Properties of Gelatin Film from Horse Mackerel (Trachurus japonicus) Scale Thuy Le, Hiroki Maki, Kigen Takahashi, Emiko Okazaki, and Kazufumi Osako
E: Food Engineering & Materials Science
Abstract: Optimal conditions for extracting gelatin and preparing gelatin film from horse mackerel scale, such as extraction temperature and time, as well as the protein concentration of film-forming solutions were investigated. Yields of extracted gelatin at 70 °C, 80 °C, and 90 °C for 15 min to 3 h were 1.08% to 3.45%, depending on the extraction conditions. Among the various extraction times and temperatures, the film from gelatin extracted at 70 °C for 1 h showed the highest tensile strength and elongation at break. Horse mackerel scale gelatin film showed the greatly low water vapor permeability (WVP) compared with mammalian or fish gelatin films, maybe due to its containing a slightly higher level of hydrophobic amino acids (total 653 residues per 1000 residues) than that of mammalian, cold-water fish and warm-water fish gelatins. Gelatin films from different preparation conditions showed excellent UV barrier properties at wavelength of 200 nm, although the films were transparent at visible wavelength. As a consequence, it can be suggested that gelatin film from horse mackerel scale extracted at 70 °C for 1 h can be applied to food packaging material due to its lowest WVP value and excellent UV barrier properties. Keywords: gelatin film, horse mackerel scale, mechanical properties, extraction temperature, extraction time
In this study, gelatin film from horse mackerel scale at various preparation conditions could be successfully prepared. Horse mackerel scale gelatin films exhibited lower water vapor permeability values than gelatin films from the skin of other fishes or mammals do. Utilization of horse mackerel scale as a raw material of edible film could be solved the environmental pollution by seafood waste during fish processing.
Practical Application:
Introduction Gelatin is a protein derived by partial hydrolysis of collagen extracted from skin, bones, connective tissues, organs, and some intestines of mammalian species (Johnston-Banks 1990; Kavoosi and others 2013). Nevertheless, concerns regarding bovine and porcine health problems in recent years lead to increased attention on fish gelatin (G´omez-Guill´en and others 2011). Gelatin has wide application in the food processing industry, such as a gelling agent in cooking, a stabilizer, thickener, or texturizer in foods (Irwandi and others 2009; G´omez-Guill´en and others 2011). Another important application of fish gelatin is in the production of film. Fish gelatin film, which is biodegradable, is considered a vital eco-friendly packaging material for reducing the environmental impact of synthetic plastic materials (Nagarajan and others 2012a). Furthermore, fish gelatin films used as a foodstuff covering have the capacity to protect food against drying, light, and oxygen (G´omez-Guill´en and others 2009). Horse mackerel (Trachurus japonicus) is one of the most important marine fishes in Japan, with an annual catch of 133915 tonnes in 2012 (Production figures of fishery and aquaculture industry, Ministry of Agriculture, Forestry and Fisheries, 2012). This fish is used as a raw material of frozen surimi (Yamanaka and Tanaka 2007) and in the preparation of raw fresh sashimi and sushi, which are popular foods in Japan (Yamanaka and Tanaka 2007). However, during fish processing, a large amount of byproduct (accounting for approximately 50% to 70% of fish weight, and consisting of
skin, scales, and bone) is produced and discarded, resulting in serious environmental problems (Kittiphattanabawon and others 2005). Utilization of marine byproducts, including scales, as raw materials in gelatin extraction represents a potential strategy to reduce the environmental pollution caused by marine waste, as well as increasing the value of the fish product. Fish gelatin films from brownstripe red snapper and bigeye snapper skin (Jongjareonrak and others 2006), blue shark skin (Limpisophon and others 2009), Nile perch skin (Muyonga and others 2004), halibut skin (Carvalho and others 2008), tuna skin (G´omez-Guill´en and others 2007), trout skin (Kim and Min 2012), and cuttlefish (Hoque and others 2011) have been characterized. However, little information is available regarding the characteristics of gelatin films from fish scale, with the exception of lizard fish scale (Wangtueai and others 2010). Thus, the purpose of this study was to develop edible film by investigating the properties of gelatin film from horse mackerel (T. japonicus) scale at different preparation conditions, that is, extraction temperature and time, of gelatin from scale, and the protein concentration of film-forming solutions (FFS).
Materials and Methods
Characterization of horse mackerel scale gelatin Fish scale preparation. Horse mackerel scale was collected in Nagasaki Prefecture, Japan. The scale was collected and transported to our laboratory under ice. The fish scale was removed of impurities manually, washed with chilled water, and then kept in MS 20141299 Submitted 7/27/2014, Accepted 1/12/2015. Authors are with plastic bags and stored in deep freezer at –30 ± 2 °C until use. Gelatin extraction. Gelatin was extracted from horse mackDept. of Food Science and Technology, Tokyo Univ. of Marine Science and Technology, Japan. Direct inquiries to author Osako (E-mail: [email protected]). erel scale following the method described by Wangtueai and Noomhorm (2009) with minor modifications. Briefly, frozen scale E734
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doi: 10.1111/1750-3841.12806 Further reproduction without permission is prohibited
Properties of horse mackerel scale film. . . Table 1–Yield of gelatin from horse mackerel scale at various high-performance liquid chromatography system (LC-10A amino extraction times and temperatures. acid analysis system, Shimadzu, Kyoto, Japan) with a Shim-pack
Extraction time (h) 15 min 30 min 1 2 3
70 1.08 ± 1.37 ± 0.09ab 2.50 ± 0.54cde 2.59 ± 0.51cdef 2.78 ± 0.57def 0.21a
80 1.81 2.57 2.90 2.93 3.45
± ± ± ± ±
0.05abc 0.40cdef 0.75def 0.56def 0.99f
90 2.18 2.73 3.08 3.18 3.21
± ± ± ± ±
0.12bcd 0.39def 0.70ef 0.72ef 0.37ef
Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
was thawed and soaked with 0.1 M NaOH solution for 6 h at a 1:8 (w/v) ratio of scale/alkali solution to move out the noncollagenous protein. The NaOH solution was changed to the new solution after 3 h, and scales were soaked in cold distilled water (DIW) to obtain a neutral pH. After the removal of noncollagenous protein, the fish scales were drained using a cheesecloth, dehydrated manually, and then immersed in 10× DIW (w/v) at various temperatures (70 °C, 80 °C, and 90 °C) for various times (15 min, 30 min, 1 h, 2 h, and 3 h) with stirring to extract the fish scale gelatin. After gelatin extraction, the coarse solids were removed by filtration with two layers of cheesecloth and the liquid was centrifuged at 16400 × g for 30 min under 20 °C. The gelatin solution in the supernatant was freeze-dried (DC 401; Yamato Scientific Co., Ltd., Japan). Extraction yield. The yield of gelatin was evaluated by the method of Limpisophon and others (2009). Approximately 1 g of the noncollagenous fish scale was weighed, and then extracted in 10× DIW (w/v) as described above. The protein concentration of the supernatant was determined following the method of Lowry (Lowry and others 1951) using bovine serum albumin as a protein standard. The yield of extraction (YD) was determined by the following equation: YD (%) =
Protein concentration (mg/mL) × 10 times of DIW (mL) × 100% Fish scales weight (mg)
Electrophoretic of gelatin. Protein pattern of extracted gelatin powder from horse mackerel scale was conducted by the method of Laemmli (1970) with minor modifications. The supernatant thus collected at each extraction temperature and time was mixed with the buffer (0.5 M Tris-HCl, pH 6.8, containing 10% (w/v) sodium dodecyl sulfate (SDS) and 20% (v/v) glycerol) in the presence of 10% (v/v) mercaptoethanol. The samples (10 μL) were loaded onto a 7.5% polyacrylamide gel (AE-6000, NPU-7.5L E-PAGEL, ATTO Co., Tokyo, Japan). The gel was fixed with the solution containing 25% (v/v) methanol and 5% (v/v) acetic acid for 30 min, stained by immersing in 0.1% (w/v) Coomassie Brilliant Blue R-250 solution (30% (v/v) methanol and 10% (v/v) acetic acid) and decolored in solution of 30% (v/v) methanol and 10% (v/v) acetic acid. Molecular weight marker (Sigma Chemical Co., St. Louis, Mo., U.S.A.) ranging from 10 to 200 kDa were used as a standard. Amino acid analysis. Twenty milligrams of extracted gelatin powder from horse mackerel scale were hydrolyzed in 6 M HCl at 110 °C for 22 h in vacuo. The hydrolysate was neutralized with 6 N and 0.6 N NaOH, and filtrated with a 0.45 μm cellulose membrane filter (Toyo Roshi Kaisha Ltd., Tokyo, Japan). The amino acid concentration in filtrate was determined by using
Amino-Li column (100 mm × 6.0 mm, Shimadzu) and Shim-pack SC-30/S0504 Li precolumn (150 mm × 4.0 mm, Shimadzu). Fourier transform infrared spectroscopy (FTIR). FTIR of gelatin powder was performed using FT-IR spectrophotometer (NICOLET 200SXV, Thermo Electron Corporation, U.S.A.). FTIR spectra was obtained from discs consisted of 2 mg of gelatin powder in approximately 100 mg potassium bromide (Muyonga and others 2004). Measurement was performed at 4000–500 cm−1 at a resolution of 2 cm−1 per point at room temperature by using Omnic 6.0 software (Thermo-Nicolet, Madison, Wis., U.S.A.).
