大西洋三文鱼和虹鳟鱼双重PCR检测方法的建立及应用

为了实现大西洋三文鱼和虹鳟鱼的物种掺假快速检测,本研究建立了一种基于双重PCR（普通和荧光）的物种鉴定方法。通过序列分析,分别基于线粒体COⅠ和Cyt b基因设计大西洋三文鱼和虹鳟鱼特异性引物,验证引物的特异性并对双重PCR反应体系进行优化,建立大西洋三文鱼和虹鳟鱼的双重PCR快速鉴定方法。利用本研究设计的引物,仅大西洋三文鱼和虹鳟鱼可以分别扩增出108 bp和207 bp的特异性条带,而其他23种非目标物种均未有扩增条带。经验证,在双重PCR反应体系中,大西洋三文鱼和虹鳟鱼引物的最佳添加量分别为0.1和0.4 μmol/L,熔解温度分别约为81.5 ℃和85 ℃。利用该方法从29份市售“三文鱼”样品中检测到6份大西洋三文鱼和3份虹鳟鱼,且与DNA条形码比对结果一致。本研究建立的大西洋三文鱼和虹鳟鱼双重PCR（普通和荧光）鉴定方法具有良好的特异性和适用性,为大西洋三文鱼和虹鳟鱼物种鉴定提供了可靠的技术手段。     

双重实时荧光PCR法鉴别大西洋鲑鱼和虹鳟鱼

目的建立双重实时荧光PCR法同时鉴别大西洋鲑鱼和虹鳟鱼的检测方法。方法在现有单一物种检测方法的基础上,应用多重实时荧光PCR检测技术对大西洋鲑鱼和虹鳟鱼同时进行检测,并对PCR反应时间和反应温度进行优化。结果该方法通过1管PCR反应实现对大西洋鲑鱼和虹鳟鱼的同时鉴别,反应时间缩短至1 h以内,方法的特异性好,灵敏度可达到0.1%(DNA质量分数)。结论该方法检测效果好、时间短、成本低,可为监管部门解决三文鱼市场监管问题提供技术手段。     

A practical quality index method (QIM) developed for aquacultured rainbow trout (Oncorhynchus mykiss)

Quality index method (QIM) was developed for whole (W) and gutted (G) rainbow trout during ice storage. Draft schemes were modified and final schemes consisted of 30 and 15 demerit points, and 12 and 14 days of shelf life was found for whole and gutted rainbow trout, respectively. Linear regression was calculated with storage time and correlation of QI scores was found to be 0.98 and 0.99 for W and G samples, respectively. Moreover, the developed QIM for W and G rainbow trout with this study is a nondestructive and rapid method for the sensory evaluation of rainbow trout.     

Differentiation of the rainbow trout (Oncorhynchus mykiss) from Atlantic salmon (Salmon salar) by the AFLP-derived SCAR

A species-specific SCAR marker for rainbow trout, which was used to detect adulteration and fraudulent labeling in Atlantic salmon products, has been developed based on the AFLP analysis and evaluated in this study. The SCAR marker could be amplified and visualized in 1% agarose gel in all tested rainbow trout samples and absent in all salmon samples. Using DNA admixtures, the detection of 1% (0.5 ng), 10% (5 ng) rainbow trout DNA in Atlantic salmon DNA for fresh and processed samples, respectively was readily achieved. The molecular approach was sensitive and demonstrated to be a rapid and reliable method for identifying frauds in salmon products and could be extended for applications of species identification in food industry.     

