Anthocyanins from jussara (Euterpe edulis Martius) extract carried by calcium alginate beads pre-prepared using ionic gelation

Accepted Manuscript Anthocyanins from jussara (Euterpe edulis Martius) extract carried by calcium alginate beads pre-prepared using ionic gelation

Ana Gabriela da Silva Carvalho, Mariana Teixeira da Costa Machado, Helena Dias de Freitas Queiroz Barros, Cinthia Baú Betim Cazarin, Mário Roberto Maróstica Junior, Miriam Dupas Hubinger PII: DOI: Reference:

S0032-5910(19)30016-6 https://doi.org/10.1016/j.powtec.2019.01.016 PTEC 14060

To appear in:

Powder Technology

Received date: Revised date: Accepted date:

20 September 2018 22 December 2018 5 January 2019

Please cite this article as: Ana Gabriela da Silva Carvalho, Mariana Teixeira da Costa Machado, Helena Dias de Freitas Queiroz Barros, Cinthia Baú Betim Cazarin, Mário Roberto Maróstica Junior, Miriam Dupas Hubinger , Anthocyanins from jussara (Euterpe edulis Martius) extract carried by calcium alginate beads pre-prepared using ionic gelation. Ptec (2019), https://doi.org/10.1016/j.powtec.2019.01.016

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ACCEPTED MANUSCRIPT Anthocyanins from jussara (Euterpe edulis Martius) extract carried by calcium alginate beads pre-prepared using ionic gelation

Ana Gabriela da Silva Carvalhoa*, Mariana Teixeira da Costa Machadob, Helena Dias de Freitas

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Queiroz Barrosa, Cinthia Baú Betim Cazarina, Mário Roberto Maróstica Juniora, Miriam Dupas

University of Campinas, School of Food Engineering, 80, Monteiro Lobato Street, 13083-862,

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a

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Hubingera

b

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Campinas, São Paulo, Brazil.

Federal Rural University of Rio de Janeiro, Department of Food Technology, 465, Highway 23,

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890-000, Seropédica, Rio de Janeiro, Brazil.

*Corresponding Author. Tel: +55 19 35214036; Fax: +55 19 35214044 E-mail

addresses:

[email protected]

(A.

G.

S.

Carvalho)*,

[email protected] (M. T. C. Machado), [email protected] (H. D. F. Q. Barros), [email protected] (C. B. B. Cazarin), [email protected] (M. R. Maróstica Junior), [email protected] (M. D. Hubinger).

ACCEPTED MANUSCRIPT ABSTRACT In order to take advantage of the functional properties of the jussara (Euterpe edulis Martius) extract, this study aimed at producing alginate hydrogel beads by ionic gelation process to carry jussara extract, increase its stability and to protect the anthocyanins from environmental conditions that interfere in stability and color of these compounds, such as the pH of the

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medium. Anthocyanins encapsulation from extract occurred by adsorption technique using

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blank alginate beads. Ionic gelation method was combined with complexation process using

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chitosan, whey protein concentrate or gelatin. Complexation technique using cationic polymers was effective in protecting these pigments during refrigerated storage. Gel strength

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of the hydrogel beads and anthocyanins retention was influenced by zeta potential and apparent viscosity of the materials used in the coating process. Chitosan presented higher zeta

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potential (+57.1 ± 2.0 mV) and apparent viscosity (0.013 ± 0.000 Pa.s) values and the beads coated with this material showed higher monomeric anthocyanins retention (8.4 ± 0.2 mg of

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cyanidin 3-glucoside equivalent/g of dry bead) than those coated with whey protein

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concentrate and gelatin (6.6 ± 0.3 and 5.2 ± 0.4 mg of cyanidin 3-glucoside equivalent/g of dry bead, respectively). Ionic gelation encapsulation process led to the formation of hydrogel

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beads containing anthocyanins, thus enabling the release profile of the compounds at specific

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pH conditions, such as the intestinal fluid.

Keywords: Sodium alginate; release profile; hydrophilic compounds; storage stability; cationic polymers.

ACCEPTED MANUSCRIPT 1. INTRODUCTION The jussara palm (Euterpe edulis Martius), found in the Brazilian Atlantic Forest, produces berries known as jussara which are a source of bioactive compounds, mainly anthocyanins [1]. The use of natural extracts containing anthocyanins as raw material by the industry has some limiting factors. Although they have a great potential for applications in

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food, pharmaceutical and cosmetic industries, they present chemical instability, a major drawback [2].

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Anthocyanins are the most abundant polyphenols in fruits and vegetables and they are

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the most significant group of visible, water-soluble and vacuolar plant pigments [3, 2].

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Anthocyanins intake was associated with protection against certain cancers, cardiovascular diseases, as well as other chronic human disorder [4], mainly due its antioxidant capacity,

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capable of reducing inflammation, lipid peroxidation, and the deleterious effects of reactive oxygen species (ROS) [5]. In this way, microencapsulation process is a promising method to

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overcome the limitations in stability of these pigments [6].

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Ionic gelation is an interesting encapsulation method that offers as advantages an easy execution and practicality, avoiding high temperatures and organic solvents. Particulate forms of gel present some useful applications: structuring, strengthening and texturizing agents in

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food matrices and the capacity of enhancing the visual acceptability of products. In addition,

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they are able to adapt in shape and size, enabling controlled release of the active in agricultural, pharmaceuticals or food products [7, 8]. By definition, hydrogels are polymeric networks with hydrophilic properties and physicals hydrogels known as “ionotropic hydrogel” are obtained by combining a polyelectrolyte with a multivalent ion of the opposite charge [9]. Materials such as sodium alginate, gellan gum, carrageenan, pectin or chitosan are dissolved in water and form an insoluble gel in the presence of multivalent ions. The gelation process occurs by diffusion of these ions into the hydrocolloid solution, in which the droplets in contact with the ionic

