双壳贝类的净化技术研究

本文综述双壳贝类净化技术研究的现状和发展前景，重点介绍双壳贝类的净化方法，为我国双壳贝类净化厂的建设提供科学依据。     

贝类中PSP毒素的脱除方法研究进展

为探讨麻痹性贝类毒素(PSP)的脱除方法,有效提高贝类食品安全性。本文从物理法、化学法以及生物法三个方面综述了PSP毒素在水产品中的脱除方法及净化效率;对温度处理,安全吸附,微生物降解,酶解转化等的研究成果进行了分析总结,以期为贝类脱除PSP毒素及贝类净化研究提供参考。     

壳聚糖及其衍生物脱除贝类中重金属的机理及应用研究进展

海洋重金属污染问题已成为全球关注的热点。因具有较强的重金属富集能力,水产贝类鲜食及其加工产品的重金属超标问题亟待解决。壳聚糖是天然的碱性多糖,具有很强的重金属螯合能力,蕴含量也非常丰富。文章分析壳聚糖及其衍生物用于吸附及脱除重金属的分子结构特征与吸附作用机制;综述其在牡蛎、扇贝等主要经济贝类中的相关研究及应用,强调以生物吸附剂脱除并净化贝类重金属的重要性;对贝类重金属脱除研究提出建议,并展望其应用前景。     

2018—2020年河北省市售贝类中麻痹性贝类毒素污染状况调查分析

目的了解2018—2020年河北省市售贝类中麻痹性贝类毒素(paralytic shellfish poison,PSP)污染状况。方法 2018年8月—2020年5月间,对河北省市售的7种双壳贝类,共508份进行检测分析。样品经0.5%乙酸水提取,石墨化碳黑固相萃取柱净化,采用高效液相色谱-串联质谱法进行检测。结果 508份样品,PSP阳性样品24份,检出率为4.7%, 15份样品超过世界卫生组织规定安全限量,超标率为3.0%。检出贝类为贻贝、毛蚶、杂色蛤、扇贝, PSP含量范围分别为217.0~13001.8μg石房蛤毒素当量(saxitoxin equivalent, STXeq/kg)、217.0~4893.2μg STXeq/kg、217.0~503.6μg STXeq/kg、217.0~11024.5μg STXeq/kg;超标贝类为贻贝、毛蚶、扇贝。贝类中检出的PSP类型有GTX1、GTX4、GTX2、GTX3、neoSTX、STX。结论河北省市售贝类麻痹性贝类毒素暴露风险整体较低,秦皇岛地区贻贝等贝类产品在4、5月份较易受到PSP污染,应持续关注,加强早期监测预警。     

神经性贝类毒素免疫亲和柱的制备与应用

通过纯化PbTX-2单克隆抗体,以琼脂糖凝胶(sepharose 4B)为载体制备神经性贝类毒素(NSP)免疫亲和柱,并建立NSP的免疫亲和柱净化-液相色谱-串联质谱分析方法。样品以含80%甲醇水溶液提取,PBS缓冲液稀释,经过NSP免疫亲和柱净化和富集后进行LCMS/MS测试分析。同时对上样液、淋洗液、洗脱液等亲和柱参数进行优化。结果表明:NSP免疫亲和柱对贝类样品有很好的净化作用,降低了贝类基质中脂肪、蛋白质、色素、分泌黏液等杂质干扰,提高了样品回收率。在40~800μg/kg范围内样品加标平均回收率为82.95%~112.95%,相对标准偏差为1.27%~2.94%。该NSP免疫亲和柱净化效果强、灵敏度高且受基质干扰小,适用于贝类中神经性贝类毒素的分析测定。     

贝类中诺如病毒的研究进展

从贝类中诺如病毒的污染现状、检测组织靶点、病毒浓缩方法及效率、检测方法有效性等多个方面评述贝类中诺如病毒的研究现状、进展、急需解决的问题和未来的发展方向,以期为制定诺如病毒检测技术标准及我国贝类进出口贸易提供参考。     

