The Role of ZnO and TiO₂ in Antimicrobial Bioplastics Based on PLA-Chitosan

Abstract

The purpose of this study was to develop bioplastics using chitosan-Poly Lactic Acid (PLA), ZnO, and TiO₂ to improve antimicrobial properties. In the preparation of the bioplastics, PLA with chitosan-ZnO or chitosan-TiO₂ were used. The antimicrobial activity, mechanical properties, thermal properties, and water vapor permeability of bioplastics were evaluated. In general, the addition of ZnO produces better properties compared to TiO₂. PLA-ZnO and chitosan have antimicrobial activity against bacteria such as Salmonella typhi, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, yeast such as Candida albicans, and fungus Aspergillus niger. PLA-chitosan-ZnO alloys have the best antimicrobial activity, even though there is no formation of new functional groups of alloys. In comparison to other similar products, these bioplastics have medium tensile strength, tensile modulus, and elongation percentages with low barrier ability to water vapor. The PLA with chitosan and ZnO or TiO₂ addition decomposed in a single stage, though the decomposition temperature increases after the addition of PLA.

Keywords: antimicrobial properties, bioplastic, PLA, TiO₂, ZnO

Introduction

Petrochemical plastics such as polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyamide (PA)  are widely used as packaging materials [1]. Since plastics take longer to degrade completely, their usage needs to be reduced in order to limit environmental pollution. According to the Ministry of Environment and Forestry (KLHK), environmental pollution due to waste generation reached 65.2 million tons in 2016, with 3.2 million tons being plastic waste disposed to the sea [2]. The plastic waste might break down into microplastics, which risks the life sustainability of marine biota since they are indigestible. Besides, plastic packages are often contaminated with food ingredients and biological substances, and therefore recycling them is impractical.

Polylactic acid (PLA) is a polymer that is chemically synthesized from agricultural sources. It is produced from L-lactic acid obtained from the fermentation of corn starch and other sources of polysaccharides [3]. PLA has a biocompatible, easily processed, and renewable, though it is not a bioactive material. Since it lacks antimicrobial properties, it is necessary to add materials that work against microbial growth, such as chitosan.

Chitosan is a cationic polysaccharide from the chitin deacetylation process that is widely used as an active packaging material due to its high biodegradability, non-toxicity, and antimicrobial properties. It is widely used as an antimicrobial agent either alone or mixed with other natural polymers. According to Bonilla [4], PLA-chitosan films have antimicrobial activity against mesophilic aerobic bacteria and coliform microorganisms. To increase its antimicrobial activity, titanium dioxide (TiO₂) or zinc oxide (ZnO) can be added to the bioplastic.

As inorganic materials, TiO₂ and ZnO are used for energy and medicine, preservatives in packaging, and antimicrobial agents, respectively [6][7]. TiO₂ has essential characteristics of excellent chemical stability, being non-toxic, low-cost, and antibacterial properties, while ZnO has antibacterial activity against gram-positive, gram-negative, and pathogenic bacteria carried by food products [7].

As food packaging, non-degradable plastic has adverse effects on the environment. However, degradable plastic with antimicrobial properties increases the safety level of packaging materials. Therefore, this research focuses on developing a new method of bioplastic production containing antimicrobial properties by combining PLA and/chitosan with TiO₂ or ZnO.

Materials and Methods

Materials

The materials for bioplastic preparation consist of  PLA (Ingeo 4060D), chitosan (Degree of deacetylation ≥ 87.5%), TiO₂ (Merck, Darmstadt Germany), ZnO (Wako pure chemical industries, Ltd.), acetic acid (AcOH) 2.5% v/v (technical grade), acetone (Merck, KGaA, Darmstadt Germany), and glutardialdehyde (Merck, Schuchardt OHG). The antimicrobial activity test media materials include H₂O, EtOH 70% (technical grade), Nutrient Broth (NB), Nutrient Agar (NA)  and Potato Dextrose Agar (PDA) (Himedia Laboratories Pvt.Ltd), Potato Dextrose Broth (GDP) (Le Pont de Claix France), and Bacteriological Agar (LP0011 Oxoid). The observation of antimicrobial activity involved the use of bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Salmonella typhi, yeast, Candida albicans, and fungus Aspergillus niger.

