Changes

''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. 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 a couple of strains found that do not produce phenols, such as the Sacch Trois strain), with most being above flavor threshold. 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 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, STA2, or STA3 genes. 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 STA1, STA2, or STA3 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 ''diastaticus'' that contain the STA 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 STA 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 strains that do not ferment dextrins. March 2018.]</ref>. When beer containing Krogarus et al. (2019) discovered that a region of genes just upstream of the STA1 gene (promoter) called 1162 bp 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 yeast region is packaged too early, it will continue needed for the STA1 gene to slowly ferment dextrins and cause over-carbonationbecome effective. When pitching a proper cell count or pitching rate They also discovered that STA1 gene is found in the Beer 2 group of yeast (see [[Saccharomyces#History_of_Domestication|History of a ''diastaticus'' Domestication]] above), and wild yeast strain into strains do not carry the wortSTA1 gene. Coincidentally, some Beer 2 yeast strains will fully ferment as quickly or nearly as quickly as any other brewers lack the genes that the Beer 1 yeaststrains do for fermenting maltotriose, while other strains may take as long as 16 days to fully yet Beer 2 yeasts ferment a simple 12maltotrios just fine; it was discovered by Krogarus et al.4°P (1.050 SG2019) wort. A highly dextrinous wort may take longer that the STA1 gene allows the Beer 2 yeasts to fully ferment. The problem of slow fermentation in already packaged beer maltotriose (although this exact mechanism is usually only a concern when ''diastaticus'' is introduced as a very small cell count, for example as an accidental contamination not known yet) <refname="krogarus_2019">[https://www.facebookbiorxiv.comorg/groupscontent/MilkTheFunk10.1101/permalink/1982499288444942/?comment_id=1983013578393513&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Caroline Whalen Taggart654681v1 A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains. Milk The Facebook post on how quickly diastaticus fermentsKristoffer Krogerus, Frederico Magalhães, Joosu Kuivanen, Brian Gibson. 02/09/2018.]</ref>2019. 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>[DOI: https://wwwdoi.mbaaorg/10.com/publications/tq/tqPastIssues1101/1983/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, 1983654681.] </ref>. Almost all strains of ''SFor more details on the Krogarus et al. cerevisiae'' var. ''diastaticus'' are able to ulilize all of the sugars found in wort (e.g. glucose, fructose, sucrose, maltose, maltotriose2019)study, although one strain tested in a recent study could not efficiently ferment maltose or maltotriose <ref name="Meier-Dörnberg_2018" see [http://>beer. ''Ssuregork. cerevisiae'' var ''diastaticus'' can grow at 37°C com/?p=4068 this Suregork Loves Beer blog post] and can also remain viable at refrigeration temperatures <ref>[httphttps://www.ebc2017facebook.com/inhaltgroups/uploadsMilkTheFunk/P095_Begrow.pdf Wade Begrow. "Recent notable microbiological contaminations of craft beer in the United States". Presentation poster at EBC 2017. Retrieved 11permalink/192697088176986046/2017.]</ref>. Some strains of ''diastaticus'' are as flocculant as typical ale strains, while others are less flocculant <ref name="Meier-Dörnberg_2018" />. One strain of ''diastaticus'', 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 ''diastaticus'' may or may not be killer neutral (more data is needed; see [[Saccharomyces#Killer_Wine_Yeast|killer wine strains]this MTF thread posted by Kristoffer Krogarus] for more information on this topic).

