Journal Pre-proof

Preparation of cellulose nanofibril/titanium dioxide nanoparticle

nanocomposites as fillers for PVA-based packaging and
investigation into their intestinal toxicity

Zhilong Yu, Wei Wang, Lin Sun, Fanbin Kong, Mengshi Lin,
Azlin Mustapha

PII: S0141-8130(19)36010-6
DOI: https://doi.org/10.1016/j.ijbiomac.2019.11.153
Reference: BIOMAC 13934

To appear in: International Journal of Biological Macromolecules

Received date: 30 July 2019

Revised date: 10 November 2019
Accepted date: 18 November 2019

Please cite this article as: Z. Yu, W. Wang, L. Sun, et al., Preparation of cellulose
nanofibril/titanium dioxide nanoparticle nanocomposites as fillers for PVA-based
packaging and investigation into their intestinal toxicity, International Journal of
Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.11.153

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© 2019 Published by Elsevier.

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Preparation of cellulose nanofibril/titanium dioxide nanoparticle

nanocomposites as fillers for PVA-based packaging and investigation into

their intestinal toxicity

Zhilong Yu1, Wei Wang1, Lin Sun1, Fanbin Kong2, Mengshi Lin1, Azlin Mustapha1*

1
Food Science Program, Division of Food Systems & Bioengineering, University of Missouri,

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Columbia, MO 65211, USA

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2
Department of Food Science & Technology, University of Georgia, Athens, GA, 30602-7610,

USA
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* Corresponding author:

A. Mustapha. Food Science Program, Division of Food Systems & Bioengineering,

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University of Missouri, Columbia, MO 65211, USA. Tel.: (573) 882-2649; E-mail:

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mustaphaa@missouri.edu.
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Abstract

The aims of this work were to synthesize cellulose nanofibril/titanium dioxide nanoparticle

(CNF/TiO2NP) nanocomposites, evaluate the use of CNF/TiO2NP nanocomposites in

PVA-based films, and investigate the intestinal toxicity of the nanocomposites. Via a mixing

method, CNF/TiO2NP nanocomposites were synthesized. The addition of the nanocomposites

significantly enhanced the tensile strength, Young’s modulus, and light barrier capacity of

of
PVA films. Moreover, a high concentration of CNF/TiO2NP nanocomposites (10 mg/mL) had

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no appreciable effect on the growth of Escherichia coli P-24, Lactobacillus acidophilus ADH,
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and Bifidobacterium animalis Bif-6 cells. The nanocomposites did not exhibit significant
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toxicity to cancerous and normal colon cells even when their concentrations increased to a
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high level of 1000 µg/mL. The results indicate that CNF/TiO2NP nanocomposites can

potentially be used as functional fillers for a PVA-based packaging system.

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Keywords: nanocellulose; titanium dioxide nanoparticle; food packaging; intestinal bacteria;

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human colon cell; toxicity

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1. Introduction

Food packaging is used to extend the shelf life of food products by maintaining food

quality during transportation and storage[1]. Food packaging materials are often chosen based

on various factors, such as cost and basic functions. In particular, plastics have been widely

used as food packaging materials because of their low cost, good barrier properties, and low

energy consumption during manufacturing[2]. Plastic packaging materials can be produced by

of
some polymers, such as polyethylene terephthalate, high density polyethylene, and poly(vinyl

ro
alcohol) (PVA). Compared with traditional plastics, PVA-based materials have advantages of
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being biodegradable and nontoxic, thus, have attracted much attention in the food industry[3].
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To develop PVA-based packaging with multiple or strengthened functions, researchers are
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interested in modifying the material by incorporating active fillers, especially nanomaterials.

For example, a PVA-based film incorporated with nanocellulose (NC) and silver
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nanoparticles was reported to show significantly improved mechanical properties and

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antibacterial efficacy against Staphylococcus aureus and Escherichia coli[4]. Zinc oxide NPs
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were used to ameliorate the water vapor barrier, light barrier, and antimicrobial effects of

PVA films[5]. In addition, incorporating both chitin nanofibers and NC into PVA films enabled

the composite to display enhanced stiffness, tensile strength, and thermostability[6].

