Evaluation of microbial diversity in sulfite-added and sulfite-free wine by culture-dependent and -independent methods.

The difference in microbiota including non-lactic acid bacteria, non-acetic acid bacteria, and wild yeast during winemaking and in the end-products between sulfite-added and sulfite-free wine, was investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and a culture-dependent method. There were differences between the microorganisms detected by PCR-DGGE and those detected by the culture-dependent method, probably because of the selectivity of culture medium and the characteristics of PCR-based method. In both the red wine and white wine, the microbial diversity of the sulfite-added wine was lower than that of the sulfite-free wine during fermentation. Tatumella terrea was detected from the fermenting must by PCR-DGGE and by the culture-dependent method, even though sulfite inhibited its growth to some extent. We confirmed that the addition of sulfite plays an important role in winemaking by inhibiting the growth of unexpected microorganisms, but on the other hand, it was revealed that some microorganisms can survive and grow in sulfite-added fermenting must. We also analyzed 15 samples of commercial wines by the PCR-DGGE method and detected various microorganisms. Among them, Sphingomonas sp., Pseudozyma sp., Ochromonas sp. and Methylophilus sp. were found for the first time in wine as far as we know. We did not identify a specific microorganism that was detected only from wines without sulfite addition. Thus, the microbiota of end-products seemed to be influenced by other factors, such as filtration before bottling, the production equipment and the storage environment.