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Preparation of a hordein-quercetin-chitosan antioxidant electrospun nanofibre film for food packaging and improvement of the film hydrophobic properties by heat treatment
Food Packaging and Shelf Life 23 (2020) 100466
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Preparation of a hordein-quercetin-chitosan antioxidant electrospun nanofibre film for food packaging and improvement of the film hydrophobic properties by heat treatment
T
Sen Li, Yan Yan, Xiao Guan*, Kai Huang School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hordein Quercetin Chitosan Electrospinning Heat treatment Antioxidant
Bio-based functional packaging materials are attracting increasing attention, but poor mechanical properties often hinder their application. This study aims to develop a novel degradable antioxidant nanomaterial with hordein, quercetin and chitosan via electrospinning. Heat treatment was applied to increase the water resistance of the blended nanofibre film. Our results showed that heat treatment significantly improved the water resistance of the blended nanofibre film without affecting its antioxidant activity. Further physiochemical analysis indicated that heat treatment might improve the water resistance of the nanomaterial by decreasing the crystallinity, reducing surface absorbed hydroxyl groups and increasing the structural stability of the blended nanofibres. Together, our results suggested that heat treatment could be an effective method to improve the water resistance of protein-based nanofibres, and the heat-treated hordein-quercetin-chitosan electrospun nanofibre film was a novel biodegradable material with excellent antioxidant activity and water resistance for food packaging.
1. Introduction In recent years, bio-based packaging materials have attracted increasing attention, as traditional plastic materials have caused serious threats to the environment (Alehosseini, Gómez-Mascaraque, MartínezSanz, & López-Rubio, 2019; Hansen & David, 2008). Electrospinning is one of the most widely used methods to generate bio-materials because it can generate nanofibres (NFs) with a more uniform diameter, higher surface area-to-volume ratio, richer composition and thinner membrane compared with other methods (Bhardwaj & Kundu, 2010; Moreira, de Morais, de Morais, da Silva Vaz, & Costa, 2018). Electrospinning is also an effective technique to develop new functional food packaging materials, as bioactive or functional substances could be easily incorporated into the materials by electrospinning. Natural polymers, such as proteins and polysaccharides, are ideal raw materials for bio-based environment-friendly materials due to their biodegradability, biocompatibility, low toxicity and renewability (Sharma, Saini, Sharma, & Sandhu, 2019; Sirviö, Kolehmainen, Liimatainen, Niinimäki, & Hormi, 2013). Hordein (HO) is the most important storage protein in barley, and it can be obtained from the byproducts of the brewing industry (Houde, Khodaei, Benkerroum, &
Karboune, 2018). The reutilization of HO is poor because of its nutritional defects (Wang & Chen, 2012). However, HO has many advantages such as biodegradability, non-toxicity, edible properties and excellent spinnability. Chitosan (CS) is another bio-based material that is frequently used for electrospinning. CS showed distinct properties, such as good compatibility, biodegradability, and low toxicity (Ahsan et al., 2018). In particular, CS has been widely used in electrospinning as a stabilizer and reducing agent, and it shows distinctive characteristics such as antioxidant and antimicrobial activity (Zhuang, Cheng, Kang, & Xu, 2010). Poor mechanical characteristics especially water resistance is an evident shortcoming that restricts the application of bio-based NF films. In our previous study, we found the water resistance of pure HO-NFs was poor as well as other protein-based NFs, and CS addition could improve the water resistance ability of HO electrospun NFs to some extent by alleviating the shrinkage of the film (unpublished data). However, the water resistance of the film is still poor, and more effort needs to be made to strengthen the water resistance of HO-based nanofibre films to widen their application in food packaging. Crosslinking is the most common method to overcome the limitations of a nanofibre film from biomaterials. Several chemical and
⁎ Corresponding author at: School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai, 200093, China. E-mail address: [email protected] (X. Guan).
https://doi.org/10.1016/j.fpsl.2020.100466 Received 25 August 2019; Received in revised form 19 December 2019; Accepted 3 January 2020 2214-2894/ © 2020 Elsevier Ltd. All rights reserved.
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centrifugation at 8500 ×g for 30 min at 4 °C, the precipitates were obtained and further freeze-dried. QU (minimum purity 98%) was purchased from the Shanghai Winherb Medical Science Co., Ltd. CS (< 200 mPa s−1, MW = 152143, 95% deacetylated), methanol (98%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt (ABTS) were acquired from the Aladdin Reagent Co., Ltd. Acetic acid (100%) and all the rest of the chemical reagents were provided from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were used without further purification, and distilled deionized water was used in all experiments. Apples and potatoes were purchased from the local supermarket.
physical crosslinking methods have been reported to improve the mechanical properties of biomolecule-based NF films. Badawy et al. found that glutaraldehyde treatment could form crosslinking bonds between chitosan and alginate that significantly decrease the degree of swelling of chitosan/alginate/gelatin polymers (Badawy, Taktak, Awad, Elfiki, & Elela, 2017). However, chemical substances, such as glutaraldehyde, epichlorohydrin, and carbodiimide, have shown toxicity or low efficiency (Reddy, Reddy, & Jiang, 2015). In contrast, physical methods are much safer to improve the water resistance of biopolymer materials. Among which, heat treatment could be a good candidate, especially for those NFs that encapsulate light sensitive chemicals. Heat treatment is the process of exposing materials to high temperature cycles, thereby affecting the rearrangement of the molecular structure (Miller, Chiang, & Krochta, 2010). Heat treatment can cause conversion of the protein secondary structure or break the secondary bonds used to maintain the spatial structure of protein (Bonwell & Wetzel, 2009). Internal sulfur-containing amino acids will be exposed when proteins undergo denaturation during heat treatment, and new intermolecular disulfide bonds will form between those amino acids and reactive groups (Wu et al., 2019). Thermal stability and conformation of proteins largely depend on amino acid composition, therefore different proteins exhibit different degrees of modification after heat treatment (Neo et al., 2014). Heat treatment has been applied to modify the functional and structural properties of proteins and protein based materials (Sun et al., 2016). The whey protein gel prepared by heat treatment showed a smaller pore size and larger gel strength (Spotti, Tarhan, Schaffter, Corvalan, & Campanella, 2017). Studies from Sullivan et al exhibited that the heat treatment of whey protein NFs could increase the thermal stability of the fibres, making the fibre mat substantially insoluble in water (Sullivan, Tang, Kennedy, Talwar, & Khan, 2014). Neo et al. found that the morphology of the gallic acid loaded zein fibres became distorted and flattened and the hydrophobicity of the fibre surface was increased (Neo et al., 2014). Quercetin (QU) is a flavonoid found in many foods and is well known for its strong antioxidant properties (Tan, Liu, Guo, & Zhai, 2011). However, poor water solubility hindered its application (Aceituno-Medina, Mendoza, Rodríguez, Lagaron, & López-Rubio, 2015; Vashisth et al., 2013). In this study, we prepared a novel antioxidant nanomaterial with HO, CS, and QU by electrospinning and evaluated the effect of heat treatment on the water resistance of the blended fibre film. We examined the influence of heat treatment on the fibre morphology, water resistance and antioxidant activity by using scanning electron microscopy analysis (SEM), water contact angle analysis, free radical scavenging analysis and an enzymatic browning test. Fourier transform infrared spectrum analysis (FTIR), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were employed to explore the molecular and physiochemical property alterations of heat-treated NFs.
