Effect of cinnamaldehyde on interfacial rheological properties of proteins adsorbed at O/W interfaces

1 Effect of cinnamaldehyde on interfacial rheological properties of proteins adsorbed at O/W interfaces Manuel Felixa,*, Jack Yangb, Antonio Guerreroa and Leonard M. C. Sagisb a Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 41011 Sevilla, Spain. b Laboratory of Physics and Physical Chemistry of Foods, Wageningen University, Bornse Weilanden 9, 6708WG Wageningen, The Netherlands. ______________________ *M. FELIX Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, 41012 Sevilla (Spain) E-mail: mf[email protected] Phone: +34 954557179; fax: +34 954556447. 2 Abstract 1 The dynamics of heterogeneous food products such as emulsions or foams can be affected 2 significantly by the interfacial properties of their interfaces. Proteins are widely used to increase 3 the stability of these food products. This work compares the interfacial properties of a model 4 protein (whey protein isolate, WPI) and silkworm pupae (SLW) adsorbed at the O/W interface. 5 A natural aldehyde (cinnamaldehyde, CNM) was used for both protein systems in order to 6 promote protein-protein interactions. Interfacial properties were characterised during protein 7 adsorption and after reaching a quasi-equilibrium state by means of oscillatory and step 8 dilatational, and oscillatory interfacial shear measurements. The results obtained from dilatational 9 and interfacial shear tests showed that the use of CNM resulted in the development of stronger 10 interfaces, with higher values for the dilatational and surface shear storage moduli, and a lower 11 loss tangent. Step-dilatation tests indicated that the addition of CNM also resulted in more 12 homogeneous interfaces. Our results show that CNM addition can enhance the surface properties 13 of SLW, to a level which is close to the properties of un-modified WPI stabilized interfaces. 14 15 16 17 18 19 20 21 22 23 24 25 Keywords: Dilatational; Interfacial shear; LAOD; Lissajous 26 3 1. Introduction 27 Many food products consist of a kinetically stable combination of two phases (A/W or O/W). 28 These products require the use of surface active agents to avoid their destabilization (which is 29 driven by thermodynamic events) (Damodaran, Parkin, & Fennema, 2007). Proteins are widely 30 used to achieve kinetic stability of food products. The food industry prefers using proteins rather 31 than low molecular weight surfactants (LMWS), since proteins have better consumer acceptance, 32 have nutritional added-value, and are able to modify rheological properties of the continuous 33 phase since they can interact with each other (Tadros, 2013). When proteins adsorb at the 34 interface, they reduce the interfacial tension and they are able to form complex fluid-fluid 35 interfaces, decreasing their lateral mobility (Fuller & Vermant, 2012). The adsorption of proteins 36 at O/W interfaces often results in their unfolding and consequently, their denaturation. After that, 37 the number of exposed hydrophobic residues, the ability to develop protein-protein interactions, 38 as well as the flexibility of the protein structure increase (Dickinson, 1989; Patino et al., 2007). 39 Chemical modification has been used to tailor the hydrophobicity or net surface charge of 40 proteins. The in-situ crosslinking of proteins at O/W interface has been carried out, mainly by the 41 use of enzymes (Fischer & Windhab, 2011; Romoscanu & Mezzenga, 2005), or by using 42 aldehydes, which are not food-grade (Gerrard, 2002). Studies using natural aldehydes (i.e. 43 cinnamaldehyde, CNM) for promoting protein-protein interactions at O/W interfaces are still 44 scarce. 45 Typically, food emulsions have been stabilized most commonly by the use of milk or egg 46 proteins. Whey proteins (WPI) from milk exhibit excellent functional properties (i.e. high surface 47 activity) since their globular proteins have a strong tendency to get adsorbed at O/W interfaces. 