Development of a rapid and simple molecular identification methodology for true sardines (Sardina pilchardus) and false sardines (Sardinella aurita) based on the real-time PCR technique

Sardines are a small pelagic species belonging to the Clupleidae family and have high commercial value because of the properties of their meat. One of the most valuable fish in this family is Sardina pilchardus, which in addition to its high commercial value is the one that should be labeled on canned sardines, the other species being labeled as sardines X. In this study, a fast real-time polymerase chain reaction (PCR) assay based on TaqMan probe technology, which amplifies a fragment of the internal transcribed spacers (ITS1 and ITS2) and the 5.8 S rRNA region, was developed for the authentication of two commercially important species of sardines, S. pilchardus and S. aurita. This methodology can be applied to a variety of products, including those that have been subjected to intensive processing treatments such as canning. The present methodology was validated and subsequently applied to 50 commercial samples in order to determine whether correct labeling is being carried out in the market. This assay combines the high specificity, sensitivity, and rapidity of fast real-time PCR with the advantages of using two different probes in the same reaction, making it a powerful tool for verifying the correct application of food labeling regulations.     

Comparison of methods in the recovery and amplificability of DNA from fresh and processed sardine and anchovy muscle tissues

An important condition for a successful PCR amplification is an efficient DNA-extraction procedure out of a complex biological matrix such as canned fish. In this study we compared six extraction methods, including commercial kit, in terms of DNA yield, purity and time requirement. Such methods were applied to distinguish small pelagic fish species (Sardina pilchardus and Engraulisencrasicolus) among commercial canned products. The quantity and quality of DNA extracted were evaluated using the ratio A₂₆₀/A₂₈₀. Data were submitted to principal component analysis (PCA) in order to assess the differences between PCR results of fresh and processed anchovy and sardine muscles. Two main PC characterised the PCR of sardine and anchovy (70% and 69% of all variance): principal component 1 (PC1) (4% and 60%) and principal component 2 PC2 (66.0% and 9%) for sardine and anchovy, respectively. According to the PC1, the PCI/SDS and Chelex extractions (in decreasing order) were positively correlated with results of PCR for both species.     

Influence of frozen storage on aptitude of sardine and dolphinfish for cold-smoking process

Stability of dolphinfish (Coryphaena hippurus) and sardines (Sardina pilchardus) of different fat content (lean and fatty sardines) during frozen storage and its suitability for cold-smoking throughout storage were evaluated in order to overcome seasonal and excess catches of these species. Dolphinfish showed a relative stability regarding protein functionality (protein solubility, apparent viscosity, water and lipid holding capacity), lipid oxidation and total volatile basic nitrogen (TVBN) accumulation, which led to high acceptability ratings of the resulting smoked product throughout frozen storage (340 days). However, both lean and fatty sardines showed a marked loss of protein functionality, which coincided with the accumulation of oxidation products and TVBN. Freezing of raw muscle may become a valuable preservation method for the smoking industry to overcome the short caught period of dolphinfish in the Balearic Islands and to make use of excess catches of both, lean and fatty sardine. High quality smoked products may be obtained from the frozen muscle during approximately twelve, four and two months of frozen storage for Dolphinfish, lean and fatty sardine, respectively.     

Effect of modified atmosphere and vacuum packaging on microbial,chemical and sensory quality indicators of fresh,filleted Sardina pilchardus at 3 °C

The self‐life, quality and safety of refrigerated sardine fillets (Sardina pilchardus) were determined at 3 °C in atmospheric air, vacuum and modified atmosphere (50% CO₂/50% N₂) packaging conditions. Microbiological, physico‐chemical and sensory parameters were utilised as quality indicators. The microbial flora of sardine comprised—according to order of occurrence—Shewanella putrefaciens, pseudomonads, Brochothrix thermosphacta, lactic acid bacteria and, finally, Enterobacteriaceae. Bacteria grew most quickly in sardines stored in air, followed by those in vacuum packaging, and the lowest counts were found in modified atmosphere packaging. The concentrations of moisture, ash, protein, fat and polyunsaturated fatty acids were not affected during the storage period compared to the pH values and the concentrations of lactate and ammonia that showed significant differences. Copyright © 2007 Society of Chemical Industry     

