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An Effective Method for Producing a Nutritive Protein Extract Powder from Shrimp-head Waste
M. Mizani, M. Aminlari and M. Khodabandeh
Food Science and Technology International 2005 11: 49
DOI: 10.1177/1082013205051271

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 An Effective Method for Producing a Nutritive Protein Extract
Powder from Shrimp-head Waste

M. Mizani,1,* M. Aminlari2 and M. Khodabandeh3

1
Department of Food Science and Technology, Science and Research Campus, Azad University, P.O. Box
14155-4933, Tehran, Iran
2
Department of Biochemistry, School of Veterinary Medicine, Shiraz University, Shiraz 71345, Iran
3
Biological Product Group, National Research Center for Genetic Engineering and Biotechnology, P.O. Box
14155-6343, Tehran, Iran

Enzymatic hydrolysis has been widely applied for production of protein hydrolysate from shrimp waste
and for purification of chitin. In the present study, shrimp (P. semisulcatus) head waste was hydrolysed,
using a commercial proteolytic enzyme, Alcalase. In order to improve protein extraction efficiency,
certain chemicals such as sodium sulphite and Triton x-100 were used along with the enzyme. When
Alcalase (12 AU/kg) used alone, the yield of protein extraction was 45.1% and by using Triton x-100
(0.01 g/kg) together with Alcalase, the yield was decreased to 39%, whereas the presence of sodium sul-
phite (200 mmol/L) with the enzyme or with the enzyme and Triton x-100 increased the level of protein
extraction to 62% and 65.1%, respectively. The resulting protein powder contained sufficient amounts
of essential amino acids to be used in feed formulations. By precipitating proteins from the resulting
protein extract at pH  3.1, the residual sulphite in protein powder was decreased by 97% and thus the
powder can be considered suitable for animal and/or aquaculture feed formulations.

Key Words: shrimp-head waste, P. semisulcatus, Alcalase, proteins, hydrolysis

INTRODUCTION source in aqua feeds and also livestock and poultry

The most important waste materials in shrimp pro-
cessing industries are head and shell wastes, which
comprise about 40 to 45% of the whole shrimp weight
(Subasinghe, 1999). Making use of such wastes has
been of interest to researchers for two reasons. First,
those wastes are highly perishable and create environ-
mental pollution (Bataille and Bataille, 1983; Tan and
Lee, 2002); second, they are rich sources of protein and
chitin. Shrimp shell waste contains a greater percent-
age of chitin (14–32%, dw basis) than head (11%, dw)
therefore it is considered a richer source of this com-
pound (Ferrer et al., 1996; Subasinghe, 1999; Syn-
owiecki and Al-Khateeb, 2000). However, shrimp-head
waste is rich in protein (50–65% dw basis). Usually,
these wastes are dried and then used as a protein
*To whom correspondence should be sent
(e-mail: mizanil_2000@yahoo.com).
Received 15 March 2004; revised 5 June 2004.
Food Sci Tech Int 2005; 11(1):049–6
© 2005 Sage Publications
ISSN: 1082-0132
DOI: 10.1177/1082013205051271
Downloaded from fst.sagepub.com at University of Alabama at Birmingham on October 23, 2014
diets in order to provide essential amino acids. Occa-
sionally, whole shrimp meal is also used for this
purpose (Gernat, 2001; Laining et al., 2001; Lien et al.,
1995; Nwanna and Daramola, 2000; Tacon, 1987). The
presence of indigestible chitin compounds in such
protein sources causes a decline in the protein effi-
ciency ratio and growth of certain farmed animals.
Usually, because of this problem, inevitably smaller
amounts of these protein compounds should be used in
feed formulations (Gernat, 2001; Laining et al., 2001).
In recent years, certain proteolytic enzymes such as
Alcalase, chymotrypsin, and papain have been used to
extract the protein and chitin parts of shrimp waste
separately and therefore, it has been possible to use
protein hydrolysate and also chitin with more desirable
physico-chemical properties and with smaller amounts
of chemical contaminants (Gagne and Simpson, 1993;
Gildberg and Stenberg, 2001; Synowiecki and Al-
Khateeb, 2000). Also, in order to improve protein
extraction and to achieve a higher degree of purity in
chitin, lactic acid fermentation has been used before
enzymatic hydrolysis (Tan and Lee, 2002).
Since such processes are costly and time consuming,
the main objective of the present research was to use a
combination of chemicals and the proteolytic enzyme
49
50
(i.e. Alcalase) to develop an efficient method for
extracting proteins from shrimp-head waste and to
produce protein powders suitable for use in feed for-
mulations.
MATERIAL AND METHODS
Alcalase® food grade (2.4L, Subtilisin carlsberg)
was provided by Novo Nordisk A/S (Bagsvaerd,
Denmark). All other reagents used were of high ana-
lytical grade and were purchased from commercial
sources.
M. MIZANI ET AL.
Methods
Chemical Analysis
Moisture and ash contents of shrimp-head waste
were determined by standard methods according to
Gildberg and Stenberg (2001).The protein content of
the extracts was measured by the Lowry method
(Lowry et al., 1951) using crystalline bovine albumin as
standard protein. The total protein content of shrimp-
head waste was calculated according to the following
formula (Chang and Tsai, 1997; Nair and Prabua,
1989):

