Pepsin-solubilized Collagen Research Articles

Extraction of collagen-II with pepsin and ultrasound treatment from chicken sternal cartilage; physicochemical and functional properties.

A simple and green approach was developed to extract the pepsin soluble collagen-II using the ultra-sonication treatment that significantly increased the extracted yield from chicken sternal cartilage (CSC). The pretreatment of raw CSC had positive effects on proximate composition. The maximum yield of pepsin soluble collagen was obtained by ultrasound treatment time 36min (UPSCII36; 3.37g) as compared to non-ultrasound treated pepsin soluble collagen at zero minutes (UPSCII0; 1.73g) (control). The amino acid profile, differential scanning calorimetry (DSC) of UPSC were significantly (p<0.05) improved by the application of ultrasound. The results showed the secondary structure of ultrasound treated PSC-II was partially altered as the ultra-sonication time prolonged. Moreover, ultrasound-treated collagen had superior functional properties such as water, oil absorption capacity, water holding capacity, foaming and emulsifying properties than non-ultrasound treated collagen. The poultry by-products CSC would be a potential source of land animal collagen-II. The utilization of ultrasound for the extraction of pepsin soluble collagen-II is a good alternative technology to expand the application of collagen at industrial level.

Aggregation Behavior of Acylated Pepsin-Solubilized Collagen Based on Fluorescence Spectrum Technology.

The aggregation behavior of collagen-based materials plays an important role in their processing because it could affect their physicochemical properties. Based on the intrinsic fluorescence characteristic of tyrosine, fluorescence spectrum technology was used to investigate the aggregation state of the acylated collagen molecules in aqueous solution. The results showed that the aggregate degree of the acylated collagen was higher than that of the native collagen due to the hydrophobic interaction. With the increase of concentrations of the acylated collagen or at NaCl higher than 40 mmol/L, the aggregate degree of the acylated collagen molecules increased. When the pH was close to the isoelectric point of the acylated collagen, the hydrophobic interaction and the hydrogen bond helped to increase the aggregation degree. However, with the increase of temperature (10-70 ℃), the aggregation state of the acylated collagen decreased gradually due to the quenching, the molecular collision, and the broken of hydrogen bonds. Furthermore, two-dimensional correlation spectroscopy (2D-COS) showed that the response order was 360 > 305 nm at various acylated collagen and NaCl (>40 mmol/L) concentrations, while the response order was 305 > 360 nm when the pH value was increased from 5.0 to 9.0. Temperature-dependent 2D-COS showed there were four bands that occurred and the response order was listed as follows: 293 > 305 > 360 > 420 nm. In brief, the results might provide an important guide for molding processes of the acylated collagen.

Collagen fibrils of sea cucumber (Apostichopus japonicus) are heterotypic

Sea cucumbers attracted increasing interest due to its nutritional functions. Collagen is the most important structural biomacromolecule in sea cucumber body wall, and is highly related to the textual properties and food quality of sea cucumber. In this study, the type of constituent collagens of sea cucumber collagen fibrils was investigated, employing a commercially important species Apostichopus japonicus as the material. Proteomics and bioinformatics analysis revealed that collagen fibrils of A. japonicas are heterotypic. Two clade A and one clade B fibrillar collagens and two FACIT collagens were identified from the fibrils. Besides, the heterogeneity was also revealed in the pepsin-solubilized collagen (PSC) of A. japonicus by using the proteomics strategy. It implied that the previous conclusions on the type of sea cucumber collagen deduced from SDS-PAGE analysis should be rechecked. The results provided novel insight into the composition of sea cucumber collagen fibrils.