2.2. Electrospinning Pure HO NFs and QU-CS-encapsulated HO complex nanofibres (HOQU-CS NFs) were produced by electrospinning. For the HO NFs, the 15% (w/v) HO powder was prepared in acid/water (9:1, v/v), which was stirred for 12 h until complete dissolution. The HO-QU-CS NFs were prepared as follows: first, the CS solution (0.4%, w/v) was prepared in acetic acid/water (9:1, v/v) and stirred for a minimum of 4 h until complete dissolution. HO powder (11%, w/v) was then dissolved in the CS solution with vigorous stirring overnight. Finally, QU powder (5%, w/w, relative to HO) was evenly dissolved in the obtained CS/HO solution under constant stirring for 8 h at room temperature. The final polymer solution was inhaled into 5 mL syringes powered by a syringe pump (Longerpump, Hebei, China) to achieve a polymer solution feed rate of 1.0 mL h−1. The stainless-steel needle of the syringe (inner diameter =0.9 mm) was connected to the positive terminal of the direct current (dc) power supply (Boher HV, Shanghai, China), and the receiving plate covered with aluminium foil was connected to the negative electrode of the power supply. The dc power supply voltage was maintained at approximately 9 kV, and the distance from the needle tip to the receiving plate was 15 cm. Eventually, the electrospun NF film was dried in a vacuum drying oven at 45 ℃ overnight. 2.3. Heat treatment HO-QU-CS NF films were put into a drying oven for heat treatment. The effects of the treatment temperature and treatment time were examined separately. The temperature was set at 90 ℃, 120 ℃, 150 ℃, and 180 ℃ with treatment for 6 h. The times were set as 3 h, 6 h, 9 h, and 12 h with treatment at 150℃. After heat treatment, water contact angle analysis was performed to analyse the hydrophobic property of NF films. 2.4. Physiochemical characterization and measurement Scanning electron microscopy analysis (SEM): The morphology of the NFs was determined by a field-emission scanning electron microscope (FEI Quanta Q400). Prior to examination, the samples were sprayed with a gold-palladium mixture under vacuum. Fibre diameters were determined using Java Image Processing Software ImageJ 1.29. The average diameter of the fibre was calculated and examined by measuring the sizes of the NFs in the scanning electron micrographs at more than 100 different places. Thermogravimetric analysis and differential thermal analysis (TGADTG): The thermal properties of the electrospun NFs were evaluated by TGA (SDT Q600, TA Instruments, USA) in the temperature range of 50 ℃-550 ℃. The heating rate was regulated at 10 ℃ /min in an argon atmosphere. Thermogravimetric analysis (TGA) and derivative thermogravimetry (DTG) curves based on the data were obtained Fourier transform infrared spectrum analysis (FTIR): The chemical interactions and composition of the electrospun nanofibre films were analysed by FTIR spectroscopy (IRTracer-100 Shimadzu, Japan). This process was carried out in the middle infrared region with a
2. Materials and methods 2.1. Materials HO protein was prepared using the ethanol methodology reported by Wang et al. (Wang et al., 2010). Briefly, barley flour and hexane were stirred for 2 h at room temperature for defatting with a flour-tohexane ratio of 1:6 (w/v). The defatted barley flour was separated from hexane by centrifugation at 5000 ×g at 25 °C for 30 min. Then, it was air-dried overnight, followed by extraction with 1 M NaCl solution (10% w/v suspension) at 55 °C for 1 h. After washing thrice with deionized water, the residue was extracted with 60% ethanol solution (v/v; 10% w/v suspension) at 60 °C for 1 h. The supernatant was obtained by centrifugation at 5000 ×g for 30 min, and ethanol was removed by evaporation using a rotary evaporator. HO was separated by cold precipitation at 4 °C overnight from the supernatants. After 2
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wavenumber range of 4000-400 cm−1 and a spectral resolution of 4 cm−1. X-ray diffraction analysis (XRD): The crystal structure of the NFs was evaluated by XRD (D8 Advance diffractometer, Bruker, Germany) employing the Cu tube as an X-ray source (λ of CuKα = 0.15418 nm). The testing speed was 0.1 s/step with a step size of 0.02°. X-ray photoelectron spectroscopy (XPS): XPS measurements were carried out with an ESCALAB 250Xi spectrometer (Thermo Fisher, USA) equipped with an Al- Kα X-ray source. Water contact angle analysis: The wettability of electrospun NFs was evaluated through the sessile drop approach. An NF film (approximately 1 cm2 area) was cut and placed on a lifting platform attached to the Optima contact angle goniometer (AST Instrument Ltd., USA). A droplet (approximately 0.1 μL) of distilled water was dropped on the surface of the fibre by adjusting a microsyringe at room temperature (approximately 24℃). The tangent line between water droplets and the contact point of the NF film surface was captured and measured in real time by CAM 100 computer simulation software. The contact angle of each sample was measured three times and averaged.
approximately 1644, 1560, and 1460 cm−1, which represented the bands of amide I (C]O), amide II (C–N, NeH), and amide III (NeH), respectively. The typical absorption bands of CS were located at 1645 cm-1 (C]O), 1428 cm−1 (CH2 bending), 1383 cm−1 (CH3 bending) and 1158 cm-1 (CeOeC). QU had a characteristic peak of -C = O groups at 1667 cm-1 and three typical peaks of benzene rings (two main peaks at 1610 cm-1 and 1520 cm-1 and a small peak between them) (Yan, Wu, Yu, Williams, & Sun, 2014). A band at 1318 cm-1 corresponds to the = CeOeC bridge. Compared with HO-NFs, the three typical HO absorption bands shifted in the blended NFs (Fig. 1b). In particular, the amide I band near 1644 cm−1 shifted significantly to 1659 cm−1. This phenomenon indicated that the addition of CS and QU possibly changed the surface molecular structure of the NFs, which resulted in the shift and deformation of the peak position. In the spectrum of blended NFs, the typical bands of QU were also shifted because the characteristic peaks of benzene rings were covered and overlapped by the intense amide bands of HO. The peak at 1667 cm−1 in the quercetin spectrum and 1645 cm-1 in the chitosan spectrum was shifted to 1659 cm−1 in the HOeQUeCS NF spectrum, which may be due to the hydrogen bond formation among the carbonyl groups of QU, carboxyl groups of HO, and hydroxyl groups of CS. The thermal stability of QU, CS, HO-NFs and HO-QU-CS NFs were further investigated by TGA-DTG, and the thermograms are shown in Fig. 1c and d. From the TGA curve, we could observe that distinct extents of weight loss were found at various temperature ranges and in different samples. The decomposition of the HO-QU-CS NFs was divided into three main stages of weight loss between 25 ℃ and 500 ℃. In the initial stage, the initial weight loss was slight (approximately 4%) below 100 ℃ due to water loss. The second decomposition stage was between 100℃ and 340℃, and this stage was related to the evaporation of HO and CS, with nearly 58% weight loss. The final weight loss between 340 ℃ and 500 ℃ resulted in 18% weight loss. The DTG curve of HO-QU-CS NFs had a small exothermic peak at 340℃, which may be due to the main thermal degradation of QU (Fig. 1d). The DTG thermograms showed that the evaporation of HO-QU-CS NFs was shifted to a higher temperature compared with that of pure HO-NFs. This result implied the successful loading of QU and CS into the HO electrospun NFs and the strong interaction among HO, QU, and CS. In addition, it is worth noting that there was still no mass weight loss of the blended NFs at 200℃, which indicated that heat treatment under 150℃ couldn’t lead to degradation of the blended NFs.
2.5. Free radical scavenging analysis The DPPH radical scavenging capacity of the NFs was examined as follows. Generally, 100 μL of the nanofibre films dissolved in acetic acid (2 mg/mL) was added into 3 mL of DPPH solution (0.1 mmol/L, in methanol). The mixed solution was maintained at room temperature for 30 min in the dark and then, the absorption value was recorded at 517 nm. All measurements were performed in triplicate. The DPPH scavenging rate was calculated by the following formula: DPPH scavenging rate (%) = (A0 −Ai) /A0 × 100
(1)
where A0 is the absorbance of the blank (0.1 mmol/L DPPH reagent) and Ai is the absorbance of the sample. The ABTS scavenging capacity of the NFs was also tested. Briefly, NF films (100 μL) dissolved in acetic acid (2 mg/mL) were mixed with 3 mL of ABTS+ solution (7.4 mmol/L ABTS mixed with 2.6 mmol/L K2S2O8). Afterwards, the mixture was incubated in the dark for 6 min. The absorbance was then examined at 734 nm. The ABTS scavenging rate was calculated by the following formula: ABTS scavenging rate (%) = (A0 −Ai) /A0 × 100
(2)
where A0 is the absorbance of the blank and Ai is the absorbance of the sample.
