Yeast

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"Fermentation is life without oxygen." –Louis Pasteur, 1876

The conversion of the fermentable carbohydrates (sugars) into ethanol and carbon dioxide gas is achieved by pitching yeast. However, other by-yeast metabolism products are also excreted into the fermenting wort and can affect the organoleptic properties (i.e., taste, color, odor and feel) of the beer. These by-products include esters, aldehydes, vicinal diketones, higher alcohols and acids, as well as sulfur compounds.[1]

In beer, glucose, fructose, maltose, and maltotriose are all consumed simultaneously. The glucose, fructose and sucrose are depleted first because they are present in lower amounts. The rate of maltotriose consumption is slowest, and so it is the last sugar to be depleted.[2]

Boiled wort ferments more quickly than raw wort.[2] Why?

Preparing yeast for fermentation

Rehydrating dry yeast

Starters for liquid yeast

Yeast produce membrane lipids only when grown aerobically. In the initial growth phase, proper oxygen management leads to proper production and storage of sterols in the yeast cell, which can be shared with subsequent daughter cells. It is possible to increase yeast ethanol tolerance by promoting synthesis of sterols, by adding oxygen (air) in the starter and during fermentation. Yeast lees deplete the oxygen content and can impact the redox potential and formation of VSCs.[3]

While oxidative stress is known to occur, is it significantly less that stress from carbon dioxide. High amounts of foam means that insufficient oxygen delivery is occurring.[4]

The oxygen content in the propagation medium has to exceed 0.15 ppm in order to allow good growth of the yeast and to guarantee pitching yeast with a good physiology.[5]

Fancy stir bar (Amazon)

Stirring helps with oxygen diffusion[6]

Smack Pack activation
Hold it upside down at an angle and when the inner pouch sinks into the corner, gently squeeze, slowly increasing pressure, until you feel the inner pouch pop.[7]

Biomass may actually be more important that cell count with regard to pitch rate.[8] However this isn't easy to measure at home. Pitching rate calculators are still useful for determining correct pitch rate.

Yeast in worts rich in glucose may not be able to adapt to metabolize maltose and maltotriose, leading to slow or stuck fermentations.[9]

slow/stuck fermentation

In general, fermentation rates increase with temperature up to a maximum value (commonly >29C), after which premature cessation is probable due, in part, to elevated ethanol toxicity (D’Amato et al. 2006).[10] Beltran et al. demonstrated that low temperature fermentations altered nitrogen transport and metabolism, and suggested that coordination between carbon and nitrogen metabolisms may be hampered. Increased fermentation temperatures also alter the nutrient requirements of Saccharomyces (Shinohara et al. 1996; Ribéreau-Gayon et al. 2000), but these effects are not well defined for most nutrients.[10]

When brewer's yeast is exposed to high concentrations of glucose, a phenomenon referred to as the "glucose effect" may be experienced with poor quality yeast, which can result in sluggish and "hung" wort fermentations.[11]

Nutrition

Deficits in FAN directly can lead to an insufficient and slow start of the fermentation, insufficient fermentation performance, and stuck fermentations.[12] Low molecular weight nitrogen compounds, especially amino acids, influence the metabolism of the yeast. In particular, they impact the production of higher alcohols and vicinal diketones.

"Nutrition" in this context refers to sources of yeast-assimilable nitrogen (YAN), necessary vitamins, and certain trace minerals. YAN is the amount of nitrogen from the combination of ammonium plus Free Amino Nitrogen (FAN), in the form of amino acids.

