The effects of zinc oxide nanoparticles on the physical, mechanical and antimicrobial properties of chicken skin gelatin/tapioca starch composite films in food packaging

Abstract

The aim of this study was to characterize chicken skin gelatin/tapioca starch composite films with varying concentrations (0–5%) of zinc oxide nanoparticles using the casting technique. The incorporation of 5% zinc oxide nanoparticles increased the water vapor permeation (1.52–1.93 × 10⁻⁷ gmm/cm²hPa) and melting temperature of the films. The tensile strength (22.96–50.43 MPa) was increased, while elongation at break decreased with increasing concentrations of zinc oxide nanoparticles. The structures of the films were also investigated via Fourier transform infrared spectroscopy. The inhibitory zones for both the gram-positive (Staphylococcus aureus) (16–20 mm) and gram-negative (Escherichia coli) (15–20 mm) bacteria were larger in the film with 5% zinc oxide. Overall, chicken skin gelatin-tapioca starch composite films with 3% zinc oxide nanoparticles were found to have the optimal formulation, demonstrating good physical, mechanical and antibacterial properties. Gelatin-based composite films with nanoparticle incorporation show strong potential for use in biodegradable food packaging materials.

Introduction

Most synthetic plastic used in food packaging is derived from petroleum, which is non-biodegradable and tends to cause environmental problems. For this reason, modern consumers prefer environmentally friendly packaging. Biocomposite films have been recently developed to reduce the usage of synthetic plastics. However, films of biological origin are often unable to provide sufficient mechanical strength and barrier protection for food (Suderman et al. 2018). Recent studies have shown that there has been a significant increase in interest among researchers in developing more environmentally friendly packaging, such as via the incorporation of bio-composite films with nano-fillers, to produce bio-nanocomposite films with greater strength (Othman 2014).

Bio-nanocomposite films produced by the addition of nano-filler into bio-composite film offer improved mechanical and barrier properties. According to Tang et al. (2012), the addition of nano-filler into the film-forming solution may increase film strength and stiffness, while elongation decreases. The use of nano-filler in bio-composite films has attracted interest from researchers, as nano-filler is not only able to improve strength and barrier properties, but can also confer antimicrobial properties to food packaging (Jamroz et al. 2019). Commonly used nanofillers in films include cellulose, chitin, chitosan, and metal and metal oxide nanoparticles (Jamroz et al. 2019).

Nano-size zinc oxide is a metal oxide that is frequently incorporated into biocomposite films, as it is considered generally recognized as safe (GRAS) by the United States Food and Drug Administration (Espitia et al. 2016). Additionally, the ability of zinc oxide to confer antibacterial properties makes it a suitable material for use in food packaging. Zinc oxide inhibits the growth of bacteria as well as spores that can survive thermal processes. Nano-size zinc oxide may provide a larger surface area, which allows the zinc oxide to have closer interaction with bacteria and thus destroy them (Rhim 2011).

The increasing interest in eco-friendly packaging has resulted in the development of bio-nanocomposite films (Rhim 2011). Gelatin-based films are a type of protein-based film used as a substitute for synthetic plastics (Loo and Sarbon 2020). There are many types of gelatins used in films, including fish gelatin (Rouhi et al. 2013) chicken skin gelatin (Nazmi et al. 2017; Soo and Sarbon 2018) and bovine gelatin (Marvizadeh et al. 2017). Although gelatin-based films are able to provide excellent mechanical properties for films, their poor barrier properties have become a major drawback. One of the most commonly used methods to reduce water vapor permeability of gelatin-based films is the incorporation of polysaccharides such as starch (Alias and Sarbon 2019).