3.2. Heat treatment promoted the water resistance of the HO-QU-CS NF film
2.6. Enzymatic browning test
Structural stability is a substantial parameter for assessing fibre materials. We found in our previous study that the HO-NF film had poor structural stability in an aqueous environment due to its poor hydrophobic property. Heat treatment was adopted in this study to improve the water resistance of the HO-QU-CS NFs films. The hydrophobic property of the blended NFs was investigated by measuring the water contact angle of the NF film surface. A comparatively large contact angle (θ > 90°) normally suggests a hydrophobic surface, and a relatively small contact angle (θ < 90°) indicates a hydrophilic surface (Yang, Wang, Lu, Yu, & Liu, 2018). Before heat treatment, the HO-QU-CS NF film had a hydrophilic surface, and water droplets quickly spread into the fibre matrices in the first 4 s. After heat treatment at 90℃, the NF film maintained a stable contact angle of 60° (Fig. 2a), and the water contact angle increased with the increase of the temperature. The contact angle increased to 78° at the temperature of 120℃ (Fig. 2b) and to 122° at the temperature of 150 ℃ (Fig. 2c). It was worth noting that the contact angle stopped increasing at 180 ℃ and remained at 122° (Fig. 2d), suggesting that 150℃ may be a proper temperature for heat treatment. The time of heat treatment was also verified at 150 ℃, and the
Surfaces of apples and potatoes at certain sizes were cut off and covered by the HO (1 cm × 2 cm) or heat-treated HO-QU-CS NF films. Then, the apple samples were stored at 26 °C for 6 h, and the potato samples were stored at 4 °C overnight. Afterwards, the NF films were removed, and the surfaces were photographed. To minimize the errors, the tests of different NF films were conducted on the same fruit, and three replicates were tested. 3. Results and discussion 3.1. Electrospinning of the HO-QU-CS NFs HO-QU-CS blended NF films were prepared by electrospinning. The morphology and structure of the films were investigated by SEM. From Fig. 1a, we could observe that the surface of the blended fibres was smooth, and the fibres showed a circular shape (Fig. 1a). FTIR analysis was adopted to investigate the existence of QU and CS in the blended NFs and determine the possible reaction between HO, QU and CS (Fig. 1b). The typical absorption bands of HO-NFs were observed at 3
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Fig. 1. Characteristic analysis of quercetin-chitosan encapsulated hordein complex nanofibres (HO-QU-CS NFs): a, scanning electron micrograph of NFs; b, FTIR spectra of CS, QU, hordein-NFs, and HO-QU-CS NFs; c, TGA thermograms of hordein-NFs, HO-QU-CS NFs, CS, and QU; d, DTG curves of hordein-NFs, HO-QU-CS NFs, CS, and QU.
treatment didn’t alter the morphological integrity of the blended NFs (Fig. 3a and b). The average diameter of the fibre was calculated and examined, and the results suggested that heat treatment reduced the fibre diameter from 393 ± 92.5 nm to 379 ± 76.4 nm (p < 0.05). Diameter distribution analysis indicated that small fibre branches increased slightly after heat treatment.
contact angles of samples with different treatment times were observed. The contact angle was 74° after treatment for 3 h (Fig. 2e) and increased when the treatment time was prolonged, as follows: 122° for 6 h and 123° for 9 h and 12 h respectively (Fig. 2f–h). For the consideration of energy savings, heat treatment was performed at 150 ℃ for 6 h. We examined the morphologies and diameters of untreated and heat-treated (150 ℃/6 h) HO-QU-CS NFs with SEM and found that heat
Fig. 2. Water contact angle analysis of HO-QU-CS NF films after heat treatment: a–d, contact angles of different treatment temperatures of 90℃, 120℃, 150℃, and 180℃, respectively; e–h, contact angles of different treatment times of 3 h, 6 h, 9 h, and 12 h, respectively. 4
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Fig. 3. The effect of heat treatment on the morphology of HO-QU-CS NFs: a, scanning electron micrographs and fibre diameter distributions of untreated fibres; b, scanning electron micrographs and fibre diameter distributions of heat-treated fibres.
conducted to control enzymatic browning, such as heating and cooling or coating the food with a functional film (Singh et al., 2018). Among them, the application of antioxidants into packaging materials could be an effective way. QU, as a flavonoid compound, has an ortho-diphenol structure. Thus, QU may function as a phenolase substrate analogue that could competitive react with phenolases, thereby reducing the rate of phenolic substrate catalysed by phenolase. In this study, the anti-enzymatic browning ability of the heattreated HO-QU-CS NF film was determined and compared with that of the HO-NF film. Our results showed that covering with a heat-treated HO-QU-CS NF film could retain the fresh colour of an apple incision after 6 h, while the apple incision was brown stained when covered with the HO-NF or untreated HO-QU-CS NF films (Fig. 4c). Similar results were also observed in the potato samples, in which large areas of browning were not observed in the potato incision covered by the heattreated NF film after 12 h, contrary to the HO-NF or untreated HO-QUCS film covered surfaces (Fig. 4d). The browning of incisions covered by untreated HO-QU-CS film might due to the poor water resistance of untreated film, as we found in our experiment that the film shrinked when covered to the incisions, so that, the anti-enzymatic browning ability of QU could not work well.
3.3. Heat treatment didn’t influence the antioxidant activity of HO-QU-CS NF films Oxidation occurs when food is produced and stored, which results in a series of particularly negative changes in the sensory properties of products (such as a rancid appearance as well as colour and texture changes), resulting in a decline in quality and economic losses. By adding antioxidants to food packaging materials, the oxidation of fat components and pigments is controlled and helps to maintain food quality (Portes, Gardrat, Castellan, & Coma, 2009). QU possesses free radical scavenging properties due to its ability to provide a proton. In this research, the free radical scavenging ability of the blended NFs was evaluated, and the influence of heat treatment was determined. From Fig. 4, we could observe that the DPPH and ABTS scavenging abilities of QU were approximately 47% and 88% respectively, and the scavenging ability of untreated blended NFs was slightly lower than pure QU, 39% for DPPH and 73% for ABTS. Moreover, the scavenging ability of heat-treated NFs didn’t alter much and was approximately 37% for DPPH and 72% for ABTS (Fig. 4a and b). These results suggested that heat treatment didn’t alter the antioxidant property of blended NFs. Enzymatic browning has a considerable effect on food quality because it can lead to undesirable brown colours, off-flavours, and undesirable changes in nutritional value (Hemachandran et al., 2017). Enzymatic browning often occurs in fruits and vegetables and is caused by postharvest processing, such as cutting, peeling, juicing or exposure to any abnormal conditions (Rasouli & Saba, 2018). When fruit tissue cells are broken, oxygen invades and highly active phenolases oxidize ortho-phenolic substrates to synthesize coloured quinone compounds (Moon et al., 2018; Supapvanich, Mitrsang, Srinorkham, Boonyaritthongchai, & Wongs-Aree, 2016). Many trials have been
3.4. The effect of heat treatment on the physiochemical properties of HOQU-CS NFs XRD is widely applied to analyse the structure and composition of electrospun NFs. Fig. 5 illustrates the XRD patterns of CS, QU, HO NFs, HOeQUeCS NFs and heat-treated NFs. Pure HOeNF appeared amorphous and had no significant characteristic peaks. Several remarkable peaks were identified for QU at diffraction angle 2θ values of 6.1°, 10.7°, 12.4°, 15.8°, 16.1°, 17.8°, 23.8°, 24.7°, 27.3°, and 28.1°, which 5
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Fig. 4. The influence of heat treatment on free the radical scavenging and anti-enzymatic browning ability of HO-CS-QU NFs: a, DPPH scavenging ability of pure QU, HO-NFs, untreated blended NFs and heat-treated blended NFs; b, ABTS scavenging ability of pure QU, HO-NFs, untreated blended NFs and heat-treated blended NFs; c, apple incisions covered with the HO-NF film, untreated blend NF film or heat-treated blended NF film for 6 h, respectively; d, potato incisions covered with the HONF film, untreated blend NF film or heat-treated blended NF film for 12 h, respectively.
