soy protein system for essential oil encapsulation with intestinal delivery

Carbohydrate Polymers 200 (2018) 15–24

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Alginate/soy protein system for essential oil encapsulation with intestinal delivery

T

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Mina Volića, Ivana Pajić-Lijakovića, Verica Djordjevića, , Zorica Knežević-Jugovića, Ilinka Pećinarb, Zora Stevanović-Dajićb, Đorđe Veljovića, Miroslav Hadnadjevc, Branko Bugarskia a

University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia c University of Novi Sad, Institute of Food Technology, Bul. cara Lazara 1, 21000 Novi Sad, Serbia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Alginate Soy protein isolate Beads Thyme oil Controlled release

Preparation of alginate-soy protein isolate (AL/SPI) complex beads containing essential oil of thyme was carried out by emulsification of thyme oil in aqueous sodium alginate solution blended with SPI solution, followed by atomization via electrostatic extrusion and gelification with calcium ions. The process parameters were optimized by variation of the alginate (1–2.5 wt.%) and SPI (0–1.5 wt.%) concentrations. Dry alginate-SPI particles exhibited wrinkle surface while shape distortion of hydrogel beads occurred with ≥1.5 wt.% alginate concentration, whereas SPI induced reduction of the particle size. Encapsulation efficiency of 72–80 % based on total polyphenols was achieved. In SGF the samples exhibited oil release of 42–55 % (due to matrix shrinkage and proteins degradation by pepsin activity), while the rest was delivered in SIF within 2.5 h simultaneously with swelling and degradation of the matrix.

1. Introduction Essential volatile oils are herbal drugs that have received substantial application in several areas including food technology, biomedicine, pharmaceuticals, and agriculture due to their significant biological usefulness. Essential oils have diverse and relevant biological activities (Bakkali, Averbeck, Averbeck, & Idaomar, 2008); even more, the studies demonstrated a synergetic effect of two or more essential oils mixed together (Gutierrez, Barry-Ryan, & Bourke, 2009). For example, the essential oil of Thyme (Thymus vulgaris) is known for its expectorant, antitussive, antibroncholytic, antispasmodic, antihelminthic, carminative, muscle relaxant and diuretic properties (Al-Asmari, Athar, Al-Faraidy, & Almuhaiza, 2017; Boskabady, Aslani, & Kiani, 2006; Boskovic et al., 2015; Sienkiewicz, Łysakowska, Denys, & Kowalczyk, 2012). In general, essential oils are complex blends of a variety of odorous compounds which are chemically unstable and susceptible to oxidative deterioration and loss of volatile compounds, so that composition of essential oils may be changed when they are exposed to light, moisture, oxygen and high temperature (Bakry et al., 2016). Therefore, essential oils are typically encapsulated in order to prevent their oxidation and volatilization, increase shelf-life, and enable their controlled release.

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Among many biopolymers used so far for the purpose of encapsulation, alginate is unique among polysaccharides regarding chemical stability, pH sensitivity, capacity to form strong gel barriers to water and gases, and biological functionality in appetite regulation (Ching, Bansal, & Bhandari, 2017; Jensen, Kristensen, & Astrup, 2012). Alginate is a linear 1,4-linked copolymer of α-L guluronic and β-D mannuronic acids and it forms gels by cross-linking with multivalent cations. Sodium alginate is insoluble at pH < 4, while it’s aqueous solution is highly viscous and stable between pH 6–9 (Zhong, Huang, Yang, & Cheng, 2010). Therefore, alginate-based microspheres crosslinked with Ca2+ exhibited controlled-release function due to the sensitivity of Ca2+/COO2 linkage to pH and other ions (Zheng, Zhou, Chen, Huang, & Xiong, 2007). However, alginate has poor emulsifying capacity (Chan, 2011); thus, the miscible blends of alginate and other biopolymers have been used to adjust drug release behavior and enhance the functions of alginate-based microspheres. These functions were related to the properties of individual components, to the composition in whole blend and interaction between the components (BelščakCvitanović et al., 2015; Zheng et al., 2007). Soybean proteins have been considered for fortification of alginate carriers for delivering drugs (Wongkanya et al., 2017; Zheng et al., 2007), plant extracts (Belščak-Cvitanović et al., 2015) and probiotics

Corresponding author. E-mail address: [email protected] (V. Djordjević).

https://doi.org/10.1016/j.carbpol.2018.07.033 Received 24 January 2018; Received in revised form 11 July 2018; Accepted 11 July 2018 Available online 20 July 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.

