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Chitosan-vanillin composites with antimicrobial properties
Accepted Manuscript Chitosan-vanillin composites with antimicrobial properties Marta Stroescu, Anicuta Stoica-Guzun, Gabriela Isopencu, Sorin Ion Jinga, Oana Parvulescu, Tanase Dobre, Mihai Vasilescu PII:
S0268-005X(15)00069-7
DOI:
10.1016/j.foodhyd.2015.02.008
Reference:
FOOHYD 2883
To appear in:
Food Hydrocolloids
Received Date: 4 November 2014 Revised Date:
30 January 2015
Accepted Date: 4 February 2015
Please cite this article as: Stroescu, M., Stoica-Guzun, A., Isopencu, G., Jinga, S.I., Parvulescu, O., Dobre, T., Vasilescu, M., Chitosan-vanillin composites with antimicrobial properties, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.02.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chitosan-vanillin composites with antimicrobial properties
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Marta Stroescu1, Anicuta Stoica-Guzun1*, Gabriela Isopencu1, Sorin Ion Jinga1, Oana
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Parvulescu1, Tanase Dobre1, Mihai Vasilescu2 1
University “Politehnica” of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu 1-3, Bucharest, 011061, Romania
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Babes-Bolyai University, Faculty of Physics, M. Kogalniceanu 1, Cluj-Napoca, 400084, Romania
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Corresponding author:
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Anicuta Stoica-Guzun
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Department of Chemical and Biochemical Engineering
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Faculty of Applied Chemistry and Materials Science
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University “Politehnica” of Bucharest
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Polizu 1-3, Bucharest, 011061, Romania
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E-mail: [email protected]
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Tel: +040 021 402 3870
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Abstract
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In this work there are described the obtaining and characterization of composite films containing
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chitosan, vanillin and Tween 60. Vanillin was used as cross-linker of chitosan. The composites
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were characterized by various methods like Fourier transform infrared (FTIR) spectroscopy,
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thermogravimetric analysis (TGA), X-ray diffraction (XRD) and solid state 13C nuclear magnetic
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resonance (CP/MAS
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chitosan and vanillin. Scanning electron microscopy (SEM) has also enlightened a strong
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interaction between the components. Tween 60 has influenced the value of water vapour
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transmission rate (WVTR), all the composites that contain Tween 60 having a low value of this
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parameter. Due to the fact that not all the vanillin is consumed in the Schiff base reaction, these
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composites could be used for vanillin delivery. After having studied the release behaviour we
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have concluded that most probably it is governed by crystal dissolution and Fick’s diffusion law.
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Antimicrobial activity was highlighted using Escherichia coli (ATCC 8737) strain, all the
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composites having a good antimicrobial activity.
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C NMR), which have proved the formation of a Schiff base between
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Keywords: chitosan, vanillin, Schiff base, Tween 60, antimicrobial, vanillin release
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Chemical compounds studied in this article
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Vanillin (PubChem CID: 1183); Tween 60 (PubChem CID: 24832100); Ethanol (PubChem CID:
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702)
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1.
Introduction
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The field of active food packaging finds itself in a constant development because of the
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consumers’ demand for food quality maintenance and safety. From this large family, what drew
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in the last few years more and more the attention of the researchers was the area of antimicrobial
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packaging systems, given that these materials could guarantee an extended food shelf-life as well
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as the improvement of food quality. An antimicrobial packaging is designed to inhibit or retard
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microbial growth on food surfaces (Appendini & Hotchkiss, 2002). In the development of such
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materials one could observe two directions: one is the incorporation of an antimicrobial agent
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(either synthetic or natural) in a biodegradable or non-biodegradable polymer matrix, the other
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being the incorporation of an antimicrobial agent into an antimicrobial polymer (Ramos,
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Jimenez, Peltzer & Garrigos, 2012; Cha, & Chinnan, 2004). What the latter is concerned, the
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polymer could display natural antimicrobial properties, or could be modified to possess enhanced
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antimicrobial properties. Chitosan, which is a bio polymer obtained from chitin by alkaline
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deacetylation, was already used in order to obtain antimicrobial food packaging (Cruz-Romero,
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Murphy, Morris, Cummins, & Kerry, 2013). Its antimicrobial and antifungal activity has also
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been demonstrated (Aider, 2010). In order to improve the properties of chitosan, cross-linking of
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the films is used, the most well-known cross-linkers being glutaraldehyde, formaldehyde,
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epichlorohydrin, ethylene glycol diglycidyl ether, glyoxal and other toxic reactive cross-linking
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agents (Simionescu, Marin, Tardei, Marinescu, Oprea, & Capatina, 2014; Chen, & Chen, 2009).
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Because of this inconvenience, new cross-linkers belonging to the class of natural substances are
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being tested, for example vanillin. This one, obtained from the bean or pod of the tropical
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Vanilla orchid, exhibits antioxidant and antimicrobial activity against bacteria, moulds and yeast
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and could be used also as food preservative (Rupasinghe, Boulter-Bitzer, Ahn, & Odumeru,
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2006; Rakchoy, Suppakul, & Jinkarn, 2009). Moreover, it is also being used as flavour and
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fragrance in food and cosmetic industry (Zhang, Jiang, Gao, & Li, 2008). Because the costs of
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natural vanillin are very high, the vanillin obtained by chemical synthesis is now used instead of
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natural vanillin. The aldehyde group of vanillin and of other natural or synthetic aldehydes could
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react with the amino groups of chitosan to form chitosan Schiff bases, which have superior
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antimicrobial properties in comparison with chitosan (Sashikala, & Syed Shafi, 2014; Marin,
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Stoica, Mares, Dinu, Simionescu, & Barboiu, 2013; Jagadish, Divyashree, Viswanath, Srinivas,
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& Raj, 2012). The chitosan Schiff base with vanillin was used for controlled release of
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resveratrol (Peng, Xiong, Li, Liu, Bai, & Chen, 2010), or as adsorbent for heavy metal ions
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(Cestari, Vieira, Matos, & dos Anjos, 2005). The presence of an emulsifier in the composition of
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the films could modify some of their properties, for example to decrease water vapour
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permeability (Ziani, Oses, Coma, & Maté, 2008). In this work, we have used polysorbate 60
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(Tween 60). Tween 60 is a polyoxyethylene type non-ionic surfactant commonly used as
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emulsifier, dispersant and stabilizer in many cosmetics as well as in some pharmaceutical
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products (Dang, Gray, Watson, Bates, Scholes, & Eccleston, 2006).
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The aim of this work is to characterize the composite films based on chitosan and vanillin
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with and without Tween 60 and to demonstrate that these composites could be used as
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antimicrobial, and also as flavour release materials. The release kinetics of vanillin from these
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films was evaluated and the emulsifier influence on the flavour release was also accomplished. To the best of our knowledge this is a first attempt to use a Schiff base between chitosan
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and vanillin as a flavour release and antimicrobial material.
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2. Materials and methods
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2.1. Reagents
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Unless otherwise stated, all chemicals and reagents were supplied by Sigma-Aldrich and
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were used without further purification. Chitosan, obtained from shrimp shells (viscosity <200
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mPa.s having deacetylation value ≥75.5%), was also supplied by Sigma-Aldrich.
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2.2. Film preparation
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Chitosan (CH) (1.5% w/w) was dissolved in aqueous acetic acid solution (1.0 % v/v) under
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magnetic stirring at 600 C. After the complete dissolution of chitosan, 1 g of synthetic vanillin 4
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was dissolved in 10 mL of ethanol and was added to the chitosan solution for the first casting
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solution. For the second formulation 3 g of vanillin were used, for the same concentration of
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chitosan solution. The mixture was maintained at 600 C for three hours and its colour changed in
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pal–yellow. In some solutions the emulsifier Tween 60 was also added. The prepared solutions
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were degassed under vacuum and were then cast in polytetrafluorethylene (PTFE) Petri dishes
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and dried at 400C in an oven for 48 h. Film samples were stored in plastic bags and held in
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desiccators at 75% RH and 25°C before further testing. Formulations were named as follows: F1
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(CH: vanillin mass ratio 1:1); F2 (CH: vanillin: Tween 60 mass ratio 1:1:0.25), F3 (CH: vanillin
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mass ratio 1:2) and F4 (CH: vanillin: Tween 60 mass ratio 1:2:0.25).
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2.3. Physico-chemical films characterization
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2.3.1. Film thickness measurement
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The samples’ thickness was measured using a digital external micrometer (Mitutoyo Co., Japan) at ten different points of the film. Average values were calculated.
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2.3.2. Scanning electron microscopy (SEM), X-ray diffraction, FTIR, Solid state nuclear
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magnetic resonance (NMR)
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Morphological observations were done using a scanning electron microscope HITACHI S-
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2600N (Hitachi Romania, Japan). The working conditions were the following: accelerating
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voltage 25 kV, WD (working distance) 13 mm and beam 30, in a good agreement with the
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physical characteristics of the sample. All samples were gold coated prior to SEM examination.
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The X-ray diffraction (XRD) measurements were conducted using a Shimadzu XRD 6000 diffractometer (Ni filtered Cu-Kα radiation, 40 kV, 30 mA and 0.02° step scan).
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The chitosan-vanillin composites were examined on a Jasco FT/IR6200 spectrometer
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(ABL& E-JASCO Romania) with Intron µ Infrared Microscope with ATR-1000-VZ objective.
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The spectra were obtained from the average of 50 scans recorded at a resolution of 4 cm-1 in a
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range from 4000 to 500 cm-1 with a TGS detector.
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Solid state
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(150.92 MHz) using a Bruker Avance 600 spectrometer (14.10 T). The samples were packed in a
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ZrO rotor with an outer diameter of 3.2 mm. The CP/MAS spectra were recorded with a contact
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time of 1.5 ms. Chemical shift of the 13C nuclei was estimated by using an external reference of
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TMS (tetramethilsilane, δ = 0 ppm). In order to distinguish peaks from spinning side bands,
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NMR measurements with two spinning rates of 8 and 14 kHz were carried out.
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2.3.3. Thermal gravimetric analysis
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The thermal behaviour of the composites was tested using thermogravimetric analysis on a
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thermal analyser (DTG-60-Shimadzu). The operating conditions were as follows: temperature
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range of 20 °C to 1000°C, with a heating rate of 10 °C/min, and air flow rate of 50 mL/min.