Fig. 5. XRD patterns of the hordein-NFs, CS, QU, HO-QU-CS NFs, and heattreated HO-QU-CS NFs.
Fig. 6. FTIR spectrum of HO-QU-CS NFs and heat-treated HO-QU-CS NFs.
were consistent with a previous report that the drug presented as a crystalline material (Chavoshpour-Natanzi & Sahihi, 2019). After electrospinning, QU characteristic peaks decreased in intensity, and the reduction became more obvious after heat treatment. According to a previous report, CS had a significant peak at 2θ = 20.4° (Stie et al., 2019). Similar with QU, the intensity of CS diffraction was also reduced after electrospinning, and it almost disappeared in the blended fibres after heat treatment (Fig. 5). These results suggested that heat treatment could decrease the crystallinity of QU and CS. Based on previous studies, we suspected that the alteration of the crystalline forms of QU and CS molecules maybe due to their thorough dispersion in the polymer matrix and the formation of new bonds with HO (Wu et al., 2008). FTIR was employed to examine the influence of heat treatment on the chemical bonds of the blended NFs. From Fig. 6, we could observe that heat-treated and untreated HOeQUeCS NFs demonstrated a
similar FTIR spectrum, but the position of some peaks shifted. The characteristic peak of the blended fibres at 3100-3650 cm−1 indicated the OeH stretching between HO and water. However, a narrowed peak was observed for the OeH stretching in the spectrum of the heat-treated blended fibres, indicating that less hydroxyl groups existed on the fibre surface. Thus, the water resistance of the blended NF film was decreased, which was consistent with the contact angle analysis. In addition, a redshift was also observed for the amide mode band. The amide I mode at 1659 cm−1 shifted to 1655 cm−1. Similarly, the amide II band at 1540 cm−1 shifted to 1529 cm−1, whereas the amide III band at 1448 cm−1 shifted to 1444 cm-1 (Fig. 6). This shift indicated that the molecular structure was more stable after heat treatment. The surface chemical characteristics of the electrospun NFs were also analysed by using XPS. The XPS survey scans of the blended NF film with and without heat treatment are shown in Fig. 7. Nitrogen and phosphorus atoms were found on the surface of the blended NFs as expected, indicating the presence of HO on the NF surface. The changes 6
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Fig. 7. X-ray photoelectron spectra of the HO-QU-CS NFs: a, untreated HO-QU-CS NF film; b, heat-treated HO-QU-CS NF film.
References
in the surface composition of the NFs after heat treatment were also evaluated by XPS. The atomic oxygen content of the heat-treated NFs was 20.65%, which was slightly lower (∼1.3%) than that of the untreated one (21.99%), while the contents of carbon and nitrogen atoms were slightly increased after heat treatment (Fig. 7). The decrease in the atomic oxygen content may be related to the decrease of the hydroxyl groups absorbed on the surface of heat-treated NFs, which was consistent with the structural changes observed in the FTIR spectrum. The above results indicated that heat treatment was an attractive modification process to improve the structural ability of a HOeQUeCS NF film by altering the crystallinity, the internal chemical bonds and its surface chemical properties.
Aceituno-Medina, M., Mendoza, S., Rodríguez, B. A., Lagaron, J. M., & López-Rubio, A. (2015). Improved antioxidant capacity of quercetin and ferulic acid during in-vitro digestion through encapsulation within food-grade electrospun fibers. Journal of Functional Foods, 12, 332–341. Ahsan, S. M., Thomas, M., Reddy, K. K., Sooraparaju, S. G., Asthana, A., & Bhatnagar, I. (2018). Chitosan as biomaterial in drug delivery and tissue engineering. International Journal of Biological Macromolecules, 110, 97–109. Alehosseini, A., Gómez-Mascaraque, L. G., Martínez-Sanz, M., & López-Rubio, A. (2019). Electrospun curcumin-loaded protein nanofiber mats as active/bioactive coatings for food packaging applications. Food Hydrocolloids, 87, 758–771. Badawy, M. E. I., Taktak, N. E. M., Awad, O. M., Elfiki, S. A., & Elela, N. E. A. (2017). Preparation and characterization of biopolymers chitosan/alginate/gelatin gel spheres crosslinked by glutaraldehyde. Journal of Macromolecular Science Part BPhysics, 56(6), 359–372. Bhardwaj, N., & Kundu, S. C. (2010). Electrospinning: A fascinating fiber fabrication technique. Biotechnology Advances, 28(3), 325–347. Bonwell, E. S., & Wetzel, D. L. (2009). Innovative FT-IR imaging of protein film secondary structure before and after heat treatment. Journal of Agricultural and Food Chemistry, 57(21), 10067–10072. Chavoshpour-Natanzi, Z., & Sahihi, M. (2019). Encapsulation of quercetin-loaded β-lactoglobulin for drug delivery using modified anti-solvent method. Food Hydrocolloids. Hansen, N. M. L., & David, P. (2008). Sustainable films and coatings from hemicelluloses: A review. Biomacromolecules, 9(6), 1493–1505. Hemachandran, H., Anantharaman, A., Mohan, S., Mohan, G., Kumar, D. T., Dey, D., ... Doss, C. G. P. (2017). Unraveling the inhibition mechanism of cyanidin-3-sophoroside on polyphenol oxidase and its effect on enzymatic browning of apples. Food Chemistry, 227, 102–110. Houde, M., Khodaei, N., Benkerroum, N., & Karboune, S. (2018). Barley protein concentrates: Extraction, structural and functional properties. Food Chemistry, 254, 367–376. Miller, K. S., Chiang, M. T., & Krochta, J. M. (2010). Heat curing of whey protein films. Journal of Food Science, 62(6), 1189–1193. Moon, K. M., Lee, B., Cho, W.-K., Lee, B.-S., Kim, C. Y., & Ma, J. Y. (2018). Swertiajaponin as an anti-browning and antioxidant flavonoid. Food Chemistry, 252, 207–214. Moreira, J. B., de Morais, M. G., de Morais, E. G., da Silva Vaz, B., & Costa, J. A. V. (2018). Electrospun polymeric nanofibers in food packaging. Impact of nanoscience in the food industry. Elsevier387–417. Neo, Y. P., Perera, C. O., Nieuwoudt, M. K., Zujovic, Z., Jin, J., Ray, S., ... GizdavicNikolaidis, M. (2014). Influence of heat curing on structure and physicochemical properties of phenolic acid loaded proteinaceous electrospun fibers. Journal of Agricultural and Food Chemistry, 62(22), 5163–5172. Portes, E., Gardrat, C., Castellan, A., & Coma, V. (2009). Environmentally friendly films based on chitosan and tetrahydrocurcuminoid derivatives exhibiting antibacterial and antioxidative properties. Carbohydrate Polymers, 76(4), 578–584. Rasouli, M., & Saba, M. K. (2018). Pre-harvest zinc spray impact on enzymatic browning and fruit flesh color changes in two apple cultivars. Scientia Horticulturae, 240, 318–325. Reddy, N., Reddy, R., & Jiang, Q. (2015). Crosslinking biopolymers for biomedical applications. Trends in Biotechnology, 33(6), 362–369. Sharma, L., Saini, C. S., Sharma, H. K., & Sandhu, K. S. (2019). Biocomposite edible coatings based on cross linked-sesame protein and mango puree for the shelf life stability of fresh-cut mango fruit. Journal of Food Process Engineering, 42(1), e12938. Singh, B., Suri, K., Shevkani, K., Kaur, A., Kaur, A., & Singh, N. (2018). Enzymatic browning of fruit and vegetables: A review. Enzymes in food technology. Springer63–78. Sirviö, J. A., Kolehmainen, A., Liimatainen, H., Niinimäki, J., & Hormi, O. E. O. (2013). Biocomposite cellulose-alginate films: Promising packaging materials. Food Chemistry, 151(4), 343–351.
