Food Sci. Biotechnol.
19(2): 553-557 (2010)
DOI 10.1007/s10068-010-0077-z
RESEARCH NOTE
Screening of Industrial Enzymes for Deproteinization of Shrimp
Head for Chitin Recovery
Angel U. Valdez-Peña, Judith D. Espinoza-Perez, Georgina C. Sandoval-Fabian, Nagamani Balagurusamy,
Adriana Hernandez-Rivera, Iliana M. De-la-Garza-Rodriguez, and Juan Carlos Contreras-Esquivel
Received: 7 October 2009 / Revised: 21 December 2009 / Accepted: 8 January 2010 / Published Online: 30 April 2010
© KoSFoST and Springer 2010
Abstract Food grade proteolytic enzymes were examined and as a result large quantities of wastes are generated
for deproteinization of shrimp head. Shrimp head was
easily deproteinized by Alcalase® and trypsin at a pH of
8.0. Alcalase was chosen as the most efficient commercial
enzyme for deproteinization of shrimp head. Alcalase
treatment of shrimp head recorded 61% of weight loss on
dry basis and a residual protein of 275 mg/g dried shrimp
head. The enzymatically deproteinized shrimp head was later
demineralized with lactic acid using microwave radiation at
400 W. The combination of enzymatic and physicochemical
treatments promoted the chitin recovery from dried shrimp
head under eco-friendly conditions.
Keywords: Litopenaues vannamei, protease, microwave,
demineralization, infrared spectroscopy
Introduction
The shrimp aquaculture sector in Mexico has been grown
Angel U. Valdez-Peña, Judith D. Espinoza-Perez, Juan Carlos Contreras-
Esquivel ( )
Research and Development Center, Coyotefoods Biopolymer and
Biotechnology Co., Simon Bolivar 851-A, Saltillo City 25000, Coahuila
State, Mexico
Tel: +52-844-416-9213; Fax: +52-844-439-0511
E-mail: coyotefoods@hotmail.com
Angel U. Valdez-Peña, Georgina C. Sandoval-Fabian
Centro de Investigacion y Asistencia en Tecnología y Diseño del Estado de
Jalisco A.C., Guadalajara City 44270, Jalisco State, Mexico
Nagamani Balagurusamy
School of Biological Sciences, Universidad Autonoma de Coahuila,
Torreon City 27104, Coahuila State, Mexico
Hernandez-Rivera, Iliana M. De-la-Garza-Rodriguez, Juan Carlos
Contreras-Esquivel
School of Chemistry, Universidad Autonoma de Coahuila, Saltillo City
25000, Coahuila State, Mexico
during its processing. High value by-products from shrimp
wastes can be recovered such like carotenoproteins (1,2), chitin
(3-5), and salts (5). Among various by-products, chitin is a
water insoluble polysaccharide constituted substantially of
β-(1→4) linked N-acetyl-D-glucosamine units (6). This
natural biopolymer exists widely in crustaceans, insects,
and microorganisms and has wide potential applications in
different areas.
With the increase in shrimp processing, the amount of
wastes generated is increasing enormously. Shrimp chitin
can be produced by chemical (7), physical (8), enzymatic
(9), or microbiological (10-12) methods. The use of corrosive
chemical agents such as NaOH or HCl in the chemical
method results in environmental pollution (13). Recovery
of chitin by biotechnological process from crustacean shell
waste consists of two fundamental steps: 1) protein degradation
with proteolytic enzyme, and 2) demineralization. However,
demineralization has been done using HCl, either after or
previous to enzymatic deproteinization step (10-12). An
eco-friendly deproteinization/demineralization of shrimp
wastes by combining fermentation with fungal (14) or
bacterial (15) strains and a further treatment with lactic
acid have been studied for chitin recovery. However the
longer fermentation period (48 and 120 hr for bacterial and
fungal strains, respectively) is a limitation. Microwave-
assisted (16,17) technologies can use mild catalysts for
chitin recovery. Recently, we developed a process for chitin
recovery using microwave-assisted technology combined
with a treatment of organic acid (17). Microwave-assisted
technology aided in time and energy saving during
deproteinization and demineralization steps of crustacean
wastes.
A variety of enzymatic procedures for deproteinization
have been developed over the years for the chitin
554
production (9, 18-21). To date, however, there has not been
reported about chitin recovery using enzymatic deproteinization
step combined with microwave-assisted demineralization
step. In this paper, 5 different food-grade commercial
proteolytic enzymes were screened for deproteinization of
shrimp head and the enzymatic process was combined with
microwave-assisted demineralization process for chitin
