3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P108 MONITORING OF ChANGES IN POPuLATION
OF yEASTS DuRING FERMENTATION OF
GRAPE MuST
DAnA VRánOVá, MICHAELA ZDEňKOVá and
REnATA VADKERTIOVá
Faculty of Chemistry, Brno University of Technology, Purkyňova
118, 612 00 Brno,Czech Republic,
vranova@fch.vutbr.cz
Introduction
Traditionally, wine fermentation has been carried out in
spontaneous way by indigenous yeasts present on the grapes
when harvested, or introduced from the equipment and cellar
during the fermentation process. At present most wine producers
carry out the fermentation process by adding a pure
Saccharomyces strains to the must to improve final wine quality
1,23 .
The strains at the species level can be identified using
conventional methods based on biochemical and physiological
characteristic or molecular techniques. Over the last
decade molecular techniques for the identification of yeast
strains have been widely studied 4,5,8 . The applicability of
techniques as the RAPD method and restriction enzyme analysis
of PCR amplified DnA fragments (PCR-RFLP) has
been demonstrated as a tool for taxonomic purposes 11,16,19 .
The fermentation of grape must into wine is a complex
microbiological process characterized by the presence of a
large number of different microorganisms. The composition
of yeasts population on grape berries and leaves plays
an important role in wine fermentations, as different genera,
species and strains with their metabolic activity influence
the sensory quality and organoleptic characteristics of wine.
3,7,8,13,15,16,18 Yeasts associated with grape or wine ecosystem
are usually classified in 15 different genera 21 : Brettanomyces/
Dekkera, Candida, Cryptococcus, Debaryomyces, Hanseniaspora/Kloeckera.
Kluyveromyces, Pichia, Metschnikowia,
Rhodotorula, Saccharomyces, Saccharomycodes, Schizosaccharomyces,
Zygosaccharomyses.
The aim of this study was monitoring of the changes in
yeast population during spontaneous alcoholic fermentation
of cider of Veltlin green cultivar. We isolated yeasts in different
period of grape cider fermentation. Identification of the
individual strains was carried out using PCR-RFLP analysis
of 5.8S-ITC region. 6,7,11,12
Experimental
S a m p l e P r e p a r a t i o n
The samples analyzed in this study were isolated from
white wine cider (“Veltlin green”) obtained from local producer
from wine region Velke Pavlovice. Control yeast strains
used in this study were from the Culture Collection of Yeasts,
Bratislava, Slovakia (CCY).
Sampling at different stages of fermentations was performed
at days: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 Aliquots
of each sample (several dilutions) were spred on to
s819
plates of wort agar. Cells were directly collected from a fresh
yeast colony using a microbiological loop.
DnA was extracted and purified by Ultra Clean Microbial
DnA Isolation Kit – obtained from Elizabeth Pharmacon
spol. s.r.o., CR. 3–10 ng of DnA in 1–2 μl of TBE buffrer
were used in a 50 μl amplification reaction.
P C R A m p l i f i c a t i o n o f t h e Y e a s t
D n A
The amplification reaction of the rDnA region spannding
the 5.8 rDnA gene and the ITS regions were carried
our under the following conditions: 50 µl of reaction mixture
contained 5–20 ng template DnA, polymerase buffer,
0.2 mM of dnTP (n = A, T, G, C) 0.5 µM of each primer
and 1 U Taq DnA polymerase. The amplification protocol
included the following steps: Initial denaturation at 94 °C
for 4 min, 25 cycles of amplification: denaturation at 94 °C
for 1 min, annealing at 48 °C for 30 sec, and extension at
72 °C for 1 min. The final extension was at 72 °C for 10 min.
Amplification products were analysed on 0.7% (w/v) agarose
gel in TBE buffer. PCR amplification was carried out in a
PTC-100 thermocycler (MJ.Research,Inc.). Upper primer:
(ITS1) 5’-TCC GTA GGT GAA CCT GCG G-3’,Lower primer:
(ITS4) 5’-TCC TCC GCT TAT TGA TAT GC-3’ (VBC
BIOTECH, GmbH) 6 . With these two primers a different products
(900, 800, 700, 500 and 450 bp) were obtained after
amplification with DnA template from species of genus Saccharomyces
(Fig. 1.).
R e s t r i c t i o n A n a l y s i s a n d G e l
E l e c t r o p h o r e s i s
PCR products (20 µl) were digested in 20 µl of reaction
volume with 1U of restriction endonuclease using the manufacturers
conditions.
The restriction enzymes used: Alu I, Hae III, Hinf I and
Mse I. (Fermentas, Lithuania)
The producst of digestion were analysed by horizontal
gel electroforesis system (OWL, USA) in 2% (w/v) agarose
gels and comparison with yeasts from CCY. After electroforesis
the gels were stained with EtBr and visualized under UV
light (Fig. 2.).
Results
The restriction fragments of amplified part of DnA from
27 yeasts isolated from wine must were compared with those
of 40 yeasts from the Collection of yeasts of Chemical institute
of Slovak Academy of Sciences in Bratislava.
Based on the identity of length and number of restriction
fragments the taxonomic assignment of yeasts was performed.
In some cases no species, but only genus assignment
was carried out. Samples no. 83, 107, 70, 9, 30, 62 and 102
were identified as Pichia fermentans, the sample no. 6 and
106 were identified as Hanseniaspora uvarum.