Replacing modified starch by inulin as prebiotic encapsulant matrix of lipophilic bioactive compounds

    Replacing modified starch by inulin as prebiotic encapsulant matrix of lipophilic bioactive compounds Giovani L. Zabot, Eric Keven Silva, Viviane M. Azevedo, M. Angela A. Meireles PII: DOI: Reference:

S0963-9969(16)30135-1 doi: 10.1016/j.foodres.2016.04.005 FRIN 6237

To appear in:

Food Research International

Received date: Revised date: Accepted date:

5 March 2016 6 April 2016 9 April 2016

Please cite this article as: Zabot, G.L., Silva, E.K., Azevedo, V.M. & Meireles, M.A.A., Replacing modified starch by inulin as prebiotic encapsulant matrix of lipophilic bioactive compounds, Food Research International (2016), doi: 10.1016/j.foodres.2016.04.005

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ACCEPTED MANUSCRIPT

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Replacing modified starch by inulin as prebiotic encapsulant matrix of

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lipophilic bioactive compounds

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LASEFI/DEA/FEA (School of Food Engineering)/UNICAMP (University of Campinas) Rua Monteiro Lobato, 80, Campinas, SP; CEP 13083-862, Brazil

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Giovani L. Zabot1*, Eric Keven Silva1, Viviane M. Azevedo2, M. Angela A. Meireles1

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Food Science Department, Federal University of Lavras (UFLA) Lavras, MG; CEP 37200-000, Brazil

*E-mail address: [email protected] (Giovani L. Zabot) Tel.: +55-19-3521-0100

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ACCEPTED MANUSCRIPT Abstract

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The purpose of this work was to replace modified starch (SF) by inulin (IN), a prebiotic carbohydrate, during emulsification assisted by ultrasound. Oregano extract was encapsulated

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using five proportions of IN and SF as wall materials. The effect of such substitution on the

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microparticles characteristics was evaluated. Attempting to contribute with the increasing

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demand for prebiotics consumption, mixing one part of SF with three parts of IN (1:3, mass basis) yielded encapsulation efficiency equal to 66 ± 1% and the largest thymol retention:

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84 ± 9%. Besides the entrapment of thymol, high amount of other compounds present in oregano extract could be entrapped in the polymeric matrix: 92 ± 1%. Reduction of the

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microparticles sizes when increasing the proportion of inulin was also observed. Comprising such results and those presented for powder morphology, surface extract, particle size

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distribution, X-ray diffraction and thermal stability, the proportion 1:3 (SF:3IN) is a favorable prebiotic encapsulant matrix for encapsulating oregano extract and retaining target bioactive compounds.

Keywords: prebiotic wall material; encapsulation efficiency; Origanum vulgare L.; X-ray diffraction; thermogravimetric analysis; thymol entrapment.

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INTRODUCTION

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Several vegetal sources have extractable substances that are industrially desirable in the food, chemical, cosmetic and pharmaceutical fields. One of these sources is oregano (Origanum

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vulgare L.), which contains thymol, carvacrol, gamma-terpinene, alpha-terpinene, alpha-

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terpineol, linalool, terpinen-4-ol, sabinene and beta-phellandrene, among others (Borgarello,

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Mezza, Pramparo, & Gayol, 2015; Ruben, Valeria, & Ruben, 2014). Antioxidant and antimicrobial activities of oregano extract are mainly associated with the presence of thymol and carvacrol

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(Asensio, Grosso, & Juliani, 2015; Falco, Roscigno, Landolfi, Scandolera, & Senatore, 2014), which are efficient against bacterial strains and lipid oxidation. Oregano extract has been used

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as flavoring agent in food and beverages, as well as in the manufacture of fungicides and insecticides (Kordali et al., 2008).

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Some sensitive compounds are unstable when exposed at light and oxygen ambient. Thus, the encapsulation of bioactive compounds is a technology used for protecting them into a physical entrapment formed by a homogeneous or a heterogeneous matrix (Costa et al., 2012). Oregano extract, for instance, was encapsulated in chitosan nanoparticles by a two-step process: oil-in-water (o/w) emulsification and ionic gelation (Hosseini, Zandi, Rezaei, & Farahmandghavi, 2013). In another study, gum Arabic and modified starch were used in the encapsulation of oregano essential oil (Botrel et al., 2012). It is seen diverse wall materials from a wide variety of natural and synthetic polymers used for encapsulation processes. However, the wall materials present different actions of protection and influence particles characteristics and retention/release of active substances. Regarding the human intake, some polymers present high energetic content, opposing the consumption of prebiotic substances. As concept 3

ACCEPTED MANUSCRIPT introduced by Gibson and Roberfroid (1995), prebiotics are nondigestible food ingredients that beneficially influence the host by stimulating the growth and/or activity of a limited number of

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bacteria species resident in the colon, and thus improve host health. This definition has been

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revised along the time, but the main features have mostly been retained.

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The great interest in the development of prebiotics is aimed at nondigestible oligosaccharides. Some of the prebiotics are the inulin-type fructans, because they provided

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evidence of their ability to change the gut flora composition after a short feeding period based

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on results from in vitro studies and human subjects (Kolida & Gibson, 2007). Inulin is a versatile fructooligosaccharide generally extracted from chicory (Pandey et al., 1999) that is applied in

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the stabilization of proteins and modified drug delivery (Mensink, Frijlink, Maarschalk, &

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Mazutti et al., 2010b).