recovery in order to achieve short reaction times.
Material and Methods
Characterization of protein hydrolyzates by gel
Material and reagents Frozen shrimp (Litopenaues filtration chromatography Chromatographic protein
vannamei) heads were obtained from El Camaron Dorado
Co. (Huatabampo City, Sonora State, Mexico). Frozen
shrimp heads were defrosted in a microwave oven (2,450
MHz, Panasonic Co., Osaka, Japan) and dried in a
convection industrial dryer (Koleff KL26; Queretaro City,
Queretaro State, Mexico) at 55oC until constant weight.
Dried sample was ground through a Waring blender and
stored under vacuum at −0.2 bar at room temperature. Blue
dextran 2000 was purchased from Amersham, Biosciences/
GE Healthcare (Uppsala, Sweden). Reference shrimp chitin
was purchased from Industrias Poseidon (Mexico City,
Mexico). Details on the commercial enzymes, shrimp head
powder and commercial chitin is given in Table 1. Unless
otherwise stated, all commercially available reagents were
purchased from Jalmex (San Nicolas de Los Garza City,
Nuevo Leon State, Mexico).
Microwave-assisted chitin recovery from enzymatically
Enzyme-assisted protein extraction from shrimp head deproteinized shrimp head A large scale microwave
A rotary device (CoyoteZyme-ABL™ Enzyme Reactor)
was designed by Coyotefoods Biopolymer and Biotechnology
Co. (Saltillo City, Coahuila State, Mexico), which can hold
10 vacuum bottles (FoodSaver®, Tila, Rye, New York, NY,
USA) of 0.5-L of capacity, variable rotational speed (1-50
rpm) and power supply of 110 V. Each bottle has a circular
acrylic frame with 2 acrylic baffles to mix slurry under
rotation. Powder shrimp head (10 g) was mixed with 50
mg of correspondent enzymes in 200 mL of 0.2 M
phosphate buffer (pH 8.0). The slurry was placed in
cylindrical containers closed under vacuum (−0.2 bar).
Table 1. Shrimp head meal, commercial food enzymes, and commercial chitin
Code Sample Company
S1 Shrimp head meal Coyotefoods
S2 Alcalase® 2.4 L FG Novozymes
S3 Flavorzyme® 500 MG Novozymes
S4 Lysozyme, Inovapure 300 Inovatech Bioproducts
S5 Papain DSM Food Specialties
S6 Trypsin VI Inovatech Bioproducts
S7 Shrimp chitin Industrias Poseidon
A. U. Valdez-Peña et al.
CoyoteZyme ABL rotary device was used for crude chitin
recovery (deproteinization) at 40 rpm for 6 hr at 37oC.
After treatment, samples were filtered through muslin cloth
and insoluble solids were washed with water and ethanol
(95%) and dried at 50oC until constant weight. Weight loss
was evaluated after drying the residual shrimp solids and
expressed as a percentage. Extracts obtained from shrimp
head were filtered under vacuum through a glass fiber filter
and acetate cellulose (0.70-µm, Millipore, Billerica, MA,
USA) membrane.
analyses were carried out using a fast protein liquid
chromatograph (FPLC, AKTA Prime, Amersham
Biosciences) system. Gel filtration chromatography of
shrimp hydrolyzates was carried out on an XK 50/60
column (GE Healthcare Biosciences AB) and packed with
Sephacryl S-300 High Resolution (GE Healthcare
Biosciences AB). Protein hydrolyzates were filtered
through 0.45-µm acetate cellulose microfilters and loaded
(500 mL) for fractionation on a FPLC system and
monitored online at Abs280 nm. Before loading the sample,
the column was equilibrated with 50 mM sodium acetate
buffer (pH 4.5) with 0.02%(w/v) NaN3 and the sample was
eluted (90 mL) with the same buffer at a flow rate of 1 mL/
min. Void and dead volume were determined using blue
dextran 2000 and cobalt chloride, respectively.
multi-mode lab-station (BatchSYNTH®; Milestone Co.,
Sorisole City, Bergemo Province, Italy) with a Teflon®
closed vessel was used for demineralization of the
enzymatically deproteinized shrimp head. Enzymatically
deproteinized shrimp head (0.5 g) was added with 100 mL
of 1 M lactic acid and the slurry was microwave-irradiated
at 400 W until its temperature rose to 121oC for 30 min
(17). After treatment, chitin slurry was filtered through
muslin cloth and insoluble solids were washed with water
and dried at 50oC until constant weight, milled with a
mortar and pestle and stored in plastic container.
State, Country Lot Source
Coahuila, Mexico 001 Litopenaues vannamei
Bagsvaerd, Denmark PLN 05324 Bacillus licheniformis
Bagsvaerd, Denmark HP 202314 Aspergillus oryzae
Abbotsford BC, Canada LG 5333 Egg
Seclin, France NR Carica papaya
Abbotsford BC, Canada PS 5182 Animal
Mexico City, Mexico NR Litopenaues vannamei
Enzymes for Deproteinization of Shrimp Head 555

