A Dye-incorporated Chitosan-based CO 2 Indicator for Monitoring of Food Quality Focusing on Makgeolli Quality during Storage

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1 Food Sci. Biotechnol. 24(3): (2015) DOI /s RESEARCH ARTICLE A Dye-incorporated Chitosan-based CO 2 Indicator for Monitoring of Food Quality Focusing on Makgeolli Quality during Storage Kyuho Lee, Xiangpeng Meng, Tae-Young Kang, and Sanghoon Ko Received May 18, 2014; revised December 29, 2014; accepted January 13, 2015; published online June 30, 2015 KoSFoST and Springer 2015 Abstract A chitosan-based CO 2 indicator that exhibits a prominent visual change depending on different CO 2 levels was developed. The partial pressure of CO 2 in the headspace of food packaging can be considered as a quality indicator of makgeolli. For fabrication of a CO 2 indicator, 0.3% brilliant blue (BB) was incorporated into chitosan dissolved in a 0.1 M HCl solution, and the ph was subsequently adjusted to 7.0. At the beginning of makgeolli storage, the indicator was opaque because chitosan is insoluble at ph 7.0. With an increase in the CO 2 level in the packaging headspace during fermentation, the indicator developed transparency due to dissolution at lower ph values caused by CO 2. BB dye encapsulated in flocculated chitosan particles was released. The transparency of the indicator was correlated with the makgeolli quality attribute of titratable acidity over the storage time. Keywords: carbon dioxide, indicator, chitosan, intelligent food packaging, makgeolli Introduction Modern consumers are focused on the quality and safety of food products. Packaging helps to protect food products from spoilage and to extend product shelf life (1). There is much study in progress regarding food quality preservation and new smart packaging technologies have been developed. Smart packaging includes a carbon dioxide indicator, oxygen indicator, or a time-temperature indicator for general use (2). Quality and freshness indicators that are Kyuho Lee, Xiangpeng Meng, Tae-Young Kang, Sanghoon Ko ( ) Department of Food Science and Technology, Sejong University, Seoul , Korea Tel: ; Fax: sanghoonko@sejong.ac.kr placed inside sealed food packages provide consumers an easy way to identify microbial spoilage of food. Microbial growth and subsequent spoilage leads to production and accumulation of carbon dioxide (CO 2 ) and the partial pressure of CO 2 in the headspace of food packages can be considered to be a quality indicator of food (3). The headspace of packaged food fills with CO 2 due to spoilage and microbial fermentation. In this study, a CO 2 indicator for the Korean traditional fermented rice wine alcoholic beverage makgeolli was studied. A chitosan-based CO 2 indicator provides information about the optimum ripeness of fermented foods (4). A visually observable changebased CO 2 indicator using transparency is effective for easy monitoring of food quality without expertise. A chitosan-based CO 2 indicator for food packaging applications was developed based on the principle of changes in the visual appearance of a chitosan solution due to ph changes with CO 2 accumulation in the headspace of food packaging during microbial fermentation of food. The operational principle is that a chitosan suspension in the headspace is opaque at a low CO 2 concentration, but becomes transparent at high concentrations due to a phdependent semi-crystalline behavior of chitosan molecules in aqueous media (3). However, the chitosan based CO 2 indicator has the drawback that the indicator becomes opaque again due to a subsequent decrease in the CO 2 concentration. Visual change-based indicators of food quality should be irreversible and correlated with food quality. Irreversibility has been successfully applied in time-temperature indicators that show an irreversible color change in response to the time-temperature history of foods or food packages. However, a CO 2 dependent food quality indicator with an irreversible function has not been reported. Therefore, an irreversible color-changeable CO 2 indicator for food quality was prepared in this study based on the ph-dependent polarity of chitosan molecules in a

