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FTIR spectroscopy studies on the spontaneous neutralization of chitosan acetate films by moisture conditioning
Accepted Manuscript Title: FTIR spectroscopy studies on the spontaneous neutralization of chitosan acetate films by moisture conditioning Authors: Reina Araceli Mauricio-S´anchez, Ricardo Salazar, J. Gabriel Luna-B´arcenas, Arturo Mendoza-Galv´an PII: DOI: Reference:
S0924-2031(17)30233-3 https://doi.org/10.1016/j.vibspec.2017.10.005 VIBSPE 2750
To appear in:
VIBSPE
Received date: Revised date: Accepted date:
16-8-2017 6-10-2017 27-10-2017
Please cite this article as: Reina Araceli Mauricio-S´anchez, Ricardo Salazar, J.Gabriel Luna-B´arcenas, Arturo Mendoza-Galv´an, FTIR spectroscopy studies on the spontaneous neutralization of chitosan acetate films by moisture conditioning, Vibrational Spectroscopy https://doi.org/10.1016/j.vibspec.2017.10.005 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.
FTIR spectroscopy studies on the spontaneous neutralization of chitosan acetate films by moisture conditioning Reina Araceli Mauricio-Sáncheza, Ricardo Salazarb, J. Gabriel Luna-Bárcenasa, Arturo Mendoza-Galvána,* a
Cinvestav-Unidad Querétaro, Libramiento Norponiente 2000, Querétaro,76230 Mexico
b
CONACyT-Universidad Autónoma de Guerrero, Av. Javier Méndez Aponte No. 1, Fracc.
Servidor Agrario, 39070 Chilpancingo de los Bravo, Guerrero, Mexico Authors e-mail addresses: Reina Araceli Mauricio-Sánchez: [email protected] Ricardo Salazar: [email protected] J. Gabriel Luna-Bárcenas: [email protected] Arturo Mendoza-Galván: [email protected] * Corresponding author. Tel.: +52-442-211-9922; fax: +52-442-211-9938 E-mail address: [email protected] (Arturo Mendoza-Galván)
1
Highlights
A reliable method for soft neutralization of chitosan acetate films is presented.
Spontaneous neutralization is controlled by water activity.
Water activity of 0.53 neutralizes the films after only five days of storage.
Chitosan films in the acetate form can be stored at low water activity.
Protonation degree can be tuned by adjusting the ambiance relative humidity.
Abstract The influence of moisture ambiance on the spontaneous neutralization of amorphous chitosan acetate films during storage at 30 ˚C and water activities (aw) in the range of 0.11 to 0.84 is studied by Fourier transform infrared (FTIR) spectroscopy. Three ranges of relative humidity affecting the chitosan acetate films are identified: i) 0.11≤aw≤0.32, produces dehydration and partial neutralization; ii) aw=0.44 and 0.55, spontaneous neutralization and rearrangement of OH bonds; iii) 0.64≤aw≤0.85, prevalence of films in the acetate form and moisture absorption. Acetate content of films as function of water activity and storage time is assessed from the band accounting for the electrostatic interaction of protonated amine groups (-NH3+) in chitosan and carboxylate ions (-COO-) of acetic acid. FTIR spectroscopy results are correlated with the equilibrium properties determined from moisture sorption isotherm. The simple method presented may help to produce chitosan acetate films with tuned protonation degree that are relevant in many applications such as biomedical, food, cosmetics, and pharmaceutical. Keywords: Chitosan acetate; FTIR spectroscopy; neutralization; water activity.
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1. Introduction Chitin is a polysaccharide formed by β-(14) N-acetyl-D-glucosamine units widely distributed in nature [1]. It is insoluble in most of common solvents but its deacetylated derivative, chitosan, is soluble in aqueous solutions of several organic acids [2,3]. Because chitosan combines properties of no toxicity, antimicrobial activity, biocompatibility, biodegradability, and renewability it has found applications in diverse fields like biomedicine, food industry, agriculture, biotechnology, cosmetics, and pharmaceutics [1]. Among the organic acids dissolving chitosan, an aqueous solution of acetic acid is the preferred one because its low toxicity and low dissociation constant help to produce chitosan acetate films. These chitosan-based films are particularly promising for applications in food industry due to suitable mechanical and barrier properties [4,5], biodegradability [6], and antimicrobial activity [7]. A comprehensive review of potential applications of chitosan-based materials in food industry can be found in recent literature [8-10]. Furthermore, applications of chitosan acetate in biomedicine have been reported to prevent infections in wounds [11] and skin burns [12]. Chitosan acetate films are characterized by the electrostatic interaction of protonated amine groups (-NH3+) and carboxylate ions (-COO-) [2]. Often, chitosan acetate films are subject to a neutralization process whereby -NH3+ groups are transformed to free amine groups (-NH2). Effects of neutralization on the physicochemical properties and cellular compatibility of chitosan films [13,14], antibacterial [15], and drug-release properties [16] have been reported. Because many applications of chitosan stem on its cationic character in acid media, the stability of the chitosan acetate salts during storage has been explored by different authors. In fully deacetylated chitosan acetate films, Demarger-Andre and Domard [2] showed that the -NH3+ -NH2 transformation progressively decreased over time. By comparing drying in vacuum and open-air conditions, those authors identified the importance of the water content on the transformation. In the case of chitosan acetate obtained from tendon chitosan, the water removing action of acid occurred after one month at 100% relative humidity [17]. Later, the crystalline transformation from a hydrated chitosan salt form to an anhydrous polymorph of chitosan was determined [18]. In another study, it was reported that storage for three weeks reduced the differences between neutralized and acetate films regardless of storage temperature 4 or 24 ºC [19]. More recently, solid state nuclear 3
magnetic resonance studies showed that chitosan acetate films stored for one month retained more acetate ions when the relative humidity was 32% than when it was 75% [20]. The exploratory studies referred in the last paragraph are indicative of the influence of the storage ambiance conditions on the stability of chitosan acetate films. Although in some cases the relative humidity during storage was not specified, apparently the removal of acetate ions is more efficient at larger values of relative humidity. However, the equilibrium form of the films, acetate or free amino chitosan, will depend on the water content in the film and the ambiance conditions. In this work, we present a systematic study on the effect of relative humidity on chitosan acetate films during storage at 30 °C. For the study, we combine Fourier transform infrared (FTIR) spectroscopy and the water sorption isotherm obtained by a gravimetric method. A moisture sorption isotherm describes the relationship between the moisture uptake in the material and the relative humidity of the air with which the material is in equilibrium at a constant temperature. The knowledge of moisture sorption characteristics of film materials would allow correctly specifying the conditions of storage and packaging, and understanding the physicochemical changes involved in product making process [21,22]. We use FTIR spectroscopy because it is a simple and highly sensitive technique to distinguish vibrational bands due to the electrostatic interaction between -NH3+ groups and carboxylate ions (-COO-) in chitosan acetate from those vibrations of free amine groups. FTIR results are correlated with the equilibrium properties determined from water sorption isotherm. The conditions for a soft and predictable neutralization of chitosan acetate films are discussed.
