Barley and yeast β-glucans as new emulsifier agents for the development of aqueous natural antifungal formulations

1 BARLEY AND YEAST β-GLUCANS AS NEW EMULSIFIER AGENTS FOR THE DEVELOPMENT OF 1 AQUEOUS NATURAL ANTIFUNGAL FORMULATIONS 2 Marta Salgado, Soraya Rodríguez-Rojo*, María José Cocero 3 High Pressure Processes Group, Department of Chemical Engineering and Environmental 4 Technology, EII Sede Mergelina, University of Valladolid, 47011 Valladolid, Spain. 5 * Corresponding author at: Soraya Rodríguez-Rojo. Tel: +34 983 423166. E-mail adress: 6 [email protected]va.es 7 8 ABSTRACT 9 Barley and yeast β-glucans were selected, together with lecithin, to encapsulate resveratrol by 10 emulsification-evaporation method to develop new and safer antifungal formulations. Different 11 emulsification techniques were used: high-shear, high pressure and high pressure and 12 temperature emulsification. Morphology, crystallinity, encapsulation efficiency and in vitro 13 antifungal activity against Botrytis cinerea of the different formulations were evaluated. No 14 significant differences between each emulsification procedure in particle size (below 90 nm) and 15 in encapsulation efficiency (70-100%) were observed; only barley β-glucan emulsions showed 16 lower efficiency due to the formation of a gel that retained most of the active compound. A 17 great influence of the emulsification method and the encapsulating material on the crystallinity 18 of the particles was observed. The highest antifungal activity (up to 53% growth inhibition) was 19 obtained by the formulations with yeast β-glucans, indicating an enhanced absorption of 20 encapsulated resveratrol through the cell wall of the fungus at the presence of (1-3, 1-6)-β21 glucans. 22 Keywords: β-glucan, lecithin, resveratrol, encapsulation, high-pressure emulsification, Botrytis 23 cinerea. 24 2 1. Introduction 25 Fungal and bacterial infections are a major concern in agriculture since they produce great losses 26 (Spadaro & Gullino, 2004). On one hand, many fruits and vegetables must be discarded because 27 they cannot be recovered once they are affected, raising an ethical issue due to the increasing 28 global demand of food derived from a bigger population (Bebber & Gurr, 2015). On the other 29 hand, producers must carry out a big investment in order to keep infections under control and 30 to minimize the damages on their crops. Therefore, it is important to develop effective products 31 which reduce the growth of pathogens and preserve the quality of food. For many years, several 32 chemical products have been used with this purpose. However, they can leave toxic residues on 33 the plant, which afterwards could affect human health and contaminate the environment (Cruz 34 Cabral, Fernández Pinto, & Patriarca, 2013). Furthermore, fungi and bacteria can develop 35 resistant strains after prolonged application (Panebianco et al., 2015). Thus, in recent years 36 there is a tendency towards the development of alternative antifungal and antibacterial 37 products from non-toxic, natural origin substances (Mari, Bertolini, & Pratella, 2003). 38 In this sense, essential oils have been extensively studied because of their antifungal properties 39 (Soylu, Kurt, & Soylu, 2010; Stević et al., 2014). The biocide activity of essential oils is mainly due 40 to their content in polyphenolic compounds that increases cell membrane permeability and 41 promotes its disruption (Tiwari et al., 2009); they also trigger defense mechanisms in the 42 infected plant: produce alkalinization of the medium, stimulate oxidative burst and induce 43 defense genes (Chang, Heene, Qiao, & Nick, 2011). Therefore, many works analyze plant 44 extracts, focusing on their phenolic content and their activity against different fungi, like 45 Aspergillus, Penicillium, Botrytis or Candida (Gatto et al., 2011; Martins, Barros, Henriques, Silva, 46 & Ferreira, 2015; Rashed, Ćirić, Glamočlija, & Soković, 2014). 47 The aim of this work is to develop a liquid antifungal formulation with a model phenolic 48 compound, resveratrol, from an oil-in-water emulsion and removal of the organic solvent. 49 3 Resveratrol is included among phytoalexins, secondary metabolites that are synthesized by 50 plants as protective agents against bacterial and fungal attacks (Adrian & Jeandet, 2012). 