Characterization of thermophilic fungal community associated with pile fermentation of Pu-erh tea.

This study aimed to characterize the thermophilic fungi in pile-fermentation process of Pu-erh tea. Physicochemical analyses showed that the high temperature and low pH provided optimal conditions for propagation of fungi. A number of fungi, including Blastobotrys adeninivorans, Thermomyces lanuginosus, Rasamsonia emersonii, Aspergillus fumigatus, Rhizomucor pusillus, Rasamsonia byssochlamydoides, Rasamsonia cylindrospora, Aspergillus tubingensis, Aspergillus niger, Candida tropicalis and Fusarium graminearum were isolated as thermophilic fungi under combination of high temperature and acid culture conditions from Pu-erh tea pile-fermentation. The fungal communities were analyzed by PCR-DGGE. Results revealed that those fungi are closely related to Debaryomyces hansenii, Cladosporium cladosporioides, A. tubingensis, R. emersonii, R. pusillus, A. fumigatus and A. niger, and the last four presented as dominant species in the pile process. These four preponderant thermophilic fungi reached the order of magnitude of 10(7), 10(7), 10(7) and 10(6)copies/g dry tea, respectively, measured by real-time quantitative PCR (q-PCR). The results indicate that the thermophilic fungi play an important role in Pu-erh tea pile fermentation.

Identification of acetic acid bacteria in traditionally produced vinegar and mother of vinegar by using different molecular techniques.

Culture-dependent and culture-independent methods were combined for the investigation of acetic acid bacteria (AAB) populations in traditionally produced vinegars and mother of vinegar samples obtained from apple and grape. The culture-independent denaturing gradient gel electrophoresis (DGGE) analysis, which targeted the V7-V8 regions of the 16S rRNA gene, showed that Komagataeibacter hansenii and Komagataeibacter europaeus/Komagataeibacter xylinus were the most dominant species in almost all of the samples analyzed directly. The culture-independent GTG5-rep PCR fingerprinting was used in the preliminary characterization of AAB isolates and species-level identification was carried out by sequencing of the 16S rRNA gene, 16S-23S rDNA internally transcribed to the spacer (ITS) region and tuf gene. Acetobacter okinawensis was frequently isolated from samples obtained from apple while K. europaeus was identified as the dominant species, followed by Acetobacter indonesiensis in the samples originating from grape. In addition to common molecular techniques, real-time PCR intercalating dye assays, including DNA melting temperature (Tm) and high resolution melting analysis (HRM), were applied to acetic acid bacterial isolates for the first time. The target sequence of ITS region generated species-specific HRM profiles and Tm values allowed discrimination at species level.

Diversity and activities of yeasts from different parts of a Stilton cheese.

Blue cheeses are very complex food matrices presenting significant spatial differentiation between sections and the Stilton variety also has a hard brown crust making its matrix even more complex. The mycobiota communities in the three sections (blue veins, white core and outer crust) of a Stilton blue cheese were studied by employing culture-independent (TRFLP, DGGE) and culture-dependent analyses. Yeasts isolated from the cheese were studied for aroma production in a dairy model system with and without the starter Lactococcus lactis and filamentous fungus Penicillium roqueforti using SPME GC-MS. Significant qualitative and quantitative differences were observed in the yeast communities between the cheese sections with all the techniques. Yarrowia lipolytica presented strong synergistic activity with P. roqueforti enhancing the production of ketone aroma compounds, characteristic of blue cheeses. Culture techniques allowed the observation of the presence and uneven distribution of two different morphological groups of Debaryomyces hansenii in the different sections and of Trichosporon ovoides but failed to isolate Candida catenulata which dominated some parts of the cheese in the culture-independent analysis. This suggests that this species may be an important early coloniser but fails to survive into the final cheese. The study indicated that the yeast flora in the cheese sections differ including isolates that could affect their aroma profiles.

Determination of yeast diversity in ogi, mawè, gowé and tchoukoutou by using culture-dependent and -independent methods.

The maize based ogi and mawè and the sorghum based gowé and tchoukoutou are traditional, spontaneously fermented products widely consumed by the population of Benin (West Africa). Yeast occurrence in the products, as sold on local markets at different locations, was studied using a combination of culture-dependent and independent methods. Number of yeasts is varied from 3.75 log10 colony forming units (cfu)/g for ogi to 5.60 log10 cfu/g for tchoukoutou. Isolated yeasts (236) were identified based on different migration profiles on denaturing gradient gel electrophoresis (DGGE) and 26S rRNA gene sequencing. Candida krusei was the yeast most frequently isolated with strongest predominance in the maize based products. Other predominant yeast present at equal or lower incidence were Clavispora lusitaniae and Saccharomyces cerevisiae in ogi and mawè, Cl. lusitaniae, Candida tropicalis and Kluyveromyces marxianus in gowè and Cl. lusitaniae, S. cerevisiae and Candida rugosa in tchoukoutou. Grouping of C. krusei isolates (164) by rep-PCR analysis indicated that several biotypes were involved in fermentation of the four products. The DGGE analysis on the DNA directly extracted from the food matrices demonstrated the presence of Dekkera bruxellensis and Debaryomyces hansenii, not detected by the culture-based approach. This is the first study combining culture-dependent and independent methods to reveal predominant yeast species and biotypes in traditional foods from Benin.

Grape berry yeast communities: influence of fungicide treatments.

The yeast communities colonising grape berry surfaces were evaluated for the influence of fungicide treatments in an organic vineyard (copper/sulphur-based products) and a conventional vineyard (commonly used fungicides). Analysis of yeast abundance and diversity was carried out on grape berries and juice during fermentation, using culture-dependent and -independent approaches. Yeast abundance was as generally reported for mature grapes and it was slight higher from grapes treated with conventional fungicides. Initial grape samples showed less yeast species diversity in the organic vineyard compared with the conventional one. In both vineyards, the dominant yeast were Candida zemplinina and Hanseniaspora uvarum (>50%), respectively, typical species that colonise surfaces of mature grape berries. Metschnikowia pulcherrima was widely found in the conventional samples while it was only occasionally found in organic ones. Saccharomyces cerevisiae was isolated only at the end of natural fermentation (conducted in sterile condition), with lower levels in the organic samples. S. cerevisiae strains showed less intraspecies diversity in the organic samples (two genotypes), in comparison with the conventional samples (six genotypes).