Imino Acid Content Research Articles

Characterization of collagen from the skin of Amur sturgeon (Acipenser schrenckii)

The collagen in Amur sturgeon skin was isolated using sodium chloride (SSC, 4.55%), acetic acid (ASC, 37.42%) and pepsin (PSC, 52.80%), respectively. The collagens appeared to be dense sheet-like film linked by random-coiled filaments under SEM. The denaturation and melting temperature were 32.15 °C and 116.62 °C for SSC, 32.78 °C and 120.66 °C for ASC, 32.46 °C and 115.42 °C for PSC, assessed by CD and DSC. SDS-PAGE showed that the collagens were mainly type I with two different α chains. The amino acid profiles of them was similar to each other with high imino acid content and hydroxylation degree. FTIR confirmed the triple helical structure of the collagens, and indicated more hydrogen bond in PSC and more intermolecular crosslinks in ASC. These results suggested that the collagens have potential in commercial applications as alternatives to mammalian collagen.

Thermal Stability of Chicken Keel Bone Collagen

Acid-soluble and pepsin-solubilized collagen were isolated from chicken keel bone and analyzed by electrophoresis and circular dichroism. The denaturation temperature of chicken keel bone was determined by circular dichroism and compared to denaturation temperature of black drum or alligator bone. Our results show that collagen from chicken keel bone is a mixture of type II and type I, has predominantly the amino acids glycine, proline and hydroxyproline, and its secondary structure is predominantly triple-helix. The onset of the denaturation temperature is 37C; at 44C, 50% of the collagen is denatured and the denaturation is complete at 48.8C making it the highest of vertebrate collagen denaturation temperatures reported in the literature. The high heat stability of chicken keel bone collagen may be in part ascribed to its imino acid content. Because of the high heat stability and low methionine content, chicken keel bone collagen is a high value-added product with several biochemical and biomedical applications. Practical Applications The denaturation temperature of chicken keel bone collagen is above body temperature and allows applications in cosmetics where other types of collagen may be denatured because of their low denaturation temperature. Chicken type II collagen is well tolerated by patients with rheumatoid arthritis and is effective in the treatment of rheumatoid arthritis. Methionine-restricted diet inhibits the growth of solid tumor such as gastric cancer, glioblastoma, medulloblastoma and neuroblastoma. Chicken type collagen may be a medical food for patients with solid tumors mentioned above.

Isolation and characterization of collagen from the cartilage of Amur sturgeon (Acipenser schrenckii)

The collagen in Amur sturgeon cartilage was isolated using sodium chloride (salt-solubilized collagen, SSC, 2.18%), followed by acetic acid (acid-solubilized collagen, ASC, 27.04%) and then pepsin (pepsin-solubilized collagen, PSC, 55.92%). These collagens appeared to be dense sheet-like film linked by random-coiled filaments under SEM. The denaturation and melting temperatures of PSC (35.71 and 123.90°C) were significantly higher than SSC (32.64 and 114.51°C) and ASC (32.98 and 120.72°C) assessed by circular dichroism and differential scanning calorimetry, which could be attributed to its high imino acid content (22.57%) and degree of hydroxylation (47.29%). Electrophoresis pattern showed that the SSC and ASC were type I collagen, while PSC was predominantly type II collagen along with other minor types. Infrared spectra confirmed their triple helical structure, and indicated more hydrogen bonding in ASC and more intermolecular crosslinks in PSC. These results provide some basis for their large-scale production and further application as alternatives to mammalian collagen.

Characteristics and gel properties of gelatin from skin of seabass (Lates calcarifer) as influenced by extraction conditions

Characteristics and gel properties of gelatin from seabass skin, as influenced by extraction conditions, were studied. Yields of gelatin extracted at 45 and 55°C for various times were 51.6–57.3% and 62.0–66.4% (dry weight basis), respectively. All gelatins contained β-chain and α-chains as the predominant components and showed a high imino acid content (198–202residues/1000 residues). Generally, the gel strength of gelatins decreased as the extraction temperature and time increased. Gelatin extracted at 45°C for 3h exhibited the highest gel strength (369g). Gelling and melting temperatures for seabass skin gelatin were 19.5–20.0 and 26.3–27.0°C, respectively. All gelatins could be set at 25°C within 30min, however gelatins extracted at 45°C had a shorter setting time than those extracted at 55°C (P<0.05). Gelatin from seabass skin showed a higher gel strength than bovine gelatin and could be used as a potential replacement for land animal gelatins.