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2.3.4. Optical properties of films
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The colour of the films was also measured using a spectrophotometer UV-2450 with an
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integrating sphere (Shimadzu, Japan). CIE lab colour scale of lightness (L), a (red–green), and b
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(yellow– blue) values were used to calculate the total colour difference (∆E), and the yellowness
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index (YI) using relations 1-3(Rhim, Wu, Weller, & Schnepf, 1999).
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∆E = ∆L2 + ∆a 2 + ∆b 2
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YI = 142.86 ⋅ b / L
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where: ∆L = Ls − Lsample ; ∆a = a s − a sample ; ∆b = bs − bsample
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The standard values used were those of the white background, namely: Ls=100.42, as=-0.12 and
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bs=0.23. Calculations were made for D-65 illuminant and 100 observer. Colour measurements
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were replicated three times for each type of film on both film sides.
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2.3.5. Swelling tests
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Swelling tests were conducted using water and ethanol-water mixture (83% vol. ethanol)
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as contact liquids. Composite films were dried to constant weight and then cut into 2 cm × 2 cm
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square shapes, then immersed in distilled water or ethanol-water mixture at room temperature for
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two hours. The experiments were done in triplicate. The mass of polymer dissolved into the 6
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distilled water or ethanol-water mixture was neglected considering the short time needed for the
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experiment. Also, the amount of vanillin, contained in the active films and released in aqueous
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media was considered to be negligible in comparison with the amount of absorbed water.
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Swelling degree (SD) was obtained by measuring the initial weight (m0 ) and the weight of the
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sample in swollen state (mτ ) , using equation 3:
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SD (%) = (mτ − m0 ) m0 ⋅ 100
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2.3.6. Water vapour transmission rate
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Water vapor transmission rate (WVTR) was determined using a modified ASTM standard
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method (ASTM E 96-95, 1995) with some modifications.
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The test cups were filled with 20 g of silica gel (desiccant) to produce a 0 % RH below the
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film. The desiccant was heated at 180 °C for at least 8 h prior to its use for the experiment. The
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cups were placed at 25 °C in a desiccator containing a saturated salt solution (natrium chloride),
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which produced an RH of 75%. A temperature and humidity sensor has measured these
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parameters during the experiment. The mass of the cups was measured periodically over a 165 h
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time interval. WVTR values were determined, by linear regression, as the slopes of the steady
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state period of the curves of weight gain versus time, for all the samples. WVTR (g m-2 day-1)
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was calculated as follows:
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=
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where: J is the slope of the weight gain curves versus time (g/day) and A is the aria of the tested
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films (m2).
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2.3.7. Antibacterial activity of the films
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Antimicrobial activity was highlighted using Escherichia coli (ATCC 8737) strain. The starter
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bacterial culture was prepared in liquid Luria-Bertrani (LB) culture media, growth at 37° for 24
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hours. The cells were filtered and suspended in sterile distilled water in order to obtain a
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inoculum having the concentration of 5 ⋅ 10 5 cel / mL . The depletion of the inoculum in amount
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of 1 µL was made in Petri dishes with nutrient broth and the disk diffusion method was used to
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emphasize the antibacterial activity. The used films were cut into 6 mm diameter disk-shaped
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samples and were sterilized using short UV irradiation. The samples were incubated at 37° for 96
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h, recording the results every 24h to 24h.
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2.4. Kinetics of vanillin release
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The release behaviour of unreacted vanillin from chitosan-vanillin composites was investigated
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by total immersion assay (Stroescu, Stoica-Guzun, & Jipa, 2013). The composite films were cut
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as circular disks having 1.5 cm diameter and were immersed in 50 mL of ethanol-water mixture
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or distilled water. The experimental vials were shaken at room temperature at a shaking rate of
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100 rpm. From time to time 0.1 mL of the solution from each vial was dipped out and was
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analysed in order to determine the vanillin content. The vanillin content was measured using an
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UV-VIS spectrometer (CINTRA 6, GBS-Australia), at a fixed wavelength of 231 nm, according
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to the standard vanillin calibration curve. To determine the equilibrium vanillin concentration,
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considered as maximum value concentration which could be released for each sample, the same
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experiments were done for 120 h. The cumulative release of vanillin was calculated with
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equation (5):
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=
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where Mt is the amount of vanillin released at time t and Me is the amount of vanillin released at
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infinite time (equilibrium). The experiments were performed in triplicate and the average values
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were reported.
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2.5. Characterization of the mechanism of vanillin release
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chitosan-vanillin composite films (Siepmann, & Peppas, 2001). The first one is the Higuchi model, which is described by the following equation:
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In this paper the following empirical models were used to describe vanillin release from
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The second model that has been used was the power law model, known also as Korsmeyer–
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Peppas model and which is described by the equation (7).
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=
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The vanillin release time curves were also analysed using the Peppas and Shalin model
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proposed to underline the contribution of diffusion and also of relaxation/erosion.
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The significance of the terms of the equations (6-8) is: Mt/Me represents the vanillin fraction
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released in time t, (the equation could be applied only for Mt/Me <0.6), kH and kp are constants
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which characterise the macromolecular network, while is the diffusional exponent
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characteristic of the release mechanism for Korsmeyer–Peppas equation. For n=0.5 the release
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mechanism is dominated by the Fickian diffusion, for n=1 the active substance release is directly
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proportional to time, for 0.5 < n <1 the release mechanism named anomalous diffusion (non-
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Fickian diffusion) is the predominant one and the value of n<0.5 indicates a pseudo-Fickian
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behaviour of diffusion (Li, Fu, & Zhang, 2014). In the Peppas-Shalin equation, K1 is the Fickian
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kinetic constant and K2 is the erosion rate constant. The value of the exponent must be 0.5
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for planar films. The magnitude of the two constants is also important, if the ratio K1/K2 is
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greater than 1, the solute release is determined mainly by diffusion; if the same ratio is less than
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1, then relaxation/erosion becomes the predominant mechanism for the active agent release. It is
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also possible to have a ratio equal to 1, which corresponds to the situation in which diffusion and
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erosion might coexist for the release mechanism.
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3. Results and discussion
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3.1. Films morphology by SEM
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From Fig. 1 one could observe a lamellar structure for the films F1 and F3, which do not contain
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any emulsifier. For film F3 the structure is rather interesting due to the fact that vanillin crystals
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are entrapped in the polymeric material. The films F2 and F4, which contain emulsifier Tween
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60 are quite different, having a more compact structure in which the vanillin crystals are not
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visible. In Fig. 2 are presented SEM pictures of pure film of chitosan and vanillin crystals, being
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thus in accordance with the observation concerning films F1 and F3 from Fig. 1.
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Fig.1.
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Fig.2.
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3.2. X-ray diffraction
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From Fig. 3a one could observe that vanillin powder exhibits a sharp peak at 2θ≅ 13$ and that
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the chitosan film has two diffraction peaks: a strong one at 2θ≅ 20.5$ and the other at 2θ≅
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10.5$ . The composite films present a sharp diffraction peak due to vanillin at 2θ≅ 13$ and a
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very weak reflection due to chitosan, which is more visible for films F2 and F4. The obtained
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results must be carefully interpreted, because chitosan could react in different conditions with
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vanillin forming chitosan–vanillin Schiff-base biopolymers, which were already reported in
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literature (Sashikala, & Syed Shafi, 2014; Marin, Simionescu, & Barboiu, 2012; Tree-udom,
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Wanichwecharungruang, Seemork, & Arayachukeat, 2011). So, the peak at 2θ≅ 13$ observed
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for all composite films could also correspond to interdigitated H-bonded vanillin layers between
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the chitosan backbones, due to imine bond formation on chitosan polymeric backbones (Marin,
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Stoica, Mares, Dinu, Simionescu, & Barboiu, 2013). Also, the very weak reflection due to
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chitosan in composite XRD patterns is in agreement with those obtained by Jin, Wang, & Bai
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(2009), and indicates a poor crystallinity of the new formed Schiff base in comparison with
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chitosan.
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Fig.3.
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These aspects will be presented in detail after the analysis of the FT-IR spectra and of the
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thermogravimetric curves.
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3.3. Fourier transform-infrared spectroscopy (FT-IR) and solid state 13C NMR analysis
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FTIR spectra of all composites (F1-F4) and of the chitosan film are presented in Fig.4. These
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spectra show the absorption characteristic bands of chitosan, vanillin and also new peaks. In 10
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comparison with spectra of pure chitosan film, possible overlapping and shift of absorption
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peaks are observed in the composite spectra. So, the peaks of chitosan at 1380 cm-1 (CH bending
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and CH3 deformation), 1154 cm-1(C-O-C symmetric stretching), 1060 cm-1 (C-O stretching
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vibration of C3-OH) and 1023 cm-1 (C-O stretching vibration of C6-OH) are also visible in the
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composite spectra having different intensities (Leceta, Guerrero, & de la Caba, 2013). The peak
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at 1644 cm-1 assigned to C=O stretching (amide I band) is shifted in the composite spectra at
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1640 cm-1. This peak (marked with (*) in Fig.4) could be considered a new one, representing
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thus a stretching vibration of the newly form C=N bond between chitosan and vanillin. There are
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also some peaks which could be assigned to free vanillin in the composite spectra, these being:
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1590 cm-1 and 1513 cm-1 assigned to stretching vibration of benzene ring and 1284 cm-1
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characteristic to C-O-C stretching vibration (Liu, Zhou, Zhang, Yu, & Cao, 2013; Raschip,
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Hitruc, Oprea, Popescu, & Vasile, 2011). From FTIR spectra of composite films one could
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conclude that vanillin has reacted with chitosan so as to form a Schiff base, but an amount of free
277
vanillin is also present in the samples. The peaks characteristic to emulsifier could not be
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enlightened because of the fact that this one is to be found only in a very small amount in the
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composition of films F2 and F4, and also because between chitosan and Tween 60 there weren’t
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created any new types of bonds. A similar conclusion was formulated when Tween 20 was used
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in the composition of chitosan films (Ziani, Oses, Coma, & Maté, 2008).
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Fig. 4.
