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Food Hydrocolloids 24 (2010) 588e594

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Compositional and physicochemical characteristics of acid solubilized collagen extracted from the skin of unicorn leatherjacket (Aluterus monoceros)
Mehraj Ahmad, Soottawat Benjakul*, Sitthipong Nalinanon
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
Article history: Received 16 November 2009 Accepted 5 March 2010 Keywords: Acid solubilized collagen Unicorn leatherjacket FTIR Viscosity Peptide maps Zeta potential

a b s t r a c t
Acid solubilized collagen (ASC) was extracted from the skin of unicorn leatherjacket (Aluterus monoceros) using 0.5 M acetic acid, followed by precipitation with 2.6 M NaCl. ASC with the yield of 4.19% (wet weight basis) was identified as type I collagen, which was composed of two a1 chains and one a2 chain. Different peptide maps were observed between ASC hydrolyzed by V8 protease and lysyl endopeptidase. The maps were also different from those of type I collagen from calf skin, suggesting the differences in amino acid sequences between both collagens. Glycine was the most predominant amino acid. ASC contained the relatively higher content of alanine, but lower contents of proline and hydroxyproline, compared with calf skin collagen. FTIR analysis showed that ASC was in triple helix structure. Tmax of ASC dispersed in 0.05 M acetic acid and deionized water were 27.7 and 35.8  C, respectively. Relative viscosity of 0.03% (w/v) ASC dissolved in 0.1 M acetic acid decreased continuously as the temperature increased from 4 to 52  C, indicating thermal destabilization or denaturation of ASC molecules. ASC had the solubility greater than 90% in very acidic pH range (pH 1e4) and the solubility decreased continuously with increasing NaCl concentrations (0e6%). Net charge of ASC and calf skin collagen became zero at pHs of 5.58 and 5.68, respectively as determined by zeta potential titration. Therefore, skin of unicorn leatherjacket can be used as an alternative collagenous source. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Fish processing industry is an important source of income for Thailand. A large amount of by-products (60e70%) including skins, bones, scales, fins, head, guts and frame is generated and is usually used as animal feed with low market value. These underutilized resources have attracted the increasing attention as the raw material for marketable value-added product such as collagen, gelatin, hydrolysate, etc (Benjakul & Morrissey, 1997; Jongjareonrak, Benjakul, Visessanguan, Nagai, & Tanaka, 2005). Collagen is one of most abundant biological macromolecule of extracellular matrix where it provides the major structural and mechanical support to tissues. It constitutes about 30% of total proteins found in multicellular organisms and contains at least 27 different types of collagen, named from type I to type XXVII, which varies considerably in their complexity and diversity of their structure (Birk & Bruckner, 2005). All members of collagen family are characterized by containing domains with repetitions of the glycine-rich tripeptides Gly-X-Y involved in the formation of

* Corresponding author. Tel.: þ66 7428 6334; fax: þ66 7421 2889. E-mail address: soottawat.b@psu.ac.th (S. Benjakul). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.03.001

primary structure of trimeric collagen triple helix. Collagen triple helix is stabilized mainly by inter- and intra-chain water mediated hydrogen bonding as well as direct inter-chain hydrogen bonding (Brodsky & Persikov, 2005). Collagen contents vary considerably with fish species, age and season (Nagai, Araki, & Suzuki, 2002). Nevertheless, collagen obtained from different species and habitats might be different in terms of molecular compositions and properties (Foegeding, Lanier, & Hultin, 1996) Collagen is one of the best biomaterials, which has wide range of applications in food industry, pharmaceuticals, biomedical, leather industry, cosmetics and tissue engineering due to its excellent biocompatibility and biodegradability (Zhang, Li, & Shi, 2006). Due to its amphoteric nature, the interaction of collagen with other molecules has the desired effect on its rheological, chemical and biochemical properties. Collagen is readily available and non-toxic and can serve as an excellent basis for biomaterials (Sionkowska, Skopinska-Wisniewska, & Wisniewski, 2009). Unicorn leatherjacket (Aluterus monoceros) belongs to the order Tetraodontiformes and is a member of the Monacanthidae family, found around the ‘Gulf of Thailand’ in subtropical oceans between latitudes 43 N and 35 S, at the depths down to 50 m. Its length varies from 30 to 76 cm. Although fish collagen has been extracted from different marine or fresh water fish with varying inhabiting

