2株鲤鱼源乳酸菌体外抗逆性研究

对2株鲤鱼源乳酸菌作为益生菌候选菌株进行体外抗逆性试验.通过耐受胆盐、耐受pH值、耐受蛋白酶和耐高温等试验,对这2株乳酸菌进行研究.其中在胆盐耐受性试验中,YL-1和YL-7在胆盐浓度0.4%、培养4 h时仍有30%以上的存活率;在pH值耐受性试验时,2株乳酸菌均可在一定的酸性和碱性条件下存活;胰蛋白酶对2株菌的存活率没有影响;在70℃的高温中,2株乳酸菌均有一定的耐受性.     

2株鲤鱼源乳酸菌体外抗逆性研究

对2株鲤鱼源乳酸菌作为益生菌候选菌株进行体外抗逆性试验。通过耐受胆盐、耐受pH 值、耐受蛋白酶和耐高温等试验, 对这2株乳酸菌进行研究。其中在胆盐耐受性试验中, YL- 1和YL- 7在胆盐浓度0. 4%、培养4 h时仍有30%以上的存活率; 在pH 值耐受性试验时, 2株乳酸菌均可在一定的酸性和碱性条件下存活; 胰蛋白酶对2株菌的存活率没有影响; 在70e 的高温中, 2株乳酸菌均有一定的耐受性。     

斜带石斑鱼消化道乳酸菌在模拟胃肠道环境中的存活

从斜带石斑鱼(Epinephelus coioides)幼鱼消化道分离到3株乳酸菌,通过抑菌试验筛选出抑菌效果较好的2株菌(MM1和MM4),经生理生化鉴定结合16SrRNA基因测序分析,MM1菌株鉴定为乳酸乳球菌(Lactococcus lactis),MM4菌株鉴定为屎肠球菌(Enterococcus faecium)。进一步研究了MM1和MM4的体外生长特性,及其对不同pH值的人工胃液、含有不同浓度胆盐的人工肠液及高温的耐受能力。结果表明,MM1和MM4均在培养6h左右进入对数生长期。对人工胃液的耐受能力相似,pH4.0和3.0的人工胃液对2株菌存活的影响较小,而pH2.0的人工胃液对2株菌的影响较大,经2h处理后两者的存活数即下降到105CFU/mL,经4h处理后的存活数低于5.0×103CFU/mL。MM1和MM4对含不同浓度胆盐的人工肠液均具有较好的耐受能力,含中低浓度胆盐(0.1%～0.3%)的人工肠液对2株菌的存活影响很小,处理3h后2株菌的存活数均维持在107CFU/mL以上;高浓度胆盐(0.5%～0.7%)处理3h后2株菌的存活数明显下降,但活菌数仍维持在105CFU/mL以上。MM1和MM4具有一定的耐高温能力,60℃处理30min二者存活数在107CFU/mL以上;80℃处理30min二者存活数在106CFU/mL以上。结论认为,2株乳酸菌具有生长速度快、抑菌能力强、对胃肠道环境和高温耐受能力较强的特点,具有作为益生菌的应用潜力。     

2株鱼源芽孢杆菌的生物学特性研究

体外研究了2株分离自斜带石斑鱼肠道的芽孢杆菌(DE5和SE5)的生长曲线、对模拟胃肠道环境的耐受能力及对潜在致病菌的抑制效果.结果显示,SE5生长速度明显快于DE5,SE5培养4 h进入对数生长期,而DE5培养8 h进入对数生长期.人工胃液耐受试验表明,DE5对人工胃液(不同pH值、胃蛋白酶)的耐受能力较强,pH 2.0的人工胃液处理4 h,其活菌数超过104cfu/ml,pH 3.0和4.0的人工胃液对DE5的生长几乎没有影响;SE5对人工胃液的耐受能力相对较差,pH 2.0的人工胃液处理2 h后未检测到活菌,但pH 3.0和4.0的人工胃液处理4 h后SE5的活菌数超过105cfu/ml.SE5和DE5对人工肠液(不同质量分数胆盐、胰蛋白酶)均有很强的耐受能力,经含0.1%～0.7%胆盐的人工肠液处理3 h后,2株菌的存活数均超过107cfu/ml,且SE5的耐受能力略强于DE5.抑菌试验显示,2株芽孢杆菌对多株潜在致病菌具有一定抑制作用.因此,2株芽孢杆菌均具有生长速度较快,对胃肠道环境耐受能力强特点,且对潜在致病菌具有一定抑制效果,具有作为益生菌应用的潜力.     

