Food Chemistry 120 (2010) 817–824

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Extraction and characterisation of pepsin-solubilised collagen from the skin of unicorn leatherjacket (Aluterus monocerous)
Mehraj Ahmad, Soottawat Benjakul *
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

a r t i c l e

i n f o

a b s t r a c t
Pepsin-solubilised collagen (PSC) was extracted from the skin of unicorn leatherjacket (Aluterus monocerous), using 0.5 M acetic acid in the presence of pepsin from albacore tuna, yellowfin tuna or porcine pepsin at a level of 20 units/g of defatted skin. Yields of 8.48 ± 0.3%, 8.40 ± 0.3% and 7.56 ± 0.4% (wet weight basis) were obtained for PSC extracted with the aid of albacore tuna pepsin (APSC), yellowfin tuna pepsin (YPSC) and porcine pepsin (PPSC), respectively. All PSCs were classified as Type I collagen containing two a1-chains and one a2-chain with no disulphide bond. The peptide maps of different PSCs hydrolysed by V8 protease and lysyl endopeptidase were different. ATR-FTIR spectra analysis revealed that PSC molecules had the compact triple helical structure stabilised mainly by the hydrogen bond. Tmax of all PSCs (31.68–31.98 °C) shifted to lower values (29.33–29.40 °C) when dispersed in 0.05 M acetic acid, indicating the conformational changes in the collagen structure induced by acid. Relative viscosity of 0.03% PSC in 0.1 M acetic acid solution decreased progressively as the temperature increased from 4 to 52 °C, indicating thermal destabilisation or denaturation of PSC molecules. All PSCs were soluble in the pH range of 1–6 and sharply decreased at neutral pH. Decreases in solubility were observed in the presence of NaCl, especially at concentrations above 2% (w/v). Therefore, the skin of unicorn leatherjacket could serve as a potential source of collagen. Ó 2009 Published by Elsevier Ltd.

Article history: Received 30 June 2009 Received in revised form 19 October 2009 Accepted 9 November 2009

Keywords: PSC Unicorn leatherjacket FTIR Viscosity Denaturation temperature

1. Introduction Collagen is the predominant protein of connective tissue in animals, making up about 30% of the total protein content (Birk & Bruckner, 2005). It possesses a unique protein sequence and is able to form insoluble fibres with a high tensile strength (Gelse, Poschl, & Aigner, 2003). Collagen has a wide range of applications in various branches of industry. Its pharmaceutical and biomedical uses include tissue engineering for implants in humans, inhibition of angiogenic diseases, treatment of hypertension, urinary incontinence and osteoarthritis (Lee, Singla, & Lee, 2001). It is also a very attractive substrate for fragrance and cosmetic applications (Tzaphlidou, 2004). Its denatured form (gelatin) has been widely used in the food industry (Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005). All members of the collagen family are characterised by domains with repetitions of the proline-rich tripeptide Gly-X-Y, involved in the formation of trimeric collagen triple helix (Muyonga, Cole, & Duodu, 2004; Ramachandran, 1988). In general, the traditional sources of collagen are bovine and porcine skins and bones. Porcine collagen is unacceptable for some religions, for example Judaism and Islam, while those from bovine
* Corresponding author. Tel.: +66 7428 6334; fax: +66 7421 2889. E-mail address: soottawat.b@psu.ac.th (S. Benjakul). 0308-8146/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.foodchem.2009.11.019

sources are at risk of contamination with bovine spongiform encephalopathy (BSE) (Fernandez-Diaz, Montero, & Gomez-Guillen, 2001). With the rapid development of seafood processing industries, huge quantities of by-products have been discarded, which may cause pollution and emit offensive odours. Hence, the comprehensive utilisation of these by-products, especially the production of value-added products, is a promising means to increase revenue for the producers and accelerate the development of the seafood processing industry. Fish skin is an important source for collagen, which can be used as a replacement for mammalian sources. In general, a low yield of collagen is obtained with traditional processes. Since pepsin has been reported to cleave peptides in the telopeptide region, the extraction of collagen partially cleaved by pepsin resulted in a higher yield (Jongjareonrak, Benjakul, Visessanguan, Nagai, & Tanaka, 2005; Nalinanon, Benjakul, Visessanguan, & Kishimura, 2007a). Due to the enormous amount of fish viscera, especially stomach, pepsin of fish origin can be recovered and used to increase the extraction efficiency of collagen from fish skin (Nalinanon, Benjakul, Visessanguan, & Kishimura, 2007b). Unicorn leatherjacket (Aluterus monocerous) belongs to the order Tetraodontiformes and is a member of the Monacanthidae family. This species has been used for fillet production, in which a large amount of skin has been produced as by-product. Due to

