Morphological and molecular identification of three geographical populations of the storage pest Liposcelis bostrychophila (Psocoptera)

Traditional morphological identification of three strains of the most widespread and important psocid pest of stored products, Liposcelis bostrychophila (Liposcelididae), was compared with the use of a molecular diagnostic method. The strains were from geographically isolated localities in Europe, Asia, and the United States. Morphological identification was based on the use of distinguishing morphological characters; data were obtained using body size measurements and scanning electron microscope (SEM) micrographs. Molecular identification was based on determining the 16S rDNA sequences and sequence similarities and then phylogenetic analyses of the three geographical populations. Both morphological and molecular methods are able to properly identify the species, but distinctively significant differences between geographical populations of L. bostrychophila were only found using the molecular method.     

Rapid molecular diagnosis of the stored-product psocid Liposcelis corrodens (Psocodea: Liposcelididae): Species-specific PCR primers of 16S rDNA and COI

Psocids of the genus Liposcelis (Psocodea: Liposcelididae) are common economically important pests in storage facilities all over the world. Stored-product psocids have small bodies and are difficult to identify to species using external morphological characteristics. Quick and accurate identification methods for psocid species serve as the foundation for pest management, quality control of food products, and settling of legal disputes from quarantine inspection. Liposcelis corrodens (Heymons) is a common stored-product pest distributed in European, American, and Oceania countries but has not been reported as established in China. To facilitate pest management and quarantine inspection to prevent importation and establishment of L. corrodens in China, methods for accurate and rapid identification need to be developed. Based on the sequencing and alignment of mitochondrial 16S rDNA and COI gene of 10 common Liposcelis species, two pairs of species-specific primers were designed according to the variation regions among species. PCR cycling parameters were developed for the amplification of specific fragments of 16S rDNA and COI genes of L. corrodens with lengths of 261 bp and 243 bp, respectively. Both primer sets have high sensitivity in target species detection and successfully identified psocids found by quarantine personnel in China as L. corrodens. The diagnostic method we have developed is reliable for identification of L. corrodens for quarantine and pest management purposes.     

Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA)

The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control.     

浓香型白酒曲药中细菌组成及系统学分析

利用免培养法(Culture independent approach)提取浓香型白酒曲药中细菌的总DNA,在分子水平上运用PCR扩增技术和16S rDNA序列同源性分析等方法测定曲药中细菌的16S rDNA基因近全序列,并建立系统发育树图.结果表明,组成该曲药中的细菌分属于Delftia、Dysgonomonas、Bacteroidetes,proteobacterium、Nocardiopsis、Pseudomonas和Arthrobacter几大类群(phylum),表现出高度的细菌多样性.     

Comparison of culture-dependent and independent techniques for characterisation of the microflora of peroxyacetic acid treated,vacuum-packaged beef

The diversity of microflora associated with peroxyacetic acid (POAA) treated and untreated beef was investigated by 16S rDNA gene cloning, DGGE analysis and conventional bacterial cultivation. Following vacuum packaging, POAA treated and untreated meat samples were stored for up to 18 weeks at −1.5 °C. Each culture independent method showed Carnobacterium spp. to predominate on both POAA treated and untreated meat. However, 16S rDNA gene analysis also detected the presence of psychrotolerant Clostridium spp. in the POAA-treated beef. Culture-dependent analysis did not distinguish Carnobacterium spp. from Lactobacilli. Although culture-dependent analysis showed an increase in the ratio of Enterobacteriaceae to lactic acid bacteria from weeks 6–18 in the POAA treated compared with the untreated meat, the numbers of Enterobacteriaceae were significantly less on POAA treated than on untreated meat. The combination of data collected by culture-dependent and independent techniques provided the most robust approach for elucidating the efficacy of chemical sanitization of chilled vacuum-packaged beef. If conventional cultivation is used for monitoring bacterial spoilage of vacuum-packaged chilled meats it is recommended that culture methods specific for Carnobacterium and Clostridium spp. should be included in order to provide a more complete indication of microbial diversity.     