Methods

Chitosan, ZnO, and TiO₂ Antimicrobial Activity Test

The antimicrobial activity test of chitosan, ZnO, and TiO₂ was carried out through modification of Mostafa [8] method using 2.5% v/v AcOH solvent. Before evaluating the antimicrobial activity against bacteria, yeast, and fungus with agar diffusion techniques, the samples were sterilized using an autoclave for 15 minutes. The inoculum preparation and microbial inoculation were prepared in liquid media, including NB for bacteria Salmonella typhi, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus and PDB for Candida albicans. NA solid media for bacteria Salmonella typhi, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus was prepared. Potato Dextrose Agar (PDA) was used for yeast Candida albicans, and the fungus Aspergillus niger. To evaluate the sample inhibition zone, 20 µL inoculum and 20 mL of media were poured into Petri dishes made in the quadrant, which were then rotated until the contents mixed evenly. Petri dishes already containing microbes were compacted and then formed wells with a diameter of 0.6 mm using cork borer. Each sample of 50 µL was poured into wells and incubated at 30 °C. The zone inhibition diameters were measured at 24, 48​​, and 72 hours.

Formulation of Chitosan-ZnO and Chitosan-TiO₂ Bioplastics

Zhang et al., with the modification method, was used in the preparation of the chitosan-ZnO and chitosan-TiO₂ film. 0.5 g chitosan and 0.05 g ZnO or TiO₂ powder was added into 20 mL AcOH 2.5% v/v to prepare chitosan-ZnO and chitosan-TiO₂ films. Each solution was stirred with a magnetic stirrer and sonicated at 120 W for 10 minutes. The stirring of the solution was carried out until a uniform solution was formed. This was followed by the addition of 0.1 mL glutardialdehyde in each solution and stirring for 4 hours at room temperature, then placed into a mold and dried [9].

Preparation bioplastics with PLA

PLA-chitosan-ZnO and PLA–chitosan-TiO₂ films were modified through the Ghozali et al. [10] method. PLA was dissolved in acetone, each 2 g, and stirred slowly at room temperature, followed by the addition of chitosan-ZnO and chitosan-TiO₂ in powder forms and stirring with a magnetic stirrer for 2 hours. Each solution was then placed in the mold and dried [10].

Antimicrobial Activity Test of Bioplastics

Bioplastic samples tested for antimicrobial activities include chitosan-ZnO, chitosan-TiO₂, PLA-chitosan-ZnO, and PLA-chitosan-TiO₂. Testing the antimicrobial activity of bioplastic samples followed the approach used in the evaluation of the antimicrobial activity of chitosan, ZnO, and TiO₂.

FTIR Analysis

Bioplastics samples of approximately 0.1 mg were placed on a plate. IR spectra were recorded with a scan count of 1 per sample in absorption mode and a spectral resolution of 4.0 cm^-1 in the range of 4000 to 400 cm^-1 at room temperature using spectrum software (Perkin Elmer, USA).

Tensile Strength Test of Bioplastics

Testing for composite products included tensile strength, which, according to ASTM D882-75b [11], was carried out by cutting the bioplastic into a size of 10 × 1 cm². Afterward, the samples were tested using the Shimadzu Universal Testing Machine and pulled at a constant speed until the films were broken. The tensile strength and elongation percentage (%E) can be determined using equations 1 and 2 as follows

(1)                                                          (2)

Where σ is tensile strength (MPa), F is a force of tensile strength (N), A is field area (mm²), % E is elongation percentage (%), ΔL is length increase (mm), and L1 is an initial length (mm)

Thermogravimetric Analysis (TGA)

Thermogravimetric analysis (TGA 4000 Perkin Elmer) was carried out based on Bonilla et al. [5] with modification to measure thermal weight loss of each sample formulation. The temperature range was set at 30 – 900 °C with a heating rate of 10 ° C/min under nitrogen flow.

