ENFOQUES OTROS ENFOQUES  
Entrevista a Sibylle Krieger-Weber

Some yeast metabolites can be used as pre-cursors for the production of aromatic compounds

Algunos metabolitos de las levaduras pueden ser usados por las bacterias malolácticas como precursores para la producción de compuestos aromáticos

Sergi Ferrer
ENOLAB. Universitat de València

Dr. Krieger studied Food Science at the University of Hohenheim, Germany, and in 1984, she received a Masters Degree entitled "Malolactic Fermentation In Wine" from LVOW Weinsberg. Upon completion of her Masters Degree, she accepted employed for two years at the QC laboratory of the central cooperative in Württemberg WZG. In 1989, Dr. Krieger was awarded a PhD from Universität Hohenheim for her original work entitled “Malolactic Starter Culture Preparations For induction Of MLF In Wine". In 1990, she completed her post doctoral studies at the New York state Agricultural Experiment Station/Cornell University, and accepted employment at Gewürzmüller GmbH Stuttgart, where she until 1998. While employed at this institution, Dr. Krieger performed the following duties: research, development and marketing of Bitec starter cultures for MLF, microbiological laboratory manager, R&D biotechnology manager, assistant manager of the Gewürzmüller quality control laboratories and manager of the Bitec malolactic bacteria starter culture plant. In 1999, Dr. Krieger joined Lallemand, where she has been responsible for malolactic bacteria starter culture research and development as well as technical support of the wine markets worldwide.     

      

From your point of view, what is the importance of the MLF nowadays?

Depending on the climate zone and the particular vintage: There are still cool climate regions like Germany, like north of France (Champagne, Loire), some regions in Northern Italy and North America where de-acidification - biological de-acidification - is important not to say crucial for the balance and overall wine quality. Just to memorize the 2010 vintage in Europe, which was a year with high must acidity in most wine growing regions and with particular high malic acid levels. Under these conditions wine pH is low and spontaneous MLF does not happen frequently and controlled induction of malolactic fermentation with robust and active ML starter cultures is the only practice to achieve MLF. As a reminder in the vinification of Champagne base wine still standard cultures are applied, which need two step acclimatization. Because direct inoculation with freeze-dried ML starter cultures will fail under these low pH, high acidity and low nutrient conditions, and only acclimatized bacteria will induce a successful MLF, which is needed for acidity balance but also to avoid a spontaneous undesired MLF during secondary yeast fermentation.

Very different to the hotter climates, like to South of Europe, California, South Africa, South America and Australia – in these climates malic acid levels are generally low and pH high – thus, importance of microbiological stability and sensory impact of plays a more and more important role. Especially in the New World wine growing regions a strict pH management in the grape must is applied to avoid spontaneous MLF during alcoholic fermentation and to induce controlled MLF post AF. That’s the New World answer to their experience with uncontrolled MLFs under high pH conditions during AF, which resulted in the past frequently in stuck AFs and high VA levels. The hotter climates in the old world practice a less strict pH managements and in some minor cases Lysozyme is applied to avoid spontaneous MLF and more recently early inoculation with selected ML bacteria 24 h after the yeast addition or at the mid or 2/3 rd of AF is applied to avoid a spontaneous MLF, which not only can trigger AF and increase VA, but also produce biogenic amines or other metabolites which can be hazardous to human health or negatively impact on the total wine quality.

 

How is it perceived around the world (I mean from the enologists’ point of view)? Do you see geographical differences?

There are certainly geographical differences. In the New World – MLF is integral part of the vinification strategy and MLF is induced with a high frequency by a known strain, despite they use numerous protocols to increase the bacteria biomass (and to dilute the seeding rate) applying pied de cuve multiplication steps or cross-seeding with tanks undergoing MLF, … that’s worth to write a book chapter on.

In the old world MLF is rather perceived a phenomenon, which arrives, when it wants, and generally does not cause obvious problems. But some regions suffer for example from quite high biogenic amines levels after MLF. This also leads to typical comments such as –“It doesn’t go when I want it to”, or more rarely: “I don’t like what it does to the wine. These winemakers tend also to wait for the spontaneous MLF to take place, and if it does not start after weeks or even month of heating and pump-over, they decide to inoculate the wine finally with selected starter cultures. But in such a wine situation it is obvious, that the wines have some extremely limiting parameters or even inherit toxic compounds which inhibit not only spontaneous bacteria but also selected bacteria.

In the rather cooler climates of the old and the new world, winemaker have the experience and accept, that spontaneous MLF doesn’t occur regularly, but MLF is desired for de-acidification and/or as a stylistic tool. In these regions inoculation with selected ML bacteria starter cultures is part of the vinification protocol. Co-inoculation – inoculation with ML bacteria shortly after the yeast, or at an early stage of vinification is getting more popular, due to the possibility to achieve an acclimatization of the bacteria during AF and to induce a quick MLF right at the end of alcoholic fermentation.

