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.     

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.     

Characteristics of Collagen from Rohu (Labeo rohita) Skin

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were isolated from rohu skin with the yield of 64.2 and 6.8% (dry weight basis), respectively. Both collagens had glycine as the major amino acid with imino acid content of 196–202 residues/1,000 residues and were characterized as type I collagen with molecular composition of (α1)₂α2-heterotrimer. Fourier transform infrared spectra of both collagens were similar, with no shift in wavenumber of all amide bands. The T_max value of ASC and PSC was 36.40 and 35.48°C, respectively. The zero surface net charge of ASC and PSC was found at pH 5.9 and 5.3, respectively.     

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.     

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.     

Extraction and Physicochemical Characterization of Pepsin Soluble Collagens from Golden Pompano (Trachinotus blochii) Skin and Bone

Pepsin-soluble collagen (PSC) was extracted and purified from wasted skin and bone of the golden pompano by acetic acid-pepsin method. The result showed that the PSCs extraction yields of skin and bone were 21.81% and 1.25% (wet weight), 62.21% and 1.78% (on the basis of lyophilized dry weight), respectively. Golden pompano skin and bone PSCs contained the typical chain of α and β dimers, and they were preliminarily judged to belong to type I collagen. The skin PSC had similar amino acid composition to bone PSC, which is rich in glycine, alanine, proline, and hydroxyproline. After addressing the pepsin, three helical structure of PSCs were intact, and their natural structures largely remained. The denaturation temperatures of skin and bone PSCs were 37.04°C and 38.23°C, respectively. Solubility results showed that the skin and bone PSCs solubility was the largest at pH = 3. The solubility of skin and bone PSCs was stable at NaCl concentrations lower than 3%. In addition, two PSCs in acid and low salt conditions had good solubility. This study demonstrated that golden pompano skin and bone could be used as good materials to extract PSC, representing an economic benefit and added value.

Abbreviations: SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; FTIR: Fourier transform infrared; DSC: Differential scanning calorimetry; XRD: X-ray diffraction     

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。     

Enzymatic solubilization of collagen in the skin of diamond squid Thysanoteuthis rhombus: application of a fungal acid protease

Enzymatic solubilization of collagen from the skin tissue of diamond squid Thysanoteuthis rhombus, an underutilized resource in Japan, was attempted using an acid protease from the fungus Rhizopus niveus. This novel approach was compared with the conventional method using porcine pepsin. Both proteases were able to solubilize most of the skin collagen (>90 % of the total collagen) by performing the treatment in 0.5 M acetic acid at 4 °C for 72 h and at an enzyme/substrate ratio (w/w) of 1/10. The SDS-PAGE patterns of the solubilized collagen preparations were quite similar to each other, and two types of collagen (major and minor collagens) were purified from each preparation by cation-exchange column chromatography. These collagen types from the porcine pepsin-solubilized collagen showed similar features to those from the Rhizopus acid protease-solubilized collagen. These results suggest that the Rhizopus acid protease, a protease of non-animal origin, is applicable for solubilizing collagen in the skin of diamond squid.     

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.     

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.

     

鱿鱼加工副产物综合利用研究进展

综述了近年来国内外利用鱿鱼皮、内脏、软骨、墨汁、鱼眼、精巢等副产物为原料开发胶原蛋白、鱼油、核酸、酶、软骨素、透明质酸、鱼精蛋白等一系列生化制品的研发状况,展望了几种鱿鱼副产物的应用前景和发展趋势.     

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.     

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。     

Physicochemical Properties and the Radical Scavenging Capacities of Pepsin-Solubilized Collagen from the Body Wall of Starfish (Asterina pectinifera)

Pepsin-soluble collagen was successfully prepared from the body wall of starfish (Asterina pectinifera) (PSCBWS). Amino acid composition suggested that the collagen might be classified as type I collagen. Ultraviolet-visible and Fourier transform infrared spectroscopic analyses showed that the PSCBWS was a high-purity collagen that maintained the intact triple-stranded helices. Physical and chemical characterizations of the PSCBWS showed an isoelectric point (pI) of 5.0 ± 0.2, superior moisture-absorption and retention capacities compared with glycerol, a minimum solubility at pH 5.0 in 0.5 M acetic acid, and a sharp decrease in solubility in the presence of low concentration of NaCl. The PSCBWS was evaluated for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and hydroxyl radicals. Results indicated that PSCBWS possessed DPPH and hydroxyl radicals scavenging capacity in a dose-dependent manner. These results indicated that PSCBWS might be used as a new collagen resource in the food and cosmetic industries.     

