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Craft Brewery Yeast Handling
Types of Brewery Organisms
Brettanomyces
Figure 1 - B. bruxellenis from DME Agar at 800x Light Magnification (Bootleg Biology, 2019)
A genus of yeast which was first documented to contribute to stock ales of the 19th century, it is responsible for part of the terroir of the tertiary fermentation of Lambic and Flanders ales. The genus consists of five species with two found in the brewery, B. bruxellensis and B. anomalus. Considered a contaminant as it can contribute undesirable flavours and aromas; it has been viewed as a new type of yeast for experimentation by craft brewers (Yakobson, 2012). It can be used to increase profile complexity. Brettanomyces is an asexual budding anamorph and can ferment in aerobic and anaerobic conditions. Brettanomyces follows similar pathways to Saccharomyces; however, Brettanomyces can produce more phenolic compounds such as 4-ethylguaciacol and 4-ethylphenol and more esters such as ethyl caproate, ethyl caprylate, and ethyl decanoate (Tyrawa, 2019).
Figure 2 - Comparison of ester & phenol production at 15°C & 22.5°C between Saccharomyces (US-05) and several Brettanomyces strains (Preiss, 2019).
Lactobacillus
Figure 3 - L. delbrueckii Grown on MRS Agar at 2000x Liquid Magnification (Bootleg Biology, 2019)
Lactobacillus is a genus of bacteria of a group known as lactic acid bacteria (LAB). Other LAB used in food fermentation include Lactococcus, Streptococcus, Pediococcus, and Leuconostoc. The metabolic pathways for LAB are glycolysis, lipolysis, and proteolysis, which is the formation of lactic acid from sugar, fat, and protein, respectively. Other fermentation products include diacetyl, acetoin, acetaldehyde or acetic acid (Bintsis, 2018). Species of lactobacillus found in a brewery setting include L. brevis, L. lindneri, and L. delbrueckii. Lactobacillus are rod-shaped, facultatively anaerobic, and gram-positive. Lactobacillus grow best near 30°C with a pH between 4.0-5.0 (White, 2012). Considered a contaminant for brewing applications, there are several styles of beer that require Lactobacillus. Examples include Berliner Weiss, Flanders Red Ale, Oud Bruin, Lambic, Gueuze; it can contribute to Witbier and Saison; and used in styles of Gose and Litchtenhainer (Strong and England, 2015). Barley malt has populations of Lactobacillus on the husks. Some brewers will use a small amount of malt to inoculate unhoped wort for souring, this can aid for mash or kettle acidification (White, 2012).
Pediococcus
Figure 4 - Pediococcus Grown on MRS Agar at 2000x Liquid Magnification (Bootleg Biology, 2019)
Pediococcus is a genus of coccocoidal or ovid shaped LAB. They are gram-positive, produce lactic acid from glycolysis, and grow best at a pH between 4.0-5.0 (Priest, 2012). They are non-motile and non-spore forming; typically, they do not produce carbon dioxide, ethanol, or acetic acid (Wade et al., 2018). Considered a contaminant apart from certain sour beers, as they contribute diacetyl, turbidity, and acid formation; as well as they form extracellular polysaccharides which appear as a “ropey” viscous substance (Priest, 2012). Due to the division mechanism of Pediococcus cells, they often appear to be in small clusters and are the only LAB in beer to do this, which allows identification with a microscope (Wade et al., 2018).
Saccharomyces
Saccharomyces is a genus of unicellular fungi known as "yeast". The most common species used can be classified as S. cerevisiae, S. pastorianus, or S. cerevisiae diastaticus. Saccharomyces perform a fermentation of short polysaccharides to produce alcohol, carbon dioxide, and flavours and aromas that are associated with beer. Strains from this genus are anamorphs, where aerobic conditions encourage cell growth, and anaerobic conditions encourage alcohol production (Van Zandycke, 2012).
Saccharomyces Cerevisiae
Figure 5 - S. cerevisiae from DME Agar at 800x Light Magnification (Bootleg Biology, 2019)
S. cerevisiae is the most common species. Critical in the fermentation of beer, wine, spirits wash, sake and in the production of bread. S. cerevisiae is categorized as a "top-fermenting" yeast as it produces a thick layer of krausen during fermentation and are used primarily for ales. S. cerevisiae carries out fermentation at temperatures of 18°C-24°C. They are known for ester production that leads to a more complex profile. Over 200 different strains of S. cerevisiae are commercially available (Dunn, 2012).
