Extracellular protease production of bacteriolytic bacteria isolated from marine environments

ABSTRACT:   Fourteen bacterial strains isolated from marine environments exhibited antagonistic action against a wide range of bacteria including Vibrio spp. A double layer agar method was used for preliminary screening to determine the relative degree of growth inhibition or bacteriolysis exhibited by the isolates. Most of the antagonistic isolates were found to be Gram-negative, motile rods and were oxidase positive, and oxidative in the oxidation and fermentation test, suggesting that they are belong to the genera Pseudomonas . The antagonistic isolates lyzed the dead cells of marine Gram-negative bacteria in both plate and liquid methods. Bacteriolytic and casein hydrolytic activities were observed in the culture supernatant of the isolates. Anion exchange column chromatography (Toyopearl DEAE-650 M) was used to purify the extracellular protease produced by an antagonistic strain A1-J25a. The active fractions of protease collected from the eluted solution also exhibited bacteriolytic activity.     

Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring¹⁴C-oleic acid release from¹⁴C-triolein.¹⁴C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200–400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40–43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that V_max=0.016 nmol/h/mg protein and that K_m=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg²⁺. These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.A part of this work was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1990, San Antonio, TX.     

Rates of fish waste production and effluent discharge from a recirculating system (biofish) under commercial conditions

Application of the regulatory principle of ‘best available technology’ (BAT) to fish farm effluent control has, to some extent, been a driving force for the development of new culture and treatment technology. In Norway today, there are a number of farms for the production of Atlantic salmon, Salmo salar L., smolts and brown trout, Salmo trutta L., fingerlings that utilize microstrainers for the removal of particles from the effluent water. At least one commercial farm also utilizes a simplified recirculation system called BIOFISH as a demonstration of new and alternative technology for the production of brown trout restocking fish. In this paper, calculated effluent discharge and rates of waste production from the biofish demostration trials are compared to literature data and to measurements on un-treated as well as microstrained effluents from the production of Atlantic salmon smolts in a traditional flow-through tank system. Rates of fish waste production in the biofish trials were obtained from mass-balance calculations based on measured concentrations of water quality parameters at several points in the system. The results of these calculations show fish waste production rates that are low, but comparable to data found in the literature. Given the level of waste treatment that takes place in the biofish tanks, the specific effluent discharge levels from those tanks, in terms of grams per kilogram biomass and grams per kilogram feed, are considerably lower than those found for salmon smolt production in traditional flow-through tanks. There are also substantial differences in hydraulic self-cleaning properties of the two systems and a corresponding difference in the distribution of effluent discharge during normal tank operation and during tank/effluent pipeline flushing. The results presented here give valuable information related to: (1) waste output characterization; (2) the long-term efficiency of commercially available particle separation systems; and (3) the capabilities of the simplified biofish recirculation technology under field conditions.     

海洋酶法利用海产品下脚料制取活性胶原肽的研究

以富含胶原成分的海产品下脚料为原料,经脱除杂蛋白、脂肪后,用海洋碱性蛋白酶水解,以Feton法检测水解产物的羟自由基清除率,提取具有抗氧化活性的胶原肽。通过温度T(℃),pH值,酶料比([E]∶[S]),料水比([S]∶[W]),时间(min)单因素实验,选取合理水平,做L18(37)正交实验,最终优化得到最佳提取条件为:温度45℃、酶料比1∶150、pH8·0、料水比1∶4、反应30min,所得胶原肽的羟自由基清除率为60·42%。与同浓度Vc、谷胱甘肽(GSH)相比,均具有较高的抗氧化活性,有着良好的开发应用前景。     

鱼类加工副产物中胶原提取技术与性质分析

鱼类加工产业发展的同时,不可避免地产生大量鱼皮、鱼鳞、鱼骨、鱼鳍、鱼鳔等不同鱼类加工副产物,如何对这些副产物进行高值加工利用引起广泛关注。目前,鱼类加工副产物的利用途径主要包括制作休闲食品、饲料鱼粉、胶原等。其中,因胶原产品的附加值空间大,以鱼类加工副产物为原料提取胶原成为当前研究的热点之一。在介绍不同鱼类加工副产物组成特点的基础上,阐述了近年来国内外以不同鱼类加工副产物为原料提取胶原的研究现状,总结分析了从不同鱼类加工副产物中提取胶原的提取率、产物纯度及性质,展望了以鱼类加工副产物为原料提取胶原的发展前景和方向,分析认为酸-酶结合法更适用于鱼胶原的提取,并具有广阔前景。     

