Changes

<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 sometimes acetic acid) [[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 ''LactoLactobacillus'' 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]].

! 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>.|-| [[Brewing Science Institute]] || L. brevis || ''Levilactobacillus brevis'' || Heterofermentative || || Shipped during log phase, so recommended to use within 2 to 3 days of receiving. Unlike yeast, BSI sells bacteria by volume, and will sell a specific 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>.|-| [[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 || ''Lactobacillus delbrueckii'' || Homofermentative || || Lactic acid bacteria producing moderate acidity and sour flavors found in Lambics, Berliner Weiss, and Sour Ales. Commercial pitches only.|- | [[Craft Cultures]] || CCYL512 || ''Levilactobacillus brevis'' || Heterofermentative || || Typically produces more lactic acid than Lactobacillus delbrueckii. Commercial pitches only.|- | [[East Coast Yeast]] || ECY32 || ''Levilactobacillus brevis'' || Heterofermentative || || Originally isolated from kefir. Bright acidity and hop-tolerant (up to 30 IBU). Fermentation temperature 60 - 80F <ref name="ecy_website">[http://www.eastcoastyeast.com/wild-stuff.html "Wild Yeast / Brettanomyces / Lactic Bacteria". East Coast Yeast website. Retrieved 04/27/2018.]</ref>. |-| [[Escarpment Laboratories]] || Lactobacillus Blend || ''Levilactobacillus brevis'' and ''Lactiplantibacillus plantarum'' || Heterofermentative/Faculatative Heterofermentative || || This blend is designed to be usable at a wide range of temperatures, and is especially suited for kettle souring/Wort Souring. We recommend pre-acidifying wort to 4.5 with lactic acid, then pitching the Lactobacillus blend in a CO2-purged kettle or fermentor at 32-42°C.|-| [[Escarpment Laboratories]] || Lactobacillus Blend 2.0 || ''Lacticaseibacillus rhamnosus'' and ''Lactiplantibacillus plantarum'' || Heterofermentative/Faculatative Heterofermentative || || This blend is a product of our ongoing research into optimizing Lactobacillus strain selection. It is a blend of our main L. plantarum strain with a strain of Lactobacillus rhamnosus. This blend has a wide temperature range ( 30ºC to 45ºC) and enhances fruit flavours in the finished beer, with tasters noting red fruit and guava aromas. It is intended for kettle/quick souring but can also be used in 0 IBU wort.|-| [[Escarpment Laboratories]] || Lactobacillus Secondary Souring Blend || ''Levilactobacillus brevis'' and ''Lacticaseibacillus paracasei'' || Heterofermentative/Faculatative Heterofermentative || || This blend of 2 hop resistant Lactobacillus strains (''L. brevis'' and ''L. paracasei'') is intended for use in long-term souring. We recommend 15 IBU or less in the first generation.|-| [[Escarpment Laboratories]] || Lactobacillus Brevis || ''Levilactobacillus brevis'' || Heterofermentative || || This strain is moderately hop-tolerant, and as such it can also be used for long-term souring of <10IBU beers. It also performs well in kettle souring/wort souring where fast and clean lactic acidity is desired. We recommend pre-acidifying wort to 4.5 with lactic acid, then pitching the Lactobacillus blend in a CO2-purged kettle or fermentor at 35-45°C.|-| [[Escarpment Laboratories]] || Lactobacillus Delbrueckii|| ''Lactobacillus Delbrueckii'' || Homofermentative || || A single strain of ''L. delbrueckii'' often used for quick souring.|-| [[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 || ''Lactobacillus delbrueckii''? <ref>[https://www.facebook.com/GigaYeast/posts/565914926872849?comment_id=567669393364069&offset=0&total_comments=1&notif_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>.|-| [[Inland Island Brewing & Consulting|Inland Island Yeast Laboratories]] || INISBC-991 || ''Levilactobacillus brevis'' || Heterofermentative || || Produces more lactic acid at higher temperatures and in low hop worts. 70-95 F Temperature Range|- | [[Inland Island Brewing & Consulting|Inland Island Yeast Laboratories]] || INISBC-992 || ''Lactobacillus delbruekii'' || Homofermentative || || Produces more lactic acid at higher temperatures and in low hop worts. 70-95 F Temperature Range|-| [[Inland Island Brewing & Consulting|Inland Island Yeast Laboratories]] || INISBC-932 || ''Limosilactobacillus fermentum'' || Heterofermentative || || |-| [[Jasper Yeast]] || JY-LPLANT || ''Lactiplantibacillus plantarum'' || Facultatively heterofermentative || || Ideal 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://jasperyeast.com/bacteria "Available Bacteria". Jasper Yeast Website.]</ref> |-| [[Jasper Yeast]] || JY-LBREV || ''Levilactobacillus brevis'' || heterofermentative || || Ideal for kettle souring. works well at 95°F-105°F. <ref name="Jasper_Lacto"/> |-| [[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&reply_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'''.|-| [[Mainiacal Yeast]] (CLOSED) || MYLP2 || ''Lactiplantibacillus plantarum'' || Facultatively heterofermentative || || Isolated 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 70-105°F <ref name="Amaral_Mainiacal" />. '''Commercial pitches only'''.|-| [[Mainiacal Yeast]] (CLOSED) || MYLD1 || ''Lactobacillus delbruekii'' || Homofermentative || || Isolated 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 a clean lactic acidity, but generally doesn't produce as much lactic acid as some other variants. It's best used co-pitched with other microbes and allowed to age. Expect this strain to be a bit lighter on the souring side leaving a tart refreshing beer. Recommended fermentation temperature is 60-85°F <ref name="Amaral_Mainiacal" />. '''Commercial pitches only'''.|-| [[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&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-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>.|-| [[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>.