仔猪肠源枯草芽孢杆菌体外耐受性研究

研究了分离的仔猪肠源枯草芽孢杆菌体外耐受性。以枯草芽孢杆菌的存活率为考察指标,通过耐酸性、耐胆盐、耐高温试验来研究菌株的体外培养特性。结果:耐酸性试验中枯草芽孢杆菌在p H为2.0的培养基中培养3 h的存活率仍能达到64.7%;耐胆盐试验中其在0.1%和0.3%胆盐培养基中培养8 h存活率均在60%以上;耐高温试验中,70℃、90℃和100℃对其处理20 min存活率分别为70.4%、29.3%和0.2%。结果表明:仔猪肠源枯草芽孢杆菌对酸及胆盐有较强耐受性,对高温有一定耐受性,试验为进一步研制仔猪肠源枯草芽孢杆菌制剂研究提供参考。     

林蛙肠道中乳酸菌的分离及鉴定

用2种方法从健康林蛙肠道菌群中分离乳酸菌,并进一步纯化及鉴定.试验结果表明,用乳酸菌半选择性培养基液体培养液先富集后分离得到了11株单一的乳酸杆菌,经过一系列生化鉴定,11株菌为同一种菌即嗜酸乳杆菌.该菌对嗜水气单胞菌具有抑制作用,抑菌圈直径为12 mm.在胆盐含量为0.3%,pH值为3时仍然生长,表明该菌株完全能适应动物胃肠道环境,作为微生态制剂用菌种具有较好的潜力.     

2株乳酸菌抑菌作用研究及安全性评价

该研究利用无菌操作技术对2株乳酸菌[乳酸菌S60 (干酪乳杆菌Lactobacillus casei)和S72 (植物乳杆菌Lactobacillus plantarum)]开展了常规生化检测、药敏实验、体外抑菌实验和动物实验,验证了其作为渔用益生菌的可行性和安全性。结果显示,2株乳酸菌均能在pH为4.5和0.1%的胆盐环境中存活,对水产动物常见病原菌嗜水气单胞菌(Aeromonas hydrophila)等均有一定的抑制作用,且抑菌物质分析结果显示,其有效抑菌物质与其代谢过程中产生的有机酸类相关。安全性评价实验结果显示2株乳酸菌无分解明胶和溶血的能力。死亡统计和血液生化指标结果显示,高达1.0×10~9 CFU·mL~(-1)的活菌腹腔注射攻毒并未引起实验鱼类[异育银鲫(Carassius auratus)]的严重死亡和机体损伤。可见2株乳酸菌作为口服益生菌具有较高的安全性。此外,耐药特性研究表明,2株乳酸菌对所用药物中的氨基糖苷类和磺胺甲唑耐受能力强,对氨苄青霉素、恩诺沙星、氟苯尼考和多西环素敏感。因此,2株乳酸菌的应用需要注意管理过程中药物的选择。     

海水对固定化乳酸菌生长的影响

为探究固定化乳酸菌对海水中不同因素的耐受性,采用涂布分离计数法测定海水中不同pH值、NaCl浓度、Pb~(2+)浓度、葡萄糖浓度对固定化乳酸菌生长的影响。结果表明:海水pH值为6.0,德氏乳杆菌、嗜热链球菌、屎肠球菌三种乳酸菌在海水中的活菌率分别为98.0%、117.6%、111.4%;在海水中添加NaCl和醋酸铅溶液均抑制乳酸菌的生长,但屎肠球菌的耐盐性与耐重金属铅的能力均比德式乳杆菌和嗜热链球菌强;乳酸菌生长的最适葡萄糖浓度为20%,过量葡萄糖会抑制其生长。综合比较三种乳酸菌在海水中的生长耐受性,得出屎肠球菌对海水中的pH值、NaCl浓度、Pb~(2+)浓度及葡萄糖变量的耐受性较强。     

