Fermentation, malolactic monitoring

Estimating microbiological population density and diversity plays important and often pivotal roles at several junctures in the winemaking process. For instance, it is frequently necessary to determine changes in microbial populations during the preparation of starter cultures, growth and decline phases of malolactic fermentation, or monitoring potential Brettanomyces infections. 

M. Esti, G. Volpe, L. Micheli, E. Delibato, D. Compagnone, D. Moscone and G. Palleschi, Electrochemical biosensors for monitoring malolactic fermentation in red wine using two strains of Oenococcus oeni, Anal. Chim. Acta, 513(1) (2004) 357-364. 

Figure 1.19 Mass spectra of acetaldehyde PFB-oxime (a), diacetyl mono PFB-oxime (b), acetoin PFB-oxime derivative (c), and o-chlorobenzaldehyde PFB-oxime (d) recorded in the GC/MS analysis of standard solution performed in positive ion chemical ionization mode using methane as reagent gas (reagent gas flow 1 mL/min ion source temperature 200 °C). Flamini et al., (2005) Monitoring of the principal carbonyl compounds involved in malolactic fermentation of wine by synthesis of 0-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine derivatives and solid-phase-microextraction positive-ion-chemical-ionization mass spectrometry analysis, Journal of Mass Spectrometry, 40, p. 1561. Copyright John Wiley Sons, Ltd. Reproduced with permission...

If the composition of a wine is monitored regularly from the end of malolactic fermentation until after bottling, the results of the anthocyanin assay decrease regularly. Free anthocyanins disappear completely after a few years, although the wine... 

When a winery considers investment in laboratory equipment, a compound microscope should be a priority. Microscopic capabilities allow winemakers to quickly monitor the progress of alcoholic and malolactic fermentations and to tentatively determine the source of microbiological problems. This chapter oudines basic microscopy as well as techniques to view wine microorganisms. 

Restriction polymorphism is not relevant for the identification of bacterial species. No profile type exists for Oenococcus oeni, for example, nor for each of the other species that are of interest in winemaking. This method seems better adapted to the differentiation of strains of the same species. It is thus easy to identify strains of O. oeni, following hydrolysis of their DNA by the Notl enzyme with rare restriction sites. This method is used to monitor development of inoculated selected strains, used during winemaking to promote malolactic fermentation. The restriction profile of the biomass collected in wine during malolactic fermentation is compared with that of the selected strain that was added (Gindreau et al., 1997 2003). 

PCR will soon have another application in our domain for the differentiation of strains of the same species. Random primers are used for the moment. In this case, the reactions amplify several zones of the bacterium genome. After electrophoresis, the amplification products furnish a profile that can be characteristic of the strain. The difficulty lies in finding the primers. The best adapted ones for recognizing strains must give a profile for each strain in a reproducible manner. Among the lactic bacteria in wine, this process has only been applied to strains of O. oeni. The main application is in monitoring selected bacteria for malolactic fermentation. 

Along with pH, temperature is certainly the factor that most strongly influences the malolactic fermentation speed of a properly vinified wine not excessively sulfited. This factor is also the most easily monitored and controlled. 

At the end of alcoholic fermentation, malic acid concentrations should be determined and monitored if necessary. Malolactic fermentation (MLF) normally occurs after the complete depletion of sugars. An early initiation of MLF is generally linked to alcoholic fermentation difficulties and insufficient sulfiting. In certain cases, the two fermentations take place simultaneously, even though the antagonistic phenomena between yeasts and bacteria tend to inhibit alcoholic fermentation. 

Since the late 1990s, it has become increasingly popular to run the wine off into barrel immediately (Section 12.7.2), as malolactic fermentation in wood has been shown to enhance aromatic complexity as well as the finesse of oak character. In fact, it is not known whether this undisputed improvement is due to the effect of bacteria on molecules released by the oak, or the fact that the new wine is stiU warm when it is put into barrel. The fact remains that, if red wines are to be barrel-aged, they should be run off into barrel as soon as possible. We now have all the necessary techniques to avoid the problems that led to the abandomnent of barrel-aging in the past blending, temperature control, analytical monitoring of fermentation in individual barrels, etc. 

The diminution of malic acid is also monitored in order to choose the ideal moment for definitively stabilizing the wine. Sulfiting at 3-8 g/hl (press wine) permits this stabilization. Before sulfiting, the wine is racked to eliminate a fraction of the bacteria with the coarse sediment. This operation should follow the complete depletion of malic acid. After its initiation, malic acid degradation is normally completed in several days. In rare cases, bacteriophages destroy bacteria (Section 6.5) they stop malolactic fermentation and several hundred milligrams of malic acid per liter may remain undegraded. In this situation, the wine must be reinoculated to restart the fermentation. 

Juice density is measured daily to monitor alcoholic fermentation kinetics. When the density drops to approximately 0.994-0.993, sngar concentrations are then measured daily to verify the completion of fermentation. Fermentation is considered complete when less than 2 g of redncing sugars per liter remain. The fermentors are then carefully topped off. Subsequent operations depend on whether malolactic fermentation is carried ont. 

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