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(Liu et al., 2018) and for designing films and fibers aimed for sausage casting industry (Harper, Barbut, Lim, & Marcone, 2013) and biomedical applications (Tansaz, Durmann, Detsch, & Boccaccini, 2017; Wongkanya et al., 2017) due to high availability and biodegradability, non-cytotoxicity and high thermal stability. Other important attributes are water holding, oil binding, and emulsifying properties. In addition, some studies indicated that blending with polysaccharides may increase water holding capacity of soy proteins (Hua, Cui, & Wang, 2003). However, the application of soy proteins in microencapsulation is still restricted, due to their limited solubility in water, and high viscosity of the corresponding dispersions at high solid content. Therefore, it is a challenge to find optimal alginate/SPI ratio able to be processed into round and uniform particles. Previous reports have shown obvious differences in drug release from alginate/SPI gel beads under given pH of stomach, intestine, and colon (Belščak-Cvitanović et al., 2015; Zheng et al., 2007) but it is still unknown how alginate/SPI blend would release a drug under conditions really mimicking GIT regarding not only pH values, but enzymes and physiological temperature; especially when having in mind that proteins are susceptible to hydrolysis by activity of gastric pepsins and pancreatic enzymes, but when entrapped within alginate matrix they are less degraded than free proteins (Koutina, Ray, Lametsch, & Ipsen, 2018; Zhang, Zhang, & McClements, 2017). An encouraging result has been published recently about superior gastric resistance property of alginate/SPI matrix (compared to a single component wall material, e.g. alginate, or SPI) based on survival of probiotic bacteria encapsulated within (Liu et al., 2018). The objective of this work is to offer a complex microsphere based on the mixture of alginate and soy protein isolate (SPI) as a carrier for essential oils; such mixture could combine the pH sensitivity of alginate with the bioactivity and emulsifying properties of SPI and might present a new system with intestinal delivery of essential oils. Commercial thyme oil was used as an oil model and alginate-SPI particles with encapsulated thyme oil were fabricated by emulsification of thyme oil in aqueous sodium alginate-SPI blend solution, followed by atomization via electrostatic extrusion and then Ca2+ crosslinking. Based on hypothesis that alginate/SPI matrix provides gastric resistance to encapsulated compounds, release kinetics in entirely simulated human gastrointestinal tract (pH and temperature conditions and enzymes of stomach and intestine) was examined and interrelated with swelling kinetics and changes in viscoelastic properties. While doing it, of importance was to reveal the contribution of each of the two constituents (i.e. alginate and protein), so blends of variable alginate (1–2.5 wt.%) and SPI (0–1.5 wt.%) concentrations were compared and the interaction between components was investigated. In addition, the effects of composition on bead size, shape, morphology and encapsulation efficiency were evaluated.

Fisher Scientific, UK. Sodium chloride was purchased from Fargon, US. All reagents were used as received. 2.2. Emulsion preparation The polymer-thyme oil emulsion was prepared as following. Soy protein isolate was hydrated in 30 mL of miliQ water at room temperature; thus obtained dispersion was adjusted to pH 8.0 with NaOH (1 mol/L) and heated at 80 °C for 40 min to denature protein. Separately, sodium alginate solution was prepared by dispersing the adequate sample in the same amount of miliQ water. This solution was kept for at least 3 h with constant stirring at room temperature, until complete dissolution of alginate. After mixing of the two prepared solutions, thyme oil was added to the ultimate alginate/soy protein solution at weight ratio 1:5 and then emulsified using Ultra-Turrax homogenizer (UltraTurrax IKA T25 digital) at 12,000 rpm for 3 min. According to the alginate and SPI contents in the solid, the beads were coded as A1/SP1 (1 wt.% of sodium alginate and 1 wt.% of SPI), A1/ SP1.5 (1 wt.% of sodium alginate and 1.5 wt.% of SPI), A1.5/SP1 (1.5 wt.% of sodium alginate and 1 wt.% of SPI), A1.5/SP1.5 (1.5 wt.% of sodium alginate and 1.5 wt.% of SPI), A2/SP1 (2 wt.% of sodium alginate and 1 wt.% of SPI), A2/SP1.5 (2 wt.% of sodium alginate and 1.5 wt.% of SPI), A2.5/SP1 (2.5 wt.% of sodium alginate and 1 wt.% of SPI) and A2.5/SP1.5 (2.5 wt.% of sodium alginate and 1.5 wt.% of SPI). 2.3. Beads preparation Ten milliliters of emulsion were extruded through a blunt, 18-gauge stainless steel needle and trickled into 20 mL of a gelling bath solution containing CaCl2·2H2O (10 g/L), with gently stirring at magnetic stirrer place. A constant flow rate of 39.3 mL/h was provided using the syringe pump (Razel, Scientific Instruments, Stamford, USA). Needle tip was positively charged and fixed at nearly 6 cm above the grounded gelling liquid. To control the potential difference between them, a high voltage unit (Model 30R, Bertan Associates, Inc., New York) of 5.0 kV, was applied. After Na+ - Ca2+ ions exchange, alginate-soy protein droplets formed insoluble hydrogel matrix with thyme essential oil entrapped in. The beads were left in the CaCl2 solution for 15 min to harden, and used for further analysis. Dry beads were obtained by air-drying of hydrogel beads at room temperature until reaching constant mass. 2.4. Characterization of liquid systems 2.4.1. Viscosity The steady shear measurements of sodium alginate/SPI (Na-A/SPI) solutions were performed using Haake Mars rheometar (Thermo Scientific Karlsruhe, Germany) equipped with parallel plate geometry (PP35S, 35 mm diameter). Flow curves were recorded at 25 °C by registering shear stress values in the range of shear rate 0–50 1/s. Shear rate was increased linearly for 2 min and afterwards shear rate was decreased linearly for 2 min. Measurements were performed in triplicates.

2. Materials and methods 2.1. Materials Sodium Alginate (Alginic Acid Sodium Salt BioChemica, Mw from 10,000 g/mol to 600,000 g/mol) with the mole fraction of mannuronic acid (FM), guluronic acid (FG), and their ratio (M/G) of 0.58, 0.42 and 1.38, respectively, was purchased from PanReac AppliChem, Germany. Commercial soy protein powder was purchased from Brenntag, Ireland. Thyme oil was kindly supplied from local pharmaceutical shop Prima Cosmetics doo. Calcium chloride dihydrate (CaCl2•2H2O) was purchased from Analytika, Ltd., Czech Republic. Pepsin (from porcine gastric mucosa) was obtained from Sigma-Aldrich, Germany. Pancreatin 4X USP grade (from porcine pancreas) was purchased from MP Biomedicals, LLC, France. Bile salts were obtained from Biolife, Italia. Sodium hydrogen carbonate was purchased from Alkaloid AD, Macedonia. Sodium hydroxide was supplied from Alfapanon, Serbia. Methanol was purchased from J.T.Baker, Netherlands. Folin-Ciocalteu’s phenol reagent was purchased from Merck KGaA, Germany. Sodium carbonate anhydrous and sodium citrate dihydrate were obtained from

