Microencapsulation of lipophilic bioactive compounds using prebiotic carbohydrates: Effect of the degree of inulin polymerization

This paper presents novel outcomes about the effect of degree of inulin polymerization (DP) on the technological properties of annatto seed oil powder obtained by freeze-drying. Inulins with two DP’s were evaluated: GR-inulin (DP ≥ 10) and HP-inulin (DP ≥ 23).

Carbohydrate Polymers 152 (2016) 775–783 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Microencapsulation of lipophilic bioactive compounds using prebiotic carbohydrates: Effect of the degree of inulin polymerization Eric Keven Silva a,∗ , Giovani L Zabot b , Matheus A Bargas a , M Angela A Meireles a a b LASEFI/DEA/FEA (School of Food Engineering)/UNICAMP (University of Campinas), Rua Monteiro Lobato, 80, Campinas, SP, CEP: 13083-862, Brazil Federal University of Santa Maria, UFSM, Rua Ernesto Barros, 1345, Cachoeira Sul, RS, 96506-322, Brazil a r t i c l e i n f o Article history: Received 18 May 2016 Received in revised form July 2016 Accepted 17 July 2016 Available online 18 July 2016 Keywords: Fructooligosaccharide Functional carbohydrate Glass transition temperature Wall material Water adsorption isotherms a b s t r a c t This paper presents novel outcomes about the effect of degree of inulin polymerization (DP) on the technological properties of annatto seed oil powder obtained by freeze-drying Inulins with two DP’s were evaluated: GR-inulin (DP ≥ 10) and HP-inulin (DP ≥ 23) Micrographs obtained by confocal microscopy were analyzed to confirm the encapsulation of bioactive compounds using both inulins, especially the encapsulation of the natural fluorescent substance ␦-tocotrienol Microparticles formed with both inulins presented the same capacity for geranylgeraniol retention (77%) Glass transitions of microparticles formed with GR-inulin and HP-inulin succeeded at 144 ◦ C and 169 ◦ C, respectively Regarding water adsorption isotherms, microparticles formed with HP-inulin and GR-inulin presented behaviors of Types II (sigmoidal) and III (non-sigmoidal), respectively Reduction of water adsorption capacity in the matrix at high relative moistures ( > 70%) was presented when HP-inulin was used At low relative moistures ( < 30%), the opposite behavior was observed © 2016 Elsevier Ltd All rights reserved Introduction The crescent interest of developing prebiotic substances is aimed at non-digestible oligosaccharides One of the prebiotics is inulin, a fructooligosaccharide generally extracted from chicory (Pandey et al., 1999), but also from other sources as artichoke (Ruiz-Aceituno, García-Sarrió, Alonso-Rodriguez, Ramos, & Sanz, 2016), and used for stabilizing proteins (Mensink, Frijlink, van der Voort Maarschalk, & Hinrichs, 2015) or converted into other functional ingredients by inulinases (Mazutti et al., 2010) Longer chain lengths make inulin as dietary fiber useful for food and pharmaceutical applications Examples of applications include as low caloric sweetener, as substance that provides solid dispersion for increasing dissolution rate, as an agent to form gels and to increase the viscosity of solutions, and as a non-digestible fiber (Mensink et al., 2015) Inulin also presents the ability to change the gut flora composition after a short feeding period based on results from in vitro studies and human subjects (Kolida & Gibson, 2007) The use of inulin depends on its degree of polymerization (DP) The molecular chain length can range some extent, commonly between and 80 (Mensink et al., 2015) The degree of inulin poly- ∗ Corresponding author E-mail address: engerickeven@gmail.com (E.K Silva) http://dx.doi.org/10.1016/j.carbpol.2016.07.066 0144-8617/© 2016 Elsevier Ltd All rights reserved merization depends on the harvest time, storage time/temperature and growing conditions (Saengthongpinit & Sajjaanantakul, 2005) Consequently, the degree of inulin polymerization determines the physicochemical characteristics to a substantial extent, as morphology (i e., crystal morphology, crystal structure and structure in solution), solubility, rheology (i e., viscosity, hydrodynamic shape and gelling), thermal characteristics and physical stability (i e., glass transition temperature, vapor sorption and melting temperature) and chemical stability (Mensink et al., 2015) Following the scenario of using inulin as prebiotic substance (Zabot, Silva, Azevedo, & Meireles, 2016), bioactive compounds extracted from vegetal sources are also of recent interest because of their functional properties Annatto seed oil is one of the sources of value-added bioactive compounds, as ␦-tocotrienol (Albuquerque & Meireles, 2012; Moraes, Zabot, & Meireles, 2015) and geranylgeraniol (Silva, Zabot, & Meireles, 2015) Tocotrienol-rich fractions obtained from annatto seeds act as natural antioxidants by inhibiting lipid oxidation of fish oil and lipid-based formula emulsions (Zou & Akoh, 2015) In addition, geranylgeraniol modulates the apoptosis of carcinogen cells (Marcuzzi et al., 2012) In this context, the objective of this study was to evaluate the influence of the degree of inulin polymerization (DP ≥ 10 and DP ≥ 23) on the physical properties of annatto seed oil microparticles obtained by freeze-drying Inulin was used as encapsulating matrix and the effect of its degree of polymerization on the recon- 776 E.K Silva et al / Carbohydrate Polymers 152 (2016) 775–783 stitution properties of the emulsions was assessed Furthermore, the morphology and ␦-tocotrienol distribution in the particles microstructures, geranylgeraniol retention by the encapsulating matrices, glass transition of microparticles and physical stability through water adsorption isotherms were also the responses taken into account when evaluating the effect of the degree of inulin polymerization were performed at 25 ◦ C The mean diameter was calculated based on the mean diameter of a sphere of similar area, superficial mean diameter (D32 ), as Eq (1) Polydispersity index (PDI) was calculated as Eq (2) All samples were analyzed through the wet method, with dispersion in water and refractive index of 1.52 D32 = Material and methods PDI = 2.1 Annatto seed oil extraction Annatto seed oil used as active material was extracted with supercritical CO2 using a pilot scale equipment (Thar Technologies, Pittsburgh, USA) containing an extraction vessel of L with internal diameter of 10.2 cm Solvent flow rate was equal to 200 g CO2 /min and the bed was maintained at 40 ◦ C and 20 MPa, as described in detail by a previous study (Silva, Gomes, Hubinger, Cunha, & Meireles, 2015) 2.2 Inulins and materials for chromatographic analyses Inulins used as active materials for encapsulating annatto seed oil were both from chicory: Orafti® GR (GR-inulin, DP ≥ 10) and Orafti® HP (HP-inulin, DP ≥ 23) (BENEO-Orafti, São Paulo, Brazil) Materials for chromatographic analyses were: methanol, ethanol and hexane (Chemco, Hortolândia, Brazil); geranylgeraniol (purity > 85%, Sigma–Aldrich, Steinhein, Germany) and ammonium acetate (P.A., Dinâmica, Campinas, Brazil) 2.3 Microencapsulation of annatto seed oil Annatto seed oil was encapsulated with GR- and HP-inulins through emulsification assisted by ultrasound with nominal power of 160 W during using a 13 mm diameter, 19 kHz ultrasonic probe (Unique, Disruptor, 800 W, Indaiatuba, Brazil) for each 30 mL of emulsion Total concentration of solids in the emulsion (emulsifying + oil) was equal to 20 g/100 g of emulsion Annatto seed oil was added to the suspensions containing GR- and HP-inulins and the concentration of oil was maintained at 20% relative to the amount of solids, that is, g of oil per 100 g of emulsion Immediately after homogenization, the emulsions were frozen in aluminum plates at −40 ◦ C for h and then subjected to freeze-drying (FD) process Drying was performed in a freeze-dryer system (Liobras, L 101, Sao Carlos, Brazil) The dried emulsions were converted into fine powders through maceration Detailed description of obtaining annatto seed oil microparticles is reported by a previous study (Silva & Meireles, 2015) 2.4 Reconstitution properties of emulsions The wettability of the powders was determined as the method reported by Fuchs et al (2006), with a few modifications The samples of powders (0.1 g) were dispersed over water surface of a Becker containing 100 mL of ultrapure water under stirring at 25 ◦ C The time spent for immersing or wetting the last particle of powder was used as wettability response The annatto seed oil microparticles obtained by freeze-drying were reconstituted in ultrapure water An amount of g of powder was mixed with g of ultrapure water and the solution was stirred during 30 s in a vortex type homogenizer (PHOENIX, AP-56 model, Araraquara, São Paulo, Brazil) at 