竹醋液与茶多酚对南美白对虾的复合保鲜研究

本文研究了竹醋液、茶多酚以及二者复配液对常见食源性致病菌大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌及副溶血性弧菌的抑制效果。考察了竹醋液及茶多酚对上述四种供试菌的最低抑菌浓度,进而用于南美白对虾的保鲜。结果表明:竹醋液与茶多酚复配液具有协同抑菌作用。竹醋液、茶多酚及二者复配液对南美白对虾均有较好的保鲜效果,其中,复配液保鲜效果最佳,将南美白对虾货架期由原来的4 d延长到了7 d。     

副溶血性弧菌在动态体外人胃仿生原位消化系统中的存活情况研究

副溶血性弧菌(Vibrio parahaemolyticus)是一种经口摄入感染的食源性致病菌,广泛存在于水产品中,然而其进入人胃后的存活情况尚属研究空白。本研究将浓度为10~7 cfu/g的副溶血性弧菌接种于三文鱼和南美白对虾中,运用体外人胃仿生原位消化系统进行消化模拟,经过120 min后,测定食物排空率、胃部pH值变化及幽门排出食糜中副溶血性弧菌的存活情况。结果显示三文鱼的胃排空滞后时间为60 min,南美白对虾为90 min,模拟消化120 min时,两种食糜均未完全排空。胃部pH值为(1.6±0.1),在食物摄入10 min后大幅上升,随着胃酸的不断分泌及食物的消化分解,其pH值开始下降,并保持在5.41左右。在食品消化120 min进入肠道后,副溶血性弧菌并没有完全被胃酸杀灭,对虾中该菌的存活率为(0.119±0.025)%,而三文鱼中存活率为(0.007±0.005)%。综上所述,副溶血性弧菌可随食物基质的消化分解通过胃排空进入肠道,从而躲避胃酸的杀灭并导致人体患病。     

一株分离自美国龙虾的麦氏弧菌的鉴定及其低温状态下细胞脂肪酸的变化

[目的]对从美国龙虾(Homarus americanus)中分离的菌株F5-1进行种属鉴定并分析其低温状态下脂肪酸组成变化.[方法]通过VITEK 2 Compact全自动微生物鉴定仪分析菌株的生理生化特征和进行药敏试验;16S rRNA基因序列同源性分析确定该菌株的系统发育学地位;通过气相色谱和质谱联用法(GC-MS)分析菌株的全细胞脂肪酸.[结果]菌株F5-1为革兰氏阴性菌,对弧菌抑制剂O/129敏感,对青霉素有耐药性;生理生化特征与麦氏弧菌(Vibrio metschnikovii)的相似性为96％,16S rRNA序列与麦氏弧菌(GenBank No.HQ658055)的相似性为99％;菌株的主要脂肪酸组成为C12∶0、C14∶0、C16∶0和C16∶1(n-7),不饱和脂肪酸(棕榈油酸)相对含量达34％,低温培养状态下,不饱和脂肪酸(棕榈油酸)相对含量增加至40％.[结论]将美国龙虾中分离的菌株F5-1鉴定为麦氏弧菌,该菌对多种药物敏感,菌细胞脂肪酸组成与来源于俄罗斯海参威某饮用水水库中分离的麦氏弧菌有较大差异.     

两株海洋蛭弧菌的分离及生物学性质

[目的]从深圳湾海泥中分离鉴定蛭弧菌,并对其生物学性质进行初步研究.[方法]通过稀释营养肉汤(dilute nutrient broth,DNB)双层平板法分离蛭弧菌,对所分离的菌株进行电镜形态观测,并进行16S rDNA测序分析,之后结合1994年版伯杰氏鉴定细菌学手册对菌株进行鉴定,最后通过生理试验对其生物学性质进行研究.[结果]从深圳湾海泥中分离出2株蛭弧菌,分别命名为5#-12和5#-sh06,它们可在20℃～35℃范围内生长,最适温度分别是25℃和30℃;生长pH范围6.1～8.6,最适pH均为7.2;2株蛭弧菌可分别裂解46和48株试验菌,各占总试验菌株数(58)的79.3%和82.8%;联合2株蛭弧菌,可裂解56株试验菌,占总试验菌株数的96.6%;同时,它们一起能将所有试验弧菌裂解.[结论]研究结果揭示了蛭弧菌作为一种生物净化因子具有极大的潜在应用价值.     

