Nanoencapsulation of green tea catechins by electrospraying technique and its effect on controlled release and in-vitro permeability

Journal of Food Engineering 199 (2017) 82e92

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Nanoencapsulation of green tea catechins by electrospraying technique and its effect on controlled release and in-vitro permeability J. Anu Bhushani a, b, Nawneet Kumar Kurrey c, C. Anandharamakrishnan a, d, * a

Centre for Food Nanotechnology, Department of Food Engineering, CSIR e Central Food Technological Research Institute, Mysore, 570 020, India Academy of Scientific and Innovative Research (AcSIR), CSIR-CFTRI Campus, Mysore, 570 020, India c Department of Biochemistry, CSIR e Central Food Technological Research Institute, Mysore, 570 020, India d Indian Institute of Crop Processing Technology, Thanjavur, 613 005, India b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 August 2016 Received in revised form 15 December 2016 Accepted 16 December 2016 Available online 18 December 2016

Zein, a biocompatible, biodegradable macromolecule was employed for nanoencapsulation of green tea catechins by electrospraying technique. For this, the electrohydrodynamic behavior of zein solution was studied and the effective electrospraying concentration of zein was optimized. Later, the influence of nanoencapsulation and core-to-wall ratio on the gastrointestinal stability and permeability of green tea catechins were investigated. Among the various concentrations of zein studied (i.e. 1%e40% w/w), 5% w/ w zein solution yielded spherical, monodisperse nanoparticles with mean diameter of 157 ± 36 nm. Nanoencapsulates with 1:50 core-to-wall ratio had highest encapsulation efficiency compared to 1:10 and 1:05 core-to-wall ratio samples. The nanoencapsulated catechins had significantly improved in-vitro gastrointestinal stability and Caco-2 cell monolayer permeability compared to unencapsulated catechins. Further, 1:50 and 1:10 samples possessed higher permeability of catechins compared to 1:05 nanoencapsulates. Hence, the study provides a one-step approach for production of green tea catechin nanoencapsulates with sustained release and enhanced permeability properties. © 2016 Published by Elsevier Ltd.

Keywords: Catechins Zein Nanoencapsulation Electrospraying Caco-2 permeability Core-to-wall ratio

1. Introduction Green tea (Camellia sinensis) catechins are known to possess antioxidant, antiangiogenic, antitumor and antiobesity properties (Cabrera et al., 2006). However, catechins undergo epimerization, degradation and oxidation reactions during food processing and storage, which limits the usage of green tea catechins in functional food development. The stability of catechins both in-vitro and invivo are affected by factors such as temperature, pH and the presence of oxygen or metal ions (Ananingsih et al., 2013). In this context, nanoencapsulation is an effective means of delivering catechins in its chemically active form. Nanoencapsulation is the process of entrapping a bioactive compound in to a protective shell with final particle diameters ranging from 10 nm to 200 nm. Nanoencapsulation improves the chemical stability of the core compound and aids in sustained release in in-vivo environment (Quintanilla-Carvajal et al., 2010). Further, nanoencapsulated food

* Corresponding author. Indian Institute of Crop Processing Technology, Thanjavur, 613 005, India. E-mail address: [email protected] (C. Anandharamakrishnan). http://dx.doi.org/10.1016/j.jfoodeng.2016.12.010 0260-8774/© 2016 Published by Elsevier Ltd.

bioactives (hydroxycitric acid, vitamin E) are shown to possess improved in-vivo bioavailability compared to their microencapsulated counterparts (Ezhilarasi et al., 2016; Parthasarathi et al., 2016). Various techniques such as nanoemulsification, coacervation, nanoprecipitation, exist for the production of colloidal nanoparticle dispersions or suspensions. However, food grade nanoparticles in dry powder form are recognized to be suitable for long term storage stability, controlled release and food incorporation applications (Ezhilarasi et al., 2013). Here, need exists in overcoming the challenges associated with the processing and handling of nanoparticles in dry form. In this context, the use of electrospraying as a nanoencapsulation technique for food bioactives can aid in development of dry nanoencapsulates (Bhushani and Anandharamakrishnan, 2014). Electrospraying is the process of liquid atomization by electrical forces. Recently, electrospraying technique has been used for the encapsulation and stabilization of food bioactives such as curcumin (Gomez-Estaca et al., 2012), folic acid (Bakhshi et al.,
rez-Masia
et al., 2015a), lycopene (Pe
rez-Masi
2013; Pe a et al.,
mez-Mascaraque et al., 2015) 2015b), epigallocatechin gallate (Go and also live cells such as Lactobacillus acidophilus (Laelorspoen

