Food Hydrocolloids 39 (2014) 68e76

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Isolation and characterization of gelatin from the skins of skipjack

tuna (Katsuwonus pelamis), dog shark (Scoliodon sorrakowah), and
rohu (Labeo rohita)
K. Shyni*, G.S. Hema, G. Ninan, S. Mathew, C.G. Joshy, P.T. Lakshmanan
Biochemistry & Nutrition Division, Central Institute of Fisheries Technology, Cochin, India

a r t i c l e i n f o a b s t r a c t

Article history: Gelatin was extracted from the skins of dog shark (Scoliodon sorrakowah), skipjack tuna (Katsuwonus
Received 27 January 2013 pelamis) and rohu (Labeo rohita) and their physico-chemical properties were measured. The skins of
Accepted 4 December 2013 shark, tuna and rohu yielded 19.7, 17.2 and 11.3% gelatin, respectively. The gel strength of dog shark
gelatin (6.67%, 10
C) was found to be higher (206 g) than tuna and rohu skin gelatins (177 g and 124 g,
Keywords: respectively). Similarly, molecular weight, viscosity, melting point, foaming properties, water holding
Gelatin
capacity, odour, colour and clarity of dog shark gelatin were in general better than the tuna and rohu skin
Skipjack tuna
gelatins. The amino acid analysis showed that hydroxyproline content in dog shark skin gelatin was the
Dog shark
Rohu
highest when compared to tuna and rohu skin gelatins.
Gel strength Ó 2013 Elsevier Ltd. All rights reserved.
Fish gelatin

1. Introduction importance (Gomez-Guillen et al., 2009). This may be due to the

Gelatin is a denatured fibrous protein derived from collagen by
partial thermal hydrolysis. It is an important functional biopolymer
that has broad applications in the food, pharmacy and photography
industries (Hao et al., 2009). The source, type of collagen and the
processing conditions will influence the properties of the resulting
gelatin. Different types of gelatins have varying thermal and
rheological properties such as Bloom strength, melting and gelling
temperatures. These properties are governed by factors such as
chain length or molecular weight distribution, amino acid
composition and hydrophobicity, etc. (Gomez-Guillen et al., 2002;
Norziah, Al-Hassan, Khairulnizam, Mordi, & Norita, 2009).
The global demand for gelatin has shown an increasing trend in
recent years. Recent reports indicate that the annual world pro-
duction of gelatin is nearly 326,000 tonnes, with pig skin derived
gelatin accounting for the highest (44%) output, followed by bovine
hides (28%), bovine bones (27%), and other sources (1%) (Ahmad &
Benjakul, 2011). Other sources, which include fish gelatin,
accounted for around 1.5% of total gelatin production in 2007, but
this percentage was double that in 2002, indicating that gelatin
production from alternative non-mammalian species had grown in
* Corresponding author. Tel.: þ91 4842666845; fax: þ91 4842668212.
E-mail address: shynicof@yahoo.co.in (K. Shyni).
shortage of the primary raw materials mostly cattle hides, bones
and pigskins (Spend Matters, 2012).
Gelatins from land animal sources are preferred over marine
sources due to their superior gel strength, melting point and vis-
cosity (Cho, Gu, & Kim, 2005). However, fish gelatin received
increasing attention as an alternative to land animal gelatin due to
religious constraints and health issues associated with the latter.
Both Judaism and Islam forbid the consumption of any pork-related
products and non-religiously slaughtered beef, while Hindus
refrain from consuming cow-related products (Karim & Bhat,
2009). In addition, gelatin from aquatic sources has been shown
to be free of infectious materials such as bovine spongiform en-
cephalopathy (Sadowska, Kolodziejska, & Niecikowska, 2003).
The fish skins and bones contribute almost 30% of the total
weight of the fish (Gomez-Guillen et al., 2002). Fish skins are a
major by-product of the fisheries and aquaculture industry. The
skin yield is highly variable according to species, fish size and
processing styles. Conversion of these wastes into value-added
products such as gelatin to yield additional income has both eco-
nomic and waste management benefits for the fish industry (Choi &
Regenstein, 2000).
A number of studies have addressed properties of fish skin
gelatins (Arnesen & Gildberg, 2007; Choi & Regenstein, 2000;
Fernandez-Diaz, Montero, & Gomez-Guillen, 2001; Gomez-
Guillen & Montero, 2001; Grossman & Bergman, 1992;

0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2013.12.008
 K. Shyni et al. / Food Hydrocolloids 39 (2014) 68e76
Gudmundsson, 2002; Gudmundsson & Hafsteinsson, 1997; Holzer,
1996; Jamilah & Harvinder, 2002; Jongjareonrak, Benjakul, Vises-
sanguan, & Tanaka, 2006; Jongjareonrak, Benjakul, Visessanguan,
Prodpran, & Tanaka, 2006; Yang et al., 2007; Zhou & Regenstein,
2005) showing that their properties differ from those of mamma-
lian gelatins. The gelatin properties were found to vary with the
method of production and between species.
Around the world, sharks are mainly used for producing shark
fins and fillets. As a result, skins are discarded along with the rest of
the carcass or treated as a by-product with low market value.
Studies have shown that cartilaginous fishes including sharks have
a higher content of collagen, the precursor of gelatin, when
compared to bony fishes (Nair, 2002). Cho et al. (2005) reported
that gelatin extracted from yellowfin tuna skin showed better
functional properties than those from other fish sources. Some
studies have ascertained that freshwater fish can have a high
gelatin yield (Grossman & Bergman, 1992; Jamilah & Harvinder,
2002; Muyonga, Cole, & Duodu, 2004a). Only a few studies have
been conducted on warm-water fish gelatin and these showed that
gelatin from these species had better functional properties than
those from cold-water fish species (Gilsenan & Ross-Murphy, 2000;
Grossman & Bergman, 1992; Leuenberger, 1991).
Dog shark and skipjack tuna are available along the south west
coast of India. Rohu is one of the major carp species, a natural
inhabitant of the freshwater sections of the rivers of India and
contributes to the inland catch. At present, the fishery is sustainable
for all the above species.
