Changes

Var. ''diastaticus'' contamination in breweries has been a recent hot topic. These strains are effectively eliminated by standard cleaning and sanitation practices, although inadequate cleaning hygiene can lead to biofilm formation which makes them more resistant to cleaning regiments <ref name="Meier-Dörnberg" />. The source of some contaminations has also been suspected to potentially come from yeast suppliers, although there is no known percentage of infections which originate from yeast suppliers versus poor hygiene in the brewery. For example, a lawsuit by Left Hand Brewing Co. accused White Labs as being the source for the brewery's ''diastaticus'' contamination issues (see also [https://www.whitelabs.com/diastaticus this statement by White Labs]) <ref>[https://www.courthousenews.com/wp-content/uploads/2017/11/Left-Hand-v-White-Labs-COMPLAINT.pdf District Court, Boulder County, Colorado. Case Number 2017CV31132. Filed 11/14/2017.]</ref>. Detection of ''S. cerevisiae'' var. ''diastaticus'' as a contaminant can be difficult. Contamination usually occurs as a secondary contaminant (meaning in the packaging system), and can come from contact with beer lines, by air circulation in the area of the packaging equipment, or by insufficient heat treatment of the packaging line. Since a very small number of cells is enough to contaminate beer, contaminations can be sporadic with only a percentage of bottles being contaminated <ref name="Meier-Dörnberg_2018" />. Documented attenuation percentages above 75% for any strain of ''S. cerevisiae'' is also an indicator that the strain could be ''diastaticus'', however, some non-diastaticus strains can also attenuate higher than 75%, so this indicator is not a reliable method to be sure that a given strain is ''diastaticus'' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2004689559559248/?comment_id=2004695002892037&reply_comment_id=2005133672848170&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Adi Hastings. Milk The Funk Facebook Group post on non-diastaticus strains with high attenuation. 02/27/2018.]</ref><ref name="mbaa_diastaticus" />. Often this contamination can only be detected by sensory testing weeks after packaging. This yeast has reportedly been detected using [http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1981.tb04005.x/pdf LCSM agar plates], although other species of wild ''Saccharomyces'' yeast can grow on this media <ref name="mbaa_diastaticus" />(~18 mins) and PCR DNA analysis is required to give a positive identification of ''diastaticus''. Additionally, the default level of CuSO₄ which is ~550 ppm (this can vary depending on manufacturer) can inhibit some strains of ''diastaticus''; Wade Begrow of Founders Brewing Co. recommends diluting the LCSM media with a basic malt media so that the CuSO₄ reaches around 200 ppm, or using LCSM plates modified with a gradient of CuSO₄ <ref name="Begrow_MBAA" /> (~22 mins in). Adding p-coumaric acid or other cinnamic acids to the LCSM agar media which can then test for POF+ yeast and then confirmed for the presence of phenols via a gas chromatography or some other method can also be used to indicate that a yeast might be ''diastaticus'' since there is a high occurrence of ''diastaticus'' to produce phenols from these cinnamic acids <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2149139905114212/?comment_id=2150763631618506&reply_comment_id=2158975484130654&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Shawn Savuto and linked references. Milk The Funk Facebook book post on POF+ correlation with ''diastaticus''. July 2018.]</ref> (see also [https://www.facebook.com/groups/MilkTheFunk/permalink/1903290776365794/ this MTF thread] on using cinnamic acids to identify phenolic off flavor strains). Cheaper methods of doing PCR are recently becoming available, and could help breweries with smaller budgets sufficiently detect this as a contaminant (see [[Laboratory_Techniques#PCR.2FqPCR|PCR Lab Techniques]]). A recent study used agar plates with 15 g/L^-1 of starch as the only nutrient with 40 mg/L^-1 bromophenol blue in anaerobic conditions to detect the fermentation of starch (a pH drop from 5.2 to 4.6-3.0 will change the color of the agar plate to blue/violet). For some of the slower growing strains, 14 days were required to verify that they were ''diastaticus'' while other strains grew as quickly as two days and most strains grew after five days. The yeast cells had to be thoroughly washed of all other carbohydrate material and starved in order to avoid false positives. Using dextrin agar plates instead of starch also led to false positives <ref name="Meier-Dörnberg_2018" />. This starch media has been recommended by Richard Preiss from [[Escarpment Laboratories]] and Justin Amaral from [[Mainiacal Yeast]] <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2149139905114212/?comment_id=2150763631618506&comment_tracking=%7B%22tn%22%3A%22R%22%7D Richard Preiss and Justin Amaral. Milk The Funk Facebook thread on plate media for diastaticus. 06/26/2018.]</ref>. Note that ''diastaticus'' cells look the same under a microscope as regular ''S. cerevisiae'', so cell morphology is not an effective way to identify ''diastaticus'' strains <ref name="Begrow_MBAA">[https://www.mbaa.com/education/webinars/Pages/webcast.aspx?vid=diastaticus Wade Begrow. "S. cerevisiae var. diasttaicus". MBAA webinar. July 2018.]</ref> (~8 minutes in). Other methods of detection include using a Durham tube/fermentation tube test to see if the beer produces CO² after fermentation, although this method does not identify the cause of the additional fermentation <ref name="Begrow_MBAA" /> (~18 mins in). More recently, Krogarus et al. (2019) developed a more precise PCR primers to detect STA1 active, STA1 non-active, and non-STA1 based on their discovered role of an STA1 promoter called ''1162 bp'' that is required for the STA1 gene to be effective at producing the glucoamylase enzyme (see [http://beer.suregork.com/?p=4068 this Suregork Loves Beer blog post] and [https://www.facebook.com/groups/MilkTheFunk/permalink/2697088176986046/ this MTF thread posted by Kristoffer Krogarus]).