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Sorption Behavior of Crawfish ChitosanFilms as Affected by Chitosan ExtractionProcesses and Solvent TypesKKKKKANDASAMYANDASAMYANDASAMYANDASAMYANDASAMY N N N N NADARAJAHADARAJAHADARAJAHADARAJAHADARAJAH, , , , , WWWWWITITITITITOONOONOONOONOON P P P P PRINYRINYRINYRINYRINYAAAAAWIWWIWWIWWIWWIWAAAAATKULTKULTKULTKULTKUL, H, H, H, H, HONGONGONGONGONG K K K K KYYYYYOONOONOONOONOON N N N N NOOOOO, S, S, S, S, SUBRAMANIAMUBRAMANIAMUBRAMANIAMUBRAMANIAMUBRAMANIAM S S S S SAAAAATHIVELTHIVELTHIVELTHIVELTHIVEL, , , , , ANDANDANDANDAND Z Z Z Z ZHIMINHIMINHIMINHIMINHIMIN X X X X XUUUUU

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: E: E: E: E: Effects of chitosan extrffects of chitosan extrffects of chitosan extrffects of chitosan extrffects of chitosan extraction praction praction praction praction processes and solvocesses and solvocesses and solvocesses and solvocesses and solvent types on sorption behavior of unplasticizent types on sorption behavior of unplasticizent types on sorption behavior of unplasticizent types on sorption behavior of unplasticizent types on sorption behavior of unplasticized cred cred cred cred crawfishawfishawfishawfishawfishchitosan films were investigated. Four different chitosans prepared from crawfish shell were dissolved in 1% v/vchitosan films were investigated. Four different chitosans prepared from crawfish shell were dissolved in 1% v/vchitosan films were investigated. Four different chitosans prepared from crawfish shell were dissolved in 1% v/vchitosan films were investigated. Four different chitosans prepared from crawfish shell were dissolved in 1% v/vchitosan films were investigated. Four different chitosans prepared from crawfish shell were dissolved in 1% v/vacetic, formic, lactic, or malic acids at 1% w/v concentration. Chitosans dissolved in acetic or formic acid formedacetic, formic, lactic, or malic acids at 1% w/v concentration. Chitosans dissolved in acetic or formic acid formedacetic, formic, lactic, or malic acids at 1% w/v concentration. Chitosans dissolved in acetic or formic acid formedacetic, formic, lactic, or malic acids at 1% w/v concentration. Chitosans dissolved in acetic or formic acid formedacetic, formic, lactic, or malic acids at 1% w/v concentration. Chitosans dissolved in acetic or formic acid formedflexible and transparent films that are desirable for packaging applications. Chitosan acetate films maintainedflexible and transparent films that are desirable for packaging applications. Chitosan acetate films maintainedflexible and transparent films that are desirable for packaging applications. Chitosan acetate films maintainedflexible and transparent films that are desirable for packaging applications. Chitosan acetate films maintainedflexible and transparent films that are desirable for packaging applications. Chitosan acetate films maintainedlower moisture contents at any relative humidity level compared with chitosan formate films. The type of chitosanlower moisture contents at any relative humidity level compared with chitosan formate films. The type of chitosanlower moisture contents at any relative humidity level compared with chitosan formate films. The type of chitosanlower moisture contents at any relative humidity level compared with chitosan formate films. The type of chitosanlower moisture contents at any relative humidity level compared with chitosan formate films. The type of chitosansignificantly influenced the sorption isotherms of chitosan formate films but not chitosan acetate films. Thesignificantly influenced the sorption isotherms of chitosan formate films but not chitosan acetate films. Thesignificantly influenced the sorption isotherms of chitosan formate films but not chitosan acetate films. Thesignificantly influenced the sorption isotherms of chitosan formate films but not chitosan acetate films. Thesignificantly influenced the sorption isotherms of chitosan formate films but not chitosan acetate films. TheGGGGGuggenheim-Anderson-de Buggenheim-Anderson-de Buggenheim-Anderson-de Buggenheim-Anderson-de Buggenheim-Anderson-de Boeroeroeroeroer, O, O, O, O, Oswin, and Cswin, and Cswin, and Cswin, and Cswin, and Caurauraurauraurie models (ie models (ie models (ie models (ie models (RRRRR22222 = 0.98, 0.95, and 0.95, r = 0.98, 0.95, and 0.95, r = 0.98, 0.95, and 0.95, r = 0.98, 0.95, and 0.95, r = 0.98, 0.95, and 0.95, respectivespectivespectivespectivespectively) could be used toely) could be used toely) could be used toely) could be used toely) could be used topredict sorption behavior of crawfish chitosan acetate and formate films.predict sorption behavior of crawfish chitosan acetate and formate films.predict sorption behavior of crawfish chitosan acetate and formate films.predict sorption behavior of crawfish chitosan acetate and formate films.predict sorption behavior of crawfish chitosan acetate and formate films.

