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[[File:Omega-lacto-microscope.jpg|thumb|Omega Yeast Labs OYL-605 Lactobacillus Blend; photo by [https://www.facebook.com/groups/MilkTheFunk/permalink/1096077917087088/ Stephen Little].]]
<div style="background-color: #fff0f0; border: 1px solid black; padding: 1ex; margin: 1ex; margin-right: 24em; min-width: 20em;">The genus of ''Lactobacillus'' has recently been broken up into 25 different genera. Portions of this wiki may still refer to the old nomenclature until we can make all the updates. For the purposes of this wiki, all new genera that were once considered to be ''Lactobacillus'' will remain on this wiki page for the foreseeable future. Abbreviations will remain the same; for example ''Lactiplantibacillus plantarum'', previously ''Lactobacillus plantarum'', is still abbreviated as ''L. plantarum''. Also note that yeast labs and probiotics manufacturers may not update their product brands to reflect the new scientific nomenclature. See [[Lactobacillus#Recent_Taxonomy_Changes|''Lactobacillus'' Recent Taxonomy Changes]] for more information. </div> '''''Lactobacillus''''' (often referred to by brewers as "Lacto") is a genus of Gram-positive, rod-shaped lactic acid bacteria (LAB) which produces acidity and sour flavors in the form of lactic acid and [[Lactobacillus#Sugar_Utilization_and_Secondary_Metabolites|secondary metabolites]] found in lambics, Berliner Weiss, sour brown ales, and gueuze. All ''Lactobacillus'' species are facultative anaerobes, which means they grow anaerobically but can also grow in the presence of oxygen and use oxygen to some degree <ref name="todar_lactics4"></ref>. They [https://www.researchgate.net/post/How_to_prepare_spore_forming_media_for_lactobacillus do not form spores]. There are more than 100 species, many of which are found in the human gastrointestinal track <ref name="todar_lactics4">[http://textbookofbacteriology.net/lactics_4.html ''Lactic Acid Bacteria''. Todar's Online Texbook of Bacteriology. Kenneth Todar, PhD. Pg. 4. Retrieved 07/28/2015.]</ref><ref name="Todar_nutgro4">[http://textbookofbacteriology.net/nutgro_4.html ''Nutrition and Growth of Bacteria''. Todar's Online Texbook of Bacteriology. Kenneth Todar, PhD. Retrieved 07/28/2015.]</ref>. In addition to beer, some species of ''Lactobacillus'' are also used to ferment yogurt, cheese, sauerkraut, pickles, wine, cider, kimchi, cocoa, and kefir <ref>[https://en.wikipedia.org/wiki/Lactobacillus ''Lactobacillus''. Wikipedia. Retrieved 07/28/2015.]</ref>. ''Lactobacillus'' can form a [[pellicle]] (need reference). See ''[[Pediococcus]]'', ''[[Brettanomyces]]'', ''[[Saccharomyces]]'', and [[Mixed Cultures]], [[Kveik#Commercial_Availability|Kveik]], and [[Nonconventional Yeasts and Bacteria]] charts for other commercially available cultures. See the [[Sour WortingWort Souring]] and [[Mixed Fermentation]] pages for brewing techniques with ''Lactobacillus''. See the [[Alternative Bacteria Sources]] section for culturing ''Lactobacillus'' from grains, yogurt, probiotics, and other sources. ==Introduction of Characteristics and Taxonomy==''Lactobacillus'' is a genus of bacteria that are considered to be a part of a broader classification of bacteria known as ''lactic acid bacteria'' (abbreviated as "LAB"). Other genera of bacteria that belong to this group and also appear in food fermentation include ''Lactococcus'', ''Streptococcus'', ''Pediococcus'', and ''Leuconostoc''. ''Lactobacillus'', as well as these other LAB genera, have three main metabolic pathways: glycolysis (fermentation of sugars), lopolysis (degradation of fat), and proteolysis (degradation of proteins). Lactic acid (specifically the conjugate base form, lactate), is the major byproduct of their fermentation. Other secondary metabolites include diacetyl, acetoin, acetaldehyde or acetic acid (some of which can contribute yogurt flavors to yogurt as well as maybe beer). While the lopolysis pathway contributes little to flavor, the proteolysis pathway produces amino acids which can be further converted into various alcohols, aldehydes, acids, esters, and sulphur compounds, many of which contribute various flavors to dairy fermentation products as well as to sour beer <ref name="Bintsis_2018">[http://www.aimspress.com/article/10.3934/microbiol.2018.4.665/fulltext.html Lactic acid bacteria as starter cultures: An update in their metabolism and genetics. Thomas Bintsis. 2018. DOI: 10.3934/microbiol.2018.4.665.]</ref>. The genus ''Lactobacillus'' contains a large number of relatively diverse species, and is the largest genus of the lactic acid bacteria group with over 50 species <ref>[https://web.archive.org/web/20070202132806/http://www.bacterio.cict.fr/l/lactobacillus.html List of Prokaryotic Names with Standing in Nomenclature - Genus Lactobacillus. J.P. Euzéby. Archive.org Wayback Machine; Feb 02, 2007.]</ref>, many of which have been identified as playing an important role in food fermentation or as probiotic species found in the human gut. The species ''Lactobacillus delbruekii'' consists of three subspecies: subsp. ''delbrueckii'', subsp. ''lactis'' and subsp. ''bulgaricus'', and have been used in yogurt fermentation. ''L. plantarum'' has one of the largest genomes among LAB. ''L. sanfranciscensis'' is the predominant LAB in sourdough cultures. ''Lactobacillus paracasei'' subsp. ''paracasei'', ''L. plantarum'', ''L. curvatus'', ''L. rahmosus'', and ''L. casei'' are often found in cheese maturation. ''L. johnsonii'' and ''L. reuteri'' strains have mostly been found in human and animal feces, suggesting that they are natural intestinal flora and are probiotic. Other species that have been used as probiotics include ''L. fermentum'', ''L. plantarum'', ''L acidophilis'' (the latter is also used in yogurt fermentation). ''Lactobacillus sakei'' subsp. ''sakei'' is used in the fermentation of sake <ref name="Bintsis_2018" />. Many of the previously mentioned species are purchased from yeast labs and used intentionally by brewers making sour beer (see [[Lactobacillus#Culture_Charts|Culture Charts]] below). ''L. acetotolerans'' has recently been claimed to also be found in many mixed fermentation sour beers, specifically in spontaneously fermented sour beers <ref>[https://www.sciencedirect.com/science/article/pii/S0740002020302471? Alexander Tyakht, Anna Kopeliovich, Natalia Klimenko, Daria Efimova, Nikita Dovidchenko, Vera Odintsova, Mikhail Kleimenov, Stepan Toshchakov, Alexandra Popova, Maria Khomyakova, Alexander Merkel. Characteristics of bacterial and yeast microbiomes in spontaneous and mixed-fermentation beer and cider, Food Microbiology. Volume 94, 2021, 103658.ISSN 0740-0020. https://doi.org/10.1016/j.fm.2020.103658.]</ref><ref>[https://www.biorxiv.org/content/10.1101/2021.07.21.453094v1 Mixed culture metagenomics of the microbes making sour beer. Renan Eugênio Araujo Piraine, Fábio Pereira Leivas Leite, Matthew L. Bochman. bioRxiv 2021.07.21.453094; doi: https://doi.org/10.1101/2021.07.21.453094.]</ref> (see also [https://www.facebook.com/groups/592560317438853/search/?q=acetotolerans MTF threads]). ===Recent Taxonomy Changes===Recently, whole genome sequencing led to the genetically driven proposal to divide the genus of ''Lactobacillus'' into either 2 subdivisions, or more radically into 10-14 subdivisions by one study <ref>[https://aem.asm.org/content/84/17/e00993-18 Comparative Genomics of the Genus Lactobacillus Reveals Robust Phylogroups That Provide the Basis for Reclassification. Elisa Salvetti, Hugh M. B. Harris, Giovanna E. Felis, Paul W. O'Toole. 2018. DOI: 10.1128/AEM.00993-18.]</ref><ref>[https://aem.asm.org/content/85/3/e02155-18 Towards a Genome-Based Reclassification of the Genus Lactobacillus. Stijn Wittouck, Sander Wuyts, Sarah Lebeer. 2019. DOI: 10.1128/AEM.02155-18.]</ref> and 23 divisions by another study accepted for publication by the International Journal of Systematic and Evolutionary Microbiology which tends to carry more authority in microbiological circles <ref name="Zheng_2020">[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijsem.0.004107 A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Jinshui Zheng et al. 2020. DOI: https://doi.org/10.1099/ijsem.0.004107.]</ref>. With the emergence of whole genome sequencing, other changes have been proposed, such as merging and splitting species of ''Lactobacillus'' <ref>[https://www.biorxiv.org/content/biorxiv/early/2019/01/31/537084.full.pdf A genome-based species taxonomy of the Lactobacillus Genus Complex. Stijn Wittouck, Sander Wuyts, Conor J Meehan, Vera van Noort, Sarah Lebeer. 2019. DOI: http://dx.doi.org/10.1101/537084.]</ref>. Renaming 200+ lactobacilli into new categories and names could also have a significant impact on the industries that use these microbes. The scale of this change has been discussed and considerations given for such industries, while the new classifications should be robust enough to withstand future scientific discoveries and should be based on genetic patterns <ref>[https://www.sciencedirect.com/science/article/pii/S0924224419303164 The potential impact of the Lactobacillus name change: the results of an expert meeting organised by the Lactic Acid Bacteria Industrial Platform (LABIP). Bruno Pot, Elisa Salvetti, Paola Mattarelli, Giovanna E. Felis. 2019. DOI: https://doi.org/10.1016/j.tifs.2019.07.006.]</ref>. Several species mergers and splits have also been identified <ref>[https://search.proquest.com/docview/2299499440?pq-origsite=gscholar A Genome-Based Species Taxonomy of the Lactobacillus Genus Complex. Wittouck Stijn; Wuyts Sander; Meehan, Conor J; van Noort Vera; Lebeer, Sarah. 2019. DOI:10.1128/mSystems.00264-19.]</ref>. The outcome of these analyses has been to update the genus of ''Lactobacillus'' into 25 distinct genera, including 23 new genera. They now include: ''Lactobacillus'', ''Paralactobacillus'', ''Acetilactobacillus'', ''Agrilactobacillus'', ''Amylolactobacillus'', ''Apilactobacillus'', ''Bombilactobacillus'', ''Companilactobacillus'', ''Dellaglioa'', ''Fructilactobacillus'', ''Furfurilactobacillus'', ''Holzapfelia'', ''Lacticaseibacillus'', ''Lactiplantibacillus'', ''Lapidilactobacillus'', ''Latilactobacillus'', ''Lentilactobacillus'', ''Levilactobacillus'', ''Ligilactobacillus'', ''Limosilactobacillus'', ''Liquorilactobacillus'', ''Loigolactobacilus'', ''Paucilactobacillus'', ''Schleiferilactobacillus'', and ''Secundilactobacillus''. This [http://lactotax.embl.de/wuyts/lactotax/ Taxonomy Tool] can be used to check which species have changed. it is important to note that species names did not change, only the genus names changed for most species <ref name="Zheng_2020" />. The International Scientific Association for Probiotics and Prebiotics has also [https://isappscience.org/new-names-for-important-probiotic-lactobacillus-species adopted this new nomenclature]. Examples of changes made to typical brewing strains:{| class="wikitable sortable"|-! style=width:30em | Previous Name ! style=width:30em | New Name <ref name="Zheng_2020" />|-| ''Lactobacillus casei'' || ''Lacticaseibacillus casei''|-| ''Lactobacillus paracasei'' || ''Lacticaseibacillus paracasei''|-| ''Lactobacillus rhamnosus'' || ''Lacticaseibacillus rhamnosus''|-| ''Lactobacillus plantarum'' || ''Lactiplantibacillus plantarum''|-| ''Lactobacillus brevis'' || ''Levilactobacillus brevis''|- | ''Lactobacillus fermentum'' || ''Limosilactobacillus fermentum''|-| ''Lactobacillus reuteri'' || ''Limosilactobacillus reuteri''|-| ''Lactobacillus acetotolerans'' || Unchanged|-| ''Lactobacillus acidophilus'' || Unchanged|-| ''Lactobacillus delbrueckii'' || Unchanged|-| ''Lactobacillus helveticus'' || Unchanged|-|} For more information on the taxonomic changes to the ''Lactobacillus'' genus, see:* [https://www.facebook.com/groups/MilkTheFunk/permalink/3542631249098397/ Post in MTF about splitting the genus into 23 new groups.]* [http://lactobacillus.ualberta.ca Name changes supported by the International Journal of Systematic and Evolutionary Microbiology] and [https://isappscience.org/new-names-for-important-probiotic-lactobacillus-species/ this science news article]. For more information on the metabolism of lactobacilli, see [[Lactobacillus#Metabolism|Metabolism]].
==Commercial Lactobacillus Cultures==
{| class="wikitable sortable"
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! Lab Name !! Mfg# Product Name !! Taxonomy !! CO2 Producer (HetHetero/HomHomo) !! Starter Note !! Fermentation/Other Notes|-| [[Bootleg Biology]]/[[Spot Yeast]] || Sour Weapon L || ''Lactiplantibacillus plantarum'' (blended strains) || Facultatively heterofermentative || || Drops the pH of wort quickly. At 98F, trial batches dropped the pH of wort to 3.0 after just 24 hours. When pitched at 84F, pH should reach 3.5 in 24 hours. Ideal to use for acidifying wort for quick/kettle sours, and is also very effective when co-pitched with a yeast strain. As with any Lactobacillus culture, we do not recommend using in worts with <s>10 or more IBUs</s> any hops (this is the up to date recommendation from Jeff Mello of Bootleg Biology; any amount of hops will inhibit ''L. plantarum'' in general) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2800217156673147/?comment_id=2800400083321521&reply_comment_id=2804474432914086&comment_tracking=%7B%22tn%22%3A%22R%22%7D Jeff Mello. Milk The Funk Facebook thread on the lack of IBU tolerance of ''L. plantarum'' and Sour Weapon L. 07/23/2019.]</ref> as that will prevent significant souring. Isolated from traditional Norwegian Kveik <ref>[http://bootlegbiology.com/2017/06/27/new-culture-pre-sale-july-5-featuring-mtf-mega-blend-sour-weapon-l/ "New Culture Pre-Sale July 5: Featuring MTF Mega Blend & Sour Weapon L!" Bootleg Biology website. 06/27/2017. Retrieved 06/05/2017.]</ref>.
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| [[Brewing Science Institute]] || L. delbrueckii brevis || Lactobacillus delbrueckii ''Levilactobacillus brevis'' || Homofermentative Heterofermentative || || A Lactobacillus Shipped during log phase, so recommended to use within 2 to 3 days of receiving. Unlike yeast, BSI sells bacteria that produces by volume, and will sell a clean lactic sournessspecific volume for the number of BBL's the brewer is souring. Store at room temperature, not cold. Optimal temperature for fermentation is 102-105°F. Bacteria is half the price of yeast. Commercial pitches only; not listed on their catalog, but is carried in stock. BSI does not have IBU tolerance data, but there has been at least one report of it being tolerant of up to 20 IBU <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2147198278641708/?comment_id=2147921045236098&reply_comment_id=2148812765146926&comment_tracking=%7B%22tn%22%3A%22R%22%7D John Rowley, Andrew Deming, and Dan Ramos. Milk The Funk Facebook group thread on BSI brevis. 06/26/2018.]</ref>.
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| [[Brewing Science Institute]] || L. delbrueckii || ''Lactobacillus delbrueckii'' || Homofermentative || || A Lactobacillus bacteria that produces a clean lactic sourness. |-| Chr. Hansen || Harvest LB-1 || ''Lactiplantibacillus plantarum'' || Faculatative Heterofermentative || The culture is ready for inoculation directly in all beverage bases without previous reactivation (freeze dried) || Harvest LB-1 is a freeze dried concentrated pure culture of ''L. plantarum''. The culture has been selected to ensure a fast and safe acidification of cereal bases, vegetable juices and other sugar beverage bases. Can acidify wort to pH 3.2 and should allow brewers to decrease pH from 5.5 to 3.5 in ~ 16 hours. Temp range of 70 – 100 ⁰F. Tolerates 8 IBU. Commercial only sizes available through [https://www.gusmerenterprises.com/catalog/brewing/brewing-processing-aids/sour-beer/harvest-lb-1/ Gusmer Brewing]. <ref>[https://www.gusmerbeer.com/wp-content/uploads/sites/8/2019/04/PI_GLOB_HarvestLB-1_718316_EN.pdf "Harvest LB-1". Chr. Hansen. Retrieved 12/19/2019.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3140287979332728/?comment_id=3140612629300263&reply_comment_id=3140685999292926 Chris Webster. Sales at Gusmer. Milk The Funk Facebook thread on Chr Hanson L. plantarum. 12/19/2019.]</ref>|-| [[Community Cultures Yeast Lab]] || Lactobacillus brevis || ''Levilactobacillus brevis'' || Heterofermentative || || For traditional and kettle souring methods, produces high lactic acid. Suggested for use in wort under 5-10 IBUs. Fermentation Temperature: 75-105F.|-| [[Community Cultures Yeast Lab]] || Lactobacillus Plantarum || ''Lactiplantibacillus plantarum'' || Facultatively heterofermentative || || Produces high levels of lactic acid for kettle souring and sour mash beers. Suggested for use in wort under 1-2 IBUs. Fermentation Temperature: 90-100F.|-| [[Craft Cultures]] || CCYL510 || L. ''Lactobacillus delbrueckii '' || Homofermentative || || Lactic acid bacteria producing moderate acidity and sour flavors found in Lambics, Berliner Weiss, and Sour Ales. Commercial pitches only.
