Journal of Chromatography A, 1105 (2006) 106–110

Analysis of free amino acids in fermented shrimp waste by

high-performance liquid chromatography
J. López-Cervantes, D.I. Sánchez-Machado ∗ , J.A. Rosas-Rodrı́guez
Departamento de Biotecnologı́a y Ciencias Alimentarias, Instituto Tecnológico de Sonora, P.O. Box 541, Cd. Obregón, Sonora, México
Available online 6 September 2005

Abstract
This work presents an HPLC method for the quantification of free amino acids in lyophilized protein fraction from shrimp waste hydrolysate
which is obtained by acid lactic fermentation and analyzed using pre-column derivatization with 9-fluorenylmethyl-chloroformate. The amino acids
were separated in a Hypersil ODS 5 ␮m column (250 mm × 4.6 mm) at 38 ◦ C. The mobile phase was a mixture of phase A: 30 mM ammonium
phosphate (pH 6.5) in 15:85 (v/v) methanol/water; phase B: 15:85 (v/v) methanol/water; and phase C: 90:10 (v/v) acetonitrile/water, with flow rate
1.2 ml/min. Fluorescence detection was used at an excitation wavelength of 270 nm and an emission wavelength of 316 nm. Method precisions for
the different amino acids were between 4.4 and 7.1% (relative standard deviation, RSD); detection limits were between 23 and 72 ng/ml; and the
recoveries were between 89.0 and 95.0%. The amino acid present at the highest concentration was tyrosine.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Free amino acids; HPLC; FMOC; Shrimp waste; Lactic fermentation

1. Introduction gives greater sensitivity than the traditional methods using

The overwhelming majority of foods contain amino acids
either in the free form or in the form of protein, partially
hydrolyzed or intact. In recent years, it has become more com-
mon to analyze for free amino acid content in foods and nutri-
tional products. This is due to the growing practice of supple-
menting nutritional products with added free amino acids [1,2].
During the past few years, the evolution of instrumental analysis
has allowed the detection and quantification of a greater number
of free amino acids with greater accuracy [3]. There is at the
same time increasing interest in the development of rapid, reli-
able and precise analytical methods. Diverse analytical methods
have been proposed for the analysis of amino acids, including
gas chromatography, HPLC and capillary electrophoresis [4].
Traditionally, the determination of amino acids has been
conducted by ion-exchange chromatography, followed by post-
column derivatization with ninhydrin. In recent years, the
analysis of amino acids using pre-column derivatization and
reversed-phase high-performance liquid chromatography (RP-
HPLC) separation of the derivatives has become widely accepted
[5]. This approach requires much shorter analysis times and
∗ Corresponding author. Tel.: +52 644 4109000; fax: +52 644 4109001.
E-mail address: dsanchez@itson.mx (D.I. Sánchez-Machado).
ion change chromatography and post-column derivatization
[6]. Typical derivatization reagents include 9-fluorenylmethyl-
chloroformate (FMOC-Cl) [7–9], orthophthalaldehyde (OPA)
[10–12], phenylisothiocyanate (PITC) [13–17], 1-fluoro-2,4-
dinitrobenzene, 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide
and dansyl-chloride. Each of these reagents has specific advan-
tages and limitations [18]. Only PITC, FMOC-Cl and OPA are
widely used in the analysis of amino acids, but PITC derivati-
zation methods have poor sensitivity compared with methods
based on fluorometric detection, while OPA does not react with
secondary amino acids. The FMOC derivatization method is
attractive as it is applicable to both primary and secondary amino
acids. The derivatization is rapid and is conduced at ambient tem-
perature, along with the resultant derivatives being very stable
and highly fluorescent [6,18].
Shrimp production in Sonora, México, mainly of genus
Penaeus, was around 40,300 t in 2004 [19]. Only 55% of the
animal is edible, the rest is composed of inedible cephalotho-
rax and exoskeleton [20–22]. This waste is rich in protein
and chitin. Lactic acid fermentation of shrimp waste has been
reported as an effective and economical technique to protect
this biomass from bacterial decomposition, and the fermented
waste formed a silage containing a protein rich liquor, a lipid
fraction and the insoluble chitin [23–28]. Generally, the shrimp
waste hydrolysate has a high content of essential amino acids,