Keywords: chitosan extraction, chitosan film, crawfish, solvent type, sorption isothermKeywords: chitosan extraction, chitosan film, crawfish, solvent type, sorption isothermKeywords: chitosan extraction, chitosan film, crawfish, solvent type, sorption isothermKeywords: chitosan extraction, chitosan film, crawfish, solvent type, sorption isothermKeywords: chitosan extraction, chitosan film, crawfish, solvent type, sorption isotherm

Introduction

In recent years, there has been an increasing interest in develop-ing antimicrobial packaging materials using biodegradable and

renewable polymers. Use of biopolymer rather than plastics as apackaging material may considerably reduce the burden on envi-ronmental pollution. Chitosan is a biodegradable carbohydratepolymer that can be derived from crustacean shells. Crawfish shellwaste is abundant in Louisiana, U.S.A., but has been relatively un-exploited as a resource for chitosan extraction compared with otherwastes of crustacea such as crab and shrimp. Chitosan has beendocumented to possess antibacterial (Sudarshan and others 1992;Yalpani and others 1992; No and others 2002a, 2002b) and antifun-gal (Allan and Hadwiger 1979; Stossel and Leuba 1984; Kendra andothers 1989; Fang and others 1994) properties, thus making it a fa-vorable biopolymer for antimicrobial film development. Chitosanextracted from crawfish shell may offer functionalities favorable forthe development of antimicrobial films (Nadarajah 2005).

Various organic acids (acetic, lactic, formic, malic, and propionicacids) have been used for preparing chitosan films (Kienzle-Sterzerand others 1982; Butler and others 1996; Rhim and others 1998;Park and others 1999; Park and others 2002). Because chitosanmust be dissolved in dilute organic acids to form films, usage oforganic acids that possess antimicrobial properties such as acetic,formic, and lactic acids would offer synergistic antimicrobial activity.As these acids are food-grade ingredients, they are safe for food-grade film formation (Begin and Van Calsteren 1999). No and oth-ers (2002a, 2002b) showed that chitosan exhibited varying levels ofantimicrobial activity against different bacteria with different or-ganic acids. They reported that chitosans dissolved in acetic, lactic,

and formic acids were generally more effective in inhibiting bacte-rial growth than in propionic and ascorbic acids.

The film-forming ability of chitosan has been reported by manyinvestigators (Muzzarelli and others 1974; Averbach 1978; Butlerand others 1996; Caner and others 1998; Wiles and others 2000).Chitosan films are described as being tough, long lasting, flexible,and very difficult to tear (Butler and others 1996). The film-formingability of crawfish chitosans and their film characteristics were re-ported by Nadarajah (2005). However, their films were very hydro-philic and sensitive to humidity. The major drawback of using suchhydrophilic films in packaging applications is that they cannot beused in direct contact with food or for direct handling (Olabarrietaand others 2001). Hence, it is a challenge to develop crawfish chito-san-based antimicrobial films that are less sensitive to humidity.

Rout and Prinyawiwatkul (2001) and Nadarajah and Prinyawi-watkul (2002) demonstrated that crawfish chitosans having differ-ent functionalities can be derived from different extraction proto-cols. This presents an opportunity for developing a variety of filmsusing different crawfish chitosans and organic acid solvents. A de-tailed study of sorption behaviors of such films would allow an ef-ficient selection of crawfish chitosan films that are less sensitive tohumidity. Hence, this study was conducted to determine the ef-fects of different chitosans and solvent types on sorption behaviorof crawfish chitosan films and to establish models to predict sorp-tion behaviors of such films.

Materials and Methods

MaterialsMaterialsMaterialsMaterialsMaterialsCrawfish shell was collected from a local seafood processing fa-

cility in Louisiana, washed with warm tap water to remove solubleorganics and remaining muscle particles, and dried in a forced-airoven at 60 °C overnight. The dried shell was ground with a centrif-ugal grinding mill (Retsch/Brinkmann ZM-1, Westbury, N.Y., U.S.A.)and sifted with 20-mesh (0.841 mm) and 40-mesh (0.425 mm)sieves. Ground shell of 0.425- to 0.841-mm particle size was usedthroughout this research to obtain reproducible and consistent

MS 20050318 Submitted 5/25/05, Revised 7/22/05, Accepted 10/21/05. Au-thors Nadarajah, Prinyawiwatkul, and Xu are with Dept. of Food Science,Louisiana State Univ. Agricultural Center, Baton Rouge, LA 70803-4200.Author No is with Dept. of Food Science and Technology, Catholic Univ. ofDaegu, Hayang, South Korea. Author Sathivel is with Fishery IndustrialTechnology Center, Univ. of Alaska Fairbanks, Kodiak, Alaska. Direct in-quiries to author Prinyawiwatkul (E-mail: wprinya@lsu.edu).

