Use of a mixer-type rheometer for predicting the stability of O/W protein-based emulsions

1 Use of a mixer-type rheometer for predicting the stability of O/W 1 protein-based emulsions 2 A. Romeroa,*, M. Felixa,V. Perez-Puyanaa, L. Choplinb, A. Guerreroa aDepartamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, 3 41012 Sevilla, Spain 4 bGEMICO, ENSIC, Université de Lorraine, Nancy, France 5 __________________ 6 Corresponding autor: 7 *A. ROMERO 8 Departamento de Ingeniería Química, 9 Universidad de Sevilla, Facultad de Química, 10 41012 Sevilla (Spain) 11 E-mail: alro[email protected] 12 Phone: +34 954557179; fax: +34 954556447. 13 Abstract 14 The present work illustrates the feasibility of performing Oil-in-Water (O/W) 15 emulsionsstabilized by different protein concentrates, as well as predicting the 16 likelihood ofemulsion destabilization over ageing time just after its preparation. To 17 achieve this objective, four protein sources (rice, crayfish, potato and albumen) and 18 four oil concentrations (450, 550, 650 and 750g·Kg-1) were used. The emulsification 19 process was monitored by the use of a mixer-type rheometer. This rheometer is a 20 valuable tool for understanding and controlling the emulsification process through the 21 measure of the viscosity of the different systems during the emulsification stage. 22 *Manuscript Click here to view linked References 2 Results reveal the importance of controlling the emulsification process to optimise the 23 properties of the final emulsion, which is highly influenced by the oil concentration. 24 Emulsions were prepared after a previousoptimization of agitation speed, protein 25 concentration and pH value. Then, emulsions were characterized by means of flow 26 properties and droplet size distribution (DSD). Eventually, a relationship has been 27 found that relates the rheological properties that relate the rheological properties and 28 the microstructure of the final emulsions during and after emulsification stage.These 29 measurements have been demonstrated to be useful in order to predict the stability of 30 protein-based emulsions. 31 Keywords: Mixer type rheometer; Droplet size distribution; Emulsification; Proteins; 32 Viscosity. 33 1. Introduction 34 An emulsion is a mixture of two immiscible liquids in which one is dispersed in the other 35 in the form of droplets. Common food emulsions are form by a mixture between oil and 36 water, and are called oil-in-water (O/W) emulsions or water-in-oil emulsions (W/O), 37 depending on the dispersed or continuous phase.The final properties (i.e. stability, 38 texture or droplet size) of an O/W emulsion strongly depend on the specific 39 characteristics of each compound of an emulsion: the dispersed phase (oil), the 40 continuous phase (water) and the interface. Each one of them has its own complexity, 41 which should be analysed to obtain useful information about further emulsion 42 properties. Among them, the interface became of particular interest in these types of 43 kinetically-stabilized systems. TheO/W interface of a food is usually stabilized by 44 proteins, low-molecular weight emulsifiers (mainly monoglycerides, phospholipid and 45 esters) or a combination of them (McClements, 2004).Characteristics of the interface 46 depend on the type and concentration of the emulsifier, which tends to be adsorbed at 47 interfacedue to its amphiphilic character. Proteins are widely used as emulsifier in food 48 emulsions, being considered as a functional ingredient in the formation and 49 3 stabilization of food emulsions and foams (McClements, 2004; Taherian, Fustier, & 50 Ramaswamy, 2006). Proteins are quickly adsorbed atO/W interface of the droplet 51 formed and form a film which protect droplets againstdestabilization phenomena 52 (Dickinson & McClements, 1995; Foegeding & Davis, 2011; Law & Kennedy, 1999).In 53 recent years, egg yolk has been replaced by alternative protein systems such 54 asvegetable proteins, mainly soybean and wheat, or, even animal proteins systems. 