 3, and accelerating voltage of 5 kV using an Everhart Thornley detector (ETD). A Gatan Alto 2500 system (Gatan UK, 25 Nuffield Way, Abingdon, Oxon, UK) mounted on the Nova SEM was used for the cryo preparation. Initially, small amounts of gels were mounted on aluminum slot stubs, frozen in liquid nitrogen slush, and, subsequently, transferred under vacuum into the Gatan chamber, were the samples were fractured with a scalpel at − 185 °C. After, the samples were transferred onto the SEM cryo stage set at − 90 °C for sublimation. After the sublimation, the samples were sputter-coated with platinum for 120 s at 15 m/A, and analyzed. All images were recorded using a working distance of 5–6 mm.

TCD =

(L∗ − L0∗)2 + (a∗ − a0∗)2 + (b∗ − b0∗)2

(6)

C ∗ab =

(a∗)2 + (b∗)2

(7)

b∗ h° = tan−1 ⎛ ∗ ⎞ ⎝a ⎠

(8)

2.8. Rheological tests, small strain oscillatory, frequency sweep tests 2.11. Statistical analyses Small strain oscillatory shear tests were carried out, with modifications, according to Chang et al. (2014), in a rheometer (AR2000 from TA Instruments, New Castle, DE, USA) using a parallel plate geometry (60 mm diameter, 1 mm gap). Initially, a 2 min resting time was used in order to equilibrate the samples and eliminate stresses produced by the sample loading. In order to avoid water evaporation, silicone oil was added on the edge of all samples. Strain sweep experiments were performed at 10 °C at a constant frequency of 1 Hz, with a strain sweep varying from 0.1 to 100%, in order to determine the linear viscoelastic regions (LVRs). The frequency sweep tests were carried out using a strain amplitude of 2% (within the determined LVR), over an angular frequency range of 0.016–1.6 Hz, at 10 °C, and the results were analyzed using a power law model given by Eqs. (2) and (3), using the Rheology Advantage Data Analysis V.5.3.1 software (TA Instruments, New Castle, USA) (Chang et al., 2014; Özkan, Xin, & Chen, 2002). ′

G′ = K′. ωn

(2)

G" = K". ωn"

(3)

The means of the treatments were compared using Tukey's tests with a 5% (p < 0.05) significance level on the SAS Software version 9.2. 3. Results and discussion 3.1. Production of cold-set mixed gels The ability of SPI to form cold-set mixed gels with XG was investigated using different concentrations of SPI, XG, and CaCl2, and the results are illustrated in Fig. 1. Self-supported gels were formed only with 15% (w/v) SPI, 0.1% (w/v) XG and 5 mM CaCl2 (formulation GXG). It is accepted that a suitable selection of the protein concentration is fundamental for the successful application of cold-set gelation methods, as it must be low enough to avoid the formation of a threedimensional network during the pre-heating process, and high enough to allow the necessary physico-chemical interactions among the protein molecules, after the salt addition, to form a gelled network (Bertrand & Turgeon, 2007; Vilela et al., 2011). In the present study, 15%(w/v) was a suitable concentration for the production of cold-set gels, as it did not allow the formation of gelled networks in the absence

where K′ and K″ are power law constants, n’ and n” are frequency exponents, and ω is the angular frequency. 3

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Low viscosity dispersion Viscous dispersion High viscosity dispersion Non self-supported gel Self-supported gel

SPI (%)

15

10

15

5 2 0. XG (%)

Cl 2(

1 0.

Ca

5

mM )

10

3 0.