VarWhen 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'' contamination in breweries has been 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 recent hot topicsimple 12.4°P (1.050 SG) wort. These strains are effectively eliminated by standard cleaning and sanitation practices, although inadequate cleaning hygiene can lead A highly dextrinous wort may take longer to biofilm formation which makes them more resistant to cleaning regiments <ref name="Meier-Dörnberg" fully ferment. The problem of slow fermentation in already packaged beer is usually only a concern when ''diastaticus'' is introduced as a very small cell count, for example as an accidental contamination <ref>[https://>www.facebook. 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 brewerycom/groups/MilkTheFunk/permalink/1982499288444942/?comment_id=1983013578393513&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Caroline Whalen Taggart. For example, a lawsuit by Left Hand Brewing CoMilk The Facebook post on how quickly diastaticus ferments. accused White Labs as being the source for the brewery's ''diastaticus'' contamination issues (see also [https: 02/09/www2018.whitelabs.com]</diastaticus this statement by White Labs]) <ref>[https://www.courthousenews.com/wp-content 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:/uploads/2017www.mbaa.com/publications/tq/tqPastIssues/1983/11Abstracts/Left-Hand-v-White-Labs-COMPLAINTtq83ab19.htm Factors That Control the Utilization Of Wort Carbohydrates by Yeast.pdf District Court, Boulder County G. G. Stewart, ColoradoI. Case Number 2017CV31132Russell, and A. Filed 11/14/2017M.]</ref>Sills. Detection MBAA Technical Quarterly, Vol. 20, No. 1, 1983.] </ref>. Almost all strains of ''S. cerevisiae'' var. ''diastaticus'' as a contaminant can be difficultare able to ulilize all of the sugars found in wort (e.g. Contamination usually occurs as a secondary contaminant (meaning in the packaging systemglucose, fructose, sucrose, maltose, maltotriose), and can come from contact with beer lines, by air circulation although one strain tested in the area of the packaging equipment, a recent study could not efficiently ferment maltose or by insufficient heat treatment of the packaging linemaltotriose <ref name="Meier-Dörnberg_2018" />. Since a very small number of cells is enough to contaminate beer, contaminations ''S. cerevisiae'' var ''diastaticus'' can grow at 37°C and can be sporadic with only a percentage of bottles being contaminated also remain viable at refrigeration temperatures <ref name="Meier-Dörnberg_2018" >[http://www.ebc2017.com/inhalt/uploads/>P095_Begrow.pdf Wade Begrow. Documented attenuation percentages above 75% for any strain "Recent notable microbiological contaminations of ''Scraft beer in the United States". Presentation poster at EBC 2017. cerevisiae'' is also an indicator that the strain could be Retrieved 11/19/2017.]</ref>. Some strains of ''diastaticus''are as flocculant as typical ale strains, however, some nonwhile others are less flocculant <ref name="Meier-Dörnberg_2018" />. One strain of ''diastaticus strains can also attenuate higher than 75%'', the Belle Saison strain from Lallemand, so this indicator is killer neutral, which means that killer wine strains will not a reliable method to be sure that a given strain kill it (it is 'not verified if Belle Saison is the same as WY3711, although it is suspected to be). Other strains of ''diastaticus'' <ref>[https://wwwmay or may not be killer neutral (more data is needed; see [[Saccharomyces#Killer_Wine_Yeast|killer wine strains]] for more information on this topic).facebook  Var.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>''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="mbaa_diastaticusMeier-Dörnberg" />. Often this contamination can only be detected by sensory testing weeks after packaging. This yeast The source of some contaminations has reportedly also been detected using [http://onlinelibrary.wiley.com/doi/10suspected 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.1002/j.2050-0416 For example, a lawsuit by Left Hand Brewing Co.1981.tb04005.x/pdf LCSM agar plates], although other species of wild accused White Labs as being the source for the brewery's ''Saccharomycesdiastaticus'' yeast can grow on this media <ref name="mbaa_diastaticus" contamination issues (see also [https://www.whitelabs.com/diastaticus this statement by White Labs]) <ref>(~18 mins) and PCR DNA analysis is required to give a positive identification of ''diastaticus''[https://www.courthousenews. Additionally, the default level of CuSO<sub>4<com/wp-content/uploads/2017/sub> which is ~550 ppm (this can vary depending on manufacturer) can inhibit some strains of ''diastaticus''; Wade Begrow of Founders Brewing Co11/Left-Hand-v-White-Labs-COMPLAINT.pdf District Court, Boulder County, Colorado. recommends diluting the LCSM media with a basic malt media so that the CuSO<sub>4 Case Number 2017CV31132. Filed 11/14/2017.]