Assessment of interfacial viscoelastic properties of Faba bean (Vicia faba) protein-adsorbed O/W layers as a function of pH

Assessment of interfacial viscoelastic properties of Faba bean (Vicia faba) 1 protein-adsorbed O/W layers as a function of pH 2 Manuel Felix1, Alberto Romero2, Cecilio Carrera3, Antonio Guerrero1,* 3 1Departamento de Ingeniería Química, Escuela Politécnica superior, Universidad de Sevilla, 41011 Sevilla, 4 Spain. 5 2Departamento de Ingeniería Química, Facultad de Física, Universidad de Sevilla, 41012 Sevilla, Spain. 6 3Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012 Sevilla, Spain. 7 8 9 10 11 12 13 ------------------- 14 *Antonio GUERRERO 15 Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 16 41011 Sevilla, Spain. 17 E-mail: [email protected] 18 Phone: +34 954557179; fax: +34 954556447. 19 Abstract 20 Interfacial rheology may be regarded as a powerful tool dominating the dynamics of complex 21 fluid-fluid interfaces. More specifically, rheological properties from shear measurements have 22 been postulated as the most useful technique for the assessment of the microstructure of 23 complex fluid-fluid interfaces, and its relationship to long-term emulsion stability. The aim of 24 this work was to evaluate the interfacial properties of Faba bean (FB) protein as a function of 25 protein concentration and pH value (3.0, 5.0 and 8.0). The comparison between results obtained 26 from dilatational and interfacial shear measurements provides accurate details related to the 27 microstructure of the protein at the O/W interface. Viscoelastic moduli of the protein adsorbed 28 at the O/W interface were obtained through dilatational measurements (Tracker, IT Concept), 29 as well as interfacial SAOS measurements, using a double wall-ring geometry fitted to a DHR3 30 rheometer (TA Intruments). Results from dilatational rheology indicate that proteins are able to 31 form a film at O/W interface, whose strength depends on the pH value (where E’ ranges from 32 2.0·10-2 Pa·m at pH 5.0 to 5.5·10-3 Pa·m at pH 8.0). However, these measurements have shown 33 to be less sensitive to pH modifications than interfacial shear viscoelastic measurements. 34 Regardless of the pH value, interfacial shear measurements can follow the evolution of the 35 viscoelastic behaviour of the O/W interfacial layer over the development of the protein gel 36 network, as protein adsorption proceeds. This evolution indicates that protein adsorption takes 37 place much faster at pH 3.0 and 5.0 than at pH 8.0. 38 39 Keywords: Faba bean protein; Interfacial Shear Rheology; Linear viscoelasticity; Pendant 40 Droplet; Protein adsorption 41 1. Introduction 42 Interfacial rheology provides key information for understanding the dynamic behaviour of 43 complex fluid-fluid interfaces, which are the basis of not only droplet formation and stability 44 in emulsions, but also structural and mechanical properties (Wijmans & Dickinson, 1998). The 45 adsorption of model globular protein molecules such as β-lactoblobulin, ovalbumin or BSA at 46 O/W has been previously studied (E Dickinson, 1999). In fact, there are successful studies in 47 which the understanding of complex interfaces is provided by their interpretation as a quasi-48 two-dimensional network of rigid spherical particles, which represent globular proteins and are 49 cross-linked (Wijmans & Dickinson, 1998). These models are based on the premise that the 50 rheological properties observed for a globular protein adsorbed at the O/W interface are similar 51 to the ones found for bulk gels (Eric Dickinson, 1998). However, in a mixture between different 52 kinds of proteins (i.e., differing in molecular weight, composition, conformation, etc.), 53 adsorption is very complex. In these cases, the competitive adsorption between different kinds 54 of proteins must be taken into account (van Aken, 2003). For competitive adsorption, models 55 only work for proteins of reagent grade (mainly BSA and β-Lactoglobulin) and molecules such 56 as