Changes

: ''This article is about sour brewing methods using commercial cultures. For other sour brewing methods, see [[Brewing Methods]].''

=Introduction=For the purposes of this article, we are defining a ''mixed 'Mixed fermentation'' ' (also referred to as "mixed culture fermentation" or more specifically "multiple species mixed fermentation") is any fermentation that consists of a combination of ''[[Saccharomyces]]'' (brewer's yeast), ''[[Brettanomyces]]'' (wild yeast), ''[[Lactobacillus]]'' (lactic acid bacteria), and ''[[Pediococcus]] cultures'' (lactic acid bacteria), or other [[Nonconventional_Yeasts_and_Bacteria|microbes that are unconventional in brewing]]. Broadly speaking, there are two styles of mixed fermentations: "sour" mixed fermentations with lactic acid bacteria (''Lactobacillus'' and "funky" /or ''Pediococcus'') and mixed fermentationswithout lactic acid bacteria. "Sour" fermentations Mixed fermentation sour beers are characterized by their higher acidity and tart flavorcaused by the production of [[Lactic Acid|lactic acid]], and require the use of a Lactic Acid Bacteria lactic acid bacteria (abbreviated as '''LAB'''; generally ''Lactobacillus '' and/or ''Pediococcus''). "Funky" fermentations These beers generally do not contain LAB, but instead use fall within a pH range of 3.0-3.7 (although [[Titratable Acidity]] is more accurate for measuring perceived sourness). Mixed fermentation without lactic acid bacteria are usually fermented with a combination of ''Saccharomyces '' and ''Brettanomyces''. Funky Mixed fermentation beers without lactic acid bacteria may be slightly tartfrom the [[Acetic Acid|acetic acid]] production of ''Brettanomyces'', but are generally not considered to be sourif well brewed because they lack lactic acid and too much acetic acid is considered a flaw. For both categories, the primary fermentation will be completed by yeasts such as ''Saccharomyces '' and/or Brettanomyces. For ''Funky Mixed FermentationsBrettanomyces'', see the [[Funky Mixed Fermentations]] page. For 100% Brettanomyces fermentations, see the [[100% Brettanomyces Fermentation]] page.

It This page will focus on information for mixed fermentation sour beers using pure laboratory cultures and where the lactic acid bacteria is important allowed to mention that sour brewing in general has very few wellco-established "rules"exist with yeast (e.g. Many methods can be used in conjunction with other not [[Brewing MethodsWort_Souring#Souring_in_the_Boiler_.28Kettle_Sour.29|Kettle Sours]], and new methodologies are constantly being developed). Many of For mixed fermentation beers without lactic acid bacteria, see the methods used are determined by the types of microbes the brewer is working with. An article of this length cannot encompass all mixed [[Brettanomyces and Saccharomyces Co-fermentation methods]] page. Instead it will provide For 100% ''Brettanomyces'' fermentations (technically not a "big picturemixed" view of fermentation), see the general methodologies[[100% Brettanomyces Fermentation]] page. 100% ''Lactobacillus'' or ''Pediococcus'' beers do not exist because they do not fully attenuate wort (see [[Lactobacillus#100. Towards this end, we divide mixed 25_Lactobacillus_Fermentation|100% ''Lactobacillus'' fermentation methods into two approaches: the traditional long fermentation method and an increasingly popular, short fermentation method]] for details). They are divided here as a device to illustrate the philosophy of each Other alternative yeast and facilitate the discussion of the techniques bacteria can also be used , however this is currently not common even for each methodologybrewers who make wild/sour beers. The distinction of these two methods is however somewhat artificial For example, indeed many brewers use elements [[Spontaneous Fermentation|spontaneous fermentation]] and [[Wild_Yeast_Isolation#Growing_and_Testing_Without_Plating|wild yeast captures]] usually contain a plethora of both approaches [[Nonconventional_Yeasts_and_Bacteria|yeast and bacteria that are not conventional to achieve their desired results. More detailed information can be found in Michael Tonsmeire's pivotal book on sour modern brewing, "American Sour Beers"]].

=''Sour'' =Mixed FermentationSour Beer -The Basics==Sour fermentations require at least one Lactic Acid Bacteria (LAB), such as ''[[Lactobacillus]] '' or ''[[Pediococcus]]'', and at least one yeast such as ''[[Saccharomyces]] '' or ''[[Brettanomyces]]''. Many yeast companies offer [[Mixed Cultures]] that provide all of the microorganisms necessary to make a sour beer. The results of these commercial mixed cultures can be as varied as the cultures themselves. For example, some of these commercial mixed cultures produce lightly tart beer that may exhibit minimal funky flavors; others may produce intense sourness and assertive funk. This is dependent on the types of microbes in the mixed culture, their ratios, how old the cultures are, and what methods the brewer uses to encourage or discourage certain flavors. The brewer must understand that all of these microbes are complex organisms (some more complex than others). Not only do different species behave differently and produce different results under different conditions, but different strains of the same species also can also behave differently and produce different results under different conditions. Just as strains of ''Saccharomyces cerevisiae '' produce different results in clean beers (e.g., California Ale yeast versus Belgian Ale yeast), strains of ''Lactobacillus '' spp. and especially ''Brettanomyces '' spp. can also vary widely.

Beer BJCP styles that can be brewed using this method include [[Berliner Weissbier]] and the subcategories of [[American Wild Ale]], which include [[Mixed-Fermentation Sour Beer]] and [[Soured Fruit Beer]] <ref>[http://www.bjcp.org/docs/2014%20BJCP%20Style%20Guidelines%20%28DRAFT%29.pdf BJCP 2014 Style Guidelines Draft.]</ref>. European Belgian sour styles such as [[Lambic]], [[Gueuze]], and [[Fruit Lambic]], technically can only be produced by [[Spontaneous Fermentation]], however for beer competitions process is less important than what the resulting beer tastes like. [[Flanders Red Ale]] and [[Oud Bruin]] styles can be brewed using pure cultures, but can also be brewed using spontaneous fermentation or a mix of using pure cultures and spontaneous fermentation.

=Traditional Method - Long Fermentation==Wort Production=Introduction==The most basic method grain bill and production for making the wort doesn't have to be complex. In fact many sour breweries produce their full line of sours from 2-3 base sour recipes which are then modified after aging by blending, the addition of fruit, dry hops or simply packaging them without alteration. For sour blonde ales, a mixed fermentation sour beer is to brew some simple wort grain bill of about 70% Pilsner malt and 30% malted wheat can be used (fresh these can be replaced by Pilsner and wheat unhopped extracts for the extract brewer. See [http://byo.com/videos/item/975-lambic-brewing Lambic Brewing by Steve Piatz] or all grain) that is low in IBU[http://www.homebrewtalk.com/showthread.php?t=322168 AmandaK'slambic-style extract recipe] for a good extract recipe). Iso-alpha acids can inhibit many species Some crystal and strains a small amount of LABroasted malts an be used for sour brown ales. Keeping the wort less than 6 IBUSome higher chain sugars or even starches can be included for beers that will be aged for a long time and include ''Brettanomyces'', or ''Brettanomyces'' and ''Pediococcus'' ''(Pediococcu''s is recommended in general, unless generally should not be used without ''Brettanomyces''. See the brewer has information about their LAB culture that indicates that they can tolerate ''[[Pediococcus]]'' page for moredetails). Performing a [[Turbid Mash hopping ]] is one technique that can be used the traditional way to limit include starches in the wort. However other methods such as steeping some oats or flaked wheat during the IBU's by about 70% boil <ref>[http://www.homebrewersassociationhomebrewtalk.orgcom/attachmentsf127/presentationsadjuncts-starches-sour-beer-448529/pdf/2014/Putting%20Some%20Numbers%20on%20First%20Wort%20and%20Mash%20Hop%20additions.pdf Putting Some Numbers on First Wort and Mash Hop additions. David Curtis. NHC 2014.Homebrewtalk Discussion started by Amos Brown aka 'Metic']</ref>. If hops are not required (commercial brewers may be required , or running off over a bag of flaked oats or wheat on the way to use hops, while homebrewers arenthe kettle can also impart starches that won't), they can be completely excluded from the recipe. This method has been used converted to sugars by some brewers before a lot was known about sour brewing microbes and before some of the more current techniques were popularized mash (such as see [[Sour WortingTurbid_Mash#Alternative_methods_to_yield_starchy_wort|alternatives to turbid mashing]], or the other methods mentioned in this article). This method step is best described by the following: Brewer X has a beer, and perhaps that beer didn't come out the way they wantedcompletely optional, or perhaps there is some other motivationhowever, but Brewer X decides that they want this beer to it may be sour. Great idea, right? Perhaps. [[Lactobacillus]] tends very beneficial to prefer simple sugars, which are required for lactic acid production. [[Pediococcus]] can break down make sure some of the larger higher chain sugars that or starches are left over available in the beer from wort if the [[Saccharomyces]] fermentation, but there may not be enough of those sugars brewer wants to create even a moderate level rely on ''Pediococcus'' for producing most of the acidity. If the beer contains more than 6 IBU'sExtract brewers can use 0.25 lbs. (0.11 kg) of Maltodextrin <ref>[http://byo.com/videos/item/975-lambic-brewing Lambic Brewing. Piatz, the bacteria may be largely inhibitedSteve. Brew Your Own Magazine. The [[BrettanomycesOctober, 2004.]] in general should create </ref>, or hot steep a nice range pound of flavors (see flaked wheat, flaked oats, or carapils malt. See [[Funky Mixed FermentationsMTF Member Recipes]]) in secondaryfor ideas on recipes, so this could benefit or the beer. However, due to recipe sections of the lack of sugars, finished beers which a brewer may attempt to sour often do not get very sour at all. Some brewers have reported good results with this technique (particularly professional brewers who use barrels; see books "American Sour Beers" by Michael Tonsmeire for more information), but in general if the brewer is going for a truly sour beer, it is better to use another methodand "Wild Brews" by Jeff Sparrows.