0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2005.08.040
 J. López-Cervantes et al. / J. Chromatogr. A 1105 (2006) 106–110
indicating a high nutritional value used for food, feed or as a
nitrogen source in growth media for micro-organisms [29]. The
shrimp waste investigation monitors the kinetics of deproteiniza-
tion of shrimp shell [20,30], nutritional components of shrimp
salt-fermented sauce [31], the possibility of producing amino
acids from shrimp shell [32] and the compositional character-
ization of a protein hydrolysate [29,33]. This study team has
no knowledge of other studies about the amino acid content in
protein fraction obtained from fermented shrimp waste.
In this work, we present an HPLC method, after derivatiza-
tion with 9-fluorenylmethyl chloroformate, for analysis of free
amino acids in lyophilized protein fraction from shrimp waste
hydrolyzate obtained by lactic acid fermentation.
2. Experimental
2.1. Standards and reagents
HPLC-grade methanol and acetonitrile were obtained from
EMD Chemicals (Darmstadt, Germany). Glacial acetic acid,
boric acid, anhydrous ammonium monohydrogen phosphate,
anhydrous dihydrogen phosphate, sodium hydroxide, EDTA
and HCl were obtained from Products Monterrey (Monterrey,
Nuevo Leon, México). Amino acids standard, hydroxylamine
hydrochloride, 9-fluorenylmethyl chloroformate (FMOC) and
(2-(methylthio)-ethanol were purchased from Sigma (St. Louis,
MO, USA). All reagents were analytical grade, unless other-
wise noted. All aqueous solutions were prepared with ultrapure
water purified with NANOpure Diamond UV system (Barnstead
International, Dubuque, Iowa, USA). Ammonium monohydro-
gen phosphate, (NH4 )2 HPO4 , stock solution (2 M), used for
preparation of HPLC eluents, was adjusted to pH 6.5 with ammo-
nium dihydrogen phosphate, NH4 H2 PO4 .
Sixteen amino acids were detected in the samples: aspartic
acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), histi-
dine (His), arginine (Arg), threonine (Thr), alanine (Ala), proline
(Pro), tyrosine (Tyr), valine (Val), methionine (Met), isoleucine
(Ile), leucine (Leu), phenylalanine (Phe) and lysine (Lys). Amino
acid standards were dissolved in 0.1 M hydrochloric acid, and
diluted to obtain different concentrations and calculated from
calibration plots. The standard curves covered the concentration
ranges of all samples. All samples were analyzed in duplicate.
Solution derivatization of amino acids. FMOC-Cl was dis-
solved in acetonitrile as a 4 mg/ml. Borate buffer was prepared
from a 250 mM boric acid solution adjusted to pH 8.5 with
1 M sodium hydroxide solution prepared from sodium pellets.
The alkaline cleavage reagent was prepared daily in 1000 ␮l
batches by mixing 680 ␮l of 850 mM sodium hydroxide solution
with 300 ␮l of 500 mM hydroxylamine hydrochoride solution
and 20 ␮l of 2-(methylthio)ethanol. The quench reagent was
acetonitrile–acetic acid (8:2).
2.2. Equipment
The HPLC system (GBC, Dandenong, Australia) was
equipped with an auto injector LC 1650, an on line solvent
degasser LC 1460, a system controller with WinChrom for chro-
107
Table 1
Gradient program employed for the separation of FMOC-amino acids
Time (min) Eluent A (%) Eluent B (%) Eluent C (%)
0 16.5 69 14.5
26 11 44 45
26.10 0 0 100
30 0 0 100
30.10 16.5 69 14.5
43 16.5 69 14.5
matography data analysis, a pump LC 1150, a column oven LC
1150, a 20-␮l injection loop (Rheodyne, Cotati, CA, USA) and
a fluorescence detector LC 5100.
Chromatographic analysis was performed using an analyti-
cal scale (4.6 mm × 250 mm) SGE Hypersil ODS C18 column
with a particle size 5 ␮m (SGE, Dandenong, Australia). HPLC
conditions were as follows: mobile phase A: 30 mM ammonium
phosphate (pH 6.5) in 15:85 (v/v) methanol:water; mobile phase
B: 15:85 (v/v) methanol:water; mobile phase C: 90:10 (v/v) ace-
tonitrile:water. The flow rate was constant at 1.2 ml/min and
the column maintained at 38 ◦ C. The detection was by fluores-
cence using the wavelengths of excitation and emission at 270
and 316 nm, respectively. The total time between injections was
43 min. The gradient program used is shown in Table 1.
2.3. Samples
Slightly thawed minced shrimp waste samples were fer-
mented at 30 ◦ C for 36 h. The silage was centrifuged to obtain the
chitin-rich fraction (sediment), the protein rich liquor (liquor)
and the lipid fraction. The protein liquor was lyophilized and
ground. After that the samples were conserved in desiccator and
in darkness until their analysis.
2.4. Sample preparation
To evaluate free amino acids, amino acids were extracted
from the dry samples with borate buffer. Specifically, 25 mg of
finely ground freeze-dried sample was placed in a volumetric
flask and diluted to 25 ml with borate buffer, to obtain a concen-
tration of 1 mg/ml. Afterwards, the samples were sonicated for
2 min for complete dissolution. The solution was then ready for
the derivatization process.
2.5. Derivatization
The method of analysis of Haynes and Sheumack was
followed with minor modifications. To derivatize, 300 ␮l of
the amino acid standard solution or of sample prepared was
deposited in vial with capacity for 1.5 ml and then 300 ␮l of
FMOC reagent was added and vortexed for 90 s. The cleavage
reagent was added (180 ␮l) and the tubes were vortexed for 15 s,
then left for 5 min at room temperature, 420 ␮l of quench reagent
was then added. The resulting solution was vortexed for 15 s and
filtered with a membrane 0.45 ␮m. A 5 ␮l sample of this solution
was injected onto the column of the HPLC system.
108 J. López-Cervantes et al. / J. Chromatogr. A 1105 (2006) 106–110