Analysis of bacterial diversity during the fermentation of inyu, a high-temperature fermented soy sauce,using nested PCR-denaturing gradient gel electrophoresis and the plate count method

The diversity of bacteria associated with the fermentation of inyu, also known as black soy sauce, was studied through the nested PCR-denaturing gradient gel electrophoresis (DGGE) of samples collected from the fermentation stages of the inyu production process. The DGGE profiles targeted the bacterial 16S rDNA and revealed the presence of Citrobacter farmeri, Enterobacter cloacae, Enterobacter hormaechei, Enterococcus faecium, Klebsiella pneumoniae, Pantoea agglomerans, Salmonella enterica, Serratia marcescens, Staphylococcus sciuri and Weissella confusa. The bacterial compositions of 4 fermented samples were further elucidated using the plate count method. The bacteria isolated from the koji-making stage exhibited the highest diversity; Brachybacterium rhamnosum, E. hormaechei, K. pneumoniae, Kurthia gibsonii, Pantoea dispersa, Staphylococcus gallinarum, Staphylococcus kloosii and S. sciuri were identified. Koji collected during the preincubation stage presented the largest cell counts, and E. hormaechei, K. pneumoniae, E. cloacae and Enterobacter pulveris were identified. In brine samples aged for 7 and 31 days, the majority of the bacteria isolated belonged to 4 Bacillus species, but 4 Staphylococcus species and Delftia tsuruhatensis were also detected. This study demonstrates the benefits of using a combined approach to obtain a more complete picture of microbial populations and provides useful information for the control or development of bacterial flora during inyu fermentation.     

新鲜哈密瓜汁中可培养细菌的分离鉴定及系统发育分析

采用稀释平板法从新鲜哈密瓜汁中分离出48株可培养细菌,通过传统的形态特征鉴定,将其归为10个类群。利用细菌通用引物PCR扩增10个类群代表菌株的16S rRNA基因,并进行序列测定。将获得的序列通过Blast在GenBank数据库中搜索相关菌株的同源性序列,采用Clustal1.81软件比对,用Mega4.1软件构建系统发育树。系统发育分析结合表型特征研究表明,新鲜哈密瓜汁中可培养细菌以芽孢杆菌属为优势微生物类群,主要为枯草芽孢杆菌Bacillus sub-tilis,其次为地衣芽孢杆菌Bacillus licheniformis、苏云金芽孢杆菌Bacillus mojavensis。此外还有铜绿假单胞菌Pseudomonas aeruginosa、洋葱伯克霍尔德氏菌Burkholderia cepaciastrain、鲍曼不动杆菌Acinetobacter baumannii,以及肠球菌属Enterococcussp、克雷伯氏菌属Klebsiellasp的菌株。     

一株牛蒡表面产纤维素酶细菌的分离鉴定

从牛蒡表面获得的多株产纤维素酶细菌中筛选到一株产酶活性较高的菌株NP10。使用生物技术方法获得了NP10长度为1419bp的16S rDNA序列。利用生物信息学方法在美国国立生物技术信息中心NCBI基因库比对分析了该序列,通过分析初步确定该菌为Bacillus sp.。本文为食品科学中进行细菌鉴定提供了方法参考,并为开发新型纤维素酶提供了资料。     

自然发酵糯玉米中细菌多样性分析及纯菌种对其加工特性的影响

目的：研究自然发酵糯玉米中细菌的菌群组成,以及其中乳酸菌对糯玉米粉加工特性的影响。方法：以地方特色的糯玉米为原料,采用MiSeq技术对自然发酵菌液中细菌16S rDNA的V3～V4区进行测序分析,获得自然发酵液中的细菌多样性。对其中的1 株优势菌株进行筛选鉴定,比较纯菌种发酵和自然发酵对糯玉米粉的糊化特性和质构特性的影响。结果：自然发酵液菌中乳杆菌（Lactobacillus）为优势菌,经形态学特征、生理生化特征及16S?rDNA基因序列分析鉴定其为发酵乳杆菌（Lactobacillus?fermentum）。经过纯种发酵后糯玉米粉的峰值黏度、衰减值和最终黏度显著提高,回生值显著降低;经过蒸制后硬度、黏附性、胶着性和咀嚼性显著增高从而改善糯玉米粉质特性。     

