Predominance of Tetragenococcus halophilus as the cause of sugar thick juice degradation

The industrial storage of sugar thick juice was simulated on a laboratory scale. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis and the application of Clone Libraries in parallel with classical microbiology were used to study the bacterial diversity and all revealed a dominance (>99%) of Tetragenococcus halophilus during storage. The degradation of thick juice correlated with the appearance of L-lactic acid and high concentrations of T. halophilus. In addition, pure cultures of T. halophilus induced degradation of sterile thick juice. A specific PCR was developed to detect T. halophilus and industrial thick juice samples from Belgium, Germany and France all contained T. halophilus, suggesting a consistent association of this organism with thick juice. T. halophilus has been known only as a halophile thus far, and this report is the first to show an association of this organism with a sugar-rich environment.     

泸型酒酿造期间酒醅中可培养细菌的分离鉴定及其种群演替规律研究

目的:为了系统的探究泸型酒酿造期间酒醅中细菌的多样性及演替变化规律,尽可能多的得到各时期可培养的占主体地位的细菌。方法:选用三种不同的培养基对各时期的酒醅样品进行分离计数,并通过16S rRNA基因序列分析法对分离得到的纯菌进行鉴定和系统发育分析。结果:分离得到258株细菌,其中250株分别属于29个种,其余8株鉴定到属(芽孢杆菌属)。在发酵过程中占优势的主体细菌主要为芽孢杆菌属,乳酸菌属细菌在发酵中期也较多。结论:泸型酒发酵过程中细菌的多样性很丰富,发酵前期细菌种类比较多,中期主要以乳酸菌属和芽孢杆菌属为主,后期芽孢杆菌属为主体。可以推测出乳酸菌属和芽孢杆菌属细菌对泸型酒的酿造形成具有特殊意义。      

Dynamic changes in the bacterial community in Moutai liquor fermentation process characterized by deep sequencing

Microbes are the major producers of alcohol and aromatic compounds in alcohol beverages. It is believed that, in addition to production techniques and grains, different micro‐ingredient compositions yield variations in aromas and tastes. The bacterial communities of the three key phases from Moutai liquor production were profiled with the Illumina sequencing platform. A total of 54 bacterial families were detected and the microbial structure showed clear differences among samples. In the starter‐making phase, Leuconostocaceae and Enterococcaceae dominated the shaping stage, and Bacillaceae dominated the mature starter and ripening stages. In the latter two phases, Bacillaceae and Lactobacillales dominated in the early stage of stack fermentation and the anaerobic stage of pit fermentation, respectively. Most of the important microbes (approximately 26 families) might have originated from the starter and greatly affected the bacterial structure of the samples during the early stages of stack fermentation. Production techniques and environmental factors, such as temperature and oxygen, also shaped the unique bacterial composition during different phases. Copyright © 2015 The Institute of Brewing & Distilling     

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.     

应用16S rDNA全序列测序技术鉴定生鲜乳中的细菌种类

采用玻璃珠法提取生鲜乳中分离的34株细菌基因组DNA,用细菌通用引物(27f/1492r)PCR扩增16S r DNA片段并测序,与Gen Bank数据库同源序列比对,系统发育树聚类分析,并结合形态学和生理学特征确定了34株菌的种属地位。结果表明,共鉴定出16个种属。3个生鲜乳样中细菌种类及其分布特点不同。其中1号样有7种细菌,以Acinetobacter sp.(不动杆菌属)为主,2号样有6种细菌,以Klebsiella sp.(克雷贝氏杆菌属)为主,3号样有6种细菌,分布较为分散。从致病性来看,分离菌株主要为条件致病菌或腐败菌,无烈性致病菌。      

Effects of lipopolysaccharide dosing on bacterial community composition and fermentation in a dual-flow continuous culture system

