Focusing on drawback in micromanipulate yeast of Hanseniaspora spp. for improvement of technological traits.

Yeasts of the genus Hanseniaspora perform alcoholic fermentation producing notably amounts of acetic acid and for this attribute usually they are not useful in winemaking [1]. On the contrary, there are strains of Hanseniaspora able to produce esters that improve the wine aromatic profile [2]. Therefore, Hanseniaspora spp. may 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 so useful to control conventional winemaking together with selected strains of Saccharomyces cerevisiae. Hanseniaspora spp. have similar life cycle to Saccharomyces cerevisiae so as for S. cerevisiae it is possible to breed via classical methods, such as micromanipulation, the best strains of Hanseniaspora spp. for the production of vinegar. This contribution underlies the drawback encountered in managing the dissection of the Hanseniaspora spp. asci. To our knowledge, no papers report about the dissection of Hanseniaspora asci and related technical problems. Different strains of Hanseniaspora spp. isolated from Calabrian grape musts were grown in YPD medium and then inoculated on agar acetate to induce sporulation. Then, a small quantity of biomass was dissolved in zymolyase solution (20U/ml) to prepare asci for the micromanipulation (MSM System 400, Singer Instrument Co Ltd., England) according to the MSM System instruction. The small cell size of Hanseniaspora strains compared to S. cerevisiae coupled with the common MSM objective (20x) and media formulation used in the asci yeast dissection did not allow to separate spores and obtain monosporal cultures. Possible proposals to overcome this technical drawback could be the use of higher magnification that anyway take into account the operational mode of the MSM (correct working distance) and a medium composition richer in agar. These two proposals 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 medium harder than normal would help in creating more friction between needle and cell that ends to easily break yeast asci. 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. [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.