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CP/MAS
C NMR spectra of chitosan, vanillin and composite film F1are presented in Fig. 5.
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The spectrum of chitosan presents all the already known characteristic peaks reported in
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literature for aliphatic carbons in the chitosan, between 20 and 110 ppm (Silva, Goodfellow,
286
Benesch, Rocha, Mano, & Reis, 2007; Krishnapriya & Kandaswamy, 2010; Chang, Chen, Ellis,
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& Tung, 2012), for example C1 ring carbon at 105 ppm, or methyl CH3 carbon at 23.76 ppm.
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Also, the vanillin spectrum is comparable with those already reported (Peña, de Ménorval,
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Garcia-Valls, & Gumí, 2011). The composite F1 spectrum exhibits all the peaks belonging to the 11
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chitosan and vanillin carbons, in addition to which one notices the presence of a new peak at
291
168.29 ppm, which could be attributed to the imine (CH=N) carbon of a new linkage formed by
292
condensation reaction of the vanillin aldehyde group with chitosan amine group (Baran, Mentes,
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& Arslan, 2015). Fig.5.
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3.4. Thermal behaviour
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The typical thermograms of composite films (F1-F4) and of the chitosan film are presented in
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Fig.6. For the films F1-F4 a percentage of weight loss up to 6-14% is observed up to 1200C (due
298
to the loss of adsorbed and bound water dehydratation) for all the films. A second weight loss up
299
to 24-32 % was observed between 2000C and 3500C and could be attributed to the thermal
300
decomposition of the Schiff base formed between chitosan and vanillin. Even if the TGA curve
301
of chitosan is similar with those of composite films, there are still some differences. The peak
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due to the partial oxidation of chitosan was observed at 278 0C, which is a higher temperature
303
than those observed in the case of composite films F1-F4.
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In Fig. 7, where there are presented the DTA curves for the composite films, one could observe
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the existence of exothermic peaks that correspond to the second weight loss. The third weight
307
loss was from 350-6000C and may be related to the slow decomposition of the carbonaceous
308
residue. Our results are in accordance with similar results obtained for thermal behavior of Schiff
309
bases obtained from the reaction of chitosan and salicylaldehyde derivatives (Estrela dos Santos,
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Dockal, & Cavalheiro, 2005).
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Fig.7.
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Analysing the exothermic peaks from Fig. 7 and basing our reasoning on the conclusions of
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Guinesi, & Cavalheiro (2006), we can appreciate that the degree of substitution (defined by the
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afore mentioned authors as the number of free amino groups in relation to the Schiff bases on the
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substituted biopolymeric matrix) of the film F1 is the greatest one, because this film has the 12
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lowest value for the exothermic decomposition peak. This result is in accordance with the fact
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that the starting composition of film F1 has a molar ratio approximately 1:1 between –NH2/–
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CHO, which is more favourable to the chemical reaction between chitosan and vanillin in
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comparison with the casting solutions used for films F3 and F4.
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3.5. Film colour
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Colour difference (∆E*) and yellowness index (YI) for the composite films F1-F4 are depicted in
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Fig. 8. The colour difference (∆E*) varies in the 44-48 units range, without sensible differences
323
between the composite films F1-F4. One can see that the films F1 and F3, which do not contain
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Tween 60, have the yellowness index in the 68-71 units range, while for the films F2 and F4,
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which contain an emulsifier, the values of YI are greater, being of 78-79 units. The yellow index
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is also an argument for the reaction between chitosan and vanillin, because films with yellow
327
colour of different intensities have been obtained for chitosan Schiff bases with vanillin and its
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derivatives (Marin, Stoica, Mares, Dinu, Simionescu, & Barboiu, 2013). The yellow colour could
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be also accentuated by the presence of the emulsifier in the composition of films F2 and F4.
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3.6. Film swelling
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In Fig. 9 there is presented the swelling degree for all the composite films in two solvents:
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ethanol-water mixture and water. At first sight the composite F3 has a very high swelling degree
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in both solvents. The swelling degrees for all the films are higher in water, probably due to the
335
presence of the unreacted chitosan in the studied samples. The presence of Tween 60 in the
336
composition of film F2 and F4 reduce the swelling degree in both solvents. When a surfactant is
337
used in a polymer casting solution, multiple interactions between surfactant and film polymers
338
are possible. In the case of Tween 60, which is a surfactant with a high Hydrophile-Lipophile
339
Balance (HLB >10), hydrogen bond interactions could be established between the hydrophilic
340
group of the surfactant and the hydrophilic group of chitosan. In this case a strong interaction
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13
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341
between polymer and surfactant could explain the decrease of the swelling degree of composite
342
films which contain Tween 60 (Maran, Sivakumar, Thirugnanasambandham, & Sridhar 2013). Fig. 9.
343
3.7. Water vapour transmission rate
345
Fig. 10 presents the WVTR values of all composite films (F1-F4) and of the chitosan film (F0).
346
For the latter we have measured a WVTR value of 636.527±4.95 g/m2day. For the composite
347
films the values obtained are in the 400-635 g/m2day range. One could observe that the
348
composite samples which contain emulsifier have the lowest value of WVTR, which could be
349
explained by the hydrophobic nature of Tween 60. The films F1 and F3 have WVTR values
350
nearest to the value of this parameter measured for chitosan film. The results could be
351
qualitatively related to the swelling degree, but not for all the films. For example, film F3 has the
352
highest swelling degree and also the highest WVTR, while film F2 has the lowest swelling
353
degree and the lowest WVTR value. The water vapour transfer mechanism could be described in
354
terms of solubility and diffusion of water vapour molecules through polymeric films. When a
355
surfactant is used, multiple interactions between surfactant and polymer matrix are possible. In
356
the studied case, the SEM images (Fig.1) reveal a more compact structure of the films that
357
contain Tween 60 in comparison with the films without surfactant. Due to the fact that Tween 60
358
has a HLB>10 there are possible interactions between its polar group and the hydrophilic film
359
matrix. Also, its hydrophobic structure could be a barrier to water molecules in the composite
360
films, as it was already observed for other composite films (Brandelero, Yamashita, &
361
Grossmann, 2010; Chen, Kuo, & Lai, 2010). Due to the fact that chitosan and vanillin have
362
reacted to form a Schiff base, it is also necessary to take into account the contribution of the
363
structure of the newly formed Schiff base (vanillin–imino-chitosan) to the WVTR measured
364
values, but this aspect is difficult to appreciate and more experimental studies are needed.
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Fig. 10.
365 366
3.8. Antibacterial activity of the films 14
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The antibacterial activity of the films against E. coli is presented in Table 1. The values of the
368
inhibition zone (IZ), expressed in millimetres, highlight a good antibacterial activity of the
369
studied films. From the data presented in Table 1 one could observe that the chitosan film has no
370
antibacterial activity. The films F2 and F4 showed antimicrobial activity from the first day of
371
incubation, the inhibition zone at this time being of 11±1.3 mm; that indicates a quick release of
372
the antimicrobial substance in the culture media (in the first 24 h) and afterwards a slow release
373
of a small quantity of antibacterial substance. The films F1 and F3 have a slow release of the
374
antibacterial substance at the beginning of the incubation. The F1 film presents a release of a
375
large amount of the active substance after 48 h. The amount released from the film F1 was
376
higher than the one obtained from the other films, because in this case the IZ was higher at the
377
end of the incubation period of the samples. The F3 film showed an intermediate release in the
378
first period of incubation and a significant greater release of active substance after 72 h of
379
incubation.
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380
Table 1.
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3.9. Kinetics of vanillin release
383
The cumulative release of vanillin was calculated using equation (5) and is represented in Fig.
384
11a for water and in 11b for an ethanol-water mixture as release media. In both media,
385
cumulative release displays a burst effect for all films. The composite films showed a lower
386
cumulative release in ethanol-water mixture than in water. These results could be explained by
387
the higher swelling degrees of the composite films in water. Moreover, the film F3 showed the
388
greatest cumulative release in both media, in accordance with its highest swelling degree.
389
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Fig.11.
≤ 0.6 was analysed
390
The portion of the vanillin release curves with a fractional release
391
according to several empirical models described by equations (6-8). Nonlinear least squares
15
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392
fitting method was used to determine the parameters in each equation. The calculated parameter
393
values along with the regression coefficients (r2) are listed in Table 2. Table 2.
395
As one could see from Table 2, we have obtained a good fit to the Korsmeyer-Peppas model
396
(r2>0.968 for vanillin release in water and r2>0.969 for ethanol-water as release medium). For the
397
Higuchi model we have also obtained high correlation coefficients (r2>0.921 for water as release
398
medium and r2>0.950 for vanillin release in ethanol-water). The exponent values for the
399
Korsmeyer-Peppas model are inferior to 0.5, which is the characteristic value for pure Fickian
400
diffusion for thin films. This is not surprising, given that the release mechanism, in such systems
401
as those studied in this paper, could be a complex one including not only diffusion, but also
402
swelling, crystal dissolution and polymer erosion. Because the values of are higher than 0.2
403
we may consider a pseudo Fickian release mechanism for all the studied films and for both
404
solvents used as release media in the experiments. Analyzing the values of correlation coefficient
405
(r2) for all the tested models we have observed that the models are comparable and we have not
406
obtained spectacular results with any of them.
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Based on the obtained results and applying the three empirical models depicted in Table 2,
408
we could presume that the release of vanillin could be controlled by diffusion through the
409
composite films. Fick's second law could be used in these conditions in order to determine the
410
diffusion coefficients of vanillin through chitosan-vanillin composite films. For this, we have
411
used one of the solutions of Fick’s second law of diffusion under non-steady state condition for
412
film/planar geometry and constant boundary conditions, which are recommended for ‘monolithic
413
systems’ (Siepmann, & Siepmann, 2012). The fraction of an active substance released in time is
414
given by the following equation:
415
416
where: Mt is the cumulative amount of the released substance at time t, Me is the amount of the
417
substance released at equilibrium (at infinite time), D is the difusion coefficient of the substance
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,
= 1 − - . ∑; <$
01 2345 67. - . 8/9 . :
(9)
5 67.
16
ACCEPTED MANUSCRIPT
418
within the polymer matrix and δ is the total thickness of the film. In this equation D is the main
419
transport parameter and was determined applying the following short time approximation of
420
equation 10:
421
= 45
48 / 7 ;
-9 .