48 After protein adsorption, WPI proteins are unfolded and their layers are characterised by a slow 49 reorganization step which involves the development of strong protein-protein interactions. 50 Eventually, a viscoelastic film is formed, in which the proteins are densely packed. Consequently, 51 WPI proteins provide emulsions with excellent stability against physicochemical destabilization 52 phenomena (Dickinson, 2003). There is however an increasing demand from consumers for the 53 4 use of non-animal protein sources. In this sense, insect proteins have gained global attention since 54 their use may reduce the environmental footprint of products. Protein extracts from insects are 55 soluble and they exhibit functional properties like water holding capacity, foaming and 56 emulsifying properties (Zielińska, Karaś, & Baraniak, 2018). Proteins from silkworm pupae 57 (SLW) have also shown nutritional and technological properties (Kim, Setyabrata, Lee, Jones, & 58 Kim, 2016). 59 Proteins adsorbed at O/W interfaces can form interfacial layers which may exhibit viscoelastic 60 or even elastic behaviour. The characteristics of these films are key for understanding the stability 61 of products stabilized by proteins, and interfacial rheology is, therefore, an essential tool for the 62 characterization of the suitability of proteins as a stabilizer for emulsions. Interfacial rheology 63 can be performed in dilatational or interfacial shear mode. Dilatational rheological measurements 64 are most commonly performed using Langmuir troughs or droplet tensiometers. Droplet 65 tensiometers create a droplet at the tip of a syringe and use the Laplace equation to calculate the 66 interfacial tension, either from the droplet profile, or the pressure in the droplet. When the area of 67 the droplet is subjected to a sinusoidal change, the elastic (or storage) and the viscous (or loss) 68 dilatational moduli can be determined from the first harmonic of the Fourier transform of the 69 oscillating interfacial tension signal (Bagley & Torvik, 1983). 70 Interfacial shear rheology has also been proposed as a tool for the characterisation of complex 71 interfaces since it can provide information about inter- and intra-molecular protein-protein 72 interactions at the O/W interface (Krägel, Derkatch, & Miller, 2008). Contrary to dilatational 73 deformations, in interfacial shear measurements, the protein concentration in the adsorbed film 74 does not change during deformation. They also give direct access to the in-plane deviatoric 75 stresses and are not affected by the bending rigidity of the interfacial film. Although previous 76 studies deal with the use of CNM to increase the stability of WPI-based nanoemulsions (Chen et 77 al., 2018; Chen, Wu, McClements, Li, & Li, 2017) and chitosan-based nanoemulsions (Tian, Lei, 78 Zhang, & Li, 2016), these authors did not characterise the changes of the interfaces when CNM 79 is present in the oil phase. They only determined the interfacial tension and analysed images of 80 the pendant drop after compression. 81 5 The aim of this work was to establish the interfacial effects of the natural aldehyde CNM, on 82 the interfacial rheological properties of O/W interfaces stabilized by WPI and SLW, and in 83 particular, to establish if this aldehyde can promote protein-protein interactions to such an extent 84 that the values for the surface rheological properties are increased. To assess these effects, 85 oscillatory dilatational and interfacial shear measurements were carried out. In addition, the 86 response of the interfaces was probed using dilatational step deformation tests. 87 2. Material and methods 88 2.1. Materials 89 BiPRO® was the whey protein isolate used for all measurements (purity 98%). It was supplied 90 by AGROPUR (Longueuil, Canada). Silkworm protein concentrate (SLW, 50.5 ± 0.3 wt.