Total protein  (total nitrogen – chitin nitrogen)  6.25

Shrimp-head Waste Preparation

Fresh whole shrimp (P. semisulcatus), supplied by The total nitrogen content of shrimp-head waste
Bushehr Fisheries (Bushehr, Iran), were subjected to was measured by the Kjeldahl method (928.08)
manual head and carapace separation, within 12 h after (AOAC, 2000). The percentage of chitin present in
capture. Shrimp heads (about 34% of the whole shrimp shrimp-head waste was determined according to the
weight) were ground in a National meat grinder (MK- method of Shahidi and Synowiecki (1991). Briefly 1 g
G5NS, Mitsushita Co., Japan). The resulting smooth shrimp-head waste sample was deproteinised by
paste (about 926.5 g/kg of shrimp-head weight), adding 20 mL of a 5% NaOH solution, and incubating
referred to as shrimp-head waste, was used as raw for 2 h at 100 °C. The suspension was filtered and the
material in the present research. The paste was precipitate was washed with deionised water. The dem-
wrapped in metallised polyethylene packaging material ineralisation step was accomplished by adding 20 mL of
and kept at –20 °C for use in further experiments 5% HCl for 2 h at room temperature, followed by fil-
(Scheme 1). tering the suspension and washing the precipitate
(chitin) with deionised water and then oven drying at
105 °C. Finally the percentage of chitin was determined
((weight of the chitin/initial weight of the
sample)  100). The chitin nitrogen was measured also
Shrimp heads
by the Kjeldahl method.
Amino acid analysis was performed after the
hydrolysis of freeze dried protein extracts (6M HCl,
Grinding (110 °C, 24 h) under nitrogen pressure. Derivatisation
Hard part remained was done with phenylisothiocyanate (PITC) reagent
in grinder according to Waters, Division of Millipore
Shrimp-head waste (PICO.TAG, 1990) and the derivatives were separated
Buffer (1:1, w/v)
With or without sulphite by reverse phase HPLC, using a PICO.TAG C18
column (150  3.9 mm, part no. 88131, USA). The tem-
Homogenisation perature of the column was maintained at 38 °C.
Alcalase Mobile phase consisted of two solvents:
and/or Triton x-100

Hydrolysis Solvent A: sodium acetate trihydrate, triethylamine,

T 40 °C, t 1 h acetonitrile, water.
Solvent B: acetonitrile, water.