Effect of extraction temperature and time, and protein concentration of FFS on the gelatin films Preparation of gelatin films. To study the effect of extraction temperature on gelatin films, gelatin powder extracted at various temperatures (70 °C, 80 °C, and 90 °C) for 1 h were diluted in DIW at 60 °C for 30 min to adjust the protein concentration of FFS to 2% (w/v). The effect of extraction time on gelatin films was investigated by dissolving gelatin powder obtained at different extraction times (15 min, 1 h, and 3 h) at 70 °C with DIW at 60 °C for 30 min to obtain a FFS protein concentration of 2% (w/v). Glycerol was used as a plasticizer, and was added to FFS at a protein concentration of 20% (w/w) with stirring at ambient temperature for 15 min. FFS solution was removed its air bubbles by using a mixer (HM-500; Keyence Co., Tokyo, Japan). The prepared FFS (4 g) was put onto a rimmed silicone plate (50 × 50 mm) and dried in a chamber (EYELA, KCL-2000A; Tokyo Rikakikai Co., Ltd., Japan) at 25 ± 0.5 °C and 50 ± 5% relative humidity (RH) for 48 h. The films thus obtained were manually peeled off. The effect of FFS protein concentration on gelatin film from horse mackerel scale was investigated by dissolved gelatin powder extracted at 70 °C for 1 h in DIW at 60 °C for 30 min to obtain FFS with protein concentrations of 1%, 2%, and 3% (w/v), determined by using a Lowry’s method (Lowry and other 1951). Glycerol as a plasticizer was added to FFS at 20% (w/w) protein concentration and stirred at room temperature for 15 min continuously. The FFS was cast and dried as previously described. Moisture content of gelatin films. Moisture content of gelatin films was analyzed by dried sample in oven at 105 °C for 24 h following the methods of AOAC (2000). Thickness. Film thickness was determined by using a dial-type thickness gauge (Series 7300; Mitsutoyo Co., Kanagawa, Japan). Every film sample was measured its thickness at 6 random positions of the film. Mechanical properties. Tensile strength (TS) and elongation at break (EAB) of film samples were measured by using an instrument for measuring physical properties (Tensipresser TTP50BX II; Taketomo Electric Inc., Tokyo, Japan) following the ASTM standard method D 882–97 (ASTM, 1999). Sampled film (20 × 45 mm) was placed in specific tensile grip with an initial grip distance of 30 mm and a cross-head speed of 1 mm/s until the films were disrupted. TS and EAB were calculated by the following equation, respectively: TS (MPa) = EAB (%) =
Maximum load (N) Initial cross − sectional area (m2 )
Film length (mm) at rupture × 100 Initial grip length of samples (mm)
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Temperature (°C)
Properties of horse mackerel scale film. . . Water vapor permeability (WVP). WVP of films was determined following the ASTM standard test method (1983) as reported by Gontard and others (1992). Each film was hold onto a glass cup (diameter of 5 cm) containing silica gel (0% RH) with silicon vacuum grease and a plastic band. The cups were placed in a desiccator with saturated water (100% RH) and put in ventilated chamber at 30 °C. The cups with films were weighed at initial time and every 1 h during 9-h period. Each film was measured its WVP with 6 replications. WVP of gelatin film (expressed as a gm−1 s−1 Pa−1 ) was calculated by following equation (McHugh and others 1993): WVP = w × x × A−1 × t −1 × P −1
E: Food Engineering & Materials Science
where w is the weight gain (g) of the cup, x is the film thickness (m), A is the area of exposed film (m2 ), t is the time of gain (s), ࢞P is the vapor pressure gradient (Pa) between inner and outer surface of the film. Light transmission. Light transmission in ultraviolet (UV) and visible ranges of gelatin films was measured at different wavelengths from 200 to 800 nm using a UV-Visible Recording Spectrophotometer (UV-160; Shimadzu Co., Kyoto, Japan) following the method described by Fang and others (2002). Electrophoretic analysis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of gelatin films from horse mackerel scale was conducted according to the method of Limpisophon and others (2009) with some modifications. Film samples (50 mg) were mixed with 5 mL of 1% SDS solution (w/v) and shaken at ambient temperature for 16 h continuously. Supernatants obtained after centrifugation at 5000 × g for 5 min at 20 °C were subjected to SDS-PAGE as described in previous section.
Statistical analysis Data in this study were presented as mean ± standard deviation with 6 replications. An analysis of variance (ANOVA) of the data was performed by using SPSS software (SPSS 11.5 for Windows).
Results and Discussion Characterization of gelatin from horse mackerel scale Extraction yield. Gelatin yields from horse mackerel scale at various extraction times and temperatures are shown in Table 1. Yields ranged from 1.08% to 3.45%, depending on the extraction condition. The yield increased by the disruption of hydrogen bonds stabilizing collagen’s triple helical structure with increasing extraction time and temperature, which may lead to converse insoluble collagen into soluble gelatin (Benjakul and others 2009). The results were supported by Limpisophon and others (2009) and Wangtueai and Noomhorm (2009), who clarified that increasing time and temperature (from 0.5 to 5 h at 45 °C to 100 °C for blue shark skin and from 1 to 5 h at 70 °C to 90 °C for lizard fish scale) during gelatin extraction were related with increasing yield of gelatin from blue shark skin and lizard fish scale, respectively (3.95% to 5.20% and 9.10% to 10.90%, respectively). The yield of gelatin from horse mackerel scale was lower than that from blue shark skin and lizard fish scale. However, horse mackerel is one of the economic marine fish species with high amount of annual catch, which can be used as a raw material to extract gelatin. SDS-PAGE. The SDS-PAGE profile of extracted gelatin powder at different extraction temperatures and times is shown in
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Table 2–Amino acid composition of gelatin from horse mackerel scale. Amino acid Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine Hydroxyproline Proline Imino acidsa Hydrophobic amino acidsb
Residues/1000 residues Horse mackerel scale gelatin 49 ± 25 ± 33 ± 71 ± 322 ± 131 ± 21 ± 17 ± 10 ± 23 ± 2 ± 18 ± 10 ± 32 ± 7 ± 51 ± 67 ± 111 ± 178 ± 653
2 2 2 3 5 3 2 2 2 3 1 2 1 3 2 4 3 7 9
Data are expressed as mean ± standard deviation (n = 3). a Indicates total hydroxyproline and proline. b Indicates total glycine, alanine, valine, methionine, isoleucine, leucine, phenylalanine, and proline.
Figure 1. High molecular weight protein was observed in gelatin powder from horse mackerel scales, consisting of α and β chains. However, the degradation of α and β chains was observed at extraction temperatures higher than 70 °C and extraction times longer than 1 h. Limpisophon and others (2009) observed the degradation of α and β chains in the protein pattern of blue shark skin gelatin at extraction temperatures higher than 80 °C. The differences in raw materials (scale and skin) can explain the observed differences in the temperature of protein degradation between horse mackerel scale gelatin (70 °C) and blue shark skin gelatin (80 °C). Amino acid analysis. The amino acid composition from horse mackerel scale gelatin powder extracted at 70 °C for 1 h is presented in Table 2. Glycine, the dominant amino acid in horse mackerel scale gelatin, accounted for 30% of total amino acids. Imino acid contents (proline and hydroxyproline) of horse mackerel scale gelatin (178 residues per 1000 residues) were greater than those from grass carp scales (94 residues per 1000 residues; Zhang and others 2011) but lower than those of lizardfish scale gelatin (205 residues per 1000 residues; Wangtueai and Noomhorm 2009) and mammalian gelatin (216 to 225 residues per 1000 residues; Avena-Bustillos and others 2006). Imino acids have a significant function in the triple helical structure of gelatin and affect the gel strength of gelatin gel (Ward and Courts 1977; Zhang and others 2011). Most fish gelatins have lower amino acid content than mammalian gelatins, explaining the poor gelling in comparison with mammalian gelatins. Notably, horse mackerel scale gelatin contained the same or slightly higher hydrophobic amino acids (total 653 residues per 1000 residues) compared to commercial mammalian (pork skin), commercial cold-water fish (from skin of haddock) and warm-water fish (from catfish skin) gelatins (total 651 residues, 647 residues and 649 residues per 1000 residues, respectively; Avena-Bustillos and others 2006). FTIR. Figure 2 shows the FTIR spectra of gelatin powder extracted at 70 °C, 80 °C, and 90 °C for 1 h (a, b, c) and
Properties of horse mackerel scale film. . . extracted at 70 °C, 80 °C, and 90 °C for 1 h appeared at 1650.49, 1652.69, and 1654.60 cm−1 , respectively. The amide I bands of gelatin extracted at 70 °C for 15 min, 1 h, and 3 h appeared at 1643.33, 1650.49, and 1653.85 cm−1 , respectively. In addition, amide III has been also associated with loss of triple-helix state of the molecules and transformation of a helical to random coil Figure 1–SDS-PAGE patterns of gelatin powder extracted at various temperatures (70 o C, 80 o C, and 90 o C for 1 h) and times (at 70 o C for 15 min, 30 min, 1 h, and 2 h). “M” means protein marker.
