WINETECH Technical Yearbook 2021

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 p- coutaric and p- coumaric acids concentrations for the CE- strain. Meaning no additional

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 study (Madsen et al ., 2017) 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.

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 (Dugelay et al ., 1993). THE RESEARCH OBJECTIVES The first objective of the study was to establish what the impact is of the specific Oenococcus oeni strain on the p- coutaric and p- coumaric acids concentrations of Pinot noir and eventually, the effect on vol at i l e phenol product ion 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 (Smith & Divol, 2016). The researchers therefore decided to add a second objective to the study, namely to determine the impact of acid hydrolysis over time on p- coutaric and p- coumaric acids concentrations of Pinot noir and eventually, the effect on volatile phenol production by Brettanomyces bruxellensis.

substrate was created for Brett. In contrast, for the wines that underwent MLF with the CE+ strain, a decrease in p- coutaric acid and an increase in p- coumaric 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 p- coutaric and p- coumaric 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 (Kallithraka et al ., 2009). Whereas in this study, almost 100% of the p- coutaric acid was converted to p- coumaric 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.

WINETECH TECHNICAL YEARBOOK 2021 | 68