PMSF (1mmol/L) Enzyme inactivation Flow rate was adjusted according to gradient table
(PICO.TAG, 1990). Eluted amino acids were moni-
tored by UV detector (Model 486) at 254 nm.
Centrifugation
10000 g, 45 min, 4°C
Protein Extraction Procedures

protein hydrolysate In order to improve the efficiency of protein extrac-

tion, three factors, each at two levels, were chosen as
Figure 1. Scheme of protein extraction procedure. follows: Alcalase ((0, 12) AU/kg, i.e. (0, 0.5)% v/w),
Downloaded from fst.sagepub.com at University of Alabama at Birmingham on October 23, 2014
 Protein Extract Powder from Shrimp-head Waste
Triton x-100 ((0, 0.01) g/kg), sodium sulphite
((0.200) mmol/L).
These amounts were chosen as the most effective
concentration of each compound, based on another
research in which the effect of each on the efficiency of
protein extraction from shrimp-head waste was studied
independently (Mizani, 2002).
The effect of eight different treatment combinations
derived from a (23) factorial experiment (using the
above mentioned three factors) on the efficiency of
protein extraction were compared. In all treatments
25 g of the ground and frozen shrimp-head waste was
homogenised in 25 mL of 0.1 mol/L Tris buffer, pH  8,
in a commercial Waring blender at a fixed speed for
2 min. Sodium sulphite, when necessary, was added to
the homogenising buffer, but Triton x-100 and
Alcalase were added to the homogenised mixture at
the beginning of the incubation process, which was
done at 40 °C for 1 h.
The adjusted temperature was lower than the
optimum temperature range for Alcalase activity
(50–60 °C) (Novo Nordisk A/S, 1991) in order to slow
down undesirable browning reactions that usually
occur in the extract. After an hour enzymatic reactions
were stopped by adding 1 mmol/L phenylmethyl-
sulphonylfluoride (PMSF) (Novozymes A/S, 2001).
The mixture was immediately centrifuged (10,000 g,
30 min, 4 °C) and the protein concentration of super-
natants was determined by the Lowry method.
The sulphite content of the protein extracts which
were obtained from sodium sulphite treatments was
determined by a method of distillation (Pearson, 1970).
In order to reduce the amount of residual sulphite in
these extracts, protein precipitation was done by drop
wise addition of dilute solution (1M) of HCl and slowly
lowering pH down to 3.1 (Friedman, 1994).
Trichloroacetic acid solution at a final concentration of
12% was also used for this purpose.
All protein extracts were then lyophilised and used
for subsequent experiments.
Statistical Analysis
The effect of combined treatments was compared
according to a factorial experiment, arranged in a com-
pletely randomised design, each with four replications,
utilising analysis of variance and the Duncan’s Multiple
Range test, as described by Steel and Torrie (1987).
RESULTS AND DISCUSSION
Results (Table 1) indicated that the prepared
shrimp-head waste was rich in protein (639.0 g/kg, dw
basis) and that it contained about 88 g/kg chitin, while
the chitin content in the head part of shrimp (before
preparation) was estimated to be about 115 g/kg. Other
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51
Table 1. Chemical composition of shrimp
(P. semisulcatus) head waste.
Shrimp-head waste
Component (g/kg, mean  SD, n  4)
Moisture 805.04  3.20
Total nitrogen (dw basis) 108.02  4.45
Crude protein (dw basis) 639.01  27.85
Chitin (dw basis) 87.83  6.83
(114.68  2.45)a
Ash (dw basis) 231.64  8.00
a
Chitin content of shrimp heads before preparation step (Figure 1).
researchers also estimated the mean content of the
chitin of shrimp heads to be 110.0 g/kg (Subasinghe,
1999). Reduction in the chitin content is related to the
shrimp preparation method (Figure 1), as in this
method the hard part remaining in the grinder (about
73.5 g/kg of the shrimp-head weight) which may
contain the major amount of chitin, was discarded and
the smooth and homogenous paste (315 g/kg of the
whole shrimp weight), which is more suitable for the
extraction of proteins, was used as raw material.
The results of the effect of different agents on the
efficacy of protein extraction from shrimp-head waste
are presented in Table 2. In group “N”, where no
chemical or enzymatic treatment was used, a protein
extraction yield of 37% was obtained. This amount of
protein solubility might be the result of proteolytic
action of the endogenous enzymes, especially serine
proteases present in shrimp-head waste (Haard, 1994).
Significant increase in protein extraction yield was
observed in Alcalase treated (group “A”, 45%) or
sodium sulphite treated samples (group “S”, 49%).
The effect of sodium sulphite on improving the effi-
ciency of protein extraction can be attributed to its
effect on reducing disulphide bonds and increasing
protein solubility, as has been previously shown in the
case of soy products (Abtahi and Aminlari, 1997).
However, Triton x-100 had no significant effect
(p  0.01), as the yield was improved by only 4% com-
pared to group “N”. It has been reported that tempera-
ture can change the properties of detergents due to the
growth of the micelle form of these agents (Neugebauer,
1990). Group “S  A”, using sodium sulphite with
Alcalase, increased protein extraction yield by 68%, as
compared with group “N”. Therefore, a positive corre-
