Characteristics of vitamin E loaded nanofibers from dextran

Transcription

1 International Journal of Food Properties ISSN: (Print) (Online) Journal homepage: Characteristics of vitamin E loaded nanofibers from dextran Milad Fathi, Maryam Nikbakht Nasrabadi & Jaleh Varshosaz To cite this article: Milad Fathi, Maryam Nikbakht Nasrabadi & Jaleh Varshosaz (2016): Characteristics of vitamin E loaded nanofibers from dextran, International Journal of Food Properties, DOI: / To link to this article: Accepted author version posted online: 10 Nov Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at Download by: [University of Toronto Libraries] Date: 11 November 2016, At: 01:23

2 Journal Title: International Journal of Food Properties Characteristics of vitamin E loaded nanofibers from dextran Milad Fathi 1*, Maryam Nikbakht Nasrabadi 1 and Jaleh Varshosaz 2 1 Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, P.O. Box , Isfahan, Iran 2 Department of Pharmaceutics, Faculty of Pharmacy and Novel Drug Delivery Systems Research Center, Isfahan University of Medical Sciences, P.O. Box , Isfahan, Iran Received: Accepted: ABSTRACT In this research, dextran nanofibers were produced as novel carriers for entrapment of vitamin E. Morphological studies indicated that developed fine fibers were in the nano range without spherical beads. Thermal behavior, chemical structure and crystalline structure of vitamin E loaded nanofibers were analyzed by DSC, FTIR and XRD which showed that vitamin was well incorporated within amorphous structure of fibers without chemical interactions. Nanofibers *. Corresponding author: Tel.: ; Fax: varshosaz@pharm.mui.ac.ir

3 were used for fortification of cheese. Sensory analysis of the fortified product was performed which showed better acceptability and texture of cheese containing nanofibers in comparison to blank and direct fortified samples. Nanofiber showed capability of water holding and increase cheese firmness. The release of vitamin E in gastrointestinal media showed that about 14% of vitamin could be released in gastric media and about 30% release in intestinal media. Finally, the results revealed that electrospinning can be used for production of dextran ultrathin fibers to entrap hydrophobic compounds with high potential for design of novel functional foods. Keywords: Electrospinning, Dextran, Nanofiber, Vitamin E, Delivery system INTRODUCTION Production and consumption of functional food is a subject of interest of both food factories and consumers. Vitamin E is a natural fat-soluble antioxidant which shows health benefits such as reducing cardiovascular disease, diabetes and cancer [1-3]. However, its utilization for food fortification is limited due to instability against heat and oxygen and low water solubility. Therefore, delivery systems should be designed to protect functional bioactives from undesirable processing, storage and consumption conditions [4-6]. Delivery vehicles should be biocompatible, biodegradable and do not have inverse effect on stability, texture, flavor or appearance of food or beverage. Some researchers have worked on encapsulation of vitamin E for food [7, 8] or pharmaceutical applications [9].

4 Nanofibers are defined as fibers with diameter in nanometer range. They showed different applications in food technology such as filtration, enhancement of mechanical properties of films and delivery of bioactives. Nanofibers are also favored since can be easily and controllably manufactured from natural polymers such that their functional and physical features are controlled and/or manipulated during their production [10]. This technique has been previously used for encapsulation of food bioactives [11] or probiotic bacteria [12]. Dextran is a bacterial polysaccharide that consists of R-l, 6 linked d-glucopyranose residues with some R-1, 2-, R-1, 3-, or R-1, 4 linked side chains. The most important property makes dextran very versatile is that it is soluble in both water and some organic solvents. Some affords has been made to produce nanofibers from dextran for tissue engineering [13] and wound dressing [14]. However, there is not any publication on encapsulation of food bioactives by dextran nanofibers. Due to instability of vitamin E and superior features of nanofibers, the objectives of this study were to encapsulate vitamin E using dextran nanofibers, investigation of physicochemical properties of produced delivery system and evaluation of potential application of vitamin loaded nanofibers for cheese fortification. MATERIALS AND METHODS Materials Dextran (produced by Leuconostoc mesenteroides, Molecular mass of 70000) and tocopheryl acetate were supplied from Sigma Chemical Company (Missouri, USA) and DSM Nutritional Products Ltd Company (Heerlen, the Netherlands), respectively. Ethanol was purchased from Merck (Darmstadt, Germany). All other chemical compounds were of analytical grade.

