King Crab Hepatopancreas Research Articles

Morphology, processing attributes, fatty acid, and amino acid composition in cooked leg meat and raw hepatopancreas of juvenile male red king crab (Paralithodes camtschaticus) after 12 months of live holding

This study investigated how diet composition (DC) (i.e., raw shrimp or dry feed alone and combinations thereof) affected the morphology, processing attributes, fatty acid (FA)- and amino acid (AA) composition in cooked leg meat and raw hepatopancreas of juvenile red king crabs (RKCs). After 12 months, the crab weight nearly doubled, and the average mortality was 10%. The DC influenced the attributes of the cooked leg meat and the raw hepatopancreas. In addition, crumbling the dry feed led to reduced nutritive conditions, especially in the 80 and 100% dry feeding groups. In the leg meat, the main FA components were PUFA, MUFA, and SFA in descending order, while for the raw hepatopancreas, the corresponding order was MUFA, PUFA, and SFA. Minor AA variations across the groups were observed in the cooked leg meat and raw hepatopancreas. A PCA revealed a grouping of selected morphological-, processing attributes, and FAs according to the 20 and 100% dry feed groups. This study did not explore the specific feed intake. Hence, whether the results reflect the DC, a lower feed intake due to the crumbling, or a combination is unknown. Overall, the results show positive prospects for live holding of juvenile RKCs.

Novel Antimicrobial Peptide from the Hepatopancreas of the Red King Crab.

Crustaceans have successfully adapted to survive in their natural habitat, rich in microorganisms, due to the presence of antimicrobial peptides (AMPs) in their organism. They achieve this adaptation despite lacking the highly specific adaptive immune system found in vertebrates. One valuable source of AMPs is the hepatopancreas, a waste product from crab fishery and its processing. Applying zymographic and spectrophotometric techniques, we discovered a small peptide (approximately 5 kDa) within a low molecular weight protein fraction extracted from the acetone powder of the red king crab hepatopancreas. This peptide hydrolyzes both M. lysodeikticus cell wall and M. lysodeikticus cell wall polysaccharide, while showing no activity against gelatin. The found peptide may be of interest for application in medicine, biotechnology, and the food industry, for example as a bio-preservative.

Investigation of lipolytic activity of the red king crab hepatopancreas homogenate by NMR spectroscopy.

The digestive gland of craboids (hepatopancreas) is rich in a huge number of various enzymes (collagenases, nucleases, hyaluronidases, proteases), which are well studied at the moment. However, little is known about crustacean lipases. In this work, using 1H NMR spectroscopy, it was found that the hepatopancreas homogenate of the red king crab Paralithodes camtschaticus demonstrates high lipolytic activity against triacetin in a wide pH range and shows moderate activity against the caprylic/capric triglyceride emulsion. Under the action of the hepatopancreas homogenate, triacylglycerols are converted into 1,2-diacylglycerol, and then into 2-monoacylglycerol and 1-monoacylglycerol. The 1-monoacylglycerol predominates in the reaction products. The use of NMR spectroscopy makes it possible to quickly detect hydrolysis products and evaluate the reaction direction.

Fatty acid profile of cooked leg meat and raw hepatopancreas of red king crab (Paralithodes camtschaticus) during three-month live holding without feeding at 5 and 10 °C

The red king crab (RKC, Paralithodes camtschaticus) is a highly-valued decapod species. Typically, RKCs undergo a period of live holding (LH), often without feeding, in onshore facilities, allowing for flexible management before export to destination markets. This study aimed to (i) gain information on the fatty acid (FA) profile of the cooked leg meat and raw hepatopancreas obtained from RKC harvested in Norwegian waters of the Barents Sea and (ii) investigate how these FA profiles are affected by LH without feeding for up to 92 days at 5 or 10 °C. Minor changes were observed in the FA profile of cooked leg meat, which retained its nutritional value in omega−3 FA content. In contrast, the FA composition of raw hepatopancreas was severely affected by the LH time, with substantial changes occurring especially between 41 and 62 days at 10 °C and between 62 and 92 days at 5 °C. Saturated and specific monounsaturated FAs (16:1n−7c and 18:1n−9t), as well as 22:5n−3c, were preferentially utilized at the beginning of the starvation period, followed by the mobilization of C18–22 unsaturated FAs. Long-chain highly-unsaturated FAs were preferentially retained during LH, especially 20:4n−6c and 20:5n−3c. The information emerging from the present study may be practically exploited for selecting or designing suitable feed for RKC during LH at different temperatures.

