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chitosan films for antimicrobial food packaging
Food Packaging and Shelf Life 22 (2019) 100396
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Development of grapefruit seed extract-loaded poly(ε-caprolactone)/ chitosan films for antimicrobial food packaging Kaiying Wang, Poon Nian Lim, Shi Yun Tong, Eng San Thian
T
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Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobial Chitosan Food packaging Grapefruit seed extract Poly(ε-caprolactone)
In this study, the packaging films were prepared by blending poly(ε-caprolactone) (PCL) with chitosan and grapefruit seed extract (GFSE) using extrusion compounding followed by compression molding. Different GFSE/ chitosan ratios (0.5, 1.0, 1.5, 2.0 and 2.5 mL/g) affected the surface morphology, mechanical and antimicrobial properties of the films. Although the addition of GFSE decreased the films’ resistance to breakage and stretching, the films retained adequate tensile strength and desirable flexibility for packaging. More notably, PCL/chitosan/ GFSE films were effective against both Escherichia coli and Pseudomonas aeruginosa and could inhibit the bacterial growth for up to 120 h when GFSE content increased to 0.5 mL/g and 1.0 mL/g, respectively. Packaging application test using salmon showed that PCL/chitosan/GFSE films were effective to inhibit the growth of E. coli in salmon during storage. Furthermore, no mold growth was observed on the bread packaged with films containing ≥1.0 mL/g GFSE after 7 days.
1. Introduction The global wide attention towards maintaining food safety and extending the shelf-life of packaged food has triggered an increased interest in developing antimicrobial packaging. In general, antimicrobial packaging is achieved by directly incorporating antimicrobial agents into packaging films, coating packaging films with antimicrobial substances, and packaging materials made up of polymers with inherent antimicrobial properties (Irkin & Esmer, 2015). Numerous antimicrobial agents have been used to develop antimicrobial food packaging films. They include organic acids such as benzoic acid (Dobiáš, Chudackova, Voldrich, & Marek, 2000), citric acid (Ramirez et al., 2017) and sorbic acid (Cagri, Ustunol, & Ryser, 2006), enzymes such as lysozyme (Mecitoǧlu et al., 2006), bacteriocins such as nisin (Jin, Liu, Zhang, & Hicks, 2009), polymers such as chitosan (Dutta, Tripathi, Mehrotra, & Dutta, 2009), plant extracts such as clove extract (da Cruz Cabral, Fernández Pinto, & Patriarca, 2013) and cinnamaldehyde (Qin, Yang, & Xue, 2016), essential oils such as thyme essential oils (Karabagias, Badeka, & Kontominas, 2011) and oregano essential oils (Emiroǧlu, Yemiş, Coşkun, & Candoǧan, 2010), as well as some nanosized metals and metallic oxides (Bumbudsanpharoke, Choi, Park, & Ko, 2019). Currently, with the consumers’ increasing awareness for food safety and preference for natural over synthetic products, natural
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antimicrobial substances are becoming attractive candidates for antimicrobial agents in food packaging. As one of the natural antimicrobial substances, grapefruit seed extract (GFSE) exhibits a wide spectrum of microbial growth inhibition against both Gram-positive and Gram-negative bacteria (Heggers et al., 2002). GFSE is generally recognized as safe (GRAS) without human toxicity and has been widely used in various industries such as food processing, pharmaceuticals and cosmetics industries (Reagor, Gusman, McCoy, Carino, & Heggers, 2002; Sakamoto, Sato, Maitani, & Yamada, 1996). The bactericidal and antioxidant activities of GFSE are mainly due to the presence of flavonoids, tocopherol and other polyphenolic compounds (Cvetnić & VladimirKnežević, 2004; Hong, Lim, & Song, 2009). It was reported that the antimicrobial substances in GFSE were stable to heat treatments (Kim, Cheong, & Jae, 1994). According to the thermogravimetric analysis results of GFSE used in this study, the onset of thermal degradation of main ingredients in GFSE was around 170 °C. Hence, GFSE was thermally stable in this study as the processing temperature was much lower than 170 °C. As an antimicrobial agent, GFSE was widely used to blend with various types of natural biopolymers to create antimicrobial films by solution casting technique (Kanmani & Rhim, 2014a, 2014b; Song, Shin, & Song, 2012; Tan, Lim, Tay, Lee, & Thian, 2015). However, the solvent evaporation process during solution casting was difficult to control, and the residue of solvent left on the films might cause safety concern when the films were used as food packaging. Moreover, the
Corresponding author. E-mail address: [email protected] (E.S. Thian).
https://doi.org/10.1016/j.fpsl.2019.100396 Received 25 February 2019; Received in revised form 30 August 2019; Accepted 4 September 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
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obtained films exhibited limited usage due to their relatively poor mechanical and obvious hydrophilic properties (Rhim, Park, & Ha, 2013). Therefore, thermoplastic polymers could be introduced as the base polymers for the preparation of antimicrobial films to improve their mechanical and physical properties. PCL is one of the thermoplastic polymers which has been used extensively for tissue engineering and it is also considered as a promising packaging material because of its non-toxicity and biodegradability. PCL is a semi-crystalline polymer that has a very low glass transition temperature (−60 °C) as well as a low melting point (59–64 °C), which leads to its superior rheological and ductile properties with easy formability by different processing methods at relatively low temperature (Woodruff & Hutmacher, 2010). Therefore, PCL becomes a preferred polymer for blending with other polymers such as starch (Alix et al., 2013), chitosan (Wu, 2005), polylactic acid (PLA) (Luyt & Gasmi, 2016) and polyhydroxybutyrate (PHB) (Correa et al., 2017) to improve the thermal properties, mechanical properties, degradability and processibility of the composites. In this study, PCL was chosen as the base polymer and GFSE as an antimicrobial agent. The temperature required to process PCL by extrusion process was considerably lower than that used to process other thermoplastic polymers, thus reducing the risk of inactivation or degradation of GFSE. However, direct blending of GFSE with PCL would pose difficulties during the processing due to the low compatibility between GFSE and PCL. Furthermore, the antimicrobial effectiveness of the composite films could also be affected. For example, PCL/lemon extract and PCL/thymol films obtained by extrusion processing, their antimicrobial activities could only be maintained for 60 h and 10 h, respectively (Del Nobile et al., 2009). Thus, suitable carriers to include GFSE are required to sustain and prolong its antimicrobial effectiveness, which could be analogous to the significance of carriers for drug delivery systems (Malafaya, Silva, & Reis, 2007). Natural biopolymers are suitable to load with natural antimicrobials such as plant extracts due to their hydrophilic nature. Recently, Wang and Rhim (2016) used starch as a carrier for GFSE to develop antimicrobial polymer films. Chitosan is another abundant natural biopolymer that is inherently antimicrobial and it has been extensively used to prepare antimicrobial films (Dutta et al., 2009) and it has been extensively used to prepare antimicrobial films. Chitosan was incorporated directly as an additive into meat products to inhibit microbial growth and to increase the shelf-life of the meat products during refrigeration (Soultos, Tzikas, Abrahim, Georgantelis, & Ambrosiadis, 2008). Besides the direct incorporation of chitosan into bulk food, it was blended with other thermoplastic polymers to obtain antimicrobial films (Van Den Broek, Knoop, Kappen, & Boeriu, 2015). Recently, chitosan nanoparticles were also used to load natural antimicrobials into food packaging against food-borne pathogens (Lin, Gu, & Cui, 2019). Therefore, chitosan is a suitable carrier for GFSE to facilitate the blending with PCL, and also stabilize the antimicrobial properties of the composite films. The main objectives of the present study were to prepare antimicrobial plastic films based on PCL, chitosan and GFSE for food packaging, and to investigate the effect of different GFSE/chitosan ratios on the film morphology, mechanical and antimicrobial properties of the composite films. Additionally, food packaging test was also conducted to evaluate the effectiveness of the composite films as an antimicrobial packaging.