Saccharomyces Pastorianus
Figure 6 - S. pastorianus from DME Agar at 800x Light Magnification (Bootleg Biology, 2019)
S. pastorianus, or lager yeast, is a hybridization of S. cerevisiae and S. eubayanus (Libkind et al., 2011). It is physiologically different from S. cerevisiae; ferments at cooler temperatures and can process melibiose. S. pastorianus typically has a higher degree of attenuation with lower levels of ester formation, establishing the profile of lagers. S. pastorianus were selected unintentionally from brewing practises resulting from the Reinheitsgebot which restricted brewing during the summer (Sherlock, 2012).
Saccharomyces Cerevisiae var Diastaticus
Figure 7 - S. diastaticus from DME Agar at 800x Light Magnification (Bootleg Biology, 2019)
S. diastaticus can attenuate higher than S. cerevisiae by consuming starch and dextrins. Complex polysaccharides escape digestion by S. cerevisiae, S. diastaticus produces glucoamylase. Glucoamylase cleaves the 1-4 linkages in amylose while leaving the 1-6 linkages in amylopectin (Andrews and Gilliland, 1952). Dextrins contribute to body and mouthfeel of the final beer, not consumable by S. cerevisiae, but are fermented when S. diastaticus is present. S. diastaticus cannot be identified from S. cerevisiae using plating methods; a polymerase chain reaction must be used to identify the glucoamylase genetic marker: STA1. S. diastaticus is an issue in brewing as STA1 is common with Saison-style yeasts. Sources include: an increase in craft-scale brewing, market demands increasing the variety of yeast strains, and lacking in equipment such as pasteurization units, kieselguhr-based filtration, and automated clean-in-place. Contamination of S. diastaticus can result in higher alcohol, phenolic flavours, over-carbonation, and final package failure. Cellar management that avoids cross-contamination of yeast and brewing wort with reduced levels of dextrins can be used to lower the mentioned risks (Ferguson, 2018).
Kveik
Kveik is a Norwegian dialect word for "yeast', and in English, it refers to a mixed culture of S. cerevisiae used for hundreds of years in traditional farmhouse brewing. As a verb in Norwegian, it means to "begin life" (Geithung, 2019). Common traits among Kveik cultures are high-flocculation, low phenols, quick fermentations while being high temperature tolerant. They are a genetically distinct group of S. cerevisiae, and have direct applications to brewing, allowing for faster fermentations and lower cooling requirements. Kveik cultures also have a high degree of alcohol tolerance. Kveik cultures produce more of ethyl caproate, ethyl caprylate, ethyl decanoate, and phenethyl acetate compared to monoculture S. cerevisiae (Preiss et al., 2018).
Figure 8 - Origin of Common Kveik Cultures (Preiss et al., 2018)
Biological Sampling
There are several areas of minimum sampling to identify beer spoilage organisms. The main goal is to evaluate cleaning procedures and update methods to remove contaminants.
|
Test |
Sample Area |
Frequency |
|
Forced Wort |
After wort chilling Prior to adding yeast |
With each batch of wort |
|
Clean-In-Place Swab |
Interior of Equipment |
After Cleaning Prior Use |
|
HLP |
Holding Vessel or Package |
After Beer Transfer |
Table 1 - Proposed Sampling Procedure
Forced Wort Test
A forced wort test is a determination of the cleanliness of the wort chiller. If the wort chiller has been appropriately maintained and cleaned to the proper degree, there should be no growth in a sample that has no inoculate.
Materials
- Sample Port
- Isopropyl Alcohol
- Butane Torch
- Sterile Sampling Container
Method
- Thoroughly clean hands and forearms; apply gloves and personal protection equipment.
- During wort transfer to the cellar, prepare materials.
- Apply isopropyl alcohol to sampling port and sterilize with the butane torch.
- Collect a sample in the sterile container.
- Leave the container in an isolated warm location.
- Inspect for haze formation (Jones, 2018).
|
Time (Days) |
Result |
|
1 |
Extremely dirty pathway. Dump beer. |
|
2-3 |
Major Contamination. May spoil beer. Do not reuse yeast. |
|
3-6 |
Minor Contamination. May not affect beer. |
|
7+ |
Very Clean. |
Table 2 - FWT Haze Results (Jones, 2018)
Clean-In-Place Swab Test
A cleaning verification method is a swab test; it can identify growth not removed by clean-in-place or clean-out-place procedures. Sampling areas that encounter beer will ensure a stable product. Several example areas are fermenter connections, hard-piping ports, the wort chiller, or any filters.
