International Journal of Food Science and Technology 2010, 45, 1807–1813 1807

Original article
Optimisation of extraction conditions and characteristics of skin
gelatin from Nile tilapia (Oreochromis niloticus)

Shaokui Zeng,1 Xiaoyan Yan,1 Wenhong Cao,1 Pengzhi Hong,1 Chaohua Zhang1* & Laihao Li2
1 Key Laboratory of Aquatic Product Advanced Processing of Guangdong Higher Education Institutes, College of Food Science & Technology,
Guangdong Ocean University, Zhanjiang, 524088, China
2 South China Sea Fishery Research Institute, Chinese Academy of Fishery Science, Guangzhou, 510300, China
(Received 21 December 2009; Accepted in revised form 4 June 2010)

Summary Response surface methodology was used to optimise gelatin extraction conditions from the skin of Nile
tilapia (Oreochromis niloticus), and characteristics of the gelatin were determined. Concentration of NaOH
(%, X1), alkaline treatment time (h, X2), concentration of HCl (&, X3) and acid treatment time (min, X4)
were chosen for independent variables. Dependent variable was yield of gelatin (%, Y). Optimal conditions
were X1 = 3.2 (%), X2 = 2.3 (h), X3 = 0.7 (&) and X4 = 84 (min), and predicted value of response
optimal conditions was Y = 20.4%. Actual value was 19.3% by verification experiments under optimal
conditions. Crude protein content of the tilapia skin gelatin was 88.5%. The content of imino acids including
proline and hydroxyproline in the gelatin was 185 residues per 1000 total amino acid residues. Its gel strength
was 260 g. The gelling and melting points were 18.0 and 22.4 C, respectively.
Keywords Amino acid, gels, response surface methodology, skin gelatin, tilapia.

China is by far the largest producer and exporter of

Introduction
Gelatin has been widely used in many industrial fields
such as food, material, pharmacy and photography,
especially in the food and pharmaceutical industries
for its unique physico-chemical properties (Jamilah &
Harvinder, 2002). Gelatin is a product of partial
hydrolysis of collagen and is usually extracted from
porcine or bovine bones and skins. In the past
10 years, considerable effort has been expended on
studying gelatin extraction from different fish species
(Kołodziejska et al., 2004, 2008; Zhou & Regenstein,
2005; Zhou et al., 2006; Cho et al., 2005; Liu et al.,
2008; Muyonga et al., 2004; Aewsiri et al., 2008;
Kwak et al., 2009). However, the production of fish
gelatin is still in its infancy, contributing only about
1% of the annual world gelatin production (Arnesen
& Gildberg, 2006). There are some limiting factors
such as insufficient availability of raw materials,
inferior rheological properties, prices. These problems
have hampered the large-scale development of fish
gelatin industry.
*Correspondent: Fax: +86 0759 2382049;
e-mail: chz2382@126.com
tilapia in the world. According to the statistics of FAO,
China’s annual production of tilapia in 2007 had risen to
nearly 1.2 million ton, which accounts for about 50% of
tilapia production of the world. Tilapia is usually
processed as fresh and frozen fillets and exported to
overseas markets. During tilapia fillet processing, large
quantities of by-products such as skin and scale are
produced. The skin is rich in collagen. If the skin can be
re-utilised to produce gelatin, it may increase the
economic value of the fish. According to previous
reports (Grossman & Bergman, 1992; Zhou et al.,
2006), gelatins extracted from tilapia skin present good
gelling properties and high gel strength. Therefore, it can
serve as an additional source of extracting gelatin.
Usually, the extraction procedures take a long time.
Jamilah & Harvinder (2002) previously reported a
procedure for extracting tilapia skin gelatin, in which
the hot-water extracting process took about 12 h.
However, Kołodziejska et al. (2008) used minced skins
of cold-water fish instead of whole skins, the extraction
time could be shortened from 12 h to 30 min. To
enhance industrial utilisation of tilapia fish gelatin, the
aim of this study is to optimise the pretreatment of the
skin so as to shorten the extraction time and to determine
the rheological properties of the gelatin.

doi:10.1111/j.1365-2621.2010.02332.x
 2010 The Authors. Journal compilation  2010 Institute of Food Science and Technology
1808 Optimisation of extraction conditions and characteristics of skin gelatin S. Zeng et al.

