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Development and characterization of biodegradable antimicrobial packaging films based on polycaprolactone, starch and pomegranate rind hybrids
Food Packaging and Shelf Life 18 (2018) 71–79
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Development and characterization of biodegradable antimicrobial packaging films based on polycaprolactone, starch and pomegranate rind hybrids
T
⁎
Saud Khalida, Long Yua,b, , Mingyue Fenga, Linghan Menga, Yuting Baic, Amjad Alia, Hongsheng Liua,b, Ling Chena a b c
Center for Polymers from Renewable Resources, School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China Sino-Singapore International Joint Research Institute, Knowledge City, Guangzhou, 510663, China School of Materials Science and Engineering, South China University of Technology, Guangzhou, 510640, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Polycaprolactone Pomegranate Starch Antimicrobial Biodegradable Film
Polycaprolactone (PCL)/starch/pomegranate rind (PR) hybrids were developed for antimicrobial packaging applications. PR was used, for the first time, as an antimicrobial compound and was incorporated directly in PCL matrix, without the extraction of any active compound from the fruit rind. The PCL-based antimicrobial films were fabricated using extrusion technique. It was observed that PCL/PR films demonstrated reasonably good antimicrobial activity at higher concentration of active compound. Addition of starch not only lowered the cost but also improved the rigidity of PCL matrix. Adding to this, starch enhanced the antimicrobial activity of PR, and provided a releasing channel for the delivery of polyphenols by attenuating the interactions between PCL and PR. Since all the materials used in this work are biodegradable and food contactable, it is expected that the developed material can be employed as food-grade antimicrobial packaging material.
1. Introduction Antimicrobial packaging is a kind of active packaging in which active compound is incorporated within the packaging material to reduce the possibility of cross-contamination by suppressing the activities of targeted microorganisms (Ahmed et al., 2017; Appendini & Hotchkiss, 2002). Several organic and inorganic chemicals with antimicrobial properties have been utilized to develop active packaging materials. They mainly include organic acids (da Rocha, Loiko, Tondo, & Prentice, 2014; Ouattara, Simard, Piette, Begin, & Holley, 2000), bacteriocins (Jin, Liu, Zhang, & Hicks, 2009; Yuan, Lv, Yang, Chen, & Sun, 2015b), plant extracts (Adilah, Jamilah, & Hanani, 2018; Marcos et al., 2014; Wang & Rhim, 2016), natural polymers (Kurek, Galus, & Debeaufort, 2014; Pelissari, Grossmann, Yamashita, & Pineda, 2009; Schnell et al., 2017), enzymes (Ozer, Uz, Oymaci, & Altinkaya, 2016; Yener, Korel, & Yemenicioglu, 2009), nanoclays (Kanmani & Rhim, 2014) and metallic oxides (Shankar, Wang, & Rhim, 2017). Recently, the utilization of natural antimicrobial compounds such as essential oils (Hafsa et al., 2016; Wu et al., 2014; Zivanovic, Chi, & Draughon, 2005), spice extracts (Arfat, Ahmed, Ejaz, & Mullah, 2018; Nisar et al., 2017)
and fruit extracts (Adilah et al., 2018; Emam-Djomeh, Moghaddam, & Yasini Ardakani, 2015; Uzunlu & Niranjan, 2017; Wang & Rhim, 2016) have attracted more attention due to their antimicrobial activity and safety. However, the high price and low decomposition temperature of such natural antimicrobial compounds makes them unsuitable for commercial applications, since the processing temperature of most of the biodegradable polymers (150–200 °C) is higher than the decomposition temperature of naturally occurring active compounds (∼100 °C) (Scaffaro, Botta, Maio, & Gallo, 2016; Tawakkal, Cran, Miltz, & Bigger, 2014). Several approaches have been proposed till date to control the loss of antimicrobial activity of active compounds during processing, including but not limited to, plasticization of polymer (Liu, Jin, Coffin, & Hicks, 2009; Wang & Rhim, 2016), microencapsulation or formation of inclusion complexes (Abarca et al., 2017; Joo, Merkel, Auras, & Almenar, 2012), nanocomposites (Correa et al., 2017; Rhim, Hong, & Ha, 2009; Yahiaoui et al., 2015), etc. However, such techniques lack commercial success either due to high processing cost (for example, microencapsulation approach), safety issues (for example, nanocomposites) or migration phenomenon (for example, plasticized polymers) of packaging materials. In this regard, development of low-
⁎ Corresponding author at: Center for Polymers from Renewable Resources, School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China. E-mail address: [email protected] (L. Yu).
https://doi.org/10.1016/j.fpsl.2018.08.008 Received 2 March 2018; Received in revised form 27 August 2018; Accepted 28 August 2018 2214-2894/ © 2018 Elsevier Ltd. All rights reserved.
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cost active packaging material will be highly beneficial for commercial applications. Poly(caprolactone) (PCL), with its low melting (60 °C approximately) and glass transition (around −60 °C) temperature, offers an ideal biodegradable matrix for natural antimicrobial agents as it can be processed at low temperature and have the potential for use in commercial applications. Moreover, the toughness of PCL is superior than most of the biodegradable polymers (Averous, 2004). However, lower modulus and rigidity of PCL limit its many applications. Starch, being a natural polymer, is abundantly available and is blended with several bio-based polymers (Liu, Xie, Yu, Chen, & Li, 2009) with an objective to either reduce the cost or to control the hydrophobicity of the base polymer (Imre & Pukanszky, 2013). Starch have been widely used as reinforcing agent to improve modulus of various biodegradable polymeric materials (Khalid et al., 2017), since starch itself is also biodegradable (Imre & Pukanszky, 2013; Yu, Dean, & Li, 2006). Actually, PCL/starch composites have been reported previously, whereby starch was utilized as filler to optimize the mechanical properties and to control the cost of PCL matrix (Campos et al., 2013; Kong et al., 2017). Starch can also be used as a delivery vehicle for the transportation of embedded molecules from the matrix (Khalil, Galland, Cottaz, Joly, & Degraeve, 2014). By playing with the hydrophobicity of PCL matrix, the transportation of embedded antimicrobial compounds can be tailored for any specific application. PCL and starch, being immiscible polymers since the former is hydrophobic and the latter is hydrophilic, are ideal candidates for developing such kind of biodegradable polymers with controlled release properties. Pomegranate (Punicagranatum L.) rind (PR) is obtained as a byproduct during processing of pomegranate juice. PR is rich in tannins and polyphenols, that demonstrate remarkable antimicrobial activity (Al-Zoreky, 2009). However, the extraction of tannins or polyphenols from the rind is an expensive step that makes it unsuitable for general applications. In this study, PR powder was used as an antimicrobial compound, and was incorporated directly in the matrix without extracting any active compound from the fruit rind. PCL-based films were prepared using extrusion technique to check the stability of active compound in actual processing conditions. Both starch and PR were also used to decrease the cost of PCL. It is expected that this study will be beneficial in designing biodegradable materials filled with naturally occurring antimicrobial compounds and can also be employed to design any other kind of active packaging material.
Fig. 1. Flow line of sample preparation.
