Carp Swim Bladder Research Articles

Silver carp swim bladder collagen derived from deep eutectic solvents: Enhanced solubility against pH and NaCl stresses

Deep eutectic solvents (DESs) are renowned in chemical and food industries for their eco-friendly extraction efficacy. Silver carp swim bladders, a collagen-rich byproduct of surimi production, are underutilized, resulting in considerable protein waste. Traditionally, collagen extraction has relied on harsh acids, contributing to environmental pollution and producing collagens with limited solubility, thus constraining their applications. This study evaluated DESs compared to conventional acids in extracting collagen, focusing on structural and solubility variations. Notably, urea-based DESs (urea-lactic acid: U-LA, 1:10, w/v) achieved the highest hydroxyproline recovery rates (∼ 94 %), comparable to acetic acid (AA, 1:20, w/v), but with half the solid-liquid ratio. Unlike acid-extracted collagen, which preserved the triple-helical structure, urea-based DESs partially disrupted this configuration by reducing intramolecular hydrogen bonding within collagen. However, these solvents simultaneously increased intermolecular hydrogen bonding. This alteration significantly enhanced collagen's solubility, maintaining over 60 % across a broad pH range (1−10) and various NaCl concentrations (0–6 %, w/v). Furthermore, urea-acetic acid (U-AA) extracted collagen exhibited the highest maximum transition temperature (solid state, Tmax = 101.94 °C) and gel strength (165 g). The findings suggest that urea-based DESs not only enhance collagen recovery rates but also its solubility and gelation properties, broadening its potential applications in cosmetics, food products, and biomaterials.

Physicochemical properties, biological activities, and peptidome profile of grass carp swim bladder collagen hydrolysates

This study investigated the physicochemical properties and biological activities of hydrolysates derived from grass carp swim bladder collagen through enzymatic hydrolysis using Papain, Alcalase, Flavourzyme, and Neutrase. The results indicated that the select of protease significantly influences the degree of hydrolysis (DH), molecular weight distribution (MW), trichloroacetic acid-nitrogen solubility index (TCA-NSI), structure, and bioactivities of the hydrolysates. The hydrolysate of Alcalase (HA) exhibited the highest DH, TCA-NSI, Fe2+ chelating activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, and angiotensin-converting enzyme (ACE) inhibitory activity. Pearson's correlation and principal component analysis (PCA) revealed strong interrelationships among the DH, TCA-NSI, β-sheet content, Fe2+ chelating activity, ACE inhibitory activity, and short peptide content within the hydrolysates (|r| > 0.80, P < 0.05). A total of 524 peptides were identified from HA, and 20 novel peptides with no toxicity, no allergenicity, and potential bioactivity were discovered by in silico strategy. Molecular docking analysis revealed that hydrophobic interactions and hydrogen bonds were the primary interaction forces between the peptides with free radicals and ACE. These findings provide a foundation for the high-value utilization of grass carp swim bladder hydrolysates as bioactive peptides in the food industry, enabling the sustainable utilization of freshwater swim bladder by-products.

Identification of a novel peptide that activates alcohol dehydrogenase from crucian carp swim bladder and how it protects against acute alcohol-induced liver injury in mice

Alcoholism is a severe threat to public health, and there are no adequate treatments for alcoholic liver disease. The aim of this study was to identify bioactive peptides derived from natural proteins that prevent acute alcohol-induced liver injury. We identified a peptide with the sequence Gly-Leu-hydroxyproline-Gly-Glu-Arg (GLpGER) from the hydrolysate of crucian carp swim bladder using size-exclusion chromatography and reversed-phase chromatography. The in vitro EC50 value of GLpGER to activate alcohol dehydrogenase (ADH) was 137.9 ± 9 µM. Molecular docking experiments indicated that the mechanism by which GLpGER activates ADH may be related to the formation of stable complexes with ADH active pockets through hydrogen bonding, and electrostatic and hydrophobic interactions. Oral administration of GLpGER one hour before acute alcohol ingestion significantly increased alcohol metabolism, manifesting as reduced incidence of the loss of righting reflex, increased alcohol tolerance time, shortened sobering time, and decreased blood alcohol concentration level. GLpGER restored liver ADH activity, maintained the typical morphology of hepatocytes, and reduced serum levels of alanine aminotransferase and aspartate aminotransferase. These findings suggest that GLpGER might reduce acute alcohol-induced liver injury and may have the potential to be developed as an anti-inebriation ingredient.