Tissue distribution and elimination of deoxynivalenol and ochratoxin A in dietary-exposed Atlantic salmon (Salmo salar)

ABSTRACT

Post-smolt Atlantic salmon (Salmo salar) were fed standard feed with added 2 or 6 mg kg^–1 pure deoxynivalenol (DON), 0.8 or 2.4 mg kg^–1 pure ochratoxin A (OTA), or no added toxins for up to 8 weeks. The experiments were performed in duplicate tanks with 25 fish each per diet group, and the feed was given for three 2-h periods per day. After 3, 6 and 8 weeks, 10 fish from each diet group were sampled. In the following hours after the last feeding at 8 weeks, toxin elimination was studied by sampling three fish per diet group at five time points. Analysis of DON and OTA in fish tissues and plasma was conducted by liquid chromatography-mass spectrometry and high-pressure liquid chromatography with fluorescence detection, respectively. DON was distributed to the liver, kidney, plasma, muscle, skin and brain, and the concentrations in liver and muscle increased significantly from 3 to 8 weeks of exposure to the high-DON diet. After the last feeding at 8 weeks, DON concentration in liver reached a maximum at 1 h and decreased thereafter with a half-life (t_1/2) of 6.2 h. DON concentration in muscle reached a maximum at 6 h and was then eliminated with a t_1/2 = 16.5 h. OTA was mainly found in liver and kidney, and the concentration in liver decreased significantly from 3 to 8 weeks in the high-OTA group. OTA was eliminated faster than DON from various tissues. By using Norwegian food consumption data and kinetic findings in this study, we predicted the human exposure to DON and OTA from fish products through carryover from the feed. Following a comparison with tolerable daily intakes, we found the risk to human health from the consumption of salmon-fed diets containing maximum recommended levels of these toxins to be negligible.     

Effect of gamma irradiation and frozen storage on chemical and sensory characteristics of rainbow trout (Oncorhynchus mykiss) fillet

The present study was conducted to evaluate the combined effect of low‐dose gamma irradiation (1, 3 and 5 kGy) and frozen storage (5 months at ?20 °C) on chemical and sensory characteristics of rainbow trout (Oncorhynchus mykiss) fillet. Our statistical analysis showed that irradiation process and frozen storage time had a significant effect (P < 0.05) on total volatile nitrogen (TVN), peroxide value (PV), thiobarbituric acid (TBA) and pH. The level of all of these factors increased with increasing frozen storage time. At the end of the fifth month of frozen storage, the lowest and the highest level of TVN, PV and TBA were corresponding to the irradiated samples at 3 and 5 kGy, respectively. In terms of the overall acceptability of their texture, odour, colour and taste, irradiated samples at 3 kGy had the best quality and remained acceptable after 5 months frozen storage. The optimum dose of gamma radiation of rainbow trout fillets according to chemical and sensory analysis was obtained at 3 kGy.     

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America

Abstract: The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5′ cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) “barcode” sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. Practical Application: This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.     

Influence of chloride and metals on silver bioavailability to Atlantic salmon (Salmo salar) and Rainbow trout (Oncorhynchus mykiss) yolk-sac fry

The effects of differing water chloride concentrations (0-10 mM) or competing metals [Cu(II), Cd(II), Zn(II), Pb(II), Co(II) (1-10,000 nM)] on Ag(I) uptake in yolk-sac fry of two salmonid species, the Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), were studied. None of the metals tested were strong competitors of Atlantic salmon yolk-sac fry whole body Ag(I) influx. Inhibition of Ag(I) influx was only seen with a 100-fold excess of Cu(II) or Cd(II) or a 1000-fold excess of Pb(II) or Co(II). At these concentrations, the degree of competition appears to be directly proportional to the conditional stability constant of the competing metal to the gill (metal-gill log K). The range of [Cl-] allowed an assessment of Ag+, AgCl(aq), and AgCl2- bioavailability. The pattern of Ag(I) uptake was similar for each fish species. At <1 mM Cl-, where the [Ag+] dominates, the Ag(I) accumulation rate was constant. Above 1 mM Cl-, where the [AgCl(aq)] is dominant and the [AgCl2-] increases, there was a decline in Ag(I) uptake rate. However, even when very little Ag+ was present (i.e., at 10 mM Cl-) Ag(I) accumulated, albeit at a lower rate. This was suggestive of passive influx by AgCl(aq) and indicated little or no entry of negatively charged silver chloride complexes. The decline in Ag(I) uptake above 1 mM Cl- demonstrated that, if Ag(I) was present as both Ag+ and AgCl(aq), salmonid Ag(I) accumulation was dominated by Ag+ uptake. Therefore, the order of bioavailability of the Ag(I) species was determined as Ag+ > AgCl(aq) > AgCl2-.     

