**1. Introduction**

Food provides nutritional support for any organism. However, most people around the world are not aware of the dangers related to the lack of proper food hygiene. According to a survey from World Health Organization (WHO), 600 million cases of foodborne diseases and 420,000 deaths were recorded worldwide in the year 2015. The common bacteria that responsible for the foodborne illness are *Norovirus*, *Listeria*, *Campylobacter jejuni*, *Salmonella*, *Staphylococcus aureus*, *Escherichia coli* [1,2].

Food contamination may happen during harvesting, processing, packaging and distribution [2]. However, packaging plays an important role to protect food from being affected by various kinds of contaminants and preserve the products from biological, chemical and physical changes while storage or during preparation. In many so-called "advanced countries", the food quality is a very important factor—they used to reject it even if there was a small change in the smell or appearance [1].

**Citation:** Sunthar, T.P.M.; Boschetto, F.; Doan, H.N.; Honma, T.; Kinashi, K.; Adachi, T.; Marin, E.; Zhu, W.; Pezzotti, G. Antibacterial Property of Cellulose Acetate Composite Materials Reinforced with Aluminum Nitride. *Antibiotics* **2021**, *10*, 1292. https://doi.org/10.3390/ antibiotics10111292

Academic Editors: Luís Melo and Andreia S. Azevedo

Received: 21 September 2021 Accepted: 20 October 2021 Published: 22 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In literature, many different techniques were applied to improve the antimicrobial properties of packaging systems [2]. Some of the methods are based on the reinforcement of volatile and nonvolatile antimicrobial compounds directly into the polymers like the application of sachet or pads that contain volatile antimicrobial compounds, applying antimicrobial coating compounds on the surface of the polymers, ions or covalent linkages of immobilization of antimicrobial agent into the polymers [3–5]. In other cases, chitosan was directly used without any modification as an antimicrobial coating film [3–6]. After consideration of the previous research, this research is aimed to implement the new and easy idea to produce the antibacterial composite material using easily degradable cellulose acetate material as the main substrate and reinforced with AlN which could replace the usage of plastic material.

Cellulose acetate (CA) is a synthetic compound which is derived from the acetylation of cellulose. Cellulose is a natural polymer obtained from plant fibers, with the chemical formula C6H7O<sup>2</sup> (OH)<sup>3</sup> [7]. However, pure cellulose has a complex structure that cannot be easily modulated by using heat or solvents. The acetylation process causes the hydrogen in the hydroxyl groups is replaced by acetyl groups (CH3CO) which turns it into cellulose acetate. CA is easier to dissolved in certain solvents like acetone or can be melted under heat and molded into solid objects, spun into fibers or cast as a film [8].

Aluminum nitride (AlN) is well known for its excellent thermal conductivity, high coefficient of thermal expansion, high electrical resistivity and high dielectric strength [9]. Besides the wide use of AlN in semiconductors, it showed antimicrobial properties such as those of Si3N<sup>4</sup> [10]. This property is due to the reactivity of the AlN with water which produces ammonia (NH3) and ammonium ions (NH<sup>4</sup> + ) that eventually kill the bacteria [10].

This research focuses on producing alternative food packaging materials, plastic tablecloths or toys by reinforcing CA with AlN. The CA/AlN composites can be produced with a lower cost and non-complicated processes. In addition, it also can show both mechanical and thermal stability with enhanced antibacterial property.

Possible future developments include degradable CA/AlN composites which might contribute to the reduction of environmentally harmful plastic waste.

#### **2. Materials and Methods**

#### *2.1. Samples Preparation*

The samples were prepared as showed in Figure 1. Cellulose acetate powder was crushed using pestle and mortar to obtain fined powder. Then the powder was mixed with acetone until completely dissolved and 0.05 mL of pure triacetin were mixed to the solution, as a plasticizer. Once the solution cleared, agglomerated AlN powder were poured into the glass vial and mixed by stirring until a homogenous mixture is obtained. The liquid is then poured inside a flat mold obtained by using polyimide tape on the Teflon plate. Then the film was casted using glass plate to fill the entire row to form a layer. The casting process were repeated 5 times to obtain a desire thickness. The film was left overnight to dry at room temperature. The film was removed from the plate after the acetone evaporated and forming a layer of the CA/AlN composites. It was then heated at 60 ◦C in a vacuum for overnight to remove the remaining solvent inside the film. Five different samples were produced with 0 wt. %, 5 wt. %, 10 wt. %, 15 wt. % and 20 wt. % of AlN, respectively, (i.e., CA content = 100 wt. % − AlN wt. %).

