Focusing on drawback in micromanipulate yeast of
Hanseniaspora spp. for improvement of technological traits
Rossana Sidari and Andrea Caridi
Department of Agraria, Mediterranea University of Reggio Calabria, Loc. Feo di Vito, I-89122 Reggio Calabria, Italy
Materials and methods
A standard protocol to micromanipulate S. cerevisiae cells considers: 1. growth of the cells in YPD medium, 2. starvation of the cells in agar acetate medium to induce sporification, 3. enzymatic lysis of asci, and 4. dissection to separate spores and obtain monosporal cultures (Fig. 3) by micromanipulator, usually using the 20x objective (Fig. 4).
Drawback
Different strains of Hanseniaspora spp. isolated from spontaneous fermentation of Calabrian grape musts were grown in YPD medium and then inoculated on agar acetate to induce sporulation. Then, a small quantity of biomass was suspended in zymolyase solution (20U/ml) to prepare asci for the micromanipulation, according to the MSM System instruction. The small cell size of Hanseniaspora strains - compared to S.
cerevisiae ones - coupled with the common used MSM objective (20x) (Fig. 5) and media
formulation used in the asci yeast dissection did not allow to separate spores and obtain monosporal cultures.
Introduction
Hanseniaspora genus includes yeasts with apiculated – lemon-shaped - cells. They are common in nature and, in details, at the first stage of grape must fermentation;
among the species, Hanseniaspora guilliermondii, predominant in the South of Italy, are characterized by 4-spored asci (Fig. 1). Hanseniaspora spp. have similar life cycle to Saccharomyces cerevisiae (Fig. 2); cells are generally diploid and can sporify without prior conjugation. So, as for S. cerevisiae, it may be possible to breed
Hanseniaspora strains via classical methods, such as micromanipulation. Yeasts of the genus Hanseniaspora perform alcoholic fermentation producing notably
amounts of acetic acid and, for this attribute, usually they are not easily suitable in winemaking [1]. On the contrary, there are strains of Hanseniaspora able to produce esters that may improve the wine aromatic profile [2]. Therefore, Hanseniaspora spp. can be used to produce wines as base for the vinegar production with high content in acetic acid and with improved flavour [3]. Moreover, following a specific improvement protocol it may be possible to obtain, using the micromanipulator, strains low-producing acetic acid and, consequently, suitable to control winemaking together with selected strains of S. cerevisiae.
This research was supported by: (1) POR CALABRIA FESR 2007/2013 - ASSE I - Obiettivo Specifico 1.1 - Obiettivo Operativo 1.1.1 - Linea di Intervento 1.1.1.2 Progetti di ricerca industriale e di sviluppo sperimentale nei settori strategici regionali - Progetto “INNOVAZIONE DI PROCESSO E NUOVI PRODOTTI PER LA VALORIZZAZIONE DEI VINI E PASSITI DA CV AUTOCTONE: ENOTRIA TELLUS” and (2) POR CALABRIA FESR 2007/2013 - ASSE I - Obiettivo Specifico 1.1 - Obiettivo Operativo 1.1.1 - Linea di Intervento 1.1.1.2 Progetti di ricerca industriale e di sviluppo sperimentale nei settori strategici regionali - Progetto “NUOVE TECNOLOGIE PER LA VALORIZZAZIONE DELLA FILIERA AGRUMICOLA REGIONALE: CITRUS CALABRIAE”
Conclusion
To our knowledge, this study is a first approach to the use of a modified micromanipulation technique on Hanseniaspora cells to improve specific technological strains. Succeeding in the above proposal would allow to obtain new Hanseniaspora strains:
a) low acetic acid producer and high ester producers able to improve the wine aromatic profile;
b) high acetic acid producer and high ester producer able to produce wines as base for the vinegar production.
Figure 2 - Hanseniaspora
guilliermondii life cycle.
PROPOSAL
MAGNIFICATION
Using higher magnification - such as a 40x objective - than magnification normally used, that anyway take into account the operational mode of the
MSM (correct working distance).
MEDIUM FORMULATION
Using a dissection medium with
higher content of agar
compared to the concentration usually used to prepare YPD agar.
The magnification and the medium formulation would work in synergy: the
higher magnification would allow
observing and identifying the cells with spores to be micromanipulated but at
the same time it implicates the
adjustment of the micromanipulation
technique; the different agar
concentration of the medium would help in creating more friction between needle and cell that ends to easily break yeast asci.
TECHNOLOGICAL APPROACH
Since the aim of our work is the technological improvement of the yeasts, an innovative approach could be:
1. obtaining cultures with spores; 2. enzymatic lysis of cultures;
3. recovering treated cells by centrifugation;
4. performing serial dilutions;
5. plating the serial dilutions in nutrient medium;
6. selecting plates with colonies number between 30-300;
7. testing the colonies and the parental strains towards common traits used for oenological yeasts. Comparing parental strain results with colony results may allow to identify cultures derived from
spore germination and then
select those with desired
characters.
Figure 4 - MSM System 400 (Singer Instrument
Co Ltd., England).
Figure 1 - Cells and 4-spored asci of
Hanseniaspora sp. strain streaked in agar acetate medium.
References
[1] A. Caridi, V. Tini, M. Benevelli, C. Zambonelli, Vini d’Italia 33, 1991, 51.
[2] M. Moreira, C. Pina, F. Mendes, J.A. Couto, T. Hogg, I. Vasconcelos, Food Control 22, 2011, 662. [3] A. Caridi, R. Sidari, 13th International Congress on Yeasts, Madison-USA, 26-30 August 2012, 137.
Figure 3 - S. cerevisiae monosporal cultures on YPD
agar obtained by micromanipulation.
Figure 5 - Hanseniaspora strain observed by
