Extraction and Characterisation of Pepsin-Solubilised Collagen from Fins, Scales, Skins, Bones and Swim Bladders of Bighead Carp

Food Chemistry xxx (2012) xxx–xxx

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Food Chemistry

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Extraction and characterisation of pepsin-solubilised collagen from ﬁns, scales, skins, bones and swim bladders of bighead carp (Hypophthalmichthys nobilis) ⇑ Dasong Liu a, Li Liang a, Joe M. Regenstein b, Peng Zhou a, a State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, China b Department of Food Science, Cornell University, Ithaca, NY 14853-7201, USA article info abstract

Article history: The objective of this study was to extract and characterise pepsin-solubilised collagens (PSC) from the Received 26 September 2011 fins, scales, skins, bones and swim bladders of bighead carp and to provide a simultaneous comparison Received in revised form 15 December 2011 of five different sources from one species. The PSC were mainly characterised as type I collagen, contain- Accepted 7 February 2012 ing two a-chains, and each maintained their triple helical structure well. The thermostability of PSC from Available online xxxx the internal tissues (swim bladders and bones) was slightly higher than that of PSC from the external tissues (fins, scales and skins). The peptide hydrolysis patterns of all PSC digests using the V8 protease Keywords: were similar. All PSC were soluble at acidic pH (1–6) and lost their solubility at NaCl concentrations above Collagen 30 g/l. The resulting PSC from the five tissues would all be potentially useful commercially. Pepsin Bighead carp Ó 2012 Elsevier Ltd. All rights reserved. Fins Scales Skins Bones Swim bladders V8 protease

1. Introduction nicity (Liu, Li, Miao, & Wu, 2009). However, the outbreaks of bovine sponge encephalopathy (BSE), transmissible spongiform encepha- Collagen is the major structural proteins in vertebrates and con- lopathy (TSE), foot-and-mouth disease (FMD) and avian influenza stitutes about 30% of the total animal’s protein (Muyonga, Cole, & have raised anxiety among some consumers of collagen and Duodu, 2004). It is unique in its ability to form insoluble fibres with collagen-derived products from these land-based animals. Further- high tensile strength and stability, and also in its right-handed tri- more, porcine collagen and other collagens from animals that were ple superhelical structure consisting of three similarly sized left- not religiously slaughtered are unacceptable to some religious and handed helical polypeptide chains with a Gly-X–Y repeating motif, ethnic groups, such as Jews and Muslims (Regenstein & Zhou, 2007). in which the X and Y positions are often occupied by proline and Therefore, the global demand for collagen from alternative sources hydroxyproline, respectively (Gelse, Pöschl, & Aigner, 2003). At such as aquatic animals has been increasing over the years. With the present, at least 29 collagen types have been identified, and each rapid development of the fish processing industry in China, large differs considerably in their sequence, structure and function quantities of by-products are generated, accounting for 50–70% (McCormick, 2009). They are widely distributed in the skins, bones, of the original raw material (Kittiphattanabawon, Benjakul, tendons, vascular system and intramuscular connective tissue, Visessanguan, Nagai, & Tanaka, 2005). Consequently, optimal use where they contribute to the stability and structural integrity of of these by-products is a promising way to protect the environment, these tissues and organs (Gelse et al., 2003). Particularly, type I col- to produce value-added products to increase the revenue to the fish lagen is found in all vertebrae connective tissues (Nagai, Suzuki, & processors, and to create new job/business opportunities. Nagashima, 2008). Research on the extraction and characterisation of collagen from Commonly isolated from by-products of land-based animals, by-products of marine animals has been reported (Ahmad & such as cows, pigs and poultry, collagen has been widely used in Benjakul, 2010; Jongjareonrak, Benjakul, Visessanguan, Nagai, & the food, pharmaceutical, and cosmetic industries because of its Tanaka, 2005; Kittiphattanabawon et al., 2005; Liu, Oliveira, & Su, excellent biocompatibility and biodegradability, and weak antige- 2010; Saito, Kunisaki, Urano, & Kimura, 2002). However, informa- tion regarding the preparation of collagen from freshwater fish is limited (Wang, Yang, Wang, & Du, 2008), and none of the work ⇑ Corresponding author. Tel./fax: +86 510 85912123. has characterised the collagen from different tissues of bighead E-mail address: [email protected] (P. Zhou). carp.