surfactants (Fainerman, Lucassen-Reynders, & Miller, 2003; Miller et al., 2000). However, 57 commercial food products contain a large number of different proteins as well as other 58 components such as starch, lipids and salts, which makes it impossible to determine the real 59 nature of the different interactions at the O/W interface. Consequently, the interpretation of the 60 behaviour of these complex fluid-fluid interfaces in terms of fundamental interactions is a very 61 difficult challenge. Recently, interfacial rheology has been used to overcome this problem, 62 studying proteins typically involved in cell exchanges or food products (Böttcher, Keppler, & 63 Drusch, 2017; Romero, Verwijlen, Guerrero, & Vermant, 2013; Schroyen, Gunes, & Vermant, 64 2017; Vandebril, Franck, Fuller, Moldenaers, & Vermant, 2010). 65 Dilatational rheology and shear rheology have been used for the interpretation of protein 66 interactions at fluid-fluid interfaces (Danov et al., 2015). The former relates changes in 67 interfacial tension when a droplet suffers oscillatory dilatations, whereas the latter uses an 68 analogous interpretation to three-dimensional bulk systems in interfacial systems (Maldonado-69 Valderrama & Patino, 2010). However, both techniques, dilatational and shear rheology, need 70 to be carried out to improve the interpretation of the protein interactions at the O/W interface. 71 Dilatational rheology has been correlated to short-term stability, whereas interfacial shear 72 measurements have been related to middle or long-term stability of emulsions (Lucassen-73 Reynders, Benjamins, & Fainerman, 2010; Romero et al., 2013). There is a variety of devices 74 for measuring interfacial shear rheology. Among them, disc, bicone or double wall ring (DWR) 75 geometries can be attached to a highly sensitive stress-controlled rheometer to characterise 76 complex fluid-fluid interfaces (Felix, Romero, Sanchez, & Guerrero, 2019; Hooghten et al., 77 2017; Narsimhan, 2016), where DWR is particularly useful, since it minimizes the effect of the 78 subphases on viscoelastic response (which can be estimated through the Boussinesq number) 79 (Felix, Romero, Vermant, & Guerrero, 2016; Vandebril et al., 2010). 80 As an alternative to traditional protein concentrates obtained by wet paths, dry-fractioning aims 81 to be more eco-friendly and cheaper than the typical acidic/alkaline solubilisation and 82 isoelectric precipitation followed by spray-drying; this new technique allows avoiding both the 83 loss of protein functionality, which usually takes place, and the waste produced in the typical 84 isoelectric precipitation (Cloutt, Walker, & Pike, 1986; Schutyser, Pelgrom, van der Goot, & 85 Boom, 2015). The protein concentrate used in this research was obtained through a 86 dry-fractioning process, which is based on the difference of size between starch and protein 87 granules. As an added benefit, this procedure requires a lower amount of energy input (Felix, 88 Lopez-Osorio, Romero, & Guerrero, 2018). Moreover, the food industry is interested in the use 89 of plant proteins as an alternative to milk or egg proteins for the development of O/W emulsions. 90 Plant proteins, such as Faba bean (FB), possess a number of benefits for being used in food 91 applications. Thus, they are cost efficient, exhibit a high nutritional value (Mattila et al., 2018) 92 and they may also help to avoid some well-known allergic problems associated to animal 93 proteins. In addition, Faba bean belongs to the legume family, which is the fifth most grown 94 crop in the world, (Du, Jiang, Yu, & Jane, 2014). 95 The overall objective of this work was to assess the interfacial properties of Faba bean protein 96 concentrate as a function of pH value (3.0, 5.0 and 8.0) in a complex fluid-fluid interfaces, 97 which would contribute to the development of FB-stabilized emulsions. The interfacial 98 behaviour was analysed by means of dilatational measurements (using a pendant drop 99 tensiometer), and interfacial shear rheology (using a DWR geometry attached to a sensitive 100 rotational stress-controlled rheometer). The comparison of results obtained by both techniques 101 may contribute to establish the potential use of FB protein system obtained by dry fractionation 102 in food products such as emulsions. 