Hydrophobization and Antimicrobial Activity of Chitosan and Paper

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Biomacromolecules 2010, 11, 88–96

Hydrophobization and Antimicrobial Activity of Chitosan and Paper-Based Packaging Material Nicolas Bordenave, Stephane Grelier, and Veronique Coma* Universite´ Bordeaux 1, INRA, CNRS, UMR 5103 US2B, 351 cours de la Libe´ration, F-33405 Talence, France Received August 20, 2009; Revised Manuscript Received November 19, 2009

This study reports the elaboration of water-resistant, antimicrobial, chitosan and paper-based materials as environmentally friendly food packaging materials. Two types of papers were coated with chitosan-palmitic acid emulsions or with a blend of chitosan and O,O′-dipalmitoylchitosan (DPCT). Micromorphology studies showed that inclusion of hydrophobic compounds into the chitosan matrix was enhanced by grafting them onto chitosan and that this led to their penetration of the paper’s core. Compared to chitosan-coated papers, the coating of chitosan-palmitic emulsion kept vapor-barrier properties unchanged (239 and 170 g · m-2 · d-1 versus 241 and 161 g · m-2 · d-1), while the coating of chitosan-DPCT emulsion dramatically deteriorated them (441 and 442 g.m-2.d-1). However, contact angle measurements (110-120° after 1 min) and penetration dynamics analysis showed that both strategies improved liquid-water resistance of the materials. Kit-test showed that all hydrophobized chitosan-coated papers kept good grease barrier properties (degree of resistance 6-8/12). Finally, all chitosancoated materials exhibited over 98% inhibition on Salmonella Typhimurium and Listeria monocytogenes.

Introduction As a result of increasing environmental concerns, there is an extensive research effort on using renewable resources to create sustainable, biodegradable, or edible food packaging systems.1–3 In this respect, paper or cardboard can be considered as environmental friendly bases for food packaging. Indeed, paper and cardboard are easy to handle and have good mechanical properties for packaging. However, other properties, as moisturebarrier for example, must be improved. This goal is generally achieved by association with other polymers. Moreover, from a general point of view, the food safety level has never been as high as today, but among the remaining risks, microbiological threat is the most important.4 Indeed, food-borne toxi-infections are mainly caused by wrong conservation or misuse of food products. So, there is a major need for packaging systems able to hinder microbial development in food. Thus, this study aimed to associate paper and chitosan, selected for its film forming and antimicrobial properties.5,6 Chitosan is a linear polysaccharide derived from chitin, a major component of crustacean and insect shells. Despite that paper and chitosan are two materials widely studied independently for food packaging applications, only a few works dealt with paper coated with chitosan7–10 and all show that chitosan decreased paper resistance to water or vapor transfer. According to these results, the authors investigated water and moisture susceptibility of paper coated with chitosan in a previous study.11 This study showed that, despite some improvement of paper properties, association of paper and chitosan was not yet suitable to food application because of water sensitivity, inherent to a majority of polysaccharides. Many works attempted to decrease water and moisture sensitivity of polysaccharides and two general solutions can be highlighted: associate polysaccharides with hydrophobic components or chemically modify polysaccharides themselves. * To whom correspondence should be addressed. E-mail: v.coma@ us2b.u-bordeaux1.fr.

Association of polysaccharides with hydrophobic components can lead to composite materials derived from emulsions or to multilayer materials.3,12,13 Numerous studies showed that multilayer materials have the best barrier properties,14–16 but this technique requires several costly processing steps and needs a decrease of interfacial tension between hydrophobic and hydrophilic surfaces. Thus, emulsion-based composite materials can be preferred and can lead to acceptable barrier properties.16–18 Barrier and mechanical properties of composite films depend on multiple parameters: type and amount of hydrophobic component used, morphology and distribution of both hydrophilic and hydrophobic phases, and drying conditions for film formation.19–21 As stated above, hydrophobization of chitosan can also be achieved by chemical modification. Chemical modifications of chitin and chitosan have been reviewed previously.22 Hydrolysis has been used to modify solubility and biological activity of chitosan. O-Hydroxyalkylation was carried out to obtain readily water-soluble chitosan. Reductive N-alkylation allowed the enhancement of chitosan chelating properties with metal ions. O- and N-carboxyalkylations allowed the synthesis of anionic chitosans. Tosylation and N-phthaloylation were used as protective methods to carry out further modifications of chitosan. Finally, acetylation and other acylations23–26 have been shown to occur primarily on amino groups and were carried out to modify hydrophilic/hydrophilic balance of chitosan, to make it soluble in organic solvents, to enhance its affinity with nonpolar compounds in separation technologies, or to engineer drug delivery systems. However, in this study, chitosan has been selected for its antimicrobial properties, mainly due to its free amino groups.27 So, esterification ought to be achieved specifically onto hydroxyl groups of chitosan and leave amino groups free, despite the higher reactivity of these latter.28–30 In this respect, chitosan had already been O,O′-acylated in methanesulfonic acid,31,32 but this reaction led to a drastic decrease of chitosan molecular weight, which should be avoided to keep film forming properties. As an alternative method, chitosan can

