Here is some useful information on preventing thiamine deficiencies from slowing your fermentations.
It is harvest time and the grapes have been picked and destemmed. Sugars are looking good, and the Yeast Assimilable Nitrogen (YAN) is on target. The recipe for a perfect vintage.
The grape juice is prepared and ready to ferment – what could go wrong? Fast-forward to the post-harvest celebration. The sounds of Méthode Cap Classique popping open can be heard over excited chatter as the bubbles flow.
The fires are lit, and everyone has the best time rejoicing in a successful harvest. Not so fast … and in stomps a sullen and puzzled winemaker. It is the fermentation tanks, and the bubbles are, in fact, not flowing.
The fermentations have become sluggish. With the risk of stuck fermentation, there is nothing to rejoice about as of yet. Perhaps there was something no one had considered. Something like vitamins, and specifically thiamine?
What is thiamine?
Thiamine (also known as vitamin B1) is one of the most essential micronutrients required by yeasts. They need this water-soluble vitamin to grow, reproduce and, more importantly for winemakers, ferment. Unlike us mammals, most yeasts, including Saccharomyces cerevisiae, can synthesise their own vitamin B1.
This is great, because thiamine and its biologically active forms are essential cofactors for central carbon (sugar) metabolism pathways. These include the ethanol-producing glycolysis, the pentose phosphate pathway for aromatic amino acids, and the organic acid-yielding tricarboxylic acid pathway. Without thiamine, several enzymes of these pathways simply cannot function.
Thiamine can also protect yeasts during stressful fermentation conditions by playing an antioxidant role. This provides a defence against free radicals and can prevent oxidation – both are welcome news for winemakers.
Impact on aroma production
Beyond its impact on fermentation, thiamine deficiency can also alter aroma compounds produced by the yeasts. This vitamin is important for amino acid breakdown, such as during the production of some aromatic compounds in the Ehrlich pathway. These include some esters, volatile fatty acids and higher alcohols.
Lower thiamine levels were shown to result in higher volatile acidity and a lower ratio of higher alcohols to fatty acids. Simply put, low thiamine was linked with increased production of unpleasant and smelly fatty acids, such as isovaleric acid and butyric acid. Generally, we would like these compounds to be limited in our wines as they impart undesirable odours like stinky cheese and rancid butter.
Other unwanted compounds that can accumulate during thiamine deficiency include α-keto acids, acetaldehyde and phosphoenolpyruvate, which are known to bind SO2. This could be an issue, as later additions of sulphites to garner enough free SO2 to preserve the ageing wine may increase total SO2 levels to above the legal limit. This is a prominent concern in stabilising noble late harvest wines or any wine from grapes with some degree of rot.
Production versus assimilation
While yeasts have their own metabolic machinery to produce thiamine, this production requires lots of energy that the yeast could use to make more yeast cells or valuable aromatic products for our wines. To conserve energy, yeasts, therefore, rely on thiamine already present in the grape must, which they can easily take up for their cellular activity. In truth, yeasts prefer this method to such an extent that when tested experimentally, the yeasts absorbed all thiamine present in the grape juice within the first six hours after inoculation.
The level of thiamine present in grape must ranges between 0.2 and 1.2 mg/L, with higher levels observed in red grape juice due to increased skin and seed contact. Further studies have revealed that yeasts can utilise even more thiamine than naturally occurring in grape must – up to 10 000 times more.
Causes of thiamine deficiency
Thiamine deficiency is one of the reasons for impaired yeast growth, premature cell death and sluggish fermentations. A tough lesson for any winemaker is that when levels are too low in grape juice, the yeast may struggle to utilise all available sugars. This could ultimately result in the dreaded stuck fermentation.
Thiamine deficiency can easily occur during winemaking. Native yeasts present in the grape juice just after crushing can deplete thiamine levels, making the situation problematic for the subsequent inoculated or primary fermenting yeast. To bypass this, adding sulphur dioxide to crushed grapes is typically an antimicrobial measure to inhibit native yeast development.
Another culprit is the growth of filamentous fungi such as Botrytis cinerea on the grape berries, which may deplete thiamine before the grapes are even harvested. However, to make matters worse, very high dosages of sulphur dioxide are also known to decrease the levels of available thiamine in the juice. Winemakers are therefore advised to add thiamine to grape musts obtained from grapes infected by Botrytis cinerea or any other fungal pathogen.
Benefits of thiamine additions
It has been proved that increased thiamine levels during fermentation improve yeast cell growth and the fermentation rate. As mentioned above, the addition can even limit the accumulation of sulphur dioxide-binding compounds during alcoholic fermentation. This can allow for a reduced requirement for SO2 additions to the grape must.
Sequential and co-inoculation regimes may cause an initial issue whereby the inoculated non-Saccharomyces yeasts assimilate all available thiamine which may cause trouble for the subsequently inoculated S. cerevisiae to complete fermentation. However, with proper planning and timing during the co-inoculation regime, these yeasts die, and their thiamine is re-released into the must, which S. cerevisiae can then assimilate. It is important to keep in mind that many non-Saccharomyces yeasts used in co-inoculation strategies are more sensitive to thiamine deficiency and may not produce the desired outcome in the wine if thiamine is limiting.
