Cellulose nanocrystals prepared from wheat bran: Characterization and cytotoxicity assessment

International Journal of Biological Macromolecules 140 (2019) 225–233

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Cellulose nanocrystals prepared from wheat bran: Characterization and cytotoxicity assessment Yaqing Xiao, Yingnan Liu, Xiaojing Wang, Mei Li, Hongjie Lei ⁎, Huaide Xu ⁎ College of Food Science and Engineering, Northwest A&F University, Yangling 712100, Shaanxi, China

a r t i c l e

i n f o

Article history: Received 28 April 2019 Received in revised form 18 August 2019 Accepted 18 August 2019 Available online 19 August 2019 Keywords: Cellulose nanocrystals Wheat bran Cytotoxicity

a b s t r a c t Wheat bran is an abundant source of cellulose and is still going to waste because of the lack of knowledge about its further exploitation and comprehensive utilisation. Here, cellulose nanocrystals (CNC) were prepared from wheat bran via sulfuric acid hydrolysis. The effects of hydrolysis time on the morphology, surface charge, yield, structure, thermal stability, physicochemical properties, and cytotoxicity of CNC were investigated. Results showed that non-cellulosic components were extensively removed by the purification process. Transmission electron microscopy confirmed that the obtained CNC displayed a needle-like shape with various dimensions. Zeta potential values of the CNC suspensions ranged from −36.5 to −39.8 mV. A hydrolysis time of 60 min resulted in CNC with the highest crystallinity (70.32%). The thermal stability of CNC shifted to lower temperature with increasing hydrolysis time. In addition, the obtained CNC exhibited interesting physicochemical properties (the water/oil retention capacities and the adsorption capacities to heavy metals) and good biocompatibility, suggesting their great potential as reinforcement for the manufacture of nanocomposites. © 2019 Elsevier B.V. All rights reserved.

1. Introduction In recent decades, the growing environmental concerns have motivated the remarkable development of environmentally friendly materials. Cellulose is the most abundant natural polymer on earth and is composed of β-1,4-linked anhydro-D-glucose units [1]. It has received extensive attention as a promising alternative material for synthetic products due to its biodegradability, biocompatibility, renewability, and nontoxicity [2]. Cellulose nanocrystals (CNC), also known as cellulose nanowhiskers or nanocrystalline cellulose, are needle-like or rodlike cellulose particles with several hundred nanometers in length and 10–30 nm in diameter [2]. The most common process for the preparation of CNC is based on acid hydrolysis, in which the amorphous regions of cellulose are preferentially hydrolyzed while the crystalline regions remain intact because of their inherent structural stability [3]. CNC exhibit very interesting properties such as large specific surface area, high crystallinity and surface activity, rich hydroxyl groups for modification, low density and cost, excellent mechanical properties and environmental benefits, which allow the application of CNC in various fields [4,5]. Although the wide application of nanotechnology has improved the performance of raw materials, it has increased human exposure to unknown risks. Cellulose is generally recognized as being safe and can be used as packaging materials or even as food ingredients due to the ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Lei), [email protected] (H. Xu).

https://doi.org/10.1016/j.ijbiomac.2019.08.160 0141-8130/© 2019 Elsevier B.V. All rights reserved.

fact that cellulose is derived from natural bio-based sources and cannot be digested by human body [6]. It indicates that the nanocellulose prepared from the natural bio-based materials may be without safety concerns. However, the biological impacts of nanomaterials on human health cannot be assessed merely based on their chemical characteristics, as their size allows them to penetrate into cells and remain in the system [7]. Furthermore, the particle morphology, degree of crystallinity, surface chemistry (zeta potential and aggregation properties), colloidal stability, and other unknown factors may cause nanomaterials to be cytotoxic [8]. Therefore, it is essential to assess the cytotoxicity and potential risks of CNC to human health. However, there are few reports in the literature regarding to the cytotoxicity assessment of CNC. Wheat bran, which is the main by-product of the wheat milling industry, represents approximately 25% of the grain weight and contains a considerable amount of cellulose (32.1%) [9]. Wheat bran is a sustainable source with an annual production of 187 and 32 million tons in the world and China, respectively, but it is mainly used as low value-added animal feed or countryside fuel [10]. Thus, an efficient reuse of this residue is of great importance not only for increasing economic returns but also for alleviating environmental concerns. Various agricultural residues, as potential sources of cellulose, have been used to prepare CNC, such as groundnut shells [4], soy hulls [5], sugarcane bagasse [11], wheat straw [12], etc. The exploitation of CNC from different kinds of cellulosic resources is necessary and relevant since the properties and applications of CNC vary with the source of the original cellulose [13]. Recently, researchers have tried to isolate and characterize nanocellulose from wheat bran [14–16]. However, this work is still in

