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.     

猪皮胶原粉的制备工艺及功能特性研究

本研究在4℃条件下用弱酸加酶法提取可溶性胶原并干燥成粉,然后对已提取过的猪皮用60%乙醇溶液作载体进行胶体磨研磨,制备不溶性胶原粉。进一步对其进行胶原含量及功能特性的测定。结果表明：猪皮胶原粉的最佳制备工艺为：采用pH2.0 醋酸+0.2% 胃蛋白酶、料液比1:15 条件下提取猪皮48h,可溶性胶原溶出率为0.602%;剩余猪皮采用60% 乙醇载体研磨后干燥可制得不溶性胶原粉。所得胶原粉各项功能特性优良,通过羟脯氨酸含量测定得出可溶性胶原粉胶原纯度为95.1%,不溶性胶原粉胶原纯度为82.9%,工艺路线易于实现规模化生产。     

Interaction of phytate with protein and minerals in a soybean–maize meal blend depends on pH and calcium addition

In order to investigate possible interactions of phytate with protein and minerals in simplified animal diets, studies were conducted on the solubility of endogenous phytate, protein and essential minerals in a soybean–maize meal blend within a physiological relevant pH range. The blend was mixed with water for 10 min and then allowed to incubate at 40 °C (30 min) after adjustment of the pH. Finally, soluble phytate, protein, zinc, manganese and iron were determined. Phytate and mineral solubility was highly influenced by pH whereas protein solubility was less affected. Addition of 5 g Ca²⁺ kg^?1 drastically reduced the solubility of phytate, zinc, manganese and iron at pH above 4.4, indicating that the formation of insoluble phytate–mineral complexes is increased in the presence of calcium. The action of pepsin increased the solubility of protein and phytate at pH below 4, indicating that insoluble phytate–protein complexes are present at low pH. Calcium had the same solubilising effect as pepsin at pH 2–4 but to a lesser degree. Copyright © 2007 Society of Chemical Industry     

Interdependence Between Heat Solubility and Pyridinoline Contents of Squid Mantle Collagen

ABSTRACT: A considerable amount of squid mantle collagen, 45% to 70% of total collagen, was not solubilized even after 30 min of heating in boiling water with its fibrous structure left intact. Pyridinoline, one of the major intermo-lecular crosslinks in matured collagen, was more predominantly included in the insoluble collagen than in the soluble one (p < 0.05). These results suggest that pyridinoline is closely related to the heat solubility of squid collagen.     

离子强度对煮制前后猪肉胶原蛋白乳化特性的影响

为研究离子强度对煮制前后猪肉胶原蛋白乳化特性的影响,测定煮制前后不同NaCl浓度（0.0、0.2、0.4、0.6 mol/L）猪肉可溶性胶原蛋白和不溶性胶原蛋白的乳化特性,包括乳化活性指数（emulsification activity index,EAI）、乳化稳定性指数（emulsion stability index,ESI）、黏度及乳化液微观结构,同时测定猪肉胶原蛋白溶解度。结果表明：随着介质离子强度的提高,猪肉可溶性胶原蛋白溶解度降低,黏度增大,EAI先升高后降低,ESI先降低后升高;猪肉不溶性胶原蛋白溶解度升高,黏度增大,EAI、ESI均呈先升高后降低趋势。改变胶原蛋白的离子强度或对胶原蛋白进行热处理,均能使猪肉可溶性胶原蛋白和不溶性胶原蛋白的乳化特性发生显著变化。     

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.     

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.     

Extraction and characterisation of pepsin‐solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus and Priacanthus macracanthus)

