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Preparation of antimicrobial active packaging film by capacitively coupled plasma treatment
LWT - Food Science and Technology 117 (2020) 108612
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Preparation of antimicrobial active packaging film by capacitively coupled plasma treatment
T
Li-Wah Wonga,1, Chih-Yao Houb,1, Chun-Chi Hsiehc, Chao-Kai Changd, Yi-Shan Wua, Chang-Wei Hsieha,e,∗ a
Department of Food Science and Biotechnology, National Chung Hsing University, 145 Xingda Rd., South Dist., Taichung City 402, Taiwan, ROC Department of Seafood Science, National Kaohsiung University of Science and Technology, 142, Haizhuan Rd., Nanzi Dist., Kaohsiung City, 81157, Taiwan, ROC c Graduate Institute of Biotechnology, National Chung Hsing University, 145 Xingda Rd., South Dist., Taichung City 402, Taiwan, ROC d College of Biotechnology and Bioresources, Da-Yeh University, 168 University Rd., Dacun, Changhua City 515, Taiwan, ROC e Department of Medical Research, China Medical University Hospital, Taichung City 404, Taiwan, ROC b
A R T I C LE I N FO
A B S T R A C T
Keywords: Active packaging Low density polyethylene Gallic acid Antimicrobial activity
This research is mainly focused on preparing gallic acid (GA) coated, antimicrobial, active packaging, low density polyethylene (LDPE) films by capacitively coupled plasma. Firstly, the suitable plasma treatment condition was 30 W treated for 60 s from the result of appearance and tensile strength. From the result of SEM and ATR-FTIR, the surface roughness increased which facilitated the GA coating and the surface turn smoother after GA coating. In the result of antimicrobial activity, these films is able to reduce the growth of Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus about 0.5–0.6 log CFU/mL when GA concentration is above 1%. Between these two microorganisms, the film has more significant inhibition on E. coli than S. aureus due to cell membrane structure differences. These results are first stage in developing antibacterial active packaging for foods preservation.
1. Introduction Active packaging is defined as “packaging in which subsidiary constituents have been deliberately included in or on either the packaging material or the package headspace to enhance the performance of the package system” (Paola Appendini, 2002). It can prolong the shelf life of products by adjusting the gas ratio, humidity, and antioxidant and antimicrobial properties. Among all kinds of active packaging, antimicrobial packaging is one of the most studied fields that can inhibit or even kill bacteria and thus enhance safety and extend shelf life. Antimicrobial packaging can take several forms such as incorporation of volatile and non-volatile antimicrobial agents directly into polymers, coating antimicrobials onto polymer surfaces, and immobilization of antimicrobials to polymers by ion or covalent linkages (Lee, 2010; Paola Appendini, 2002; Stefania Quintavalla, 2002; Vartiainen, Rättö, & Paulussen, 2005; Zhang et al., 2006). During sale and storage periods, the antimicrobial will be slowly released from the packaging material and inhibit the microorganisms in the foods (Higueras, Lopez-Carballo, Hernandez-Munoz, Gavara, & Rollini,
2013), and extend the shelf life of foods (Stefania Quintavalla, 2002). Polyethylene (PE) films are the most widely used polyolefin among many types of polymers due to their good chemical resistance, transparency, flexibility, and low cost (Zheng et al., 2016). However, polyethylene is chemically inert material and does not possess any functionality, which limits its application in active packaging (Ozdemir, Yurteri, & Sadikoglu, 1999; Theapsak, Watthanaphanit, & Rujiravanit, 2012). So, surface modification of PE film is an important pretreatment to activate PE's surface and able to bonding with preservatives to create an active packaging (Ozdemir et al., 1999). Surface modification can improve the applicability of plastics by affecting the topmost layer of the surface (Govindarajan & Shandas, 2014). In order to modify the polymer surface, there are many kinds of methods such as wet chemical modification, plasma treatment, and UV radiation (Lee, 2010). Wet chemical modification is commonly used in industry, which requires a chemical reagent such as a strong acid or a strong base to create reactive functional groups on the surface of a polymer (Govindarajan & Shandas, 2014; L.; Karam et al., 2016; Ozdemir et al., 1999). However, the waste discharged is not
∗
Corresponding author. Department of Food Science and Biotechnology, National Chung Hsing University, 145 Xingda Rd., South Dist., Taichung City 402, Taiwan, ROC. E-mail address: [email protected] (C.-W. Hsieh). 1 These authors contributed equally to this study and share first authorship. https://doi.org/10.1016/j.lwt.2019.108612 Received 19 April 2019; Received in revised form 8 August 2019; Accepted 9 September 2019 Available online 10 September 2019 0023-6438/ © 2019 Published by Elsevier Ltd.
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The chamber was pumped down to 0.0643 Torr and the air gas was introduced into the chamber with 5 sccm gas flow. When the pressure become constant, switch on the power source and adjust to certain power value. They were treated with different power (0–90 W) and treatment time (15–150 s) to determine the most appropriate treatment condition. Later, the LDPE films were treated according to the evaluation result of mechanical properties. After plasma treatment, the LDPE films were immediately immersed in different concentrations of GA solution with continuous stirring for 24 h, followed by washing for 3 cycles with distilled water to remove excess GA and then air dried at room temperature overnight (Theapsak et al., 2012).
environmentally friendly. Plasma contains photons, electrons, molecules, atoms, and free radicals, which destroy covalent bonds and initiate many chemical reactions (Kim, Lee, & Min, 2014; Riccardi et al., 2003; Shao et al., 2010). Besides, plasma treatment can also insert new functional groups on the surface, improve hydrophilicity and affect mechanical properties which has proven to increase the applicability of polymeric materials and convert them into higher value products (De Geyter, Morent, Leys, Gengembre, & Payen, 2007; Honarvar et al., 2017; Chan, Ko, & Hiraoka, 1996; Wongsawaeng, Khemngern, & Somboonna, 2017). In the study of plasma-modified polymer films, low-pressure plasma is a kind of plasma treatment that is used in the mainstream because it has a low power input and can generate plasma with a small flow of gas which in turn consumes less energy and processes more efficiently than atmospheric plasma. In addition, in a vacuum environment, the concentration of reactive species is higher, and electrons have a larger average free path (0.1 cm at low pressure and ~5.7 × 10−8 cm at atmospheric pressure) with higher energy (Andreas Schutze et al., 1998; Kikani et al., 2013). In addition, plasma treatment is relatively environmentally friendly compared to wet chemical modification (Honarvar et al., 2017). Few studies of antibacterial packaging bags developed using plasma treatment and coating an antibacterial agent such as lysozyme or sodium benzoate have been performed. These methods can effectively inhibit the growth of microorganisms, thereby extending the shelf life of food (Lee, 2010). The most common problem in food packaging is microbial recontamination in subsequent processing (Fernandez-Saiz, Lagaron, Hernandez-Munoz, & Ocio, 2008), which degrades the quality of the product and even harms the health of consumers. Common foodborne pathogens include Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Such microorganisms can cause food poisoning, causing symptoms such as vomiting and diarrhea. Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA) is a biologically active phytochemical that is widely found in plants, nuts, and teas (Arcan & Yemenicioğlu, 2009; Lu, Nie, Belton, Tang, & Zhao, 2006). GA has an inhibitory effect on foodborne pathogen - E. coli and S. aureus. (Chanwitheesuk, Teerawutgulrag, Kilburn, & Rakariyatham, 2007). Also, it has been suggested to possess strong antioxidant activities and is able to provide protection against oxidative damage cause by free radicals such as hydroxyl (HO•) and hydrogen peroxide (H2O2). Therefore, this study aimed to improve the surface activity of PE film by plasma treatment technology to bond with GA, develop an optimum condition for creating an active packaging film containing GA, and evaluate its antimicrobial effect on E. coli and S. aureus.