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| [[Craft Cultures]] || CCYL512 || L. ''Levilactobacillus brevis '' || Heterofermentative || || Typically produces more lactic acid than Lactobacillus delbrueckii. Commercial pitches only.
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| [[White LabsEast Coast Yeast]] || WLP677 ECY32 || L. delbrueckii (potentially misidentified) ''Levilactobacillus brevis'' || Heterofermentative <ref name="mtf_wiki_shaner">[http://www|| || Originally isolated from kefir.milkthefunkBright acidity and hop-tolerant (up to 30 IBU).com/wiki/100%25_Lactobacillus_Fermentation Milk The Funk Wiki. 100% Lactobacillus Fermentation Test by Lance Shaner.]</ref>temperature 60 - 80F <ref name="tmf_culturesecy_website">[http://www.themadfermentationisteastcoastyeast.com/p/commercialwild-culturesstuff.html ''Commercial "Wild Yeast / Brettanomyces, Lactobacillus, and Pediococcus Descriptions''/ Lactic Bacteria". The Mad Fermentationist Blog. Michael TonsmeireEast Coast Yeast website. Retrieved 304/427/20152018.]</ref> . |-| no stir plate, room temp [[Escarpment Laboratories]] || Lactobacillus Blend ||Incubate at > 90°F ''Levilactobacillus brevis'' and < 117°F for 5-7 days for greater lactic acid production. Cell count: 50-80 million cells''Lactiplantibacillus plantarum'' || Heterofermentative/mL (1.75-2.8 billion cells in Faculatative Heterofermentative || || This blend is designed to be usable at a 35 mL homebrew vial) <ref name="WL_cellcounts">Private correspondence with White Labs Customer Service wide range of temperatures, and Dan Pixley. 10/29/2015.</ref>. Not a good strain is especially suited for kettle souring, but can produce a "soft" acidity over a longer period of time <ref>[https://wwwWort Souring.facebookWe recommend pre-acidifying wort to 4.com/groups/MilkTheFunk/permalink/1212455192116026/?comment_id=1212475888780623&reply_comment_id=1212476575447221&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation 5 with Andrew Addkison on MTF. 01/12/2016.]</ref>. White Labs claims that it is tolerant to up to 20 IBUlactic acid, although growth starts to become inhibited then pitching the Lactobacillus blend in a CO2-purged kettle or fermentor at 15 IBU <ref name="WL_datasheet" /><ref>[http://www.themadfermentationist.com/p/commercial-cultures.html "Commercial Brettanomyces, Lactobacillus, and Pediococcus Descriptions; Commercial Yeast Laboratories." The Mad Fermentationist blog. Michael Tonsmeire. Retrieved 12/12/2016.]</ref>. Generally heat tolerant, but sours faster between 10032-110°F <ref name="WL_datasheet">[www.whitelabs42°C.com/sites/default/files/R%26D%20Wild%20Yeast%20and%20Bacteria%20Experiments_2.pdf "R&D Wild Yeast and Bacteria Experiments". White Labs data sheet. Retrieved 05/16/2017.]</ref>
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| [[White LabsEscarpment Laboratories]] || WLP672 Lactobacillus Blend 2.0 || L. brevis ''Lacticaseibacillus rhamnosus'' and ''Lactiplantibacillus plantarum'' || Heterofermentative <ref name="mtf_wiki_shaner"></ref><ref name="nick">[https://www.facebook.com/groups/MilkTheFunk/permalink/1029638267064387/?comment_id=1030638553631025&offset=0&total_comments=24 Conversation with Nick Impellitteri from The Yeast Bay on the MTF Facebook Group. 3/4/2015.]</ref> Faculatative Heterofermentative || No stir plate, room temp|| Produced by [[The Yeast Bay]]This blend is a product of our ongoing research into optimizing Lactobacillus strain selection. It is a blend of our main L. More hop tolerant than other Lacto strains, however TYB advises to use wort plantarum strain with less than 10 IBUa strain of Lactobacillus rhamnosus. White Labs data sheet shows that growth is inhibited to 82% at 5 IBU, and 60% at 10 IBU <ref name="WL_datasheet" />. Temperature This blend has a wide temperature range: 70-95°F (greatly inhibited at 110°F30ºC to 45ºC) <ref name="WL_datasheet" />; 80% attenuation (this may not reflect actual attenuation of wort and enhances fruit flavours in a real brewery; see reference <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1031115430250004/?comment_id=1031244193570461&offset=0&total_comments=33 Conversation the finished beer, with Michael Soo tasters noting red fruit and Nick Impellitteri on the Milk The Funk Facebook Groupguava aromas. 3/5/2015.]</ref>). <ref>[http://www.theyeastbay.com/wild-yeast-and-bacteria-products/wlp672-lactobacillus-brevis The Yeast Bay website. Retrieved 3/2/2015.]</ref> Cell count: 50-80 million cells/mL (1.75-2.8 billion cells It is intended for 35 mL homebrew vials) <ref name="WL_cellcounts"><kettle/ref>quick souring but can also be used in 0 IBU wort.
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| [[WyeastEscarpment Laboratories]] || 5335 Lactobacillus Secondary Souring Blend || L. buchneri ''Levilactobacillus brevis'' and ''Lacticaseibacillus paracasei'' || Heterofermentative <ref name="mtf_wiki_shaner"></ref> Faculatative Heterofermentative || 1 liter starter for a 5 gallon batch of beer, 1.020 DME sterile wort, no stir plate, no O2, starter at 90°F if possible 5-7 days || Incubate at 90°F for 5-7 days for greater lactic acid productionThis blend of 2 hop resistant Lactobacillus strains (''L. Cell count: 1brevis'' and ''L.0 x 10<sup>8</sup> (100 millionparacasei'') cells/mL (10 billion cells is intended for use in a 100 mL homebrew pouch) <ref name="wyeast_cellcounts">[https://drivelong-term souring.google.com/folderview?id=0B8CshC9nxYHdZmE4MmoyLXA2WVk&usp=sharing Wyeast Specifications 2015 Retail Products. 2015.]</ref>We recommend 15 IBU or less in the first generation.
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| [[WyeastEscarpment Laboratories]] || 5223-PC Lactobacillus Brevis || L. ''Levilactobacillus brevis '' || Heterofermentative <ref name="mtf_wiki_shaner"></ref><ref name="nick"></ref> || no stir plate, room temp is fine || Heterofermentative (produces lactic acid, ethanol and CO2), more This strain is moderately hop -tolerant. Does well at room temperature. AVAILABLE ONLY FROM JULY THROUGH SEPTEMBER 2014 (Michael Dawson from Wyeast indicated that this culture may return at some point). Jamie Daly indicated on MTF that he got almost no sourness after 24 hours at 100°F (37.8°C). He lowered the temperature to 90°F-95°F (32.2°C-35°C) for 36 hours, and the pH of the wort went down to 3.29. Thus, Jamie recommends 90°F-95°F (32.2°Cas such it can also be used for long-35°C) for 60 hours for better term souring; avoid warmer temperatures. He also aerated his starter of L. brevis (2L starter of 1.020 DME) and set it on a stir plate at 95°F <ref name="brevis_aeration">[http://www10IBU beers.ncbi.nlm.nih.govIt also performs well in kettle souring/pmc/articles/PMC547135/ Growth Response of Lactobacillus brevis to Aeration wort souring where fast and Organic Catalystsclean lactic acidity is desired. J. R. Stamer and B. O. Stoyla. Appl MicrobiolWe recommend pre-acidifying wort to 4. Sep 1967; 15(5): 1025–1030.]</ref>. The beer wort was not aeratedwith lactic acid, and then pitching the fermenter was flushed with CO2. These methods need verification. Cell count: 1.0 x 10<sup>8</sup> (100 million) cells/mL (10 billion cells Lactobacillus blend in a 100 mL homebrew pouch) <ref name="wyeast_cellcounts"></ref>CO2-purged kettle or fermentor at 35-45°C.
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| [[Omega Yeast LabsEscarpment Laboratories]] || OYL-605 Lactobacillus Delbrueckii|| L. brevis, <span style="text-decoration: line-through;">delbrueckii</span>, and plantarum blend ''Lactobacillus Delbrueckii'' || Hetero/Hetero <ref name="mtf_wiki_shaner"></ref> Homofermentative || 1 liter starter for a 5 gallon batch of beer at room temperature for 24-48 hours. No stir plate unless kept anaerobic. || Quick souring. Pitch into 65°F-95°F <ref name="adi_oyl605"></ref>. Holding temperature is not required. No longer contains delbruekii <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1065268213501392/?comment_id=1065669443461269&offset=0&total_comments=18&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Raymond Wagner A single strain of Oso Brewing Co on Milk The Funk''L. 4/30/2015.]</ref>. Dondelbrueckii''t use any hops if possible. 2 IBU is a good target if hops must be often used <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1092523807442499/?comment_id=1092571350771078&offset=0&total_comments=6&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Lance Shaner on MTF in regards to IBU tolerance of OYL-605. 6/15/2015.]</ref>. Contains ~150 billion cells per homebrew pitch <ref name="sbb2.0">[http://sourbeerblog.com/lactobacillus-2-0-advanced-techniques-for-fast-quick souring-beer/ Lactobacillus 2.0 – Advanced Techniques for Fast Souring Beer. Sour Beer Blog. Matt Miller. 11/18/2015. Retrieved 11/19/2015.]</ref>.
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| [[Escarpment Laboratories]] || Lactobacillus Plantarum || ''Lactiplantibacillus plantarum'' || Facultatively heterofermentative || || A single strain of L. plantarum that performs well in kettle souring/sour worting where fast and clean lactic acidity is desired.|-| [[Escarpment Laboratories]] || The Kveik Ring: Lactobacillus paracasei || ''Lacticaseibacillus paracasei'' || Facultatively heterofermentative || || Isolated from [https://www.garshol.priv.no/download/farmhouse/kveik.html#kv5 Terje Raftevold's Hornindal Kveik]. Works well in kettle/quick souring. Temp: 30-40ºC // Acid Profile: Light to moderate (final pH 3.4-3.6) // If co-pitching with yeast, give the Lacto a 24 hour head start. Potentially a one time offer for May 2021 <ref>[https://www.facebook.com/escarpmentlabs/posts/4007824289310386:0 Escarpment Labs Facebook Page. Retrieved 05/06/2021.]</ref>.|-| Fermentis || SafSour™ LP 652 || ''Lactiplantibacillus plantarum'' || Faculatative Heterofermentative || No starter recommended for dried format || An optimum dosing rate of 10 g/hL provides a lactic fermentation within 24h – 36h in non-hopped wort <ref>[https://fermentis.com/en/fermentation-solutions/you-create-beer/safsour-lp-652 SafSour™ LP 652. Fermentis website. Retrieved 03/14/2020.]</ref>. See also Fermentis presentation [https://www.youtube.com/watch?v=ThuTjHnnYqk here] for impacts on temperature, starting gravity, aerobic/anaerobic fermentation, and sensory impact of different strains of ''S. cerevisiae'' when kettle souring.|-| Fermentis || SafSour™ LP 1 || ''Levilactobacillus brevis'' || Heterofermentative || No starter recommended for dried format || An optimum dosing rate of 10 g/hL provides a lactic fermentation. It is recommended to pitch directly into the non-hopped wort at the temperature of 32°C (+/- 5°C) <ref>[https://fermentis.com/en/product/safsour-lb-1/ SafSour™ LP 1. Fermentis website. Retrieved 10/20/2021.]</ref>. See also Fermentis presentation [https://www.youtube.com/watch?v=ThuTjHnnYqk here] for impacts on temperature, starting gravity, aerobic/anaerobic fermentation, and sensory impact of different strains of ''S. cerevisiae'' when kettle souring.|-| [[Fermmentos Labs]] (Brazil - CLOSED) || FB7 Pure Sour || ''Lactiplantibacillus plantarum'' and ''L. brevis'' || Facultatively heterofermentative /Heterofermentative || || Designed for kettle souring. Optimal temperatures of 20-25°C <ref name="fermmentos_catalog_2017">[https://fermmentolabs.com.br/wp-content/uploads/2017/07/Cat%C3%A1logo_Fermmento_Labs_TWTF.pdf Fermmentos Labs Catalog. Retrieved 12/21/2017.]</ref>.|-| [[Fermmentos Labs]] (Brazil - CLOSED) || FB12 Lactos || ''Lactobacillus delbruekii'' and ''Lacticaseibacillus rhamnosus'' || Homofermentative || || Designed for kettle souring. Optimal temperatures of 25-30°C <ref name="fermmentos_catalog_2017" />.|-| [[GigaYeast]] (CLOSED) || GB110 || L. ''Lactobacillus delbrueckii''? <ref>[https://www.facebook.com/GigaYeast/posts/565914926872849?comment_id=567669393364069&offset=0&total_comments=1¬if_t=feed_comment From Gigayeast, Inc. on Facebook, 12/3/2014: "Appears to be L. delbrueckii."]</ref> || Heterofermentative || For a 5 gallon batch of beer use 2 liters at 1.040 with high quality yeast nutrient. Keep as close to 86°F (30°C) as possible for 3-4 days with frequent rousing (no stir plate) <ref>Personal Communication with Jim Thompson.</ref>. || Lactic Acid Bacteria are inhibited by hops, high gravity and low temperatures. You can adjust sourness by increasing or decreasing these variables. More than 7 IBU, gravity above 1050 or temps below 65 F will increase the time to sour or lead to reduced overall souring. Contains ~200 billion cells per homebrew pitch <ref name="sbb2.0"></ref>.
We recommend brewing with GB110 in one of three ways. I) “Hot Start”: Pitch GB110 to wort at 98 F with little or no hops for 48-72 hrs. Wort may be soured before kettle boil or after. If soured before kettle boil, boil with hop additions as usual. If soured after kettle boil cool wort and pitch yeast. II) “Co-Pitch”: Pitch GB110 into a primary with yeast of your choice at 68-72 F. Wort that is less than 1050 and 7 IBU will typically be very sour in 2-3 weeks. III) “Secondary”: Pitch GB110 after primary fermentation for an aged sour. Souring by this method typically requires several months. Adding simple sugars or fruit etc. will enhance souring in the secondary <ref>[http://www.gigayeast.com/fast-souring-lacto GigaYeast Webpage. Retrieved 7/22/2015.]</ref>. Sometimes referred to as GigaYeast's "Fast Acting Lacto". This strain is hop sensitive <ref name="steve_smith">[https://www.facebook.com/groups/MilkTheFunk/permalink/1068326413195572/?comment_id=1069411906420356&offset=0&total_comments=12&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Steve Smith of GigaYeast on MTF. 05/08/2015.]</ref>.