2. Materials and methods 2.1 Casting of chitosan acetate films Chitosan from crab shells of medium molecular weight (Sigma–Aldrich), acid acetic, and sodium hydroxide (J. T. Baker) were used as received. The films were cast by dissolving 1.5% w/w of chitosan in 1% w/w aqueous acetic acid solution with continuous stirring for 24 h to promote dissolution. This solution was poured into a Petri dish and allowed to evaporate at 60 °C for 16 h to obtain the chitosan acetate films. In these films, the amino side group is protonated (NH3+) and strongly interacts with (–COO-) groups of acetic acid ions leading to chitosan acetate. Small pieces (11 cm2) of the films were 4
prepared for the measurements. NaOH-neutralization was accomplished by soaking an asprepared chitosan acetate film in a 0.1M NaOH solution for 3 min and washed with deionized water until neutral pH was reached. 2.2 Water sorption isotherm The sorption isotherm of water was determined using a static equilibrium method. First, chitosan acetate films (0.6 g) were placed in vacuum desiccators containing P2O5 for two weeks to achieve near to zero moisture. Next, the samples were distributed in separate desiccators containing saturated salt slurries in the range of water activity (aw) from 0.11 to 0.85 calculated according to [23]. The samples were held at 30 °C for 7-14 days until equilibrium was reached (difference of two consecutive weights less than 0.001 g). Water sorption was analyzed using the Guggenheim-Anderson-De Boer (GAB) equation [24]:
M
M 0Ckaw , 1 kaw 1 kaw Ckaw
(1)
where M is the moisture content of the sample on dry basis; M0 is the monolayer moisture content; C is the Guggenheim constant, given by C= c’exp (hm-hn)/RT where c’ is a constant; hm is the heat of sorption of the first layer; hn is the heat of sorption of the multilayer; R is the gas constant; T is the absolute temperature; and k=k’exp((h1-hn)/RT) is a constant correcting properties of multilayer molecules with respect to bulk liquid, where k’ is the equation constant and h1 is the heat of condensation of pure water. The values of the parameters in GAB model (M0, C and k) were determined by non-linear regression fitting of Eq. (1) to the experimental data using the Kaleidagraph 4.0 package (Synergy Software, Perkiomen, USA). The goodness of the fit was evaluated using the average of the relative percentage difference between experimental and predicted values of the moisture content or mean relative deviation modulus (P) defined as: P %
100 N Mei Mci Me , N i 1 i
(2)
where Mei is the experimental moisture content of the i-th observation; Mci is the predicted moisture content; and N is the number of observations. It is generally accepted that a good fit is obtained when P < 10% [25]. 2.3 Evolution of chitosan acetate films during storage and characterization techniques
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To investigate the effect of water activity and storage time on chitosan acetate films, transmission FTIR spectroscopy was applied to study the changes in the bulk of the films as a function of time over a period of eighteen days. Thus, dedicated films of the appropriate thickness were prepared to avoid signal saturation. Films thicknesses of about 12.3 m were achieved by pouring 2.5 ml in Petri dishes 6 cm in diameter. The films were placed in desiccators containing saturated salt slurries at 30 °C as referred in section 2.2. FTIR measurements were performed using a Spectrum GX system (Perkin–Elmer) in the range 4000–400 cm-1 with a resolution of 4 cm-1 and averaged over 24 scans. For chitosan in powdered form the FTIR absorption spectrum was collected with the KBr method. The film thickness was determined by scanning electron microscopy (Phillips/XL30-ESEM) from cross sectioned films coated with a 4 nm thick evaporated gold film. X-ray diffraction patterns were acquired with a Rigaku/Dmax2100 diffractometer equipped with Cu radiation (1.5406 Å) at a scanning rate of 1.0 degree/min.
