Toxicity of puffer fish cultured in netcages

During 1990 to 2003, the toxicity of the liver in 4,515 specimens of the puffer fish Takifugu rubripes (torafugu) cultured in netcages or on land were investigated by means of mouse bioassay and liquid chromatography-mass spectrometry (LC/MS). Other tissues (skin, muscles, gonads, etc.) were also investigated in some of them. All the livers and other parts examined were found to be non-toxic. The peak corresponding to tetrodotoxin (TTX) was not detected in the samples by LC/MS analysis for TTX (< 0.1 MU/g). These results show that puffer fish fed on a non-toxic diet in netcages do not become intoxicated.     

High-resolution mass spectrometry analysis of tetrodotoxin (TTX) and its analogues in puffer fish and shellfish

Tetrodotoxin (TTX) is an emerging toxin in the European marine environment. It has various known structural analogues. It acts as a sodium channel blocker; the ability of each analogue to bind to the sodium channel varies with the particular structure of each analogue. Thus, each analogue will vary in its toxic potential. TTX analogues co-occur in food samples at variable concentrations. An LC-MS method was developed for the identification and quantitation of several analogues of TTX using an LTQ-Orbitrap XL mass spectrometer. The LTQ-Orbitrap XL mass spectrometer facilitates high mass accuracy measurement up to 100,000 full width at half maximum (FWHM). Using high resolution at 100,000 FWHM allows for the identification of TTX and its analogues in various matrices, including puffer fish and molluscan shellfish samples (Δ ppm = 0.28–3.38). The confirmation of characteristic fragment ions of TTX and its analogues was achieved by determining their elemental formulae via high mass accuracy. A quantitative method was then developed and optimised using these characteristic fragment ions. The limit of quantitation (LOQ) of the method was 0.136 µg g^–1 (S/N = 10) and the limit of detection (LOD) was 0.041 µg g^–1 (S/N = 3) spiking TTX standard into TTX-free mackerel fish extracts. The method was applied to naturally contaminated puffer fish and molluscan shellfish samples to confirm the presence of TTX and its analogues.     

养殖河豚鱼毒素的研究

为了探究养殖河豚鱼可食用性,本实验对养殖河豚鱼的食用安全进行了研究,提取河豚鱼的肝脏、肌肉、血液、性腺为研究对象,用液相色谱法测定TTX的含量。结果表明,养殖(红鳍东方豚、暗纹东方豚、菊黄东方豚、黄鳍东方豚)肝脏、血液无毒或弱毒,肌肉无毒,可作为食用鱼种,而野生的暗纹东方豚均有剧毒,不可食用。     

鼠李糖乳酸杆菌对河豚肝脏中河豚毒素毒性的影响

以野生河豚肝脏中的河豚毒素（tetrodotoxin,TTX）为研究对象,研究活化与热灭活处理的鼠李糖乳杆菌对其毒性的消减作用。将活化的与热灭活处理的鼠李糖乳杆菌作为材料对TTX进行脱除,采用酶联免疫吸附剂测定（enzyme-linked immunosorbnent assay,ELISA）、层析试纸检测和小鼠生物法分析TTX消减率的变化,采用气相色谱-质谱分析发酵处理后的肝脏二十碳五烯酸（eicosapentaenoic acid,EPA）与二十二碳六烯酸（docosahexaenoic acid,DHA）含量变化。竞争性ELISA结果显示加热灭活后的鼠李糖乳杆菌对TTX的消减率高达82.16%;而未经灭活处理的鼠李糖乳杆菌对TTX的消减率为70.05%,表明热灭活处理的鼠李糖乳杆菌消减效果更佳。此外,经鼠李糖乳杆菌发酵7 d后野生河豚肝脏的TTX消减率为93.27%,气相色谱-质谱检测结果显示发酵后肝脏内的EPA与DHA含量分别减少了11.93%、22.50%。综上所述,鼠李糖乳杆菌能消减河豚肝脏中的TTX,经鼠李糖乳杆菌发酵处理后消减效果更佳,本研究结果可为河豚内脏组织的毒性消减提供参考。     

鲀毒鱼毒素检测及物种鉴定技术研究进展

鲀毒鱼引起的河鲀毒素(tetrodotoxin,TTX)中毒是中国沿海地区食物中毒致死的主要原因之一.河鲀毒素中毒发病迅速,至今尚无特效药,因此,通过检测TTX含量或鉴定携带TTX的物种可以更好地进行TTX中毒的风险分析、管理和控制.本文综述了鲀毒鱼TTX的检测技术以及鲀毒鱼物种鉴定的方法,以期能预警并减少鲀毒鱼引起的...     