2.3. Characterization of the film 2.3.1. Tensile strength This experiment was carried out according to (Theapsak et al., 2012) with slight modifications. Tensile strength of the untreated and plasma-treated films was detected by a texture profile analyzer (TA.XT2i, Stable Micro System, Haslemere, Surrey, UK) at 25 °C. The films were cut into a rectangular shape (1 cm × 3 cm) and equipped with a 500 N load cell. A strain rate of 10 mms−1 and a gauge length of 100 mm were employed according to ASTM D882-91 standard test method. 2.3.2. Surface morphologies and chemical composition of the film The morphologies of the films were observed by a scanning electron microscope (TM 1000, Hitachi, Japan). Before testing, all the samples were dried, attached to a double-sided carbon tape with a conducting sample holder and coated with gold under vacuum using a sputtering system (Sadeghnejad, Aroujalian, Raisi, & Fazel, 2014). Surface chemical composition of the films was observed using an attenuated total Reflection-Fourier transform infrared spectroscope (ATR-FTIR, Thermo Nicolet 6700). The ATR-FTIR spectra were investigated between wavenumber 4000 to 650 cm−1 with 64 scans at a resolution of 4 cm−1 (Theapsak et al., 2012). 2.4. Assessment of the antimicrobial activity of the film The antimicrobial activity of the samples against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) was determined qualitative and quantitatively. The cell cultures were performed by inoculating 100 μL of the pre-cultures in 10 mL nutrient broth and incubated at 37 °C for 24 h. The cell cultures of each microorganism were then diluted using 0.85% sterile NaCl aqueous solution by a factor of 103 and 104 for qualitative and quantitative antibacterial assessments (L. Karam et al., 2016; Theapsak et al., 2012). The qualitative antibacterial test was carried out using a modified agar diffusion assay. Plate count agar medium was seeded with the microorganisms then the film was placed on the agar surface and incubated at 37 °C for 24 h. The antibacterial activity was assessed as an inhibition of the microorganism growth under the film. The quantitative inhibitory effect of the film was carried our according to the standard test method for determining the antimicrobial activity of immobilized antimicrobial agents under dynamic contact conditions (ASTM E 2149–01). Each film (9 cm × 7 cm) was put in 10 mL of the microorganism cell suspension and shake in a shaking incubator under 37 °C at a shaking speed of 150 rpm for 3 h 100 μL of the suspension was dipped and spread on the plate count agar in petri dishes. Bacterial growth was visualized after incubation under 37 °C for 10 h.
2. Materials and methods 2.1. Chemicals and reagents Gallic acid monohydrate with 99.5% purity was purchased from Scharlau. Folin-Ciocalteu reagent was procured from Panreac. Sodium carbonate was obtained from Katayama Chemical. Two foodborne pathogens used in this study were purchased from Bioresource Collection and Research Centre (BCRC) of the Food Industry Research and Development Institute in Hsinchu, Taiwan: Escherichia coli BCRC 10675 and Staphylococcus aureus BCRC 15211. 2.2. Preparation of gallic acid coated LDPE film Low density polyethylene (LDPE) was obtained from a market in Taiwan. LDPE films were cut into 9 cm × 7 cm size, cleaned with alcohol to remove dust or oily compounds on the film surface and dried before plasma treatment. Plasma treatment was used to prepare GA coated LDPE films with antimicrobial activity. At first, the LDPE films were treated in a plasma reactor using cold radiofrequency plasma (13.56 MHz) fitted with a capacitively coupled, parallel-electrode system with an automatic matching device (JUNSUN TECH CO., LTD).
2.5. Release rate of GA from the film during storage The release rate of GA in alcohol was measured by the method of Pankaj and Buonocore with slight modifications (Giovanna Giuliana Buonocore, Panizza, Bove, Battaglia, & Nicolais, 2003; Pankaj, Bueno2
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Table 1 Appearance of LDPE film under different plasma treatment.
properties of LDPE film under different plasma treatment time and power. In Table 1, the results show that LDPE film treated under the same power higher than 60 W but with longer treatment time has more shrinkage on the film. This situation becomes more severe as the power increases and even reduces the applicability. To be more precise, to choose the condition used in the following experiment, we used tensile strength as the indicator as it is the most important consideration in the food packaging (Honarvar et al., 2017). Tensile strength of the LDPE control film was 17.28 MPa. It showed a reduction trend after 150 s of plasma treatment and was 13.72 MPa, 11.18 MPa, 10.39 MPa, 10.79 MPa, 8.38 MPa, and 20.84 MPa after 15 W, 30 W, 45 W, 60 W, 75 W, and 90 W plasma treatment, respectively. In general, the films showed reduction in mechanical properties as the treatment time increased in the cases of 15–75 W plasma, which is in agreement with the work reported by Theapsak et al. (Theapsak et al., 2012). Under the same treatment time, the tensile strength of LDPE film that underwent different plasma treatment did not show a constant decrease trend. In each treatment time group, LDPE film treated by 90 W had the largest tensile strength, which increased as the treatment time increased. This could be due to the formation of polar groups and interfacial roughness (Honarvar et al., 2017). Although the
Ferrer, Misra, O'Neill, et al., 2014). All samples were cut into 2 cm × 4 cm size and immersed in a 50 mL centrifuge tube which containing 40 mL 95% alcohol. Each tube was shaken under 25 °C and observe for 48 h 1 mL of the solution was taken from the tube and analyzed with Folin-Ciocalteu method to determine the concentration of GA. The sampling interval is short in the beginning and then is extended later. 2.6. Statistical analysis All of the data is expressed as means ± standard deviations. Statistical data processing is achieved by means of dispersion analysis with SPSS 20 software. Statistical analysis was performed with one-way ANOVA and Duncan's multiple range tests, and statistical significance was defined at p < 0.05. 3. Results and discussion 3.1. Characterization plasma treated GA coated LDPE film Table 1 and Table 2 show the outer appearance and mechanical 3
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Table 2 Effect of plasma treatment on tensile strength of LDPE film. Power (Watt)
Treatment time (s) 0
Tensile strength (MPa)
15 30 45 60 75 90
17.28 17.28 17.28 17.28 17.28 17.28
30 ± ± ± ± ± ±
0.63 0.63 0.63 0.63 0.63 0.63
aA aA aA aA aA aA
15.17 14.30 13.47 13.92 12.92 16.15
60 ± ± ± ± ± ±
0.49 0.55 0.61 0.29 0.05 0.11
aA
15.08 13.31 12.20 11.26 11.83 16.18
abA bA bA bA bA
90 ± ± ± ± ± ±
0.72 0.97 0.31 0.17 0.38 0.96
aA abA bcA cA bcA abA
14.63 12.84 12.09 11.16 10.49 17.93
120 ± ± ± ± ± ±
0.37 0.41 0.28 0.02 0.05 0.82
aB bBC bcBC cC cdC abA
14.20 12.64 12.08 10.90 10.15 22.38
150 ± ± ± ± ± ±
0.56 0.40 0.16 0.42 0.27 0.88
aB bBC bcBC cBC cdC aA
13.72 ± 0.85 aB 11.18 ± 0.17 bBC 10.39 ± 0.34 cBC 10.77 ± 0.35 cBC 8.38 ± 0.24 dC 20.84 ± 0.98 abA
All values are expressed as mean ± standard deviation where n = 3. Different lowercase letters indicate statistically significant differences within the same row and different uppercase indicate statistically significant differences within the same column where p < 0.05.
