Characterization of marine derived collagen extracted from the by-products of bigeye tuna (Thunnus obesus)

International Journal of Biological Macromolecules 135 (2019) 668–676

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Characterization of marine derived collagen extracted from the byproducts of bigeye tuna (Thunnus obesus) Raju Ahmed a,b, Monjurul Haq a,c, Byung-Soo Chun a,⁎ a b c

Department of Food Science and Technology, Pukyong National University, 45 Yongso-ro, Nam-Gu, Busan 48513, Republic of Korea Department of Fisheries, Ramna, Dhaka 1217, Bangladesh Department of Fisheries and Marine Bioscience, Jashore University of Science and Technology, Jashore 7408, Bangladesh

a r t i c l e

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Article history: Received 12 February 2019 Received in revised form 12 May 2019 Accepted 29 May 2019 Available online 30 May 2019 Keywords: Collagen Bigeye tuna Skin Scale Bone By-products

a b s t r a c t Fish collagen is gaining immense interest because the use of mammalian collagen is restricted due to disease transmission and religious issues. So, collagen was extracted and characterized from three different parts (skin, scale, and bone) of bigeye tuna using the acid and pepsin extraction methods. The yield of acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) in skin were 13.5 ± 0.6% and 16.7 ± 0.7%, respectively, on a dry basis. The yields of PSC in scale and bone were 4.6 ± 0.3% and 2.6 ± 0.3%, respectively, while ASCs were in negligible amount. All the extracted collagens were type I collagen and a high level of imino acids (227–232/1000 residues) was found in all the extracted collagens. The thermal transition temperature (31.6–33.7 °C) and thermal denaturation temperature (31.1–32.2 °C) were higher than those of many temperate- and cold-water fish collagens. All collagens were highly soluble at acidic pH. The isoelectric points were 6.1, 6.4, 5.4, and 5.5 for skin-ASC, skin-PSC, scale-PSC, and bone-PSC, respectively. Therefore, the high collagen contents, especially in the skin, and higher thermal properties of the extracted collagens suggested that they have great potential for use as an alternate for mammalian collagen. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Collagen (CL) is the predominant protein in vertebrates, where it constitutes approximately one-third of total protein. Collagen is available in the connective tissues of the skins, scales, and bones of animals. It is constructed of three polypeptide chains intertwined to form a unique triple helical structure that provides mechanical stability, elasticity, and strength to the organisms [1]. The collagen monomer is an approximately 300 nm-long cylindrical protein with a 1.6-nm diameter [2]. Each polypeptide chain of a collagen molecule consists of the repeated amino acid sequence Gly-X-Y, where the most common amino acids are proline and hydroxyproline [3]. Type I collagen, the most studied among 29 different forms of collagen reported to date, is exclusively applied in food, pharmaceuticals, biomedical products, and cosmetics [4,5]. Commercial collagen is generally derived from porcine and bovine skins and bones. However, the application of these land-based mammalian collagens has been limited because of the emergence of some transmissible diseases, especially bovine spongiform encephalopathy (BSE), foot and mouth diseases (FMD), and avian influenza [6]. The application of this collagen is also restricted due to the constraints from Muslim, Jewish, and Hindu religions [6,7]. In this context, marine collagens ⁎ Corresponding author. E-mail address: [email protected] (B.-S. Chun).

https://doi.org/10.1016/j.ijbiomac.2019.05.213 0141-8130/© 2019 Elsevier B.V. All rights reserved.

have been attracting increasing attention as an alternative to terrestrial-based collagens since they are free from the risk of diseases and religious restrictions. Considerable amounts of by-products are produced; as much as 70% of the original materials from fish processing factories in the form of skins, scales, bones, viscera, gills, and heads [4]. These materials have traditionally been used in low-value products, such as animal feed, fertilizers, and others [8,9]. Fish by-products, such as skin, scale, and bone are known to be abundant sources of collagens [10,11]. Tuna are a widely distributed and commercially important fish species; 7.7 million metric tons were produced globally in 2014 [12]. Bigeye tuna (Thunnus obesus Lowe, 1839) is exclusively used in the sashimi and canned tuna industries, where only white meats are used resulting in large amounts of by-products [4,13]. Generally, warm-water fish species possess collagen of higher thermal stability than those of cold-water species [4]. Since bigeye tuna is distributed in the subtropical and tropical areas of the Atlantic, Indian, and Pacific Oceans, it is expected that the collagen of this fish might have good thermal stability. A number of studies on the characterization of collagen extracted from various fish byproducts have been published [5,14,15]. However, studies on the collagen extracted from this fish species are scarce despite considerable production as well as utilization in processing industries and consequently generation of large amounts of by-products. None of the available studies has extracted and characterized collagen obtained from the different

