Influence of the welan gum biopolymer concentration on the rheological properties, droplet size distribution and physical stability of thyme oil/W emulsions

Influence of the welan gum biopolymer concentration on the rheological properties, 1 droplet size distribution and physical stability of thyme oil/W emulsions 2 M. José Martin-Piñero, M. Carmen García, José Muñoz, Maria-Carmen Alfaro- 3 Rodriguez* 4 Departamento de Ingeniería Química. Facultad de Química. Universidad de Sevilla. C/ 5 Profesor García González, s/n. 41012 Sevilla. Spain 6 * Corresponding author 7 e-mail: [email protected] 8 Tel: +34 954 55 64 47 9 Fax: +34 954 55 64 41 10 Abstract. 11 The objective of this work is to obtain a stable and natural antimicrobial delivery system. 12 Thus, the effect of the addition of a natural polysaccharide such as welan gum on the 13 linear viscoelastic properties, flow behaviour, droplet size distribution and physical 14 stability of thyme oil/W emulsions formulated with a wheat-derived surfactant was 15 studied. All emulsions obtained show submicron diameters regardless of the 16 concentration of welan. Emulsion without gum shows Newtonian behaviour under steady 17 shear. Meanwhile, emulsions containing welan gum show a weak gel-like behaviour with 18 higher G’ and G” values on increasing the content of gum in the emulsion. Their flow 19 curves illustrate a shear thinning behaviour with much greater viscosity than that 20 exhibited by emulsions without gum. This behaviour fits well to the Cross model. The 21 main destabilization process of thyme oil/W emulsion without gum is creaming versus 22 flocculation and coalescence in emulsions containing welan. Rheology, diffraction laser 23 and multiple light scattering techniques have proved that welan gum is an important 24 rheological modifier for thyme oil/W ecological emulsions, it being possible to control 25 the rheological properties of these emulsions by adjusting the concentration of gum. 26 However, welan gum does not improve the physical stability of these emulsions. 27 Keywords: Welan gum, Thyme oil, Emulsions, Rheology, Physical stability. 28 1.- Introduction. 29 Nowadays, many industries are looking for new ecofriendly and sustainable products, 30 which can be used in different applications. Ecological emulsions are one of these 31 products. Finding the right formulation which fulfils environmental and non-toxicity 32 requirements is essential for researchers within both the research and industrial fields. 33 As far as formulations are concerned, one of the most important ingredients in emulsions 34 is the surfactant. Surfactants are amphiphilic molecules formed by a polar head and a 35 lipophilic tail which can be grouped by the charge of their polar head groups: cationic, 36 anionic, amphoteric (zwitterionic) or non-ionic. In addition, surfactants can also be 37 classified by their hydrophilic lipophilic balance (HLB) value. The range of HLB values 38 is from 0 to 20. HLB <9 refers to lipophilic surfactants and HLB> 11 to hydrophilic. 39 Therefore, generally for emulsions of type O / W the emulsifiers have an HLB between 40 8 and18 and for W / O emulsions of 3-8 [1]. It should be noted that the use of emulsifiers 41 whose hydrophilic lipophilic balance (HLB) value is close to the required HLB for oil 42 could enable the formulation of stable emulsions. In this work, a non-ionic surfactant, 43 specifically the alkyl poly pentoside commercially known as Appyclean 6548 (D- 44 xylofuranose, oligomeric tetradecyl and octodecyl glycoside, C14, C18 alcohol) is used. 45 This surfactant possesses an HLB value of 9.25 which has been shown to be the optimum 46 HLB value necessary to obtain stable thyme essential oil emulsions [2]. Appyclean 6548 47 is a newly-developed surfactant derived from renewable raw materials such as wheat 48 biomass. The alkyl poly pentoside surfactants are made of pentosides derived from 49 hemicellulose [3], which is not digestible by human intestinal digestive enzymes. 50 However, this kind of food fiber could be utilized by members of the intestinal microbial 51 microbiota like a prebiotic product.[4]. In addition, this surfactant fulfils the requirements 52 to be used in ecolabel formulations. 