Purification and characterization of molecular species of collagen in the skin of skate (Raja kenojei)

Two types, one major and the other minor, of collagen were obtained from pepsin-solubilized collagen of the skate (Raja kenojei) skin. The major collagen was identified as Type I collagen because of a similar characteristic to those of Type I collagens from the skate muscle and cartilage. On the other hand, the SDS–PAGE pattern and peptide maps of the minor collagen were different from those of Type V and XI collagens of the skate muscle and cartilage, respectively. However, the minor collagen was similar, in amino acid composition, to Type V collagen from the skate muscle. These results suggest the existence of a molecular subspecies of Type V collagen in skate skin which is different from the muscle Type V collagen in primary structure or subunit composition.     

Characterization of Fish Muscle Type I Collagen

Muscle Type I collagen of five teleosts was characterized with respect to its thermal stability and subunit composition. The muscle collagen exhibited a higher denaturation temperature, Td, in solution and a higher degree of proline hydroxylation, compared with skin Type I collagen of the respective species. Moreover, the Td values seemed to increase with the increasing upper limit of environmental temperature of fish habitats. The subunit composition also varied with fish species; an α1α2α3 heterotrimer existed in eel and common mackerel and an (α1)₂α2 heterotrimer in saury, while (α1)₂α2 and α1α2α3 heterotrimers appeared to be major and minor components, respectively, in chum salmon and carp.     

Cellular Distribution of Lactate Dehydrogenase in Chicken Breast Muscle

SUMMARY— The distribution of lactate dehydrogenase (LDH) in chicken breast muscle was studied by preparing subcellular fractions by homogenization and differential centrifugation under conditions known to cause the enzyme to be associated with the particulate structures. The LDH was widely distributed among the subcellular fractions with the outer cell membrane and the mitochondrion having especially high activities associated with them.
A 4-hr aging period of the whole, excised muscle had only a minor effect on the subcellular distribution of the enzyme. The major change in aged muscle was an increase of enzymic activity in the soluble, supernatant fractions. Although certain possible artifacts have been ruled out, it is not completely certain that the particle-associated LDH is a true reflection of the situation in vivo. There is at least, however, a reproducible pattern to the binding of LDH to the individual subcellular fractions when chicken breast muscle is homogenized under the specified conditions.     

Skin Color Changes of Squids Todarodes pacificus and Loligo bleekeri During Chilled Storage

ABSTRACT: Squid skin color is one of many important factors to evaluate market values. The effects of chilling and hypoxia treatments on skin color of Japanese common squid Todarodes pacificus and spear squid Loligo bleekeri were investigated to prevent highly fresh squids from abnormal fading skin color with inappropriate commercial value losses. Skin color of the squids gradually faded away during ice-cold storage. Measurements of chromato-phores expansion showed that skin color can be maintained well after death by keeping the bodies well-oxygenated and avoiding direct contact with ice.     

Identification and Characterization of Molecular Species of Collagen in Fish Skin

ABSTRACT: Pepsin-solubilized collagen (PSC) prepared from the skin of 3 fish species—common horse mackerel, yellow sea bream, and tiger puffer—were separated into 2 fractions, major and minor, by ammonium sulfate precipitation. These collagen fractions were further purified by phosphocellulose column chromatography. From the results of SDS-PAGE, peptide mapping, and amino-acid analysis, the purified major and minor collagens were identified to be type I and V collagens, respectively. These results suggest that type V collagen might be widely present in fish skin as a minor collagen.     

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.     

