Heat Sealing Properties Of Fish Gelatin Film

HEAT SEALING PROPERTIES OF FISH GELATIN FILM

INDAH KURNIASARI

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY
FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014

STATEMENT LETTER OF MANUSCRIPT AND SOURCES OF
INFORMATION*
Hereby i genuinely stated that the manuscript entitled Heat Sealing
Properties of Fish Gelatin Film is an authentic work of mine under supervision
of academic conselor and never being presented in any forms and universities. All
information taken and quoted from published or unpublished works of the writters
has been mentioned in texts and attached in the bibliography at the end of this
manuscript.
Hereby i bestow the copyright of my manuscript to Bogor Agricultural
University and Kasetsart University.

Bogor, Desember 2014

Indah Kurniasari
F24100049

ABSTRACT
INDAH KURNIASARI. Heat Sealing Properties of Fish Gelatin Film. Supervised
by DR. ELVIRA SYAMSIR and DR. KANOKRAT LIMPISOPHON.
All fish gelatins have been observed to exhibit good film forming
properties, yielding transparent, nearly colourless, water soluble, and highly
extensible films. The objectives of this research are to analyze heat sealing
property of gelatin film at different temperature and time. The film prepared from
fish gelatin and use glycerol as plasticizer. Protein of gelatin determined in the
beginning for preparation of gelatin film. In preliminary experiment used five
level of glycerol (15% glycerol, 20% glycerol, 25% glycerol, 30% glycerol, and
35% glycerol) to determine one level of glycerol which will use for futher study
of sealing property. The result showed that 25% of glycerol determined will used
for sealing study. Result determined based on properties of the film which is
color, mechanical properties, and moisture content. In the next experiment, the
film will be sealed in different combination temperature (100 0C, 110 0C, and 120

0
C) and time (1 s, 2 s, and 3 s). Then it will be analyzed the color, transparency,
opacity and seal strength. Result showed that there was no significant different in
color, transparency and opacity when it sealed. But the film have transparent
appearance and excellent barrier properties against UV light like before it sealed.
Temperature and time have effect on seal strength of the film (p0.05). Highest seal
strength was observed at temperature 110 0C and time 3 s for 25% glycerol
concentration of the film.
Keywords: fish gelatin film, heat seal, seal strength, temperature, time

HEAT SEALING PROPERTIES OF FISH GELATIN FILM

INDAH KURNIASARI

Manuscipt
submitted as a partial fulfillment of the requirement for degreee of
Sarjana Teknologi Pertanian
at the Departement of Food Science and Technology

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY

FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014

PREFACE
Praise to Allah for the the mercy, the graciousness, and the guidance
throughout the research and manuscript completion. The research entitled “Heat
Sealing Properties of Fish Gelatin Film” was carried out in Kasetsart University
from May to September 2014.
By completion of this research and manuscript, the author would like to
express great appreciation and sincere thanks to:
1. Beloved Mama, Papa, Dedek Ratna and Adek Yesa for their endless love,
care, and support.
2. Dr. Elvira Syamsir, STP, M. Si, as academic advisor, for her enormous help
academically throughtout the completion of my manuscript.
3. Dr. Kanokrat Limpisophon for her valuable and inspiring advices, support,
loves, and also her time providing me as advisee at Kasetsart University.
4. Dr. Ir. Sukarno, M. Sc and Dian Herawati, STP, M. Si, as examiners, for their
time, correction, input and helps.

5. Ditjen DIKTI for financial support during research and all comitte of AIMS
Exchange Program both in IPB (Bapak Eko, Ibu Dias, Ibu Antung, Bapak
Pungki, Mbak Tika) and KU (Lecture of Food Technology, Dr. Sasitorn
Tongchitpakdee and other staff of international Division Affairs) for chance
doing research in KU and chance knowing another part of the world.
6. Bapak Lutfi Rauf as ambassador of Indonesia for Thailand and Bapak
Yunardi Yusuf as attache Educational and Culture for their kindness and help.
7. All member of Lipid and Protein Laboratory (Phii Pat, Phii Joice, Phii Booky,
Phii Kai, Phii Tan, Phii Dear, Phii Tong, Phii Nan, Phii Milk, Titu, Phii
Oeuang, Phii Dao, Phii Koi, and Phii Ob) for their guidance, kindness,
happiness, crazyness, and wonderful memories during stay in Thailand.
8. All of staff laboratory (Phii Pong, Phii Ant, Phii Tae, Phii Jum, Phii Neng)
also all of staff in Department of Food Science and Technology (Mbak Ina,
Mbak Anie, Mbak May, Ibu Novi, etc) for their time to help.
9. Al-Banatters and friends for love, care, support and beautiful friendship
during survive studying in foodtech.
10. AIMS Student Thailand 2014 (Dyah, Lingga, Gunawan, Elvan) and Indonesia
students (Mbak Ida, Mbak Dwita, Mas Iwan, Mbak Yuntia, Mbak Hesti, Mas
Wildan, Mbak Asya, Mbak Sari, Kak Alfa, Bu Erni, Mbak Indy, Mas Aidil)
for wonderful memories and adventure during in Thailand.