Effect of ultrasound power on extraction kinetic model, and physicochemical and structural characteristics of collagen from chicken lung

The effects of ultrasound power on extraction kinetic model, and physicochemical and structural characteristics of collagen from chicken lung were studied. Ultrasound power caused a significant increase in extraction rate and equilibrium concentration, with the maximum extraction yield (31.25%) at 150 W. The experimental data were consistent with the predicted ones in this empirical equation, in which the percentage error differences was 0.026–4.159%. Besides, ultrasound treatment did not affect their triple-helical structure. The thermal stability of pepsin-soluble collagen by ultrasound pre-treatment (UPSC) was higher, due to the higher imino acid content (20.76%). UPSC also exhibited better solubility and fibril forming capacity. Overall, the kinetic model of UPSC from chicken lung could serve the purpose of obtaining collagen, which displayed a potential alternative source to mammal collagens for application in food, biomaterials and biomedical fields.Graphical abstract

Preparation of self-assembled collagen fibrillar gel from tilapia skin and its formation in presence of acidic polysaccharides

Fibrillar gel of pepsin-solubilized collagen from tilapia skin was prepared by self-assembly in neutral phosphate buffer at 28 °C. Then effects of acidic polysaccharides, such as sodium alginate (SA), chondroitin sulfate (CS), and hyaluronic acid (HA), on the formation and properties of self-assembled fibrillar gel were investigated. SA and CS prolonged gelling time, whereas HA had no obvious effect. SA made fibril network denser, while CS and HA induced the presence of larger ordered structures. All the acidic polysaccharides broadened the D-periodicity of fibrils. SA and HA increased the maximum mechanical strength of gel to 39.64 and 34.49 kN/m2, respectively, significantly higher than that of pure collagen gel (14.53 kN/m2), while that only 17.20 kN/m2 after CS introduced. HA had no evident effect on enzymatic resistance, while SA and CS decreased. Therefore, tilapia skin collagen with HA has a higher potential as a biomaterial than that with CS or SA.

Extraction of crude collagen from &lt;em&gt;Pterygoplichthys pardalis&lt;/em&gt; skin and determination of functional properties of its hydrolysates

Collagen has a great demand and fish skin is a safe alternative source of collagen. Pterygoplichthys pardalis is an endemic, freshwater fish with no economic benefit. The study was conducted to extract crude collagen from the skin of P. pardalis using a simple method and identify the functional properties of its hydrolysates. Acid and pepsin soluble collagens were extracted from skin using citric acid (CA) and ethylene diamine tetraacetic acid (EDTA). Selected crude collagens were hydrolysed using pepsin, protease and trypsin enzymes (1:100). Extracted crude collagens and best hydrolysates were selected using 8 and 15% SDS-PAGE. Hydrolysates were analysed for antioxidant, Fe(II) chelating and antibacterial activities. CA treatment showed significantly (P&lt;0.05) higher yield than EDTA treatment. Antioxidant properties, metal chelation and antibacterial activities were not significantly (P&gt;0.05) different. Hydrolysates showed significantly (P&lt;0.05) higher functional properties than crude collagen in both treatments. These results conclude that collagen hydrolysates from P. pardalis have good antioxidant, metal chelating and antibacterial properties.

Pepsin-Soluble Collagen from the Skin of Lophius litulo: A Preliminary Study Evaluating Physicochemical, Antioxidant, and Wound Healing Properties.

The structure of pepsin-solubilized collagen (PSC) obtained from the skin of Lophius litulon was analyzed using the sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). SDS-PAGE results showed that PSC from Lophius litulon skin was collagen type I and had collagen-specific α1, α2, β, and γ chains. FTIR results indicated that the infrared spectrum of PSC ranged from 400 to 4000 cm−1, with five main amide bands. SEM revealed the microstructure of PSC, which consisted of clear fibrous and porous structures. In vitro antioxidant studies demonstrated that PSC revealed the scavenging ability for 2,2-diphenyl-1-picrylhydrazyl (DPPH), HO·, O2−·, and ABTS·. Moreover, animal experiments were conducted to evaluate the biocompatibility of PSC. The collagen sponge group showed a good biocompatibility in the skin wound model and may play a positive role in the progression of the healing process. The cumulative results suggest that collagen from the skin of Lophius litulon has potential applications in wound healing due to its good biocompatibility.