The Measurement of Water Vapor Permeability

Water Vapor Transmission Rate (WVTR) and Water Vapor Permeability (WVP) were determined through the cup method, according to ASTM E 95-96 1995 [12]. Water-filled Petri dishes were covered with aluminum foil depending on their sizes. The central part of the aluminum foil paper was perforated by 10% of the area of the petri dish, and the bioplastics were affixed to which the permeability value was to be measured. Petri dishes were weighed and then put into an oven at 37 ± 0.5 ˚C every 1 hour for 7 hours. Each sample was measured three times, and the loss of water mass was determined based on time until a steady-state was reached. WVTR and WVP values were calculated using equations 1 and 2, respectively.

WVTR                             (1)   WVP                                   (2)

where WVTR is water vapor transmission rate (gs^-1 m^-2), WVP  is water vapor permeability (gs^-1 m^-1 Pa^-1), flux is slope in linear regression, d is film thickness (m), S is saturated air pressure at 37 ° C (6266,134 Pa), R₁  is RH in Petri dishes (100%),  and R₂ is RH at 37 ˚C (81%).

Results and Discussion

Antimicrobial Activity of bioplastics

Antimicrobial activity can be determined based on the formation of inhibition zones on microbial growth media. Figure 1 shows the antimicrobial activity of the initial samples with AcOH 2.5% v/v as a control, which also has antimicrobial activity. Chitosan might inhibit the growth rate of Escherichia coli, Bacillus subtilis, Salmonella typhi, and Staphylococcus aureus. In general, chitosan and its derivatives are strong antimicrobial agents that inhibit the growth of Gram-positive and Gram-negative bacteria, fungi, and viruses. Under acidic conditions, the C-2 position of the chitosan glucosamine monomer has a positive charge, which interacts easily with the negative charge of microbial cell membranes and causes leakage of proteins and intracellular microbial constituents [13]. Antimicrobial mechanism of chitosan involves penetrating the nucleus, binding to DNA, and inhibiting mRNA synthesis, inhibiting the production of amino acid [14].

Fig.1 Antimicrobial activity of initial samples: AcOH 2.5% v/v () AcOH, 2.5% v/v + TiO₂ (), AcOH 2.5% v/ v + ZnO () and AcOH 2.5% v/v + chitosan ( )

According to Wang et al. [15], TiO₂ has antimicrobial ability to mediate photocatalysis under UV light. Reactive oxygen species (ROS), such as OH, H₂O₂, and O₂^– which are essential in destroying microbial cells are produced with enough energy. However, in this study, the results obtained indicate TiO₂ produces antimicrobial activity in the absence of UV light and inhibit the growth of Escherichia coli, Bacillus subtilis, and Candida albicans since it has significant activity on Escherichia coli [16]. Additionally, ZnO inhibits the growth of Escherichia coli, Bacillus subtilis, and Aspergillus niger. The antimicrobial mechanism of ZnO involves (i) production of ROS, (ii) destabilization of microbial membranes having direct contact with ZnO particles to the cell wall, and (iii) intrinsic antimicrobial Zn²⁺ ion released by ZnO in water media [17].

The antimicrobial activity of the bioplastics refers to the ability of the film to inhibit the growth of microbes, both bacteriostatic and fungistatic. The antimicrobial activity of bioplastic is measured from the diameter of the inhibitor zone formed, as shown in Fig. 2. PLA-chitosan bioplastic does not have antimicrobial activity since chitosan powder is not activated by acetic acid. Chitosan-TiO₂ only inhibits the growth of Escherichia coli bacteria, a finding supported by Mihindukulasuriya [16], which established that TiO₂ has significant activity on Escherichia coli. In this study, chitosan-ZnO bioplastics have very high antimicrobial activity in which the growth of almost all pathogenic bacteria can be inhibited. This result corresponds with Li et al. [18], which stated that chitosan-ZnO inhibits the growth of bacteria Bacillus subtilis, Escherichia coli, and Staphylococcus aureus.

Fig. 2 Antimicrobial activities of bioplastics: PLA-chitosan (), chitosan-ZnO (), chitosan-TiO₂ (), PLA-chitosan-ZnO (), and PLA-chitosan-TiO₂ ()

PLA-chitosan-ZnO is among bioplastic alloys with the best antimicrobial activity, which can be shown by the inhibition growth activity in all tested microbes. Chitosan-ZnO and PLA-chitosan-ZnO bioplastics have the highest growth inhibition ability in the fungus, Aspergillus niger, while PLA-chitosan-TiO₂ only inhibit the growth of Salmonella typhi.