2 906 Lee et al. hydrophobic dye and water. The taste of makgeolli becomes sour due to fermentation in the product bottle. Organic acids are produced by the actions of yeasts and lactic acid bacteria (LAB) during fermentation of makgeolli. Organic acids generally decrease the ph and increase the acidity over time during storage. Therefore, ph is a major indicator of makgeolli fermentation while titratable acidity (TA) is an important factor that affects drinkability during storage. Excessive fermentation lowers the quality of makgeolli because an excessive amount of organic acids cause a sour taste. Microbial growth leads to production and accumulation of CO 2 and the partial pressure of CO 2 in the headspace of a makgeolli bottle can be considered as a product quality indicator. A visually observable change in a CO 2 indicator is effective for monitoring of food quality by consumers. Thus, an irreversible color change-based CO 2 indicator that is placed inside a makgeolli package can be used to indicate product quality and freshness. The aim of this study was to develop a chitosan-based irreversible optical CO 2 indicator to show changes based on CO 2 levels in the headspace of food packaging. An irreversible responsive dye-incorporated chitosan-based CO 2 indicator was developed for use during the ripening process of makgeolli. Materials and Methods Materials Chitosan powder (degree of deacetylation 84%, molecular weight (Mw) 22 kda) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Coomassie Brilliant Blue G-250 (BB) ultra-pure grade was purchased from Amresco Inc. (Solon, OH, USA). Analytical grade hydrochloric acid, glacial acetic acid, sodium hydroxide, and methanol were purchased from Daejung Chemicals & Metals Co., Ltd (Shiheung, Korea). Fabrication of an irreversible CO 2 indicator Chitosan powder (3 g) in 100 ml of distilled water was stirred for 2 h after adjustment to ph 5.5 by 0.1 M hydrogen chloride to produce a chitosan solution that was filtered using a 3 µm membrane filter. BB powder (0.3 g) was added to a mixture of 100 ml of acetic acid, 450 ml of methanol, and 450 ml of distilled water to produce a BB solution. A BBincorporated chitosan-based CO 2 indicator solution was prepared by mixing both the chitosan solution and the BB solution at a ratio of 10:2 (v/v) followed by stirring at 60 g using a magnet stirrer (MS-200; Daihan Scientific Co., Ltd., Wonju, Korea) for 1 h. The BB-incorporated chitosanbased CO 2 indicator solution was collected using reneutralization by adjusting the ph to 7.0 using NaOH, followed by centrifugation using a 5810R centrifuge (Eppendorf Instruments GmbH, Hamburg, Germany) at 8,000 g for 10 min. Five g of the precipitate after centrifugation was dispersed in 100 ml of distilled water, then homogenized using a shear homogenizer (Wise Mix HG-15A with the DH.WHG02118 homogenizer tool; Daihan Scientific Co., Ltd.) at 20x for 2 min. The irreversible color change-based chitosan-based CO 2 indicator that is dependent on CO 2 levels in the headspace of food package was developed. Briefly, a hydrophobic dye was encapsulated at a neutral ph in flocculated chitosan molecules, which were then subsequently ionized and dissolved as the ph was lowered due to an increasing CO 2 concentration. As a result, the dye was released and an apparent visual change occurred. Even with ph recovery to neutral conditions, either naturally or intentionally, the dye released was not re-incorporated into chitosan molecules and no visual change occurred due to ph recovery. After the indicator itself was studied and verified, the visual indicator was applied to unsterilized fermented makgeolli. Measurement of the ph and turbidity of the irreversible CO 2 indicator The ph was measured using an SP-2100 Laboratory ph/orp Meter (Suntex Instruments Co., Ltd., New Taipei, Taiwan). The turbidity of the irreversible CO 2 indicator was measured using a UV-visible spectrophotometer (DU730; Beckman Coulter, Inc., Fullerton, CA, USA) at 680 nm. Measurement of the encapsulation efficiency The encapsulation efficiency was calculated as: Encapsulation efficiency (%)= x y 100 z where x=the absorbance value of a mixture of the chitosan solution and the BB solution, y=the absorbance value of the chitosan solution, and z=the absorbance value of the BB solution. The encapsulation efficiency was 46.4%. Inclusion of the irreversible CO 2 indicator in makgeolli packaging A commercial unsterilized makgeolli (750 ml, Jangsu Makgeolli) with live LAB was purchased from a local supermarket in Seoul, Korea in March of A volume of 350 ml was divided into small packages (2215 cm) of nylon/polyethylene/linear low-density polyethylene (Nylon/PE/LLDPE) film with a thickness of 110 m and an approximate CO 2 permeability of <10 ml/m 2 h atm. The package was almost impermeable to other gasses. The irreversible CO 2 indicating solution (5 ml) was sealed using a hand sealing machine (Model XP-300/5, C- type; Hana Corporation Ltd., Gimcheon, Korea) into square (5 cm 5 cm) plastic sachets made of low density polyethylene (LDPE) film with a thickness of 50 m and an approximate CO 2 permeability of 175 ml/m 2 h atm. The