Real-Time PCR Quantification of Protease-Producing Bacteria in Traditional Chinese Fish Sauce

This study aims to isolate, identify, and quantify protease-producing bacteria in traditional Chinese fish sauce. Fifty-five protease-producing bacteria were isolated from 10 fermented fish sauce samples in five growth media based on their morphology and caseinolytic activity. These isolates were identified through their 16S rDNA gene sequences. BLAST analysis of 16S rDNA sequences revealed that 46 and 9 strains belonged to Bacillus sp. and Virgibacillus halodenitrificans, respectively. We used EvaGreen dye in real-time PCR assay to target the partial bacterial 16S rDNA gene sequences of the 55 strains from fish sauce. Two primer pairs (A and B) were designed. Primer pair B was more suitable than primer pair A for real-time PCR assay, in which the optimum annealing temperature was 60 °C. The significance of the developed method was proven by the highly linear characteristic of the standard curve that relates lower threshold cycle (Ct) values and 16S rDNA copy numbers of the standard DNA sample. This method was used to calculate the number of protease-producing bacteria in fish sauce. The minimum level of detection (1.44?×?10³ copies/μL) was also verified. The concentration of protease-producing bacteria in fish sauce was estimated to be (1.47?±?0.73)?×?10³ colony-forming units (cfu)/mL by real-time PCR assay and showed a percentage of positive results correctly assigned of 91.8 % when compared to the plate culture method used as reference. The results may serve as a foundation for future studies on the microbial succession and variation of microorganisms during fish sauce fermentation.     

市售鲫鱼体内菌株及其抗生素耐药性分新

分离健康鲫鱼体内菌株,通过显微镜观察和16S rDNA分析对菌株进行鉴定,并进行产纤维素酶和淀粉酶菌株筛选。分离到的菌株分别属于假单胞菌、气单胞菌、希瓦氏菌等。在美国国家生物信息中心NCBI Genbank基因库中提交了其中两个菌株16S rDNA序列,登录号是:FJ796224和FJ796225。筛选到多株产淀粉酶菌株,其中产酶活性较高的菌株命名为Aeromonas veronii F2。在鲫鱼肠道没有筛选到产纤维素酶菌株。抗生素检测分析显示,鲫鱼体内抗卡那霉素、氨苄青霉素和氯霉素三种药物的细菌有11.29%,显示鲫鱼体内菌株抗药性比从田野环境中获得的菌株高5~6倍,这需要引起人们的关注。     

啤酒有害乳酸菌PCR引物的设计与验证

根据乳酸菌16SrDNA序列的特征，设计合成了针对啤酒有害乳酸菌的通用引物，在16SrDNA基因水平上采用聚合酶链式反应（PCR）技术鉴定了啤酒中59株有害乳酸菌的种类。同时根据鉴定结果设计了8种乳酸菌的特异引物，PCR技术验证结果表明该8种特异引物能够准确鉴定乳酸菌。     

鮰鱼肌肉组织中不动杆菌的分离及鉴定

将鮰鱼在4℃冷库中取得背部肌肉组织,进行真空包装,在4℃冰箱中贮藏7 d,分离得到2株细菌,分别编号为by 7-2、bs 7-3,进行细菌形态学观察、革兰氏染色、生化鉴定、16S rDNA测序及负染等。结果表明:菌株by 7-2在LB琼脂培养基上为圆形、湿润、白色菌落,菌株bs 7-3在LB琼脂培养基上为圆形、湿润、乳白色菌落;革兰氏染色镜检表明,2株细菌均为革兰氏阴性菌;负染结果表明,by 7-2为短杆菌,bs 7-3为长杆菌,且二者均无鞭毛;生化鉴定结果显示,菌株by 7-2和bs 7-3硝酸盐还原、精氨酸双水解酶、乙酰胺酶、分解木糖、分解麦芽糖、分解甘露醇、DNA酶和氧化酶实验均为阴性,菌株bs 7-3的利用柠檬酸盐和O-F实验为阳性,菌株by 7-2利用柠檬酸盐和O-F实验为阴性;进一步通过16S rDNA测序及系统发育树分析显示,by 7-2与约翰逊不动杆菌(Acinetobacter johnsonii)亲缘关系最近,相似度达99.90%,bs 7-3与抗辐射不动杆菌(Acinetobacter radioresistens)亲缘关系最近,相似度达99.79%。因此,菌株by 7-2为约翰逊不动杆菌,bs 7-3为抗辐射不动杆菌。     