Chemical and biological characteristics of hydrolysate of crucian carp swim bladder: Focus on preventing ulcerative colitis

The incidence of ulcerative colitis (UC) is increasing worldwide. Currently, no curative treatment is available. Finding a food-based method for prevention of UC would be significant. In this study, the hydrolysate of crucian carp swim bladder (HCSB) was prepared using a simulated gastrointestinal digestion. 178 peptides in HCSB were identified by nano liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry. In a model of hydrogen peroxide-induced oxidative stress in an intestinal epithelial cell line (IEC-6), HCSB protects IEC-6 cells by increasing antioxidant enzyme activity and reducing the intracellular ROS content. HCSB also represses lipopolysaccharide-induced inflammation of IEC-6 cells. In vivo studies showed that prophylactic administration of HCSB reduces the severity of dextran sodium sulfate-induced UC in mice by reducing myeloperoxidase activity, production of proinflammatory cytokines, and by inhibiting activation of the NF-κB pathway. These results indicate that HCSB has the potential to prevent ulcerative colitis.

Identification and characterization of DPP-IV inhibitory peptides from silver carp swim bladder hydrolysates

Dipeptidyl peptidase IV (DPP-IV) has an important role in blood glucose metabolism. Silver carp swim bladder protein was hydrolyzed using papain, bromelain, Alcalase 2.4 L, Neutrase, and Flavourzyme. The swim bladder hydrolysate obtained using 5 h hydrolysis of Neutrase showed the highest DPP-IV inhibition (81 ± 2%) at 5 mg/mL. This hydrolysate was purified sequentially using ultrafiltration, gel filtration chromatography, and RP-HPLC. Peptide sequences from the RP-HPLC fractions were identified using liquid chromatography tandem mass spectrometry. The most potent DPP-IV inhibitory peptide WGDEHIPGSPYH (IC50: 0.35 ± 0.01 mM) showed an uncompetitive inhibition mode. Docking analysis showed that the binding site of WGDEHIPGSPYH was located inside the cavity of the DPP-IV monomer, which was close to the catalytic site. WGDEHIPGSPYH and its hydrolysate IPGSPY were transported intact across a Caco-2 cell monolayer. These two peptides were tested for their effects on Caco-2 and INS-1 cells and both showed good inhibition for soluble DPP-IV and promoted insulin secretion.

Modern status of studies of carp swim bladder inflammation (SBI)

Modern status of studies of carp swim bladder inflammation (SBI)

Physico-functional and Anti-oxidative Properties of Carp Swim Bladder Gelatin and Brown Seaweed Fucoidan Based Edible Films

Films were prepared with fish gelatin extracted from swim bladder of rohu (Labeo rohita) and fucoidan extracted from brown seaweed (Padina tetrastromatica) using 3% gelatin and sorbitol (30% of protein) as plasticizer. Fucoidan was incorporated at concentrations of 5, 10, 15 and 20% of protein concentration to form GF-5, GF-10, GF-15 and GF-20 films, respectively. Results indicated that the average thickness of films ranged from 61.60 to 66.25 μm. Tensile strength (TS) increased from 55.37 to 63.37 MPa and elastic modulus (EM) from 1278.40 to 1482.20 MPa with the increase in fucoidan. WVTR and water solubility of films decreased from 917.44 to 634 g/m2/day and 91–82%, respectively with the addition of fucoidan. GF-50 films possessed lesser whiteness (39.59) compared to control films (55.79). FTIR spectra and SEM analysis clearly depicted the interaction of fucoidan with gelatin. GF-20 films exhibited high DPPH radical scavenging activity (46.26%), ABTS radical scavenging activity (49.95%) and Ferric reducing power (0.12 absorbance units) than other films. Thus, incorporation of fucoidan in fish gelatin films enhanced anti-oxidative properties besides improving mechanical and barrier properties, to serve as an active biodegradable edible film.