A composite material with at least one nano-sized filler is called a nanocomposite[7]. In

recent years, the applications of nanocomposites as food packaging materials have been

widely studied. Emerging nanofillers used for food packages include NC and metallic

nanoparticles, such as titanium dioxide nanoparticles (TiO2NPs). NC, defined as the cellulose

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extract or product with nano-scale structures, is subdivided into cellulose nanocrystals (CNCs)

and cellulose nanofibrils (CNFs)[8]. NC can be fillers used for enhancing the mechanical

property, water vapor barrier ability, and oxygen barrier capacity of nanocomposites [9].

Moreover, TiO2NPs are titanium dioxide particles with a diameter of 1-100 nm and possess

remarkable photocatalytic and ultraviolet-blocking abilities. With both NC and TiO2NPs

added, nanocomposites are expected to exhibit the active functions deriving from both the

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two nanomaterials, which can be utilized to prepare active food packaging. However, there

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are still safety concerns about the use of nanomaterials in food packages, because unique
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surface structures and physiochemical properties of nanomaterials may cause unexpected
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toxicological effects[10,11]. Once loaded in a packaging matrix, nano-sized fillers may migrate
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from packages into foods and pose a risk to consumer health[12]. Thus, it is essential to

explore the toxicity of nanocomposites made of NC and TiO2NPs.

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In this study, cellulose nanofibril/titanium dioxide nanoparticle (CNF/TiO2NP)

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nanocomposites were synthesized using a mixing method and then incorporated in

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PVA-based packaging films. The toxicity of the nanocomposites to intestinal bacteria and

human colon cells was investigated. The CNF/TiO2NP nanocomposites were analyzed with

transmission electron microscopy (TEM) and Zetasizer Nano ZS. PVA-based nanocomposite

films were characterized using a colorimeter and a texture analyzer, UV-visible spectroscopy,

and scanning electron microscopy (SEM). In addition, the effects of CNF/TiO2NP

nanocomposites on the growth of intestinal bacteria were evaluated and further investigated

via TEM observations. The toxicity of the nanocomposites to human colon normal and

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cancerous cells was measured by cell proliferation tests.

2. Materials and methods

2.1. Materials

TiO2NP powders (anatase, >99%, 10 nm) were purchased from Nanostructured &

Amorphous Materials Inc. (Houston, TX, USA). CNFs from wood pulp (width, 20-50 nm)

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were purchased from the University of Maine. PVA (M.W. 85,000-124,000), transferrin,

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insulin, cholera toxin, and hydrocortisone were obtained from Sigma-Aldrich (St. Louis, MO,
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USA). Escherichia coli P-24, Lactobacillus acidophilus ADH, and Bifidobacterium animalis
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Bif-6 were provided by the Food Microbiology Laboratory, University of Missouri, Columbia,
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MO, USA. Tryptic soy broth, Lactobacillli MRS broth, tryptic soy agar, and Lactobacilli

MRS agar were purchased from Difco Laboratories (Detroit, MI, USA). High quality fetal
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bovine serum (FBS), DMEM:F12 medium, human colon epithelial normal cells (FHC;
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CRL-1831), and human colorectal adenocarcinoma cells (Caco-2; HTB-37) were purchased
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from ATCC (Rockville, MD, USA).

2.2. Preparation of CNF/TiO2NP nanocomposites

CNF/TiO2NP nanocomposites were synthesized according to previous methods with

some modifications[13,14]. Briefly, a CNF suspension was prepared by dispersing CNFs (0.3 g)

in deionized water (49.7 g) with gentle stirring. Then, 0.03, 0.15, and 0.3 g of TiO2NP

powders (viz., 10%, 50% and 100% based on the weight of the CNFs) were slowly added into

the CNF suspension. After the mixtures were stirred for 30 min and sonicated for 30 min,

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three types of CNF/TiO2NP composite suspensions were obtained: CNFs impregnated with

10% of TiO2NPs (CT10), CNFs impregnated with 50% of TiO2NPs (CT50), and CNFs

impregnated with 100% of TiO2NPs (CT100). Each CNF/TiO2NP nanocomposite film was

obtained by casting the composite suspension (20 g) in a sterile plastic petri dish (10 cm × 1.5

cm), followed by air-drying at room temperature.

