Novel Inulin Electrospun Composite Nanofibers: Prebiotic and Antibacterial Activities.

Inspired by the rampant digestive disorders and the vast bacterial infections, this study aimed at fabricating nanofibers made of inulin/polyvinyl alcohol (PVA) composite nanofibers (CNFs) using the electrospinning technique and testing their prebiotic and antibacterial activities. The inulin/PVA CNFs were tested for prebiotic activity with Lactobacillus species while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used to assess the antibacterial potentiality. During the fabrication of the CNFs, different electrospinning parameters have been carefully controlled, in order to produce nanofibers with relatively uniform diameter, fewer beads, and high integrity. The different parameters included variable solution concentration (material ratio varied from 14 to 20 wt %), applied voltage (varied from 15 to 25 kV), and solution flow (ranged between 0.005 and 0.5 mL/min). The chemical characteristics, thermal stability, and morphology of the formed CNFs were comprehensively characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. Selected CNFs, showing the best diameter uniformity and integrity, were tested for the prebiotic and antimicrobial activity. A 38% increase in prebiotic activity of CNFs, compared to their bulk solution, was observed. The antibacterial activity of the selected CNFs was enhanced, from ∼40% (pure inulin) to 70% (inulin/PVA CNFs) against E. coli and 45% against S. aureus. This study investigates the prebiotic and antibacterial activities of PVA/inulin CNFs and provides the foundation for inulin/PVA CNF use in the healthcare sector, as in disinfectants and/or digestive disorders.

Introduction

Digestive disorders affect millions of people worldwide with potential impact on human health and patient’s quality of life. Most digestive disorders arise from the introduction of nonendogenous pathogenic bacteria to the gastrointestinal tract. Usually, these disorders are treated with invasive procedures and/or chemotherapeutic agents. Recently, it has become popular to treat digestive disorders by herbal therapy, especially when traditional therapy fails to treat the disorders. The interest in natural herbal products arises from demands for functional food and drug-resistant infection.[1] Functional food such as prebiotics can modify the human gut microbiota by suppression of pathogenic bacteria and stimulation of beneficial bacteria.[2] Modification and stimulation of the gut microbiota is important in treating various digestive disorders and improving human health.[3]

Inulin is a natural product reported to have potential for prebiotic activity. It is commonly found in chicory, dahlia, bananas, and tubers. Inulin has several medical applications, as in the control of high blood fat, cholesterol, weight loss, constipation, and kidney function test. In food industry, inulin is used to replace sugar in diabetic patients. It is also used in ethanol production.[4] Inulin is able to resist enzymes in human salivary glands, stomach, and small intestines and only fermented by the microflora in the colony.

Nowadays, nanofibers are at the forefront of nanotechnology and play an important role in human life. There are various methods for nanofiber preparation including chemical, electrochemical, and electrospinning techniques. Electrospinning has become the most convenient and attractive method, given the ease and level of control of the formed nanofibers. Electrospinning is characterized by its simplicity, ability to control fiber diameter and shape, and low cost of nano–microfiber fabrication. Electrospun nanofibers possess superior properties compared to nanofibers fabricated by other methods and other nanomaterial forms where they show high porosity, high surface area per unit mass, and so forth. A wide variety of natural and synthetic polymers have successfully been electrospun and have attracted great attention because of their controllable properties and potential applications in various fields. Examples of these applications are biomedical scaffolds, drug delivery, wound dressings, batteries, and protective clothing.[5]

The properties of the electrospun nanofibers such as fiber diameter, fiber morphology, uniformity, and porosity are dependent on various parameters. These parameters range from processing, solution, and ambient parameters.[6] Processing parameters, which are considered as the controllable parameters, include the applied voltage, solution flow rate, and the tip-to-collector distance. Appropriate manipulation of these parameters would produce nanofibers with the desired diameters and morphology.[7] Moreover, solution parameters include polymer molecular weight, solution viscosity, and solution conductivity. These parameters have significant effect on the electrospinning ability of the polymer solution. In addition, ambient parameters such as temperature and humidity have significant role in the successful fabrication of nanofibers.[8]

