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Saccharomyces

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''Saccharomyces cerevisiae'' var. ''diastaticus'' is a variant of ''S. cerevisiae'' that can ferment certain types of starches and dextrins, and has been identified as a contaminant in breweries and is responsible for a few large recalls. This nomenclature has been called into question (classifying it as a true variant based on one phenotype is not typical in microbiology), and terms like "STA1+ strains of ''S. cerevisiae''" or "diastatic ''cerevisiae''" have been proposed as a more scientifically correct designation <ref name="Omega_diastaticus_2020">[https://omegayeast.com/news/improved-functional-assays-and-risk-assessment-for-sta-strains-of-saccharomyces-cerevisiae Improved Functional Assays and Risk Assessment for STA+ Strains of Saccharomyces cerevisiae. Laura T. Burns, Christine D. Sislak, Nathan L. Gibbon, Nicole R. Saylor, Marete R. Seymour, Lance M. Shaner, and Patrick A. Gibney. 2020. Awaiting peer review and publication.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3308119659216225/?comment_id=3308312722530252&reply_comment_id=3308849332476591 Kristoffer Krogerus. Milk The Funk Facebook group post on diastaticus diastatic ''cerevisiae'' nomenclature. 02/27/2020.]</ref>. These strains generally do not produce flavors that are considered unpleasant. For example, acetaldehyde and sulfur dioxide are produced in very low amounts compared to other brewing strains. However, all ''diastaticus'' strains tested in one study produced phenols (there have been With the exception of a couple of strains found that do not produce phenols, such as the "Sacch Trois " strain, almost all diastatic ''cerevisiae'' strains produce phenols (POF+), with most being above which are considered an off-flavor thresholdin many beer styles. Most strains also produced significant amounts of isoamyl acetate (banana ester) and other fruity esters, making them taste very similar to German wheat strains. A small number of strains also produce above flavor threshold levels of diacetyl <ref name="Meier-Dörnberg_2018">[https://www.ncbi.nlm.nih.gov/pubmed/29518233 Saccharomyces cerevisiae variety diastaticus friend or foe? Spoilage potential and brewing ability of different Saccharomyces cerevisiae variety diastaticus yeast isolates by genetic, phenotypic and physiological characterization. Meier-Dörnberg T, Kory OI, Jacob F, Michel M, Hutzler M. 2018. DOI: 10.1093/femsyr/foy023.]</ref>. This variant is often viewed as a contaminant because of its ability to over-attenuate. A survey of contamination reports in the last ten years at European breweries (50% of which were German breweries, which are obligated by law to report such contaminations) found an increase in reports from 2015, 2016, and 2017. 71% of the contamination incidents originated from the packaging systems (bottling/canning lines). These contaminations were tracked down to the filler environment and/or biofilms in the pipework system of the filler which stemmed from hygienic problems. As such, sometimes contaminations can be sporadic with some bottles being contaminated while others are not. The other 29% of the contaminations were tracked down to primary contaminations in the brewhouse, fermentation cellar, and storage cellar <ref name="Meier-Dörnberg">[https://www.mbaa.com/publications/tq/tqPastIssues/2017/Pages/TQ-54-4-1130-01.aspx Incidence of Saccharomyces cerevisiae var. diastaticus in the Beverage Industry: Cases of Contamination, 2008–2017. Tim Meier-Dörnberg, Fritz Jacob, Maximilian Michel, and Mathias Hutzler. 2017. MBAA Technical Quarterly; http://dx.doi.org/10.1094/TQ-54-4-1130-01.]</ref>.