5 Methods Preparation of vitamin-loaded dextran nanofibers: Initially the spinnability of solutions with dextran concentrations of and g/ml was investigated. Despite systematic variations of the applied voltage (9-15 kv), flow rate ( ml/h) and tip-to-target distance (5-20 cm), no fibers were obtained (Fig. 1a) except the sample with g/ml dextran under voltage of 14 kv, flow rate of 1 ml/h and distance of 15 cm which was mainly composed of spherical beads (Fig. 1,b). Dextran solutions were prepared by dissolving 1 g and 1.25 g dextran in 1 ml of 80:20 v/v water : ethanol solution and thoroughly mixed overnight [15]. Vitamin-loaded solutions were prepared by dissolving 1 mg vitamin E in each 2 ml of the biopolymer solution. For preparing vitamin-loaded nanofibers, the solutions were delivered to insulin syringe (with an inner diameter of mm) placed in a programmable syringe pump (Vita Teb Co., Iran). The flow rate pump was adjusted to 1 ml per hour. In this research two voltage levels of 13 and 15 kv were applied by a positive high-voltage supply (Vita Teb Co., Iran). The distance between needle tip and collector was set to 15 cm. Morphological study: The morphology of particles was examined using SEM (Quanta 200, FEI). Samples were mounted on aluminum stubs using double sided carbon tape and sputtercoated with gold under an argon atmosphere [16]. XRD analysis: XRD diffraction analyses were carried out in order to characterize crystalline products. X-ray diffraction patterns of samples were obtained using power X-ray diffractometer

6 (Philips, Xpert-Pro, The Netherlands) with Cu Ka 1 radiation generated at 30 ma and 50 kv. The samples were run over the most informative range from 3 to 90 of 2θ [14]. FTIR analysis: Certification of the encapsulation of vitamin E in the nanofiber and formation of any possible interactions between vitamin and fibers was performed by Fourier transform infrared spectroscopy. Infrared spectra were scanned by a Fourier transform infrared spectroscopy (WQF-510; Bomem, Canada) and recorded in the wave range of cm 1 at a resolution of 4 cm 1. Sample powder was mixed with KBr with sample to KBr ratio of 1:100[17]. DSC analysis: Investigation of thermal behavior of nanofiber is important to evaluate their stability under different temperatures. Thermal analysis of the fibers was studied by differential scanning calorimetry (DSC) (TA instruments, model DSC 204). Approximately 5 mg of particles were loaded in standard aluminum pans. The analysis was carried out at temperature heating rate of 5 C/min and temperature range of C. Release study: Percentage of surface vitamin was determined by rapid shaking of nanofiber in chloroform for 2 min. Encapsulation efficiency was determined by HPLC method at wavelength of 290 nm. Vitamin E release profiles from the nanofibers were studied in gastric (ph:1.2) and intestinal (ph:7.4) simulated solutions applying dialysis bag method at 37 C. Nanofiber solution was sealed into dialysis bag (Sigma, Canada) with a 12 kda cutoff. The bag was then placed into

7 48 ml gastric buffer for 2 h. It was subsequently subjected to 48 ml intestinal buffer for 6 h. At specified intervals, the amount of released vitamin was determined by HPLC method at wavelength of 290 nm. To study the release kinetic of vitamin E in the gastric and intestinal media, the release data were kinetically modeled by zero-order, first-order, Higuchi and Rigter- Peppas models (Eqs. 1-4) [18]: C=[1-exp(-kt)]100 where C is vitamin release percentage at time t and k is kinetic constant. Sensory analysis Fortified cheese was produced to study the potential application of produced vitamin E loaded nanofibers. Three cheese samples were produced as blank, direct fortified vitamin E and vitamin E loaded nanofiber. The recommended dietary allowance (RDA) for vitamin E is 10 mg α- tocopherol equivalents (TE) per day for men and 8 mg TE/d for women [19]. Vitamin containing cheeses were enriched under which their vitamin addition be 20% of daily need in each serving sample (100 g cheese). Vitamin E or nanofibers were added to the cheese matrix and manually (1 (2 (3 mixed with the salt. Cheese making was performed according to procedure reported by Elsamani et al [20]. Each of 25 panelists (older than 18 years) tested a sample of each cheese and recorded