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its thick skin, it is therefore a potential source of collagen, especially when fish pepsin is implemented as the extraction aid. The objective of this study was to isolate and characterise the pepsinsolubilised collagen (PSC) from the skin of unicorn leatherjacket (A. monocerous), with the aid of different pepsins including tuna and porcine pepsins. 2. Materials and methods 2.1. Chemicals Bovine haemoglobin, b-mercaptoethanol (b-ME), V8 protease from Staphylococcus aureus (EC3.4.21.19), L-tyrosine, bovine serum albumin, pepsin from porcine stomach mucosa (EC3.4.23.1; powderised, 750 U/mg dry matter), and Type I collagen from calf skin were purchased from Sigma Chemical Co. (St. Louis, MO). High molecular weight markers were purchased from GE Healthcare UK (Aylesbury, UK). Sodium dodecyl sulphate (SDS), trichloroacetic acid, Folin–Ciocalteu’s phenol reagent, acetic acid and tris(hydroxylmethyl)aminomethane were obtained from Merck (Darmstadt, Germany). Lysyl endopeptidase from Achromobacter lyticus (EC3.4.21.50) was procured from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). 2.2. Fish skin and stomach preparation The skin of unicorn leatherjacket (A. monocerous) was obtained from Sea Wealth Frozen Food Co., Ltd., Songkhla, Thailand. Stomachs of albacore tuna (Thunnus alalunga) and yellowfin tuna (Thunnus albacares) were obtained from Tropical Canning Public Co., Ltd., Songkhla, Thailand. The samples were packed in polythene bags and kept in ice using a solid/ice ratio of 1:2 (w/w) and transported to the Department of Food Technology, Prince of Songkla University, Hat Yai within 1 h. The skin was removed manually and the cleaned skin was washed with iced tap water (0–2 °C). Prepared skin was then cut into small pieces (0.5 Â 0.5 cm2). Both prepared skin and stomachs were placed in polyethylene bags and stored at À20 °C until use. The storage time was less than 2 months. 2.3. Preparation of stomach extract The frozen stomachs of both types of tuna were thawed using running water (26–28 °C) until the core temperature reached À2 to 0 °C. The sample was cut into pieces (1 Â 1 cm2) and finely ground in liquid nitrogen using a National Model MX-T2GN blender (Taipei, Taiwan), according to the method of Klomklao, Benjakul, and Visessanguan (2004). Stomach powder was suspended in 50 mM sodium phosphate buffer (pH 7.2) at a ratio of 1:10 (w/v). The mixture was stirred continuously at 4 °C for 30 min. The suspension was centrifuged at 7700g for 30 min at 4 °C using a Beckman Model Avanti JE centrifuge (Beckman Coulter, Inc., Fullerton, CA) to remove the tissue debris. The supernatant was collected and referred to as ‘stomach extract’. The protein content in the stomach extract was measured as per the method of Lowry, Rosebrough, Farr, and Randall (1951), using bovine serum albumin as a standard. 2.4. Assay of proteolytic activity Prior to assay, the stomach extracts from albacore tuna or yellowfin tuna or porcine pepsin were adjusted to pH 2 with 1 M HCl and the mixtures were allowed to stand at 4 °C for 10 min, as described by Nalinanon et al. (2007a). The treated stomach extracts were centrifuged at 5000g for 10 min at 4 °C, using a refrigerated centrifuge. The acidified supernatants were collected and used as the sources of activated pepsin.