11. P2 team for the greatest colaboration during laboratory class and all of
foodtech ’47 student (‘DOA IBU’) for being together in good and bad time
Last but not least, hopefully this manuscript is useful for the readers and
gives a real contribution in food science development.
Bogor, December 2014

Indah Kurniasari

TABLE OF CONTENT
LIST OF TABLE

x

LIST OF FIGURE

x

LIST OF APPENDIX

x

INTRODUCTION

1

Background

1

Objective

3

RESEARCH METHODOLOGY

3

Materials

3

Instruments

3

Method

3

Method of Analysis

4

RESULT AND DISCUSSION

6

Protein content of fish gelatin

6

Effect of glycerol concentration on properties of the film

6

Effect of sealing time and temperature on properties of film

10

CONCLUSION AND RECOMMENDATION

14

Conclusion

14

Recommendation

14

REFERENCES

15

APPENDIX

17

AUTHOR BIOGRAPHY

26

LIST OF TABLE
1. Effect of glycerol concentration on color of 5% protein fish gelatin film
2. Effect of glycerol concentration on thickness, tensile strength and
elongation at break
3. Effect of glycerol concentration on moisture content of 5% protein fish
gelatin film
4. Effect of sealing temperature and time on color of 25% glycerol

concentration of the film
5. Effect of sealing temperature and time on seal strength of 25% glycerol
concentration of the film

6
9
10
11
14

LIST OF FIGURE
1. Thickness of 5% protein fish gelatin film in various level of glycerol
2. Tensile strength of 5% protein fish gelatin film in various level of
glycerol
3. Elongation at break of 5% protein fish gelatin film in various level of
glycerol
4. Moisture content of 5% protein fish gelatin film in various level of
glycerol
5. Fish gelatin film in various level of glycerol
6. Effect of sealing temperature and time before and after sealed on

transparency of 25% glycerol concentration of the film
7. Effect of sealing temperature and time before and after sealed on opacity
of 25% glycerol concentration of the film

8
8
8
10
10
12
13

LIST OF APPENDIX
1 . Protein standard curve for fish gelatin
2 . Protein concentration of fish gelatin
3 . Nutritional information of fish gelatin
4 . Statistical analysis of color
5 . Statistical analysis of thickness
6 . Statistical analysis of tensile strength
7 . Statistical analysis of elongation at break
8 . Statistical analysis of moisture content
9 . Statistical analysis of color on sealed film
10. Light transmission before sealed of 25% glycerol concentration of the
film
11. Light transmission after sealed of 25% glycerol concentration of the film
12. Effect of sealing temperature and time before sealed on transparency and
opacity of 25% glycerol concentration of the film
13. Effect of sealing temperature and time before sealed on transparency and
opacity of 25% glycerol concentration of the film
14. Statistical analysis of seal strength

17
17
17
17
18
18
19
20
20
22
22
23
23
24

INTRODUCTION
Background
Gelatin is a protein with a broad range of functional properties and
applications, including film-forming ability, and is obtained by hydrolyzing
collagen of animal and fish skin. The properties and film-forming ability of
gelatins are directly related to the molecular weight, i.e., the higher the average
molecular weight, the better the quality of the gelatin. The molecular weight
distribution depends mainly on the degree of collagen cross-linking and the
extraction procedure. However, the physical properties of gelatins are related not
only to the molecular weight distribution but also to the amino acid composition
(Gómez-Estaca et al., 2009). It has comprising multifunctionalities like gelling,
thickening, water-binding, emulsifying stabilizing, foaming, film forming and
fining characteristics. It is also suitable for production of biodegradable packaging
materials, with the appropriate film forming properties and good barriers against
oxygen and aromas at low and intermediate relative humidity (Hosseini et al.,
2013). It forms thermo reversible gels through the formation of hydrogen bond
stabilized triple helices when its solution is cooled. Again on heating it melts
above 40°C. Hydrogen bond stabilization is followed by rearrangement of
individual molecular chains into ordered, helical arrangement, or collagen fold
and association of two or three ordered segments to create crystallites (Zaman et
al., 2012).
Fish skin, which is a major by product of the fish-processing industry,
causing waste and pollution, could provide a valuable source of gelatin. Gelatin
films from the skins of a warm-water fish species, such as the Nile perch, have
been reported to exhibit stress and elongation at break similar to that of bovine
bone gelatin. Fish gelatin film, however, exhibits lower water vapor permeability
than bovine gelatin. The lower WVP values (compared to those from bovine or
porcine) reported for films based on fish gelatins from several species, can be
explained interms of the amino acid composition. Fish gelatins are known to have
much higher hydrophobicity due to lower proline and hydroxyproline contents, as
the hydroxyl group of hydroxyprolineis normally available to form hydrogen
bonds with water (Karim and Bhat, 2009). All fish gelatins have been observed to
exhibit good film forming properties, yielding transparent, nearly colourless,
water soluble, and highly extensible films. The physical properties of fish-gelatin
films are highly dependent on gelatin attributes, which are in turn dependent not
only on intrinsic properties related to the fish species used but also on the process
employed to manufacture the gelatin. The mechanical and barrier properties of
these films depend largely on the physical and chemical characteristics of the
gelatin, especially the amino acid composition, which is highly species specific,
and the molecular weight distribution, which depends mainly on processing
conditions (Gómez-Guillén et al., 2009).
Hydrocolloid films (such as protein, polysaccharide, and alginate) have
good barrier properties to oxygen, carbon dioxide, and lipids but not to water
vapor. Most hydrocolloid film also possesses superb mechanical properties, which
are quite useful for fragile food products (Bourtoom et al., 2006). Protein-based

