Changes

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

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") as 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 Fermentations", 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 Fermentation It is important to mention that mixed fermentation brewing in general has very few well- The Basics==Sour fermentations require at least one Lactic Acid Bacteria (LAB)established rules and definitions. While we may categorize techniques for the sake of keeping some sort of manageable structure to this wiki, such as ''many methods can be used in conjunction with other [[LactobacillusBrewing Methods]]'' or ''[[Pediococcus]]'', and at least one yeast such as ''[[Saccharomyces]]'' brewers sometimes use same/different terminology for the same/different things (for example, the use of the term "wild beer" by professional brewers can mean "any mixed fermentation beer", or [[Brettanomyces]]can also mean "mixed fermentation beer brewed with wild caught microbes"). Many yeast companies offer [[Mixed Cultures]] New methodologies are constantly being developed that provide all combine elements of the microorganisms necessary more established techniques, as well as slight changes to make a sour beerestablished techniques. Definitions equally evolve over time. The results Many of the methods used are determined by the types of these commercial mixed cultures can be as varied as microbes the cultures themselvesbrewer is working with. For example, some An article of these commercial this length cannot encompass all mixed cultures produce lightly tart beer that may exhibit minimal funky flavors; others may produce intense sourness and assertive funkfermentation methods. This is dependent on the types Instead it will provide a "big picture" view of microbes in the general methodologies. Towards this end, we divide mixed culturefermentation methods into two approaches: the traditional long fermentation method and an increasingly popular, their ratios, how old short fermentation method. They are divided here as a device to illustrate the cultures are, philosophy of each and what methods facilitate the discussion of the brewer uses to encourage or discourage certain flavorstechniques used for each methodology. The brewer must understand that all distinction of these microbes are complex organisms (some more complex than others). Not only do different species behave differently and produce different results under different conditionstwo methods is however somewhat artificial, but different strains indeed many brewers use elements of the same species also can also behave differently and produce different both approaches to achieve their desired results under different conditions. Just as strains Examples of how techniques can overlap to create new techniques can also be found in Michael Tonsmeire''Saccharomyces cerevisiae'' produce different results in clean beers (es pivotal book on mixed fermentation brewing, "American Sour Beers".g., California Ale yeast versus Belgian Ale yeast), strains An archive of ''Lactobacillus'' spp. various sour beer terminology discussions and especially ''Brettanomyces'' spp. debates can also vary widelybe found [[Sour_Beer_Terminology|here]].

==Mixed Fermentation Sour 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. 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>. 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.

The most basic method for making a mixed fermentation sour beer is to brew some simple wort (fresh extract or all grain) that is low in IBU's. Iso-alpha acids can inhibit many species and strains of LAB. Keeping the wort less than 6 IBU's is recommended in general, unless the brewer has information about their LAB culture that indicates that they can tolerate more. Mash hopping is one technique that can be used to limit the IBU's by about 70% <ref>[http://www.homebrewersassociation.org/attachments/presentations/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.]</ref>. If hops are not required (commercial brewers may be required to use hops, while homebrewers aren't), they can be completely excluded from the recipe. The wort is often mashed at a high temperature to encourage the inclusion of complex carbohydrates in the final wort. The wort is then primary fermented with a ''Saccharomyces'' strain to achieve the majority of attenuation, leaving behind the complex carbohydrates. The primary fermentation is then inoculated with a mixed culture of ''Brettanomyces'', ''Lactobacillus'' and ''Pediococcus'', either by moving the wort into barrels with active cultures or by inoculating the primary fermentation vessel (i.e. glass carboy when the method is used by home brewers). This inoculation then starts a secondary fermentation of the remaining complex carbohydrates which follows a slow progression between the microbes that are primarily active. This secondary fermentation may not readily show apparent signs of active fermentation as in the primary fermentation but is often accompanied by the slow evolution of CO2 in the first 8 weeks and the eventual formation of a [[pellicle]] which may form quickly or very slowly depending on the presence of oxygen. This method is still the most commonly used by commercial producers of modern and traditional Belgian sour beer, with variations on the process occurring widely. While still widely used by homes brewers, fast fermentation methods such as [[Sour WortingWort Souring]] and other methods mentioned in this article are ever increasing in their use.

