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Development of Bio-nanocomposite Films Made from Pectin/ZnO Nanoparticles to Inhibit Fungal Growth on Strawberry Fruit

Nugraha Edhi Suyatma1,a, Yutaka Ishikawa2,b, and Hiroaki Kitazawa2,c 1Department of Food Science and Technology, Bogor Agricultural University, Bogor, 16680, Indonesia 2Food Packaging Laboratory, National Food Research Institut, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642 Japan Corresponding author: nugrahaedhi@ ipb.ac.id, Tel. +62-813-86 035 135

Keywords: Bio-nanocomposite film; Pectin; ZnO nanoparticles; Strawberry fruit;

Abstract The main objective of this study was to develop bio-nanocomposite

film/coating made from pectin as film matrix and zinc oxide nanoparticles (ZnO-NPs) as nano filler. Pectin/ZnO bio-nanocomposite films were fabricated at 5 levels of ZnO-NPs, i.e., 0.0, 0.5, 1.0, 2.0 and 5.0% (w/w). The effects of ZnO-NPs incorporation on improving the mechanical properties and water resistance of the films were investigated. ZnO-NPs were successfully incorporated into pectin films by nanodispersion technique followed by casting method. The presence of ZnO-NPs inside pectin films was observed clearly by SEM. The improvement in tensile strength could be achieved with ZnO-NPs incorporation without obvious loss in elasticity. Potential antimicrobial activity of pectin- ZnO nanocomposite films was proved in the absence of mold after exposing them at 97% RH and room temperature for 14 days, whereas the growth of mold had been observed in pure pectin film after 3 days of exposure. Results suggested that it would be favorable to prepare pectin/ZnO nanocomposite film by using ZnO-NPs at the amount of 2% (w/w) in the future application. The application of pectin-ZnO nanocomposite as edible coating of strawberry could inhibit the mold decay until two weeks at 5oC of storage, a week longer than that of control.

Introduction In recent years, antimicrobial packaging has attracted much attention from the

food industries thanks to the increase in consumer demand for preservative-free products [1]. Moreover, there has been a growing interest in developing biodegradable packaging materials to replace petroleum based polymers [2]. As a by-product of fruit processing industries, biopolymer pectin is both inexpensive and abundantly available, which is thus an excellent candidate for applying to eco- friendly biodegradable packaging. Unfortunately, films prepared with pure pectin did not provide satisfactory functionality due to lack of mechanical properties and water resistance.

Frequently applied method to improve the strength and water resistance as well as barrier properties of the natural polymers is to blend them with another polymer. Previous work appeared to blending pectin with starch [3], polyvinyl alcohol [4], soy flour [5], gelatin [6], chitosan and hydroxypropyl methylcellulose [7], and ethylcellulose [8], but all these blends did not exhibit preferable results in terms of water resistance and mechanical strength improvements. Hence, there is a challenge to improve functional packaging

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properties of pectin films by a new technique, i.e. nanoreinforcement by using nanoparticles as reinforcing agent.

Sorrentino et al. [9] have reported that nano-reinforcements are useful for biopolymers, because of their usually poor performance when compared to conventional petroleum-based polymers. The incorporation of nano-sized reinforcements to biopolymers may open new possibilities for improving not only their properties but also their cost-price-efficiency. Nanocomposites represent a new alternative to the neat polymers and conventional composites for improving polymer properties, such as barrier capability, mechanical strength, as well as potential bioactively antimicrobial and antioxidant effects [10].

Nanocomposites consist of a polymer matrix or continuous phase and a discontinuous phase or filler, with at least one dimension smaller than 100 nm [10]. Recently, zinc oxide nanoparticles (ZnO-NPs) have been paid great attentions as a kind of nano fillers. ZnO is a safe chemical substance, which has already been commonly used as a source for Zn supplement and fortification in the food industry [11]. Furthermore, it was found that ZnO-NPs exerted antimicrobial activities. He and co-workers [12] reported the antifungal activities of ZnO-NPs against Botrytis cinerea and Penicillium expansum, between which the former might cause severe post-harvest diseases for fruits and vegetables. ZnO-NPs also showed bacteriostatic activities against Escheriachia coli [13] and Lactobacillus plantarum [14]. Thanks to its beneficial properties, ZnO-NPs were selected as nano fillers into pectin films in thi present study. Strawberry fruit was selected as application target of the obtain bio-nanocomposite coating due to its high metabolic activity and suffers from high possibility of microbial contamination, resulting in short shelf life, fungal decay, colour change, and off-flavour.

The main goal of this study is to enhance functional packaging properties of pectin film prepared by blending pectin with zinc oxide nanoparticles (ZnO-NPs). Pectin/ZnO bio-nanocomposite films were fabricated at 5 levels of ZnO-NPs, i.e., 0.0, 0.5, 1.0, 2.0 and 5.0% (w/w). The effects of ZnO- NPs incorporation on improving the mechanical properties and water resistance of the obtained films were investigated. Furthermore, the application of bio-nanocomposite coating as edible coating was tested to inhibit fungal growth on Strawberry fruit.