surface area and roughness increased (Asadinezhad et al., 2010). After GA coating, the surface tended to become smoother with the increasing concentration of GA. Besides, several particles could be observed on the surfaces with GA coating, which was also observed in Ahn's study (Ahn, Gaikwad, & Lee, 2016). The surface chemical modification is shown in Fig. 2. The PE is almost completely composed of methylene (CH2) groups which have four main peaks corresponding to methylene stretching at 2920 and 2850 cm−1 and methylene deformations at 1464 and 719 cm−1 (Theapsak et al., 2012). After plasma treatment, new peaks at 1720 cm−1 corresponding to C]O stretching vibration and the region of 3200–3800 cm−1 corresponding to hydroxyl group (-OH) vibration occurred (Theapsak et al., 2012). GA's main characteristic bands occur at 3282 cm−1 and 3496 cm−1 and at 1612 cm−1 which corresponding to hydroxyl (-OH) stretching and C]O stretching, respectively (Devendra Singh, Semalty, & Semalty, 2011). From Fig. 2(b), films without plasma treatment did not show any characteristic peaks of GA after washing 3 cycles. As for Fig. 2(c), absorbance at wavenumber 1612 cm−1 is getting stronger as the GA concentration increases. Also, there were new peaks observed between 1400 and 1000 cm−1 which
tensile strength was improved for the 90 W treated group, the appearance of the LDPE film treated above 45 W was not suitable for packaging application. The results of Table 2 indicate that treatment under 30 W for 30 and 60 s showed no significant difference from control. Among these two treatments, the condition selected was 30 W treatment for 60 s. This is due to the slower aging effects in samples which are exposed in plasma for a longer time (Sanchis, Blanes, Blanes, Garcia, & Balart, 2006). Also, under same 60 s treatment time, surface roughness in film treated with 30 W is higher than film treated with 15 W. The increase in surface roughness will facilitate the coating of preservatives (Deshmukh & Bhat, 2016). Thus, with the combination of the results of Tables 1 and 2, the condition selected was 30 W treatment for 60 s. SEM analysis was used to measure the changes in surface morphology of the LDPE films after coating of GA. The SEM images of untreated and 30 W plasma treated film for 60 s are shown in Fig. 1. A smooth and uniform surface was observed for the untreated film, which exhibited increase in roughness when undergoing plasma treatment as plasma treatment yielded an etched character with an irregular shaped texture. This phenomenon increased the efficiency of coating as the
Fig. 1. Surface characterization of (a) control (b) 30 W plasma treated for 60 s PE film and plasma treated and coated with different concentrations of GA (c) 0.25% (d) 0.5% (e) 0.75% (f) 1% and (g) 2% observed under 10,000 × magnification. 4
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Fig. 2. ATR-FTIR spectra of (a) control and plasma treated film (30 W, 60 s) (b) no plasma treated with different concentrations of GA: 0.25%, 0.5%, 0.75%, 1%, and 2% and (c) 30 W plasma treated for 60 s PE film coated with different concentrations of GA: 0.25%, 0.5%, 0.75%, 1%, and 2%.
3.2. Assessment of the antimicrobial activity of the film
may be due to the formation of oligomeric or possibly dimeric GA structures (Neo et al., 2013). Furthermore, the bands at 3282 cm−1 are broader which indicates that GA may bind to PE film through the –OH end.
Packaging plays an important role in food preservation, especially antimicrobial activity, as microorganisms are one of the main influences on the shelf life of food. After GA adsorbed on surfaces, the 5
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Table 3 Effect of PE film with different GA (0, 0.25, 0.5, 0.75, 1, 2%) with or without plasma pre-treatment (30 W, 60 s) on Escherichia coli (n = 3).
and S. aureus after immersing in the cultures solution for 3 h. In Fig. 4, the viability of S. aureus is about 3.98–4.10 log CFU/mL in no plasma treated group while decrease from 3.98 to 3.48 log CFU/mL in plasma treated group as the concentration of GA increase from 0 to 2%. The similar trend can also be observed in Fig. 5. In Fig. 5, the viability of E. coli is about 3.83 to 3.79 log CFU/mL in no plasma treated group while decrease from 3.83 to 3.18 log CFU/mL in plasma treated group as the concentration of GA increase from 0 to 2%. Both of these results showed that after plasma treatment, the antibacterial activity of the film is strengthened as the concentration of GA increase. These result are in corresponding to the result of Karam's study which antibacterial activity of nisin is increased after the film is plasma treated (L. Karam et al., 2016). Besides that, in Karam et al. (2013)'s research, LDPE was treated with N2 plasma, polyacrylic acid and Ar/O2 plasma then coating nisin, which result 0.5–1.1 log of reduction and proven that through surface modification, the antibacterial activity is able to be improved (Layal Karam et al., 2013). Our result shows 0.5–0.6 log CFU/mL of reduction in both microorganisms which is similar to Karam's study. From the result, we can observed that these films have more significant inhibition on E. coli than S. aureus due to cell membrane structure differences (Borges, Ferreira, Saavedra, & Simoes, 2013). Lipid content
antibacterial activity was assessed qualitatively by an agar diffusion assay against E. coli and S. aureus. For E. coli in Table 3, the control films had no antibacterial activity because no inhibition was observed for the untreated films. The homogeneity of observed activity was evaluated. The plasma treated GA coated film shows uniform inhibition activity under the film. This phenomenon is more obvious as the GA concentration increases. As for the result of S. aureus shown in Table 4, the control films and plasma treated without GA coated film had no antibacterial activity because no inhibition phenomenon were observed. The homogeneity of observed activity was evaluated. As for the plasma treated and GA coated film, it shows spot-like irregular antibacterial activity and is strictly confined under film. From the result of these two microorganisms, it indicated that the GA is absorbed on the film and possessed antimicrobial activity toward these two microorganisms. This result is consistent with the result of Karam's study (L. Karam et al., 2016) which showed antibacterial activity on plasma treated nisin coated film only. Fig. 3 and Fig. 4 show the effect of different concentrations of GA with or without plasma pre-treated LDPE film on E. coli and S. aureus. The result showed a reduction trend for the plasma treated GA coated LDPE films while there is no significant changes in viability of E. coli
Table 4 Effect of PE film with different GA (0, 0.25, 0.5, 0.75, 1, 2%) with or without plasma pre-treatment (30 W, 60 s) on.