If this method ===Aeration===Questions often arise regarding if and when wort aeration should be done. It is usedwell documented that ''[[Saccharomyces]]'' uses oxygen to biosynthesize lipids, which include fatty acids and sterols, for their cell membranes. The cell membrane regulates the flow of nutrients into the cell and waste out of the cell, and allows the yeast to reproduce. Each time a yeast cell doubles during growth, the parent cell gives approximately half of its lipids to the daughter cell. The more sugar available to the yeast, the more they will reproduce, and thus the more lipids they require. Therefore, without a healthy cell membrane and a build up of lipids, the cell can die or produce weak daughter cells, potentially resulting in a range of off-flavors, especially in higher gravity beers <ref name="Aquilla">[https://www.morebeer.com/articles/how_yeast_use_oxygen "The Biochemistry of Yeast, it generally requires 6" by Tracy Aquilla. Morebeer Website. 07/25/2013. Retrieved 04/13/2016.]</ref><ref name="danstar">[http://www.danstaryeast.com/articles/aeration-and-starter-versus-wort Aeration And Starter Versus Wort. Danstar Website. Retrieved 04/13/2016.]</ref>. In the brewing of non-12+ monthsmixed fermentation beers, which is required for aerating both the Brett yeast starter and Pedio the wort before pitching the yeast is generally considered mandatory to break down the remaining complex sugarsbrewing process.

== Wort Production==The grain bill and production for Brewers have historically had concerns about aerating wort that has either been pre-soured with lactic acid bacteria (if the wort doesn't have to be complex. For sour blonde ales lactic acid bacteria is still alive) or if it will receive a simple grain bill co-pitch of about 70% Pilsner malt lactic acid bacteria, ''[[Brettanomyces]]'', and 30% malted wheat can be used ''[[Saccharomyces]]'' (these can be replaced by Pilsner see [[Mixed_Fermentation#Reusing_a_Sour_Yeast_Cake|Reusing a Sour Yeast Cake]], [[Mixed_Fermentation#Multi-Stage_Fermentation|Multi-Stage Fermentation]] and wheat unhopped extracts for the extract brewer[[Wort Souring]]). See These concerns, however, are largely unfounded. Most species of ''[http://byo[Lactobacillus]]'' are either not affected by oxygen, or benefit slightly.com/videos/item/975-lambic-brewing Lambic Brewing [[Butyric Acid]] production by Steve Piatz''Lactobacillus'' is not a concern (see [[Lactobacillus#Effects_of_Oxygen|''Lactobacillus'', effects of oxygen]] for a good extract recipedetails). Some crystal and a small amount species/strains of roasted malts an ''[[Pediococcus]]'' might be used for sour brown ales. Some higher chain sugars or even starches can be included for beers that will be aged for a long time and include Brettanomycesinhibited by oxygen, or Brettanomyces and Pediococcus but not all (Pediococcus generally should not be used without Brettanomyces. See the see ''[[Pediococcus#Growth_and_Environment|Pediococcus]] page '' for more details). Performing a ''[[Turbid MashBrettanomyces]] is the traditional way to include starches '' creates acetic acid in the wortpresence of oxygen, however other methods such as steeping some oats or flaked wheat during , in the boil can also impart starches that wonpresence of a healthy pitch of ''t be converted to sugars by the mash <ref>[http://www.homebrewtalk.com/f127/adjuncts-starches-sour-beer-448529/ Homebrewtalk Discussion started by Amos Brown aka [Saccharomyces]]'Metic']</ref>, which rapidly consumes the oxygen, this is probably also not a concern. This step is completely optionalAdditionally, however it may be very beneficial to make sure some higher chain sugars or starches are available in oxygen greatly improves the wort if the brewer wants to rely on Pediococcus vitality and cell count of ''Brettanomyces'' and a small amount is required for producing most of the acidity. Extract brewers can use 0.25 lbs. effective ''Brettanomyces'' growth and fermentation (0.11 kgsee [[Brettanomyces Propagation Experiment]]) of Maltodextrin <ref>[http://byo.com/videos/item/975-lambic-brewing Lambic Brewing. PiatzThus, Steve. Brew Your Own Magazine. October, 2004.as long as a healthy pitch of ''[[Saccharomyces]]</ref>'' is present, or hot steep aerating wort for mixed fermentation should lead to a pound of flaked wheat, flaked oats, or carapils malthealthy fermentation and good results.

==Bug Inoculation==Once Many brewers, however, do not aerate their wort when either pre-souring the wort is produced and chilledwith a pure culture of lactic acid bacteria, the mixed culture can be pitched as normal. If using pitching fresh wort on top of a mixed culture from a commercial yeast labcake, it is usually recommended to not make a yeast starter with it because the starter will throw off the intended ratios of the microbes. Instead of buying co-pitching a single mixed culturesuch as Wyeast Roeselare, or pitching a brewer can create their own mixed culture by combining their own ratios custom mix of microbes from single cultures of yeast and bacteriamultiple sources <ref>[https://www.facebook. A single com/groups/MilkTheFunk/permalink/1182597671768445/?qa_ref=qd Conversation on MTF about oxygenating wort for mixed culture can be supplemented by adding pure cultures from different yeast labs as wellfermentation. Another suggestion that often helps produce a higher quality sour beer is to supplement the mixed culture with a [[Commercial Sour Beer Inoculation]11/22/2015.]</ref>. Many such brewers have reported success without aerating. In generalWe, the more diversity of microbestherefore, recommend that the more complex a sour beer can potentially be. Using a [[Sour Worting]] method brewer investigate and experiment with their process in conjunction with this method can help increase acidity that may order to decide whether or not otherwise be produced by some commercial blends (Wyeast Roeselare aeration is known for this characteristic, for example)desired.

If the brewer is pitching a separate liquid culture of ''[[Saccharomyces]]'', it is recommended to create a starter on a stir plate and alternatively dose it with oxygen. This will allow the cell membranes to build enough lipids for their cells walls and give them the greatest chance of fermenting the wort without off-flavors <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1284106178284260/?comment_id=1284141108280767&comment_tracking=Stages %7B%22tn%22%3A%22R3%22%7DConversation with Mark Trent on MTF regarding aerating starters/wort for mixed culture fermentations. 04/13/2016.]</ref>. If the wort has been pre-soured, it might be beneficial to propagate the yeast starter with a portion of the soured wort equal to the portion of starter wort in order to acclimate the yeast to the has conditions (see [[Saccharomyces#Fermentation_Under_Low_pH_Conditions|''Saccharomyces'' fermentation under low pH conditions]]). Dried yeast is grown and processed in such a way that they contain enough lipids to support a healthy fermentation of Fermentation=====Primary Fermentation==5% ABV or less without the need for aeration (this may be dependent on manufacturer; see the yeast manufacturer's website for their individual recommendations). Aeration should be considered for beers above 5% <ref name="danstar"></ref>. Mixed cultures can also benefit from a starter if they are expired or haven't been stored correctly (To dosee [[Mixed_Cultures#Starters_and_Other_Manufacturer_Tips|mixed culture starters]]).

