Molecular Identification of Commercial Spicy Pollack Roe Products by PCR-RFLP Analysis

ABSTRACT: Spicy pollack roe products are a popular seafood item made from fish eggs that should be made with salt-cured mature roes of walleye pollack Theragra chalcogramma . Because of high demand and poor catch of walleye pollack, however, spicy pollack roe products are often susceptible to substitution with roes of closely related codfish. In this study, a simple method identifying the ingredients of commercial spicy pollack roe products was developed to differentiate walleye pollack from codfish substitutes such as gray cod Gadus macrocephalus using PCR-RFLP (Restriction Fragment Length Polymorphism) analysis. PCR amplification of the mitochondrial cytochrome b gene yielded single fragments commonly from pollack and cod. Direct digestion of the PCR products with Mph 11031 restriction enzyme showed an unique restriction fingerprint only in pollack. This PCR-RFLP analysis enabled the reliable identification of commercial spicy pollack roe products made by only pollack roes from products padded with cod roes. It thus can be useful to expose substitution of pollack roes with lower valued codfish roes in commercial spicy pollack roe products.     

Effect of processing on amine formation and the lipid profile of cod (Gadus morhua) roe

The processing of fish roe leads to changes in its chemical composition, the extent of which depends on the techniques and additives employed. This study aimed to investigate the effects of ripening temperature and the use of sodium benzoate and citric acid on the quality of ripened cod roe, with respect to the contents of volatile base nitrogen (VBN), trimethylamine (TMA), biogenic amines (BA) and on the lipid composition. In comparison with fresh roes, ripened roes presented higher contents of VBN, TMA, BA and the proportion of free fatty acids regardless of the temperature and additives used during the ripening process. The greatest increases were observed in the samples ripened at 17 °C without additives, in which histamine was detected at 8.8 mg/100 g. A low ripening temperature was the main factor responsible for minimising changes in the cod roe composition. The addition of sodium benzoate as a preservative or citric acid to decrease the pH value had a significant effect in maintaining the quality of the cod roes, mainly at high ripening temperature.     

Assessment of quality of cod roes and relationship between quality and maturity stage

Cod roe landings are subject to great variability regarding their quality and subsequent price for the food industry. A scheme was developed for the classification of cod roe into four quality grades (A, B, C and D) based on appearance and texture attributes as described by cod roe traders. Evaluation of the maturity stage of 80 commercial cod ovaries through the frequency distribution of oocyte diameter and the percentage presence of empty follicular sacks revealed that cod ovaries with commercial value are at stage 4 (ripe or vitellogenic), stage 5 (spawning) and stage 6 (spent) of development. The designated maturity stages were found to be closely related to the described quality grades. This relationship implied that vitellogenic ovaries (stages 4a, 4b and 4c) were of A quality, early spawning ovaries (stage 5a) were most often classed as B quality, advanced and late spawning ovaries (stage 5b and 5c) were of C quality, whilst spent ovaries (stage 6) were of the lowest quality grade (D). Several physicochemical characteristics recorded (eg pH, membrane features) were also found to be related to the quality and the maturity stage of the ovaries. There is a significant relationship between roe quality and moisture content, with roes of higher quality having a lower moisture content. © 1999 Society of Chemical Industry     

DNA‐Based Methods for the Identification of Commercial Fish and Seafood Species

ABSTRACT: The detection of species substitution has become an important topic within the food industry and there is a growing need for rapid, reliable, and reproducible tests to verify species in commercial fish and seafood products. Increases in international trade and global seafood consumption, along with fluctuations in the supply and demand of different fish and seafood species, have resulted in intentional product mislabeling. The effects of species substitution are far‐reaching and include economic fraud, health hazards, and illegal trade of protected species. To improve detection of commercial seafood fraud, a variety of DNA‐based techniques have been developed, including Multiplex PCR, FINS, PCR‐RFLP, PCR‐RAPD, PCR‐AFLP, and PCR‐SSCP, which are all based on polymorphisms in the genetic codes of different species. These techniques have been applied in the differentiation of many types of fish and seafood species, such as gadoids, salmonids, scombroids, and bivalves. Some emerging technologies in this field include the use of real‐time PCR, lab‐on‐a‐chip, and DNA microarray chips. In this review article, the major DNA‐based methods currently employed in the authentication of commercial fish and seafood species are discussed and future trends are highlighted. Examples of commercial applications and the use of online database resources are also considered.     