副溶血性弧菌的致病性及其快速检测

副溶血性弧菌(Vibrio parahaemolyticus,VP)是革兰阴性嗜盐性细菌,隶属弧菌科中的弧菌属,它是一种人畜共患病菌[1].     

甲烷利用细菌HG06的筛选鉴定及其对三种有毒物质降解的研究

通过传统微生物学方法,从广西玉林市广西玉林师范学院西校区池塘里土壤中筛选到一株甲烷利用细菌HG06,能以甲烷为惟一碳源和能源的无机培养基上生长.通过形态观察及16S rRNA编码序列同源性比较分析,该菌株初步鉴定为甲基孢囊菌.同时对HG06菌株在不同温度、pH值的生长条件进行研究,初步确定了HG06的最适生长的温度为32℃,最适pH值为7.0.TCE最高的耐受浓度为30mg/L;苯胺最高的耐受浓度为1 500mg/L;苯酚最高的耐受浓度为800mg/L.该菌在环保上有一定的应用价值.     

微生态制剂改善对虾养殖池塘底质的效果

研究了在117 d的养殖周期中微生态制剂对南美白对虾池塘底质的改良效果.结果表明,与对照组相比,施用微生态制剂可使底质中总氮、总磷和硫化物的含量显著下降;总菌数量无显著变化,而芽孢杆菌、氨化细菌以及硫氧化细菌、硫还原细菌、弧菌数量差异显著,其中弧菌数量在施用微生态制剂处理和对照条件下分别为3.65×10³ cfu·g^-1和1.16×10⁵ cfu·g^-1.表明施用微生态制剂可以减少氮、磷、硫等营养物质的积累,改善池塘底质的菌相,为南美白对虾的健康养殖提供良好的池塘底质环境.     

南美白对虾肠道微生物群落的分子分析

采用分子生物学手段16S rDNA克隆文库方法对实验室养殖条件下的南美白对虾肠道细菌进行了多样性研究。用限制性片段长度多态性(RFLP)方法从文库中筛选出可能不同细菌来源的克隆子12个,测定其16S rDNA片段核甘酸序列,将所获得的序列与GenBank数据库进行BLAST比对,结果表明:南美白对虾肠道的16S rDNA克隆文库中126个克隆子分属2个不同的细菌类群:变形细菌(Proteobacteria)和厚壁细菌(Firmicutes),其中厚壁细菌为优势菌群占到75.4%,且与最相似序列同源性均低于94%;变形细菌占到24.6%,与最相似序列同源性均高于98%,分别为希瓦氏菌属(Shewanella),泛菌属(Pantoea),Aranicola属,假单胞菌属(Pseudomonas)和弧菌属(Vibrio)。     

一株高效解无机磷细菌BS06的鉴定及其解磷能力分析

【目的】对一株来源于广西甘蔗根际土壤的高效解无机磷细菌BS06的分类和解磷能力进行探讨,以期为解磷微生物在广西甘蔗生产上的开发和应用提供理论依据。【方法】通过形态观察、生理生化测定及16S rRNA基因序列同源性分析,进一步结合种特异的recA基因序列分析对解磷菌BS06进行分类鉴定;通过改变无机磷培养基中的碳源、氮源对菌株解磷能力的影响,分析菌株的解磷特性;通过盆栽试验了解菌株对甘蔗品种粤糖00236、桂糖28磷素吸收的影响。【结果】分类鉴定结果表明菌株BS06属于洋葱伯克霍尔德菌(Burkholderia cepacia);菌株在以乳糖为碳源条件下具有较强的解磷能力,其发酵液中水溶性磷含量为262.71 mg/L;在以硝酸钠为氮源条件下有较强解磷能力,其发酵液中水溶性磷含量达到305.85 mg/L;接种BS06菌株显著促进甘蔗组培苗的生长并提高甘蔗植株的含磷量。【结论】解磷细菌BS06具有较大的开发利用潜力。     

