Viscoelastic properties, microstructure and stability of high-oleic O/W emulsions stabilised by crayfish protein concentrate and xanthan gum

Viscoelastic properties, microstructure and stability of high-oleic O/W 1 emulsions stabilised by crayfish protein concentrate and xanthan gum 2 Manuel Felix*, Alberto Romero, Antonio Guerrero 3 Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012 4 Sevilla, Spain. 5 Abstract 6 This work is focused on the evaluation of crayfish protein concentrates in the formation and 7 stabilization of low-fatty and high-oleic emulsions, as a function of pH, with the presence of a 8 polysaccharide (Xanthan Gum) as stabilizer. Interfacial O/W characterisation is carried out in 9 order to determine the protein concentration in which the saturation of the interface takes place, 10 protein adsorption kinetics, as well as to predict the emulsions stability. Emulsion 11 characterization is based on rheological characterization and the microstructure is evaluated 12 through droplet size distributions (DSD) analysis and Confocal Laser Scanning Microscopy 13 (CLSM). In addition, backscattering measurements are relevant to describe the destabilization 14 mechanism Results indicate that emulsion ability and stability are highly sensitive to the pH 15 value. In our conditions, the best emulsifying properties corresponds to emulsion carried out at 16 pH 3, which shows unflocculated unimodal distributions, remaining unaltered in size over 2 17 months. However, the occurrence of coalescence takes place at pH 5 and 8. In addition, results 18 support the key role of the interaction between proteins and polysaccharide (Xanthan gum, XG), 19 which form a gel-like network. 20 Keywords: Protein adsorption, Crayfish protein, Droplet Size Distribution (DSD), Emulsion, 21 Linear viscoelasticity, Xanthan Gum. 22 ------------------- 23 *Manuel FELIX 24 Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012 25 Sevilla, Spain. 26 E-mail: [email protected] 27 Phone: +34 954557179; fax: +34 954556447. 1. Introduction 28 Every year the food and agricultural industry produces a big amount of surpluses and wastes 29 which are discarded or used as low added value by-products (David & Livney, 2016). A 30 particularly relevant example of this environmental problem, found in western Andalusia (in 31 the south of Spain), is associated to the red-swamp crayfish. This crustacean was introduced in 32 the second half of the twentieth century and has been considered a fairly invasive species, 33 undergoing a fast widespread growth due to favourable weather conditions, abundant food and 34 the lack of predators (Kirjavainen & Westman, 1999). As a result of this rapid growth, a strong 35 local crayfish industry has been developed at the marshes of the Guadalquivir River (Geiger, 36 Alcorlo, Baltanas, & Montes, 2005). A currently attractive way to valorise the by-products 37 generated by this industry consists of taking benefits from their high protein quality and content. 38 In fact, crayfish constitutes an excellent source of high nutritional ingredients that include not 39 only high-quality proteins, rich in essential amino acids, but also healthy lipids such as ω-3 40 polyunsaturated fatty acids and other bioactive components such as astaxanthin (Hamed, 41 Ozogul, & Regenstein, 2016; Miki, 1991). 42 Moreover, the food industry has a great interest in the production of oil-in-water (o/w) 43 emulsions stabilised by high-quality proteins in order to develop new and healthier formulations 44 for food emulsions such as salad dressings. Proteins have been widely used as emulsifier for 45 oil-in-water (O/W) emulsions since they facilitate the breakage of oil drops and prevent droplets 46 coalescence over emulsification and storage by increasing repulsion forces between droplets 47 (Dickinson, 2003; McClements, 2004). Traditionally, egg yolk has been used as emulsifier in a 48 wide variety of sauces. However, many other alternative proteins such as plant proteins have 49 been used as substitutes of egg yolk in the stabilization of O/W emulsions (Batista et al., 2011; 50 Najafi, Hosaini, Mohammadi-Sani, & Koocheki, 2016; Walstra, 1993). More recently, 51 replacement by proteins from fish is being extensively studied (Choi, Hur, Choi, Konno, & 52 Park, 2009; Morales-Medina, Tamm, Guadix, Guadix, & Drusch, 2016; Romero, Cordobés, 53 Puppo, Guerrero, & Bengoechea, 2008). 