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its infancy; Moreover, the cytotoxicity of wheat bran CNC is unclear, which limits its application. Therefore, in this study, CNC were prepared from wheat bran via sulfuric acid hydrolysis (Fig. 1). The effects of hydrolysis time on the morphology, surface charge, yield, structure, thermal stability, and physicochemical properties (the water/oil retention capacities and the adsorption capacities to heavy metals) of CNC were investigated. Furthermore, the cytotoxicity of CNC was evaluated on the Caco-2 cells. This study aims to investigate the viability of this underutilized agrowaste as a simple and low-cost source of CNC, as well as the safety and biocompatibility of the obtained CNC as a reinforcing agent in the nanocomposite field. 2. Materials and methods 2.1. Materials Wheat bran was provided by Shaanxi Shanfu Flour Co., Ltd. (China). Caco-2 cells were obtained from the American Type Culture Collection (ATCC) and were provided by the Chinese Academy of Sciences (CAS) Kunming Cell Bank (Yunnan, China). Cell culture reagents were purchased from Thermo Fisher Scientific (USA). All other chemicals used in this work were of analytical grade. 2.2. Preparation of CNC 2.2.1. Extraction of cellulose Raw wheat bran was rinsed with tap water to remove dust and impurities, dried in an air-circulating oven at 60 °C for 24 h, and ground with a grinder to pass through a 40-mesh sieve. The ground wheat bran was washed with ethanol at room temperature (25 °C) for 12 h to remove lipid fractions and dried naturally. Then, the resulting material was hydrolyzed by 0.6% (w/v) α-amylase (4000 U/g) at a ratio of 1:20 under mechanical stirring at 70 °C for 60 min to remove the starch [17], followed by drying at 60 °C for 24 h. The residue was treated with 5% (w/v) NaOH solution (ratio of 1:20) under mechanical stirring at 70 °C for 4 h. After washing with excess deionized water, the insoluble residue was delignified with 1.5% (w/v) NaClO2 solution at a pH of 3–4 (adjusted by acetic acid). The bleaching process was repeated four times at 70 °C for 2 h under constant agitation. Finally, the obtained white

Pretreatment

product was thoroughly washed with deionized water to reach a pH value approximately 7.0, and freeze-dried to obtain wheat bran cellulose. 2.2.2. Isolation of CNC CNC were isolated from the wheat bran cellulose by classical acid hydrolysis method as described by Bano and Negi [4] with some modifications. Wheat bran cellulose was dispersed in a 64% (w/w) H2SO4 solution (ratio of 1:20) under constant stirring at 45 °C for 30 min, 60 min, or 90 min (CNC30, CNC60, or CNC90). Subsequently, the suspension was diluted 10-fold with cold deionized water to quench the hydrolysis reaction and centrifuged (12,000 rpm, 10 min, and 10 °C) to remove the excess acid. The resultant precipitate was washed several times by centrifugation and then continuously dialyzed (membrane molecular cut of 8000–14,000 Da) against deionized water to fully remove free acid molecules until a neutral pH was reached. Finally, the obtained CNC suspension was sonicated for 20 min in an ice bath and freeze-dried for further characterization. 2.3. Characterization 2.3.1. Scanning electron microscopy (SEM) The surface morphology of the wheat bran after different treatments was observed by a scanning electron microscope (S-4800, Hitachi, Japan) at an accelerating voltage of 10 kV. The sample was coated with gold before observation to prevent charging. 2.3.2. Chemical composition The chemical composition (cellulose, hemicellulose, and lignin contents) of wheat bran at different stages of treatment was determined according to the Van Soest method [18,19]. An average of three replicates was calculated for each sample. 2.3.3. Transmission electron microscopy (TEM) Approximately 5 μL of the diluted CNC suspension was deposited on a carbon-coated copper grid (230 meshes) and allowed to dry at room temperature. The sample was observed by a transmission electron microscope (JEM-1230, JEOL, Japan) operating at 80 kV. The dimensions (length and diameter) of CNC were measured from over 100 random particles using Nano Measurer software.

Alkali treatment & Bleaching

Acid hydrolysis

Fig. 1. Schematic illustration of the preparation of cellulose nanocrystals (CNC) from wheat bran.

Y. Xiao et al. / International Journal of Biological Macromolecules 140 (2019) 225–233

2.3.4. Zeta potential Zeta potential measurement was performed using a Nano-ZS Zetasizer (ZEN 3600, Malvern Instruments, UK) with water as the dispersant. The calculation was based on the electrophoretic mobility of the suspension, which was converted to the zeta potential. 2.3.5. Yield of CNC A specific amount of CNC suspension was placed into a weighing bottle to be dried in an oven at 105 °C until a constant mass was reached. The yield of CNC was calculated using Eq. (1), as described by Kian et al. [20]. Yield ð%Þ ¼