R. Ahmed et al. / International Journal of Biological Macromolecules 135 (2019) 668–676

body parts of bigeye tuna. So, the present study was focused on the extraction yield and comparative characterization of collagen extracted from different by-products of bigeye tuna. Therefore, the aim of this research was to extract collagen from the skin, scale, and bone of bigeye tuna by using acid and pepsin and to characterize various physiochemical and thermal properties of the acid (ASC) and pepsin-soluble collagen (PSC). 2. Materials and methods 2.1. Chemicals Pepsin from porcine gastric mucosa (EC 3.4.23.1, CAS 9001-75-6; 250 units/mg dry matter) and sodium dodecyl sulfate (CAS 151-21-3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Standard Type I collagen from bovine tendon (Cat. no. 5162) and high molecular-weight marker (Cat. no. 26619) were purchased from Advanced BioMatrix (San Diego, CA, USA) and Thermo Fisher Scientific, Waltham, MA, USA, respectively. All other chemicals and reagents were of analytical or HPLC grade. 2.2. Raw materials Bone and skin attached with scale of bigeye tuna (average size of 35 ± 5 kg) harvested from the Pacific Ocean by Dongwon Fisheries Co., Ltd. (Busan, Korea) were transported to the laboratory in an insulated box. Then, the sample was prepared and the oil was removed from the samples using the supercritical carbon dioxide extraction method [16]. Until further used for collagen extraction, the de-oiled materials were stored at −20 °C. 2.3. Extraction of collagen Collagen was extracted from tuna skin, scale, and bone following previously described methods [17,18] with slight modifications. All steps of the extraction process were performed below 4 °C. Noncollagenous proteins were removed by treating the de-oiled samples with 10 volumes of 0.1 M NaOH with continuous stirring for 24 h, changing the alkaline solution at 8 h intervals. Next, the samples were rinsed with chilled distilled water several times until the wash water reached neutral pH. Then, only the scale and bone samples were treated with 10 volumes of 0.5 M EDTA (pH 7.5) for 3 days, changing the solution daily. The decalcified materials were then washed with chilled distilled water until the pH reached approximately 7. Subsequently, the skin, scale, and bone samples were treated with 40 volumes of 0.5 M acetic acid for 3 days with continuous stirring. The supernatant was collected after centrifugation (Hanil Combi-514R centrifuge, Hanil Science Industrial Co., Incheon, Korea) at 18,000 ×g for 30 min and the residue was re-extracted under the same conditions. The two supernatants were mixed, and then salted out using NaCl at a final concentration of 2 M. The precipitates were harvested by centrifugation at 15,000 ×g for 30 min and then dissolved in a small volume of 0.5 M acetic acid. The solution was dialyzed against 0.1 M of acetic acid for 24 h and distilled water for 48 h. The resulting dialysate was lyophilized and assigned as “acid-soluble collagen, ASC”. For pepsin digestion, the undissolved residues remaining from the acid extraction were treated with 0.5% pepsin of 250 units/mg dry matter (w/v) (0.2 g pepsin/g of substrate) in 40 volumes of 0.5 M acetic acid for 48 h with continuous stirring. The supernatants were collected after centrifugation at 18,000 ×g for 30 min and the remainders were retreated with the same solution for 48 h. To terminate the pepsin reaction, the supernatants obtained were dialyzed against 0.02 M Na2HPO4 (pH 7.2) in a dialysis membrane with molecular weight cutoff of 30 kDa for 24 h with a change of solution every 6 h. The retentate obtained was centrifuged at 18,000 ×g for 30 min. The pellet was

