Application of PCR-SSCP to Species Identification of Fishery Products

A method of DNA analysis has been developed to verify authenticity of labelled raw material of canned fish or in products made from closely related fish species (tuna, eel, salmon, trout and sturgeon). Short segments (123–358 bp) of the mitochondrial cytochrome b gene were amplified by the polymerase chain reaction (PCR) and analysed by single strand conformation polymorphism (SSCP) to get species-specific patterns of single-stranded DNA (ssDNA). DNA strands were separated by polyacrylamide gel electrophoresis and visualised by silver staining. Differentiation between four eel species was possible. Each of three types of sturgeon caviar gave a characteristic pattern of ssDNA. Canned sardine, herring, tuna and other species expressed specific bands of ssDNA. © 1997 SCI.     

Fish species identification in canned tuna by PCR-SSCP: validation by a collaborative study and investigation of intra-species variability of the DNA-patterns

Analysis of single strand conformation polymorphism (SSCP) of an amplicon (123 bp) obtained by polymerase chain reaction (PCR) of the mitochondrial cytochrome b gene was used to identify the fish species in canned tuna. Single-stranded DNA (ssDNA) was separated by polyacrylamide gel electrophoresis, and visualised by silver staining. The reliability of the method was tested by a collaborative study in which eight European laboratories participated. Seven unknown samples (five from individual species and two mixtures of two tuna species) of canned tuna had to be identified by comparison with reference material. From a total of 72 cases, 65 (90.3%) were assigned correctly. Intra-species variability of SSCIP patterns was found in the case of Katsuwonus pelamis and Sarda sarda. As specimens from various fishing grounds gave two or three different patterns of ssDNA, the possibility of some variability of the DNA patterns has to be considered in SSCP analysis of these species.     

Comparison of aroma characteristics of 16 fish species by sensory evaluation and gas chromatographic analysis

The aroma properties of fish broths prepared from 16 fish species (10 saltwater, three freshwater, two anadromous and one brackish water species) were described quantitatively by reference to 10 sensory attributes. Principal component analysis (PCA) and cluster analysis of sensory attributes classified the fish into four groups. Group 1, characterised by a strong ‘green’ odour, comprised all three freshwater species (loach, pond smelt and carp), two saltwater whitefish species (snapper and conger) and eel. Group 2 included migratory coastal species (sardine, banded blue‐sprat and mackerel) and was distinguished by strong ‘fish oil’ and ‘grilled fish’ notes. Group 3 consisted of swordfish, sablefish and salmon, which exhibited a strong ‘fried chicken’ note. Group 4 included flounder, cod, tuna and goby, which were scored high for ‘cooked fish’, ‘roasted soy sauce’, ‘canned tuna’ and ‘sweet’ aromas. Partial least squares regression (PLSR) models derived from selected influential peaks in the gas chromatograms of the volatile components in the broths for each attribute were highly predictive (R² ≥ 0.936). The selected peaks corresponded well to each sensory attribute. © 2003 Society of Chemical Industry     

A SURVEY OF HISTAMINE LEVELS IN COMMERCIALLY PROCESSED SCOMBROID FISH PRODUCTS12

Analysis of 100 samples of each of 3 types of commercially processed scombroid fish products obtained at the retail level showed mean histamine concentrations (mg/100 g) of 3.58 for chunk light tuna, 1.50 for albacore tuna, and 2.56 for mackerel. Only 2 samples of chunk light tuna and 2 of mackerel contained greater than 10 mg% histamine. The highest level of histamine found in albacore tuna was 5.35 mg/100 g, while 99% ofthe samples had less than 5 mg% histamine. Chunk light tuna samples contained up to 28.0 mg/100 g, and mackerel samples contained up to 31.5 mg/100 g; 92% of the chunk light tuna and 95% of the mackerel had less than 5% histamine. The histamine levels found in these products were far below any level associated with food poisoning incidents.     