% 91 proteins) was supplied by FeedStimulants (the Netherlands), this protein concentrate was defatted 92 before use, removing other surface active agents apart for protein (e.g. phospholipids) . Medium 93 chain triglycerides (MCT) oil was used as non-polar phase. It was supplied by IOI Oleochemical 94 (France), containing C8 and C10 fatty acids (≥ 98%) and C6 fatty acid (≤ 2%), being free of 95 surface active contaminants. Chemical reagents (i.e. HCl, NaOH, NaH2PO4, cinnamaldehyde) 96 were purchased from Sigma–Aldrich company (St. Louis, USA). The solutions were prepared 97 using Milli-Q grade water. 98 2.2. Methods 99 2.2.1. Preparation of protein solutions 100 Taking into account protein content and solubility, WPI and SLW solutions were prepared at 101 1 wt.% soluble protein (0.1 mg/mL) in 50 mM phosphate buffer (pH 7.0). Glassware was carefully 102 cleaned to avoid the presence of surface-active agents by rinsing it several times with deionized 103 and Mili-Q water. The surface tension of the last wash water was determined using a pendant 104 droplet tensiometer, obtaining a value between 69 and 72 mN/m. Protein dispersions were gently 105 stirred in new plastic containers at 500 rpm over 30 min at room temperature, and subsequently 106 they were centrifuged for 15 min at 15,000 g. Pellets were discarded and supernatants (1 wt.% 107 6 protein solutions) were stored at 4 ºC overnight. Protein solutions were always used the day after 108 preparation. 109 2.2.2 Droplet tensiometry measurements 110 Droplet tensiometry measurements were carried out using a Tracker automatic droplet 111 tensiometer from Teclis scientific (Civrieux d'Azergue, France). An axisymmetric droplet with a 112 volume of 8 μL and a surface area of 18 mm2 was created at the tip of a vertical needle, which 113 was connected to a motorized syringe. The droplet profile was digitized every 0.01 s using a 114 digital camera connected to a computer. Droplet profiles were processed using the Laplace 115 equation (Castellani, Al-Assaf, Axelos, Phillips, & Anton, 2010). All experiments were carried 116 out using a low-absorbance glass cuvette (8 ml). Droplet experiments were carried out in absence 117 of CNM and adding 2 wt.% CNM to the oil phase. These experiments were performed at 20.0 ± 118 0.1 °C. 119 Dilatational properties in the protein adsorption phase 120 Interfacial tension was determined over a period of 10,800 s to monitor the protein adsorption 121 kinetics. Oscillatory dilatational experiments were carried out during the protein adsorption 122 phase, every 500 s. The oscillations (five cycles) were carried out at 5 % amplitude and at a 123 frequency of 0.1 Hz, which was tested to be within the linear viscoelastic regime (results not 124 shown). 125 Dilatational properties in the quasi-equilibrium phase 126 Oscillatory dilatational experiments were carried out to obtain the mechanical spectra of the 127 protein-stabilized O/W interfaces after reaching a quasi-equilibrium state (10,800 s). Frequency 128 sweep tests were performed from 0.075 Hz to 0.1 Hz at 5% amplitude, to obtain the frequency 129 dependence of the interfacial viscoelastic moduli (E’i and E’’i). The dependence of the E’i on 130 frequency was analysed with the following power law equation: 131 𝐸𝐸𝑖𝑖 ′=𝑘𝑘·𝜔𝜔𝑛𝑛′ (1) 132 where 𝜔𝜔 is the frequency and 𝑛𝑛′ is the slope in a double logarithmic representation. 133 7 Step dilatation tests were also performed after the protein adsorption phase. In these tests the 134 interfaces were subjected to a step expansion of 10 or 20 % of the area, and subsequently the 135 relaxation of the interfacial tension was monitored for 1,000 s. After this length of time, the 136 interfacial tension values were no longer changing significantly (p < 0.05) and the area was 137 compressed by 10 or 20 % (compression step). Again, the evolution of the interfacial tension was 138 monitored for 2,000 s. Results were fitted using Matlab R17a (Mathworks, USA) to the following 139 exponential equation, which combines a Kohlrausch-Williams-Watts stretched exponential term 140 with a regular exponential term (Sagis et al., 2019): 141 𝛾𝛾(𝑡𝑡)=𝑎𝑎𝑎𝑎−(𝑡𝑡/𝜏𝜏1)𝛽𝛽+𝑏𝑏𝑎𝑎−𝑡𝑡/𝜏𝜏2+𝑐𝑐 (2) 142 here γ is the interfacial tension, 𝜏𝜏1 is the initial relaxation time, β is the stretch exponent, 𝜏𝜏2 is a 143 characteristic time for ageing processes, which are also present when the interfacial area is not 144 perturbed by a step dilatation. The parameters a, b, and c are constants. 