Identification of extracellular DNase-producing bacterial populations on catfish fillets during refrigerated storage

Food spoilage is a major problem faced by consumers across the globe. As an enzyme that degrades DNA, DNase production on fish tissue seemed likely to aid in fish spoilage. Based on physical characteristics, bacteria producing extracellular DNase were isolated on selective media. 16S rDNA sequences were obtained identifying isolates as bacteria belonging to Aeromonas spp., Serratia spp., Shewanella spp., and Rahnella spp. Aeromonas spp. were the predominant bacteria isolated in this study; this statistically suggests that Aeromonas spp. are dominant in DNase-producing bacterial populations on catfish tissue. Results obtained in this study suggest that extracellular DNase-producing bacteria play a large role in catfish spoilage and support the need for further research on the role of Aeromonas spp. in fish spoilage. Rahnella spp. was isolated from catfish fillets in this study and identified, for the first time, as DNase producing bacteria.     

Analysis of a change in bacterial community in different environments with addition of chitin or chitosan

The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands of the 31 species indicated that 20 species belonged to the division Proteobacteria, and that the genus Cellvibrio was apparently predominant among them (7/20). The 16S rRNA sequences of the 16 deduced species (16/31) showed less than 98% similarities to those of previously identified bacteria, indicating that the species were derived from unidentified bacteria. The total community DNAs extracted from bacterial cells adsorbed on the surface of flakes of chitin and chitosan placed in a river, a moat, or soil were subjected to PCR-DGGE to examine the extent of diversity of chitinolytic bacteria among different environments. The predominant species significantly differed between the chitin and chitosan placed in the river and moat, but not so much between those placed in the soil. The large difference between the diversities of the three bacterial communities indicated that a wide variety of bacteria including unidentified ones are involved in the degradation of chitin and chitosan in the above-mentioned natural environments.     

气调醇化过程中片烟细菌群落结构的变化

气调醇化是片烟醇化的一种方法。微生物尤其是细菌在片烟醇化过程中对烟叶的吸食品质起着至关重要的作用。为研究气调醇化过程中片烟细菌群落结构变化规律,利用Illumina MiSeq测序平台比较气调防霉杀虫阶段（S1）、气调醇化阶段（S2）及气调保质阶段（S3）共36份样品的细菌16S rDNA序列的多样性。结果表明,从S1到S3的过程中,片烟中细菌的物种丰富度和多样性水平呈增加趋势。S1阶段片烟的优势种群为鞘氨醇单胞菌属、假单胞菌属和甲基杆菌属,其占比在整个气调醇化过程中呈逐渐下降趋势;S2阶段的优势种群为芽孢杆菌属、鞘氨醇单胞菌属和伯克霍尔德菌属,其中芽孢杆菌属和伯克霍尔德菌属在整个气调醇化过程中占比呈先上升后下降的趋势;S3阶段细菌种群分布较为均匀。非度量多维尺度分析结果表明,3个醇化阶段的样品可以较明显地区分开来。可见,气调醇化过程中片烟的细菌种群组成非常丰富,不同醇化阶段活跃的微生物类群有所不同,鞘氨醇单胞菌属、假单胞菌属和甲基杆菌属主要在醇化前期参与了烟叶醇化过程,而芽孢杆菌属和伯克霍尔德菌属则主要在醇化后期发挥着微生物醇化的作用。     

Isolation and identification of Alicyclobacillus acidocaldarius by 16S rDNA from mango juice and concentrate

