Isolation and characterization of collagens from the skin of giant grouper (Epinephelus lanceolatus)

ABSTRACT

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of giant groupers (Epinephelus lanceolatus) with yields of 39.51 and 19.12%, respectively. ASC and PSC consisted of two different α chains (α1 and α2) and were characterized to be type I collagen with no disulfide bond. The imino acid contents of the ASC and PSC from giant grouper skin were 189 and 181 per 1,000 residues, respectively. The maximum endothermic temperatures (T_max) of ASC and PSC measured by differential scanning calorimetry (DSC) were 31.71 and 31.33°C, respectively. The denaturation temperatures of ASC and PSC measured by viscometry were 29.84 and 29.05°C, respectively. The maximum solubility in 0.5 M acetic acid was observed at pH 5 and pH 6 for ASC and PSC, respectively. A sharp decrease in solubility was observed for both ASC and PSC in the presence of NaCl above 3% (w/v).     

Characteristics and Select Functional Properties of Collagen from Golden Pompano (Trachinotus ovatus) Skins

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) from golden pompano skins were extracted and characterized. The molecular weight of ASC was about 130 kDa for α₁ and 115 kDa for α₂, which were slightly higher than those of PSC. Similar amino acid composition and Fourier transform infrared (FTIR) spectra were observed in both collagens, but slight differences were found in the peptide maps of collagen digested by V8 protease and trypsin. The denaturation temperatures (Tds) of ASC and PSC calculated from the reduced viscosity were 31.8 and 30.0°C, while the transition temperature (T_m) of ASC and PSC analyzed by DSC were 33.0 and 32.0°C, respectively. ASC has a lag phase, a growth phase, and a plateau phase in the turbidity–time curve, while PSC does not have similar phenomenon. It was found that the fibril gel of ASC could be formed at 25°C, leading to improved thermal stability.     

草鱼鱼鳞胶原蛋白的提取及其部分生物学性能

以草鱼鱼鳞为原料, 分别提取鱼鳞中的酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC), 着重开展了其包括热稳定性、体外酶降解性以及胶原海绵材料特性在内的相关研究, 并与哺乳动物来源的猪皮胶原(PC)相比较。实验结果表明, 制备所得的3种胶原蛋白均为典型的Ⅰ型胶原并具有完整的三螺旋结构; PC的热变性温度(41.6 ℃)明显高于ASC(34.8 ℃)和PSC(35.2 ℃); 3种胶原蛋白的体外酶降解性能受水解酶的种类、胶原蛋白提取方法、胶原蛋白来源、胶原蛋白受热历史以及蛋白的自组装程度影响。胶原蛋白酶、胰蛋白酶和木瓜蛋白酶对淡水鱼胶原均具有不同程度的降解能力, 但胶原蛋白酶的降解能力最强; 相同条件下, 3种胶原蛋白体外酶降解率依次为ASC>PSC>PC; 经热变性处理后胶原蛋白的体外酶降解率明显提高而经体外自组装处理后其体外酶降解率均出现不同程度的降低; 3种胶原样品冻干后得到的胶原海绵材料具有不同的机械性能和组织结构, ASC和PSC海绵是一种多孔但拉伸承受力较弱的海绵材料, 而PC则与之相反。     

多棘海盘车体壁胶原蛋白的研究

采用两种不同的方法从海盘车体壁中提取出酸溶性胶原蛋白（ＡＳＣ）和胃蛋白酶促溶的胶原蛋白（ＰＳＣ）,得率分别为１０．９０％、６１．４３％。将ＡＳＣ、ＰＳＣ的氨基酸组成、理化性质与脊椎动物及其它无脊椎动物胶原蛋白进行比较,结果表明,ＡＳＣ、ＰＳＣ是典型的胶原蛋白。在此基础上对ＡＳＣ、ＰＳＣ进行了ＳＤＳ－ＰＡＧＥ电泳,进一步表明制品的纯度较高,并且它们在分子大小、构型及性质上没有显著差异。     

Extraction and Characterization of Pepsin Soluble Collagen from the Body Wall of Sea Cucumber Acaudina leucoprocta

