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Different conditions for alkaline hydrolysis of chitin and its application to produce biopolymeric films

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Different conditions for alkaline hydrolysis of chitin and its application to produce biopolymeric films
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   IV Congreso Internacional de Ciencia y Tecnología de los Alimentos, Córdoba, Argentina, 14 a 16 de Noviembre de 2012 Different conditions for alkaline hydrolysis of chitin and its application to produce biopolymeric films Moura J.M. 1 , Farias B.S. 1 , Rodrigues D.A.S.  1 , Moura C. M.  1 , Pinto L.A.A.  1 1: FURG  –   Federal University of Rio Grande, School of Chemistry and Food, Unit Operation Laboratory, Rio Grande, RS, Brazil. jaquefurg@gmail.com Resumen: La quitina y el quitosano son biopolímeros, producidas por fuentes renovables autóctonas, y sus  propiedades han sido explotados para aplicaciones industriales y tecnológicas. En la actualidad, la  preocupación de la industria alimentaria es la eliminación de los envases de plástico con tiempo de degradación muy lenta, lo que causa daños al medio ambiente. Una alternativa a los envases de plástico es el quitosano, principalmente debido a su biodegradabilidad. El quitosano con diferentes características se puede obtener por reacción de desacetilación de quitina, y las condiciones de reacción interfieren directamente en sus aplicaciones. Este trabajo objetiva producir quitosano en diferentes condiciones de hidrólisis alcalina  para obtener películas biodegradables. La metodología de diseño experimental fue empleado para verificar los efectos del tamaño de la quitina (1 y 5 mm), solución de NaOH: relación de quitina (20:1 y 60:1, v/w), la concentración de NaOH (40 y 45%, w/w) y el tiempo de reacción (90 y 240 min) en el peso molecular y grado de desacetilación. Las muestras de quitosano se disolvieron en una solución de ácido acético (1%, v/v)  para la producción de películas. Estas se caracterizan de acuerdo con la permeabilidad al vapor de agua, resistencia a la tracción y estiramiento. Palabras clave: quitina, quitosano, desacetilación, películas biopolímeros. Abstract: Chitin and chitosan are biopolymers, produced by renewable native sources, and its properties have been exploited for industrial and technological applications. Currently, a preoccupation of food industry is the disposal of plastic packing with very slow degradation time, which causes damage to the environment. An alternative to plastic packing is chitosan, mainly due its biodegradability. Chitosan with different characteristics can be obtained by chitin deacetylation reaction, and the reaction conditions interfere directly in its applications. This work aimed to produce chitosan under different conditions of alkaline hydrolysis and to obtain biodegradable films. The experimental design methodology was employed to verify the effects of chitin size (1 and 5 mm), NaOH solution:chitin ratio (20:1 and 60:1, v/w), NaOH concentration (40 and 45%, w/w) and reaction time (90 and 240 min) on the molecular weight and deacetylation degree. Chitosan samples were then dissolved in acetic acid solution (1%, v/v) for the film production. These were characterized according to the water vapor permeability, tensile strength and elongation. Keywords:  chitin, chitosan, deacetylation, biopolymeric films. INTRODUCTION Chitosan is a polysaccharide composed by units of glucosamine and N-acetylglucosamine, and can be obtained by N-deacetylation of chitin, which is the most abundant natural polymer after cellulose (Tolaimate et al.  2000). Basically, the conversion of chitin into chitosan involves the removal of acetyl groups of chitin molecular chain resulting in an amino group (NH 2 ) which has higher versatility. The deacetylation degree (DD) and molecular weight (M w ) are two important parameters for chitosan characterization influencing its  physicochemical and functional proprieties (Li and Xia 2011).   Usually, chitosan is prepared using highly concentrated solutions of sodium hydroxide (40 to 60%), which tends to promote the polymer degradation (Ottøy et al.  1996). This hydrolysis reaction removes the acetyl groups of chitin, remaining amino groups along the macromolecule, however this is not complete deacetylation, resulting in different proportions of these groups along the chain. The molecular weight is mainly influenced by the reaction time, temperature and atmospheric conditions. Thus, depending of the reaction conditions, chitosan can have different characteristics in relation DD and M w , which influence in the final application of the polymer (Chen and Hwa 1996). Due its DD (>60%) and biodegradability, chitosan has several applications, such as, water treatment,  production of contact lenses, artificial gastric protection and films for fruits and vegetables preservation (Zeng et al  . 