2
edible films offer alternative packaging without adversely affecting the
environmental costs. However, edible films are not meant to totally replace
synthetic packaging or to limit moisture, aroma and lipid migration between food
components where traditional packaging cannot function. For instance, proteinbased edible films can be used for versatile food products to reduce loss of
moisture, to restrict absorption of oxygen, to lessen migration of lipids, to
improve mechanical handling properties, to provide physical protection, or to
offer an alternative to commercial packaging materials. It has impressive gas
barrier properties compared with those prepared from lipids and polysaccharides.
When they are not moist, the O2 permeability of soy protein-based film was 500,
260, 540 and 670 times lower than that of low-density polyethylene,
methylcellulose, starch and pectin respectively. In addition, the mechanical
properties of protein-based edible films are also better than those of
polysaccharide and fat-based films. This is because proteins have a unique
structure which confers a wider range of functional properties, especially a high
intermolecular binding potential (Wittaya, 2012).
Protein films would be a suitable food packaging material for preventing the
growth of aerobic microbial and lipid oxidation in lipid enriched foods because it
shows impressive gas barriers. It may be able to partially replace some of the
conventional synthetic packaging materials used to preserve and protect foods.
These protein-based edible films should not be used alone, since contamination
during food handling could occur, but would be used to wrap foods inside a
secondary synthetic package during food distribution and storage. The wraps
could also be used in the home to cover leftovers in the refrigerator, peeled fruit
mixtures or as a sandwich bag for lunch. Because such wraps are biodegradable
and may even be eaten, they are not harmful to the environment. This attribute
could also reduce waste disposal costs. Applications of several protein-based
edible films, such as corn zein on nut and fruit products, casein emulsion film on
fruit, and whey protein films on fruit products (Wittaya, 2012).
Heat sealing is widely used to join polymer films. During the heat sealing
process, 2 films are pressed together between heated plates or dies. The surface of
the crystalline polymer melts, due to heat. The application of pressure results in
the interfacial interactions across the joint surfaces, which require time. This is a
necessary step to give sufficient seal strength to the sealed film. Upon cooling, a
heat-sealed joint is produced due to recrystallization of the polymer. The joint
formation on the polymer surface is dependent on the surface chemistry of the
material. Temperature, pressure, and dwell time are considered important process
variables which affect seal strength. Measurement of seal strength is typically
used as an indicator of seal quality (Kim and Ustunol, 2001).

3
Objectives
The objectives of this research are
1. To analyze effect of glycerol concentration on properties of the film
2. To analyze effect of sealing time and temperature on properties of film

RESEARCH METHODOLOGY
Materials
Gelatin is obtained from commercial fish gelatin (Rousselot® 275 FG8).
The chemical required for the sample preparation and analysis including glycerol,
bovine serum albumin (BSA), biuret reagent, and distillate water.

Instruments
The equipments for this research are analytical balance, magnetic water bath
stirrer, magnetic stirrer, ultrasonic cleaner, silicone resin plate, ventilated oven,
spectrophotometer, vortex, Mitutoyo dial thickness gage micrometer, TA.XT plus
texture analyzer, ultra scan XE hunter lab, “MTMS” Kit M08-08, oven, silica gel,
and desiccator.

Method
Effect of glycerol concentration on properties of the film
Gelatin film preparation was modified from methods of Limpisophon et al.,
(2009). Gelatin powder was dissolved at 60 0C for 30 min to obtain the filmforming solution (FFS) with 5% (w/v) protein concentration. Glycerol as a
plasticizer was added into FFS at the concentration of 15%, 20%, 25%, 30% and
35% (w/w) of protein with continuous stirring at room temperature for 20 min.
The air bubbles in the FFS were removed by a ultrasonic cleaner. De-aerated FFS
was cast onto a rimmed silicone resin plate and dried in a ventilated oven at 25 ±
0.5 0C and 50 ± 5% relative humidity (RH) for 24 h. Films were then conditioned
at 25 ± 0.5 0C and 50 ± 5% RH for 48 h before analysis. Physical properties of the
film were then analyzed, which are color, film thickness, tensile strength,
elongation at break and moisture content. This analyze result use to determine one
level of glycerol for further study of sealing properties.
Effect of sealing time and temperature on properties of film
Gelatin film preparation was modified from methods of Limpisophon et
al., (2009). Gelatin powder was dissolved at 60 0C for 30 min to obtain the filmforming solution (FFS) with 5% (w/v) protein concentration. Glycerol as a

4
plasticizer was added into FFS at the concentration of 25% (w/w) of protein with
continuous stirring at room temperature for 20 min. The air bubbles in the FFS
were removed by a ultrasonic cleaner. De-aerated FFS was cast onto a rimmed
silicone resin plate and dried in a ventilated oven at 25 ± 0.5 0C and 50 ± 5%
relative humidity (RH) for 24 h. Film samples were cut into strips and sealed.
Films were then conditioned at 25 ± 0.5 0C and 50 ± 5% RH for 48 h before
analysis. Properties of the film were then analyzed, which are color, transparency,
opacity, and seal strength.

