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Nanostructured active chitosan-based films for food packaging applications: Effect of graphene stacks on mechanical properties
Measurement 90 (2016) 418–423
Contents lists available at ScienceDirect
Measurement journal homepage: www.elsevier.com/locate/measurement
Nanostructured active chitosan-based films for food packaging applications: Effect of graphene stacks on mechanical properties Christian Demitri ⇑, Vincenzo Maria De Benedictis, Marta Madaghiele, Carola Esposito Corcione, Alfonso Maffezzoli University of Salento, Department of Engineering for Innovation, Via Monteroni, Campus Ecotekne, 73100 Lecce, Italy
a r t i c l e
i n f o
Article history: Received 10 December 2015 Received in revised form 19 April 2016 Accepted 5 May 2016 Available online 6 May 2016 Keywords: Chitosan Cinnamaldehyde Graphene Antifungal Activity Food Packaging
a b s t r a c t Bioactive food-preserving materials are based on the use of a natural antimicrobial compound loaded in a carrier material, which is able to trigger its release when requested and to modulate the rate of release, thus using either toxic or inhibitory properties against pathogens or bacteria due to food decomposition. In this study, the Schiff base formation for chitosan functionalization was achieved by the reaction of chitosan with cinnamaldehyde at different concentrations. Cinnamaldehyde is an aromatic a,b-unsaturated aldehyde, and the major component in essential oils from some cinnamon species. It has been shown to exert antimicrobial action against a large number of microorganisms including bacteria, yeasts, and mould. The formation of the Schiff base is reversible under suitable conditions, and this might allow the release of the active cinnamaldehyde from chitosan, used as the carrier. The reaction kinetics was investigated by means of rheological measurements, while infrared spectroscopy was used to assess the efficacy of the functionalization. The addition of nanometric graphene stacks to the cinnamaldehyde-functionalized chitosan films was evaluated with the aim to increase the mechanical properties of the film. Finally, the films were tested for antifungal properties with bread slices against a selected mould line. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Recently, different authors have been focused the attention on the development of natural innovative and active materials for food packaging applications [1]. It is well known that there are different processes involved in extending the shelf life of food. These involve controlled moisture transfer between inner and external environment, high permeability to certain substances, temperature control, structural reinforcement of food and coat flavor compounds, reduction of oxygen partial pressure in the package, extended release of organic agents like antimicrobial substances, antioxidants [2]. Active packagings are barriers that perform interactive action with their contents, releasing protective substances for the food or absorbing others that accelerate the deterioration process. Active packagings are divided into absorbers, reactors and releasers [3]. Absorbers absorb the harmful substances such as ethylene, oxygen and moisture [4]. Reactors provide a reaction, exothermic or endothermic, which allows the heating or cooling of the product contained therein. The releasers release active sub-
⇑ Corresponding author. E-mail address: [email protected] (C. Demitri). http://dx.doi.org/10.1016/j.measurement.2016.05.012 0263-2241/Ó 2016 Elsevier Ltd. All rights reserved.
stances for the preservation of the product [5]. The increased perception of consumers towards products in which chemical preservatives have been removed has shifted the focus in using natural active agents. It is known that the essential oils extracted from plants and spices are excellent antioxidants and antimicrobials [6]. These compounds are often incorporated in the package by impregnation of the film, or by means of functionalization of the polymer substrate. To this aim, different techniques are commonly used in order to obtain functionalized films of incorporating natural active agents into natural polymers. Even if many types of synthetic polymer formulations have been industrially manufactured, natural polymers, especially polysaccharides, are growing in interest in a range of technological fields [7–12]. Between the polysaccharides, chitosan has been widely studied due to its excellent film-forming nature [13], antimicrobial properties, physical and mechanical properties, its biocompatibility and biodegradability [14]. Chitosan is a natural polymer obtained by deacetylation of chitin. It is a linear polycationic polysaccharide soluble only in acidic environment (pKa 6.5). The aim of this study is to develop an innovative method to achieve an improvement in food safety by controlling the proliferation of both native or food-spoilage microorganisms by reducing the use of synthetic preservatives.
C. Demitri et al. / Measurement 90 (2016) 418–423
These active systems are based on the use of natural antifungal agents incorporated in carrier materials or devices, that trigger the release once necessary, control the rate of release, having either lethal or inhibitory actions against pathogens or microorganisms comprised in foodstuffs. This study is focused on the modification of chitosan with cinnamaldehyde and nanometric sonicated expanded graphite stacks (EGS). Cinnamaldehyde is an aromatic a,b-unsaturated aldehyde, and the major component in essential oils from some cinnamon species. In literature there are many studies focused on active packaging, for example cassava starch and gliadin [15], functionalized with cinnamaldehyde, which showed both antimicrobial and fungicide effectiveness of cinnamaldehyde [16]. It has been shown to exert antimicrobial and antifungal action against a wide number of microorganisms including bacteria, yeasts, and mould [17]. The functionalization of chitosan occurs via Schiff base formation [18]. The essential aspect of this reaction mechanism is its reversibility. Schiff base is reversible under suitable conditions, and this might allow the release of the active cinnamaldehyde from chitosan, used as the carrier [19]. Moreover the addition of nanometric stacks was evaluated in terms of enhancement of the mechanical properties.
419
any modification at a resolution of 4 cm 1, by 64 scans, at transmittance range from 4000 cm 1 to 400 cm 1.