流式细胞术在双壳贝类免疫研究中的应用

双壳贝类作为重要的水产品经济物种和生态指示物种在全球范围内得到广泛养殖,由于其具有重要的经济价值及在生态毒理学中的环境指示作用,其免疫能力逐渐受到越来越多的关注。双壳贝类先天免疫的主要参与者是血细胞,血细胞参与细胞凋亡、吞噬、抗氧化和免疫黏附等细胞免疫,同时还能分泌大量的溶菌酶、细胞因子、补体系统、抗菌肽等参与双壳贝类的体液免疫,因此,血细胞参数如细胞活力、吞噬能力、粘附能力、总循环血细胞计数、过氧化物酶活性等的检测成为探索双壳贝类免疫研究的重要内容。流式细胞术(flow cytometry, FCM)以其高效、快速、高灵敏度、客观性强等特点成为双壳贝类免疫功能研究的重要手段。本文结合国内外近年来关于流式细胞术在双壳贝类免疫研究中的应用,介绍了FCM的发展历史、双壳贝类免疫学的研究进展及FCM在双壳贝类免疫研究的应用,同时,讨论了流式细胞术在进行贝类总血细胞计数、细胞分类、吞噬作用、细胞凋亡等免疫参数的检测中的应用进展 。提出流式细胞术在处理和检测样品中凾待解决的问题及对未来流式细胞术作为贝类免疫参数检测技术发展展望,以期流式细胞术在未来贝类免疫学的进一步研究中发挥重要作用。     

速食贝类蛋花汤生产技术研究

以低值海产贝类为原料,结合真空冷冻干燥技术制备了海产贝类蛋花汤产品。在配料中添加30mg/L的乳酸链球菌素可以将产品中的菌落总数控制在1000cfu/g以内;添加5.00%的海藻糖可将固态的贝类海鲜蛋花汤的复水率提高到90%以上;研究中确定了贝类蛋花汤的生产工艺和配方,制定了贝类海鲜蛋花汤的感官、理化和微生物指标,制得的海产贝类蛋花汤产品口味鲜美,营养更加丰富。     

贝类生物保鲜技术研究进展

贝类产品味道鲜美,营养蛋白质含量丰富,深受消费者的喜爱。贝类产品大多为滤食性动物,携带有大量的微生物,易发生腐败变质,极大地影响了贝类产品品质。与传统的保鲜剂不同,生物保鲜剂具有安全绿色、无毒无害、易降解等优点已成为当今贝类保鲜领域研究的热点。本文简要总结了贝类产品中的细菌菌相构成及其影响因素,假单胞菌属和希瓦氏菌是贝类产品的特定腐败菌。综述了乳酸链球菌素、壳聚糖、ε-聚赖氨酸、生物酶等天然保鲜剂对贝类产品品质和货架期的影响,比较了上述保鲜剂在贝类产品保鲜中的效果差异,探讨了复配生物保鲜剂和与其他技术相结合的保鲜技术在贝类产品保鲜中的应用,并展望了未来生物保鲜技术在贝类产品保鲜中应用的研究方向。     

QuEChERS净化技术结合高效液相色谱-串联质谱法测定食用贝类产品中4种脂溶性贝类毒素

建立了QuEChERS净化技术结合高效液相色谱-串联质谱法测定食用贝类产品中4种脂溶性贝类毒素(OA,SPX1,DTX1,AZA1)的快速方法。贝类样品匀质后用85%乙腈水萃取,采用QuEChERS净化技术对其进行富集净化后,用HPLC-MS/MS进行检测。以乙腈-水溶液(2 mmo/L乙酸铵,0.1%甲酸)为流动相进行梯度洗脱,经C18色谱柱分离,在多反应监测(MRM)模式下扫描。采用标准曲线外标法定量,OA和DTX1的方法检出限为10μg/kg,在1.0~100μg/L范围内线性关系良好;AZA1和SPX1的方法检出限为1.0μg/kg,在1.0~20μg/L范围内线性关系良好,相关系数(r2)均大于0.999。高、中、低三个添加水平的平均回收率在81.9%~93.1%之间,RSD小于5%。应用该方法对进出口的贻贝、北极贝、象拔蚌、牡蛎等15个样品进行检测,发现5个样品的SPX1测定结果为阳性。该方法操作简便、灵敏度高、重现性好,适用于贝类产品中脂溶性贝类毒素的检测。     