Methods of Analysis
Protein analysis of fish gelatin
Amount of protein was determined by using biuret assay according to
AOAC (1995). Dilution series of calibration standard was prepared in the buffer
or solvent used to prepare the sample. In separate test tubes, add 1 ml of protein
containing sample and of each dilution of the calibration standard to 4 ml biuret
reagent and vortex. The solutions were incubated at room temperature for 20
minutes. Absorbance of the sample, calibration standards, and reference standard
were measured at 550 nm. Calibration plot was prepared by graphing absorbance
at 550 nm values for standards versus protein concentration (µg protein/ml).
Protein concentration of the sample was determined by interpolation from the plot.
Color
Color of film was determined according Nuthong et al., (2009) using a Ultra
Scan XE Hunter Lab and expressed as L* (lightness), a* (redness/greenness) and
b* (yellowness/blueness) values.
Film thickness
Film thickness was measured based on Limpisophon et al., (2009) with
Mitutoyo dial thickness gage micrometer. Six measurements were taken at
random positions.
Tensile strength and elongation at break (Limpisophon et al., 2009)
Tensile strength (TS) and percentage elongation at break (EAB) were
determined by using a TA.XT plus texture analyzer according to Limpisophon et
al., (2009). Rectangular strips (20 x 50 mm) were prepared from each film to
determine their mechanical properties. Average thickness of each film strip
wasused to estimate the cross-sectional area of the sample. Initial grip separation
and mechanical cross head speed were set at 30 mm and 1 mm/s, respectively. TS
(MPa) was calculated by the following equation:

5
TS(MPa) = Fmax/A
where F max = maximum load (N) needed to pull the sample apart;A = crosssectional area (m2) of the samples; EAB (%) was calculated by following
equation:
EAB (%) = (E/30) x 100
where E = film elongation (mm) at the moment of rupture; 30 = initial grip length
(mm) of samples.
Moisture Content
Moisture content determine according Rubilar et al., (2013). To determine
the moisture content of films, about 50 mg of film were dried at 105 0C during 24
h (until the equilibrium weight was attained). The weight loss of the sample was
determined, and moisture content was calculated using the following equation:

where Mi and Mf are the masses of initial and dried samples, respectively. Three
replicates were obtained for each sample.
Transparency and opacity
The barrier properties of gelatin films against ultraviolet (UV) and visible
light were measured according Limpisophon et al., (2009) at selected wavelength
between 200 and 800 nm using a UV–Visible Recording Spectrophotometer. The
transparency of the films was calculated according to Han and Floros (1997) by
the following equation:
Transparency (A/mm) = - log T/x
where A = absorbance at each wavelength; T = transmittance (%) at each
wavelength; x = film thickness (mm). According to the equation, high
transparency indicates opaque.
The opacity of the films was calculated the following equation:
Opacity (%) = 100% - T
where T = transmittance (%) at each wavelength.
Seal strength determination
Film samples were cut into strips. Two film strips were placed on top of one
another, and an area of 1.5 x 0.75 cm (at the edge of the film) was heat-sealed at
100, 110, or 120 0C for 1, 2 or 3 s of dwell time at 0.3 MPa pressure, using a
“MTMS” Kit M08-08. All sealed film samples were conditioned for 48 h under the
test conditions prior to determining seal strength. Seal strength of the heat-sealed
films was determined according to Standard ASTM F-88 (2009), using TA.XT

6
plus texture analyzer, at 25 ± 0.5 0C and 50 ± 5% RH. Each leg of the sealed film
was clamped to the machine, with each end of the sealed film held perpendicularly
to the direction of the pull. Seal strength was calculated as follows:
Seal strength (N/m) = peak force/film width
The maximum force required to cause seal failure was reported as seal strength in
Newtons/meter (N/m).
Statistical analysis
Statistical analysis used completely randomized experimental design for
effect of glycerol concentration on properties of the film and factorial design for
effect of sealing time and temperature on properties of film. Analyses of variance
(ANOVA) were used to analyze data and mean comparisons were run by
Duncan’s multiple range tests using SPSS programs (version 20.0) at p 0.05).

7
Table 1. Effect of glycerol concentration on color of 5% protein fish gelatin film
Sample 5% protein
L*
a*
b*
15% glycerol
96.47 ± 0.05a -0.09 ± 0.02a 0.74 ± 0.04a
20% glycerol

96.46 ± 0.10a

-0.09 ± 0.03a

0.73 ± 0.03a

25% glycerol

96.48 ± 0.13a

-0.09 ± 0.03a

0.76 ± 0.06a

30% glycerol

96.52 ± 0.11a

-0.09 ± 0.04a

0.76 ± 0.05a

35% glycerol
96.60 ± 0.14a -0.09 ± 0.03a 0.74 ± 0.07a
1
Data are expressed as mean ± standard deviation
2
Same superscripts indicate data is not significantly different (p > 0.05)
Film thickness, tensile strength and elongation at break
Film thickness is used to calculate the tensile strength of the film. Figure 1
and Table 2 showed the thickness of 5% protein fish gelatin film in various level of
glycerol. Addition of glycerol concentration influence to film thickness. The higher
level of glycerol added so the film thickness would be higher. This result is similar
to Zhang and Han (2006) that the thickness of the film increased as the amount of
plasticizers increased. This phenomena happen because addition of glycerol
increase total amount of solids in the film so the thickness also increase. Thickness
of the film consecutively are 67.69 µm (15% glycerol), 69.58 µm (20% glycerol),
72.13 µm (25% glycerol), 75.56 µm (30% glycerol), and 81.34 µm (35% glycerol).
Based on statistical analysis, thickness of each glycerol concentration is not
different significantly (P > 0.05).
Effect of glycerol concentration on tensile strength is presented in Figure 2
and Table 2. TS of the film consecutively are 79.62 MPa (15% glycerol), 66.71
MPa (20% glycerol), 49.57 MPa (25% glycerol), 37.34 MPa (30% glycerol), and
27.57 MPa (35% glycerol). TS of the film decreased when glycerol concentration
increased. This due to the use of glycerol reduce brittleness by lowering the intermolecular interactions between adjacent chains of the biopolymers (Nuthong et al.,
2009), so TS of the film decrease when glycerol concentration increase. According
Jongjareonrak et al., (2006) glycerol has the relatively small molecule with
hydrophilic characteristic which could be easily inserted between protein chains and
establish hydrogen bonds with amide group and amino acid side chains of proteins.
When glycerol was incorporated in the gelatin film network, direct interactions and
the proximity between protein chains were reduced. Based on the statistical analysis,
TS of each glycerol concentration is different significantly (P < 0.05).
Effect of glycerol concentration on elongation at break is presented in Figure
3 and Table 2. EAB of the film consecutively are 9.07% (15% glycerol), 11.38%
(20% glycerol), 39.66% (25% glycerol), 49.28% (30% glycerol), and 56.11% (35%
glycerol). The higher glycerol concentration, so EAB of the film would be higher.
This is because of plasticizing effect of glycerol is high, so it can improve the
flexibility of the film (Vanin et al., 2005). Increasing plasticizer concentration in a
film-forming solution produces a film that is more stretchable by reducing the
interactions between the biopolymer chains (Gómez-Guillén et al., 2009). The
presence of plasticizer causes a reduction of intermolecular interaction and also
increases the mobility of macromolecules, leading to the increase in EAB of films