Histological Distribution and Characterization of Collagen in European eel (Anguilla anguilla) Muscle

ABSTRACTThe histologic structure of eel muscle was observed, and the acid solubilized collagen (ASC) and pepsin solubilized collagen (PSC) from eel muscle were prepared and examined. The collagenous fiber amount in both head and tail seemed to be higher than that in the trunk. The Glu, Asp, Arg, Ala, Leu, and Lys contents accounted for about 54% of total amino acids in the eel muscle. There were no significant differences in the amino acid composition and ultraviolet spectra between ASC and PSC. The molecular weights of α1 subunit in both collagens were 129 and 123 kDa, respectively; but their molecular weights of α2 subunit were 113 kDa. According to peptide mapping analysis, it was found that there were obvious differences in primary structures between ASC and PSC. The amide A band positions of ASC and PSC appeared at 3320 cm−1 and 3312 cm−1 of Fourier transform infrared spectra, respectively. No obvious difference was found in the thermal stability of both collagens, probably due to the synergistic effect of molecular weight and hydrogen bonds.

Characterization of pepsin-solubilised collagen recovered from mackerel (Scomber japonicus) bone and skin using subcritical water hydrolysis

This study was aimed at isolation of pepsin-solubilised collagen (PSC) from Mackerel (Scomber japonicus) bone and skin in order to effectively valorise these abundant wastes. The yield of PSC (8.10%) from skin was considerably higher than that from bone (1.75%). Based on the protein patterns, both PSCs were type Ι, and consisted of two α-chains. Fourier-transform infrared spectra demonstrated that PSCs from the bone and skin exhibited a triple-helical structure. The denaturation temperatures (Td) of the PSCs from bone and skin were 27 and 30 °C, respectively. Low-molecular-weight peptides (<1650 Da) were generated from both PSCs after subcritical water hydrolysis treatment. Glycine accounted for 30% of the total amino acids identified in both PSC hydrolysates. The antioxidant activities of both PSC hydrolysates were significantly higher than those of the isolated PSCs. Therefore, PSC hydrolysates can be used as a functional ingredient in the food, cosmetic and pharmaceutical industries.

Extraction and characterization of collagen from sea cucumber (Holothuria cinerascens) and its potential application in moisturizing cosmetics

To increase the utilization of sea cucumbers and focus on the development and screening of natural raw materials for moisturizing cosmetics, we used the body wall of sea cucumber Holothuria cinerascens as raw materials for extracting pepsin-solubilized collagen. We compared the collagen with that from the skin of tilapia and pig. SDS-PAGE profiles revealed that, the collagen from the three species were type I. Sea cucumber had the smallest molecular weight (80–90 kDa). Maximum peak at 230 nm on UV–visible spectroscopy profiles revealed that the three collagens contained amino acids with C=O, COOH, and CONH2 groups. FTIR spectroscopy showed the three collagens with amide A, I, II, III peaks generated by the main functional groups in the proteins. The dominant amino acids of the three collagens were glycine (31%), proline (9–12%) and alanine (10–12%). The three collagens exhibited better moisture-retention and moisture-absorption capacity than glycerol, so the collagen molecules are rich in hydrophilic groups, which has potential application in cosmetic formulations. Further studies are needed to retain the properties of extracted collagen during processing or other applications because the commercial value of the industrial by-products of sea cucumber could be increase and thus reduce the environmental impact caused by the disposal of this material.

Acid-Pepsin Soluble Collagen from Saltwater and Freshwater Fish Scales

Extraction and characterization of acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from scales of Giant groupers (saltwater fish) and Nile tilapia (freshwater fish) were carried out in this research. Due to a higher protein content in scales, collagen yield extracted from the Giant groupers scales was higher than that of the Nile tilapia scales. The yield increased as extraction time increased for both ASC and PSC and pepsin extraction resulted in higher yields than acid extraction. Even though there were differences in collagen yields, collagen characteristics were independent of the scale sources but some differences were observed for the ASC and PSC. The peptide hydrolysis patterns of the ASC showed a wide range of molecular weights whereas all of the PSC had similar molecular weight of around 42 kDa. FTIR spectra showed that all the collagens remained the triple helical structure though ASC might be self-aggregated. From zeta potential analysis, net charge of zero was found at pH 3.2-4.0 and the dynamic light scattering suggested that the average particle sizes at pH 11-12 were around 100-200 nm. The denaturation temperatures (T(d)s) in a range of 35-42 degrees C indicated that the collagens were considerably thermally stable.

