Optimization of Chitin Extraction from Shrimp Shells

First of all, the nitrogen content was measured by elemental analysis and the percent-age of proteins calculated from the following equation where P% represents the percentage of protein

Optimization of Chitin Extraction from Shrimp Shells

Aline Percot, Christophe Viton, and Alain Domard*

Laboratoire des Mate´riaux Polyme`res et des Biomate´riaux, UMR-CNRS 5627, Baˆtiment ISTIL,

Domaine Scientifique de la Doua, 15 Bd Andre´ Latarjet, 69622 Villeurbanne Cedex, France

Received June 25, 2002; Revised Manuscript Received October 4, 2002

The aim of this paper is to define optimal conditions for the extraction of chitin from shrimp shells The

kinetics of both demineralization and deproteinization with, in the latter case, the role of temperature are

studied The characterization of the residual calcium and protein contents, the molecular weights, and degrees

of acetylation (DA) allows us to propose the optimal conditions as follows The demineralization is completely

achieved within 15 min at ambient temperature in an excess of HCl 0.25 M (with a solid-to-liquid ratio of

about 1/40 (w/v)) The deproteinization is conveniently obtained in NaOH 1 M within 24 h at a temperature

close to 70°C with no incidence on the molecular weight or the DA In these conditions, the residual

content of calcium in chitin is below 0.01%, and the DA is almost 95%

Introduction

Sources of chitin are estimated to be as abundant as those

for cellulose with a yearly production of approximately 1010

-1012T.1Chitin is a polysaccharide corresponding to linear

copolymers ofβ(1f4)-linked 2-amino-2-deoxy-D-glucan and

2-acetamido-2-deoxy-D-glucan Chitin, especially its main

derivative chitosan, has numerous applications, for example,

in agriculture, biomedicine, paper making, and food

indus-tries.2Some applications require specific architectures, and

the effectiveness of the polymers for these applications was

shown to depend on the molecular weight distribution and

the degree of acetylation (DA).3-5A cost-effective, fast, and

easily controlled industrial process for producing chitins of

high molecular weight and DA still remains to be developed

The main sources of raw material for the production of

chitin are cuticles of various crustaceans, principally crabs

and shrimps However, chitin in biomass is closely associated

with proteins, minerals, lipids, and pigments They all have

to be quantitatively removed to achieve the high purity

necessary for biological applications Although many

meth-ods can be found in the literature for the removal of proteins

and minerals, detrimental effects on the molecular weight

and DA cannot be avoided with any of these extraction

processes.6 Therefore, a great interest still exists for the

optimization of the extraction to minimize the degradation

of chitin, while, at the same time, bringing the impurity levels

down to a satisfactory level for specific applications

Demineralization is generally performed by acids including

HCl, HNO3, H2SO3, CH3COOH, and HCOOH but HCl

seems to be the preferred reagent and is used with a

concentration between 0.275 and 2 M for 1-48 h at

temperatures varying from 0 to 100 °C.1 Madhavan and Ramachandran Nair7 have shown that the viscosity of the chitosan obtained decreases with the treatment time in HCl, suggesting a decrease of the molecular weight with time Deproteinization of chitin is usually performed by alkaline treatments, although other effective reagents have been reported Typically, raw chitin is treated with approximately

1 M aqueous solutions of NaOH for 1-72 h at temperatures ranging from 65 to 100°C.1An interesting alternative method involves the enzymatic degradation of proteins However, the residual protein satisfactory level, ranging approximately from 1% to 7%, remains higher and the reaction time is longer compared to that of the chemical way These drawbacks make the enzymatic method8 unlikely to be applied industrially before progress is made in making the process more efficient

In this first paper on the study of the production of chitin,

we propose an optimized method of extraction for the production of biological-grade chitin with both high molec-ular weights and DA The kinetics of demineralization and deproteinization using two classical methods have been studied to achieve these objectives The purity level of chitin was followed by the evaluation of the calcium and protein contents as a function of the reaction time In parallel, the influence of both treatments on the preservation of the chitin structure was studied from the control of the molecular weight and DA after each step

Materials and Methods Raw Materials and Preparation Shells of marine shrimp

Parapenaeopsis stylifera were provided by France-Chitine.