4. Conclusion In this study, QU- and CS-loaded HO-NFs were successfully prepared through electrospinning. Heat treatment resulted in the improvement of the water resistance of the NF film without damaging its antioxidant activity. In addition, the heat-treated NF film also delayed the rate of enzymatic browning of the incised apple and potato surfaces. Physiochemical analysis indicated that heat treatment could result in the crosslinking of the electrospun blended NFs and alter their physiochemical properties. Thus, heat treatment could be an effective method to improve the mechanical characteristics of protein-based NFs. Our study also suggested that the heat-treated HO-QU-CS NF film could be a good candidate functional material for food packaging. Conflicts of interest There are no conflicts to declare. CRediT authorship contribution statement Sen Li: Methodology, Investigation, Funding acquisition, Writing original draft. Yan Yan: Investigation, Formal analysis. Xiao Guan: Conceptualization, Supervision, Funding acquisition. Kai Huang: Validation, Writing - review & editing. Acknowledgements This work was supported by Talent Youth Scientific Program of National Grain Industry (LQ2018204), Domestic Science and Technology Cooperation Projects of Shanghai (19395800200) and Medical-Engineering Cross Fund of USST (10-19-308-502). 7
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652–665. Wang, Y., & Chen, L. (2012). Electrospinning of prolamin proteins in acetic acid: The effects of protein conformation and aggregation in solution. Macromolecular Materials and Engineering, 297(9), 902–913. Wang, C., Tian, Z., Chen, L., Temelli, F., Liu, H., & Wang, Y. (2010). Functionality of barley proteins extracted and fractionated by alkaline and alcohol methods. Cereal Chemistry, 87(6), 597–606. Wu, M., Cao, Y., Lei, S., Liu, Y., Wang, J., Hu, J., ... Yu, H. (2019). Protein structure and sulfhydryl group changes affected by protein gel properties: Process of thermal-induced gel formation of myofibrillar protein. International Journal of Food Properties, 22(1), 1834–1847. Wu, T.-H., Yen, F.-L., Lin, L.-T., Tsai, T.-R., Lin, C.-C., & Cham, T.-M. (2008). Preparation, physicochemical characterization, and antioxidant effects of quercetin nanoparticles. International Journal of Pharmaceutics, 346(1–2), 160–168. Yan, J., Wu, Y. H., Yu, D. G., Williams, G. R., & Sun, S. Y. (2014). Electrospun acid-base pair solid dispersions of quercetin. RSC Advances, 4(102), 58265–58271. Yang, P., Wang, Y., Lu, L., Yu, X., & Liu, L. (2018). Surface hydrophobic modification of polyurethanes by diaryl carbene chemistry: Synthesis and characterization. Applied Surface Science, 435, 346–351. Zhuang, X., Cheng, B., Kang, W., & Xu, X. (2010). Electrospun chitosan/gelatin nanofibers containing silver nanoparticles. Carbohydrate Polymers, 82(2), 524–527.
Spotti, M. J., Tarhan, Ö., Schaffter, S., Corvalan, C., & Campanella, O. H. (2017). Whey protein gelation induced by enzymatic hydrolysis and heat treatment: Comparison of creep and recovery behavior. Food Hydrocolloids, 63, 696–704. Stie, M. B., Jones, M., Sørensen, H. O., Jacobsen, J., Chronakis, I. S., & Nielsen, H. M. (2019). Acids ‘generally recognized as safe’affect morphology and biocompatibility of electrospun chitosan/polyethylene oxide nanofibers. Carbohydrate Polymers, 215, 253–262. Sullivan, S. T., Tang, C., Kennedy, A., Talwar, S., & Khan, S. A. (2014). Electrospinning and heat treatment of whey protein nanofibers. Food Hydrocolloids, 35, 36–50. Sun, C., Dai, L., He, X., Liu, F., Yuan, F., & Gao, Y. (2016). Effect of heat treatment on physical, structural, thermal and morphological characteristics of zein in ethanolwater solution. Food Hydrocolloids, 58, 11–19. Supapvanich, S., Mitrsang, P., Srinorkham, P., Boonyaritthongchai, P., & Wongs-Aree, C. (2016). Effects of fresh Aloe vera gel coating on browning alleviation of fresh cut wax apple (Syzygium samarangenese) fruit cv. Taaptimjaan. Journal of Food Science and Technology, 53(6), 2844–2850. Tan, Q., Liu, W., Guo, C., & Zhai, G. (2011). Preparation and evaluation of quercetinloaded lecithin-chitosan nanoparticles for topical delivery. International Journal of Nanomedicine, 2011, 1621–1630 default. Vashisth, P., Nikhil, K., Pemmaraju, S. C., Pruthi, P. A., Mallick, V., Singh, H., ... Pruthi, V. (2013). Antibiofilm activity of quercetinen capsulated cytocompatible nanofibers against Candida albicans. Journal of Bioactive and Compatible Polymers, 28(6),
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Food Packaging and Shelf Life journal homepage: www.elsevier.com/locate/fpsl
Preparation of a hordein-quercetin-chitosan antioxidant electrospun nanofibre film for food packaging and improvement of the film hydrophobic properties by heat treatment
T
Sen Li, Yan Yan, Xiao Guan*, Kai Huang School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hordein Quercetin Chitosan Electrospinning Heat treatment Antioxidant
Bio-based functional packaging materials are attracting increasing attention, but poor mechanical properties often hinder their application. This study aims to develop a novel degradable antioxidant nanomaterial with hordein, quercetin and chitosan via electrospinning. Heat treatment was applied to increase the water resistance of the blended nanofibre film. Our results showed that heat treatment significantly improved the water resistance of the blended nanofibre film without affecting its antioxidant activity. Further physiochemical analysis indicated that heat treatment might improve the water resistance of the nanomaterial by decreasing the crystallinity, reducing surface absorbed hydroxyl groups and increasing the structural stability of the blended nanofibres. Together, our results suggested that heat treatment could be an effective method to improve the water resistance of protein-based nanofibres, and the heat-treated hordein-quercetin-chitosan electrospun nanofibre film was a novel biodegradable material with excellent antioxidant activity and water resistance for food packaging.
1. Introduction In recent years, bio-based packaging materials have attracted increasing attention, as traditional plastic materials have caused serious threats to the environment (Alehosseini, Gómez-Mascaraque, MartínezSanz, & López-Rubio, 2019; Hansen & David, 2008). Electrospinning is one of the most widely used methods to generate bio-materials because it can generate nanofibres (NFs) with a more uniform diameter, higher surface area-to-volume ratio, richer composition and thinner membrane compared with other methods (Bhardwaj & Kundu, 2010; Moreira, de Morais, de Morais, da Silva Vaz, & Costa, 2018). Electrospinning is also an effective technique to develop new functional food packaging materials, as bioactive or functional substances could be easily incorporated into the materials by electrospinning. Natural polymers, such as proteins and polysaccharides, are ideal raw materials for bio-based environment-friendly materials due to their biodegradability, biocompatibility, low toxicity and renewability (Sharma, Saini, Sharma, & Sandhu, 2019; Sirviö, Kolehmainen, Liimatainen, Niinimäki, & Hormi, 2013). Hordein (HO) is the most important storage protein in barley, and it can be obtained from the byproducts of the brewing industry (Houde, Khodaei, Benkerroum, &
Karboune, 2018). The reutilization of HO is poor because of its nutritional defects (Wang & Chen, 2012). However, HO has many advantages such as biodegradability, non-toxicity, edible properties and excellent spinnability. Chitosan (CS) is another bio-based material that is frequently used for electrospinning. CS showed distinct properties, such as good compatibility, biodegradability, and low toxicity (Ahsan et al., 2018). In particular, CS has been widely used in electrospinning as a stabilizer and reducing agent, and it shows distinctive characteristics such as antioxidant and antimicrobial activity (Zhuang, Cheng, Kang, & Xu, 2010). Poor mechanical characteristics especially water resistance is an evident shortcoming that restricts the application of bio-based NF films. In our previous study, we found the water resistance of pure HO-NFs was poor as well as other protein-based NFs, and CS addition could improve the water resistance ability of HO electrospun NFs to some extent by alleviating the shrinkage of the film (unpublished data). However, the water resistance of the film is still poor, and more effort needs to be made to strengthen the water resistance of HO-based nanofibre films to widen their application in food packaging. Crosslinking is the most common method to overcome the limitations of a nanofibre film from biomaterials. Several chemical and
⁎ Corresponding author at: School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai, 200093, China. E-mail address: [email protected] (X. Guan).
https://doi.org/10.1016/j.fpsl.2020.100466 Received 25 August 2019; Received in revised form 19 December 2019; Accepted 3 January 2020 2214-2894/ © 2020 Elsevier Ltd. All rights reserved.
