CHAPTER 6

EFFECTS OF PLASTICIZERS ON THE PROPERTIES OF EDIBLE FILMS

FROM SKIN GELATIN OF BIGEYE SNAPPER AND
BROWNSTRIPE RED SNAPPER

6.1 Abstract

The effects of type and concentration of plasticizers on the mechanical

properties (tensile strength, TS and elongation at break, EAB), water vapor permeability,
light transmission, transparency and color of fish skin gelatin edible films from bigeye
snapper (Priacanthus marcracanthus) and brownstripe red snapper (Lutjanus vitta) were
investigated. At the same plasticizer concentration, fish skin gelatin films from both species
plasticized with glycerol (Gly) showed the greatest EAB (P<0.05), whereas ethylene
glycol (EG) plasticized films showed the highest TS (P<0.05). Films prepared from
brownstripe red snapper skin gelatin exhibited slightly greater TS than those of bigeye
snapper skin gelatin (P<0.05) when Gly and sorbitol (Sor) were used. EG, polyethylene
glycol 200 (PEG 200) and polyethylene glycol 400 (PEG 400) affected the mechanical
properties of both films differently. Films generally became more transparent and EAB,
water vapor permeability (WVP), as well as light transmission of films increased, but TS
and yellowness decreased with increasing plasticizer concentrations.

6.2 Introduction

Edible film is defined as a thin, continuous layer of edible material, which

can extend the shelf-life and improve the quality of almost any food system by serving as a
barrier to mass transfer or as a mechanical protection (Torres, 1997; McHugh, 2000). The
incorporation of antibiotics or antioxidants to edible films or coating has gained
considerable interest owing to the increased potential to retard the deterioration by
microorganism or oxidation at the food product surface (Guilbert et al., 1996).
In general, edible films and coating materials are derived from renewable
sources such as carbohydrates, lipids and proteins (Lim et al., 1999). Among these
materials, proteins are generally superior to polysaccharides in their ability to form films
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with greater mechanical and barrier properties (Cuq et al., 1998). However, the stand
alone protein-based films are brittle (Tanaka et al., 2001). Therefore, plasticizers are
commonly added to polymeric films in order to reduce the protein-protein chains
interactions stabilizing the films network, resulting in the increased mobility of protein
molecules (Gontard et al., 1992; Banker, 1966). However, plasticizers generally cause
the increased gas and water vapor permeability of films (Tanaka et al., 2001). Thus,
plasticizers must be added at a certain amount to obtain the films with improved flexibility
without significant decrease of barrier properties to mass transfer (Tanaka et al., 2001;
Sothornvit and Krochta, 2001). Hydrophilic plasticizers, such as glycerol (Gly),
polyethylene glycol (PEG) and sorbitol (Sor) are generally used for protein-based films to
improve mechanical properties (Sothornvit and Krochta, 2001). However, the differences
in composition, size, structure and shape of plasticizers directly influence their ability to
function in the film network (Orliac et al., 2003).
The effect of plasticizers, both types and concentrations, on the properties
of protein-based films have been intensively studied on pea proteins (Gueguen et al.,
1998), fish water-soluble proteins (Tanaka et al., 2001), -lactoglobulin (Sothornvit and
Krochta, 2001) and sunflower proteins (Orliac et al., 2003). Nevertheless, no
information regarding the effect of plasticizers on properties of gelatin-based edible films
has been reported. Therefore, the objective of this investigation was to study the effect of
various types and concentrations of plasticizers on the mechanical, barrier and physical
properties of edible film from the gelatin of bigeye snapper and brownstripe red snapper
skins, which are the wastes generated during surimi processing.