by Heal Force® SNW Ultrapure Water System, and the resulting water conductivity was 18.2 MΩ cm, with pH value of 7. Commercial polyethylene (PE) packaging films were obtained from Glad® Ziploc. 2.2. Preparation of GFSE/chitosan mixture The chitosan powder was sieved in the Ro-Tap® Sieve Shaker before mixing with GFSE. Chitosan was mixed with GFSE in deionized water and stirred at 24 °C using a magnetic stirrer to form a suspension liquid. The suspension liquid was subjected to freeze-drying using a lab scale lyophilizer to remove water and well-mixed powdery mixture of GFSE/ chitosan was obtained. The volume of GFSE was incorporated in various proportions with respect to the weight of chitosan (vol./wt.): 0.5, 1.0, 1.5, 2.0 and 2.5 mL/g. 2.3. Film preparation Feedstock for the composite films were prepared by mixing the powdery mixture of GFSE/chitosan and PCL resins using a counter rotating twin-roll milling machine at 85 °C, of which the base polymers were composed of 15 wt.% chitosan and 85 wt.% PCL. The extruded PCL/chitosan/GFSE blends were then compressed by a hydraulic press machine at 85 °C under a pressure of 300 MPa. Film samples were obtained once the temperature cooled to room temperature and they were preconditioned in a dry cabinet controlled at 24 °C for at least 48 h before characterization. The different compositions of the films are summarized in Table 1 with the corresponding nomenclatures. 2.4. Film characterization 2.4.1. Morphology of the film surface and cross-section The morphology of film surface and cross-section was observed using field emission scanning electron microscope (FE-SEM, Hitachi S4300, Japan). To prepare the samples for cross-section observations, the films were frozen with liquid nitrogen and then cryo-fractured. Prior to loading into the FE-SEM chamber, the samples were coated with a layer of gold in a sputter coater (JEOL JFC-1200, Japan) for 40 s at 10 mA. 2.4.2. Mechanical properties Mechanical properties were analyzed by using a tensile tester (Model 3345, Instron, USA) with a load cell of 100 N. The mechanical properties of the film samples were determined by the standard method ASTM D882-12. Film samples in rectangular shape of 5 mm in width and 60 mm in length were prepared. Film thickness was measured using a hand-held digital micrometer (Mitutoyo, Mitutoyo Corporation, Japan). Measurements of film thickness were taken randomly at five different locations of each film and the mean thickness was calculated. The film samples were attached between the grips with an initial separation of 10 mm, and the crosshead speed was set at 10 mm/min. Five replicates of each film were evaluated in aspects of their Young’s Modulus, ultimate tensile strength (UTS) and elongation at break (% Ebreak). Commercial PE films were also tested as a reference for commonly used packaging material. Table 1 Formulation of composite films.a
2. Materials and methods 2.1. Materials Poly(ε-caprolactone) (PCL, average Mn 80,000, 2-Oxepanone homopolymer, 6-Caprolactone polymer) in bead form and chitosan (CHT, MW 190–310 kDa, Deacetylation degree of 75–85%) in powder form were purchased from Sigma-Aldrich Pte. Ltd., Singapore. Food grade grapefruit seed extract (GFSE) in aqueous form was purchased from ABC Techno Inc. (Tokyo, Japan). Deionized water was produced
Films
GFSE/chitosan (vol./wt.)
Concentration of GFSE in films
PCL/chitosan GC0.5 GC1.0 GC1.5 GC2.0 GC2.5
0 mL/g 0.5 mL/g 1.0 mL/g 1.5 mL/g 2.0 mL/g 2.5 mL/g
0 wt. % 7.0 wt. % 13.0 wt. % 18.4 wt. % 23.1 wt. % 27.3 wt. %
a
2
The weight ratio between PCL and chitosan was fixed at 85/15.
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Fig. 1. FE-SEM micrographs of (a) surface and (b) cross-section of PCL/chitosan, GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5 films.
samples was carried out sequentially in increasing concentrations of ethanol: 25, 50, 75, 95, 100% v/v, 5 min in each solution. The film samples were dried in a clean cabinet before being viewed under FESEM. The specimens were sputter-coated with a thin layer of gold prior to FE-SEM analysis.
2.4.3. FTIR analysis Fourier transform infrared (FTIR) spectra of film samples were analyzed using an attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (Bruker Vertex 70, USA). Samples were placed on the radial exposing stage and the spectra were recorded at wavenumber ranging from 4000 to 500 cm−1 with the resolution of 4 cm−1.
2.4.6. Microbiological analysis in salmon packaging Fresh sliced salmon were cut into 2 × 3 × 1 cm pieces and sterilized under UV before bacterial reaction. 100 μL E. coli was spread evenly on the salmon surface with an L-shaped spreader. The initial inoculation level of E. coli in the salmon samples was 4.49 ± 0.10 log CFU/g. The salmon samples were packaged by direct contact with the sterilized films and then sealed by a heat sealer. Samples packaged in commercial PE films were used for comparison. All of the samples were stored at 4 °C for 6 days and changes in the microbial growth at every three days were evaluated. Each salmon sample was transferred to a centrifuge tube with 10 mL of peptone water and homogenized for 3 min by using a vortex mixer. Obtained suspensions were filtered by sterile medical gauze and 100 μL was taken and diluted serially. Then, 20 μL of each diluted sample was surface-plated onto TSA and then incubated at 37 °C for another 24 h before the CFU were enumerated.
2.4.4. Release of GFSE from PCL/chitosan/GFSE composite films The evaluation of the release rate of GSFE from PCL/chitosan/GFSE composite films (GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5) was carried out according to the method described by Wang and Rhim (2016). 40 pieces of each film sample with size of 2 × 2 cm were immersed in 40 mL deionized water and stirred slowly using a magnetic stirrer. The temperature of the solutions was maintained at 24 °C and 5 mL of each sample was collected at 24 h intervals for 7 days. UV–vis spectrophotometer (Shimadzu UV-3600, Japan) was used to monitor the released polyphenolic compounds of GFSE by measuring the absorbance of the solution at 262 nm. 2.4.5. In-vitro study on antimicrobial activity of films Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were inoculated into 5 mL of peptone water and Mueller Hinton Broth (MHB), respectively, and subsequently incubated at 37 °C for 18 h. Sterilized films (1 × 1 cm) were placed into well-plate containing 2 mL of corresponding broth, and the wells were immediately inoculated with the E. coli and P. aeruginosa culture prepared to an initial concentration of 104 CFU/mL. The well-plates were then incubated at 37 °C for 6, 24, 72 and 120 h. To determine the bacterial populations adhered on films after incubation, testing films were taken out of the well-plate and immersed in fresh broth. The broth was then treated by ultrasonic to obtain bacterial suspensions from the testing films. Serial dilutions of the resultant bacterial suspensions were surface-plated (20 μL) onto Tryptic Soy Agar (TSA) and Mueller Hinton Agar (MHA) plates, respectively, and then incubated at 37 °C for another 24 h before the CFU were enumerated. A separate experiment was conducted to observe the adherent bacteria on the surface of film samples after 24 h culture. After the film samples were taken out of the well-plates, the films were first fixed with 10% formaldehyde solution for 8 h, and then the dehydration of fixed
2.4.7. Microbiological analysis in bread packaging Preservative-free bread of 2-day shelf life was cut into 4 × 4 × 1 cm pieces and each piece was packaged by direct contact with the sterilized films and then sealed by a heat sealer. Three replicates were prepared for each film. Packaged bread samples were stored at 24 °C, 70% RH for 7 days. Changes in the mold growth on the surface of bread were recorded by photo-taking. Bread samples were packaged by PE films for comparison.
2.5. Statistical analysis All quantitative results were shown as mean ± standard deviation. Analysis of variance (ANOVA) was performed on those results, and the significant differences between mean values were determined by Duncan’s multiple range tests using SPSS 25 statistical software. P values < 0.05 were considered statistically significant. 3
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3. Results and discussion
In this study, the presence of GFSE balanced the stiffness of chitosan particles when they were mixed together, as a result, adjacent PCL became less restrained, and thus the flexibility of composite films was improved. Moreover, Table 2 showed that the value of Young’s Modulus was approaching to tested commercial PE films as GFSE content increased, which revealed the desirable flexibility for packaging. However, the addition of GFSE negatively influenced the UTS and %Ebreak of PCL/chitosan/GFSE films, and %Ebreak was reduced by 48.89% from GC1.0 to GC1.5 films. Usually, adding plasticizer during polymer processing would result in the decease of Young’s Modulus but increase of %Ebreak. In this study, the decrease of %Ebreak was mainly due to the discontinuity of films caused by the phase separation between PCL and chitosan. Similarly, the decreased UTS was due to the weakened interfacial adhesion between chitosan and PCL. As a result, the cohesion of the polymer network forces was affected and the resistance to breakage and stretching was decreased. Similar phenomena were also observed when GFSE was incorporated into agar-based and chitosanbased composite films (Kanmani & Rhim, 2014a; Rubilar et al., 2013). However, it is interesting to notice that increasing GFSE content did not lead to significant deterioration of mechanical properties as Young’s Modulus and UTS decreased slightly when GFSE ≥ 2.0 mL/g, and UTS of PCL/chitosan/GFSE films were still within the range of commonly used plastic films (Bastarrachea, Dhawan, & Sablani, 2011), which was mainly attributed to the superior ductility and stress crack resistance inherited from PCL. Therefore, it could be concluded that adequate mechanical properties of PCL/chitosan/GFSE composite films used for packaging were maintained, and composite films with lower GFSE content exhibited better mechanical properties.
