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.     

Isolation of collagen from tiger pufferfish parts and its solubility in dilute acetic acid

Collagen was extracted from several tissues (muscle, skin, bone, alimentary tract, gill, fin, hepatopancreas, and air bladder) of the tiger pufferfish Takifugu rubripes, and the content and solubility of the collagen extracted from each tissue were examined. Collagen content in ordinary muscle was 0.95 ± 0.07 % of wet tissue, which is lower than that reported for other fish species even though tiger pufferfish meat has a tough texture. The solubility of collagen extracted from the muscle and skin was relatively high, and collagen accounted for 47.2 ± 7.8 and 70.8 ± 8.1 % of wet tissue, respectively. In contrast, the solubility of the collagen extracted from bone was the lowest of all the tissues examined, being only 5.7 ± 0.8 % of total wet tissue. The extent of hydroxylation of proline and lysine residues was also examined. In most tissues, the extent of hydroxylation of the lysine residue in insoluble collagen was higher than that of acid-soluble collagen, indicating that hydroxylysine contributes to the stability of collagen. This is the first report of collagen contents, solubility, and extent of hydroxylation of proline and lysine residues in collagen extracted from different tissues of one organism. It is possible that hydroxylysine-derived collagen cross-links play a critical role in the stability of collagen in dilute acetic acid.     

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).     

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     

The proteolytic digestive activity and growth during ontogeny of Parachromis dovii larvae (Pisces: Cichlidae) using two feeding protocols

The proteolytic digestive activity and growth of Parachromis dovii larvae during the ontogeny were evaluated in a recirculation system using two feeding strategies during a 28-day period. Larvae were reared using two feeding protocols (three replicates each): (A) Artemia nauplii (at satiation), fed from exogenous feeding [8 days after hatching (DAH)] until 15 DAH followed by nauplii substitution by formulated feed (20 % day^?1) until 20 DAH and then formulated feed until 28 DAH; (B) formulated feed (100 % BW daily) from exogenous feeding until 28 DAH. Levels of acid (pepsin type) and alkaline digestive proteases as well as growth and survival of larvae were measured along the feeding period. Survival was high and similar between treatments: 98.9 ± 0.0 for Artemia, 97.3 ± 0.0 % for formulated feed. The specific growth rate for length and weight was higher in larvae fed with Artemia nauplii than in larvae reared with formulated feed: 3.4 ± 0.1 versus 1.8 ± 0.1 % day^?1 for body length (P = 0.009) and 12.2 ± 0.1 versus 6.5 ± 0.3 % day^?1 for body weight (P = 0.002). The acid and alkaline proteolytic activity was detected, in both treatments, from the beginning of the experiment, at 8 DAH. The total enzymatic activity (U larva^?1) for acid and alkaline proteases was higher in larvae reared with Artemia after 12 DAH, whereas the specific enzymatic activity was similar for both enzyme types in the two treatments. The results suggest that P. dovii larvae were capable to digest formulated diets from the beginning of exogenous feeding and that they could be reared with formulated feeds. However, the formulated feed used should be nutritionally improved because of the poor growth obtained in this research.     

Identification and primary structures of eel type I collagen proα1, proα2 and proα3

Many bony fish type I collagens have a characteristic third chain designated as α3(I). However, much less is known about the primary structure and distinction of the proα(I) chains. Their cDNAs were cloned by RT-PCR from the muscle tissue of Japanese eel, Anguilla japonica. Three cDNAs coding for the triple-helical domain of fibrillar collagen were identified as proα1(I), proα2(I) and proα3(I) chains by sequencing selected tryptic peptides isolated from eel type I collagen subunit chains, α1(I), α2(I) and α3(I). Eel proα3(I) had high amino acid sequence identity (81 %) to its proα1(I). The distribution of seven Cys residues in the C-propeptide of proα3(I) was identical to that of proα1(I). There was a third Cys residue at the 1,268th position from the N-terminus in proα1(I), though a supposed Cys residue at the 1,264th position in proα3(I) was replaced by a Ser residue. Similar replacement has been observed in the proα3(I) chains of trout and zebrafish. These combined results suggest that replacement of the Cys residue allows for the identification of fish collagen proα(I) previously not identified as proα3(I).     

Hatching and nutritional quality of Artemia cysts progressively deteriorates as a function of increased exposure to hydration/dehydration cycles

Nauplii hatching from Artemia cysts are crucial in larviculture nutrition. Artemia cysts may be exposed to repeated hydration/dehydration (H/D) cycles pre-harvesting or during processing and storage. To observe the effect of these cycles on cyst quality, Artemia franciscana cysts were exposed to a comprehensive set of various H/D treatments, differing in the number of cycles (1, 2, or 3) and the duration of the freshwater hydration period (2 or 4 h). Cyst quality was assessed using the criteria of immediate relevance for aquaculture use, such as hatching percentage directly after H/D treatment and after ?18 °C storage up to 1 month, longevity of axenically hatched starved nauplii, cyst and naupliar energy content, and (for the most extreme H/D treatment) cyst and naupliar fatty acid and vitamin C content. Repeated H/D cycles resulted in significantly (P < 0.05) decreased cyst hatching, reduced starved naupliar longevity and individual energy content, loss in vitamin C and fatty acid content, and moreover a close correlation between these parameters as a function of progressive H/D treatments. This is of immediate relevance for aquaculture nutrition, as commercial Artemia cysts may have gone through an unknown sequence of H/D cycles in nature or in the processing line, which affects the nutritional quality of the nauplii used in larviculture operations.     