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Sorption behavior of crawfish chitosan films . . .

results. All chemicals used were analytical grade purchased fromSigma Chemical Co. (St. Louis, Mo., U.S.A.).

Production of chitosansProduction of chitosansProduction of chitosansProduction of chitosansProduction of chitosansProduction of chitosans from crawfish shell involved deproteiniza-

tion (designated as DP), demineralization (DM), decoloration (DC),and deacetylation (DA) (No and Meyers 1995; No and others 2000).The shell was deproteinized with 1 N NaOH at 65 °C for 1 h at a solid/solvent ratio of 1:10 (w/v). Following deproteinization, the shell wascollected on a 100-mesh sieve, washed to neutrality in running tapwater, rinsed with deionized water, and filtered to remove excessmoisture. Demineralization was accomplished with 1 N HCl at anambient temperature for 30 min at a solid/solvent ratio of 1:15 (w/v).The residue was then washed and filtered as described previously.For decoloration, the chitin residue was mixed with acetone at a solid/solvent ratio of 1:10 (w/v) for 10 min, filtered, dried for 2 h at an am-bient temperature, followed by bleaching with 0.315% NaOCl for 5min at a solid/solvent ratio of 1:10 (w/v). The decolored chitin waswashed and filtered as described previously. Deacetylation wasachieved by treating the decolored chitin under a condition of 121°C/15 psi with 50% NaOH for 30 min at a solid/solvent ratio of 1:10(w/v). The resulting chitosan was collected, washed as describedpreviously, and dried at 60 °C for 4 h in a forced-air oven.

To study sorption behavior of crawfish chitosan films, 4 differenttypes of chitosan samples were prepared and designated as fol-lows: (1) DPMCA = deproteinized + demineralized + decolorized +deacetylated (chitosan prepared according to the previously men-tioned 4 steps); (2) DPMA = deproteinized + demineralized +deacetylated (chitosan prepared without the “decolorized” step);(3) DMCA = demineralized + decolorized + deacetylated (chitosanprepared without the “deproteinized” step); and (4) DMA = dem-ineralized + deacetylated (chitosan prepared without the “depro-teinized” and “decolorized” steps).

Characterization of chitosanCharacterization of chitosanCharacterization of chitosanCharacterization of chitosanCharacterization of chitosanMoisture and ash contents (%) were determined by the AOAC

standard methods 930.15 and 942.05, respectively (AOAC 1995).Nitrogen (%) was determined using an elemental analyzer (EA1110, CE Instrument, Rodano-Milan, Italy). Viscosity (cP) was de-termined with a Brookfield viscometer, model LVDV-II+ (BrookfieldEngineering Laboratories, Stoughton, Mass., U.S.A.). Chitosan so-lution was prepared in 1% (v/v) acetic acid at a 1% (w/v) concentra-tion. Viscometric measurements were made using a small sampleadapter on solution (8 mL) at 25 ± 2 °C with values reported in cen-tipoise (cP) units. Degree of deacetylation (%) was determinedaccording to a colloid titration method (Toei and Kohara 1976) us-ing N/400 potassium polyvinyl sulfate (f = 1.004, Wako Pure Chem-icals, Osaka, Japan). For the determination of viscosity-averagemolecular weight (MW ) of chitosan, chitosan was dissolved in asolution of 0.1 M acetic acid and 0.2 M NaCl and an automated so-lution viscometer (relative viscometer model Y501, Viscotek Corp.,Houston, Tex., U.S.A.) was used to measure the intrinsic viscosity[�]. The Mark-Houwink equation [�] = KMWa was used to calculatethe molecular weight and reported as kDa. Values of empirical vis-cometric constants K and a of 1.81 × 10–3 cm3/g and 0.93, respective-ly, were used (Kurata and Tsunashima 1989).

