Collagen as the Major Edible Component of Sea Cucumber (Stichopus japonicus)

The body wall collagen of an edible sea cucumber, Stichopus japonicus, was studied with respect to its chemical composition and subunit structure. About 70% of the total body wall protein was accounted for by highly insoluble collagen fibers. The disaggregation with β‐mercaptoethanol, 0.1 M NaOH treatment, and limited pepsin digestion of these collagen fibers resulted in complete solubilization. The solubilized collagen was isolated and characterized; it had 2 distinct subunits, αl and α2, which formed (α1)₂α2 heterotrimers and was rich in glutamic acid when compared with other fibrillar collagens. The unique textural properties of cooked sea cucumber seem to be due to thermal denaturation of the insoluble collagen fibers.     

Collagen of the skin of ocellate puffer fish (Takifugu rubripes)

Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from ocellate puffer fish skin and partially characterized. With respect to the pepsin-solubilized collagen, it was a heterotrimer with a chain composition of (α1)₂α2. The patterns of peptide fragments were different from skin collagens of other species. The denaturation temperature was 28 °C, about 9 °C lower than that of porcine skin collagen. On the other hand, the yields of acid-solubilized and pepsin-solubilized collagens were very high, 10.7% and 44.7%, respectively, on a dry weight basis. These results suggest that ocellate puffer fish skin has potential as an alternative source of collagen for use in various fields.     

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.     

Biochemical properties of acid-soluble collagens extracted from the skins of underutilised fishes

Acid-soluble collagens (ASCs) were extracted from the skins of several underutilised fishes, namely dusky spinefoot (Siganus fuscescens), sea chub (Kyphosus bigibbus), eagle ray (Myliobatis tobijei), red stingray (Dasyatis akajei) and yantai stingray (Dasyatis laevigata). The yields of the ASCs from skins of dusky spinefoot and sea chub were about 3.4–3.9%, and from ray species were about 5.3–5.7%, on a dry weight basis. According to the electrophoretic pattern, ASCs consisted of two different α-chains (α1 and α2) and were classified as type I collagen. However, the molecular weights of α2-chain for ray species were lower than those of bony fishes. ASC from ray species contained a higher content of imino acids than those from dusky spinefoot and sea chub. The denaturation temperatures (T_d) of ray species were about 33 °C, which was about 5 °C higher than those of dusky spinefoot and sea chub. The high T_d of ray species suggested the possibility of its utilisation as a substitute for mammalian collagen.     

PREPARATION AND CHARACTERIZATION OF SEVERAL FISH BONE COLLAGENS

As a part of the study regarding an effective use of underutilized resources, type I collagen was prepared from several fish bones and partially characterized. The yield of collagen was fairfy high with values ranging from about 41 to 54% on the basis of lyophilized dry weight. The pattern of lysyl endopeptidase peptide fragments from fish bone collagen obtained from different fish specie were very similar. However, the submit structures were quite different and were as follows: Japanese sea bass; (α1)₂α2, horse mackerel; (α1)₃ and ayu; α1 α2 α3. These results suggest a wide distribution of molecular forms in fish bone collagen.     

Properties of collagen from skin,scale and bone of carp (Cyprinus carpio)

Carp (Cyprinus carpio) is one of the main species of freshwater fish produced in China. Acid-soluble collagens (ASC) were prepared from carp skin, scale and bone. The yields of skin ASC, scale ASC and bone ASC are 41.3%, 1.35% and 1.06% (on the dry weight basis), respectively. SDS–PAGE pattern showed that ASCs of carp skin, scale and bone were all type I collagen, which were composed of two α₁ and one α₂ chains. The molecular weight of α₂ chain is 116 KDa. The amino acid composition and peptide maps of ASCs were similar to each other, but they were totally different from those of cod skin ASC. Denaturation temperatures (T_d) of ASCs were around 28 °C. Fourier transform infrared spectroscopy proved that ASCs were integrate and native. The results suggest that carp skin, scale and bone collagens have potential as an alternative source of collagen for use in various fields.     

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.     

尖吻鲈鱼鳞和鱼皮胶原蛋白的提取及其理化特性分析

以尖吻鲈鱼鳞和鱼皮为原料,提取并分离纯化酶溶性胶原蛋白,通过十二烷基硫酸钠-聚丙烯酰胺凝胶 电泳（sodium dodecyl sulfate-polyacrylamide gel electropheresis,SDS-PAGE）、氨基酸组成分析、差示扫描量热 （differential scanning calorimetry,DSC）、傅里叶变换红外光谱、X射线衍射和Zeta电位以及溶解度研究,分析 和比较了其主要理化性质。冷冻干燥后鱼鳞和鱼皮胶原蛋白得率（干质量）分别为2.3 g/100 g和47.3 g/100 g; SDS-PAGE结果显示两种胶原蛋白构型均为[α1(Ⅰ)]2α2(Ⅰ),初步判断属于Ⅰ型胶原蛋白;DSC结果显示鱼鳞和鱼 皮胶原蛋白热变性温度（Td）分别为37.54 ℃和36.74 ℃;傅里叶变换红外光谱和X射线衍射结果显示胶原蛋白经 胃蛋白酶处理后仍能保持其完整的三股螺旋结构;Zeta电位结果显示鱼鳞和鱼皮胶原蛋白等电点分别为pH 6.40和 pH 6.64;溶解度研究结果显示两种胶原蛋白在酸性条件和低NaCl质量浓度下均表现出良好的溶解性。     

Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonia cromis) and sheepshead seabream (Archosargus probatocephalus)

Acid-soluble collagen (ASC) and pepsin-solubilized collagen (PSC) were isolated from the bones and scales of black drum (Pogonia cromis) and sheepshead seabream (Archosargus probatocephalus) caught in the Gulf of Mexico. ASC and PSC were analyzed for molecular weight by SDS–PAGE, amino acid composition, secondary structure, and denaturation temperature. The molecular masses of the collagen subunits were about 130 kDa for α₁ and 110 kDa for α₂, respectively. The amino acid composition of the PSCs was closer to that of calf skin ASC than to that of cod skin ASC. The melting temperatures of ASC and PSC were >34 °C. Intrinsic viscosity of the PSCs was similar to the intrinsic viscosity of collagen from fish species such as hake, cod, and catfish. Black drum and sheepshead bone and scale collagens were typical type-I collagens and may find applications in the functional food, cosmetic, biomedical, and pharmaceutical industries.     

Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus)

The compositions and some properties of acid-soluble collagens (ASC) of the skin and bone of bigeye snapper, (Priacanthus tayenus) were investigated. The collagens were extracted with the yields of 10.94% and 1.59% on the basis of wet weight, from skin and bone, respectively. Similar electrophoretic patterns of collagens from the skin and bone were observed. Both collagens comprised two different α chains, α1 and α2 and were classified as type I collagen. However, peptide maps of collagen from the skin and bone of bigeye snapper, digested by V8 protease and lysyl endopeptidase, revealed differences between collagens from skin and bone, and both were completely different from those of calf skin collagen. Collagen rehydrated in acetic acid had lower T_max and enthalpy than those rehydrated in deionised water, suggesting a conformational change caused by acid. Collagens form the skin and bone had the highest solubility at pH 2 and 5, respectively. No changes in solubility were observed in the presence of NaCl up to 3% (w/v). However, a sharp decrease in solubility was found with NaCl above 3% (w/v).     

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.     

Isolation and Characterization of Collagen from the Body Wall of Sea Cucumber Stichopus monotuberculatus

To exploit a new collagen resource from the body wall of tropical sea cucumber, pepsin‐solubilized collagen of Stichopus monotuberculatus (PSC‐Sm) was isolated and characterized with UV‐vis spectra, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), amino acid composition, enzyme‐digested peptide maps, Fourier transform infrared spectroscopy (FTIR), maximum transition temperature (T_m), and solubilities. The maximum absorbance of PSC‐Sm was exhibited at 218 nm in UV‐vis spectra. The triple helical structure and activity of PSC‐Sm could be indicated by FTIR. SDS‐PAGE showed that the triple helix of PSC‐Sm was formed as (α₁)₃ by 3 α₁ chain homologous with molecular weight of 137 kDa. The T_m of PSC‐Sm and calf skin collagen (CSC) were 30.2 and 35.0 ºC, respectively, which consistent with the result of FTIR that CSC contained more stable triple‐helix than PSC‐Sm. Peptide maps were different between PSC‐Sm and CSC, indicating the differences in their amino acid compositions and sequences. The maximum and minimum solubilities of PSC‐Sm were observed at pH 2.0 and 4.0, respectively. A sharp decrease in solubility appeared when NaCl concentration was between 3% and 5%. These results showed that collagen from S. monotuberculatus had the type I collagen characteristics and good thermal stability, and therefore, it could be used as an alternative resource of collagen.     

Characterization of collagen from Japanese sea bass caudal fin as waste material

In an investigation into making more effective use of underutilized resources, Japanese sea bass caudal fin collagen was prepared and partially characterized. It was separated in two fractions, acid-solubilized collagen (ASC) and acid-insoluble collagen (AIC). The yields of ASC and AIC were about 5.2% and 36.4% respectively, on a dry weight basis. AIC consisted of only one chain and has a chain composition of (1)₃ homotrimer. The patterns of peptide fragments of ASC and AIC were different from that of porcine skin Type I collagen. The denaturation temperatures of these collagens were about 28 °C, about 9 °C lower than that of porcine skin Type I collagen. Japanese sea bass caudal fin has a potential as an alternative source of mammalian collagen for use in various fields such as foods, medicines, and cosmetics.     

