Contamination of shellfish from Shanghai seafood markets with paralytic shellfish poisoning and diarrhetic shellfish poisoning toxins determined by mouse bioassay and HPLC

This paper reports the results of investigations of shellfish toxin contamination of products obtained from Shanghai seafood markets. From May to October 2003, 66 samples were collected from several major seafood markets. Paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) toxins in shellfish samples were monitored primarily by a mouse bioassay, then analysed by HPLC for the chemical contents of the toxins. According to the mouse bioassay, eight samples were detected to be contaminated by PSP toxins and seven samples were contaminated by DSP toxins. Subsequent HPLC analysis indicated that the concentrations of the PSP toxins ranged from 0.2 to 1.9 µg/100 g tissues and the main components were gonyautoxins 2/3 (GTX2/3). As for DSP, okadaic acid was detected in three samples, and its concentration ranged from 3.2 to 17.5 µg/100 g tissues. Beside okadaic acid, its analogues, dinophysistoxins (DTX1), were found in one sample. According to the results, gastropod (Neverita didyma) and scallop (Argopecten irradians) were more likely contaminated with PSP and DSP toxins, and most of the contaminated samples were collected from Tongchuan and Fuxi markets. In addition, the contaminated samples were always found in May, June and July. Therefore, consumers should be cautious about eating the potential toxic shellfish during this specific period.     

VARIED ASSAYS FOR PSP TOXINS IN HEAT SHOCKED PHILIPPINE GREEN MUSSELS (PERNA VIRIDIS)

Toxin assays namely: the mouse bioassay, the receptor binding assay (RBA) and, the immuno-chromatography assay using MIST Alert™ rapid test kit were used to determine the concentrations of Paralytic Shellfish Poisoning (PSP) toxins from untreated and heat shocked Philippine green mussels, Perna viridis contaminated with Pyrodinium bahamense var. compressum. Toxin levels ranging from 4–15 μg STXeq/100 g sample were quantified in the mussel samples analyzed using RBA. Higher levels of PSP toxins at about 30 μg STX eq/100 g sample were recorded using mouse bioassay, which was attributed to interfering factors that could induce mouse death resulting in false positive reactions. The MIST Alert™ test kit showed positive reaction in the samples evaluated based on the reported average profile of PSP toxin analogues at about 40 μg STX eq/100 g sample. The test heat treatments did not elicit definitive change in the PSP toxin profiles of heat shocked mussels relative to the untreated samples.     

Determination of the variability of both hydrophilic and lipophilic toxins in endemic wild bivalves and carnivorous gastropods from the Southern part of Chile

The aim of this study was to analyse and determine the composition of paralytic shellfish poisoning (PSP) toxins and lipophilic toxins in the Region of Aysén, Chile, in wild endemic mussels (Mytilus chilensis, Venus antiqua, Aulacomya ater, Choromytilus chorus, Tagelus dombeii and Gari solida) and in two endemic carnivorous molluscs species (Concholepas concholepas and Argobuccinum ranelliforme). PSP-toxin contents were determined by using HPLC with fluorescence detection, while lipophilic toxins were determined by using LC-MS/MS. Mean concentrations for the total of PSP toxins were in the range 55–2505 μg saxitoxin-equivalent/100 g. The two most contaminated samples for PSP toxicity were bivalve Gari solida and carnivorous Argobuccinum ranelliforme with 2505 ± 101 and 1850 ± 137 μg saxitoxin-equivalent/100 g, respectively (p < 0.05). The lipophilic toxins identified were okadaic acid, dinophysistoxin-1 (DTX-1), azaspiracid-1 (AZA-1), pectenotoxin-2 (PTX-2) and yessotoxins (YTX). All analysed molluscs contained lipophilic toxins at levels ranging from 56 ± 4.8 to 156.1 ± 8.2 μg of okadaic acid-equivalent/kg shellfish together with YTX at levels ranging from 1.0 ± 0.1 to 18 ± 0.9 μg of YTX-equivalent/kg shellfish and AZA at levels ranging from 3.6 ± 0.2 to 31 ± 2.1 μg of AZA-equivalent/kg shellfish. Furthermore, different bivalves and gastropods differ in their capacity of retention of lipophilic toxins, as shown by the determination of their respective lipophilic toxins levels. In all the evaluated species, the presence of lipophilic toxins associated with biotransformation in molluscs and carnivorous gastropods was not identified, in contrast to the identification of PSP toxins, where the profiles identified in the different species are directly related to biotransformation processes. Thus, this study provides evidence that the concentration of toxins in the food intake of the evaluated species (Bivalvia and Gastropoda class) determines the degree of bioaccumulation and biotransformation they will thereafter exhibit.     

