广东汕尾患病黑稚鲍微生物胞外产物的分析

从广东汕尾一鲍鱼养殖厂的患病黑稚鲍中分离筛选到56株菌,并对之进行致病毒力因子(胞外酶及溶血作用)的分析.结果表明,在56株菌中有16株具有较强的分泌胞外蛋白酶、明胶酶、磷脂酶、脂肪酶和淀粉酶的能力,占总菌株数的28.6%;而有溶血现象的仅有6株,占总菌株数的10.7%,且溶血能力不强.其中,8株具有很强的产胞外蛋白酶的能力,3株具有很强的产胞外明胶酶的能力,21株具有产胞外磷脂酶的能力,5株具有产溶血素的能力.此外,同时可分泌4种以上胞外产物的菌株为22株,占39.3%;同时分泌6种胞外产物的菌株为3株.综合考虑每种胞外产物的潜在致病作用以及菌株分泌多种胞外产物的能力,初步确认16株菌为潜在的致病菌.     

近江牡蛎养殖水体中细菌产酶能力的研究

为了解近江牡蛎养殖水体中细菌的产酶特性 ,筛选出有应用潜力的有益菌 ,本实验从近江牡蛎养殖水体中分离到 32株菌。革兰氏染色表明 ,10株为革兰氏阳性菌 ,2 2株革兰氏阴性菌。在此基础上研究了它们蛋白酶、脂肪酶、淀粉酶和纤维素酶的产酶能力。结果表明 ,6 1.9% ( 17株 )的菌株能分泌蛋白酶 ,71.9% ( 2 3株 )菌株能分泌脂肪酶或淀粉酶 ,34.4 % ( 11株 )的菌株产纤维素酶。这些产酶菌株均以革兰氏阴性菌为主。在这 32株菌中 ,产四种酶的有 5株菌 ,产三种酶的有 9株菌 ,产两种酶的有 9株菌 ,产一种酶的有 9株菌 ,也就是说 ,所有的菌株都能产酶。由此表明近江牡蛎养殖水体中细菌在该环境物质循环中的重要地位。     

健康和患病稚黑鲍中微生物产胞外酶的分析比较

2005年初自福建漳浦鲍鱼场患病以及健康稚黑鲍中各分离到27和56株异养细菌,在此基础上研究了它们产胞外蛋白酶、淀粉酶、明胶酶、磷脂酶及溶血能力。结果表明,虽然来自健康鲍的菌株产胞外酶的数量明显大于来自病鲍的菌株,但来自病鲍的菌株具有强分泌胞外酶能力的比例明显大于来自于健康鲍的菌株。综合考虑每一种胞外酶的潜在致病作用,以及菌株分泌多种胞外酶的能力,最后确认10株菌为潜在的致病菌。     

产胞外多糖的近海海洋细菌的筛选

海洋微生物是海洋生物的重要组成部分，海洋微生物可产生大量不同于陆生生物的活性物质，是重要的海洋药物资源。实验所用泥样采集于黄海近海海岸，对其进行海洋细菌的分离与纯化，获得了25株纯化的海洋细菌。采用苯酚-硫酸法和乙醇沉淀法检测这些菌株的发酵液中是否含有胞外多糖，进一步筛选出胞外多糖的高产菌株。实验结果表明，胞外多糖产量高于0．22g／L的菌株共7株，占总测定菌株数的28％，胞外多糖产量高于0．20g／L的菌株共13株，占总测定菌株数的52％。有48％的菌株胞外多糖产量介于0．19g／L和0．20g／L之间。其中以MBN003中多糖含量为最高，产量达到0．2305g／L。此研究结果表明MBN003菌株具有较高的产胞外多糖能力，可以选用MBN003作为胞外多糖提取物研究的来源。     