6.3 Materials and Methods

Chemicals

Glycerol (Gly), sorbitol (Sor), ethylene glycol (EG), and polyethylene

glycol (PEG 200 and PEG 400) were purchased from Wako Pure Chemical Industries,
Ltd. (Tokyo, Japan).
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Fish skin preparation

Bigeye snapper (Priacanthus macracanthus) and brownstripe red snapper

(Lutjanus vitta) with an average total length of 22-25 cm were caught from Songkhla
coast along the Gulf of Thailand, stored in ice and off-loaded after 24-36 h of capture.
Upon arrival to the dock in Songkhla, fish were stored in ice with a fish/ice ratio of 1:2
(w/w) and transported to the Department of Food Technology, Prince of Songkla
University, Hat Yai. Fish were washed with a tap water. Skins were then removed, descaled
and cut into small pieces (0.5x0.5 cm). Prepared skins were kept on ice prior to gelatin
extraction.

Extraction of fish skin gelatin

Gelatin was extracted from fish skin according to the method of Sarabia et
al. (2000) with a slight modification. Skins were soaked in 0.2 M NaOH with a
skin/solution ratio of 1:10 (w/v) at 4oC with a gentle stirring. The solution was changed
every 30 min for 3 times to remove noncollagenous proteins and pigments. Alkaline treated
skins were then washed with tap water until neutral or faintly basic pHs of wash water were
obtained. The skins were then soaked in 0.05 M acetic acid with a skin/solution ratio of
1:10 (w/v) for 3 h at room temperature (25oC) with a gentle stirring to swell the
collagenous material in fish skin matrix. Acid treated skins were washed as previously
described. The swollen fish skins were soaked in distilled water with a skin/water ratio of
1:10 (w/v) at 45oC for 12 h with a continuous stirring to extract gelatin from the skin
matter. The mixture was then filtered using two layers of cheese clothes. The resultant
filtrate was freeze-dried and the dry matter was referred to as gelatin powder.

Preparation of fish skin gelatin films

Gelatin powder was mixed with distilled water to obtain the film-forming
solution (FFS) with the protein concentration of 3% (w/v). Gly, Sor, EG, PEG 200 and
PEG 400 at various concentrations (25, 50, 75% of protein) were used as plasticizers. To
reduce the degradation of bigeye snapper gelatin caused by heat-activated proteinase, 10
mM EDTA was added into FFS. The FFS from both gelatins were incubated at 60oC for
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30 min in a water bath with an occasional stirring. The air bubbles in solution were
removed by a Hybrid mixer (HM-500; Keyance Co., Tokyo, Japan). De-aerated film
forming solution (40.01 g) was cast onto a rimmed silicone resin plate (50 mm x 50
mm) and dried with a ventilated oven (Environmental chamber model H110K-30DM;
Seiwa Riko Co., Tokyo, Japan) at 250.5oC and 505% relative humidity (RH) for 24
h. Dried films obtained were manually peeled off. The films were conditioned for 48 h at
250.5oC and 505% RH prior to analyses, however the film thickness was determined
without conditioning.

Film thickness

Film thickness was measured to the nearest 5 m with a micrometer (Dial

Pipe Gauge, Peacock Co., Tokyo, Japan). Nine measurements were taken at random
positions. Precision of the thickness measurements was 5%.

Color

Color of gelatin films was measured in the L*, a*, b* mode of CIE using a
colorimeter (Model ColorFles, HunterLab Reston, VA, USA). Color measurement was
carried out in ten replicates for each treatment.

Mechanical properties

Tensile strength (TS) and elongation at break (EAB) of gelatin films were
determined using an Instron (Model TTP-50BXII; Taketomo Electric Co., Ltd. Tokyo,
Japan). Ten samples were measured for each treatment.

Water vapor permeability

Water vapor permeability (WVP) of films was measured using a modified

ASTM method (ASTM, 1989) as described by Shiku et al. (2004). Films were sealed
onto a glass permeation cup containing silica gel (0% RH) with silicone vacuum grease
and an O-ring to hold the film in place. The cups were then placed in a desiccator saturated
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with water vapor at 30oC. The cups were weighed at 1 h intervals over a 7 h period and
WVP of films was calculated as follows (McHugh et al., 1993):
WVP = wxA-1t-1(P2-P1)-1
where w is the weight gain of the cup (g), x is the film thickness (m), A is the area of
exposed film (m2), t is the time of gain (s), and (P2-P1)-1 is the vapor pressure
differential across the film (Pa).
The WVP was expressed as g.m-1s-1Pa-1. A total of five samples were
determined for each treatment.