25 ◦ C Droplet size distribution and mean diameter of the emulsion droplets, after reconstitution and before drying, were determined by light scattering technique using laser diffraction (Mastersizer 2000 Malvern Instruments Ltd, Malvern, UK) The measurements ni di ni di (d90 − d10 ) d50 (1) (2) Where: di is the mean diameter of the droplets; ni is the number of droplets; and d10 , d50 and d90 are the diameters at 10%, 50% and 90% of cumulative volume, respectively 2.5 Scanning electron microscopy (SEM) Micrographs were taken in a scanning electron microscope with Energy Dispersive X-ray Detector (SEM) (Leo 440i, EDS 6070, SEM/EDS: LEO Electron Microscopy/Oxford, Cambridge, England) Analyses were performed with kV accelerating voltage and 50 pA beam current for obtaining the micrographs 2.6 Confocal scanning laser microscopy (CSLM) CSLM analysis was performed using a Zeiss LSM 780-NLO confocal on an Axio Observer Z.1 microscope (Carl Zeiss AG, Germany) with a 40 × objective Images were taken by exciting ␦-tocotrienol molecules with lasers at 488 nm wavelength, without any previous preparation of the samples as a consequence of the fluorescent properties of ␦-tocotrienol, similarly to the procedure described by a previous study (Silva, Zabot, Cazarin, Maróstica Jr., & Meireles, 2016) 2.7 Geranylgeraniol retention Geranylgeraniol content in annatto seed oil (before and after encapsulation) was determined by high-performance liquid chromatography (HPLC) Chromatographic analyses were accomplished using an HPLC-PDA (Waters, Alliance E2695, Milford, USA) system, consisting of a separation module with an integrated column heater, an autosampler and a photodiode array (PDA) detector Separation of geranylgeraniol was fulfilled using a fused-core type column (Kinetex, C18 , 100 mm × 4.6 mm × 2.6 ␮m; Phenomenex, Torrance, USA) An aliquot of 10 ␮L of each sample diluted to 500 ppm (w/w) in hexane (Chemco, Hortolandia, Brazil) and filtered using nylon membrane (0.45 ␮m) was injected A solution of methanol:ammonium acetate 50 mM (90:10, v/v) was the mobile phase The column was maintained at 40 ◦ C Mobile phase flow rate was mL/min and the analytical run time was Detector wavelength range was 200–400 nm Geranylgeraniol was detected at 210 nm and at 2.4 min, and its quantification was performed using external standard calibration curve Intending to quantify geranylgeraniol entrapped in the microparticles after using inulins with two degrees of polymerization, four procedures were carried out to break the structure for releasing such compound: I Centrifugation: Approximately 0.1 g of particles from each treatment was mixed with mL of ultra-pure water The samples were maintained static during 24 h; thereafter, they were manually agitated for reconstituting the emulsions Aliquots of 0.5 mL were transferred to an Eppendorf tube of mL containing 1.3 mL of hexane The mixtures were then centrifuged at 5000 rpm for 20 and at 10,000 rpm for This procedure was performed to break the emulsion and to capture the E.K Silva et al / Carbohydrate Polymers 152 (2016) 775–783 777 bioactive material The superior phase was collected and the geranylgeraniol content was analyzed as following the analytical procedures used for pure annatto seed oil (oil before encapsulation) II Diffusion in hexane and extraction assisted by ultrasound: Approximately 0.1 g of particles from each treatment was mixed with 20 mL of hexane The samples were maintained static during 24 h for attaining the diffusion of geranylgeraniol In the sequence, the samples were sonicated at 600 W during using a 13 mm diameter, 19 kHz ultrasonic probe (Unique, Disruptor, 800 W, Indaiatuba, Brazil) Afterwards, an aliquot was taken and subjected to chromatographic analyses following the same procedures developed with pure oil III Diffusion in ethanol and extraction assisted by ultrasound: This procedure was accomplished similarly to procedure II The only one difference was the substitution of hexane by ethanol IV Diffusion in ethanol:water solution and extraction assisted by ultrasound: This procedure was accomplished similarly to procedures II and III The only one difference was the substitution of hexane or ethanol by ethanol:water (1:1, w/w) solution Table Reconstitution properties of annatto seed oil powders 2.8 Differential scanning calorimetry (DSC) Minitab 16® software was used to perform