M. Ahmad et al. / Food Hydrocolloids 24 (2010) 588e594


environment, the information regarding the collagen from the skin of unicorn leatherjacket, which is one of the species used for fillet production in Thailand, especially for export, is scarce. The aim of this study was to isolate and characterize acid solubilized collagen (ASC) from skin of unicorn leatherjacket in comparison with commercial calf skin collagen. 2. Materials and methods 2.1. Chemicals

spectrophotometric method (Bergman & Loxley, 1963). The factor of 10.25 obtained from amino acid composition of ASC was used to convert the hydroxyproline content to collagen. ASC was then subjected to analyses. 2.4. Amino acid analysis Collagen samples were hydrolyzed under reduced pressure in 4 M methane sulfonic acid containing 0.2% (v/v) 3-2(2-aminoethyl) indole at 115  C for 24 h. The hydrolysates were neutralized with 3.5 M NaOH and diluted with 0.2 M citrate buffer (pH 2.2). An aliquot of 0.4 ml was applied to an amino acid analyzer (MLC-703; Atto Co., Tokyo, Japan). The degree of hydroxylation of proline was calculated as the percentage of hydroxyproline from total imino acids. 2.5. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed by the method of Laemmli (1970). Collagen samples were suspended in 5% (w/v) SDS prior to incubation at 85  C for 1 h. The mixture was centrifuged at 4,000g for 5 min at room temperature to remove undissolved debris. Solubilized samples were mixed at 1:1 (v/v) ratio with the sample buffer (0.5 M TriseHCl, pH 6.8, containing 4% (w/v) SDS, 20% (v/v) glycerol and bromophenol blue) in the presence or absence of 10% (v/v) bME. The mixtures were boiled in boiling water for 2 min. Samples (15 mg protein) were loaded onto polyacrylamide gels comprising 7.5% separating gel and 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., Richmond, CA, USA). 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 the mixture of 30% (v/v) methanol and 10% (v/v) acetic acid. High molecular weight protein markers were used to estimate the molecular weight of proteins. Calf skin type I collagen was used as a standard. 2.6. Peptide mapping of collagen Peptide mapping of ASC and calf skin collagen was performed according to the method of Saito, Kunisaki, Urano, and Kimura (2002) with a slight modification. The samples (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 ml of the reaction buffer (0.1 M sodium phosphate, pH 7.2) containing 5 mg of V8 protease from S. aureus or 0.05 mg of lysyl endopeptidase from A. lyticus to collagen solutions, the reaction mixture was incubated at 37  C for 25 and 50 min for V8 protease and lysyl endopeptidase hydrolysis, respectively. The reaction was terminated by submerging the reaction mixture in the boiling water for 3 min. Peptides generated by the protease digestion were determined using SDS-PAGE using 7.5% separating gel and 4% stacking gel. High molecular weight protein markers were used to estimate the molecular weight of peptides. 2.7. ATR-FTIR analysis ASC from the skin of unicorn leatherjacket and calf skin collagen were subjected to Attenuated 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 Technology Inc., Madison, WI, USA) was used. Prior to analysis, calf skin type I collagen (Sigma chemical Co.) was dissolved in 10 volumes of 0.5 M acetic acid. The mixture was stirred at 4  C for 12 h and then dialyzed against 20

b-mercaptoethanol (b-ME), V8 protease from Staphylococcus aureus (EC, bovine serum albumin, calf skin type I collagen and lysyl endopeptidase from Achromobacter lyticus (EC were purchased from Sigma Chemical Co. (St. Louis, MO, USA). High molecular weight protein marker was purchased from GE Healthcare UK (Buckinghamshire, UK). Sodium dodecyl sulfate (SDS), Folin-Ciocalteu’s phenol reagent, acetic acid and tris (hydroxymethyl) aminomethane were obtained from Merck (Darmstadt, Germany).
2.2. Fish skin preparation The skin of unicorn leatherjacket (A. monocerous) was obtained from Sea Wealth Frozen Food Co., Ltd., Songkhla, Thailand. The skin was kept in ice using a skin/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 remaining flesh was removed manually from the skin. The cleaned skin was washed with iced tap water (0e2  C). Prepared skin was then cut into small pieces (0.5 Â 0.5 cm2) using the scissor, placed in polyethylene bags and stored at À20  C until use. The storage time was less than 2 months. 2.3. Extraction of acid solubilized collagen (ASC) All the procedures were carried out at 4  C. ASC from the skin of unicorn leatherjacket was extracted following the method of Nalinanon, Benjakul, Visessanguan, and Kishimura (2007) with a slight modification. Skin was soaked in 0.1 M NaOH with a skin/ solution ratio of 1:20 (w/v) with the continuous stirring to remove non-collagenous proteins for 6 h using an overhead stirrer model W20.n (IKAÒ-Werke GmbH & CO.KG, Stanfen, Germany). 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 (w/v) for 18 h with a change of solution every 6 h. Defatted skin was thoroughly washed with 15 volumes of cold water (4e5  C). To extract ASC, the prepared skin was mixed with 0.5 M acetic acid with a skin/solution ratio of 1:15 (w/v). The mixture was stirred continuously for 48 h. The mixture was filtered with two layers of cheesecloth to remove undissolved debris. The undissolved debris was re-extracted under the same conditions. Both filtrates were combined and precipitated using 2.6 M NaCl in the presence of 0.05 M tris(hydroxymethyl) aminomethane (pH 7.0). The resultant precipitate was collected by centrifuging at 20,000g using a Beckman model Avanti J-E centrifuge (Beckman coulter, Inc., Palo Alto, CA, USA) for 60 min at 4  C. The pellet was dissolved in 10 volumes of 0.5 M acetic acid and dialyzed against 20 volumes of 0.1 M acetic acid and distilled water, respectively. The dialyzate was finally freeze-dried using a Scanvac Model Coolsafe 55 freeze drier (Coolsafe, Lynge, Denmark). The yield of ASC was calculated and expressed as dry matter/wet weight of skin. Additionally, the yield of ASC was calculated based on the original collagen content in the skin. Hydroxyproline content in the skin was determined by


