Wild Yeast Isolation

From Milk The Funk Wiki
Revision as of 15:46, 1 March 2019 by DanABA (talk | contribs)
Jump to: navigation, search
Picture of the first step to catching wild yeast by David Thornton of SouthYeast Labs and Carolina Bauernhaus Ales.

Wild yeast isolation, sometimes known as "yeast wrangling" or more formally "bioprospecting", refers to the process of catching wild, native yeast (autochthonous yeast), and isolating it with agar plates as a pure culture. This article will also contain information on growing up a captured culture in wort rather than isolating yeast cultures on agar plates. Culturing wild yeast and bacteria should not be confused with Spontaneous Fermentation because many of the microbes that might make a flavor impact during spontaneous fermentation are killed off during the bioprospecting processes. For general lab techniques, see Laboratory Techniques.

Special thanks goes out to Bryan Heit from Sui Generis blog and Jeff Mello from Bootleg Biology for providing the information in this article.

Isolating Pure Cultures

Preparation

  • Sui Generis Blog video guides on setting up a clean workspace, building and using an alcohol lamp, and aseptic techniques.
  • Bootleg Biology's guide on creating agar plates using wort, agar powder, and yeast nutrient.
  • Sui Generis Blog guide to more types of agar plates, and video tutorial.

Catching

  • Bootleg Biology defines three methods of catching a wild yeast.
  • Sui Generis Blog video tutorial for catching wild yeast from fruit or from the air.
  • David Thornton of SouthYeast Labs describes his favorite method of catching wild yeast/bacteria: "Most of our cultures are taken from over-ripened fruit macerated in its own juice and left to ferment 24 hours at the location in a small Erlenmeyer with a perforated cap. Always grows something, and almost always get an alcohol fermenting strain, and of the alcohol fermenting strains id say 10% are worth brewing with. I like this method because I can stir my loop and do a streak on regular UBA aerobically, then pull favorable looking colonies for yeast and Lactobacillus and do a quadrant streak on selective media for isolation." [1]. See also the MTF 'The podcast' episode with David Thornton.
  • Monitor for mold growth. If mold begins to grow on the growth media, scrape it out and continue on with the isolation process. This should be safe since isolating the yeast will separate it from any potential mycotoxins that developed during the initial catching. Do not consume any growth media that has had mold grow on it (see Mold). One suggested method is to remove any materials (fruit, leaves, etc.) that were used as an inoculant as soon as visible fermentation happens; this can help avoid mold growth by removing floating material [2].
  • In general, summer and spring are more successful times to catch wild yeast, however any time of year can be successful. Simply choose fruits or flowers that bloom/grow during that particular season. For example, in spring, flowers and fruit blossoms are a good source. In summer, wild summer fruit can be a good source (raspberries, strawberries, cherries, etc.). In the fall, apples and grapes can be a good source. In winter, juniper berries [3].
  • A new study showed that wild yeast actually exists more so in leaf matter on the ground than it does on bark or fruit. See the "Where (Do) the Wild Yeast Roam" video by Bryan from Sui Generis blog below and the below studies. Unless isolating microbes, this method could be dangerous unless the starter medium pH is lowered to be under 4.5 and enough ethanol (vodka) is added to reach 3-5% ABV to kill potential pathogens. Purging the flask with CO2 might help prevent mold growth.
  • David Thornton's use of foam as stoppers.
  • Bryan of Sui Generis Blog instructions for using a jar of wort and cheesecloth to catch microbes in the air:

"1) Prepare wort with an OG of 1.040, with enough lactic acid added to reduce the pH to 4.5. I'll often split this off of a normal brew day, prior to my hop addition, or you can make it from DME/LME. I am usually filling a 1L wide-mouthed pickel jar with the wort. I fill the jar to just below the threads.

2) I place the jar, covered with sanitised cheesecloth, in a place where I want to collect [microbes] from - usually I aim for an area with either growing plants (garden, etc), or in an area with lots of deciduous trees, as I've had the best success in these areas. I put the jars out in the evening, once the afternoon heat has passed, and gather them the next morning. I don't worry about having the wort hot and letting it "cool" to mimic a cool-ship as I've found this to not matter. In fact, I often pre-can (using a pressure canner) a few of these, and take them with me camping or to the cottage, to gather the local yeasts. You could even "guerrilla capture" yeast by sneaking these jars into local orchards/farms/etc...not that I'd ever do that ;-)

3) After collecting the (hopefully inoculated) jars, I remove the cheesecloth and replace it with the jar's lid + an airlock fitted into a hole I've drilled. I then place the jar in a dark warm place and let it sit for 3-4 weeks before I sniff test it. If it smells OK I will typically brew with it within the next 2 weeks.