Materials and Method Materials. Pectin from citrus peel was purchased from Wako Pure Chemical

Industries Ltd (Japan) in powder form. Zinc oxide (ZnO) nanoparticles with an average particle size of 20 nm and other chemicals, were also purchased from Wako Pure Chemical Industries Ltd (Japan).

Preparation of Pectin-ZnO Nanocomposite films. Pure pectin film was prepared by dispersing pectin in distilled water (8 g/400 g water). Pectin-ZnO nanocomposites were fabricated at 4 levels, of ZnO nanoparticles (0.5, 1.0, 2.0, and 5.0%, w/w). When preparing 1.0% of ZnO nanoparticles in 8 g of pectin, 80 mg of ZnO nanoparticles were dispersed in distilled water, intended as pectin solvent, and homogenized using a high-speed homogenizer (Kinematica GmbH, Kriens/Luzern, Switzerland). Particle size of dispersed ZnO-NPs in water was observed with Laser Diffraction Particle Size Analyzer (Model SALD-2100, Shimadzu, Japan). After that, pectin powder was added into solution containing ZnO-NPs. In this blending system, pectin polymer could serve as capping agent of ZnO-NPs to avoid aggregation. The final solutions were poured into Poly (tetra fluoro ethylene) (PTFE) mold with a size of 25 x 25 cm2, followed by oven drying at

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45oC. The dried films were peeled off from the casting surface and preconditioned at room temperature for 3 days prior to testing.

Measurement of Opacity. The opacity of the films were determined in triplicate using X-Rite SP60 (Color Techno System Co., Japan). Opacity was calculated by the software of the equipment (X-Rite SP60), as the relationship between the opacity of each sample over the black standard and that over the white standard.

Scanning Electron Microscopy. Scanning electron micrographs were obtained using a Jeol JSM-5600LV (JEOL, Japan). Film specimens were scratched on the top surface before being mounted on aluminium stub covered with double-sided carbon tape and sputter coated with gold to enhance surface conductivity. All specimens were viewed in a scanning electron microscope at 5 kV.

Measurement of Mechanical Properties. Mechanical properties of the films were determined using a tensile testing machine (Instron 4411, U.S.A.). ASTM D882 method (ASTM Intl. 2001) was used with minor modifications for measuring tensile strength (TS) and percent elongation at break (E%). The thickness of the film specimens were measured with a digital micrometer (Digimatic Micrometer, Mitutoyo, Japan) prior to testing, which was needed to calculate the cross- sectional area. During tensile testing, each film specimen was mounted between the grips of the tensile testing machine, and tested with an initial grip separation of 30 mm and a cross-head speed of 1 mm/s. Measurements were repeated at least 7 times. A microcomputer was used to record the stress–strain curves. The tensile strength was expressed as the maximum force at break per initial cross sectional area of the film and the elongation as a percentage of the original length.

Measurement of Water Absorption. Samples with a size of 3 × 3 cm2 were first preconditioned in a desiccator containing P2O5 for 3 days before testing. After weighing (Mo), they were placed in a desiccator containing saturated K2SO4 solution to ensure a RH of 97%. The samples were weighed using a four-digit balance (0.0000 mg) until a constant weight was reached (Mf). The water absorption (WA) of the samples was calculated as: WA (%) = [(Mf-Mo)/Mo] X 100; where Mf is the weight of the sample after exposure at 97% RH when a constant weight is reached, and Mo is the weight of the sample before exposure.

Application trial as edible coating. Bio-nanocomposite solution prepared by mixing of pectin and 2% of ZnO-NPs was intended as edible coating of fresh strawberry fruit. Strawberry fruits from local market (Tsukuba, Japan) with uniform colour and size were selected and used for the experiment.

Statistical analysis. SPSS (Statistical Product and Service Solutions) version 17.0 was used for all statistical analyses. A one-way ANOVA and Duncan post hoc test, when needed, were used to describe results at a significance level of p<0.05. Values are presented as the average from at least three measurements.

Results and Discussion The ZnO nanoparticles used in this study have the average diameter of 20 nm

according to the producer. Particle size distribution of the dispersed ZnO-NPs in distilled water is presented in Fig. 1 with the median found at around 25 nm. It seems there was a slightly agglomeration of ZnO nanoparticles in water after dispersing process so there were ZnO-NPs having diameter size of 100 nm.

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Film Opacity To elucidate the transparency of the obtained films, % opacity (OP) was measured.

OP values of the films are shown in Fig. 2. From statistical analysis, it was found that the incorporation of ZnO- NPs affected significantly opacity of the pectin films (p<0.05). However, the significant effect was only found in the film containing 5% of ZnO-NPs.

