Preparation and characterization of pea protein isolate-pullulan blend electrospun nanofiber films

Journal Pre-proof Preparation and characterization of pea protein isolate-pullulan blend electrospun nanofiber films

Xi wen Jia, Ze yu Qin, Jing xin Xu, Bao hua Kong, Qian Liu, Hao Wang PII:

S0141-8130(19)36591-2

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.216

Reference:

BIOMAC 13997

To appear in:

International Journal of Biological Macromolecules

Received date:

20 August 2019

Revised date:

25 November 2019

Accepted date:

26 November 2019

Please cite this article as: X.w. Jia, Z.y. Qin, J.x. Xu, et al., Preparation and characterization of pea protein isolate-pullulan blend electrospun nanofiber films, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.11.216

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

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Preparation and characterization of pea protein isolate-pullulan

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blend electrospun nanofiber films

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Xi wen Jia, Ze yu Qin, Jing xin Xu, Bao hua Kong, Qian Liu*, Hao Wang*

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College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang

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150030, China

Corresponding author. Tel: +86-451-55191847; fax: +86-451-55190577 E-mail address: [email protected] (Q. Liu)

Corresponding author. Tel: +86-451-55191847; fax: +86-451-55190577 E-mail address: [email protected] (H. Wang)

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Abstract: The objective of this work was to fabricate and characterize food-grade pea protein isolate (PPI) and carbohydrate polymer pullulan (PUL) nanofiber films by using green electrospinning technology. The effect of the blend ratios on the PPI/PUL solution properties (e.g. viscosity, surface tension and electrical conductivity) and morphology of the resulting electrospun nanofibers was investigated. The presence of

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PUL in the blends resulted in decreased apparent viscosity (P<0.05), stable surface

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tension (42.09~46.26 mN/m) (P<0.05) and lower conductivity of the solutions

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(P<0.05), which were due to a better chain entanglement and decrease in the

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polyelectrolyte protein character, respectively, both factors were needed for uniform

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nanofiber (around 203 nm) formation. Rheological evaluation indicated a

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pseudoplastic behavior for all formulations. The Fourier transform infrared spectral changes and XRD patterns indicated that the protein and polysaccharide were well

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tangled in nanofibers. The results of the differential scanning calorimetry (DSC) indicate that thermal stability of the electrospun nanofiber films were improved in comparison to pure PUL. Finally, in order to expand the application range of the electrospun nanofiber films in future, thermal crosslinking method was conducted and water contact angles (WCAs) of the thermal treated nanofiber films exhibited better hydrophobic properties compared to the un-crosslinking samples.

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Keywords: Electrospinning, Pea protein isolate, Pullulan, Characterization, Thermal

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crosslinking, Hydrophobic property

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1. Introduction In recent years, electrospinning technology has become the most effective and simplest method for directly and continuously preparing nanofibers under mild operation mode [1]. Electrospun nanofibers exhibit many advantages such as high porosity, high specific surface area, high gas permeability and small fiber pore size

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compared to the conventional fibers [2-3]; therefore, these materials have started to be

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widely used in various fields such as biology, medicine, materials as well as food

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science [4-6].

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In biomedical applications such as wound dressing and tissue scaffolding for

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organ regeneration, the raw materials for preparing nanofibers by electrospinning

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technology are mainly chemical synthetic polymers (polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, etc.) [7]. In food science, however, these materials are

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not edible. Therefore, aiming to reduce usage of organic solvent to minimize environmental impact, the green electrospinning has been proposed, which means using biopolymers (such as proteins and polysaccharides) as the matrix materials and water as the sole solvent to prepare nanofibers [8-9]. Compared to nanofibers prepared from synthetic polymer solutions by electrospinning, bionanofibers have remarkable advantages in terms of uniformity, crystallinity, biodegradability, biocompatibility and sustainability, which are expected to be widely used in the food industry [10]. Proteins form the major ingredient of the human body, and are often in

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themselves, valuable dietary supplements and functional food enhancers [11]. At present, whey proteins, egg albumin, soy protein isolate, gelatin, and zein have been used as raw materials for electrospun nanofibers under different conditions [11-16]. However, scientists are still trying to explore more sources driven by the demand of nutritional complete proteins and to get more edible electrospinning systems.