LC-MS/MS method for the determination of tetrodotoxin (TTX) on a triple quadruple mass spectrometer

Tetrodotoxin (TTX), often referred to as the ‘puffer fish’ poison, is a marine toxin and it has been identified as the agent responsible for many food poisoning incidents around the world. It is a neurotoxin that blocks voltage-gated sodium channels, resulting in respiratory paralysis and even death in severe cases. It is known to occur in many different species of fish and other organisms. The toxin is mainly found in the Southeast Asia region. Worryingly, TTX is starting to appear in European waters. It is suspected that this is a consequence of Lessepsian migration, also known as the Erythrean invasion. Therefore, straightforward and reliable extraction and analytical methods are now urgently required to monitor seafood of European origin for TTX. This paper provides a versatile, dependable and robust method for the analysis of TTX in puffer fish and trumpet shellfish using LC-MS/MS. A three-stage approach was implemented involving: (1) the screening of samples using fast multiple reaction monitoring (MRM) mass spectral analysis to identify quickly positive samples on a triple quadrupole mass spectrometer (QqQMS/MS), the API 3000; (2) a Fourier-transform (FT)-MS full-scan analysis of positive samples to collect qualitative data; and (3) a method with a longer chromatography run to identify and quantitate the positive samples using the QqQMS. The quantitative LC-QqQMS method delivered excellent linearity for solvent-based standards (0.01–7.5 µg ml^–1; R² ≥ 0.9968) as well as for matrix-matched standards (0.05–37.50 µg g^–1; R² ≥ 0.9869). Good inter-day repeatability was achieved for all the relevant analytes with %RSD values (n = 9) ranging from 1.11% to 4.97% over a concentration range of 0.01–7.5 µg ml^–1. A sample clean-up procedure for the puffer fish and trumpet shellfish was developed to ensure acceptable and reproducible recoveries to enable accurate and precise determination of TTX in a myriad of tissues types. Blank mackerel matrix was used for the TTX standard spiking studies in order to calculate the recoveries of the toxin during the extraction procedure. The recovery was 61.17% ± 5.42% for the extraction protocol. MS/MS studies were performed on a linear-trap quadruple-Orbitrap mass spectrometer (LTQ-Orbitrap) to obtain high-mass-accuracy data of the target analytes and their characteristic fragment ions in the puffer fish and trumpet shellfish samples. This facilitated identification of TTX and its associated analogues. These high-mass-accuracy studies facilitated the development of a rapid MRM-based quantitative method for TTX determination on the LC-QqQMS.     

Development of competitive indirect ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the tissues of wild puffer fish in the waters of south-east China

A monoclonal antibody against tetrodotoxin (TTX) was produced from the hybridoma cell line T6D9, which was established by the fusion of Sp2/0 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with the TTX–keyhole limpet hemocyanin (KLH) conjugate. This monoclonal antibody belongs to the IgG1 subclass; the affinity constant of the antibody is 2.4 × 10^?8 mol l^?1. The relative cross-reactivity of the antibody with TTX was 100%, but with saxitoxin, KLH and bovine serum albumin (BSA) it was less than 1%, respectively. The titre of the antibody in ascites was 6.4 × 10⁶; the reference working concentration was 1:1.2 × 10⁵. By using this monoclonal antibody, a competitive indirect enzyme-linked immunoabsorbant assay (ELISA) for the analysis of TTX was developed. The linear portion of the dose–response curve of TTX concentration was in range 5–500 ng ml^?1. The limit of detection was 5 ng ml^?1 according 10% inhibition with TTX to anti-TTX monoclonal antibody. The concentration of TTX inhibiting 50% of antibody binding was about 50 ng ml^?1. The recoveries from TTX spiked samples were 79.5–109.5%. In addition, the toxicity of some wild puffer fish specimens captured from south-east China and the Yangzi River in Jiangsu province was determined. The results indicate that the toxicity and toxin tissue distribution vary in different species of wild puffer fish.     