6
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Fig. 3. Antimicrobial activity of PE film with different concentrations of GA (0, 0.25, 0.5, 0.75, 1, 2%) with or without plasma pre-treatment (30 W, 60 s) on S. aureus. a~d Means followed by different lowercase are significantly different at p < 0.05.
that intracellular acidification changes permeabilization of cell membrane, which causes leakage of the intracellular content, leading to cell death (Borges et al., 2013). Fig. 5 shows the release rate of GA from untreated and plasma
of the cell wall of Gram-negative bacteria is higher than Gram-positive cell wall (Salton, 1953). Thus, the phenolic acid-lipid interaction is able to explain the higher susceptibility of the Gram-negative bacteria. Mechanism for the antimicrobial activity of GA are based on the fact
Fig. 4. Antimicrobial activity of PE film with different concentrations of GA (0, 0.25, 0.5, 0.75, 1, 2%) with or without plasma pre-treatment (30 W, 60 s) on E. coli. a~e Means followed by different lowercase are significantly different at p < 0.05. 7
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Fig. 5. Release rate of GA from plasma treated and untreated film with 1% GA coated immersed in 95% alcohol under different time where the experiment was carried out in triplicate. ãb Means followed by different lowercase are significantly different at p < 0.05.
treated film with 1% GA coated immersing in 95% alcohol after storage for 48 h under 25 °C. The results showed that plasma treated film has a faster release rate of GA than untreated film by approximately 150%. The increase in the release rate of GA after plasma treatment may be due to the etching effect and increase of surface roughness which will reduce film thickness and exposed GA more easily. This phenomenon is in similar with Pankaj (2014)'s result (Pankaj, Bueno-Ferrer, Misra, Milosavljević, et al., 2014). This result is speculated to be one of the reason that causes the increase of the antibacterial activity of the film after plasma treatment.
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4. Conclusions In this study, we used tensile strength as a processing condition indicator and chose the appropriate working condition, which is 30 W treatment for 60 s. Antimicrobial film was developed by first using plasma treatment and following coating step. These treatment film showed spot like irregular antimicrobial activity against E. coli and S. aureus and has higher antimicrobial activity against E. coli than against S. aureus. During 48 h of storage time, GA is slowly released and able to maintain its antimicrobial activity. However, further research on various types of food model preservation such as fresh fruits, vegetables or sashimi are required to understand the application of this active packaging. Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement Funding: This work was supported by the Ministry of Science and Technology, Republic of China (grant no. 106-2221-E−005-093-MY3) and Q-YO BIO-technology. 8
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2013.634129. Paola Appendini, J. H. H. (2002). Review of antimicrobial food packaging. Innovative Food Science & Emerging Technologies, 3, 113–126. Riccardi, C., Barni, R., Selli, E., Mazzone, G., Massafra, M. R., Marcandalli, B., et al. (2003). Surface modification of poly(ethylene terephthalate) fibers induced by radio frequency air plasma treatment. Applied Surface Science, 211(1–4), 386–397. https:// doi.org/10.1016/s0169-4332(03)00265-4. Sadeghnejad, A., Aroujalian, A., Raisi, A., & Fazel, S. (2014). Antibacterial nano silver coating on the surface of polyethylene films using corona discharge. Surface and Coatings Technology, 245, 1–8. https://doi.org/10.1016/j.surfcoat.2014.02.023. Salton, M. R. (1953). Studies of the bacterial cell wall: IV. The composition of the cell walls of some gram-positive and gram-negative bacteria. Biochimica et Biophysica Acta, 10, 512–523. Sanchis, M. R., Blanes, V., Blanes, M., Garcia, D., & Balart, R. (2006). Surface modification of low density polyethylene (LDPE) film by low pressure O2 plasma treatment. European Polymer Journal, 42(7), 1558–1568. https://doi.org/10.1016/j.eurpolymj. 2006.02.001. Schutze, A., Jeong, J. Y., Babayan, S. E., Park, J., Selwyn, G. S., & Hicks, R. F. (1998). The atmospheric-pressure plasma jet: A review and comparison to other plasma sources. IEEE Transactions on Plasma Science, 26(6), 1685–1694. Shao, T., Zhang, C., Long, K., Zhang, D., Wang, J., Yan, P., et al. (2010). Surface modification of polyimide films using unipolar nanosecond-pulse DBD in atmospheric air. Applied Surface Science, 256(12), 3888–3894. https://doi.org/10.1016/j.apsusc.2010. 01.045. Stefania Quintavalla, L. V. (2002). Antimicrobial food packaging in meat industry. Meat Science, 62, 373–380. Theapsak, S., Watthanaphanit, A., & Rujiravanit, R. (2012). Preparation of chitosancoated polyethylene packaging films by DBD plasma treatment. ACS Applied Materials and Interfaces, 4(5), 2474–2482. https://doi.org/10.1021/am300168a. Vartiainen, J., Rättö, M., & Paulussen, S. (2005). Antimicrobial activity of glucose oxidase-immobilized plasma-activated polypropylene films. Packaging Technology and Science, 18(5), 243–251. https://doi.org/10.1002/pts.695. Wongsawaeng, D., Khemngern, S., & Somboonna, N. (2017). Environmentally-friendly RF plasma treatment of Thai silk fabrics with chitosan for durable antibacterial property. Engineering Journal, 21(1), 29–43. https://doi.org/10.4186/ej.2017.21.1.29. Zhang, W., Chu, P. K., Ji, J., Zhang, Y., Ng, S. C., & Yan, Q. (2006). Surface antibacterial characteristics of plasma-modified polyethylene. Biopolymers, 83(1), 62–68. https:// doi.org/10.1002/bip.20527. Zheng, Y., Miao, J., Zhang, F., Cai, C., Koh, A., Simmons, T. J., ... Linhardt, R. J. (2016). Surface modification of a polyethylene film for anticoagulant and anti-microbial catheter. Reactive and Functional Polymers, 100, 142–150. https://doi.org/10.1016/j. reactfunctpolym.2016.01.013.