Proteolytic activity of acidified stomach extract from both tuna and pepsin powder from porcine stomach mucosa was determined using haemoglobin as a substrate, as per the method of Nalinanon et al. (2007a), with a slight modification. To initiate the reaction, 200 ll of stomach extract were added into the assay mixture containing 200 ll of 2% haemoglobin, 200 ll of distilled water and 625 ll of McIlvaine’s buffer (0.2 M Na-phosphate and 0.1 M Na citrate, pH 2.0). Appropriate dilution was made to ensure that the amount of enzyme was not excessive for the available substrate in the assay system. The reaction was conducted at 50 °C for 20 min. To terminate the enzymatic reaction, 200 ll of 50% (w/v) trichloroacetic acid (TCA) were added. Unhydrolysed protein substrate was allowed to precipitate for 15 min at 4 °C, followed by centrifuging at 4725g for 10 min at room temperature (26–28 °C) using a MIKRO 20 centrifuge (Hettich Zentrifugen Tuttlingen, Germany). The oligopeptide content in the supernatant was measured by the Lowry method (Lowry et al., 1951), using tyrosine as a standard. One unit of activity was defined as the amount releasing 1 lmol of tyrosine per min. A blank was performed in the same manner, except that the acidified stomach extract was added into the reaction mixture after the addition of 50% TCA (w/v). 2.5. Extraction of pepsin-solubilised collagen (PSC) All procedures were carried out at 4 °C with gentle stirring. The collagen from the skin of unicorn leatherjacket was extracted following the method of Nalinanon et al. (2007b) with slight modification. Skin was soaked in 0.1 M NaOH with a skin/solution ratio of 1:20 (w/v), with continuous stirring to remove non-collagenous proteins for 6 h. The solution was changed every 2 h. The alkaline-treated skin was then washed with cold water until the pH of the wash water became neutral or faintly basic. Residual fat in the skin was removed using 10% (v/v) butyl alcohol with a sample/solution ratio of 1:10 for 18 h with a change of solution every 6 h. Defatted skin was thoroughly washed with 15 volumes of cold water (4–5 °C). To extract PSC, the prepared skin was soaked in 0.5 M acetic acid with a solid/solution ratio of 1:15 (w/v) for 48 h in the presence of acidified stomach extracts or acidified porcine pepsin at a level of 20 units/g of defatted skin. The mixture was filtered with two layers of cheesecloth to remove undissolved debris. The filtrate was added with NaCl to obtain a final concentration of 2.6 M in the presence of 0.05 M tris(hydroxymethyl) aminomethane (pH 7.0). The resultant precipitate was collected by centrifuging at 20,000g for 60 min at 4 °C. The pellets were dissolved in 10 volumes of 0.5 M acetic acid and dialysed against 9 volumes of 0.1 M acetic acid and distilled water, respectively. The dialysate was finally freeze-dried using a Model Coolsafe 55, Scanvac, (Coolsafe, Lynge, Denmark). The yield of PSC was calculated and expressed as dry matter/wet weight of skin. PSC was then subjected to analysis. 2.6. Amino acid analysis PSC samples were hydrolysed under reduced pressure in 4 M methanesulfonic acid containing 0.2% (v/v) 3-2(2-aminoethyl)indole at 115 °C for 24 h. The hydrolysates were neutralised with 3.5 M NaOH and diluted with 0.2 M citrate buffer (pH 2.2). An aliquot of 0.4 ml was applied to an amino acid analyser (MLC703; Atto Co., Tokyo, Japan). 2.7. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) SDS–PAGE was performed by the method of Laemmli (1970). The PSC samples were dissolved in 5% SDS and the mixtures were incubated at 85 °C for 1 h. The mixture was centrifuged at 4000g

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for 5 min at room temperature to remove undissolved debris. Solubilised samples were mixed at 1:1 (v/v) ratio with the sample buffer (0.5 M Tris–HCl, pH 6.8, containing 4% SDS and 20% glycerol) in the presence or absence of 10% b-ME. The mixtures were boiled in boiling water for 2 min. Samples (15 lg protein) were loaded onto polyacrylamide gels comprising a 7.5% running gel and a 4% stacking gel and subjected to electrophoresis at a constant current of 15 mA/gel, using a Mini Protein II unit (Bio-Rad Laboratories, Inc., Hercules, CA). After electrophoresis, gel was stained with 0.05% (w/v) Coomassie Blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid and destained with 30% (v/v) methanol and 10% (v/v) acetic acid. High molecular weight markers were used to estimate the molecular weight of proteins. Type I calf skin collagen was used as a standard.