54 The lifetime and stability of a final emulsion are strongly correlated to the droplet size 55 distribution (DSD) and the interactions between particles in the dispersed phase, which depend 56 on the final pH of the emulsion because it may alter the protein surface and therefore, it may 57 modify the properties of the previously formed interfaces (McClements, 2004). In addition, the 58 rheology of the continuous phase, which may be strongly influenced by the use of a 59 polysaccharide as stabilizer (thickening agent), plays an essential role in emulsion stability 60 (Tadros, 2004). Thus, polysaccharides are commonly incorporated to the aqueous continuous 61 phase of O/W, because they can provide stability against destabilization phenomena (mainly 62 creaming and flocculation). The thickening efficiency of a stabilizer depends on its 63 concentration, the size and shape of its molecules and its interactions with protein molecules, 64 since all of them contribute to the formation of a gel-like polymer network (McClements, 2004; 65 Quintana, Califano, Zaritzky, Partal, & Franco, 2002). Knowledge of the nature of these 66 interactions is relevant since they can often be used to tailor novel structures and 67 physicochemical properties. Thus, by modulating the composition and environmental 68 conditions of the aqueous media it is possible to control the interactions between the 69 biopolymers and therefore to create microstructures that can improve emulsion stability to 70 environmental stresses (Aguilar et al., 2011; McClements, 2004). On the other hand, non-71 adsorbed polysaccharide particles may induce flocculation in emulsions when they exceed a 72 particular concentration because of an osmotic effect that may involve emulsion destabilization 73 (Dickinson & McClements, 1995; Walstra, 1993). 74 Previous results on crayfish protein-based emulsions have been focused on the relationship 75 between rheological properties, microstructure and stability of highly concentrated emulsions 76 using crayfish flour as the only stabiliser (Romero, Cordobes, & Guerrero, 2009; Romero et al., 77 2008). However, it was compulsory to carry out the emulsification process at high pH, in order 78 to obtain stable emulsions. Therefore, further addition of acid was necessary to achieve the 79 target pH for salad dressing-like food products. On the other side, heat treatment was assessed 80 as a method for the stabilisation of crayfish flour-based emulsions (Romero, Cordobes, Puppo, 81 et al., 2009). However, the above-mentioned strategy for emulsion stabilisation (by increasing 82 the rheological properties through the addition of a polysaccharide) has not yet been studied for 83 crayfish protein. Moreover, the incorporation of a polysaccharide is essential for obtaining 84 reduced-calorie dressings by reducing the oil content in food products. This has become 85 particularly important since the current occidental lifestyle has brought about a noticeable 86 excess in the dietary fat intake and there is an increasing interest among consumers in reduced-87 calorie diets. The selection of xanthan gum as the polysaccharide is based on its wide use in 88 food products since it is most often found in commercial salad dressings and sauces. This broad 89 functionality is related to its ability to modify rheological properties, stabilising efficiency, 90 compatibility with food ingredients and low cost. Moreover, unlike other gums, it is very stable 91 under a wide range of temperatures and pH (Garcıa-Ochoa, Santos, Casas, & Gómez, 2000). It 92 is precisely this stability to pH what makes xanthan gum particularly suitable for use in protein-93 based emulsions since it contributes to compensate the high dependence of protein on pH, 94 which in fact is one of the most important variables affecting emulsion stability. Hence, it is 95 important to assess the emulsion behaviour in a wide range of pH, but still within the window 96 for food applications. 