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et al., 2014). Compared to the commercial spray drying method, electrospraying technique offers various advantages for encapsulation of bioactives (Table 1). However, due to the scalability concerns associated with electrospraying method, its use for production of food-grade nanoencapsulates is still in its laboratory level. Scaling up can be achieved by modifying the nozzle set-up, controlling the temperature and humidity of the electrospraying chamber and increasing the feed flow rate applied. These initiatives would broaden the scope for commercialization of this technology. Electrospraying technique utilizes a wide range of food grade proteins for encapsulation of bioactives. Among them, zein is a hydrophobic, biocompatible, biodegradable prolamine rich protein from corn. It is classified by the U.S. Food and Drug Administration (FDA) as a generally recognized as safe (GRAS) polymer for pharmaceutical and food applications (Shukla and Cheryan, 2001). It is used as an encapsulating material due to its film forming property, thermal resistance, and oxygen and moisture barrier properties (Corradini et al., 2014). Additionally, the stability and release properties of the core material are largely dependent on the physicochemical properties of wall material and the core-to-wall ratio used for encapsulation purpose (Rajam and Anandharamakrishnan, 2015). With this background, the major objectives of the study were, (i) to characterize the electrohydrodynamic behavior of zein solution at various concentrations and select the best concentration for nanoencapsulation purpose, and (ii) to study the effect of nanoencapsulation and core-to-wall (catechins-to-zein) ratio on the controlled release and Caco-2 cell monolayer permeability properties of green tea catechins. 2. Materials and methods 2.1. Materials Dried green tea leaves (‘Tetley’ green tea, Tata Global Beverages Ltd., Bengaluru, India) were procured from local market. Zein (from maize), (
)-epigallocatechin (EGC), (
)-epicatechin (EC), (
)-epigallocatechin gallate (EGCG), (
)-epicatechin gallate (ECG), Corning® Transwell® polycarbonate membrane cell culture inserts (12 mm dia, 0.4 mm pore size with cell growth area of 1.12 cm2) and lucifer yellow dipotassium salt were obtained from Sigma-Aldrich chemical co. (St. Louis, Missouri, USA). Minimum essential medium eagle, fetal bovine serum, antibiotics, Hank's balanced salt solution (HBSS), non-essential amino acids were obtained from HiMedia chemicals (HiMedia Laboratories Pvt. Ltd., Mumbai, India).

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Other chemicals, reagents and solvents used were of analytical grade. 2.2. Extraction and isolation of catechins The extraction of polyphenols from commercial green tea leaves were performed using the method as reported by Bhushani et al. (2016). The green tea leaves were microwave extracted (2450 MHz; 1000 W) at leaves to water ratio of 1:30 at 80  C for 10 min. The catechins were isolated using the procedure of Dong et al. (2011) with slight modifications. The green tea extract (100 ml) was washed with chloroform (50 ml) thrice and the obtained aqueous layer was extracted thrice with ethyl acetate (50 ml). The green tea catechins isolated powder was obtained by drying the ethyl acetate phases under reduced pressure at 60 οC. This isolated catechin powder was stored at 4 οC until further use and was used as the catechin core material in the study. 2.3. Preparation of electrospraying feed solutions Zein solutions were prepared at different concentrations ranging from 1.0% to 40% w/w by dissolving it in 80% aqueous ethanol using a magnetic stirrer. For nanoencapsulation, catechins powder was incorporated to the optimized concentration of zein solution at different core-to-wall ratios of 1:50, 1:10 and 1:05. 2.4. Characterization of electrospraying solutions Viscosity of the emulsions were determined at 25 ± 1  C using a stress-controlled rheometer (Haake RheoStress 6000, Thermo Scientific, Karlsruhe, Germany) by applying a shear rate in a linear manner from 0.1 to 250 s
1. Analysis was carried out in triplicates and apparent viscosity of the emulsions was calculated at shear rate of 100 s
1. The zero shear rate viscosity of all the solutions was obtained by using the Carreau-Yasuda model. Later, the specific viscosity (hsp) of the solutions was calculated using the following equation:

hsp ¼

ðho
hs Þ

(1)

hs

where, ho is the zero shear rate viscosity and hs is the viscosity of dispersing solvent i.e. 80% aqueous ethanol (1.96 ± 0.03 mPa s). According to the de Gennes's scaling concept, a theoretical relationship between the polymer rheology and its

Table 1 Comparison of electrospraying and spray drying techniques for encapsulation of food bioactives. Operational parameters

Electrospraying

Spray drying

Temperature Use of solvents

Non-thermal process Water as well as organic solvents However, solvent recovery at industrial scale set-up is mandatory to avoid explosion risks. Spherical with fine pores, Tailor-made structures Monodisperse, non-aggregated, nano, sub-micron and micron size particles can be produced. The particle collection and handling system has to be precisely monitored to obtain nanopowders. No denaturation of proteins or degradation of bioactives

Involves high inlet and outlet temperatures Water Organic solvents not preferred due to possible risk of solvent vapor explosion. Spherical, dented or with blow holes

Morphology of particles Particle size and product recovery

Product quality Encapsulation efficiency Wall materials used

Energy consumption

High Wide range of polymers and biopolymers Control on wall material thickness possible using coaxial electrospraying set-up Low energy required

Heterogenous particle size distribution due to aggregation, only micron sized particles can be produced. Product collection is simple due to the micron size. Degradation of bioactives might occur due to high temperature process Medium to high Polymers and biopolymers suitable for atomization and high temperature drying High energy required

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electrohydrodynamic property can be derived from a logarithmic plot of the polymer's specific viscosity as a function of its concentration. Hence, the specific viscosity of the solutions were calculated to determine the concentration exponents for viscosity of zein to identify the four different concentration regimes namely dilute, semidilute unentangled, semidilute entangled and concentrated regimes. The predicted exponents for polymer concentration in semidilute unentangled regime is 1.25 and in the semidilute entangled regime ranges from 3.75 to 4.8 depending on the solvent quality (McKee et al., 2004). The electrical conductivity of the feed solutions was measured using digital conductivity meter (CyberScan, PCD650, Eutech Instruments Pte Ltd, Singapore) at room temperature (26 ± 2  C) and the average of three measurements were reported. 2.5. Electrospraying process The electrospraying equipment used for the study consist of three major components namely (i) the syringe pump, (ii) the electrified needle, and (iii) the movable grounded collector plate, enclosed within a chamber (supplementary Fig. S1). The polymer solutions were introduced in a 10 ml syringe connected with a polytetrafluoroethylene (PTFE) tube to the stainless steel needle (internal diameter, 0.394 mm) and electrosprayed at a constant flow rate of 0.2 ml/h with a voltage of 16 kV at a distance of 8 cm between the needle tip and collector. The process was carried out at ambient conditions and the dried nanoencapsulates collected on a stainless steel plate were stored in a vacuum desiccator before