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| [[RVA Inland Island Brewing & Consulting|Inland Island Yeast LabsLaboratories]] || RVA 600 INISBC-991 || ''Levilactobacillus brevis'' || Heterofermentative ||| L| Produces more lactic acid at higher temperatures and in low hop worts. rhamnosus GG 70-95 F Temperature Range|- | [[Inland Island Brewing & Consulting|Inland Island Yeast Laboratories]] || INISBC-992 || ''Lactobacillus delbruekii'' || Homofermentative || No starter necessary per RVA || Homofermentative Lacto strain found Produces more lactic acid at higher temperatures and in probiotics; sensitive to hops; does well at room temperaturelow hop worts. 70-95 F Temperature Range|-| [[Inland Island Brewing & Consulting|Inland Island Yeast Laboratories]] || INISBC-932 || ''Limosilactobacillus fermentum'' || Heterofermentative || ||
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| [[SouthYeast LabsJasper Yeast]] || Lactobacillus 1 JY-LPLANT || Unknown ''Lactiplantibacillus plantarum'' || Heterofermentative Facultatively heterofermentative || || SourceIdeal for kettle souring. Optimum temperature is 100°F-110°F. L. plantarum is hop sensitive, we advise not to use any hops until souring is satisfactory. <ref name="Jasper_Lacto">[https: Spontaneously infected beer (South Carolina)//jasperyeast. Best suits Light sours, gose, farmhouse saison (mediumcom/high acidity)bacteria "Available Bacteria". Jasper Yeast Website.]</ref>
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| [[SouthYeast LabsJasper Yeast]] || Lactobacillus 2 JY-LBREV || Unknown ''Levilactobacillus brevis'' || Homofermentatative heterofermentative || || Source: Prickly pear fruit (South Carolina)Ideal for kettle souring. Best suits strong sours, and lambic (high acidity)works well at 95°F-105°F.<ref name="Jasper_Lacto"/>
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| [[Inland Island Jasper Yeast]] || Lactobacillus blend || ''Lactiplantibacillus plantarum'' and ''Levilactobacillus brevis'' || Facultatively heterofermentative/heterofermentative || || Ideal for kettle souring. Optimum temperature is 95°F-110°F. L. plantarum is hop sensitive, we advise not to use any hops until souring is satisfactory. <ref name="Jasper_Lacto"/>|-| [[Jasper Yeast]] || JY-LACID || ''Lactiplantibacillus acidophilus''|| Homofermentative || || Optimum temperature is 95°F-105°F. <ref name="Jasper_Lacto"/>|-| Lallemand || WildBrew Sour Pitch || ''Lactiplantibacillus plantarum'' <ref>[https://www.facebook.com/Lallemandyeasts/photos/a.941604692537326.1073741829.939455986085530/1656901501007638/?type=3&comment_id=1657229347641520&reply_comment_id=1657231934307928&comment_tracking=%7B%22tn%22%3A%22R5%22%7D Post on the Lallemand Facebook page. 09/22/2017. Retrieved 09/22/2017.]</ref> || Facultatively heterofermentative || || See [https://www.facebook.com/groups/MilkTheFunk/permalink/1790290834332456/ this information from Scott Lucas on MTF]. This culture comes in a dry (desiccated) format. Although the [http://www.lallemandbrewing.com/product-details/wildbrew-sour-pitch manufacturer's website] claims this strain is tolerant of 4 IBU, we recommend that brewers treat this strain like any other strain of ''L. plantarum'' and do not expose it to any hops until the desired acidity has been produced (for example, see [[Wort Souring]]). Recommended temperature: 95-100°F (35-38°C) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1882643148430557/?comment_id=1883360638358808&reply_comment_id=2017197581641779&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Caroline Smith (rep from Lallemand). Milk The Funk Facebook group regarding Lallemand WildBrew Sour Pitch. 03/09/2018.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1988756664485871/?comment_id=1990170714344466&reply_comment_id=2017220451639492&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Caroline Smith (rep from Lallemond). Milk The Funk Facebook group regarding Lallemand WildBrew Sour Pitch and IBU tolerance. 03/09/2018.]</ref>. Reports in MTF on hop tolerance are mixed: Thomas Delaney reported no souring with 4 IBU, while Warren Knowles reported slower souring to 3.2 in 48 hours with ~5 IBU. Adam Stout reported a pH drop from 5.4 to 5.0 over 24 hours with ~3-5 IBU. Knowles, Matt Lange, and Matt Waugh reported light THP for a few days that went away or dimished by serving time but otherwise clean acidity and good results <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1988756664485871/ Various Milk The Funk members. MTF post on the Lallemand WildBrew Sour Pitch product. 02/14/2018.]</ref>. The homebrew pitch is enough for two 5 gallon/19 liter batches, but the package is not vacuum-sealable. It is recommended to seal the original sachet without vacuum sealing, and then double bag it into a bvacuum-sealable package and store in the freezer. This will help prevent contamination (see reference for anecdotes of saving left-over open packages of this product) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3204419266252932/?comment_id=3205432392818286 Gianmaria Ricciardi, Lallemand Brewing sales representative. Milk The Funk thread on saving Lallemand Wild Brew opened packages. 01/14/2020.]</ref>. |-| Lallemand || WildBrew Helveticus || ''Lactobacillus helveticus'' || Homofermentative || || Temp range: 38°C - 45°C (100°F - 113°F). Hop tolerance: In lab tests, growth was inhibited at 4ppm iso-alpha acid, but they recommend no hops during kettle souring. The pH range is 3.0-3.5 (within 36 hours). Dosage: 10g/hL <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3016721145022746/?comment_id=3016866791674848& Consultingreply_comment_id=3017521094942751 Joan Montasell from Lallemand Brewing. Milk The Funk Facebook thread on Lallemand WildBrew Helveticus. 11/01/2019.]</ref>. [https://www.facebook.com/groups/MilkTheFunk/permalink/3016721145022746/ MTF thread on availability and personal experiences using it.]|-| [[Mainiacal Yeast]] (CLOSED) || MYLP1 || ''Lactiplantibacillus plantarum'' || Facultatively heterofermentative || || Isolated from flowers at King Richards Faire in Massachusetts. It produces a clean lactic sour and prefers it a little cooler however does sour more quickly at its higher temps. Recommended fermentation temperature is 70-90°F <ref name="Amaral_Mainiacal">Private correspondence with Justin Amaral by Dan Pixley. 01/24/2018.]</ref>. '''Commercial pitches only'''.|-|Inland Island [[Mainiacal Yeast Laboratories]] (CLOSED) || MYLP2 || ''Lactiplantibacillus plantarum'' || Facultatively heterofermentative || || INISBCIsolated from grains going through the steeping process at Blue ox Malthouse. It produces a clean lactic sour and is a viable option for kettle souring, co-pitching, or post fermentation. Recommended fermentation temperature is 70-991 105°F <ref name="Amaral_Mainiacal" />. '''Commercial pitches only'''.|-| [[Mainiacal Yeast]] (CLOSED) || MYLD1 || ''Lactobacillus delbruekii'' || Homofermentative || || LIsolated from a spontaneous beer, this strain likes it warm but not to hot. It does not sour as quickly and will require some longer aging times to reach terminal pH. It produces a clean acidity with a hint of farmhouse like straw. Recommended fermentation temperature is 65-90°F <ref name="Amaral_Mainiacal" />. '''Commercial pitches only'''. |-| [[Mainiacal Yeast]] (CLOSED) || MYLB2 || ''Levilactobacillus brevis '' || Heterofermentative || || Produces more a clean lactic acidity, but generally doesn't produce as much lactic acid at higher temperatures as some other variants. It's best used co-pitched with other microbes and in low hop wortsallowed to age. Expect this strain to be a bit lighter on the souring side leaving a tart refreshing beer. 70 Recommended fermentation temperature is 60-95 F Temperature Range85°F <ref name="Amaral_Mainiacal" />. '''Commercial pitches only'''.|- | [[Inland Island Omega Yeast Labs]] || OYL-605 || ''Levilactobacillus brevis'', <span style="text-decoration: line-through;">''delbrueckii''</span>, and ''plantarum'' blend || Hetero/Hetero <ref name="mtf_wiki_shaner"></ref> || 1 liter starter for a 5 gallon batch of beer at room temperature for 24-48 hours. No stir plate unless kept anaerobic. || Quick souring. Pitch into 65°F-95°F <ref name="adi_oyl605"></ref>. Holding temperature is not required. No longer contains delbruekii <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1065268213501392/?comment_id=1065669443461269&offset=0&total_comments=18&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Raymond Wagner of Oso Brewing Co on Milk The Funk. 4/30/2015.]</ref>. Don't use any hops if possible. 2 IBU is a good target if hops must be used <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1092523807442499/?comment_id=1092571350771078&offset=0&total_comments=6& Consultingcomment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Lance Shaner on MTF in regards to IBU tolerance of OYL-605. 6/15/2015.]</ref>. Contains ~150 billion cells per homebrew pitch <ref name="sbb2.0">[http://sourbeerblog.com/lactobacillus-2-0-advanced-techniques-for-fast-souring-beer/ Lactobacillus 2.0 – Advanced Techniques for Fast Souring Beer. Sour Beer Blog. Matt Miller. 11/18/2015. Retrieved 11/19/2015.]</ref>. This product is vegan <ref>Adi Hastings. Private correspondance with Dan Pixley. 08/17/2018.</ref>.|-|Inland Island [[Propagate Lab]] || MIP-911 || ''Levilactobacillus brevis'' || Heterofermentative || || Acidifies unhopped wort in 48 hours at 100°F <ref>[http://www.propagatelab.com/mip911-lactobrevis Propagate Lab. MIP-911. Retrieved 06/20/2020.]</ref>.|-| [[Propagate Lab]] || MIP-912 || ''Lactobacillus delbruekii'' || Homofermentative || || Acidifies over an extended period of time; used in barrel aging <ref>[http://www.propagatelab.com/mip912-lactodelbureckii Propagate Lab. MIP-912. Retrieved 06/20/2020.]</ref>.|-| [[Propagate Lab]] || MIP-913 || ''Lacticaseibacillus casei'' || Facultative Heterofermentative || || |-| [[Propagate Lab]] || MIP-914 || ''Lactiplantibacillus plantarum'' || Facultative Heterofermentative || || Acidifies unhopped wort in 48 hours at 100°F <ref>[http://www.propagatelab.com/mip-914lactoplantarum Propagate Lab. MIP-914. Retrieved 06/20/2020.]</ref>.|-| [[RVA Yeast LaboratoriesLabs]] || RVA 600 || ''Lacticaseibacillus rhamnosus'' || Homofermentative || No starter necessary per RVA || Homofermentative Lacto strain found in probiotics; sensitive to hops; does well at room temperature. |-| [[SouthYeast Labs]] (CLOSED) || Lactobacillus 1 || Unknown || Heterofermentative || || Source: Spontaneously infected beer (South Carolina). Best suits Light sours, gose, farmhouse saison (medium/high acidity).|-| [[SouthYeast Labs]] (CLOSED) || Lactobacillus 2 || Unknown || Homofermentatative || || INISBCSource: Prickly pear fruit (South Carolina). Best suits strong sours, and lambic (high acidity).|-992 | [[The Yeast Bay]] || Lactobacillus Blend || ''Lactiplantibacillus plantarum'', and 2 strains of ''Levilactobacillus brevis'' || Heterofermentative || || The Lactobacillus Blend includes three strains: ''Lactobacillus plantarum'', ''Lactobacillus brevis'' and a second strain of ''Lactobacillus brevis'' isolated from an accidentally soured blonde ale from a Mexican craft brewery. Quickly produces acidity across a wide range of temperatures. It can be used on its own for kettle souring prior to pitching yeast to create acidity quickly, or co-pitched with yeast to create sourness over time. It will produce a pronounced and rounded acidity. The Yeast Bay recommends holding the IBU on the low end (< 2-3) if you'd like to use this blend to create acidity in a shorter time frame. Higher IBUs may result in souring, but the strain of ''L. delbruekii brevis'' isolated from the Mexican craft brewery is hop tolerant up to about 15-20 IBU. Temperature: 70-90°F. Cell count: 50-80 million cells/mL (1.75-2.8 billion cells for 35 mL homebrew vials) <ref name="WL_cellcounts"></ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1280135442014667/?comment_id=1280341068660771&reply_comment_id=1280498695311675&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Nick Impellitteri on MTF regarding TYB Lactobacillus Blend cell counts. 04/08/2016.]</ref>. Recommended temperature range for fastest acid production for kettle souring is 85-90°F, although if kept in the 70's it should produce good acidification in 48-72 hours. A major drop off of in acid production is seen above 90°F <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1616265398401668/?comment_id=1617001948328013&comment_tracking=%7B%22tn%22%3A%22R%22%7D Impellitteri, Nick. Milk The Funk Facebook group. 03/17/2017.]</ref>.|-| [[The Yeast Bay]] || TYB282 || ''Lactiplantibacillus brevis'' || Homofermentative Heterofermentative || || Produces more TYB282 is a single strain of Lactobacillus brevis isolated out of an unintentionally soured golden ale produced by a Mexican craft brewery.This strain produces a clean lactic acidity (down to ~pH 3.16-3.18) in unhopped wort within 36 hours at a temperature of ~72-77 F. The higher the temperature (up to 90 F is what we've tested), the faster the acid production. Recommended for kettle souring, as it grows rather quickly and produces acidity fast with no detectable off flavors. The Yeast Bay has tested this strain at ~20 IBU and it was able to reduce the pH of beers down to 3.30 pH when co-pitched with a farmhouse yeast. It might create acidity at higher temperatures IBU's (Nick suggests maybe up to 30 IBU); however, this has not been tested yet <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2907190232642505/?comment_id=2907235245971337 Nick Impellitteri. Milk The Funk Fcaebook group post on TYB282 hop tolerance. 09/12/2019.]</ref>. Temperature: 70-90 ºF. |-| [[White Labs]] || WLP677 || ''Lactobacillus delbrueckii'' (might be misidentified <ref>[http://masterbrewerspodcast.com/085-lactic-acid-bacteria-case-study Tim Lozen. Master Brewers Association podcast interview on lactic acid bacteria case study. 04/23/2018.]</ref>) || Heterofermentative <ref name="mtf_wiki_shaner">[http://www.milkthefunk.com/wiki/100%25_Lactobacillus_Fermentation Milk The Funk Wiki. 100% Lactobacillus Fermentation Test by Lance Shaner.]</ref><ref name="tmf_cultures">[http://www.themadfermentationist.com/p/commercial-cultures.html ''Commercial Brettanomyces, Lactobacillus, and Pediococcus Descriptions''. The Mad Fermentationist Blog. Michael Tonsmeire. Retrieved 3/4/2015.]</ref> || no stir plate, room temp ||Incubate at > 90°F and < 117°F for 5-7 days for greater lactic acid production. Cell count: 50-80 million cells/mL (1.75-2.8 billion cells in low a 35 mL homebrew vial) <ref name="WL_cellcounts">Private correspondence with White Labs Customer Service and Dan Pixley. 10/29/2015.</ref>. Not a good strain for kettle souring, but can produce a "soft" acidity over a longer period of time <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1212455192116026/?comment_id=1212475888780623&reply_comment_id=1212476575447221&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation with Andrew Addkison on MTF. 01/12/2016.]</ref>. White Labs claims that it is tolerant to up to 20 IBU, although growth starts to become inhibited at 15 IBU <ref name="WL_datasheet" /><ref>[http://www.themadfermentationist.com/p/commercial-cultures.html "Commercial Brettanomyces, Lactobacillus, and Pediococcus Descriptions; Commercial Yeast Laboratories." The Mad Fermentationist blog. Michael Tonsmeire. Retrieved 12/12/2016.]</ref>. Generally heat tolerant, but sours faster between 100-110°F <ref name="WL_datasheet">[http://www.whitelabs.com/sites/default/files/R%26D%20Wild%20Yeast%20and%20Bacteria%20Experiments_2.pdf "R&D Wild Yeast and Bacteria Experiments". White Labs data sheet. Retrieved 05/16/2017.]</ref>|-| [[White Labs]] || WLP672 || ''Levilactobacillus brevis'' || Heterofermentative <ref name="mtf_wiki_shaner"></ref><ref name="nick">[https://www.facebook.com/groups/MilkTheFunk/permalink/1029638267064387/?comment_id=1030638553631025&offset=0&total_comments=24 Conversation with Nick Impellitteri from The Yeast Bay on the MTF Facebook Group. 3/4/2015.]</ref> || No stir plate, room temp|| Produced by [[The Yeast Bay]]. More hop wortstolerant than other Lacto strains, however TYB advises to use wort with less than 10 IBU. White Labs data sheet shows that growth is inhibited to 82% at 5 IBU, and 60% at 10 IBU <ref name="WL_datasheet" />. Temperature range: 70-95 F Temperature Range95°F (greatly inhibited at 110°F) <ref name="WL_datasheet" />. <ref>[http://www.theyeastbay.com/wild-yeast-and-bacteria-products/wlp672-lactobacillus-brevis The Yeast Bay website. Retrieved 3/2/2015.]</ref> Cell count: 50-80 million cells/mL (1.75-2.8 billion cells for 35 mL homebrew vials) <ref name="WL_cellcounts"></ref>. This strain can take several days to acidify unhopped wort, and as such is not recommended by MTF for kettle sours. This strain benefits from magnesium nutrient additions, and is slightly inhibited by zinc nutrient additions <ref>[https://www.mdpi.com/2218-273X/10/12/1599 Chemical Composition of Sour Beer Resulting from Supplementation the Fermentation Medium with Magnesium and Zinc Ions. Aneta Ciosek, Katarzyna Fulara, Olga Hrabia, Paweł Satora, and Aleksander Poreda. 2020. DOI: https://doi.org/10.3390/biom10121599.]</ref>.
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| [[Inland Island Brewing & Consulting|Inland Island Yeast LaboratoriesWyeast]] || INISBC-932 5335 || L. fermentum ''Lentilactobacillus buchneri'' || Heterofermentative <ref name="mtf_wiki_shaner"></ref> || 1 liter starter for a 5 gallon batch of beer, 1.020 DME sterile wort, no stir plate, no O2, starter at 90°F if possible 5-7 days || Incubate at 90°F for 5-7 days for greater lactic acid production. Cell count: 1.0 x 10<sup>8</sup> (100 million) cells/mL (10 billion cells in a 100 mL homebrew pouch) <ref name="wyeast_cellcounts">[https://drive.google.com/folderview?id=0B8CshC9nxYHdZmE4MmoyLXA2WVk&usp=sharing Wyeast Specifications 2015 Retail Products. 2015.]</ref>.