The objectives of this study were to evaluate the effects of lipopolysaccharide (LPS) dosing on bacterial fermentation and bacterial community composition (BCC), to set up a subacute ruminal acidosis (SARA) nutritional model in vitro, and to determine the best sampling time for LPS dosing in a dual-flow continuous culture system. Diets were randomly assigned to 6 fermentors in a replicated 3 × 3 Latin square with three 11-d experimental periods that consisted of 7 d for diet adaptation and 4 d for sample collection. Treatments were control diet (CON), wheat and barley diet (WBD) to induce SARA, and control diet + LPS (LPSD). Fermenters were fed 72 g of dry matter/d. The forage:concentrate ratio of CON was 65:35. The WBD diet was achieved by replacing 40% of dry matter of the CON diet with 50% ground wheat and 50% ground barley. The LPS concentration in LPSD was 200,000 endotoxin units, which was similar to that observed in cows with SARA. The SARA inducing and LPS dosing started at d 8. The BCC was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform (Illumina Inc., San Diego, CA). The LPSD and CON maintained pH above 6 for the entire experimental period, and the WBD kept pH between 5.2 and 5.6 for 4 h/d, successfully inducing SARA. Digestibility of neutral detergent fiber and crude protein in LPSD were not different from WBD but tended to be lower than CON. Lipopolysaccharide dosing had no effect on pool of VFA concentrations and profiles but decreased bacterial N; the pattern changes of VFA and LPS in LPSD started to increase and be similar to WBD 6 h after LPS dosing. Pool of LPS concentration was around 11-fold higher in WBD and 4-fold higher in LPSD than CON. In the solid fraction, the BCC of LPSD was different from WBD and tended to be different from CON. In the liquid fraction, the BCC was different among treatments. The LPS dosing increased the relative abundance of Succinimonas, Anaeroplasma, Succinivibrio, Succiniclasticum, and Ruminobacter, which are main gram-negative bacteria related to starch digestion. Our results suggest that LPS dosing does not affect pH alone. However, LPS could drive the development of SARA by affecting bacteria and bacterial fermentation. For future studies, samples are suggested to be taken 6 h after LPS dosing in a dual-flow continuous culture system.     

清香型大曲细菌群落结构的比较分析

通过构建细菌16SrRNA基因文库、RFLP指纹图谱分析、测序和系统发育学分析,分别对汾酒清茬曲、后火曲和红心曲的细菌群落结构进行比较。结果表明,清香型3种大曲细菌均分属于芽孢杆菌纲、放线菌纲及变形菌纲,但具体种属组成存在一定差异,其中清茬曲的优势细菌菌群为乳杆菌属和葡萄球菌属(占全部克隆子的比例分别为37.78%,22.22%);后火曲的优势细菌菌群为乳杆菌属、葡萄球菌属、芽孢杆菌属、魏斯氏菌属(占全部克隆子的比例分别为35%,16.67%%,16.67%,13.33%);红心曲的优势细菌菌群为乳杆菌属、芽孢杆菌属、高温放线菌属(占全部克隆子的比例分别为26.67%,28.89%,17.78%)。     

内蒙古酸马奶中乳酸菌多样性的研究

采用16S rRNA基因全序列测定和聚类分析技术,对酸马奶中的乳酸菌进行了准确鉴定并构建了乳酸菌的系统发育树.然后对乳酸菌菌群进行了多样性分析,结果显示,酸马奶中的优势乳酸菌分别为:Lactobacillus plantarum(10%),Lactobacillus brevis(8%),Lactobacillus casei(7.8%),Enterococcus faecium(17%),Enterococcus faecalis(14%),Lactococcuslactis(19%),Lactobacillus acidlophilus(5%),Lactobacillus paracasei(2%),Lactobacillus delbrueckii subsp.bulgaricus(4%),Lactobacillus helveticus(4%),Enterococcus durans(4%),Leuconostoc mesenteroides (4%),Leuconostoc garlicum(1%),Streptococcus thermophilus(1%).     

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses

BACKGROUND: Fermented rice flour (khao‐khab, a non‐glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao‐khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty‐five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S–23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product. Copyright © 2011 Society of Chemical Industry     

Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia

Spontaneous milk fermentation has a long history in Mongolia, and beneficial microorganisms have been handed down from one generation to the next for use in fermented dairy products. The objective of this study was to investigate the diversity of lactic acid bacteria (LAB) communities in fermented yak, mare, goat, and cow milk products by analyzing 189 samples collected from 13 different regions in Mongolia. The LAB counts in these samples varied from 3.41 to 9.03 log cfu/mL. Fermented yak and mare milks had almost identical mean numbers of LAB, which were significantly higher than those in fermented goat milk but slightly lower than those in fermented cow milk. In total, 668 isolates were obtained from these samples using de Man, Rogosa, and Sharpe agar and M17 agar. Each isolate was considered to be presumptive LAB based on gram-positive and catalase-negative properties, and was identified at the species level by 16S rRNA gene sequencing, multiplex PCR assay, and restriction fragment length polymorphism analysis. All isolates from Mongolian dairy products were accurately identified as Enterococcus faecalis (1 strain), Enterococcus durans (3 strains), Lactobacillus brevis (3 strains), Lactobacillus buchneri (2 strains), Lactobacillus casei (16 strains), Lactobacillus delbrueckii ssp. bulgaricus (142 strains), Lactobacillus diolivorans (17 strains), Lactobacillus fermentum (42 strains), Lactobacillus helveticus (183 strains), Lactobacillus kefiri (6 strains), Lactobacillus plantarum ssp. plantarum (7 strains), Lactococcus lactis ssp. lactis (7 strains), Leuconostoc lactis (22 strains), Leuconostoc mesenteroides (21 strains), Streptococcus thermophilus (195 strains), and Weissella cibaria (1 strain). The predominant LAB were Strep. thermophilus and Lb. helveticus, which were isolated from all sampling sites. The results demonstrate that traditional fermented dairy products from different regions of Mongolia have complex compositions of LAB species. Such diversity of LAB provides useful information for further studies of probiotic strain selection and starter culture design, with regard to the industrial production of traditional fermented milk.     