2.4.2. Raman spectroscopy Raman spectroscopy was performed by using XploRA Raman spectrometer from Horiba Jobin Yvon on AlgProtOil samples. Raman scattering was excited by a frequency-doubled Nd/YAG laser at a wavelength of 532 nm (maximum output power of 20–25 mW) equipped with a 1200 lines/mm grating, spectra were recorded by applying exposure time 5 s and scanning the sample 10 times, using 100% filter. Spectral resolution was 4 cm−1 and calibration was checked by 520.47 cm−1 line of silicon. Spectra were recorded using the 50x objective lens. The spectral range was in the intervals from 200 and 3250 cm−1, where the region from 1800 to 2770 cm−1 was excluded. Spectra acquisitions were managed by the LabSpec software (Horiba Jobin Yvon), and analyzed by Origine Pro 8.6 software (OriginLab, 16

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Northampton, MA, USA). In order to take possible sample inhomogeneity into account, for each sample at least ten Raman spectra were recorded, and then the average spectrum was used for a representative spectrum for each sample.

Weight change (%) =

(Final weight − Initial weight) × 100 Initial weight

(3)

2.8. Folin-Ciocalteu method 2.5. Characterization of beads

Total phenol content was determined spectrophotometrically (UV1800 SHIMADZU spectrophotometer, USA MFG INC), using FC reagent, according to a method of Malićanin et al. (2014) with slight modifications. Concisely, the reaction mixture contained 100 μL of sample, 900 μL of methanol-in-water solution (10:90), and 250 μL of the FolinCiocalteu reagent (1:2) freshly prepared and 750 μL of 5% sodium carbonate. The sample was centrifuged for 1 min at 4000 rotations per minute (IKA centrifuge, USA). After 2 h of reaction at room temperature, the absorbance at 765 nm was measured and used to calculate the released oil content, using previously prepared standard curve. Measurements were performed in triplicate.

2.5.1. Optical microscopy The images of the beads after gelation were captured for size and shape analysis. The shape of the alginate beads were quantified by using the dimensionless shape indicators Aspect ratio (AR) and Sphericity factor (SF) described by the following equations, respectively (Chan, Boon-Beng, Ravindra, & Poncelet, 2009):

AR=dmax/dmin

(1)

SF=(dmax−dmin )/(dmax + dmin)

(2)

where dmax denotes maximum diameter of the beads while dmin refers to minimum diameter. The measurements were performed by using digital light microscope (Motic BA210 Series) with an image analyzer (Motic Images Plus 2.0 ML).

2.9. Rheological measurements of beads Carriers were characterized by small strain dynamic oscillatory tests using Haake Mars rheometar (Thermo Scientific Karlsruhe, Germany) equipped with parallel plate geometry (PP35S, 35 mm diameter) which was serrated in order to avoid slippage. Mechanical spectra (frequency sweeps) were recorded over the range 0.1–10 Hz at 5 Pa stress (which was within the linear viscoelastic region) at 37 °C. The monolayer of beads was placed at the measuring plate. Measurements were performed in triplicates.

2.5.2. Scanning electron microscopy (SEM) The surface morphology of the air-dried beads was analyzed by SEM. Prior the analysis, beads were sputter-coated (Polaron SC502 Sputter Coater) with gold/platinum alloy (15/85). After a precipitation of a thin gold/palladium layer, beads were examined using a TESCAN MIRA 3 XMU (Cranberry Township, USA) field-emission scanning electron microscope (FE-SEM), at an accelerating voltage of 20 kV.

3. Results and discussion

2.6. Release studies in simulated gastrointestinal tract (GIT) model

3.1. Viscosity

In order to simulate human GI tract, the release of thyme oil from alginate-soy protein beads with encapsulated thyme oil was studied for 1 h at 37 °C in gastric solutions, and for 4 h at 37 °C in pancreatic solution or until complete degradation of the beads. Gastric and pancreatic solutions were freshly prepared prior the analysis, following the procedure of Wu et al. (2017). Firstly, 4387.5 mg of NaCl and 157.5 mg of NaHCO3 were dissolved in 500 mL flask of water, thus preparing gastric juice. Thus obtained mixture was adjusted to pH 1.6 by adding 5.0 M HCl. Ultimately 92.4 mg of pepsin was added to the solution, thus preventing enzyme deactivation from strong acid. Pancreatic juice was prepared similarly, dissolving 2250 mg of NaHCO3 and 851.4 mg of bile salts in 500 mL of water. Thus obtained mixture was adjusted to pH 6.2, and then, 283.8 mg of pancreatin was added. Wet alginate-soy protein beads with encapsulated thyme oil (3.0 g) were kept in the flask containing 50 mL of gastric solution for 1 h under mild magnetic stirring, thus simulating gut movement. Repeatedly, aliquots of a sample were taken at predetermined time intervals. After 1 h the beads were filtered and immediately transferred in the 50 mL flask containing pancreatic solution and sampling was continued for next 4 h. The total phenol content of aliquots was determined using the method described below.