0

(A)

(B)

Fig. 1. Visual phase diagram (A) of mixed systems produced with different concentrations of SPI, XG and CaCl2, classified using visual patters (B) as: low viscosity dispersion, viscous dispersion, high viscosity dispersion, non self-supported gel and self-supported gel.

generally involves compaction and strengthening of these systems (Yang, Liu, & Tang, 2013), however it may only be confirmed through rheological tests and microscopy images. Systems prepared with the replacement of 75% of water by SLM dispersion presented higher values of WHC, hence it was selected for the production, further characterization of EFG and for the investigation of the stability of curcumin encapsulated in SLM incorporated in EFG.

of salt, however it formed self-supported gels in the presence of 5 mM CaCl2. This salt concentration was necessary for the gelation process, as Ca2 + neutralized electrostatic repulsions among the molecules and formed salt bridges among protein aggregates. On the other hand, the increase of CaCl2 concentration was apparently deleterious to the gelation process as observed by other authors (Bryant & McClements, 1998; Maltais et al., 2008). The structures formed during the cold-set gelation of the protein-polysaccharide systems resulted from the competition between gelation and phase separation, both largely affected by the gelation rate and charge neutralization (Jong et al., 2009; Jong & van de Velde, 2007; Vilela et al., 2011). Therefore, whereas low alterations of ionic strength may allow the development of organized structures with a lower possibility of phase separation, the excess of salt in the system may promote phase separation, leading to the weakening of the protein-polysaccharide gels (Bertrand & Turgeon, 2007; Jong et al., 2009; Jong & van de Velde, 2007; Vilela et al., 2011). As observed in Fig. 1(A), such situation was observed in systems produced with 15 mM of CaCl2. Similarly, self-supported gels were not obtained in systems with XG concentrations higher than 0.1% (w/v), probably because under these conditions the volume occupied by the polysaccharide in the structures became too large and did not allow a synergistic balance between the gelation process and the phase separation of protein aggregates and polysaccharide molecules (Chang et al., 2014; Jong et al., 2009). According to Jong and van de Velde (2007), the properties of gels built from aggregated protein strands depend on the number of effective strands, which is largely affected by micro-phase separations. Therefore, depending on the characteristics of the biopolymers used, increases in polysaccharide concentrations in mixed gels may lead to a decrease of effective strands, to phase inversions (polysaccharide continuous gel), or even result in a discontinuous protein network, with no effective strands and, consequently, a liquid aspect (Jong & van de Velde, 2007). Therefore, the visual phase diagram showed in Fig. 1(A) indicated that the formulation GXG was the most appropriate for the production of EFG, through the incorporation of different amounts of stable SLMs (average diameter of approximately 0.6 μm during 60 days of storage) to the systems. It was verified that EFG obtained with replacement of 50% and 100% of water by SLM dispersion exhibited weaker structures, in comparison to the systems produced with 75% replacement. Besides, the incorporation of SLM also affected the WHC of the systems. Whereas non-filled gels presented WHC of 63.1%, the EFG produced with the replacement of 50%, 75% and 100% of water by SLM dispersion presented WHC of, respectively, 67.9%, 68.20% and 62.7%. The increases in WHC with the addition of lipid droplets to EFGs

3.2. Characterization of cold-set mixed gels In order to assess the effect of SLM incorporation in the microstructure of gels prepared with the selected formulation GXG, non-filled gels and EFG were characterized through CLSM and cryo-SEM, and the results are illustrated in Fig. 2. The CLSM images indicated that whereas the non-filled gels produced with formulation GXG (Fig. 2A) exhibited only one phase, the EFG (Fig. 2D) presented a clear phase separation (Jong & van de Velde, 2007). Although many authors have identified the organization observed in non-filled gels as interpenetrate networks, this term may not be completely appropriate, if the non-gelling properties of XG are considered (Saha & Bhattacharya, 2010). In this case, the matrix was formed by the SPI, and, during the gelation process, the XG molecules were able to penetrate and accommodate inside that structure, but not to form another gelled matrix. On the other hand, the presence of SLM increased the thermodynamic incompatibility between the components, not allowing the formation of a homogeneous distribution of XG molecules throughout the gel matrix and resulting in the accumulation of polysaccharide in certain regions of the EFG structure, which is clearly visible in the gels illustrated in Fig. 2(D). It is known that at the pH condition used in the present study (pH 7), both protein and XG are negatively charged, therefore they exhibit electrostatic repulsion between their chains, which generally result in phase separation. However, even in cases of thermodynamically unstable mixed biopolymer systems, the phase separation process may not necessarily occur due to energy barriers associated with the restriction of the motion of the molecules in the network (Bryant & McClements, 2000), as previously discussed. Through the cryo-SEM images (Fig. 2B and C), it was observed that the non-filled gels exhibit a denser matrix, with small pores, and a more homogeneous structure than that observed in the EFG samples (Fig. 2E and F), which show a heterogeneous matrix, with many distinct regions, different structural properties and a more porous structure. The cryo-SEM images also showed the SLM were distributed throughout the EFG and appeared to be physically connected to the matrix. This physical interaction between the gelled matrix and the SLM may have been 4