lation was observed between Alcalase and sodium sul-
phite. It is possible that the proteolytic activity of the
enzyme makes disulphide bonds more accessible for
the sulphite. Although Alcalase is a detergent-compati-
ble protease (Anwar and Saeemudiin, 2000), when
Triton x-100 was used with this enzyme (group
“T  A”) the amount of extracted protein decreased
14% compared with group “A”. This may happen
52
Table 2. Improvement of protein extraction yield at 40 °C from shrimp-head waste in presence of effective
concentration of different agents.
Extracted protein
Contentb
Group yielda (g, mean  SD, n  4)
N 46.2  1.2
S 61.0  1.1
T 48.2  0.5
A 56.5  0.9
ST 67.0  0.5
SA 77.5  0.7
TA 48.6  0.8
STA 81.5  0.8
a
N  no treatment, T  Triton x-100 (0.01 g/kg), A  Alcalase (12 AU/kg), S  sodium sulphite (200 mmol/L)
b
Protein extracted from 1 kg of “shrimp-head waste”, by each treatment.
c
The yield is calculated as the percentage of the total protein content of “shrimp head waste” (124.6 0  0.50 g/kg).
because Alcalase is known as a protease with a higher
tendency for hydrophobic sites (Adler-Nissen, 1986).
On the other hand, Triton x-100 with HLB  13.5
(Neugebauer, 1990) covers the same parts of protein
structure and makes them unavailable to the enzyme.
When Alcalase and sodium sulphite was used in
combination (group “S  A”), the protein extraction
yield increased to 62%; however, when Triton x-100
was added to the mixture, (group “S  T  A”) only
5% improvement was achieved as compared with the
group “S  A”. (The amount of extracted protein was
increased from 77.5 g/kg to 81.5 g/kg.)
Gildberg and Stenberg (2001) suggested a process
for the hydrolysis of shrimp-waste proteins with
Alcalase at 40 °C and for 2 h. In this process, the total
nitrogenous compounds extracted from shrimp waste
were estimated at 68.5%, out of which 51.5% was
related to proteinaceous nitrogen. In comparison, in
the present study, using chemical agents together with
Alcalase, the amount of proteinaceous nitrogen
extracted in the treatments (groups “S  A” and
“S  T  A”) was increased up to 59% and 62%
respectively. Also, this efficiency of extraction was
achieved after 1 h of hydrolysis (as compared to 2 h in
the previous method).
Table 3 shows the results of amino acid analysis of
the protein extracts obtained from the treatments of
group “S  A” and “S  T  A”. Based on this data,
these extracted proteins could be considered as suit-
able protein sources and can provide essential amino
acids for various fish species according to the mean
value of the required level of each amino acid sug-
gested by Wilson (2003). It should be mentioned that
histidine is the first limiting amino acid of these
sources, as in the case of whole shrimp meal (Tacon,
1987) which might be compensated for by other
protein sources in the diet. Both of the protein extracts
can also be considered superior to whole shrimp meal
Downloaded from fst.sagepub.com at University of Alabama at Birmingham on October 23, 2014
M. MIZANI ET AL.
In comparison to “N”
(%) Protein extractionc (%)
– 37.0
32.0 48.7
4.3 38.5
22.3 45.1
45.0 53.5
67.8 62.0
5.2 39.0
76.4 65.1
because they are free of chitinous materials, which are
indigestible for most animals and the presence of which
causes a decline in growth (Gernat, 2001; Laining et al.,
2001). There was no significant difference between the
amount of each amino acid of the treatments group
“S  A” and “S  T  A” (Table 3).
In the present study, it was shown that the colour of
protein extracts obtained from different treatments
varied from pink to dark brown. Under the heating
condition used (i.e. 40 °C) in the treatments the unde-
sirable browning reactions might occur slowly such that
these reactions result in the development of low intens-
ity of dark colour in all of the protein extracts with the
exception of treatments in which sodium sulphite was
used (groups “S”, “S  A” and “S  T  A”). In the
presence of sodium sulphite, these reactions were
inhibited, and the desirable pink colour of the raw
material was preserved. This can be considered as
another advantage of using sodium sulphite in the
treatments. Therefore, the resulting protein powders
can be used in feed formulations for farmed animals
and also for fabricated sea foods such as shrimp ana-
logue or shrimp crackers in order to provide essential
amino acids as well as carotenoids.
According to the FDA regulations, the residual
amount of sulphite in different food products had to be
controlled (Warner et al., 2000). So in shrimp-head
waste protein powder (group “S  A”) one must take
the regulations into consideration. The initial level of
residual sulphite (expressed as SO2) was
73.5  1.22 g/kg which reduced to 1.5  0.57 g/kg after
protein precipitation at pH  3.1. This method of redu-
cing the amount of sulphite had already been recom-
mended for soy proteins (Friedman, 1994). Not only is
the presence of this amount of sulphite in the resulted
protein powder harmless, but it can also act as a
preservative in formulated feed (Vinelli, 2003).
It should be mentioned that this method made it
 Protein Extract Powder from Shrimp-head Waste 53
Table 3. Amino acid composition of freeze dried shrimp-head waste hydrolysates compared to required levels
for various fish species (mean  SD, n  3).
Hydrolysates

