Characterization of novel nanoemulsions, with improved properties, based on rosemary essential oil and biopolymers

1 This is the peer reviewed version of the following article: Martin-Piñero, M.J., García, M.C., Santos, J., Alfaro-Rodriguez, M.-C. and Muñoz, J. (2020), Characterization of novel nanoemulsions, with improved properties, based on rosemary essential oil and biopolymers. J Sci Food Agric, 100: 3886-3894., which has been published in final form at https://doi.org/10.1002/jsfa.10430 . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited 2 3 Characterization of novel nanoemulsions, with improved properties, based on 4 rosemary essential oil and biopolymers. 5 M José Martin-Piñeroa, M Carmen Garcíaa, Jenifer Santosa, Maria-Carmen Alfaro- 6 Rodrigueza* and José Muñoza. 7 a Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, C/ 8 P. García González, 1, E41012, Sevilla, Spain. 9 * Corresponding author: Maria-Carmen Alfaro-Rodriguez; Tel.: +34 954 557180; fax: 8 +34 954 556447; E-mail address: [email protected] 9 Abstract 10 BACKGROUND: Nowadays, it is of great interest to develop stable and sustainable 11 formulations that act as nanocarriers of active ingredients. In this work, the droplet size 12 distribution, rheology and physical stability of nanoemulsions with improved properties 13 containing rosemary essential oil and biopolymers as a function of the concentration of 14 these polysaccharides was investigated. 15 RESULTS: Mean diameters below 150 nm were achieved, indicating nanostructures 16 were obtained. Regardless of gum type, a gel-like structure and a shear thinning behaviour 17 was achieved. In addition, an increase of G’, G” and viscosity and a decrease of J0, J1, J2, 18 1 and 2 with increasing gum concentration were observed, due to the formation of a 19 three-dimensional network in the aqueous phase. Slight differences between 20 nanoemulsions containing welan or xanthan were found. Creaming, depletion 21 flocculation and gel aggregation were the main destabilization processes at low, 22 intermediate and high gum concentration, respectively. 0.4 wt% gum nanoemulsion 23 exhibited the best physical stability. 24 CONCLUSION: These stable and sustainable nanoemulsions with improved rheological 25 properties contribute to the development of biodegradable and non-toxic food or 26 agrochemical products. 27 28 Keywords: Nanoemulsion, rosemary essential oil, biopolymer, rheology, physical 29 stability. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 1. INTRODUCTION 67 In recent years, numerous studies about nanoemulsions have been developed due to the 68 interest of these formulations as delivery systems for lipophilic active compounds. 69 However, nanoemulsions are Newtonian and lack the desired rheological properties for 70 some applications. Thus, for example in the case of an emulsion-based agrochemical or 71 food product, this product should possess a non-Newtonian behaviour with high viscosity 72 at rest in order to prevent its destabilization during lifetime, but at the same time, with 73 low viscosity under an applied stress in order to facilitate the handling. In addition, a gel- 74 like structure is also interesting due to the fact that is correlated with a higher stability of 75 emulsions against creaming. This rheological behaviour can be obtained with these 76 structured nanoemulsions which can be used not only in agrochemical or food 77 applications but also in pharmaceutic, medical or cosmetic applications. Currently, 78 another aspect to consider when formulating a product is the growing interest of 79 consumers in the use of natural products and sustainable raw materials. 80 Natural polymers, actually strongly demanded due not only to their thickening, stabilizing 81 or emulsifying properties, but also their biocompatibility, biodegradability and non- 82 toxicity, can be used to enhance the rheological properties and improve the shelf life of 83 nanoemulsions. Specifically, xanthan gum advanced performance and welan gum were 84 used in this study. 