DNA检测技术在鳕鱼及其制品鉴定中的应用

鳕鱼是经济价值高和营养价值高的重要食用鱼类,但由于市场上鱼类俗名混乱问题,以及不同鱼种之间价值的巨大差异,错误标识鳕鱼产品或以次充好的现象时有发生,有可能因为含有过敏原成分或有毒成分造成食品安全风险。因此,需要快速可靠的鳕鱼成分鉴定方法和来保障市场监管。目前,鳕鱼及其制品的鉴定方法主要为DNA检测技术。本文主要介绍了DNA指纹图谱PCR-RFLP技术、DNA条形码技术、环介导等温扩增技术和实时荧光定量PCR技术的原理、优缺点、在鳕鱼及其制品鉴定中的应用,最后讨论了鳕鱼及其制品鉴定方法的发展趋势和监管建议。     

On the specificity of tuna-directed primers in PCR-SSCP analysis of fish and meat

Single strand conformation polymorphism (SSCP) of an amplicon (148 bp) obtained by polymerase chain reaction (PCR) of the mitochondrial cytochrom b gene used to identify tuna species was studied with other fish and animal species. Single-stranded DNA (ssDNA) patterns of two to four strong bands were obtained with blue ling, carp, haddock, mackerel, mackerel shark, saithe, catfish, Alaska pollack, and skipjack which, however, differed from those obtained with tuna samples. Other fish species resulted in weak (cod, spined dogfish) or no ssDNA bands (Atlantic salmon, halibut, herring, pike-perch, plaice, redfish, sprat, trout). Samples from animals other than fish resulted in strong ssDNA bands differing from those of tuna and from each other (crayfish; cattle, European rabbit, fallow deer, hare, horse, red deer, roe deer; goose, turkey), in bands differing from tuna but not from each other (domestic goat/sheep, domestic pig/wild boar), or in weak bands (octopus, shrimp; chicken, duck). Increasing the stringency of PCR caused a more pronounced difference between strong and weak ssDNA bands. Inter-laboratory reproducibility of the method was good.     

Nuclear DNA barcodes for cod identification in mildly-treated and processed food products

Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group. DNA-based approaches (using either nuclear or mitochondrial DNA) are most commonly used in this case, due to their high specificity and to the high resilience of the target molecules to food processing techniques. In this article, we identified, using an automated screening approach, novel barcode regions and their associated primers in the nuclear genome, to be used for the efficient identification of Gadoids. The barcode regions were tested on official and commercial samples, raw or mildly treated products, like frozen, or salted, as well as pre-cooked complex mixtures and processed samples, using next-generation sequencing (NGS) technique. The method proposed could complement existing fish identification strategies in establishing an efficient framework to detect and prevent frauds along the food chain.     

Quantification of total viable bacteria on fish fillets by using ethidium bromide monoazide real-time polymerase chain reaction

Real-time PCR based on universal primers for amplification of a highly conserved bacterial 16S rDNA sequence was utilized in conjunction with the treatment of extracted bacterial cells with ethidium bromide monoazide (EMA) for the differential enumeration of viable and dead cells on cod fillets. Amplification of DNA from dead bacterial cells was successfully inhibited by EMA, whereas the DNA from viable cells was readily amplified. The detection range of the EMA real-time PCR assay was linear from 1 x 10(1) to 1 x 10(5) mixed bacterial genomic targets per PCR derived from broth cultures of fish tissue. The minimum detection limit of bacteria was found to be 1 x 10(1) genomic units/real-time PCR, equivalent to 1 x 10(5) CFU per gram of tissue. The EMA real-time PCR allowed construction of a standard curve obtained by plotting the log of genomic targets from strictly viable cells against resulting PCR cycles (Ct values) that facilitated quantification of total viable bacteria from fish fillets. The log of the total number of genomic DNA targets from EMA treated cells and plate counts from six randomly procured cod fillets were found not to be statistically different with the exception of one fillet. The process of freezing and thawing fillet tissue resulted in a drop in mean colony forming units (CFU) detected by plate counts from log 8.5+/-0.2 to log 8.1+/-0.1. A similar reduction in genomic targets from 8.5+/-0.1 to 8.0+/-0.16 was detected by EMA real-time PCR.     

