Characteristics of Collagen from Rohu (Labeo rohita) Skin

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were isolated from rohu skin with the yield of 64.2 and 6.8% (dry weight basis), respectively. Both collagens had glycine as the major amino acid with imino acid content of 196–202 residues/1,000 residues and were characterized as type I collagen with molecular composition of (α1)₂α2-heterotrimer. Fourier transform infrared spectra of both collagens were similar, with no shift in wavenumber of all amide bands. The T_max value of ASC and PSC was 36.40 and 35.48°C, respectively. The zero surface net charge of ASC and PSC was found at pH 5.9 and 5.3, respectively.     

Extraction and Characterization of Pepsin Soluble Collagen from the Body Wall of Sea Cucumber Acaudina leucoprocta

Sea cucumber Acaudina leucoprocta is a potential alternative collagen source. However, the high levels of heavy metals contained in the body wall restricts its utilization. In this work, an efficient method was established to remove the heavy metals accumulated in the body wall of A.leucoprocta by demineralizing with 0.2 M ethylenediaminetetraacetic acid (EDTA). The resulting body wall of A.leucoprocta was then used for extracting pepsin-soluble collagen (PSC) with pepsin proteolysis. The PSC from the body wall of A.leucoprocta was obtained with a yield of 43.99 ± 0.65% (dry weight) and high purity. Maximum and minimum solubility for the isolated PSC in 0.5 M acetic acid was observed at pH 2.66 and 4.43, respectively. The solubility was remarkably decreased in the presence of NaCl. The denaturation temperature of PSC rehydrated in 0.5 M acetic acid was measured as 25.4°C. The PSC was characterized as type I collagen, which consists of three α₁ chains without α₂ chain. Interestingly, α₁ chain in PSC showed two isoforms with the pI values of 4.02 and 4.29. The heavy metals existing in PSC were all below the contaminant limit of edible gelatin. The PSC isolated from the body wall could be an alternative to mammalian collagens.     

Physicochemical Properties and Angiotensin I Converting Enzyme Inhibitory Peptides of Freshwater Fish Skin Collagens

ABSTRACT

The objectives of this study were to characterize physicochemical properties of two collagens, tilapia skin (TS) and hybrid catfish skin (HS). Angiotensin-converting enzyme (ACE) inhibitory peptides from extracted TS and HS collagen using pepsin were also determined. HS collagen had a higher amount of imino acid than TS collagen, while TS contained higher amounts of tyrosine (Tyr) and lysine (Lys). Fourier-transform infrared spectra of both collagens showed predominant helix structure. The HS collagen hydrolysate prepared by pepsin was fractionated using sequential ultrafiltration membranes, and the fraction with molecular weight (MW) <5 kDa showed the highest ACE inhibitory activity (p < .05). After cation exchange and two steps size exclusion chromatography, peptides showed ACE inhibitory activity of 72.06%. This study revealed that ACE inhibitory peptides derived from HS collagen could be developed as a functional food with potential antihypertensive properties.     

Isolation and characterization of collagens from the skin of giant grouper (Epinephelus lanceolatus)

ABSTRACT

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of giant groupers (Epinephelus lanceolatus) with yields of 39.51 and 19.12%, respectively. ASC and PSC consisted of two different α chains (α1 and α2) and were characterized to be type I collagen with no disulfide bond. The imino acid contents of the ASC and PSC from giant grouper skin were 189 and 181 per 1,000 residues, respectively. The maximum endothermic temperatures (T_max) of ASC and PSC measured by differential scanning calorimetry (DSC) were 31.71 and 31.33°C, respectively. The denaturation temperatures of ASC and PSC measured by viscometry were 29.84 and 29.05°C, respectively. The maximum solubility in 0.5 M acetic acid was observed at pH 5 and pH 6 for ASC and PSC, respectively. A sharp decrease in solubility was observed for both ASC and PSC in the presence of NaCl above 3% (w/v).     

Physicochemical Properties of Pepsin-Solubilized Collagen Extracted from Nile Tilapia (Oreochromis niloticus) Scale

ABSTRACT

The aim was to investigate the physicochemical properties of pepsin-solubilized collagen (PSC) extracted from fish scales of Nile tilapia. The results indicated that Nile tilapia scales are rich in collagen. Therefore, the scales are a promising cost-effective collagen source. The conversion of hydroxyproline to collagen was 12.9. Based on the patterns of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), PSC comprised at least two different α chains, α1 and α2, and was classified to be type I with no disulfide. Fourier transform infrared spectroscopy spectrum showed that the bands of amide A, I, and II of PSC were found at 3,301, 1,631, and 1,239 cm^?1 wavelength. The content of imino acids was 187 residues per 1,000 total amino acid residues. Maximum solubility was observed at pH 4, and minimum was at pH 7. Almost no change in solubility was observed in the presence of NaCl up to 2% (w/v), and the decrease was more pronounced with increasing NaCl concentration. The temperature of denaturation was 35.4°C. Results show that PSC has physicochemical properties that can be applied in the cosmetics, biomedical, pharmaceutical, and food industries.     