Preparation of chitosan filmsPreparation of chitosan filmsPreparation of chitosan filmsPreparation of chitosan filmsPreparation of chitosan filmsEach chitosan (DPMCA, DPMA, DMCA, and DMA) was dis-

solved in 1% (v/v) solvent (acetic, formic, lactic, or malic acid) at 1%concentration (w/v) by stirring at an ambient temperature for 30min, and then in a boiling water bath for 10 min. The solution wasfiltered through glass-wool to remove undissolved particles. Film-

forming solution (300 mL) was poured onto a teflon-coated plate(31 × 31 cm) and allowed to dry at ambient conditions. Dried filmswere carefully peeled out and stored in desiccators containing asaturated solution of NaBr to produce a constant relative humidityof 57% at 25 °C. A micrometer (Model 293-766, Mitutoyo, Tokyo,Japan) was used to measure the film thickness to the nearest 0.001mm. An average of 10 random measurements on each sample wasrecorded.

Sorption experimentsSorption experimentsSorption experimentsSorption experimentsSorption experimentsMoisture isotherms of chitosan films were determined gravimet-

rically (Hatakcyama and Hatakcyama 1998) at 25 °C. To avoid pos-sible curing effects that may arise due to heating, films were driedat 25 °C for 3 wk in a hermetically sealed desiccator containingDrierite desiccant (W.A. Hammond Drierite Co. Ltd., Xena, Ohio).

Dried film samples were weighed, placed on separate glass con-tainers, and kept in a vacuum desiccator containing a saturated saltsolution of known water activity (Robinson and Stokes 1959). Therange of water activities from 0.112 to 0.927 was studied using sat-urated salt solutions of lithium chloride, potassium acetate, mag-nesium chloride, sodium bromide, potassium iodide, sodium ni-trate, potassium chloride, and potassium nitrate. The desiccatorwith samples was kept under temperature-controlled environmentat 25 °C. Equilibrium was considered after 3 wk as initial experi-ments revealed that sample weight did not change by more than 2mg of water/g of chitosan by dry weight (Labuza 1984) after the 3rdwk. The amount of water adsorbed on the film sample was deter-mined by re-weighing the film.

Modeling sorption isothermModeling sorption isothermModeling sorption isothermModeling sorption isothermModeling sorption isothermSix sorption isotherm models, Oswin (1946), Smith (1947), Halsey

(1948), Henderson (1952), Caurie (1970), and Guggenheim-Ander-son-De Boer (GAB model) (Anderson 1946; De Boer 1953; Guggen-heim 1966) were used to fit the experimental sorption isothermdata (Table 1).

Statistical analysisStatistical analysisStatistical analysisStatistical analysisStatistical analysisAll experiments were carried out in 3 separate replicates and

average values were reported. Linear regression analysis (PROCREG) was performed to obtain regression models for measured re-sponses and to calculate the best fitted values of constants forequations (Table 1) (SAS Inst. 2002). The GAB model parameterswere calculated with a nonlinear regression program using theWater Analysis Series v97.4 software (Webb Tech Pty Ltd, Australia)developed by Professor T.P. Labuza, Univ. of Minnesota, St. Paul,Minn., U.S.A. The suitability of the equations was evaluated andcompared among models using the coefficient of regression (R2).The PROC ANOVA procedure (SAS Inst. 2002) with a nested factorialmodel was applied to test the differences among isotherms. Signif-icance of difference was defined at P < 0.05.

Results and Discussion

Characteristics of crawfish chitosanCharacteristics of crawfish chitosanCharacteristics of crawfish chitosanCharacteristics of crawfish chitosanCharacteristics of crawfish chitosanThe molecular weight (Mw) values of chitosans ranged from 454

to 1462 kDa with the largest value observed for DPMA. Comparingthe Mw values between DPMCA and DPMA as well as betweenDMCA and DMA, it was evident that the decoloration (DC) stepdrastically broke down the chitosan polymer chain, thus reducingits Mw. Furthermore, its effect was greater than the deproteinization(DP) step in reducing Mw of chitosan. The viscosity of 1% chitosansolution was well correlated with Mw values. The degree of deacety-lation values of all 4 chitosans were slightly different and in the

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range of 84.2% to 86.7%. Excluding the DC or DP step did not affectthe ash (0.26% to 0.36%) and nitrogen (7.2% to 7.36%) content ofchitosans. Results from Table 2 revealed that different extractionprocesses used to produce chitosans resulted in varying molecularweights and viscosities, which may, in turn, result in varying filmforming and sorption properties.

