Inulin Nanofibers

Abstract

Electrospun Polyvinyl Alcohol (PVA)/Inulin composite nanofibers (CNFs) are provided using electrospinning technique and tested for their prebiotic and antibacterial activities. The PVA/Inulin electrospun CNFs were tested for prebiotic activity with Lactobacillus sp. and for antibacterial activity against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). A number of electrospinning parameters such as solution concentration, PVA: Inulin mixing ratio, solution flow rate and applied voltage were carefully varied and the best PVA/Inulin electrospun CNFs (bead free) were selected for prebiotic and antibacterial tests. The concentration of the composite solution varied between 14-20%, the flow rate ranged between 0.005-0.5 mL/min and the applied voltage used ranged between 15-20 Kv. The structural properties and morphology of the PVA/Inulin electrospun CNFs were fully characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and scanning electron microscopy (SEM).

Claims

1. A composition of electrospun composite nanofibers, comprising cross-linked polyvinyl alcohol (PVA) and inulin electrospun nanofibers, wherein the inulin is 4 to 10% of the total weight of the composite nanofibers.

2. The composition as set forth in claim 1, wherein the PVA is 8% to 12% of the total weight of the composite nanofibers.

3. The composition as set forth in claim 1, wherein the composite nanofibers are produced at a range of 300 nm to 640 nm.

4. The composition as set forth in claim 1, wherein the composite nanofibers are chemically crosslinked by glutaraldehyde.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1A-C show according to an exemplary embodiment of the invention in FIGS. 1A-B SEM images of PVA/Inulin electrospun CNFs fabricated from 15% (w/w) blend solution at voltage of 16 kv and flow rate of 0.1 mL/min. Corresponding histogram showing the fiber diameter distribution (FIG. 1C).

[0029] FIGS. 2A-C show according to an exemplary embodiment of the invention in FIG. 2A Total viable count, FIG. 2B pH, and FIG. 2C Optical density of Lactobacillus sp. culture containing PVA electrospun Nanofibers, inulin solution, PVA/Inulin electrospun CNFs, and water after 24 hours of incubation.

[0030] FIG. 3 shows according to an exemplary embodiment of the invention growth curve of Lactobacillus sp. culture containing PVA/Inulin electrospun CNFs.

[0031] FIGS. 4A-B show according to an exemplary embodiment of the invention in FIG. 4A an inhibition zone of PVA electrospun Nanofibers, inulin solution, PVA/Inulin electrospun CNFs, antibiotic and water with E. coli, in FIG. 4B an inhibition zone of PVA electrospun Nanofibers, inulin solution, PVA/Inulin electrospun CNFs, antibiotic and water with S. aureus.

[0032] FIGS. 5A-B show according to an exemplary embodiment of the invention an inhibition curve of E. coli (FIG. 5A) and S. aureus (FIG. 5B) culture of PVA/Inulin electrospun CNFS (Optical density).

DETAILED DESCRIPTION

[0033] The objective of this invention is the fabrication of nanofibers of Inulin, a naturally occurring polysaccharide, with enhanced prebiotic and antibacterial activities. The description is divided into: [0034] (i) Fabrication of electrospun composite nanofibers (CNFs) from Inulin and Poly vinyl alcohol (PVA) using an electrospinning technique, [0035] (ii) Characterization of the electrospun CNFs (morphological by scanning electron microscopy (SEM) and spectroscopically by Fourier Transform Infrared (FT-IR) Spectroscopy), [0036] (iii) Cross-linking the successfully electrospun CNFs by physical and chemical methods to select the most efficient cross-linking method that could keep the mesh structures of the fabricated CNFs when dissolved in other solvents, and [0037] (iv) Testing the electrospun CNFs (both cross-linked and non cross-linked) for their prebiotic and antibacterial activities.

[0038] In the first set of CNFs fabrication experiments by the Electrospinner, Inulin couldn't be directly electrospun into uniform nanofibers at any concentration, except after mixing with PVA polymer to improve the spinning ability of inulin. A wide variety of concentrations of the PVA/Inulin blend solutions of (14, 15, 16, 18 &20) % w/w were prepared. In addition, applied voltages of (16-20 kv) and flow rates of (0.005 to 0.5 mL/min.) were used.

[0039] Nanofibers electrospun from PVA/Inulin blend solution of concentration 15% w/w at voltage 16 kv and flow rate 0.1 mL/min were selected to be the best parameters to produce smooth, uniform and beads-free nanofibers.

[0040] The electrospun CNFs have been tested for their prebiotic and antibacterial with three types of bacteria: Lactobacillus sp., gram positive and gram negative (E. coli and S. aureus bacteria).