2008). Chitosan is a promising biopolymer in relation to the development of packaging, due its good mechanical properties, antimicrobial, nontoxic, biocompatible and excellent capability to form films   IV Congreso Internacional de Ciencia y Tecnología de los Alimentos, Córdoba, Argentina, 14 a 16 de Noviembre de 2012 (No et al.  2007). However, these films are highly permeable to water vapor, due to the highly hydrophilic nature (Fernandez-Saiz et al  . 2009). Both characteristics of the films produced from the biopolymer are influenced by the main parameters that characterize chitosan, i.e. its deacetylation degree and molecular weight. The aim of this work was to study the conditions of chitin alkaline hydrolysis to produce chitosan with different molecular weight and deacetylation degree. For this purpose, an experimental factorial design methodology, 2 4-1 , was used. The studied factors were: chitin size (1 and 5 mm), NaOH solution: chitin ratio (20:1 and 60:1, v/w), NaOH concentration (40 and 45%, w/w) and reaction time (90 and 240 min), being the responses, molecular weight and deacetylation degree. Different chitosans were then used to produce films which were characterized in relation water vapor permeability, tensile strength and elongation. MATERIALS AND METHODS Material Chitin was obtained from shrimp wastes (  Penaeus brasiliensis ) by demineralization, deproteinization and deodorization steps. Chitin was tray dried (5.0 to 6.0% wet basis) according to Weska et al.  2007. Experimental procedure In order to study the different conditions of deacetylation reaction, the experimental factorial design methodology was used. The levels and factors are presented in Table 1. Table 1 : Matrix factorial experimental design with factors in the actual and coded. (A), (B), (C) e (D): factors in coded form . These levels and factors were determined according to preliminary tests and values reported in the literature (Chen and Hwa 1996, Tolaimate et al.  2000, Galed et al  . 2005, Trung et al  . 2006, Weska et al  . 2007, Zhou et al.  2008, Baskar and Kumar 2009, Alvarenga et al  . 2010, Moura et al.  2011). Runs were performed at random. Results were analyzed using Statistic version 7 (StatSoft Inc., USA) software. Film Production The film was produced by “ casting  ” technique; this consists in dissolving the chitosan solution in 0.1 M acetic acid by dispersing powder of chitosan (1.0% w/w) for 2 h stirring (752A FISATOM, Brazil) at room temperature. An appropriate volume (50 mL) of the film-forming solution was poured onto a Plexiglas, in order to keep constant the total amount of polymer deposited. The films were obtained by solvent evaporation in an oven with air circulation at 40 °C for about 24 h. Finally, the film samples were removed from plates and conditioned in desiccators to 25 ± 1 °C for at least 48 h prior to testing. Before testing, the thickness of the film samples was measured by a digital micrometer (MDC-25S, Mitutoya Corp., Tokyo, Japan). Mean thickness was calculated from ten measurements taken at different locations on film sample, according to Ferreira et al  . 2009. Analytical methods Deacetylation degree (DD) The deacetylation degree was carried out by the linear potentiometric method. This analysis occurred by dissolving 0.25 g of chitosan in 20 mL of HCl solution, 0.1 N, and completing until 100 mL with distilled water. The solution was placed under constant agitation in a magnetic stirrer at 600 rpm and then pH was adjusted (MB10-Marte-Brazil), to 2.0 with standard NaOH solution, 0.1 M, being considered as the titration starting point. Titration was performed until the chitosan solution reached a pH of approximately 6.5 (range Run Chitin size  NaOH:quitina ratio  NaOH Concentration Reaction time A* mm B* mL g -  C* % D* min 1 -1 1 -1 20:1 -1 40 -1 90 2 +1 5 -1 20:1 -1 40 +1 240 3 -1 1 +1 60:1 -1 40 +1 240 4 +1 5 +1 60:1 -1 40 -1 90 5 -1 1 -1 20:1 +1 60 +1 240 6 +1 5 -1 20:1 +1 60 -1 90 7 -1 1 +1 60:1 +1 60 -1 90 8 +1 5 +1 60:1 +1 60 +1 240   IV Congreso Internacional de Ciencia y Tecnología de los Alimentos, Córdoba, Argentina, 14 a 16 de Noviembre de 2012 of chitosan non-protonation) (Tan et al  . 1998 and Jiang et al  . 