Materials
- Dry or wet swabs
- Sterile saline
- Sterile wort samples, 10mL in transport tubes.
- Incubator
- Microscope with slides
- Gram Stain Reagents
Method
- Thoroughly clean hands and forearms; apply gloves and personal protection equipment.
- With dry swabs, remove the packaging and apply sterile saline. With wet swabs, remove from container ensuring no contact with the container rim.
- With a substantial amount of pressure apply the swab to an area less than 10cm2.
- Place swab in wort sample and enclose.
- Incubate the sample at 30°C for three days and monitor for growth.
- If growth occurs, prepare for gram staining and mount to a wet slide (Manufacturing.net, 2014).
Hsu's Lactobacillus Pediococcus (HLP) Test
HLP media detects Lactobacillus and Pediococcus and designed to simplify procedures. It is a low-cost preparation that contains the yeast inhibitor Actidione and an oxygen scavenger. It is semi-solid and semi-aerobic.
Materials- HLP Media
- Water Bath
- Erlenmeyer Flask
- Sterile test tubes
- Pipettes
- Incubator
- Microscope with slides
- Gram Stain Reagents
Method
- Prepare HLP media as per package instructions in an Erlenmeyer Flask. Heat gently until homogeneous.
- Seal and store at 45°C in the water bath.
- Transfer 15mL of media into test tubes. Media must be above 25°C for use.
- Gather 1mL of beer sample and pipette into the media, disperse across the media.
- Seal and incubate at 28-30°C for 48-72h. Check for growth.
- If growth occurs, prepare for gram staining and mount to a wet slide (Pellettieri, 2015).
Rapid Method: ATP Bioluminescence
A swab method that uses light to detect biological activity but cannot identify the organisms present. A sample is collected and inserted into a monitor giving near-instantaneous results. The reaction involves ATP from the microorganisms reacting with Luciferin and oxidizing to produce bioluminescence which is measured. ATP bioluminescence provides a quick pass/fail analysis of cleaning procedures and can be used to prior to disinfecting or for ensuring the equipment is safe for beer (Bolton and Quain, 2013).
Pilot Batch Propagation
Sterilization
When brewing, the equipment is typically cleaned and sanitized. When preparing a fresh sample for yeast growth, a sterile environment is needed to ensure that unwanted micro-organisms do not propagate alongside. Sterile materials can be achieved with a wet environment for 15m at 121°C or in a dry environment for 2h at 160°C (White and Zainasheff, 2010).
Figure 9- Flow Diagram of a Proposed Laboratory Propagation of Brewing Yeast (Bolton & Quain, 2008)
Method
- Clean and sanitize an area of the workspace. Light the Bunsen burner or alcohol lamp. Use sterile microbiological techniques:
- Only use equipment that has been adequately heat-sterilized.
- Work in proximity (<10cm) to the open flame.
- Flame all openings of any glass or metal containers.
- Do not flame the opening of any disposable containers. (White and Zainasheff, 2010)
- Prepare a 20mL sterile solution of 1.040SG Wort. Use a 50mL test tube. This may be autoclaved
- Identify the yeast colonies on the plate for harvesting.
- Using a flamed inoculation loop:
- Open the plate.
- Touch the flamed inoculation loop to the agar to cool.
- Collect a single entire colony.
- Close the plate.
- Open the container of wort and deposit the colony into the medium.
- Seal the wort container (WC), place upright. Store at ambient temperatures for 24-48 hours.
- Manually agitate the wort, gather a sample and perform a diluted sample cell count using methylene blue (White and Zainasheff, 2010).
- Prepare a 200mL sterile solution of 1.040SG Wort. Use a 500mL Erlenmeyer flask. This may be autoclaved (Bolton & Quain, 2012).
- When the secondary WC is at ambient temperature:
- Agitate the primary WC and vent.
- Empty the primary WC into the secondary (White and Zainasheff, 2010).
- Place the secondary WC on a stir plate with a sterile stir bar. Agitate for 72 hours. Biological activity should be evident within 12-24 hours (Bolton and Quain, 2012).
- Prepare a 3L sterile solution of 1.040SG Wort. Use a 5-10L Erlenmeyer flask. Autoclave the wort for sterility. Repeat Step 9 for the tertiary WC combined with aeration (Bolton and Quain, 2012).
- Agitate the WC, gather a sample, and perform a diluted sample cell count using methylene blue.