This variant STA1+ strains of ''S. cerevisiae'' can produce extracellular glucoamylase (also called [https://en.wikipedia.org/wiki/Alpha-glucosidase alpha-glucosidase], which is the same enzyme that ''[[Brettanomyces]]'' produces to break down starches and dextrins). This enzyme is released outside of the cell and can break down the α-1,4 linkages of starches and dextrins releasing glucose that is then fermented by the yeast. The capability to produce this enzyme is encoded by the ''STA1'' gene, which is a fusion of two other genes that are present separately in all ''S. cereivisaecerevisiae'' yeasts, ''FLO11'' and ''SGA1'' (the ''STA2'' and ''STA3'' genes are the same as ''STA1''; they were initially found on different chromosomes and so they received different names, but they are all the same gene <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2697088176986046/?comment_id=2697419373619593&reply_comment_id=2698451940183003&comment_tracking=%7B%22tn%22%3A%22R%22%7D Kristoffer Krogerus. Milk The Funk Facebook thread post on the significance of STA2 and STA3 genes in diastaticus strains. 06/01/2019.]</ref>). Not all strains containing one of these genes produce the glucoamylase enzyme or are as effective as others at metabolizing dextrins <ref>[https://link.springer.com/article/10.1007%2FBF00365634 STA10: A gene involved in the control of starch utilization by Saccharomyces. Julio Polaina, Melanie Y. Wiggs. 1983.]]</ref><ref>[http://onlinelibrary.wiley.com/doi/10.1002/yea.1102/full Structural analysis of glucoamylase encoded by the ''STA1'' gene of Saccharomyces cerevisiae (var. diastaticus). Ana Cristina Adam, Lorena Latorre-Garcia, Julio Polaina. 2004.]</ref>. It has been reported by some microbiologists that most brewing strains that contain the ''STA1'' gene do produce the glucoamylase enzyme <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1935201836508021/?comment_id=1936604203034451&reply_comment_id=1937166892978182&comment_tracking=%7B%22tn%22%3A%22R7%22%7D Richard Preiss. Milk the Funk thread about ''STA1'' gene correlation to glucoamylase production. 12/31/2017.]</ref><ref name="mbaa_diastaticus">[http://masterbrewerspodcast.com/068-diastaticus-part-1 Matthew Peetz of Inland Island and Tobias Fischborn of Lallemand. "Master Brewers Association Podcast" 12/25/2017.]</ref>(~16 mins). A study that surveyed 18 strains of ''diastaticusS. cerevisiae'' that contain the ''STA1'' gene found that only one was not able to ferment dextrins <ref name="Meier-Dörnberg_2018" />. Richard Preiss has also reported that WLP351 has the ''STA1'' gene, but is not able to ferment dextrins <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1888017211226484/?comment_id=2013050695389801&reply_comment_id=2013355312026006&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Richard Preiss. Milk The Funk Facebook group thread on diastaticus diastatic strains that do not ferment dextrins. March 2018.]</ref>. Krogerus et al. (2019) discovered that a region of 1162 base pairs just upstream of the ''STA1'' gene called "a promoter gene" is missing in strains that test positive for the ''STA1'' gene but do not test positive for fermenting starches, dextrins, or secreting the enzyme. They were able to demonstrate that this promoter gene region is needed for the ''STA1'' gene to become expressed. They also discovered that ''STA1'' gene is found in the Beer 2 group of yeast (see [[Saccharomyces#History_of_Domestication|History of Domestication]] above), and wild ''S. cerevisiae'' strains do not carry the ''STA1'' gene. Coincidentally, Beer 2 yeast strains lack the genes that the Beer 1 yeast strains do for fermenting maltotriose, yet Beer 2 yeasts ferment maltotriose just fine; it was discovered by Krogerus et al. (2019) that the ''STA1'' gene allows the Beer 2 yeasts to ferment maltotriose (although this exact mechanism is not known yet). It was proposed that the ''STA1'' gene evolved in the Beer 2 yeast strains as a means to take advantage of grain fermentation as an evolutionary advantage, and the existence of strains that are missing the promoter gene could be because humans later started selecting for strains that didn't dry the beer out too much <ref name="krogarus_2019">[https://link.springer.com/article/10.1007/s00253-019-10021-y A deletion in the ''STA1'' promoter determines maltotriose and starch utilization in ''STA1+'' Saccharomyces cerevisiae strains. Kristoffer Krogerus, Frederico Magalhães, Joosu Kuivanen, Brian Gibson. 2019. DOI: https://doi.org/10.1101/654681.]</ref>. For more details on the Krogerus et al. (2019) study, 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 Krogerus].