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Fig. 2. Confocal laser scanning micrographs (CLSM)* (A, D) and cryo-MEV (B, C, E, F) images of non-filled (A, B, C) and emulsion filled gels (D, E, F) with formulation GXG. *The structures formed by SPI, LBG and SLM were identified at CLSM images, respectively, by dark red, yellowish-green and light red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

that the incorporation of SLM increased the strength of the matrices, as previously discussed, but also resulted in a small decrease in the of elasticity of the systems, probably due to the disruption of the microstructural organization of the gel due to phase separation. The strengthening of the gels with the incorporation of SLM was also confirmed by the results obtained from the uniaxial compression tests (Table 1). Depending on the effect of the droplets/particles incorporation to the gels, fillers are generally classified as active or inactive. It is well accepted that the addition of active fillers may lead to an increase or decrease in Eg, a decrease in fracture strain (εF), and an increase in fracture stress (σF), whereas the addition of inactive fillers leads to a decrease in Eg, an increase or decrease in εF, and a decrease in σF (Oliver et al., 2015). In the present work, however, the results showed that EFG presented higher values of Eg, σF and εF, indicating that SLM behaved as active fillers inside the matrix, as previously discussed, but they did not act as structure breakers, probably due to a good distribution of the droplets in the gelled matrix and the absence or small degree of aggregation of particles (Liu et al., 2015; Oliver et al., 2015), which was confirmed by cryo-SEM images (Fig. 2).

a result from the different sizes of the polar heads of the surfactants applied (larger for Tween 80 than Span 80), which may have produced inhomogeneity in the interface, in which some groups of the protein and/or polysaccharide chains were probably able to anchor. The effects of these different microstructures in the rheological properties of the gels were investigated by small strain oscillatory tests. From the results of strain sweep tests, showed in Fig. 3(A), the linear viscoelastic regions of the samples were determined, and a strain amplitude of 2% was selected for the development of frequency sweep tests. The mechanical spectra of non-filled gels and EFG are illustrated in Fig. 3(B), and they both show values of G′ higher than those of G″, as expected for gel-like systems. Although the systems presented a similar behavior, with a low dependency on frequency, the EFG presented higher values of G′ and G″ in comparison to the non-filled gels, indicating that the presence of SLM strengthened the gel system. These results were confirmed through the power law parameters, which are reported in Table 1. EFG systems had higher values of K′ and K″, reflecting the increase on strength of the matrices with the incorporation of SLM and lower values of n″. The low values of n′ and n″ confirmed that the viscoelastic parameters obtained were slightly frequency-dependent, which, according to Spotti et al. (2016), indicates that both systems may be classified as strong gels, resembling covalent or chemical gels. Results illustrated in Fig. 3(C) indicate that both non-filled and EFG had a tan δ (G″/G′) having a slight frequency dependence, which was more evident at the low frequency range. A comparison of the two systems revealed only a small difference between non-filled gels and EFG, however, the addition of SLM increased tan δ and also led to a small increase in the frequency dependence of this parameter. Low values of tan δ, with low frequency dependence, are generally associated to more elastic systems (Rocha, Teixeira, Hilliou, Sampaio, & Gonçalves, 2009). In this context, these results indicated

3.3. Stability of curcumin encapsulated in SLM incorporated in cold-set EFG Curcumin was encapsulated in SLM incorporated to EFG using the selected formulation GXG. The curcuminoid was also incorporated in non-filled gels (as free curcumin). The visual appearance of both systems is shown in Fig. 4. The addition of non-encapsulated curcumin to the gels resulted in systems with a brownish color and with an appearance of a non self-supported gel. The weakening of the gel with the addition of free curcumin may be explained by the complexation of this bioactive with SPI. According to Tapal and Tiku (2012) curcumin molecules may bind to non-polar regions of SPI by hydrophobic interactions and this phenomenon may be used to enhance the water solubility 5