2.2. Sample preparation Fig. 1 shows the step-by-step guide that was followed to prepare samples. Initially, starch was dried in a vacuum drier (Yingkou Liaohe pharmaceutical & chemical equipment Co Ltd, Yingkou, China) at a speed of 90 rpm, −0.1 MPa of vacuum, 140 °C temperature for 4 h, followed by the addition of stearic acid (0.2% concentration on w/w basis); vacuum drier was run again to coat the surface of starch granules with stearic acid at 140 °C for 1 h at a speed of 110 rpm without creating any vacuum pressure. Pomegranate rind was ground using herbal medicine disintegrator (FW177, Tianjin Taisite Instrument Co Ltd, Tianjin, China) and was then passed through 180 mesh sieve size. Sieved PR was then dried at 37 °C for 10–12 h in air drier (DHG 9145 A, Shanghai Right Instrument Co Ltd, Shanghai, China). PR was then surface treated with stearic acid in a lab scale high speed mixer (SRL W10/25) at 1200 rpm for 3 min. All the samples were hermetically sealed and were stored in air-tight container under controlled conditions of temperature and humidity to avoid any probability of moisture uptake.
2. Materials and methods
2.3. Processing
2.1. Materials
2.3.1. Extrusion PCL/starch/PR composites were prepared using twin screw extruder (Rheomex PTW 24/40p, Ø30, screw diameter D = 24 mm, screw length L = 28D) with 150 mm wide sheet die. PCL, starch and PR were mixed manually, and the composition of each specimen is given in Table 1. Samples were fed through hopper using 80 rpm screw speed. There were eight temperature controlling zones along the barrel of extruder and the maximum temperature of barrel was set as 80 °C while the temperature of die was set as 75 °C A separate hauling device was used to collect the extruded sheets from the die.
Polycaprolactone (MW: 37000; & MFI: 24) was obtained from Taisu Plastic Industrial Ningbo Co Ltd (Ningbo, China). Cornstarch (amylose/ amylopectin ratio was 26/74) was obtained from Foshan Nanhai Suiyang Food material Co Ltd (Foshan, China). Stearic acid, with a melting point of 67–70 °C, was procured from Shanghai Lingfeng Chemical Reagen t Co (Shanghai, China). Pomegranate rind powder (bulk density: 52 g/100 mL) was obtained from Shaanxi Honghao Biotech Co Ltd (Xian, China). Both culture media (brain heart infusion-BHI agar, HCM105; and lactose broth-LB agar, HCM055) and microbial culture (Staphylococcus aureus: ATCC 29213; and Eschericia coli: ATCC 25922) were procured from Guangdong Huankai Microbial Sci & Tech Co Ltd (Guangzhou, China). Sodium carbonate (MW = 105.99), gallic acid (MW = 170.12; purity = 99%) and Folin-Ciocalteu’s phenol reagent (2 M Acid) were procured from Guangzhou Chemical Reagent Factory (Guangzhou, China), Guangzhou Topwork Chemical Co Ltd (Guangzhou, China) and Sinopharm Chemical Reagent Co Ltd (Ningbo, China), respectively.
2.3.2. Hot pressing Samples were pressed using hot-plate machine (Guangzhou Shunchuang Rubber Machinery Factory, Guangzhou, China) with an objective to control the thickness and to flatten the extruded specimens. All the samples were covered with fiber coated Teflon sheets prior processing them in hot plate machine in order to avoid thermal degradation of the material. Temperature and pressure of 70 °C and 2100 bar, respectively, was used while the thickness of all the samples was adjusted to around 0.300 mm. All the samples were stored in room 72
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Ciocalteu’s phenol reagent (2 M with respect to acid) in a glass tube. Mixture was allowed to react for a short duration (6–7 min) followed by the addition of 1 mL of 7% Na2CO3 solution and 0.8 mL of distilled water in dark. The glass tube was then incubated at room temperature in dark for 2 h. Absorption was measured at 760 nm using a UV–vis spectrophotometer (Evolution 201, Thermo Fisher Scientific, USA) to monitor the release of polyphenolic compounds from film samples. Standard curve was prepared using gallic acid.
Table 1 Specimen code and composition of each specimen. Sr. No.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Description
Control Starch as filler
Pomegranate rind (PR) as filler
Starch and PR as filler at 20% concentration Starch and PR as filler at 40% concentration
Specimen code
S100/0/0 S90/10/0 S80/20/0 S70/30/0 S60/40/0 S90/0/10 S80/0/20 S70/0/30 S60/0/40 S80/15/5 S80/10/10 S80/5/15 S60/35/5 S60/30/10 S60/25/15 S60/20/20
Composition (%) PCL
Starch
PR
100 90 80 70 60 90 80 70 60 80 80 80 60 60 60 60
– 10 20 30 40 – – – – 15 10 5 35 30 25 20
– – – – – 10 20 30 40 5 10 15 5 10 15 20
2.4.6. Mechanical analysis Tensile properties were evaluated in accordance with ASTM D88212 guidelines using Instron 5565 apparatus. Dog bone shaped specimens, with a testing section of 120 mm length and 10 mm width, were cut from all the hybrid sheets. Samples were run using a cross-head speed of 200 mm min−1 at room temperature (25 °C). All the samples were conditioned for 48 h prior testing their mechanical properties. Seven specimens of all the samples were tested and the average value of each sample is reported. 2.5. Statistical analysis
temperature before characterization.
Statistical analysis was performed with Analytical Software STATISTIX 8.1 (Tallahassee FL 32317, USA). The analysis of variance (ANOVA) and Fisher's LSD multiple comparisons was performed to detect significant differences in properties of samples. The significance level (P ≤ 0.05) was used.
2.4. Characterization 2.4.1. Particle size analysis Particle size of starch and pomegranate rind powder was calculated using Mastersizer 2000 of Melvin instruments (Worcestershire, UK), following ISO 13320:2009 guidelines. A refractive index of 1.33 was used to calculate the particle size of fillers and the samples were added to circulating distilled water until an obscuration of > 10% was recorded.
3. Results and discussion 3.1. Appearance and morphology The appearance and morphology of filler provide useful information about the material properties. Fig. 2 shows the visual appearance and morphology of cornstarch and pomegranate rind powder (PR). Cornstarch is white, while PR is yellowish brown in color. Apparently, cornstarch and PR have similar powdery appearance. However, morphological analysis under SEM revealed that cornstarch and PR have different particle shape and size. Cornstarch granules were polygon in shape and their particle size varied from 7.86μm (D10) to 28.92μm (D90), with a median particle size of 16.25μm (D50), as widely reported (Chen, Yu, Chen, & Li, 2006, 2009). Contrary to this, PR exhibited variable morphology. PR particles were found to be irregular in appearance with fibrous pieces showing large distribution in particle size. The particle size of PR varied from 6.71μm (D10) to 105.97μm (D90), while the median particle size was found to be 32.99μm (D50). The median volume distribution of PR particles (D4,3 = 46.49μm) was larger than that of cornstarch granules (D4,3 = 18.26μm). Moreover, the PR particles were found to be agglomerated together, unlike cornstarch granules, and it was difficult to separate such particles from one another due to irregular shape and larger surface area. Fig. 3 shows the photos of the PCL-based films filled with different concentration of cornstarch and PR. Pure PCL film (S100/0/0) was reasonably transparent in color. Addition of filler, either starch or PR, alone or in combination, attenuated the transparency of the PCL matrix. As expected, the transparency of PCL was reduced with the incorporation of filler.