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Hinrichs, 2015) or converted in other functional ingredients by inulinases (Mazutti et al., 2010a;

Although inulin has several applications in diverse areas, including in the food area for decades, the use of inulin as wall material in the food field is a few exploited. Wall materials commonly used in the encapsulation of bioactive compounds are gums, modified starches, whey proteins and dextrins (Chranioti, Nikoloudaki, & Tzia, 2015; Khazaei, Jafari, Ghorbani, & Kakhki, 2014; Silva, Gomes, Hubinger, Cunha, & Meireles, 2015a; Zandi, Mohebbi, Varidi, & Ramezanian, 2014). However, such substances do not present functional activities as inulin does. Looking for the substitution of gums, maltodextrin or starches by prebiotic materials, some researchers are studying the use of inulin. Recently, Fernandes, Borges, and Botrel (2014a) evaluated the effects of the partial or total replacement of gum Arabic by inulin on the characteristics of rosemary essential oil microencapsulated by spray-drying. Saénz, Tapia, 4

ACCEPTED MANUSCRIPT Chávez, and Robert (2009) also reported the ability of inulin for microencapsulation of bioactive compounds from cactus pear fruit. Thus, the use of inulin can favor the application of oregano

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extract in functional foods.

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Accompanying the global changes and demands, the market of ingredients and additives

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supplied to the food industry is recently overcoming the challenge of substituting synthetic substances by natural compounds. In the past few decades, considerable efforts have been

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devoted to developing functional products that promote healthiness and well-being. One of

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these recent efforts in the food field stands for using functional encapsulating matrices with the objective to protect bioactive compounds. Based on this trend of consuming compounds with

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functional activities, this scientific study proposes the substitution of a high caloric substance

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(modified starch) by a prebiotic carbohydrate with low content of calories (inulin) for the

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encapsulation of bioactive compounds. The objective was to evaluate the main characteristics of microparticles containing oregano extract formed with five proportions of modified starch and inulin as wall materials, aiming to use inulin to a feasible extent. The main characteristics assessed were: extract entrapment efficiency, encapsulation efficiency, surface extract, thymol retention, particle size distribution, micrographs of microparticles, X-ray diffraction and thermal stability of microparticles.

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MATERIAL AND METHODS Obtaining oregano extract by supercritical fluid extraction Supercritical fluid extraction has been increasingly applied to obtain several bioactive

compounds (Moraes, Zabot, & Meireles, 2015; Zabot, Moraes, Carvalho, & Meireles, 2015). 5

ACCEPTED MANUSCRIPT Then, oregano extract was obtained with supercritical CO2 at 50°C and 20 MPa (Diaz, 2010) using the SFE-2×1L equipment described in previously studies (Zabot, Moraes, & Meireles, 2014;

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Zabot, Moraes, Petenate, & Meireles, 2014). Firstly, dry oregano leaves (acquired from local

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market) were comminuted until mean diameter size of 0.9 ± 0.1 mm. After, 480 g of milled

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leaves was loaded into the extraction vessel of 1 L. The extraction bed was pressurized with CO2 and maintained at static extraction during 20 min. Dynamic extraction started with constant CO2

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flow rate of 28 g/min during 120 min, indicating a solvent mass to feed mass (S/F) ratio of 7. The

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extract was collected and immediately stored for further applications. Five extractions assays were performed aiming to reach sufficient extract for the emulsification step. Wall materials

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Wall materials used for encapsulating oregano extract were as follows: Snow-Flake (SF)

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E6131 chemically modified starch (Ingredion Brasil Ingredientes Industriais Ltda., Mogi Guaçu, SP, Brazil) (SF wall material) and chicory inulin (IN) Orafti®GR (with a degree of polymerization higher than 10) supplied by Beneo-Orafti (São Paulo, Brazil) (IN wall material). Silva et al. (2015a) reported the characterization of these wall materials used in this study. 2.3

Emulsions of oregano extract The emulsions characteristics were evaluated after partially replacing the modified

starch (SF) for inulin (IN). The procedure was accomplished as follows: SF was added to ultrapure water supplied by a Milli-Q Advantage water purifier system (Millipore, Bedford, USA) and the biopolymer suspension was prepared 24 h before the emulsification process. The suspension was maintained static at approximately 25°C during 24 h to ensure the complete

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ACCEPTED MANUSCRIPT saturation of the molecules of SF. After this period, IN was dissolved in ultra-pure water at 90°C and mixed with SF according to the defined concentration (Table 1) for each essay. After

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saturation, 20 wt.% of oregano extract (relative to the mass of total solids) was added to the

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suspension, that is, 5 g of extract/100 g of emulsion. The concentration of total solids in the

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emulsion (emulsifying + extract) was 25 g/100 g of emulsion.

Aliquots of 40 mL of the suspensions were sonicated using a 13 mm diameter and 19 kHz

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ultrasonic probe (Unique, Disruptor, 800 W, Indaiatuba, Brazil) for obtaining the emulsions. The

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probe height contacting the emulsions was standardized to 40 mm (Silva et al., 2015a). For ultrasonication, the same equivalent energy density reported by Silva, Zabot, and Meireles

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(2015c) was used, representing a nominal power of 760 W during 7 min. The experimental runs

Characterization of emulsions

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2.4

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were carried out in duplicate, accounting 10 runs.

2.4.1 Droplet size distribution Droplet size distribution and mean diameter of the emulsion droplets were determined by light scattering technique using laser diffraction (Mastersizer 2000 Malvern Instruments Ltd, Malvern, UK). The mean diameter was calculated based on the mean diameter of a sphere of similar area, superficial mean diameter (D[3,2]), as Equation (1). Polydispersity index (PDI) was calculated as Equation (2). All samples were analyzed in triplicate, using the wet method, with dispersion in water and refractive index of 1.52.

