Production of high-quality chitin and chitosan from preconditioned shrimp shells

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Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 81:1113–1118 (2006) DOI: 10.1002/jctb.1437 Production of high-quality chitin and chitosan from preconditioned shrimp shells Nguyen Van Toan, 1,2Chuen-How Ng, 1,3 Kyaw Nyein Aye, 1,4 Trung Si Trang 1,5 and Willem F. Stevens 1,6 1 Bioprocess Technology, Asian Institute of Technology, PO Box 4, Klong Luang, Pathumthani 12120, Thailand 2 International University, Ho Chi Minh City, Vietnam 3 Primex Ltd, Reykjavik, Iceland 4 Yangon Technical University, Yangon, Myanmar 5 University of Fisheries, Nha Trang, Vietnam 6 Centre for Chitin and Chitosan Biomaterials, Chulalongkorn University, Bangkok, Thailand Abstract: Chitin and chitosan with improved characteristics were produced from shrimp shell waste preconditioned by limited decay or by treatment with 0.016 mol L 1 benzoic acid. Preconditioned shrimp shells were transparent, had a clean surface and were susceptible to demineralization and deproteinization using 0.68 mol L 1 HCl and 0.62 mol L 1 NaOH, respectively. The ash and protein residues in the final chitosan were about 0.2% and 0.4%, respectively, the viscosity was up to 7000 cps, and the solubility and transparency nearly 100%. In comparison with treatment at ambient temperature (30 C) without preconditioning, the chemical consumption, the duration of the treatment, ash and protein residues was reduced to 75–25%, whereas viscosity and absence of insolubles improved by a factor of 2–3. 2006 Society of Chemical Industry Keywords: chitosan; chitin; preconditioned shrimp shell; limited decay; benzoic acid INTRODUCTION The cationic biopolymer chitin can be isolated very effectively from shrimp shell by removal of protein, minerals and low molecular components using alkali and strong acid. This method is applied at the industrial scale but has two major disadvantages: it lacks sustainability and it damages the final product, particularly during treatment at elevated temperature. Prolonged alkali treatment leads to aldol condensation products, while acid treatment causes fission in the chitin backbone by hydrolysis of the β -glucosydic bonds in the chitin chain. In a search for treatments that might precondition the shell solid material to make it more susceptible to protein and calcium removal, two methods have emerged that seem to be extremely useful for application at a large scale for chitin production. The methods are (1) limited decay and (2) treatment with acetic acid or benzoic acid. Data on limited decay have been originally obtained in studies in our laboratory on the application of Lactobacillus fermentation to remove protein and calcium carbonate from crude waste of shrimp shell. 1 In control studies without added inoculum, autofermentation phenomena were observed that led to a fast decay of the waste but also to a high degree of deproteination and demineralization. The decay process, probably initiated by enzymes produced by the shrimp intestinal microflora, generates putrescent compounds causing an awful smell and making the shrimp non-amenable for processing. The intestinal bacteria, however, also produce proteases and organic acid that lead to solubilization of protein and minerals. In the present study we have tried to find acceptable conditions in which autofermentation assisted in the isolation of chitin to remove protein and minerals but decay and damage to the chitin molecule were minimal or did not occur. The other approach has been to look for addition of a food-grade compound to the shrimp shell slurry prior to deproteination and deacetylation that leads to preconditioning of the waste and facilitates further treatment. One of the compounds chosen for the study is benzoic acid, which is known for its bacteriostatic and fungistatic action and is effective in keratolysis of cornified epithelium. If the keratolytic effect were to apply in the layered structure of protein, Ca 2+ and chitin in shrimp skeletal biomaterial, the shell after benzoic acid treatment might be more susceptible to alkali deproteination and/or HCl demineralization. In this paper successful application of both limited decay and preconditioning using acetic acid and benzoic acid are reported. Correspondence to: Nguyen Van Toan, School of Biotechnology, International University, Block 6, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam E-mail: [email protected] (Received 16 May 2005; revised version received 12 August 2005; accepted 12 August 2005) Published online 24 February 2006 2006 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2006/$30.00 1113

Transcript of Production of high-quality chitin and chitosan from preconditioned shrimp shells

Page 1: Production of high-quality chitin and chitosan from preconditioned shrimp shells