55 Among other alternatives, rice, potato and crayfish protein systems could be 56 mentioned. 57 Rice industry produces every year a large amount of by-products which involve an 58 undesirable environmental effect. Thus, around 100 million tons of them are produced 59 every year around the world (Li, Liu, Liao, & Yan, 2010). Unfortunately, these by-60 products are used in low-value-added applications, being mainly incinerated to obtain 61 electricity or used for animal feeding (Njie & Reed, 1995).Another by-product from food 62 industry comes from the potato protein corresponding to the potato skin. Potato 63 proteinconcentrate from industrial wastes are generally subjected toextreme conditions 64 to increase the protein solubility, as a consequence, proteins are extensively 65 denaturated. However, some emulsifyingof these potato concentrates have been found 66 (Romero et al., 2011; van Koningsveld et al., 2006). On the other hand, protein 67 surpluses not only may come from plants but also they may derive from an animal 68 source. In Andalusia (southern region of Spain), there is a relevant example, which is 69 associated to the crayfish Procambarusclarkii. It was introduced some years ago and 70 suffered a fast widespread growth, being considered as an invasive species, 71 (Kirjavainen & Westman, 1999). Nowadays, a strong local industry produces a big 72 amount of surpluses and wastes. Emulsions have been made from this protein surplus 73 (Felix, Romero, & Guerrero, 2017; Romero, Cordobes, & Guerrero, 2009). 74 During the emulsification process, the strain and rupture of the dropletsare controlled 75 by a local capillary number (Ca), which establishes the relationship between the shear 76 stress and the interfacial tension. Hence, there is a critical value for this relationship 77 4 that depends on the viscosity ratio of disperse and continuous phases. Above the 78 critical value, shear forces exceed surface tension and the breakup of droplets takes 79 place. However, below this critical value the recoalescence process is more 80 relevant(Janssen & Meijer, 1995). 81 On the other hand, the stability of an emulsion is the key factor considered for 82 performing an emulsion since these are only kinetically stable. An emulsion is stable 83 when the number, size distribution and spatial distribution of droplets do not change 84 over time. Thus, the destabilization of emulsions mainly depends on the initial size of 85 droplets, the rheology of the continuous phase and the interactions among particles, 86 which are responsible for the flocculation and further coalescence of droplets (Sahin & 87 Sumnu, 2006). Destabilization phenomena are related to the presence of attractive 88 interactions (Van der Waals), electrostatic repulsions and steric interactions. Then, if 89 attractive interactions are weak, droplets tend to form a reversible structure which 90 favours the stability. However, if attractive forces are strong the destabilization of 91 emulsions may take place through the droplet flocculation and further 92 coalescence(Tadros, 2013). At equilibrium, proteins adsorbed at O/W are able to form 93 a film which has a double function. On the one hand, it favours the electrostatic 94 repulsions. In addition, it may act as a mechanic barrier which avoids the droplet 95 coalescence, due to its viscoelastic properties (Tadros, 2013). Then, high values of 96 electrostatic potentials are desired for the stability of this kind of dispersed system 97 (Franco, Guerrero, & Gallegos, 1995). 