2.11. Viscosity of collagen solution Collagen was dissolved in 0.1 M acetic acid to obtain a concentration of 0.03% (w/v). The solution (500 ml) was subjected to viscosity measurement using a Brookfield Synchrolectic viscometer (model DVII+, Brookfield Eng. Labs Inc., Stoughton, MA) with spindle No. 1 and speed of 100 rpm. Collagen solution was heated from 4 to 52 °C with a heating rate of 4 °C/min. At the designated temperature, the solution was held for 30 min prior to viscosity determination. The relative viscosity was calculated in comparison to that obtained at 4 °C. 2.12. Collagen solubility The collagen solubility was determined by the method of Montero, Jimenez-Colmenero, and Borderias (1991) with slight modification. Collagen was dissolved in 0.5 M acetic acid to obtain a final concentration of 3 mg/ml and the mixture was stirred at 4 °C until collagen was completely solubilised. 2.12.1. Effect of pH on collagen solubility Collagen solution (8 ml) was transferred to a centrifuge tube and the pH was adjusted with either 6 N NaOH or 6 N HCl to obtain a final pH ranging from 1 to 10. The volume of solutions was made up to 10 ml by deionised water previously adjusted to the same pH as the collagen solution. The solution was centrifuged at 20,000g for 30 min at 4 °C. Protein content in the supernatant was measured by the Lowry method (Lowry et al., 1951) using bovine serum albumin as a standard. Relative solubility was calculated in comparison to that obtained at the pH rendering the highest solubility. 2.12.2. Effect of NaCl on collagen solubility Collagen (6 mg/ml) was dissolved in 0.5 M acetic acid. Solution (5 ml) was added with 5 ml of NaCl in 0.5 M acetic acid at various concentrations (0%, 2%, 4%, 6%, 8%, 10% and 12% (w/v)), in which the final concentrations of 0%, 1%, 2%, 3%, 4%, 5% and 6% were obtained. The mixture was stirred continuously at 4 °C for 30 min, followed by centrifuging at 20,000g for 30 min at 4 °C. Protein content in the supernatant was measured and the relative solubility was calculated as previously described. 2.13. Statistical analyses All experiments were performed in triplicate and a completely randomised design (CRD) was used. Data were presented as means ± standard deviation and a probability value of <0.05 was considered significant. Analysis of variance (ANOVA) was performed and mean comparisons were done by Duncan’s multiple range tests. Analysis was performed using SPSS 11.0 for Windows (SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Yields of PSC from the skin of unicorn leatherjacket The yields of APSC and YPSC extracted with the aid of albacore tuna and yellowfin tuna stomach extract were 8.48 ± 0.3% and 8.40 ± 0.3% (wet weight basis), respectively. A lower yield (7.56 ± 0.4%) was obtained for PPSC extracted with the aid of porcine pepsin. Without pepsin, much lower yield (4.19 ± 0.4%) was obtained. The skin was not completely solubilised in 0.5 M acetic acid. The dense covalent cross-linking through the condensation of aldehyde groups at the telopeptide regions of collagen chains and the inter-molecular cross-linked collagen were not readily solubilised by acetic acid (Jongjareonrak et al., 2005). The results sug-

2.8. Peptide mapping of collagen Peptide mapping of PSC was performed according to the method of Saito, Kunisaki, Urano, and Kimura (2002) with slight modification. The sample (0.2 mg) were dissolved in 0.1 ml of 0.1 M sodium phosphate (pH 7.2) containing 0.5% (w/v) SDS. After the addition of 10 ll of the sample buffer containing 5 lg of V8 protease from S. aureus or 0.05 lg of lysyl endopeptidase from A. lyticus to collagen solutions, the reaction mixture was incubated at 37 °C for 25 and 5 min for V8 protease and lysyl endopeptidase, respectively. The reaction was terminated by submerging the reaction mixture in boiling water for 3 min. Peptides generated by the protease digestion were determined by SDS–PAGE using 7.5% separating gel and 4% stacking gel. High molecular weight markers (GE Healthcare UK) were used to estimate the molecular weight of peptides.

2.9. ATR-FTIR analysis PSC samples from the skin of unicorn leatherjacket were subjected to attenuate total reflectance/Fourier-transform infrared spectroscopy (ATR/FTIR). FTIR spectrometer (Model Equinox 55, Bruker, Ettlingen, Germany) equipped with a horizontal ATR trough plate crystal cell (45° ZnSe; 80 mm long, 10 mm wide and 4 mm thick) (PIKE Technologies Inc., Madison, WI) was used. For spectral analysis, the PSC samples were placed onto the crystal cell and the cell was clamped into the mount of the FTIR spectrometer. The spectra in the range of 600–4000 cmÀ1 with automatic signal gain were collected in 32 scans at a resolution of 4 cmÀ1 and were compared against a background spectrum recorded from the clean empty cell at 25 °C.

2.10. Differential scanning calorimetry Differential scanning calorimetry (DSC) of the PSC samples was run following the method of Rochdi, Foucat, and Renou (2000) with slight modification. The samples were rehydrated by adding deionised water or 0.05 M acetic acid to dried samples at a solid/solution ratio of 1:40 (w/v). The mixtures were allowed to stand for 2 days at 4 °C. DSC was performed using a differential scanning calorimeter (Perkin Elmer, Model DSC7, Norwalk, CA). Temperature calibration was run using the Indium thermogram. The samples were accurately weighed into aluminium pans and sealed. The samples were scanned at 1 °C/min over the range of 20–50 °C using iced water as the cooling medium. An empty pan was used as the reference. Total denaturation enthalpy (DH) was estimated by measuring the area in the DSC thermogram. The maximum transition temperature (Tmax) was estimated from the thermogram.