Bighead carp, native to Asia, is one of the ‘‘four major cultured modifications. All procedures were performed in a walk-in chill fish species’’ along with black carp, grass carp and silver carp room at a temperature no higher than 4 °C. grown in China (Wang, Yang et al., 2008), and it has been intro- duced into more than 70 other countries in Europe, South America, 2.3.1. Fin, bone and scale collagen and North America. In the United States, it is regarded as a highly To remove the non-collagenous proteins and pigments, the fins destructive invasive species. and bones (0.5 kg) were soaked in 0.1 M NaOH at a sample/alkaline Pepsin is able to cleave the cross-linked regions at the telopep- solution ratio of 1:10 (w/v). The mixture was stirred for 36 h using tide without damaging the integrity of the triple-helix and hence the C-MAG HS7 magnetic stirrer (IKA Werke GmbH & Co. KG, the extraction of collagen with limited pepsin digestion contrib- Staufen, Germany), changing the alkaline solution every 12 h. Sub- utes to a higher yield (Heu et al., 2010). To make better use of this sequently, the treated samples were decalcified with 0.5 M EDTA fish, the present study was conducted to extract and characterise (pH 7.5) at a sample/EDTA solution ratio of 1:10 (w/v) for 5 days, pepsin-solubilised collagens (PSC) from bighead carp fins, scales, with the EDTA solution being changed every day. Then the residues skins, bones and swim bladders and to provide a simultaneous were suspended in 10% (v/v) butyl alcohol to remove fat at a comparison of five different sources from one species at one time. sample/solution ratio of 1:10 (w/v) for 36 h with a change of solution every 12 h. After being fully washed with cold distilled water, 2. Materials and methods the residues were extracted with 0.5 M acetic acid containing 0.1% (w/v) pepsin at a sample/solution ratio of 1:10 (w/v) for 3 days. The 2.1. Chemicals and enzymes suspension was then centrifuged at 12,500g for 40 min at 4 °C using an Avanti J-E centrifuge (Beckman Coulter, Inc., Indianapolis, All chemicals used were of analytical grade. Pepsin from porcine IN, USA), and the supernatant was salted-out by adding NaCl to a gastric mucosa (EC 3.4.23.1; 2 crystallised and lyophilised; final concentration of 2.0 M. The resultant precipitate was 2800 U/mg dry matter based on haemoglobin hydrolysis; Sigma– collected by centrifugation at 12,500g for 40 min at 4 °C and then Aldrich Co., St. Louis, MO, USA); Staphylococcus aureus V8 protease dissolved in 0.5 M acetic acid at a sample/solution ratio of 1:10 (EC 3.4.21.19; lyophilised; 500–1000 U/mg based on the hydrolysis (w/v). The resulting solution was salted-out again with NaCl at a of N-t-Boc-L-glutamic acid a-phenyl ester; Sigma–Aldrich Co.); final concentration of 2.0 M, and the precipitate was collected bovine serum albumin (Sigma–Aldrich Co.); high molecular weight and redissolved in 0.5 M acetic acid under the same conditions (MW) markers (manufacturer’s specified MW: myosin, 220 kDa; employed above. The final solution was dialysed against cold dis- a2-macroglobulin, 170 kDa; b-galactosidase, 116 kDa; transferrin, tilled water using a dialysis bag with a nominal manufacturer’s 76 kDa; glutamic dehydrogenase, 53 kDa) were obtained from GE specified MW cut-off of 7 kDa (Shanghai Green Bird Science and Healthcare UK Ltd. (Amersham, Buckinghamshire, UK); Precision Technology Development Co., Shanghai, China) and then lyophi- Plus Protein All Blue Standards (Bio-Rad Laboratories, Inc., Hercules, lised using the Labconco freeze dryer (Labconco Corp., Kansas, CA, USA) consisted of a cocktail of pre-stained proteins with the MO, USA). The yield was calculated on the basis of the wet weight manufacturer’s specified MW of 10, 15, 20, 25, 37, 50, 75, 100, of the fins and bones, respectively. 150, and 250 kDa; sodium dodecyl sulphate (SDS), N,N,N0,N0-tetra- The procedures for extraction of PSC from the scales (0.5 kg) methyl ethylene diamine (TEMED), tris(hydroxymethyl)amino- were slightly different in that the times for decalcification was methane, acrylamide and bisacrylamide were purchased from shorten to 3 days, and the step for removing fat was omitted. Sangon Biotech Co., Ltd. (Shanghai, China). 2.3.2. Skin and swim bladder collagen 2.2. Preparation of fish fins, scales, skins, bones and swim bladders The skins and swim bladders (0.125 kg) were soaked in 0.1 M NaOH for 36 h, with the alkaline solution being changed every Live farmed bighead carps (Hypophthalmichthys nobilis), with 12 h. During alkaline treatment, the skins and swim bladders weights ranging from 2 to 3 kg, were obtained in the spring from would swell, and a sample/alkaline solution ratio of 1:30 (w/v) a local market in Wuxi, Jiangsu Province, China. They were trans- was used to ensure the mixture could be stirred by the magnetic ported to the laboratory in water within 30 min. Upon arrival at stirrer and a sample/butyl alcohol solution ratio of 1:30 (w/v) the laboratory, the fish were stunned by a sharp blow to the head was also used during the following defatting treatment. The depro- with a wooden stick. Then, the caudal fins, scales, skins, bones teinised residues were suspended in 10% (v/v) butyl alcohol for (vertebral columns with intervening meat remaining) and swim 36 h with a change of solution every 12 h. The defatted residues bladders were manually removed with a filleting knife and each were thoroughly washed with cold distilled water, and then sus- were washed with cold distilled water. The clean caudal fins, skins pended in 0.5 M acetic acid containing 0.1% (w/v) pepsin for 3 days. 2 and swim bladders were cut into small pieces (0.5 0.5 cm ) using During the extracting process, the viscosity of the solution would a scissor, and the bones were also cut into small pieces (0.5 cm in greatly increase, and a sample/solution ratio of 1:40 (w/v) was used length) and then broken using a hammer. All the prepared samples to ensure the solution stay with the appropriate viscosity and can were kept on ice prior to collagen extraction. Three different lots of be stirred by the magnetic stirrer. The subsequent procedures were bighead carp with 3 weeks intervals between purchases were used performed essentially as those applied to the fins and bones. and the extraction was performed once for each lot. It was not possible to determine if the three lots were from the same or different 2.4. Characterisation of collagen farms. The approximate moisture contents of the fins, scales, skins, bones and swim bladders were determined by drying at 105 °C for 2.4.1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis 24 h. (SDS–PAGE) SDS–PAGE was done according to the method of Laemmli 2.3. Extraction of collagen from fins, scales, skins, bones and swim (1970) using a 4% stacking gel and a 5% resolving gel made in the bladders lab. PSC were dissolved in 0.02 M sodium phosphate buffer (pH 7.2) containing 1% (w/v) SDS and 3.5 M urea to obtain a final con- The extraction of collagen was done according to the methods centration of 2 mg/ml, and then mixed with an equal volume of of Nagai and Suzuki (2000), Ogawa et al. (2004), and Matmaroh, sample buffer (0.5 M Tris–HCl, pH 6.8, containing 4% (w/v) SDS Benjakul, Prodpran, Encarnacion, and Kishimura (2011) with slight and 20% (v/v) glycerol). Then 20 ll of the sample (20 lg protein)