Physicochemical and Functional Properties of Type I Collagens in Red Stingray (Dasyatis akajei) Skin.

Collagen is widely used in the pharmaceutical, tissue engineering, nutraceutical, and cosmetic industries. In this study, acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the skin of red stingray, and its physicochemical and functional properties were investigated. The yields of ASC and PSC were 33.95 ± 0.7% and 37.18 ± 0.71% (on a dry weight basis), respectively. ASC and PSC were identified as type I collagen by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis, possessing a complete triple helix structure as determined by UV absorption, Fourier transform infrared, circular dichroism, and X-ray diffraction spectroscopy. Contact angle experiments indicated that PSC was more hydrophobic than ASC. Thermal stability tests revealed that the melting temperature of PSC from red stingray skin was higher than that of PSC from duck skin, and the difference in the melting temperature between these two PSCs was 9.24 °C. Additionally, both ASC and PSC were functionally superior to some other proteins from terrestrial sources, such as scallop gonad protein, whey protein, and goose liver protein. These results suggest that PSC from red stingray skin could be used instead of terrestrial animal collagen in drugs, foods, cosmetics, and biological functional materials, and as scaffolds for bone regeneration.

New Strategy to Cope with Common Fishery Policy Landing Obligation: Collagen Extraction from Skins and Bones of Undersized Hake (Merluccius merluccius).

In order to promote sustainable fishing practices within European fishing fleets and to avoid the large waste of valuable fish biomass through the practice of fish discarding, the new reform of the common fisheries policy includes the obligation of landing all species under total allowable catch (TAC) regulations. The new policy also prohibits the use of specimens under minimum conservation reference size for direct human consumption. In this context, it is necessary to find new uses for undersized fish, which might help to alleviate the costs associated with the landing obligation but without prompting the creation of a market. European hake (EH) (Merluccius merluccius), which is one of the most important commercial fish species for the Spanish fishing industry, with a total TAC for 2018 of 37,423 t, is used for this study. Consistent with the current policy framework and taking into account the commercial importance of this species, the aim of this work is to study a new strategy for the extraction of collagen from the skin and bone fraction of Merluccius merluccius undersized discards. Three collagen fractions are successfully isolated for the first time from the skin of M. merluccius skin and bone discarded raw material: acid-soluble collagen (ASC) fraction 1 and pepsin-soluble collagen (PSC) fraction 2 from the skin and ASC fraction 3 from bones. The total collagen yield of the process is 13.55 ± 3.18% in a dry basis (g collagen/100 g of skin and bone fraction (SBF)) and 47.80 ± 9.83% (g collagen/100 g of collagen determined by the hydroxyproline content in SBF). The three fractions are further characterized by using different physical and chemical analysis techniques, with the conclusion drawn that the triple helix structure is preserved in the three fractions, although ASC fractions (F1 and F3) present more or stronger hydrogen bonds than the PSC fraction (F2). With the process herein presented, deboned and skinned hake specimens could represent an interesting source of high quality type I collagen, which could be useful as a raw material for the biomedical, cosmetic, and nutraceutical industries.

Extraction and Physicochemical Characterization of Pepsin Soluble Collagens from Golden Pompano (Trachinotus blochii) Skin and Bone