Physico-chemical characteristics and fibril-forming capacity of carp swim bladder collagens and exploration of their potential bioactive peptides by in silico approaches

This study explores the sustainable valorization of carp swim bladder by-products. The high-value molecule collagens were successfully extracted from carp swim bladder with a yield of ∼60% (dry weight basis) and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, UV-spectrum and Fourier transform infrared spectroscopy. The extracted collagens showed the fibril-forming ability and high denaturation temperature (38–39°C). Furthermore, the extracted collagens subjected to preparing bioactive collagen hydrolysates with potential antioxidant activities. In vitro and In silico approaches (PepDraw, BIOPEP, PeptideRanker, Pepcalc and ToxinPred) were employed to evaluate the potential of carp collagens as a potential source of bioactive peptides. Furthermore, primary structure, biological potential, physicochemical, sensory and toxicity characteristics of the theoretically release antioxidative collagen peptides were predicted. Overall, the present study highlights the carp collagens hydrolysate could be a promising precursor of bioactive peptides for developing functional food or nutraceutical products.

A Novel Training Model for Retroperitoneal Laparoscopic Dismembered Pyeloplasty

To create and evaluate an inexpensive novel model for training in retroperitoneal laparoscopic dismembered pyeloplasty. The retroperitoneal cavity was simulated by five boards fixed together by detachable hinges; the extent of operating difficulty could be adjusted by changing the angle of inclination. The middle of a plasticine model kidney was imbedded into a metal clip, and a carp swim bladder was fitted to the kidney to model the dilated pelvis. The "ureter" was a 10-cm long porcine ureter. Semicircular-shaped simulator, laparoscopic camera system, scissors, needle holder, and surgical forceps specific for training were used. Ten rounds of intensive training of the "ureteropelvic" anastomosis procedure was provided for five laparoscopic surgeons. Each trainee's mean time of achieving dismembered pyeloplasty in three patients was recorded; the corresponding index in the last three ureteropelvic junction obstruction operations performed by each person before intensive training was retrospectively revised. Subjective evaluation was also performed, and a general self-efficacy (GSE) scale was applied to evaluate the self-confidence of trainees. This model reproduced the technical complexity of laparoscopic dismembered pyeloplasty. The operative time significantly declined from 41.84 +/- 3.00 minutes to 25.04 +/- 2.37 minutes after intensive training. GSE scores after training were also higher than before (27.60 +/- 1.14 vs 22.20 +/- 1.79). Correlation between operative time and GSE was significant. After training, all participants were more skillful in performing ureteropelvic anastomosis. This is a small study with a middle-term follow-up. The operation also cannot be simulated completely because the ureter is separate from the renal pelvis in the beginning. This model demonstrates satisfying fidelity and practicality; it could easily be built and reused, and could result in lower costs than those incurred using the traditional model.

Collagenolytic activity of a soil actinomycete.

A soil actinomycete hydrolyzed collagen extracted from bovine Achilles tendon, calf skin, carp swim-bladder and rat tail tendon. Glucose, mannose, aspartic acid and asparagine increased its collagenolytic activity which was optimum at 28 °C and at pH 7.2 – 7.5. Metallic ions, NaEDTA, cysteine, 4-chloromercuribenzoate, glutathione and sodium azide were inhibitory.

Native collagen has a two-bonded structure

A number of structural models that differ sterically and in interpeptide hydrogen-bonding pattern have been put forward for the native collagen molecule. These have proven remarkably difficult to distinguish by X-ray diffraction and other methods. The various models have generally been divided into a “one-bonded” class (one hydrogen bond per triad of amino acid residues) and a “two-bonded” class (nearly two bonds per triad). To discriminate between the models, we have applied hydrogen-tritium exchange methods to native soluble ichthyocol (carp swim bladder) collagen. Free and hydrogen-bonded peptide groups were recognized by their hydrogen exchange rates. Under two different solution conditions, a set of peptide hydrogens was found to display exchange rates nearly identical to those previously calibrated for free peptides in model systems and, therefore, was taken to represent free peptides. The remaining hydrogens exchanged much more slowly and were assigned to internally hydrogen-bonded peptide groups. The numbers of free and of internally bonded peptide hydrogens were measured independently in different kinds of exchange experiments. They coincide with the numeration required for two-bonded models. The results appear to exclude one-bonded models for ichthyocol collagen but cannot distinguish between the earlier two-bonded model, which contains two direct peptide to peptide hydrogen bonds per triad, and the more recent proposal involving one direct bond and one cross-bridging water molecule. The results, together with other data in the literature, indicate that native collagens from other species may also exist in a two-bonded structure, though some synthetic sequence polypeptides related to collagen do clearly assume a one-bonded structure. The results are relevant also to the general problem of structure analysis by hydrogen exchange methods. It has previously been demonstrated that free peptide hydrogens exchange at precisely predictable rates in small molecules, in oligo- and polypeptides, and in a globular protein. The present data extend this conclusion to a fibrous protein. Thus the techniques used here may be quite generally applied to distinguish and to count free and internally bonded peptide groups in protein and polypeptide systems.

