观赏鱼类疾病及其诊断方法

观赏鱼因经长期驯化变种而来比较娇贵,且所处环境也相对稳定,一旦管理不善很容易引起疾病。本文主要讨论了观赏鱼饲养过程中常见疾病的种类、发病原因、诊断方法等,重点介绍了:免疫学诊断技术、核酸诊断技术、核酸技术与免疫学相结合的方法。免疫学诊断技术包括单克隆抗体技术、荧光抗体技术等。核酸诊断技术包括核酸杂交技术、PCR(聚合酶链式反应)技术、16S rRNA检测技术。核酸技术与免疫学相结合的方法有PCR-ELISA法等。     

Quality aspect of fish sauce prepared from underutilized fatty Japanese anchovy and rabbit fish

ABSTRACT:   To explore the potential utility of underutilized fish in the fish sauce industry, fish sauces were prepared from both raw and heat-induced meat of Japanese anchovy Engraulis japonica and rabbit fish Siganus fuscescens using wheat malt, and their quality aspects and sensory properties were assessed. Proximate composition of the fish meat represents protein as the major component (16.0–17.8%), other than moisture. Analyses of free amino acids, peptides including oligopeptides, and organic acids contents in fish sauces revealed suitability of both raw and heat-induced meat of the selected species in commercial fish sauce production. The variations in taste of raw meat fish sauces were species-specific, but the taste of fish sauces from heat-induced meat were similar.     

鱼露特征挥发性化合物的研究进展

研究了鱼露浓郁独特的风味及现代快速发酵工艺所存在的风味品质问题,并综述了近20年来的鱼露特征挥发性化合物的研究,结果表明探究鱼露特征挥发性化合物的形成是可行的。结果显示,气味指纹技术与气相色谱—人工嗅辨仪—质谱(GC-O-MS)联用可以探讨鱼露特征风味的形成;鱼露的气味是融合了奶酪味、氨味、腌渍味、酸味、鱼腥臭味和肉香味的复杂刺激性气味,含硫与含氮化合物是特征挥发性化合物;鱼的种类、嗜盐微生物、酶解途径对特征挥发性化合物形成都有很大影响。研究表明,鱼露的挥发性风味是非常复杂的体系,挥发性化合物的形成不是简单因素作用或个别化合物转化导致的。鱼露挥发性化合物研究可以解决鱼露风味品质提升的难题。     

微生物促进传统鱼酱油发酵和品质改善的研究进展

鱼酱油是一种在东亚和东南亚地区被广泛使用的海鲜调味品,一般是以新鲜海鱼或虾为原料,并拌入大量的食盐(一般20%~30%),在开放环境下经过长期缓慢发酵而制成,具有味道鲜美、营养丰富等特点。传统的鱼酱油生产存在发酵周期过长、生物胺积累、产品品质不一致等问题,利用特定的微生物发酵剂有望解决上述问题。文章在概述传统鱼酱油生产工艺的基础上,着重介绍了传统鱼酱油中的产酶微生物种类,以及利用微生物促进鱼酱油发酵和改善鱼酱油产品品质的研究和应用情况。     

Genotypic diversity among Shewanella spp. collected from freshwater fish

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC‐PCR and pulsed‐field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC‐PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.     

Polymerase chain reaction-restriction fragment length polymorphism analysis for species identification of hairtail fish fillets from supermarkets in Japan

Hairtail fillets are marketed as both fresh and frozen forms in retail outlets within Japan. The present study was undertaken to develop a rapid and reliable method for identification of the hairtail species in commercial fillets. A total of 64 fillet samples, caught from various localities within Japan and purchased from various supermarkets, were analyzed. Polymerase chain reaction (PCR) amplification of a portion of the mitochondrial DNA encoding 16S ribosomal RNA gene and subsequent restriction digestion of the amplified products, using Vspl endonuclease, generated different restriction patterns indicating two different species of hairtails in the fillet samples. The results indicated that the commercial hairtail fillets, labeled and marketed as ‘Tachiuo’ in Japan, were comprised of two species of hairtails with Trichiurus japonicus (commonly called Tachiuo) accounting for 47% and Trichiurus sp. 2 (commonly called Tenjikutachi) accounting for 53% of the analyzed samples. This simple and inexpensive PCR-restriction fragment length polymorphism analysis can be routinely applied to determine species composition of hairtails in commercial fillets.     

Taste effects of oligopeptides in a Vietnamese fish sauce

ABSTRACT: A Vietnamese fish sauce Nuoc mam used in the present study was rich in oligopeptides of which nitrogen content accounted for 20% of total nitrogen. The high-molecular-weight peptide fractions fractionated by ion-exchange chromatographies and ultrafiltration enhanced sweetness and umami as well as sourness and bitterness of the fish sauce, and increased several flavor characteristics including continuity, first taste and aftertaste. Thus, it is apparent that large amounts of peptides produced during the long-term fermentation of fish sauce are responsible for the complicated taste of the sauce. From each fraction, 17 peptides in total were isolated and determined for their amino acid sequences. These di-, tri- and tetra-peptides synthesized by solid- or liquid-phase method gave any one of bitter, sour, umami taste, or practically no taste in the absence of salt. In the presence of 0.3% NaCl, however, almost all peptides showed sweet and umami tastes. Other than these 17 peptides, there existed many other kinds of peptides in the fish sauce. Thus, these peptides are thought to contribute to the overall taste of the fish sauce.     

