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.     

The effects of scallop shell extract on collagen synthesis

ABSTRACT:   Previous observations showed that scallop shells contain organic components that have various useful applications for skin. In this study, the effect of the organic components of scallop shell (scallop shell extract) on collagen metabolism is investigated. Collagen metabolism is tightly controlled by the collagen degrading matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). Treatment of human skin fibroblast cells with the scallop shell extract increased the mRNA expression levels of type I collagen, MMP-1 and TIMP-1, suggesting that the scallop shell extract may activate collagen metabolism in skin fibroblast cells. Sirius red staining and the colorimetric quantification of collagen in fibroblast cells demonstrated that the scallop shell extract increased collagen content by approximately 1.3-fold. In vivo studies also revealed that the topical application of the scallop shell extract to rat dorsal skin increased the collagen content in the skin tissue section. These results suggest that the scallop shell extract may be effective for the treatment of photoaged and aging skin, which undergo collagen loss.     

Characterization of the quantitatively major collagen in the mantle of oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

ABSTRACT:   A quantitatively major collagen was isolated from the pepsin-solubilized collagen preparation of the mantle by differential salt precipitation and phosphocellulose column chromatography, and its constituent α components (α1 and α2) were purified by phosphocellulose column chromatography. The subunits were demonstrated to be genetically distinct from each other by peptide mapping and amino acid analysis. The amino acid composition calculated from those of the α1 and α2 components in 2 : 1 ratio coincided well with that of the major collagen from the mantle. These results suggest that the major collagen in the mantle of the oyster may have a heterotrimer structure (α1)₂α2.     

罗非鱼皮营养成分分析及鱼皮明胶提取工艺的探讨

以罗非鱼皮为原料，对其营养成分进行分析，并通过正交试验方法探讨罗非鱼皮明胶的提取工艺，同时对明胶的理化及微生物指标进行分析。结果表明，罗非鱼皮中粗蛋白含量高达33．14％，其中胶原蛋白含量为27．8％，占其粗蛋白的83．9％；鱼皮提取明胶的适宜工艺条件为在2．5％（W／V）NaOH溶液中浸泡3．5h，然后在0．02％（V／V）HCl浸泡约3h，熬胶温度为55℃，提取得率为26．5％；所得鱼皮明胶的凝胶强度达468g，粗蛋白含量为82．46％，菌落总数为1．3×10^5CFU·g^-1，大肠菌群结果报告为阴性。     

Size selectivity of a trammel net for oval squid Sepioteuthis lessoniana

ABSTRACT:   The size selectivity of a trammel net for herded oval squid Sepioteuthis lessoniana in Tateyama Bay, Chiba Prefecture, was estimated by comparison between the mantle length frequency distributions of oval squid caught by a trammel net and by a set net. The measured mesh sizes of the inner net of the trammel net and of the final section of the set net were 85.3 and 11.3 mm, respectively. In the trammel net fishery where oval squid are herded into the net, most of the oval squid are caught in the bag-shaped inner net. Hence, the logistic function was employed for the size selectivity curve of the trammel net. The 'share each length's catch total' (SELECT) model was implemented for the estimation of the selectivity curve. The size selectivity r ( l ) of the trammel net for the oval squid was expressed as a logistic function of the mantle length l : r ( l ) = exp(−18.57 + 0.88  l )/[1 + exp(−18.57 + 0.88  l )]. From these logistic parameter estimates, the 50% selection mantle length and selection range ( L ₇₅– L ₂₅) were calculated as 21.07 and 2.49 cm, respectively. The selection probability of oval squid whose mantle girth was equivalent to the mesh perimeter of the inner net was 0.09. Accordingly, oval squid of a girth smaller than the mesh perimeter were likely to pass through the mesh to escape from the net.     

The glycerinated body wall of the sea cucumber as a suitable preparation for electron microscopic and physiological studies of ‘catch mechanism’

ABSTRACT:   The body wall of the sea cucumber changes its stiffness by ionic environments. The stiff state can be held for a long time, and the mechanism concerned is known as 'catch mechanism'. In the present study, the direct effects of ions on the mechanism using the glycerinated body wall treated with 50% glycerin to clarify how the ions effect changes of stiffness were examined. The glycerinated body walls contained collagen fibers and some broken cells in the connective tissue ultrastructurally. Cell membranes were not clearly present in the broken cells, and cell organelles were dispersed around the cells. The glycerinated body walls went into a limp state during addition of 10 mM ethylenediaminetetraacetic acid (EDTA), and showed height elongation rate in this study's experimental system. In contrast, the elongation rate decreased by the addition of 10 mM CaCl₂, that is, the body wall came to a stiff state. This stiff state could be considered as equivalent to 'catch state' of glycerinated body wall. Collagen fibers in those samples showed more compact arrangements at 10 mM CaCl₂ treatment than the one of 10 mM EDTA ultrastructurally. These features and physiological results suggested that EDTA and/or CaCl₂ from outside affect directly to the main part of the 'catch' mechanism in the glycerinated body wall.     