Characterization of the microflora of the human axilla

It is widely accepted that axillary malodour is attributable to the microbial biotransformation of odourless, natural secretions into volatile odorous products. Consequently, there is a need to understand the microbial ecology of the axilla in order that deodorant products, which control microbial action in this region, can be developed in the appropriate manner. A detailed characterization of the axillary microflora of a group of human volunteers has been performed. The axillary microflora is composed of four principal groups of bacteria (staphylococci, aerobic coryneforms, micrococci and propionibacteria), and the yeast genus Malassezia. Results indicated that the axillary microflora was dominated by either staphylococcal or aerobic coryneform species. Comparisons between axillary bacterial numbers and levels of axillary odour demonstrated the greatest association between odour levels and the presence of aerobic coryneforms in the under-arm. As the taxonomy of cutaneous aerobic coryneforms is poorly understood, a further study was conducted to characterize selected axillary aerobic coryneform isolates. Using the molecular technique of 16S rDNA sequencing, selected genomic sequences of a number of axillary aerobic coryneform isolates were obtained. Comparisons with sequence databases indicated the likely presence of a range of Corynebacterium species on axillary skin, although the majority of isolates were most similar to either Corynebacterium G-2 CDC G5840 or C. mucifaciens DMMZ 2278. Although for a panel of individuals differences in the carriage of Corynebacterium species were noted, similar species were carried by a number of panellists. All isolates examined in this limited evaluation failed to demonstrate the capability to metabolize long-chain fatty acids (LCFAs) to shorter chain, more volatile products. The application of this modern molecular phylogenetic technique has increased understanding of the diversity of aerobic coryneform carriage in the axilla, and on human skin. The application of this technique in other studies to assess the ethnic differences in cutaneous bacterial ecology, or the effects on the microflora of specific product use, will assist in the future development of novel deodorant systems.     

河北平泉农田土壤放线菌物种多样性筛查

针对河北平泉县典型的农田土壤类型,采用基于全长16S rDNA序列系统发育分析的免培养技术,并结合放线菌门特异引物的筛查,揭示了土壤中放线菌的物种多样性。构建的放线菌16S rRNA克隆文库中的596个放线菌序列归属于29个属,17个科,12个亚目和2个亚纲,并确定了其优势菌属,其中芽球菌属(Blastococcus)(占放线菌总克隆数的12.75%),类诺卡氏菌属(Nocardioides)(9.39%),节杆菌属(Arthrobacter)(8.72%),小月菌属(Microlunatus)(5.37%),酸土单胞菌属Aciditerrimonas(4.69%),沉积岩杆菌属Ilumatobacter(3.35%)。本文为深入理解放线菌的生态功能,及更深层次的放线菌资源的研究、开发和利用提供宝贵的科学资料和丰富的物种信息资源。      

Assessment of rumen bacteria in dairy cows with varied milk protein yield

The present study was conducted to assess rumen bacteria in lactating cows with different milk protein yield, aiming to understand the role of rumen bacteria in this trait. Cows with high milk protein yield (high milk yield and high milk protein content, HH; n = 20) and low milk protein yield (low milk yield and low milk protein content, LL; n = 20) were selected from 374 mid-lactation Holstein dairy cows fed a high-grain diet. Measurement of the rumen fermentation products showed that the concentrations of ruminal total volatile fatty acids, propionate, butyrate, and valerate and the proportion of isobutyrate were higher in the HH cows than in the LL cows. Amplicon sequencing analysis of the rumen bacterial community revealed that the richness (Chao 1 index) of rumen microbiota was higher in the LL cows than in the HH cows. Among the 10 predominant bacterial phyla (relative abundance being >0.10%, present in >60% of animals within each group), the relative abundance of Proteobacteria was 1.36-fold higher in the HH cows than in the LL cows. At the genus level, the relative abundance of Succinivibrio was significantly higher and that of Clostridium tended to be higher in the LL cows than in the HH cows. Sharpea was 2.28-fold enriched in the HH cows compared with the LL cows. Different relationships between the relative abundances of rumen microbial taxa and volatile fatty acid concentrations were observed in the HH and the LL animals, respectively. Succinivibrio and Prevotella were positively correlated with acetate, propionate, and valerate in the LL cows, whereas Sharpea was positively correlated with propionate and valerate concentrations in the HH cows. Collectively, our results revealed that rumen bacterial richness and the relative abundances of several bacterial taxa significantly differed between dairy cows with high and low milk protein yields, suggesting the potential roles of rumen microbiota contributing to milk protein yield in dairy cows.     