3.1. Morphology of film surface and cross-section The FE-SEM images of film surface and cross-section obtained for PCL/chitosan and PCL/chitosan/GFSE composite films (GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5) are shown in Fig. 1. Noticeable morphological differences were observed between the PCL/chitosan and PCL/ chitosan/GFSE films. It was found that the surface of PCL/chitosan film was homogenous. When GFSE was incorporated, the surface morphology of the films changed considerably, in which increased pits were distributed all over the surface, especially with higher GFSE content. At higher magnification of GC2.5 (inset, Fig. 1a), the aggregation of chitosan particles was found at those pits. Similar microstructures were also observed when plasticized chitosan was incorporated in polyolefin (Matet, Heuzey, Ajji, & Sarazin, 2015), low density polyethylene (Tan, 2016) and metallocene polyethylene (Matet, Heuzey, & Ajji, 2014). Accordingly, those pits could be attributed to the presence of non-volatile plasticizer glycerol in GFSE. Those pits with exposed chitosan on the film surface could facilitate the release of GFSE out of the composite films as GFSE was mainly carried by chitosan due to their hydrophilic nature. The morphology of the cross-section was observed to be heterogeneous, with chitosan in irregular flake-shape embedded in PCL matrix (as pointed by arrows in Fig. 1b). The addition of GFSE to chitosan-PCL blends led to the interfacial debonding of polymers, since the cavities between chitosan particles and PCL matrix became more obvious in the cross-section. So, the chitosan-PCL interactions were modified when GFSE was present in the films. Similarly, the polymer phase separation between PCL and starch was also found when polyethylene glycol (PEG) was added into starch-PCL blends (Ortega-Toro, Muñoz, Talens, & Chiralt, 2016). This could be attributed to the fact that GFSE, as well as PEG has a higher chemical affinity with polysaccharides such as chitosan and starch than with PCL, because of their predominantly hydrophilic character. The addition of GFSE resulted in a poorer interfacial adhesion between chitosan and PCL, and this could lead to the possibility of having weaker tensile strength and elongation at break for PCL/chitosan/GFSE as compared to PCL/chitosan films. In all the composite films, the distribution of chitosan particles in the cross-section images appeared to be compliant enough to adapt their geometry to the stresses applied during processing, which was attributed to the superior rheological property of melting PCL (Woodruff & Hutmacher, 2010). The superior rheological property allows melting PCL to form plastic flow easily under shear stress and pressure during extrusion and compression molding, which made it easier to distribute the applied force during processing (Vlachopoulos & Strutt, 2003). As a result, the distribution of chitosan observed in the cross-section was controlled by the flow pattern of melting PCL, and this phenomenon was also reported by Correlo et al. (2005) in the study of chitosan blended with different melting aliphatic polyesters.
3.3. FTIR analysis The FTIR spectra of the PCL, PCL/chitosan and GC1.0 films are shown in Fig. 2. The FTIR spectra of the PCL/chitosan and GC1.0 films were very similar to that of PCL film, which indicated that chemical structures were preserved without forming appreciable chemical bonding between PCL, chitosan and GFSE. The intensive peak at 1720 cm−1 was related to the stretching vibration of carbonyl. The weak band at 2945 cm−1 was attributed to the asymmetric stretching of eCH2− group. The peaks at 1160 cm−1 represented the CeOeC stretching vibration of PCL. An obvious difference of the FTIR spectra between the films with and without GFSE was that a broad band appeared at the wavenumber between 3600 cm−1 and 3000 cm−1 of the GFSE-loaded film. The broad band represented stretching vibration of free hydroxyl, which was one of the characteristic FTIR band of GFSE (Wang & Rhim, 2016). 3.4. Release of GFSE from PCL/chitosan/GFSE composite films The UV–vis spectra of the deionized water immersed with different PCL/chitosan/GFSE films on day 7 are shown in Fig. 3a. A broad absorption band between 240–280 nm with absorption peak wavelength at 262 nm was found, and the absorbance at 262 nm changed notably along with the increasing GFSE content in composite films. These findings were consistent with related research in which it was reported that for most natural polyphenols, the UV–vis spectra exhibited an absorption band ranging from 240 to 280 nm (Anouar, Gierschner, Duroux, & Trouillas, 2012). Meanwhile, an absorption band with absorption peak wavelength at 320 nm to identify the existence of chitosan (Oh, Chun, & Murugesan, 2019) was not found in Fig. 3a, as no distinct peak was observed after 280 nm and the absorbance afterwards were reduced to zero, which indicated that the release of chitosan from films was negligible as compared to GFSE. Thus, GFSE was the main antimicrobial agent released out of PCL/chitosan/GFSE films and its release rate was evaluated by measuring the absorbance at 262 nm for 7 days. As shown in Fig. 3b, released GFSE increased significantly along with the increasing GFSE content in composite films until it reached
3.2. Mechanical properties Mechanical properties such as ultimate tensile strength (UTS), elongation at break (%Ebreak), and Young’s Modulus of PCL/chitosan, PCL/chitosan/GFSE and PE films, as well as their thicknesses are shown in Table 2. With the incorporation of GFSE, Young’s Modulus decreased from 364.23 ± 11.66 to 160.21 ± 22.92 MPa. The significant decrease of Young’s Modulus was due to the presence of plasticizer glycerol in GFSE as similar phenomena were observed by Martino, Pollet, and Avérous (2011) in the study of plasticized chitosan/PCL composite films, and Matet et al. (2015) in the study of plasticized chitosan/ polyolefin composite films, in which they reported that the decrease of Young’s Modulus was due to the successful chitosan plasticization by the addition of glycerol. Kanmani and Rhim (2014a, 2014b) also reported that GFSE could attenuate the stiffness of polysaccharide-based films, which implied that GFSE conveyed the function as a plasticizer. 4
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Table 2 Mechanical properties of films.* Films
Thickness (mm)
Young's Modulus (MPa)
UTS (MPa)
Ebreak (%)
PE PCL/chitosan GC0.5 GC1.0 GC1.5 GC2.0 GC2.5
0.05 0.12 0.12 0.12 0.12 0.12 0.12
177.05 364.23 293.26 256.99 225.93 160.69 160.21
14.26 ± 0.39a 13.31 ± 0.91b 11.42 ± 0.45c 10.90 ± 0.35 cd 10.14 ± 0.39d 8.91 ± 0.26e 8.81 ± 0.44e
250.09 421.80 394.23 324.26 165.72 163.59 147.80
± ± ± ± ± ± ±
0.00b 0.00a 0.01a 0.00a 0.00a 0.00a 0.01a
± ± ± ± ± ± ±
0.58e 11.66a 13.52b 7.21c 18.03d 10.48e 22.92e
± ± ± ± ± ± ±
25.93c 38.37a 47.08a 21.56b 27.33d 5.92d 2.53d
* Mean values with different letters within the same column are significantly different by Duncan’s multiple range test at p < 0.05.
which it was blended with, and thereby influencing the effectiveness of the antimicrobial activity. 3.5. In-vitro study on antimicrobial activity of films The amount of E. coli and P. aeruginosa adhered on PCL/chitosan and PCL/chitosan/GFSE films with different GFSE content from 6 to 120 h was calculated by reading the number of bacteria colonies plated on agar. As log reduction assay results shown by Fig. 4, the bacterial populations with PCL/chitosan films increased significantly within the first 24 h and reached the plateau afterwards, which indicated that they were not effective against E. coli and P. aeruginosa. Although chitosan possesses inherent antimicrobial properties, it did not exert its antimicrobial effect with PCL/chitosan films. One possible reason could be that the chitosan particles were mostly trapped under PCL surface as shown in Fig. 1, as a result, they could not release out and react on bacteria. In comparison, the GFSE-loaded films showed evident antimicrobial activities against both E. coli and P. aeruginosa when GFSE content reached above 0.5 mL/g and 1.0 mL/g, respectively, as no viable bacterium was detected within the first 24 h and the effectiveness could last for 120 h. The log reduction results were consistent with the release profile of GFSE in Fig. 3b, as adequate amount of GFSE was released out within the first 24 h and it was sustained at a high level for 7 days. The antimicrobial effectiveness was mainly derived from different polyphenols in GFSE released from the films, such as flavan-3-ols and flavonols, which are able to suppress some microbial virulence factors such as inhibition of biofilm formation and reduction of bacterial swarm motility (Daglia, 2012). As shown by FE-SEM images in Fig. 4a, observable E. coli were attached on PCL/chitosan and GC0.5 Films, while no bacterium was found on the surface of GC1.0 film. However, E. coli on GC0.5 film showed different morphology as compared to those on PCL/chitosan film. E. coli on PCL/chitosan film were all rod-shaped, which were consistent with E. coli in normal state, and
Fig. 2. FTIR spectra of (a) PCL (b) PCL/chitosan and (c) GC1.0 films.