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 81:1113–1118 (2006)DOI: 10.1002/jctb.1437

Production of high-quality chitin and chitosanfrom preconditioned shrimp shellsNguyen Van Toan,1,2∗ Chuen-How Ng,1,3 Kyaw Nyein Aye,1,4 Trung Si Trang1,5

and Willem F. Stevens1,6

1Bioprocess Technology, Asian Institute of Technology, PO Box 4, Klong Luang, Pathumthani 12120, Thailand2International University, Ho Chi Minh City, Vietnam3Primex Ltd, Reykjavik, Iceland4Yangon Technical University, Yangon, Myanmar5University of Fisheries, Nha Trang, Vietnam6Centre for Chitin and Chitosan Biomaterials, Chulalongkorn University, Bangkok, Thailand

Abstract: Chitin and chitosan with improved characteristics were produced from shrimp shell wastepreconditioned by limited decay or by treatment with 0.016 mol L−1 benzoic acid. Preconditioned shrimpshells were transparent, had a clean surface and were susceptible to demineralization and deproteinizationusing 0.68 mol L−1 HCl and 0.62 mol L−1 NaOH, respectively. The ash and protein residues in the finalchitosan were about 0.2% and 0.4%, respectively, the viscosity was up to 7000 cps, and the solubilityand transparency nearly 100%. In comparison with treatment at ambient temperature (30 ◦C) withoutpreconditioning, the chemical consumption, the duration of the treatment, ash and protein residues wasreduced to 75–25%, whereas viscosity and absence of insolubles improved by a factor of 2–3. 2006 Society of Chemical Industry

Keywords: chitosan; chitin; preconditioned shrimp shell; limited decay; benzoic acid

INTRODUCTIONThe cationic biopolymer chitin can be isolated veryeffectively from shrimp shell by removal of protein,minerals and low molecular components using alkaliand strong acid. This method is applied at theindustrial scale but has two major disadvantages: itlacks sustainability and it damages the final product,particularly during treatment at elevated temperature.Prolonged alkali treatment leads to aldol condensationproducts, while acid treatment causes fission in thechitin backbone by hydrolysis of the β-glucosydicbonds in the chitin chain. In a search for treatmentsthat might precondition the shell solid material tomake it more susceptible to protein and calciumremoval, two methods have emerged that seem tobe extremely useful for application at a large scale forchitin production. The methods are (1) limited decayand (2) treatment with acetic acid or benzoic acid.

Data on limited decay have been originally obtainedin studies in our laboratory on the application ofLactobacillus fermentation to remove protein andcalcium carbonate from crude waste of shrimpshell.1 In control studies without added inoculum,autofermentation phenomena were observed that ledto a fast decay of the waste but also to a high degreeof deproteination and demineralization. The decay

process, probably initiated by enzymes produced bythe shrimp intestinal microflora, generates putrescentcompounds causing an awful smell and making theshrimp non-amenable for processing. The intestinalbacteria, however, also produce proteases and organicacid that lead to solubilization of protein and minerals.In the present study we have tried to find acceptableconditions in which autofermentation assisted in theisolation of chitin to remove protein and minerals butdecay and damage to the chitin molecule were minimalor did not occur.

The other approach has been to look for additionof a food-grade compound to the shrimp shell slurryprior to deproteination and deacetylation that leadsto preconditioning of the waste and facilitates furthertreatment. One of the compounds chosen for the studyis benzoic acid, which is known for its bacteriostaticand fungistatic action and is effective in keratolysis ofcornified epithelium. If the keratolytic effect were toapply in the layered structure of protein, Ca2+ andchitin in shrimp skeletal biomaterial, the shell afterbenzoic acid treatment might be more susceptible toalkali deproteination and/or HCl demineralization.

In this paper successful application of both limiteddecay and preconditioning using acetic acid andbenzoic acid are reported.

∗ Correspondence to: Nguyen Van Toan, School of Biotechnology, International University, Block 6, Linh Trung Ward, Thu Duc District, HoChi Minh City, VietnamE-mail: [email protected](Received 16 May 2005; revised version received 12 August 2005; accepted 12 August 2005)Published online 24 February 2006

2006 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2006/$30.00 1113

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MATERIALS AND METHODSThe shrimp shell waste used in this study was donatedby Thai Royal Frozen Seafood Processing Co. Ltdin Samutsakon, Thailand. The shrimp shells werepeeled and collected in the factory in large plastic bagssurrounded by ice to keep the temperature close to0 ◦C and to avoid direct exposure to sunlight during2 h transportation. Immediately after arrival at theinstitute, the shells were used for chitin and chitosanisolation.