51 Resveratrol is present in many plants, although grapes have the highest concentration 52 (Fernández-Mar, Mateos, García-Parrilla, Puertas, & Cantos-Villar, 2012). Antifungal activity of 53 resveratrol has been previously reported (Jung et al., 2005), also against Botrytis cinerea (Adrian, 54 Jeandet, Veneau, Weston, & Bessis, 1997), which is a widely extended fungus that affects several 55 fruits and plants in postharvest stage, causing great losses (Williamson, Tudzynski, Tudzynski, & 56 Van Kan, 2007). Besides, structural changes in conidia upon resveratrol application were 57 observed (disrupted plasma membrane, disorganized cytoplasm withdrawn from conidial wall), 58 which could explain the mode of action of resveratrol leading to cell death (Adrian & Jeandet, 59 2012). However, fungal laccases produce extracellular oxidation of resveratrol and its 60 degradation (Chang et al., 2011; Rivera-Hoyos et al., 2013; Timperio, D’Alessandro, Fagioni, 61 Magro, & Zolla, 2012). Therefore, a proper formulation is required in order to encapsulate 62 resveratrol and protect it from laccases until the target. In our previous work, solid particles of 63 resveratrol encapsulated on barley β-glucans were obtained by emulsification-evaporation and 64 further drying of the suspensions (Salgado, Rodríguez-Rojo, Alves-Santos, & Cocero, 2015). 65 These particles showed antifungal activity against B. cinerea. However, a liquid formulation 66 would be preferred because it is easier to apply in situ. Resveratrol has been previously 67 formulated as liquid suspension in different materials such as lipids, and synthetic and natural 68 polymers (proteins and polysaccharides), mainly for pharmaceutic, cosmetic or food-related 69 applications. These suspensions were created by different methods, including evaporation 70 (Pujara, Jambhrunkar, Wong, McGuckin, & Popat, 2017), antisolvent precipitation and 71 electrostatic deposition (Xulin Huang et al., 2017), ionic gelation (Jeon, Lee, & Lee, 2016), or 72 ultrasounds (Caddeo et al., 2016), among others. 73 Lipids (e.g. fatty acids, lecithin) are commonly used as surfactants because they can incorporate 74 both hydrophilic and lipophilic active compounds, are non-toxic and easily absorbed through 75 4 biological membranes due to the formation of liposomes, which are very similar to cell 76 membranes (Varona et al., 2013). Polysaccharides are also used because they enhance the 77 stability of the emulsion through an increase of viscosity that reduces the movement of the 78 droplets within the emulsion (García, Alfaro, Calero, & Muñoz, 2014). Among polysaccharides, 79 β-glucans, polymers of D-glucose linked by glycosidic bonds in positions (1-3), have been 80 selected because they are present in several fungi, bacteria and some kind of plants, including 81 barley or oat. Therefore, it is hypothesized that, since β-glucans are present in the cell wall of B. 82 cinerea (Tenberge, 2007), they may improve the absorption of the encapsulated active 83 compound and thus increase its effectiveness, besides acting as surfactant. β-glucans have 84 different structure depending on their origin. For instance, cereal β-glucans have (1-4) 85 branching, whereas yeast β-glucans (YBG) have (1-6) branching (Zhu, Du, & Xu, 2016). Also, the 86 variation on their structure provides them different physical and biological properties. For 87 instance, branched (1-3),(1-6)-β-glucans have stronger immune modulatory properties than (188 3),(1-4)-β-glucans (Mikkelsen, Jespersen, Mehlsen, Engelsen, & Frøkiær, 2014). Moreover, many 89 works report the ability of β-glucans as elicitors in plants to induce the formation of secondary 90 metabolites and trigger defense responses (Almagro, García-Pérez, Belchí-Navarro, Sánchez91 Pujalte, & Pedreño, 2016; Aziz et al., 2003). 92 β-glucans have been used as encapsulating material for anthocyanins by coacervation (Xiong, 93 Melton, Easteal, & Siew, 2006), for proteins through the production of cryogels (Lazaridou, 94 Kritikopoulou, & Biliaderis, 2015) and for resveratrol by spray-drying and PGSS-drying (Salgado 95 et al., 2015). In emulsion, they have been previously used as stabilizers (Burkus & Temelli, 2000; 96 Kontogiorgos, Biliaderis, Kiosseoglou, & Doxastakis, 2004; Thammakiti, Suphantharika, 97 Phaesuwan, & Verduyn, 2004). Barley β-glucans (BBG) were found to reduce surface tension in 98 aqueous solutions, reaching a constant value of 50-55 mN/m at 2 g/L (unpublished work). 