Sea cucumber Acaudina leucoprocta is a potential alternative collagen source. However, the high levels of heavy metals contained in the body wall restricts its utilization. In this work, an efficient method was established to remove the heavy metals accumulated in the body wall of A.leucoprocta by demineralizing with 0.2 M ethylenediaminetetraacetic acid (EDTA). The resulting body wall of A.leucoprocta was then used for extracting pepsin-soluble collagen (PSC) with pepsin proteolysis. The PSC from the body wall of A.leucoprocta was obtained with a yield of 43.99 ± 0.65% (dry weight) and high purity. Maximum and minimum solubility for the isolated PSC in 0.5 M acetic acid was observed at pH 2.66 and 4.43, respectively. The solubility was remarkably decreased in the presence of NaCl. The denaturation temperature of PSC rehydrated in 0.5 M acetic acid was measured as 25.4°C. The PSC was characterized as type I collagen, which consists of three α₁ chains without α₂ chain. Interestingly, α₁ chain in PSC showed two isoforms with the pI values of 4.02 and 4.29. The heavy metals existing in PSC were all below the contaminant limit of edible gelatin. The PSC isolated from the body wall could be an alternative to mammalian collagens.     

Characterization of Collagen from Different Discarded Fish Species of the West Coast of the Iberian Peninsula

ABSTRACT

Skin collagen of six discarded fish species was analyzed and compared. Acid soluble collagen (ASC) was extracted; a characteristic sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile for type I collagen was obtained, except for Chimaera mostrosa. Contents of collagen calculated from HPro (31.85% average) were higher than those determined from ASC extracts (17.75% average), with Galeus spp. being the species with the higher percentage. Amino acid analysis revealed the typical composition of collagen, with very few differences among species. Specific profiles were obtained after protease digestion. Denaturation temperature of ASC correlated well with imino and hydroxyproline contents.

Results demonstrate the feasibility of using the obtained collagens in different industrial applications.     

Characterization of Acid-Soluble Collagen From Bone of Pacific Cod (Gadus macrocephalus)

Acid-soluble collagen (ASC) was isolated from Pacific cod (Gadus macrocephalus) bone. The ASC was rich in glycine. The amount of imino acid was lower than that of calf skin collagen, as was the transition temperature (48.6°C). Electrophoresis revealed two different α chains (α₁ and α₂), β-component, and γ-component. Fourier transform infrared spectroscopy measurement showed that ASC was in triple-helix structure. ASC had a solubility greater than 90% in a very acidic pH range (pH 1–4), and the solubility decreased with increasing NaCl concentrations up to 3%. Lyophilized ASC had a network ultrastructure with lace-like fibers, similar to calf skin collagen sponge.     

Chemical and Thermal Properties of Freshwater Prawn (Macrobrachium rosenbergii) Meat

Chemical compositions and thermal properties of cultured freshwater prawn meat (FPM) were studied. FPM contained 83.2% protein (dry basis), 62.7% of which was myofibrillar protein. Pepsin-soluble collagen (PSC) and insoluble collagen (ISC) contents were 0.63 and 0.32%, respectively. Both collagens were similar to type V collagen from porcine placenta. Glutamic acid/glutamine, arginine, aspartic acid/?asparagine, and lysine were abundant amino acids in FPM. Glycine, proline, hydroxyproline, and aspartic acid/?asparagine were predominant in both collagens. FPM exhibited thermal transition temperatures (T_max) of 48.3 and 64.7°C, whereas T_max of PSC and ISC were 43.0 and 46.0°C, respectively. Textural changes in FPM during post-mortem storage on ice are plausibly dependent upon its compositional and thermal properties.     