Duplex real-time PCR for authentication of anglerfish species

A duplex real-time PCR assay based on TaqMan probe technology was developed for the authentication of two commercially important anglerfish species (Lophius budegassa and L. piscatorius). This technique uses the cytochrome oxidase subunit I and allows the unambiguous identification of this species. It is notable for its simplicity, rapidity, highest potential for automation and minor risk of contamination. The TaqMan real-time PCR is currently the most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of these species. The method can be applied to all kind of products as fresh, frozen, and precooked products. The developed methodology, which uses two specific primer–probe sets, has been validated and subsequently applied to 40 commercial samples labeled as any anglerfish species in order to determinate whether the species used for their manufacturing correspond to these species. The methodology herein developed is appropriate to clarify questions related with the correct labeling of commercial products, traceability in commercial trade, and fisheries control.     

Consumption of raw and fried sardine (Clupea pilchardus) as protein source of diets: effects on iron metabolism in rats

The influence of fried fish consumption on dietary iron bioavailability in rats was studied. Sardines (Clupea pilchardus), raw and fried in olive oil, were used whole or defatted to prepare the samples. Five diets containing casein and the different samples as protein source were prepared. After in vitro digestion of the diets a greater proportion of soluble iron was found in the casein diet compared with the sardine diets. Five groups of rats were fed the diets over 28 days. Body weight and food intake were monitored weekly, and during days 21–28 an iron balance study was carried out. No significant differences were observed in food intake, body weight and protein efficiency ratio between groups. Absorbed and retained iron and apparent iron digestibility were higher in rats fed the sardine diets with respect to the control group, except in the whole fried sardine group. Metabolic efficiency and retention efficiency were increased in the four sardine groups. The frying process led to significant changes in the overall digestive and nutritive efficiency of dietary iron, which were different depending on whether the diets contained whole sardine or its protein. It was concluded that the consumption of sardines, whether raw or fried in olive oil, enhances dietary iron availability. © 2002 Society of Chemical Industry     

Chemical,enzymatical and textural changes during marination and storage period of sardine (<Emphasis Type="Italic">Sardina pilchardus</Emphasis>) marinades

In this study frozen fillets of sardine (Sardina pilchardus) were used to make marinades. The marinating process was performed in 7% acetic acid and 14% sodium chloride in barrels. The fish:solution ratio was (1.5:1). After the marination process, sardine fillets were packed in glass jars in two different formulations fish:solution ratio of (1.5:1); the first formulation contained 2% acetic acid and 4% sodium chloride with tomato sauce and spices and the other was 2% citric acid and 4% sodium chloride with lemon and also the same spices. Pasteurization process had been applied on half of the jars at 70 °C for 20 min. Chemical, enzymatical and textural changes during marination and 6 months storage period of sardine marinades were determined. The results obtained in proteolytic activities correlated well with the observed texture measurements according to time of storage. A decrease in the histidine content and an increase in glutamic acid and aspartic acid contents of marinated sardines were determined. Histamine levels were lower than the toxic limit (100 mg/kg) during the marination and storage period of sardine marinades.     

Determination of bisphenol A in canned fish by sol–gel immunoaffinity chromatography,HPLC and fluorescence detection

The paper presents a highly selective analysis method for the determination of bisphenol A (BPA) in canned fish. The procedure consists of sample clean-up by sol–gel immunoaffinity chromatography followed by high performance liquid chromatography and fluorescence detection. BPA concentrations were determined in nineteen tuna, sardine and mackerel cans by analysing the solid and the liquid parts of the contents separately. In different tested matrices limits of detection (S/N=3) ranged from 0.2 ng/g (sardines) to 1.8 ng/ml (oil) and limits of quantification (S/N=6) from 0.4 ng/g to 3.8 ng/ml, respectively. In the solid part (fish) very low BPA levels (2–4 ng/g) were found in mackerels, the highest level (59 ng/g) in tuna. In oil significantly higher BPA concentrations were found than in brine. In all samples BPA concentrations were significantly lower than the Specific Migration Level of 0.6 mg/kg for BPA migration into food established by the EU Commission in 2004.     

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.     