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Pea protein isolate (PPI), as a by-product of pea starch, not only has balanced

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amino acid composition, but also has prominent lysine content [17]. At the same time,

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it also has good dispersion, stability, fluidity and other water-soluble characteristics as

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well as good gel, is a high value-added protein resources [18-19]. However,

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electrospinning for aqueous solutions of proteins, in either their native or denatured

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state, is not an effective process because of its molecules cannot exhibit sufficient entanglements or interchain associations [14]. In our pre-experiment, solutions of

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various concentrations of PPI alone could not be effectively electrospun, even at high level of protein content (>30% w/v) under different denaturation conditions (heating or pH changing). Therefore, the strategies to generate electrospun nanofibers are based on the use of blending with other spinnable polymers [20]. Pullulan (PUL) is a commonly used spinner material among many generally recognized as safe (GRAS) polysaccharides [10]. The unique linear structure makes PUL good adhesion and excellent film forming properties [21-22]. In the electrospinning process, PUL can bind to proteins by hydrogen bonding, thereby improving the spinnability of the

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proteins by changing the properties of the polymer solutions [23-24], and therefore some studies have focused on the use of blends of proteins with PUL that are mutually compatible for formation of electrospun nanofibers [22, 25-27]. In this work, the PPI/PUL composite nanofiber films were fabricated by green electrospinning technology. Multiple polymer solution characterizations were

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conducted including pH value, surface tension, electrical conductivity and apparent

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viscosity. Nanofiber film characterizations were conducted by scanning electron

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microscopy (SEM), fourier transform infrared spectroscopy (FTIR), differential

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scanning calorimetry (DSC), X-ray diffraction (XRD). The method of thermal

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crosslinking was used to improve the hydrophobicity of nanofiber films, which were

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proved by the measurement of water contact angle (WCA). These biocomposite films are envisaged as potential encapsulation matrices of bioactives for functional food and

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pharmaceutical applications as well as structural components for fabrication of composite materials with specific properties. 2. Materials and methods

2.1. Materials and chemicals Pea protein isolate (PPI), which contains 80% protein (based on the total wet weight basis) was purchased from (Anhui, China). Pullulan (PUL) was supplied by Hai Yang Bai Chuan Biological Technology Co., Ltd, Tianjin, China). All other reagents and chemicals were of analytical grade.

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2.2. Production of nanofibers Firstly, pure PUL powder was dissolved in distilled water. Then the pure PPI powder was added to mix with PUL solutions. The total polymer content in solution was kept constant at 22.5% (w/v). The two polymeric materials were blended at different proportions, PPI:PUL at 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and

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0:100 (w/w). Pure PPI solution (PPI: PUL = 100:0) was excluded from the

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experiment simply because only droplets were produced during the process of

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electrospinning. Each solution was stirred on a digital stirrer (DSX-60, Instrument

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Motor Corporation Co., Ltd., Hangzhou, China) for 3 h and 500 r·min-1 at room

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electrospinning process.

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temperature (25°C) to ensure complete solubilization of the two materials before the

The electrospinning apparatus (DFS-001, Kaiweixin Technology Co., Ltd.,

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Beijing, China) consists of high DC voltage power supply, a syringe and a grounded aluminium foil collector. The polymer solution was placed into the syringe and a 18 kV voltage was applied between the needle tip and the collector (grounded aluminium foil). The flow rate was 0.4 mL/h and the distance between the needle and the fiber collector was 10 cm. The experiments were conducted at room temperature (25°C). Crosslinking was achieved using thermal crosslinking strategy. The electrospun nanofibers of PPI/PUL blends were subjected to step-wise thermal treatment in Midea electric oven with independent temperature control for upper and lower tubes

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(T4-L326F, Midea Kitchen and Bathroom Appliance Manufacturing Co., Ltd., Guangdong, China) for 3 h at 120℃. The fibers with PPI/PUL before crosslinking were denoted as PPI50PUL50, PPI40PUL60, PPI30PUL70 and PPI20PUL80, while the corresponding fibers after crosslinking were denoted as TC PPI50PUL50, TC PPI40PUL60, TC PPI30PUL70 and TC PPI20PUL80, respectively.