ijfoodmicro.2013.06.003. Honarvar, Z., Farhoodi, M., Khani, M. R., Mohammadi, A., Shokri, B., Ferdowsi, R., et al. (2017). Application of cold plasma to develop carboxymethyl cellulose-coated polypropylene films containing essential oil. Carbohydrate Polymers, 176, 1–10. https://doi.org/10.1016/j.carbpol.2017.08.054. Karam, L., Casetta, M., Chihib, N. E., Bentiss, F., Maschke, U., & Jama, C. (2016). Optimization of cold nitrogen plasma surface modification process for setting up antimicrobial low density polyethylene films. Journal of the Taiwan Institute of Chemical Engineers, 64, 299–305. https://doi.org/10.1016/j.jtice.2016.04.018. Karam, L., Jama, C., Mamede, A.-S., Fahs, A., Louarn, G., Dhulster, P., et al. (2013). Study of nisin adsorption on plasma-treated polymer surfaces for setting up materials with antibacterial properties. Reactive and Functional Polymers, 73(11), 1473–1479. https://doi.org/10.1016/j.reactfunctpolym.2013.07.017. Kikani, P., Desai, B., Prajapati, S., Arun, P., Chauhan, N., & Nema, S. K. (2013). Comparison of low and atmospheric pressure air plasma treatment of polyethylene. Surface Engineering, 29(3), 211–221. https://doi.org/10.1179/1743294413y. 0000000111. Kim, J. E., Lee, D. U., & Min, S. C. (2014). Microbial decontamination of red pepper powder by cold plasma. Food Microbiology, 38, 128–136. https://doi.org/10.1016/j. fm.2013.08.019. Lee, K. T. (2010). Quality and safety aspects of meat products as affected by various physical manipulations of packaging materials. Meat Science, 86(1), 138–150. https://doi.org/10.1016/j.meatsci.2010.04.035. Lu, Z., Nie, G., Belton, P. S., Tang, H., & Zhao, B. (2006). Structure-activity relationship analysis of antioxidant ability and neuroprotective effect of gallic acid derivatives. Neurochemistry International, 48(4), 263–274. https://doi.org/10.1016/j.neuint.2005. 10.010. Neo, Y. P., Ray, S., Jin, J., Gizdavic-Nikolaidis, M., Nieuwoudt, M. K., Liu, D., et al. (2013). Encapsulation of food grade antioxidant in natural biopolymer by electrospinning technique: A physicochemical study based on zein-gallic acid system. Food Chemistry, 136(2), 1013–1021. https://doi.org/10.1016/j.foodchem.2012.09.010. Ozdemir, M., Yurteri, C. U., & Sadikoglu, H. (1999). Physical polymer surface modification methods and applications in food packaging polymers. Critical Reviews in Food Science and Nutrition, 39(5), 457–477. https://doi.org/10.1080/ 10408699991279240. Pankaj, S. K., Bueno-Ferrer, C., Misra, N. N., Milosavljević, V., O'Donnell, C. P., Bourke, P., ... Cullen, P. J. (2014). Applications of cold plasma technology in food packaging. Trends in Food Science & Technology, 35(1), 5–17. https://doi.org/10.1016/j.tifs.2013. 10.009. Pankaj, S. K., Bueno-Ferrer, C., Misra, N. N., O'Neill, L., Jiménez, A., Bourke, P., et al. (2014). Surface, thermal and antimicrobial release properties of plasma-treated zein films. Journal of Renewable Materials, 2(1), 77–84. https://doi.org/10.7569/jrm.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Preparation of antimicrobial active packaging film by capacitively coupled plasma treatment
T
Li-Wah Wonga,1, Chih-Yao Houb,1, Chun-Chi Hsiehc, Chao-Kai Changd, Yi-Shan Wua, Chang-Wei Hsieha,e,∗ a
Department of Food Science and Biotechnology, National Chung Hsing University, 145 Xingda Rd., South Dist., Taichung City 402, Taiwan, ROC Department of Seafood Science, National Kaohsiung University of Science and Technology, 142, Haizhuan Rd., Nanzi Dist., Kaohsiung City, 81157, Taiwan, ROC c Graduate Institute of Biotechnology, National Chung Hsing University, 145 Xingda Rd., South Dist., Taichung City 402, Taiwan, ROC d College of Biotechnology and Bioresources, Da-Yeh University, 168 University Rd., Dacun, Changhua City 515, Taiwan, ROC e Department of Medical Research, China Medical University Hospital, Taichung City 404, Taiwan, ROC b
A R T I C LE I N FO
A B S T R A C T
Keywords: Active packaging Low density polyethylene Gallic acid Antimicrobial activity
This research is mainly focused on preparing gallic acid (GA) coated, antimicrobial, active packaging, low density polyethylene (LDPE) films by capacitively coupled plasma. Firstly, the suitable plasma treatment condition was 30 W treated for 60 s from the result of appearance and tensile strength. From the result of SEM and ATR-FTIR, the surface roughness increased which facilitated the GA coating and the surface turn smoother after GA coating. In the result of antimicrobial activity, these films is able to reduce the growth of Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus about 0.5–0.6 log CFU/mL when GA concentration is above 1%. Between these two microorganisms, the film has more significant inhibition on E. coli than S. aureus due to cell membrane structure differences. These results are first stage in developing antibacterial active packaging for foods preservation.
1. Introduction Active packaging is defined as “packaging in which subsidiary constituents have been deliberately included in or on either the packaging material or the package headspace to enhance the performance of the package system” (Paola Appendini, 2002). It can prolong the shelf life of products by adjusting the gas ratio, humidity, and antioxidant and antimicrobial properties. Among all kinds of active packaging, antimicrobial packaging is one of the most studied fields that can inhibit or even kill bacteria and thus enhance safety and extend shelf life. Antimicrobial packaging can take several forms such as incorporation of volatile and non-volatile antimicrobial agents directly into polymers, coating antimicrobials onto polymer surfaces, and immobilization of antimicrobials to polymers by ion or covalent linkages (Lee, 2010; Paola Appendini, 2002; Stefania Quintavalla, 2002; Vartiainen, Rättö, & Paulussen, 2005; Zhang et al., 2006). During sale and storage periods, the antimicrobial will be slowly released from the packaging material and inhibit the microorganisms in the foods (Higueras, Lopez-Carballo, Hernandez-Munoz, Gavara, & Rollini,
2013), and extend the shelf life of foods (Stefania Quintavalla, 2002). Polyethylene (PE) films are the most widely used polyolefin among many types of polymers due to their good chemical resistance, transparency, flexibility, and low cost (Zheng et al., 2016). However, polyethylene is chemically inert material and does not possess any functionality, which limits its application in active packaging (Ozdemir, Yurteri, & Sadikoglu, 1999; Theapsak, Watthanaphanit, & Rujiravanit, 2012). So, surface modification of PE film is an important pretreatment to activate PE's surface and able to bonding with preservatives to create an active packaging (Ozdemir et al., 1999). Surface modification can improve the applicability of plastics by affecting the topmost layer of the surface (Govindarajan & Shandas, 2014). In order to modify the polymer surface, there are many kinds of methods such as wet chemical modification, plasma treatment, and UV radiation (Lee, 2010). Wet chemical modification is commonly used in industry, which requires a chemical reagent such as a strong acid or a strong base to create reactive functional groups on the surface of a polymer (Govindarajan & Shandas, 2014; L.; Karam et al., 2016; Ozdemir et al., 1999). However, the waste discharged is not
∗
Corresponding author. Department of Food Science and Biotechnology, National Chung Hsing University, 145 Xingda Rd., South Dist., Taichung City 402, Taiwan, ROC. E-mail address: [email protected] (C.-W. Hsieh). 1 These authors contributed equally to this study and share first authorship. https://doi.org/10.1016/j.lwt.2019.108612 Received 19 April 2019; Received in revised form 8 August 2019; Accepted 9 September 2019 Available online 10 September 2019 0023-6438/ © 2019 Published by Elsevier Ltd.