Of the available thiamine in grapes, around 75% is found in grape juice, while 25% is in the skin and insoluble pulp. Due to this reason, all thiamine is made available in red grape musts (extended skin contact), but only around 80% is reported to be available in white grape juice. Yeasts love thiamine, so supplementation is a common practice in most winemaking countries.
Inactive dry yeasts and other complex yeast nutrients are commonly used in cellars to supplement the available thiamine levels in the juice. Unfortunately, while thiamine can be accurately quantified in a well-equipped laboratory, cellars have no “quick-and-dirty” method to help them estimate thiamine levels in grape juice.
Supplementing with thiamine
To ensure that the wine fermentation is completed, current regulations allow up to 60 mg/hL of thiamine hydrochloride to be added to grape musts (OIV resolution OENO 50/2000). Similarly, thiamine hydrochloride can be added to base wines before secondary fermentation to produce sparkling wines. Optimal conditions for thiamine uptake occur at around a pH of 4.5 and between 27 – 37°C.
However, the stability of thiamine decreases as temperatures increase beyond 25°C. While the wine fermentation environment does not quite meet these conditions, good efficiency can still be obtained. To achieve this, the addition of thiamine is best done at the onset of yeast growth.
This can either be at the beginning of the fermentation, at inoculation or somewhere in-between to avoid a potential stuck fermentation. Alternatively, thiamine is also present in various complex yeast nutrients commercialised by several manufacturers of oenological products, so their addition will also help in case of a deficiency.
Further research on vitamins
For more information on thiamine and its role in winemaking, the reader is invited to read the comprehensive mini-review published in 2021 by Labuschagne and Divol. While thiamine may be one of the best-studied, the other vitamins, such as biotin and niacin, also play essential roles during fermentation. Research on vitamins in winemaking is a constantly developing field.
New information on the other vitamins and their impact on our wines is being uncovered daily. There is a lot still to learn on precisely what keeps our yeasts and wines bubbling away.
Abstract
Thiamine (vitamin B1) is a vital yeast growth and metabolism nutrient. Yeasts favour the assimilation of thiamine from the grape must before they start producing their own. Yeasts can store up to 10 000 times more thiamine than is available in the grape must.
This storage process conserves energy, which the yeast can use to produce metabolic compounds, such as ethanol and valuable aromas. Thiamine deficiency is a real concern during winemaking as this can easily occur due to indigenous or contaminant organisms consuming all available thiamine before inoculation. Deficiency can result in a stuck fermentation and the production of undesirable aromatic compounds.
Supplementation of thiamine at the onset of yeast growth has been shown to improve the fermentation rate and result in healthier yeasts. Added benefits include a reduced requirement for SO2 additions and an antioxidant effect on the wine.
Key points
- Thiamine is an essential vitamin for yeast growth during fermentation.
- Yeasts can synthesise thiamine, but prefer to take it up from the must.
- Botrytis infection and non-Saccharomyces yeast growth can cause thiamine shortages in grape must.
- Thiamine shortages can lead to the accumulation of SO2 binding compounds, the formation of unwanted aromas such as volatile acidity, and stuck fermentations.
- Thiamine should ideally be added at yeast inoculation or as soon as possible after the onset of fermentation.
- There is a legal limit for thiamine additions to grapes musts of 60 mg/hL.
References
Burdock, G.A., 2010. Flavor Ingredients, 6th Edn Boca Raton. FL: Fenaroli’s Handbook CRC Press.
Hall, A.P., Brinner, L., Amerine, M.A. & Morgan, A.F., 1956. The B vitamin content of grapes, musts and wines. Journal of Food Science 21, pp. 362 – 371.
Jackson, R.S., 2008. Wine science: principles and applications. Academic Press.
Labuschagne, P.W.J. & Divol, B., 2021. Thiamine: a key nutrient for yeasts during wine alcoholic fermentation. Applied Microbiology and Biotechnology 105(3), pp. 953 – 973.
Nakamura, I., Ohmura, Y., Nagami, Y., Kamihara, T. & Fukui, S., 1982. Thiamin accumulation and growth inhibition in yeasts. Microbiology 128(11), pp. 2601 – 2609.
OIV, 2020. International code of oenological practices. International Organisation of Vine and Wine, Paris. Available at: https://www.oiv.int/en/technical-standards-and-documents/oenological-products/oenological-codex-products-used-in-oenology-per-code-sheet [Accessed 23 July 2022].
Wolak, N., Kowalska, E., Kozik, A. & Rapala-Kozik, M., 2014. Thiamine increases the resistance of baker’s yeast Saccharomyces cerevisiae against oxidative, osmotic and thermal stress, through mechanisms partly independent of thiamine diphosphate-bound enzymes. FEMS Yeast Research 14(8), pp. 1249 – 1262.
– For more information, contact Prof Benoit Divol at divol@sun.ac.za.
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