Development of Chitosan-TiO₂ Nanocomposite for Packaging Film and its Ability to Inactive Staphylococcus Aureus

Introduction

Plastic is widely used as food pacaking due to elastic, lightweight, not easily broken, transparent, waterproof and easy to carry. However, plastics made from petroleum has led to serious ecological problems due to their non biodegradability.^1,2 In the recent years, there has been an increasing interest in developing biodegradable plastics from renewable resources.

One of renewable plastic is bioplastic because the compounds inside are derived from plants like cellulose, lignin, starch and animal like casein, protein, lipid and chitosan.³ Chitosan represents biodegradable^4,5 and biocompatible cationic polysaccharide with premium film forming ability, mechanical strength, flexibility and also a non toxic.^4,6 Unfortunately, bioplastic from chitosan has a disadvantage that is its lower mechanical properties compared to fossil based plastics.⁷ A modification in the form of nanocomposite can be a good solution to improve the mechanical properties of chitosan-based bioplastic.⁸

The synthesis of nanocomposite was carried out by incorporating nanofillers such as silica, clay, and titanium dioxide (TiO₂) in chitosan which made it possible not only to improve mechanical and barrier properties but also to provide other functions in food packaging applications.^9,10 TiO₂ is a photocatalyst and has been widely utilized in many applications such as hydrogen generation, water purification, and decomposition of pollutants, air purification, and also in medical applications, self cleaning and self-disinfecting materials.^11-15 TiO₂ nanoparticle has been used as an effective antibacterial agent because its widespread availability, broad-spectrum antibiosis and low cost. It has excellent physic-chemical properties, physical and chemical stability, good dispersing properties, strong oxidizing power, fast electron transfer rate and good biocompatibility.^16,17

The migration of the active compounds into the food stuff is the major disadvantage of antimicrobial package. This is especially undesirable, when the general trend is to limit the presence of additives in processed food. Therefore, chitosan and TiO₂ are ideal candidates for food packaging due to its nontoxicity. Furthermore, there are few reports on the synergistic effect between TiO₂ nanoparticles and chitosan regarding its biodegradability and antibacterial activity especially on Staphylococcus aureus. The aim of this study was to prepare chitosan-TiO₂ nanocomposite and to characterize these films for their structure, morphology, mechanical properties and antibacterial activity.

Materials and Method

Synthesis of Chitosan-TiO₂ Nanocomposite

1 g of chitosan (DD 85-89% Biochitosan) was added to 1% (v/v) 100 mL glacial acetic acid. The mixture was stirring using an overhead stirrer for 3 hours at room temperature. Then, the solution was homogenized for 30 minutes. Furthermore, TiO₂ nanoparticles were added with variations in the composition (0 g, 0.1 g, 0.2 g, 0.5 g, and 1 g). The solution was stirred using an overhead stirrer for 4 hours at room temperature and then the solution was homogenized for 1 hour. The solution was poured into a glass plate and dried at a temperature of 80°C. After obtaining bioplastic, SEM characterization, FTIR analysis, mechanical strength test, antibacterial activity test and biodegradable plastic analysis were carried out.

Biodegradability Analysis

Biodegradation analysis was carried out by the method of sample burial in a mixture of soil and compost. The simplest quantitative method to determine the biodegradation of a polymer is by measuring its mass loss. Mass loss measurement was done by weighing the polymer mass before and after the biodegradation process for a certain time interval. In this study, six plastic samples were used with dimensions of 4 x 4 cm and weighed regularly 1 week for 2 months.

Antimicrobial Activity

Staphylococcus aureus was first rejuvenated, then the NA media and nanocomposite with predetermined variations were sterilized. Serial dilution of Staphylococcus aureus was performed by using of 10³ and 10⁴ dissolution. After that, the media and bacteria were poured on the petri dish. The process was followed by incubation for 24 hours in a dark room with UV irradiation. After the incubation, the number of bacterial colonies were calculated by CFU method (colony forming units).

Results and Discussion

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis

FTIR is a useful technique for the detection of chemical bonds in the materials. The FTIR spectra of chitosan and chitosan-TiO₂ nanocomposites are shown in Figure 1. It shows the similarity of the resulting spectrum.

Figure 1: FTIR pattern of chitosan bioplastic and chitosan-TiO2 nanocomposites. Click here to view figure

 

Figure 1 shows the similarity of the resulting spectrum. This similarity shows that most functional groups owned by Cs are also owned by Cs-TiO₂ bioplastics. If two ingredients are mixed (Cs and TiO₂), a physical mixture can be formed and even chemical interactions can occur which are characterized by characteristic changes at the peak of the spectrum of bioplastic analysis results on FTIR. Compared to pure chitosan (Cs), hydroxyl bonds, amino and amide groups experience changes in the Cs-Ti spectrum. It indicated there are interaction between Cs and TiO₂.⁴ The interaction of Cs and TiO₂ is characterized by the formation of Cs-TiO₂ bonds in the wavelength range of 450-950 cm^-1.