M. Ahmad et al. / Food Hydrocolloids 24 (2010) 588e594

volumes of 0.1 M acetic acid for 24 h, followed by dialysis using 20 volumes of distilled water for 24 h. The dialyzed sample was freezedried. For spectra analysis, the freeze-dried samples were placed onto the crystal cell and the cell was clamped into the mount of FTIR spectrometer. The spectra in the range of 400e4000 cmÀ1 with automatic signal gain were collected in 32 scans at a resolution of 4 cmÀ1 and were ratioed against a background spectrum recorded from the clean empty cell at 25  C. 2.8. Differential scanning calorimetry Differential scanning calorimetry (DSC) of collagen samples was run following the method of Rochdi, Foucat, and Renou (2000) with a slight modification. Rehydration was done by adding deionized water or 0.05 M acetic acid to dried samples at a sample/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, USA). Temperature calibration was run using the Indium thermogram. The samples were accurately weighed into aluminum pans and sealed. The samples were scanned at 1  C/min over the range of 20e50  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. 2.9. Viscosity of collagen solution

0e6% (w/v) 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. 2.11. Measurement of z- potential ASC and calf skin type _ collagen were dissolved in 0.5 M acetic acid at a concentration of 0.5 mg/ml. The mixture was stirred at 4  C for 12 h. The z-potential of each sample (20 ml) was measured using a zeta potential analyzer (ZetaPALS, Brookhaven Instruements Co., Holtsville, NY, USA). z-potential of samples adjusted to different pHs with 1.0 M nitric acid or 1.0 M KOH using an autotitrator (BI-ZTU, Brookhaven Instruments co., Holtsville, NY, USA) was determined. pH rendering zero z- potential was obtained from the titration curve. 2.12. Statistical analysis All experiments were performed in triplicate and a completely randomized design (CRD) was used. Analysis of variance (ANOVA) was used and mean comparison was performed using Duncan’s multiple range tests (Steel & Torrie, 1980). Analysis was performed using an SPSS package (SPSS 11.0 for windows, SPSS Inc, Chicago, IL, USA). 3. Results and discussion 3.1. Yield

ASC 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 synchorolectic viscometer (model DVIIþ, Brookfield Eng Labs Inc., Stoughton, MA, USA) with the spindle No.1 and speed of 100 rpm. ASC solution was heated from 4 to 50  C with a heating rate of 4  C/min (Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005). At the designated temperature, the solution was held for 30 min prior to viscosity determination. The relative viscosity was calculated in comparison with that obtained at 4  C. Td was defined as the temperature causing 50% decrease in the relative viscosity of collagen solution. 2.10. Collagen solubility

Acid solubilized collagen (ASC) was extracted from the skin of unicorn leatherjacket with the yield of 4.19 Æ 0.4% (based on wet weight of skin). Skin contained the collagen at the level of 22.67 g/ 100 g. Therefore, the yield based on the initial collagen presented in the skin or % recovery was 18.47%. The skin was not completely solubilized with 0.5 M acetic acid even with two repetitions of extraction. This result suggested a high amount of cross-links at the telopeptide region as well as other inter-molecular cross-links, leading to low solubility of collagen in acid (Zhang et al., 2007). The presence of cross-links was more likely associated with the tough and elastic skin of this species (Ahmad & Benjakul, 2010). 3.2. Amino acid compositions

