Hake Muscle Research Articles

A simple and rapid method for the identification and quantification of malachite green and its metabolite in hake by HPLC–MS/MS

Aquaculture is the fastest growing animal food-producing sector in the world and similarly to any other type of food production systems, the use of veterinary drugs is unavoidable and common, but it needs to be controlled. Special attention should be paid to the use of illegal substances such as malachite green. This substance was found in the last three control monitoring plans conducted in Europe. Methods for the detection of malachite green in commonly farmed species, including salmon, trout, carp, shrimp and panga can be easily found but not emerging species such as hake. Hake is becoming an emerging species and nowadays, farmed hake can be found in some markets.This study presents an HPLC–MS/MS method for the simultaneous determination of malachite green and its metabolite, leucomalachite green, in hake muscle. The recoveries, decision limit, and limit of quantification of malachite and leucomalachite green were greater than 70%, below 2 μg kg−1, between 0.5 and 1 μg kg−1, respectively.

Differential biomagnification of PCB, PBDE, Hg and Radiocesium in the food web of the European hake from the NW Mediterranean

Consumption of marine organisms represents one of the main exposure sources of contaminants for human populations. To obtain a global view of the contamination in commercial fish in the NW Mediterranean Sea, we analysed four types of priority contaminants (PCBs, PBDEs, Hg and 137Cs) in the European hake, Merluccius merluccius, from the Gulf of Lions in relation with organism’s trophic level (δ15N). All contaminants presented a significant increase in concentration in hake muscle with trophic level. However, obvious differences between contaminants were evidenced. Biomagnification factors (BMF and FWMF) along the hake food web were higher for Hg and CB-153 than for BDE-47 and 137Cs, and increase in contaminant concentration with trophic level occurred at different rates depending on contaminants. Such differences of biomagnification patterns can be related to physico-chemical properties of the different contaminants.

Novel Tools for Conservation Genomics: Comparing Two High-Throughput Approaches for SNP Discovery in the Transcriptome of the European Hake

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).

Lipid Damage Inhibition in Hake by Active Packaging Film with Natural Antioxidants

The capacity of the natural antioxidant to retard oxidation of polyunsaturated fatty acids in hake (Merluccius merluccius) muscle was investigated. Hake fillets were packaged in low‐density polyethylene with the natural antioxidant (sample C1 = 7 mg/dm2 film and sample C2 = 24 mg/dm2 film) or without the antioxidant (control sample) and frozen for 12 months at −20°C. The results confirm the efficacy of an active packaging with a natural antioxidant derived from barley husks to slow the progress of lipid hydrolysis and increase oxidative stability in hake muscle. Determination of peroxide value, conjugated dienes, free fatty acids, thiobarbituric acid reactive substances (TBARS) and anisidine value (AV) proved suitable for studying lipid hydrolysis and primary and secondary lipid oxidation over time. After 6 months of frozen storage, the TBARS levels were lower in hake packed with low‐density polyethylene with the natural antioxidant (C1 and C2) than in the control sample; at month 12, the TBARS values of C1 and C2 were lower (16 and 21%, respectively) than the control samples. AVs observed in samples C1 and C2 at month 9 were lower than those observed in the control sample at month 12. Copyright © 2011 John Wiley & Sons, Ltd.

Temperature influence on post-prandial metabolic rate of sub-Antarctic teleost fish

The influence of temperature on the aerobic metabolism and the energetic cost of food intake (Specific Dynamic Action; SDA) have been investigated in four species of Sub-Antarctic teleosts. The species were the notothenioids Paranotothenia magellanica, Patagonotothen sima and Harpagifer bispinis and the zoarcid Austrolycus depressiceps. Individuals were captured in the vicinity of Ushuaia Bay. Experimental temperatures were 10, 4 and 2 degrees C, which correspond to summer, winter and extreme winter respectively. Individual respirometry chambers and calorimetric techniques were used. Different food items were provided: crustaceans (isopods and amphipods) and Argentinean hake muscle. Interspecific analysis was done on species fed with isopods. A rapid increase in oxygen consumption was registered after meals, indicating a typical SDA response. The Duration of the SDA was longer at low temperatures. The extra energy spent during the process itself, and when expressed as a percentage of consumed food energy, decreased with decreasing temperature. The SDA Coefficient was higher for H. bispinis that were fed with isopods. We suggest that decreases in temperature diminish the metabolic cost and extend SDA. Energy-saving mechanisms could be an evolutionary advantage to minimize the energetic cost of living at low sub-Antarctic temperatures. A general model of exponential decay is suggested for the duration of SDA and Temperature, based on the present study and compiled from literature data.