The common procedures for chitin productionstart either with deproteination (Fig. 1A) or withdemineralization (Fig. 1B). For preconditioning, twomethods have been used (Fig. 1C, D). In procedure Cthe shrimp shell was allowed to decay naturally at theambient temperature for 24–48 h before subjectingthe material to chitin processing. This limited decayshould last long enough to cause the beneficial effectsdescribed in this paper. Usually that is after 48 h. Thegeneration of an awful smell and the appearance ofvery clean shell material (Fig. 3) are the operationalcriteria. Decay during 24 h is usually, but not always,too short. Decay during 72 h results in complete decayof the shrimp waste including that of the chitin. Thepresence of organic acids in the fermentation fluid wasinvestigated by gas chromatography.5

In procedure D, the shrimp shell waste was soakedin 0.05 mol L−1 acetic acid or 0.016 mol L−1 benzoicacid solution for 8 h.

The preconditioned shrimp shell was demineralizedwith 0.68 mol L−1 HCl solution (1:5 w/v) at ambienttemperature (28–32 ◦C) in procedure C for 12 h andin procedure D for 6 h. The residue was washed andsoaked in tap water for 6–8 h. It was then dewateredand deproteinized with 0.62 mol L−1 NaOH solution(1:5 w/v) at ambient temperature for 20 h (procedureC) or 16 h (procedure D).

The chitin obtained from the above processes wasdeacetylated in 12.5 mol L−1 NaOH (1:5 w/v) solutionat 65 ◦C for 20 h. After deacetylation, the chitosanwas washed and dried in sunlight and assayed formoisture content, ash content, protein content, degreeof deacetylation, viscosity, solubility, turbidity andmolecular weight.2

The protein content in the chitosan sample wasdetermined using the micro-biuret method.3 Thedegree of deacetylation was analyzed by first-derivativeultraviolet (UV) spectrophotometry.4 Weight averagemolecular weight was determined by gel permeationchromatography (Waters GPC) with a differentialrefractometer detector.2 Dextrans of various molecularweights ranging from 9.9 × 103 to 2 × 106 were usedas standards.

The crystallinity of chitosan in powder form wasmeasured by Rigaku X-ray diffractometer, model‘Miniflex’ (USA), using nickel-filtered CuKα at ascanning speed of 2◦ min−1 over the 2θ range from 5◦to 30◦.

Shrimp shell Shrimp shell

Chitin

(C)Limited Decay30°C, 24-48 h

(D)Benzoic acid (0.016 mol L−1)

orAcetic acid (0.05 mol L−1)

30°C, 8 h

(B)Demineralization1.1 mol L−1 HCl,

30°C, 12 h

(A)Deproteination

1 mol L−1 NaOH,

30°C, 24 h

Deacetylation,12.5 mol L−1, NaOH,

65°C, 20 h

Chitosan

Deproteination1 mol L−1 NaOH,

30°C, 24 h

Demineralization1.1 mol L−1 HCl,

30°C, 12 h

Demineralization0.68 mol L−1 HCl,

30°C, 12 h (C),30°C, 6 h (D)

Deproteination0.62 mol L−1 NaOH,

30°C, 20 h (C)30°C, 16 h (D)

Deacetylation,12.5 mol L−1, NaOH,

65°C, 20 h

Chitin

Chitosan

Figure 1. Comparison between the common process (A and B) and revised process (C and D) including preconditioning for the production of chitinand chitosan. Between all steps washing is carried out to remove all soluble materials, including released protein, mineral salts and non-usedchemical reagent.

