3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
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Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology<br />
Table I<br />
Yeasts identification during fermentation of wine cider<br />
Different stages of fermentation<br />
[days]<br />
number of<br />
samples<br />
Identification<br />
1<br />
83, 107<br />
106<br />
Pichia fermentans<br />
Hanseniaspora uvarum<br />
3<br />
9, 70<br />
6<br />
Pichia fermentans<br />
Hanseniaspora uvarum<br />
104 Rhodosporium sp.<br />
5 30 Pichia fermentans<br />
40 Saccharomyces sp.<br />
7<br />
7<br />
62, 102<br />
Saccharomyces mangini<br />
Pichia fermentans<br />
95A Saccharomyces cerevisiae<br />
9 95B, 96 Pichia sp.<br />
22, 87 Saccharomyces mangini<br />
11 67, 58 Saccharomyces mangini<br />
13 90, 72 Saccharomyces mangini<br />
15 73, 88 Saccharomyces mangini<br />
17 63 Saccharomyces mangini<br />
19<br />
18, 97<br />
46<br />
Saccharomyces mangini<br />
Metschnikowia pulcherrima<br />
After restriction analysis with enzyme HinfI the sample<br />
no. 106 was identified as Pichia fermentans.<br />
nevertheless, the detailed analysis of restriction fragments<br />
with enzyme AluI and microscopic analysis led to the<br />
attribution to Hanseniaspora uvarum. (TableI)<br />
After the restriction analysis of the sample no. 40 was<br />
assigned to the genus Saccharomyces without the determination<br />
of species. The sample no. 7 was identified as Saccharomyces<br />
mangini.Using the restriction analysis with enzymes<br />
HinfI, HaeIII and AluI the adherence to the genus Saccharomyces<br />
was proved by the samples 18, 22, 58, 63, 67, 72,<br />
73, 87, 88, 90 and 97. The analysis of these samples with<br />
enzyme MseI enabled the species identification. The simila-<br />
Fig. 1. Example of agarose gel with PCR products of an<br />
amplification of the 5.8S-ITS rDNA (S24–S39 = CCY yeasts,<br />
100 + – size marker<br />
s820<br />
rity with the collection species Saccharomyces mangini was<br />
evidenced.<br />
The restriction analysis using enzyme HinfI proved the<br />
similarity with the genus of Pichia in the case of the sample<br />
no. 96. Applying endonuclease HinfI the probe no. 96 was<br />
consequently assigned as Pichia fermentans. The submission<br />
to the genus of Pichia was determined also by the sample<br />
no. 95B using the enzyme HaeIII. By the sample no.95A the<br />
similarity with the collection species Saccharomyces cerevisiae<br />
was proved. The result of restriction analysis using the<br />
enzymes HinfI, MseI, HaeIII and AluI documented the identity<br />
with the collection species Metschnikowia pulcherrima<br />
by the sample no. 46.<br />
Fig. 2. Example of digestion products of the amplificates obtained<br />
using the restriction enzyme HinfI (S20–S34 = CCY yeasts,<br />
67, 72, 88, 96, 102 – wine yeasts,K – size marker 100pb)<br />
Conclusions<br />
The result of the analysis is the identification of yeasts<br />
of genus Pichia, Hanseniaspora and Rhodotorula in the initiation<br />
stages of fermentation and that of Saccharomyces in<br />
the further stages of fermentation. The species assignment is<br />
Pichia fermentans and Hanseniaspora uvarum. The unambiguous<br />
taxonomic assignment can be performed with Saccharomyces<br />
mangini. By other yeasts of genus Saccharomyces<br />
the determination of species was not possible.<br />
It was proved that in the stage of late time of must fermentation<br />
and of the formation of young wine the yeasts of<br />
genus Saccharomyces prevail. The presence of Saccharomyces<br />
cerevisiae was proved. The interesting result was identification<br />
of yeasts species Metschnikowia pulcherrima. This<br />
yeasts appears in the first stages of fermentation.<br />
The method PCR-RFLP is suitable for taxonomic assignment<br />
of yeasts of different genus. nevertheless, for the exact<br />
assignment the selection of primers and restriction enzymes<br />
plays the important role.