2.0 mL/g, which implied that there was a threshold level of free GFSE that could be released out of composite films as some GFSE might be trapped in PCL matrix. Different PCL/chitosan/GFSE films showed similar trends in the release test, in which absorbance at 262 nm of all the samples increased significantly within the first 24 h, and then the absorbance increased steadily with a slower rate until it reached a plateau. Meanwhile, the release rate of GFSE from PCL/chitosan/GFSE composite films was shown to be faster as compared to the films of which GFSE was incorporated into low density polyethylene (LDPE) and polylactic acid (PLA) matrix (Wang & Rhim, 2016). Therefore, the release rate of the antimicrobial agent from composite films was not only affected by its content, but also correlated to the polymer matrix
Fig. 3. (a) UV–vis spectra of deionized water immersed with PCL/chitosan/GFSE films on day 7 (b) Release of GFSE from GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5 films. 5
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Fig. 4. Log reduction assay results of PCL/chitosan, GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5 films against (a) E. coli (b) P. aeruginosa.
the surface of bacteria was smooth and intact. E. coli on GC0.5 film were out of shape and the cell outline was ambiguous, and similar phenomena were also reported in the research which investigated the effectiveness of graphene-based antibiotics against E. coli (Li et al., 2014). This could be attributed to the membrane disruption of E. coli, which indicated their poor living state. GC0.5 film was less effective to P. aeruginosa as compared to E. coli. In Fig. 4b, P. aeruginosa colonies were found on PCL/chitosan and GC0.5 films, while no viable bacterium could be observed on GC1.0 film. The reduced bacterial populations indicated that the antimicrobial activities of PCL/chitosan/GFSE films could maintain after the processing at 85 °C. Overall, in-vitro study demonstrated that PCL/chitosan/GFSE films exhibited excellent bacteriocidal and bacteriostatic abilities against food-borne pathogens E. coli and P. aeruginosa when GFSE content reached 1.0 mL/g.
Table 3 Changes in the populations of E. coli (log CFU/g) in salmon during storage at 4 °C.*,** Films
0 day
PE PCL/chitosan GC0.5 GC1.0 GC1.5 GC2.0 GC2.5
4.49 4.49 4.49 4.49 4.49 4.49 4.49
± ± ± ± ± ± ±
3 days 0.10Ac 0.10Ac 0.10Ac 0.10Ab 0.10Ab 0.10Ab 0.10Ac
7.22 6.00 6.09 5.70 5.68 5.72 5.65
± ± ± ± ± ± ±
6 days 0.06Ab 0.00Bb 0.12Ba 0.00Ca 0.06Ca 0.14Ca 0.09Ca
7.77 7.40 5.25 4.97 5.11 5.04 5.04
± ± ± ± ± ± ±
0.10Aa 0.00Aa 0.07Bb 0.29Bb 0.35Bab 0.37Bab 0.06Bb
* Mean values with different capital letters within the same column are significantly different by Duncan’s multiple range test at p < 0.05. ** Mean values with different small letters within the same row are significantly different by Duncan’s multiple range test at p < 0.05.
3.6. Food packaging test nutrient in salmon, so E. coli grew extensively on the surface as well as inside salmon. As a result, the growth of bacteria was much faster than the diffusion of GFSE, and thus the antimicrobial activity of GFSE was effective mainly on the surface of salmon. After 6 days of storage, the populations of E. coli in the salmon packaged with PE and PCL/chitosan films increased gradually and reached up to 7.77 ± 0.10 log CFU/g and 7.40 ± 0.00 log CFU/g, respectively, whereas the populations of E.
3.6.1. Microbiological analysis in salmon packaging As shown in Table 3, changes in the populations of E. coli (log CFU/ g) in salmon at 4 °C for 6 days were significant. The initial populations of E. coli inoculated on salmon were 4.49 ± 0.10 log CFU/g. The populations of E. coli in the salmon increased within the first three days. The high rate of microbial growth at the beginning was due to the rich 6
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Fig. 5. Bread samples packaged by (a) PE (b) PCL/chitosan (c) GC0.5 (d) GC1.0 (e) GC1.5 (f) GC2.0 and (g) GC2.5 films at 24 °C, 70% RH for 7 days.
coli in those salmon packaged with films containing ≥0.5 mL/g GFSE was ≥2.52 log CFU/g lower as compared with those packaged by PE films. A significant bacterial reduction from 3rd day to 6th day of those samples packaged with PCL/chitosan/GFSE films was found, because more GFSE was diffused into salmon to counter against the bacteria while the growth rate of E. coli slowed down as nutrient level in salmon was reduced. Therefore, PCL/chitosan/GFSE films were effective against the growth of E. coli on packaged salmon during storage.
to the adequate mechanical properties. In-vitro study demonstrated that PCL/chitosan/GFSE films exhibited excellent bacteriocidal and bacteriostatic abilities against different food-borne pathogens when GFSE content reached above 1.0 mL/g. Packaging application test using salmon and bread showed that PCL/chitosan/GFSE films would be greatly beneficial for ensuring food safety and extending the shelf-life of food. In consideration of effective antimicrobial activity, prior mechanical properties as well as cost effectiveness of raw materials, GC 1.0 was suggested as the optimum composition among the PCL/chitosan/ GFSE films.
3.6.2. Microbiological analysis in bread packaging Fig. 5 shows the mold growth on the bread samples packaged by PE, PCL/chitosan and PCL/chitosan/GFSE films on 7th day. Visible mold appeared on the surface of the bread packaged by PE films on 3rd day. Delayed development of mold was observed on the samples packaged by PCL/chitosan and GC0.5 films on 4th day and 5th day, respectively. No mold growth was observed on the samples packaged by films with higher GFSE content (GC1.0, GC1.5, GC2.0, GC2.5) on 7th day. Although bread packaged by PCL/chitosan and GC0.5 films were affected, the mold growth on the samples packaged by PCL/chitosan and GC0.5 films did not spread extensively on 7th day as compared to that of PE films, which suggested that the presence of chitosan and lower GFSE content could also inhibit mold growth to some extent. The fungicidal properties of PCL/chitosan/GFSE films and the prolonged effectiveness as compared to PCL/chitosan films could be due to the synergistic effect from GFSE and chitosan as reported by Xu et al. (2007), in which combined treatment of GFSE and chitosan was found to have longer effectiveness to inhibit gray mold on grapes as compared to using GFSE or chitosan alone. Results from the packaging application test using bread indicated that PCL/chitosan/GFSE films of GFSE content ≥1.0 mL/g exhibited desirable antifungal properties and these films showed great potential to extend the shelf-life of bread.
Acknowledgements This work was supported by the Ministry of Education Academic Research Fund, Singapore [Grant Number R-265-000-592-114]. The author Kaiying Wang would like to thank China Scholarship Council, China for supporting her study in National University of Singapore. References Alix, S., Mahieu, A., Terrie, C., Soulestin, J., Gerault, E., Feuilloley, M. G. J., et al. (2013). Active pseudo-multilayered films from polycaprolactone and starch based matrix for food-packaging applications. European Polymer Journal, 49(6), 1234–1242. Anouar, E. H., Gierschner, J., Duroux, J.-L., & Trouillas, P. (2012). UV/visible spectra of natural polyphenols: A time-dependent density functional theory study. Food Chemistry, 131(1), 79–89. Bastarrachea, L., Dhawan, S., & Sablani, S. S. (2011). Engineering properties of polymeric-based antimicrobial films for food packaging. Food Engineering Reviews, 3(2), 79–93. Bumbudsanpharoke, N., Choi, J., Park, H. J., & Ko, S. (2019). Zinc migration and its effect on the functionality of a low density polyethylene-ZnO nanocomposite film. Food Packaging and Shelf Life, 20 100301. Cagri, A., Ustunol, Z., & Ryser, E. T. (2006). Antimicrobial, mechanical, and moisture barrier properties of low pH whey protein-based edible films containing p-aminobenzoic or sorbic acids. Journal of Food Science, 66(6), 865–870. Correa, J. P., Molina, V., Sanchez, M., Kainz, C., Eisenberg, P., & Massani, M. B. (2017). Improving ham shelf life with a polyhydroxybutyrate/polycaprolactone biodegradable film activated with nisin. Food Packaging and Shelf Life, 11, 31–39. Correlo, V. M., Boesel, L. F., Bhattacharya, M., Mano, J. F., Neves, N. M., & Reis, R. L. (2005). Properties of melt processed chitosan and aliphatic polyester blends. Materials Science and Engineering A, 403(1–2), 57–68. Cvetnić, Z., & Vladimir-Knežević, S. (2004). Antimicrobial activity of grapefruit seed and pulp ethanolic extract. Acta Pharmaceutica, 54(3), 243–250. da Cruz Cabral, L., Fernández Pinto, V., & Patriarca, A. (2013). Application of plant derived compounds to control fungal spoilage and mycotoxin production in foods.