2.3. Characterization of CNF/TiO2NP nanocomposites

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2.3.1. TEM analysis

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TEM micrographs were acquired using a JEOL JEM-1400 TEM (JEOL Ltd., Tokyo,
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Japan). Each composite suspension (100 μg/mL) was dropped on a 200-mesh copper/carbon
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grid and then air-dried before characterization.
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2.3.2. Zeta potential measurement

Each composite suspension was diluted with deionized water to a final concentration of
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50 μg/mL. Then, a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) was used to determine
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the Zeta potential of the composite samples. Each measurement was conducted with a
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minimum of 12 runs at 25 ± 0.1°C.

2.4. Application of CNF/TiO2NP nanocomposites in PVA-based films

2.4.1. Film preparation

A PVA solution (3%, w/v) was prepared in an autoclave at 121°C for 30 min and then

stirred at room temperature. After glycerol (1%, w/v) was added to the solution, CNFs (0.3%,

w/v) were incorporated and stirred for 30 min. Then, TiO2NPs (0.03, 0.15, and 0.3%, w/v)

were slowly added in the mixture with mild stirring. The mixture was stirred for 30 min and

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sonicated for another 30 min. The film-making solutions (30 g) were casted in square petri

dishes (120 mm × 120 mm) and air-dried at 37°C for 72 h. Finally, five types of PVA-based

films were obtained: PVA film without CNFs and TiO2NPs (P), PVA film with CNFs but

without TiO2NPs (PC), PVA film with CNFs and 1% of TiO2NPs (PCT1), PVA film with

CNFs and 5% of TiO2NPs (PCT5), and PVA film with CNFs and 10% of TiO2NPs (PCT10).

2.4.2. Color determination

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A Model CR-410 colorimeter (Konica Minolta Sensing, Inc., Japan) was used to

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determine the L*, a*, and b* values of film samples. The total color difference (E) was

calculated according to the following formula:

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E = [(L* – L0*)2 + (a* – a0*)2 + (b* – b0*)2]1/2
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where L*, a*, and b* are color parameters obtained from film samples, and L0*, a0*, and b0*

are 97.76, -0.01, and 1.92, respectively.

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2.4.3. Water vapor permeability (WVP)

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The WVP of film samples was measured according to a reported method[15]. Each glass
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jar filled with 15 g of anhydrous calcium chloride was covered with a film sample, and then

the jar was placed in a desiccator to which was added saturated sodium chloride solution

(75% relative humidity). After the weight of the jars was recorded at 0, 1, 2, 4, 6, 8, 10, and

12 h at 25°C, the WVP of each film was calculated with the following equation:

WVP (g·m-1·s-1·Pa-1) = (m × n)/(a × t ×△p)

where m is the change in jar weight (g), n is film thickness (m), a is the exposed area of the

film (m2), t is time (s), and △p is the partial pressure difference existed between the two

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sides of film sample (Pa).

2.4.4. Mechanical properties

Tensile strength and Young’s modulus of film samples were evaluated using a TA-XT2i

texture analyzer (Texture Technologies Corp., UK). The width and gauge length of each film

were 5 and 30 mm, respectively. The stretching speed of the upper grip was set to 1 mm/s.

2.4.5. Light barrier capacity

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UV-visible spectra of film samples (wavelength, 200-800 nm) were obtained through a

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Cary 50 Bio UV-visible spectrophotometer (Varian, Inc., USA). The absorbance at 600 nm
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was recorded. The O value, which is positively associated with film opacity, was calculated
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according to the following formula:
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O value = A/N

where A is the absorbance of film sample at 600 nm and N is the film thickness (mm)
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determined via an electronic digital caliper (Model, 14-648-17; Control Company, USA).
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2.4.6. SEM analysis

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A Quanta 600 FEG environmental SEM (FEI Company, USA) was used to acquire

SEM micrographs of the film samples. Each sample was affixed to an aluminum standard

stub and analyzed by SEM with the following parameters: low-vacuum mode, 60 Pa; detector,

ETD-SE; working distance, 7 mm; objective aperture, 30 μm; spot size for imaging, 3.0.

2.5. Evaluation of the toxicity of nanocomposites

2.5.1. Toxicity to intestinal bacteria

E. coli was grown in tryptic soy broth, while L. acidophilus and B. animalis were

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prepared in Lactobacillli MRS broth. In brief, 100 mg of film samples in square shape was

added to 10 mL of a bacterial solution (cell concentration, ～107 CFU/mL), and the mixture

was mixed well. After incubation at 37°C with shaking, the bacterial number in the mixture

was measured at 0, 4, 10, and 24 h. To enumerate bacteria, the mixture at each time point was

collected, diluted using peptone water, and plated in agar media. After the intestinal bacteria

were incubated at 37°C for 24 h aerobically (E. coli) or anaerobically (L. acidophilus and B.