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gested that skin of unicorn leatherjacket contained a large amount of cross-links, leading to a low solubility of collagen in acid (Foegeding, Lanier, & Hultin, 1996; Zhang et al., 2007). With the further limited hydrolysis by pepsin, the cross-links at the telopeptide region were most likely cleaved as shown by the higher extractability. Collagen could be solubilised into the extracting solution in the presence of both tuna stomach extracts as well as porcine pepsin to a greater extent, compared with that extracted without pepsin. The results are in agreement with Nalinanon et al. (2007b) who reported the increased efficiency of collagen extraction with the aid of pepsin from bigeye snapper stomach or porcine pepsin. The differences in the collagen yield were governed by the sources of pepsin. Different pepsins might cleave telopeptide regions of collagens from skin of unicorn leatherjacket to different degrees. Due to the superior extraction efficiency of pepsins from both albacore tuna or yellowfin tuna, they could be used as promising replacers for porcine pepsin. 3.2. Amino acid composition of PSC from the skin of unicorn leatherjacket Amino acid composition of PSC extracted from the skin of unicorn leatherjacket is presented in Table 1. For all PSCs extracted with the aid of pepsins from different sources including APSC, YPSC and PPSC, glycine was the most predominant amino acid in all collagens. Generally, glycine represents nearly one-third of the total residues and occurs at every third residue in collagen except for the first 14 amino acid residues from the N-terminus and the first 10 amino acids from the C-terminus (Burghagen, 1999). Alanine was found as the second most abundant amino acid in all collagens. All collagens contained no cysteine and negligible tryptophan content. The imino acid content (hydroxyproline and proline) of APSC, YPSC and PPSC was 187, 169, 192 residues/1000 residues, respectively. Thus, APSC and PPSC contained the higher imino acid content, compared with YPSC. Calf skin collagen contained 215 residues/1000 residues (Giraud-Guille, Besseau, Chopin, Durand, & Herbage, 2000). The degree of hydroxylation of proline was calculated to be around 40% in PSC from the skin of unicorn leatherjacket. The degree of hydroxylation of proline residues plays an

important role in stabilising the triple helix of collagen (Ramachandran, 1988). Hydroxylation of proline in APSC and PPSC was slightly higher than that in YPSC, suggesting a slightly more complex structure of both PSCs than that from YPSC as shown by the higher degree of hydroxylation. APSC and PPSC generally showed the similar amino acid composition, but had some differences in comparison with YPSC. APSC and PPSC had the higher contents of glycine and alanine than did YPSC. On the other hand, the former possessed lower contents of aspartic acid, asparagine, glumatic acid, glutamine, valine, leucine and lysine than the latter. The result suggested that pepsin used for collagen extraction had an influence on the amino acid composition of resulting collagen. 3.3. Protein pattern of PSC from the skin of unicorn leatherjacket Protein patterns of PSC from the skin of unicorn leatherjacket determined under reducing and non-reducing conditions are illustrated in Fig. 1. All PSCs consisted of two a1-chains and one a2chain. High molecular weight components, including b-, c-components as well as the cross-linked constituents were also observed in all PSCs. PSCs isolated from the skin of unicorn leatherjacket were most likely classified as Type I collagen, which consisted of a heterotrimer of two identical a1-chains and one a2-chain (Muyonga et al., 2004). A slight difference in the protein pattern was noticeable in YPSC, compared with APSC and PPSC. Slight degradation of major proteins with the concomitant formation of low molecular weight peptides was noticeable when pepsin from yellowfin tuna stomach was used. It was suggested that pepsin from yellowfin tuna stomach was able to hydrolyse tropocollagen, as indicated by the decreased band intensity of b- and a-chains. It was noted that molecular weights of a-chains of all collagens were slightly lower than that of ASC. The results are in accordance with Nalinanon et al. (2007a) who reported that molecular weights of achains of collagen from bigeye snapper skin extracted with the aid of pepsin from the same fish species decreased slightly when compared with ASC. Hence, it can be inferred that pepsin most likely cleaved some peptides in the telopeptide region. As a consequence, a slight decrease in molecular weight of PSC was noticeable, in comparison with that of ASC. Generally, the higher molecular weight components including c-chain were found at a lower content in PSC, compared with those of ASC. This result indicated that pepsin used in this study could cleave inter-molecular cross-links

Table 1 Amino acid composition of the pepsin-solubilised collagen from the skin of unicorn leatherjacket (expressed as residues/1000 residues). Amino acid Aspartic acid/asparagine Threonine Serine Glutamic acid/glutamine Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine Tryptophan Hydroxyproline Proline Imino acid APSC 49 27 38 74 320 140 0 22 12 8 18 2 12 4 28 6 53 0 81 106 187 YPSC 55 30 39 86 290 134 0 26 13 12 27 5 15 4 33 8 53 1 72 97 169 PPSC 47 27 36 72 325 141 0 21 12 7 16 2 12 4 27 6 53 0 83 109 192

Non-reducing

Reducing

212kDa 170kDa 116kDa

β α1 α2

76kDa 70kDa

53kDa

M

I

ASC

A

Y

P

ASC

A

Y

P

APSC: pepsin solubilised collagen using albacore tuna pepsin, YPSC: pepsin solubilised collagen using yellowfin pepsin and PPSC: pepsin solubilised collagen using porcine pepsin.