Prospects of Red King Crab Hepatopancreas Processing: Fundamental and Applied Biochemistry

Since the early 1980s, a large number of studies on enzymes from the red king crab hepatopancreas were conducted. They have been relevant both from a fundamental point of view in terms of studying the enzymes of marine organisms and in terms of rational natural resource management aimed to obtain new valuable products from the processing of crab fishing waste. Most of these works were performed by Russian scientists due to the area and amount of waste of red king crab processing in Russia (or the Soviet Union). However, the close phylogenetic kinship and the similar ecological niches of commercial crab species and the production scale of the catch provide the bases for the successful transfer of experience in the processing of the red king crab hepatopancreas to other commercial crab species caught worldwide. This review describes the value of recycled commercial crab species, discusses processing problems, and suggests possible solutions for these issues. The main emphasis is made on hepatopancreatic enzymes as the most salubrious products of red king crab waste processing.

Fatty Acid Composition in the Hepatopancreas of the Barents Sea Red King Crab

The fatty acid composition in the hepatopancreas of the red king crab from the Barents Sea was studied. Polyunsaturated fatty acids dominated among the fatty acids analyzed (11 792 ± 592 μg/g, 45.3 ± 0.7% of the total fatty acid concentration). Saturated fatty acids and monounsaturated fatty acids were found in approximately equal proportions accounting for 7825 ± 454 μg/g (28.3 ± 0.4%) and 7661 ± 510 μg/g (26.4 ± 0.6%), respectively. Significant interannual and intersexual differences were found in the fatty acid profiles of the crabs studied. The crab size, shell age, and number of injured legs did not affect the fatty acid compositions in the hepatopancreas. The high concentrations of essential fatty acids belonging to the omega-3 and omega-6 classes indicate that the hepatopancreas of the Barents Sea red king crab could be used as a source of extraction of these valuable substances.

The effect of hepatopancreas homogenate of the Red king crab on HA-based filler.

In this study, several methods were used to analyze the hydrolysis of hyaluronic acid (HA)-based cosmetic fillers by the hepatopancreas homogenate of the Red king crab. The results show that the homogenate and commercially available hyaluronidases have similar hydrolysis activities on the fillers. Atomic force microscopy images reveal that the HA fillers consist mainly of spherical-like particles, which are converted into filamentous structures as a result of hydrolysis by the Red king crab hepatopancreas homogenate. Turbidimetric analysis of the hydrolysis process shows that HA aggregation with acidic albumin exhibits a bell-shaped dependence on reaction time. Analysis of the hydrolysis process by nuclear magnetic resonance shows that HA degradation lasts several days. The maximum rate of the reaction is detected in the 1st h of incubation. The data confirm that the purified homogenate of the Red king crab hepatopancreas exerts hyaluronidase activity on HA-based cosmetic fillers; therefore, it may be considered as a potential therapeutic agent for treating filler complications.

Soybean Trypsin Inhibitors: Selective Inactivation at Hydrolysis of Soybean Proteins by Some Enzymatic Complexes

Inactivation of the Kunitz and Bowman-Birk soybean trypsin inhibitors at hydrolysis of soybean proteins by enzymatic complexes was studied. These complexes are derived from king crab hepatopancreas, cod fish pyloric caeca, and a commercial enzymatic complex of protosubtilin. Analysis of the soybean protein hydrolysates showed that these enzymatic complexes completely digested the Kunitz trypsin inhibitor (60–70% of the total trypsin inhibitors) and had almost no effect on the Bowman-Birk trypsin and chymotrypsin inhibitor. All of the enzymatic complexes contain different sets of enzymes with different proteolytic specificity. This allow to make the conclusion that Bowman-Birk inhibitor is highly resistant to proteolysis and is not inactivated at enzymatic hydrolysis.

Plant protein hydrolysates as fish fry feed in aquaculture. Hydrolysis of rapeseed proteins by an enzyme complex from king crab hepatopancreas

Rapeseed proteins were processed by an enzyme complex isolated from king crab hepatopancreas in order to obtain a hydrolysate for use as fish fry feed. The amino acid composition of the obtained protein preparation was close to the amino acid composition of fishmeal traditionally used in the production of fish feed. SDS-PAGE, HPLC, and mass spectrometric analysis of the products of enzymatic hydrolysis of rapeseed proteins showed that the proteins were hydrolyzed to a high degree. The composition of the hydrolysates depended on the hydrolysis time and included free amino acids (27% of the total weight of the protein mix after 3 h of hydrolysis and 56% after 21 h of hydrolysis), short peptides (2 to 20 amino acid residues), and small amounts of protein fragments with a molecular weight of approximately 14 kDa, as shown by by SDS-PAG electrophoresis.