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extracted at 70 °C for 15 min, 1 and 3 h (d, e, f). Amide I band, wavenumber between 1600 and 1700 cm−1 , was the most useful in studies on the secondary structure of gelatin (Nikoo and others 2011). Amide I band arises from C = O stretching vibration of the peptidic bond in the Gly-X-Y tripeptide sequence with in-plane NH bending. Amide I bands of gelatin from horse mackerel scale
Figure 2–FTIR spectra of gelatin extracted at various temperature and time.
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Properties of horse mackerel scale film. . . Table 3–Effect of gelatin-extraction temperature and time on thickness, tensile strength (TS), elongation at break (EAB), and water vapor permeability (WVP) of gelatin film. TS (MPa)
EAB (%)
WVP (×10-10 gm-1 Pa-1 s-1 )
± 0.52c ± 0.36b ± 0.34a
36.48 ± 3.66c 19.95 ± 1.46b 13.14 ± 1.66a
46.09 ± 7.44c 25.56 ± 2.73b 19.74 ± 4.25a
0.98 ± 0.07a 1.07 ± 0.08ab 1.11 ± 0.07b
± 0.56b ± 0.52c ± 0.4a
35.62 ± 2.44bc 36.48 ± 3.66b 28.84 ± 2.19a
43.04 ± 6.78bc 46.09 ± 7.44b 33.32 ± 8.79a
1.01 ± 0.02a 0.98 ± 0.07a 1.14 ± 0.07b
Thickness (µm) Extraction temperature (for 1 h) 70 °C 23.58 80 °C 22.44 90 °C 21.57 Extraction time (at 70 °C) 15 min 22.75 1h 23.58 3h 22.05
Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
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structure due to denaturation of collagen to gelatin (Muyonga and others 1993). The amide III bands could be observed at 1238.69, 1240.67, and 1242.69 cm−1 with extraction temperature of 70 °C, 80 °C, and 90 °C for 1 h, respectively. With gelatin extracted at 70 °C for 15 min, 1 h, and 3 h, amide III bands appeared at 1235.71, 1238.69, and 1241.98 cm−1 , respectively. The increase in frequency shift of amide I and amide III region with increasing extraction temperate and time is responsible for the denaturation of α-helix in amide I and III by uncoupling of intermolecular cross-links and interruption of intramolecular bonding when increasing extraction temperature or time during gelatin extracted (Kittiphattanabawon and others 2012). From the results, we can conclude that characterization of gelatin can be affected by extraction conditions. The increasing of extraction temperature and time led to increased extraction yields. However, the triple helix structure of gelatin powder can be destroyed with increasing temperature and time and it may affect physical and mechanical properties of the gelatin film. The effects of extraction condition on the gelatin film were studied as next step.
Effect of extraction time and temperature on properties of gelatin film from horse mackerel scale Moisture content of gelatin films. The moisture content of gelatin films ranged from 17.39% to 17.49%, depending on the preparation conditions of films. However, no statistical differences were found among the films. Thickness. The thickness of films prepared from horse mackerel scale gelatin at various extraction conditions differed (Table 3). An extraction temperature greater than 70 °C and an extraction time longer than 1 h resulted in a slightly decreased film thickness. A possible reason may be that increases in extraction temperature and time led to an increased degree of gelatin hydrolysis. Hoque and others (2011) observed a slight decrease in thickness in gelatin films with a higher degree of hydrolysis. This result is in agreement with Nagarajan and others (2012a), who reported that the thickness of film made from splendid squid (Loligo formosana) gelatin decreased with increasing extraction temperature. Mechanical properties. TS and EAB of gelatin films at various extraction temperatures and times are presented in Table 3. Under identical extraction time (1 h), the gelatin film extracted at 70 °C had the highest TS (36.48 ± 3.66 MPa) and EAB (46.09 ± 7.44%) values when compared with gelatin films extracted at higher temperatures (80 °C and 90 °C); which were 19.95 ± 1.46 MPa and 13.14 ± 1.66 MPa, respectively, for TS, and 25.56 ± 2.73% and 19.74 ± 4.25%, respectively, for EAB. At an extraction temperature of 70 °C, the TS and EAB values of gelatin film extracted for 15 min (35.62 ± 2.44 MPa and 43.04 ± 6.78%, E738 Journal of Food Science r Vol. 80, Nr. 4, 2015
respectively) did not significantly differ from those for 1 h extraction (36.48 ± 3.66 MPa and 46.09 ± 7.44%, respectively). However, both values decreased to 28.84 ± 2.19 MPa and 33.32 ± 8.79%, respectively, when heated for 3 h. Thus, TS and EAB of gelatin film decreased with increasing extraction temperature and time. It is known that the mechanical properties of gelatin film depend on the triple helical structure of gelatin. Hoque and others (2011) reported that gelatin films are mainly stabilized by weak forces, including hydrogen bonding and hydrophobic interaction. These weak bonds are likely destroyed by the increasing extraction temperature and time, leading to the disruption of gelatin’s triple helical structure and the formation of shorter peptide chains (Nagarajan and others 2012b), and resulting in the alteration of TS and EAB of gelatin films. WVP of gelatin film. The WVP of gelatin films extracted under various conditions is shown in Table 3. Under identical extraction time (1 h), WVP increased with increasing extraction temperature; the gelatin film extracted at 70 °C showed the lowest WVP value (0.98 ± 0.07 × 10−10 gm−1 Pa−1 s−1 ). Increase in extraction time from 15 min to 3 h at 70 °C resulted in increased WVP value (1.01 ± 0.02 × 10−10 gm−1 Pa−1 s−1 to 1.14 ± 0.07 × 10−10 gm−1 Pa−1 s−1 ). WVP appears to be determined by the strength of protein molecule interactions within the network of the film (Tongnuanchan and others 2011). The triple helical structure of gelatin may be broken down to small peptide chains with increased extraction time and temperature. These small peptide chains can be easily inserted into the protein network, leading to a decrease in density of intermolecular interactions between gelatin chains, and a subsequent increase in the WVP of gelatin films. The slightly higher hydrophobic amino acids content may be the reason for low WVP of the films, when compared to films produced from other fish or land-based animal gelatins (Limpisophon and others 2009). Light transmission of gelatin film (%). UV and visible light transmission of films from horse mackerel scale gelatin prepared at various temperatures and times is presented in Table 4. Gelatin films extracted at various temperatures (70 °C to 90 °C) for 1 h showed transmission in the visible range (350 to 800 nm) and UV range (200 to 280 nm) from 23.68 ± 1.33% to 75.77 ± 2.03% and from 12.15 ± 0.61% to 16.35 ± 2.51%, respectively. Transmission in the visible range and UV range of gelatin film extracted at 70 °C for different times was from 29.03 ± 1.91% to 79.08 ± 6.24% and from 15.97 ± 2.83% to 20.92 ± 0.57%, respectively. However, at the wavelength of 200 nm, the transmission of these films could not be measured, regardless of extraction temperature or time. A possible explanation is that the amino acid composition of gelatin consists of aromatic amino acids with a phenol ring, such as phenylalanine, tryptophan, and tyrosine, could prevent UV
Properties of horse mackerel scale film. . . Table 4–Effect of gelatin-extraction temperature and time on light transmission (%) of gelatin films. Light transmission at different wavelength (nm) 200 Extraction temperature (for 1 h) 70 °C 0.3 80 °C 0.3 90 °C 0.3 Extraction time (at 70 °C) 15 min 0.3 1h 0.3 3h 0.3
280
350
400
500
600
800
16.35 ± 2.51a 13.68 ± 0.55b 12.15 ± 0.61b
30.42 ± 2.78a 27.30 ± 2.73b 23.68 ± 1.33c
52.70 ± 1.14a 45.17 ± 1.74b 42.20 ± 0.99c
62.60 ± 1.59a 59.18 ± 0.62b 49.82 ± 1.33c
69.43 ± 1.12a 63.58 ± 0.41b 57.85 ± 0.91c
75.77 ± 2.03a 68.55 ± 1.74b 64.25 ± 2.04c
20.92 ± 0.57a 16.35 ± 2.51b 15.97 ± 2.83b
35.98 ± 1.53a 30.42 ± 2.78b 29.03 ± 1.91b
61.01 ± 2.35a 52.70 ± 1.14b 52.35 ± 0.96b
68.77 ± 3.20a 62.60 ± 1.59b 59.85 ± 3.28b
75.93 ± 7.97a 69.43 ± 1.12b 61.13 ± 2.70c
79.08 ± 6.24a 75.77 ± 2.03a 68.88 ± 4.00b
transmission, similar to that observed at brownstripe red snapper and bigeye snapper skin gelatin film (Jongjareonrak and others 2006). Thus, the gelatin film prevented UV light transmission effectively. These results suggested the potential preventive effect of gelatin films on the retardation of lipid oxidation induced by UV light. SDS-PAGE. SDS-PAGE profiles of gelatin films produced under various extraction conditions are depicted in Figure 3. From the protein pattern, the main constituents of horse mackerel gelatin film were α and β chains. Moreover, with increasing time and temperature of gelatin extracted, these main constituents were degraded, especially with an extraction temperature higher than 70 °C and an extraction time longer than 1 h. These results show that physical and mechanical properties of gelatin film can be affected by extraction conditions of gelatin. TS and EAB of films decreased with increasing extraction temperature and time. On the contrary, WVP increased with increasing temperature and time during extraction. The degradation of protein patterns of gelatin film with increasing extraction temperature and time can be observed in Figure 3 and this result is the similar tendency with SDS profile of gelatin powder extracted at different temperature and time (shown in Figure 1). The optimum
temperature and time for extracting gelatin to prepare the film having high mechanical properties (TS and EAB) was 70 °C and 1 h, respectively. Gelatin powder extracted at 70 °C for 1 h was used for next step.