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10000

Non-filled gel - G''

Non-filled gel - G' Non-filled gel - G" EFG - G' EFG - G'' Model

10000

EFG - G'

G', G'' (Pa)

G' G" (Pa)

EFG - G''

1000

100

10 0.1

1

10

100

Fig. 3. Results of strain sweep tests (A) and frequency sweep tests (B, C) of non-filled and emulsion-filled gels with formulation GXG.

1000

100

10 0.01

Strain (%)

0.1

1

10

Angular frequency (Hz)

(A)

(B)

Non-filled gel

1

Tanδ

EFG

0.1 0.01

0.1

1

10

Angular frequency (Hz)

(C)

120

Table 1 Power law parameters and uniaxial compression test results of the gels and emulsion filled produced with different formulations.

G′ = K′.ω

n′

G″ = K″.ω

Eg (kPa) σF (kPa) εF

n″

Non-filled gels K′ n’ R2 K″ n″ R2

b

591.8 ± 16.0 0.1100 ± 0.0022a 0.997 99.2 ± 4.2b 0.0872 ± 0.0052a 0.924 1.14 ± 0.16b 0.31 ± 0.02b 0.15 ± 0.02b

EFG

b

80

1306.1 ± 108.0a 0.1044 ± 0.0033a 0.995 237.8 ± 2.8a 0.0730 ± 0.0065b 0.871 2.29 ± 0.30a 0.59 ± 0.06a 0.26 ± 0.02a

Parameter

Parameters

a 100

a

b

60

b

a

a

a* b*

b

L* 40 C*ab h°

20

0

Non-filled gel Means followed by different lowercase letters in the same line are statistically different (p < 0.05).

-20

a

EFG a Formulation

Fig. 4. Visual aspects and colorimetric parameters of the non-filled gels with non-encapsulated curcumin (free curcumin) and the EFG produced with the incorporation of curcumin-loaded solid lipid microparticles. Averages followed by different lowercase letters, to the same parameter, are statistically different (p < 0.05).

and stability of curcumin, which depend on the concentrations of each component. In the present study, however, due to the high concentrations of SPI and the low concentrations of curcumin, the complexation between those ingredients probably resulted in a decrease of proteinprotein interactions and affected the competition between processes of gelation and phase separation, which was discussed above, leading to the weakening of the gelled structure. The brownish color observed in gels obtained with incorporation of free curcumin also highlighted the importance of the encapsulation of this bioactive compound. It is known that curcumin is unstable at neutral-basic conditions and presents a fast decomposition into compounds like vanillin and ferulic acid (Kumavat et al., 2013). According to Bernabé-Pineda, Ramírez-Silva, Romero-Romob, González-Vergara, and Rojas-Hernández (2004) if the pH is adjusted to neutral-basic conditions, the curcumin molecule is deprotonated, originating red solutions. In this context, the brownish color of the non-filled systems with free curcumin may be explained by the decomposition of the bioactive at a pH of 7 during the cold-set gelation. On the other hand,

EFG containing encapsulated curcumin had a strong yellowish color, which is expected from curcumun (Anand et al., 2007; Basnet & SkalkoBasnet, 2011). The color differences between the gels, observed by the colorimetric parameters, are also illustrated in Fig. 4, especially b*, which takes positive values for yellowish and negative values for bluish colors, whereas L*, is a measurement of luminosity (Pathare et al., 2013). It was confirmed that gels with free curcumin had less yellowish tones and reduced luminosity in comparison to the gels containing encapsulated curcumin, evidencing the reduction of curcumin concentration when it is in a free form, mainly due to accelerated oxidative processes. As expected, differences between values of a*, b* and L* also reflected in the values of Chroma and Hue angle. 6

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Table 2 Values of the colorimetric parameters of EFG produced with curcumin-loaded SLM, during the storage period. Period of storage (days)