97 In addition, consumers are increasingly more selective in the nutritional quality of the oil which 98 takes part of the regular diet. In this sense, dietary recommendations from FAO/WHO continue 99 to support increased monounsaturated intake along with the reduction in consumption of 100 saturated fats and trans-fatty acids, or hydrogenated oils (FAO/WHO, 2008). Thus, the use of 101 high-oleic sunflower oil is a good choice since it contributes to achieve all these requirements. 102 This research provides scientific knowledge related to the elaboration and the stability of low-103 fat and high oleic emulsions or salad dressings in a wide range of pH values. Despite the fact 104 that this market is well-stablished, nowadays consumers claim for the purity and authenticity 105 of raw materials used in these products whose quality is in question. The use of a natural source 106 for the elaboration of these products constitutes an alternative to traditional food additives, 107 which are currently undesired. The use of Xanthan gum allows an extended stability of these 108 food products, the selection of this polysaccharide is based on its wide use in food products, 109 which is related to its low cost. 110 The main objective of this study was the evaluation of the rheological, microstructural and 111 stability behaviour of crayfish protein concentrate as emulsifier for low-fat and high-oleic O/W 112 emulsions in combination with a stabiliser such as xanthan gum. This is a primary requirement 113 contributing to assess the actual potentials of crayfish protein in the formulation of functional 114 food emulsions. A wide range of pH was analysed in order to evaluate the contribution of 115 relevant protein-polysaccharide interactions. 116 First of all, it was necessary to optimize some of the parameters (protein concentration and pH 117 value) that would lead to long-term stable emulsions. In this context, interfacial measurements 118 were performed in order to facilitate the prediction of emulsion stability. 119 A characterization of the rheological properties and microstructure of crayfish-based emulsions 120 was carried out since they provide relevant information on emulsion stability. 121 The rheological characterization was focused on the linear viscoelastic properties of the 122 emulsions, determined by means of small-amplitude oscillatory shear (SAOS). Microstructure 123 was evaluated through Droplet Size Distributions (DSD) analysis and Confocal Laser Scanning 124 Microscopy (CLSM). In addition, backscattering measurements were carried out to provide 125 useful information on the destabilization mechanisms of these emulsions. 126 2. Material and methods 127 2.1. Materials 128 Preparation of Crayfish protein concentrate: the crayfish (CF) meat was separated from the shell 129 by grinding and sieving and supplied as CF pulp by ALFOCAN (Isla Mayor, Seville, Spain). 130 CF pulp was homogenized and subjected to centrifugation at 15,000 x g for 15 minutes in a 131 Centromix II-BL (Selecta, Spain), obtaining three different phases: a heavy phase, CF1P (c.a. 132 20 wt.%), an intermediate phase (CF2P), which consists of mainly water (c.a. 70 wt.%) and a 133 light phase, CF3P (c.a. 10 wt.%). The CF2P was the selected phase because it is the water 134 soluble protein fraction and it represents the highest protein content. Finally, the intermediate 135 phase (CF2) was freeze-dried in a BETA 1-8 LD Plus Series (CHRIST, Germany) to obtain a 136 powder-fraction rich in proteins. This phase will be named CF2L. The protein content of the 137 CF2L was determined in quadruplicate as % N x 6.25 using a LECO CHNS-932 nitrogen micro 138 analyser (Leco Corporation, St. Joseph, MI, USA). In the same way, lipid, moisture and ash 139 contents were determined according to A.O.A.C. (2000). Results reveal that composition 140 corresponds to 78.6±0.5, 6.8±0.1, 5.1±0.3 and 9.5±0.6 of proteins, moisture, lipid and ash 141 content, respectively. 142 Xanthan Gum (XG) was purchased from Panreac Química, S.A. (Barcelona, Spain) and high 143 oleic sunflower (Capicua) was supplied by Coreysa company (Seville, Spain). The water used 144 was distilled for the emulsion preparation and has the MilliQ grade for interfacial 145 measurements. 