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Ash content was determined by the standardAOAC method.6 Various physicochemical criteriawere investigated for chitosan in a 1% solution in0.35 mol L−1 acetic acid. Turbidity was assessed usinga turbidimeter (Model 2100P portable turbidimeter,HACH Company, USA) and viscosity by a BrookfieldModel DV-VII+ viscometer. Solubility was measuredusing the transglucosidase method:7 the pH of50 mL 1% (w/v) chitosan solution was adjustedto 4.8 with 30% (w/v) sodium acetate and mixedwith transglucosidase L-500 (Genencor International,500 µL). After incubation at 60 ◦C for 24 h, theinsoluble material was collected by filtration usinga pre-weighed Whatman GF/C filter paper (1.2 µm).The filter paper was dried and weighed and the amountof insolubles was calculated from its weight gain.

RESULTSEffect of limited decay on chitin productionAt present, the main sources for industrial isolationof chitin are the exoskeletons of crustaceans, mainlycrab and shrimp. In these materials, the chitin is ina tight-layered complex bound to protein and inor-ganic material, mostly CaCO3. Various procedureshave been adopted to isolate shrimp chitin and removethe unwanted materials. Demineralization is achievedby treatment with HCl and deproteination by treat-ment with NaOH8 but other methods have beenproposed as well. Alternatively, chitin can be pro-duced by Lactobacillus fermentation, which removesprotein and calcium carbonate.1 Most proceduresare carried out at elevated temperature (70–90 ◦C),although ambient temperature (28–32 ◦C) is to bepreferred in order to avoid thermo-chemical damage tothe chitin.8 Deproteination is usually applied prior todemineralization.9,10 The choice of processing condi-tions highly affects the quality of chitin and of chitosan,its deacetylated derivative.11 In the research presentedhere, shrimp shell has been preconditioned by limiteddecay at ambient temperature before conducting thenormal chitin isolation process. For comparison, chitinwas isolated at ambient temperature without precondi-tioning (Fig. 1). In the limited decay procedure, fresh

shrimp shells were stored without any treatment atambient temperature (28–32 ◦C). As expected, shrimpshell produced putrescent compounds after 24–48 hof decay and released a bad smell. It was observed thatafter partial decay about 40% of protein is not boundto the solid and can be removed easily by washingthe material with tap water. Without decay, washingcan remove about 20% of the protein. The limiteddecay resulted in a facilitation of the subsequent dem-ineralization. After treatment of a limited decayedsample with five concentrations of HCl, ash content islower in all cases in comparison with similarly treatednon-decayed sample.

Data on mineral removal are shown in Table 1.Demineralization and deproteination were carriedout using 0.68 mol L−1 instead of 1.1 mol L−1 HCland with 0.62 mol L−1 instead of 1 mol L−1 NaOH,respectively. Surprisingly, the limited decay affectedneither the amount nor the quality of chitin in thebiowaste. The characteristics of chitosan producedfrom limited-decay shrimp shell (LD-CTS) andcontrol chitosan (C-CTS) are listed in Table 2. Thedata presented are after decay during 48 h. Shorterdecay time resulted in less evident change. Longerdecay resulted in deterioration of the chitin material.

Effect of organic acid on chitin productionThe processing liquid produced during limiteddecay was collected and analyzed by using gas

Table 1. Ash content of chitin samples (CT) treated with various HCl

concentrations: effect of limited decay chitin (LD-CT) versus control

(C-CT)

Ash content (%) in chitinHCl concentration(mol L−1) C-CT LD-CT

0.55 2.25 ± 0.04 0.91 ± 0.020.69 1.98 ± 0.04 0.70 ± 0.030.82 1.75 ± 0.03 0.68 ± 0.010.96 1.38 ± 0.05 0.62 ± 0.051.10 1.11 ± 0.01 0.55 ± 0.04

Duration of the treatment was 6 h.The ash content of the original shrimp shell was 24.5%.

Table 2. Characteristics of chitosan produced from common method (C-CTS), limited decayed shrimp shell (LD-CTS) and chitosan produced after

treatment with acetic acid (AA-CTS) or benzoic acid (BA-CTS)