cDNA cloning of oyster matrix metalloproteinase and its possible involvement in hypoxic adaptation

ABSTRACT:   We have cloned a cDNA encoding the matrix metalloproteinase (MMP) from oyster Crassostrea gigas . The clone contains a 1797 bp open reading frame encoding a protein of 599 amino acids. Oyster MMP (Cg-MMP) has a transmembrane domain at its C-terminal and a furin/prohormone convertase cleavage site at the end of a propeptide domain, which are commonly observed in membrane-type MMPs (MT-MMPs). This suggests that Cg-MMP is an MT-MMP. The deduced amino acid sequence of oyster MMP shares approximately 30% identity with human MT4-MMP and MMPs from fruit fly and hydra. Cg-MMP mRNA was detected in the gill, mantle and adductor muscle, and more intense signals in the northern blot analysis were recognized in the gill and adductor muscle. Similar tissue distribution was observed for tissue inhibitor of metalloproteinase (Cg-TIMP) in oyster. In response to hypoxic stress, the abundance of Cg-MMP mRNA was elevated in the gill, while that of Cg-TIMP mRNA remained almost constant. These findings suggest that promotion of collagen metabolism may be implicated in the hypoxic adaptation in oyster.     

Culture of mantle epithelial cells expressing shell matrix proteins from scallop Patinopecten yessoensis

ABSTRACT:   In molluscs, mantle epithelial cells secrete organic matrix proteins to form shells. In this study, we established a culture of mantle epithelial cells by using the mantle pallial layer of scallops. We aimed to identify the mantle epithelial cells expressing scallop shell matrix proteins and establish a culture system of epithelial cells. After the mantle pallial layer was carefully isolated from the mantle tissue, explant culture was performed at 4°C. Most cells that migrated from the explant tissue were round cells. Most of the adhered cells retained round morphology, while some of the cells adhered to the dish and showed morphology similar to that of epithelial-like and fibroblast-like cells. When the cultured cells were immunostained with a polyclonal antibody against the shell matrix protein, the antibody recognized many of the adhered cells. An estimation of the number of epithelial cells revealed that approximately 70% of the adhered cells were epithelial cells. This is the first report to describe epithelial cells in cultured mantle cells, which express shell matrix proteins. This culture system may be a useful method for characterization of the mantle epithelial cells.     

Physiochemical and Textural Alterations in Indian Squid (Loligo duvauceli) Mantle During Frozen Storage and Cooking

Textural variations in squid mantle and the role of proteins on texture during frozen storage and cooking were investigated. Myofibrillar protein (62.36%) and pepsin soluble collagen (10.70%) accounted for the major fraction of total protein (22.17%). The histochemistry of mantle tissue showed a mesh-like arrangement of myofibrillar proteins with a collagenous dermal layer and feebly passing collagen through myotome bundles. Texture profile analysis of unfrozen mantle suggested the first phase of hardening at 50°C with hardness 1 (H1) of 11.53 kgf and hardness 2 (H2) of 9.68 kgf; and the second phase of hardening with optimal texture and maximum juiciness at 70°C (H1, 8.11 kgf; H2, 7.13 kgf) that varied with extended frozen storage. Increased frozen storage and cooking led to protein denaturation and formation of new low molecular weight proteins as evidenced in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); these possibly influenced the functional and microstructural properties of the tissue.     