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centrifugation at 8500 ×g for 30 min at 4 °C, the precipitates were obtained and further freeze-dried. QU (minimum purity 98%) was purchased from the Shanghai Winherb Medical Science Co., Ltd. CS (< 200 mPa s−1, MW = 152143, 95% deacetylated), methanol (98%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt (ABTS) were acquired from the Aladdin Reagent Co., Ltd. Acetic acid (100%) and all the rest of the chemical reagents were provided from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were used without further purification, and distilled deionized water was used in all experiments. Apples and potatoes were purchased from the local supermarket.
physical crosslinking methods have been reported to improve the mechanical properties of biomolecule-based NF films. Badawy et al. found that glutaraldehyde treatment could form crosslinking bonds between chitosan and alginate that significantly decrease the degree of swelling of chitosan/alginate/gelatin polymers (Badawy, Taktak, Awad, Elfiki, & Elela, 2017). However, chemical substances, such as glutaraldehyde, epichlorohydrin, and carbodiimide, have shown toxicity or low efficiency (Reddy, Reddy, & Jiang, 2015). In contrast, physical methods are much safer to improve the water resistance of biopolymer materials. Among which, heat treatment could be a good candidate, especially for those NFs that encapsulate light sensitive chemicals. Heat treatment is the process of exposing materials to high temperature cycles, thereby affecting the rearrangement of the molecular structure (Miller, Chiang, & Krochta, 2010). Heat treatment can cause conversion of the protein secondary structure or break the secondary bonds used to maintain the spatial structure of protein (Bonwell & Wetzel, 2009). Internal sulfur-containing amino acids will be exposed when proteins undergo denaturation during heat treatment, and new intermolecular disulfide bonds will form between those amino acids and reactive groups (Wu et al., 2019). Thermal stability and conformation of proteins largely depend on amino acid composition, therefore different proteins exhibit different degrees of modification after heat treatment (Neo et al., 2014). Heat treatment has been applied to modify the functional and structural properties of proteins and protein based materials (Sun et al., 2016). The whey protein gel prepared by heat treatment showed a smaller pore size and larger gel strength (Spotti, Tarhan, Schaffter, Corvalan, & Campanella, 2017). Studies from Sullivan et al exhibited that the heat treatment of whey protein NFs could increase the thermal stability of the fibres, making the fibre mat substantially insoluble in water (Sullivan, Tang, Kennedy, Talwar, & Khan, 2014). Neo et al. found that the morphology of the gallic acid loaded zein fibres became distorted and flattened and the hydrophobicity of the fibre surface was increased (Neo et al., 2014). Quercetin (QU) is a flavonoid found in many foods and is well known for its strong antioxidant properties (Tan, Liu, Guo, & Zhai, 2011). However, poor water solubility hindered its application (Aceituno-Medina, Mendoza, Rodríguez, Lagaron, & López-Rubio, 2015; Vashisth et al., 2013). In this study, we prepared a novel antioxidant nanomaterial with HO, CS, and QU by electrospinning and evaluated the effect of heat treatment on the water resistance of the blended fibre film. We examined the influence of heat treatment on the fibre morphology, water resistance and antioxidant activity by using scanning electron microscopy analysis (SEM), water contact angle analysis, free radical scavenging analysis and an enzymatic browning test. Fourier transform infrared spectrum analysis (FTIR), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were employed to explore the molecular and physiochemical property alterations of heat-treated NFs.
2.2. Electrospinning Pure HO NFs and QU-CS-encapsulated HO complex nanofibres (HOQU-CS NFs) were produced by electrospinning. For the HO NFs, the 15% (w/v) HO powder was prepared in acid/water (9:1, v/v), which was stirred for 12 h until complete dissolution. The HO-QU-CS NFs were prepared as follows: first, the CS solution (0.4%, w/v) was prepared in acetic acid/water (9:1, v/v) and stirred for a minimum of 4 h until complete dissolution. HO powder (11%, w/v) was then dissolved in the CS solution with vigorous stirring overnight. Finally, QU powder (5%, w/w, relative to HO) was evenly dissolved in the obtained CS/HO solution under constant stirring for 8 h at room temperature. The final polymer solution was inhaled into 5 mL syringes powered by a syringe pump (Longerpump, Hebei, China) to achieve a polymer solution feed rate of 1.0 mL h−1. The stainless-steel needle of the syringe (inner diameter =0.9 mm) was connected to the positive terminal of the direct current (dc) power supply (Boher HV, Shanghai, China), and the receiving plate covered with aluminium foil was connected to the negative electrode of the power supply. The dc power supply voltage was maintained at approximately 9 kV, and the distance from the needle tip to the receiving plate was 15 cm. Eventually, the electrospun NF film was dried in a vacuum drying oven at 45 ℃ overnight. 2.3. Heat treatment HO-QU-CS NF films were put into a drying oven for heat treatment. The effects of the treatment temperature and treatment time were examined separately. The temperature was set at 90 ℃, 120 ℃, 150 ℃, and 180 ℃ with treatment for 6 h. The times were set as 3 h, 6 h, 9 h, and 12 h with treatment at 150℃. After heat treatment, water contact angle analysis was performed to analyse the hydrophobic property of NF films. 2.4. Physiochemical characterization and measurement Scanning electron microscopy analysis (SEM): The morphology of the NFs was determined by a field-emission scanning electron microscope (FEI Quanta Q400). Prior to examination, the samples were sprayed with a gold-palladium mixture under vacuum. Fibre diameters were determined using Java Image Processing Software ImageJ 1.29. The average diameter of the fibre was calculated and examined by measuring the sizes of the NFs in the scanning electron micrographs at more than 100 different places. Thermogravimetric analysis and differential thermal analysis (TGADTG): The thermal properties of the electrospun NFs were evaluated by TGA (SDT Q600, TA Instruments, USA) in the temperature range of 50 ℃-550 ℃. The heating rate was regulated at 10 ℃ /min in an argon atmosphere. Thermogravimetric analysis (TGA) and derivative thermogravimetry (DTG) curves based on the data were obtained Fourier transform infrared spectrum analysis (FTIR): The chemical interactions and composition of the electrospun nanofibre films were analysed by FTIR spectroscopy (IRTracer-100 Shimadzu, Japan). This process was carried out in the middle infrared region with a
2. Materials and methods 2.1. Materials HO protein was prepared using the ethanol methodology reported by Wang et al. (Wang et al., 2010). Briefly, barley flour and hexane were stirred for 2 h at room temperature for defatting with a flour-tohexane ratio of 1:6 (w/v). The defatted barley flour was separated from hexane by centrifugation at 5000 ×g at 25 °C for 30 min. Then, it was air-dried overnight, followed by extraction with 1 M NaCl solution (10% w/v suspension) at 55 °C for 1 h. After washing thrice with deionized water, the residue was extracted with 60% ethanol solution (v/v; 10% w/v suspension) at 60 °C for 1 h. The supernatant was obtained by centrifugation at 5000 ×g for 30 min, and ethanol was removed by evaporation using a rotary evaporator. HO was separated by cold precipitation at 4 °C overnight from the supernatants. After 2
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wavenumber range of 4000-400 cm−1 and a spectral resolution of 4 cm−1. X-ray diffraction analysis (XRD): The crystal structure of the NFs was evaluated by XRD (D8 Advance diffractometer, Bruker, Germany) employing the Cu tube as an X-ray source (λ of CuKα = 0.15418 nm). The testing speed was 0.1 s/step with a step size of 0.02°. X-ray photoelectron spectroscopy (XPS): XPS measurements were carried out with an ESCALAB 250Xi spectrometer (Thermo Fisher, USA) equipped with an Al- Kα X-ray source. Water contact angle analysis: The wettability of electrospun NFs was evaluated through the sessile drop approach. An NF film (approximately 1 cm2 area) was cut and placed on a lifting platform attached to the Optima contact angle goniometer (AST Instrument Ltd., USA). A droplet (approximately 0.1 μL) of distilled water was dropped on the surface of the fibre by adjusting a microsyringe at room temperature (approximately 24℃). The tangent line between water droplets and the contact point of the NF film surface was captured and measured in real time by CAM 100 computer simulation software. The contact angle of each sample was measured three times and averaged.