2.5. Morphology: SEM, TEM and X-ray SEM micrographs were made using a Jeol JSM-6550F instrument. Transmission electron microscopy (TEM) images were taken by a Hitachi H-9000NAR model instrument operated at an accelerating voltage of 100 kV. Samples were prepared by placing a single drop of the suspensions (diluted in deionized water and sonicated before use) onto carbon coated copper grids, dried in air and loaded into the electron microscope chamber. Wide Angle X-ray diffraction (WAXD) patterns were collected on a PW 1729 Philips, using Cu Ka radiation in reflection mode (k = 0.154 nm). Samples of natural graphite (NG), expandable graphite (GIC), expanded graphite (EG), expanded and sonicated graphite (EGS) were step-scanned at room temperature from values of 2h ranging from 1.3–60°. The samples were held in the diffractometer using a socket glass sample holder.
2.6. Mechanical tests 2. Materials and methods 2.1. Materials Chitosan (CS) with low molecular weight (50–190 kDa, deacetylation degree: 75–85%), cinnamaldehyde (CA) and glacial acetic acid were purchased from Sigma Aldrich (Milan, Italy) and used as received. Expandable graphite (GIC) was supplied by Anthracite Industries (Sunbury, USA). 2.2. Preparation of EG Expanded graphite (EG) was gained by expansion and exfoliation of GIC by heating up to 600 °C for 2 min. EG particles were then added to chitosan solution in order to obtain an EGSchitosan dispersion [20].
The mechanical properties of the CSCA_EGS specimens were investigated with Z1.0 TH material testing machine (Zwick Roell, Germany), equipped with a 100 N load cell. Specimens for uniaxial tensile tests were cut into strips 10.0 mm wide and 60 mm long, from films with 0.3 mm of thickness, and mounted between the clamps of the tensile tester. The upper clamp was connected to the load cell and to the movable crosshead. Tests were performed under displacement control, with a displacement rate of 1 mm/ min. The elastic modulus (E), fracture strength (r) and elongation at break (e) were elaborated and recorded by means of TestXpert II software. The elastic modulus was calculated as the initial slope of the stress–strain curve. Three samples from each group were tested to obtain average and standard deviation values of E, r and e. 2.7. Preliminary antifungal activity tests
2.3. Preparation of a Schiff base of EGS-chitosan dispersion The modified EGS-chitosan dispersion via Schiff base was obtained by suspending 2 g of CS powder in 100 ml of acetic acid solution (0.1 M). The flasks were immersed in a thermostatic bath at 25 °C and the suspension was stirred gently for two hours with a mechanical stirrer until complete dissolution. Then the EGS dry powders at different concentrations (1.5%, 3%, 6% w/w of dry polymer) were added to chitosan solution under constant stirring. The modification of chitosan via Schiff base was achieved by the reaction with cinnamaldehyde at different concentrations (0.1%, 0.25%, 0.5% w/w of dry polymer) under controlled temperature (25 °C). The gels were cast in Teflon trays and dried at 25 °C to obtain films. 2.4. Characterization of the Schiff base The study of kinetics reaction was followed via viscosity measurements made with a parallel plate rheometer (ARES, Scientific Rheometric) at 25 °C, from 0.1 to 100 s 1. For each CA concentration, independent measurements were performed in triplicate, and the results were expressed as the average, including standard deviation. A Fourier transform infrared spectrometer (FTIR-6300 Jasco, Easton, MD, USA) was used to assess the efficacy of the functionalization. These analyses were carried out on the samples without
Rhizopus stolonifer was isolated and grown for 2 weeks on agar plates (potato dextrose). Spores were scrubbed from the agar surface plate by means of a glass rod. Then a small volume of sterile solution containing a surfactant (0.5% v/v, Triton X-100) was added. The obtained suspension was gently shaken for 15 s to break any conidial chains and then filtered to remove mycelial fragments. The concentration of the spore was determined using a haemocytometer and adjusted with sterile water to ca. 5 105 spores/ml, and stored on ice until use. In vitro tests were carried out by inoculating fresh cut white bread slices with a suspension of the selected spores, prepared as described above. Cinnamaldehyde-functionalized EGS-chitosan films with different CA concentrations were used to wrap the slices after each inoculation. In order to prevent water evaporation, each sample was further stored in dark conditions (37 °C) in sealed polyethylene bags. 2.8. Statistical analysis Two-way analysis of variance (ANOVA), performed by means of StatView software, was used to assess the effect of both cynnamaldehyde (CA) and expanded graphite (EGS) concentrations on the mechanical properties of the films. Fisher’s Protected Least Significant Difference (PLSD) tests were applied to compare sets of data. Significance was accepted with p < 0.05.