L*

a*

1 3 7 15 22 30 60

67.24 68.72 68.36 67.44 66.77 66.47 61.64

± ± ± ± ± ± ±

− 8.45 − 9.18 − 9.15 − 9.09 − 8.53 − 8.19 − 3.56

0.54a 0.96a 0.92a 1.15a 1.69a 1.34a 3.05b

b* ± ± ± ± ± ± ±

0.44a 0.17a 0.34a 0.26a 0.77a 0.79a 2.27b

62.02 60.61 61.71 61.43 68.95 69.05 56.59

Chroma (C*ab) ± ± ± ± ± ± ±

1.91b 1.19bc 3.13b 2.06b 2.67a 2.89a 2.45c

62.68 61.34 62.40 62.14 69.59 69.45 57.06

± ± ± ± ± ± ±

Hue Angle (h°)

1.93b 1.20bc 3.13b 2.06b 2.65a 2.90a 2.37c

97.90 98.83 98.55 98.35 96.62 96.62 93.40

± ± ± ± ± ± ±

0.28abc 0.15a 0.31a 0.25ab 0.68bc 0.64c 2.36d

Total color difference (TCD) – 2.12 1.83 1.97 7.03 7.19 8.97

± ± ± ± ± ±

0.20c 0.48c 0.50c 0.85b 0.27b 1.98a

Averages followed by different lowercase letters in the same column are statistically different (p < 0.05).

structural alterations of the gel systems, confirmed through results of TPA and WHC, which are illustrated in Fig. 5. Gels are considered very complex materials, as they exhibit highly nonlinear, transient and nonequilibrium mechanical and chemical behavior occurring in a metastable state (Teece, Faers, & Bartlett, 2011). According to Renard, van de Velde, and Visschers (2006), due to their metastable state, the structure of gels changes with time, either spontaneously (i.e. ageing phenomena) or due to external forces. In the present work, it was confirmed that the ageing phenomena resulted in significantly changes on the characteristics of the EFG systems, especially hardness. An increase in hardness was observed during the first seven days of storage, which may be explained by structural rearrangements leading to a coarsening of the systems. Different studies found in the literature state that ageing commonly involves a gradual coarsening of the structures and a change of gels' firmness (Chang & Leong, 2014; Renard et al., 2006; Teece et al., 2011). According to Alting, Hamer, Kruif, and Visschers (2003), who investigated the properties of acid-induced cold-set WPI-gels, open clusters of aggregates formed due to a decrease of electrostatic repulsion, during the second step of the cold-set gelation process, are thermodynamically unstable in comparison to a denser cluster of protein. Therefore, the initial weak and fragile structure of these clusters can be partly stabilized by the formation of additional covalent disulfide bonds, leading to locally denser clusters and a more turbid gel. The authors also discussed that those rearrangements may lead to the formation of larger pores and a more permeable microstructure affecting the WHC of the systems, which was not verified in the present work. Conversely, after the 7th day of storage, it was observed a decrease of hardness and a small increase of WHC until the 30th day of storage. After this period, the EFG collapsed, presenting the appearance of non self-supported gels, which did not allow TPA and WHC determinations. Such phenomenon may be explained by alterations on the interactions among the SLM and the gel matrix on the EFG. As previously discussed, SLM produced with palm stearin as the lipid phase and stabilized with Tween 80–Span 80 seemed to be physically connected to the matrix. After the 7th day of storage, the

