Isolation and characterisation of collagen extracted from the skin of striped catfish (Pangasianodon hypophthalmus)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of striped catfish (Pangasianodon hypophthalmus) were isolated and characterised. The yields of ASC and PSC were 5.1% and 7.7%, based on the wet weight of skin, respectively, with the accumulated yield of 12.8%. Both ASC and PSC comprising two different α-chains (α1 and α2) were characterised as type I and contained imino acid of 206 and 211 imino acid residues/1000 residues, respectively. Peptide maps of ASC and PSC hydrolysed by either lysyl endopeptidase or V8 protease were slightly different and totally differed from those of type I calf skin collagen, suggesting some differences in amino acid sequences and collagen structure. Fourier transform infrared (FTIR) spectra of both ASC and PSC were almost similar and pepsin hydrolysis had no marked effect on the triple-helical structure of collagen. Both ASC and PSC had the highest solubility at acidic pH. A loss in solubility was observed at a pH greater than 4 or when NaCl concentration was higher than 2% (w/v). T_max of ASC and PSC were 39.3 and 39.6 °C, respectively, and shifted to a lower temperature when rehydrated with 0.05 M acetic acid. Zeta potential studies indicated that ASC and PSC exhibited a net zero charge at pH 4.72 and 5.43, respectively. Thus, ASC and PSC were slightly different in terms of composition and structure, leading to somewhat different properties.     

Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were extracted from scale of spotted golden goatfish (Parupeneus heptacanthus) with the yields of 0.46% and 1.20% (based on dry weight basis), respectively. Both ASC and PSC were characterised as type I collagen, containing α₁ and α₂ chains. β and γ components were also found in both collagens. Based on FTIR spectra, the limited digestion by pepsin did not disrupt the triple helical structure of collagen. ASC and PSC contained glycine (336–340 residues/1000 residues) as the major amino acid and had imino acids of 186–189 residues/1000 residues. Maximal transition temperatures (T_max) were 41.58 and 41.01 °C for ASC and PSC, respectively. From zeta potential analysis, net charge of zero was found at pH 4.96 and 5.39 for ASC and PSC, respectively. Both collagens exhibited high solubility in acidic pH (2–4) and were soluble in the presence of NaCl at concentration up to 20 and 30 g/l for ASC and PSC, respectively.     

Isolation and characterisation of collagens from the skin of largefin longbarbel catfish (Mystus macropterus)

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the skin of largefin longbarbel catfish (Mystus macropterus) with yields of 16.8% and 28.0%, respectively, on the basis of dry weight. Both ASC and PSC contained α1 and α2 chains and the amino acid composition of collagen was close to that of calf skin type ? collagen. The intrinsic viscosities of ASC and PSC were 14.9 dl/g and 14.5 dl/g, respectively. Similar ultraviolet and FTIR spectra of ASC and PSC were observed. However, peptide maps of ASC and PSC, hydrolysed by trypsin, revealed some differences in primary structures between the two fractions. Denaturation temperatures of ASC and PSC were 32.1 °C and 31.6 °C, respectively. The higher T_m showed that it is possible to use largefin longbarbel catfish skin collagen as an alternative source of vertebrate collagens for industrial purposes.     

Isolation and characterisation of acid-solubilised collagen from the skin of Nile tilapia (Oreochromis niloticus)

Acid-solubilised collagen (ASC) was extracted from the skin of Nile tilapia (Oreochromis niloticus) and characterisation was studied. The results indicated that the yield of ASC was 39.4% on the basis of dry weight. This ASC was rich in glycine (35.6%). The amount of imino acids, proline and hydroxyproline, in ASC was 210 residues per 1000 residues. The ultraviolet (UV) absorption spectrum of ASC showed that the distinct absorption was at 220 nm. ASC showed transition curve at maximum temperature (T_max) of 32.0 °C in 0.05 M acetic acid, about 12 °C lower than that of calf skin collagen. Maximum solubility (0.75 mg/ml) in 0.5 M acetic acid was observed at pH 3. Solubility reached the minimum at pH 7. A sharp decrease in solubility was observed in 2% (w/v) NaCl or above. Biochemical studies indicated that ASC was composed of the α1α2α3 heterotrimers.     