The present study was undertaken to extract and characterize
gelatin from the skins of skipjack tuna (Katsuwonus pelamis) from
the family scombridae, dog shark (Scoliodon sorrakowah) from the
family of carcharhinidae, a cartilaginous fish and rohu (Labeo
rohita) from the family of cyprinidae, a freshwater fish.
2. Materials and methods
2.1. Raw material
The species used for the study were skipjack tuna (K. pelamis),
dog shark (S. sorrakowah), and rohu (L. rohita). The marine fishes
were landed from long-line day boats that iced the fish on board, at
the local fish landing centre in Cochin during May 2011, from the
Arabian sea. The length of the fishes were measured and recorded,
i.e., tuna (92  2.4 cm, 17 in number) shark (73  2.9 cm, 23 in
number). The skinning of the fish was carried out by the fisherman
at the market under supervision. The iced skin in prime quality was
brought to the laboratory. Rohu (39  6.6 cm, 55 in number) freshly
caught and dead, in iced condition, was procured from a local fish
farm and skinning was carried out at laboratory in the iced
condition.
The skins were cut into 2e3 cm2 pieces using a scalpel and
washed with ice cold tap water. They were then placed in poly-
ethylene bag with added glaze water (10%) and stored at 20
C
until use. The storage time was less than 2 months. All chemicals
used unless otherwise noted were of analytical grade (Merck KGaA
Chemical & Pharmaceutical Company, Darmstadt, Germany;
SigmaeAldrich Corporation, MO, USA).
2.2. Gelatin extraction
Based on preliminary extraction trials, it was decided to follow
the gelatin extraction method of Gudmundsson and Hafsteinsson
(1997). Thawed skins were thoroughly washed and treated with
warm water (38e40
C) for 10 min to remove superfluous material
and reduce the fat content. Before gelatin extraction, skins were
soaked in 0.1 M NaOH at ambient temperature (w27
C) with a
69
skin/solution ratio of 1:10 (w/v) for 2 h. The alkaline solution was
changed every 1 h to remove non-collagenous proteins and pig-
ments. Alkaline-treated skins were washed with tap water until
the wash water was neutral or faintly basic. The pH of wash water
was monitored using Cyberscan 510 pH meter (Eutech In-
struments Pte Ltd, Singapore). The skins were then soaked in
0.2 M acetic acid with a skin/solution ratio of 1:10 (w/v) for 24 h
with gentle stirring at 4
C. The acidic solution was changed every
12 h to swell the collagenous material in the fish skin matrix. Acid-
pretreated skins were washed thoroughly with tap water until the
wash water became neutral. The skins were then subjected to a
final wash with distilled water to remove any residual matter. The
final extraction was carried out in distilled water at 45
C for 12 h
with a skin/water ratio of 1:10 (w/v). The clear extract obtained
was filtered through a Buchner funnel with Whatman filter paper
No. 4 (Sunshine Instruments, Coimbatore, Tamil Nadu, India). Fat
separation was done using a simple fat separating funnel (India-
MART, Maharashtra, India). The extract was poured in to the
funnel and was allowed to settle for few seconds. Within few
seconds, suspended fat formed a layer over the gelatin extract.
Then the gelatin was allowed to flow slowly through the outlet
valve of the funnel. The valve was carefully closed on reaching the
level of suspended fat layer. The clear extract obtained was
concentrated by evaporation under vacuum at 5
C, with a flash
evaporator (Buchi rotavapor R215, Buchi Labortechnik, Flawil,
Switzerland). The concentrated viscous solution was frozen in an
air blast freezer (Icematic T10, CastelMAC SpA, Castelfranco Veneto
(TV), Italy) at 40
C and then freeze-dried with freeze drier
(Gamma 1e16 LSC, Osterode am Harz, Germany) in two steps. The
solution was subjected to main drying for 8 h at set shelf tem-
perature of 20
C and set pressure of 0.01 mbar. The final drying
was done for 2 h at set shelf temperature of 25
C and set pressure
of 0.01 mbar at a condenser temperature of 55
C.
2.3. Yield of gelatin
The yields of the gelatins obtained were calculated as:
Dry wt: of gelatin
% Yield ðwet wt: basisÞ ¼  100
Wet wt: of skins
2.4. Determination of proximate composition
Moisture, lipid, ash and protein were determined by AOAC
(1995) methods 950.46, 960.39, 900.2A and 928.08, respec-
tively. Protein digestion was done as described by Eastoe and
Eastoe (1952) to ensure complete hydrolysis of collagen. A con-
version factor of 5.4 was used for calculating the protein content
from the Kjeldahl nitrogen content since collagen, the main
protein in skin, contains approximately 18.7% nitrogen (Eastoe &
Eastoe, 1952). This is an estimate and is based on mammalian
gelatin.
2.5. Determination of pH
The pH values of raw fish skins and gelatin solutions were
measured using the British Standard Institution method (BSI, 1975).
For determining the pH of the skins, samples were chopped and
blended for 5 min at ambient temperature (37
C) by vigorous
shaking (ICS-BLENDER, Hyderabad, Andhra Pradesh, India) in
distilled water to form a 1% (w/v) skin suspension. For the gelatin
solution, a 1.0% (w/v) gelatin solution was prepared by adding 1 g of
gelatin in 99 ml of distilled water. The mixture was heated to 45
C
70 K. Shyni et al. / Food Hydrocolloids 39 (2014) 68e76
for 5 min, for dissolving gelatin. The solution was allowed cool
down to room temperature before pH measurement. The pH was
measured using a Cyberscan 510 pH meter (Eutech Instruments Pte
Ltd, Singapore).
2.6. Gelatin colour and gel clarity
Instrumental colour measurements of the dry gelatin samples
were made using a HunterLab MiniScan XE Plus Spectrocolorimeter
(Hunter Associates Laboratory Inc., Reston, VA, USA) based on three
colour co-ordinates, namely L* (lightness), a* (redness/greenness)
and b* (yellow ness/blueness). The equipment was standardized
using a white tile and black glass. The gelatin sample was filled into
the glass sample cup and the cup was fed into the instrument.
Instrumental colour measurement readings were taken from the
instrument monitor. Visual observations for colour were also noted.
