PaPer 366 Ital. J. Food Sci., vol. 27 - 2015 - Keywords: gelatin, gel strength, extraction, temperature, swim bladder, yellowfin tuna - CharaCteristiCs of gelatin from swim bladder of yellowfin tuna (Thunnus albacores) as influenCed by extraCting temperatures o. Kaewdang1, s. benjaKul1*, t. prodpran2, t. Kaewmanee3 and h. Kishimura4 1Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 2Department of Material Product Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 3Department of Food Science and Nutrition, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand 4Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan *Corresponding author: Tel. 66 7428 6334, Fax 66 7455 8866 email: soottawat.b@psu.ac.th AbstrAct Gelatin was extracted from the swim bladder of yellowfin tuna (Thunnus albacores) at differ- ent temperatures (60, 70 and 80°c) with the extraction yields of 35.6%, 41.1% and 47.3% (dry weight basis), respectively. the α-chains of gelatin decreased with increasing extraction tempera- tures. similar amino acid compositions were noticeable among all gelatins, in which glycine con- stituted the major amino acid. Imino acids ranged from 169 to 172 residues/1,000 residues. the gel strength of gelatin extracted at lower temperature was higher than that of gelatins extracted at higher temperatures. Gelling and melting temperatures for swim bladder gelatin were 11.07- 15.24 and 20.36-22.33°c, respectively. Higher gelling and melting points were observed for gel- atin extracted at lower temperatures. Microstructure of gel of gelatin extracted at 60°c was finer with smaller voids, compared with others. FtIr spectra of obtained gelatins revealed the signifi- cant loss of molecular order of the triple-helix. thus, extraction temperatures showed the direct impact on characteristics of gelatin from swim bladder. mailto:soottawat.b@psu.ac.th Ital. J. Food Sci., vol. 27 - 2015 367 IntroductIon Gelatin is a fibrous protein obtained by par- tial denaturation or hydrolysis of collagen. Gela- tin represents biopolymer with many applications in food, materials (for edible and biodegradable packaging), cosmetic, pharmaceutical and pho- tographic industries (JELLouLI et al., 2011). the source, type of collagen and processing conditions have the influence on the properties of the result- ing gelatin (KIttIPHAttAnAbAWon et al., 2010). different types of gelatins have varying thermal and rheological properties such as gel strength, melting and gelling temperatures (bEnJAKuL et al., 2012). these properties are governed by sev- eral factors such as chain length or molecular weight distribution, amino acid composition and hydrophobicity, etc. (GÓMEZ-GuILLÉn et al., 2002; norZIAH et al., 2009). commercial gelatins are produced mainly from porcine and bovine skins and bones by alkaline or acidic extraction (bEnJAKuL et al., 2009). Howev- er, both Judaism and Islam forbid the consump- tion of any pork-related products, while Hindus do not consume cow-related products. Addition- ally, bovine gelatin has a high risk for bovine spongiform encephalopathy (nAGArAJAn et al., 2012). Furthermore, the need to exploit the fish processing byproducts has led to the production of gelatin as an alternative to mammalian coun- terpart (GÓMEZ-GuILLÉn et al., 2011). Fish gela- tin has been extracted mainly from fish skin such as seabass (sIntHusAMrAn et al., 2014), cobia (sILVA et al., 2014) skipjack tuna, dog shark and rohu (sHYnI et al., 2014) and unicorn leatherjack- et (KAEWruAnG et al., 2013), etc. Among fish processing industries, canned tuna industry is economically important. tuna includ- ing yellowfin, skipjack and tongol have been the important species for canning in thailand with a large volume of raw materials used. Approximate- ly two-thirds of the whole fish are utilized and the remainings involving the viscera, head, bone and swim bladder become the byproducts (KLoMKLAo et al., 2004). Fish swim bladders can be used for production of “isinglass” (WEbEr et al., 2009). re- cently, KAEWdAnG et al., (2014) reported that alka- line pretreatment was essential for gelatin extrac- tion from yellowfin tuna swim bladder. However, no information on the effect of extracting temper- ature on characteristics and properties of gelatin has been reported. therefore, the objectives of this investigation were to extract and characterize gel- atin from the swim bladder of yellowfin tuna us- ing different extraction temperatures. 