Use of microbial transglutaminase and sodium alginate in the preparation of restructured fish models using cold gelation: Effect of frozen storage

Changes in physicochemical properties of raw-appearing restructured models made from hake (Merluccius capensis) muscle, using cold gelation technology by addition of sodium alginate and microbial transglutaminase (MTGase), were studied during frozen storage at − 15 °C. Among the more interesting results, addition of MTGase produced more protein aggregation in models processed by muscle homogenization and thus the protein network that formed in the gel was better than in samples with added sodium alginate. All samples presented enhanced changes in mechanical properties such as gel strength in the course of frozen storage. The protein network that formed was better organized as shown by the ultrastructure analysis and by protein aggregation data from the beginning of storage, and as a result Water Binding Capacity was greater in restructured models processed by muscle homogenization. Some of the models studied presented lipid oxidation during frozen storage, slightly more in the ones with MTGase added. The industrial relevance of the present work is to establish bases for using fish fillet trimmings and minced muscle to prepare restructured products that can be commercialized in a raw state and that could also support frozen storage in order to prolong the self-life in the market. The models studied here are not final products but the base with which to prepare them. One possibility of them is to process this kind of products with technologies normally used in whole fish muscle such as smoked fish fillets, carpaccios or marinated products. In addition, these raw restructured products can be commercialized to be cooked as a normal fresh fish fillet or pieces of fillet in a variety of preparations, and also products that contain a high amount of salt. Samples elaborated by binding larger muscle fragments imitate well the original fibrousness of the muscle so that it could be used to elaborate products similar to hamburgers or others that will be fried in batter.

Changes in Red Hake (Urophycis chuss) Muscle Induced by Different Freezing Strategies

The effect of single and double freezing at −20°C on fresh and aged red hake with two different holding times of the thawed fillets between the first and second freezing treatments was determined. In addition, twice-frozen fresh red hake was treated with two antioxidation systems to assess their effectiveness in reducing lipid oxidation. Fresh, untreated red hake at 0 time was used for comparison. Significant dimethylamine (DMA) production occurred with all frozen samples, and the differences were not marked after 7-month storage. With a storage time of 3 months, however, there were differences between the treatments in both the fresh and aged samples with the once-frozen sample producing the least DMA, the twice-frozen sample with the 24-h holding time between freezings the most, and the twice-frozen sample with a 6-h holding time between freezings giving intermediate values. All samples showed a greater than 50% loss in protein solubility as determined by lithium chloride extraction after 3 months of storage, and the protein solubility declined further when samples were tested at 7 months. Samples with the best (once-frozen fresh) and worst (twice-frozen with 24-h holding time between freezings of 5-day aged muscle) treatments had the highest thiobarbituric acid-reactive substances (TBARS) values. Both antioxidant treatments were effective in reducing TBARS development.

Effect of high hydrostatic pressure on mortality and allergenicity ofAnisakis simplexL3 and on muscle properties of infested hake

AbstractBACKGROUND:High pressure (HP) ranging from 100 to 350 MPa (1–15 min) was applied toAnisakis simplexlarvae and parasitised hake (Merluccius merluccius) muscle. The aim of the study was to kill the larvae to prevent human anisakidosis, to evaluate the effect onA. simplexallergens and to minimally alter fish muscle quality.RESULTS:The larvae were killed at pressures ≥ 200 MPa and times ≥ 1 min, producing alterations in the larva body and ruptures in the cuticle when observed by scanning electron microscopy. Nevertheless, Ani s 4 andA. simplexcrude antigens were recognised by immunoblotting and immunohistochemistry at all HPs assayed. Small changes in colour and texture were observed in fish muscle under all pressure/time conditions. Major changes were observed visually at 300 MPa, where the muscle appeared as slightly cooked. Apparent viscosity of muscle homogenates decreased significantly at longer times or higher applied pressure. No changes were detected at 200 MPa in the electrophoretic pattern of proteins treated with or without β‐mercaptoethanol, suggesting that disulfide bonds were not formed.CONCLUSION:Application of HP at 200 MPa for up to 5 min would killA. simplexlarvae, avoiding infestation of the consumer and causing small changes in the hake muscle perceived sensorially. However, HP‐treatedA. simplex‐parasitised fish would still be a potential hazard for consumers allergic to the larvae. Copyright © 2009 Society of Chemical Industry

Study of two different cold restructuring processes using two different qualities of hake (Merluccius capensis) muscle, with addition of microbial transglutaminase

