Certain selected wine bacteria act as biocontrol tool against Brettanomyces

Biocontrol of contaminating microorganisms with certain selected wine bacteria is a secure and biological option for winemakers. By carefully managing alcoholic fermentation with properly rehyrdrated and nourished yeast, and by using a selected wine bacteria in co-inoculation or in sequential inoculation, the population of Brettanomyces, and subsequently the production of phenolic off-odors is better controlled.

What is biocontrol ?

 

The concept of biological control or protection also called « biocontrol » is a method known and applied since the beginning of agriculture even without knowing it. Agriculture development was necessarily linked with the need to protect cultures with various methods including biocontrol. With the development of chemistry applied to the agri-food sector biological control tools were put aside for many years. With a growing awareness of the environmental and health issues due to the use of chemicals, alternative methods such as biocontrol were studied again with improved knowledge and scientific approach. Biocontrol appears to be a great natural way to protect crops against pests and diseases.

 

Where does it originate ?

 

The principle of biocontrol is founded on the management of the balance of negative populations more than their eradication. Biocontrol tools encourage the use of natural mechanisms and interactions to the relations between species in a media. 

In food processing, the concept of food preservation with protective flora is more then 40 years old concept. The purpose was to extend shelf life and to limit the risks of a microbiological contamination: a great alternative to chemical food preservatives. Now the applications fields are various and we can find biocontrol tools in cheese, bread, beverages, etc. 

 

What does it mean in wine ?

 

In wine, the use of SO2 was an amazing improvement regarding product quality mainly because of its antifungal and antibacterial activity. But nowadays there is an increasing willingness for reducing chemicals in winemaking and SO2 is obviously one of them. It remains an excellent tool to reduce spoilage population during the whole winemaking process, such as oxidative yeast flora, spoilage bacteria, and of course Brettanomyces which is still the number one enemy in wine. But recent studies (Curtin et al. 2012 ; Vigentini et al. 2013 ; Albertin et al. 2017) showed that there is a high genetic diversity in Brettanomyces and among them there is a significant number of Brettanomyces bruxellensis strains able to resist and survive to SO2 (figure 1). AWRI (Curtin et al., 2012) demonstrate that 85% of the Brettanomyces population of their study was able to survive and grow at 0,6 mg/L of molecular SO2. Albertin et al. (in 2016) identified 34% of B. bruxellensis strains among a collection of 33 strains as highly SO2 resistant.

 

 

Can selected bacteria be used as biocontrol ?

 

This kind of resistance would result in very high levels of final SO2 in wines to reach the lethal level needed to eradicate such Brettanomyces population. Those results show the limits of SO2 as a Brettanomyces control tool and highlight the need of alternative biocontrol tools to handle this risk. Yeast early inoculation (Gerbaux et al., 2004) is already known as a very efficient biocontrol tool against Brettanomyces growth. More recently it was found that some effective selected wine bacteria can also be considered as an efficient biocontrol tool to handle Brettanomyces contamination and volatile phenols levels in wine. These volatile compounds and mainly 4-ethylguaiacol (4-EG) and 4-ethylphenol (4-EP) are responsible of off-flavors in wines. The proper inoculation of selected wine bacteria practice is also recognized by OIV (RESOLUTION OIV-OENO 462-2014) to reduce the growth of Brettanomyces during the winemaking process. It has been found that certain selected wine bacteria are considered as efficient biocontrol tools against spoilage micro-organisms to protect the wine quality.

 

The results

 

Biocontrol with selected wine bacteria against growth of Brettanomyces

 

Previous studies showed the clear impact of early inoculation of MLB on the reduction in final volatile phenols levels (Gerbaux et al. 2009 ; Pillet et al. 2011). It is known that the lag period between the end of alcoholic fermentation and the start of malolactic fermentation (MLF) is critical for spoilage microorganisms such as contaminant bacteria and Brettanomyces. In 2014, OIV recognized the co-inoculation of selected yeasts and selected lactic bacteria could help to reduce this lag phase and consequently limit the development of Brettanomyces. Recent research projects have studied if in addition to the reduction of this lag phase, some selected bacteria can have a direct inhibition on Brettanomyces growth. Research done in collaboration with IFV in Burgundy in 2015 and 2016 in Pinot showed the dynamics of the microorganism’s population in wines inoculated just after alcoholic fermentation with Brettanomyces and then with various selected bacteria (1g/hL) at temperature between 16 and 18°C.

The results showed that there is a clear inhibition of Brettanomyces growth from selected bacteria for both vintages. In the figure 2 we can see the Brettanomyces population measurements in 2016 with 3 different selected bacteria compared to control where no bacteria were inoculated and in which MLF occurs spontaneously. The results were the same when Brettanomyces was at 103 cfu/mL.