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animalis), bacterial enumeration was conducted. E. coli was plated in tryptic soy agar,

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whereas L. acidophilus and B. animalis were plated in Lactobacillli MRS agar.

2.5.2. TEM analysis of intestinal bacteria

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Intestinal bacteria treated with CT100 film (10 mg/mL) for 24 h were fixed in a 0.1 M
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Na-cacodylate buffer (pH 7.35) with 2% formaldehyde and 2% glutaraldehyde. After the cell

pellets were enrobed in HistoGel and fixed with 1% osmium tetroxide, samples were
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dehydrated by a graded series of ethanol, transitioned into acetone, infiltrated with Epon resin,
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and polymerized at 60°C overnight. Then, 75-nm thin sections were mounted on copper grids
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and post-stained. TEM micrographs were obtained with a JEOL JEM-1400 TEM (JEOL Ltd.,

Japan) at 80 kV.

2.5.3. Toxicity to human colon cells

Caco-2 cells were grown in Eagle’s minimum essential medium (supplemented with 1%

of antibiotic antimycotic solution and 20% of high quality FBS) and incubated at 37°C with

5% CO2. When the cells reached 80% confluence, they were harvested with trypsin and

seeded into 96-well plates (volume, 100 µL/well; density, 105 cells/mL). After a 48-h

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incubation, the cells in each well were washed with phosphate-buffered saline and incubated

with fresh medium (150 µL) containing the CNF/TiO2NP nanocomposites (0, 50, 100, 250,

500, and 1000 µg/mL). After a 24-h exposure, the cells in each well were washed with

phosphate-buffered saline. Then, fresh medium (100 µL) and MTT reagent (10 µL) were

added. After incubation at 37°C for 4 h, each well was filled with 100 µL of detergent reagent.

The 96-well plates were left in the dark for 4 h, before absorbance at 570 nm and 655 nm (as

of
reference) were measured. Cell-free wells were used as the blank; the wells with cells

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incubated with fresh medium were used as the control. The cell viability of Caco-2 cells was

calculated with the following formula:

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Cell viability (%) = (As – Ab)/(Ac – Ab) × 100%
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where Ac is the absorbance of the control at 570 nm, As is the absorbance of the treatment

group at 570 nm, and Ab is the absorbance of blank at 655 nm.

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FHC cells were cultured in DMEM:F12 medium supplemented with cholera toxin (10
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ng/mL), hydrocortisone (100 ng/mL), transferrin (0.005 mg/mL), insulin (0.005 mg/mL),
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HEPES (10 mM), high quality FBS (10%, v/v), and antibiotic antimycotic solution (1%, v/v).

The cells were incubated at 37°C with 5% CO2. When the cells reached 80% confluence, they

were harvested with trypsin and seeded into 96-well plates (volume, 100 µL/well; density,

104 cells/mL). After an incubation period of 48 h, the cells in each well were washed with

phosphate-buffered saline and incubated with fresh medium (150 µL) containing the

CNF/TiO2NP nanocomposites (0, 50, 100, 250, 500, and 1000 µg/mL). After an exposure of

24 h, the cells in each well were washed with phosphate-buffered saline, and then fresh

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medium (100 µL) and WST-8 mixture (10 µL) were added. After incubation at 37°C for 4 h,

absorbance was determined at 450 nm. Cell-free wells were used as the blank while the wells

with cells incubated in fresh medium were used as the control. The cell viability was

calculated with the following equation:

Cell viability (%) = (As – Ab)/(Ac – Ab) × 100%

where Ac is the absorbance of the control at 450 nm, As is the absorbance of the treatment

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group at 450 nm, and Ab is the absorbance of the blank at 450 nm.