Excellent gas barrier properties can be achieved when polysaccharides (chitin, chitosan, starch, pectin, modified cellulose, carrageenan and xanthan gum) are used in the production of films. This helps prevent the oxidation of food (Alias and Sarbon 2019). Numerous studies on the incorporation of starches such as tapioca starch (Loo and Sarbon 2020), potato starch (Alias and Sarbon 2019) and corn starch (Mali et al. 2006) in gelatin films have been conducted to improve the properties of gelatin films. It is believed that the incorporation of starch and nano-filler into gelatin film-forming solutions can produce suitable replacements for non-biodegradable plastic (Rhim 2011). Previously, chicken skin gelatin films with tapioca starch incorporation were successfully developed and characterized (Loo and Sarbon 2020). The properties of these films can be improved by the addition of nanoparticles. Therefore, the objective of this study was to formulate bio-nanocomposite films from chicken skin gelatin with incorporation of tapioca starch and zinc oxide nanoparticles. The physical, mechanical and antibacterial properties of the bio-nanocomposite films were also investigated.

Materials and methods

Materials

The chicken skin gelatin used in this study was extracted from fresh chicken skins obtained from Jang Maju Enterprise, Kuala Terengganu, Malaysia. The zinc oxide dispersion (< 130 nm) was purchased from Sigma Aldrich. The tapioca starch was purchased from a local market in Kuala Terengganu. All other chemicals used for analysis were of analytical grade.

Methods

Chicken skin preparation

Frozen chicken skins were thawed at 4–5 °C before the experiments were conducted. The skins were cut into pieces and dried in a cabinet drier (Protech FSD-380, Tech-Lab Scientific Sdn. Bhd., Selangor, Malaysia). The dried chicken skins were ground with a blender before being defatted using Soxtec method (AOAC 2006). After defatting, the dried chicken skins were weighed and stored in a chiller for further use.

Gelatin extraction

Chicken skin gelatin was extracted based on the method described by Soo and Sarbon (2018). Firstly, 15 g of defatted dried chicken skin was added into 200 ml of 0.15% (w/v) sodium hydroxide. The mixture of defatted chicken skin and sodium hydroxide solution was stirred continuously for 30 min before centrifugation (CR 22 N, Hitachi, Tokyo, Japan) at 3500×g for 10 min at room temperature. This treatment was carried out 3 times. The alkaline treated pellets were rinsed with distilled water and centrifuged once with 200 ml of distilled water. Then, the resultant pellets were treated with 200 ml of 0.15% (w/v) sulphuric acid, followed by 200 ml of 0.7% (w/v) citric acid. The pre-treatment steps were repeated 3 times on the resulting pellets. The resulting pellets were finally washed with distilled water. The final extraction of chicken skin gelatin was conducted overnight in distilled water at 45 °C. The mixture was filtered with a filter funnel and Smith filter paper (no. 101). The extracted gelatin was concentrated using a rotary evaporator until its volume was reduced to 1/10 of the original volume and then freeze-dried. The freeze-dried gelatin powder was ground, weighed and stored for further use.

The yield of gelatin was calculated based on the dried weight of chicken skin and expressed as follows:

$$\text{Yield}(\%)=\frac{\text{Weight of gelatin powder (g)}}{\text{Dried weight of chicken skin gelatin (g)}}\times 100$$

Preparation of bio-nanocomposite film

Chicken skin gelatin/tapioca starch/zinc oxide (ZnO) composite films were prepared by a casting method following Loo and Sarbon (2020) with the incorporation of zinc oxide nanoparticles. Different concentrations of ZnO nanoparticles were added into water to obtain Formulation 1 (0% ZnO), Formulation 2 (1% ZnO), Formulation 3 (3% ZnO), and Formulation 4 (5% ZnO), w/w of total solid. Each solution mixture was stirred for 1 h at 60 °C. Then, the ZnO solution was placed into the sonicating bath for 30 min to ensure the homogeneity of the solution. Chicken skin gelatin powder (4 g) and tapioca starch (0.4 g) were simultaneously dissolved separately in distilled water at 45 °C and 85 °C, respectively. Then, the chicken skin gelatin solution (pH 6.0) was added into tapioca starch solution and stirred constantly, followed by the addition of ZnO solution. Lastly, glycerol (30% (w/w of gelatin)) was added into the film forming solution (FFS) at 45 °C with constant stirring for 30 min. Approximately 25 g of bio-nanocomposite solution was cast in a Petri dish. The films were dried in a cabinet dryer until thoroughly dry.