Tetrodotoxin poisoning due to smooth-backed blowfish Lagocephalus inermis and toxicity of L. inermis caught off the Kyushu coast, Japan

Food poisoning due to ingestion of a puffer fish occurred in Nagasaki Prefecture, Japan, in October 2008, causing neurotoxic symptoms similar to those of tetrodotoxin (TTX) poisoning. In the present study, we identified the species, toxicity, and toxins using the remaining samples of the causative puffer fish. The puffer fish was identified as smooth-backed blowfish Lagocephalus inermis by nucleotide sequence analysis of the 16S rRNA and cytochrome b gene fragments of muscle mitochondrial DNA. The residual liver sample showed toxicity as high as 1,230 mouse unit (MU)/g by bioassay and TTX was detected by liquid chromatography/mass spectrometry analysis. We therefore concluded that the food poisoning was due to TTX caused by consumption of the toxic liver of L. inermis. This is the first report that the liver of L. inermis caught in Japanese waters is strongly toxic, with levels exceeding 1,000 MU/g. In this context, we re-examined the toxicity of L. inermis collected off the coast of Japan. Of 13 specimens assayed, 12 were toxic, although the toxicity varied markedly among individuals and tissues. Because the intestine and ovary of L. inermis have been considered non-toxic, it is particularly noteworthy that these organs were determined to be toxic, with a maximum toxicity of 43.6 MU/g and 10.0 MU/g, respectively. Furthermore, kidney, gallbladder, and spleen, whose toxicity has been unknown, were frequently found to be weakly toxic with levels ranging from 10 to 99 MU/g. Therefore, further study is needed to re-examine the toxicity of smooth-backed blowfish L. inermis in the coastal waters of Japan.     

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.     

Determination of Tetrodotoxin by Capillary Isotachophoresis

A method is described for the quantification of tetrodotoxin (TTX) in crude extracts of puffer fish by capillary isotachophoresis. Conditions for isotachophoresis consisted of a leading electrolyte of 5 mM potassium acetate, pH 6.0, containing 0.2% Triton X-100 and 0.5 vol of dioxane, and a terminating electrolyte of 10 mM β-alanine adjusted to pH 4.5 with acetic acid. It was possible to quantify the TTX content of crude unfractionated puffer extracts, and obtain values similar to those obtained by mouse bioassays which are time consume.     

Rapid screening of tetrodotoxin in urine and plasma of patients with puffer fish poisoning by HPLC with creatinine correction

A rapid and simple detection method for tetrodotoxin (TTX) in urine and plasma of patients with puffer fish poisoning was developed using commercially pre-packed solid-phase extraction (SPE) cartridges (C18 and weak cation exchange columns) and subsequent analyses by HPLC with UV detection. The detection limit of the standard TTX, TTX-spiked urine and plasma samples were all 10 ng/ml and the average TTX recovery in urine and plasma samples after SPE were 90.3 ± 4.0 and 87.1 ± 2.9%, respectively. It was noticed that the creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation apparently correlated much better with the severity of poisoning than the urinary TTX concentration without adjusting for variations in concomitant creatinine excretion.     

