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Production and characterization of catechin-loaded electrospun nanofibers from Azivash gum- polyvinyl alcohol
Carbohydrate Polymers 235 (2020) 115979
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Production and characterization of catechin-loaded electrospun nanofibers from Azivash gum- polyvinyl alcohol
T
Seyedeh Zahra Hoseyni, Seid Mahdi Jafari*, Hoda Shahiri Tabarestani, Mohammad Ghorbani, Elham Assadpour, Moslem Sabaghi Faculty of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Azivash gum Polyvinyl alcohol Catechin Encapsulation Electrospinning
In this study, Response Surface Methodology was used to optimize the electrospinning process parameters including voltage, distance, and flow rate in order to obtain catechin-loaded electrospun nanofibers from Azivash (Corchorus olitorius. L) gum-polyvinyl alcohol with the minimum diameter of nanofibers. The optimum electrospinning conditions were applied for catechin encapsulation at different loading concentrations (500, 1000, 2000 and 3000 mg L−1). According to the results, increase in catechin concentration led to increment in polymer solution viscosity. However, electrical conductivity decreased and mean diameter of nanofibers increased from 89 nm to 371 nm. There was a robust interaction between the catechin and polymer matrix; also addition of catechin improved thermal stability of nanofibers. In general, at higher catechin levels, despite increasing loading capacity, encapsulation efficiency was significantly reduced (p < 0.05). Optimum nanofibers loaded with 500 and 1000 mg L−1 catechin can be considered to apply in active food packaging and pharmaceutical applications.
1. Introduction Nanotechnology has affected many aspects of agriculture and the food industry by creating new features of materials and structures. Nanoencapsulation is a novel technique for protecting and transferring bioactive compounds to the target point with the help of polymeric or lipidic walls (Assadpour & Jafari, 2019; Rezaei, Fathi, & Jafari, 2019). In this process, the entrapped material can be released under specific conditions at a controlled rate (Nedovic, Kalusevic, Manojlovic, Levic, & Bugarski, 2011). There are various methods for nanoencapsulation of bioactive compounds such as nano spray drying, nanoemulsification, coacervation, nanoprecipitation and nanoliposomal production, each of which has disadvantages such as the use of high temperatures, toxicity and solvent residues (Katouzian & Jafari, 2016; Rafiee, Nejatian, Daeihamed, & Jafari, 2019). Electrospun fibers can also be considered as carriers for food additives and other bioactive ingredients during food processes to produce functional foods (Ramakrishna et al., 2010; Weiss, Gaysinsky, Davidson, & McClements, 2009; Wongsasulak, Patapeejumruswong, Weiss, Supaphol, & Yoovidhya, 2010). Methods of nanofibers production include drawing, template synthesis, phase separation, self-assembly and electrospinning (Kriegel, Arrechi, Kit, McClements, &
⁎
Weiss, 2008). Among these methods, electrospinning is the most proficient method for the production of nanofibers and microfibers from all types of natural biopolymer solutions (Rostami, Yousefi, Khezerlou, Aman Mohammadi, & Jafari, 2019). As the fiber diameter decreases from micrometer to nanometer, the functional properties of fibers improve (McCann, Li, & Xia, 2005). In this regard, due to advantages such as elongation, low thickness, high surface-to-volume ratio, high porosity, non-contact production method and increased efficiency relative to other production procedures, the formation of electrospun nanofibers has been recognized as an innovative and attractive strategy for encapsulation purposes (Khoshnoudi-Nia, Sharif, & Jafari, 2020). In electrospinning process, polymeric fluid for fiber production is exposed to very high voltage electric fields and then charged fluid forms a fluid jet that moves to the negative pole of a collector plate (Kanafchian, Valizadeh, & Haghi, 2011). Polysaccharides are divided into three groups based on their behavior during the electrospinning process: those with capability of fiber formation, only jet formation, and the third category without jet formation ability (Stijnman, Bodnar, & Tromp, 2011). Gums are a broad group of polysaccharides used as a new source of biopolymers for the production of nanofibers (Taheri & Jafari, 2019). Azivash (Corchorus olitorius L.) is an edible and medicinal plant found in tropical countries
Corresponding author. E-mail address: [email protected] (S.M. Jafari).
https://doi.org/10.1016/j.carbpol.2020.115979 Received 10 November 2019; Received in revised form 9 February 2020; Accepted 10 February 2020 Available online 11 February 2020 0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
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undesirable conditions due to high loading capacity, ability to produce various compounds at the same time, simplicity of operation and costeffectiveness. Neo et al. (2013) investigated nanoencapsulation of gallic acid in zein polymer matrix through electrospinning process; addition of gallic acid due to intermolecular reactions increased viscosity of polymer solution and thus increased diameter of nanofibers but had no effect on antioxidant properties (Neo et al., 2013). Shao et al. (2018) reported nanoencapsulation of tea polyphenols in carboxymethyl cellulose and pullulan mixture due to increased polymer chain entanglement and reduced electrical conductivity of electrospinning increased viscosity of polymer solution (Shao et al., 2018). The purpose of the present research is to apply the electrospinning process as a simple, fast and single-step method for nanoencapsulation of catechin in Azivash gum-PVA nanofibers and investigating the effect of catechin concentration on polymeric properties and morphology of nanofibers for food packaging and nutraceutical delivery applications.
of Asia and Africa. The leaf gum of Azivash is non-toxic with a high molecular weight about 940 kDa (Yamazaki, Kurita, & Matsumura, 2008). Azivash gum is capable of jet formation but does not form fibers. It is reported that the hydrocolloid viscosity of Azivash at 0.5 % (w/w) concentrations is higher than two other commercial hydrocolloids namely agar gum and ceratonia siliqua seed gum, under similar conditions (Yamazaki et al., 2008). Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer, nontoxic, and biodegradable with good mechanical properties. The presence of hydroxyl groups in the PVA structure gives it an ability to form a gel network which facilitates the electrospinning process (Fahami & Fathi, 2018). Some recent studies have concentrated on a combination of PVA as an aid material in the electrospinning process with various gums. In this regard, Fahami and Fathi (2018) used cress seed gum in combination with PVA to produce electrospun nanofibers; these researchers succeeded in producing the lowest diameter nanofibers from cress seed mucilage: PVA at the ratio of 60:40 so that PVA incorporation improved the stability of nanofibers (Fahami & Fathi, 2018). Similar results were reported for almond gum (Rezaei, Tavanai, & Nasirpour, 2016). In another study conducted by Kurd et al. (2017), the electrospinning of basil seed gum in combination with PVA was proposed to protect bioactive compounds. They showed the mixing ratio of 70:30 at different concentrations of basil seed gum, produced nanofibers with diameter range of 179–390 nm (Kurd, Fathi, & Shekarchizadeh, 2017). Islam and Karim (2010) also used combination of PVA (10 % w/v) and alginate gum (2 w/v) to produce electrospun nanofibers with high thermal stability and good mechanical properties. Sousa et al. (2015) used PVA (10 % w/w) combined with agar gum (1 w/v). They showed that PVA (70): agar gum (30) was the optimum ratio for nanofiber production (Sousa et al., 2015). Padil, Senan, Wacſawek, and Ŀerník (2016) used Arabic, karaya and Kondagogu gums mixed with PVA for electrospinning. At optimum ratios of PVA blend with these gums (70:30, 80:20, 10:90), non-spherical nanofibers with mean diameter of 240, 220 and 210 nm were obtained, respectively (Padil et al., 2016). In the electrospinning setup, the main factors that govern the morphology and properties of the nanofibers are processing parameters, polymer properties, and spinning solution properties (Kanafchian et al., 2011). The polymer solution in electrospinning must have appropriate molecular weight to provide sufficient viscosity for stable jet formation; higher concentration and molecular weight increases entanglement of polymer chains needed for a fiber strand to be drawn out of the droplet and amplified toward collector plate. However, concentrations above threshold lead to the formation of thicker fibers, and concentration < threshold results in formation of beads or very fine fibers along with the spindle structures (Jafari, 2017). Shao, Niu, Chen, and Sun (2018) investigated the effect of electrospinning parameters, voltage and flow rate on the diameter of the nanofibers made from carboxymethyl cellulose-pullulan and tea polyphenols; as the voltage increased from 19 to 21 kV, diameter of the nanofibers reduced from 187 to 92 nm, but beaded-nanofibers were created with higher increase in voltage up to 24 kV. Furthermore, nanofiber diameter increased from 106 to 161 nm with increasing volume flow rate from 0.36 to 0.6 mL h−1 (Shao et al., 2018). Rezaei et al. (2016) reported the effect of process conditions on the production of almond gum nanofibers with PVA; they stated as voltage increases from 12 to 18 kV due to the stronger electrostatic field, jet elongation and drawing fiber, mean diameter decreases (Rezaei et al., 2016). Application of synthetic antioxidants in food products due to potential health risks has a legal restriction, so researchers are considering to replace synthetic antioxidants with natural ones. Phenolic compounds such as tea catechins are a good choice for this purpose (Sabaghi, Maghsoudlou, Khomeiri, & Ziaiifar, 2015). Catechin is unstable under processing conditions such as temperature, pH and enzymes or storage conditions such as light and oxygen (Shi et al., 2018). Production of nanofibers containing catechin via electrospinning process could be suggested as an effective method for their protection from
2. Materials and methods Azivash leaves were purchased from a local market (Gorgan, Iran(, Polyvinyl alcohol with, Mw = 124 K Da was purchased from Sigma Aldrich (USA), deionized distilled water (D.D.W) from Zolal company (Iran), green tea catechin from Chemsavers Company (USA), ethanol 99 %, isopropanol and other chemicals were provided by Merck (Germany). 2.1. Azivash gum extraction The leaves of Azivash were powdered after drying at ambient temperature and screened through a mesh (0.425 mm sieve size). The powder was kept in low-density polyethylene bags and stored in a dry and cool place until future applications. Extraction of gums from Azivash leaf was done according to the method of Brummer et al. (2003) with slight modification. Azivash leaf powder was dissolved in distilled water at a ratio of 1:30 (w/v) on a magnetic stirrer (Alpha, D500, Iran) at 700 rpm (ambient temperature for 45 min). The mixture was passed through a filter cloth to separate slurry. For purification, this solution was centrifuged at 4042 g for 40 min (Centurion Scientific, K240, UK). The extracted gum was mixed with ethanol at -18 °C at a ratio of 30:70 (v/v) and then rinsed three times with pure isopropanol at 50:50. Finally, it was dried by freeze dryer (Operon, FDB5503, South Korea) under vacuum (0.01 Pa and -48 °C) until completely dried. The extraction efficiency of gum from Azivash leaf was 5.4 ± 0.4 % d.w (Brummer, Cui, & Wang, 2003). 2.2. Preparation of Azivash gum-PVA solutions Azivash gum (AZG) solutions with a concentration of 2 g L −1 were prepared by dissolving AZG in 100 mL D.D.W at 40 °C using a magnetic stirrer at 700 rpm for 12 h to obtain a homogenized solution. Then the solution was left in a refrigerator (4 °C) overnight for complete hydration. PVA solution (80 g L−1) was mixed with D.D.W at 80 °C under magnetic stirrer 700 rpm for 4 h. Polymeric solutions were prepared by mixing AZG solutions with PVA solutions at ratio of 70:30 for 12 h using a magnetic stirrer at 700 rpm. Finally, polymeric solutions were combined with different concentrations of catechin (500, 1000, 2000, and 3000 mg L−1). 2.3. Properties of AZG-PVA solutions for electrospinning 2.3.1. Apparent viscosity Viscosity test was performed using a rotational viscometer (Brookfield, DV-II + pro, USA). Shear rate, shear stress and viscosity of AZG-PVA solutions containing different catechin levels (0, 500, 1000, and 2000 mg L−1) were measured. Samples were poured into a cylinder (16 mL capacity: ULA-31Y Brookfield) for viscosity measurement at 2
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Vegall, Germany. Nanofibers were coated with a thin layer of gold sheet. Images were taken at an acceleration voltage of 30 kV and magnification of 10,000 times. The mean diameter of 100 fibers was determined using Digimizer software version 2018.