ABSTRACTPepsin-soluble collagen (PSC) was extracted and purified from wasted skin and bone of the golden pompano by acetic acid-pepsin method. The result showed that the PSCs extraction yields of skin and bone were 21.81% and 1.25% (wet weight), 62.21% and 1.78% (on the basis of lyophilized dry weight), respectively. Golden pompano skin and bone PSCs contained the typical chain of α and β dimers, and they were preliminarily judged to belong to type I collagen. The skin PSC had similar amino acid composition to bone PSC, which is rich in glycine, alanine, proline, and hydroxyproline. After addressing the pepsin, three helical structure of PSCs were intact, and their natural structures largely remained. The denaturation temperatures of skin and bone PSCs were 37.04°C and 38.23°C, respectively. Solubility results showed that the skin and bone PSCs solubility was the largest at pH = 3. The solubility of skin and bone PSCs was stable at NaCl concentrations lower than 3%. In addition, two PSCs in acid and low salt conditions had good solubility. This study demonstrated that golden pompano skin and bone could be used as good materials to extract PSC, representing an economic benefit and added value.Abbreviations: SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; FTIR: Fourier transform infrared; DSC: Differential scanning calorimetry; XRD: X-ray diffraction

Biochemical study of type I collagen purified from skin of warm sea teleost Mahi mahi (Coryphaena hippurus), with a focus on thermal and physical stability.

Acid- and pepsin-soluble collagen were purified from the skin of mahi mahi (mmASC and mmPSC). The Pro+Hyp content of the latter (185/1,000 residues) was highest among all marine teleost fishes. Fourier transform infrared spectroscopy and Circular Dichroism (CD) analysis showed the typical structure of type I collagen. The ratio of positive over negative peak intensity calculated from the CD spectrum was approximately 1.19 in mmPSC, which is remarkably high, and indicates the stability of the triple helix. The denaturation temperatures (Td ) of mmASC and mmPSC were the highest (29.5 and 28.8°C, respectively) among the marine teleost fishes previously studied. atomic force microscope and scanning electron microscope images showed that even after pretreatment, the fibrils presented their structure and fiber orientation. These results indicate the robustness of both collagens, which can be attributed to the high value of Pro+Hyp stabilizing the helix structure of the collagen molecule. Practical applications While Mahi mahi is highly valuable for its meat, other parts such as skin is not fully utilized in seafood industry. On the contrary, it has been empirically shown that the skin of Mahi mahi has high thermal stability, thus, the skin has been used for leather products in some areas located in the tropical and subtropical zones. In this study, we focused on collagen a major component in skin and investigated the structure and the biochemical characteristics of it. Some results showed that collagen from skin has high physical stability. The collagen from skin of Mahi mahi will be a new fishery resource which could be used as a material for collagen products.

Collagen from Turkey (Meleagris gallopavo) tendon: A promising sustainable biomaterial for pharmaceutical use

Collagen is the major fibrillar component and protein in both human and animal connective tissue. It is applied in medical preparations, e.g. wound dressings and tissue engineering. Meat and poultry production industries result in large amounts of organic waste, rich in collagen. Our aim was to isolate and characterize pepsin soluble collagen from turkey tendon. Structural analysis indicated the presence of α-chains from both collagen type I and III, β-dimers and γ-trimers, consistent with the estimated molecular weight of 477.3 kDa. Circular dichroism spectroscopy confirmed an intact triple helix. The collagen demonstrated excellent thermal stability, with denaturation temperatures (Tmax) at 38.5 °C and 44.5 °C and partial refolding after extensive heating. Biocompatibility was confirmed through cell viability tests. The collagen was investigated for its potential drug carrier ability. Freeze dried collagen scaffolds containing prilocaine hydrochloride and riboflavin were prepared in the presence or absence of photo-crosslinking. Photochemical crosslinking was confirmed by SEM and enhanced mechanical properties were observed. Scaffolds had a significant slower in vitro release of the active ingredient than a reference solution. Altogether, our study suggests collagen from turkey tendon as a promising sustainable biomaterial for pharmaceutical use.