8 their responses on a 7-point hedonic scale (1, dislikes; 7, likes very much) for flavor, after taste, texture (firmness), mouth-feel, color (yellowness), homogeneity and total acceptance [21]. Data were subjected to analyze of variance and means were compared using LSD test. RESULTS AND DISCUSSION Electrospinning of vitamin E loaded dextran solutions In this work dextran nanofibers were formed for encapsulation of vitamin E. As mentioned above under low concentrations ( and g/ml) no appropriate fibers were formed. However, for higher polymer concentrations deposition of particles and beads and formation of continuous fibers was observed [15]. Vitamin E loaded dextran solutions were spun with concentrations 1 and 1.25 g/ml extruded at 1ml/h under voltage of 13 and 15 kv and distance of 15 cm from collector. These results are consistent with those reported by Deitzel et al. (2001) indicated that the electrospun membrane of PEO was a mixture of fibers and beads when the solution viscosity was low [22]. The fibers are produced when the product of solution intrinsic viscosity ( ) and biopolymer concentration ( c ), known as the Berry s number, Be = [ ] c, is greater than a certain critical value ( Be cr ) which is characteristic property of each biopolymer [23]. Below this critical value the jets of polymer solution are unstable and expected to break up and form droplets. However, for high viscosity solutions, the jets are broken up and riven into filaments to the collecting plate and form fibers [24].

9 Morphology of vitamin-loaded dextran nanofibers Fig. 2 demonstrated SEM images of the electrospun dextran fibers. The SEM images of the vitamin loaded fibers did not showed presence of beads, vitamin crystals or other types of the aggregates over the surface of the fibers which implied that the vitamin E was incorporated well within the electrospun fibers. Similar results were reported for poly(vinyl alcohol) nanofiber loaded with fish oil under low oil concentrations. While fibers produced from solutions with a fish oil load higher than 1.5% (w/w), showed spindle-like enlargements interspersed along the fibers [25]. The average diameter of dextran fibers was approximately 400 nm-1000 nm. Higher concentration of dextran at higher applied voltage led to thinner and more fine fibers (400 nm) (Fig. 2 a). The applied voltage should be sufficient to overcome the surface tension at the tip of the Taylor cone, in order to initiate the ejection of the charged jet [26]. Using of higher voltage to certain point, initially leads to formation of smaller fiber diameter, then thicker fibers are formed. Thus there is an ideal applied voltage that results in a minimum fiber diameter depending on the polymer and interacting parameters [27]. Under a fixed applied voltage higher amounts of polymer in the solution led to thinner and more uniform fibers. Changing the polymer concentration can vary the solution viscosity. Moreover, the surface tension coefficient depends on the polymer and solvent [24]. It should be noted that there is also an optimum concentration under which beads or nanoparticles might be formed and upper which solution is not spinnable [27]. Similar diameter sizes were obtained for polyurethane dextran [28] and whey protein [29] nanofibers.

10 XRD analysis Vitamin E loaded dextran nanofibers and pure dextran powder were subjected to XRD analysis in order to further illustrate the physical structure and distribution of the vitamin E in the nanofibers (Fig. 3). The crystal structure depends on characteristics of the polymer (molecular weight) [30], process parameters (applied voltage and flow rate) and the solvent medium (evaporation rate and polymer solvent interaction) [30]. Dextran powder had typical broad peaks around 2θ of 20 and 45, since it has an amorphous structure. The electrospun dextran fibers were amorphous as denoted by the absence of a characteristic diffraction peak and have the same peaks proving that loading the vitamin did not change the nature of the dextran nanofibers. It could be due to low weight proportion of vitamin to dextran. It also indicated that there was a good compatibility between dextran and vitamin E [14]. FTIR analysis FTIR spectra were analyzed to confirm the composition of the fibers. Fig. 4 reveals the infrared spectra of pure dextran powder, pure vitamin E and vitamin loaded dextran fibers over the wavenumber range from 400 to 4000 cm 1. Several characteristic bands of dextran are observed e.g. C-H bending at 760 and 846 cm 1, C-O-C stretching band at 1005 cm -1, C O stretching at 1274 cm 1, CH 3 bending at 1435 cm 1, CH stretching at 2922 cm 1, CH3 stretching at 3089 cm 1 and OH stretching at 3335 cm 1. FTIR spectrum of vitamin E showed characteristic C=O stretching vibration at around cm -1, C-O formation at 1205 cm -1, C=C formation at 1683 cm -1 and C-H alkanes group at 2925 cm -1. Absorption band of vitamin E at cm -1