146 2.2. Interfacial characterization 147 2.2.1. Air-Water interface characterization 148 Measurements of the surface pressure were performed using the Wilhelmy plate method on a 149 Sigma (KSV, Helsinki, Finland). The measurements were carried out at different protein 150 concentration (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 5.0 wt.%) and at three pH values (3, 5 and 151 8). For this purpose, the recipient was filled with each of the buffer systems employed for 152 emulsion preparation and the temperature was kept at 20 ºC by water circulation from a 153 thermostat Bioblock Ministat (Illkirch, France). Measurements were continued along time until 154 to reach at equilibrium value. 155 2.2.2. Oil-Water interface characterization 156 Initially, an interfacial O/W characterisation was carried out by means of droplet pendant in 157 order to determine protein adsorption, as well as to predict the emulsion stability. Transient 158 interfacial pressure and interfacial dilatational measurements were carried out using a pendant 159 drop tensiometer from TRACKER (IT Concept, France). In this sense, an axisymmetric drop 160 was formed at the tip of the needle of a syringe whose verticality was controlled by a computer. 161 The drop profile was digitized and analysed through a CCD camera coupled to a video image 162 profile digitizer board connected to a computer. Drop profiles were processed according to the 163 Laplace equation as was described by Castellani, Al-Assaf et al. (2010). A single batch of 164 commercial n-hexadecane oil was used to measure oil–water kinetic adsorption. The n-165 hexadecane oil was purified by elution on a silica column to extract the polar lipids. 166 In addition, the viscoelastic moduli of protein adsorption layers were determined at 10% strain 167 amplitude under non-equilibrium at 0.126 rad/s and equilibrium conditions at different 168 frequencies (ranging from 0.031 to 0.314 rad/s) once the pseudo-steady state was achieved after 169 30,000 s (results obtained experimentally). All the experiments were carried out, at least in 170 duplicate, using an optical glass cuvette (8 ml), containing the oil phase, which was 171 thermostated at 20.0 ± 0.1 °C. 172 2.3. Emulsions preparation 173 Emulsions has been prepared according to the procedure followed by Ettoumi et al. (2016) for 174 legume protein-based food emulsions. First of all, Xanthan Gum (XG) stock solution was 175 prepared by dissolving 3wt.% XG powder in water. Then, high mechanical agitation was 176 applied overnight and after that, the evaporated water was added to keep constant the gum 177 concentration. Finally, 0.1 wt.% NaN3 was added to preserve the gum dispersion. The stock 178 solution was diluted to prepare XG solutions at 0.25 wt%. Systems were kept refrigerated at 5 179 ⁰C for at least 48 h to ensure the complete polysaccharide hydration. 180 Emulsions were prepared by gradually blending high oleic sunflower oil (50 wt.%) with the 181 proportional aqueous CF2L protein dispersion (at each pH value) using the high-shear mixer 182 Ultraturrax T-50 (Staufen, Germany). Both phases were mixed for 2 min at 5,000 rpm (pre-183 emulsion). Subsequently, the pre-emulsions were passed through the high-pressure valve 184 homogenizer EmulsiFlex-C5 once at 200 KPa (Avestin, Germany). Final emulsions were 185 prepared by diluting concentrated emulsions with XG solution. The concentrated emulsion and 186 XG gum solutions were mixed manually using a spatula (final emulsion). Final emulsion 187 contained 50 wt% high-oleic sunflower oil, 47 wt% aqueous CF2L protein dispersion (2 or 3 188 wt% CF2L) and 0.25 wt.% XG. Three selected pH values were studied (3, 5 and 8). The stability 189 of emulsions without any XG is very poor. In fact, the phase separation took place after 1 day 190 in absence of XG. 0.25 wt. % XG was selected as a suitable concentration since it provided 191 kinetic stability to emulsions, without inducing droplet flocculation to a marked extent. 192 2.4. Emulsions characterization 193 2.4.1. Droplet size distribution measurements 194 Droplet size distribution (DSD) was determined with the particle size analyser of laser 195 diffraction Mastersizer X (Malvern, United Kingdom). To avoid the measure of flocculated 196 drops, 1 wt.