Comparison of Physicochemical Characteristics and Fibril Formation Ability of Collagens Extracted from the Skin of Farmed River Puffer (Takifugu obscurus) and Tiger Puffer (Takifugu rubripes).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of river puffer (ASC-RP and PSC-RP) and tiger puffer (ASC-TP and PSC-TP) were extracted and physicochemically examined. Denaturation temperature (Td) for all the collagens was found to be 25.5–29.5 °C, which was lower than that of calf skin collagen (35.9 °C). Electrophoretic patterns indicated all four samples were type I collagen with molecular form of (α1)2α2. FTIR spectra confirmed the extracted collagens had a triple-helical structure, and that the degree of hydrogen bonding in ASC was higher than PSC. All the extracted collagens could aggregate into fibrils with D-periodicity. The fibril formation rate of ASC-RP and PSC-RP was slightly higher than ASC-TP and PSC-TP. Turbidity analysis revealed an increase in fibril formation rate when adding a low concentration of NaCl (less than 300 mM). The fibril formation ability was suppressed with further increasing of NaCl concentration, as illustrated by a reduction in the turbidity and formation degree. SEM analysis confirmed the well-formed interwoven structure of collagen fibrils after 24 h of incubation. Summarizing the experimental results suggested that the extracted collagens from the skin of river puffer and tiger puffer could be considered a viable substitute to mammalian-derived collagens for further use in biomaterial applications.

Isolation and Characterization of Acid-soluble Collagen and Pepsin-soluble Collagen from the Skin of Hybrid Sturgeon

By using the wastes fish skin of sturgeon processed as a raw material, a macromolecule biomaterial of collagen was extracted. Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were successfully isolated from the skin of hybrid sturgeon with two extraction methods. The yields of ASC and PSC based on the wet weight of skin were 5.73 ± 0.11% and 10.26 ± 0.39%, respectively. The denaturation and melting points of ASC (26.83 °C and 110.49 °C) and PSC (26.54 °C and 102.99 °C) were assessed by Circular dichroism (CD) and Differential scanning calorimetry (DSC). ASC and PSC appeared to be dense sheet-like film linked by random-coiled filaments under scanning electron microscopy (SEM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR) confirmed that both the ASC and PSC were Type I collagen and maintained a complete triple helix structure. These results indicated that both ASC and PSC possessed good biological activity and could be widely used in medical biomaterials and other fields.

Physicochemical, antioxidant properties of giant croaker (Nibea japonica) swim bladders collagen and wound healing evaluation

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were obtained from Nibea japonica swim bladders. The denaturation temperature (Td) of ASC and PSC was approximately 33.8 °C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR) analyses indicated that ASC and PSC contained triple-helical type I collagen when compared to rat tail collagen type I. Moreover, the microstructure of collagen sponges was uniform and porous. In addition, ASC and PSC exhibited antioxidant properties and in vitro scratch assays showed that PSC at various concentrations (0, 12.5, 25, and 50 μg/mL) had significant effects on the scratch closure rate. Furthermore, collagen sponge from Nibea japonica swim bladders exhibited an increased efficacy of wound healing when compared to the control mice. The levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in the collagen sponge treated mice were significantly decreased when compared to the control group. Thus, our results suggested that collagen sponge from Nibea japonica swim bladders has potential wound healing applications.

Characterization of marine derived collagen extracted from the by-products of bigeye tuna (Thunnus obesus)

Fish collagen is gaining immense interest because the use of mammalian collagen is restricted due to disease transmission and religious issues. So, collagen was extracted and characterized from three different parts (skin, scale, and bone) of bigeye tuna using the acid and pepsin extraction methods. The yield of acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) in skin were 13.5 ± 0.6% and 16.7 ± 0.7%, respectively, on a dry basis. The yields of PSC in scale and bone were 4.6 ± 0.3% and 2.6 ± 0.3%, respectively, while ASCs were in negligible amount. All the extracted collagens were type I collagen and a high level of imino acids (227–232/1000 residues) was found in all the extracted collagens. The thermal transition temperature (31.6–33.7 °C) and thermal denaturation temperature (31.1–32.2 °C) were higher than those of many temperate- and cold-water fish collagens. All collagens were highly soluble at acidic pH. The isoelectric points were 6.1, 6.4, 5.4, and 5.5 for skin-ASC, skin-PSC, scale-PSC, and bone-PSC, respectively. Therefore, the high collagen contents, especially in the skin, and higher thermal properties of the extracted collagens suggested that they have great potential for use as an alternate for mammalian collagen.