Pepsin-solubilized Collagen Research Articles

Changes in muscle collagen content during post mortem storage of farmed sea bream (Sparus aurata): 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.

Fish Skin Type I Collagen

ABSTRACT The acid soluble collagens (ASC) and pepsin soluble col-lagens (PSC) of albacore tuna and silver-line grunt skin, by-products from frozen fish fillet industry, had been extracted, purified and characterized. Analysis of the collagens by SDS-PAGE indicated the absence of a disulfide bond, which is a characteristic of type I collagen. They also contained different subunits, a, β and ?. At least two different a chains, a1 and a2, were presented. The proportion of ? trimer in ASC was higher than in PSC. This caused reduced solubility of ASC, whereas, PSC were lower molecular weight and had fewer crosslinks, which resulted in increased solubility. Collagen water solubility was lower than in 0.5 M acetic acid, but the solubility increased as the temperature increased. Denaturation temperature (Td) of silver-line grunt collagen was slightly higher than albacore tuna collagen, but about 7–9°C lower than calfskin collagen.

Isolation and characterization of collagen from bigeye snapper (Priacanthus macracanthus) skin

AbstractAcid‐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 Tmax 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

Characterization of the quantitatively major collagen in the mantle of oyster Crassostrea gigas

A quantitatively major collagen was isolated from the pepsin-solubilized collagen preparation of the mantle by differential salt precipitation and phosphocellulose column chromatography, and its constituent α components (α1 and α2) were purified by phosphocellulose column chromatography. The subunits were demonstrated to be genetically distinct from each other by peptide mapping and amino acid analysis. The amino acid composition calculated from those of the α1 and α2 components in 2∶1 ratio coincided well with that of the major collagen from the mantle. These results suggest that the major collagen in the mantle of the oyster may have a heterotrimer structure (α1)2 α2.

Collagen changes in refrigerated sea bass muscle treated with pyrophosphate and stored in modified- atmosphere packaging

Changes in collagen of sea bass muscle treated with and without pyrophosphate (PP) were monitored during storage in modified-atmosphere packaging (MAP) (80% CO2, 10% O2 and 10% N2) at 4 °C. No changes in acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) of sea bass muscle with and without PP treatment were observed during storage in MAP up to 21 days. However, a slight decrease in insoluble collagen (ISC) of a sample kept in MAP was found. For sea bass muscle stored under an air atmosphere ASC increased, whereas PSC and ISC decreased with a concomitant loss in the firmness

Isolation and characterisation of acid and pepsin-solubilised collagens from the skin of Brownstripe red snapper ( Lutjanus vitta)

Acid-solubilised collagen (ASC) and pepsin-solubilised collagen (PSC) were successfully extracted from the skin of Brownstripe red snapper ( Lutjanus vitta) with yields of 9% and 4.7%, respectively, on the basis of wet weight. Both ASC and PSC consisted of two different α chains (α1 and α2), and were characterised to be type I with no disulfide bond. PSC had a lower content of high molecular weight cross-links, than did ASC. Peptide maps of ASC and PSC hydrolysed by V8 protease and lysyl endopeptidase showed some differences in peptide patterns between the two fractions and were totally different from those of calf skin collagen type I, suggesting differences in amino acid sequences and collagen conformation. Maximum solubility in 0.5 M acetic acid was observed at pH 3 and pH 4 for ASC and PSC, respectively. A sharp decrease in solubility was observed in the presence of NaCl, above 2% and 3%, (w/v) for ASC and PSC, respectively. T max values of both collagen fractions were similar and shifted to a lower value in the presence of acetic acid, suggesting some changes in the collagen structure caused by acid induction.

AGE-related cross-linking of collagen is associated with aortic wall matrix stiffness in the pathogenesis of drug-induced diabetes in rats