ðM2 −M3 Þ  V1  100 M1  V2

ð1Þ

where M1 is the mass of dried wheat bran cellulose (g), M2 is the total mass of oven-dried CNC and the weighing bottle (g), M3 is the mass of the weighing bottle (g), V1 is the total volume of the obtained CNC suspension (mL), and V2 is the volume of the CNC suspension to be ovendried (mL). 2.3.6. Fourier transform infrared spectroscopy (FTIR) Changes in the functional groups were detected using a FTIR spectrometer (Vertex 70, Bruker, Germany) with 16 scans and at a resolution of 4 cm−1 over 4000–500 cm−1. 2.3.7. X-ray diffraction (XRD) The crystallinity of samples was evaluated using an X-ray diffractometer (D8 Advance, Bruker, Germany) equipped with Cu Kα radiation (λ = 0.154 nm) at a scan rate of 5°/min from 10° to 60°. The crystallinity index (CrI) was determined using Eq. (2). CrI ð%Þ ¼

I002 −Iam  100 I002

ð2Þ

where I002 represents the maximum intensity of the (002) lattice diffraction peak located at a diffraction angle of approximately 2θ = 22° and Iam represents the lowest intensity of the amorphous part located at a diffraction angle of approximately 2θ = 18° [21]. 2.3.8. Thermogravimetric analysis (TGA) TGA and its derivative thermogravimetric (DTG) analysis were performed by employing a thermogravimetric analyzer (DTG-60A, Shimadzu, Japan). Approximately 5 mg of the dried sample was heated from 50 °C to 800 °C at a heating rate of 10 °C/min under a nitrogen atmosphere with a gas flow of 50 mL/min. 2.3.9. Water retention capacity (WRC) and oil retention capacity (ORC) The WRC and ORC were determined using the method of Rupérez and Saura-Calixto [22] with slight modifications. First, the mass of the clean centrifuge tube (M1) was determined. Then, 0.5 g of dried sample (M2) was dispersed in 30 mL of deionized water and poured into the centrifuge tube. The tube was stirred and left for 1 h at room temperature, followed by centrifugation at 8000 rpm for 20 min. The supernatant was discarded, and the centrifuge tube containing wet sample (M3) was accurately weighed again. The WRC was calculated using Eq. (3). The ORC was measured according to the above protocol but using commercial olive oil instead of deionized water. WRC ðg=gÞ ¼

M3 −ðM1 þ M2 Þ M2

ð3Þ

227

5.5 by HCl and NaOH. The adsorption experiment was conducted at 37 °C by agitating 0.05 g of dried sample with 10 mL of ion solution in a test tube using a thermostatic shaker bath at a speed of 120 rpm for 3 h. Subsequently, the supernatant was collected by centrifugation (4000 rpm, 5 min) and measured for its metal ion concentrations by an atomic absorption spectrophotometer (ZEEnit 700P, Analytik Jena, Germany). The adsorption capacity of the adsorbent was calculated using Eq. (4), as described by Lu et al. [17]. q ðmg=gÞ ¼

ðC0 −Ce ÞV M

ð4Þ

where M is the mass of dried sample (g), V is the volume of ion solution (L), and C0 and Ce are the initial and equilibrium metal ion concentrations (mg/L), respectively. 2.4. Cytotoxicity of CNC 2.4.1. Cell culture Caco-2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in an atmosphere of 5% CO2 and 90% relative humidity at 37 °C. When the cells reached 80–90% confluence, they were subcultured using 0.25% trypsin-EDTA. The cells at passages 25–35 were harvested for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 2.4.2. Cell viability assay CNC solution were prepared at different concentrations (50, 100, 500, 1000, 2000, and 5000 μg/mL) by suspending the CNC in supplemented DMEM. Caco-2 cells (1.0 × 104 cells/well in 96-well plates) were cultured in 200 μL of supplemented DMEM for 24 h. Then, the medium was replaced with 200 μL of CNC solution and the cells were cultured for 24 h. A blank sample (supplemented DMEM without cells) and a positive control (supplemented DMEM with cells) were also tested. Next, the supernatant was removed, 200 μL of MTT solution (0.5 mg/mL in supplemented DMEM) was added to each well, and the cells were incubated for an additional 4 h. After the supernatant was removed, 200 μL of dimethyl sulfoxide (DMSO) was added to each well. The culture plates were shaken on a shaker at 37 °C for 30 min in order to completely solubilize purple formazan crystals. Absorbance values were read at 490 nm and the cell viability was calculated using Eq. (5). Cell viability ð%Þ ¼

AExp −Ablank  100 Apositive −Ablank

ð5Þ

where AExp is the absorbance of CNC sample, Ablank is the absorbance of blank sample, and Apositive is the absorbance of positive control. 2.5. Statistical analysis Data analysis was completed using the General Linear Models procedure of Statistix 8.0 (Analytical Software, St. Paul, USA). The analysis of variance with Tukey's test was employed to determine the significant differences among different treatments (p b .05). The results were presented as the mean ± standard error from at least three independent experiments. 3. Results and discussion 3.1. Morphological analysis