3. Results and discussion 3.1 Structural and molecular characterization of chitosan and chitosan acetate Fig. 1 shows the FTIR absorption spectra of three samples: chitosan powder, typical chitosan acetate film, and NaOH-neutralized chitosan film. The spectral range between 2500 and 1750 cm-1 has been omitted because chitosan does not present vibrational bands in that range. The spectrum of chitosan powder used for film preparation shows the characteristics bands of chitosan reported by other authors [1,26,27]. Namely, an asymmetric broadband due to OH vibrations extending from 3670 to 2600 cm-1; bands of NH stretching modes at 3370 and 3300 cm-1 as well as those of CH groups at 2930 and 2880 cm-1 appear superimposed to the OH broadband; amide I at 1656 cm-1, -NH2 bending at 1580 cm-1; bands of CHn groups at 1420 (CH2 deformation), 1380 (-CH3 symmetric deformation), and 1320 cm-1 (amide III and CH2 wagging); absorption bands in the range 1260-800 cm-1 belong to the glycosidic ring, in particular, the band at 1156 cm-1 corresponds to the glycosidic linkage. On the other hand, the spectrum of chitosan acetate is distinguished by the shift of the amide I band to 1640 cm-1 and two strong bands at 1560 and 1410 cm-1 due to vibrations of the carboxylate ion –COO- [17,19], the former often is identified with the protonated amine group of chitosan in acid media. The small but sharp 6
band at 655 cm-1 corresponds to the vibrational mode OCO(δ) of acetic acid [28]. For clarity, the bands belonging to characteristic vibrations of acetate groups are identified with arrows. Finally, the spectrum of the NaOH-neutralized chitosan film shows the same bands as chitosan powder evidencing the elimination of acetate groups. The degree of acetylation was calculated from the absorbance ratio of bands at 1320 and 1420 cm-1 [26] obtaining 22.6% for chitosan powder and 21.6% for the neutralized film. Figure 1 Fig. 2 shows the XRD data of chitosan powder and a chitosan acetate film. As can be noticed chitosan shows two diffraction peaks at 2=9.94 and 20.01º corresponding to interplanar distances of 8.891 and 4.436 Å, respectively. The latter values agree with results reported by other authors [29]. On the other hand, the XRD data of as-prepared chitosan acetate film clearly reveal an amorphous structure. Figure 2
3.2 Time evolution and relative humidity effects on FTIR spectra of chitosan acetate films Fig. 3 shows the infrared absorption spectra of as-prepared chitosan acetate films and after eighteen days of storage at 30 °C in ambiances of controlled water activities between 0.11 and 0.85. To minimize swelling or shrinking effects in the films due to sorption or desorption of molecules, the spectra were normalized respect to the most intense band at 1080 cm-1 of the glycosidic ring. That normalization assumes that chitosan chains do not degrade during the experiment. Indeed, bands in the range of 1200 to 850 cm-1 are practically unaffected with the exemption of the sample stored at aw=0.44 where small variations are observed, which are probably due to conformational changes. As can be noticed in the infrared spectra of Fig. 3, the most notorious changes are observed in the range of 1700 to 1400 cm-1 which corresponds to the superposition of vibrations of amide I, protonated amine groups (-NH3+), carboxylate ions (-COO-), and free amine groups (-NH2). Also, evident changes can be seen in the stretching bands of OH groups in the range of
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3700 to 2500 cm-1 and more subtle changes appear between 850 and 400 cm-1. The details are discussed below. In terms of relative humidity, the changes observed in the broadband of OHstretching vibrations after eighteen days can be classified in three groups: i) low values, 0.11≤aw≤0.32, the band becomes narrower and less strong indicating the loss of water molecules; ii) intermediate values, aw=0.44 and 0.55, the band becomes narrower and stronger; iii) higher values, 0.64≤aw≤0.85, the band increases in strength due to the gain of water molecules. We should remark that the as-prepared films contain certain amount of water contributing to the broadening of the OH band. Thus, depending on the relative humidity of ambiance, equilibrium is attained with the exchange of solvent molecules and the rearrangement of bonds in the films. The narrowing of the OH band from the smallwavenumber side for aw=0.44 and 0.55 in Fig. 3, indicates that some weak OH bonds (of low frequency) increase in strength giving rise to a more well-defined value of the vibrational frequency in the 3430-3370 cm-1 range. Thus, the narrowing of OH band can be associated to increasing hydrogen bonding between chitosan chains. As was mentioned before in this section, the most notorious effect of relative humidity after the eighteen days of observation occurs in the range of 1700 to 1400 cm-1. That is, the three bands at 1640, 1560, and 1410 cm-1 decrease in intensity at different extent depending on water activity. That decrease is small for the lowest (aw=0.11) and the higher (aw=0.64, 0.75, and 0.85) values of relative humidity. In fact, the bands associated to -COO- ions remain almost intact indicating the equilibrium of chitosan acetate with the ambient at these conditions. For aw=0.22 and 0.32 the effect is rather moderate. However, for aw=0.44 and 0.55 the bands at 1560 and 1410 cm-1 characteristics of –COO- groups and the protonated amino group (NH3+) disappear. Changes in intensity of the characteristic bands of chitosan acetate are accompanied by shifts in their central frequency. After the eighteen days of observation the amide I band shifts from 1640 to 1656 cm-1, whereas the bands initially at 1560 and 1410 cm-1 shift to 1580 and 1420 cm-1, respectively. Furthermore, the band of the OCO(δ) mode of acetic acid located at 655 cm-1 disappears in films stored at aw=0.44 and 0.55 and remains almost unaffected in samples at the lowest and the higher values of relative humidity investigated in this work. It is noteworthy that the FTIR spectra of films at aw=0.44 and 0.55 after eighteen days of storage in Fig. 3 have 8