Blood fatty acids in the development and correction of metabolic syndrome

to investigate the composition of plasma fatty acids (FA) and red blood cells and the level of eicosanoids in patients with metabolic syndrome (MS) and to assess whether metabolic disturbances may be corrected during a cycle use of an ω-3 polyunsaturated fatty acid (PUFA). Examinations were made in 46 patients, including Group 1 (a control group) of 15 persons without MS components; Group 2 of 31 patients with MS, Group 3 of 16 MS patients who had taken an ω-3 PUFA for 6 months, and Group 4 of 15 MS patients who had received the drug for 12 months. The composition of plasma FA and red blood cells was analyzed on a gas-liquid chromatograph. An enzyme immunoassay was used to measure the serum levels of tumor necrosis factor-α (TNF-α) and eicosanoids (thromboxane B2, 6-keto-prostaglandin F1α, leukotriene B4). A biologically active additive from the king crab (Paralithodes camtschatica) hepatopancreas was used as a source of ω-3 PUFA. Having a higher proportion of linoleic and α-linolenic acids in the plasma, the patients were found to have decreased levels of ω-3 and ω-6 PUFAs (linoleic and α-linolenic, arachidonic, and eicosapentaenoic acids) and a larger proportion of Mead acid and saturated FAs (myristic and stearic acids) in the red blood cells, suggesting that that cellular blood FA transfer was impaired and FAs were absorbed by cells. Their serum samples showed the high levels of leukotriene B4, 6-keto-prostaglandin F1α, and thromboxane A2. The long-term (6- and 12-month) use of ω-3 PUFA from the king crab hepatopancreas had a positive impact in modifying the lipid FA composition of red blood cells and in eliminating deficiencies of physiologically important ω-3 and ω-6 PUFAs in the blood cells. The findings suggest that FAs and their metabolites play an important role in the pathogenesis of MS and that dietary ω-3 PUFA should be incorporated into a package of preventive and therapeutic measures for MS.

Duplex-specific nuclease (DSN): A method for the rehabilitation of low-copy number DNA profiles

A continual challenge in the field of forensic DNA analysis is the amplification and interpretation of degraded and low-copy number (LCN) DNA obtained from amounts of limited biological evidence. It has been well established that DNA profiles obtained from the amplification of low quality, degraded, and/or LCN DNA samples are often of limited value due to the frequent occurrence of preferential amplification during polymerase chain reaction (PCR). The by-products of preferential PCR amplification are often observed as inter- and intra-locus peak imbalance, allelic dropout, and/or locus dropout. These are all artifacts that are identified during the interpretation phase of analysis rather than by improving the quality of the DNA present. While it is theoretically possible to obtain a complete DNA profile from a single cell, in reality, profiles obtained from suboptimal amounts of DNA are difficult to interpret and frequently inconsistent when replicated. Inspired by advances in next-generation sequencing techniques, we propose a methodology for simultaneously normalizing the abundance of PCR products across all short tandem repeat (STR) loci using the DNA exonuclease, duplex-specific nuclease (DSN). DSN is an enzyme isolated from the hepatopancreas of Red King (Kamchatka) crab that possesses a strong affinity for digesting double stranded DNA (dsDNA) and has limited activity toward single stranded DNA (ssDNA). Degraded DNA known to display peak imbalance and allele dropout was amplified using AmpFlSTR® Identifiler® Plus for 28 cycles. Following amplification, samples were denatured at 99.9°C for 5min and incubated with one unit of DSN at 62°C in a 28μl volume for 1min. Nuclease activity was terminated through the addition of equal volume of 10mM EDTA and 95°C incubation for 2min. Following DSN treatment, 21 of 30 alleles within the known profile exhibited some improvement in peak height balance. The findings obtained support the potential use of DSN treatment as a method for normalizing STR profiles and improving the quality of data from degraded and low quantity DNA samples.

Anticoagulative and anticomplementary activity of endogenous inhibitor preparation from hepatopancreas of red king crab (paralithosed camtschaticus) towards human blood

Serpins (SERine Protease INhibitors)--is large and diverse group of proteins with similar structures, which can inhibit both serine and cysteine proteases by an irreversible suicide mechanism. A novel serpin from hepatopancreas of Red King Crab (Paralithosed camtschaticus) was obtained and was studied its effect on the process of human blood plasma clotting. The investigated serpin shows a noticeable anticoagulative activity, which increases dramatically in the combined action with heparine. Though the inhibitor has almost no effect on thrombin, it inhibits C1s (C1-esterase). We studied the action of the serpin from P. camtschaticus on C1s via its competitive inhibition by C1 inhibitor and the novel enzyme. The calculated inhibition constant of the serpin from P. camtschaticus towards C1s is 2.02 +/- 0.71 M. Unlike C1 inhibitor, the novel serpin from P. camtschaticus doesn't suppress fibrinolysis and at the same time prevents blood clotting. These features may be of interest for medical purposes.