Clarity was determined by measuring transmittance (%T) at
620 nm in spectrophotometer (Spectronic Instruments Inc.,
Rochester, N.Y., USA) through 6.67% (w/v) gelatin solutions which
were heated at 60
C (Julabo TW20 water bath, Allentown, PA, USA)
for 1 h (Avena-Bustillos et al., 2006).
2.7. Determination of odour
Sensory evaluation was conducted using a seven member expert
panel according to the method of Muyonga et al. (2004a). The
samples were prepared in test tubes with screw caps, by dissolving
0.5 g of gelatin in 7 ml of distilled water, thus obtaining a solution
containing approximately 6.67% gelatin. The tubes were then held
in a water bath at 50
C for dissolving, with the screw caps closed.
Panelists were instructed to remove the screw caps, sniff the con-
tents and identify the odour they perceived as well as to indicate
the odour intensity, using a six point scale. i.e., 0 ¼ no odour,
1 ¼ very mild and only perceivable on careful assessment, 2 ¼ mild
but easily perceivable, 3 ¼ strong but not offensive, 4 ¼ strong and
offensive, 5 ¼ very strong and very offensive.
2.8. Determination of viscosity
The viscosity of the gelatin (6.67% concentration at 60
C) was
measured using a Brookfield digital viscometer (model DV-E,
Brookfield Engineering, Middleboro, MA, USA) equipped with a
No. 1 spindle at 30  0.5
C. The measured values were obtained
directly in centipoises (cP) from the instrument.
2.9. Determination of gel strength
The gel strength (Bloom) was determined according to British
Standard 757:1975 method (BSI, 1975), by using a texture analyzer
(Lloyd Instruments, Model LRX Plus, Sussex, U.K.). A solution con-
taining 6.67% (w/v) gelatin was prepared by mixing 7.50 g of gelatin
and 105 ml of distilled water in a Bloom bottle (Schott Duran,
Mainz, Rhineland-Palatinate, Germany). The mixture was swirled
and left to stand at room temperature for 1 hr, for allowing the
gelatin to absorb water and swell. The Bloom bottles were then
transferred to a water bath (Julabo TW20, Allentown, PA, USA)
maintained at 65
C and held for 25 min with occasional swirling to
dissolve the gelatin. The bottles were taken out of the water bath,
allowed to cool for 15 min at room temperature and then placed in
a cold-water bath (circulating bath, Haake D3 Model, Berlin, Ger-
many) maintained at 10  0.1
C and held at this temperature for
16e18 h before the determination of the gel strength. The Bloom
bottle was placed centrally under the plunger (Delrin probe, which
is an acetal homopolymer, 12.7 mm diameter, black in colour) of the
instrument. The Bloom strength was determined with a load cell of
5 kg and crosshead speed of 0.5 mm/s. The maximum force (g) was
determined when the probe penetrated to a depth of 4 mm into the
gel.
The test determines the weight (in grams) needed by a probe
(normally with a diameter of 0.5 inches) to deflect the surface of the
gel 4 mm without breaking it. The result is expressed as Bloom
(grams). Gelatin, if it gels, is usually between 30 and 300 Bloom.
2.10. Determination of amino acid composition
A dry sample (100 mg) of gelatin was weighed and taken in a
test tube. 10 ml 6 N HCl was added to in to the test tube. Then the
tubes were filled with nitrogen, sealed and hydrolyzed at 110
C for
24 h. After the hydrolysis, the contents were quantitatively trans-
ferred into a round bottom flask through Whatman filter paper. No.
42. The contents were flash evaporated to dryness. Distilled water
was added and flash evaporated again to remove traces of HCl. The
process was repeated thrice. The residue was then dissolved and
made up to 5e10 ml (known volume) with 0.05 M HCl. The sample
was then filtered through a membrane filter (Thermo Scientific,
MK, Buckinghamshire, England) with a nominal pore size of
0.45 mm. The sample (20 ml) was injected into the amino acid
analyzer (HPLCeLC 10 AS, Hitachi L-2130 Elite La Chrome, Tokyo,
Japan) and the amino acid composition was determined as per the
method of Ishida, Fugita, and Asai (1981). The system consists of a
binary pump (L-7100), autosampler (L-2200), column oven (L-
2350), fluorescent (FL) detector (L-2485) and a post-column deri-
vatisation unit fitted with a peristaltic pump. The column used is a
cation exchange column (Shodex, CX Pak, 4.6  15 mm) connected
with a guard column. The eluents used were buffer A (13.3 g tri-
sodium citrate, 70 ml ethanol, 12.8 ml citric acid monohydrate,
3.74 g NaCl, 4 ml Brij (SigmaeAldrich Corporation, MO, USA), pH 3.2
made up to 1 l with distilled water) and buffer B (117.6 g trisodium
citrate and 24.8 g boric acid, 500 ml distilled water, 45 ml 4 N
NaOH, pH to 10 and make up to 2 l with distilled water). The sep-
aration is effected using a gradient programme (0 min-100% A,
30 min-50% B). The eluted amino acids are subjected to post-
column derivatisation with ortho-phthalaldehyde (OPA) reagent
delivered using a peristaltic pump at the rate of 0.5 ml/min at 30
C.
Detection was done using a FL detector with absorption wave
length of 340 nm and emission wave length of 450 nm. Amino acid
standards for collagen hydrolyzate (Sigma A-9531) were run to
calculate the concentration of amino acids in the sample. Base line
auto-zero was made through software D-2000 Elite, Hitachi. The
method showed a linear response (R2 0.99) in this range. The
identification of amino acids in the sample is done using D-2000
Chromatography Data Station Software. Tryptophan analysis was
carried out according to colorimetric method (Sastry & Tammuru,
1985).
2.11. Determination of molecular weight distribution
Electrophoretic patterns of the different gelatins were analyzed
according to the method of Laemmli (1970). The samples were
dissolved in 50 g/l SDS solution. The mixtures were then heated at
85
C for 1 h, followed by centrifugation at 8500  g for 5 min at
room temperature using a microcentrifuge (MIK-RO20, Hettich
Zentrifugen, Germany) to remove undissolved debris if any. Solu-
bilized samples were mixed (1:1 v/v) with the sample buffer
(0.5 mol/l TriseHCl, pH 6.8 containing 40 g/l SDS, 200 ml/l glycerol
in the presence or absence of 100 ml/l b mercaptoethanol). The
mixtures were loaded onto a polyacrylamide gel having a 7.5%
resolving gel (10% SDS: 100 ml, acrylamide: 2.5 ml, ammonium
persulphate (APS) 10%: 50 ml, distilled water: 4.85 ml, TEMED: 5 ml,
TriseHCl, 1.5 M: 2.5 ml) and a 4% stacking gel (10% SDS: 100 ml,
 K. Shyni et al. / Food Hydrocolloids 39 (2014) 68e76
acrylamide: 1.33 ml, APS 10%: 50 ml, distilled water: 6.1 ml, tetra-
methylethylenediamine (TEMED): 10 ml, TriseHCl, 1.5 M: 2.5 ml).
