Long-term stability of RNA isolated from muscle of red seabream (Pagrus major) during ice storage.

Recovering high-quality intact RNA from postmortem tissue is of major concern for gene expression studies. However, it is difficult to perform RNA extraction from aquacultured fish immediately after death, as rapid and accurate skills are needed for the procedure. The objective of this study was to quantitatively assess the integrity of total RNA extracted from muscle, liver, and digestive tract tissues of red seabream stored in ice as whole bodies, at a range of time points up to 10 days postmortem, using RNA integrity number (RIN) and quantitative PCR (qPCR). The RIN of total RNA in muscle remained over 8.0 for 5 days postmortem. The RINs in the liver and digestive tract were under 5.0 at 2 days postmortem. The mRNA levels of tissue inhibitor metalloproteinase 2 (TIMP-2) and ß-actin, measured using qPCR in muscle, decreased to 87.8% at 1 day postmortem and to 45.5% at 2 days postmortem, from that at 0 days postmortem. In the liver and digestive tract, the mRNA levels were not significantly changed until 1 day postmortem. These results indicate that RNA, especially from fish muscle, can be maintained at high quality for several days postmortem solely by storing the fish body in ice.

Effect of preheating temperature on the microstructure of walleye pollack surimi gels under the inhibition of the polymerisation and degradation of myosin heavy chain.

BACKGROUND: The physical attribute of heat-induced gel texture is highly dependent on the microstructure of the gel. In this study the microstructures of walleye pollack surimi gels preheated at various temperatures with and without inhibitors (ethylenediamine-N,N,N',N'-tetraacetic acid, iodoacetamide and leupeptin) were observed with a natural scanning electron microscope. RESULTS: Without inhibitors, gels preheated at 30 °C showed a fine and uniform network structure together with the highest polymerisation of myosin heavy chain (MHC) and the highest gel strength. At 60 °C, gels exhibited a broken, disrupted and loose cluster-like structure together with the highest degradation of MHC and the lowest gel strength. Under the inhibition of polymerisation and degradation of MHC a fine network was observed up to 40 °C during preheating. However, after a second step of heating at 80 °C the microstructures were disrupted and resembled each other regardless of the preheating temperature. CONCLUSION: Heat-induced gel formation is related to the polymerisation and degradation of MHC and the microstructure of the gel during preheating. Gelation occurred during setting even under the inhibitory condition, and the formation of covalent bonding by transglutaminase is not essential to the formation of a three-dimensional network during setting but is essential to the gel strength enhancement effect of setting by subsequent heating at 80 °C.

Mangrove crab intestine and habitat sediment microbiomes cooperatively work on carbon and nitrogen cycling.

Mangrove ecosystems, where litter and organic components are degraded and converted into detrital materials, support rich coastal fisheries resources. Sesarmid (Grapsidae) crabs, which feed on mangrove litter, play a crucial role in material flow in carbon-rich and nitrogen-limited mangrove ecosystems; however, the process of assimilation and conversion into detritus has not been well studied. In this study, we performed microbiome analyses of intestinal bacteria from three species of mangrove crab and five sediment positions in the mud lobster mounds, including the crab burrow wall, to study the interactive roles of crabs and sediment in metabolism. Metagenome analysis revealed species-dependent intestinal profiles, especially in Neosarmatium smithi, while the sediment microbiome was similar in all positions, albeit with some regional dependency. The microbiome profiles of crab intestines and sediments were significantly different in the MDS analysis based on OTU similarity; however, 579 OTUs (about 70% of reads in the crab intestinal microbiome) were identical between the intestinal and sediment bacteria. In the phenotype prediction, cellulose degradation was observed in the crab intestine. Cellulase activity was detected in both crab intestine and sediment. This could be mainly ascribed to Demequinaceae, which was predominantly found in the crab intestines and burrow walls. Nitrogen fixation was also enriched in both the crab intestines and sediments, and was supported by the nitrogenase assay. Similar to earlier reports, sulfur-related families were highly enriched in the sediment, presumably degrading organic compounds as terminal electron acceptors under anaerobic conditions. These results suggest that mangrove crabs and habitat sediment both contribute to carbon and nitrogen cycling in the mangrove ecosystem via these two key reactions.

Participation of cysteine protease cathepsin L in the gel disintegration of red bulleye (Priacanthus macracanthus) surimi gel paste.