===Secondary FermentationMicrobe Inoculation===Once the wort is produced and chilled, the mixed culture can be pitched as normal. If using a mixed culture from a commercial yeast lab, a starter is generally not needed. If the culture is old or a larger volume is needed, generally a normal starter can be made for mixed cultures without fear of "throwing off the balance of microbes" (To dosee [[Mixed_Cultures#Manufacturer_Tips|The Yeast Bay starter tips]] as an example; Bootleg Biology and Omega Yeast Labs also recommend starters for mixed cultures for larger batches <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1336235339738010/?comment_id=1336237029737841&comment_tracking=%7B%22tn%22%3A%22R%22%7D Discussion on MTF regarding mixed culture starters. 06/23/2016.]</ref>). Instead of buying a single mixed culture, a brewer can create their own mixed culture by combining their own ratios from single cultures of yeast and bacteria. A single mixed culture can be supplemented by adding pure cultures from different yeast labs as well. Another suggestion that often helps produce a higher quality sour beer is to supplement the mixed culture with a [[Commercial Sour Beer Inoculation]]. In general, the more diversity of microbes, the more complex a sour beer can potentially be. Using a [[Wort Souring]] method in conjunction with this method can help increase acidity that may not otherwise be produced by some commercial blends (Wyeast Roeselare is known for this characteristic, for example).

===Aging=Staggered Versus Co-Pitching====Aging is generally required for mixed fermentations that include Brettanomyces. Expect an aging time of at least 9 months, but preferably 12-18 months or longer. Sour beer should be aged in an environment that minimizes high temperatures and exposure to oxygen. Drastic temperature fluctuations and changes in atmospheric pressure will cause a vacuum inside of the fermentation vessel causing water airlocks to "suck back" air into the fermenter. This could potentially contribute to acetic acid and ethyl acetate (nail polish aroma in high concentrations) production by Brettanomyces, however the development of a pellicle can help protect against this. Filling the carboy to the neck will also help minimize the surface area of the beer that can be exposed to air. Avoid over sampling the beer (once every 3 months at the very most). It should also be noted that micro-oxygenation is helpful for creating certain flavors in sour beer, and many homebrewers have reported not having any issues with over exposure to oxygen using water-based airlocks.

=Modern Method Staggered pitching versus co-pitching can have a significant impact on the final flavor profile of the beer. While there is a lot of information regarding the fermentation profile of various microbes used in sour brewing, the impact of co-fermentation is less understood. Butler et al., partnered with Gilded Goat Brewing Company, analyzed the differences between co- Fast Fermentationpitching ''S. cerevisiae'', a strain of ''B. bruxellensis'', and a strain of ''L. plantarum'' (Sample A), versus pitching the ''S. cerevisiae'' and ''B. bruxellensis'' first and then the ''L. plantarum'' three days later (sample B), versus pitching the ''L. plantarum'' first and then the ''S. cerevisiae'' and ''B. bruxellensis'' three days later (Sample C). The three different beers were aged for a month and a half before packaged. Sample A was characterized as tasting the most balanced and consumers preferred it. Sample B was preferred the least and was characterized as having more "funk" flavor. Sample C had a distinctly sharp lactic sourness that overwhelmed the flavor from the ''Brettanomyces'', despite having slightly less lactic acid and a slightly lower titratable acidity than Sample A. Each of the three different fermentation profiles had a different sensory fingerprint with different measurements for proteins, titratable acidity (slight differences), lactic acid (slight differences), polyphenols, turbidity, color, and residual sugar, indicating that when individual species are introduced to ferment the wort, that it potentially has a wide impact on many different aspects of the beer. See the full poster [https://docs.wixstatic.com/ugd/695caf_8f98746d2f6942ff8810b298ef219eb9.pdf by Butler et al. here], as well as [https://www.facebook.com/groups/MilkTheFunk/permalink/2507876332573899/?comment_id=2507958592565673&reply_comment_id=2508072125887653&comment_tracking=Introduction%7B%22tn%22%3A%22R2%22%7D clarifications and corrections] to the "Conclusion" statements in the poster by Charlie Hoxmeier of Gilded Goat Brewing Company <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2503719276322938/?comment_id=2503738896320976&comment_tracking=(Intro needed; define "Fast Fermentation")%7B%22tn%22%3A%22R%22%7D Kelley Freeman. Milk The Funk Facebook group post. 02/09/2019.]</ref>. These results may or may not be repeatable with different strains or other variables, but it does demonstrate that co-pitching and staggered pitching produce measurably different results.

==Wort Production==The grain bill for Depending on the ethanol tolerance of the lactic acid bacteria strains present in the culture, the presence of ethanol can have a short fermentation sour can be based negative impact on nearly any stylelactic acid bacteria. The wort For example, howeverin one published study, is generally designed to be highly fermentableat 11% ABV, in contrast to the low fermentability strain of wort ''L. brevis'' used in the long ferment method. This is because the order and timing of microbial inoculation is used to control acidity and fermentation characters rather study didn't grow as well than depending on the selective availability of carbohydrates to particular microbes as in the long ferment method. A few modifications to grain bills can be made to increase the fermentability of the wort lower ABV samples, and accomplish the full attenuation of resulting lactic acid content was lower as well ion the wort in a relatively short period of time11% ABV beer <ref>[https://www.biorxiv.org/content/10.1101/2022.03.07. These modifications include lowering or removing crystal malts from the recipe 483260v1 Beer ethanol and mashing for 90iso-α-120 minutes at 149Facid level affect microbial community establishment and beer chemistry throughout wood maturation of beer. Extract brewers can steep 2Sofie Bossaert, Tin Kocijan, Valérie Winne, Johanna Schlich, Beatriz Herrera-3 lbsMalaver, Kevin J. Verstrepen, Filip Van Opstaele, Gert De Rouck, Sam Crauwels, Bart Lievens. bioRxiv 2022.03.07.483260; doi: https://doi. of crushedorg/10.1101/2022.03.07.483260.]</ref>. Therefore, malted 6-row at 149F adding the lactic acid bacteria earlier in their kettle with their extract the fermentation process versus later in order to increase higher ABV beers will most likely impact the fermentability of their wortfinal beer's lactic acid content.

