Encapsulation of resveratrol on lecithin and β-glucans to enhance its action against Botrytis cinerea

ENCAPSULATION OF RESVERATROL ON LECITHIN AND β-GLUCANS TO ENHANCE ITS 1 ACTION AGAINST BOTRYTIS CINEREA 2 Marta Salgadoa, Soraya Rodríguez-Rojoa,*, Fernando Manuel Alves-Santosb, María 3 José Coceroa 4 a High Pressure Processes Group, Department of Chemical Engineering and 5 Environmental Technology, EII Sede Mergelina, University of Valladolid, 47011 6 Valladolid, Spain. 7 b Sustainable Forest Management Research Institute, Department of Plant Production 8 and Forest Resources, University of Valladolid-INIA, Avda. Madrid, 57. Campus La 9 Yutera, 34004 Palencia, Spain. 10 * Corresponding author at: Soraya Rodríguez-Rojo. Tel: +34 983 423166. Fax: +34 98311 423013. E-mail adress: [email protected] 12 Salgado, M., Rodríguez-Rojo, S., Alves-Santos, F. M., & Cocero, M. J. (2015). Encapsulation of resveratrol on lecithin and Β-glucans to enhance its action against botrytis cinerea. Journal of Food Engineering, 165, 13-21. https://doi.org/10.1016/j.jfoodeng.2015.05.002 ABSTRACT 13 β-glucans and soy lecithin were used as encapsulating materials for resveratrol and 14 tebuconazole, in order to test their antifungal activity against Botrytis cinerea. First an 15 oil-in-water emulsion was formed and afterwards the emulsion was dried by spray-16 drying or by particles from gas saturated solutions-drying (PGSS-drying). β-glucans were 17 precipitated also by supercritical anti-solvent (SAS), but it was not a suitable drying 18 process for this material. Particles were characterized regarding particle size, 19 morphology, crystallinity, encapsulation efficiency and in vitro activity against Botrytis 20 cinerea. Although the emulsions with β-glucans had bigger droplet size than the ones 21 with lecithin, there was no difference on particle size for each encapsulating material, it 22 was just dependent on the drying process. For all the materials and drying processes, 23 completely amorphous particles were obtained, in spite of the crystalline form of the 24 pure active compounds. Concerning the antifungal activity, the encapsulation of 25 tebuconazole did not improve its action, because it was already very effective. Pristine 26 resveratrol did not reduce fungal growth, but it was inhibited between 50-70% with 27 encapsulated resveratrol, which implies the production of an effective fungicide against 28 Botrytis cinerea totally from natural origin substances. 29 Keywords: resveratrol, β-glucan, lecithin, spray-drying, PGSS-drying, fungicide. 30 1. INTRODUCTION 31 Botrytis cinerea is a pathogenic fungus causing gray mold, which affects several fruits 32 and plants all over the world. Once a product is attacked by Botrytis cinerea, it cannot 33 be recovered, so it produces great economic losses (Williamson et al., 2007). Also, one 34 of the most important problems when handling with Botrytis cinerea is that it infects the 35 plants during the blooming, but it is not noticeable until the ripening (Timperio et al., 36 2012). Commonly, chemicals are used to fight against this disease, for instance azoles 37 (Stehmann and de Waard, 1996), anilinopyrimidines, phenylpyrroles or hydroxyanilides 38 (Rosslenbroich and Stuebler, 2000). However, resistant strains are developed quickly by 39 the fungus (Elad et al., 1995; Pappas, 1997). Furthermore, as the treatment against 40 Botrytis cinerea must be applied in full-grown products, or even post-harvest, there is a 41 tendency towards replacing the use of toxic substances by natural, environmentally 42 friendly products (Ali et al., 2015). 43 When a plant is attacked by a pathogen, it develops a chemical response which involves 44 the production of some proteins, phytoalexins and other phenolic compounds 45 (Langcake and Pryce, 1976; Montero et al., 2003; Timperio et al., 2012) that show 46 antifungal activity (Mendoza et al., 2013). The main phytoalexin produced is resveratrol, 47 which is found in high quantities on grape skin (Casas et al., 2010; Zheng et al., 2011). 