The electrophoresis was done till the base line reached bottom of
the gel, at a constant current of 20 mA per gel using a Bio-Rad Tetra
Mini Protean II unit (Bio-Rad Laboratories, Inc., Richmond, CA, USA)
with gel documentation system. After electrophoresis, gels were
fixed with a mixture of 500 ml/l methanol and 100 ml/l acetic acid
for 30 min, followed by staining with 0.5 ml/l Coomassie blue R-
250 (ultra-pure grade) in 150 ml/l methanol and 50 ml/l acetic acid
for 1 h. Finally, they were destained with a mixture of 300 ml/l
methanol and 100 ml/l acetic acid for 1 hr and destained again with
the same solution for 30 min. High molecular weight protein
marker (Sigma Chemical Co., St. Louis, MO, USA) with a molecular
weight range of 36,000e205,000 Da was used to estimate the
molecular weight of proteins. The molecular weight distribution of
sigma marker (Da) used in the study is
 Myosin, rabbit muscle e 205,000
 b-Galactosidase, Escherichia coli e 116,000
 Phosphorylase b, rabbit muscle e 97,000
 Fructose-6-phosphate Kinase, rabbit muscle e 84,000
 Albumin, bovine serum e 66,000
 Glutamic dehydrogenase, bovine liver e 55,000
 Ovalbumin, chicken egg e 45,000
 Glyceraldehyde-3-phosphate dehydrogenase, rabbit muscle e
36,000
2.12. Determination of melting point of gelatin
The melting point measurement was done according to the
method of Wainewright (1977). Gelatin solutions, 10% (w/w), were
prepared and a 5 ml aliquot of each sample was transferred to a
small culture tube (12  75 mm). The samples were degassed in
vacuum desiccators (Heraeus vacutherm, Hohenpriessnitz, Sach-
sen, Germany) for 5 min. The tubes were then covered with Par-
afilm (Pechiney Plastic Packaging, Inc., Chicago, IL, USA) and heated
in a water bath at 60
C for 15 min. The tubes were immediately
cooled in ice-chilled water and matured at 10
C for 18 h. Five drops
of a mixture of 75% chloroform and 25% red food colour (Magnil
Dye Chem, Mumbai, Maharashtra, India) was placed on the surface
of the gel. The gels were put in a water bath (Haake D3, Lab
Extreme, Inc., Kent City, MI, USA) at 10
C and the bath was heated
at the rate of 0.2
C/min. The temperature at which the dye drops
began to move freely down the gel was taken as the melting point.
2.13. Determination of setting point and setting time
The method used for the determination of setting point (SP) and
setting time (ST) of gelatin was that described by Muyonga et al.
(2004a). Gelatin solutions of 10% (w/v) dissolved in thin wall
(12  75 mm) test tubes were prepared in the same way as
described for the Bloom samples. For setting point determination,
the dissolved samples from the warm-water bath were transferred
to a circulating water bath held at 40
C (Haake D3). The bath was
then cooled at the rate of 2
C/min. A thermometer was inserted
into the sample and lifted out at 15 s intervals. The temperature of
the mixture at which the gelatin solution no longer dripped from
the tip of the thermometer was recorded as the setting
temperature.
Setting time was determined on samples prepared in the same
way as those for the determination of the setting temperature.
Samples were transferred to the water bath maintained at 10
C
(Haake D3). A rod was inserted into the gelatin solution and raised
71
at intervals of 15 s. The time at which the rod could not detach from
the gelatin sample was recorded as the setting time.
2.14. Determination of foaming properties
Foam formation ability (FA) and foam stability (FS) of gelatin
were determined by the procedure of Cho et al. (2004). Gelatin
solution, 1 g/100 ml was put in a beaker and swollen at 60
C. The
foam was prepared by homogenizing at 10,000 rpm for 5 min in a
homogenizer (Euro Turrax T20b, IKA Labortechnik, Staufen, Ger-
many). The homogenized solution was then poured into a 250 ml
measuring cylinder. The FA was calculated as the ratio of volume of
foam to the initial volume of liquid. The foam stability was calcu-
lated as the ratio of the initial volume of foam to the final volume of
foam after 30 min.
2.15. Determination of water holding capacity and fat binding
capacity
Fat binding capacity (FBC) and water holding capacity (WHC) of
gelatin were determined as per the procedure of Cho et al. (2004).
For measuring FBC, 1 g of gelatin powder was placed in a
centrifuge tube and weighed (tube with gelatin). Then, 10 ml sun-
flower oil was added, and held at room temperature for 1 h. During
this period, the gelatin solutions were mixed with a Vortex mixer
(CM-101 Plus, REMI Instruments, Maharashtra, India) for 5 s every
15 min. The gelatin solutions were then centrifuged at 450  g
(Model CPR 24, REMI Instruments, Maharashtra, India) for 20 min
with cylinder bottom centrifuge of 20 ml capacity (REMI In-
struments, Maharashtra, India). The upper phase was removed by
tilting the centrifuge tube to 45
angle and draining on to a filter
paper for 30 min. The FBC was calculated as the weight of the
contents of the tube after draining divided by the weight of the
dried gelatin, and expressed as the weight % of dried gelatin.