≤ 0.6
(10)
The results obtained for the diffusion coefficient of vanillin are presented in Table 3 along with
423
correlation coefficient values. From Table 3 one could see that diffusion coefficients calculated
424
for Fickian diffusion are in the range 10-9 and 10-10 cm2/s for almost all the tested films and for
425
the two solvents used as release media. The exception is due to the film F3. When water
426
constituted the release medium the value of vanillin diffusion coefficient through the composite
427
film F3 was determined as 1.208x10-8 cm2/s, being the highest for all the films tested, and in
428
ethanol-water as release medium the same coefficient has the value 7.541x10-9 cm2/s, this one
429
being also the highest value of all the coefficients calculated. If we analyse the swelling data, we
430
may observe that the film F3 has the highest swelling rate of all the tested films and in both
431
tested solvents as release media. The explanation comes probably from the fact that water
432
diffuses very fast in the polymer matrix, which enhances the diffusion/dissolution of vanillin. If
433
we analyse the coefficient of determination calculated for Fickian diffusion, the results obtained
434
are high enough, yet not very high, being in the range 0.955 to 0.988. The analysis of the
435
correlation coeffcient reveals a good correlation, but not greater than those obtained when the
436
empirical models were tested. This could be an indication that the release mechanism is more
437
complicated than the simple diffusion, even if diffusion could be one of the phenomena implied
438
in vanillin release from composite films. From the experiments of swelling, a high swelling
439
degree of the composite films was measured, and under these conditions the probability that the
440
release mechanism be governed only by Fick’s second law of diffusion is low. Also, from SEM
441
images there could be observed the existence of vanillin crystals in the structure of film F3, for
442
example. In this context, a model which considers that the total amount released is due to
443
diffusion and crystals dissolution seems to be a realistic one. In this model, the fractional amount
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17
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444
released is considered to be formed by the sum of a fraction due to diffusion, which could be
445
calculated using a solution of the Fick’s law and a fraction due to vanillin crystals dissolution
446
(Uz, & Altınkaya, 2011; Berens, & Hopfenberg, 1978). The fraction of vanillin release using this
447
model is described by equation 11.
448
449
where: Mt is the cumulative amount of the released substance at time t, Me is the amount of the
450
substance released at equilibrium, XF is the fraction of vanillin released by Fickian diffusion and
451
kd is the dissolution constant of vanillin crystals. This equation has three main parameters: XF, D
452
and kd, which were determined by minimizing the difference between the experimental data and
453
the model prediction given by equation (11). The results obtained using the dissolution crystals
454
model are presented in Table 3.
-.
∑; <$
01 2345 67. - . 8/9 . : 5 67.
A + 51 − >? 7B1 − CD5−E 7F
(11)
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,
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= >? @1 −
Table 3.
455
One can observe from Table 3 that the values of vanillin diffusion coefficients were higher for
457
the composite films than for the Fickian diffusion, because in the latter model only a fraction of
458
the vanillin is released through diffusion. If we compare all the composites, film F3 represents
459
once again the exception, being characterised by the highest vanillin diffusion coefficients. The
460
dissolution constants have values in the range 10-4 and 10-6 s-1, the highest value being obtained
461
also for vanillin release from F3 composite film when water is used as release medium. The
462
values of dissolution constants are comparable with those obtained by Uz, & Altınkaya (2011)
463
for potassium sorbate release from cellulose acetate based mono and multilayer films. The
464
correlation coefficient calculated for the crystals dissolution model is now higher, being in the
465
range 0.980-0.998 and indicating thus that this model describes better the vanillin release
466
mechanism from composite films.
467
4. Conclusion
468
New composite films containing chitosan-vanillin and Tween 60 were prepared using different
469
ratios between components. All the films have a yellow-pal colour. The SEM micrographs reveal
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18
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that the structures of the films with emulsifier are different from those of the films without
471
emulsifier. The films without emulsifier have a lamellar structure with vanillin crystals entrapped
472
in the films. The water vapour transmission rate is lower for the films containing Tween 60.
473
FTIR spectra proved that chitosan and vanillin have formed a Schiff base, but because of the
474
mild conditions used for solution casting films, a certain amount of vanillin has remained
475
unreacted. For this reason, the release of vanillin was also measured in order to use these films as
476
flavour release materials. Among the empirical models used to fit the experimental data, the
477
most suitable was the one which took into account the crystals dissolution and the vanillin
478
Fickian diffusion. All the prepared films have proven a good antimicrobial activity against E.
479
coli, this being also an indirect proof for the Schiff base vanillin–imino–chitosan formation.
480
These films could be used as antimicrobial materials, but also as flavour release films for food or
481
cosmetic products packaging.
482
485
Financial support from UEFSCDI–Romania (Research Grant–SABIOM-No. 114/2012) is gratefully acknowledged.
486
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484
Acknowledgement
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488
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Zhang, Q.-F., Jiang, Z.-T., Gao, H.-J., & Li, R. (2008). Recovery of vanillin from aqueous
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ACCEPTED MANUSCRIPT Ziani, K., Oses, J., Coma, V., & Maté, J. I. (2008). Effect of the presence of glycerol and
599
Tween 20 on the chemical and physical properties of films based on chitosan with different
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ACCEPTED MANUSCRIPT Our reference: FOOHYD 2883
Table captions Table 1. Inhibition zones obtained by the agar diffusion method against E. coli for the
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composite films F1-F4 and for the chitosan film (F0). Table 2. Vanillin release kinetic model parameters of F1-F4 composites.
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Table 3. Parameters of Fick model and model with crystals dissolution for vanillin release.
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Our reference: FOOHYD 2883
2
Figure captions
3
Fig.1. SEM pictures of vanillin-chitosan composites F1-F4.
4
Fig.2. SEM pictures of pure chitosan film and vanillin powder.
5
Fig.3. XRD patterns of: a) vanillin powder and chitosan film and b) composite films F1-F4.
6
Fig.4. FTIR spectra of composite films (F1-F4) and chitosan (absorption characteristic peaks of
7
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1
(#)-chitosan; (•)-vanillin and (*)-Schiff base vanillin–imino–chitosan.
Fig.5. 13C NMR spectra of: (a) chitosan, (b) vanillin and (c) composite film F1.
9
Fig.6. TGA curves of: (a) chitosan, (b) films (F1, F2) and (c) films (F3, F4).
SC
8
Fig.7. DTA curves of composite films F1-F4.
11
Fig.8. Total colour difference (∆E*) and yellowness index (YI) for the composite films F1-F4.
12
Fig.9. Comparison between swelling degrees of composite films F1-F4 in different solvents
13
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ethanol-water mixture (///) and water (III).
Fig.10. WVTR values for composite films (F1-F4) and chitosan film (F0).
15
Fig.11. Vanillin profile release from composite films: a) in water and b) in ethanol-water as
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17
release media.
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14
1
ACCEPTED MANUSCRIPT Table 1.
Inhibition zone (mm) Sample 48 h
96 h
F0
No effect
No effect
No effect No effect
F1
6.0±1.2
7.0±1.0
15±1.8
15±1.5
F2
11±1.3
11±1.2
11±1.8
12±1.3
F3
8.0±1.2
8.0±1.5
8.0±1.3
12±1.2
F4
11±1.5
13±1.2
13±1.7
13±1.2
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72 h
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ACCEPTED MANUSCRIPT Table 2. Higuchi eqn
Korsmeyer-Peppas eqn n
kp (min-n)
r2
0.468 0.230 0.235 0.406
0.065 0.231 0.236 0.085
0.971 0.982 0.968 0.983
0.070 0.233 0.225 0.083
0.028 0.992 0.432 0.042 F1/ew 0.022 0.985 0.473 0.028 F2/ew 0.065 0.950 0.375 0.112 F3/ew 0.030 0.984 0.376 0.064 F4/ew *w-water and ew-ethanol-water mixture
0.992 0.983 0.969 0.989
0.041 0.033 0.109 0.065
0.981 0.921 0.968 0.976
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F1/w F2/w F3/w F4/w
kH (min-1) 0.057 0.072 0.065 0.057
kd krx104 (min-m) (min-m)
m
r2
10.50 4.07 64.01 4.56
0.450 0.228 0.231 0.406
0.972 0.986 0.969 0.979
1.29 0.59 3.36 0.99
0.433 0.432 0.379 0.376
0.990 0.983 0.970 0.993
SC
r2
Peppas-Sahlin eqn
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Samples/ Release medium*
ACCEPTED MANUSCRIPT
Table 3. Fick model
Model with crystal dissolution Dissolution Fraction constant of vanillin k(s-1) released by Fickian diffusion XF
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Sample/ Release medium
Coefficient of determination r2
Diffusion coefficient DCS (cm2/s)
F1/w F2/w F3/w F4/w F1/ew F2/ew F3/ew F4/ew
1.181x10-9±3.30x10-11 1.684x10-9±8.60x10-11 1.208x10-8±2.60x10-9 2.272x10-9±1.80x10-10 3.430x10-10±2.59x10-11 1.783x10-10±1.64x10-11 7.541x 10-9±1.00x10-10 5.549x10-10±5.33x10-11
0.978 0.955 0.978 0.983 0.979 0.988 0.978 0.962
2.414x10-9±1.20x10-10 1.089x10-8±3.72x10-9 5.304x10-7±4.70x10-9 4.291x10-9±7.74x10-10 1.259x10-9±1.37x10-10 1.774x10-9±3.33x10-10 1.661x10-8±3.17x10-9 6.212x10-9±2.83x10-10
Coefficient of determination r2
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w/water ew/ethanol-water
Diffusion coefficient DF (cm2/s)
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7.94x10-6±1.52x10-7 1.34x10-5±1.67x10-6 1.54x10-4±1.55x10-5 7.86x10-6±1.44x10-7 2.91x10-6±1.34x10-7 5.14x10-6±1.88x10-7 4.64x10-6±2.99x10-7 3.26x10-6±1.24x10-7
0.725±0.003 0.619±0.003 0.353±0.008 0.806±0.002 0.531±0.004 0.360±0.047 0.864±0.038 0.449±0.025
0.980 0.986 0.996 0.986 0.991 0.996 0.980 0.990
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Highlights
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• Chitosan-vanillin composite films with and without Tween 60 were synthetized. • Antimicrobial properties of composites were investigated against Escherichia coli. • The formation of a Schiff base between chitosan and vanillin was proved. • The release kinetics of vanillin was also studied.