3 Dye-incorporated Chitosan Based CO 2 Indicator 907 Fig. 1. (A) A dye-incorporated chitosan-based CO 2 indicator installed in makgeolli packaging and (B) the basic operating principle. irreversible CO 2 indicator sachet bag was placed in the headspace of a makgeolli package at a distance sufficient to avoid direct contact with the makgeolli, and the package was sealed using a heat treatment (Fig. 1A). For positive controls, makgeolli was replaced with 3 diluted water and packaged with the same indicator sachets. Quality measurement of makgeolli during storage Properly sealed makgeolli packages were stored at 4 o C for 10 days in a refrigerating chamber (Model FR-B232F; Daewoo Electronics, Incheon, Korea) and at 25 o C for 6 days. Makgeolli samples were collected every day and ph and TA values were measured as quality indices. For measurement of ph, makgeolli samples were stirred at 60 g using a magnet stirrer (MS-200; Daihan Scientific Co., Ltd.) for 10 min to allow CO 2 degassing, then the ph was measured. For TA measurement, the method of regulation for liquor analysis (5) was followed. Makgeolli samples were titrated using 0.1 N NaOH and TA values were calculated as: TA (%) = 0.1 N NaOH (ml) 0.1 N NaOH factor Sample (ml) All measurements were repeated 3 times. Results and Discussion Design of an irreversible CO 2 indicator The operational principle of a dye-incorporated chitosan based CO 2 indicator for irreversible indication of food quality is shown in Fig. 1B. Hydrophobic color dye is encapsulated in and on the flocculates of chitosan particulates under neutral conditions. A possible scenario of action involves a ph reduction due to dissolution of CO 2 in the indicator, and the chitosan molecules in the flocculated particulates become positively charged. As a result, positively charged chitosan molecules are solubilized under acidic aqueous conditions. Subsequently, clearly visible flocculates of chitosan particulates disappear from the indicator, which then presents an homogeneous appearance with invisible chitosan particulates due to dissociation of flocculates. In addition, a color dye encapsulated in and on the flocculates of chitosan particulates is released and is dispersed homogeneously throughout the indicator. Other action scenario involves hydrophobic color dye that is released and is never re-encapsulated in or on chitosan particulates, even if the ph of the indicator is recovered to neutral conditions. In other words, an irreversible indication occurs in the dye-incorporated chitosan-based CO 2 indicator. As the ph of the indicator is recovered to neutral conditions, either intentionally or unintentionally, the dissolved chitosan particulates and the color dye molecules that were released under acidic conditions by CO 2 in the food package never re-associate to the initial encapsulated forms. Chitosan molecules cannot re-encapsulate the color dye since the released color dye molecules have a lyophilic affinity to the acidic aqueous conditions. Therefore, the color dye released by the aqueous indicator does not re-combine with chitosan particulates to the initial indicator state under neutral conditions. Subsequently, the homogeneous appearance of the indicator due to invisible chitosan molecules and particulates is maintained even after re-neutralization of the ph (6,7). Herein, Coomassie Brilliant Blue G-250 (BB) was used as a color dye. BB is a medium lyophilic to aqueous medium under neutral conditions that is highly lyophilic in a methanol/water mixture under acidic conditions. BB also has a capacity for rapid binding to chitosan molecules. The indicator solution was a water and methanol mixture under neutral conditions in order to improve irreversibility of the indicator. As the ph of the indicating solution was reduced, the lyophilicity of BB under acidic alcoholic conditions was increased (8,9). This ph-dependent lyophilicity of BB is a unique property that can be used to achieve irreversibility of the chitosan based CO 2 indicator.