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

Pepsin-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     

Molecular species of collagen in muscular and vertebral parts of white sturgeon Acipenser transmontanus

The molecular species of collagen in the muscular and vertebral parts of white sturgeon Acipenser transmontanus were examined by biochemical techniques. Pepsin-solubilized collagens were prepared from these parts, and fractionated into major and minor collagen fractions by differential salt precipitation at acidic or neutral pH. Collagens contained in these fractions showed several features characteristic of fish type I and V collagens, respectively, in SDS-PAGE patterns and the amino acid compositions of both parts. These results suggest that at least two molecular species, corresponding to type I and V collagens, are distributed in the muscular and vertebral parts of white sturgeon.     

Hot-water solubility of mantle collagens in several cephalopod molluscs

The mantle muscles of five cephalopod species, Todarodes pacificus, Photololigo edulis, Sepioteuthis lessoniana, Sepia esculenta, and Sepia longipes, were extracted with 0.1 M NaOH to prepare crude collagen fiber, called ‘residue after alkali extraction’ (RS-AL). Solubility of the collagens in water was examined in the temperature range 20–90°C. The collagens showed a similar tendency in solubility, which gradually increased depending on the treating temperature, and the values at 40–90°C were constantly less than 47.0% for all the species examined. In addition, the collagens were estimated to denature in the approximate temperature range of 37.5–42.5°C. These results suggest that the collagens in RS-AL from these species may have relatively high resistance to hot-water extraction even after their denaturation.     

Application of ultrasonic treatment to extraction of collagen from the skins of sea bass Lateolabrax japonicus

This study investigates an efficient method to extract collagen from the skins of sea bass. The ultrasonic treatment (20 kHz with amplitude of 20–80 %) was applied to fish skin for 24 h after the addition of 0.01, 0.05, and 0.1 M acetic acid (1:200 sample/acetic acid, w/v). As a result, the rate (Ki) of the collagen yield increased depending on the amount of acid added, the duration of ultrasonic treatment and the amplitude of the ultrasonic waves. The subunit compositions of the extracted components were analyzed by SDS-PAGE, and the main components were determined as collagen, including the α1 (α3), α2, β, and γ chains. And in addition to collagen, some unknown components were also observed after a longer period of ultrasonic treatment. Therefore, we had to optimize the efficient extraction conditions for pure collagen while minimizing the creation of unknown components. The most effective extraction condition for collagen by ultrasonic treatment was 80 % amplitude with 0.1 M acetic acid for 3 h of treatment. It was found that the component extracted by the ultrasonic treatment was indeed collagen, since there were no changes in the main components of collagen after pepsin treatment.     

Characterization of protease inhibitors of seminal plasma of cyprinids

Anti-proteinase activity was demonstrated in the seminal plasma of cyprinid fish species (bream, chub, ide, dace, asp, goldfish, roach, common carp) using electrophoretic techniques combined with a detection method based on inhibition of bovine trypsin. We found species-specific protease inhibitors in the seminal plasma of cyprinids. At least three bands of protease inhibitors with different migration rates could be identified by native PAGE. Higher variability was characterized for bands with slower migration rates. Visualization of inhibitors after SDS-PAGE under non-reducing conditions allowed estimation of their molecular weights. Apparent molecular weights were within the range of 51–59 and 47–54 kDa for the bands with slower and moderate migration rates, respectively. The molecular weight of fast migration bands for roach and common carp were estimated to 23 and 30 kDa, respectively. Inhibitors of common carp seminal plasma differed in their affinity toward serine proteases. Three inhibitors in common carp seminal plasma could be visualized using cod and bovine trypsin, but only two inhibitors (of high molecular weight) were recognized with chymotrypsin. There were differences in anti-proteinase activity and seminal plasma protein concentration in relation to the origin of common carp seminal plasma (breeding lines) and time of milt collection (spawning vs. post-spawning season).     