BACKGROUND: Fish collagen has been paid increasing attention as an alternative to the mammalian counterpart owing to the abundance of fish skin as a processing by‐product. Generally, the low yield of collagen extracted using the typical acid solubilisation process has led to the use of mammalian pepsin as an aid for increasing the yield. Alternatively, fish pepsin, especially from tuna stomach, can be used for the extraction of pepsin‐solubilised collagen (PSC). Therefore the objective of this study was to extract and characterise PSC from the skin of bigeye snapper, a fish widely used for surimi production in Thailand. RESULTS: PSCs from the skin of two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus, were extracted with the aid of tongol tuna (Thunnus tonggol) pepsin and porcine pepsin. PSCs from the skin of both species extracted using porcine pepsin had a higher content of β‐chain but a lower content of α‐chains compared with those extracted using tuna pepsin. All PSCs contained glycine as the major amino acid and had an imino acid (proline and hydroxyproline) content of 189–193 residues per 1000 residues. Transition temperatures of PSCs were in the range 30.6–31.3 °C. Fourier transform infrared spectra revealed some differences in molecular order between PSCs extracted using porcine pepsin and tuna pepsin. Nevertheless, the triple‐helical structure of PSCs was not affected by pepsin digestion. Zeta potential analysis indicated that PSCs from P. tayens and P. macracanthus possessed zero net charge at pH 7.15–7.46 and 5.97–6.44 respectively. CONCLUSION: Tongol tuna pepsin could be used as a replacement for mammalian pepsin in PSC extraction. However, a slight difference in PSC properties was found. Copyright © 2009 Society of Chemical Industry     

Tuna pepsin: characteristics and its use for collagen extraction from the skin of threadfin bream (Nemipterus spp.)

ABSTRACT:  Pepsin from the stomach of albacore tuna, skipjack tuna, and tongol tuna was characterized. Pepsin from all tuna species showed maximal activity at pH 2.0 and 50 °C when hemoglobin was used as a substrate. Among the stomach extract of all species tested, that of albacore tuna showed the highest activity (40.55 units/g tissue) ( P < 0.05). Substrate-Native-PAGE revealed that pepsin from albacore tuna and tongol tuna consisted of 2 isoforms, whereas pepsin from skipjack tuna had only 1 form. The activity was completely inhibited by pepstatin A, while EDTA (ethylenediaminetetraacetic acid), SBTI (soybean trypsin inhibitor), and E-64 (1-( L -trans-epoxysuccinyl-leucylamino)-4-guanidinobutane) exhibited negligible effect. The activity was strongly inhibited by SDS (sodium dodecyl sulfate) (0.05% to 0.1%, w/v). Cysteine (5 to 50 mM) also showed an inhibitory effect in a concentration dependent manner. ATP, molybdate, NaCl, MgCl₂, and CaCl₂ had no impact on the activity. When tuna pepsin (10 units/g defatted skin) was used for collagen extraction from the skin of threadfin bream for 12 h, the yield of collagen increased by 1.84- to 2.32-fold and albacore pepsin showed the comparable extraction efficacy to porcine pepsin. The yield generally increased with increasing extraction time ( P < 0.05). All collagen obtained with the aid of tuna pepsin showed similar protein patterns compared with those found in acid-solubilized collagen. Nevertheless, pepsin from skipjack tuna caused the degradation of α and β components. All collagens were classified as type I with large portion of β-chain. However, proteins with molecular weight (MW) greater than 200 kDa were abundant in acid-solubilized collagen.     

Effect of Enzymatic Digestion, pH and Molecular Weight on the Iron Solubilizing Properties of Chicken Muscle

The effects of a simulated gastrointestinal pH, enzymatic digestion and molecular weight (MW) on the iron solubilizing properties of a heated dilute salt insoluble fraction of chicken muscle were examined. The solubility of 50 ppm added FeCl₃ increased linearly from 0–260 min during pepsin digestion. The total soluble iron reached a maximum concentration following a 120 min pepsin-30 min pancreatin digestion, with pepsin digestion products ranging in MW from 6200–2500. Solubilization capacity, defined as an in vitro measure of total iron bioavailability, did not correlate to binding by free sulfhydryl groups. The soluble low molecular weight iron chelates found may explain, in part, the mechanism by which the “meat factor” enhances iron bioavailability.     

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.     

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.     

