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.     

Physicochemical properties of acid-soluble collagens from different tissues of large yellow croaker (Larimichthys crocea)

Acid-soluble collagens (ASCs) were extracted and characterised from different tissues of large yellow croaker (Larimichthys crocea). The yields of ASCs in bones, skins, scales and muscles were 3.22 ± 0.47%, 42.30 ± 1.15%, 2.82 ± 0.31% and 0.89 ± 0.12%, respectively. SDS-PAGE and ATR-FTIR analysis showed that all ASCs were type I collagen with intact triple helical structure and consisted of α1,3-chain, α2-chain, β-chain and γ-chain. Quantitative analysis of SDS-PAGE revealed that the ratio of β-chains in ASC from scales was lower than other tissues. The imino acid contents of ASC from bones, skins, scales and muscles were 169, 167, 162 and 173 residues/1000 residues, respectively. The maximum transition temperature (T_m) of ASC from scales was 30.15 °C, lower than the other three ASCs, indicated that the thermal stability of collagen was affected by both the content of imino acid and β-chains. The results also implied that thermal stability of ASC might exist the tissue specificity among different fish species.     

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.     

Biochemical properties of skin collagens isolated from black carp (Mylopharyngodon piceus)

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted with the yield of 15.5 and 26.5% on the basis of dry weight from the skin of black carp (Mylopharyngodon piceus). ASC and PSC were similar in amino compositions and subunit compositions, but they were slightly different in the secondary structure. Both of ASC and PSC, either rehydrated in distilled water or in acetic acid, had a lower denaturation temperature than pig skin collagen (PPSC), but their denaturation temperature were higher than that of collagens from other freshwater fish. Intrinsic viscosity of different collagens decreased in the order of PPSC>PSC>ASC. Both of ASC and PSC showed only partial degradation in collagenase solution, but they revealed more sensitive to collagenase compared with PPSC. Black carp skin collagens did not induce a significant cytotoxic effect according to the results of in vitro cytotoxicity test.     

草鱼鱼鳞、鱼皮和鱼骨酸溶性胶原蛋白特性对比研究

从草鱼鱼鳞、鱼皮和鱼骨中提取酸溶性胶原蛋白(ASC),对其特性进行对比分析。紫外光谱和电泳图谱结果显示,3种ASC紫外特征吸收峰出现位置与I型胶原蛋白紫外特征吸收峰出现的位置基本一致,分子结构都至少含有两条α链;傅里叶红外光谱显示,草鱼鱼鳞、鱼皮和鱼骨ASC在酰胺A带的N-H伸缩均以氢键形成缔合体,酰胺Ⅰ带和酰胺Ⅲ带的特征吸收峰表明鱼鳞和鱼骨ASC的α-螺旋结构保存较完整,鱼皮ASC的α-螺旋结构已经被破坏;3种ASC在热稳定性方面存在一定差异,热变性温度鱼骨ASC(34.5℃)鱼皮ASC(32℃)鱼鳞ASC(31℃),可能与胶原存在的组织和所处的环境有关。     

Characterization of acid- and pepsin-soluble collagens from flatfish skin

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from flatfish skin were characterized. The yield of PSC was 85.5%, which was higher than that of ASC at 57.3%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns showed that both the ASC and PSC were consisted of α1 and α2 chains, and β-chain. According to the results of Fourier transform infrared (FTIR) spectroscopy and hydroxylation, no difference in the helical structure between ASC and PSC was found. Thermal denaturation temperature (TDT) of flatfish skin collagen was 26.6°C for ASC and 26.7°C for PSC, which were similar to those of temperate fishes, while were lower compared to tropical fishes, such as the conger eel and Nile perch. There was no solubility difference between ASC and PSC at various pH and NaCl concentrations. Therefore, PSC will be preferable compared to ASC for commercial uses because of its higher yield.     

Compositional and physicochemical characteristics of acid solubilized collagen extracted from the skin of unicorn leatherjacket (Aluterus monoceros)

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 α₁ chains and one α₂ 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. T_max 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 1–4) and the solubility decreased continuously with increasing NaCl concentrations (0–6%). 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Partially Purified Collagen from Refiner Discharge of Pacific Whiting Surimi Processing

The physicochemical properties of acid‐soluble collagen (ASC) from refiner discharge and the partially purified collagen (PPC) from both the refiner discharge and the fish skin were evaluated. Yield of collagen from refiner discharge was 34% higher in PPC than ASC. Mercury, lead, cadmium, and chromium contents of PPC from refiner discharge were not detected. There was no difference in the pattern of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) between ASC and PPC from refiner discharge. PPC from refiner discharge showed better functional properties than that from skin and was similar to ASC: whiteness, solubility, emulsifying activity, and cooking stability. Therefore, PPC from refiner discharge could be used as a new resource.     