Encapsulation efficiency ð%Þ ¼

2.8. Hygroscopicity analysis The hygroscopicity of samples was analyzed as per the procedure of Fritzen-Freire et al. (2012). Isolated catechins, pure zein powder, electrosprayed zein nanoparticles and nanoencapsulated catechins (1:50, 1:10, 1:05) of known weight were placed in a desiccator with relative humidity maintained at 75%. After seven days, the samples were weighed again and the hygroscopicity was calculated on the basis of the absorbed moisture content. The results were expressed as gram of absorbed moisture per 100 g of sample (g/100 g). 2.9. Encapsulation efficiency The catechin content in the samples was analyzed by reversed phase high-performance liquid chromatography (RP-HPLC) (Waters, manual injector, 515 HPLC pump and 2489 UV/visible detector, Milford, MA, USA) according to the method described by ElShahawi et al. (2012). Mobile phases A and B containing 5% (v/v) acetonitrile with 0.035% (v/v) trifluoroacetic acid and 50% (v/v) acetonitrile with 0.025% (v/v) trifluoroacetic acid, respectively were eluted using a gradient profile at a flow rate of 1.0 ml/min. The chromatographic separation was performed by a C18 column (Kromasil, 4.6  250 mm, 5 mm, 100 Å) maintained at 32 οC and eluted compounds were detected at a wavelength of 205 nm. The analysis was performed in triplicates and mean values were used for calculating the encapsulation efficiency of individual catechin isomers (EGC, EC, EGCG and ECG) using the following formula:

Catechins content in the nanoencapsulates  100 Catechins content in the feed solution

(2)

being packed in polyethylene pouches.

2.6. Morphology and particle size analysis Scanning electron microscopy (Leo 435 VP, Leo Electronic Systems, U.K.) was performed to analyze the morphology of the electrosprayed samples. The samples were sputter coated with gold (2 min, 2 mbar) and the micrographs were observed at 15 kV in a vacuum of 9.75  10
5 Torr. For morphological characterization of the nanoencapsulates that were exposed to gastrointestinal conditions (simulated gastric juice and phosphate buffer at pH 6.8), the particles retrieved from simulation solution were dried overnight at 40  C and were followed by the procedure for SEM analysis. The particle sizes of the electrosprayed samples were determined from the SEM images in their original magnification using Digimizer software (version 4.3.4, Medcalc software) by scanning at least 200 particles.

2.7. Fourier-transform infrared analysis The interaction between polymer and catechins in the nanoencapsulates were studied using fourier-transform infrared (FTIR) spectrometer (Spectrum Two, PerkinElmer, Massachusetts, USA). Pure zein powder, electrosprayed zein nanoparticles and catechins encapsulated zein nanoparticles were placed on the spectrometer and spectra recorded in the wavenumber range of 4000e400 cm
1.

2.10. Stability in simulated gastro-intestinal conditions The unencapsulated and nanoencapsulated catechins were exposed to simulated gastrointestinal conditions according to the procedure described by Aceituno-Medina et al. (2015) and their invitro stability (or release) was assessed. For gastric digestion, 4 mg of sample was diluted with 5 ml of distilled water and acidified to pH 2 using 6 M HCl under constant agitation. Later, 0.6 ml of pepsin from porcine (160 mg/ml) in 0.1 M HCl was added to the solution and made up to 10 ml using distilled water. Gastric digestion was initiated and sample incubated in water bath at 37  C for 2 h. After gastric digestion, the pH of the digesta was increased to pH 5 with 0.045 M NaHCO3 and 2.4 ml of pancreatin-bile solution (pancreatin 4 mg/ml, bile 25 mg/ml in 0.1 M NaHCO3) was added. The intestinal phase was initiated by increasing the pH of the solution to pH 7 by 0.005 M NaHCO3 and incubated at 37  C for 2 h under constant stirring. During the gastrointestinal digestion, aliquots of digesta were withdrawn at specific time intervals (30, 60, 90, 120, 150, 180, 210, 240 min), the reaction was stopped by cooling in ice, followed by centrifugation of the sample at 16,000 rpm for 15 min. The supernatant was appropriately diluted and used for detection of released polyphenols by spectrophotometry. In UVeVis spectrophotometry, the total polyphenol content (TPC) of the samples was determined using gallic acid as standard, according to the method described by the International Organization for Standardization

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(ISO 14502-1: 2005). Briefly, 1.0 mL of the appropriately diluted digested samples (or distilled water in case of blank) was transferred in triplicates to separate tubes containing 5.0 mL of a 1/10 dilution of Folin-Ciocalteu's reagent in water. After 5 min, 4.0 mL of a sodium carbonate solution (7.5% w/v) was added. The tubes were then allowed to stand at room temperature for 60 min before absorbance at 765 nm was measured. The TPC was expressed as mg of gallic acid equivalents (GAE) per total volume of digesta.