99 However, to the authors’ knowledge, there is just one work in which they were used as 100 surfactant of emulsions, but it was found out that the active compound (quercetin) precipitated 101 5 in crystals inside the polymer, so to avoid this, lecithin was added as co-surfactant (Gonçalves et 102 al., 2015). 103 In this work, β-glucans were used as encapsulating material for the production of liquid 104 formulations of resveratrol by emulsification-evaporation. Also lecithin was used, both alone 105 and mixed with the β-glucans, in order to enhance the formation of the emulsion. Different 106 emulsion techniques were used: high-shear, high pressure and high pressure and temperature 107 emulsification. The latter methods were performed because they were reported to provide 108 higher encapsulation efficiencies (de Paz, Martín, Mateos, & Cocero, 2013). Finally, the 109 antifungal activity of the formulations with resveratrol was tested against B. cinerea. 110 111 2. Materials and methods 112 2.1. Materials 113 As encapsulating materials, two different types of β-glucans were used: barley (1-3, 1-4)-β114 glucans (75% purity; 125 kDa, determined as indicated in (Salgado et al., 2015) Glucagel, kindly 115 supplied by DKSH, France) and (1-3, 1-6)-β-glucans from yeast Saccharomyces cerevisae (64% 116 purity, determined as indicated in section 2.2.1. β-glucan content; L-Naturae Nutraceutical, 117 kindly supplied by Naturae, Spain). Soybean lecithin (Glama-sot, SOTYA S.A., Spain) was also 118 used as encapsulating material. Ethyl acetate (99%), malt extract agar, acetic acid glacial and 119 sodium acetate were purchased from Panreac (Spain). Resveratrol with 98% purity was 120 purchased from Pure Bulk (USA). Pullulan standards were purchased from Shodex. 121 2.2. Solubilization and characterization of YBG 122 BBG were soluble in water, so they could be directly dissolved in water and used in 123 emulsification. However, YBG were insoluble in water, therefore a pretreatment was required 124 in order to solubilize them. For that purpose, a 170 mL stainless steel vessel was used. Beside 125 6 YBG, sodium acetate (1.41 mg/g of β-glucan) and glacial acetic acid (12.2 μL/g of β-glucan) were 126 charged into the vessel at concentrations reported in (Cox, 2008), with a solid to liquid ratio of 127 1:10 (w:w). The vessel was heated with an electric external resistance and kept at 135° C and 128 0.28 MPa for 4.5 hours. Samples of the product were obtained at 3, 3.5, 4 and 4.5 h. Further 129 description of the equipment can be found in (Sánchez-Bastardo, Romero, & Alonso, 2017) . 130 2.2.1. β-glucan content 131 The quantity of β-glucan on the solubilized YBG was analyzed by means of the “β-Glucan Assay 132 Kit (Yeast & Mushroom)” from Megazyme (Ireland). Following the described procedure in the 133 kit, the percentage of total glucans and α-glucans was determined spectrophotometrically 134 (Shimadzu UV-2550), and the percentage of β-glucans was calculated as the difference between 135 the total and the α-glucans. 136 2.2.2. Molecular weight of the β-glucans 137 Molecular weight of YBG was measured by size exclusion chromatography, with a guard column 138 (Shodex SB-G), a column (Shodex SB-804 HQ, particle size 10 μm, 7.8x300 mm) and a differential 139 refractive index detector (410, Waters Corporation). The column was kept at 35º C and flow rate 140 of the mobile phase (0.1 M NaNO3 and 0.02% NaN3) was set at 0.4 mL/min. Pullulan standards 141 (5.9-708 kDa) were used. 142 2.3. Preparation of emulsions 143 2.3.1. High shear emulsification 144 An oil-in-water emulsion was formed, containing resveratrol dissolved in ethyl acetate (7.5 g/L) 145 as organic phase and the encapsulating material dissolved in the aqueous phase, previously 146 saturated with ethyl acetate. Different concentrations were tested in the aqueous phase (10, 15 147 and 20 g/L) for each of the encapsulating materials used: BBG, solubilized YBG and lecithin, and 148 mixtures of lecithin and each of the glucans (50% in weight). 149 7 Both liquid phases were mixed in a ratio 1:9 (v:v) at 800 rpm for 5 min and then the emulsion 150 was fed into a 200 mL rotor-stator machine (IKA LABOR PILOT 2000/4) and processed for 4 151 minutes at 4200 rpm, as described in (Salgado et al., 2015). 