10.1021/bm9009528  2010 American Chemical Society Published on Web 12/08/2009

Antimicrobial Activity of Chitosan

be O,O′-acylated in biphasic reactive media, at interface between acidic water-phase containing protonated chitosan and organic phase containing acylating agent.33,34 Nevertheless, the hydrophobization of chitosan by grafting long alkyl chains could also modify grease-barrier properties of polysaccharide-based materials, and then these latter should be evaluated. As a consequence, this paper aims to study the comparative effects on chitosan-coated papers of these two hydrophobization strategies (association with fatty acids O,O′-acylated chitosan). Moisture and water susceptibility of subsequent materials were measured and confronted with micromorphology characterization. Additionally, grease-barrier properties of the designed materials were investigated and their antimicrobial effects were evaluated to ensure their remaining potential as antimicrobial food packaging materials.

Materials and Methods Materials. Raw Materials. Chitosan 652 (deacetylation degree higher than 85%, Mw ) 165 kDa) was provided by France Chitine (Marseille, France). Acetic acid (assay >99.5%) was provided by Sigma-Aldrich (St. Quentin Fallavier, France). In the project framework, papers were provided by Ahlstrom (Ascoflex 40, noncalendered, one side minerally pretreated, 40 g m-2, 48 µm thick, Grenoble, France) and Stora Enso (Performa Nature 320, noncalendered, one side minerally pretreated, 320 g m-2, 344 µm thick, Helsinki, Finland). Chemicals. Absolute ethanol was provided by Fluka (L’Isle d’Abeau Chesnes, France). Acetone, ethanol, methanol, and dichloromethane were HPLC grade and provided by SDS (Val de Reuil, France). Phosphorus pentoxide, palmitoyl chloride (assay >98%), and lauric, palmitic, and stearic acids (assays >97.5%) were provided by SigmaAldrich (Saint Quentin Fallavier, France). Sodium hydroxide (granules, assay >99%) and hydrochloric acid (assay >37%) were provided by VWR (Fontenay sous bois, France). Bacterial Growth Media and Bacterial Strains. Following growth media were used under liquid form or solid form (by addition of 12 g L-1 of DIFCO agar, provided by Sigma-Aldrich, Saint Quentin Fallavier, France) in Nutritive or Tryptose Broths (Difco 234000 and Difco 262200, noted NB and TB respectively, both provided by DIFCO). Tested bacterial strains were Salmonella typhimurium (Institut Pasteur 5858) grown in BN and agar-BN, and Listeria monocytogenes (Institut Pasteur 82110) grown in TB and agar-TB. Methods. 1. Samples Preparation. 1.1. Preparation of Homogeneous Chitosan Films. Chitosan film forming solutions (2% (w/w)) were obtained by dispersing chitosan in a 1% aqueous acetic acid solution. After mixing, the solution was degassed under vacuum and cast in a polyethylene Petri dish and then dried at room relative humidity (RH) and temperature for 12 h. The films were then conditioned in a controlled atmosphere (23 ( 1 °C and 50 ( 5% RH) for at least 5 days before the property measurements were taken. 1.2. Preparation of Composite Chitosan-Based Films. Chitosan base solutions were made from 2/2/32/64 parts of chitosan/acetic acid/ absolute ethanol/water (w/w/w/w), stirred for 2 h at 500 rpm. On one hand, fatty acid was incorporated in chitosan base solution that was previously heated up to 70 °C. The amounts of fatty acids incorporated were 0.04, 0.08, 0.16, and 0.24 moles per mole of anhydroglycosidic unit of chitosan. On the other hand, long alkyl chain grafted chitosan was incorporated directly in the other unheated chitosan base solution: 0.715 g of chitosan and 0.285 g of O,O′-dipalmitoylchitosan (DPCT) with degrees of substitution (DS) ) 0.281. All the solutions were homogenized with an Ultraturax homogenizer (IKA-Werke, Paris, France) at maximum speed for 1 min and slightly degassed under vacuum. The resulting solutions were cast in polyethylene Petri dishes and then dried at room RH and temperature for 12 h. Films were then conditioned in a controlled atmosphere (23 ( 1 °C