Evaluation of weakly acidic electrolyzed water and modified atmosphere packaging on the shelf life and quality of farmed puffer fish (Takifugu obscurus) during cold storage

The combined effect of weakly acidic electrolyzed water (WAEW) and modified atmosphere packaging (MAP) treatments on the quality of puffer fish (Takifugu obscurus) during cold storage was studied on aspects of microbiological activity, texture, total volatile basic nitrogen (TVB-N), trimethylamine (TMA), free amino acids (FAAs), thiobarbituric acid reactive substance (TBARS), ATP-related compounds and K value, volatile organic compounds (VOCs), and organoleptic properties. As a result, significantly (p < .05) higher inhibitory effects on total viable counts (TVC), H₂S-producing bacteria (including Shewanella putrefaciens), Pseudomonas spp., and lactic acid bacteria (LAB) were observed in WAEW-treated puffer fish packaged in 60%CO₂/5%O₂/35%N₂ atmosphere than that in air package and vacuum package with/without WAEW-treated samples. In addition, chemical results showed that WAEW together with MAP treatments were highly efficient in maintaining lower TVB-N, TMA, and TBARS values in refrigerated puffer fish. Moreover, the presence of WAEW combined with MAP treatments showed positive effects on retarding the relative content of fishy flavor compounds, such as 1-octen-3-ol, 1-penten-3-ol, hexanal, heptanal, nonanal, decanal, (E)-2-octenal, and 2,3-butanedione. As a whole, the combined effect of WAEW and MAP on refrigerated puffer fish is advisable to maintain better quality and extend the shelf life.     

河豚毒素及其发酵生产的研究进展

河豚毒素(tetrodotoxin,TTX) 常见于河豚体内,是一种毒性很强的氨基全氢化喹唑啉化合物。它是世界上最致命的生物毒素之一,同时也是对神经有选择性作用的强效麻醉药和戒毒良药。因此,科研人员在弄清TTX的产生途径及产生菌后,正在竭力研究其生产工艺及高产方式,以使其显著的医药学价值为人类所用。本文综述TTX 的起源、理化性质及其生物活性等基本特性,并重点介绍近年的研究方向和热点问题,即TTX 的产生菌和发酵生产等方面的研究。     

Puffer fish-based commercial fraud identification in a segment of cytochrome b region by PCR–RFLP analysis

To identify the mislabeled or fraudulently substituted toxic puffer fish in thermally processed fish products, a polymerase chain reaction (PCR) method using restriction sites and sequence analysis has been developed in this study. A 376-bp fragment of the cytochrome b gene was produced after PCR amplification. Fish tissue samples were prepared under autoclaving conditions at 121 °C for 10–90 min at 10 min intervals. DNA fragments could not be detected after 90 min of autoclaving at 121 °C. For PCR product digestion, BsaJ I, Aci I, Hinf I, Taq I, and Sap I endonucleases were used to yield species-specific profiles for the identification of puffer fish species from 60 commercial market samples. Results from this study showed that the restriction fragment length polymorphism technique can be used to identify 17 puffer fish species from commercial products even after severe thermal processing.     

Effects of combined treatment of electrolysed water and chitosan on the quality attributes and myofibril degradation in farmed obscure puffer fish (Takifugu obscurus) during refrigerated storage

The obscure puffer fish (Takifugu obscurus) is underutilized as a food fish. To improve the quality of puffer fish flesh, the fish samples were preserved after treating them with electrolysed water (EW), a combination of EW and chitosan (EW/chitosan), or a combination of EW and carboxymethyl chitosan (EW/CMC). The effects of these treatments were compared by analysing changes in the total viable count of aerobic bacteria, firmness, total volatile basic nitrogen, trimethylamine, pH, the thiobarbituric acid and sensory attributes of the refrigerated puffer fish. Changes in the myofibril length of puffer fish flesh was also studied, which is related to flesh softening. The findings suggest that during storage, EW/chitosan treatment is more effective than both EW and EW/CMC treatments in inhibiting microbial growth and myofibril degradation, and in maintaining the quality of obscure puffer fish flesh. This could extend the shelf life by 50% of the original shelf-life of about 4 days of the fish samples.     