Use of eye fluid refractive index in sardine (<Emphasis Type="Italic">Sardina pilchardus</Emphasis>) as a freshness indicator

In this study the usage of eye fluid refractive index as a freshness indicator for sardine (Sardina pilchardus) was investigated. Eye fluid refractive index measurements and quality control analyses of sardine during storage at 0°C and +4°C were performed at 24h intervals. According to the sensory analysis results, the sardines stored at 0°C and +4°C had a shelf life of 6 and 4days, respectively. The changes in eye fluid refractive index values during storage at 0°C were not significant while the changes at +4°C were significant (p<0.01). TVB-N and TMA-N values significantly (p<0.01) increased. No microbiological growth was observed at 0°C, however the increase in microorganism counts was significant at +4°C. As a result, eye fluid refractive index measurements can be used as a quality criterion for sardine freshness, when stored at +4°C.     

Organochlorine pesticides in canned tuna and sardines on the Serbian market

The aim of this study was to determine the level of organochlorine (OC) pesticides in 57 samples of canned tuna and 31 samples of canned sardines in vegetable oil, collected from supermarkets in Serbia. OC pesticides α-HCH, β-HCH, δ-HCH, dichlorodiphenyltrichloroethane (DDT), DDE, DDD, dielderin, endosulfane I, endosulfane II, endosulan sulfate, endrin, endrin ketone, heptachlor, heptachlor epoxide, lindane, aldrin, metoxichlor, cis-chlordane and trans-chlordane were determined using a GS-MS method. The highest concentrations (µg kg^?1, arithmetic means) in canned tuna were for δ-HCH (60.6 ± 97.0) and p, p´-DDT (55.0 ± 25.1), while the corresponding values in canned sardines were for δ-HCH (90.7 ± 102.7) and endosulfane II (78.0 ± 145.9). Mean level for the sum of endosulfans was above the maximum limit in canned sardines (85.0 µg kg^?1). Also, dieldrin (39.7 µg kg^?1) was measured above the ML.     

The effects of soluble gas stabilisation on the quality of packed sardine fillets (Sardina pilchardus) stored in air,VP and MAP

Sardine (Sardina pilchardus) is a species that for its abundance assumes great importance in the Portuguese fishing sector. In order to contribute for a better utilisation of this species, the aim of this study was to investigate the effect of the pre‐treatment with soluble gas stabilisation (SGS) (100% CO₂ at 2 bar, during 15 and 30 min) on the quality and shelf‐life of sardine fillets, packed in air (AP), vacuum (VP) and modified atmosphere (MAP: 5% O₂/35% CO₂/60% N₂). During the chilled storage, the quality changes were evaluated by sensory evaluation, chemical and microbiological analysis. The total volatile basic nitrogen content remained almost constant, between 16 and 19 mg N/100 g muscle, during the storage period, for all samples. The TBARs values increased with storage time, for all batches and storage conditions. The application of SGS treatment to sardine fillets, resulted in a bacteriostatic effect, contributing to the improvement of the microbiological quality of fillets. Considering a sensory criteria, the shelf‐life of SGS pre‐treated sardine fillets was found to be 5 days in AP and MAP while in VP‐treated fillets a shelf‐life of 8 days was reported. At sensory rejection, sardine fillets presented a K‐value of 30% in AP and MAP batches and 40% in VP batch.     

Enzymatic oxidative activity in sardine (Sardina pilchardus) and herring (Clupea harengus) during chilling and correlation with quality

 Enzymatic oxidative activity of two fatty fish species, sardine (Sardina pilchardus) and herring (Clupea harengus), was studied during chilled storage. Lipoxygenase enzyme activity was isolated and tested by measuring the hydroperoxides produced after induced oxidation of arachidonic and docosahexaenoic fatty acids. The most abundant degradation products of the hydroperoxides formed were 12- and 16-hydroxy acids which were detected by HPLC. Lipoxygenases were concentrated in the skin tissue of fish, and were active for up to 48 h of chilled storage. The pro-oxidative activity due to haem proteins continued for longer than that due to lipoxygenase. Trends of fluorescent formation resulting from interaction between oxidation products and biological amino constituents were compared with the pro-oxidative activities to establish correlations with quality loss during chilling. Received: 29 December 1998 / Revised version: 14 March 1999     

Authentication of gadoids from highly processed products susceptible to include species mixtures by means of DNA sequencing methods

