MALDI-TOF-MS对金桔表面肠杆菌科微生物分布的研究

为了评估金桔的食用风险,试验采用基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF-MS)调查金桔表面肠杆菌科微生物分布情况,筛选致病微生物,同时采用与生化鉴定和16S rDNA测序鉴定辅助鉴定结果。实验共分离6种的肠杆菌科细菌,分别为日沟维肠杆菌、分散泛菌、肺炎克雷伯菌、阪崎肠杆菌、生癌肠杆菌、阴沟肠杆菌,其中分散泛菌、阴沟肠杆菌和肺炎克雷伯菌出现频率较高;对三种鉴定方法进行比较分析,MALDI-TOF-MS鉴定结果与另外两种与属水平一致,种水平有偏离;MALDI-TOF-MS对分离微生物进行聚类分析表明同属微生物亲缘较近,同种微生物峰谱图仍有差异。试验结果表明,金桔表面具有肠道致病微生物,有一定食用风险,建议加强农产品致病菌常规的检测及风险评估力度;MALDI-TOF-MS方法可分辨率高且能提供基于蛋白质水平归类分析可以用于农产品微生物的快速检测。     

电脑智能在肠杆菌科细菌分析鉴定中的应用

为肠杆菌科细菌分析的需要 ,应用C 语言 ,编制了《华顺微生物分析鉴定智能系统》(HUASUN) ,该系统的V2 0版主要由肠杆菌科细菌分析组件组成 ,该组件以伯杰氏细菌分类鉴定手册、美国临床微生物学手册和全国临床检验操作规程等为参考基础 ,编制肠杆菌科细菌分析鉴定组件 (ent15和ent4 8)。该版本可鉴定分析肠杆菌科的常见的 2 1个种类 ,包括了大部分临床上最新的分离菌种。与常用的VITEK、API、ATB及国内开发的常见鉴定系统比较 ,该系统可鉴定分析的肠杆菌科细菌最多。生化鉴定项目由常用的 15个生化检验项目 (ent15 )和 4 8个生化检验项目(ent4 8)两部分组成。在ent4 8中 ,可任意选择 4 8个生化中的试验项目和个数 ,组成鉴定系列 ,进行鉴定分析 ,这一智能化特征在VITEK、ATB、API及国内开发的常见鉴定系统中是没有的。该版本对细菌鉴定过程中的异常生化及进一步区分的生化项目可作智能化选择分析。该系统的V2 0版还包括了沙门氏菌血清型分析和细菌性食物中毒分析组件。     

1例奶茶中污染菌的分析鉴定

某企业生产的1批奶茶贮存1~2个月后发现约70%产品变质,出现pH值轻微下降,但不产气,肉眼观察不到分层、沉淀等感官指标的变化,检测发现有细菌的污染,变质产品中细菌数高达106cfu/mL。分离到2株典型污染菌,经过16S rDNA序列比对,鉴定为微杆菌属(Microbacterium sp.)。该属微生物能耐受85℃、10min~30min,是从巴士杀菌乳和喷雾干燥奶粉中分离到的最耐热的非芽孢菌之一,常导致乳制品中出现该类微生物的大量污染。一般30℃、3d以上才能形成肉眼可识别的菌落,常规检测中因未形成可识别的菌落或容易被忽略。显微形态上有着特殊的排列方式,容易与微小的球菌混淆。该类微生物的污染往往因乳石未彻底清洁造成,能在货架期缓慢生长,导致产品变质,隐蔽性强,需要引起乳制品及相关行业的重视。     

奶粉中阪崎肠杆菌PCR和荧光PCR检测方法的研究

建立了奶粉中可致婴幼儿高死亡率的阪崎肠杆菌的PCR和荧光PCR检测方法。利用细菌16S和23SrDNA的保守区设计通用引物,对6株阪崎肠杆菌16S-23SrDNA间区序列(ITS)进行扩增和测序,在比对阪崎肠杆菌ITS序列的基础上,设计了11条PCR和荧光PCR检测引物,组合成30对PCR引物,并筛选出一对种特异性引物,建立了奶粉中阪崎肠杆菌PCR和荧光PCR检测方法。用10株阪崎肠杆菌,18株近源菌株验证实验表明,本文所建立的PCR和荧光PCR方法特异性强;加菌实验表明,奶粉样品中阪崎肠杆菌检测低限为(2.2～5.4)CFU/100g,灵敏度高;新建的PCR和荧光PCR方法与FDABAM(美国食品及药品管理局微生物分析手册)方法比对实验表明,三种方法的检测结果完全一致。由于PCR和荧光PCR检测方法快速、可靠,因此可替代传统检验方法。     