Physicochemical changes of pepsin-solubilized and insoluble collagen in jumbo squid (Dosidicus gigas) muscle after cooking process

Collagen is the major connective tissue (CT) protein and one of the main constituents of the jumbo squid (Dosidicus gigas). Therefore, physicochemical changes of pepsin-solubilized collagen (PSC) and insoluble collagen (IC) were studied after cooking (100°C/30 min) of muscle (mantle, fins, and arms). Different pyridinoline (Pyr) contents (the major cross-linking molecule in collagen fibers) were found in the fresh muscle of the three anatomical regions. After the cooking process, a decrease from 10 to 30% in the thermal resistance of collagen was observed, depending on the anatomical region and fraction evaluated. Furthermore, the electrophoretic profile, Fourier transform infrared (FTIR) spectroscopy, and the amino-acid profile revealed that structural changes occurred in the two different collagen fractions caused by the thermal process, and the changes were greater in the mantle. Under the conditions applied in this study, collagen fractions from the squid arms showed more stability during the cooking process due to the high cross-linking degree of their fibers.     

Effect of Excitation Wavelength on the Separation of Types I and III Collagen by Fiber Optic Fluorimetry

The fluorescence of bovine tendon and adipose tissue and purified Types I and III collagen was measured with fiber optics. Fluorescence of Type I collagen was stronger than for Type III collagen over a range from 410 to 470 nm. Around 510 and 520 nm, however, both Type I and Type III collagen exhibited a similar degree of weak fluorescence. Fluorescence of intact tendons and adipose tissue resembled that of Type I and Type III collagen, respectively. The 510/440 nm ratio of fluorescence was used to identify Types I and III collagen. The optimum excitation wavelength for identification was 370 nm. Type IV (basement membrane) collagen had a similar fluorescence to Type III collagen so that fluorescence from these two sources would be combined in measurements made on intact samples of meat.     

Three-dimensional observation of connective tissue of bovine masseter muscle under concentrate- and roughage-fed conditions by using immunohistochemical/confocal laser-scanning microscopic methods

ABSTRACT:  We investigated changes in connective tissue components of masseter (MA) muscle in Japanese black heifers ( n = 6) in concentrate- and roughage-fed groups (groups C and R, respectively). Body weight, at slaughter, of experimental heifers in group C (272.3 ± 22.3 kg) was higher ( P < 0.05) than that of group R (213.8 ± 27.5 kg). However, muscle weight and myofiber diameter (superficial and deep layers) of MA muscle did not differ between groups C and R. In contrast, total mastication duration of group R was longer ( P < 0.05) than that of group C. MA muscle of groups C and R was composed only of type I myofiber. Using immunohistochemical/confocal laser-scanning microscopy, type I collagen was observed mainly in perimysium, and type V and VI collagen were observed in perimysium and endomysium of both groups. Type IV collagen and laminin were observed only in the endomysium in both groups. However, type III collagen and fibronectin were strongly apparent in the perimysium and endomysium in group R. Connective tissue components in the perimysium of groups C and R were observed to form plate-shaped layers. On the other hand, honeycomb-shaped connective tissue components were seen in the endomysium-surrounded muscle fibers. In particular, fibronectin was strongly observed in the perimysium and endomysium in group R. These results indicate that there are different developmental changes among connective tissue components in MA muscle in response to mastication. The immunohistochemical/confocal laser-scanning microscopic method is useful to investigate the structural relationship among connective tissue components in skeletal muscle.     

Characterization of collagen from Japanese sea bass caudal fin as waste material

In an investigation into making more effective use of underutilized resources, Japanese sea bass caudal fin collagen was prepared and partially characterized. It was separated in two fractions, acid-solubilized collagen (ASC) and acid-insoluble collagen (AIC). The yields of ASC and AIC were about 5.2% and 36.4% respectively, on a dry weight basis. AIC consisted of only one chain and has a chain composition of (1)₃ homotrimer. The patterns of peptide fragments of ASC and AIC were different from that of porcine skin Type I collagen. The denaturation temperatures of these collagens were about 28 °C, about 9 °C lower than that of porcine skin Type I collagen. Japanese sea bass caudal fin has a potential as an alternative source of mammalian collagen for use in various fields such as foods, medicines, and cosmetics.     