2.3.10. Determination of the adsorption of heavy metals In the experiment, 50 mg/L Cu2+, Cd2+, and Pb2+ ion solutions were prepared by diluting the stock solutions of CuSO4·5H2O, 3CdSO4·8H2O, and Pb(NO3)2, respectively. The pH of each ion solution was adjusted to

Fig. 2 shows the appearance of wheat bran at each stage of treatment. The color of raw wheat bran changed from light brown to dark brown after alkali treatment (Fig. 2A–D), and it became white after

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A

B

C

D

E

F CNC30

CNC60

CNC90

Fig. 2. Photographs of (A) raw, (B) ground, (C) α-amylase-treated, (D) alkali-treated, and (E) bleached wheat bran as well as (F) CNC suspensions.

bleaching (Fig. 2E). The color changes are attributed to the removal of hemicelluloses, lignin, and other non-cellulosic components (e.g., waxes, pectins, and pigments) during the purification process. Visually, the acid hydrolysis conditions used led to stable aqueous suspensions of CNC being obtained (Fig. 2F). The microscopic morphology of samples was observed using SEM (Fig. 3). Raw wheat bran exhibited a smooth surface and compact structure (Fig. 3A), which may be because some non-cellulosic components act as a binder around the cellulose fibrils [4]. SEM image of the ground wheat bran revealed the presence of starch granules (Fig. 3B), resulting from the contact of wheat bran with the endosperm. After α-amylase and alkali treatments, the fiber surface became rough and irregular (Fig. 3C and D). These two treatments are thus crucial to promote the swelling of the fiber structure and to improve the penetration of the bleaching agent [23]. It was worth noting that bleaching step resulted in partial defibrillation and an opening of fiber bundles (Fig. 3E).

Moreover, the obtained cellulose fibrils displayed a network structure, and the fibrils diameter was reduced to a great extent. 3.2. Chemical composition Generally, the contents of cellulose (30–50%), hemicellulose (15–35%), and lignin (10–25%) in lignocellulosic biomass vary among species [24,25]. The chemical composition of wheat bran at different stages of treatment is summarized in Table 1. The original wheat bran consisted of 31.1% cellulose, 34.3% hemicellulose, and 16.3% lignin, proving that wheat bran can be a potential source of cellulose and hence nanocellulose. The obtained results are comparable with the literature data, where wheat bran contains 32.1% cellulose, 29.2% hemicellulose, and 16.4% lignin [9]. Indeed, lignin is known as the hardest component to remove from lignocellulosic biomass [24]. As shown in Table 1, alkali treatment was

Fig. 3. SEM images of (A) raw, (B) ground, (C) α-amylase-treated, (D) alkali-treated, and (E) bleached wheat bran.

Y. Xiao et al. / International Journal of Biological Macromolecules 140 (2019) 225–233 Table 1 Chemical composition of wheat bran at different stages of treatment. Samples

Cellulose (%)

Hemicellulose (%)

Lignin (%)

Raw wheat bran Alkali treated wheat bran Bleached wheat bran

31.1 ± 1.0c 64.6 ± 1.1b 91.3 ± 0.4a

34.3 ± 1.2a 11.1 ± 0.6b 2.4 ± 0.4c

16.3 ± 1.2a 14.4 ± 0.9a 1.6 ± 0.8b

229

.05), which reached 91.3%, suggesting that the applied treatments are suitable for cellulose extraction. The cellulose content (91.3%) obtained in this study was higher than the previous reports of sugarcane bagasse (87.4%) [11] and garlic straw (86%) [23], but lower compared to oat husk (94.1%) [26] and rice husk (96%) [21]. The differences should be attributed to the cellulose source and extraction conditions.

Means with different lowercase letters (a–c) in the same column indicate significant differences (p b .05).

3.3. Particle size

efficient in removing hemicellulose, whereas most of the lignin and residual hemicellulose were removed by continuous bleaching. After purification, the cellulose content significantly increased as expected (p b

TEM images presented individual needle-shaped nanocrystals and some agglomerates (Fig. 4A1–C1), confirming that the preparation of CNC from wheat bran was successful. Crystalline spherical nanocellulose with a diameter around 80–100 nm have previously

Fig. 4. TEM images and corresponding length, diameter, and aspect ratio distribution histograms of (A) CNC30, (B) CNC60, and (C) CNC90.