98 Apart from the colloidal stability, the rheology of emulsions is influenced by several 99 structural parameters, where the nature of the interactions among particles has 100 particular relevance. Moreover, other parameters such as the size and the shape of 101 particles, the polydispersity and rheology of the continuous medium also determine the 102 rheological response significantly (Ma & Barbosa-Cánovas, 1995). The stability of an 103 emulsion can be determined through the use of rheological techniques, sincethe 104 destabilization phenomena which takes place in an emulsion such as coalescence, 105 5 creaming, phase inversion or sedimentation provoke changes in the rheology 106 properties of emulsions (Sanchez, Berjano, Guerrero, Brito, & Gallegos, 1998; Tadros, 107 2013). Rheological measurements are useful because it is possible to relate these 108 properties withthe microstructure of the emulsions, allowing a prediction of emulsion 109 stabilities. Especially interesting are flow measurements in steady and transitory states. 110 Thus, since the first one contributes to describe the real response of emulsions during 111 its transport, the second one reflects the way in which the structural construction or 112 destruction takes place, reflecting also the shear effect(Gallegos, Franco, & Partal, 113 2004; Guerrero, Partal, Berjano, & Gallegos, 1996; Mezger, 2006). More specifically, 114 the study of the rheological properties during the emulsification stage allows getting 115 knowledge about the shear-induced droplet formation. This characterisation at an early 116 stage is of crucial importance to predict the properties and the long-term stability of the 117 final emulsions. 118 The aim of this study was to compare the use of four different proteins (Rice, Crayfish, 119 Potato and Albumen) in O/W emulsion stabilization at four different oil concentrations 120 (450, 550, 650 and 750 oil grams per Kg emulsion). The protein-stabilized emulsions 121 were rheologically characterised over emulsification stage after being processed by a 122 mixer-type rheometer. The rheological characterisation of the emulsification stage put 123 forward the utility of this rheometer as a valuable tool for understanding and controlling 124 the emulsification process, which further will influence the final viscoelastic properties 125 of final emulsions. In addition, a further objective of this work was to evaluate the 126 influence of oil concentration (450, 550, 650 and 750 g·kg-1) in viscoelastic properties 127 and stability, when each protein system was used.In this sense, the characterization of 128 the emulsions, particularly its viscosity and particle size distribution (Droplet Size 129 Distribution, DSD), was carried out the day after emulsion preparation, as well as, after 130 one month in order to determine the stability of the emulsions performed. 131 6 2. Materials and methods 132 2.1. Materials 133 Four different protein systems were used. Thus, the rice protein concentrate, from rice 134 husks, was provided by Remy Industries (Leuven-Wijgmaal, Belgium),the crayfish flour 135 was obtained from ALFOCAN S.A. (Isla Mayor, Seville, Spain), Potato Protein Isolate 136 PPI was supplied by Protastar (Reus,Barcelona, Spain) and Albumen protein isolate 137 was delivered by Proanda (Barcelona, Spain). Table 1 shows the compositionfor each 138 protein concentrate system supplied. Sunflower oil was purchased in a local market 139 and used without further purification. 