Depuration and Relaying: A Review on Potential Removal of Norovirus from Oysters

Pollution of coastal waters can result in contamination of bivalve shellfish with human enteric viruses, including norovirus (NoV), and oysters are commonly implicated in outbreaks. Depuration is a postharvest treatment involving placement of shellfish in tanks of clean seawater to reduce contaminant levels; this review focuses on the efficacy of depuration in reducing NoV in oysters. There have been many NoV outbreaks from depurated oysters containing around 10³ genome copies/g oyster tissue, far exceeding the median infectious dose (ID50). Half of the published NoV reduction experiments showed no decrease in NoV during depuration, and in the remaining studies it took between 9 and 45.5 d for a 1‐log reduction—significantly longer than commercial depuration time frames. Surrogate viruses are more rapidly depurated than NoV; the mean number of days to reduce NoV by 1 log is 19, and 7.5 d for surrogates. Thus, surrogates do not appear to be suitable for assessing virological safety of depurated oysters; data on reduction of NoV infectivity during depuration would assist evaluations on surrogate viruses and the impact of methods used. The longer persistence of NoV highlights its special relationship with oysters, which involves the binding of NoV to histo‐blood group‐like ligands in various tissues. Given the persistence of NoV and on‐going outbreaks, depuration as currently performed appears ineffective in guaranteeing virologically safe oysters. Conversely, relaying oysters for 4 wk is more successful, with low NoV concentrations and no illnesses associated with products. The ineffectiveness of depuration emphasizes the need for coastal water quality to be improved to ensure oysters are safe to eat.     

A semiquantitative approach to estimate Norwalk-like virus contamination of oysters implicated in an outbreak

Gastroenteritis outbreaks linked to shellfish consumption are numerous and Norwalk-like viruses (NLVs) are frequently the responsible causative agents. However, molecular data linking shellfish and clinical samples are still rare despite the availability of diagnostic methods. In a recent outbreak we found the same NLV sequence in stool and shellfish samples (100% identity over 313 bp in the capsid region), supporting the epidemiological data implicating the shellfish as the source of infection. A semiquantitative approach using most-probable-number-RT-PCR (MPN-RT-PCR) demonstrated the presence of a hundred of RT-PCR units per oyster. Follow-up of the oysters in the harvest area, for approximately 2 months, showed persistence of NLV contamination of the shellfish at levels up to a thousand RT-PCR units per oyster prior to depuration of the shellfish. This finding is useful in beginning to understand shellfish contamination and depuration for use in future hazard analyses.     

Depuration dynamics of viruses in shellfish

The consumption of shellfish has been associated with viral infections even in cases where shellfish complied with the current regulation, which is based on bacterial analysis. In this study, depuration rates of potential indicators and human viruses have been analysed in order to study the use of complementary parameters for evaluating the microbiological quality of depurated shellfish. Depuration of naturally highly polluted mussels has been evaluated and analyses for Escherichia coli, Clostridium perfringens, somatic coliphages, F-RNA phages and bacteriophages infecting Bacteroides fragilis RYC2056 and HSP40, human adenovirus, enterovirus have been done. Seawater of the depuration tank was disinfected by UV irradiation, ozone and passed through a skimmer and a biological filter. The correct functioning of the depuration tank was monitored by the quantification of total organic carbon (TOC), NH4+ and total aerobic bacteria in the seawater. To study the relation between the bacteriophages and the human viruses analysed, a logistic regression model was applied. F-RNA phages are significantly related to human adenoviruses and enteroviruses. Thus, they can be used as a complementary parameter for evaluating the efficiency of the depuration treatment. Somatic coliphages are also significantly associated with enteroviruses. Bacteriophages infecting B. fragilis HSP40 were analysed by the double-agar-layer (DAL) method, which quantifies infectious viruses, and by nested PCR, which detects the presence of the genome of these phages. The highest sensitivity of the molecular techniques was demonstrated and the results obtained are an indicator of a close relation between positive results by PCR and the presence of infectious viral particles in shellfish. All shellfish samples were negative for human viruses by PCR after 5 days of depuration treatment and the results obtained applying a regression model also showed negative results or nearly for F-RNA phages and bacteriophages infecting B. fragilis RYC2056. Thus, in this specific depuration treatment, 5 days may be necessary to assess the sanitary quality of shellfish.     