2003). Detailed calculations can be verified in the Jiang et al  . 2003 research. Molecular weight (M w ) Viscometry is a simple and rapid method for the determination of molecular weight; the constants α and K in the Mark-Houwink equation have been determined in 0.1 M acetic acid and 0.2 M sodium chloride solution. The intrinsic viscosity is expressed as   0.93w3 α w M1081.1M.K = η     (1) where, η  is intrinsic viscosity (mL g -1 ), M w  is viscosity average molecular weight (kDa), K and α  are constants that depend on the solvent polymer system. The charged nature of chitosan in acid solvents and chitosan’s propensity to form aggregation complexes require care when applying these constants (Roberts and Domszy 1982). Furthermore, converting chitin into chitosan lowers the molecular weight, changes the deacetylation degree, and thereby alters the charge distribution, which in turn influences the agglomeration (Ravi Kumar 2000). Water vapor permeability (WVP)  After the storage period, the film thickness was measured by a digital micrometer (Mitutoya Corp., MDC-25S, Japão) with 0.0010 mm of resolution. Mean thickness was calculated from ten measurements taken at different locations on biofilm samples, according to Ferreira et al.  2009. Water vapor permeability (WVP) of  biofilms was determined gravimetrically at 25ºC, using the ASTM standard method E96/E96M-05. Samples of each biofilm in the form of discs (diameter = 50 mm) were fixed with paraffin cell permeation of aluminum, containing anhydrous calcium chloride. These cells were placed in desiccators at 25ºC and 75% relative humidity. By increasing the mass of anhydrous calcium chloride (measured in intervals of 24 h for 7 days), it was possible to determine the water vapor transferred through the biofilm according to Equation 2. The water vapor permeability (WVP) of films was evaluated by a gravimetric test according to E00996-00 (ASTM 2001a), using Equation 2. PAL tw WPV ab   (2) where, w ab  is the amount of adsorbed water (g), t is the time (days), L is the average biofilm thickness (mm), A is the area of the exposed biofilm surface (m 2 ) and ∆P is the partial vapor pressure difference across the  biofilm (Pa). Mechanical properties (TS e E) Tensile strength (TS) and elongation percentage (E) at break point were measured uniaxially by stretching the specimen in one direction using a Texture Analyzer (Stable Microsystems SMD, TA.XP2i, UK) according to the ASTM D-882-02, (ASTM, 2001b) standard, with a 50 N load cell. Samples of biofilms were cut into 25 mm wide and 100 mm long strips. The initial grip separation and crosshead speed were set to 50 mm and 50 mm min -1 , respectively. RESULTS AND DISCUSSION Table 2 shows the experimental design results for the molecular weight and deacetylation degree of chitosan  produced under different reaction conditions.   IV Congreso Internacional de Ciencia y Tecnología de los Alimentos, Córdoba, Argentina, 14 a 16 de Noviembre de 2012 Table 2:  Experimental design results for chitosan M w  and DD.   Run Chitin size (mm)  NaOH:chitin ratio (mL g -1 )  NaOH Concentration (%) Reaction time (min) Molecular Weight (kDa) *  Deacetylation degree (%) *  1 1 20:1 40 90 144.6±1.2 74.6±0.8 2 5 20:1 40 240 156.4±4.4 78.6±0.1 3 1 60:1 40 240 192.7±0.4 84.0±0.1 4 5 60:1 40 90 162.0±5.3 72.8±0.8 5 1 20:1 60 240 247.4±0.4 83.4±1.5 6 5 20:1 60 90 175.8±1.3 69.0±0.2 7 1 60:1 60 90 175.8±1.8 87.5±0.6 8 5 60:1 60 240 142.1±2.2 94.0±0.5 *mean values±standard error (n=3). Figures 1 and 2 show the Pareto chart with the main effects for the responses molecular weight and deacetylation degree, respectively. p=,05 Standardized Effect DBCA   Figure 1 : Pareto graph for the molecular weight. p=,05 Standardized EffectACBD   Figure 2 : Pareto graph for the deacetylation degree. In Figures 1 and 2 it can be observed that all effects were significant (p≤0.05), being the chitin size, the more  pronounced influencing relation to the molecular weight response. This behavior can be attributed to the reagent migration on the surface of the material, which is facilitated in lowered particle sizes. On the contrary, at higher chitin sizes, lower degradation of the polymer chain occurs. In Figure 2 it was verified that the more important factors in relation to the deacetylation degree were the NaOH:chitin ratio and reaction time. This can be explained due to the NaOH excess and high exposure time, favoring the removal of acetyl groups from chitin amide groups. Table 3 shows the values of molecular weight and deacetylation degree of the chitosan obtained in different experimental conditions, as well as the values of water vapor permeability, tensile strength and elongation of the films produced.   