Visual Appearance of Bioplastic Films

Chitosan-ZnO film in Fig.3a is more transparent and brighter than chitosan-TiO₂ shown in Fig.3b. This is because during the dissolution, ZnO dissolves in acetic acid to produce the yellowish color while TiO₂ forms a white solution. This color formation is also caused by the migration of C─H bonds of chitosan in reaction with AcOH. 2.5% v/v.

Fig.3 Bioplastics : Chitosan-ZnO (a), Chitosan-TiO₂ (b), PLA-chitosan (c), PLA-chitosan-ZnO (d), and PLA-chitosan-TiO₂ (e)

Alloy is a microscopic combination of two or more substances with different phases into a new material that has better properties than its constituents. In the preparation of an alloy of bioplastics consisting of PLA, chitosan, ZnO and TiO₂, the chitosan-ZnO or chitosan-TiO₂ was prepared before the films were mashed and dissolved with PLA. The alloy of PLA-chitosan bioplastic, as shown in Fig.3c, has a flat surface with white color, though it is fragile and brittle. The bioplastic of PLA-chitosan-ZnO alloy has a slightly wavy and pores surface with orange color, as shown in Fig. 3d. The Bioplastic of PLA-chitosan-TiO₂ alloy (Fig. 3e) has uneven pores surface with beige color. The formation of pores is caused by the addition of chitosan-ZnO or chitosan-TiO₂ powder in PLA.

 FTIR Spectra

The absorption of the infrared radiation makes the molecules agitated to a higher energy state like in other types of energy absorption. It is a quantized process in which molecules only absorb certain frequencies as the energy of infrared radiation. The absorption is related to energy changes at 8 to 40 kJ /mol, and the radiation corresponds to a range that includes the frequency of vibrational stretching and bonding of most covalent molecules. In the absorption process, infrared radiation frequencies matching the natural vibrational frequencies of the molecules are absorbed. The energy absorbed increases the amplitude of the vibrational motion of molecule bonds [19]. Fig. 4 shows the FTIR spectra of the chitosan, chitosan-ZnO, and chitosan-TiO₂ bioplastics.

Fig.4 FTIR spectra of chitosan (a), chitosan-ZnO (b), and chitosan-TiO₂ (c) bioplastics

The FTIR spectrum of pure chitosan (Fig. 4a) shows the peak at a wavenumber of 2920.44 cm^-1, which can be identified as absorption ─OH. The addition of ZnO and TiO₂ facilitates each Zn and Ti⁴⁺ ions to immediately form a coordination bond with ─OH and -NH from chitosan [20]. It causes a shift in the absorption peak of -OH in the chitosan-ZnO (Fig. 4b) and chitosan-TiO₂ (Fig. 4c) bioplastics towards the 3252 cm^-1 and 3269.02 cm^-1 wavenumbers, respectively. This is also evident from the differences in the absorption peak width of each bioplastic.

The absorption peak in the range of wavenumber of 580-400 cm^-1 originates from O─Zn─O vibrations [18]. In the chitosan-ZnO bioplastic, the peak absorption of O─Zn─O is at the wavenumber of 415.28 cm^-1, as shown in Fig.4b. The absorption peaks in the range fingerprint of wavenumbers of 700-400 cm^-1 are derived from Ti─O absorption peaks [20]. A peak of absorption of Ti─O at the wavenumber of 443.11 cm^-1 appears in the chitosan-TiO₂. The FTIR spectra confirm that chitosan-ZnO and chitosan-TiO₂ interact chemically with the proposed reaction, as shown in Fig.5.