| [[Brewing Science InstituteRVA Yeast Labs]] || L. delbrueckii RVA 600 || Lactobacillus delbrueckii ''Lacticaseibacillus rhamnosus'' || Homofermentative || No starter necessary per RVA || A Lactobacillus bacteria that produces a clean lactic sournessHomofermentative Lacto strain found in probiotics; sensitive to hops; does well at room temperature.

| [[White SouthYeast Labs]] (CLOSED) || WLP677 Lactobacillus 1 || L. delbrueckii Unknown || Heterofermentative <ref name="tmf_cultures">[http|| || Source://wwwSpontaneously infected beer (South Carolina).themadfermentationist.com/p/commercial-cultures.html ''Commercial Brettanomyces Best suits Light sours, Lactobacillusgose, and Pediococcus Descriptions''. The Mad Fermentationist Blog. Michael Tonsmeire. Retrieved 3/4/2015.]<farmhouse saison (medium/ref> || no stir plate, room temp ||Incubate at > 90°F and < 117°F for 5-7 days for greater lactic acid productionhigh acidity).

| [[White SouthYeast Labs]] (CLOSED) || WLP672 Lactobacillus 2 || L. brevis Unknown || Heterofermentative <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> Homofermentatative || No stir plate, room temp|| Produced by [[The Yeast Bay]]. More hop tolerant than other Lacto strains, however TYB advises to use wort with less than 10 IBU. Temperature rangeSource: 70-95°F; 80% attenuation Prickly pear fruit (this may not reflect actual attenuation of wort in a real brewery; see reference <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1031115430250004/?comment_id=1031244193570461&offset=0&total_comments=33 Conversation with Michael Soo and Nick Impellitteri on the Milk The Funk Facebook Group. 3/5/2015.]</ref>South Carolina). <ref>[http://www.theyeastbay.com/wild-yeast-Best suits strong sours, and-bacteria-products/wlp672-lactobacillus-brevis The Yeast Bay websitelambic (high acidity). Retrieved 3/2/2015.]</ref>

| [[WyeastThe Yeast Bay]] || 5335 Lactobacillus Blend || L. buchneri ''Lactiplantibacillus plantarum'', and 2 strains of ''Levilactobacillus brevis'' || Heterofermentative || 1L starter|| The Lactobacillus Blend includes three strains: ''Lactobacillus plantarum'', 1''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.020 DME sterile wortIt can be used on its own for kettle souring prior to pitching yeast to create acidity quickly, no stir plateor 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, no O2, starter at but the strain of ''L. brevis'' isolated from the Mexican craft brewery is hop tolerant up to about 15-20 IBU. Temperature: 70-90°F if possible 5. 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 85-7 days || Incubate at 90°F for 5, although if kept in the 70's it should produce good acidification in 48-7 days for greater lactic 72 hours. A major drop off of in acid productionis 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>.

