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. 2001 Nov;67(11):5113-21.
doi: 10.1128/AEM.67.11.5113-5121.2001.

Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages

L Cocolin  1 , M Manzano, C Cantoni, G Comi
Affiliations

Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages

L Cocolin et al. Appl Environ Microbiol. 2001 Nov.

Abstract

In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family Micrococcaceae and meat contaminants, such as Brochothrix thermosphacta and Enterococcus sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required.

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Figures

FIG. 1
FIG. 1
Microbial population dynamics, as determined by classical methods, and pH curve during natural fermentation of sausages.
FIG. 2
FIG. 2
DGGE profiles of the PCR products obtained from the control strains. (A) 30 to 50% denaturant gradient; (B) 40 to 60% denaturant gradient. Lanes 1, L. brevis DSM 20054; lanes 2, L. casei DSM 20011; lanes 3, L. alimentarius DSM 20249; lanes 4, L. plantarum DSM 20174; lanes 5, L. curvatus subsp. curvatus DSM 20019; lanes 6, L. sake DSM 6333; lanes 7, S. intermedius DSM 20373; lanes 8, S. carnosus subsp. carnosus DSM 20501; lanes 9, S. simulans DSM 20322; lanes 10, S. xylosus DSM 6179; lanes 11, K. kristinae DSM 20032; lanes 12, K. varians DSM 20033.
FIG. 3
FIG. 3
L. sake and L. curvatus trends during ripening of naturally fermented sausages.
FIG. 4
FIG. 4
DGGE profiles of the DNA amplicons obtained directly from fermented sausages. Profiles obtained at zero time and after 3, 10, 20, 30, and 45 days of fermentation are shown. (A) 40 to 60% denaturant gradient; (B) 30 to 50% denaturant gradient. Bands indicated by numbers were excised and after reamplification subjected to sequencing.
FIG. 5
FIG. 5
DGGE profiles of the RNA amplicons obtained directly from fermented sausages. Profiles obtained at zero time and after 3, 10, 20, 30, and 45 days of fermentation are shown. (A) 40 to 60% denaturant gradient; (B) 30 to 50% denaturant gradient. Bands indicated by numbers were excised and after reamplification subjected to sequencing.
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