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2.3. Characterization of the polymer solutions

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pH value of the polymer solutions was measured with a potentiometer

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(METTLER TOLEDO, FE20K, Zurich, Switzerland). The surface tension of the

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polymer solutions was determined by Wilhelmy Plate method using a tensiometer

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(DCAT21, Dataphysics, Stuttgart, Germany). The conductivity of the polymer

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solutions was determined using a digital conductivity meter (METTLER TOLEDO, Delta 326, Zurich, Switzerland).

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Rheological behavior of the polymer solutions was measured using a dynamic shear rheometer (DHR-1, TA Instruments Inc., New Castle, USA) equipped with two parallel plates (diameter 60 mm) set at 0.5 mm apart. The shear rate range from 0.1 s-1 to 100 s-1 at 25 ± 1℃. The shear stress (τ) and shear rate (γ∙) data were collected. The data were fitted to the power law model (Eq. (1)):

  k( ) n where, τ is the shear stress (Pa), γ∙ is the shear rate (s-1), k is the consistency coefficient (Pa·sn ) and n is flow behavior index. Using the values of flow behavior

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index and consistency coefficient, apparent viscosities were calculated using (Eq. (2)) for different shear rates:   k  n1

where, η is apparent viscosity (Pa·s). 2.4. Scanning electron microscopy (SEM)

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The morphology of the electrospun fibers was examined using SEM (SN-3400,

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Hitachi, Tokyo, Japan) after sputtering the samples with a goldepalladium mixture

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under vacuum. All SEM experiments were carried out at an accelerating voltage of

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5.0 kV. Fiber diameters of the electrospun fibers were measured by ImageJ-DiameterJ

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software from the SEM images obtained at a magnification of 5000×.

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2.5. Fourier transform infrared analysis (FTIR) To determine the composition and possible interaction between the fibers

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components, the infrared spectrums of the fibers were registered using a spectrophotometer (FTIR, ALPHA-T spectrometer, Bruker, Billerica, USA). Scans were taken over a spectral range of 4000~600 cm-1 with 4 cm-1 wavenumber resolution and an average of 16 scans. 2.6. Differential scanning calorimetry (DSC) Thermal analysis of the pure pea protein isolate and PUL as well as the electrospun fibers were investigated using DSC (TA Instruments Q2000, New Castle, USA). Measurements were carried on a DSC Q2000 with a sealed empty pan as the

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reference and under a nitrogen atmosphere (30 mL/min). Samples of about 5 mg were sealed in aluminum pans. The samples were heated in a single cycle from 20 to 250℃ at a heating rate of 10 ℃/min. 2.7. X-ray diffraction (XRD) The crystal structures of the PPI/PUL nanofibers were investigated by X-ray

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diffraction (XRD) using the X’Pert Pro diffractometer (PA Nalytical B.V., Almelo,

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The Netherlands) with a CuK a radiation source operated at a tube voltage of 40 kV

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and a tube current of 35 mA. A diffraction range of 10~60° (2θ) was selected. The

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2.8. The water contact angles (WCAs)

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experiments were carried out in triplicate.

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sessile

drop

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The water contact angles (WCAs) of the PPI/PUL nanofibers was measured by method

using

a

contact

angle

goniometer

(XG-CAM,

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Xuanyichuangxi Industrial Equipment Co., Ltd., Shanghai, China). The PPI/PUL nanofibers (2×3 cm2) were placed on a glass slide. A drop of distilled water (3 µL) was dropped on the fiber mat surface, and drops were video recorded. The WCAs were calculated from the images at the time of 3 s and Five Point fitting was applied on measurements. The measurements were performed five times for each sample at different locations on the surface. The maximum and minimum values were removed from the data and the final result was analyzed based on the rest three which expressed as mean ± standard deviations (SD).

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2.9. Statistical analysis For each batch of samples, measurements of related traits were carried out in triplicate technical replication. All data were expressed as mean ± standard deviations (SD) and were analysed using the General Linear Models procedure of the Statistix 8.1 software package (Analytical Software, St Paul, USA). One-way analysis of

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variance (ANOVA) was performed (P<0.05) between means using the Tukey

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procedures.

