TASK OF FOOD PROCESSING MATERIALS

ISOLATION AND SYNTHESIS OF FISH

SCALES GELATIN KAKAP
WHITE (Lates calcarifer) crosslinked
GAMMA IRRADIATED WITH INDUCTION
TECHNIQUE

Name :Nur Lailatul Hidayah.

NIM : 14050394023.

S1 PENDIDIKAN TATA BOGA

JURUSAN PENDIDIKAN KESEJAHTERAAN KELUARGA

UNIVERSITAS NEGERI SURABAYA

2014/2015
 ABSTRACT

ISOLATION AND SYNTHESIS OF CROSSLINKED

GELATIN OF WASTE LATES CALCARIFER SCALES USING
GAMMA IRRADIATION INDUCTION TECHNIQUE. In order to
use the fisheries waste, isolation of gelatin has been carried out from
Lates calcarifer scales and then synthesized to be cross-linked gelatin
by induction of gamma irradiation at irradiation dose of 0-50 kGy.
Chemical structures change of irradiated gelatin was characterized by
using UV-Vis and FT-IR spectrophotometer. The viscosity of gelatin
was measured by using viscosimeter. The results showed that
radiation dose correlate linearly to absorbance and viscosity of gelatin
solution at doses of 10-30 kGy. While at doses 40-50 kGy, gelatin
solution was transformed from solution to gel with significant change
in FT-IR spectrum. Based on this result, gamma irradiation technique
should be considered as a safe and quick technique to synthesis cross-
linked gelatin.

Keywords: Fish scale, Gelatin, Gamma irradiation, Crosslinking

 INTRODUCTION

Waste from fish processing known reaches 75% of the total weight of
fish [1]. the waste such as bone, skin and fish scales are considered as low-value
materials. Waste fishing is expected to reduce the volume of waste while
providing added value to the product generated. Gelatin is one of the material
contained in the prospective fishery waste to be developed.

At this time most of the commercial gelatin extracted from pigs

and cows. the prohibition consume pork for Muslims and cows for the people
Hindu as well as the outbreak of mad cow disease, bovine spongiform
encephalopathy triggered the development of The new source for the
isolation of gelatin, one of which is fishery waste. Therefore, fish
gelatin has manufactured from leather Oreochromis mossambicus,
Oreochromis nilotica, Priacanthus macracanthus, Lutjanus vitta Johnius
dussumeiri and Decapterus macrosoma, as well as skin and bone Lates
niloticus.
In addition to skin and fish bones, fish scales is one one form of waste
that can be processed fishery as a source of gelatin with a relative quantity large.
Indonesia as an archipelago have a great chance to develop gelatin fish. Lates
calcarifer is one type of fish. consumption in Indonesia that waste can be used as a
source of gelatin. Gelatin is a product of collagen partially hydrolyzed consists of
a mixture of chains polidispersi polypeptide with a molecular weight of more than
30 kDa. Gelatin is one of the materials prospective because it is biodegradable and
biocompatible in physiological environment that can used as a biomaterial.
Various types of gelatin are generally used as food, films, adhesives, moisturizer
and medical devices. In the field of biomaterials, gelatin has now used as capsule
material, wound dressings and scaffold in tissue engineering. gelatin in generally
in the form of sol at temperatures> 40 0C and transition hydrogel occurs if the
temperature is lowered past the temperature sol-gel transition of the gelatin. Sol-
gel transition is as thermo-reversible process that takes place non-covalent bonds
such as hydrogen bonds and ionic bonds between gelatin molecules.
Thermoplastic properties of gelatin are important in field of food as in the
formation of jelly and not feasible in the field of medical devices due hydrogel
formed can not maintain shape at body temperature (37 oC). because that,
to overcome this has been done research to synthesize crosslinked gelatin. In
general, the synthesis of cross-linked gelatin carried out by the reaction of clams
using aldehyde derivative compounds are toxic such as Glutaral dehida, formal
dehydeandacetaldehyde.
Gamma and electron beam irradiation is tools (tool) is the most popular
utilized for sterilization in health, preservation food. In addition, both devices are
can be used for surface coating and manufacture of heat shrinkable cable. Both
types of The energy source at this time has been utilized Further to the
crosslinking process and degradation monomers / polymers. Advantages of
gamma radiation and beam Among other electron is relatively quick process, not
residue left, no need catalyst or initiator and irradiation dose can be adjusted as
necessary. Therefore, in this study will be used gamma radiation to synthesize
crosslinked gelatin of gelatin solution. Based on the description of things above,
the study was conducted synthesis of isolated fish scales gelatin into gelatin
crosslinked using gamma irradiation and investigate the physico-chemical
character.
 METHODS EXPERIMENT