the analysis of variance for verifying the effects of the degrees of inulin polymerization on the microparticles characteristics Differences between average values were compared using Tukey’s test with 5% of significance (p-value < 0.05) DSC results were analyzed by descriptive approach DSC analysis was performed using calorimeter equipment (TA 60, Shimadzu Corporation, Kyoto, Japan) for determining the glass transition temperature (Tg) of microparticles and inulins used as wall material Approximately mg of sample, under an atmosphere with 10 mL/min of nitrogen, was submitted to two heating gradients for constructing DSC curves: 25 ◦ C to 110 ◦ C with scanning rate of 10 ◦ C/min and isothermal period of (first run); 25 ◦ C to 250 ◦ C (second run) Tg was observed in the second run at the midpoint of the glass transition range (Botrel, de Barros Fernandes, Borges, & Yoshida, 2014) 2.9 Water adsorption isotherms Water adsorption isotherms of microparticles containing annatto seed oil, formed with both degrees of polymerization, were determined by the gravimetric static method (Silva, Borges, da Costa, & Queiroz, 2015; Silva, Fernandes, Borges, Botrel, & Queiroz, 2014) The samples were conditioned in phosphorous pentoxide previously of performing the assays, until constant weight Such procedure was fulfilled to assure that only the adsorption phenomenon could be observed for all relative moistures evaluated in the experiment Afterwards, the microparticles were stored at 30 ◦ C under some relative moisture conditions (11.28% to 90.20%) provided by seven saturated saline solutions (lithium chloride, magnesium chloride, potassium carbonate, sodium bromide, sodium chloride, potassium chloride and barium chloride) (Greenspan, 1977) The parameters of the Guggenheim–Anderson–de Boer (GAB) model (Eq (3)) (Van den Berg, 1984) were fitted using the experimental data of equilibrium moisture The Quasi-Newton nonlinear regression was used with a convergence criterion of 10−4 on the Statistica software (Statsoft, 8.0, 2007) Xeq = Xm CKaw (1 − Kaw ) (1 − Kaw + CKaw ) (3) Where: Xeq is the equilibrium moisture (g water/100 g dry solids); Xm is the monolayer moisture content (g water/100 g dry solids); aw is the water activity (dimensionless); C is a constant representing the adsorption on the first monolayer; K is a constant representing the adsorption of molecules of water on multilayers Microparticles Wettability (s) 16 ± 57 ± GR-FD HP-FD D32 (␮m) PDI FE RE FE RE 2.4 ± 0.5 3.7 ± 0.1 5.9 ± 0.2 4.5 ± 0.2 2.0 ± 0.1 5±2 7±2 5±1 FE: fresh emulsion; RE: reconstituted emulsion The fit of the model parameters was assessed based on the coefficient of determination (R2 ) and the mean relative percentage deviation modulus (E) (Eq (4)): E= 100 n n i=1 |Y − Yˆ | Y (4) Where: Y is the observed value; Yˆ is the model estimated value; n is the number of observed data 2.10 Statistical analysis Results and discussion 3.1 Reconstitution properties of annatto seed oil emulsions The evaluation of reconstitution properties of particulate systems allows predicting the best conditions for applying and distributing encapsulated active compounds In this sense, the wettability is one of the more important physical properties taken into account when the characteristics of reconstituted powder products are evaluated, because the wettability expresses the capacity of the product to adsorb water Then, the responses of wettability of annatto seed oil microparticles formed with inulins of two degrees of polymerization (GR-inulin, DP ≥ 10; HP-inulin, DP ≥ 23) as encapsulating matrices are presented (Table 1) The use of GR-inulin (lower DP) enabled the formation of microparticles with higher instantisation velocity This result could be associated with the differences on the molecular chain, because larger molecular chains could act as physical barrier against water penetration, thus reducing the velocity of water absorption Fernandes, Borges, and Botrel (2014) evaluated the wettability of rosemary essential oil microparticles obtained by spray-drying The authors used blends of inulin/Arabic gum and inulin/modified starch, both with the same mass proportion of wall materials (1:1) The findings indicated the inulin/Arabic gum blend presented instantisation at 93 ± s, while inulin/modified starch blend presented instantisation at 131 ± s Droplet size distributions of annatto seed