When beer containing this yeast is packaged too early, it will continue to slowly ferment dextrins and cause over-carbonation. When pitching a proper cell count or pitching rate of a ''diastaticus'' diastatic yeast strain into the wort, some strains will fully ferment as quickly or nearly as quickly as any other brewers yeast, while other strains may take as long as 16 days to fully ferment a simple 12.4°P (1.050 SG) wort. A highly dextrinous wort may take longer to fully ferment. The problem of slow fermentation in already packaged beer is usually only a concern when diastatic ''diastaticuscerevisiae'' is introduced as a very small cell count, for example as an accidental contamination <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1982499288444942/?comment_id=1983013578393513&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Caroline Whalen Taggart. Milk The Facebook post on how quickly diastaticus diastatic ferments. 02/09/2018.]</ref>. The enzyme produced by these strains is heat stable and can continue to work on starches and dextrins even after the yeast is killed by heat pasteurization <ref>[https://www.mbaa.com/publications/tq/tqPastIssues/1983/Abstracts/tq83ab19.htm Factors That Control the Utilization Of Wort Carbohydrates by Yeast. G. G. Stewart, I. Russell, and A. M. Sills. MBAA Technical Quarterly, Vol. 20, No. 1, 1983.] </ref>. Almost all diastatic strains of ''S. cerevisiae'' var. ''diastaticus'' are able to ulilize all of the sugars found in wort (e.g. glucose, fructose, sucrose, maltose, maltotriose), although one strain tested in a recent study could not efficiently ferment starch, maltose, (80%) or maltotriose (45%) <ref name="Meier-Dörnberg_2018" />. STA1+ strains of ''S. cerevisiae'' var ''diastaticus'' can grow at 37°C and can also remain viable at refrigeration temperatures <ref>[http://www.ebc2017.com/inhalt/uploads/P095_Begrow.pdf Wade Begrow. "Recent notable microbiological contaminations of craft beer in the United States". Presentation poster at EBC 2017. Retrieved 11/19/2017.]</ref>. Some strains of diastatic ''diastaticuscerevisiae'' are as flocculant as typical ale strains, while others are less flocculant <ref name="Meier-Dörnberg_2018" />. One strain of diastatic ''diastaticuscerevisiae'', the Belle Saison strain from Lallemand, is killer neutral, which means that killer wine strains will not kill it (it is not verified if Belle Saison is the same as WY3711, although it is suspected to be). Other strains of diastatic ''diastaticuscerevisiae'' may or may not be killer neutral (more data is needed; see [[Saccharomyces#Killer_Wine_Yeast|killer wine strains]] for more information on this topic).
Var. Diastatic ''diastaticuscerevisiae'' 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'' diastatic yeast 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 STA1+ strains of ''S. cerevisiae'' var. ''diastaticus'' as a contaminant can be difficult(see "Detection Methods" below). 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 one viable cell that is able to divide can be 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''diastatic, however, some non-diastaticus diastatic strains can also attenuate higher than 75%, so this indicator is not a reliable method to be sure that a given strain is ''diastaticus'' STA1+ <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 diastatic 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.
=====Detection Methods===== This yeast can 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 STA1+ strains of ''diastaticusS. cerevisiae''. Additionally, the default level of CuSO<sub>4</sub> 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 CuSO<sub>4</sub> reaches around 200 ppm, or using LCSM plates modified with a gradient of CuSO<sub>4</sub> <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). [[Omega Yeast Labs]] reported that a slight alteration to the classic LCYM media recipe had significantly more reliable detection than classic LCYM and the proprietary Weber diastaticus agar for all STA1 positive strains in Omega's collection, including detecting slow growing strains within 2-3 days and strains with the non-active promoter genes as per Krogerus et al (2019) and limited false positives; see [[Laboratory_Techniques#Saccharomyces|''Saccharomyces'' agar plates]] for the recipe and [https://www.facebook.com/groups/MilkTheFunk/permalink/2874530432575152/ this MTF thread] by Laura Burns from Omega Yeast Labs.
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<sup>-1</sup> of starch as the only nutrient with 40 mg/L<sup>-1</sup> 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 CO<sup>2</sup> after fermentation, although this method does not identify the cause of the additional fermentation <ref name="Begrow_MBAA" /> (~18 mins in). More recently, Krogerus et al. (2019) developed 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, however, PCR and qPCR have limited detection rates of 10<sup>-4</sup> and 10<sup>-5</sup> cells (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 Krogerus]).
=====Commercial Strains===== WY3711 saison yeast has been determined to be ''S. cerevisiae'' var. ''diastaticus'' STA1+ <ref name="preiss_diastaticus" />. Since yeast cannot be patented, many yeast labs are thought to offer this strain to customers. In the [[Saccharomyces#Commercial_Farmhouse.2FBelgian_Strains_of_Saccharomyces|commercial culture]] lists below where we believe a lab is selling this strain or another strain of ''diastaticus'' yeast, we note that it is determined or suspected to be ''diastaticus''.
White Labs now reports that the strains

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