4. Conclusions In this study, we prepared PCL/chitosan/GFSE films without additional chemical reagents by using extrusion compounding and compression molding technique to address the prevailing consumers concerns about the safety of packaged food products. The integrity of PCL/ chitosan/GFSE films as packaging materials was maintained according 7
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International Journal of Food Microbiology, 166(1), 1–14. Daglia, M. (2012). Polyphenols as antimicrobial agents. Current Opinion in Biotechnology, 23(2), 174–181. Del Nobile, M. A., Conte, A., Buonocore, G. G., Incoronato, A. L., Massaro, A., & Panza, O. (2009). Active packaging by extrusion processing of recyclable and biodegradable polymers. Journal of Food Engineering, 93(1), 1–6. Dobiáš, J., Chudackova, K., Voldrich, M., & Marek, M. (2000). Properties of polyethylene films with incorporated benzoic anhydride and/or ethyl and propyl esters of 4-hydroxybenzoic acid and their suitability for food packaging. Food Additives & Contaminants, 17(12), 1047–1053. Dutta, P. K., Tripathi, S., Mehrotra, G. K., & Dutta, J. (2009). Perspectives for chitosan based antimicrobial films in food applications. Food Chemistry, 114(4), 1173–1182. Emiroǧlu, Z. K., Yemiş, G. P., Coşkun, B. K., & Candoǧan, K. (2010). Antimicrobial activity of soy edible films incorporated with thyme and oregano essential oils on fresh ground beef patties. Meat Science, 86(2), 283–288. Heggers, J. P., Cottingham, J., Gusman, J., Reagor, L., McCoy, L., Carino, E., et al. (2002). The effectiveness of processed grapefruit-seed extract as an antibacterial agent: II. Mechanism of action and in vitro toxicity. Journal of Alternative and Complementary Medicine, 8(3), 333–340. Hong, Y. H., Lim, G. O., & Song, K. B. (2009). Physical properties of Gelidium corneumgelatin blend films containing grapefruit seed extract or green tea extract and its application in the packaging of pork loins. Journal of Food Science, 74(1), C6–C10. Irkin, R., & Esmer, O. K. (2015). Novel food packaging systems with natural antimicrobial agents. Journal of Food Science and Technology, 52(10), 6095–6111. Jin, T., Liu, L., Zhang, H., & Hicks, K. (2009). Antimicrobial activity of nisin incorporated in pectin and polylactic acid composite films against Listeria monocytogenes. International Journal of Food Science & Technology, 44(2), 322–329. Kanmani, P., & Rhim, J. W. (2014a). Antimicrobial and physical-mechanical properties of agar-based films incorporated with grapefruit seed extract. Carbohydrate Polymers, 102(1), 708–716. Kanmani, P., & Rhim, J. W. (2014b). Development and characterization of carrageenan/ grapefruit seed extract composite films for active packaging. International Journal of Biological Macromolecules, 68, 258–266. Karabagias, I., Badeka, A., & Kontominas, M. G. (2011). Shelf life extension of lamb meat using thyme or oregano essential oils and modified atmosphere packaging. Meat Science, 88(1), 109–116. Kim, Y. M., Cheong, N. E., & Jae, D. Y. (1994). Characterization of an antimicrobial chitinase purified from grapefruit seed extract. Korean Journal of Plant Pathology, 10, 277–283. Li, J., Wang, G., Zhu, H., Zhang, M., Zheng, X., Di, Z., et al. (2014). Antibacterial activity of large-area monolayer graphene film manipulated by charge transfer. Scientific Reports, 4, 4359. Lin, L., Gu, Y., & Cui, H. (2019). Moringa oil/chitosan nanoparticles embedded gelatin nanofibers for food packaging against Listeria monocytogenes and Staphylococcus aureus on cheese. Food Packaging and Shelf Life, 19, 86–93. Luyt, A. S., & Gasmi, S. (2016). Influence of blending and blend morphology on the thermal properties and crystallization behaviour of PLA and PCL in PLA/PCL blends. Journal of Materials Science, 51(9), 4670–4681. Malafaya, P. B., Silva, G. A., & Reis, R. L. (2007). Natural–origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications. Advanced Drug Delivery Reviews, 59(4), 207–233. Martino, V. P., Pollet, E., & Avérous, L. (2011). Novative biomaterials based on chitosan and poly(ε-Caprolactone): Elaboration of porous structures. Journal of Polymers and the Environment, 19(4), 819–826. Matet, M., Heuzey, M. C., & Ajji, A. (2014). Morphology and antibacterial properties of plasticized chitosan/metallocene polyethylene blends. Journal of Materials Science,
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Development of grapefruit seed extract-loaded poly(ε-caprolactone)/ chitosan films for antimicrobial food packaging Kaiying Wang, Poon Nian Lim, Shi Yun Tong, Eng San Thian
T
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Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobial Chitosan Food packaging Grapefruit seed extract Poly(ε-caprolactone)
In this study, the packaging films were prepared by blending poly(ε-caprolactone) (PCL) with chitosan and grapefruit seed extract (GFSE) using extrusion compounding followed by compression molding. Different GFSE/ chitosan ratios (0.5, 1.0, 1.5, 2.0 and 2.5 mL/g) affected the surface morphology, mechanical and antimicrobial properties of the films. Although the addition of GFSE decreased the films’ resistance to breakage and stretching, the films retained adequate tensile strength and desirable flexibility for packaging. More notably, PCL/chitosan/ GFSE films were effective against both Escherichia coli and Pseudomonas aeruginosa and could inhibit the bacterial growth for up to 120 h when GFSE content increased to 0.5 mL/g and 1.0 mL/g, respectively. Packaging application test using salmon showed that PCL/chitosan/GFSE films were effective to inhibit the growth of E. coli in salmon during storage. Furthermore, no mold growth was observed on the bread packaged with films containing ≥1.0 mL/g GFSE after 7 days.
1. Introduction The global wide attention towards maintaining food safety and extending the shelf-life of packaged food has triggered an increased interest in developing antimicrobial packaging. In general, antimicrobial packaging is achieved by directly incorporating antimicrobial agents into packaging films, coating packaging films with antimicrobial substances, and packaging materials made up of polymers with inherent antimicrobial properties (Irkin & Esmer, 2015). Numerous antimicrobial agents have been used to develop antimicrobial food packaging films. They include organic acids such as benzoic acid (Dobiáš, Chudackova, Voldrich, & Marek, 2000), citric acid (Ramirez et al., 2017) and sorbic acid (Cagri, Ustunol, & Ryser, 2006), enzymes such as lysozyme (Mecitoǧlu et al., 2006), bacteriocins such as nisin (Jin, Liu, Zhang, & Hicks, 2009), polymers such as chitosan (Dutta, Tripathi, Mehrotra, & Dutta, 2009), plant extracts such as clove extract (da Cruz Cabral, Fernández Pinto, & Patriarca, 2013) and cinnamaldehyde (Qin, Yang, & Xue, 2016), essential oils such as thyme essential oils (Karabagias, Badeka, & Kontominas, 2011) and oregano essential oils (Emiroǧlu, Yemiş, Coşkun, & Candoǧan, 2010), as well as some nanosized metals and metallic oxides (Bumbudsanpharoke, Choi, Park, & Ko, 2019). Currently, with the consumers’ increasing awareness for food safety and preference for natural over synthetic products, natural
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antimicrobial substances are becoming attractive candidates for antimicrobial agents in food packaging. As one of the natural antimicrobial substances, grapefruit seed extract (GFSE) exhibits a wide spectrum of microbial growth inhibition against both Gram-positive and Gram-negative bacteria (Heggers et al., 2002). GFSE is generally recognized as safe (GRAS) without human toxicity and has been widely used in various industries such as food processing, pharmaceuticals and cosmetics industries (Reagor, Gusman, McCoy, Carino, & Heggers, 2002; Sakamoto, Sato, Maitani, & Yamada, 1996). The bactericidal and antioxidant activities of GFSE are mainly due to the presence of flavonoids, tocopherol and other polyphenolic compounds (Cvetnić & VladimirKnežević, 2004; Hong, Lim, & Song, 2009). It was reported that the antimicrobial substances in GFSE were stable to heat treatments (Kim, Cheong, & Jae, 1994). According to the thermogravimetric analysis results of GFSE used in this study, the onset of thermal degradation of main ingredients in GFSE was around 170 °C. Hence, GFSE was thermally stable in this study as the processing temperature was much lower than 170 °C. As an antimicrobial agent, GFSE was widely used to blend with various types of natural biopolymers to create antimicrobial films by solution casting technique (Kanmani & Rhim, 2014a, 2014b; Song, Shin, & Song, 2012; Tan, Lim, Tay, Lee, & Thian, 2015). However, the solvent evaporation process during solution casting was difficult to control, and the residue of solvent left on the films might cause safety concern when the films were used as food packaging. Moreover, the
Corresponding author. E-mail address: [email protected] (E.S. Thian).