Isolation and characterization of acid-soluble collagen from the scales of marine fishes from Japan and Vietnam

Acid-soluble collagen (ASC) was successfully extracted from the scales of lizard fish (Saurida spp.) and horse mackerel (Trachurus japonicus) from Japan and Vietnam and grey mullet (Mugil cephalis), flying fish (Cypselurus melanurus) and yellowback seabream (Dentex tumifrons) from Japan. ASC yields were about 0.43–1.5% (on a dry weight basis), depending on the species. The SDS–PAGE profile showed that the ASCs were type I collagens, and consisted of two different α chains, α1 and α2, as well as a β component. ASC of horse mackerel from Vietnam contained a higher imino acid level than that from Japan. ASC denaturation temperature (Td) ranged from 26 to 29°C, depending on fish species and imino acid content (p<0.01). Maximal solubility of individual collagens was observed at pHs 1–3. Collagen solubility decreased sharply at NaCl concentrations >0.4M, regardless of fish type.

Preparation and characterisation of type I and V collagens from the skin of Amur sturgeon (Acipenser schrenckii)

The collagen in Amur sturgeon was extracted by pepsin digestion and separated into two fractions, P2.4 (92.40%) and P4.0 (2.16%), by sodium chloride precipitation. SDS–PAGE and amino acid profile suggested that the P2.4 and P4.0 might be classified as type I collagen (PSC-I) and type V collagen (PSC-V), respectively. These collagens appeared to be dense sheet-like film linked by random-coiled filaments under SEM. The denaturation and melting temperatures of PSC-V (35.92 and 122.86°C) were significantly higher than PSC-I (32.52 and 116.01°C) assessed by CD and DSC, which could be attributed to its higher imino acid content (23.43%) and degree of hydroxylation (52.18%). FTIR confirmed their triple helical structure, and indicated more intermolecular crosslinks in PSC-I and more hydrogen bond in PSC-V. These results provide some basis for their large-scale production and further application as alternatives to mammalian collagen.

Characterization of wami tilapia (Oreochromis urolepis hornorum) skin gelatin: microbiological, rheological and structural properties

Gelatin was extracted from the skin of tilapia (Oreochromis urolepis hornorum) and its microbiological, rheological and structural properties were characterized. The tilapia skin gelatin presented typical molecular weight distribution of type I collagen with contents of imino acids (proline + hydroxyproline) of 21.67%. Gel strength and viscosity values were 221 ± 5.68 g and 5.98 ± 0.34 cP, respectively, with the maturation time of 18 ± 1 h, and both parameters increased with the maturation time. Melting and gelling points of 25 degrees C and 21 degrees C, respectively, were obtained for tilapia skin gelatin. The gelatin presented microbiological standards in accordance with the Brazilian Legislation.

Physical and chemical properties of gelatin from the skin of cultured Amur sturgeon (Acipenser schrenckii )

To expand the usefulness of cultured Amur sturgeon, Acipenser schrenckii, its skin was used to explore the production of gelatin. After acetic acid pre-treatment (0.05 m for 3 h), gelatin was extracted at temperatures of 50 or 70°C for 1 or 6 h. Gelatin yield ranged from 9.42 to 12.47% (wet weight basis) (P < 0.05). With increasing extraction time and temperature, the content of imino acids (proline + hydroxyproline), gel strength and L*-value (lightness) decreased, while the a*-value (redness) and b*-value (yellowness) of gelatin gel increased (P < 0.05). Electrophoretic analysis revealed that α-chains and β-chains were predominant components in all extracted gelatins. Higher molecular weight proteins (γ-chain) were also observed. Gelling and melting temperatures of gelatin were 13.6–14.6°C and 20.3–22.6°C, respectively. Circular dichroism (CD) spectra and Fourier transform infrared (FTIR) spectroscopy revealed the triple helix loss in gelatin (A1235 (AIII)/A1451 < 1). Extraction conditions caused secondary structure changes in the gelatin. More likely due to the differences in the culture water temperature, gelatin exhibited gelling and melting temperatures intermediate between those of cold- and warm-water fish gelatins. The obtained gelatin can be used in food products or in the production of bioactive compounds.

