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.     

Isolation and Characterization of Pepsin-Soluble Collagen from the Skin of Peru Squid (Dosidicus gigas)

Pepsin-soluble collagen (PSC) was isolated from Peru squid (Dosidicus gigas) skin and physicochemical properties of the PSC were determined. The PSC exhibited a maximum absorbance at 220 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested the collagen containing α1 and α2 chain was classified as type I collagen. Amino acid composition indicated that the collagen had lower amino acid content than that of mammalian collagen. Denaturation temperature (T_d) of the PSC was 26.8°C. The PSC had relatively high solubility in alkaline condition or NaCl concentrations below 2%. Fourier transform infrared spectroscopy investigations showed the existence of helical arrangements of collagen. The lyophilized collagen had a uniform and regular network structure. These results suggested that Peru squid skin was a potential source of collagen for further research and application.     

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.     

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.     

Partial characterization of digestive proteases in tropical gar Atractosteus tropicus juveniles

Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6 ± 12.7 g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55 °C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71 rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4 kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.     

Amino acids composition and protein quality evaluation of marine species and meals for feed formulations in cephalopods

The amino acid composition and protein levels of three species of cephalopods (Octopus vulgaris, Loligo gahi and Todarodes sagittatus), the natural diets of common octopus (O. vulgaris) and different kinds of meals were determined in order to optimise the content of these nutrients in artificial feeds. Arginine, leucine and lysine were the most abundant essential amino acids in cephalopods, while glutamate and aspartate represented the main non-essential amino acids. Arginine and leucine were the limiting amino acid in most samples, with maximum Chemical Score values for mussel (79–98 %), squid (84 %) and crustaceans (65–91 %); medium for fish (41–70 %); and minimum for meals (29–64 %). Mussel, squid, crustaceans and fish showed a high essential amino acid index according to Oser (OI: 88–99 %) suggesting a suitable amino acid balance. The protein from animal meals (fish and krill) covered all the essential amino acids except arginine and lysine in fish meal. The vegetable meal presented the worst amino acid balance (OI: 74–89 %) with several deficiencies in essential amino acids, including arginine, threonine, lysine and methionine. Supplementation with arginine or leucine and protein complementation of crustaceans and bivalves with fish or animal meal are proposed as alternatives for improving the performance of protein in feed for cephalopods.     

Effects of plasticizer type and concentration on the physicochemical properties of edible film from squid <Emphasis Type="Italic">Todarodes pacificus</Emphasis> mantle muscle

Physical properties of edible film from squid Todarodes pacificus mantle muscle plasticized with different plasticizers (glycerol, sorbitol, glucose, and fructose) at various concentrations (10%, 20%, and 30% w/w of protein) were determined. The results showed that tensile strength (TS) significantly decreased (P < 0.05) while elongation at break significantly increased (P < 0.05) upon plasticizer addition. Water vapor permeability (WVP) significantly increased (P < 0.05) upon addition of glycerol or sorbitol but significantly decreased (P < 0.05) upon addition of glucose or fructose. Among the plasticizers used in this study, glycerol showed the greatest ability to decrease film TS. However, glycerol-plasticized film was less transparent than those with other plasticizers. Addition of glucose or fructose seemed to decrease the WVP of the film via a Maillard reaction. However, change in film color also occurred to a greater degree than for glycerol- or sorbitol-plasticized film.     

ATP depletion inhibits retraction of chromatophores induced by GABA in cephalopod Todarodes pacificus

Color changes in cephalopods are generated by the expansion or retraction of chromatophores located under the dermis. The behavior of the chromatophores is regulated by neurotransmitters; l-glutamate (l-Glu) is an excitatory transmitter that causes the chromatophores to expand. To date, serotonin [5-hydroxytryptamine (5-HT)] is the only neurotransmitter known to stimulate retraction of chromatophores. We found that the chromatophores in the Japanese squid Todarodes pacificus were regulated by γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, and that GABA caused expanded chromatophores to retract. We also found that adenosine 5′-triphosphate (ATP) concentrations in skin samples remained stable at their initial values for more than 24 h after the death of each squid; therefore, the chromatophores could respond to both l-Glu and GABA during that period. Furthermore, we attempted to reduce the levels of ATP by storing skin sample in sodium azide solution. The chromatophores in sodium azide-treated skin samples were induced to expand by l-Glu, but these expanded chromatophores could not be induced to retract by GABA. Based on these observations, we conclude that ATP is essential for retraction, but not expansion, of chromatophores.     