Biochemical Characterization of Collagen in Myocommata and Endomysium Fractions of Carp and Spotted Mackerel Muscle

Myocommata and endomysium fractions were prepared from the different parts of body muscle of carp and spotted mackerel. Both type I and V collagens were detected in the myocommata and endomysium fractions of both fish. The relative concentration of type V collagen to type I collagen was higher in the endomysium fraction than in the myocommata fraction. Both type I and V collagens were less soluble in the endomysium fraction than in the myocommata fraction. These results indicated that the biochemical property of the collagen in the pericellular connective tissue was different from that of the collagen in the interstitial connective tissue of fish.     

Isolation and characterization of lecithin from squid (Todarodes pacificus) viscera deoiled by supercritical carbon dioxide extraction

Marine lecithin was isolated and characterized from squid (Todarodes pacificus) viscera residues deoiled by supercritical carbon dioxide (SC-CO(2)) extraction. SC-CO(2) extraction was carried out to extract the oil from squid viscera at different temperatures (35 to 45 °C) and pressures (15 to 25 MPa). The extraction yield was higher at highest temperature and pressure. The major phospholipids of squid viscera lecithin were quantified by high-performance liquid chromatography (HPLC). Phosphatidylcholine (PC; 80.5% ± 0.7%) and phosphatidylethanolamine (PE; 13.2% ± 0.2%) were the main phospholipids. Thin layer chromatography (TLC) was performed to purify the individual phospholipids. The fatty acid compositions of lecithin, PC and PE were analyzed by gas chromatography (GC). A significant amount of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were present in both phospholipids of PC and PE. Emulsions of lecithin in water were prepared through the use of a homogenizer. The oxidative stability of squid viscera lecithin was high in spite of its high concentration of long-chain polyunsaturated fatty acids. PRACTICAL APPLICATION: Squid viscera are discarded as a waste by fish processing industry. Since lecithin from squid viscera contains higher amounts of polyunsaturated fatty acids, it may have promising effect to use in food, pharmaceutical, and cosmetic industries.     

Collagen content in farmed Atlantic salmon (Salmo salar,L.) and subsequent changes in solubility during storage on ice

To elucidate whether collagen is an important factor for fish flesh quality, the collagen content and its changes in solubility during storage on ice in muscle of farmed Atlantic salmon (Salmo salar, L.) were measured. The contents of acidsoluble, pepsin-soluble and insoluble collagen in white muscle were determined in fresh fish muscle and at the end of 5, 10 and 15 days storage on ice. Total collagen was found to be 0.66% of fresh weight, with a relative distribution of 6% acidsoluble, 93% pepsin-soluble and 1% insoluble collagen. During storage on ice, a progressive change in solubility of muscle collagen was found. For insoluble collagen, significantly lower values were detected at day 15 compared to day 0. A minor, but even increase in acid-soluble collagen was found from day 0, while no changes were seen in pepsin-soluble collagen during storage. These results show that collagen fibres of farmed Atlantic salmon have a high solubility in acid and salt solutions and contain few cross-links. Some cleavage of intermolecular cross-links seems to occur during storage on ice.     

Analysis of radiolytic products of lipid in irradiated dried squids (Todarodes pacificus)

The lipid portion of dried squids (Todarodes pacificus) was extracted, and its hydrocarbons and 2-alkylcyclobutanones were separated using a florisil column. Both compounds were identified by gas chromatography and mass spectrometry and used to investigate the production of radiation-induced hydrocarbons and 2-alkylcyclobutanones. Concentrations of the hydrocarbons and 2-alkylcyclobutanones increased linearly with the radiation dosage. The major hydrocarbons in the irradiated dried squids were pentadecane and 1-tetradecene, which originated from palmitic acid. The amount of pentadecane was the highest among the radiation-induced hydrocarbons in the dried squids. The major 2-alkylcyclobutanone in the irradiated dried squids was 2-dodecylcyclobutanone, which was formed from the large amount of palmitic acid. 2-Tetradecylcyclobutanone, which may be produced from stearic acid in sample lipids, was also detected. Radiation-induced hydrocarbons and 2-alkylcyclobutanones were detected at > or = 0.5 kGy. These compounds were not detected in dried squids that were not irradiated. Radiation-induced hydrocarbons can be used as a detection marker for irradiated dried squids; however, the amount of 2-alkylcyclobutanones produced was not enough to be used as a marker. Radiolytic products of lipids, such as hydrocarbons or 2-alkylcyclobutanones. can be used to monitor food safety for consumers, ensuring proper irradiation labeling in foods and quarantine treatment in international trade.     