27 °C. Three equations including Herschel Bulkley (Eq. (1)), Power law (Eq. (2)) and Casson (Eq. (3)) were used to fit the experimental data of shear stress to shear rate using MATLAB software (R2010a) for various electrospinning solutions.
τ = τ0H + KH Y nH
(1)
γ np
(2)
τ = Kp
τ 0.5 = τ0C 0.5 + ɳcγ 0.5
2.6.2. Attenuated Total Reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) ATR-FTIR spectrum in the transient state was determined using IR spectrophotometer (Equinox 55-LSI 01, Bruker, UK) in a range of 500 to 4000 cm−1 (Rezaei et al., 2016).
(3) n
where K is the consistency coefficient (Pa.s ), τ0 is the yield stress (Pa), n is the flow behavior index, ɳc is plastic viscosity and γ is the shear rate (s−1).
2.6.3. Thermogravimetric analysis (TGA) Thermal stability/degradation behavior of nanofibers was investigated using a thermogravimetric analyzer (TGA 8000™, Perkin Elmer, USA) at a heating rate of 10 °C min−1 in a nitrogen-containing space at temperature range of 30–700 °C (Varsha, Bajpai, & Navin, 2010).
2.3.2. Electrical conductivity To measure electrical conductivity, 20 mL of different AZG-PVA solutions were poured in a beaker and electrical conductivity was measured with a conductivity meter (Lutron, TM-947SD, Taiwan) (Fahami & Fathi, 2018).
2.7. Statistical analysis 2.4. Electrospinning process To investigate the effect of electrospinning process factors (voltage, flow rate and tip needle-to-collector), Response Surface Methodology (RSM) was applied using constant central composite design (α = 1) by Design Expert software (version 10.7). For this purpose, voltage (16−20 kV), tip needle-to-collector (10−14 cm) and flow rate (0.50.9 mL h−1) were considered as independent variables and diameter changes as the response. Finally, optimum conditions for the device to produce nanofibers with minimum mean diameter of nanofibers was, voltage 20 kV, needle to collector distance 11 cm and flow rate 0.5 mL h−1. Statistical analysis was performed using One-way ANOVA by SAS version 9 (p > 0.05). Charts were plotted using Excel 2013 and modeling of data was done using MATLAB 2010.
In this process, a 10 mL syringe was filled by polymeric solutions and pumped through a G18 needle of the electrospinning device. Positive and negative electrodes were attached to the needle and collector (rotating cylinder), respectively. In order to evaluate the effect of processing factors on the morphology of fibers, response surface methodology (RSM) was applied using faced-center central composite design (α = 1) by Design Expert software (version 10.7). For this purpose, voltage (16−20 kV), needle-to-collector distance (10−14 cm) and flow rate (0.5-0.9 mL h−1) were considered as independent variables and fiber size as the response factor. Finally, optimum conditions for electrospinning to produce nanofibers with the minimum mean diameter were obtained as voltage 20 kV, needle to collector distance 11 cm and flow rate of 0.5 mL h−1.
3. Results and discussion
2.5. Encapsulation efficiency and loading capacity of catechin-loaded nanofibers
3.1. The Effect of process variables on the mean diameter of electrospun fibers
Encapsulation efficiency was determined through determination of unloaded catechin via washing 15 mg nanofibers with 15 mL of 99 % ethanol and measuring the amount of catechin released from the surface of nanofibers. Then after complete dissolution of the nanofibers in water, total catechin content was measured via the Folin-Ciocalteu method. Finally, encapsulation efficiency (Eq. (4)) and loading capacity (Eq. (5)) of catechin was determined (Qi et al., 2010).
With increasing the voltage and decreasing the flow rate and needleto-collector distance, diameter of nanofibers decreased (Table 1A). Voltage of 20 kV, needle to collector distance of 11 cm and flow rate of 0.5 mL h−1 were the optimum conditions suggested by RSM to produce
Encapsulation efficiency Total catechin content− Surface catechin content = × 100 Total catechin content
loading capacity=
Total weight of catechin × 100 weight of the nanofibers
Table 1A Design of experiments and experimental results based on RSM. Run
Factor 1 A: Voltage (kV)
Factor 2 B: Distance (cm)
Factor 3 C: Flow rate (mL h−1)
Response Diameter (nm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
20.00 20.00 20.00 18.00 16.00 18.00 20.00 18.00 18.00 18.00 18.00 18.00 20.00 18.00 16.00 16.00 18.00 16.00 16.00 18.00
14.00 10.00 10.00 12.00 10.00 12.00 12.00 12.00 12.00 14.00 12.00 12.00 14.00 12.00 14.00 10.00 12.00 12.00 14.00 10.00
0.90 0.50 0.90 0.70 0.90 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.50 0.50 0.50 0.50 0.90 0.70 0.90 0.70
198 97 155 138 172 147 126 137 141 144 146 157 114 120 161 157 145 172 185 142
(4)
(5)
2.5.1. Determination of catechin content by Folin-Ciocalteu method In this method, 0.25 mL of each solution was mixed with 1.25 mL Folin-Ciocalteu reagent and after 8 min, 1 mL of sodium carbonate solution (75 g L−1) was added. The test tubes were shaken in water bath at 40 °C and the absorbance was recorded after 60 min using a spectrophotometer at 765 nm. Amount of catechin (g kg−1) was calculated according to the standard curve equation (y = 0.0775 + 0.9696, R2 = 0.9725) (Kodama, Gonçalves, Lajolo, & Genovese, 2010). 2.6. Analysis of produced electrospun nanofibers loaded with catechin 2.6.1. Scanning electron microscopy (SEM) The morphology of nanofibers was investigated by SEM Tescan, 3
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Table 1B ANOVA for quadratic model (Response: Diameter). Source
Sum of Squares
df
Mean Square
F-value
p-value
Model A-VOLTAGE B-DISTANCE C-FLOW RATE AB AC BC A² B² C² Residual Lack of Fit Pure Error Cor Total Fit Statistics Std. Dev. Mean C.V. %
9874.25 2464.90 624.10 4243.60 231.13 1326.13 153.13 369.46 85.96 66.27 885.95 610.62 275.33 10760.20
9 1 1 1 1 1 1 1 1 1 10 5 5 19
1097.14 2464.90 624.10 4243.60 231.13 1326.13 153.13 369.46 85.96 66.27 88.60 122.12 55.07
12.38 27.82 7.04 47.90 2.61 14.97 1.73 4.17 0.9703 0.7480
0.0003 0.0004 0.0241 < 0.0001 0.1373 0.0031 0.2180 0.0684 0.3478 0.4074
significant
2.22
0.2013
not significant
R² Predicted R² Adeq Precision
0.9177 0.5269 14.0482
9.41 147.70 6.37
(D) and flow rate (FR) on fiber diameter (Y) was the following equation with the correlation coefficient (R2 = 91.77) and (p-value = 0.0003).