4 908 Lee et al. Incorporation of BB in chitosan flocculates for the CO 2 indicator Surface phenomena of ph-dependent chitosan molecules play roles in incorporation of BB and subsequent release behavior. Flocculation of chitosan molecules with reduced charges occurs under neutral conditions. The ph of 7.0 in a mixture of acetic acid, methanol, and distilled water suggested that spontaneous formation of flocculates among chitosan particulates (less lyophilic) is inevitable with a decrease in the specific surface area. The binding sites for both hydrogen bonding and van der Waals attractions for BB entrapment are the acetamido and amino groups in chitosan molecules (10). In addition, BB molecules with both polar and non-polar components may have superior adsorption capacities due to the highly porous structure of chitosan particles (11). Chitosan is used widely as an adsorbtion material for bonding organic species and some metal cations because amino (-NH 2 ) and hydroxy (-OH) groups can serve as coordination and reaction sites (12-14). Flocculates of chitosan particulates were formed at ph 7 in a mixture of acetic acid, methanol, and distilled water in which BB molecules were localized in and on flocculates due to reduced lyophilicity of BB against the mixture. BB may be less lyophilic to a mixture of acetic acid, methanol, and distilled water, but is relatively easily incorporated on and in chitosan particulates. In other words, BB molecules are supposed to bind to chitosan molecules spontaneously, whereas they are unstable electrostatically under neutral conditions in a mixture where strong van der Waals forces existed among chitosan molecules. As a result, chitosan molecules were close to each other, resulting in subsequent flocculation. The behavior of the CO 2 indicator involving dissociation of chitosan flocculates and BB release Chitosan flocculates (lyophobic at ph 7) were dissociated at ph 5 in a mixture of acetic acid, methanol, and distilled water. The surface of the chitosan particulates was charged due to ionization of the amino groups under acidic conditions. The flocculates of chitosan particulates under initial neutral conditions were larger than flocculates of small molecules due to dissociation of flocculates under acidic conditions as the ph was decreased due to accumulation of CO 2 in the indicator medium. The specific interface area of positively charged chitosan particulates in the dispersion medium was as high as 100 more than for chunk-sized flocculates of chitosan particulates under neutral conditions, resulting in physical stabilization of the chitosan particulates in the indicator medium and homogeneous dispersion with invisibility. Electrostatic repulsion of charged chitosan particulates under acidic conditions prevented flocculation of chitosan molecules. Ionization of chitosan molecules under acidic conditions decreased the colloidal size of the particulates and, therefore, improved the dispersion stability, resulting in the homogeneous appearance of the indicator medium. The thermodynamic stability of the lyophilic chitosan particulates under acidic conditions indicated that the Gibbs energy was less than for the chunk-sized flocculates of chitosan particulates under neutral conditions. Under acidic conditions, dispersions of positively charged chitosan particulates or molecules spontaneously form the homogeneous appearance with invisible chitosan particulates due to dissociation from macroscopic flocculates. When the ph of the indicator medium was reduced to acidic conditions, the flocculates of chitosan particulates were dissociated into small particulates, resulting in an increase in both the specific surface area and the number of particles. The entropy of the small chitosan particulates under acidic conditions after accumulation of CO 2 in the indicator medium was larger than for flocculates of chitosan particulates under initial neutral conditions. Consequently, the surface Gibbs energy of the small chitosan particulates under acidic conditions was small (favorable reaction). Irreversibility of the BB-incorporated chitosan-based CO 2 indicator The absorbance and visual appearance of the irreversible CO 2 indicator are shown, respectively, in Figs. 2 and 3 when serially adjusted from ph 7.0, to 5.0 and 7.0. The indicator had a relatively high absorbance (0.259 abs) before ph modification with many precipitates visible to the naked eye. The indicator showed a transparent blue color and a low absorbance value of abs at ph 5.0. The indicator showed a similar absorbance value of abs after re-neutralization at ph 7.0. The indicator did not return to the original absorbance and visibility values, but remained a slightly turbid blue. Thus, chitosan did not reunite with BB because chitosan particles have stronger binding among themselves than with BB. In irreversibility testing, chitosan particulates were small enough to indicate that the ph of the indicator was recovered to neutral conditions