85 Xanthan gum (XG) is a natural high molecular weight anionic polysaccharide produced 86 by aerobic fermentation of sugars by the microorganism Xanthomonas campestris.1 There 87 are numerous products in different fields which use xanthan gum. Cleaners, coatings, 88 polishes and products in the agricultural and food industry also use xanthan gum.2 89 Specifically, advanced performance food grade xanthan gum is suitable for sauces, milk 90 and cream products or beverages. 91 Welan gum is an anionic extracellular polysaccharide produced by the micro-organism 92 Sphingomonas sp., namely from Alcaligenes sp., ATCC 31555.3 Although welan gum is 93 rarely used in emulsions, in the last few years, there have been a number of studies 94 concerning its use in this type of formulations. For example, welan gum may be used as 95 an ingredient in food products in which it can act as a thickening, suspending, binding, 96 emulsifying, stabilizing and viscosifying agent.3 97 The high antioxidant and antibacterial activity of REO against L. monoctytogenes 98 bacteria and also against Escherichia coli, Salmonella indiana and Listeria innocua has 99 been attributed to the presence of its main components such as αpinene, 1,8-cineole, 100 camphor, myrcene, camphene, borneol and verbenone.4–6 REO is used in a wide range of 101 applications, such as in food, in cosmetics, in nutraceuticals, agrochemical, 102 phytomedicines and aromatherapeutical products.7,8 103 In this study, a non-ionic surfactant known as Appyclean 6548 was used. This surfactant 104 belongs to the alkyl polypentoside (APP) group. Appyclean is a family of 105 environmentally friendly surfactants possessing an Ecocert certificate, which are very 106 desirable for using in green formulations. Rosemary oil/water emulsions has been 107 demonstrated to have high physical stability using Appycelan 6548 (D-pentose, 108 oligomeric C14/18 alkylglycoside) as surfactant.9 However, this system exhibits low 109 viscosity, a Newtonian flow behaviour and lacks a linear viscoelastic region. In addition, 110 this emulsion suffers a destabilization by creaming. It is to be expected that the addition 111 of gum causes the formation of a network capable of delaying the creaming and providing 112 viscoelasticity to the nanoemulsion. Thus, the development of sustainable nanoemulsions 113 with desirable rheological characteristic could be achieved by varying the concentration 114 of gum. Therefore, the main objective of this work was to study the influence of the 115 concentration of two different polysaccharides (welan and xanthan advanced 116 performance gum) on the droplet size distribution, rheology and physical stability of 117 nanoemulsions formulated with REO and Appyclean 6548, a non-ionic and renewable 118 bio-based surfactant. These stable and sustainable nanoemulsions with improved 119 rheological properties can be useful to the food and agrochemical industries as food 120 preservative or insecticide due to the antimicrobial properties of the rosemary oil.10,11 121 Furthermore, they can be useful to develop lipophilic active-loaded products with novel 122 applications. 88 89 2. MATERIALS AND METHODS. 90 Materials. 91 All components of nanoemulsions are derived from natural sources. Thus, the surfactant 92 is wheat-derived, while the dispersed phase is rosemary essential oil (REO) which is 93 distilled from Rosemarinus officianalis L. REO was supplied by Sigma Aldrich (Spain) 94 and Appyclean 6548 was purchased from Wheatoleo (France). Additionally, xanthan gum 95 (KELTROL® Advanced Performance) of molecular weight about 2·106 /mol and welan 96 gum (K1A96) of molecular weight about 6.6· 105 g/mol were used as stabilizing 𝑖 𝑖 97 biopolymers. Both gums were kindly provided by CP Kelco Company (San Diego, USA). 98 Ultrapure water was used to prepare the aqueous phase. 99 Emulsification Procedure. 100 The aqueous phases were prepared by dissolving 0.1/100 g sodium azide in ultrapure 101 water cleansed using a Milli-Q water purification system. The dispersed phases were 102 formed by the emulsifier, Appyclean 6548 (4/100 g), and rosemary oil (20/100 g). 