nanofibers with the minimum mean diameter. The predicted results for the optimum conditions were in agreement with the experimental values. According to the results of the present study, the most important parameter in reducing the diameter of electrospun fibers was voltage (Table 1B). Creation of electrostatic repulsive forces due to increased voltage, overcomes the viscosity and surface tension forces of the polymer solution, causing the polymer jet to be pulled out of the capillary needle and formation of thinner fibers (Thenmozhi, Dharmaraj, Kadirvelu, & Kim, 2017). In general, the results showed that by increasing the voltage and simultaneously decreasing the flow rate, fiber diameter was decreased significantly. Similarly, Lin et al. (2008) concluded that increasing the applied voltage until 29.7 kV led to the reduction of polyether sulfon fibers diameter, but voltage goes beyond this range due to reduced flight time and by influencing on the electrostatic forces, fiber diameter increases (Lin et al., 2008). Kurd et al. (2016) obtained optimum voltage of 18 kV in the electrospinning process of polyvinyl alcohol-basil seed mucilage for producing smaller diameter uniform fibers; their results revealed with increasing voltage to 23 kV, fiber diameter increased. They concluded higher ratio of gum in polymer mixture and voltage increased the diameter of nanofibers (Lin et al., 2008). In the present investigation, the lowest average diameter of nanofibers was obtained at voltage of 20 kV and a flow rate of 0.5 mL h−1. At constant distance, increasing the voltage, overcomes the electrostatic repulsion force on droplets and elongation polymer jet, thus the mean diameter of fiber begins to decrease consequently (Ghorani & Tucker, 2015; Thenmozhi et al., 2017). At high volume flow rates and more applied voltage, high feed rate of solution is ejected from the tip of needle, thereby increasing the diameter of fibers or formed beaded fibers; so a high flow rate produces higher diameter fibers (Ballengee & Pintauro, 2011; Soleimanifar, Jafari, & Assadpour, 2020). In the present study, increasing flow rate from 0.5 to 0.9 mL h-1 increased the diameter of the nanofibers. Zong et al. (2002) reported that at high volumetric flow intensities, time for solvent evaporation is too low, leading to large bead formation and fiber breakage (Zong et al., 2002). According to suggested parameters via RSM, at a lower volume flow rate and higher voltage to obtain minimum mean diameter nanofibers, minimum distance from needle to collector must be increased slightly (Fig. 1). The flight time increases as the distance between the needle and the collector increases; as a result, the solvent evaporates well, thus reduces bead formation and mean diameter of fibers (Ahn et al., 2006; Jafari, 2017). Finally, according to the analysis of multivariable variance, predicted model for the effect of voltage (V), distance of needle-to-collector
Y= 141.6 1-15.70V+7.90D+20.6FR + 12.88V*FR
(6)
Based on the numerical optimization results, the lowest mean diameter of the electrospun nanofibers, 95.76 nm was predicted at a flow rate of 0.5 mL h−1, voltage of 20 kV and needle to collector distance of 11 cm; with applying the suggested processing conditions predicted by RSM, a mean diameter of 89.44 nm was obtained experimentally. 3.2. Effect of different levels of catechin on the properties of AZG-PVA solutions 3.2.1. Apparent viscosity Determination of material flow behavior is essential in designing processes such as fluid flow, pumping power, and predictability of the electrospinning potential of polymeric solutions (Maghsoudlou, Sabaghi, & Kashiri, 2018; Sousa et al., 2015). The viscosity of electrospinning solutions in combination with different concentrations of catechin (0, 500, 1000, 2000 and 3000 mg L−1) are shown in Fig. 2. The addition of catechin caused a significant change in viscosity from 14.2 MPa in catechin-free solution to 14.9 MPa in 500 mg L−1 catechin solution (p < 0.05). Peres et al. (2011) reported a similar result in the nanoencapsulation of catechin in maltodextrin-Arabic gum solutions (Peres et al., 2011). The results of Shao et al. (2018) showed that the addition of tea polyphenols to CMC-pullulan solution increased the viscosity of the polymeric solution. In general, it can be concluded that the increase in the viscosity of the solution is due to higher entanglement of polymer chains (Shao et al., 2018). In our study, the viscosity increased significantly (p < 0.05) at higher catechin levels from 14.9 MPa (500 mg L−1 catechin) to 18.1 MPa (3000 mg L−1 catechin). Previous research has shown that polyphenolic compounds derived from green tea extract enhanced the film structure due to interaction with polymers and creation of new bonds in polymer structure (Stijnman et al., 2011). Investigation of rheological behavior of electrospinning solutions revealed pseudoplastic behavior; with increasing shear rate, the apparent viscosity decreased, indicating shear thinning behavior of nanofiber solutions with different concentrations of catechin. Shear stressshear rate data were fitted with Power law, Herschel- Bulkley and Casson models. The Power law model is typically used to investigate the behavior of non-Newtonian fluids. According to the power law, all solutions showed shear thinning behavior. Higher catechin concentrations in AZG-PVA solutions decreased the flow behavior (n) index from 0.8679 to 0.7686 and consistency coefficient (K) from 0.221 to 4
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Fig. 1. Optimizing the diameter of electrospun nanofibers via device parameters (voltage, flow rate and distance of the tip needle to the collector) using response surface methodology.
0.4072 mPa.s, respectively. Rheological parameters obtained by fitting the Casson model showed that higher catechin levels in nanofibers solutions increased the Casson's viscosity from 1.18 to 1.461 mPa.s. Our results revealed that the Herschel- Bulkley equation was the best model to determine the flow behavior of nanofiber solutions (Table S1 in supplementary materials). This model was selected as the best model with the highest value of R2 to RMSE (Table S1) and experimental data were fitted to this model (Fig. 3). The Herschel-Bulkley model is commonly used to investigate the behavior of non-Newtonian fluids. Rheological parameters of Fitting Herschel- Bulkley model on experimental data of shear stress to shear rate at different concentrations of catechin are given in Table S2. Through examining the flow behavior index, it can be deduced that all
samples showed shear thinning behavior. Increasing the concentration of catechin in nanofiber solutions decreased the flow behavior index from 0.9019 in control to 0.6043 in 3000 mg L−1 catechin-containing samples. The data obtained from the Herschel-Bulkley model showed a direct effect on the increase in the coefficient of consistency from 0.1871 to 0.8774 mPa by catechin concentration. The increase in the consistency coefficient is due to the interaction between the polymeric solution and natural antioxidant catechin (Siripatrawan & Harte, 2010). 3.2.2. Electrical conductivity The electrical conductivity of nanofiber solutions significantly (P < 0.05) decreased at higher catechin levels from 375 μS cm−1 (0 mg L−1) to 348 μS. cm−1 (3000 mg L−1), as shown in Fig. 4. 5
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Fig. 4. Effect of different concentrations of catechin (0, 500, 1000, 2000 and 3000 mg L−1) in nanofiber solutions on their electrical conductivity.
viscosity and higher molecular entanglement and thereby, high resistance to jet elongation at the point of flight during the electrospinning process (Fahami & Fathi, 2018; Sabaghi et al., 2015). According to the results of SEM, 500 and 1000 mg L−1 catechin levels are the most suitable concentrations for the successful electrospinning process (Fig. 5).
Fig. 2. The effect of different concentrations of catechin (500, 1000, 2000 and 3000 mg L−1) on the viscosity of nanofiber solutions.
Similarly, Shao et al. (2018) reported that the addition of tea polyphenols to pullulan-CMC decreases the electrical conductivity of nanofiber solutions. This decrease in electrical conductivity at higher catechin levels may be due to metal ions chelating properties of green tea catechins that chelate metal ions in gum solution through the formation of surface complexes (Ndlovu & Afolayan, 2008; Sabaghi et al., 2015)
3.3.2. Encapsulation efficiency and loading capacity The results of the encapsulation efficiency and loading capacity of nanofibers loaded with different concentrations of catechin is depicted in Fig. 7. It is obvious that encapsulation efficiency significantly decreased from 99 % (catechin level of 500 mg L−1) to 78.4 % (catechin level of 2000 mg L−1), but further increase in catechin concentration to 3000 mg L−1 improved the percentage of encapsulation efficiency to 81 %. Actually, after electrospinning of solutions containing 3000 mg L−1 catechin, a transparent film was obtained; and as catechin enclosed in the inner structure of the film, a higher encapsulation efficiency was obtained. Also, with increasing catechin concentration, loading capacity improved significantly (p < 0.05) from 0.59 % (500 mg L−1 catechin) to 2.93 % (3000 mg L-1 catechin). As revealed in Fig. 7, increasing the loading capacity of catechin to about 5-fold changed the appearance and microstructure of nanofibers. ATR-FTIR spectroscopy confirmed interaction between catechin (especially at 3000 mg L−1) and polymer solutions during electrospinning (Section 3.3.3). In accordance with the results of the present study, Neo et al. (2013) found that at a higher gallic acid concentration in the electrospinning process of zein, nanoencapsulation efficiency decreased. The same trend was also reported about decreasing encapsulation efficiency and increasing the loading capacity of limonene via electrospinning in Alyssum homolocarpum seed gum (Khoshakhlagh,
3.3. Properties of the catechin-loaded Azivash gum-PVA electrospun nanofibers 3.3.1. Morphology of nanofibers SEM results (Fig. 5) revealed that the mean diameter of the electrospun nanofibers increased from 89 nm (control sample) to 371 nm (containing 3000 mg L−1 catechin) with the rise in catechin concentration. The simultaneous effects of viscosity and electrical conductivity of nanofibers on their diameter is shown in Fig. 6. As can be seen, due to increases in viscosity and decrease in electrical conductivity, the mean diameter of the electrospun nanofibers increased. On the other hand, catechin concentration above 2000 mg L−1 resulted in a reduction of density and a higher mean diameter of nanofibers along with fiber discoloration from white to transparent sheets. Increasing diameter of nanofibers at higher catechin content might be due to increased
Fig. 3. Shear stress-shear rate (left) and viscosity-shear rate changes (right) in nanofiber solutions incorporating different concentrations of catechin (0, 500, 1000, 2000 and 3000 mg L−1). 6
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Fig. 5. Scanning electron microscopy of nanofibers loaded with different concentrations of catechin (0, 500, 1000, 2000 and 3000 mg L−1); voltage 30 kV, 10,000×.