S0268-005X(15)00069-7
DOI:
10.1016/j.foodhyd.2015.02.008
Reference:
FOOHYD 2883
To appear in:
Food Hydrocolloids
Received Date: 4 November 2014 Revised Date:
30 January 2015
Accepted Date: 4 February 2015
Please cite this article as: Stroescu, M., Stoica-Guzun, A., Isopencu, G., Jinga, S.I., Parvulescu, O., Dobre, T., Vasilescu, M., Chitosan-vanillin composites with antimicrobial properties, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.02.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chitosan-vanillin composites with antimicrobial properties
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Marta Stroescu1, Anicuta Stoica-Guzun1*, Gabriela Isopencu1, Sorin Ion Jinga1, Oana
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Parvulescu1, Tanase Dobre1, Mihai Vasilescu2 1
University “Politehnica” of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu 1-3, Bucharest, 011061, Romania
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Babes-Bolyai University, Faculty of Physics, M. Kogalniceanu 1, Cluj-Napoca, 400084, Romania
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Corresponding author:
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Anicuta Stoica-Guzun
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Department of Chemical and Biochemical Engineering
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Faculty of Applied Chemistry and Materials Science
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University “Politehnica” of Bucharest
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Polizu 1-3, Bucharest, 011061, Romania
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E-mail: [email protected]
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Tel: +040 021 402 3870
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Abstract
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In this work there are described the obtaining and characterization of composite films containing
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chitosan, vanillin and Tween 60. Vanillin was used as cross-linker of chitosan. The composites
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were characterized by various methods like Fourier transform infrared (FTIR) spectroscopy,
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thermogravimetric analysis (TGA), X-ray diffraction (XRD) and solid state 13C nuclear magnetic
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resonance (CP/MAS
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chitosan and vanillin. Scanning electron microscopy (SEM) has also enlightened a strong
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interaction between the components. Tween 60 has influenced the value of water vapour
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transmission rate (WVTR), all the composites that contain Tween 60 having a low value of this
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parameter. Due to the fact that not all the vanillin is consumed in the Schiff base reaction, these
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composites could be used for vanillin delivery. After having studied the release behaviour we
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have concluded that most probably it is governed by crystal dissolution and Fick’s diffusion law.
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Antimicrobial activity was highlighted using Escherichia coli (ATCC 8737) strain, all the
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composites having a good antimicrobial activity.
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C NMR), which have proved the formation of a Schiff base between
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Keywords: chitosan, vanillin, Schiff base, Tween 60, antimicrobial, vanillin release
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Chemical compounds studied in this article
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Vanillin (PubChem CID: 1183); Tween 60 (PubChem CID: 24832100); Ethanol (PubChem CID:
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702)
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1.
Introduction
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The field of active food packaging finds itself in a constant development because of the
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consumers’ demand for food quality maintenance and safety. From this large family, what drew
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in the last few years more and more the attention of the researchers was the area of antimicrobial
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packaging systems, given that these materials could guarantee an extended food shelf-life as well
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as the improvement of food quality. An antimicrobial packaging is designed to inhibit or retard
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microbial growth on food surfaces (Appendini & Hotchkiss, 2002). In the development of such
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materials one could observe two directions: one is the incorporation of an antimicrobial agent
67
(either synthetic or natural) in a biodegradable or non-biodegradable polymer matrix, the other
68
being the incorporation of an antimicrobial agent into an antimicrobial polymer (Ramos,
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Jimenez, Peltzer & Garrigos, 2012; Cha, & Chinnan, 2004). What the latter is concerned, the
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polymer could display natural antimicrobial properties, or could be modified to possess enhanced
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antimicrobial properties. Chitosan, which is a bio polymer obtained from chitin by alkaline
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deacetylation, was already used in order to obtain antimicrobial food packaging (Cruz-Romero,
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Murphy, Morris, Cummins, & Kerry, 2013). Its antimicrobial and antifungal activity has also
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been demonstrated (Aider, 2010). In order to improve the properties of chitosan, cross-linking of
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the films is used, the most well-known cross-linkers being glutaraldehyde, formaldehyde,
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epichlorohydrin, ethylene glycol diglycidyl ether, glyoxal and other toxic reactive cross-linking
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agents (Simionescu, Marin, Tardei, Marinescu, Oprea, & Capatina, 2014; Chen, & Chen, 2009).
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Because of this inconvenience, new cross-linkers belonging to the class of natural substances are
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being tested, for example vanillin. This one, obtained from the bean or pod of the tropical
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Vanilla orchid, exhibits antioxidant and antimicrobial activity against bacteria, moulds and yeast
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and could be used also as food preservative (Rupasinghe, Boulter-Bitzer, Ahn, & Odumeru,
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2006; Rakchoy, Suppakul, & Jinkarn, 2009). Moreover, it is also being used as flavour and
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fragrance in food and cosmetic industry (Zhang, Jiang, Gao, & Li, 2008). Because the costs of
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natural vanillin are very high, the vanillin obtained by chemical synthesis is now used instead of
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natural vanillin. The aldehyde group of vanillin and of other natural or synthetic aldehydes could
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react with the amino groups of chitosan to form chitosan Schiff bases, which have superior
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antimicrobial properties in comparison with chitosan (Sashikala, & Syed Shafi, 2014; Marin,
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Stoica, Mares, Dinu, Simionescu, & Barboiu, 2013; Jagadish, Divyashree, Viswanath, Srinivas,
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& Raj, 2012). The chitosan Schiff base with vanillin was used for controlled release of
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resveratrol (Peng, Xiong, Li, Liu, Bai, & Chen, 2010), or as adsorbent for heavy metal ions
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(Cestari, Vieira, Matos, & dos Anjos, 2005). The presence of an emulsifier in the composition of
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the films could modify some of their properties, for example to decrease water vapour
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permeability (Ziani, Oses, Coma, & Maté, 2008). In this work, we have used polysorbate 60
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(Tween 60). Tween 60 is a polyoxyethylene type non-ionic surfactant commonly used as
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emulsifier, dispersant and stabilizer in many cosmetics as well as in some pharmaceutical
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products (Dang, Gray, Watson, Bates, Scholes, & Eccleston, 2006).
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The aim of this work is to characterize the composite films based on chitosan and vanillin
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with and without Tween 60 and to demonstrate that these composites could be used as
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antimicrobial, and also as flavour release materials. The release kinetics of vanillin from these
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films was evaluated and the emulsifier influence on the flavour release was also accomplished. To the best of our knowledge this is a first attempt to use a Schiff base between chitosan
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and vanillin as a flavour release and antimicrobial material.
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2. Materials and methods
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2.1. Reagents
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Unless otherwise stated, all chemicals and reagents were supplied by Sigma-Aldrich and
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were used without further purification. Chitosan, obtained from shrimp shells (viscosity <200
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mPa.s having deacetylation value ≥75.5%), was also supplied by Sigma-Aldrich.
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2.2. Film preparation
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Chitosan (CH) (1.5% w/w) was dissolved in aqueous acetic acid solution (1.0 % v/v) under
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magnetic stirring at 600 C. After the complete dissolution of chitosan, 1 g of synthetic vanillin 4
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was dissolved in 10 mL of ethanol and was added to the chitosan solution for the first casting
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solution. For the second formulation 3 g of vanillin were used, for the same concentration of
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chitosan solution. The mixture was maintained at 600 C for three hours and its colour changed in
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pal–yellow. In some solutions the emulsifier Tween 60 was also added. The prepared solutions
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were degassed under vacuum and were then cast in polytetrafluorethylene (PTFE) Petri dishes
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and dried at 400C in an oven for 48 h. Film samples were stored in plastic bags and held in
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desiccators at 75% RH and 25°C before further testing. Formulations were named as follows: F1
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(CH: vanillin mass ratio 1:1); F2 (CH: vanillin: Tween 60 mass ratio 1:1:0.25), F3 (CH: vanillin
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mass ratio 1:2) and F4 (CH: vanillin: Tween 60 mass ratio 1:2:0.25).
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2.3. Physico-chemical films characterization
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2.3.1. Film thickness measurement
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The samples’ thickness was measured using a digital external micrometer (Mitutoyo Co., Japan) at ten different points of the film. Average values were calculated.
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2.3.2. Scanning electron microscopy (SEM), X-ray diffraction, FTIR, Solid state nuclear
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magnetic resonance (NMR)
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2600N (Hitachi Romania, Japan). The working conditions were the following: accelerating
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voltage 25 kV, WD (working distance) 13 mm and beam 30, in a good agreement with the
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physical characteristics of the sample. All samples were gold coated prior to SEM examination.
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The X-ray diffraction (XRD) measurements were conducted using a Shimadzu XRD 6000 diffractometer (Ni filtered Cu-Kα radiation, 40 kV, 30 mA and 0.02° step scan).
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The chitosan-vanillin composites were examined on a Jasco FT/IR6200 spectrometer
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(ABL& E-JASCO Romania) with Intron µ Infrared Microscope with ATR-1000-VZ objective.
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The spectra were obtained from the average of 50 scans recorded at a resolution of 4 cm-1 in a
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range from 4000 to 500 cm-1 with a TGS detector.
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Solid state
CP/MAS
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(150.92 MHz) using a Bruker Avance 600 spectrometer (14.10 T). The samples were packed in a
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ZrO rotor with an outer diameter of 3.2 mm. The CP/MAS spectra were recorded with a contact
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time of 1.5 ms. Chemical shift of the 13C nuclei was estimated by using an external reference of
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TMS (tetramethilsilane, δ = 0 ppm). In order to distinguish peaks from spinning side bands,
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NMR measurements with two spinning rates of 8 and 14 kHz were carried out.
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2.3.3. Thermal gravimetric analysis
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The thermal behaviour of the composites was tested using thermogravimetric analysis on a
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thermal analyser (DTG-60-Shimadzu). The operating conditions were as follows: temperature
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range of 20 °C to 1000°C, with a heating rate of 10 °C/min, and air flow rate of 50 mL/min.