Thermal Properties

The melting temperatures of the films were determined using a differential scanning calorimeter (DSC) (Perkin Elmer, Norwalk, CT, USA) following Soo and Sarbon (2018). A segment of film (5 mg) was weighed in an aluminium pan. An empty pan was used as reference. The pan was hermetically sealed and then heated at a scanning rate of 10 °C/min from 10 °C to 150 °C. Nitrogen gas at a rate of 20 ml/min was used to flush the sample. From the thermogram, the endothermic peak was observed to determine the melting point (T_m) of the film.

Water vapor permeability

The water vapor permeability (WVP) was measured using the method proposed by Jahit et al. (2016). Approximately 10 g silica gel (0% RH) was weighed and placed in a circular aluminium cup. Then, the cup containing silica gel was sealed with film. The cup was weighed together with film to calculate the initial weight before placed in a desiccator filled with distilled water. The weight of the cup was measured at 1 h intervals for 8 h. The WVP was calculated as follows:

$${\text{Water vapour permeability (gmm/m}}^{\text{2}}\text{sPa) =}\frac{\mathrm{\Delta }w\times \text{X}}{\text{A}\times \text{t}\times \mathrm{\Delta }\text{P}}$$

where Δw is the change in cup weight (g), X is the average film thickness (mm), A is the permeation surface area (cm²), t is time and ΔP is the partial pressure difference between silica gel and distilled water (Pa).

Mechanical properties

Tensile strength (TS) and elongation at break (EAB) of the films were determined using the method described by Nur Hazirah et al. (2016). TS and EAB of bio-nanocomposite films were measured using TA. XT2i Texture analyser (Stable Micro System, Godalming, United Kingdom). The film was cut into 10 × 70 mm sections. The film thickness was measured at five different positions using a micrometre (Mitutoyo, Japan) to obtain the average thickness used to determine the elastic behaviour of films. The films were conditioned at room temperature and 0% RH. Each film strip was fixed between the grip pairs with a 50 N load cell 50 mm away from the film strips. The speed of the crosshead was set at 1 mm/s. Then, the upper grid was moved until the load cell broke the film.

Tensile strength (TS) was calculated using the formula:

$$\text{Tensile strenth}(\text{Mpa})=\frac{\text{Maximum load needed to pull the sample apart}(\text{N})}{\text{Film width}(\text{mm})\times \text{Film thickness}(\text{mm})}$$

Elongation at break (EAB) was calculated using the formula:

$$\text{Elongation at break}\left(\%\right)=\frac{\text{Elongation length when the film breaks}(\text{mm})}{\text{Initial length of film}(\text{mm})}\times 100$$

Functional group determination by Fourier transform infrared spectroscopy (FTIR)

The infrared spectra of the films were measured by using Thermo Nicolet iS10 Spectrometer (Thermo Fisher Scientific, Massachusetts, USA) with deuterated triglycerine sulphate (DTGS) as a detector. The background spectrum (without a sample) was collected after the plate was cleansed with acetone, followed by affixing the film samples to the plate and recording the spectra obtained. The sample scanning frequencies were in rhe range of 4000 to 650 with a spectra resolution of 4 cm⁻¹ over 32 scans. Each sample was measured in triplicate and averaged per formulation. The peaks of amide A, amide I, amide II and aliphatic alcohol were identified by software and assigned according to Nazmi et al. (2017).

Antibacterial properties

The effects of bacteria inhibition were measured using disc diffusion method as described by Kim and Kim (2007) with slight modification. The growth rates of 2 pathogenic bacteria, one gram-negative bacteria (E. coli) and one gram-positive bacteria (Staphylococcus aureus), were observed. Both bacteria were cultured on Tryptone Soya Agar (TSA) at 37 °C for 18 h. Mueller–Hinton agar was poured and allowed to solidify in Petri dishes. A single colony of bacteria culture was inoculated and spread evenly onto Petri dishes using a sterilized swab. The dishes containing bacteria culture were pre-incubated at 37 °C for 10 min. The films were cut into a circular shape with 4.5 mm diameters. Then, the films were placed onto the agar containing bacteria culture using sterilized forceps. The plates were incubated at 37 °C for one day. The zones of inhibition were measured using a ruler and expressed in mm.