The grain bill and production for 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 simple grain bill of about 70% Pilsner malt and 30% malted wheat can be used (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 [http://www.homebrewtalk.com/showthread.php?t=322168 AmandaK's lambic-style extract recipe] for a good extract recipe). Some crystal and a small amount of roasted malts an 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 ''Brettanomyces'', or ''Brettanomyces'' and ''Pediococcus'' ''(Pediococcu''s generally should not be used without ''Brettanomyces''. See the ''[[Pediococcus]]'' page for more details). Performing a [[Turbid Mash]] is the traditional way to include starches in the wort. However other methods such as steeping some oats or flaked wheat during the boil <ref>[http://www.homebrewtalk.com/f127/adjuncts-starches-sour-beer-448529/ Homebrewtalk Discussion started by Amos Brown aka 'Metic']</ref>, or running off over a bag of flaked oats or wheat on the way to the kettle can also impart starches that won't be converted to sugars by the mash (see [[Turbid_Mash#Alternative_methods_to_yield_starchy_wort|alternatives to turbid mashing]]). This step is completely optional, however , it may be very beneficial to make sure some higher chain sugars or starches are available in the wort if the brewer wants to rely on ''Pediococcus'' for producing most of the acidity. Extract brewers can use 0.25 lbs. (0.11 kg) of Maltodextrin <ref>[http://byo.com/videos/item/975-lambic-brewing Lambic Brewing. Piatz, Steve. Brew Your Own Magazine. October, 2004.]</ref>, or hot steep a pound of flaked wheat, flaked oats, or carapils malt. See [[MTF Member Recipes]] for ideas on recipes, or the recipe sections of the books "American Sour Beers" by Michael Tonsmeire and "Wild Brews" by Jeff Sparrows.

Questions often arise regarding if and when wort aeration should be done. It is well 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 outside 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. ThusTherefore, 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," 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>. Therefore in In the brewing of non-mixed fermentation beers, aerating both the yeast starter and the wort before pitching the yeast is generally considered mandatory to the brewing process.  Brewers have historically had concerns about aerating wort that has either been pre-soured with lactic acid bacteria (if the lactic acid bacteria is still alive) or if it will receive a co-pitch of lactic acid bacteria, ''[[Brettanomyces]]'', and ''[[Saccharomyces]]'' (see [[Mixed_Fermentation#Reusing_a_Sour_Yeast_Cake|Reusing a Sour Yeast Cake]], [[Mixed_Fermentation#Multi-Stage_Fermentation|Multi-Stage Fermentation]] and [[Wort Souring]]). These concerns, however, are largely unfounded. Most species of ''[[Lactobacillus]]'' are either not affected by oxygen, or benefit slightly. [[Butyric Acid]] production by ''Lactobacillus'' is not a concern (see [[Lactobacillus#Effects_of_Oxygen|''Lactobacillus'', effects of oxygen]] for details). Some species/strains of ''[[Pediococcus]]'' might be inhibited by oxygen, but not all (see ''[[Pediococcus#Growth_and_Environment|Pediococcus]]'' for details). ''[[Brettanomyces]]'' creates acetic acid in the presence of oxygen, however, in the presence of a healthy pitch of ''[[Saccharomyces]]'', which rapidly consumes the oxygen, this is probably also not a concern. Additionally, oxygen greatly improves the vitality and cell count of ''Brettanomyces'' and a small amount is required for effective ''Brettanomyces'' growth and fermentation (see [[Brettanomyces Propagation Experiment]]). Thus, as long as a healthy pitch of ''[[Saccharomyces]]'' is present, aerating wort for mixed fermentation should lead to a healthy fermentation and good results.  Many brewers, however, do not aerate their wort when either pre-souring the wort with a pure culture of lactic acid bacteria, pitching fresh wort on top of a mixed culture yeast cake, co-pitching a mixed culture such as Wyeast Roeselare, or pitching a custom mix of microbes from multiple sources <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1182597671768445/?qa_ref=qd Conversation on MTF about oxygenating wort for mixed culture fermentation. 11/22/2015.]</ref>. Many such brewers have reported success without aerating. We, therefore, recommend that the brewer investigate and experiment with their process in order to decide whether or not aeration is 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=%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 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 (see [[Mixed_Cultures#Starters_and_Other_Manufacturer_Tips|mixed culture starters]]).