53 In this work, thyme essential oil is used as oil phase. This essential oil is classified as a 54 GRAS product by the United States Food and Drug Administration [5]. Thyme Vulgaris, 55 has very high antioxidant activity due to its main components: thymol, carvacrol, γ- 56 terpinene, myrcene, linalool, pcymene, limonene, 1,8-cineole, and α-pinene. Among 57 other essential oils, thyme oil has relatively strong antimicrobial activity which it makes 58 it suitable for use in the food field [6,7], Thyme oil is found to be effective against 59 Salmonella typhimurium L. monocytogenes, A. hydrophila and autochthonous flora 60 spoilage in meat products [8]. Nevertheless, thyme essential oil emulsions might undergo 61 one or several destabilization mechanisms, such as flocculation, coalescence, Ostwald 62 ripening or creaming. The incorporation of biopolymers into the continuous phase of 63 emulsions decreases or prevents the movement of droplets and increases the viscosity of 64 the continuous phase, reducing the creaming destabilization process. The use of natural 65 polysaccharides in food or cosmetic applications as thickening, stabilizing and 66 emulsifying agents is due to their biocompatibility, biodegradability and non-toxicity. In 67 this study, welan gum is used as a polysaccharide in order to enhance the rheological 68 properties of emulsions. 69 Welan is an anionic extracellular polysaccharide, secreted by the micro-organism 70 Sphingomonas sp., namely from Alcaligenes sp., ATCC 31555, a gram negative 71 microorganism. It is used in a wide variety of applications in several fields such as food, 72 coating materials, medicine and the petroleum and cement industries [9]. Specifically, 73 this gum can be used as an ingredient in food products like jellies, beverages or dairy 74 products in which it can act as a thickening, suspending, binding, emulsifying, stabilizing 75 and viscosifying agent [10]. Structurally, it is composed of l-mannose, l-rhamnose, d- 76 glucose, and d-glucuronic acid.[11]. The chemical structure is essential in order to explain 77 the behaviour in aqueous phase. For example, although gellan, welan or rhamsan, all of 78 which are derived from Sphingomonas sp, are structurally close, they have different 79 behaviours in different mediums. Thus, while gellan can make stable gels in the presence 80 of salts, rhamsan and welan give only weak gels or very viscous solutions with a high 81 degree of thermal, pH and salinity stability [12]. 82 Despite the advantages of this environmentally friendly polysaccharide, there are only a 83 few studies dealing with the use of welan gum in oil-in-water emulsions. In addition, there 84 have been no previous studies concerning the physical stability and rheological behaviour 85 of thyme essential oil emulsions prepared with welan gum. 86 In this study, we investigate the influence of welan gum concentration to improve the 87 rheological properties of emulsions using thyme essential oil as dispersed phase. 88 Additionally, the effect on the physical stability of emulsions is studied. All the 89 components selected to formulate the emulsions are environmentally friendly materials 90 (thyme essential oil, welan and appyclean 6548). The combination of rheology, laser 91 diffraction and multiple light scattering techniques demonstrated that these results have 92 important implications for the design and utilisation of emulsions as antimicrobial 93 delivery systems in the food industry. 94 2.-Materials and Methods. 95 2.1. Materials. 96 Thyme essential oil (938 g/l), supplied by Bordas Chinchurreta S.A, and a wheat-derived 97 surfactant called Appyclean 6548 obtained from Wheatoleo are used as oil phase and 98 emulsifier respectively. Sodium azide (0.1wt%) is used to avoid microbiological 99 contamination. Welan gum (K1A96) is used as supplied by CP Kelco Company (San 100 Diego, USA). 101 2.2. Emulsification Procedure. 