鲤鱼鱼鳞胶原蛋白ASC与PSC的比较

以鲤鱼鱼鳞为原料,研究了酸法和酶法提取的鱼鳞胶原蛋白ASC和PSC的异同.经过SDS-凝胶电泳和氨基酸分析可以看出,2种蛋白在分子构型、氨基酸组成等方面的差异并不明显,经过蛋白酶K有限酶解后的图谱证明了这一点.但从差热分析,以及蛋白质受热的分解变化来看,二者之间在热稳定性方面存在一定的差异.推测差距产生的原因与蛋白质制备过程中,胃蛋白酶的作用有关,胶原蛋白两端的非螺旋区域受到酶的作用而分解,非螺旋区域对三螺旋的稳定性起着重要作用,非螺旋区域的分解大大降低了PSC对热的耐受性.     

金枪鱼鱼骨胶原肽的制备及抗氧化活性研究

为制备金枪鱼鱼骨胶原肽,并对其抗氧化活性进行研究,利用酶解、超滤、凝胶色谱和反相高效液相色谱制备抗氧化胶原肽,采用氨基酸序列分析仪测定其氨基酸序列,利用质谱(ESIMS)确定其分子量,采用羟自由基、DPPH自由基、ABTS自由基和超氧阴离子自由基清除实验和脂质过氧化抑制实验对胶原肽抗氧化能力进行评价。结果显示,金枪鱼鱼骨胶原蛋白经胃蛋白酶和胰蛋白酶2步酶解和分离纯化得到1个十肽(TFCH-P2),经氨基酸序列分析和质谱(ESIMS)确定其氨基酸序列为Gly-Pro-Ala-Gly-Pro-Ala-Gly-Glu-Gln-Gly(GPAGPAGQEG),分子量为839.87 u([M+H]+840.68 u)。体外抗氧化实验结果表明,GPAGPAGQEG对羟自由基(EC500.18 mg/mL)、DPPH自由基(EC500.97 mg/mL)、ABTS自由基(EC500.52 mg/mL)和超氧阴离子自由基(EC500.38 mg/mL)具有良好的清除作用;GPAGPAGQEG亦显示出良好的脂质过氧化抑制作用。研究表明,胶原肽GPAGPAGQEG抗氧化活性良好,可以用于抗氧化相关的功能食品、药物或者食品添加剂。     

Isolation and Characterization of Pepsin-Soluble Collagen from the Skin of Peru Squid (Dosidicus gigas)

Pepsin-soluble collagen (PSC) was isolated from Peru squid (Dosidicus gigas) skin and physicochemical properties of the PSC were determined. The PSC exhibited a maximum absorbance at 220 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested the collagen containing α1 and α2 chain was classified as type I collagen. Amino acid composition indicated that the collagen had lower amino acid content than that of mammalian collagen. Denaturation temperature (T_d) of the PSC was 26.8°C. The PSC had relatively high solubility in alkaline condition or NaCl concentrations below 2%. Fourier transform infrared spectroscopy investigations showed the existence of helical arrangements of collagen. The lyophilized collagen had a uniform and regular network structure. These results suggested that Peru squid skin was a potential source of collagen for further research and application.     

鱼鳞胶原蛋白研究

采用酸性和酶提取法从鱼鳞中提取酸溶性和酶促溶性Ⅰ型胶原蛋白，经ＳＤＳ－ＰＡＧＥ电泳显示，所提取的胶原蛋白电泳区带与Ⅰ型标准品相同。氨基酸分析表明，ＡＳＣ和ＰＳＣ是典型的胶原蛋白，热稳定性测定ＡＳＣ的Ｔｄ为３２．３℃，ＰＳＣ的Ｔｄ为２７．８℃。     

Purification and immunochemical detection of a quantitatively major collagen in the dermis of sea cucumber Apostichopus japonicus

Pepsin-solubilized collagen (PSC) was prepared from the dermis of sea cucumber Apostichopus japonicus (green type) by performing pepsin digestion to collagen fiber pretreated with disaggregating solution (0.1 M Tris–HCl, pH 8.0, containing 0.5 M NaCl, 0.05 M ethylenediaminetetraacetic acid (EDTA), and 0.2 M 2-mercaptoethanol) and 0.1 M NaOH. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the PSC clearly showed two alpha bands under phosphate buffer system in the presence of 3.5 M urea. An antiserum was raised against chromatographically purified major molecular species in the PSC, and immunoblot analyses were performed for the soluble fractions at 4 M guanidine hydrochloride treatment and disaggregation as well as the collagen fiber before and after treatment. These fractions and collagen fibers showed quite similar band patterns to that of the PSC, showing mainly two alpha bands. These combined results suggest that the major molecular species of collagen contains at least two distinct alpha components and that the effect of pepsin digestion is relatively small on the structure of this collagen type.