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| [[Escarpment LaboratoriesWyeast]] || Lactobacillus Blend 5223-PC || L. ''Levilactobacillus brevis and L. plantarum '' || Heterofermentative <ref name="mtf_wiki_shaner"></ref><ref name="nick"></ref> || no stir plate, room temp is fine || This blend is designed Heterofermentative (produces lactic acid, ethanol and CO2), more hop tolerant. Does well at room temperature. Jamie Daly indicated on MTF that he got almost no sourness after 24 hours at 100°F (37.8°C). He lowered the temperature to be usable at a wide range 90°F-95°F (32.2°C-35°C) for 36 hours, and the pH of temperaturesthe wort went down to 3.29. Thus, and is especially suited Jamie recommends 90°F-95°F (32.2°C-35°C) for 60 hours for kettle better souring; avoid warmer temperatures. He also aerated his starter of L. brevis (2L starter of 1.020 DME) and set it on a stir plate at 95°F <ref name="brevis_aeration">[http:/sour worting/www.ncbi.nlm. We recommend pre-acidifying wort nih.gov/pmc/articles/PMC547135/ Growth Response of Lactobacillus brevis to 4Aeration and Organic Catalysts. J. R. Stamer and B. O. Stoyla. Appl Microbiol.Sep 1967; 15(5 with lactic acid): 1025–1030.]</ref>. The beer wort was not aerated, then pitching and the Lactobacillus blend fermenter was flushed with CO2. These methods need verification. Cell count: 1.0 x 10<sup>8</sup> (100 million) cells/mL (10 billion cells in a CO2-purged kettle or fermentor at 32-42°C100 mL homebrew pouch) <ref name="wyeast_cellcounts"></ref>.
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| [[Escarpment LaboratoriesWyeast]] || Lactobacillus brevis 4335 || L. brevis ''Lactobcillus delbruekii'' || Heterofermentative || || This strain There are [https://www.google.com/search?safe=off&rlz=1C1CHBF_enUS741US743&ei=BAuPW-WyL8OAk-4PuoGryA8&q=wyeast+4335&oq=wyeast+4335&gs_l=psy-ab.3..0i30.16026.16026..16511...0.0..0.83.83.1......0....1..gws-wiz.......0i71.9JdIoR14NT8 various references on the internet during the mid to late 2000's] to a product called "Wyeast 4335 ''Lactobacillus delbruekii''", however, this product is moderately hop-tolerantno longer offered by Wyeast. When asked about this product, the Wyeast customer support reported that the "4335" product was renamed to "5335" fifteen years ago, and the "5335" and "4335" products are the same culture. It is unclear why "4335" was labeled as such ''L. delbruekii'', but it can also be used for long-term souring of is likely that it was originally misidentified <10IBU beersref>[https://www.facebook. It also performs well in kettle souringcom/groups/MilkTheFunk/permalink/2265479906813544/sour worting where fast ?comment_id=2267884689906399&comment_tracking=%7B%22tn%22%3A%22R%22%7D Chris Cates private correspondance with Wyeast customer service representative. Milk The Funk Facebook thread on the origin and clean lactic acidity is desireddisappearance of WY4335 ''L. delbruekii''. We recommend pre-acidifying wort to 409/04/2018.5 with lactic acid, then pitching the Lactobacillus blend in a CO2-purged kettle or fermentor at 35-45°C]</ref>.
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===General Advice===
====Starters and Pitching Rate====
In addition to the starter information given in the [[Lactobacillus#Manufacturer_Tips|Manufacturer Tips]] above, this section includes general advice for ''Lactobacillus'' starters for homebrewers and commercial brewers. For growing ''Lactobacillus'' in a lab environment, or from an initial grouping of cells from a plate/slant or smaller cell count, MRS media is the most efficient growth media. However, for full pitches of ''Lactobacillus'' in beer/wort, brewers probably donwon't want to add that much MRS media to their beer since MRS media has a distinct odor and smell that would not be desirable in beer <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1152135114814701/?comment_id=1152674674760745&offset=0&total_comments=9&comment_tracking=%7B%22tn%22%3A%22R0%22%7DConversation with Lance Shaner and Nick Impellitteri on Lacto starters on MTF. 09/22/2015.]</ref>. Therefore, growing ''Lactobacillus'' in a wort-based starter media is recommended for building full pitches of ''Lactobacillus''. We recommend using an Erlenmeyer flask dedicated to growing ''Lactobacillus'' in order to lower the potential for yeast contamination. We also recommend using [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's Starter Procedures]]. See [[Lactobacillus#External_Resources|External Resources]] for additional starter guides.
A good pitching rate for ''Lactobacillus'' is 100-125 billion cells per 5 gallons of wort, although pitching rates for ''Lactobacillus'' are more forgiving than they are for yeast. Having a manufacturer supply a cell count or doing a cell count manually after growth in a starter, and then pitching an exact cell count is the best approach to achieving the desired pitching rate. However, counting cells of ''Lactobacillus'' under a microscope can be difficult to achieve due to the small size of bacteria cells, so starter volumes are generally used instead when talking about pitching rates for ''Lactobacillus'' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1068323126529234/?comment_id=1068337639861116&offset=0&total_comments=47&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation on MTF with Bryan of Sui Generis Blog. 5/6/2015.]</ref>. Pitching ~0.5-1 liter per ~20 liters of wort (~0.75-1 gallon per barrel) of ''Lactobacillus'' starter is the general guideline(see below). Note that the exact advisable pitching rates starter volumes of commercial cultures may differ from manufacture to manufacturer <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1077639885597558/ MTF thread started by Brad Primozic. 5/29/2015.]</ref>, and their recommendations might differ from ours. Achieving exact pitching rates for ''Lactobacillus'' is generally not necessary because over-pitching does not have a negative impact, and under-pitching is also more forgiving than under-pitching yeast (under-pitching risks producing less acid than desired for some strains of ''Lactobacillus''). For these reasons, and the difficulty in estimating cell counts based on starter media type, strain, and other variables, a pitching calculator for ''Lactobacillus'' does not currently exist. However, estimations based on starter volume and freshness should be adequate.
The amount of growth that occurs in a starter is an highly variable depending on the type of starter media, and the actual cell count is unknown unless the brewer can do a cell count. Matt Miller of Sour Beer Blog and Richard Preiss of Escarpment Yeast Labs advise that if ideal growth (1-2 billion cells/mL) can be achieved (for example by using [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's starter procedure]] or MRS media), then pitching as little as 100-125 mL of fresh ''Lactobacillus'' starter for 5 gallons of beer can achieve desirable acidity within 24 hours <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1180630378631841/?comment_id=1180733761954836&reply_comment_id=1180740128620866&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Richard Preiss on MTF regarding Lacto starters. 11/19/2015.]</ref><ref name="sbb2.0"></ref>. Moderately ideal growth in a starter results in around 500 million cells/mL, resulting in a 400 mL starter for 5 gallons, and low growth results in around 100 million cells/mL, resulting in a 2 liter starter for 5 gallons, according to Matt Miller (see [http://sourbeerblog.com/lactobacillus-2-0-advanced-techniques-for-fast-souring-beer/ Matt Miller's article] for more details) <ref name="sbb2.0"></ref>. If the amount of growth cannot be accurately determined by cell counting under a microscope, then pitching 0.5-1 liter of fresh ''Lactobacillus'' starter culture per 5 gallons of wort ensures that an adequate number of cells will be pitched.
Another thing to consider is that achieving a pH of 4 as fast as possible is advisable for preventing off-flavors from contaminating microbes <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1180630378631841/?comment_id=1181674265194119&reply_comment_id=1181715048523374&comment_tracking=%7B%22tn%22%3A%22R7%22%7D Conversation with Bryan of Sui Generis Blog on MTF regarding speed of acid production with Lacto. 11/20/2015.]</ref>. Larger pitch rates tend to achieve a lower pH faster <ref name="bryan_lacto_starters"></ref>. Therefore, unless using [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's starter procedure]], pitching 0.5-1 liters of starter for 5 gallons of wort is advisable in general. Even when using [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's starter procedure]], over-pitching ''Lactobacillus'' is not a concern (pitching an overly massive starter wort could produce undesirable flavors), so the same pitch rate of 0.5-1 liters for 5 gallons should still be considered unless the brewer is confident that high growth rate has been achieved in the starter, in which case 100-125 mL for 5 gallons of wort/beer should be adequate (again, exact pitching rates for ''Lactobacillus'' are generally not as important as they are for yeast pitching rates). Other factors that might affect the effectiveness of a volume based starter is the species/strain of the ''Lactobacillus'' being used, how much yeast contamination has occurred, and how old the ''Lactobacillus'' starter is. Some species/strains may require a larger volume of starter, as well as if yeast has contaminated the starter or wort (see [[Lactobacillus#100.25_Lactobacillus_Fermentation|100% Lactobacillus Fermentation]]). If a ''Lactobacillus'' culture is older than 1 month, then a fresh starter should be made. Keeping a separate Erlenmeyer flask for ''Lactobacillus'' starters can help to prevent yeast contamination <ref>Private correspondence with Richard Preiss to Dan Pixley. 11/20/2015.]</ref>, as well as using sterilization equipment such as an autoclave or pressure cooker.
Starter mediums that brewers have used include unhopped DME wort starters and apple juice starters. These tend to be adequate for many brewers. However, [https://eurekabrewing.wordpress.com/2015/05/18/evaluate-starter-media-to-propagate-lactobacillus-sp/ Samuel Aeschlimann from Eureka Brewing Blog] showed that using DME with a little bit of apple juice, chalk, and yeast nutrients provides close to optimal cell densities that match MRS media cell densities. See [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's Starter Procedures]]. Specific nutrients that will increase growth is thiamine (vitamin B1), or a combination of thiamine and riboflavin (vitamin B2). For example, it has been shown that thiamine is required in order for ''L. brevis'' to efficiently convert pyruvate into lactic acid and ethanol. The addition of these nutrients can help encourage growth <ref>[http://onlinelibrary.wiley.com/doi/10.1002/jib.385/full The influence of thiamine and riboflavin on various spoilage microorganisms commonly found in beer. Barry Hucker, Melinda Christophersen, Frank Vriesekoop. 2017.]</ref>. For example, the addition of vitamin H (biotin) was found to greatly increase the growth of ''L. plantarum'' in a lab setting <ref>[http://dspace.nau.edu.ua/handle/NAU/34046 Reshetnyak, L., Bondarchuk, N. (2018). The influence of biothin on growth and development of bacteria lactobacillus plantarum. Proceedings of the National Aviation University, 74 (1), 130 - 135.]</ref>.
Although more experiments and probably needed, agitation is believed to be an important factor for both yeast and bacteria in general. Gentle stirring on a stir plate or orbital shaker, or frequent gentle manual agitation leads to faster growth and a higher number of organisms. Agitation keeps the microbes in solution. It also maximizes the microbes' access to nutrients and disperses waste evenly. In a non-agitated starter, the microbes are limited to the diffusion rate of nutrients, leading to a slower and more stressful growth <refname="bryan_agitation">[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1174865305875015&reply_comment_id=1176092372418975&total_comments=1&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Bryan of Sui Generis Blog about starters and agitation. 11/09/2015.]</ref>. If agitation is not possible for whatever reason, a successful starter can be made without agitation. Sam Aeschlimann reported good success with ''Lactobacillus'' starters that are not agitated <ref name="Sam_starter2"></ref>.
Although ''Lactobacillus'' are tolerant of oxygen and oxygen usually does not negatively affect their growth (except in the case of ''L. plantarum'', which has been shown to produce small amounts of acetic acid when exposed to oxygen and glucose is not present <ref name="Quatravaux_plantarum">[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2006.02955.x/full Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters. S. Quatravaux, F. Remize, E. Bryckaert, D. Colavizza, J. Guzzo. 2006]</ref><ref name="microbewiki_plantarum"></ref>), it is also generally not needed (an exception to this may be ''L. brevis'', which has been shown to increase growth rates in the presence of oxygen <ref name="brevis_aeration"></ref>). Therefore, it is generally best practice to prevent aerating the starter with an airlock for ''Lactobacillus'' starters. If exposure to air occurs, and the starter does not smell like it has been contaminated by the exposure, then the starter can still be used.
* Improperly dried ''Lactobacillus'' can lead to a thick sedimentation in starter media. See [https://www.facebook.com/groups/MilkTheFunk/permalink/1979622492065955/?comment_id=1981516635209874&comment_tracking=%7B%22tn%22%3A%22R%22%7D this advice from Mark Riester].
* For information on mixed culture starters, see [[Mixed_Cultures#Starters_and_Other_Manufacturer_Tips|Mixed Culture Starters]].
[[File:BootlegBiologyLactoStarter.jpg|thumb|300px|Visual comparison of ''Lactobacillus'' growth under CaCO3 and no CaCO3; photo provided by [http://bootlegbiology.com/ Jeff Mello of Bootleg Biology]. See [https://www.facebook.com/BootlegBiology/photos/a.148869931970401.1073741829.124634287727299/452222674968457/?type=3 this page] for details about this image.]]
The recipe for this starter wort is: '''1.040 SG (10°P) Dried Malt Extract wort with 10% apple juice + 20 1.5-2 grams of chalk (CaCO3) per liter + yeast nutrients'''(originally, Aeschlimann recommended 20 grams of chalk, but we now recommend a much smaller amount of chalk; see below for details). This starter wort might be as effective without the 10% apple juice addition, but this has not been tested as far as we know. Regarding the use of chalk, it is the preferred buffer because it does not react is relatively nonreactive with CO2 (unlike CO<sub>2</sub> compared to something like baking soda), so it won't be consumed by exposure to air or due to CO2 CO<sub>2</sub> production by the Lacto''Lactobacillus''. It also has a pKa (maximum buffering capacity) of around 4.6, which is ideal for ''Lactobacillus'' growth. The fact that it easily precipitates out also makes it ideal to use as a buffer <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1180630378631841/?comment_id=1181674265194119&reply_comment_id=1181743348520544&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Bryan of Sui Generis Blog regarding the use of chalk as a buffer in Lacto starters. 11/20/2015.]</ref>. Jeff Mello from [[Bootleg Biology]] and , Nick Impellitteri from [[The Yeast Bay]], and Bryan from [https://suigenerisbrewing.blogspot.com/ Sui Generis blog] suggest that using the smaller amount of 1.5-2 grams of CaCO3 per liter is preferable because that amount is easier to precipitate out of the starter and avoid pitching into the beer (the growth differences from using less chalk has not been tested though) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1369904163037794/?comment_id=1370329352995275&reply_comment_id=1372184639476413&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Jeff Mello on MTF regarding using less chalk in LAB starters. 08/10/2016.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1619935741367967/?comment_id=1619986154696259&reply_comment_id=1619991214695753&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Impellitteri, Nick. Milk The Funk Facebook group. 03/19/2017.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1457469187614624/?comment_id=1457541770940699&reply_comment_id=1457620514266158&comment_tracking=%7B%22tn%22%3A%22R%22%7D Bryan from Sui Generis. MTF Thread on using 1.5g/L of chalk for Lactobacillus starters. 11/02/2016.]</ref>. To create a 1 liter starter for 20 liters of wort, follow these directions:
# Add 100 grams of DME to around 900 mL of water and heat pasteurize/boil as you would normally do for a starter. This should make 1.040 SG (10°P) starter wort.
# Cool the DME wort to the desired incubation temperature (see step 4), and add 100 mL of pasteurized apple juice, 20 1.5-2 grams of chalk (CaCO3), and half a teaspoon of yeast nutrients. The chalk won't dissolve into solution, so don't worry about it. <ref name="sam_starter">[https://eurekabrewing.wordpress.com/2015/05/18/evaluate-starter-media-to-propagate-lactobacillus-sp/ Evaluate starter media to propagate Lactobacillus sp., Eureka Brewing Blog, by Samuel Aeschlimann.]</ref>. Boiling the apple juice might destroy some of the nutrients in the apple juice that assist ''Lactobacillus'' in its growth, and since it is pasteurized boiling it is not necessary <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1152787381416141/?comment_id=1153930734635139&reply_comment_id=1153949097966636&total_comments=4&comment_tracking=%7B%22tn%22%3A%22R6%22%7D Conversation with Samuel Aeschlimann and Jason Pappas on MTF about the effects of boiling apple juice. 09/24/2015.]</ref>. # Best practice is that starters should not be aerated, although there may be an exception to this for ''L. brevis'' <ref name="brevis_aeration"></ref>. Some people prefer to stir their starter with an airlock in order to keep the bacteria in suspension, others do not use a stir plate and keep the starter still. Agitation might improve growth rates by dispersing waste and nutrients equally <ref name="bryan_agitation" />. One advantage to not using a stir plate at least until the brewer is more familiar with their culture is that the top of the starter will begin to clear when the starter is done if the starter was kept still.
# The starter should be held at the temperature best suited for the culture as shown in the [[Lactobacillus#Culture_Charts|Culture Charts]].