Effect of Lactococcus lactis and Enterococcus faecium on growth performance,digestive enzymes and immune response of grouper Epinephelus coioides

The effect of dietary administration of Lactococcus lactis MM1 and Enterococcus faecium MM4, two strains of lactic acid bacteria isolated from the gut of juvenile grouper Epinephelus coioides, on the growth, feed utilization, digestive enzymes and immune response of juvenile E. coioides was evaluated. The fish were fed for 60 days with three different diets: control diet (without probiotic), diet P1 supplemented with 1.0 × 10⁸ CFU g^?1L. lactis or diet P2 with 1.0 × 10⁸ CFU g^?1E. faecium. Although growth and feed intake decreased slightly, significant improvement of feed utilization was observed in fish fed diet P1 and diet P2 for 60 days. The hepatopancreatic protease activities in the two probiotic treatments were significantly higher than that in the control. The phagocytic activity, serum superoxide dismutase activity and complement component 4 (C4) and IgM levels were not significantly affected by diet P1 and diet P2. Serum complement component 3 (C3) levels in the two treatments were significantly higher than that in the control. Significant increased serum lysozyme activity was observed in fish fed the diet P1, while significant decreased lysozyme activity was observed in fish fed the diet P2 compared with the fish fed the control diet. There was a significant interaction effect between diet and administration date for the complement C4. The study demonstrates potential for L. lactis MM1 and E. faecium MM4 to improve the feed utilization and modulate the immune function in E. coioides.     

斑点叉尾鮰源嗜麦芽寡养单胞菌胞外蛋白酶的纯化及性质研究

采用硫酸铵盐析,DEAE Sephadex A－50凝胶层析等方法从嗜麦芽寡养单胞菌胞外产物中提纯了一种分子量为45.7 ku的单一多肽蛋白酶。该酶对小鼠和斑点叉尾鮰具有明显的致死作用,其LD50分别为4.33 μg•g-1体重和3.49 μg•g-1体重。酶的最适温度为20 ℃,热稳定性差,100 ℃作用15 min酶活完全丧失,最适pH为9.0,PMSF对酶活无影响,部分金属离子如Ca2+、Hg2+、Cu2+能使酶活性明显下降,而Co2+对酶有一定程度的激活作用,EDTA能完全抑制酶活性,表明该酶为一种金属蛋白酶。图1表4参29     

Biochemical and physiological characteristics and plasmid profiles of Aeromonas hydrophila strains, isolated from freshwater fish and from fresh water

Abstract. Twenty-five Aeromonas hydrophila strains, isolated from freshwater fish and from freshwater samples were identified, using API 20NE and CH50. They were screened for the one plasmid (20 kb), and a third group (four strains) contained two or three plasmids. A 20 kb plasmid was common to all plasmid-positive strains. Although their plasmid profile differs, all groups are very similar with regard to their biochemical and physiological characterization. However, the differences observed using clustering can be explained by characteristics, some of which have been reported as virulence associated. No relationship between these factors and the presence of plasmids is found. The possibility of differentiating A. hydrophila strains on the basis of biochemical characteristics using a cluster analysis is considered.     

鱼蛋白酶水解物的钙螯合修饰及其功能活性研究

以低值鱼蛋白为原料通过复合酶水解法和钙修饰法获得了蛋白质酶水解物的修饰产物并对其功能活性进行了初步研究.结果表明,未脱脂的鱼蛋白酶水解物钙螯合修饰的最适宜条件为蛋白质水解度为5%、螯合pH为7.0、螯合温度为20℃、螯合完成时间为15min;无水乙醇分级分离可获得三种螯合组分,水不溶组分(CA)、50%无水乙醇不溶性组分(CB)和80%无水乙醇不溶性组分(CC);红外光谱分析表明,CA的钙紧密地与氨基和羰基基团结合形成螯合修饰物,并以钙为中心形成五元环,而CB和CC组分中钙只与羰基紧密结合形成环状结构;在这些螯合物组分中,CA具有最高的抗氧化活性,且高达α-生育酚的94%,而CC具有最高的抗大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌的活性,并发现抗菌活性大小与组分的水溶性和分子中电子中继系统的电子缓冲能力有关.本文的研究可望对海洋低值鱼蛋白质的高效利用和蛋白质的酶水解物经螯合修饰后作为食品添加剂在食品工业中的应用奠定理论基础.     