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3. Results and discussion 3.1. Characterization of the Schiff base Fig. 1 shows the IR spectra of cinnamaldehyde, chitosan and chitosan–CA mixtures at different reaction time points. Cinnamaldehyde shows a characteristic absorption at 1683 cm 1, related to the vibration stretching of the carbon–oxygen double bond [16]. Chitosan has reactive amino groups that can be used to modify its properties under mild reaction conditions (i.e. low temperature and no toxic compounds or solvents). The presence of these groups is an indication of the possibility to perform several chemical modifications, such as the formation of Schiff bases by chemical reaction with aldehyde. The peaks of interest for the reaction of chitosan with cinnamaldehyde are those that fall to 1649 cm 1 and 1559 cm 1, corresponding respectively to the stretching of the carbon–oxygen double bond of the amide bond and the bending of the nitrogen–hydrogen of the amine. After the reaction between chitosan and cinnamaldehyde, FTIR was used to confirm the structure of the Schiff base of chitosan. The FTIR spectra highlighted the presence of the absorption peak of imine, derived by reaction from amino group of chitosan and carbonyl of aldehyde. Among chitosan characteristic bands, new absorption peaks appeared at 1635 cm 1 corresponding to the C@N vibration characteristic of imines. Moreover, the characteristic peaks associated with the stretching of the carbon–oxygen double bond and the peaks corresponding to the stretching of the CAH bond aldehyde disappeared.
Fig. 1. FTIR spectra of cinnamaldehyde (CA), chitosan (CS), and chitosan–cinnamaldehyde (CS–CA) compound (0.5% CA) at different reaction time points (0 and 24 h).
Rheological analyses of chitosan–cinnamaldehyde (CS–CA) solutions were performed at different time points in order to monitor the progress of the CS–CA reaction. As a result of the Schiff base formation, the solution viscosity (at a fixed shear rate) gradually increased up to a plateau value, which indicated the formation of a physically entangled hydrogel and was assumed as representative of the maximum extent of CS functionalization yielded for each CA concentration. The results showed that the CS solution with 0.1% CA reached its maximum viscosity after approximately 72 h, while those with 0.25% CA and 0.5% CA were both found to achieve their viscosity plateau after 8 h. This suggested that the reaction rate was particularly increased when increasing the cinnamaldehyde concentration from 0.1% to 0.25%, while further additions of CA did not seem to affect the kinetics. Moreover, while chitosan solutions typically behaved as pseudoplastic fluids, functionalized chitosan showed a hybrid behavior (Fig. 2). At low shear rates, the material displayed a typical dilatant behavior, which then converted to pseudoplastic at high shear rates. Such a peculiar dependence of the viscosity on the shear rate might be likely due to the formation of micelles ascribable to the hydrophobicity of cinnamaldehyde [21]. Micelles result from a molecular rearrangement of CS following CA functionalization and may be disrupted at high shear rates.
3.2. Morphology The morphology of expandable graphite flakes, before (GIC) and after (EG) expansion and ultrasonication (EGS), is reported in Fig. 3A–C, respectively. GIC platelets had a diameter of 400–600 lm (Fig. 3A) [22]. EG particles showed a worm-like or an accordion-like shape, made of multiple nano-scaled platelets. EGS particles had an average particle diameter of about 10 lm, as shown in Fig. 3C. Fig. 3D reported the TEM image of the resulting structure of chitosan–EGS mixtures after sonication. It is evident the formation of a multi-layered structure with separate sheets and dimensions of about 500 nm. The results of WAXD, implemented on GIC, EG and EGS, are reported in Fig. 4. GIC pattern displayed a peak centered at 2h = 26.02° (d = 0.336 nm) with a shoulder at 2h = 26.54°. EG curve showed a peak at 2h = 26.05° with a minor intensity. EGS curve evidenced the disappearance of the peak at 2h = 26.05°, confirming the disaggregation of intercalated galleries.
Fig. 2. Viscosity of chitosan solutions following functionalization with cynnamaldehyde (CA), as a function of CA concentration (0.10%, 0.25%, 0.50% CA).
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Fig. 3. Electron microscopy images of GIC (a), EG (b), EGS (c), chitosan–EGS (d) respectively. The scale bars in A, B and C are 200 lm. The scale bar in D is 200 nm.
Table 1 Mechanical properties of CSCA_EGS samples. Results are reported as mean ± standard deviation (n = 3). Sample
CS0.1CA CS0.1CA_EGS (1.5 wt%) CS0.1CA_EGS (3 wt%) CS0.1CA_EGS (6 wt%) CS0.25CA CS0.25CA_EGS (1.5 wt%) CS0.25CA_EGS (3 wt%) CS0.25CA_EGS (6 wt%) CS0.5CA CS0.5CA_EGS (1.5 wt%) CS0.5CA_EGS (3 wt%) CS0.5CA_EGS (6 wt%)
Fig. 4. WAXD patterns of GIC, EG, EGS fillers.