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C0 is the initial concentration of compound in the donor chamber (mg/ml). After the transport experiment, the integrity of the monolayers was measured again using Lucifer yellow as marker as described in section 2.11.1. It was observed that the Papp values were 0.86 ± 0.056  10
6 cm/s, which was not statistically different from those values before the transport assay, indicating the integrity of the cell monolayer throughout the experiment. 2.12. Statistical analysis

2.11. Caco-2 cell permeability of nanoencapsulates The permeability of catechins in its free form and nanoencapsulated form were assessed using the Caco-2 cell model. 2.11.1. Cell culture The Caco-2 cell line (human epithelial colorectal adenocarcinoma) was obtained from the National Centre for Cell Science (NCCS, Pune, India). The cells were cultured in minimum essential medium eagle (MEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% non essential amino acids and 1% penicillin (10,000 Units/mL) and streptomycin (10,000 mg/mL). Cells were incubated at 37  C in a humidified atmosphere with 5% CO2 in air and subcultured at 70e90% confluence by trypsinisation with 0.05% trypsin-EDTA and resuspended in medium. Subsequently, the cells were seeded at a density of 2.6  105 cells/cm2 onto transwell inserts (polycarbonate membrane, 12 mm i.d., 0.4 mm pore size, 1.12 cm2 growth area) placed in 12 well plate. Cells in transwells were allowed to grow and differentiate to form a confluent monolayer for 21 days and the growing medium was replaced three times a week. The cells used in this study were between passages 45 to 55. After 21 days, Lucifer yellow was used as a paracellular marker to assess the integrity of the formed monolayer using the procedure as stated in Bhushani et al. (2016). The cell monolayers used in the study had Papp values in the range of 0.705 ± 0.06  10
6 cm/s, indicating formation of intact monolayer. 2.11.2. Transport assay The transport experiment was performed according to the procedure described by Hubatsch et al. (2007). For the assay, media was removed from transwells and the cell monolayers were washed thrice with HBSS buffer (pH 7.4, 37  C). Free and nanoencapsulated catechins were subjected to gastric digestion (as described in 2.10) and the gastric digesta appropriately diluted with HBSS buffer (final pH 6.4) were used in the study for measuring the apical to basolateral permeability of the catechins. Briefly, 0.5 mL of the diluted sample and 1.2 mL of pH 7.4 HBSS buffer were loaded on the apical and basolateral sides of the transwell system, respectively. The samples were incubated at 37  C with continuous shaking at 65 rpm. Aliquots (0.6 ml) of samples were collected from the basolateral compartment at regular intervals of 30, 60, 90 and 120 min and replaced by the same volume of HBSS buffer. All the samples were stabilized immediately with one tenth volume of preservative solution (20% ascorbic acid and 0.01% sodium EDTA dissolved in sodium phosphate buffer, pH 3.6) and analyzed for catechins content by RP-HPLC method. The apparent permeability (Papp) of the compounds was calculated using the following equation:


dQ 1 : Apparent permeability Papp ¼ dt A:Co

(3)

where, Papp is the apparent permeability coefficient (cm/sec), dQ/dt is the linear appearance rate of the compound on the receiver end based on its cumulative transport for 2 h (mg/sec), A is the surface area of the cell monolayer (1.12 cm2 for 12 mm dia transwells) and

Results are expressed as mean ± standard deviation (SD) of triplicate values. Analysis of variance (ANOVA) of the data was performed using SPSS version 16.0 for windows (SPSS Inc., Chicago, IL, USA) and significant differences were evaluated by Tukey's post hoc test with a confidence interval of 95%. 3. Results and discussion 3.1. Optimization of zein concentration for production of nanoparticles In this study, the concentration of zein was optimized at fixed instrumental conditions that enabled electrospraying at stable cone-jet mode i.e. 16 kV of electric potential, 8 cm distance between needle tip and collector and 0.2 ml/h of feed rate. The feed solutions were characterized for its rheology and conductivity and the solution concentration was optimized based on the product morphology and particle size. 3.1.1. Rheology The rheogram of zein solutions at different concentrations (1.0%e40.0% w/w) are presented in supplementary Fig. S2. The zein solutions exhibited Newtonian behavior as the viscosity did not change with an increase in shear rate. Similar reports have been published for zein solutions prepared using aqueous ethanol (Fu and Weller, 1999; Li et al., 2012). Further, it was observed that an increase in the zein concentration was associated with an exponential increase in apparent viscosity of the solutions (Table 2). For instance, the viscosity of 1.0% zein solution was 2.20 ± 0.01 mPa s and it increased to 32.64 ± 0.43 mPa s for 20% and furthermore to 660.55 ± 3.96 mPa s for 40% zein solution. To understand the effect of zein concentration (C) on viscosity, the specific viscosity of the solutions were calculated as described in Eqn. (1). As described earlier, from the concentration dependence of viscosity, four distinct solution regimes namely dilute, semidilute unentangled, semidilte entangled and concentrated regimes can be identified. However, in the current study, the dilute and concentrated regimes were not determined as rheology was not performed below 1%w/w and above 40%w/w concentrations, respectively. The plot of zein concentration versus specific viscosity showed a linear increase until 10% w/w concentration, after which there was a non-linear increase (Fig. 1). The semidilute unentangled and semidilute entangled regimes had slope of 1.35 and 3.82, respectively, which were in good agreement with the theoretical prediction range. From the crossover of the semidilute unentangled and semidilute entangled regimes, the onset of critical chain entanglement concentration (Ce) was found to be 12.51%wt. Briefly, in the semidilute unentangled regime, overlap of polymer chains occurs, whereas entanglement is absent, however, in the semidilute entangled regime, significant overlap which causes constraint in the individual polymer chain motion occurs (Kong and Ziegler, 2012). The critical entanglement concentration (Ce) of zein plays a significant role in its electrohydrodynamic behavior and has a bearing on the morphology of the end product. For the production of nanoparticles by electrospraying, it is appropriate to use polymer