Effect of fish feed processing conditions on digestive protease activities, free amino acid pools, feed conversion efficiency and growth in Atlantic salmon (Salmo salar L.)

The responses of the digestive proteases trypsin and chymotrypsin and protein metabolism to differences in feed protein quality were investigated in Atlantic salmon (Salmo salar L.). Two sets of experimental feeds were produced. Each set of high and low quality feeds was provided to either 150 g or 2 kg salmon. Protein in the high quality feeds had significantly higher percentages of free (reactive) sulphydryl (SH) groups than the corresponding feeds based on low quality meals. After 90 days feeding, groups given high and low quality feeds did not differ in their specific growth rates (SGR) in either experiment. However, feed conversion efficiency (FCE) was significantly different between the high and low quality feed groups in 2 kg salmon, where the difference between the high and low feed protein qualities was larger, 10% versus 4% SH/[SH + (S–S)] in 150 g salmon. Higher FCE was preceded by significantly higher trypsin and chymotrypsin specific activities on day 60. SGR, in general, changed after the first month and was stable during the last 2 months in both experiments. Concurrently, both trypsin (T) and chymotrypsin (C) decreased with an increased activity ratio of trypsin to chymotrypsin (T/C ratio), and resulting in significantly lower T/C ratio on day 90 in salmon feeding on high quality feeds in both sizes of fish. Differences in FCE were associated with significant differences in levels of total free amino acids (TFAA) in the plasma and the white muscle, as well as in the ratio of essential to non‐essential free amino acids (EAA/NEAA ratio), free hydroxyproline, and RNA in the white muscle. Interestingly, after 3 days starvation (day 93), 5–7 h postprandial EAA/NEAA ratio in the plasma was significantly lower in the high quality diet groups in both experiments. Trypsin specific activity inversely correlated with muscle TFAA levels in 2 kg salmon, concurrent with higher muscle levels of RNA, lower free hydroxyproline and higher FCE in fish fed higher quality diets.     

鱼源气单胞菌的毒力基因检测、分型及致病力

为调查鱼源气单胞菌毒力基因与其致病力的相关性,以2009--2018年从不同患病鱼分离的173株气单胞菌为研究对象,通过检测毒力相关基因、测定溶血活性、腹腔注射感染异育银鲫等方法开展评价.通过管家基因gyrB分子鉴定结果显示,173株气单胞菌中维氏气单胞菌(119/173,68.9％)和嗜水气单胞菌(50/173,28...     

添加外源酶类对腌制罗非鱼品质的影响

该研究利用中性蛋白酶、Protamex1.5L和碱性蛋白酶酶解罗非鱼 (Oreochromis mossambicus) 鱼皮,研究了各种酶解产物对钙离子 (Ca²⁺) 的结合能力,比较了酶解物结合Ca²⁺后的抗氧化能力,并分析了结合物的傅里叶红外光谱 (FT-IR)。结果显示,Protamex1.5L水解罗非鱼皮2 h的酶解产物具有最高的Ca²⁺结合率 (87.79%);3种罗非鱼皮酶解产物均存在抗氧化性,其中Protamex1.5L酶解罗非鱼皮产物的1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率、·OH自由基清除率和还原力最高分别为51.46%、17.26%和0.09;罗非鱼皮酶解物 (TSH) 结合Ca²⁺后,钙离子结合物 (TSH-Ca) 的DPPH自由基清除率和羟基自由基清除率减弱,还原力增强;FT-IR分析结果表明了Ca²⁺与TSH中氨基氮原子和羰基氧原子发生了结合。

     