48 Further, it has been found to be active against different fungi (Aldred et al., 2008; Santos 49 et al., 2006), including Botrytis cinerea (Adrian et al., 1997). It was observed that, when 50 resveratrol is produced spontaneously to fight against fungi, it is present within the cell 51 wall of the plant to be in contact with the pathogen (Adrian and Jeandet, 2012), or it is 52 even excreted out from the cell in liquid culture (Donnez et al., 2011) to improve the 53 contact between resveratrol and the fungus. Based on this findings, resveratrol has been 54 selected as promising antifungal of natural origin. Nevertheless, when applied externally 55 in the plant to fight Botrytis cinerea, resveratrol should be encapsulated in a carrier that 56 reaches easily the cell wall of the fungi and improves its penetration. 57 Encapsulation of active substances into polymeric matrices provides a controlled release 58 of the compound, since the encapsulating material can act as a carrier for the active 59 compound into the target, as well as protection against thermal or oxidative 60 degradation. Recently, some works focused on the treatment of Botrytis cinerea with 61 chitosan (Badawy and Rabea, 2009; Wu et al., 2005; Xu et al., 2007), which is one of the 62 components of the cell wall of the fungus. Furthermore, the encapsulation of essential 63 oil into chitosan improved its antifungal action against Botrytis cinerea (Mohammadi et 64 al., 2015). Taking into account that also β-glucans are present on the cell wall of Botrytis 65 cinerea (Tenberge, 2007), this polysaccharide is going to be use as carrier in the present 66 work. β-glucans are composed by D-glucose monomers linked by glycosidic bonds. 67 Depending on the bonds they have, they present different structure and branching, 68 which provides them their properties, such as solubility or biological activity (Bae et al., 69 2013). To the authors’ knowledge, there is just one previous study using β-glucans 70 against Botrytis cinerea (Aziz et al., 2003). In this work, linear β-1,3-glucans from 71 seaweeds were used as antifungal compound, and they reduced the growth of the 72 fungus by 50% in 4 days with a concentration of 1 g/L. However, β-glucans have never 73 been used as encapsulating material, although some studies have demonstrated that, 74 when they are added to an emulsion, they increase its stability (Burkus and Temelli, 75 2000; Kontogiorgos et al., 2004). 76 Encapsulation processes based on supercritical fluids techniques have attracted great 77 interest in the last years, because they allow better control of particle size and 78 morphology and reduce the contamination of the product by working in an inert 79 atmosphere or by an improved removal of the organic solvents (Martín and Cocero, 80 2008). Among the micronization processes with supercritical fluids, particles from gas 81 saturated solutions-drying (PGSS-drying) and supercritical anti-solvent (SAS) are 82 performed in this work. 83 PGSS-drying is a supercritical fluid process suitable for the production of particles, in 84 which the suspension is saturated with CO2 at high pressure and temperature prior to 85 the atomization. The sudden decompression of the fluid in the nozzle promotes the 86 desorption of the CO2 from the droplets, breaking them and enhancing the drying 87 process, so it can be performed at lower temperature than with conventional spray-88 drying (SD), which prevents the product from thermal degradation (Rodríguez-Rojo et 89 al., 2013). Also, all the process is a closed system inerted with CO2, so it is appropriate 90 for drying substances that are oxidized easily (Martín and Weidner, 2010). Few works 91 used this technology for the production of particles. They were mainly focused on the 92 encapsulation of antioxidants and essential oils (de Paz et al., 2012; Meterc et al., 2008; 93 Varona et al., 2013). 94 Alternatively, in SAS, the encapsulating material and the active compound are dissolved 95 in an organic solvent, and the liquid is introduced through a nozzle in a high pressure 96 vessel containing CO2 in supercritical conditions. At these conditions, CO2 is highly 97 soluble on organic solvents. When the droplets of the liquid stream are put in contact 98 with the supercritical CO2, the organic solvent is saturated with the CO2, so the solubility 99 of the solute decreases and this promotes its precipitation (Rodríguez-Rojo et al., 2013). 