中国市售配方粉中阪崎肠杆菌和其它肠杆菌的污染状况

目的:调查和研究我国市售配方粉中阪崎肠杆菌和其它肠杆菌的污染状况。方法:采用传统的微生物学分离和鉴定方法检测212份市售配方粉中阪崎肠杆菌和其它肠杆菌。试验采用无菌水增菌、EE肉汤选择性增菌、VRBGA平板选择性分离、TSA平板分离培养和生化鉴定等。结果:从11份配方粉中分离到阪崎肠杆菌(占检测样品总数的5.19%),从103份样品中检出肠杆菌(占检测样品总数的48.58%)。检出的肠杆菌包括:泛菌属某些种、肺炎克雷伯氏菌、阴沟肠杆菌、醋酸钙-鲍曼复合不动杆菌、阪崎肠杆菌、非脱羧勒克氏菌、伤口埃希氏菌和大肠埃希氏菌等。结论:配方粉中肠杆菌的污染可能会导致婴幼儿,特别是婴儿的严重感染。本研究结果将有助于针对我国市售配方粉的卫生预防和控制。     

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.     

我国首例因Asaia sp.污染而导致含果汁及果肉饮料变质的案例分析

某含椰果粒椰汁饮料发生变质,外观正常,经检测发现细菌严重超标。污染菌通过16S rDNA序列比对鉴定为Asaia sp.,这是该属微生物第一次在我国检出。该菌能耐受pH值3.0~3.5的酸性环境,能耐受75℃、30min或80℃、10min热处理,耐受0.8g/kg浓度的山梨酸钾。尤其值得重视的是:该菌在36℃~37℃平板计数琼脂培养基上不生长,所以根据GB 47892-2010食品安全国家标准食品微生物检验菌落总数测定规定的方法无法检出该污染菌。因其耐酸、耐热、耐防腐剂和多种抗生素、产生物膜能力较强的特性,Asaia sp.细菌可能对水果、果汁等产品的安全性造成不可忽视的影响。     

建立一种快速检测坂歧肠杆菌的方法

目的:建立快速检测婴儿配方奶制品(IFM)中坂歧肠杆菌的方法。方法:选择ATCC坂歧肠杆菌标准株,对不同的增菌性培养基、选择性培养基和显色培养基进行研究;结合VITEK仪和API20E细菌鉴定系统,构建快速检测配方奶制品中坂歧肠杆菌的方法。结果:建立的快速检测配方奶制品中坂歧肠杆菌的方法,所需检验流程为72h,方法灵敏度为2CUF/g,能有效区别于阴沟杆菌、产气杆菌等肠杆菌科细菌;方法应用稳定;操作简单、方法可靠,适宜规模化检测。结论:本研究认为,所建立的快速检测配方奶制品中坂歧肠杆菌的方法适宜检验检疫的工作要求,能有效地发现配方奶制品中坂歧肠杆菌的污染情况。     

冷却猪肉中腐败微生物鉴定及其消长规律的研究

目的对冷却猪肉中腐败微生物进行鉴定,研究其在0～4℃条件下贮藏时的消长规律。方法采用选择性培养基对冷却猪肉中的腐败微生物进行分离培养,利用Biolog微生物自动鉴定系统对菌株进行鉴定。结果共鉴定出11株具有代表性的细菌:肠杆菌4株,假单胞菌1株,热杀索丝菌1株,不动杆菌1株,乳酸菌2株,葡萄球菌2株。冷却猪肉中腐败微生物初始菌相结构为:热杀索丝菌54.9%,肠杆菌科8.7%,假单胞菌属3.6%,乳酸菌属29.5%,葡萄球菌/微球菌0.6%,霉菌/酵母菌2.7%。在0～4℃条件下贮藏时,热杀索丝菌、肠杆菌科和假单胞菌属是冷却猪肉中的优势腐败菌,假单胞菌属和肠杆菌科在菌相结构中的比例增长最高,特别是假单胞菌属的数量增长最快。结论鉴定出了冷却猪肉中的主要腐败微生物,确定了其初始菌相和优势腐败菌。     