养殖河豚鱼的加工及河豚毒素消减技术研究概述

河豚鱼肉质鲜嫩,味道鲜美,享誉古今中外。我国东南沿海地区、日本等许多国家和地区均有吃河豚的习惯,但河豚鱼的内脏、表皮、卵巢等部位含有河豚毒素,因食用河豚鱼导致的中毒事件时有发生。因此,有效消减控制河豚毒素对河豚鱼产业发展有重要意义。该文主要介绍了我国河豚鱼食用历史和消费现状,对养殖河豚鱼深加工制品种类做详细阐述,重点探讨了河豚毒素消减方法和消减机理的研究情况,以期对河豚毒素的消减控制提供参考。     

Profiling of volatile compounds of Phyllostachys pubescens shoots in Taiwan

This study examined the influence of heating temperature and duration on volatile aromatic components of spring and winter Phyllostachys pubescens shoots using SPME. Results from GC–MS analyses revealed that the main constituents in both bamboo shoots at ambient temperature include methoxy-phenyl oxime, followed by n-hexanol and 3Z-hexenal, which gives a fresh green aroma. Comparing the different compounds, between spring and winter shoots, revealed that spring bamboo shoots at ambient temperature comprise 12.30% methyl salicylate, which provides protection against insect attack, and 9.71% epi-cedrol; while winter bamboo shoots comprise 17.00% 1-octen-3-ol, which produces a distinct mushroom aroma. After heating at 100 °C for 60 min, a marked increase in relative content of benzyl salicylate (43.30%) and a significant decrease in methyl salicylate content in spring bamboo shoots were observed; while the major compound in winter bamboo shoots was n-heneicosane (78.09%) and the content of specific 1-octen-3-ol significantly decreased.     

基于食品防护的出口河豚鱼加工过程控制的研究

基于食品防护和HACCP原理对出口河豚鱼加工过程控制的研究,为出口河豚鱼加工企业科学化控制加工过程提供借鉴。应用危害分析的方法,对出口河豚鱼加工过程中的生物性、化学性和物理性的食品安全危害进行识别和分析,应用关键控制点判断树的方法确定关键控制点并制定控制措施。出口河豚鱼加工过程中的关键控制点如原料验收、挑选、排盘、金属探测可以通过实施HACCP计划加以控制;而对于人为蓄意破坏而引起的危害则通过实施食品防护计划包括企业内外部安全、加工过程安全、贮存和运输过程安全等11个方面加以控制。通过实施HACCP计划和食品防护计划,可对出口河豚鱼加工过程中食品安全危害进行有效地控制,从而确保出口河豚鱼的质量。     

Chemical variation in the essential oil ofEphedra sinica from Northeastern China

Hydro-distillated volatile oils of Ephedra sinica Stapf. from six populations of Inner Mongolia in Northeastern China were analyzed by using GC/MS. Ninety-nine compounds were identified in the oils and a relatively high variation in their contents was found. The main constituents of the essential oils were α-terpineol (19.28–52.23%), p-vinylanisole (0.59–11.64%), 3-methyl-2-buten-1-ol (0–5.44%), tetramethylpyrazine (0.63–8.99%), terpine-4-ol (1.17–4.37%), α-linalool (1.62–5.15%), phytol (1.24–15.73%), γ-eudesmol (0–7.77%), and eudesm-7(11)-en-4-ol (0.41–6.13). Six populations were divided into two chemotypes based on cluster analysis and principal component analysis (PCA); one rich in α-terpineol and p-vinylanisole, and the other rich in phytol, γ-eudesmol, and eudesm-7(11)-en-4-ol.     

The composition of fatty acids in the tissues of Tunisian swordfish (Xiphias gladius)

The objective of this study was to compare the composition of fatty acids stored in the various parts of swordfish. Muscle and organ sections of a series of swordfish samples were collected. Their chemical analysis allowed the classification of swordfish as a semi-fatty fish, with its byproducts totalling 35.6% of the total fatty acids (TFA) and its white and red muscles (MR) 7.2%. The highest contents were found in the liver (26%), the gonads (4.7%) and the red muscle (RM) (4.5%). The UFA/SFA ratio as recommended by nutritionists is 3; in the liver, skin and RM samples this ratio was, respectively, 3.5, 2.8 and 2.7. There is a high level of unsaturated fatty acids (UFA) in swordfish. More than 40% of the muscular tissues are made up of polyunsaturated fatty acids (PUFA) of the n-3 series (EPA + DHA). The byproducts contain more than 30% of monounsaturated acids characterised by the fatty acids of the n-9 series and particularly by oleic acid.