145 2.2.3. Shear measurements 146 Interfacial shear rheology was carried out using an AR-G2 rheometer from TA Instruments (New 147 Castle, USA), with a double-wall-ring geometry (DWR). The protein solutions were poured in a 148 double wall cup and the ring was positioned at the air/water interface. Immediately afterwards, 149 MCT oil was carefully added on top of the aqueous phase to avoid any disturbance of the 150 interface. All shear experiments were carried out using oil phases without CNM and oil containing 151 2 wt.% CNM. All these experiments were carried out at 20.0 ± 0.1 °C. 152 Shear properties during the adsorption phase 153 Interfacial viscoelastic moduli were obtained during the protein adsorption phase (10,800 s) 154 within the linear viscoelastic regime (LVR). These measurements were carried out in stress-155 controlled mode, with an amplitude of 1·10-6 Pa·m for the oscillation stress and a frequency of 156 0.01 rad/s (which was confirmed to be in the LVR by performing strain sweep tests). 157 Shear properties in the quasi-equilibrium state 158 Small amplitude oscillatory measurements (SAOS) were carried out after reaching the quasi-159 equilibrium state (10,800 s) to obtain the mechanical spectra of the O/W interfaces. Frequency 160 8 sweep tests were performed from 0.075 Hz to 0.1 Hz at 1·10-6 Pa·m stress amplitude (which was 161 confirmed to be in the LVR by performing strain sweep tests), to obtain the interfacial viscoelastic 162 shear moduli (G’i and G’’i). The dependence of the G’i on frequency was analysed with the 163 following power-law equation: 164 𝐺𝐺𝑖𝑖 ′=𝑘𝑘·𝜔𝜔𝑛𝑛′ (3) 165 The Boussinesq Number (Bo) was calculated to assess the relative contributions of the adjoining 166 bulk phases and the interface to the total measured stress (Vandebril, Franck, Fuller, Moldenaers, 167 & Vermant, 2010). This number is defined as 168 Bo =𝜂𝜂𝑖𝑖 ∗ (𝜂𝜂𝑂𝑂+𝜂𝜂𝑊𝑊)∙ 𝑅𝑅 (6) 169 where 𝜂𝜂𝑖𝑖 ∗ is the interfacial complex viscosity (units Pa·s·m), and 𝜂𝜂𝑂𝑂 and 𝜂𝜂𝑊𝑊 are the viscosity for 170 the bulk phases (oil and water) and 𝑅𝑅 is the characteristic experimental length scale (0.7 mm) of 171 the DWR geometry. The lowest observed value for Bo was 27.2, and since Bo >> 1, interfacial 172 stresses dominated the bulk response in all shear experiments (Vandebril et al., 2010). 173 2.3. Statistical analysis 174 At least three replicates were carried out for each measurement. Statistical analyses were 175 performed by analysis of variance (ANOVA) using the 95 % confidence limit of the mean. SPSS 176 22 statistical software for Windows (Chicago, USA) was used for this analysis. Standard 177 deviations were calculated for some selected parameters. 178 3. Results and discussion 179 3.1 Droplet measurements 180 Dilatational properties during the protein adsorption phase 181 Figure 1 shows the values obtained for the transient interfacial tension of the O/W interface 182 during the protein adsorption phase (t < 10,800 s), with and without cinnamaldehyde (CNM), for 183 WPI (Fig. 1A) and SLW (Fig. 1B). The kinetics of protein adsorption is characterised by an initial 184 fast decrease in interfacial tension values. This fast decrease was followed by a much slower 185 reduction in the interfacial tension, according to previous studies these results can be related to 186 9 different stages of protein adsorption (from protein diffusion to protein penetration, unfolding and 187 rearrangement) (Manuel Felix, Romero, & Guerrero, 2017; Pizones Ruiz-Henestrosa, Carrera 188 Sanchez, Pedroche, Millan, & Rodriguez Patino, 2009). These results illustrate that for WPI 189 stabilized interfaces, CNM has only a marginal effect