The stability of the encapsulated curcumin in EFG was monitored during 60 days of storage through colorimetry, as evaluations of colorimetric parameters may be considered simple but powerful alternatives for indirect measurement of pigment content in food products (Pathare et al., 2013). Results presented in Table 2 showed that the parameters L* and a* (which takes positive values for reddish and negative values for greenish colors) remained constant for 30 days. On the other hand, the parameter b* presented significant alterations after 15 days. The behavior of these parameters was also reflected by the values of Chroma, Hue angle and TCD. Values of Chroma, which are considered to be a quantitative attribute of colorfulness, increased after 15 days of storage and significantly decreased after 30 days, indicating that the decrease of color intensity of the samples may only be perceived by humans after 30 days of storage (Pathare et al., 2013). Similarly, the values of Hue angle exhibited significant changes in comparison to the first day of storage, only between days 30 and 60. Hue angle is considered to be a qualitative attribute of color, and it is related to the differences in absorbance at different wavelengths. According to the literature, angles of 90°, 180° and 270° represent, respectively, yellow, green and blue hues, so the results indicated that the samples had a greenish yellow color, characteristic of the curcumin molecule, during all the storage period (López et al., 1997; Pathare et al., 2013). Another important parameter estimated was the TCD, which stands for, in this case, the magnitude of color difference between the day of sampling and the first day of storage. This parameter allows the analytical classification of the colors as very distinct (TCD > 3), distinct (1.5 < TCD < 3) and colors with small difference (TCD < 1.5) (Adekunte, Tiwari, Cullen, Scanell, & O'Donnell, 2010). In this context, it was confirmed that the samples exhibited distinct colors at the first 15 days of storage and very distinct colors only after the 22nd day. Evaluation of those parameters indicated that until the 15th day of storage the encapsulated curcumin had a relatively high stability. Conversely, after this period the concentration of the bioactive on the EFG started to decrease. Those results may be a consequence of

a a

Parameter

0.8

a

a ab

0.6

ab

a

b

80

a

a b

b 0.4

100

a

ab

a

0.2

c

cd

WHC (%)

1

c

bc

abc

a

ab c

60 40 20

d 0

0

1

3

7

15

22

30

1

3

7 15 22 Storage period (days)

Storage period (days) Hardness (N)

Springiness

Cohesiveness

(A)

(B) 7

30

Fig. 5. Results of TPA (A) and WHC (B) of EFG produced with curcumin-loaded SLM, during the storage period. Averages followed by different lowercase letters, to the same parameter, are statistically different (p < 0.05).

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São Paulo for the fellowship of Marina Bispo. The authors also thank Agropalma and Danisco for donating the palm stearin and xanthan gum, respectively, the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABiC) at the State University of Campinas (Unicamp) for the access to the Inverted Microscope LSM 780 NLO-Zeiss (Zeiss, Germany), and Dr. Robert Seiler from Life Science Microscopy Facility in Purdue University for his assistance in cryo-SEM.

structural rearrangements of the gel may have harmed the anchoring of some groups of the protein and/or the polysaccharide chains on the spaces existent in the interface of the particles, decreasing the strength and the hardness of the gels. The decrease of the matrix-SLM interactions probably allowed an increase of the matrix-water interactions, as observed through the small increase of WHC. Probably due to this process, after the 30th day of storage, the interactions among SLM and the biopolymer matrix were reduced, and the fillers started to act as structural defects of the matrix, drastically reducing the number of effective strands and resulting in a discontinuous mixed network, with a more liquid appearance. Recently, different researchers have reported the sudden collapse of aged gels (Bartlett, Teece, & Faers, 2012; Buscall et al., 2009; Chang & Leong, 2014; Teece et al., 2011). In their research, Teece et al. (2011) explained that, during the mechanisms of gels ageing, the relatively stable network, capable of supporting its own weight, starts to yield and clear interfaces appear. Then, those interfaces start to grow smoothly, as the gel shrinks and a rapid collapse occurs, when phase separation is almost completed and the interface approaches one final equilibrium plateau. According to these authors, collapse is one of the most dramatic macroscopic manifestations of gel ageing, and requires more attention, especially because a quantitative prediction of gel stability is an important issue in the formulation of many commercial products. Although in the present work a collapse of the structures was observed, the EFG systems did not exhibit continuous shrinking, instead a gradual alteration resulting in decrease of hardness was observed. In this context, those results highlighted the importance of the investigation of the ageing process of EFG, especially considering the complexity and specificities of the different systems, and the lack of studies approaching this important issue. Although structural alterations during storage time led to the collapse of EFG indicating that formulation improvements are required, the high stability of encapsulated curcumin up to 15 days of storage, and the relatively low rate of color changes up to the 30th day, indicate that the encapsulation of this bioactive compound in SLM incorporated in an EFG may be considered a potential alternative for future applications on delivery efficient of bioactive compounds.

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