可食性涂膜对南美白对虾微冻冷藏的影响

为改善南美白对虾易于黑变及货架期短的问题,采用M1(4-己基间苯二酚(4-HR)0.05g/L＋壳聚糖1.5g/L+乳酸链球菌素(Nisin)0.2g/L)和M2(4-己基间苯二酚(4-HR)0.05g/L＋酪蛋白酸钠1.5g/L＋聚丙烯酸钠0.5g/L＋乳酸链球菌素(Nisin)0.2g/L)两种保鲜剂配方对南美白对虾涂膜保鲜,(－3±1)℃条件贮藏。结果表明：相对于对照组,南美白对虾经M1和M2两种保鲜剂处理后,在(－3±1)℃贮藏过程中菌落总数、挥发性盐基氮(TVB-N)和丙二醛(MAD)增加缓慢,黑变被有效抑制。M1组有最佳抑菌效果,M2组有最佳的感官评分,M1和M2两种保鲜剂均可将货架期延长1倍多。     

EFFECTS OF FEED DIETS ON DIGESTIVE PROTEASES FROM THE HEPATOPANCREAS OF WHITE SHRIMP (Penaeus vannamei)

Protease activities in the hepatopancreas extract (HP) from white shrimp (Penaeus vannamei) fed one of seven test diets for 30 days were evaluated by several methods. The test diet contained 85% of a reference ration for shrimp and 15% of either anchovy meal, tuna waste meal, deboned white fish meal, langostilla meal, soybean meal, and two menhaden meals as a protein replacer. One of the menhaden fish meals tested (B) had the lowest quality as a shrimp feed based on amino acid analysis. SDS-PAGE zymograms of HP from each of the seven diet groups showed similar proteins activity patterns with casein as substrate. The degree of hydrolysis of casein, measured by pH-stat, was also the same for HP from the seven diet groups (P > 0.05). However, total protease activity measured by azocasein hydrolysis (units/g HP) was higher for the diet group fed the test ration containing tuna waste as a replacer (P > 0.05). Trypsin and chymotrypsin activities measured with synthetic substrates (units/mg protein; units/g HP) from animals reared on the diet with menhaden meal B replacer were greater than in the other diet groups (P < 0.05). This study shows that a relatively small amount (15%) of a specific protein replacer in white shrimp rations can influence the protease activity of shrimp HP. Given that digestive proteases such as trypsin can leach into the muscle of postharvest shrimp and thereby cause softening of the meat, the impact of the rearing diet on postharvest shelf-life should be considered along with the standard measures of feed quality that are used by the fish farmer, i.e. animal growth and heatlh.     

Do saxitoxin-like substances have a role in scombrotoxicosis?