Effect of protein concentration of FFS on gelatin film Mechanical properties. Mechanical properties of gelatin films at different protein concentrations are presented in Table 5. TS of gelatin film increased from 19.40 ± 4.75 MPa to 36.77 ± 4.13 MPa and EAB increased from 38.76 ± 9.77% to 48.77 ± 9.23% with increasing FFS protein concentration. The greater FFS protein content might result in increased aggregation of proteins forming the film, resulting in improved flexibility and mechanical properties of films (Jongjareonrak and others 2006). The increasing trend of TS and EAB of horse mackerel scale gelatin films was similar to gelatin films from brownstripe red snapper and bigeye snapper skin (Jongjareonrak and others 2006). WVP. The effect of various FFS protein concentrations on the WVP values of gelatin films is shown in Table 5. WVP of gelatin films increased from 0.46 ± 0.05 to 1.51 ± 0.07 × 10−10 gm−1 Pa−1 s−1 with increasing FFS protein concentration. Notably, greater WVP values were observed in gelatin films with
Figure 3–SDS-PAGE patterns of gelatin films at different extraction temperatures (70 o C, 80 o C, and 90 o C for 1 h) and times (at 70 o C for 15 min, 1 h, and 3 h). “M” means protein marker.
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Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
Properties of horse mackerel scale film. . . Table 5–Effect of FFS∗ protein concentration on thickness, tensile strength (TS), elongation at break (EAB), and water vapor permeability (WVP) of gelatin films. Protein concentration 1% 2% 3%
Thickness (µm)
TS (MPa)
EAB (%)
WVP (×10-10 gm-1 Pa-1 s-1 )
10.65 ± 0.33a 23.58 ± 0.52b 38.48 ± 0.71c
19.40 ± 4.75a 36.48 ± 3.66bc 36.77 ± 4.13b
38.76 ± 9.77a 46.09 ± 7.44ab 48.77 ± 9.23b
0.46 ± 0.05a 0.98 ± 0.07b 1.51 ± 0.07c
∗ FFS means film-forming solution. Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
Table 6–Effect of FFS∗ protein concentration on the light transmission (%) of gelatin films. Light transmission at different wavelength (nm)
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Protein concentration 1% 2% 3%
200
280
350
0.3 0.3 0.3
23.18 ± 16.35 ± 2.51a 11.78 ± 1.57a
0.89b
400
34.30 ± 30.42 ± 2.78a 36.43 ± 1.91a
1.51b
500
59.40 ± 52.70 ± 1.14a 47.17 ± 0.96a
1.07b
600
69.68 ± 62.60 ± 1.58b 55.23 ± 1.79a 0.88c
800
75.13 ± 69.43 ± 2.12a 78.63 ± 1.24c
0.82b
80.62 ± 0.87b 75.77 ± 2.03a 79.98 ± 1.43b
∗ FFS means film-forming solution. Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
increased thickness. WVP values of gelatin films from horse mackerel scale (0.98 ± 0.07 × 10−10 gm−1 Pa−1 s−1 ) were lower than gelatin films from brownstripe red snapper and bigeye snapper skin (1.22 ± 0.11 × 10−10 gm−1 Pa−1 s−1 and 1.33 ± 0.04 × 10−10 gm−1 Pa−1 s−1 , respectively; Jongjareonrak and others 2006) at identical FFS protein concentration (2%). We cannot find critically reason for the lower WVP values of horse mackerel gelatin film when compare with others gelatin film at similar protein content in FFS. However, one reason may be explained by the slightly higher amount of hydrophobic amino acids. The gelatin film of the present study contained a slightly higher amount of hydrophobic amino acids compared with commercial mammalian, cold-water fish and warm-water fish gelatins (Avena-Bustillos and others 2006). Light transmission of gelatin film (%). Light transmission of gelatin films from horse mackerel scale at different protein concentration of FFS is shown in Table 6. Light transmission at selected wavelength between 280 and 800 nm decreased with the increasing of FFS protein concentration. A likely explanation is that gelatin films with greater thickness would absorb the light more effectively than those with lower thickness, leading to low light transmission in selected wavelength. SDS-PAGE. SDS-PAGE of films prepared with different FFS protein concentrations showed that gelatin films contained α1 and α2 chain, as well as a β chain; furthermore, there were no differences in protein profiles among the films (data not shown). Limpisophon and others (2009) reported no differences in the protein patterns of blue shark skin gelatin films prepared with various FFS protein concentrations. Increasing in the protein concentration of FFS lead to the increase in TS and EAB of the film. However, WVP of the film also increased at higher FFS protein concentration. Increasing of WVP is disadvantage to apply gelatin film as a packaging material in food industry. From the above, it is concluded that gelatin film can be prepared from horse mackerel scale, and that its mechanical properties (TS and EAB) can be modified according to the intended purpose. However, further investigation is needed, especially to control WVP of the film to apply in food industry. E740 Journal of Food Science r Vol. 80, Nr. 4, 2015
Conclusion The properties of horse mackerel scale gelatin film were affected by gelatin extraction temperature and time, as well as FFS protein concentration. An extraction temperature higher than 70 °C and an extraction time longer than 1 h resulted in a deterioration of mechanical properties and an increase in WVP of the gelatin film. Gelatin films from horse mackerel scale exhibited lower WVP values than gelatin films from the skin of other fishes or mammals do. Thus, it was demonstrated that film can be produced from horse mackerel scale gelatin extracted at 70 °C for 1 h, and that the film properties can be modified according to the intended purpose as packaging material in food industry. However, further investigation is needed to extend the application range in food industry.
Acknowledgment This work was supported by JSPS KAKENHI Grant Number 25450302.
Author Contributions K. Osako designed the study and interpreted the results. H. Maki and K. Takahashi analyzed amino acid composition of gelatin. E. Okazaki gave some advices on discussion in this study. T. Le collected test data and drafted the manuscript.
Conflict of Interest Statement None of the authors have a conflict of interest.
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Fang Y, Tung MA, Britt IJ, Yada S, Dalgleish DG. 2002. Tensile and barrier properties of edible films made from whey proteins. J Food Sci 67(1):188–93. doi: 10.1111/j.13652621.2002.11381. G´omez-Guill´en MC, Ihl M, Bifani V, Silva A, Montero P. 2007. Edible films made from tunafish gelatin with antioxidant extracts of two different murta ecotypes leaves (Ugni molinae Turcz). Food Hydrocolloids 21(7):1133–43. doi: 10.1016/j.foodhyd.2006.08.006. G´omez-Guill´en MC, P´erez-Mateos M, G´omez-Estaca J, L´opez-Caballero E, Gim´enez B, Montero P. 2009. Fish gelatin: a renewable material for developing active biodegradable films. Trends Food Sci Tech 20:3–16. doi: 10.1016/j.tifs.20068.10.002. G´omez-Guill´en MC, Gim´enez B, L´opez-Caballero ME, Montero MP. 2011. Functional and bioactive properties of collagen and gelatin from alternative sources: a review. Food Hydrocolloids 25:1813–27. doi: 10.1016/j.foodhyd.2011.02.007. Gontard N, Guilbert S, Cuq JL. 1992. Edible wheat gluten films: influence of the main process variables on film properties using response surface methodology. J Food Sci 57(1):190–5. doi: 10.1111/j.1365-2621.1992.05453. Hoque MS, Benjakul S, Prodpran T. 2011. Effects of partial hydrolysis and plasticier content on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin. Food Hydrocolloids 25:82–90. doi: 10.1016/j.foodhyd.2010.05.008. Irwandi J, Faridayanti S, Mohamed ESM, Hamzah MS, Torla HH, Che Man YB. 2009. Extraction and characterization of gelatin from different marine fish species in Malaysia. Intl Food Res J 16:381–9. Johnston-Banks FA. 1990. Gelatin. In: P. Harris, editor. Food gel. London: Elsevier Applied Science Publisher. p 233–89. Jongjareonrak A, Benjakul S, Visessanguan W, Prodpran T, Tanaka M. 2006. Characterization of edible films from skin gelatin of brownstripe red snapper and bigeye snapper. Food Hydrocolloids 20:492–501. doi: 10.1016/j.foodhyd.2005.04.007. Kavoosi, G, Dadfar, SM, Purfard, AM. 2013. Mechanical, physical, antioxidant, and antimicrobial properties of gelatin films incorporated with thymol for potential use as nano wound dressing. J Food Sci 78(2):244–50. doi: 10.1111/j.1750-3841.2013.12015. Kim D, Min SC. 2012. Trout skin gelatin-based edible film development. J Food Sci 77(9):240–6. doi: 10.1111/j.1750-3841.2012.02880. Kittiphattanabawon P, Benjakul S, Visessanguan W, Nagai T, Tanaka M. 2005. Characterisation of acid soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chem 89:363–72. doi: 10.1016/j.foodchem.2004.02.042. Kittiphattanabawon P, Benjakul S, Visessanguan W, Shahidi F. 2012. Effect of extraction temperature on functional properties and antioxidative activities of gelatin from shark skin. Food Bioprocess Technol 5:2646–54. doi: 10.1007/s11947-010-0427-0 .
