Can malolactic fermentation enhance volatile phenol production by Brettanomyces?

by | Apr 1, 2021 | Oenology research, Winetech Technical

The short answer

Yes, it can. Researchers from Oregon State University found that the choice of malolactic fermentation (MLF) starter culture can influence Brettanomyces spoilage in Pinot noir wines.1 The reason? Certain lactic acid bacteria (LAB) display enzymatic activities that can generate more substrate for Brettanomyces to convert to volatile phenols.

Practical advice

In cellars with a risk of Brettanomyces:

  • If commercial MLF starter cultures are used, avoid using cinnamoyl esterase positive (CE+) strains in Pinot noir.
  • Avoid spontaneous malolactic fermentation (MLF) in Pinot noir since the bacteria conducting the MLF can potentially be CE+.

The long answer

Brettanomyces forms 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) in a two-step process from pcoumaric acid and ferulic acid, respectively. These hydroxycinnamic acids are naturally present in grape juice, but mostly bound to tartaric acid to form the esters: pcoutaric and fetaric acids. Brettanomyces cannot metabolise the tartaric acid esters; it can only metabolise the “free” form of the hydroxycinnamic acids to form the dreaded, foul smelling ethyl phenols. That means, for Brett to really flourish, it needs to get by with a little help from its (microbial) “friends”, in this case: lactic acid bacteria. Certain strains of lactic acid bacteria (LAB) possess cinnamoyl esterase (CE) enzymatic activity that can hydrolyse the tartaric acid esters, to release the free forms of the hydroxycinnamic acids, i.e. Brett substrate.

It should be noted that commercial wine enzyme preparations can sometimes also contain CE activities that can lead to increased concentrations of free hydroxycinnamic acids.2

The research objectives

The first objective of the study was to establish what the impact is of the specific Oenococcus oeni strain on the pcoutaric and pcoumaric acids concentrations of Pinot noir and eventually, the effect on volatile phenol production by Brettanomyces bruxellensis.

However, it has been found by previous studies that chemical hydrolysis, happening slowly over time as the wine ages, can also release hydroxycinnamic acids from their esterified forms. Brett is a “survivor” and lurks in wines, while it ages until conditions gradually become more favourable, which includes more substrates becoming available.3

The researchers therefore decided to add a second objective to the study, namely to determine the impact of acid hydrolysis over time on pcoutaric and pcoumaric acids concentrations of Pinot noir and eventually, the effect on volatile phenol production by Brettanomyces bruxellensis.

 Project layout

The bacteria used in the study comprised Viniflora oenos (Chr. Hansen) and Enoferm Alpha (Lallemand). Both are commercial MLF starter cultures. The former is a CE+ strain and the latter a CE- strain.

Small scale winemaking was conducted using a commercial yeast and yeast nutrient. After alcoholic fermentation, the wines were pressed and sterile filtered before inoculation with the two MLF starter cultures, respectively. After the completion of MLF, portions of the wines had pH and alcohol adjustments before being sterile filtered and bottled. Wines were stored at 13 and 21°C for 180 days. The aim of the pH and alcohol adjustments, as well as the two different storage temperatures, was to evaluate the impact of these factors on chemical hydrolysis.

After 180 days of storage, wines were inoculated with a Brettanomyces culture from the AWRI culture collection. Volatile phenol concentrations were quantified after eight weeks.

Results and discussion

Malolactic fermentation was completed for both strains within 21 days. There was no significant change in the pcoutaric and pcoumaric acids concentrations for the CE- strain. Meaning no additional substrate was created for Brett. In contrast, for the wines that underwent MLF with the CE+ strain, a decrease in pcoutaric acid and an increase in pcoumaric acid concentrations were noted. Meaning an increase in substrate for Brett was created.