POTENTIAL OF IRRADIATION TECHNOLOGY FOR IMPROVED SHELLFISH SANITATION

Ionizing radiation is shown capable of serving as an effective sanitizing treatment improving the sanitary quality of shellfish and providing an increased margin of safety for shellfish consumers. ⁶⁰Co irradiation of the hard-shelled clam , Mercenaria mercenaria, and the oyster , Crassostrea virginica, significantly reduced virus carriage numbers without unduly affecting shellfish survival rates or desirable organoleptic qualities. A D₁₀ value of 2 kGy was determined for depletion of hepatitis A virus in clams and oysters as measured by in situ hybridization fluorescent foci and cytopathology enumeration methods. A D₁₀ value of 2.4 kGy was determined for depletion of rotavirus SA11 in clams and oysters as measured by a plaque forming unit enumeration method. Study results showed ionizing radiation capable of providing an extra, highly effective safeguard of shellfish sanitary quality when combined with traditional depuration treatment. Data drawn from other studies is introduced which shows D₁₀ values as low as 1.0 kGy effectively eliminate Vibrio cholerae, and V. parahemolyticus, from shellfish.     

Removal of Escherichia coli, Enterococcus fecalis, coliphage MS2, poliovirus, and hepatitis A virus from oysters (Crassostrea virginica) and hard shell clams (Mercinaria mercinaria) by depuration

Filter-feeding bivalve mollusks (shellfish) can bioaccumulate pathogenic microorganisms in up to 1000-fold higher levels than overlying waters, and therefore disease risks are associated with consuming raw or partially cooked shellfish. Many of these shellfish-borne diseases are due to enteric bacteria and viruses associated with fecal contamination. To control shellfish-borne diseases, guidelines for shellfish harvest waters and shellfish meat have been devised, which include cleansing of contaminated shellfish by depuration in controlled systems, heat pasteurization, or relay to clean waters. This study examines the depuration of oysters (Crassostrea virginica) and hard shell clams (Mercinaria mercinaria) in a flow-through depuration system under variable temperature (12 °C, 18 °C, and 25 °C), salinity (8 ppt, 18 ppt, and 28 ppt), turbidity (< 1 NTU, 10 NTU, and 20 NTU), pH (pH 7 and pH 8), and algae conditions (0 cells/mL and 50,000 cells/mL), with constant dissolved oxygen (5-7 mg/L). Oysters and hard shell clams were artificially contaminated with enteric microorganisms: Escherichia coli, Enterococcus faecalis, coliphage MS2, Poliovirus type-1 and Hepatitis A virus HM-175 (HAV), then depurated in 5-day trials with daily sampling. In oysters, optimizing environmental parameters of water temperature improved E. coli, MS2, poliovirus and HAV depuration, and optimized salinity improved E. coli, E. faecalis, and MS2 depuration rates. In hard shell clams, salinity improved E. coli and E. faecalis depuration rates. Adjusting turbidity, pH or algae did not improve microorganism depuration in either oysters or hard shell clams, with the exception of turbidity on E. faecalis in hard shell clams. Microorganism depuration rates in oysters from greatest to least were: MS2 > E. coli > E. faecalis > poliovirus > HAV, and in clams depuration rates from greatest to least were: E. coli > E. faecalis > HAV > MS2 > poliovirus. Because E. coli and E. faecalis were removed at faster rates than HAV and poliovirus, these fecal bacteria appear to be poor process indicators of the virological quality of depurated oysters and hard shell clams.     