For measuring WHC, 1 g of gelatin powder was placed in a
centrifuge tube and weighed (tube with gelatin). Distilled water
(50 ml) was added, and held at room temperature for 1 h. During
this period, the gelatin solutions were mixed with a Vortex mixer
(CM-101 Plus, REMI Instruments, Maharashtra, India) for 5 s every
15 min. The gelatin solutions were then centrifuged at 450  g
(Heraeus Multifuge 3SR Plus, Thermo Scientific, MK, Buck-
inghamshire, England) for 20 min with 600 ml bucket centrifuge
(Thermo Scientific, MK, Buckinghamshire, England). The upper
phase was removed and the centrifuge tube was drained for 30 min
on a filter paper after tilting to 45
angle. WHC was calculated as
the weight of the contents of the tube after draining divided by the
weight of the dried gelatin, and expressed as the weight % of dried
gelatin.
2.16. Statistical data analysis
One way analysis of variance was used to find the effect of
different species on yield and functional properties of gelatin. Once
ANOVA was found significant, Tukey’s test was used as a post-hoc
test to compare the means of different species. The statistical
analysis was carried out using SAS. 9.3 software (SAS Institute Inc,
Cary, NC, USA). The mean value of three replications and its stan-
dard deviations are shown in the tables. Letters are placed as a
superscript along with the mean to indicate significant difference
between the means in all the tables except the amino acid table, i.e.,
means with different superscripts are statistically significant at 1%
level of significance (p < 0.01).
72 K. Shyni et al. / Food Hydrocolloids 39 (2014) 68e76
3. Result and discussion
3.1. Proximate composition of fish skins
The protein, moisture and ash content values of three selected
fish skins after cleaning, are tabulated in Table 2. All three fish skins
had considerably higher ash contents (2.03e4.39%) compared to
that of the Red tilapia (0.51%), Walking catfish (0.52%) and Striped
catfish (0.46%) (Jamilah, Tan, Umi Hartina, & Azizah, 2011).
Compared to the other two species, ash content in rohu skins was
lower, probably due to perfect de-scaling. De-scaling was not car-
ried out for shark and tuna skins since the scales were too tiny for
tuna and was deeply embedded in the shark skin. Shark skins
contained comparatively higher amount of protein (27.7%) than
other two skins. The skins of rohu had highest moisture content,
followed by shark and tuna skins. Subcutaneous fat was highest
(18.3%) for tuna skins when compared with shark and rohu skins.
This can be explained as the site of lipid storage in shark is liver,
whereas it is skin and viscera in tuna. The results of lipid can be
correlated with the moisture content in reverse order in the above
species.
3.2. Yield of gelatin
Gelatin yield is calculated as g of gelatin per 100 g of clean skins.
The yield of shark skin gelatin (SSG), tuna skin gelatin (TSG) and
rohu skin gelatin (RSG) were significantly different (p < 0.01) and
were 19.7  0.04%, 11.3  0.03% and 17.2  0.03%, respectively. It
was observed that shark skins tend to swell more in the alkaline
and acidic solutions compared to the skins of rohu and tuna.
Therefore, shark skins gave a better yield; possibly due to increased
opening of cross-links during swelling. As a cartilaginous fish, shark
has comparatively more connective tissue and hence more collag-
enous material than bony fishes. This can be correlated with the
high yield of gelatin from shark skins.
Gelatin processing has three steps, alkali pretreatment, acid
pretreatment and hot water extraction. The alkali & acid treatment
removes non-collagenous protein and the sample swells in the acid
solution. The hot water extraction uses thermohydrolysis to solu-
bilize gelatin which is then separated. The lower yield observed in
rohu could be due to the leaching of collagen during the washing
treatment or insufficient denaturation of collagen during extraction
(Jamilah & Harvinder, 2002). The crude protein content and hy-
droxyproline content were also lower in rohu skins.
3.3. Proximate composition of gelatin
The proximate compositions of extracted gelatins are tabulated
in Table 1. The gelatin extracted from skins of the three species
showed protein as the major component and protein content varied
between 88.4 and 90.1%. Jongjareonrak, Benjakul, Visessanguan,
Prodpran, et al. (2006) reported a protein content of 87.9% and
Table 1
Proximate composition and pH of skins and gelatins from shark, rohu and tuna.
Sample Components and pH
Moisture
Skins Shark 68.4  0.43
Rohu 76.5  0.45
Tuna 56.5  0.09
Gelatin Shark 8.7  0.25b
Rohu 9.3  0.36b
Tuna 10.9  0.24a
Values are given as mean  standard deviation of triplicate. Values with the same superscript letters within a column are not significantly different (p < 0.01).
88.6% in gelatin extracted from the skins of bigeye snapper and
brown eye snapper, respectively. The gelatin from the skins of adult
Nile perch also contained 88% protein when extracted at 50
C
(Muyonga et al., 2004a). Moisture content in all the samples was
well below the prescribed limit of 15% (GME, 2005) for edible
gelatin. At 6e8% moisture, gelatin is very hygroscopic and it be-
comes difficult to determine the physico-chemical attributes with
accuracy (Cole, 2000). The gelatin samples extracted were almost
free of fat. This showed that the simple de-fattening method fol-
lowed here had eliminated the fat content as desired. The gelatins
were found to be low in ash content, well below the recommended
maximum of 2.6% (Jones, 1977).
3.4. The pH values of gelatin samples
The pH of extracted gelatins varied between 4.17 and 4.34 as
shown in Table 1, indicating their category as Type B. It has been
reported that alkali pretreatment results in Type B gelatin with pH
in the range of 4e5 (Baziwane & He, 2003) and in the present study
an alkali pretreatment was employed during the extraction of
gelatin. Wide variations in the pH of skins gelatin of cod (2.7e3.9)
(Gudmundsson & Hafsteinsson, 1997), red tilapia (3.05) and black
tilapia (3.91) (Jamilah & Harvinder, 2002), freshwater carps (4.01e
4.88) (Ninan, Abubacker, & Jose, 2011; Ninan, Jose, & Abubacker,
2011) and bigeye snapper (6.44) (Binsi, Shamasundar, Dileep,
Badii, & Howell, 2009) have been reported. This was, most likely
due to the different pretreatments employed during the extraction
involving both alkaline and acid treatments.
It has also been reported that for Type B gelatin, the viscosity is
minimum and gel strength is maximum at pH 5.0 (Cole, 2000)
signifying the importance of pH for gelatin’s rheological properties.
Hence from the manufacturer’s point of view it is advantageous to
manufacture gelatin with a pH of 5.0. However, due to the strong
buffering capacity of gelatin, this pH may not be the most advan-
tageous for the customer.