The complex microspheres based on alginate and soy protein isolate were prepared by mixing sodium alginate solution with soy protein isolate solution; thus obtained Na-A/SP blend solution was subjected to extrusion process under the electrostatic field to produce droplets which were exposed to crosslinking process with Ca2+ in the collecting bath. The final size of the calcium alginate /SPI beads is influenced by the viscosity of corresponding Na-A/SP blend solutions. The viscosity of liquid systems Na-A/SP vs. shear rate is shown on Fig. 1. The liquid blend Na-A/SP systems obtained from 1 and 1.5 wt.% alginate showed nearly Newtonian behavior, while further increase in

2.7. Swelling studies Swelling studies were carried out using wet beads (immediately after preparation). Hydrogel beads of pure calcium–alginate and alginate-SPI of different polymer concentration were kept immersed in simulated gastric and pancreatic fluids, the same as in release studies. At fixed time intervals beads were separated from the medium using stainless steel colander and the mass of beads was measured. The weight change of the beads was calculated using the following empirical relationship:

Fig. 1. Viscosity vs. shear rate for liquid Na-A/SP systems. 17

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Raman spectra. As regarding the spectrum of alginate-soy proteinthyme oil sample, the region from 1000–1666 cm−1 corresponds to the proteins (Fig. 2a). The typical Raman signature of protein in the alginate-soy protein-thyme oil sample (Fig. 2a) shows C]O stretching vibration and NeH wagging in the peptide bonds from Amide I at 1666 cm−1, CH2 scissoring at 1445 cm−1 and Amide III at 1245 cm−1, the bands originated from CeN stretching vibration and NeH band (Alleavitch et al., 1990; Lin-Vien, Colthup, Fateley, & Grasselli, 1991; Zhu, Zhu, Fan, & Wan, 2011). A sharp and low intensity aromatic ring breathing peak around 1003 cm−1 and low intensity one at 1608 cm−1 (Fig. 2a,c) arises probably from phenylalanine amino acid residues in the sample (Lee et al., 2013; Zhu et al., 2011), as soybean crude protein content. The main components of thyme essential oil are phenols such as: thymol, carvacrol, p-cymene (with benzene ring in their structure) and γ-terpinene, having ring of six carbons with two C]C (Tawaha & Hudaib, 2012). The Raman spectra of all mentioned components were reported by Schulz, Quilitzsch, & Krüger, 2003; Schulz, Ozkan, Baranska, Krüger, & Ozcan, 2005. The Raman spectrum of thyme essential oil (Fig. 2d) shows the most important bands at 1619, 1450, 1378, 1087, 803 and 736 cm−1. The band at 1619 cm−1 is assigned to the ring quadrant stretching mode of p-cymene. The band specific of thyme oil at 1450 cm−1 is assigned to CH3/CH2 bending modes. While, the band at 1378 cm−1 is assigned to a CH3 bending mode from attached to an aromatic ring. The band in the range 1600–1000 cm−1 (e.g. at 1087 cm−1) is attributed to CeH bending vibrations from benzenes ring (Larkin, 2011). The very weak band at 952 cm−1 is assigned to ring deformations of p-cymene, while signals at 736 cm−1 and 803 cm−1 (out-of-plane CH wagging vibrations) have higher intensity, and they are attributed to thymol (Schulz & Baranska, 2007; Schulz et al., 2003, 2005; Vargas Jentzsch, Ramos, & Ciobotă, 2015). As regarding the spectrum of the emulsion of thyme oil in alginateSPI water solution, some peaks typical for thyme compounds also appeared there (such as at 1087, 952, 803 cm−1), while other signals (e.g. band at 736 cm−1) were of low intensity or were not recognized. On the hand, some peaks characteristic for pure alginate (2940, 1160 and 846 cm−1) were masked while others characteristic for protein attenuated (2932 cm−1) or entirely diminished (1445 cm−1) in the spectrum of alginate-SPI-thyme oil suggesting interactions between the three constituents and aggregation of protein molecules (involving the disruption of hydrogen bonds). Our assumption is that thyme oil compounds were more likely surrounded by and interacted with protein molecules, as SPI is more hydrophobic than alginate. According to Rowley, Madlambayan, and Mooney, (1999), covalent (amide) bond can be formed between the terminal amide in a peptide molecule and the carboxyl group of alginate. Others reported that essential oils such as carvacol induce protein agglomeration (Arancibia, López-Caballero, Gómez-Guillén, & Montero, 2014; Arfa, Chrakabandhu, Preziosi-Belloy, Chalier, & Gontard, 2007). Based on FTIR analysis, Zheng et al. (2007) suggested hydrogen bonding between alginate and SPI in the complex microspheres, while Wang et al. (2006) claimed about good compatibility between the alginate and soy protein isolate molecules.

alginate concentration led to shear tinning behavior. As expected, increase in Na-A concentration from 1 wt.% to 2.5 wt.% at the same amount of SPI led to a significant increase in viscosity. The increase in SPI content induced increase in viscosity, but only in case of 2.5 wt.% alginate blends. 3.2. Raman spectroscopy Raman vibrational spectroscopic analysis was performed on alginate-soy protein-thyme oil emulsion (before crosslinking) and compared to as received sodium alginate, SPI and thyme oil to identify characteristic Raman bands corresponding to the chemical structures (Fig. 2). The presence of β-D mannuronic (M) and α-L guluronic (G) acids of alginate can be identified through characteristic Raman bands in the vibrational spectra (Fig. 2a,b). CeH stretching vibrations appear as strong and wide peaks in Raman spectra, located at around 2930 cm−1. In the spectral region from 1270–1500 cm−1 three bands (1345, 1421 and 1458 cm−1) are assigned to the CeH, CeOeH deformation vibration. While bands around 1125 and 1160 cm−1 are assigned to the CeO and CeC and CeOeC stretching vibration, characteristic of G units (Driskell et al., 2005; Sartori, Finch, Ralph, & Gilding, 1997), because each band may be due to contributions of two or more types of motions (Cardenas-Jiron, Leal, Matsuhiro, & OsorioRoman, 2011; Synytsya, Copikova, Matejka, & Machovic, 2003). The region between 750 and 950 cm−1, so called the ‘finger print’ region of alginate samples, includes a band at 846 cm−1 assigned to skeletal stretching of CeC and deformation modes (Frisch et al., 2003), while bands below 700 cm−1 are related to the deformation of pyranosyl rings and CeOeC vibration of glycosidic linkage in alginate (CamposVallette et al., 2009; Cardenas-Jiron et al., 2011; Ivleva, Wagner, Horn, Niessner, & Haisch, 2009; Synytsya et al., 2003). According to some studies (Cardenas-Jiron et al., 2011; Pielesz, 2007; Sartori et al., 1997) characteristic bands of guluronic and mannuronic units in Raman spectra are in the region from approximately 1000 to 1100 cm−1 (Pereira, Sousa, Coelho, Amado, & Ribeiro-Claro, 2003; Pielesz, 2007; Sartori et al., 1997), while others such as Cardenas-Jiron et al. (2011) report about these bands in the ‘finger print’ region, from 800 to 960 cm−1. Raman spectrum of SPI is also presented in Fig. 2c. Since proteinaceous materials produce a high fluorescence continuum (Vandenabeele et al., 2000), that region has been excluded in presented