152 2.3.2. Precipitation from a pressurized emulsion 153 High pressure emulsification was also performed with the same aqueous and organic phases 154 aforementioned, at 6 MPa and ambient temperature. The aqueous solution was pressurized 155 with a Dosapro Milton Royal D (18 mL/min, 20 g surfactant/L), whereas a Jasco PU-2080 plus 156 pump was used for feeding the organic phase (2 mL/min, 7.5 g resveratrol/L). After 157 pressurization, both streams were put in contact in a T-mixer, where the emulsion was formed, 158 and afterwards the product was recovered. 159 2.3.3. Precipitation from a hot pressurized emulsion 160 This method of encapsulation was previously used (de Paz et al., 2012) to accelerate the mass 161 transfer kinetics to the time scales of the precipitation process, thus improving the control over 162 the precipitation. Briefly, a solution or suspension of the active compound in the organic phase 163 is put in contact with another stream of pure organic solvent at high temperature and 164 immediately afterwards this flow is mixed with the cold water phase which contains the 165 surfactant. This causes the emulsification of the organic phase and the precipitation of the active 166 compound into the polymer. The emulsion is thus formed by the combined anti-solvent and 167 cooling effect of the water, and the active compound does not undergo thermal degradation 168 during the process because due to the configuration of the equipment, the exposition time to 169 high temperature is lower than 2 seconds. A detailed explanation about the equipment used 170 can be found in (de Paz et al., 2012). 171 The operation starts with the pressurization of the 3 streams up to 6.0-6.5 MPa and heating up 172 of the organic solvent to the desired temperature (in this case, 85-90ºC). When the operating 173 conditions were reached, the feed was changed from pure water and ethyl acetate to the 174 8 solutions used in this work: a solution of ethyl acetate with 7.5 g resveratrol/L at 2.75 mL/min, 175 hot ethyl acetate at 4.25 mL/min and a solution of 20 g surfactant/L in water at 20 mL/min. The 176 operating conditions were selected based on a previous work in which quercetin was 177 encapsulated on starch, lecithin and β-glucans (Gonçalves et al., 2015). 178 2.4. Formation of suspensions 179 Suspensions were obtained for all the different emulsification methods by removing the organic 180 solvent by vacuum evaporation (Heidolph) at 60º C, 75 rpm and a vacuum of 0.08 MPa. 181 Afterwards, big agglomerates of polymer and non-encapsulated crystals of resveratrol were 182 removed by centrifugation at 7800 rpm (6461 g) for 10 min. 183 2.5. Characterization of emulsions and suspensions 184 2.5.1. Droplet size distribution 185 Droplet and particle size distribution of the emulsions and suspensions was measured by laser 186 diffraction (Mastersizer 2000, Malvern) by diluting the emulsions on a solution of water 187 saturated with ethyl acetate and the suspensions only in water. Particle size was measured both 188 before and after centrifugation of the suspensions. Results are expressed as % in number. The 189 droplet and particle sizes reported correspond to the value of d(0.5). 190 2.5.2. CryoTEM 191 CryoTEM analysis of the aqueous solutions of the different β-glucans and their mixtures with 192 lecithin were performed to check how their chains associated themselves, in a JEOL JEM-FS2200 193 HRP 200 kV TEM with electron filtering. In brief, 4 μL of sample was deposited on a rack C-Flat 194 1.2/1.3, which was previously hydrophilized by a plasma cleaner. A blotting is performed on 195 either side of the filter grid during 4 s and then liquid ethane is introduced there in order to 196 freeze the samples, avoiding the formation of crystals (Gatan Cryoplunge 3). Samples are 197 maintained in liquid nitrogen until their transfer to the holder (Gatan Cryotransfer 626). 198 9 2.5.3. X-ray diffraction 199 X-ray diffraction (XRD) measurements were performed on a Bruker Discover D8 diffractometer 200 to check the crystallinity of the particles in the final suspensions, using the Cu Kα radiation (λ = 201 0.15406 nm). The suspensions were further centrifuged at 20000 rpm for 30 min (Beckman) in 202 order to recover the encapsulated particles. Afterwards, this sediment was dried in a freeze203 drier (LyoQuest -55, Telstar) for 48 hours and the final powder was analyzed by XRD. The 204 scattering intensities were measured over an angular range of 5 < 2θ < 70 for all the samples, 205 with a step size of 0.02°. 