8
(Jongjareonrak et al., 2006). Based on the statistical analysis, EAB of each glycerol
concentration is different significantly (P < 0.05).
Based on tensile strength and EAB result, film with 15% and 20% level of
glycerol are more brittle than another level. However, film with 30% and 35%
level of glycerol are more sticky than another level. But, 25% level of glycerol
has characterictic between them, it isn’t brittle and sticky. Because of that, 25%
level of glycerol use for sealing experiment. Compare with syntetic film, fish
gelatin films are stronger and less flexibility than LDPE. Sample with 15%
glycerol has tensile strength close to PE, TS sample with 20% glycerol close to
PVDC and TS sample with 25% glycerol close to OPP but less flexibility than
those synthetic films. Eventhough, PE and PVDC have less flexibility than
sample with 25% glycerol, 30% glycerol and 35% glycerol.

Figure 1. Thickness of 5% protein fish gelatin film in various level of glycerol

Figure 2. Tensile strength of 5% protein fish gelatin film in various level of
glycerol

Figure 3. Elongation at break of 5% protein fish gelatin film in various level of
glycerol

9
Table 2. Effect of glycerol concentration on thickness, tensile strength and
elongation at break
Sample 5%
Thickness (µm) Tensile strength (MPa)
EAB (%)
protein
15% glycerol
67.69 ± 3.14a
79.62 ± 1.74d
9.07 ± 4.07a
20% glycerol

69.58 ± 4.91a

66.71 ± 7.87c

11.38 ± 7.65a

25% glycerol

72.13 ± 3.14a

49.57 ± 0.28b

39.66 ± 13.94b

30% glycerol

75.56 ± 4.98a

37.34 ± 1.63a

49.28 ± 11.89b

35% glycerol

81.34 ± 2.68a

27.57 ± 4.98a

56.11 ± 13.38b

Syntetic film3
LDPE

16.5 ± 0.9

> 1000

OPP

50.7 ± 8.2

73 ± 27

PE

81.6 ± 3.2

19 ± 6

PVDC
65.6 ± 10.8
18 ± 5
1
Data are expressed as mean ± standard deviation
2
Different superscripts indicate data significantly different (p < 0.05)
3
LDPE (low-density polyethylene), OPP (oriented polypropylene), PE (polyester),
PVDC (polyvinylidene chloride) (Shiku et al., 2003)

Moisture content
Effect of glycerol concentration on moisture content of 5% protein fish
gelatin film showed in Figure 4 and Table 3. Moisture content has the role in
resistant of the film to humidity. Moisture content of the film consecutively are
13.26% (15% glycerol), 14.16% (20% glycerol), 15.12% (25% glycerol), 16.19%
(30% glycerol), and 17.10% (35% glycerol). Increase of glycerol concentration
cause the moisture content of the film increase. Increase moisture content due to
hygroscopic plasticizer property, therefore the higher level glycerol added into
film forming solution so the film would be more hygroscopic. If the films were
more hygroscopic so it would be easy to absorb water, thereby moisture content of
the film would increase (Vanin et al., 2005). Glycerol also retains water in the
film matrix due to its hydrophilic nature. Higher concentrations of plasticizer
favor the adsorption of water molecules, which is mainly attributed to the
predisposition of plasticizers to form hydrogen bonds (O-H) (Cerqueira et al.,
2012). Based on the statistical analysis, moisture content of each glycerol
concentration is different significantly (P < 0.05).

10

Figure 4. Moisture content of 5% protein fish gelatin film in various level of
glycerol
Table 3. Effect of glycerol concentration on moisture content of 5% protein fish
gelatin film
Sample 5% protein Moisture content (%)
15% glycerol
13.26 ± 0.51a
20% glycerol

14.16 ± 0.18b

25% glycerol

15.12 ± 0.37c

30% glycerol

16.19 ± 0.68d

35% glycerol
17.10 ± 0.44e
1
Data are expressed as mean ± standard deviation
2
Different superscripts indicate data significantly different (p < 0.05)

15% glycerol

20% glycerol

20% glycerol

25% glycerol

30% glycerol

35% glycerol

Figure 5. Fish gelatin film in various level of glycerol

Effect of sealing time and temperature on properties of film
Color
Effect of sealing time and temperature on color of 25% glycerol
concentration of the film was presented in Table 4. The appearance of sealed film
is clear, green and yellow. This result almost the same with the film before sealed,
but the lightness of sealed film decreased. Color of sealed film is not significantly
different on each sealing time and temperature (p > 0.05). It means that sealing
time and temperature don’t have effect to color.