approximately 1644, 1560, and 1460 cm−1, which represented the bands of amide I (C]O), amide II (C–N, NeH), and amide III (NeH), respectively. The typical absorption bands of CS were located at 1645 cm-1 (C]O), 1428 cm−1 (CH2 bending), 1383 cm−1 (CH3 bending) and 1158 cm-1 (CeOeC). QU had a characteristic peak of -C = O groups at 1667 cm-1 and three typical peaks of benzene rings (two main peaks at 1610 cm-1 and 1520 cm-1 and a small peak between them) (Yan, Wu, Yu, Williams, & Sun, 2014). A band at 1318 cm-1 corresponds to the = CeOeC bridge. Compared with HO-NFs, the three typical HO absorption bands shifted in the blended NFs (Fig. 1b). In particular, the amide I band near 1644 cm−1 shifted significantly to 1659 cm−1. This phenomenon indicated that the addition of CS and QU possibly changed the surface molecular structure of the NFs, which resulted in the shift and deformation of the peak position. In the spectrum of blended NFs, the typical bands of QU were also shifted because the characteristic peaks of benzene rings were covered and overlapped by the intense amide bands of HO. The peak at 1667 cm−1 in the quercetin spectrum and 1645 cm-1 in the chitosan spectrum was shifted to 1659 cm−1 in the HOeQUeCS NF spectrum, which may be due to the hydrogen bond formation among the carbonyl groups of QU, carboxyl groups of HO, and hydroxyl groups of CS. The thermal stability of QU, CS, HO-NFs and HO-QU-CS NFs were further investigated by TGA-DTG, and the thermograms are shown in Fig. 1c and d. From the TGA curve, we could observe that distinct extents of weight loss were found at various temperature ranges and in different samples. The decomposition of the HO-QU-CS NFs was divided into three main stages of weight loss between 25 ℃ and 500 ℃. In the initial stage, the initial weight loss was slight (approximately 4%) below 100 ℃ due to water loss. The second decomposition stage was between 100℃ and 340℃, and this stage was related to the evaporation of HO and CS, with nearly 58% weight loss. The final weight loss between 340 ℃ and 500 ℃ resulted in 18% weight loss. The DTG curve of HO-QU-CS NFs had a small exothermic peak at 340℃, which may be due to the main thermal degradation of QU (Fig. 1d). The DTG thermograms showed that the evaporation of HO-QU-CS NFs was shifted to a higher temperature compared with that of pure HO-NFs. This result implied the successful loading of QU and CS into the HO electrospun NFs and the strong interaction among HO, QU, and CS. In addition, it is worth noting that there was still no mass weight loss of the blended NFs at 200℃, which indicated that heat treatment under 150℃ couldn’t lead to degradation of the blended NFs.
2.5. Free radical scavenging analysis The DPPH radical scavenging capacity of the NFs was examined as follows. Generally, 100 μL of the nanofibre films dissolved in acetic acid (2 mg/mL) was added into 3 mL of DPPH solution (0.1 mmol/L, in methanol). The mixed solution was maintained at room temperature for 30 min in the dark and then, the absorption value was recorded at 517 nm. All measurements were performed in triplicate. The DPPH scavenging rate was calculated by the following formula: DPPH scavenging rate (%) = (A0 −Ai) /A0 × 100
(1)
where A0 is the absorbance of the blank (0.1 mmol/L DPPH reagent) and Ai is the absorbance of the sample. The ABTS scavenging capacity of the NFs was also tested. Briefly, NF films (100 μL) dissolved in acetic acid (2 mg/mL) were mixed with 3 mL of ABTS+ solution (7.4 mmol/L ABTS mixed with 2.6 mmol/L K2S2O8). Afterwards, the mixture was incubated in the dark for 6 min. The absorbance was then examined at 734 nm. The ABTS scavenging rate was calculated by the following formula: ABTS scavenging rate (%) = (A0 −Ai) /A0 × 100
(2)
where A0 is the absorbance of the blank and Ai is the absorbance of the sample.
3.2. Heat treatment promoted the water resistance of the HO-QU-CS NF film
2.6. Enzymatic browning test
Structural stability is a substantial parameter for assessing fibre materials. We found in our previous study that the HO-NF film had poor structural stability in an aqueous environment due to its poor hydrophobic property. Heat treatment was adopted in this study to improve the water resistance of the HO-QU-CS NFs films. The hydrophobic property of the blended NFs was investigated by measuring the water contact angle of the NF film surface. A comparatively large contact angle (θ > 90°) normally suggests a hydrophobic surface, and a relatively small contact angle (θ < 90°) indicates a hydrophilic surface (Yang, Wang, Lu, Yu, & Liu, 2018). Before heat treatment, the HO-QU-CS NF film had a hydrophilic surface, and water droplets quickly spread into the fibre matrices in the first 4 s. After heat treatment at 90℃, the NF film maintained a stable contact angle of 60° (Fig. 2a), and the water contact angle increased with the increase of the temperature. The contact angle increased to 78° at the temperature of 120℃ (Fig. 2b) and to 122° at the temperature of 150 ℃ (Fig. 2c). It was worth noting that the contact angle stopped increasing at 180 ℃ and remained at 122° (Fig. 2d), suggesting that 150℃ may be a proper temperature for heat treatment. The time of heat treatment was also verified at 150 ℃, and the
Surfaces of apples and potatoes at certain sizes were cut off and covered by the HO (1 cm × 2 cm) or heat-treated HO-QU-CS NF films. Then, the apple samples were stored at 26 °C for 6 h, and the potato samples were stored at 4 °C overnight. Afterwards, the NF films were removed, and the surfaces were photographed. To minimize the errors, the tests of different NF films were conducted on the same fruit, and three replicates were tested. 3. Results and discussion 3.1. Electrospinning of the HO-QU-CS NFs HO-QU-CS blended NF films were prepared by electrospinning. The morphology and structure of the films were investigated by SEM. From Fig. 1a, we could observe that the surface of the blended fibres was smooth, and the fibres showed a circular shape (Fig. 1a). FTIR analysis was adopted to investigate the existence of QU and CS in the blended NFs and determine the possible reaction between HO, QU and CS (Fig. 1b). The typical absorption bands of HO-NFs were observed at 3
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Fig. 1. Characteristic analysis of quercetin-chitosan encapsulated hordein complex nanofibres (HO-QU-CS NFs): a, scanning electron micrograph of NFs; b, FTIR spectra of CS, QU, hordein-NFs, and HO-QU-CS NFs; c, TGA thermograms of hordein-NFs, HO-QU-CS NFs, CS, and QU; d, DTG curves of hordein-NFs, HO-QU-CS NFs, CS, and QU.
treatment didn’t alter the morphological integrity of the blended NFs (Fig. 3a and b). The average diameter of the fibre was calculated and examined, and the results suggested that heat treatment reduced the fibre diameter from 393 ± 92.5 nm to 379 ± 76.4 nm (p < 0.05). Diameter distribution analysis indicated that small fibre branches increased slightly after heat treatment.
contact angles of samples with different treatment times were observed. The contact angle was 74° after treatment for 3 h (Fig. 2e) and increased when the treatment time was prolonged, as follows: 122° for 6 h and 123° for 9 h and 12 h respectively (Fig. 2f–h). For the consideration of energy savings, heat treatment was performed at 150 ℃ for 6 h. We examined the morphologies and diameters of untreated and heat-treated (150 ℃/6 h) HO-QU-CS NFs with SEM and found that heat
Fig. 2. Water contact angle analysis of HO-QU-CS NF films after heat treatment: a–d, contact angles of different treatment temperatures of 90℃, 120℃, 150℃, and 180℃, respectively; e–h, contact angles of different treatment times of 3 h, 6 h, 9 h, and 12 h, respectively. 4
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Fig. 3. The effect of heat treatment on the morphology of HO-QU-CS NFs: a, scanning electron micrographs and fibre diameter distributions of untreated fibres; b, scanning electron micrographs and fibre diameter distributions of heat-treated fibres.