Nutritive value of copra cake meal fermented with Rhizopus spp. and its use as a protein source in practical diets for rabbitfish (Siganus javus)

The aims of this study were to evaluate fermentation of copra cake meal with Rhizopus spp. to enhance its nutritive value and to determine the optimum supplementation levels of the fermented meal on the growth performance and feed utilization of the rabbitfish, Siganus javus. Copra cake meal was fermented with Rhizopus spp. and included in four iso-nitrogenous diets at 0, 150, 300, and 450 g/kg. Rabbitfish (88.3 ± 1.7 g initial body mass) were fed with the test diets at 2.5%–3.0% of biomass/d for 12 weeks. After fermentation, the crude protein content of copra cake increased significantly (P < 0.05) from 218 ± 16 g/kg (mean ± SD) to 293 ± 4 g/kg, while lipid decreased from around 127 ± 4 g/kg to 60 ± 11 g/kg. Total amino acid content of copra cake meal increased after fermentation from 176 ± 12 g/kg to 207 ± 18 g/kg. Based on the third-order (cubic) polynomial regression, inclusion of fermented copra cake meal (FCCM) in test diets had significant (P < 0.05) effects on all parameters observed except for feed intake and survival rate. Fish fed the FCCM0 and FCCM150 diets had a similar protein retention (PR) but significantly differed (P < 0.05) from the diets containing higher FCCM inclusion rates. The breakpoint of two regressions fitted to specific growth rate (SGR) determined that the optimum inclusion rate is 137 g FCCM /kg diet, and at this level, protein from soybean meal can be included at approximately 315 g/kg diet for grow-out of rabbitfish, S. javus, in floating net cages.     

Effects of Freshness and Heating Temperature on the Discoloration of Squid Meat Products

ABSTRACT

Squid processed products such as dried-seasoned squid turn brown during storage, causing a change in quality. The main cause of this browning is melanoidin produced by the Maillard reaction. In this study, four types of squid were processed at different heating temperatures between 50 and 90°C and examined for suppression of the production of D-ribose, the main component of melanoidin. Furthermore, the color change of the squid meat with each heat treatment during storage of 32 days at 30°C was investigated. Bigfin reef squid and cuttlefish with a relatively high freshness decreased D-ribose generation as the heating temperature increased, and the browning during storage also decreased accordingly. In contrast, Humboldt squid with a low freshness did not generate new D-ribose at any heating temperature during storage. Nevertheless, the browning was reduced as the heating temperature increased. It is thought that the Maillard reaction was also reduced by the heat denaturation of muscular proteins. These results suggest that browning due to the Maillard reaction of squid meat is greatly affected by initial freshness and heating temperature.     

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.     

Characteristics of divalent minerals extracted from liver of Japanese common squid Todarodes pacificus under various experimental conditions

In relation to the problem of how to utilize the waste from squid processing, we determined the proximate and mineral compositions of liver of the Japanese common squid Todarodes pacificus, and characterized the behavior of divalent minerals, focusing in particular on cadmium removal. The squid liver contained fat (44.0 g/100 g dry matter), protein (13.5 g/100 g dry matter), and ash (2.11 g/100 g dry matter). It also contained the macrominerals sodium, potassium, magnesium, and calcium, as well as the trace minerals iron, zinc, cadmium, and copper. Low- and high-pH treatments (pH 2 and 12) removed the cadmium entirely, but the trace minerals zinc, iron, and copper, as well as soluble protein, which are all important for proper functioning of the human organism, were also removed in large quantities. High percentages of soluble minerals and soluble protein were found in the low molecular weight (MW < 10,000) fraction. The solubility of zinc and copper were strongly related to the solubility of cadmium, but there was no relationship between the solubilities of magnesium and cadmium. On the other hand, while treatment with either 0.9 % NaCl or 100 mM phosphate buffer (pH 7.4) also removed the cadmium completely, it left some useful compounds (trace minerals and soluble protein) in the solid part, so these treatments may represent appropriate methods for removing cadmium from squid processing waste.     

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     

鱿鱼加工副产物综合利用研究进展

综述了近年来国内外利用鱿鱼皮、内脏、软骨、墨汁、鱼眼、精巢等副产物为原料开发胶原蛋白、鱼油、核酸、酶、软骨素、透明质酸、鱼精蛋白等一系列生化制品的研发状况,展望了几种鱿鱼副产物的应用前景和发展趋势.     

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).     

The improvement of growth,digestive enzyme activity and disease resistance of white shrimp by the dietary citric acid

A 45-day feeding trial was conducted to evaluate the effects of dietary citric acid on growth, digestive enzyme and disease resistance of white shrimp, Litopenaeus vannamei. Shrimp with initial body weight of 5.57 ± 0.21 g were fed with basal diet supplemented with 0.0 g kg^?1 (control), 1.0, 2.0, 3.0, 4.0 and 5.0 g kg^?1 citric acid. Results showed that weight gain was increased by 15.9 % and feed conversion ratio was decreased by 0.17 by 2.0 g kg^?1 dietary citric acid compared with control group (P < 0.05). Intestinal protease activity of shrimp fed 2.0 g kg^?1 citric acid was significantly higher (P < 0.05) than that of control group. No significant difference was found in intestinal amylase activity among treatments (P > 0.05). The activities of serum phenoloxidase, superoxide dismutase and lysozyme in 2.0 and 3.0 g kg^?1 citric acid group were significantly higher, and accumulative mortalities of the two groups on the fourth day after injection of Vibrio alginolyticus were significantly lower than those of control group (P < 0.05). Results above demonstrated that dietary citric acid could improve growth performance, immunity and resistance against V. alginolyticus. The supplementation level of citric acid in diet was suggested to be 2.0–3.0 g kg^?1 for white shrimp.     