103 2. Material and methods 104 2.1. Materials 105 The Faba bean(FB) protein concentrate (HerbaPro F65) used within the framework of this 106 research was provided by Herba Ingredients (San José de la Rinconada, Seville, Spain). This 107 protein concentrate was obtained from directly milling dried beans from FB, and, subsequently, 108 the powder obtained was submitted to a densification process. This process consists on the 109 application of a centrifugal counter-flow. After that, particles where the drag force is higher 110 than the centrifugal force are refused and a light effluent, which corresponds to the protein-rich 111 fraction, is generated (Felix et al., 2018). The sunflower oil was purchased from a local producer 112 (Coreysa, Seville, Spain) and other chemical reagents were purchased from Sigma-Aldrich (St. 113 Louis, Missouri, USA). 114 2.2. Protein characterisation 115 The protein content was determined in quadruplicate as % N x 6.25 (Vioque, Alaiz, & Girón-116 Calle, 2012) using a LECO CHNS-932 nitrogen micro analyser (Leco Corporation, St. Joseph, 117 MI, USA). The lipid, moisture and ash contents of the FB protein concentrate were determined 118 in quadruplicate by A.O.A.C. (2000) approved methods. 119 2.3. Interfacial characterization 120 2.3.1. Determination of interfacial pressure at equilibrium 121 Measurements to determine the saturation concentration of O/W interface were performed 122 using a Wilhelmy plate connected to a Sigma D701 tensiometer (KSV, Helsinki, Finland). The 123 measurements were carried out at different protein concentrate amounts (0.1, 0.25, 0.5, 0.75, 124 1.0, 2.0, 3.0, 4.0 and 5.0 wt.%) and at three pH values (3.0, 5.0 and 8.0). Thus, protein 125 dispersions were prepared in 40 mL vessels, the pH was adjusted by adding 2M HCl or NaOH 126 and the temperature was kept at 20 ºC overnight to allow the complete adsorption of the protein 127 at the interface. Then, the Wilhelmy plate was placed at the water/air interface and the vessel 128 was filled up with sunflower oil (generating the O/W interface). The interfacial pressure was 129 measured after obtaining constant interfacial tension values. 130 2.3.2. Pendant droplet measurements 131 Pendant droplet measurements were carried out in order to determine protein adsorption 132 kinetics. Transient interfacial pressure and interfacial dilatational measurements were carried 133 out using the TRACKER pendant-droplet tensiometer (IT Concept, France). An axisymmetric 134 drop was formed at the tip of the needle of a syringe whose verticality was controlled by a 135 computer. The droplet profile was digitized and analysed through a CCD camera coupled to a 136 video image profile digitizer board connected to a computer. Drop profiles were processed 137 according to the Laplace equation as was described by Castellani, Al-Assaf et al. (2010). 138 In addition, the viscoelastic moduli of protein adsorption films were determined at 10% strain 139 amplitude under non-equilibrium at 0.1 Hz, as well as at different frequencies (ranging from 140 0.0075 to 0.1 Hz) once the pseudo-steady state was achieved after 10,800 s. All the experiments 141 were carried out, at least in triplicate. The oil was contained in an optical glass cuvette (8 mL), 142 whereas the protein solution was contained in a syringe (200 μL). Both elements were 143 thermostated at 20.0 ± 0.1 °C (Sánchez & Patino, 2005). 