Diabetes mellitus is major risk factor for cardiovascular disease, and atherosclerosis accounts for most of the morbidity and mortality of diabetic patients. To examine the effects of diabetes on the vessel wall, we examined the association of collagen cross-linking in relation to matrix stiffness of the descending aorta in streptozotocin-induced diabetic rats. The matrix stiffness of the vessel was determined by measuring the tensile properties of the tissue. Seven weeks following the establishment of diabetes, both control and diabetic rats were killed and the descending aortas were excised and analyzed. The findings from biomechanical analysis indicated a significant increase in maximum load (26%), stress (22%), Young's modulus of elasticity (60%), and toughness (32%) in diabetic aortas compared to control. In contrast, the maximum strain of the diabetic rat aorta was significantly reduced by 20% compared to control rats, suggesting stiffening of the blood vessel. The results from biochemical analysis showed that the amount of total collagen increased by 21% in diabetic tissues compared to the control. The sequential extractions of collagen showed that the diabetic specimens yielded 34% more neutral salt-soluble collagen (NSC) than the control. The amount of pepsin-soluble collagen was 31% less in diabetic tissues than in the control group, whereas the amount of insoluble collagen (ISC) increased by 56%. A significant accumulation in advanced glycation end products (AGEs) were seen in pepsin- and collagenase-soluble collagen in diabetic vessel. Furthermore, the altered biomechanical properties of the vessel wall were strongly correlated with the biochemistry of collagen. Overall, these results provide evidence that the diabetic state is associated with the changes in collagen biochemistry and in the biomechanics of the blood vessel.

Interactions between ice storage time, collagen composition, gaping and textural properties in farmed salmon muscle harvested at different times of the year

Atlantic salmon were sampled in June, September and February of the consecutive year and were stored on ice for up to 14 days in order to test the effect of harvest time and subsequent ice storage on meat quality. Texture and gaping frequency were analysed and were related to colour, protein degradation, collagen solubility, collagen types and final pH as well as lipid oxidation in the fillets to test possible interactions between harvest time and quality degradation during storage. In February, the connective tissue contained more soluble collagen and less insoluble collagen, as well as more of both types I and V collagen, than in the samples collected in June. During ice storage, fish became softer with a concomitant increase in the number of fish displaying very high gaping. pH increased during ice storage and fillet colour became lighter and redder, while yellowness changed in the fattier fillets upon ice storage. Ice storage resulted in changes in pepsin-soluble collagen (PSC) depending on harvest time as did both types I and V collagen. The softer the fish, the higher the gaping score and the more insoluble collagen, the less gaping occurred.

Identification and characterization of molecular species of collagen in ordinary muscle and skin of the Japanese flounder Paralichthys olivaceus

Pepsin-solubilized collagens prepared from ordinary muscle and skin tissues of the Japanese flounder (Paralichthys olivaceus) were separated into two fractions, major and minor, by ammonium sulfate precipitation. Collagens in these fractions were further purified by cation-exchange column chromatography. The results of SDS–PAGE, peptide mapping and amino acid analysis of collagen in both fractions suggested that the purified major and minor collagens might be classified as types I and V collagens, respectively. Each type of collagen was fundamentally similar, among the ordinary muscle and the skin, in amino acid composition and peptide maps.

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 α 1 and 110 kDa for α 2, 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.

Biochemical and immunochemical detection of types I and V collagens in tiger puffer Takifugu rubripes

Crude collagen fractions, prepared by alkali extraction from several parts (muscle, liver, swim bladder, bone, fin, gill, alimentary tract) of tiger puffer Takifugu rubripes, were analysed by biochemical and immunochemical techniques to examine the distribution of collagen types (types I and V collagens) among these parts. Collagens from these parts showed quite similar SDS–PAGE patterns and similar peptide maps. Immunoblot analyses were performed for these collagens using antisera against types I and V collagens isolated from pepsin-solubilized collagen of skin, revealing the existence of the positive components for these antisera in all of them. The results suggest that types I and V collagens may be widely distributed in these parts of tiger puffer.

Collagen from diamondback squid (Thysanoteuthis rhombus) outer skin.

Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from diamondback squid outer skin and partially characterized. The yields of acid-solubilized and pepsin-solubilized collagens were about 1.3 and 35.6%, respectively, on a dry weight basis. Pepsin-solubilized collagen was heterotrimer with a chain composition of ala2a3. The patterns of peptide fragments were different from that of porcine skin collagen. Denaturation temperature was 27.5 degrees C, about 10 degrees C lower than that of porcine collagen. The amino acid composition of pepsin-solubilized collagen from diamondback squid outer skin was similar to that from cuttlefish outer skin. This squid is big among squid species, and its skin is thick. It is clear that diamondback squid outer skin has a potential as an alternative source of collagen to bovine skin and bone. At present, collagen using aquatic materials such as skin (cod and a deep-sea fish) and scale (sea bream and anchovy) is the development stage in the related industries. Unless the problem of BSE infection in land animals is resolved aquatic materials as an alternative source of collagen will attract much attention in the cosmetic and medical fields.