The tiny brown shrimp, which is common all over the Indian Ocean, originates in our case from the port of Jakham (India)

* To whom correspondence should be addressed.

10.1021/bm025602k CCC: $25.00 © 2003 American Chemical Society

located on the Arabian Sea The shrimps were kept on ice

for 2 or 3 days before being peeled; the shells were scraped

free of loose tissue and washed individually in lightly salted

water The following procedure was chosen by the producer

as an optimal treatment for a long time preservation of the

raw material The shells were then separated from

cephal-othoraxes, salted (10 kg of NaCl per 500 g of shell), and

dried in the sun (25-30°C) for 3 days Prior to use, the

shrimp shells were washed thoroughly in distilled water until

the conductivity reached that of water The shells were then

freeze-dried and cryo-ground under liquid nitrogen The

powder thus obtained was sieved, and the fraction below 80

µm was used hereafter.

Characterization of Shrimp Shells and Intermediary

Products The water content in the obtained powders was

estimated by thermogravimetric analysis using a Du Pont

Instrument TGA 2000 with 10-20 mg samples and a

temperature ramp of 2°C/min

The percentage of proteins remaining in chitin was

determined by two different methods First of all, the nitrogen

content was measured by elemental analysis and the

percent-age of proteins calculated from the following equation

where P% represents the percentage of proteins remaining

in the obtained powder and N%represents the percentage of

nitrogen measured by elemental analysis with 6.9

corre-sponding to the theoretical percentage of nitrogen in fully

acetylated chitin and 6.25 corresponding to the theoretical

percentage of nitrogen in proteins In the case of the crude

shrimp shells, no accurate values could be obtained because

of the great amount of calcium carbonate present For this

special case, the amount of proteins was measured using the

amino acid analysis Two milligrams of sample was

hydro-lyzed with HCl 6 M in the presence of trifluoroacetic acid

for 45 min at 150°C under vacuum The samples were then

solubilized in a buffer, and an aliquot was used for analysis

on a Beckman system 6300 amino acid analyzer The

percentage of proteins was calculated from the total amino

acid weight

The lipid content was estimated after a Soxlhet extraction

with chloroform/methanol (2/1, v/v) and subsequent

gravi-metric analysis of the shrimp shells

The ash content was determined by slowly heating a

sample to 900 °C with stages at 120 and 340 °C and

weighing the remaining product after cooling in a desiccator

For the minerals, in addition to ashes analysis, levels of

calcium and magnesium were analyzed by inductively

coupled plasma atomic emission spectroscopy (ICP-AES)

and ICP mass spectrometry (ICP-MS) Prior to analysis, the

solid samples were digested in concentrated sulfuric acid in

a microwave reactor until complete dissolution had occurred

Determination of the Degree of N-Acetylation (DA) of

Chitin Chitin samples were dissolved in DCl/D2O (20%

w/w) with vigorous stirring for 8 h at 50°C These conditions

were necessary to sufficiently depolymerize chitin, thus

allowing the full solubilization of the polymer

The spectra were recorded on a Bruker AC 200

spectrom-eter (200 MHz for1H) at 298 K The DA was calculated, as

proposed by Hirai et al.,9from the ratio of the methyl proton

signal of (1f4)-2-acetamido-2-deoxy-β-D-glucan residues with reference to the H-2 to H-6 proton signals of the whole structure

For samples containing proteins, solid-state 13C NMR spectroscopy was used Indeed, in this case, the too high amount of proteins makes too difficult the interpretation of the proton NMR spectra The spectra were obtained on lyophilized samples with CP-MAS techniques (cross polar-ization, magic angle spinning) using a Bruker DSX400 instrument working at 100.6 MHz Typical conditions were

as follows: 90 RF pulse, 4.5µs; contact time, 2.5 ms; pulse

repetition, 2s; MAS rate, 5 kHz; 4096 scans were acquired The measurements were performed at room temperature The

DA was calculated by comparison between the integrated areas of the methyl group carbon (δ 24 ppm) and the

C2-C6 signals (δ 56-105 ppm).9

Determination of the Intrinsic Viscosity of Chitin.