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ACCEPTED MANUSCRIPT k

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(1)

n D i 1

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(2)

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D90  D10 D50

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PDI 

i

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D[3,2] 

n D

Where: Di is the mean diameter of the droplets; ni is the number of droplets; and D10, D50 and

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D90 are the particle diameters at the 10th, 50th and 90th percentile of particles undersized,

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respectively. Powder formation by freeze-drying

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Immediately after formed, the emulsions were frozen in aluminum plates at -40°C for 3 h

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and then subjected to freeze-drying (FD) process. Drying was performed in a freeze-dryer

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system (Liobras, L 101, Sao Carlos, Brazil). The dried emulsions were converted into fine powders through maceration. The experimental runs were performed in duplicate. 2.6

Particles characterization 2.6.1 Moisture

Moisture content of microparticles obtained after partially replacing modified starch by inulin was determined gravimetrically in a forced circulation oven at 105°C until reaching constant weight (AOAC, 1997). 2.6.2 Extract entrapment efficiency Extract entrapment efficiency (ETE) means the total extract (TE) entrapped by the encapsulating matrix relative to the extract added into the emulsion. Oregano extract was 8

ACCEPTED MANUSCRIPT determined by distillation of microparticles in a Clevenger type system for 3 h (Jafari, He, & Bhandari, 2007). The extract from the aqueous phase was transferred to ethyl ether by

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partitioning. Afterwards, the solvent was evaporated at 25°C for 24 h. The amount of TE in the

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microparticles was obtained by a gravimetrical measurement, taking into account the mass of

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extract recorded in the burette of Clevenger system and the mass of extract obtained after

matrix (Lin, Lin, & Hwang, 1995). TE  100 Extract added into the emulsion

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ETE (%) 

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evaporating the ethyl ether. Equations (3) was used to calculate ETE into the encapsulating

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2.6.3 Surface extract

(3)

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Approximately 1.5 g of microparticles was added to 15 mL of hexane and the solution

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was manually stirred for obtaining the free extract and the surface extract (SE) content. The solvent plus bulk extract solution was filtered through Whatman filter (pore diameter: 11 μm). The collected particles were washed twice with hexane (20 mL). Hexane was evaporated at 25°C for 24 h. SE was calculated by mass difference between the initial mass of the clean flask and the final mass of the flask containing the extract (Carvalho, Silva, & Hubinger, 2014). The responses were expressed as percentage of extract adhered on the microparticles surface. 2.6.4 Extract encapsulation efficiency Encapsulation efficiency (EE) is defined as the ratio between the extract inside the encapsulating matrix and total extract present in the microparticles. Equation (4) was used to calculate EE (Tonon, Pedro, Grosso, & Hubinger, 2012).

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ACCEPTED MANUSCRIPT EE (%) 

TE  SE  100 TE

(4)

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2.6.5 Thymol retention

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Thymol contents in the pure oregano extract and in the microparticles were determined

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by gas chromatography (GC) with a flame ionization detector (FID). Chromatographic analyses were performed in the GC–FID system (Shimadzu, CG17A, Kyoto, Japan) equipped with a

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capillary column of fused silica DB-5 (J&W Scientific, 30 m × 0.25 mm × 0.25 μm, Folsom, USA).

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Oregano extracts were diluted to 5 mg/mL in ethyl acetate (Synth, Diadema, Brazil), filtered through nylon membrane filters (0.45 μm) and 1 μL of each sample was injected into the

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column. The sample split ratio was 1:20 and the carrier gas (Helium, 99.9% purity, White

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Martins, Campinas, Brazil) flowed at 1.1 mL/min. Injector and detector temperatures were

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220°C and 240°C, respectively. Column was heated from 60°C to 246°C at 3°C/min and maintained at 246°C for 3 min, following Kovats methodology (Adams, 2007). Approximately 0.1 g of each sample of microparticles was mixed with 4 mL of ultra-pure water for reconstituting the emulsion and determining thymol content in the microparticles. Each solution was maintained static for 24 h. Afterwards, an aliquot of 0.5 mL of each reconstituted emulsion in contact with 1.3 mL of ethyl acetate was centrifuged at 5,000 rpm for 20 min and afterwards at 10,000 rpm for 5 min. This procedure was performed to breakdown the emulsion droplets and to capture the bioactive material. We collected the supernatant and analyzed the thymol content following the analytical procedures used for pure oregano extract, as described before. Thymol retention (mass basis) was calculated according to Equation (5).

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Thymol entrapped in the microparticles  100 Thymol added into the emulsions

(5)

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2.6.6 Particle size distribution

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Size distribution and mean diameter of microparticles were determined at 25°C by light

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scattering technique using laser diffraction (Mastersizer 2000 Malvern Instruments Ltd, Malvern, UK). The mean diameter was determined based on the mean diameter of a sphere of

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the same volume, the Brouckere diameter (D[4,3]) (Equation (6)). The samples were analyzed by

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wet method, with dispersion in ethanol (purity > 99.5%). k

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n D i

(6)

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i 1

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D[ 4 ,3] 

n D

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Where: Di is the mean diameter of the particles; and ni is the number of particles. 2.6.7 Scanning electron microscopy (SEM) Micrographs were taken in a scanning electron microscope with Energy Dispersive X-ray Detector (Leo 440i, EDS 6070, SEM/EDS: LEO Electron Microscopy/Oxford, Cambridge, England) after coating with a thin gold film with the aid of a sputter coater (Emitech, K 450, Kent, UK). Analyses were performed with 5 kV accelerating voltage and 50 pA beam current for obtaining the micrographs. 2.6.8 X-ray diffraction X-ray diffraction (XRD) analysis of oregano microparticles and pure wall materials was performed on a Shimadzu XRD-6000 equipment (Shimadzu, Tokyo, Japan) using a graphite 11

ACCEPTED MANUSCRIPT crystal monochromator with filter radiation of Cu-Kα1 (λ = 1.5406 Å) at 30 kV and 30 mA. Samples were analyzed in angles from 5° to 40° angle in 2θ with a step of 0.02° (1.2°/min)

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(Fernandes, Borges, Botrel, & Oliveira, 2014).