100 The aim of this work is to develop the formulation of a product against Botrytis cinerea 101 based entirely on natural substances, namely resveratrol, β-glucans and soy lecithin. Soy 102 lecithin has been used as comparison for the β-glucans because it is has been 103 demonstrated that encapsulation essential oils in lecithin can be improved their 104 bactericidal action since it can form liposomes in aqueous media and interact with cells 105 (Varona et al., 2013). Besides, it is biocompatible and non-toxic, and thus it is commonly 106 used in formulation of emulsions in applications related with food processing (de Paz et 107 al., 2012). First, an oil-in-water emulsion of resveratrol on β-glucan, lecithin or a mixture 108 of both substances is produced and afterwards it is dried by conventional SD or by PGSS-109 drying. Further, tebuconazole, a triazole commonly used in plant protection (Yang et al., 110 2014), is used to compare the action of resveratrol. Besides, β-glucan particles are also 111 obtained by precipitation by SAS. The particles formed by all these methods are 112 characterized and tested in vitro against Botrytis cinerea. 113 114 2. MATERIALS AND METHODS 115 2.1. Materials 116 As encapsulating materials, barley (1-3, 1-4)-β-glucans (75% purity; Glucagel, kindly 117 supplied by DKSH, France) and soybean lecithin (Glama-sot, SOTYA S.A., Madrid, Spain) 118 were used. A molecular weight of 125 kDa was determined by size exclusion 119 chromatography at the conditions indicated on Section 2.3. 120 Ethyl acetate (99%) and malt extract agar were purchased from Panreac (Spain), and 121 dimethylsulfoxide (DMSO) from Sigma-Aldrich (Spain). Resveratrol with 98% purity was 122 purchased from Pure Bulk (USA). Tebuconazole was kindly supplied by Aragonesas Agro 123 (Spain), both as pure powder (technical grade) and the commercial product Orius 20EW 124 (an oil-in-water emulsion with 20% w/v of tebuconazole). 125 2.2. Formation of particles 126 First, an oil-in-water emulsion was formed (IKA Labor Pilot), with the fungicide 127 (tebuconazole or resveratrol) dissolved in ethyl acetate (7.5 g/L) as organic phase, 128 whereas the aqueous phase, saturated with ethyl acetate, contained the different 129 matrixes used (15 g/L): β-glucans, soybean lecithin or a mixture of both of them (50% in 130 weight). Both liquid phases were mixed in a ratio 1:9 at 750 rpm for 5 minutes and then 131 the resulted emulsion was fed into a rotor-stator machine (IKA® LABOR PILOT 2000/4), 132 with 200 mL capacity, and processed at 4200 rpm for 4 minutes. These operating 133 conditions were selected based on some previous works (Varona et al., 2009), where it 134 was found a reduction in droplet size with increasing surfactant concentration, 135 homogenization velocity and time. Afterwards, the organic solvent was removed by 136 vacuum evaporation (Heidolph) at 60º C, 75 rpm and a vacuum of 0.08 MPa, and finally 137 the suspensions were dried either by SD or by PGSS-drying. 138 By SD (Gea Niro Mobile Minor), the suspension (1 L/h) was introduced into the drying 139 chamber through a rotary atomizer (compressed air at 0.6 MPa). Droplets were formed 140 and water was removed from them by a stream of hot air (130º C at the inlet and 85º C 141 at the outlet). The dry particles were recovered in a cyclone. 142 By PGSS-drying the suspension was pumped and put into contact with 10 kg/h of 143 preheated and pressurized CO2 (Milton Roy membrane pump) at 9.5 MPa and 125º C in 144 a 150 mm static mixer filled with 4 mm glass beads, so that the liquid was saturated with 145 CO2. Gas to liquid ratio was set at 30 (weight basis). Then this stream was expanded 146 through a nozzle (Spraying Systems, 500 µm) in the drying chamber, which was kept at 147 65-70º C. The conditions selected for the drying process were based on a previous work 148 (Varona et al., 2011), which contains further details about the experimental device. 149 Figure 1 shows a simplified diagram of the formation of particles by SD and PGSS-drying, 150 with the previous creation of the emulsion and suspension. 151 FIGURE 1 152 153 For the production of β-glucan particles by SAS, DMSO containing β-glucans in a 154 concentration of 2-10 g/L was pumped (HPLC pump, Gilson, model 850) and introduced 155 in a vessel with CO2. Preliminary tests at 20º C showed limited solubility of β-glucans in 156 ethyl acetate, acetone, ethanol and tetrahydrofuran (lower than 2.6 g/L for every 157 solvent), and higher in DMSO (14.8 g/L), so it was the solvent selected. Prior to introduce 158 the solution, the vessel must be conditioned just with CO2 until the required pressure 159 and temperature are achieved. Pressure was always set at 10-11 MPa to avoid the 160 formation of a liquid phase in the precipitation vessel. After all the suspension was 161 pumped, CO2 was still passed through the vessel in order to make sure that there was 162 no remaining DMSO inside. Then, the system was decompressed and the particles 163 formed were recovered from a filter placed at the outlet of the vessel. A scheme of the 164 process is shown in Figure 2. 