ACCEPTED MANUSCRIPT solution provide instantaneous formation of spherical gel structures containing the active material [7, 10, 11]. Beads produced by ionic gelation have a porous gel matrix, which allows a fast and easy diffusion of water or other fluids into or out of the particle structure [7, 8]. Taking advantage of this property, hydrogels are materials commonly used for encapsulation due to their high capacity of adsorption of water and biological fluids [12]. As presented by

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Chan, Yim, Phan, Mansa and Ravindra [12], the encapsulation of liquid extracts such as concentrated herbal aqueous extracts can be performed through adsorption of the extracts by

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pre-fabricated calcium-alginate hydrogel beads. The adsorption method to encapsulate

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hydrophilic compounds may be an alternative technique to avoid losses of core material into

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the ionic solution. Hydrogel beads are immersed in a concentrated solution and the active compounds from the extract diffuse into the blank beads. Furthermore, according to Chan,

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Yim, Phan, Mansa and Ravindra [12] Ca-alginate hydrogel structure was shown to be a compatible matrix for encapsulating biochemical active compounds extracted from natural

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sources.

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However, the porosity of the particles decreases the encapsulation efficiency by the ionic gelation. This is a major drawback for such encapsulation process and several strategies have been developed to encapsulate hydrophilic compounds from natural plant extracts.

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Combined techniques of ionic gelation and complexation with cationic polyelectrolytes have

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been proposed with the aim of reducing the porosity and diffusion process of the beads. The complexation is a result of the electrostatic interaction between the anionic charges of the polysaccharides and the cationic charges of the polymers. Electrostatic complexes are usually reversible, depending on the pH and the ionic strength [13]. Materials such as chitosan and proteins can provide higher protection to the encapsulated compounds, preventing the diffusion of the hydrophilic compounds through the pores of the gel matrix, by an electrostatic interaction with anionic charges of the surfaces of the gel particles [14, 15, 16]. Nevertheless, the ionic gelation process is still a challenge for hydrophilic compounds, such as anthocyanins

ACCEPTED MANUSCRIPT pigments, in relation to the encapsulation efficiency, diffusion of compounds, interaction between polymers and hydrophilic actives and controlled release properties of the core material [17]. To the best of our knowledge, it is the first time that the encapsulation of anthocyanins from jussara pulp (Euterpe edulis Martius) by ionic gelation is described. Our research group studied the jussara extract preparation and the encapsulation of

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its bioactive compounds. This extract is a rich source of anthocyanins as cyanidin 3-rutinoside and cyanidin 3-glucoside [18] and, therefore, the aim of this work was to encapsulate aqueous

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jussara extract by ionic gelation process. Jussara extract was prepared through aqueous

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extraction process and encapsulation of anthocyanins from this extract was performed by

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adsorption of pre-prepared calcium alginate beads. The samples were subjected to complexation by electrostatic interaction using chitosan and proteins. The hydrogel beads

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were characterized in relation to total solids content, average size of beads, gel strength, monomeric anthocyanin content and antioxidant capacity, microstructure, storage stability

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and release profile in simulated gastric and intestinal conditions.

2.1 Materials

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2. MATERIAL AND METHODS

Hydrogel beads were prepared using sodium alginate (GRINDSTED® Alginate FD 175)

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with a high content of guluronic acid (above 60%) supplied by Danisco Brasil Ltda (Cotia, São

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Paulo). Complexation process was performed using chitosan (low molecular weight, deacetylation, 75%–85%) supplied by Sigma-Aldrich Brazil Ltda, whey protein concentrate (WPC) supplied by Fonterra Co-operative Group Limited (Auckland, New Zealand) and gelatin powder (Rousselot® 225H 30, with Bloom 222) supplied by Rousselot Gelatinas do Brasil SA (Amparo, São Paulo). Concentrated jussara extract was prepared according to the methodology of da Silva Carvalho, Costa Machado, Silva, Sartoratto, Rodrigues and Hubinger [18] using jussara pulp (Euterpe edulis Martius). Physicochemical composition of the concentrated jussara extract

ACCEPTED MANUSCRIPT showed 97.42 ± 0.04 (wet basis) of moisture content using AOAC [19] method; 14.15 ± 0.74 (dry basis) of fat using the method of Bligh and Dyer [20]; 43.08 ± 2.30 (dry basis) of total sugar according to the method of Dubois, Gilles, Hamilton, Rebers and Smith [21] with modifications of Hodge and Hofreiter [22]; 34.25 ± 1.58 (dry basis) of fiber obtained by difference; 5.03 ± 0.60 (dry basis) of total protein by AOAC [19] method and 3.49 ± 0.50 (dry basis) by AOAC [19]

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method.

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2.2 Production of hydrogel beads containing anthocyanins from jussara extract Sodium alginate at a concentration of 2% w/w (2 g/100 g) was dispersed in Milli-Q

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water, remaining in the hydration process overnight for complete dispersion. Alginate

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hydrogel beads were prepared according to Souza, Gebara, Ribeiro, Chaves, Gigante and Grosso [23], with some modifications. The alginate dispersion was dripped into a calcium

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chloride solution (2% w/v, 2g/100 mL), using a double fluid atomizer with 0.5-mm-diameter nozzle with a distance of 12 cm between the nozzle atomizer and the calcium chloride

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solution. Feed flow rate was 2.5 mL/min using a peristaltic pump (Masterflex Cole Parmer

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Instruments, Chicago, USA). The beads were maintained in a calcium chloride solution for 30 min under magnetic stirring; after that, they were filtered and washed to remove excess calcium and to stop the gelation process.