oil emulsions stabilized by GR- and HP-inulins in fresh state (before freeze-drying) and reconstituted state are presented (Fig 1) For both treatments, the patterns were different when the fresh and reconstituted emulsions are compared In addition, bimodal distributions are seen The responses of D32 and PDI (Table 1) corroborate the performance of GR-FD and HP-FD microparticles, which the responses were not satisfactory, mainly with respect to the reconstitution properties Such responses are associated with the lack of superficial activity of inulin molecule, regardless of its degree of polymerization Inulin alone as wall material cannot form interfacial films over the annatto seed oil droplets The main mechanism for emulsions stabilization 778 E.K Silva et al / Carbohydrate Polymers 152 (2016) 775–783 Fig Droplet size distributions of fresh and reconstituted emulsions and visual aspect of emulsions stored during 24 h after reconstitution is linked to the inulin action as thickener of continuous phase, creating a physical barrier against the coalescence of oil droplets (Silva, Gomes, et al., 2015) The visual aspect of emulsions stored during 24 h after reconstitution is also presented (Fig 1) The emulsion stabilized with HP-inulin showed stability against phase separation after the reconstitution However, the emulsion stabilized with GRinulin was unstable during the storage period The findings could be associated with the degree of inulin polymerization, because the highest degree led to the formation of a thickener continuous phase, more efficient for reducing the coalescence of oil droplets (Silva & Meireles, 2015) 3.2 Microstructural analysis The morphology of annatto seed oil microparticles formed with two degrees of polymerization (GR: DP ≥ 10; HP: DP ≥ 23) is presented after performing SEM and CSLM analyses (Fig 2) SEM is an important analysis used for characterizing the microstructure of particulate products With the micrographs, the effects of the process on microparticles formation and the effects of composition of wall materials on the morphological characteristics of the powder products can be assessed Likewise, CSLM analysis is an important tool for characterizing microstructures based on the natural fluorescent properties of compounds present in the structure or based on the addition of substances with such properties The main advantage of the technique relies on the possibility of observing the internal structure of microparticles without fragmenting the material The results obtained after performing both analyses (SEM and CSLM) are complementary, whereas they characterize the morphology and distribution of the bioactive compounds on the microstructure of the particles Some scientific studies report these analyses as a combined form of characterization of powder products containing encapsulated bioactive compounds (Carvalho, da Costa Machado, da Silva, Sartoratto, Rodrigues, & Hubinger, 2016; Silva, Azevedo, Cunha, Hubinger, & Meireles, 2016) The regions in fluorescent green (Fig 2) indicate the distribution of ␦-tocotrienol in the microparticles structure, which means annatto seed oil was distributed through the encapsulating matrix Albuquerque and Meireles (2012) reported annatto seed oil is the richest source of ␦-tocotrienol, containing approximately 15 g ␦tocotrienol/100 g oil Therefore, we infer both inulins were able to entrap ␦-tocotrienol inside the matrix (qualitative analysis) Regardless the degree of inulin polymerization, all microparticles presented irregular morphology, typical of freeze-dried materials (Chranioti, Chanioti, & Tzia, 2016) The morphology is a result of breaking the spongy structure formed after removing ice crystals in the encapsulating system through sublimation Regarding the microstructure, no differences were observed after using inulins with different molecular chain lengths E.K Silva et al / Carbohydrate Polymers 152 (2016) 775–783 779 Fig SEM and CSLM micrographs of annatto seed oil microparticles 3.3 Geranylgeraniol retention One of the main responses when evaluating the efficiency of an encapsulating matrix is the ability of retaining the target bioactive compound into the encapsulating system This ability assures the efficiency of an expected action after applying the microparticles in a determined product In this sense, the influence of the degree of inulin polymerization