Table 1 Levels of independent variables used for tilapia skin gelatin

Materials and methods extraction and observations (Expt.) of gelatin yield (%) together with
predictions (Pred.) from the response surface model
Materials
Coded levels of variable Response (Y)
Oreochromis niloticus skin was provided by a local fish-
processing factory (Zhanjiang Global Aquatic Product Run no. X1 X2 X3 X4 Expt. Pred.
Co., Ltd., Zhanjiang, Guangdong, China). The skin was
1 )1 )1 )1 )1 12.91 14.89
mechanically separated from fresh tilapia while process-
2 )1 )1 )1 1 17.77 18.03
ing frozen fillets. The residue of adherent tissue such as
3 )1 )1 1 )1 11.47 14.89
scale was removed manually. The cleaned skin was 4 )1 )1 1 1 17.87 18.01
stored in )18 C for about 30 days until used. Gelatin 5 )1 1 )1 )1 15.62 14.89
extracted from the skin of porcine (9764) was purchased 6 )1 1 )1 1 17.41 18.03
from Amresco Co. (Solon, OH, USA). All reagents used 7 )1 1 1 )1 14.70 14.89
were of analytical grade. 8 )1 1 1 1 18.51 18.03
9 1 )1 )1 )1 10.74 14.89
10 1 )1 )1 1 15.62 18.03
Determination of proximate components 11 1 )1 1 )1 14.98 14.89
12 1 )1 1 1 21.33 18.03
Moisture (oven-drying procedure), crude protein and ash
13 1 1 )1 )1 13.30 14.89
contents were estimated by the AOAC official methods
14 1 1 )1 1 16.60 18.03
(Horwitz, 2000). The analyses were replicated three times. 15 1 1 1 )1 15.55 14.89
16 1 1 1 1 17.06 18.03
Extraction of gelatin 17 )2 0 0 0 18.29 14.93
18 2 0 0 0 12.69 14.93
The cleaned skin was treated with 10 volumes (v ⁄ w) of 19 0 )2 0 0 18.79 17.01
NaOH solution (1–5%, w ⁄ v) for 1–5 h at room 20 0 2 0 0 16.31 17.01
temperature (28–30 C) to remove the non-collagen 21 0 0 )2 0 18.99 20.13
protein and pigments with gentle stirring. The solution 22 0 0 2 0 18.58 20.13
23 0 0 0 )2 13.11 10.63
was changed when it became black. Alkaline-treated
24 0 0 0 2 15.50 16.91
skins were washed with distilled water until neutral.
25 0 0 0 0 18.82 20.13
Then the skin was soaked in HCl solution (0.1–0.9&) 26 0 0 0 0 19.20 20.13
for 30–120 min with gentle stirring and washed with 27 0 0 0 0 20.22 20.13
distilled water until neutral. For hot-water extraction, 6 28 0 0 0 0 22.88 20.13
volumes (v ⁄ w) of distilled water were added and heated 29 0 0 0 0 21.41 20.13
at temperature 60 C for 3 h. The extracted solution was 30 0 0 0 0 22.65 20.13
centrifuged at 900 g for 30 min. The upper phase was
X1, concentration of NaOH, %; X2, alkaline treatment time, h; X3,
vacuum-filtered with a Whatman No. 1 filter paper
concentration of HCl, &; X4, acid treatment time, min; Y, yield of
(Whatman International Ltd., Maidstone, UK). The
gelatin, %.
filtered solution was vacuum-concentrated to about
10% (w ⁄ v) at 60 C and dried at 1.4 m s)1 for 24 h in
a hot-air dryer (Shanghai Laboratory Instrument Works treatment time (h, X2), concentration of HCl (&, X3)
Co., Ltd, China) at 60 C. The powder obtained was and acid treatment time (min, X4) were chosen for
referred to as tilapia skin gelatin (TSG). independent variables. The range and centre point
values of four independent variables were based on the
results of preliminary experiments. After chemical
Experimental design
treatment, gelatin was extracted with water at 60 C
Central composite design (CCD) (Box & Wilson, 1951) for 3 h. The yield of gelatin (%, Y) was selected as the
was adopted in the optimisation of pretreatment the dependent variable. Experimental runs were randomised
tilapia skin. The samples were prepared according to a to minimise the effects of unexpected variability in the
4-factor, 5-level central composite design. CCD in the observed responses.
experimental design consists of twenty-four factorial
points, of which 8 are axial points (a = ±2) and six are
Analysis of data
replicates of the central point (Table 1). Processing fish
gelatin included two important processes – chemical The response surface regression (RSREG) procedure of
treatment and hot-water extraction. In general, a mild the Statistical Analysis System software (Version 8.01;
chemical treatment is used prior to gelatin extraction. In SAS Institute Inc., Cary, NC, USA) was used to fit the
our study, concentration of NaOH (%, X1), alkaline following quadratic polynomial equation