Brewers have historically had concerns about aerating ===Microbe Inoculation===Once the wort that has either been pre-soured with lactic acid bacteria (if 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 lactic acid bacteria culture is still alive) old or if it will receive a co-pitch larger volume is needed, generally a normal starter can be made for mixed cultures without fear of "throwing off the balance of lactic acid bacteria, ''[[Brettanomyces]]'', and ''[[Saccharomyces]]'' microbes" (see [[Mixed_FermentationMixed_Cultures#Reusing_a_Sour_Yeast_CakeManufacturer_Tips|Reusing a Sour The Yeast Cake]], [[Mixed_Fermentation#Multi-Stage_Fermentation|Multi-Stage FermentationBay starter tips]] as an example; Bootleg Biology and Omega Yeast Labs also recommend starters for mixed cultures for larger batches <ref>[[Sour Worting]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>). These concerns, however, are largely unfounded. Most species Instead of ''[[Lactobacillus]]'' are either not effected by oxygenbuying a single mixed culture, or benefit slightly. [[Butyric Acid]] production a brewer can create their own mixed culture by ''Lactobacillus'' is not a concern (see [[Lactobacillus#Effects_of_Oxygen|''Lactobacillus'', effects combining their own ratios from single cultures of oxygen]] for details)yeast and bacteria. Some species/strains of ''[[Pediococcus]]'' might A single mixed culture can be inhibited supplemented by oxygen, but not all (see ''[[Pediococcus#Growth_and_Environment|Pediococcus]]'' for details)adding pure cultures from different yeast labs as well. ''[[Brettanomyces]]'' creates acetic acid in Another suggestion that often helps produce a higher quality sour beer is to supplement the presence of oxygen, however in the presence of mixed culture with a healthy pitch of ''[[SaccharomycesCommercial Sour Beer Inoculation]]'', which rapidly consumes the oxygen, this is probably also not a concern. AdditionallyIn general, oxygen greatly improves the vitality and cell count more diversity of ''Brettanomyces'' (see [[Brettanomyces Propagation Experiment]])microbes, the more complex a sour beer can potentially be. Thus, as long as Using a healthy pitch of ''[[SaccharomycesWort Souring]]'' method in conjunction with this method can help increase acidity that may not otherwise be produced by some commercial blends (Wyeast Roeselare is presentknown for this characteristic, aerating wort for mixed fermentation should lead to a healthy fermentation and good resultsexample).

Many brewers, however, do not aerate their wort when either pre====Staggered Versus Co-souring the wort with a pure culture of lactic acid bacteria, pitching fresh wort on top of a mixed culture yeast cake, co-pitching a mixed culture such as Wyeast Roeselare, or pitching a custom mix of microbes from multiple sources <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1182597671768445/?qa_refPitching====qd Conversation on MTF about oxygenating wort for mixed culture fermentation. 11/22/2015.]</ref>. Many such brewers have reported success without aerating. We therefore recommend that the brewer investigate and experiment with their process in order to decide whether or not aeration is desired.

If 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 brewer impact of co-fermentation is less understood. Butler et al., partnered with Gilded Goat Brewing Company, analyzed the differences between co-pitching ''S. cerevisiae'', a separate liquid culture strain of ''[[Saccharomyces]]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), it is recommended to create 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 starter on month and a stir plate half before packaged. Sample A was characterized as tasting the most balanced and alternatively dose consumers preferred it with oxygen. This will allow 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 cell membranes to build enough lipids three different fermentation profiles had a different sensory fingerprint with different measurements for their cells walls proteins, titratable acidity (slight differences), lactic acid (slight differences), polyphenols, turbidity, color, and give them residual sugar, indicating that when individual species are introduced to ferment the greatest chance wort, that it potentially has a wide impact on many different aspects of fermenting the wort without off-flavors 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=%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/12841061782842602503719276322938/?comment_id=12841411082807672503738896320976&comment_tracking=%7B%22tn%22%3A%22R322R%22%7DConversation with Mark Trent on MTF regarding aerating starters/wort for mixed culture fermentations7D Kelley Freeman. Milk The Funk Facebook group post. 0402/1309/20162019.]</ref>. If the wort has been pre-souredThese results may or may not be repeatable with different strains or other variables, but it might be beneficial to propogate 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 does demonstrate that co-pitching and processed in such a way that they contain enough lipids to support a healthy fermentation of 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)staggered pitching produce measurably different results. Aeration should be considered for beers above 5% <ref name="danstar"></ref>.