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2.6.9 Thermogravimetric analysis

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Thermal stabilities of oregano microparticles and pure wall materials were evaluated

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through thermogravimetric analysis (TGA) in TG-DTA H Shimadzu 60 equipment (Shimadzu Corporation, Kyoto, Japan). Analyses were performed in nitrogen atmosphere flowing at

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10 mL/min and with heating from 25°C to 600°C at 10°C/min. The thermograms (TG) and their respective derivatives (DTG) were graphically presented between 80°C and 350°C aiming to

Statistical analysis

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offer the information for practical operations using oregano microparticles.

Minitab 16® software was used to perform the analysis of variance for verifying the effects of replacing modified starch by inulin on the particles characteristics. Differences between average values were compared using Tukey’s test with 5% of significance (p < 0.05). Microparticle morphology, TGA and XRD were analyzed by descriptive approach.

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RESULTS AND DISCUSSION Oregano extract After performing the supercritical fluid extraction from oregano leaves in five replicates,

65 g of bulk extract was obtained, yielding 2.7 ± 0.2 g extract/100 g of raw material.

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ACCEPTED MANUSCRIPT Chromatographic analysis of the bulk extract indicated thymol content of 69 ± 3 mg/g extract, accounting approximately 2 g of thymol per kg of raw material. Droplet size distribution of the emulsions

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Different values of D[3,2] and PDI were obtained for the assays using different proportions

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of modified starch and inulin (Table 1).The largest D[3,2] was observed when inulin alone was

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used as encapsulating matrix, because inulin does not have itself superficial activity to adsorbing on the water-oil interface, that is, inulin does not present properties of forming an interfacial

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film for protecting integrally the core material. Otherwise, when combining 3 parts of inulin and 1 part of modified starch (mass basis, SF:3IN assay, Table 1) the lowest D[3,2] was observed.

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Although inulin does not have the ability to adsorb on the water-oil interface, thereby reducing the interfacial tension (enthalpy) and the total free energy of the system, inulin acts as

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a thickening agent of the continuous phase (water), thus promoting stability to phase separation by a physical mechanism of entrapment of the oil droplets (Silva & Meireles, 2015). Then, the properties of the Snow-Flake modified starch (SF), like having superficial activity, associated with the properties of Orafti®GR inulin (IN) and also summed to the high intensity ultrasonic homogenization led to a system with D [3,2] equal to 2.3 ± 0.1 μm. Such assay (SF:3IN) presented the smallest PDI value: 2.4 ± 0.3. PDI values express the degree of size distribution and polydispersity. Studies dealing with drying fine emulsions (small droplets size of dispersed phase) point out a trend of increasing the extract encapsulation efficiency by reducing D[3,2] (Silva, Zabot, Cazarin, Maróstica, & Meireles, 2016; Soottitantawat, Yoshii, Furuta, Ohkawara, & Inko, 2003). This implies that a fine emulsion is likely more stable and, when submitted to drying processes, could retain a significant content of compounds. The possible mechanism that affects 13

ACCEPTED MANUSCRIPT the encapsulation efficiency is associated with the higher loss of bioactive compounds when larger emulsions are dried. This could be explained by the breakdown of the large emulsion

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droplets during the drying process. Consequently, the encapsulation efficiency is reduced and

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the surface extract is increased. Then, the phenomenon of breaking down the emulsions

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droplets is likely less significant when D[3,2] is smaller. More information about encapsulation efficiency for the different proportions of inulin and modified starch is presented into

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Section 3.4.

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The shapes of the distribution curves were also different for the five assays (Fig. 1). The narrowest distribution was obtained for SF:3IN, whilst irregular shapes were obtained for the

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other modified starch:inulin proportions. SF:3IN assay presented a more pronounced peak with droplet size smaller than 10 μm. Although there was another smaller peak, SF:3IN presented a

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size distribution in volume closer to a monomodal distribution than the other assays. According to Dickinson, Radford, and Golding (2003), monomodal distribution (with no associated phase separation apparent in the creaming tube) is an indicative of a stable emulsion, and a bimodal distribution is an indicative of irreversible flocculation. Kinetic stability of emulsions is achieved through the addition of small-molecule surfactants or biopolymers (proteins, carbohydrates and gums). These compounds can decrease interfacial tension, induce steric or electrostatic interactions and promote changes in the viscosity of the overall system, thereby improving the stability of the emulsion (Silva, Rosa, & Meireles, 2015b). Thus, when inulin was applied together with modified starch, water-oil interface tension and final size of the droplets were reduced.

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Powder morphology, particle size distribution and moisture Microparticles morphology influences other properties as bulk density, retention and

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release of bioactive compounds. Morphologies of microparticles containing oregano extract

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were most similar to shriveled flakes with small fissures and openings on the surfaces (Fig. 2).

