Identification and Distribution of Bacillus Species in Doenjang by Whole-Cell Protein Patterns and 16S rRNA Gene Sequence Analysis
ABSTRACT Many bacteria are involved in fermentation of doenjang and Bacillus species are known to perform significant roles. Although the SDS-PAGE technique has been frequently used for classification and identification of bacteria in various samples, there has been no investigation of the microbial diversity in doenjang. This study aims to investigate the identification and distribution of dominant Bacillus species in doenjang using SDS-PAGE profiles of whole cell proteins and 16S rDNA sequencing. SDS-PAGE of whole cell proteins of the reference Bacillus strains yielded differential banding patterns that could be considered to be highly specific fingerprints. Bacterial strains isolated from doenjang samples were grouped using whole cell protein patterns, which were confirmed by the analysis of 16S rDNA sequencing. B. subtilis was found to be the most dominant strain in most of the samples, and B. licheniformis and B. amyloliquefaciens were less frequently detected. The results obtained in this study showed that a combined identification method, SDS-PAGE patterns of whole cell proteins and subsequent 16S rDNA sequence analysis, could successfully identify Bacillus species isolated from doenjang.
- SourceAvailable from: Maria Anita Mendes
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- "These methods are time-consuming and do not provide an insight into the physiological properties of the microorganism.    Koichi Tanaka, the winner of the Nobel Prize in Chemistry in 2002, developed a method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry "
ABSTRACT: RATIONALEDue to increases in greenhouse gas emissions, it is necessary to explore renewable sources of energy. Interesting alternatives are biofuels derived from microalgae. One challenge is the development of a detailed microalgae database compiling species identifications and characterizations that would facilitate microalgae selection for biomass production. Mass spectrometric (MS) analysis using a matrix-assisted laser desorption/ionization (MALDI) source is an advanced technique that enables advancement in this biological area. In this work a MALDI time-of-flight (TOF)MS method for the rapid identification of proteins in whole cells of selected microalgae species was studied. Furthermore, the efficiency of different matrix and solvent systems was tested. MS analyses were performed using an UltrafleXtreme MALDI-TOF mass spectrometer operating in linear positive ion mode.METHODS Mass spectra were acquired in a mass range from 4000 to 20,000 Da with ions generated from Smartbeam laser irradiation using a frequency of 2000 Hz, a PIE 100 ns and a lens 7 kV. The voltage was 25 kV for the first ion source and 23 kV for the second. Each spectrum was generated by averaging of 10,000 laser shots and the laser irradiance was set at 95–100%.RESULTSSimilar mass spectra were obtained for all matrices (SA, HCCA, DHB and sDHB); however, the use of the sDHB matrix resulted in spectrum profiles with a greater amount number of proteins, a better signal/noise (S/N) ratio and higher intensities for the majority of microalgae analyzed. Trifluoroacetic acid (TFA) content was also studied and the best results in terms of S/N ratio, number of proteins and signal intensities were obtained with 0.1% TFA in the matrix solvent. The addition of isopropanol did not produce improvement in the quality of spectrum profiles.CONCLUSIONS Therefore, the optimal matrix for the analysis of protein from intact microalgae cells is sDHB with TA50 as the matrix solvent and without isopropanol. These conditions allow the acquisition of high quality spectrum profiles. Copyright © 2014 John Wiley & Sons, Ltd.Rapid Communications in Mass Spectrometry 02/2015; 29(3). DOI:10.1002/rcm.7110 · 2.64 Impact Factor
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- "Cellular protein samples were prepared according to Kim et al. (2010). Flasks (250 ml) each containing 50 ml LB broth were inoculated with the bacterial species and incubated overnight at 20, 25, 30, 35, 40, 45 and 50 @BULLETC by shaking until OD 600 ~0.4, "
ABSTRACT: Three gram positive bacterial isolates, isolated from soil and identified as Bacillus licheniformis, Bacillus circulans and Geobacillus thermoglucosidasius were grown at 20, 35, and 50 o C then subjected to cellular fatty acids analysis. Although in different amounts, the fatty acids (10:0), (12:0), (14:0), (16:0) and (17:0) were detected in cells of the three bacterial isolates obtained from all the incubation temperatures. Increasing temperature from 20 to 50 o C raised the proportion of the saturated fatty acids by 26.10%, 09.89% and 29.61% in B. licheniformis, B. circulans and G. thermoglucosidasius, respectively. Cellular protein contents and protein banding pattern on SDS-PAGE of the three isolates were estimated at 20, 25, 30, 35, 40, 45 and 50 o C. The highest amount of protein concentration in all isolates was obtained at 20ºC. In contrast, the highest number of protein bands was not obtained from these treatments.
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- "Culture-independent microbial techniques, such as PCRdenaturing gradient gel electrophoresis (DGGE) has proven much more reliable, fast, and economical in profiling complex bacterial community structure, dynamics and evolution of microbial populations of fermented foods (Giraffa and Neviani, 2001; Randazzo et al., 2002; Cocolin et al., 2007; Jany and Barbier, 2008). PCR-DGGE has been successfully employed to studying the microbial community structures and dynamics of fermented vegetal protein seeds such as doenjang and meju (Cho and Seo, 2007; Kim et al., 2009; Lee et al., 2010) as found in Asia. More recently, barcoded pyrosequencing of V1/V2 region of 16S rRNA gene was used in assessing the microbial community of doenjang (Nam et al., 2012). "
ABSTRACT: In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.Frontiers in Microbiology 01/2012; 3:436. DOI:10.3389/fmicb.2012.00436 · 3.94 Impact Factor