鲐鱼鱼精中5_脱氧核苷酸的分离工艺

论述了用桔青霉PenicilliumcitrinumM71菌株,经液体培养制得的5＇-磷酸二酯酶降解鲐鱼鱼精DNA成5＇-脱氧核苷酸的分离工艺.分离采用201×8阴离子交换树脂,具体条件为柱床高l05mm,柱床直径45mm,样品浓度213mg@mL-1,洗脱流速0.5mL@cm-2@min-1.分离结果表明,采用0.005MHCl+0.04MNaCl作洗脱刺,流速为0.7mL@cm-2@min-1时,四种5'-脱氧核苷酸组分能完全被洗脱下来,且呈一个大峰,同其它成分分开,再先后采用0.0018MHCl、0.0028MHCl、0.036MNaCl(pH6.0)、0.005MHCl+0.02MNaCl作洗脱剂时,则能分别将dCMP、dAMP、TMP、dGMP完全分离.     

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.)

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.     

Induction and characterization of a lysogenic bacteriophage of Lactococcus garvieae isolated from marine fish species

This study investigated the presence of prophages in Lactococcus garvieae isolated from several marine fish species in Japan. Representative strains of 16 bacterial genotypes (S1–S16) selected from more than 400 L. garvieae isolates were used to induce lysogenic bacteriophages. These strains were treated with 500 ng mL^?1 freshly prepared mitomycin C. A cross‐spotting assay was performed to validate the lysogenic and indicator strains. The lysogenic strains were selected for isolation and concentration of the phages. Phage DNA was digested with EcoRI for biased sinusoidal field gel electrophoresis analysis. Polymerase chain reaction (PCR) was used to detect integrated prophage DNA. Of the 16 representative bacterial genotypes, 12 strains integrated prophages as indicated by the PCR assay, and 10 phages were detected and isolated using two indicator bacterial strains. Analysis of genomic DNA showed that these phages were homologous and named as PLgT‐1. Transmission electron microscopy revealed that the morphology of PLgT‐1 was consistent with the virus family Siphoviridae. PCR analysis of the prophage DNA revealed that all of the S1 genotype strains were lysogenic (30/30), but none of the S16 genotype strains were lysogenic (0/30). This is the first study to investigate lysogenic bacteriophages from L. garvieae.     

Dominant pathogenic species of mesophilic aeromonads isolated from diseased and healthy fish cultured in Poland

Aeromonas isolates were collected from cultured fish, characterized phenotypically and identified to species using 16S rDNA. The pathogenicity of all isolates was assayed on the basis of haemolytic and proteolytic activity and challenge tests were performed for isolates from healthy fish. A total of 131 Aeromonas isolates were obtained and identified as follows: A. hydrophila (13), A. bestiarum (23), A. salmonicida (motile biogroup) (19), A. caviae (2), A. sobria (18), A. veronii bt. sobria (42), A. jandaei (1), A. encheleia (11) and A. allosaccharophila (2). All isolates of A. hydrophila and A. bestiarum and most isolates of A. salmonicida and A. veronii were classified as pathogenic. Aeromonas hydrophila was isolated only from diseased trout except for one isolate obtained from carp fry. The other potentially pathogenic Aeromonas species were present in diseased as well as healthy fish. The pathogenicity of isolates from healthy fish was correlated with their enzymatic activity and was also tested by challenge experiments. The dominant pathogenic species were A. veronii bt. sobria, A. bestiarum and A. salmonicida in common carp and A. hydrophila in rainbow trout.     

比较基因组学分析鱼源荧光假单胞菌的致腐和适应性

为探究荧光假单胞菌的强致腐和环境适应性,实验通过分析2株海水鱼源荧光假单胞菌的蛋白酶活性和挥发性盐基氮(TVB-N)的形成,并采用比较基因组学方法解析致腐和适应机制。结果显示,荧光假单胞菌PF07和PF08在冷藏鱼汁中的蛋白酶活性强,积累较多TVB-N。经全基因组测序、组装和功能注释后,得到PF07基因组长度为6.13 Mb,GC含量61.4%。通过泛基因组分析可知,PF07与PF08核心基因共4 980个,独特基因分别有516和470个。COG和KEGG注释显示2株致腐菌基因组中参与氨基酸代谢基因占比最高,PF07独特基因参与无机离子转运代谢居多。碳水化合物活性酶CAZy注释表明,2株荧光假单胞菌基因组中糖苷转移酶与糖苷水解酶基因均占比最高,还鉴定得到大量分解碳水化合物、蛋白质和脂质等多种底物的酶和相关蛋白基因,特别有碱性金属蛋白酶AprA、多胺ABC转运蛋白渗透酶PotC、精氨酸与鸟氨酸脱羧酶等多种降解蛋白酶。另外,2株致腐菌分布有rpoS、rpoN和rpoD多种σ因子。2株鱼源荧光假单胞菌表现出强的致腐性,研究通过比较基因组学方法解析荧光假单胞菌分布多种编码蛋白酶和腐胺形成的氨基...     