165 FIGURE 2 166 167 2.3. Molecular weight of β-glucans 168 Molecular weight of the β-glucans was measured by size exclusion chromatography, 169 with a guard column (Waters Ultrahydrogel Guard Column), a column (Ultrahydrogel 170 500, Waters Corporation) and a differential refractive index detector (410, Waters 171 Corporation). The column was kept at 35º C and flow rate of the mobile phase (0.1 M 172 NaNO3 and 0.02% NaN3) was set at 0.4 mL/min. 173 2.4. Morphology of the particles 174 The morphology of the particles was analyzed by scanning electron microscopy (SEM, 175 JEOL JSM-820, 20 kV, 23-mm working distance) at vacuum conditions. Prior to the 176 analysis, the samples were covered with gold in an argon atmosphere. 177 2.5. Particle size distribution 178 Particle size distribution of the particles and emulsions produced was measured by laser 179 diffraction (Mastersizer 2000, Malvern). In order to perform the measurement, the 180 emulsions were suspended on a solution of water saturated with ethyl acetate, whereas 181 the suspensions were suspended only in water. On the contrary, the dried particles were 182 measured within a stream of air at 0.2 MPa. 183 2.6. DSC 184 Differential scanning calorimetry (DSC) profiles of the particles produced by SAS were 185 obtained using a Mettler Toledo 822e calorimeter. Heating rate was 10º C/min in the 186 temperature range of -40 to 280º C. An empty pan was used as reference. The 187 equipment was calibrated with indium. 188 2.7. X-ray diffraction 189 X-ray diffraction (XRD) measurements were performed on a Bruker Discover D8 190 diffractometer to check the crystallinity of the obtained particles, using the Cu Kα 191 radiation (λ = 0.15406 nm). The scattering intensities were measured over an angular 192 range of 5 < 2θ < 70 for all the samples, with a step size of 0.02º. 193 194 195 commercial oil-in-water emulsion (Orius), and the particles of lecithin (Lec), a mixture of 339 lecithin and β-glucans (LecbG) and β-glucans (bG) produced by SD (SD) or PGSS-drying 340 (PGSS). 341 FIGURE 7 342 343 All the particles obtained produced a great inhibition of the fungal growth (between 75 344 and 80% compared to the control sample). However, no significant differences were 345 observed with the commercial tebuconazole products. Therefore, although the 346 encapsulated particles were very effective against Botrytis cinerea growth, they did not 347 improve the action of powder and oil-in-water commercial tebuconazole, because it is 348 already very active against this fungus. Figure 7 shows some images of the Petri dishes 349 of Botrytis culture as control (b), with commercial tebuconazole powder (c) and with the 350 particles of tebuconazole encapsulated on β-glucan obtained by SD (d) and by PGSS-351 drying (e). It is noticed that growth is reduced when applying both pure tebuconazole 352 and the particles, compared to the control dishes, but there are not significant 353 differences among them. 354 For the activity of resveratrol, it was considered that previous works (Adrian et al., 1997) 355 reported growth inhibition of Botrytis cinerea with resveratrol concentration between 356 60 and 160 mg/L, so 100 mg/L of resveratrol was chosen for the in vitro tests. Figure 8 357 (a) reveals the results corresponding to the growth of Botrytis cinerea in the presence 358 of pure resveratrol (Resv), pure soy lecithin (Lec), pure β-glucans (bG) and a mixture of 359 both substances (LecbG), and the particles created with these encapsulating materials 360 and pure resveratrol by SD (SD) or PGSS-drying (PGSS). 361 FIGURE 8 362 363 No effect was observed on fungal growth when applying pure resveratrol, although 364 growth inhibition at that concentration range was reported in previous works (Adrian et 365 al., 1997). The main difference is that here it was applied as pure crystals, whereas in 366 that work it was dissolved in an ethanolic solution. However, the growth was reduced 367 between 50 and 70% by all the particles of resveratrol produced in this work. This means 368 that the encapsulation of resveratrol enhanced its antifungal activity. Small differences 369 were noticed between both drying processes for every encapsulating material, although 370 particles of lecithin were more effective by SD, while particles of β-glucans resulted 371 slightly better by PGSS-drying. 