Study in collaboration with
the University of Modena and Reggio Emilia

YEASTS FOR SPARKLING WINES

ADAPTIVE EVOLUTION AND BREEDING STRATEGIES FOR SELECTING OPTIMISED STARTERS

Sparkling wines have a very prominent position in the wine industry and their growing popularity among consumers, who are increasingly focused on product quality, also stimulates research towards the selection of starter wines optimised for the sparkling process.

The production of sparkling wine and the use of selected yeasts

The production of sparkling wine in bottles or autoclaves is a well-established practice even in small wineries. However, this practice, if not performed correctly, is the cause of various failures.

Among the most common undesirable effects are:

bottle explosion due to excessive pressure;
a wine with a bitter aftertaste and too much sediment;
a wine that remains hazy in the bottle;
a wine that does not referment;
smells of reduced.

To overcome these problems and produce a good sparkling wine, it is necessary to have a good understanding of the mechanisms of fermentation and to pay close attention to all stages of production, from the vinification of the grapes to the choice of sparkling method, but above all, a suitable starter culture must be chosen.
Using unsuitable yeasts can compromise the final product, slowing down or abnormally blocking the process

Refermentation and undesirable effects on yeasts

Refermentation, whether in the bottle or autoclave, is a rather limiting process for yeasts, as they are subjected to stressful conditions such as:

a pH between 2.8 and 3.5;
an ethanol concentration of 11% to 12%;
a high partial pressure of CO₂ (up to 7-8 atm);
a high concentration of organic acids;
a high sulphur dioxide concentration of approximately 50-80 mg/L;
low fermentation temperatures, between 10 and 15 °C.

In addition, the lack of oxygen prevents the synthesis of sterols and unsaturated fatty acids, altering the functionality of the yeast cell membrane.
Scientific evidence confirms the undesirable effects on yeasts, in terms of reduced activity and fermentation vigour, due to the accumulation of CO₂during the process.

Weak organic acids and their influence on cell metabolism

To better understand these effects, one must consider the action of weak organic acids, which influence the metabolism of the cell in a similar way to carbon dioxide.
In fact, CO₂, even in low concentrations, forms carbonic acid, i.e. a weak acid. If one evaluates the acid dissociation constants of carbonic acid and other weak organic acids, one can see that they have roughly similar values, so the dissociative state of these molecules in intra- and extracellular matrices will be reasonably similar.
Weak organic acids at a pH value of around 3-3.5 are found in the indissociated form XCOOH, in this state they are able to freely enter the interior of the cell where the pH is 7.4. At this value they dissociate into the carboxyl anion XCOOH^- and H⁺ ion. The carboxyl anions are unable to passively leave the membrane and accumulate within the cytoplasm. This accumulation induces various cellular responses to stress and, if left unchecked, can even induce apoptosis.

Adaptive evolution strategies for yeast selection

It follows that a potential selection of yeast cultures suitable for sparkling winemaking can be achieved with adaptive evolution strategies, using weak organic acids, such as different concentrations of acetic acid, sorbic acid or benzoic acid, as a stress factor. In general, approaches based on evolutionary principles are very effective in obtaining strains with specific phenotypes required in industry. The key to the evolutionary adaptation approach is the genetic variability generated by recombination processes, which increase the probability of selecting improved strains for the characteristics of interest.

In particular, meiotic recombination processes are particularly effective in the case of heterozygous strains, since the shuffling of genes makes it possible to obtain a different set of haploid cells. In practice, crossing over is exploited as a source of new genetic variability. This approach can also be applied in breeding programmes to obtain both intra- and interspecific hybrids.

The new yeast strain obtained through interspecific breeding

The evolutionary approach can also be applied in breeding programmes to obtain both intra- and interspecific hybrids. In a first work carried out by the research group of the Unimore Microbial Culture Collection (UMCC), the evolutionary approach, previously described, allowed to obtain a new strain of Saccharomyces cerevisiae, deposited with the code UMCC 2949, having the phenotypic characteristics of interest such as, resistance to weak organic acids.
In order to validate the experimental hypothesis of the correspondence with resistance even at high CO₂ concentrations, sparkling wine tests were carried out in prototype autoclaves, in which the selected strain performed well.
In order to further improve the UMCC 2949 strain, also giving it greater resistance to low temperatures, the interspecific breeding strategy was subsequently applied, crossing the S. cerevisiae strain with the UMCC 2633 strain of the S. uvarum species. The colonies resulting from the different crosses between the spores of the two parental strains were characterised at the molecular level, by means of “colony PCr” and RFLP (Restriction Fragment Length Polymorphism) analysis of the ITs-5,8s region of the rNA, in order to confirm the formation of the hybrids (see Figure 1 below).

Figure 1

Schematic representation of the interspecific breeding strategy:

a) direct spore-spore crossing between S. cerevisiae and S. uvarum strains;

b) molecular characterisation of the cultures derived from the crosses;

c) phenotypic screening;

d) fermentation tests to assess the performance of the hybrid.

The fermentation performance of the new yeast

Subsequently, the selected hybrid, named UMCC 3008, was tested on selective grounds to verify tolerance to 12% and 15% ethanol, under two different temperature conditions, 15 and 27 °C respectively, and resistance to acetic acid at 50 mM and 75 mM concentrations. The fermentation performance of the UMCC 3008 hybrid was preliminarily evaluated in microvinification tests carried out in flasks with sucrose-supplemented base wine at a final concentration of 30g/L. Finally, a BIGGY agar growth medium test was carried out for a qualitative estimate of hydrogen sulphide production.

RESEARCH RESULTS A

Phenotypic screening showed the following results for the new hybrid UMCC 3008 and the strain UMCC2949 (see Table 1 below):

tolerance to ethanol under the conditions tested;
resistance to acetic acid concentrations of up to 50 mM;
low H₂S production;
good tolerance to selective pressures applied in phenotypic screening;
excellent fermentative fitness in microvinification trials.

Therefore, it is considered that this hybrid and its ancestore are an excellent candidate as a starter cultures to be validated through further experimentation in order to confirm their fermentation performance and their suitability for the production of sparkling wines.

Codice UMCC	Species	Ethanol 12%		Ethanol 15%		Acetic acid 50 mM	Acetic acid 75 mM	H₂S (Biggy agar)
		15°	27°	15°	27°	27°	27°	27°
UMCC 2949	*S. cerevisiae*	+	+	+	+	+	+	medium
UMCC 2633	*S. uvarum*	+	+	-	-	+	-	low
UMCC 3008	*S. cerevisiae x S. uvarum*	+	+	+	+	+	-	low

Table 1
Screening for resistance to ethanol, performed on YPDA medium with 12% or 15% ethanol added (incubation at 15°C and 27°C) and acetic acid, performed on Yeast Nitrogen Base (YNB) minimal medium with 50mM or 75mM acetic acid added, in addition, qualitative evaluation of H₂S production on BIGGY agar medium, after 6 days of plate incubation.

DISCOVER THE NEW YEAST FERMOL PERLE

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SPARKLING WINE RANGE BROCHURE

Study in collaboration withthe University of Modena and Reggio Emilia