Isolation and characterization of collagen from bigeye snapper (Priacanthus macracanthus) skin

Acid‐solubilized collagen (ASC) and pepsin‐solubilized collagen (PSC) were isolated from the skin of bigeye snapper (Priacanthus macracanthus) with yields of 64 and 11 g kg^?1 wet weight, respectively. Both ASC and PSC were characterized as type I collagens with no disulfide bonds. Peptide maps of ASC and PSC digested by V8 protease and lysyl endopeptidase showed some differences in peptide patterns and were totally different from those of calf skin collagen. The maximum solubility was observed at pH 4 and 5 for ASC and PSC, respectively. A sharp decrease in solubility of both collagens in acetic acid was found with NaCl concentration above 30 g l^?1. Thermal transitions of ASC and PSC in deionized water were observed with T_max of 30.37 and 30.87 °C, respectively, and were lowered in the presence of acetic acid (0.05 mol kg^?1 solution). Therefore, ASC was a major fraction in bigeye snapper skin and it exhibited some different characteristics to PSC. Copyright © 2005 Society of Chemical Industry     

Isolation and Characterisation of collagen from the skin of brownbanded bamboo shark (Chiloscyllium punctatum)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of brownbanded bamboo shark (Chiloscyllium punctatum) were isolated and characterised. The yield of ASC and PSC were 9.38% and 8.86% (wet weight basis), respectively. Based on protein patterns and TOYOPEARL^® CM-650M column chromatography, both collagens contained α- and β-chains as their major components. These were characterised as type I collagen with the cross-link of α2-chain. As digested by V8-protease and lysyl endopeptidase, peptide maps of both ASC and PSC were similar, but differed from that of type I collagen from calf skin. Fourier transform infrared (FTIR) spectra of both collagens were similar and pepsin hydrolysis had no effect on triple-helical structure of collagen. Transition temperature (T_max) of ASC and PSC were 34.45 and 34.52 °C, respectively, as determined by differential scanning colorimetry (DSC). From zeta potential study, the isoelectric points of ASC and PSC were estimated to be 6.21 and 6.56, respectively. Therefore, the skin of brownbanded bamboo shark could serve as an alternative source of collagen for different applications.     

Type I collagen from the skin of ornate threadfin bream (Nemipterus hexodon): Characteristics and effect of pepsin hydrolysis

Type I collagen from the skin of ornate threadfin bream (Nemipterus hexodon) was purified and characterised. Purified type I collagen contained [α1(I)]₂α2(I) as the dominant component with the co-presence of α1(I)α2(I)α3(I). It was rich in glycine and alanine with high content of imino acids (188 residues/1000 residues). The maximum transition temperature (T_m) and the total denaturation enthalpy (ΔH) of purified type I collagen was 33.35 °C and 0.819 J/g, respectively. The isoelectric point (pI) of purified type I collagen was estimated to be 6.40. After hydrolysis of purified type I collagen using pepsin, the band intensity ratios of α1/α2-chains were increased (P < 0.05). The cross-linked components were effectively hydrolysed by pepsin 1 and 2 from skipjack tuna stomach and porcine pepsin at 4 °C without the cleavage of β- and α-chains. At 50 °C, they were more susceptible to porcine pepsin hydrolysis, followed by pepsin 2 and 1, respectively.     

Collagen characteristics of farmed Atlantic salmon with firm and soft fillet texture

The possible role of collagen in texture variations among Atlantic salmon (Salmo salar L.) grown under commercial conditions at a Norwegian farm was studied. The texture was determined instrumentally as the breaking strength, and collagen and its salt (SSC), acid (ASC), pepsin (PSC) and insoluble (IF) fractions were analysed in order to determine the collagen aggregation degree. The collagen solubility and its overall amino acid (AA) composition showed no correlation to the breaking strength, but a positive correlation was observed between the breaking strength and glycine (r = 0.74) and alanine (r = 0.87) contents of the PSC fraction. Salmon with high breaking strength had higher T_peak (temperature of transition) and ΔH (enthalpy of transition), and the collagen seemed to have triple helix structures mainly stabilized by covalent associations as compared to salmon with low breaking strength. The glycosylation degree was also positively correlated to the breaking strength (r = 0.88, p ? 0.05). It is concluded that firmness of salmon muscle was not related to the total amount of collagen in the muscle, but rather higher collagen stability.     