杂交鲍稚鲍"漫爬病"潜在病原菌胞外产物的分析及种类鉴定

从发病的杂交鲍(皱纹盘鲍×盘鲍)稚鲍中共分离27株菌株,并对其分泌胞外产物(包括酪蛋白酶、明胶酶、淀粉酶、脂肪酶、磷脂酶和溶血素)的能力进行了分析。试验结果表明,具有分泌胞外蛋白酶、明胶酶、淀粉酶、脂肪酶、磷脂酶和溶血素能力的菌株分别占总菌株数的81.5%(22/27)、74.1%(20/27)、18.5%(5/27)、0%(0/27)、11.1%(3/27)、29.6%(8/27)。其中,1#、14#菌株具有很强的产胞外蛋白酶能力,而明胶酶的最大分泌者为2#、7#、9#、17#菌株,淀粉酶的为11#、14#菌株,磷脂酶的为11#、15#菌株,溶血素的为菌株10#、21#。此外,10#、11#、15#菌株可同时分泌4种胞外产物,而12#、14#、18#、21#、23#、24#菌株可同时分泌3种胞外产物。综合考虑每一种胞外酶的潜在致病作用,以及菌株分泌多种胞外酶的能力,初步确认11株菌为潜在的致病菌:1#、2#、7#、9#、10#、11#、12#、14#、15#、17#、21#。用API条带法对它们进行了种类鉴定,11株菌中巴斯德氏菌和假单胞菌所占的比例分别为45.5%、27.3%。     

草鱼和银鲫肠道产消化酶细菌的研究

检测了分别从草鱼(Ctenopharyngodon idellus)和银鲫(Carassius auratus gibelio)肠道中分离的180株细菌的蛋白酶、脂肪酶、淀粉酶和纤维素酶的产酶能力。结果显示,两种鱼肠道内可分泌胞外消化酶的细菌包括Aero-monas(气单胞菌属,Aer.)、Vibrio(弧菌属,Vib.)、Bacillus(芽孢杆菌属,Bac.)、Pseudomonas(假单胞菌属,Pse.)四个种属的细菌,Aer.在其中占主要优势,45.71%的Aer.可分泌胞外消化酶。草鱼可分泌上述四种胞外消化酶的菌株共有33株,占肠道菌总数的36.67%;银鲫43株,占47.78%。产酶菌的分布上,草鱼中肠内产消化酶细菌数量显著多于前肠和后肠(P<0.05),前、中、后肠分别是6株、20株和7株;银鲫中肠和后肠数量差异不显著,前肠分布最少。草鱼分泌蛋白酶、脂肪酶、淀粉酶和纤维素酶的菌株分别有21株(23.33%)、10株(11.11%)、30株(33.33%)和16株(17.78%)。银鲫肠道内未检测到可分泌纤维素酶的细菌,蛋白酶、脂肪酶和淀粉酶菌株的数量分别是21株、37株和17株。可见鱼类肠道细菌对食饵消化有重要作用。     

工厂化循环水养殖条件下云纹石斑鱼消化道产酶菌的分离鉴定

采用16S r DNA-PCR菌群分离鉴定的方法,对循环水养殖条件下云纹石斑鱼(Epinehelus moara)幼鱼的胃、幽门盲囊、前肠、中肠和后肠的菌群结构进行了鉴定,用产酶菌筛选培养法对产消化酶的菌株进行了分离鉴定,并测试了各菌株消化酶的活力。研究发现,云纹石斑鱼幼鱼消化道内可培养的主要菌群为假单胞菌属(Pseudomonas)、微小杆菌属(Exiguobacterium)、不动杆菌属(Acinetobacter)、寡养单胞菌属(Stenotrophomonas)和葡萄球菌属(Staphylococcus),其中产消化酶的菌株占可培养菌的55.6%。在产酶菌中,同一株菌产3种酶的有5株;产2种酶的有9株;中肠和后肠的菌株数最为丰富,胃次之,幽门盲囊和前肠菌群种类较少;产脂肪酶的菌株都集中在中肠。产消化酶的菌株主要以产蛋白酶和淀粉酶为主,且产酶量丰富,产蛋白酶活力最高达(87.732±1.134)U/m L;淀粉酶活力为(77.176±0.599)~(73.458±0.574)U/m L;产纤维素酶的菌仅一株,且酶活力较低。分析得知,消化道的菌群结构直接影响了外源性消化酶的种类与活性。本研究为工厂化循环水养殖条件下产酶有益菌的筛选提供了理论依据。     