conducted to control enzymatic browning, such as heating and cooling or coating the food with a functional film (Singh et al., 2018). Among them, the application of antioxidants into packaging materials could be an effective way. QU, as a flavonoid compound, has an ortho-diphenol structure. Thus, QU may function as a phenolase substrate analogue that could competitive react with phenolases, thereby reducing the rate of phenolic substrate catalysed by phenolase. In this study, the anti-enzymatic browning ability of the heattreated HO-QU-CS NF film was determined and compared with that of the HO-NF film. Our results showed that covering with a heat-treated HO-QU-CS NF film could retain the fresh colour of an apple incision after 6 h, while the apple incision was brown stained when covered with the HO-NF or untreated HO-QU-CS NF films (Fig. 4c). Similar results were also observed in the potato samples, in which large areas of browning were not observed in the potato incision covered by the heattreated NF film after 12 h, contrary to the HO-NF or untreated HO-QUCS film covered surfaces (Fig. 4d). The browning of incisions covered by untreated HO-QU-CS film might due to the poor water resistance of untreated film, as we found in our experiment that the film shrinked when covered to the incisions, so that, the anti-enzymatic browning ability of QU could not work well.
3.3. Heat treatment didn’t influence the antioxidant activity of HO-QU-CS NF films Oxidation occurs when food is produced and stored, which results in a series of particularly negative changes in the sensory properties of products (such as a rancid appearance as well as colour and texture changes), resulting in a decline in quality and economic losses. By adding antioxidants to food packaging materials, the oxidation of fat components and pigments is controlled and helps to maintain food quality (Portes, Gardrat, Castellan, & Coma, 2009). QU possesses free radical scavenging properties due to its ability to provide a proton. In this research, the free radical scavenging ability of the blended NFs was evaluated, and the influence of heat treatment was determined. From Fig. 4, we could observe that the DPPH and ABTS scavenging abilities of QU were approximately 47% and 88% respectively, and the scavenging ability of untreated blended NFs was slightly lower than pure QU, 39% for DPPH and 73% for ABTS. Moreover, the scavenging ability of heat-treated NFs didn’t alter much and was approximately 37% for DPPH and 72% for ABTS (Fig. 4a and b). These results suggested that heat treatment didn’t alter the antioxidant property of blended NFs. Enzymatic browning has a considerable effect on food quality because it can lead to undesirable brown colours, off-flavours, and undesirable changes in nutritional value (Hemachandran et al., 2017). Enzymatic browning often occurs in fruits and vegetables and is caused by postharvest processing, such as cutting, peeling, juicing or exposure to any abnormal conditions (Rasouli & Saba, 2018). When fruit tissue cells are broken, oxygen invades and highly active phenolases oxidize ortho-phenolic substrates to synthesize coloured quinone compounds (Moon et al., 2018; Supapvanich, Mitrsang, Srinorkham, Boonyaritthongchai, & Wongs-Aree, 2016). Many trials have been
3.4. The effect of heat treatment on the physiochemical properties of HOQU-CS NFs XRD is widely applied to analyse the structure and composition of electrospun NFs. Fig. 5 illustrates the XRD patterns of CS, QU, HO NFs, HOeQUeCS NFs and heat-treated NFs. Pure HOeNF appeared amorphous and had no significant characteristic peaks. Several remarkable peaks were identified for QU at diffraction angle 2θ values of 6.1°, 10.7°, 12.4°, 15.8°, 16.1°, 17.8°, 23.8°, 24.7°, 27.3°, and 28.1°, which 5
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Fig. 4. The influence of heat treatment on free the radical scavenging and anti-enzymatic browning ability of HO-CS-QU NFs: a, DPPH scavenging ability of pure QU, HO-NFs, untreated blended NFs and heat-treated blended NFs; b, ABTS scavenging ability of pure QU, HO-NFs, untreated blended NFs and heat-treated blended NFs; c, apple incisions covered with the HO-NF film, untreated blend NF film or heat-treated blended NF film for 6 h, respectively; d, potato incisions covered with the HONF film, untreated blend NF film or heat-treated blended NF film for 12 h, respectively.
Fig. 5. XRD patterns of the hordein-NFs, CS, QU, HO-QU-CS NFs, and heattreated HO-QU-CS NFs.
Fig. 6. FTIR spectrum of HO-QU-CS NFs and heat-treated HO-QU-CS NFs.
were consistent with a previous report that the drug presented as a crystalline material (Chavoshpour-Natanzi & Sahihi, 2019). After electrospinning, QU characteristic peaks decreased in intensity, and the reduction became more obvious after heat treatment. According to a previous report, CS had a significant peak at 2θ = 20.4° (Stie et al., 2019). Similar with QU, the intensity of CS diffraction was also reduced after electrospinning, and it almost disappeared in the blended fibres after heat treatment (Fig. 5). These results suggested that heat treatment could decrease the crystallinity of QU and CS. Based on previous studies, we suspected that the alteration of the crystalline forms of QU and CS molecules maybe due to their thorough dispersion in the polymer matrix and the formation of new bonds with HO (Wu et al., 2008). FTIR was employed to examine the influence of heat treatment on the chemical bonds of the blended NFs. From Fig. 6, we could observe that heat-treated and untreated HOeQUeCS NFs demonstrated a
similar FTIR spectrum, but the position of some peaks shifted. The characteristic peak of the blended fibres at 3100-3650 cm−1 indicated the OeH stretching between HO and water. However, a narrowed peak was observed for the OeH stretching in the spectrum of the heat-treated blended fibres, indicating that less hydroxyl groups existed on the fibre surface. Thus, the water resistance of the blended NF film was decreased, which was consistent with the contact angle analysis. In addition, a redshift was also observed for the amide mode band. The amide I mode at 1659 cm−1 shifted to 1655 cm−1. Similarly, the amide II band at 1540 cm−1 shifted to 1529 cm−1, whereas the amide III band at 1448 cm−1 shifted to 1444 cm-1 (Fig. 6). This shift indicated that the molecular structure was more stable after heat treatment. The surface chemical characteristics of the electrospun NFs were also analysed by using XPS. The XPS survey scans of the blended NF film with and without heat treatment are shown in Fig. 7. Nitrogen and phosphorus atoms were found on the surface of the blended NFs as expected, indicating the presence of HO on the NF surface. The changes 6
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Fig. 7. X-ray photoelectron spectra of the HO-QU-CS NFs: a, untreated HO-QU-CS NF film; b, heat-treated HO-QU-CS NF film.
References
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4. Conclusion In this study, QU- and CS-loaded HO-NFs were successfully prepared through electrospinning. Heat treatment resulted in the improvement of the water resistance of the NF film without damaging its antioxidant activity. In addition, the heat-treated NF film also delayed the rate of enzymatic browning of the incised apple and potato surfaces. Physiochemical analysis indicated that heat treatment could result in the crosslinking of the electrospun blended NFs and alter their physiochemical properties. Thus, heat treatment could be an effective method to improve the mechanical characteristics of protein-based NFs. Our study also suggested that the heat-treated HO-QU-CS NF film could be a good candidate functional material for food packaging. Conflicts of interest There are no conflicts to declare. CRediT authorship contribution statement Sen Li: Methodology, Investigation, Funding acquisition, Writing original draft. Yan Yan: Investigation, Formal analysis. Xiao Guan: Conceptualization, Supervision, Funding acquisition. Kai Huang: Validation, Writing - review & editing. Acknowledgements This work was supported by Talent Youth Scientific Program of National Grain Industry (LQ2018204), Domestic Science and Technology Cooperation Projects of Shanghai (19395800200) and Medical-Engineering Cross Fund of USST (10-19-308-502). 7
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