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and 50 ( 5% RH) for at least five days before the property measurements were taken. 1.3. Preparation of Chitosan and Paper-Based Materials. Stora Enso and Ahlstrom papers were coated with the different chitosan-based film forming solutions mentioned above using a coating table (K101 Control Coater, Erichsen, Rueil-Malmaison, France) at room temperature and equipped with a 120 µm blade. A 5 mL aliquot of chitosan-based solution was layered on untreated side of each Ahlstrom and Stora Enso papers (on 210 × 297 mm format sheets), leading to a dry deposit of ∼1.6 g.m-2. The coated materials were subsequently dried at room RH and temperature for 12 h and conditioned at 23 ( 1 °C and 50 ( 5% RH for 5 days before achieving properties assessments. 2. Chitosan Modification. This method was adapted from Vasnev et al.34 Chitosan (4 g) was dissolved in 100 mL of 4% acetic acid aqueous solution in a reactor equipped with mechanical stirring (2000 rpm) and a refrigerating system. A solution made from 40 mL of dichloromethane and palmitoyl chloride (1.2 mol per mol of anhydroglycosidic unit from chitosan) was poured in an intensely stirred chitosan solution. A total of 91 mL of a 1 M KOH solution was gradually added over 20 min to neutralize the pH of the reactive media. The emulsion was stirred for an additional 40 min and destroyed by heating up to 50 °C for 30 min to evaporate dichloromethane. Modified chitosan was precipitated by adjusting the pH of the solution to 8.5-9 and recovered by centrifugation. Palmitoyl acid traces were removed by Soxhlet extraction with dichloromethane for 24 h. Modified chitosan was then washed 3 times with methanol and, subsequently, with water and centrifuged repeatedly until pH of supernatant had neutral pH. The resulting product was dried overnight under vacuum with phosphorus pentoxide. The DS of recovered products were calculated as the number of chains grafted per anhydroglycosidic unit of chitosan (total anhydroglycosidic units of chitosan are the sum of 2-amino-2-deoxy-Dglucopyranose and 2-acetamido-2-deoxy-D-glucopyranose units). Two DS were targeted: around 0.08 and 0.3. FTIR spectroscopy was then used for characterization because of products insolubility in deutered solvents. 3. Chemical AnalysessFTIR Spectroscopy. Infrared spectra of the products were carried out in KBr tablets (1% w/w of product in KBr), with a resolution of 4 cm-1 and 100 scans per sample on a ThermoNicolet AVATAR 370 apparatus. DPCT DS was determined using a characteristic band from long alkyl chains at 2918 cm-1 (C-H asymmetric stretching). For that determination, several chitosan/palmitic acid blends with known ratios were made and analyzed by FTIR. Absorbance of 2918 and 895 cm-1 bands were noted A2918 and A895, respectively; a molar amount of palmitic acid and of anhydroglycosidic units in blends was noted: npalm.ac. and nglyc., respectively. (A2918)/(A895) and (npalm.ac.)/(nglyc.) exhibited a linear relationship: (A2918)/(A895) ) 12.81 × (npalm.ac.)/(nglyc.) + 1.85, with R2 ) 0.9976. 4. Materials Analyses. 4.1. Scanning Electron Microscopy (SEM). Film and paper samples were cut and placed onto specimen supports to obtain surface and transversal views. After the gold and palladium coating procedure, samples were viewed using a Jeol 840A microscope (Jeol Europe, Croissy-sur-Seine, France). 4.2. Fourier Transform Infrared (FTIR) Microspectroscopy. FTIR microspectroscopy analyses were performed to study the coated papers. Reflection spectra were recorded by a ThermoNicolet AVATAR 370 FTIR coupled with a Nicolet Centaurus IR microscope and treated by OMNIC software (Thermo-Nicolet, Courtaboeuf, France), between 400 and 4000 cm-1 using 50 scans with a resolution of 4 cm-1. The area of analysis was a square with 100 µm side centered on the middle point of the cross-section of the analyzed materials. 4.3. Material Thickness. Material thickness was determined with a micrometer Lorentzen & Wettre SE050 (Lorentzen & Wettre, SaintGermain-en-Laye, France) from six measurements per material. 4.4. Water Vapor Transmission Rate (WVTR). WVTR of chitosanbased materials was evaluated using the NF-ISO-2528 (1995) procedure.