alcohol, Azivash gum, and catechin during electrospinning (Fig. 8). Hydroxyl groups (OeH) were detected in PVA at 3276 cm−1. The symmetric and asymmetric stretching CH2 correspond to the bands of 2864 and 2942 cm−1, respectively. The peak of 1166 cm−1 due to stretching of acetate groups and 818 cm−1 due to CeC vibrations were revealed in structure. Similar findings for PVA have been reported by (Maghsoudlou et al., 2018). The characteristic peak of catechin was observed at 1136 cm−1 (alcohol, CeOeH) and 1519 cm−1 (C]C aromatic ring) (Lin, Feng, Lai, Lin, & Chen, 2014). In Azivash gum powder, the peak area of 1602 cm−1 was related to symmetric stretching carboxyl and peak of 1023 cm−1 and 1416 cm−1 was related to symmetric stretching of CeO groups of uronic acid. Peaks in the 900−1250 cm−1 region due to CeO, CeOeC vibrations of the glycosidic bond and CeOeH bonds. The peak area of 1732 cm−1 was due to the symmetric stretching of the CeOO group of acetyl and carboxylic acid, which could be indicative of the presence of both uronic acid and carboxylic acid in the gum structure. The peak observed at 2992 cm−1 was related to C–H junctions with stretching vibrations including C–H, C–H2 and C–H3 in C-6 glycosidic units (Rezaei et al., 2016). Peaks of region 3100 cm−1 are due to the stretching vibrations of OeH in gum structure, which are caused by intermolecular and intramolecular hydrogen bonds. In general, these two peaks are considered to be prominent functional groups in the gum structure. The change in peaks of basic functional groups is due to an interaction between the nanofiber component (Martins et al., 2012). As shown in Fig. 8, the band intensities increased between 784 cm−1 to 907 cm−1 when adding gum to the PVA solution. In electrospun nanofibers, the intensity of bands increased in the region of 1212−1309 cm−1; the presence of gum increased glycosidic vibrations
Fig. 6. Relationship between the electrical conductivity and viscosity of nanofiber solutions incorporating different concentrations of catechin (500, 1000, 2000 and 3000 mg L−1) along with the diameter of electrospun nanofibers.
Koocheki, Mohebbi, & Allafchian, 2017; Neo et al., 2013). Considering the high encapsulation efficiency (up to 99 %) at 500 mg L−1 catechin (Fig. 6), it is confirmed that electrospinning can be suggested as a single-step and effective method for nanoencapsulation of catechin.
3.3.3. ATR-FTIR results ATR-FTIR spectroscopy was performed to identify any chemical interactions and changes in functional groups regarding polyvinyl 7
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Fig. 7. Encapsulation efficiency and loading capacity of nanofibers containing different concentrations of catechin (500, 1000, 2000 and 3000 mg L−1). Non-similar letters indicate significant differences in treatments at 95 % confidence level.
Fig. 9. Thermogravimetric analysis of Azivash gum powder (—), polyvinyl alcohol nanofibers (-.-. -), Azivash gum- polyvinyl alcohol nanofibers (-), and Azivash gum-polyvinyl alcohol nanofibers containing catechin at a concentration of 1000 mg L−1 (…).
gum powder, respectively. It should be noted that the second stage for PVA began at 338 °C and the rate of thermal degradation reached 92.63 % of the initial weight. The third stage of thermal degradation began at about 348 °C, which might be due to the depolymerization of polymer chains. Under these conditions, sample containing 1000 mg L−1 catechin and the control reached 39 % and 25 % of initial weight. This stage for PVA began at 529 °C and the rate of thermal degradation reached 22.16 % of its initial weight. Arrieta et al. (2014) showed that the addition of catechin into polypropylene films increased thermal resistance compared to the control samples (Arrieta et al., 2014). The presence of tea catechin in fibers increased the thermal degradation temperature. In general, the thermal stability of fibers containing catechin was higher than that of catechin free fibers (Fig. 9). Results of Shao et al. (2018) showed green tea extract significantly increased the thermal stability of PVA films compared to the control sample. According to ATR-FTIR spectroscopy, it can be concluded that interaction between polymer solutions (Azivash gum-PVA) and catechin is mainly hydrogen bonding which caused adhesion between molecule chains and relatively higher thermal resistance of functional nanofibers.
Fig. 8. Fourier transform infrared (FTIR) spectra of electrospun nanofibers.
of CeO, CeCeO and CeOeH bands. The addition of catechin to the nanofibers increased the intensity of peaks associated with the OeH group (3276 cm−1 region), probably due to the interaction of hydroxyl groups in the catechin structure with PVA-Azivash gum mixture. Terao et al. (2009) obtained similar results with the addition of catechin into PVA film (Terao et al., 2009). ATR-FTIR spectra showed that with increasing catechin concentration, more interaction between catechin (especially at 3000 mg L−1) and polymer wall occurred which is also reflected in SEM results.
4. Conclusion 3.3.4. Thermal stability results The thermogravimetric analysis of Azivash gum powder, Azivash gum –PVA nanofibers (control), and Azivash gum-PVA-1000 mg L−1 catechin are shown in Fig. 9. According to thermograms, three main stages were observed in samples. Weight loss in the first stage of the thermal process which could be due to the removal of moisture and other volatile compounds from the sample (Varsha et al., 2010); it was a minimum for catechin-loaded Azivash gum-PVA fibers. The second stage began at 280 °C and about 86 %, 81 % and 63 % weight loss were recorded for catechin-loaded Azivash gum-PVA fiber, control fiber and
In the present study, nanoencapsulation of catechin in nanofibers of Azivash gum- polyvinyl alcohol was performed via electrospinning. Results of numerical optimization (RSM) of three variables, voltage, flow rate and distance of needle to the collector with the aim of achieving the minimum diameter of final nanofibers revealed the optimum electrospinning conditions as a flow rate of 0.5 mL h−1, voltage=20 kV and distance of needle to the collector = 11 cm. Highest voltage overcame electrostatic repulsion force on the droplets and elongation polymer jet; also lower flow rate and optimum distance from 8
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needle to collector caused enough time for solvent evaporation, so decreased nanofiber diameter. Increasing catechin concentration from 500 to 2000 mg L−1, due to the entrapment of catechin in the inner structure of the films increased the encapsulation efficiency. Higher catechin levels from 500 to 3000 mg L-1 resulted in about 5-fold increase in the loading capacity of nanofibers, which affected the appearance and microstructure of nanofibers. As the concentration of catechin increased, more interaction was found between hydroxyl groups in the catechin structure and the polymer wall. Catechin improved the thermal resistance of nanofibers due to interaction with the polymer solution through hydrogen bonds and increased adhesion between molecular chains. Based on the results of this study, the electrospinning process can be suggested as a single-step method with minimum deleterious effects for nanoencapsulation of bioactive compounds such as phenolic compounds in order to design active food packaging and for pharmaceutical applications.