on geranylgeraniol retention was evaluated, because geranylgeraniol presents therapeutic properties associated with its biological activities as anti-inflammatory and anticancer agent Annatto seed oil, before encapsulation, presented 25.0 ± 0.6 g geranilgeraniol/100 g oil Recovering geranilgeraniol from annatto seed oil microparticles aiming to perform its quantification was fulfilled through two dis- 780 E.K Silva et al / Carbohydrate Polymers 152 (2016) 775–783 Fig Effect of the degree of inulin polymerization on geranylgeraniol retention US: ultrasonication tinct mechanisms: i) emulsions were reconstituted by suspending the microparticles in water followed by centrifugation to break the colloidal system and to capture the compound; ii) suspension of microparticles was done in different organic solvents for diffusing the compound followed by breaking the particles microstructures using ultrasonication The influence of the degree of inulin polymerization on geranylgeraniol retention could be seen (Fig 3), where it ranged from 40% to 74% for GR-FD and from 2% to 77% for HP-FD Breaking the reconstituted emulsions by centrifugation for extracting annatto seed oil was not an efficient method to quantify geranylgeraniol, because high viscosities of emulsions acted as an effective physical barrier to entrap the oil The lowest extraction found for HP-FD corroborates this observation, because the viscosity of the emulsion increases with increasing the degree of inulin polymerization (Silva & Meireles, 2015; Silva, Gomes et al., 2015) Diffusing the oil in organic solvent followed by sonication of the matrix for releasing the oil was the most efficient method for quantifying geranylgeraniol retained in the microparticles Diffusion in ethanol or ethanol:water solution did not influence the geranylgeraniol retention (p-value = 0.725) The lowest retention found after using hexane as diffusion medium can be associated with the lowest polarity of this solvent when comparing it with the other diffusion media Furthermore, the interaction of hexane with the encapsulating matrix is low and not enough to break the structure during ultrasonication Microparticles presented the same capacity of geranylgeraniol retention (p-value = 0.616) Our results corroborate the entrapment efficiencies reported by Silva and Meireles (2015) The authors reported that the degree of inulin polymerization did not influence the oil retention in the microparticles, since GR-inulin and HP-inulin resulted in the same entrapment efficiency of annatto seed oil 3.4 Glass transition temperature (Tg) Determining Tg of dehydrated products is an important step for characterizing particulate systems (Gomes da Costa, Silva, Toledo Hijo, Azevedo, & Borges, 2015) Conditions of temperature above Tg provide more molecular mobility, accelerating the reaction rates Conditions of temperature below Tg provide higher stability of the products against deterioration during storage (Oikonomopoulou, Fig DSC curves of pure inulins and annatto seed oil microparticles Krokida, & Karathanos, 2011) Silva and Meireles (2015) evaluated X-ray diffractograms of GR- and HP-inulins, as well as GR-FD and HP-FD microparticles GR- and HP-inulins were characterized as amorphous materials and both microparticles were characterized as amorphous materials with crystalline regions Semicrystalline biopolymers can present three thermal transition characteristics: a) glass transition to amorphous fraction; b) fusion of the crystalline fraction; c) transition as a consequence of crystallization (Teac˘a, Bodỵrl˘au, & Spiridon, 2013) When evaluating encapsulating systems, the more relevant phase transition is the glass transition, because Tg of amorphous materials is defined as the temperature which the material changes from amorphous state to gummy (elastic) state Therefore, after the transition, the microparticles coating structure that entrap (protect) the annatto seed oil loose its efficiency as encapsulating matrix, releasing the active material DSC curves of pure inulins and microparticles containing annatto seed oil (Fig 4) were obtained after the cooling period at second scanning stage, aiming to erase the thermal history over Tg (Haque, Kawai, & Suzuki, 2006) Overall, glass transition is evaluated in terms of the moisture of the material, because water acts as plasticizing, thus reducing Tg as a