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The chamber was pumped down to 0.0643 Torr and the air gas was introduced into the chamber with 5 sccm gas flow. When the pressure become constant, switch on the power source and adjust to certain power value. They were treated with different power (0–90 W) and treatment time (15–150 s) to determine the most appropriate treatment condition. Later, the LDPE films were treated according to the evaluation result of mechanical properties. After plasma treatment, the LDPE films were immediately immersed in different concentrations of GA solution with continuous stirring for 24 h, followed by washing for 3 cycles with distilled water to remove excess GA and then air dried at room temperature overnight (Theapsak et al., 2012).
environmentally friendly. Plasma contains photons, electrons, molecules, atoms, and free radicals, which destroy covalent bonds and initiate many chemical reactions (Kim, Lee, & Min, 2014; Riccardi et al., 2003; Shao et al., 2010). Besides, plasma treatment can also insert new functional groups on the surface, improve hydrophilicity and affect mechanical properties which has proven to increase the applicability of polymeric materials and convert them into higher value products (De Geyter, Morent, Leys, Gengembre, & Payen, 2007; Honarvar et al., 2017; Chan, Ko, & Hiraoka, 1996; Wongsawaeng, Khemngern, & Somboonna, 2017). In the study of plasma-modified polymer films, low-pressure plasma is a kind of plasma treatment that is used in the mainstream because it has a low power input and can generate plasma with a small flow of gas which in turn consumes less energy and processes more efficiently than atmospheric plasma. In addition, in a vacuum environment, the concentration of reactive species is higher, and electrons have a larger average free path (0.1 cm at low pressure and ~5.7 × 10−8 cm at atmospheric pressure) with higher energy (Andreas Schutze et al., 1998; Kikani et al., 2013). In addition, plasma treatment is relatively environmentally friendly compared to wet chemical modification (Honarvar et al., 2017). Few studies of antibacterial packaging bags developed using plasma treatment and coating an antibacterial agent such as lysozyme or sodium benzoate have been performed. These methods can effectively inhibit the growth of microorganisms, thereby extending the shelf life of food (Lee, 2010). The most common problem in food packaging is microbial recontamination in subsequent processing (Fernandez-Saiz, Lagaron, Hernandez-Munoz, & Ocio, 2008), which degrades the quality of the product and even harms the health of consumers. Common foodborne pathogens include Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Such microorganisms can cause food poisoning, causing symptoms such as vomiting and diarrhea. Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA) is a biologically active phytochemical that is widely found in plants, nuts, and teas (Arcan & Yemenicioğlu, 2009; Lu, Nie, Belton, Tang, & Zhao, 2006). GA has an inhibitory effect on foodborne pathogen - E. coli and S. aureus. (Chanwitheesuk, Teerawutgulrag, Kilburn, & Rakariyatham, 2007). Also, it has been suggested to possess strong antioxidant activities and is able to provide protection against oxidative damage cause by free radicals such as hydroxyl (HO•) and hydrogen peroxide (H2O2). Therefore, this study aimed to improve the surface activity of PE film by plasma treatment technology to bond with GA, develop an optimum condition for creating an active packaging film containing GA, and evaluate its antimicrobial effect on E. coli and S. aureus.
2.3. Characterization of the film 2.3.1. Tensile strength This experiment was carried out according to (Theapsak et al., 2012) with slight modifications. Tensile strength of the untreated and plasma-treated films was detected by a texture profile analyzer (TA.XT2i, Stable Micro System, Haslemere, Surrey, UK) at 25 °C. The films were cut into a rectangular shape (1 cm × 3 cm) and equipped with a 500 N load cell. A strain rate of 10 mms−1 and a gauge length of 100 mm were employed according to ASTM D882-91 standard test method. 2.3.2. Surface morphologies and chemical composition of the film The morphologies of the films were observed by a scanning electron microscope (TM 1000, Hitachi, Japan). Before testing, all the samples were dried, attached to a double-sided carbon tape with a conducting sample holder and coated with gold under vacuum using a sputtering system (Sadeghnejad, Aroujalian, Raisi, & Fazel, 2014). Surface chemical composition of the films was observed using an attenuated total Reflection-Fourier transform infrared spectroscope (ATR-FTIR, Thermo Nicolet 6700). The ATR-FTIR spectra were investigated between wavenumber 4000 to 650 cm−1 with 64 scans at a resolution of 4 cm−1 (Theapsak et al., 2012). 2.4. Assessment of the antimicrobial activity of the film The antimicrobial activity of the samples against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) was determined qualitative and quantitatively. The cell cultures were performed by inoculating 100 μL of the pre-cultures in 10 mL nutrient broth and incubated at 37 °C for 24 h. The cell cultures of each microorganism were then diluted using 0.85% sterile NaCl aqueous solution by a factor of 103 and 104 for qualitative and quantitative antibacterial assessments (L. Karam et al., 2016; Theapsak et al., 2012). The qualitative antibacterial test was carried out using a modified agar diffusion assay. Plate count agar medium was seeded with the microorganisms then the film was placed on the agar surface and incubated at 37 °C for 24 h. The antibacterial activity was assessed as an inhibition of the microorganism growth under the film. The quantitative inhibitory effect of the film was carried our according to the standard test method for determining the antimicrobial activity of immobilized antimicrobial agents under dynamic contact conditions (ASTM E 2149–01). Each film (9 cm × 7 cm) was put in 10 mL of the microorganism cell suspension and shake in a shaking incubator under 37 °C at a shaking speed of 150 rpm for 3 h 100 μL of the suspension was dipped and spread on the plate count agar in petri dishes. Bacterial growth was visualized after incubation under 37 °C for 10 h.