102 The continuous phases are prepared by dissolving 0.1 wt% sodium azide in ultrapure 103 water cleansed using a Milli-Q water purification system. The emulsifier, Appyclean 104 6548 (6wt%) is melted and dissolved in the dispersed phase (thyme oil – 40wt%) due to 105 the fact that it is solid and immiscible in water. The mixture of oil and surfactant is heated 106 to 70ºC for 30 minutes and then cooled to 25ºC. Once at this temperature, a total mass of 107 200 g of the coarse emulsions is prepared in two steps using a rotor-stator device equipped 108 with a mesh screen (Silverson L5M) at 2000 rpm. Firstly, the mixture of solvent and 109 surfactant is gradually added for 180s to the aqueous phase. Subsequently, a batchwise 110 procedure is carried out for 30s. High pressure homogenization is carried out with a 111 microfluidization system (M110P with an F12Y interaction chamber, Microfluidics, 112 USA) at 2500psi. Coarse emulsions are passed once through the microfluidization 113 system. 114 Finally, the gum is added very slowly to the final emulsions. The emulsions are 115 mechanically stirred using an Ika-Visc MR-D1 (Ika, Germany), for two hours at 500rpm. 116 Five emulsions are prepared using different gum concentrations (0, 0.05, 0.1, 0.15, 0.2 117 wt%). Two replicates of each emulsion with a final weight of 200g are prepared. 118 2.3. Rheological properties. 119 The rheological characterization involved stress and frequency sweeps in small amplitude 120 oscillatory shear experiments (SAOS) and stepwise steady shear flow tests. Haake- 121 MARS controlled-stress rheometer (Thermo-Scientific, Germany) is used to carry out the 122 rheological experiments. Flow curves are performed using a 60/1º double cone geometry 123 for emulsions without gums. Emulsions with gums are measured using a serrated plate- 124 plate sensor (60 mm). Flow curves are carried out in a range of 0.05 to 19 Pa. Stress 125 sweeps are performed in a range of 0.05 to 19 Pa at 1 Hz. Equilibration time prior to 126 rheological tests is 300s. All measurements are performed at 25 °C ± 0.1 °C using a C5P 127 Phoenix circulator (Thermo) and in duplicate, the values shown being the average of the 128 two replicates. 129 Microscopy 130 An optical microscope Axio Scope A1 (Carl Zeiss) with a 40x objective is used to observe 131 the microstructure of the emulsions at room temperature. 132 2.4. Droplets size distribution measurements. 133 Size distribution of oil droplets is determined by the laser diffraction technique 134 (Mastersizer 2000, Malvern, Worcestershire, United Kingdom). Sauter diameter (D3,2), 135 volume-weighted mean diameter (D4, 3) and span are the parameters selected to discuss 136 the results. D3,2 and D4,3 values are obtained from the following equation: 137 𝐷3,2 = ∑𝑛𝑖𝑑𝑖 3𝑁 𝑖=1 ∑𝑛𝑖𝑑𝑖 2𝑁 𝑖=1 (𝐸𝑞.1) 𝐷4,3 = ∑𝑛𝑖𝑑𝑖 4𝑁 𝑖=1 ∑𝑛𝑖𝑑𝑖 3 𝑁 𝑖=1 (𝐸𝑞.2) 138 where ni is the number of droplets with diameter di. On the other hand, span values are 139 given by: 140 𝑆𝑝𝑎𝑛 = 𝐷(𝑣,0.9)−𝐷(𝑣,0.1) 𝐷(𝑣,0.5) (𝐸𝑞.3) 141 where D(ν, 0.9), D(ν, 0.5) and D(ν, 0.1) are the highest droplet size contained in 90%, 142 50% and 10% of volume of dispersed phase, respectively. 143 2.5. Physical Stability. 144 The physical stability of thyme essential oil emulsions is evaluated for 15 days at 30ºC 145 by means of a Turbiscan Lab Expert device (Formulaction, France) using the multiple 146 light scattering technique. Emulsion stability is studied by analysing the variation of the 147 backscattering (BS) profiles as a function of length of tube containing the sample, and 148 time. This technique makes it possible to determine the major mechanisms of 149 destabilization in each sample as well as the kinetics of the destabilization process. 150 2.6. Statistical Analysis 151 A one-way analysis of variance (ANOVA) was used to determine the occurrence of 152 significant differences among the obtained results. For this purpose, OriginPro 8 software 153 is used. 154 3.- Results and Discussion. 155 3.1. Rheological properties 156 3.1.1. Small amplitude oscillatory shear tests 157 Figure 1 shows stress sweeps at 1Hz as a function of welan gum concentration. Thanks 158 to this test, the linear viscoelastic range (LVR) can be measured, which indicates stress 159 region within which the response of the material does not depend on the applied stress. 