Immunostick Colorimetric ELISA Assay for the Identification of Smoked Salmon,Trout and Bream

A colorimetric ELISA using immunostick tubes has been developed for the rapid identification of smoked salmon (Salmo salar), trout (Oncorhynchus mykiss) and bream (Brama raii). The assay uses polyclonal antibodies raised in rabbits against muscle-soluble proteins of smoked salmon (anti-SSP), trout (anti-TSP) and bream (anti-BSP) and rendered species-specific by blocking them with the heterologous species of smoked fish. The blocked antibodies were used for the immunorecognition of the smoked fish samples bound to the paddles of immunostick tubes and the immunocomplex detected with peroxidase-conjugated goat anti-rabbit immunoglobulins. The blue colour developed by conversion of the peroxidase substrate allows unequivocal identification of S salar, O mykiss and B raii. © 1997 SCI.     

The effects of natural antioxidants and lighting conditions on the quality of salmon (Salmo salar) fillets packaged in modified atmosphere

The effect of lighting conditions (darkness, a low‐UV colour‐balanced lamp and supermarket fluorescents), along with the application of natural antioxidants (rosemary extract and ascorbic acid) on shelf‐life of salmon (Salmo salar) fillets packaged in modified atmosphere and stored at 1 ± 1 °C was studied. Darkness and lighting with low‐UV colour‐balanced lamps led to an extension of shelf‐life compared with conventional light, as assessed by a* values, lipid oxidation (TBARS value) and sensory evaluation. The application of natural antioxidants on the surface of MAP salmon fillets gave rise to a delay of lipid oxidation as well as an improvement of the sensory quality, mainly in the case of conventional lighting conditions. Copyright © 2005 Society of Chemical Industry     

Rapid PCR-RFLP Method for the Identification of Marine Fish Fillets (Seabass, Seabream, Umbrine, and Dentex)

A rapid and reliable PCR‐RFLP method was optimized to identify marine fish fillets. Seabass, seabream, umbrine, and dentex were considered in the study. After DNA extraction and PCR, the 359 bp amplification products, obtained from gene encoding the cytochrome b, were subjected to restriction enzyme analysis. All the enzymes tested were not able to distinguish all the 5 species at the same time, but the combination of the results obtained from the digestion HaeIII and NlaIII can be used to differentiate the fish fillets considered. The method described is sensitive, rapid, and reliable, and it could be used to expose fraudulent substitutions with less valuable fish.     

Effects of freezing and thawing processes on the quality of Atlantic salmon (Salmo salar) fillets

ABSTRACT:  High-pressure processing is finding a growing interest in the food industry. Among the advantages of this emerging process is the ability to favorably freeze and thaw food. This study aims at comparing the effect of different freezing and thawing processes on the quality of Atlantic salmon fillets. Atlantic salmon ( Salmo salar ) samples were frozen by Pressure-Shift Freezing (PSF, 200 MPa, −18 °C) and Air-Blast Freezing (ABF, −30 °C, 4 m/s). Samples were stored 1 mo at −20 °C and then subjected to different thawing treatments: Air-Blast Thawing (ABT, 4 °C, 4 m/s), Immersion Thawing (IMT, 20 °C), and Pressure-Assisted Thawing (PAT, 200 MPa, 20 °C). Changes in texture, color, and drip loss were investigated. The toughness of the PSF samples was higher than that of the ABF sample. The modification of color was more important during high-pressure process than during the conventional process. The PSF process reduced thawing drip compared with ABF. The presence of small ice crystals in the pressure-shift frozen sample is probably the major reason leading to the reduced drip volumes. The freezing process was generally much more influent on quality parameters than the thawing process. These results show the interaction between freezing and thawing processes on selected quality parameters.     