11 is attributed to the terminal hydroxyl group and pick at cm -1 is due to the C O stretching. All the characteristic peaks of dextran and vitamin E were detectable in nanofibers spectra, although they showed overlap. However, due to low weight ratio of vitamin, its characteristic bands did not have much effect on spectra of nanofibers in comparison to dextran. The band of hydrogen bond observed at 3470 cm 1 and the C-OH and C-O stretching modes detected at 1470 and 1460 cm -1, respectively. The weak bands in the calculated spectrum at 1660 and 1570 cm -1 theoretically correspond to symmetric and asymmetric aromatic ring stretches, respectively [14, 17, 31]. DSC analysis Based on the SEM results it is observed that treatment fabricated from the 1.25 g/ml dextran solution under voltage of 15 kv had the best size, therefore this sample was selected for DSC analysis. DSC thermograms at the range of 20 to 350 C are presented in Fig. 5. The thermogram of tocopheryl acetate exhibited presence of an endothermic event around 340 ºC which was a characteristic of the thermal behavior of this material [32]. Dextran showed a wide endothermic pick at 320 C followed by an exothermic pick. Due to low weight proportion of vitamin to dextran the characteristic pick of vitamin E in thermogram of vitamin E loaded nanofiber was weak but yet detectable while its pick started from lower temperatures which was due to its overlap with dextran pick. It could be stated that that no particular chemical interaction between vitamin E and dextran occurred which means that vitamin was well dispersed in the fiber matrix and the crystallinity of the polymer was not affected by vitamin E incorporation. Aforementioned

12 XRD data also showed that the crystalline structure of dextran remained intact; FTIR results agreed with DSC data that not any adverse reactions were formed between dextran and vitamin. Release study Encapsulation efficiency of vitamin E loaded nanofiber was 98.3%. It could be high solubility of vitamin in dextran. The profile vitamin E release from nanofibers was depicted in Figure 6. The release percentages were about 6%, 35% in the gastric and intestine media, respectively. It is indicated that produced fiber can postpone vitamin release in acidic gastric media. Vitamin release from produced nanofibers was kinetically studied using zero-order, first-order and Higuchi models. Table 1 showed model parameters and their corresponding coefficients of determination. As can be observed, for gastric, intestine and whole release media zero order model showed the best fitness. It is indicated that release of vitamin is not concentration dependent and just varied with release time. Sensory analysis Sensory analysis was conducted for three cheese samples, i.e. blank cheese, direct vitamin E fortified cheese and vitamin E loaded nanofiber fortified cheese. The nanofiber sample which showed smaller diameter was selected sensory analysis. The results indicated that means of all studied features (except for color) were significantly different (P<0.01). Average and standard error of sensory properties are tabulated in Table 2. These data indicated that properties of nanofiber containing cheese sample were all better than direct fortification of vitamin E (except

13 for after taste). Interesting results obtained for texture of cheese samples which showed higher firmness of nanofiber containing sample in comparison to blank and direct fortified cheeses. These results could be attributed to the texturizing effect of produced nanofibers that showed gelling properties. Finally, total acceptance data suggested that the consumer acceptability attributes of cheese containing vitamin E-loaded nanofibers is higher than other samples. CONCLUSION Dextran nanofibers as a delivery system for vitamin E were fabricated by the electrospinning method. The polymer concentration and applied voltages were the main important factors affecting the electrospinnability of the bulk solutions and the morphology of the electrospun nanofibers. Uniform and thin dextran fibers with an average diameter of nanometer-scale (~400 nm) were obtained from 1.25 g/ml of dextran solution under voltage of 15 kv. The results from SEM, FTIR, XRD and DSC demonstrated that vitamin E had good compatibility with dextran and were able to distribute uniformly within the polymer fiber matrix. The fibers were in amorphous structure. Sensory analysis showed that produced nanofibers can enhance homogeneity, texture and acceptance of fortified cheese in comparison direct vitamin E fortified sample. These findings demonstrated the potential application of nanofibers for entrapment of bioactives and enrichment of food and beverage.