Please cite this article in press as: Liu, D., et al. Extraction and characterisation of pepsin-solubilised collagen from fins, scales, skins, bones and swim bladders of bighead carp (Hypophthalmichthys nobilis). Food Chemistry (2012), doi:10.1016/j.foodchem.2012.02.032 D. Liu et al. / Food Chemistry xxx (2012) xxx–xxx 3 was loaded in each well. The high MW markers were used to esti- Sampler accessory (Thermo Electron Corp., Madison, WI, USA). mate the MW of the bands. After electrophoresis, the gels were Lyophilised PSC were placed onto the single reflection germanium stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 50% crystal cell with a 45° crystal angle. The signals were automatically (v/v) methanol and 6.8% (v/v) glacial acetic acid for 5 h and de- collected for 64 scans over the range of 4000–400 cm1 at a reso- stained using 7.5% (v/v) of glacial acetic acid and 5% (v/v) methanol lution of 2 cm1 and were compared to a background spectrum for about 9 h with a change of solution every 3 h. The semi- collected from the clean empty cell at room temperature. quantitative analysis of band intensity was done using the Gel Doc EZ Imager with the Image Lab 3.0 software (Bio-Rad Laborato- 2.4.6. Collagen solubility test ries, Inc.). The effects of pH and NaCl on PSC solubility were done according to the method of Montero, Jiménez-Colmenero, and Borderìas 2.4.2. Determination of amino acid composition (1991). PSC were rehydrated in 0.5 M acetic acid at 4 °C for 12 h A 100 mg sample of PSC was hydrolysed in 8 ml of 6 M HCl in an to obtain final concentrations of 3 and 6 mg/ml. evacuated and sealed tube with a volume of 15 ml at 110 °C for To investigate the effect of pH on collagen solubility, 8 ml of 22 h. The hydrolysate was evaporated to dryness in a vacuum collagen solutions (3 mg/ml) were adjusted with either 6 M HCl evaporator (Shanghai Jing Hong Laboratory Instrument Co., or 6 M NaOH to obtain a final pH ranging from 1 to 10 in incre- Shanghai, China) at 50 °C and then diluted with 0.02 M HCl to ments of 1 pH unit. Then the volume of the solution was made 25 ml. An Agilent 1100 Series high performance liquid chromatog- up to 10 ml by adding 0.5 M acetic acid that was previously ad- raphy system (Agilent Technologies, Inc., Santa Clara, CA, USA) was justed to the same pH as the sample solution tested. The solution used with a C18 ODS HYPERSIL column (250 mm 4.6 mm i.d., was centrifuged at 20,000g at 4 °C for 30 min using the Sigma 5 lm particle size; Agilent Technologies, Inc.). Amino acid analysis 3K15 centrifuge (Sigma Laborzentrifugen GmbH, Osterode am was done using pre-column derivatisation with o-phthalaldehyde Harz, Germany). The protein content in the supernatant was mea- and fluorenylmethyl chloroformate. The individual amino acid sured according to the method of Lowry, Rosebrough, Farr, and content was based on the area of the corresponding peak as deter- Randall (1951) using weighed bovine serum albumin as a standard. mined by the software on the elution curves of the sample and The relative solubility was calculated in comparison to that standard (Sigma–Aldrich Co.), and the sample’s amino acid compo- obtained at the pH exhibiting the highest solubility. sition is expressed as residues per 1000 total residues recovered by To investigate the effect of NaCl on collagen solubility, 5 ml of the amino acid analyser. collagen solutions (6 mg/ml) were mixed with 5 ml of 0.5 M acetic acid containing NaCl with various concentrations of 0, 20, 40, 60, 2.4.3. Peptide hydrolysis patterns 80, 100 and 120 g/l, respectively. The subsequent procedures were The peptide hydrolysis patterns