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2.3.4. Optical properties of films
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The colour of the films was also measured using a spectrophotometer UV-2450 with an
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integrating sphere (Shimadzu, Japan). CIE lab colour scale of lightness (L), a (red–green), and b
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(yellow– blue) values were used to calculate the total colour difference (∆E), and the yellowness
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index (YI) using relations 1-3(Rhim, Wu, Weller, & Schnepf, 1999).
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∆E = ∆L2 + ∆a 2 + ∆b 2
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YI = 142.86 ⋅ b / L
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where: ∆L = Ls − Lsample ; ∆a = a s − a sample ; ∆b = bs − bsample
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The standard values used were those of the white background, namely: Ls=100.42, as=-0.12 and
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bs=0.23. Calculations were made for D-65 illuminant and 100 observer. Colour measurements
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were replicated three times for each type of film on both film sides.
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2.3.5. Swelling tests
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0.5
(1) (2)
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Swelling tests were conducted using water and ethanol-water mixture (83% vol. ethanol)
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as contact liquids. Composite films were dried to constant weight and then cut into 2 cm × 2 cm
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square shapes, then immersed in distilled water or ethanol-water mixture at room temperature for
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two hours. The experiments were done in triplicate. The mass of polymer dissolved into the 6
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distilled water or ethanol-water mixture was neglected considering the short time needed for the
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experiment. Also, the amount of vanillin, contained in the active films and released in aqueous
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media was considered to be negligible in comparison with the amount of absorbed water.
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Swelling degree (SD) was obtained by measuring the initial weight (m0 ) and the weight of the
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sample in swollen state (mτ ) , using equation 3:
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SD (%) = (mτ − m0 ) m0 ⋅ 100
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2.3.6. Water vapour transmission rate
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Water vapor transmission rate (WVTR) was determined using a modified ASTM standard
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method (ASTM E 96-95, 1995) with some modifications.
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The test cups were filled with 20 g of silica gel (desiccant) to produce a 0 % RH below the
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film. The desiccant was heated at 180 °C for at least 8 h prior to its use for the experiment. The
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cups were placed at 25 °C in a desiccator containing a saturated salt solution (natrium chloride),
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which produced an RH of 75%. A temperature and humidity sensor has measured these
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parameters during the experiment. The mass of the cups was measured periodically over a 165 h
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time interval. WVTR values were determined, by linear regression, as the slopes of the steady
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state period of the curves of weight gain versus time, for all the samples. WVTR (g m-2 day-1)
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was calculated as follows:
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=
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where: J is the slope of the weight gain curves versus time (g/day) and A is the aria of the tested
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films (m2).
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2.3.7. Antibacterial activity of the films
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Antimicrobial activity was highlighted using Escherichia coli (ATCC 8737) strain. The starter
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bacterial culture was prepared in liquid Luria-Bertrani (LB) culture media, growth at 37° for 24
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hours. The cells were filtered and suspended in sterile distilled water in order to obtain a
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inoculum having the concentration of 5 ⋅ 10 5 cel / mL . The depletion of the inoculum in amount
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of 1 µL was made in Petri dishes with nutrient broth and the disk diffusion method was used to
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emphasize the antibacterial activity. The used films were cut into 6 mm diameter disk-shaped
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samples and were sterilized using short UV irradiation. The samples were incubated at 37° for 96
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h, recording the results every 24h to 24h.
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2.4. Kinetics of vanillin release
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The release behaviour of unreacted vanillin from chitosan-vanillin composites was investigated
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by total immersion assay (Stroescu, Stoica-Guzun, & Jipa, 2013). The composite films were cut
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as circular disks having 1.5 cm diameter and were immersed in 50 mL of ethanol-water mixture
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or distilled water. The experimental vials were shaken at room temperature at a shaking rate of
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100 rpm. From time to time 0.1 mL of the solution from each vial was dipped out and was
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analysed in order to determine the vanillin content. The vanillin content was measured using an
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UV-VIS spectrometer (CINTRA 6, GBS-Australia), at a fixed wavelength of 231 nm, according
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to the standard vanillin calibration curve. To determine the equilibrium vanillin concentration,
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considered as maximum value concentration which could be released for each sample, the same
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experiments were done for 120 h. The cumulative release of vanillin was calculated with
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equation (5):
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=
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where Mt is the amount of vanillin released at time t and Me is the amount of vanillin released at
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infinite time (equilibrium). The experiments were performed in triplicate and the average values
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were reported.
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2.5. Characterization of the mechanism of vanillin release
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In this paper the following empirical models were used to describe vanillin release from
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The second model that has been used was the power law model, known also as Korsmeyer–
212 213
Peppas model and which is described by the equation (7).
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=
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The vanillin release time curves were also analysed using the Peppas and Shalin model
215
proposed to underline the contribution of diffusion and also of relaxation/erosion.
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The significance of the terms of the equations (6-8) is: Mt/Me represents the vanillin fraction
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released in time t, (the equation could be applied only for Mt/Me <0.6), kH and kp are constants
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which characterise the macromolecular network, while
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characteristic of the release mechanism for Korsmeyer–Peppas equation. For n=0.5 the release
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mechanism is dominated by the Fickian diffusion, for n=1 the active substance release is directly
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proportional to time, for 0.5 < n <1 the release mechanism named anomalous diffusion (non-
224
Fickian diffusion) is the predominant one and the value of n<0.5 indicates a pseudo-Fickian
225
behaviour of diffusion (Li, Fu, & Zhang, 2014). In the Peppas-Shalin equation, K1 is the Fickian
226
kinetic constant and K2 is the erosion rate constant. The value of the exponent
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for planar films. The magnitude of the two constants is also important, if the ratio K1/K2 is
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greater than 1, the solute release is determined mainly by diffusion; if the same ratio is less than
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1, then relaxation/erosion becomes the predominant mechanism for the active agent release. It is
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also possible to have a ratio equal to 1, which corresponds to the situation in which diffusion and
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erosion might coexist for the release mechanism.
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3. Results and discussion
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3.1. Films morphology by SEM
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From Fig. 1 one could observe a lamellar structure for the films F1 and F3, which do not contain
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any emulsifier. For film F3 the structure is rather interesting due to the fact that vanillin crystals
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are entrapped in the polymeric material. The films F2 and F4, which contain emulsifier Tween
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60 are quite different, having a more compact structure in which the vanillin crystals are not
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visible. In Fig. 2 are presented SEM pictures of pure film of chitosan and vanillin crystals, being
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thus in accordance with the observation concerning films F1 and F3 from Fig. 1.
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Fig.1.
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Fig.2.
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3.2. X-ray diffraction
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From Fig. 3a one could observe that vanillin powder exhibits a sharp peak at 2θ≅ 13$ and that
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the chitosan film has two diffraction peaks: a strong one at 2θ≅ 20.5$ and the other at 2θ≅
246
10.5$ . The composite films present a sharp diffraction peak due to vanillin at 2θ≅ 13$ and a
247
very weak reflection due to chitosan, which is more visible for films F2 and F4. The obtained
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results must be carefully interpreted, because chitosan could react in different conditions with
249
vanillin forming chitosan–vanillin Schiff-base biopolymers, which were already reported in
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literature (Sashikala, & Syed Shafi, 2014; Marin, Simionescu, & Barboiu, 2012; Tree-udom,
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Wanichwecharungruang, Seemork, & Arayachukeat, 2011). So, the peak at 2θ≅ 13$ observed
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for all composite films could also correspond to interdigitated H-bonded vanillin layers between
253
the chitosan backbones, due to imine bond formation on chitosan polymeric backbones (Marin,
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Stoica, Mares, Dinu, Simionescu, & Barboiu, 2013). Also, the very weak reflection due to
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chitosan in composite XRD patterns is in agreement with those obtained by Jin, Wang, & Bai
256
(2009), and indicates a poor crystallinity of the new formed Schiff base in comparison with
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chitosan.
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Fig.3.
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These aspects will be presented in detail after the analysis of the FT-IR spectra and of the
260
thermogravimetric curves.
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3.3. Fourier transform-infrared spectroscopy (FT-IR) and solid state 13C NMR analysis
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FTIR spectra of all composites (F1-F4) and of the chitosan film are presented in Fig.4. These
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spectra show the absorption characteristic bands of chitosan, vanillin and also new peaks. In 10
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comparison with spectra of pure chitosan film, possible overlapping and shift of absorption
265
peaks are observed in the composite spectra. So, the peaks of chitosan at 1380 cm-1 (CH bending
266
and CH3 deformation), 1154 cm-1(C-O-C symmetric stretching), 1060 cm-1 (C-O stretching
267
vibration of C3-OH) and 1023 cm-1 (C-O stretching vibration of C6-OH) are also visible in the
268
composite spectra having different intensities (Leceta, Guerrero, & de la Caba, 2013). The peak
269
at 1644 cm-1 assigned to C=O stretching (amide I band) is shifted in the composite spectra at
270
1640 cm-1. This peak (marked with (*) in Fig.4) could be considered a new one, representing
271
thus a stretching vibration of the newly form C=N bond between chitosan and vanillin. There are
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also some peaks which could be assigned to free vanillin in the composite spectra, these being:
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1590 cm-1 and 1513 cm-1 assigned to stretching vibration of benzene ring and 1284 cm-1
274
characteristic to C-O-C stretching vibration (Liu, Zhou, Zhang, Yu, & Cao, 2013; Raschip,
275
Hitruc, Oprea, Popescu, & Vasile, 2011). From FTIR spectra of composite films one could
276
conclude that vanillin has reacted with chitosan so as to form a Schiff base, but an amount of free
277
vanillin is also present in the samples. The peaks characteristic to emulsifier could not be
278
enlightened because of the fact that this one is to be found only in a very small amount in the
279
composition of films F2 and F4, and also because between chitosan and Tween 60 there weren’t
280
created any new types of bonds. A similar conclusion was formulated when Tween 20 was used
281
in the composition of chitosan films (Ziani, Oses, Coma, & Maté, 2008).
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Fig. 4.
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CP/MAS
C NMR spectra of chitosan, vanillin and composite film F1are presented in Fig. 5.