See also:* [http://brulosophy.com/2018/05/14/mixed-fermentation-combined-vs-staggered-microbe-pitch-exbeeriment-results/ Brülosophy blind triangle test between co-pitched vs staggered pitched mixed fermentation sour beer]. See also the [http://brulosophy.com/2018/05/14/mixed-fermentation-combined-vs-staggered-microbe-pitch-exbeeriment-results/ associated Brülosophy podcast episode].* [[Mixed_Fermentation#Multi-Stage_Fermentation|Multi-Stage Fermentation]] below.* [[Brettanomyces_and_Saccharomyces_Co-fermentation#Review_of_Scientific_Analysis|Cofermentation of ''Brettanonyces'' and ''Saccharomyces'']].* [[Mixed_Fermentation#Souring_Without_Brettanomyces|Mixed Fermentation Without ''Brettanomyces'']].* [[Lactobacillus#Effects_on_Mixed_Fermentation|Effects of ''Lactobacillus'' on cofermentation]]. ===Stages of Fermentation===[[File:Long_Fermentation.jpg|thumb|upright=2.5|Conceptual graph of traditional microbe and wort dynamics|Conceptual graph of traditional souring microbe and wort dynamics. Y-axis for each microbe group depicts relative activity which combines in a conceptual sense: growth, acidification of wort, attenuation and production of flavor compounds. Plot drawn by Drew Wham based on concepts discussed in American Sour Beer <ref> Tonsmeire, M. (2014). American Sour Beers. Brewers Publications </ref> and Wild Brews <ref> Sparrow, J. (2005). Wild Brews: Beer Beyond the Influence of Brewer's Yeast. Brewers Publications</ref> . ]]====Primary Fermentation====Primary fermentation by ''Saccharomyces'' is generally conducted in the same way for a sour beer as for a non-sour beer. Depending on the intended final result the brewer might select a neutral ale strain (WLP 001/Wyeast 1056, WLP036/Wyeast 1007) to provide a neutral background for the souring microbes to act on. Alternatively, the brewer may use a Belgian strain or a saison/farmhouse strain (see ''[[Saccharomyces]]'' page for a comprehensive list) to increase the ester and/or phenol characters of the beer which can then be acted on by ''[[Brettanomyces]]''. Primary fermentation with ''Saccharomyces'' also tends to lend to more glycerol production which increases the beer's mouthfeel (''Brettanomyces'' generally does not produce much glycerol <ref>[http://www.milkthefunk.com/wiki/Brettanomyces#Secondary_Metabolites Brettanomyces; Secondary Metabolites. MTF Wiki. Retrieved 06/23/2016]</ref>). However, the role of glycerol in creating mouthfeel is debatable in the wine world <ref>[https://www.winesandvines.com/features/article/68760 Tim Patterson. "Many Roads to Mouthfeel". Wines & Vines Magazine. Nov 2009. Retrieved 03/23/2018.]</ref>. This primary fermentation can take place in any vessel suitable for a normal ''Saccharomyces'' fermentation. In general, it is best practice to maintain fermentation temperature control as suggested by the yeast lab for the strain of ''Saccharomyces'' selected for this step, although strict temperature control might not be completely necessary as long as the primary fermentation remains within the suggested temperature range of the selected ''Saccharomyces'' strain (the goal is to avoid off-flavor production from the ''Saccharomyces'' fermentation, although a higher amount of esters might be desirable and ''Brettanomyces'' can clean up some off-flavors like diacetyl in small amounts). Once active fermentation has subsided the mostly attenuated wort can then be moved on to the secondary fermenting vessel. There is some variation in common practice as to whether or not the primary fermentation yeast should be carefully settled out, moving over bright clear beer only, or if unsettled cloudy high yeast population wort is moved to the secondary vessel. New Belgium moves their lager primary fermented beer after centrifuging, indicating that this centrifuged beer exhibits cleaner characters from secondary fermentation faster than un-centrifuged beer, allowing the resulting sour beer to be ready for packaging more quickly <ref> The Sour Hour Episode 2 with Lauren Salazar from New Belgium Brewing Company.</ref>. Concerns of yeast autolysis, however, have generally been minimized by most brewers (see [[Mixed_Fermentation#Secondary_Fermentation|Secondary Fermentation]]). During both primary and secondary fermentation, a complex set of interactions occurs between the various yeast and bacteria species. Much of this is as yet unknown scientifically. For example, the production of [[Lactic Acid|lactic acid]] by lactic acid bacteria not only stresses and limits growth of ''S. cerevisiae'', but it can also turn off "glucose repression", meaning that instead of consuming simple sugars first, ''S. cerevisiae'' stops choosing which sugar types to consume first and consumes all sugar types indiscriminately. This can result in under-attenuation problems in the short run, but also more residual sugars for ''Brettanomyces'' (see [[Lactic Acid|lactic acid]] for details). Another example is that co-fermenting with brewer's yeast and ''Lactobacillus'' can create a different flavor profile than if they are staggered with a kettle souring method (see [[Lactobacillus#Effects_on_Mixed_Fermentation|effects of ''Lactobacillus'' on mixed fermentation]]). Another example is that some studies support that in nitrogen rich substrates, ''S. cerevisiae'' will synthesize simpler amino acids from the more complex nitrogen sources, and those amino acids contribute to the sustained survival of both ''Lactobacillus'' and ''Brettanomyces'' in the more stressful, post-fermentation environment <ref>[https://academic.oup.com/femsyr/article-abstract/17/4/fox018/3867021/The-influence-of-Dekkera-bruxellensis-on-the?redirectedFrom=fulltext The influence of Dekkera bruxellensis on the transcriptome of Saccharomyces cerevisiae and on the aromatic profile of synthetic wine must. Janez Kosel Neža Čadež Dorit Schuller Laura Carreto Ricardo Franco-Duarte Peter Raspor. 2017.]</ref><ref>[http://www.sciencedirect.com/science/article/pii/S2405471217303903?via%3Dihub Yeast Creates a Niche for Symbiotic Lactic Acid Bacteria through Nitrogen Overflow. Olga Ponomarova, Natalia Gabrielli, Daniel C.Sévin, Michael Mülleder, Katharina Zirngibl, Katsiaryna Bulyha, Sergej Andrejev, Eleni Kafkia, Athanasios Typas, Uwe Sauer, Markus Ralser, Kiran Raosaheb Patil. 2017.]</ref> (see also [https://www.facebook.com/groups/MilkTheFunk/permalink/1856920997669439/ this MTF thread]). ====Secondary Fermentation====After primary fermentation, the mostly attenuated beer is sometimes moved to a secondary fermentation vessel (and sometimes not; read below). In commercial production secondary fermentation is often conducted in wine barrels (mostly because it is messy to conduct primary fermentation in barrels), however, home brewers can accomplish this phase in glass or plastic carboys with low oxygen permeability. A mixed culture of ''Brettanomyces'', ''Lactobacillus'' and ''Pediococcus'' is then introduced to the beer. If barrels are being used then these microbes may simply come from the walls of the barrel, originating from a previous batch. Alternatively, the brewer might inoculate the wort with a mixed culture directly, either with a house culture or by introducing the dregs of bottled sour beer. Upon their introduction, these new microorganisms begin converting the longer chain sugars left over from the primary fermentation. These sugars are primarily converted into alcohol and lactic acid, increasing the degree of attenuation and lowering the pH of the beer. This also corresponds with a decrease in ''S. cerevisiae'' cell counts, and the release of amino acids and vitamins from yeast autolysis helps feed lactic acid bacteria and ''Brettanomyces'' <ref name="Hubbe">[https://www.facebook.com/groups/MilkTheFunk/1407620505932826/ Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016.]</ref>. Other flavor-impacting secondary metabolites are also produced, depending on the strains used. For example, if the beer contains ''Brettanomyces'' this often results in the production of a high amount of fruity esters such as ethyl acetate and ethyl lactate, as well as "funky" phenols and other flavor compounds specific to ''Brettanomyces'' (see [[Brettanomyces#Secondary_Metabolites|''Brettanomyces'' secondary metabolites)]]. In the presence of oxygen, acetic acid is also produced by ''Brettanomyces'' (and acetic acid bacteria if they are present) which in low amounts can be complementary, adding to the complexity of the beer. In one study on mixed fermentation sour beer with one strain each of ''L. brevis'', ''B. bruxellensis'', and S-04, researchers found that diacetyl that was formed around month 2 had disappeared after another 4 months of aging, indicating that diacetyl, if present in earlier stages of fermentation, can age out of mixed fermentation beer. They also reported that the antioxidant phenol, guaiacol, was present above flavor threshold during all stages of aging from 2-12 months, and [[Isovaleric Acid]] was formed after 12 months of aging <ref>[https://www.mdpi.com/2076-2607/11/7/1681 Postigo, V.; García, M.; Arroyo, T. Study of a First Approach to the Controlled Fermentation for Lambic Beer Production. Microorganisms 2023, 11, 1681. https://doi.org/10.3390/microorganisms11071681.]</ref>. These flavor compounds are essential to the flavor profile of mixed fermentation sour beer. For example, traditional Berliner Weisse was fermented with a mixed culture containing ''Brettanomyces'', and this was considered the most important aspect of achieving the fruity ester character of that beer style historically (see [https://docs.google.com/spreadsheets/d/1CNrO46TPSFpjhO3HX1-CbKK5rhFd7uGWdpONf7AJAlU/edit#gid=0 Benedikt Koch's table comparing esters of traditional Berliner Weisse versus kettle soured Kindl Weisse and Belgian gueuze]). Some brewers (including homebrewers and professional brewers) do not find it necessary to move the mostly attenuated beer from the primary fermentation vessel to a secondary aging vessel. Instead, the mixed culture is pitched directly into the primary fermenter. While yeast autolysis is a concern in regular brewing, it is arguably not a cause for concern in mixed fermentations that contain ''Brettanomyces''. Lambic brewers, for example, perform a primary fermentation in barrels and leave the beer in the barrels during the beer's entire aging process, which is usually 1-3 years <ref>[http://www.lambic.info/Brewing_Lambic#Barrels Lambic.info Wiki. Brewing Lambic. Retrieved 6/8/2015.]</ref>. Yeast autolysis releases trehelose, acids, and other compounds, which are metabolized by ''Brettanomyces'' <ref>[http://www.mbaa.com/districts/michigan/events/Documents/2011_01_14BrettanomycesBrewing.pdf Brettanomyces in Brewing the horse the goat and the barnyard. Chad Yakobson. 1/14/2011.]</ref>. Maintaining a [[Solera]] may be an exception to this (see the [[Solera]] page for details). The advantage of not moving the beer into a secondary vessel is that less overall oxygen is introduced into the beer (oxygen exposure will contribute to more acetic acid and then ethyl acetate production), and might be the best option if the brewer does not have a closed/CO2 system to prevent exposure to oxygen during transferring. Some [[Brettanomyces#Nitrogen_Metabolism|evidence suggests]] that the nutrients released by yeast autolysis are beneficial to ''Brettanomyces'', so leaving the beer on the yeast cake might even be more desirable than not. Some sour beer brewers strive to achieve autolysis in their beers with the belief that it could improve mouthfeel and react with other compounds to produce favorable flavors, similar to how autolysis is sometimes desired in winemaking in the form of [https://www.thekitchn.com/wine-words-lees-aging-179813 lees aging] or [https://www.thekitchn.com/wine-words-btonnage-191331 bâtonnage ]<ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3177292242298968/ Lars Meiner, Richard Preiss, and Alex Seitz. milk The Funk Facebook group thread on autolysis. 01/03/2019.]</ref>. Co-pitching all of the microbes to begin with, including the primary ''Saccharomyces'' culture, can produce different results than staggering the pitches of individual species over time. For example, many brewers pitch a single mixed culture that contains ale yeast, ''Brettanomyces'', and lactic acid bacteria. Other brewers, such as Vinnie Cilurzo at Russian River, prefer to pitch their ale yeast first, and then pitch ''Brettanomyces'' and/or lactic acid bacteria after the primary fermentation <ref>Tonsmeire, Michael. "American Sour Beers: InnovativeTechniques for Mixed Fermentations". Brewers Publications. 2014. Pgs 100-101.</ref>. See this [http://brulosophy.com/2018/05/14/mixed-fermentation-combined-vs-staggered-microbe-pitch-exbeeriment-results/ Brulosophy experiment comparing co-pitching versus staggered pitching] (note that oxygen exposure during the staggered pitch and other variables in this experiment could account for some of the differences between the two beers) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2093241697370700/?comment_id=2093637253997811&comment_tracking=%7B%22tn%22%3A%22R%22%7D Dan Pixley and Zach Taggart. Milk The Funk Facebook thread on the "MIXED FERMENTATION: COMBINED VS. STAGGERED MICROBE PITCH" blog post. 05/14/2018.]</ref>. It is not unusual to see a slight rise in pH during the secondary or aging phases. For example, Santeri Tenhovirta's in his masters thesis he measured the pH of several species of ''Lactobacillus'' that were pitched into wort for 2 days, followed by US-05. Tenhovirta reported a slight pH rise of about 0.3 from day 150 until day 300-330. According to Kunze and Bamforth, an increase in pH towards the end of fermentation or aging could be caused by yeast autolysis <ref name="Tenhovirta_masters">[https://helda.helsinki.fi/handle/10138/303018 The Effects of Lactic Acid Bacteria Species on Properties of Sour Beer. Santeri Tenhovirta; master thesis in Food Science from the University of Helsinki. 