急性pH和碳酸盐碱度对缢蛏存活率、Na+/K+-ATPase活性及血淋巴吞噬能力的影响

采用静态毒理学实验方法,分析了2种规格缢蛏(小规格SSc和大规格LSc)在pH和碳酸盐碱度(C_A)急性胁迫条件下的存活率,Na~+/K~+-ATPase(NKA)活性以及血淋巴的吞噬能力。结果显示,当C_A浓度为2.5 mmol/L、pH值为7.5～9.5时,2种规格缢蛏的存活率均接近100%;当pH值大于9.5时,2种规格缢蛏的存活率均显著下降。当C_A浓度为0～44.58mmol/L、pH值为9.0～10.0时,随着C_A浓度的上升,缢蛏的存活率明显下降;在pH值为9.5条件下,LSc的鳃组织NKA活性,随着C_A浓度的上升而升高,LSc血淋巴的吞噬能力随着C_A浓度的上升而下降。由此可见,缢蛏在高pH或高C_A下表现出较强的耐受性,但高pH和高C_A协同胁迫下对缢蛏的存活率具有较大的影响,研究结果为进一步探索缢蛏在盐碱地的养殖提供了一定的理论参考。     

817肉鸡肠道乳酸菌及生理特性研究

为筛选817肉鸡同源性乳酸菌菌株,从健康30日龄肉鸡盲肠内容物中分离出LB1、LB2、LB3和LB4四种疑似乳酸菌,经革兰氏染色镜检,生化分析及糖发酵试验进行鉴定,并通过耐酸性、耐胆盐性及抑菌性生理特性研究。结果表明:4种菌株均初步判定为乳酸菌属,LB3和LB4在0.3%胆盐浓度的菌落存活数显著高于其他菌株(p<0.05);LB4和LB1在pH4.0酸度的存活菌数显著高于其他菌株(p<0.05);LB4抑菌圈直径最大,抑菌效果最佳。经综合评定,LB4菌株生理特性最佳,可作为乳酸菌制剂菌种备选。     

棘胸蛙蝌蚪“腹水病”病原菌分离与鉴定

自腹部肿大、腹水和出血等症状的患病棘胸蛙蝌蚪内脏中分离到4株细菌,人工感染确定菌株JK-3为致病菌。经生化鉴定,菌株JK-3符合肺炎克雷伯菌特征; 16S rDNA基因序列比对及系统发育分析表明,菌株JK-3与肺炎克雷伯菌标准株(ATCC700721/NR_074913.1)的同源性达99.5%。结果表明,菌株JK-3为肺炎克雷伯菌,是引起棘胸蛙蝌蚪腹部肿大、腹水和出血等症状疾病的病原。药敏试验结果显示,肺炎克雷伯菌JK-3对美满霉素、丁胺卡那、复达欣、哌拉西林、庆大霉素、先锋必素、先锋Ⅳ敏感,对其余19种药物均耐药或中度耐药。     

Bacteria associated with early life stages of the great scallop, <Emphasis Type="BoldItalic">Pecten maximus</Emphasis>: impact on larval survival

