Utilisation of nitrogenous compounds by commercial non-Saccharomyces yeasts

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.

Project 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 thermotolerance (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 happened 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 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 and thereby allowed fermentations to almost reach dryness.
• Frootzen fermentations contained the highest concentrations of 2-phenylethyl-acetate (floral aroma) and isoamyl acetate (fruity, banana aroma).

Significance of the study:
The study confirms once again the importance of measuring YAN before making any decisions with regards to fermentation. The study also reveals that non-Sacch yeasts can compete with the S. cerevisiae yeasts for nutrients, thereby affecting the possibility of fermentations to complete. It is therefore very wise to supplement musts, where non-Sacch yeasts initiated the fermentation, with yeast nutrients.

Saccharomyces cerevisiae strains are starved for nitrogen during 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. However, when non-Sacch yeasts start the fermentation, the YAN can be very low by the time the commercial 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 and depending on the prevailing must conditions, an organic nutrient, a complex yeast nutrient or plain DAP will be the best options. The standard addition of DAP to all musts, regardless of YAN, micronutrient content, or yeasts conducting the fermentation, is extremely unscientific and not conducive to optimising wine quality.

Reference:
Kelly J. Prior, Florian F. Bauer, Benoit Divol (2019). The utilisation of nitrogenous compounds by commercial non-Saccharomyces yeasts associated with wine. Food Microbiology Vol. 79 (75-84). https://doi.org/10.1016/j.fm.2018.12.002