The utilisation of nitrogenous compounds

PHOTO: Shutterstock.

Aim of the study

The study investigated the preferences and order of uptake of nitrogen sources for various commercially available non-Saccharomyces yeasts. The influence on growth and fermentation kinetics, as well as aroma formation, was also investigated. The study was, however, restricted to major secondary aromas only (i.e. those arising from the metabolism of sugar and certain amino acids). It is important to note that for some of the yeasts mentioned in the study, the aim of their selection for commercialisation was not their fermentative performance, but rather their specific enzymatic activities or metabolisms. The study results provide winemakers with insights into the nutritional dynamics of multi-yeast fermentations.

Experimental layout

Five commercial yeasts were used in the study:

• EC 1118 – Saccharomyces cerevisiae (Lallemand).
• Viniflora Frootzen – Pichia kluyveri (Chr. Hansen).
• Biodiva TD291 – Torulaspora delbrueckii (Lallemand).
• Flavia MP346 – Metschnikowia pulcherrima (Lallemand).
• Viniflora Concerto – Lachancea thermotolerans (Chr. Hansen).

Fermentations were performed in three different synthetic grape musts with a total YAN of 200 mg/L:

• Treatment 1 – Amino acids in equal amounts of assimilable nitrogen levels plus ammonium.
• Treatment 2 – Amino acids in equal amounts of assimilable nitrogen levels without ammonium.
• Treatment 3 – Grape must-like nitrogen concentrations.

Pure culture fermentations of all five yeasts were conducted, as well as co-inoculation treatments, where EC 1118 was inoculated 48 hours after non-Sacch inoculation.

An additional treatment included filtering out the non-Sacch yeasts before EC 1118 inoculation.

Main results

• In the pure culture fermentations, only EC 1118 fermented to dryness.
• Nitrogen source uptake started before the onset of active growth for all strains.
• EC 1118 had the highest CO₂ release (most active fermentation), followed by Concerto and Biodiva.
• Ammonia did not affect CO₂ release. There were also no significant differences in residual sugar with or without the presence of ammonia.
• All strains consumed glucose faster that fructose.
• EC 1118, Concerto and Biodiva had similar sugar consumption rates in the first 48 hours, which were much faster than Frootzen and Flavia.
• Depending on the yeast species, ammonia consumption occurred at different rates and different amounts.
• 18 hours after inoculation, EC 1118, Concerto and Biodiva consumed almost all of the ammonia (less than 5 mg/L remaining). Frootzen completed ammonia consumption 24 hours after inoculation and Flavia took longer than 48 hours after inoculation.
• Amino acid consumption started at about six hours after inoculation for all yeasts. Amino acids were taken up in different order of preference for the different yeasts.
• Interestingly, Frootzen and Flavia first released certain amino acids before uptake began.
• EC 1118, Concerto and Biodiva took up most amino acids by 48 hours after inoculation leaving the synthetic must with a YAN of 6 mg/L or less (treatment 3).
• After 48 hours, Frootzen and Flavia synthetic musts contained YAN’s of 54 and 41 mg/L (treatment 3).
• The more natural must-like medium (treatment 3) did not provide better fermentation support compared to treatments 1 and 2.
• When EC 1118 was co-inoculated 48 hours after Frootzen and Flavia inoculations, the fermentations went to dryness in the filtered (wild yeasts were removed before S. cerevisiae inoculation) and unfiltered synthetic musts. This indicates the favourable impact of these yeasts on EC 1118 to complete the fermentation (with 54 and 41 mg/L YAN).
• In the cases of Concerto and Biodiva/EC 1118 co-inoculations, the filtered must got stuck, indicating not enough nutrients for EC 1118 to complete the fermentation. In the unfiltered must, the non-Sacch yeasts seemed to release some nutrients back into the medium, thereby allowing fermentations to reach dryness. This is most certainly linked to autolysis of the non-Sacch as they die off after Sacch inoculation.
• Frootzen fermentations contained the highest concentrations of 2-phenylethyl-acetate (floral) and isoamyl acetate (fruity, banana).

In conclusion

The study confirms once again the importance of measuring YAN before making any decisions with regard to fermentation. The study also reveals that non-Sacch yeasts do compete with the S. cerevisiae yeasts for nutrients in a species-dependent manner, thereby sometimes affecting the possibility of fermentation to complete and the aromatic outcome. It is therefore very wise to supplement musts with yeast nutrients, whenever non-Sacch yeasts initiated the fermentation.

Saccharomyces cerevisiae strains are starved for nitrogen during the final stages of commercial production in order to stop budding formation before drying, since budding yeasts are very susceptible to heat damage. In order to grow in must, commercially-produced yeasts need nitrogen, which will be in the must as a result of the natural YAN. When non-Sacchs start the fermentation, the YAN can be very low by the time the commercial S. cerevisiae yeast is inoculated and not supportive of complete fermentations. All yeasts have different requirements for nitrogenous compounds. Suppliers are able to provide guidance on their yeasts’ requirements. The standard addition of DAP to all musts, regardless of YAN and micronutrient content of musts, and yeasts conducting the fermentation, is extremely unscientific and not conducive to optimising wine quality.

Acknowledgements

The study was funded by Winetech and the National Research Foundation.

Reference

Prior, K.J., Bauer, F.F. & Divol, B., 2019. The utilisation of nitrogenous compounds by commercial non-Saccharomyces yeasts associated with wine. Food Microbiology 79, 75 – 84. https://doi.org/10.1016/j.fm.2018.12.002.

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