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The coating polymers (chitosan, WPC or gelatin) were dispersed in Milli-Q water at

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a concentration of 1% w/w (1g/100g) and kept in hydration process overnight. Gelatin was heated up to 65 °C until complete dispersion. These dispersions were acidified with acetic acid (60% v/v, 60 mL/100 mL) to pH 3.50. To determine the zeta potential of the materials, sodium alginate, chitosan, WPC and gelatin were dispersed in Milli-Q water at the concentration of approximately 0.2% (0.2g/100g). For chitosan, WPC and gelatin samples the pH was adjusted up to 3.50 using acetic acid 60% (v/v), and sodium alginate was analyzed at its natural pH of 6.50. The samples were placed into the measurement chamber of a microelectrophoresis instrument (Nano ZS

ACCEPTED MANUSCRIPT Zetasizer, Malvern Instruments Ltd., Worcestershire UK) to determine the zeta potential. The measurements were performed in triplicate. Viscosity of the dispersions of alginate 2% (2g/100g), chitosan, WPC and gelatin 1% (1g/100g and pH 3.50) and concentrated jussara extract was determined by flow curves. The assays were carried out using a rheometer, Model AR 1500ex, TA Instruments (New Castle,

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United State) at 25 °C. Double concentric cylinders geometry was used for the measurements with a gap of 500 μm for the dispersions and 1000 μm for the extract. The apparent viscosity

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was obtained from the relationship between the shear stress and the shear rate.

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The time of adsorption of the extract and its complexation with cationic

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polyelectrolytes was determined after a kinetics study in relation to beads’ weight gain and total solids. Anthocyanins from concentrated jussara extract (2.5 g of solids/100 mL) were

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adsorbed for 90 min under constant stirring at room temperature by blank alginate hydrogel beads. Alginate beads and jussara extract were maintained in a ratio of 1:2 (w/w). After that,

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the beads containing anthocyanins were maintained for 10 min in either a chitosan or protein

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(whey protein concentrate or gelatin) dispersion in a ratio of 1:2 (w/w), at room temperature, for coating. The beads were filtered and analyzed. 2.3 Hydrogel beads characterization Total solids content

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2.3.1

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Total solids content was determined gravimetrically by drying in a vacuum oven at 70 ° C; approximately 2 g of sample from each assay were weighed and placed in petri dishes until constant weight. This analysis was performed in triplicate [19]. 2.3.2

Average size of beads Hydrogel beads were visualized using a Nikon optical microscope (AZ100 model,

Tokyo, Japan) with 0.5X of magnification, according to the methodology of Li, Kim, Chen, & Park [24]. After the acquisition of the images, the beads diameter was obtained individually from approximately 200 images per sample, in duplicate. The mean diameter of the samples

ACCEPTED MANUSCRIPT was expressed as D[4,3] (De Brouckere mean diameter) and the frequency (%) of beads distribution with the same diameter, varying from 0 to 2 mm. 2.3.3 Gel strength of the hydrogel beads Maximum force required for compression represents the maximum strength of the surface of the beads under compression by probe, thus indicating the hardness of the samples.

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Uniaxial compression tests were performed on a TA-XT Plus Texture Analyzer (Stable Micro Systems, UK) using an acrylic cylindrical plate probe with 35 mm diameter, lubricated with

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silicone oil to avoid friction with the samples. For the analysis, 6 beads were compressed to

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80% of their initial height at a velocity of 0.5 mm/s. The maximum compressive strength was

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expressed in N/mm2. These measurements have been made immediately after samples’ preparation and in triplicate.

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2.3.4 Monomeric anthocyanins content and antioxidant capacity The wet samples with extract and the ones coated with chitosan, WPC or gelatin

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were placed in ethylenediaminetetraacetic acid (EDTA) solution in order to chelate and,

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therefore, remove the calcium ions and promote the disorganization of the gel structure in order to release the monomeric anthocyanins present in the beads. In this way, 0.8 g of samples were dispersed in 24.5 mL of EDTA (0.2 M) and kept under stirring in a TE-420 model

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orbital shaker, Tecnal (Piracicaba, São Paulo) at 100 rpm for 90 min at 25 °C. The solution rate

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between alginate beads and EDTA and the extraction time of anthocyanins for quantification were defined after preliminary trials. Monomeric anthocyanins content was determined by a spectrophotometric pH differential method described by Giusti and Wrolstad [25]. Aliquots of the samples were placed in buffers of pH 1.0 and 4.5, of potassium chloride (0.025 M) and sodium acetate (0.4 M) respectively. After 15 min at room temperature, the values for absorbance were measured at 510 nm and 700 nm using a Unico SQ2800 UV–Vis spectrophotometer (Unico, New Jersey, USA). Monomeric anthocyanins content was calculated as cyanidin 3-glucoside equivalent using the molar absorptivity of 26,900 L/mol.cm

ACCEPTED MANUSCRIPT and molecular weight of 449.2 g/mol. The results were expressed as mg of cyanidin 3glucoside equivalent/g of dry beads. This analysis was performed in triplicate. Antioxidant capacity of the wet samples was determined by an Oxygen Radical Absorbance Capacity (ORAC) assay. For this analysis, 25 μL of diluted sample, 150 μL of fluorescein solution diluted in a phosphate buffer (pH 7.4) and 25 μL of AAPH (2,20’-Azobis (2-

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amidino-propane) dihydrochloride) were added to black microplates, in the dark. Trolox was used as a standard and readings were made on a microplate reader (Synergy HT, Biotek) with

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the following fluorescent filters: excitation wavelength of 485 nm and emission wavelength of

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520 nm. ORAC values were obtained in μmol of Trolox equivalent/L using the standard curves

μmol of Trolox equivalent/g of dry beads [26].