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been prepared from wheat bran, accompanied by similar aggregation behavior [16]. Such aggregation of CNC is probably ascribed to their very small size and high specific surface area that leads to them stacking with each other via strong hydrogen bonds or van der Waals forces [1,5]. However, all CNC revealed good stability in a colloidal suspension after a static duration of 24 h (Fig. 2F). Fig. 4(A2–C4) shows the length (L), diameter (D), and aspect ratio (L/D) distributions of CNC obtained from TEM images. The statistics of the average dimensions of the CNC are listed in Table 2. It was clear that an increase in the acid hydrolysis time resulted in a significant decrease in the average length and diameter of the CNC (p b .05). The reduction of the dimensions is connected to the destruction of amorphous regions and even partial crystalline regions of cellulose, as seen by XRD analysis (mentioned below). The diameter of the CNC in this study is obviously different with that of cellulose nanofibers (200–1000 nm) prepared from wheat bran using electrospinning method [15], which may indicate the different properties and applications of the two nanocelluloses. In addition, the average aspect ratio gradually increased with an increase in the hydrolysis time, similar to the results reported by Nsor-Atindana et al. [27]. The aspect ratio of nanocellulose affects their performance. Generally, nanocrystals with a high aspect ratio have great potential applications as a reinforcing agent in nanocomposites [23], which suggests that CNC90 and CNC60 can provide a better reinforcing effect than CNC30 at the same filler loading level. 3.4. Zeta potential and yield Zeta potential is a critical parameter to indicate the tendency for stability or aggregation in the dispersion. A nanocellulose suspension is considered to be stable when the zeta potential value is either less than −30 mV or higher than 30 mV or 25 mV, where the nanoparticles have enough charge to repulse each other and thus prevent aggregation [28]. Clearly, all of the CNC suspensions exhibited high and negative zeta potential values ranging from −36.5 to −39.8 mV (Table 2), demonstrating their ability to remain stable in an aqueous suspension. The use of sulfuric acid during hydrolysis grafts negatively charged sulfate groups onto the nanocrystals surface, which induces the formation of a negative electrostatic layer covering the CNC and contributes to stabilizing the colloidal suspension via strong electrostatic repulsion [11,27]. The CNC yield obviously decreased with increasing hydrolysis time from 37.11% for CNC30 to 28.27% for CNC90 (Table 2), probably because a longer hydrolysis time disintegrates more glycosidic bonds that exist in the amorphous regions or even crystalline regions of cellulose. The sulfuric acid concentration is one of the crucial factors affecting the yield of CNC. In general, CNC with high crystallinity (N80%) and a yield ranging from 21 to 38% can be obtained using 64% sulfuric acid [24]. It is necessary to improve the production efficiency of CNC by optimizing acid hydrolysis or implementing new processes since a low yield of CNC limits their commercial availability. 3.5. FTIR and XRD The chemical structures of raw wheat bran, wheat bran cellulose, and CNC samples were analyzed by FTIR (Fig. 5A). The prominent peaks appeared at 3415 cm−1 and 2914 cm−1 are assigned to the

O\\H stretching vibration and symmetric C\\H vibration, respectively [28]. The peak near 1642 cm−1 corresponds to the O\\H bending of the absorbed water [3]. These bands observed in all spectra are strongly associated with the cellulose structure [21], suggesting that the cellulose component of wheat bran was not removed during chemical treatment. The absence of peaks at 1523 cm−1 (C_C aromatic skeletal vibration) and 1254 cm−1 (=C-O-C axial asymmetric strain) were determined in the spectra of the cellulose and all CNC samples, possibly due to the significant elimination of hemicellulose and lignin by the purification process [13]. Besides, some small peaks at 1321 cm−1 (O\\H bending), 1157 cm−1 (C-O-C glycoside bonds asymmetrical stretching), 1104 cm−1 (C-OH stretching), and 1052 cm−1 (C-O-C pyranose ring vibration) corresponding to the cellulose characteristic were more intense after chemical treatment due to the higher exposure of cellulose [20,28]. FTIR spectra support the results of chemical composition (Table 1). It was worth noting that a new peak appeared at 820 cm−1 in the CNC spectra, which is related to the C-O-S bonding vibration from sulfate groups [20]. XRD analysis was conducted to evaluate the crystalline structure of samples (Fig. 5B). Characteristic diffraction peaks were observed at approximately 16.2°, 22.4°, and 34.7°, corresponding to the (110), (002), and (004) crystallographic planes, respectively [3]. These crystallographic planes represent the typical structural type of cellulose I in the cellulosic samples [1]. Compared to the raw material, the diffraction intensity at the main crystalline peak (22.4°) following chemical treatment was clearly increased. The increase in cellulosic crystallinity is expected to improve their potential mechanical properties and reinforcing capability [23]. In addition, CNC60 presented the highest crystallinity index of 70.32%, followed by CNC90 (66.74%) and CNC30 (66.67%). Compared to CNC60, the slight decrease in the crystallinity of CNC90 resulted from the attack of cellulose crystalline regions by the acid during hydrolysis. Previous research has reported that CNC are highly crystalline between 54 and 88% [29]. The crystallinity indexes of CNC obtained from wheat bran are comparable with the values of sweet potato residue (72.53%) [17] and garlic straw (68.80%) [23]. 3.6. Thermal stability TGA and DTG curves of samples are shown in Fig. 5C and D, respectively. Lignocellulosic biomass is characterized by multistep degradative behavior [1,4]. In all cases, the initial weight loss below 150 °C was mainly related to water evaporation, while the second step ranging from 200 to 380 °C corresponded to cellulose degradation including the depolymerization, dehydration, and decomposition of glycosidic units. The oxidation and breakdown of charred residue into low molecular weight gaseous components above 380 °C were responsible for the third degradation step. The main degradation temperature of wheat bran cellulose (352.97 °C) was higher than that of the raw material (294.77 °C). This is attributed to the removal of hemicellulose and lignin which have lower degradation temperatures compared to cellulose [21]. However, all CNC samples displayed a lower degradation temperature than the obtained cellulose, suggesting that sulfuric acid hydrolysis induces a decrease in thermal stability. Furthermore, CNC30 exhibited the highest degradation temperature (302.92 °C), followed by CNC60 (299.83 °C) and CNC90