# Reference the above [[Lactobacillus#Culture_Charts|Culture Charts]] for how long the starter should be incubated for before pitching(24-48 hours is a general rule of thumb). If a stir plate is not used, one indication that the starter is done will be when the top of the starter begins to clear (turbidity is an indication that the culture is growing, and once the top portion of the starter starts to clear then that is a sign that growth has stopped) <ref name="Sam_starter2">[https://www.facebook.com/groups/MilkTheFunk/permalink/1131778916850321/?comment_id=1131806746847538&offset=0&total_comments=6&comment_tracking=%7B%22tn%22%3A%22R2%22%7D Conversation with Sam Aeschlimann of Eureka Brewing Blog on MTF. 08/20/2015.]</ref>.
# The chalk is not desirable to pitch into the beer because of its buffering effect. The chalk will sediment within hours of being added to the starter, or if a stir plate is used, a couple of hours after the stir plate is turned off <ref>[https://en.wikipedia.org/wiki/Stokes%27_law Stokes' law. Wikipedia. retrieved 09/24/2015.]</ref><ref name="Sam_starter2"></ref>. The ''Lactobacillus'' should stay in suspension for at least a day or two after the starter is done, so swirling the starter isn't necessary, although it is certainly an option. If the starter is swirled, allow a couple of hours for the chalk to sediment out again. After the chalk sediments to the bottom of the flask, pour all of the liquid from the top of the starter into the wort/beer, and leave the chalk sediment behind. Avoid cold crashing the starter because it can have an adverse effect on the bacteria's health <ref name="bryan_lacto_starters">[http://suigenerisbrewing.blogspot.ca/2015/05/lacto-starters.html "Lacto Starters." Bryan from Sui Generis Blog. Retrieved 6/15/2015.]</ref><ref name="sam_starter"></ref>.
======Notes On Safety======It is well documented that many pathogens can grow in wort when the pH is above 4.5 and ethanol is not present or very low. While it may be possible for pathogens to grow in the environment created by adding chalk to the starter, the chances of this are very low. There are a few reasons for this low risk. Firstly, typical brewing sanitation regimes and the use of commercial pure cultures of ''Lactobacillus'' should prevent unwanted microbes from contaminating the starter media. Additional steps can be taken with [[Quality Assurance|quality assurance]] to ensure the purity of the starter. Secondly, once the starter is added to wort and the wort drops below a pH of 4.6, any contaminating microbes will be killed. In the case of [[Wort_Souring#Souring_in_the_Boiler_.28Kettle_Sour.29|kettle souring]], any contaminating pathogens will be killed during the second boiling step. Finally, yeast fermentation will ensure that pathogens are not able to survive once the pH levels drop below 4.6 and ethanol is produced. For example, a similar pattern of pathogenic bacteria being killed by yeast growth and/or lactic acid bacteria growth can be seen in [[Spontaneous_Fermentation#Microbial_Succession_During_Fermentation|spontaneous fermentation]] where enteric bacteria are often inherently present during the early stages of fermentation, but are quickly killed as the pH drops and ethanol levels rise. Storing the growth media anaerobically with a pH above 4.5 for more than 3-4 days could result in a very small risk of botulism growth. Therefore, if the starter is going to be stored anaerobically for more than a few days, a target pH under 4.6 should be achieved to prevent the growth of botulism or other pathogenic contaminants. Additionally, the starter should be stored cold to further inhibit the growth of most potential contaminating microorganism species. For more information on the general risks of pathogens in beer/wort/starters, see the [[Wild_Yeast_Isolation#Safety|Wild Yeast Isolation Safety wiki page]], the [[Mold|Mold wiki page]], [https://suigenerisbrewing.com/index.php/2017/01/05/fact-of-fiction-can-pathogens-survive-in-beer-the-rdwhahb-edition/ this Sui Generis Blog post], and [https://beerandwinejournal.com/botulism/ this article on the small risk of botulism in wort that is stored for more than a few days by Chris Colby]. ======Modified Versions======* Microbiologist Dr. Matt Humbard uses a modified version of the above recipe: combine 10% apple juice (no preservative) with 1.010 SG wort and a tablespoon of calcium carbonate for every gallon. Grow for 2 days at ~90°F, and then cool to refrigeration temperature and store it cold until ready to use (pitching cold is fine). To maintain the culture long term, every ~4 weeks stir the culture and take 10-15% of the liquid culture and add it to a new batch of starter media, and grow the new starter for 2 days as previously instructed. As long as yeast does not contaminate the process, the lactic acid bacteria can be maintained this way indefinitely <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3154764734551719/?comment_id=3154792411215618&reply_comment_id=3154838211211038 Dr. Matt Humbard. Milk The Funk Facebook post on maintaining a lactic acid bacteria culture. 12/25/2019.]</ref>.* Nick Impellitteri from [[The Yeast Bay]] shared his formulation for a 1 liter formulation: 25 g Dextrose, 10 g Fermaid O, 2.5 g CaCO3, 100 mL apple juice (no preservatives), 20 mL tomato juice (no preservatives), 1 mL tween 80; QS with DI water to 1L; pH ~6.2-6.3 <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3360639773964213/?comment_id=3360874993940691 Nick Impellitteri. Milk The Funk Facebook group post on his Lactobacillus growth media formulation. 03/20/2020.]</ref>. See also:* [http://suigenerisbrewing.blogspot.ca/2015/05/lacto-starters.html ''Lacto Starters'', by Bryan of Sui Generis blog] for additional information on ''Lactobacillus'' starters.* For media for growing from stocks, see [[Laboratory_Techniques#Lactobacillus.2FPediococcus|Laboratory Techniques]].
====Cell Growth====
<blockquote>"I typically grow it by itself anaerobically in [http://www.neogen.com/Acumedia/pdf/ProdInfo/7406_PI.pdf MRS media]. Seems to work very well and results in good growth. I've personally had the best success with MRS media (only for growing from frozen stocks; MRS is not food grade and the fermentation byproduct should be decanted or pelleted; MRS should not be used for large scale propagation) and in an anaerobic environment, though I know some ''Lactobacillus'' strains grow aerobically just fine. The problem with growing lactic acid bacteria is the acid they produce will eventually inhibit their own growth. MRS contains a buffer to help combat the drop in pH as a result of LAB metabolism, which keeps the pH around 6-6.5 (I think) for optimal growth. I usually grow them at 35 C, but sometimes incubator space is at a premium (like right now) and I just [use a stir plate with an airlock]" <ref>[https://wwwdrive.facebookgoogle.com/groups/MilkTheFunk/permalink/1031115430250004/open?comment_idid=1031228363572044&offset=0&total_comments=24 1UzqB-Bq4K_4q0rHzYXntJwiIp6XInuWBZLKueYJW8dE Conversation with Nick Impellitteri on Milk The Funk Facebook group. 3/5/2015.]</ref>. - Nick Impellitteri from [[The Yeast Bay]] on general Lactobacillus cell growth</blockquote>
Maximum cell densities of ''Pediococcus'' and ''Lactobacillus'' are around 1-9 billion cells/mL, depending on the available nutrients (amino acids and FAN) in the growth media <ref name="Peyer">[http://www.asbcnet.org/publications/journal/vol/2015/Pages/ASBCJ-2015-0811-01.aspx Growth Study, Metabolite Development, and Organoleptic Profile of a Malt-Based Substrate Fermented by Lactic Acid Bacteria. Lorenzo C. Peyer, Emanuele Zannini, Fritz Jacob, and Elke K. Arendt. 2015.]</ref><ref>[https://www.reddit.com/r/Homebrewing/comments/3qp7b7/advanced_brewers_round_table_neva_parker_white/cwh7iqq Neva Parker, Reddit thread. 10/29/2015.]</ref>. Cell growth rates concur with a drop in pH and a rise in [[Titratable_Acidity|titratable acidity]]. Brewer's wort has shown to be a nutritionally adequate growth medium for ''Lactobacillus''. Both growth and the the lowering of pH begin to stabilize around 12-48 hours (assuming the ''Lactobacillus'' does not have any yeast to compete with). Titratable acidity will also rise drastically during growth, but will also continue to rise after growth has completed. The maximum growth that a particular species or strain is capable of might be explained by its pH tolerance, and thus its ability to produce more acid. For example, ''L. plantarum'' has been shown to grow in a very low pH environment (3.37-3.0 pH, depending on strain) due to their ability to better control large pH gradients between the cytoplasma and the external environment. This has been shown in the [[Lactobacillus#Commercially_available_Lactobacillus_strains_and_their_pH_change_over_time|above data provided by Matt Humbard]], as well as this reference <ref name="Peyer"></ref>. Thomas Hübbe's masters thesis showed that a strain of ''L. brevis'' had a spike of growth after 50 hours, and then a small dip in cell count after 96 hours, at which time the cell count remained consistent for at least 528 hours <ref name="Hubbe"></ref>.
All [https://en.wikipedia.org/wiki/Prokaryote prokaryotes], which includes all bacteria, are categorized based on the levels of oxygen in their environment in which they can grow and how they utilize oxygen if at all <ref name="Todar_nutgro4"></ref>. ''Lactobacillus'' species are usually considered to be "facultative anaerobes" (or "facultative aerobes") <ref name="todar_lactics4"></ref>, however, they are a special case. Facultative anaerobes usually make energy from oxygen if it is present via the [https://en.wikipedia.org/wiki/Oxidative_phosphorylation oxidative phosphorylation pathway], but otherwise engage in anaerobic fermentation <ref>[http://www.ncbi.nlm.nih.gov/books/NBK21208/ Biochemistry. 5th edition. Berg JM, Tymoczko JL, Stryer L. 2002. Chapter 18.]</ref><ref>[http://inst.bact.wisc.edu/inst/index.php?module=book&type=Effects of Oxygen=user&func=displayarticle&aid=111 Virtual Microbiology Textbook. Department of Bacteriology, University of Wisconsin-Madison. Retrieved 12/02/2015.]</ref>. ''Lactobacillus'' species can utilize oxygen, but not through the oxidative phosphorylation pathway. They use an alternative pathway instead. This pathway uses flavine-containing oxidases and peroxidases to carry out the oxidation of NADH2 using O2 <ref name="bergey">Bergey's Manual of Systematic Bacteriology, 2nd edition. pg 471</ref><ref>Correspondence with Bryan of Sui Generis Blog from Dan Pixley. 12/01/2015.</ref>. Lactobacilli, therefore, are unique in that they blur the line between facultative anaerobes and another class of prokaryotes known as "aerotolerant anaerobes". Aerotolerant anaerobes do not use oxygen to generate energy but can grow in the presence of oxygen.
====Hop Tolerance====
There are some other options; I've purified (but didn't keep - doh) some pretty resistant strains from grain by by making plates where you half-fill a plate, on an angle, with a high-IBU wort, and then overlay that with a no-IBU wort. This gives you a gradient plate, with low-IBUs on the end where the hopped-wort layer is thinnest and high IBUs where it is thickest. Some of those strains were resistant to over 30IBU, but being early in my yeast farming days I didn't bother keeping those <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1002795743081973/?comment_id=1003625646332316&offset=0&total_comments=16&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation 1 with Bryan of Sui Generis Blog on Milk The Funk regarding Lactobacillus hop tolerance. 01/19/2015.]</ref>.
Hops contain multiple compounds which are bacteriostatic. Alpha acids are the best understood, but other compounds such as beta acids, a number of polyphenols (e.g. xanthohumol), and even some of the aromatic oils (e.g. humulene) have been found to have some inhibitory effects on ''lactobacilli''. The later compounds (especially the beta acids) are why aged hops retain inhibitory characteristics, despite being nearly devoid of alpha acids. In all cases these compounds appear to inhibit the bacteria in the same way - all of these compounds contain fairly large, flat-ish, hydrophobic regions. These regions do not "like" to be in water, and thus will be driven into the hydrophobic core of the bacterial plasma membrane. This opens minute holes in the membrane which prevents the bacteria from maintaining ion (in particular, proton) gradients, leading to suppression of growth and even death of the bacteria.
Hop resistance is generally due to the induced expression of "multi-drug transport" (MDT) genes, which are "pumps" that recognize the general chemical signature of membrane-disruptive compounds, and then pump them out of the cell. Other mechanisms may also be involved - a few papers have identified changes in the lipid make-up of the plasma membrane, which may increase stability. This change also occurs in response to alcohol (to improve stability), so its not clear if that particular change has anything to do with hop resistance.
</blockquote>
Hop tolerance is not only species dependent, but is also strain dependent. For example, a dissertation by F.J. Methner measured the pH drop of wort that started at a pH of 5.55 from day 3 to day 14 for several strains of ''L. brevis'' at different IBU levels (7,9,11,13 and 18 IBU's). One strain of ''L. brevis'' eventually got down to a pH of 3.8 at day 14 with 7 IBU's, while another strain got down to 3.3 pH at day 14 (with other strains in-between those numbers). At 18 IBU, the relatively hop intolerant ''L. brevis'' strain got down to only 4.2 pH, while another strain got down to 3.7. In general, the higher the IBU, the slower the pH drop. Interestingly, another species called ''L. coryniformis'' was shown to be more hop tolerant than ''L. brevis''. ''L. coryniformis'' dropped the 18 IBU wort down to 3.6 pH over 14 days <ref name="Methner">[https://www.facebook.com/groups/MilkTheFunk/permalink/1537381402956735/ Methner, F.D. Uber Die Aromabildung beim berliner weissebier unter besonderer berucksichtigung von sauren and estern (data reported and translated by Benedikt Rausch Koch on Milk THe Funk Facebook group). 1987.]</ref>.
Methner's data is shown below; graphs created by Benedikt Rausch Koch <ref name="Methner" />. Y axis = pH, X axis = days.
<gallery>
File:Methner 18IBU7IBU.JPG|'''7 IBU''' File:Methner 18IBU9IBU.JPG|'''9 IBU''' File:Methner 18IBU11IBU.JPG|'''11 IBU''' File:Methner 18IBU13IBU.JPG|'''13 IBU'''
File:Methner 18IBU.JPG|'''18 IBU'''
</gallery>
See also:
* [[Hops#Antimicrobial_Properties|Hops antimicrobial propertiesAntimicrobial Properties]] and [[Hops#Inhibiting_Lactic_Acid_Bacteria|Dry Hopping Inhibits ''Lactobacillus'']].
* [http://www.garshol.priv.no/blog/337.html How hops prevent infection, by Lars Garshol].
* [https://www.youtube.com/watch?v=J2g5P7ZlGn4 Per Buer's Video Demonstration of how dry hopping inhibits ''Lactobacillus''.]
====Storage====
Major microbe labs will often store bacteria in a -80°C laboratory freezer in a media/glycerol solution (any standard media and 20-50% glycerol <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2710723275622536/?comment_id=2712853618742835&comment_tracking=%7B%22tn%22%3A%22R%22%7D Dr. Bryan Heit, Shawn Savuto, and Dr. Matt Humbard. Milk The Funk Facebook post about storing bacteria with glycerol. 06/08/2019.]</ref>), however, this option is generally not practical for brewers. For dried ''Lactobacillus'', such as probiotics or [[Dry Yeast for Sour Ales BlackManYeast]] products, [http://suigenerisbrewing.blogspot.com/ Bryan of Sui Generis Blog's] states that lab studies have shown that they can lose viability ~80 times faster at room temperature than when stored at refrigeration temperatures. Therefore, it is recommended to store dried ''Lactobacillus'' at refrigeration temperatures. Short term storage of liquid cultures (less than 2 months) should also be stored refrigerated. Consider making a starter before using a culture that is not fresh.
Liquid cultures become stressed by two factors: storage in an acidic environment, and storage without sugar <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1154970097864536/?comment_id=1156656834362529&offset=0&total_comments=12&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Bryan of Sui Generis Blog on stress factors in storing Lacto. 09/28/2015.]</ref>. Sugar storage creates more acid as the ''Lactobacillus'' ferments it, so it may not be ideal unless the ''Lactobacillus'' is continually fed. Ideally for longer term storage, liquid cultures of ''Lactobacillus'' should be stored frozen with 20% glycerol, or refrigerated as slants with water or mineral oil. Also, there is anecdotal evidence that certain species may survive better at room temperature. Bryan hypothesizes that stable temperatures may be more important than storing at an "ideal" temperature <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1067614393266774/ Conversation with Bryan of Sui Generis Blog on Milk The Funk. 05/04/2015.]</ref>. For instructions on how to make slants at home capable of storing any microbe for potentially 2+ years, [http://suigenerisbrewing.blogspot.com/2015/11/easy-home-yeast-banking-and-video.html see Bryan video on Sui Generis Brewing (requires a pressure cooker)].
A practical option for brewers without a pressure cooker is to store the liquid culture with a few grams of a buffering chemical such as calcium carbonate (chalk), potassium phosphate, calcium sulfate (gypsum), or calcium hydroxide (pickling lime). The exact amounts should be adjusted to reach a pH of about 4.0-6.0 for the entire solution (begin with 1 or 2 grams per liter, and adjust as needed) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1095449350483278/?comment_id=1095492120479001&offset=0&total_comments=23&comment_tracking=%7B%22tn%22%3A%22R6%22%7D Conversation with Adi Hastings on MTF. 6/20/2015.]</ref>.