140 2.2. Emulsification Process 141 Emulsions were prepared in a mixer-type rheometer. This device consists of a 142 cylindrical vessel equipped with a double helical ribbon impeller installed in a RS150 143 controlled stress rheometer, as described by(Nzihou, Bournonville, Marchal, & Choplin, 144 2016). Different O/W protein-based emulsionswere prepared into the mixer-type 145 rheometer (rotating at 5 rad/s), where an Ultra Turrax T-25 homogenizer from IKA at 146 15,000 rpm was used to apply mechanical energy for the emulsification process. The 147 final pH of all emulsions was 3.0, and the protein concentration used was 30 g·kg- 148 1.Emulsions were characterized in terms of the influence of the type of protein 149 concentrate used as well as the oil concentration (450, 550, 650 and 750 g·kg-1). The 150 emulsification sequence consists of 30 s with only Ultra Turrax (UT) agitation, 420 s 151 with UT homogenization and oil addition, followed by 60 s with UT agitation and 290 s 152 without homogenization in order to stabilize the viscosity value. 153 2.3. Characterization of emulsions 154 2.3.1. Rheological measurements 155 During emulsification 156 A mixing-type rheometer was used to determining rheological changes over the 157 emulsification process, more specifically, viscosity (η) along the emulsification time. 158 7 This mixing-type rheometer has been already validated for the in-situ measurement of 159 the viscosity during the emulsification process (Romero, Perez-Puyana, Marchal, 160 Choplin, & Guerrero, 2017). Note that parameters are included in an analytical method, 161 based on the Couette analogy, which allows as to extract absolute viscosity-shear rate 162 data in non-conventional geometries (Aït-Kadi, Marchal, Choplin, Chrissemant, & 163 Bousmina, 2002; Nzihou et al., 2016). 164 After emulsification 165 Steady state shear flow tests were carried out in a RF II rheometer from 166 Rheometric Scientific (USA) the day after emulsion preparation and 30 days later. The 167 measurements were performed at 25 ºC from 0.1 to 10 s-1and the geometry used was 168 parallel plates (dia: 25 mm) with a rough surface, avoiding sample slide, and a gap 169 between plates of 1 mm.The data obtained were fitted to a Power-law model (Ostwald 170 de Waele), obtaining the viscosity (η) as a function of the shear rate ( ). The following 171 expression is used for this model: 172 WhereK and n are the consistency and flow index, respectively. The value of viscosity, 173 η at 1 s-1 was selected in order to compare emulsions. 174 2.3.2. DSD measurements 175 Measurements of Droplet Size Distribution (DSD) were performed in a laser light 176 scattering apparatus (Malvern Mastersizer 2000, UK). For this purpose, 0.5 mL of 177 emulsion was taken and diluted in 11.5 of 0.05 M, pH Tris-HCL buffer with 1 % SDS. 178 SDS was used as a disaggregating material in order to facilitate disruption of floccules 179 (drops agglomeration), often achieved by repulsive droplet/droplet interactions 180 (Hasenhuettl & Hartel, 2008; Nzihou et al., 2016). Values of the Sauter mean diameter 181 (d3,2)which is inversely proportional to the specific surface area of droplets, were 182 obtained as follows: 183 8 Whereniis the number of droplets with a diameter di. 184 Uniformity parameter (U) also was calculated from DSD measurements. This 185 parameter is an index of polydispersity of the DSD and it is defined as follows: 186 (3) Whered(v,0,5) is the median for the distribution, and Vi is the volume of droplets with a 187 diameter di. 188 Both studies (rheological and DSD measurements)were carried out along time (1 and 189 30 days after emulsification) in order to study the effect ofprotein concentrates and oil 190 concentration on the emulsion stability. Emulsions were stored at 5 ºC, being subjected 191 to the same thermorheological history before performing any tests. 