Characteristics of crawfish chitosan filmsCharacteristics of crawfish chitosan filmsCharacteristics of crawfish chitosan filmsCharacteristics of crawfish chitosan filmsCharacteristics of crawfish chitosan filmsAll crawfish chitosans (DPMCA, DPMA, DMCA, DMA) yielded

clear solutions with acetic, formic, lactic, or malic acid and theyformed films. The films formed with different chitosan types butwith the same acid resembled each other and showed no apparentvisual differences among them. The characteristics of these craw-fish chitosan films are described in Table 3. Although all crawfishchitosans exhibited film-forming ability with all the acids used, onlythose films formed with acetic or formic acids were flexible, trans-parent, and desirable for packaging purposes. The films formedwith lactic or malic acid were highly hydrophilic and became asticky mass or clump upon peeling from the teflon-coated plate.Compared with acetic and formic acids, lactic and malic acids havemore hydroxyl groups that could have made the crawfish chitosanfilm more hydrophilic. These observations indicate that the filmforming ability of crawfish chitosan is influenced by the solventtypes but not by chitosan types.

All 4 crawfish chitosans, with acetic or formic acids, yielded flex-ible films without added plasticizers. According to Nadarajah andothers (2005a), crawfish chitosan films with tensile strength of 135.8MPa and elongation of 37.16% that resemble flexibility of plasticpackaging materials can be formed without any plasticizers. Addi-tion of plasticizers minimizes or eliminates brittleness of films.However, plasticizers also adversely affect film properties by makingthem more hygroscopic and contributing to greater moisture ab-sorption (Lawton 1992). Furthermore, addition of plasticizers entailsa decrease in barrier properties of edible packaging (Butler andothers 1996; Shaw and others 2002). Hence, the ability of crawfishchitosans to form flexible films, without any added plasticizer, isadvantageous for making the films that are less sensitive to humid-ity. According to Cagri and others (2001), the organic acids used assolvents may render plasticizing effects because these acids aresmall molecules with hydroxyl groups. In this study, acetic and for-mic acids may have provided plasticizing effects.

Thickness of acetate and formate films ranged from 0.02 mm to0.03 mm. There was no significant difference in film thickness withregard to the solvent type, although some films differed in thick-ness with regard to the type of chitosan used. While most of thefilms had a mean thickness in the range of 0.020 mm to 0.021 mm,formate films formed with DPMCA chitosan and acetate filmsformed with DMA chitosan had significantly higher thickness (P <0.05) of 0.027 mm and 0.026 mm, respectively.

Sorption isothermsSorption isothermsSorption isothermsSorption isothermsSorption isothermsThe moisture sorption characteristics of chitosan films are im-

portant for ascertaining their integrity and potential uses at vary-ing humidity conditions. According to the film properties (Table 3),only chitosan acetate and formate films were studied for their sorp-tion isotherms. The sorption isotherms developed for all crawfishchitosan acetate or formate films exhibited the classical sigmoidalcurve (Figure 1 and 2), which is called a type II isotherm in referenceto the Brunauer-Emmett-Teller (BET) classification (Brunauer1945). Most biological products follow a sigmoid curve representingthe type II isotherm of the BET classification (Labuza 1984).

For crawfish chitosan films (Figure 1 and 2), the moisture sorp-tion content increased with increased water activity, and the in-crease became more pronounced and exponential at elevated wateractivity levels. This observation is in agreement with that reportedby Despond and others (2001) for chitosan films formed with 1%acetic acid (but the acid was removed later). The pronounced in-crease in sorption uptake at higher water activity levels indicatedswelling effects in the chitosan films. Swelling causes changes in themicrostructure of film and increases moisture sorption, and watervapor would act as a plasticizer inside the chitosan matrix (Rogers

Table 1—Isotherm models used for fitting experimental data

Isotherm Modela Eq nr

Oswin (1946) 1

Smith (1947) m = mb – ma × [ln(1 – aw)] 2Halsey (1948) ln(m) = a + b × {ln[–1n(aw)]} 3

Henderson (1952) 4

Caurie (1970) 5

GAB 6

aSee nomenclature for abbreviations.

Table 2—Physicochemical properties of chitosans preparedfrom crawfish shella

Molecular Visco- Mois- Nitro-Chitosan weight sity Deacetyl- ture Ashc genc

typeb (kDa) (cP) ation (%) (%) (%) (%)

DPMCA 454d 35d 86.7a 2.19c 0.36a 7.35aDPMA 1462a 1164a 86.1ab 2.74b 0.26a 7.20aDMCA 950c 259c 84.6bc 2.16c 0.32a 7.22aDMA 1054b 273b 84.2c 4.77a 0.34a 7.36aaMeans in the same column with different letters differ significantly (P <0.05).bDPMCA = deproteinized + demineralized + decolorized + deacetylated; DPMA= deproteinized + demineralized + deacetylated; DMCA = demineralized +decolorized + deacetylated; DMA = demineralized + deacetylated.cDry basis.