Characteristics C-CTSa LD-CTSb AA-CTS BA-CTS

Ash content (%) 0.56 ± 0.20 0.65 ± 0.22 0.52 ± 0.15 0.18 ± 0.25Protein content (%)c 0.55 ± 0.02 0.54 ± 0.25 0.56 ± 0.22 0.40 ± 0.03Solubility (%) 99 ± 0.01 98 ± 0.52 99 ± 0.1 99 ± 0.05Degree of deacetylation (%) 88 ± 0.01 87 ± 0.02 88 ± 0.3 88 ± 0.02Turbidityd (NTU) 25 ± 4.00 30 ± 0.65 20 ± 5.0 13 ± 5Viscosity (cps) 2650 ± 300 5100 ± 270 4500 ± 300 7000 ± 100Molecular weight (daltons ×10−6) 1.71 ± 0.02 1.75 ± 0.02 NDe 1.74 ± 0.01

a Prepared according to procedure in Fig. 1(A).b Limited decay (LD) during 48 h.c Protein content: non-treated shrimp shell 23.5%, after washing with water 18.1%, after limited decay 14.1%, in LD-CT 0.72%.d Chitosan 1% (w/v) dissolved in 0.35 mol L−1 acetic acid. NTU, nephelometric turbidity unit.e Not determined.

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chromatography.5 Various organic acids appeared tobe generated in the liquid such as acetic, propionic,butyric, isobutyric, isovaleric and lactic acid, withacetic acid in the highest concentration. Incubation offresh shrimp shell waste in diluted acetic acid solution(0.05 mol L−1) over 8h was found to be effective inmimicking the effect of preconditioning by limiteddecay. Successful demineralization and deproteinationcould be achieved using rather low acid andalkali concentrations (0.68 mol L−1 and 0.62 mol L−1,respectively). The smell of the fermented productwas much milder and the chitosan produced had agood quality. The characteristics of this acetic acidtreated chitosan (AA-CTS) are shown in Table 2.Other organic acids mentioned above were also usedto mimic the decay process but did not result in achitosan product of good quality.

Effect of benzoic acid on chitin productionAfter the successful application of limited decay andacetic acid treatment, preconditioning with benzoicacid was assessed for the facilitation of deminer-alization and deproteination. Shrimp shells soakedin 0.016 mol L−1 benzoic acid solution for 8 h at28–32 ◦C appeared to be free of hanging-on con-taminants and had a very clean and transparentappearance. The difference in appearance of nor-mal shrimp shell and the benzoic acid preconditionedshrimp shell is shown in Figs 2 and 3. After precondi-tioning by benzoic acid, effective demineralization anddeproteination can be achieved at ambient tempera-ture using lower concentrations of HCl and NaOH.Characteristics of chitosan produced from precon-ditioned shell by benzoic acid (BA-CTS) are listedin Table 2 and compared with control chitosan (C-CTS).

After preconditioning, 0.68 mol L−1 HCl can reducethe ash content to even less than 0.2% within 6 h oftreatment. The deproteination process is facilitated aswell. In 0.62 mol L−1 NaOH, it took not more than16 h to obtain a protein residue of about 0.4%.

It seems that benzoic acid treatment causes a looserstructure of chitin–protein–calcium complexes in the

Figure 2. Appearance of shrimp shell as received from theshrimp-peeling factory, after washing but before any treatment.

Figure 3. Appearance of shrimp shell after preconditioning usingbenzoic acid and after washing.

shell, resulting in a release of non-chitin componentsand a better accessibility for the action of HCl andNaOH.

DISCUSSIONRecently Aye and Stevens described the pretreatmentof fresh shrimp shell waste by washing with acidifiedwater.12 The pretreatment aims to remove looselybound protein prior to deproteination. In thepreconditioning described in this paper, the skeletalmatrix structure is first weakened by limited decay orby benzoic acid, followed by the removal of solubilizedprotein by washing with tap water. Consequently,less chemical treatment is needed in the subsequentdeproteination. In its turn this will lead less exposureof the chitin and chitosan to acid and alkali, to ahigher product quality and a reduction of the proteinand chemical content of the wastewater. The removedprotein can be converted into a protein by-productapplicable as human or feed protein supplement.

Limited decay (LD) does not lead to a lowerquality or yield of chitosan. All characteristic propertiesof LD-CTS are as good as, or better than, thoseof the control (C-CTS). The viscosity of dissolvedLD-CTS is nearly two times higher than that ofC-CTS, showing the high quality of the LD-CTS.Data on the removal of protein and minerals fromthe decayed shrimp shell (shown in Table 2) and theless stringent conditions to achieve demineralizationand deproteination have consolidated the argumentconcerning the loose structure of the shrimp shellmatrix after preconditioning. In spite of the lessstringent extraction conditions, minerals and proteincould be removed efficiently to very satisfying residuallevels of much less than 1% (Table 2).