ASC solubility at different pH and NaCl concentration was determined by the method of Kittiphattanabawon et al. (2005). 2.10.1. Effect of pH on collagen solubility ASC 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 solubilized. The pH of ASC solution (8 ml) was adjusted with either 6 N NaOH or 6 N HCl to obtain the final pH ranging from 1 to 10. The volume of solutions was made up to 10 ml by deionized water previously adjusted to the same pH as the collagen solution. The solution was centrifuged at 20,000g for 30 min at 4  C. The supernatants were 10-fold diluted using distilled water. Protein content in the diluted supernatants was determined by the Lowry method using bovine serum albumin as a standard (Lowry, Rosebrough, Farr, & Randall, 1951). Relative solubility was calculated in comparison with that obtained at the pH rendering the highest solubility. 2.10.2. Effect of NaCl on collagen solubility ASC (6 mg/ml) was dissolved in 0.5 M acetic acid. ASC solution (5 ml) was added with 5 ml of NaCl in 0.5 M acetic acid at various concentrations (0e12%; w/v), in which the final concentrations of

Amino acid compositions of ASC from the skin of unicorn leatherjacket and calf skin collagen are presented in Table 1. Glycine is the major amino acid in both 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 residues from the C-terminus (Burghagen, 1999). Alanine was found as the second abundant amino acid. However ASC from unicorn leatherjacket skin had the higher content of alanine than that of calf skin collagen. No cysteine was found in both collagens. Tryptophan was observed in only calf skin collagen, but was not found in ASC. The imino acid content (hydroxyproline and proline) of ASC was 190 residues/1000 residues, while calf skin collagen contained 215 residues/1000 residues. Therefore, ASC had the lower imino acid content than calf skin collagens, which was in accordance with the report of Jongjareonrak et al. (2005). The degree of hydroxylation of proline was calculated to be 42.6% in ASC. Ramachandran (1988) reported that the degree of hydroxylation of proline residues plays an important role in stabilizing the triple helix of collagen. The results suggested that ASC might have the lower thermal stability than mammalian collagen. ASC contained the higher content of

M. Ahmad et al. / Food Hydrocolloids 24 (2010) 588e594 Table 1 Amino acid composition of ASC from skin of unicorn leatherjacket and calf skin collagen (expressed as residues/1000 residues). Amino acids 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 Unicorn leatherjacket skin collagen 45 27 33 74 321 141 0 21 13 9 17 4 13 5 27 6 53 0 81 109 190 Calf skin collagen 45 18 33 75 330 119 0 21 6 11 23 3 3 7 26 5 50 3 94 121 215


indicating that there was no disulfide bond in ASC. This result was in accordance with the absence of cysteine in both collagens (Table 1). It was noted that ASC from the skin of unicorn leatherjacket contained a high amount of cross-linked proteins. This might reflect the high content of cross-links in their skin, which resulted in the difficulty of acid solubilization of collagen from the skin of this species. Cross-linking of a-chains was also observed in collagens from other fish species such as brownbanded bamboo shark (Kittiphattanabawon, Benjakul, Visessanguan, Kishimura, & Shahidi, 2009). 3.4. Peptide mapping of collagen Lysyl endopeptidases from A. lyticus and V8 protease from S. aureus have been employed for peptide mapping of collagens (Kittiphattanabawon et al., 2005). The peptide maps of ASC digested by both enzymes are shown in Fig. 2. When V8 protease was used, the band intensities of g- and b-chains disappeared. Band intensity of a-chains decreased with the coincidental increase in lower molecular weight peptides. However, calf skin collagen was resistant to hydrolysis by V8 protease. Thus, ASC was more susceptible to hydrolysis, compared to calf skin collagen by V8 protease. The result suggested that the primary structure of ASC from skin of unicorn leatherjacket was different from that of calf skin collagen, particularly in terms of sequence of amino acids as well as amino acid composition (Table 1). V8 protease shows a high specific preference for glutamic acid and aspartic acid residues of proteins (Jongjareonrak et al., 2005). For peptide map of ASC digested by lysyl endopeptidase, all major components including a-, b- and g-chains as well as high MW cross-links were completely hydrolyzed. It was noted that calf skin collagen was digested by lysyl endopeptidase, whereas it was resistant to V8 protease. Also it was found that the patterns of peptides were different between peptide map of ASC digested by V8 and lysyl endopeptidase. The result reconfirmed that ASC from unicorn leatherjacket skin had the different sequence and composition of amino acids as well as the degree of cross-linking, in comparison with calf skin type _ collagen. 3.5. Differential scanning calorimetry DSC thermograms of ASC from the skin of unicorn leatherjacket dispersed in 0.05 M acetic acid or deionized water are depicted in
Original V8 protease Lysyl endopeptidase