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CRediT authorship contribution statement Seyedeh Zahra Hoseyni: Data curation, Investigation. Seid Mahdi Jafari: Conceptualization, Project administration, Resources, Supervision. Hoda Shahiri Tabarestani: Validation, Visualization, Supervision. Mohammad Ghorbani: Formal analysis. Elham Assadpour: Formal analysis. Moslem Sabaghi: Methodology. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2020.115979. References Ahn, Y., Park, S., Kim, G., Hwang, Y., Lee, C., Shin, H., et al. (2006). Development of high efficiency nanofilters made of nanofibers. Current Applied Physics, 6(6), 1030–1035. Arrieta, M. P., Castro-Lopez, M.d. M., Rayón, E., Barral-Losada, L. F., López-Vilariño, J. M., López, J., et al. (2014). Plasticized poly (lactic acid)–poly (hydroxybutyrate)(PLA–PHB) blends incorporated with catechin intended for active food-packaging applications. Journal of Agricultural and Food Chemistry, 62(41), 10170–10180. Assadpour, E., & Jafari, S. M. (2019). A systematic review on nanoencapsulation of food bioactive ingredients and nutraceuticals by various nanocarriers. Critical Reviews in Food Science and Nutrition, 59(19), 3129–3151. Ballengee, J., & Pintauro, P. (2011). Morphological control of electrospun Nafion nanofiber mats. Journal of the Electrochemical Society, 158(5), B568–B572. Brummer, Y., Cui, W., & Wang, Q. (2003). Extraction, purification and physicochemical characterization of fenugreek gum. Food Hydrocolloids, 17(3), 229–236. Fahami, A., & Fathi, M. (2018). Fabrication and characterization of novel nanofibers from cress seed mucilage for food applications. Journal of Applied Polymer Science, 135(6), 45811. Ghorani, B., & Tucker, N. (2015). Fundamentals of electrospinning as a novel delivery vehicle for bioactive compounds in food nanotechnology. Food Hydrocolloids, 51, 227–240. Islam, Md. S., & Karim, M. (2010). Fabrication and characterization of poly(vinyl alcohol)/ alginate blend nanofibers by electrospinning method. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 366(1–3), 135–140. Jafari, S. M. (2017). Nanoencapsulation technologies for the food and nutraceutical industries. Academic Press. Kanafchian, M., Valizadeh, M., & Haghi, A. K. (2011). Electrospun nanofibers with application in nanocomposites. The Korean Journal of Chemical Engineering, 28(2), 428–439. Katouzian, I., & Jafari, S. M. (2016). Nano-encapsulation as a promising approach for targeted delivery and controlled release of vitamins. Trends in Food Science & Technology, 53, 34–48. Khoshakhlagh, K., Koocheki, A., Mohebbi, M., & Allafchian, A. (2017). Development and characterization of electrosprayed Alyssum homolocarpum seed gum nanoparticles for encapsulation of d-limonene. Journal of Colloid and Interface Science, 490, 562–575. Khoshnoudi-Nia, S., Sharif, N., & Jafari, S. M. (2020). Loading of phenolic compounds into electrospun nanofibers and electrosprayed nanoparticles. Trends in Food Science & Technology, 95, 59–74. Kodama, D. H., Gonçalves, A. E.d. S. S., Lajolo, F. M., & Genovese, M. I. (2010). Flavonoids, total phenolics and antioxidant capacity: Comparison between commercial green tea preparations. Food Science and Technology (Campinas), 30(4), 1077–1082. Kriegel, C., Arrechi, A., Kit, K., McClements, D., & Weiss, J. (2008). Fabrication, functionalization, and application of electrospun biopolymer nanofibers. Critical Reviews in Food Science and Nutrition, 48(8), 775–797. Kurd, F., Fathi, M., & Shekarchizadeh, H. (2017). Basil seed mucilage as a new source for electrospinning: Production and physicochemical characterization. International Journal of Biological Macromolecules, 95, 689–695. Lin, Y.-H., Feng, C.-L., Lai, C.-H., Lin, J.-H., & Chen, H.-Y. (2014). Preparation of
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Production and characterization of catechin-loaded electrospun nanofibers from Azivash gum- polyvinyl alcohol
T
Seyedeh Zahra Hoseyni, Seid Mahdi Jafari*, Hoda Shahiri Tabarestani, Mohammad Ghorbani, Elham Assadpour, Moslem Sabaghi Faculty of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Azivash gum Polyvinyl alcohol Catechin Encapsulation Electrospinning
In this study, Response Surface Methodology was used to optimize the electrospinning process parameters including voltage, distance, and flow rate in order to obtain catechin-loaded electrospun nanofibers from Azivash (Corchorus olitorius. L) gum-polyvinyl alcohol with the minimum diameter of nanofibers. The optimum electrospinning conditions were applied for catechin encapsulation at different loading concentrations (500, 1000, 2000 and 3000 mg L−1). According to the results, increase in catechin concentration led to increment in polymer solution viscosity. However, electrical conductivity decreased and mean diameter of nanofibers increased from 89 nm to 371 nm. There was a robust interaction between the catechin and polymer matrix; also addition of catechin improved thermal stability of nanofibers. In general, at higher catechin levels, despite increasing loading capacity, encapsulation efficiency was significantly reduced (p < 0.05). Optimum nanofibers loaded with 500 and 1000 mg L−1 catechin can be considered to apply in active food packaging and pharmaceutical applications.
1. Introduction Nanotechnology has affected many aspects of agriculture and the food industry by creating new features of materials and structures. Nanoencapsulation is a novel technique for protecting and transferring bioactive compounds to the target point with the help of polymeric or lipidic walls (Assadpour & Jafari, 2019; Rezaei, Fathi, & Jafari, 2019). In this process, the entrapped material can be released under specific conditions at a controlled rate (Nedovic, Kalusevic, Manojlovic, Levic, & Bugarski, 2011). There are various methods for nanoencapsulation of bioactive compounds such as nano spray drying, nanoemulsification, coacervation, nanoprecipitation and nanoliposomal production, each of which has disadvantages such as the use of high temperatures, toxicity and solvent residues (Katouzian & Jafari, 2016; Rafiee, Nejatian, Daeihamed, & Jafari, 2019). Electrospun fibers can also be considered as carriers for food additives and other bioactive ingredients during food processes to produce functional foods (Ramakrishna et al., 2010; Weiss, Gaysinsky, Davidson, & McClements, 2009; Wongsasulak, Patapeejumruswong, Weiss, Supaphol, & Yoovidhya, 2010). Methods of nanofibers production include drawing, template synthesis, phase separation, self-assembly and electrospinning (Kriegel, Arrechi, Kit, McClements, &
⁎
Weiss, 2008). Among these methods, electrospinning is the most proficient method for the production of nanofibers and microfibers from all types of natural biopolymer solutions (Rostami, Yousefi, Khezerlou, Aman Mohammadi, & Jafari, 2019). As the fiber diameter decreases from micrometer to nanometer, the functional properties of fibers improve (McCann, Li, & Xia, 2005). In this regard, due to advantages such as elongation, low thickness, high surface-to-volume ratio, high porosity, non-contact production method and increased efficiency relative to other production procedures, the formation of electrospun nanofibers has been recognized as an innovative and attractive strategy for encapsulation purposes (Khoshnoudi-Nia, Sharif, & Jafari, 2020). In electrospinning process, polymeric fluid for fiber production is exposed to very high voltage electric fields and then charged fluid forms a fluid jet that moves to the negative pole of a collector plate (Kanafchian, Valizadeh, & Haghi, 2011). Polysaccharides are divided into three groups based on their behavior during the electrospinning process: those with capability of fiber formation, only jet formation, and the third category without jet formation ability (Stijnman, Bodnar, & Tromp, 2011). Gums are a broad group of polysaccharides used as a new source of biopolymers for the production of nanofibers (Taheri & Jafari, 2019). Azivash (Corchorus olitorius L.) is an edible and medicinal plant found in tropical countries
Corresponding author. E-mail address: [email protected] (S.M. Jafari).
https://doi.org/10.1016/j.carbpol.2020.115979 Received 10 November 2019; Received in revised form 9 February 2020; Accepted 10 February 2020 Available online 11 February 2020 0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
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undesirable conditions due to high loading capacity, ability to produce various compounds at the same time, simplicity of operation and costeffectiveness. Neo et al. (2013) investigated nanoencapsulation of gallic acid in zein polymer matrix through electrospinning process; addition of gallic acid due to intermolecular reactions increased viscosity of polymer solution and thus increased diameter of nanofibers but had no effect on antioxidant properties (Neo et al., 2013). Shao et al. (2018) reported nanoencapsulation of tea polyphenols in carboxymethyl cellulose and pullulan mixture due to increased polymer chain entanglement and reduced electrical conductivity of electrospinning increased viscosity of polymer solution (Shao et al., 2018). The purpose of the present research is to apply the electrospinning process as a simple, fast and single-step method for nanoencapsulation of catechin in Azivash gum-PVA nanofibers and investigating the effect of catechin concentration on polymeric properties and morphology of nanofibers for food packaging and nutraceutical delivery applications.