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2.5.4. TEM observation of colon cells
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Colon cells were seeded in 12-well plates containing gold-coated sapphire disks
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(diameter, 3 mm) and incubated for 48 h. After that, the cells were treated with CT10, CT50,
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and CT100 for 24 h and cryo-immobilized by high-pressure freezing. Super quick freeze

substitution (FS) was performed over a period of 2.5 h; and the FS cocktail contained acetone
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with 0.5% (w/v) of osmium tetroxide, 0.1% (w/v) of imidazole, 0.1% (w/v) of uranyl acetate,
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and 4% (v/v) of ultrapure water. After the infiltration with Epon/Araldite, samples were
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prepared as 75-nm thin sections and mounted on formvar/carbon-coated slot grids. TEM

images were collected with a JEOL JEM-1400 TEM (JEOL Ltd., Japan) at 80 kV.

2.6. Statistical analysis

The data were collected from at least three parallel experiments and shown as means ±

standard deviations. Differences among means were analyzed using one-way analysis of

variance (ANOVA) with Tukey’s test by Minitab 18 (Minitab, Inc., State College, PA, USA).

Means were considered to be significantly different if the probability values were less than

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0.05 (P < 0.05).

3. Results and discussion

3.1. Characterization of CNF/TiO2NP nanocomposites

TEM micrographs of CNFs and CNF/TiO2NP nanocomposites are shown in Fig. 1.

Nano-sized cellulose fibrils were observed in Fig. 1A-D. For CT10, CT50, and CT100 (Fig.

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1B-D), TiO2NPs were attached on the surface of CNFs. Among these three treatments, CT100

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was loaded with the highest number of TiO2NPs. In addition, Table 1 shows the zeta potential
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of TiO2NPs, CNFs, and CNF/TiO2NP nanocomposites. TiO2NPs and CNFs possessed
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positive and negative surface charges, respectively. With more TiO2NPs loaded, the
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nanocomposites exhibited less net negative charges on the surface. The results indicate that

the combination of CNFs and TiO2NPs is largely due to electrostatic interactions, which
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agrees with a previous study. The study showed that the cellulose/TiO2NP nanocomposite
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was formed via electrostatic interactions between the positively charged groups of TiO2NPs
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and the dissociated carboxyl groups of oxidized cellulose[16,17].

3.2. Characterization of PVA films with CNF/TiO2NP nanocomposites

3.2.1. Film color

Fig. 2 shows the images of the PVA film samples, and the color parameters of each film

as displayed in Table 2. Compared with Film P, Film PC showed a significantly higher b*

value. With more TiO2NPs incorporated, PVA films exhibited lower L* and a* values and

larger b* and △E values. These results suggest that the incorporation of CNFs makes the

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PVA film yellower, while the filling of TiO2NPs leads to a darker, greener, and yellower color

of the film. Some studies showed that the color of packaging materials could be modified by

adding NC or TiO2NPs. The alginate-based and starch/chitosan-based films turned yellower

after the incorporation of NC[18]. In addition, with more TiO2NPs filled, starch-based films

displayed higher △E; the color change was highly associated with the crystallite type of

TiO2NPs, film thickness, and particle size of TiO2NPs[19].

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3.2.2. Water vapor barrier property

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Table 2 displays the WVP of the PVA films. The addition of CNFs did not cause an
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appreciable change in the films’ WVP (P > 0.05). Comparably, the presence of TiO2NPs (up
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to 10%) in the PVA films did not significantly alter the WVP of the films (P > 0.05). The
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result suggests that incorporating CNF/TiO2NP nanocomposites cannot significantly affect

the WVP of the PVA films. Previous studies showed that the addition of NC or TiO2NP
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lowered the WVP of PVA films. This was because both NC and TiO2NPs could form
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hydrogen bonds with PVA polymers and hinder the diffusion of water vapor in the film
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matrix[20,21]. However, when the concentrations of NC or TiO2NPs were high enough, the

hydrophilic nature of cellulose and the hygroscopicity of TiO2NPs might increase the WVP of

PVA films[21,22]. Thus, the concentrations of CNF/TiO2NP nanocomposites in PVA films may

be too high to bring a notable decrease in WVP.

3.2.3. Mechanical property

Effects of CNF/TiO2NP nanocomposites on the mechanical properties of PVA films are

shown in Fig. 3A and B. After the incorporation of CNFs, tensile strength and Young’s

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modulus of PVA films were increased by 204% and 167%, respectively. The tensile strength

of PVA films was not significantly changed with the addition of TiO2NPs. Compared with the

PC film, the PCT1 film showed a significantly improved Young’s modulus (P < 0.05).