all the characteristic features of the spectrum belonging to the NaOH-neutralized film in Fig. 1. Therefore, storing chitosan acetate films in an ambiance at the appropriate relative humidity can be used as a soft neutralization process avoiding the use of any hydroxide. The soft neutralization produces chitosan films stiffer than chitosan acetate films and less brittle than the NaOH-neutralized ones. The study of thermal, mechanical, polycationic, and other functional properties of chitosan films softly neutralized is part of our ongoing research and will be discussed elsewhere. Figure 3 The time evolution of the absorbance band at 1560 cm-1 that identifies the carboxylate ion of chitosan acetate films as function of time and water activity is shown in Fig. 4. The data were normalized respect to the band at 1080 cm-1 of glycosidic bond. For comparison, the absorbance of the -NH2 bending at 1580 cm-1 of chitosan corresponding to the NaOH-neutralized film is located with the dashed line. In is clear that storing chitosan acetate films at aw=0.44 and 0.55 the -NH3+ -NH2 transformation is accomplished. Particularly, storing chitosan acetate films for one day at aw=0.55 produces a 77% reduction of acetate ions and after five days the content is only 7%. In fact, Demarger-Andre and Domard [2] obtained similar results for fully deacetylated chitosan exposed to an open-air ambiance of unspecified relative humidity. In that work, the authors pointed out that such transformation can be a drawback for applications of chitosan as a biomaterial where the secondary reactions of remaining acetic acid in chitosan acetate can be important. However, the soft-neutralized chitosan films may be used in applications as an inert resin [18]. On the other hand, the films retain the acetate form when they are stored at low values of aw (0.11). Although the acetate form of chitosan is retained at high values of aw0.64, fungi growth and the development of Millard reactions degrading chitosan are very likely. In summary, depending on storage parameters of relative humidity and time, different protonation degrees of chitosan acetate can be achieved which opens new routes to investigate possible applications. Figure 4 9
3.3 Moisture sorption at equilibrium and neutralization of chitosan acetate films Fig. 5 shows the water sorption isotherm at 30 °C of chitosan acetate films. As can be seen, the GAB equation describes adequately the experimental data over the whole range of water activity investigated. The GAB parameters determined from non-linear regression analysis that describe water sorption were: C =4.01, k =1.01, and Mo=4.64 g water/100 g dry solid (0.0464 g/gds). The values of the statistical parameters measuring the goodness of the fitting were r2 =0.99 and P =6.3%, which indicates a good fit [25]. As can be noticed in Fig. 5, the moisture content in the monolayer of acetate films Mo=0.0464 g/gds nearly corresponds to aw=0.33. According to Rahman and Labuza [30] the value of Mo represents the amount of water bound to specific polar sites in the dehydrated material and at that water content a storage product should be stable against degradation reactions. On the other hand, as the Guggenheim constant C is a measure of the interaction between water molecules and the sorption sites, whereas the value of k discriminates between water molecules in a structured multilayer or bulk water, the combined analysis of these parameters gives insight on the mechanism of water sorption in similar systems [24]. For example, the reported sorption isotherm at 30 ºC for chitosan powder measured in desorption were described with GAB parameters C=12.56, k=0.93, and Mo=0.191 kg/kgds [31]. A comparison of the Guggenheim constant C shows that water in acetate chitosan films (this work) has a less strong bonded monolayer than in chitosan powder. Additionally, as in both cases k1 the monolayer has properties similar to bulk water. Furthermore, reported GAB parameters for the moisture sorption of NaOH-neutralized chitosan films at 20 ºC were C=15.17, k=0.879, and Mo=0.06 g/gds [32]. The latter parameters indicate a stronger binding and a more structured multilayer of water molecules in chitosan films (at 20 °C) than in the acetate chitosan films investigated in the present work (at 30 °C). Figure 5 To rationalize the interpretation of both FTIR spectroscopy and water sorption results, we stress that the data in Fig. 5 correspond to the moisture equilibrium content in 10
the films for a dry basis. Whereas, the FTIR spectra were acquired on as-prepared films which corresponds to a wet basis (no drying was applied). According to the interpretation of FTIR data in Sec. 3.2, at low values of relative humidity, 0.11≤aw≤0.32, dehydration occurs accompanied with the partial loss of acetate groups, that is, the hydrated films loss weight to establish the equilibrium implying that the initial moisture content of the asprepared films was higher than 4.64%, i.e. the value of Mo. For water activities, aw=0.44 and 0.55 just above Mo, the transformation of chitosan acetate to its neutralized form was observed. On the other hand, to attain equilibrium at the higher values of relative humidity (aw0.64, 11.6% moisture content), the films should increase their water content inhibiting the lost of water and acetate ions. Therefore, depending on the processing parameters used to obtain the films, the relative humidity needed for spontaneous neutralization would be different. In fact, Madeleine-Perdrillat et al. [20] reported that chitosan acetate films stored at 20 ºC for one month retained acetate ions when the relative humidity was 32% but the content of acetate ions decreases by 73.2% at relative humidity 75%. This means that for their processing conditions, ranges i) and ii) of water activity defined in 3.2 were shifted to higher values of aw.
4. Conclusions The time evolution of chitosan acetate films at different water activities was assessed by FTIR spectroscopy. Spontaneous neutralization of chitosan acetate films takes place at aw=0.44 and 0.55 in a few days. This represents an alternative soft method for neutralization without damage of the films. The specific values of aw for spontaneous neutralization are just above the moisture content of the monolayer-bulk transition. Furthermore, for applications using chitosan acetate films, they remain in their original form stored at low water activity 0.11. Intermediate protonation degrees in chitosan acetate films can be tuned adjusting storage time and relative humidity.
Acknowledgments The authors acknowledge the technical assistance of M. A. Hernández-Landaverde and E. Urbina-Álvarez for XRD and SEM data acquisition.
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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Figure Captions Fig. 1. Absorbance infrared spectra of (a) chitosan powder, (b) as-prepared chitosan acetate film, and (c) chitosan film NaOH-neutralized.
Fig. 2. X-ray diffraction pattern of (a) chitosan powder and (b) as-prepared chitosan acetate film.
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Fig. 3. Absorbance infrared spectra of as-prepared chitosan acetate films and after eighteen days as function of water activity (aw). The spectra were normalized respect to the band at 1080 cm-1 of the glycosidic ring.
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Fig. 4. Normalized absorbance of the carboxylate band at 1560 cm-1 respect to the band of glycosidic ring (1080 cm-1) of chitosan acetate films as function of time and water activity (aw). Solid lines are a guide to the eye. The corresponding value of NaOH-neutralized chitosan is located with the dashed line.