14 REFERENCES 1. Yang, Y. and D.J. McClements, Encapsulation of vitamin E in edible emulsions fabricated using a natural surfactant. Food Hydrocolloids, (2): p Sylvester, P.W., et al., Tocotrienol combination therapy results in synergistic anticancer response. Frontiers in bioscience (Landmark edition), : p Songsermsakul, P., E. Pornphairin, and S. Porasuphatana, Comparison of antioxidant activity of grape seed extract and fruits containing high β-carotene, vitamin C, and e. International Journal of Food Properties, (3): p Fathi, M., M. Mozafari, and M. Mohebbi, Nanoencapsulation of food ingredients using lipid based delivery systems. Trends in food science & technology, (1): p Fathi, M., A. Martin, and D.J. McClements, Nanoencapsulation of food ingredients using carbohydrate based delivery systems. Trends in food science & technology, (1): p Dasgupta, N., et al., Fabrication of Food Grade Vitamin E Nanoemulsion by Low Energy Approach, Characterization and Its Application. International Journal of Food Properties, (3): p Quintanilla-Carvajal, M.X., et al., Effects of microfluidisation process on the amounts and distribution of encapsulated and non-encapsulated α-tocopherol microcapsules obtained by spray drying. Food Research International. 63, Part A: p Tackenberg, M.W., et al., Orange terpenes, carvacrol and α-tocopherol encapsulated in maltodextrin and sucrose matrices via batch mixing. Journal of Food Engineering, : p

15 9. Laouini, A., et al., Characterization of different vitamin e carriers intended for pulmonary drug delivery. International Journal of Pharmaceutics, (1-2): p Rezaei, A., A. Nasirpour, and M. Fathi, Application of Cellulosic Nanofibers in Food Science Using Electrospinning and Its Potential Risk. Comprehensive Reviews in Food Science and Food Safety, (3): p Lemma, S.M., et al., Controlled release of retinyl acetate from β-cyclodextrin functionalized poly(vinyl alcohol) electrospun nanofibers. Journal of Agricultural and Food Chemistry, (13): p Fung, W.-Y., K.-H. Yuen, and M.-T. Liong, Agrowaste-based nanofibers as a probiotic encapsulant: Fabrication and characterization. Journal of Agricultural and Food Chemistry, (15): p Dahlin, R.L., F.K. Kasper, and A.G. Mikos, Polymeric nanofibers in tissue engineering. Tissue Engineering - Part B: Reviews, (5): p Unnithan, A.R., et al., Wound-dressing materials with antibacterial activity from electrospun polyurethane dextran nanofiber mats containing ciprofloxacin HCl. Carbohydrate polymers, (4): p Jiang, H., et al., Optimization and characterization of dextran membranes prepared by electrospinning. Biomacromolecules, (2): p Kriel, H., R.D. Sanderson, and E. Smit, Coaxial electrospinning of miscible PLLA-core and PDLLA-shell solutions and indirect visualisation of the core-shell fibres obtained. Fibres & Textiles in Eastern Europe, (2): p. 91.

16 17. Jalaja, K., et al., Modified dextran cross-linked electrospun gelatin nanofibres for biomedical applications. Carbohydrate polymers, : p Fathi, M., et al., Hesperetin-loaded solid lipid nanoparticles and nanostructure lipid carriers for food fortification: Preparation, characterization, and modeling. Food and Bioprocess Technology, (6): p Murphy, S.P., A.F. Subar, and G. Block, Vitamin E intakes and sources in the United States. The American journal of clinical nutrition, (2): p Elsamani, M.O., et al., Biochemical, microbial and sensory evaluation of white soft cheese made from cow and lupin milk. LWT - Food Science and Technology, (1): p Bermúdez-Aguirre, D. and G.V. Barbosa-Cánovas, Quality of selected cheeses fortified with vegetable and animal sources of omega-3. LWT-Food Science and Technology, (7): p Deitzel, J., et al., The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer, (1): p Jaworek, A., et al., Nanocomposite fabric formation by electrospinning and electrospraying technologies. Journal of Electrostatics, : p Fong, H., I. Chun, and D. Reneker, Beaded nanofibers formed during electrospinning. Polymer, (16): p García-Moreno, P.J., et al., Encapsulation of fish oil in nanofibers by emulsion electrospinning: Physical characterization and oxidative stability. Journal of Food Engineering, : p