Evidence is presented which establishes that mackerel fed in captivity can, by relay from contaminated shellfish via sand eels, accumulate paralytic shellfish poisons (PSP) in the edible flesh at a level (250 micrograms saxitoxin equivalents per kg) similar to that in the contaminated shellfish. Data from ELISAs performed independently in two laboratories show that commercial mackerel fillets which have been associated with incidents of scombrotoxicosis contained 0.02-1.30 micrograms saxitoxin equivalents per kg, concentrations some two to four orders of magnitude below that normally detectable by the mouse bioassay. The doses, expressed as saxitoxin equivalents, administered inadvertently during volunteer testing of such fillets ranged up to 0.5 ng/kg bw, at least four orders of magnitude less than the fatal oral dose for an adult. The doses associated with the rapid induction of nausea/vomiting and/or diarrhoea, 0.11-1.0 ng/kg bw, could not be distinguished from the doses which failed to produce such symptoms in susceptible volunteers (up to 0.5 ng/kg bw). Factors that might explain this lack of correlation between dose (saxitoxin equivalents) and volunteer response are discussed along with previously published reports of PSP relay through the food web. It is suggested that the relay of algal toxins, particularly PSP, but possibly in combination with diarrheic shellfish poisons, may be responsible for scombrotoxicosis.     

烫漂工艺对白对虾品质的影响

研究了白对虾烫漂预处理中的品质变化。采用NaCl溶液对白对虾进行烫漂,探讨在100℃条件下,烫漂溶液浓度和烫漂时间对白对虾失重率、色泽、质构和烫漂残液的影响。试验表明:2%NaCl、烫漂1 min为合理参数,在此条件下,白对虾失重率达10.34%,烫漂液浊度94NTU,每克白对虾流失蛋白质<0.1g,硬度和弹性分别为1 813g、0.842g,彩度为28.07,模糊综合评价等级为优,失重率高,营养损失少,色泽、组织状态均较理想。     

南美白对虾复合生物保鲜剂的优选

为解决南美白对虾易腐败以及化学保鲜剂可能引起的食品安全等问题,通过正交试验,将壳聚糖、茶多酚、乳酸链球菌素(Nisin)进行复配,优选出一种复合生物保鲜剂,并验证了其在南美白对虾防腐保鲜中的效果。结果表明：复合生物保鲜剂的优化质量分数配比为壳聚糖1.5%、茶多酚0.1%、Nisin 0.02%;经该复合生物保鲜剂处理后的南美白对虾在(4 ± 1)℃贮藏过程中挥发性盐基氮(TVB-N)在第8 天还未达到30mg/100g,细菌总数在第9天才达到5 × 105 个/g,保持了较好的感官品质,货架期由4d 延长到了7～8d。处理1kg 对虾新增成本为0.17 元,具有较好的应用价值。     

Effect of cinnamaldehyde on melanosis and spoilage of Pacific white shrimp (Litopenaeus vannamei) during storage

BACKGROUND: Shrimp is a very perishable product and postmortem changes occur rapidly. Sulfiting agents were once and are still widely used as a preservative in the shrimp industry. However, the application of sulfite in shrimp may pose a risk to human health. Thus development of a natural preservative as a sulfite alternative to extend the shelf life of Pacific white shrimp is urgently needed. RESULTS: The effects of cinnamaldehyde essential oil (1 and 5 g kg^?1) on the shelf life of Pacific white shrimp stored at 4 °C were investigated. As the concentration of cinnamaldehyde increased, residual polyphenoloxidase (PPO) enzyme activity decreased. Kinetic analysis showed that cinnamaldehyde was a noncompetitive inhibitor for the oxidation of L ‐DOPA (L ‐3,4‐dihydroxyphenylalanine) by PPO of Pacific white shrimp. Based on this study, shrimp treated with 5 g kg^?1 cinnamaldehyde possessed the lowest aerobic plate count, total volatile basic nitrogen, and pH values in all treatments after 10 days of storage. According to the results of L*, cinnamaldehyde showed inhibitory activity toward the formation of melanosis. CONCLUSION: Treatment with cinnamaldehyde could improve the sensory properties and extend the shelf life of Pacific white shrimp to 8 days. Therefore, cinnamaldehyde could be used as a promising natural preservative for inhibiting melanosis and preventing the growth of microbes during the chilled storage of Pacific white shrimp. Copyright © 2012 Society of Chemical Industry     

Determination of Paralytic Shellfish Poisoning (PSP) toxins in dietary supplements by application of a new HPLC/FD method