In terms of acid hydrolysis, there was no significant change between the pcoutaric and pcoumaric concentrations after 180 days, regardless of pH, alcohol and storage temperature. This only means that under the conditions tested in this research project it did not happen, since previous research under different conditions does indicate that it can happen. One such study has indicated that after nine months of ageing up to 60% of the initial hydroxycinnamic acid esters remain.4 Whereas in this study, almost 100% of the pcoutaric acid was converted to pcoumaric acid within six months, indicating that enzymatic hydrolysis is both faster and more efficient than chemical hydrolysis.

Wines that underwent MLF with the CE- strain had lower 4-EP and 4-EG concentrations than wines that underwent MLF with the CE+ strain. Therefore, the wines with the higher volatile phenol concentrations reflect the wines with the higher concentrations of available substrates for Brettanomyces growth.

What this study tells us

If a Pinot noir wine has a risk of being contaminated by Brettanomyces, it is probably wise to avoid CE+ MLF starter cultures during production.

It is also wise to avoid spontaneous MLF on Pinot noir at risk of Brettanomyces spoilage, since one has no idea what LAB will be performing the MLF. It could involve various bacteria species and strains that could have CE activities.

What this study does not tell us

It does not tell us to avoid CE+ MLF starter cultures altogether. Many starter cultures that are CE+ are used with great success worldwide, including Viniflora oenos. It is only to be avoided in wines at risk of Brettanomyces spoilage.

It also does not tell us to avoid CE+ strains in all red cultivars, only Pinot noir. A previous study5 done on Cabernet Sauvignon comparing the impact of a CE+ and a CE- strain on volatile phenol formation by Brett, revealed no difference between the two strains in terms of final volatile phenol levels. There was, however, an initial difference in the free hydroxycinnamic acid concentrations, with the CE+ strain wine containing higher concentrations.

Other red cultivars, such as Cabernet Sauvignon, generally have more colour (anthocyanin) than Pinot noir. Hydroxycinnamic acids, such as pcoumaric acid, can form complexes with anthocyanins to form stable pigments, thereby making the hydroxycinnamic acids unavailable for Brett metabolism. In fact, studies have noted decreases in pcoumaric acid over time, independent of Brettanomyces growth, which they attributed to co-pigmentation with anthocyanins. Unfortunately, it has been shown that Pinot noir has very low levels of co-pigmentation, which was confirmed in the Oregon State University study.

Practical advice

The same as for the short answer.

Acknowledgements

The author would like to thank Prof. Maret du Toit and Prof. Benoit Divol from the South African Grape and Wine Research Institute, Department of Viticulture and Oenology, Stellenbosch University for a critical review of this article.

References

  1. Du Bois, A., Shelton, M., Qian, M. & Osborne, J., 2021. Effect of malolactic fermentation and ageing on the concentration of pcoumaric acid of Pinot noir wine and the consequence for volatile phenol production by Australian Journal of Grape and Wine Research 27: 81 – 86. https://doi.org/10.1111/ajgw.12464
  2. Dugelay, I., Sapis, J.C., Baumes, R.L. & Bayonnove, C.L., 1993. Role of cinnamoyl esterase activities from enzyme preparations on formation of volatile phenols during winemaking. Journal of Agricultural and Food Chemistry 41: 2093 – 2096.
  3. Smith, B.D. & Divol, B., Oct. 2016. Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. Food Microbiology 59: 161 – 175. https://doi.org/10.1016/j.fm.2016.06.008
  4. Kallithraka, S., Salacha, M.I. & Tzourou, I., Mar. 2009. Changes in phenolic composition and antioxidant activity of white wine during bottle storage: Accelerated browning test versus bottle storage. Food Chemistry 113(2): 500 – 505. https://doi.org/10.1016/j.foodchem.2008.07.083
  5. Madsen, M.G., Edwards, N., Petersen, M.A., Mokwena, L., Swiegers, J.H. & Arneborg, N., 2017. Influence of Oenococcus oeni and Brettanomyces bruxellensis on hydroxycinnamic acids and volatile phenols of aged wine. American Journal of Enology and Viticulture 68, 23 – 29. https://www.ajevonline.org/content/68/1/23

– For more information, contact Karien O’Kennedy at karien@winetech.co.za.

 

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