144 2.3.3. Interfacial shear rheological properties 145 The protein solution was placed in a double wall cup and then a double-wall-ring (DWR) was 146 carefully laid at the air/water interface. Subsequently, sunflower oil was carefully added to 147 avoid any disturbance of the interface. The ring was connected to a highly sensitive magnetic 148 air-bearing stress-controlled rheometer (DHR-3, TA Instruments, USA) (Vandebril et al., 149 2010). Prior to frequency sweep tests, stress swept tests were performed to determine the linear 150 viscoelastic region (LVR). Then, mechanical spectra were obtained by means of frequency 151 tests, which were carried out within the linear viscoelastic range. The frequency sweep tests 152 were performed during the protein adsorption (frequency range from 0.314 to 6.14 rad/s) and 153 after reaching the pseudo-equilibrium state (frequency range from 0.06 to 20 rad/s). In addition, 154 time sweep experiments were carried out over 10,800 s at 0.1 Hz to obtain the interfacial 155 mechanical spectra of protein adsorption for elucidating protein-protein interactions. All 156 experiments were carried out, at least in duplicate and the systems were thermostated at 20.0 ± 157 0.1 °C by coupling the double wall cup directly onto the Peltier bottom plate. Moreover, to 158 neglect the contribution of the subphase in interfacial small amplitude oscillatory shear 159 measurements (i-SAOS), the Boussinesq number (Bo) was calculated for all the systems 160 studied. This number can be expressed in terms of a proportion between the viscosities of the 161 interface and the bulk (𝜂𝜂𝑠𝑠 and 𝜂𝜂𝑏𝑏, respectively): 162 𝐵𝐵𝐵𝐵 = 𝜂𝜂𝑠𝑠 𝑎𝑎·𝜂𝜂𝑏𝑏 (1) 163 where 𝑎𝑎 is a characteristic length (0.07 mm for DWR geometry). 164 Bo was above 100 in all cases (Bo > 100). These values indicate that the response obtained is 165 solely related to the interfacial contribution (Erni et al., 2003; Vandebril et al., 2010). 166 2.4. Statistical analysis 167 At least three replicates of each measurement were carried out. Measurement uncertainty was 168 determined by means of standard deviation. Moreover, significant differences (p<0.05) were 169 analysed by means of ANOVA tests (Excel statistical package). 170 3. Results and discussion 171 3.1. Protein system characterisation 172 The chemical composition of FB protein concentrate has been reported in a previous study 173 (Felix et al., 2018). The protein content obtained after the dry-fractioning process reaches up to 174 56.4 ± 0.1 wt.%. Moreover, the densification process let to a concentrate of low fat (4.6 ± 0.3 175 wt.%), moisture (4.3 ± 0.8 wt.%) and ash (4.7 ± 0.1 wt.%) contents. The carbohydrate content 176 (estimated by difference) was up to 30 wt.%. 177 3.2 Characterisation of the O/W interface 178 3.2.1. Determination of interfacial pressure at equilibrium 179 Initially, values of interfacial pressure measured after equilibrium (24 h protein adsorption) are 180 determined by means of a Wilhelmy plate connected to a tensiometer. Interfacial pressure (Π) 181 values are calculated from the interfacial tension values (σ) as follows: 182 𝛱𝛱=𝜎𝜎0− 𝜎𝜎 (2) 183 Where 𝜎𝜎0 is the O/W interfacial tension value measured in the absence of surface active agents 184 (52 mN/m) and σ the experimental interfacial tension value. 185 Results from interfacial pressure as a function of pH (3.0, 5.0 and 8.0) and FB protein 186 concentrate concentration (from 0.1 to 5 wt.%) are plotted in Fig. 1. As can be observed, the 187 increase of protein concentration involved an increase in interfacial pressure, until it reached a 188 constant value. This is the typical behaviour found in other protein/surfactant systems such as 189 milk proteins (Niño & Patino, 1998) or soy proteins (Ortiz, Sánchez, Niño, Añon, & Patino, 190 2003). The interfacial pressure increases when the protein concentration concentration is below 191 the saturation concentration of the O/W interface. However, once the interface is saturated, an 192 increase of protein concentration in the bulk does not involve a higher number of protein 193 molecules at the O/W interface. Consequently, the interfacial pressure does not rise anymore 194 (Sánchez & Patino, 2005). Therefore, according to the results obtained, the saturation 195 concentration for the systems at pH 5.0 and 8.0 is reached at 2 wt.