% of SDS was added to the measured dispersion ad stirred at the same time. The 197 mean droplet diameter was calculated as follows: 198 𝐷𝐷[𝑀𝑀,𝑁𝑁]=�∫𝐷𝐷𝑀𝑀𝑛𝑛(𝐷𝐷)𝑑𝑑𝐷𝐷 ∫𝐷𝐷𝑁𝑁𝑛𝑛(𝐷𝐷)𝑑𝑑𝐷𝐷� 1 𝑀𝑀−𝑁𝑁 (1) Where M and N acquire the value of 3 and 2 (D[3,2]), respectively, for Sauter diameter. The 199 values of 4 and 3, for M and N, respectively, for the mean Volumetric droplet size (D[4,3]). 200 Then the upside term is referred to the volumetric size and the downside is referred to the area. 201 From DSD, uniformity parameter also was obtained. The uniformity ratio is an index of 202 polidispersity of the different droplet sizes and is defined by the following expression: 203 𝑈𝑈= ∑𝑉𝑉 𝑖𝑖 |𝑑𝑑(𝑣𝑣, 0,5)−𝑑𝑑 𝑖𝑖 | 𝑑𝑑(𝑣𝑣, 0,5)∑𝑉𝑉𝑖𝑖 (2) where d(v,0,5) is the median for the distribution, and Vi is the volume of droplets with a diameter 204 di. 205 2.4.2. Linear viscoelasticity 206 The AR-2000 rheometer (TA instruments, USA) was used for all rheological measurements. 207 Firstly, strain sweep tests were performed to define the linear viscoelastic region. Subsequently, 208 frequency sweep tests were carried out from 0.01 to 20 Hz, at constant strain amplitude 209 (between 0.01 and 0.30%, within the linear viscoelastic region). 210 Firstly, stress sweeps test were performed at three different frequencies (0.62, 6.20 and 12.52 211 rad/s) for all systems studied to estimate the dynamic linear viscoelastic range (LVR). 212 Subsequently, frequency sweep tests were carried out at constant stress, within the LVR, from 213 0.06 to 100 rad/s. Serrate plates of 40 mm were used in frequency sweep tests to avoid slipping 214 effect. 215 2.4.3. Backscattering measurements 216 Two parameters (D[4,3] and Uniformity index) were calculated from DSD measurements 362 according to equations (1) and (2) and presented in Table 2 as a function of ageing time. At pH 363 3, no noticeable increase in D[4,3] took place over time, which may be attributed to the higher 364 elastic interface observed in Fig. 3. However, droplet size showed an apparent increase over 365 ageing time for emulsions prepared at pH 5 and 8, most probably caused by coalescence as 366 reported by Quintana et al. and Romero et al. (2002; 2008; respectively). Furthermore, the 367 uniformity parameter is a useful tool that reflects the polydispersity of the samples, for which 368 low values are desirable. The smallest uniformity values were found for CF2L protein 369 emulsions at pH 3, coinciding with highly stable unimodal distributions. The rest of systems 370 showed U vales higher than 0.8 corresponding to broad bimodal (and even trimodal) 371 dispersions. It is interesting to remark that emulsions showing low polydispersity (i.e. at pH 3) 372 did not show any appreciable flocculation, whereas high polydispersity values seem to be 373 obtained after adding SDS to significantly flocculated emulsions (results not shown). 374 3.3.2. Viscoelastic properties 375 Dynamic frequency sweep tests were carried out for all the emulsions studied to determine the 376 frequency dependence of storage and loss moduli, well within the linear viscoelastic range 377 (LVR). The mechanical spectra obtained were plotted in Fig. 4B, as a function of pH, for 378 emulsions at 0.25 wt.% XG concentration. CF2L-based emulsions exhibited a gel-like 379 behaviour where G’ is always above G’’, regardless of the pH value, showing a moderate 380 dependence on frequency. This behaviour corresponded to the plateau region of the mechanical 381 spectrum and has been previously found for a wide variety of polydisperse systems. Occurrence 382 of a plateau zone has been related to the development of macromolecular entanglements either 383 in polymer melt rheology (Fetters, Lohse, Richter, Witten, & Zirkel, 1994) or in concentrated 384 polymer solutions (Nandan, Kandpal, & Mathur, 2004). Tasneem, Siddique et al. (2014) 385 reported the development of a plateau region for emulsions stabilised by a polysaccharide, 386 attributing this behaviour to the formation of a polysaccharide gel network. Other food 387 emulsions (Bengoechea, Cordobes, & Guerrero, 2006; Quintana et al., 2002) or aqueous 388 dispersions of protein-polysaccharide systems (Aguilar et al., 2011) also showed this 389 entanglement region. As may be seen in Fig. 4B, an increase in pH leads to a progressive 390 weakening of the gel-like network of the emulsion. This effect may be