Wort produced from a high percentage of malt tends to supply necessary vitamins in levels well beyond what is needed for fermentation (including biotin, inositol, and pantothenate). The levels left after fermentation as to the overall nutritional value of beer. Malt also tends to add the required levels of copper, iron, zinc, and magnesium.[13]

Nitrogen is generally plentiful in wort and typically does not require supplementation for beer production.[1] The concentration of the amino acids isoleucine, valine, phenylalanine, glycine, alanine, tyrosine, lysine, histidine, arginine and leucine, are considered important, as these are an important part of the complex system regulating the biosynthesis of flavour-active compounds formed by yeast.[1] However, if supplementation is desired, a mixture of amino acids is more favorable to growth than when ammonium ions are the source of nitrogen.[1] Phenolic yeast may have a higher nitrogen requirement.[1]

Yeast consume at least 100-140ppm FAN in wort. Since proline cannot be utilized, wort has to contain 200-220ppm FAN. Inadequate nutrition can result in reduced yeast propagation and a delay in fermentation and maturation, and ultimately the retention of undesirable "young beer" off-flavors. Higher modified malts produce more FAN.[14] If adjuncts are used, the brewer should consider using a protein rest (45-50°C) (see Mashing) or adding yeast nutrient.

Worts that are prepared with reasonable percentages of malt tend to be rich in amino acids. Low FAN levels are undesirable in wort. The traditional rule is that serious problems (long lags, high diacetyl, etc) can result from FAN below 150-175ppm. A 12°P malt wort will typically have 225-275ppm FAN, which is ideal.[13] As a general rule, it is usually desirable to keep FAN levels below 350ppm, something that can be achieved with a suitable mashing schedule.

Too much or too little FAN can increase diacetyl production during fermentation.[13]

See also: Protein

The levels of vitamins present in conventional (all-malt) worts rarely or never limit yeast growth and fermentation.[9]

Review:

Measuring YAN
We can measure YAN via with a few reagents and a pH meter:

things

The Formol titration has the advantage of measuring both ammonia and FAN amino acids. However, the method over-titrates proline (which yeast cannot use) and under-titrates arginine, which the yeast do use. Our research suggested that this generally balances out [in wine].[3]

YAN Target
The optimal YAN level for wine is generally 250-350ppm (nitrogen).[15][16][17]

Nutrient tables:

Too much YAN (>350mg/L) can induce an overpopulation of yeast, which will increase stress conditions and produce undesirable characteristics such as off-flavors or stuck fermentation.[18] To ferment 1g/L of sugar, yeast need 1mg/L of YAN. (1°Brix = 10g/L sugar) For good population growth, a minimum of 150mg/L YAN is needed.

YAN target depends heavily on yeast strain and fermentation conditions (e.g. initial sugar, temperature, fermentation aeration).[17][16]

  • YAN requirement for clean/fruity flavour has only been determined in Chardonnay: low YAN juices gave more complex aromas whereas moderate YAN gave cleaner and more fruity aromas in young wines.[17]
  • Large additions of inorganic nitrogen (DAP) can increase risk of ester taint (ethyl acetate) formation.[17]

Proficient YAN guidelines should be based on starting Brix (Butzke and Dukes, 1998). For instance, a starting brix of 21 requires 200 mg N/L whereas as starting brix of 25 requires 300 mg N/L.[19]

DAP is 21% nitrogen by weight. Adding DAP during active fermentation will help the yeast remove existing sulfide within a few hours, but only if the ABV is under 7%.[15]

Ammonia (in the form of DAP) can prevent the appearance of aromatic degradation products from amino acids. Amino acids are an important source of yeast esters, which can add to complexity and wine quality. Thus, the supply of nitrogen must be available to allow a continuous re-synthesis of these proteins. If that does not occur, the yeast lose the ability to conduct the fermentation. Nitrogen addition may be effective in avoiding problem fermentations until about two-thirds of the sugar is utilized. Cells which have passed the point of transcriptional responsiveness will not respond to added nutrients.[3]

Equal proportions of ammonium to amino nitrogen and moderate initial concentrations of DAP (100 to 150 mg N/l) result in the lowest sulfide formation after peak fermentation.[20]

