AGRE-0012 Control of Malolactic Fermentation Development of Lactic Acid Bacteria for Direct Inoculation into Wine

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Biological Conversion : Biotechnology : ECLAIR Cluster III - Carbohydrates

Direct innoculation of wine with lactic acid starter cultures

SUMMARY

Malolactic fermentation (MLF) is an essential process in the production of high quality wine. Modern hygienic standards and technical developments have decreased the spontaneous evolution of MLF, hence increasing the demand for starter cultures based on the lactic acid bacteria Leuconostoc oenos which is often responsible for the spontaneous MLF. Commercial preparations of freeze dried L. oenos, which have been available for several years, are not always satisfactory. This project has achieved its main goal, which was to develop cultures of L. oenos which are able to survive, grow and perform malolactic fermentation after direct inoculation into wine. Some strains are now being developed towards commercialisation.

INTRODUCTION

Malolactic fermentation (MLF) is an essential process in the production of high quality wine. Modern hygienic standards and technical developments have decreased the spontaneous evolution of MLF increasing the demand for starter cultures. The highly specialised lactic acid bacteria Leuconostoc oenos is often responsible for spontaneous MLF, and it is recognised as the best organism for induction of MLF. Commercial preparations of L. oenos have been available for several years. However, at the time this project was initiated under the ECLAIR programme, the only available industrial preparations were freeze dried cultures which could only be used after reactivation. Such application, which is not always successful, takes a significant time and is labour intensive.

Malic acid and fermentation in wine production.

OBJECTIVES

The objective of this project was to develop malolactic starter cultures of L. oenos for direct inoculation into wine after alcoholic fermentation, which initiate growth and MLF without a significant lag phase.

METHODOLOGY

Malolactic bacteria have been isolated from different wine-growing regions. In particular, strains of Leuconostoc oenos were chosen, since this species is known to produce active malolactic fermentations, and screened for their growth and activity in wine; ie the ability of the selected strains to survive direct inoculation into wine and to grow and perform malolactic fermentation. The metabolism of the selected strains, in terms of substrates (sugars, organic acids) used as well as products formed, was investigated and correlated with the impact of inoculation on the organoleptic characteristics of the wine. Further work defined the genetic determinants of factors which adapted such cultures to the wine environment, enabling optimisation with respect to various downstream processes.

ACTIVITIES

More than 850 bacterial strains were isolated from French, Spanish, Italian and Portuguese wines undergoing MLF. Using DNA hybridisation techniques, 77 % of the strains were identified as L. oenos. These were screened for prophages, by treatment with mitomycin C, and the phage spectra established.

The subsequent selection of 11 interesting strains was based on metabolic studies which assessed the following criteria: survival and growth in wine; resistance towards specific toxic components of wine; malolactic activity and production of metabolites; growth potential under industrial culture conditions. Following further studies of sugar metabolism and resistance, 5 of the strains were selected. At the same time more basic studies of the mechanisms of bacterial adaptation to the wine environment and its genetic control, as reflected in the composition of the cell membranes, were carried out. Cultivation conditions were then optimised by studies of the growth dynamics of the selected strains. Downstream processes, which influence the survival of the bacteria through the preparation and the direct inoculation into wine, were studied. Finally, the cultures were tested in vinification trials in France, Portugal, Italy and Spain.

RESULTS AND CONCLUSION

The main goal of the project, to develop malolactic cultures of Leuconostoc oenos which are able to survive, grow and perform malolactic fermentation after direct inoculation into wine,. has been achieved. At the same time, considerable additional information has been obtained covering aspects of phage, growth dynamics, sugar and acid metabolism, the effects of the toxic components in the wine and the interaction between yeast and bacteria. The importance of changes in membrane composition that can be induced by the medium has increased considerably, but a clear causal relation to the adaptation of cultures to the high ethanol environment has not yet been established. DNA studies revealed different genetic groups among the selected strains, but the genetic background of the adaptation to the wine environment and how this adaptation may be stabilised or increased is still not understood. However, substantial modifications were observed in the tRNA when the malolactic fermentation was stimulated.

EXPLOITATION PLANS

Chr. Hansen's has commenced a development project to commercialise the selected strains. These will be the first malolactic cultures for direct inoculation after the alcoholic fermentation.

PARTICIPANTS

Institut d'Oenologie, University of Bordeaux II (France), Estacao Vitivinicola Nacional and Centro de Tecnologia Química e Biológica (Portugal), the Istituto di Genetica, University of Bologna (Italy).