基质辅助激光解吸电离飞行时间质谱技术分离鉴定生鲜猪肉中沙门氏菌/大肠杆菌类似菌

目的利用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry,MALDI-TOF-MS)技术鉴别生鲜猪肉中大肠杆菌和沙门氏菌的常规检验中出现的类似菌。方法在郑州市不同地区分批次采集164份生鲜猪肉样品,按照国标方法进行增菌培养,选择性培养基HE和EMB对细菌进行分离纯化。通过MALDI-TOF-MS技术对疑似菌落进行鉴定;提取被检菌株基因组DNA,根据MALDI-TOF-MS鉴定出的细菌种类,参考Gen Bank中相关菌株的已知基因序列分别设计引物,进行PCR扩增、测序和序列比对,以验证MALDI-TOF-MS鉴定结果。结果经MALDI-TOF-MS方法鉴定,从68个疑似菌株中分别检出柠檬酸杆菌(Citrobacter)6株、奇异变形杆菌(Proteobacteria)5株、肺炎克雷伯氏菌(Klebsiella pneumoniae)9株、绿脓杆菌(Pseudomonas aeruginosa)1株、阴沟肠杆菌(Enterobacter cloacae)3株、不动杆菌(Acinetobacter)1株。PCR结果与MALDI-TOF-MS鉴定结果完全一致。结论 MALDI-TOF-MS可用于生鲜猪肉检验过程中大肠杆菌和沙门氏菌的分离鉴定。本次共检出6种在HE和EMB培养基上与沙门氏菌/大肠杆菌菌落特征类似的肠杆菌科其他细菌,说明在生鲜猪肉中存在着一定程度的条件致病性肠道细菌污染,因此具有一定的食品安全风险。     

苦笋复合饮料的研制

单纯的苦笋饮料 ,其味甘苦单一 ,适口性差。把苦笋、胡萝卜、柑桔花三者配合使用 ,通过正交试验进行配方优选 ,结果表明 ,最佳风味调配浓度为 :苦笋汁 10 % 胡萝卜汁 10 % 柑桔花汁 3 % ,由此调制成的苦笋复合饮料营养丰富 ,具有独特的风味及一定的保健作用。     

Identification of corynebacterium bovis and other coryneforms isolated from bovine mammary glands

Bovine mastitis remains the most economically important disease in dairy cows. Corynebacterium bovis, a lipid-requiring Corynebacterium spp., is frequently isolated from the milk of infected mammary glands of dairy cows and is associated with reduced milk production. A total of 212 coryneform bacteria isolated from the milk of dairy cows were obtained from mastitis reference laboratories in the United States and Canada. All isolates had been presumptively identified as Corynebacterium bovis based on colony morphology and growth in the presence of butterfat. Preliminary identification of the isolates was based on Gram stain, oxidase, catalase, and growth on unsupplemented trypticase soy agar (TSA), TSA supplemented with 5% sheep blood, and TSA supplemented with 1% Tween 80. Of the 212 isolates tested, 183 were identified as Corynebacterium spp. based on preliminary characteristics. Of the strains misidentified, one was identified as a yeast, two as Bacillus spp., 11 as Enterobacteriaceae, 18 as staphylococci, one as a Streptococcus spp., and one as an Enterococcus spp. Eighty-seven coryneforms were selected for identification to the species level by direct sequencing of the 16S rRNA gene, the Biolog system and the API Coryne system. Fifty strains were identified as C. bovis by 16S rRNA gene similarity studies: the Biolog and API Coryne systems correctly identified 54.0 and 88.0% of these strains, respectively. The other coryneforms were identified as other Corynebacterium spp., Rhodococcus spp., or Microbacterium spp. These data indicate that the coryneform bacteria isolated from bovine mammary glands are a heterogeneous group of organisms. Routine identification of C. bovis should include Gram-stain, cell morphology, catalase production, nitrate reduction, stimulated growth on 1% Tween 80 supplemented media, and beta-galactosidase production as the minimum requirements.     

Microbiological quality of sous and tamarind, traditional drinks consumed in Jordan