of PSC were studied according done essentially the same as those described for the effect of pH. to the method of Saito et al. (2002). PSC samples (0.2 mg) were dissolved in 0.1 ml of 0.1 M sodium phosphate buffer (pH 7.2) 2.5. Statistical analysis containing 0.5% (w/v) SDS, and then 10 ll of the same buffer containing 5 lgofS. aureus V8 protease was added to the protein Statistical analysis was done using SAS version 8.0 (1999, SAS solution. The reaction mixture was incubated at 37 °C for 25 min Institute, Inc., Cary, NC, USA). Analysis of variance (ANOVA) using and then placed in boiling water for 3 min to terminate the reac- the General Linear Model procedure and the difference between tion. Peptides generated by the protease digestion were separated means using the Duncan test were determined at an a level of 0.05. using the SDS–PAGE method of Laemmli (1970) with a 4% stacking gel and 7.5% resolving gel. The Precision Plus Protein All Blue 3. Results and discussion Standards were used as protein markers. 3.1. Yield 2.4.4. Differential scanning calorimetry (DSC) DSC studies were done using the Q2000 Series DSC (TA Table 1 shows the yields of PSC from five different tissues of big- Instruments, Inc., New Castle, DE, USA) equipped with a Refriger- head carp. Higher yields were obtained from the skins and swim ated Cooling System 90 and TA Universal Analysis 2000 software. bladders than for the fins, scales and bones (P < 0.05). Other The instrument was calibrated for temperature and enthalpy using researchers have reported the yields (wet weight basis) of fish indium as the standard and the measurements were done while collagen for other species as follows: bone and skin collagen of big- samples were constantly purged with ultrahigh-purity nitrogen eye snapper (1.6% and 10.9%, respectively) (Kittiphattanabawon (Wuxi Taihu Gas Factory, Wuxi, China) at 50 ml/min. The samples et al., 2005), scale collagen of silver carp (1.45%) (Zhang, Duan, were prepared according to the method of Kittiphattanabawon Ye, & Konno, 2010), skin collagen of unicorn leatherjacket (7.6%) et al. (2005) with slight modification. PSC were rehydrated in (Ahmad & Benjakul, 2010), and skin collagen of bigeye snapper 0.05 M acetic acid at a sample/solution ratio of 1:40 (w/v) for (Priacanthus tayenus and Priacanthus macracanthus) (7.7% and 2 days at 4 °C. Then rehydrated PSC samples (10 ± 0.5 mg) were accurately weighted into aluminium pans (TA Instruments, Inc.), hermetically sealed and scanned from 20 to 50 °C at a heating rate Table 1 of 1 °C/min. An empty sealed aluminium pan was used as the ref- Moisture contents (wet weight basis) of the fins, scales, skins, bones and swim bladders of bighead carp, and the yield of PSC based on dry and wet weight basis of erence. The maximum transition temperature (Tmax) was recorded the raw material. by the software as the peak temperature of each endothermic peak and the total denaturation enthalpy (DH, J/g sample material) for Tissues Moisture (%) Yield (%) each peak was determined by measuring the corresponding area Dry weight basis Wet weight basis under each endothermic peak. Fins 61.8 ± 0.8c 5.1 ± 0.6b 2.0 ± 0.2c Scales 58.1 ± 0.4d 2.7 ± 0.2b 1.1 ± 0.1c 2.4.5. Attenuated total reflectance-Fourier transform infrared Skins 71.0 ± 1.4b 60.3 ± 4.4a 17.5 ± 1.3a d b c (ATR-FT-IR) spectroscopy Bones 57.1 ± 0.2 2.9 ± 0.1 1.3 ± 0.1 Swim bladders 75.2 ± 1.4a 59.0 ± 8.1a 14.6 ± 2.0b The ATR-FT-IR spectra of PSC were obtained using a Nicolet Nexus 470 FT-IR spectrometer equipped with a Smart OMNI- a–d Means in a column followed by different letters differ significantly (P < 0.05).

