A new putative Zygosaccharomyces yeast species isolated from traditional balsamic vinegar

The taxonomic status and species number of the genus Zygosaccharomyces have rapidly changed in the last years. In this study, two new osmotolerant Zygosaccharomyces strains isolated from traditional balsamic vinegar, viz. ABT301 and ABT601, were investigated to elucidate their taxonomic relationships with Zygosaccharomyces rouxii species. A multi-gene sequence approach was employed, including regions of the rDNA repeat [5.8S, two internal transcribed spacers (ITS) and the 26S D1/D2 domain], COX2 mitochondrial gene and two nuclear genes (SOD2 and HIS3). Cloning and sequence analysis of 5.8S-ITS rDNA revealed that these strains bear an unusual polymorphism for this region. Three highly divergent 5.8S-ITS sequences were detected, one identical to Z. rouxii, the other two showing some relatedness to Z. mellis. Sequence and gene number polymorphism was also observed for the protein-encoding nuclear genes SOD2 and HIS3, as two copies for each gene different from those found in Z. rouxii were detected. Analysis of the D1/D2 26S domain showed that ABT301 and ABT601 have only one type of D1/D2 sequence statistically different from that of Z. rouxii. The findings obtained in this work suggest that the genomic background of strains ABT301 and ABT601 is different from the other Zygosaccharomyces species. We speculated that they could belong to a new putative species related to Z. rouxii.     

基于Illumina MiSeq测序技术分析大鲵肉冷藏过程中微生物菌群演替规律

为探究大鲵肉冷藏过程中菌相组成及特定腐败微生物,对托盘包装大鲵肉不同冷藏时间（4 ℃,0、2、4、6、8 d）的挥发性盐基氮和菌落总数进行评价,在此基础上采用Illumina MiSeq测序技术探究其微生物菌群变化与多样性。结果表明,大鲵肉在冷藏过程中菌落总数和挥发性盐基氮呈上升趋势,分别在第6天和第8天超标。高通量测序结果显示,大鲵肉中微生物丰度随着冷藏时间的延长呈降低趋势。在门水平上进行群落组成分析,细菌的优势菌门从冷藏前期（0、2 d）的拟杆菌门、厚壁菌门逐渐转变为中（4 d）、后期（6、8 d）的变形菌门。在属水平上细菌的优势菌属从冷藏前期主要的拟杆菌属、Faecalibacterium逐渐转变为中、后期的假单胞菌属、气单胞菌属、Hafnia-Obesumbacterium、沙雷氏菌属。主坐标分析显示0-2、4、6、8 d之间微生物菌群差异较大,2 个主坐标叠加解释度达80.92%;LEfSe分析表明,引起大鲵肉各冷藏期差异显著的菌门主要为变形菌门、放线菌门、拟杆菌门、纤维杆菌门、厚壁菌门,菌属主要为假黄单胞菌属、Bauldia、沙雷氏菌属、不动杆菌属、气单胞菌属、假单胞菌属、ambiguous_taxa、Hafnia-Obesumbacterium、Rikenellaceae_RC9_gut_group、Prevotella_9、拟杆菌属、纤维杆菌属、毛螺菌属、Faecalibacterium、Clostridium_sensu_stricto_1。进化分析表明大鲵肉冷藏过程中微生物菌群演替与冷藏时间具有较强相关性。综合分析,引起冷藏期间大鲵肉腐败变质的微生物主要为假单胞菌属、气单胞菌属、Hafnia-Obesumbacterium和沙雷氏菌属。该研究为今后大鲵肉冷藏过程中靶向抑菌及货架期延长提供了参考。     

产琼胶酶菌株Agarivorans sp.FG15的筛选鉴定与酶学性质

从中国南海热带海洋环境中分离纯化出产琼胶酶菌株FG15,并以16S rDNA进行分子鉴定,分析其酶学性质。FG15为革兰氏阴性球菌,对硫酸链霉素,氨苄青霉素,羧苄青霉素和氯霉素都不敏感。通过16S rDNA序列分析和BLAST同源性比对发现:FG15菌株的16S rDNA序列与船蛆杆菌(Teredinibacter turnerae),噬琼胶菌属(Agarivorans sp.)对应序列同源性最高,为95%。利用MEGA 5.0软件构建系统发育树,FG15与噬琼胶菌属(Agarivorans sp.)亲缘关系最近,同源性高达99%。因此,可以初步鉴定FG15为噬琼胶菌属(Agarivorans sp.)。酶学性质的研究表明:FG15所产琼胶酶的最适温度为36℃,在20~45℃之间热稳定性较好;最适pH为7.5,在pH 7.0~8.0之间酸碱稳定性较好;产酶主要为胞外酶;琼脂酶的动力学参数米氏常数Km为4.978mg/mL,最大反应速率Vmax为10.33μmol/(L·min)。     