**Figure 1.** Experimental procedure of CA/AlN composites. **Figure 1.** Experimental procedure of CA/AlN composites.

#### *2.2. Sample Characterization 2.2. Sample Characterization*

#### 2.2.1. Laser Microscopy 2.2.1. Laser Microscopy

The surface morphology of the sample was analyzed with the aid of a confocal scanning laser microscope (Laser Microscope 3D and Profile measurements, Keyence, VK × 200 Series, Osaka, Japan) equipped with a numerical aperture between 0.30 and 0.95. It has the x-y stage and autofocus function for z range. The micrographs were collected ranging from 10× to 150× to evaluate macroscopic and microscopic roughness of the samples. 25 images randomly selected from the surface of the map and the micrographs were then analyzed using Keyence Color 3D Laser Microscope VK-X100/X200 series VK Analyzer software (Keyence, Osaka, Japan). The surface morphology of the sample was analyzed with the aid of a confocal scanning laser microscope (Laser Microscope 3D and Profile measurements, Keyence, VK × 200 Series, Osaka, Japan) equipped with a numerical aperture between 0.30 and 0.95. It has the x-y stage and autofocus function for z range. The micrographs were collected ranging from 10× to 150× to evaluate macroscopic and microscopic roughness of the samples. 25 images randomly selected from the surface of the map and the micrographs were then analyzed using Keyence Color 3D Laser Microscope VK-X100/X200 series VK Analyzer software (Keyence, Osaka, Japan).

#### 2.2.2. Fourier Transformed Infrared Spectroscopy

Horiba/Jobin-Yvon, Kyoto, Japan) software.

2.2.2. Fourier Transformed Infrared Spectroscopy Fourier Transformed Infra-Red spectroscopy (FTIR) spectra were collected at room temperature using an FTIR spectrometer (ATR-FTIR, FTIR-4700 with ATR PRO ONE equipped with a diamond prism, Jasco Co., Tokyo, Japan) with a Michelson 28-degree interferometer with corner-cube mirrors with a range between 250,000 and 5 cm−1. The aperture size was 200 × 200 µm2. the acquisition time was fixed to 30 s. The instrument was operated using (Spectra Manager, JASCO, Tokyo, Japan) software. A total of three samples of each type were scanned from 400 and 4000 cm−1 at 5 different locations. The spectra were analyzed with (OriginLab Co., Northampton, MA, USA, and LabSpec, Fourier Transformed Infra-Red spectroscopy (FTIR) spectra were collected at room temperature using an FTIR spectrometer (ATR-FTIR, FTIR-4700 with ATR PRO ONE equipped with a diamond prism, Jasco Co., Tokyo, Japan) with a Michelson 28-degree interferometer with corner-cube mirrors with a range between 250,000 and 5 cm−<sup>1</sup> . The aperture size was 200 <sup>×</sup> <sup>200</sup> <sup>µ</sup>m<sup>2</sup> the acquisition time was fixed to 30 s. The instrument was operated using (Spectra Manager, JASCO, Tokyo, Japan) software. A total of three samples of each type were scanned from 400 and 4000 cm−<sup>1</sup> at 5 different locations. The spectra were analyzed with (OriginLab Co., Northampton, MA, USA, and LabSpec, Horiba/Jobin-Yvon, Kyoto, Japan) software.