of Asia and Africa. The leaf gum of Azivash is non-toxic with a high molecular weight about 940 kDa (Yamazaki, Kurita, & Matsumura, 2008). Azivash gum is capable of jet formation but does not form fibers. It is reported that the hydrocolloid viscosity of Azivash at 0.5 % (w/w) concentrations is higher than two other commercial hydrocolloids namely agar gum and ceratonia siliqua seed gum, under similar conditions (Yamazaki et al., 2008). Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer, nontoxic, and biodegradable with good mechanical properties. The presence of hydroxyl groups in the PVA structure gives it an ability to form a gel network which facilitates the electrospinning process (Fahami & Fathi, 2018). Some recent studies have concentrated on a combination of PVA as an aid material in the electrospinning process with various gums. In this regard, Fahami and Fathi (2018) used cress seed gum in combination with PVA to produce electrospun nanofibers; these researchers succeeded in producing the lowest diameter nanofibers from cress seed mucilage: PVA at the ratio of 60:40 so that PVA incorporation improved the stability of nanofibers (Fahami & Fathi, 2018). Similar results were reported for almond gum (Rezaei, Tavanai, & Nasirpour, 2016). In another study conducted by Kurd et al. (2017), the electrospinning of basil seed gum in combination with PVA was proposed to protect bioactive compounds. They showed the mixing ratio of 70:30 at different concentrations of basil seed gum, produced nanofibers with diameter range of 179–390 nm (Kurd, Fathi, & Shekarchizadeh, 2017). Islam and Karim (2010) also used combination of PVA (10 % w/v) and alginate gum (2 w/v) to produce electrospun nanofibers with high thermal stability and good mechanical properties. Sousa et al. (2015) used PVA (10 % w/w) combined with agar gum (1 w/v). They showed that PVA (70): agar gum (30) was the optimum ratio for nanofiber production (Sousa et al., 2015). Padil, Senan, Wacſawek, and Ŀerník (2016) used Arabic, karaya and Kondagogu gums mixed with PVA for electrospinning. At optimum ratios of PVA blend with these gums (70:30, 80:20, 10:90), non-spherical nanofibers with mean diameter of 240, 220 and 210 nm were obtained, respectively (Padil et al., 2016). In the electrospinning setup, the main factors that govern the morphology and properties of the nanofibers are processing parameters, polymer properties, and spinning solution properties (Kanafchian et al., 2011). The polymer solution in electrospinning must have appropriate molecular weight to provide sufficient viscosity for stable jet formation; higher concentration and molecular weight increases entanglement of polymer chains needed for a fiber strand to be drawn out of the droplet and amplified toward collector plate. However, concentrations above threshold lead to the formation of thicker fibers, and concentration < threshold results in formation of beads or very fine fibers along with the spindle structures (Jafari, 2017). Shao, Niu, Chen, and Sun (2018) investigated the effect of electrospinning parameters, voltage and flow rate on the diameter of the nanofibers made from carboxymethyl cellulose-pullulan and tea polyphenols; as the voltage increased from 19 to 21 kV, diameter of the nanofibers reduced from 187 to 92 nm, but beaded-nanofibers were created with higher increase in voltage up to 24 kV. Furthermore, nanofiber diameter increased from 106 to 161 nm with increasing volume flow rate from 0.36 to 0.6 mL h−1 (Shao et al., 2018). Rezaei et al. (2016) reported the effect of process conditions on the production of almond gum nanofibers with PVA; they stated as voltage increases from 12 to 18 kV due to the stronger electrostatic field, jet elongation and drawing fiber, mean diameter decreases (Rezaei et al., 2016). Application of synthetic antioxidants in food products due to potential health risks has a legal restriction, so researchers are considering to replace synthetic antioxidants with natural ones. Phenolic compounds such as tea catechins are a good choice for this purpose (Sabaghi, Maghsoudlou, Khomeiri, & Ziaiifar, 2015). Catechin is unstable under processing conditions such as temperature, pH and enzymes or storage conditions such as light and oxygen (Shi et al., 2018). Production of nanofibers containing catechin via electrospinning process could be suggested as an effective method for their protection from
2. Materials and methods Azivash leaves were purchased from a local market (Gorgan, Iran(, Polyvinyl alcohol with, Mw = 124 K Da was purchased from Sigma Aldrich (USA), deionized distilled water (D.D.W) from Zolal company (Iran), green tea catechin from Chemsavers Company (USA), ethanol 99 %, isopropanol and other chemicals were provided by Merck (Germany). 2.1. Azivash gum extraction The leaves of Azivash were powdered after drying at ambient temperature and screened through a mesh (0.425 mm sieve size). The powder was kept in low-density polyethylene bags and stored in a dry and cool place until future applications. Extraction of gums from Azivash leaf was done according to the method of Brummer et al. (2003) with slight modification. Azivash leaf powder was dissolved in distilled water at a ratio of 1:30 (w/v) on a magnetic stirrer (Alpha, D500, Iran) at 700 rpm (ambient temperature for 45 min). The mixture was passed through a filter cloth to separate slurry. For purification, this solution was centrifuged at 4042 g for 40 min (Centurion Scientific, K240, UK). The extracted gum was mixed with ethanol at -18 °C at a ratio of 30:70 (v/v) and then rinsed three times with pure isopropanol at 50:50. Finally, it was dried by freeze dryer (Operon, FDB5503, South Korea) under vacuum (0.01 Pa and -48 °C) until completely dried. The extraction efficiency of gum from Azivash leaf was 5.4 ± 0.4 % d.w (Brummer, Cui, & Wang, 2003). 2.2. Preparation of Azivash gum-PVA solutions Azivash gum (AZG) solutions with a concentration of 2 g L −1 were prepared by dissolving AZG in 100 mL D.D.W at 40 °C using a magnetic stirrer at 700 rpm for 12 h to obtain a homogenized solution. Then the solution was left in a refrigerator (4 °C) overnight for complete hydration. PVA solution (80 g L−1) was mixed with D.D.W at 80 °C under magnetic stirrer 700 rpm for 4 h. Polymeric solutions were prepared by mixing AZG solutions with PVA solutions at ratio of 70:30 for 12 h using a magnetic stirrer at 700 rpm. Finally, polymeric solutions were combined with different concentrations of catechin (500, 1000, 2000, and 3000 mg L−1). 2.3. Properties of AZG-PVA solutions for electrospinning 2.3.1. Apparent viscosity Viscosity test was performed using a rotational viscometer (Brookfield, DV-II + pro, USA). Shear rate, shear stress and viscosity of AZG-PVA solutions containing different catechin levels (0, 500, 1000, and 2000 mg L−1) were measured. Samples were poured into a cylinder (16 mL capacity: ULA-31Y Brookfield) for viscosity measurement at 2
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Vegall, Germany. Nanofibers were coated with a thin layer of gold sheet. Images were taken at an acceleration voltage of 30 kV and magnification of 10,000 times. The mean diameter of 100 fibers was determined using Digimizer software version 2018.
27 °C. Three equations including Herschel Bulkley (Eq. (1)), Power law (Eq. (2)) and Casson (Eq. (3)) were used to fit the experimental data of shear stress to shear rate using MATLAB software (R2010a) for various electrospinning solutions.
τ = τ0H + KH Y nH
(1)
γ np
(2)
τ = Kp
τ 0.5 = τ0C 0.5 + ɳcγ 0.5
2.6.2. Attenuated Total Reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) ATR-FTIR spectrum in the transient state was determined using IR spectrophotometer (Equinox 55-LSI 01, Bruker, UK) in a range of 500 to 4000 cm−1 (Rezaei et al., 2016).
(3) n
where K is the consistency coefficient (Pa.s ), τ0 is the yield stress (Pa), n is the flow behavior index, ɳc is plastic viscosity and γ is the shear rate (s−1).
2.6.3. Thermogravimetric analysis (TGA) Thermal stability/degradation behavior of nanofibers was investigated using a thermogravimetric analyzer (TGA 8000™, Perkin Elmer, USA) at a heating rate of 10 °C min−1 in a nitrogen-containing space at temperature range of 30–700 °C (Varsha, Bajpai, & Navin, 2010).
2.3.2. Electrical conductivity To measure electrical conductivity, 20 mL of different AZG-PVA solutions were poured in a beaker and electrical conductivity was measured with a conductivity meter (Lutron, TM-947SD, Taiwan) (Fahami & Fathi, 2018).
2.7. Statistical analysis 2.4. Electrospinning process To investigate the effect of electrospinning process factors (voltage, flow rate and tip needle-to-collector), Response Surface Methodology (RSM) was applied using constant central composite design (α = 1) by Design Expert software (version 10.7). For this purpose, voltage (16−20 kV), tip needle-to-collector (10−14 cm) and flow rate (0.50.9 mL h−1) were considered as independent variables and diameter changes as the response. Finally, optimum conditions for the device to produce nanofibers with minimum mean diameter of nanofibers was, voltage 20 kV, needle to collector distance 11 cm and flow rate 0.5 mL h−1. Statistical analysis was performed using One-way ANOVA by SAS version 9 (p > 0.05). Charts were plotted using Excel 2013 and modeling of data was done using MATLAB 2010.
In this process, a 10 mL syringe was filled by polymeric solutions and pumped through a G18 needle of the electrospinning device. Positive and negative electrodes were attached to the needle and collector (rotating cylinder), respectively. In order to evaluate the effect of processing factors on the morphology of fibers, response surface methodology (RSM) was applied using faced-center central composite design (α = 1) by Design Expert software (version 10.7). For this purpose, voltage (16−20 kV), needle-to-collector distance (10−14 cm) and flow rate (0.5-0.9 mL h−1) were considered as independent variables and fiber size as the response factor. Finally, optimum conditions for electrospinning to produce nanofibers with the minimum mean diameter were obtained as voltage 20 kV, needle to collector distance 11 cm and flow rate of 0.5 mL h−1.
3. Results and discussion
2.5. Encapsulation efficiency and loading capacity of catechin-loaded nanofibers
3.1. The Effect of process variables on the mean diameter of electrospun fibers
Encapsulation efficiency was determined through determination of unloaded catechin via washing 15 mg nanofibers with 15 mL of 99 % ethanol and measuring the amount of catechin released from the surface of nanofibers. Then after complete dissolution of the nanofibers in water, total catechin content was measured via the Folin-Ciocalteu method. Finally, encapsulation efficiency (Eq. (4)) and loading capacity (Eq. (5)) of catechin was determined (Qi et al., 2010).