Table 3—Characteristics of crawfish chitosan films formedwith different solventsa

Solvent Film properties

Acetic acid A transparent, yellow tinted, flexible, non-sticky film with smooth shiny surface and slight acidic odor

Formic acid A transparent, yellow tinted, flexible, non-sticky film with smooth shiny surface without acidic odor

Lactic acid Highly sticky films that shrink upon peeling, becoming a sticky mass/clump and do not give acidic odor

Malic acid Highly sticky films that shrink upon peeling, becoming a sticky mass/clump and do not give acidic odor

a1% w/v chitosan in 1% v/v solvent.

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Sorption behavior of crawfish chitosan films . . .

1985; Wiles and others 2000). The swelling effects were more prom-inent with chitosan formate films than chitosan acetate films.When the water activity level was raised from 0.743 to 0.936, theamount of water sorbed by chitosan acetate films increased by138% (dry basis) on average (Figure 1). However, a much higherincrease (192%, dry basis) was observed for chitosan formate films(Figure 2). This indicates that chitosan formate films adsorbed morewater than chitosan acetate films at elevated water activity levels.

Unplasticized films tend to become brittle and lose their integrity,especially at lower water activity levels. In the present study, al-though crawfish chitosan films were formed without added plasticiz-ers, both chitosan acetate and formate films maintained their flexi-bility at all water activity levels (0.112 to 0.927). Both chitosan acetateand formate films had a tendency to become more yellowish in colorwith a prolonged storage at elevated humidity conditions (data notshown). Similar observations were reported by Caner and others(1998) and Wiles and others (2000) for different chitosan films.

The chitosan acetate films prepared from different crawfish chi-tosans exhibited similar isotherms (Figure 1), in spite of the differ-ences in some physicochemical properties (Table 2). The PROCANOVA procedure, performed with nested water activity levels, re-vealed that there was no significant difference (P > 0.05) among theisotherms of chitosan acetate films due to different types of chito-sans. Unlike the chitosan acetate films, chitosan formate filmsformed with different types of crawfish chitosans resulted in slightlydifferent (P < 0.05) isotherms (Figure 2). The chitosan formate filmsmade with the DPMCA chitosan had lower (P < 0.05) moisture con-tents compared with those made with the DMCA or DMA chitosans.Although not apparent at lower aw levels (aw < 0.43), for a given aw

level and the chitosan used, chitosan formate films had a higher (P< 0.05, based on the nested factorial model) moisture content thanacetate films, except for DPMCA acetate and DPMCA formate films.Thus, the solvent types had more effects on the film sorption behav-ior. Compared with the chitosan formate films, the chitosan acetatefilms, attaining a lower moisture content throughout the water activ-ity continuum, are more suitable for developing packaging films thatare less humidity sensitive. Because all acetate films produced similarisotherms, acetate films could be produced more economically usingthe DMA chitosan as it takes less chemical usage and process time fortheir extraction, thus a lower production cost.

At elevated aw levels of 0.74 to 0.84, acetate films prepared from91.7%, 84.0%, and 73.0% deacetylated chitosans (2% chitosan in 1%acetic acid) by Wiles and others (2000) absorbed a moisture contentof 21.4% to 34.1% (dry basis). Compared with those films, our craw-fish chitosan acetate films absorbed less moisture content of 17.12%to 29.43% (dry basis). This indicated that crawfish chitosan acetatefilms were less sensitive to humidity at elevated aw levels. Similarly,the crawfish chitosan formate films contained lower moisture con-tents at water activity levels between 0.74 and 0.84 compared withthe films made with 84.0% and 73.0% deacetylated chitosans report-ed by Wiles and others (2000). Although these differences could beattributed to the chitosan types and amounts of chitosans used toform films, the possibility of producing chitosan films that are lesshumidity sensitive using crawfish chitosans was evident.

Sorption model analysisSorption model analysisSorption model analysisSorption model analysisSorption model analysisThe isotherm models and the constants calculated by linear fit-

ting of isotherm equations are summarized in Table 4 and 5. A good

Figure 1—Moisturesorption isothermsof chitosan acetatefilms at 25 °C. Insertshows effects ofwater activity onclustering of watermolecules in craw-fish chitosan acetatefilms. Nc = clusternumber.