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dissolved in 0.5 M acetic acid. The solute was precipitated by salt and dialyzed as described for ASC. 2.4. Characterization of collagen 2.4.1. SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was conducted by using previous method [19] with some modifications, using a Mini-PROTEAN apparatus (Bio-Rad Laboratories, Hercules, CA, USA). The collagen samples (1 mg/ml) were suspended in 0.02 M sodium phosphate buffer (pH 7.5), and then mixed with the same volume of sample buffer (1 M Tris–HCl, pH 6.8, containing 10% SDS, 25% glycerol, and 0.05% bromophenol blue), in the presence or absence of 5% β-mercaptoethanol. The samples were then dissolved by heating at 90 °C for 5 min. Next, the undissolved debris was removed by centrifuging at 8500 ×g for 10 min. Then, 20 μl of each sample (10 μg protein) was loaded onto a polyacrylamide gel that consisted of a 6.5% resolving gel and 4% stacking gel. The electrophoresis was conducted at a constant voltage of 120 V for 1 h. After electrophoresis, the gel was stained with 0.1% (w/v) Coomassie Blue R-250 in 10% (v/v) acetic acid and 40% (v/v) methanol for 2 h and subsequently destained with a solution containing 10% acetic acid and 20% methanol. A high molecular-weight protein marker was used, and type I collagen from bovine tendon was used as the standard collagen. The protein band intensities were quantified by the public domain digital analysis software ImageJ software (ImageJ k 1.45, National Institute of Health, MD, USA). 2.4.2. Fourier transform infrared spectroscopy Fourier transform infrared (FTIR) spectra were obtained using a published method [5]. A 2-mg collagen sample was mixed with 100 mg potassium bromide (KBr) and formed into a tablet, which was mounted in the sample holder of the FTIR spectrometer. All spectra were obtained using an infrared spectrophotometer (Jasco-4100, Jasco Corporation, Tokyo, Japan) with a resolution of 2 cm−1 in the range of 400–4000 cm−1. 2.4.3. Amino acid composition Twenty milligrams of collagen sample dissolved in 2 ml of 6 M HCl was evacuated, vacuum-sealed and hydrolyzed at 110 °C for 24 h. The hydrolysate was analyzed on an amino acid auto-analyzer (S430, SYKAM, Eresing, Germany). A cation separation column (LCA K07/Li; 4.6 × 150 mm) was used with a column temperature of 37–74 °C and buffer pH range of 2.90–7.95. The mobile phase was 5 mM p-toluene sulphonic acid solution at a flow rate of 0.45 ml/min. A mixture of 5 mM p-toluene sulphonic acid, 20 mM of Bis-Tris and 100 mM of EDTA was used as the post-column reagent at a flow rate of 0.25 ml/min. The excitation and emission wavelengths were maintained at 440 and 570 nm. 2.4.4. Determination of denaturation temperature The denaturation temperature (Td) was determined following the previous method [20] with slight modification, using a viscometer (Brookfield DVII+Pro EXTRA, Middleboro, MA, USA). Collagen samples were dissolved in 0.5 M acetic acid at a concentration of 0.03% and a 7ml sample was taken into a small sample adaptor. The viscosity was measured at various temperatures ranging from 20 to 45 °C with incubation for 10 min at each temperature. Data were taken three times at each point. The fractional viscosity was determined at each temperature using the following equation: Fractional viscosity ¼

Maximum viscosity−Measured viscosity Maximum viscosity−Minimum viscosity

These fractional viscosities were plotted against the temperature and the denaturation temperature was determined as the temperature at which the fractional viscosity was 0.5.