threonine, methionine and phenylalanine but lower leucine than did calf skin collagen. Thus, ASC from unicorn leatherjacket skin was different from calf skin collagen in terms of amino acid compositions, which might determine the differences in properties between both collagens. 3.3. SDS-polyacrylamide gel electrophoresis Protein patterns of ASC from the skin of unicorn leatherjacket under reducing and non-reducing conditions are shown in Fig. 1. ASC consisted of two a-chains (a1 and a2). The band intensity of a1-chain was approximately 2-fold higher than that of a2. High molecular weight components, particularly b-components, as well as other cross-linked molecules with higher molecular weight were also observed in ASC. Protein pattern of ASC generally was similar to those of the type I collagen from calf skin, indicating that ASC from the skin of unicorn leatherjacket was most likely type I collagen. No differences in the electrophoretic patterns of ASC were observed, when analyzed in the presence and absence of b-ME,

220kDa 170kDa 116kDa 76kDa 70kDa 53kDa

Fig. 1. SDS-PAGE pattern of ASC from the skin of unicorn leatherjacket under reducing (R) and non-reducing (N) conditions. M: high molecular weight markers; I: calf skin type I collagen and ASC: acid solubilized collagen.




Fig. 2. Peptide maps of ASC from the skin of unicorn leatherjacket and calf skin collagen digested by V8 protease and lysyl endopeptidase. M: high molecular weight markers; I: calf skin type _ collagen and ASC: acid solubilized collagen from the skin of unicorn leatherjacket.


M. Ahmad et al. / Food Hydrocolloids 24 (2010) 588e594

Fig. 4. Fourier transform infrared spectra of ASC from the skin of unicorn leatherjacket and calf skin collagen (CSC).

Fig. 3. DSC thermogram of ASC from the skin of unicorn leatherjacket dispersed in 0.05 M acetic acid and in deionized water.

Fig. 3. Endothermic peaks with the maximum transition temperatures (Tmax) at 27.2 and 35.8  C were observed for ASC rehydrated in 0.05 M acetic acid and deionized water, respectively. For ASC rehydrated in acetic acid, Tmax shifted to a lower temperature (27.2  C), compared with that found in the sample rehydrated with deionized water. The result suggested that the hydrogen bonds which stabilize the secondary structure of collagen were partially disrupted by acetic acid. 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). In the presence of acetic acid, the hydrogen bonding might be disrupted to some extent due to electrostatic repulsion of ASC molecules and the breakage of hydrogen bond stabilizing the collagen molecules, resulting in the decrease in Tmax in acidic solution. Tmax of ASC was much lower than that of calf skin collagen (40.8  C) (Kittiphattanabawon et al., 2005). The lower Tmax of ASC from the skin of unicorn leatherjacket might be related to the lower content of imino acids than that of the calf skin collagen. The higher the imino acid content, the more thermal stability was obtained (Zhang et al., 2007). In addition, a higher enthalpy (DH ¼ 2.11 J/g) was also observed for ASC rehydrated in deionized water, compared with that of ASC dispersed in 0.05 M acetic acid (DH ¼ 0.55 J/g). The differences in DH among ASC rehydrated with two different media might be governed by hydrogen bonding in collagen structure. 3.6. ATR-FTIR analysis FTIR spectra of ASC from the skin of unicorn leatherjacket and calf skin collagen are shown in Fig. 4. For ASC of unicorn leatherjacket, five characteristic amide bands representing amide A, amide B, amide _, amide __ and amide ___ were found at the wavenumbers of 3294.86, 2920.87, 1646.24, 1549.08, and 1236.76 cmÀ1, respectively. For calf skin collagen, the corresponding amide bands were observed at the wavenumbers of 3295.61, 2933.94, 1635.76, 1545.29 and 1235.57 cmÀ1. The absorption spectrum of ASC from Nile perch skin was situated in the amide region including amide I (1651e1662 cmÀ1), amide II (1540e1560 cmÀ1) and amide III (1230e1242 cmÀ1) (Muyonga, Cole, & Duodu, 2004). The amide I band is originated from C¼O stretching vibrations coupled to NeH bending vibrations, CN stretch and CCN deformation (Bandekar, 1992). Dialyzed calf skin