%, whereas at pH 3.0 it is 196 reached at 1 w.%, approximately. These data are important, since all further interfacial 197 experiments were carried out at these FB protein concentrate concentrations, at which the 198 interface is saturated. On the other hand, according to Felix et al. (2018), the pH value 3.0 is 199 below the isoelectric point (IEP) of the protein (3.5). The lower concentration found for pH 3.0 200 for interfacial saturation may be related to protein unfolding, which takes place at this extreme 201 pH value. Thus, at pH 3.0, the unfolded protein can cover the interface completely when the 202 bulk concentration is lower (O. E. Pérez, Sánchez, Pilosof, & Rodríguez Patino, 2008). 203 Furthermore, the highest value of interfacial pressure after reaching the pseudo equilibrium was 204 found for the system at pH 8.0, whereas equilibrium values for the systems at pH 5.0 and 3.0 205 were very similar. These results may be related to the closeness of the minimum solubility of 206 the FB protein concentrate, which involves a lower amount of soluble proteins available to be 207 adsorbed at the interface and, therefore, would lead to lower values of surface pressure. Chang 208 et al. (2015) found similar pH-influence for other legume proteins. Moreover, they hypothesised 209 that interfacial tension is related to an optimum balance between the charge and hydrophobicity 210 of the protein. Thus, according to previous results the efficiency of FB proteins to stabilise the 211 interface at pH 8.0 may be related to the increase in surface charges, as reported previously 212 (Felix et al., 2018). Thus, the zeta potential values found for FB concentrate were ca. 15, -20 213 and -35 mV at pH 3.0, 5.0 and 8.0, respectively. In addition, a reduction of its hydrophobicity 214 where the first frequency sweep after 10 min of protein adsorption indicates that the interfacial 363 shear response is dominated by the viscous component at low frequency. The evolution of the 364 viscoelastic moduli over the adsorption time shows a crossover point, which shifts along protein 365 adsorption from low to high frequencies. Initially, these results may not be considered 366 consistent with the ones obtained from dilatational rheology (where the crossover point was not 367 observed). Results from interfacial shear rheology seem to indicate the ability of the adsorbed 368 proteins to exhibit cohesive interactions with neighbouring molecules (Narsimhan, 2016). In 369 any case, after reaching the pseudo-equilibrium value (i.e. after 180 min) all interfacial O/W 370 protein-based films showed a gel-like response, regardless of the pH value. These results from 371 interfacial shear rheology not only demonstrate that it is possible to monitor the adsorption 372 kinetics of the O/W interface; they also allow following the development of the protein gel 373 network at the interfacial layer by measuring the mechanical spectra during time (rheo-kinetics 374 of the process). 375 Comparison between dilatational rheology and interfacial shear rheology 376 Table 1 compares the tan (δ) values from dilatational and shear measurements (tan(δ)s,0.1 dand 377 tan(δ)s,0.1 s, respectively), as well as the dilatational and shear interfacial elastic moduli 378 (E′(δ)s,0.1 d and G′(δ)s,0.1 d, respectively) obtained at 0.1 Hz after the adsorption of FB protein at 379 the O/W interface. First of all, the low tan (δ)s,0.1 values obtained (either (tan(δ)s,0.1 d or 380 tan(δ)s,0.1 𝑠𝑠) confirm the predominant gel-like behaviour of the interface. However, this marked 381 solid character is different, and depends on the method used. Thus, tan(δ)s,0.1 d values are equal 382 or lower than 0.10 at any pH value. However, tan(δ)s,0.1 𝑠𝑠 values oscillate between 0.2 and 0.3. 