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When applying SF alone in the emulsion, small particles (< 20 μm) deposited on the surface of large particles (> 20 μm) were observed. When the content of IN in the emulsion was increased,

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microparticles with rough surfaces were observed, some of them similar to round particles and

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others similar to walled particles or porous plates exhibiting pointed structures. Microparticles formed by freeze-drying typically present irregular shapes and porous

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structures. Besides, the superficial area is higher than that formed when drying emulsions by spray-drying. The pores seen in the microparticles (Fig. 2) raised in the same moment the ice

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crystals were formed during freeze-drying process. Similar morphologies of particles containing annatto seed oil obtained with inulin as wall material by freeze-drying process are reported (Silva & Meireles, 2015). Even though the microparticles were formed through spray-drying process, aggregates are observed in the micrographs of quercetin + inulin samples (SunWaterhouse, Wadhwa, & Waterhouse, 2013). The partial replacement of gum Arabic by inulin led to microparticles containing rosemary oil with a spherical shape after submitting the emulsion to spray-drying process (Fernandes et al., 2014a). In this work, microparticles obtained with inulin alone presented a higher proportion of spherical shapes, most likely because inulin provided elasticity during the drying step. SF:3IN assay demonstrated micrographs closer to IN assay, but the micrographs of the other assays were different when comparing with IN assay. In such way, the spherical shape of the structures 15

ACCEPTED MANUSCRIPT formed in the process was partially lost. Thus, the morphological variations are tightly dependent of the shell-forming materials.

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Particle size distribution in volume (Fig. 2) indicates that most of microparticles

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containing oregano extract were distributed between the ranges of 1 μm to 1,000 μm. Both

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distributions were monomodal, and the sizes of microparticles were smaller when the proportion of inulin increased. Results of D[4,3] (Table 2) corroborate the results of particle size

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distribution (Fig. 2). SF:3IN and IN assays presented the smallest particle sizes: 151 ± 12 μm and

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137 ± 1 μm, respectively. No significant differences were observed for D[4,3] among the treatments, except when SF was used alone (highest Brouckere diameter). Silva and Meireles

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(2015) reported D[4,3] values equal to 101 ± 3 μm and 158 ± 5 μm after characterizing

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microparticles containing annatto seed oil formed with inulin alone and presenting degrees of

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polymerization higher than 10 and 23, respectively. When using gum Arabic as wall material, microparticles obtained through freeze-drying presented D[4,3] equal to 171 ± 24 μm (Silva et al., 2015c). Production of particles with high average size using high proportions of modified starch can be associated to the high viscosity of this polymer (Jafari, Assadpoor, He, & Bhandari, 2008). The uniformity of the microparticle size distribution can be evaluated by the PDI values (Table 2). No significant differences (p-value = 0.629) of PDI were observed among the assays studied in this paper. Silva and Meireles (2015) and Fernandes et al. (2014a) reported PDI values in the same range of those reported in this study. Even though the mean value of moisture was the lowest for SF:3IN assay, no significant differences (p-value = 0.924) of moisture were observed among the assays (Table 2). Comprising the results presented and discussed in this section, the partial replacing of modified starch by inulin has been favorable, because the mean 16

ACCEPTED MANUSCRIPT diameter size was reduced, as well as the shape and distribution size of the microparticles were more uniform. Encapsulation efficiency and thymol retention

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3.4

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Different values of encapsulation efficiency, thymol retention, surface extract and

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entrapment efficiency (Fig. 3) were obtained for the microparticles containing oregano extract.

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One of the most important parameters of quality for the encapsulation of bioactive compounds is the encapsulation efficiency (EE), because it indicates the relative amount of active that is

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protected inside the matrix against adverse conditions that could cause its degradation. In this study, EE regards the oregano extract encapsulated or protected inside the polymeric matrix. EE

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ranged from 27 ± 4% (IN) to 76 ± 10% (SF). No significant differences (p-value = 0.142) were observed for EE among the four assays containing SF (SF; 3SF:IN; SF:IN; SF:3IN). SF:3IN assay, for

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instance, yielded EE equal to 66 ± 1%. The structural properties of the wall materials used in the encapsulation process represent the main factor on the retention of active substances. In this context, EE was higher when the matrix contained SF (alone or proportionally mixed with IN). Modified starch is known by its efficient emulsifying properties and retention of bioactive compounds (Jafari et al., 2008; Silva et al., 2015a; Simsek, Ovando-Martinez, Marefati, Sjӧӧ, & Rayner, 2015). But, looking forward the increasing demand for the consumption of functional products (as products presenting prebiotic properties), a considerable EE was obtained when replacing some content of SF by IN. Using three parts of IN together with one part of SF resulted in EE statistically similar to that obtained using SF alone. Therefore, the mechanism of increasing the viscosity of the continuous phase provided by IN, thus entrapping the droplets and avoiding the coalescence of the emulsion (stabilization through physical barriers), combined with the 17

ACCEPTED MANUSCRIPT mechanism of steric stabilization provided by SF led to a stable system that protected a high amount of oregano extract as a consequence of the synergic effect of the wall materials. The

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lowest proportion of starch in the mixture with inulin (SF:3IN) was sufficient to form an efficient

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interfacial film.

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There are no reports on scientific literature we could straightly compare our findings about EE. Even so, a high EE of polyphenols from purple cactus pear (Opuntia ficus-indica) was

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obtained using the blend constituted of soybean protein isolate and inulin as encapsulating

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matrix (Robert, Torres, García, Vergara, & Sáenz, 2015). An EE of approximately 88% was reported for the encapsulation of annatto seed oil using the blend constituted of whey protein

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isolate and inulin (Silva et al., 2016). Otherwise, EE of rosemary essential oil was reduced when

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inulin was mixed with gum Arabic. Despite of this, the presence of inulin improved the

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wettability and reduced the hygroscopicity of particles under high relative humidity (Fernandes et al., 2014a). Once inulin does not have superficial activity and its charge density is low, we consider the use of a small quantity of a carbohydrate with high emulsification capacity (SF, for instance) together with inulin as a great combination for reaching good results of EE, as obtained for SF:3IN assay (Fig. 3).