284
The spectrum of chitosan presents all the already known characteristic peaks reported in
285
literature for aliphatic carbons in the chitosan, between 20 and 110 ppm (Silva, Goodfellow,
286
Benesch, Rocha, Mano, & Reis, 2007; Krishnapriya & Kandaswamy, 2010; Chang, Chen, Ellis,
287
& Tung, 2012), for example C1 ring carbon at 105 ppm, or methyl CH3 carbon at 23.76 ppm.
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Also, the vanillin spectrum is comparable with those already reported (Peña, de Ménorval,
289
Garcia-Valls, & Gumí, 2011). The composite F1 spectrum exhibits all the peaks belonging to the 11
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chitosan and vanillin carbons, in addition to which one notices the presence of a new peak at
291
168.29 ppm, which could be attributed to the imine (CH=N) carbon of a new linkage formed by
292
condensation reaction of the vanillin aldehyde group with chitosan amine group (Baran, Mentes,
293
& Arslan, 2015). Fig.5.
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3.4. Thermal behaviour
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The typical thermograms of composite films (F1-F4) and of the chitosan film are presented in
297
Fig.6. For the films F1-F4 a percentage of weight loss up to 6-14% is observed up to 1200C (due
298
to the loss of adsorbed and bound water dehydratation) for all the films. A second weight loss up
299
to 24-32 % was observed between 2000C and 3500C and could be attributed to the thermal
300
decomposition of the Schiff base formed between chitosan and vanillin. Even if the TGA curve
301
of chitosan is similar with those of composite films, there are still some differences. The peak
302
due to the partial oxidation of chitosan was observed at 278 0C, which is a higher temperature
303
than those observed in the case of composite films F1-F4.
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In Fig. 7, where there are presented the DTA curves for the composite films, one could observe
306
the existence of exothermic peaks that correspond to the second weight loss. The third weight
307
loss was from 350-6000C and may be related to the slow decomposition of the carbonaceous
308
residue. Our results are in accordance with similar results obtained for thermal behavior of Schiff
309
bases obtained from the reaction of chitosan and salicylaldehyde derivatives (Estrela dos Santos,
310
Dockal, & Cavalheiro, 2005).
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Fig.7.
312
Analysing the exothermic peaks from Fig. 7 and basing our reasoning on the conclusions of
313
Guinesi, & Cavalheiro (2006), we can appreciate that the degree of substitution (defined by the
314
afore mentioned authors as the number of free amino groups in relation to the Schiff bases on the
315
substituted biopolymeric matrix) of the film F1 is the greatest one, because this film has the 12
ACCEPTED MANUSCRIPT
lowest value for the exothermic decomposition peak. This result is in accordance with the fact
317
that the starting composition of film F1 has a molar ratio approximately 1:1 between –NH2/–
318
CHO, which is more favourable to the chemical reaction between chitosan and vanillin in
319
comparison with the casting solutions used for films F3 and F4.
320
3.5. Film colour
321
Colour difference (∆E*) and yellowness index (YI) for the composite films F1-F4 are depicted in
322
Fig. 8. The colour difference (∆E*) varies in the 44-48 units range, without sensible differences
323
between the composite films F1-F4. One can see that the films F1 and F3, which do not contain
324
Tween 60, have the yellowness index in the 68-71 units range, while for the films F2 and F4,
325
which contain an emulsifier, the values of YI are greater, being of 78-79 units. The yellow index
326
is also an argument for the reaction between chitosan and vanillin, because films with yellow
327
colour of different intensities have been obtained for chitosan Schiff bases with vanillin and its
328
derivatives (Marin, Stoica, Mares, Dinu, Simionescu, & Barboiu, 2013). The yellow colour could
329
be also accentuated by the presence of the emulsifier in the composition of films F2 and F4.
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Fig 8.
330
3.6. Film swelling
332
In Fig. 9 there is presented the swelling degree for all the composite films in two solvents:
333
ethanol-water mixture and water. At first sight the composite F3 has a very high swelling degree
334
in both solvents. The swelling degrees for all the films are higher in water, probably due to the
335
presence of the unreacted chitosan in the studied samples. The presence of Tween 60 in the
336
composition of film F2 and F4 reduce the swelling degree in both solvents. When a surfactant is
337
used in a polymer casting solution, multiple interactions between surfactant and film polymers
338
are possible. In the case of Tween 60, which is a surfactant with a high Hydrophile-Lipophile
339
Balance (HLB >10), hydrogen bond interactions could be established between the hydrophilic
340
group of the surfactant and the hydrophilic group of chitosan. In this case a strong interaction
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13
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341
between polymer and surfactant could explain the decrease of the swelling degree of composite
342
films which contain Tween 60 (Maran, Sivakumar, Thirugnanasambandham, & Sridhar 2013). Fig. 9.
343
3.7. Water vapour transmission rate
345
Fig. 10 presents the WVTR values of all composite films (F1-F4) and of the chitosan film (F0).
346
For the latter we have measured a WVTR value of 636.527±4.95 g/m2day. For the composite
347
films the values obtained are in the 400-635 g/m2day range. One could observe that the
348
composite samples which contain emulsifier have the lowest value of WVTR, which could be
349
explained by the hydrophobic nature of Tween 60. The films F1 and F3 have WVTR values
350
nearest to the value of this parameter measured for chitosan film. The results could be
351
qualitatively related to the swelling degree, but not for all the films. For example, film F3 has the
352
highest swelling degree and also the highest WVTR, while film F2 has the lowest swelling
353
degree and the lowest WVTR value. The water vapour transfer mechanism could be described in
354
terms of solubility and diffusion of water vapour molecules through polymeric films. When a
355
surfactant is used, multiple interactions between surfactant and polymer matrix are possible. In
356
the studied case, the SEM images (Fig.1) reveal a more compact structure of the films that
357
contain Tween 60 in comparison with the films without surfactant. Due to the fact that Tween 60
358
has a HLB>10 there are possible interactions between its polar group and the hydrophilic film
359
matrix. Also, its hydrophobic structure could be a barrier to water molecules in the composite
360
films, as it was already observed for other composite films (Brandelero, Yamashita, &
361
Grossmann, 2010; Chen, Kuo, & Lai, 2010). Due to the fact that chitosan and vanillin have
362
reacted to form a Schiff base, it is also necessary to take into account the contribution of the
363
structure of the newly formed Schiff base (vanillin–imino-chitosan) to the WVTR measured
364
values, but this aspect is difficult to appreciate and more experimental studies are needed.
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Fig. 10.
365 366
3.8. Antibacterial activity of the films 14
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The antibacterial activity of the films against E. coli is presented in Table 1. The values of the
368
inhibition zone (IZ), expressed in millimetres, highlight a good antibacterial activity of the
369
studied films. From the data presented in Table 1 one could observe that the chitosan film has no
370
antibacterial activity. The films F2 and F4 showed antimicrobial activity from the first day of
371
incubation, the inhibition zone at this time being of 11±1.3 mm; that indicates a quick release of
372
the antimicrobial substance in the culture media (in the first 24 h) and afterwards a slow release
373
of a small quantity of antibacterial substance. The films F1 and F3 have a slow release of the
374
antibacterial substance at the beginning of the incubation. The F1 film presents a release of a
375
large amount of the active substance after 48 h. The amount released from the film F1 was
376
higher than the one obtained from the other films, because in this case the IZ was higher at the
377
end of the incubation period of the samples. The F3 film showed an intermediate release in the
378
first period of incubation and a significant greater release of active substance after 72 h of
379
incubation.
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380
Table 1.
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3.9. Kinetics of vanillin release
383
The cumulative release of vanillin was calculated using equation (5) and is represented in Fig.
384
11a for water and in 11b for an ethanol-water mixture as release media. In both media,
385
cumulative release displays a burst effect for all films. The composite films showed a lower
386
cumulative release in ethanol-water mixture than in water. These results could be explained by
387
the higher swelling degrees of the composite films in water. Moreover, the film F3 showed the
388
greatest cumulative release in both media, in accordance with its highest swelling degree.
389
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Fig.11.
≤ 0.6 was analysed
390
The portion of the vanillin release curves with a fractional release
391
according to several empirical models described by equations (6-8). Nonlinear least squares
15
ACCEPTED MANUSCRIPT
392
fitting method was used to determine the parameters in each equation. The calculated parameter
393
values along with the regression coefficients (r2) are listed in Table 2. Table 2.
395
As one could see from Table 2, we have obtained a good fit to the Korsmeyer-Peppas model
396
(r2>0.968 for vanillin release in water and r2>0.969 for ethanol-water as release medium). For the
397
Higuchi model we have also obtained high correlation coefficients (r2>0.921 for water as release
398
medium and r2>0.950 for vanillin release in ethanol-water). The exponent values
399
Korsmeyer-Peppas model are inferior to 0.5, which is the characteristic value for pure Fickian
400
diffusion for thin films. This is not surprising, given that the release mechanism, in such systems
401
as those studied in this paper, could be a complex one including not only diffusion, but also
402
swelling, crystal dissolution and polymer erosion. Because the values of
403
we may consider a pseudo Fickian release mechanism for all the studied films and for both
404
solvents used as release media in the experiments. Analyzing the values of correlation coefficient
405
(r2) for all the tested models we have observed that the models are comparable and we have not
406
obtained spectacular results with any of them.
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Based on the obtained results and applying the three empirical models depicted in Table 2,
408
we could presume that the release of vanillin could be controlled by diffusion through the
409
composite films. Fick's second law could be used in these conditions in order to determine the
410
diffusion coefficients of vanillin through chitosan-vanillin composite films. For this, we have
411
used one of the solutions of Fick’s second law of diffusion under non-steady state condition for
412
film/planar geometry and constant boundary conditions, which are recommended for ‘monolithic
413
systems’ (Siepmann, & Siepmann, 2012). The fraction of an active substance released in time is
414
given by the following equation:
415
416
where: Mt is the cumulative amount of the released substance at time t, Me is the amount of the
417
substance released at equilibrium (at infinite time), D is the difusion coefficient of the substance
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,
= 1 − - . ∑; <$
01 2345 67. - . 8/9 . :
(9)
5 67.