Table 2 Characterization of the average dimensions, zeta potential, and yield of CNC. Samples

Length, L (nm)

Diameter, D (nm)

Aspect ratio, L/D

Zeta potential (mV)

Yield (%)

CNC30 CNC60 CNC90

644.77 ± 225.20a 568.81 ± 229.66b 486.18 ± 177.36c

33.80 ± 9.83a 21.57 ± 9.71b 16.94 ± 7.30c

20.39 ± 8.40b 30.01 ± 13.88a 32.11 ± 13.19a

−36.5 ± 0.8b −39.8 ± 1.0a −39.6 ± 1.2a

37.11 ± 1.43a 35.12 ± 0.95a 28.27 ± 1.54b

Means with different lowercase letters (a–c) in the same column indicate significant differences (p b .05).

Y. Xiao et al. / International Journal of Biological Macromolecules 140 (2019) 225–233 1104 1052

A

B

231

22.4

16.2

Intensity (a. u.)

e d c b a

4000 100

e d c b a

1642 1523 1254

3000 2000 -1 Wavenumber (cm )

1000

10

C

0.0

80

Weight (%)

34.7

820 2914 3415

60 40 20

c

0

a e b

d

Derivative weight (mg/min)

Transmittance (%)

1157 1321

20

30 40 2θ (degree)

50

60

D

-0.2

a

-0.4

e d c

-0.6 -0.8

b

-1.0 -1.2 -1.4 -1.6 -1.8

100

200

300 400 500 600 Temperature (ºC)

700

800

100

200

300 400 500 600 Temperature (ºC)

700

800

Fig. 5. (A) FTIR spectra, (B) XRD patterns, (C) TGA curves, and (D) DTG curves of (a) raw wheat bran, (b) wheat bran cellulose, (c) CNC30, (d) CNC60, and (e) CNC90.

(294.98 °C). In other words, there was a slight shift of the degradation to lower temperature with increasing hydrolysis time. The lower thermal stability is possibility due to the combined effect of the large specific surface area of CNC, which leads to a larger exposure of the surface area to heat, and the introduction of sulfate groups onto the surface of the CNC, which leads to a reduction in the activation energy of degradation [4,28]. 3.7. Physicochemical properties 3.7.1. WRC and ORC As shown in Table 3, both the WRC and ORC of wheat bran cellulose were significantly increased in comparison with the ground wheat bran (p b .05), whereas wheat bran cellulose revealed much lower WRC and ORC than those of the CNC samples. Insoluble dietary fiber, including cellulose, generally contains some functional groups such as carboxylic acids, phenolics, ketones, aldehydes, and ether linkages, which possess a strong affinity to bind water or oil [30]. The chemical treatment used in this study can improve the exposure of these functional groups or binding sites, in turn leading to the enhancement of the WRC and ORC. On the other hand, a reduction in the particle size can increase the specific surface area and space-enlarging effect of the fiber, thus enhancing the physical entrapment of water and oil, as reported by Lu

et al. [17]. Besides, the binding abilities are also related to the porosity, crystallinity, and surface properties of the fiber [30]. The higher WRC and ORC of CNC indicate their potential applications as low-calorie bulk ingredients in some foodstuffs requiring moisture or oil retention. 3.7.2. Analysis of the adsorption of heavy metals The adsorption of heavy metals by samples is presented in Table 3. Comparing to the ground wheat bran, the adsorption capacities of wheat bran cellulose for Cu2+, Cd2+, and Pb2+ ions were significantly decreased by 0.41, 1.09, and 0.33 mg/g, respectively (p b .05), possibly due to the destruction of the fiber surface structure and porous tissue skeleton by alkali and bleaching processes [17]. Obviously, CNC were more effective than the ground wheat bran and wheat bran cellulose for the adsorption of metal ions (p b .05), and the adsorption capacities gradually increased with an increase in the hydrolysis time. The pattern of adsorption for metal ions is likely ascribable to the active groups and bonds existing on the fiber surface, such as O\\H, -COOH, C_O, and C\\H [17,31]. Acid hydrolysis would expose more surface area and internal active groups, thus making the CNC absorb more metal ions. Solution pH has an important impact on the adsorption process. The presence of excess H+ ions at low pH competes with the metal ions for active sites; and at high pH, low adsorption capacity is mainly due to the formation of soluble hydroxyl complexes [32]. In this study, adsorption

