Analysis of bacterial diversity during the fermentation of inyu, a high-temperature fermented soy sauce, using nested PCR-denaturing gradient gel electrophoresis and the plate count method

2013 ◽  
Vol 33 (2) ◽  
pp. 252-261 ◽  
Author(s):  
Chia-Li Wei ◽  
Shiou-Huei Chao ◽  
Wen-Bin Tsai ◽  
Pei-Shan Lee ◽  
Nai-Hung Tsau ◽  
...  
Keyword(s):  
High Temperature ◽  
Bacterial Diversity ◽  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Soy Sauce ◽  
Plate Count ◽  
Count Method ◽  
Gradient Gel Electrophoresis ◽  
Plate Count Method

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

Molecular Analysis of Spoilage-Related Bacteria in Pasteurized Milk during Refrigeration by PCR and Denaturing Gradient Gel Electrophoresis

2009 ◽  
Vol 72 (3) ◽  
pp. 572-577 ◽  
Author(s):  
HONGFEI HE ◽  
JIN DONG ◽  
CHIN NYEAN LEE ◽  
YONG LI
Keyword(s):  
Shelf Life ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Plate Count ◽  
Pasteurized Milk ◽  
Milk Samples ◽  
Gradient Gel Electrophoresis ◽  
Aerobic Plate Count ◽  
Species Specific

Bacterial diversity in fluid milk products has been extensively studied in order to improve milk quality. Here, we illustrate the utility of viable counts and PCR–denaturing gradient gel electrophoresis (DGGE) for monitoring the microbial spoilage of pasteurized milk during shelf life. Five pasteurized milk samples stored at 4°C were examined at 10 and 5 days before expiration and on the expiration day. With bacterial DNA extracted directly from the samples, PCR-DGGE analysis indicated that Pseudomonas became dominant in four samples. Meanwhile, the aerobic plate count of these four samples exceeded the regulatory limit of 20,000 CFU/ml at 5 days before expiration, and the rapid psychrotrophic count markedly surpassed the aerobic plate count on the expiration day. Streptococcus and Buttiauxella spp. were detected in several samples. Sequence analysis of DGGE fragments revealed high diversity among Pseudomonas spp. in the milk samples. P. putida and P. migulae grew to high numbers during refrigerated storage. Further identification of Pseudomonas at the species level was facilitated by PCR and multiplex PCR using species-specific primers; consequently, P. fluorescens and P. fragi were observed. These results highlight an important role of Pseudomonas in the shelf life of pasteurized milk.

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