International Journal of Food Science and Technology 2010  2010 The Authors. Journal compilation  2010 Institute of Food Science and Technology

X
4 X
4 3 X
X 4
Y ¼ b0 þ bi Xi þ bii X2i þ
i¼1 i¼1 i¼1 j¼iþ1
where Y is dependent variable (yield of gelatin), b0 is
intercept, bi, bii, bij are regression coefficients and Xi
indicate the linear terms, Xi2 for the quadratic terms for
a single variable, Xi Xj for the interaction terms. For the
model calculated from the linear regression, analysis of
variance (anova) was performed. The R2 value, the
residual error, the pure error (calculated from the
repeated measurements) and the lack of fit were calcu-
lated. The lack of fit indicated whether the calculated
response surface represents the true surface. The sum of
squares (SS) of the lack of fit was the SSresidual – SSpure
error, with its significance being tested with an F-test,
which was given by the relationship: F = MSlack of
fit ⁄ MSpure error, where MS was the mean sum of squares
(Myers & Montgomery, 2002).
Yield of extracted gelatin
The yield was calculated as follows:
Yieldð%Þ ¼ dried gelatin (g)
 100=weight of wet skin used (g):
Amino acid analysis
Dry gelatin was dissolved in 6 m HCl solution and
hydrolysed at 110 C for 24 h. The solvent was analysed
by an amino acid analyzer (HITACHI 835-50 Amino
Acid Analyzer, Tokyo, Japan).
SDS–polyacrylamide gel electrophoresis (SDS–PAGE)
SDS–PAGE was performed as described by Laemmli
(1970) using 7.5% separating gel and 4.0% stacking gel.
TSG, porcine skin gelatin (PSG), molecular weight
marker proteins (44.3–200 kDa; Biodee Biotechnology
Co., Ltd, Beijing, China) and acid-solubilised collagen
from tilapia skin (produced in our laboratory) were
applied and subjected to electrophoresis. After electro-
phoresis, the gel was stained with Coomassie brilliant
blue R-250.
Determination of gel strength
According to the method of Gómez-Guillén et al. (2002),
the gel strengths of TSG and PSG were determined on a
6.67% gel (w ⁄ v), formed by dissolving the dry gelatin in
distilled water at 60 C and cooling the solution in a
refrigerator at 7 C (maturation temperature) for
16–18 h. The gel strength was determined at 7 C using
samples with 3.3 cm diameter and 6 cm height on a Tms-
pro Testing Machine (Food Technology Corporation,
 2010 The Authors. Journal compilation  2010 Institute of Food Science and Technology
Optimisation of extraction conditions and characteristics of skin gelatin S. Zeng et al. 1809
Maries, VA, USA) with a load cell of 1000 N, cross-
bij Xi Xj ð1Þ head speed 0.5 mm s)1 and equipped with a 1.27-cm-
diameter flat-faced cylindrical plunger. The maximum
force (in grams), taken when the plunger had penetrated
4 mm into the gelatin gels, was recorded. The determi-
nation was run in triplicate.
Dynamic viscoelastic properties
Dynamic viscoelastic properties of TSG and PSG were
measured by a concentric cylinder geometry of the
rheometer (Rheostress 1 RS30; HAAKE Co., Ltd,
Karlsruhe, Germany). The gelatin solution (6.67%,
w ⁄ v) was prepared in distilled water at 60 C. The
measurement was performed during cooling and heating
from 40 to 5 C and from 5 to 40 C, respectively. The
temperature ramps were done at a scan rate of
0.5 C min)1, frequency 1 Hz, and oscillating applied
stress of 3.0 Pa. The elastic modulus (G¢; Pa), the
viscosity modulus (G¢¢; Pa) and the phase angle (d) were
plotted as a function of temperature.
Determination of gelling and melting points
Gelling and melting points were determined by the
method of Gudmundsson (2002). The gelling point was
evaluated from the intersection point, where the elastic
modulus (G¢; Pa) and the viscosity modulus (G¢¢; Pa)
during the cooling process. The melting point was
determined during the heating process in the same
manner as for the gelling point.
Results and discussion
Diagnostic checking of the fitted model
The RSREG procedure for SAS software was employed
to fit the quadratic polynomial equation to the exper-
imental data. All the coefficients of linear (X1; X2; X3;
X4), quadratic (X12; X22; X32; X42) and interaction terms
were calculated for significance with t-statistic, and the
estimated coefficients of the model are presented in
Table 2. The coefficients of linear X4, quadratic X12 and
X42 were highly significant (P < 0.01). The X22 was also
significant (P < 0.05). On the other hand, the coeffi-
cients of X32, all linear terms except X4 and all
interaction terms were not significant (P > 0.05). To
develop the fitted response surface model equation, all
insignificant terms (P > 0.05) were eliminated, and the
final regression equation was formed:
Y ¼ 20:13þ1:57X4 1:30X1 2 0:78X2 2 1:59X4 2 ; ð2Þ
where Y is the predicted yield of gelatin real value, X1,
X2 and X4 are the coded values of variable concentration
of NaOH, alkaline and acid treatment time, respectively.
International Journal of Food Science and Technology 2010
1810 Optimisation of extraction conditions and characteristics of skin gelatin S. Zeng et al.