 

 

The inoculation and the growth of those 3 selected bacteria limit significantly the development of Brettanomyces even if the Brettanomyces contamination was strong at the beginning. Final levels of Brettanomyces in presence of the selected bacteria was more or less the same level as initial (between 102 and 103 cfu/mL) whereas in the control with indigeneous bacteria (spontaneous MLF), final level of Brettanomyces is much higher (105) with a peak at 106 cfu/mL, i.e. a difference of more than 2 to 4 log.

Volatile phenols were measured at the end of the experiment in all the treatments (figure 3) and the wine in which MLF occurred spontaneously showed high levels of both 4-ethylphenol and 4-ethylgaïacol, above the perception threshold whereas the wines in which the selected bacteria were inoculated had very low levels of both phenols, close to zero.

 

 

Those differences in volatile phenols are clearly linked with the differences in population and show once again the real benefit of a biocontrol of Brettanomyces thanks to the inoculation of some selected bacteria, also in the wine quality.

This work was confirmed by another study in 2016 in Pinot Noir. Four different selected bacteria were used in coinoculation (24hrs after yeast inoculation ) in a must initially contaminated with 1.7 x103 cfu/mL of Brettanomyces MLF finished in 20-40 days whereas the uninoculated control finished in more than 60 days.

Fig 4a and fig 4b illustrate the evolution of population of Brettanomyces and bacteria : in the case of co-inoculation with selected bacteria (fig 4a : results shown are an average of all the treatments with the selected bacteria) and in the case of the control - spontaneous (fig 4b). We can clearly see on the figure 4a that there is no growth of Brettanomyces population (even with this strong contamination) and this contaminant population decreases from the beginning to the end in opposition of the population of the selected bacteria growing at the same time. At the opposite in the figure 4b, Brettanomyces population maintains a high level until the 11th days (date of the rack off) and there is a regrowth due to the slow development of spontaneous bacteria population. Final levels are significantly different between the wines in co-inoculation and the control : there are 10 times more Brettanomyces in the control than the co-inoculated wines (more than one log ufc/mL of difference). Those results confirm the strong competition between our selected bacteria and a population of contaminant yeast such as Brettanomyces, especially co-inoculation thanks an easier growth of the selected bacteria and an excellent survivability of those bacteria.

 

 

Biocontrol of certain selected bacteria against release of off-flavors precursors

 

With Brettanomyces, 4-ethylguaiacol (4-EG) and 4-ethylphenol (4-EP) are produced during the biotransformation of the hydroxycinnamic acids, p-coumaric acid and ferulic acid, which are precursors naturally present in grapes in the bound or free form. The transformation of these free precursors into 4-EG and 4-EP (figure 5) occurs in two steps: first with the cinnamate decarboxylase enzyme, followed by the vinylphenol reductase enzyme. But before Brettanomyces can use those precursors, they must be release from their bound form to the free form by the action of cinnamyl esterase.

 

 

Certain lactic bacteria, including Oenococcus oeni, have this cinnamyl esterase enzymatic activity and could therefore increase the quantity of free precursors, made usable by Brettanomyces to produce volatile phenols (Burns and Osborne 2013). Their results showed that depending on the wine bacteria used for MLF, different concentraton of free precursors resulted.

This signifies that when using a wine bacteria, it must be cinnamyl esterase negative in order to avoid the production of the precursors of volatile phenols that would be used by Brettanomyces.

All our wine bacteria have been screened and we can confirm that they are all phenol negative as shown in Figure 6, from a study done by J.Osborne (OSU).

 

 

In summary

 

Biocontrol of contaminating microorganisms with certain selected wine bacteria is a secure and biological option for winemakers. By carefully managing alcoholic fermentation with properly rehyrdrated and nourished yeast, and by using a selected wine bacteria in co-inoculation or in sequential inoculation, the population of Brettanomyces, and subsequently the production of phenolic off-odors is better controlled. When the level of contamination from Brettanomyces is high, it is better to co-inoculate since the biocontrol will start right at the beginning of fermentation.

Furthermore, our selected wine bacteria have been confirmed as phenol-negative, and can't supply to Brettanomyces the precursors to produce the off odors, 4-ethyl phenol and 4-ethyl guaiacol.

Biocontrol throught the protection of our selected wine bacteria, along with denying Brettanoymces its precursors are winning combination to respect the wine typicity by letting it fully be expressed and without the 'Brett' faults.

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Author:
Lallemand Oenologie
Winemaking with wine yeast and bacteria
Sud-Ouest - France

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