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2.4.5. Thermal transition measurement The thermal transition of collagen sample was measured following the method of Zeng et al. [21] using a differential scanning calorimeter (DSC Q200 V24.4 Build 116, TA Instruments, Water LLC, MA, USA). The lyophilized collagens were rehydrated in 40 volumes of deionized water for 48 h at 4 °C. The rehydrated samples (5–10 mg) were accurately weighed into aluminum pans that were sealed, then scanned over the range of 20–50 °C with a heating rate of 1 °C/min. An empty sealed aluminum pan was used as the reference. The instrument was calibrated for temperature and enthalpy using indium as the standard, and samples were constantly purged with ultrahigh-purity nitrogen at 50 ml/min. The instrument was equilibrated at 20 °C for 5 min before scanning; ice water was used as a cooling medium. The results were analyzed using TA Universal Analysis 2000 V4.5A software (TA Instruments). 2.4.6. Solubility of collagens Collagen solubility was determined at various pHs using the method described by Liu et al. [22] with slight modifications. The samples were dissolved in 0.5 M acetic acid at a concentration of 0.3% (w/v) with gentle stirring at 4 °C for 12 h. The collagen solution (8 ml) was placed in a centrifuge tube. Then, the pH was adjusted to different levels, ranging from 2 to 10, using 6 M HCl or 6 M NaOH. The final volume was brought to 10 ml with distilled water that had previously been adjusted to the same pH as the collagen solution tested. The solutions were gently stirred at 4 °C for 30 min and left overnight. Next, the supernatants were collected after centrifugation for 30 min at 10,000 ×g. Protein content was determined using the Lowry method [23] with bovine serum albumin as the protein standard. Relative solubility was determined in comparison with that obtained at the pH level providing highest solubility. 2.4.7. Zeta potential analysis Zeta potential analysis was performed according to the method of Benjakul et al. [24] with some modifications. The sample was dissolved in 0.5 M acetic acid to a final concentration of 0.05%. The mixture was continuously stirred at 4 °C for 6 h using a magnetic stirrer at 300 rpm. Zeta potential of the collagen samples was measured on a zeta potential analyzer (Zetasizer Nano-ZS90, Malvern Instrument Ltd., Malvern, Worcestershire, UK). The solution (20 ml) was transferred to an autotitrator (MPT-2, Malvern Instrument), in which the pH was adjusted to 3–9 using either 1 M KOH or 1 M nitric acid. The isoelectric point (pI) was determined at the pH where the zeta potential was zero. 2.5. Statistical analysis Values of different parameters studied are presented as the mean ± standard deviation of triplicates. Statistical analysis was done by oneway analysis of variance (ANOVA) using IBM SPSS statistics (Version 20.0 software, SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Yield of collagen The yield of ASC and PSC from the skin of bigeye tuna were 13.5 ± 0.6% and 16.7 ± 0.7%, respectively (dry matter basis). The yield of PSC from scale and bone were estimated to be 4.6 ± 0.3% and 2.6 ± 0.3%, respectively (dry matter basis), while the yield of ASC from scale and bone were very low (0.05 ± 0.01% and 0.1 ± 0.02%, respectively). A collagen molecule, which contains non-helical parts in its two terminal ends, known as the telopeptide regions, possesses a cross-linked structure. The degree of cross-linking at the terminal ends of collagen limits its solubility in acid solution [25]. The lower yield of scale-ASC and bone-ASC from bigeye tuna indicted that the degree of cross-linking among

collagen molecules might be stronger in scale and bone than in skin. During pepsin digestion, collagen was cleaved specifically at the telopeptide region; thus, the yield of collagen increased [22,26]. The yields of collagen from the scale and bone were lower than that from the skin of bigeye tuna. Similar results were observed in previous studies, which revealed the yields of collagen from scales of red drum fish (ASC, 0.6%; PSC 4.3%) [5] and silver carp (1.5%) [27], and from bones of bigeye snapper (1.6%) [28], and carp (1.1%) [12]. Differences among these collagen yields have been reported to vary with the species, age, size, starvation condition, structure and composition of tissue, as well as the extraction procedure [26,29]. 3.2. Characterization of collagens 3.2.1. SDS-PAGE patterns Similar electrophoretic patterns were found for bovine tendon and bigeye tuna collagens (Fig. 1). The reducing and non-reducing conditions (with or without beta-mercaptoethanol) showed similar electrophoretic patterns for the same collagen, indicating non-existence of interchain disulfide bonds. All the collagen samples comprised α1and α2-chains with their high molecular cross-linked dimers (βchains) and small amounts of trimers (γ-chains). This electrophoretic pattern was similar to that of standard type I collagen from bovine tendon [5,25]. The ratio of α1- to α2-chain of the extracted collagens was above 2 and differed slightly from each other; might be due to the presence of different quantity of α3-chain in collagen trimers. The α3-chain was not identified during electrophoresis because it migrated to the α1 position under the electrophoretic conditions used [30]. It was reported that the α3-chain was not identified during electrophoresis because it migrated to the α1 position under the electrophoretic conditions used [30]. These results were consistent with others found in collagens extracted from the skins of albacore tuna [31] and arabesque greenling [32] the scales of red drum fish [5], and the bones of bigeye tuna [33]. The band intensity ratio of cross-linked component (especially βcomponent) to total non-cross-linked monomer chains (α1 and α2) of skin-ASC (2.5) was higher than that of skin-PSC (1.6). Thus, it could be concluded that the ASC contains greater amounts of high molecular cross-linking components than those of PSC. Ogawa et al. [46] explained this result by conversion of some β- and γ-components to αcomponent in PSC by the action of pepsin. It was also reported that pepsin eliminates the cross-link-containing telopeptide, and concomitantly one β-component is converted to two α-components. The molecular weights of the α- and β-components of bone- and scale-PSCs were higher than that of skin collagen. These results indicated that the major collagen extracted from skin, scale, and bone of bigeye tuna were type I collagen. 3.3. FTIR All the collagen samples showed characteristic amide A and B bands, as well as amide I, II, and III peaks. The amide A band which usually occurs between 3400 and 3440 cm−1, arises from a free N\\H stretching vibration with hydrogen bonding [34]. This band was found at 3299, 3301, 3299, 3298, and 3297 cm−1 for standard Type I collagen, skinASC, skin-PSC, scale-PSC, and bone-PSC, respectively (Fig. 2). The position of the free N\\H stretching vibration can migrate to lower frequencies (3300 cm−1) when the NH group of a peptide bond is involved in a hydrogen bond. The amide A band of bone-PSC occurred at lower wavenumber than that of other extracted collagens, indicating involvement of stronger hydrogen bond in bone-PSC. The lowest transmittance (78%) of bone-PSC also indicated higher degree of hydrogen bond intensities (Fig. 2). The amide B band, which arises from the asymmetrical stretching of CH2 groups [35] was found at 2922–2931 cm−1. The amide I peak is often associated with the analysis of protein secondary structure and is usually found between 1600 and 1700 cm−1 [33]. This spectrum of frequencies arises from the stretching vibration of the