206 2.5.4. Encapsulation efficiency 207 Quantification of encapsulated resveratrol was done by HPLC with a guard column (Bio-Sil C18 208 HL 90-5, 4.6 x 30 mm, particle size 5 µm, Bio-Rad), a column (Symmetry C18, 4.6 x 150 mm, 209 particle size 5 µm, Waters) and a UV detector (λ = 306 nm). The column was kept at 25º C and 210 flow rate of the mobile phase (acetonitrile and water in ratio 1:3, with 0.2% formic acid) was set 211 at 0.8 mL/min. Encapsulation efficiency was determined as the ratio between the quantity of 212 resveratrol in the centrifuged suspension and the amount of resveratrol in the initial emulsion. 213 Calibration range of resveratrol was between 10 and 40 ppm. Prior to analysis, the centrifuged 214 suspensions were diluted (0.6 mL of sample in 10 mL) and filtered (0.22 μm). The analysis was 215 performed in triplicate, and the data were analyzed by t-Student´s test (unpaired samples, 216 unequal variances) with a significance p-value of 0.05. 217 2.6. B. cinerea culture 218 For the in vitro culture of B. cinerea, the fungus was isolated from vines in our university (Campus 219 La Yutera, Universidad de Valladolid, Palencia, Spain) and it was grown on malt extract agar. 220 After autoclaving, the agar was poured into Petri dishes containing the corresponding sample or 221 control, and when it solidified, the fungus was placed in the center of the surface. The quantity 222 of sample on each plate was determined so as to have a concentration of resveratrol of 100 223 16 Encapsulation efficiency was much lower with BBG because during centrifugation a gel-like 334 structure was formed and it retained most of the compounds. No significant differences were 335 observed between each emulsification method with any of the encapsulating materials. In some 336 previous works much higher encapsulation efficiencies were achieved by high pressure and 337 temperature emulsification (80%) than by high-shear emulsification (8%) in the formulation of 338 β-carotene with modified starch (de Paz et al., 2013). However, in that case the increase was 339 due to the higher solubility of the active compound in the hot organic solvent, whereas in this 340 work the concentration of resveratrol in the organic phase was kept constant in all the 341 experiments. 342 3.3. B. cinerea culture 343 For the samples by high-shear emulsification, no significant differences were observed between 344 each product at the lowest concentrations of surfactant (10 and 15 g/L), since none of them had 345 effect on the fungal growth (results not shown). However, some inhibition was achieved with 346 the suspensions at 20 g/L of encapsulating material, also for the samples by high-pressure and 347 high-pressure and temperature emulsification. Figure 6 presents fungal growth area when 348 applying the suspensions of resveratrol formulated with lecithin (Lec), a mixture of lecithin and 349 BBG (LecBBG), BBG, a mixture of lecithin and YBG (LecYBG) and YBG. The results shown here 350 correspond to the centrifuged suspensions except in the case of BBG. These had much lower 351 encapsulation efficiency, so higher quantity of sample was required to perform the analysis and 352 therefore it interfered in the growth of the fungus due to the dilution of the agar. Therefore, the 353 bars named BBG in Figure 6 correspond to the suspensions without centrifugation. 354 17 355 Figure 6. Growth area of B. cinerea for the centrifuged suspensions of resveratrol. Light gray: 356 high-shear emulsification. Dots: high pressure emulsification. Dark gray: high pressure and 357 temperature emulsification. *: suspensions without centrifugation. Significantly different 358 results are considered for p<0.05. 359 360 The formulation of resveratrol with lecithin and with the mixture of lecithin and BBG had no 361 effect on fungal growth, although some inhibition was achieved with BBG without mixing (up to 362 23%). The greatest growth reduction corresponded to the suspensions of YBG, ranging the 363 inhibition between 44 and 53%. However, this effect was almost unnoticeable for the mixture 364 of YBG with lecithin: no inhibition was observed by high pressure emulsification, and up to 15% 365 for the other methods. Except in this case, no significant difference was noticed between each 366 emulsification method regardless of the encapsulating material. Pure resveratrol did not 367 produce any inhibition of fungal growth. 