Quantification of total and specific gram-negative histamine-producing bacteria species in fish using an MPN real-time PCR method

Quantification of histamine-producing bacteria (HPB) is necessary in order to elucidate the role that HPB play in scombrotoxin (histamine) fish poisoning. We report here the evaluation of a real-time PCR method for the quantification of total and specific Gram-negative HPB species in fish using a most probable number (MPN) format. The species-specific real-time PCR assay was 100% inclusive for independently detecting Morganella morganii, Enterobacter aerogenes, Raoultella planticola/ornithinolytica and Photobacterium damselae and did not cross react with other histamine- or non- histamine-producing bacteria. The efficiency of the reactions in the absence and presence of Spanish mackerel enrichment containing 1 × 10⁶ CFU/ml of background microflora were 93-104 and 92-99%, respectively. The MPN-real-time PCR assay accurately quantified total and specific HPB in spiked mahi-mahi (Coryphaena hippurus) and Spanish mackerel (Scomberomorus maculates) samples. These methods were used to quantify total and specific HPB in naturally contaminated, decomposing mahi-mahi, Spanish mackerel and tuna (Thunnus albacares) samples. The results of this study indicate that MPN-real-time PCR assays can be used to accurately enumerate total and specific HPB in fish samples. These assays can be applied to assess the effectiveness of mitigation strategies and understand the relationship between HPB and histamine production in decomposing fish.     

Species-specific multiplex real-time PCR assay for identification of deer and common domestic animals

A multiplex real-time PCR method for discriminating deer and common domestic species, including cattle, goat, horse, donkey, pig, and chicken was developed. Species-specific primer pairs were designed and used to produce different size DNA fragments with diverse melting temperature (T _m ) values. The specificity and sensitivity of these primer pairs were separately confirmed using simplex real-time PCR analysis. Multiplex real-time PCR analysis was performed using combined primers, yielding distinct melting curve profiles for each species. The sensitivity limit of the multiplex PCR method was evaluated. Trace DNA of other species in deer DNA could be identified. Common deer products, including blood, meat, and antler were tested using this multiplex PCR method and different species of deer and domestic animals were identified. The rapid multiplex real-time PCR assay described herein is a specific, sensitive, and reliable method for high-throughput authentication of deer and domestic animal products.     

Detection of fish DNA in ruminant feed by PCR amplification

The Japanese Government has prohibited the use of seafood protein, as well as mammalian protein, in ruminant feed. There is an official method to detect meat and bone meal, but no method is yet available to detect fishmeal in ruminant feed. We tried to develop a suitable method to detect fishmeal in ruminant feed, similar to the official method "PCR detection of animal-derived DNA in feed". Our previously reported primers (fishcon5 and fishcon3-1) showed low sensitivity, so we designed new primers based on a DNA sequence from yellowfin tuna mitchondrial DNA. Among the primers, FM5 and FM3 specifically detected fish DNA (sardine, yellowfin tuna, skipjack tuna, chub mackerel, Pacific saury, salmon, rainbow trout, Japanese anchovy, codfish and Japanese horse mackerel) from fish meat, and did not amplify DNA from animals and plants. The sensitivity for detection of the presence of fishmeal in ruminant feed was 0.01-0.001%.     

Taurine content of raw and processed fish fillets/portions

The health benefits of seafood are well recognised and fish and fish products are increasingly being advocated as functional foods. Taurine is also well recognised as beneficial to cardiovascular health, and seafood is a good source of this compound. This study investigated the taurine content of different fish species and also the use of vacuum tumbling and injection procedures for introducing additional taurine into fish. The taurine content of fish purchased in supermarkets was in the order plaice (146), cod (108), mackerel (78) and farmed salmon (60 mg/100 g fresh weight). Spot-sample tests on 14 other fish species showed a wide range (6–176 mg/100 g fresh weight) in taurine contents. Vacuum tumbling and injection in/with a taurine/sodium tripolyphosphate solution were used successfully to enrich tuna cubes (800 mg/100 g fresh weight) and salmon sides (891 mg/100 g fresh weight), respectively, with taurine thus making them (potentially) functional foods. The added taurine was well retained in processed tuna cubes and did not adversely affect the sensory acceptability of the samples. Taurine retention in cooked taurine-enriched tuna cubes was best for grilling followed by microwave heating and steaming.     