Chitin samples were solubilized at about 0.25 mg/mL in

N,N-dimethylacetamide (DMAc) containing 5% lithium chloride (LiCl).10,11The viscosity was measured using an automatic capillary viscometer, Viscologic TI 1 SEMATech (diameter 0.8 mm), at 25°C

Kinetics of Demineralization Demineralization was

carried out in dilute HCl solutions Typically, the shrimp shells were soaked in HCl 0.25 M at ambient temperature with various solution-to-solid ratios under constant stirring The demineralization kinetics were followed by monitoring the pH as a function of time in the supernatant The characteristics of the obtained chitin as a function of the demineralization time were studied by retrieving a repre-sentative sample of known volume from the dispersion of shrimp shell particles at 2, 6, 13, 30, 60, 180, 360, and 1440 min using a syringe with a large needle The heterogeneous samples were then filtered under vacuum on paper, and the supernatant was analyzed for the pH and the calcium content (by ICP-AES) The recovered demineralized shrimp shell powder was washed to neutrality and freeze-dried Following the demineralization step, the partly demineralized shrimp shells were deproteinized with a solution of NaOH 1 M under vigorous stirring using 30 mL of solution per gram of demineralized shells After 24 h of reaction, the solid samples were washed to neutrality and freeze-dried The calcium content in the purified chitin was measured by ICP-AES

Kinetics of Deproteinization The kinetic studies of

deproteinization were performed on demineralized shrimp shell powder (Figure 1) Quantitative demineralization was carried out in HCl 1 M at a room temperature corresponding

to 22 ( 1°C with a solution-to-solid ratio of 10 mL/g After

24 h, the demineralized shrimp shell powder was removed

by filtration, washed to neutrality, and freeze-dried Depro-teinization kinetics studies were then performed by addition

of NaOH 1 M to decalcified powder at a solution-to-solid ratio of 15 mL/g, and a representative sample was taken at

5, 20, 60, 180, 300, and 1440 min The partly deproteinized shrimp shell powder was removed in each sample by filtration, washed to neutrality, and freeze-dried, while the supernatant was analyzed for the protein content

The release of proteins in the supernatant was followed

by the measurement of the absorbance at 280 nm,

charac-teristic of the tryptophan residues present in the protein

composition The absorbance was measured on an Uvikon

(UV-vis.) spectrophotometer 941 (Kontron Instruments)

Shrimp shell proteins were recovered by a classical process

of deproteinization on demineralized shrimp shells in NaOH

1 M for 24 h with a solution-to-solid ratio of 15 mL/g (Figure

1) After filtration, the supernatant containing the proteins

was dialyzed against pure water for 1 week and lyophilized

The protein content in the recovered powder was determined

by amino acid analysis and found to be close to 60% (w/w)

These proteins were used to plot a calibration curve giving

a straight line with the following equation

where abs corresponds to the UV absorbance measured at

280 nm, Cpcorresponds to the protein concentration in mg/

mL, and r corresponds to the correlation coefficient.

The weight of proteins released could be then expressed

as a weight percentage of the initial weight of decalcified

shrimp shells

The deproteinization was also studied as a function of

temperature In this case, NaOH 1 M was added to

de-mineralized shrimp shells with a solution-to-solid ratio of

15 mL/g The deproteinization was carried out for 24 h at

various temperatures The protein content was measured in

the supernatant and in the obtained chitin

Results and Discussion

The overall process for the preparation of shrimp chitin

is given in Figure 1 The raw shrimp shells contain about

20% of chitin and other components reported in Table 1

While CaCO3 is the major inorganic component, some

magnesium is also present in a low proportion

Demineral-ization (usually performed in concentrated acid) and

depro-teinization (in aqueous NaOH) are therefore the critical steps

of chitin extraction It is generally agreed that the processing conditions strongly affect the molecular weight and DA of chitin As a rule, as the acidic conditions for demineralization (pH, time, and temperature) become harsher, the molecular weight of the products thus obtained becomes lower Indeed, chitin is an acid-sensitive material and can be degraded by several pathways: hydrolytic depolymerization, deacetyla-tion, and heat degradation leading to physical property modifications