Exploration of the antibacterial proteins in pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> induced by bacterial inoculation

The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 10¹² colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.     

Effect of replacement of fish oil with camelina (Camelina sativa) oil on growth, lipid class and fatty acid composition of farmed juvenile Atlantic cod (Gadus morhua)

Camelina (Camelina sativa) oil was tested as a replacement for fish oil in diets for farmed Atlantic cod (Gadus morhua). Camelina differs from other plant oilseeds previously used in aquaculture with high lipid (40 %), α-linolenic acid (40 %), antioxidants and low proportions of saturated fats. Dietary treatments were fed to cod (19 g fish^?1 initial weight) for 9 weeks and included a fish oil control (FO), 40 % (CO40) and 80 % (CO80) replacement of fish oil with camelina oil. There was no effect of replacing fish oil with camelina oil included at levels up to 80 % on the growth performance. Cod fed CO80 stored more lipid in the liver (p < 0.01), including more neutral lipid (p < 0.05) and triacylglycerol (p < 0.05). Cod fed CO80 decreased in total polyunsaturated fatty acids (PUFAs) in muscle compared to CO40 and FO (p < 0.05), increased in monounsaturated fatty acids (p < 0.01), decreased in total ω3 fatty acids (FO > CO40 > CO80; p < 0.01) and increased in total ω6 fatty acids (FO < CO40 < CO80; p < 0.01). In the liver, long-chain (LC) PUFA such as 20:4ω6, 20:5ω3, 22:5ω3 and 22:6ω3 decreased when fish oil was removed from the diet (p < 0.05), and increased in 18-carbon fatty acids (p < 0.01). Camelina oil can reduce the amount of fish oil needed to meet lipid requirements, although replacing 80 % of fish oil reduced LC PUFAs in both tissues. A comparison of BF₃ and H₂SO₄ as catalysts to transmethylate cod liver and muscle lipids revealed small but significant differences in some fatty acid proportions.     

Regulation of adipocytes lipolysis by n-3 HUFA in grass carp (Ctenopharyngodon idellus) in vitro and in vivo

N-3 highly unsaturated fatty acids (n-3 HUFA) have been shown to inhibit body fat accumulation in animals. To clarify the mechanism of this fat-lowering effect of n-3 HUFA in grass carp (Ctenopharyngodon idellus), two experiments were conducted. In experiment 1, isolated grass carp mature adipocytes were incubated with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at different concentrations for 6 h. The release of glycerol to the medium was detected, and the expression of the lipolysis-related genes was analyzed. In experiment 2, a 95-day feeding trial was conducted with two diets formulated with either lard oil (as control) or fish oil (supplying n-3 HUFA as treatment) as the main lipid source. The glycerol and free fatty acid (FFA) released from the isolated adipocytes of both groups were detected after the feeding period. The expression of select lipolysis-related genes in adipose tissue was also analyzed. The results from experiment 1 showed that the release of glycerol was significantly increased by DHA and EPA (P < 0.05). Moreover, the expression of lipolysis-related genes, such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), tumor necrosis factor α (TNFα) and leptin, was also significantly elevated in the treatment group (P < 0.05). Experiment 2 demonstrated that glycerol and FFA release from the isolated adipocytes were significantly higher in the treatment group compared to the control group (P < 0.05). The expression level of ATGL, HSL, TNFα and leptin in the treatment group was significantly higher than in the control group (P < 0.05). The present results provide novel evidence that n-3 HUFAs could regulate grass carp adipocyte lipolysis in vitro or in vivo, and the effect might be in part associated with their influence on the expression of lipolysis-related genes and lipolysis-related adipokines genes.     