Tom Belgrano offers these additional steps in order to remove the residual sugars from a storage solution, as well as raise the pH <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1154970097864536/?comment_id=1155560504472162&offset=0&total_comments=12&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Tom Belgrano on MTF regarding storing Lacto. 09/28/2015.]</ref>:
Commercial brewers who are attempting to re-use pure ''Lactobacillus'' cultures for kettle souring will often pull a portion of the soured wort before boiling it. The soured wort with living ''Lactobacillus'' is stored for later use in future batches. Cold storage is probably preferred. This can be difficult to accomplish because the residual sugars in the wort can easily attract a yeast contamination or other contaminations (see [[Lactobacillus#100.25_Lactobacillus_Fermentation|100% Lactobacillus fermentation]]) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1457254734302736/?comment_id=1457291207632422&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Bryan of Sui Generis blog on storing Lactobacillus in wort. 11/02/2016.]</ref>. Storing buffered wort above 4.5 pH anaerobically could provide the potential for botulism toxin formation (see [http://beerandwinejournal.com/botulism/ this Beer and Wine Journal article by Dr. Chris Colby]). Autoclaving, pressure cooking, or [https://en.wikipedia.org/wiki/Tyndallization tyndallizing] the wort before adding the ''Lactobacillus'' culture to it would provide a sterile media free of potential botulism spores <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1447181778643365/?comment_id=1447997755228434&reply_comment_id=1457273457634197&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation with Logan Blancett regarding tyndallization and botulism toxin. 11/02/2016.]</ref>, but may not be a practical process for the brewer. It might be possible to achieve a pH of 4.0-4.5 using CaCO3 (chalk) to buffer the pH in a range that extends the viability of the ''Lactobacillus'', and also makes the wort safe for storage (this needs more data) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1415038668524343/ Sharing Dr. Roy Ventullo's experiment results comparing Samuel Aeschlimann's "Eureka" Lactobacillus starter versus traditional MRS media on MTF. 09/23/2016.]</ref> (amounts of CaCO3 needed). Using chalk as a buffer would be similar to using the [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Eureka starter method]] above; the chalk should settle to the bottom of the vessel, and can be decanted off of.
See also:
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1981733585188179/ Mark Reister details some approaches to using lyoprotectants to dry ''Lactobacillus''.]
====Selecting for on Agar====
''Lactobacillus'' species can be isolated on agar plates by using a selective media called Rogosa SL. Rogosa SL agar contains ammonium citrate, sodium acetate, a relatively low pH, and acetic acid, which select for ''Lactobacillus'' while inhibiting mold and other bacteria. This medium will not inhibit yeast on it's its own, however. If inhibiting yeast is a requirement then add 10 mg/L cycloheximide to inhibit most yeast <ref>[https://web.archive.org/web/20160508021144/http://basementbrewlab.com/lab/lab-media/sla/ Rogosa SL Agar (Lactobacillus). Basement Brew Lab. Dustin MetzgerMichael. Retrieved 12/23/2015.]</ref>.
* See [https://web.archive.org/web/20160508021144/http://basementbrewlab.com/lab/lab-media/sla/ Rogosa SL Agar (Lactobacillus); Basement Brew Lab, by Dustin Metzger] for more information.
====Effects on Mixed Fermentation====
== Commercially available Lactobacillus strains and their pH change over time ==
All data provided by [http://phdinbeer.com/2015/08/05/beer-microbiology-lactobacillus-ph-expeirment/ Matt Humbard]. Similar results were reported by Lance Shaner's [[Lactobacillus_Fermentation|100% Lactobacillus Fermentation]] experiment. See also the associated [https://byo.com/article/brewing-with-lactobacillus/ write up in BYO Magazine].
=== pH change at 86°F ===
All metabolism by Lactobacillus, including growth, will require sugar to be consumed and lactate (lactic acid) to be produced. Two categories of metabolism exist, '''homolactic''' and '''heterolactic'''. In summary, homolactic fermentation produces only lactic acid, while heterolactic fermentation produce lactic acid, CO2, and ethanol/acetic acid <ref name="Todar">[http://textbookofbacteriology.net/lactics_2.html Todar's Online Texbook of Bacteriology. Kenneth Todar, PhD. Retrieved 05/06/2015.]</ref>.
See also:
* [https://www.facebook.com/groups/MilkTheFunk/posts/5589875397707295/?comment_id=5590160227678812 Richard Preiss and Dr. Bryan Heit's layman explanation of ''L. plantarum'' having an extracellular electron transport chain.]
====Homolactic====
====Heterolactic====
Heterolactic metabolism is described as the cell catabolizing one molecule of glucose into one molecule of CO2CO<sub>2</sub>, one molecule of glyceraldehyde phosphate, and one molecule of acetyl phosphate. The molecule of glyceraldehyde phosphate is reduced to one molecule of lactate, and the acetyl phosphate is reduced to one molecule of ethanol (or one molecule of acetic acid instead of ethanol, depending on its growing environment <ref name="Raunak">[https://raunakms.wordpress.com/2011/01/30/lactic-acid-bacteria/ Lactic Acid Bacteria. Raunak Shrestha. Retrieved 6/7/2015.]</ref>). Heterolactic fermentation allows the fermentation of hexoses and pentoses <ref>[https://books.google.com/books?id=eZjIfud742wC&pg=PA33&lpg=PA33&dq=facultative+heterofermentative&source=bl&ots=QQYpzpsrvC&sig=kkyP7wUjgWiE2UV2qkIaRyxMMGA&hl=en&sa=X&ei=K_d0VYDJPLX9sATv_IXgAQ&ved=0CDIQ6AEwBDgK#v=onepage&q=facultative%20heterofermentative&f=false Handbook of Dough Fermentations. Karel Kulp, Klaus Lorenz. CRC Press, May 20, 2003. Pg 33.]</ref>. Heterolactic fermentation follows the Phosphoketolase Pathway, which is a branch of the Pentose Phosphate Pathway (also called the "Phosphogluconate Pathway") <ref name="Effects on Food Properties"></ref><ref>[https://books.google.com/books?id=ZKzzCAAAQBAJ&pg=PA157&lpg=PA157&dq=is+Pentose+Phosphate+Pathway+pathway+the+same+as+phosphoketolase+pathway&source=bl&ots=5-uJY2vpKx&sig=Q-2yFtjWIXGXQnZvgvWZ66OayGc&hl=en&sa=X&ved=0ahUKEwix0fmi64HKAhUI82MKHQMzDcsQ6AEINTAE#v=onepage&q=is%20Pentose%20Phosphate%20Pathway%20pathway%20the%20same%20as%20phosphoketolase%20pathway&f=false Understanding Bacteria. S. Srivastava. 2013. Pg 157.]</ref><ref>[http://textbookofbacteriology.net/metabolism_3.html Todar's Online Textbook of Bacteriology. Diversity of Metabolism in Procaryotes (page 3). Retrieved 12/29/2015.]</ref><ref>[https://books.google.com/books?id=CKEgLmqfbRQC&pg=PA180&lpg=PA180&dq=is+Pentose+Phosphate+Pathway+pathway+the+same+as+phosphoketolase+pathway&source=bl&ots=p_01NAx1Aq&sig=wijMw9u4nBJ2oiH0JjHZfSxrgPU&hl=en&sa=X&ved=0ahUKEwix0fmi64HKAhUI82MKHQMzDcsQ6AEIMjAD#v=onepage&q=is%20Pentose%20Phosphate%20Pathway%20pathway%20the%20same%20as%20phosphoketolase%20pathway&f=false Microbiology. Daniel V. Lim. 2003. Pg 180.]</ref>. When different substrates are available to heterolactic fermenting ''Lactobacillus'', such as fructose or oxygen, acetate (acetic acid) can be produced instead of ethanol <ref name="Peyer"></ref>. It has been observed for at least one strain of ''L. brevis'', which only performs heterolactic fermentation, that the amount of CO<sub>2</sub> produced by this fermentation in brewer's wort was negligible. This is most likely due to the small amount of sugar consumed by ''Lactobacillus'' <ref name="Ciosek_2019" />. See [[Lactobacillus#100.25_Lactobacillus_Fermentation|100% ''Lactobacillus'' fermentation]] for more information.
===Categories of Lactobacillus===
! Obligatory Heterofermentative !! Facultatively Heterofermentative
|-
| L. acidophilus || L. brevis || L. casei <ref name="Toh_2013">[https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075073 Genomic Adaptation of the Lactobacillus casei Group. Toh et al. 2013.]</ref>
|-
| L. delbruekii || L. buchneri || L. curvatus
| L. lactis <ref name="fao">[http://www.fao.org/docrep/x0560e/x0560e10.htm Fermented Fruits and Vegetables. A Global Perspective. Food and Agriculture Organization of the United Nations. Chapter 5, Bacterial Fermentations. Retrieved 11/15/2015.]</ref> || L. cellobiosus <ref name="fao"></ref> || L. coryniformis <ref name="fao"></ref>
|-
| L. leichmannii <ref name="fao"></ref> || L. confusus <ref name="fao"></ref> || L. paracasei <ref name="Toh_2013" />
|-
| Streptococcus bovis <ref name="fao"></ref> || L. coprophilus <ref name="fao"></ref> ||
''Lactobacillus'' generally prefers glucose, fructose, and maltose, and does not ferment maltotriose. Some species may prefer certain types of sugars over others. For example ''L. plantarum'' ferments glucose first, and then fructose if it is available. ''L. reuteri'' ferments maltose first, while ''L. brevis'' feeds on maltose, glucose, and fructose. Disaccharides such as sucrose and maltose enter the cells through specific types of membrane transport proteins called permeases, and are broken down into monosaccharides through phosphorolysis before they enter the normal carbohydrate metabolic pathway <ref name="peyer_review"></ref>. Peak sugar consumption without competition from yeast is typically 48 hours, and very little alcohol or CO2 is produced (around 0.10-0.30% ABV, far less than the 0.5% required for non-alcoholic drinks). Consumption of sugars occurs mainly during the 48 hour growth period, but also occurs after growth has stopped. No more than 0.5-1°P worth of sugar is consumed by ''Lactobacillus''. Rather than high residual sugar concentration being the limiting factor on growth it is thought that low pH and other metabolic byproducts weaken and finally stop the growth of ''Lactobacillus'' <ref name="Peyer"></ref>. For a chart and in depth discussion on what types of sugars are fermentable by different species of ''Lactobacillus'', as well as charts on secondary metabolites, see [http://phdinbeer.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/ Matt Humbard's ''Physiology of Flavors in Beer – Lactobacillus Species'' blog article].
A small number of strains of ''Lactobacillus'' can also break down polysaccharides and starches. They are referred to as "amylolytic LAB". They generally belong to the species ''Lb. manihotivorans'', ''L. fermentum'', ''L. amylovorus'', ''L. amylophilus'', ''L. plantarum'' or ''L. amylolyticus''. This seems to be associated with a gene called "amyA", which encodes for extracellular alpha-amylase activity, as well as alpha-glucosidase, neopullulanase, amylopectin phosphorylase, and maltose phosphorylase. This activity is limited by high amounts of glucose, maltose, or sucrose <ref name="peyer_review"></ref>. Some species can also produce beta-glucosidase capable of breaking down monoglycosides (see [[Glycosides]]), or beta-galactosidase which breaks down lactose and other [https://en.wikipedia.org/wiki/Galactoside galactocides], but not diglycosides. The activity of both alpha and beta-glucosidase enzymes are stable at low pH ranges of 3-4, are generally encouraged by increasing percentages of alcohol all the way up to 12% v/v, and are optimal at 35-45°C (depending on strain) <ref>[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2005.02707.x/full Screening of Lactobacillus spp. and Pediococcus spp. for glycosidase activities that are important in oenology. A. Grimaldi, E. Bartowsky, V. Jiranek. 2005. DOI: 10.1111/j.1365-2672.2005.02707.x.]</ref><ref>[http://www.foodandnutritionjournal.org/volume8number3/effect-of-nutritional-factors-on-growth-behaviour-proteolytic-%CE%B2-glucosidase-and-%CE%B2-galactosidase-activities-of-lactobacillus-cultures-during-soy-drink-fermentation/ Effect of Nutritional Factors on Growth Behaviour, Proteolytic, β-Glucosidase and β-Galactosidase Activities of Lactobacillus Cultures during Soy-Drink Fermentation. Sujit Das, Birendra Kumar Mishra, and Subrota Hati. 2020.]</ref>. ====100% ''Lactobacillus'' Fermentation====
The amount of CO2 produced is very small in heterofermentative species. Lance Shaner's experiment on testing [[100% Lactobacillus Fermentation]] showed of Omega Yeast Labs noted that although ''L. brevis'pure cultures''' of WLP677is classified as obligatory heterofermentative, WLP672, Wyeast 5335, Wyeast 5223-PC, and the ''L. plantarum'' from human eye cannot detect any CO2 production in the Omega Yeast Lactobacillus blend (OYL-605, could not fully attenuate a 1).037 SG wort Lance still needs to test this blend to see if it produces any CO2 at all. The most attenuative There have been reliable reports of pure ''Lactobacillusbrevis'' culture, WLP677, was only able to attenuate down to 1cultures producing a layer of bubbles on the surface of wort if roused <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1354678291227048/?comment_id=1354678411227036&reply_comment_id=1355288821165995¬if_t=group_comment_reply¬if_id=1468974761019794# Conversation with Richard Preiss on MTF regarding pure Lactobacillus fermentation. 07/19/2016.03255 SG]</ref>. It is likely clear though that all species and strains any type of ''Lactobacillus'' available to brewers , regardless of whether it is heterofermentative or homofermentative, cannot fully attenuate wortproduce a krausen. In addition, this study showed at most a 0.29% ABV in 100% Krausens are sometimes seen even with the use of commercially available ''Lactobacillus'' fermentations (attributed to WLP677). See [[100% Lactobacillus Fermentation]] for more informationcultures and good sanitation techniques. If a higher attenuation krausen develops in wort when it is the only culture that is achievedpitched, this is indicative of cross -contamination of yeast is most likely ''Saccharomyces'' or ''Brettanomyces'' in either the wort or the cause''Lactobacillus'' culture itself <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1083842231643990/?comment_id=1084646124896934&offset=0&total_comments=26&comment_tracking=%7B%22tn%22%3A%22R8%22%7D Discussion with Lance Shaner on MTF. 6/7/2015.]</ref>. Thomas Hübbe's masters thesis also supports that In addition to this, heterolactic fermentation by ''Lactobacillus'' attenuates less than can only produce 10-20% of the sugars in wort ethanol that Saccharomyces can produce <ref name="HubbePhysioLacto">[http://phdinbeer.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/ Humbard, Matt. Physiology of Flavors in Beer – Lactobacillus Species. Retrieved 6/14/2015.]</ref>, therefore a high level of attenuation cannot be achieved by ''Lactobacillus'' and is again a sign of cross contamination by yeast. Take a gravity reading and if the wort gravity has dropped more than 1°P (.004 specific gravity points) then this is due to a yeast fermentation.
* See also [[Lactobacillus_Fermentation|100% Lactobacillus Fermentation]].
===Primary/Secondary Metabolites===
====Primary Metabolites====
Lactic acid is the primary metabolite for ''Lactobacillus'', as well as CO2 and ethanol/acetate (acetic acid) in heterofermentative species. Acid production is at it's highest during the exponential growth phase, but continues into the stationary and decline phases. Typically just under 50% of the lactic acid produced is L-lactic acid (more nutritionally relevant) while the slight majority is D-lactic acid <ref name="Peyer"></ref>. The amount of lactic and acetic acids produced varies from species to species. For example, the referenced study showed that ''L. plantarum'' produces more than twice the amount of lactic acid than ''L. brevis'', and ''L. reuteri'' produced slightly more lactic acid than ''L. brevis''. ''L. reuteri'' produced around twice as much acetic acid than ''L. brevis'', and ''L. plantarum'' produced very little acetic acid. The small amount of acetic acid produced by ''L. plantarum'' in this study was explained by oxygen exposure during sampling, while the obligate heterofermentative species (''L. reuteri'' and ''L. brevis'') produced acetic acid as a direct result of their heterolactic fermentation <ref name="Peyer"></ref>. Santeri Tenhovirta's master thesis where he soured wort with several species of ''Lactobacillus'' without purging the air out of the headspace, followed by fermenting it unpasteurized with US-05, reported 8 mg/ml of lactic acid in the ''L. rhamnosus'' sample, 7 mg/ml in the ''L. plantarum'' and ''L. alimentarius'' samples, and 5 mg/ml in the ''L. brevis'' and ''L. buchneri'' samples. None of the samples had significant acetic acid production, except for ''L. brevis'' which produced 0.3 mg/ml and the ''L. buchneri'' which produced 1.3 mg/ml <ref name="Tenhovirta_masters" />.