7.1%, respectively) (Benjakul et al., 2010). The differences in yields & Hultin, 1996), which is in agreement with the previous reports might be attributed to the differences in fish species, biological for skin collagen from bigeye snapper (Benjakul et al., 2010), and conditions and preparative methods (McCormick, 2009; Muyonga skin, scale and bone collagen from deep-sea redfish and carp et al., 2004; Regenstein & Zhou, 2007). The above-mentioned data (Duan, Zhang, Du, Yao, & Konno, 2009; Wang, An et al., 2008). also indicated that the scales, skins, bones, fins and swim bladders However, another heterotrimer of type I collagen containing three of bighead carp may be useful sources of collagen with skins and non-identical a-chains, a1(I)a2(I)a3(I), was found in the skin and swim bladders having the most commercial potential all else being muscle collagen of rainbow trout (Saito et al., 2001). Since the a3 equal. However, scale collagen may have a unique niche in the chain had a similar MW to a1 chain and could not be separated kosher market, where only scaled fish are kosher and thus scale from the a1 chain using the electrophoretic conditions employed, collagen is clearly derived from kosher fish (Regenstein, Chaudry, it was not possible to determine if the a3 existed in PSC from dif- & Regenstein, 2003). ferent tissues of bighead carp (Kittiphattanabawon et al., 2005). Moreover, skin collagen from the chordate giant red sea cucumber