A bacteriological study was carried out at a scallop (Pecten maximus) hatchery near Bergen, western Norway following a severe increase in mortality rates during the larval stages of the scallops. No larvae survived to settling, except for those in groups treated prophylactically with chloramphenicol. In order to identify pathogenic strains of bacteria, we performed a challenge test on 10- to 16-day-old larvae using isolated bacterial strains from the hatchery. Infection with six of these strains produced mortalities that were not statistically different from that resulting from infection with the known pathogen Vibrio pectenicida. However, about 5% of the strains tested in the challenge experiment produced higher motility rates than found in the unchallenged control group, indicating a possible probiotic effect. On the basis of 16S rDNA analysis on these strains, the phylogenetic tree indicated two groups of apparent pathogens: (1) one strain, LT13, grouped together with Alteromonas/Pseudoalteromonas; (2) a cluster of strains grouped together with Vibrio splendidus (LT06, LT21, LT73, PMV18 and PMV19). Strain LT13 was isolated from cultures of the microalga Chaetoceros calcitrans used for feed, while the other strains were isolated from larval cultures. Transmission electron microscopy showed intracellular bacteria that resembled bacteria in the groups Chlamydiaceae and Rickettsiaceae.     

具有结合胆酸盐作用卵形鲳鲹蛋白酶解物的制备和分子量分布研究

以卵形鲳鲹(Trachinotus ovatus)鱼肉为原材料,以酶解产物与胆酸盐的体外结合率为指标,从5种常用食品用蛋白酶中筛选最优蛋白酶。进而采用正交法优化酶解条件,以提高酶解产物的胆酸盐结合率,并采用GPC法分析产物中蛋白肽的分子量分布情况。结果表明,胰蛋白酶酶解产物(100 mg·m L~(-1))与胆酸钠、甘氨胆酸钠和牛磺胆酸钠等3种胆酸盐的体外结合能力均好于其他4种蛋白酶,结合率分别达到42.1%、33.5%和30.1%,分别相当于降血脂药物考来烯胺散(20 mg·m L~(-1))的78.3%、74.4%和76.8%;经过正交试验优化的制备卵形鲳鲹蛋白肽的工艺条件为酶解时间2 h,加酶量2 000 U·g~(-1),料液比1∶4(g·m L~(-1)、p H 8、37℃)。该条件下酶解产物(20 mg·m L~(-1))对甘氨胆酸钠体外结合率达到同浓度阳性对照物(考来烯胺散)的48.3%,酶解产物中相对分子量3 k D以下的肽类组分占总蛋白水解物的77.30%。     

Susceptibility of three strains of blue catfish,Ictalurus furcatus (Valenciennes), to Ichthyophthirius multifiliis

This study compared the susceptibility of three blue catfish strains (D&B, USDA 101 and USDA 102) to the parasite Ichthyophthirius multifiliis (Ich). In Trial I, a cohabitation study (all strains stocked communally) was conducted and fish were exposed to theronts at 0, 200, 1000, 5000 or 25 000 theronts fish^?1, respectively. All fish died when exposed to theronts at 5000 or 25 000 theronts fish^?1. When exposed to 1000 theronts fish^?1, USDA 102 strain of blue catfish showed significantly lower mortality (78.5%) compared to USDA 101 and D&B strains (92.7% and 100%). In Trial II, the same three strains of blue fish were evaluated for their susceptibility to Ich with strains challenged in separate tanks by adding Ich theronts at 0, 200 and 1000 theronts fish^?1, respectively. All D&B and USDA 101 blue catfish died; however, 42.3% of USDA 102 strain survived the infection when exposed to 1000 theronts per fish. The results indicate that there are differences among strains of blue catfish for susceptibility to Ich, and these differences will be useful in the development of improved catfish germplasm for commercial aquaculture.     

