DNA-based identification of fish species implicated in Puffer fish poisoning

A method for identification of fish species using three different mitochondrial DNA regions, 16S rRNA, cytochrome b and cytochrome c gene fragments, was investigated. The combined use of all three regions enabled reliable species identification in not only raw fish, but also dried, seasoned and boiled fish, products. Furthermore, the method was applicable even to vomitus from a patient involved in a puffer fish poisoning incident. However, further improvement is necessary to discriminate between closely related species such as Takifugu rubripes and T. chinensis, because they showed close similarity in the nucleotide sequences in the three gene fragments analyzed in this study.     

Photobacterium phosphoreum caused a histamine fish poisoning incident

An incident of histamine fish poisoning (HFP) occurred due to the consumption of iwashi maruboshi (dried sardine) in Osaka, Japan in March 2002. A histamine-producing bacterial strain, YS4-7, was isolated from iwashi maruboshi that contained 1700 mg of histamine per kilogram. This strain was identified as Photobacterium phosphoreum by biochemical examinations and partial sequencing of 16S rDNA. P. phosphoreum YS4-7 showed greater capability as a histamine producer at 4 and 12 degrees C than Morganella morganii JCM 1672. Strain YS4-7 produced 546 mg of histamine per kilogram in a sardine homogenate stored for 12 h at 20 degrees C. M. morganii, Raoultella planticola and Hafnia alvei have been isolated from fish implicated in HFP incidents, whereas this is the first report of P. phosphoreum being the causative bacterium in a sporadic case of histamine food poisoning.     

Histamine contents of fermented fish products in Taiwan and isolation of histamine-forming bacteria

Twenty-seven imported fermented fish products from Southeast Asian countries and sold in the supermarkets in Taiwan, including fish sauce, fish paste and shrimp paste, were tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, total volatile basic nitrogen, trimethylamine, and aerobic plate count in all samples ranged from 4.8% to 6.5%, 16.2% to 45.3%, 51 to 275 mg/100 g, 5.4 to 53.9 mg/100 g and 1.0 to 4.2 log CFU/g, respectively. The average content for each of eight different biogenic amines in all samples was less than 90 ppm, except for histamine which has an average content of 394 ppm in fish sauce, 263 ppm in fish paste, and 382 ppm in shrimp paste. Most of the tested fermented fish products (92.6%) had histamine levels greater than the FDA guideline of 50 ppm, while seven of them (25.9%) contained >500 ppm of histamine. Although Bacillus coagulans and Bacillus megaterium were identified as the two histamine-producing bacteria capable of producing 13.7 and 8.1 ppm of histamine, respectively, in trypticase soy broth broth supplemented with 1.0% l-histidine, they were not determined to be the main contributors to histamine accumulation in these fermented fish products.     

鱼露前期发酵过程中组胺变化规律研究

鱼露是我国沿海地区以及东南亚各国的传统调味品.鱼露在前期发酵过程中,会产生各种胺类物质,其中以组胺为主.文章采用不同种类的海鱼(金枪鱼、缇鱼、巴浪鱼、鲒鱼)、不同发酵温度(室温,35℃,45℃)、不同腐败程度、以及不同NaCl含量(15%,25%,35%,45%)四种条件下发酵鱼露,定期检测其中组胺的含量,得出组胺变化规律图.结果表明,随着鱼露发酵时间的延长,组胺含量先升高后降低,但不同的发酵条件组胺的含量及变化规律有所不同.     

Histamine levels in commercially important fresh and processed fish of Oman with reference to international standards

Histamine was investigated in fresh (378), frozen (441), canned (290) and dried (24) fish samples of scombroid and non-scombroid species of Oman using a high performance liquid chromatography with a fluorescence detector. Of the 1133 fish samples tested, histamine was detected in 551 samples with a detection rate of about 41.8% among fresh fish, 61.0% of frozen fish, 78.9% of canned fish and 91.6% of dried fish samples and the mean histamine levels were 2.6, 5.8, 3.1 and 104 mg kg⁻¹, respectively. A total of 3.7% and 0.79% of the total samples exceeded the FDA and EU regulatory limits for histamine. Imported dried anchovies contained high histamine levels. The study confirms that post catching and commercialisation practices of seafood are adequate, warranting good quality fish and may not cause histamine risk to consumer in terms of human diet. While necessary monitoring may be done for imported dried fish products.     