2. Materials and methods 2.1. Chemicals and reagents Gallic acid monohydrate with 99.5% purity was purchased from Scharlau. Folin-Ciocalteu reagent was procured from Panreac. Sodium carbonate was obtained from Katayama Chemical. Two foodborne pathogens used in this study were purchased from Bioresource Collection and Research Centre (BCRC) of the Food Industry Research and Development Institute in Hsinchu, Taiwan: Escherichia coli BCRC 10675 and Staphylococcus aureus BCRC 15211. 2.2. Preparation of gallic acid coated LDPE film Low density polyethylene (LDPE) was obtained from a market in Taiwan. LDPE films were cut into 9 cm × 7 cm size, cleaned with alcohol to remove dust or oily compounds on the film surface and dried before plasma treatment. Plasma treatment was used to prepare GA coated LDPE films with antimicrobial activity. At first, the LDPE films were treated in a plasma reactor using cold radiofrequency plasma (13.56 MHz) fitted with a capacitively coupled, parallel-electrode system with an automatic matching device (JUNSUN TECH CO., LTD).
2.5. Release rate of GA from the film during storage The release rate of GA in alcohol was measured by the method of Pankaj and Buonocore with slight modifications (Giovanna Giuliana Buonocore, Panizza, Bove, Battaglia, & Nicolais, 2003; Pankaj, Bueno2
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Table 1 Appearance of LDPE film under different plasma treatment.
properties of LDPE film under different plasma treatment time and power. In Table 1, the results show that LDPE film treated under the same power higher than 60 W but with longer treatment time has more shrinkage on the film. This situation becomes more severe as the power increases and even reduces the applicability. To be more precise, to choose the condition used in the following experiment, we used tensile strength as the indicator as it is the most important consideration in the food packaging (Honarvar et al., 2017). Tensile strength of the LDPE control film was 17.28 MPa. It showed a reduction trend after 150 s of plasma treatment and was 13.72 MPa, 11.18 MPa, 10.39 MPa, 10.79 MPa, 8.38 MPa, and 20.84 MPa after 15 W, 30 W, 45 W, 60 W, 75 W, and 90 W plasma treatment, respectively. In general, the films showed reduction in mechanical properties as the treatment time increased in the cases of 15–75 W plasma, which is in agreement with the work reported by Theapsak et al. (Theapsak et al., 2012). Under the same treatment time, the tensile strength of LDPE film that underwent different plasma treatment did not show a constant decrease trend. In each treatment time group, LDPE film treated by 90 W had the largest tensile strength, which increased as the treatment time increased. This could be due to the formation of polar groups and interfacial roughness (Honarvar et al., 2017). Although the
Ferrer, Misra, O'Neill, et al., 2014). All samples were cut into 2 cm × 4 cm size and immersed in a 50 mL centrifuge tube which containing 40 mL 95% alcohol. Each tube was shaken under 25 °C and observe for 48 h 1 mL of the solution was taken from the tube and analyzed with Folin-Ciocalteu method to determine the concentration of GA. The sampling interval is short in the beginning and then is extended later. 2.6. Statistical analysis All of the data is expressed as means ± standard deviations. Statistical data processing is achieved by means of dispersion analysis with SPSS 20 software. Statistical analysis was performed with one-way ANOVA and Duncan's multiple range tests, and statistical significance was defined at p < 0.05. 3. Results and discussion 3.1. Characterization plasma treated GA coated LDPE film Table 1 and Table 2 show the outer appearance and mechanical 3
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Table 2 Effect of plasma treatment on tensile strength of LDPE film. Power (Watt)
Treatment time (s) 0
Tensile strength (MPa)
15 30 45 60 75 90
17.28 17.28 17.28 17.28 17.28 17.28
30 ± ± ± ± ± ±
0.63 0.63 0.63 0.63 0.63 0.63
aA aA aA aA aA aA
15.17 14.30 13.47 13.92 12.92 16.15
60 ± ± ± ± ± ±
0.49 0.55 0.61 0.29 0.05 0.11
aA
15.08 13.31 12.20 11.26 11.83 16.18
abA bA bA bA bA
90 ± ± ± ± ± ±
0.72 0.97 0.31 0.17 0.38 0.96
aA abA bcA cA bcA abA
14.63 12.84 12.09 11.16 10.49 17.93
120 ± ± ± ± ± ±
0.37 0.41 0.28 0.02 0.05 0.82
aB bBC bcBC cC cdC abA
14.20 12.64 12.08 10.90 10.15 22.38
150 ± ± ± ± ± ±
0.56 0.40 0.16 0.42 0.27 0.88
aB bBC bcBC cBC cdC aA
13.72 ± 0.85 aB 11.18 ± 0.17 bBC 10.39 ± 0.34 cBC 10.77 ± 0.35 cBC 8.38 ± 0.24 dC 20.84 ± 0.98 abA
All values are expressed as mean ± standard deviation where n = 3. Different lowercase letters indicate statistically significant differences within the same row and different uppercase indicate statistically significant differences within the same column where p < 0.05.
surface area and roughness increased (Asadinezhad et al., 2010). After GA coating, the surface tended to become smoother with the increasing concentration of GA. Besides, several particles could be observed on the surfaces with GA coating, which was also observed in Ahn's study (Ahn, Gaikwad, & Lee, 2016). The surface chemical modification is shown in Fig. 2. The PE is almost completely composed of methylene (CH2) groups which have four main peaks corresponding to methylene stretching at 2920 and 2850 cm−1 and methylene deformations at 1464 and 719 cm−1 (Theapsak et al., 2012). After plasma treatment, new peaks at 1720 cm−1 corresponding to C]O stretching vibration and the region of 3200–3800 cm−1 corresponding to hydroxyl group (-OH) vibration occurred (Theapsak et al., 2012). GA's main characteristic bands occur at 3282 cm−1 and 3496 cm−1 and at 1612 cm−1 which corresponding to hydroxyl (-OH) stretching and C]O stretching, respectively (Devendra Singh, Semalty, & Semalty, 2011). From Fig. 2(b), films without plasma treatment did not show any characteristic peaks of GA after washing 3 cycles. As for Fig. 2(c), absorbance at wavenumber 1612 cm−1 is getting stronger as the GA concentration increases. Also, there were new peaks observed between 1400 and 1000 cm−1 which
tensile strength was improved for the 90 W treated group, the appearance of the LDPE film treated above 45 W was not suitable for packaging application. The results of Table 2 indicate that treatment under 30 W for 30 and 60 s showed no significant difference from control. Among these two treatments, the condition selected was 30 W treatment for 60 s. This is due to the slower aging effects in samples which are exposed in plasma for a longer time (Sanchis, Blanes, Blanes, Garcia, & Balart, 2006). Also, under same 60 s treatment time, surface roughness in film treated with 30 W is higher than film treated with 15 W. The increase in surface roughness will facilitate the coating of preservatives (Deshmukh & Bhat, 2016). Thus, with the combination of the results of Tables 1 and 2, the condition selected was 30 W treatment for 60 s. SEM analysis was used to measure the changes in surface morphology of the LDPE films after coating of GA. The SEM images of untreated and 30 W plasma treated film for 60 s are shown in Fig. 1. A smooth and uniform surface was observed for the untreated film, which exhibited increase in roughness when undergoing plasma treatment as plasma treatment yielded an etched character with an irregular shaped texture. This phenomenon increased the efficiency of coating as the
Fig. 1. Surface characterization of (a) control (b) 30 W plasma treated for 60 s PE film and plasma treated and coated with different concentrations of GA (c) 0.25% (d) 0.5% (e) 0.75% (f) 1% and (g) 2% observed under 10,000 × magnification. 4
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Fig. 2. ATR-FTIR spectra of (a) control and plasma treated film (30 W, 60 s) (b) no plasma treated with different concentrations of GA: 0.25%, 0.5%, 0.75%, 1%, and 2% and (c) 30 W plasma treated for 60 s PE film coated with different concentrations of GA: 0.25%, 0.5%, 0.75%, 1%, and 2%.