A few other pieces of advice:

1) This works best in the spring and fall - e.g. when nighttime temperatures are getting below ~10C/50F. When its warmer I've found my success rate drops from >80% to ~50%. This doesn't work well if the temperature is going below freezing, as the small volume of wort will inevitably freeze.

2) A small amount of "pure" alcohol can be added to improve your chances - e.g. add enough vodka or other unflavoured cheap booze (whiskey, unflavoured rum, etc) to bring your starters alcohol content up to 3-5%; this will suppress a lot of the unwanted [microbes]. For example, in my case I typically have ~900ml of wort in my 1L jar. To get 5% ABV I would make up 790 ml of wort (at ~1.045), to which I would add 110 ml of 40% ABV vodka. This will give ~5% alcohol, and keep the gravity above 1.035. Obviously, you add the alcohol after the wort has been boiled/canned/etc as you won't want to boil it off.

3) Hoped wort can be used as well, although my success at getting yeast + lacto with hopped wort is poor...I usually only get yeast, even with a meagre 3-5 IBU.

4) Raccoons, possums, rats, squirrels, deer, and pretty much every other fur-bearing animal (and some of the feather-bearing kinds as well) love unfermented wort, so try to place your jar somewhere they cannot get at it - while still maintaining airflow. We've got wild turkeys in the back, and those f***ers can fly (editor's note: language, Bryan!), so its a real challenge to keep them out." [4]

Isolating

Identifying

Growing and Testing

Storage

Slants are a good option for longer term storage (3 months to potentially 2+ years). For instructions on how to make slants at home capable of storing any microbe for potentially 2+ years, see Bryan's video on Sui Generis Brewing (requires a pressure cooker).

David Thornton's "Yeast Bootcamp"

This method was developed by David Thornton of SouthYeast Labs and Carolina Bauernhaus for collecting wild Saccharomyces yeast and lactic acid bacteria. David described his method in detail on the Milk The Funk "The Podcast" episode #005. The following is a summary of this process:

  1. Start with 75 mL of 6° Plato wort with yeast nutrient in a 250 mL flask. Add the sample (fruit, flower, bee, etc). If it's fruit, squish it up. Set the flask on a shaker or stirplate at 80-90F with a foam stopper (the foam stoppers can be autoclaved/boiled and create an aerobic environment; aluminum foil should work as a substitute) for 12-48 hours. Look for frothing and as soon as that happens put an airlock on the flask, but keep on stirplate to assist fermentation.
  2. After the 24-48 hours, decant and discard the liquid and leave the biomass at the bottom. Add 75 mL of new starter media to the flask (with the biomass still at the bottom of the flask) that is 8-10* Plato with yeast nutrient, and purge the flask with CO2. Put an airlock on it on a stirplate and grow until the sugars have fermented.
  3. Again, decant and discard the liquid and leave the biomass at the bottom of the flask. Purge the flask (with the biomass still at the bottom) with CO2, and add 75 mL of 20° Plato wort with yeast nutrient and an airlock, and place on a stirplate or shaker. Look for the wort to ferment down to around 2 Plato or so.
  4. After the 20° Plato wort has finished fermenting, samples from the liquid (the liquid contains the more active yeast versus the biomass at the bottom of the flask) are streaked onto media to isolate the strains that have survived.

Agar that David uses (see also Growth Media on the wiki for other media types that might also be useful):

  • Wallenstein labs nutrient: serves as a catch-all.
  • HLP: isolate Lactobacillus and Pediococcus.
  • LMDA: color indicators for acid production (then do a gram stain to see if it is positive or negative; Lactobacillus versus Acetobacter).
  • Lin’s Cupric Sulfate Medium: selects for wild Saccharomyces.

Other notes:

  • Measure pH to make sure it gets down to under 4.5; this helps monitor for fermentation activity.
  • Generally this method selects for 1-3 strains of yeast with usually a lactic acid bacteria strain.
  • The anaerobic nature of this method selects against mold growth.
  • This method may not select for wild Brettanomyces since aerobic growth is limited. This method could possibly be altered to support the growth of Brettanomyces by allowing for small amounts of oxygen exposure after the initial aerobic step.
  • Agitation during growth helps both aerobic and anaerobic microbes grow by evenly distributing nutrients and waste. David has used something as simple as setting flasks on his washing machine to keep the growth media agitated.