Further increasing the content of TiO2NPs in PVA films led to a slight decrease in Young’s

modulus for PCT5. This implies that the strength and stiffness of PVA films can be

significantly ameliorated with the addition of CNF/TiO2NP nanocomposites.

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NC can be used as a filler in packaging materials to reduce pores in the film and to

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construct more compact film structures[15]. It was reported that incorporating 8% of CNFs in
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a PVA film matrix made the Young’s modulus of the film increase by 59%. CNFs and PVA
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were compatible with each other because of the formation of hydrogen bonds deriving from
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their hydroxyl groups[23]. Moreover, when sufficient TiO2 particles were added in

biodegradable films, the nanocomposites were strengthened by electrostatic interactions or

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O-Ti-O bonding[24]. This explains why the film PCT1 exhibited a significantly higher
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Young’s modulus than the film PC. However, further increasing the TiO2 content in the films
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could cause unhomogeneous distribution of agglomerated particles, disrupt the film matrix,

and weaken film mechanical properties[25].

3.2.4. Light barrier capacity

Effects of CNF/TiO2NP nanocomposites on the light barrier property of PVA films are

shown in Fig. 3C and D. With the addition of CNFs and CNF/TiO2NP nanocomposites, the

transmittance of PVA films notably decreased at the wavelength of 200-800 nm. For the

PCT5 and PCT10 films that were filled with a high number of TiO2NPs, their film

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transmittance dropped to almost zero. Moreover, incorporation of both CNFs and TiO2NPs

significantly increased the O value. The PCT10 film with the highest content of TiO2NPs

displayed the largest O value. The results indicate that adding CNF/TiO2NP nanocomposites

can significantly enhance the light barrier capacity of PVA films; with more TiO2NPs loaded,

the PVA film can exhibit a stronger light-blocking ability.

CNFs and TiO2NPs were previously used to improve light barrier properties of

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packaging materials. Starch/chitosan films filled with CNFs showed decreased film

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transmittance in the UV and visible regions[26]. The filling effect of CNFs mainly resulted in
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an improved film barrier property. Another study showed that the opacity of a
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biopolymer-based film was strengthened by incorporating TiO2NPs[24]. Increasing the TiO2NP
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concentration in the film caused a lower transmittance at 800 nm and a higher crystallinity,

which was greatly due to the agglomeration of TiO2NPs.

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3.2.5. SEM observation

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SEM micrographs of CNF/TiO2NP nanocomposites are shown in Fig. 4. The filling of

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CNFs gave rise to a rougher surface of PVA films (Fig. 4A-E). With TiO2NPs added in the

film, many agglomerates appeared on the surface. The PCT10 film displayed the highest

number of agglomerates compared with PCT1 and PCT5. As shown in Fig. 4F-J, cross

sections of PVA films became less smooth and thicker after the incorporation of CNFs and

CNF/TiO2NP nanocomposites, but all constituents in the film matrix appeared highly

compatible with one another. These results suggest that CNF/TiO2NP nanocomposites are

well incorporated in the PVA film. The TiO2NPs are functional in their agglomerate form,

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which is likely because TiO2NPs are more hydrophobic compared with other film

constituents and, thus, aggregate to reduce energy dissipated in the film system[27]. The

agglomeration of TiO2NPs may affect the mechanical properties and light barrier capacity of

PVA films. With more TiO2NPs loaded in PVA films, more agglomerates were formed to

block visible and UV light; and 1% of TiO2NP agglomerates strengthened the stiffness of

PVA films without significantly reducing film homogeneity.

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3.3. Toxic effect of CNF/TiO2NP nanocomposites on intestinal bacteria

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Effects of CNF/TiO2NP nanocomposites on the growth of intestinal bacteria are shown
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in Fig. 5. As displayed in Fig. 5A, the number of E. coli P-24 cells incubated with and
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without CNF/TiO2NP nanocomposites increased to ～9 log CFU/mL at 4 h and then was
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maintained at a stable level. No significant changes in cell number were observed with the

treatment of CT10, CT50, and CT100 films. Similar results were found for L. acidophilus
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ADH in Fig. 5B. For both the control and treatment groups, the number of B. animalis Bif-6
ur

increased to >9 log CFU/mL at 10 h and remained steady (Fig. 5C). The results indicate that a
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high amount of CNF/TiO2NP nanocomposites (10 mg/mL) does not show appreciable effects

on the growth of the intestinal bacteria.