Fig. 2. HPLC profile of a mixture of amino acids standards.

Fig. 1. Example HPLC profile of amino acids in lyophilized protein fraction
from shrimp waste hydrolysate.
2.6. Statistical analysis
For the descriptive and regression statistical analyses, the
computer program used was SPSS 11.0 for Windows (SPSS
Inc., Chicago, IL). The relative standard deviation (RSD, %) is
the ration standard deviation/average value in percentage.
3. Results and discussion
3.1. Derivatization procedure
By comparison with other derivatization methods for HPLC
analysis of amino acids, FMOC derivatization requires a rel-
atively short time both for derivatization and for passage of
FMOC derivatives through the column [9]. Initially, we per-
formed derivatization assays for the optimization of sample
alkalinization. Ultrapure water and borate buffer (pH 7.8 and
8.5) were tested. The better results were achieved by using borate
buffer pH 8.5. Trials indicated that the derivatized amino acids
were stable for 72 h when stored at ambient temperature. The
derivatization of amino acid with FMOC-Cl requires alkaline
conditions, pH ≥ 8.0 [6,9]. The total time required for derivati-
zation was 8 min.
The sample preparation method was simple and consisted
of diluting the shrimp waste hydrolyzate powder in the selected
solvent (borate buffer pH 8.5). Some authors have suggested that
sample quantity depends on protein content [13,14,18]. For the
determination of the amount of sample to use in the chromato-
graphic analysis, assays were made in different concentrations
from sample powder (0.5, 1.0, 1.5, 2.0, 3.0 and 5.0 mg/ml) with
different time lengths of sonification (0, 2 and 4 min), all of them
dissolved in borate buffer. The process indicated that acceptable
results are achievable with 0.5 and 1.0 mg/ml. For the analy-
sis of all samples, we decided to use 1.0 mg/ml and 2 min of
sonification.
3.2. Identification and separation
The HPLC method proposed allows simultaneous analysis of
16 amino acids in 43 min, including 8 min for elution of FMOC
amino acid derivatives, which elute after the derivatized amino
acids. The different amino acids were identified by comparison

Table 2
Results of linear regression analysis of calibration data
Amino acid Range (␮g/ml) Intercept (b)a Slope (a)a Standard error constant (Sb ) Standard error slope (Sa ) r2