Fig. 1. SDS–PAGE pattern of pepsin-solubilised collagen from the skin of unicorn leatherjacket under reducing and non-reducing conditions. M: high molecular weight markers, I: Type I calf skin collagen, ASC: acid-solubilised collagen, A: pepsin-solubilised collagen using albacore tuna pepsin, Y: pepsin-solubilised collagen using yellowfin tuna pepsin and P: pepsin-solubilised collagen using porcine pepsin.

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of a-chains. It was demonstrated that two types of inter-molecular bonds, side-to-side and end-to-end bond, in calf collagen can easily be cleaved by pepsin, whereas head-to-tail bond is relatively pepsin resistant (Zimmermann, Pikkarainen, Fietzek, & Kuhn, 1970). Drake, Davison, Bump, and Schmitt (1966) reported that most of intra- and inter-molecular cross-links found in collagen occur through the telopeptide region. No differences in the electrophoretic patterns of PSC were noticeable in the presence and absence of b-ME. This indicated that there was no disulphide bond in PSCs. Some of the telopeptides of calf skin tropocollagen are vulnerable to pepsin actions, since intra-molecular cross-links are broken on pepsin digestion and fragments comprising a small fraction of the molecule (approximately 1%) become dialysable (Drake et al., 1966). 3.4. Peptide map of PSC from the skin of unicorn leatherjacket Lysyl endopeptidases from A. lyticus and S. aureus V8 protease have been employed for peptide mapping of collagens (Kittiphattanabawon et al., 2005). The peptide maps of PSC digested by both enzymes are shown in Fig. 2. When V8 protease was used, the band intensities of b- and a-chains decreased with the concomitant increase in lower molecular weight peptides. Among all PSCs, YPSC was more susceptible to hydrolysis by V8 protease. This was evidenced by the almost total disappearance of all major components such as a-, b-chains, etc. When comparing between APSC and PPSC, the former was more prone to hydrolysis by V8 protease than was the latter. V8 protease shows a high specific preference for glutamic acid and aspartic acid residues of proteins (Kittiphattanabawon et al., 2005). Due to higher content of aspartic acid and glutamic acid (Table 1), YPSC was more prone to hydrolysis by V8 protease than APSC and PPSC. When lysyl endopeptidase was used, similar results were observed. YPSC underwent hydrolysis to the highest extent. PPSC was more resistant to hydrolysis, compared with APSC. However, different peptide maps were noticeable when two different enzymes were used for digestion. The differences in peptide maps between the different PSCs generated by lysyl endopeptidase or V8 protease digestion suggested that there might be some differences in their primary structures, especially the a-helix strand, in terms of sequence as well as amino acid composition (Jongjareonrak et al., 2005). Thus the degree of hydrolysis by protease might be different, due to variation

in the collagen structure and composition. Peptide maps of collagens were reported to differ among sources and species (Mizuta, Yamasa, Miyagi, & Yoshinaka, 1999). Thus, PSCs prepared with the aid of different pepsins might be different in terms of domain or cross-links, as shown by the difference in peptide maps generated by limited digestion under suitable conditions for each enzyme. 3.5. Thermal denaturation of PSC from the skin of unicorn leatherjacket Thermal transitions of PSC from the skin of unicorn leatherjacket dispersed in 0.05 M acetic acid or deionised water are depicted in Fig. 3. Endothermic peaks with maximum peak temperatures (Tmax) of 29.36 ± 0.7, 29.34 ± 0.5 and 29.33 ± 0.5 °C were found in APSC, YPSC and PPSC dispersed in 0.05 M acetic acid, respectively. When PSC were suspended in deionised water, Tmax values of 31.73 ± 0.8, 31.68 ± 0.7 and 31.98 ± 0.8 °C were observed, respectively. It was noted that Tmax shifted to a lower temperature (p < 0.05), when PSC was dispersed in acetic acid. The results suggested that intra-molecular hydrogen bonds stabilising the triple helix structure of collagen might be disrupted at some levels, in the presence of acetic acid, mainly due to the repulsion of collagen molecules in acidic solution. This alteration resulted in the decrease in Tmax. For the same medium used for suspending PSC, similar Tmax was noticeable among all PSC samples. Nevertheless, differences in DH were noticeable among all three samples. In acetic acid, YPSC showed the lowest DH, while PPSC had the highest

Original

V8 protease

Lysyl endopeptidase

212kDa 170kDa 116kDa

76kDa 70kDa

53kDa

M

A

Y

P

A

Y

P

A

Y

P

Fig. 2. Peptide maps of pepsin-solubilised collagens from the skin of unicorn leatherjacket digested by V8 protease and lysyl endopeptidase. M: high molecular weight markers, A: pepsin-solubilised collagen using albacore tuna pepsin, Y: pepsin-solubilised collagen using yellowfin pepsin and P: pepsin-solubilised collagen using porcine pepsin.