192 2.4. Statistical analysis 193 Three replicates of each rheological and DSD measurement were carried out. All the 194 data were reported as means ±95% confidence limits. Statistical analyses were 195 performed using t-test and one-way analysis of variance (ANOVA, p<0.05). 196 3. Results 197 3.1. Emulsification process 198 Figure 1 shows the results of viscosity obtained from the mixer-type rheometerover 199 emulsification process for all the protein systems studied (rice, crayfish, potato and 200 albumen) at 650 g·kg-1 oil concentration. As it may be observed, the viscosity of 201 emulsions has a quite marked dependence on the emulsification stage. Thus, over the 202 first 30 s, where the only component is the protein concentrate dispersed into water, 203 the viscosity does not change over mixing time. However, after starting the addition of 204 oil (from 30s), the viscosity of the systems increases in any case until reach a 205 maximum value. In this stage, two different behaviours can be found. On the one hand, 206 rice and albumen-based emulsions reach a plateau value before finishing theoil 207 9 addition. On the other hand, potato and crayfish-based emulsions increase their 208 viscosity as the amount of oil increases. Finally, the following 60 s with UT agitation 209 does not yield any significant change in emulsions viscosity, while the latest 290 s 210 without homogenization induce a decrease in viscosity only for the potato-based 211 emulsion, probably as a consequence of a damping process after the significant raise 212 during the oil addition. The profiles of viscosity reveal significant differences which 213 depend not only on the emulsification stage, but also it depends on the protein used. 214 Thus, the viscosity of the potato-based emulsion is the highest, whereas the one found 215 for rice-based emulsions is the lowest. Crayfish and albumen-based emulsions show 216 an intermediate behaviour. These differences are probably due to not only the different 217 protein amount and type of protein but also the presence of other components in the 218 composition (i.e. lipids or starches) as well as the different behaviour at the pH value 219 used (3.0). Anyway, viscosity of emulsions is a key factor for the stability of these 220 systems, since it decrease the destabilization kinetic (Tadros, 2013). Thus, systems 221 which initially show a higher viscosity suggest a higher long-term stability.In this sense, 222 depending on the protein system, it is possible to optimize the emulsification time with 223 the corresponding power saving, selecting the proper time in order to reach the 224 maximum viscosity. 225 3.2. Emulsion characterisation 226 Figure 2 shows flow curves for all protein-based emulsions studied (Rice, Crayfish, 227 Potato and Albumen) at four different oil concentration: 450 g·kg-1 (A), 500 g·kg-1(B), 228 650 g·kg-1 (C) and 750 g·kg-1 (D)the day after emulsion preparation. All steady state 229 flow curves exhibit a very shear-thinning behaviour, which is characteristic for O/W 230 emulsions(Tadros, 2004). As for the influence of the dispersed phase, this figure 231 reveals that the concentration of the oil has a marked effect on the viscosity of the 232 system. Thus, an increase in oil concentration induces an increase in the viscosity of 233 the protein-based emulsionsin any case. This oil-dependence of the viscosity has been 234 16 http://doi.org/10.1205/026387604323142694 400 Pal, R. (1992). Rheology of polymer‐thickened emulsions. Journal of Rheology, 36(7), 401 1245–1259. http://doi.org/10.1122/1.550310 402 Pearce, K. N., & Kinsella, J. E. (1978). Emulsifying properties of proteins: evaluation of 403 a turbidimetric technique. Journal of Agricultural and Food Chemistry, 26(3), 716–404 723. http://doi.org/10.1021/jf60217a041 405 Romero, A., Beaumal, V., David-Briand, E., Cordobes, F., Guerrero, A., & Anton, M. 406 (2011). Interfacial and Oil/Water Emulsions Characterization of Potato Protein 407 Isolates. 