3.2. Assessment of the antimicrobial activity of the film
may be due to the formation of oligomeric or possibly dimeric GA structures (Neo et al., 2013). Furthermore, the bands at 3282 cm−1 are broader which indicates that GA may bind to PE film through the –OH end.
Packaging plays an important role in food preservation, especially antimicrobial activity, as microorganisms are one of the main influences on the shelf life of food. After GA adsorbed on surfaces, the 5
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Table 3 Effect of PE film with different GA (0, 0.25, 0.5, 0.75, 1, 2%) with or without plasma pre-treatment (30 W, 60 s) on Escherichia coli (n = 3).
and S. aureus after immersing in the cultures solution for 3 h. In Fig. 4, the viability of S. aureus is about 3.98–4.10 log CFU/mL in no plasma treated group while decrease from 3.98 to 3.48 log CFU/mL in plasma treated group as the concentration of GA increase from 0 to 2%. The similar trend can also be observed in Fig. 5. In Fig. 5, the viability of E. coli is about 3.83 to 3.79 log CFU/mL in no plasma treated group while decrease from 3.83 to 3.18 log CFU/mL in plasma treated group as the concentration of GA increase from 0 to 2%. Both of these results showed that after plasma treatment, the antibacterial activity of the film is strengthened as the concentration of GA increase. These result are in corresponding to the result of Karam's study which antibacterial activity of nisin is increased after the film is plasma treated (L. Karam et al., 2016). Besides that, in Karam et al. (2013)'s research, LDPE was treated with N2 plasma, polyacrylic acid and Ar/O2 plasma then coating nisin, which result 0.5–1.1 log of reduction and proven that through surface modification, the antibacterial activity is able to be improved (Layal Karam et al., 2013). Our result shows 0.5–0.6 log CFU/mL of reduction in both microorganisms which is similar to Karam's study. From the result, we can observed that these films have more significant inhibition on E. coli than S. aureus due to cell membrane structure differences (Borges, Ferreira, Saavedra, & Simoes, 2013). Lipid content
antibacterial activity was assessed qualitatively by an agar diffusion assay against E. coli and S. aureus. For E. coli in Table 3, the control films had no antibacterial activity because no inhibition was observed for the untreated films. The homogeneity of observed activity was evaluated. The plasma treated GA coated film shows uniform inhibition activity under the film. This phenomenon is more obvious as the GA concentration increases. As for the result of S. aureus shown in Table 4, the control films and plasma treated without GA coated film had no antibacterial activity because no inhibition phenomenon were observed. The homogeneity of observed activity was evaluated. As for the plasma treated and GA coated film, it shows spot-like irregular antibacterial activity and is strictly confined under film. From the result of these two microorganisms, it indicated that the GA is absorbed on the film and possessed antimicrobial activity toward these two microorganisms. This result is consistent with the result of Karam's study (L. Karam et al., 2016) which showed antibacterial activity on plasma treated nisin coated film only. Fig. 3 and Fig. 4 show the effect of different concentrations of GA with or without plasma pre-treated LDPE film on E. coli and S. aureus. The result showed a reduction trend for the plasma treated GA coated LDPE films while there is no significant changes in viability of E. coli
Table 4 Effect of PE film with different GA (0, 0.25, 0.5, 0.75, 1, 2%) with or without plasma pre-treatment (30 W, 60 s) on.
6
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Fig. 3. Antimicrobial activity of PE film with different concentrations of GA (0, 0.25, 0.5, 0.75, 1, 2%) with or without plasma pre-treatment (30 W, 60 s) on S. aureus. a~d Means followed by different lowercase are significantly different at p < 0.05.
that intracellular acidification changes permeabilization of cell membrane, which causes leakage of the intracellular content, leading to cell death (Borges et al., 2013). Fig. 5 shows the release rate of GA from untreated and plasma
of the cell wall of Gram-negative bacteria is higher than Gram-positive cell wall (Salton, 1953). Thus, the phenolic acid-lipid interaction is able to explain the higher susceptibility of the Gram-negative bacteria. Mechanism for the antimicrobial activity of GA are based on the fact
Fig. 4. Antimicrobial activity of PE film with different concentrations of GA (0, 0.25, 0.5, 0.75, 1, 2%) with or without plasma pre-treatment (30 W, 60 s) on E. coli. a~e Means followed by different lowercase are significantly different at p < 0.05. 7
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Fig. 5. Release rate of GA from plasma treated and untreated film with 1% GA coated immersed in 95% alcohol under different time where the experiment was carried out in triplicate. ãb Means followed by different lowercase are significantly different at p < 0.05.
treated film with 1% GA coated immersing in 95% alcohol after storage for 48 h under 25 °C. The results showed that plasma treated film has a faster release rate of GA than untreated film by approximately 150%. The increase in the release rate of GA after plasma treatment may be due to the etching effect and increase of surface roughness which will reduce film thickness and exposed GA more easily. This phenomenon is in similar with Pankaj (2014)'s result (Pankaj, Bueno-Ferrer, Misra, Milosavljević, et al., 2014). This result is speculated to be one of the reason that causes the increase of the antibacterial activity of the film after plasma treatment.
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4. Conclusions In this study, we used tensile strength as a processing condition indicator and chose the appropriate working condition, which is 30 W treatment for 60 s. Antimicrobial film was developed by first using plasma treatment and following coating step. These treatment film showed spot like irregular antimicrobial activity against E. coli and S. aureus and has higher antimicrobial activity against E. coli than against S. aureus. During 48 h of storage time, GA is slowly released and able to maintain its antimicrobial activity. However, further research on various types of food model preservation such as fresh fruits, vegetables or sashimi are required to understand the application of this active packaging. Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement Funding: This work was supported by the Ministry of Science and Technology, Republic of China (grant no. 106-2221-E−005-093-MY3) and Q-YO BIO-technology. 8
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