3.2. SDS–PAGE of collagen mainly consisted of [a1(I)]3 homotrimer (Liu et al., 2010). Additionally, a minor component with an approximate MW of Fig. 1 shows the electrophoretic patterns of PSC from the fins, 170 kDa was observed between the a- and b-chains for PSC from scales, skins, bones and swim bladders of bighead carp. All PSC the fins and scales of bighead carp, and the electrophoretic pat- were mainly composed of two different a-chains (a1 and a2). The terns were quite similar to that of type V collagen isolated from initial estimates of the staining ratio of a1 to a2 were 2.12, 2.11, carp and spotted mackerel muscle (Sato, Yoshinaka, Sato, & Tomita, 2.12, 2.13 and 2.13 for PSC from the fins, scales, skins, bones and 1989). According to the studies of Sato et al. (1989), type V collagen swim bladders, respectively. Using an approximate MW of may exist in the molecular form of a1(V)a2(V)a3(V) and

126 kDa for a1 and 116 kDa for a2, the MW corrected ratios for [a1(V)]2a2(V), and the minor component between the a- and b- the five different samples were 1.95, 1.94, 1.95, 1.96 and 1.96, thus chains has been tentatively postulated to be a mixture of a1(V) suggesting the ratio of a1 being approximately twofold higher than and a3(V) chains, while the a2(V) chain would be overlapped by that of a2. Generally, the mobility of the a-chains was similar the a1(I) chain. Similar components have also been reported for among the PSC from all five sources, indicating a similarity in their the PSC from snakehead scales (Liu et al., 2009), carp muscles (Sato subunit MW. Cross-links, contained in the telopeptide region of et al., 1988) and Japanese flounder muscles and skins (Nishimoto, tropocollagen, were removed by pepsin (Kittiphattanabawon, Sakamoto, Mizuta, & Yoshinaka, 2005). Mizuta, Hwang, and Benjakul, Visessanguan, & Shahidi, 2010); therefore, only small Yoshinaka (2003) also reported that PSC from the pectoral fin amounts of higher MW components, including b-chains (dimers cartilage of skate was believed to consist of three types of collagen, of the a-chains), as well as the inter- and intra-molecular cross- type I and type II as the major ones and type XI as the minor one. linked components were observed in all PSC. It was noted that PSC from the swim bladders showed fewer high MW components. 3.3. Amino acid composition of collagen Moreover, PSC from the fins, scales, skins and bones contained two different b-chains (Saito, Takenouchi, Kunisaki, & Kimura, 2001), The amino acid composition, expressed as residues per 1000 to- while only one major b-chain was observed in PSC from the swim tal residues recovered, is presented in Table 2. All PSC had glycine bladders. as the most abundant amino acid, and were also rich in alanine, In higher vertebrates, type I collagen is widely known as the proline and glutamic acid/glutamine, in descending order, while major fibrillar collagen that is found in most organs, and also negligible amounts of cysteine and tryptophan were found. Also, was the first collagen to be identified (Saito et al., 2001). Based all PSC contained hydroxyproline and hydroxylysine, the unique on the subunit composition and electrophoretic mobility (Sato, amino acids found in collagen. In general, glycine represents about Yoshinaka, Sato, Itoh, & Shimizu, 1988), it was suggested that the one-third of the total residues and is normally spaced at every PSC from all five sources were mainly composed of type I collagen, third residue in collagen except for the first 10 or so amino acid a heterotrimer containing two identical a1-chains and one residues at the C-terminus and the last 14 or so at the N-terminus a2-chain in the molecular form of [a1(I)]2a2(I) (Foegeding, Lanier, (Jongjareonrak et al., 2005; Kittiphattanabawon et al., 2005). Glycine, as the smallest amino acid with only a hydrogen atom side chain, allows the three helical a-chains to form the final superhelix (Bae et al., 2008). The amino acid profiles of all PSC were quite similar to those of type I collagen purified from carp muscle (Sato et al., 1988). However, slight differences in amino acid compositions were also observed for PSC from different tissues of bighead carp. PSC from the swim bladders and scales showed the highest amounts of valine and glycine, respectively. Moreover, the proline content was highest in PSC from the fins, scales and bones, while hydroxyproline was lowest in PSC from the fins and scales. The amino acid compositions are expected to affect the properties of collagen from different sources. The total imino acids (proline and hydroxyproline) content of PSC from all five tissues of bighead carp (156–175 residues per 1000 total residues) were lower than those of calf skin collagen (about 215 residues per 1000 total residues) and pig skin collagen (about 220 residues per 1000 total residues) (Zhang et al., 2007). The imino acids contribute to the thermal stability of collagens, which is one of the most important characteristics determining their potential use (Regenstein & Zhou, 2007). The pyrrolidine rings Fig. 1. SDS–PAGE patterns of PSC from the fins (FI), scales (SC), skins (SK), bones (BO) and swim bladders (SW) of bighead carp. The first lane is the protein standards of proline and hydroxyproline would impose restrictions on (Std). changes in the secondary structure of the polypeptide chain, and

Table 2 Amino acid compositions of PSC from the ﬁns, scales, skins, bones and swim bladders of bighead carp (residues/1000 total amino acid residues recovered).

Amino acid Fins Scales Skins Bones Swim bladders Aspartic acid/asparagine 50.3 47.6 49.4 50.4 47.1 Glutamic acid/glutamine 77.3 80.1 77.9 78.1 77.7 Serine 34.0 33.0 33.7 37.0 30.6 Histidine 4.3 5.5 3.8 4.0 3.5 Glycine 339 350 341 332 331 Threonine 27.6 27.1 27.5 26.3 28.4 Arginine 54.6 54.4 55.0 53.4 53.2 Alanine 122 125 126 120 123 Tyrosine 3.4 2.9 3.0 3.7 2.9 Cysteine 0.3 0.4 0.6 0.6 0.6 Valine 22.3 20.6 21.1 24.1 33.9 Methionine 14.9 15.7 15.1 14.0 14.5 Phenylalanine 14.7 13.9 14.5 15.4 14.5 Isoleucine 12.6 11.9 11.4 13.3 10.9 Leucine 23.5 22.1 22.0 22.8 20.8 Lysine 27.7 27.7 29.0 26.4 26.9 Tryptophan 0.0 0.0 0.0 0.0 0.0 Proline 107 100 92 100 95 Hydroxylysine 5.5 5.6 4.1 5.7 5.5 Hydroxyproline 58.8 56.2 73.2 73.8 80.5 Imino acids 166 156 165 174 175 Total 1000.0 1000.0 1000.0 1000.0 1000.0 hence help to maintain the triple helical structure (Bae et al., 2008; Nagai et al., 2008). Moreover, hydroxyproline also contributes to the stabilisation of the triple helical structure by the formation of interchain hydrogen bond through the hydroxyl group (Kittiphattanabawon et al., 2005). Generally, the collagens obtained from hot-water and warm-water fishes have higher total imino acid contents and hydroxyproline contents than those from cold-water and ice-water fishes (Regenstein & Zhou, 2007). Bighead carp is native to the large rivers and lakes of Asia, and the total imino acid contents and hydroxyproline contents of PSC from this fish is comparable to those of collagens from other warm-water fish species previously studied (Regenstein & Zhou, 2007).