Extraction and characterisation of pepsin-solubilised collagen from the skin of unicorn leatherjacket (Aluterus monocerous)

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 α₁-chains and one α₂-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. T_max 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.     

Acid‐soluble and pepsin‐soluble collagens from grass carp (Ctenopharyngodon idella) skin: a comparative study on physicochemical properties

In this study, acid‐soluble (ASC) and pepsin‐soluble (PSC) collagens with triple helical structures were successfully extracted from the skin of grass carp (Ctenopharyngodon idella) by two different extraction approaches. SDS‐PAGE pattern revealed that ASC and PSC are type I collagens with typical α1, α2 and β‐chains. In addition, the intensity of χ‐chain (trimer) in ASC was higher than that of PSC, representing the presence of the high proportion of intra‐ and intermolecular cross‐links of extracted collagens with large molecular weight using the acid method. Differential scanning calorimetry (DSC) results demonstrate that T_d (69.04 °C) of ASC was higher than T_d (62.20 °C) of PSC. Both ASC and PSC had the highest solubility at acidic pHs or at a low concentration of NaCl (<2%, w/v). The results of FTIR suggested the ASC and PSC maintained in the helical secondary structure at high degree.     

Characterization of acid-soluble collagen from the skin of walleye pollock (Theragra chalcogramma)

Acid-soluble collagen (ASC) was extracted from the skin of walleye pollock (Theragra chalcogramma) and partially characterized. It exhibited a maximum absorbance at 220 nm, but little absorbance near to 280 nm. Amino acid composition and SDS-PAGE suggested that the collagen might be classified as type I collagen. Moreover, FTIR investigations showed the existence of helical arrangements of collagen. The denaturation temperature (T_d) and shrinkage temperature (T_s) were 24.6 °C and 47 °C, respectively, both lower than those of mammalian collagens. However, T_d of walleye pollock skin collagen was higher than that of cod skin collagen reported previously. These results indicate that walleye pollock skin is a potential source of collagen and provide the theoretical basis for further research.     

人工养殖大鲵皮胶原蛋白的性质研究

在低温4 ℃条件下,采用酸法（0.5 mol/L乙酸）和酶法（胃蛋白酶）对人工养殖大鲵皮中的胶原蛋白进行了提取,对提取纯化后的两种不同类型的胶原蛋白--酸溶性胶原蛋白（acid-soluble collagen,ASC）和胃蛋白酶酶促溶性胶原蛋白（pepsin-soluble collagen,PSC）其各项理化性质进行了研究。结果表明：ASC和PSC均包含2 条α链及它们的二聚体β链,为Ⅰ型胶原蛋白;ASC和PSC中甘氨酸含量最高,其次为谷氨酸和脯氨酸,胱氨酸含量最低;ASC和PSC中亚氨基酸含量分别为144和173（以残基/1 000残基计）,两者热变性温度分别为23.5 ℃和26.5 ℃,表明PSC的热稳定性较优;ASC和PSC的紫外最大吸收峰分别位于233.0 nm和232.0 nm波长处,符合胶原蛋白的特征;ASC和PSC的红外特征吸收频率相似,均含有酰胺Ⅱ带、酰胺Ⅲ带及在两酰胺带间的一系列吸收峰,表明ASC和PSC中胶原蛋白的三螺旋结构较为完整。     

Studies on collagen from the skin of channel catfish (Ictalurus punctaus)

Acid-soluble and pepsin-soluble collagens (ASC and PSC) were extracted from the skin of channel catfish (Ictalurus punctaus) and partially characterized . The collagen obtained in the experiment contained more than 23% glycine as the most abundant amino acid. The denaturation temperature of acid-soluble collagen was 32.5 °C, about 5 °C lower than that of the porcine skin collagen. SDS–PAGE showed that the collagens were composed of two distinct alpha chains, which is similar to the porcine type I collagen. The contents of the skin ASC and PSC, on a dry weight basis, were 25.8% and 38.4%, respectively. These results suggest that channel catfish skin has potential as a supplement to the sources of vertebrate collagens.     