Collagen changes in refrigerated sea bass muscle treated with pyrophosphate and stored in modified- atmosphere packaging

Changes in collagen of sea bass muscle treated with and without pyrophosphate (PP) were monitored during storage in modified-atmosphere packaging (MAP) (80% CO₂, 10% O₂ and 10% N₂) at 4 °C. No changes in acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) of sea bass muscle with and without PP treatment were observed during storage in MAP up to 21 days. However, a slight decrease in insoluble collagen (ISC) of a sample kept in MAP was found. For sea bass muscle stored under an air atmosphere ASC increased, whereas PSC and ISC decreased with a concomitant loss in the firmness     

Detection of five novel GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of blacktip shark (Carcharhinus limbatus) were isolated and characterized. The yield of ASC (20.01%) was much higher than that of PSC isolated from the residue of ASC extraction (0.86%). Both collagens had protein as their major constituent with the trace amounts of ash and fat. Based on protein patterns and TOYOPEARL^® CM-650M column chromatography, both collagens contained α- and β-chains as their main components and were characterized as type I collagen with the cross-link of α2-chain. Similar peptide maps of both collagens, digested by either V8 protease or lysyl endopeptidase, were observed but they were totally different from those of type I collagen from calf skin hydrolyzed by the same enzyme. Thermal transition temperature (T _max) of ASC and PSC were 34.23 and 34.37 °C, respectively. Fourier-transform infrared spectra suggested that both collagens were in triple-helical structure. From zeta potential analysis, isoelectric points (pI) of ASC and PSC were estimated to be 6.78 and 7.02, respectively. Thus, blacktip shark skin may serve as an alternative source of collagen and acid solubilization process could be implemented with ease and high yield.     

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     

The rheological and structural properties of fish collagen cross-linked by N-hydroxysuccinimide activated adipic acid

Fish skin collagen, with a concentration (6 mg/ml) higher than its critical aggregation concentration (∼0.5 mg/ml), was cross-linked using N-hydroxysuccinimide activated adipic acid (NHS-AA). Dynamic rheological measurement indicated that the storage modulus, loss modulus, complex viscosity and dynamic denaturation temperature increased while the loss tangent decreased with increasing the degree of cross-linking (DC). In addition, collagens with different DC exhibited a significant difference in their activation energies. It was found that collagens with DC in three intervals (DC < ∼25%, ∼25% < DC < ∼50% and DC > ∼50%, respectively) possessed remarkably different rheological behaviors each other. That is, collagens with DC < ∼25% exhibited a liquid-like behavior, while collagens with DC > ∼25% tended to exhibit a solid-like behavior, and especially a sudden loss of flow was observed for collagen with DC > ∼50%. Similar trends were observed from the features in differential scanning calorimetry (DSC) thermograms of collagen. That is, denaturation temperature (T_d) was slightly increased and the endothermic peak became broader as DC < ∼25%, T_d was increased gradually as DC was further increased, while a sudden increase in T_d was observed and the endothermic peak became sharp for collagen with DC > ∼50%. The three-step changes in the rheological behaviors and DSC thermograms of collagens might be due to the unique aggregation behavior of collagen in solution.     

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.     

Biochemical and thermo-mechanical analysis of collagen from the skin of Asian Sea bass (Lates calcarifer) and Australasian Snapper (Pagrus auratus), an alternative for mammalian collagen

Australasia has a large fish industry, and fish skin by-products from the processing industry could be used for the commercial production of fish collagen. The aim of this study was to characterize collagen extracted from the Asian sea bass (Australian barramundi) (Lates calcarifer) and snapper (Pagrus auratus) skin as an alternative to mammalian-derived collagen in gelatin products. The acid-soluble fractions of collagen from Asian sea bass and snapper skin were extracted and yielded about 8 and 7.5 % collagen (on a dry weight basis), respectively. The electrophoretic and chromatography patterns indicated that both collagens comprise of α1, α2, α3, and β chains, corresponding to the properties of calf skin collagen type I. Amino acid analysis and peptide mapping of digested collagen suggested differences in their amino acid sequences and collagen primary structure. Fourier transform infrared spectroscopy demonstrated that the helical structure of collagen was completely maintained in Asian sea bass and partially in snapper. Transition temperatures for the completion of the melting process in the two collagen networks were confirmed with differential scanning calorimetry and dynamic oscillatory rheology to be about 29 °C. Zeta potential analysis identified the isoelectric points (pI values) of collagen from Asian sea bass and snapper skin at pH 6.90 and 7.75, respectively. Thus, Asian sea bass and snapper skin could be an important alternative source of collagen to replace mammalian collagen for industrial applications.     

Isolation and characterisation of collagen from the ribbon jellyfish (Chrysaora sp.)

Pepsin‐solubilised collagen from the ribbon jellyfish (Chrysaora sp., morphotype 1) umbrella (JPSC) was isolated and characterised. The yield of collagen varied (9–19%, based on ash‐free dry weight) depending on the amount of pepsin used. Type II collagen was the major component of extracted collagen. The peptide map of JPSC differed from that of standard collagen type II, which indicates their different primary structures. FTIR spectra of JPSC, however, did not differ significantly from those of type II collagen. The T_max of JPSC was 37.38 °C, which is higher than that of other marine collagens. Glycine was the main amino acid in JPSC (320 residues per 1000 residues), followed by glutamic acid, alanine, proline, aspartic acid and hydroxyproline. The isoelectric point of JPSC was 6.64. These results indicate that this jellyfish species has the potential to be a marine source of type II collagen that can be used in place of land‐based sources.