Laemmli UK. 1970. Cleavage of structural protein during assembly head of bacteriophage T4. Nature 277:680–5. doi: 10.1036/226680a0. Limpisophon K, Tanaka M, Weng W, Abe S, Osako K. 2009. Characterization of gelatin films prepared from under-utilized blue shark (Prionace glauca) skin. Food Hydrocolloids 23:1993– 2000. doi: 10.1016/j.foodhyd.2009.03.147. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with Folin phenol reagent. J Biol Chem 193:256–75. McHugh TH, Avena-Bustillos R, Krochta JM. 1993. Hydrophilic edible films: modified procedure of water vapor permeability and explanation of thickness effects. J Food Sci 58(4): 899–903. doi: 10.1111/j.1365-2621.1993.09387. Muyonga JH, Cole CGB, Duodu KG. 2004. Fourier transform infrared (FTIR) spectroscopic study of acid soluble collagen and gelatin from skins and bone of young and adult Nile perch (Lates niloticus). Food Chem 86(3):325–32. doi: 10.1016/j.foodchem.2003.09.038. Nagarajan M, Benjakul S, Prodpran T, Songtipya P. 2012a. Properties of film from splendid squid (Loligo formosana) skin gelatin with various extraction temperatures. Intl J Biol Macromol 51:489–96. doi: 10.1016/j.ijbiomac.2012.06.017. Nagarajan M, Benjakul S, Prodpran T, Songtipya P, Kishimura H. 2012b. Characteristics and functional properties of gelatin from splendid squid (Loligo formosana) skin as affected by extraction temperatures. Food Hydrocolloid 29:389–97. doi: 10.1016/j.foodhyd.2012.04.001. Nikoo M, Xu X, Benjakul S, Xu G, Ramirez-Suarez JC, Ehsani A, Kasankala LM, Duan X, Abbas S. 2011. Characterization of gelatin from the skin of farmed Amur sturgeon Acipenser schrenckii. Int Aquat Res 3:135–44. Tongnuanchan P, Benjakul S, Prodpran T, Songtipya P. 2011. Characteristics of film based on protein isolate from red tilapia muscle with negligible yellow discoloration. Intl J Biol Macromol 48(5):758–67. doi: 10.1016/j.ijbiomac.2011.02.017. Wangtueai S, Noomhorm A. 2009. Processing optimization and characterization of gelatin from lizardfish (Saurida spp.) scales. LWT – Food Sci Technol 42:825–34. doi: 10.1016/j.lwt.2008.11.014. Wangtueai S, Noomhorm A, Regenstein JM. 2010. Effect of microbial transglutaminase on gel properties and film characteristics of gelatin from lizardfish (Saurida spp.) scales. J Food Sci 75:731–9. doi: 10.1111/j.1750-3841.2010.01835. Ward AG, Courts A. 1977. The science and technology of gelatin. New York: Academic Press. Yamanaka H, Tanaka M. 2007. Suisanbutsu no riyou. 2nd ed. Tokyo: Seizando shoten (in Japanese). p 18–28. Zhang F, Xu S, Wang Z. 2011. Pre-treatment optimization and properties of gelatin from freshwater fish scales. Food Bioproducts Process 89:185–93. doi: 10.1016/j.fbp.2010.05.003.
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Properties of horse mackerel scale film. . .
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Abstract: Optimal conditions for extracting gelatin and preparing gelatin film from horse mackerel scale, such as extraction temperature and time, as well as the protein concentration of film-forming solutions were investigated. Yields of extracted gelatin at 70 °C, 80 °C, and 90 °C for 15 min to 3 h were 1.08% to 3.45%, depending on the extraction conditions. Among the various extraction times and temperatures, the film from gelatin extracted at 70 °C for 1 h showed the highest tensile strength and elongation at break. Horse mackerel scale gelatin film showed the greatly low water vapor permeability (WVP) compared with mammalian or fish gelatin films, maybe due to its containing a slightly higher level of hydrophobic amino acids (total 653 residues per 1000 residues) than that of mammalian, cold-water fish and warm-water fish gelatins. Gelatin films from different preparation conditions showed excellent UV barrier properties at wavelength of 200 nm, although the films were transparent at visible wavelength. As a consequence, it can be suggested that gelatin film from horse mackerel scale extracted at 70 °C for 1 h can be applied to food packaging material due to its lowest WVP value and excellent UV barrier properties. Keywords: gelatin film, horse mackerel scale, mechanical properties, extraction temperature, extraction time
In this study, gelatin film from horse mackerel scale at various preparation conditions could be successfully prepared. Horse mackerel scale gelatin films exhibited lower water vapor permeability values than gelatin films from the skin of other fishes or mammals do. Utilization of horse mackerel scale as a raw material of edible film could be solved the environmental pollution by seafood waste during fish processing.
Practical Application:
Introduction Gelatin is a protein derived by partial hydrolysis of collagen extracted from skin, bones, connective tissues, organs, and some intestines of mammalian species (Johnston-Banks 1990; Kavoosi and others 2013). Nevertheless, concerns regarding bovine and porcine health problems in recent years lead to increased attention on fish gelatin (G´omez-Guill´en and others 2011). Gelatin has wide application in the food processing industry, such as a gelling agent in cooking, a stabilizer, thickener, or texturizer in foods (Irwandi and others 2009; G´omez-Guill´en and others 2011). Another important application of fish gelatin is in the production of film. Fish gelatin film, which is biodegradable, is considered a vital eco-friendly packaging material for reducing the environmental impact of synthetic plastic materials (Nagarajan and others 2012a). Furthermore, fish gelatin films used as a foodstuff covering have the capacity to protect food against drying, light, and oxygen (G´omez-Guill´en and others 2009). Horse mackerel (Trachurus japonicus) is one of the most important marine fishes in Japan, with an annual catch of 133915 tonnes in 2012 (Production figures of fishery and aquaculture industry, Ministry of Agriculture, Forestry and Fisheries, 2012). This fish is used as a raw material of frozen surimi (Yamanaka and Tanaka 2007) and in the preparation of raw fresh sashimi and sushi, which are popular foods in Japan (Yamanaka and Tanaka 2007). However, during fish processing, a large amount of byproduct (accounting for approximately 50% to 70% of fish weight, and consisting of
skin, scales, and bone) is produced and discarded, resulting in serious environmental problems (Kittiphattanabawon and others 2005). Utilization of marine byproducts, including scales, as raw materials in gelatin extraction represents a potential strategy to reduce the environmental pollution caused by marine waste, as well as increasing the value of the fish product. Fish gelatin films from brownstripe red snapper and bigeye snapper skin (Jongjareonrak and others 2006), blue shark skin (Limpisophon and others 2009), Nile perch skin (Muyonga and others 2004), halibut skin (Carvalho and others 2008), tuna skin (G´omez-Guill´en and others 2007), trout skin (Kim and Min 2012), and cuttlefish (Hoque and others 2011) have been characterized. However, little information is available regarding the characteristics of gelatin films from fish scale, with the exception of lizard fish scale (Wangtueai and others 2010). Thus, the purpose of this study was to develop edible film by investigating the properties of gelatin film from horse mackerel (T. japonicus) scale at different preparation conditions, that is, extraction temperature and time, of gelatin from scale, and the protein concentration of film-forming solutions (FFS).