In this study we investigate the spoilage of ultra high temperature UHT mango juice as well as a carbonated fruit juice blend to identify organisms contributing to the spoilage. The mango concentrate, the final product, as well as the other ingredients used during manufacturing, were tested for the presence of Alicyclobacillus by polymerase chain reaction (PCR) and sequencing analyses. Microbiological examination of the mango pureé and spoiled fruit juices, using YSG agar [yeast extract 2 g, glucose 1 g, soluble starch 2 g, pH 3.7 (adjust with 2N H₂SO₄), H₂O 1000 mL, bacto agar 15 g] incubated at 55 °C, detected sporeforming, acid dependent and thermotolerant bacteria. The hyper variable region of the 16S rDNA was amplified. The nucleotide sequence of the PCR fragments was determined using the ABI Prism 310 automated DNA sequencer and the collected sequencing data were analysed and compared with the non‐redundant database using NCBI‐BLAST. Alicyclobacillus acidocaldarius were isolated and identified by 16S rDNA gene sequences analyses. The results indicated that the mango purèe, as well as the final product of mango juice and the fruit juice blend, were positive for Alicyclobacillus. The preventative measures of low pH, pasteurization of mango juice and the subsequent use of aseptic packaging were not regarded as sufficient to prevent the outgrowth of Alicyclobacillus spoilage organisms.     

The full-length mitochondrial rDNA sequence of Tyrophagus longior (Astigmata: Acaridae) and phylogeny of astigmatid mites based on mitochondrial rDNA sequences

Mites of the genus Tyrophagus are economically important as they are polyphagous stored product pests and cause medically allergic reactions to human. Considering that examining the phylogenetic relationship among Astigmata species might help to understand their evolutionary process, we sequenced and analyzed the mitochondrial (mt) rDNA of the Tyrophagus longior and compared with those of six other astigmatid mites, as well as assessed the phylogenetic relationship among astigmatid species based on the complete mt rDNA sequences. Sequence analysis showed that the mt rDNA sequences of seven Astigmata species have high A + T contents (average value: 72.0% for 12S rRNA and 72.4% for 16S rRNA, respectively). The genetic distance and transition/transversion (TS/TV) of the seven Astigmata species reached interspecies level. In addition, the predicted stem-loop secondary structure of T. longior mt rDNA closely resembled those of other reported acariform mites. Finally, phylogenetic analyses based on complete sequences of 12S rRNA and 16S rRNA genes revealed that a close relationship between the families Acaridae and Pyroglyphidae, and supported the monophyly of the Acaridae as well as the Astigmata. These results are consistent with traditional classifications. Therefore, the mt rRNA genes are suitable for inferring phylogenetic relationship among astigmatid mites in this study, although the differentiation among the seven astigmata species has approached interspecies level.     

熟制鲍鱼残留细菌的分离及16S rDNA鉴定

为控制熟制鲍鱼残留的细菌污染,采用平板划线分离技术对烘制加工后鲍鱼中残留的主要菌群进行分离,并通过菌落形态观察、革兰氏染色分析和16S rDNA 序列比对等方法对其进行鉴定. 结果表明,残留在烘制加工后鲍鱼的主要菌群有希瓦氏菌属、不动杆菌属、库特氏杆菌属、海洋类香菌、蜡样芽孢杆菌、肠杆菌属、彭氏变形杆菌等.     

部分茶饮料原辅料中优势菌-克罗诺杆菌(原阪崎肠杆菌)的分离和鉴定

克罗诺杆菌(原阪崎肠杆菌)是新兴的条件致病菌,主要通过污染的婴儿配方奶粉引起2岁以下婴幼儿严重疾病甚至死亡.在对茶饮料原辅料192份样品进行细菌总数测定和优势菌16S rDNA序列分析时,发现2批次绿茶和l批次果葡糖浆中优势菌疑似条件致病菌-克罗诺杆菌,而且其数量较高(4.0× 102cfu/g～104cfu/g).进而通过rpoB序列分析,将4个分离株鉴定为阪崎克罗诺杆菌Cronobacter sakazakii(原阪崎肠杆菌Enterobacter sakazakii).结果表明在常见的食品原料-果葡糖浆和绿茶中存在克罗诺杆菌的污染,rpoB分型方法在对克罗诺杆菌属细菌的鉴定到种上表现出高度特异性,灵敏而准确.