Growing and Testing Without Plating

While using agar plates to isolate yeast colonies is the most effective way to culture wild yeast, it is not the only way. Wild yeast should first be caught using DME wort as outlined in one of Bootleg Biology's methods. Hopping the wort will help decrease bacteria if that is desired. The wort's pH should be lowered to 4.5 or lower with lactic acid to avoid bacteria as much as possible. Molds may still grow in the yeast starter even with the lower pH; wort that has grown mold should not be consumed because mycotoxins can contaminate the wort (see Mold). Keeping the foraged fruit, flowers, etc. under the surface of the wort will help reduce the chances of mold growth. Some brewers recommend submerging the fruit/flowers in the wort for a few minutes and then removing them to prevent them from attracting mold growth [7]. If the yeast cannot be separated from wort that mold grew on then it should be thrown out. Signs of a small krausen within 1-3 days is a good sign that viable wild yeast has been collected. After another few days, the yeast will start dropping to the bottom of the collection vessel [8].

Once the wort has fermented out (allowing 2 weeks total is a good rule of thumb), decant the beer and pitch the collected yeast into 500ml of starter wort, again lowering the pH of the starter wort to 4.5 with lactic acid. After the starter wort has been fermented, the yeast should have enough of a population to ferment out a 1 gallon batch of wort. Keep the recipe of the wort simple, and in the 1.050 gravity range. The yeast cell count should be high enough at this point to out-compete bacteria and lower the pH of the wort within a few days. The pH of the 1 gallon batch, therefore, does not need to be lowered, although the brewer may choose to do so anyway. For safety reasons, test to make sure that the beer fully attenuated and has a pH of 4.5 or less. Allow for one month after fermenting the 1 gallon batch of beer before sampling. If it smells like feces or vomit, do not sample it and throw it away. If the beer smells ok, feel free to sample the beer to see if the wild yeast produced a good tasting beer. David Thornton from SouthYeast Labs estimates that only about 10% of yeast that can fully attenuate produces favorable results, so failures are to be expected (keep trying!) [1]. Fermenting a few batches of beer at different temperatures is a useful method for identifying the ideal fermentation temperature range for the yeast.

Once a wild culture has been determined to be something that the brewer wants to keep for future batches, yeast harvesting methods can be used to maintain the culture. See Wyeast's article on yeast harvesting and harvesting from an over-built yeast starter.

See also:

  • Mold for identifying mold and safety issues with mold. See this MTF thread for tips on avoiding mold when bioprospecting.
  • Microbes cannot be accurately identified based on what their pellicle looks like. See Pellicle for more information.

Notes on Fermentation Timeframes

Some wild captures might take a long time to ferment. Most wild caught, non-isolated cultures that can ferment the sugars in wort take 2-4 weeks to ferment, although some can take longer and some can take less time (even just a couple of days). This is impossible to predict due to the impossibility of predicting what microbes are in a non-isolated wild culture. For example, wild Brettanomyces might take a long time to finish fermenting (up to 8 months to reach a stable gravity, according to Matt Spaanem). In some experiences, krausen will form for a few days, and then drop, and then form again (presumably as another microbe starts fermenting). Others ferment long and slow over a month or so. Once re-pitched with higher cell counts, often the fermentation times will hasten, but other times they will not. The best piece of advice is to take gravity readings to determine when the fermentation is finished instead of relying on visual indicators. Even though there could be a krausen and signs of fermentation, if the fermentation is going slow then it is possible that the sugars in the wort are not being fermented effectively (this might improve upon re-pitching as this should help select for microbes that can withstand this environment; see reference for more details on MTF members' experiences) [9].

Safety

An often asked question when it comes to tasting beer fermented from a wild culture that wasn't plated and isolated is, "When is it safe to drink?" To our knowledge, no studies have shown when a wild caught beer is scientifically ~99.9% safe to drink. However, several studies can give us some guidance on this, as well as Bryan from Sui Generis Blog.