372 Figure 8 contains images of the growth in Petri dishes of some samples. Similar growth 373 is observed between the control sample (b), the one with pure resveratrol powder (c) 374 and the one with pure β-glucan (d). However, the growth area is smaller for the particles 375 of resveratrol encapsulated on β-glucans by SD (e), and even smaller for the ones by 376 PGSS-drying (f). 377 Pure β-glucans did not affect the growth of the fungus, but lecithin reduced it by 40% 378 (figure 8 (a)). Still, the mixture of both compounds had also no effect on growth 379 inhibition. Taking this into account, the effectiveness of the particles encapsulated on 380 lecithin relays greatly on lecithin. Nevertheless, the action of the particles encapsulated 381 on β-glucans and a mixture of lecithin and β-glucans is related to the synergistic effect 382 of these substances and resveratrol, because neither of them showed effect when 383 applied alone, as well as to the change of resveratrol from crystals to amorphous state 384 after processing, determined by XRD. Therefore, the shell material improved the 385 absorption of resveratrol, which in this way was active against the Botrytis cinerea. 386 387 4. CONCLUSIONS 388 Resveratrol and tebuconazole were formulated as emulsions with lecithin, β-glucans 389 and mixtures thereof. Formulations were successfully dried by SD and PGSS-drying. 390 However, SAS was not a suitable process to obtain β-glucan particles in the conditions 391 tested, because the product had quantities of remaining organic solvent up to 30% 392 (w/w). Final particle size by SD was in the range of 10 µm and in PGSS-drying in the range 393 of 100 µm, showing that the drying processes and not the carrier material determined 394 the final particle size. As first there was an emulsification step, the loading of active 395 compound in the particles was very similar for both drying processes. Furthermore, the 396 interaction of the active compounds with the carriers produced their precipitation in the 397 encapsulated particles in amorphous state instead of their usual crystalline form. This 398 change in crystallinity improved the antifungal activity of resveratrol, which showed no 399 effect against Botrytis cinerea as pure crystals, but reduced the fungal growth between 400 50 and 70% with all resveratrol particles. 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Droplet size distribution of the emulsions (a) and suspensions (b) of resveratrol with different encapsulating material. Figure 4. SEM images of β-glucan particles by SD (a), PGSS-drying (b) and by SAS (exp. F: 5 g/L in DMSO, precipitation at 10 MPa, 35º C, 2 mL/min; c and d). Figure 5. Droplet size distribution of the particles produced by SD and PGSS-drying with different encapsulating material. Figure 6. Physical mixture and SD particles of resveratrol and lecithin. Figure 7. Growth area of Botrytis cinerea for the products of tebuconazole (a) and images of the growth area in the Petri dishes: control (b), commercial tebuconazole (c), tebuconazole and βglucan particles by SD (d) and by PGSS-drying (e). Figure 8. Growth area of Botrytis cinerea for the products of resveratrol (a) and images of the growth area in the Petri dishes: control (b), pure resveratrol (c), β-glucans (d), resveratrol and βglucan particles by SD (e) and by PGSS-drying (f). FIGURES Figure 1. Formation of particles by SD and PGSS-drying. Figure 8. Growth area of Botrytis cinerea for the products of resveratrol (a) and images of the growth area in the Petri dishes: control (b), pure resveratrol (c), β-glucans (d), resveratrol and β-glucan particles by SD (e) and by PGSS-drying (f). b) c) d) e) f) a) TABLE CAPTIONS Table 1. SAS experiments. Table 2. Fungicide loading for the different materials and drying processes. TABLES Table 1. SAS experiments. Exp β-glucan concentration (g/LDMSO) Dissolution flow (mL/min) CO2 flow (kg/h) T (ºC) A 2 2 2.5 34-35 B 5 2 2.5 37 C 5 2 2.5 38 D 5 2 2.5 50 E 5 2 2.5 34-35 F 5 2 5 34-35 G 5 4 2.5 34-35 H 10 2 2.5 34-35 Table 2. Fungicide loading for the different materials and drying processes. % loading Tebuconazole Resveratrol SD PGSS-drying SD PGSS-drying Lecithin 86.4 ± 1.1 79.1 ± 1.9 71 ± 4 75.3 ± 0.2 Lecithin and β-glucans 82.6 ± 1.6 72 ± 9 65 ± 7 67 ± 2 β-glucans 57 ± 2 49 ± 14 61.3 ± 1.2 94 ± 2