Although HCl may be the cause of detrimental effects on the intrinsic properties of the purified chitin, it remains the most commonly used decalcifying agent in both laboratory and industrial scale production of chitin It is generally used

at a concentration of 1 M Chang and Tsai12 determined optimal demineralization conditions for the shell of pink

shrimp (Solenocera melantho) measuring the calcium level

in the obtained product but without taking into account the chitin characteristics that they obtained In this paper, a concentration of HCl of 0.25 M, below all values reported

in the literature was tested to minimize the hydrolysis of the polymer The solution-to-solid ratio was kept above 10 mL/g

to obtain a homogeneous mixture with a large excess of solution The demineralization occurs when the acid reacts with the calcium carbonate according to the following simple equation:

Figure 1 Overall process for the preparation of chitin from salted shrimp shells.

abs ) Cp× 1.7 r2) 0.999 73 (2)

Table 1 Composition of Crude Shrimp Shells, Demineralized

Shrimp Shells (24 h in HCl 1 M), and Chitin (24 h in NaOH 1 M)

composition

shrimp shells (wt %)

demineralized shrimp shells (wt %)

chitin (wt %)

crude fat 6(2 ash (as oxide) 35.49(0.04

magnesium 1 a

a Measured by ICP-MS b Measured by ICP-AES c Measured by amino acid analysis d Measured by elemental analysis.

Therefore, pH increases till the end of the reaction Rhazi et

al.13have proposed the use of acidimetric titration to follow

the demineralization process, and the end of the reaction is

related to the persistence of the acidity in the medium In

Figure 2A, we can see a fast increase of pH as a function of

the reaction time In this experiment, 20 mL of HCl 0.25 M

was initially added to 1 g of shrimp shells After 30 s, 98%

of the added acid had already reacted After 2 h, the pH of

the solution was neutral, and 10 mL of HCl 0.25 M was

further added to the solution, leading successively to a

decrease of pH and then a rapid increase The added acid

thus reacted with the remaining calcium carbonate, and after

3 h, the pH of the solution reached a plateau at 5.5 Still

another 10 mL of acid was added to the solution We may

now consider that the acid added was in excess because the

pH remained acidic and stable at 1.8 even after 4 h From

these results, we may estimate the amount of acid necessary

to carry out the reaction to completion and we may also

follow the kinetics of the reaction Then, a similar experiment

was carried out from a unique addition of acid in excess:

40 mL of HCl 0.25 M was added to 1 g of shrimp shell

powder, and from Figure 2B, we can see that the reaction

was complete after only 15 min (pH remained acidic and

constant with time) After 18 h, another addition of 10 mL

of HCl 0.25 M was made to make sure that the reaction was

indeed complete pH remained acidic at a value of 1.4 in a

similar manner as in Figure 2A after the second addition of

HCl To verify that the measure of pH could actually be

used to accurately follow the demineralization kinetics, we

performed an experiment in the same conditions while

samples of the mixture were collected periodically with a

large syringe HCl 0.25 M in excess was then added to a

shrimp shell powder

Figure 3A shows that pH measurement increases in the supernatant with the calcium concentration Because the latter was very high in the supernatant, the samples had to be diluted before ICP-AES analysis This might have caused additional experimental errors on the results These results were used to calculate the number of molecules of H3O+

(nH 3 O+) having reacted with the calcium carbonate in relation

with the number of calcium ions (nCa) released from the shrimp shell powder The following ratio was obtained:

As expected, the experimental result is about 2 and cor-roborates eq 3 The difference between the theoretical and obtained value results from the uncertainty of the pH measurement and of the calcium concentration We also determined the amount of calcium remaining in the decalci-fied shrimp shell as a function of time For the mineralization

of the sample, several methods were tested: solubilization

of dry ashes in acid, mineralization of the sample in acid with heating at 900°C, and digestion of the sample in sulfuric acid in a microwave reactor With the first two methods, there was always an insoluble fraction and the obtained results were not reproducible Only the digestion in a microwave oven gave accurate results Both the decalcified samples and the obtained chitin were mineralized in this reactor In Figure 3B are reported the kinetics of deminer-alization in relation with the calcium content in the material Two minutes after the beginning of the process, the calcium content in the samples is about 168µg/g of powder, while

the lowest amount possible to be reached with the process

is near 108 ( 11µg/g after 24 h of treatment All of the

samples obtained were deproteinized in NaOH 1 M for 24 h

at ambient temperature The calcium content in the chitin obtained was measured (Figure 3B) The nondemineralized

chitin sample (t ) 0) contains 0.28 g of calcium per gram

Figure 2 Kinetics of demineralization: (A) HCl was initially in default,

and then, 10 mL of HCl 0.25 M was successively added at 120 and

180 min; (B) HCl was initially in excess, and then, 10 mL of HCl 0.25

M was added after 18 h.