PREPARATION AND CHARACTERIZATION OF PEPSIN-SOLUBILIZED TYPE I COLLAGEN FROM THE SCALES OF SNAKEHEAD (OPHIOCEPHALUS ARGUS)

Pepsin-solubilized collagen prepared from the scales of snakehead ( Ophiocephalus argus ) was separated into two fractions, major and minor, by NaCl precipitation. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition, and secondary structure showed that the major collagen was typical type I collagen; in contrast, the minor collagen might be classified as type V collagen from the SDS-PAGE patterns and precipitation properties by NaCl. A sharp decrease in solubility of type I collagen was observed at the NaCl concentration of 40 g/L. The maximum and the minimum solubilities of collagen were observed at pH 3 and 8, respectively. Peptide maps of type I collagen digested by trypsin and V8 protease were different from those of calfskin and fish skin collagens. The imino acid content of type I collagen was lower than those of mammalian collagens and so did its denaturation temperature that was 30.3C obtained by viscosity measurement.

PRACTICAL APPLICATIONS

Collagen has been widely utilized as a material for foods, cosmetics, and pharmaceuticals. However, the use of collagen-derived products from land animals (e.g., bovine and pig) has been called into question because of foot-and-mouth disease crisis etc. Aquatic animal offals, which are readily available and inexpensive, seem to be safe sources for extraction of collagen. This work reports on preparation and characterization of collagen from snakehead scales, which will have potential in supplementing the skins and bones of land animals as an important collagen resource for use in functional food, biomedical, and cosmetic industries.     

Effects of gamma radiation on sensory qualities, microbiological and chemical properties of salted and fermented squid

The effects of gamma radiation on sensory quality, microbial population, and chemical properties of salted and fermented squid were investigated. Squid (Todarodes pacificus) was sliced, washed, and then salted with 5, 10, and 20% (wt/wt) sodium chloride. Salted squid was irradiated with dosages of 0, 2.5, 5.0, and 10 kGy of gamma radiation and fermented at 15 degrees C for 50 days. Proximate composition, salinity, water activity, sensory evaluation, and total microbiological populations were examined. Chemical analyses providing information on degree of fermentation, such as amino nitrogen (AN), volatile basic nitrogen (VBN), trimethylamine (TMA), and hypoxanthine (Hx) were also conducted. Irradiated squid was not different in proximate composition, salinity, and water activity from nonirradiated squid. Sensory evaluation scores, total bacteria populations, and pH values were variable depending on salt concentration and irradiation dose. During fermentation, AN, VBN, TMA, and Hx contents increased rapidly as the salt concentration and irradiation dose decreased. Specifically, these chemical compounds of salted and fermented squid prepared with 10% salt and 10 kGy of gamma radiation maintained the appropriate level of fermentation. The present results showed that the combination of low salt concentration (10%) and gamma radiation was effective in processing salted and fermented squid and extending its shelf life compared to control (20% of salt) without adding any food additives.     