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2.3.5 Microstructure

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(2.5 and 100.0 μmol of Trolox equivalent/L) for each assay and the results were expressed in

2.3.5.1 Scanning electron microscopy

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Alginate beads were dried at 30 °C for 24 h in a drying oven. The samples were

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observed in a Scanning Electron Detector microscope with Energy Dispersive X-ray, LEO 440i — 6070 (LEO Electron Microscopy/Oxford, Cambridge, England), operating at 15 kV and an electron beam current of 50 pA. The samples were fixed directly on door-metallic specimens

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(stubs) of 12 mm diameter and 10 mm height and then subjected to metallization (sputtering)

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with a thin layer of gold in a Sputter Coater EMITECH K450 model (Kent, United Kingdom) with a thickness of 200 Å. After metallization, the samples were observed with magnifications of 200; 1500 and 3000x. Image acquisition was performed by the LEO software, version 3.01. 2.3.5.2 Optical microscopy The morphology of the wet alginate beads was observed in an optical microscope Nikon (AZ100 model, Tokyo, Japan) with the 0.5x objective lenses. 2.3.5.3 Confocal laser scanning microscopy

ACCEPTED MANUSCRIPT The confocal microscopy analysis of the wet alginate beads was performed based on the fluorescence properties of natural bioactive compounds, mainly anthocyanins, present in the jussara extract. The samples were analyzed by Zeiss Inverted Zeiss Axio Observer Z.1 (LSM780-NLO, Carl Zeiss AG, Germany) with the 10x objective lenses. The images were observed with an excitation laser of 514 nm and reading range between 547 and 640 nm.

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2.4 Storage stability of anthocyanins under refrigeration conditions Anthocyanins stability in alginate beads during refrigerated storage was determined

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according to the method of Belscak-Cvitanovic, Stojanovic, Manojlovic, Komes, Cindric,

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Nedovic and Bugarski [15], with some modifications. The samples were stored for 4 weeks at 5

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°C. Monomeric anthocyanin quantification was performed according to the method described in Item 2.3.4.

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2.5 Release profile of anthocyanins from alginate beads in simulated gastric and intestinal conditions

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The simulated gastric and intestinal fluids were prepared according to Sanguansri, Day,

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Shen, Fagan, Weerakkody, Jiang Cheng, Rusli and Augustin [27], with modifications. The simulated gastric fluid was prepared from the dispersion of 3.2 g of pepsin and 2 g of sodium chloride in distilled water. 7 mL of hydrochloric acid (36% v/v, 36 mL/100mL) were added and,

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then, the obtained volume was completed to 1000 mL with distilled water. The final pH of the

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simulated gastric fluid was maintained at 1.2 with HCl solution (6 M). The simulated intestinal fluid was prepared by dispersion of 1.25 g of pancreatin and 6.8 g of monopotassium phosphate in distilled water. 77 mL of sodium chloride (0.2 M) were added and the obtained volume was completed to 1000 mL with distilled water. The pH of the solution was adjusted to 7.4 with sodium hydroxide (5 M). Both fluids were prepared at 37 °C. Release profile of anthocyanins out of beads under simulated gastrointestinal conditions in vitro was performed according to the methodology used by Belscak-Cvitanovic, Busic, Barisic, Vrsaljko, Karlovic, Spoljaric, Vojvodic, Mrsic and Komes [28], with some

ACCEPTED MANUSCRIPT modifications. For the analyses, 6 g of beads were suspended in 60 mL of simulated gastric fluid (pH 1.2) and kept under stirring at 100 rpm in an orbital shaker, at 37 °C, for 120 min. The beads were filtered and suspended in 60 mL of simulated intestinal fluid (pH 7.4) and kept under stirring at 100 rpm for 120 min, at 37 °C. Aliquots of fluids were collected at set times (5; 10; 15; 20; 40; 60; 80; 100 and 120 min) and the monomeric anthocyanin quantification was

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performed according to the method described in Item 2.3.4. Simulated gastric and intestinal fluids after 120 min of incubation were evaluated in

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relation to the color using a colorimeter (Ultra Scan Vis 1043, Hunter Lab, Reston, EUA) with

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CIELab scale (L*, a* and b*), in reflectance mode, with D65 as an illuminant and a 10° observer

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angle as a reference system. The color measurements were expressed in relation to lightness L* and the chromaticity parameters a* and b*.

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2.6 Statistical analysis

The results of zeta potential, apparent viscosity, total solids content, average size of

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bead, gel strength, monomeric anthocyanins content and antioxidant capacity of the hydrogel

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beads were statistically analyzed by one-way Analysis of Variance, using the software trial edition (Minitab 16.1.0, Minitab Inc., State College, PA, U.S.A.). Statistical analysis was performed employing the Tukey test, where differences between means were considered at a

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95% confidence level (p≤0.05).

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3 RESULTS AND DISCUSSION 3.1 Preparation of alginate beads containing anthocyanins Preliminary results showed that the dispersion of sodium alginate in the jussara

extract (2 g alginate in 100 g extract) in acid medium (approximately pH 3.0) led to the formation of a weak undesirable textured gel. The dripping of this mixture of alginate and extract in 2% calcium chloride (2 g/100 mL) formed fragile particles, without defined formats and with a big loss of anthocyanins for the solution of calcium chloride, due to the diffusion process of the hydrophilic compounds into the surrounding medium. As a consequence, we

ACCEPTED MANUSCRIPT chose to embed blank alginate beads in anthocyanins from jussara extract, for a better retention of these hydrophilic compounds. The preparation process of the samples by ionic gelation was carried out with the concern of keeping the stability of the anthocyanins and, at the same time, take into account solubility and stability limitations of the coating polymers and alginate. The pH value of the

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extract has been maintained at 3.0 for better stability of the anthocyanins. The coating dispersions were kept at acid pH (3.5). For the coatings, the acid medium was also important,

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since chitosan is soluble in dilute acid solutions. The isoelectric point of the WPC solution is

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approximately 4.35, with the zeta potential varying as a function of pH, obtaining positive

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charges at pH 3.0 (20.61 ± 4.99 mV) and negative at pH 7.0 (-19.70 ± 3.00 mV) [16]. In addition, gelatin in acid medium with pH value lower than 5.0, shows the formation of positive charges

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[29], as also observed in this work (Table 1).

All samples were prepared at ambient temperature conditions, not affecting the

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anthocyanins or coating materials stability during the ionic gelation process.