Figure 3 Kinetics of demineralization in HCl 0.25 M at ambient

temperature: (A) determination of the pH (9) and the calcium concentration (0) in the supernatant as a function of time; (B) calcium content in the demineralized shrimp shell (b) and in the corresponding chitin (O) as a function of time.

nH

of deproteinized shrimp shells If we consider the percentage

of impurities (proteins and lipids) eliminated during the

deproteinization step, we can estimate that nCa eliminated

during the demineralization step corresponds to nCarecovered

in the supernatant more or less 10% In Figure 3B, we can

observe a slight decrease of the calcium content with the

demineralization time, and the calcium content in the

obtained chitin is about 75µg/g after complete

demineral-ization

All of these results show that the demineralization times

reported in the literature are too long Thus, with an excess

of HCl 0.25 M (with a solid-to-solvent ratio of about 1/40

(w/v) corresponding to 10 times more H3O+than necessary),

the reaction of demineralization is mostly complete within

15 min Then, the excess time will only contribute to the

degradation of chitin

Table 2 shows the variation of the intrinsic viscosity of

chitin as a function of demineralization time A rapid increase

of [η] was observed with time This increase parallels the

demineralization kinetics as can be seen in Figure 3B The

samples recovered before the end of the extraction process

(2 and 6 min) are characterized by a substantial amount of

minerals remaining in the chitin, insoluble in the solvent used

for viscometric experiments As a consequence, this added

weight causes a significant error on the true chitin

concentra-tion thus reducing the apparent viscosity of the mixture

compared to a pure sample of a similar chitin After 13 min,

the calcium ratio in chitin becomes negligible, and then, [η]

remains stable for 3 h, followed by a decrease in the

viscosity In the latter case, the acid hydrolysis of chitin is

the only mechanism occurring in the media Table 2 also

shows the variation of DA as a function of time The values

obtained were above 95% for all the samples, and no

significant decrease was observed with time Then, we may

consider that in HCl 0.25 M the treatment has no particular

effect on the DA of chitin As a consequence, we may

conclude that in native shrimp shells the DA should be close

to 96% ( 3% This value is approximately 6% over that of

squid pens (to be published)

Deproteinization Deproteinization by alkaline treatment

was shown to be much less damaging to the chitin structure

compared to the acidic treatment involved in the

deminer-alization.1For this reason, we decided to use the conventional

procedure for the deproteinization of our samples Thus, it

was carried out in NaOH 1 M using a solution-to-solid ratio

of 15 mL/g The amount of proteins extracted from the demineralized shrimp shell powder was followed as a function of time from the variation of the absorbance at 280

nm, characteristic of tryptophan This method differs from the conventional method that involves the determination of proteins remaining in chitin (using the nitrogen content) or the determination of the protein content in the supernatant

by a colorimetric method such as the Lowry assay.14Hunt and Nixon15also examined the ultraviolet absorption spectra

of the alkaline supernatants for tryptophan To plot a more reliable calibration curve, we extracted proteins or peptides from demineralized shrimp shells and used these proteins to plot a calibration curve As expected, the absorbance at 280

nm increases linearly with the amount of proteins and the equation of the curve (see Experimental Section) can be used

to calculate the concentration of proteins in the supernatant NaOH 1 M was added to a demineralized shrimp shell powder (demineralization proceeded in HCl 1 M at room temperature with a solution-to-solid ratio of 10 mL/g) with

a solution-to-solid ratio of 15 mL/g at ambient temperature With a large syringe, we took samples from the mixture, filtered the solution, and measured the absorbance in the supernatant Figure 4 shows the increase of the amount of proteins released in the supernatant at ambient temperature

as a function of the deproteinization time At the same time,

we can observe a decrease of the percentage of proteins in the recovered chitin To compare the results obtained by both methods, we transformed the protein concentration in the supernatant into a protein weight and then into the percentage

of remaining proteins assuming that after 24 h the whole of the proteins was extracted (Figure 4) These calculated percentages are similar to those obtained by elemental analysis Although both methods gave the same results, taking into account the experimental errors, the absorbance method was a much simpler method to follow the depro-teinization process as a function of time The reaction is complete (percentage of proteins below 2%) after 6 h