368 According to these results, the presence of β-glucans in the formulation enhances the fungicide 369 action of resveratrol with respect to the formulation with lecithin. Varona et al., 2013 studied 370 the penetration of liposomes formed by lecithin into cell membranes of different bacteria. In 371 that work, liposomes showed ability to cross cell walls when they are constituted by 372 phospholipid layers, as happens in gram-negative bacteria. However, they could not cross into 373 the cell when there were some other barriers, as in the case of the gram-positive bacteria. B. 374 0 1000 2000 3000 4000 5000 6000 Control Lec LecBBG BBG* LecYBG YBG Growth area (mm2) 18 cinerea has a two-layer cell wall composed of chitin and β-glucans (Tenberge, 2007). It is possible 375 that liposomes could not penetrate into that barrier and deliver resveratrol inside, and thus the 376 formulations with lecithin had no antifungal effect. On the contrary, resveratrol is better 377 absorbed within the cell wall of the fungus when it is encapsulated in β-glucans, especially YBG, 378 which contains the same β-glucan type as the fungal cell wall (Zhu et al., 2016), so the inhibitory 379 effect on fungal growth is bigger. Nevertheless, the inhibitory effect achieved with the liquid 380 formulations was not comparable to the one obtained with solid particles of the same products 381 reported in a previous work (Salgado et al., 2015). Some studies reported an elicitor effect of β382 glucans on the production of resveratrol by some plants, like Vitis vinifera (Vuong, Franco, & 383 Zhang, 2014). Thus, it is possible that when the formulations with β-glucans are applied in vivo, 384 the concentration of resveratrol increases, and this could cause a greater fungal growth 385 inhibition. 386 387 4. Conclusions 388 A liquid formulation of resveratrol was developed and tested against B. cinerea, by three 389 different emulsification methods: high-shear, high-pressure and high pressure and temperature 390 emulsification. As encapsulating materials, BBG and YBG were used, both alone and mixed with 391 lecithin. YBG showed good encapsulating properties regarding encapsulation efficiency (74392 84%), besides providing 50% inhibition of fungal growth, the best for all the products tested. On 393 the contrary, BBG were not as good as encapsulating material because their chains entangle and 394 form a net instead of single droplets, thus having low encapsulation efficiency (31-54%). Also 395 they showed lower antifungal activity (around 20% inhibition). The formulation with lecithin also 396 showed high encapsulation efficiency and small particle size, although it did not inhibit fungal 397 growth. When mixing β-glucans with lecithin, a reduction on particle size was observed, 398 although the mixing also induced the formation of crystals of resveratrol inside the encapsulated 399 19 particles in comparison with β-glucans alone. Therefore, it was concluded that the formulation 400 of resveratrol with β-glucans improved the action against B. cinerea, probably through an 401 enhanced absorption of the active compound by the fungus. Comparing the emulsification 402 methods developed in this work, there were not big differences between them on encapsulation 403 efficiency and particle size. Nevertheless, by high pressure and temperature emulsification, 404 amorphous resveratrol was obtained inside the particles, whereas it formed crystals with the 405 other methods. Despite this, the effect on the reduction of fungal growth was similar by all the 406 emulsification methods for each material, without significant differences between them. As β407 glucans appear as a promising effective encapsulating material to improve the antifungal activity 408 against B. cinerea, the formulations could be developed with other polyphenols in future works. 409 It would be also interesting to assess if they are also effective against other fungal species. 410 411 412 Acknowledgments 413 Authors acknowledge the European project SHYMAN FP7-NMP-2011-LARGE-280983 and project 414 PIP 063/147181 from Fundación General of the University of Valladolid for financial support. M. 415 Salgado thanks to Ministerio de Educación, Cultura y Deporte for her Formación de Profesorado 416 Universitario grant. S. Rodríguez-Rojo acknowledges to Ministerio de Economía, Industria y 417 Competitividad and Universidad de Valladolid for her Juan de la Cierva fellowship. 418 419 20 REFERENCES 420 Adrian, M., & Jeandet, P. (2012). 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