2. MAtErIALs And MEtHods 2.1. Chemicals All chemicals were of analytical grade. sodium dodecyl sulphate (sds), coomassie blue r-250 and N,N,N’,N’-tetramethylethylenediamine (tE- MEd) were procured from bio-rad Laborato- ries (Hercules, cA, usA). High-molecular-weight markers were purchased from GE Healthcare uK Limited (buckinghamshire, uK). Food grade bovine bone gelatin with the bloom strength of 150-250 g was obtained from Halagel (thailand) co., Ltd., (bangkok, thailand). 2.2. Collection and preparation of swim blad- der swim bladders of yellowfin tuna (Thunnus al- bacares) were obtained from tropical canning Public co., Ltd., songkhla, thailand. swim blad- ders with the length of 8-12 cm were placed in polyethylene bags, inserted in ice using a sam- ple/ice ratio of 1:2 (w/w) and transported to the department of Food technology, Prince of songkla university, songkhla. upon arrival, swim bladders were washed with distilled water and cut into pieces with the length of approxi- mately 2 cm. the prepared samples were then placed in polyethylene bag and frozen at -20°c. the samples were stored at -20°c until used. the storage time was not longer than 3 months. Prior to extraction, frozen swim bladders were thawed using running water until the tempera- ture was 0-2°c 2.3. Extraction of gelatin from swim bladder Prior to gelatin extraction, swim bladders were pretreated with alkaline solution as per the method of KAEWdAnG et al. (2014). Pre- pared swim bladders were added with the mixed alkaline solution (na 2 co 3 :naoH; 7:3) having the concentration of 4% (w/v) at a ratio of 1:10 (w/v). the mixture was stirred for 12 h at room temperature (28-30°c) using an overhead stir- rer model W20.n (IKA®-Werke GmbH & co.KG, stanfen, Germany). the alkaline solution was changed every 6 h. the residues were washed with tap water until a neutral or faintly basic pH was obtained. to extract gelatin, alkali pretreated samples were soaked in distilled water with different temperatures (60, 70 and 80°c) using a swim bladder/water ratio of 1:5 (w/v) in a tempera- ture-controlled water bath (W350, Memmert, schwabach, Germany) for 24 h with a contin- uous stirring at a speed of 150 rpm. the mix- tures were then filtered using a buchner fun- nel with a Whatman no. 4 filter paper (What- man International, Ltd., Maidstone, England). the filtrates were freeze-dried using a freeze- dryer (coolsafe 55, scanLaf A/s, L ynge, den- mark). the dry gelatin extracted from swim bladder from yellowfin tuna at 60, 70 and 80 °c was referred to as ‘G60’, ‘G70’ and ‘G80’, respectively. All gelatin samples were weighed, calculated for extraction yield and subjected to analyses. 368 Ital. J. Food Sci., vol. 27 - 2015 2.4. Analyses 2.4.1. Yield Gelatin yield was calculated by the following equation. Weight of dry gelatin (g) x 100 Yield (%) = ______________________________________ Weight of initial dry swim bladder (g) where the weight of dry swim bladder was cal- culated by subtracting moisture content de- termined by AoAc (2000) from the initial wet weight. 2.4.2. sds-polyacrylamide gel electrophore- sis (sds-PAGE) sds–PAGE was performed by the method of LAEMMLI (1970). samples were dissolved in 5% sds solution. the mixtures were then heated at 85°c for 1 h using a temperature controlled water bath model W350 (Memmert, schwabach, Ger- many). the mixtures were centrifuged at 8,500 g for 5 min using a microcentrifuge (MIKro20, Het- tich Zentrifugan, 170 Germany) to remove undis- solved debris. solubilized samples were mixed at 1:1 (v/v) ratio with the sample buffer (0.5 M tris– Hcl, pH 6.8, containing 5% sds and 20% glycer- ol). samples were loaded onto a polyacrylamide gel made of 7.5% separating gel and 4% stack- ing gel and subjected to electrophoresis at a con- stant current of 20 mA/gel. After electrophoresis, the gels were stained with 0.05% (w/v) coomas- sie blue r-250 in 50% (v/v) methanol and 7.5% (v/v) acetic acid for 30 min. Finally, they were destained with a mixture of 50% (v/v) metha- nol and 7.5% (v/v) acetic acid for 30 min and destained again with a mixture of 5% (v/v) meth- anol and 7.5% (v/v) acetic acid for 1 h. High-mo- lecular-weight protein markers were used to es- timate the molecular weight of proteins. 