Natural polymers are frequently electrospun into nanofibers, and many examples of such natural polymers have been used to fabricate nanofibers such as collagen,[9] gelatin,[10] chitosan,[11] cellulose acetate,[12,13] and hyaluronic acid.[14] They can be electrospun alone or in combination with other polymers either natural or synthetic. On the other hand, synthetic polymers are more widely used to fabricate nanofibers of desired properties. Synthetic polymers include polyvinyl alcohol (PVA),[15,16] polyethylene oxide,[17] poly(lactic acid-co-glycolic acid),[18] and polylactide.[19]

PVA was selected in this study to assist the fabrication of composite nanofibers (CNFs) when mixed with inulin. PVA was selected because it is a water-soluble polymer and approved by Food and Drug Administration (FDA) for use in medical applications.[20,21] Additionally, PVA is a biocompatible, nontoxic, and biodegradable polymer.[22,23] Additionally, it is known for its fiber-forming capability and is widely used in electrospinning of various blended fibers.[24] After careful control of the processing and solution parameters, the CNFs with the best uniformity and integrity were selected for prebiotic and antibacterial testing against both Gram positive and Gram negative bacteria.

Results and Discussion

Chemical Analysis

Fourier Transform Infrared Analysis

The chemical structure of the fabricated inulin/PVA CNFs was confirmed by Fourier transform infrared (FT-IR) spectroscopy. Its representative FT-IR spectra with comparison to its corresponding starting materials are shown in Figure . The characteristic IR absorption bands of O–H stretching of both inulin and PVA found around 3350–3400 and 3300–3500 cm–1, respectively, were found to be overlapped forming a broader O–H absorption peak in the resulting inulin/PVA CNFs. This might be because of the hydrogen bonding formed between the OH groups of both blended materials. Additionally, the characteristic peaks of inulin found around 1650 cm–1 and of PVA around ∼1442 and ∼1716 cm–1 remained the same in the resulted blended inulin/PVA CNFs with slight shifting in the peaks, and this is might be because of the physical interaction between the two materials.

Figure 1

FT-IR spectrum for inulin/PVA CNFs in comparison with its starting materials.

Thermogravimetric Analysis

The thermograms under a nitrogen atmosphere of the inulin/PVA CNFs in comparison with the blended materials, inulin and PVA, are shown in Figure . The thermogravimetric analysis (TGA) thermogram of the inulin/PVA CNFs showed a great enhancement in the thermal stability of the inulin extract at which it started to decompose at higher temperature range ≈ 338.9 °C, in contrast to that of the pure inulin extract, which showed lower decomposition temperature range ≈ 235 °C. Moreover, the CNF decomposition temperature was observed to be closer to that of the PVA, which was found to be around ∼323.19 °C. These results could be because of inulin protection created by its incorporation within the PVA nanofibers, in addition to the physical and chemical interactions created between both the inulin and PVA.

Figure 2

TGA thermogram of inulin/PVA CNFs in comparison with its starting materials.

Loading capacity determination using UV–vis spectroscopy was carried out to determine the percentage of inulin inside the PVA nanofibers. The results showed that each 100 mg of PVA contained 15.08 g of inulin, and the entrapment efficiency was determined and found to be 87.52%.

Morphological Analysis

As mentioned previously, different parameters have been changed through the nanofiber preparation to achieve the most uniform nanofibers with least beads. Scanning electron microscopy (SEM) has been used to investigate the effect of different parameters on the morphology of the obtained nanofibers.