11
Table 4. Effect of sealing temperature and time on color of 25% glycerol
concentration of the film
Temperature (°C) Time (s)
L*
a*
b*
a
92.00 ± 0.68
1s
-0.24 ± 0.09a
1.70 ± 0.05a
100 °C
2s
92.08 ± 0.55a
-0.24 ± 0.06a
1.71 ± 0.10a
3s
92.04 ± 0.67a
-0.22 ± 0.12a
1.70 ± 0.14a
1s
92.73 ± 0.48a
-0.26 ± 0.11a
1.77 ± 0.08a
110 °C
2s
92.53 ± 0.65a
-0.25 ± 0.15a
1.76 ± 0.08a
3s
92.72 ± 0.56a
-0.24 ± 0.11a
1.76 ± 0.10a
1s
92.43 ± 0.37a
-0.23 ± 0.04a
1.71 ± 0.05a
120 °C
2s
91.92 ± 0.42a
-0.30 ± 0.06a
1.67 ± 0.15a
3s
92.40 ± 0.68a
-0.32 ± 0.03a
1.74 ± 0.12a
1
Data are expressed as mean ± standard deviation
2
Same superscripts indicate data not significantly different (p > 0.05)
Transparency and opacity
Effect of sealing time and temperature before and after sealed on
transparency of 25% glycerol concentration of the film were presented in Figure 6.
Transparency used to know the barrier properties of sealed film against ultraviolet
and visible light. Lower transparency value showed that film more transparent and
clear. From the figure, transparency value on visible light range is very low that
indicate the film transparent. Transparency (A600/mm) of some synthetic films,
LDPE (low-density polyethylene) 3.05, OPP (oriented polypropylene) 1.67, PE
(polyester) 1.51, and PVDC (polyvinylidene chloride) 4.58 (Shiku et al., 2003).
Transparency of the film before or after seal are more transparent than pea protein
isolate edible film (16.71 ± 0.93) (Choi and Han, 2002) and close to some syntetic
film (OPP and PE). These result can indicate that the film are transparent and
clear enough for use as packaging.
Based on data, transparency is high and little increased after sealed in uv
range than before sealed, but it decreased in visible range. The higher transparency
value showed that film has better barrier properties. It means that the film have
excellent barrier properties against UV light, which induces lipid oxidation in the
food system. This result same to Mu et al., (2012) that showed protein-based film
have excellent barrier properties. The transparency was high at 280 nm from 3.82 –
19.68, contrast with in visible light that have transparency value less than 1.
Protein films are considered to have very good UV barrier properties, owing to
their high content of aromatic amino acids that absorb UV light (Hamaguchi et al.,
2007). Transparency of film before and after sealed was not significantly different
on each sealing time and temperature (p > 0.05). It means that sealing time and
temperature don’t have effect to transparency.

12
Before sealed

After sealed

Figure 6. Effect of sealing temperature and time before and after sealed on
transparency of 25% glycerol concentration of the film
Effect of sealing time and temperature before and after sealed on opacity
of 25% glycerol concentration of the film were presented in Figure 7. Opacity was
used also to know the barrier properties of sealed film against ultraviolet and
visible light. Based on data, opacity was high and little increased after sealed in uv
range, but it little decreased in visible range. The same result were also reported
by Elango et al., (2014) with respect to red snapper gelatin film and grouper
gelatin film. The opacity was high in the UV range (90-100%) than visible light
region for the fish gelatin films. High opacity in the UV range of 200-280 nm
indicated that the fish gelatin films can effectively prevent UV light - induced
lipid oxidation when applied in food systems. It means that uv barrier properties
of sealed film better than before it was sealed. Opacity of film before and after
sealed was not significantly different on each sealing time and temperature (p >
0.05). It means that sealing time and temperature don’t have effect to opacity.

13
Before sealed

After sealed

Figure 7. Effect of sealing temperature and time before and after sealed on opacity
of 25% glycerol concentration of the film
Seal strength
Effect of sealing temperature and time on seal strength of 25% glycerol
concentration of the film was presented in Table 5. Seal strength usually used as
indicator of seal quality (Kim and Ustunol, 2001). Sealing temperature and time
influence significantly (P < 0.05) on seal strength. The higher temperature cause
the seal strength increase (Yuan et al., 2006). Increase of time also cause seal
strength increase. But combination time and temperature didn’t affect
significantly on seal strength. The result is higher than whey protein film
(Hernandez & Krochta, 2009), Whey Protein Isolate/Lipid Emulsion Edible Films
(Kim and Ustunol, 2001), syntetic polymer LDPE, but lower than LLDPE. The
maximum achievable heat-seal strength for laminated film using LDPE (low
density polyethylene) and LLDPE (linear low density polyethylene) as sealant
material is 598 N/m and 4020 N/m (Yuan et al., 2006). Seal strengths of heat-

14
sealed synthetic polymers was >730 N/m (Kim and Ustunol, 2001). Highest seal
strength was observed at temperature 110 0C and time 3 s for 25% glycerol
concentration of the film.
Table 5. Effect of sealing temperature and time on seal strength of 25% glycerol
concentration of the film
Temperature (°C)

Time (s)
1

Seal strength (N/m)
442.20 ± 247.16

100

2

970.71 ± 62.81

3

1226.33 ± 230.33

1

1484.29 ± 331.85

2

1682.91 ± 243.02

3

1706.94 ± 246.26

1

1251.01 ± 168.37

2

1446.37 ± 78.97

3

1703.84 ± 111.52

110

120

CONCLUSION AND RECOMMENDATION
Conclusion
Increase levels of glycerol causes increase in thickness, elongation at break
and moisture content but decrease in tensile strength. Different sealing
temperature and time didn’t affect significantly on color, transparency, and
opacity. Compare with film before sealed, color of the film was decreased after it
sealed. But it was still clear and very transparent. The film has good barrier
properties against ultraviolet (UV) light even after it sealed. Different sealing
temperature and time have effect on seal strength. Seal strength will increase with
increase of temperature. Increase of time also make seal strength increase. But
combination temperature and time didn’t have significant effect on seal strength.
Recommendation
Recommendation for this study, use small range of temperature to get the
optimum temperature for sealing and use another plasticizer as comparison.