This study was conducted to evaluate the microbiological quality of sous (a drink prepared by extracting dried roots of Glycyrrhiza glabra) and tamarind (a drink prepared by infusing Tamarindus indica dried pulp), traditional drinks consumed in Jordan. Twenty-one samples of sous and 44 samples of tamarind were collected from the local market in Amman, Jordan. Water is the major component of the drinks. Sous drink is characterized by having an alkaline pH (range, 6.6 to 9.9; mean, 8.6), whereas tamarind drink has an acidic pH (range, 1.8 to 3.7; mean, 2.8). The drinks are not processed for safety before serving, and at some vendors drinks are not properly refrigerated. The mean counts for aerobic bacteria, lactic acid bacteria, and yeasts in sous drink samples were 5.9, 5.0, and 3.8 log CFU/ml, respectively; those in tamarind drink samples were 4.0, <1, and 5.8 log CFU/ml, respectively. The lactic acid bacteria isolated were Enterococcus raffinosus, Enterococcus hirae, Enterococcus durans, Lactobacillus acidophilus, and Lactobacillus buchneri. The yeast isolates in sous drink were from the genera Candida, Filobasidium, Hanseniaspora, Lodderomyces, Pichia, and Williopsis, and those in tamarind drink were from Arthroascus, Brettanomyces, Candida, Debaromyces, Filobasidiella, Hanseniaspora, Klavispora, Lodderomyces, Pichia, Saccharomycodes, Trichosporon, and Zygosaccharomyces. Enterobacteriaceae were detected in two sous samples and were identified as Enterobacter sakazakii and Erwinia sp., and in two tamarind samples and were identified as Citrobacter freundii and Klebsiella pneumoniae. Salmonella was detected in one sous and one tamarind sample. Pseudomonas aeruginosa was detected in only one sous sample. These findings highlight the importance of application of hygienic practices throughout preparation and vending of drinks, starting with raw ingredients and continuing through preparation, storage, display, and serving.     

一例茶饮料污染源的分析

某企业生产的一批茶饮料中发现某些产品变质,3份样品出现pH值下降,或产气胀罐现象,或有肉眼可观察到浑浊、变色等感官指标的变化。检测发现引起腐败变质的主要是细菌,其数量≥104 CFU/mL,4株主要污染菌菌株经16S rDNA序列比对,鉴定为假单胞菌属(Pseudomonas sp.)、肠杆菌属(Enterobacter sp.)、芽胞杆菌属(Bacillus sp.)和短芽孢杆菌(Brevibacillus sp.)。样品中还有数量很低(101～102 CFU/mL左右)的霉菌检出,经形态观察初步鉴定为交链孢霉(Alternaria spp.)、枝孢霉(Cladosporium spp.)、曲霉(Aspergillus spp.)和青霉(Penicillium spp.)。该案例中霉菌虽然不是主要的腐败变质菌,但是其菌相组成与空气中主要的霉菌菌相一致,显示该污染的源头可能是空气,细菌的组成也能支持这个推论。实践中,通过更换无菌空气过滤器,确实解决了污染问题,证实了该推论。     

High-pressure processing of Turkish white cheese for microbial inactivation

High-pressure processing (HPP) of Turkish white cheese and reduction of Listeria monocytogenes, total Enterobacteriaceae, total aerobic mesophilic bacteria, total molds and yeasts, total Lactococcus spp., and total Lactobacillus spp. were investigated. Cheese samples were produced from raw milk and pasteurized milk and were inoculated with L. monocytogenes after brining. Both inoculated (ca. 10(7) to 10(8) CFU/g) and noninoculated samples were subjected to HPP in a high-pressure food processor at 50 to 600 MPa for 5 and 10 min at 25 degrees C. Reductions in L. monocytogenes, total aerobic mesophilic bacteria, Lactococcus spp., and Lactobacillus spp. in both pasteurized- and raw-milk cheese samples and reductions in total molds and yeasts and total Enterobacteriaceae counts in raw-milk cheese samples increased with increased pressure (P < or = 0.05). The maximum reduction of the L. monocytogenes count, ca. 4.9 log CFU/g, was obtained at 600 MPa. Because of the highly inhibitory effect of pasteurization, the total molds and yeasts and total Enterobacteriaceae counts for the cheese samples produced from pasteurized milk were below the detection limit both before and after HPP. There was no significant difference in inactivation of L. monocytogenes, total aerobic mesophilic bacteria, Lactococcus spp., and Lactobacillus spp. under the same treatment conditions for the raw milk and pasteurized milk cheeses and for 5- and 10-min treatment times (P > 0.05). No significant change was detected in pH or water activity of the samples before and after HPP. Our findings suggest that HPP can be used effectively to reduce the microbial load in Turkish white cheese.     

Rapid propidium monoazide PCR assay for the exclusive detection of viable Enterobacteriaceae cells in pasteurized milk