collagen had the lower wavenumber at amide _ region, as compared to ASC, suggesting the more interaction of C¼O with adjacent chains via hydrogen bond of the former. Generally, the lower wavenumber of bands corresponds to higher hydrogen bonding potential (Li, Liu, Gao, & Chen, 2004). It was presumed that intramolecular interaction between a-chains in calf skin tropocollagen was more pronounced than ASC. Previous research suggested the following secondary structures in the amide I region: b-turn, 1660e1700 cmÀ1; a-helix, 1645e1659 cmÀ1; irregular structure, 1640e1644 cmÀ1; and b-sheet or extended structure, 1620e1640 cmÀ1 (Farrell, Wickham, Unruh, Qi, & Hoagland, 2001). For undialyzed calf skin collagen, an additional peak at amide _ region with the wavenumber of 1728.90 cmÀ1 was found (data not shown). After the excessive dialysis, acetic acid or other acids used for collagen solubilization were completely removed. This was confirmed by the disappearance of peak at 1728.90 cmÀ1, which more likely represented C¼O stretching of the carboxyl group from acetic acid or other acids (Kaminska & Sionkowska, 1996). The lower wavenumber of amide II peak was also found in calf skin collagen as compared to ASC, indicating that NeH of calf skin collagen was more involved in bonding with the adjacent a-chains. The amide II_ represented the combination peaks between CeN stretching vibrations and NeH deformation from amide linkages as well as absorptions arising from wagging vibrations from CH2 groups from the glycine backbone and proline side-chains (Jackson, Choo, Watson, Halliday, & Mantsch, 1995). This amide ___ was detected around the wavenumber of 1236.76 and 1235.57 cmÀ1 for ASC and calf skin collagen, respectively. The ratio of amide III and 1451.30 cmÀ1 was 0.86 and 0.85 for ASC and calf skin collagen, respectively. In general, the ratio of 1 indicated the triple helical structure (Plepis, Goissis, & Das, 1996). Therefore, there was similarity in helical structure between both samples. Nevertheless, calf skin collagen might have the higher intra- and inter-molecular cross-links, especially the higher hydrogen bonding in stabilizing the triple helix, as evidenced by the higher Tmax of calf skin collagen, compared with that of ASC. The amide A band, arising from the stretching vibrations of NeH group, appeared at 3294.86 and 3295.61 cmÀ1 in ASC and calf skin collagen, respectively. Doyle, Blout, and Bendit (1975) reported that a free NeH stretching vibration commonly occurs in the range of 3400e3440 cmÀ1. The amide B was observed at 2920.87 and 2933.94 cmÀ1 for ASC and calf skin collagen, respectively, corresponding to asymmetric stretch vibration of ¼CeH as well as eNHþ. The shift of amide B of 3 calf skin collagen to higher wavenumber was probably associated with free NHþ group of lysine residues or of N-termini. Thus, FTIR 3 spectrum of ASC from the skin of unicorn leatherjacket had slight

M. Ahmad et al. / Food Hydrocolloids 24 (2010) 588e594


3.8. Viscosity of collagen solution One of the physicochemical characteristics of collagen is its high viscosity. Collagen solutions are highly structured systems which even at low concentrations are characterized by a high degree of viscosity due to stronger electrostatic repulsion among the collagen molecular chains in solution (Zhang, Liu, & Li, 2009). Collagen is irreversibly destroyed as a result of the thermal denaturation. The triple helix structure of collagen stabilized by hydrogen bonds was converted into the random coil configuration by the process of thermal depolymerization, which was accompanied by a change in physical properties, such as viscosity, sedimentation, diffusion, light scattering and optical activity (Gurdak, Booth, Roberts, Rouxhet, & Dupont-Gillain, 2006). The continuous decreases in relative viscosity of ASC in 0.1 M acetic acid were observed when the temperature increased up to 52  C (P < 0.05) (Fig. 6). At higher temperature, the rearrangement of all chains, a, b or g chains, might take place and the less compact configuration was obtained, leading to the decrease in viscosity of solution. From the result, Td of ASC was estimated to be 31.16  C. The high viscosity can be attributed to the high proportion of b and g chains with a higher

Fig. 5. Zeta potential of ASC from the skin of unicorn leatherjacket and calf skin collagen (CSC) at different pHs. Bars represent the standard deviation (n ¼ 3).

differences in functional groups and inter- and intra-molecular interaction, compared with calf skin collagen type I.

3.7. Measurement of z- potential The z- potential values of ASC and calf skin collagen type _ measured as a function of pH are shown in Fig. 5. The surface net charge of ASC and calf skin collagen became zero at pH of 5.58 and 5.68, respectively. Protein molecules in an aqueous system have zero net charge at their isoelectric points (pI), in which the positive charges are balanced out by the negative charges (Bonner, 2007). Thus, pIs of ASC and calf skin collagen were estimated to be 5.58 and 5.68, respectively. The pIs of ASC and calf skin collagen were in acidic range, possibly due to the higher density of carboxyl groups. It was noted that the molecular charge of ASC remained very low in the alkaline region. This might be associated with the low solubility of ASC in the alkaline pH range, in which the repulsion force between the molecules with the negative charge was not sufficient to cause the complete solubilization. The slight difference in the pI of ASC and calf skin collagen might be caused by the differences in amino acid composition and configuration between collagens.