Characterization of Acid and Pepsin Soluble Collagen from the Skin of Horse Mackerels (Magalaspis cordyla) and Croaker (Otolithes ruber)

Acid and pepsin-soluble collagen was isolated from the skin of horse mackerel (Magalaspis cordyla) and croaker (Otolithes ruber) using 0.5 M acetic acid followed by precipitation with 0.9 M NaCl. The yields of acid soluble collagen were 17.3 ± 0.4 and 21.9 ± 0.6% and pepsin soluble collagen (22.5 ± 0.8 and 25.7 ± 0.3%) as per wet weight basis, respectively. The extracted acid soluble collagen and pepsin soluble collagen were characterized by SDS-PAGE, amino acid composition and FTIR spectroscopy. SDS-PAGE showed that all the extracted collagen contained two alpha components (α 1 and α 2) and one beta component (β); all the collagens were typical type I and maintained their triple helical structures well with slight molecular structure differences. The amino acid profiles of these collagens were similar with a low imino acid content. Therefore, there is a possibility to use horse mackerel and croaker skin collagen as an alternative source for commercial collagen and may find applications in the food, cosmetic, biomedical, and pharmaceutical industries.

Sequence recombination improves target specificity in a redesigned collagen peptideabc-type heterotrimer

Stability of the collagen triple helix is largely governed by its imino acid content, namely the occurrence of proline and 4R-hydroxyproline at the X and Y positions, respectively, of the periodic (Gly-X-Y)(n) sequence. Although other amino acids at these positions reduce stability of the triple helix, this can be partially compensated by introducing intermolecular side-chain salt bridges. This approach was previously used to design an abc-type heterotrimer composed of one basic, one acidic, and one neutral imino acid rich chain (Gauba and Hartgerink, J Am Chem Soc 2007;129:15034-15041). In this study, an abc-type heterotrimer was designed to be the most stable species using a sequence recombination strategy that preserved both the amino acid composition and the network of interchain salt bridges of the original design. The target heterotrimer had the highest T(m) of 50 °C, 7 °C greater than the next most stable species. Stability of the heterotrimer decreased with increasing ionic strength, consistent with the role of intermolecular salt bridges in promoting stability. Quantitative meta-analysis of these results and published stability measurements on closely related peptides was used to discriminate the contributions of backbone propensity and side-chain electrostatics to collagen stability.

Isolation and characterization of acid soluble collagens and pepsin soluble collagens from the skin and bone of Spanish mackerel (Scomberomorous niphonius)

The objective of this study was to extract and characterize acid soluble collagens (ASCs) and pepsin soluble collagens (PSCs) from the skin and bone of Spanish mackerel (Scomberomorous niphonius) and to provide a simultaneous comparison of the four collagens. The yields of ASC-S (ASC from skin), PSC-S (PSC from skin), ASC-B (ASC from bone) and PSC-B (PSC from bone) were 13.68 ± 0.35, 3.49 ± 0.24, 12.54 ± 0.83 and 14.27 ± 0.66% (on wet weight basis), respectively. The four collagens contained glycine (341.6–352.6 residues/1000 residues) as the major amino acid and the contents of imino acids were between 177.1 and 184.3 residues/1000 residues. Amino acid composition, SDS-PAGE and FTIR confirmed that ASC-S, ASC-B and PSC-B were mainly composed of type I collagen with slight molecular structure differences, and PSCs had lower content of high-molecular weight cross-links than that of ASCs. The denaturation temperatures of ASC-S, PSC-S, ASC-B and PSC-B were 15.12, 14.66, 18.02 and 16.85 °C, respectively, which were much lower than those of collagens from the mammalian and tropical fish species due to low imino acid contents. All the collagens were soluble at acidic pH (1–4) and lost their solubility when the NaCl concentrations were above 2% (w/v). The four lyophilized collagens displayed a uniform and regular network ultrastructure based on the ultrastructural analysis. The isolated collagens from Spanish mackerel could serve as an alternative source of collagens for further application in food and neutraceutical industries.