Furthermore, thymol retention was the largest in such assay (SF:3IN): 84 ± 9%. Significant differences (p-value = 0.001) were observed when comparing thymol retention among the five assays (Fig. 3) because using IN alone as wall material yielded the lowest retention: 16 ± 8%. Although no significant differences (p-value = 0.204) were observed when comparing thymol retention among the four assays containing SF (SF; 3SF:IN; SF:IN; SF:3IN), the mean value for SF:3IN was larger than that obtained for SF alone. Therefore, the partial 18

ACCEPTED MANUSCRIPT substitution of SF by IN seems to be suitable for retaining thymol. Results of retention capacity are associated with results of EE. Greater EE led to greater thymol retention, which indicates a

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lower loss of such bioactive compound. Over again, the synergic effects of IN (it allows

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stabilizing the droplets in the emulsion through incrementing the viscosity) with SF (it presents

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superficial activity) entrapped a high percentage of the target compound into the microcapsules structure.

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Some studies have shown that decreasing the emulsion droplets size causes an increase

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of the EE and a better retention of active substances (Botrel, Borges, Fernandes, & Carmo, 2014; Soottitantawat et al., 2003). However, those reports do not state a rule. There are reports that

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larger particles have an increased EE (Jafari et al., 2008). Silva and Meireles (2015) obtained

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D[3,2] equal to 1.6 ± 0.1 μm using inulin with degree of polymerization higher than 10 (GR-IN) and

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D[3,2] equal to 5.1 ± 0.4 μm using inulin with degree of polymerization higher than 23 (HP-IN) as wall materials in the encapsulation of annatto seed oil. However, EE and entrapment efficiency (ETE) of annatto seed oil were higher for HP-IN system, justly the system which the mean droplets size was higher.

In the case of ETE of oregano extract, significant differences (p-value = 0.006) were observed among the assays. SF:3IN yielded the highest ETE: 92 ± 1% (Fig. 3). Besides the entrapment of thymol, a high amount of other compounds present in oregano extract could be entrapped in the polymeric matrix. Our findings are in agree with other systems, like 94% of ETE for microparticles containing extract from amaranth seeds (Ott et al., 2015) and approximately 95% of ETE for microparticles containing oil from annatto seeds (Silva & Meireles, 2015). Otherwise, our findings for ETE are better than those reported by Chen, Zhong, Wen, 19

ACCEPTED MANUSCRIPT McGillivray, and Quek (2013). The authors reported the encapsulation of limonene with a blend of whey protein isolate and soluble corn fiber at 1:1 (w/w) ratio. Microparticles obtained

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through freeze-drying presented ETE equal to 54 ± 2%.

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In the case of surface extract, significant differences (p-value = 0.005) were observed

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among the assays, because IN assay led to a high surface extract value equal to 53 ± 9%, approximately 3 times higher than the surface extract obtained at SF assay: 16 ± 7%. Results

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obtained for surface extract could be associated to the lack of superficial activity of the inulin

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molecules, as corroborated by other studies (Fernandes et al., 2014a; Silva & Meireles, 2015). Thus, in the IN assay, the target component was not effectively encapsulated, but only adhered

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to the external surface of the wall material. In such condition, a lower amount of non-

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encapsulated extract and a higher amount of surface extract is an indicative of a weak

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protection against oxidation. Comprising the results presented and discussed in this section, the total replacing of SF by IN is not advantageous. However, we consider the proportion 1:3 (SF:3IN) as a polymeric matrix surely favorable for encapsulating oregano extract and retaining the target compounds. 3.5

X-ray diffraction The analysis of X-ray diffraction was performed to verify the structural properties of the

microparticles obtained by gradually replacing SF by IN. Different patterns are seen in the diffractogram of samples (Fig. 4). Once sharp peaks define crystalline materials and broad peaks define amorphous materials (Caparino et al., 2012), the results could be interpreted as a mix of them, that is, amorphous materials with crystalline regions (semicrystalline structures). The encapsulation of oregano extract presented a remarkable influence on crystallinity when IN 20

ACCEPTED MANUSCRIPT alone was used as wall material. A typical pattern of amorphous material of inulin powder (IN – wall material) is seen, as also reported by other studies (Fernandes, Borges, Botrel, & Oliveira,

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2014b; Silva & Meireles, 2015). However, the diffractogram of IN assay showed peaks at 7.9°,

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12.0°, 16.4°, 17.7°, 21.4°, 23.8° and 37.7°. The presence of oregano compounds adhered or

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encapsulated into the matrix influenced the organizational structure of the microparticles, thus interfering on the intensity of the diffraction. Otherwise, most of these peaks were absent in the

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diffractogram of SF assay. Therefore, replacing SF by IN indicates a tendency of increasing the

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crystallinity of the microparticles obtained by FD. Change in structure can be associated to a physical interaction between molecules of wall materials and the oregano extract, which

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suggests that the bioactive compounds could be encapsulated in the polymeric matrix.

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Crystallization of compounds within the polymeric matrix causes slower drug release as

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higher activation energy is required to dissolve crystals when compared to amorphous powder (Wu, Zhang, & Watanabe, 2011). Amorphous materials are generally more soluble, even though they are more hygroscopic than materials with a crystalline structure (Yu, 2001). Based on this context, we could infer that microparticles containing oregano extract encapsulated with inulin can preserve for a longer time the active substances into the core, thus releasing them slowly and uniformly. Regarding the peaks at 15.0°, 17.6° and 22.8° for modified starch (SF – wall material), they disappeared when oregano extract was added, suggesting that the long-range ordered structure of normal modified starch was affected during the encapsulation and micronization processes. Comprising the results presented and discussed in this section, the presence of inulin in the polymeric matrix increases the crystallinity degree of the powder products, as reflected in the shape and intensity of the diffraction peaks. 21

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Thermal stability Thermal stabilities of microparticles containing oregano extract and pure wall materials

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were assessed through the mass lost during controlled heating. The microparticles started

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losing mass at approximately 34°C, while the pure wall materials started losing mass at

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approximately 64°C for modified starch and 70°C for inulin. Thermograms (TG) and their respective derivatives (DTG) are displayed between 80°C and 350°C aiming to evidence the

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remarkable mass variations (Fig. 5). Peaks seen in the DTG curves identify the beginning and

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ending of a thermal degradation event.