3.4. Peptide hydrolysis patterns of collagen

Peptide hydrolysis patterns of PSC from different tissues of bighead carp digested using the V8 protease are shown in Fig. 2. After the digestion by the V8 protease, the bands of b-chains as well as other high MW components almost entirely disappeared. Moreover, the decreases in band intensity of both the a-chains

(a1 and a2) were observed with a concomitant generation of lower MW peptide fragments ranging broadly from 20 to 100 kDa. Gen- Fig. 2. Peptide hydrolysis patterns of PSC after V8 protease digestion from the fins (FI), scales (SC), skins (SK), bones (BO) and swim bladders (SW) of bighead carp. The erally, similar MW peptide fragments were generated for PSC from first lane is the protein standards (Std). all five sources. However, scale collagen from snakehead (Liu et al., 2009) and skin collagen from blacktip shark (Kittiphattanabawon considered less seriously than the peptide hydrolysis patterns, et al., 2010) using the same hydrolysis system showed different which are probably reflecting the primary structures of PSC. peptide hydrolysis patterns from those for bighead carp in the present study. The V8 protease exhibits a high degree of specificity for glutamic and aspartic acid residues of protein, and the differ- 3.5. Thermal stability of collagen ence in peptide hydrolysis patterns of collagen from different species has been attributed to differences in their primary structure The maximum transition temperature (Tmax) and total denatur- (Jongjareonrak et al., 2005; Kittiphattanabawon et al., 2005; Liu ation enthalpy (DH) of PSC from different tissues of bighead carp et al., 2009, 2010). Therefore, it may be suggested that the amino rehydrated in 0.05 M acetic acid are shown in Table 3. When the acid sequences as reflected by V8 protease digestion of PSC from raw material was subjected to the limited digestion by pepsin, the different tissues of bighead carp were generally similar, but dif- the triple helical structure was still predominant in the PSC ferent from those of collagen from other fish species previously (Jongjareonrak et al., 2005). Slightly higher Tmax were observed studied. Due to the limitations of the current method for measur- for PSC from the swim bladders and bones (37.3 and 36.4 °C, ing amino acid compositions with a 22 h digestion and the poten- respectively) than for PSC from the fins, scales and skins (35.6, tial impurities in the PSC, the slight differences in amino acid 35.2 and 35.7 °C, respectively) (P < 0.05). Moreover, the DH of compositions in the present study, especially when each amino PSC from the swim bladders and bones (1.39 and 1.35 J/g, respec- acid is expressed as residues per 1000 total residues, should be tively) was also slightly higher than that of PSC from the fins, scales

Table 3

Maximum transition temperature (Tmax) and total denaturation enthalpy (DH) of PSC from the ﬁns, scales, skins, bones and swim bladders of bighead carp rehydrated in 0.05 M acetic acid.