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Table 2 Apparent viscosity, zero shear rate viscosity and conductivity of zein solutions at various concentrations and nanoencapsulates at different core-to-wall ratios. Apparent viscosity (mean ± SD) (mPa.s) Zein Solution (%wt) 1.0 2.20 ± 0.01 2.0 2.54 ± 0.02 2.5 2.69 ± 0.10 3.0 2.88 ± 0 4.0 3.36 ± 0.01 5.0 3.82 ± 0.04 6.0 4.27 ± 0.01 7.5 5.36 ± 0.05 8.0 5.81 ± 0.04 10.0 6.99 ± 0.06 12.0 10.18 ± 0.09 15.0 15.60 ± 0.16 18.0 23.52 ± 0.17 20.0 32.64 ± 0.43 25.0 67.70 ± 1.87 30.0 127.27 ± 2.08 35.0 316.25 ± 2.79 40.0 660.55 ± 3.96 Nanoencapsulates (core:wall ratio) 1:50 3.84 ± 0.09 1:10 3.91 ± 0.04 1:05 3.77 ± 0.02

Zero shear rate viscosity (mean ± SD) (mPa.s)

Conductivity (mean ± SD) (mS/cm)

2.19 ± 0.01 2.53 ± 0.01 2.66 ± 0.10 2.88 ± 0.01 3.35 ± 0.02 3.80 ± 0.05 4.26 ± 0.01 5.34 ± 0.07 5.80 ± 0.06 6.96 ± 0.09 10.17 ± 0.08 15.53 ± 0.25 23.83 ± 0.17 32.73 ± 0.42 68.71 ± 3.36 132 ± 1.55 405.45 ± 3.46 835.3 ± 2.54

186.10 ± 0.42 340 ± 0.42 408.85 ± 0.64 476.05 ± 0.78 594.70 ± 1.13 700.05 ± 0.21 800.15 ± 0.49 931.10 ± 2.97 976.35 ± 3.04 1115 ± 2.83 1242 ± 2.82 1356.5 ± 2.12 1467.50 ± 2.12 1485.50 ± 2.12 967.40 ± 4.38 938.80 ± 0.42 762.05 ± 0.35 684.85 ± 1.76

3.80 ± 0.08 3.88 ± 0.03 3.74 ± 0.03

715.70 ± 2.83 725.65 ± 1.06 699.05 ± 1.06

Values reported are mean ± SD of the measurements (n ¼ 3).

stable cone-jet mode and result in production of droplets with multimodal size distribution. Also, high conductivity of samples leads to high charge density in the Taylor cone and lead to breakup of the jet in to polydisperse spray (Bock et al., 2012; Guo et al., 2016; Subramanian et al., 2005). Hence, the optimal conductivity range of the samples can be determined in correspondence to the morphology of the electrosprayed particles at the stable cone-jet mode.

Fig. 1. Plot of zein concentration (%wt) versus specific viscosity (hsp) indicating the semidilute unentangled and semidilute entangled solution regimes.

solutions below its critical concentration. On the other hand, a minimum entanglement concentration is required to electrospin the polymer solution by preventing the breaking up of polymer jet during electrospinning (Bock et al., 2011). 3.1.2. Conductivity The conductivity of zein solutions increased with increasing concentration and followed a linear trend until 8% w/w concentration (R2 ¼ 0.99). However, beyond 8% w/w concentration, a linear increase in conductivity with respect to concentration was not observed. This can be due to the onset on the polymer chain overlap in the solution and as the polymer solution approaches the critical entanglement concentration, the conductivity of the solution reduces. Further, beyond 20% w/w concentration, there was a decrease in the solution conductivity (Table 2). This decrease in conductivity can be attributed to the decrease in the water content for ionization of molecules at higher polymer concentrations. Similar decrease in conductivity with increase in polyvinyl alcohol concentration in chondroitin sulfate/polyvinyl alcohol composite solution has been reported recently (Guo et al., 2016). It should be noted that an increase in conductivity will reduce the region of

3.1.3. Morphology The distinct changes in the morphology of electrosprayed zein with change in concentration are shown in Fig. 2. When the solution concentration was low (i.e. less than 4.0% w/w), the electrospray droplets deform in to irregular shapes and during evaporation of solvent, due to the absence of sufficient intermolecular entanglements in the polymer chains they cannot attain compact spherical structures. Hence, irregular, complex shaped particles with bidisperse spherical forms are produced (Fig. 2aee). However, when the solution concentration was optimum, (i.e. 5e6% w/w) this limitation was overcome by the polymer droplets and compact, spherical particles are formed (Fig. 2feg). At 5% w/w concentration, spherical nanoparticles with smooth surface were formed with mean particle diameter, minimum and maximum diameters of 157.5 ± 35.6 nm, 64 nm and 273 nm, respectively. At high polymer concentration (i.e. more than 15% w/w), continuous fibers or beaded structures were observed which can be attributed to the increase in the molecular chain entanglement in the polymer jet that prevented Coulombic fission and led to stable elongation of the jet (Fig. 2lep). Fiber formation at zein solutions above 20%wt concentration has been reported in previous works (Li et al., 2009; Moomand and Lim, 2015). Further, work by Neo et al. (2012) also reported the formation of smooth fibers at zein concentrations of approximately 2 times the entanglement concentration i.e. at 24% wt concentration. Apart from these, varied morphologies with wide range of particle diameters were observed at concentration ranging from 7.5% w/w to 12.0% w/w (Fig. 2hek). For instance, the minimum and maximum particle diameters were 90 nm and 657 nm respectively, for 7.5% w/w zein solution. This is attributable to the increase in

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Fig. 2. Morphology of electrosprayed zein particles at concentrations of (a) 1.0%, (b) 2.0%, (c) 2.5%, (d) 3.0%, (e) 4.0%, (f) 5.0%, (g) 6.0%, (h) 7.5%, (i) 8.0%, (j) 10.0%, (k) 12.0%, (l) 15.0%, (m) 18.0%, (n) 20.0%, (o) 25.0% and (p) 40.0% electrosprayed at a flow rate of 0.2 ml/h with a voltage of 16 kV at a needle to collector distance of 8 cm.