Economic importance of gadoids such as fishing resource, and their conservation status, necessitates the development of techniques that allow unequivocal authentication of products made from them. Amplification of a fragment of mitochondrial cytochrome b (cyt b) marker and subsequent phylogenetic analysis were carried out to authenticate these products and assure their correct labeling. Also, SNP analysis that allows detection of species mixtures of Gadus genus was developed. For this, two fragments of the cyt b gene were amplified and sequenced, one of 464 bp and another internal fragment to this of 263 bp to allow the authentication of gadoid species in highly processed products. Obtained sequences were aligned and analyzed in order to assess the presence of informative variable positions and a maximum of 14 SNP were identified and selected. These allow detection and identification of species mixtures belonging to this genus. The developed methodologies were validated and applied to 25 commercial samples. The main novelty of this work lies in the fact that is the only work that allows the detection of species mixtures of the Gadus genus and is the only one that allows the authentication of highly processed products up to date. Furthermore, this methodology allows identifying more of 15 species of gadoids and can be applied to all kinds of seafood products. Therefore, this molecular tool can be applied in questions related to check the fulfillment of labeling regulations for seafood products, verify the correct traceability in commercial trade and for fisheries control.     

Effect of Delayed Processing on Changes in Histamine and Other Quality Characteristics of 3 Commercially Canned Fishes

ABSTRACT: Changes in histamine and other quality characteristics were examined in 3 commercially important fishes, tuna ( Katsuwonus pelamis ), seerfish ( Scomberomorus commersonii ), and sardines ( Sardinella gibbosa ) through the canning process, at 3 different stages (raw, precooked, and canned) immediately on receipt and also after a delay of 6 h at ambient temperature (30 ± 2 °C). Tuna and seerfish remained sensorially acceptable when processed after delay, whereas sardines exhibited slight ammoniacal/putrid odor. TMA-N and TVB-N contents were low in precooked fish compared with their fresh counterparts, whereas in canned fish, both compounds increased significantly ( P < 0.05). Histamine content in the fish held for 6 h increased to 14, 17, and 8 ppm in tuna, seerfish and sardine, respectively, and never exceeded the maximum permissible limit of 50 ppm prescribed by the U.S. Food and Drug Administration. In precooked and canned fish, histamine was lower than in their raw counterparts and found to be within the range of 1.6 to 8.0 ppm in precooked and 1.2 to 4.3 ppm in canned fish. Holding the fishes destined for canning at 30 ± 2 °C for 6 h, therefore, was found to be safe from histamine toxicity problems.     

Proximate composition,cholesterol and fatty acids profile of canned sardines (Sardinella brasiliensis) in soybean oil and tomato sauce

Different brands of sardines canned in soybean oil and tomato sauce, that are commercialized in Brazil, had their proximate composition, cholesterol content and fatty acids composition analyzed. Protein contents were equivalent to the values found for sardines in natura, ranging from 19.8 to 24.4%. High variations of the total lipids content (5.30–16.8%) were verified; the highest levels were found for sardines canned in soybean oil. The cholesterol content ranged from 50.4 to 65.1 mg/100 g. The highest levels of essential C18:2n − 6 and C18:3n − 3 fatty acids were found in sardines canned in soybean oil. The EPA (C20:5n − 3) and DHA (C22:6n − 3) concentrations ranged from 5.39 to 15.1% and from 3.89% to 9.51%, respectively, and the highest levels were observed in sardines canned in tomato sauce.     

Changes in the halophilic amine forming bacterial flora during salt-drying of sardines (Sardinella gibbosa)

The incidence of halophilic amine forming bacteria in fresh sardines was around 20%, which on salting reached a maximum level of 84% and finally decreased during the drying process. No amine forming bacteria were detected in salt-dried sardines after final drying. The critical stage for the survival of amine forming bacteria in salt-dried sardine was between salting and initial stage (first day) of drying. Micrococcus luteus dominated during the salting process, while Pseudomonas and Alcaligenes species were the major amine forming bacteria during the drying stage. The amine forming bacteria isolated from the salt-dried sardines were able to produce only cadaverine and putrescine. However, no histamine forming bacteria was detected in this study.     