Over the past years the importance of food additives and the development of so-called novel food increased permanently. Especially, the application of dietary supplements was on the rise. Then, more and more new products based on plants hitherto not used for human consumption were launched. Algae products containing valuable amounts of essential nutrients such as amino acids and trace elements play a decisive role. On the other hand, some algae including the blue-green algae (cyanobacteria) are capable of synthesizing harmful substances depending on species and provenience. Therefore, methods must be available to evaluate possible risks caused by toxins in algae-based dietary supplements. There are different groups of toxins related to marine algae and cyanobacteria. However, both marine algae and cyanobacteria are able to produce Paralytic Shellfish Poisoning (PSP) toxins which are potential neurotoxins. Hence, analytical methods for PSP determination have to be developed. The method for PSP toxin determination described below is based on ion-pair chromatography of the underivatized PSP toxins followed by post-column oxidation and fluorescence detection (FD). The determination of very low amounts of PSP toxins in different matrices of novel food is possible. In addition, the method allows to compare PSP profiles of various algae-based dietary supplements.     

南美白对虾与南极磷虾复合虾糜的凝胶特性

为了改善南极磷虾虾糜的品质，该研究以南美白对虾与南极磷虾为原料，按照不同质量比例（0:1、1:1、3:2、7:3、4:1）进行混合，研究了南美白对虾与南极磷虾不同比例对复合虾糜凝胶质构、持水率、蒸煮损失、色泽、流变性能和微观结构的影响。结果表明：纯南极磷虾虾糜形成凝胶能力差，南美白对虾与南极磷虾比例从1:1到4:1，复合虾糜的硬度、凝胶强度和持水率分别增加了18.28%、168.64%和5.55%，储能模量（G''）和损耗模量（G"）显著提高；白度值和蒸煮损失分别降低了4.92%和22.81%。扫描电镜结果显示，随着南美白对虾比例的提高，复合虾糜凝胶的孔洞逐渐均匀，致密。南美白对虾与南极磷虾以3:2的比例混合所得复合虾糜，其凝胶性能最好。研究结果为南极磷虾虾糜制品的开发提供理论支撑。     

T-2毒素对凡纳滨对虾肌肉品质特性和自溶作用强度的影响

通过以0.0、0.5、1.2、2.4、4.8、12.2 mg/kg T-2毒素微胶囊毒饵料20 d染毒凡纳滨对虾,采用感官评价、理化分析及显微观察法测定T-2毒素对凡纳滨对虾肌肉品质特性和自溶作用强度的影响。结果表明,T-2毒素暴露对生鲜对虾色泽具有显著性影响(P0.05),通过理化分析得出低剂量作用时对虾肌肉硬度和咀嚼性明显升高(P0.05),随后肌肉组织硬度、弹性、内聚性、胶黏性和咀嚼性显著降低(P0.05);肌肉蒸煮损失率呈现先下降后上升趋势,显微结构观察肌纤维间隙不断变大,肌纤维密度下降,肌纤维碎片不断增加,肌肉品质劣化。随T-2毒素暴露剂量升高,氨基态氮溶出量逐渐降低、可溶性蛋白溶出量先升高后降低,可溶性固形物的溶出量显著增加(P0.05),肌原纤维长度呈现显著性下降(P0.05),扫描电子显微镜下观察肌原纤维断裂严重,自溶强度增加。     

The problem of selective determination of PSP toxins in mussels

Levels of paralytic shellfish poisoning (PSP) toxins in shellfish are routinely determined by mouse bioassay. In order to improve the qualitative and quantitative determination of PSP toxins, chromatographic techniques with fluorescence detection have been developed. These HPLC methods and the HPLC/MS coupling were used to determine a second PSP toxin which was found, in addition to saxitoxin, in canned Spanish mussels. These canned mussels were rejected in 1986 by the German food control because PSP concentrations were too high. It has been shown that these samples contained mainly dc-saxitoxin.     