Table 3 Physicochemical properties of ground wheat bran, wheat bran cellulose, and CNC. Samples

Water retention capacity (g/g)

Oil retention capacity (g/g)

Adsorption of heavy metals (mg/g) Cu2+

GWB WBC CNC30 CNC60 CNC90

c

4.32 ± 0.44 8.68 ± 0.48b 9.38 ± 0.31ab 9.79 ± 0.20a 9.68 ± 0.32a

c

3.72 ± 0.12 5.26 ± 0.42b 6.39 ± 0.23a 6.28 ± 0.35a 6.41 ± 0.39a

Cd2+ c

3.32 ± 0.16 2.91 ± 0.12d 4.27 ± 0.12b 4.45 ± 0.19b 5.82 ± 0.13a

Pb2+ d

4.21 ± 0.27 3.12 ± 0.09e 6.50 ± 0.13c 6.80 ± 0.09b 8.22 ± 0.10a

Means with different lowercase letters (a–e) in the same column indicate significant differences (p b .05). GWB: ground wheat bran. WBC: wheat bran cellulose.

6.74 ± 0.11c 6.41 ± 0.04d 7.56 ± 0.06b 7.66 ± 0.09ab 7.71 ± 0.05a

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experiment was performed at an initial pH of 5.5, because a maximum biosorption of heavy metals by modified orange peel was achieved at such pH value [31]. The adsorption capacity order for each adsorbent is Cu2+ b Cd2+ b Pb2+, and a possible explanation can lie in the charge to mass ratios of the three metal ions, but further studies are necessary to elucidate the adsorption mechanism. In any event, the higher adsorption capacity of CNC suggests their potential applications as adsorbent ingredients in some foodstuffs requiring heavy metal adsorption. 3.8. Cytotoxicity assessment To evaluate any potential cytotoxicity of CNC and their possible impact on human health, Caco-2 cells were exposed to different concentrations of CNC for 24 h, and the cell viability was measured using an MTT assay. The effects of CNC30, CNC60, and CNC90 on the viability of Caco-2 cells are displayed in Fig. 6. It was observed that cell viability slightly decreased with increasing in CNC concentration. Compared to the positive control group (100% cell viability), the cell viability was not significantly affected after exposure to all the CNC samples at concentrations of 50, 100, 500, and 1000 μg/mL (p N .05). The cell viability decreased significantly upon contact with CNC90 (88.09%) at 2000 μg/mL (p b .05), although CNC30 (92.81%) and CNC60 (93.11%) did not significantly decrease the cell viability at this concentration (p N .05). However, concentrations of CNC higher than 2000 μg/mL were obviously cytotoxic to Caco-2 cells (p b .05). For all CNC samples, 1000 μg/mL is the maximum concentration that can be used without impairing Caco-2 cells viability. This value is lower than the result reported by Tibolla et al. [6], who found that the cellulose nanofibers isolated from banana peel by enzymatic treatment did not show cytotoxic effects toward Caco-2 cells at a concentration of 2000 μg/mL. However, in another study, Tibolla et al. [33] observed that the cellulose nanofibers prepared using the combination of chemical and mechanical treatments were cytotoxic to Caco-2 cells above 500 μg/mL (1000–5000 μg/mL). The cellulose nanofibers obtained from waste brown algae by TEMPO-mediated oxidation exhibited no cytotoxicity to COS-7 cells at 1000 μg/mL [34]. The differences are possibly dependent on the cellulose source, preparation processes, particle size, aspect ratio, and surface chemistry (zeta potential and aggregation properties) of nanocellulose. The use of such high CNC concentration (1000 μg/mL) as reinforcing agents in packaging materials is not expected [33]. Furthermore, once incorporated into packaging films, CNC are presumably unable to migrate out of the film matrices, therefore posing virtually no danger to the consumers or environment. Previous study has evaluated the effect of the incorporation of wheat bran nanocellulose into the chitosan/glycerol films [14]. Based on their interesting physicochemical properties

Fig. 6. Effects of CNC on Caco-2 cells viability after cultivation for 24 h. Means with different lowercase letters in the same concentration indicate significant differences among samples (p b .05). * Asterisks denote a significant difference relative to the positive control group (p b .05).