Effect of Concentration

The concentration of the solutions used for electrospinning varied between 15 and 20% w/w. During electrospinning of the solutions, dropping of the solution occurred continuously at a concentration of 20 and 18% with no fiber formation. However, upon decreasing the concentration to 16% (Figure a), fibers were formed but dropping still occurred. Additionally, decreasing the concentration further to 15% (Figure b) resulted in more uniform fibers and solution dropping decreased. Accordingly, upon decreasing the solution concentration, nanofiber formation was improved and solution dropping decreased. Furthermore, as the concentration decreased below 15%, more uniform and less beaded nanofibers were formed. These results were found to be in disagreement with the results stated by Mit-uppatham, Nithitanakul, and Supaphol, (2004),[25] where solution dropping occurred at low concentration, and increasing concentration resulted in the formation of smooth nanofibers. At low concentrations, the viscoelastic force is not large enough, resulting in the break-up of the solution drop into smaller droplets (Mit-uppatham et al., 2004).[25]

Figure 3

SEM images of inulin/PVA CNF electrospun at a solution flow rate less than 0.1 mL/min and at applied voltage 16–18 kV from different inulin/PVA blend solution concentration (w/w): (a) 15, (b) 16, (c) 18, and (d) 20% (w/w).

Effect of Applied Voltage

Based on the previous studies, the applied voltage is one of the most important and crucial variables that affects the morphology of fabricated CNFs as reported by Deitzel, Kleinmeyer, Harris, & Beck Tan, (2001).[26] As the applied voltage was increased from 16 to 20 kV (Figure ), the monodispersity of the fabricated CNFs decreased and there were broadening of the distribution of the CNF diameter. In addition, increasing the applied voltage above 16 kV resulted in the appearance of few beads, similar to previous reports (Zong, Kim, Fang, Ran, Hsiao, & Chu, 2002).[27] This could be attributed to the formation of a Taylor cone from more amount of the solution because of driving more solution through the syringe nozzle. Also, the increase in the applied voltage causes rapid removing of the solution from the needle tip (Deitzel et al., 2001).[26] These results are in agreement with the results previously reported by Li et al. (2007)[28] and Kegere et al. (2019)[36] similarly reporting that at higher applied voltage, a broad distribution in the nanofiber diameter is produced where on the other hand, lower applied voltage assists uniform nanofiber formation with narrower diameter distribution.

Figure 4

SEM images (a,c,e,g,i) and their corresponding histograms (b,d,f,h,j) of inulin/PVA CNFs from 15% w/w blend solution at different voltages; 16 (a,b), 17 (c,d), 18 (e,f), 19 (g,h), and 20 kV (i,j) at a solution flow rate of 0.1 mL/min.

Therefore, in the current work, several applied voltage values ranging between 16 and 20 kV were varied; however, 16 kV resulted in the best monodispersity and a narrower distribution of the nanofiber diameter. As a result, 16 kV applied voltage was the best voltage to fabricate uniform, monodisperse nanofibers with a narrower diameter distribution.

Effect of the Solution Flow Rate

The solution flow rate was one of the key factors affecting the uniformity and formation of beads in the resulted nanofibers. It was observed that too low flow rate of 0.005 mL/min resulted in a slightly branched and beaded nonuniform fabricated nanofibers as shown in Figure a. Moreover, a high flow rate above 0.01 mL/min caused difficulty in electrospinning of the composite solutions because of the overwhelming flow of the solution as well as its high viscosity, resulting also in bead formation as shown in Figure e–i. Interestingly, it was found that adjusting the solution flow rate at 0.01 mL/min resulted in the disappearance of branching in addition to more uniform nanofibers with much less beads as shown in Figure c.

Figure 5

SEM images (a,c,e,g,i) and their corresponding histograms (b,d,f,h,j) of inulin/PVA CNFs electrospun from 15% w/w blend solution at different solution flow rates; 0.005 (a,b), 0.01 (c,d), 0.05 (e,f), 0.1 (g,h), and 0.5 mL/min (i,j) at an applied voltage of 16 kV.

Physical Analysis

Inulin/PVA CNFs were found to immediately dissolve when immersed in water, thus cross-linking of the electrospun CNFs was required to prevent their water dissolution. Inulin/PVA CNFs, electrospun from 15% w/w blend solution, were physically and chemically cross-linked to select the best cross-linking method. To investigate the effect of the different cross-linking applied on the solubility stability of the obtained nanofibers, the water immersion test was carried out as mentioned previously.