Materials and Methods
Characterization of horse mackerel scale gelatin Fish scale preparation. Horse mackerel scale was collected in Nagasaki Prefecture, Japan. The scale was collected and transported to our laboratory under ice. The fish scale was removed of impurities manually, washed with chilled water, and then kept in MS 20141299 Submitted 7/27/2014, Accepted 1/12/2015. Authors are with plastic bags and stored in deep freezer at –30 ± 2 °C until use. Gelatin extraction. Gelatin was extracted from horse mackDept. of Food Science and Technology, Tokyo Univ. of Marine Science and Technology, Japan. Direct inquiries to author Osako (E-mail: [email protected]). erel scale following the method described by Wangtueai and Noomhorm (2009) with minor modifications. Briefly, frozen scale E734
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R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12806 Further reproduction without permission is prohibited
Properties of horse mackerel scale film. . . Table 1–Yield of gelatin from horse mackerel scale at various high-performance liquid chromatography system (LC-10A amino extraction times and temperatures. acid analysis system, Shimadzu, Kyoto, Japan) with a Shim-pack
Extraction time (h) 15 min 30 min 1 2 3
70 1.08 ± 1.37 ± 0.09ab 2.50 ± 0.54cde 2.59 ± 0.51cdef 2.78 ± 0.57def 0.21a
80 1.81 2.57 2.90 2.93 3.45
± ± ± ± ±
0.05abc 0.40cdef 0.75def 0.56def 0.99f
90 2.18 2.73 3.08 3.18 3.21
± ± ± ± ±
0.12bcd 0.39def 0.70ef 0.72ef 0.37ef
Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
was thawed and soaked with 0.1 M NaOH solution for 6 h at a 1:8 (w/v) ratio of scale/alkali solution to move out the noncollagenous protein. The NaOH solution was changed to the new solution after 3 h, and scales were soaked in cold distilled water (DIW) to obtain a neutral pH. After the removal of noncollagenous protein, the fish scales were drained using a cheesecloth, dehydrated manually, and then immersed in 10× DIW (w/v) at various temperatures (70 °C, 80 °C, and 90 °C) for various times (15 min, 30 min, 1 h, 2 h, and 3 h) with stirring to extract the fish scale gelatin. After gelatin extraction, the coarse solids were removed by filtration with two layers of cheesecloth and the liquid was centrifuged at 16400 × g for 30 min under 20 °C. The gelatin solution in the supernatant was freeze-dried (DC 401; Yamato Scientific Co., Ltd., Japan). Extraction yield. The yield of gelatin was evaluated by the method of Limpisophon and others (2009). Approximately 1 g of the noncollagenous fish scale was weighed, and then extracted in 10× DIW (w/v) as described above. The protein concentration of the supernatant was determined following the method of Lowry (Lowry and others 1951) using bovine serum albumin as a protein standard. The yield of extraction (YD) was determined by the following equation: YD (%) =
Protein concentration (mg/mL) × 10 times of DIW (mL) × 100% Fish scales weight (mg)
Electrophoretic of gelatin. Protein pattern of extracted gelatin powder from horse mackerel scale was conducted by the method of Laemmli (1970) with minor modifications. The supernatant thus collected at each extraction temperature and time was mixed with the buffer (0.5 M Tris-HCl, pH 6.8, containing 10% (w/v) sodium dodecyl sulfate (SDS) and 20% (v/v) glycerol) in the presence of 10% (v/v) mercaptoethanol. The samples (10 μL) were loaded onto a 7.5% polyacrylamide gel (AE-6000, NPU-7.5L E-PAGEL, ATTO Co., Tokyo, Japan). The gel was fixed with the solution containing 25% (v/v) methanol and 5% (v/v) acetic acid for 30 min, stained by immersing in 0.1% (w/v) Coomassie Brilliant Blue R-250 solution (30% (v/v) methanol and 10% (v/v) acetic acid) and decolored in solution of 30% (v/v) methanol and 10% (v/v) acetic acid. Molecular weight marker (Sigma Chemical Co., St. Louis, Mo., U.S.A.) ranging from 10 to 200 kDa were used as a standard. Amino acid analysis. Twenty milligrams of extracted gelatin powder from horse mackerel scale were hydrolyzed in 6 M HCl at 110 °C for 22 h in vacuo. The hydrolysate was neutralized with 6 N and 0.6 N NaOH, and filtrated with a 0.45 μm cellulose membrane filter (Toyo Roshi Kaisha Ltd., Tokyo, Japan). The amino acid concentration in filtrate was determined by using
Amino-Li column (100 mm × 6.0 mm, Shimadzu) and Shim-pack SC-30/S0504 Li precolumn (150 mm × 4.0 mm, Shimadzu). Fourier transform infrared spectroscopy (FTIR). FTIR of gelatin powder was performed using FT-IR spectrophotometer (NICOLET 200SXV, Thermo Electron Corporation, U.S.A.). FTIR spectra was obtained from discs consisted of 2 mg of gelatin powder in approximately 100 mg potassium bromide (Muyonga and others 2004). Measurement was performed at 4000–500 cm−1 at a resolution of 2 cm−1 per point at room temperature by using Omnic 6.0 software (Thermo-Nicolet, Madison, Wis., U.S.A.).
Effect of extraction temperature and time, and protein concentration of FFS on the gelatin films Preparation of gelatin films. To study the effect of extraction temperature on gelatin films, gelatin powder extracted at various temperatures (70 °C, 80 °C, and 90 °C) for 1 h were diluted in DIW at 60 °C for 30 min to adjust the protein concentration of FFS to 2% (w/v). The effect of extraction time on gelatin films was investigated by dissolving gelatin powder obtained at different extraction times (15 min, 1 h, and 3 h) at 70 °C with DIW at 60 °C for 30 min to obtain a FFS protein concentration of 2% (w/v). Glycerol was used as a plasticizer, and was added to FFS at a protein concentration of 20% (w/w) with stirring at ambient temperature for 15 min. FFS solution was removed its air bubbles by using a mixer (HM-500; Keyence Co., Tokyo, Japan). The prepared FFS (4 g) was put onto a rimmed silicone plate (50 × 50 mm) and dried in a chamber (EYELA, KCL-2000A; Tokyo Rikakikai Co., Ltd., Japan) at 25 ± 0.5 °C and 50 ± 5% relative humidity (RH) for 48 h. The films thus obtained were manually peeled off. The effect of FFS protein concentration on gelatin film from horse mackerel scale was investigated by dissolved gelatin powder extracted at 70 °C for 1 h in DIW at 60 °C for 30 min to obtain FFS with protein concentrations of 1%, 2%, and 3% (w/v), determined by using a Lowry’s method (Lowry and other 1951). Glycerol as a plasticizer was added to FFS at 20% (w/w) protein concentration and stirred at room temperature for 15 min continuously. The FFS was cast and dried as previously described. Moisture content of gelatin films. Moisture content of gelatin films was analyzed by dried sample in oven at 105 °C for 24 h following the methods of AOAC (2000). Thickness. Film thickness was determined by using a dial-type thickness gauge (Series 7300; Mitsutoyo Co., Kanagawa, Japan). Every film sample was measured its thickness at 6 random positions of the film. Mechanical properties. Tensile strength (TS) and elongation at break (EAB) of film samples were measured by using an instrument for measuring physical properties (Tensipresser TTP50BX II; Taketomo Electric Inc., Tokyo, Japan) following the ASTM standard method D 882–97 (ASTM, 1999). Sampled film (20 × 45 mm) was placed in specific tensile grip with an initial grip distance of 30 mm and a cross-head speed of 1 mm/s until the films were disrupted. TS and EAB were calculated by the following equation, respectively: TS (MPa) = EAB (%) =
Maximum load (N) Initial cross − sectional area (m2 )
Film length (mm) at rupture × 100 Initial grip length of samples (mm)
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Temperature (°C)
Properties of horse mackerel scale film. . . Water vapor permeability (WVP). WVP of films was determined following the ASTM standard test method (1983) as reported by Gontard and others (1992). Each film was hold onto a glass cup (diameter of 5 cm) containing silica gel (0% RH) with silicon vacuum grease and a plastic band. The cups were placed in a desiccator with saturated water (100% RH) and put in ventilated chamber at 30 °C. The cups with films were weighed at initial time and every 1 h during 9-h period. Each film was measured its WVP with 6 replications. WVP of gelatin film (expressed as a gm−1 s−1 Pa−1 ) was calculated by following equation (McHugh and others 1993): WVP = w × x × A−1 × t −1 × P −1
E: Food Engineering & Materials Science
where w is the weight gain (g) of the cup, x is the film thickness (m), A is the area of exposed film (m2 ), t is the time of gain (s), ࢞P is the vapor pressure gradient (Pa) between inner and outer surface of the film. Light transmission. Light transmission in ultraviolet (UV) and visible ranges of gelatin films was measured at different wavelengths from 200 to 800 nm using a UV-Visible Recording Spectrophotometer (UV-160; Shimadzu Co., Kyoto, Japan) following the method described by Fang and others (2002). Electrophoretic analysis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of gelatin films from horse mackerel scale was conducted according to the method of Limpisophon and others (2009) with some modifications. Film samples (50 mg) were mixed with 5 mL of 1% SDS solution (w/v) and shaken at ambient temperature for 16 h continuously. Supernatants obtained after centrifugation at 5000 × g for 5 min at 20 °C were subjected to SDS-PAGE as described in previous section.
Statistical analysis Data in this study were presented as mean ± standard deviation with 6 replications. An analysis of variance (ANOVA) of the data was performed by using SPSS software (SPSS 11.5 for Windows).
Results and Discussion Characterization of gelatin from horse mackerel scale Extraction yield. Gelatin yields from horse mackerel scale at various extraction times and temperatures are shown in Table 1. Yields ranged from 1.08% to 3.45%, depending on the extraction condition. The yield increased by the disruption of hydrogen bonds stabilizing collagen’s triple helical structure with increasing extraction time and temperature, which may lead to converse insoluble collagen into soluble gelatin (Benjakul and others 2009). The results were supported by Limpisophon and others (2009) and Wangtueai and Noomhorm (2009), who clarified that increasing time and temperature (from 0.5 to 5 h at 45 °C to 100 °C for blue shark skin and from 1 to 5 h at 70 °C to 90 °C for lizard fish scale) during gelatin extraction were related with increasing yield of gelatin from blue shark skin and lizard fish scale, respectively (3.95% to 5.20% and 9.10% to 10.90%, respectively). The yield of gelatin from horse mackerel scale was lower than that from blue shark skin and lizard fish scale. However, horse mackerel is one of the economic marine fish species with high amount of annual catch, which can be used as a raw material to extract gelatin. SDS-PAGE. The SDS-PAGE profile of extracted gelatin powder at different extraction temperatures and times is shown in
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Table 2–Amino acid composition of gelatin from horse mackerel scale. Amino acid Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine Hydroxyproline Proline Imino acidsa Hydrophobic amino acidsb
Residues/1000 residues Horse mackerel scale gelatin 49 ± 25 ± 33 ± 71 ± 322 ± 131 ± 21 ± 17 ± 10 ± 23 ± 2 ± 18 ± 10 ± 32 ± 7 ± 51 ± 67 ± 111 ± 178 ± 653
2 2 2 3 5 3 2 2 2 3 1 2 1 3 2 4 3 7 9
Data are expressed as mean ± standard deviation (n = 3). a Indicates total hydroxyproline and proline. b Indicates total glycine, alanine, valine, methionine, isoleucine, leucine, phenylalanine, and proline.
