Novel insights into microbial community dynamics during the fermentation of Central European ice wine.

Culture-dependent and culture-independent strategies were applied to investigate the microbiota of autumn undamaged and damaged berries, winter berries and ice wine must samples of Grüner Veltliner (Veltlínske zelené) from Small Carpathian wine-producing region. One hundred twenty-six yeasts and 242 bacterial strains isolated from several microbiological media (YPD, PDA, R2A, GYC, MRS and MRS-T) were clustered by ITS-PCR and subsequent Qiaxcel electrophoresis. Representatives of each cluster were identified by sequencing. The extracellular hydrolytic properties and intracellular activities of esterase and ß-glucosidase of isolates were assayed. The culture-independent approach permitted the analysis of extracted DNA and RNA coupling DGGE fingerprinting with construction of clone libraries (bacterial and fungal; DGGE-cloning). The combination of the two approaches provided comprehensive data that evidenced the presence of a complex microbiota in each analyzed sample. RNA and DNA analyses facilitated differentiation of living microorganisms from the entire microbiota. Diverse microbial communities colonized the autumn and winter berries. Generally, the combination of results obtained by the methods suggested that the must samples contained mainly Saccharomyces cerevisiae, Metschnikowia spp., Hanseniaspora uvarum, Lactococcus lactis and Leuconostoc spp. The strains exhibited interesting esterase and ß-glucosidase properties, which are important for aroma formation in wine. Fermentation strategies utilising these microorganisms, could be attempted in the future in order to modulate the ice wine characteristics.

The microbial diversity of water kefir.

The microbial diversity of water kefir, made from a mixture of water, dried figs, a slice of lemon and sucrose was studied. The microbial consortia residing in the granules of three water kefirs of different origins were analyzed. A collection of 453 bacterial isolates was obtained on different selective/differential media. Bacterial isolates were grouped with randomly amplified polymorphic DNA (RAPD)-PCR analyses. One representative of each RAPD genotype was identified by comparative 16S rDNA gene sequencing. The predominant genus in water kefirs I and II was Lactobacillus, which accounted for 82.1% in water kefir I and 72.1% in water kefir II of the bacterial isolates. The most abundant species in water kefirs I and II were Lactobacillus hordei and Lb. nagelii followed by considerably lower numbers of Lb. casei. Other lactic acid bacteria (LAB) were identified as Leuconostoc mesenteroides and Lc. citreum in all three water kefirs. The most abundant species in water kefir III was Lc. mesenteroides (28%) and Lc. citreum (24.3%). A total of 57 LAB belonging to the species of Lb. casei, Lb. hordei, Lb. nagelii, Lb. hilgardii and Lc. mesenteroides were able to produce exopolysacchrides from sucrose. Non LABs were identified as Acetobacter fabarum and Ac. orientalis. The Acetobacter species were more prevalent in consortium III. Cluster analyses of RAPD-PCR patterns revealed an interspecies diversity among the Lactobacillus and Acetobacter strains. Aditionally, Saccharomyces cerevisiae, Lachancea fermentati, Hanseniaospora valbyensis and Zygotorulaspora florentina were isolated and identified by comparison of partial 26S rDNA sequences and FTIR spectroscopy.

Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese.

In order to investigate the microflora of Slovakian bryndza cheese (a cheese containing unpasteurized or pasteurized ewes' milk component) by a culture-independent method, DNA was extracted directly from 7 bryndza samples and analysed by an innovative method. Using the universal prokaryotic and fungal primers, ribosomal DNA internal transcribed spacer (ITS) regions with variable length were amplified. The standard universal reverse primer L1 aligning to bacterial 23s rDNA was found unsuitable for some lactic acid bacteria and other species based on in silico analysis. Therefore, L1 primer was replaced by a combination of novel primers GplusR and GminusR aligning to the adjacent, more conserved DNA region. The amplification profiles were visualised by both standard electrophoresis and by fluorescent capillary gel electrophoresis. From representative samples, major amplicons were excised from the gel, cloned and sequenced. Sequencing revealed that the samples contained Lactobacillus delbrueckii, Lactobacillus brevis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus raffinolactis, Streptococcus macedonicus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum. These results represent an extension of the knowledge on the microflora of Slovakian bryndza cheese. The introduced automated ribosomal DNA intergenic spacer analysis of the bacterial and fungal genomes proved to be very effective in the application of studying microflora of cheese.