2.4.2. ATR FTIR analysis FTIR spectra were obtained using Nicolet IS50 FTIR (Thermo Fisher Scientific Co Ltd, Shanghai, China) at a wavelength of 5000 to 5 cm−1 in attenuated total reflectance (ATR) mode. 2.4.3. Microstructure characterization SEM (ZEISS, Oberkochen, Germany) was used to investigate the interface between matrix and filler in broken tensile bar specimens and to study the microstructure of starch granules and PR. All the samples were put on metal stubs, previously covered with double sided adhesive, and coated with gold using Eiko Sputter Coater under vacuum. 2.4.4. Determination of anti-microbial properties BHI agar and LB agar medium were used to simulate the application environment for the growth of S.aureus and E.coli, respectively. Samples were cut into 6.0 ± 0.1 mm diameter disks using a circular knife. Film disks were then placed on agar plates that had been seeded previously with 0.1 mL of a bacterial suspension containing 106 CFU/mL of the targeted micro-organism. The control sample was placed in the center of agar media to contrast the anti-microbial properties of the samples. The plates were incubated at 37 °C. Afterwards, the results of inhibition zones were calculated by subtracting the area of inhibition zone with that of the area of sample, after incubation. The tests were performed in triplicate.
3.2. ATR FTIR analysis 2.4.5. Polyphenol releasing test Total phenolic contents in samples were measured using FolinCiocalteu method. Briefly, 15 pieces of each film sample having 2.54 mm diameter were cut using circular knife and were immersed in distilled water. Samples were stored in room temperature and the content inside the beaker was mixed using vortex mixer for 5–7 min each time before and after procuring the sample from the beaker. 100μl of sample was mixed with 400μl of distilled water and 100μl of Folin-
Fig. 4 shows the FTIR spectra of PCL films and the films filled with cornstarch and PR. It was observed that the interactions between PCL and PR were due to stretching vibrations of CeH (2942 & 2864 cm−1), C]O (1722 cm−1) and CeO (1163 cm−1) bonds. On the other hand, the interactions between PCL and starch were due to stretching vibrations of C]O and CeO bonds, while sp3CeH stretching didn’t play any role in blending PCL and starch. Except these 3 peaks, all the other 73
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Fig. 2. Macrostructure and morphology of cornstarch (A, A-1 & A-2) and pomegranate rind (B, B-1 & B-2).
peaks of PCL were retained in all the specimens with a slight variation in intensity which indicated that the chemical structure of PCL was preserved in all the samples, without forming any appreciable chemical bonding between PCL and starch or between PCL and PR. It can also be concluded that the interactions between PCL and PR powder were stronger than that between PCL and starch, as PR affected the intensity of 4 different wavelengths of PCL while starch only affected the intensity of 2 different wavelengths of PCL. The results indicated that the interface and bond between PCL and PR was stronger than that between PCL and starch. 3.3. Antimicrobial properties Fig. 5 shows the inhibition zone of samples against Staphylococcus aureus, after 24 h of incubation. As expected, no inhibition zone was observed with the addition of starch as filler in PCL matrix. On the other hand, PCL/PR films demonstrated inhibition zone only at higher concentration of PR. The possible reason for the absence of inhibition zone at lower concentration of PR in PCL matrix might be the tight bonding
Fig. 4. ATR-FTIR spectra of film specimens (S100/0/0: PCL film; S60/40/0: PCL/starch film; S60/0/40: PCL/PR film).
Fig. 3. Visual appearance of PCL films filled with cornstarch and pomegranate rind (PR) particles (A: starch as filler; B: PR as filler; C: starch and PR as filler at 20% concentration; and D: starch and PR as filler at 40% concentration). 74
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Fig. 5. Inhibition zone of samples against S. aureus after 24 h of incubation at 37 °C (A: starch as filler; B: pomegranate rind-PR as filler; C: starch and PR as filler at 20% concentration; and D: starch and PR as filler at 40% concentration). Table 2 Area of inhibition zone of samples against S. aureus after 24 h of incubation.1. Sr. No.
Description
Specimen code
Area of inhibition zone (mm2)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Control Starch as filler
S100/0/0 S90/10/0 S80/20/0 S70/30/0 S60/40/0 S90/0/10 S80/0/20 S70/0/30 S60/0/40 S80/15/5 S80/10/10 S80/5/15 S60/35/5 S60/30/10 S60/25/15 S60/20/20
0.00g 0.00g 0.00g 0.00g 0.00g 0.00g 33.17 ± 2.34e 35.74 ± 2.45e 100.66 ± 8.45b 0.00g 16.26 ± 2.52f 47.68 ± 3.41c 0.00g 74.20 ± 4.75c 98.56 ± 4.25b 115.37 ± 4.15a
Pomegranate rind (PR) as filler
Starch and PR as filler at 20% concentration Starch and PR as filler at 40% concentration
Fig. 6. Inhibition zone of pomegranate rind-filled PCL film samples against E.coli after 24 h of incubation at 37 °C.
1
Each value is mean of three replicates with the standard deviation. Any two means in the same column followed by the same letter are not significantly (P ≤ 0.05) different by Fisher’s LSD multiple comparison tests.
matrix. It must also be noted that the results of this study aren’t comparable with previous studies, as the antimicrobial compound utilized in previous studies were expensive pure active compounds extracted from PR. Contrary to this, PR was directly incorporated in the PCL matrix without the extraction of any active compound, in this study, in order to reduce the cost of antimicrobial polymer. There is possibility of degradation of active compounds within PR due to repeated heating (drying, extrusion, hot pressing) and shear mixing, that needs to be investigated further. On the other hand, with the combined addition of
of filler in the matrix. Table 2 is showing the diameter of inhibition zone of samples observed after 24 h of incubation. In one of the previous study, pomegranate peel extract (PPE) filled chitosan demonstrated inhibition zone (2.5 ± 0.29 mm) against S.aureus, even at lower concentration of PPE (10%) (Yuan et al., 2015b). Antimicrobial properties can be expected from chitosan-based polymers due to its inherent antimicrobial characteristics against S. aureus, unlike PCL
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Fig. 7. Phenolic content of PCL films as a function of pomegranate rind and starch. Table 3 Mechanical properties of PCL-based films filled with cornstarch and pomegranate rind (PR).1. Sr. No.
Description
Specimen code
Thickness (mm)
Elongation (%)
Young’s Modulus (MPa)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Control Starch as filler
S100/0/0 S90/10/0 S80/20/0 S70/30/0 S60/40/0 S90/0/10 S80/0/20 S70/0/30 S60/0/40 S60/35/5 S60/30/10 S60/25/15 S60/20/20
0.322 0.291 0.374 0.286 0.343 0.251 0.318 0.301 0.321 0.303 0.417 0.362 0.324
333.49 ± 30.15a 253.50 ± 45.00b 225.53 ± 44.30b 181.86 ± 21.51c 107.02 ± 18.95d 47.65 ± 16.97fg 14.83 ± 3.92gh 10.30 ± 6.06h 5.01 ± 1.86h 87.13 ± 13.94de 69.50 ± 6.74ef 33.76 ± 5.52fgh 20.14 ± 3.19gh
400.29 423.53 447.66 470.28 468.97 434.44 505.25 523.34 573.16 567.55 580.36 584.40 510.61
PR as filler
Combined addition of starch and PR as filler
± ± ± ± ± ± ± ± ± ± ± ± ±
25.46 f 18.85ef 20.50ef 28.88cde 36.50de 25.07ef 35.69cd 45.55bc 34.12ab 26.51ab 35.75a 37.19a 33.39cd
Yield strength2 (MPa)
Tensile strength (MPa)
16.27 ± 2.52a 9.33 ± 0.52bcd 8.48 ± 0.38cde 8.38 ± 0.25cde 7.63 ± 0.40e 10.36 ± 0.47b 9.74 ± 0.55bc 8.36 ± 1.17cde 7.80 ± 0.64de 8.08 ± 1.07cde 7.06 ± 0.82e 7.47 ± 1.25e 8.02 ± 0.64de
28.25 ± 3.54a 18.73 ± 0.66b 17.83 ± 0.63b 11.31 ± 0.40de 8.69 ± 0.27f 14.56 ± 0.52c 11.42 ± 0.40d 9.60 ± 0.34ef 9.18 ± 0.32f 9.67 ± 0.34def 8.18 ± 0.29f 8.38 ± 0.26f 8.41 ± 0.26f
1 Each value is mean of three replicates with the standard deviation. Any two means in the same column followed by the same letter are not significantly (P ≤ 0.05) different by Fisher’s LSD multiple comparison tests. 2 Yield strength was calculated at 0.2% offset of stress-strain curves.