The effects of conditioning on meat collagen: Part 3-Evidence for proteolytic damage to endomysial collagen after conditioning

The effects of conditioning on the endomysial fraction of bovine meat were investigated. Solubility studies on endomysial connective tissue and analysis of insoluble endomysial fractions remaining after conditioning were carried out. Yields of soluble endomysial fractions represented, on average, 94·5% of total extracted endomysial material for unconditioned muscles compared with 97·5% for conditioned muscles. Soluble endomysial fractions contained, on average, 0·13% collagen from unconditioned muscles and 0·22% collagen from conditioned muscles. The main peptide components observed on analysis of insoluble endomysial fractions after CN Br digestion were derived from types I and III collagen. Changes observed on the peptide maps, evident as the appearance of a number of new bands from conditioned samples, appeared to be muscle specific. The amount of type III collagen relative to type I decreased on conditioning, indicating that endomysial type III collagen was preferentially destroyed during conditioning. Two-dimensional polyacrylamide gel electrophoresis maps revealed new peptide material in the molecular weight range 40 000 to 50 000 on conditioning. These data provide direct evidence of the effect of proteolytic processing on endomysial collagen during conditioning.     

Collagen contribution to meat toughness: Theoretical aspects

One of the major changes in connective tissues during heating is the transformation of the quasi-crystalline structure of collagen into a random-like structure. This molecular change induces a shortening of these tissues and gives them a rubber-like behaviour. In this state, their mechanical properties are dependent on the total number of cross-linked chains present per volume, which can be estimated from the number and the functionality of each cross-link present in the sample. The number of cross-linked chains per volume of meat explains a large amount of the tenderness variation, produced by muscle type, animal age, type, and sex in different species. During heating collagen fibres and fibrils shortening produces a pressure which is also dependent on the total amount of cross-linked chains present per volume, but also on the morphology of endomysial and perimysial envelopes. In meat, during heating, collagen fibres and fibrils thermal shortening is restricted by muscle fibres and muscle fibre bundles. This restriction, which depends on several muscle fibre characteristics, has a strong effect on the final elastic modulus of connective tissues, by changing the respective amount of crystalline and rubber-like fractions in collagen fibres and fibrils after heating. The implications of this phenomenon in tenderness variations are discussed.     

Histological Changes in Collagen Related to Textural Development of Prawn Meat During Heat Processing

Heat processing enhanced firmness and degree of shrinkage deformation of kuruma prawn Penaeus japonicus meat. In histological experiments, most collagen fiber in perimysium, epimysium, and subcuticular connective tissue maintained structures after heat-processing in hot water at 70°C for 30 min. Crude collagen fiber fractions (residues after alkali extraction, RS-AL) prepared from the muscle were measured for hot-water solubility and compared with the RS-AL from the muscle of carp Cyprinus carpio. Collagen in the RS-AL of prawn muscle had very low hot-water solubility (about 23.5% at 70°C) compared with that of carp. These results suggested that collagen had important functions in promoting thermal shrinkage and hardening of prawn meat and in maintaining mechanical strength of heat-processed meat.     

Collagen contribution to meat toughness: Theoretical aspects

One of the major changes in connective tissues during heating is the transformation of the quasi-crystalline structure of collagen into a random-like structure. This molecular change induces a shortening of these tissues and gives them a rubber-like behaviour. In this state, their mechanical properties are dependent on the total number of cross-linked chains present per volume, which can be estimated from the number and the functionality of each cross-link present in the sample. The number of cross-linked chains per volume of meat explains a large amount of the tenderness variation, produced by muscle type, animal age, type, and sex in different species. During heating collagen fibres and fibrils shortening produces a pressure which is also dependent on the total amount of cross-linked chains present per volume, but also on the morphology of endomysial and perimysial envelopes. In meat, during heating, collagen fibres and fibrils thermal shortening is restricted by muscle fibres and muscle fibre bundles. This restriction, which depends on several muscle fibre characteristics, has a strong effect on the final elastic modulus of connective tissues, by changing the respective amount of crystalline and rubber-like fractions in collagen fibres and fibrils after heating. The implications of this phenomenon in tenderness variations are discussed.