Table 2 Coefficients of the response surface model of tilapia skin Response surface
gelatin yield
Figure 1 shows the estimated response function and the
Term Coefficient P value effects of the independent variables X1, X2, X4 on
the dependent variable Y. Fig. 1a, b and c suggest that
Intercept 20.1341 <0.0001
the yield of tilapia skin gelatin increases with the
X1 )0.5117 0.1775
X2 0.0458 0.9017
increase in concentration of NaOH, alkaline and acid
X3 0.4450 0.2384
treatment time to optimum point. When concentration
X4 1.5700 0.0003 of NaOH (X1), alkaline and acid treatment time (X2, X4)
X12 )1.2973 0.0005 further increase, the yield of gelatin decreased. Over
X2X1 )0.3988 0.3847 treatment, skin with alkaline and acid could derive
X22 )0.7823 0.0225 protein degradation to other low-molecular weight
X3X1 0.8638 0.0681 fragments. This resulted in the loss of extracted collagen
X3X2 )0.3575 0.4349 through leaching during the series of washing steps
X32 )0.4735 0.1508 (Jamilah & Harvinder, 2002). For gelatin manufacture,
X4X1 )0.0513 0.9102
the degree of collagen cross-linking is a key factor. Acid
X4X2 )0.7550 0.1076
X4X3 0.2025 0.6567
treatment would destroy the covalent and non-covalent
X42 )1.5935 <0.0001
crosslink and would result in collagen swelling
(Stainsby, 1987). Subsequent heating cleaves hydrogen
X1, concentration of NaOH, %; X2, alkaline treatment time, h; X3, and covalent bonds, leading to the conversion into
concentration of HCl, &; X4, acid treatment time, min. gelatin by a helix-to-coil transition (Djabourov et al.,
1993). Many previous studies demonstrated that acid
Table 3 Analysis of variance (anova) for the response surface model of concentration is very important for skin gelatin extrac-
tilapia skin gelatin yield tion (Gómez-Guillén & Montero, 2001; Giménez et al.,
2005; Kasankala et al., 2007). However, in our results,
Sources DF SS MS F value P value acid concentration (X3) is not significant. This may be
Regression 14 258.4624 18.4616 5.72 0.0002 related to the method used for pretreatment of the skin.
Linear 4 70.2439 17.5610 5.44 0.0036 The pretreatment by alkaline solution followed by an
Quadratic 4 161.8740 40.4685 12.54 <0.0001 acid solution in our study, not only removed the non-
Cross-product 6 26.3445 4.3908 1.36 0.2758 collagenous proteins but also provided the proper pH
Residual 21 67.7713 3.2272 – – condition for extraction (Zhou & Regenstein, 2005).
Lack of fit 10 40.4304 4.0430 1.63 0.2183
Pure error 11 27.3409 2.4856 – –
Total 35 326.2337 – – – Verification of predicted gelatin yield under optimal
conditions
Statistical testing of the regression model has been Skin gelatin production was conducted under optimal
done by the Fisher’s statistical test for anova (Table 3). conditions (concentration of NaOH 3.2%, alkaline
The probability (P) value of the regression model was treatment time 2.3 h, concentration of HCl 0.7& and
<0.01, with no significant lack of fit (P > 0.05), acid treatment time 84 min) to verify the predicted
indicating that the model is well adapted to the gelatin yield in the response surface model. The
response. The determination coefficient (R2 = 0.7923) extraction was carried out in distilled water at temper-
was satisfactory. The predicted value agreed well with ature 60 C for 3 h. Then, the solution was separated
the experimental one. and dried. The results of the three time repeated
experiments indicated that the actual value of gelatin
yield was 19.3 ± 2.3%, which is close to predicted
Conditions for optimum responses
value (20.4%).
Four independent variables, concentration of NaOH,
alkaline treatment time, concentration of HCl and acid
Chemical composition
treatment time were chosen as the critical factors of the
CCD model. The coded values of X1, X2, X3 and X4 for The contents of moisture, crude protein and ash in TSG
the optimised model calculated by response optimisers were 10.7, 88.5 and 0.6%, respectively. Amino acid
were 0.1202, )0.3080, 0.5360 and 0.3514, respectively. composition of the gelatin from tilapia skin is shown in
The actual values of X1, X2, X3 and X4 were 3.2%, 2.3 h, Table 4. The results indicated that the amino acid
0.7& and 84 min, respectively. The predicted value of composition of TSG was quite similar to that of warm-
extraction yield of gelatin was 20.4%, which is a water fish gelatins such as megrim and Dover sole
maximum. (Gómez-Guillén et al., 2002; Giménez et al., 2005). The