films into distilled water were evaluated and the results are shown in Fig. 7. Concentration of polyphenols increased abruptly on the first day and then showed a relatively linear pattern from the second day. Interestingly, the phenolic content of film samples was found to be lower in the absence of starch as compare to those film samples that were filled with starch granules. This indicated that the starch granules provided passage to release polyphenols from the PCL matrix. It was observed in section 3.2 (ATR-FTIR analysis) that the interactions between PCL and PR were stronger than that between PCL and starch. It can be assumed that part of polyphenols couldn’t come out from PCL matrix in PCL/PR films due to stronger interactions between PCL and PR than that of PCL and starch. In one of the previous study, it was observed that starch triggered the release of active compound from the matrix by reducing the hydrophobicity of the base polymer (Bie et al., 2013). It can be concluded therefore that the release rate of active compound from the PCL matrix can be optimized by varying the concentration of starch in the material, depending on the intended application.
starch and PR as filler within PCL matrix, the PR particles demonstrated reasonably good antimicrobial activity against targeted micro-organism even at lower concentration (see Table 2). It can be assumed that the addition of starch attenuated the interactions between PCL and PR (see Fig. 4) and provided passage to release PR particles from PCL matrix. This is a very useful finding and can be utilized to optimize the performance of active packaging materials. Nevertheless, incorporating active compound at higher concentration increases the cost of the material and deteriorates the mechanical properties of the matrix, simultaneously. Inhibition zone of PCL-based films, filled with PR particles, were also tested against E. coli to check their efficacy against this Gram-negative bacterium. No inhibition zone was detected, regardless of the concentration of PR particles in PCL matrix (see Fig. 6). Generally, the Gram-positive bacteria are considered to be more sensitive against antimicrobial compounds as compare to that of Gram-negative bacteria (Emam-Djomeh et al., 2015). This is generally attributed to the differences in the structure of their cell walls, as the cell walls of Gram-negative bacteria contain lipopolysaccharides, which may prevent active components from reaching the cytoplasmic membrane. The present results were in agreement with previous studies in which E.coli was found to be resistant against pomegranate peel extract-incorporated chitosan- (Yuan, Lv, Yang, Chen, & Sun, 2015a) or PCL-based (Uzunlu & Niranjan, 2017) films.
3.5. Mechanical properties Packaging materials should have enough strength and stiffness to be self-supporting and resist handling damage. Tensile properties of the films were tested at room temperature after 48 h of conditioning, and their results are shown in Table 3 and Fig. 8. Pure PCL film (S100/0/0) demonstrated high elongation (> 300%). However, lower modulus and rigidity of PCL limit its certain applications (Imre & Pukanszky, 2013). Addition of filler, either starch or PR, improved the rigidity of PCL at
3.4. Polyphenol releasing test Releasing of polyphenols from PCL/PR and PCL/cornstarch/PR 76
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Fig. 8. Tensile properties of PCL-based films filled with cornstarch and pomegranate rind-PR (A: starch as filler; B: PR as filler; and C: starch and PR as filler).
3.6. Microstructures and interface
the expense of its elongation and strength. Starch granules were found to be more efficient in resisting the elongation of matrix, while PR particles were found to be more efficient in improving the rigidity of PCL. Likewise, PCL films filled with PR particles demonstrated higher strength than that of films filled with starch granules. Irregular surface provided larger surface area to PR particles for interaction with matrix than that of smooth-surfaced polygon-shaped cornstarch granules that resulted in higher rigidity or modulus and strength of PCL/PR films. On the other hand, higher inter-particle distance among starch granules resulted in less heterogeneous dispersion and higher toughness or elongation of PCL/starch films. It was also observed that the combined addition of starch and PR, as filler, demonstrated relatively better mechanical properties, especially toughness, than that of adding individual constituent in PCL at same concentration of filler. It can be therefore concluded that the mechanical properties of PCL matrix can be optimized by the combined addition of starch and PR, as filler.
Cross-sections of tensile fractured specimen were analyzed using SEM, and the results are shown in Fig. 9. The fractured surface of pure PCL (S100/0/0) was relatively smooth in appearance. Appearance of small fibrous deformations in PCL matrix can be attributed to its high ductility. On the other hand, starch granules and PR particles can be clearly distinguished within the matrix in the composite system. Debonded starch granules can be observed on the fractured surface of PCL/starch blend. Polygonal or roughly spherical surface of starch granules can better resist the deformation of matrix. As a result of that, higher ductility or elongation was observed for PCL/starch blends as compare to PCL/PR blends (see Fig. 8). Contrary to this, pomegranate rind demonstrated layered structure whereby PR particles were found to be constrained within PCL matrix. Irregular shape, sharp edges and larger surface area of PR particles can be used to explain the high strength and rigidity of PCL/PR films as compare to PCL/starch films 77
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Fig. 9. Cross-sectional images of tensile fractured specimen (S100/0/0: PCL matrix; S80/20/0: PCL/starch blend; S80/0/20: PCL/pomegranate rind (PR) blend; and S80/10/10: PCL/starch/PR film).
References
(see Table 3). Moreover, the poor interface between PCL and starch provided a passage in the form of gaps to release PR particles from the matrix (corresponding with the results in Figs. 4 and 7).
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4. Conclusion Antimicrobial packaging materials were successfully developed based on PCL/starch/PR hybrids using extrusion technique. It was observed that PCL is an effective biodegradable matrix that can be utilized to develop active packaging materials, since it can be processed at low temperature (～80 °C) that is suitable to process naturally occurring active compounds. PCL/starch/PR hybrid films demonstrated reasonably good antimicrobial activity against S. aureus. Cornstarch can be incorporated as functional filler in developing such materials, as it not only provided passage to embedded active compound but also improved the mechanical properties of the matrix. The PCL/starch/PR hybrid films have reasonably low price since both starch and PR decreased the cost of the material. Since all the materials used in this work are food contactable and biodegradable, it is expected that this material can be used as food grade active packaging material for commercial applications.