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Sorption behavior of crawfish chitosan films . . .

agreement between experimental and predicted data was ob-served with GAB, Oswin, and Caurie models with the R2 values of0.93 to 0.98. Consequently, these models are more appropriate forthe prediction of equilibrium moisture content of crawfish chitosanacetate and formate films. The GAB isotherm model allowed for themost accurate prediction of all chitosan films with the R2 values of0.97 to 0.98. The GAB equation adequately describes moisturesorption of many foods and natural polymers (Taoukis and others1988). Despond and others (2001) reported the adequacy of theGAB model for the description of films made of chitosan with Mw of200 kDa. Among the models tested in this study, the Halsey modeloffered the poorest fit for the prediction of sorption behavior ofcrawfish chitosan films. The goodness of fit of the models were inthe order of GAB > Oswin, Caurie > Smith, Henderson > Halsey.

Clustering of water moleculesClustering of water moleculesClustering of water moleculesClustering of water moleculesClustering of water moleculesThe pronounced upward curvatures, observed in the isotherms

of crawfish chitosan films at elevated water activities (Figure 1 and2), indicate the clustering of water molecules taking place in poly-mer matrices. According to Despond and others (2001), at lowerwater activity levels, water is distributed mainly through the poly-mer matrix, and probably sorbed on the active sites of hydrogenbonds. As water activity increases, water molecules predominantlycluster on the hydrogen bonding sites, likely resulting in plasticiza-tion of the polymer. Water molecules that initially enter the poly-mer structure may open the structure to make it easier for subse-quent water molecules to sorb in the neighborhood of the initiallysorbed molecules. As a result, a pronounced upward curvature isobserved in the adsorption isotherms of polymers.

The GAB model and the water clustering theory of Zimm andLundberg (1956) were used to infer the water-clustering phenom-enon in chitosan films (Despond and other 2001). Zimm and Lun-dberg (1956) developed an equation to calculate a cluster integralfrom the sorption isotherm. The clustering function (G11/v1) is acharacteristic quantity that enables the calculation of tendency ofthe (water) molecules to cluster in the given polymer matrix. Theclustering function is defined as:

(7)

where G11 is the cluster integral, calculated from the molecular pairdistribution, v1 is the partial molar volume of the penetrant (forexample, water), �1 is the volume fraction, and a1 is the activity ofcomponent 1. For an ideal solution, the activity coefficient (a1/ �1)does not vary with concentration, that is, the activity is proportionalto the volume fraction, and, therefore, Eq. 7 becomes:

(8)

For nonrandom mixing solutions, however, the activity coefficientdecreases with increasing �1 so that G11/v1 is greater than –1. The

Figure 2—Moisturesorption isotherms ofchitosan formate filmsat 25 °C. Insert showseffects of wateractivity on clusteringof water molecules incrawfish chitosanformate films. Nc =cluster number.

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Sorption behavior of crawfish chitosan films . . .

extent of clustering in the solution is indicated by the extent towhich G11/v1 exceeds –1. The quantity �1G11/v1 is the mean numberof excess water molecules in the neighborhood of a given watermolecule. The average number of solvent molecules in a cluster canbe described by the following equation:

(9)

where Nc is the cluster number. For an ideal solution where there isno clustering of water, Nc is equal to 1. The Nc values greater than1 represent clustering of water molecules. Positive Nc values indi-cate that the solute increases the free volume of the polymer matrix,increasing the sorption capacity, diffusivity, and permeability. Bycombining Eq. 6 (Table 1), 7, and 9, Nc can be expressed as follows(Zhang and others 1999):

(10)

where mo is the monolayer moisture content, and k and C are theGAB constants. To deduce this equation, the variable m in Eq. 6(Table 1) was transformed from gravimetric to volumetric fraction�1 using densities of crawfish chitosan films.

The Nc values deduced from the Eq. 10 indicate that when water

activity exceeds 0.57, clustering of water molecules takes place inboth crawfish chitosan acetate and formate films (inserts of Figure1 and 2). Despond and others (2001) reported that chitosan films(made with 1% chitosan in 1% acetic, but acid was removed later bytreating the films with ammonia/methanol solution and rinsing withwater) showed clustering of water molecules at aw levels exceeding0.60, which is similar to the present study. These observations maysuggest that chitosan films made with or without organic acidshave similar aw levels for clustering of water molecules to takeplace.

According to Saravacos and Maroulis (2001), addition of plasti-cizers would lead to clustering of water at lower water activity levels.Hence, it can be expected that clustering of water may occur at awater activity level below 0.57, if plasticizers are added to formcrawfish chitosan films. According to Nadarajah and Prinyawiwat-kul (2002) and Nadarajah and others (2005b), acetate, formate,lactate, or malate films formed with different types of crawfish chi-tosans with plasticizers resulted in highly hydrophilic films, whichwere undesirable for packaging. These indicate that unplasticizedcrawfish chitosan films may serve as better candidates for packag-ing development.