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An aluminum cup containing anhydrous CaCl2 desiccant was sealed by the tested material (50 cm2 exchange area) with paraffin wax at 50 °C and placed in fixed controlled atmosphere (50 ( 5% RH and 23 ( 1 °C). The coated side of the paper faced outward during WVTR measurements. Starting 2 h after the experiment setup, cups were weighed every 2 h for 24 h. Weight variations of cups were plotted versus time. WVTR (g · m-2 · d-1) was determined from the slope of this plot linear fit according to the following equation: WVTR ) (∆m × 24)/(∆t × S), where ∆m is the weight (g) of H2O vapor passing through a material of area S (m2) during time ∆t (h). Control cups, without the anhydrous CaCl2 desiccant were conducted in parallel. Their weight gain or loss was used as a correcting factor of the tested cups. WVTR was calculated from 3 measurements and given at atmospheric pressure (1 atm) with a RH gradient of 0-50%. 4.5. Water Contact Angle Measurements. Contact angle between the material and a distilled water droplet was measured according to TAPPI T458 cm-94 (1994) and using a goniometer Kru¨ss DSA 10Mk2 (Kru¨ss, Palaiseau, France) equipped with a camera and a recording system. The resulting angle was calculated from 10 measurements. 4.6. Penetration Dynamics Analysis (PDA). PDA was performed with an EMTEC PDA C02 apparatus (Emtec Electronic, Leipzig, Germany35). A high-frequency/low-energy ultrasound transmitter coated with water and the corresponding ultrasound receiver coated with the tested paper were brought into contact. Ultrasound waves are transmitted through water with different intensity losses depending on the crossed material (water, air, occurrence of paper fibers occasioning diffusion, etc.).36–38 PDA curves plot the percentage of transmission of the ultrasonic signals versus time. Transmission is set at 100% at maximum of each curve, the time corresponding to the maximum of each curve is defined as the “wetting time” of the material, and the slope of the curve after this maximum is proportional to the speed of penetration of water through the material. Each curve is the mean of three experiments. 4.7. Grease Barrier Properties. Grease barrier properties were evaluated according to TAPPI T559 pm-96 standard, also known as “kit test”. This test gives an index of resistance of a material against grease on a scale of 12 level (level 1 is the least resistant, 12 is the most resistant). Each resistance index is the mean of three repetitions. 5. Biological Assessments. 5.1. Bacterial Strains Precultures. Overnight precultures were performed as follows: L. monocytogenes and S. Typhimurium were grown in Tryptose broth (Difco 262200) and Nutrient broth (Difco 234000) at 37 °C for 18 h, respectively. 5.2. BioactiVity Assessments of Films. For L. monocytogenes and S. Typhimurium, 100-300 colony forming units (CFU) from 18 h preculture were spread onto the surface of agarose culture media in 50 mm diameter Petri dishes. A 90 mm diameter circular sample of tested material was then carefully fitted onto agarose surface, avoiding any air bubbles. This system was incubated at 37 °C for 48 h. All samples and controls were tested three times. Counting of colonies could not be performed because of papers opacity. Thus, after this 48 h incubation, paper-based material and agarose gel were introduced together in a Stomacher sachet with 10 mL of liquid growth media. From this sachet, kneaded for 5 min, 10 mL of liquid growth media were extracted and diluted by a 106 factor with sterile physiological water. A total of 100 µL of resulting solution were inoculated on new agarose culture media and incubated at 37 °C for 48 h prior to counting. Each inoculation, each dilution, and each final bacterial growth was repeated three times. Thus, for each material, the inhibition result is the average result of 27 countings. Moreover, papers coated with HPC (coating solution composed of 2/2/32/64 parts of HPC/acetic acid/absolute ethanol/water) were tested in parallel as references. Inhibition was calculated as follows:

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Figure 1. Percentage of WVTR decrease of composite chitosan-stearic acid and chitosan-palmitic acid films from homogeneous chitosan films as a reference, depending on the free fatty acid content of films.

inhibition(%) ) CFU in reference - CFU in tested sample × 100 CFU in reference 6. Statistical Treatment. All experiments were repeated at least three times. Data are mean values given with a Student’s confidence interval at 95% probability (p < 0.05).