Designed Coiled Coils Promote Folding of a Recombinant Bacterial Collagen

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

PREPARATION AND CHARACTERIZATION OF GELATIN EXTRACTED FROM THE SKINS OF ROHU (LABEO ROHITA) AND COMMON CARP (CYPRINUS CARPIO)

ABSTRACT Gelatin was extracted from the skins of rohu and Common carp, and its physicochemical properties were studied. The yield of gelatin obtained from the skin of rohu and Common carp were 12.93 and 12%, respectively. The corresponding viscosity values were 6.06cP and 5.96cP. Rohu and Common carp skin gelatin had high content of imino acids i.e., 20.49 and 19.16%, respectively. Rohu gelatin had a significantly higher (P < 0.05) melting point than that of Common carp skin gelatin. The setting temperature observed for the gels from rohu and Common carp was 18.52 and 17.96C, respectively. Rohu and Common carp skin gelatins were found to have a mild but easily perceivable odor. The gelatins from the skin of rohu and common carp had a snowy white appearance and were light-textured. Rohu skin gelatin had significantly higher gel strength compared with Common carp skin gelatin. Foam formation ability and form stability of Common carp gelatin was significantly lower (P < 0.05) than that of Rohu gelatin. Rohu skin gelatin had the high fat-binding capacity. In this study, the functional properties observed for gelatins from the skin of rohu and Common carp are similar, if not better than many of the gelatins from other fish sources, and can be used as potential substitutes for the same in many applications. PRACTICAL APPLICATIONS Among the cultured carps, rohu (Labeo rohita) and Common carp (Cyprinus carpio) contribute significantly to the Aquaculture production in India. These species are presently being increasingly used for the preparation of mince and fillet-based value added products, with skin forming a major portion of the fishery waste thus generated. Hence, based on the availability and commercial importance and with the objective of efficient utilization of fish skin waste, these species were chosen for studies on gelatin extraction. It was observed that the physicochemical and functional properties for rohu and Common carp skin gelatins are similar, if not better than many of the gelatins from other fish sources and can be used as potential substitutes for the same in many applications. There is a future scope for developing binary blends of these gelatins with animal gelatins that are completely compatible and commercially useful in many applications.

Physical, Mechanical, and Barrier Properties of Carp and Mammalian Skin Gelatin Films

Films of 0.11 to 0.13 mm thickness were prepared using gelatins from the skins of cultured freshwater carp species and mammalian gelatins viz., porcine and bovine skin gelatin. A comparative study was made on the physical, mechanical, and barrier properties of these films. The amino acid composition, gel strength, clarity, and gel setting point of the gelatins were also determined. Carp skin gelatins had a lower imino acid content (19.16% to 20.86%) than mammalian skin gelatins (22.91% to 23.7%). Grass carp gelatin had gel strength of 230.2 B that is comparable to the reported value for bovine skin gelatin (227.2 B). The bloom values of rohu and common carp skin gelatins were 188.6 B and 181.3 B, respectively, which were significantly lower than mammalian gelatins. Mammalian gels have significantly higher (P < 0.05) setting temperatures (23.7 to 24.2 °C) than carp skin gelatins. Tensile strength (TS) was lowest for films from common carp and rohu skin gelatin (490 and 497 kg/cm(2), respectively) and highest for porcine skin gelatin film. The degree of transparency (L*) was significantly higher for films from grass carp, bovine hide, and pork skin gelatin films. Carp skin gelatin films had significantly lower water vapor permeability (WVP) and oxygen permeability (OP) than mammalian skin gelatin films, which indicated that carp skin gelatin based films have superior barrier properties than mammalian skin gelatin films.

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 (Δ H) 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.