Pitch Rate = Style Pitching Rate x Volume (L) x Wort Gravity (*P)
225 x 10^9 Cells = 0.75 x 20L x 15*P
- A quaternary WC may be required if not enough cells are present:
- Chill and decant the spent wort from the tertiary WC keeping the condensed yeast. Yeast may be kept cold for up to two weeks (White and Zainasheff, 2010). Allow the temperature to rise to ambient.
- If there is not ample yeast, prepare a sterile solution of 1.040SG Wort. Use a 5-10L Erlenmeyer flask. This may be autoclaved. Repeat Step 9 for the quaternary WC for 24-48h (Palmer, 2017).
- When the final wort propagation is at ambient temperature, add to the pilot batch of wort.
References
Andrews, B. and Gilliland, R. 1952. Super-Attenuation of Beer: A Study of Three Organisms Capable of Causing Abnormal Attenuations. Journal of the Institute of Brewing, 58(3), pp.189-196.
Boulton, C. and Quain, D., 2013. Brewing Yeast and Fermentation. Hoboken: Wiley.
Bintsis, T., 2018. Lactic acid bacteria as starter cultures: An update in their metabolism and genetics. AIMS Microbiology, 4(4), pp.665-684.
Bootleg Biology. 2019. DIYeast: Microbe Portrait Gallery. [online] Available at: https://bootlegbiology.com/diy/microbe-portrait-gallery/ [Accessed 16 Nov. 2019].
Dunn, B., 2012. Ale Yeast. In: The Oxford Companion to Beer, 1st ed. New York: Oxford University Press, pp.33.
Ferguson, N., 2018. Understanding (Over)attenuation, Carbonation, and Bursting: AKA Understanding Diastaticus.
Geithung, I., 2019. In Norway, Kveik Means More Than Just Yeast. [video] Available at: https://www.youtube.com/watch?v=MCC3tBS2j_Q [Accessed 16 Nov. 2019].
Jones, N. 2018. Yeast Management on a Budget.
Libkind, D., Hittinger, C., Valerio, E., Goncalves, C., Dover, J., Johnston, M., Goncalves, P. and Sampaio, J., 2011. Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast. Proceedings of the National Academy of Sciences, 108(35), pp.14539-14544.
Manufacturing.net. 2014. How to Swab For Bacterial Contamination in Your Brewery. [online] Available at: https://www.manufacturing.net/operations/blog/13190255/how-to-swab-for-bacterial-contamination-in-your-brewery [Accessed 16 Nov. 2019].
Pellettieri, M. (2015). Quality Management: Essential Planning for Breweries. 1st ed. Boulder, Colorado: Brewers Publications, p.81.
Palmer, J. 2017. How to Brew. 4th ed. Boulder, Colorado: Brewers Publications.
Preiss, R., Tyrawa, C., Krogerus, K., Garshol, L. and van der Merwe, G., 2018. Traditional Norwegian Kveik Are a Genetically Distinct Group of Domesticated Saccharomyces cerevisiae Brewing Yeasts. Frontiers in Microbiology, 9.
Preiss, R., 2019. Don't Forget The Brett. [online] Escarpment Laboratories. Available at: https://www.escarpmentlabs.com/single-post/2019/08/14/Dont-forget-the-Brett [Accessed 16 Nov. 2019].
Priest, F., 2012. Pediococcus. In: The Oxford Companion to Beer, 1st ed. New York: Oxford University Press, pp.644.
Sherlock, G., 2012. Lager Yeast. In: The Oxford Companion to Beer, 1st ed. New York: Oxford University Press, pp.535.
Strong, G. and England, K. (2015). 2015 Style Guidelines. [online] Beer Judge Certification Program. Available at: https://bjcp.org/docs/2015_Guidelines_Beer.pdf [Accessed 16 Nov. 2019].
Tyrawa, C. et al., 2019. The temperature dependent functionality of Brettanomyces bruxellensis strains in wort fermentations. Journal of the Institute of Brewing, 125(3), pp.315–325.
Van Zandycke, S., 2012. Yeast. In: The Oxford Companion to Beer, 1st ed. New York: Oxford University Press, pp.858-861.
Wade, M., Strickland, M., Osborne, J. and Edwards, C., 2018. Role of Pediococcus in Winemaking. Australian Journal of Grape and Wine Research, 25(1), pp.7-24.
White, C. and Zainasheff, J., 2010. Yeast. Boulder, Colorado: Brewers Publications.
White, C., 2012. Lactobacillus. In: The Oxford Companion to Beer, 1st ed. New York: Oxford University Press, pp.531.
Yakobson, C., 2012. Brettanomyces. In: The Oxford Companion to Beer, 1st ed. New York: Oxford University Press, pp.157-158.