</subref> reaches around 200 ppm, or using LCSM plates modified with a gradient . Detection of CuSO₄ <ref name="Begrow_MBAA" /> (~22 mins in)''S. cerevisiae'' var. Adding p-coumaric acid or other cinnamic acids to the LCSM agar media which ''diastaticus'' as a contaminant can then test for POF+ yeast be difficult. Contamination usually occurs as a secondary contaminant (meaning in the packaging system), and then confirmed for the presence of phenols via a gas chromatography can come from contact with beer lines, by air circulation in the area of the packaging equipment, or some other method can also be used by insufficient heat treatment of the packaging line. Since a very small number of cells is enough to indicate that a yeast might contaminate beer, contaminations can be ''diastaticus'' since there is sporadic with only a high occurrence percentage of ''diastaticus'' to produce phenols from these cinnamic acids <refbottles being contaminated <ref name="Meier-Dörnberg_2018" />[https://www.facebook.com/groups/MilkTheFunk/permalink/2149139905114212/?comment_id=2150763631618506&reply_comment_id=2158975484130654&comment_tracking= Documented attenuation percentages above 75%7B%22tn%22%3A%22R0%22%7D Shawn Savuto and linked referencesfor any strain of ''S. Milk The Funk Facebook book post on POF+ correlation with cerevisiae'' is also an indicator that the strain could be ''diastaticus''. July 2018.]</ref> (see , however, some non-diastaticus strains can also [https://www.facebook.com/groups/MilkTheFunk/permalink/1903290776365794/ this MTF thread] on using cinnamic acids attenuate higher than 75%, so this indicator is not a reliable method to identify phenolic off flavor strains)be sure that a given strain is ''diastaticus'' <ref>[https://www. 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]])facebook.com/groups/MilkTheFunk/permalink/2004689559559248/?comment_id=2004695002892037&reply_comment_id=2005133672848170&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Adi Hastings. A recent study used agar plates Milk The Funk Facebook Group post on non-diastaticus strains with 15 ghigh attenuation. 02/27/L2018.]<sup/ref>-1<ref name="mbaa_diastaticus" /sup> of starch as the . Often this contamination can only nutrient with 40 mgbe detected by sensory testing weeks after packaging. This yeast has reportedly been detected using [http:/L^-1 bromophenol blue in anaerobic conditions to detect the fermentation of starch (a pH drop from 5onlinelibrary.2 to 4wiley.6-3com/doi/10.0 will change the color of the agar plate to blue1002/violet)j.2050-0416. For some of the slower growing strains1981.tb04005.x/pdf LCSM agar plates], 14 days were required to verify that they were ''diastaticusalthough other species of wild ''Saccharomyces'' while other strains grew as quickly as two days and most strains grew after five days. The yeast cells had can grow on this media <ref name="mbaa_diastaticus" />(~18 mins) and PCR DNA analysis is required to be thoroughly washed give a positive identification of all other carbohydrate material and starved in order to avoid false positives''diastaticus''. Using dextrin agar plates instead Additionally, the default level of starch also led to false positives CuSO_{4<ref name="Meier-Dörnberg_2018" /sub>. This starch media has been recommended by Richard Preiss from [[Escarpment Laboratories]] and Justin Amaral from [[Mainiacal Yeast]] 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<refsub>[https:4} reaches around 200 ppm, or using LCSM plates modified with a gradient of CuSO<sub>4</www.facebooksub> <ref name="Begrow_MBAA" /> (~22 mins in).com/groups/MilkTheFunk/permalink/2149139905114212/?comment_id=2150763631618506&comment_tracking=%7B%22tn%22%3A%22R%22%7D Richard Preiss Adding p-coumaric acid or other cinnamic acids to the LCSM agar media which can then test for POF+ yeast and Justin Amaral. Milk The Funk Facebook thread on plate media for diastaticus. 06/26/2018.]</ref>. Note 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'' cells look the same under since there is a microscope as regular high occurrence of ''S. cerevisiaediastaticus'', so cell morphology is not an effective way to identify ''diastaticus'' strains produce phenols from these cinnamic acids <ref name="Begrow_MBAA">[https://www.mbaafacebook.com/educationgroups/MilkTheFunk/webinarspermalink/Pages2149139905114212/webcast.aspx?vidcomment_id=diastaticus Wade Begrow2150763631618506&reply_comment_id=2158975484130654&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Shawn Savuto and linked references. "S. cerevisiae var. diasttaicus". MBAA webinarMilk The Funk Facebook book post on POF+ correlation with ''diastaticus''. July 2018.]</ref> (~8 minutes in)see also [https://www.facebook. Other methods of detection include using a Durham tubecom/fermentation tube test to see if the beer produces CO<sup>2<groups/MilkTheFunk/permalink/1903290776365794/sup> after fermentation, although this method does not MTF thread] on using cinnamic acids to identify the cause phenolic off flavor strains). Cheaper methods of the additional fermentation <ref name="Begrow_MBAA" /> doing PCR are recently becoming available, and could help breweries with smaller budgets sufficiently detect this as a contaminant (~18 mins 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 inanaerobic 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 (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]).