Fig. 3. DSC thermogram of pepsin-solubilised collagen from the skin of unicorn leatherjacket dispersed in 0.05 M acetic acid (a) and in deionised water (b). APSC: pepsin-solubilised collagen using albacore tuna pepsin, YPSC: pepsin-solubilised collagen using yellowfin pepsin and PPSC: pepsin-solubilised collagen using porcine pepsin.

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DH (p < 0.05). For PSC dispersed in deionised water, the lowest DH was found in PPSC, compared with APSC and YPSC (p < 0.05). The differences in Tmax and DH among all PSC samples might be governed by the composition and sequence of amino acid as well as the tertiary structure of collagen. Tmax values of PSC samples from the skin of unicorn leatherjacket were much lower than that of pig skin collagen (37 °C) (Ikoma, Kobayashi, Tanaka, Walsh, & Mann, 2003) and that of calf skin collagen (40.8 °C) (Komsa-Penkova, Koyonava, Kostov, & Tenchov, 1999). It was found that PSC from the skin of unicorn leatherjacket showed higher Tmax values than collagen from other fish species, such as bigeye snapper (28.68 °C), ocellate puffer fish (28.0 °C) and grass carp (24.6 °C) (Kittiphattanabawon et al., 2005; Nagai, Araki, & Suzuki, 2002; Zhang et al., 2007). Thermal stability of collagen is influenced by the imino acid content. The higher the imino acid content, the more stability is obtained. The molecular structure of collagen is maintained mainly by restrictions on changes in the secondary structure of the polypeptide chain, imposed by the pyrrolidine rings of proline and hydroxyproline, and also partially stabilised by the hydrogen bond through the hydroxyl group of hydroxyproline (Zhang et al., 2007). Factors influencing the change of collagen include the content of imino acid, degree of hydroxylation of proline and the content of the Gly-Pro-Hyp sequence (Burjanadze, 1999). The thermal stability of the collagen triple helix is attributed to the hydrogen-bonded networks, mediated by water molecules, which connect the hydroxyl group of hydroxyproline in one strand to the main chain amide or carboxyl groups of another chain (Babu & Ganesh, 2001). Therefore, the differences in hydroxyproline content might determine the thermal transition temperatures of collagens from different fish species.
3.6. ATR-FTIR of PSC from the skin of unicorn leatherjacket FTIR spectra of PSCs from the skin of unicorn leatherjacket are depicted in Fig. 4. The absorption bands in the spectra of all PSC samples were situated in the amide band region such as amide I (1600–1700 cmÀ1), amide II (1500–1600 cmÀ1) and amide III (1200–1300 cmÀ1) as previously reported by Muyonga et al. (2004). For the spectrum of APSC, the characteristic absorption bands at wave numbers of 3293.06, 3079.61, 1631.76, 1546.71 and 1235.36 cmÀ1 were observed, whereas YPSC showed main absorption bands at wave numbers of 3294.37, 3079.74, 1639.71,

1545.34 and 1235.14 cmÀ1. The characteristic absorption bands of PPSC were noticeable at wave numbers of 3294.37, 3085.53, 1634.77, 1546.30 and 1235.67 cmÀ1. The amide I bands are originated from C@O stretching vibrations coupled to N–H bending vibrations, CN stretch and CCN deformation (Bandekar, 1992). For amide I band, APSC had the lowest wave number, indicating the interaction of C@O with the adjacent chains, while YPSC had the highest wave number. The amide II bands of all PSCs were obtained at similar wave number, representing N–H bending vibrations coupled to C–N stretching vibrations. Generally, the shift to the lower wave number showed the existence of hydrogen bonds in each collagen (Li, Liu, Gao, & Chen, 2004). The amide III represented the combination peaks between N–H deformation and C– N stretching vibrations and was involved with the triple helical structure of collagen (Muyonga et al., 2004). The triple helical structure of collagens extracted with the aid of pepsin was confirmed from the IR ratio between amide III and 1450 cmÀ1 which was approximately 1. The other bands, arising from the stretching vibrations of N–H group, of a medium to weak intensity, appeared at 3293.06, 3294.37 and 3294.37 cmÀ1, corresponding to amide A, which occurs commonly in the range of 3280–3300 cmÀ1. The amide B with similar wave number (3079.61 cmÀ1, 3079.74 cmÀ1) was observed for APSC and YPSC. Higher wave number of 3085.53 cmÀ1 was noticeable for PPSC. Amide B corresponds to asymmetric stretch vibration of @C–H as well as ÀNHþ . 3 Fourier-transform infrared study indicated that PSC from skin of unicorn leatherjacket had some differences in functional groups and inter- and intra-molecular interaction. Thus, the pepsin used might affect the resulting PSC to some degree. 3.7. Viscosity of PSC from the skin of unicorn leatherjacket Relative viscosities of PSCs in 0.1 M acetic acid subjected to heat-treatment at different temperatures are depicted in Fig. 5. Collagen solutions are highly structured systems associated with the high viscosity. The collagen is irreversibly destroyed as a result of thermal denaturation. Viscosity of APSC and YPSC decreased continuously when heated up to 32 °C. Rate of decrease was retarded in the temperature range of 36–52 °C. The relative decrease of viscosity up to 32 °C might be due to continuous disruption of hydrogen bonds, which stabilised the structure of collagen. Nevertheless, PPSC had the marked decrease at temperature above 32 °C.