Identification of Ingredient in Mullet Roe Products by the Real-Time PCR Method

Mullet roe is a product of high economic value in a number of Asian countries, particularly Taiwan. However, actual mullet roe is commonly adulterated by the addition of other species, such as escolar and oilfish. The purpose of this study was to develop a method to detect the ingredient of mullet roe products. Based on the TaqMan real-time PCR assay, we designed the specific primers-probe set (mullet) that targets the mitochondrial 16S ribosomal RNA gene. Meanwhile, the positive amplification control is designed based on the eukaryotic 18S rRNA gene. The PCR amplicon used to identify fish species in processed roe products is smaller than 200 bp. Method specificity was evaluated by analyzing tissue samples of 29 food fish and 9 puffer fish species. No indications of cross-reactivity toward non-target species were observed. Sensitivity and linearity tests were conducted using five-fold serial dilutions of target DNA from processed mullet roe, and we determined that our proposed method has a sensitivity of 1.2 ng. Further tests on a random survey of commercial fish roe products demonstrated the efficacy of the technique in the detection of mullet DNA. The real-time PCR methods developed in this study could be used to verify the labeling of actual mullet roe products.     

Application of DNA‐Based Methods to Identify Fish and Seafood Substitution on the Commercial Market

ABSTRACT: Fish and seafood substitution has become an important concern in domestic and international marketplaces, in part due to increased international trade, per capita seafood consumption, and production of processed foods. In many cases, seafood substitution is a form of economic deception, where highly prized species are substituted with those of lesser value. To prevent illegal species substitution, a number of DNA‐based methods have been developed to detect fish and seafood species in commercial products. These methods, along with common gene targets, have been reviewed previously in this journal. The current article is meant to build upon earlier discussions by providing a comprehensive review of the application of these DNA‐based methods to the discovery of fish and seafood substitution on the commercial market. Popular food uses, potential substitution cases, and peer‐reviewed research articles published to date are discussed for all major species groups of concern, including flatfish, gadoids, scombroids, salmonids, percoids, sturgeons, sharks, eels, and bivalves. The use of DNA‐based methods to monitor commercial whale meat products is also reviewed.     

Comparison of DNA Extraction and PCR Setup Methods for Use in High-Throughput DNA Barcoding of Fish Species

DNA barcoding is a sequencing-based method that can be used for the identification of fish species in a regulatory setting. The objective of this study was to compare modified versions of three DNA extraction kits (i.e., Qiagen DNeasy Blood and Tissue Kit, Sigma-Aldrich Extract-N-Amp Kit; and Life Technologies MagMax-96 DNA Multi-Sample Kit) and two polymerase chain reaction (PCR) setup methods (manual vs. automated) for use in DNA barcoding, with a focus on minimizing time, costs, and labor. DNA was extracted from 83 fish products using each of the three kits and the results were compared based on sequencing success and sequencing quality parameters. A subset of 14 fish products was also tested in triplicate to compare PCR setup methods. Initially, reduced sequencing success was observed with the MagMax Kit (88 %) compared to the other two kits (95–96 %); however, after PCR and sequencing were repeated for DNA samples that initially failed, all three methods showed very high sequencing success (98–99 %). Overall, the modified Extract-N-Amp Kit offered the greatest reduction in time and costs, while the DNeasy Blood and Tissue Kit produced sequences with the highest quality and highest initial success rates. Automation of the PCR setup process resulted in slightly greater success (100 %) compared to manual PCR setup (98 %), and reduced the potential for human error that may result from manual pipetting. The results of this study demonstrate the advantages of incorporating rapid and/or automated methods into the DNA barcoding workflow, especially with regard to high-throughput operations.     