Application of HPLC for the Determination of PSP Toxins in Shellfish

A high performance liquid chromatographic (HPLC) procedure for determination of the toxins associated with paralytic shellfish poisoning (PSP) is compared to the standard AOAC mouse bioassay method on 100 shellfish samples representing a variety of species. For those samples with toxin content below the detection limit of the bioassay (35 μg saxitoxin (STX)/100g) HPLC analysis indicated a similar low level with a range of <10 to 56 μg STX/100g (n = 60). A correlation coefficient of 0.92 was determined for the 40 samples exhibiting toxicity in the bioassay (i.e., >35 μg STX/100g). Among the advantages of the HPLC method over the bioassay are significantly better sensitivity, greater sample through-put, and ability to determine the levels of each individual PSP toxin.     

南美白对虾虾头主要自溶酶的分离纯化及鉴定

以水解试验为导向,采用硫酸铵分级沉淀、Sephadex G-100凝胶层析、DEAE-DE52阴离子交换柱层析等手段分离纯化南美白对虾虾头主要自溶酶,同时采用抑制剂试验及SDS-PAGE对纯化蛋白酶进行鉴定。结果表明,南美白对虾虾头主要自溶酶经过纯化后比活力高达2.2×106U/g,纯化倍数为96.90。丝氨酸蛋白酶抑制剂苯甲基磺酰氟(PMSF)对主要自溶酶的抑制率高达91.3%;SDS-PAGE显示该酶为单一电泳带,且在电泳中与标准胰蛋白酶移动距离相同,表明南美白对虾虾头内的主要自溶酶为胰蛋白酶。     

Inhibition of melanosis formation in Pacific white shrimp by the extract of lead (Leucaena leucocephala) seed

Lead (Leucaena leucocephala) seed extract was prepared using distilled water as a medium. An extraction yield of 26.16 g/100 g of seed was obtained after extraction at room temperature for 12 h. Total phenolic and mimosine contents in the lead seed extract powder (LSEP) were 17.4 g GAE/100 g and 8.8 g/100 g, respectively. LSEP at different concentrations (0.05%, 0.1%, 0.25%, 0.5%, and 1%, w/v) showed inhibitory activity towards polyphenoloxidase (PPO) of Pacific white shrimp in a dose dependent manner. When the whole Pacific white shrimp were treated with 0.25% and 0.5% (w/v) LSEP, the shrimp treated with 0.5% LSEP had the lower melanosis score throughout the storage of 12 days and showed a higher score for colour and odour, as well as overall likeness, compared with the control (without treatment) and 1.25% sodium metabisulphite treated samples at day 12 (P < 0.05). Meat of shrimps treated with LSEP at both levels had the increase in mimosine content up to 8 days, suggesting the migration of mimosine into shrimp muscle during extended storage. Therefore, 0.5% LSEP can be used as a novel melanosis inhibitor for Pacific white shrimp.     

Quality and shelf life assessment of Pacific white shrimp (Litopenaeus vannamei) freshly harvested and stored on ice

Pacific white shrimps (Litopenaeus vannamei) are an important shrimp aquaculture species worldwide. To quantify the quality and shelf life of untreated shrimp is imperative prior to the application of preservative treatments. In this paper, the quality and shelf life of Pacific white shrimp freshly harvested from three different farms and stored on ice for up to 12 days was investigated. The titratable acidity (TA) of shrimp specimens exhibited significant decreases (P < 0.05) whereas the metric chroma (C), total colour difference (TCD), aerobic plate count (APC), trimethylamine (TMA-N) and total volatile basic – nitrogen (TVB-N), peroxide value (PV) and p-anisidine value (AnV) exhibited significant increases during iced storage (P < 0.05). The TMA-N and TVB-N were significantly correlated whereas temporal TMA-N/TVB-N ratio increased considerably (P < 0.05). While the PV and AnV significantly correlated (P < 0.05), the temporal PV/AnV ratio depicted how primary and secondary lipid oxidation of Pacific white shrimp could relate during iced storage of 12 days. The shelf life of ice stored Pacific white shrimps was determined to be 8 days. The information gained by this study could serve as baseline for preservative treatments applied to fresh shrimps.     