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Fig. 5. Moisture sorption isotherm of chitosan acetate films at 30 °C. Experimental data (symbols) and fitting with GAB equation (solid line).
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S0924-2031(17)30233-3 https://doi.org/10.1016/j.vibspec.2017.10.005 VIBSPE 2750
To appear in:
VIBSPE
Received date: Revised date: Accepted date:
16-8-2017 6-10-2017 27-10-2017
Please cite this article as: Reina Araceli Mauricio-S´anchez, Ricardo Salazar, J.Gabriel Luna-B´arcenas, Arturo Mendoza-Galv´an, FTIR spectroscopy studies on the spontaneous neutralization of chitosan acetate films by moisture conditioning, Vibrational Spectroscopy https://doi.org/10.1016/j.vibspec.2017.10.005 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.
FTIR spectroscopy studies on the spontaneous neutralization of chitosan acetate films by moisture conditioning Reina Araceli Mauricio-Sáncheza, Ricardo Salazarb, J. Gabriel Luna-Bárcenasa, Arturo Mendoza-Galvána,* a
Cinvestav-Unidad Querétaro, Libramiento Norponiente 2000, Querétaro,76230 Mexico
b
CONACyT-Universidad Autónoma de Guerrero, Av. Javier Méndez Aponte No. 1, Fracc.
Servidor Agrario, 39070 Chilpancingo de los Bravo, Guerrero, Mexico Authors e-mail addresses: Reina Araceli Mauricio-Sánchez: [email protected] Ricardo Salazar: [email protected] J. Gabriel Luna-Bárcenas: [email protected] Arturo Mendoza-Galván: [email protected] * Corresponding author. Tel.: +52-442-211-9922; fax: +52-442-211-9938 E-mail address: [email protected] (Arturo Mendoza-Galván)
1
Highlights
A reliable method for soft neutralization of chitosan acetate films is presented.
Spontaneous neutralization is controlled by water activity.
Water activity of 0.53 neutralizes the films after only five days of storage.
Chitosan films in the acetate form can be stored at low water activity.
Protonation degree can be tuned by adjusting the ambiance relative humidity.
Abstract The influence of moisture ambiance on the spontaneous neutralization of amorphous chitosan acetate films during storage at 30 ˚C and water activities (aw) in the range of 0.11 to 0.84 is studied by Fourier transform infrared (FTIR) spectroscopy. Three ranges of relative humidity affecting the chitosan acetate films are identified: i) 0.11≤aw≤0.32, produces dehydration and partial neutralization; ii) aw=0.44 and 0.55, spontaneous neutralization and rearrangement of OH bonds; iii) 0.64≤aw≤0.85, prevalence of films in the acetate form and moisture absorption. Acetate content of films as function of water activity and storage time is assessed from the band accounting for the electrostatic interaction of protonated amine groups (-NH3+) in chitosan and carboxylate ions (-COO-) of acetic acid. FTIR spectroscopy results are correlated with the equilibrium properties determined from moisture sorption isotherm. The simple method presented may help to produce chitosan acetate films with tuned protonation degree that are relevant in many applications such as biomedical, food, cosmetics, and pharmaceutical. Keywords: Chitosan acetate; FTIR spectroscopy; neutralization; water activity.
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1. Introduction Chitin is a polysaccharide formed by β-(14) N-acetyl-D-glucosamine units widely distributed in nature [1]. It is insoluble in most of common solvents but its deacetylated derivative, chitosan, is soluble in aqueous solutions of several organic acids [2,3]. Because chitosan combines properties of no toxicity, antimicrobial activity, biocompatibility, biodegradability, and renewability it has found applications in diverse fields like biomedicine, food industry, agriculture, biotechnology, cosmetics, and pharmaceutics [1]. Among the organic acids dissolving chitosan, an aqueous solution of acetic acid is the preferred one because its low toxicity and low dissociation constant help to produce chitosan acetate films. These chitosan-based films are particularly promising for applications in food industry due to suitable mechanical and barrier properties [4,5], biodegradability [6], and antimicrobial activity [7]. A comprehensive review of potential applications of chitosan-based materials in food industry can be found in recent literature [8-10]. Furthermore, applications of chitosan acetate in biomedicine have been reported to prevent infections in wounds [11] and skin burns [12]. Chitosan acetate films are characterized by the electrostatic interaction of protonated amine groups (-NH3+) and carboxylate ions (-COO-) [2]. Often, chitosan acetate films are subject to a neutralization process whereby -NH3+ groups are transformed to free amine groups (-NH2). Effects of neutralization on the physicochemical properties and cellular compatibility of chitosan films [13,14], antibacterial [15], and drug-release properties [16] have been reported. Because many applications of chitosan stem on its cationic character in acid media, the stability of the chitosan acetate salts during storage has been explored by different authors. In fully deacetylated chitosan acetate films, Demarger-Andre and Domard [2] showed that the -NH3+ -NH2 transformation progressively decreased over time. By comparing drying in vacuum and open-air conditions, those authors identified the importance of the water content on the transformation. In the case of chitosan acetate obtained from tendon chitosan, the water removing action of acid occurred after one month at 100% relative humidity [17]. Later, the crystalline transformation from a hydrated chitosan salt form to an anhydrous polymorph of chitosan was determined [18]. In another study, it was reported that storage for three weeks reduced the differences between neutralized and acetate films regardless of storage temperature 4 or 24 ºC [19]. More recently, solid state nuclear 3
magnetic resonance studies showed that chitosan acetate films stored for one month retained more acetate ions when the relative humidity was 32% than when it was 75% [20]. The exploratory studies referred in the last paragraph are indicative of the influence of the storage ambiance conditions on the stability of chitosan acetate films. Although in some cases the relative humidity during storage was not specified, apparently the removal of acetate ions is more efficient at larger values of relative humidity. However, the equilibrium form of the films, acetate or free amino chitosan, will depend on the water content in the film and the ambiance conditions. In this work, we present a systematic study on the effect of relative humidity on chitosan acetate films during storage at 30 °C. For the study, we combine Fourier transform infrared (FTIR) spectroscopy and the water sorption isotherm obtained by a gravimetric method. A moisture sorption isotherm describes the relationship between the moisture uptake in the material and the relative humidity of the air with which the material is in equilibrium at a constant temperature. The knowledge of moisture sorption characteristics of film materials would allow correctly specifying the conditions of storage and packaging, and understanding the physicochemical changes involved in product making process [21,22]. We use FTIR spectroscopy because it is a simple and highly sensitive technique to distinguish vibrational bands due to the electrostatic interaction between -NH3+ groups and carboxylate ions (-COO-) in chitosan acetate from those vibrations of free amine groups. FTIR results are correlated with the equilibrium properties determined from water sorption isotherm. The conditions for a soft and predictable neutralization of chitosan acetate films are discussed.