attributed to 391 modifications in the interactions among protein side chains and polysaccharide molecules. At 392 low pH, positive charges are predominant at protein surfaces that are forming interfacial films 393 at the O/W interfaces. As a consequence, attractive interactions may be developed with the 394 negatively charged carboxyl groups of XG macromolecules dispersed in the continuous phase 395 (Calero, Munoz, Cox, Heuer, & Guerrero, 2013). As a result, electrostatic interactions tend to 396 reinforce emulsion network structures. Similar results were reported for O/W emulsions 397 stabilised by potato protein isolate and chitosan (CH), at pH higher than the IEP, also resulting 398 in attractive electrostatic interactions (Calero et al., 2013). These authors described how CH 399 strands were linked to protein segments located at the oil droplet surfaces and how, in this way, 400 oil droplets were trapped into the CH network that is formed at the continuous phase. This 401 rheological behaviour is directly related to the above-described stability found at pH 3 and may 402 also explain the absence of flocculation at this pH. However, after increasing pH up to a value 403 close to the IEP no further attractive electrostatic interactions take place, which results in an 404 apparent reduction of the emulsion viscoelastic properties. On the other hand, a further increase 405 in pH led to occurrence of repulsive electrostatic interactions among protein and polysaccharide 406 molecules, as protein surfaces are negatively charged, eventually leading to a weakening of the 407 emulsion gel-like network. It may be also recalled that the amount of protein required to saturate 408 the interface (for the same area) was higher near the IEP, which may also contribute to the 409 higher viscoelastic response obtained as compared to pH 8. 410 Table 2 shows the values for the elastic modulus and tan δ obtained at π/2 rad/s (G’π/2) for all 411 the emulsions studied. The strength of the gel-like network obtained at pH 3, which is the 412 strongest of all, is a consequence of the above-mentioned electrostatic attractive interactions 413 among protein and polysaccharide molecules. Thus, the gel-like strength found for this protein 414 emulsions can be attributed not only to the polysaccharide network but also to the interaction 415 among protein and polysaccharide molecules, as a consequence of the formation of a highly 416 flocculated droplet network (Kontogiorgos, Biliaderis, Kiosseoglou, & Doxastakis, 2004). As 417 for the ageing time, a decrease of G’ was found for all the systems studied over the elapsed 418 time. Emulsions prepared at pH 3 exhibit less decrease in droplet size and, as a consequence, 419 are more stable according to DSD measurements. As regards tan δπ/2, the lowest values were 420 found for the emulsion at pH 5, which are related to the lower particle size. However, at this 421 pH tan δπ/2 increases over ageing time. Ageing time does not involve any increase in this 422 parameter only at pH 8, indicating the absence of changes in structural forces at this pH value. 423 3.3.3. Light scattering measurements 424 Consecutively to the emulsification process, and as a part of the stability study, light scattering 425 diffraction was measured, reflecting both short and long-term stability in the emulsions 426 containing 0.25 wt.% XG. Back Scattering (BS) measurements were performed in order to 427 determine the destabilization mechanism of CF2L emulsions. Fig. 5 shows the evolution of the 428 relative Back Scattering (ΔBS) as a function of time obtained within the middle part of the tube. 429 ΔBS was defined as follows: 430 ∆𝐵𝐵𝐵𝐵=𝐵𝐵𝐵𝐵 0 −𝐵𝐵𝐵𝐵 𝑡𝑡 (5) where BS0 and BSt are the mean values for the BS profile obtained at the initial time and time t, 431 respectively. Once again, CF2L-based high oleic emulsions at pH 3 do not suffer any significant 432 coalescence destabilisation in the studied ageing. On the other hand, ΔBS values decrease 433 dramatically at pH 5 and 8, as a consequence of the coalescence process, which was more 434 noticeable at pH 5. 435 3.3.4. Optical measurements 436 Fig. 6 shows the confocal laser scanning microscopy (CLSM) images for CF2L-based high 437 oleic and low-oil content emulsions. Pictures were taken for emulsions containing 0.25 wt.