Physical measurement of the quality of fresh scad (Trachurus trachurus) and rainbow trout (Oncorhynchus mykiss) during ice storage using the RT Freshmeter

The RT Freshmeter was used to obtain a quality index of two Portuguese fish, scad ( Trachurus trachurus ) and farmed rainbow trout ( Oncorhynchus mykiss ). The curves obtained were comparable to those previously published using the GR Torrymeter. However variations were lower during the first phase of storage and it was observed that the curves could be divided into two distinct regions. The slope of the linear regression was higher during the first 5 days of storage, representing the first region of the curve, in both fish studied.
It is proposed that the two different rates of change of the quality index obtained with the meter reflect the two predominant spoilage mechanisms, enzymic and microbiological. The RT Freshmeter measurements are compared to organoleptic changes.
Preliminary results obtained with the RT Freshmeter on rainbow trout fillets without skin indicate that this instrument is not suitable for use with this product due to large variations and curve slopes close to zero.     

Effect of heating fatty fish: Baltic herring (Clupea harengus membras), European sprat (Sprattus sprattus) and rainbow trout (Oncorhynchus mykiss) on lipid oxidation and contents of eicosapentaenoic and docosahexaenoic acids

In this study, analyses were carried out to establish the impact of heating three species of fatty fish: trouts, herrings and sprats, on the lipids oxidation and on contents of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. The comminuted fish tissue was heated at restricted access of oxygen at temperature of 60, 100 and 160 °C for 15–120 min. Lipids, extracted with the Bligh‐Dyer method, were determined for peroxide value (PV) and anisidine value (AV). The Fatty acid methyl esters were prepared directly from the tissue, whereas contents of EPA and DHA were determined with the GC/MS method. Depending on the temperature applied, 15‐min heating of fish meat tissue caused 30–80% decrease of PN and 20–40% decrease of AV, on average. Generally, the continued heat treatment caused successive decreases in both PV and AV and the rate of this process was observed to increase along with an increasing temperature. The heating of trout and sprats at temperature of 60, 100 and 160 °C even for 1–2 h did not evoke losses either in EPA or in DHA content. In turn, in the case of herring caught during the pre‐spawning season, ca. 90–120 min of heat treatment contributed to ca. 20–25% decrease in contents of these fatty acids.     

Development of a genetic method for the identification of salmon,trout, and bream in seafood products by means of PCR–RFLP and FINS methodologies

In the present study, two alternative methods for identifying 13 salmon, trout and bream species were developed. Both of them are based on polymerase chain reaction (PCR) amplification of a cytochrome b gene fragment. Subsequently, different techniques were assayed to assign the PCR amplicons previously obtained to particular species. The first one is based on the restriction fragment length polymorphism (RFLP) and includes three endonucleases for generating species-specific restriction profiles, while the second one is based on the phylogenetic analysis of DNA sequences. The main novelty of this work lies in the applicability of the developed methods to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 25 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those incorrectly labeled (16%). Therefore, these methods are useful to check the fulfillment of labeling regulations for seafood products, verify the correct traceability in commercial trade, and for fisheries control.     

Determination of astaxanthin stereoisomers and colour attributes in flesh of rainbow trout (Oncorhynchus mykiss) as a tool to distinguish the dietary pigmentation source