Figure 1 shows that Cs spectrum had OH and NH functional groups which absorption peaks at wavelengths 3419.79 cm^-1, CH groups at wavelengths 2924.09 cm^-1, groups C=O at wavelengths 1616.35 cm^-1, CO group at wavelength 1257.59 cm^-1, and CN group at wavelength 1039.63 cm^-1. While the Cs-Ti spectrum was a combination of the peaks of chitosan and TiO₂ groups. It indicated that there was a cross-linking process between chitosan and TiO₂. The OH functional group overlaps with NH at a wavelength of 3439.08 cm^-1, the R-NH-R group at a wavelength of 1600.92 cm^-1, the deformation group OH at a wavelength of 1442.75 cm-¹, the deformation group Cs-TiO₂ at a wavelength of 947.05 cm^-1.

Scanning Electron Microscopy (SEM) Characterization

In an attempt to study microstructural changes in the Cs-Ti nanocomposites, SEM was conducted to visualize the surface topography for different samples. The SEM images of chitosan bioplastic (Cs), nanocomposite chitosan-0.1 g TiO₂ (Cs-0.1Ti), chitosan-0.2 g TiO₂ (Cs-0.2 Ti), chitosan-0.3 g TiO₂ (Cs-0.3 Ti), chitosan-0.5 g TiO₂ (Cs-0.4 Ti) and chitosan-0.5 g TiO₂ (Cs-0.5 Ti) can be seen at Figure 2. Addition of TiO₂ on bioplastic strongly influenced the morphology of the Cs-Ti nanocomposites as evidenced in Figure 2.

Figure 2: SEM images of (a) Cs (b) Cs-0.1 Ti (c) Cs-0.2 Ti (d) Cs-0.3Ti (e) Cs-0.4 Ti (f) Cs-0.5 Ti. Click here to view figure

 

Figure 2(a) shows bioplastics without the addition of TiO₂ have homogeneous, smooth and clean morphology without granules. It is different to the SEM images of Cs-Ti nanocomposites. Figure 2 (b-e) indicated that the addition of TiO₂ created morphology of bioplastic had an irregular granule distribution which shows the distribution of TiO₂ in the matrix. SEM images of nanocomposites Cs-Ti shows irregular shapes of TiO₂ distribution in the matrix.

In Figures 2(b) to Figure 2(f) show that the surface of the nanocomposite had a homogeneous, less smooth surface, granules and looks less than perfect. The irregularity of the nanocomposite surface was due to the addition of TiO₂ in the chitosan matrix. It indicated that the distribution of granules in the matrix increases with the addition of TiO₂. It can be seen in Figure 2(f) that Cs-0.5Ti had denser granule distribution than the other nanocomposites with different TiO₂ compositions. A study reported that the homogeneous dispersion in matrix polymers is one of the most important factors in creates a good composite performance.¹⁸

Tensile Strength and Elongation

The effect of TiO₂ addition on nanocomposites to the value of tensile strength (TS)  and elongation (E) can be seen at Table 1.

Table 1: The effect of TiO₂ addition on nanocomposites to the value of tensile strength and elongation.

No	Mixture	Tensile Strength (MPa)	Error of TS	Elongation (%)	Error of elongation
1	Cs	63.86	±0.001	4.66	±0.0001
2	Cs-0.1Ti	12.35	±0.001	2	±0.0001
3	Cs-0.2Ti	10.21	±0.001	1.66	±0.0001
4	Cs-0.3Ti	7.85	±0.001	0.59	±0.0001
5	Cs-0.4Ti	4.97	±0.001	0.57	±0.0001
6	Cs-0.5Ti	0.24	±0.001	0.12	±0.0001

 

Table 1 shows the addition of TiO₂ on nanocomposites lead to the decreasing value of TS and E. The results showed the highest TS and E at the addition of 0.1 gr TiO₂ (Cs-0.1 Ti). This decrease was due to the presence of granules in the matrix which caused the matrix to be less homogeneous, whereas to get a large TS, a homogeneous molecular structure was needed. The higher the addition of TiO₂ caused the composition of chitosan which is originally homogeneous to change its molecular structure to become irregular.

Biodegradable Analysis

Samples (4x4cm) were tested for biodegradation by measuring their weight every week. A decrease in mass from nanocomposites can be seen in Table 2 and Figure 3.

Table 2: Biodegradable analysis of Cs-Ti nanocomposites.