BACKGROUND: Endogenous proteases, among them cysteine-type proteases, are reported to contribute to gel disintegration, resulting in kamaboko of poor quality. Severe gel disintegration occurs in red bulleye surimi gel paste. The objective of this study was to clarify the participation of cysteine protease cathepsin L in the gel disintegration of red bulleye surimi. The surimi was made into kamaboko with and without cathepsin L inhibitors. To confirm its hydrolysis action, crude cathepsin L was also extracted and added to the surimi to make kamaboko. RESULTS: The gel strength of kamaboko obtained by both one-step (50 degrees C, 2 h) and two-step (50 degrees C, 2 h + 80 degrees C, 20 min) heating was very low in the absence of inhibitors. Protease inhibitors E-64 and leupeptin were found to enhance the gel strength considerably. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the hydrolysis of kamaboko was promoted by crude cathepsin L and inhibited by E-64 and leupeptin. The gel strength of two-step heated kamaboko was increased from 12 to 110 and 130 g cm(-2) by E-64 and leupeptin respectively at a concentration of 0.2 g kg(-1) surimi. CONCLUSION: Endogenous cathepsin L of red bulleye surimi participates in gel disintegration during kamaboko processing. It does so by degrading the myosin heavy chain of actomyosin and consequently hindering the gelation of red bulleye surimi.

Correlation of proline, hydroxyproline and serine content, denaturation temperature and circular dichroism analysis of type I collagen with the physiological temperature of marine teleosts.

Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.

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

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

Occurrence of two distinct types of tissue inhibitor of metalloproteinases-2 in teleost fish.

We have cloned for the first time two cDNAs encoding distinct types of tissue inhibitor of metalloproteinases-2 (TIMP-2) from teleost fish, Japanese flounder, and designated these types as jfTIMP-2a and jfTIMP-2b. The open reading frames of the jfTIMP-2a and jfTIMP-2b cDNAs are composed of 663 and 657 nucleotides and 221 and 219 amino acids, respectively. Both jfTIMP-2s contain 12 cysteine residues, which might form six disulfide bonds as in other animals' TIMP-2s. The predicted full-length amino acid sequence of jfTIMP-2a has lower identity to jfTIMP-2b (63%) than to those of human (74%) and chicken (73%) TIMP-2s, but higher than to those of other human TIMPs (TIMP-1: 39%, TIMP-3: 43%, TIMP-4: 45%), indicating that jfTIMP-2a is a common TIMP-2, while jfTIMP-2b is unique to Japanese flounder. However, the C-terminal region including the last three disulfide bonds of jfTIMP-2b has higher amino acid identity to those of other animal TIMP-2s than to that of jfTIMP-2a. Reverse-transcribed polymerase chain reaction (RT-PCR) analysis showed the mRNAs of jfTIMP-2a and jfTIMP-2b to be ubiquitously expressed in all tissues examined, but with different expression patterns. These findings suggest that the two distinct jfTIMP-2s might perform different functions in teleost tissues.

Enhancement of the gelation properties of hairtail (Trichiurus haumela) muscle protein with curdlan and transglutaminase.

The effects of curdlan in combination with microbial transglutaminase on the gelling properties of hairtail muscle protein were investigated. When curdlan of 4g/100g paste was combined with transglutaminase at a concentration of 0.4units/g meat paste, the gel strength, water holding capacity and the whiteness of the heated gel were improved. Textural profiles, such as hardness, springiness, cohesiveness, guminess and chewiness, reached their peaks as well. The increased band intensity of cross-linked proteins, accompanied by weakened myosin heavy chain, was observed from the SDS-PAGE pattern, indicating that curdlan might activate the formation of more ε-(γ-glutamyl) lysine cross-links induced by transglutaminase, especially at the level of 0.4units/g paste, leading to a denser gel matrix.

Existence of cathepsin L and its characterization in red bulleye surimi.

Cathepsin L could not be removed completely during conventional actomyosin extraction and still remained in the actomyosin of red bulleye surimi. Sepharose 6B gel filtration profile showed that the main peak of cathepsin L was separated from that of actomyosin suggesting the enzyme was non-binding to actomyosin. The fractions showing the main activity of cathepsin L were pooled and mentioned as L(mix). Optimal pH of cathepsin L in actomyosin and L(mix) was 5.0 and the optimum temperature of L(mix) was 45 degrees C. Stability of L(mix) was closely related to temperature and pH. At optimum temperature 45 degrees C and optimum pH 5.0, activity of cathepsin L remained 76.8% after 120 min. At acidic pH 4.0, it remained 25% of its original activity after incubation at 45 degrees C for 120 min. At neutral pH 7.0, incubated at 45 degrees C, cathepsin L decreased the activity to 50% within 30 min and remained only 3% after 120 min. When incubated at low temperature 25 degrees C, cathepsin L kept 83-85% of its original activity at pH 4.0, 5.0 and 7.0. Effect of NaCl concentration on cathepsin L was related to pH. At neutral pH 7.0, in 0.6 M NaCl solution, cathepsin L activity was about 85% that of pH 5.0, indicating that cathepsin L might contribution to the gel deterioration of red bulleye surimi even in neutral condition. Studies of substrate specificity and effects of activators and inhibitors confirmed L(mix) to be a thiol-type cysteine protease.