3.3. Mechanical properties Results of the tensile tests performed on CSCA_EGS specimens, i.e. elastic modulus (E), fracture strength (r) and elongation at break (e), are summarized in Table 1. Two-factor ANOVA showed that both CA and EGS concentrations significantly affected the elastic modulus of the films (p = 0.004 for CA concentration, p = 0.001 for EGS concentration). In particular, for given CA concentrations, the addition of EGS led
Mechanical properties E (GPa)
r (MPa)
e (%)
2.0 ± 0.4 2.2 ± 0.2 2.5 ± 0.5 3.0 ± 0.7 2.2 ± 0.3 2.6 ± 0.4 3.0 ± 0.5 3.3 ± 0.1 2.5 ± 0.3 3.0 ± 0.4 3.5 ± 0.5 3.8 ± 0.6
40 ± 6 62 ± 5 70 ± 8 84 ± 6 45 ± 7 65 ± 4 72 ± 7 85 ± 5 50 ± 7 72 ± 8 78 ± 5 90 ± 6
11 ± 3 9±2 8±3 6±4 13 ± 3 11 ± 2 10 ± 3 8±4 15 ± 3 13 ± 2 12 ± 3 9±4
to significant increases of E when the added amount was at least 3% (0% vs. 3%, p = 0.005; 0% vs. 6%, p = 0.001; 1.5% vs. 6%, p = 0.005). At the same time, at fixed EGS concentrations, increasing CA amounts led to higher moduli, although no significant difference was detected between samples with 0.1% and 0.25% CA (p = 0.13). CA and EGS concentrations were also found to distinctly increase the fracture strength of the films (p = 0.02 for CA concentration, p < 0.001 for EGS concentration), despite the effect of CA being less pronounced than that of EGS. While relevant increases of r were only observed between sets of samples with 0.1% and 0.5% CA (p = 0.006), addition of EGS caused significant increments of the fracture strength, with the only exception of samples with
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Fig. 5. Effectiveness of chitosan films at different cinnamaldehyde concentration (0.10 CA, 0.25 CA, 0.50 CA) against Rhizopus Stolonifer. Chitosan and PET bags were used as controls.
1.5% and 3% EGS concentration that displayed similar strength values (p = 0.052). As regards the elongation at break, the number of samples tested (n = 3) was too low to detect any clear effect of both CA concentration (power = 0.58) and EGS concentration (power = 0.56). However, a slight decrease of e was observed either when increasing the EGS amount or when decreasing the CA concentration. This could be related to changes in chitosan chain mobility resulting from the formation of micelles following CS–CA reaction, as well as the strong interaction between EGS and the polymer backbone [23–25]. In the overall, the results confirmed that the presence of expanded graphite in the CS–CA films led to higher values of tensile strength and modulus, likely due to the increase in chain entanglements. The latter were also favored, although to a lesser extent, by higher degrees of chitosan functionalization with cynnamaldehyde. 3.4. Antifungal activity test on white bread slices The antifungal activity of the cinnamaldehyde functionalized chitosan films was studied at different cinnamaldehyde concentrations (0.10%, 0.25% and 0.5%). The antifungal potential activity of the films was assessed qualitatively in terms of fungal growth. The results are presented in Fig. 5. The test showed an increased efficiency against moulds by increasing the cinnamaldehyde concentration. Exposure of films to humid environments, which are commonly found in foodstuffs and promote fungal growth, triggered the release of the cinnamaldehyde and, thereby, its antifungal activity. Small concentrations of cynnamaldehyde are sufficient to functionalize chitosan and are effective against the natural proliferation of moulds. This efficacy against fungi and moulds confirms that there is a gradual release of the active agent under appropriate conditions (see Fig. 5). 4. Conclusions The proposed study demonstrated that cinnamaldehyde and graphite stacks induce antifungal activity and increased
mechanical properties in chitosan substrates. The fungicidal effect was successfully evaluated by means of in vitro experiments against R. stolonifer showing an increased inhibition effect by increasing cinnamaldehyde concentration. On the other side, the presence of graphite nano-stacks significantly increased the mechanical properties of the composite material. The combination of these two properties can promote the use of these materials as potential active packaging in the food industry.
References [1] M.C. Cruz-Romero, T. Murphy, M. Morris, E. Cummins, J.P. Kerry, Antimicrobial activity of chitosan, organic acids and nano-sized solubilisates for potential use in smart antimicrobially-active packaging for potential food applications, Food Control 34 (12) (2013) 393–397. [2] P. Dutta, S. Tripathi, G. Mehrotra, J. Dutta, Perspectives for chitosan based antimicrobial films in food applications, Food Chem. 114 (2009) 1173–1182. [3] L. Vermeiren, F. Devlieghere, M. van Beest, N. de Kruijf, J. Debevere, Developments in the active packaging of foods, Trends Food Sci. Technol. 10 (3) (1999) 77–86. [4] J.P. Smith, H.S. Ramaswamy, B.K. Simpson, Developments in food packaging technology. Part II. Storage aspects, Trends Food Sci. Technol. 1 (7) (1990) 111– 118. [5] T.P. Labuza, W.M. Breene, Applications of ‘‘active packaging” for improvement of shelf-life and nutritional quality of fresh and extended shelf-life foods, J. Food Process. Preserv. 13 (1989) 1–69. [6] A. Souza, G. Goto, J. Mainardi, A. Coelho, C. Tadini, Cassava starch composite films incorporated with cinnamon essential oil: antimicrobial activity, microstructure, mechanical and barrier properties, LWT – Food Sci. Technol. 