AbstractBACKGROUND: Microbial transglutaminase (MTGase) can improve the mechanical and functional properties of restructured fish products without the need of thermal gelation. The present study seeks to determine whether, for different setting times, MTGase activity in restructured hake muscle made with pieces or with homogenised muscle can be affected by the quality of the protein in the raw materials.RESULTS: As regards mechanical properties, samples of both qualities subjected to the two different processes attained a suitable consistency after setting for 24 h at 5 °C. The quality of the protein in the sample is important when pieces are used for restructuring, but not when sample is homogenised. Also, there were strong correlations between residual MTGase activity up to 12 h and mechanical properties and electrophoretic band density. Water binding capacity (WBC) was not significantly altered by MTGase addition.CONCLUSION: The experimental combination of 10 g kg−1 of MTGase, 15 g kg−1 of sodium chloride and 7.5 g kg−1 of sodium caseinate was suitable for the production of minimally processed raw restructured fish products made with two different qualities of fish protein and two different restructuring processes. In the restructured products made with pieces, the protein quality significantly affected final properties, but in finely homogenised product the protein quality was less important. Copyright © 2009 Society of Chemical Industry

Influence of alginate and microbial transglutaminase as binding ingredients on restructured fish muscle processed at low temperature

AbstractBACKGROUND: In view of the increasing demand for fresh products in Western countries recently, there is considerable interest in commercialising restructured fish products having the appearance of fresh fish. A number of methods have been studied for the purpose of inducing cold gelification. Two of the most common methods, namely addition of alginates and addition of transglutaminases, have been studied mainly in connection with meat products. The present study deals with the use of alginate and transglutaminase as additives in cold gelification of minced hake (Merluccius capensis) muscle. The experiments were targeted on the effects of concentration and combined effects of additional additives on physicochemical characteristics and mechanical properties.RESULTS: As regards mechanical properties, the effectiveness of sodium alginate was improved by addition of a low concentration (1 g kg−1) of calcium chloride (CaCl2), whereas a higher concentration (10 g kg−1) reduced the binding ability of the alginate. The presence of sodium caseinate (15 g kg−1) in combination with microbial transglutaminase (MTGase) was important in helping to increase the work of penetration in fish gels induced at low temperature. Examination of the chemical properties of the muscle gels showed that sodium alginate did not establish covalent protein–protein bonds, while MTGase dramatically increased the number of covalent bonds formed between adjacent muscle proteins.CONCLUSION: With both ingredients, thermostable fish gels of good quality were produced at temperatures below 10 °C. Gels induced by sodium alginate were considerably improved by addition of 1 g kg−1 CaCl2. However, gels induced by MTGase were better suited for the preparation of restructured products. Copyright © 2008 Society of Chemical Industry

Concentration of antioxidant compounds and lipid peroxidation in the liver and white muscle of hake (Merluccius merluccius L.) in the Adriatic sea

Specimens of a marine freshwater fish - hake (Merluccius merluccius L.) - were collected at the localities of Platamuni and Valdanos (Montenegro, South Adriatic) in the spring of 2003. Our results showed higher concentrations of LPO, Vit C, and Vit E in the liver in comparison with white muscle. The concentration of LPOin both tissues was higher, while that of Vit E was lower at Valdanos compared to Platamuni. These differences in parameters of oxidative stress are partly due to differences in temperature and the concentrations of nitrites, nitrates, and detergents in the waters of Valdanos compared to Platamuni.

Development of an indirect α‐actinin‐based immunoassay for the evaluation of protein breakdown and quality loss in fish species subjected to different chilling methods

Summary α‐Actinin release from the myofibrillar protein fraction to the sarcoplasm can be considered as an accurate proteolysis index in seafood muscle. The main objective of the present study was to develop a specific enzyme‐linked immunosorbent assay (ELISA), based on the use of a monoclonal antibody against α‐actinin to evaluate the degree of proteolysis in two different chilled fish species – European hake (Merluccius merluccius) and turbot (Psetta maxima) – kept under two different storage systems: flake ice and slurry ice. Comparison with sensory assessment, K‐value and sarcoplasmic protein profiles was carried out. A different degree of proteolysis could be observed in both fish species; thus, the immunoassay was shown to be useful in monitoring the protein degradation events in hake muscle especially under flake ice storage. In the case of turbot, as very low proteolysis development could be obtained, the assay was not suitable for assessing quality changes. A different break point of immunoassay values for each fish species is suggested.