Pasteurized milk is a complex food and contains numerous PCR inhibitors and can often contain high levels of dead Enterobacteriaceae cells, depending on the condition of food sanitation. Usually, propidium monoazide (PMA) or ethidium monoazide PCR techniques decrease the number of dead bacteria by up to 3.5 log to the associated dead bacteria with no treatment. However, this difference could be insufficient to completely inhibit DNA amplification in the PCR from 10(6) cells of dead Enterobacteriaceae bacteria/mL, potentially contaminated in pasteurized milk. Actually, such potentially high levels of dead Enterobacteriaceae cells in milk has prevented milk researchers from applying PMA- or ethidium monoazide PCR to the assay of viable Enterobacteriaceae cells in milk. We, therefore, developed a rapid PMA real-time PCR whose minimum levels of detection were 1.5 log cfu/PCR for Cronobacter muytjensii and Escherichia coli, and 2.5 log cfu/PCR for Salmonella enteritidis without DNA purification in milk matrices. The PMA real-time PCR allowed us to specifically detect viable Enterobacteriaceae cells (5-10 cfu/mL) in pasteurized milk (20 mL) within 7.5h of total testing time, following the hygienic guidelines for pasteurized milk in the United States and European Union. The long DNA amplification (mainly 2,451 bp) of the 16S-23S rRNA gene was completely suppressed in highly contaminated dead Enterobacteriaceae cells (7.5 log cfu of Cronobacter muytjensii) in 20 mL of pasteurized milk by 23-μM PMA treatment. Although the contamination of the PCR reaction with 5% milk usually causes great inhibition, our method led to the successful elongation of PCR from viable Enterobacteriaceae cells still in the pasteurized milk matrices finally corresponding to 2 to 4 mL of milk PCR inhibitors without a DNA purification step. To comply with current customer demands for chilled pasteurized milk at the most excellent possible quality, our new technique could enable laboratory persons in a factory to conduct rapid milk coliform testing before shipping from a factory.     

Mixed extracts of green tea and orange peel encapsulated and impregnated on black tea bag paper to be used as a functional drink

Herbal drinks have been considered among the known groups of functional drinks. The aim of this study was to encapsulate green tea and orange peel extracts using double emulsion followed by complex coacervation to be used in the preparation of a functional drink. All of the microcapsule formulations were spherically prepared without cracks in the walls and their sizes increased by decreasing the ratio of the core to the wall. Microcapsules under the best conditions of this study were used to coat the paper of a bag containing black tea. The results showed that theaflavin and its ratio to thearubigin as the quality indices for black tea have increased in comparison with the sample without encapsulation. This study resulted in the production of encapsulated mixed extracts of green tea and orange peel that can be used later for the preparation of a functional drink.     

奶茶的制作研究

对奶茶的制作进行了研究，根据主料茶叶、牛奶搭配组合的情况，确定使用纯牛奶制作奶茶较好，再根据奶茶的特性、颜色、滋味等，选择适合的辅料进行搭配，最后加上装饰与点缀，制作了8款能表现各种茶叶特性，并符合现代人口味，具有营养保健功能和市场发展潜力的奶茶。     

橙汁的果汁含量测定及与其6种特征指标相关性的比较

测定了19个橙汁样品的果汁含量,并比较了GB/T16771-1997《橙、柑、桔汁及其饮料中果汁含量的测定》中所规定的6个特征指标(钾、总磷、氨基酸态氮、L-脯氨酸、D-异柠檬酸、总黄酮)含量与果汁含量的相关性。结果表明,6个特征指标的含量和果汁含量都具有不同程度的相关性,其中钾含量与果汁含量相关性最大,相关系数达0.6873;氨基酸态氮含量与果汁含量相关性最小,相关系数为0.3768,但均未达到显著相关水平。     

Enterococcus and Lactobacillus contamination of raw milk in a farm dairy environment

Enterococci and lactobacilli are ubiquitously found in the intestinal microflora of humans and animals. The aim of the present study was to determine the importance of bovine faeces as a source of these organisms in raw milk. One hundred and fifty six putative enterococci and 362 lactobacilli were isolated from bovine faeces (n=26), cows' teats, raw milk, the milking machine and the milking environment on one farm. The clonal relationships of each group were investigated using Pulsed-Field Gel Electrophoresis and representatives of the different clusters were identified by repetitive DNA element (rep)-PCR fingerprinting, protein profiling, phenylalanyl-tRNA synthase (pheS) sequence analysis or 16S rDNA gene sequencing. Lactobacilli were present at approximately 3 orders of magnitude greater than enterococci in the bovine faeces. The majority of the bovine faecal enterococcal isolates were identified as Aerococcus viridans. Seven teat isolates belonged to a potential novel Aerococcus sp. and one bovine faecal isolate to a potential second novel Aerococcus sp. The lactobacilli present in the bovine faeces were predominantly Lactobacillus mucosae and Lactobacillus brevis, with small numbers of Lactobacillus plantarum. Only one Enterococcus (a strain of E. casseliflavus) out of 76 and one Lactobacillus (a strain of L. parabuchneri/kefir) out of 247 of the bovine faecal isolates was found in the milk. The major source of these bacteria in the milk was the milking equipment.