54 (2013) 346–352. [7] A. Sannino, M. Madaghiele, C. Demitri, F. Scalera, A. Esposito, V. Esposito, et al., Development and characterization of cellulose-based hydrogels for use as dietary bulking agents, J. Appl. Polym. Sci. 115 (2010) 1438–1444. [8] M.G. Raucci, M.A. Alvarez-Perez, C. Demitri, A. Sannino, L. Ambrosio, Proliferation and osteoblastic differentiation of hMSCs on cellulose-based hydrogels, J. Appl. Biomater. Funct. Mater. 10 (2012) 302–307. [9] C. Demitri, F. Scalera, M. Madaghiele, A. Sannino, A. Maffezzoli, Potential of cellulose-based superabsorbent hydrogels as water reservoir in agriculture, Int. J. Polym. Sci. 2013 (2013) 6. [10] C. Demitri, A. Giuri, M.G. Raucci, D. Giugliano, M. Madaghiele, A. Sannino, et al., Preparation and characterization of cellulose-based foams via microwave curing, Interf. Focus 4 (2014). [11] G. Cannazza, A. Cataldo, E. De Benedetto, C. Demitri, M. Madaghiele, A. Sannino, Experimental assessment of the use of a novel superabsorbent polymer (SAP) for the optimization of water consumption in agricultural irrigation process, Water 6 (2014) 2056–2069. [12] M. Madaghiele, F. Marotta, C. Demitri, F. Montagna, A. Maffezzoli, A. Sannino, Development of semi-and grafted interpenetrating polymer networks based
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[20] C.E. Corcione, F. Freuli, A. Maffezzoli, The aspect ratio of epoxy matrix nanocomposites reinforced with graphene stacks, Polym. Eng. Sci. 53 (2013) 531–539. [21] S. Dimida, C. Demitri, V.M. De Benedictis, F. Scalera, F. Gervaso, A. Sannino, Genipin-cross-linked chitosan-based hydrogels: reaction kinetics and structure-related characteristics, J. Appl. Polym. Sci. 132 (28) (2015). [22] C. Donghwan, L. Sangyeob, Y. Gyeongmo, H. Fukushima, T.L. Drzal, Macromol. Mater. Eng. 290 (3) (2005) 79–187. [23] X. Zhao, Q.H. Zhang, D.J. Chen, P. Lu, Enhanced mechanical properties of graphene-based poly(vinyl alcohol) composites, Macromolecules 43 (2014) 2357–2363. [24] S. Casciaro, F. Conversano, S. Musio, E. Casciaro, C. Demitri, A. Sannino, Full experimental modelling of a liver tissue mimicking phantom for medical ultrasound studies employing different hydrogels, J. Mater. Sci. – Mater. Med. 20 (4) (2009) 983–989. [25] F. Conversano, G. Soloperto, A. Greco, A. Ragusa, E. Casciaro, F. Chiriacò, C. Demitri, G. Gigli, A. Maffezzoli, S. Casciaro, Echographic detectability of optoacoustic signals from low-concentration PEG-coated gold nanorods, Int. J. Nanomed. 7 (2012) 4373.
Contents lists available at ScienceDirect
Measurement journal homepage: www.elsevier.com/locate/measurement
Nanostructured active chitosan-based films for food packaging applications: Effect of graphene stacks on mechanical properties Christian Demitri ⇑, Vincenzo Maria De Benedictis, Marta Madaghiele, Carola Esposito Corcione, Alfonso Maffezzoli University of Salento, Department of Engineering for Innovation, Via Monteroni, Campus Ecotekne, 73100 Lecce, Italy
a r t i c l e
i n f o
Article history: Received 10 December 2015 Received in revised form 19 April 2016 Accepted 5 May 2016 Available online 6 May 2016 Keywords: Chitosan Cinnamaldehyde Graphene Antifungal Activity Food Packaging
a b s t r a c t Bioactive food-preserving materials are based on the use of a natural antimicrobial compound loaded in a carrier material, which is able to trigger its release when requested and to modulate the rate of release, thus using either toxic or inhibitory properties against pathogens or bacteria due to food decomposition. In this study, the Schiff base formation for chitosan functionalization was achieved by the reaction of chitosan with cinnamaldehyde at different concentrations. Cinnamaldehyde is an aromatic a,b-unsaturated aldehyde, and the major component in essential oils from some cinnamon species. It has been shown to exert antimicrobial action against a large number of microorganisms including bacteria, yeasts, and mould. The formation of the Schiff base is reversible under suitable conditions, and this might allow the release of the active cinnamaldehyde from chitosan, used as the carrier. The reaction kinetics was investigated by means of rheological measurements, while infrared spectroscopy was used to assess the efficacy of the functionalization. The addition of nanometric graphene stacks to the cinnamaldehyde-functionalized chitosan films was evaluated with the aim to increase the mechanical properties of the film. Finally, the films were tested for antifungal properties with bread slices against a selected mould line. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Recently, different authors have been focused the attention on the development of natural innovative and active materials for food packaging applications [1]. It is well known that there are different processes involved in extending the shelf life of food. These involve controlled moisture transfer between inner and external environment, high permeability to certain substances, temperature control, structural reinforcement of food and coat flavor compounds, reduction of oxygen partial pressure in the package, extended release of organic agents like antimicrobial substances, antioxidants [2]. Active packagings are barriers that perform interactive action with their contents, releasing protective substances for the food or absorbing others that accelerate the deterioration process. Active packagings are divided into absorbers, reactors and releasers [3]. Absorbers absorb the harmful substances such as ethylene, oxygen and moisture [4]. Reactors provide a reaction, exothermic or endothermic, which allows the heating or cooling of the product contained therein. The releasers release active sub-
⇑ Corresponding author. E-mail address: [email protected] (C. Demitri). http://dx.doi.org/10.1016/j.measurement.2016.05.012 0263-2241/Ó 2016 Elsevier Ltd. All rights reserved.