3.3. Particle size and shape Although all samples were produced by electrostatic extrusion device under the same process conditions, they dramatically differ in size and shape (Table 1). The size of hydrogel beads ranged between ∼1.3 mm and ∼2.4 mm (mean diameter), while dried particles reduced in size compared to their hydrogel counterparts (mean diameter 0.6–1.4 mm). The shape was estimated from Aspect ratio (AR) and Sphericity factor (SF), where AR varies from unity for a perfect sphere

Fig. 2. Raman spectra of emulsion of thyme oil in alginate-SPI bland (a) and its components (alginate (b), SPI powder (c) and thyme oil (d)) in spectral range from 200 to 3250 cm−1.

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Table 1 Dimensions (Maximum diameter and Minimum diameter), shape indicators (Aspect ratio and Sphericity factor) and encapsulation efficiency of alginate-SPI complex beads with encapsulated thyme oil. System code

Wet beads dmin(μm) ± s.d.

A1.5/SP0 A1/SP1 A1/SP1.5 A1.5/SP1 A1.5/SP1.5 A2/SP1 A2/SP1.5 A2.5/SP1 A2.5/SP1.5

1627 1312 1256 1631 1419 n.d. 1250 n.d. 1130

± ± ± ± ±

Dry beads dmax(μm) ± s.d.

55a 32bc 51cd 51a 39b

± 75cd ± 70d

1629 1330 1295 1752 1576 n.d. 1570 n.d. 2016

± ± ± ± ±

56b 45c 42c 23b 13b

± 87b ± 134a

dmin(μm) ± s.d. 577 720 780 829 730 923 570 620 550

± ± ± ± ± ± ± ± ±

34d 45bc 13b 23ab 65b 24a 40d 38cd 43d

AR = dmax/dmin

SF = (dmax-dmin)/(dmax + dmin)

dmax(μm) ± s.d.

ARw

ARd

SFw

SFd

623 ± 44e 838 ± 54d 896 ± 55d 1000 ± 67d 920 ± 64d 1746 ± 102b 750 ± 44e 1880 ± 113a 1450 ± 112c

1.001 1.014 1.031 1.074 1.111 n.d. 1.256 n.d. 1.784

1.080 1.164 1.149 1.206 1.260 1.892 1.316 3.032 2.636

0.001 0.007 0.015 0.036 0.052 n.d. 0.113 n.d. 0.282

0.038 0.076 0.069 0.093 0.115 0.308 0.136 0.504 0.450

EE (%) ± s.d.

72 ± 1.5 76 ± 2 80 ± 2 80 ± 1.1 79 ± 1.3 77 ± 1.5 76 ± 2 75.5 ± 1.6 77 ± 2.6

*Values with the same letter in each column showed no statistically significant difference (p < 0.05; Tukey's test). dmax- maximum diameter, dmin – minimum diameter, ARw – Aspect ratio of wet beads, ARd – Aspect ratio of dry beads, SFw – sphericity factor of wet beads, SFd – sphericity factor of dry beads, EE (%) – encapsulation efficiency, s.d. – standard deviations, n.d.-not determined.

3.4. Encapsulation efficiency

to approaching infinity for an elongated particle, while SF is the deformation factor and varies from 0 for a perfect sphere to approaching unity for an elongated object. According to Chan et al. (2009), the aspect ratio gives good description of large deformations but small distortion are suppressed, while the sphericity factor can describe the relative change of the shape more efficiently. As can be seen, complex hydrogel (wet) beads obtained with alginate concentration up to 1.5 wt.% can be considered as spherical (SF ≤ 0.05), further increase in alginate concentration led to increased deformation of the particles, while mixtures with alginate concentration above 2.5 wt.% were impossible to be processed by using our apparatus. This result is a consequence of an increase in viscosity of Na-A/ SPI blend solution and promoted shear-thinning behavior with an increase in alginate concentration above 2 wt.% as described previously (Section 3.1). According to literature, extrusion of high viscosity polymer solutions (i.e., high concentration polymer solutions) produces deformed particles having elongated shape of eggs or drops (Levic et al., 2015; Prüsse et al., 2008). In general, higher polymer concentrations result in more intensive inter- and intra-chain interactions which could reduce their mobility and cause creation of elongated droplet forms during processing. There is no systematic dependence of shape on SPI concentration, which is in accordance with viscosity measurements for 1.5 and 2 wt.% alginate formulations. Here it should be stressed that deformation and orientation of protein clusters and conformational changes of alginate chains induced by electrostatic and gravitational force, depend on several features, i.e. on the nature of inter- and intrachain interactions, local charges per volume of droplets, polymer concentration and their cause-consequence relations. Regarding the impact of SPI on size, there was statistically significant reduction of particles size with an increase in amount of SPI at the same concentration of alginate. Tansaz, Durmann, Detsch, & Boccaccini (2017) also showed that SPI inclusion induced reduction of alginate-SPI hydrogel microcapsules, but those were fabricated by pneumatic extrusion technique. A possible explanation is that denatured and unfolded SPI protein clusters are also able to generate crosslinkages with Ca2+ ions (Abaee, Mohammadian, & Jafari, 2017), making matrix to be as more tight as more proteins are there. After dehydration, a reduction in bead roundness was observed and the process caused the beads to shrink.