Characteristics of Gelatin from Giant Grouper (Epinephelus Lanceolatus) Skin

Gelatin extraction from the skin of giant grouper (Epinephelus lanceolatus) was conducted by acid process with a yield of 20.27 g/100 g wet skin sample. The characteristics of extracted gelatin from giant grouper was investigated in this study, and further compared to that from commercial tilapia. Results showed that when compared to commercial tilapia, giant grouper had lower levels of bloom strength and foam formation ability, but greater values of viscosity, foam stability, and lightness (L*) on gelatin skin. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed three high-bands intensities of major protein components of giant grouper skin gelatin, representing α1-chain, α2-chain, and β-components, and was similar to that of standard calf skin collagen type I. Compared to giant grouper, commercial tilapia contained extra proteins with molecular weight less than 70 kDa on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis of both skin gelatins.     

鮰鱼鱼皮胶原的提取及性质研究

利用醋酸及胃蛋白酶从鮰鱼鱼皮中提取了胶原,并对其物理与化学特性进行了探讨研究。结果表明:提取的胶原属于典型的Ⅰ型胶原,胶原的三股螺旋分子组成为[α1(Ⅰ)]2α2(Ⅰ)。氨基酸组成及变性温度的分析说明提取的鮰鱼皮胶原由于其自身亚氨基酸含量的特征,使得它的稳定性远远高于其他鱼类的皮胶原,但仍然比大多数哺乳类动物皮胶原稳定性要弱。紫外扫描光谱、傅立叶红外光谱及圆二色性研究结果证明提取的胶原三股螺旋结构保持完整。     

PHYSICAL AND CHEMICAL PROPERTIES OF EPIMYSIAL ACID-SOLUBLE COLLAGEN FROM MEATS OF VARYING TENDERNESS

SUMMARY: Components of the molecular structure of epimysial acid-soluble collagen (ASC) from meats of varying tenderness were studied by several methods. The ASC was studied since it contains an appreciable amount of intramolecular cross-links but is still soluble. Though the amount of total collagen in epimysial tissue was found to have no correlation with meat tenderness, the molecular studies indicated some correlation of the type of epimysial ASC with meat tenderness. Sucrose density-gradient ultracentrifugation analyses of denatured epimysial ASC suggested that this type of collagen contains a lesser degree of cross-links when obtained from tender meat samples. Viscosity measurements were found to be correlated to tenderness of meat in a manner similar to the results obtained by ultracentrifugation, in that the intrinsic viscosity of epimysial ASC from tender meat was lower than that from tough meat, indicating differences among the relative sizes of the collagen molecules from the different samples. Partial amino acid analyses of the epimysial ASC samples via gas chromatography showed no differences in the levels of aspartic, serine or hydroxyproline, amino acids which may be involved in the ester type of cross-links. Results of chemical estimation of the ester type of cross-links indicated that the proportions of esters in the various ASC samples were similar. The amount of lysine was found to be significantly higher ( P < 0.05) in epimysial ASC from tough meat compared to tender meat, suggesting an increased potential of the aldehydic-type of cross-link, which is formed via an aldol condensation of two aldehydes derived biosynthetically from lysine. This was strengthened by the results obtained in the chemical estimation of the aldehydic-type of cross-link, in that the epimysial ASC of tough meat contained significantly more aldehyde than did that of tender meat ( P <0.05).     

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 acid and pepsin-solubilized collagens from the skin of balloon fish (Diodon holocanthus)

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were successfully extracted from the skin of balloon fish (Diodon holocanthus) with yields of 4% and 19.5% respectively (based on dry weight). According to the electrophoretic patterns, both the ASC and PSC consisted of two different α chains (α1 and α2), were characterized to be type I, and contained imino acid of 179 and 175 residues/1000 residues, respectively. The PSC had a lower content of high-molecular weight cross-links than the ASC. The ultraviolet (UV) absorption spectrum of collagens showed that the distinct absorption was between 210 and 230 nm. A maximum solubility in 0.5 M acetic acid was observed at pH 1–5, and a sharp decrease in solubility above 4% (w/v) in both the ASC and PSC was observed in the presence of NaCl. The denaturation temperatures (T_d) of the ASC and PSC measured by viscometry were 29.01 °C and 30.01 °C, respectively. The maximum temperatures (T_max) of the ASC and PSC were 29.64 °C and 30.30 °C, respectively.