Physical Cross-Linking Using Heat

Physical cross-linking of inulin/PVA CNFs was performed by heating the inulin/PVA CNFs at various temperatures (from 80 to 140 °C) for 10 min. Then, the CNFs were immersed in warm distilled water at 37 °C for 2 and 24 h. Interestingly, the inulin/PVA electrospun CNFs dissolved immediately, and the solution turned clear, once they were immersed in water. On the other hand, the cross-linked inulin/PVA CNFs shrunk and hardened but did not dissolve. Moreover, the water stability improved where the weight loss % decreased as indicated in Table and Figure a.

Table 1

Weight Loss % of Physically Cross-Linked Inulin/PVA CNFs after Water Immersion

heating temperature (°C)	weight loss % (2 h)	weight loss % (24 h)
80	43.1	49.5
100	30	36
120	28.7	34.1
140	22.7	28.3

Figure 6

(a) % weight loss of physically crosslinked inulin/PVA electrospun nanofibers after immersion in distilled water for 2 and 24 h. Different heating temperatures were tested: (A) 80, (B) 100, (C) 120, and (D) 140 °C. (b) % weight loss of chemically crosslinked inulin/PVA electrospun nanofibers after immersion in distilled water for 2 and 24 h. Different exposure times were tested: (A) 30, (B) 60, (C) 90, and (D) 120 min.

By examining the morphology of physically cross-linked inulin/PVA CNFs at different temperatures from 80 to 140 °C, using SEM, they appeared to be curved, and some fibers were merged together to form bundles but still retained the fibrous structure as shown in Figure . These results were found to be in agreement with the results reported by Kang et al. (2010), in which the heat treatment of PVA nanofibers increased their water resistance.

Figure 7

SEM images of physically cross-linked PVA/inulin electrospun CNFs at different temperatures: (a) 80, (b) 100, (c) 120, and (d) 140 °C.

Chemical Cross-Linking Using Glutaraldehyde

Inulin/PVA CNFs were chemically cross-linked by exposing the CNFs to glutaraldehyde (GA) solution vapor at different exposure time intervals (from 30 to 120 min). Following 2 and 24 h of immersion in warm distilled water, the chemically cross-linked CNFs did not dissolve but they shrunk and hardened. However, after chemical cross-linking of the CNFs with GA, the water stability did not improve, as determined by the unchanged weight loss before the chemical cross-linking as indicated in Table and Figure b.

Table 2

Weight Loss % of Chemically Cross-Linked Inulin/PVA CNFs after Water Immersion

exposure time (min)	weight loss % (2 h)	weight loss % (24 h)	weight loss % (7 days)	weight loss % (14 days)	weight loss % (28 days)
30	54.6	63.1	71.8	72.1	74.6
60	51.2	60.7	65	67.2	70
l90	49.7	58.1	63.4	64.5	69.4
120	44.2	56	60	60.7	68.9

Similar to the physical cross-linking, the chemically cross-linked electrospun CNFs had a curved morphology, and bundles were formed by the merged fibers while retaining the fibrous structure as shown in Figure .

Figure 8

SEM images of chemically cross-linked PVA/inulin electrospun CNFs with different exposure durations: (a) 30, (b) 60, (c) 90, and (d) 120 min.

Comparing both cross-linking methods, the water stability was improved with the physical cross-linking and provided the optimal cross-linking protocol for inulin/PVA CNFs. However, the chemical cross-linking was not a feasible method, as the water stability of nanofibers did not improve.

Microbiological Assessment of Inulin/PVA CNFs

Prebiotic Activity

The prebiotic activity of the electrospun CNFs was tested against Lactobacillus spp., and the colony forming units (CFU) enumeration was recorded (Figure a). An increase in the total viable count following 24 h incubation with inulin/PVA CNFs, reaching 4 × 103 CFU/mL (3 × 103 CFU/mL for the control). However, no increase in the viable count of inulin solution was observed (remained at 2.9 × 103 CFU/mL). In addition, the pH and the optical density (OD) of the culture inoculated with the tested materials were measured before incubation and following 24 h incubation. The results revealed that cultured inulin/PVA CNF solution showed a decrease in pH to 5.7 compared to 6.3 for the control. However, the inulin solution showed no decrease in the pH and remained at 6.2 (Figure b). Note that the inulin/PVA CNFs recorded the highest OD reading, following 24 h incubation, among the tested samples (Figure c).