zein viscosity and associated increase in solution conductivity. Briefly, due to the increased conductivity of the samples, the emerging droplet from the needle tip reaches the Rayleigh limit faster and satellite droplets are ejected alongside the primary droplets (Festag et al., 1998; Shenoy et al., 2005). Similar results have been reported for poly(vinylidene fluoride) films prepared by electrospraying technique (Rietveld et al., 2006). On comparing the morphology results with the rheology and conductivity profiles of the corresponding zein solutions, it can be seen that electrospraying occurred best at concentrations far below the critical entanglement concentration i.e. 12.51%wt. Smooth, spherical structures were obtained at 5% w/w and 6% w/w zein solutions. However, 5% w/w had a narrow size distribution width and lower mean particle diameter compared to 6% w/w. Thus, 5% w/ w was optimized as the appropriate concentration for electrospraying of zein solutions to obtain nanoparticles. 3.2. Morphological and physical properties of nanoencapsulated catechins Green tea catechins were incorporated in to the zein solution at optimized concentration (i.e. 5% w/w) at three different core-towall ratios namely 1:50, 1:10 and 1:05 respectively. 3.2.1. Solution properties The viscosity and conductivity values of the catechins incorporated solution are given in Table 2. There was a slight decrease in the viscosity of the 1:05 solution (3.77 ± 0.02 mPa s) compared to 1:50 (3.84 ± 0.09 mPa s) and 1:10 (3.91 ± 0.04 mPa s) solutions,

however, the difference was not statistically significant at p < 0.05. On the other hand, there was a significant (p < 0.05) difference in the conductivity of the three solutions with 1:10 (725.65 ± 1.06 mS/ cm) and 1:05 (699.05 ± 1.06 mS/cm) possessing high and low conductivities respectively. This can be explained by the high catechin content and protein-polyphenol interaction occurring in the 1:05 system that causes a decrease in the viscosity and a subsequent decrease in the conductivity of the solution. 3.2.2. Morphology Electrosprayed-nanoencapsulated particles exhibited significantly (p < 0.05) higher particle diameters than plain 5% w/w zein solution (Table 3). The mean ± SD particle diameter of 1:50, 1:10 and 1:05 nanoencapsulates were 188 ± 62 nm; 187 ± 62 nm and 174 ± 59 nm, respectively. Among these, 1:05 nanoencapsulates possessed lowest mean particle diameter, which is in agreement with its relatively low apparent viscosity values. Also, the maximum particle diameters were in the higher range (413e495 nm) for nanoencapsulates compared to plain 5% w/w zein nanoparticles (273 nm). The morphology of the nanoencapsulates (as seen in Fig. 3) revealed reduction in sphericity and roundness with negligible agglomeration as against the zein nanoparticles. Report from a previous study indicated that there is no difference in the morphology and particle size of zein nanocapsules after the incorporation of curcumin, which could be attributed to the low core-to-wall ratio (1:500) and hydrophobic nature of curcumin (Gomez-Estaca et al., 2012). However, in the current study, catechins, being hygroscopic in nature; might have interfered in the drying rate of zein during the flight from needle tip

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J.A. Bhushani et al. / Journal of Food Engineering 199 (2017) 82e92 Table 3 Particle size of electrosprayed zein particles at various concentrations and nanoencapsulates at different core-to-wall ratios. Average (±SD) diameter (nm) Zein solution concentration (%wt) 1.0 NA 2.0 NA 2.5 NA 3.0 NA 4.0 NA 5.0 157.5 ± 35.6 6.0 169.2 ± 64.7 7.5 206 ± 103.4 8.0 241.1 ± 78.8 10.0 360.1 ± 163.8 12.0 307.7 ± 147.7 15.0 336.6 ± 200.8 18.0 299.7 ± 248.9 20.0 180.5 ± 51 25.0 231.7 ± 87.7 30.0 533.1 ± 91.4 35.0 853.7 ± 142.3 40.0 1142.8 ± 367 Nanoencapsulates (core:wall ratio) 1:50 188.1 ± 61.6 1:10 187.4 ± 61.9 1:05 174.3 ± 59.1

Min diameter (nm)

Max diameter (nm)

NA NA NA NA NA 64 40 90 69 108 88 83 47 104 95 112 297 417

NA NA NA NA NA 273 405 657 571 1074 853 1057 1462 464 582 812 1207 2424

49 78 90

495 440 413

NA - not applicable, as the particles had irregular morphology.

Fig. 3. Morphology and particle size distribution of nanoencapsulated catechins at core-to-wall ratios of (a) 1:50, (b) 1:10 (Image magnification: 20,000 X, inset 80,000 X) and (c) 1:05 (Image magnification: 10,000X, inset 80,000 X).

to collector. Nevertheless, the nanoencapsulates possessed smooth structures with no structure collapse or surface dents. 3.2.3. Hygroscopicity Hygroscopicity of encapsulates is chiefly influenced by surface area and particle morphology. A powder is considered nonhygroscopic if the hygroscopicity value is less than 10%. One approach to reduce the hygroscopicity of sample is by encapsulating it with high molecular weight wall materials (Bhandari et al., 2013). Hygroscopicity of pure zein powder was estimated to be 12.84 ± 0.73 g/100 g, whereas, it was only 6.97 ± 0.34 g/100 g for electrosprayed zein nanoparticles (at 5% w/w). With our results, we infer the decreased hygroscopicity of electrosprayed zein

nanoparticles compared to pure zein powder can be attributed to the following reasons. In the current study, the initial moisture content of zein nanoparticles (6.15%) was higher than that of pure zein powder (4.01%). Hence, the higher water concentration gradient between the pure zein powder and the surrounding air at 75%RH might have increased the hygroscopicity of pure zein powder compared to electrosprayed nanoparticles. Further, electrospraying technique produced compact spherical zein nanostructures which might possess lower water vapor and oxygen permeability compared to pure zein powder (Lawton, 2002). The hygroscopicity of catechins was 13.12 ± 0.53 g/100 g. Among the nanoencapsulates, 1:05 sample possessed highest hygroscopicity of 7.5 ± 0.24 g/100 g, followed by 1:50 and 1:10 samples with

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3.3. Encapsulation efficiency

Fig. 4. Comparative FTIR spectra of (a) isolated catechins; (b) electrosprayed zein nanoparticles; (c) to (e) nanoencapsulates at core-to-wall ratio of 1:50, 1:10 and 1:05 respectively.