竹荪提取物对食源性细菌的抑菌特性研究

利用4种方法获得棘托竹荪和长裙竹荪的提取物,以5种食源性致病细菌单增李斯特菌、金黄色葡萄球菌、肠炎沙门菌、大肠埃希菌O157H7、副溶血性弧菌为供试菌,比较其MIC(最低抑菌浓度)和MBC(最低杀菌浓度)。超声波辅助破碎竹荪干粉,制取水、乙醇和石油醚为提取介质的浸提物以及竹荪挥发油,利用液体培养基连续稀释法对5种供试菌进行抑菌实验。所有提取物对供试菌都具有抑菌活性;长裙竹荪提取物的综合抑菌能力高于棘托竹荪提取物;水提物和挥发油的抑菌效果最好;2种竹荪提取物对革兰阴性菌和革兰阳性菌的抑菌作用存在互补效应。竹荪提取物对5种食源性细菌的生长有较好的抑制作用,可应用2种竹荪的提取物复配,开发天然食品防腐剂。     

Detection and identification of Vibrio species using whole-cell protein pattern analysis

Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE wholecell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.     

Improved recovery of pathogenic Vibrio parahaemolyticus from oysters using colony hybridization following enrichment

The traditional streak plating and alternative spread-plating methods were compared for detection of pathogenic Vibrio parahaemolyticus (Vp) in oyster enrichments. We found the alternative method to be more efficient: it was quicker (2d vs. 3d) and had a significantly (p < 0.05) greater detection rate than streak plating.     

荧光定量PCR法检测副溶血弧菌tlh和tdh基因的表达差异

副溶血弧菌是广泛存在于近海区域,盐湖和海产品中的食源性致病菌,会引起大规模的食物中毒。TLH(不耐热溶血毒素)和TDH(耐热直接溶血毒素)是副溶血弧菌最主要的毒力基因,通过比较毒力基因的表达量可以间接比较同种菌株在不同应激条件下以及不同菌株之间的毒力差异。本文以在不同条件下培养的3株Vp为材料,分别提取其总RNA,以16S rRNA为内标基因,运用荧光定量PCR技术检测副溶血弧菌TLH和TDH基因在不同应激条件下的表达差异。结果表明:不同菌株和同种菌株在不同应激条件下tlh、tdh基因表达差异均显著;tlh的最适表达条件分别为5%盐度和20°C;tdh的最适表达条件分别为1%盐度和25°C。运用SPSS软件对实验结果进行统计学分析表明:菌株对tlh表达的影响大于盐度大于温度;菌株对tdh表达的影响大于温度大于盐度。     

Serological method for identification of Vibrio parahaemolyticus from marine samples

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

Serological method for identification of Vibrio parahaemolyticus from marine samples.

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

Occurrence of pathogenic vibrios in coastal areas of France

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

Environmental Vibrio parahaemolyticus DNA signatures validation

Insignia is a novel DNA computational system which uses highly efficient algorithms to compare bacterial genomes and to identify specific DNA signatures to distinguish a target bacterium, or group of bacteria, from all other known bacterial species. It is currently being validated using different bacterial groups, including Vibrio spp. In this study, the genomic analysis by Insignia was conducted on Vibrio parahaemolyticus, a halophilic gram-negative bacteria which constitutes a leading cause of seafood-borne disease. Insignia was used to identify 37 V. parahaemolyticus-specific signatures and to design PCR assays to validate the representative signature sequences by TaqMan essays. The 37 assays targeted loci distributed around the genome and detected genes coding for hypothetical proteins and for proteins involved in adhesion, starvation and virulence. A panel of V. parahaemolyticus environmental strains isolated from the North Adriatic Sea (Italy) and from the Black Sea (Georgia) was used to validate the selected signatures. The signature assays revealed both sensitive and specific and the method allowed a more accurate identification of the tested bacterial strains at the species level when compared to biochemical and PCR standard methods. Using Insignia, it was possible to distinguish two different groups among the strains previously identified as V. parahaemolyticus: most of the strains were included in a "V. parahaemolyticus-like group" showing nearly all of the signatures assayed while a small group of 10 strains contained only a few of the signatures tested. By sequencing the 16S rDNA of this latter group, it was confirmed that they were not V. parahaemolyticus but in fact belonged to other Vibrio species. No significant genome-wide differences were detected between the strains isolated in Italy and in Georgia though the very different geographical origin.