https://doi.org/10.1016/j.fpsl.2019.100396 Received 25 February 2019; Received in revised form 30 August 2019; Accepted 4 September 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
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obtained films exhibited limited usage due to their relatively poor mechanical and obvious hydrophilic properties (Rhim, Park, & Ha, 2013). Therefore, thermoplastic polymers could be introduced as the base polymers for the preparation of antimicrobial films to improve their mechanical and physical properties. PCL is one of the thermoplastic polymers which has been used extensively for tissue engineering and it is also considered as a promising packaging material because of its non-toxicity and biodegradability. PCL is a semi-crystalline polymer that has a very low glass transition temperature (−60 °C) as well as a low melting point (59–64 °C), which leads to its superior rheological and ductile properties with easy formability by different processing methods at relatively low temperature (Woodruff & Hutmacher, 2010). Therefore, PCL becomes a preferred polymer for blending with other polymers such as starch (Alix et al., 2013), chitosan (Wu, 2005), polylactic acid (PLA) (Luyt & Gasmi, 2016) and polyhydroxybutyrate (PHB) (Correa et al., 2017) to improve the thermal properties, mechanical properties, degradability and processibility of the composites. In this study, PCL was chosen as the base polymer and GFSE as an antimicrobial agent. The temperature required to process PCL by extrusion process was considerably lower than that used to process other thermoplastic polymers, thus reducing the risk of inactivation or degradation of GFSE. However, direct blending of GFSE with PCL would pose difficulties during the processing due to the low compatibility between GFSE and PCL. Furthermore, the antimicrobial effectiveness of the composite films could also be affected. For example, PCL/lemon extract and PCL/thymol films obtained by extrusion processing, their antimicrobial activities could only be maintained for 60 h and 10 h, respectively (Del Nobile et al., 2009). Thus, suitable carriers to include GFSE are required to sustain and prolong its antimicrobial effectiveness, which could be analogous to the significance of carriers for drug delivery systems (Malafaya, Silva, & Reis, 2007). Natural biopolymers are suitable to load with natural antimicrobials such as plant extracts due to their hydrophilic nature. Recently, Wang and Rhim (2016) used starch as a carrier for GFSE to develop antimicrobial polymer films. Chitosan is another abundant natural biopolymer that is inherently antimicrobial and it has been extensively used to prepare antimicrobial films (Dutta et al., 2009) and it has been extensively used to prepare antimicrobial films. Chitosan was incorporated directly as an additive into meat products to inhibit microbial growth and to increase the shelf-life of the meat products during refrigeration (Soultos, Tzikas, Abrahim, Georgantelis, & Ambrosiadis, 2008). Besides the direct incorporation of chitosan into bulk food, it was blended with other thermoplastic polymers to obtain antimicrobial films (Van Den Broek, Knoop, Kappen, & Boeriu, 2015). Recently, chitosan nanoparticles were also used to load natural antimicrobials into food packaging against food-borne pathogens (Lin, Gu, & Cui, 2019). Therefore, chitosan is a suitable carrier for GFSE to facilitate the blending with PCL, and also stabilize the antimicrobial properties of the composite films. The main objectives of the present study were to prepare antimicrobial plastic films based on PCL, chitosan and GFSE for food packaging, and to investigate the effect of different GFSE/chitosan ratios on the film morphology, mechanical and antimicrobial properties of the composite films. Additionally, food packaging test was also conducted to evaluate the effectiveness of the composite films as an antimicrobial packaging.
by Heal Force® SNW Ultrapure Water System, and the resulting water conductivity was 18.2 MΩ cm, with pH value of 7. Commercial polyethylene (PE) packaging films were obtained from Glad® Ziploc. 2.2. Preparation of GFSE/chitosan mixture The chitosan powder was sieved in the Ro-Tap® Sieve Shaker before mixing with GFSE. Chitosan was mixed with GFSE in deionized water and stirred at 24 °C using a magnetic stirrer to form a suspension liquid. The suspension liquid was subjected to freeze-drying using a lab scale lyophilizer to remove water and well-mixed powdery mixture of GFSE/ chitosan was obtained. The volume of GFSE was incorporated in various proportions with respect to the weight of chitosan (vol./wt.): 0.5, 1.0, 1.5, 2.0 and 2.5 mL/g. 2.3. Film preparation Feedstock for the composite films were prepared by mixing the powdery mixture of GFSE/chitosan and PCL resins using a counter rotating twin-roll milling machine at 85 °C, of which the base polymers were composed of 15 wt.% chitosan and 85 wt.% PCL. The extruded PCL/chitosan/GFSE blends were then compressed by a hydraulic press machine at 85 °C under a pressure of 300 MPa. Film samples were obtained once the temperature cooled to room temperature and they were preconditioned in a dry cabinet controlled at 24 °C for at least 48 h before characterization. The different compositions of the films are summarized in Table 1 with the corresponding nomenclatures. 2.4. Film characterization 2.4.1. Morphology of the film surface and cross-section The morphology of film surface and cross-section was observed using field emission scanning electron microscope (FE-SEM, Hitachi S4300, Japan). To prepare the samples for cross-section observations, the films were frozen with liquid nitrogen and then cryo-fractured. Prior to loading into the FE-SEM chamber, the samples were coated with a layer of gold in a sputter coater (JEOL JFC-1200, Japan) for 40 s at 10 mA. 2.4.2. Mechanical properties Mechanical properties were analyzed by using a tensile tester (Model 3345, Instron, USA) with a load cell of 100 N. The mechanical properties of the film samples were determined by the standard method ASTM D882-12. Film samples in rectangular shape of 5 mm in width and 60 mm in length were prepared. Film thickness was measured using a hand-held digital micrometer (Mitutoyo, Mitutoyo Corporation, Japan). Measurements of film thickness were taken randomly at five different locations of each film and the mean thickness was calculated. The film samples were attached between the grips with an initial separation of 10 mm, and the crosshead speed was set at 10 mm/min. Five replicates of each film were evaluated in aspects of their Young’s Modulus, ultimate tensile strength (UTS) and elongation at break (% Ebreak). Commercial PE films were also tested as a reference for commonly used packaging material. Table 1 Formulation of composite films.a
2. Materials and methods 2.1. Materials Poly(ε-caprolactone) (PCL, average Mn 80,000, 2-Oxepanone homopolymer, 6-Caprolactone polymer) in bead form and chitosan (CHT, MW 190–310 kDa, Deacetylation degree of 75–85%) in powder form were purchased from Sigma-Aldrich Pte. Ltd., Singapore. Food grade grapefruit seed extract (GFSE) in aqueous form was purchased from ABC Techno Inc. (Tokyo, Japan). Deionized water was produced
Films
GFSE/chitosan (vol./wt.)
Concentration of GFSE in films
PCL/chitosan GC0.5 GC1.0 GC1.5 GC2.0 GC2.5
0 mL/g 0.5 mL/g 1.0 mL/g 1.5 mL/g 2.0 mL/g 2.5 mL/g
0 wt. % 7.0 wt. % 13.0 wt. % 18.4 wt. % 23.1 wt. % 27.3 wt. %
a
2
The weight ratio between PCL and chitosan was fixed at 85/15.
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Fig. 1. FE-SEM micrographs of (a) surface and (b) cross-section of PCL/chitosan, GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5 films.
samples was carried out sequentially in increasing concentrations of ethanol: 25, 50, 75, 95, 100% v/v, 5 min in each solution. The film samples were dried in a clean cabinet before being viewed under FESEM. The specimens were sputter-coated with a thin layer of gold prior to FE-SEM analysis.
2.4.3. FTIR analysis Fourier transform infrared (FTIR) spectra of film samples were analyzed using an attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (Bruker Vertex 70, USA). Samples were placed on the radial exposing stage and the spectra were recorded at wavenumber ranging from 4000 to 500 cm−1 with the resolution of 4 cm−1.
2.4.6. Microbiological analysis in salmon packaging Fresh sliced salmon were cut into 2 × 3 × 1 cm pieces and sterilized under UV before bacterial reaction. 100 μL E. coli was spread evenly on the salmon surface with an L-shaped spreader. The initial inoculation level of E. coli in the salmon samples was 4.49 ± 0.10 log CFU/g. The salmon samples were packaged by direct contact with the sterilized films and then sealed by a heat sealer. Samples packaged in commercial PE films were used for comparison. All of the samples were stored at 4 °C for 6 days and changes in the microbial growth at every three days were evaluated. Each salmon sample was transferred to a centrifuge tube with 10 mL of peptone water and homogenized for 3 min by using a vortex mixer. Obtained suspensions were filtered by sterile medical gauze and 100 μL was taken and diluted serially. Then, 20 μL of each diluted sample was surface-plated onto TSA and then incubated at 37 °C for another 24 h before the CFU were enumerated.
2.4.4. Release of GFSE from PCL/chitosan/GFSE composite films The evaluation of the release rate of GSFE from PCL/chitosan/GFSE composite films (GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5) was carried out according to the method described by Wang and Rhim (2016). 40 pieces of each film sample with size of 2 × 2 cm were immersed in 40 mL deionized water and stirred slowly using a magnetic stirrer. The temperature of the solutions was maintained at 24 °C and 5 mL of each sample was collected at 24 h intervals for 7 days. UV–vis spectrophotometer (Shimadzu UV-3600, Japan) was used to monitor the released polyphenolic compounds of GFSE by measuring the absorbance of the solution at 262 nm. 2.4.5. In-vitro study on antimicrobial activity of films Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were inoculated into 5 mL of peptone water and Mueller Hinton Broth (MHB), respectively, and subsequently incubated at 37 °C for 18 h. Sterilized films (1 × 1 cm) were placed into well-plate containing 2 mL of corresponding broth, and the wells were immediately inoculated with the E. coli and P. aeruginosa culture prepared to an initial concentration of 104 CFU/mL. The well-plates were then incubated at 37 °C for 6, 24, 72 and 120 h. To determine the bacterial populations adhered on films after incubation, testing films were taken out of the well-plate and immersed in fresh broth. The broth was then treated by ultrasonic to obtain bacterial suspensions from the testing films. Serial dilutions of the resultant bacterial suspensions were surface-plated (20 μL) onto Tryptic Soy Agar (TSA) and Mueller Hinton Agar (MHA) plates, respectively, and then incubated at 37 °C for another 24 h before the CFU were enumerated. A separate experiment was conducted to observe the adherent bacteria on the surface of film samples after 24 h culture. After the film samples were taken out of the well-plates, the films were first fixed with 10% formaldehyde solution for 8 h, and then the dehydration of fixed
2.4.7. Microbiological analysis in bread packaging Preservative-free bread of 2-day shelf life was cut into 4 × 4 × 1 cm pieces and each piece was packaged by direct contact with the sterilized films and then sealed by a heat sealer. Three replicates were prepared for each film. Packaged bread samples were stored at 24 °C, 70% RH for 7 days. Changes in the mold growth on the surface of bread were recorded by photo-taking. Bread samples were packaged by PE films for comparison.