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3.2 Hydrogel beads characterization

Total solids content and average size of the hydrogel beads

According to Table 2, the total solids content of the alginate hydrogel beads

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containing extract was higher than the content of the other beads, which was expected due to

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the adsorption capacity of the porous matrix of the alginate beads. Jussara extract occupied the empty spaces of the structure of the alginate beads through the adsorption process, adding solids to the samples. Beads prepared with alginate only presented the lowest solids content. The coating process provided by the electrostatic interaction among alginate beads (negative charges) and the polymers of chitosan, WPC or gelatin (positive charges) led to a loss of solids (9.0, 13.0 and 11.0% for chitosan, WPC and gelatin, respectively) due to diffusion to the hydrophilic external solution. The loss of solids was observed gravimetrically by weight loss in a vacuum oven at 70 °C.

ACCEPTED MANUSCRIPT The moisture content of the samples was higher than 92%. Similar results were observed in other studies for particles produced by ionic gelation using materials such as pectin and alginate. Hydrogel beads produced by external ionic gelation are hydrophilic and present porous polymeric structure capable of adsorbing high-water content in their threedimensional networks, filling the space between the macromolecules. Ability of hydrogels to

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adsorb water is a consequence of the hydrophilic functional groups associated with the polymer skeleton [9, 28].

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Alginate hydrogel beads’ average diameters varied from 1.0 to 1.2 mm in relation to

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D[4,3] (De Brouckere mean diameter), as presented in Table 2. The particle size of alginate

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hydrogel beads around 1 mm was consistent to other studies that reported the size of calcium alginate beads produced by extrusion dripping technique is generally bigger than 1 mm, as

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observed by Busic, Belscak-Cvitanovic, Cebin, Karlvic, Kovac, Spoljaric, Mrsic and Komes [30] and Arriola, Chater, Wilcox, Lucini, Rocchtti, Dalmina, Pearson and Amboni [31]. The droplet

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size and hence the final bead is influenced by the needle diameter of the dripping system,

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viscosity and flow rate of the feed material [7]. Hydrogel beads showed a monomodal distribution as seen in Figure 1A. The curve represented by alginate only showed a displacement to the right, having a tendency for larger

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diameter beads when compared to the beads containing extract. These beads presented

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similar curves when compared among themselves. The process of dripping technique without the use of compressed air may have contributed to obtain homogeneous particle size distribution and spherical shape. According to Chan, Lee, Ravindra and Poncelet [32] the dripping technique is a well-known method for producing mono-dispersed and round calcium alginate macrobeads. 3.2.2

Gel strength of the hydrogel beads

Maximum gel strength of the alginate hydrogel beads is presented in Figure 1B. Beads with alginate only showed higher gel strength in comparison to other beads containing

ACCEPTED MANUSCRIPT jussara extract and coating polymers. Jussara extract may have acted as a plasticizing agent, decreasing gel strength and increasing the flexibility of alginate beads. According to Batista Reis, Souza, Silva, Martins, Nunes and Druzian [33], the free sugars present in vegetable extracts and fruit pulps can act as plasticizers in starch films. In addition, Chan, Yim, Phan, Mansa and Ravindra [12] reported that calcium-alginate hydrogel with a high mannuronic

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content became more fragile after encapsulating an herbal aqueous extract. Coating polymers also had lower gel strength than beads with alginate only. Similarly, chitosan, WPC or gelatin

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could have acted as plasticizing agents.

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The main role of the plasticizing agents is to improve the flexibility of the polymers.

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They are low molecular weight compounds capable of occupying intermolecular spaces of the polymer chains, reducing the forces among them. In the same way, these agents alter the

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three-dimensional organization of the polymers, reducing the energy required for molecular movement and formation of the hydrogen bonds among chains [34].

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In relation to the rheological behavior of the coating polymers, WPC showed the

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lowest apparent viscosity at a shear rate of 100 s-1 (Table 1), what could have contributed to a higher accommodation of the molecules into the alginate beads thereby providing the gel with a higher strength. Dispersions of higher viscosity, as those made of chitosan and gelatin,

Monomeric anthocyanins content and antioxidant capacity

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3.2.3

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according to Table 1, provided weaker gels.

Anthocyanins degradation may happen because of their interaction with oxygen, high temperatures or light and because of changes in pH values. For instance, there are changes in the pH along the entire gastrointestinal tract: in the saliva, the pH is around 6.4; in the stomach, 1.5 and in the intestine’s microsurface, 5.3 [35]. These changes in pH can destabilize the anthocyanins present in foods after intake. Therefore, encapsulation appears to be effective in protecting these compounds [36].

ACCEPTED MANUSCRIPT Hydrogel beads composed of alginate only showed higher monomeric anthocyanins content, this being considered the initial anthocyanins content and, therefore, the content present in the samples before the coating process. A loss in anthocyanins was observed after coating using chitosan, WPC or gelatin. Beads coated with gelatin presented the greatest loss, of around 50%; for WPC and chitosan, the loss was 36 and 18.5%, respectively, as shown in

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Table 2. Beads with chitosan showed lower monomeric anthocyanins losses during the coating

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process, compared to WPC and gelatin dispersions. This material (chitosan) showed higher

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values of zeta potential, as shown in Table 1, and higher values of positive charge may have

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contributed to a faster and stronger electrostatic interaction process, resulting in lower losses of hydrophilic compounds by diffusion, compared to WPC and gelatin coatings. These results

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confirm that the choice of the coating agent is a critical step during anthocyanins encapsulation [37].

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Hydrogels are normally prepared from polar monomers. Jussara extract and the

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hydrogel beads were prepared in aqueous solutions, and due to the porous structure of the beads and the easy diffusion of these bioactive compounds in hydrophilic conditions, especially because of their polar characteristic, a loss in anthocyanins was expected.

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Antioxidant capacity by Oxygen Radical Absorbance Capacity (ORAC) assay of the

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beads followed the same trend observed for the monomeric anthocyanins content, as displayed in Table 2. Alginate hydrogel beads containing extract only showed the highest antioxidant capacity, followed by the samples coated with chitosan, WPC and gelatin. In this way, the antioxidant capacity of the beads was directly correlated to the amount of anthocyanins present in the beads. Alginate hydrogel beads and samples coated with chitosan were more efficient in incorporating and protecting the anthocyanins, resulting in higher antioxidant capacity values in relation to samples containing WPC and gelatin.