5.88 ± 0.19 g/100 g and 4.76 ± 0.14 g/100 g respectively. From the hygroscopicity values, it can be stated that 1:10 nanoencapsulates conferred highest protection to catechins from the simulated external environment with 75% relative humidity. All the nanoencapsulates were non-hygroscopic as the absorbed moisture content is less than 10% weight of the sample. 3.2.4. FTIR spectroscopy The comparative FTIR spectra of isolated catechins; electrosprayed zein nanoparticles and nanoencapsulates are represented in Fig. 4. With respect to the functional groups of the core molecule, i.e. catechins, the broad band around 3400 - 2600 cm
1 region belongs to the aliphatic and aromatic CeH, phenolic and alcoholic OeH stretching. Band observed at 1608.33 cm
1 in the spectra of isolated catechins is due to the aromatic C]C stretching. The amide A band corresponding to the stretching of the NeH and OeH bonds of the amino acids of zein protein appeared at 2961.12 cm
1 and 3288.32 cm
1, respectively for zein nanoparticles. After nanoencapsulation of catechins, the nanoencapsulates possessed mild shift in the bands. The amide A bands were observed at 2960.95 cm
1 and 3288.88 cm
1 for 1:50; 2961.53 cm
1 and 3289.27 cm
1 for 1:10 and 2962.27 cm
1 and 3295.89 cm
1 for 1:05 nanoencapsulates, respectively. These shifts indicate the formation of hydrogen bonds caused due to the interaction between amide group in zein and hydroxyl group in catechins. Similar shift in amide A bands has been observed by Sun et al. (2015) when zeinquercetagetin composite colloidal nanoparticles were prepared. The typical absorption band observed for amide I group in zein nanoparticles was shifted from 1644.67 cm
1 to 1644.77 cm
1, 1645.42 cm
1 and 1647.1 cm
1 for 1:50, 1:10 and 1:05 nanoencapsulates, respectively. This broadening of the amide I band due to C]O stretch vibrations suggests that the interaction between zein and catechins are stabilized by hydrogen and hydrophobic interactions.

The encapsulation efficiencies of nanoencapsulates are given in Table 4. The encapsulation efficiencies of the individual catechins (EGC, EC, EGCG and ECG) were above 85% for all the nanoencapsulates. These high encapsulation efficiencies can be attributed to the film forming property of zein and the non-thermal electrospray based encapsulation process. Among the four isomers of catechins, EGC had the highest encapsulation efficiency of 97.44± 0.70% for 1:50 samples, followed by ECG, EGCG and EC. With respect to EGC and EC, there was no significant difference in the encapsulation efficiency among the three nanoencapsulates. However, the encapsulation efficiency of EGCG was significantly (p < 0.05) high in 1:50 (95.527 ± 1.08%), followed by 1:10 (92.75± 1.2%) and 1:05 (89.96± 0.96%) nanoencapsulates. The overall high encapsulation efficiency in 1:50 and 1:10 nanoencapsulates is due to the low core-to-wall ratio which enabled complete entrapment of catechins during the flight time of the electrospray droplets. This difference in the encapsulation efficiency had an effect on the antioxidant potential of the nanoencapsulates as detailed in supplementary file (supplementary Fig. S3). The significance of core-to-wall ratio on microencapsulation efficiency of Lactobacillus plantarum has been reported by Rajam and Anandharamakrishnan (2015). Also, previous works on electrospray based encapsulation of food bioactives such as curcumin, EGCG and folic acid have reported encapsulation efficiencies above 85e90% and have attributed it to mild operating conditions
rezof electrospraying technique (Gomez-Estaca et al., 2012; Pe
et al., 2015a; Go
mez-Mascaraque et al., 2015). Masia 3.4. Release of catechins with respect to time in simulated gastrointestinal conditions The release and subsequent stability of the released polyphenols

Fig. 5. Release and stability of polyphenols in nanoencapsulated and free forms under simulated gastrointestinal conditions. Table 4 Encapsulation efficiency of nanoencapsulates. Nanoencapsulates (core:wall ratio)

1:50 1:10 1:05

Encapsulation efficiency (%) EGC

EC

EGCG

ECG

97.45 ± 0.70a 96.45 ± 1.20a 94.68 ± 1.49a

89.71 ± 1.93a 89.54 ± 1.00a 86.84 ± 1.25a

95.53 ± 1.08a 92.75 ± 1.20b 89.96 ± 0.96c

95.62 ± 0.97a 93.07 ± 1.49ab 90.67 ± 1.51b

Data reported are mean ± SD of the measurements (n ¼ 3). Values in the same column with different superscripts are significantly different at p < 0.05.