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Fig. 1. SDS-PAGE pattern of collagens extracted from bigeye tuna under non-reducing (Lanes 3–6) and reducing conditions (Lanes 7–10). Lane 1: MW protein markers; Lanes 2–6: Standard Type I collagen, acid-soluble collagen from skin (skin-ASC), pepsin-soluble collagen from skin (skin-PSC), pepsin-soluble collagen from scale (scale-PSC) and pepsin-soluble collagen from bone (bone-PSC), respectively, under non-reducing condition; Lanes 7–10: Same as 3–6, but under reducing condition.

carbonyl group (C_O band) in the polypeptide backbone. The amide I peak frequencies of the collagens studied were between 1635 and 1639 cm−1. The transmittance value of amide I peak was lowest for bone collagen (56.5%), followed by scale-PSC (61%), skin-ASC (61%), standard (67%) and skin-PSC collagen (68%) (Fig. 2). The lowest transmittance value suggested the higher intensities of the respective

bands. The bone collagen showed lowest transmittance value at almost all the major band positions. Amide II peak, which arises from N\\H bending vibrations coupled to C\\N stretching vibrations, usually occurs near 1550 cm−1. Collagen contains an exceptional amino acid composition in which glycine and proline are the most noticeable residues. These two amino acids

Fig. 2. FTIR spectra of standard Type I collagen and bigeye tuna collagens from skin, scale, and bone.

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Table 1 FTIR spectra important peak locations and assignment for standard Type I and extracted collagens from bigeye tuna. Region

Amide A Amide B – – Amide I Amide II – – – Amide III – –

Peak wavenumber (cm−1)