Infrared spectroscopy Fourier transforms-infrared

attenuated total reflectance (FT-IR/ATR) spectra were
recorded using a FT-IR spectrometer (Spectrum GX;
Perkin-Elmer, Waltham, MA, USA). This equipment was
operated at 4/cm resolution. The mirror velocity was 0.08/
cm and 35 interferograms were co-added before Fourier
transformation. Spectra were collected from 4,000 to 650/
cm and normalized in a way that the absorption band at
about 1,008/cm was equal 1. Normalization did not change
signals proportion in the original spectra.
Protein and ash content Protein content present in
deproteinized shrimp head after enzymatic treatment was
determined by the modified method of Takiguchi . (22)
et al

and the released amino acids were assayed by adopting the

method of Lowry . (23). A visible spectrophotometer
et al

(Genesys 20; Thermo Scientific-Fisher, Waltham, MA,

USA) was used for protein assays and a pH-meter
(Corning, Vernon Hills, IL, USA) was used to verify the
pH of the solutions. Deproteinized shrimp head powders
were ashed in a muffle at 550oC for 2 hr.

Results and Discussion

Screening of commercial proteolytic enzymes for

shrimp head deproteinization Commercial proteolytic Fig. 1. Weight loss of shrimp head after 6 hr of enzymatic
treatment with commercial grade proteolytic enzymes (A) and
enzymes were screened for deproteinization of shrimp
head. After enzymatic treatments, dry weight content,
residual insoluble protein, IR spectroscopy, and ashes were
determined from the insoluble solids of dried shrimp head.
As shown in Fig. 1A, shrimp head was susceptible to
weight loss with a broad range of food grade commercial
enzymes. Enzyme sample S2 and S6 showed a decrease in
dry weight content of 61 and 48%, respectively, with a
concomitant high proteolytic activity. Protein hydrolysis
occurred during enzymatic treatment and the hydrolyzed
protein is released into the liquid medium. Results on
efficiency of Alcalase enzyme for protein removal from
crustacean’s wastes in this study is in agreement with
previous reports (18,21,24). In these studies mentioned,
decalcified crustacean discards were used for enzymatic
treatment. Meanwhile, in this work, enzyme deproteinization
was carried out on non-decalcified wastes. In contrast to
alkali process (3,7), the enzyme method does not promote
complete removal of proteins. Synowiecki and Al-Khateeb
(18) reported that preliminary demineralization increased
the permeability for enzyme penetration. On the contrary,
decalcification of crustacean wastes with HCl may change
structure and consequently affect the active sites for
proteases (25).
All enzymatically treated samples showed the presence
of minor quantity of endogenous protein in dry insoluble
endogenous protein and ash content present in shrimp head
meal treated with commercial grade proteolytic enzymes (B).
See Table 1 for sample code.
solids (Fig. 1B). But S2 treatment recorded the lowest
protein content in insoluble solids, which indicated its high
efficiency for deproteinization of shrimp head waste (24).
However the protein removal was not complete by any
enzyme preparation. The integument of shrimp is comprised
of a rigid exoskeleton (26). The ash content of the
insoluble solids after enzyme treatment was in the range of
17.85 to 26.58% (Fig 1B). The increased ash content in
samples treated with S2 could be related to a decrease in
protein content. These results suggested that the enzymatic
treatments were not capable of releasing minerals from
proteins-chitin complex. The advantage of enzymatic
treatment on non-demineralized crustacean wastes promotes
the release of peptides and amino acids alone into liquid
phase and thus can facilitate a subsequent downstream
processing.
Chitin obtained from shrimp head after enzymes
treatment (S2-S6), control sample (S1) and commercial
chitin (S7) were analyzed in the region 4,000-650/cm using
