Molecular fingerprinting by PCR-denaturing gradient gel electrophoresis reveals differences in the levels of microbial diversity for musty-earthy tainted corks

Abstract

The microbial community structure of cork with marked musty-earthy aromas was analyzed using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Cork stoppers and discs were used for DNA extraction and were analyzed by using selective primers for bacteria and fungi. Stoppers clearly differed from discs harboring a different fungal community. Moreover, musty-earthy samples of both types were shown to have a specific microbiota. The fungi Penicillium glabrum and Neurospora spp. were present in all samples and were assumed to make only a small contribution to off-odor development. In contrast, Penicillium islandicum and Penicillium variabile were found almost exclusively in 2,4,6-trichloroanisole (TCA) tainted discs. Conversely, Rhodotorula minuta and Rhodotorula sloofiae were most common in cork stoppers, where only small amounts of TCA were detected. Alpha- and gammaproteobacteria were the most commonly found bacteria in either control or tainted cork stoppers. Specific Pseudomonas and Actinobacteria were detected in stoppers with low amounts of TCA and 2-methoxy-3,5-dimethylpyrazine. These results are discussed in terms of biological degradation of taint compounds by specific microorganisms. Reliable and straightforward microbial identification methods based on a molecular approach provided useful data to determine and evaluate the risk of taint formation in cork.

FIG. 1.

Negative image composition of SybrGold-stained DGGE gels of environmental ITS1 genes of fungi obtained with the specific primer set ITS-1F-ITS2. The dendrogram on top is based on a similarity matrix (Dice index) calculated from a presence-absence binary matrix of bands. Grouping was based on the UPGMA method. Bands indicated with an arrow head were excised and sequenced. REF, reference position markers for gel comparison; D_ME-TCA, TCA tainted cork discs; D_C, control untainted cork discs.

FIG. 2.

Negative image composition of SybrGold-stained DGGE gels of environmental ITS1 genes of fungi obtained with the specific primer set ITS-1F-ITS2 from cork stoppers. The dendrogram on top is based on a calculated similarity matrix (Dice index) from a presence-absence binary matrix of bands. Grouping has been made by using a UPGMA method. Bands indicated with an arrow head were excised and sequenced. REF, reference position markers for gel comparison; ME, ME tainted cork stoppers; C, control untainted cork stoppers.

FIG. 3.

Negative image composition of SybrGold-stained DGGE gels of environmental bacterial 16S rRNA genes obtained with the specific primer set 357F-907R from cork stoppers. The dendrogram on top is based on a calculated similarity matrix (Dice index) from a presence-absence binary matrix of bands. Grouping has been made by using a UPGMA method. Bands indicated with an arrow head were excised and sequenced. REF, reference position markers for gel comparison; ME, ME tainted cork stoppers; C, control untainted cork stoppers.

FIG. 4.

NJ tree generated from the alignment of Penicillium spp. ITS1 sequences (188 to 201 bp). Sequences and accession numbers of selected organisms retrieved from GenBank appear in boldface. Samples numbering corresponds to DGGE bands in Fig. 1 and 2. Bootstraps values >60% are indicated at branch nodes. ITS1 sequence from Eurotium repens (AY373890) was used as outgroup. Origin of sequences is depicted by shaded dots on the right side of each name: white, control discs; gray, TCA tainted discs; black, cork stoppers.