16
ACCEPTED MANUSCRIPT
418
within the polymer matrix and δ is the total thickness of the film. In this equation D is the main
419
transport parameter and was determined applying the following short time approximation of
420
equation 10:
421
= 45
48 / 7 ;
-9 .
≤ 0.6
(10)
The results obtained for the diffusion coefficient of vanillin are presented in Table 3 along with
423
correlation coefficient values. From Table 3 one could see that diffusion coefficients calculated
424
for Fickian diffusion are in the range 10-9 and 10-10 cm2/s for almost all the tested films and for
425
the two solvents used as release media. The exception is due to the film F3. When water
426
constituted the release medium the value of vanillin diffusion coefficient through the composite
427
film F3 was determined as 1.208x10-8 cm2/s, being the highest for all the films tested, and in
428
ethanol-water as release medium the same coefficient has the value 7.541x10-9 cm2/s, this one
429
being also the highest value of all the coefficients calculated. If we analyse the swelling data, we
430
may observe that the film F3 has the highest swelling rate of all the tested films and in both
431
tested solvents as release media. The explanation comes probably from the fact that water
432
diffuses very fast in the polymer matrix, which enhances the diffusion/dissolution of vanillin. If
433
we analyse the coefficient of determination calculated for Fickian diffusion, the results obtained
434
are high enough, yet not very high, being in the range 0.955 to 0.988. The analysis of the
435
correlation coeffcient reveals a good correlation, but not greater than those obtained when the
436
empirical models were tested. This could be an indication that the release mechanism is more
437
complicated than the simple diffusion, even if diffusion could be one of the phenomena implied
438
in vanillin release from composite films. From the experiments of swelling, a high swelling
439
degree of the composite films was measured, and under these conditions the probability that the
440
release mechanism be governed only by Fick’s second law of diffusion is low. Also, from SEM
441
images there could be observed the existence of vanillin crystals in the structure of film F3, for
442
example. In this context, a model which considers that the total amount released is due to
443
diffusion and crystals dissolution seems to be a realistic one. In this model, the fractional amount
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17
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444
released is considered to be formed by the sum of a fraction due to diffusion, which could be
445
calculated using a solution of the Fick’s law and a fraction due to vanillin crystals dissolution
446
(Uz, & Altınkaya, 2011; Berens, & Hopfenberg, 1978). The fraction of vanillin release using this
447
model is described by equation 11.
448
449
where: Mt is the cumulative amount of the released substance at time t, Me is the amount of the
450
substance released at equilibrium, XF is the fraction of vanillin released by Fickian diffusion and
451
kd is the dissolution constant of vanillin crystals. This equation has three main parameters: XF, D
452
and kd, which were determined by minimizing the difference between the experimental data and
453
the model prediction given by equation (11). The results obtained using the dissolution crystals
454
model are presented in Table 3.
-.
∑; <$
01 2345 67. - . 8/9 . : 5 67.
A + 51 − >? 7B1 − CD5−E 7F
(11)
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,
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SC
= >? @1 −
Table 3.
455
One can observe from Table 3 that the values of vanillin diffusion coefficients were higher for
457
the composite films than for the Fickian diffusion, because in the latter model only a fraction of
458
the vanillin is released through diffusion. If we compare all the composites, film F3 represents
459
once again the exception, being characterised by the highest vanillin diffusion coefficients. The
460
dissolution constants have values in the range 10-4 and 10-6 s-1, the highest value being obtained
461
also for vanillin release from F3 composite film when water is used as release medium. The
462
values of dissolution constants are comparable with those obtained by Uz, & Altınkaya (2011)
463
for potassium sorbate release from cellulose acetate based mono and multilayer films. The
464
correlation coefficient calculated for the crystals dissolution model is now higher, being in the
465
range 0.980-0.998 and indicating thus that this model describes better the vanillin release
466
mechanism from composite films.
467
4. Conclusion
468
New composite films containing chitosan-vanillin and Tween 60 were prepared using different
469
ratios between components. All the films have a yellow-pal colour. The SEM micrographs reveal
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18
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that the structures of the films with emulsifier are different from those of the films without
471
emulsifier. The films without emulsifier have a lamellar structure with vanillin crystals entrapped
472
in the films. The water vapour transmission rate is lower for the films containing Tween 60.
473
FTIR spectra proved that chitosan and vanillin have formed a Schiff base, but because of the
474
mild conditions used for solution casting films, a certain amount of vanillin has remained
475
unreacted. For this reason, the release of vanillin was also measured in order to use these films as
476
flavour release materials. Among the empirical models used to fit the experimental data, the
477
most suitable was the one which took into account the crystals dissolution and the vanillin
478
Fickian diffusion. All the prepared films have proven a good antimicrobial activity against E.
479
coli, this being also an indirect proof for the Schiff base vanillin–imino–chitosan formation.
480
These films could be used as antimicrobial materials, but also as flavour release films for food or
481
cosmetic products packaging.
482
485
Financial support from UEFSCDI–Romania (Research Grant–SABIOM-No. 114/2012) is gratefully acknowledged.
486
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484
Acknowledgement
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488
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ACCEPTED MANUSCRIPT Ziani, K., Oses, J., Coma, V., & Maté, J. I. (2008). Effect of the presence of glycerol and
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24
ACCEPTED MANUSCRIPT Our reference: FOOHYD 2883
Table captions Table 1. Inhibition zones obtained by the agar diffusion method against E. coli for the
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composite films F1-F4 and for the chitosan film (F0). Table 2. Vanillin release kinetic model parameters of F1-F4 composites.
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Table 3. Parameters of Fick model and model with crystals dissolution for vanillin release.
ACCEPTED MANUSCRIPT
Our reference: FOOHYD 2883
2
Figure captions
3
Fig.1. SEM pictures of vanillin-chitosan composites F1-F4.
4
Fig.2. SEM pictures of pure chitosan film and vanillin powder.
5
Fig.3. XRD patterns of: a) vanillin powder and chitosan film and b) composite films F1-F4.
6
Fig.4. FTIR spectra of composite films (F1-F4) and chitosan (absorption characteristic peaks of
7
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1
(#)-chitosan; (•)-vanillin and (*)-Schiff base vanillin–imino–chitosan.
Fig.5. 13C NMR spectra of: (a) chitosan, (b) vanillin and (c) composite film F1.
9
Fig.6. TGA curves of: (a) chitosan, (b) films (F1, F2) and (c) films (F3, F4).
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Fig.7. DTA curves of composite films F1-F4.
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Fig.8. Total colour difference (∆E*) and yellowness index (YI) for the composite films F1-F4.
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Fig.9. Comparison between swelling degrees of composite films F1-F4 in different solvents
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ethanol-water mixture (///) and water (III).
Fig.10. WVTR values for composite films (F1-F4) and chitosan film (F0).
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Fig.11. Vanillin profile release from composite films: a) in water and b) in ethanol-water as
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release media.
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Inhibition zone (mm) Sample 48 h
96 h
F0
No effect
No effect
No effect No effect
F1
6.0±1.2
7.0±1.0
15±1.8
15±1.5
F2
11±1.3
11±1.2
11±1.8
12±1.3
F3
8.0±1.2
8.0±1.5
8.0±1.3
12±1.2
F4
11±1.5
13±1.2
13±1.7
13±1.2
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ACCEPTED MANUSCRIPT Table 2. Higuchi eqn
Korsmeyer-Peppas eqn n
kp (min-n)
r2
0.468 0.230 0.235 0.406
0.065 0.231 0.236 0.085
0.971 0.982 0.968 0.983
0.070 0.233 0.225 0.083
0.028 0.992 0.432 0.042 F1/ew 0.022 0.985 0.473 0.028 F2/ew 0.065 0.950 0.375 0.112 F3/ew 0.030 0.984 0.376 0.064 F4/ew *w-water and ew-ethanol-water mixture
0.992 0.983 0.969 0.989
0.041 0.033 0.109 0.065
0.981 0.921 0.968 0.976
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F1/w F2/w F3/w F4/w
kH (min-1) 0.057 0.072 0.065 0.057
kd krx104 (min-m) (min-m)
m
r2
10.50 4.07 64.01 4.56
0.450 0.228 0.231 0.406
0.972 0.986 0.969 0.979
1.29 0.59 3.36 0.99
0.433 0.432 0.379 0.376
0.990 0.983 0.970 0.993
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Peppas-Sahlin eqn
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Table 3. Fick model
Model with crystal dissolution Dissolution Fraction constant of vanillin k(s-1) released by Fickian diffusion XF
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Sample/ Release medium
Coefficient of determination r2
Diffusion coefficient DCS (cm2/s)
F1/w F2/w F3/w F4/w F1/ew F2/ew F3/ew F4/ew
1.181x10-9±3.30x10-11 1.684x10-9±8.60x10-11 1.208x10-8±2.60x10-9 2.272x10-9±1.80x10-10 3.430x10-10±2.59x10-11 1.783x10-10±1.64x10-11 7.541x 10-9±1.00x10-10 5.549x10-10±5.33x10-11
0.978 0.955 0.978 0.983 0.979 0.988 0.978 0.962
2.414x10-9±1.20x10-10 1.089x10-8±3.72x10-9 5.304x10-7±4.70x10-9 4.291x10-9±7.74x10-10 1.259x10-9±1.37x10-10 1.774x10-9±3.33x10-10 1.661x10-8±3.17x10-9 6.212x10-9±2.83x10-10
Coefficient of determination r2
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w/water ew/ethanol-water
Diffusion coefficient DF (cm2/s)
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7.94x10-6±1.52x10-7 1.34x10-5±1.67x10-6 1.54x10-4±1.55x10-5 7.86x10-6±1.44x10-7 2.91x10-6±1.34x10-7 5.14x10-6±1.88x10-7 4.64x10-6±2.99x10-7 3.26x10-6±1.24x10-7
0.725±0.003 0.619±0.003 0.353±0.008 0.806±0.002 0.531±0.004 0.360±0.047 0.864±0.038 0.449±0.025
0.980 0.986 0.996 0.986 0.991 0.996 0.980 0.990
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Highlights
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• Chitosan-vanillin composite films with and without Tween 60 were synthetized. • Antimicrobial properties of composites were investigated against Escherichia coli. • The formation of a Schiff base between chitosan and vanillin was proved. • The release kinetics of vanillin was also studied.