With increasing the voltage and decreasing the flow rate and needleto-collector distance, diameter of nanofibers decreased (Table 1A). Voltage of 20 kV, needle to collector distance of 11 cm and flow rate of 0.5 mL h−1 were the optimum conditions suggested by RSM to produce
Encapsulation efficiency Total catechin content− Surface catechin content = × 100 Total catechin content
loading capacity=
Total weight of catechin × 100 weight of the nanofibers
Table 1A Design of experiments and experimental results based on RSM. Run
Factor 1 A: Voltage (kV)
Factor 2 B: Distance (cm)
Factor 3 C: Flow rate (mL h−1)
Response Diameter (nm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
20.00 20.00 20.00 18.00 16.00 18.00 20.00 18.00 18.00 18.00 18.00 18.00 20.00 18.00 16.00 16.00 18.00 16.00 16.00 18.00
14.00 10.00 10.00 12.00 10.00 12.00 12.00 12.00 12.00 14.00 12.00 12.00 14.00 12.00 14.00 10.00 12.00 12.00 14.00 10.00
0.90 0.50 0.90 0.70 0.90 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.50 0.50 0.50 0.50 0.90 0.70 0.90 0.70
198 97 155 138 172 147 126 137 141 144 146 157 114 120 161 157 145 172 185 142
(4)
(5)
2.5.1. Determination of catechin content by Folin-Ciocalteu method In this method, 0.25 mL of each solution was mixed with 1.25 mL Folin-Ciocalteu reagent and after 8 min, 1 mL of sodium carbonate solution (75 g L−1) was added. The test tubes were shaken in water bath at 40 °C and the absorbance was recorded after 60 min using a spectrophotometer at 765 nm. Amount of catechin (g kg−1) was calculated according to the standard curve equation (y = 0.0775 + 0.9696, R2 = 0.9725) (Kodama, Gonçalves, Lajolo, & Genovese, 2010). 2.6. Analysis of produced electrospun nanofibers loaded with catechin 2.6.1. Scanning electron microscopy (SEM) The morphology of nanofibers was investigated by SEM Tescan, 3
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Table 1B ANOVA for quadratic model (Response: Diameter). Source
Sum of Squares
df
Mean Square
F-value
p-value
Model A-VOLTAGE B-DISTANCE C-FLOW RATE AB AC BC A² B² C² Residual Lack of Fit Pure Error Cor Total Fit Statistics Std. Dev. Mean C.V. %
9874.25 2464.90 624.10 4243.60 231.13 1326.13 153.13 369.46 85.96 66.27 885.95 610.62 275.33 10760.20
9 1 1 1 1 1 1 1 1 1 10 5 5 19
1097.14 2464.90 624.10 4243.60 231.13 1326.13 153.13 369.46 85.96 66.27 88.60 122.12 55.07
12.38 27.82 7.04 47.90 2.61 14.97 1.73 4.17 0.9703 0.7480
0.0003 0.0004 0.0241 < 0.0001 0.1373 0.0031 0.2180 0.0684 0.3478 0.4074
significant
2.22
0.2013
not significant
R² Predicted R² Adeq Precision
0.9177 0.5269 14.0482
9.41 147.70 6.37
(D) and flow rate (FR) on fiber diameter (Y) was the following equation with the correlation coefficient (R2 = 91.77) and (p-value = 0.0003).
nanofibers with the minimum mean diameter. The predicted results for the optimum conditions were in agreement with the experimental values. According to the results of the present study, the most important parameter in reducing the diameter of electrospun fibers was voltage (Table 1B). Creation of electrostatic repulsive forces due to increased voltage, overcomes the viscosity and surface tension forces of the polymer solution, causing the polymer jet to be pulled out of the capillary needle and formation of thinner fibers (Thenmozhi, Dharmaraj, Kadirvelu, & Kim, 2017). In general, the results showed that by increasing the voltage and simultaneously decreasing the flow rate, fiber diameter was decreased significantly. Similarly, Lin et al. (2008) concluded that increasing the applied voltage until 29.7 kV led to the reduction of polyether sulfon fibers diameter, but voltage goes beyond this range due to reduced flight time and by influencing on the electrostatic forces, fiber diameter increases (Lin et al., 2008). Kurd et al. (2016) obtained optimum voltage of 18 kV in the electrospinning process of polyvinyl alcohol-basil seed mucilage for producing smaller diameter uniform fibers; their results revealed with increasing voltage to 23 kV, fiber diameter increased. They concluded higher ratio of gum in polymer mixture and voltage increased the diameter of nanofibers (Lin et al., 2008). In the present investigation, the lowest average diameter of nanofibers was obtained at voltage of 20 kV and a flow rate of 0.5 mL h−1. At constant distance, increasing the voltage, overcomes the electrostatic repulsion force on droplets and elongation polymer jet, thus the mean diameter of fiber begins to decrease consequently (Ghorani & Tucker, 2015; Thenmozhi et al., 2017). At high volume flow rates and more applied voltage, high feed rate of solution is ejected from the tip of needle, thereby increasing the diameter of fibers or formed beaded fibers; so a high flow rate produces higher diameter fibers (Ballengee & Pintauro, 2011; Soleimanifar, Jafari, & Assadpour, 2020). In the present study, increasing flow rate from 0.5 to 0.9 mL h-1 increased the diameter of the nanofibers. Zong et al. (2002) reported that at high volumetric flow intensities, time for solvent evaporation is too low, leading to large bead formation and fiber breakage (Zong et al., 2002). According to suggested parameters via RSM, at a lower volume flow rate and higher voltage to obtain minimum mean diameter nanofibers, minimum distance from needle to collector must be increased slightly (Fig. 1). The flight time increases as the distance between the needle and the collector increases; as a result, the solvent evaporates well, thus reduces bead formation and mean diameter of fibers (Ahn et al., 2006; Jafari, 2017). Finally, according to the analysis of multivariable variance, predicted model for the effect of voltage (V), distance of needle-to-collector
Y= 141.6 1-15.70V+7.90D+20.6FR + 12.88V*FR
(6)
Based on the numerical optimization results, the lowest mean diameter of the electrospun nanofibers, 95.76 nm was predicted at a flow rate of 0.5 mL h−1, voltage of 20 kV and needle to collector distance of 11 cm; with applying the suggested processing conditions predicted by RSM, a mean diameter of 89.44 nm was obtained experimentally. 3.2. Effect of different levels of catechin on the properties of AZG-PVA solutions 3.2.1. Apparent viscosity Determination of material flow behavior is essential in designing processes such as fluid flow, pumping power, and predictability of the electrospinning potential of polymeric solutions (Maghsoudlou, Sabaghi, & Kashiri, 2018; Sousa et al., 2015). The viscosity of electrospinning solutions in combination with different concentrations of catechin (0, 500, 1000, 2000 and 3000 mg L−1) are shown in Fig. 2. The addition of catechin caused a significant change in viscosity from 14.2 MPa in catechin-free solution to 14.9 MPa in 500 mg L−1 catechin solution (p < 0.05). Peres et al. (2011) reported a similar result in the nanoencapsulation of catechin in maltodextrin-Arabic gum solutions (Peres et al., 2011). The results of Shao et al. (2018) showed that the addition of tea polyphenols to CMC-pullulan solution increased the viscosity of the polymeric solution. In general, it can be concluded that the increase in the viscosity of the solution is due to higher entanglement of polymer chains (Shao et al., 2018). In our study, the viscosity increased significantly (p < 0.05) at higher catechin levels from 14.9 MPa (500 mg L−1 catechin) to 18.1 MPa (3000 mg L−1 catechin). Previous research has shown that polyphenolic compounds derived from green tea extract enhanced the film structure due to interaction with polymers and creation of new bonds in polymer structure (Stijnman et al., 2011). Investigation of rheological behavior of electrospinning solutions revealed pseudoplastic behavior; with increasing shear rate, the apparent viscosity decreased, indicating shear thinning behavior of nanofiber solutions with different concentrations of catechin. Shear stressshear rate data were fitted with Power law, Herschel- Bulkley and Casson models. The Power law model is typically used to investigate the behavior of non-Newtonian fluids. According to the power law, all solutions showed shear thinning behavior. Higher catechin concentrations in AZG-PVA solutions decreased the flow behavior (n) index from 0.8679 to 0.7686 and consistency coefficient (K) from 0.221 to 4
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Fig. 1. Optimizing the diameter of electrospun nanofibers via device parameters (voltage, flow rate and distance of the tip needle to the collector) using response surface methodology.
0.4072 mPa.s, respectively. Rheological parameters obtained by fitting the Casson model showed that higher catechin levels in nanofibers solutions increased the Casson's viscosity from 1.18 to 1.461 mPa.s. Our results revealed that the Herschel- Bulkley equation was the best model to determine the flow behavior of nanofiber solutions (Table S1 in supplementary materials). This model was selected as the best model with the highest value of R2 to RMSE (Table S1) and experimental data were fitted to this model (Fig. 3). The Herschel-Bulkley model is commonly used to investigate the behavior of non-Newtonian fluids. Rheological parameters of Fitting Herschel- Bulkley model on experimental data of shear stress to shear rate at different concentrations of catechin are given in Table S2. Through examining the flow behavior index, it can be deduced that all
samples showed shear thinning behavior. Increasing the concentration of catechin in nanofiber solutions decreased the flow behavior index from 0.9019 in control to 0.6043 in 3000 mg L−1 catechin-containing samples. The data obtained from the Herschel-Bulkley model showed a direct effect on the increase in the coefficient of consistency from 0.1871 to 0.8774 mPa by catechin concentration. The increase in the consistency coefficient is due to the interaction between the polymeric solution and natural antioxidant catechin (Siripatrawan & Harte, 2010). 3.2.2. Electrical conductivity The electrical conductivity of nanofiber solutions significantly (P < 0.05) decreased at higher catechin levels from 375 μS cm−1 (0 mg L−1) to 348 μS. cm−1 (3000 mg L−1), as shown in Fig. 4. 5
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Fig. 4. Effect of different concentrations of catechin (0, 500, 1000, 2000 and 3000 mg L−1) in nanofiber solutions on their electrical conductivity.
viscosity and higher molecular entanglement and thereby, high resistance to jet elongation at the point of flight during the electrospinning process (Fahami & Fathi, 2018; Sabaghi et al., 2015). According to the results of SEM, 500 and 1000 mg L−1 catechin levels are the most suitable concentrations for the successful electrospinning process (Fig. 5).
Fig. 2. The effect of different concentrations of catechin (500, 1000, 2000 and 3000 mg L−1) on the viscosity of nanofiber solutions.