103 Emulsions were prepared following the protocol reported by Martin-Piñero.9 Finally, in 104 order to obtain the structured nanoemulsion, the gum was added very slowly into the final 105 emulsions at room temperature. The emulsions were mechanically stirred using an Ultra- 106 turrax device (IKA Janke and kunkel, IKA-Labortechnik) for two hours up to a maximum 107 of 520 x g (800rpm). A total of 11 samples were prepared using different kinds of 108 biopolymer (xanthan and welan gums) at different concentrations (0, 0.1/100, 0.2/100, 109 0.3/100, 0.4/100, 0.5/100 g). 110 Droplet size distribution (DSD) measurements. 111 Size distribution of oil droplets was determined by the laser diffraction technique 112 (Mastersizer 2000, Malvern, Worcestershire, United Kingdom). Aging time evolution of 113 the volume-weighted mean diameter (D4,3) of the emulsion droplets and the span values 114 were studied for 28 days. D4,3 values were obtained from the following equation: 115 115 𝐷4,3 = 𝑁 𝑖=1 𝑁 𝑖=1 𝑛 𝑖 𝑑 4 𝑛 𝑖 𝑑 3 (Eq. 1) 116 where ni is the number of droplets with diameter di. On the other hand, span values are 117 given by: 118 118 𝑆𝑝𝑎𝑛 = 𝐷(𝑣,0.9)−𝐷(𝑣,0.1) 𝐷(𝑣,0.5) (Eq. 2) 119 where D(ν, 0.9), D(ν, 0.5) and D(ν, 0.1) are the highest droplet size contained in 90%, 120 50% and 10% of volume of dispersed phase, respectively. 121 Physical Stability. 122 The physical stability of rosemary essential oil nanoemulsions was evaluated for 28 days 123 at 30ºC by a Turbiscan Lab Expert device (Formulaction, Toulouse, France) using the 124 multiple light scattering technique. Emulsion stability was studied by analysing the 125 variation of the backscattering (BS) profiles as a function of time. The intensity of the ∑ ∑ 126 scattered light makes it possible to determine the major mechanism of destabilization in 127 each sample as well as the kinetics of the destabilization process. 128 Rheology. 129 Flow curves were performed with a Modular Advanced Rheometer System (Haake 130 MARS, Thermo Electron Corp., Germany) and a double cone geometry (60/1º) for 131 nanoemulsions with gum concentration lower than 0.3/100 g. Nanoemulsions with 132 0.4/100 g and 0.5/100 g of gums were measured using a serrated plate-plate sensor (60 133 mm). Flow curves were carried out in a range of 0.05 to 20 Pa. 134 Frequency sweeps were obtained from 0.05 to 15 rad/s at fixed shear stress within the 135 linear viscoelastic region. Creep experiments were performed for constant shear stresses 136 (0.05 Pa) for 3600 s. 137 Equilibration time prior to rheological tests was 300s. All measurements were performed 138 at 20 °C ± 0.1 °C using a C5P Phoenix circulator (Thermo‐Scientific, Germany). 139 All measurements were carried out in triplicate and the values shown are the average of 140 the three replicates. The standard deviation of all measurements was lower than 5-10% 141 for all the samples. 142 Statical analysis. 143 A general linear model – one-way ANOVA – was used to determine significant 144 differences (P≤0.05) between samples, followed by the post-hoc Tukey HSD test. All 145 statistical treatments were done with the software Infostat, version 2017 (Facultad de 146 Ciencias Agropecuarias, Universidad Nacional de Cordoba, Cordoba, Argentina). 147 148 3. RESULTS AND DISCUSSION. 149 3.1.Droplet Size Distribution 150 Figure 1 shows as inset, by way of example, the droplet size distribution of the emulsions 151 containing 0.4/100 g WG and 0wt% gum as reference, both 24 hours after preparation. 152 As can be observed, 0wt% gum emulsion is monomodal (span = 0.74±0.01) and the 153 volumetric weight diameter is 149 ± 6 nm. By contrast, the addition of 0.4/100 g of 154 biopolymer provokes a shift of DSD towards bigger droplets and the distribution was 155 bimodal. In order to gain a deep insight into the results obtained, measurements of laser 156 diffraction at different conditions of dispersion (introducing ultrasounds) as a function of 157 time in the measuring cell were carried out. Results obtained are shown in Figure 1. As 158 can be observed, the droplet size distribution shifted towards smaller droplet sizes and 159 was monomodal. These facts revealed that the emulsion was initially flocculated and 160 proved that the second peak corresponded to air bubbles, which were removed. Therefore, 161 ultrasonics break-up the agglomerates, making it possible to obtain the droplet size of the 162 primary droplets. It should be noted that the addition of gum did not significantly change 163 the DSD compared to the emulsion without gum. However, these results demonstrate the 164 ability of both xanthan gum and welan gum to promote flocculation. 