Results and Discussion One of the objectives of this study was to improve the resistance of chitosan and paper-based materials toward water, that is, increase their hydrophobic character, by mixing chitosan with fatty acids or by grafting the equivalent hydrophobic moieties directly onto chitosan. Thus, a preliminary study was conducted on chitosan films to determine the amount of hydrophobic components needed. Preliminary Study. Chitosan-based composite films were made with lauric, palmitic and stearic acid and their WVTR were compared with respect to the amount of fatty acid incorporated in chitosan. Films elaborated with lauric acid showed poor properties, probably due to the melting point of this acid which is too close to room temperature. For both stearic and palmitic acid, WVTR decreases with increasing amounts of fatty acids and finally reaches a plateau corresponding to ∼90% decrease of WVTR from homogeneous chitosan films (Figure 1). This result is consistent with previous studies.39,17 From these results, it was stated that the amount of fatty acid necessary to decrease drastically WVTR of chitosan films was 0.08 mol of fatty acid per mol of anhydroglycosidic unit of chitosan, either with palmitic or stearic acids. Thus, palmitic acid was preferred to stearic acid due to the cost of chloride derivatives of these compounds for further grafting, and 0.08 mol of palmitic acid per mol of anhydroglycosidic unit of chitosan was set as the reference amount of hydrophobic component to be added or grafted to chitosan for further paper coatings. Chitosan was selected with respect to its antimicrobial and film-forming properties. Thus, chemical modifications of chitosan leading to its hydrophobization should not be done on amine groups, and biphasic reactive medium (CHCl3-H2O) with



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Figure 2. FTIR spectra of chitosan and DPCT.

mild conditions was preferred to methanesulfonic acid method31,32 because of potential degradations of chitosan. 1. Hydrophobization of Chitosan. The aim of that part of the study was to prepare an DPCT to form films and to coat papers, according to the requirements listed above. FTIR spectra obtained (Figure 2) were similar to those exhibited by Tong et al.,32 and the band at 895 cm-1 (N-H angular bending out-of-plane) was identified as a possible reference band for quantification of grafting. The intensity of this band could be affected by grafting palmitoyl functions on amino groups of chitosan. In this case, the ratio of the 895 cm-1 and 1654 cm-1 (CdO stretching of amides in N-acetyl groups) band intensities would decrease from chitosan to grafted chitosan. Because this ratio is identical for chitosan and acylated chitosan, the chitosan degree of acetylation remained unchanged, free amino groups had not been converted into amido groups with palmitoyl chloride, and acylated chitosan could be referred as O,O′-acylated chitosan. Thus, the 895 cm-1 band can be used as a reference for DS determination of the DPCT products. DPCT were obtained with DS ) 0.071 ( 0.002 and DS ) 0.281 ( 0.002, respectively. As hypothesized, hydrophobic components as palmitic acid and DPCT could provide water resistance to chitosan- and paperbased materials. Thus, DPCT with DS ) 0.071 was designed to be compared with chitosan-palmitic acid blend containing 0.08 mol of palmitic acid per mol of anhydroglycosidic units of chitosan. Unfortunately, this hydrophobized chitosan no longer exhibited film-forming properties and, thus, could not form a continuous biopolymeric matrix anymore when coated on papers. DPCT with a DS of 0.281 was then associated with chitosan to allow the same type of organization in the material. In this case, chitosan acylation was no longer considered as a mean to hydrophobize chitosan, but as a mean to enhance chitosan and palmitic acid compatibility.28,30 As a consequence, the following study aims to compare properties of materials made from chitosan-palmitic acid and from chitosan-DPCT (DS ) 0.281) with a global ratio of 0.08 moles of palmitoyl chains per mole of anhydroglycosidic units in the blend. 2. Hydrophobization of Chitosan- and Paper-Based Materials. 2.1. Coatings Analyses. Papers and papers coated with chitosan were studied in a previous work that showed that, instead of forming a layer on top of the papers, chitosan penetrated deeply into paper’s cores, embedding cellulose fibers and filling the paper’s pores.11 Readers should refer to that work to see SEM micrographs and chemical surface analyses of homogeneous chitosan films and coatings. The same effect was

Figure3.(a)SEMmicrographoftheuppersurfaceofthechitosan-palmitic acid composite film (magnification: 1000×). (b) SEM micrograph of the lower surface of the chitosan-palmitic acid composite film (magnification: 2000×).

observed here, and attention was focused on the distribution of hydrophobic components added to coating solutions. 2.1.1. SEM Observations. Figure 3a,b and Figure 4a,b are SEM micrographs of chitosan-palmitic acid and chitosan-DPCT films, respectively. Both of them experienced phase separation during the drying process but to different extents. Indeed, Figure 3a shows that a large amount of palmitic acid migrated toward the film surface and formed aggregates (∼10 µm diameter), as observed by Greener and Fennema with methylcellulose,14 while no aggregates could be observed from the bottom side of the film (Figure 3b). On the other hand, Figure 4a and b show that even if some micrometer-size aggregates of palmitoylchitosan are observable, they are less numerous than in Figure 3a, and most of these aggregates are distributed over the surface as microglobules (∼300 nm diameter). Moreover, the surface shows a roughness, probably due to inclusion of hydrophobic aggregates into the chitosan matrix. All these observations confirm that grafting palmitoyl chains onto chitosan allows better compatibility between chitosan and hydrophobic component.