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。     

凡纳滨对虾肠道内产消化酶益生菌的分离与筛选

为获得具有消化酶活性且安全的益生菌,从凡纳滨对虾肠道中初步分离得到576株细菌,对菌株进行产蛋白酶、淀粉酶和脂肪酶能力的定性及定量测试,筛选出产酶种类多且产酶能力强的菌株11株。对筛选出的11株菌进行了幼虾浸浴实验、药敏性实验和溶血性实验,以评价其生物安全性。将11株菌的菌悬液添加到凡纳滨对虾幼虾的养殖水体中进行浸浴实验,浸浴结束后用鳗弧菌进行刺激,测定不同浸浴组幼虾相关免疫基因的相对表达量,以确定其对幼虾的保护效果。综合消化酶活性、菌株对幼虾的保护效果及生物安全性,筛选得到4株效果较好的菌株。菌株的16S r DNA分子鉴定结果表明,细菌1号、2号和4号分别与芽孢杆菌(Bacillus sp.PCSAS2-38,GQ284495.1)、蜡样芽孢杆菌(B.cereus strain N419,JN400121.1)及苏云金芽孢杆菌(B.thuringiensis strain EA26.1,KC758847.1)的相似性均为100%,9号菌株与荚膜红细菌(Rhodobacter capsulatus strain PSB-03,FJ866782.1)相似性达到99%,为后续益生菌制剂的开发奠定了前期基础。     

黄海希瓦氏菌胞外产物特性研究

为研究黄海希瓦氏菌(S. smarflavi)AP629胞外产物(ECPs)的致病性,用平板玻璃纸法提取胞外产物。结果显示,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)发现ECPs蛋白条带主要在114 kDa、66 kDa、39kDa、36 kDa、79 kDa;ECPs具有酪蛋白酶、淀粉酶、卵磷脂蛋白酶和脂肪酶活性,无明胶酶活性;一些金属离子和化学试剂对菌株AP629ECPs的酶活有影响,EDTA、PMSF、SDS、MnCl_2作用浓度分别为10、5、5、5 mM时,ECPs酶活受到不同程度抑制;Ca~(2+)和Mg~(2+)则提高ECPs的酶活,可分别提高3%～19%和12%～28%的酶的活性。菌株AP629的胞外产物人工感染小白鼠和仿刺参试验结果表明,黄海希瓦氏菌AP629的胞外产物不显示致病性。该文为揭示黄海希瓦氏菌AP629发病机理奠定了基础。     

Transmission of protease genes into a protease-deficient mutant of Aeromonas salmonicida in river sediments

Abstract. The transformation of Aeromonas salmonicida with DNA fragments from bacterial cell-free sonicates was investigated with intraspecific, interspecific band intergeneric fish pathogenic bacteria including Aeromonas salmonicida, Aeromonas hydrophila, Pseiidomonas fluorescens and Vibrio anguillarum strains as donor bacteria. A phenotypic marker for transformation was extracellular protease production since a protease-deficient mutant NTG-1 induced from pathogenic A. salmonicida strain A-7301 by mutagenesis was used as a recipient. This mutant was non-pathogenic to rainbow trout. The mutant was incubated with each sonicate at 20°C for 20 days with a nutrient-poor medium containing a trace (5 μg/ml each) of both humic acid and tryptone in the presence of clean river sand (100 g/100 ml medium) corresponding with an environment of rivers. During the incubation, the survival of mutant NTG-1 cells was observed and protease positive NTG-1 cells were isolated from each culture. The protease production of the isolates was due to the transmission of protease genes of the donor strains. The activity of proteases produced by the transformants extra-cellularly was determined. These transformants induced with the sonicates of the parent strain, intraspecific strain and with the sonicates of the interspecific A. hydrophila strain were pathogenic to rainbow trout, whereas the transformants derived with the sonicates of the intergeneric strains P. fluorescens and V. anguiUarum showed non-pathogenicity, although all the donor strains, with the exception of the P. fluorescens strain, were pathogenic. These findings are interesting since they demonstrate that trausformation in A. salmonicida occurs with considerable ease even intergenencally and interspecifically, as well as intraspecifically in river environments, and that there is a large difference in the lethal toxicity of extracellular protease produced by these bacteria.     