IV Congreso Internacional de Ciencia y Tecnología de los Alimentos, Córdoba, Argentina, 14 a 16 de Noviembre de 2012 Table 3:  Water vapor permeability values, tensile strength and elongation of the films prepared from chitosan produced in different conditions as experimental design. Run Molecular weight (kDa) Deacetylation degree (%) Water vapor  permeability (g mm kPa -1  dia -1 m -2 )* Tensile strength (MPa)* Enlongation (%)* 1 144.6±1.2 74.6±0.8 2.48±0.34 a  22.60±1.27 a,b  72.50±1.56 a  2 156.4±4.4 78.6±0.1 2.74±0.06 a  25.25±0.49 a,,c  86.65±0.35 3 192.7±0.4 84.0±0.1 2.46±0.13 a  31.50±0.28 a,,c  119.30±0.99 4 162.0±5.3 72.8±0.8 2.51±0.04 a  24.30±0.85 a,,c  91.60±1.13 5 247.4±0.4 83.4±1.5 1.77±0.07 33.00±0.71 c  119.30±1.98 6 175.8±1.3 69.0±0.2 2.59±0.01 a  19.10±0.28 a  102.80±0.42 c  7 175.8±1.8 87.5±0.6 2.49±0.04 a  30.25±0.78  b,c  105.00±0.42 c  8 142.1±2.2 94.0±0.5 2.82±0.03 a  27.80±1.98 a,b,c  77.90±2.97 a     *Mean±standard error (n=3). Equal letters in the same column (p>0.05). Different letters in the same column (p<0.05). It can be observed in Table 3 that the films obtained from chitosans with high molecular weights (247.4 kDa) showed lower values of water vapor permeability. Similar behavior was found by Leceta, et al.  (2012) and Kerch, et al  . (2011), which studied the functional properties of films based on chitosan and the effect of storage time and temperature on the structure, mechanical properties and barrier films, respectively. Both studies were performed with chitosan of low and high molecular weight. This behavior can be explained by the structural organization of the polymer, because the larger the polymer chain, the structure is more cohesive film, which hinders the passage of water vapor. The mechanical properties are largely associated with the distribution and intermolecular and intramolecular interactions in the network created in the films of chitosan, and so, from the obtained data, it can be seen that the higher molecular weight provided greater interaction between chains, since the tensile strength values were higher when the films were prepared with higher molecular weight chitosans. It can be observed that the deacetylation degree has virtually no influence on the films characteristics, since this is a parameter that is more directly related to the polymer reactivity. CONCLUSION Chitosans obtained from different deacetylation conditions presented molecular weights and deacetylation degrees ranging from142 to 247 kDa and from 69 to 94%, respectively. The molecular weight increase caused a decrease of 59% in water vapor permeability of the chitosan films, and an increase in elongation and tensile strength. Thus, the more adequate conditions of deacetylation reaction to produce biopolymeric films were chitin size of 1 mm, NaOH:chitin ratio of 20:1 (v/w), NaOH concentration of 60% and reaction time of 240 min. BIBLIOGRAPHY Alvarenga ES, Oliveira CP, Bellato CR. 2010. An approach to understanding the deacetylation degree of chitosan. Carbohydrate Polymers, 80:1155  –  1160. ASTM. 2001a. American Society for Testing and Materials. Standard test methods for water vapor transmission of materials. Standard Designations: E96/E96M-05. In Annual book of ASTM, 406  –  413. ASTM. 2001b. American Society for Testing and Materials. Standard test method for tensile properties of thin plastic sheeting. Standard D882-02. In Annual book of ASTM, 162  –  170. Baskar D, Kumar TSS. Effect of deacetylation time on the preparation, properties and swelling behavior of chitosan films. Carbohydrate Polymers, 78:767  –  772. Chen RH, Hwa H. 1996. Effect of molecular weight of chitosan with the same deacetylation degree on the thermal, mechanical, and permeability properties of the prepared membrane. Carbohydrate Polymers, 29:353-358. Fernandez-Saiz P, Lagaron JM, Ocio MJ. 2009. Optimization of the biocide properties of chitosan for its application in the design of active films of interest in the food area. Food Hydrocolloids, 23:913-921. Ferreira CO, Nunes CA, Delgadillo I, Lopes-da-Silva JA. 2009. Characterization of chitosan  –  whey protein films at acid pH. Food Research International, 42:807  –  813. Galed G, Miralles B, Paños I, Santiago A, Heras A. 2005. N-Deacetylation and depolymerization reactions of chitin/chitosan: Influence of the source of chitin. Carbohydrate Polymers, 62:316  –  320.

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