2019.]</ref><ref>Bamforth, CW. 2001. pH in brewing: An overview. Master Brewers Association of the Americas Technical Quarterly 38(1):1-9.</ref>. ====Aging====Aging is generally required for mixed fermentations that include ''Brettanomyces''. The necessary/ideal amount of aging time will depend on many factors including the microbes pitched, the pitching rate, wort composition, storage temperature, and the desired final beer. Keep in mind that the beer will also continue to develop once packaged. For more straightforward beers with highly attenuative primary strains (like tart saisons), a reasonable final product with tartness and ''Brettanomyces'' character can be reached in a few months. For more complex and/or acidic beers (such as Flemish reds or beers inspired by lambics) you may expect an aging time of at least 9 months, but quite possibly as long as 12-18 months or longer. In general longer aging will allow more complex expression of the spectrum microbes present. Some brewers will package a beer after the finishing gravity has stabilized (see [[Packaging]]), and allow the beer to fully develop in the bottle. Keep in mind that some volatile flavor compounds, such as sulfur-based compounds, may volatilize off at a faster rate in a fermenter (especially a shallow fermenter such as a barrel) than they would in a sealed bottle, and bottling too early can result in over-carbonation.  Sour beer should be aged in an environment that minimizes high temperatures and exposure to oxygen. Avoid temperatures over 85°F (29.5°C) and under 55°F (13°C). Drastic temperature fluctuations and changes in atmospheric pressure will cause a vacuum inside of the fermentation vessel causing water airlocks to "suck back" air into the fermenter. This could potentially contribute to [[Acetic Acid]] and [[Ethyl acetate]] (nail polish aroma in high concentrations) production by ''Brettanomyces'', and these off-flavor metabolites are considered permanent. Filling the carboy to the neck or topping up carboys or barrels after primary fermentation will also help minimize the surface area of the beer that can be exposed to air. Topping up and flushing with CO₂ might also help reduce the risk of mold growth <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2640657275962470/?comment_id=2641402822554582&reply_comment_id=2642128842481980&comment_tracking=%7B%22tn%22%3A%22R%22%7D Lars Meiner. Milk The Funk Facebook group thread on preventing mold growth on krausen. 05/02/2019.]</ref> on any krausen material that has dried on the sides of the fermentation vessel after primary fermentation. Avoid oversampling the beer (once every 3 months at the very most). Using an "S airlock" has the benefit of showing if there is positive, negative, or equalized pressure in the fermenter, which could possibly assist in showing whether suck-back is a problem (see the Mark Trent YouTube video below). One way ventilated silicone bungs can be used for barrels or other waterless type airlocks (such as the [http://www.better-bottle.com/products_master.html BetterBottle "DryTrap"] or kegs with a [http://seanterrill.com/2015/06/25/build-a-better-spunding-valve/ spunding valve]; see also [[Sanke Fermenter]]) that allow gases to escape the fermenter but not enter from the environment, and [https://beer.thegremlyn.com/2019/09/04/grems-the-gas-reducing-multivessel-system/ Colin Burton's homebrew setup for connecting multiple cornelius kegs to a single spunding valve]. Topping up barrels with fresh beer every 3-6 months might help reduce acetic acid and ethyl acetate, and humidity and temperature control can help reduce evaporation (see [[Barrel#Using_Barrels_for_fermentation_and.2For_aging|Barrel]]). It should also be noted that micro-oxygenation is helpful for creating certain flavors in sour beer, and many homebrewers have reported not having any issues with overexposure to oxygen using water-based airlocks. For example, a small amount of oxygen helps [[Brettanomyces]] growth, and a small level of acetic acid is desirable for the complexity of long-aged sour beers <ref>[https://www.milkthefunk.live/podcast Richard Preiss. Interview on Milk The Funk "The Podcast" Episode #000. 12/13/2017.]</ref> (~30 minutes in). Higher levels of acetic acid are sometimes desirable for [[Flanders Red Ale]] style beers.  :Mark Trent's demonstration of how easy it is for temperature changes to cause a vacuum and suck-back air into a vessel::<youtube>aSgp0E96HCU</youtube> Different airlocks and vessel materials diffuse oxygen at different rates. For example, a set of experiments published by Dr. Enrich L. Gibbs at BetterBottle™ showed that rubber stoppers prevented oxygen transfer more effectively than silicone stoppers, plastic stoppers, and both the "3 piece" airlock and "S" airlock. Solid bungs, however, can build pressure inside the fermenter as the beer slowly ferments, and can pop off due to the pressure (and can cause messes if the vessel becomes pressurized too much). Weekly degassing for a few months while the beer ages is one option with solid bungs. Another option is to rack the beer to a keg and age it in a sealed environment, however, pressure can build up in kegs as well so they should occasionally be partially degassed (some gas should remain in the keg to maintain the seal for corny kegs). See the [http://www.better-bottle.com/pdf/ClosuresOxygenPassageStudy.pdf BetterBottle™ paper for more information]. Raj Apte found that HDPE buckets let in far more oxygen than carboy setups, and taking into account the high surface area to volume ratio in homebrew setups versus full-size barrels, oxygen exposure over time on the homebrew level can be a difficult issue to solve. For example, Apte attempted using wooden dowels as stoppers in carboys and found that it let in about the same amount of oxygen as wooden tanks at Rodenbach. However, the swelling of the wooden dowels led some people to crack or destroy glass carboys (therefore this method is not recommended) <ref name="Apte">[http://web.archive.org/web/20100410025103/http://www2.parc.com/emdl/members/apte/flemishredale.shtml "How to Make Sour Ale: an inquiry". Raj B Apte. 2004. Retrieved 1/1/2016.]</ref>. If signs of oxygen exposure appear (the growth of a [[pellicle]], the smell of acetic acid or ethyl acetate, etc.), it might be wise to package the beer sooner rather than later, assuming the gravity is stable.  In regards to buckets, MTF members have reported using HDPE buckets successfully for beers aged even 2+ years and soleras. We recommend using a higher quality HDPE bucket with a lid that has a gasket that seals. Avoid plastic-on-plastic lids and screw on lids <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1710698425625031/ Various MTF members. Milk the Funk Facebook group. 06/05/2017.]</ref>. {| class="wikitable sortable"|-! Tank <ref name="Apte" /> !! Volume in Liters !! O₂ cc/Liter per Year |-| Burgundy barrel || 300 || 8.5|-| Rodenbach tank, wood, small || 12,000 || 0.86|-| Rodenbach tank, wood, large || 20,000 || 0.53|- | HDPE bucket || 20 || 220|-| Homebrew barrel || 40 || 23|-| Glass carboy, 30cm vinyl immersion tube || 20 || 0.31|-| Glass carboy, silicone stopper || 20 || 17|-| Glass carboy, wood stopper (not recommended) || 20 || 0.10|} Headspace and fermenter size are also concerns when it comes to aging beer with living ''Brettanomyces''. This includes sour beers, [[Brettanomyces_and_Saccharomyces_Co-fermentation|non-sour beers with ''Saccharomyces'' and ''Brettanomyces'']], and [[100%25_Brettanomyces_Fermentation|100% ''Brettanomyces'' beers]] that are aged. The larger the headspace, the more air will be sucked in when a vacuum occurs. The smaller the fermenter, the more headspace becomes a problem. Smaller vessels, in general, have a larger surface area to volume ratio. Therefore, they have more potential for exposure to oxygen. A large headspace in a smaller vessel exacerbates this problem, therefore it is advised to top up small fermenters and flush them with CO₂ after primary fermentation or if significant evaporation occurs during aging. For example, a 1-gallon jug should be filled all the way to the neck if possible. A 5-gallon carboy could also be filled to the neck, but a little more headspace is permissible since it is a larger volume. Barrels are porous and the liquid inside them slowly evaporates. Some brewers combat this by topping up their barrels on a regular basis; this also helps keep the top staves from drying out (higher humidity can help limit evaporation; see the [[Barrel#Using_Barrels_for_fermentation_and.2For_aging|Barrel]] page). One misconception about aging beers is the claim that CO₂ is heavier than air and forms a blanket that protects the beer from oxygen. This is not true unless CO₂ is constantly being produced from the beer. The [https://en.wikipedia.org/wiki/Ideal_gas_law Ideal Gas Law] states that unlike solids or liquids of different densities, gasses of different densities eventually mix. See [http://beerandwinejournal.com/can-co2-form-a-blanket/ Dr. Chris Colby's explanation of this on Beer and Wine Journal.] and this [https://www.youtube.com/watch?v=_oLPBnhOCjM science video documentary demonstration of how gasses eventually mix] (note that the molecular weight of bromine used in the video is 160 g/mol and the weight of CO₂ is 44.01 g/mol, so CO₂ would diffuse into air faster than bromine <ref>[https://pubchem.ncbi.nlm.nih.gov/compound/Dibromine Bromine. PubChem. Retrieved 1/1/2016.]</ref><ref>[https://pubchem.ncbi.nlm.nih.gov/compound/280 Carbon Dioxide. PubChem. Retrieved 1/1/2017.]</ref>).  See also:* [https://www.youtube.com/watch?v=boLqmFIzUZ0 Dan Pixley's video on "Airlocks 101 for Sour Beers".]* [http://www.faybutler.com/pdf_files/HowHoseMaterialsAffectGas3.pdf The oxygen permeability of different plastics.]* [[Eccentric Beekeeper Sampling Syringe and Purging Wand]] for tools that help limit oxygen exposure during sampling.* [http://beachwoodbbq.com/pdf/BBAIBLTBLENDERY.pdf "Brewing Beer in America Inspired By the Belgian Lambic Tradition" by Ryan Fields, head brewer and blender at Beachwood Blendery.]* [https://www.homebrewtalk.com/threads/turning-your-fermonster-into-a-complete-closed-transfer-system-for-cheap.68099 Dominick Patrick's HBT thread, "Turning your Fermonster into a complete closed transfer system for cheap!"] ==Modern Method - Fast Fermentation=====Introduction===The short fermentation method refers to an approach for making sour beers that involves successive inoculation of microbes by the brewer to a wort designed for faster attenuation. This approach accomplishes the souring and full attenuation of the wort in a shorter time frame than the traditional method. In the traditional or long ferment method, the selective availability of carbohydrates to particular microbes allows the activity of those microbes to occur in a natural succession. As the microbes with better competitive ability run out of metabolic resources microbial groups with lower competitive advantage, but wider access to metabolic resources, begin their primary activity. In the short fermentation method, the brewer controls the phases of microbial activity. This allows the brewer to introduce the microbes with the lowest competitive ability to the wort first, allowing them to act on the simplest sugars and establishing their population in the absence of better competing microbes. The order of primary microbial activity in the short fermentation method is, therefore, often the opposite of the order typically observed in the long fermentation method. Further, this approach allows the brewer to maintain temperature profiles that are optimal for each microbial phase. Since the phases are controlled by the brewer, there isn't a need for the longer chain sugars that are generally included in the wort designed for long fermentation because the microbes with lower competitive ability have already been established in the beer by the time the better competing microbes are introduced. For this reason, the beer can fully attenuate within 3-4 weeks of it's final inoculation in many cases. It is important to note however that care should be taken in the decision to bottle these beers in such a short time frame (See bottling section below). Once the beer has reached full attenuation the beer can be packaged, in some cases this can be only 6-8 weeks from brew day (see [[Mixed_Fermentation#Fermentation_in_Less_Than_3_Months|Mixed Fermentation In Less than 3 Months]]). A number of biochemical reactions that effect flavor and aroma may still take place over a period of weeks, to months or even years, however, most of these reactions do not involve the production of carbon dioxide so these reactions may take place in the bottle. ===Wort Production===The grain bill for a short fermentation sour can be based on nearly any style. In contrast to the low fermentability wort used in the long ferment method, the wort used in the short fermentation method is generally designed to be highly fermentable. This is because the order and timing of microbial inoculation, rather than natural succession of the microbial community, is used to control acidity and fermentation characters. A few modifications to grain bills can be made to increase the fermentability of the wort and accomplish the full attenuation of the wort in a relatively short period of time. These modifications include lowering or removing crystal malts from the recipe and mashing for 90-120 minutes at 149°F/65°C. Extract brewers can steep 2-3 lbs. of crushed, malted 6-row or 2-row base pale malt at 149F in their kettle with their extract in order to increase the fermentability of their wort. ===Multi-Stage Fermentation===

