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.     

Chemical compositions and characterisation of skin gelatin from farmed giant catfish (Pangasianodon gigas)

Gelatin was extracted from the skin of farmed giant catfish (Pangasianodon gigas) with a yield of 20.1 g/100 g skin sample on the basis of wet weight. The chemical composition and properties of gelatin were characterised. The gelatin had high protein (89.1 g/100 g) but low fat (0.75 g/100 g) content and contained a high number of imino acids (proline and hydroxyproline) (211 residues per 1000 residues). Giant catfish skin gelatin had a slightly different amino acid composition than calf skin gelatin. The bloom strength of the gelatin gel from giant catfish skin gelatin (153 g) was greater than that of calf skin gelatin (135 g) (P < 0.05). Viscosity, foam capacity and foam stability of gelatin from giant catfish skins were in general greater than those of the gelatin from calf skin tested. SDS-PAGE of giant catfish skin gelatin showed a high band intensity for the major protein components, especially, α-, β- and γ-components and was similar to that of standard calf skin collagen type I.     

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.     

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.     

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.     

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.     

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.     

Chemical compositions and characteristics of farm raised giant catfish (Pangasianodon gigas) muscle

Proximate composition, chemical and physical properties of dorsal, ventral and lateral line cuts of farm raised giant catfish were determined. Protein, fat and ash content of the different cuts averaged 16.88, 4.45 and 1.24 g/100 g, respectively. Dorsal contains higher protein concentrations (19 g/100 g) than other two parts (p < 0.05). Ventral showed the highest hydroxyproline content (0.83 mg/g). Differences in lipid composition and fatty acid profiles were found among different cuts with highest phospholipids in the dorsal and highest triglyceride in both ventral and lateral line (p < 0.05). All the meat cuts contained high saturated fatty acid, followed by mono- and polyunsaturated fatty acid. High muscle hardness and toughness was found in the dorsal than that in the ventral (p > 0.05). The highest content of myoglobin and total pigment in lateral line resulted in the highest redness index (a*/b*) of this part. Three major nitrogenous compositions classified based on solubility in giant catfish muscle were myofibrillar, sarcoplasmic and alkaline soluble proteins.     

斑点叉尾鮰鱼皮胶原蛋白的理化特征研究

选用斑点叉尾鮰（Ictalurus punctatus）鱼皮为材料,用乙酸和乙酸-胃蛋白酶,分别提取鱼皮中的酸溶性胶原蛋白（acid-solubilise collagen,ASC）和酶溶性胶原蛋白（pepsin-solubilise collagen,PSC）,并对其理化性质特征进行研究。研究发现,提取得到的ASC纯度高达93.11%,PSC纯度高达93.46%;紫外吸收分析表明,ASC和PSC的吸收峰值均在233nm处;蛋白图谱中两种胶原蛋白均由两种不同的α链（α1）2α2组成,具备I型胶原蛋白的特征;ASC和PSC的傅里叶红外图谱相似,具有完整的三螺旋结构;ASC的变性温度为34.2℃,PSC的变性温度为33.9℃。      

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.     

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 characterization of collagen from the skin of deep-sea redfish (Sebastes mentella)

ABSTRACT:  To make more effective use of underutilized resources, acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were isolated from the skin of deep-sea redfish ( Sebastes mentella ) and characterized for their potential in commercial applications. The yield of ASC (47.5%) was lower compared to PSC (92.2%), but the purity of ASC was significantly higher. The intrinsic viscosity of ASC (15.9 dL/g) was greater than PSC (14.6 dL/g), indicating a higher average molecular weight of ASC on account of the high proportion of polymers of collagen. The denaturation temperatures of ASC and PSC were 16.1 and 15.7 °C, respectively, suggesting the triple helical structure of PSC was still predominant. The amino acid profiles of ASC and PSC were similar with lower imino acid content than most other species, which might be the reason for the lower denaturation temperature. SDS-PAGE and FTIR showed that both ASC and PSC were type I mainly with slight structure differences. ASC held its triple helical structure well, and possessed a higher extent of intermolecular cross-link. While the structure of PSC was changed slightly due to the loss of N- and C-terminus domains, the triple helical structure was still predominant as a result of the formation of more and/or stronger hydrogen bond.     

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.     