Tissues Tmax (°C) DH (J/g) Fins 35.6 ± 0.1c 1.23 ± 0.02d Scales 35.2 ± 0.1d 1.25 ± 0.01d Skins 35.7 ± 0.1c 1.32 ± 0.01c Bones 36.4 ± 0.1b 1.35 ± 0.01b Swim bladders 37.3 ± 0.0a 1.39 ± 0.02a a–d Means in a column followed by different letters differ significantly (P < 0.05). and skins (1.23, 1.25 and 1.32 J/g, respectively) (P < 0.05). The DH was very dependent on how the baseline was drawn and may be sensitive to impurities in the various preparations. Therefore, although the data showed two different groups of DH values, it was not clear that these represented real variations in the materials. Fig. 3. FT-IR spectra of PSC from the fins (FI), scales (SC), skins (SK), bones (BO) and swim bladders (SW) of bighead carp. The Tmax values of PSC from the five tissues of bighead carp were lower than that of calf skin collagen (40.8 °C) (Duan et al., 2009), but higher than those of collagens from the skins of dusky 1 spinefoot (28.7 °C) (Bae et al., 2008) and the skins of arabesque absorbance in the range of 1600–1700 cm was mainly related greenling (15.4 °C) (Nalinanon, Benjakul, & Kishimura, 2010). The to the C@O stretching vibration along the polypeptide backbone, and it can be a sensitive marker of the peptide’s secondary struc- Tmax values of collagen from different sources have been correlated with the contents of imino acids, temperature of normal habitat, ture (Muyonga et al., 2004). The amide I band of all PSC were ob- 1 seasons and age (Matmaroh et al., 2011; Regenstein & Zhou, served at a wavenumber of 1648 cm . Furthermore, the amide II 2007; Singh, Benjakul, Maqsood, & Kishimura, 2011). Collagen band, which is caused by the N–H bending vibration coupled with 1 from calf skin had a higher imino acid content (about 215 residues a C–N stretching vibration, normally occurs at 1550–1600 cm , per 1000 total residues) than did collagen from bighead carp and the shift to lower wavenumbers suggests the existence of (156–175 residues per 1000 total residues), and hence showed a hydrogen bonds (Ahmad & Benjakul, 2010; Duan et al., 2009). 1 higher Tmax value (Duan et al., 2009). Generally, collagens from fish The amide II band of all PSC were observe at 1546–1551 cm .In 1 species with a high habitat temperature had higher amounts of addition, the amide III band (1220–1320 cm ) is associated with imino acids and they also had higher thermal stability than those N–H deformation and C–N stretching vibrations, and an absorption 1 from fish with a low habitat temperature (Matmaroh et al., 2011; ratio of approximately 1 between the amide III and the 1454 cm Regenstein & Zhou, 2007). band indicates that the triple helical structure of collagen is intact (Ahmad & Benjakul, 2010; Benjakul et al., 2010; Heu et al., 2010). Many fish collagens had Tmax values lower than 30 °C and hence had lower thermal stability than mammalian collagens (Bae et al., The ratios of 1.04, 1.07, 1.12, 1.01 and 1.08 were obtained for the PSC from the fins, scales, skins, bones and swim bladders, respec- 2008). However, Tmax values above 30 °C have also been reported for collagens from the skins of some fish species, such as ornate tively, suggesting that the triple helical structures were well threadfin bream (33.4 °C) (Nalinanon et al., 2010), striped catfish maintained. (35.3 °C) (Singh et al., 2011), brownstripe red snapper (30.5 °C) (Jongjareonrak et al., 2005), and bigeye snapper (P. tayenus and P. 3.7. Effect of pH and NaCl concentration on collagen solubility macracanthus) (31.3 and 31.2 °C, respectively) (Benjakul et al., 2010). The comparatively high T values of these fish collagens max The effect of pH and NaCl concentration on the solubility of PSC indicated their high heat resistance and great structural stability, from different tissues of bighead carp in 0.5 M acetic acid is shown which might be beneficial when using them as potential substi- in Fig. 4. All PSC were solubilised to a greater extent in the acidic tutes for mammalian collagen. pH range from 1 to 6 (P < 0.05). Similar results were reported for PSC from the skin of unicorn leatherjacket (Ahmad & Benjakul, 3.6. ATR-FT-IR spectra 2010). As the pH goes above 6, a sharp decrease in solubility was observed (P < 0.05). Generally, low solubility was observed in the The FT-IR spectra of PSC from the different tissues of bighead neutral and alkaline pH range. However, the solubility of PSC from carp and the major peaks with their corresponding assignments the skin of brownstripe red snapper reached a maximum and min- are shown in Fig. 3. The amide A band is related to a free N–H imum at pH 4 and 7, respectively (Jongjareonrak et al., 2005). The stretching vibration that commonly occurs in the range of 3400– differences in pH affecting the solubility of collagens have been 3440 cm1; however, when the NH group of a peptide is part of a attributed to the variations in molecular properties and conforma- hydrogen bond, the position of the amide A band will be shifted tions among the collagens (Bae et al., 2008; Jongjareonrak et al., to a lower wavenumber, approximately 3300 cm1 (Doyle, Bendit, 2005). & Blout, 1975). The amide A band of PSC from the fins, scales, skins, All PSC showed similar solubility patterns with various NaCl bones and swim bladders were found at wavenumbers of 3321, concentrations. The solubility of all PSC remained constant in the 3317, 3318, 3318 and 3318 cm1, respectively. The amide B bands presence of NaCl up to 30 g/l (P > 0.05), and sharply decreased when were observed at wavenumbers of 2925–2935 cm1, correspond- the NaCl concentration was 40 g/l (P < 0.05), after which solubility ing to the asymmetrical stretch of CH2 (Muyonga et al., 2004). remained at a constant low level (P > 0.05). The decrease in The wavenumbers of the amide I, amide II and amide III bands solubility of PSC with the increase of NaCl concentration might are directly associated with the configuration of collagen (Heu be attributed to the salting-out effect (Bae et al., 2008; et al., 2010). The amide I band with the characteristic strong Kittiphattanabawon et al., 2005). These solubility behaviours of

Acknowledgements

This research was partly supported by the National Natural Science Foundation of China (30901123) and the 111 Project (B07029).

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