 Materials :
The materials used are waste fish scales barramundi (Lates calcarifer) were
obtained from the Center for Processing and Products Research and Marine
Biotechnology Fisheries, Arcadia. Chemicals used in the form of acetic acid
(glacial) 100% (pro analysis, Merck) and potassium bromide (pro analysis,
Merck).

 Tool :
Spektrofotometer Ultra Violet - Visible (UV-Vis),(Shimadzu, UVmini-1240),
Spektrofotometer Fourier Transform - Infra Red (Shimadzu, Prestige-21), Sealer
Machine (Polystar, 401 HM), Viscosimeter (Tokyo Seiki Co.Ltd., Visconic
EMD), Shaker With Water Bath (Kottermann Labortechnik, D-3162 type 3047),
Autoklaf (Rayva) and Irradiator Co-60 (Iradiator , IRKA).

Process Issolation of gelatine .

Barramundi fish scales waste cleared from impurities on the surface,
washed with water faucets, and soaked in a solution of detergent during 24 hours.
Then the scales of a fish in a solution of detergent dishaker for 1 hour. The
treatment was repeated three times and last fish scales rinsed clean with distilled
water. Scales clean fish dried in an oven at a temperature 70 oC. Gelatin in fish
scales extracted using 0.5 M acetic acid solvent at a temperature of 121 o C
pressure 1 atm to 1.5 atm for 20 minutes. After the process extraction, the
extracted solution was cooled ago separated from fish scales.

Synthesis of crosslinked gelatin.

The extracted gelatin solution with a concentration 12.5% filtered with filter
paper No. 42 uses vacuum pump. After the solution is packaged as 50 mL in
plastic bag size (9 x 11) cm 2 use a sealer machine. Synthesis of gelatin binds
cross with gamma ray irradiation at a dose of 10 kGy,20 kGy, 30 kGy, 40 kGy,
and 50 kGy (dose rate of 7 kGy / h) under conditions of room temperature and
atmospheric air in Application Center Isotopes and Radiation Technology
(PATIR) BATAN.

Characterization of FT-IR spectrophotometer .

Characterized gelatin sample irradiated FT-IR spectrophotometer
(Shimadzu, Prestige-21) in PATIR-BATAN. Samples were scanned 20 times with
a resolution of 2 cm in the wave number range of 4000 cm up to 400 cm. samples
of gelatin characterized in the form of a solution directly with FT-IR
spectrophotometer, while samples gel is characterized in the form of
films.Background standard used is a solution of 0.5 M acetic acid to the sample
solution in the form and potassium bromide to the sample in the form of films.FT-
IR spectrum of 0 kGy dose used for confirm the test materials as gelatin and also
as the standard spectrum of gelatin without irradiation.

Measurement of UV-Vis Spectrophotometer .

Absorbance shaped gelatin irradiation solution was measured using a
spectrophotometer UV-Vis (Shimadzu, UVmini 1240) in PATIR-BATAN.
Measurements were taken at wavelengthinter vals 200 nm to 400 nm at 25 oC.
Correction baseline absorption spectrum using an acid solution 0.5 M acetate.

Determination of Viscosity
Gelarin solution viscosity is determined using viskosimeter (Tokyo Keiki Co.
Ltd., Visconic EMD) in PATIR-BATAN. tool viskosimeter working at 0.5 rpm
and a temperature of 30 oC. Tests carried out 10 replications.