Fig. 6. Relative viscosity of ASC from the skin of unicorn leatherjacket at different temperatures. Bars represent the standard deviation (n ¼ 3).

Fig. 7. Relative solubility (%) of ASC from the skin of unicorn leatherjacket as affected by different pHs (a) and NaCl concentration (b). Bars represent the standard deviation (n ¼ 3).


M. Ahmad et al. / Food Hydrocolloids 24 (2010) 588e594 Burghagen. (1999). Collagen. In H. D. Belitz, & W. Grosch (Eds.), Food chemistry (2nd ed.). (pp. 540e547) Berlin: Springer. Doyle, B. B., Blout, E. R., & Bendit, E. G. (1975). Infrared spectroscopy of collagen and collagen like polypeptides. Biopolymers, 14, 937e957. Farrell, H. M., Wickham, E. D., Unruh, J. J., Qi, P. X., & Hoagland, P. D. (2001). Secondary structural studies of bovine caseins: temperature dependence of b-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization. Food Hydrocolloids, 15, 341e354. Foegeding, E. A., Lanier, T. C., & Hultin, H. O. (1996). Collagen. In O. R. Fennema (Ed.), Food chemistry (3rd ed.). (pp. 902e906) New York: Marcel Dekker, Inc. Gurdak, E., Booth, J., Roberts, C. J., Rouxhet, P. G., & Dupont-Gillain, C. C. (2006). Influence of collagen denaturation on the nanoscale organization of adsorbed layers. Journal of Colloid and Interface Science, 302, 475e484. Jackson, M., Choo, L., Watson, P. H., Halliday, W. C., & Mantsch, H. H. (1995). Beware of connective tissue proteins: assignment and implications of collagen absorptions in infrared spectra of human tissues. Biochima et Biophysica Acta, 1270, 1e6. Jongjareonrak, A., Benjakul, S., Visessanguan, W., Nagai, T., & Tanaka, M. (2005). Isolation and characterization of acid and pepsin-solubilized collagens from the skin of brown stripe red snapper (Lutjanus vitta). Food Chemistry, 93, 475e484. Kaminska, A., & Sionkowska, A. (1996). Effect of UV radiation on the infrared spectra of collagen. Polymer Degradation and Stability, 51, 19e26. Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Kishimura, H., & Shahidi, F. (2009). Isolation and characterisation of collagen from the skin of brownbanded bamboo shark (Chiloscyllium punctatum). Food Chemistry, 119, 1519e1526. Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Nagai, T., & Tanaka, M. (2005). Characterization of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chemistry, 89, 363e372. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature, 277, 680e685. Li, H., Liu, B. L., Gao, L. Z., & Chen, H. L. (2004). Studies on bull frog skin collagen. Food Chemistry, 84, 65e69. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193, 256e275. Muyonga, J. H., Cole, C. G. B., & Duodu, K. G. (2004). Characterization of acid soluble collagen from skins of young and adult Nile perch (Lates niloticus). Food Chemistry, 85, 81e89. Nagai, T., Araki, Y., & Suzuki, N. (2002). Collagen of the skin of ocellate puffer fish (Takifugu rubripes). Food Chemistry, 78, 173e177. Nalinanon, S., Benjakul, S., Visessanguan, W., & Kishimura, H. (2007). Use of pepsin for collagen extraction from skin of bigeye snapper (Priacanthus tayenus). Food Chemistry, 104, 593e601. Ogawa, M., Portier, J. R., Moody, M. W., Bell, J., Schexnayder, M. A., & Losso, J. N. (2004). Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonia cromis) and sheepshead sea bream (Archosargus probatocephalus). Food Chemistry, 88, 495e501. Plepis, A. M. D. G., Goissis, G., & Das, G. D. K. (1996). Dielectric and pyroelectric characterization of anionic and native collagen. Polymer Engineering and Science, 36, 2932e2938. Ramachandran, G. N. (1988). Stereochemistry of collagen. Journal of Peptide and Protein Research, 31, 1e16. Rochdi, A., Foucat, L., & Renou, J. (2000). NMR and DSC studies during thermal denaturation of collagen. Food Chemistry, 69, 295e299. Saito, M., Kunisaki, N., Urano, N., & Kimura, S. (2002). Collagen as the major edible component of sea cucumber (Stichopus japonicus). Journal of Food Science, 67, 1319e1322. Sionkowska, A., Skopinska-Wisniewska, J., & Wisniewski, M. (2009). Collagenesynthetic polymer interactions in solution and in thin films. Journal of Molecular Liquids, 145, 135e138. Steel, R. G. D., & Torrie, J. H. (1980). Principles and procedures of statistics: A biometrical approach (2nd ed.). New York: McGraw-Hill. (633 pp.). Wong, D. W. S. (1989). Mechanism and theory in food chemistry. New York: Van Nostrand Reinhold Company Inc. Zhang, Z. K., Li, G. Y., & Shi, B. (2006). Physicochemical properties of collagen, gelatin and collagen hydrolysate derived from bovine limed split wastes. Journal of the Society of Leather Technologists and Chemists, 90, 23e28. Zhang, M., Liu, W., & Li, G. (2009). Isolation and characterisation of collagens from the skin of largefin longbarbel catfish (Mystus macropterus). Food Chemistry, 115, 826e831. Zhang, Y., Liu, W. T., Li, G. Y., Shi, B., Miao, Y. Q., & Wu, X. H. (2007). Isolation and partial characterization of pepsin-soluble collagen from the skin of grass carp (Ctenopharyngodon idella). Food Chemistry, 103, 906e912.