Antioxidant activity of several marine skin gelatins

Gelatins obtained from the skins of tuna and halibut, and from the tunics of jumbo flying squid were hydrolysed by Alcalase to produce antioxidant peptides. Hydrolysis yielded an increase in the antioxidant capacity of gelatins of around two-fold when measured by the Fe reducing capacity (FRAP) method and even more when measured by the ABTS radical scavenging method. When both squid and tuna gelatins were hydrolysed with different enzymes (collagenase, trypsin, pepsin), Alcalase and pepsin gave the hydrolysates with the highest and lowest ABTS radical scavenging ability, respectively. FRAP assay showed that the squid hydrolysates prepared using Alcalase were the most effective in reducing ferric ions, whereas trypsin gave rise to the tuna hydrolysates with the highest iron reducing ability. When the amino acid composition of the gelatins was related to the antioxidant properties, ABTS radical scavenging was observed to be negatively correlated to the total content of hydrophobic amino acids and imino acids in all the samples, while Fe reducing power (FRAP) was strongly correlated with Hyl and degree of hydroxylation.

Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

Original article: Optimisation of extraction conditions and characteristics of skin gelatin from Nile tilapia (Oreochromis niloticus)

SummaryResponse surface methodology was used to optimise gelatin extraction conditions from the skin of Nile tilapia (Oreochromis niloticus), and characteristics of the gelatin were determined. Concentration of NaOH (%, X1), alkaline treatment time (h, X2), concentration of HCl (‰, X3) and acid treatment time (min, X4) were chosen for independent variables. Dependent variable was yield of gelatin (%, Y). Optimal conditions were X1 = 3.2 (%), X2 = 2.3 (h), X3 = 0.7 (‰) and X4 = 84 (min), and predicted value of response optimal conditions was Y = 20.4%. Actual value was 19.3% by verification experiments under optimal conditions. Crude protein content of the tilapia skin gelatin was 88.5%. The content of imino acids including proline and hydroxyproline in the gelatin was 185 residues per 1000 total amino acid residues. Its gel strength was 260 g. The gelling and melting points were 18.0 and 22.4 °C, respectively.

Study on the properties of gelatins from skin of carp ( Cyprinus carpio) caught in winter and summer season

The objective of this work was to compare the physiochemical (molecular weight distribution and amino acid composition) and rheological (viscosity property, gel strength and melting point) properties of gelatins from skins of carp caught in winter to those obtained for the summer equivalents. Gelatins from winter and summer fish skins were extracted at 60, 70 and 80 °C. SDS-PAGE patterns for gelatins extracted under the same conditions showed that the degradation of gelatins from winter fish skins were more severe than that of the summer ones. The imino acid contents of the winter and summer gelatins extracted at 60 °C were very similar, showing 190 and 188 residues/1000 residues, respectively. The gelatins from summer fish presented higher melting points and gel strengths, as well as better viscosity properties than the winter equivalents (P < 0.05). The differences in the rheological properties between winter and summer gelatins may be explained by different thermostability of interstitial collagen molecules (from which gelatins were derived) in the two seasons.

Collagens from the skin of arabesque greenling (Pleurogrammus azonus) solubilized with the aid of acetic acid and pepsin from albacore tuna (Thunnus alalunga) stomach

Due to the low extraction efficiency of collagen from fish skin by the typical acid solubilization process, pepsin has been widely used to aid further extraction of collagen from the residue. The aim of this study was to characterize collagen from the skin of arabesque greenling extracted with the aid of albacore tuna pepsin, in comparison with collagen obtained from the acid solubilization process. Acid-solubilized collagen (ASC) from the skin of arabesque greenling was extracted with acetic acid. Pepsin-solubilized collagen (PSC) was further extracted from the skin residue with the aid of pepsin from albacore tuna. The yields of ASC and PSC were 303 and 140 g kg(-1) (dry weight), respectively. Both collagens contained alpha- and beta-chains as their major components and were characterized as type I collagen. Both collagens contained glycine as a major amino acid and had imino acid content of 157-159 residues per 1000 residues. The degradation induced by lysyl endopeptidase and V8-protease was more pronounced in PSC compared with ASC. Maximal transition temperatures of both collagens were in the range of 15.4-15.7 degrees C. Fourier transform infrared spectra revealed some differences in molecular order between ASC and PSC. Nevertheless, the triple-helical structure of PSC was still predominant. Based on zeta-potential, pI of ASC and PSC was estimated to be 6.31 and 6.38, respectively. Isolation of collagens from the skin of arabesque greenling could be achieved by acid or albacore tuna pepsin solubilization. However, there was a slight difference in properties between ASC and PSC.