  • The addition of hops in the starter wort will inhibit or kill some Gram-positive bacteria pathogens (L. monocytogenes and S. aureus) [10][11].
  • A low pH is not the only requirement for making beer safe. A combination of alcohol, low pH, and hops will ensure that pathogens cannot grow (survival is another matter) [12][13].
  • Two studies have shown that E. coli can survive in moderate strength beer (~5% ABV, 4.3 pH) for at least 28 days (more than 30 days in one study) if the beer is stored cold (39-41°F or 4-5°C) [10][14]. Therefore, the wild culture should be stored at room temperature for a least a month before tasting. Additionally, the pH of the beer should be less than 4.6, and alcohol should be present for at least one month before tasting. The smell of feces or vomit indicates that bad bacteria may be present; if so dump it out [15].
  • In unfermented wort, E. coli O157:H7 and Salmonella typhimurium were shown to grow at a pH of 4.3, but stopped growing at a pH of 4.0 [10]. However, some of these food poisoning bacteria can survive the low pH of the stomach (~2.0) long enough to cause illness, so relying on a low pH alone is not adequate. Bryan of Sui Generis Blog recommends a pH of 4.5 or less, the use of well sanitized equipment, and monitoring the starter for the rapid onset of fermentation without putrid smells. Some oxidative yeasts that are the first "barriers" to bacterial growth may be inhibited at 4.0 pH [11].
  • The risk of botulism toxin is extremely low, but not zero. Generally, C. botulism is not able to grow and produce toxin in low protein (non-meats) substances at a pH lower than 4.6 [16][17]. However, proteins in the wort may allow growth at a lower pH than 4.6 (studies have not been done on wort, only on meats) [11]. The presence of more than 5% oxygen in the wort will also reduce the risk [18].
  • To our knowledge, there have been no documented cases of food poisoning from beer [12]. However: "It is well established that at least a dozen human pathogens can survive in fully fermented beer; moreover, sour-brewing (and home brewing in general) offers an additional opportunity for pathogens to gain a foothold or produce toxins which persist into the final product. Reality is that the source of most food poisonings is never established. A few industries (milk, meat, canning, etc) are under close scrutiny, and as such we have firm numbers for rates of food-borne illnesses from those industries. Neither commercial nor homebrewing is subject to that monitoring, so any cases of disease arising from contaminated products from either source would likely go unidentified." - Bryan of Sui Generis Blog on MTF [11]. For more information on the potential for pathogens surviving in beer, see this Sui Generis Blog article..
  • In the words of Bryan from Sui Generis Blog: "If you have an immunodeficiency or are immunosuppressed: Obviously, don't take medical advice off the internet. Talk to your medical doctor to see if you are at risk of infection - especially if you're condition/treatment increases your risk of fungal infections. If so, or if you are concerned, stick to beers that are fermented using commercial strains of Saccharomyces yeast." [12]
  • See also this thread on MTF.
  • See also this MTF thread on the mechanisms of botulism poisoning, and how it applies to different brewing processes.
  • Further explanation of what makes microbes "pathogenic" by Bryan from Sui Generis Blog and a related MTF discussion.
  • If mold grows on growth media and the yeast can be isolated cleanly, then this is not an issue, however, do not consume beer or wort that has had mold growing on it. If the yeast is not being isolated on agar plates, then throw out the capture sample and do not use it. See Mold for identifying mold and safety issues with mold.
  • An example of identifying a pathogenic species of Candida (C. lusitaniae) in a wild capture.
  • Do not taste wort soured with malted grain; grain soured wort has been found to grow pathogens. Wait until fermentation with yeast is finished. See Grain for more information.

Wild Brettanomyces

Brettanomyces is notoriously difficult to bioprospect from the wild. While it has been alleged by many that the natural environment for Brettanomyces is the skins of fruit or the bodies of insects, it wasn't until 2007 that science was able to show that Brettanomyces survives on the skins of grapes, although it does so in a very low population and a possibly "viable but not culturable" state. Renouf et al. (2007) and Comitini et al. (2019) demonstrated that an "enrichment Brettanomyces bruxellensis" media called EBB is more efficient at first growing up Brettanomyces before trying to culture it on DBDM. Brettanomyces was allowed to grow for 80 days in the EBB media, and then streaked onto DBDM for selection for Brettanomyces (other wild yeast such as Hanseniaspora and Pichia grew much more readily than Brettanomyces that was cultured from wine grapes).