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℃。     

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.     

基于垂直水温因子的秘鲁外海茎柔鱼春、夏季栖息地分布

为了研究温度变动对茎柔鱼(Dosidicus gigas)资源丰度和栖息地的分布的影响, 利用 2006—2015 年秘鲁外海春夏季节(9 月至翌年 2 月)茎柔鱼渔业捕捞数据结合中上层垂直水温数据(0 m, 50 m, 100 m 和 150 m)建立栖息地适宜性(habitat suitability index, HSI)模型, 分析秘鲁外海茎柔鱼渔场以及栖息地的时空分布。通过计算适宜栖息地内的茎柔鱼资源量占比, 并用 2014—2015 年的数据进行验证。结果发现, 基于垂直水温因子和算术平均法的栖息地模型可以较好模拟出茎柔鱼栖息地适宜性指数。空间相关分析结果显示, 秘鲁外海水域各水层水温与栖息地适宜性呈现负相关关系。茎柔鱼的 CPUE 和适宜栖息地面积的变化相对平稳, 没有明显的年间和月间差异, 两者之间呈现显著的正相关关系。茎柔鱼的渔场重心和栖息地重心存在显著的年间和月间变化, 均呈现向东南方向移动的趋势, 同时春季适宜栖息地面积与夏季相比明显减少。茎柔鱼渔场的经纬度重心的月间和年际变化与栖息地经纬度重心的移动具有一致性, 两者之间呈现明显的正相关。研究表明, 茎柔鱼的资源丰度与适宜栖息地密切相关, 其适宜栖息地存在明显的年间和月间变化, 这可能是造成秘鲁外海茎柔鱼时空分布变动的重要原因。     

Characteristics and Functional Properties of Ovary from Squid Loligo Formosana

The characteristics and functional properties of the ovary from Loligo formosana were studied. Moisture (72.07 ± 0.24%) was dominant, followed by protein (18.64 ± 0.51%) and carbohydrate (7.44 ± 0.2%). Ash (1.39 ± 0.03%) and lipids (0.46 ± 0.5%) were found as the minor constituents. Albumin (79.02 ± 0.79%) was the major protein of the squid ovary, followed by glutelin-1 (8.31 ± 0.62%) and globulin (6.68 ± 0.08%). Nevertheless, prolamin and glutelin-2 constituted approximately 1% of the total proteins. Based on the electrophoretic studies, albumin had the largest band intensity. The squid ovary was rich in non-essential amino acids (52.26%) and high in hydrophobic amino acids (48.03%). It was also rich in polyunsaturated fatty acid (PUFA, 43.76 ± 0.84%), followed by saturated fatty acid (SFA, 39.36 ± 0.12%) and monounsaturated fatty acid (MUFA, 12.94 ± 0.55%). Ovary lipids had a high amount of docosahexaenoic acid (C22:6) (28.59%). At pH 3, the squid ovary powder (SOP) had the maximum solubility (96.39%), whereas the lowest solubility (38.33%) was observed at pH 9. The foaming capacity and stability of SOP were increased with increasing concentration up to 8% (p < 0.05). The globulin fraction showed the higher foaming capacity, as compared to albumin and glutelin-1 fractions. The squid ovary had good nutritional value and possessed excellent foaming properties. Therefore, the squid ovary could serve as a novel food additive or ingredient.     

Effect of dietary marine lipids on female white bass ova compositions and progeny survival

We evaluated white bass ovum fatty acid composition as well as embryonic and larval survival after varying n-3 and n-6 long-chain polyunsaturated fatty acid (LC-PUFA) concentrations in maternal diets. Diets containing graded levels (0, 33, 66, or 100%) of squid to menhaden oils were fed daily to apparent satiation to female white bass for 8 weeks prior to spawning. Embryonic survival was negatively related to maternal squid oil intake (P = 0.015, R ² = 0.970). Squid oil-fed broodstock produced ova with decreased 20:5n-3 and increased C₁₈ polyunsaturated fatty acid concentrations, largely reflecting the fatty acid profile of squid oil. Within ovum phospholipid, accumulation of 18:2n-6 may have altered biological function resulting in the lower embryonic survival among ova produced from the squid oil-fed broodstock. Our data suggest the importance of feeding white bass broodstock diets high in total n-3 LC-PUFA (at least 4.0% dry matter), and 20:5n-3-rich lipid sources such as menhaden oil can be effectively utilized by female white bass to produce quality ova.