Light transmission

The barrier properties of gelatin films against ultraviolet (UV) and visible
light were measured at selected wavelengths between 200 and 800 nm, using a UV-
Visible Recording spectrophotometer (Model UV-1601, Shimadzu Co., Australia)
according to the method of Fang et al. (2003). The transparency of films was calculated
by the following equation (Han and Floros, 1997):
transparency = -logT600/x
where T600 is the transmittance at 600 nm and x is the film thickness (mm).

Statistical analysis

Data were subjected to analysis of variance (ANOVA). Comparison of

means was carried out by Duncans multiple-range test (Steel and Torrie, 1980). Analysis
was performed using a SPSS package (SPSS 10.0 for window, SPSS Inc, Chicago, IL).

6.4 Results and Discussion

Mechanical properties

Fish skin gelatin films from both species showed the different TS and EAB
when various types and concentrations of plasticizers were used (Table 14). Generally, TS
of bigeye snapper and brownstripe red snapper skin gelatin films decreased with increasing
plasticizer concentrations (P<0.05). Hydrophilic plasticizer is able to insert between protein
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Table 14. Mechanical properties and WVP of fish skin gelatin films as affected by
plasticizer types and concentrations

*Values are given as mean SD from ten determinations. **Values are given as mean
SD from five determinations. ***Different letters in the same column indicate significant
differences (P < 0.05). Different capital letters under the same plasticizer and gelatin
source indicate significant differences (P < 0.05). ND : Non-detected (films were too
brittle to peel off).