Matt Miller outlines a ''"three stage fermentation'' " process on his blog article [http://sourbeerblog.com/understanding-brewing-blending-lambic-style-kriek/ Understanding, Brewing, and Blending a Lambic Style Kriek] <ref>[http://sourbeerblog.com/understanding-brewing-blending-lambic-style-kriek/ Understanding, Brewing, and Blending a Lambic Style Kriek. Miller, Matt. Retrieved 1/24/2015.]</ref>. See the article for a much more detailed process. Matt was also interviewed about his process by James Spencer on the [http://traffic.libsyn.com/basicbrewing/bbr01-22-153stepkriek.mp3 BasicBrewing Radio podcast]. In summary, his process is as follows:# Produce a low or no hopped wort (see the [[Mixed_Fermentation#Standard_Method|Standard Method]] above).# After boiling the wort, cool it to 11090-120°F (4332.3-48.9°C), and run it into the fermenter. The exact temperature depends on the culture being used (see the [[Lactobacillus#Culture_Charts|''Lactobacillus'']] page for recommended temperatures).# If possible, purge the fermenter of oxygen with CO2. Pitch a pure culture of ''[[Lactobacillus]]'', and if possible hold the temperature between 11090-120°F (4332.3-48.9°C) for 2-4 days (see the [[Sour_WortingWort_Souring#Souring_in_the_Primary_Fermenter|Souring in the Primary Fermenter]] page for more details).# After 21-4 3 days, or after the desired pH is achieved (generally between a pH of 3.0-3.7), cool the wort to 65-70°F (18.3-21.1°C), oxygenate the wort, and pitch a starter of ''[[Saccharomyces]]''.