Effects of freeze-thaw cycles of Pacific white shrimp (Litopenaeus vannamei) subjected to radio frequency tempering on melanosis and quality

This study was conducted to investigate the effects of different tempering methods and freeze-thaw cycles on melanosis and quality parameters of pacific white shrimp. Frozen pacific white shrimps tempered with radio frequency tempering (RFT) were compared to that in water tempering (WT) and refrigerator tempering (RT) in terms of temper loss, total volatile base nitrogen (TVBN), polyphenol oxidase (PPO) activity, melanosis, total sulfhydryl contents, differential scanning calorimetry (DSC), and texture properties after 0, 1, 3, 5 freeze-thaw cycles. Results showed that crushed ice was effective as an effective surrounding medium for six layers of frozen shrimp reaching −2 °C within 6 min in RFT. For quality attributes, the temper loss of samples tempered with radio frequency is lower than that of RT and WT after all freeze-thaw cycles, and RFT resulted in the lowest TVBN value (9.17 mgN/100 g) of shrimps after the 5th freeze-thaw cycle. The PPO activity and melanosis of samples increased as the number of freeze-thaw cycles increased, and RFT effectively inhibited the development of melanosis. After the 3rd freeze-thaw cycle, the enthalpy change (△H) and the sulfhydryl content (0.16 mmol/gprot) of radio frequency tempered samples was significantly higher (p < 0.05) than that of WT and RT. RFT retained the hardness and chewiness of shrimp samples in all freeze-thaw cycles. Therefore, RFT effectively inhibited melanosis and reduce protein oxidation in Pacific white shrimp during freeze-thaw cycles with its fast and uniform heating characteristics.     

Semiautomated Method for the Analysis of PSP Toxins in Shellfish

The method uses an autoanalyzer continuous flow reaction system to oxidize toxin in standard acid extracts of shellfish, prepared for mouse bioassay, to derivatives which are detected by fluorescence. Oxidation is by periodic acid under alkaline (NH₄OH) conditions and is followed by acidification by acetic acid. Concentrations of 10 μg/100g toxin and above can be measured with good reproducibility and accuracy: coefficient of variation was 9.5% for samples with 60 μg/100g or greater. Correlation with the mouse bioassay was 0.82 for 204 samples (toxin from 0–2000 μg/100g). The method is proposed to screen shellfish samples for PSP toxins with only samples falling into the range 60–250 μg/100g being subject to the more tedious and expensive mouse bioassay.     

Total lipid,fatty acid composition and lipid oxidation of Indian white shrimp (Fenneropenaeus indicus) fed diets containing different lipid sources

BACKGROUND: Seafood is an important constituent of the human diet. In Iran, Indian white shrimp (Fenneropenaeus indicus) is the major cultured shrimp species as a result of market demand, local availability and growth rate. It is mainly reared using commercial feed. The purpose of this study was to evaluate the effects of replacing 50% of the fish oil by vegetable oils in shrimp feed on total lipid, fatty acid composition and lipid oxidation of shrimp muscle. RESULTS: No significant differences in total lipid content (6.1–7.3 g kg^?1) were found between edible tissues of shrimp fed different diets. The major fatty acids in shrimp muscle were palmitic, oleic, lionoleic, stearic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Higher levels of EPA and DHA were observed in muscle of shrimp fed a diet containing fish oil. Oxidative rancidity, measured as thiobarbituric acid reactive substances, for all shrimps did not exceed 0.2 mg malonaldehyde kg^?1 muscle tissue, which was low and acceptable. CONCLUSION: This study had shown that the fatty acid composition of feed directly affects the fatty acid composition of Indian white shrimp muscle. Farmed Indian white shrimp can be considered as a species of low fat and shrimp muscle was quite stable to oxidation during storage. Copyright © 2009 Society of Chemical Industry