虾夷扇贝组织中微量元素的分布特性

研究了虾夷扇贝闭壳肌、外套膜、内脏团、瓣鳃和性腺中Cu、Fe、Mn、Zn、Pb和Cd等元素的分布特性。试验结果表明,内脏团中的Cu显著(P<0.05)高于其他各组织,其他各组织间差异不显著(P>0.05);闭壳肌中Zn的含量显著(P<0.05)高于内脏团、外套膜、瓣鳃和性腺中的含量;Fe含量内脏团和瓣鳃中显著(P<0.05)高于性腺、闭壳肌和外套膜;各组织中Mn的分布特性为瓣鳃>闭壳肌>性腺>内脏团>外套膜;Pb的分布特性为内脏团>闭壳肌>性腺>外套膜>瓣鳃;内脏团中的Cd占全贝总量的67%,显著高于其他组织。因此,约占全贝质量10%的内脏团蓄积了较高含量的Cu、Fe、Pb和Cd,尤其是Cu和Cd(分别约占全贝蓄积总量的71%和67%),食用时去掉内脏团,可保证虾夷扇贝的食用安全和较高的食用及营养价值。     

A formulated diet for Atlantic halibut (Hippoglossus hippoglossus, L.) larvae

A diet for Atlantic halibut-larvae was formulated taking into account the fact that marine-fish larvae have a limited ability to assimilate protein and lipid. Dietary protein consisted of a free amino-acid premix (7.2% of crude protein), predigested-squid mantle (7.2%), squid mantle (8.6%) and cod-muscle mince (77.0%). Lipid sources were soyabean lecithin (33% of crude lipids), crude phospholipids extracted from cod roe (10%) and sardine oil (57%). Larvae were weaned onto the experimental diet at wet-body weights of 0.07, 0.10 or 0.16 g, respectively. The experimental diet was fed for 31, 25 or 17 days, respectively, and the experiment was terminated on the same calendar day for all groups. A control group was fed with Artemia nauplii enriched with DHA Selco™ from 0.07 g. Survivals ranged from 78% in larvae transferred at 0.10 g to 96% in those transferred at 0.16 g and in the control group. Daily specific-growth rates (SGR) were 3.1 ± 0.07, 3.3 ± 0.11 and 2.2 ± 0.01% day⁻¹ in larvae transferred at 0.07, 0.10 and 0.16 g, respectively, while growth in the control group was 5.1% day⁻¹. It was concluded that weaning of Atlantic-halibut larvae is feasible from 0.7 g (approximately 20 days post first-feeding) when the formulated diet contains predigested protein and ample amounts of phospholipids.     

Changes in meat texture of three varieties of squid in the early stage of cold storage

ABSTRACT: The present study examined the changes in texture and protein components during cold storage of different squid varieties. Raw oval squid, Japanese common squid and arrow squid were sliced fresh and the muscles were stored at 4°C for 0, 4, 8, 12, 18, 24, 48 and 120 h. The rheological measurements, protein components and amounts of collagen were examined. The adhesiveness of each squid increased significantly in the early stage of cold storage. In all varieties, penetration decreased at 4 h, which is considered to be rigor mortis, then increased. The amounts of total collagen, 20°C water-soluble collagen and 70°C water-soluble collagen did not change significantly in each variety during cold storage. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) pattern showed that the 580 kDa component gradually disappeared up to 48 h. The correlations between the amounts of 580 kDa component and adhesiveness or firmness were high. Models of fit based on chemical kinetics accurately expressed the behavior of adhesiveness, firmness and penetration showing that 63.2% of adhesiveness changes occurred in 13–19 h and that 63.2% of firmness changes occurred in 18–24 h.     

酶法提取鱿鱼皮胶原蛋白工艺条件的研究

研究了酶法提取鱿鱼皮胶原蛋白的工艺条件。根据5种蛋白酶水解液中羟脯氨酸（HYP）的含量,确定了胰蛋白酶和木瓜蛋白酶这2种提取率高的酶为水解用酶,采用L16（4^5）正交试验确定了这2种酶水解鱿鱼皮以制备胶原蛋白的最佳酶解条件。结果表明,胰蛋白酶和木瓜蛋白酶水解鱿鱼皮以制备胶原蛋白的最佳温度、加酶量、底物浓度、pH、时间分别为55℃、1200U·g^-1、1：20、pH8.0、4h和50℃、3200U·g^-1、1：20、pH6.0、6h。2种蛋白酶提取的胶原蛋白含量分别为11.08%和11.36%,提取率分别为95.16%和97.56%。     

扇贝裙边氨基酸营养粉的制备工艺研究

为了对扇贝裙边进行综合利用,本文选择由中性蛋白酶、胰蛋白酶和复合蛋白酶3种酶组合成3对混合酶对扇贝裙边进行酶水解实验,以水解液中游离氨基酸态氮为考察指标,采用正交实验设计研究了加酶量、酶解时间、酶解温度及酶的种类对酶解效果的影响,从而优化了扇贝裙边的酶法水解工艺条件。以最佳水解工艺条件获得的酶解液经喷雾干燥制成的氨基酸营养粉,经检测,其粗蛋白含量为82.89%,粗脂肪含量为1.55%,总糖含量为6.82%。该氨基酸营养粉中氨基酸种类齐全,每100g粗蛋白中氨基酸总量达79.06%,其中游离氨基酸总量达51.53%。     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).     