stances for the preservation of the product [5]. The increased perception of consumers towards products in which chemical preservatives have been removed has shifted the focus in using natural active agents. It is known that the essential oils extracted from plants and spices are excellent antioxidants and antimicrobials [6]. These compounds are often incorporated in the package by impregnation of the film, or by means of functionalization of the polymer substrate. To this aim, different techniques are commonly used in order to obtain functionalized films of incorporating natural active agents into natural polymers. Even if many types of synthetic polymer formulations have been industrially manufactured, natural polymers, especially polysaccharides, are growing in interest in a range of technological fields [7–12]. Between the polysaccharides, chitosan has been widely studied due to its excellent film-forming nature [13], antimicrobial properties, physical and mechanical properties, its biocompatibility and biodegradability [14]. Chitosan is a natural polymer obtained by deacetylation of chitin. It is a linear polycationic polysaccharide soluble only in acidic environment (pKa 6.5). The aim of this study is to develop an innovative method to achieve an improvement in food safety by controlling the proliferation of both native or food-spoilage microorganisms by reducing the use of synthetic preservatives.
C. Demitri et al. / Measurement 90 (2016) 418–423
These active systems are based on the use of natural antifungal agents incorporated in carrier materials or devices, that trigger the release once necessary, control the rate of release, having either lethal or inhibitory actions against pathogens or microorganisms comprised in foodstuffs. This study is focused on the modification of chitosan with cinnamaldehyde and nanometric sonicated expanded graphite stacks (EGS). Cinnamaldehyde is an aromatic a,b-unsaturated aldehyde, and the major component in essential oils from some cinnamon species. In literature there are many studies focused on active packaging, for example cassava starch and gliadin [15], functionalized with cinnamaldehyde, which showed both antimicrobial and fungicide effectiveness of cinnamaldehyde [16]. It has been shown to exert antimicrobial and antifungal action against a wide number of microorganisms including bacteria, yeasts, and mould [17]. The functionalization of chitosan occurs via Schiff base formation [18]. The essential aspect of this reaction mechanism is its reversibility. Schiff base is reversible under suitable conditions, and this might allow the release of the active cinnamaldehyde from chitosan, used as the carrier [19]. Moreover the addition of nanometric stacks was evaluated in terms of enhancement of the mechanical properties.
419
any modification at a resolution of 4 cm 1, by 64 scans, at transmittance range from 4000 cm 1 to 400 cm 1.
2.5. Morphology: SEM, TEM and X-ray SEM micrographs were made using a Jeol JSM-6550F instrument. Transmission electron microscopy (TEM) images were taken by a Hitachi H-9000NAR model instrument operated at an accelerating voltage of 100 kV. Samples were prepared by placing a single drop of the suspensions (diluted in deionized water and sonicated before use) onto carbon coated copper grids, dried in air and loaded into the electron microscope chamber. Wide Angle X-ray diffraction (WAXD) patterns were collected on a PW 1729 Philips, using Cu Ka radiation in reflection mode (k = 0.154 nm). Samples of natural graphite (NG), expandable graphite (GIC), expanded graphite (EG), expanded and sonicated graphite (EGS) were step-scanned at room temperature from values of 2h ranging from 1.3–60°. The samples were held in the diffractometer using a socket glass sample holder.
2.6. Mechanical tests 2. Materials and methods 2.1. Materials Chitosan (CS) with low molecular weight (50–190 kDa, deacetylation degree: 75–85%), cinnamaldehyde (CA) and glacial acetic acid were purchased from Sigma Aldrich (Milan, Italy) and used as received. Expandable graphite (GIC) was supplied by Anthracite Industries (Sunbury, USA). 2.2. Preparation of EG Expanded graphite (EG) was gained by expansion and exfoliation of GIC by heating up to 600 °C for 2 min. EG particles were then added to chitosan solution in order to obtain an EGSchitosan dispersion [20].
The mechanical properties of the CSCA_EGS specimens were investigated with Z1.0 TH material testing machine (Zwick Roell, Germany), equipped with a 100 N load cell. Specimens for uniaxial tensile tests were cut into strips 10.0 mm wide and 60 mm long, from films with 0.3 mm of thickness, and mounted between the clamps of the tensile tester. The upper clamp was connected to the load cell and to the movable crosshead. Tests were performed under displacement control, with a displacement rate of 1 mm/ min. The elastic modulus (E), fracture strength (r) and elongation at break (e) were elaborated and recorded by means of TestXpert II software. The elastic modulus was calculated as the initial slope of the stress–strain curve. Three samples from each group were tested to obtain average and standard deviation values of E, r and e. 2.7. Preliminary antifungal activity tests
2.3. Preparation of a Schiff base of EGS-chitosan dispersion The modified EGS-chitosan dispersion via Schiff base was obtained by suspending 2 g of CS powder in 100 ml of acetic acid solution (0.1 M). The flasks were immersed in a thermostatic bath at 25 °C and the suspension was stirred gently for two hours with a mechanical stirrer until complete dissolution. Then the EGS dry powders at different concentrations (1.5%, 3%, 6% w/w of dry polymer) were added to chitosan solution under constant stirring. The modification of chitosan via Schiff base was achieved by the reaction with cinnamaldehyde at different concentrations (0.1%, 0.25%, 0.5% w/w of dry polymer) under controlled temperature (25 °C). The gels were cast in Teflon trays and dried at 25 °C to obtain films. 2.4. Characterization of the Schiff base The study of kinetics reaction was followed via viscosity measurements made with a parallel plate rheometer (ARES, Scientific Rheometric) at 25 °C, from 0.1 to 100 s 1. For each CA concentration, independent measurements were performed in triplicate, and the results were expressed as the average, including standard deviation. A Fourier transform infrared spectrometer (FTIR-6300 Jasco, Easton, MD, USA) was used to assess the efficacy of the functionalization. These analyses were carried out on the samples without
Rhizopus stolonifer was isolated and grown for 2 weeks on agar plates (potato dextrose). Spores were scrubbed from the agar surface plate by means of a glass rod. Then a small volume of sterile solution containing a surfactant (0.5% v/v, Triton X-100) was added. The obtained suspension was gently shaken for 15 s to break any conidial chains and then filtered to remove mycelial fragments. The concentration of the spore was determined using a haemocytometer and adjusted with sterile water to ca. 5 105 spores/ml, and stored on ice until use. In vitro tests were carried out by inoculating fresh cut white bread slices with a suspension of the selected spores, prepared as described above. Cinnamaldehyde-functionalized EGS-chitosan films with different CA concentrations were used to wrap the slices after each inoculation. In order to prevent water evaporation, each sample was further stored in dark conditions (37 °C) in sealed polyethylene bags. 2.8. Statistical analysis Two-way analysis of variance (ANOVA), performed by means of StatView software, was used to assess the effect of both cynnamaldehyde (CA) and expanded graphite (EGS) concentrations on the mechanical properties of the films. Fisher’s Protected Least Significant Difference (PLSD) tests were applied to compare sets of data. Significance was accepted with p < 0.05.