Figure 9

(a) Total viable count, (b) pH, and (c) OD of Lactobacillus sp. culture containing PVA nanofibers, inulin solution, inulin/PVA CNFs, and water following 24 h incubation, and (d) growth curve of Lactobacillus sp. culture containing inulin/PVA CNFs.

It is likely that the pH of inulin/PVA CNFs decreased because of bacterial fermentation (Gibson & Roberfroid, 1995),[29] and these findings are in agreement with the results reported by Zubaidah and Akhadiana (2013),[30] where inulin showed a decrease in the pH of fermented bacteria. Also, there was an increase in the total viable count of the Lactobacillus sp., because of prebiotic activity of the inulin. In Figure d, the growth curve showed that the growth of the culture containing inulin/PVA CNFs is not substantially greater than the growth of the control.

Antibacterial Activity

The antibacterial activity of PVA/inulin nanofibers was evaluated by the disc diffusion method and CFU method (in the Supporting Information Figures S1 and S2). The antimicrobial tests revealed zero activity for pure PVA and high activity for inulin against Escherichia coli. Inulin/PVA CNFs showed significant activity against E. coli (Figure ). However, both the pure PVA and inulin had little activity against Staphylococcus aureus, contrary to the inulin/PVA CNFs, which showed moderate activity against S. aureus (Figure ). The antimicrobial activity of inulin and PVA/inulin nanofibers could be because of the fructose units present in inulin, which have a natural antibacterial property, resulting from fructose interference with the physiology of the bacterial cell membranes and inability of the bacteria to break it down. Although there was comparable results for both pure inulin and PVA/inulin nanofibers, it is accepted that incorporation of inulin in nanofibers greatly enhances the stability and thus enables controlled release.

Figure 10

Antibacterial activity of PVA, inulin, PVA/inulin, and vancomycin against E. coli and S. aureus.

These results iterate the crucial role of the material shape in bulk form versus nanoform on both prebiotic and antibacterial effects. It is evident that one cannot ignore this fact when fabricating nanocoatings and antibacterial scaffolds with superior properties.

Conclusions

In this study, electrospinning of inulin/PVA composite solution was carried out, and the prebiotic and antibacterial activities were evaluated. A careful investigation to optimize the concentration of the electrospun solutions, electrospinning applied voltage, and solution flow rate was carried out. Inulin solution was not able to be directly electrospun into uniform nanofibers at any concentration. However, an improved spinning ability of inulin solutions was observed, upon mixing inulin with the PVA polymer. In this regards, uniform electrospun CNFs of inulin/PVA were successfully produced by electrospinning, and fabricating inulin/PVA electrospun CNFs was successful.

After thorough investigation and characterization of the produced inulin/PVA electrospun CNFs, the prebiotic and antibacterial activities were assessed. The results of the prebiotic activity of the inulin/PVA electrospun CNFs showed a 33% increase in the Lactobacillus sp. growth compared with inulin solution. Additionally, inulin/PVA CNFs showed higher antibacterial activity with E. coli and S. aureus compared with inulin solution. This clearly shows the unique antibacterial effect of the nanoscale transformation of inulin solution versus inulin nanofibers and their high application potentiality in diverse biomedical settings.

Materials and Methods

Materials

PVA (DP = 2800) and GA solution (25 wt % in H2O) were purchased from Sigma-Aldrich, Germany. Inulin from dahlia tubers was purchased from Sigma-Aldrich, USA. Difco Luria Bertani (LB) agar medium and Difco LB broth medium were purchased from Becton Dickinson Company, USA. The bacterial strains used in this study are: S. aureus ATCC 6538, E. coli, ATCC 8739, and Lactobacillus sp. ATCC 33222.