3.5. Gelatin colour and gel clarity
Both colour and clarity of a gelatin gel are important aesthetic
properties, depending on the application for which the gelatin is
intended. Instrumental colour measurements of gelatins are tabu-
lated in Table 2. The colour of gelatin depends on the raw materials
used and method of extraction (Ockerman & Hansen, 1999). In
general, colour does not influence the functional properties. How-
ever light colour is preferred because it is easier to incorporate
gelatins into any food systems without imparting any strong colour
attribute to the product. According to the instrumental colour
measurement, shark skins gelatin was significantly lighter/whiter
than the other two samples, Rohu skins gelatin was the greenest
and it was significantly (p < 0.01) less yellowish when compared to
shark and tuna gelatins. By visual observation, shark and tuna
Protein Fat Ash pH
27.7  0.36 0.16  0.02 4.19  0.03 6.23  0.01
18.8  0.06 2.93  0.05 2.03  0.04 6.37  0.02
20.5  0.26 18.3  0.11 4.39  0.03 6.31  0.01
90.1  0.30a 0.42  0.05b 0.72  0.06a 4.34  0.01a
89.2  0.49b 0.48  0.08b 0.73  0.07a 4.17  0.01c
88.4  0.12b 0.82  0.05a 0.68  0.06a 4.29  0.01b
 K. Shyni et al. / Food Hydrocolloids 39 (2014) 68e76 73

Table 2 gelatin has been observed in the pH range of 6e8 (Stainsby, 1987).
Instrumental colour, visual observation and transmittance of shark skin gelatin The above results thus indicate that natural variations in the vis-
(SSG), rohu skin gelatin (RSG) and tuna skin gelatin (TSG).
cosity can be expected from different fish species, although the
Sample L* a* b* Colour Transmittance extraction methods do play a role.
SSG 82.8  0.40a 1.65  0.04b 18.3  0.21a Pearly 44.8  0.72a
white
RSG 78.1  0.62b 0.75  0.01c 5.36  0.04b Shiny 44.4  0.59a
3.8. Gel strength values of gelatin samples
whitish
TSG 75.3  0.60c 9.13  0.04a 18.4  0.17a Pearly 35.5  0.87b Gel strength (Bloom) is the most important physical property of
white a gelatin. The gel strengths of shark, rohu and tuna gelatins were
Values are given as mean  standard deviation of triplicate. Values with the same 206,124 and 177 g, respectively. All three samples were significantly
superscript letters within a column are not significantly different (p < 0.01). (p < 0.01) different (Table 3). The gel strength of fish gelatin has
been reported in a wide range of 124e426 Bloom, compared to
200e300 Bloom for bovine or porcine gelatin (Karim & Bhat, 2009).
gelatins appeared pearly white while rohu gelatin was shiny
This may be due to many factors such as pH, molecular weight
whitish.
distribution and amino acid content. Hydroxyproline contents of
While shark and rohu gelatins showed good transmittance (%T),
shark, rohu and tuna gelatins were about 9.85%, 6.78% and 7.86%, of
tuna gelatin showed significantly (p < 0.01) poor %T values
the total amino acids (Table 4), while that of bovine gelatin was 14%
(Table 3). The turbidity and dark colour of gelatin is commonly
(Nalinanon, Benjakul, Visessanguan, & Kishimura, 2008). The gel-
caused by inorganic, proteinaceous and mucosubstance contami-
ling properties of gelatin are influenced by the source of raw ma-
nants introduced or not removed during its extraction (Eastoe &
terials, which vary in proline and hydroxyproline contents
Leach, 1977). Muyonga et al. (2004a) stated that the efficiency of
(Jongjareonrak, Benjakul, Visessanguan, Prodpran, et al., 2006). The
the filtration process during gelatin extraction affected the degree
main difference between fish and mammalian gelatins is the imino
of clarity of gelatin solution.
acid content, where the mammalian gelatins have higher amounts
(Gudmundsson, 2002). Imino acids played a role in gel formation.
3.6. Odour of gelatin samples The hydroxyl groups of hydroxyproline play a part in the stability of
the helix by inter-chain hydrogen bonding via a bridging water
Sensory evaluation showed a difference in odour between gela- molecule as well as direct hydrogen bonding to the carbonyl group
tins. The shark and rohu gelatins were found to be free of fishy odour (Wong, 1989). Gelatin, with the shorter chain lengths cannot form
and had a mild putrid odour (hedonic score of 1.83  0.29 and as strong a gel due to the lower inter-junction zones
2.17  0.29, respectively), whereas the tuna gelatin had a detectable (Intarasirisawat et al., 2007). The quality of gelatin is generally
fishy odour with a hedonic score of 3 which was significantly higher determined by the gel strength or Bloom value, including low
(p < 0.01) than shark and rohu gelatins. Muyonga et al. (2004a) re- (<150), medium (150e220) and high Bloom (220e300) (Johnston-
ported that the gelatins prepared from the skins and bones of Nile Bank, 1983). Gudmundsson and Hafsteinsson (1997) suggested that
perch by activated carbon treatment were found to have a mild the gel strength may depend on the isoelectric point and may be
putrid odour. Strong fishy odour was reported for freeze-dried controlled, to certain extent, by adjusting the pH.
gelatin prepared from the skins of black tilapia (Jamilah & Harvinder,
2002). Choi and Regenstein (2000) observed that activated carbon
treatment at the final stage of extraction can further reduce the 3.9. Amino acid composition
odour and improve the acceptability of gelatin.
The amino acid composition of gelatin extracted from the skins
of shark, rohu and tuna are shown in Table 4. It is expressed as g of
3.7. Viscosity values of gelatin samples

Viscosity is the second most important commercial property of Table 4

gelatin after gel strength (Ward & Courts, 1977). The viscosity for Amino acid composition of shark skin gelatin (SSG), rohu skin gelatin (RSG) and tuna
the samples was in the range of 2.5e5.6 cP and all the samples were skin gelatin (TSG).