The studies on intestinal toxicity showed that the antimicrobial effect of positively

charged TiO2NPs was primarily due to the induced reactive oxygen species (ROS)[28]. The

TiO2 particles first adhered to the negatively charged surface of bacteria through electrostatic

interactions[29]; TiO2NPs produced ROS to damage bacterial cell membrane, increase the

permeability of cell membrane, and cause the leakage of cytoplasmic contents[30]; after that,

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TiO2NPs penetrated into the cytoplasm, binded to organelles, and resulted in further damage

to biological macromolecules[31]. Nevertheless, the CNF/TiO2NP nanocomposites did not

exhibit negative effects on the growth of intestinal bacteria in our study. This phenomenon

was investigated via TEM analysis, and TEM micrographs of intestinal bacteria with and

without the treatment of CNF/TiO2NP nanocomposites are shown in Fig. 6. E. coli P-24, L.

acidophilus ADH, and B. animalis Bif-6 cells in the absence and presence of CNF/TiO2NP

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nanocomposites displayed normal cell morphology and intact cell membranes. In Fig. 6B, D,

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and F, TiO2NPs were released from CNFs and adhered to the surface of the intestinal bacteria.
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No TiO2NPs were present inside the cells; and many TiO2NPs were still attached on the
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surface of CNFs (shown in the red squares in Fig. 6B). The data demonstrate that only a
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small number of TiO2NP agglomerates migrate from the nanocomposites to the bacterial

surface, which largely limits the toxic effect of CNF/TiO2NP nanocomposites. In addition,
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some studies showed that the TiO2NPs exposed to light could induce more ROS to cause a
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higher level of lipid peroxidation, cellular respiration disruption, and cell mortality[31,32]. For
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this reason, absence of photoactivation might also be responsible for the current toxicity

results.

3.4. Toxic effect of CNF/TiO2NP nanocomposites on human colon cells

Effects of CNF/TiO2NP nanocomposites on the viability of human colon cells are

shown in Fig. 7. Compared with the control, CT10, CT50, and CT100 (concentration,

50-1000 µg/mL) did not significantly affect the number of Caco-2 viable cells (Fig. 7A-C);

likewise, they had no appreciable effect on the viability of FHC cells (Fig. 7D-F). The results

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suggest that CNF/TiO2NP nanocomposites have no significant toxicity to cancerous and

normal colon cells even when their concentrations were up to 1000 µg/mL.

The ever-increasing use of TiO2NPs in the food industry raises a safety concern;

therefore, researchers have studied the toxicity of the nanomaterial to human intestinal cells.

When Caco-2 cells were incubated with TiO2NPs, the particles were present at the apical side

and in the cells[33]. TiO2NPs penetrated Caco-2 cells mainly via EGFR-associated endocytosis,

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and they induced the inflammatory response within the cells. For low concentrations of

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TiO2NPs (10 and 100 µg/mL), they did not affect the epithelial integrity and viability of
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human colon cells, but they altered microvillar organization and increased intracellular-free
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calcium[34]. However, high concentrations of TiO2NPs (>200 µg/mL), by damaging cell
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membranes and decomposing intracellular constituents, caused a concentration-dependent

decrease in the viability of human colon cells[35]. In addition, the toxicity of TiO2NPs to colon
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cells increased through photoactivation, because photoactivated TiO2NPs were able to induce
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higher levels of ROS that result in more damage to cells[36]. Nevertheless, our results
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demonstrate CNF/TiO2NP nanocomposites do not significantly reduce the viability of human

colon cells. To explore its mechanism, we observed the morphology of Caco-2 cells in the

presence and absence of CT10, CT50, and CT100 (1000 µg/mL) via TEM analysis (data not

shown). Cells in the control and treatment groups showed normal membranes, mitochondria,

and nuclei, and no TiO2NPs were observed on the membrane or within the cells. A possible

explanation is that most TiO2NPs firmly adhere onto CNFs, and few can be released from the

nanocomposites, which greatly impairs the toxicity of CNF/TiO2NP nanocomposites to colon

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cells.