A Comparative Study of Glycopeptides Derived from Selected Vertebrate Collagens: A POSSIBLE ROLE OF THE CARBOHYDRATE IN FIBRIL FORMATION

Abstract Glycopeptides from carp swim bladder, guinea pig skin, and human skin collagens were prepared by sequential collagenase-trypsin digestion. Gel filtration on Sephadex G-25 permitted the isolation of a low molecular weight fraction containing most of the hydroxylysyl glycosides present in the parent collagen. Application of this fraction to a Dowex 50X-8 ion exchange column consistently resulted in distinctive glycopeptide patterns characteristic of each collagen. The glycohexapeptide, Gly-Met-Hyl (Gal-Glc)-Gly-His-Arg, which had been previously characterized in digests of soluble guinea pig skin collagen, was found in significant quantities in all three collagens examined, but the carp and human collagens each revealed an additional major glycopeptide. The subtractive Edman procedure was used in establishing amino acid sequences, and alkaline hydrolysis was used to confirm the composition and mode of attachment of the carbohydrate side chains. The predominant glycopeptide isolated from carp swim bladder collagen was Gly-Ile-Hyl(Gal-Glc)-Gly-His-Arg, while the structure of the unique glycopeptide isolated from human skin collagen was Gly-Phe-Hyl(Gal-Glc)-Gly-Ile-Arg. A possible role of the carbohydrate side chains in the organization of fibrils is discussed.

Invertebrate collagens: Isolation, characterization and phylogenetic aspects

Acid-soluble collagens were extracted from the sea anemone Actinia equina and from the liver fluke Fasciola hepatica and purified by repeated reprecipitation. They share the following properties with collagens from highly developed animals: digestibility by collagenase; important characteristics of amino acid composition (one-third glycine, high imino acid content, presence of hydroxyproline and hydroxylysine); a firmly attached hexose moiety; sedimentation as an extremely sharp boundary in the native state; a high negative value for the specific optical rotation; the ability of forming cross-striated fibrils and segments as seen in the electron microscope. The molecular weight of the collagen from Actinia is in accord with that of vertebrate collagen (310,000). For the collagen isolated from Fasciola a higher value (500,000) was obtained. The subunits of the latter collagen were very labile and could not be characterized in the ultracentrifuge or by disc-electrophoresis. The length and the highly differentiated cross-striation pattern of the long spacing segments from the two invertebrate collagens were found to be identical with those of segments from calf skin collagen and carp swim bladder collagen. This indicates invariance of the tertiary and quaternary structures of the collagen molecule during evolution, and probably reflects also invariance of fundamental features of the amino acid sequence. Two further evolutionary aspects of collagen are discussed. Evidence for only one α-subunit was found in collagen from Actinia, whereas two or three are present in the collagens of highly developed animal phyla. This apparently confirms a hypothesis on the divergent evolution of the collagen α-chain gene. The thermal stability of the collagen molecule in solution depends on the environmental conditions of the animal from which the collagen was prepared. There is no relationship between this property, however, and the phylogenetic stage of the animal.