===Bug Inoculation===Once Depending on the ethanol tolerance of the lactic acid bacteria strains present in the wort is produced and chilledculture, the mixed culture presence of ethanol can be pitched as normalhave a negative impact on lactic acid bacteria. If using a mixed culture from a commercial yeast labFor example, in one published study, at 11% ABV, a starter is generally not neededthe strain of ''L. If brevis'' used in the culture is old or a larger volume is neededstudy didn't grow as well than as in lower ABV samples, generally a normal starter can be made for mixed cultures without fear of "throwing off and the balance of microbes" (see [[Mixed_Cultures#Manufacturer_Tips|The Yeast Bay starter tips]] resulting lactic acid content was lower as an example; Bootleg Biology and Omega Yeast Labs also recommend starters for mixed cultures for larger batches well ion the 11% ABV beer <ref>[https://www.facebookbiorxiv.comorg/groupscontent/MilkTheFunk10.1101/permalink2022.03.07.483260v1 Beer ethanol and iso-α-acid level affect microbial community establishment and beer chemistry throughout wood maturation of beer. Sofie Bossaert, Tin Kocijan, Valérie Winne, Johanna Schlich, Beatriz Herrera-Malaver, Kevin J. Verstrepen, Filip Van Opstaele, Gert De Rouck, Sam Crauwels, Bart Lievens. bioRxiv 2022.03.07.483260; doi: https:/1336235339738010/?comment_id=1336237029737841&comment_tracking=%7B%22tn%22%3A%22R%22%7D Discussion on MTF regarding mixed culture startersdoi. 06org/2310.1101/20162022.03.07.483260.]</ref>). Instead of buying a single mixed cultureTherefore, a brewer can create their own mixed culture by combining their own ratios from single cultures of yeast and adding the lactic acid bacteriaearlier in the fermentation process versus later in higher ABV beers will most likely impact the final beer's lactic acid content. A single  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 culture can be supplemented by adding pure cultures from different yeast labs as wellfermentation sour beer]. Another suggestion that often helps produce a higher quality sour beer is to supplement See also the [http://brulosophy.com/2018/05/14/mixed culture with a -fermentation-combined-vs-staggered-microbe-pitch-exbeeriment-results/ associated Brülosophy podcast episode].* [[Commercial Sour Beer InoculationMixed_Fermentation#Multi-Stage_Fermentation|Multi-Stage Fermentation]]below. In general, the more diversity * [[Brettanomyces_and_Saccharomyces_Co-fermentation#Review_of_Scientific_Analysis|Cofermentation of microbes, the more complex a sour beer can potentially be''Brettanonyces'' and ''Saccharomyces'']].* [[Mixed_Fermentation#Souring_Without_Brettanomyces|Mixed Fermentation Without ''Brettanomyces'']]. Using a * [[Sour WortingLactobacillus#Effects_on_Mixed_Fermentation|Effects of ''Lactobacillus'' on cofermentation]] 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).

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. As always In general, it is best practice to maintain fermentation temperature control is of critical importance and temperature profiles for this fermentation step should match those 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 un-settled 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]).

After primary fermentation , the mostly attenuated beer is sometimes moved to a secondary fermentation vessel(and sometimes not; read below). Often in traditional 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 "bugs" 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 might help fuel 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>.

Some brewers (mostly homebrewers) do It is not find it necessary unusual to move see a slight rise in pH during the mostly attenuated beer into a secondary vesselor aging phases. InsteadFor example, Santeri Tenhovirta's in his masters thesis he measured the mixed culture is pitched directly into the primary fermenter. While yeast autolysis is a concern in regular brewing, it is not a cause for concern in mixed fermentations that contain pH of several species of ''BrettanomycesLactobacillus''. Lambic brewers, that were pitched into wort for example2 days, perform a primary fermentation in barrels, and leave the beer in the barrels during the beer's entire aging process, which is usually 1followed by US-3 years <ref>[http://www.lambic.info/Brewing_Lambic#Barrels Lambic.info Wiki05. Brewing LambicTenhovirta reported a slight pH rise of about 0. Retrieved 6/8/2015.]</ref>3 from day 150 until day 300-330. Yeast autolysis releases trehelose, acids, According to Kunze and other compoundsBamforth, which are metabolized an increase in pH towards the end of fermentation or aging could be caused by ''Brettanomyces'' yeast autolysis <refname="Tenhovirta_masters">[httphttps://wwwhelda.mbaahelsinki.comfi/districtshandle/michigan10138/events/Documents/2011_01_14BrettanomycesBrewing303018 The Effects of Lactic Acid Bacteria Species on Properties of Sour Beer.pdf Brettanomyces Santeri Tenhovirta; master thesis in Brewing Food Science from the horse the goat and the barnyardUniversity of Helsinki. Chad Yakobson. 1/14/20112019.]</ref><ref>Bamforth, CW. Maintaining a [[Solera]] may be an exception to this (see the [[Solera]] page for details)2001. pH in brewing: An overview. The advantage Master Brewers Association of not moving the beer into a secondary vessel is that less overall oxygen is introduced into the beer Americas Technical Quarterly 38(oxygen exposure will contribute to more acetic acid and then ethyl acetate production1), and may be the best option if the brewer does not have a closed:1-9.</CO2 system to prevent exposure to oxygen during transferringref>.