Asp 6.6–53.0 9607.5 12477 5319.6 174.2 0.9993

Glu 5.4–42.7 8752.1 4821.5 3827.4 155.4 0.9993
Ser 5.3–42.6 9427.9 10368 4934.3 201.2 0.9991
His 5.7–68.6 3614.2 264.84 1087.9 28.1 0.9999
Gly 6.4–51.2 11387.0 20488 7222.3 244.8 0.9991
Thr 5.9–47.5 7275.9 8247.2 3264.3 119.2 0.9994
Ala 4.8–57.8 10415.0 15035 5728.1 175.6 0.9991
Pro 5.0–59.5 6441.4 6468.8 2287.8 68.1 0.9997
Tyr 4.8–57.1 3228.1 5446.2 1727.8 49.7 0.9995
Arg 4.5–53.8 4187.4 2303.3 781.8 25.7 0.9999
Val 6.2–49.6 7641.9 6947.8 4317.8 151.0 0.9992
Met 5.6–67.9 3572.1 1591.4 721.9 19.1 0.9999
Ile 5.4–42.9 7201.0 4825.2 2451.4 99.2 0.9997
Leu 6.2–74.4 7212.2 5014.6 3128.4 109.4 0.9995
Phe 5.2–62.4 5998.2 8826.4 2569.2 73.0 0.9996
Lys 6.7–53.6 8778.1 14047 5357.9 173.4 0.9992

r2 : determination coefficient.
a y = ax + b.
 J. López-Cervantes et al. / J. Chromatogr. A 1105 (2006) 106–110 109

with retention times for amino acid stock solutions. For determi- Table 4
nation of retention times, the reference standards were injected Recovery of amino acids added lyophilized protein fraction from shrimp waste
hydrolysate
both individually and as a mixture. For optimization of elution
conditions, this team also experimented with different eluent Amino acid Mean value Amount addition Amount found Recovery (%)
proportions (i.e. A:B:C proportions) during the stages of elution, Asp 17.7a 13.0a 29.5a 90.8b
four mobile phase flow rates (0.8, 1.0, 1.20 and 1.40 ml/min) Glu 26.2 10.8 35.9 90.5
and three different column temperatures (34, 36 and 38 ◦ C). Ser 13.5 10.2 23.2 95.0
Finally, better reproducibility is obtained at 38 ◦ C, 1.20 ml/min His 12.1 9.9 21.3 93.8
Gly 28.0 12.1 39.5 94.6
with mobile phase compositions shown in Table 1. An example Thr 19.0 10.6 29.0 94.2
of an HPLC separation of the amino acids from a lyophilized Ala 29.8 9.3 38.3 91.4
from shrimp waste hydrolysate is shown in Fig. 1. Fig. 2 shows Pro 25.9 9.5 34.7 93.2
the HPLC separation of a standard solution. The concentration Tyr 40.6 9.1 49.1 93.6
of the amino acids standard solution injected ranged from 17.9 Arg 23.0 8.6 31.2 94.6
Val 21.4 11.8 32.2 91.4
(Arg) to 26.8 (Lys) ␮g/ml. Met 13.2 10.5 22.9 92.5
Ile 16.2 10.2 25.8 94.3
Leu 29.3 11.8 40.0 90.7
3.3. Analytical characteristics Phe 23.4 9.9 32.5 91.9
Lys 15.4 13.0 27.0 89.0
The relationship between concentrations and peak area is a Results expressed as mg/g dry mass.
shown by a, b and r2 in Table 2, where a and b are the coefficient b Percent recovery = (amount found − mean value)/(amount added) × 100.
of the regression equation y = ax + b, x being the concentration
of the analyte and y the peak area and r2 is the determination
Accuracy was estimated by means of recovery assays. For
coefficient of the equation. In all cases, the relationships between
evaluation of recovery, six samples of powdered shrimp waste
concentration and peak area were linear over the range tested,
hydrolysate were spiked with known concentrations of amino
with coefficients of determination greater than 0.999. All curves
acids prior to extraction, derivatization and quantitation. Recov-
are based on the analyses of at least four dilutions of the corre-
ery was in all cases good, considering the concentrations and
sponding amino acid standard.
type of analytes, in line with a previous evaluation of a similar
Method precisions (i.e. between-run coefficients of variation,
method for analysis of free amino acids in foods [14]. Recovery
eight runs of a single sample) ranged from 4.4% (Thr) to 7.1%
ranged from 89.0% (Ala) to 95.0% (Gly). Table 4 shows the
(His, Lys) (Table 3); mean reproducibility (6.0%) can be con-
recovery of amino acids.
sidered good.
Detection limits (defined as three times the signal-to-noise
ratio), in line with American Chemical Society guidelines [34]) 3.4. Sample characteristics
ranged from 23 ng/ml (Lys) to 72 ng/ml (Tyr) (Table 4), in line
with values reported in related previous studies [9]. The practical applicability of the method was assessed by the
analysis of 16 samples. Amino acid profile content in the sam-