-chains and disrupts the hydrogen bonding, which stabilizes the film network (Gontard et
al., 1993). When plasticizer was incorporated in the gelatin film structure, the interactions
and the proximity between protein chains were reduced. However, films containing EG at
different concentrations had no differences in TS (P>0.05). Sunflower protein films
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plasticized with EG at 40% and 50% of protein showed the similar mechanical properties
(Orliac et al., 2003). Among all plasticizers used, EG resulted in the highest TS of skin
gelatin films from both species (P<0.05). For both species, gelatin films plasticized with
Gly and Sor had the drastic decrease in TS, when plasticizers added increased from 25% to
75% (P<0.05). The decreases in TS of gelatin type A films (Lim et al., 1999) and
myofibrillar protein films from Atlantic sardine (Cuq et al., 1997a) were also observed
with increasing glycerol content. Arvanitoyannis et al. (1998b) reported that TS of
chitosan/gelatin blended films decreased with increasing Gly and Sor contents.
From the result, TS of brownstripe red snapper skin gelatin films was
slightly greater than that of bigeye snapper skin gelatin films (P<0.05) when Gly and Sor
were used. EG, PEG 200 and PEG 400 affected the mechanical properties of both films
differently. Bigeye snapper skin gelatin films plasticized with 25% PEG 200 and PEG 400
were too brittle to peel off. At level of 50%, PEG 200 and PEG 400 addition rendered the
films with high TS, suggesting the strong network of protein film. Nevertheless, TS
decreased drastically with addition of 75% PEG 200 or PEG 400 (P<0.05). In contrast,
brownstripe red snapper skin gelatin films plasticized with 25% PEG 200 or PEG 400
were formed and TS slightly decreased with increasing plasticizers concentration up to
75%. Generally, TS of skin gelatin films from both species plasticized with PEG 400 was
greater than that of films plasticized with PEG 200 at any concentration used (P<0.05).
Plasticizers with the smaller size are more efficient in interacting with protein molecules to
decrease elastic modulus (EM), TS and to increase EAB of films (Sothornvit and
Krochta, 2001). Similar result were observed for -lactoglobulin films (Sothornvit and
Krochta, 2001) and methylcellulose films (Donhowe and Fennema, 1993) plasticized
with PEG having various molecular weights.
EAB of gelatin films containing plasticizers with different types and
concentrations is shown in Table 14. In general, EAB of films increased with increasing
plasticizers concentrations from 25% to 75% of protein (P<0.05). The presence of
plasticizer causes a reduction of interaction forces between protein chains and also increases
the movement of macromolecules (Gontard et al., 1993), leading to the increase in EAB
of films. However, EAB of gelatin films was not affected by EG. From the result, EAB of
gelatin films from both species plasticized with Gly was greater than those films plasticized
by Sor, EG, PEG 200 and PEG 400 at any concentration used (P<0.05). This result was
in agreement with Sothornvit and Kroachta (2001) who reported that Gly was the most
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effective plasticizer among all plasticizers tested (Sor, Sucrose, PEG 200 and PEG 400)
for -lactoglobulin films. Gly is the smallest straight chain molecule and is the most
hygroscopic among all plasticizers tested (Sothornvit and Krochta, 2001). Thus, Gly
could be easily inserted between protein chains and attracted more water to the protein films
structure, resulting in the increase in its effectiveness as a plasticizer (Sothornvit and
Krochta, 2001; Gontard et al., 1993). When the effect of Sor and PEG 200 on EAB was
compared, PEG 200 showed higher plasticizer efficiency as evidenced by the greater EAB,
compared with Sor (P<0.05) (Table 14). Sor and PEG 200 are similar in molecular
weight, shape and number of oxygen atoms in the molecules (Sothornvit and Krochta,
2001). The differences in plasticizing effect between both plasticizers were possibly due to
the different availability of oxygen atoms for hydrogen bonding (Sothornvit and Krochta,
2001). The differences in TS and EAB of films plasticized with different plasticizers
suggested that the appropriate plasticizer for an individual protein film system depends on
the nature, size and the structure of plasticizer as well as the compatibility between protein
molecules and plasticizers.
From the results, the differences in TS and EAB between bigeye snapper
and brownstripe red snapper skin gelatin films plasticized with various types and
concentrations of plasticizers could possibly due to the different compositions, particularly
in term of amino acid compostion and size of protein chains between both gelatins
(Muyonga et al., 2004b; Paschoalick et al., 2003). This might result in the different film
formation.

Water Vapor Permeability

WVP of fish skin gelatin films of both species plasticized with different
types and concentrations of plasticizers are shown in Table 14. In general, WVP of the
films increased when the plasticizer concentration increased (P<0.05). With increasing Gly
concentration from 25% to 50%, WVP of gelatin films from both species increased (P
<0.05). However, WVP of films slightly increased with further increase of plasticizers
concentration up to 75% of protein (P<0.05). For other plasticizers, the constant increases
were found in the range of 25-75%. The increase in plasticizer concentration resulted in
the increase of WVP of films from gluten (Gontard et al., 1993), sunflower proteins
(Orliac et al., 2003), chitosan/gelatin blended (Arvanitoyannis et al., 1998b), muscle
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proteins (Paschoalick et al., 2003), fish water soluble proteins (Tanaka et al., 2001),
gelatin (Sobral et al., 2001b) and methyl cellulose/soluble starch blends (Arvanitoyannis
and Biliaderis, 1999). The insertion of plasticizers between chains of macromolecules
increases the free volume of the system and favors the mobility of polymeric chains.
Consequently, films network structure became less dense and more permeable (Gontard et
al., 1993). Since all plasticizers used are hydrophilic, an increase in their concentrations
enhanced the absorption of more water to the network and increased water vapor transfer
(Orliac et al., 2003). However, the different rate of water vapor transmission was
observed among gelatin films plasticized with different types and levels of plasticizers. This
might be due to the differences in the hygroscopic nature of the plasticizers used, which
caused the different ability to attract water to the film network system (Leung, 1986).