 RESULTS AND DISCUSSION

Synthesis of crosslinked gelatin raw material scales barramundi (Lates
calcarifer) and gelatin extraction results are shown in figure 1 Extraction of raw
materials produced clear yellow solution with a distinctive aroma fish. The
solution is still in the sol phase at room temperature. FT-IR measurement results
of extraction solution (gelatin irradiation dose of 0 kGy) confirm that the solution
is gelatin, gelatin control shows FT-IR spectra A typical fish gelatin, where there
are 3 peak area, namely:
peak area 1 (3600 cm to 2700 cm) includes bond amide A and amide B peak area
2 (1900 cm to 900 cm) includes bonding amide I, amide II and amide III and 3
peak area (900 cm to 400 cm) covers IV amide bond, amide V and amide VI.
These results consistent with the FT-IR spectra of fish gelatin obtained by
previous researchers.

Synthesis of crosslinking in the gelatin can be done .

Through physical and chemical processes that are relatively the same. The
process of formation of crosslinking in gelatin solution occurs in two stages. In
solution, gelatin chain showed that molecular aggregation behavior are the
random coil structure. Gelatin chains on rich segment imino acid content then
renaturation process of forming segment experienced a triple helix-like structure
tropokolagen.

In the random coil segments are amorph.

Chains are cross-linked to each other form a braided structure of three-
dimensional (3D) with phase change of the gelatin solution (sol) into a
gel.Synthesis of crosslinked gelatin as a physics carried out by lowering the
temperature of gelain solution below the transition temperature sol-gel or raise
concentration of gelatin to pass the critical concentration aggregation. Physically
crosslinking occurs because presence of hydrogen bonding, Van der Waals bonds
and bondionic. Chemically crosslinking occurs due formation of covalent bonds
that can be done with the addition of cross-linker or induced by irradiation
gamma.
In this study, cross-linked gelatin has synthesized by gamma irradiation
induction. gamma irradiation at a dose of 40 kGy and 50 kGy causes gelatin
experienced a phase change from sol to gel at room temperature. The phase
change indicates formation of intermolecular crosslinking gelatin form a 3D
braided structure. The crosslinking thought to occur due to the formation of
chemical bonds covalent. This will be confirmed based on the results FT-IR
characterization of irradiated gelatin gel.Gamma irradiation at doses up to 30 kGy
not cause a phase change in the gelatin solution. Allegedly crosslinking has been
formed between molecules gelatin but not forming 3-dimensional fabric of the
thorough in solution. Crosslinking that occurred this is partially confirmed by the
change UV-Vis absorbance spectra and the viscosity of the solution gelatin
irradiation.

UV-Vis spectrum of Gelatin.

UV-Vis spectra measurements in this study used to prove the formation of
bonds cross in the gelatin solution irradiation at doses up to 30 kGy. The increase
in absorbance of the solution of collagen in UV-Vis spectra due to a treatment-
related with the increase in the degree of cross-linking of collagen molecules in
the solution. UV-Vis spectrum of the solution gelatin irradiation 0 kGy to 30 kGy.
At the irradiation dose up to 30 kGy, the pattern spectrum UV-Vis irradiation
gelatin did not change either on the wavelength and peak shape absorbance. This
indicates that irradiation gamma does not cause transformation of gelatin into
other compounds. This result is consistent with the results previous studies of
gelatin from fish bigeye spapper. Peak UV-Vis absorbance spectra of gelatin
occurs at a wavelength of 235 nm with a value absorbance increased with an
increase in radiation dose.
Absorbance gelatin solution 0 kGy irradiation dose, 10 kGy, 20 kGy and 30
kGy, respectively for 1.80 cm by 2.27 cm by 2.39 cm and 2.46 cm. Increased
gelatin solution absorbance correlated with radiation dose following linear
equation Y = 1,91+ 0,02X with r = 0.91. This proves that the bond cross in the
gelatin molecule also occurs at doses irradiation up to 30 kGy although gelatin
solution has not undergone a phase change. Crosslinking in a gelatine solution
irradiation was also confirmed based on the increase in viscosity of the solution.