We also characterized the chitins obtained to evaluate the effect of deproteinization on the molecular weight of chitin and its DA In Table 3, we notice the rapid increase of the intrinsic viscosity of chitin with time The phenomenon is

Table 2 Characterization of the Extracted Chitin upon

Demineralization (HCl 0.25 M) at Ambient Temperature a

demineralization

time (min) [η] (mL/g) DA (%) b

a The characterization was performed after a deproteinization

pro-ceeded for 24 h in NaOH 1 M under vigorous stirring using 30 mL of

solution per gram of demineralized shells The intrinsic viscosity [η] and

DA are given as a function of time b Measured by liquid NMR.

Figure 4 Kinetics of deproteinization in NaOH 1 M at ambient

temperature Variation of the protein concentration in the supernatant, expressed in mg/mL (O), and remaining in the obtained chitin (measured by elemental analysis), expressed in protein % (9) is shown The measured protein concentration in the supernatant can also be used to calculate the percentage of proteins remaining in the obtained chitin (b).

similar to that for the demineralization step Thus, until 3 h,

chitin is polluted by a nonnegligible amount of partially

hydrolyzed proteins remaining insoluble in water This

phenomenon affects the viscosity by reducing the amount

of chitin really present in solution and certainly also by a

lower contribution to the viscosity of these proteins Beyond

3 h, the protein content becomes negligible and the intrinsic

viscosity is then stable, even after 24 h of treatment Because

NaOH is a deacetylating agent, the DA of chitin was

measured as a function of time by solid-state NMR Because

of the presence of proteins, the integrations of the various

carbons were overestimated and the DAs measured for times

below 1 h were erroneous For high deproteinization times

and fully deproteinized samples, no significant difference

could be observed among all of the samples (taking into

account the experimental errors)

The role of temperature on the deproteinization process

was estimated from reactions performed for 24 h at various

temperatures In Figure 5, the protein concentration in the

supernatant is plotted as a function of temperature We

observe an increase of the amount of proteins extracted with

temperature, while the percentage of proteins remaining in

the obtained chitin decreases Temperature seems to be a

critical parameter for the deproteinization with respect to the

purity of the obtained chitin Differences between both results

can be expected because the deproteinization process carries

on during the washing step In the case of elemental analysis, negative values were obtained for almost pure products because of the low precision of the method

In Table 4, the intrinsic viscosity of the various chitin samples obtained after 24 h of deproteinization in NaOH 1

M are given as a function of the deproteinization temperature The product obtained at 15°C remains polluted by nonhy-drolyzed or very weakly hynonhy-drolyzed proteins, and as a consequence, the determination of the viscosity is wrong For the deproteinizations carried out at higher temperatures, the obtained chitin is purer and the viscosities are stable with time As expected, the deproteinization conditions do not involve the hydrolysis of the chain even at 70°C However, the alkaline treatment can induce the hydrolysis of the

N-acetyl linkage and decrease the DA The DA was estimated

by solid NMR (data not shown) No deacetylation was noticed, even for the reaction performed at 70°C Then, it

is possible to increase the rate of deproteinization by an increase of temperature without any influence on DA up to

70 °C In a paper in preparation, we will further describe the kinetics and thermodynamic parameters of the depro-teinization steps

Conclusion

The demineralization process of shrimp shells can be simply followed by the measurement of the variation of pH

in the supernatant, and the increase of pH is easily related

to the calcium release The study of the variation of pH also allows us to follow the kinetics of demineralization and then

to estimate the optimal reaction time and to foresee the exact amount of acid necessary to perform a complete reaction thus minimizing the hydrolysis of the glycosidic bonds We notice that the demineralization times reported in the literature are too long Indeed, the reaction is mostly complete within 15 min in the conditions used in this paper Another interesting result is that the DA of obtained chitin remains stable with this mild acidic treatment

In NaOH 1 M as reactive, the deproteinization process is slow and several hours are necessary to perform a satisfying deproteinization This behavior has to be related both to the