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3. Results and discussion 3.1. Characterization of the Schiff base Fig. 1 shows the IR spectra of cinnamaldehyde, chitosan and chitosan–CA mixtures at different reaction time points. Cinnamaldehyde shows a characteristic absorption at 1683 cm 1, related to the vibration stretching of the carbon–oxygen double bond [16]. Chitosan has reactive amino groups that can be used to modify its properties under mild reaction conditions (i.e. low temperature and no toxic compounds or solvents). The presence of these groups is an indication of the possibility to perform several chemical modifications, such as the formation of Schiff bases by chemical reaction with aldehyde. The peaks of interest for the reaction of chitosan with cinnamaldehyde are those that fall to 1649 cm 1 and 1559 cm 1, corresponding respectively to the stretching of the carbon–oxygen double bond of the amide bond and the bending of the nitrogen–hydrogen of the amine. After the reaction between chitosan and cinnamaldehyde, FTIR was used to confirm the structure of the Schiff base of chitosan. The FTIR spectra highlighted the presence of the absorption peak of imine, derived by reaction from amino group of chitosan and carbonyl of aldehyde. Among chitosan characteristic bands, new absorption peaks appeared at 1635 cm 1 corresponding to the C@N vibration characteristic of imines. Moreover, the characteristic peaks associated with the stretching of the carbon–oxygen double bond and the peaks corresponding to the stretching of the CAH bond aldehyde disappeared.
Fig. 1. FTIR spectra of cinnamaldehyde (CA), chitosan (CS), and chitosan–cinnamaldehyde (CS–CA) compound (0.5% CA) at different reaction time points (0 and 24 h).
Rheological analyses of chitosan–cinnamaldehyde (CS–CA) solutions were performed at different time points in order to monitor the progress of the CS–CA reaction. As a result of the Schiff base formation, the solution viscosity (at a fixed shear rate) gradually increased up to a plateau value, which indicated the formation of a physically entangled hydrogel and was assumed as representative of the maximum extent of CS functionalization yielded for each CA concentration. The results showed that the CS solution with 0.1% CA reached its maximum viscosity after approximately 72 h, while those with 0.25% CA and 0.5% CA were both found to achieve their viscosity plateau after 8 h. This suggested that the reaction rate was particularly increased when increasing the cinnamaldehyde concentration from 0.1% to 0.25%, while further additions of CA did not seem to affect the kinetics. Moreover, while chitosan solutions typically behaved as pseudoplastic fluids, functionalized chitosan showed a hybrid behavior (Fig. 2). At low shear rates, the material displayed a typical dilatant behavior, which then converted to pseudoplastic at high shear rates. Such a peculiar dependence of the viscosity on the shear rate might be likely due to the formation of micelles ascribable to the hydrophobicity of cinnamaldehyde [21]. Micelles result from a molecular rearrangement of CS following CA functionalization and may be disrupted at high shear rates.
3.2. Morphology The morphology of expandable graphite flakes, before (GIC) and after (EG) expansion and ultrasonication (EGS), is reported in Fig. 3A–C, respectively. GIC platelets had a diameter of 400–600 lm (Fig. 3A) [22]. EG particles showed a worm-like or an accordion-like shape, made of multiple nano-scaled platelets. EGS particles had an average particle diameter of about 10 lm, as shown in Fig. 3C. Fig. 3D reported the TEM image of the resulting structure of chitosan–EGS mixtures after sonication. It is evident the formation of a multi-layered structure with separate sheets and dimensions of about 500 nm. The results of WAXD, implemented on GIC, EG and EGS, are reported in Fig. 4. GIC pattern displayed a peak centered at 2h = 26.02° (d = 0.336 nm) with a shoulder at 2h = 26.54°. EG curve showed a peak at 2h = 26.05° with a minor intensity. EGS curve evidenced the disappearance of the peak at 2h = 26.05°, confirming the disaggregation of intercalated galleries.