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 straight forward 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'', however the development of a pellicle is believed by some brewers to help protect against thisand 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 over sampling 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 over exposure 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.

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 up 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 farm 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>.

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, the gasses will 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>).

# After boiling the wort, cool it to 90-120°F (32.3-48.9°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).# Pitch a pure culture of ''[[Lactobacillus]]'', and if possible hold the temperature between 90-120°F (32.3-48.9°C) for 2-4 days (see the [[Sour_WortingWort_Souring#Souring_in_the_Primary_Fermenter|Souring in the Primary Fermenter]] page for more details).

# 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.

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 bugs yeast/bacteria to clear up, the sooner it will be ready.

==Finishing Mixed Fermentations==See also:=== 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 in even an old yeast cake (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>[httphttps://www.themadfermentationistfacebook.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 2SouthernBrewersConference/19videos/2015.]<881235365357984/ref>. Using a young yeast cake is also a viable option, Devin Bell and may carry over more surviving ''Saccharomyces'' cells as well as more viable cells of the other various microbes. In general, washing the yeast cake is not necessary. 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 Dan Pixley presentation at SBC 2017 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 12/22/2016quick souring.] (~52 mins in)</ref>.

Some ===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 will harvest yeast at the same time or staggered with pitching ''Lactobacillus'' first for a certain amount of trub from their fermenters day or two and then brewers yeast (500mL Cascade Brewing is known for examplethe 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 use only this 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 to inoculate 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 new batch 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 beerthan 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. This will allow  ===="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 brewer yeast character to control be expressed more so than with kettle sours. In the amount case of dead trub material pitching ''L. plantarum'' after fermentation with saison yeast, Bell reported that goes into the new beerturned 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. Michael Tonsmeire often advises Devin clarified that the brewer also pitches his "best success" is pitching ''S. cerevisiae'' saison strain with a fresh culture selection of ''Brettanomyces'' for primary fermentation. After 5-7 days of fermentation, he pitches ''SaccharomycesL. plantarum'' (2 shots of GoodBelly or 1 package of Omega Labs OYL-605 for 5-6 gallons of beer) <ref>[httphttps://www.themadfermentationistfacebook.com/2009groups/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. 06/30/112017.]</brewing-sour-beerref>. Once terminal gravity is reached (1.002-at-home1.html Tonsmeire004), Michaelhe bottles right away. The Mad Fermentationistbottles can be served at 8 weeks in the bottle, but start to peak at 24 weeks <ref>[https://www. Brewing Sour Beer at Homefacebook.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. Comments section. Retrieved 201/1926/20152017.]</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].

Some brewers have good success reusing a yeast cake or a portion of a yeast cake by leaving the wort in contact with the old yeast cake for 1-4 daysSee also:* [https://www. After 1-4 days, a fresh culture of ''Saccharomyces'' is pitched to finish the fermentationfacebook. The 1-4 day head start gives the souring bacteria a head start com/SouthernBrewersConference/videos/881235365357984/ Devin Bell and results in a low pH beer. The harvested yeast cake can also be pitched Dan Pixley presentation at the same time as a fresh culture of SaccharomycesSBC 2017 on quick souring, or afterwardincluding more details on this method. The decided timing ]* [[Alternative_Bacteria_Sources#Culturing_from_Probiotics|General tips on when using probiotics to pitch the harvest sour yeast cake will effect the acidity of the sour beer: early pitching of the make sour yeast cake generally produces a more acidic beer, and later pitching generally produces a less acidic beer]].

As ==Finishing Mixed Fermentation Sour Beer=====Determining When It Is Done===Unless the brewer has worked with all methods, the species and strains same blend of the microbes being used should always and wort recipe, it is difficult to give an exact time frame on when a mixed fermentation beer might be taken into considerationready. Experimentation Anywhere from three to twelve months (and repeated processes should sometimes longer) is a reasonable amount of time, but when any given beer will be carefully employed by ready for packaging within this time depends on many factors including the brewer in order to find microbes pitched, their health over time, wort composition, temperatures during the aging time, etc. The best results 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 their culturespackaging. For example, using different strains of ''Saccharomyces cerevisiae'' as The second factor is how does the primary fermenter could produce widely different resultsbeer taste? If it tastes good, and the use of 10-30 IBU's in the wort gravity is stable, then it can be used 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 inhibit dissipate out of the lactic acid bacteria if they become too strong and produce too much acidityfermenter slowly over time.

* [[Sour WortingBrettanomyces and Saccharomyces Co-fermentation]]* [[Wort Souring]]