17 26. Wongsasulak, S., et al., Electrospinning of food-grade nanofibers from cellulose acetate and egg albumen blends. Journal of Food Engineering, (3): p Castillo, Y.S., Design of experimentation to systematically determine the interaction between electrospinning variables and to optimize the fiber diameter of electrospun poly (d, l-lactide-co-glycolide) scaffolds for tissue engineered constructs Unnithan, A.R., et al., Wound-dressing materials with antibacterial activity from electrospun polyurethane dextran nanofiber mats containing ciprofloxacin HCl. Carbohydrate Polymers, (4): p Sullivan, S.T., et al., Electrospinning and heat treatment of whey protein nanofibers. Food Hydrocolloids, : p de Oliveira Mori, C.L., et al., Electrospinning of zein/tannin bio-nanofibers. Industrial Crops and Products, : p Kumar, Y.S., et al., Microgravity biosynthesized penicillin loaded electrospun polyurethane dextran nanofibrous mats for biomedical applications. Colloids and Surfaces A: Physicochemical and Engineering Aspects, : p Almeida, M.M.d., et al., Stability evaluation of tocopheryl acetate and ascorbyl tetraisopalmitate in isolation and incorporated in cosmetic formulations using thermal analysis. Brazilian Journal of Pharmaceutical Sciences, (1): p

18 Figure 1. Scanning electron microscopic images of dextran solutions at concentration of a) g/ml and b) g/ml dextran under the voltage of 14 kv, flow rate of 1 ml/h and the distance of 15 cm at 25 C with magnification of 500, 1000 and 2000 from left to right.

19 Figure 2. SEM images of vitamin E- loaded dextran nanofibers [a) 1.25 and b) 1 g/ml dextran under 15 kv and c) 1.25 and d) 1 g/ml dextran under 13 kv with magnification of 500, 1000 and 2000 from left to right].

20 Figure 3. X-ray diffraction of dextran powder (dp) and vitamin E- loaded nanofibers with1) 1.25 and 2) 1 g/ml dxtran at 15 kv and 3) 1.25 and 4) 1 g/ml dxtran at 13 kv.

21 Figure 4. FTIR spectra of vitamin E- loaded nanofibers, vitamin E and dextran powder.

22 Figure 5. DSC curves of vitamin E (a), pure dextran powder (b) and vitamin E loaded dextran nanofiber fabricated from 1.25 g/ml dextran solution under voltage of 15 kv (c).

23 Fig. 6. Release profile of vitamin E from dextran nanofibers in gastrointestinal media.

24 Table 1. Model parameters of vitamin E release from dextran nanofibers. Model Zero order First order Higuchi Release media Model parameter and coefficient of determination Gastric Intestinal Whole release media media media k R k R k R

25 Table 2. Sensory evaluation of blank cheese, direct vitamin E fortified cheese and vitamin E loaded nanofiber fortified cheese. Characteristics Blank cheese Direct vitamin E fortified Vitamin E loaded nanofiber cheese fortified cheese Flavor 3.8±0.29 a 2.6±0.22 b 3.7±0.30 a After taste 3.9±0.26 a 2.6±0.23 b 3.5±0.18 a Texture (firmness) 2.9±0.28 b 3±0.26 b 4.1±0.21 a Mouth-feel 3±0.21 b 2.7±0.26 b 3.6±0.30 a Color (yellowness) NS 3.8± ±0.31 4±0.26 Homogeneity 4±0.26 a 2.8±0.25 b 4.5±0.27 a Total acceptance 3.2±0.20 b 2.7±0.26 b 4.1±0.17 a * Rows followed by different letters are statistically significant at P=0.01; NS Not statistically significant