Vitamins and Trace Elements

Thiamine
Used as a co-enzyme for fermentation. It stimulates yeast growth, speeds up fermentation, and decreases undesirable fermentation byproducts, notably acetaldehyde.[21]
Biotin
Biotin is the most important vitamin for yeast (Fig.2) It is involved in almost all enzyme reactions that create the compounds yeast are made of: proteins, DNA, carbohydrates, and fatty acids. Biotin deficiency results in slow yeast growth and stuck fermentations.[22]
Zinc
It is needed in the micro molar (10-3M) range in wort. Zinc is important in the cell cycle (reproduction), and is a cofactor for alcohol dehydrogenase, the enzyme responsible for alcohol production. Other metal ions can not substitute in place of zinc. Supplementation of zinc into brewers worts generally has the effect of speeding up fermentation, as well as preventing stuck fermentations.[22]

Typical vitamin requirements for yeast include biotin, nicotinic acid, vitamin B, and pantothenic acid.[22]

Essential vitamins: 250 µg/l Ca-pantothenate, 250 µg/l thiamin a HCl, 25 µg/l pyridoxine, 2 µg/l biotin.[20]

0.2 mg/L folic acid, 200 mg/L myo-inositol, 4 mg/L pyridoxine, 4 mg/L nicotinic acid, 1 mg/L thiamin, 0.4 mg/L riboflavin and 0.250 mg/L pantothenic acid.[23]

Staggered Nutrients

  • Higher initial juice/must YAN values increase fermentation rate and heat production.[17]
  • DAP can be added in divided doses to give a more moderate rate of fermentation.[17]
  • Higher initial juice/must YAN values or DAP additions can increase the risk of residual YAN in finished wines.[17]

Amino acids are brought into the yeast cell through transport across the cell membrane. The presence of alcohol and ammonium ions (i.e., DAP) inhibit amino acids from being brought into the cell. This is why winemakers are advised NOT to add DAP at inoculation or at the beginning of fermentation, as yeast can actively absorb organic nitrogen in the juice (aqueous) environment.[24] Once alcohol concentrations begin to increase, as a result of primary fermentation progression, transport of amino acids from the wine into the yeast cell will be inhibited. Therefore, the primary source of nitrogen will then come from inorganic sources, such as DAP.[24][citation needed] Higher concentrations of the inorganic component of YAN can lead to a high initial biomass of yeast. This is a problem because the rapid increase in yeast populations can lead to starvation by the majority of the yeast by mid- to late-fermentation, especially if there is not enough nutrition to fulfill all of the yeast during fermentation. Yeast starvation leads to yeast stress, and one of the stress responses by yeast is the production and release of hydrogen sulfide. Therefore, having a high YAN at the start of fermentation may cause hydrogen sulfide issues in the wine by the time fermentation is complete.[24]

Yeast don’t need all the nutrients at the same time:

  • During the growth phase, yeast need vitamins, minerals and nitrogen. The presence of alcohol and/or ammonium ions inhibits transport of amino acids through cell membranes and reduces their consumption.[18]
  • To optimize their absorption and efficiency, amino acids should be added at inoculation, before ammonium ions. At this stage, yeast can assimilate amino acids to build ‘healthy’ cells which are resistant to stress conditions and produce aromas.[18]
  • At 1/3 of sugar depletion, yeast start to become stressed and the assimilation of nitrogen is lower. To complete fermentation and increase their alcohol resistance, they need fast and easy nutrients to absorb (ammonium ions) and survival factors (sterols and unsaturated fatty acids) with oxygen.[18]
  • In case of strong nitrogen deficiency, must needs to be corrected by an addition of ammonium ions 24-48 hours after inoculation (after the addition of amino acids).[18]
  • The nutrient additions should be split between inoculation and no later than 1/3 sugar depletion.[18]
  • Late nutrient additions are ineffective for yeast activity and can promote development of spoilage organisms, appearance of off-flavors and formation of biogenic amines.[18]

Nutrient Products

An innovative new method of mineral delivery is Servomyces, which White Labs represents in North America. It is produced in a patented process, by which brewers yeast is grown in the presence of metal ions, including zinc and magnesium, and then dried and killed. When the dry, dead yeast (Servomyces) is added to brewery fermentations (in very minute quantities), the effect is dramatic to fermentation speed and to yeast performance/viability. [25]