ACCEPTED MANUSCRIPT Gonçalves, Moeenfard, Rocha, Alves, Estevinho and Santos [38] observed that chlorogenic acid microparticles with modified chitosan or sodium alginate showed higher antioxidant capacity than the free core compound, in part due to the influence of the antioxidant capacity of these biopolymers. 3.3 Microstructure

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Particles microstructure was analyzed by scanning electron, optical and confocal

relation to their morphology. Scanning electron microscopy (SEM)

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3.3.1

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microscopy. The purpose of these microscopies was to better characterize the particles in

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Scanning electron microscopy (SEM) analysis showed the behavior of the coatings and their superficial effect on the alginate samples. Figure 2 (A1, A2, A3, B1, B2, B3, C1, C2, C3,

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D1, D2, D3, E1, E2 and E3) reveals the SEM microphotographs of the hydrogel beads. The beads exhibited spherical shape and irregular surface after the drying process. Hydrogel beads

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morphology (Figure 2 A1, A2 and A3 and B1, B2 and B3, of alginate only and alginate with

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extract, respectively) presented cracks and, after coating process with chitosan, WPC or gelatin, a surface free of cracks was observed. Beads with chitosan coating had more wrinkled surfaces (Figure 2 C1, C2 and C3) compared to the ones coated with WPC and gelatin, which

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provided the beads with a smoother surface, as seen in Figure 2 (D1, D2 and D3 and E1, E2 and

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E3, for WPC and gelatin, respectively). The presence of cracks in dried beads can favor oxygen transfer and its interaction with the active compounds and lead to their degradation. As observed in the gel strength analysis, the plasticizing effect of the chitosan, WPC

or gelatin may have improved the flexibility of the alginate beads, since no cracks were observed on their surfaces after drying. Furthermore, the addition of the jussara extract may have also contributed to the plasticizing effect on the beads, since the beads containing alginate only (Figure 2 A1, A2 and A3) presented a structure with more cracks than samples of alginate with extract (Figure 2 B1, B2 and B3).

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Optical and confocal laser scanning microscopy

Through optical microscopy of hydrogel beads it was possible to analyze the shape and quantify the particle size, as can be seen in Figure 2 (A4, B4, C4, D4 and E4). The beads samples showed uniform spherical shape, homogeneous size distribution as already verified in Figure 1A and a direct correlation between the measures of the images visualized (scale bar 1

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mm) by optical microscopy with the mean diameter D[4,3] (around 1 mm), showed in the Table 2. The alginate beads presented differences in coloration due to the coating process using

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chitosan, WPC or gelatin. Samples coated with chitosan showed a bright intense red color

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(Figure 2 C4), similar to the beads containing extract only (Figure 2 B4). WPC provided an

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opaque red color (Figure 2 D4) and the beads coated with gelatin presented a pink color (Figure 2 E4). The color change in the hydrogel beads is related to the loss of anthocyanins

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during the coating process.

The presence of anthocyanins as an indication of encapsulation efficiency in beads

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was verified by confocal laser scanning microscopy. Intense fluorescence of alginate beads was

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observed in the presence of jussara extract (Figure 2 B5, C5, D5 and E5) in relation to hydrogel bead with alginate only (Figure 2 A5). The coating process did not influence the confocal analysis of the beads. There was a homogeneous distribution of the jussara extract in the

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alginate matrix (green area), indicating possible retention of anthocyanins in all structures of

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the bead, as seen in Figure 2 (B5, C5, D5 and E5). Similar images were reported by da Silva Carvalho, da Costa Machado, da Silva, Sartoratto, Rodrigues and Hubinger [18] for particles produced by spray drying containing anthocyanins from jussara extract. These authors observed homogeneous distribution of these pigments throughout the microparticle, therefore showing the encapsulation efficiency of this extract. The confocal laser scanning microscopy can be seen as a qualitative analysis that shows the encapsulation efficiency. The distribution of the core material on the particle structure is an indicator of its encapsulation [39].

ACCEPTED MANUSCRIPT 3.4 Storage stability of anthocyanins under refrigeration conditions Although the initial content of anthocyanins among samples was not equal due to the coating process, alginate beads with extract only exhibited higher anthocyanins loss (varying from 10.3 ± 0.5 to 5.1 ± 0.5 mg of cyanidin 3-glucoside equivalent/ g dry particle) in comparison to other samples coated with chitosan, WPC and gelatin (varying from 8.4 ± 0.2 to

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5.2 ± 0.5; 6.6 ± 0.3 to 4.7 ± 0.5 and 5.2 ± 0.4 to 4.2 ± 0.3 mg of cyanidin 3-glucoside equivalent/ g dry particle, respectively) during the storage period, as displayed in Figure 3A.

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. The preparation of alginate beads by ionic gelation is a simple process, the gels

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produced by this method present pores, which can accelerate the permeation of oxygen

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through the matrix or to facilitate the release of the active compound inserted into the gel. Therefore, the presence of these pores may present limitations on the functional aspect of gels

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in the form of particles, reducing the efficiency of encapsulation [40, 8]. Combined process, as ionic gelation and complexation technique using cationic polyelectrolytes have been studied

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on improving the functionality of encapsulated particles [41, 16]. The zeta potential analysis

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showed the differences in charge of coating materials and alginate (Table 1), indicating an electrostatic interaction among alginate, of anionic character, and the polymers (chitosan, WPC or gelatin), of cationic character. As observed in this work, the coating process of alginate

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hydrogel beads provided improvements in the functionality of the samples, due to lower