====Secondary Metabolites====
Both primary and secondary metabolites play a large role in the flavor and aroma profile of wort fermented with ''Lactobacillus''. Secondary metabolites are compounds that are not directly related to the growth of an organism, but often assist with survival <ref>[http://www.ncbi.nlm.nih.gov/pubmed/11036689 The natural functions of secondary metabolites. Demain AL, Fang A. 2000.]</ref>. These secondary metabolites are produced by the pathways mentioned above, and different strains probably regulate the enzymes involved in various pathways differently and produce different secondary metabolites <ref>Private correspondence with Richard Preiss from Dan Pixley. 12/29/2015.</ref>. Thus, different species and strains can produce a wide variety of flavors and aromas (compare this to food grade lactic acid in which none of these secondary metabolites exist). These secondary metabolite are the result of carbohydrate fermentation and amino acid metabolism <ref name="peyer_review"></ref>. Major secondary metabolites Different species can have varying ranges of flavor intensities, especially citrus flavor, which one study that soured wort and then fermented with US-05 two days later (not kettle soured) reported was higher for ''L. plantarum'', ''L. alimentarius'', ''L. brevis'', and ''L. buchneri'' versus ''L. rhamnosus''. Vinuous, malty, and sour flavors were also reported to vary in intensity based on species. ''L. buchneri'' was reported in the study to be the most vinuous, followed by (in descending order of intensity) ''L. brevis'', ''L. plantarum'', and ''L. alimentarius''. The sour flavor was reported highest for ''L. plantarum'' and ''L. alimentarius'', followed by (in descending order of intensity) ''L. brevis'', ''L. buchneri'', and finally ''L. rhamnosus''. ''L. rhamnosus'' had the most raspberry flavor, while the other species had a similar level of moderate raspberry flavor. All of the species tested had similar levels of apple flavor (moderate), acetic flavor (low), butyric (very low to absent, even though the fermenting vessels were never purged of oxygen), bitter aftertaste (low), and yeasty (low) <ref name="Tenhovirta_masters" />.
An example in-house experiment at Bell's Brewery by Timothy Lozen demonstrated similar flavor differences in wort soured with a selection of different strains: ''L. brevis'' from one study showed that Bell's Brewery, ''L. casei'' (White Labs), ''L. plantarumbuchneri'' produced significantly more diacetyl(White Labs), acetoin ''L. delbruekii'' subsp. ''bulgarius'' (yogurt-like flavorATCC #11842), and acetaldehyde than ''L. reuteriplantarum'' (Goodbelly), and ''L. brevisrossiae''(White Labs). These three compounds were associated The wort soured with dairy-related notes of "buttery", "lactic", and "yogurt" flavors identified during sensory testing <ref name="Peyer"></ref>''L. Some LAB can release these compounds through bucherni'' was the catabolism most preferred with a score of citric acid5.9, followed closely by ''L. casei'' which is found in wortscored 5. Ester production is generally insignificant3, although significant ester formation has been found during malolactic fermentation in red wines, and ethyl acetate has been found to be produced in malt based beverages <ref name="peyer_review">[http://www''L.sciencedirectbrevis'' wich scored 4.com/science/article/pii/S0924224415300625 Lactic Acid Bacteria as Sensory Biomodulators for Fermented Cereal-Based Beverages9, ''L. Lorenzo Cplantarum'' which scored 4. Peyer 6, Emanuele Zannini , Elke Kand ''L. Arendtrossiae'' which scored 4. 2016.]</ref>3. Some strains may also produce fusel alcohols and other off-flavorsThe ''L. For example the referenced study found an accumulation of the fusel alcohol n-Porponal in the sample of delbruekii''Lsubsp. reuteri'', and a small decrease of isovaleric acid coupled bulgarius'' scored lowest on preference with a small increase score of [https:1.6, and was characterized as "cheesy//enpukey and funky".wikipedia.org/wiki/Hexanoic_acid hexanoic acid] by ''L. breviscasei''was characterized as the most sour, while ''L. plantarumbuchneri'', was characterized as the most citrusy and ''L. reuteriplantarum'' (only 0.25-0.32 mg/L was found, and characterized as the flavor threshold most fruity. The different strains also produced a range of hexanoic acid is 5titratable acidity and diacetyl with ''L.4 mg/casei'' producing the most and ''L <ref>[http://www.leffingwelldelbruekii'' subsp.com/odorthre.htm Leffingwell & Associates website''bulgarius'' the lowest titratable acidity and the most diacetyl. Odor Thresholds. Retrieved 12/30/2015.]</ref>) <ref name="Peyer"></ref>''L buchneri'' produced the most alcohol at 0. Heterofermentative species can also produce [[Tetrahydropyridine|tetrahydropyridines (THP)]], which is the cause of "mousy" off-flavors 64% ABV <ref name="Costellolozen_2017">[httphttps://pubswww.acsmbaa.orgcom/meetings/archive/2017/doiproceedings/absPages/1092.1021/jf020341r Mousy Off-Flavor aspx Timothy Lozen. "A comparison of Wine: Precursors and Biosynthesis selected lactic acid bacteria for use in the production of the Causative N-Heterocycles 2-Ethyltetrahydropyridine, 2-Acetyltetrahydropyridine, sour wort and 2-Acetyl-1-pyrroline by Lactobacillus hilgardii DSM 20176beer. " Peter J. Costello and Paul A. HenschkePresentation by Bell's Brewery for the 2017 Master Brewers Conference. 20022017.]</ref>. Aldehydes (2-methyl-1-propanal, 2-methyl-1-butanal, 3-methyl-1-butanal) and their associated non-fusel alcohols (2-methyl-1-propanol, 2-methyl-1-butanol, and 3-methyl-1-butanol) can be produced from amino acids such as leucine, isoleucine, and valine to form fruity flavors <ref name="peyer_review"><See also [https://ref>www. A few species, especially most strains of ''Lmasterbrewerspodcast. fermentum'', and some strains of ''L. delbrueckii subsp. bulgaricus'', can produce ropiness in the form of exopolysaccharides, similar to [[Pediococcus]com/085 MBAA podcast #85 with Tim Lozen] <ref name="peyer_review"></ref>.
Another study showed that ''L. plantarum'' produced significantly more diacetyl, acetoin (yogurt-like flavor), and acetaldehyde than ''L. reuteri'' and ''L. brevis''. These three compounds were associated with dairy-related notes of "buttery", "lactic", and "yogurt" flavors identified during sensory testing <ref name="Peyer"></ref>. Some LAB can release these compounds through the catabolism of citric acid, which is found in wort. Ester production is generally insignificant, although significant ester formation has been found during malolactic fermentation in red wines, and ethyl acetate has been found to be produced in malt based beverages <ref name="peyer_review">[http://www.sciencedirect.com/science/article/pii/S0308814617302911#t0005 Dongmo et alS0924224415300625 Lactic Acid Bacteria as Sensory Biomodulators for Fermented Cereal-Based Beverages. Lorenzo C. (2017)] found 56 volatile flavor compoundsPeyer , including various estersEmanuele Zannini , alcohols, ketones, aldehydes, acids, ethers compounds, sulfur compounds, heterocyclic compounds, phenols, terpenes, lactones, and several unidentified compoundsElke K. Arendt. Key compounds 2016.]</ref><ref name="Tenhovirta_masters" />. Acetaldehyde produced by from ''LactobacillusL. plantarum''helps to produce pyranoanthocyanins that stabilize wine' include acetaldehyde, β-Damascenone, furaneol, phenylacetic acids red color <ref>[https://www.sciencedirect.com/science/article/pii/S0963996918302084 Acetaldehyde released by Lactobacillus plantarum enhances accumulation of pyranoanthocyanins in wine during malolactic fermentation. Shaoyang Wanga, 2-phenylethanolSiyu Lic, 4-vinylguaiacolHongfei Zhaoa, sotolonPan Gua, methionalYuqi Chena, vanillinBolin Zhanga, acetic acid, nor-furaneol, guaiacol Baoqing Zhu. 2018. https://doi.org/10.1016/j.foodres.2018.03.032]</ref>. Some strains may also produce fusel alcohols and ethyl 2other off-methylbutanoateflavors. Acetaldehyde was For example the most impactful aroma compound referenced study found followed by propanan accumulation of the fusel alcohol n-1-ol Porponal in the sample of ''L. reuteri'', and γ-dodecalactonea small decrease of isovaleric acid coupled with a small increase of [https://en.wikipedia. Acetaldehyde was generally produced in much higher amounts (~23-64 µgorg/wiki/L) Hexanoic_acid hexanoic acid] by the select strains of ''L. plantarumbrevis'', while ''L. amylolyticusplantarum'' , and ''L. brevisreuteri'' produced (only 10.525-3 µg0.32 mg/L. In factwas found, and the levels flavor threshold of all of these compounds differed significantly based on the species and strainhexanoic acid is 5. The selected strains of ''4 mg/L<ref>[http://www. brevis'' were associated as having worse aromas that were dominated by methional (cooked potatoes), acetic acid (vinegar), and nor-furaneol (caramel-like)leffingwell.com/odorthre.htm Leffingwell & Associates website. Odor Thresholds. The ''LRetrieved 12/30/2015. plantarum'' strains selected were identified as producing more positive aromas from compounds such as β-damascenone (apple]</fruit juiceref>), furaneol (strawberry), 2-phenylethanol (rose<ref name="Peyer"></caramel) and ethyl 2-methylbutanoate (citrus) ref>. Small but significant amounts of linalool and geraniol were Heterofermentative species can also found, which are normally terpenes found in produce [[HopsTetrahydropyridine|hopstetrahydropyridines (THP)]]. Vanillan , which is formed from ferulic acid by some ''Lactobacillus'' species as well as ''Oenococcus oeni'' the cause of "mousy" off-flavors <ref name="DongmoCostello">[http://wwwpubs.sciencedirectacs.comorg/sciencedoi/articleabs/pii10.1021/S0308814617302911 Key volatile aroma compounds jf020341r Mousy Off-Flavor of lactic acid fermented malt based beverages – impact Wine: Precursors and Biosynthesis of lactic acid bacteria strains. Sorelle Nsogning Dongmothe Causative N-Heterocycles 2-Ethyltetrahydropyridine, Bertram Sacher2-Acetyltetrahydropyridine, Hubert Kollmannsberger, Thomas Beckerand 2-Acetyl-1-pyrroline by Lactobacillus hilgardii DSM 20176. 2017Peter J. Costello and Paul A. Henschke. doi:http://dx2002.doi.org]</10ref>.1016 Aldehydes (2-methyl-1-propanal, 2-methyl-1-butanal, 3-methyl-1-butanal) and their associated non-fusel alcohols (2-methyl-1-propanol, 2-methyl-1-butanol, and 3-methyl-1-butanol) can be produced from amino acids such as leucine, isoleucine, and valine to form fruity flavors <ref name="peyer_review"></jref>.foodchem A few species, especially most strains of ''L.2017fermentum'', and some strains of ''L.02.091delbrueckii'' subsp.''bulgaricus'', can produce ropiness in the form of exopolysaccharides, similar to [[Pediococcus]]<ref name="peyer_review"></ref>.
The type of grain that the ''Lactobacillus'' is fermented in may also play a role in the types and amounts of secondary metabolites that are produced. One study compared volatile acids produced by a probiotic strain of ''L. plantarum'' (NCIMB 8826) when fermented in oats, barley, malted barley, and wheat. In oats, there was slight increase in oleic acid and linoleic acid and a decrease when fermented in wheat, barley, or malted barley. In malted barley, there were small increases in flavor active compounds such as furfural ("almond" flavor), 2-ethoxyethyl acetate and isoamyl alcohol, but little to none detected when fermented in oats, wheat, or unmalted barley. Acetic acid production was higher in barley and malted barley than it was in oats and wheat. Many other organic acids in the oats, wheat, barley, and malted barley were supposedly taken up by the ''L. plantarum'' during fermentation. In barley, there were trace amounts of new acids created that were not already in the barley itself <ref>[http://www.sciencedirect.com/science/article/pii/S0308814609004373 Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-based substrates Ivan Salmeron, Pablo Fuciños, Dimitris Charalampopoulos, Severino S. Pandiella. 2009.]</ref>. Some species of ''Lactobacillus'', including ''L. lactis'' and ''L. plantarum'', produce diacetyl (which can be reduced to acetoin and 2,3-butanediol) as an intermediate metabolite from consuming sugar, citrate, and amino acids. However, citrate levels are rather low in malted barley (but higher in sorghum), and diacetyl production has been observed to be very low in barley and oat-based worts <ref name="peyer_review"></ref>. Aging has a large impact on the aromas and flavors produced by ''Lactobacillus'' fermentation over time and is typically influenced by the temperature of the environment, oxygen exposure, and the byproducts of fermentation. Generally, fermentation has a positive effect on preserving some aroma and flavor compounds. Other compounds may change, causing aroma and flavor changes. For example, one study characterized wort freshly fermented with ''L. plantarum'' as "butter" and honey", and when aged as "yogurt" and "sour". In the same study, ''L. reuteri'' was characterized as "sour" when fresh, and "honey" and "pungent" when aged. ''L. brevis'' was characterized as "soy sauce" when fresh, and "yeasty" and "cider" when aged <ref name="Peyer"></ref>. Many strains of ''Lactobacillus'' and other lactic acid bacteria can produce tannase, which is an enzyme that breaks down a certain class of tannins called "hydrolizable tannins" (for example, tannic acid). The enzymatic breakdown of tannins provides a food source for the ''Lactobacillus''. In the cited study, a strain of ''L. plantarum'' was selected out of 47 other tannase producing LAB as being the highest producer of this enzyme. Although the optimum pH for tannase is 5-8, it is also at least 50% active at a pH of 3-7 and a temperature of 15-30°C. Tannase has been produced as a product for removing haze in food products such as iced tea, wine, and beer <ref>[http://www.asbcnet.org/publications/journal/vol/2016/Pages/ASBCJ-2016-4298-01.aspx Purification and Characteristics of Tannase Produced by Lactic Acid Bacteria, Lactobacillus plantarum H78. Mari Matsuda, Yayoi Hirose, and Makoto Kanauchi. 2016.]</ref><ref>[http://www.beveragedaily.com/R-D/New-enzyme-aims-to-take-the-haze-out-of-iced-tea "http://www.beveragedaily.com/R-D/New-enzyme-aims-to-take-the-haze-out-of-iced-tea". Beveragedaily.com. Guy Montague-James. 04/04/2011. Retrieved 011/09/2016.]</ref>. Some ''Lactobacillus'' strains could , therefore , have a positive effect on beer clarity by breaking down some haze forming tannins <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1464383586923184/?comment_id=1465361093492100&reply_comment_id=1465496463478563&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Review of this entry by Mike Lentz via MTF. 11/10/2016.]</ref>.
See the [[Lactobacillus#100.25_Lactobacillus_Fermentation|Elke Arendt video presentation above]] on the referenced study, starting at ~14:45.
[[File:Mark Horsley Foam.jpg|thumb|300px|[https://www.facebook.com/groups/MilkTheFunk/permalink/1145033525524860/ Photo provided by Mark Horsley. Left is a 5.30 pH wort and the right is a 4.75pH wort. Both were kettle soured with ''L. delbrueckii''. Both reached 3.30 pH post ferment with finishing gravity of 1.7 Plato.]]]