Characteristics and Select Functional Properties of Collagen from Golden Pompano (Trachinotus ovatus) Skins

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) from golden pompano skins were extracted and characterized. The molecular weight of ASC was about 130 kDa for α₁ and 115 kDa for α₂, which were slightly higher than those of PSC. Similar amino acid composition and Fourier transform infrared (FTIR) spectra were observed in both collagens, but slight differences were found in the peptide maps of collagen digested by V8 protease and trypsin. The denaturation temperatures (Tds) of ASC and PSC calculated from the reduced viscosity were 31.8 and 30.0°C, while the transition temperature (T_m) of ASC and PSC analyzed by DSC were 33.0 and 32.0°C, respectively. ASC has a lag phase, a growth phase, and a plateau phase in the turbidity–time curve, while PSC does not have similar phenomenon. It was found that the fibril gel of ASC could be formed at 25°C, leading to improved thermal stability.     

Effects of ultrasonic treatment on collagen extraction from skins of the sea bass Lateolabrax japonicus

In this study we investigated the effects of ultrasonic wave treatment on the extraction yield of acid-soluble collagen from sea bass skins. Two extraction methods were compared: a 24 h acid treatment using 0.5 M acetic acid (1:200 sample/acid, w/v) and an extraction using ultrasonic treatment after the addition of a 0.5 M acetic acid solution. The results indicated that the extraction yield of collagen increased with the ultrasonic treatment, with the extraction rate increasing rapidly at higher amplitudes of ultrasonic treatment. The subunit compositions of the collagen extracted by ultrasonic treatment were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which revealed that the α1(α3), α2, and β chains of collagen were present early in the ultrasonic treatment. An unknown component, believed to be a product of collagen degradation induced by the ultrasonic treatment, was detected only after a longer treatment time. The component extracted by the ultrasonic treatment was determined to be collagen based on the finding that there were no changes in the main components of collagen, specifically, the α1(α3), α2, and β chains, following pepsin treatment.     

Enzymatic solubilization of collagen in the skin of diamond squid Thysanoteuthis rhombus: application of a fungal acid protease

Enzymatic solubilization of collagen from the skin tissue of diamond squid Thysanoteuthis rhombus, an underutilized resource in Japan, was attempted using an acid protease from the fungus Rhizopus niveus. This novel approach was compared with the conventional method using porcine pepsin. Both proteases were able to solubilize most of the skin collagen (>90 % of the total collagen) by performing the treatment in 0.5 M acetic acid at 4 °C for 72 h and at an enzyme/substrate ratio (w/w) of 1/10. The SDS-PAGE patterns of the solubilized collagen preparations were quite similar to each other, and two types of collagen (major and minor collagens) were purified from each preparation by cation-exchange column chromatography. These collagen types from the porcine pepsin-solubilized collagen showed similar features to those from the Rhizopus acid protease-solubilized collagen. These results suggest that the Rhizopus acid protease, a protease of non-animal origin, is applicable for solubilizing collagen in the skin of diamond squid.     

Protein Hydrolysates and Ultrafiltration Fractions Obtained from Krill (Euphausia superba): Nutritional,Functional, Antioxidant,and ACE-Inhibitory Characterization

ABSTRACT

Krill (Euphausia superba) was hydrolyzed by proteolytic enzymes in order to produce multifunctional bioactive peptides, and their functional properties were evaluated. Krill protein hydrolysate (KPH) by pepsin with 4-h hydrolysis showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities. The solubility and foaming properties of KPH were higher than those of the unhydrolyzed krill protein at a wide range of pHs. KPH was further fractionated based on molecular weight. The 1- to 3-kDa peptide fraction exhibited the highest DPPH scavenging activity (IC₅₀ value of 0.5 mg/mL), oxygen radical absorbance capacity (497.39 ± 4.31 µM TE/mg fraction), 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid cation radical scavenging activity (48.41 ± 0.23 µM TE/mg fraction), and reducing power (110.40 ± 2.07 µM TE/mg fraction). However, the < 1-kDa peptide fraction exhibited a higher ACE inhibitory activity than that of other fractions. The 1- to 3- and < 1-kDa peptide fractions are rich in aromatic and hydrophobic amino acids, respectively.     

Characterization of Collagen from Different Discarded Fish Species of the West Coast of the Iberian Peninsula

ABSTRACT

Skin collagen of six discarded fish species was analyzed and compared. Acid soluble collagen (ASC) was extracted; a characteristic sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile for type I collagen was obtained, except for Chimaera mostrosa. Contents of collagen calculated from HPro (31.85% average) were higher than those determined from ASC extracts (17.75% average), with Galeus spp. being the species with the higher percentage. Amino acid analysis revealed the typical composition of collagen, with very few differences among species. Specific profiles were obtained after protease digestion. Denaturation temperature of ASC correlated well with imino and hydroxyproline contents.

Results demonstrate the feasibility of using the obtained collagens in different industrial applications.     