Light transmission

Transmission of UV and visible light at selected wavelength of 200-800

nm to fish skin gelatin films for both species is shown in Table 15. Generally, films
prepared from the skin gelatin of both fish species exhibited the low transmission to light in
the UV ranges (200-280 nm). At the same level of plasticizer used, films plasticized with
PEG 200 and PEG 400 showed the lower light transmission at 280 nm, compared with
those plasticized with Gly, Sor and EG. The differences in light transmission at 280 nm of
films plasticized with various types of plasticizers might be due to the different molecular
weight, composition, size, nature and some properties of plasticizers used that might
interfere with the light transmission properties of the films (Orliac et al., 2003). From the
result, the addition of plasticizers at the higher content resulted in the increase in light
transmission of films for both UV and visible ranges. Plasticizers used are the transparent
compound (Paschoalick et al., 2003), which could increase the light transmission of the
film. This result was in accordance with Pascoalick et al. (2003) who observed that the
muscle protein films from Nile tilapia became more transparent with the increase in
plasticizer contents.
The transparency of fish skin gelatin films was in the range of 1.03 2.40
(Table 15), indicating that the films were fairly transparent. Films plasticized with PEG
200 and PEG 400 were more transparent, compared with those plasticized with Gly, Sor
and EG at the same concentration used. The different characteristics of plasticizers used
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might cause the differences in film transparency (Orliac et al., 2003). It was noticeable
that films became more transparent with further increase of plasticizer concentrations. The
similar result was reported in edible films from Nile tilapia muscle protein plasticized with
increasing glycerol concentration (Paschoalick et al., 2003). The transparency of fish skin
gelatin films obtained in this study was greater than those of pea protein isolate films (Choi
and Han, 2002), myofibrillar protein films (Shiku et al., 2003), fish water soluble
protein films (Hamaguchi et al., 2003) and surimi films (Shiku et al., 2004).

Table 15. Light transmission (%T) and transparency of fish skin gelatin films as affected
by plasticizer types and concentrations

ND: Non-detected (films were too brittle to peel off)

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Table 16. Color of fish skin gelatin films as affected by plasticizer types and
concentrations

1
Values are given as mean SD from ten determinations. **Different letters in the same
column indicate significant differences (P<0.05). Different capital letters under the same
plasticizer and gelatin source indicate significant differences (P<0.05). ND: Non-detected
(films were too brittle to peel off).

Color

Color of fish skin gelatin films from both species plasticized with different
types and concentrations of plasticizers is presented in Table 16. From the result, b* values
(-b* = blueness, +b* = yellowness) of films decreased with increasing plasticizer
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concentrations from 25% to 75% of protein (P<0.05), whereas L* (lightness) and a*

values (-a* = greenness, +a* = redness) slightly increased (P<0.05). The decrease in b*
value of film plasticized with greater concentration of plasticizers might be owing to the
dilution of the proteins by colorless plasticizers (Paschoalick et al., 2003). The result was
in accordance with Paschoalick et al. (2003) who reported that the color values of muscle
protein films from Nile tilapia decreased linearly with increasing plasticizer concentration.
Additionally, the increase in plasticizer content might enhance the reflection of light at the
film surface, leading to the increased L* value of film added with some plasticizer,
especially Gly.

6.5 Conclusion

Transparent edible films were successfully prepared from fish skin gelatin
of brownstripe red snapper and bigeye snapper. The properties of fish skin gelatin films
from both fish species were affected by types and concentrations of plasticizers used. Films
plasticized with Gly showed the highest EAB among all plasticizers at the same
concentration used, whereas EG plasticized films showed the highest TS. EAB, WVP and
light transmission increased but TS and yellowness decreased with increasing plasticizers
concentration. The properties of gelatin films from both species were affected by
plasticizers differently.