Fig. 4. Fourier-transform infrared spectra of pepsin-solubilised collagen from the skin of unicorn leatherjacket. APSC: pepsin-solubilised collagen using albacore tuna pepsin, YPSC: pepsin-solubilised collagen using yellowfin pepsin and PPSC: pepsinsolubilised collagen using porcine pepsin.

Fig. 5. Relative viscosity of the solution of pepsin-solubilised collagen from the skin of unicorn leatherjacket at different temperatures. APSC: pepsin-solubilised collagen using albacore tuna pepsin, YPSC: pepsin-solubilised collagen using yellowfin pepsin and PPSC: pepsin-solubilised collagen using porcine pepsin.

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The high viscosity can be attributed to the high proportion of band c-chains, which favoured the entanglement of molecules (Ogawa et al., 2004). Collagen denatures completely at a temperature above 40 °C to a mixture of random coils of single, double and triple strands (Wong, 1989). Denaturation of collagen structure due to heat-treatment is associated with changes in viscosity. Among all PSCs, APSC showed the highest viscosity (22.8 cP at 4 °C), followed by YPSC (21.1 cP at 4 °C) and PPSC (19.6 cP at 4 °C), respectively. APSC might contain a higher proportion of inter- and intramolecular cross-links stabilising the structure of collagen. 3.8. Effects of pH and NaCl on collagen solubility Fig. 6 shows the effect of pH and NaCl on the solubility of PSC from the skin of unicorn leatherjacket. All PSCs were solubilised in the pH range of 1–6, with the highest solubility at pH 2 (p < 0.05). The decrease in solubility was observed in the pH range of 7–8 (p < 0.05). The dissolved protein was found to precipitate in this pH range. The observation of precipitation and aggregation at a particular pH can be attributed to hydrophobic interaction among

collagen molecules, which increases at pI, and the total net charge of protein molecules becomes zero (Jongjareonrak et al., 2005). Slight increase in solubility was noticeable at pH 9 and 10. This was probably due to the repulsive effect of collagen molecules at pH above pI. The differences in the pH determining the solubility of collagens were caused by the differences in the molecular properties and conformations of collagens (Kittiphattanabawon et al., 2005). All PSCs had a slight decrease in solubility at NaCl concentrations of 0–2%. Drastic decrease was observed at NaCl concentration higher than 2% (Fig. 6b). At NaCl concentration of 4%, APSC showed the highest solubility (p < 0.05), followed by YPSC and PPSC, respectively. At high NaCl concentration, salting-out effect might occur. As a result, salt might bind with water and collagen molecules might more likely undergo precipitation. The differences in amino acid composition and structure might have an impact on solubility, especially at high NaCl concentration. Thus, the collagen extracted by different sources of pepsin might have different molecular properties, leading to the varied characteristics of collagens. 4. Conclusion Collagens (PSC) could be extracted from the skin of unicorn leatherjacket with the aid of pepsin from tuna stomach. The collagens extracted with the aid of albacore tuna pepsin were characterised as Type I collagen, exhibiting similar properties to the PSC extracted by using porcine pepsin. PSC were solubilised in acidic pH range, while NaCl at higher concentration could lower their solubility. Thus, the applications should be maximised based on their characteristics. Acknowledgements The authors would like to express their sincere thanks to Graduate School, Prince of Songkla University and TRF senior research scholar program for the financial support. References
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Fig. 6. Relative solubility (%) of pepsin-solubilised collagen from the skin of unicorn leatherjacket as affected by different pH (a) and NaCl (b) concentrations. APSC: pepsin-solubilised collagen using albacore tuna pepsin, YPSC: pepsin-solubilised collagen using yellowfin pepsin and PPSC: pepsin-solubilised collagen using porcine pepsin.

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