In general, high gel porosity has been ascribed to alginate beads which cause leakage of the loaded drugs and drug diffusion from the gel network to the aqueous medium. To reduce oil loss during preparation of the alginate particles, other polymers (such as chitosan) have been frequently used in combination with sodium alginate. In our study, encapsulation efficiency of total polyphenols varied in a narrow range, 75–80 %, depending on the content of SPI and alginate, while plain alginate provided EE of 72%. Systematic dependence of encapsulation efficiency was not observed either on alginate or SPI concentration. Soliman, El-Moghazy, Mohy, El-Din, and Massoud, (2013) have reported the increase in encapsulation efficiency of three different types of essential oils (cinnamon oil, clove oil and thyme oil) from about 70–80 % for 1 wt.% alginate to about 90% for 2 wt.% alginate; this result was explained by that increasing of alginate concentration led to forming a dense network structure with cohesive vacancies (pores) that entrap the essential oils droplets. In our case this effect of alginate concentration on encapsulation efficiency failed probably because protein molecules partially occupied the free volume within the polymer matrix available for oil. The values reported here are close to other literature data on encapsulation of essential oils in alginate-based systems. For a sake of comparison, Lertsutthiwong, Noomun, Jongaroonngamsang, Rojsitthisak, and Nimmannit, (2009) have reported the percent recovery of turmeric oil in alginate nanocapsules of 52%, whereas the recoveries ranged from 61.9 to 68.5 % with inclusion of chitosan. Natrajan, Srinivasan, Sundar, and &Ravindran, (2015) have reported encapsulation efficiency of 71.1% of the total turmeric oil and 86.9% of the lemongrass oil for alginate-chitosan nanocapsules. 3.5. SEM analysis Fig. 3 shows the SEM images of air-dried alginate-SPI complex beads. From the figures with low magnification (images A1, B1, C1, D1), it is obvious that the samples with 1 wt.% alginate are round in comparison to pronounced elongated shape structures of the beads obtained with 2.5 wt.% alginate, and the elongation is more pronounced in case of A2.5/SP1 than A2.5/SP1.5, which is in consistence with shape indicators of these dry beads (Table 1). According to the surface observations (images A2, B2, C2, D2), all samples exhibited

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Fig. 3. SEM images of alginate-SPI beads with encapsulated thyme oil (A1, B1, C1, D1) and their surfaces (A2, B2, C2, D2) for the samples A1/SP1 (A1, A2), A1.5/SP1 (B1, B2), A2.5/SP1 (C1, C2) and A2.5/SP1.5 (D1, D2).

determined by the covalent binding of protein amides to alginate, and these bonds are dependent on the peptide sequence (i.e. type of protein). 3.6. Swelling behavior of beads The weight change of pure calcium-alginate and alginate-SPI beads in SGF and SIF at 37 °C is shown in Fig. 4. In SGF, shrinking of the beads occurred in the first 20 min mainly due to lose of negative charge of alginate carboxylate groups in acid solutions (i.e., stomach phase), which weakens the electrostatic repulsion between the chains (Pasparakis & Bouropoulos, 2006). Another reason for weight loss is that some of the protein molecules were released and digested due to the susceptibility to pepsin hydrolysis (Zhang, Zhang, Zou, & McClements, 2016; Zhang, Zhang, Zou, & McClements, 2017). The samples exhibited weight loss of ∼40% which is in accordance with the reports on shrinkage degree of alginate in SGF (Pasparakis & Bouropoulos, 2006). After this time interval, the beads remained intact due to electrostatic interactions operating between protonated –NH+3 groups of proteins (pI = 4.6) and unionized −COOH groups of alginate. Then, when transferred in the pancreatic juice, the beads began to uptake water. As the samples swelled up to ∼60 to ∼420 % of the initial mass, they began to disintegrate and dissolve after 5 to 25 min depending on the composition. The mechanism of alginate swelling can be explained by ion-exchange of monovalent Na+ ions from the saline solution with the Ca2+ ions present within the polymannuronate and polyguluronate blocks of alginate chains. As a result, a decrease in crosslinking density occurs mainly due to decreased electrostatic attraction between charged carboxylate groups (i.e. −COO groups) of alginate and less densely charged hydrated sodium ions. Simultaneously, soy protein molecules had diffused out of the hydrogel beads and were digested. Zhang et al.