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3. Results and discussion

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3.1. Electrospinning solution properties

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Solution parameters such as viscosity, conductivity, and surface tension

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significantly affect the electrospun nanofiber morphology. Table. 1 summarized various parameters which included component ratio of PPI/PUL, pH value, electrical

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conductivity, surface tension that affected the electrospinning process and nanofiber morphology obtained. For the spinnability of different solutions, the higher PPI content of solutions (≥70% (w/w)) did not produce fibers but drops since PPI was a kind of globular protein which leaded to low entanglement between molecules. Conversely, mixed solutions with high PUL ratio (>50% (w/w)) showed good spinnability due to the unique linear linkage pattern of PUL, which endowed PUL with adhesive property and capacity to form composite nanofibers. And the result was consistent with previous reports that the PUL content in blended solutions was able to

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influence productivity as well as nanofiber morphology [22, 28]. For the conductivity of different solutions, it can be observed that increasing the amount of the PUL led to the decrease of the conductivity (8.98~0.63 mS/cm). This was mainly due to the interaction between PPI and PUL, which resulted in the decreased polyelectrolyte properties of proteins [16, 25].

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Apparent viscosity affects the extent of the polymer molecular entanglements

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within the solutions [29-32]. The flow curves were shown in Fig. 1 and all solutions

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obeyed the Power Law. The apparent viscosity of solutions decreased with the

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increase of shear rate, which meant the shear thinning behavior of Non-Newtonian

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fluid. Table. 2 showed the consistency index (k) and the flow behavior index (n) of

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the solutions that contain different ratio of PPI and PUL. As shown in Table. 2, when the PPI content increased, the consistency index (k) of the solutions decreased

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(P<0.05). Relatively low flow behavior index values of solutions (0.416~0.987) also confirmed the shear thinning behavior, which indicated that the protein molecules and polysaccharide molecules were intertwined in the blended solutions because of the hydrogen bonding between protein molecules and polysaccharide molecules [31-32]. 3.2. Morphology of nanofibers Fig. 2 showed the morphologies and diameter distributions of the PPI/PUL electrospun nanofibers of varying weight ratios. During the electrospinning process, increase of PUL concentration promoted the formation of smoother nanofibers with

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higher diameters (Fig. 2E-2H), this was due to fact that the increase of PUL amount improved molecular entanglements through the interactions between both biopolymers, which stabilized the polymer jet [22]. The pure PUL nanofibers showed an average diameter of 294 ± 102 nm. As PPI was mixed with PUL in water solution, the average diameter of the hybrid nanofibers decreased gradually, for instance, when

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the weight ratio of PPI increased from 20% (Fig. 2G) to 70% (Fig. 2B), the nanofiber

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diameters gradually decreased from 203 nm to 76 nm, which suggested that the

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protein molecules, particularly of globular conformation, cannot exhibit sufficient

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entanglements or interchain associations for electrospinning [14, 20, 33-34].

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3.3. FTIR analysis

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Fig. 3 showed the infrared spectra of PPI and PUL powders and electrospun nanofiber films obtained from different ratios of PPI/PUL. For the PUL powder, a

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broad peak located around 3342 cm-1 was assigned to O-H stretching, this absorption band was affected by the inter-molecular or intra-molecular hydrogen bonds. The peak around 2926 cm-1 corresponded to the C-H stretching, while the peaks between 1156 cm-1 and 1028 cm-1 were assigned to the C-O. The absorption band at 1079 cm-1 corresponded to the polysaccharide (1→4) glycosidic bonds stretching vibration, while the peaks at 849 cm-1 and 928 cm-1 appeared to originate from the α-glucopyranosyl units and α-(1,6) glycosidic bonds, respectively [35]. In the PPI powder spectrum, a maximum band was placed at around 3288 cm-1, which

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With the increase of PPI proportion, on one hand, a shift to lower wave number in the

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FTIR range of the O-H and N-H stretching vibrations (i.e. from ~3000 to ~3500 cm-1)

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and the widening of the band from ~3000 to ~3500 cm-1 were observed (Fig. 3A). On

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the other hand, the bands of the Amide I and Ⅱ moved to higher wave number (Fig.