165 Figure 1 166 The influence of gum concentration and ageing time (1 and 28 days) on the values of D4,3 167 and span of nanoemulsions are shown in Table 1. These parameters were selected because 168 they were very sensitive to the aggregation of droplets and, therefore, to detect 169 destabilization by coalescence or Ostwald ripening. As can be observed, nanoemulsions 170 containing welan gum exhibited similar volumetric mean diameter 24 hours after 171 preparation, except for the one formulated with 0.4/100 g, which presented the highest 172 values of both D4,3 and span. Similar results were found for nanoemulsions containing 173 xanthan gum but it was the nanoemulsion with 0.5/100 g of gum which exhibited the 174 highest values of both parameters. Ageing time has an influence on distribution 175 parameters for nanoemulsions containing 0.3/100 g and 0.5/100 g of WG and for 176 nanoemulsions with 0.3/100 g of APXG. For these nanoemulsions, an increase in 177 volumetric mean diameter and span occurred with time, suggesting the occurrence of a 178 destabilization process by coalescence.12,13 It is noteworthy that the increase in droplet 179 size observed is not caused by flocculation due to the fact that the aggregates were broken 180 up before the measurement was carried out, as mentioned above. 181 181 182 3.2. SAOS tests 183 Regardless of type of gum used, all nanoemulsions exhibited a measurable linear 184 viscoelastic range except the one with 0.1/100 g of biopolymer. For this reason, results at 185 this concentration of gum are not shown. The effects of gum concentration on storage, 186 G‘, and loss, G”, moduli as a function of frequency for microfluidized rosemary O/W 187 emulsions, aged for 24 hours, are shown in Figure 2. All nanoemulsions showed that G’ 188 was larger than G” over the whole measured frequency range, indicating that the elastic 189 component was predominant in the viscoelasticity. Furthermore, a slight frequency 190 dependence of both moduli was found. These profiles are consistent with a weak gel-like 191 viscoelastic structure and they indicate more semi-solid-like than liquid-like behaviour. 192 As expected, G´ and G´´ increased with gum concentration, consistent with a tri- 193 dimensional macromolecular network with more elastic and viscous behaviour. As occurs 194 in gum solutions with gel-like structure, both moduli have an exponential relationship 195 with the gum concentration which was fitted to the following equations: 196 log 𝐺′ = 𝑙𝑜𝑔𝑎 + 𝑏 ∗ 𝑙𝑜𝑔𝐶 (Eq. 3) 197 log 𝐺" = 𝑙𝑜𝑔𝑎′ + 𝑏′ ∗ 𝑙𝑜𝑔𝐶 (Eq. 4) 198 199 where b and b’ are the power law index of G’ and G”, respectively, and a and a’ are elastic 200 coefficient and viscous coefficient, respectively. As can be observed in Table 2, a and a’ 201 values were higher for nanoemulsions containing welan gum, indicating that these 202 nanoemulsions are more elastic and viscous than nanoemulsions containing xanthan gum. 203 Likewise, nanoemulsions with welan gum were more dependent on the concentration, as 204 can be deduced from the higher values of the b and b’ parameters. 205 Figure 2 206 Table 2 207 From the frequency sweeps, values for loss tangent were calculated. tan  is defined as 208 the ratio of loss modulus and storage modulus, which is used to detect differences in the 209 temporary structure of samples. 210 210 𝑡𝑎𝑛𝛿 = 𝐺´´ 𝐺´ (Eq. 5) 211 When the loss tangent approaches zero, the elastic structure of the system predominates, 212 whereas if it exceeds 1 value the system is considered viscous. As shown in Figure 3A, 213 the elastic component predominates over the viscous one over the whole frequency range 214 with 0.23< tan <0.33 and 0.29< tan <0.68 for nanoemulsions with APXG and WG, 215 respectively. The tan values were lower than 1, indicating that the molecular network 216 was strong and permanent.14 In all cases, 0.1< 𝑡𝑎𝑛𝛿 < 1 indicating that these 217 nanoemulsions do not behave as true gels, but as weak gels such as discussed above. 