Fig.5 Interactions in the chitosan-ZnO (a) and chitosan-TiO₂ (b) films

Interaction of chitosan and metal ions forms complex compounds (Fig.5). Chitosan is an efficient chelating agent that bind metals to remove toxic produced by microorganisms. A regular absorption of each type of bond, such as N─H, C─H, O─H, C─X, C═O, C─O, C─C, C═C, C≡C, and C≡N, is only found in certain parts of the infrared vibration region. A small absorption range can be determined for each type of bond, though other types of bonds form absorption outside this range. Absorption in the range of 3000 ± 150 cm⁻¹ is often attributed to the presence of C─H bond in the molecules.  However, the absorption in the range of 1715 ± 100 cm ⁻¹ is caused by the presence of the C═O bond (carbonyl groups) in the molecules. The same range applies to each type of bond [19].

Fig. 6 FTIR spectra: PLA (a), PLA-chitosan (b), PLA-chitosan-ZnO (c), dan PLA-chitosan-TiO₂ (d) bioplastics

The spectra of bioplastics in the presence of additional PLA is shown in Fig 6. The regions of interest for PLA and the composites are 1780, and 1680 cm^-1 for the C=O and 3600-3000 cm^-1 for the O-H stretch. The peaks of about 1750 and 1180 cm^-1 belong to the C=O and the C-O-C stretching of PLA [21]. In general, all PLA bioplastics contain these two absorption peaks, and therefore the interactions between PLA and chitosan, chitosan-ZnO, and chitosan-TiO₂ do not occur chemically. Their occurred interactions are only physically in the alloy form.

Mechanical Properties of Bioplastics

Mechanical properties, Tensile Strength (TS), percentage elongation (% E), and modulus Young depend on bioplastic composition and constituent component features. By measuring these properties, the limitation of allowed external pressure in the bioplastic can be determined. Tensile strength is the maximum value of the bioplastic holding a given load before breaking. The elongation increases to the maximum length after the bioplastics are pulled up to break. The results of the tensile strength of bioplastics are summarized in Table 1.

Table 1.  Mechanical properties of bioplastics

The addition of ZnO and TiO₂ to chitosan increases the tensile strength value and the elongation at break, respectively. TS of Chitosan-ZnO bioplastics is higher than that of chitosan-TiO₂. Based on the result, the chitosan-ZnO bioplastics is hard and brittle, while the chitosan-TiO₂ film is hard and tough. These results are in line with Cazon et al. [22], which stated that the TS of polysaccharide-based films, such as chitosan, is similar to the TS of synthetic polymers. The TS of chitosan mixed with different molecular weights and solvents ranged from 6.7 to 150.2 N/mm² [22]. Chitosan-ZnO and chitosan-TiO₂ bioplastics fall in this range.

Pure PLA has a higher tensile strength than elongation at break. The addition of chitosan, ZnO, and TiO2 decreases tensile strength but increases the elongation at break. The mixing of PLA and chitosan results in a low TS of PLA-chitosan while the addition of ZnO and TiO₂ increases its TS to 193.64% and 102.91%, respectively. The TS of PLA-chitosan-ZnO and PLA-chitosan-TiO₂ bioplastics are lower than that of the TS of chitosan-ZnO and chitosan-TiO₂ due to the porous and slightly bumpy surface. Therefore, the PLA-Chitosan, PLA-Chitosan-ZnO, and PLA-Chitosan-TiO₂ bioplastics are soft and weak.

Modulus young measures the stiffness of solid material and defines the relationship between stress (force per unit area) and strain (proportional deformation) in a material in the linear elasticity regime of uniaxial deformation. It indicated chitosan-ZnO of 10.496 N/mm², and this means the film is more resistant to tensile pressure compared to others. As stated before, the hard and brittle structure of chitosan-film affects this value.

Thermal Properties of Bioplastics

The thermogravimetry (TGA) test determines the changes in sample weight-loss as a function of temperature variation. It relies on a high degree of precision in three measurements, including weight, temperature, and temperature changes. The principle of TGA helps to measure the average velocity of mass changes of a material/sample as a function of temperature or time in a controlled atmosphere condition.

This method characterizes an ingredient or sample by its mass loss or the occurrence of decomposition, oxidation, or dehydration. The mechanism of mass changes in the TGA in the material demonstrates a loss or increase in mass. The process of mass loss occurs because of the decomposition process due to the breaking of chemical bonds, evaporation (loss of volatility as temperature increase), reduction of the interaction of the material with reducing agents, and also desorption. The increase in mass is caused by the oxidation process, which involves the interaction of the material with the oxidizing agent and absorption [24]. The thermal stability of chitosan, chitosan-ZnO, and chitosan-TiO₂ was as shown in Table 2.