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3C). As reported by Aceituno-Medina et al. [16], these results were attributed to the

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protein and the polysaccharide interactions through hydrogen bonding. 3.4. Differential scanning calorimetry analysis (DSC)

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The thermal stability of the PPI and PUL powders and hybrid nanofiber films were evaluated by DSC. PPI powder (Fig. 4g) showed an endothermic peak at 180℃ corresponding to its melting point (Tm), PUL powder (Fig. 4a) showed its Tm at 149℃. The pure PUL electrospun nanofibers (endothermic peak of 164℃ in Fig. 4b) showed greater thermal stability than the PUL powder, which indicated the electrospinning process could improve the intermolecular interactions, these results agreed with the previous report by Aceituno-Medina et al. [16]. For nanofibers obtained from blends of natural polymers, Tm value shifted to a higher temperature

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with the increase of the PPI content, for example, the PPI:PUL 20:80, 30:70, 40:60 and 50:50 electrospun nanofiber films (Fig. 4 c, d, e and f) showed Tm values at 167℃, 171℃, 186℃ and 187℃, respectively, this can be attributed to the interactions through hydrogen bonding between amino and carboxyl groups of PPI and hydroxyl group of PUL [37-38]. The PPI:PUL nanofiber films (Fig. 5 d, e, and f) showed

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slightly better thermal stability than that of the pure PPI powder which concurred with

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the interactions between the protein and the polysaccharide chains mentioned by the

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3.5. X-ray diffraction analysis (XRD)

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FTIR spectroscopy analysis.

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Fig. 5 showed the XRD patterns of PPI powder, PUL powder and nanofiber films

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of different PPI/PUL proportions. The PPI and PUL powders showed broader peak at 19.3° and 18.3°, respectively and the diffraction peak appeared as an amorphous

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structure. After electrospinning process, the XRD pattern of the nanofiber films exhibited wider peaks and small angular peak shift compared with the powder, which caused by intermolecular force between PPI and PUL, these results were in concordance with the previous study that electrospinning process could hinder the crystallization and facilitate the formation of amorphous structure of polymers [39]. 3.6. The water contact angles (WCAs) After finishing the characterization of different nanofiber films, we found that the nanofiber films were too hydrophilic to be used in food packaging materials.

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Therefore, thermal crosslinking method was conducted to improve the hydrophobicity of the samples. The WCAs of PPI/PUL nanofiber films at 3 s before and after cross-linking by thermal were shown in Fig. 6, before crosslinking, the WCAs of the PPI/PUL nanofiber films were increased from 15.1° to 38.0° with the reduction of PUL. The reason for the relatively low WCAs was that the water could infiltrate and

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fill up most of the hollows and pores formed by the hydrophilic protein and

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polysaccharide, forming a surface which was partly solid and liquid [40-41]. After

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thermal crosslinking treatment, the WCAs of the corresponding nanofiber films

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increased from 36.8° to 89.8°. Moreover, for the same ratio of PPI/PUL nanofiber

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films, the WCAs of cross-linked ones were more than twice that of the uncrosslinked

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samples, which due to the fact that the PPI and PUL molecules interacted with each other by hydrogen bonding formed by hydrophilic groups, resulted in more

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hydrophobic surfaces [42]. 4. Conclusions

Food grade nanofiber films based on aqueous solutions of PPI/PUL without using any synthetic polymers were fabricated using the green electrospinning technique. The addition of PUL to the blend reduced the apparent viscosity and electrical conductivity of the solution, which was conducive to the formation of uniform continuous nanofibers with a diameter of 203 nm. FTIR spectroscopy confirmed that both protein and polysaccharide constituents were present in the nanofiber structures,

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which was consistent with the strong interaction between the amino group of the protein and the hydroxyl group of PUL. The electrospun PPI/PUL nanofibers exhibited better thermal stability than that of pure PPI or pullulan. The WCAs showed that the hydrophobicity of thermally crosslinked nanofiber films was significantly improved. The edible nanofiber films obtained by the green electrospinning process

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could be potentially used as antibacterial packaging matrix materials to extend the

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shelf life of foods or a promising delivery system for value added enriched and

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fortified food products.