The main factor to affect the polymer backboneof the chitin molecule is HCl, which cleaves the β-1,4-glucosydic bonds in chitin. As a consequence, thefinal product—chitosan—will have a lower molecularweight and, after dissolution, a lower viscosity. Theshrimp shell matrix in LD-CTS contains less boundprotein. It can be demineralized using less exposure

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to HCl. This will result in a chitosan solution with ahigher viscosity.

Benzoic acid is known for its bacteriostatic andfungistatic properties and is effective in keratolysis ofcornified epithelium. This acid has been widely usedin food conservation as preservative and in pharma-ceutical applications, especially for enhancement ofcutaneous and percutaneous absorption.13 It has beenshown that benzoic acid softens keratin, loosens corni-fied epithelium and causes swelling even of viablecells. In a test of percutaneous absorption throughskin of hairless rat, 10 different chemical compoundswere studied including dexamethasone, hydrocorti-sone, dehydroepidiandrosterone, testosterone, acetyl-salicylic acid, sodium salicylate, caffeine, benzoic acid,manitol and thiourea. The data showed that the mostpenetrating molecule, benzoic acid, penetrated 50times faster than dexamethasone. Benzoic acid softensthe horny layer and enhances the permeability of thestratum corneum.

Exoskeletal chitin–protein complexes give protec-tion against chitinases and mild alkaline conditions.The more strongly the protein is bound to chitin,the harder for NaOH to penetrate and to detach theprotein from the solid shell material. Benzoic acidseems to weaken the exoskeletal complex matrix ofprotein, resulting in the release of a protein frac-tion that otherwise remains bound to the solid waste.Benzoic acid seems to allows NaOH solution topenetrate and facilitate the breakdown of protein com-ponents in the matrix. The effects of benzoic acid onthe chitin and chitosan products are similar to theeffect of limited decay. The viscosity of the benzoicacid treated chitosan (BA-CTS) in solution is veryhigh—7000 cps—compared with 2650 cps of con-trol chitosan (C-CTS). The BA-CTS also has a morecompact structure as compared with C-CTS, proba-bly due to a more intact backbone. Determination ofthe crystallinity of BA-CTS and C-CTS using X-raydiffraction shows a higher crystallinity for the former(Fig. 4).

The degree of deacetylation, solubility and molecu-lar weight of BA-CTS and C-CTS is similar. The tur-bidity of the BA-CTS solution (13 NTU) is about halfthat of the C-CTS (25 NTU). This is a consequence

5 10 15 20 25 30

Figure 4. X-ray diffraction spectra of C-CTS (bottom) and BA-CTS(top). The diffraction peaks of BA-CTS are higher and sharper thoseof C-CTS.

of the very low protein content and very low mineralcontent in the BA-CTS sample. Similar to the limiteddecay, benzoic acid treatment facilitates the chemicalprocessing of shrimp shell waste. In this case, there isno negative effect on the final product either.

Comparing preconditioning by limited decay andbenzoic acid conditioning, the latter is to be preferred.The limited decay treatment produces an awful smell,albeit only during the first phase of demineralization.During benzoic acid conditioning, the product hasan acceptable, even attractive smell. Moreover, thebenzoic conditioning takes a shorter time (8 h insteadof 48 h) and produces chitosan solution with highviscosity and low turbidity.

CONCLUSIONThe study demonstrates that high-quality chitin andchitosan were produced from shrimp shells afterpreconditioning by limited decay or by benzoic acidtreatment. Benzoic acid (0.016 mol L−1) helps byshortening the subsequent chemical treatment andby minimizing the need for chemicals. It avoids thegeneration of a bad smell and enhances the qualityof chitosan product. In this way, chitosan can beproduced at lower cost and in a more environmentallyfriendly way. Due to less aggressive demineralizationand deproteination procedure chitosan with highviscoelastic properties is obtained.

ACKNOWLEDGEMENTSThe authors would like to express their sincere thanksto the late Prince de Lignac for his generous financialsupport. They would like to extend their thanks to DrNi Tar Nwe for useful comments and to Thai RoyalFrozen Seafood Processing Co. Ltd, Samutsakon,Thailand for the supply of raw materials.

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