Mercury and Fatty Acids in Canned Tuna, Salmon, and Mackerel

ABSTRACT: Canned tuna ( n = 240), salmon ( n = 16), and mackerel ( n = 16) were analyzed for mercury and fatty acids. Average mercury levels were 188, 45, and 55 ppb, respectively, and below the FDA Action Level of 1000 ppb. "Light tuna in water" contained lower mercury (x = 54 ppb) compared with "white/albacore tuna in water," which contained higher eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) x= 711 mg/100 g wet tissue). Mercury residues in salmon (x = 45 ppb) and mackerel (x = 55 ppb) were lower than in tuna products, but the EPA/DHA levels were higher (salmon, ×= 1623 mg/100 g wet tissue; mackerel, ×= 851 mg/100 g wet tissue). Information from this study will help women of childbearing age to limit their intake of mercury while obtaining healthy fats from fish.     

Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene

Polymerase chain reaction (PCR) based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene was applied to the specific identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus). The use of a common reverse primer, together with forward specific primers for red deer, fallow deer, and roe deer, allowed the selective amplification of the desired cervid sequences. The specificity of each primer pair was verified by PCR analysis of DNA from various game and domestic meats. The assay can be useful for the accurate identification of meats from cervid species, avoiding mislabeling or fraudulent species substitution in meat products.     

Concentration and Exposure Assessment of Mercury in Commercial Fish and Other Seafood Marketed in Oman

The results of this study present analytical data of the mercury levels in several fish and shellfish species to create awareness among individuals of the risks associated with consuming fish contaminated with mercury. Mercury concentrations varied from a mean of 0.02 mg/kg in Indian mackerel to 0.19 mg/kg in shark in both fresh and frozen fish, from 0.02 mg/kg in sardines to 0.18 mg/kg in skipjack tuna in canned fish, and from 0.02 mg/kg in Indian mackerel to 0.79 mg/kg in shark in dried fish. Shellfish contained a slightly higher amount of mercury than fresh or frozen fish with a mean of 0.09 mg/kg. Trophic position, followed by habitat, was the most important factors for variability in mercury concentrations in fish and shellfish. The maximum safe weekly intake (MSWI) values of mercury were significantly higher for herbivores than for carnivores. The MSWI value for total mercury in the case of consuming most (72%) fish species was more than 5 kg; however, the MSWI value was never more than 5 kg in most (66%) shellfish species. Risks were identified upon consumption of 120 g of dried shark when exceeding the provisional tolerable weekly intake threshold (1.6 μg/kg) for methylmercury. Therefore, fish‐eating populations should reduce the quantity of dried shark to efficiently diminish the exposure to mercury.     

Characterization of Mexican Fishes According to Fatty Acid Profile and Fat Nutritional Indices

The fatty acid profile of crevalle jack was compared with that of Atlantic bluefin tuna, Atlantic Spanish mackerel, red snapper, and Nile tilapia. Crevalle jack and Atlantic Spanish mackerel showed similarities in their fatty acid profiles, with the saturated fatty acid > monounsaturated fatty acid > polyunsaturated fatty acid pattern. Atlantic bluefin tuna and red snapper followed a similar pattern to each other, with the polyunsaturated fatty acid > saturated fatty acid > monounsaturated fatty acid pattern, and the pattern for Nile tilapia was saturated fatty acid > monounsaturated fatty acid ≈ polyunsaturated fatty acid. In winter, the DHA+EPA content of crevalle jack (493.2 mg/100 g fillet) was 37% lower than Atlantic bluefin tuna and Atlantic Spanish mackerel, 35% higher than for the red snapper, and 2.8 fold higher than for the Nile tilapia. The best nutritional indices were for Atlantic bluefin tuna > red snapper > Atlantic Spanish mackerel > crevalle jack > Nile tilapia.     