Methods

As inulin is considered a very interesting material to be tested and cannot be electrospun on its own to form nanofibers, PVA was used in combination with inulin to fabricate the required nanofibers. Both pure PVA and inulin/PVA solutions in distilled water were prepared to be used for nanofiber fabrication and testing of their physical and chemical properties and microbiological activities. First different concentrations of the solutions were prepared to examine the optimal one for electrospinning experiments. Then, different types of crosslinking agents were applied to the obtained CNFs to investigate their effects on the resulting nanofiber stability. Finally, various electrospinning parameters including applied voltage and solution flow rate were varied to achieve the most uniform fibers with least bead formation.

Preparation of PVA Nanofibers

Various concentrations of PVA solutions (e.g., 15, 16, 18, and 20% w/w) were prepared by dissolving the PVA in distilled water using a magnetic stirrer at temperatures ≤100 °C for a period of less than 90 min to acquire a homogenous solution.

Each of the as-prepared PVA solutions was electrospun into nanofibers using a commercial electrospinner (E-Spin Tech, India) with a solution 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. Electrospinning parameters were adjusted as follows: high voltages between 16 and 20 kV and solution flow rates of 0.005–0.5 mL/min. Aluminum foil sheets were used to cover the copper plate collector, and the distance from the tip of the needle to the collector was adjusted to 10 cm. Electrospinning of all solution mixtures was carried out at room temperature. The preparation of PVA nanofibers is dependent upon the parameters of the electrospinning, such as the solution concentration, the solution flow rate, and applied voltage.

Preparation of Inulin/PVA CNFs

The different concentrations of inulin/PVA blend solutions were prepared by adding 2–5% inulin in the form of aqueous solution to each of the as-prepared PVA solutions mentioned previously and left to stir until obtaining the required homogenous blended solution.

Each of the as-prepared solutions of inulin/PVA with different weight blending ratios was electrospun into nanofibers using the same commercial electrospinner with the same procedures used in the preparation of the PVA nanofibers. The preparation of inulin/PVA CNFs is dependent also on the parameters of the electrospinning, such as the solution concentration, the solution flow rate, and the applied voltage.

Preparation of Cross-Linked Inulin/PVA CNFs

Physical and chemical cross-linking of the most acceptable inulin/PVA CNFs was carried out to obtain the most reliable cross-linking method. GA solution was used for chemical cross-linking of the inulin/PVA CNFs. The CNFs were placed inside a closed desiccator occupied with the vapors of 50 mL of GA solution. Exposure time to GA vapor varied from 30 to 120 min and was then thermally treated for 24 h in an oven at 70 °C under vacuum for enhancement of cross-linking and removal of unreacted GA (Abdelgawad, Hudson, & Rojas, 2014). Physical cross-linking was performed by thermal treatment of the inulin/PVA CNFs in a vacuum oven (Jelotech, OV-11, and Korea) at temperatures from 80 to 140 °C for 10 min (Kang et al., 2010).[23]

Analysis

Chemical Analysis

Chemical characterization was carried out to obtain information about the chemical nature of the materials as well as to determine the effect of blending them into a composite to obtain useful information on the type of bonding and functional group changes that are useful in their applications. This test was done by FT-IR spectroscopy (Nicolet 380-Thermo Scientific, USA). Condensed discs consisting of the material (powder/nanofibers) and potassium bromide (KBr) were subjected to infrared radiation, and absorption peaks were obtained. These peaks which gave characteristic chemical details of the chemical composition of the material were processed. Additionally, a thermogravimetric analyzer (TGA Q50, TA Instruments) was employed to reveal the thermal stability profiles of the inulin/PVA CNFs in comparison with its starting materials in order to study the thermal behavior resulted from the formulation formed. To achieve this, few milligrams of each material were placed in a platinum pan and tested under a 60 mL/min purge of nitrogen gas. The samples were then heated up to 700 °C at a rate of 10 °C/min. UV–vis spectroscopy (Varian Cary 500, Agilent Technologies, USA) was used to study the loading capacity and determine the percentage of inulin in PVA nanofibers, following the procedure reported by Shetta et al.[35] Exploiting differences in solubility with temperature PVA/inulin nanofibers were loaded into dialysis bags, immersed in distilled water, and stirred at 400 rpm at room temperature for 2 h. The resulting distilled water containing inulin, which dissolves in water at room temperature, was analyzed using UV–vis spectroscopy. The amount of inulin was estimated by the suitable calibration curve of pure inulin in distilled water with an r2 of 0.995. The loading capacity was determined by eq .