The presence of carotenoids in animal tissue reflects their sources along the food chain. Astaxanthin, the main carotenoid used for salmonid pigmentation, is usually included in the feed as a synthetic product. However, other dietary sources of astaxanthin such as shrimp or krill wastes, algae meal or yeasts are also available on the market. Astaxanthin possesses two identical asymmetric atoms at C-3 and C-3' making possible three optical isomers with all-trans configuration of the chain: 3S,3'S, 3R,3'S, and 3R,3'R. The distribution of the isomers in natural astaxanthin differs from that of the synthetic product. This latter is a racemic mixture, with a typical ratio of 1:2:1 (3S,3'S:3R,3'S:3R,3'R), while astaxanthin from natural sources has a variable distribution of the isomers deriving from the different biological organism that synthesized it. The high-performance liquid chromatographic (HPLC) analysis of all-trans isomers of astaxanthin was performed in different pigment sources, such as red yeast Phaffia rhodozyma, alga meal Haematococcus pluvialis, krill meal and oil, and shrimp meal. With the aim to investigate astaxanthin isomer ratios in flesh of fish fed different carotenoid sources, three groups of rainbow trout were fed for 60 days diets containing astaxanthin from synthetic source, H. pluvialis algae meal and P. rhodozyma red yeast. Moreover, the distribution of optical isomers of astaxanthin in trout purchased on the Italian market was investigated. A characteristic distribution of astaxanthin stereoisomers was detected for each pigment sources and such distribution was reproduced in the flesh of trout fed with that source. Colour values measured in different sites of fillet of rainbow trout fed with different pigment sources showed no significant differences. Similarly, different sources of pigment (natural or synthetic) produced colour values of fresh fillet with no relevant or significant differences. The coefficient of distance computed amongst the feed ingredient and the trout fillet astaxanthin stereoisomers was a useful tool to identify the origin of the pigment used on farm.     

A trout (Oncorhynchus mykiss) perfusion model approach to elucidate the role of blood removal for lipid oxidation and colour changes in ice-stored fish muscle

Whole body saline-perfused rainbow trout (Oncorhynchus mykiss) was ice-stored for 4 weeks and compared with unwashed/washed minces from unbled and bled trout in terms of rancid odour, peroxide value (PV), thiobarbituric acid reactive substances (TBARS) and redness loss. Muscle from saline-perfused fish, which had 72% less total haem, was deficient in rancid odour during the whole storage, while bled (54% less haem) and unbled samples developed rancid odour already after ~4 and 2 days; higher intensity without bleeding. PV/TBARS also developed in the order unbled > bled > perfused samples; however, PV/TBARS were not as completely prevented as rancid odour after perfusion. Saline washing (3 × 3 volumes) of unbled mince removed 84% haem and yielded the second most stable sample while saline washing (1 × 1 volumes) destabilised unbled mince, despite 64% haem removal. Concurrent antioxidant removal during washing of minces obviously counteracted the effect of blood removal and washing fish mince with small volumes of solution should be used with great care.     

Analysis of incurred crystal violet in Atlantic salmon (Salmo salar L.): comparison between the analysis of crystal violet as an individual parent and leucocrystal violet and as total crystal violet after oxidation with 2,3-dichloro-5,6-dicyanobenzoquinone

Due to on-going concern about the occurrence of triphenylmethane dye residues in fish destined for human consumption, a depletion study of crystal violet in salmon was carried out. Atlantic salmon less than 12 months old were exposed to crystal violet in fresh water at 15°C and subsequently sampled at 1, 7, 14, 28, 63 and 91 days after exposure. The salmon were then analysed by two analytical methods. In the first method, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) was used to oxidise leucocrystal violet to its parent form. Total parent crystal violet was then analysed by LC-MS/MS. In the second method, crystal violet and leucocrystal violet were analysed individually by LC-MS/MS without oxidation. Both methods gave comparable results for total crystal violet concentrations, with a correlation of r ^2?=?0.69. Statistical treatment for 88 incurred salmon samples showed no significant difference between the two sets of results with t?=?1.68 and t _crit?=?1.99. Up to 98% of crystal violet was metabolised to its leuco form in the salmon after 1 day of exposure and could be detected at significant concentrations (approximately 20?µg?kg^–1) 91 days after exposure. The depletion data also suggest that crystal violet has a half-life of approximately 15–16 days in salmon.