4. Conclusions

CNF/TiO2NP nanocomposites were synthesized via a facile mixing method based on

electrostatic interactions between TiO2NPs and CNFs. The CNF/TiO2NP nanocomposites

significantly improved the mechanical properties and light barrier capacity of PVA films; and

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they showed no appreciable toxic effect on both intestinal bacteria and human colon cells.

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Consequently, the nanocomposites are biocompatible and have great potential to be used as

functional fillers in food packaging materials.

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Acknowledgments

This research was partially supported by the Robert T. Marshall Scholarship and the
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Nanotechnology Program of USDA National Institute of Food and Agriculture

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(2016-67021-24994). We thank Dr. Bongkosh Vardhanabhuti for her assistance in the

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analysis using Zetasizer Nano ZS.

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Figure captions

Fig. 1. TEM micrographs of CNFs (A) and CNF/TiO2NP nanocomposites (B: CT10; C:

CT50; D: CT100). TiO2NPs coated on CNFs are pointed by red arrows in Fig. 1B-D.

Fig. 2. Images of PVA-based films (A: P; B: PC; C: PCT1; D: PCT5; E: PCT10) above a

background of a red flower.

Fig. 3. Mechanical properties (A: tensile strength; B: Young’s modulus) and light barrier

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capacity (C: film transmittance; D: O value) of PVA-based films.

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Fig. 4. SEM images of surfaces (A: P; B: PC; C: PCT1; D: PCT5; E: PCT10) and cross
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sections (F: P; G: PC; H: PCT1; I: PCT5; J: PCT10) of PVA-based films. Scale bar: A-J, 50
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μm. Magnification: A-J, 1000×.
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Fig. 5. Toxic effects of CNF/TiO2NP nanocomposites on intestinal bacteria (A: E. coli P-24;
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B: L. acidophilus ADH; C: B. animalis Bif-6).

Fig. 6. TEM micrographs of E. coli P-24 (A: control; B: treatment with CT100 film), L.
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acidophilus ADH (C: control; D: treatment with CT100 film), and B. animalis Bif-6 cells (E:
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control; F: treatment with CT100 film). TiO2NPs present on the surface of intestinal bacteria

are pointed by red arrows. TiO2NPs on CNFs are shown in red squares.

Fig. 7. Effect of CNF/TiO2NP nanocomposites on the viability of Caco-2 (A: CT10; B: CT50;

C: CT100) and FHC (D: CT10; E: CT50; F: CT100) colon cells.

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Table 1. Zeta potential of TiO2NPs, CNFs, and CNF/TiO2NP nanocomposites

Sample TiO2 CNF CT10 CT50 CT100

Zeta potential -36.50 ± -29.90 ± -25.70 ± -22.27 ±

10.80 ± 0.72
(mV) 1.13 0.92 2.10 1.21

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Table 2. Thickness, color, and WVP of PVA-based films

Sample Thickness L* a* b* △E WVP

(mm) (×10-11
g.m-1.s-1.Pa-1)

P 98.61 ± 0.00 ± 2.11 ± 0.88 ± 8.26 ± 0.70a

0.09 ± 0.02a
0.25a 0.01a 0.06a 0.23a

PC 98.18 ± -0.04 ± 2.60 ± 0.82 ± 7.75 ± 0.64a

0.12 ± 0.01b
0.24a 0.02a 0.04b 0.10a

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PCT1 97.15 ± -0.04 ± 2.80 ± 1.07 ± 8.80 ± 0.77a

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0.12 ± 0.01b
0.08b 0.01a 0.01c 0.04ab
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PCT5 96.92 ± -0.19 ± 2.97 ± 1.36 ± 8.40 ± 1.84a
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0.12 ± 0.01b
0.24bc 0.02b 0.13c 0.23b
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PCT10 96.44 ± -0.27 ± 3.51 ± 2.09 ± 7.81 ± 1.00 a

0.12 ± 0.01b
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0.01c 0.01c 0.08d 0.07c

The data with different lowercase letters in each vertical column are significantly different
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(P < 0.05).
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Highlights

 CNF/TiO2NP nanocomposites were prepared using a mixing method

 Adding the nanocomposites enhanced the mechanical properties of PVA-based films
 Filling the nanocomposites improved the light barrier capacity of PVA-based films
 The nanocomposites did not display significant toxic effects on intestinal bacteria
 The nanocomposites did not exhibit notable toxicity to human colon cells

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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7