160 G´ (storage modulus) and G´´ (loss modulus) remain practically constant until a critical 161 stress (𝜏𝐶 ) is reached. From this point, generally, both moduli sharply decrease. However, 162 in these emulsions loss modulus values increase before the decrease. This behaviour is 163 observed in other systems and it can be explained by considering that a reorganization of 164 the structure occurs before the nonlinear region is reached [13]. The critical stress value 165 is important because from this point the deformation in the structure is irreversible with 166 increasing stress [12]. This parameter is often known as yield stress although it should be 167 noted that its value depends on the evaluation technique. 168 As can be observed in Figure 1, the linear viscoelastic range, and thus the critical stress 169 values, are clearly dependent on gum concentration. The critical stress values for 170 emulsions are 0.06, 0.14, 0.36 and 0.59 Pa at 0.05 wt%, 0.1 wt%, 0.15 wt% and 0.2 wt% 171 of welan, respectively. This result indicates that increasing gum concentration in the 172 emulsions increases the resistance to shear, which implies a higher stability of the 173 emulsion under the stress amplitude. In addition, the storage modulus and the loss 174 modulus values at the LVR slightly increase with increasing welan concentration, which 175 means an increase in the structural strength of the emulsions. Emulsions without gum do 176 not present a linear viscoelastic response in the range of shear stress amplitude studied. 177 Figure 1 178 In order to obtain more information concerning the linear viscoelastic properties, figure 179 2 illustrates the changes in G’ and G“as a function of the frequency and the concentration 180 of welan gum for thyme oil/W emulsions aged for 24h. The results of these experiments 181 provide insights into the emulsion structure because these tests are non-destructive and 182 do not interfere with it. According to Steffe (1996) [14,15] and other authors, in weak 183 gels, storage and loss moduli exhibit a slight dependence on frequency, and G‘ values 184 exceed G“ values in the whole frequency range studied. Emulsions formulated with 185 thyme essential oil containing different concentrations of welan gum present clear 186 viscoelastic properties with a predominance of the elastic over the viscous component at 187 all frequencies and with both moduli practically independent of frequency. Therefore, 188 these emulsions behave as weak gel-like materials. 189 Figure 2 190 The dependence of the storage modulus on the frequency can be quantitatively described 191 by a power law type equation: 192 𝐺´(𝜔)= 𝐾´.𝜔𝑛´ 𝐸𝑞.(4) 193 where K´ is the intercept and n´ is the slope in the double logarithmic plot of G´ against 194 frequency. 195 The curves of G´as a function of angular frequency fit fairly well to the power law 196 (R2>0.99). The fitting parameters are plotted in figure 3. The values for the slopes of G´ 197 (n´) are similar for all welan gum concentrations, except for 0.1wt% which exhibits a 198 slight maximum value. This result indicates that no change in the structure occurs but 199 only a change of consistency of the system connected with the addition of welan gum 200 (thickening effect). The value of this slope also makes it possible to classify the behaviour 201 of system. It is known that n´> 0 denotes weak gels whereas n´=0 corresponds to strong 202 gels [16]. Our emulsions present n´ values between 0.1 and 0.08 so it can be concluded 203 that these emulsions exhibit a weak gel-like behaviour. It is worth noting that a plateau 204 zone is obtained for the emulsion containing 0.05 wt% welan gum. The plateau zone is 205 Figure 9 347 Figure 10 348 Table 2 349 350 4.-Conclusions. 351 Thyme essential oil-in-water emulsions of submicron range were obtained with 352 microfluidizer M100P at 2500psi-one pass using a bio-based surfactant called Appyclean 353 6548. In order to improve the rheological properties of emulsions, welan gum 354 polysaccharide was included in the emulsion formulation at different concentrations. 