International Journal of Food Science and Technology 2010  2010 The Authors. Journal compilation  2010 Institute of Food Science and Technology
 Optimisation of extraction conditions and characteristics of skin gelatin S. Zeng et al.
(a)
(b)
(c)
Figure 1 The response surface for optimisation of gelatin extraction
from tilapia skin. X1 (concentration of NaOH, %); X2 (alkaline
treatment time, h); X4 (acid treatment time, min); Y (yield of gelatin, %).
content of amino acids including proline and hydroxy-
proline in TSG was 18.5%, which was lower than that in
PSG (23%) and tuna fin gelatin (36%) (Aewsiri et al.,
 2010 The Authors. Journal compilation  2010 Institute of Food Science and Technology
1811
2008). Ledward (1986) has reported that the stability of
the triple helical structures in gelatins is dependent on
the content of imino acids, as regions rich in proline and
hydroxyproline are likely to be involved in the forma-
tion of nucleation zones. A gelatin with high contents of
proline, hydroxyproline and alanine shows better visco-
elastic properties than others with low contents in these
amino acids (Gómez-Guillén et al., 2002; Sarabia et al.,
2000). The content of alanine in TSG was 12.6%. Thus,
TSG could be expected to have good rheological
properties.
Molecular weight distribution
The protein patterns of TSG and PSG are presented in
Fig. 2. In the case of PSG, the distinct band corre-
sponding to b component (200 kDa) in the electro-
phoretic pattern was not visible. Over the whole length
of the gel, only a smudged band was visible. For TSG,
the distinct bands corresponding to b component of
tilapia skin collagen and to the one a chain (100 kDa)
in the electrophoretic pattern were visible. Additional
smudged bands with molecular weight lower than
100 kDa appeared below a chain. They could be the
products of hydrolysis of elementary chains of collagen.
According to Gómez-Guillén et al. (2002), damage or
partial loss of a1 chains can occur during the extraction
procedure. The difference of molecular weight distribu-
tions of TSG and PSG may be because of the different
content of covalent cross-links in the native collagen.
The results of Normand et al. (2000) suggested that
neither high nor low molecular weight chains contribute
to the elasticity. Besides, large amounts of b- and c-
chains have been shown to negatively affect some of the
functional properties of fish gelatins, such as lowering
melting and setting points (Muyonga et al., 2004).
Therefore, TSG may have a lower gel strength, gelling
and melting points than PSG. This is most probably
because of a different molecular weight distribution as
well as the presence of different hydrolytic fragments
(Eysturskarð et al., 2009).
Gel strength
The gel strength of TSG was 260 ± 2.1 g, which was
lower than that of PSG (367 ± 1.9 g). The result was
similar to the reports of Grossman & Bergman (1992)
and Zhou et al. (2006). Songchotikunpan et al. (2008)
also reported th at the gelatin from Nile tilapia skin
showed high gel strength (328 g). Their differences could
be explained by differences in manufacturing process
used. The study of Kwak et al. (2009) suggested that gel
strength of shark (Isurus oxyrinchus) cartilage gelatins