2.4.3. Amino acid analysis Amino acid composition of gelatin samples was analyzed according to the method of nAGA- rAJAn et al. (2012) with a slight modification. the samples were hydrolyzed under reduced pressure in 4 M methanesulphonic acid contain- ing 0.2% (v/v) 3-2(2-aminoethyl) indole at 115 °c for 24 h. the hydrolysates were neutralized with 3.5 M naoH and diluted with 0.2 M citrate buffer (pH 2.2). An aliquot of 0.04 ml was ap- plied to an amino acid analyzer (MLc-703; Atto co., tokyo, Japan). 2.4.4. Fourier transform infrared (FtIr) spec- troscopic analysis FtIr spectra of the gelatin samples were ob- tained using a FtIr spectrometer (EQuInoX 55, bruker, Ettlingen, Germany) equipped with a deuterated L-alanine tri-glycine sulphate (dLAtGs) detector. A horizontal attenuated to- tal reflectance accessory (HAtr) was mounted into the sample compartment. the internal re- flection crystal (Pike technologies, Madison, WI, usA), made of zinc selenide, had a 45° angle of incidence to the Ir beam. spectra were acquired at a resolution of 4 cm-1 and the measurement range was between 400 and 4,000 cm-1 (mid- Ir region) at room temperature. Automatic sig- nals were collected in 32 scans at a resolution of 4 cm-1 and were ratioed against a background spectrum recorded from the clean empty cell at 25°c. Analysis of spectral data was carried out using the oPus 3.0 data collection software pro- gramme (bruker, Ettlingen, Germany). 2.4.5. determination of gel strength Gelatin gel was prepared by the method of KIt- tIPHAttAnAbAWon et al. (2010). Gelatin was dis- solved in distilled water (60 °c) to obtain a final concentration of 6.67% (w/v). the solution was stirred until the gelatin was completely solubi- lized and then transferred to a cylindrical mold with 3 cm diameter and 2.5 cm height. the so- lution was incubated at the refrigerated temper- ature (4°c) for 18 h prior to analysis. the gel strength was determined at 8-10°c using a texture analyzer (stable Micro system, surrey, uK) with a load cell of 5 kg and cross- head speed of 1 mm/s. A 1.27 cm diameter flat- faced cylindrical teflon® plunger was used. the maximum force (grams), taken when the plung- er had penetrated 4 mm into the gelatin gels, was recorded. 2.4.6. determination of gelling and melting temperatures Gelling and melting temperatures of gela- tin samples were measured following the meth- od of borAn et al. (2010) using a controlled stress rheometer (rheostress rs 75, HAAKE, Karlsruhe, Germany). the gelatin solution (6.67%, w/v) was prepared in the same manner as described previously. the solution was pre- heated at 35°c for 30 min. the measuring ge- ometry included a 3.5 cm parallel plate and the gap was set at 1.0 mm. the measurement was performed at a scan rate of 0.5°c/min, frequen- cy of 1 Hz, oscillating applied stress of 3 Pa dur- ing cooling from 35 to 5°c and heating from 5 to 35°c. the gelling and melting temperatures were calculated, where tan δ became 1 or δ was 45°. 2.4.7. Microstructure analysis of gelatin gel the microstructure of gelatin gel was visu- alized using a scanning electron microscopy (sEM). Gelatin gels having a thickness of 2-3 mm were fixed with 2.5% (v/v) glutaraldehyde in 0.2 Ital. J. Food Sci., vol. 27 - 2015 369 M phosphate buffer (pH 7.2) for 12 h. the sam- ples were then rinsed with distilled water for 1 h and dehydrated in ethanol with a serial con- centration of 25%, 50%, 70%, 80%, 90% and 100% (v/v). dried samples were mounted on a bronze stub and sputter-coated with gold (sput- ter coater sPI-Module, West chester, PA, usA). the specimens were observed with a scanning electron microscope (JEoL JsM-5800 LV, to- kyo, Japan) at an acceleration voltage of 20 kV. 2.4.8. determination of color of gelatin gel the color of gelatin gels (6.67% w/v) was measured with a Hunter lab colorimeter (color Flex, Hunter Lab Inc., reston, VA, usA). L*, a* and b* values indicating lightness/brightness, redness/greenness and yellowness/blueness, respectively, were recorded. the colorimeter was warmed for 10 min and calibrated with a white standard. the total difference in color (ΔE*) was calculated according to the following equation. (GEnnAdIos et al., 1996): where ΔL*, Δa* and Δb* are the differences be- tween the corresponding color parameter of the sample and that of the white standard (L* = 93.6, a*= -0.94 and b* = 0.40). 