# Add one or more cultures of ''[[Brettanomyces]]''. Optionally, also pitch a culture of ''[[Pediococcus]] '' and/or bottle dregs from commercial sours (see [[Commercial_Sour_Beer_Inoculation|Commercial Sour Beer Inoculation]] for more details on using commercial bottle dregs). For more ''funky'' Brett flavors, do not make a starter for the Brett. '''(Editor's note: new information suggests that the pitching rate for ''Brettanomyces'' in a mixed fermentation probably does not impact flavor. See [[Brettanomyces secondary fermentation experiment]] for more details).''' Also optionally, these additional microbes can be co-pitched along with the [[Saccharomyces]] during step 5.

# For the last two months of aging, fruit, spices, and/or oak can be added directly into the fermenter(see [[Soured Fruit Beer]] and [[Soured_Herb,_Spice,_and_Vegetable_Beer|Soured Herb, Spice, and Vegetable Beer]]). Also , consider [[Blending]] with other sour beers. ===Fermentation in Less Than 3 Months===Some brewers have been experimenting with mixed fermentations that can finish within 3 months. This approach to mixed fermentations takes some knowledge of the cultures being used and is considered to be an advanced topic. In general, use cultures that don't produce a lot of off-flavors early on in fermentation. For example, Omega Yeast Lab's ''Lactobacillus'' blend (OYL-605) and The Yeast Bay's Lorchristi ''Brettanomyces'' blends are good choices. '''Warning: if mixed fermentation beers are bottled too early, they can result in bottle bombs or gushing bottles.''' Gareth Young offers his advise to brewers wanting to try mixed fermentations that finish within 3 months <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1262504037111141/?comment_id=1262518747109670&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Gareth Young on MTF regarding fast mixed fermentations. 03/24/2016.]</ref>: <blockquote>I typically turn around funky beers, especially roughly saison-ish things, pretty fast. One of the things I like about them is the way they change over time, so I like being able to drink them when they're very young, then drinking them sporadically over the next months as they develop. Looking through stuff I've posted here, I've found one that I posted about 5 weeks from brew day, and it was good a while before that. It was also dry-hopped and had honey added, which meant it took longer. https://www.facebook.com/groups/MilkTheFunk/permalink/1108334109194802/ Here's some stuff about my process: 1) Lower ABV stuff will be quicker. It doesn't need to be super low, but below 6% is probably wise. 2) Make your wort very fermentable. The more fermentable, the quicker it will hit a stable gravity. I do this by mashing at 63 for two hours, then not sparging too hot, so it's becoming more fermentable all the way to the kettle. Also, don't use any grains with unfermentable (or slow-fermentable) sugars. Just base malt, and maybe some oats/wheat or something. 3) Pitch plenty of yeast. I'd use a culture with lots of healthy ''Saccharomyces'' and lots of ''Brettanomyces'', and maybe bacteria, right at the start of primary. This lets ''Brettanomyces'' character develop more quickly and helps you hit a stable gravity quicker. I always do this, and there are ''Brettanomyces'' aromas coming out of the airlock almost immediately. If you reuse this culture, you'll start selecting for things that get the job done quickly. 4) Pitch your culture low, but then let it rip, and warm it up a bit towards the end of fermentation, if necessary. That should get most of the sugars fermented pretty fast but without the off flavours you can get from pitching hot. Brettanomyces can often clear those up, but it takes time. The less mistakes you leave for your yeast/bacteria to clear up, the sooner it will be ready. 5) Bottle as soon as you hit a stable gravity. If you do it right, your final gravity should be 0.998-1.002 pretty quick. If you bottle soon, I find you don't get any [[Tetrahydropyridine|THP]] (presumably because there's enough healthy ''Saccharomyces'' around to ferment the priming sugar without producing it). It was only when I decided I wanted my beer to be a bit clearer, and so started leaving it in primary until it cleared, that I started regularly seeing small amounts of THP developing early in bottle conditioning. For 4-5%-ish beers, the soonest I've started drinking was 1.5 weeks, but I've started in 2 weeks quite a few times (edit: 4 weeks sometimes). Typically, they hit the final gravity I want within a week, then I bottle them and they're properly carbonated in another week (sometimes less). They obviously develop and get "brettier" and more refined over time, but they're still good, and still funky that quickly.</blockquote> See also:* [https://www.facebook.com/SouthernBrewersConference/videos/881235365357984/ Devin Bell and Dan Pixley presentation at SBC 2017 on quick souring.] ===Souring Without ''Brettanomyces''===Methods of creating sour beer without using ''Brettanomyces'' are also considered a form of mixed fermentation. In general, these methods include pitching a pure culture of ''Lactobacillus'' along with brewers yeast at the same time or staggered with pitching ''Lactobacillus'' first for a day or two and then brewers yeast (Cascade Brewing is known for the latter process <ref>Tonsmeire, Michael. "American Sour Beers: Innovative Techniques for Mixed Fermentations". Brewers Publications, Jun 15, 2014. Pg 125.</ref>). In some cases, the brewer's yeast (''Saccharomyces cerevisiae'' or ''Saccharomyces pastorianus'') can be pitched first, and then the ''Lactobacillus'' is pitched (see "Reverse MTF Method" below). Since ''Brettanomyces'' is removed from the process, these methods tend to create a sour beer in a shorter amount of time, but without the complex ester and phenol profile of ''Brettanomyces''. While these types of beers may be less complex than beers with ''Brettanomyces'', they produce a different beer than [[Wort Souring|Kettle soured]] beers. Several studies have shown that co-fermentation of brewers yeast and lactic acid bacteria produces an objectively different beer than kettle souring or adding pure lactic acid. Pre-acidifying with lactic acid bacteria fermentation can also negatively affect the primary yeast fermentation, but other studies have shown that it can also result in a faster fermentation time but with less attenuation and less yeast growth. See the [[Lactobacillus#Effects_on_Mixed_Fermentation|''Lactobacillus'' Effects on mixed Fermentation]] wikipage for more information on these studies.  ===="Reverse MTF Method"====Devin Bell reported getting a good level of sourness by co-pitching probiotics with ''L. plantarum'' or Omega Labs OYL-605 with yeast, or even after primary fermentation (also known colloquially as the "Reverse MTF Method"). By allowing the yeast to ferment for two or three days before adding ''Lactobacillus'' for souring, it is claimed that this method allows the yeast character to be expressed more so than with kettle sours. In the case of pitching ''L. plantarum'' after fermentation with saison yeast, Bell reported that the beer turned out like a sour saison, where as co-pitched makes for a better Berliner Weisse or Gose style beer without the "saison" yeast character. This has also improved head retention in his beers. Using no hops seems to be required in order to get acid production from the ''L. plantarum'' after primary fermentation. Devin clarified that his "best success" is pitching ''S. cerevisiae'' saison strain with a selection of ''Brettanomyces'' for primary fermentation. After 5-7 days of fermentation, he pitches ''L. plantarum'' (2 shots of GoodBelly or 1 package of Omega Labs OYL-605 for 5-6 gallons of beer) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1739156616112545/?comment_id=1739183316109875&reply_comment_id=1739749539386586&comment_tracking=%7B%22tn%22%3A%22R%22%7D Devin Bell. Milk The FunkFacebook group. 06/30/2017.]</ref>. Once terminal gravity is reached (1.002-1.004), he bottles right away. The bottles can be served at 8 weeks in the bottle, but start to peak at 24 weeks <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1562696320425243/?comment_id=1562702310424644&comment_tracking=%7B%22tn%22%3A%22R0%22%7D MTF discussion with Devin Bell on "reverse MTF kettle sour. 01/26/2017.]</ref>. See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1217518078276404/ this thread by Devin Bell] and [https://www.facebook.com/groups/MilkTheFunk/permalink/1728489143845959/ this thread by (Zach) Caroline Whalen Taggart]. See also:* [https://www.facebook.com/SouthernBrewersConference/videos/881235365357984/ Devin Bell and Dan Pixley presentation at SBC 2017 on quick souring, including more details on this method.]* [[Alternative_Bacteria_Sources#Culturing_from_Probiotics|General tips on using probiotics to make sour beer]]. ==Finishing Mixed Fermentation Sour Beer=====Determining When It Is Done===Unless the brewer has worked with the same blend of microbes and wort recipe, it is difficult to give an exact time frame on when a mixed fermentation beer might be ready. Anywhere from three to twelve months (and sometimes longer) is a reasonable amount of time, but when any given beer will be ready for packaging within this time depends on many factors including the microbes pitched, their health over time, wort composition, temperatures during the aging time, etc. The best guide is a long-term stable gravity: if the beer's gravity has remained stable between several readings over a month or two, then the beer may be ready for packaging. The second factor is how does the beer taste? If it tastes good, and the gravity is stable, then it can be packaged. If the beer does not seem to have a mature flavor or has off-flavors that need to age out, then feel free to age it longer. Some off-flavors will change even when bottled or kegged, but others (such as sulfur-based compounds) will need to dissipate out of the fermenter slowly over time. ===Bottling and Kegging===See the [[Packaging]] page. ===Reusing a Sour Yeast Cake===Reusing a sour yeast cake can often provide great results. Brewers have reported success re-pitching on very old yeast cakes (2+ years) without getting off flavors from yeast autolysis. After several months, ''[[Saccharomyces]]'' tends to die off due to the low pH in a sour beer. The bacteria and ''[[Brettanomyces]]'' tend to survive the lower pH, and their cell counts can be high depending on how old the yeast cake is (interestingly, ''Brettanomyces'' remains more viable over time if it was co-fermented with ''S. cerevisiae'' than if it was fermented by itself; i.e. [[100%25_Brettanomyces_Fermentation|100% ''Brettanomyces'' beers]] <ref name="Hubbe"></ref>). By pitching new wort on an old sour yeast cake, these microbes (particularly the [[Lactobacillus]]) have access to the simple sugars in the wort <ref>[http://www.themadfermentationist.com/2009/11/brewing-sour-beer-at-home.html Tonsmeire, Michael. The Mad Fermentationist. Brewing Sour Beer at Home. Last paragraph in the "Inoculation" section. Retrieved 2/19/2015.]</ref>. Using a young yeast cake is also a viable option, and may carry over more surviving ''Saccharomyces'' cells as well as more viable cells of the other various microbes. In general, [[Laboratory_Techniques#Yeast_Rinsing.2FWashing|rinsing or washing the yeast cake]] is not necessary ([https://www.youtube.com/watch?v=9LXEAZbei_8 acid washing] can kill the bacteria). The beer itself can also be used as an inoculate and might be more desirable so as to avoid trub. If the beer has sat in a barrel ''Acetobacter'' and other unwanted microbes might be more present on the surface of the pellicle, and would remain after racking the beer out of the barrel, so some professional brewers advise using beer as an inoculate for this reason <ref>[https://youtu.be/IGzoh4brILA?t=52m30s Yakobson, Chad. Interview on Craft Commander. 12/20/2016. Retrieved 12/22/2016.] (~52 mins in)</ref>. If the yeast cake is particularly old, perhaps say older than 1 year, or has a very low pH (low 3's), then making a starter with the slurry will help guarantee the viability of the microbes. Such a starter can be treated the same as a [[Mixed_Cultures#Starters_and_Other_Manufacturer_Tips|mixed culture starter]] that can be assumed to not have any viable ''Saccharomyces''.