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Figure 4. (a) SEM micrograph of the upper surface of the chitosan-DPCT composite film (magnification: 2000×). The black square is the zoomin zone represented in Figure 3b. (b) SEM micrograph of the upper surface of the chitosan-DPCT composite film (magnification: 9000×). Zoom in from Figure 3a.

These observations on films were related to the SEM observation of coated papers: SEM micrographs of Ahlstrom paper coated with chitosan-palmitic acid composite and with chitosan-DPCT are shown on Figure 5a and b, respectively. Micromorphology of composite-coated papers studied here is consistent with micromorphology of chitosan-coated papers studied previously and with observations made on films. Indeed, composite-coated papers show chitosan embedding cellulose fibers and filling paper pores and also exhibited two kinds of hydrophobic component distributions: ∼10 µm diameter aggregates on top of the paper in the case of chitosan-palmitic acid composite, and smaller and dispersed aggregates in the case of chitosan-DPCT composite. Similar structures were observed on Stora Enso paper coated with the same chitosan-based hydrophobic composite solutions (views not shown). So, it can be expected that the same phase-separation phenomenon happened both in film-forming and in paper-coating processes. As

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Figure5.(a)SEMmicrographoftheuppersurfaceofthechitosan-palmitic acid coated Ahlstrom paper (magnification: 1000×). (b) SEM micrograph of the upper surface of the chitosan-DPCT coated Ahlstrom paper (magnification: 1000×).

a consequence, no palmitic acid should penetrate through the paper core in the first case, and DPCT should be detected in the paper core in the second case. Consequently, chitosan-palmitic acid coated papers should have a higher surface hydrophobicity than chitosan-coated papers, and chitosan-DPCT composite papersshouldhaveamicroporousstructuresimilartochitosan-DPCT films. These statements were then confirmed by FTIR microspectroscopy. 2.1.2. FTIR Microspectroscopy. To evaluate the penetration of hydrophobic components in papers cores, a characteristic IR band at 2915 cm-1 was used, which corresponds to C-H elongation in alkyl chains. It allows detecting the presence of C16 alkyl chains belonging to hydrophobic components (palmitic acid and DPCT). Stora Enso paper coated with chitosan-palmitic acid was analyzed by surface and side view (Figure 6a). These spectra identified palmitic acid on the top surface of the paper (C-H elongation band at 2915 cm-1), but no characteristic band could be detected in the core of the paper. The same analyses were


Figure 6. (a) FTIR microspectroscopy spectra of Stora Enso paper coated with chitosan-palmitic acid composite: coated surface (top), core of the paper from side cut (bottom). (b) FTIR microspectroscopy spectra of the core of Stora Enso paper coated with chitosan-DPCT composite.

conducted on Ahlstrom paper leading to similar results (data not shown). Thus, it can be assumed that chitosan-palmitic acid composite solution underwent phase separation during the coating process. As a consequence, those papers can be considered schematically as a matrix of cellulose fibers embedded with chitosan with a distribution of palmitic acid globules (diameter ∼10 µm) on top surface of the materials (∼180 mg of palmitic acid per square meter of paper). Papers coated with chitosan and DPCT were analyzed according to the same procedure (Figure 6b). Side IR spectra of Stora Enso coated paper exhibited characteristic bands of DPCT. Even though their intensity was rather low, their presence allows concluding that the hydrophobic component penetrated through the core of the paper. Same analyses were conducted on Ahlstrom paper leading to similar results (data not shown). It confirms also that this component was probably carried by chitosan through paper, due to their compatibility, and that chitosan-DPCT emulsion remained stable during coating process. Finally, the two types of materials produced in this study exhibited dramatically different microstructures. The following of this study aimed to understand the consequences of these different microstructures on macroproperties of the materials.