Figure 1. High molecular weight protein was observed in gelatin powder from horse mackerel scales, consisting of α and β chains. However, the degradation of α and β chains was observed at extraction temperatures higher than 70 °C and extraction times longer than 1 h. Limpisophon and others (2009) observed the degradation of α and β chains in the protein pattern of blue shark skin gelatin at extraction temperatures higher than 80 °C. The differences in raw materials (scale and skin) can explain the observed differences in the temperature of protein degradation between horse mackerel scale gelatin (70 °C) and blue shark skin gelatin (80 °C). Amino acid analysis. The amino acid composition from horse mackerel scale gelatin powder extracted at 70 °C for 1 h is presented in Table 2. Glycine, the dominant amino acid in horse mackerel scale gelatin, accounted for 30% of total amino acids. Imino acid contents (proline and hydroxyproline) of horse mackerel scale gelatin (178 residues per 1000 residues) were greater than those from grass carp scales (94 residues per 1000 residues; Zhang and others 2011) but lower than those of lizardfish scale gelatin (205 residues per 1000 residues; Wangtueai and Noomhorm 2009) and mammalian gelatin (216 to 225 residues per 1000 residues; Avena-Bustillos and others 2006). Imino acids have a significant function in the triple helical structure of gelatin and affect the gel strength of gelatin gel (Ward and Courts 1977; Zhang and others 2011). Most fish gelatins have lower amino acid content than mammalian gelatins, explaining the poor gelling in comparison with mammalian gelatins. Notably, horse mackerel scale gelatin contained the same or slightly higher hydrophobic amino acids (total 653 residues per 1000 residues) compared to commercial mammalian (pork skin), commercial cold-water fish (from skin of haddock) and warm-water fish (from catfish skin) gelatins (total 651 residues, 647 residues and 649 residues per 1000 residues, respectively; Avena-Bustillos and others 2006). FTIR. Figure 2 shows the FTIR spectra of gelatin powder extracted at 70 °C, 80 °C, and 90 °C for 1 h (a, b, c) and
Properties of horse mackerel scale film. . . extracted at 70 °C, 80 °C, and 90 °C for 1 h appeared at 1650.49, 1652.69, and 1654.60 cm−1 , respectively. The amide I bands of gelatin extracted at 70 °C for 15 min, 1 h, and 3 h appeared at 1643.33, 1650.49, and 1653.85 cm−1 , respectively. In addition, amide III has been also associated with loss of triple-helix state of the molecules and transformation of a helical to random coil Figure 1–SDS-PAGE patterns of gelatin powder extracted at various temperatures (70 o C, 80 o C, and 90 o C for 1 h) and times (at 70 o C for 15 min, 30 min, 1 h, and 2 h). “M” means protein marker.
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extracted at 70 °C for 15 min, 1 and 3 h (d, e, f). Amide I band, wavenumber between 1600 and 1700 cm−1 , was the most useful in studies on the secondary structure of gelatin (Nikoo and others 2011). Amide I band arises from C = O stretching vibration of the peptidic bond in the Gly-X-Y tripeptide sequence with in-plane NH bending. Amide I bands of gelatin from horse mackerel scale
Figure 2–FTIR spectra of gelatin extracted at various temperature and time.
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Properties of horse mackerel scale film. . . Table 3–Effect of gelatin-extraction temperature and time on thickness, tensile strength (TS), elongation at break (EAB), and water vapor permeability (WVP) of gelatin film. TS (MPa)
EAB (%)
WVP (×10-10 gm-1 Pa-1 s-1 )
± 0.52c ± 0.36b ± 0.34a
36.48 ± 3.66c 19.95 ± 1.46b 13.14 ± 1.66a
46.09 ± 7.44c 25.56 ± 2.73b 19.74 ± 4.25a
0.98 ± 0.07a 1.07 ± 0.08ab 1.11 ± 0.07b
± 0.56b ± 0.52c ± 0.4a
35.62 ± 2.44bc 36.48 ± 3.66b 28.84 ± 2.19a
43.04 ± 6.78bc 46.09 ± 7.44b 33.32 ± 8.79a
1.01 ± 0.02a 0.98 ± 0.07a 1.14 ± 0.07b
Thickness (µm) Extraction temperature (for 1 h) 70 °C 23.58 80 °C 22.44 90 °C 21.57 Extraction time (at 70 °C) 15 min 22.75 1h 23.58 3h 22.05
Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
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structure due to denaturation of collagen to gelatin (Muyonga and others 1993). The amide III bands could be observed at 1238.69, 1240.67, and 1242.69 cm−1 with extraction temperature of 70 °C, 80 °C, and 90 °C for 1 h, respectively. With gelatin extracted at 70 °C for 15 min, 1 h, and 3 h, amide III bands appeared at 1235.71, 1238.69, and 1241.98 cm−1 , respectively. The increase in frequency shift of amide I and amide III region with increasing extraction temperate and time is responsible for the denaturation of α-helix in amide I and III by uncoupling of intermolecular cross-links and interruption of intramolecular bonding when increasing extraction temperature or time during gelatin extracted (Kittiphattanabawon and others 2012). From the results, we can conclude that characterization of gelatin can be affected by extraction conditions. The increasing of extraction temperature and time led to increased extraction yields. However, the triple helix structure of gelatin powder can be destroyed with increasing temperature and time and it may affect physical and mechanical properties of the gelatin film. The effects of extraction condition on the gelatin film were studied as next step.
Effect of extraction time and temperature on properties of gelatin film from horse mackerel scale Moisture content of gelatin films. The moisture content of gelatin films ranged from 17.39% to 17.49%, depending on the preparation conditions of films. However, no statistical differences were found among the films. Thickness. The thickness of films prepared from horse mackerel scale gelatin at various extraction conditions differed (Table 3). An extraction temperature greater than 70 °C and an extraction time longer than 1 h resulted in a slightly decreased film thickness. A possible reason may be that increases in extraction temperature and time led to an increased degree of gelatin hydrolysis. Hoque and others (2011) observed a slight decrease in thickness in gelatin films with a higher degree of hydrolysis. This result is in agreement with Nagarajan and others (2012a), who reported that the thickness of film made from splendid squid (Loligo formosana) gelatin decreased with increasing extraction temperature. Mechanical properties. TS and EAB of gelatin films at various extraction temperatures and times are presented in Table 3. Under identical extraction time (1 h), the gelatin film extracted at 70 °C had the highest TS (36.48 ± 3.66 MPa) and EAB (46.09 ± 7.44%) values when compared with gelatin films extracted at higher temperatures (80 °C and 90 °C); which were 19.95 ± 1.46 MPa and 13.14 ± 1.66 MPa, respectively, for TS, and 25.56 ± 2.73% and 19.74 ± 4.25%, respectively, for EAB. At an extraction temperature of 70 °C, the TS and EAB values of gelatin film extracted for 15 min (35.62 ± 2.44 MPa and 43.04 ± 6.78%, E738 Journal of Food Science r Vol. 80, Nr. 4, 2015
respectively) did not significantly differ from those for 1 h extraction (36.48 ± 3.66 MPa and 46.09 ± 7.44%, respectively). However, both values decreased to 28.84 ± 2.19 MPa and 33.32 ± 8.79%, respectively, when heated for 3 h. Thus, TS and EAB of gelatin film decreased with increasing extraction temperature and time. It is known that the mechanical properties of gelatin film depend on the triple helical structure of gelatin. Hoque and others (2011) reported that gelatin films are mainly stabilized by weak forces, including hydrogen bonding and hydrophobic interaction. These weak bonds are likely destroyed by the increasing extraction temperature and time, leading to the disruption of gelatin’s triple helical structure and the formation of shorter peptide chains (Nagarajan and others 2012b), and resulting in the alteration of TS and EAB of gelatin films. WVP of gelatin film. The WVP of gelatin films extracted under various conditions is shown in Table 3. Under identical extraction time (1 h), WVP increased with increasing extraction temperature; the gelatin film extracted at 70 °C showed the lowest WVP value (0.98 ± 0.07 × 10−10 gm−1 Pa−1 s−1 ). Increase in extraction time from 15 min to 3 h at 70 °C resulted in increased WVP value (1.01 ± 0.02 × 10−10 gm−1 Pa−1 s−1 to 1.14 ± 0.07 × 10−10 gm−1 Pa−1 s−1 ). WVP appears to be determined by the strength of protein molecule interactions within the network of the film (Tongnuanchan and others 2011). The triple helical structure of gelatin may be broken down to small peptide chains with increased extraction time and temperature. These small peptide chains can be easily inserted into the protein network, leading to a decrease in density of intermolecular interactions between gelatin chains, and a subsequent increase in the WVP of gelatin films. The slightly higher hydrophobic amino acids content may be the reason for low WVP of the films, when compared to films produced from other fish or land-based animal gelatins (Limpisophon and others 2009). Light transmission of gelatin film (%). UV and visible light transmission of films from horse mackerel scale gelatin prepared at various temperatures and times is presented in Table 4. Gelatin films extracted at various temperatures (70 °C to 90 °C) for 1 h showed transmission in the visible range (350 to 800 nm) and UV range (200 to 280 nm) from 23.68 ± 1.33% to 75.77 ± 2.03% and from 12.15 ± 0.61% to 16.35 ± 2.51%, respectively. Transmission in the visible range and UV range of gelatin film extracted at 70 °C for different times was from 29.03 ± 1.91% to 79.08 ± 6.24% and from 15.97 ± 2.83% to 20.92 ± 0.57%, respectively. However, at the wavelength of 200 nm, the transmission of these films could not be measured, regardless of extraction temperature or time. A possible explanation is that the amino acid composition of gelatin consists of aromatic amino acids with a phenol ring, such as phenylalanine, tryptophan, and tyrosine, could prevent UV