Fig. 4. Weight change of pure Ca-alginate and alginate-SPI beads in simulated human gastrointestinal conditions.

wrinkle free surface, and the pleats were more pronounced for A2.5/ SP1 and A2.5/SP1.5 than for other two samples, indicating that alginate concentration rather than protein concentration had most profound effect on morphological characteristics. Belščak-Cvitanović et al. (2015) have also observed rugged and inhomogeneous surface of alginateprotein (whey proteins, bovine serum albumine, calcium caseinate, soy proteins, hemp proteins) beads encapsulating green tea extract, but they assigned the globular structures (observed under very high magnification) to proteins claiming that surface morphology was

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(2016) determined that diffusion of whey protein molecules from alginate microgels occurred at a higher rate than in an acidic environment. In another study, Zhang et al. (2017) determined that whey protein molecules entrapped within alginate matrix were digested more easily in intestinal medium than in the stomach, since the pore size of swollen alginate gels became appreciably larger than the dimensions of digestive enzymes (Sarkar et al., 2015), which allowed proteases to easily move through the biopolymer network. The maximum swelling was dependent on the sodium alginate concentration, and it was close to 90% for the A1.5/SP1.5, 320% for the A2/SP1.5 and 420% for the A2.5/SP1.5. Also, the time of maximum swelling attainment was increased with alginate concentration. Del Gaudio, Colombo, Colombo, Russo, and Sonvico, (2005) revealed that pure alginate beads of different concentrations behave in the same manner as herein, but in their study swelling profiles was determined in SIF without enzymes and with no previous treatment by SGF. Bajpai and Sharma (2004) have proved that acidic-treated crosslinked alginate gel swells less compared to untreated alginate, since alginate matrix, when exposed to low pH, is converted into alginic acid which results in lowering of degree of crosslinking and hence faster degradation. Namely, when acidic/treated beads are put in the buffer of pH 7.4, the beads swell at faster rate but do not attain a higher water uptake value due to loosely bound structure of the beads which is unable to retain large amount of water within the matrix. From the Fig. 4 it is evident that proteins contribute to faster increase in water uptake. More intensive water uptake induced by higher density of alginate-protein network can be explained by the fact that in SIF both the digested proteins and the alginate molecules should have regained their negative charge (pH was well above the pKa values of the anionic carboxyl groups); this resulted with electric repulsion between them and induced swelling of the alginate-protein matrix. Similarly, Hébrard et al. (2010) observed the same effect of whey proteins after 1 h incubation of alginate/whey protein beads in SIF without enzymes, but on the other hand, in SGF weight change of their beads was not observed at all. However, it should be stressed here, that these results only refer to weight change of a bulk of particles, but the information about the samples erosion due to degradation process should not be derived solely from them since swelling process may occur simultaneously with proteins release and change in microstructure.

Fig. 5. Release of thyme oil from alginate-SPI beads in simulated human gastrointestinal conditions.

temperature, while our release experiment was designed to entirely mimic GIT system regarding temperature (37 °C), enzymes and pHs. According to the release curves in Fig. 5, increase in alginate concentration led to slower release of thyme oil (statistically significant for A1.5/SP1 vs. A1/SP1 with p < 0.0001, A2/SP1 vs. A1/SP1 with p < 0.0001, A2/SP1.5 vs. A1.5/SP1.5 with p = 0.02 and A2.5/SP1.5 vs. A1.5/SP1.5 with p = 0.0006). There are other papers reporting a decrease in the rate and extent of release with increase in the polymer concentration in alginate formulations, despite the fact that those formulations with higher alginate content were more swollen (Coppi, Iannuccelli, Leo, Bernabei, & Cameroni, 2002; Soni, Kumar, & Namdeo, 2010), as also obtained herein (Fig. 4); a lower release rate have been attributed to the increase in the density of the polymer matrix with increased polymer concentration. On the other hand, the effect of SPI is under question; it clearly contributes to retarding of release when looking at blank alginate, A1.5/SP0, vs. either A1.5/SP1 (p < 0.0001) or A1.5/SP1.5 (p = 0.006); however, it seems that above 1 wt.% the effect of SPI becomes reverse (statistically significant for A1.5/SP1 vs. A1.5/SP1.5 with p = 0.049). Apparently, the release curves do not comply with swelling curves (Fig. 4). We hypothesize that oil droplets were inhomogeneously distributed within the matrix and were arranged such to have interfacial protein layers. We also expect that the effect of protein concentration on oil liberation was determined by two opposite tendencies, one is intensification of release with increase in protein content due to the swelling (Fig. 4) and restriction of release with increase in protein content due to the hydrophobic oil/protein interactions (alkali-heat modified soy proteins, such as those used here, are even more hydrophobic than native unmodified SPI). Liu, Chen, and Tang, (2014) have determined that soy oil (as an oil model) in SPI emulsion exhibited a high tendency to flocculate. On the other hand, swelling process in SIF was mainly determined by electric repulsion between alginate and SP anionic chains (as discussed above).

3.7. In vitro release The release profiles of thyme essential oil from alginate-SPI beads in SGF and SIF at 37 °C are shown in Fig. 5. In the first 10 min, in gastric solution, release of thyme oil from beads is characterized by high initial rate. This initial burst effect has been attributed mainly by fast diffusion of non-encapsulated compounds from the polymer matrix surface (Huang & Brazel, 2001). The percentage of the released content in the simulated conditions of stomach was 42–55 %, depending on the formulation. The rest of encapsulated polyphenols was liberated in the simulated intestine with the delay of 2.5 h (with the exception of A1/SP1), suggesting that the A/SPI complex beads have a function of controlled release in the site of intestine, while pure alginate spheres are characterized by the rapid cleavage (steady state after 75 min). Similarly, Zheng et al. (2007) have investigated complex microspheres based on alginate (3 wt.%) and SPI (25–75 % of the total weight) as a drug carrier (theophylline was used as a model drug); their microspheres released up to 15% of a drug in the gastric solution and they were able to carry through intestinal pH solution releasing up to ∼45% of the drug and then up to ∼80% in the colonic solution. However, in their study the release studies were performed in pH-gradient solutions, without any enzyme and at room

3.8. Mathematical modelling of release kinetics Thyme oil release kinetics in gastric or pancreatic juices is nonlinear and could be described by logistic equation (Cambel, 1993) as follows:

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Table 2 Relaxation time in gastric (τRG) and pancreatic juices (τRP) for alginate-soy protein beads of various compositions characterized by Xs/a parameter. System code