Hyaluronic-acid-based semi-interpenetrating materials

In order to enhance the mechanical performances of hyaluronic acid (HA) without compromising its biological activity, HA has been interpenetrating with a fibrillar collagen scaffold. The semi-interpenetrating materials were obtained by mixing HA with different molecular weight and a pepsin-solubilized collagen (atelocollagen) solution, and then by inducing collagen fibrillogenesis. Results indicate that molecular weight of HA significantly influences the mechanical properties of the semi-interpenetrating materials and more specifically stronger material results from the use of low-molecular-weight (LMW) HA. According to the dynamic mechanical data the composite collagen-LMW HA has a higher elastic modulus than collagen, whereas the opposite is true for the high-molecular-weight (HMW) HA. This result highlights the role of specific interactions that occur between collagen and HA during the gel formation in controlling the network mechanical stability. LMW HA may, probably, interact more strongly with collagen during the fibrillogenesis process than HMW HA due to the higher mobility of the chains and the weaker homologous interactions. Moreover, morphological observations showed that LMW HA is intimately interdispersed within the collagen network and completely coated the fibrils, which act as mechanical support.

Partial characterization of collagen in several bivalve molluscs

Histological distribution and biochemical properties of collagen were examined in the edible tissues of five bivalve molluscan species. Collagen fibre was mainly distributed in the muscle connective tissues (epimysium, perimysium, and endomysium) of both mantles and adductors, and in the inner connective tissue matrix of the mantles. The collagen contents of the edible tissues varied considerably; the mantles showed much higher collagen content than the adductors. Soluble collagen could be recovered from the crude collagen fraction by a pepsin digestion method and was referred to as pepsin-solubilized collagen (PSC). The PSC showed a quite distinct SDS–PAGE pattern from those of collagen fractions prior to pepsin digestion for some species, suggesting considerable effects of pepsin digestion on the structure of the triple-helical domain.

Biochemical properties of black drum and sheepshead seabream skin collagen.

Acid-soluble collagen (ASC) and pepsin solubilized collagen (PSC) were isolated from the skins of black drum (Pogonias cromis) and sheepshead seabream (Archosargus probatocephalus) harvested in the Gulf of Mexico coastal waters. The yields of ASCs on dry basis from black drum and sheepshead were estimated at 2.3 and 2.6%, and the yields of PSCs were 15.8 and 29.3%, respectively. Analyses of molecular weight profile, amino acid composition, and secondary structure showed that the skin collagens from both species were typical type-I collagen. The molecular mass of alpha(1) and alpha(2) subunits, as determined by SDS-PAGE using Tris-Acetate gels, was 127 kDa and 116 kDa, respectively. The amino acid composition of ASC and PSC for both species was closer to calf skin ASC than to cod skin ASC. Thermal denaturation temperatures, measured by melting point using circular dichroism, gave the following values: black drum ASC, 34.2 degrees C; sheepshead ASC, 34.0 degrees C; black drum PSC, 35.8 degrees C; sheepshead PSC, 34.3 degrees C. The literature value for the heat stability of calf skin collagen is 36.3 degrees C. The potentials of collagens from black drum and sheepshead skins in the functional food, healthcare, and pharmaceutical industries are discussed.

Effect of an aqueous extract of Phaseolus vulgaris on the properties of tail tendon collagen of rats with streptozotocin-induced diabetes.

Changes in the structural and functional properties of collagen caused by advanced glycation might be of importance for the etiology of late complications in diabetes. The present study was undertaken to investigate the influence of oral administration of aqueous pod extract (200 mg/kg body weight) of Phaseolus vulgaris, an indigenous plant used in Ayurvedic Medicine in India, on collagen content and characteristics in the tail tendon of streptozotocin-diabetic rats. In diabetic rats, collagen content (117.01 6.84 mg/100 mg tissue) as well as its degree of cross-linking was increased, as shown by increased extent of glycation (21.70 0.90 g glucose/mg collagen), collagen-linked fluorescence (52.8 3.0 AU/ mol hydroxyproline), shrinkage temperature (71.50 2.50 C) and decreased acid (1.878 0.062 mg hydroxyproline/100 mg tissue) and pepsin solubility (1.77 0.080 mg hydroxyproline/100 mg tissue). The alpha/ ratio of acid- (1.69) and pepsin-soluble (2.00) collagen was significantly decreased in streptozotocin-diabetic rats. Administration of P. vulgaris for 45 days to streptozotocin-diabetic rats significantly reduced the accumulation and cross-linking of collagen. The effect of P. vulgaris was compared with that of glibenclamide, a reference drug administered to streptozotocin-diabetic rats at the dose of 600 g/kg body weight for 45 days by gavage. The effects of P. vulgaris (collagen content, 64.18 1.97; extent of glycation, 12.00 0.53; collagen-linked fluorescence, 33.6 1.9; shrinkage temperature, 57.0 1.0; extent of cross-linking - acid-soluble collagen, 2.572 0.080, and pepsin-soluble collagen, 2.28 0.112) were comparable with those of glibenclamide (collagen content, 71.5 2.04; extent of glycation, 13.00 0.60; collagen-linked fluorescence, 38.9 2.0; shrinkage temperature, 59.0 1.5; extent of cross-linking - acid-soluble collagen, 2.463 0.078, and pepsin-soluble collagen, 2.17 0.104). In conclusion, administration of P. vulgaris pods had a positive influence on the content of collagen and its properties in streptozotocin-diabetic rats.