significantly (p < 0.01) different. The viscosity was lowest in rohu Amino acid Residues/100 residues
gelatin compared to shark and tuna gelatins (Table 3). Viscosity is SSG RSG TSG
partially controlled by molecular weight and molecular size dis-
Aspartic 3.64  0.09 4.39  0.10 4.08  0.17
tribution (Sperling, 1985). The viscosities of most of the commercial
Threonine 2.08  0.19 2.38  0.11 2.33  0.13
gelatins have been reported to be up to 13.0 cP (Johnston-Banks, Glutamic 7.69  0.27 6.30  0.36 7.26  0.23
1990). Jamilah and Harvinder (2002) reported the viscosity values Glycine 32.8  062 33.4  0.74 33.2  0.55
of 3.2 cP and 7.12 cP for red and black tilapia, respectively, whereas Alanine 10.9  0.30 11.7  0.20 12.2  0.36
Cystine 0 0 0
for channel catfish, it was 3.23 cP (Yang et al., 2007). The changes in
Valine 2.52  0.24 3.06  0.25 2.95  0.22
pH are known to influence the viscosity and minimum viscosity for Methionine 1.07  0.15 1.27  0.12 1.24  0.07
Isoleucine 1.58  0.05 0.77  0.03 1.09  0.07
Leucine 2.17  0.17 2.14  0.18 2.04  0.09
Table 3 Tyrosine 0.08  0.03 0.25  0.07 0.26  0.03
Viscosity and Bloom values of shark skin gelatin (SSG), rohu skin gelatin (RSG) and Phenylalanine 1.58  0.10 1.93  0.09 1.37  0.09
tuna skin gelatin (TSG). Lysine 2.77  0.13 2.48  0.25 2.57  0.12
Hydroxylysine 0.73  0.10 0.70  0.08 0.77  0.08
Sample Viscosity Bloom
Histidine 0.86  0.07 0.67  0.12 0.88  0.08
SSG 5.60  0.10a 206  3.51a Arginine 5.29  0.09 5.47  0.13 4.76  0.15
RSG 2.50  0.00c 124  3.61c Proline 9.90  0.11 11.6  0.17 10.1  0.12
TSG 4.37  0.06b 177  3.51b Hydroxyproline 9.85  0.33 6.78  0.17 7.86  0.27
Serine 3.61  0.11 4.11  011 4.36  0.11
Values are given as mean  standard deviation of triplicate. Values with the same
Total 99.1 ± 0.74 99.4 ± 0.63 99.4 ± 0.50
superscript letters within a column are not significantly different (p < 0.01).
74 K. Shyni et al. / Food Hydrocolloids 39 (2014) 68e76

amino acids per 100 g of solids. All gelatins had glycine as their
major amino acid (32.8e33.4) and had relatively high content of
alanine (10.9e12.2). For imino acids, all samples had proline and
hydroxyproline content of 9.90e11.6 and 6.78e9.85, respectively. It
was observed that the level of tryptophan was below the detectable
limit in all three samples. The preparation of amino acid monomers
from gelatin through the acid hydrolysis process leads to the
destruction of indole ring of tryptophan. Hence the detection of
tryptophan is not possible in HPLC. The absence can also be resulted
from acid hydrolysis during the gelatin extraction process
(Chapman & Hall, 1997; Jamilah & Harvinder 2002). Cysteine and
tryptophan are not commonly present in collagen and gelatin
(Foegeding, Lanier, & Hultin, 1996; Jongjareonrak, Benjakul,
Visessanguan, & Tanaka, 2006; Muyonga, Cole, & Duodu, 2004b;
Yata, Yoshida, Fujisawa, Mizuta, & Yoshinaka, 2001). Regenstein
and Zhou (2007) reported that glycine, alanine, proline and hy-
droxyproline are four of the most abundant amino acids in gelatin.
The samples had an imino acid content of 17.96e19.75% of
protein. Kasankala, Xue, Weilong, Hong, and He (2007) reported an
imino acid content of 19.47% for gelatin prepared from the skin of
carp. Imino acid content of Nile perch gelatin was 21.5% (Muyonga
et al., 2004a). Grossman and Bergman (1992) reported an imino
acid content of 17% and 5%, for gelatin from cod and tilapia,
respectively. Mammalian gelatins generally contain 30% imino
acids (Poppe, 1992). Johnston-Banks (1990), reported that the
imino acids, especially hydroxyproline, impart considerable rigidity
to the collagen structure and a relatively limited imino acid content
might affect the dynamic properties of gelatin. Maximum gel
strength was observed for shark skin gelatin which shows that
Plate I. Protein pattern of gelatin gels from of shark skin gelatin (SS), rohu skin gelatin
hydroxyproline is the major determinant of stability due to its
(RS) and tuna skin gelatin (TS) run alongside of the high molecular weight marker
hydrogen bonding ability through its hydroxyl group, although (HM).
proline is also important (Burjandze, 1979; Ledward, 1986).

3.10. Molecular weight distribution
The gelatin preparations from the skins of three species were
analyzed using SDS-PAGE, to characterize differences in molecular
weight distribution (Plate I). For gelatin gels a1 and a2 chains were
found as the major components for all the samples. However, b
component (covalently linked a chain dimer) with molecular
weight w200 kDa was observed in shark gelatin and it was absent
in the other two samples. The presences of peptides with molecular
weight below 100 kDa were slightly noticeable in all three samples.
That may be the results of heat induced cleavages of protein chains
that occurred during the extraction process (Muyonga et al.,
2004a). The result suggested that b chains of shark skin gelatin
were more tolerant to thermal hydrolysis than those of tuna and
rohu skin gelatin.
3.11. Melting point of gelatin
The melting point of shark and tuna gelatins was significantly
higher (p < 0.01) than that of rohu gelatin (Table 5). This can be
correlated with the higher hydroxyproline content in these gela-
tins. Melting point increases with the maturation time and it has
been observed that the levels of hydroxyproline contribute to the
melting point characteristics (Choi & Regenstein, 2000;
Gudmundsson, 2002). It is known that fish gelatins have lower
melting points than mammalian gelatins. Norland (1990) and
Gudmundsson (2002) had observed melting points of 29.7
C and
32.3
C, for bovine and porcine gelatin, respectively. The melting
points observed in the present study are far higher than those re-
ported for cold-water fishes such as cod (13.8
C), hake (14
C)
(Gomez-Guillen et al., 2002) and hoki (16.6
C) (Mohtar, Perera, &
Quek, 2010). However, these melting points were lower than that
of black tilapia (28.9
C) (Jamilah & Harvinder, 2002) which is a
warm-water fish. Fish gelatins with lower melting temperatures
had a better release of aroma and offered stronger flavour and
useful in product development to control the texture and flavour
release during mastication (Zhou, Steven, & Regenstein, 2006).