钠盐替代物对马苏大马哈鱼子酱品质的影响

鱼籽主要用于鱼子酱生产，为保证食用口感和贮藏品质，加工中用高含量的氯化钠进行盐渍。为解决加工过程中钠盐的使用量较高问题，以马苏大马哈鱼（Oncorhynchus masou）鱼籽为原料，以微生物菌落总数为指标，通过单因素试验、Plackett-Burman（PB）试验、最佳陡坡试验和响应面优化试验，优化最佳钠盐替代物复合盐配方。结果表明：乳酸钾、氯化钾、氯化镁的替代比例分别为17.64%、14.61%、34.36%时，对鱼子酱品质的影响最小，能在一定程度上改善鱼子酱pH值、水分活度及色差，抑制微生物的生长。因此，将钠盐替代物复合盐用于鱼子酱的制备，可以提高产品质量和安全性，具有良好的应用潜力。     

Factors affecting chemical‐based purification of DNA from Saccharomyces cerevisiae

Extraction of high molecular weight chromosomal DNA from yeast cells is a procedure that is performed frequently for experiments involving polymerase chain reaction (PCR), Southern blotting and other DNA analysis techniques. We have investigated several parameters affecting DNA yield and quality, using a simple chemical‐based purification procedure that was modelled on alkaline lysis methods developed for bacterial cells. The three major steps of the procedure, cell lysis, protein removal and DNA precipitation, were optimized by testing the impacts of several chemicals, including sodium dodecyl sulphate (SDS), sodium hydroxide, Tris buffer, sodium acetate and potassium acetate. Other parameters, such as the effect of elevated temperatures on cell lysis, were also investigated. A rapid, optimized protocol was derived for the purification of DNA from small cell cultures that can be readily digested with restriction enzymes and used as a template for PCR. Average yield was calculated to be approximately 1.7 µg DNA/10⁸ cells, which is similar to the theoretical maximum amount obtainable from haploid yeast cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of Fish and Oil Nature on Frying Process and Nutritional Product Quality

ABSTRACT: The modifications on a lean fish (cod—Gadus morhua) and a fatty fish (farmed salmon—Salmo salar) after the application of pan-frying using 2 types of oil with different lipid profile (extra virgin olive oil and sunflower oil) was the aim of this study. Fat content and total energetic value increased significantly after the frying process only in the lean fish, without relevant changes in the fatty fish. Extra virgin olive oil led to a higher fat absorption rate than sunflower oil in both fish. Frying hardly affected the lipid profile of farmed salmon regardless the oil used, however it drastically changed in fried cod compared to raw cod. Omega-6/omega-3 ratio increased from 0.08 in raw cod to 1.01 and 6.63 in fried cod with olive oil and sunflower oil, respectively. In farmed salmon, the omega-6/omega-3 ratio was 0.38 (raw), and 0.39 to 0.58 in fried salmon. The amount of EPA + DHA slightly decreased with frying in salmon, and increased in cod. The type of oil has more influence in the nutritional fish quality for the lean fish compared to that of the fatty fish. The use of extra virgin olive oil was efficient to avoid a significant increase of the lipid oxidation intensity during frying in cod but not in salmon. Practical Application: Food modifies its composition and nutritional value with the application of cooking technologies. As most food table composition tables are based on raw food products, this article contributes with interesting data on pan-fried fish composition, which may improve the approach to achieve a real intake of healthy nutrients as omega 3 fatty acids.     

Frozen Storage of Unwashed Cod (Gadus morhua) Frame Mince with and without Kidney Tissue

Removal of kidney material was essential for a higher quality cod frame mince. The removal of kidney tissue before deboning eliminated typical “chemical” and “petroleum” type flavors and resulted in a white, less red, and higher quality unwashed frame mince. Those samples without kidney tissue had good frozen storage stability, (particularly at -40°C) and could be used as an ingredient in apropriate meat products. The length of iced storage of frames or whole cod had little effect on frame mince quality during frozen storage.     