Age of Farmed Atlantic Salmon at Seawater Transfer Affects Muscle Collagen Content and Solubility at Harvest

ABSTRACTSalmon released to seawater as different smolt types differed significantly in the total collagen content and the solubility of the collagen present. A 11/2 smolt contained more total collagen than 1/2 and 1 smolt types. Less insoluble collagen and more of the pepsin soluble collagen was generally present in 11/2 smolt type as compared with both 1/2 and 1 types, with the exception of prerigor type 1 salmon. No differences between smolt types in acetic soluble collagen were present. Fish with the higher amount of collagen also had the lowest end pH. Although the bled body weight differed, no correlation between body weight and collagen content or its solubility was found. Fish with higher end pH had lower total collagen and pepsin soluble collagen (r = ‐0.77, P = 0.0001 and r = ‐0.51, P = 0.04, respectively, and more insoluble collagen (r = 0.72, P = 0.002).

Studies on bullfrog skin collagen

Pepsin-soluble collagens were prepared from bullfrog skin and partially characterized. This study revealed interesting differences in the frog skin collagen when compared with known vertebrate collagens. This may be attributed to the position of the amphibians in the vertebrate hierarchy. Therefore, detailed investigations on various physicochemical properties, such as molecular weight, amino acid composition, denaturation temperature, UV–Vis and IR spectra of bullfrog skin collagen were carried out. The study confirms the structural relationship of collagen to habitat and function. Moreover bullfrog skin has potential as an alternative source of collagen to calf skin and bone for use in various fields.

Characterization of molecular species of collagen in muscles of Japanese amberjack, Seriola quinqueradiata

Pepsin-solubilized collagens prepared from the muscle tissues (ordinary and dark muscles) of Japanese amberjack were separated into two fractions, major and minor, by ammonium sulphate precipitation. Collagens in these fractions were further purified by cation-exchange column chromatography. The results of SDS-PAGE, peptide mapping, and amino acid analysis suggested that the purified major and minor collagens might be classified as type I and V collagens, respectively. Each type of collagen was fundamentally similar, among the ordinary and dark muscles, in amino acid compositions and peptide maps.

Modulatory effect of Coccinia indica on aortic collagen in streptozotocin-induced diabetic rats.

1. The effects of Coccinia indica, an indigenous plant used in Ayurvedic medicine in India, on aortic collagen content and its characteristics were assessed in streptozotocin (STZ) diabetic rats. 2. Rats were made diabetic with a single intraperitoneal injection of STZ (45 mg/kg). Blood glucose, hydroxyproline, collagen, extent of glycation, collagen-linked fluorescence, soluble pattern of pepsin-soluble collagen, shrinkage temperature, alpha/beta ratio of type I collagen and type I/type III collagen ratio were determined in rats treated with C. indica leaf extract (CLEt; 200 mg/kg for 45 days using an oral intragastric tube). 3. In diabetic rats, the collagen content, as well as the degree of cross-linking, was increased, as evidenced by increased shrinkage temperature and decreased pepsin solubility. The alpha/beta ratio of type I collagen and the type I/type III collagen ratio of pepsin-soluble collagen were significantly decreased in STZ diabetic rats. 4. In conclusion, administration of CLEt for 45 days to STZ diabetic rats significantly reduced the accumulation and cross-linking of collagen. The effects of C. indica (collagen content 23.87 +/- 1.52 mg/100 mg tissue (t value = 6.80), extent of cross-linking 0.893 +/- 0.072 mg hydroxyproline/100 mg tissue (t value = 9.0)) were comparable with those of glibenclamide (collagen content 26.18 +/- 1.65 mg/100 mg tissue (t value = 4.58), extent of cross-linking 0.787 +/- 0.057 mg hydroxyproline/100 mg tissue (t value = 7.1)), a reference drug.