3.12. Setting point and setting time of gelatin
Setting point denotes the temperature at which setting/gelling
process begins, which involves the transition from random coil to
triple helical structure of gelatin (Haug, Draget, & Smidsrod, 2004).
Setting points of shark and tuna gelatins were significantly higher
(p < 0.01) than that of rohu gelatin (Table 5). Bovine and porcine
gelatins have considerably higher setting and melting points than
most fish gelatins (Choi & Regenstein, 2000; Gilsenan & Ross-
Murphy, 2000; Gudmundsson, 2002; Leuenberger, 1991). Setting
points of cold-water fish gelatin was in general lower than warm-
water fish/mammalian gelatins. This difference is related to the
imino acid composition. Setting and melting temperatures are also
influenced by the change in ionic strength and pH of gelatin. They
decreased with the increase in ionic strength of >0.5 mol/L, which
Table 5
Melting point (MP), setting point (SP) and setting time (ST) parameters for shark
skin gelatin (SSG), rohu skin gelatin (RSG) and tuna skin gelatin (TSG).
Sample MP (
C) SP (
C) ST (s)
SSG 25.8  0.29a 20.8  0.29a 93.3  1.15c
RSG 18.2  0.29c 13.8  0.29c 114  1.53a
TSG 24.2  0.29b 18.7  0.29b 103  1.15b
Values are given as mean  standard deviation of triplicate. Values with the same
superscript letters within a column are not significantly different (p < 0.01).
 K. Shyni et al. / Food Hydrocolloids 39 (2014) 68e76
was probably due to the reduced electrostatic interaction pre-
venting ionic inter-chain bridging and gelation of fish gelatin (Haug
et al., 2004).
Setting time required for shark skin gelatin was significantly
(p < 0.01) lower than rohu and tuna gelatins (Table 5). Gelling
temperatures reported for Nile perch bone gelatin were 18.5e
19.0
C and gelling time of 90 s (Muyonga et al., 2004a). Similarly,
Ninan, Abubacker, et al. (2011) and Ninan, Jose, et al. (2011)
observed gelling temperatures of 18.5
C and 18.0
C and gelling
times of 106 and 103 s in rohu and common carp skin gelatin which
differed from the result of the present study. That may be attributed
to the difference in age and size of the fish or because of the dif-
ference in extraction conditions.
3.13. Foaming properties
Foaming ability (FA) and foam stability (FS) are another impor-
tant property of gelatin for commonly used foods such as marsh-
mallows (Zuniga & Aguilera, 2009). Foaming ability and foam
stability of the gelatins are shown in Table 6. The foaming capacity
of shark gelatin was found to be significantly higher (p < 0.01) than
both tuna and rohu gelatins. Foam formation is generally controlled
by transportation, penetration and reorganization of protein mol-
ecules at the airewater interface. A protein must be capable of
migrating rapidly to the airewater interface, unfolding and rear-
ranging at the interface to show good foaming properties (Halling,
1981). Foam stability depends on the nature of the film and in-
dicates the extent of protein interaction within the matrix
(Mutilangi, Panyam, & Kilara, 1996). Foam stability was significantly
lower (p < 0.01) in rohu gelatin when compared to shark and tuna
gelatins. Foams with higher concentration of proteins were denser
and more stable because of an increase in the thickness of inter-
facial films (Zayas, 1997). It has been suggested that reduced foam
formation and stability may be due to aggregation of proteins
which interfere with interactions between the protein and water
needed for foam formation (Kinsella, 1977).
3.14. Water holding capacity and fat binding capacity
Water holding and fat binding capacities are functional prop-
erties that are closely related to texture by the interaction between
components such as water, oil and other components (Cho et al.,
2004). Water holding capacity and fat binding capacity of gelatins
are shown in Table 6. Water holding capacity is significantly higher
(p < 0.01) in shark gelatin. Higher water holding capacity is mainly
related to the higher amounts of hydrophilic amino acids and
higher hydroxyproline content (Ninan, Abubacker, et al., 2011;
Ninan, Jose, et al., 2011). Fat binding capacity is significantly
higher (p < 0.01) in tuna gelatin. The degree of exposure of the
hydrophobic residues and the high amount of tyrosine were found
responsible for the high fat binding capacity (Ninan, Abubacker,
et al., 2011; Ninan, Jose, et al., 2011).
Table 6
Foaming ability (FA), foam stability (FS), water holding capacity (WHC) and fat
binding capacity (FBC) of shark skin gelatin (SSG), rohu skin gelatin (RSG) and tuna
skin gelatin (TSG).
Sample FA (%) FS (%) WHC FBC
SSG 21.5  0.45a 17.6  0.35a 256  4.51a 347  5.51b
RSG 17.4  0.15c 10.5  0.25c 163  4.00c 360  29.19b
TSG 19.2  0.36b 14.4  0.25b 214  3.61b 452  6.51a
Values are given as mean  standard deviation of triplicate. Values with the same
superscript letters within a column are not significantly different (p < 0.01).
75
4. Conclusion
It may be concluded that there are considerable differences
between yield and functional properties of gelatin from the skins of
dog shark, skipjack tuna and rohu. The characteristics of the gela-
tins extracted through this process indicate that they have good
yield and functional properties. There is, therefore, a potential for
exploitation of processing waste for gelatin extraction from these
species. The potential is higher for dog shark skins than the other
two species because dog shark skins give higher gelatin yield and
the skin gelatin showed better functional properties.
The ability to form weak gels may find new applications for fish
gelatin and it could possibly be used in refrigerated products and in
products where low gelling temperatures are required
(Gudmundsson, 2002).
Acknowledgements
The authors are thankful to the Director, Central Institute of
Fisheries Technology (Kochi), Kerala, India for according permission
to publish the paper. The authors also wish to express their grati-
tude to the Department of Biotechnology, Ministry of Science and
Technology, Government of India, for the financial support given for
this research work.
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