3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
Table I
Yeasts identification during fermentation of wine cider
Different stages of fermentation
[days]
number of
samples
Identification
1
83, 107
106
Pichia fermentans
Hanseniaspora uvarum
3
9, 70
6
Pichia fermentans
Hanseniaspora uvarum
104 Rhodosporium sp.
5 30 Pichia fermentans
40 Saccharomyces sp.
7
7
62, 102
Saccharomyces mangini
Pichia fermentans
95A Saccharomyces cerevisiae
9 95B, 96 Pichia sp.
22, 87 Saccharomyces mangini
11 67, 58 Saccharomyces mangini
13 90, 72 Saccharomyces mangini
15 73, 88 Saccharomyces mangini
17 63 Saccharomyces mangini
19
18, 97
46
Saccharomyces mangini
Metschnikowia pulcherrima
After restriction analysis with enzyme HinfI the sample
no. 106 was identified as Pichia fermentans.
nevertheless, the detailed analysis of restriction fragments
with enzyme AluI and microscopic analysis led to the
attribution to Hanseniaspora uvarum. (TableI)
After the restriction analysis of the sample no. 40 was
assigned to the genus Saccharomyces without the determination
of species. The sample no. 7 was identified as Saccharomyces
mangini.Using the restriction analysis with enzymes
HinfI, HaeIII and AluI the adherence to the genus Saccharomyces
was proved by the samples 18, 22, 58, 63, 67, 72,
73, 87, 88, 90 and 97. The analysis of these samples with
enzyme MseI enabled the species identification. The simila-
Fig. 1. Example of agarose gel with PCR products of an
amplification of the 5.8S-ITS rDNA (S24–S39 = CCY yeasts,
100 + – size marker
s820
rity with the collection species Saccharomyces mangini was
evidenced.
The restriction analysis using enzyme HinfI proved the
similarity with the genus of Pichia in the case of the sample
no. 96. Applying endonuclease HinfI the probe no. 96 was
consequently assigned as Pichia fermentans. The submission
to the genus of Pichia was determined also by the sample
no. 95B using the enzyme HaeIII. By the sample no.95A the
similarity with the collection species Saccharomyces cerevisiae
was proved. The result of restriction analysis using the
enzymes HinfI, MseI, HaeIII and AluI documented the identity
with the collection species Metschnikowia pulcherrima
by the sample no. 46.
Fig. 2. Example of digestion products of the amplificates obtained
using the restriction enzyme HinfI (S20–S34 = CCY yeasts,
67, 72, 88, 96, 102 – wine yeasts,K – size marker 100pb)
Conclusions
The result of the analysis is the identification of yeasts
of genus Pichia, Hanseniaspora and Rhodotorula in the initiation
stages of fermentation and that of Saccharomyces in
the further stages of fermentation. The species assignment is
Pichia fermentans and Hanseniaspora uvarum. The unambiguous
taxonomic assignment can be performed with Saccharomyces
mangini. By other yeasts of genus Saccharomyces
the determination of species was not possible.
It was proved that in the stage of late time of must fermentation
and of the formation of young wine the yeasts of
genus Saccharomyces prevail. The presence of Saccharomyces
cerevisiae was proved. The interesting result was identification
of yeasts species Metschnikowia pulcherrima. This
yeasts appears in the first stages of fermentation.
The method PCR-RFLP is suitable for taxonomic assignment
of yeasts of different genus. nevertheless, for the exact
assignment the selection of primers and restriction enzymes
plays the important role.