Statistical analysis

All analyses were performed in triplicate and the data were presented by mean ± standard deviation. Then, the data obtained were analysed using One-way Analysis of Variance (ANOVA) with Minitab-14.0 software. Fisher’s Test was used to compare the mean with α < 0.05 confidence level.

Results and discussion

Thermal properties

Table 1 show the melting temperatures (T_m) of chicken skin gelatin/tapioca starch composite films incorporated with different concentration (0–5%) of zinc oxide nanoparticles. The T_m increases from Formulations A to D. Based on Table 1, there were two endothermic peaks for film Formulations A and B. Meanwhile, film Formulations C and D had only one single endothermic peak in the thermogram.

Table 1.

Melting temperature and water vapour permeability of chicken skin gelatin/tapioca starch composite film at different concentration of zinc oxide nanoparticles

Film formulation	Tm (°C)		WVP (× 10−7 gmm/cm2hPa)
	1st peak	2nd peak
A	61.86 ± 1.93b	96.68 ± 4.55a	1.52 ± 0.11b
B	64.37 ± 0.44b	87.58 ± 1.73b	1.46 ± 0.06b
C	94.87 ± 1.63a	–	1.24 ± 0.03c
D	93.42 ± 1.37a	–	1.93 ± 0.03a

A is film with 0% zinc oxide nanoparticles; B is film with 1% zinc oxide nanoparticles; C is film with 3% zinc oxide nanoparticles; and D is film with 5% zinc oxide nanoparticles. Data reported are mean ± standard deviation.

The different superscript letter^abc within column indicate significance different (p < 0.05)

Both Formulations A and B showed double melting behaviour, and the melting transition progressively evolved towards single melting with increasing nanoparticles content. The presence of 2 endothermic peaks represented the existence of 2 different ordered structures in the chicken skin gelatin/tapioca starch composite film, with one dominated by chicken skin gelatin and the other dominated by tapioca starch (Loo and Sarbon 2020). Meanwhile, the single endothermic peak in film Formulations C and D indicated the relative homogeneity of films when more zinc oxide nanoparticles were added (Tongdeesoontorn et al. 2011). However, the presence of multiple endothermic peaks was due to the polymorphism in films while the merging of endothermic peaks into one might be the result of an altered balance between crystals formed in the sample (Bikiaris 2010). The increment of T_m was mainly due to the formation of hydrogen bond between nanoparticles with other components in the polymer matrix. Compact and homogeneous structures are formed when more bonds are formed between the components and nanoparticles.

The findings in the current study are in contrast to those from a study by Sahraee et al. (2017), which found that the T_m of bio-nanocomposite film was increased with the increased concentration of nanoparticles. However, above certain concentrations, the T_m of film decreased. Additionally, the formation of bonding between nanoparticles and components increases the degree of crystallinity of bio-nanocomposite films and therefore, more heat energy is needed to break the bonding of films (Sahraee et al. 2017). Conversely, the decrease in T_m of bio-nanocomposite films could be attributed to the agglomeration of nanoparticles in the film. The agglomeration of nanoparticles reduced the interaction of nanoparticles in the biopolymer matrix. Thus, the T_m of bio-nanocomposite film decreased when too many nanoparticles were added.

Water vapor permeability (WVP)

The effects of zinc oxide nanoparticles concentration on water vapor permeability (WVP) of chicken skin gelatin-tapioca starch composite films are shown in Table 1. The WVP values decreased from Formulation A to C. At 5% zinc oxide nanoparticles, WVP increased drastically. There was a significant difference (p < 0.05) between all formulations, except between Formulations A and B.