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Acknowledgements

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This study was supported by the Heilongjiang Postdoctoral Financial Assistance

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and characterization of food-grade electrospun fibers from amaranth protein and pullulan blends. Food Res. Int. 54 (1) (2013) 667-674. [26] A. Blanco-Padilla, A. López-Rubio, G. Loarca-Piña, L. Gómez-Mascaraque, S. Mendoza, Characterization, release and antioxidant activity of curcumin-loaded amaranth protein-pullulan electrospun fibers. LWT-Food Sci. Technol. 63 (2) (2015) 1137-1144. [27] M. Aceituno-Medina, S. Mendoza, J. M. Lagaron, A. López-Rubio, Photoprotection of folic acid upon encapsulation in food-grade amaranth

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(Amaranthus hypochondriacus L.) protein isolate-pullulan electrospun fibers. LWT-Food Sci. Technol. 62 (2) (2015) 970-975. [28] J. Aneli, G. Zaikov, O. Mukbaniani, Physical principles of the conductivity of electrically conductive polymer composites (Review). Mol. Cryst. Liq. Cryst. 554 (1) (2012) 167-187.

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[29] A. Aydogdu, G. Sumnu, S. Sahin, A novel electrospun hydroxypropyl Morphology

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physicochemical properties. Carbohydr. Polym. 181 (2017) 234-246.

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[30] N. Maftoonazad, M. Shahamirian, D. John, H. Ramaswamy, Development and

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(2019) 393-402.

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nanocomposite mats incorporated with cinnamaldehyde. Mater. Sci. Eng: C. 94

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[31] S. Sukigara, M. Gandhi, J. Ayutsede, M. Micklus, F. Ko, Regeneration of bombyx mori silk by electrospinning-part 1: Processing parameters and geometric properties. Polymer. 44 (19) (2003) 5721-5727. [32] C. Kriegel, A. Arecchi, K. Kit, D. J. Mcclements, J. Weiss, Fabrication, functionalization, and application of electrospun biopolymer nanofibers. Crit. Rev. Food Sci. Nutr. 48 (8) (2008) 775-797.

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[33] P. Nieuwland, P. Geerdink, P. Brier, P. V. D. Eijiden, J. T. M. M. Henket, M. L. P. Langelaan, N. Stroeks, H. C. Deventer, A. H. Martin, Food-grade electrospinning of proteins. Innovative Food Sci. Emerging Technol. 20 (2013) 269-275. [34] S. T. Sullivan, C. Tang, A. Kennedy, S. Talwar, S. A. Khan, Electrospinning and heat treatment of whey protein nanofibers. Food Hydrocoll. 35 (1) (2014) 36-50.

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nanofibers: Preparation and property characterization for antibacterial activity.

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[36] J. L. Kong, S. N. Yu, Fourier transform infrared spectroscopic analysis of protein

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secondary structures. Acta Biochim. Biophys. Sin. 39 (8) (2007) 549-559.

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[37] B. Dhandayuthapani, U. M. Krishnan, S. Sethuraman, Fabrication and characterization of chitosan-gelatin blend nanofibers for skin tissue engineering.

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J. Biomed. Mater. Res., Part B. 94 (1) (2010) 264-272. [38] C. Salas, M. Ago, L. A. Lucia, O. J. Rojas, Synthesis of soy protein-lignin nanofibers by solution electrospinning. React. Funct. Polym. 85 (2014) 221-227. [39] R. Zhao, X. Li, B. Sun, Y. Zhang, D. W. Zhang, Z. H. Tang, X. S. Chen, C. Wang, Electrospun chitosan/sericin composite nanofibers with antibacterial property as potential wound dressings. Int. J. Biol. Macromol. 68 (2014) 92-97.

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[40] L. L. Deng, Y. Li, F. Q. Feng, H. Zhang, Study on wettability, mechanical property and biocompatibility of electrospun gelatin/zein nanofibers cross-linked by glucose. Food Hydrocoll. 87 (2019) 1-10. [41] Z. W. Ma, M. Kotaki, T. Yong, W. He, S. Ramakrishna, Surface engineering of electrospun polyethylene terephthalate (PET) nanofibers towards development of

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[42] B. Satilmis, T. Uyar, Fabrication of thermally crosslinked hydrolyzed polymers

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membranes. Macromol. Chem. Phys. 220 (2018) 1-11.