Table 2. Temperature decomposition of bioplastics and their PLA alloys

The addition of ZnO and TiO₂ in chitosan reduces and increases the decomposition temperature in stage 2, respectively. The chitosan-based bioplastics have a similar decomposition model in which they are decomposed in two stages, as shown in Table 2. The first mass loss below 100 °C is associated with water loss and shows very small shape as presented in the TGA curve. There is a lot of mass loss between temperatures of 200 °C and 300 °C, as shown in Fig.7A. This is due to dehydration of the saccharide ring complex, depolymerization, and decomposition of the acetate polymer unit and its deacetylation [20]. The addition of PLA to the chitosan-ZnO bioplastics and chitosan-TiO₂ causes an increase in decomposition temperature, as shown in Table 2. PLA-chitosan, PLA-chitosan-ZnO, and PLA-chitosan-TiO₂ decompose in one stage in the temperature range of 270 to 310 °C, as shown in Fig.7B.

Fig. 7 A. TGA curves of chitosan (■), chitosan-ZnO (■), chitosan TiO₂ (■); B. PLA (■), PLA-chitosan (■), PLA-chitosan-ZnO (■), PLA-chitosan-TiO₂ (■)

Water Vapor Permeability

Water vapor transmission rate (WVTR) describes the ability of a material to hold water vapor into the plastic. This transmission value affects the water vapor permeability (WVP), which is the ability of a material to pass water vapor in an area unit. This value is affected by the pore size and hydrophilicity of the bioplastic constituent component. Fig 8 shows the WVP of bioplastics.

Fig.8 A.Water vapor permeability of chitosan-ZnO (   ), chitosan-TiO₂(   ), bioplastics;  B. PLA-chitosan (    ), PLA-chitosan-ZnO (   ), and PLA-chitosan-TiO₂ (    )

The WVP values of chitosan-ZnO and chitosan-TiO₂ bioplastics are 4,146 x 10^-9 (gs^-1 m^-1 Pa^-1) and 4,263 x 10^-9 (gs^-1 m^-1 Pa^-1). Chitosan-ZnO has a lower WVP value than chitosan-TiO₂. A hard and brittle property of chitosan-ZnO allows water vapor to pass through it easily. In contrast, the chitosan-TiO₂ film is hard and tough, and therefore water vapor hardly passes through it. WVP values of chitosan-ZnO and chitosan-TiO₂ is greater than wvp of pure chitosan, which is 1.05 x 10^-14 (gs^-1 m^-1 Pa^-1) [22].

The lowest WVP is found in PLA-chitosan, which has a great barrier against water vapor. However, the PLA-Chitosan-ZnO has the highest WVP, which means it has a low ability to resist water vapor passage. PLA-Chitosan-ZnO and PLA-Chitosan-TiO₂ have a high WVP due to the formation of pores on the film surface, which allows easy passage of water vapor. The WVP value of bioplastic produced is higher than that of commercial plastic and pure PLA.  A low-density polyethylene has a WVP value of (7.3-9.7) x 10^-13 (gs^-1 m^-1 Pa^-1) [22], while PLA has WVP value 2.47 x 10^-11 (gs^-1 m^-1 Pa^-1) [25].

Conclusions

Generally, ZnO has better antimicrobial activity than TiO₂. The addition of ZnO to PLA and chitosan show antimicrobial activity against the bacteria Salmonella typhi, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, yeast; Candida albicans, and fungus; Aspergillus niger. The best antimicrobial activity of bioplastic alloys is PLA-chitosan-ZnO. The FTIR spectrum of PLA-chitosan-ZnO shows no formation of new functional groups, but a combination of each material to form an alloy. The tensile strength, modulus Young and % E of alloy are lower compared to chitosan-ZnO and chitosan-TiO₂ bioplastics. Decomposition temperature increases after the addition of PLA to the chitosan-ZnO bioplastics and chitosan-TiO₂. The PLA-chitosan-ZnO has low ability against water vapor.

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