6株水产动物源气单胞菌安徽分离株的毒力基因的克隆与序列分析

根据气单胞菌主要黏附素基因（ahal）、溶血素基因（hly）和细胞兴奋性肠毒素基因（alt）完整开放阅读框（0RF）设计3对特异性引物，对6株气单胞菌安徽分离株进行ahal、hly和alt基因的PCR扩增、克隆和测序。测序结果显示，安徽分离株aim、hly和alt基因的ORF大小分别为1056--1068bp、1482bp和1104N1107bp，各编码351-355、493和367-368个氨基酸。序列分析显示：（1）属于alt^＋aim^＋hly^＋毒力基因型的3个安徽分离株间aha1核苷酸序列和推测的氨基酸序列同源性均很高，分别为98.8％-99．3％和97％-97．6％，且与国内参考株mh／Trionyx sinensis／Guangzhou（Guangdong）／AF276639的同源性高达98．5％-99％和96.8％-97．6％。而alt^＋ahal^＋hly^-HA6安徽分离株与国外参考株Ah／Fish／Barcelona（Spain）／AF183931间的核苷酸序列和氨基酸序列同源性较高，分别为95.8％和97．2％。（2）不同表型种气单胞菌安徽分离株之间及其与国内外其他分离株之间的hly核苷酸序列和推测的氨基酸序列同源性均较高，分别介于88．4％-100％之间和90.8％-98．7％之间。（3）5个安徽分离株（RA16、CA1、HA6、HA7和GA1）之间及其与惟一参考株之间的alt核苷酸序列和推测的氨基酸序列同源性分别高达93．5％-99．9％和80．2％-98．3％，但与BA17分离株间的核苷酸序列同源性（81．6％-81．7％）和推测的氨基酸序列同源性（77．5％-84．9％）均较低。这些结果表明，气单胞菌ahal和alt基因在不同毒力基因型间存在一定差异性，hly基因在不同表型种间存在较高保守性，该基因可作为研制气单胞菌基因工程亚单位疫苗的候选成分。     

Low-temperature preservation (at 4 °C) of marine rotifer Brachionus

Mass production of rotifers is essential as live food during the larval rearing season, but a major problem of rotifer culture is unpredictable culture collapse. If mass‐produced rotifers could be kept alive at low temperature for an extended period of time, they could be supplied as live food to cultured marine fish larvae without interruption. Four experiments were performed to test this possibility in six strains of Brachionus plicatilis O. F. Müller and eight strains of Brachionus rotundiformis Tschugunoff. The results showed that: (1) B. rotundiformis strains were less tolerant to 4 °C than B. plicatilis strains. Among the B. rotundiformis strains, the strains known as SS type were the most susceptible and showed the lowest survival. (2) Exchange of culture media during the incubation at 4 °C in B. plicatilis and B. rotundiformis resulted in higher survival than not changing the culture media, but there were no differences in the regression slope with or without changing the culture media. (3) Acclimation at 15 °C for 96 h for B. plicatilis and B. rotundiformis before transfer to 4 °C resulted in higher survival rates than acclimation at 10 °C. (4) The combination of frequent exchange of culture media and acclimation significantly improved the survival of B. plicatilis and B. rotundiformis compared with controls that were maintained at 4 °C without exchange of the culture media. Large‐scale trials using B. plicatilis (Kamiura strain) cultured in 30‐L tanks were conducted in a hatchery at a density of 2000–20 000 individuals mL^?1. Rotifers were transferred directly from 25 °C to 4 °C. About 50% of the rotifers at 20 000 individuals mL^?1 survived after 14 days at 4 °C. These preserved rotifers could be cultured at 20 °C, recovering within 4 days.     

Lactic acid bacteria effective for regulating the growth of contaminant bacteria during the fermentation of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Phaeophyta)

ABSTRACT:   Lactic acid fermentation of seaweed is a recent topic and quite limited information is available on culture conditions. To know the suitable strains for use as a starter culture for seaweed fermentation, 14 lactic acid bacteria (LAB) strains, including 11 species, were tested in culture conditions prepared with or without salt. A commercial product of Undaria pinnatifida powder was used as a substrate for fermentation without sterilizing. Starter-suitability of the LAB strains was assessed from their predominance after culture. Among the tested strains, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus rhamnosus showed high (>90%) predominance in their cultures, while control cultures prepared without inoculation of LAB did not show any detectable growth of acid producing bacteria and spoiled. A total of 102 strains not showing acid producing activity were isolated and characterized from spoiled cultures, and all the isolates were observed as Bacillus strains, including 64 strains (62.7%) and 16 strains (15.7%) of a Bacillus cereu s-related and B. fusiformis -related species, respectively. The Undaria substrate before fermentation contained culturable microorganisms at 1.4–3.1 × 10² CFU/g, but the Bacillus cereu s-related strain was not a major composition, suggesting a concern of selective growth of the Bacillus cereu s-related strain during the spoiled fermentation.     