微生物亚硝化抑制剂对风干肠发酵成熟期间微生物菌落变化的影响及其感官评价分析

为研究微生物亚硝化抑制剂（microbial nitrosation inhibitor,MNI）对风干肠发酵和成熟过程中微生物群落动态变化及感官特性的影响,该试验设计4组风干肠,MNI组：添加MNI;MNIP组：添加MNI并接入PRO-MIX5商业发酵剂（木糖葡萄球菌、清酒乳杆菌、类植物乳杆菌）;FBFAP组：发酵牛骨调味基料和复配抗氧化剂（fermented beef flavorings and compound antioxidant,FBFA）并接入PRO-MIX5;CK组。结果表明：在发酵阶段,4组风干肠乳酸菌数逐渐增多,MNI组和MNIP肉馅分别在发酵的15 h、12.5 h达到发酵终点即pH降到5.4~5.6,乳酸菌数分别为8.99 lg cfu/g、8.91 lg cfu/g,显著高于CK组发酵终点（22 h）的乳酸菌数8.88 lg cfu/g（p<0.05）。到成熟终点时,革兰氏阴性腐败微生物在MNI组的相对丰度占比最低（19.35%）,对肠杆菌科和假单胞菌的抑制率分别为30.43%、27.22%,仅次于MNIP组37.00%、33.79%,优于CK组和FBFAP组。综合分析,MNI能作为促生长因子,促进肉馅中乳酸杆菌属菌的生长,在接种有发酵剂PRO-MIX5的风干肠中加入MNI对腐败微生物的抑制效果优于FBFAP。单独添加MNI或MNI与PRO-MIX5协同作用均能提高风干肠微生物安全性和感官性能,说明MNI在提高风干肠安全品质方面具有广阔的应用前景。     

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.     

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。     

产4-羟基-2,5-二甲基-3(2H)-呋喃酮酵母菌的分离、筛选及分子生物学鉴定

以菠萝、草莓等水果为分离源,分离、筛选利用D-果糖产香料HDMF酵母菌株,并对其高产菌株进行分子生物学鉴定。采用稀释涂布法分离酵母菌株,高效液相色谱法检测HDMF产量,26SrDNAD1/D2区序列分析及系统发育分析鉴定菌株。共分离得到46株酵母,5株可利用D-果糖产HDMF;产量较高的两株菌为:C5(6.84mg/L)、P3(10.96mg/L),分别约为已报道毕赤氏酵母属(Pichia capsulata)利用L-(+)-鼠李糖产HDMF的3倍和5倍;26S rDNA D1/D2区序列分析及系统发育分析结果显示,P3与Pichia caribbica(毕赤酵母属),C5与Hanseniaspora sp.(有孢汉逊酵母属)相似性均在99%以上,分子生物学法鉴定P3为Pichia caribbica,C5为Hanseniaspora sp.     

一株乳酸菌的鉴定及其在酸豆乳加工中的应用

对从酸菜汁中分离到的一株乳酸菌菌株L4进行鉴定,通过传统的形态学及生理生化特性分析,初步确定菌株L4为乳酸杆菌属(Lactobacillus)。通过序列比对及构建系统发育树,表明L4与植物乳杆菌(Lactobacillus plantarum)相似性在90%以上,鉴定菌株L4为植物乳杆菌。对菌株L4在豆乳中发酵的酸化能力、后酸化能力及耐受酸能力进行了研究。结果表明,该菌株在豆乳中37℃发酵,15h酸度值为58°T左右;用此菌株发酵的豆乳在4℃贮藏期间也很稳定,20d内酸度变化范围极小,约为4°T;耐受酸能力较强,在pH值为3.0条件下处理120min,菌数存活率为91.2%。     

绿衣观音土曲细菌DNA的提取

采用土壤DNA提取试剂盒和SDS-酶裂解法分别提取观音土曲的细菌DNA。基于SDS-酶法裂解原理的提取方法获得的结果比较理想,此方法获得的基因组片段大于15kb;A260/A280为1.82,A260/A230为1.54,DNA纯度很高;提取的DNA不经纯化能直接用于细菌16S rDNA的扩增。该法提取的DNA能直接用于后续的分子生物学实验操作。     

16S rDNA序列关键位点的信息论分析

16S rDNA中含有较多的菌种特异性信息,但是如何使用这些信息进行菌种鉴定却因不同菌种间序列的特征变化缺少规律而变得困难。作者使用信息论方法对那些种内较保守,种间变异明显的关键位点进行研究。结果表明,16S rDNA序列中仅有少数位点对菌种的分类是重要的。基于少数关键位点的相似度比传统的全序列鉴定有着更好的统计学特性。