[0041] To the best of our knowledge, this is the first study to report on the successful fabrication of electrospun nanofibers using inulin and more importantly testing the fabricated CNFs for their prebiotic and antibacterial with three types of bacteria. The parameters of the electrospinning such as the applied voltage, concentration of the PVA/Inulin blend solution as well as the flow rate were varied and adjusted to give the most acceptable PVA/Inulin electrospun CNFs. The choice of PVA as the main polymer to be mixed with inulin was mainly due to its chemical stability at room temperature along with its unique physical properties, which made it one of the most acceptable polymers that is mainly used in fiber fabrication.

Preparation of PVA/Inulin Electrospun CNFs

[0042] A wide variety of concentrations of the PVA/Inulin blend solutions of (14, 15, 16, 18 &20) % w/w were prepared. The concentration of PVA was kept constant (10 grams) in all samples, and the inulin concentration was varied between 4-10 grams to obtain PVA: Inulin ratios between 2.5:1 to 1:1, leading to total mixture concentration of 14-20 (w/w %). PVA aqueous solutions were prepared by weighing PVA in distilled water. Then the solutions were stirred with a magnetic stirrer at temperatures 100 C. for a period of less than 90 min to acquire a homogenous solution. Inulin aqueous solutions were prepared by dissolving inulin in distilled water at room temperature. Then aqueous solutions of PVA and inulin were added to either to obtain blend solution of PVA/Inulin.

Electrospinning Parameters of PVA/Inulin CNFs

[0043] Electrospinning was carried out by using a commercial electrospinner (E-Spin Tech, India) with a syringe pump and a high voltage power supply (Gamma High Voltage power supply, USA). The solutions were loaded into a plastic syringe connected to a sharp tip needle, which was grounded by a crocodile clip.

[0044] Electrospinning parameters were adjusted as follows; high voltages 16-20 kv and flow rates of 0.005-0.5 mL/min. Aluminum foil sheets were used to cover copper plate collector, and the distance from the tip of the needle to the collector was adjusted to 10 cm. Electrospinning of all solutions mixtures were carried out at room temperature. PVA/Inulin electrospun CNFs were successfully produced by electrospinning using 15% w/w blend solution at 16 kv applied voltage and a flow rate 0.1 mL/min.

[0045] The parameters for producing PVA/Inulin electrospun CNFs were: [0046] Effect of solution concentration on the morphology of PVA/Inulin electrospun CNFs. To observe the changes in fiber formation, and to select the parameters that produce smooth, uniform and beads-free CNFs. [0047] Effect of applied voltage on the morphology of PVA/Inulin electrospun CNFs. To observe the changes in nanofiber formation and nanofibers morphology upon varying the applied voltage. And to select the parameters that produce CNFs with desired morphology. [0048] Effect of solution flow rate on the morphology of PVA/Inulin electrospun CNFs. To observe the changes in nanofiber formation and nanofibers morphology upon varying the flow rate. And to select the parameters that produce CNFs with desired morphology.

[0049] FIGS. 1A-B show SEM images of PVA/Inulin electrospun CNFs fabricated after a number of assessments to accomplish the most acceptable electrospinning parameters.

Cross-Linking of PVA/Inulin CNFs

[0050] Physical and chemical cross-linking of the PVA/Inulin electrospun CNFs (smooth, uniform and bead free) were carried out to obtain the most reliable cross-linking method.

[0051] 1. Physical Cross-Linking

[0052] Physical cross-linking was performed by thermal treatment of the electrospun PVA/Inulin CNFs in a vacuum oven (Jelotech, OV-11, Korea) at temperatures from 80 C. to 140 C. for 10 minutes.

[0053] 2. Chemical Cross-Linking

[0054] Glutaraldehyde solution (GA) was used for chemical cross-linking of the PVA/Inulin electrospun CNFs. The electrospun CNFs were placed inside a desiccator occupied with the vapors of 50 mL of GA solution. Exposure time to GA vapor varied from 30 to 120 minutes and then were thermally treated for 24 hours in an oven at 70 C. under vacuum.

Water Immersion Test

[0055] To investigate the efficiency of both cross-linking methods on the PVA/Inulin electrospun CNFs, the stability of the electrospun CNFs in warm distilled water at 37 C. for 24 hours was tested. Then the electrospun CNFs were immediately weighed after removing the surface water with filter paper. The weight of the dry cross-linked composite nanofibers was calculated by determining the weight loss according to equation (1). The weight before immersion in water (w.sub.i) and after immersion in water and drying (w.sub.f) were measured.