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degradation of anthocyanins during the storage period. Chitosan, WPC or gelatin were efficient in protecting the jussara extract carried by the alginate beads. The gelatin beads presented a more stable behavior during the analysis of whole

stability under refrigeration conditions. Beads coated with gelatin showed a final loss of 20% in relation to the initial anthocyanins content, whereas there was a loss of 50, 38 and 29% for beads composed of alginate only and coated with chitosan and WPC, respectively. Gelatin is a protein with the property of forming thermoreversible elastic gels at room temperature, in low concentrations. Therefore, for solutions of concentration higher than 1% (1 g/100 mL) at

ACCEPTED MANUSCRIPT temperatures below 40 °C, a sol-gel transition occurs with progressive increases in viscosity and elasticity. However, with an increase in temperature, the gel melts into the liquid state [42]. This gel like behavior may have contributed to a better anthocyanins protection in the hydrogel beads, due to its increased viscosity as compared to WPC and chitosan coatings. Moura, Berling, Germer, Alvim and Hubinger [43] observed that increasing the

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storage temperature (5, 15 and 25 ° C) reduced the stability of anthocyanins from hibiscus (Hibiscus sabdariffa L.) extract encapsulated in pectin beads. Storage under refrigeration

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condition was used in this work as a food preservation technique due to the high moisture

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content of the hydrogel beads, above 92% (Table 2). In this case, the lowest storage

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temperature (5 °C) also contributed to stability and conservation of the anthocyanins and prevents the growth of microorganisms in alginate beads during the analysis time.

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3.5 Release profile of anthocyanins from alginate beads in simulated gastric and intestinal conditions

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The bioavailability of anthocyanins in plasma (< 6% of initial dose) after the intake of

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a meal rich in these compounds is limited [44], which is generally attributed to their low solubility, low stability, low permeability, to an active efflux process and to the metabolism of the gastrointestinal tract as well [45]. In this way, the encapsulation of bioactive ingredients

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can improve bioavailability by enhancing their solubility in water and their release in a specific

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milieu, resulting, thus, in better absorption by the human body [46]. Samples coated with chitosan presented lower anthocyanins release in relation to

the other samples in the first 20 min. In general, the release of anthocyanins increased in the first 40 min, remaining in equilibrium after this period, until 120 min of analysis. The release profile of anthocyanins indicated that the porosity of alginate hydrogel was not reduced completely to prevent the diffusion of anthocyanins compounds from the beads, since more 50% of this pigment was release in the first 20 min. Alginate beads containing extract only showed a final percentage of released anthocyanins to the gastric phase of 76% and the

ACCEPTED MANUSCRIPT samples covered with chitosan, WPC and gelatin of 73, 71 and 70%, respectively, as displayed in Figure 3B. The sample beads, after release in simulated gastric fluid, revealed spherical shape and red color. In the intestinal phase, the integrity of the alginate beads was maintained in the first 20 min and, after this period, the beads completely disintegrated due to the pH 7.4. The

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analysis of the monomeric anthocyanins content from the simulated gastric fluid suggests the existence of such pigments in the intestinal fluid, mainly for the hydrogel beads with extract

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only and those coated with chitosan, both presenting positive values for the parameter a*

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(0.58 ± 0.05 and 0.27 ± 0.05 in intestinal phase for beads with extract only and coated with

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chitosan, respectively) (CIELab scale), indicative of red color.

According to CIElab (L* and a*) parameters, after 120 min, the samples from gastric

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and intestinal fluids presented a large reduction in intensity of the red color, mainly for the particles coated with WPC and gelatin, showing negative values for the parameter a* (7.95 ±

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0.23 in gastric phase to -0.59 ± 0.04 in intestinal phase and 9.54 ± 0.38 in gastric phase to -0.71

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± 0.06 in intestinal phase for beads coated with WPC and gelatin, respectively). The intestinal fluid revealed a tendency to white color (higher L*) when compared to the gastric fluid (2.49 ± 0.20 in gastric phase to 14.59 ± 0.30 in intestinal phase, 2.38 ± 0.19 in gastric phase to 15.04 ±

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0.61 in intestinal phase, 3.26 ± 0.23 in gastric phase to 14.04 ± 0.49 in intestinal phase, 5.41 ±

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0.45 in gastric phase to 14.03 ± 0.57 in intestinal phase, for beads with extract, coated with chitosan, WPC and gelatin, respectively). This behavior may have been caused by the pH conditions. The stability of the anthocyanins is highly dependent on the pH, being unstable in neutral conditions [47]. Alginate beads, when placed in a phosphate buffer (pH 7.4), pass through a process of exchange of Ca2+ ions, that are linked to -COO- groups of alginate, characterized mainly by polymannuronate sequences, with the Na+ ions present in the saline solution. As a result, the electrostatic repulsion between the negative charges of the -COO- groups increases, causing

ACCEPTED MANUSCRIPT chain relaxation and increased swelling of gel. The exchange between the Na+ and Ca2+ ions alters the structure of the gel beads and allows for interaction of the phosphate buffer ions with calcium, thus forming calcium phosphate and making the medium cloudy. In the late stages of the swelling process, the Ca2+ ions that are linked to the –COO- groups of the polyguluronate units, forming the “egg box” structure, also begin to be exchanged for the Na+

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ions of the phosphate buffer. Since the polyguluronate sequences have strong autocooperativity binding to calcium ions, promoting stable cross-links within the gel structure, the

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gel beads begin to disintegrate when the calcium ions in the “egg box” structure decay and

CONCLUSION

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4

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diffuse into the medium. In this way, the alginate beads lose weight and dissolve [48].

The results obtained by the alginate hydrogels containing monomeric anthocyanins

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were very promising, indicating the potential use of these particles as carriers of compounds with functional properties and capacity of application in the food industry. The production

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process of alginate beads with a porous structure allowed the adsorption and, thus, the

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encapsulation of the anthocyanins from jussara extract. Coating process by electrostatic interaction of polymers (chitosan, WPC or gelatin) on the surface of the particles brought structural alterations to the samples that provided greater protection of the bioactive

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compounds during the storage stability and release study.