     

Changes in muscle collagen content during <Emphasis Type="Italic">post mortem</Emphasis> storage of farmed sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>): influence on textural properties

The changes in collagen content and its solubility in sea bream muscle were studied for variable storage times following the death of the fish, and these variables were related to the evolution of physical parameters important for consumer acceptance: firmness and water-holding capacity (WHC). The results show that the collagen content in muscle diminished slightly over storage time and that this variable was directly related to firmness but inversely related to the water-holding capacity. With regard to collagen solubility, a decline was detected in acid-soluble collagen (ASC) in the first few post mortem hours, perhaps related to the end of rigor mortis that occurs at these stages. Pepsin-soluble collagen (PSC) increased, while insoluble collagen (ISC) decreased from 96 h, coinciding with a loss of firmness. This softening can be explained as a result of specific collagenases acting on the insoluble fraction of the collagen.     

Physicochemical Properties of Pepsin-Solubilized Collagen Extracted from Nile Tilapia (Oreochromis niloticus) Scale

ABSTRACT

The aim was to investigate the physicochemical properties of pepsin-solubilized collagen (PSC) extracted from fish scales of Nile tilapia. The results indicated that Nile tilapia scales are rich in collagen. Therefore, the scales are a promising cost-effective collagen source. The conversion of hydroxyproline to collagen was 12.9. Based on the patterns of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), PSC comprised at least two different α chains, α1 and α2, and was classified to be type I with no disulfide. Fourier transform infrared spectroscopy spectrum showed that the bands of amide A, I, and II of PSC were found at 3,301, 1,631, and 1,239 cm^?1 wavelength. The content of imino acids was 187 residues per 1,000 total amino acid residues. Maximum solubility was observed at pH 4, and minimum was at pH 7. Almost no change in solubility was observed in the presence of NaCl up to 2% (w/v), and the decrease was more pronounced with increasing NaCl concentration. The temperature of denaturation was 35.4°C. Results show that PSC has physicochemical properties that can be applied in the cosmetics, biomedical, pharmaceutical, and food industries.     

Pyridinoline concentrations in muscular and skin collagen of fish and relationship between collagen solubility and pyridinoline concentration in fish muscular collagen

Pyridinoline (Pyr), one of the mature crosslinks of collagen, was determined in muscular collagen of three species of fish. The amounts of muscular Pyr in red sea bream, yellowtail, and tiger puffer were 3.4, 8.8, and 50.3 mmol/mol collagen, respectively, indicating that the Pyr concentration in muscular collagen differs greatly among fish species. The Pyr concentration in tiger puffer muscular collagen was the greatest, but it was only one-fourth that in rabbit muscle. As in mammalian skin collagen, Pyr was not detected in skin collagen of red sea bream and yellowtail. However, tiger puffer skin contained Pyr (3.75 mmol/mol collagen). The presence of Pyr would have a relationship to some features of tiger puffer skin, such as mechanical strength and thickness. Pyr concentrations in acid-soluble collagen (ASC), pepsin-solubilized collagen (PSC), and insoluble collagen (ISC) in muscles of three species of fish were determined. Pyr was found in ISC > PSC > ASC, from the highest to the lowest concentration, and the concentration in ISC was 45–200 times that in ASC. Therefore, Pyr crosslinks that are formed between collagen molecules would have a close relationship to collagen solubility.     