Assignment

Type I collagen

Skin-ASC

Skin-PSC

Scale-PSC

Bone-PSC

3299 2924 2855 1743 1635 1547 1449 1396 1340 1239 1071 8,56

3301 2927 / / 1639 1546 1451 1396 1337 1240 1055 8,67

3299 2931 / / 1639 1546 1451 1396 1336 1240 1066 8,69

3298 2926 / / 1639 1546 1451 1392 1333 1239 1058 8,65

3297 2926 / / 1639 1545 1451 1392 1333 1239 1069 8,55

contribute to the spectral characteristics of 1200–1400 cm−1 that were observed in all the extracted collagens (Table 1) [36]. Amide III peak usually occurs at 1320–1220 cm−1 and arises from the NH bend coupled with the CN stretch, which is responsible for collagen helical integrity [37]. The collagen triple helix structure is maintained primarily by restrictions on changes in the secondary structure of the polypeptide chain, imposed by the pyrrolidine rings of proline and hydroxyproline. In addition, the hydroxyl group of hydroxyproline also helps stabilize the triple helix structure by interchain hydrogen bonding via a bridging water molecule, as well as direct hydrogen bonding to a carboxyl group [44]. All the extracted collagens showed amide III peaks at around 1240 cm−1. The IR ratios between the amide III and 1454 cm−1 of the extracted collagens were about 1.17, which was around 1.0, confirming the existence of a triple helix [38]. 3.4. Amino acid composition The amino acids of extracted collagens were dominated by glycine (215–227/1000 residues), followed by proline (140–150/1000 residues), glutamic acid (99–102/1000 residues), alanine (92–99/1000 residues), and hydroxyproline (82–87/1000 residues) (Table 2), which

N\ \H stretch coupled with hydrogen bond CH2 asymmetric stretch CH3 symmetric stretch Presence of lipid C_O stretch/hydrogen bond coupled with COO– NH bend coupled with CN stretch CH2 bend COO– symmetrical stretch CH2 wagging of proline NH bend coupled with CN stretch C\ \O stretch Skeletal stretch

were comparable to those of other fish and mammalian collagens. Although glycine (215–227/1000 residues) was the most prevalent amino acid in bigeye tuna collagen, the amount was significantly lower than that of most other collagens, such as carp (332–336/1000 residues) [12] and balloon fish (340–353/1000 residues) [20], but similar to that of Nile tilapia scale (195/1000 residues) [39], Nile perch skin (211–221/1000 residues) [29], and yellowfin tuna skin (251/1000 residues) [37]. Ideally, collagen contains one-third glycine of the total amino acids residues. The lower content of glycine in this study might be attributed to the interference by some other proteins. Hydroxyproline and hydroxylysine, also indicative amino acids of collagen [18], were found in all the extracted collagen. The extracted collagens contained a higher level of imino acids (227–232/1000 residues) than standard Type I collagen from calf skin (216/1000 residues) [37], pig skin (220/1000 residues) [15], yellowfin tuna (205/1000 residues) [37], striped catfish (217/1000 residues) [40], brownstrip red snapper (221/1000 residues) [25], squid skin (225/1000 residues) [38], but lower than the tilapia skin (254/1000 residues) [42]. However, the hydroxyproline content (82–87/1000 residues) of the extracted collagens was lower than that of calf (99/1000 residues) or pig (97/1000 residues) collagen (Table 2). The total imino acid content was higher in the skin

Table 2 Amino acid compositions of collagen from skin, scale, and bone of bigeye tuna compared with those of calf and pig skin collagens. Amino acids

Residues/1000 residues Skin-ASC

Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Hydroxylysine Hydroxyproline Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine Imino acids

a

99.76 ± 3.40 81.64a ± 2.67 40.28b ± 1.45 1.04b ± 0.40 99.31a ± 3.58 227.23a ± 6.78 7.17b ± 0.73 9.43a ± 1.34 82.13b ± 3.76 11.23b ± 1.87 25.86b ± 1.43 33.45b ± 2.84 21.34a ± 2.56 20.76a ± 3.65 150.16a ± 4.87 36.04ab ± 2.18 31.44a ± 1.46 4.34b ± 0.82 21.98b ± 2.87 232.45a ± 6.72

Skin-PSC a

97.24 ± 2.31 80.45a ± 2.38 42.48ab ± 1.56 1.14b ± 0.067 99.43a ± 4.66 221.64b ± 8.34 7.46b ± 0.28 9.29a ± 1.04 82.36b ± 4.27 12.67b ± 1.42 27.10a ± 1.82 33.36b ± 3.45 20.47a ± 3.97 20.20a ± 2.65 148.25a ± 5.43 38.87a ± 1.78 33.87a ± 3.65 5.47ab ± 1.02 23.48ab ± 1.86 230.34a ± 7.67

Scale-PSC b

92.30 ± 2.94 75.13b ± 3.57 44.28a ± 2.42 2.23a ± 0.38 102.02a ± 4.24 221.45b ± 7.35 8.53ab ± 0.61 9.44a ± 1.76 87.31a ± 3.39 14.26a ± 1.35 29.35a ± 1.49 32.26b ± 1.98 19.43a ± 2.18 20.16a ± 3.43 142.36b ± 2.56 39.25a ± 3.36 33.45a ± 1.45 6.23a ± 0.68 25.43a ± 2.29 229.12a ± 8.34