| [[WyeastThe Yeast Bay]] || 5223-PC TYB282 || L. ''Lactiplantibacillus brevis '' || Heterofermentative <ref name="nick"></ref> || no stir plate, room temp is fine || Heterofermentative (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 acid, ethanol and CO2), more hop tolerantacidity (down to ~pH 3. Does well at room temperature16-3. AVAILABLE ONLY FROM JULY THROUGH SEPTEMBER 2014 (Michael Dawson from Wyeast indicated that this culture may return at some point18). Jamie Daly indicated on MTF that he got almost no sourness after 24 in unhopped wort within 36 hours at 100°F (37a temperature of ~72-77 F.8°C). He lowered The higher the temperature (up to 90°F-95°F (3290 F is what we've tested), the faster the acid production.2°C-35°C) Recommended for 36 hourskettle 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 the wort went beers down to 3.2930 pH when co-pitched with a farmhouse yeast. Thus, Jamie recommends 90°F-95°F It might create acidity at higher IBU's (32.2°C-35°CNick suggests maybe up to 30 IBU) for 60 hours for 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 however, this has not been tested yet <ref name="brevis_aeration">[httphttps://www.ncbifacebook.nlm.nih.govcom/groups/pmcMilkTheFunk/articlespermalink/PMC5471352907190232642505/ Growth Response of Lactobacillus brevis to Aeration and Organic Catalysts?comment_id=2907235245971337 Nick Impellitteri. J. R. Stamer and B. O. StoylaMilk The Funk Fcaebook group post on TYB282 hop tolerance. Appl Microbiol. Sep 1967; 15(5): 1025–103009/12/2019.]</ref>. The beer wort was not aerated, and the fermenter was flushed with CO2. These methods need verificationTemperature: 70-90 ºF.

| [[Omega Yeast White Labs]] || OYLWLP677 || ''Lactobacillus delbrueckii'' (might be misidentified <ref>[http://masterbrewerspodcast.com/085-605 lactic-acid-bacteria-case-study Tim Lozen. Master Brewers Association podcast interview on lactic acid bacteria case study. 04/23/2018.]</ref>) || L. brevis, Heterofermentative <span styleref name="text-decoration: line-through;mtf_wiki_shaner">delbrueckii[http://www.milkthefunk.com/wiki/100%25_Lactobacillus_Fermentation Milk The Funk Wiki. 100% Lactobacillus Fermentation Test by Lance Shaner.]</spanref><ref name="tmf_cultures">[http://www.themadfermentationist.com/p/commercial-cultures.html ''Commercial Brettanomyces, Lactobacillus, and plantarum blend || HeteroPediococcus Descriptions''. The Mad Fermentationist Blog. Michael Tonsmeire. Retrieved 3/4/2015.]</Hetero ref> || 1 liter starter at room temperature for 24-48 hours. no stir plate , room temp || Quick souringIncubate at > 90°F and < 117°F for 5-7 days for greater lactic acid production. Pitch into 65°FCell count: 50-90°F80 million cells/mL (1.75-2.8 billion cells in a 35 mL homebrew vial) <ref name="WL_cellcounts">Private correspondence with White Labs Customer Service and Dan Pixley. 10/29/2015. Holding temperature is not required</ref>. No Not a good strain for kettle souring, but can produce a "soft" acidity over a longer contains delbruekii period of time <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/10652682135013921212455192116026/?comment_id=10656694434612691212475888780623&offsetreply_comment_id=0&total_comments=181212476575447221&comment_tracking=%7B%22tn%22%3A%22R22R3%22%7D Conversation with Raymond Wagner of Oso Brewing Co Andrew Addkison on Milk The FunkMTF. 401/3012/20152016.]</ref>. Don't use any hops if possible. 2 White Labs claims that it is tolerant to up to 20 IBU, although growth starts to become inhibited at 15 IBU is a good target if hops must be used <ref name="WL_datasheet" /><ref>[httpshttp://www.facebookthemadfermentationist.com/groupsp/MilkTheFunkcommercial-cultures.html "Commercial Brettanomyces, Lactobacillus, and Pediococcus Descriptions; Commercial Yeast Laboratories." The Mad Fermentationist blog. Michael Tonsmeire. Retrieved 12/permalink12/10925238074424992016.]</?comment_id=1092571350771078&offset=0&total_comments=6&comment_trackingref>. Generally heat tolerant, but sours faster between 100-110°F <ref name="WL_datasheet">[http://www.whitelabs.com/sites/default/files/R%7B26D%22tn20Wild%2220Yeast%3A20and%22R120Bacteria%22%7D Conversation with Lance Shaner on MTF in regards to IBU tolerance of OYL-60520Experiments_2.pdf "R&D Wild Yeast and Bacteria Experiments". White Labs data sheet. 6Retrieved 05/1516/20152017.]</ref>.