Morphological Analysis

Morphological studies were carried out to determine the integrity of the fibers being formed, their fiber diameter, bead formation, fiber alignment, and length. This test was carried out by a scanning electron microscope (FESEM, Leo Supra 55-Zeiss Inc., Germany). SEM samples were prepared by cutting aluminum foil containing nanofibers into 0.5 cm2 and mounting them on stubs for SEM imaging.

Physical Analysis

Physical characterization was carried out to determine the stability of nanofibers and the ability to resist degradation by water or during processing and storage for cross-linked nanofibers. Noncross-linked nanofibers are known to easily disintegrate in solvents and may easily lose their integrity as a result. This is minimized by crosslinking the nanofibers either by chemical (GA) or physical (heat) means.

Water Immersion Test

Nanofibers were immersed in warm distilled water at 37 °C for 2 and 24 h. Then, the CNFs were immediately weighed after removing the surface water with a filter paper. The degree of weight loss of cross-linked CNFs was calculated according to eq (Yuan, Mo, Wang, Li, Zhang, & Shen, 2012).[31] The weight before immersion in water (wi) and after immersion in water and drying (wf) was measured.

Microbiological Assessment

Inulin, as a natural food ingredient (Niness, 1999), possesses prebiotic activity by balancing the bacterial population in human gut health (Fotiadis, Stoidis, Spyropoulos, & Zografos, 2008).[32] The prebiotic activity of the fabricated PVA/inulin CNFs was tested with Lactobacillus spp. The antibacterial activity of the fabricated CNFs was tested against both S. aureus (Gram positive) and E. coli (Gram negative). All the experiments were carried out in triplicates. Inulin was weighed and sterilized under a UV lamp at 365 nm for 30 min and then dissolved in sterile distilled water. The previously prepared circular discs of PVA and PVA/inulin CNFs were weighed individually. All of the weighed CNFs were placed in sterile Petri dishes and left for 30 min under a UV lamp at 365 nm (Berthomieu & Hienerwadel, 2009).[33]

Prebiotic Activity

The prebiotic activity of inulin nanofibers was examined with Lactobacillus spp. (ATCC 33222) by measuring bacterial growth using total bacterial viable count and OD of bacteria grown in liquid cultures, and any pH changes were recorded. Individual bacterial strains were incubated overnight in a shaking incubator at 37 °C after introducing a single colony to fresh LB broth. Serial dilutions of the overnight cultures were performed to attain dilution factors of 10–1 to 10–6. In brief, 1 mL of the culture was introduced to 9 mL of fresh LB broth (Mandal, DebMandal, Pal, & Saha, 2010),[34] and the test tubes were incubated with PVA nanofibers, PVA/inulin CNFs, inulin solution, control, and water. Following 24 h incubation at 37 °C of the plated cells, the CFU were counted. The total viable counts were carried out again for all tubes after 24 h incubation. Additionally, the OD and pH were measured at zero and 24 h following incubation (Li, Q.; Jia, Z.; Yang, Y.; Wang, L.; Guan, Z. ICSD ’07. IEEE International Conference on Solid Dielectrics, 2007).[28]

Antimicrobial Assessment

The CNFs were examined for their antibacterial activity against E. coli (ATCC 8739) and S. aureus (ATCC 6538) by the disc diffusion method. The cultured bacteria were inoculated separately and after the agar solidification; plugs were cut out with the cork-borer (10 mm diameter). The sterile CNF discs were placed on the surface of seeded agar while solutions were inoculated into the agar well. The plates were solidified at 4 °C for 2 h and then incubated at 37 °C for 24 h, and the zones of inhibition were measured.