Scanning Electron Microscopy (SEM) In an attempt to verify the presence of ZnO-NPs in the pectin films, scanning

electron microscopy (SEM) was used to visualize the surface of all prepared pectin-ZnO nanocomposite films. To observe ZnO-NPs inside the films, the top surface of the films were scratched gently prior to observation (Fig. 3A). The observation of cross-section films was technically very difficult due to the thinness of the films obtained in this work. Fig. 3B represents the microstructure of pectin film without ZnO-NPs. There was no round particle observed in this picture, indicating no ZnO- NPs inside. From Fig. 3C-F, round particles were clearly observed, which suggested that ZnO-NPs were successfully incorporated into the pectin films. However, the diameter of ZnO particles inside the pectin films seemed larger than 100 nm, which could be tentatively attributed to the case that the observed ZnO-NPs in Fig. 3C-E might be coated by pectin as well as gold and were not in pure condition. It was also found that more ZnO particles were observed in the films with increasing amounts of ZnO-NPs added (Fig. 3B-F), indicating that more ZnO-NPs used could result in more incorporation of the nanoparticles into the films under the present concentration tested in this work.

Mechanical properties It was expected to improve the tensile strength of pectin films by incorporating

with ZnO-NPs. Tensile strength (TS) is a measure of integrity and heavy-duty use potential of the films, and elongation at break (EB) is a quantitative representative of the ability to stretch for the films. Results of the tensile tests are shown in Fig. 4. Statistical analysis revealed that the incorporation of ZnO-NPs was highly significant factors to the mechanical properties of the tested films (p< 0.01). The incorporation of ZnO- NPs could raise TS and slightly reduce EB of pectin films.

Water absorption Water absorption can be used as a parameter to evaluate water sensitivity of the

materials. Generally, the polymer with higher water absorption is more sensitive to water. Fig. 5 shows water absorption of the obtained films. It was observed that the water absorption of pectin films declined with increasing incorporation of ZnO-NPs indicating that it was beneficial water sensitivity reduction of the films by incorporating with ZnO-NPs. In addition, statistics showed that the decrease of water absorption was significantly achieved while using relatively higher addition of ZnO-NPs, that is, the amounts of 2.0 and 5.0% tested in the present work.

Potency of antimicrobial activity of the nanocomposite films were observed during water absorption test, stored at high humidity (RH > 97%) which was a favorable environment for the growth of fungi. It was noticeable that no growth of fungi was observed on the surface of pectin films containing 1.0; 2.0 and 5.0% of ZnO-NPs, whereas intensive fungal growth occurred on the surface of pectin films without ZnO-NPs. On the surface of pectin films containing 0.5% of ZnO- NPs, the fungi proliferated slightly on the films (Fig. 6).

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Application trials as Strawberry coating As additional experiment, we have applied pectin solution containing 2% of ZnO-NPs as edible coating of fresh strawberry fruit (Fig. 7). The results showed that in pectin-ZnO coated strawberry there was no coincidence of fungal growth until 7 days after stored at 5oC whereas in uncoated and pure pectin coated strawberry fruits the fungal growth was observed after 3 days of storage. All strawberry fruits without coating and with pure pectin coating were not acceptable to consume after 7 days of storage.

Conclusion ZnO-NPs were successfully incorporated into pectin films by nanodispersion

technique and casting method. The presence of ZnO-NPs inside pectin films was observed clearly by using SEM. The improvement in tensile strength has been achieved significantly with 0.5% of ZnO-NPs without too much lost in elasticity. Water absorption of pectin-ZnO nanocomposite films was lower than that of pure pectin film indicating water resistance of films has been improved. Potential antimicrobial capacity of pectin-ZnO films was proved with the absent of mold formation whereas it has been observed in pure pectin films after 3 days of exposition at high humidity condition (RH 97%). The significant change in film opacity was only found in the use of 5% of ZnO as nanofiller. Thus, it is suggested in using ZnO-NPs at the amount of 2% (w/w) in fabrication of pectin/ZnO bio- nanocomposite films.

Acknowledgements This research has been carried out at National Food Research Institute (NFRI),

Tsukuba, Japan under the United Nations University-Kirin Fellowship Programme in 2012-13, sponsored by Kirin Holdings Co. Ltd. Tokyo, Japan.

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Figures

Figure 1. Particle size of dispersed ZnO-NPs

in water. Figure 2. Opacity of film pectin/ZnO

nanocomposites.

Figure 3. SEM photos of pectin-ZnO nanocomposite films. A represents the top surface

of the film after gently scratching, B represents pure pectin film. C, D, E and F represent pectin films containing 0.5, 1.0, 2.0, and 5.0% of ZnO-NPs,

respectively.

Figure 4. Mechanical properties of film

pectin/ZnO Figure 5. Water absorption of film

pectin/ZnO

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Figure 6. Inhibition of fungal growth on the surface of pectin films containing ZnO-NPs

during water absorption test.

Day

0

Control (without coating) Coating with pectin Coating with pectin-ZnO

Day

7

Figure 7. Appearance of strawberry fruit after seven days of storage at temperature of

5oC.

0% ZnO-NP 0.5% ZnO-NP 1.0% ZnO-NP 2.0% ZnO-NP 5.0% ZnO-NP