average molecular weight (Ogawa et al., 2004). Heat-treatment at high temperature can break down the hydrogen bonds, which stabilize collagen structure (Wong, 1989), leading to the dissociation of collagen molecules. 3.9. Effects of pH and NaCl on collagen solubility Fig. 7(a) shows the effect of pH on the solubility of ASC from skin of unicorn leatherjacket. ASC was soluble in the pH ranging from 1 to 4 and the highest solubility was observed at pH 2 (P < 0.05). The lowest solubility was observed at pH 6e7 (P < 0.05), which was in agreement with the pH rendering the net charge of zero. At the pH near the isoelectric point, ASC and calf skin collagen molecules are unstable and tend to coagulate or flocculate due to increased hydrophobic interaction among collagen molecules (Jongjareonrak et al., 2005). The higher solubility at lower pH could arise from the increased repulsive force among collagen molecules. At alkaline pH, the slight increase in solubility was also observed (P < 0.05). ASC had the decrease in solubility in the presence of salt. The slight decrease in solubility was observed in the presence of 1e2% and the sharp decrease was found as the salt concentration increased up to 6% (P < 0.05). At 6% NaCl, the solubility of 36.91% was obtained. The lowered solubility of ASC was mainly due to the ‘salting out’ effect. 4. Conclusion ASC could be extracted from unicorn leatherjacket skin. The collagen consisted of two a-chains (a1and a2) and were characterized as type I collagen. ASC was in triple helical structure and was solubilized in acidic pH range and lost its solubility as the concentration of salt increased. The applications of ASC should be maximized based on its characteristics. Due to the low yield of ASC from the skin of unicorn leatherjacket, the improvement of extraction method should be further studied. Acknowledgements The authors would like to express their sincere thanks to Graduate School, Prince of Songkla University and the TRF senior research scholar program for the financial support. References
Ahmad, M., & Benjakul, S. (2010). Extraction and characterisation of pepsinsolubilised collagen from the skin of unicorn leatherjacket (Aluterus monoceros). Food Chemistry, 120, 817e824. Babu, R. I., & Ganesh, K. N. (2001). Enhanced triple helix stability of collagen peptides with 4R-Aminoprolyl (Amp) residues: relative roles of electrostatic and hydrogen bonding effects. Journal of American Chemical Society, 123, 2079e2080. Bandekar, J. (1992). Amide modes and protein conformation. Biochimica et Biophysica Acta (BBA) e Protein Structure and Molecular Enzymology, 1120, 123e143. Benjakul, S., & Morrissey, M. T. (1997). Protein hydrolysates from Pacific whiting solid wastes. Journal of Agricultural and Food Chemistry, 45, 3423e3430. Bergman, I., & Loxley, R. (1963). Two improved and simplified methods for the spectrophotometric determination of hydroxyproline. Analytical Chemistry, 35, 1961e1965. Birk, D. E., & Bruckner, P. (2005). Collagen suprastructures. Topics in Current Chemistry, 247, 185e205. Bonner, P. L. R. (2007). Protein purification. Cornwall, UK: Taylor & Francis Group. Brodsky, B., & Persikov, A. V. (2005). Molecular structure of the collagen triple helix. Advances in Protein Chemistry, 70, 301e339.

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