R chitin structure of difficult access to the reactives and to the various kinds of possible interactions with proteins (paper

in preparation) The measurement of the absorbance of the supernatant at 280 nm is a reliable, simple method to follow the protein release The reaction rate can be increased by an increase of temperature The effect of temperature on the intrinsic characteristics of the obtained chitin was studied,

Table 3 Characterization of the Extracted Chitin upon

Deproteinization in NaOH 1 M at Room Temperature a

deproteinization

time (min) [η] (mL/g) DA (%) b

a This step was performed after a demineralization proceeded for 24 h

in HCl 1 M at room temperature with a solution-to-solid ratio of 10 mL/g.

The intrinsic viscosity [η] and DA are given as a function of time.

b Measured by solid NMR.

Figure 5. Deproteinization in NaOH 1 M for 24 h at various

temperatures Measurement of the protein concentration in the

supernatant at the end of the reaction (O) and determination of the

amount of proteins remaining in the obtained chitin (measured by

elemental analysis) (9) as a function of the deproteinization

temper-ature is shown.

Table 4 Variation of the Intrinsic Viscosity [η] and DA of the Extracted Chitins upon Deproteinization (NaOH 1 M) for 24 h at Various Temperatures

deproteinization temperature ( ° C) [η] (mL/g) DA a (%)

a Measured by solid NMR.

and as expected, the deproteinization time and temperature

have no influence on both the molecular weight and DA

using NaOH 1 M with a temperature and a reaction time

below 70°C and 24 h, respectively

Acknowledgment This work belongs to the CARAPAX

project financially supported by the EC through the 5th

PCRD

References and Notes

(1) Roberts, G A F In Chitin chemistry; Roberts, G A F., Ed.;

Macmillan Press Ltd.: London, 1992.

(2) Ravi Kum, M N V React Funct Polym 2000, 45, 1.

(3) Muzzarelli, R A A.; Tanfani, F.; Emanuelli, M.; Chiurazzi, E.; Piani,

M In Chitin in Nature and Technology; Muzzarelli, R A A.,

Jeuniaux, C., Gooday, G W., Eds.; Plenum Press: New York, 1986;

p 469.

(4) Vander, P.; Vårum, K M.; Domard, A.; El Gueddari, N E.;

Moerschbacher, B M Plant Physiol 1998, 118, 1353.

(5) Ho¨rner, V.; Pittermann, W.; Wachter, R In AdVances in Chitin

Science; Proceedings of the 7th International Conference on Chitin/

Chitosan; Domard, A.; Roberts, G A F.; Vårum, K M., Eds; Jacques Andre´ Publisher: Lyon, France, 1997; p 671.

(6) Kurita, K.; Tomita, K.; Tada, T.; Ishii, S.; Nishimura, S.; Shimoda,

K J Polym Sci., Part A: Polym Chem 1993, 31, 485.

(7) Madhavan, P.; Ramachandran Nair, K G Fish Technol 1974, 11,

50.

(8) Shirai, K.; Palella, D.; Castro, Y.; Guerrero-Legaretta, I.;

Saucedo-Castan˜eda, G.; Huerta-Ochoa, S.; Hall, G M In AdVances in Chitin Science; Proceedings of the third Asia-Pacific Chitin and Chitosan

Symposium; Hirano, S.; Tokura, S.; Kurita, K.; Hsing-Chen, C.;

Rong, H C., Eds; Rong H C., Hsing, C C Publisher: 1998, p 103.

(9) Hirai, A.; Odani, H.; Nakajima, A Polym Bull 1991, 26, 87.

(10) Teng, W L.; Khor, E.; Tan, T K.; Lim, L Y.; Tan, S C Carbohydr.

Res 2001, 332, 305.

(11) Austin, P R German Patent 2,707,164, 1977.

(12) Chang, K L B.; Tsai, G J Agric Food Chem 1997, 45,

1900-1904.

(13) Rhazi, M.; Desbrie`res, J.; Tolaimate, A.; Alagui, A.; Vottero P Polym.

Int 2000, 49, 337.

(14) Peterson, G L Anal Biochem 1977, 83, 346.

(15) Hunt, S.; Nixon, M Comp Biochem Physiol 1981, 68B, 535.

BM025602K