Under higher pH conditions – with some delay – co-inoculation strategies are also gaining more popularity, but for other reasons: early dominance of the selected strains and effective suppression of the undesired spontaneous bacteria flora, acclimatization to very high alcohol levels and for stylistic impact – more fruit driven wine styles.

 

 How can the climate change affect the enological practices related to MLF?

pH and alcohol belong to the main factors impacting not only on MLF and bacteria viability and malolactic activity, but also on the composition of the microbial flora in must and wines, And these two parameters, pH and alcohol level, are highly affected by the climate.

At low pH (< pH 3.1), under cool climate conditions,  MLF does not occur easily and if does, then it is mainly driven by Oenococcus oeni strains, which have generally a less critical potential with regard to negative impact on wine quality and safety. Under these conditions mostly MLF is induced by selected ML starter cultures and the lower the pH the more specific will be also the acclimatization protocols. Thus, there is little risk of “real” spoilage microorganism and even within yeast population there is a little diversity

At low to moderate pH (3.1. to 3.4): spontaneous ML doesn’t always occurs, again it is mainly driven by O. oeni strains and inoculation is frequent. Brettanomyces can occur and spoil the wine especially, if there is a gap between the end of alcoholic fermentation and the start of malolactic fermentation. Thus, if Brettanomyces infections are a known phenomena in a winery, fast inoculation with selected bacteria should be practiced along with a prompt stabilization of the wine post MLF.

At moderate to high pH (pH 3.5-3.6) spontaneous MLF occur frequently, and in certain cases critical non-Oenococcus lactic acid bacteria strains will drive the ML fermentation. Control is difficult since very often spontaneous MLF occurs uncontrolled before the end of AF. The winemakers, especially in the old world, tend to control the situation often by higher SO2 addition, which can, if used in too excessive amounts avoid completely spontaneous MLF, but also limit success of an induced MLF. This situation will give way to Brettanomyces, which can tolerate higher levels of SO2 and which often lives in perfect symbiosis with Pediococcus sp. and/or hetero-fermentative Lactobacillus strains, such as Lactobacillus hilgardii. The symbiosis of these microbes, does not only lead to undesired aromatic phenols (“Brett” aroma), but also high VA levels and biogenic amines. Winemakers in the new world tend to decrease the pH to have a better control over the microbial flora. Lysozyme or co-inoculation strategies can be used as tools to control better the situation. Early stabilization of the wines after malolactic fermentation is obligatory to avoid secondary infection with Brettanomyces or other LAB or acetic acid bacteria.

High pH (pH > 3.6) normally allows a very fast MLF during or right after alcoholic fermentation, very often driven by obligatory hetero-fermentative Lactobacillus strains such as Lb. brevis (high VA and biogenic amine producer) and/or Pediococcus sp., which can produce biogenic amines our undesired exo-polysaccharides. The microbial situation is difficult to control and the only limiting factor is in most cases the very high alcohol content in these wines. pH and SO2 management is needed effectively manage this situation, in combination with earlier inoculation strategies to dominate the microflora and to adapt the selected bacteria to the high alcohol levels. Good management practices for the alcoholic fermentation (robust yeast strain and good nutrition strategies for the yeast) are crucial as fast and efficient stabilization of the wines post AF.

 

Nowadays, ‘couples’ of yeasts and bacteria are becoming more popular. Is it a choice or a tendency for the future?

The yeast, which is running the alcoholic fermentation defines to a large extend the wine matrix the bacteria will have to work in. In two senses this can be important for the out-come of the MLF and the wine quality:

    • Some yeast can produce specific metabolites which can make the environment more difficult up to un-fermentable for the ML bacteria: high SO2 production, production of medium chain fatty acids, anti-microbial peptides, and high levels of succinic acid or phenyl ethanol, or depletion of key-nutrients such as amino acids, vitamins and minerals.
    • Other yeast can stimulate MLF by production of metabolites or enrichment of the environment by early autolysis and release of key nutrients.

Or, and that’s most recent findings:

    • Some yeast metabolites can be used as pre-cursors for the production of aromatic compounds such as esters, which can increase for example the red fruit berry aromas in red wines.

Thus, we will have in future more recommendation for the best pairing of yeast and bacteria for different reasons:

    • Best pairing of yeast and bacteria for best success of both fermentation under most difficult conditions.
    • Best pairing of yeast and bacteria for specific stylist goals.
    • Or to achieve a desired aroma profile of the wine.
    • Most probably this will also include a defined nutrition strategy.