2. Materials and methods 2.1 Casting of chitosan acetate films Chitosan from crab shells of medium molecular weight (Sigma–Aldrich), acid acetic, and sodium hydroxide (J. T. Baker) were used as received. The films were cast by dissolving 1.5% w/w of chitosan in 1% w/w aqueous acetic acid solution with continuous stirring for 24 h to promote dissolution. This solution was poured into a Petri dish and allowed to evaporate at 60 °C for 16 h to obtain the chitosan acetate films. In these films, the amino side group is protonated (NH3+) and strongly interacts with (–COO-) groups of acetic acid ions leading to chitosan acetate. Small pieces (11 cm2) of the films were 4
prepared for the measurements. NaOH-neutralization was accomplished by soaking an asprepared chitosan acetate film in a 0.1M NaOH solution for 3 min and washed with deionized water until neutral pH was reached. 2.2 Water sorption isotherm The sorption isotherm of water was determined using a static equilibrium method. First, chitosan acetate films (0.6 g) were placed in vacuum desiccators containing P2O5 for two weeks to achieve near to zero moisture. Next, the samples were distributed in separate desiccators containing saturated salt slurries in the range of water activity (aw) from 0.11 to 0.85 calculated according to [23]. The samples were held at 30 °C for 7-14 days until equilibrium was reached (difference of two consecutive weights less than 0.001 g). Water sorption was analyzed using the Guggenheim-Anderson-De Boer (GAB) equation [24]:
M
M 0Ckaw , 1 kaw 1 kaw Ckaw
(1)
where M is the moisture content of the sample on dry basis; M0 is the monolayer moisture content; C is the Guggenheim constant, given by C= c’exp (hm-hn)/RT where c’ is a constant; hm is the heat of sorption of the first layer; hn is the heat of sorption of the multilayer; R is the gas constant; T is the absolute temperature; and k=k’exp((h1-hn)/RT) is a constant correcting properties of multilayer molecules with respect to bulk liquid, where k’ is the equation constant and h1 is the heat of condensation of pure water. The values of the parameters in GAB model (M0, C and k) were determined by non-linear regression fitting of Eq. (1) to the experimental data using the Kaleidagraph 4.0 package (Synergy Software, Perkiomen, USA). The goodness of the fit was evaluated using the average of the relative percentage difference between experimental and predicted values of the moisture content or mean relative deviation modulus (P) defined as: P %
100 N Mei Mci Me , N i 1 i
(2)
where Mei is the experimental moisture content of the i-th observation; Mci is the predicted moisture content; and N is the number of observations. It is generally accepted that a good fit is obtained when P < 10% [25]. 2.3 Evolution of chitosan acetate films during storage and characterization techniques
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To investigate the effect of water activity and storage time on chitosan acetate films, transmission FTIR spectroscopy was applied to study the changes in the bulk of the films as a function of time over a period of eighteen days. Thus, dedicated films of the appropriate thickness were prepared to avoid signal saturation. Films thicknesses of about 12.3 m were achieved by pouring 2.5 ml in Petri dishes 6 cm in diameter. The films were placed in desiccators containing saturated salt slurries at 30 °C as referred in section 2.2. FTIR measurements were performed using a Spectrum GX system (Perkin–Elmer) in the range 4000–400 cm-1 with a resolution of 4 cm-1 and averaged over 24 scans. For chitosan in powdered form the FTIR absorption spectrum was collected with the KBr method. The film thickness was determined by scanning electron microscopy (Phillips/XL30-ESEM) from cross sectioned films coated with a 4 nm thick evaporated gold film. X-ray diffraction patterns were acquired with a Rigaku/Dmax2100 diffractometer equipped with Cu radiation (1.5406 Å) at a scanning rate of 1.0 degree/min.
