Biobased Composite Films from Chitosan and Lignin: Antioxidant

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Bio-based composite films from chitosan and lignin: antioxidant activity related to structure and moisture Kevin Crouvisier-Urion, Philippe R. Bodart, Pascale Winckler, Jesus Raya, Regis D. Gougeon, Philippe Cayot, Sandra Domenek, Frederic Debeaufort, and Thomas Karbowiak ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00956 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 4, 2016

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ACS Sustainable Chemistry & Engineering

Bio-based composite films from chitosan and lignin: antioxidant activity related to structure and moisture Kevin Crouvisier-Urion1, Philippe R Bodart1,2 , Pascale Winckler1, Jésus Raya3, Régis D Gougeon1, Philippe Cayot1, Sandra Domenek4, Frédéric Debeaufort1 and Thomas Karbowiak1,*

1

Univ. Bourgogne Franche-Comté, Agrosup Dijon, PAM UMR A 02.102, F-21000, Dijon,

France 2

IUT A - Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq, France

3

Université de Strasbourg, Institut de Chimie, CNRS, 67008 Strasbourg, France

4

UMR Ingénierie Procédés Aliments, INRA, AgroParisTech, Université de Saclay, 91300

Massy, France

* Corresponding author: Tel: +33 3 80 77 23 88 Fax: +33 3 80 77 40 47 e-mail: [email protected]

ACS Paragon Plus Environment

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Abstract Composite films based on chitosan and lignin biopolymers were investigated for their mechanical, barrier, surface and antioxidant properties and linked to an extensive microscopic analysis of their external and internal structure. In particular, the fluorescence properties of lignins were exploited, using two-photon microscopy, to achieve a 3D representation of its distribution within the chitosan matrix. The lignin incorporation generated small aggregates homogenously distributed in the film. The aggregates slightly weakened the network as reflected by the mechanical properties. Lignin as an antioxidant provided to the film a radical scavenging activity, essentially governed by a surface activity mechanism. Accordingly, the film surface showed a chemical reorganization induced by the presence of lignin as highlighted by surface hydrophobicity and X-ray photoelectron spectroscopy. On the molecular scale, solid state NMR also revealed the absence of covalent bonds between lignin and chitosan and the establishment, but to a small extent, of low energy dipole-dipole interactions. Finally, lignin is a promising compound for a good added-value due to radical scavenging in a chitosan matrix.

Keywords: sustainable material, active packaging, natural resources, 3D-structure


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INTRODUCTION Large developments in renewable packaging have been appearing for fifteen years (bio-plastic mainly from natural sources) to reach about 70 % of bio-based plastic market innovations.1 These bio-based polymers represent 3.5 billion tons in 2011 and the worldwide production is estimated to reach 12 billion tons in 2020.2 Industrialists and academia attempt to create new bioplastics made from wastes or co-products from agricultural and food industries: fruit and vegetable pulp, stone powder (nuts), leather waste, algae, shellfish powder. These extracted polymers can be used as films, bottle trays or thermoforming slabs for further transformation by packaging producers.3, 4 Among bio-polymers, chitosan is a heteropolysaccharide mainly extracted from insect or crustacean shells, or from some mushroom cell walls. This polymer is derived from Ndeacetylation of chitin, which is the second most abundant natural polymer after cellulose, with a global industrial production around 1010-1012 ton per year.5 Deacetylation of amide groups (R-NH-CO-CH3) induces the formation of ammonium groups (R-NH3+; pKa≈6.5) which provides chitosan good film forming properties and an improved water solubility.6 Solubility is not only dependent on the degree of deacetylation that favors charge-dipole interactions with water, but also on the molecular weight, pH and the acid used for solubilization.7 Indeed, at pH < pKa, the amino groups are cationic and a counter-ion is used to stabilize the film forming solution or gel. Among the different anions that can be used to that purpose (such as lactic acid, acetic acid, formic acid, propionic acid …)8-11, lactic acid is well suited due to its non-volatility at room temperature. Moreover, in its use as a film (or packaging), chitosan displays some interesting properties in terms of antimicrobial/antifungal effects based on the presence of cationic groups which create electrostatic interactions with anionic groups present at the external