The WVP values of the bio-nanocomposite films produced in the present study were slightly lower than the WVP values of chicken skin gelatin-tapioca starch composite film (3.34 × 10⁻⁷ g mm cm⁻² h^−1 Pa−1) (Loo and Sarbon 2020). The lower WVP of bio-nanocomposite films was due to formation of hydrogen bonds between chicken skin gelatin, tapioca starch and zinc oxide. Leading to the formation of a compact structure with smaller inter-chain spaces in the film matrix. The compact structure of bio-nanocomposite films reduced the diffusion of water from the environment through the films. However, at 5% zinc oxide concentration, WVP was found to be increased significantly. This increase in WVP in this study might be due to uneven distribution of nanoparticles in bio-nanocomposite film, leading to the aggregation of nanoparticles in the film. Mueller et al. (2011) explained that the dispersion of large particle of zinc oxide in the film matrix is relatively poor as compared to nanometric size zinc oxide, thus reducing the barrier properties of the nano-composite film.

The findings in this study were similar to the results obtained by Arfat et al. (2014) for fish skin gelatin/fish protein isolate film with different concentrations of zinc oxide nanoparticles incorporation. The difference in WVP value may be due to different components used in the films, leading to the formation of different bonding between the main components, as well as components with nanoparticles. The water barrier properties of the film were improved when nanoparticles were added, as this formed a more compact structure and reduced water vapor diffusion rate through the film (Arfat et al. 2014). A tortuous pathway was formed when nanoparticle was added into the film, which resists the permeation of water vapor through the film.

The gelatin film in this study had lower WVP values (1.24–1.93 × 10⁻⁷ gmm/cm²hPa) as compared to the reported results for commercial PVC film, which recorded a value of 2.61 × 10⁻⁷ gmm/cm²hPa (Kaewprachu et al. 2015). This might be due to the presence of zinc oxide nanoparticles dispersed in the polymer matrix which contributed to the delayed transmission of water vapor through the matrix.

Mechanical properties

The effects of zinc oxide nanoparticles concentration on tensile strength (TS) and elongation at break (EAB) of chicken skin gelatin-tapioca starch composite film are presented in Table 2. The reinforcement of zinc oxide nanoparticles ranged from 0 to 5% on the film formulation caused a significant increase in TS value of bio-nanocomposite film whereas the EAB value of film decreased. There was a significant increase (p < 0.05) in TS values between all formulations, except between Formulations B and C.

Table 2.

Tensile strength and elongation at break of chicken skin gelatin-tapioca starch composite film at different concentration of zinc oxide nanoparticles

Film formulation	Tensile strength (MPa)	Elongation at break (%)
A	22.96 ± 0.65c	71.37 ± 1.96a
B	31.08 ± 0.89b	50.19 ± 4.36b
C	34.24 ± 3.30b	50.34 ± 2.92b
D	50.43 ± 0.59a	20.56 ± 1.86c

A is film with 0% zinc oxide nanoparticles; B is film with 1% zinc oxide nanoparticles; C is film with 3% zinc oxide nanoparticles; and D is film with 5% zinc oxide nanoparticles. Data reported are mean ± standard deviation

The different superscript letter^abc within column indicate significance different (p < 0.05)

The increasing trend in TS value of chicken skin gelatin/tapioca starch composite films was attributed to the reinforcement of zinc oxide nanoparticles in the composite films that allow the formation of stronger intermolecular forces between the polymer chains (Al-Hassan and Norziah 2012). The molecular force increases when zinc oxide nanoparticles react with the hydroxyl group of gelatin and starch to form both hydrogen bonds and covalent bonds (Marvizadeh et al. 2017). The nano-filler with greater surface area promoted the interaction between nanoparticles and polymer matrix and thus enhanced the formation of cross linking between polymer chains (Rhim 2011). Thus, films with high concentration of zinc oxide nanoparticles showed a more compact film matrix structure, leading to the formation of stronger films (Al-Hassan and Norziah 2012).