2.5. Statistical analysis All quantitative results were shown as mean ± standard deviation. Analysis of variance (ANOVA) was performed on those results, and the significant differences between mean values were determined by Duncan’s multiple range tests using SPSS 25 statistical software. P values < 0.05 were considered statistically significant. 3
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3. Results and discussion
In this study, the presence of GFSE balanced the stiffness of chitosan particles when they were mixed together, as a result, adjacent PCL became less restrained, and thus the flexibility of composite films was improved. Moreover, Table 2 showed that the value of Young’s Modulus was approaching to tested commercial PE films as GFSE content increased, which revealed the desirable flexibility for packaging. However, the addition of GFSE negatively influenced the UTS and %Ebreak of PCL/chitosan/GFSE films, and %Ebreak was reduced by 48.89% from GC1.0 to GC1.5 films. Usually, adding plasticizer during polymer processing would result in the decease of Young’s Modulus but increase of %Ebreak. In this study, the decrease of %Ebreak was mainly due to the discontinuity of films caused by the phase separation between PCL and chitosan. Similarly, the decreased UTS was due to the weakened interfacial adhesion between chitosan and PCL. As a result, the cohesion of the polymer network forces was affected and the resistance to breakage and stretching was decreased. Similar phenomena were also observed when GFSE was incorporated into agar-based and chitosanbased composite films (Kanmani & Rhim, 2014a; Rubilar et al., 2013). However, it is interesting to notice that increasing GFSE content did not lead to significant deterioration of mechanical properties as Young’s Modulus and UTS decreased slightly when GFSE ≥ 2.0 mL/g, and UTS of PCL/chitosan/GFSE films were still within the range of commonly used plastic films (Bastarrachea, Dhawan, & Sablani, 2011), which was mainly attributed to the superior ductility and stress crack resistance inherited from PCL. Therefore, it could be concluded that adequate mechanical properties of PCL/chitosan/GFSE composite films used for packaging were maintained, and composite films with lower GFSE content exhibited better mechanical properties.
3.1. Morphology of film surface and cross-section The FE-SEM images of film surface and cross-section obtained for PCL/chitosan and PCL/chitosan/GFSE composite films (GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5) are shown in Fig. 1. Noticeable morphological differences were observed between the PCL/chitosan and PCL/ chitosan/GFSE films. It was found that the surface of PCL/chitosan film was homogenous. When GFSE was incorporated, the surface morphology of the films changed considerably, in which increased pits were distributed all over the surface, especially with higher GFSE content. At higher magnification of GC2.5 (inset, Fig. 1a), the aggregation of chitosan particles was found at those pits. Similar microstructures were also observed when plasticized chitosan was incorporated in polyolefin (Matet, Heuzey, Ajji, & Sarazin, 2015), low density polyethylene (Tan, 2016) and metallocene polyethylene (Matet, Heuzey, & Ajji, 2014). Accordingly, those pits could be attributed to the presence of non-volatile plasticizer glycerol in GFSE. Those pits with exposed chitosan on the film surface could facilitate the release of GFSE out of the composite films as GFSE was mainly carried by chitosan due to their hydrophilic nature. The morphology of the cross-section was observed to be heterogeneous, with chitosan in irregular flake-shape embedded in PCL matrix (as pointed by arrows in Fig. 1b). The addition of GFSE to chitosan-PCL blends led to the interfacial debonding of polymers, since the cavities between chitosan particles and PCL matrix became more obvious in the cross-section. So, the chitosan-PCL interactions were modified when GFSE was present in the films. Similarly, the polymer phase separation between PCL and starch was also found when polyethylene glycol (PEG) was added into starch-PCL blends (Ortega-Toro, Muñoz, Talens, & Chiralt, 2016). This could be attributed to the fact that GFSE, as well as PEG has a higher chemical affinity with polysaccharides such as chitosan and starch than with PCL, because of their predominantly hydrophilic character. The addition of GFSE resulted in a poorer interfacial adhesion between chitosan and PCL, and this could lead to the possibility of having weaker tensile strength and elongation at break for PCL/chitosan/GFSE as compared to PCL/chitosan films. In all the composite films, the distribution of chitosan particles in the cross-section images appeared to be compliant enough to adapt their geometry to the stresses applied during processing, which was attributed to the superior rheological property of melting PCL (Woodruff & Hutmacher, 2010). The superior rheological property allows melting PCL to form plastic flow easily under shear stress and pressure during extrusion and compression molding, which made it easier to distribute the applied force during processing (Vlachopoulos & Strutt, 2003). As a result, the distribution of chitosan observed in the cross-section was controlled by the flow pattern of melting PCL, and this phenomenon was also reported by Correlo et al. (2005) in the study of chitosan blended with different melting aliphatic polyesters.
3.3. FTIR analysis The FTIR spectra of the PCL, PCL/chitosan and GC1.0 films are shown in Fig. 2. The FTIR spectra of the PCL/chitosan and GC1.0 films were very similar to that of PCL film, which indicated that chemical structures were preserved without forming appreciable chemical bonding between PCL, chitosan and GFSE. The intensive peak at 1720 cm−1 was related to the stretching vibration of carbonyl. The weak band at 2945 cm−1 was attributed to the asymmetric stretching of eCH2− group. The peaks at 1160 cm−1 represented the CeOeC stretching vibration of PCL. An obvious difference of the FTIR spectra between the films with and without GFSE was that a broad band appeared at the wavenumber between 3600 cm−1 and 3000 cm−1 of the GFSE-loaded film. The broad band represented stretching vibration of free hydroxyl, which was one of the characteristic FTIR band of GFSE (Wang & Rhim, 2016). 3.4. Release of GFSE from PCL/chitosan/GFSE composite films The UV–vis spectra of the deionized water immersed with different PCL/chitosan/GFSE films on day 7 are shown in Fig. 3a. A broad absorption band between 240–280 nm with absorption peak wavelength at 262 nm was found, and the absorbance at 262 nm changed notably along with the increasing GFSE content in composite films. These findings were consistent with related research in which it was reported that for most natural polyphenols, the UV–vis spectra exhibited an absorption band ranging from 240 to 280 nm (Anouar, Gierschner, Duroux, & Trouillas, 2012). Meanwhile, an absorption band with absorption peak wavelength at 320 nm to identify the existence of chitosan (Oh, Chun, & Murugesan, 2019) was not found in Fig. 3a, as no distinct peak was observed after 280 nm and the absorbance afterwards were reduced to zero, which indicated that the release of chitosan from films was negligible as compared to GFSE. Thus, GFSE was the main antimicrobial agent released out of PCL/chitosan/GFSE films and its release rate was evaluated by measuring the absorbance at 262 nm for 7 days. As shown in Fig. 3b, released GFSE increased significantly along with the increasing GFSE content in composite films until it reached
3.2. Mechanical properties Mechanical properties such as ultimate tensile strength (UTS), elongation at break (%Ebreak), and Young’s Modulus of PCL/chitosan, PCL/chitosan/GFSE and PE films, as well as their thicknesses are shown in Table 2. With the incorporation of GFSE, Young’s Modulus decreased from 364.23 ± 11.66 to 160.21 ± 22.92 MPa. The significant decrease of Young’s Modulus was due to the presence of plasticizer glycerol in GFSE as similar phenomena were observed by Martino, Pollet, and Avérous (2011) in the study of plasticized chitosan/PCL composite films, and Matet et al. (2015) in the study of plasticized chitosan/ polyolefin composite films, in which they reported that the decrease of Young’s Modulus was due to the successful chitosan plasticization by the addition of glycerol. Kanmani and Rhim (2014a, 2014b) also reported that GFSE could attenuate the stiffness of polysaccharide-based films, which implied that GFSE conveyed the function as a plasticizer. 4
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Table 2 Mechanical properties of films.* Films
Thickness (mm)
Young's Modulus (MPa)
UTS (MPa)
Ebreak (%)
PE PCL/chitosan GC0.5 GC1.0 GC1.5 GC2.0 GC2.5
0.05 0.12 0.12 0.12 0.12 0.12 0.12
177.05 364.23 293.26 256.99 225.93 160.69 160.21
14.26 ± 0.39a 13.31 ± 0.91b 11.42 ± 0.45c 10.90 ± 0.35 cd 10.14 ± 0.39d 8.91 ± 0.26e 8.81 ± 0.44e
250.09 421.80 394.23 324.26 165.72 163.59 147.80
± ± ± ± ± ± ±
0.00b 0.00a 0.01a 0.00a 0.00a 0.00a 0.01a
± ± ± ± ± ± ±
0.58e 11.66a 13.52b 7.21c 18.03d 10.48e 22.92e
± ± ± ± ± ± ±
25.93c 38.37a 47.08a 21.56b 27.33d 5.92d 2.53d
* Mean values with different letters within the same column are significantly different by Duncan’s multiple range test at p < 0.05.