Changes in purine content of tilapia surimi products during processing

ABSTRACT:   Changes in purine-related compounds of tilapia surimi product during processing were investigated. The washing step could result in about 60% decrease of total purine content in tilapia mince during processing. The main released purine substance was inosine monophosphate. The major reducing effect was conducted in the first 10 min during washing. No significant changes were observed after washing for 20 and 30 min. The lowest total purine content of tilapia surimi product was obtained with repeating the washing step twice. Thus, this procedure could reduce the purine content of tilapia mince from a high purine content level to a middle level. The gel strength of tilapia surimi product increased with increasing washing duration within 30 min. However, tilapia surimi product with a middle purine content and acceptable gel strength might be produced by washing twice in 10 min during processing.     

Taurine Supplementation of All-plant Protein Diets for Rainbow Trout (Oncorhynchus mykiss)

Abstract.— Taurine has been demonstrated to be conditionally indispensable for several carnivorous fish species. Current trends in trout production include decreasing levels of fish-meal content in feeds, along with faster growing strains of fish. Taurine may be a limiting nutrient in support of elevated planes of growth for rainbow trout. A 9-wk feeding trial was conducted using a factorial treatment design with protein source (fish meal or plant) and taurine supplementation (four levels) as the main effects. The fish-meal diet series included 23% herring meal and contained 1.76% total sulfur amino acids (TSAA). The plant diet series did not contain any animal proteins and substituted protein from soy protein concentrate in place of the herring-meal protein and contained 1.5% TSAA. Taurine was supplemented at 0, 5, 10, and 15 g/kg dry diet to each of the diets in the plant series and the fish-meal series of diets. All diets were formulated to contain 43.8% crude protein and 20% lipid with an estimated physiological fuel value of 4.2 kcal/g. Fifteen fish were stocked in each of 24 tanks with a mean initial weight of approximately 26.8 g per fish. The unsupplemented fish-meal diet contained 2 g/kg taurine, and the unsupplemented plant diet had taurine levels below the detection limit of 0.1 g/kg diet. Taurine supplementation improved growth, feed conversion ratios, protein retention efficiencies, and energy retention efficiencies of fish fed the plant protein diets. No effects of taurine supplementation were observed for these response factors in fish fed the fish-meal series diets. This study demonstrates that taurine supplementation may be necessary for rainbow trout fed plant-protein-based feeds.     

The dietary linoleic and linolenic fatty acids requirements of the prawn Penaeus monodon

The dietary requirement of the prawn Penaeus monodon for linoleic (LOA) and linolenic (LNA) fatty acids was examined in the absence of other long-chain polyunsaturated and highly unsaturated fatty acids (PUFA-20:2, 20:3, 22:2, 22:3 and HUFA-18:4, 20:4, 20:5, 22:4, 22:5, 22:6, respectively). Incremented dietary amounts of LOA (7, 14, 21, 28 and 35% of total fatty acids) and LNA (0, 7, 14, 21 and 28% of total fatty acids) were examined in a 5 × 5 factorial growth experiment lasting 50 days. An additional diet containing both PUFA and HUFA (cod-liver oil) was provided as a reference. The total lipid content (excluding sterols) of each of the 26 diets was maintained at 70 g kg⁻¹ of dry diet. The fatty acid composition of the neutral lipid was manipulated by blending different plant oils and supplementing with purified free fatty acids to provide the desired fatty acid composition upon addition to the total diet. At the end of the 50-day growth experiment, the prawn digestive gland (DG) was quantitatively analysed for lipid and fatty acid content. Prawns fed the reference diet increased in weight (mean ± SEM) by 214 ± 6%. Growth was generally greater when combinations of LOA and LNA were used. The best growth (213 ± 17%) was obtained with the diet containing a fatty acid content of 14% LOA and 21% LNA. This growth was comparable to that of the reference diet. The digestibility of the total lipid in the diet was usually higher when both fatty acids were present. The lipid content of the DG was highest in prawns fed diets containing both LOA and LNA, similar to the growth response. The fatty acid composition of the prawn's DG lipid reflected the fatty acid composition of the diet. However, the maximum assimilation of LNA in the DG lipid (14.2% of DG lipid fatty acids) was about half that of LOA (32.5% of DG lipid fatty acids).