15

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17
APENDICES

Absorbance

Apendix 1. Protein standard curve for fish gelatin
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000

y = 0.0317x + 0.0619
R² = 0.9999

0

2

4
6
8
Concentration (mg/ml)

10

12

Apendix 2. Protein concentration of fish gelatin
Absorbance
Replication
1
2

Protein concentration

Duplication 1 Duplication 2 Duplication 1 Duplication 2
0.340
0.350

0.340
8.7729
0.340
9.0883
Average (mg/10 mg gelatin)
Protein concentration in 100 g gelatin (%)

Apendix 3. Nutritional information of fish gelatin
Nutritional information (for 100 g)
Moisture
11 g
Proteins
88 g
Total fat
0g
Total carbohydrates
0g
Dietary fiber
0g
Sodium
80 mg
Potassium
1 mg
Calcium
10 mg
Magnesium
1 mg
Cholesterol
0 mg
Vitamins
0 mg
352 kcal
Energy value
1473.5 kJ

8.7729
8.7729

Protein
concentration
(mg/10 mg
gelatin)
8.7729
8.9306
8.8517
88.52 ± 0.11

18
Apendix 4. Statistical analysis of color
ANOVA
Sum of Squares

L

a

b

df

Mean Square

Between Groups

,026

4

,007

Within Groups

,063

5

,013

Total

,089

9

Between Groups

,000

4

,000

Within Groups

,004

5

,001

Total

,004

9

Between Groups

,002

4

,000

Within Groups

,011

5

,002

Total

,013

9

F

Sig.
,525

,724

,030

,998

,173

,943

Apendix 5. Statistical analysis of thickness
ANOVA
thickness
Sum of Squares
Between Groups
Within Groups
Total

df

Mean Square

232,833

4

58,208

75,840

5

15,168

308,673

9

F
3,838

Sig.
,086

Apendix 6. Statistical analysis of tensile strength
ANOVA
tensile strength
Sum of Squares
Between Groups
Within Groups
Total

df

Mean Square

3593,868

4

898,467

92,466

5

18,493

3686,334

9

F
48,584

Sig.
,000

19

Post Hoc Tests
tensile strength
Duncan
sample 5% protein gelatin

N

Subset for alpha = 0.05
1

2

35% glycerol

2

27,5700

30% glycerol

2

37,3400

25% glycerol

2

20% glycerol

2

15% glycerol

2

3

4

49,5650
66,7150
79,6200

Sig.

,072

1,000

1,000

1,000

Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 2.000.

Apendix 7. Statistical analysis of elongation at break
ANOVA
elongation at break
Sum of Squares
Between Groups
Within Groups
Total

df

Mean Square

3766,779

4

941,695

589,638

5

117,928

4356,417

9

Post Hoc Tests
elongation at break
Duncan
sample 5% protein gelatin

N

Subset for alpha = 0.05
1

2

15% glycerol

2

9,0700

20% glycerol

2

11,3800

25% glycerol

2

39,6650

30% glycerol

2

49,2750

35% glycerol

2

56,1100

Sig.

,840

Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 2.000.

,200

F
7,985

Sig.
,021

20
Apendix 8. Statistical analysis of moisture content
ANOVA
moisture content (%)
Sum of Squares
Between Groups

Mean Square

28.308

4

7.077

2.184

10

.218

30.492

14

Within Groups
Total

df

F

Sig.

32.403

.000

Post Hoc Tests
moisture content (%)
Duncan
sampel 5% gelatin

N

Subset for alpha = 0.05
1

15% glycerol

3

20% glycerol

3

25% glycerol

3

30% glycerol

3

35% glycerol

3

2

3

4

5

13.2567

Sig.

14.1633
15.1200
16.1933
17.0967
1.000

1.000

1.000

1.000

1.000

Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 3.000.

Apendix 9. Statistical analysis of color on sealed film
Tests of Between-Subjects Effects
Dependent Variable: L
Source

Type III Sum

df

Mean Square

F

Sig.

of Squares
2,403a

8

,300

,909

,531

230105,754

1

230105,754

696180,650

,000

temp

1,813

2

,907

2,743

,091

time

,266

2

,133

,402

,675

temp * time

,324

4

,081

,245

,909

Error

5,949

18

,331

Total

230114,107

27

8,353

26

Corrected Model
Intercept

Corrected Total

a. R Squared = ,288 (Adjusted R Squared = -,029)

21
Tests of Between-Subjects Effects
Dependent Variable: a
Source

Type III Sum of

df

Mean Square

F

Sig.