#### 2.2.3. Raman Spectroscopy

2.2.3. Raman Spectroscopy Raman spectra of the samples were collected with the aid of triple monochromator (T-64000, Jobin-Ivon/ Horiba Group, Kyoto, Japan) equipped with a charge coupled device (CCD) detector. The excitation source used is 532 nm Nd:YVO4 diode pumped solidstate laser (SOC JUNO, Showa Optronics Co. Ltd., Tokyo, Japan). In total, 25 randomly picked locations were investigated with spectrograph center wavelength 2500 cm−1, grating 300 gr/mm, exposure time 4 s and average of 3. The resulting spectra were averaged. Raman spectral acquisition and pre-processing of raw data such as baseline subtraction, smoothing, normalization and fitting were acquired utilizing commercially available software (LabSpec, Horiba/Jobin-Yvon, Kyoto, Japan and Origin 8.5, OriginLab Co., North-Raman spectra of the samples were collected with the aid of triple monochromator (T-64000, Jobin-Ivon/Horiba Group, Kyoto, Japan) equipped with a charge coupled device (CCD) detector. The excitation source used is 532 nm Nd:YVO4 diode pumped solidstate laser (SOC JUNO, Showa Optronics Co. Ltd., Tokyo, Japan). In total, 25 randomly picked locations were investigated with spectrograph center wavelength 2500 cm−<sup>1</sup> , grating 300 gr/mm, exposure time 4 s and average of 3. The resulting spectra were averaged. Raman spectral acquisition and pre-processing of raw data such as baseline subtraction, smoothing, normalization and fitting were acquired utilizing commercially available software (LabSpec, Horiba/Jobin-Yvon, Kyoto, Japan and Origin 8.5, OriginLab Co., Northampton, MA, USA).

#### ampton, MA, USA). *2.3. Mechanical and Thermal Properties*

#### *2.3. Mechanical and Thermal Properties*  2.3.1. Mechanical Properties

2.3.1. Mechanical Properties Tensile mechanical testing was conducted using a MCT 2150 Desktop Tensile-Compression Tester (AND Discover Precision, Tokyo, Japan) using a 500 N load cell at a strain Tensile mechanical testing was conducted using a MCT 2150 Desktop Tensile-Compression Tester (AND Discover Precision, Tokyo, Japan) using a 500 N load cell at a strain rate of 50 mm min−<sup>1</sup> . For testing, CA/AlN composite samples were cut using a standardized dumbbell shaped tensile sample cutter with an overall length of 35 mm, gauge length of 10 mm, distance between shoulders 12 mm, grip section 4.5 mm, width of grip section

6 mm, reduced section 12 mm. In total, 5 samples were tested with each concentration (0, 5, 10, 15, and 20 wt. %). The result was analyzed using Excel and OriginLab (Co., Northampton, MA, USA).

#### 2.3.2. Thermal Properties

The thermal properties of the CA composite were investigated using a differential scanning calorimeter (DSC) (TA Q200, TA Instruments Japan Inc., Tokyo, Japan) with a heating/cool/heat cycle program. The sample was heated at 10 ◦C min−<sup>1</sup> from −30 to 300 ◦C and cooled at 5 ◦C min−<sup>1</sup> under a nitrogen atmosphere with a gas flow rate of 50 <sup>µ</sup>L·<sup>h</sup> −1 . Each sample was measured three times.

#### *2.4. In Vitro Testing*

The antibacterial analysis was conducted at the Kyoto Prefectural University of Medicine. Gram-positive bacteria, *Staphylococcus epidermidis* (14990TM ATCC® purchased from American Type Culture Collection (ATCC)) and Gram-negative bacteria, *Escherichia coli* (*E. coli*, ATCC® 25922™)*,* were cultured using a brain heart infusion (BHI) liquid medium. The initial 1.8 <sup>×</sup> <sup>10</sup><sup>10</sup> CFU/mL was subsequently diluted to 1.8 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/mL using a phosphate-buffered saline (PBS, NACALAI TESQUE.INC, Kyoto, Japan) solution to mimic ion blood concentrations. The samples with dimensions of 1 cm × 1 cm were sterilized prior to the experiment using a UV sterilizer for 24 h. Then the samples were incubated at 37 ◦C for 12 and 24 h.

#### 2.4.1. Microbial Viability Assay (WST)

WST is well known technique to measure the bacterial metabolism by calorimetric detection. In this experiment, the WST-8 kit (Microbial Viability Assay Kit-WST, Dojindo, Kumamoto, Japan) was used as a calorimetric indicator which releases a watersoluble formazan dye upon reduction in the presence of electron mediator. The amount of the formazan dye generated is linearly related to the number of living microorganisms. The solution is subjected to microplate readers (EMax, Molecular Devices, Sunnyvale, CA, USA) upon collecting the OD value related to living cells. Three samples were used to calculate the average values.