Anticoagulant and anticomplement effects of an endogenous inhibitor from hepatopancreas of red king crab (Paralithosed camtschaticus) on human blood

Serpins are the superfamily of serine and cysteine protease inhibitors (SERine Protease Inhibitors) acting by an irreversible suicide mechanism. A novel serpin from hepatopancreas of red king crab (Paralithosed camtschaticus) was isolated and its effect on the process of human blood plasma coagulation was investigated. The investigated serpin exhibited a significant anticoagulant effect, which dramatically increased in the combination with heparin. The study of the crab serpin on C1s (C1 esterase) revealed its competition with the C1 inhibitor from blood plasma. Although the inhibitor weakly influenced thrombin activity, inhibition constant for C1s was (2.02 ± 0.71) 10−7 M. Unlike the C1 inhibitor the novel red king crab serpin does not inhibit fibrinolysis but inhibits blood coagulation. This creates certain clinical perspectives.

Peculiarities of substrate hydrolysis by endopeptidases from hepatopancreas of king crab

Kinetic parameters of hydrolysis of peptide and protein substrates by psychrophilic endopeptidases from hepatopancreas of the king crab Paralithodes camtschaticus (PC), in particular, by trypsin, collagenolytic protease, and metalloprotease, were measured at different temperatures. The PC trypsin was shown to hydrolyze Bz-Arg-pNA in the temperature range studied (4-37 degrees C) 19 times more effectively than bovine trypsin. The rate constants of hydrolysis of Glp-Ala-Ala-Leu-pNA by the PC collagenolytic protease increased approximately by one order of magnitude along with temperature decrease, while Km decreased by 3.5 times. The effective values of Km for the hydrolysis of azocasein by the metalloprotease insignificantly depend on temperature. We proposed that electrostatic interactions of negative charges around the cavity of active site are critical for the effective hydrolysis of substrates by endopeptidases of the PC hepatopancreas.

Brachyurins--serine collagenolytic enzymes from crabs

The properties of brachyurins, proteolytic enzymes belonging to a new subfamily of chymotrypsin-like proteases, are considered. These enzymes, found in various species of crustacean, exhibit mixed substrate specificity and a marked collagenolytic activity. The enzymatic and physicochemical properties of brachyurins I and their primary and spatial structures are discussed in detail. A separate chapter is devoted to the preparations of collagenases from the hepatopancreas of king crab: their action on the damaged skin and use in medicine.

Isolation and primary structure of trypsin from the red king crab Paralithodes camtschaticus

Trypsin from hepatopancreas of the Paralithodes camtschaticus crab was isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. The cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the Penaeus vannamei shrimp appeared to be the most close in its primary structure to that of the king crab (70% identity).

Proteolysis in vitro as the Way of Modification of the Protein Trophic Component

The protein hydrolysates of two types, fodder and for microbiological medium, have been obtained in vitro, using protein containing wastes of Iceland scallop fishery (WSF) and enzyme preparation from the red king crab hepatopancreas. The degree of protein degradation and composition of obtained hydrolysates were analyzed. Intensive protein hydrolysis was necessary to obtain the microbiological diagnostic cultural media that fit trophic requirements for 12 microbial test-cultures. Addition of the fodder protein hydrolysate to diet of juvenile salmons Salmo salar during mixed feeding produces a positive effect on their survival; the 5 and 20% replacement of fish flour by the fodder hydrolysate results in a reduction of fish mortality by 21 and 57%, respectively. The living weight of chickens increased by 15% after substitution of the 10% fish flour by the fodder WSF hydrolysate in their diet. The results obtained can be explained by a more effective assimilation of partly hydrolyzed proteins in comparison with native ones.

Enzymatic hydrolysis of proteins from crustaceans of the Barents Sea

Enzymatic preparations from king crab hepatopancreas were shown to be capable, in principle, of producing protein hydrolysates. Hydrolysis of protein-containing waste of deep-water prawn and king crab occurs most successfully at pH 8.0-8.5 and 50-55 degrees C for 5-6 h in the presence of 6 g enzyme per kg substrate. The total chemical composition of the hydrolysates, the molecular weight distributions of proteins and polypeptides, and the contents of free amino acids were studied in dry hydrolysates.

A Protein Hydrolysate Enzymatically Produced from the Industrial Waste of Processing Icelandic Scallop Chlamys islandica

Dry hydrolysate was prepared from protein-containing waste of Icelandic scallop processing by means of a proteinase complex from king crab hepatopancreas. The resulting product contains no less than 80% free amino acids and oligopeptides. Predominant are aspartic acid, leucine, isoleucine, arginine, and lysine, which account for more than 50% of the free amino acids. The potential of using the protein hydrolysate as a nutrient for microorganism cultivation is estimated. It is demonstrated that the hydrolysate can be used for growing test cultures.