德宏水牛乳饼中乳酸菌的分离鉴定及发酵性能研究

德宏水牛奶乳饼是云南特有的乳制品,微生物资源丰富,是乳酸菌的重要来源,也是分离筛选优良发酵剂的天然基础。本文采用16S rRNA基因序列分析法和纯培养法对15份水牛奶乳饼中乳酸菌种属进行分离鉴定,通过对菌株产酸、产香、发酵乳活菌计数等比较,以筛选出具有优良发酵特性的乳酸菌。结果表明:15份水牛奶乳饼样品中共分离鉴定出57株乳酸菌（3个属,6个种和1个亚种）,其中Lactobacillus fermentum和Lactobacillus oris为优势菌属,占总菌属的36.84%和24.56%。57株乳酸菌发酵特性比较后得到两株优势菌株MGR3-1和MBR1-1。此研究为后续开发和应用优良发酵剂提供理论基础。      

Molecular detection of bacteria in calcium carbonate powder used in cosmetic formulations

Given that a variety of bacterial species may occur in the calcium carbonate powder used for cosmetic formulations, an understanding of their diversity and abundance is necessary to accurately assess the contamination of the finished product. 16S rRNA was PCR-amplified from genomic DNA extracted from three different calcium carbonate powder grades, and these amplicon libraries were sequenced using deep amplicon sequencing technology. The resulting libraries contained 4149-6688 16S rRNA reads per sample with a length of 327-342 bp. Classification into genus of pyrosequencing reads of the dominant bacterial species found in calcium carbonate powders was used to confirm the absence of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella spp. and Escherichia coli. The analysis described here can be used to determine the microbial diversity of calcium carbonate powder or the presence of any 'indicator microorganisms' in raw materials as well as in cosmetic products. This work provides guidance for prioritizing subsequent culturable and quantitative analysis, ensuring that potentially significant microorganisms are not left out of risk estimations.     

Molecular identification of mesophilic and psychrotrophic bacteria from raw cow's milk

The aim of this study was to use molecular techniques to assess the microbiota of eight raw cow's milk samples at biotype and species level. Sixty-six isolates from raw milk samples were screened by Randomly amplified polymorphic DNA–PCR (RAPD–PCR) biotyping and representative strains of RAPD–PCR profiles were identified by 16S rRNA gene sequencing. Pseudomonas spp. were the most commonly occurring contaminants along with Enterobacteriaceae such as Hafnia alvei, Serratia marcescens and Citrobacter freundii. Moreover, Gram-positive isolates belonging to the genera Staphylococcus and Lactococcus were also found. Experiments of growth at different temperatures showed that more than 50% of the Gram-negative isolates could grow at chill temperatures and that 65% of the Pseudomonas spp. strains grew at 7 °C within 5 days. Only 13 Gram-negative isolates displayed proteolytic activity on milk agar, suggesting that not all the biotypes of milk contaminating species are able to perform this spoilage-associated activity. Among the Gram negative, the proteolytic strains were mainly Peudomonas spp. that displayed the activity at both 7 °C and 20 °C. A reliable molecular identification of raw milk microbiota is important for the study of the microbiological quality of raw milks and for the assessment of the ecology at species level in order to develop improved systems, preventing contamination and having the best conditions for the storage of milk.     

16S rDNA克隆文库法分析生鲜牛乳中 细菌种群的多样性

目的 利用16S rDNA基因文库分析法, 对生鲜牛乳中微生物的种群多样性进行研究。方法 提取生鲜牛乳中总微生物基因组DNA为模板, 将扩增到的生鲜牛乳样品的16S rDNA的PCR产物与pMD~18T载体连接, 构建其菌群的16S rDNA文库。对随机选取的56个阳性克隆子进行序列测定和BLAST比对。结果 文库序列分析表明, 有53个克隆子分属3个不同的类群, 即厚壁菌类群、变形菌类群和异常球菌-栖热菌类群, 其余3个克隆子属于未知类群。其中, 优势细菌类群为厚壁菌类群(86%), 在该类群中, 尤以无乳链球菌为主要代表, 约占所有克隆子的82%; 其他类群为γ~变形菌类群(7.1%)和异常球菌-栖热菌门类群(1.8%)。系统发育分析表明, 未知类群在分类地位上更接近与厚壁菌类群。结论 生鲜牛乳中微生物具有多样性, 无乳链球菌为其中的优势种群, 同时生鲜牛乳中还存在葡萄球菌等致病菌, 或不动杆菌和肠道菌等条件致病菌。