and those chunks of chitosan flocculates did not reappear. After re-neutralization of the ph in the indicating medium, chitosan particulates and molecules has reduced charges, but did not recover spontaneously to form a phase separation of chitosan particulates, which were more thermodynamically stable under re-neutralization conditions than under the initial neutral conditions with respect to the flocculation of chitosan molecules. In general, re-neutralization of the ph in the indicator medium made chitosan particulates less lyophilic due to a decrease in the surface charge, resulting in unfavorable reactions due to an increase in the surface Gibbs energy value. However, chitosan particulates did not recover spontaneously for reflocculated into larger particles. The number of small-size chitosan particulates was slightly increased or retained (not

5 Dye-incorporated Chitosan Based CO 2 Indicator 909 Fig. 2. Effect of ph on a dye-incorporated chitosan-based CO 2 indicator for absorbance. (A) initial ph 7.0, (B) ph 5.0 due to CO 2 exposure, and (C) ph 7.0 due to re-neutralization Fig. 4. Changes in (A) ph and (B) TA during makgeolli storage at 4 C for 10 days and 25 C for 6 days. Fig. 3. Irreversibility of a dye-incorporated chitosan-based CO 2 indicator with changes in ph values. (A) ph 7.0, (B) ph 5.0, and (C) ph 7.0 re-flocculated) even after re-neutralization of the ph to 7.0, although enlargement of particles was favorable due to a decrease in the specific surface area under lyophobic conditions for reduced charge chitosan molecules at ph 7.0. In addition, as a result of steric repulsion between chitosan particulates, close contact between colloidal chitosan particulates was hindered, which prevented enlargement of particulates, resulting in stabilization of particulates against flocculation. The TA and ph values of makgeolli during storage Changes in ph and TA values of makgeolli stored at 4 o C for 10 days and at 25 o C for 6 days are shown in Fig. 4. At 25 o C, the ph of makgeolli was initially 3.39, but a significant increase was observed thereafter. Active fermentation of makgeolli was observed at the beginning of storage and, correspondently, the ph increased up to 3.98 within 60 h. At 4 o C, the initial 4.17 ph of makgeolli steadily increased up to The TA profile changed depending on the progress of fermentation (Fig. 4). At 25 o C, the initial TA of makgeolli was approximately 0.19% and steadily increased up to 2.0% during storage. The TA of makgeolli increased more rapidly at 25 o C than at 4 o C since fermentation of makgeolli was faster at 25 o C. The TA of makgeolli stored at 4 o C was initially 0.18%, remained constant for 24 h, increased slowly up to 0.39% after 96 h, then decreased to Similarly, at 25 o C, the TA began decreasing after 48 h. The decrease in the TA was caused by malolactic fermentation, which is a kind of bioconversion of organic acids by LAB. During fermentation of makgeolli, the LAB Lactobacillus sp. and Leuconostoc sp. transformed malic acid (containing 2 carboxyl groups) to lactic acid (containing 1 carboxyl group) (15,16). As a result, the number of carboxyl groups decreased and,

6 910 Lee et al. Fig. 5. Changes in the gas content in the headspace of makgeolli packaging over time in (A) makgeolli and (B) distilled water at 4 o C for 10 days and in (C) makgeolli and (D) distilled water at 25 o C for 6 days. correspondingly, the TA decreased during the middle stages of storage. MLF is known to contribute to taste and flavor improvements in makgeolli due to decreases in organic acid and acetaldehyde production over time (16). Headspace gas composition measurement Variations in the headspace gas composition in makgeolli packages storage at 4 o C for 6 days and 25 o C for 10 days are shown in Fig. 5. Positive control results for distilled water are shown in Fig. 5B and D. During fermentation, yeast converts glucose (sugar) into CO 2, which accumulates, while the O 2 content decreases until exhaustion. In addition, ethanol and carbon dioxide, which contribute to yeast metabolism, make a smaller contribution to wine flavor than organic acids and higher alcohols (17). The CO 2 content showed an increasing trend during makgeolli fermentation. An increasing CO 2 content was observed in the first 24 h at 4 o C and in the first 6 h at 25 o C with final CO 2 concentrations of 70% at 4 o C and 90% at 25 o C, on average, respectively. Positive control samples showed no changes in the headspace gas content. Signal response of the BB-incorporated chitosan based CO 2 indicator during makgeolli storage Changes in ph values of the BB-incorporated chitosan-based CO 2 indicator during makgeolli storage at 4 o C for 10 days and 25 o C for 6 days are shown in Fig. 6A. The BB-incorporated chitosan-based CO 2 indicator installed in the headspace of makgeolli packaging showed a rapid ph decrease during the post-fermentation process. At 25 o C, the ph of the indicator in makgeolli packaging declined to 5.01 at 6 h after storage, subsequently increased slightly, and was stable at approximately 5.1 within 24 h. Similarly, at 4 o C, the ph decreased rapidly to close to 5.0, then subsequently remained stable without any significant change. The ph of the BBincorporated chitosan-based CO 2 indicator was not effective for representation of makgeolli quality. The change of TA (a main sensory factor for makgeolli) over storage time was slower than for the ph in the indicator. The initial transparency values of BB-incorporated chitosan-based CO 2 indicators in makgeolli packaging during storage at 4 o C for 10 days and 25 o C for 6 days were