infrared spectroscopy to distinguish chemical structure and
composition of materials (Fig. 2). Chitin, minerals, and
556
Fig. 2. Infrared spectra of shrimp head insoluble solids after
enzymatic treatment with commercial grade proteolytic
enzymes. See Table 1 for sample code.
proteins are the major components in shrimp head waste
(3,13). The FTIR spectra of analyzed samples recorded
differences especially in the range of 1,200-950/cm
corresponding to carbohydrate finger-print feature (Fig. 2).
The control sample (S1) showed a characteristic infrared
spectrum of natural composite of proteins and carbohydrates.
Insoluble solids from sample S2 and S6 showed intensified
signals at 1,200 to 950/cm, which indicated that the
carbohydrate finger print represented the major presence of
chitin after enzymatic treatment. The gel filtration
chromatographic profile of enzymatic hydrolyzates and
control samples were evaluated. Control sample showed
narrow and well separated peaks. Meanwhile all enzymatic
filtrates showed the presence of different fractions
indicating hydrolysis proteins during extraction process
(data no shown). Alcalase (S2) treated samples showed a
A. U. Valdez-Peña et al.
marked difference in their protein profile. These results
demonstrated the ability of the enzyme for protein
hydrolysis and consequent release of peptides and amino
acids. High content of essential amino acids in Alcalase
treated shrimp wastes have been reported earlier (18). This
enzyme is widely used in food processing for production of
several types of protein hydrolyzates (27-29).
Microwave-assisted chitin recovery from enzymatically
deproteinized shrimp head The shrimp powder prepared
with S2 treatment was submitted to microwave-assisted
demineralization for chitin recovery. The yield of chitin
was found to be 22% and ash content around 0.2%.
Product quality was monitored using FT-IR spectroscopy.
The chitin obtained by the combined enzymatic and
microwave treatment showed major peaks around 3,424/
cm characteristic of amide I, 3,268/cm (O-H stretching),
2,920/cm (C-H stretching), 1,651/cm (N-H) stretching,
1,466/cm (C-H bending), 1,234/cm (C-O-H bending), and
1,008/cm (C-O-C stretching). Infrared spectra of chitin
mainly composed of the absorption band corresponding to
α-chitin (30). Assignments of signals in the infrared
spectra for laboratory chitin were consistent with the
previous reports (17,30) and commercial chitin (Fig. 2).
However, the spectral features of the chitin obtained after
enzymatic treatment varied mainly in the 1,185 to 1,780/
cm region. Recently, Kwon et al. (31) has been reported a
new process for chitin recovery from crab shell using
ultrasound demineralization step in HCl combined with
microwave-assisted deproteinization step with NaOH. In
our work, we used microwave-assisted technology for
demineralization of deproteinized shrimp head employing
lactic acid in short reaction times. Apart from
demineralization, this technique promoted the elimination
of residual protein present in enzymatically deproteinized
shrimp head.
In summary, combination of enzymatic deproteinization
and microwave-assisted demineralization demonstrated the
recovery of high grade chitin under environment friendly
conditions. Recovered chitin can be used as raw material
for production of -acetyl-D-glucosamine, chitin-oligomers,
N
and chitosan for agricultural, biocontrol, feed, food, and
medical applications. Optimization process of enzymatic
deproteinization of shrimp head using Alcalase is in
progress.
Acknowledgments This work was supported by the
National Council of Science and Technology (CONACYT,
Mexico) under Mixed Funds (Fondos Mixtos) from
Quintana Roo State, Mexico (Project number: 2004-01-
QROOC03-04-023) and SAGARPA-CONACYT (2005-
12532). A.U. Valdez-Peña acknowledges the graduate
scholarship from CONACYT.
Enzymes for Deproteinization of Shrimp Head
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