Fig. 2. Viscosity of chitosan solutions following functionalization with cynnamaldehyde (CA), as a function of CA concentration (0.10%, 0.25%, 0.50% CA).
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Fig. 3. Electron microscopy images of GIC (a), EG (b), EGS (c), chitosan–EGS (d) respectively. The scale bars in A, B and C are 200 lm. The scale bar in D is 200 nm.
Table 1 Mechanical properties of CSCA_EGS samples. Results are reported as mean ± standard deviation (n = 3). Sample
CS0.1CA CS0.1CA_EGS (1.5 wt%) CS0.1CA_EGS (3 wt%) CS0.1CA_EGS (6 wt%) CS0.25CA CS0.25CA_EGS (1.5 wt%) CS0.25CA_EGS (3 wt%) CS0.25CA_EGS (6 wt%) CS0.5CA CS0.5CA_EGS (1.5 wt%) CS0.5CA_EGS (3 wt%) CS0.5CA_EGS (6 wt%)
Fig. 4. WAXD patterns of GIC, EG, EGS fillers.
3.3. Mechanical properties Results of the tensile tests performed on CSCA_EGS specimens, i.e. elastic modulus (E), fracture strength (r) and elongation at break (e), are summarized in Table 1. Two-factor ANOVA showed that both CA and EGS concentrations significantly affected the elastic modulus of the films (p = 0.004 for CA concentration, p = 0.001 for EGS concentration). In particular, for given CA concentrations, the addition of EGS led
Mechanical properties E (GPa)
r (MPa)
e (%)
2.0 ± 0.4 2.2 ± 0.2 2.5 ± 0.5 3.0 ± 0.7 2.2 ± 0.3 2.6 ± 0.4 3.0 ± 0.5 3.3 ± 0.1 2.5 ± 0.3 3.0 ± 0.4 3.5 ± 0.5 3.8 ± 0.6
40 ± 6 62 ± 5 70 ± 8 84 ± 6 45 ± 7 65 ± 4 72 ± 7 85 ± 5 50 ± 7 72 ± 8 78 ± 5 90 ± 6
11 ± 3 9±2 8±3 6±4 13 ± 3 11 ± 2 10 ± 3 8±4 15 ± 3 13 ± 2 12 ± 3 9±4
to significant increases of E when the added amount was at least 3% (0% vs. 3%, p = 0.005; 0% vs. 6%, p = 0.001; 1.5% vs. 6%, p = 0.005). At the same time, at fixed EGS concentrations, increasing CA amounts led to higher moduli, although no significant difference was detected between samples with 0.1% and 0.25% CA (p = 0.13). CA and EGS concentrations were also found to distinctly increase the fracture strength of the films (p = 0.02 for CA concentration, p < 0.001 for EGS concentration), despite the effect of CA being less pronounced than that of EGS. While relevant increases of r were only observed between sets of samples with 0.1% and 0.5% CA (p = 0.006), addition of EGS caused significant increments of the fracture strength, with the only exception of samples with
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Fig. 5. Effectiveness of chitosan films at different cinnamaldehyde concentration (0.10 CA, 0.25 CA, 0.50 CA) against Rhizopus Stolonifer. Chitosan and PET bags were used as controls.
1.5% and 3% EGS concentration that displayed similar strength values (p = 0.052). As regards the elongation at break, the number of samples tested (n = 3) was too low to detect any clear effect of both CA concentration (power = 0.58) and EGS concentration (power = 0.56). However, a slight decrease of e was observed either when increasing the EGS amount or when decreasing the CA concentration. This could be related to changes in chitosan chain mobility resulting from the formation of micelles following CS–CA reaction, as well as the strong interaction between EGS and the polymer backbone [23–25]. In the overall, the results confirmed that the presence of expanded graphite in the CS–CA films led to higher values of tensile strength and modulus, likely due to the increase in chain entanglements. The latter were also favored, although to a lesser extent, by higher degrees of chitosan functionalization with cynnamaldehyde. 3.4. Antifungal activity test on white bread slices The antifungal activity of the cinnamaldehyde functionalized chitosan films was studied at different cinnamaldehyde concentrations (0.10%, 0.25% and 0.5%). The antifungal potential activity of the films was assessed qualitatively in terms of fungal growth. The results are presented in Fig. 5. The test showed an increased efficiency against moulds by increasing the cinnamaldehyde concentration. Exposure of films to humid environments, which are commonly found in foodstuffs and promote fungal growth, triggered the release of the cinnamaldehyde and, thereby, its antifungal activity. Small concentrations of cynnamaldehyde are sufficient to functionalize chitosan and are effective against the natural proliferation of moulds. This efficacy against fungi and moulds confirms that there is a gradual release of the active agent under appropriate conditions (see Fig. 5). 4. Conclusions The proposed study demonstrated that cinnamaldehyde and graphite stacks induce antifungal activity and increased
mechanical properties in chitosan substrates. The fungicidal effect was successfully evaluated by means of in vitro experiments against R. stolonifer showing an increased inhibition effect by increasing cinnamaldehyde concentration. On the other side, the presence of graphite nano-stacks significantly increased the mechanical properties of the composite material. The combination of these two properties can promote the use of these materials as potential active packaging in the food industry.
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