Similarly, Shao et al. (2018) reported that the addition of tea polyphenols to pullulan-CMC decreases the electrical conductivity of nanofiber solutions. This decrease in electrical conductivity at higher catechin levels may be due to metal ions chelating properties of green tea catechins that chelate metal ions in gum solution through the formation of surface complexes (Ndlovu & Afolayan, 2008; Sabaghi et al., 2015)
3.3.2. Encapsulation efficiency and loading capacity The results of the encapsulation efficiency and loading capacity of nanofibers loaded with different concentrations of catechin is depicted in Fig. 7. It is obvious that encapsulation efficiency significantly decreased from 99 % (catechin level of 500 mg L−1) to 78.4 % (catechin level of 2000 mg L−1), but further increase in catechin concentration to 3000 mg L−1 improved the percentage of encapsulation efficiency to 81 %. Actually, after electrospinning of solutions containing 3000 mg L−1 catechin, a transparent film was obtained; and as catechin enclosed in the inner structure of the film, a higher encapsulation efficiency was obtained. Also, with increasing catechin concentration, loading capacity improved significantly (p < 0.05) from 0.59 % (500 mg L−1 catechin) to 2.93 % (3000 mg L-1 catechin). As revealed in Fig. 7, increasing the loading capacity of catechin to about 5-fold changed the appearance and microstructure of nanofibers. ATR-FTIR spectroscopy confirmed interaction between catechin (especially at 3000 mg L−1) and polymer solutions during electrospinning (Section 3.3.3). In accordance with the results of the present study, Neo et al. (2013) found that at a higher gallic acid concentration in the electrospinning process of zein, nanoencapsulation efficiency decreased. The same trend was also reported about decreasing encapsulation efficiency and increasing the loading capacity of limonene via electrospinning in Alyssum homolocarpum seed gum (Khoshakhlagh,
3.3. Properties of the catechin-loaded Azivash gum-PVA electrospun nanofibers 3.3.1. Morphology of nanofibers SEM results (Fig. 5) revealed that the mean diameter of the electrospun nanofibers increased from 89 nm (control sample) to 371 nm (containing 3000 mg L−1 catechin) with the rise in catechin concentration. The simultaneous effects of viscosity and electrical conductivity of nanofibers on their diameter is shown in Fig. 6. As can be seen, due to increases in viscosity and decrease in electrical conductivity, the mean diameter of the electrospun nanofibers increased. On the other hand, catechin concentration above 2000 mg L−1 resulted in a reduction of density and a higher mean diameter of nanofibers along with fiber discoloration from white to transparent sheets. Increasing diameter of nanofibers at higher catechin content might be due to increased
Fig. 3. Shear stress-shear rate (left) and viscosity-shear rate changes (right) in nanofiber solutions incorporating different concentrations of catechin (0, 500, 1000, 2000 and 3000 mg L−1). 6
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Fig. 5. Scanning electron microscopy of nanofibers loaded with different concentrations of catechin (0, 500, 1000, 2000 and 3000 mg L−1); voltage 30 kV, 10,000×.
alcohol, Azivash gum, and catechin during electrospinning (Fig. 8). Hydroxyl groups (OeH) were detected in PVA at 3276 cm−1. The symmetric and asymmetric stretching CH2 correspond to the bands of 2864 and 2942 cm−1, respectively. The peak of 1166 cm−1 due to stretching of acetate groups and 818 cm−1 due to CeC vibrations were revealed in structure. Similar findings for PVA have been reported by (Maghsoudlou et al., 2018). The characteristic peak of catechin was observed at 1136 cm−1 (alcohol, CeOeH) and 1519 cm−1 (C]C aromatic ring) (Lin, Feng, Lai, Lin, & Chen, 2014). In Azivash gum powder, the peak area of 1602 cm−1 was related to symmetric stretching carboxyl and peak of 1023 cm−1 and 1416 cm−1 was related to symmetric stretching of CeO groups of uronic acid. Peaks in the 900−1250 cm−1 region due to CeO, CeOeC vibrations of the glycosidic bond and CeOeH bonds. The peak area of 1732 cm−1 was due to the symmetric stretching of the CeOO group of acetyl and carboxylic acid, which could be indicative of the presence of both uronic acid and carboxylic acid in the gum structure. The peak observed at 2992 cm−1 was related to C–H junctions with stretching vibrations including C–H, C–H2 and C–H3 in C-6 glycosidic units (Rezaei et al., 2016). Peaks of region 3100 cm−1 are due to the stretching vibrations of OeH in gum structure, which are caused by intermolecular and intramolecular hydrogen bonds. In general, these two peaks are considered to be prominent functional groups in the gum structure. The change in peaks of basic functional groups is due to an interaction between the nanofiber component (Martins et al., 2012). As shown in Fig. 8, the band intensities increased between 784 cm−1 to 907 cm−1 when adding gum to the PVA solution. In electrospun nanofibers, the intensity of bands increased in the region of 1212−1309 cm−1; the presence of gum increased glycosidic vibrations
Fig. 6. Relationship between the electrical conductivity and viscosity of nanofiber solutions incorporating different concentrations of catechin (500, 1000, 2000 and 3000 mg L−1) along with the diameter of electrospun nanofibers.
Koocheki, Mohebbi, & Allafchian, 2017; Neo et al., 2013). Considering the high encapsulation efficiency (up to 99 %) at 500 mg L−1 catechin (Fig. 6), it is confirmed that electrospinning can be suggested as a single-step and effective method for nanoencapsulation of catechin.
3.3.3. ATR-FTIR results ATR-FTIR spectroscopy was performed to identify any chemical interactions and changes in functional groups regarding polyvinyl 7
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Fig. 7. Encapsulation efficiency and loading capacity of nanofibers containing different concentrations of catechin (500, 1000, 2000 and 3000 mg L−1). Non-similar letters indicate significant differences in treatments at 95 % confidence level.
Fig. 9. Thermogravimetric analysis of Azivash gum powder (—), polyvinyl alcohol nanofibers (-.-. -), Azivash gum- polyvinyl alcohol nanofibers (-), and Azivash gum-polyvinyl alcohol nanofibers containing catechin at a concentration of 1000 mg L−1 (…).
gum powder, respectively. It should be noted that the second stage for PVA began at 338 °C and the rate of thermal degradation reached 92.63 % of the initial weight. The third stage of thermal degradation began at about 348 °C, which might be due to the depolymerization of polymer chains. Under these conditions, sample containing 1000 mg L−1 catechin and the control reached 39 % and 25 % of initial weight. This stage for PVA began at 529 °C and the rate of thermal degradation reached 22.16 % of its initial weight. Arrieta et al. (2014) showed that the addition of catechin into polypropylene films increased thermal resistance compared to the control samples (Arrieta et al., 2014). The presence of tea catechin in fibers increased the thermal degradation temperature. In general, the thermal stability of fibers containing catechin was higher than that of catechin free fibers (Fig. 9). Results of Shao et al. (2018) showed green tea extract significantly increased the thermal stability of PVA films compared to the control sample. According to ATR-FTIR spectroscopy, it can be concluded that interaction between polymer solutions (Azivash gum-PVA) and catechin is mainly hydrogen bonding which caused adhesion between molecule chains and relatively higher thermal resistance of functional nanofibers.
Fig. 8. Fourier transform infrared (FTIR) spectra of electrospun nanofibers.
of CeO, CeCeO and CeOeH bands. The addition of catechin to the nanofibers increased the intensity of peaks associated with the OeH group (3276 cm−1 region), probably due to the interaction of hydroxyl groups in the catechin structure with PVA-Azivash gum mixture. Terao et al. (2009) obtained similar results with the addition of catechin into PVA film (Terao et al., 2009). ATR-FTIR spectra showed that with increasing catechin concentration, more interaction between catechin (especially at 3000 mg L−1) and polymer wall occurred which is also reflected in SEM results.
4. Conclusion 3.3.4. Thermal stability results The thermogravimetric analysis of Azivash gum powder, Azivash gum –PVA nanofibers (control), and Azivash gum-PVA-1000 mg L−1 catechin are shown in Fig. 9. According to thermograms, three main stages were observed in samples. Weight loss in the first stage of the thermal process which could be due to the removal of moisture and other volatile compounds from the sample (Varsha et al., 2010); it was a minimum for catechin-loaded Azivash gum-PVA fibers. The second stage began at 280 °C and about 86 %, 81 % and 63 % weight loss were recorded for catechin-loaded Azivash gum-PVA fiber, control fiber and
In the present study, nanoencapsulation of catechin in nanofibers of Azivash gum- polyvinyl alcohol was performed via electrospinning. Results of numerical optimization (RSM) of three variables, voltage, flow rate and distance of needle to the collector with the aim of achieving the minimum diameter of final nanofibers revealed the optimum electrospinning conditions as a flow rate of 0.5 mL h−1, voltage=20 kV and distance of needle to the collector = 11 cm. Highest voltage overcame electrostatic repulsion force on the droplets and elongation polymer jet; also lower flow rate and optimum distance from 8
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needle to collector caused enough time for solvent evaporation, so decreased nanofiber diameter. Increasing catechin concentration from 500 to 2000 mg L−1, due to the entrapment of catechin in the inner structure of the films increased the encapsulation efficiency. Higher catechin levels from 500 to 3000 mg L-1 resulted in about 5-fold increase in the loading capacity of nanofibers, which affected the appearance and microstructure of nanofibers. As the concentration of catechin increased, more interaction was found between hydroxyl groups in the catechin structure and the polymer wall. Catechin improved the thermal resistance of nanofibers due to interaction with the polymer solution through hydrogen bonds and increased adhesion between molecular chains. Based on the results of this study, the electrospinning process can be suggested as a single-step method with minimum deleterious effects for nanoencapsulation of bioactive compounds such as phenolic compounds in order to design active food packaging and for pharmaceutical applications.
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