Three degradation stages are observed during the thermal decomposition. The first one,

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which offers useful information for practical applications, comprises mass loss ranging from 3% (SF wall material) to 12% (SF assay), corresponding to temperatures below 220°C. In this stage,

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loss of volatiles and water succeeded with increasing the temperature (Martínez-Camacho et al., 2010), where microparticles with higher proportion of inulin, as in SF:3IN assay, lost slightly less mass than the other microparticles up to 220°C. Then, microparticles formed with a blend of inulin and modified starch can show more thermal stability than microparticles formed with inulin or modified starch alone submitted to heating in the range of 25-220°C (first stage). The extent of the first stage of thermal decomposition is longer than other systems, as 25-200°C for microparticles rich in tocotrienols and geranylgeraniol (Silva & Meireles, 2015) and 30-213°C for carboxy functionalized cellulosic nanoparticles (Sharma & Varma, 2014). The second stage (maximum degradation) comprises mass loss ranging from 51% to 74%, corresponding to temperatures in the range of 220-330°C. In this stage, other compounds not lost in the first stage were thermally decomposed, which mostly includes the wall materials and 22

ACCEPTED MANUSCRIPT compounds effectively encapsulated in the polymeric matrix. Maximum decomposition rate (DTGMAX) was observed for the seven samples (Fig. 5B) at: 266°C (IN), 280°C (IN – Wall material),

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300°C (SF:3IN), 310°C (SF:IN), 311°C (3SF:IN), 315°C (SF – Wall material) and 321°C (SF). The

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third stage comprises mass loss ranging from 22% to 33% for temperatures above 330°C. In this

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stage, carbonaceous inert material (not decomposed in the other stages) was thermally decomposed. Then, comprising the results presented and discussed in this section, partially

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replacing modified starch by inulin is favorable when microparticles are handled up to 220°C,

CONCLUSION

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4

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especially for the SF:3IN assay.

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The use of 25 wt.% of a conventional carbohydrate (modified starch) presenting high

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emulsification capacity together with 75 wt.% of inulin is considered to be a promising combination of polymeric prebiotic matrix for reaching great results about the characteristics of microparticles containing bioactive compounds. In order to give some emulsifying capabilities to the encapsulant matrix, the mixture of modified starch and inulin in the proportion of 1:3 (SF:3IN) showed to be effective for encapsulating oregano extract and retaining thymol. Furthermore, the presence of inulin in the polymeric matrix increased the crystallinity degree of the microparticles and provided great thermal stability when handling temperatures up to 220°C. Thus, this blend could be a potential wall material for industrial fields based on the prebiotic properties of inulin and based on the consumer seeking for food with additional health benefits. Therefore, this study brings forward novel outcomes that are interesting for defining

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ACCEPTED MANUSCRIPT excellent supports of sensible bioactive compounds to be applied, specially but not only, in the

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food industry.

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Acknowledgements

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Authors are grateful to CNPq (470916/2012-5) and FAPESP (2012/10685-8 and 2015/13299-0) for the financial support. Giovani L. Zabot thanks FAPESP (2014/15685-1) for the

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postdoctoral assistantship. Eric Keven Silva thanks CNPq (140275/2014-2) for the Ph.D.

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scholarship and FAPESP (2015/22226-6) for the postdoctoral assistantship. M. A. A. Meireles

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thanks CNPq (301301/2010-7) for the productivity grant.

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Martínez-Camacho, A. P., Cortez-Rocha, M. O., Ezquerra-Brauer, J. M., Graciano-Verdugo, A. Z., Rodriguez-Félix, F., Castillo-Ortega, M. M., . . . Plascencia-Jatomea, M. (2010). Chitosan composite films: Thermal, structural, mechanical and antifungal properties. Carbohydrate Polymers, 82(2), 305-315.

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Mazutti, M. A., Zabot, G., Boni, G., Skovronski, A., Oliveira, D. d., Luccio, M. D., . . . Maugeri, F. (2010b). Optimization of inulinase productionby solid-state fermentation in a packed-bedbioreactor. Journal of Chemical Technology and Biotechnology, 85, 109-114.

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Mensink, M. A., Frijlink, H. W., Maarschalk, K. v. d. V., & Hinrichs, W. L. J. (2015). Inulin, a flexible oligosaccharide. II: Review of its pharmaceutical applications. Carbohydrate Polymers, 134, 418428.

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Moraes, M. N., Zabot, G. L., & Meireles, M. A. A. (2015). Extraction of tocotrienols from annatto seeds by a pseudo continuously operated SFE process integrated with low-pressure solvent extraction for bixin production. Journal of Supercritical Fluids, 96, 262-271.

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Ott, C., Lacatusu, I., Badea, G., Grafu, I. A., Istrati, D., Babeanu, N., . . . Meghea, A. (2015). Exploitation of amaranth oil fractions enriched in squalene for dual delivery of hydrophilic and lipophilic actives. Industrial Crops and Products, 77, 342-352.