Identification and primary structures of eel type I collagen proα1, proα2 and proα3

Many bony fish type I collagens have a characteristic third chain designated as α3(I). However, much less is known about the primary structure and distinction of the proα(I) chains. Their cDNAs were cloned by RT-PCR from the muscle tissue of Japanese eel, Anguilla japonica. Three cDNAs coding for the triple-helical domain of fibrillar collagen were identified as proα1(I), proα2(I) and proα3(I) chains by sequencing selected tryptic peptides isolated from eel type I collagen subunit chains, α1(I), α2(I) and α3(I). Eel proα3(I) had high amino acid sequence identity (81 %) to its proα1(I). The distribution of seven Cys residues in the C-propeptide of proα3(I) was identical to that of proα1(I). There was a third Cys residue at the 1,268th position from the N-terminus in proα1(I), though a supposed Cys residue at the 1,264th position in proα3(I) was replaced by a Ser residue. Similar replacement has been observed in the proα3(I) chains of trout and zebrafish. These combined results suggest that replacement of the Cys residue allows for the identification of fish collagen proα(I) previously not identified as proα3(I).     

Physicochemical Properties and Angiotensin I Converting Enzyme Inhibitory Peptides of Freshwater Fish Skin Collagens

ABSTRACT

The objectives of this study were to characterize physicochemical properties of two collagens, tilapia skin (TS) and hybrid catfish skin (HS). Angiotensin-converting enzyme (ACE) inhibitory peptides from extracted TS and HS collagen using pepsin were also determined. HS collagen had a higher amount of imino acid than TS collagen, while TS contained higher amounts of tyrosine (Tyr) and lysine (Lys). Fourier-transform infrared spectra of both collagens showed predominant helix structure. The HS collagen hydrolysate prepared by pepsin was fractionated using sequential ultrafiltration membranes, and the fraction with molecular weight (MW) <5 kDa showed the highest ACE inhibitory activity (p < .05). After cation exchange and two steps size exclusion chromatography, peptides showed ACE inhibitory activity of 72.06%. This study revealed that ACE inhibitory peptides derived from HS collagen could be developed as a functional food with potential antihypertensive properties.     

Molecular species of collagen in muscular and vertebral parts of white sturgeon Acipenser transmontanus

The molecular species of collagen in the muscular and vertebral parts of white sturgeon Acipenser transmontanus were examined by biochemical techniques. Pepsin-solubilized collagens were prepared from these parts, and fractionated into major and minor collagen fractions by differential salt precipitation at acidic or neutral pH. Collagens contained in these fractions showed several features characteristic of fish type I and V collagens, respectively, in SDS-PAGE patterns and the amino acid compositions of both parts. These results suggest that at least two molecular species, corresponding to type I and V collagens, are distributed in the muscular and vertebral parts of white sturgeon.     

Physicochemical and Reactive Oxygen Species Scavenging Properties of Collagen and Collagen Hydrolysates from Farmed Globefish (Fugu rubripes) Bone

Globefish (Fugu rubripes) bone collagen (GBC) was isolated and characterized. Electrophoresis and Fourier transform infrared (FTIR) spectra indicated that GBC was high-purity type I collagen with an intact triple-helical structure. Amino acid analysis revealed that GBC contained higher glycine content (364 residues/1,000 residues) than that of other fishery bones, and the imino acid content was 189 residues/1,000 residues. The denaturation temperature (T_d) of GBC was 27.1°C. GBC displayed a rapid fibril-forming rate and well-defined fibril morphology in vitro. Globefish bone collagen hydrolysates (GBCH) were optimized by neutral protease through response surface methodology (RSM) with the highest hydroxyl radical (?OH) scavenging capacity. The optimal hydrolysis condition was a reaction temperature of 48.4°C, time of 120 min, and enzyme/substrate ratio of 2520 U/g. Three fractions were collected by ultrafiltration, and the fraction GBCH-III (Mw<1 kDa) possessed the strongest ?OH scavenging ability. Reactive oxygen species (ROS) scavenging capacities were evaluated with two model systems: ?OH and nitric oxide (NO?). The ROS scavenging capacities increased in a dose-dependent manner. GBCH-III proved to be a more effective ?OH scavenger than reduced glutathione (GSH). These findings demonstrated that GBC and GBCH have potential as an alternative source of collagen-derived materials for use in pharmaceutical, nutraceutical, and cosmetic industries.