鲽鱼骨胶原蛋白的结构及流变学特性

采用酸法和酶法从鲽鱼骨中提取制备得到酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC),对ASC与PSC进行紫外吸收(UV)、红外吸收(FTIR)、亚基组成(SDS-PAGE)、热稳定性(DSC)、微观结构(SEM)以及流变学特性(DHR)分析。UV显示:ASC与PSC分别在227 nm与226 nm处有最大紫外吸收峰。FTIR图谱表明:ASC与PSC保持了三螺旋结构。SDS-PAGE电泳图表明:ASC与PSC是Ⅰ型胶原蛋白。DSC测定ASC与PSC的热变性温度分别为52.24℃与49.65℃。SEM显示:ASC与PSC呈现交织状纤维网状结构。DHR表明:1%的ASC溶液或PSC溶液在振荡频率0.1~10 Hz范围主要表现为弹性且具有较高的凝胶稳定性。     

Characterization and subunit composition of collagen from the body wall of sea cucumber Stichopus japonicus

Pepsin-solubilized collagen (PSC) without telopeptides was prepared from the body wall of the sea cucumber Stichopus japonicus and isolated by selective precipitation with NaCl. The PSC exhibited a maximum absorbance at 220 nm. The subunit of PSC was isolated by Sephacryl S-300 HR. The results of SDS-PAGE suggested that purified collagen from S. japonicus was a 1α trimer (about 135 kDa) while 1α chain resembling α₁ chain of type I collagen of vertebrate. The thermal stability temperature (T_s) was 57.0 °C as measured by DSC, about 5.0 °C lower than that of type I collagen of calf. Peptide mapping and amino acid analysis of PSC also revealed the difference between invertebrate and vertebrate. However, the presence of (α₁)₃ trimers was evident.     

Isolation and characterization of acid soluble collagens and pepsin soluble collagens from the skin and bone of Spanish mackerel (Scomberomorous niphonius)

The objective of this study was to extract and characterize acid soluble collagens (ASCs) and pepsin soluble collagens (PSCs) from the skin and bone of Spanish mackerel (Scomberomorous niphonius) and to provide a simultaneous comparison of the four collagens. The yields of ASC-S (ASC from skin), PSC-S (PSC from skin), ASC-B (ASC from bone) and PSC-B (PSC from bone) were 13.68 ± 0.35, 3.49 ± 0.24, 12.54 ± 0.83 and 14.27 ± 0.66% (on wet weight basis), respectively. The four collagens contained glycine (341.6–352.6 residues/1000 residues) as the major amino acid and the contents of imino acids were between 177.1 and 184.3 residues/1000 residues. Amino acid composition, SDS-PAGE and FTIR confirmed that ASC-S, ASC-B and PSC-B were mainly composed of type I collagen with slight molecular structure differences, and PSCs had lower content of high-molecular weight cross-links than that of ASCs. The denaturation temperatures of ASC-S, PSC-S, ASC-B and PSC-B were 15.12, 14.66, 18.02 and 16.85 °C, respectively, which were much lower than those of collagens from the mammalian and tropical fish species due to low imino acid contents. All the collagens were soluble at acidic pH (1–4) and lost their solubility when the NaCl concentrations were above 2% (w/v). The four lyophilized collagens displayed a uniform and regular network ultrastructure based on the ultrastructural analysis. The isolated collagens from Spanish mackerel could serve as an alternative source of collagens for further application in food and neutraceutical industries.     

Use of pepsin for collagen extraction from the skin of bigeye snapper (Priacanthus tayenus)

Extraction and some properties of pepsin-solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus) were investigated. Addition of bigeye snapper pepsin (BSP) at a level of 20 kUnits/g of defatted skin resulted in an increased content of collagen extracted from bigeye snapper skin. The yields of collagen from bigeye snapper skin extracted for 48 h with acid and with BSP were 5.31% and 18.74% (dry basis), respectively. With pre-swelling in acid for 24 h, collagen extracted with BSP at a level of 20 kUnits/g of defatted skin for 48 h had a yield of 19.79%, which was greater than that of collagen extracted using porcine pepsin at the same level (13.03%). The skin collagen was characterised to be type I with no disulfide bond. Electrophoretic study revealed slight differences in molecular weight between acid-solubilised collagen and all pepsin-solubilised collagens. The molecular weights of α1 and α2 chains in acid-solubilised collagen were estimated to be 120 and 112 kDa, respectively, whereas α1 and α2 chains of pepsin-solubilised collagens had molecular weights of 118 and 111 kDa, respectively. The result suggested that these pepsin-solubilised collagens might undergo partial cleavage in the telopeptide region by pepsin treatment. The maximum transition temperature (T_max) of acid-solubilised collagen was observed at 32.5 °C, which was slightly higher than that of pepsin-solubilised collagens (by about 1 °C). Generally, all collagens were highly solubilised in the pH range of 2–5 and sharply decreased at the neutral pH. No changes in solubility were observed in the presence of NaCl up to 3% (w/v) and the decrease was more pronounced with increasing NaCl concentration.     