Flavor compounds

During active biomass accumulation, H2S and ester production may occur. In this case, ester formation is stimulated by the presence of nitrogen, indicating that biosynthetic reactions are the source of these compounds. Once active growth has diminished and ethanol is accumulating, amino acid degradation occurs and, at this time, additional esters and fusel compounds may be produced. The fusel compounds come from the degradation of amino acids as nitrogen sources via the Ehrlich pathway.[26] https://wineserver.ucdavis.edu/industry-info/enology/fermentation-management-guides/wine-fermentation/characters -- discussion of VSCs, esters, and aldehydes

If the total fusel alcohol level is below 300 mg/L, the wine is described as fruity and pleasant containing peach and apricot aromas. Above 400 mg/L, the wine becomes pungent with a strong foul chemical taste and aroma. In wine, the total produced varies within this range, from less than 100 to greater than 500 mg/L. The individual compounds vary from 10-140 mg/L. The amounts formed show a strong stain dependence.[26]

in general, when acetaldehyde is found in wine it comes from the chemical re-oxidation of ethanol during aging and oxygen exposure of the wine. Acetaldehyde levels in wine range from 1 – 160 mg/L. It has a putative threshold of 100 mg/L. It is described most often as sherry-like in wines, but has bruised apple and nutty notes as well. The higher aldehydes derived from amino acids can have strong nutty and rancid nutty notes at high concentrations and are made under the same chemical conditions conducive to acetaldehyde formation. At lower concentrations, they may confer notes of coffee, chocolate or stone fruits.[26]

With respect to ethyl acetate formation, the amounts made by Saccharomyces are inconsequential but the amounts formed by the acetic acid bacteria and Hanseniaspora uvarum can be so high that these compounds will not be lost from the wine during fermentation or subsequent processing. If the odor of ethyl acetate is noticed during a cold soak or other pre-fermentation treatment, that treatment should be halted and the fermentation inoculated immediately.[26]

Fusel alcohols are produced from the carbon skeletons of amino acids, and the effect of fusel alcohols on the finished product can be quite negative if present above or near their respective flavor thresholds.[13]

In many breweries producing South- ern German-style wheat beer, otherwise known as weissbier, after the installation of new cylindroconical fermentors, it is common for the beers to exhibit a noticeable decline in the bouquet characteristic of the style, which consists of primarily of compounds like isoamyl acetate (banana ester)[2]. The reason behind this somewhat diminished weissbier aroma is, among others, the high rate of yeast reproduction, which reduces the amount of the acetyl-coenzyme A available for ester formation. In addition, the high hydrostatic pressure in vertical vessels moderates the production of higher alcohols, thus reducing the numbers of reactants for the formation of esters. In short, the higher the liquid level is in a fermentation tank, the stronger the convection and homogenization, which results in a reduction in the formation of esters (fig. 4).[27]

The estery notes in beer have been observed to become more pronounced as the ratio of glucose to maltose tips in favor of glucose.[27] Alcoholic fermentation with yeast in the presence of high concentrations of glucose leads to a delay in the onset of maltose metabolism after an initial rapid decline in the extract content of the wort (similar to a "second lag phase"). This explains the plateau in the extract curve. During this time, the yeast are scarcely reproducing and are compensating with the synthesis of maltose permease and maltase. The diminished yeast reproduction results in overflow of the acetyl-CoA pool and thus greater ester formation and fruitier beers.

Yeast fermentation of maltose is repressed by glucose.[28]

This also comes into play when using adjuncts in brewing.