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Anthocyanins were diffused into the hydrophilic medium during coating process and this loss was influenced by the viscosity and zeta potential of the agents used. Chitosan was more cationic, had a greater viscous dispersion compared to WPC and gelatin and helped retention of more pigment in the alginate structure. Beads covered with this material showed higher antioxidant capacity than WPC and gelatin dispersions, proving to be a good option to keep antioxidant capacity of hydrogel beads containing anthocyanins. The coating process using proteins and chitosan was effective in protecting monomeric anthocyanins, as observed during refrigerated storage and release profiles in the

ACCEPTED MANUSCRIPT simulated gastrointestinal fluid. Alginate beads containing extract only showed a decrease in the anthocyanins content when compared to the chitosan-, WPC- and gelatin-added samples during the analysis of the stability period. In the release study, samples with alginate and extract only presented higher anthocyanins release in the simulated gastric fluid. In the intestinal phase, the integrity of the beads was maintained for approximately 20 min for all

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samples, with later release of the remainder of the anthocyanins and disintegration, due to the basic medium.

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ACKNOWLEDGEMENTS

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We acknowledge the financial support of CNPq (156674/2015-7, 449506/2014-2,

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304475/2013-0 and 140280/2013-8), EMU (2009/54137-1) and FAPESP (2011/06083-0, 2011/51707-1, 2009/50593-2 and 2004/08517-3) and the access to the confocal microscopy

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equipment of the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC - UNICAMP) co-funded by FAPESP (08/57906-3) and CNPq (573913/2008-0).

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CONFLICT OF INTEREST

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The authors declare no conflict of interest. REFERENCES

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1: A: Particle size distribution of alginate hydrogel. B: Evaluation of gel strength of the alginate beads. Figure 2: External microphotographs and optical and confocal microscopy of alginate hydrogel beads. External microphotographs of alginate hydrogel bead: A1 with 200x of magnification; A2 with

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1.500x of magnification and A3 with 3.000x of magnification. A4 and A5: optical and confocal microscopy, respectively.

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External microphotographs of alginate hydrogel bead containing extract: B1 with 200x of magnification; B2 with 1.500x of magnification and B3 with 3.000x of magnification. B4 and B5:

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optical and confocal microscopy, respectively.

External microphotographs of alginate hydrogel bead containing extract and chitosan: C1 with

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200x of magnification; C2 with 1.500x of magnification and C3 with 3.000x of magnification. C4 and C5: optical and confocal microscopy, respectively.

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External microphotographs of alginate hydrogel bead containing extract and WPC: D1 with 200x of magnification; D2 with 1.500x of magnification and D3 with 3.000x of magnification. D4 and D5: optical and confocal microscopy, respectively.

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External microphotographs of alginate hydrogel bead containing extract and gelatin: E1 with

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200x of magnification; E2 with 1.500x of magnification and E3 with 3.000x of magnification. E4 and E5: optical and confocal microscopy, respectively. Figure 3: A: Behavior of samples stored under refrigeration conditions: anthocyanins loss

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conditions.

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during four weeks. B: Release profile of anthocyanins from alginate beads in simulated gastric

ACCEPTED MANUSCRIPT Table 1: Characterization of sodium alginate, chitosan, WPC and gelatin in relation

Raw material

Zeta potential (mV)*

pH

Apparent viscosity**

Sodium alginate

-90.6 ± 2.1

6,5

1.083 ± 0.010

Chitosan

+57.1 ± 2.0a

3,5

0.013 ± 0.000a

WPC

+19.9 ± 0.7c

3,5

0.001 ± 0.000c

Gelatin

+22.3 ± 0.6b

3,5

0.003 ± 0.000b

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to zeta potential and apparent viscosity.

*For the analysis of the zeta potential 0.2% (0.2 g/100 g) of sodium alginate, chitosan,

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WPC and gelatin were dispersed in Milli-Q water. **Concentration of materials for the analysis of the rheological behavior: 2% (2 g/100 g) of sodium alginate and 1% (1 g/100 g) of coating polymers (chitosan, WPC and

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gelatin). Apparent viscosity in Pa.s considering the shear rate of 100 s -1.

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Different small letters in the same column indicate a significant difference (p≤0.05).

ACCEPTED MANUSCRIPT Table 2: Alginate hydrogel beads characterization in relation to the total solids content, average size of bead (D[4,3]), monomeric anthocyanin content and antioxidant capacity. Total solids content (%)

D[4,3] (mm)

Monomeric anthocyanin content*

Alginate

5.6 ± 0.1d

1.19 ± 0.02a

-

-

lginate + extract

7.7 ± 0.2a

1.13 ± 0.01a

10.3 ± 0.5a

1606.0 ± 67.6a

te + extract + chitosan

7.0 ± 0.1b

1.18 ± 0.05a

8.4 ± 0.2b

844.6 ± 53.0b

ate + extract + WPC

6.7 ± 0.3c

1.15 ± 0.01a

6.6 ± 0.3c

614.5 ± 43.4c

ate + extract + gelatin

6.8 ± 0.2b,c

1.12 ± 0.01a

5.2 ± 0.4d

265.6 ± 5.2d

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Hydrogel beads

Different small letters in the same column indicate a significant difference (p≤0.05).

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* mg of cyanidin 3-glucoside equivalent/g of dry bead.

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** μmol of Trolox equivalent/g of dry bead.

Antioxidant capacity (O

ACCEPTED MANUSCRIPT Graphical abstract

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The manuscript aimed to produce alginate hydrogel beads containing anthocyanins from jussara (Euterpe Edulis Martius) extract by ionic gelation. Apart that the hydrogel beads were subjected to complexation by electrostatic interaction using chitosan and proteins.

ACCEPTED MANUSCRIPT HIGHLIGHTS Beads with jussara extract showed high anthocyanin content and antioxidant capacity. Chitosan, WPC and gelatin were used as coating polymers of the alginate beads. Coating polymers were effective in protecting pigments during storage stability.

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RI

PT

In the intestinal phase, the integrity of the beads has been maintained up to 20 min.

Figure 1

Figure 2

Figure 3