Identification, characterization and deduced amino acid sequence of the dominant protease from Kudoa paniformis and K. thyrsites: A unique cytoplasmic cysteine protease

Kudoa paniformis and Kudoa thyrsites (Myxozoa: Myxosporea) infections are associated with severe proteolysis of host muscle tissue post-mortem. The present study was undertaken to identify and characterize the protease responsible for myoliquefaction and determine mechanisms controlling protease function in vivo. N-terminal sequence analysis of partially purified protease from hake muscle infected with K. paniformis and K. thyrsites revealed a 23 amino acid sequence that aligned with cysteine proteases. Enzyme inhibition assays confirmed the presence of an essential active site cysteine residue. Using the above K. paniformis amino acid sequence data, a corresponding cDNA sequence from K. thyrsites plasmodia was elucidated revealing a cathepsin L proenzyme (Kth-CL). The translated amino acid sequence lacked a signal sequence characteristic of lysosomal and secreted proteins suggesting a unique cytoplasmic location. Only the proenzyme form of Kth-CL was present in Atlantic salmon muscle anti-mortem but this form became processed in vivo when infected muscle was stored at 4 degrees C. The proenzyme of Kth-CL showed uninhibited activity at pH 6.0, negligible activity at pH 6.5 and no measurable activity at pH 7.0 whilst the processed protease showed stability and function over a broad pH range (pH 4.5-8.8). The pH dependent processing and function of Kth-CL was consistent with histidine residues in the proregion playing a critical role in the regulation of Kth-CL.

RELATION BETWEEN THE FREE AMINO ACIDS, ANSERINE AND THE TOTAL VOLATILE BASIC NITROGEN PRODUCED IN MUSCLE OF HAKE (MERLUCCIUS MERLUCCIUS, L.) DURING ICED STORAGE

This work studied the development of free amino acids (FAAs) and dipeptide anserine as quality indices for gutted hake stored in ice for 25 days. The correlation of these compounds was determined with total volatile basic nitrogen (TVBN) which has been used as a quality index, for fish stored in ice. The most abundant free amino acids in hake muscle were found to be threonine, glycine, alanine, glutamic acid, β-alanine methylhistidine. lysine and the dipeptide, anserine. The only hydrophobic free ammo adds which exhibit significant differences (P<0.05) throughout storage was tryptophan. moreover, this amino acid exhibited a very high correlation (r=0.951) with TVBN. A significant decrease in anserine (P<0.05) correlated with the increases in 1-methylhistidine and β-alanine throughout storage. These changes also exhibited a very high correlation with TVBN. Therefore, 1-methylhistidine, β-alanine anserine and tryptophan could be used as quality parameters for hake stored in ice.

Evaluation of Various Infused Cryoprotective Ingredients for Their Freeze–Thaw Stabilizing and Texture Improving Properties in Frozen Red Hake Muscle

Various cryoprotective ingredients were evaluated for their freeze-thaw stabilizing and texture improving properties during 6-mo frozen storage when red hake (Urophycis chuss) fillets were injected with sorbitol-sodium tripolyphosphate (STPP) (40% to 60% : 3%), 1.5% alginate, or 0.75% alginate with soy protein isolate (SPI)-sorbitol-STPP (5% to 10% : 40% : 3%). Injection of 10% SPI, 1.5% alginate, or 0.75% alginate-5% SPI effectively improved water binding and retarded the freeze-induced texture changes when drip and cooking loss, centrifugal expressible moisture, protein extractability, SDS-PAGE profile, and Instron and sensory texture were assessed. The sorbitol and STPP combination was not as effective as alginate and SPI. Freeze-susceptible whitefish can be injected with proper cryoprotective ingredients for improved frozen storability.

Esterase activities and lipid peroxidation levels in offshore commercial species of the NW Mediterranean Sea

There is a lack of information on monitoring neurotoxicity in offshore commercial species. To help fill this gap, we sampled hake ( Merluccius merluccius) and Norway lobster ( Nephrops norvegicus) in fishing grounds off the coast of l’Ametlla de Mar (NW Mediterranean) in June 2005 at a depth of 100 m and 400 m. Additionally, at 400 m depth, two other fish species, Micromesistius poutassou and Phycis blennoides were included. Neurotoxicity markers such as Colinesterases (ChEs), namely acethyl- (AChE), butyryl- (BChE), propionyl- (PrChE) and carboxilesterase (CbE) were measured in muscle. Lipid peroxidation (LP), a marker of oxidative damage, was also included. The results are discussed in relation to the animal’s sex, size and fishing depth. A comparison of esterases and LP levels between muscle and liver of hake and between muscle and hepatopancreas of Norway Lobster was made. AChE was dominant in muscle and CbE in hepatopancreas. No differences between fish species were seen for AChE. However, N. norvegicus, presented lower levels of ChEs and LP. A size-dependence in ChEs was seen for M. merluccius, with larger animals showing significantly lower activities ( p < 0.05). Sex-dependence was seen in N. norvegicus for most esterases, except AChE, with males displaying higher activities ( p < 0.05). A sampling-depth effect was also seen in the crustacea, with animals from 100 m generally presenting lower esterase activities and higher LP levels.