were different with drying methods. In addition, the
source and type of collagen will influence the properties
of the resulting gelatins. According to the report of Zeng
International Journal of Food Science and Technology 2010
1812 Optimisation of extraction conditions and characteristics of skin gelatin S. Zeng et al.
Table 4 Amino acid composition of gelatin from tilapia skin (residues
per 1000 total amino acid residues)
Amino acids
Aspartic acid
Threonine
Serine
Glutamic acid
Glycine 339
Alanine 126
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Proline 101
Hydroxyproline
et al. (2009), tilapia skin collagen molecule composed of
three a-chains and its denaturation temperature was
high up to 32.0 C. Badii & Howell (2006) confirmed
that hydrophobic amino acids (Ala, Val, Leu, Ile, Pro,
Phe, and Met) could also contribute to the high bloom
value of tilapia gelatin. Montero & Gómez-Guillén
(2000) suggested that the hydrophobic amino acid
composition and distribution influence the physical
properties of gelatin even more than the imino acids
content. The contents of Ala, Val and Leu in TGS were
higher than that in the reports of Gómez-Guillén et al.
(2002) and Sarabia et al. (2000). These may be the
reasons for such high gel strength of TSG. Besides, the
high bloom values of both gelatins in our study were
related to the low temperature during curing.
Gelling and melting points
The gelling and melting points can be judged from the
sharp decrease in phase angle (Fernández-Dı́az et al.,
2003). The gelling and melting points of TSG were
18.0 ± 0.3 and 22.4 ± 0.2 C, respectively. They were
lower than that of PSG (25.8 ± 0.1 and 32.4 ± 0.1 C,
respectively). The results were similar to the volumes of
previous literatures (Gudmundsson, 2002; Jamilah &
Harvinder, 2002).
Conclusion
The yield of gelatin extracted from Oreochromis niloticus
skin was high up to 19.3 ± 2.3%. SDS–PAGE analysis
showed that the gelatin was much less degraded than
gelatin from porcine skin. The gelatin showed high
strength (260 ± 2.1 g). It could not form a gel at 20 C
International Journal of Food Science and Technology 2010
200.0 kDa
48
27 116.0 kDa
39
71 97.2 kDa
19
66.4 kDa
9
9
25
4
13
44.3 kDa
26
5 1 2 3 4
55
Figure 2 Protein patterns of tilapia and porcine skin gelatin. (1)
84 molecular weight markers; (2) acid-solubilised collagen from tilapia
skin; (3) porcine skin gelatin (PSG); (4) tilapia skin gelatin (TSG).
and showed a lower melting temperature in comparison
with porcine skin gelatin. Low gelling temperature
(22.4 C) of TSG may offer new potential applications
for dry products such as for micro-encapsulation.
Acknowledgments
Project supported by the Foundation for Agriculture
Project of Guangdong (Grant No. 2008A020100006,
2009B020201003) and the Important Projects in Key
Fields in Guangdong and Hongkong (Grant No.
2009A020700004)*. Authors also thank Engineer Wang
Zhongming with Rousselot (Guangdong) Gelatin Co.,
Ltd. for gelling and melting points determination.
*Correction added on 16 August 2010, after first Online
Publication. Grant No. 2009498D14 was changed to Grant
No. 2009A020700004.
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