consequence of reducing inter and intramacromolecular forces (Chuang, Panyoyai, Shanks, & Kasapis, 2015) The samples were analyzed at the conditions of moisture and aw from FD process, as described by Silva and Meireles (2015), searching for the characterization of products obtained on their natural conditions According to DSC curves (Fig 4), the phenomena of glass transition of GR, HP, GR-FD and HP-FD samples succeeded at 132 ◦ C, 157 ◦ C, 144 ◦ C and 169 ◦ C, respectively After analyzing the pure biopolymers, increasing the degree of polymerization increased Tg of inulin The molecular chain length of inulin did not influence Tg of microparticles Although differences higher than 25 ◦ C in the Tg of pure inulins were observed, the presence of annatto seed oil approximated the physical properties of the systems Ronkart, Paquot, Fougnies, Deroanne, and Blecker (2009) evaluated the moisture content over Tg of inulin (mean DP = 23) The authors concluded the glass transition of inulin succeeded between 150 ◦ C and 160 ◦ C for moisture contents below wt.% (dry mass basis), similarly to our findings E.K Silva et al / Carbohydrate Polymers 152 (2016) 775–783 781 Fig Water adsorption isotherms of annatto seed oil microparticles 3.5 Water adsorption isotherms of annatto seed oil microparticles Studying the isothermal behavior of water adsorption of annatto seed oil microparticles allows predicting the best storage conditions for maintaining long shelf life of products Water adsorption of dehydrated products is the main factor of degradation of such products Increasing moisture content, after water adsorption, can cause several changes on the encapsulating matrix For instance, microparticles can agglomerate or Tg can reduce, compromising the efficiency of the polymeric matrix Water adsorption isotherms of annatto seed oil at 30 ◦ C for each degree of inulin polymerization are presented (Fig 5) HPFD samples presented behavior of Type II: sigmoidal According to classification of Brunauer, Deming, and Teller (1940), this behavior is typical of porous or macro porous materials which present high adsorption energy Otherwise, GR-FD samples presented behavior of Type III: non-sigmoidal This behavior is typical of porous or macro porous materials which present low adsorption energy The behaviors corroborate the differences on the equilibrium moistures observed for microparticles with relative moisture higher Fig Images of annatto seed oil microparticles after water adsorption at seven equilibrium relative moistures than 30% In the same conditions of pressure and temperature, GRFD samples presented larger capacity of water adsorption than that observed for HP-FD samples Then, water adsorption in the inulin with smaller molecular chain expends lower energy After comparing the behavior of the curves, reduction of water adsorption capacity in the matrix at high relative moistures ( > 70%) was verified when the degree of inulin polymerization is increased (higher molecular length) However, the opposite behavior was observed at low relative moistures ( < 30%) Similar findings were reported by Schaller-Povolny, Smith, and Labuza (2000) when evaluating four degrees of inulin polymerization (mean DP equal to 5, 9, 11 and 23) over water adsorption isotherms at 23 ◦ C This effect is coupled with the molecular length and arrangement of inulin, 782 E.K Silva et al / Carbohydrate Polymers 152 (2016) 775–783 Table Parameters fitted for GAB model Acknowledgements Microparticles Xm (g water/100 g dry solids) C K R2 E (%) GR-FD HP-FD 5.47 5.38 5.33 30.94 0.844 0.737 0.995 0.997 4.41 0.22 which the shortest chain contains more hydroxyl groups available to bond with water GAB model was used to fit the parameters that represent the experimental data because such model can successfully describe water isothermal adsorption of powder products (Rao & Labuza, 2012; Zhou, Liu, Chen, Chen, & Labuza, 2014) Thus, the parameters of GAB model were fitted and the fit was evaluated statistically (Table 2) According to high values of coefficient of determination (R2 ≥ 0.995) and low values of mean relative percentage deviation modulus (E ≤ 4.41), the curves fitted to the adsorption isotherms were suitable to describe the behavior of the experimental data After fitting the parameters of GAB model, Xm was obtained This parameter comprises the content of water that is strongly adsorbed