7 Dye-incorporated Chitosan Based CO 2 Indicator 911 other microorganisms (20,21). The BB-incorporated chitosanbased CO 2 indicator is an off-on model dualistic indicating system. Matching of the indicator dissolution point with the initial point of fermentation or the optimal freshness point was the focus of this study. In conclusion, a dye-incorporated chitosan-based CO 2 indicator is transparent and blue when released from chitosan under acidic conditions during fermentation of stored makgeolli. In addition, the visual chitosan-based CO 2 indicator can indicate partial pressure changes of CO 2 and has potential for application in monitoring of the optimal ripeness of fermented foods and for monitoring the freshness or microbial spoilage status of a wide variety of foods during storage, transportation, and distribution. Acknowledgments This research was supported by the Agriculture Research Center (ARC, SB120) program of the Ministry for Food, Agriculture, Forestry and Fishries, Korea. Disclosure The authors declare no conflict of interest. References Fig. 6. Changes in the (A) ph and (B) transparency of a dyeincorporated chitosan-based CO 2 indicator during makgeolli and distilled water storage at 4 o C for 10 days and 25 o C for 6 days and 43.7%, respectively (Fig. 6B). Lee (18) reported that, due to an increasing CO 2 level in indicator sachets and carbonic acid formation, a decrease in indicator ph and dissolution of chitosan and BB dye particles occurred. The solution then became transparent with a color change to dark blue. The elapsed time to reach the maximum transparency of indicators was different under different storage conditions, although transparency values increased up to a maximum value of approximately 80%. The elapsed time to reach the maximum transparency of the indicator was 48 h at 25 o C and 216 h at 4 o C. As a result, the transparency of the BB-incorporated chitosan-based CO 2 indicator responded properly to the makgeolli quality. The visual change to a clear blue appearance of the indicator was accompanied by a change in the TA value over the storage time. Therefore, excessive fermentation of makgeolli can be indicated using the indicator installed in the headspace of a makgeolli bottle. CO 2 is a by-product of yeast metabolism during makgeolli fermentation and CO 2 accumulates during fermentation. However, the specific mechanism for the effect between CO 2 and bacteria is not well known (19). Also, CO 2 inhibits growth of yeast and 1. Meng X, Kim S, Puligundla P, Ko S. Carbon dioxide and oxygen gas sensors-possible application for monitoring quality, freshness, and safety of agricultural and food products with emphasis on importance of analytical signals and their transformation. J. Korean Soc. Appl. Bi. 57: (2014) 2. Ahvenainen R, Hurme E. Active and smart packaging for meeting consumer demands for quality and safety. Food Addit. Contam. 14: (1997) 3. Puligundla P, Jung J, Ko S. Carbon dioxide sensors for intelligent food packaging applications. Food Control 25: (2012) 4. Jung J, Lee K, Puligundla P, Ko S. Chitosan-based carbon dioxide indicator to communicate the onset of kimchi ripening. LWT-Food Sci. Technol. 54: (2013) 5. Korea National Tax Service Liquor Analysis Regulation. National Tax Service Technical Service Institute, Seoul, Korea. pp (2013) 6. Rinaudo M. Chitin and chitosan: Properties and applications. Prog. Polym. Sci. 31: (2006) 7. Pillai CKS, Paul W, Sharma CP. Chitin and chitosan polymers: Chemistry, solubility, and fiber formation. Prog. Polym. Sci. 34: (2009) 8. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: (1976) 9. Kang D, Gho YS, Suh M, Kang C. Highly sensitive and fast protein detection with coomassie brilliant blue in sodium dodecyl sulfatepolyacrylamide gel electrophoresis. B. Korean Chem. Soc. 23: (2002) 10. Giles CH, Hassan ASA. Adsorption at organic surfaces V-a study of the adsorption of dyes and other organic solutes by cellulose and chitin. J. Soc. Dyers Colour. 74: (1958) 11. Wong YC, Szeto YS, Cheung WH, McKay G. Equilibrium studies for acid dye adsorption onto chitosan. Langmuir 19: (2003) 12. Chang M-Y, Juang R-S. Adsorption of tannic acid, humic acid, and dyes from water using the composite of chitosan and activated clay. J. Colloid Interf. Sci. 278: (2004)

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