Acknowledgements The authors from SCUT, China, would like to acknowledge the research fund National Key R&D Program of China (2018YFD0400700), NSFC (31571789) and 111 Project (B17018). Saud Khalid and Amjad Ali would like to thank the China Scholarship Council and South China University of Technology for providing opportunity and support during their stay in China. The authors would also like to thank Muhammad Saeed Khan, Xinyang Bao (both are Ph.D. student at School of Food Science and Engineering, South China University of Technology) and anonymous reviewers for providing useful suggestions in improving the quality of this manuscript. 78
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Development and characterization of biodegradable antimicrobial packaging films based on polycaprolactone, starch and pomegranate rind hybrids
T
⁎
Saud Khalida, Long Yua,b, , Mingyue Fenga, Linghan Menga, Yuting Baic, Amjad Alia, Hongsheng Liua,b, Ling Chena a b c
Center for Polymers from Renewable Resources, School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China Sino-Singapore International Joint Research Institute, Knowledge City, Guangzhou, 510663, China School of Materials Science and Engineering, South China University of Technology, Guangzhou, 510640, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Polycaprolactone Pomegranate Starch Antimicrobial Biodegradable Film
Polycaprolactone (PCL)/starch/pomegranate rind (PR) hybrids were developed for antimicrobial packaging applications. PR was used, for the first time, as an antimicrobial compound and was incorporated directly in PCL matrix, without the extraction of any active compound from the fruit rind. The PCL-based antimicrobial films were fabricated using extrusion technique. It was observed that PCL/PR films demonstrated reasonably good antimicrobial activity at higher concentration of active compound. Addition of starch not only lowered the cost but also improved the rigidity of PCL matrix. Adding to this, starch enhanced the antimicrobial activity of PR, and provided a releasing channel for the delivery of polyphenols by attenuating the interactions between PCL and PR. Since all the materials used in this work are biodegradable and food contactable, it is expected that the developed material can be employed as food-grade antimicrobial packaging material.
1. Introduction Antimicrobial packaging is a kind of active packaging in which active compound is incorporated within the packaging material to reduce the possibility of cross-contamination by suppressing the activities of targeted microorganisms (Ahmed et al., 2017; Appendini & Hotchkiss, 2002). Several organic and inorganic chemicals with antimicrobial properties have been utilized to develop active packaging materials. They mainly include organic acids (da Rocha, Loiko, Tondo, & Prentice, 2014; Ouattara, Simard, Piette, Begin, & Holley, 2000), bacteriocins (Jin, Liu, Zhang, & Hicks, 2009; Yuan, Lv, Yang, Chen, & Sun, 2015b), plant extracts (Adilah, Jamilah, & Hanani, 2018; Marcos et al., 2014; Wang & Rhim, 2016), natural polymers (Kurek, Galus, & Debeaufort, 2014; Pelissari, Grossmann, Yamashita, & Pineda, 2009; Schnell et al., 2017), enzymes (Ozer, Uz, Oymaci, & Altinkaya, 2016; Yener, Korel, & Yemenicioglu, 2009), nanoclays (Kanmani & Rhim, 2014) and metallic oxides (Shankar, Wang, & Rhim, 2017). Recently, the utilization of natural antimicrobial compounds such as essential oils (Hafsa et al., 2016; Wu et al., 2014; Zivanovic, Chi, & Draughon, 2005), spice extracts (Arfat, Ahmed, Ejaz, & Mullah, 2018; Nisar et al., 2017)
and fruit extracts (Adilah et al., 2018; Emam-Djomeh, Moghaddam, & Yasini Ardakani, 2015; Uzunlu & Niranjan, 2017; Wang & Rhim, 2016) have attracted more attention due to their antimicrobial activity and safety. However, the high price and low decomposition temperature of such natural antimicrobial compounds makes them unsuitable for commercial applications, since the processing temperature of most of the biodegradable polymers (150–200 °C) is higher than the decomposition temperature of naturally occurring active compounds (∼100 °C) (Scaffaro, Botta, Maio, & Gallo, 2016; Tawakkal, Cran, Miltz, & Bigger, 2014). Several approaches have been proposed till date to control the loss of antimicrobial activity of active compounds during processing, including but not limited to, plasticization of polymer (Liu, Jin, Coffin, & Hicks, 2009; Wang & Rhim, 2016), microencapsulation or formation of inclusion complexes (Abarca et al., 2017; Joo, Merkel, Auras, & Almenar, 2012), nanocomposites (Correa et al., 2017; Rhim, Hong, & Ha, 2009; Yahiaoui et al., 2015), etc. However, such techniques lack commercial success either due to high processing cost (for example, microencapsulation approach), safety issues (for example, nanocomposites) or migration phenomenon (for example, plasticized polymers) of packaging materials. In this regard, development of low-
⁎ Corresponding author at: Center for Polymers from Renewable Resources, School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China. E-mail address: [email protected] (L. Yu).
https://doi.org/10.1016/j.fpsl.2018.08.008 Received 2 March 2018; Received in revised form 27 August 2018; Accepted 28 August 2018 2214-2894/ © 2018 Elsevier Ltd. All rights reserved.
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cost active packaging material will be highly beneficial for commercial applications. Poly(caprolactone) (PCL), with its low melting (60 °C approximately) and glass transition (around −60 °C) temperature, offers an ideal biodegradable matrix for natural antimicrobial agents as it can be processed at low temperature and have the potential for use in commercial applications. Moreover, the toughness of PCL is superior than most of the biodegradable polymers (Averous, 2004). However, lower modulus and rigidity of PCL limit its many applications. Starch, being a natural polymer, is abundantly available and is blended with several bio-based polymers (Liu, Xie, Yu, Chen, & Li, 2009) with an objective to either reduce the cost or to control the hydrophobicity of the base polymer (Imre & Pukanszky, 2013). Starch have been widely used as reinforcing agent to improve modulus of various biodegradable polymeric materials (Khalid et al., 2017), since starch itself is also biodegradable (Imre & Pukanszky, 2013; Yu, Dean, & Li, 2006). Actually, PCL/starch composites have been reported previously, whereby starch was utilized as filler to optimize the mechanical properties and to control the cost of PCL matrix (Campos et al., 2013; Kong et al., 2017). Starch can also be used as a delivery vehicle for the transportation of embedded molecules from the matrix (Khalil, Galland, Cottaz, Joly, & Degraeve, 2014). By playing with the hydrophobicity of PCL matrix, the transportation of embedded antimicrobial compounds can be tailored for any specific application. PCL and starch, being immiscible polymers since the former is hydrophobic and the latter is hydrophilic, are ideal candidates for developing such kind of biodegradable polymers with controlled release properties. Pomegranate (Punicagranatum L.) rind (PR) is obtained as a byproduct during processing of pomegranate juice. PR is rich in tannins and polyphenols, that demonstrate remarkable antimicrobial activity (Al-Zoreky, 2009). However, the extraction of tannins or polyphenols from the rind is an expensive step that makes it unsuitable for general applications. In this study, PR powder was used as an antimicrobial compound, and was incorporated directly in the matrix without extracting any active compound from the fruit rind. PCL-based films were prepared using extrusion technique to check the stability of active compound in actual processing conditions. Both starch and PR were also used to decrease the cost of PCL. It is expected that this study will be beneficial in designing biodegradable materials filled with naturally occurring antimicrobial compounds and can also be employed to design any other kind of active packaging material.
Fig. 1. Flow line of sample preparation.