The mean value of

Essential amino acids S  A (g/kg protein) S  T  A (g/kg protein) requirementa,b

His 13.72  0.20 14.01  0.10 20 (15–25)

Ile 32.21  0.11 31.80  0.20 25.5 (20–31)
Leu 49.80  0.51 48.90  0.13 39.5 (33–46)
Lys 53.79  0.10 53.44  0.30 47 (32–62)
Met  Cys 30.70  0.50 29.96  0.10 29.5 (19–40)
Phe  Tyr 59.20  0.22 58.70  0.15 54 (43–65)
Thr 33.02  0.20 33.32  0.20 35 (20–50)
Trp nd nd 8 (5–11)
Arg 50.30  0.11 49.80  0.30 49 (33–65)
Val 35.54  0.61 36.60  0.30 31 (22–40)
a
According to Wilson (2003).
b
The values in parenthesis represent the required level of each amino acid.
nd: Not determined.

possible to precipitate only 50% of the proteins exist-
ing in the extract, so it did not succeed in increasing the
overall protein recovery, because the procedure used
to eliminate sulphite resulted in further loss of protein
during precipitation. Therefore it is necessary to find
an efficient method for sulphite removal. Also it might
be more useful to apply other reducing compounds
such as N-acetyl cysteine as a substitute for sodium sul-
phite or a mixture of both in the extraction process so
that the result will be nutritionally safer (Friedman,
1994) and more efficient.
ACKNOWLEDGEMENTS
The help by Ms Seyhoon for the amino acid analysis
conducted in the Food Laboratory of Gamma Irradia-
tion Center of Atomic Energy Organization, Tehran,
Iran is greatly appreciated.
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