3. Results and discussion 3.1 Structural and molecular characterization of chitosan and chitosan acetate Fig. 1 shows the FTIR absorption spectra of three samples: chitosan powder, typical chitosan acetate film, and NaOH-neutralized chitosan film. The spectral range between 2500 and 1750 cm-1 has been omitted because chitosan does not present vibrational bands in that range. The spectrum of chitosan powder used for film preparation shows the characteristics bands of chitosan reported by other authors [1,26,27]. Namely, an asymmetric broadband due to OH vibrations extending from 3670 to 2600 cm-1; bands of NH stretching modes at 3370 and 3300 cm-1 as well as those of CH groups at 2930 and 2880 cm-1 appear superimposed to the OH broadband; amide I at 1656 cm-1, -NH2 bending at 1580 cm-1; bands of CHn groups at 1420 (CH2 deformation), 1380 (-CH3 symmetric deformation), and 1320 cm-1 (amide III and CH2 wagging); absorption bands in the range 1260-800 cm-1 belong to the glycosidic ring, in particular, the band at 1156 cm-1 corresponds to the glycosidic linkage. On the other hand, the spectrum of chitosan acetate is distinguished by the shift of the amide I band to 1640 cm-1 and two strong bands at 1560 and 1410 cm-1 due to vibrations of the carboxylate ion –COO- [17,19], the former often is identified with the protonated amine group of chitosan in acid media. The small but sharp 6
band at 655 cm-1 corresponds to the vibrational mode OCO(δ) of acetic acid [28]. For clarity, the bands belonging to characteristic vibrations of acetate groups are identified with arrows. Finally, the spectrum of the NaOH-neutralized chitosan film shows the same bands as chitosan powder evidencing the elimination of acetate groups. The degree of acetylation was calculated from the absorbance ratio of bands at 1320 and 1420 cm-1 [26] obtaining 22.6% for chitosan powder and 21.6% for the neutralized film. Figure 1 Fig. 2 shows the XRD data of chitosan powder and a chitosan acetate film. As can be noticed chitosan shows two diffraction peaks at 2=9.94 and 20.01º corresponding to interplanar distances of 8.891 and 4.436 Å, respectively. The latter values agree with results reported by other authors [29]. On the other hand, the XRD data of as-prepared chitosan acetate film clearly reveal an amorphous structure. Figure 2
3.2 Time evolution and relative humidity effects on FTIR spectra of chitosan acetate films Fig. 3 shows the infrared absorption spectra of as-prepared chitosan acetate films and after eighteen days of storage at 30 °C in ambiances of controlled water activities between 0.11 and 0.85. To minimize swelling or shrinking effects in the films due to sorption or desorption of molecules, the spectra were normalized respect to the most intense band at 1080 cm-1 of the glycosidic ring. That normalization assumes that chitosan chains do not degrade during the experiment. Indeed, bands in the range of 1200 to 850 cm-1 are practically unaffected with the exemption of the sample stored at aw=0.44 where small variations are observed, which are probably due to conformational changes. As can be noticed in the infrared spectra of Fig. 3, the most notorious changes are observed in the range of 1700 to 1400 cm-1 which corresponds to the superposition of vibrations of amide I, protonated amine groups (-NH3+), carboxylate ions (-COO-), and free amine groups (-NH2). Also, evident changes can be seen in the stretching bands of OH groups in the range of
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3700 to 2500 cm-1 and more subtle changes appear between 850 and 400 cm-1. The details are discussed below. In terms of relative humidity, the changes observed in the broadband of OHstretching vibrations after eighteen days can be classified in three groups: i) low values, 0.11≤aw≤0.32, the band becomes narrower and less strong indicating the loss of water molecules; ii) intermediate values, aw=0.44 and 0.55, the band becomes narrower and stronger; iii) higher values, 0.64≤aw≤0.85, the band increases in strength due to the gain of water molecules. We should remark that the as-prepared films contain certain amount of water contributing to the broadening of the OH band. Thus, depending on the relative humidity of ambiance, equilibrium is attained with the exchange of solvent molecules and the rearrangement of bonds in the films. The narrowing of the OH band from the smallwavenumber side for aw=0.44 and 0.55 in Fig. 3, indicates that some weak OH bonds (of low frequency) increase in strength giving rise to a more well-defined value of the vibrational frequency in the 3430-3370 cm-1 range. Thus, the narrowing of OH band can be associated to increasing hydrogen bonding between chitosan chains. As was mentioned before in this section, the most notorious effect of relative humidity after the eighteen days of observation occurs in the range of 1700 to 1400 cm-1. That is, the three bands at 1640, 1560, and 1410 cm-1 decrease in intensity at different extent depending on water activity. That decrease is small for the lowest (aw=0.11) and the higher (aw=0.64, 0.75, and 0.85) values of relative humidity. In fact, the bands associated to -COO- ions remain almost intact indicating the equilibrium of chitosan acetate with the ambient at these conditions. For aw=0.22 and 0.32 the effect is rather moderate. However, for aw=0.44 and 0.55 the bands at 1560 and 1410 cm-1 characteristics of –COO- groups and the protonated amino group (NH3+) disappear. Changes in intensity of the characteristic bands of chitosan acetate are accompanied by shifts in their central frequency. After the eighteen days of observation the amide I band shifts from 1640 to 1656 cm-1, whereas the bands initially at 1560 and 1410 cm-1 shift to 1580 and 1420 cm-1, respectively. Furthermore, the band of the OCO(δ) mode of acetic acid located at 655 cm-1 disappears in films stored at aw=0.44 and 0.55 and remains almost unaffected in samples at the lowest and the higher values of relative humidity investigated in this work. It is noteworthy that the FTIR spectra of films at aw=0.44 and 0.55 after eighteen days of storage in Fig. 3 have 8