to the bonding sites on the microparticles surface Xm is important for the physical stability of particulate systems against lipid oxidation, enzymatic activity, non-enzymatic browning and changes in the structural characteristics Therefore, Xm is considered to be the suitable moisture content for extending the shelf life of products (Rao & Labuza, 2012) C and K constants of GAB model are associated with the monolayer and monolayer properties, respectively Ranges of 5.67 ≤ C < ∞ and 0.24 < K ≤ indicate that GAB model properly describes water adsorption isotherms (Lewicki, 1997) Increasing K by decreasing the degree of inulin polymerization corroborates the change of the shape of isothermal curve from Type II to Type III After the equilibrium of microparticles with water vapor in seven relative moistures, the images of the samples were taken (Fig 6) No visible phase transition (i e., liquefaction) was observed in each matrix, even though at high relative moistures However, the GR-FD microparticles displayed caking after submitted to relative moisture higher than 32%, whilst HP-FD microparticles were stable at all conditions of relative moisture Conclusion The microparticles formed and presented in this paper are highlighted as a consequence of their singular properties, which have been not enough studied on scientific community Such microparticles are totally functional, because the bioactive compounds encapsulated and the encapsulating matrix are therapeutic substances to be applied in food-related and pharmaceutical-related products Considering the technical approach, after comparing inulins with two degrees of polymerization (GR: DP ≥ 10; HP: DP ≥ 23) for encapsulating annatto seed oil, the conclusion was that the polymeric matrix influenced the microparticles properties When the emulsions were reconstituted, the emulsion stabilized with HP-inulin showed stability against phase separation 24 h after the reconstitution Both inulins could entrap annatto seed oil; therefore, the microparticles presented the same capacity of geranylgeraniol retention (77%) Furthermore, for water activities higher than 0.3, water isothermal adsorption was lower when using HP-inulin Images of HP-FD microparticles indicated they were more stable at all conditions of relative moisture (11% to 90%) Therefore, HP-inulin is inferred to be a potential encapsulating agent of bioactive compounds extracted from vegetal sources, as annatto seeds Authors are grateful to CNPq (470916/2012-5) and FAPESP (2012/10685-8; 2015/13299-0) for the financial support Eric Keven Silva thanks CNPq (140275/2014-2) for the Ph.D scholarship and FAPESP (2015/22226-6) for the postdoctoral scholarship Giovani L Zabot thanks FAPESP (2014/15685-1) for the postdoctoral scholarship M Angela A Meireles thanks CNPq (302423/2015-0) for the productivity grant In addition, the authors would thank the access to equipment and assistance provided by the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the University of Campinas; INFABIC is co-funded by FAPESP (08/57906-3) and CNPq (573913/2008-0) References Albuquerque, C L C., & Meireles, M A A (2012) Defatting of annatto seeds using supercritical carbon dioxide as a pretreatment for the production of bixin: Experimental, modeling and economic evaluation of the process Journal of Supercritical Fluids, 66, 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Zhou, P., Liu, D., Chen, X., Chen, Y., & Labuza, T P (2014) Stability of whey protein hydrolysate powders: Effects of relative humidity and temperature Food Chemistry, 150, 457–462 Zou, L., & Akoh, C C (2015) Antioxidant activities of annatto and palm tocotrienol-rich fractions in fish oil and structured lipid-based infant formula emulsion Food Chemistry, 168, 504–511  ... scanning calorimetry (DSC) Minitab 16® software was used to perform the analysis of variance for verifying the effects of the degrees of inulin polymerization on the microparticles characteristics... particulate products With the micrographs, the effects of the process on microparticles formation and the effects of composition of wall materials on the morphological characteristics of the powder products... product to adsorb water Then, the responses of wettability of annatto seed oil microparticles formed with inulins of two degrees of polymerization (GR -inulin, DP ≥ 10; HP -inulin, DP ≥ 23) as encapsulating