Comparative genomic insights into the taxonomy of Edwardsiella tarda isolated from different hosts: Marine,freshwater and migratory fish

Edwardsiella tarda, a Gram‐negative member of the family Enterobacteriaceae, has been isolated from many animal species worldwide, especially fish species. Its broad host range indicates the diversity in taxonomy, which attracted the attention of many researchers. Here, we added genome of E. tarda strain isolated from freshwater fish to comparative genomics study for the first time. We sequenced and assembled the genome of E. tarda ASE201307 which was isolated from freshwater Asian swamp eel. ASE201307 genome contained a single circular chromosome of 3.68M with G+C 57.09% content. Comparative genomics including SNP calling, synteny block, Core/Pan genes analysis and phylogeny analysis was conducted among ASE201307 and other Edwardsiella strains isolated from different fish species. Results of SNP analysis and synteny block demonstrated the close relative of ASE201307, FL95.01 and DT which were all isolated from freshwater fish. In further analysis heat map of dispensable genes and phylogenetic tree, all E. tarda strains were divided into two groups. One was isolated from freshwater fish and the other was isolated from marine/migratory fish. Based on all studies above, we proposed the living environment of hosts as a new taxonomic character and divided E. tarda isolated from diseased fish into freshwater group and marine/migratory group.     

Genotypic diversity,and molecular and pathogenic characterization of Photobacterium damselae subsp. piscicida isolated from different fish species in Taiwan

Photobacteriosis, caused by Photobacterium damselae subsp. piscicida (Phdp), is a serious disease in marine fish species worldwide. To date, the epidemiological characterization of this pathogen in Taiwan remains limited. In this study, we collected 39 Phdp isolates obtained from different farmed fish for phenotypic and genotypic analysis. Phenotype bioassays using API-20E and API-20NE systems showed that the Phdp is a homogeneous group. However, genotyping using the pulsed-field gel electrophoresis (PFGE) technique revealed genetic variability among Phdp isolates when 13 and 11 different PFGE band patterns were obtained with SmaI and NotI as restriction enzymes, respectively. Phylogenetic analysis using 16S rDNA and the Fur gene clustered Taiwanese isolates and other species of P. damselae in the same clade. In contrast, the ToxR phylogenetic tree, a powerful discriminatory marker, separated the two subspecies. Furthermore, the virulence-associated genes, AIP56, P55, PDP_0080, Sod and Irp1, were detected from all isolates. Virulence testing with nine representative isolates in cobia (Rachycentron canadum) and Asian sea bass (Lates calcarifer) showed that some were highly pathogenic with 80%–100% mortality rates. This study provides epidemiological data of Phdp infections in farmed fish in Taiwan, which is necessary to develop comprehensive prevention and control strategies for the disease.     

Removal of cadmium from scallop processing waste by washing with weak acid solution and utilization of useful constituents for organic fertilizer manufacturing

ABSTRACT:   Hepatopancreases were washed four times with 2% acetic acid solution. In both the washed hepatopancreases and the supernatant of the washing solution, the residual cadmium (Cd) concentration was in accordance with the required standard for organic fertilizers. Hepatopancreases do not naturally contain high levels of nitrogen, but they do contain several amino acids which are useful for cultivation, and these were retained during the washing process. Although the condensed precipitate of the washing solution contained a considerable amount of Cd, Cd elution levels at the time of final disposal met judgment standard for special management industrial waste as a result of kneading and immobilizing with chelating adsorbent for treatment of contaminated soil. The volume of the precipitate of the washing solution was one-fifth of the original hepatopancreas, and the solid waste was suitable for burial at a final disposal site without further treatment.