2.5. Statistical analysis All experiments were run in triplicate, using three different lots of samples. data were sub- jected to analysis of variance (AnoVA) and mean comparisons were carried out using a duncan’s multiple range test (stEEL and torrIE, 1980). statistical analysis was performed using the sta- tistical Package for social sciences (sPss for windows: sPss Inc., chicago, IL, usA). 3. rEsuLts And dIscussIon 3.1. Extraction yield Yield of gelatin from the swim bladder of yel- lowfin tuna extracted at various temperatures was different. Increasing yield was obtained when the extraction temperatures increased (P < 0.05). Yield of 35.6%, 41.1% and 47.3% (on dry weight basis) was found for G60, G70, and G80, respectively. this result was in agreement with KAEWruAnG et al. (2013), duAn et al. (2011) and KIttIPHAttAnAbAWon et al. (2010) who re- ported the increasing yield of gelatin as the ex- traction temperature increased with higher tem- peratures, the bondings stabilizing α-chains in the native mother collagen were destroyed to a higher extant. As a consequence, the triple he- lix structure became amorphous and could be extracted into the medium with ease, leading to the higher yield (sIntHusAMrAn et al., 2014). In addition, the higher energy applied could induce thermal hydrolysis of peptide chains, resulting in the formation of shorter peptides. As a result, those small peptides could be easily extracted into water. the yield and characteristics of gel- atin are associated with the type of raw materi- al and gelatin extraction process, including the pretreatment process and extraction tempera- tures. (nAGArAJAn et al., 2012; KIttIPHAttAn- AbAWon et al., 2010; MontEro and GÓMEZ- GuILLÉn, 2000). 3.2. Protein patterns Protein patterns of gelatin from the swim blad- der of yellowfin tuna extracted at different tem- peratures are shown in Fig. 1. the band inten- sity of α 1 -chain and α 2 -chain decreased with in- creasing extraction temperature. the decreas- es in α 1 -chain band intensity were observed in G70 and G80, in comparison with that found in G60. Among all gelatin samples, G80 possessed the lowest α-chain band intensity. this might be caused by the degradation induced by the ther- mal process. therefore, the extraction tempera- tures played a major role in protein components of resulting gelatin. KIttIPHAttAnAbAWon et al. (2010) reported that the gelatins extracted from the skins of brownbanded bamboo shark and blacktip shark with higher extraction tempera- ture contained more peptides with the MW less than α-chain and the lower proportion of high MW (greater than β-chain) fractions, compared with those obtained from lower temperature ex- traction. Gelatins from splendid squid skin with higher extraction temperatures contained a low- er band intensity of the α-chains than those ob- tained with lower extraction temperature (nAGA- Fig. 1 - Protein patterns of gelatins from the swim blad- der of yellowfin tuna extracted at different temperatures. M: high molecular weight markers. G60, G70 and G80 rep- resent gelatin extracted from swim bladder at 60, 70 and 80°c, respectively. 370 Ital. J. Food Sci., vol. 27 - 2015 rAJAn et al., 2012). on the other hand, gelatin from skin of unicorn leatherjacket extracted at higher temperature (65-75°c) had α-chain re- tained at higher level than that extracted at low- er temperature (KAEWruAnG et al., 2013). this was due to the thermal inactivation of indige- nous proteases in the skin of unicorn leather- jacket at high temperature. Generally, gelatins with a higher content of α-chains showed better functional properties including gelling, emulsi- fying and foaming properties (GÓMEZ-GuILLÉn et al., 2002). In general, the formation of pep- tide fragments is associated with lower viscosi- ty, low melting point, low setting point, high set- ting time, as well as decreased bloom strength of gelatin (MuYonGA et al., 2004a). the results suggested that G70 and G80, which were ex- tracted at higher temperatures, had the shorter chains as indicated by lower content of α-chain. 