218 218 N N 219 All nanoemulsions containing gum as thickener showed a clear minimum in tan  value 220 except for the emulsion with 0.2/100 g WG. From this minimum it is possible to calculate 221 the plateau modulus, G 0: 15 222 222 G 0  [G´] (Eq. 6) N tanmin 223 223 224 The higher the G 0 value, the higher the entanglement density between polymeric 225 molecules.16 As expected (Figure 3B), an increase in gum concentration caused an 226 increase in the plateau modulus value, indicating an increase in the entanglement density. 227 It should be noted that the values of plateau modulus are slightly higher in nanoemulsions 228 containing welan gum than those containing xanthan gum. 229 All results are consistent with a double helix conformation for both gums. However, it 230 seems strange that welan gum, despite having lower molecular weight than xanthan, 231 provides nanoemulsions with greater viscoelasticity. This result could be explained by 232 the fact that welan gum adopts a different distribution pattern.17 233 Figure 3 234 The evolution with ageing time of G′ values at an intermediate frequency within the 235 frequency range studied (6.8 rad/s) for all nanoemulsions is illustrated in Figure 4. Fig. 4 236 shows that for the first days nanoemulsions exhibited a slight tendency to a decrease in 237 G′, made more marked by increasing the concentration of gum in the emulsion. 238 Subsequently, the values remained constant. A decrease in G’ with ageing time could be 239 attributed to an increase in droplet size, either by coalescence or by flocculation, which 240 provokes a decrease in the area of contact between droplets, weakening the interactions 241 between them and, therefore, a decrease in elastic properties. 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Influence of 484 primary homogenization step on microfluidized emulsions formulated with 485 thyme oil and Appyclean 6548. J Ind Eng Chem 66:203–208 (2018). 486 486 487 Figures 488 488 489 Figure 1. Droplet size distribution for emulsions containing 0.4wt% of WG as function of 490 ultrasound time in the measurement cell and as inset, 0.4 wt% WG without ultrasound and 0 491 wt% gum 492 493 494 495 496 Figure 2. Influence of gum concentration on the mechanical spectra for nanoemulsions 497 containing a)WG or b) APXG. Temperature = 20 °C. 498 499 500 501 Figure 3. A) Influence of frequency on loss tangent of rosemary essential oil nanoemulsions 1- 502 day aged containing a) WG or b) APXG as a function of the gum concentration. B) Plateau 503 modulus of rosemary essential oil nanoemulsions versus gum concentration as a function of the 504 type of gum. 505 506 507 Figure 4. Influence of aging time on the elastic modulus at 6.8 rad/s of rosemary essential oil 508 nanoemulsions containing WG or APXG as a function of gum concentration. Temperature = 20 509 °C. 510 511 512 Figure 5. Creep compliance versus time of rosemary essential oil nanoemulsions containing WG 513 or APXG as a function of the gum concentration. Shear stress= 0.05 Pa. Temperature 20 °C. 514 515 516 517 Figure 6. Flow curves of rosemary essential oil nanoemulsions containing different 518 concentrations of APXG. Continuous lines correspond to the Cross model fitting equation. 519 Temperature = 20 °C. 520 521 522 Figure 7. BS profiles versus measuring cell height of rosemary essential oil nanoemulsions 523 containing a) APXG and b) WG at different concentrations of gum ( 1 ( 0.1/100g), 2 (0.2/100g), 524 3 (0.3/100 g), 4 (0.4/100 g) and 5 (0.5/100 g)) as a function of aging time. 525 526 527 Figure 8. Turbiscan stability index versus aging time of rosemary essential oil nanoemulsions 528 containing WG (closed symbols) or APXG (open symbols) as a function of the gum concentration. 529 Temperature = 200C 530 531 Tables 532 Table 1. Volume mean diameter and span at 1 and 28 days of Rosemary essential oil 533 nanoemulsions containing WG or APXG. Mean value ± standard deviation values by a 534 different superscripts within raw were significantly different (P<0.05). Gum concentration (wt%) Gum D4,3 - Day 1 (nm) D4,3 - Day 28 (nm) Span (Day 1) Span (Day 28) 0.1 APXG 147±1a - 1.1±0.4a - WG 148±1a - 1.09±0.02a,b - 0.2 APXG 149±5a 150±4a 0.86±0.08a,b 1.0±0.1a,b WG 145±2a 146±1a 0.771±0.01a 0.744±0.03a 0.3 APXG 154±2a 204±12a 0.8±0.1a 1.7±0.1b