The TS values of bio-nanocomposite films in the current study (22.90–50.43 MPa) were higher than chicken skin gelatin (0.98 MPa) (Nazmi et al. 2017). The TS values of chicken skin gelatin/tapioca starch composite films reached a maximum value of 50.43 MPa at 5% zinc oxide nanoparticles concentration. The improvement in TS of chicken skin gelatin-tapioca starch composite films may be due to the formation of strong intermolecular bonding. Similarly, Marvizadeh et al. (2017) showed that the TS of bovine gelatin/tapioca starch/zinc oxide nanorod increased with increasing zinc oxide nanorod concentration. However, the TS value for bio-nanocomposite films in the present study was higher than the TS values of bio-nanocomposite films produced by Marvizadeh et al. (2017) at 34.24 and 18.69 MPa, respectively, when 3.0 and 3.5% of zinc oxide nanoparticles were added. This might be due to the higher level of imino acids found in chicken skin gelatin as compared to bovine gelatin that encourage the formation of triple helix structure between the polymer chains (Sarbon et al. 2013). The development of a triple helix structure improves the film-forming properties of gelatin. In addition, the TS value of chicken skin gelatin-tapioca starch composite films have recorded slightly lower TS values (22.96–50.43%) as compared to PVC commercial films (4.50–59.50 MPa) (Kaewprachu et al. 2015; Rawdkuen et al. 2020). The incorporation of zinc oxide nanoparticles most likely acts as a reinforcing filler in the gelatin matrix, thereby enhancing the strength of the resulting films (Arfat et al. 2014). Thus, chicken skin gelatin composite films can compete with synthetic packaging because of their comparable strength and strong potential for use as a substitution for plastics in the film packaging sector.

The reduction in EAB values of chicken skin gelatin-tapioca starch composite films was caused by the incorporation of zinc oxide nanoparticles in the films, which restricts the movement of macromolecules and decreases the elongation ability of the films (Kumar et al. 2010). The incorporation of nanoparticles in film enhances the formation of hydrogen bond between the nanoparticles and components in film, resulting in the formation of compact structure (Sahraee et al. 2017). The reinforcement of nanoparticles in film reduces the availability of glycerol to interact with starch and gelatin (Mueller et al. 2011). The nanoparticles in film become associated with glycerol, and thus glycerol loses its plasticizing effect.

Similar trends of decreasing EAB value with the increasing nanoparticles concentration have been reported by Marvizadeh et al. (2017) (bovine gelatin-tapioca starch-zinc oxide films) and Arfat et al. (2017) (fish skin gelatin films incorporated with silver-copper nano-particles). As compared to the EAB values of films based on bovine gelatin/tapioca starch/zinc oxide (8.02–18.45%) and fish skin gelatin/silver-copper nanoparticles (54.20–32.90%), the EAB values of bio-nanocomposite films in the current study were higher, ranging from 20.56 to 71.37%. Higher EAB values were obtained by chicken skin gelatin based films, perhaps due to different sources of gelatin containing different amounts of amino acid (Nazmi et al. 2017). The hydroxyl groups in chicken skin gelatin improve the intermolecular interaction between the chicken skin gelatin, tapioca starch and zinc oxide nanoparticles. However, as compared to PVC commercial film, the chicken skin gelatin composite films have resulted in lower EAB value which were recorded at 2.50–241.94% and 20.56–71.73%, respectively (Kaewprachu et al. 2015; Rawdkuen et al. 2020). The reduction of film extensibility may be attributed to the restricted motion of gelatin molecules due to interfacial interactions between the gelatin and zinc oxide nanoparticles.

Functional group by Fourier Transform Infrared Spectroscopy (FTIR)

The structural properties of gelatin films were determined using Fourier Transform Infrared Spectroscopy (FTIR). Based on Table 3, FTIR spectra of chicken skin gelatin/tapioca starch composite films incorporated with different concentrations (0–5%) of zinc oxide nanoparticles showed that amide A, amide I, amide II and aliphatic alcohol bands were detected with wavelengths in a range from 3285.53–3292.62 cm⁻¹, 1628.86–1632.68 cm⁻¹, 1542.95–1552.15 cm⁻¹ and 1138.56–1159.70 cm⁻¹, respectively.

Table 3.