Gelatin Viscosity Determination

Gelatin is a biopolymer compounds with relatively high molecular weight.
Increased viscosity polymer molecules in solution as a result of a treatment on the
environmental conditions remain associated with increase the molecular weight of
the polymer.Gelatin solution viscosity changes as a result of irradiation gamma
irradiation at doses up to 30 kG. viscosity of gelatin solution irradiation increases
with increasing radiation dose at doses up to 30 kGy. Increased viscosity,is
linearly correlated with the dose of radiation follows the equation Y = 41.10 +
1.089 X with r = 0.96. This confirms the increase in weight gelatin molecules due
to the formation of the crosslinking caused by induction of gamma radiation.
crosslinking in a gelatine molecules causes the formation of chains.

FT-IR spectrum of Gelatin.

The effects of radiation on the formation of cross-linking the molecular
structure of gelatin in solution is known based on changes in the FT-IR spectrum
of the sample.Effects was analyzed according to the change in bond I amide bond
amide I (1900 cm to 1650 cm) is the most important absorption peaks in the
analysis of protein structures using spectroscopic FT-IR as much as 80% of the
peak absorption derived from the C = 0 stretch vibration of peptide bonds of
proteins sensitive to small variations in the geometry of the molecule and the
pattern of hydrogen bonds. decrease in intensity absorption at this peak is caused
by the loss of secondary structures and random coil formation. FT-IR spectrum of
irradiated gelatin solution FT-IR spectrum of irradiated gelatin gel irradiation at a
dose of 40 kGy and 50 kGy changed significant compared to the control gelatin
(solution form). Seen that I amide bond peak intensity increased absorbance
accompanied by a shift of the wave number 1650 cm to 1700 cm. Increased
intensity amide I absorbance peak is indicative occurrence of crosslinking through
the carboxyl group (C = O) the peptide bond which led to branching and
elongation of molecules or gelatin. In addition, the first peak area irradiation dose
of 40 kGy gelatin and 50 kGy narrowing and shift wave numbers compared to the
control gelatin (0 kGy). Area I gelatin controls were in the range 3600 cm to 2700
cm while gelatin irradiation dose 40 kGy and 50 kGy were in the range 3250 cm
up to 3800 cm.
This indicates that crosslinking gamma irradiation-induced clusters also
involves hidoksil (-OH) on gelatin. Hydroxyl group is reactive groups on the
side chain gelatin molecules form crosslinks to form structures helical and
tropokolagen the gelatin chain.
 The transition from the sol to a solution of collagen gel crosslinking due to
chemically induced.At this early stage in which the degree of crosslinking was
low, the intramolecular crosslinking of collagen aggregate involving lysine
residues form a spiral chain. Furthermore, when the degree of crosslinking
increases, bond intramolecular cross occurs and intermoluker in a gelatine
molecules in solution to form system is a mixture of interwoven collagen that has
not inter-connected and the aggregate weight of collagen with diverse molecules.
At this stage, the collagen is still are sol phase. When the degree of crosslinking in
solution past the critical point the sol to gel transition, aggregate collagen cross-
linked to form a 3D braided structure are interconnected. Aggregate mobility
decreases dramatically resulting in the transition from sol to gel. In this study,
gelatin irradiation at a dose of 10 kGy up to 30 kGy is still in the solution phase
because of the degree of crosslinking in the gelatin solution is still low and has not
reached a critical point sol-gel transition.

Crosslinking formation under these conditions involving the hydroxy

groups in lysine and arginine residues.Crosslinking occurs between hiroksimetil-
lysine with hydroxymethyl-arginine to form a bridge dimetilen-ether between the
two groups of the amino acids. it is seen in the FT-IR spectra of the irradiated
gelatin which showed narrowing of the I peak area (area hydroxyl absorbance)
without accompanying changes in the amide I absorbance peak intensity
compared gelatin control.
 CONCLUSION

Gamma irradiation can be used to change gelatin solution into a gel form.
Increasing irradiation dose up to 50 kGy resulted in viscosity increased gelatin
solution and gelatin solution changed to gel.
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