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Conflict of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or

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the review of, the manuscript entitled.

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Fig.1 Viscosity-shear rate profiles of different pea protein isolate (PPI)-pullulan (PUL) blended solutions. Fig.2 SEM images of electrospun pea protein isolate (PPI)-pullulan (PUL) blend structures of varying weight ratios (both polymeric constituents were dissolved in

of

distilled water) : A) 80:20, B) 70:30, C) 60:40, D) 50:50, E) 40:60, F) 30:70, G) 20:80,

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H) 0:100.

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Fig.3 Infrared absorbance spectra of pure pea protein isolate (PPI) and pullulan (PUL)

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as well as of different electrospun PPI-PUL blend structures.

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Fig.4 DSC scans of different electrospun pea protein isolate (PPI)-pullulan (PUL)

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blend structures: a) PUL and g) PPI powder, b) Fibers from 0:100 (PPI/PUL), c)

(PPI/PUL).

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Fibers from 20:80 (PPI/PUL), d) 30:70 (PPI/PUL),e) 40:60 (PPI/PUL) and f)50:50

Fig.5 X-ray profiles of different pea protein isolate (PPI)-pullulan (PUL) nanofibers Fig.6 Water contact angles of the pea protein isolate (PPI)-pullulan (PUL) nanofibers before and after thermal crosslinking (TC).

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Table 1 Changes of pH value, conductivity, surface tension and spinnability of different pea protein isolate (PPI)-pullulan (PUL) blended solutions PPI/PUL

Conductivity

Surface tension Spinnability x

pH value (w/w)

(mS/cm)

(mN/m)

7.25 ± 0.01e

8.98 ± 0.19a

52.68 ± 2.45b

-

70:30

7.29 ± 0.02d

8.24 ± 0.05b

41.91 ± 1.20d

-

60:40

7.32 ± 0.02cd

7.95 ± 0.14b

39.87 ± 0.26d

-/+

50:50

7.37 ± 0.01ab

6.91 ± 0.03c

42.09 ± 0.39d

+

40:60

7.39 ± 0.01a

5.75 ± 0.11d

46.26 ± 0.70c

+

30:70

7.35 ± 0.01bc

4.93 ± 0.24e

46.20 ± 0.65c

+

20:80

7.33 ± 0.03cd

3.11 ± 0.04f

45.32 ± 1.08c

+

0:100

6.88 ± 0.01f

0.63 ± 0.01g

63.78 ± 0.38a

+

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80:20

Values are given as the mean ± SD from triplicate determinations;

a-g

indicates that

different letters in the same column differ significantly (P < 0.05). x

means the spinnability of different pea protein isolate (PPI)-pullulan (PUL) blended

solutions. The “-” represents the low spinnability, and the “+” represents the high spinnability.

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Table 2 Rheological properties of different pea protein isolate (PPI)-pullulan (PUL) blended solutions PPI/PUL (w/w)

K (Pa·sn)

80:20

58.01 ± 0.01a

70:30

34.51 ± 0.50b

60:40

19.36 ± 1.17c

0.67 ± 0.01b

50:50

7.83 ± 0.91d

0.88 ± 0.03a

40:60

4.70 ± 0.76e

0.89 ± 0.04a

0:100

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20:80

0.42 ± 0.07c 0.64 ± 0.09b

2.96 ± 0.32ef

0.94 ± 0.02a

2.73 ± 0.14f

0.97 ± 0.01a

0.47 ± 0.01g

0.99 ± 0.01a

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30:70

n

Values are given as the mean ± SD from triplicate determinations; different letters in the same column differ significantly (P < 0.05). k: consistency index; n: flow behavior index.

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a-g

indicates that

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Highlights • The PPI/PUL composite nanofibers were fabricated by electrospinning technology. • Impact of electrospinning parameters on PPI/PUL nanofiber films was investigated. • The electrospun PPI/PUL fibers exhibited better thermal stability than that of pure PPI or pullulan.

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• Thermal crosslinking method can improve the hydrophobic properties of the

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PPI/PUL electrospun nanofiber films.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6