all the characteristic features of the spectrum belonging to the NaOH-neutralized film in Fig. 1. Therefore, storing chitosan acetate films in an ambiance at the appropriate relative humidity can be used as a soft neutralization process avoiding the use of any hydroxide. The soft neutralization produces chitosan films stiffer than chitosan acetate films and less brittle than the NaOH-neutralized ones. The study of thermal, mechanical, polycationic, and other functional properties of chitosan films softly neutralized is part of our ongoing research and will be discussed elsewhere. Figure 3 The time evolution of the absorbance band at 1560 cm-1 that identifies the carboxylate ion of chitosan acetate films as function of time and water activity is shown in Fig. 4. The data were normalized respect to the band at 1080 cm-1 of glycosidic bond. For comparison, the absorbance of the -NH2 bending at 1580 cm-1 of chitosan corresponding to the NaOH-neutralized film is located with the dashed line. In is clear that storing chitosan acetate films at aw=0.44 and 0.55 the -NH3+ -NH2 transformation is accomplished. Particularly, storing chitosan acetate films for one day at aw=0.55 produces a 77% reduction of acetate ions and after five days the content is only 7%. In fact, Demarger-Andre and Domard [2] obtained similar results for fully deacetylated chitosan exposed to an open-air ambiance of unspecified relative humidity. In that work, the authors pointed out that such transformation can be a drawback for applications of chitosan as a biomaterial where the secondary reactions of remaining acetic acid in chitosan acetate can be important. However, the soft-neutralized chitosan films may be used in applications as an inert resin [18]. On the other hand, the films retain the acetate form when they are stored at low values of aw (0.11). Although the acetate form of chitosan is retained at high values of aw0.64, fungi growth and the development of Millard reactions degrading chitosan are very likely. In summary, depending on storage parameters of relative humidity and time, different protonation degrees of chitosan acetate can be achieved which opens new routes to investigate possible applications. Figure 4 9
3.3 Moisture sorption at equilibrium and neutralization of chitosan acetate films Fig. 5 shows the water sorption isotherm at 30 °C of chitosan acetate films. As can be seen, the GAB equation describes adequately the experimental data over the whole range of water activity investigated. The GAB parameters determined from non-linear regression analysis that describe water sorption were: C =4.01, k =1.01, and Mo=4.64 g water/100 g dry solid (0.0464 g/gds). The values of the statistical parameters measuring the goodness of the fitting were r2 =0.99 and P =6.3%, which indicates a good fit [25]. As can be noticed in Fig. 5, the moisture content in the monolayer of acetate films Mo=0.0464 g/gds nearly corresponds to aw=0.33. According to Rahman and Labuza [30] the value of Mo represents the amount of water bound to specific polar sites in the dehydrated material and at that water content a storage product should be stable against degradation reactions. On the other hand, as the Guggenheim constant C is a measure of the interaction between water molecules and the sorption sites, whereas the value of k discriminates between water molecules in a structured multilayer or bulk water, the combined analysis of these parameters gives insight on the mechanism of water sorption in similar systems [24]. For example, the reported sorption isotherm at 30 ºC for chitosan powder measured in desorption were described with GAB parameters C=12.56, k=0.93, and Mo=0.191 kg/kgds [31]. A comparison of the Guggenheim constant C shows that water in acetate chitosan films (this work) has a less strong bonded monolayer than in chitosan powder. Additionally, as in both cases k1 the monolayer has properties similar to bulk water. Furthermore, reported GAB parameters for the moisture sorption of NaOH-neutralized chitosan films at 20 ºC were C=15.17, k=0.879, and Mo=0.06 g/gds [32]. The latter parameters indicate a stronger binding and a more structured multilayer of water molecules in chitosan films (at 20 °C) than in the acetate chitosan films investigated in the present work (at 30 °C). Figure 5 To rationalize the interpretation of both FTIR spectroscopy and water sorption results, we stress that the data in Fig. 5 correspond to the moisture equilibrium content in 10
the films for a dry basis. Whereas, the FTIR spectra were acquired on as-prepared films which corresponds to a wet basis (no drying was applied). According to the interpretation of FTIR data in Sec. 3.2, at low values of relative humidity, 0.11≤aw≤0.32, dehydration occurs accompanied with the partial loss of acetate groups, that is, the hydrated films loss weight to establish the equilibrium implying that the initial moisture content of the asprepared films was higher than 4.64%, i.e. the value of Mo. For water activities, aw=0.44 and 0.55 just above Mo, the transformation of chitosan acetate to its neutralized form was observed. On the other hand, to attain equilibrium at the higher values of relative humidity (aw0.64, 11.6% moisture content), the films should increase their water content inhibiting the lost of water and acetate ions. Therefore, depending on the processing parameters used to obtain the films, the relative humidity needed for spontaneous neutralization would be different. In fact, Madeleine-Perdrillat et al. [20] reported that chitosan acetate films stored at 20 ºC for one month retained acetate ions when the relative humidity was 32% but the content of acetate ions decreases by 73.2% at relative humidity 75%. This means that for their processing conditions, ranges i) and ii) of water activity defined in 3.2 were shifted to higher values of aw.
4. Conclusions The time evolution of chitosan acetate films at different water activities was assessed by FTIR spectroscopy. Spontaneous neutralization of chitosan acetate films takes place at aw=0.44 and 0.55 in a few days. This represents an alternative soft method for neutralization without damage of the films. The specific values of aw for spontaneous neutralization are just above the moisture content of the monolayer-bulk transition. Furthermore, for applications using chitosan acetate films, they remain in their original form stored at low water activity 0.11. Intermediate protonation degrees in chitosan acetate films can be tuned adjusting storage time and relative humidity.
Acknowledgments The authors acknowledge the technical assistance of M. A. Hernández-Landaverde and E. Urbina-Álvarez for XRD and SEM data acquisition.
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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Figure Captions Fig. 1. Absorbance infrared spectra of (a) chitosan powder, (b) as-prepared chitosan acetate film, and (c) chitosan film NaOH-neutralized.
Fig. 2. X-ray diffraction pattern of (a) chitosan powder and (b) as-prepared chitosan acetate film.
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Fig. 3. Absorbance infrared spectra of as-prepared chitosan acetate films and after eighteen days as function of water activity (aw). The spectra were normalized respect to the band at 1080 cm-1 of the glycosidic ring.
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Fig. 4. Normalized absorbance of the carboxylate band at 1560 cm-1 respect to the band of glycosidic ring (1080 cm-1) of chitosan acetate films as function of time and water activity (aw). Solid lines are a guide to the eye. The corresponding value of NaOH-neutralized chitosan is located with the dashed line.
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Fig. 5. Moisture sorption isotherm of chitosan acetate films at 30 °C. Experimental data (symbols) and fitting with GAB equation (solid line).
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