Antibacterial activity of essential oil vapor for histamine-producing bacteria

In this study, we evaluated the antibacterial activity of essential oil vapors against histamine-producing bacteria Morganella morganii NBRC3848 and Raultella planticola NBRC3317. We measured the minimum inhibitory dose (MID) of 14 essential oils towards these two strains. Allyl isothiocyanate (AIT) and salicylaldehyde (SA) vapors showed higher antibacterial activity than the other 12 essential oil vapors. Both AIT and SA vapors suppressed growth of total aerobic bacteria and histamine-producing bacteria in bigeye tuna and mackerel meat during storage at 12°C. These vapors also inhibited histamine accumulation in bigeye tuna meat and mackerel meat. Thus, application of AIT and SA vapors is effective for preventing increase of histamine-producing bacteria and histamine formation in fish meat.     

Mercury content in Chilean fish and estimated intake levels.

The intake of fish products is a major public health concern due to possible methyl mercury exposure, which is especially toxic to the human nervous system. This pilot study (n = 46) was designed to determine mercury concentrations in fish products for national consumption (Chilean jack mackerel, hake, Chilean mussel, tuna) and for export (salmon, Patagonian toothfish, swordfish, southern hake), and to estimate the exposure of the general population. The fish products were collected from markets in Talcahuano, Puerto Montt and Santiago. Samples were analyzed at the National Environmental Center by cold vapor atomic absorption spectrophotometry. Mercury levels in swordfish and one canned tuna sample exceeded levels prescribed by national and international standards. The remaining two export products (Patagonian toothfish, also known as Chilean sea bass, and salmon) complied with international limits, which are more demanding than Chilean regulations. Theoretical estimates of mercury intake varied from 0.08 to 3.8 microg kg(-1) bw day(-1) for high fish consumers, exceeding the provisional tolerable intake for tuna, Chilean seabass, Chilean jack mackerel and swordfish. This group appears to be at the greatest risk from mercury contamination among the Chilean population.     

Glutathione Peroxidase Activity During Storage of Fish Muscle

Activity of fish muscle glutathione peroxidase, which presumably protects muscle from oxidative deterioration during storage and processing, was found in both Japanese jack mackerel and skipjack tuna. Activity of the peroxidase and level of reduced glutathione (enzyme substrate) decreased during 5 days storage at 4°C. Lipid hydroperoxides were substantially formed in the fish muscles during the storage.     

Quality assessment of edible vegetable oils used as liquid medium in canned tuna

An experimental investigation was conducted to assess the quality of vegetable oils used as liquid medium in preserved fish. Sixteen tuna preserves were tested, including two in extra virgin olive oil, nine in olive oil (refined olive oil plus virgin olive oil) and five in refined seed oil. Next to the traditional routine analyses prescribed by the EEC Regulation N° 2568/91 (useful for a preliminary characterisation of oils used as liquid medium), the content of trans isomers of unsaturated fatty acids was determined and the analysis of polar compounds was performed by HPSEC (high performance size-exclusion chromatography). The acid composition showed the presence of highly unsaturated fatty acids, typical of fish lipids. Trans isomers were always absent in extra virgin olive oils, whereas they were always present in olive oils and in refined seed oils. Triglyceride oligopolymers, oxidised triglycerides and diglycerides, measured by means of the HPSEC analysis of polar compounds, ranged within 0.13-1.07%, 0.51-2.36% and 0.96-4.02%, respectively. The HPSEC analysis of polar compounds and the determination of trans isomers enabled better assessment of the quality of different types of oils used as liquid medium for canned tuna.     