which it was blended with, and thereby influencing the effectiveness of the antimicrobial activity. 3.5. In-vitro study on antimicrobial activity of films The amount of E. coli and P. aeruginosa adhered on PCL/chitosan and PCL/chitosan/GFSE films with different GFSE content from 6 to 120 h was calculated by reading the number of bacteria colonies plated on agar. As log reduction assay results shown by Fig. 4, the bacterial populations with PCL/chitosan films increased significantly within the first 24 h and reached the plateau afterwards, which indicated that they were not effective against E. coli and P. aeruginosa. Although chitosan possesses inherent antimicrobial properties, it did not exert its antimicrobial effect with PCL/chitosan films. One possible reason could be that the chitosan particles were mostly trapped under PCL surface as shown in Fig. 1, as a result, they could not release out and react on bacteria. In comparison, the GFSE-loaded films showed evident antimicrobial activities against both E. coli and P. aeruginosa when GFSE content reached above 0.5 mL/g and 1.0 mL/g, respectively, as no viable bacterium was detected within the first 24 h and the effectiveness could last for 120 h. The log reduction results were consistent with the release profile of GFSE in Fig. 3b, as adequate amount of GFSE was released out within the first 24 h and it was sustained at a high level for 7 days. The antimicrobial effectiveness was mainly derived from different polyphenols in GFSE released from the films, such as flavan-3-ols and flavonols, which are able to suppress some microbial virulence factors such as inhibition of biofilm formation and reduction of bacterial swarm motility (Daglia, 2012). As shown by FE-SEM images in Fig. 4a, observable E. coli were attached on PCL/chitosan and GC0.5 Films, while no bacterium was found on the surface of GC1.0 film. However, E. coli on GC0.5 film showed different morphology as compared to those on PCL/chitosan film. E. coli on PCL/chitosan film were all rod-shaped, which were consistent with E. coli in normal state, and
Fig. 2. FTIR spectra of (a) PCL (b) PCL/chitosan and (c) GC1.0 films.
2.0 mL/g, which implied that there was a threshold level of free GFSE that could be released out of composite films as some GFSE might be trapped in PCL matrix. Different PCL/chitosan/GFSE films showed similar trends in the release test, in which absorbance at 262 nm of all the samples increased significantly within the first 24 h, and then the absorbance increased steadily with a slower rate until it reached a plateau. Meanwhile, the release rate of GFSE from PCL/chitosan/GFSE composite films was shown to be faster as compared to the films of which GFSE was incorporated into low density polyethylene (LDPE) and polylactic acid (PLA) matrix (Wang & Rhim, 2016). Therefore, the release rate of the antimicrobial agent from composite films was not only affected by its content, but also correlated to the polymer matrix
Fig. 3. (a) UV–vis spectra of deionized water immersed with PCL/chitosan/GFSE films on day 7 (b) Release of GFSE from GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5 films. 5
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Fig. 4. Log reduction assay results of PCL/chitosan, GC0.5, GC1.0, GC1.5, GC2.0 and GC2.5 films against (a) E. coli (b) P. aeruginosa.
the surface of bacteria was smooth and intact. E. coli on GC0.5 film were out of shape and the cell outline was ambiguous, and similar phenomena were also reported in the research which investigated the effectiveness of graphene-based antibiotics against E. coli (Li et al., 2014). This could be attributed to the membrane disruption of E. coli, which indicated their poor living state. GC0.5 film was less effective to P. aeruginosa as compared to E. coli. In Fig. 4b, P. aeruginosa colonies were found on PCL/chitosan and GC0.5 films, while no viable bacterium could be observed on GC1.0 film. The reduced bacterial populations indicated that the antimicrobial activities of PCL/chitosan/GFSE films could maintain after the processing at 85 °C. Overall, in-vitro study demonstrated that PCL/chitosan/GFSE films exhibited excellent bacteriocidal and bacteriostatic abilities against food-borne pathogens E. coli and P. aeruginosa when GFSE content reached 1.0 mL/g.
Table 3 Changes in the populations of E. coli (log CFU/g) in salmon during storage at 4 °C.*,** Films
0 day
PE PCL/chitosan GC0.5 GC1.0 GC1.5 GC2.0 GC2.5
4.49 4.49 4.49 4.49 4.49 4.49 4.49
± ± ± ± ± ± ±
3 days 0.10Ac 0.10Ac 0.10Ac 0.10Ab 0.10Ab 0.10Ab 0.10Ac
7.22 6.00 6.09 5.70 5.68 5.72 5.65
± ± ± ± ± ± ±
6 days 0.06Ab 0.00Bb 0.12Ba 0.00Ca 0.06Ca 0.14Ca 0.09Ca
7.77 7.40 5.25 4.97 5.11 5.04 5.04
± ± ± ± ± ± ±
0.10Aa 0.00Aa 0.07Bb 0.29Bb 0.35Bab 0.37Bab 0.06Bb
* Mean values with different capital letters within the same column are significantly different by Duncan’s multiple range test at p < 0.05. ** Mean values with different small letters within the same row are significantly different by Duncan’s multiple range test at p < 0.05.
3.6. Food packaging test nutrient in salmon, so E. coli grew extensively on the surface as well as inside salmon. As a result, the growth of bacteria was much faster than the diffusion of GFSE, and thus the antimicrobial activity of GFSE was effective mainly on the surface of salmon. After 6 days of storage, the populations of E. coli in the salmon packaged with PE and PCL/chitosan films increased gradually and reached up to 7.77 ± 0.10 log CFU/g and 7.40 ± 0.00 log CFU/g, respectively, whereas the populations of E.
3.6.1. Microbiological analysis in salmon packaging As shown in Table 3, changes in the populations of E. coli (log CFU/ g) in salmon at 4 °C for 6 days were significant. The initial populations of E. coli inoculated on salmon were 4.49 ± 0.10 log CFU/g. The populations of E. coli in the salmon increased within the first three days. The high rate of microbial growth at the beginning was due to the rich 6
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Fig. 5. Bread samples packaged by (a) PE (b) PCL/chitosan (c) GC0.5 (d) GC1.0 (e) GC1.5 (f) GC2.0 and (g) GC2.5 films at 24 °C, 70% RH for 7 days.
coli in those salmon packaged with films containing ≥0.5 mL/g GFSE was ≥2.52 log CFU/g lower as compared with those packaged by PE films. A significant bacterial reduction from 3rd day to 6th day of those samples packaged with PCL/chitosan/GFSE films was found, because more GFSE was diffused into salmon to counter against the bacteria while the growth rate of E. coli slowed down as nutrient level in salmon was reduced. Therefore, PCL/chitosan/GFSE films were effective against the growth of E. coli on packaged salmon during storage.
to the adequate mechanical properties. In-vitro study demonstrated that PCL/chitosan/GFSE films exhibited excellent bacteriocidal and bacteriostatic abilities against different food-borne pathogens when GFSE content reached above 1.0 mL/g. Packaging application test using salmon and bread showed that PCL/chitosan/GFSE films would be greatly beneficial for ensuring food safety and extending the shelf-life of food. In consideration of effective antimicrobial activity, prior mechanical properties as well as cost effectiveness of raw materials, GC 1.0 was suggested as the optimum composition among the PCL/chitosan/ GFSE films.
3.6.2. Microbiological analysis in bread packaging Fig. 5 shows the mold growth on the bread samples packaged by PE, PCL/chitosan and PCL/chitosan/GFSE films on 7th day. Visible mold appeared on the surface of the bread packaged by PE films on 3rd day. Delayed development of mold was observed on the samples packaged by PCL/chitosan and GC0.5 films on 4th day and 5th day, respectively. No mold growth was observed on the samples packaged by films with higher GFSE content (GC1.0, GC1.5, GC2.0, GC2.5) on 7th day. Although bread packaged by PCL/chitosan and GC0.5 films were affected, the mold growth on the samples packaged by PCL/chitosan and GC0.5 films did not spread extensively on 7th day as compared to that of PE films, which suggested that the presence of chitosan and lower GFSE content could also inhibit mold growth to some extent. The fungicidal properties of PCL/chitosan/GFSE films and the prolonged effectiveness as compared to PCL/chitosan films could be due to the synergistic effect from GFSE and chitosan as reported by Xu et al. (2007), in which combined treatment of GFSE and chitosan was found to have longer effectiveness to inhibit gray mold on grapes as compared to using GFSE or chitosan alone. Results from the packaging application test using bread indicated that PCL/chitosan/GFSE films of GFSE content ≥1.0 mL/g exhibited desirable antifungal properties and these films showed great potential to extend the shelf-life of bread.
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