Isolation and characterization of collagen from the cartilages of brownbanded bamboo shark (Chiloscyllium punctatum) and blacktip shark (Carcharhinus limbatus)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the cartilages of brownbanded bamboo shark (BBS; Chiloscyllium punctatum) and blacktip shark (BTS; Carcharhinus limbatus) were isolated and characterized. ASC and PSC extracted from BBS cartilage showed the yields of 1.27 and 9.59 g/100 g (Based on dry weight), respectively, while yields of ASC and PSC from BTS cartilage were 1.04 and 10.30 g/100 g (based on dry weight), respectively. All collagens had protein as a major constituent with the trace amount of ash and fat. They contained glycine as the major amino acid with high contents of alanine, proline and hydroxyproline. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns and subunit compositions, all collagens more likely comprised 2 types of collagen, type I and II, and contained α- and β-chains as the major components. Peptide maps of those collagens from both species digested by V8-protease and lysyl endopeptidase were different and were completely different from those of type I collagen from calf skin. Thermal transition temperature of ASC from those collagens (36.28-36.73 °C) was slightly higher than their corresponding PSC (34.56-35.98 °C). From zeta potential analysis, isoelectric points (pI) of collagen from the cartilages of BBS and BTS were estimated to be from 6.53 to 7.03 and from 6.96 to 7.26, respectively. Fourier transform infrared (FTIR) spectra of both ASC and PSC were quite similar, suggesting that pepsin hydrolysis did not affect the secondary structure of collagen, especially triple-helical structure.     

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.     

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.     

Extraction and properties of gelatin from channel catfish (Ietalurus punetaus) skin

Response surface method was used to determine the optimum operating conditions for extracting the gelatin from channel catfish skin. The optimal conditions for maximum gel strength are 68.8 h for the time of treatment with calcium hydroxide solution, 43.2 °C for the extraction temperature, 5.73 h for the extraction time with hot water. The gelatin from channel catfish skin showed a high gel strength, 276±5 g. Compare to porcine skin gelatin, the gelatin from channel catfish skin has different amino acids composition and a lower thermo-stability.     

Characteristics of gelatin from the skin of unicorn leatherjacket (Aluterus monoceros) as influenced by acid pretreatment and extraction time

Gelatins from the skin of unicorn leatherjacket (Aluterus monoceros) pretreated with different acids (0.2 M acetic acid or 0.2 M phosphoric acid) and extracted with distilled water at 45 °C for various times (4 and 8 h) were characterized. Yields of 5.23–9.18 or 6.12–11.54% (wet weight basis) were obtained for gelatins extracted from the skin pretreated with 0.2 M acetic acid or 0.2 M phosphoric acid, respectively. Extracted gelatins contained α₁ and α₂ chains as the predominant components and some degradation peptides. The absorption bands of gelatins in FTIR spectra were mainly situated in the amide band region (amide I, amide II and amide ???) and showed the significant loss of molecular order of triple helix. Gelatin samples had a relative solubility greater than 90% in the wide pH ranges (1–10). The gel strength of gelatin from skin pretreated with phosphoric acid (GPA) was higher than that of gelatin from skin pretreated with acetic acid (GAA). Both GPA and GAA had the lower gel strength than that of commercial bovine gelatin (P < 0.05). Net charge of GAA and GPA became zero at pHs of 6.64–7.15 and 6.78–7.26, respectively, as determined by zeta potential titration. Emulsifying and foaming properties of GAA and GPA increased with increasing concentrations (1–3%, w/v). Those properties were governed by pretreatments and extraction time. Thus gelatin can be successfully extracted from unicorn leatherjacket skin using the appropriate acid pretreatment and extraction time.     

Isolation and partial characterization of pepsin-soluble collagen from the skin of grass carp (Ctenopharyngodon idella)

Pepsin-soluble collagen was extracted from the skin of grass carp (Ctenopharyngodon idella) with a yield of 46.6%, on a dry weight basis. Electrophoretic patterns showed that the collagen contained α1 and α2 chains, similar to those of calf skin collagen. The imino acid content of the collagen from grass carp skin was much lower than those of mammalian’s collagens, as also were the transition temperature and denaturation temperature which were only 24.6 °C and 28.4 °C respectively. Peptide maps of the collagen digested by trypsin and V8 protease showed different peptide fragments from those of calf skin collagen and other fish skin collagens, suggesting differences in amino acid sequences and collagen conformation. In addition, the lyophilized collagen sponge from grass carp skin had a uniform and regular network structure, just like calf skin collagen sponge. These results suggest that grass carp skin has potential for use as a supplementary source of collagen.     

Characterisation of acid-soluble collagen from skin of silver carp (Hypophthalmichthys molitrix)

Acid-soluble collagen (ASC) from the skin of silver carp (Hypophthalmichthys molitrix) was isolated and some properties of ASC were investigated. SDS–PAGE patterns showed ASC from silver carp skin was type ? collagen. Sulfopropyl-Toyopearl 650(M) column chromatography indicated that ASC from silver carp skin was composed of three kinds of α chains, α₁, α₂ and α₃. Hydroxyproline and proline content of ASC from silver carp skin was 192 residues/1000 residues, which was similar to that of ASC from carp skin. Denaturation temperature (T_d) of ASC from silver carp skin was around 29 °C. The results showed that some properties of ASC from silver carp skin were similar to those of ASC from carp skin. However, the peptide map of ASC from silver carp skin digested by pepsin was distinguished with that of ASC from carp skin.