Conclusions

Crawfish chitosans formed flexible and transparent films withacetic or formic acid without any added plasticizers. All these

films maintained their flexibility at all water activity levels. Filmsformed with lactic or malic acid were very hydrophilic, which was

Table 4—Constants of sorption models for chitosan acetatefilms

ChitosanIsotherm typea Constantsb R2

Oswin a nDPMCA 0.115 0.459 0.97DPMA 0.116 0.393 0.93DMCA 0.115 0.459 0.97DMA 0.107 0.450 0.94

Smith ma mbDPMCA 0.134 0.021 0.95DPMA 0.111 0.036 0.91DMCA 0.125 0.021 0.96DMA 0.118 0.025 0.95

Halsey A bDPMCA –2.46 –0.60 0.95DPMA –2.41 –0.52 0.93DMCA –2.45 –0.59 0.93DMA –2.52 –0.59 0.92

Henderson A BDPMCA 14.41 1.47 0.93DPMA 24.18 1.73 0.89DMCA 109.86 2.17 0.97DMA 14.02 2.21 0.94

Caurie C moDPMCA 0.162 0.709 0.97DPMA 0.151 0.771 0.93DMCA 0.162 0.709 0.97DMA 0.155 0.691 0.94

GAB C kb moDPMCA 16.63 0.90 0.062 0.98DPMA 27.77 0.93 0.069 0.98DMCA 9.61 0.87 0.069 0.98DMA 13.25 0.88 0.068 0.98

aDPMCA = deproteinized + demineralized + decolorized + deacetylated; DPMA= deproteinized + demineralized + deacetylated; DMCA = demineralized +decolorized + deacetylated; DMA = demineralized + deacetylated.bSee nomenclature for abbreviations.

Table 5—Constants of sorption models for chitosan formatefilms

ChitosanIsotherm typea Constantsb R2

Oswin a nDPMCA 0.111 0.501 0.95DPMA 0.127 0.471 0.96DMCA 0.125 0.483 0.94DMA 0.127 0.460 0.94

Smith ma mbDPMCA 0.146 0.010 0.94DPMA 0.159 0.016 0.94DMCA 0.169 0.009 0.93DMA 0.161 0.013 0.93

Halsey A bDPMCA –2.52 –0.65 0.93DPMA –2.37 –0.62 0.95DMCA –2.40 –0.64 0.94DMA –2.36 –0.61 0.95

Henderson A BDPMCA 79.00 1.99 0.95DPMA 79.05 2.11 0.96DMCA 73.67 2.06 0.94DMA 86.20 2.17 0.94

Caurie C moDPMCA 0.167 0.667 0.95DPMA 0.173 0.734 0.96DMCA 0.174 0.730 0.95DMA 0.171 0.746 0.94

GAB C kb moDPMCA 9.53 0.92 0.060 0.98DPMA 14.08 0.92 0.066 0.97DMCA 15.15 0.93 0.063 0.98DMA 14.36 0.92 0.065 0.98

aDPMCA = deproteinized + demineralized + decolorized + deacetylated; DPMA= deproteinized + demineralized + deacetylated; DMCA = demineralized +decolorized + deacetylated; DMA = demineralized + deacetylated.bSee nomenclature for abbreviations.

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Sorption behavior of crawfish chitosan films . . .

undesirable for packaging purposes. Chitosan acetate films wereless sensitive to humidity compared with formate films. The sol-vent types exerted more effects on the sorption behavior of crawfishchitosan films than the chitosan types. The sorption behavior ofcrawfish chitosan films could be described by the GAB, Oswin, orCaurie models, whereas the GAB model offered the best prediction.Clustering of water molecules take place in crawfish chitosan filmswhen water activity exceeded 0.57. Selection of chitosan extractionmethods, solvent types, and avoidance of plasticizers are critical todevelop chitosan films that are less humidity sensitive. The un-plasticized crawfish chitosan acetate films, which were flexible,transparent, and less humidity sensitive, would serve as a bettercandidate for the development of antimicrobial packaging films.Because all acetate films produced similar isotherms, acetate filmscould be produced more economically using the DMA chitosan asit takes less chemical usage and process time for their extraction,thus a lower production cost.

Nomenclaturea, b, c, k, ma, mb, n, A, B, and C = sorption isotherm constantsa1 = activity of component 1aw = water activityG11 = cluster integralm = moisture content (dry-basis)mo = monolayer moisture content (dry-basis)Nc = cluster numberv1 = partial molar volume of the penetrant�1 = volume fraction

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