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in the simulated gastrointestinal conditions were evaluated for nanoencapsulates and compared with unencapsulated catechins (Fig. 5). As uniaxial electrospraying was employed, it can be predicted that final morphologies of the nanoencapsulates were in the form of nanosphere, with catechins dispersed uniformly in the zein matrix. Hence, the immediate release of catechins observed in the gastric phase is attributable to the surface erosion and pore formation in the zein matrix. The pepsin digestion of nanoencapsulates and the subsequent morphological change is shown in the inset image (Fig. 5). However, after 30 min incubation at intestinal phase, there was an increase in the total polyphenol content. This is due to the effect of pancreatin on partially digestion zein. Similar effect of pepsin and pancreatin on zein microspheres
pez and Murdan (2006). After has been reported by Hurtado-Lo 30 min of intestinal incubation, a steady state is obtained in the intestinal release (i.e. after 150 min) in all the nanoencapsulates. This might be due to the enhanced swelling of the nanoencapsulates in the simulated gastrointestinal fluid that might have increased the diffusion path length within the nanoparticles (Wongsasulak et al., 2014). The swelling of nanoencapsulate exposed to pH 6.8 (phosphate buffer, mimicking intestinal pH) for 2 h is depicted in the inset figure (Fig. 5). Work by Bisharat (2012) also revealed that the release of drugs coated with zein occurred mainly via diffusion through the aqueous channels formed by the erosion of the zein coating in the presence of digestive enzymes. In contrast to the encapsulated samples, the unencapsulated catechins showed a marked decrease from 769.90 to 369.25 mg/total volume of digesta in the polyphenol content after exposure to intestinal conditions. This is due to the degradation of catechins at alkaline or near neutral pH conditions of the intestinal phase. Similar decrease in the green tea polyphenol content at intestinal pH has been reported in previous works (Tenore et al., 2015; Green et al., 2007). The results show evidence of sustained release of

catechins from the nanoencapsulates in the gastric and intestinal conditions with minimal degradation. It can be stated that 1:50 and 1:10 nanoencapsulates possess better gastrointestinal stability and release property due to enhanced protection of the sample attributed to their low core-to-wall ratios compared to 1:05 nanoencapsulates. 3.5. Caco-2 cell permeability The apparent permeability (Papp) coefficients of the unencapsulated and nanoencapsulated catechins are shown in Fig. 6. The low Papp values (less than 1  10
6 cm/s) of the individual catechin isomers indicate its poor permeability across the in-vivo intestinal membrane. The permeability of the nanoencapsulated samples were significantly (p < 0.05) higher than the unencapsulated catechins. Among the four catechins, EGC (1.21 ± 0.07  10
6 cm/s) had highest permeability in 1:05 nanoencapsulates, EGCG (0.85 ± 0.04  10
6 cm/s) in 1:10 and EC (0.84 ± 0.07  10
6 cm/s) and ECG (0.81 ± 0.02  10
6 cm/s) in 1:50 nanoencapsulates. Other than EGC, the permeability coefficients differed significantly (p < 0.05) among the nanoencapsulates with varying core-to-wall ratios. It was observed that the overall permeability profile of 1:50 and 1:10 nanoencapsulates were higher than 1:05 nanoencapsulates while excluding the non-significant difference in EGC. As stated earlier, the zein nanoparticles undergo pepsin mediated erosion during the gastric digestion and when the partially eroded samples are subjected to Caco-2 cell permeability, the combined effect of erosion and diffusion mechanism takes place in releasing the catechins from the wall matrix. Similar erosion-diffusion mechanism has been reported for the in-vitro release of alpha tocopherol from electrospun zein-chitosan composite fibers (Wongsasulak et al., 2014). This release property is employed in the

Fig. 6. Caco-2 cell permeability of (a) EGC; (b) EC; (c) EGCG and (d) ECG in unencapsulated and nanoencapsulated forms. * represents significance at p < 0.05 and NS represents non-significance among the groups according to Tukey's multiple comparison test.

J.A. Bhushani et al. / Journal of Food Engineering 199 (2017) 82e92

pharmaceutical field for the controlled release of drugs entrapped in zein based micro/nano fibers (Zhang et al., 2015). Also, the nanoencapsulation protects the catechins from sudden exposure to near neutral pH of the apical compartment in the transwell system. Earlier studies have shown an enhancement in the Caco-2 cell permeability of green tea catechins after nanoencapsulation process (Tang et al., 2013; Bhushani et al., 2016). 4. Conclusion Electrospraying was employed as a nanoencapsulation technique for improving the stability and release properties of green tea catechins. Zein was used as a wall material and the optimum electrospraying concentration was determined using rheology, conductivity and morphology studies. The critical entanglement concentrations of the zein solutions in 80% aqueous ethanol was found to be 12.51%wt. Zein at 5%wt produced spherical, monodisperse nanoparticles with diameter of 157.5 ± 35.6 nm. However, after nanoencapsulation of catechins at 1:50, 1:10 and 1:05 core-towall ratios, the particle diameter increased by approximately 30 nm compared to plain zein nanoparticles. Nanoencapsulates with 1:50 and 1:10 core-to-wall ratios had the highest encapsulation efficiency, gastrointestinal stability and Caco-2 cell monolayer permeability compared to 1:05 samples. Hence, it was evident that electrospray aided nanoencapsulation process improved the release and permeability properties of catechins, especially at lower core-to-wall ratios (1:50 or 1:10) compared to higher core-to-wall ratio (1:05) or as catechins in free form. Acknowledgments Authors wish to thank the Director, CSIR-Central Food Technological Research Institute, for his constant support and encouragement. We wish to acknowledge the Ministry of Food Processing Industries (Grant number: SERB/MOFPI/ 0043/2012), Government of India for providing financial support for this work. We thank Mr. K. Anbalagan, Sr. Technician, CSIR-CFTRI, for his kind technical support for SEM analysis. The author (Anu Bhushani) thanks the University Grants Commission (UGC) for awarding Research Fellowship. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jfoodeng.2016.12.010. References
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