Virulence of Flavobacterium branchiophilum in experimentally infected salmonids

Abstract. The ability of selected strains of Flavobacterium branchiophilum to attach to and colonize the gills of five species of teleosts (four salmonid and one cyprinid) and cause mortality was investigated. In virulence studies with F. branchiophilum strain LAB4a, cumulative mortality was dose-dependent in rainbow trout, Oncorhynchus mykiss (Walbaum), and ranged from 0 to 75%. However, regardless of dose, the relative amount of gill-associated F. branchiophilum antigen increased 1 h after challenge. The gill-associated F. branchiophilum antigen in fish which survived (moribund fish) increased by four to six times compared to that detected on the gills 1 h after challenge. The gill-associated antigen on moribund fish was not significantly different from that on the gills of dead fish. Flavobacterium branchiophilum strain LAB4a also attached to the gills of brook trout, Salvelinus fontinalis (Mitchell), rainbow trout, chinook salmon, Oncorhynchus tshawytscha (Walbaum), Arctic charr, Salvelinus alpinus (L.), and common shiners, Luxilus cornutus (Mitchell), following a 1-h bath exposure, and caused mortality in all species. The virulence of eight strains of F. branchiophilum for rainbow trout was examined. Both virulent and avirulent strains adhered to the gills following bath exposure (fimbriae were observed on all strains), but only virulent strains had the capacity to further colonize the gills and cause mortality.     

Use of cloned cDNA probes for diagnosis of infectious pancreatic necrosis virus infections

Abstract. A dot-blot hybridization test has been developed for the detection of infectious pancreatic necrosis virus (IPNV) in infected fish. For this purpose, cloning of the dsRNA of the West Buxton strain of IPNV was carried out. Two cDNA clones (WB and A4) were characterized for use as diagnostic probes and corresponded to IPNV genome segments A and B. respectively. Clone WB1, with an insert of 812 base pairs, showed an 87 and 77% nuclcotidc sequence homology with the corresponding sequences of Jasper and N1 strains, respectively. Clone A4, with an insert size of 596bp, presented a nuclcotidc sequence homology of 90 and 80% with the corresponding sequences of the Jasper and Sp strains, respectively. Both probes were able to detect 15 ng of purified dsRNA, and were highly efficient in detecting the RNA of American IPNV strains. However, the A4 probe was less effective than WB1 in hybridizing to RNA from European and Spanish strains of IPNV. Both probes detected IPNV RNA in cells 4–8h post-infection with the homologous West Buxton strain, 8–12h post-infection with other American strains and 24h post-infection with the European strains of IPNV. The method was less sensitive in detecting IPNV RNA directly in infected fish tissues. However, the present authors obtained a 100% effectiveness to detect viral RNA in cells inoculated with fish tissues confirmed by conventional diagnostic methods as being infected with IPNV. Therefore, the hybridization test is appropriate if combined with conventional diagnostic procedures, e.g. applying the dot blot hybridization test on tissue cultures 12–24 h after inoculation with infected fish tissue homogenates.     

两种芽孢杆菌体外体内拮抗气单胞菌的试验

将蜡样芽孢杆菌Y1和巨大芽孢杆菌BM1259混合培养,结合平板活菌计数、抑菌活性测定及体内拮抗试验,研究其对水产动物气单胞菌的抑菌效果。体外拮抗试验结果表明,这两种芽孢杆菌均能明显抑制嗜水气单胞菌NQ的增殖,抑制率分别为53.74%和56.12%。不同温度下的抑菌活性试验显示,2种芽孢杆菌的抑菌活性温度分别为32℃和29℃。体内拮抗试验结果表明,巨大芽孢杆菌能显著降低异育银鲫肠道气单胞菌的数量。提示巨大芽孢杆菌可作为潜在的水产专用微生态制剂。