3.3. Amino acid composition Amino acid compositions of gelatins from the swim bladder of yellowfin tuna extracted at dif- ferent temperatures are shown in table 1. Gly- cine was the predominant amino acid in all gel- atin samples, ranging from 305 to 314 resi- dues/1000 residues. this implied that gela- tin obtained was derived from its mother colla- gen. collagen consists of one-third glycine in its molecule (bALtI et al., 2011). It was noted that G80 had the higher glycine content than G60 and G70. the higher glycine in G80 might be caused by free glycine, which was released to a high extent during extraction at high tempera- ture. Alanine (121-122 residues/1000 residues) was found at high content. Alanine plays a role in viscoelastic property of gelatin (GIMÉnEZ et al., 2005). Low contents of cysteine (1 residues/1000 residues), tyrosine (5-6 residues/1000 residues), histidine (7-8 residues/1000 residues) and hy- droxylysine (10 residues/1000 residues) were observed in all gelatin samples. For imino acids, all gelatins contained proline and hydroxypro- line contents of 95–99 and 72–74 residues/1000 residues, respectively. rEGEnstEIn and ZHou (2007) reported that glycine, alanine, proline and hydroxyproline are four of the most abundant amino acids in gelatin. the properties of gelatin are largely influenced by the amino acid com- position and their molecular weight distribution (GÓMEZ-GuILLÉn et al., 2009). When comparing the content of imino acids (proline and hydroxy- proline), gelatin from swim bladder had the lower imino acid content than those from seabass skin (198-202 residues/1000 residues) (sIntHusAM- rAn et al., 2014) and from carp skin (188-190 residues/1000 residues) (duAn et al., 2011). the imino acid content of fish collagens and gelatins correlates with the water temperature of their normal habitat (FoEGEdInG et al., 1996; rE- GEnstEIn and ZHou, 2007). It has been known that imino acid content, especially hydroxypro- line content, affected functional properties of gel- atin, especially gelling property (AEWsIrI et al., 2008; bEnJAKuL et al., 2009). therefore, amino acid composition of gelatin from swim bladder was governed by extraction temperature. 3.4. Fourier transform infrared (FTIR) spec- troscopy FtIr spectra of gelatins extracted using differ- ent temperatures are shown in Fig. 2. FtIr spec- troscopy has been used as a well-established technique to monitor the functional groups and secondary structure of gelatin (KonG and Yu, table 1 - Amino acid compositions of gelatins from the swim bladder of yellowfin tuna extracted at different temperatures. Amino acids Number of residues/1000 residues G60 G70 G80 Alanine 121 121 122 Arginine 52 52 53 Aspartic acid/asparagine 49 48 46 Cysteine 1 1 1 Glutamic acid /glutamine 80 80 78 Glycine 307 305 314 Histidine 7 8 7 Isoleucine 14 14 13 Leucine 29 30 28 Lysine 26 26 26 Hydroxylysine 10 10 10 Methionine 17 16 16 Phenylalanine 16 16 16 Hydroxyproline 74 72 73 Proline 95 99 99 Serine 41 41 40 Threonine 30 30 30 Tyrosine 6 6 5 Total 1000 1000 1000 Imino acids 169 171 172 Fig. 2 - Atr-FtIr spectra of gelatins from the swim blad- der of yellowfin tuna extracted at different temperatures (see Fig. 1 caption). Ital. J. Food Sci., vol. 27 - 2015 371 2007). the absorption bands were situated in the amide region. the absorption in the amide-I re- gion, owing to c=o stretching vibration, is prob- ably the most useful for infrared spectroscopic analysis of the secondary structure of proteins (bEnJAKuL et al., 2009). It depends on the hy- drogen bonding and the conformation of the pro- tein structure (bEnJAKuL et al., 2009; urIArtE- MontoYAEtAL et al., 2011). G60, G70 and G80 exhibited the amide-I bands at the wavenumbers of 1652.8, 1653.7 and 1652.9 cm-1, respective- ly. the characteristic absorption bands of G60, G70 and G80 in amide-II region were noticeable at the wavenumbers of 1544.6, and 1545.5 and 1543.5 cm-1, respectively. Amide-II arises from bending vibration of n-H groups and stretching vibrations of c-n groups. In addition, amide- III was detected at the wavenumbers of 1241.9, 1241.3 and 1240.8 cm-1 for G60, G70 and G80, respectively. the amide-III represents the com- bination peaks between c-n stretching vibra- tions and n-H deformation from amide linkages as well as absorptions arising from wagging vi- brations from cH 2 groups from the glycine back- bone and proline side-chains (JAcKson et al., 1995). G80 had the lowest amplitude, whereas G60 exhibited the highest amplitude at amide- III region. this indicated that the greater disor- der of molecular structure due to transforma- tion of an α-helical to a random coil structure occurred at higher temperature. these chang- es were associated with loss of triple-helix