Bone-PSC b

91.63 ± 3.30 79.28a ± 4.81 44.48a ± 2.49 2.45a ± 0.72 101.17a ± 4.85 215.45b ± 8.75 9.45a ± 1.65 10.36a ± 1.45 87.22a ± 4.54 14.24a ± 1.49 27.86a ± 2.46 39.34a ± 2.36 19.12a ± 2.93 20.19a ± 2.26 140.28b ± 3.81 38.36a ± 2.57 33.40a ± 3.06 6.43a ± 1.01 24.24a ± 1.50 226.85a ± 5.39

Yellow fin tuna skin⁎

Calf skin⁎

Pig skin⁎⁎

92.39 76.38 69.91 1.30 113.92 251.28 8 0 80 11 27 32 0 15 125 41 35 0 24 205

78.82 73.28 68.73 1.08 109.35 241.65 11 0 99 17 33 4 0 24 117 35 20 6 28 216

114.82 48.874 44.32 0 72.27 341.843 5 7 97 11 22 27 6 12 123 33 15 1 22 220

Values are presented as means ± standard deviation of triplicates. Means with the different superscript letter in each row differ significantly (P b 0.05) according to Duncan's Multiple Range tests. ⁎ Woo et al. [37]. ⁎⁎ Li et al. [15].

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collagen than in scale and bone collagen, while the hydroxyproline content was higher in scale/bone collagen than skin collagen. The collagens from different sources vary in amino acid compositions that ultimately affect the properties of collagens [18]. In general, the collagens of tropical fish contain a greater amount of imino acids and show higher thermal stability [4]. The higher level of imino acids in collagens obtained

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from bigeye tuna, in comparison with those of cold-water fish (154–165/1000 residues), were consistent with the report that tropical fish contains a higher level of imino acid than cold-water species [32]. The extracted collagens also contained 9–10 hydroxylysine residues per 1000 residues, which suggested that the partial cross-linking had formed via covalent bonds.

Fig. 3. Thermal transition curves measured by DSC (a) and Thermal denaturation curves measured by viscosity (b) of skin, scale, and bone collagen from bigeye tuna.

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3.5. Thermal stability The maximum temperatures (Tmax) corresponding denaturation temperatures, of skin-ASC, skin-PSC, scale-PSC and bone-PSC were 32.1, 33.7, 31.6 and 32.3 °C, respectively (Fig. 3a). These results were similar to the Tmax values of some tropical and subtropical fishes, such as giant grouper (31.7 °C) [42], bigeye snapper (31 °C) [28], and brownbanded bamboo shark (34.5 °C) [17]. In contrast, the Tmax of bigeye tuna collagen was much higher than that of the collagens of several temperate- and cold-water fishes, including arabesque greenling (15.5 °C) [32], chum salmon (19.4 °C) [43], carp (28 °C) [12], bullhead shark (25 °C), chub mackerel (25.6 °C), and Japanese seabass (26.5 °C) [44]. However, the Tmax of collagen from bigeye tuna was lower than that of terrestrial animals, such as porcine skin (40 °C) and calf skin (37 °C) [46]. The collagen helical structure disintegrates into random coils when the hydrogen bonds break down with increasing temperature. As a result, the physical characteristics of collagen, such as viscosity, diffusion, sedimentation, optical activity, and light scattering change with increasing temperature [47]. For this reason, the thermal stability of collagen is determined by using DSC and viscosity measurements. Fig. 3(b) shows the changes in fractional viscosity with increasing temperature. The denaturation temperatures obtained from fractional curve were 31.2, 32.8, 31.1, and 31.4 °C, for skin-ASC, skin-PSC, scale-PSC, and bone-PSC, respectively. The denaturation temperature, determined by DSC or viscosity measurements, of skin-PSC was a little bit higher than that of skinASC, suggesting that the triple helical structure of the collagen was not disturbed by pepsin digestion, though a slight amount of lowermolecular-weight α-chains was found in skin-PSC (Fig. 1). The higher Tmax of PSC was explained by elimination of the telopeptide region, which is related with a highly ordered and compact structure [48]. This observation was consistent with the studies conducted with the skins of bigeye snapper [25], brownbanded bamboo shark [17], and balloon fish [20]. Thermal stability is one of the most important properties of collagens that determine their potential application [26] and is associated with the imino acid content, habitat temperature, and body