FTIR bands of chicken skin gelatin/tapioca starch composite film at different concentration of zinc oxide nanoparticles

Film formulations	Amide A (cm−1)	Amide I (cm−1)	Amide II (cm−1)	Aliphatic Alcohol (cm−1)
	Stretching vibration of C–N bands and NH groups of bound amide, vibration of C–H groups of glycine	C=O stretching	Bending vibration N–H group, stretching vibration of C–N group	Hydroxyl group (–OH), CO stretching
A	3292.08 ± 0.27a	1629.28 ± 0.03abc	1542.95 ± 1.65a	1139.64 ± 0.08a
B	3292.62 ± 0.49a	1629.25 ± 0.12abc	1543.04 ± 1.59ac	1139.74 ± 0.18a
C	3289.11 ± 9.23a	1632.68 ± 3.82b	1552.15 ± 7.05bc	1159.70 ± 14.82b
D	3285.53 ± 12.22a	1628.86 ± 0.74c	1551.24 ± 4.72c	1138.56 ± 1.49a

A is film with 0% zinc oxide nanoparticles; B is film with 1% zinc oxide nanoparticles; C is film with 3% zinc oxide nanoparticles; and D is film with 5% zinc oxide nanoparticles. Data reported are mean ± standard deviation

The different superscript letter^abc within column indicate significance different (p < 0.05)

Amide A band is generally associated with the N–H stretching vibration and shows the existence of hydrogen bonds (Nazmi et al. 2017). The addition of zinc oxide nanoparticles into chicken skin gelatin/tapioca starch composite films as shown in Table 3 have shifted toward a lower wavelength, from 3292.08 to 3285.53 cm⁻¹, as compared to the gelatin film control. The formation of intramolecular forces such as hydrogen bond between zinc oxide nanoparticles with chicken skin gelatin has occupied the functional group of gelatin matrix, thus lowering free hydrogen group that can form hydrophilic bonding with water and causing a shift to a lower wavelength. These results are in agreement with findings by Nazmi et al. (2017) which observed a lower wavelength due to intermolecular interaction between hydroxyl group from gelatin and carboxyl group from CMC. In addition, when the NH group of a peptide is involved in a hydrogen bond, the position is shifted to lower frequencies (Duan et al. 2009).

The amide I band, with characteristic frequencies in the range of 1600–1700 cm⁻¹, is mainly associated with the stretching vibrations of the carbonyl groups (C=O bond) along the polypeptide backbone and is a sensitive marker of polypeptide secondary structure like gelatin (Nazmi et al. 2017). For the amide I band, the absorption peak of control gelatin film resulted in higher value as compared to absorption peak of chicken skin gelatin/tapioca starch composite films incorporated with zinc oxide nanoparticles except for film added with 3% of zinc oxide nanoparticles. The addition of zinc oxide nanoparticles introduced more intramolecular forces such as hydrogen and covalent bonding due to hetero molecular interaction on the C=O group and thus resulted in a lower wave number.

Meanwhile, amide II is associated with N–H bending coupled with C–N stretching (Suderman et al. 2018). The incorporation of zinc oxide into gelatin-based film have shifted to higher value which were observed within the range of 1543.04–1552.15 cm⁻¹ as compared to control chicken skin gelatin film (1542.95 cm⁻¹). The changes might be due to the interaction and cross-linking of proteins in the gelatin with the zinc oxide nanoparticles which altered the secondary structure of gelatin polypeptide chains. A similar occurrence was noted by Nazmi et al. (2017), who observed that chicken skin gelatin film incorporated with CMC shifted to a higher value as compared to single gelatin film due to alteration of the secondary structure of gelatin polypeptide chains caused by the addition of CMC.

The aliphatic alcohol group content in the film reflected the position of the OH stretch absorption and CO stretching (Suderman et al. 2018). The results demonstrated that all chicken skin gelatin composite films showed no shifts as compared to the control films, except for the film with 3% zinc oxide nanoparticles. This might be due to the similar hydroxyl groups composition of chicken skin gelatin, tapioca starch and glycerol present in each film formulation. The results were in line with those of a study by Suderman et al. (2018), which found no significant differences in aliphatic alcohol groups between gelatin films due to the standardized amount of glycerol used.