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surface of microorganisms, especially proteins and lipopolysaccharides.12 These properties make chitosan a promising candidate for packaging film formation. Supplementary added-value can be brought to the material by the addition of antioxidant molecules. Some studies have already been carried out incorporating for example α-tocopherol13 or ferulic acid.14, 15 However these natural antioxidants are too expensive for a use in packaging applications. Lignin is the most abundant aromatic polymer and accounts for 18-35 % of the dry wood weight.16 It has a very complex poly-phenolic structure extracted from biomass, constituted by three subunits (coumaryl, coniferyl and syringyl alcohols). Lignin displays a hyper-branched structure as well as a large polydispersity. This large polydispersity may confers to lignins some cytotoxic effects, with reported values of IC50 (concentration necessary to kill 50% of initial cells) around 600-650 µg/mL and up to 1200 µg/mL for lignosulphonates17-19. Nevertheless, these cytotoxic concentrations are very high compared to the classical range of concentrations usually used to obtain an antioxidant effect (about 17-fold higher)18. Nowadays, the annual lignin production comes close to 108-109 tons.20 Only 2.5 % of it is being used in the paper industry21 (Kraft paper) and 1 % as additive in industry (wood panel, biodispersant, epoxy resin …).22 The very large majority of lignin is burnt as low cost fuel. As a waste from the paper industry, lignin is cheap and widely available. Recent works already focused on its use as an added-value compound for thermoplastic or packaging film by developing sustainable composites using different kind of matrix presented in the review of Kumar Thakur et al (2014)23-27. Inclusion of lignin in biodegradable polymer matrix is one of these applications and was already study on PLA28, 29 or wheat gluten film.30 The presence of free phenolic hydroxyl groups in the structure, which act as radical scavengers (Ar-OH, MeO-Ar-OH, (MeO)2-Ar-OH), confers antioxidant activity to lignin.31,

32

Classically, active


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packaging films targeting antioxidant effects rely on the migration of the radical scavenger to the food for their activity. However, some evidence exists in the literature that the radical scavenging activity could also be present on the film surface without migration. Both mechanisms, migration 33, 34

and surface activity, are beneficial for the overall performance of the materials.

The objective of this study is to convert industrial by-products into a performing active packaging for food preservation, using chitosan, for its film forming capacity, and lignin, for its antioxidant properties. The functionality of this system is based on the barrier properties of chitosan and on the lignin activity against oxidation in order to limit the oxygen transfer and reactivity of free radicals in the packed food, based on antioxidant molecule migration or surface activity. It is thus essential to understand the influence of lignin incorporation on the structure of chitosan-lignin films and on the related functional properties (mechanical, surface, barrier and antioxidant) of such composite films, paying particular attention to the role of water.18, 35 EXPERIMENTAL SECTION Materials Chitosan was obtained from France Chitin (ref 652, Marseille, France; deacetylation degree of 90 %, viscosity of 43 cps at 27°C, data from supplier) and used as received. The weight-average molecular weight Mw is 274 kDa (±32), the number-average molecular weight Mn is 163 kDa (±9).18 Lignin powder was extracted by an alkali process from sugar cane (Saccharum munja) and provided by Granit SA (Switzerland). Lignin “Sarkanda” had a G/S (Guaiacyl / Syringyl) unit ratio of 1.04 and a low content of free monomers, being around 0.5 mg.g-1 of mainly ferulic acid and p-coumaric acid.33


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Ethylene glycol (107-21-1 Sigma Aldrich, 99.5 % purity, Germany), glycerol (56-81-5 Sigma Aldrich, 99.5 % purity, Germany), diiodomethane (75-11-6 Sigma Aldrich, 99 % purity, Germany) and formamide (75-12-7 Sigma Aldrich, 99.5 % purity, Germany) were chosen as liquids for contact angle measurements. Ethanol (64-17-5 Sigma Aldrich, 96 % purity, Germany) was used for lignin dissolution. Lactic acid (50-21-5 Merck, 90 % purity, Germany) was used for preparing the chitosan film forming solution. Film preparation Chitosan-lignin composite films were produced by the solvent casting method after mixing an aqueous solution of chitosan and an ethanol solution of lignin under appropriate conditions of concentration and pH. Chitosan aqueous acidic solution was prepared by dispersing 2 % (w/w) chitosan in a 1 % (w/w) lactic acid aqueous solution (final pH≈6) and stirred at 350 rpm during 24 h at 20 °C. Lignin was first solubilized in ethanol at a concentration of 0.69, 1.38 and 2.08 % (w/w) to achieve, upon mixing with the chitosan solution, a final concentration of 10, 20 and 30 % wlignin/wchitosan, respectively. The lignin solution was stirred at 350 rpm during 24 h at 20 °C. These two solutions (chitosan and lignin) were then mixed to obtain at a water/ethanol ratio of 70:30 (w/w) and stirred 24 h at 20 °C and 350 rpm. 40 mL of this final film forming solution were then poured in a polystyrene Petri dish (13.5 cm diameter) and dried during 24 h at 25 °C and 40 % relative humidity (RH) in a climatic chamber (WTC, Binder). Films were finally peeled off from the Petri dish and stored until water sorption