哈维弧菌vhh基因缺失株的构建及其相关生物学特性研究

哈维弧菌(Vibrio harveyi)是水生动物的重要病原,为研究哈维弧菌溶血素基因vhh缺失后对其生物学特性的影响,该研究利用同源重组技术构建了V.harveyi 345的vhh基因缺失突变株,并比较了野生株和突变株的生物学特性变化。结果显示,vhh基因的缺失不影响菌株的生长、胞外蛋白酶分泌、过氧化氢(H2O2)和铜离子(Cu2+)的压力感应、铁的吸收利用、15种抗生素抗性和生物膜形成等生物学特性,但会导致菌株游动和涌动显著增强;另发现,vhh基因虽然在野生菌株内高表达,对绵羊红细胞却未表现出溶血活性。结果表明该基因负调控着菌株的运动能力。该研究为认识哈维弧菌vhh基因功能研究提供新的资料。     

Enzyme producing bacterial flora isolated from fish digestive tracts

Isolationand enumeration of aerobic bacterial flora in the gastrointestinal tract of nineculturable freshwater teleosts, namely catla, rohu, mrigal, silver carp, grasscarp, common carp, tilapia, walking catfish and murrel have been carried out.Amylolytic, cellulolytic, lipolytic and proteolytic microflora were identifiedfrom the culture plate using selective media. The isolates were qualitativelyscreened on the basis of their extracellular enzyme producing ability. Theselected strains were further quantitatively assayed for amylase, cellulase,lipase and protease activities. Protease activity was exhibited by almost allthe bacterial isolates, while strains isolated from tilapia, grass carp andcommon carp showed considerable amylolytic and cellulolytic activities. Maximumactivity of lipase was exhibited by a strain isolated from silver carp. Thestudy indicates that there is a distinct microbial source of the digestiveenzymes – amylase, cellulase, lipase and protease, apart from endogenoussources in fish gut. The information generated from the present investigationmight contribute towards better feed formulations for carp at low cost,incorporating the enzyme producing bacterial isolates as probiotics.     

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.     

Identification of gut‐associated amylase,cellulase and protease‐producing bacteria in three species of Indian major carps

Isolation and enumeration of amylase, cellulase and protease‐producing autochthonous bacteria in the proximal intestine (PI) and distal intestine (DI) of three species of Indian major carps, catla (Catla catla), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita), were investigated using the conventional culture‐based technique. Population levels of amylolytic strains were the highest in the PI of catla and the lowest in the DI of rohu. The highest viable count of cellulase and protease‐producing bacteria was recorded in the DI and PI of mrigal respectively. Among the bacteria isolated, 10 strains (five from PI and five from DI) were selected as potent enzyme producers according to a quantitative enzyme assay. The chosen strains were further identified by 16S rRNA gene sequence analysis. The five strains isolated from catla showed high similarity to Citrobacter sp. clone W2, Enterobacter sp. JA24, Bacillus coagulans strain TR, uncultured bacterial clone Hel3bc04 and Bacillus cereus strain UST2006‐BC004. The four strains isolated from mrigal were most closely related to Bacillus sp. KCd2, uncultured bacterial clone Hel3bd09, B. cereus strain BU040901‐020 and Citrobacter freundii strain YRL11, while the strain isolated from rohu probably belonged to Bacillus sp. GV.     