355 Emulsion without gum does not exhibit linear viscoelastic region. All the emulsions with 356 gum show a weak-gel like behaviour with stronger interactions as the welan concentration 357 in the emulsion increases. Flow curves of emulsions containing gum exhibit a shear 358 thinning behaviour and they are fitted to the Cross-model equation. The viscosity of these 359 emulsions also increases with concentration of welan. Conversely, the emulsion without 360 gum shows Newtonian behaviour. Multiple light scattering indicates that creaming is the 361 main destabilization mechanism for emulsion without gum. Creaming is decreased by the 362 better rheological properties of emulsions with polysaccharide. However, an increase in 363 droplet size, probably due to flocculation, occurs, this being significant at 0.2wt% welan 364 gum. This emulsion shows the highest kinetics of destabilization. From the results 365 obtained, it can concluded that welan gum is an excellent rheological modifier of thyme 366 essential oil/W emulsions, making it possible to control the viscosity of the emulsion by 367 adjusting its concentration. However, welan gum is not effective in increasing the 368 physical stability of these emulsions. The main destabilization process is flocculation, 369 which finally provokes phase separation. 370 Acknowledgements 371 The financial support received (Project CTQ2015-70700-P) from the Spanish 372 Ministerio de Economía y Competitividad and from the European Commission 373 (FEDER Programme) is kindly acknowledged. 374 References 375 [1] K.J. Vander, L.L. Schramm, The effect of shear and oil/water ratio on the 376 required hydrophile-lipophile balance for emulsification, J. Surfactants Deterg. 5 377 (2002) 19–24. doi:10.1007/s11743-002-0200-6. 378 [2] M.J. Martin, L.A. Trujillo, M.C. Garcia, M.C. 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Fitting parameters of the first-order kinetic equation for the BS in the 20−25 mm 460 zone of the measuring cell versus aging time as a function of welan gum concentration. 461 Welan gum concentration (wt%) BSE (%) BS0 − ΔBSE (%) K (s-1) R2 0 -1.05 1.17 4.38 0.95 0.05 -1.36 1.35 1.69 0.98 0.1 -2.65 2.54 2.74 0.99 0.15 -2.10 2.04 3.77 0.97 0.2 -38.38 40.20 4.03 0.97 462 Table captions 463 Table 1. Fitting parameters to the Cross model for emulsions studied. Standard deviation 464 of the mean (2 replicates) for 𝜂∞ <2%, 𝜂0 < 10%, 𝛾󰇗 < 1% and n < 5% . 465 Table 2. Fitting parameters of the first-order kinetic equation for the BS in the 20−25 mm 466 zone of the measuring cell versus aging time as a function of welan gum concentration. 467 Figure captions 468 Figure 1. Oscillatory shear stress sweeps as a function of welan concentration. 469 Temperature = 25 °C 470 Figure 2. Mechanical spectra of emulsions as a function of welan gum concentration. 471 Standard deviation for the mean is also plotted. Temperature = 25 °C 472 Figure 3. Fitting parameters to the power law equation for all the emulsions studied. K´ 473 is the intercept and n´ is the slope in the double logarithmic plot of G´ against frequency 474 as a function of welan gum concentration. 475 Figure 4. Storage modulus versus welan gum concentration. Continuous lines illustrate 476 the data fitting to the power law. Standard deviation for the mean is also plotted. 477 Temperature = 25 °C. 478 Figure 5. Flow curves of emulsions aged for 24 as a function of welan gum concentration. 479 Continuous lines illustrate the data fitting to the Cross equation. Temperature = 25 °C. 480 Standard deviation of the mean (two replicates) for η < 8%. 481 Figure 6. Droplet size distributions for emulsions aged for 24 h as a function of welan 482 gum concentration. 483 Figure 7. Sauter mean diameters, volumetric mean diameters and span for all emulsions 484 aged for 24 h as a function of welan gum concentration. 485 Figure 8. Photomicrographs for all emulsions prepared as a function of welan gum 486 concentration a) without gum, b) 0.05wt%, c) 0.1wt%, d) 0.15wt% and e) 0.2wt% at 24 487 h of aging time. 488 Figure 9. Backscattering versus measuring cell height as a function of time and reference 489 mode (insets) for emulsions made a)without gum, b)0.1wt% welan gum and c) 0.2wt% 490 welan gum, at 25°C. 491 Figure 10. Destabilization kinetics in the 20−25 mm zone monitored over 15 days for all 492 studied emulsions as a function of welan gum concentration. 493 494 495