Histamine formation by Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce.

We examined histamine formation in cultures of Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce. T. muriaticus formed histamine in low acidity (pH 5.8), O2 limiting conditions with optimal NaCl and glucose concentrations of 5-7% (w/v) and above 1%, respectively. Histamine formation could not be prevented even at 20% (w/v) NaCl, indicating that NaCl could not prevent histamine formation by this bacterium. A conspicuous amount of histamine accumulated only during the late stationary phase regardless of the growth conditions. Studies of cell suspension experiments confirmed the results obtained from cultured cells.     

Liquid chromatography-tandem mass spectrometry determination of the toxicity and identification of fish species in a suspected tetrodotoxin fish poisoning

Suspected tetrodotoxin (TTX) poisoning was associated with eating unknown fish in April 2009 in Taiwan. After ingestion of the fish, symptoms of the victim included perioral paresthesia, nausea, vomiting, ataxia, weakness of all limbs, respiration failure, and death within several hours. The toxicity in the remaining fish was determined, with the mice exhibiting symptoms of neurotoxin poisoning. The implicated fish and deceased victim tissues were analyzed for TTX by liquid chromatography-tandem mass spectrometry. The urine, bile, cerebrospinal fluid (spinal cord), pleural effusion, and pericardial effusion of the victim contained TTX. In addition, the partial cytochrome b gene of the implicated fish was determined by PCR. The DNA sequence in the partial 465-bp cytochrome b gene identified the implicated fish as Chelonodon patoca (puffer fish). These results indicate that people should avoid eating unknown fish species from fish markets where harvested fish may include toxic species.     

Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies.

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a black jack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, > or = 1 and > 1 MUg g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or-2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H]+ ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.     

Screening Test for Histamine in Fish

A rapid screening method is described that can detect histamine in fish. The method uses a two–step sequential enzyme system. First, diamine oxidase catalyzes the breakdown of histamine with formation of hydrogen peroxide. Hydrogen peroxide is then detected by formation of crystal violet from the leuco base in the presence of peroxidase. The two reactions can be combined and peroxidase can be used to control the tightness of the screening. The method can be used for raw or heat-processed fish.     

Skin-lightening products revisited

Skin colour typology depends on the amount and location of its chromophores. Among them, eumelanins derived from 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), and phaeomelanins are of utmost importance. These biomolecules result from the multi-step enzymatic and non-enzymatic conversion of tyrosine into melanins. Pigmentation disorders are multiple and depend on alterations in the density in active melanocytes, and on specific abnormalities of any of the complex melanogenesis mechanisms. This review presents some of the main skin-lightening agents with respect to their mechanisms of action and side-effects. Some of the novel compounds may lead to new perspectives in the fields of dermatology and cosmetology. The methods commonly used to assess efficacy of skin-lightening products rely on in vitro models including cell-free enzymatic assays, melanocyte cultures and reconstructed epidermis bioassays. Animal models have little relevance. By contrast, human testing with the support of instrumental evaluations is the most informative.     

Histamine and Tyramine Content of Yeast Products

SUMMARY— Chromatographic estimations of tyramine and histamine in "Marmite" and four other yeast extracts showed a tyramine content from 0.1 to 1.6 mg/g and a histamine content of 0.2 to 2.8 mg/g. "Marmite" contained the largest amounts of both amines, but' three samples of this extract showed a wide range in amine content. These variations in amine content of yeast extracts are discussed in terms of their manufacture. Pharmacological activity in one of the yeast extracts ("Marmite") on the rat's blood pressure and on the intact bronchioles and isolated ileum of the guinea pig was due to the presence of tyramine and histamine. The clinical relevance of a high histamine content is discussed.     