temperature [28]. The thermal stability of the triple helical structure of collagen is attributed to hydrogen-bonded networks, mediated by water molecules, which connect the hydroxyl groups of hydroxyprolines in one strand to the main chain amide carboxyl of another chain. Thus, the thermal stability of the collagens increases with increasing number of imino acids, especially hydroxyproline content determines the thermal stability of collagens from different species. Although the total imino acid content was higher in bigeye tuna collagens (227–232/1000 residues) than in mammalian collagen (216–220/1000 residues), bigeye tuna collagen showed lower thermal stability. Bigeye tuna collagen showed lower thermal stability than mammalian collagen due to lower hydroxyproline content in bigeye tuna collagens (82–87/ 1000 residues) than mammalian collagens (97–99/1000 residues) (Table 2). This result was supported by previous studies, which suggested that the denaturation temperature was reliant on the hydroxyproline content, rather than proline residues [4,45]. The thermal stabilities of scale and bone collagen were different, although they showed similar imino acid compositions. The higher thermal stability of bone might be attributed to the higher molecular weight of its αand β-chains (Fig. 1), lower transmittance value at major FTIR bands (Fig. 2) and/or its content of the Gly-Pro-Hyp sequence. 3.6. Solubility of collagen at different pHs All the extracted collagens showed high solubility under acetic conditions, with the highest solubility at pH 6, except scale-PSC, which showed maximum solubility at pH 5 (Fig. 4). The solubility decreased sharply from the maximum point and reached its lowest point at pH 7 for all the collagens. However, in the alkaline pH range, from 7 to 10, solubility was found to increase slightly with increasing pH. Loss of solubility at particular pH might be attributed to an increase in hydrophobichydrophobic interaction among collagen molecules, and the total net charge become zero, especially at pI [25]. The solubility was increased slightly from pH 8 to 10 because of the repulsive effect of the collagen molecules [28]. In contrast, the collagen might undergo denaturation, to some extent, at very highly acidic conditions (e.g., pH 2), leading to impaired solubility.

Fig. 4. Relative solubility of the bigeye tuna collagens at different pHs.

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Fig. 5. Zeta potential at different pH values of bigeye tuna collagens extracted from skin, scale, and bone.

3.7. Zeta potential Net charges of the extracted collagens were found zero at pH 6.1, 6.4, 5.4, and 5.5 for skin-ASC, skin-PSC, scale-PSC, and bone-PSC, respectively (Fig. 5). Thus, these pH were assumed to their pIs. The pI refers to the pH at which the net charge of a protein molecule is zero [49]. Zeta potential value, which represents the surface charge of a protein molecules, increased as the pH was lower from the pI. The higher surface charge leads to intermolecular repulsions and thus results in stable suspension, while the lower surface charge may lead to precipitation. At the pI of protein, its structure is more hydrophobic, more compact and less stable due to absence of inter-particle repulsive forces. Hence, proteins can easily aggregate and precipitate at their pIs. The pIs of all the extracted collagen were observed at acidic pHs. The pIs of scale (5.4) and bone (5.5) collagens were more acidic than those of skin collagens (6.1–6.4). This result might be associated with the higher content of acidic amino acids, such as aspartic acid and glutamic acid in scale and bone collagen than skin collagens (Table 2). Collagen obtained from various sources showed different pIs, e.g., 5.5–5.7 for clown featherback skin [14], 6.2–6.6 for brownbanded bamboo shark skin [17], and 9.92 for snakehead scale [22]. This differences might be caused by the difference in their amino acid compositions and sequence of amino acid residues, particularly on surface domain.

4. Conclusions The collagens isolated in this study from the skin, scale, and bone of bigeye tuna using acetic acid and pepsin were type I collagen. The yield of collagen was considerably higher from skin, followed by scale and bone. All the isolated collagens contained a high amount of imino acids; thus, they exhibited higher thermal properties than those of temperate fish species. Collagen triple helical was not affected during pepsin extraction. However, there were slight differences in the properties of collagens obtained from various body parts. Therefore, the collagen extracted from the by-products of bigeye tuna could be used in various commercial applications as an alternative to terrestrial collagen.

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