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2.2. Moisture and Water SensitiVity of Coated Materials. Moisture sensitivity was evaluated by the WVTR. Liquid water sensitivity was determined by measurement of droplet contact angle and by penetration dynamics analysis. 2.2.1. Water Vapor Transfer Rate. Data from all materials are shown in Table 1 (see Supporting Information). They show that all chitosan/DPCT-based materials exhibited higher WVTR than chitosan/palmitic acid-based materials. Indeed, while palmitic acid allowed decreasing WVTR of chitosan films by 90%, the decrease with DPCT was only by 36.5%, despite materials having the same amount of hydrophobic component. This can be interpreted as a consequence of the differences among the microstructures observed previously. Indeed, the distribution of palmitic acid on top of the films may reduce adsorption of vapor at the surface and then reduce WVTR globally. On the other hand, films made from chitosan and DPCT showed better compatibility between chitosan and the hydrophobic component. This might have prevented a major reduction of adsorption of vapor at the materials surface and might have facilitated diffusion of vapor through films, respectively. Indeed, the inclusion of DCPT in the chitosan matrix might have led to preferential channels for vapor diffusion. However, this effect is even more obvious on coated papers. Indeed, on one hand, addition of palmitic acid left WVTR almost unchanged compared to chitosan-coated paper (see Table 1 of the Supporting Information). For these materials, which are much thicker than films, diffusion of vapor through the coatedpaper network may be the main factor driving WVTR, compared to surface adsorption and desorption. Then, surface distribution of palmitic acid and a subsequent change in vapor adsorption on the surface do not lead to a global change of WVTR. On the other hand, incorporation of DPCT led to a dramatic increase of WVTR values: 441 ( 20 and 442 ( 33 g · m-2 · d-1, for Ahlstrom and Stora Enso papers, that is, +83% and +174%, respectively, from values obtained with chitosan-coated papers. As a result, despite filling of papers pores and coating material hydrophobicity, these values of WVTR are comparable with WVTR of reference papers without coating. It can be assumed that adsorption of vapor on surface of chitosan-DPCT coated papers was unchanged compared to chitosan-coated papers due to the low surface distribution of the hydrophobic component. Thus, the data obtained mean that the vapor diffusion rate through chitosan-DPCT coated papers and reference papers are comparable, that is, global porosity is comparable for those two types of materials. This observation may confirm that chitosan-DPCT still adopt the same microstructure in the film state or when coated on papers with the presence of preferential channels for vapor diffusion. This is correlated with the work of Wu et al.40 who observed lamellar structures for N- and O-acylated chitosan powders. 2.2.2. Sensitivity to Liquid Water. Contact angles between materials and water droplets were measured and are reported in Table 2 (see Supporting Information). Addition of palmitic acid or DPCT led to angles all above 110° and that were stable over time, thus, it led to a significant decrease of surface tension of all materials. The relation between these measurements and the observed microstructure (major surface distribution of hydrophobic components) is obvious. Even in the case of esterified chitosan where phase separation was less important, it is still consistent with results from Aburto et al.41 who observed a decrease in surface tension of polyethylene films by incorporation of esterified amylose. However, there is no significant difference in contact angle values between chitosanpalmitic acid and chitosan-DPCT based films and coated

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Figure 7. (a) Penetration dynamics analyses curves of reference and hydrophobized chitosan-based coated papers. (b) Zoom in from (a), on time 0-25 s, of penetration dynamics analyses curves of reference and hydrophobized chitosan-based coated papers.

papers. This study of static contact angles was completed by a dynamic analysis of liquid water penetration through papers. PDA curves are shown on Figure 7a,b for Ahlstrom and Stora Enso papers coated with chitosan, chitosan-palmitic acid, and chitosan-DPCT. It can be observed from Figure 7a that water penetration rates of chitosan-coated papers and chitosan-palmitic acid coated papers are similar, while they are higher for papers coated with chitosan and DPCT. Moreover, Figure 7b shows that the addition of palmitic acid in chitosan led to a significant increase of wetting times on coated papers compared to papers coated with chitosan only (from ∼0 to 12 s on Ahlstrom and from 1-8 s on Stora Enso). The effect of DPCT is similar, but wetting times are slightly shorter than with palmitic acid (3 and 4 s for Ahlstrom and Stora Enso papers, respectively). Thus, data concerning papers coated with chitosan and palmitic acid are consistent with previous statements: they can be considered as a matrix of cellulose fibers embedded with chitosan with a distribution of palmitic acid globules on top surface of sheets. As a consequence, palmitic acid on surface make wetting of this surface slower. Nevertheless, both papers coated with chitosan, with or without palmitic acid have the same internal microstructure, so once surface is wet and water penetrated the core of the paper, water crosses both materials at the same rate. This confirms the observations made from WVTR and contact angle measurements: addition of palmitic acid only provides surface hydrophobization. On the other hand, addition of esterified chitosan did not bring the expected outcomes. Indeed, water sensitivity is apparently