state as a result of denaturation of collagen to gela- tin (MuYonGA et al., 2004b). the result recon- firmed the higher degradation of gelatin extract- ed at higher temperatures. Amide-A band, arising from the stretching vi- brations of the n-H group, appeared at 3338.3, 3339.1 and 3339.3 cm-1 for G60, G70 and G80, respectively. Amide-A represents nH-stretch- ing coupled with hydrogen bonding. normal- ly, a free n-H stretching vibration is found in the range of 3400-3440 cm-1 (MuYonGA et al., 2004b). When the n-H of a peptide is involved in a hydrogen bond, the position shifts to lower frequencies (doYLE et al. 1975). In amide-A re- gion, the lower wavenumber was found in G60, suggesting the hydrogen bonding involvement of n-H in α-chain. on the other hand, the low- er wavenumber with the concomitantly higher amplitude of amide-A observed in G80 could be associated with the higher degradation of gel- atin and higher free amino groups. the amide b was observed at 3082.1, 3080.9 and 3081.8 cm-1 for G60, G70 and G80, respectively. Amide b corresponds to asymmetric stretch vibration of =c-H as well as –nH 3 +. thus, the secondary structure of gelatins obtained from the swim bladder of yellowfin tuna was affected to some degree by extraction temperature. 3.5. Gel strength Gel strength of gelatin from the swim bladder of yellowfin tuna extracted at different temperatures is presented in Fig 3. G60, G70 and G80 had the gel strength of 72, 64 and 51 g, respectively. the difference in gel strength between the samples could be due to the differences in intrinsic charac- teristics, especially molecular weight distribution. Protein degradation resulted in the formation of peptides with shorter chain length, which might show the lower ability to from the junction zone or anneal each other. the longer chains in G60 could undergo aggregation to form gel network more effectively than G70 and G80. As a result, a stronger gel network could be formed as indi- cated by the higher gel strength. bloom strength of commercial gelatins ranges from 100 to 300, but gelatins with bloom values of 250-260 are Fig. 3 - Gel strength of gelatin from the swim bladder of yel- lowfin tuna with different temperatures. different upper- case letters on the bars denote significant differences (P< 0.05). bars represent the standard deviations (n = 3). (see Fig. 1 caption). table 2 - Gelling and melting temperatures and gel color of gelatin from the swim bladder of yellowfin tuna extracted at dif- ferent temperatures. Samples Melting point Gelling point Colour (C˚) (C˚) L* a* b* ΔE* G60 22.33±0.42A 15.24±0.27A 27.98±0.57C -2.07±0.02C 8.21±0.11C 66.09±0.57A G70 22.05±0.45A 14.86±0.24A 42.79±0.47B -0.76±0.10B 16.79±0.24B 53.39±0.42B G80 20.36±0.27B 11.07±0.58B 45.79±0.78A -0.34±0.05A 19.03±0.20A 51.32±0.80C Mean ±SD (n = 3). Different uppercase superscripts in the same column indicate significant differences (P < 0.05). 372 Ital. J. Food Sci., vol. 27 - 2015 the most desirable (HoLZEr, 1996). different gel strength was reported for gelatin from skin of dif- ferent species including splendid squid (85–132 g) (nAGArAJAn et al., 2012), brownbanded bam- boo shark and blacktip shark (206–214 g) (KIt- tIPHAttAnAbAWon et al., 2010) and bigeye snap- per (108 g) (bInsIA et al., 2009). 3.6. Gelling and melting temperatures the gelling temperatures of all the gelatin samples were in the range of 11.07-15.24°c (ta- ble 2). thermal transitions were monitored by changes in the phase angle (δ) of dissolved gel- atins during cooling (35-5°c) and subsequent heating (5-35°c). It was found that G80 had the lowest gelling point (11.07°c) (P < 0.05), while no difference in gelling point were observed between G60 and G70 (P > 0.05). In general, fish gela- tin is not able to form gel at room temperature (norLAnd, 1990). It has been known that imino acid content is an essential factor governing ge- lation of getatin (GILsEnAn and ross-MurPHY, 2000). However, the similar amino acid content was observed among all samples (169-172 resi- dues/1000 residues). the result indicated that the gelling temperature was affected by the ex- traction temperature, more likely related with varying chain length. As a thermoreversible gel, gelatin gel starts melting when the temperature increases above a certain point, which is called the gel melting point (KArIM and bHAt, 2009). the melting