     

After all these years, which do you consider has been the impact of malolactic recombinant yeasts? And in the future?

In the past, although such a yeast had been already introduced in some markets e.g. USA and Eastern Europe – NONE. The yeast is not appearing in the wineries. Two explanations will apply:

    • The consumer doesn’t accept GMOs in winemaking.
    • Using a yeast degrading malic acid results in a wine without malic acid, but not in a wine with MLF.

The last point will also play an important role in a possible future application, when GMOs might be accepted for application in wine:

    • Malolactic fermentation is not only the degradation of malic acid to lactic acid and CO2.
    • Malolactic fermentation slightly modifies the wine pH.
    • Malolactic fermentation will also impact on the sensory profile of a wine by the production of secondary metabolites such as diacetyl, ethyl lactate, butandiol, polysaccharides, fruit esters, volatile sulfur compounds … depending on the ML strain applied or the inoculation strategy involved.
    • Some strain can add complexity and increase the mouthfeel of the wines.
    • A wine after MLF has a higher microbiological stability, due to the consumption of key nutrient by the ML bacteria, which is conducting the MLF, or by the production of inhibitory compounds towards other microorganisms. Wines after MLF are e.g. less suitable for a Brettanomyces infection.

To conclude: also in the future the impact of Malolactic recombinant yeasts, even when officially authorized, will be limited and minor.

 

The actual use of malolactic bacteria as starters in cellars is still far from that of yeasts. Do you think that oenologists are changing their mind, or will this difference be kept in the future?

The difference will become smaller - that for sure, and much more selected ML bacteria will be applied, but I doubt that we will achieve the global use as we have it for the selected wine yeast.

    • There is a defined amount of wine, which will be not considered for undergoing MLF, such as certain aromatic white wines with a higher acid profile, some fruit and acid driven fresh rosé wines.
    • Since MLF is always the second fermentation and come after alcoholic fermentation, spontaneous MLF still can interfere and drive the malolactic fermentation before a wine can be inoculated. Spontaneous MLF will be always the strongest competitor to a controlled MLF induced with selected ML starter cultures.

We can see a clear trend to use more selected ML bacteria starter cultures. This can be also explained with the much better quality of the ML starter culture nowadays compared to the liquid starter cultures, which had been used in the late 80ies and early 90ies. Today’s freeze-dried ML starter cultures are easy to use and robust with regard to the resistance to the environmental limiting parameters. The most used starter strains will successfully work over a large range of limiting wine conditions.

And for quality and security reasons we need and we will have much more induced and controlled MLFs:

    • Good manufacturing practices, which will apply, and ask for minimizing the levels of hazardous compounds in wine such as biogenic amines, acetaldehyde, and high SO2 levels.
    • Specific aroma profiles or styles e.g. “buttery Chardonnay” or red berry fruit driven Cabernet Sauvignon wines will ask for specific yeast and bacteria combinations and vinification strategies.
    • Increasing energy cost, which ask for cost reduction and to minimized use of energy sources – thus, e.g. to avoid long-term cellar heating while waiting for a spontaneous MLF and to aim for a fast stabilization of the wine post MLF.
    • Security that the winemaker controls the fermentation and not the microbes.
    • Big discounters ask for a constant quality and aroma profile of their wines, thus it will become crucial to control both fermentations the AF and the MLF to assure the defined quality the consumer asks for.

 

A rapid recommendation on how to manage properly a MLF?

  • Select the appropriate ML starter culture, which will work best at your most limiting wine conditions.
  • Manage your pH and avoid pHes far above pH 3.6.
  • Manage your alcoholic fermentation and avoid yeast stress.
  • Select the appropriate yeast strain, which is compatible with your ML strain.
  • Co-inoculation will allow you to overcome limiting wine conditions and to achieve an early dominance of the selected ML bacteria strain.
  • Avoid high temperature > 24 °C when alcohol levels are > 14.5%vol or SO2 levels > 50 ppm.
  • Don’t forget bacteria nutrition, especially when the wine had been highly clarified, a yeast was used for alcoholic fermentation, which has a high nutrient demand or other limiting wine conditions apply.

 

What needs of innovation do you detect in this area? What the future will bring about MLF?

  • Robust ML bacteria strains which work at most limiting wine conditions, when inoculated directly without acclimatization.
  • ML bacteria strains with very specific sensory properties to contribute in a specific way to the wine aroma profile.
  • ML bacteria starter strains with a fast dominance.
  • Non-Oenococcus starter cultures.
  • Lactic acid bacteria starter cultures, which can eliminate or degrade toxic compound in wine.
[21.12.11]
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