A few species/strains of ''Lactobacillus'' can create all of the amino acids that they need for growth. These species are known as '''prototrophic'''. However, most species/strains can only produce some of the amino acids required for growth and must obtain the other amino acids from their environment. These species are known as '''auxotrophic''' for the amino acids that they cannot produce themselves <ref>[http://www.ncbi.nlm.nih.gov/pubmed/17993552 Phenotypic and genotypic analysis of amino acid auxotrophy in Lactobacillus helveticus CNRZ 32. Christiansen JK, Hughes JE, Welker DL, Rodríguez BT, Steele JL, Broadbent JR. 2007.]</ref>. Auxotrophic ''Lactobacillus'' can break down proteins into simpler amino acids in their environment in order to consume the amino acids that they cannot make themselves, including foam forming proteins in beer, through a process called '''proteolysis''' <ref name="Todar"></ref>. Proteolysis is the breakdown of various proteins into smaller polypeptides or amino acids through the use of various protease enzymes . This process is a large part of cheese and yogurt fermentation <ref>[https://ir.library.oregonstate.edu/xmlui/bitstream/handle/1957/26351/ShellhammerThomasH.FoodScience.TextureProteolysisViable.pdf?sequence=1 Texture, proteolysis and viable lactic acid bacteria in commercial Cheddar cheeses treated with high pressure. Cheryl Wick, Uwe Nienaber, Olga Anggraeni, Thomas H Shellhammer and Polly D Courtney. 2002. Retrieved 7/7/2015.]</ref><ref name="Haq_Mukhtar">[http://www.researchgate.net/profile/Hamid_Mukhtar4/publication/230245468_PROTEASE_BIOSYNTHESIS_FROM_LACTOBACILLUS_SPECIES_FERMENTATION_PARAMETERS_AND_KINETICS/links/00b7d52c403b158288000000.pdf Protease Biosynthesis from Lactobacillus Species: Fermentation Parameters and Kinetics. Ikram-Ul_Haq and Hamid Mukhtar. Jan 2007. Retrieved 7/7/2015.] </ref><ref>[https://en.wikipedia.org/wiki/Proteolysis Proteolysis. Wikipedia. Retrieved 7/7/2015.]</ref>. Both homofermentative and heterofermentative species have been observed to have proteolytic activity , and the degree of proteolytic activity differs from strain to strain and even more so from species to species. These differences are most likely due to differences in gene expression as well as differences in optimal conditions between strains and species <ref>[https://www.frontiersin.org/articles/10.3389/fmicb.2018.02354/full Production of Bioactive Peptides by Lactobacillus Species: From Gene to Application. Cyril Raveschot, Benoit Cudennec, François Coutte, Christophe Flahaut, Marc Fremont, Djamel Drider, and Pascal Dhulster. 2018.]</ref><ref>[https://books.google.com/books?id=qMreBwAAQBAJ&pg=PA133&lpg=PA133&dq=lactobacillus+homofermentative+proteolytic&source=bl&ots=bjxq9rGdha&sig=pCz4WeKek3zTv5oL6Rui1dUuEqw&hl=en&sa=X&ei=AJqcVeT0O8XkoAS0-oC4CQ&ved=0CF0Q6AEwBw#v=onepage&q=lactobacillus%20homofermentative%20proteolytic&f=false Brewing Microbiology. Fergus Priest. Springer Science & Business Media, Jun 29, 2013. Pg 133.]</ref>. This process is a large part of cheese and yogurt fermentation. ''Lactobacillus'' species that have been identified as breaking down proteins (mostly in cheese or yogurt) include ''Lactobacillus bulgaricus'', ''Lactobacillus rhamnosus'', ''Lactobacillus casei'', ''Lactobacillus paracasei'', ''Lactobacillus helveticus'', ''Lactobacillus delbrueckii'', ''Lactobacillus brevis'', ''Lactobacillus cellobiosus'', ''Lactobacillus fermentum'', and ''Lactobacillus plantarum'' <ref name="Haq_Mukhtar"></ref>. Results of using various species/strains appears to demonstrate that different species/strains are worse for degrading head retention proteins than others. For example, it's been reported that B.H. Meyer says that souring with ''L. delbruekii'' creates better head retention than souring with other species such as ''L. brevis''. <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1105663546128525/?comment_id=1105772812784265&offset=0&total_comments=107&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Kristen England on MTF. 7/7/2015.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1096427697052110/?comment_id=1096496983711848&offset=0&total_comments=45&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Jace Marti on MTF about L. delbruekii head retention problems. 6/21/2015.]</ref>. Different strains of the same species may also have different levels of proteolytic activity <ref>[http://www.ncbi.nlm.nih.gov/pubmed/8568030 Comparison of proteolytic activities in various lactobacilli. Sasaki M, Bosman BW, Tan PS. 1995.]</ref>.
Proteolytic activity has been shown to decrease as pH falls below 5.0 for some species of ''Lactobacillus'' <ref name="Haq_Mukhtar"></ref>. In order to combat poor head retention in beers that are soured with ''Lactobacillus'', it has been suggested by German brewing scientist, Burghard Hagen Meyer, to [[Sour_Worting#How_to_Pre-Acidify|lower the pH of the wort to 4.5-4.8]] with food grade lactic acid or phosphoric acid before pitching ''Lactobacillus'' <ref name="Gail">[https://sourbrewster.wordpress.com/2012/09/18/berliner-weisse-the-old-time-kettle-souring-technique/ ''Berliner Weisse – the old-time kettle-souring technique.'' Brewing Sour blog, by Gail Ann Williams.September 18, 2012. Retrieved 7/7/2015.]</ref><ref>[http://ingenuitybrew.blogspot.com/2013/06/berliner-weisse-test.html ''Berliner Weisse Test''. Ingenuity Brew Blog. June 4, 2013. Retrieved 7/7/2015.]</ref><ref>[http://www.themadfermentationist.com/2012/06/100-lactobacillus-berliner-weisse.html ''100% Lactobacillus Berliner Weisse.'' The Mad Fermentationist Blog, by Michael Tonsmeire. June 25, 2012. Retrieved 7/7/2015.]</ref>. Additionally, ingredients that increase head retention such as unmalted chit, malted wheat, and carafoam have been used to help combat poor head retention in beers soured by ''Lactobacillus'' <ref name="Gail"></ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1105839682777578/?comment_id=1105942362767310&offset=0&total_comments=27&comment_tracking=%7B%22tn%22%3A%22R8%22%7D Conversation with Richard Preiss on MTF. 7/7/2015.]</ref>. Professional brewer [https://www.facebook.com/groups/MilkTheFunk/permalink/1105839682777578/ Kristen England of Bent Brewstillery] tested [http://www.mbaa.com/districts/MidSouth/mash/Documents/2014%20PDF%20Hop%20Product%20Applications%20Presentation.pdf?Mobile=1&Source=%2Fdistricts%2FMidSouth%2Fmash%2F_layouts%2Fmobile%2Fview.aspx%3FList%3D85ff6384-aa05-491f-8ef3-d2e9eea713f2%26View%3Da234a10f-469b-4edd-a8a9-9a96b00750ee%26CurrentPage%3D1 Hexa Iso Hop Extract] by dosing at 4 times the recommended dosage and found that it greatly increased head retention in a Berliner Weisse (3.5% abv, pH 3.1, TA ~1, BU 5), with a minor taste difference. Kristen recommends experimenting with lower dosages to avoid too much flavor impact.
Another method that has been reported to help with head retention when [[Sour WortingWort Souring]] (ex., kettle souring) is to add a pound of DME per 5 gallons of wort during the heat pasteurization process (after the wort has been soured with ''Lactobacillus''). One could also steep specialty grains such as wheat malt, chit malt, carafoam, or carapils, and add the extract into the kettle during the heat pasteurization or boiling process. This will add back head formation proteins that were lost during the ''Lactobacillus'' fermentation <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1069228273105386/?comment_id=1069266549768225&offset=0&total_comments=24&comment_tracking=%7B%22tn%22%3A%22R8%22%7D Conversation with Gareth Young on MTF. 05/08/2015.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1137179879643558/?comment_id=1137362989625247&offset=0&total_comments=8&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Paul Finney on MTF in regards to head retention of Berliner Weisse. 08/29/2015.]</ref>.
See also [https://www.facebook.com/groups/MilkTheFunk/permalink/1069228273105386/?hc_location=ufi This discussion with Gareth Young on MTF].
[[File:Hexa Iso Foam Test.jpg|none|thumb|500px|Kristen England of Bent Brewstillery's Hexa Iso Hop Extract results (Hop Iso added to the left beer, nothing added to the right)]]
See also:
* [https://www.masterbrewerspodcast.com/215 John Paul Maye on the MBAA podcast explaining tetra and hexa hop extract products.]
===Bacteriocins===
While most species of ''Lactobacillus'' do not produce bacteriocins, many strains of ''L. acidophilus'' are well known for being able to produce bacteriocins, including probiotics and yogurt strains. Bacteriocins are similar to the [[Saccharomyces#Killer_Wine_Yeast|toxins that some wine yeast strains produce]]; however, bacteriocins are toxins target other bacteria. The bacteriocin that ''L. acidophilus'' produces is a narrow spectrum class II bacteriocin, '''lactacin B''' ("narrow spectrum" means that this toxin kills a very narrow range of closely related Gram-positive bacteria). Species that are susceptible to the lactacin B toxin include species that are genetically closely related to ''L. acidophilus''. These species include ''L. leichmanii'', ''L. bulgaricus'', ''L. delbruekii'', ''L. lactis'', and ''L. helveticus''. Species that are insensitive to the toxin because they are more distantly related genetically are ''L. plantarum'', ''L. casei'', ''L. viridescens'', and ''L. fermentum''. Some strains of sensitive species might be insensitive to the toxin from certain strains of ''L. acidophilus'' but sensitive to others. The toxin does not affect a wide range of bacteria, nor yeast species <ref>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC242543/ Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. S F Barefoot and T R Klaenhammer. 1983.]</ref><ref>[https://link.springer.com/article/10.1007/s12602-017-9326-2 Lack of Heterogeneity in Bacteriocin Production Across a Selection of Commercial Probiotic Products. J. W. Hegarty, C. M. Guinane, R. P. RossC. Hill, P. D. Cotter. 2017.]</ref>. Some species of ''[[Pediococcus]]'' can also create bacteriocins (see this [https://www.facebook.com/BootlegBiology/photos/a.148869931970401.1073741829.124634287727299/465185997005458/?type=1&theater Bootleg Biology Facebook post]).
Some strains of ''L. plantarum'' cultured from Tibetan yaks have been found to have varying levels of bacteriocin activity as well. These strains produced a "class II enterocin" that inhibits the growth of ''E. coli'' and ''Staphylococcus aureus''. A strain of ''Pediococcus pentocaseus'' was also found to produce this enteriocin <ref>[https://www.sciencedirect.com/science/article/pii/S0882401017314559 Antibacterial activity of Lactobacillus plantarum isolated from Tibetan yaks. Lei Wang, Hui Zhang, Mujeeb Ur Rehman, Khalid Mehmood, Xiong Jiang, Mujahid Iqbal, Xiaole Tong, Xing Gao, Jiakui Li. 2017.]</ref>. One strain of ''L. plantarum'' isolated from kombucha was found to produce a bacteriocin called ''SLG10'' which has a broad spectrum of killing power, including affecting both Gram-positive and Gram-negative bacteria and preventing biofilm formation in other bacteria. This bacteriocin works by increasing the permeability of the cell walls of competing bacteria, which causes the target cells to excrete potassium and eventually die <ref>[https://www.sciencedirect.com/science/article/pii/S0956713519305122 Isolation, purification, and structural identification of a new bacteriocin made by Lactobacillus plantarum found in conventional kombucha. Jinjin Pei, Wengang Jin, A.M.Abd El-Aty, Denis A. Baranenko, Xiaoying Gou, Hongxia Zhang, Jingzhang Geng, Lei Jiang, Dejing Chen, Tianli Yue. 2019.]</ref>.
===Gluten Reduction===
In recent years, there has been an increase in the number of people with gluten intolerance or perceived/believed gluten intolerance whose resulting gluten-free diets that are unbalanced from low fat from carbohydrates, and this has lead to research on whether or not ''Lactobacillus'' fermented grain beverages or beers can produce gluten reduced beverages. Some lactic acid bacteria produce specific peptidase enzymes during growth that break down gluten by cleaving bonds between the amino acids in gluten, similar to how lactic acid bacteria can break down head retention proteins (see [[Lactobacillus#Foam_Degradation|Foam Degradation above]]) <ref name="Bradauskiene_2019">[http://agris.fao.org/agris-search/search.do?recordID=LV2019000334 Fermentation with Lactobacillus strains for elimination of gluten in wheat (Triticum aestivum) by-products. Vijole Bradauskiene, Lina Vaiciulyte-Funk, Edita Mazoniene, Darius Cernauskas. 2019. DOI: http://doi.org/10.22616/FoodBalt.2019.029.]</ref>.
Taubman et al. published a paper in the MBAA Technical Quarterly that reported that a strain of ''L. brevis'', ''L. curvatus'', ''L. plantarum'', and ''Pediococcus pentosaceus'' that were found to reduce gluten in sourdough bread making had a similar functionality when fermenting wort. They found that these strains reduced gluten to undetectable levels in 5-7 weeks. However, they lost the ability to reduce gluten when co-fermented with yeast, probably due to competition from the yeast. The wort that was fermented with only one of the lactic acid bacteria strains and no yeast resulted in an unpleasant fermented beverage. The researchers also reported analyzing commercial sour beers and finding some with reduced levels of gluten, but did not offer an explanation on how to accomplish this <ref>[https://www.mbaa.com/publications/tq/tqPastIssues/2018/Pages/TQ-55-1-0305-01.aspx Microbial Gluten Reduction in Beer Using Lactic Acid Bacteria and Standard Process Methods. Brett F. Taubman, Stephan Sommer, Jacob Edwards, Travis Laws, Logan Hamm, and Brenton A. Frank. 2018. DOI: https://doi.org/10.1094/TQ-55-1-0305-01.]</ref><ref>[http://masterbrewerspodcast.com/094-microbial-gluten-reduction-in-beer-using-lactic-acid-bacteria-and-standard-process-methods "094: Microbial Gluten Reduction in Beer Using Lactic Acid Bacteria and Standard Process Methods". Master Brewers Association Podcast. June 2018.]</ref>. The researchers hypothesized that the cause of the off-flavors in the 100% ''Lactobacillus'' fermentations were due to oxygen and hydrogen sulfide in the headspace of the fermenters and that further experiments with purging the oxygen and hydrogen sulfide from the head space should be done, however, previous research has shown that wort fermented with only ''Lactobacillus'' does not fully attenuate which leaves ample amounts of residual sugar available for contaminants to potentially produce off-flavors (assuming they can withstand the low pH produced by the lactic acid bacteria fermentation). Performing long fermentations with only ''Lactobacillus'' are generally not recommended due to the residual sugar left by 100% ''Lactobacillus'' fermentation. For example, it is recommended to [[Wort_Souring#Souring_in_the_Boiler_.28Kettle_Sour.29|kettle sour]] within 24-48 hours in order to lower the risk of off-flavor development. Attenuation/ethanol/final gravity measurements were not reported in this study.
Another study by [http://agris.fao.org/agris-search/search.do?recordID=LV2019000334 Bradauskiene et al. (2019)] looked at the reduction of gluten by four different probiotic ''Lactobacillus'' strains. They first performed a "wet fractionation" which is a method of separating solids from the liquid wort. This is a method that has been shown to physically remove a portion of gluten from the liquid and is done by centrifuging the wheat/water mixture. Separate fractions of starch, fiber, and bran were obtained and then each separately centrifuged. The centrifuged liquid of each fraction (starch, fiber, and bran) was split into 4 different fermenters and each fermented with a different ''Lactobacillus'' strain (two strains of ''L acidophilus'', one strain of ''L. plantarum'', and one strain of ''L. brevis''). The results of the study showed that different strains reduce gluten by different amounts after 24 hours of fermentation, but overall the amount of gluten reduction was too small to achieve the 20 mg/kg of gluten that is required to label something as "gluten free". The gluten content was reduced by a significant amount in the fiber portion which was initially 7800 mg/kg and went down to 2200-2800 mg/kg depending on the strain used with the greatest reduction by one of the ''L. acidophilus'' strains. The starch portion had low gluten to begin with at 80 mg/kg and was reduced to 12-30 mg/kg. The bran fraction had around 33750 mg/kg, but the gluten reduction was not reported for it. While some strains of ''Lactobacillus'' could be used to make a starch-only containing liquid gluten-free, they were unable to achieve enough gluten reduction with the fiber and bran fractions of wheat <ref name="Bradauskiene_2019" />.
===Probiotics===
See [[Alternative Bacteria Sources]].
===Biogenic Amines===
(To do)
* [https://www.facebook.com/groups/MilkTheFunk/permalink/3278367452191446/ MTF thread].
See also:
* [[Brettanomyces#Biogenic_Amines|Biogenic amines from ''Brettanomyces'']]
* [[Spontaneous_Fermentation#Biogenic_Amines|Biogenic amines in lambic]]
==See Also==
* [[Mixed Cultures]]
* [[Mixed Fermentation]]
* [[Sour WortingWort Souring]]
* [[Scientific Publications]]
* [[Pediococcus]]
* [https://www.microbes.info/resource-topic/lactobacillus-eubacteria-microorganisms-general-microbiology Microbes.info links to species databases and microbiology resources.]
* [https://matthumbard.wordpress.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/ Physiology of Flavors in Beer – Lactobacillus Species] - Matt Humbard's overview of different species of Lactobacillus physiology, discussion on homofermentative vs heterofermentative physiology, which species can ferment different types of sugars, and secondary metabolites. Extensive data points included.
* [http://phdinbeer.com/2015/08/05/beer-microbiology-lactobacillus-ph-expeirment/ Beer Microbiology – Lactobacillus pH experiment] - Matt Humbard's experiment to determine final pH of individual ''Lactobacillus'' strains used in the graphs on this page. See also the [https://byo.com/article/brewing-with-lactobacillus/ published article in BYO Magazine].
* [http://www.fivebladesbrewing.com/lactobacillus-starter-guide/ Lactobacillus Starter Guide by Derek Springer.] - Information about starters for both pure strains, as well as culturing from grains.
* [https://eurekabrewing.wordpress.com/2015/05/18/evaluate-starter-media-to-propagate-lactobacillus-sp/ Evaluate starter media to propagate Lactobacillus sp., Eureka Brewing Blog, by Samuel Aeschlimann.] - This experiment showed that growing Lactobacillus in '''10°P DME, 10% apple juice + CaCO3 (20 g L-1) + yeast nutrients''' lead to the best growth results, and close to expensive MRS media growth results.