Myosporidium merluccius n. g., n. sp. Infecting Muscle of Commercial Hake (Merluccius sp.) from Fisheries near Namibia

A new species of Microsporidia classified to a new genus was observed in the trunk muscle of commercial hake (Merluccius capensis/paradoxus complex) from Namibian fisheries. Macroscopic examination revealed thin and dark filaments inserted among muscle fibers. Inside the filaments were many sporophorous vesicles with about 30-50 spores per vesicle. The shape of the spore was pyriform and the extruded polar filament was of moderate length (up to 4.29 microm, n=12). This new species of Microsporidia is described using macrophotography, microphotography, staining, and transmission electron microscopy (TEM), as well as molecular methods. Its 16S rRNA was found to be similar to that of Microsporidium prosopium Kent et al., 1999, while both sequences were quite different from 16S rRNA sequences known for other Microsporidia. Nevertheless, this new species is separated morphologically from M. prosopium by the presence of 11-12 anisofilar coils and the formation of the xenoma at the site of infection. Type species.

LIPID CHANGES IN FROZEN STORED FILLETS FROM PRE- AND POSTSPAWNED HAKE (MERLUCCIUS HUBBSI MARINI)

The lipid composition of frozen stored fillets from pre- and postspawned hake was studied. The total lipid (TL) content in the chloroform/methanol extract from unfrozen postspawned hake was four times higher than that of prespawned fish. After freezing, the TL content of postspawning hake muscle remained unchanged whereas the TL extracted from prespawning fish muscle increased about 90%. The TL extractability of muscle from fish in both different gonadal conditions was not affected by frozen storage. Lipolysis in frozen stored fillets from prespawned hake occurs principally by hydrolytic action on phospholipids (PL), and phosphatidylcholine was the main PL hydrolyzed. Triacylglycerols were the main substrates hydrolyzed in frozen stored fillets from postspawned hake. Freezing and frozen storage affected polyenoics and n-3 fatty acids (FA). The decrease in the contents of n-3 FA in fillets from postspawned hake was lower than that observed in fillets from prespawned fish.

Raman spectroscopic study of structural changes in Hake (Merluccius merluccius L.) muscle proteins during frozen storage.

This paper examines changes in the structure and functionality of fish muscle proteins at frozen storage temperatures known to render very different practical storage lives (-10 and -30 degrees C). Apparent viscosity and dimethylamine (DMA) content showed drastic temperature-related differences during storage. Raman spectroscopy revealed the occurrence of some structural changes involving secondary and tertiary protein structures. The changes in secondary structure were quantified, showing an increase of beta-sheet at the expense of alpha-helix structure. The nuC-H stretching band near 2935 cm(-)(1) increased in intensity, indicating denaturation of the muscle proteins through the exposure of aliphatic hydrophobic groups to the solvent. These structural changes were more pronounced at -10 degrees C but occurred at both storage temperatures, whereas changes in apparent viscosity and DMA only occurred in storage at -10 degrees C. The possible utility of these structural changes for quality assessment is discussed.

Enhanced shelf-life of chilled European hake ( Merluccius merluccius) stored in slurry ice as determined by sensory analysis and assessment of microbiological activity

Slurry ice, a biphasic system consisting of small spherical ice crystals surrounded by seawater at subzero temperature, was evaluated as a new chilled storage method for whole European hake ( Merluccius merluccius), a gadoid fish species of remarkable commercial interest, and this method was compared with a control batch stored for 19 days in traditional flake ice. The results obtained in the sensory analysis indicated a significant extension of shelf-life – from 5 days (flake ice batch) to 12 days (slurry ice batch) – in the latter batch, mainly deriving from a better maintenance of both external and fresh odour, the appearance of the gills and consistency. The slurry ice batch exhibited a significantly lower increase in pH as compared to the flake ice batch, this indicating a better control of the former over alkalinising microflora. Thus, significantly lower counts of total aerobes and proteolytic bacteria were also attained in hake muscle stored in slurry ice, microbial numbers reaching average differences of ≈1 log unit before 12 days of storage and above 2 or 3 log units, respectively, when this period was extended up to 19 days. The formation of total volatile base-nitrogen and trimethylamine were also significantly lower in the slurry ice batch after 12 days of storage. According to the parameters assessed, storage of European hake in slurry ice extends the shelf-life of this species and allows a better maintenance of sensory and microbiological quality.