See:

Influence of water minerals

Harvesting

Storage

  • Under beer, jars vs vials
  • Isotonic saline
  • Slants or plates
  • Temperature
  • Periodic feeding

Articles to be reviewed

References

  1. ↑ a b c d e Ferreira, Inês M., and Guido, Luís F. "Impact of Wort Amino Acids on Beer Flavour: A Review." Fermentation. 2018, 4, 23.
  2. ↑ a b https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.2010.tb00425.x
  3. ↑ a b c https://www.apps.fst.vt.edu/extension/enology/EN/133.html
  4. ↑ https://www.mbaa.com/publications/tq/tqPastIssues/2005/Abstracts/TQ-42-0128.htm
  5. ↑ Van Landschoot, A., et al. "Effect of pitching yeast preparation on the refermentation of beer in bottles." Cerevisia, vol. 29, no. 3, 2004, pp. 140–146.
  6. ↑ https://www.cmbe.engr.uga.edu/bche4510/assign/Interlude.pdf
  7. ↑ https://www.homebrewtalk.com/forum/threads/smack-pack-issues.676389/#post-8813147
  8. ↑ "Wiki Kwiki #005 - Lance Shaner of Omega Yeast Labs" (at ~30 minutes) Milk the Funk podcast, December 2019.
  9. ↑ a b Briggs DE, Boulton CA, Brookes PA, Stevens R. Brewing Science and Practice. Woodhead Publishing Limited and CRC Press LLC; 2004.
  10. ↑ a b https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1745-4557.2010.00365.x
  11. ↑ Stewart GG. Adjuncts. In: Stewart GG, Russell I, Anstruther A, eds. Handbook of Brewing. 3rd ed. CRC Press; 2017.
  12. ↑ Pahl R, Meyer B, Biurrun R. Wort and Wort Quality Parameters. In: Bamforth CW, ed. Brewing Materials and Processes: A Practical Approach to Beer Excellence. Academic Press; 2016.
  13. ↑ a b c d Fix, George. Principles of Brewing Science. 2nd ed., Brewers Publications, 1999.
  14. ↑ Kunze, Wolfgang. "3.2 Mashing." Technology Brewing & Malting. Edited by Olaf Hendel, 6th English Edition ed., VBL Berlin, 2019, p. 230.
  15. ↑ a b Kaiser, K. "Controlling Reductive Wine Aromas." Brock University CCOVI lecture series. 1 Feb 2010.
  16. ↑ a b "Approximate YAN Contribution for the Important Yeast Nutrients."
  17. ↑ a b c d e f g "Yeast Assimilable Nitrogen (YAN)." The Australian Wine Research Institute. Accessed online March 2020.
  18. ↑ a b c d e f g "Yeast Nutrition for a Successful Fermentation." Enartis Vinquiry. Technical Harvest Newsletter. Volume 4. September 2014.
  19. ↑ https://midwestwinepress.com/2014/11/01/back-basics-preventing-rotten-eggs-aka-reduction/
  20. ↑ a b Butzke, CE and Park, SK. "Impact of Fermentation Rate Changes on Potential Hydrogen Sulfide Concentrations in Wine." J. Microbiol. Biotechnol. 2011. 21(5). pp. 519–524
  21. ↑ https://pennsylvaniawine.com/wp-content/uploads/2017/04/Yeast-Nutrition.pdf
  22. ↑ a b c White, C. "Yeast Nutrients Make Fermentations Better."
  23. ↑ Bohlscheid, JC., et al. "The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations." Journal of Applied Microbiology. 102 2007. 390-400.
  24. ↑ a b c Gardner, DM. "Starting your fermentation right: nutrient supplementation." Penn State Extension Wine & Grapes U. 2016.
  25. ↑ https://www.jstrack.org/brewing/Yeast_nutrition_article.pdf
  26. ↑ a b c d https://wineserver.ucdavis.edu/industry-info/enology/fermentation-management-guides/wine-fermentation/characters
  27. ↑ a b Sacher B, Becker T, Narziss L. Some reflections on mashing – Part 2. Brauwelt International. 2016;6:392-397.
  28. ↑ Meussdoerffer F, Zarnkow M. Starchy raw materials. In: Esslinger HM, ed. Handbook of Brewing: Processes, Technology, Markets. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA; 2009.
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