Table 3 Table 5
Retention times, method precision and detection limits for the different amino Free amino acid content (mg/g dry mass) in lyophilized protein fraction from
acids considered shrimp waste hydrolysate

Amino Retention Method precision (n = 8) Detection Amino acid Concentration (mg/g dry mass)
acid times (min) limit (ng/ml)
Mean ± SD RSD (%) Average Maximum Minimum
(mg/g dry mass)
Asp 17.5 ± 4.08a 26.3 9.9
Asp 5.04 ± 0.25 19.3 ± 1.20 6.2 53 Glu 24.0 ± 4.83 31.9 15.6
Glu 5.50 ± 0.28 28.0 ± 1.53 5.5 59 Ser 13.6 ± 3.42 18.4 7.4
Ser 11.70 ± 0.52 13.7 ± 0.75 5.5 42 His 11.8 ± 1.67 15.3 8.9
His 11.74 ± 0.51 10.7 ± 0.78 7.1 62 Gly 28.4 ± 8.57 38.3 8.3
Gly 12.55 ± 0.55 27.0 ± 1.32 4.9 30 Thr 31.6 ± 7.17 39.7 18.4
Thr 13.04 ± 0.56 17.8 ± 0.78 4.4 47 Ala 20.4 ± 9.19 35.6 8.6
Ala 13.85 ± 0.57 31.4 ± 1.76 5.6 35 Pro 20.2 ± 5.59 28.9 9.8
Pro 14.54 ± 0.56 21.7 ± 1.16 5.4 46 Tyr 56.9 ± 17.81 76.2 16.4
Tyr 15.64 ± 0.56 45.1 ± 3.00 6.7 72 Arg 27.0 ± 4.23 35.4 19.4
Arg 17.49 ± 0.63 23.9 ± 1.55 6.5 68 Val 24.8 ± 1.84 27.6 20.9
Val 18.28 ± 0.56 22.7 ± 1.34 5.9 46 Met 9.3 ± 0.57 10.4 8.5
Met 18.07 ± 0.60 10.4 ± 0.72 6.9 60 Ile 18.8 ± 1.87 21.9 14.8
Ile 20.89 ± 0.61 17.5 ± 1.09 6.2 52 Leu 29.5 ± 1.85 33.2 26.9
Leu 21.07 ± 0.59 33.3 ± 1.95 5.9 52 Phe 21.6 ± 2.13 24.6 17.2
Phe 22.26 ± 0.59 23.2 ± 1.28 5.5 66 Lys 23.8 ± 3.92 34.2 18.4
Lys 33.96 ± 0.46 22.0 ± 1.55 7.1 23 a Means ± standard deviations (n = 16).
110 J. López-Cervantes et al. / J. Chromatogr. A 1105 (2006) 106–110
ples is shown in Table 5. Free amino acid contents ranged from
9.3 mg/ml dry mass (Met) to 56.9 mg/g dry mass (Tyr); glycine
and leucine are the other major amino acids found. Tyrosine was
the most abundant amino acid, which has also been reported
in protein from shrimp shell [20]. Similar results have been
obtained previously for other protein hydrolysate from shrimp
waste produced by commercial protease [29]. Free amino acids
are one of the most important fractions of non-protein, and many
of these, such as alanine, glutamic acid and glicine are respon-
sible for flavour and taste. Alanina and glicine have a sweet
taste, and glutamic acid has the umami taste, typical of crus-
taceans [35]. Additionally, free amino acids have also been used
as quality indicators in various fish and crustacean species [36].
4. Conclusions
The HPLC method presented here allows the simultaneous
analysis of 16 free amino acids in protein hydrolysate from
shrimp waste. This methodology is easy and fast, the derivates
were stable and both primary and secondary amino acids could
be detected. The method validation is satisfactory, and the
method’s viability has been confirmed by the analysis of var-
ious hydrolysate samples.
Acknowledgements
This work was financed under project no. ITSON-EXB-36
from the Program of Profesor’s Improvement from the Public
Education Department (PROMEP). Michael A. Bryan, MBA
and Native English Professor, offered suggestions and improve-
ments in the wording of this manuscript.
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