Characterization and Quantitative Determination of the Hydroxylysine-linked Carbohydrate Units of Several Collagens

Abstract The hydroxylysine-containing glycopeptides from collagenase-Pronase digests of bovine tendon collagen were found to have glucose and galactose as their monosaccharide components. The ratio of galactose to glucose in these glycopeptides was greater than unity and after alkaline hydrolysis both glucosylgalactosylhydroxylysine and galactosylhydroxylysine were found. In order to determine the content of glucosylgalactosylhydroxylysine, galactosylhydroxylysine, and unsubstituted hydroxylysine present in tendon collagen as well as in several other collagens, analyses for these components were performed on the amino acid analyzer after alkaline hydrolysis. The other collagens studied were obtained from rat skin, rat tail tendon, carp swim bladder, rabbit sclera, and corneas from rabbits and calves. From most of these sources both the citrate-soluble and citrate-insoluble collagens were studied. In all of the proteins studied both glucosylgalactosylhydroxylysine and galactosylhydroxylysine were found, with the galactosylhydroxylysine making up from 46% of the total hydroxylysine-linked carbohydrate units in calf skin citrate-soluble collagen to 11% in carp swim bladder citrate-insoluble collagen. The citrate-insoluble collagens tended to contain a greater percentage of glucosylgalactosylhydroxylysine units than the citrate-soluble collagens from the same source. The percentage of the total hydroxylysine residues substituted by the glycosidic linkages of these units varied from 5% in rat tail tendon citrate-soluble collagen (0.4 carbohydrate unit/1000 amino acid residues) to 56% in the rabbit cornea citrate-insoluble collagen (5.8 carbohydrate units/1000 amino acid residues). All these collagens were shown to contain markedly fewer hydroxylysine-linked carbohydrate units than basement membranes, suggesting that higher levels of hydroxylysine-linked carbohydrate are reflected in lower degrees of morphological organization as seen under the electron microscope. The glucosylgalactosylhydroxylysine and galactosylhydroxylysine obtained from the alkaline hydrolyses of the various collagens had the same migration on paper and ion exchange chromatography as the 2-O-α-d-glucosyl-O-β-d-galactosylhydroxylysine and O-β-d-galactosylhydroxylysine previously isolated from the bovine renal glomerular basement membrane. For structural studies glycopeptides containing the disaccharide units were obtained from the collagenase-Pronase digest of ichthyocol. The preferential release of glucose during graded acid hydrolysis of these glycopeptides and the isolation of a disaccharide with galactose on the reducing end indicated a glucosylgalactose sequence in these carbohydrate units. The disaccharide was split by α-glucosidase but was resistant to the action of β-glucosidase. Periodate oxidation, galactose oxidase treatment, and methylation of the glycopeptides indicated that the glucose is linked glycosidically to carbon-2 of the galactose. These results are consistent with a structure for the disaccharide of 2-O-α-d-glucopyranosyl-d-galactose, which is identical with that found for the glomerular basement membrane. In addition to glucose and galactose, the collagens contained small amounts of other sugar components including mannose, fucose, hexosamines, and sialic acids. In the collagenase-Pronase digests of ichthyocol and tendon collagen, glycopeptides containing these sugars could be separated from most of the hydroxylysine-containing glycopeptides by gel filtration.

Localization and Partial Characterization of an Aldehydic Component in Tropocollagen

1. An alternative simplified procedure is described for the isolation of a 30-residue peptide obtained by digestion with collagenase of carp swim bladder tropocollagen (ichthyocol). The peptide contains an aldehydic component as a 2,4-dinitrophenylhydrazone. The aldehyde dinitrophenylhydrazone is present in stoichiometric proportion to the amino acids in the peptide. 2. This peptide is shown to be devoid of free terminal amino groups by lack of reactivity with ninhydrin or phenylisothiocyanate. Since it is prepared by treatment with collagenase and yet devoid of any terminal α-amino group including that of a glycine residue, the peptide most likely arises from a blocked amino-terminal position of a tropocollagen chain. 3. The aldehyde-bearing component most likely is not bound to any of the following amino acid side chains for the reasons given: (a) to side chain carboxyl groups of glutamyl or aspartyl residues in ester linkage, since treatment with aqueous hydrazine does not release the dinitrophenylhydrazone from the peptide; (b) to the hydroxyl group of a serine residue by means of a glycosidic-like attachment, since mild treatment with NaOH does not promote β elimination in the serine; (c) to side chains of either threonine, hydroxylysine, hydroxyproline, lysine, arginine, or histidine, since residues of these are not present in the peptide; (d) to the hydroxyl side chain of a tyrosine residue, since this amino acid, while present in the aldehyde-dinitrophenylhydrazone-peptide obtained following digestion with collagenase, is absent from the aldehyde-dinitrophenylhydrazone-peptide obtained by further digestion with Pronase. 4. The peptide obtained with collagenase is cleaved further into at least seven peptides which have been partially characterized. A yellow heptapeptide containing the aldehydic component as a 2,4-dinitrophenylhydrazine derivative is also without free amino groups, and this locates the aldehyde-bearing component within 7 residues of a blocked amino terminus of a tropocollagen chain. 5. Ultraviolet absorption spectra of derivatives of the aldehyde component, and its tendency to undergo acid-catalyzed α,β-dehydration to α,β-unsaturated aldehyde derivatives, suggest that its first 3 carbon atoms are involved in a β-hydroxyaldehyde structure. The aldehyde-bearing component must contain an additional functional group with which it makes covalent attachment to the protein. 6. It is suggested that the aldehyde component, by virtue of its chemical nature, reactivity, stoichiometry, and location in the protein, may be involved in the maturation and cross-linking of soluble tropocollagen to form insoluble mature tissue collagen.