tem- peratures of gelatin gel from swim bladder were in the range of 20.36-22.33°c. G80 had the low- est melting point (20.36°c) (P < 0.05). neverthe- less, G60 and G70 showed similar melting points (P > 0.05). typical melting points for fish gela- tins ranged from 11 to 28°c (KArIM and bHAt, 2009). GÓMEZ-GuILLÉn et al. (2002) reported that melting points of cod, hake, sole and me- grim were 13.8, 14, 19.4 and 18.8°c, respec- tively. Melting points of red and black tilapia skin gelatins were 22.4 and 28.9°c, respective- ly (JAMILAH and HArVIndEr, 2002). there was a relationship between melting point and molecu- lar weight of gelatin. Low molecular weight gela- tins melt at lower temperature than high molec- ular weight counterparts (GILsEnAn and ross- MurPHY, 2000). the results suggested that lower melting point of G80 was attributed to the low- er molecular weight of peptide chains. temper- ature of the environment also affects the gel- ling and melting temperatures of gelatin (Gud- Mundsson, 2002). Poorer gel strength of G80 (Fig. 3) was in accordance with lower gelling and melting points. 3.7. Microstructures of gelatin gels the microstructures of gelatin gels from swim bladder with different extraction temperatures are illustrated in Fig. 4. In general, the conformation Fig. 4 - Microstructures of gelatin gel from the swim bladder of yellowfin tuna extracted at different temperatures. Mag- nification: 3000 (see Fig. 1 caption). Ital. J. Food Sci., vol. 27 - 2015 373 and chain length of the proteins in gel matrix di- rectly contributed to the gel strength of gelatin (bEnJAKuL et al., 2009). Gelatin extracted at 60°c showed the finest gel network with small voids. conversely, the coarser networks with the larg- er voids were found in gel of the gelatin extract- ed at higher temperatures. the fine gel structure of gelatin extracted at lower temperature was in accordance with the higher gel strength (Fig. 3). It has been known that the microstructure of the gel is related to the physical properties. the gel- atin gel network was governed by the pretreat- ment conditions (YAnG et al., 2008) and gelatin concentration (YAnG and WAnG, 2009). Gelatin extracted at lower temperatures had the lower degradation, in which proteins with higher chain length were present. As a result, junction zones could be formed to a greater extent. this led to the high aggregation with a strong and ordered network. In the first stage of gel network forma- tion, there is competition between intramolecular folding and intermolecular aggregate formation (YAnG and WAnG, 2009). For gelatin extracted at lower temperature, longer chains might under- go aggregation to a higher extent. thus, the ar- rangement of peptides in the network during ge- lation as determined by chain length directly af- fected gel properties of gelatin. 3.8. Color color of the gelatin gel from swim bladder with different extraction temperatures expressed as L*, a* and b* is shown in table 2. Gel of gelatin extracted at lower temperatures (G60) showed the lower L*-value (lightness) than others (G70 and G80) (P < 0.05). the higher redness (a*-val- ue) and yellowness (b*-value) were found in the latters (P < 0.05). Generally, the increases in L*, a* and b*-value of gelatin increased with increas- ing extraction temperatures. For yellowness (b*- value), an increase was observed in all gelatin gels when the extraction temperatures increased (P < 0.05). this might be due to a non-enzymat- ic browning reaction taken place at the higher temperature, especially when the extraction time increased (AJAndouZ and PuIGsErVEr, 1999). Among all the gelatin samples, those extracted at a lower temperature (60˚c) showed the highest total difference in the color value (ΔE*) (66.09) with the lowest lightness (L*-values). these re- sults showed that the extraction temperatures had the impact on color of gelatin extracted from the swim bladder of yellowfin tuna. 4. concLusIon swim bladder from yellowfin tuna could be an alternative source of gelatin. Gelatin extracted at a higher temperature had the highest extrac- tion yield, but possessed the poorer gel proper- ties. Extraction conditions also affected the color of resulting gelatin. the appropriate extraction temperature for gelatin from swim bladder was 60 °c, providing the highest gel strength. 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