=Finishing Mixed Fermentations=== Reusing Some brewers will harvest a Sour Yeast Cake==Reusing a sour yeast cake can often provide great results. Brewers have reported success repitching on very old yeast cakes certain amount of trub from their fermenters (2 years500mL for example) without getting off flavors from yeast autolysis. After several months, [[Saccharomyces]] tends and use only this amount to die off due to the low pH in inoculate a sour new batch of beer. The bacterias and [[Brettanomyces]] tend This will allow the brewer to survive control the amount of dead trub material that goes into the lower pH, and their cell counts can be high in even an old yeast cakenew beer. By pitching new wort on an old sour yeast cake, these microbes (particularly Michael Tonsmeire often advises that the [[Lactobacillus]]) have access to the simple sugars in the wort brewer also pitches a fresh culture of ''Saccharomyces'' <ref>[http://www.themadfermentationist.com/2009/11/brewing-sour-beer-at-home.html Tonsmeire, Michael. The Mad Fermentationist. Brewing Sour Beer at Home. Last paragraph in the "Inoculation" Comments section. Retrieved 2/19/2015.]</ref>. In general, washing the yeast cake is not necessary.

Some brewers will harvest In general, it is advised to pitch a certain amount fresh culture of trub from their fermenters (500ml for example), and use only this amount ''Saccharomyces'' to inoculate a new batch ferment the bulk of beerthe wort sugars. This will allow can be done before adding the brewer to control sour yeast cake, or at the amount same time. Some brewers have good success reusing a yeast cake or a portion of dead trub material that goes into a yeast cake by leaving the wort in contact with the old yeast cake for 1-4 days before pitching a fresh beerculture of ''Saccharomyces''. Michael Tonsmeire often advises that the brewer also pitch After 1-4 days, a fresh culture of ''Saccharomyces <ref>[http://www'' is then pitched to finish the fermentation.themadfermentationist The 1-4 day head start gives the souring bacteria a head start and results in a low pH beer.com/2009/11/brewing- The decided timing on when to pitch the harvest sour yeast cake will affect the acidity of the sour beer: early pitching of the sour-yeast cake generally produces a more acidic beer-at-home.html Tonsmeire, Michaeland later pitching generally produces a less acidic beer. The Mad FermentationistIt might also be possible to not pitch any fresh yeast and rely completely on the sour yeast cake to fully attenuate the wort. Brewing Sour Beer at HomeIn this case, it might take 3-10 days for the fermentation to begin because this approach would be relying on the ''Brettanomyces'' that is alive in the yeast cake, and the growth phase of ''Brettanomyces'' can take a week or so. Comments sectionIf relying only on the sour yeast cake to fully attenuate the wort, making a starter for the yeast cake is preferable to ensure that the microbes in the yeast cake are viable. Retrieved 2/19/2015 A [[Pellicle|pellicle]] might also develope early, depending on what types of microbes are in the yeast cake and if they have a tendency to develop pellicles or not. Oxygenate as normal whenever the ''Saccharomyces'' is pitched to ensure a healthy ''Saccharomyces'' fermentation (''Brettanomyces'' also benefits from small amounts of oxygen, and oxygen does not greatly effect lactic acid bacteria; see [[Mixed_Fermentation#Aeration|Aeration]]</ref>above).

Some brewers have good success reusing a yeast cake or a portion As with all methods, the species and strains of a yeast cake the microbes being used should always be taken into consideration. Experimentation and repeated processes should be carefully employed by leaving the wort brewer in contact with order to find the old yeast cake best results for 3-4 daystheir cultures. After 3-4 daysFor example, a fresh culture using different strains of ''Saccharomyces is pitched to finish cerevisiae'' as the primary fermenter could produce widely different results, and the fermentation. The 3use of 10-4 day head start gives 30 IBU's in the souring bacterias a head start and results in a low pH beer. The harvested yeast cake wort can also be pitched at the same time as a fresh culture of Saccharomyces, or afterwards. The decided timing on when used to pitch the harvest sour yeast cake will effect inhibit the outcome of the sour beer. Early pitching of the sour yeast cake generally produces a more acidic beer, lactic acid bacteria if they become too strong and later pitching generally produces a less acidic beerproduce too much acidity.

As with all methods, ====Storing a Yeast Cake or Sample====The brewer may find that it isn't possible to re-use a yeast cake or a portion of the species and strains sour beer to inoculate a new batch of the microbes being used should always be taken into considerationbeer/wort right away. Experimentation and repeated processes The yeast cake or beer sample should be carefully employed by stored in a glass jar in the brewer fridge at a stable temperature in order to find preserve the best results microbes for their culturesas long as possible. For exampleAfter more than 3-6 months of storage (depends on how hardy the culture is), using different strains of Saccharomyces cerevisiae a starter should be made for the yeast cake/beer to make up for viability loss. Viability is less important for ''Brettanomyces'' if it is not being used as the primary fermenter since pitching rate in secondary appears not to have a large effect (see [[Brettanomyces_secondary_fermentation_experiment|''Brettanomyces'' secondary experiment]]), but viability is usually more important for the bacteria, which will be stressed from the long storage. Adding ~2 grams of chalk per liter of the slurry might help to buffer the pH and preserve the viability of all of the microbes for longer (more data is needed to prove this hypothesis). If the yeast slurry/beer sample is not very old and the brewer thinks that there might be residual sugars that could be fermented by the ''Brettanomyces'', leave the lid of the jar slightly loose in case a slow fermentation happens (this can produce widely different resultsoccur even at refrigeration temperatures). Another option that some brewers do is to keep the culture at room temperature and "feed" the culture a small amount of wort every few weeks <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1924825847545620/?comment_id=1925173547510850&comment_tracking=%7B%22tn%22%3A%22R%22%7D Per Karlsson. Milk The Funk thread on storing mixed cultures. 12/20/2017.]</ref>. This requires a vessel with an airlock.

==When to BottleQuality Assurance and Avoiding Cross Contamination==(To do)See [[Quality_Assurance|Quality Assurance]].

==BottlingSee Also==(To do)===Additional Articles on MTF Wiki===* [[Mixed Cultures]]* [[Alternative Bacteria Sources]]* [[Brettanomyces and Saccharomyces Co-fermentation]]* [[Wort Souring]]* [[Lactobacillus]]* [[Pediococcus]]* [[Brettanomyces]]* [[Saccharomyces]]* [[Spontaneous Fermentation]]* [[Commercial Sour Beer Dregs Inoculation]]* [[Brewing Methods]]* [[Quality Assurance]]

==References==