Direct Application of the Polymerase Chain Reaction for Quantification of Total Bacteria on Fish Fillets

A rapid, conventional PCR assay that quantifies total bacteria from fish fillets is described. The methodology is based on the use of the universal primers DG74 and RW01 to amplify a 370-bp conserved sequence of the 16S rRNA gene of Gram-positive and Gram-negative bacteria. The intensity of amplified DNA bands (from mixed bacterial flora of fish fillets) in agarose gels was found to be linear from 5 × 10² to 1 × 10⁵ CFU/PCR. Differential centrifugation of tissue homogenates followed by sample dilution was successful in eliminating PCR inhibition by tissue components. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of bacterial numbers for rapidly assessing the total number of bacteria per gram of fish tissue by PCR.     

PCR detection of soy ingredients in bread

Bread may contain soy ingredients to variable extends. The nature of this soy may be genetically modified, or the ingredient may cause allergic reactions in sensitive patients. PCR is a powerful tool to detect the presence of soy in food products. Major prerequisite for a PCR analysis is DNA of good quality and quantity. The persistence of soy DNA during bread making was evaluated using different amounts of different soy ingredients. Agarose gel electrophoresis of the samples taken during the baking process reveal that DNA is degraded, but subsequent PCR detection remains possible. Results, however, greatly depend on the type of ingredient and the amount present. Lower detection limits were obtained for full-fat and defatted soybean flours than those for toasted soybean flour and soy fibre samples. Samples taken at different places in the bread, however, reveal that sampling strategy may influence the PCR results.     

C NMR as a tool for authentication of different gadoid fish species with emphasis on phospholipid profiles

The aim of this study was to evaluate if phospholipid profiles obtained by ¹³C nuclear magnetic resonance (NMR) spectroscopy is characteristic enough to separate species of lean gadoid fish. ¹³C NMR data were obtained from muscle lipids of five categories of lean gadoid fish, namely, north-east arctic cod and Norwegian coastal cod (Gadus morhua), haddock (Melanogrammus aeglifinus), saithe (Pollachius virens), and pollack (P. pollachius). A total of 27 fish caught at the same location on the Norwegian coast in the traditional fishing season (March/April) in 2006 were analysed. The sn-2 position specificity of 22:6n-3 (docosahexaenoic acid, DHA) in phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) for the different species/stocks were investigated, and the full ¹³C NMR spectra applied in multivariate analysis. Stereospecific distribution calculations showed significant differences among species in the distribution of 22:6n-3 in PC and PE, and the pollack group displayed the lowest values for 22:6n-3 in sn-2 position, both in PC and PE. This first screening showed that by using the ¹³C NMR fingerprint of muscle lipids, linear discriminant analysis gave a correct classification rate of 78% according to the five categories of lean gadoid fish, while successful classification (100%) was achieved with Bayesian belief networks (BBN) predictions.     

Two-Dimensional <Emphasis Type="Italic">J</Emphasis>-Resolved NMR Analyses of Fish and Its Products via Spatially Encoded Intermolecular Double-Quantum Coherences

Nuclear magnetic resonance (NMR) spectroscopy provides a powerful tool for food analyses due to its high-throughput information related to component analyses and its non-invasive property without altering detected samples. Current liquid NMR approaches, however, are subject to the influence of field inhomogeneity in direct measurements of biological foods, generally resorting to techniques of tissue extractions or magic-angle spin to eliminate the inhomogeneity. Therefore, we propose an NMR approach based on intermolecular double-quantum coherences (iDQCs) and spatial encoding technique to fast recover high-resolution two-dimensional (2D) J-resolved spectra in inhomogeneous fields. Inheriting from the immunity to field inhomogeneity of iDQCs and the enhancement in acquisition efficiency of the spatial encoding technique, the proposed method can resist the field inhomogeneity in biological foods without superfluous pretreatments and time-consuming shimming procedures, and yield satisfactory 2D J-resolved information for analyses within minutes. Fish and its products, which are closely related to human daily life, capture extensive attentions with the rapid development of the aquaculture. At first, experiments on cod liver oils are implemented in a purposely deshimmed magnetic field to demonstrate the feasibility of the proposed approach. Then, caviars and an intact fish are tested to further show the applicability of the proposed approach for direct measurements on intact biological food samples. Herein, the proposed method may constitute an effective technique in analyzing fish and its products.