Xs/a

τRG (min)

τRP (min)

A1.5/SP0 A1.5/SP1.5 A2/SP1.5 A2.5/SP1.5 A1.5/SP1 A2/SP1 A2.5/SP1 A1/SP1

0 1 0.75 0.6 0.67 0.5 0.4 1

4 ± 0.5a 6.5 ± 0.3b 6.4 ± 0.3b 8 ± 0.3c 7.3 ± 0.5b,c 7.5 ± 0.5b,c 6.7 ± 0.3b 7.2 ± 0.5b,c

7 ± 0.3a 17 ± 0.5b 21 ± 0.5c 25 ± 0.5d 31 ± 1e 31 ± 1e 34 ± 1f 6.8 ± 0.5a

*Values with the same letter in each column showed no statistically significant difference (p < 0.05; Tukey's test).

∂ν(t) =μν(t)-bν(t)2 ∂t

Fig. 6. Thyme oil release from A2.5/SP1.5 beads: experimental data (dots) and model prediction (curve) according to Eq. (5).

(4)

where ν(t) is the volume of released oil, μ is the kinetic constant (specific rate of oil release) equal to μ=1/τR , τR is the relaxation time to environmental conditions. The first term on the right hand side represents the kinetic term while the second accounts for the resistance effects to oil release caused by oil-protein interactions. Model Eq. (4) could be solved analytically starting from initial and boundary conditions: (1) at t=0 , the released oil volume is ν(0)=ν0 and (2) whenν(t eq )=νeq for corresponding environmental conditions. The boundary condition points to relation between model parameters as: b= μ/νeq . The solution could be expressed as:

ν(t)=

Fig. 7. Storage modulus vs. time for A1.5/SP1.5 hydrogel beads.

νeq ν0 eμt νeq−ν0 + ν0 eμt

(5)

3.9. Rheological studies

Model parameters for various compositions of beads are presented in Table 2, where parameter Xs/a represents the mass ratio of SPI to alginate. The volume of released oil within gastric and pancreatic juices was compared with the values obtained by the mathematical model prediction. The predicted oil volume was calculated using Eq. (5). One representative data set is shown in Fig. 6. As shown in Fig. 6, the model prediction values for the volume of released oil correlated well with the experimental data, with relative error of 5%. The optimal model parameters obtained by this fitting procedure that enable the best comparison with the experimental data are shown in Table 2. Model parameters are given in the context of characteristic times, i.e. relaxation times for systems adaptation to environmental conditions. Specific rate of oil release μ is equal to μ=1/τR . A dimensionless criterion is formulated as Y=μ G/μ P=τRP /τRG to compare specific rates of oil release within gastric and pancreatic juices. Experimental data indicates that specific rate of oil release in gastric juice is 3 to 5 times higher than in pancreatic juice (Y = 3–5). This is a consequence of the initial burst effect at the beginning of the release process. Furthermore, when compared the samples with the same protein concentration of either 1 wt.% or 1.5 wt.%, those with higher alginate concentration expressed higher relaxation time in SIF (statistically significant for A1.5/SP1 vs. A1/SP1 vs. A2.5/SP1 and for A1.5/SP1.5 vs. A2/SP1.5 vs. A2.5/SP1.5), while in SGF the impact of alginate concentration was not statistically consistent. On the other hand, at the same alginate concentration of either 1.5 wt.% or 2 wt.%, the relaxation time in SGF and SIF decreased with increase in protein concentration. The exception from the rule ‘more protein-lower relaxation time’ can be observed at the alginate concentration of 2.5 wt.% (A2.5/SP1.5 vs. A2.5/SP1) but only in SGF.

Disintegration of the beads in SGF and SIF leads to their softening which can be quantified by rheological measurements. Fig. 7 shows change of storage modulus with time of immersing of the beads in SGF followed by SIF for one representative sample. As can be seen, weakening of the A/SPI matrix was happening gradually during the entire period in SGF, although decrease of the total weight stopped after the first 20 min (Fig. 4) as well as thyme oil release (Fig. 5). Softening of the matrix is a consequence of alginate chain relaxation process which occurred due to a decrease in crosslinking density, i.e., reduced electrostatic attraction between alginate chains and due to the repulsion of positively charged proteins (pI around 4.6), which resulted in expansion of matrices and allowed easy access of H+ and enzymes. After the transfer to SIF, storage modulus of the beads declined rapidly within the first 10 min, which is matching with their most intense swelling (Fig. 4) and loss of oil (Fig. 5) while further weakening of the matrix was gradual. The effect of alginate and protein concentration as well as the impact of temperature (37 °C versus 20 °C) on storage and loss moduli for alginate-SPI hydrogel beads is shown in Supplementary material; the experimentally determined rheological parameters were compared with those which were calculated according to modified fractional Kelvin-Voigt model equation (given in Supplementary material). 4. Conclusion Alginate-SPI complex beads can be used for encapsulation of thyme oil using a three-step procedure of o/w emulsification, electrostatic extrusion and gelification. The characteristics of the beads containing essential oils are dependent on concentration of both, alginate and SPI. The inclusion of soy protein increases swellability of the matrix in SIF and prolongs release but only up to 1 wt.% concentration. Low concentration of alginate is required to obtain spherical beads by electrostatic extrusion, but higher concentrations of alginate provide more 22

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retarded release of essential oils. The oil release process occurs simultaneously with the matrix swelling and softening but the three processes are not correlated in a straightforward manner. This study supports the hypothesis that alginate/SPI is a potential delivery system with tailored release of essential oil. Further studies are envisaged which would investigate chemical profile, as well as antimicrobial and antioxidant potential of the formulations developed here.

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