Characteristics of collagen from the skin of clown featherback (Chitala ornata)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of clown featherback (Chitala ornata) were isolated and characterised. Yields of ASC and PSC were 27.64 and 44.63% (dry weight basis) with total collagen recovery of 82.08%. Both collagens contained glycine as the major amino acid with relatively high content of proline, hydroxyproline and glutamic acid/glutamine. Nevertheless, they had the low content of cysteine, histidine and tryrosine. The collagen was characterised as type I, comprising (α1)₂α2‐heterotrimer. Pepsin‐aided process did not affect triple‐helical structure of PSC as determined by FTIR spectra. Thermal transition temperature of ASC (36.28 °C) was slightly higher than that of PSC (35.23 °C). However, no differences in isoelectric point (5.54–5.68) between ASC and PSC were observed. Therefore, collagen from the skin of clown featherback could be successfully extracted for further applications.     

Collagens from the skin of arabesque greenling (Pleurogrammus azonus) solubilized with the aid of acetic acid and pepsin from albacore tuna (Thunnus alalunga) stomach

BACKGROUND: Due to the low extraction efficiency of collagen from fish skin by the typical acid solubilization process, pepsin has been widely used to aid further extraction of collagen from the residue. The aim of this study was to characterize collagen from the skin of arabesque greenling extracted with the aid of albacore tuna pepsin, in comparison with collagen obtained from the acid solubilization process. RESULTS: Acid‐solubilized collagen (ASC) from the skin of arabesque greenling was extracted with acetic acid. Pepsin‐solubilized collagen (PSC) was further extracted from the skin residue with the aid of pepsin from albacore tuna. The yields of ASC and PSC were 303 and 140 g kg^?1 (dry weight), respectively. Both collagens contained α‐ and β‐chains as their major components and were characterized as type I collagen. Both collagens contained glycine as a major amino acid and had imino acid content of 157–159 residues per 1000 residues. The degradation induced by lysyl endopeptidase and V8‐protease was more pronounced in PSC compared with ASC. Maximal transition temperatures of both collagens were in the range of 15.4–15.7 °C. Fourier transform infrared spectra revealed some differences in molecular order between ASC and PSC. Nevertheless, the triple‐helical structure of PSC was still predominant. Based on ζ‐potential, pI of ASC and PSC was estimated to be 6.31 and 6.38, respectively. CONCLUSION: Isolation of collagens from the skin of arabesque greenling could be achieved by acid or albacore tuna pepsin solubilization. However, there was a slight difference in properties between ASC and PSC. Copyright © 2010 Society of Chemical Industry     

Structure and characteristics of acid and pepsin-solubilized collagens from the skin of cobia (Rachycentron canadum)

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of cobia (Rachycentron canadum). The yields of ASC and PSC were 35.5% and 12.3%, respectively. Based on the protein patterns and carboxymethyl-cellulose chromatography, ASC and PSC were composed of α1α2α3 heterotrimers and were characterised as type I collagen with no disulfide bond. Their amounts of imino acids were 203 and 191 residues per 1000 residues, respectively. LC-MS/MS analysis demonstrated the high sequences similarities of ASC and PSC. Fourier transform infrared spectroscopy spectra showed that the amide I, II and III peaks of PSC were obtained at a lower wave number compared with ASC. The thermal denaturation temperatures of ASC and PSC, as measured by viscometry, were 34.62 and 33.97°C, respectively. The transition temperatures (T(max)) were 38.17 and 36.03°C, respectively, as determined by differential scanning calorimetry (DSC). Both collagens were soluble at acidic pH and below 2% (w/v) NaCl concentration.