肌肉胶原蛋白特性对嫩度的影响

本试验对不同月龄苏尼特羊不同部位的肌肉胶原蛋白特性进行了分析。其结果表明,随着月龄的增加,总胶原蛋白、可溶性胶原蛋白、不溶性胶原蛋白的含量都呈增加的趋势,而可溶性胶原蛋白的溶解度却在随月龄的增加呈下降的趋势。相关分析表明,肌肉的剪切值与总胶原蛋白、可溶性胶原蛋白为极显著正相关(P<0.01),胶原蛋白溶解度与剪切值成极显著(P<0.01)负相关。这些结果表明,通过测定肌肉胶原蛋白的含量可以较客观的评价肉的嫩度。     

兔皮胶原蛋白的加工特性

以兔皮胶原蛋白为原料,对其加工特性进行系统研究。结果表明,兔皮胶原蛋白具有较强的吸水性,达到14.89 m L/g;其在酸性环境中有很高的溶解性,在p H值为3时,溶解度最高,在碱性环境中溶解度降至50%左右;离子质量浓度对胶原蛋白的溶解度有明显影响,其在Na Cl质量浓度为0~2 g/100 m L时保持相对稳定,在Na Cl质量浓度由2 g/100 m L增加至4 g/100 m L过程中急剧下降,而Na Cl质量浓度大于4 g/100 m L后,胶原蛋白溶解度变化不再明显;当胶原蛋白的质量浓度小于1 g/100 m L时,兔皮胶原蛋白的乳化性随着胶原蛋白质量浓度的增加逐渐增加,但质量浓度超过1 g/100 m L时,乳化性降低,乳化稳定性随胶原蛋白质量浓度的变化呈现与乳化性相反的趋势;低质量浓度的胶原蛋白溶液在p H 3~6过程中,乳化性和乳化稳定性均呈下降趋势,随后,p H 6~9时乳化性和乳化稳定性缓慢增加后保持稳定;离子质量浓度在0.00~7.02 g/100 m L范围内,随离子质量浓度的增加,乳化性呈现出先升高后降低的趋势,而乳化稳定性则呈现先增加随后保持稳定的趋势,在离子质量浓度为5.85 g/100 m L时,胶原蛋白的乳化性与乳化稳定性较好。     

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。     

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.     

猪皮胶原的提取及其结构表征

分别以鲜猪皮和经过自制CO2 超临界处理器处理过的猪皮为原料 ,以醋酸钠 -盐酸为缓冲液 ( pH =2 .0 ) ,利用国产胃蛋白酶提取猪皮胶原。通过测定分子量、等电点以及利用DSC、IR等方法 ,对所提取的猪皮胶原结构进行了表征。结果表明 :从经过自制CO2 超临界处理器处理过的猪皮中提取的猪皮胶原的纯度 ,要比从未经自制CO2 超临界处理器处理过的猪皮中提取的猪皮胶原的纯度高很多 ,其它性质基本不变 ;同时两者都最大程度地保持了猪皮胶原的三股螺旋结构 ,因而适合用作生物医用材料及原料。     

Fish scale collagen. Preparation and partial characterization

Fish scale was decalcified and disaggregated and then collagen was prepared by limited pepsin digestion. The yields of collagens were very high on a dry weight basis; sardine 50.9%, red sea bream 37.5% and Japanese sea bass 41.0%, respectively. These scale collagens were heterotrimers with a chain composition of (α1)₂α2. Although the denaturation temperature of the collagen was lower than land animal collagen, fish scales will have potential as an important collagen source for use in various industries.     

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     

Heat-induced changes in the proportion of types I and III collagen in bovine Longissimus dorsi

Intramuscular collagen (IMC) was isolated from the Longissimus dorsi of six Simmental bulls, 17 months of age, to evaluate the effect of heating on the proportion of types I and III collagen. Cyanogen bromide (CNBr) peptides were prepared from unheated IMC and the soluble and insoluble fractions of IMC heated to 70°C for 70 min or 90°C for 140 min. Percentage of type III collagen was determined by densitometric scans of the CNBr peptides, αl(I)CB8 and αl(III)CB8, as resolved by SDS-PAGE. Percentage of collagen solubilized was greater (P < 0·05) at 90°C than at 70°C. The 70°C and 90°C insoluble IMCs were similar (P > 0·05) for percentage of type III, but both had a greater (P < 0·05) percentage of type III than unheated IMC, indicating that type I is more heat labile than type III. Heat-soluble IMC contained both α and β components and the CNBr peptides of 70°C soluble IMC were mostly type I. These results indicated that heating intramuscular collagen from bulls mainly solubilized type I collagen. Improved tenderness associated with increased heat solubility of collagen may be more closely related to heat-induced solubilization of type I than of type III collagen.