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equilibrium (at least one week) at 20 °C and 33 % or 75 % RH, using saturated salt solutions of MgCl2 or NaCl, respectively. Mechanical properties The mechanical behavior of films was assessed by uniaxial stretching tests at room conditions (21°C and 35 % RH) using a texture analyzer (TA HD plus, Texture Technologies, Hamilton, MA, USA) calibrated with 2 kg mass. The method used was based on the NF EN ISO 527-1 standard with the following modifications: an initial gauge length of 7 cm, a load cell of 100 kg and a crosshead speed of 1 mm⋅s-1. The Young modulus (YM, GPa), the tensile strength (TS, MPa) and the elongation at break (E, %) were determined from stress-strain curves. Film samples (10 x 2.5 cm) were prepared in triplicate using a precision cutter (JDC, Thwing Albert Instrument Company, West Berlin, NJ, USA). They were tested for different RH storage (33 and 75 %) and lignin contents (0, 10, 20 and 30 wt %). Gas barrier properties The permeability of films to oxygen (O2) and nitrogen (N2) was determined using a manometric method on a permeability testing apparatus (GDP-C permeameter, Brugger Feinmechanik GmbH, Munich, Germany) working at 75 % RH and 25 °C. Film thickness was measured with a Digimatic micrometer (Mitutoyo, Japan). The gas permeation system was firstly outgassed under primary vacuum. Then, after one hour of vacuum, permeability measurement started with a gas flow of ≈100 cm3⋅min-1 on one side of the film sample, and the pressure increase was recorded over time on the other side. Permeability was determined from the steady state. Analyses were carried out in triplicate.


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Water vapor permeability The water vapor permeability (WVP) was determined based on the ASTM E96-80 standard (1980) for packaging films and modified by Debeaufort et al (1993)36. A 30-100 % gradient of relative humidity was selected. The experiment was carried out in a climatic chamber at 30 % RH and 25°C (KBF 240 Binder). Prior to measurement, film samples were placed between two Teflon rings on the top of a glass cell containing distilled water (to fix 100 % RH). From the steady state, WVP (g.m-1.s-1.Pa-1) was calculated using the following equation: =

× ×

×

(1)

where ∆m/∆t is the weight of moisture loss per unit of time (g.s-1), A the film surface area exposed to the moisture gradient (9.08 x 10-4 m²), e the film thickness (m) and ∆p the water vapor partial pressure difference between both sides of the sample (Pa). Four replicates were done. Contact angle and surface tension measurements Contact angle with various liquids (water, ethylene glycol, glycerol, formamide and diiodomethane) was measured, by goniometry (Digidrop, GBX, Bourg de Peage, France), for both sides of the films (the one in contact with the casting support during drying and the other one exposed to the air), using the sessile drop method at 20°C. The contact angle value reported corresponds to the equilibrium point between the initial very fast spreading step and the second step related to the evaporation of the liquid. Then the surface tension was calculated, according to the Young-Laplace equation (Eq 2):

= + ×

(2)


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where , and are the surface tensions at the solid/gas, solid/liquid and liquid/gas interfaces, respectively. Five drops having a volume ≈ 1 µL were deposited on each film surfaces for each liquid. The surface tension of the film, composed of a polar ( ) and a dispersive ( ) component, was determined using the Owens & Wendt method37 (Eq 3):

!"#

=

"$ "%$ "#

+ &

(3)

where and are the polar and the dispersive components of the liquid tested, respectively. Water, ethylene glycol, glycerol, formamide and diiodomethane were used as probe liquids. According to Ström et al.38 and Fowkes39, their liquid polar contributions ( ) are 51.0, 16.8, 26.4, 18.7 and 0 mN⋅m-1, and their corresponding dispersive contributions ( ) are 21.8, 30.9, 37.0, 39.5 and 50.8 mN⋅m-1, respectively.38, 39 Antioxidant properties The Radical Scavenging Activity (RSA) of films was measured using the free stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•).40 100 mg film (≈10 cm²) were introduced into 10 mL DPPH solution in ethanol at a concentration of 50 mg⋅L-1. As chitosan is not soluble in ethanol, the film integrity was fully preserved during the duration of the experiment, which makes possible the determination of the efficient antioxidant activity of films.13 The reaction of the DPPH• with an antioxidant, symbolized as a phenolic compound (Ar-OH) in the present case, occurs according to the following mechanisms: i) a HAT mechanism (hydrogen