Squares
Corrected Model

,025a

8

,003

,379

,918

Intercept

1,763

1

1,763

213,785

,000

temp

,010

2

,005

,591

,564

time

,003

2

,002

,187

,831

temp * time

,012

4

,003

,368

,828

Error

,148

18

,008

Total

1,937

27

,173

26

Corrected Total

a. R Squared = ,144 (Adjusted R Squared = -,236)

Tests of Between-Subjects Effects
Dependent Variable: b
Source

Type III Sum of

df

Mean Square

F

Sig.

Squares
,029a

8

,004

,355

,931

80,152

1

80,152

7762,232

,000

temp

,021

2

,010

1,012

,383

time

,002

2

,001

,119

,889

temp * time

,006

4

,001

,144

,963

Error

,186

18

,010

Total

80,367

27

,215

26

Corrected Model
Intercept

Corrected Total

a. R Squared = ,136 (Adjusted R Squared = -,248)

22
Apendix 10. Light transmission before sealed of 25% glycerol concentration of the film
Temperature
Time (s) X (mm)
(⁰C)
1
0.066
100
2
0.072
3
0.065
1
0.079
110
2
0.086
3
0.070
1
0.068
120
2
0.085
3
0.086

200
A
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

T
100.000
100.000
100.000
100.000
100.000
100.000
100.000
100.000
100.000

280
A
1.022
1.052
0.967
1.312
1.266
1.013
1.266
1.291
1.295

350

T
9.506
8.872
10.789
4.875
5.420
9.705
5.420
5.117
5.070

A
0.140
0.139
0.135
0.157
0.152
0.137
0.152
0.156
0.155

T
72.444
72.611
73.282
69.663
70.469
72.946
70.469
69.823
69.984

400
A
0.105
0.103
0.102
0.111
0.108
0.102
0.108
0.112
0.111

T
78.524
78.886
79.068
77.446
77.983
79.068
77.983
77.268
77.446

500
A
0.085
0.083
0.083
0.087
0.085
0.082
0.085
0.088
0.087

T
82.224
82.604
82.604
81.846
82.224
82.794
82.224
81.658
81.846

600
A
0.080
0.078
0.078
0.082
0.080
0.078
0.080
0.082
0.081

T
83.176
83.560
83.560
82.794
83.176
83.560
83.176
82.794
82.985

800
A
0.076
0.073
0.074
0.077
0.075
0.073
0.075
0.077
0.076

T
83.946
84.528
84.333
83.753
84.140
84.528
84.140
83.753
83.946

Apendix 11. Light transmission after sealed of 25% glycerol concentration of the film
Temperature
Time (s) X (mm)
(⁰C)
1
0.081
100
2
0.080
3
0.080
1
0.083
110
2
0.079
3
0.078
1
0.071
120
2
0.073
3
0.079

A = Absorbance
T = Transmittance (%)

200
A
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

T
100.000
100.000
100.000
100.000
100.000
100.000
100.000
100.000
100.000

280
A
1.307
1.355
1.368
1.325
1.236
1.225
1.164
1.205
1.273

350
T
4.934
4.415
4.281
4.729
5.804
5.957
6.860
6.232
5.337

A
0.141
0.149
0.136
0.136
0.125
0.124
0.136
0.137
0.137

T
72.320
71.024
73.139
73.089
75.009
75.162
73.164
72.940
72.958

400
A
0.098
0.102
0.090
0.092
0.083
0.082
0.096
0.099
0.092

T
79.806
79.149
81.262
80.951
82.512
82.815
80.106
79.663
80.944

500
A
0.075
0.077
0.067
0.069
0.064
0.062
0.078
0.078
0.071

T
84.053
83.746
85.762
85.223
86.379
86.652
83.596
83.582
84.838

600
A
0.069
0.071
0.061
0.064
0.059
0.057
0.074
0.074
0.067

T
85.245
84.983
86.800
86.217
87.252
87.633
84.362
84.326
85.696

800
A
0.066
0.069
0.059
0.062
0.058
0.056
0.073
0.073
0.065

T
85.821
85.405
87.319
86.637
87.580
87.932
84.499
84.614
86.063

23
Apendix 12. Effect of sealing temperature and time before sealed on transparency and opacity of 25% glycerol concentration of the film
Temperature
(⁰C)

Time (s)
1
2
3
1
2
3
1
2
3

100

110

120

200
T
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00

O
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00

280
T
15.31
14.59
15.62
15.29
14.91
14.58
14.96
15.03
15.02

350
O
90.49
91.12
90.34
91.14
91.09
90.29
90.35
91.13
91.12

T
2.06
1.93
2.07
2.05
2.00
1.96
2.04
2.01
2.01

400
O
27.14
27.39
26.64
27.72
27.72
26.97
27.31
27.72
27.72

T
1.57
1.47
1.57
1.54
1.53
1.47
1.54
1.53
1.53

500
O
21.39
21.48
20.93
21.65
22.02
20.93
21.39
21.92
21.84

T
1.27
1.14
1.28
1.27
1.21
1.18
1.24
1.21
1.24

600
O
17.68
17.30
17.40
18.15
17.78
17.21
17.68
17.78
18.06

T
1.19
1.09
1.20
1.18
1.13
1.12
1.17
1.14
1.16

800
O
16.73
16.53
16.44
17.02
16.82
16.44
16.73
16.82
17.01

T
1.13
1.04
1.14
1.11
1.06
1.05
1.11
1.08
1.09

O
15.96
15.76
15.67
16.15
15.86
15.47
15.96
15.95
16.05

Apendix 13. Effect of sealing temperature and time after sealed on transparency and opacity of 25% glycerol concentration of the film
Temperature
(⁰C)

Time (s)

100

110

120

1
2
3
1
2
3
1