Antigenicity of rat collagen; reverse anaphylaxis induced in rats by anti-rat collagen serum.

Reverse anaphylactic shock was induced in rats by intravenous injection of serum containing complement-fixing antibodies, obtamed by immunization of rabbits with purified preparations of rat tail collagen. Normal rabbit serum or serum containing antibodies to collagen from tunica of carp swim bladder was without effect. The clinical and pathological findings resembled those described by previous workers studying direct and reverse anaphylactic reactions induced in the rat with other antigens. Thrombocytopenia, leukopenia with rebound leukocytosis, delayed blood coagulability, lowering of serum complement and the development of a refractory state to the antibody after the initial shock-all compatible with anaphylaxis-were demonstrated. The reacting substance was found to be in the gamma globulin fraction of the serum, and the antibody titer appeared to be correlated with the degree of shock and vascular injury. Collagen injury in rats dying from acute shock or in those injected with repeated sublethal doses was not demonstrated by the methods employed; techniques by which lesions of collagen might be induced are discussed. Absorption experiments indicated that anti-rat collagen rabbit serum is specifically directed to a substance, apparently collagen, in the rat; it is likely that the combination of the antibody and collagen induces anaphylactic shock. These studies, made in vivo, provide further evidence of antigenic and immunological differences between acid-soluble rat and fish collagens.

Optical rotation and helical polypeptide chain configuration in collagen and gelatin.

The optical rotation phenomena exhibited by a citrate-extracted fraction of ichthyocol (from carp swim bladder), as well as by the parent gelatin derived therefrom, have been studied. Dispersion data for all cases follow a single-term Drude equation, but the variations with state are adequately expressed by simple reference to changes in [alpha](D) as follows:- 1. The native collagen fraction, dispersed in 0.15 M citrate buffer at pH 3.7 in the cold (11 degrees C.), yields a high negative specific rotation, [alpha](D), near -350 degrees . 2. During equilibration at 40 degrees C., which causes conversion to a monodisperse parent gelatin, the rotation drops to about -110 degrees . 3. Gelation at 2 degrees C. results in a partial regain of rotation to around -290 degrees . This mutarotation is reversible, depending on temperature. 4. In the range 0.02 to 0.28 per cent the native ichthyocol and the warm gelatin solutions show little concentration dependence, but with the cold gelatin solutions the specific rotation increases with concentration. Gelatin films formed by cold evaporation yield high specific rotation (ca. -620 degrees ), but those formed by hot evaporation retain low optical activity. 5. Since this same collagen-gelatin system has been investigated physicochemically, it is possible to relate molecular changes to the observed variations in optical rotation. Conclusions are similar to those of Robinson (1953), who studied other gelatins: high negative rotation is believed related to a native collagen polypeptide configuration, herein specified as helical (from x-ray diffraction considerations) and destroyed by heating. The possible roles of intermolecular interactions and of prevalent pyrrolidine constituents in influencing the helical configuration and optical activity are discussed.

Particle size and shape in a citrate extract of ichthyocol

Viscosity, sedimentation, and light-scattering data indicate that an acid-soluble component of carp swim bladder (ichthyocol), capable of reconstitution into typical collagen-like fibrils, disperses as long thin filaments (protofibrils) of weight one to two million, containing about one amino acid residue per angstrom of contour length.