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atom transfer, Eq 4) and ii) stabilization of phenoxy radical (Ar-O°) by resonance or condensation of (Eq 5): DPPH•+ Ar-OH → DPPHH + Ar-O•

(4)

[Ar-O° ↔…] / Ar-O•+ Ar-O• → Ar-O-O-Ar

(5)

The reaction kinetics was followed by the disappearance of the DPPH• reactant as given by the absorbance

measurement

at

515

nm

(Biochrom

WPA

Lightwave

II

UV/Visible

spectrophotometer). For measurement, 2 mL of the reaction solution was put into the measuring vial and the solution was returned after absorbance reading. Between each measurement, samples were maintained in closed vials and in darkness under stirring (200 rpm) during 140 h. The reduction in radical scavenging activity (in %) was expressed as follows (Eq 6): '()* = 100 −

./012 34056/7 ./012

× 100

(6)

where Asample(t) is the absorbance measured for the DPPH• solution containing the film sample at time t and Ablank(t) is the absorbance of the DPPH• solution at the same time. This correction takes into account the kinetics of auto-degradation of DPPH•. Thus, the RSA value represents the antioxidant capacity of the film sample. Kinetics were realized at least in triplicate. The release of lignin monomers or oligomers from the film into the ethanol medium was also monitored under the same experimental conditions using 200 mg film in 10 mL ethanol solution under stirring (200 rpm) during 72 h at room temperature. The release of lignin constituents was detected by UV spectroscopy measurement of the solution at 285 nm.41, 42 These ethanol extracts


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were also tested for their radical scavenging activity using 5 mL mixed with 5 mL of DPPH• solution at 100 mg/L to obtain a 10 mL of final DPPH• solution at 50 mg/L. Environmental Scanning Electron Microscopy The film microstructure was observed by environmental scanning electron microscopy (ESEM, Phillips XL 30 ESEM, Japan). Film samples, previously equilibrated at 33 % RH, were cut with razor blades and fixed on the ESEM support using double side adhesive tape. The surface was observed at 1000 magnification using an accelerating voltage of 8 kV and an absolute pressure of 230 Pa, allowing the presence of water vapor in the microscope chamber, corresponding to about 30 % RH. Cross-section of the films (angle of 90° to the surface) were observed in SEM (Jeol JSM 7600 F, USA) for a better resolution with a vacuum of 9.10-6 Pa, an accelerating voltage of 1 kV and observation were realized at 1000 magnification too. Epifluorescence Using the intrinsic fluorescence properties of lignin, epifluorescence technique was used to explore the repartition of lignin inside the chitosan films and to detect the presence of aggregates. Auto-fluorescence of lignin was observed by epifluorescence (Nikon TE 2000, Japan) with an excitation wavelength of 340 nm43,

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for an emission range of 435-485 nm (Dapi-5060C-000

filter set). Observations were realized on films which were previously casted onto a coverslip for this purpose and stored at 75 % RH. Two-photon microscopy Two-photon microscopy was performed to achieve a 3D representation of the internal structure of the films with the lignin distribution through the chitosan polymer matrix. They were


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collected on a Nikon A1-MP scanning microscope (Nikon, Japan). Imaging was carried out with a Plan Apo IR x 60 objective (NA: 1.27, Water Immersion, Nikon, Japan) at a scanning speed of 1 frame per second. An IR laser (Chameleon, Coherent) was used to provide a 750 nm excitation. Auto-fluorescence emission of lignin was collected on four detection channels (FF01492/SP-25 (400-492nm), FF03-525/50-25 (500-550 nm), FF01-575/25-25 (563-588 nm), FF01629/56-25 (601-657 nm). Increasing laser intensity was used along the depth of film for 3D images to compensate thickness. For this method, the films were previously casted onto a coverslip and stored at 100 % RH prior observation. X-ray Photoelectron Spectroscopy (XPS) The XPS analyses were performed with a PHI Versaprobe 5000 apparatus using monochromatic Al Kα X-ray. A 50W Al Kα radiation (1486.7 eV) was used as the X-ray source. The average circular spot size was 200 µm diameter. High-resolution (Pass energy = 58 eV) 45 degrees emission angle integrated scans were acquired. Measurements were carried out at room temperature inside an ultra-high vacuum compartment (base pressure of 2.10-7 Pa). The use of low-energy (

Biobased Composite Films from Chitosan and Lignin: Antioxidant

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