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higher for chitosan-DPCT than for chitosan-palmitic acid coated paper. Once again, the microstructure of chitosan-esterified/ chitosan composite may be responsible for these results, probably because of the amphiphilic character of esterified chitosan. Indeed, the lamellar structure observed by Wu et al.40 on N- or O-acylated chitosans was probably due to hydropho bic-hydrophobic interaction between the long alkyl chains and to the orientation of these latter, perpendicular to the polysaccharidic backbone. Then, this type of microstructure could create preferential ways for water conduction through materials, these latter being detrimental for the hydrophobization of paper coated with chitosan. Unexpectedly, the structure adopted by chitosanDPCT composite in films was kept in papers despite the coating process and led to unexpected low performances. 2.3. Grease-Barrier Properties. Grease resistance of different papers and coated papers are shown in Table 2 of the Supporting Information. The Stora Enso paper showed higher resistance to grease than the Ahlstrom paper, probably due to its higher thickness. However, these reference papers showed global low resistance to grease. Coating of chitosan noticeably increased papers grease resistance, according to the results obtained by Kjellgren et al.8 and Ham-Pichavant et al.42 Chitosan/lipid interactions have been observed by several authors,40,43 and they could partially explain slower grease transfers through papers. Addition of palmitic acid did not significantly change grease resistance degrees of papers compared to chitosan-coated papers. Therefore, the influence on grease resistance of the incorporation of esterified chitosan on coating solutions was studied. O-acylated chitosans were shown to have higher affinity than chitosan with fatty components,32 but grease barrier properties remained unchanged despite this coating. This could be explained by the very little amount of material coated on papers. 3. Antimicrobial Properties of Chitosan and PaperBased Materials. Antimicrobial activity of coated papers is shown in Table 3 (see Supporting Information) as percentages of inhibition of S. Typhimurium and L. monocytogenes by contact with tested materials. Two factors could influence biological activity of tested materials: the presence of acetic acid in materials and the filling of paper pores by coating solutions that could limit gaseous exchanges and artificially increase results of antimicrobial activity. A preliminary study, performed in the same conditions and using hydroxypropylcellulose (HPC) as a biologically inert polymer to fill papers pores instead of chitosan, showed that these two factors had no influence on the bacterial growth. Chitosan coated on paper showed almost 100% inhibition on selected bacterial strains, despite its penetration through the core of the papers. To our knowledge, no previous study had investigated bioactivity conferred by chitosan to paper-based packaging materials, and this result looks promising for the development of chitosan and paper associations as antimicrobial packaging materials. The impact of the addition of fatty acid or the chemical modification of chitosan by O-acylation was then studied. Incorporation of palmitic acid did not have any influence on the biological activity of paper-based materials. The inhibition slightly below 100% could be explained by imperfect contact of materials on agar plate, due the important increase of their surface hydrophobicity. However, it does not seem to be a limitation for the activity of these materials. Moreover, this result is consistent with those obtained by Devlieghere et al.44 who showed that chitosan kept its antimicrobial activity on Candida


lambica in liquid media emulsified with fatty components (sunflower oil), despite potential complexation between these two components. The same efficiency was shown by papers coated with chitosan-DPCT. Obviously, this antimicrobial activity is mainly due to chitosan itself and to its free amino groups, but it can be hypothesized that preventing the amidation of chitosan-free amino groups by palmitoyl chloride might have helped to maintain the antimicrobial activity of the materials. However, antimicrobial activity of DPCT itself shall be tested in further experiments.

Conclusion Two strategies were studied to improve the hydrophobic character of chitosan-coated papers: association of chitosan with a fatty acid and compatibilization of this fatty acid by grafting onto hydroxyl moieties of chitosan. Concerning chemical modification of chitosan, despite much higher reactivity of amine moieties than hydroxyl groups toward ester chlorides, chitosan was selectively esterified on hydroxyl groups on C3 and C6. Concerning materials properties, chitosan/grafted chitosan blend did not improve vapor-barrier properties of coated papers because of a porous microstructure. However, both methods managed to improved liquid water sensitivity, and they allowed keeping good grease-barrier properties and excellent antimicrobial effects on S. Typhimurium and L. monocytogenes, leading to antimicrobial surface-hydrophobic papers for potential food packaging applications. Supporting Information Available. Detailed data about material characterizations (thickness, WVTR, contact angles, grease resistance, bioactivity). This material is available free of charge via the Internet at http://pubs.acs.org.

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Hydrophobization and Antimicrobial Activity of Chitosan and Paper

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