Lactic acid bacteria dynamics during spontaneous fermentation of cocoa beans verified by culture-independent denaturing gradient gel electrophoresis

Cocoa is naturally fermented in the field before the cocoa seeds are removed for processing. We assessed the dynamics of lactic acid bacteria during cocoa fermentation in Bahia, Brazil. During five days of fermentation, temperature and pH were measured and beans were collected for genomic DNA extraction every 12 h. The DNA was used as a template for amplification with Lac1-Lac2 and Lac3-Lac2 for denaturing gradient gel electrophoresis analyses. pH values ranged from 3.34 to 4.98, while the temperature varied from 23° to 50°C. Lac1-Lac2 primers permitted detection of 11 operational taxonomic units. Twenty-eight operational taxonomic units were obtained with the primer pair Lac3-Lac2. It was observed that there were variations between the numbers of operational taxonomic units throughout the process, probably because of changes in pH and temperature. The greatest similarity in amplified samples was obtained with the primers Lac3-Lac2.     

Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.     

变性梯度凝胶电泳（DGGE）在微生物多样性中的研究

变性梯度凝胶电泳（DGGE）技术能够再现微生物菌群多样性,从而获得微生物的动态信息,并解决了传统方法的片面性,在分析环境微生物群落多样性方面的应用发展迅速。通过对DGGE图谱的分析,可获得待测样品的生物信息。因此充分了解DGGE技术在微生态领域的利用,有助于检测和分析丰富的微生物多样性结构,提高微生物资源开发。     

Detection by denaturing gradient gel electrophoresis of ammonia-oxidizing bacteria in microcosms of crude oil-contaminated mangrove sediments

Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay.     

Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing

This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol–chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae.     

Microbial community analysis in crab ponds by denaturing gradient gel electrophoresis

In order to investigate the role of microbial community in aquatic ecology and biogeochemical cycles, the bacterial community in crab ponds was investigated and the effects of aeration and season on the bacterial community were also assessed. Total DNAs from the water samples were amplified with universal primers and the amplicons were then resolved by denaturing gradient gel electrophoresis. Bands from the resulting profiles were excised and sequenced. Cluster analysis of the resulting profiles showed that the microbial community was affected by aeration and season. The microbial community between the surface and bottom of the water was very similar. A total of fifteen bands were obtained in this study. Three of them were 91–99% similar to uncultured bacterium clones. Three were 95–99% similar to uncultured Verrucomicrobia bacteria. Three were 97–100% similar to Actinobacterium sp.. Two were similar to Candidatus Limnoluna rubra with similarity 96 and 99%, respectively. Four were 99% similar to Rhodococcus sp., 100% similar to Sporosarcina sp., 100% similar to Stenotrophomonas sp., and 98% similar to Hydrogenophaga sp., respectively. The concentrations of dissolved oxygen, total nitrogen, total phosphorous, nitrite, and ammonia and pH values were significantly affected by season while only the pH value and the concentrations of dissolved oxygen and total nitrogen were significantly affected by aeration.     

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis

Aims: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9 × 10⁶ CFU LAB g^?1) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1 : 1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8 h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat‐to‐goat variations did not affect the LAB communities found in the rumen fluid. Conclusion: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.     

Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology

Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important.     

用变性梯度凝胶电泳分析PCR克隆的突变率

用变性梯度凝胶电泳技术比较分析了分别由Taq DNA聚合酶和Pfu DNA聚合酶催化扩增的产物克隆入pUCm-T/DH5a系统中产生的重组子,发现包含突变的重组子分别为21．50％和3．50％,前为后的6．15倍。转化为每100nt净扩增长度的突变率分别为7．44％和1．21％。     

Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis.

In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence of each fragment. Analysis of the DNA fragment distribution as a function of fragment size allows the DNA size to be calculated. This method has been applied to calculate three microbial genome sizes: Mycoplasma capricolum, 724 kb; Acholeplasma laidlawii, 1646 kb; and Hemophilus influenzae, 1833 kb.     

Detection of mutations in GC-rich DNA by bisulphite denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) in combination with PCR and 'GC-clamping' has proven highly efficient as a method for detection of DNA sequence differences. Due to strand dissociation phenomena, however, its use has been limited to the analysis of sequences with a relatively low content of GC pairs. This paper describes how treatment of template DNA with sodium bisulphite drastically lowers the melting temperature of very GC-rich sequences and renders them amenable to DGGE analysis. We demonstrate the use of bisulphite DGGE for rapid and efficient detection of mutations in the p16(INK4/CDKN2) tumour suppressor gene.     

Online tool for analysis of denaturing gradient gel electrophoresis profiles

We present an online tool (EquiBands, http://www.univie.ac.at/IECB/limno/equibands/EquiBands.html) that quantifies the matching of two bands considered to be the same in different samples, even when samples are applied to different denaturing gradient gel electrophoresis gels. With an environmental example we demonstrate the procedure for the classification of two bands of different samples with the help of EquiBands.     

Detection by denaturing gradient gel electrophoresis of a new polymorphism in the apolipoprotein B gene

Summary The apolipoprotein B gene is subject to mutations that may be important in coronary heart diseases. We have used polymerase chain reaction and denaturin gradient gel electrophoresis to characterize a single nucleotide substitution in the apolipoprotein B gene. This mutation affects amino acid 4311 of the protein and converts asparagine to serine. It was found in 24% of the 81 unrelated individuals analyzed. Moreover, another mutation was detected by sequencing in a single individual.     

Microbial succession during a composting process as evaluated by denaturing gradient gel electrophoresis analysis

Microbial succession during a laboratory-scale composting process of garbage was analysed by denaturing gradient gel electrophoresis (DGGE) combined with measurement of physicochemical parameters such as temperature, pH, organic acids, total dissolved organic carbon and water-soluble humic substance. From the temperature changes, a rapid increase from 25 to 58 degrees C and then a gradual decrease, four phases were recognized in the process as follows; mesophilic (S), thermophilic (T), cooling (C) and maturing (M). The polymerase chain reaction-amplified 16S rDNA fragments with universal (907R) and eubacterial (341F with GC clamp) primers were subjected to DGGE analysis. Consequently, the DGGE band pattern changed during the composting process. The direct sequences from DGGE bands were related to those of known genera in the DNA database. The microbial succession determined by DGGE was summarized as follows: in the S phase some fermenting bacteria, such as lactobacillus, were present with the existing organic acids; in the T phase thermophilic bacillus appeared and, after the C phase, bacterial populations were more complex than in previous phases and the phylogenetic positions of those populations were relatively distant from strains so far in the DNA database. Thus, the DGGE method is useful to reveal microbial succession during a composting process.     

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.     

GC fractionation enhances microbial community diversity assessment and detection of minority populations of bacteria by denaturing gradient gel electrophoresis

Effectively and accurately assessing total microbial community diversity is one of the primary challenges in modern microbial ecology. This is particularly true with regard to the detection and characterization of unculturable populations and those present only in low abundance. We report a novel strategy, GC fractionation combined with denaturing gradient gel electrophoresis (GC-DGGE), which combines mechanistically different community analysis approaches to enhance assessment of microbial community diversity and detection of minority populations of microbes. This approach employs GC fractionation as an initial step to reduce the complexity of the community in each fraction. This reduced complexity facilitates subsequent detection of diversity in individual fractions. DGGE analysis of individual fractions revealed bands that were undetected or only poorly represented when total bacterial community DNA was analyzed. Also, directed cloning and sequencing of individual bands from DGGE lanes corresponding to individual G+C fractions allowed detection of numerous phylotypes that were not recovered using a traditional random cloning and sequencing approach.     

Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.