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Industrial & Engineering Chemistry Research, 47(17), 441 6412–6417. http://doi.org/10.1021/ie8002232 442 443 444 18 FIGURE CAPTIONS 445 Figure 1: Rheological characterization of o/w emulsions over emulsification stage at 446 650 g·Kg-1 oil concentration and for all protein systems studied: (rice, crayfish, potato 447 and albumen). 448 Figure 2: Flow curves for all protein-based emulsions studied (rice, crayfish, potato 449 and albumen) at four different oil concentrations: (A) 450 g·Kg-1, (B) 550 g·Kg-1, (C) 650 450 g·Kg-1 and (D) 750 g·Kg-1 the day after preparation. 451 Figure 3: Droplet Size Distributions (DSD) for all protein-based emulsions analysed 452 (rice, crayfish, potato and albumen) at four different oil concentrations: (A) 450 g·Kg-1, 453 (B) 550 g·Kg-1, (C) 650 g·Kg-1 and (D) 750 g·Kg-1. 454 455 TABLE CAPTIONS 456 Table 1: Composition of different protein type systems: rice, crayfish, potato and 457 albumen. 458 Table 2: Parameters from flow curves (η1 and n) as well as from DSD profiles (d3,2 and 459 U) for all protein-based emulsions studied (rice, crayfish, potato and albumen) at four 460 different oil concentrations (450, 550, 650 and 750 g·Kg-1) the day after the emulsion 461 preparation and after a storage of 30 days. 462 Table 1 Protein System Protein (g·Kg-1) Carbohydrates (g·Kg-1) Ash (g·Kg-1) Lipids (g·Kg-1) Moisture (g·Kg-1) Rice 782 ± 12 64 ± 3 52 ± 1 24 ± 3 78 ± 4 Crayfish 647 ± 12 2 ± 1 134 ± 2 183 ± 8 34 ± 3 Potato 801 ± 23 59 ± 6 8 ± 1 31 ± 4 101 ± 2 Albumen 730 ± 11 4 ± 1 60 ± 3 130 ± 5 76 ± 2 Table 1 Table 2 Oil Concentration Protein system η1 (Pa·s) n d3,2 (µm) U 1 day 30 days 1 day 30 days 1 day 30 days 1 day 30 days 45% Rice 0.3 ± 0.1a 0.1 ± 0.1b 0.47 ± 0.02a 0.49 ± 0.03a 13.42 ± 0.04a 18.72 ± 0.52b 1.83 ± 0.09a 2.59 ± 0.06b Crayfish 6.9 ± 0.1A 2.9 ± 0.2B 0.40 ± 0.04A 0.37 ± 0.01A 7.37 ± 0.19A 15.01 ± 0.50B 1.28 ± 0.60A 1.56 ± 0.01B Potato 6.1 ± 0.2α 2.4 ± 0.1β 0.41 ± 0.01α 0.49 ± 0.02β 6.89 ± 0.05α 20.12 ± 0.04β 1.16 ± 0.05α 2.45 ± 0.01β Albumen 23.1 ± 0.2П 14.5 ± 0.2Ф 0.34 ± 0.03П 0.44 ± 0.02Ф 16.79 ± 0.19П 22.99 ± 0.45Ф 0.37 ± 0.01П 0.35± 0.01П 55% Rice 3.9 ± 0.1c 2.1 ± 0.1d 0.37 ± 0.02b 0.37 ± 0.03b 7.43 ± 0.02c 8.10 ± 0.06d 0.43 ± 0.01c 0.55 ± 0.01d Crayfish 50.4 ± 0.2C 19.5 ± 0.3D 0.31 ± 0.02B 0.36 ± 0.02C 5.29 ± 0.08C 6.34 ± 0.01D 0.76 ± 0.03C 1.75 ± 0.01D Potato 43.2 ± 0.4γ 32.3 ± 0.5δ 0.21 ± 0.02γ 0.24 ± 0.02 γ 5.73 ± 0.04γ 6.46 ± 0.01δ 0.81 ± 0.01γ 0.86 ± 0.01δ Albumen 46.9 ± 0.3Ѓ 38.8 ± 0.2Σ 0.19 ± 0.02Ѓ 0.24± 0.01Ѓ 13.70 ± 0.63Ѓ 15.27 ± 0.34Ѓ 0.24 ± 0.02Ф 0.41± 0.02Ѓ 65% Rice 21.7 ± 0.2e 13.8 ± 0.7f 0.27 ± 0.02c 0.32 ± 0.02c 5.27 ± 0.03e 5.31 ± 0.02e 0.67 ± 0.01e 0.68 ± 0.01e Crayfish 91.3 ± 0.5E 43.0 ± 0.5F 0.35 ± 0.01C 0.28 ± 0.02C 4.46 ± 0.06E 4.61 ± 0.01E 0.87 ± 0.18C 0.96 ± 0.01E Potato 202.2 ± 2.6ε 174.4 ± 4.3ζ 0.31 ± 0.02δ 0.34 ± 0.03δ 4.29 ± 0.03ε 4.36 ± 0.01ζ 0.85 ± 0.14δ 1.00 ± 0.01α Albumen 90.1 ± 0.3Ω 71.6 ± 0.7Θ 0.09 ± 0.01Ω 0.13 ± 0.01Θ 9.69 ± 0.14Ω 9.70± 0.04 Ω 0.37 ± 0.02П 0.44± 0.01Ѓ 75% Rice 138.9 ± 5.8g 119.5 ± 3.7h 0.31 ± 0.03c 0.32 ± 0.02c 4.84 ± 0.05f 4.93 ± 0.04f 1.11 ± 0.01f 1.15 ± 0.01f Crayfish 460.9 ± 2.7G 330.5 ± 2.4H 0.16 ± 0.04D 0.25 ± 0.03D 2.44 ± 0.02F 2.57 ± 0.03F 0.62 ± 0.01F 0.78 ± 0.01C Potato 282.3 ± 3.6η 267.9 ± 2.9 η 0.38 ± 0.02ε 0.37 ± 0.02ε 2.37 ± 0.03η 2.43 ± 0.01η 1.02 ± 0.06α 1.07 ± 0.03α Albumen 179.7 ± 4.5Γ 132.8 ± 2.2Ξ 0.05 ± 0.01Γ 0.09 ± 0.02Ξ 8.43 ± 0.03Γ 8.73± 0.06 Γ 0.26 ± 0.01Ф 0.45± 0.01Ѓ Table 2 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image