% 438 XG, at the three pH values studied and at different ageing time (1, 7 and 14 days). 439 Since proteins exhibit auto-fluorescence, a high light intensity corresponds to the presence of 440 protein. CLSM images illustrate how protein is preferentially located at the oil/water interfaces, 441 although some small aggregates may be also detected. 442 As may be observed, the smallest drops are initially found in the emulsion at pH 5. However, 443 at pH 5 and 8, emulsion droplets undergo a dramatic increase in their sizes after the first week, 444 being the consequence of coalescence. Only droplets at pH 3 held their initial size, being more 445 stable over the selected ageing time (14 days). The presence of protein aggregates seemed to be 446 more apparent at pH 5 and 8, where coalescence was relevant, as less protein is necessary to 447 cover the reduced interfacial area. These pictures also confirmed results shown in previous 448 sections (DSD and rheological measurements). Thus, oil/water interfaces seem to be better 449 defined at pH 3. The lower interfacial equilibrium tension at pH 5 allowed the formation of 450 smaller drops, however the stronger interface found at pH 3 favoured the stability of the 451 systems. CLSM images for emulsions after 1 day at pH 8 exhibited a rather inhomogeneous 452 DSD, characterised by a poorly defined interface. 453 4 . Concluding remarks 454 Interfacial pressure measurements evidenced a strong dependence of protein adsorption 455 behaviour at the O/W interfacial layers, where the best results were obtained at pH 5. This 456 behaviour may be related to the absence of net charge close to the IEP. On the other hand, the 457 presence of charges at CF2L protein surfaces, either positives or negatives, gave rise to a 458 reduction in its interfacial activity. 459 The results of CF2L adsorbed O/W interfaces obtained from interfacial dilatational rheology 460 measurements correspond to an apparent 2D gel-like behaviour, exhibiting more predominant 461 elastic response at pH 3 that could be associated to a more tightly packed interface. However, 462 dilatational measurements cannot determine the nature of the interactions or predict the stability 463 of the interface. 464 The CF2L-based emulsion at pH 5 exhibited lower droplet sizes, as a result of its higher surface 465 pressure. These results should be reflected in the stability of the emulsion. However, the highest 466 stability corresponded to emulsions prepared at pH 3, which showed unflocculated unimodal 467 distributions remaining unaltered in size over 2 months. This behaviour may be a consequence 468 of the relevant role of the electrostatic interactions among positively charged protein surfaces 469 and anionic xanthan gum macromolecules. In fact, the results obtained for linear viscoelastic 470 properties of CF2L-based emulsions seemed to be governed by protein-polysaccharide 471 interactions that in turn depend on pH. These results support the key role of the polysaccharide 472 in the formation of the gel-like network, either through formation of extensive flocculation (at 473 pH 5 and 8) or through complexation with protein, favoured at pH 3. This scheme turned out to 474 be directly related to DSD and stability results, since higher droplet sizes induced by 475 coalescence were obtained for emulsions prepared at pH 5 and 8, which exhibited much 476 stronger dependence on ageing time. 477 Furthermore, light scattering measurements and CLSM image confirmed the emulsion stability 478 behaviour found with DSD measurements as a function of pH. The former type of 479 measurements put forward occurrence of coalescence at pH 5 and 8. 480 Acknowledgments 481 This work is part of a research project sponsored by Andalousian Government, (Spain) (Project 482 TEP-6134). The authors acknowledge also to the Microscopy Service (CITIUS-Universidad de 483 Sevilla) for providing full access and assistance. 484 References 485 A.O.A.C. (2000). Official Methods of Analysis. E.E.U.U.: Association of Official Analytical 486 Chemist. 487 Aguilar, J. M., Batista, A. P., Nunes, M. C., Cordobes, F., Raymundo, A., & Guerrero, A. 488 (2011). 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