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Pandey, A., Soccol, C. R., Selvakumar, P., Soccol, V. T., Krieger, N., & Fontana, J. D. (1999). Recent developments in microbial inulinases: Its production, properties, and industrial applications. Applied Biochemistry and Biotechnology, 81(1), 35-52. Robert, P., Torres, V., García, P., Vergara, C., & Sáenz, C. (2015). The encapsulation of purple cactus pear (Opuntia ficus-indica) pulp by using polysaccharide-proteins as encapsulating agents. LWT - Food Science and Technology, 60, 1039-1045. Ruben, O., Valeria, N., & Ruben, G. N. (2014). Antioxidant activity of fractions from oregano essential oils obtained by molecular distillation. Food Chemistry, 156, 212-219. Saénz, C., Tapia, S., Chávez, J., & Robert, P. (2009). Microencapsulation by spray drying of bioactive compounds from cactus pear (Opuntia ficus-indica). Food Chemistry, 114(2), 616-622. Sharma, P. R., & Varma, A. J. (2014). Functionalized celluloses and their nanoparticles: Morphology, thermal properties, and solubility studies. Carbohydrate Polymers, 104, 135-142. Silva, E. K., Gomes, M. T. M. S., Hubinger, M. D., Cunha, R. L., & Meireles, M. A. A. (2015a). Ultrasoundassisted formation of annatto seed oil emulsions stabilized by biopolymers. Food Hydrocolloids, 47, 1-13. Silva, E. K., & Meireles, M. A. A. (2015). Influence of the degree of inulin polymerization on the ultrasound-assisted encapsulation of annatto seed oil. Carbohydrate Polymers, 133, 578-586. Silva, E. K., Rosa, M. T. M. G., & Meireles, M. A. A. (2015b). Ultrasound-assisted formation of emulsions stabilized by biopolymers. Current Opinion in Food Science, 5, 50-59.

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ACCEPTED MANUSCRIPT Silva, E. K., Zabot, G. L., Cazarin, C. B. B., Maróstica, M. R., & Meireles, M. A. A. (2016). Biopolymerprebiotic carbohydrate blends and their effects on the retention of bioactive compounds and maintenance of antioxidant activity Carbohydrate Polymers, 144, 149-158.

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Zabot, G. L., Moraes, M. N., Carvalho, P. I. N., & Meireles, M. A. A. (2015). New proposal for extracting rosemary compounds: Process intensification and economic evaluation. Industrial Crops and Products, 77, 758-771. Zabot, G. L., Moraes, M. N., & Meireles, M. A. A. (2014). Influence of the bed geometry on the kinetics of rosemary compounds extraction with supercritical CO2. Journal of Supercritical Fluids, 94, 244254. Zabot, G. L., Moraes, M. N., Petenate, A. J., & Meireles, M. A. A. (2014). Influence of the bed geometry on the kinetics of the extraction of clove bud oil with supercritical CO2. Journal of Supercritical Fluids, 93, 56-66. Zandi, M., Mohebbi, M., Varidi, M., & Ramezanian, N. (2014). Evaluation of diacetyl encapsulated alginate–whey protein microspheres release kinetics and mechanism at simulated mouth conditions. Food Research International, 56, 211-217.

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ACCEPTED MANUSCRIPT Figure Captions

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Figure 1: Droplet size distributions of the oregano extract emulsions: influence of modified

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starch:inulin proportion.

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Figure 2: SEM images and particle size distribution of the oregano extract microparticles.

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Figure 3: Influence of modified starch:inulin proportion on the surface extract, entrapment

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efficiency, encapsulation efficiency and thymol retention.

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Figure 4: X-ray diffraction patterns of oregano extract microparticles and wall materials.

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Figure 5: (A) Thermograms (TG) of the thermal decomposition and (B) its derivative (DTG) of

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oregano extract microparticles and wall materials.

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ACCEPTED MANUSCRIPT Table 1: Influence of modified starch:inulin proportion on the mean superficial diameter (D[3,2])

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and polydispersity index (PDI) of droplets of oregano extract emulsion.

Assay

PDI (-)

2.8 ± 0.1

20.7 ± 0.1

4.1 ± 0.6

20.7 ± 0.5

5

2.9 ± 0.3

14.1 ± 0.2

5

2.3 ± 0.1

2.4 ± 0.3

5

4.5 ± 0.6

23 ± 2

Inulin

SF

20

-

5

3SF:IN

15

5

5

SF:IN

10

10

SF:3IN

5

15

IN

-

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Modified Starch

(g/100 g of emulsion)

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D[3,2] (μm)

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Wall material (g/100 g of emulsion) Oregano extract

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Moisture (wt.%)

D[4,3] (μm)

SF

4 ± 2a

392 ± 44a

3SF:IN

4 ± 1a

198 ± 14b

SF:IN

4 ± 2a

176 ± 3b

3.4 ± 0.1a

SF:3IN

4 ± 1a

4.1 ± 0.1a

IN

5 ± 1a

137 ± 1b

4.0 ± 0.6a

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Emulsion

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modified starch:inulin proportions.

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151 ± 12b

PDI (-) 4 ± 1a 3.2 ± 0.2a

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Averages with the same letters in the same column do not differ statistically (p < 0.05).

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Figure 1

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Figure 3

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Figure 5

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Graphical abstract

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ACCEPTED MANUSCRIPT HIGHLIGHTS Modified starch (SF) was replaced by inulin (IN) as encapsulating matrix of bioactive compounds;

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Microparticles containing oregano extract were formed with SF and IN;



Mixing one part of SF with three parts of IN (SF:3IN) yielded 66% of encapsulation efficiency;

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SF:3IN yielded 84% of thymol retention;

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92% of oregano extract could be entrapped in the polymeric matrix;

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