Properties of horse mackerel scale film. . . Table 4–Effect of gelatin-extraction temperature and time on light transmission (%) of gelatin films. Light transmission at different wavelength (nm) 200 Extraction temperature (for 1 h) 70 °C 0.3 80 °C 0.3 90 °C 0.3 Extraction time (at 70 °C) 15 min 0.3 1h 0.3 3h 0.3
280
350
400
500
600
800
16.35 ± 2.51a 13.68 ± 0.55b 12.15 ± 0.61b
30.42 ± 2.78a 27.30 ± 2.73b 23.68 ± 1.33c
52.70 ± 1.14a 45.17 ± 1.74b 42.20 ± 0.99c
62.60 ± 1.59a 59.18 ± 0.62b 49.82 ± 1.33c
69.43 ± 1.12a 63.58 ± 0.41b 57.85 ± 0.91c
75.77 ± 2.03a 68.55 ± 1.74b 64.25 ± 2.04c
20.92 ± 0.57a 16.35 ± 2.51b 15.97 ± 2.83b
35.98 ± 1.53a 30.42 ± 2.78b 29.03 ± 1.91b
61.01 ± 2.35a 52.70 ± 1.14b 52.35 ± 0.96b
68.77 ± 3.20a 62.60 ± 1.59b 59.85 ± 3.28b
75.93 ± 7.97a 69.43 ± 1.12b 61.13 ± 2.70c
79.08 ± 6.24a 75.77 ± 2.03a 68.88 ± 4.00b
transmission, similar to that observed at brownstripe red snapper and bigeye snapper skin gelatin film (Jongjareonrak and others 2006). Thus, the gelatin film prevented UV light transmission effectively. These results suggested the potential preventive effect of gelatin films on the retardation of lipid oxidation induced by UV light. SDS-PAGE. SDS-PAGE profiles of gelatin films produced under various extraction conditions are depicted in Figure 3. From the protein pattern, the main constituents of horse mackerel gelatin film were α and β chains. Moreover, with increasing time and temperature of gelatin extracted, these main constituents were degraded, especially with an extraction temperature higher than 70 °C and an extraction time longer than 1 h. These results show that physical and mechanical properties of gelatin film can be affected by extraction conditions of gelatin. TS and EAB of films decreased with increasing extraction temperature and time. On the contrary, WVP increased with increasing temperature and time during extraction. The degradation of protein patterns of gelatin film with increasing extraction temperature and time can be observed in Figure 3 and this result is the similar tendency with SDS profile of gelatin powder extracted at different temperature and time (shown in Figure 1). The optimum
temperature and time for extracting gelatin to prepare the film having high mechanical properties (TS and EAB) was 70 °C and 1 h, respectively. Gelatin powder extracted at 70 °C for 1 h was used for next step.
Effect of protein concentration of FFS on gelatin film Mechanical properties. Mechanical properties of gelatin films at different protein concentrations are presented in Table 5. TS of gelatin film increased from 19.40 ± 4.75 MPa to 36.77 ± 4.13 MPa and EAB increased from 38.76 ± 9.77% to 48.77 ± 9.23% with increasing FFS protein concentration. The greater FFS protein content might result in increased aggregation of proteins forming the film, resulting in improved flexibility and mechanical properties of films (Jongjareonrak and others 2006). The increasing trend of TS and EAB of horse mackerel scale gelatin films was similar to gelatin films from brownstripe red snapper and bigeye snapper skin (Jongjareonrak and others 2006). WVP. The effect of various FFS protein concentrations on the WVP values of gelatin films is shown in Table 5. WVP of gelatin films increased from 0.46 ± 0.05 to 1.51 ± 0.07 × 10−10 gm−1 Pa−1 s−1 with increasing FFS protein concentration. Notably, greater WVP values were observed in gelatin films with
Figure 3–SDS-PAGE patterns of gelatin films at different extraction temperatures (70 o C, 80 o C, and 90 o C for 1 h) and times (at 70 o C for 15 min, 1 h, and 3 h). “M” means protein marker.
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Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
Properties of horse mackerel scale film. . . Table 5–Effect of FFS∗ protein concentration on thickness, tensile strength (TS), elongation at break (EAB), and water vapor permeability (WVP) of gelatin films. Protein concentration 1% 2% 3%
Thickness (µm)
TS (MPa)
EAB (%)
WVP (×10-10 gm-1 Pa-1 s-1 )
10.65 ± 0.33a 23.58 ± 0.52b 38.48 ± 0.71c
19.40 ± 4.75a 36.48 ± 3.66bc 36.77 ± 4.13b
38.76 ± 9.77a 46.09 ± 7.44ab 48.77 ± 9.23b
0.46 ± 0.05a 0.98 ± 0.07b 1.51 ± 0.07c
∗ FFS means film-forming solution. Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
Table 6–Effect of FFS∗ protein concentration on the light transmission (%) of gelatin films. Light transmission at different wavelength (nm)
E: Food Engineering & Materials Science
Protein concentration 1% 2% 3%
200
280
350
0.3 0.3 0.3
23.18 ± 16.35 ± 2.51a 11.78 ± 1.57a
0.89b
400
34.30 ± 30.42 ± 2.78a 36.43 ± 1.91a
1.51b
500
59.40 ± 52.70 ± 1.14a 47.17 ± 0.96a
1.07b
600
69.68 ± 62.60 ± 1.58b 55.23 ± 1.79a 0.88c
800
75.13 ± 69.43 ± 2.12a 78.63 ± 1.24c
0.82b
80.62 ± 0.87b 75.77 ± 2.03a 79.98 ± 1.43b
∗ FFS means film-forming solution. Data are expressed as mean ± standard deviation (n = 6). Different superscripts in the same column indicate statistical differences (P < 0.05).
increased thickness. WVP values of gelatin films from horse mackerel scale (0.98 ± 0.07 × 10−10 gm−1 Pa−1 s−1 ) were lower than gelatin films from brownstripe red snapper and bigeye snapper skin (1.22 ± 0.11 × 10−10 gm−1 Pa−1 s−1 and 1.33 ± 0.04 × 10−10 gm−1 Pa−1 s−1 , respectively; Jongjareonrak and others 2006) at identical FFS protein concentration (2%). We cannot find critically reason for the lower WVP values of horse mackerel gelatin film when compare with others gelatin film at similar protein content in FFS. However, one reason may be explained by the slightly higher amount of hydrophobic amino acids. The gelatin film of the present study contained a slightly higher amount of hydrophobic amino acids compared with commercial mammalian, cold-water fish and warm-water fish gelatins (Avena-Bustillos and others 2006). Light transmission of gelatin film (%). Light transmission of gelatin films from horse mackerel scale at different protein concentration of FFS is shown in Table 6. Light transmission at selected wavelength between 280 and 800 nm decreased with the increasing of FFS protein concentration. A likely explanation is that gelatin films with greater thickness would absorb the light more effectively than those with lower thickness, leading to low light transmission in selected wavelength. SDS-PAGE. SDS-PAGE of films prepared with different FFS protein concentrations showed that gelatin films contained α1 and α2 chain, as well as a β chain; furthermore, there were no differences in protein profiles among the films (data not shown). Limpisophon and others (2009) reported no differences in the protein patterns of blue shark skin gelatin films prepared with various FFS protein concentrations. Increasing in the protein concentration of FFS lead to the increase in TS and EAB of the film. However, WVP of the film also increased at higher FFS protein concentration. Increasing of WVP is disadvantage to apply gelatin film as a packaging material in food industry. From the above, it is concluded that gelatin film can be prepared from horse mackerel scale, and that its mechanical properties (TS and EAB) can be modified according to the intended purpose. However, further investigation is needed, especially to control WVP of the film to apply in food industry. E740 Journal of Food Science r Vol. 80, Nr. 4, 2015
Conclusion The properties of horse mackerel scale gelatin film were affected by gelatin extraction temperature and time, as well as FFS protein concentration. An extraction temperature higher than 70 °C and an extraction time longer than 1 h resulted in a deterioration of mechanical properties and an increase in WVP of the gelatin film. Gelatin films from horse mackerel scale exhibited lower WVP values than gelatin films from the skin of other fishes or mammals do. Thus, it was demonstrated that film can be produced from horse mackerel scale gelatin extracted at 70 °C for 1 h, and that the film properties can be modified according to the intended purpose as packaging material in food industry. However, further investigation is needed to extend the application range in food industry.
Acknowledgment This work was supported by JSPS KAKENHI Grant Number 25450302.
Author Contributions K. Osako designed the study and interpreted the results. H. Maki and K. Takahashi analyzed amino acid composition of gelatin. E. Okazaki gave some advices on discussion in this study. T. Le collected test data and drafted the manuscript.
Conflict of Interest Statement None of the authors have a conflict of interest.
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Properties of horse mackerel scale film. . .