Changes in extractive components and microbial proliferation during fermentation of fish sauce from underutilized fish species and quality of final products

To make use of underutilized fish species and produce high-quality condiments substitutable for those of mammalian origin, fish sauces were produced on a small industrial scale from three fish species, the flyingfish Cypselurus agoo agoo, the small dolphinfish Coryphaena hippurus, and the deepsea smelt Glossanodon semifasciatus, using salt and koji mold. During 180 days of fermentation, the pH decreased to approximately 4.5 and total nitrogen and free amino acids increased to approximately 2 g/100 mL and 6000 mg/100 mL, respectively. Halophilic and extreme halophilic bacterial counts dramatically increased from 10⁶ to 10⁸ colony forming units/mL between days 14 and 30 and decreased the reafter. In the final products, the pH and salt concentration decreased to between soy sauce and a Vietnamese fish sauce, Nuoc mam. The total amino acid and organic acid contents were almost the same as those of Nuoc mam and soy sauce. Sensory evaluation found these products to have lower smell, saltiness, and bitterness, and higher sweetness and umami taste than Nuoc mam. During fermentation, the histamine content increased in one or two of three fermentation tanks for each fish species. As a result, the histamine content of the final products was higher than that in Nuoc mam.     

Using genetic methods for analysis of fish meals and feeds employed in Greek mariculture

Large quantities of high protein fish meals are needed to sustain cultured species and thus the impact to marine ecosystem has been highly discussed. The aim of this study was to apply a PCR‐cloning methodology for a robust insight into the composition of commercial fish meals and feeds for farmed species of the Greek mariculture, assessing the risk posed by aquaculture to marine ecosystems but also the risk posed by commercial fish feeds to the increase in trophic level of species farmed in Greece. 89% of the sequences were identified to species level and only 11% to genus/family level. Overall, a total of 49 taxa were identified (44 fish species/taxon, five non‐fish species/taxon). Even though small pelagic fish like Engraulis sp. were the main portion, a wide range of species constituted the fish meals and feeds. Plant and animal species were also detected as an alternative protein source. Feed products employed in Greek mariculture still contain large portions of fish meals which increase the mean trophic level of farmed species causing a farming up trend. The results emphasize that such molecular methodologies are needed to certify aquafeeds allowing fish feed producers to demonstrate their commitment to sustainable aquaculture.     

条斑星鲽和圆斑星鲽线粒体12S rRNA与16S rRNA基因序列及其系统分析

采用PCR产物直接测序法分别测定了条斑星鲽和圆斑星鲽线粒体基因组序列,并对12SrRNA和16S rRNA基因核苷酸全序列进行分析,条斑星鲽和圆斑星鲽线粒体12S rRNA的核苷酸序列长度分别为948 bp和949 bp,16S rRNA均为1716 bp。试验结果表明,由12S rRNA和16S rRNA基因所构建的两个系统树的结构基本一致,21种鱼主要分为3个大的分支:鲤形目、鲶形目聚为1支,鲑形目独立聚为1支,鲈形目和鲽形目聚为1大支。鲆鲽类与鲈形目的鲹科、鲷科鱼类聚类在一支,且与鲹科的日本竹荚鱼、大西洋竹荚鱼构成姊妹群,支持鲆、鲽类是从鲈形目分化出来的观点,而同属于鲽形目的鳎科鱼类塞内加尔鳎在12S rRNA树中成为一个独立的分支,在16S rRNA树中聚到鲈形目和鲽形目的分支中,并且与鲈形目关系更近些,鳎类是1个特殊类群,其分类地位有待进一步探讨。线粒体基因组序列已提交到GenBank中,序列号为:DQ403797和EF025506。     

一株病原发光杆菌的分离与鉴定

从三疣梭子蟹育苗场发病且大量死亡的大眼幼体中分离到优势生长的菌株SX1,人工感染试验证明,SX1对健康三疣梭子蟹大眼幼体及Ⅲ期幼蟹有较强的致病性;对菌株SX1进行了形态特征、理化特性等表型生物学特性检验,并进行了菌株SX1的16S rRNA、gyrB和rpoA 3种基因的同源性检索与系统发育学分析。结果显示,菌株SX1具有发光杆菌属的特征,且16S rRNA、gyrB和rpoA 3种基因序列均与发光杆菌具有较高的同源性,在系统发育树中与Photobacteriumganghwense聚为一个分支,自举数据值达100%。综合菌株SX1的形态特征、理化特性及3种基因的同源性检索与系统发育学分析,研究表明菌株SX1与P.ganghwense亲缘关系更近,鉴定SX1为P.ganghwense且为本次引起三疣梭子蟹大眼幼体大量死亡的病原菌。     