A comparative study on innate immunity parameters in the epidermal mucus of Indian major carps

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish from relevant infections. The present study is aimed at understanding and comparing the mucosal immunity of Indian major carp, that is, Labeo rohita, Catla catla and Cirrhinus mrigala, by evaluating different immune parameters such as protein content, lysozyme, myeloperoxidase, proteases and alkaline phosphatase activity in the skin mucus. The protein content of mucus sample of these species was compared, and the highest protein content was found in C. catla among the three Indian major carp species. The levels of proteases (40 ± 0.211 units/ml) and myeloperoxidase OD450 nm (1.525 ± 0.108) were found to be highest in the skin mucus of C. catla. However, lysozyme levels were highest in the skin mucus of L. rohita (10.95 ± 0.330 μg/ml), and C. mrigala had the highest alkaline phosphatase activity (30.74111 ± 0.680 U/l). Besides the enzyme activities, the epidermal mucus samples of three Indian major carp species were also tested and compared for the antibacterial activity against seven bacterial strains. Skin mucus of C. catla showed highest antibacterial activity among the three Indian major carps against all the seven bacterial strain, except that Micrococcus lysodeikticus and Vibrio cholerae, however, showed highest activity against mucus of C. mrigala. Also, the epidermal mucus from all the three species successfully agglutinated three freshwater pathogens, viz. Aeromonas hydrophila, Pseudomonas fluorescens and Edwardsiella tarda, with agglutination titres being the highest for Labeo rohita for all the pathogens. The epidermal mucus samples from the Indian major carp species showed haemagglutination activity and successfully lysed human, chicken and goat RBCs showing highest activity in C. catla. These results provide information for a better understanding of the role of epidermal mucus and its components in the innate immune system of Indian major carps.     

Effects of Freezing and Frozen Storage on Protein Functionality and Texture of Some Cephalopod Muscles

The aim of this study was to investigate the effects of freezing and frozen storage on protein functionality and texture of squid (Loligo vulgaris), octopus (Octopus vulgaris), and cuttlefish (Sepia officinalis) muscles. Squid, octopus, and cuttlefish samples were cut into pieces of 4 × 4 cm. These pieces were packed in polyethylene bags. The bags were frozen in a blast freezer at ?45°C until the thermal center reached ?18°C. Frozen samples were stored in a deep freezer at ?18°C for 30 days. After freezing and during frozen storage, total soluble protein and water holding capacity decreased and total free amino acid and cooking loss increased in all cephalopod muscles. According to instrumental texture analysis results, freezing and frozen storage affected textural characteristics of squid and cuttlefish but not of octopus. Sensory hardness and chewiness values of all cephalopods increased after freezing, but elasticity values did not change. There were no significant differences between storage days in hardness values of squid and octopus. However, significant differences in hardness values of cuttlefish were observed between the 1st day of storage and the last day.     

Physicochemical and Structural Properties of Recovered Elastin from Jumbo Squid (Dosidicus gigas) By-Products

Physicochemical and structural properties of soluble jumbo squid (Dosidicus gigas) elastin recovered from skin by-products were evaluated. The molecular weight of isolated elastin was ~40 kDa with an isoelectric point (pI) between 9 and 10. Aspartic, glutamic, arginine, proline, glycine, and lysine amino acids were the most abundant in squid elastin, whereas the hydroxyproline absence, ~0.7% cysteine content, and the calculated 0.35 isoleucine/leucine ratio were used as purity index. Total and reactive sulfhydryl contents were similar (247.0 ± 5.1 vs 242.0 ± 7.5 μmol mg^?1 of protein, P ≥ 0.05) in purified squid elastin but surprisingly higher than previously reported in other elastins. On the other hand, the secondary structures of squid elastin analyzed by Fourier transform infrared spectroscopy (FTIR) were ~45% β-sheets, ~15% α-helices, ~10% β-turns, and ~30% undefined structures. In addition, squid elastin experienced glass transition at 82.01 ± 0.01ºC, denaturation temperature at 110.45 ± 0.64ºC, and aggregation at 197.5 ± 0.23ºC. In conclusion, the prevalence of charged amino acids and pI of squid elastin can facilitate its solubilization in hydrophilic systems, whereas the secondary structure profile and thermostability are desirable features in proteins used for biopolymer designs such as food biofilms or barrier systems.