Copper Chelation Assay For Histamine in Tuna

A reported copper chelation method for histamine determination was modified for use in decomposing yellowfin tuna steaks. The assay consisted of histamine extraction with hot methanol, purification by rapid cation exchange chromatography, and addition of copper and a dye to the purified sample to form an easily visualized red complex with the histamine. This method detected histamine in the low mg% range in yellowfin tuna steaks with an accuracy comparable to the standard fluorometric histamine assay. It is an alternative to the standard assay when a visual test is desirable or a fluorometer is not available.     

Histamine formation by enterococci in goat cheese

Cheeses were made of heat-treated goat milk inoculated with large numbers of a histamine-producing strain of Streptococcus faecium or a non-histamine-producing strain of S. faecalis. Every second week during ripening (91 days) the cheeses were sampled for histamine analyses and bacteriological analyses. The numbers of enterococci remained high throughout the whole period of investigation and the maximum amount of histamine detected was 8.2 micrograms/g in one of the cheeses. The enterococci seem to have no relevance from a histamine intoxication point of view in cheeses of this kind.     

Histamine Production in Soy Extended Tuna Salads

Histamine production in tuna salads extended with textured soy protein (TSP) was evaluated. Salads were inoculated with five known histamine-producing bacteria and held at 8°C, 24°C, and 37°C for up to 48 hr. Addition of 30% TSP to tuna salads resulted in higher initial pH and favorable growth conditions for microorganisms and histidine decarboxylase activity. Addition of 15% TSP provided an initial pH for maximal enzyme and histamine production but somewhat slower microbial growth. Tuna salad extended with either 15% or 30% TSP developed toxic levels of histamine (>50 mg/l00 g) when held at either 24° or 37°C for 6 hr. Nonextended tuna salads did not develop toxic levels of histamine even when inoculated with known histamine-producing bacteria and held at 24° or 37°C for 48 hr.     

DPPH antioxidant assay revisited

Scavenging of DPPH free radical is the basis of a common antioxidant assay. A number of protocols have been followed for this assay resulting in variation in the results of different laboratories. We present a perspective of the protocols followed by different workers with incongruity in their results and recommend a standard procedure within the sensitivity range of spectrophotometry. Three common standard antioxidants viz. ascorbic acid, BHT and propyl gallate have been used in this study. The IC₅₀ values for ascorbic acid and propyl gallate were 11.8 μM and 4.4 μM in methanol and 11.5 μM and 4.7 μM in buffered methanol as reaction medium, respectively. The free radical scavenging by BHT was markedly influenced by the reaction medium. The IC₅₀ values were 60.0 μM and 9.7 μM when the reaction was done in methanol and buffered methanol, respectively.     

Stoichiometry of coagulation revisited

The roles of particles and natural organic matter (NOM) in determining coagulant (alum) doses in potable water treatment were investigated at two pH conditions (6 and 7). The concentrations of NOM and colloidal silica particles in raw water were systematically varied separately and in combination, and the impacts of these two classes of contaminants on the minimum effective alum doses were investigated using observations of turbidity and dissolved organic carbon (DOC) in laboratory jar tests. At both pHs, coagulant requirements for the removal of these contaminants by sedimentation and filtration were dominated by the DOC concentration in the raw water. The presence of low NOM concentrations (0.75-1.5 mg of C/L) decreased the minimum effective alum dose dramatically for waters low in silica particles, possibly by promoting the precipitation of aluminum hydroxide and/or Al-NOM solids, whose removal would otherwise be limited by low collision opportunities. Strong stoichiometric relationships were observed between DOC and coagulant demand at both pHs regardless of silica particle concentration. Silica contributed to coagulant demand only at very high particle concentrations.