(the water/oil retention capacities and the adsorption capacities to heavy metals) and good biocompatibility, the CNC obtained from wheat bran has great application potential as a reinforcing agent for food packaging materials. 4. Conclusions In this study, CNC were successfully prepared from wheat bran via sulfuric acid hydrolysis. Non-cellulosic components were extensively removed by alkali treatment and bleaching. The increase in the hydrolysis time resulted in decreases in the dimensions (length and diameter) and yield, and an increase in the aspect ratio of the CNC. CNC suspensions were considered stable because their zeta potential values were in good ranges. For a hydrolysis time of 60 min, CNC exhibited a needle-like shape with high crystallinity. Moreover, the obtained CNC showed interesting physicochemical properties and good biocompatibility. It can be concluded that the CNC obtained from wheat bran have great potential to be used as reinforcement agents for the manufacture of renewable nanocomposites and also for diversified applications. Acknowledgements The authors are grateful for the financial support from the Key Research and Development Plan of Shaanxi Province [grant number 2019ZDLNY04-03]. References [1] N. Kasiri, M. Fathi, Production of cellulose nanocrystals from pistachio shells and their application for stabilizing Pickering emulsions, Int. J. Biol. Macromol. 106 (2018) 1023–1031. [2] H. Du, W. Liu, M. Zhang, C. Si, X. Zhang, B. Li, Cellulose nanocrystals and cellulose nanofibrils based hydrogels for biomedical applications, Carbohydr. Polym. 209 (2019) 130–144. [3] F. Luzi, D. Puglia, F. Sarasini, J. Tirillò, G. Maffei, A. Zuorro, R. Lavecchia, J.M. Kenny, L. Torre, Valorization and extraction of cellulose nanocrystals from North African grass: Ampelodesmos mauritanicus (Diss), Carbohydr. Polym. 209 (2019) 328–337. [4] S. Bano, Y.S. Negi, Studies on cellulose nanocrystals isolated from groundnut shells, Carbohydr. Polym. 157 (2017) 1041–1049. [5] W.P. Flauzino Neto, H.A. Silvério, N.O. Dantas, D. Pasquini, Extraction and characterization of cellulose nanocrystals from agro-industrial residue–soy hulls, Ind. Crop. Prod. 42 (2013) 480–488. [6] H. Tibolla, F.M. Pelissari, J.T. Martins, E.M. Lanzoni, A.A. Vicente, F.C. Menegalli, R.L. Cunha, Banana starch nanocomposite with cellulose nanofibers isolated from banana peel by enzymatic treatment: In vitro cytotoxicity assessment, Carbohydr. Polym. 207 (2019) 169–179. [7] C. Gómez H, A. Serpa, J. Velásquez-Cock, P. Gañán, C. Castro, L. Vélez, R. Zuluaga, Vegetable nanocellulose in food science: a review, Food Hydrocoll. 57 (2016) 178–186. [8] A.B. Seabra, J.S. Bernardes, W.J. Fávaro, A.J. Paula, N. Durán, Cellulose nanocrystals as carriers in medicine and their toxicities: a review, Carbohydr. Polym. 181 (2018) 514–527. [9] W. Zhang, L. Lai, P. Mei, Y. Li, Y. Li, Y. Liu, Enhanced removal efficiency of acid red 18 from aqueous solution using wheat bran modified by multiple quaternary ammonium salts, Chem. Phys. Lett. 710 (2018) 193–201. [10] Y. Zhang, X. Song, Y. Xu, H. Shen, X. Kong, H. Xu, Utilization of wheat bran for producing activated carbon with high specific surface area via NaOH activation using industrial furnace, J. Clean. Prod. 210 (2019) 366–375. [11] F.B.D. Oliveira, J. Bras, M.T.B. Pimenta, A.A.D.S. Curvelo, M.N. Belgacem, Production of cellulose nanocrystals from sugarcane bagasse fibers and pith, Ind. Crop. Prod. 93 (2016) 48–57. [12] N. Ji, C. Liu, M. Li, Q. Sun, L. Xiong, Interaction of cellulose nanocrystals and amylase: its influence on enzyme activity and resistant starch content, Food Chem. 245 (2018) 481–487. [13] H.A. Silvério, W.P. Flauzino Neto, N.O. Dantas, D. Pasquini, Extraction and characterization of cellulose nanocrystals from corncob for application as reinforcing agent in nanocomposites, Ind. Crop. Prod. 44 (2013) 427–436. [14] M.R. de Andrade, T.B. Rocha Nery, T.I. de Santana, E. Santana, I.L. Leal, L.A. Pereira Rodrigues, J.H. de Oliveira Reis, J.I. Druzian, B.A. Souza Machado, Effect of cellulose nanocrystals from different lignocellulosic residues to chitosan/glycerol films, Polymers 11 (4) (2019) 658. [15] M.F. Yazdanbakhsh, A. Rashidi, M.K. Rahimi, R. Khajavi, H. Shafaroodi, Alphacellulose extraction from wheat bran for preparing cellulose nanofibers, Am. J. Oil Chem. Technol. 5 (1) (2017) 46–52. [16] Claes Nilsson, Preparation and Characterization of Nanocellulose From Wheat Bran, Department of Chemical Engineering, Lund University, Sweeden, 2017 (Master Thesis).

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