2.2. Sample preparation Fig. 1 shows the step-by-step guide that was followed to prepare samples. Initially, starch was dried in a vacuum drier (Yingkou Liaohe pharmaceutical & chemical equipment Co Ltd, Yingkou, China) at a speed of 90 rpm, −0.1 MPa of vacuum, 140 °C temperature for 4 h, followed by the addition of stearic acid (0.2% concentration on w/w basis); vacuum drier was run again to coat the surface of starch granules with stearic acid at 140 °C for 1 h at a speed of 110 rpm without creating any vacuum pressure. Pomegranate rind was ground using herbal medicine disintegrator (FW177, Tianjin Taisite Instrument Co Ltd, Tianjin, China) and was then passed through 180 mesh sieve size. Sieved PR was then dried at 37 °C for 10–12 h in air drier (DHG 9145 A, Shanghai Right Instrument Co Ltd, Shanghai, China). PR was then surface treated with stearic acid in a lab scale high speed mixer (SRL W10/25) at 1200 rpm for 3 min. All the samples were hermetically sealed and were stored in air-tight container under controlled conditions of temperature and humidity to avoid any probability of moisture uptake.
2. Materials and methods
2.3. Processing
2.1. Materials
2.3.1. Extrusion PCL/starch/PR composites were prepared using twin screw extruder (Rheomex PTW 24/40p, Ø30, screw diameter D = 24 mm, screw length L = 28D) with 150 mm wide sheet die. PCL, starch and PR were mixed manually, and the composition of each specimen is given in Table 1. Samples were fed through hopper using 80 rpm screw speed. There were eight temperature controlling zones along the barrel of extruder and the maximum temperature of barrel was set as 80 °C while the temperature of die was set as 75 °C A separate hauling device was used to collect the extruded sheets from the die.
Polycaprolactone (MW: 37000; & MFI: 24) was obtained from Taisu Plastic Industrial Ningbo Co Ltd (Ningbo, China). Cornstarch (amylose/ amylopectin ratio was 26/74) was obtained from Foshan Nanhai Suiyang Food material Co Ltd (Foshan, China). Stearic acid, with a melting point of 67–70 °C, was procured from Shanghai Lingfeng Chemical Reagen t Co (Shanghai, China). Pomegranate rind powder (bulk density: 52 g/100 mL) was obtained from Shaanxi Honghao Biotech Co Ltd (Xian, China). Both culture media (brain heart infusion-BHI agar, HCM105; and lactose broth-LB agar, HCM055) and microbial culture (Staphylococcus aureus: ATCC 29213; and Eschericia coli: ATCC 25922) were procured from Guangdong Huankai Microbial Sci & Tech Co Ltd (Guangzhou, China). Sodium carbonate (MW = 105.99), gallic acid (MW = 170.12; purity = 99%) and Folin-Ciocalteu’s phenol reagent (2 M Acid) were procured from Guangzhou Chemical Reagent Factory (Guangzhou, China), Guangzhou Topwork Chemical Co Ltd (Guangzhou, China) and Sinopharm Chemical Reagent Co Ltd (Ningbo, China), respectively.
2.3.2. Hot pressing Samples were pressed using hot-plate machine (Guangzhou Shunchuang Rubber Machinery Factory, Guangzhou, China) with an objective to control the thickness and to flatten the extruded specimens. All the samples were covered with fiber coated Teflon sheets prior processing them in hot plate machine in order to avoid thermal degradation of the material. Temperature and pressure of 70 °C and 2100 bar, respectively, was used while the thickness of all the samples was adjusted to around 0.300 mm. All the samples were stored in room 72
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Ciocalteu’s phenol reagent (2 M with respect to acid) in a glass tube. Mixture was allowed to react for a short duration (6–7 min) followed by the addition of 1 mL of 7% Na2CO3 solution and 0.8 mL of distilled water in dark. The glass tube was then incubated at room temperature in dark for 2 h. Absorption was measured at 760 nm using a UV–vis spectrophotometer (Evolution 201, Thermo Fisher Scientific, USA) to monitor the release of polyphenolic compounds from film samples. Standard curve was prepared using gallic acid.
Table 1 Specimen code and composition of each specimen. Sr. No.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Description
Control Starch as filler
Pomegranate rind (PR) as filler
Starch and PR as filler at 20% concentration Starch and PR as filler at 40% concentration
Specimen code
S100/0/0 S90/10/0 S80/20/0 S70/30/0 S60/40/0 S90/0/10 S80/0/20 S70/0/30 S60/0/40 S80/15/5 S80/10/10 S80/5/15 S60/35/5 S60/30/10 S60/25/15 S60/20/20
Composition (%) PCL
Starch
PR
100 90 80 70 60 90 80 70 60 80 80 80 60 60 60 60
– 10 20 30 40 – – – – 15 10 5 35 30 25 20
– – – – – 10 20 30 40 5 10 15 5 10 15 20
2.4.6. Mechanical analysis Tensile properties were evaluated in accordance with ASTM D88212 guidelines using Instron 5565 apparatus. Dog bone shaped specimens, with a testing section of 120 mm length and 10 mm width, were cut from all the hybrid sheets. Samples were run using a cross-head speed of 200 mm min−1 at room temperature (25 °C). All the samples were conditioned for 48 h prior testing their mechanical properties. Seven specimens of all the samples were tested and the average value of each sample is reported. 2.5. Statistical analysis
temperature before characterization.
Statistical analysis was performed with Analytical Software STATISTIX 8.1 (Tallahassee FL 32317, USA). The analysis of variance (ANOVA) and Fisher's LSD multiple comparisons was performed to detect significant differences in properties of samples. The significance level (P ≤ 0.05) was used.
2.4. Characterization 2.4.1. Particle size analysis Particle size of starch and pomegranate rind powder was calculated using Mastersizer 2000 of Melvin instruments (Worcestershire, UK), following ISO 13320:2009 guidelines. A refractive index of 1.33 was used to calculate the particle size of fillers and the samples were added to circulating distilled water until an obscuration of > 10% was recorded.
3. Results and discussion 3.1. Appearance and morphology The appearance and morphology of filler provide useful information about the material properties. Fig. 2 shows the visual appearance and morphology of cornstarch and pomegranate rind powder (PR). Cornstarch is white, while PR is yellowish brown in color. Apparently, cornstarch and PR have similar powdery appearance. However, morphological analysis under SEM revealed that cornstarch and PR have different particle shape and size. Cornstarch granules were polygon in shape and their particle size varied from 7.86μm (D10) to 28.92μm (D90), with a median particle size of 16.25μm (D50), as widely reported (Chen, Yu, Chen, & Li, 2006, 2009). Contrary to this, PR exhibited variable morphology. PR particles were found to be irregular in appearance with fibrous pieces showing large distribution in particle size. The particle size of PR varied from 6.71μm (D10) to 105.97μm (D90), while the median particle size was found to be 32.99μm (D50). The median volume distribution of PR particles (D4,3 = 46.49μm) was larger than that of cornstarch granules (D4,3 = 18.26μm). Moreover, the PR particles were found to be agglomerated together, unlike cornstarch granules, and it was difficult to separate such particles from one another due to irregular shape and larger surface area. Fig. 3 shows the photos of the PCL-based films filled with different concentration of cornstarch and PR. Pure PCL film (S100/0/0) was reasonably transparent in color. Addition of filler, either starch or PR, alone or in combination, attenuated the transparency of the PCL matrix. As expected, the transparency of PCL was reduced with the incorporation of filler.