Using EBB, Comitini et al. (2019) found that after 80 days of incubation in the EBB medium, half of grapes samples from a vineyard with several varieties of grapes had wild Brettanomyces on their skins. The researchers also tested the air, the trailers, and the winery where the grapes were processed. They found no Brettanomyces in the air or the trailers, but did find a couple of valves that were used to transfer must have Brettanomyces. Out of the four varieties of grapes sampled, Merlot grapes did not have any Brettanomyces growing on them, indicating that the variety of the grape might play a role in the ability of Brettanomyces to survive on grape skins. The researchers found a wide range of strains that matched the strains found in the winery, indicating that the Brettanomyces strains did originate from the grape skins [19][20].

See also:

MTF Wild Capture Swap

DeWayne Schaaf organizes an occasional swap of wild caught microbes on MTF.

  • Fall 2017 swap: currently under way. See this thread to sign up.

See Also

Additional Articles on MTF Wiki

External Resources

Links

Books

Videos

References

  1. ↑ 1.0 1.1 Conversation with David Thornton on MTF. 09/06/2015.
  2. ↑ Caroline Whallen Taggart on removing fruit to prevent mold. Milk The Funk Facebook group. 09/05/2017.
  3. ↑ Milk The Funk thread on the best seasons for bioprospecting. 05/11/2017.
  4. ↑ Bryan of Sui Generis blog. Milk The Funk Facebook group post on catching wild yeast. 09/10/2017.
  5. ↑ "Old School Identification of Wild Yeasts & Bacteria". Sui Generis blog. 04/10/2013. Retrieved 02/14/2018.
  6. ↑ Shawn Savuto. Milk The Funk Facebook thread on books on identifying microbes. 04/11/2018.
  7. ↑ Jeff Porn. Milk The Funk Facebook group. 06/06/2017.
  8. ↑ Collecting Wild Yeast. Manoaction. Homebrewtalk. Oct 30, 2012.
  9. ↑ DeWayne Schaaf, Christophe Boebion, Tobias Häberli, Justin Amaral, Bryan from Sui Generis blog, Matt Spaanem, Josh Regenold, Guy Greenhalgh, Jason Pallett, Caroline Whalen Taggart. Milk The Facebook group on how long fermetnation takes for wild caught cultures. 08/01/2017.
  10. ↑ 10.0 10.1 10.2 Growth and survival of foodborne pathogens in beer. Menz G., Aldred P, Vriesekoop F . Oct 2011.
  11. ↑ 11.0 11.1 11.2 11.3 Conversation with Bryan of Sui Generis Blog on MTF regarding when it is safe to taste wild beer. 7/6/2015.
  12. ↑ 12.0 12.1 12.2 Fact or Fiction? Can Pathogens Survive in Beer? Sui Generis Blog.
  13. ↑ The growth and survival of food-borne pathogens in sweet and fermenting brewers' wort. Menz G1, Vriesekoop F, Zarei M, Zhu B, Aldred P. May 2010.
  14. ↑ Survival of foodborne pathogenic bacteria (Bacillus cereus, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Listeria monocytogenes) and Bacillus cereus spores in fermented alcoholic beverages (beer and refined rice wine). Kim SA1, Kim NH, Lee SH, Hwang IG, Rhee MS. March 2014.
  15. ↑ Conversation with Bryan of Sui Generis Blog on MTF regarding when it is safe to taste wild beer. 7/5/2015.
  16. ↑ Toxin Production by Clostridium Botulinum in Media at pH Lower Than 4.6. Nobumasa, Tanaka. Journal of Food Protection®, Number 3, February 1982, pp. 214-284, pp. 234-237(4)
  17. ↑ Preventing Foodborne Illness: Clostridium botulinum. University of Florida IFAS Extension. Retrieved 7/5/2015.
  18. ↑ Growth and toxin production by Clostridium botulinum in steamed rice aseptically packed under modified atmosphere. Kasai Y, Kimura B, Kawasaki S, Fukaya T, Sakuma K, Fujii T. May 2005.
  19. ↑ Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries. Vincent Renouf, Aline Lonvaud-Funel. 2007. DOI: https://doi.org/10.1016/j.micres.2006.02.006.
  20. ↑ Occurrence of Brettanomyces bruxellensis on grape berries and in related winemaking cellar. Francesca Comitini1, Lucia Oro, Laura Canonico, Valentina Marinelli, Maurizio Ciani. 2019. DOI: 10.3389/fmicb.2019.00415.
Retrieved from "http://www.milkthefunk.com/w/index.php?title=Wild_Yeast_Isolation&oldid=11726"