Prevalence of enterovirus and hepatitis A virus in bivalve molluscs from Galicia (NW Spain): inadequacy of the EU standards of microbiological quality

A study of the presence of hepatitis A virus (HAV) and enterovirus (EV) in shellfish from the northwestern coast of Spain, one of the most important mussel producers in the world, was carried out employing dot-blot hybridization and RT-PCR techniques. In addition, bacterial contamination of the samples was evaluated by Escherichia coli (EC) counts, according to the European Union (EU) standards of shellfish microbiological quality. Shellfish samples included raft-cultured and wild mussels, as well as wild clams and cockles. Bacterial counts showed that the majority of samples (40.8%) could be classified as moderately polluted following the EU standards, and therefore should undergo depuration processes. However, differences in bacterial contamination were observed between cultured mussel and wild shellfish. Thus, percentage of clean samples (<230 EC/100 g shellfish) was clearly higher in cultured mussels (49.1%) than in wild mussels (22.8%) or clams and cockles (10.7%). HAV was detected in 27.4% and EV in 43.9% of the samples that were analyzed. Simultaneous detection of both viral types occurred in 14.1% of the samples. Statistical tests of dependence (chi-square test) showed no relationship either between viral and bacterial contamination, or between the presence of HAV and EV. Comparative analysis of hybridization and RT-PCR for viral detection yielded different results depending on the virus type that was studied, RT-PCR being effective for HAV but not for EV detection. The obtained results reinforce once again the inadequacy of bacteriological standards to assess viral contamination and suggest that although virological analysis of shellfish is possible by molecular techniques, interlaboratory standardization and validation studies are needed before the routine use in monitoring shellfish microbiological safety.     

Assessment of edible marine species in the Adriatic Sea for contamination from polychlorinated biphenyls and organochlorine insecticides

It is estimated that 90% of human exposure to persistent organic pollutants is through food, and fish and shellfish represent an important source of contamination for polychlorinated biphenyls (PCBs) and organochlorine insecticides. To evaluate the levels of seafood contamination coming from the central Adriatic Sea, Italy, a study involving several pools of shellfish, crustaceans, and fish was carried out. Several marine species were selected by their abundance, wide distribution, and common use in the Italian diet and sampled and analyzed during 2004. The concentration of total (sigma) PCBs exceeded that of total dichlorodiphenyltrichloroethanes (DDTs) in all samples. Atlantic mackerel showed the highest concentrations of PCBs, ranging from 514 to 1772 ng/g of fat weight, and DDTs, ranging from 52 to 656 ng/g of fat weight. The lowest concentrations of PCBs and DDTs were found in cephalopods and mussels. Despite this, to protect human health from these pollutants, legal limits have been established for fish and shellfish for DDTs but not PCBs. The most common representative PCB congeners, in all species, were PCB 153 and PCB 138; the most common DDT was p,p'-dichlorodiphenyldichloroethylene.     

Investigation into the importance of the stomatal pathway in the exchange of PCBs between air and plants

The transfer of persistent organic pollutants (POPs) from air to vegetation is an important air-surface exchange process that affects global cycling and can result in human and wildlife exposure via the terrestrial food chain. To improve understanding of this process, the role of stomata in uptake of gas-phase polychlorinated biphenyls (PCBs) was investigated using Hemerocallis x hybrida "Black Eyed Stella", a plant with a high stomatal density. Uptake of PCBs was monitored over a 72-h period in the presence and absence of light. Uptake rates were significantly greater in illuminated (stomata open) plants than unilluminated (stomata closed) plants for 18 of the 28 measured PCB congeners (p < 0.05). Depuration of PCBs was monitored in a subsequent experiment over a period of 3 weeks. Levels after 3 weeks of depuration time were still much higher than the concentration prior to contamination. Tri- and tetrachlorinated PCBs showed the greatest depuration, with less than 20% and 50% of accumulated PCBs respectively remaining, while approximately 70% of higher chlorinated PCB congeners remained in the plants at the end of the experiment. Treatments with/without light (to control stomatal opening during uptake) and with/without abscisic acid (ABA) application (to control stomatal opening during depuration) were compared. After contamination indoors for 3 days, there was a significantly higher concentration of PCBs (p < 0.05) in the light contaminated plants than the dark-contaminated plants for 13 of the 28 measured PCB congeners. The ABA treatment affected depuration of PCB-18 only. "Light/ABA-treated" plants had a significantly slower depuration rate for PCB-18 than "light/untreated", "dark/ABA-treated", and "dark/untreated" plants (p < 0.05). The results of the study indicate that there is a stomatal effect on the rate of exchange of PCBs between Hemerocallis leaves and air.