Sample	Initial mass (gr)	Final mass (gr)	Mass. loss (%)
Cs	0.05	0.02	60
Cs-0.1 Ti	0.1	0.05	50
Cs-0.2 Ti	0.1	0.05	50
Cs-0.3 Ti	0.14	0.09	35.71
Cs-0.4 Ti	0.15	0.10	33.33
Cs-0.5 Ti	0.17	0.12	29.41

 

Table 2 shows the decreasing mass of bioplastics and loss of mass after 2 months of hoarding process. Visually, the results of nanocomposites hoarded have changed, hoarded samples are brownish and appear to shrink even appear to be torn for sample without the addition of TiO₂. This is clearly different from the initial conditions of bioplastics that are still smooth, clear for pure and white chitosan when TiO₂ is added. The level of plastic biodegradation can be seen from the percentage mass loss of bioplastic material after being hoarded for a certain period.

Figure 3: Biodegradable analysis of Cs-Ti nanocomposites. Click here to view figure

 

Figure 3 shows the addition of TiO₂ affected the degradation process even though there was still a process of reduced mass. The decrease in mass tends to be long and slightly in line with the increasing number of TiO₂ compositions added. This results in a smaller percentage of mass loss and shows that Cs-Ti nanocomposites took a long time to be completely degraded. It is due to the anti bacterial properties of Cs-Ti which causes the decomposition process of the nanocomposites to be inhibited.¹⁹

Antimicrobial Activity of Cs-Ti Nanocomposites

In a series of experiments, the antibacterial activities of chitosan and Ti-Cs nanocomposites were assessed using the S. aureus microbiological test system as described earlier. The results of this work can be seen at Table 3.

Table 3: Antimicrobial activity of Cs-Ti nanocomposites.

Sample	UV Radiation			Without UV Radiation
	CFU (mL-1)	Survival Ratio (%)	CFU (mL-1)		Survival Ratio (%)
Control	2.9 x 105	100	2.9 x 105		100
Cs	2 x 104	6.90	2 x 104		3.45
TiO2	0	0	0		0
Cs-0.1Ti	0	0	0		0
Cs-0.2Ti	0	0	0		0
Cs-0.3Ti	0	0	0		0
Cs-0.4Ti	0	0	0		0
Cs-0.5Ti	0	0	0		0

 

Table 3 shows the ability of Cs bioplastic to inhibit bacterial but it could not reduce S. aureus bacteria to zero. One of the antimicrobial properties of CS is a positively charged amino group that interacts with a negatively charged microbial cell membrane, which causes the destruction of proteins and intracellular constituents of microorganisms. Cs has been shown to be more effective against gram-negative bacteria than gram-positive bacteria.¹⁸

Table 3 shows the presence of TiO₂ in Ti-Cs composite increasing the ability to kill  bacteria to zero. The survival of S. aureus bacteria was significantly impaired due to the addition of TiO₂ nanoparticles. CFU calculations performed after 24 hours of incubation under UV irradiation or without UV irradiation have shown similar results, namely successfully killing all S. aureus bacteria added. This study have in common results with the previous experiments conducted by other researchers.^18,20

The iluminated TiO₂ capable of killing bacteria entirely is because CS-Ti positively charged interacts with membranes of charged lipid bacteria negatively affecting cell permeability, blocking cell growth and survival thus causing bacterial death. Biological organisms are killed by various reactive species such as hydroxyl radicals, hydrogen peroxide, or superoxide, which are produced in the photocatalytic process of TiO₂ nanoparticles. When a TiO₂ nanoparticle is irradiated with UV it will experience the generation of electrons in the conduction band and form a hole (h⁺) in the valence band. Interaction of holes with water molecules will produce hydroxyl radicals •OH. Radical •OH is an oxidizing agent from organic compounds. From this photocatalysis process, reactive radical species •OH and •O₂ can be released which are strong oxidative substances to degrade organic compounds from the composition of bacterial cell walls.²¹ When there was no UV irradiation but TiO₂ addition is carried out, it shows that TiO₂ nanoparticles can kill bacteria entirely, but it is not clear how the mechanism is. It can be assumed that the mechanism that occurs is the same as Ag which both have antibacterial activity because Ag has a positive charge that can interact with a negative charge on bacteria. So that without UV irradiation, TiO₂ can also act as an antibacterial.

Conclusion

Bioplastics can be synthesized with a mixture of chitosan and TiO₂ with the interaction between chitosan and TiO₂ nanoparticles as seen in the FTIR test results. The surface morphology of chitosan-TiO₂ nanocomposites showed the distribution of granules which indicated that 25 nm TiO₂ nanoparticles were scattered in the matrix. Tensile strength and elongation decrease with the addition of TiO₂ nanoparticles, Cs-0.1Ti was the best result compared to other variations of TiO₂ addition. Cs-Ti nanocomposites has been proven to effectively kill Staphylococcus aureus bacteria entirely with or without UV irradiation. Bioplastics with the addition of TiO₂ could inhibited biodegradable process.

Acknowledgements

The authors would like to thank to the Islamic Development Bank for financial assistance under IDB-UNTIRTA Research Grant project No. 593/UN43.9/PL/2018.

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