基于模糊神经网络的池塘溶解氧预测模型

从患“红斑病”的虾夷马粪海胆病灶处分离出一种可疑病原菌，编号为0205。经感染实验证实，此菌可通过体表创面感染海胆，引起“红斑”症状出现。发病与水温关系密切，高温易发病，低温发病率降低。0205菌革兰氏阴性无芽孢，短杆近球形，具单极毛，生长需盐。对其进行系统鉴定归为弧菌属，其生理生化特性与伯捷氏细菌系统第9版中所列模式种均不相同。经16SrRNA gene序列分析及同源性检索发现该菌与GenBank中AJ002566菌同源性为99．8％，AJ002566菌由德国学者Yakimov直接提交其16SrRNAgene序列1～1476bp，无分类位置。     

南极微生物产低温蛋白酶菌株的筛选、分子鉴定及部分酶活特性

从南极获得的260株低温细菌中筛选到107株具有蛋白酶活性菌株，其中5株菌所产蛋白酶的活性高于45U／mL。对其进行16S rRNA基因序列的同源性和系统发育分析，结果表明，菌株NJ276、NJ5—9、NJ16—70、NJ345属于假交替单胞菌属(Pseudoalteromonas)，NJ341属于科尔韦尔氏属(Colwellia)。对其中NJ276、NJ341、NJ16—70、NJ345这4株产蛋白酶南极嗜冷菌的生长及分泌蛋白酶的部分酶活特性进行研究，结果表明：(1)4株菌最适生长、产酶温度均为10℃左右；培养2～5d，嗜冷菌生长、产酶量一直处于较高的状态。(2)4株南极嗜冷菌分泌的蛋白酶的酶活反应最适pH值为9。(3)菌株NJ276、NJ5—9分泌的蛋白酶最适酶活温度为50℃；菌株NJ341、NJ345分泌的蛋白酶最适酶活温度为40℃；在0℃时蛋白酶活性是最高活性的30％左右，蛋白酶热稳定性较差，因此菌株NJ341、NJ345分泌的蛋白酶属于低温蛋白酶。     

1株军曹鱼病原弧菌的鉴定及其系统发育树分析

从养殖的患病军曹鱼身上分离出1株病原菌JT2，革兰氏阴性，短杆状，有极生鞭毛，能运动，菌落半透明。经回归感染实验，证明该菌为军曹鱼病原菌。进行了常规生理生化实验，并用API-ID32E系统鉴定该菌。再经Biolog-GN细菌鉴定系统鉴定，结果JT2与鲨鱼弧菌（Vibrio carchariae）相似度最高。为了进一步确定该菌的分类学地位，测定了其16SrDNA序列，分析了相关细菌的同源性，构建其系统发育树。结果表明，该菌株与V．carchariae的亲缘关系最近。综合上述几种方法的鉴定结果，最后鉴定该株菌为鲨鱼弧菌V．carchariae。     

Identification of dimethylarsinic acid as the major arsenic compound in fish sauce by liquid chromatography/electrospray ionization-mass spectrometry

ABSTRACT:   Arsenobetaine, the major arsenic compound in marine animals, is substantially non-toxic. It is, however, possible that arsenobetaine undergoes bacterial transformation during manufacturing of fermented fishery products. In the present study, therefore, three types of fish sauce were examined for arsenic concentrations and species compared with those in the raw materials (sardine, Japanese sandfish and Japanese common squid). Arsenic concentrations of the three types of fish sauce were almost equivalent to those in their raw materials, suggesting no accumulation of arsenic during fermentation. Arsenic speciation was performed by a combination of cation-exchange liquid chromatography (LC) and electrospray ionization (ESI)-single quadrupole mass spectrometry (MS). Optimal conditions of LC/ESI-MS were established to analyze seven arsenic compounds (arsenate, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, trimethylarsine oxide, arsenocholine and tetramethylarsonium ion) found in biological samples. When analyzed by LC/ESI-MS, the major arsenic compound in the raw materials was arsenobetaine as expected, while not arsenobetaine but dimethylarsinic acid was identified as the major arsenic compound in the three types of fish sauce. These results suggest that arsenobetaine in the raw materials is converted to dimethylarsinic acid but not to arsenate with high toxicity by bacterial actions during manufacturing of fish sauce.     

1株鲢病原弧菌的分离鉴定

从养殖的患病鲢(Hypophthalmichthys molitrix)组织分离出1株病原菌CQWL002,革兰氏阴性,短杆状,菌落半透明。经回归感染试验,证明该菌为鲢的病原菌。用API系统结合常规生理生化实验对该菌进行初步鉴定。为了进一步确定该菌的分类学地位,测定了其16SrDNA序列,分析了相关细菌的同源性,构建其系统发育树。结果表明,该菌株与河流弧菌(Vibrio fluvialis)的亲缘关系最近。综合上述几种方法的鉴定结果,最后鉴定株菌CQWL002为河流弧菌。