2.4.2. ATR FTIR analysis FTIR spectra were obtained using Nicolet IS50 FTIR (Thermo Fisher Scientific Co Ltd, Shanghai, China) at a wavelength of 5000 to 5 cm−1 in attenuated total reflectance (ATR) mode. 2.4.3. Microstructure characterization SEM (ZEISS, Oberkochen, Germany) was used to investigate the interface between matrix and filler in broken tensile bar specimens and to study the microstructure of starch granules and PR. All the samples were put on metal stubs, previously covered with double sided adhesive, and coated with gold using Eiko Sputter Coater under vacuum. 2.4.4. Determination of anti-microbial properties BHI agar and LB agar medium were used to simulate the application environment for the growth of S.aureus and E.coli, respectively. Samples were cut into 6.0 ± 0.1 mm diameter disks using a circular knife. Film disks were then placed on agar plates that had been seeded previously with 0.1 mL of a bacterial suspension containing 106 CFU/mL of the targeted micro-organism. The control sample was placed in the center of agar media to contrast the anti-microbial properties of the samples. The plates were incubated at 37 °C. Afterwards, the results of inhibition zones were calculated by subtracting the area of inhibition zone with that of the area of sample, after incubation. The tests were performed in triplicate.
3.2. ATR FTIR analysis 2.4.5. Polyphenol releasing test Total phenolic contents in samples were measured using FolinCiocalteu method. Briefly, 15 pieces of each film sample having 2.54 mm diameter were cut using circular knife and were immersed in distilled water. Samples were stored in room temperature and the content inside the beaker was mixed using vortex mixer for 5–7 min each time before and after procuring the sample from the beaker. 100μl of sample was mixed with 400μl of distilled water and 100μl of Folin-
Fig. 4 shows the FTIR spectra of PCL films and the films filled with cornstarch and PR. It was observed that the interactions between PCL and PR were due to stretching vibrations of CeH (2942 & 2864 cm−1), C]O (1722 cm−1) and CeO (1163 cm−1) bonds. On the other hand, the interactions between PCL and starch were due to stretching vibrations of C]O and CeO bonds, while sp3CeH stretching didn’t play any role in blending PCL and starch. Except these 3 peaks, all the other 73
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Fig. 2. Macrostructure and morphology of cornstarch (A, A-1 & A-2) and pomegranate rind (B, B-1 & B-2).
peaks of PCL were retained in all the specimens with a slight variation in intensity which indicated that the chemical structure of PCL was preserved in all the samples, without forming any appreciable chemical bonding between PCL and starch or between PCL and PR. It can also be concluded that the interactions between PCL and PR powder were stronger than that between PCL and starch, as PR affected the intensity of 4 different wavelengths of PCL while starch only affected the intensity of 2 different wavelengths of PCL. The results indicated that the interface and bond between PCL and PR was stronger than that between PCL and starch. 3.3. Antimicrobial properties Fig. 5 shows the inhibition zone of samples against Staphylococcus aureus, after 24 h of incubation. As expected, no inhibition zone was observed with the addition of starch as filler in PCL matrix. On the other hand, PCL/PR films demonstrated inhibition zone only at higher concentration of PR. The possible reason for the absence of inhibition zone at lower concentration of PR in PCL matrix might be the tight bonding
Fig. 4. ATR-FTIR spectra of film specimens (S100/0/0: PCL film; S60/40/0: PCL/starch film; S60/0/40: PCL/PR film).
Fig. 3. Visual appearance of PCL films filled with cornstarch and pomegranate rind (PR) particles (A: starch as filler; B: PR as filler; C: starch and PR as filler at 20% concentration; and D: starch and PR as filler at 40% concentration). 74
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Fig. 5. Inhibition zone of samples against S. aureus after 24 h of incubation at 37 °C (A: starch as filler; B: pomegranate rind-PR as filler; C: starch and PR as filler at 20% concentration; and D: starch and PR as filler at 40% concentration). Table 2 Area of inhibition zone of samples against S. aureus after 24 h of incubation.1. Sr. No.
Description
Specimen code
Area of inhibition zone (mm2)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Control Starch as filler
S100/0/0 S90/10/0 S80/20/0 S70/30/0 S60/40/0 S90/0/10 S80/0/20 S70/0/30 S60/0/40 S80/15/5 S80/10/10 S80/5/15 S60/35/5 S60/30/10 S60/25/15 S60/20/20
0.00g 0.00g 0.00g 0.00g 0.00g 0.00g 33.17 ± 2.34e 35.74 ± 2.45e 100.66 ± 8.45b 0.00g 16.26 ± 2.52f 47.68 ± 3.41c 0.00g 74.20 ± 4.75c 98.56 ± 4.25b 115.37 ± 4.15a
Pomegranate rind (PR) as filler
Starch and PR as filler at 20% concentration Starch and PR as filler at 40% concentration
Fig. 6. Inhibition zone of pomegranate rind-filled PCL film samples against E.coli after 24 h of incubation at 37 °C.
1
Each value is mean of three replicates with the standard deviation. Any two means in the same column followed by the same letter are not significantly (P ≤ 0.05) different by Fisher’s LSD multiple comparison tests.
matrix. It must also be noted that the results of this study aren’t comparable with previous studies, as the antimicrobial compound utilized in previous studies were expensive pure active compounds extracted from PR. Contrary to this, PR was directly incorporated in the PCL matrix without the extraction of any active compound, in this study, in order to reduce the cost of antimicrobial polymer. There is possibility of degradation of active compounds within PR due to repeated heating (drying, extrusion, hot pressing) and shear mixing, that needs to be investigated further. On the other hand, with the combined addition of
of filler in the matrix. Table 2 is showing the diameter of inhibition zone of samples observed after 24 h of incubation. In one of the previous study, pomegranate peel extract (PPE) filled chitosan demonstrated inhibition zone (2.5 ± 0.29 mm) against S.aureus, even at lower concentration of PPE (10%) (Yuan et al., 2015b). Antimicrobial properties can be expected from chitosan-based polymers due to its inherent antimicrobial characteristics against S. aureus, unlike PCL
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Fig. 7. Phenolic content of PCL films as a function of pomegranate rind and starch. Table 3 Mechanical properties of PCL-based films filled with cornstarch and pomegranate rind (PR).1. Sr. No.
Description
Specimen code
Thickness (mm)
Elongation (%)
Young’s Modulus (MPa)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Control Starch as filler
S100/0/0 S90/10/0 S80/20/0 S70/30/0 S60/40/0 S90/0/10 S80/0/20 S70/0/30 S60/0/40 S60/35/5 S60/30/10 S60/25/15 S60/20/20
0.322 0.291 0.374 0.286 0.343 0.251 0.318 0.301 0.321 0.303 0.417 0.362 0.324
333.49 ± 30.15a 253.50 ± 45.00b 225.53 ± 44.30b 181.86 ± 21.51c 107.02 ± 18.95d 47.65 ± 16.97fg 14.83 ± 3.92gh 10.30 ± 6.06h 5.01 ± 1.86h 87.13 ± 13.94de 69.50 ± 6.74ef 33.76 ± 5.52fgh 20.14 ± 3.19gh
400.29 423.53 447.66 470.28 468.97 434.44 505.25 523.34 573.16 567.55 580.36 584.40 510.61
PR as filler
Combined addition of starch and PR as filler
± ± ± ± ± ± ± ± ± ± ± ± ±
25.46 f 18.85ef 20.50ef 28.88cde 36.50de 25.07ef 35.69cd 45.55bc 34.12ab 26.51ab 35.75a 37.19a 33.39cd
Yield strength2 (MPa)
Tensile strength (MPa)
16.27 ± 2.52a 9.33 ± 0.52bcd 8.48 ± 0.38cde 8.38 ± 0.25cde 7.63 ± 0.40e 10.36 ± 0.47b 9.74 ± 0.55bc 8.36 ± 1.17cde 7.80 ± 0.64de 8.08 ± 1.07cde 7.06 ± 0.82e 7.47 ± 1.25e 8.02 ± 0.64de
28.25 ± 3.54a 18.73 ± 0.66b 17.83 ± 0.63b 11.31 ± 0.40de 8.69 ± 0.27f 14.56 ± 0.52c 11.42 ± 0.40d 9.60 ± 0.34ef 9.18 ± 0.32f 9.67 ± 0.34def 8.18 ± 0.29f 8.38 ± 0.26f 8.41 ± 0.26f
1 Each value is mean of three replicates with the standard deviation. Any two means in the same column followed by the same letter are not significantly (P ≤ 0.05) different by Fisher’s LSD multiple comparison tests. 2 Yield strength was calculated at 0.2% offset of stress-strain curves.