[00001]$\begin{array}{cc}\mathrm{wt}.Math.\mathrm{loss}.Math..Math.\%=\frac{w_i-w_f}{w_i}100& \left(1\right)\end{array}$

[0056] Prebiotic Activity

[0057] Prebiotic activity was carried out using Lactobacillus sp. to assess the growth activity by calculating the i) total viable counts, ii) pH, iii) optical density (OD), and iv) growth curve. The results of the prebiotic activity of the PVA/Inulin electrospun CNFs showed an increase in the lactobacillus growth from 2.910.sup.3 cfu/mL (with inulin) to 4.010.sup.3 cfu/mL (increased by 37.9%) (FIGS. 2A-C). The inhibition curve showed that there was decrease in the growth of S. aureus, and a slight decrease of E. coli (FIG. 3).

[0058] The pH and the OD of the culture inoculated with the tested material were measured before incubation and after 24 hours incubation. PVA/Inulin electrospun CNFs solution decreased the pH to 5.7 compared to 6.3 for the control. The inulin solution showed no decrease in the pH, and remained at 6.2 (FIG. 2B). Additionally, the PVA/Inulin electrospun CNFs recorded the highest OD reading, following 24 hours incubation, among the tested samples (FIG. 2C). The results indicate that the PVA/Inulin electrospun CNFs exhibited higher prebiotic activity than inulin solution alone.

[0059] In FIG. 3, the growth curve showed that the growth of the culture containing PVA/Inulin electospun CNFs is not substantially greater than the growth of the control.

[0060] Antibacterial Activity

[0061] To the best of our knowledge, the antibacterial activity of inulin hasn't been previously reported. The antibacterial activity of prebiotics generally and inulin particularly occurs only after their fermentation by the probiotics. Fermentation of the prebiotics produces short chain fatty acids that decrease the pH of the gut environment, which the pathogenic bacteria can't tolerate.

[0062] Water, PVA electrospun Nanofibers and inulin solution didn't show any visible zone of inhibition with both E. coli and S. aureus. This confirms that they don't exhibit antibacterial activity. Surprisingly, PVA/Inulin electrospun CNFs showed high antibacterial activity with E. coli and S. aureus compared with inulin solution. The PVA/Inulin electrospun CNFs exhibited inhibition zone of 18.3 mm with both E. coli and S. aureus. On the other hand, inulin solution didn't exhibit any inhibition zone with both E. coli and S. aureus. This shows the unique antibacterial effect of the nanoscale transformation of inulin solution versus inulin nanofibers.

[0063] The inhibition curve showed that there was decrease in the growth of S. aureus in FIG. 5b, and a slight decrease of E. coli in FIG. 5A. The growth of the culture containing PVA/Inulin electospun CNFs with S. aureus is significantly less than the growth of the control. This confirms the antibacterial activity of PVA/Inulin electospun CNFs with S. aureus.

[0064] From the results presented, we concluded that PVA/Inulin electrospun CNFs possess an enhanced prebiotic activity. Moreover unlike inulin, the PVA/Inulin electrospun CNFs possess antibacterial activity with both Gram-negative E. coli and Gram-positive S. aureus.

[0065] One of the main reasons behind the enhanced prebiotic and antibacterial activities of the electrospun composite nanofibers is ascribed to the increased surface area to volume ratio of the electrospun nanofibers available for interaction with bacteria. These results are in agreement with the results reported by L. Qi et al., similarly reporting that chitosan nanoparticles exhibited higher antibacterial activity than chitosan due to the larger surface area of chitosan nanoparticles. Qi, L.; Xu, Z.; Jiang, X.; Hu, C.; Zou, X. Carbohydr. Res. 2004, 339, 2693-2700.

Advantages

[0066] The composite nanofibers are expected to possess many advantages compared to their original non-electrospun solutions. The advantages of our electrospun CNFs are mainly: (i) their enhanced prebiotic activity, and (ii) enhanced antibacterial activity, which are directly related to the large surface area per unit mass of the fabricated electrospun composite nanofibers, and the availability of more binding sites on their surfaces towards the two types of bacteria.

[0067] Electrospun CNFs are mainly composed of natural materials, which are not harmful for your human consumption, and offer enhanced prebiotic and antibacterial activity with minimal use of synthetic chemicals compared to their non-electrospun solutions. These nanofibers have a wide variety of possible applications against different types of bacteria.

[0068] Uses

[0069] The electrospun CNFs could be used for the treatment of digestive disorders, antiseptic sprays or bandages' fillers for wound infections, and many different types of bacterial infections. These electrospun CNFs could also be used as surface nano-coatings inside hospitals, sterile areas and pharmaceutical facilities.

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