383 It is worth mentioning that other low values for the dilatational viscoelastic parameters were 384 previously obtained for a variety of protein-stabilized interfacial layers (Romero et al., 2012; 385 Romero, Beaumal, David-Briand, Cordobes, Anton, et al., 2011; Romero, Beaumal, David-386 Briand, Cordobes, Guerrero, et al., 2011). In addition, the comparison of G′(δ)s,0.1 d values 387 indicates that not only strong interactions at the O/W interface take place, but also the formation 388 of a densely packed interface (Erni, Windhab, & Fischer, 2011; Freer et al., 2004). In any case, 389 this parameter corroborates the strong dependence on pH exerted by the FB protein system, 390 where the net contribution of protein-protein interactions to the interfacial film is similar at pH 391 3.0 and 5.0 (protein surface positively or null net charged), whereas at pH 8.0 the protein 392 interactions are weaker (protein surface negatively charged). This effect may be related to the 393 dependence of surface charges and hydrophobicity on pH values, with the interfacial activity 394 of FB proteins being reduced at alkaline pH. 395 4. Concluding remarks 396 The use of the dry-fractioning process carried out avoids the waste of large amounts of water 397 as well as the exposition of protein to extreme pH values and their undesirable side effects, 398 providing a FB protein concentrate (56.4 wt.%) with high interfacial activity. The results from 399 interfacial pressure (П) after reaching the equilibrium at the O/W interface indicate that the 400 effect of pH on surface charges and surface hydrophobicity (H0) has a remarkable influence on 401 interfacial properties. The higher surface pressure was found in the system at pH 8.0, where the 402 surface is high negatively charged. 403 The results from the dilatational viscoelastic measurements indicate that proteins are able to 404 form a film with a gel-like behaviour at the O/W interface, whose strength depends, once again, 405 on the pH value. However, these measurements have shown to be less sensitive to pH 406 modifications than interfacial shear viscoelastic measurements. Moreover, the evolution of 407 viscoelastic properties can be more clearly followed by this later technique, which shows that 408 protein adsorption takes place much faster at pH 3.0 and 5.0 than at pH 8.0. Regardless of the 409 pH value, i-SAOS measurements can follow the evolution of the viscoelastic behaviour of the 410 O/W interfacial layer over the development of the protein gel network. Thus, interfacial 411 rheokinetics can be useful to determine the sol-gel transition at the interfacial layer. 412 In summary, these results confirm the relevance of performing both interfacial tension and 413 interfacial shear measurements at the O/W interface. The former measurements are relevant for 414 the emulsification process, whereas the later allow following the development of the protein 415 microstructure at the interface (i.e. interfacial rheo-kinetics) which is regarded to be useful for 416 emulsion stability. Additionally, they contribute to confirm that Faba bean protein concentrate 417 obtained by dry fractionation can provide strong O/W interfaces, where the strongest interface 418 was obtained at pH 3.0. 419 5. Acknowledgments 420 The authors acknowledge to Andalusian Government for hiring this research under the research 421 project TEP-6134. Authors also acknowledge to Herba Ingredients for supplying the protein 422 powder used in this research. Eventually, the authors also acknowledge University of Seville 423 for the VPPI-US grant (Ref.-II.5) to Manuel Felix. 424 6. References 425 A.O.A.C. (2000). Official Methods of Analysis. (W. Horwitz, Ed.) (11th ed.). Wisconsin: 426 Association of Official Analytical Chemist. 427 Baldursdottir, S. G., Fullerton, M. S., Nielsen, S. H., & Jorgensen, L. (2010). Adsorption of 428 proteins at the oil/water interface—Observation of protein adsorption by interfacial shear 429 stress measurements. Colloids and Surfaces B: Biointerfaces, 79(1), 41–46. 430 https://doi.org/http://dx.doi.org/10.1016/j.colsurfb.2010.03.020 431 Barnes, H. A. (2000). A Handbook of Elementary Rheology. University of Wales, Institute of 432 Non-Newtonian Fluid Mechanics. 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