films into distilled water were evaluated and the results are shown in Fig. 7. Concentration of polyphenols increased abruptly on the first day and then showed a relatively linear pattern from the second day. Interestingly, the phenolic content of film samples was found to be lower in the absence of starch as compare to those film samples that were filled with starch granules. This indicated that the starch granules provided passage to release polyphenols from the PCL matrix. It was observed in section 3.2 (ATR-FTIR analysis) that the interactions between PCL and PR were stronger than that between PCL and starch. It can be assumed that part of polyphenols couldn’t come out from PCL matrix in PCL/PR films due to stronger interactions between PCL and PR than that of PCL and starch. In one of the previous study, it was observed that starch triggered the release of active compound from the matrix by reducing the hydrophobicity of the base polymer (Bie et al., 2013). It can be concluded therefore that the release rate of active compound from the PCL matrix can be optimized by varying the concentration of starch in the material, depending on the intended application.
starch and PR as filler within PCL matrix, the PR particles demonstrated reasonably good antimicrobial activity against targeted micro-organism even at lower concentration (see Table 2). It can be assumed that the addition of starch attenuated the interactions between PCL and PR (see Fig. 4) and provided passage to release PR particles from PCL matrix. This is a very useful finding and can be utilized to optimize the performance of active packaging materials. Nevertheless, incorporating active compound at higher concentration increases the cost of the material and deteriorates the mechanical properties of the matrix, simultaneously. Inhibition zone of PCL-based films, filled with PR particles, were also tested against E. coli to check their efficacy against this Gram-negative bacterium. No inhibition zone was detected, regardless of the concentration of PR particles in PCL matrix (see Fig. 6). Generally, the Gram-positive bacteria are considered to be more sensitive against antimicrobial compounds as compare to that of Gram-negative bacteria (Emam-Djomeh et al., 2015). This is generally attributed to the differences in the structure of their cell walls, as the cell walls of Gram-negative bacteria contain lipopolysaccharides, which may prevent active components from reaching the cytoplasmic membrane. The present results were in agreement with previous studies in which E.coli was found to be resistant against pomegranate peel extract-incorporated chitosan- (Yuan, Lv, Yang, Chen, & Sun, 2015a) or PCL-based (Uzunlu & Niranjan, 2017) films.
3.5. Mechanical properties Packaging materials should have enough strength and stiffness to be self-supporting and resist handling damage. Tensile properties of the films were tested at room temperature after 48 h of conditioning, and their results are shown in Table 3 and Fig. 8. Pure PCL film (S100/0/0) demonstrated high elongation (> 300%). However, lower modulus and rigidity of PCL limit its certain applications (Imre & Pukanszky, 2013). Addition of filler, either starch or PR, improved the rigidity of PCL at
3.4. Polyphenol releasing test Releasing of polyphenols from PCL/PR and PCL/cornstarch/PR 76
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Fig. 8. Tensile properties of PCL-based films filled with cornstarch and pomegranate rind-PR (A: starch as filler; B: PR as filler; and C: starch and PR as filler).
3.6. Microstructures and interface
the expense of its elongation and strength. Starch granules were found to be more efficient in resisting the elongation of matrix, while PR particles were found to be more efficient in improving the rigidity of PCL. Likewise, PCL films filled with PR particles demonstrated higher strength than that of films filled with starch granules. Irregular surface provided larger surface area to PR particles for interaction with matrix than that of smooth-surfaced polygon-shaped cornstarch granules that resulted in higher rigidity or modulus and strength of PCL/PR films. On the other hand, higher inter-particle distance among starch granules resulted in less heterogeneous dispersion and higher toughness or elongation of PCL/starch films. It was also observed that the combined addition of starch and PR, as filler, demonstrated relatively better mechanical properties, especially toughness, than that of adding individual constituent in PCL at same concentration of filler. It can be therefore concluded that the mechanical properties of PCL matrix can be optimized by the combined addition of starch and PR, as filler.
Cross-sections of tensile fractured specimen were analyzed using SEM, and the results are shown in Fig. 9. The fractured surface of pure PCL (S100/0/0) was relatively smooth in appearance. Appearance of small fibrous deformations in PCL matrix can be attributed to its high ductility. On the other hand, starch granules and PR particles can be clearly distinguished within the matrix in the composite system. Debonded starch granules can be observed on the fractured surface of PCL/starch blend. Polygonal or roughly spherical surface of starch granules can better resist the deformation of matrix. As a result of that, higher ductility or elongation was observed for PCL/starch blends as compare to PCL/PR blends (see Fig. 8). Contrary to this, pomegranate rind demonstrated layered structure whereby PR particles were found to be constrained within PCL matrix. Irregular shape, sharp edges and larger surface area of PR particles can be used to explain the high strength and rigidity of PCL/PR films as compare to PCL/starch films 77
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Fig. 9. Cross-sectional images of tensile fractured specimen (S100/0/0: PCL matrix; S80/20/0: PCL/starch blend; S80/0/20: PCL/pomegranate rind (PR) blend; and S80/10/10: PCL/starch/PR film).
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4. Conclusion Antimicrobial packaging materials were successfully developed based on PCL/starch/PR hybrids using extrusion technique. It was observed that PCL is an effective biodegradable matrix that can be utilized to develop active packaging materials, since it can be processed at low temperature (～80 °C) that is suitable to process naturally occurring active compounds. PCL/starch/PR hybrid films demonstrated reasonably good antimicrobial activity against S. aureus. Cornstarch can be incorporated as functional filler in developing such materials, as it not only provided passage to embedded active compound but also improved the mechanical properties of the matrix. The PCL/starch/PR hybrid films have reasonably low price since both starch and PR decreased the cost of the material. Since all the materials used in this work are food contactable and biodegradable, it is expected that this material can be used as food grade active packaging material for commercial applications.
Acknowledgements The authors from SCUT, China, would like to acknowledge the research fund National Key R&D Program of China (2018YFD0400700), NSFC (31571789) and 111 Project (B17018). Saud Khalid and Amjad Ali would like to thank the China Scholarship Council and South China University of Technology for providing opportunity and support during their stay in China. The authors would also like to thank Muhammad Saeed Khan, Xinyang Bao (both are Ph.D. student at School of Food Science and Engineering, South China University of Technology) and anonymous reviewers for providing useful suggestions in improving the quality of this manuscript. 78
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