on the time-evolution of the interfacial 190 tension. Initially, the interfacial tension decreases at a slightly slower rate, and at the end of the 191 adsorption stage, the interfacial tension is lower when CNM is added by only about 1 mN/m. 192 For SLW the difference is more substantial. The addition of CNM to the oil phase results in a 193 significant decrease in the interfacial tension at the end of the adsorption stage. Several studies 194 indicate that cinnamaldehyde exhibits the ability to interact with proteins, causing protein 195 crosslinking (Balaguer, Gomez-Estaca, Gavara, & Hernandez-Muñoz, 2011; Shrager, 196 Strickholm, & Macey, 1969). This leads to the formation of larger protein species and additional 197 rearrangements in the O/W interface (Balaguer et al., 2011). This could have resulted in a more 198 densely packed structure, with a lower interfacial tension (Dickinson, 1999). However, in WPI 199 stabilized interfaces the protein is already quite segregated and aggregated (Sagis et al., 2019) and 200 additional cross-linking does not lead to more adsorption, which would explain the insignificant 201 drop in interfacial tension. 202 The evolution of dilatational moduli of the O/W interfaces (𝐸𝐸𝑖𝑖 ′ and 𝐸𝐸𝑖𝑖 ′′, respectively) obtained 203 from oscillatory measurements during the protein adsorption phase (t < 10,800 s), with and 204 without CNM, can be observed in Figure 2 for WPI (Fig. 2A) and SLW (Fig. 2B). 205 The dilatational properties show an increase in the elastic modulus as a function of time, 206 whereas the loss modulus undergoes a small decrease. This tendency is more marked at the 207 beginning of the protein adsorption phase and it becomes more gradual after a while. 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LWT, 91, 168–174. 430 https://doi.org/https://doi.org/10.1016/j.lwt.2018.01.058 431 432 433 19 Figure Captions 434 Figure 1: interfacial tension over protein adsorption (10,800 s) at O/W interface with 435 and without CNM for WPI (A) and SLW (B) protein systems. 436 Figure 2: interfacial elastic and viscous moduli (E’i and E’’i, respectively) obtained from 437 dilatational measurements over protein adsorption (10,800 s) at O/W interface with 438 and without CNM for WPI (A) and SLW (B) protein systems. 439 Figure 3: interfacial dilatational moduli as a function of frequency obtained from 440 measurements after 10,800 s adsorption at O/W interface with and without CNM for 441 WPI (A) and SLW (B). 442 Figure 4: interfacial tension for droplet stabilized by 1 % WPI after reaching the quasi 443 equilibrium state (10,800 s) and being subjected at + 10 % expansion and – 20 % 444 compression in the area over 1,000 s. 445 Figure 5: elastic and viscous moduli ( 𝐺𝐺′𝑖𝑖 and 𝐺𝐺′′𝑖𝑖, respectively) obtained from 446 dilatational measurements over protein adsorption (10,800 s) at O/W interface with 447 and without CNM for WPI (A) and SLW (B) protein systems. 448 Figure 6: mechanical spectra obtained from interfacial shear measurements after protein 449 adsorption (10,800 s) at O/W interface with and without CNM for WPI (A) and SLW 450 (B) protein systems. 451 452 20 Table 1 453 Dilatational Shear System n’ tan δi,0.05 n’ tan δi,0.05 WPI 0.15 ± 0.01a 0.13 ± 0.01a 0.94 ± 0.03a 0.28 ± 0.01a WPI-CNM 0.10 ± 0.01b 0.10 ± 0.01b 0.52 ± 0.05b 0.27 ± 0.01a SLW 0.24 ± 0.02c 0.17 ± 0.02c 0.29 ± 0.02c 2.59 ± 0.11b SLW-CNM 0.19 ± 0.01d 0.20 ± 0.01d 0.17 ± 0.01d 0.58 ± 0.05c 454 Table 1: parameters (n’ and tan δi) from mechanical spectra obtained by means of 455 dilatational and shear measurements with and without CNM for WPI and SLW protein 456 systems adsorbed at O/W interface. Different letters within a column indicate 457 significant differences (p < 0.05). Square correlation coefficients (R2) obtained from 458 linear regression for n’ parameter were in all cases ≥ 0.98 459 460 461 21 Figure 1 462 463 464 465 22 Figure 2 466 467 468 469 23 Figure 3 470 471 472 473 474 24 Figure 4 475 476 477 478 479 25 Figure 5 480 481