EXPOSURE TIME ON BACTERIA FLORA/COUNT AND SHELF LIFE OF CANNED SARDINE (SARDINELLA PILCHARDUS) UNDER AMBIENT AND COLD STORAGE CONDITIONS

This study is aimed at investigating the exposure time on bacteria flora/count and shelf life of conned sardine (Sardinella pilchardus) under ambient and cold storage conditions. Twenty‐five cans with an average weight of 165.05 g of the Titus (with an expiration date of 4 years (September 30, 2004 to September 30, 2008 of batch no. 1432) were purchased and stored at the ambient temperature of 27C and cold (?4C) storage conditions as samples for 12 weeks (precisely between June 15 and September 10, 2005 when the experiment lasted). Proximate analysis of the samples was taken at the beginning of the experiment and at the end for both the ambient and cold stored (after an exposure time of 24 h). Initial baseline and biweekly studies were carried out for 12 weeks for: (1) organoleptic (odor, taste, texture, appearance, rigidity of flesh, color and reaction of fish with can); (2) chemical (trimethylamine [TMA], peroxide values [PVs] and thiobarbituric acid [TBA]); and lastly (3) microbiological analysis for bacteria count and identification of the bacteria on the samples from each storage environment after an exposure time of 24 h in each case. All the chemical parameters (TMA, TBA and PV) were significantly (P < 0.05) correlated with exposure/storage time. Correlation coefficients r = 0.60, 0.66 and 0.54 were low in all classes therefore indicating spoilage rate increases slightly with exposure time/storage period. The highest PV (0.023–0.715), TBA (0.057–1.056) and TMA (1.01 × 10³–3.63 × 10³) ranges were recorded for canned sardines stored at ambient temperature of 27C. However, these are still within acceptable tolerance limit (i.e., organoleptic score of 4–7). Organoleptic assessment with average scores of 5.5 and 6.0 was recorded for cold and ambient stored samples. No viable bacteria count was recorded for cold‐stored samples throughout the experiment. However, the range initial 1.0 × 10⁵ and final 5.0 × 10⁴ cfu/g total plate counts recorded for ambient storage were still below the minimum bacteria count for spoilage that could cause significant or deleterious effect that could result in food poisoning. Traces of the following bacterial species were recorded at ambient temperatures: (1) Bacillus subtilis (1.2 × 10⁴ cfu/g); (2) Streptococcus faecium (9.0 × 10³ cfu/g); (3) Proteus vulgaricus (7.0 × 10⁵ cfu/g); (4) Pediococcus halophilus (6.0 × 10⁵ cfu/g); (5) Micrococcus acidophilus (4.0 × 10³ cfu/g); (6) Streptococcus lactis (4.0 × 10³ cfu/g); and (7) Aerobacter aerogenes (4.0 × 10³ cfu/g), while fungal species Aspergillus terreus (1.0 × 10³ cfu/g) and Aspergillus niger (3.0 × 10³ cfu/g) were recorded also for samples stored at ambient temperature of 27C. Hence, in view of this, the 4‐year recommended expiration date may be upheld for canned sardine (S. pilchardus fish products in oil sources) provided the hazard analysis and critical control points, and closely monitored. It is, therefore, recommended that exposure of canned sardine in oil should not exceed 12–24 h under whatever food storage temperatures to avoid food poisoning.     

PCR-RFLP Analysis of Mitochondrial DNA for Identification of Snail Meat Species

Several species of land snails, including Helix pomatia and Helix lucorum are consumed as food products. The main source of commercial competition is an imported African snail, Achatina fulica. The only way to distinguish between these species has been morphologically. We hypothesized a reliable method for identifying canned snails could be based on using PCR-RFLP analysis of mito-chondrial DNA. The molecular weights of the amplified fragments were perfectly identical, regardless of low extraction (fresh snails or cooked and canned samples). The whole amplified products (16S rRNA and 12S rRNA) made it possible to check any fraudulent label and identify the three species using four restriction enzymes (RFLP).     

Analysis and risk assessment of arsenic,cadmium and lead in two fish species (Sardina pilchardus and Xiphias gladius) from Algerian coastal water

ABSTRACT

In this study, the levels of arsenic (As), cadmium (Cd) and lead (Pb) have been determined in the flesh of two species of fish, sardine (Sardina pilchardus) and swordfish (Xiphias gladius) fished in the Algerian coast. Quantification of As, Cd and Pb was carried out using an ICP-MS method and the results were compared with the thresholds set by national and international regulatory bodies. In a further step, the risk to consumers was assessed using estimated daily intake (EDI), target hazard quotient (THQ) and hazard index (HI).The average concentration of As and Pb was higher in sardine (1.82; 0.10 mg kg^?1 w.w., respectively), than in swordfish (1.10 mg kg^?1 w.w.; not determined), whereas the concentration recorded for Cd was the same for both species (0.01 mg kg^?1 w.w.). These concentrations are below maximum limits set in regulations. The THQs and HI were widely below 1. The consumption of these fish does not pose risk to the consumers.