Characterization of digestive enzymes protease and alpha‐amylase activities in the thick‐lipped grey mullet (Chelon labrosus,Risso 1827)

Activities of pepsin, trypsin, chymotrypsin, alpha‐amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick‐lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS‐PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha‐amylase and no lipase activity recorded in pre‐adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.     

Pyridinoline concentrations in muscular and skin collagen of fish and relationship between collagen solubility and pyridinoline concentration in fish muscular collagen

Pyridinoline (Pyr), one of the mature crosslinks of collagen, was determined in muscular collagen of three species of fish. The amounts of muscular Pyr in red sea bream, yellowtail, and tiger puffer were 3.4, 8.8, and 50.3 mmol/mol collagen, respectively, indicating that the Pyr concentration in muscular collagen differs greatly among fish species. The Pyr concentration in tiger puffer muscular collagen was the greatest, but it was only one-fourth that in rabbit muscle. As in mammalian skin collagen, Pyr was not detected in skin collagen of red sea bream and yellowtail. However, tiger puffer skin contained Pyr (3.75 mmol/mol collagen). The presence of Pyr would have a relationship to some features of tiger puffer skin, such as mechanical strength and thickness. Pyr concentrations in acid-soluble collagen (ASC), pepsin-solubilized collagen (PSC), and insoluble collagen (ISC) in muscles of three species of fish were determined. Pyr was found in ISC > PSC > ASC, from the highest to the lowest concentration, and the concentration in ISC was 45–200 times that in ASC. Therefore, Pyr crosslinks that are formed between collagen molecules would have a close relationship to collagen solubility.     

Presence of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) and Hysterothylacium aduncum (Rudolphi, 1802) (Nematoda; Anisakidae) in runts of farmed Atlantic salmon,Salmo salar L

One hundred farmed Atlantic salmon, Salmo salar L., were examined for the presence of nematodes by digestion of tissue in HCl–pepsin solution. All fish were sampled from one cage in a fish farm on the Norwegian south‐west coast. Fifty harvest quality salmon, that is, salmon for human consumption (mean 5.4 kg, variation 3.0–7.6 kg), were sampled at the processing line while 50 salmon runts (mean 1.1 kg, variation 0.4–1.8 kg), discarded due to poor performance, were sampled from the discard bin after the grading station. Runts are individual fish with clear signs of poor performance over time and abnormal appearance and are thus not processed for human consumption. No nematodes were found in the musculature or viscera of the 50 harvest quality salmon. In total, 75 nematodes were found in 10 (20%) of the runts; 53 nematodes in the viscera and 22 in the musculature. Nematodes in the musculature were identified as Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878), while nematodes in the viscera were identified as A. simplex and Hysterothylacium aduncum (Rudolphi, 1802).     

Antioxidant and protective effects of a peptide (VTAL) derived from simulated gastrointestinal digestion of protein hydrolysates of Magallana gigas against acetaminophen-induced HepG2 cells

Oxidative stress is an automatic mechanism responsible for the commencement and continuance of liver injury. In this study, an antioxidative peptide Val-Thr-Ala-Leu (VTAL) was purified from simulated gastrointestinal digestion of protein hydrolysates of the triploid oyster Magallana gigas. Significant antioxidant activity was identified, as well as a protective effect against acetaminophen (APAP)-induced human liver cancer (HepG2) cells. The results suggested that the antioxidant activity improved in a dose-dependent manner. The highest cell viability (88.105?±?3.62%) was observed in 15 mM APAP-induced cells when treated with 25 μg/mL M. gigas peptide [M.g (pep)]. The peptide sequences include hydrophobic amino acids, which could be responsible for its chemoprotective and antioxidant activities. Treatment with M.g (pep) significantly promoted the proliferation of HepG2 cells, thus protecting them against APAP and imbuing them with significant antioxidant capacity. M.g (pep) could be beneficial for treating drug-induced oxidative stress and liver damage. Additionally, M.g (pep) could serve as an alternative to synthetic antioxidant drugs.

     

Assessment of enzymatic efficiency on protein digestion in the tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The present study develops an experimental procedure aimed to estimate the efficiency of protein digestion in fish by measuring both gut transit rate and total amount of the main intestinal proteases (trypsin and chymotrypsin). The selected species was the Nile tilapia (Oreochromis niloticus). Total time for digestion, calculated through the estimation of gut transit rate using differently colored feeds, was 7.15 h. Mean production of trypsin and chymotrypsin was 15.94 and 24.11 mU in the proximal intestine and much lower (2,39, 4.90 mU) in the distal intestine. The enzyme efficiency, calculated from the average enzyme activity and time of residence of the digesta in each intestinal section, points to the major role of proximal intestine in protein digestion for this species. Results are discussed in relation to the main features characterizing digestion in stomachless fish.