Antibacterial properties

Table 4 shows the inhibition zones of chicken skin gelatin/tapioca starch/zinc oxide bio-nanocomposite films against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The result obtained showed that the concentration of zinc oxide nanoparticles in film formulations influenced the inhibition zone of both S. aureus and E. coli. The diameter of film is 4.5 mm. Zone of inhibition for both S. aureus and E. coli was increased as concentration of zinc oxide nanoparticles increased. Chicken skin gelatin/tapioca starch composite film (Formulation A) did not show any inhibitory effect on S. aureus and E. coli. In contrast, maximum antibacterial activity against S. aureus and E. coli was achieved when 5% of zinc oxide nanoparticles were incorporated in the film formulation, showing maximum zones of inhibition of 16–20 mm and 15–20 mm, respectively.

Table 4.

Inhibitory zone of chicken skin gelatin/tapioca starch composite film at different concentration of zinc oxide nanoparticles against Gram-positive bacteria and Gram-negative bacteria

Film formulation	Zone of inhibition (mm)
	Gram-positive bacteria (S.aureus)	Gram-negative bacteria (E.coli)
A	0	0
B	8–11	8–10
C	10–15	11–15
D	16–20	15–20

A is film with 0% zinc oxide nanoparticles; B is film with 1% zinc oxide nanoparticles; C is film with 3% zinc oxide nanoparticles; and D is film with 5% zinc oxide nanoparticles. Data reported as range in mm

The increment of the inhibitory zone for both gram-positive and gram-negative bacteria was attributed by the reinforcement of nano-sized zinc oxide in film formulations which facilitate the penetration of zinc oxide through the cell wall and cell membrane of bacteria. The size of zinc oxide eases its penetration through bacterial cell walls, followed by effective interaction with the interior components of bacteria, thus enhancing the antibacterial properties of the films (Azeredo 2009). Zn²⁺ ions have a lethal effect on bacterial cells, as they disrupts the enzymatic system of the bacteria and inhibit both the active transport and amino acid metabolism of the bacteria (Sirelkhatim et al. 2015).

The similar trend of increasing inhibitory zone with the addition of nanoparticles in film formulation was showed by Ji et al. (2016) (corn starch/zinc oxide bio-nanocomposite films), Agustin and Padmawijaya (2017) (banana peel starch/chitosan/zinc oxide bio-nanocomposite films) and Arfat et al. (2015) (fish skin gelatin/fish protein isolate/zinc oxide bio-nanocomposite films). The researchers demonstrated that the antimicrobial effect of bio-nanocomposite films increased when more nanoparticles were added to the film. The inhibitory effect of bacteria may be explained using several mechanisms, including the destabilization of microbial membrane by direct contact of nanoparticles with bacteria, release of zinc ions and formation of ROS (Sirelkhatim et al. 2015). The production of zinc ions and ROS interrupts the metabolism of the bacteria and contributes to the antimicrobial properties of the film.

Conclusion

In conclusion, the properties of chicken skin gelatin/tapioca starch composite films were significantly influenced by the addition of zinc oxide nanoparticles. The reinforcement of zinc oxide nanoparticles improved the films antibacterial and thermal properties, as well as water resistance. Additionally, the addition of zinc oxide nanoparticles increased tensile strength (TS) while reducing elongation at break (EAB). Overall, the film with 3% of zinc oxide nanoparticles was found to have the optimal formulation, since it has the highest melting temperature, the lowest water vapor permeability (WVP), and moderate TS and EAB. The structures of the composite films were also strengthened by the presence of amide A, amide I, amide II and aliphatic alcohol. Therefore, the incorporation of zinc oxide nanoparticles can be used to enhance physical, mechanical and antibacterial properties of chicken skin gelatin/tapioca starch composite films, making these composite films suitable for packaging materials in the food industry.

Author contributions

Sarbon NM was involved in the conceptualization, designed and supervision of the research. Lee, SW and Said NS carried out the laboratory experiments and investigations and analysed the data. All authors contributed to writing the paper. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The Authors declare no Competing Financial or Non-Financial Interests.