Characterization and Functional Properties of Gelatin Extracted from Chinese Giant Salamander (Andrias Davidianus) Skin

The characteristics and functional properties of gelatin obtained from Chinese giant salamander (Andrias davidianus) skin were investigated. After alkaline and acetic acid pre-treatment, yield of the gelatin by hot water extraction (60°, 8 h) was 17.63 g/100 g (wet basis), and the gelatin contained 89.46 g/100 g protein, 0.3 g/100 g fat, and 0.41 g/100 g ash (dry basis). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results of the gelatin showed clear band pattern for collagen subunits,β- and α-chains. The gelatin contained 18.85% imino acids, 28.62% hydrophobic amino acids, and 19.68% essential amino acids, which is slightly lower than porcine skin gelatin (PSG). The gelatin exhibited thermo-reversible properties, with gel strength of 192.50 ± 11.47 g, rupture distance of 10.17 ± 0.20 mm, gelling temperature of 4.98°, and melting temperature of 18.04°, Emulsifying activity index (EAI) of the gelatin diminished with increasing concentrations from 10 to 40 mg/mL (p < .05), whereas the emulsifying stability index (ESI) improved significantly (p < .05). Higher concentrations (30 and 40 mg/mL) could significantly enhance the gelatin’s foam expansion (FE) and foam stability (FS) (p < .05). Water absorption ability and oil absorption ability of the gelatin were 7.1 g water/g gelatin and 9.8 g oil/g gelatin, respectively. These results imply that the gelatin with good interfacial properties could potentially be used as a novel ingredient in food emulsion systems.

Abbreviations: CGSS, Chinese giant salamander skin; CSG, Chinese giant salamander skin gelatin; PSG, porcine skin gelatin; EAI, emulsion activity index; ESI, emulsion stability index; FE, foam expansion; FS, foam stability; WAC, water absorption capacity; OAC, oil absorption capacity.     

Enrichment of the nematode Panagrolaimus sp., a potential live food for marine aquaculture,with essential n-3 fatty acids

The nematode Panagrolaimus sp. (strain NFS-24-5) may have potential as a live food organism for larvae of several marine species. It can be mass-produced in liquid culture and is desiccation tolerant enabling long-term storage and transportation. Nematodes lack the essential fatty acid docosahexaenoic acid (DHA), so this investigation examined an enrichment procedure by incubating nematodes in S.presso^® (INVE Aquaculture, Belgium), a commercial enrichment product. Mass-produced nematodes from liquid cultures on yeast cells were cleaned and then exposed to concentrations of 0.1–3 % S.presso^® for 24 h at 200,000 nematodes ml^?1. Nematode viability was >90 % after the treatment. Following enrichment percentage, total lipid ranged from 23.6 to 33.3 % of nematode dry matter, and nematodes incubated in 3 % S.presso^® had a significantly higher lipid percentage than untreated controls. Enrichment was successful in increasing the percentage of DHA, and a maximum value of 5.8 % of total fatty acids was achieved. The results of enrichment of Panagrolaimus sp. (strain NFS-24-5) following treatment with 3 % S.presso^® should promote the carrying out of feeding trials to test the efficacy of the nematodes as a live food for larval marine fish and crustaceans.     

Effects of dietary β-1,3/1,6-glucan on the antioxidant and digestive enzyme activities of Pacific red snapper (Lutjanus peru) after exposure to lipopolysaccharides

The effect of β-1,3/1,6-glucan, derived from yeast, on growth, antioxidant, and digestive enzyme performance of Pacific red snapper Lutjanus peru before and after exposure to lipopolysaccharides (LPS) was investigated. The β-1,3/1,6-glucan was added to the basal diet at two concentrations (0.1 and 0.2 %). The treatment lasted 6 weeks, with sampling at regular intervals (0, 2, 4, and 6 weeks). At the end of this period, the remaining fish from either control or β-glucan-fed fish were injected intraperitoneally with LPS (3 mg kg^?1) or with sterile physiological saline solution (SS) and then sampled at 0, 24, and 72 h. The results showed a significant increase (P < 0.05) in growth performance after 6 weeks of feeding with β-glucan. Superoxide dismutase (SOD) activity in liver was significantly higher in diets containing 0.1 % β-glucan in weeks 4 and 6, compared to the control group. β-Glucan supplementation at 0.1 and 0.2 % significantly increased aminopeptidase, trypsin, and chymotrypsin activity. At 72 h after injection of LPS, we observed a significant increase in catalase activity in liver from fish fed diets supplemented with 0.1 and 0.2 % β-glucan; SOD activity increased in fish fed with 0.1 % β-glucan in relation to those injected with SS. Feed supplemented with β-1,3/1,6-glucan increased growth, antioxidant activity, and digestive enzyme activity in Pacific red snapper.     

Characterization of Blue Whiting Skin Gelatines Extracted After Pretreatment with Different Organic Acids

Gelatines were extracted from blue whiting (Micromesistius poutassou) skins after pretreatment with different organic acids (acetic, citric, lactic, malic, and tartaric acids). The effect of the pretreatment on the chemical composition and the rheological properties of extracted gelatines were analyzed. It was observed that acetic acid pretreatment resulted in significantly (p < 0.05) higher extraction yield compared to the rest of the gelatines. All gelatines, regardless of the organic acid used in the pretreatment, had similar chemical composition (p > 0.05). The amino acid analysis showed that acetic acid pretreated skins resulted in gelatines with higher imino acid levels with respect to the other pretreatments. Acetic and tartaric acid derived gelatine gels showed highly interconnected protein networks and better viscoelastic properties, in terms of storage modulus, compared to the other pretreatments.