Determination of K-value and histamine of mackerel and tuna, by using the oxygen-sensor method

Determination of K-value (fish freshness index) and histamine (Hm) of mackerel and tuna during storage at various temperatures was done by using the oxygen-sensor method. Hm in mackerel increased even at low temperature (5 degrees C), and reached the Defect Action Level (DAL) of the U.S. FDA, 5 mg/100 g, before the K-value approached the inedible level. As the storage temperature was increased, the rate of Hm formation increased remarkably and the amount of Hm in fish meat passed through the DAL, and reached the real hazard level, AL (50 mg/100 g), in a short time. The initial Hm level of raw tuna was too low to determine by our oxygen-sensor method, and the final Hm level after 8 days' storage at under 5 degrees C was only 0.6 mg/100 g. But, when the storage temperature was elevated, the rate of Hm formation increased rapidly, as in mackerel, and the Hm level reached the DAL, then the AL, in a short time. Simultaneous determination of K-value and Hm is recommended for accurate and reliable quality inspection of fish and fish products.     

CHANGES IN BIOGENIC AMINES OF MAJOR PORTUGUESE BLUEFISH SPECIES DURING STORAGE AT DIFFERENT TEMPERATURES

Changes in histamine (Him), cadaverine (Cad), putrescine (Put), agmantine (Agm) and volatile basic nitrogen (VBN) contents were examined in sardine, Atlantic horse mackerel, chub mackerel and Atlantic mackerel, during ice storage and storage at room temperature. Him formation as well as other amines varied greatly with species of fish and storage conditions. The levels of Him, Cad, Put and VBN increased gradually in all the fish species as decomposition progressed, regardless of storage temperatures. In iced fish, amine production was considerably reduced and Him concentration was, in general, lower than 100 mg/Kg. During ice storage amines increased slowly until day 7, after which a significant rise was detected. In comparison with the other fish species higher levels of Him, Cad and Put were determined in Atlantic mackerel. At room temperature Him, Cad and Put were produced at the highest concentrations in chub mackerel, followed by sardine, Atlantic mackerel and Atlantic horse mackerel. Him concentration maximum exceeded allowable limits for human consumption in the first three species after 24h of storage at room temperature. No correlation was observed for Him or other amine levels and the degree of fish decomposition. Thus, the use of Him or other amines as a freshness index of the studied fish species was not considered appropriate.     

An Integrated Quantitative Correlation of Textural Profiles of Fish

The textural properties of cooked muscle of five fish species, skipjack, flying fish, common horse mackerel, plaice and channel rock fish, were evaluated by a consumer panel. The data thus obtained were analyzed by a Multivariate Analysis Method, using quantitative categories, which normally would be considered qualitative. Although an analysis of the data of the five species as singular components of the total proved unsuccessful, satisfactory discrimination was attained by comparing correlation ratios of the data of each pairing of the fish species, each species paired against the other. Based on these ratios, an integrated correlation indicating the textural profile differences between the totality of five fish species by means of a multidimensional diagram was determined.     

国产与进口鱼油品质分析比较

以国产及进口鱼油为研究对象,分别对其感官指标、理化指标、不良物质含量及脂肪酸组成进行分析比较。结果表明:国产鱼油鱼腥味明显重于进口鱼油,且分层明显;国产鱼油在酸值和过氧化值指标上也普遍高于进口鱼油,不良物质含量也高于进口鱼油,尤其在苯并芘及二噁英等指标上;脂肪酸组成中,国产鳀鱼油DHA含量大于EPA,而进口鳀鱼油EPA含量大于DHA,且国产鱼油多不饱和脂肪酸含量低于进口鱼油;国产金枪鱼油与进口金枪鱼油在理化指标和脂肪酸组成等方面差异不大。国产鱼油中,杂鱼油与金枪鱼油、鳀鱼油在感官指标、理化指标及不良物质含量方面差异不大,但在脂肪酸组成上差异显著,杂鱼油饱和脂肪酸含量明显高于其他两种鱼油,利用价值最低。