刺参(Apostichopus japonicus)大水面养殖池塘环境中优势益生菌筛选及其特性分析

2014年12月-2015年12月,在大连地区刺参(Apostichopus japonicus)大水面养殖池塘进行了春、夏、秋、冬四季有益菌分离筛选,从其水体和底泥中共分离得到66株细菌.以刺参“腐皮综合征”主要病原菌——灿烂弧菌(Vibrio splendidus)和假交替单胞菌(Pseudoalteromonas nigrifaciens)为指示菌进行拮抗作用实验,利用选择培养基对菌株产淀粉酶和蛋白酶的能力进行测定,最后通过安全性实验得到潜在益生菌株YQ-2.结果显示,该菌株对灿烂弧菌和假交替单胞菌有较强的抑制作用,抑菌圈分别达到22 mm和24 mm;对淀粉和蛋白选择培养基水解圈的直径达到22 mm和36mm.安全性实验显示,该菌株无论是在108 CFU/ml浸浴还是投喂108 CFU/g的粉末饲料感染,30 d内供试刺参没有发病和死亡现象,健康程度好,且相对于对照组的体重明显增长,108 CFU/g粉末饲料投喂组的相对增长率达到39.31％.此外,本研究对YQ-2菌株的生理生化指标、16S rDNA序列进行了分析,其同源性与枯草芽孢杆菌Bacillus subtilis strain KLP2015相似度达99％,故将该菌株鉴定为枯草芽孢杆菌.该株枯草芽孢杆菌在大水面刺参池塘四季水体中数量为140-280 CFU/ml,高于其他菌株;同时,该菌株在水体中还具有较高的优势度,优势度分别为4.2％、3.5％、2.6％、4.6％,冬、春季节的优势度明显高于夏、秋季节;它属于土著分离菌株,对引起刺参腐皮综合征的2株病原菌具有较强的抑制作用,这对刺参大水面生态养殖具有较大的应用潜力.     

饲料添加益生菌对多病原阳性的凡纳滨对虾生长与存活的影响

采用假交替单胞菌(Pseudoalteromonas sp.) KL-3 2010和微小杆菌(Exiguobacterium sp.) KL-C2 2014作为益生菌，进行凡纳滨对虾(Litopenaeus vannamei)投喂实验，研究上述菌株对带毒对虾的生长与存活的影响。假交替单胞菌KL-3 2010对致急性肝胰腺坏死副溶血弧菌(Vibrio parahemolyticus) (VPAHPND 20130629002S01)有拮抗作用和胞外蛋白酶活性，微小杆菌KL-C2 2014有胞外蛋白酶活性。待试对虾经检测为白斑综合征病毒(WSSV)、致急性肝胰腺坏死病副溶血弧菌(VPAHPND)和虾肝肠胞虫(EHP)弱阳性。经过为期60 d的养殖实验，结果显示，与投喂普通颗粒饲料的对照组相比，投喂假交替单胞菌KL-3 2010的对虾存活率提高了213%±43% (P<0.01)；投喂微小杆菌KL-C2 2014的对虾平均生长率提高了105.5%±28.1% (P<0.05)；交替投喂2株菌的对虾存活率提高了184%±52% (P<0.05)，平均生长率提高了70.6%±32.8%。肠道可培养优势菌研究表明，2株益生菌的投喂显著影响了对虾肠道优势菌群的种类。本研究为带毒虾苗的养殖提供一种有效的病害防控和促生长的手段。     

Properties of extracellular ice-nucleating substances from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> MACK-4 and its effect on the freezing of some food materials

ABSTRACT:   Pseudomonas fluorescens MACK-4, isolated from mackerel surface, has an ice-nucleating activity (INA). In addition to bacterial cells, this strain could also produce an extracellular ice-nucleating substance (INS) with a maximal INA at pH 6.0. The extracellular INS (EINS) was stable at pH 6–9 during 1-h incubation with 3–5% of saccharides including maltose, trehalose and sucrose at 15°C. However, glycerol dramatically lowered the INA in both bacterial cells and the EINS. The addition of either the EINS or bacterial cells significantly elevated the ice-nucleating temperatures of pure water, full-cream milk, and 10% starch solution, but not orange juice and mackerel mince. The EINS produced from this strain could serve as crystal nuclei and accelerate ice crystallization during freezing.     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.     

Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus (Bloch)

A 750-bp internal fragment of the alkaline serine protease gene (asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5'- and 3'- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD(50) of 0.25 microg protein g(-1) body weight.