| [[GigaYeastWhite Labs]] || GB110 WLP672 || L. delbrueckii?''Levilactobacillus brevis'' || Heterofermentative <ref name="mtf_wiki_shaner"></ref><ref name="nick">[https://www.facebook.com/GigaYeastgroups/MilkTheFunk/permalink/posts1029638267064387/565914926872849?comment_id=5676693933640691030638553631025&offset=0&total_comments=1&notif_t=feed_comment From Gigayeast, Inc. 24 Conversation with Nick Impellitteri from The Yeast Bay on the MTF Facebook, 12Group. 3/34/2014: "Appears to be L2015. delbrueckii."]</ref> || Heterofermentative || no No stir plate, room temp ||Incubate at 98°F for 48-72 hours; Produced by [[The Yeast Bay]]. More hop tolerant than other Lacto strains, however TYB advises to use low wort with less than 10 IBU wort. Sometimes referred White Labs data sheet shows that growth is inhibited to as GigaYeast's 82% at 5 IBU, and 60% at 10 IBU <ref name="Fast Acting LactoWL_datasheet"/>. This strain is hop sensitive Temperature range: 70-95°F (greatly inhibited at 110°F) <ref name="steve_smithWL_datasheet"/>. <ref>[httpshttp://www.facebooktheyeastbay.com/groupswild-yeast-and-bacteria-products/wlp672-lactobacillus-brevis The Yeast Bay website. Retrieved 3/MilkTheFunk2/permalink2015.]</1068326413195572ref> Cell count: 50-80 million cells/?comment_id=1069411906420356&offset=0&total_commentsmL (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&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation /1599 Chemical Composition of Sour Beer Resulting from Supplementation the Fermentation Medium with Steve Smith of GigaYeast on MTFMagnesium and Zinc Ions. Aneta Ciosek, Katarzyna Fulara, Olga Hrabia, Paweł Satora, and Aleksander Poreda. 2020. DOI: https://doi. 05org/0810.3390/2015biom10121599.]</ref>.

| [[RVA Yeast LabsWyeast]] || RVA 600 5335 || L. rhamnosus GG ''Lentilactobacillus buchneri'' || Homofermentative Heterofermentative <ref name="mtf_wiki_shaner"></ref> || No 1 liter starter for a 5 gallon batch of beer, 1.020 DME sterile wort, no stir plate, no O2, starter necessary at 90°F if possible 5-7 days || Homofermentative Lacto strain found Incubate at 90°F for 5-7 days for greater lactic acid production. Cell count: 1.0 x 10⁸ (100 million) cells/mL (10 billion cells in probiotics; sensitive to hops; does well at room temperaturea 100 mL homebrew pouch) <ref name="wyeast_cellcounts">[https://drive.google.com/folderview?id=0B8CshC9nxYHdZmE4MmoyLXA2WVk&usp=sharing Wyeast Specifications 2015 Retail Products. 2015.]</ref>.

| [[SouthYeast LabsWyeast]] || Lactobacillus 1 5223-PC || Unknown ''Levilactobacillus brevis'' || Heterofermentative <ref name="mtf_wiki_shaner"></ref><ref name="nick"></ref> || no stir plate, room temp is fine || SourceHeterofermentative (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 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°C-35°C) for 60 hours for 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: Spontaneously infected beer //www.ncbi.nlm.nih.gov/pmc/articles/PMC547135/ Growth Response of Lactobacillus brevis to Aeration and Organic Catalysts. J. R. Stamer and B. O. Stoyla. Appl Microbiol. Sep 1967; 15(South Carolina5): 1025–1030.]</ref>. Best suits Light soursThe beer wort was not aerated, gose, farmhouse saison and the fermenter was flushed with CO2. These methods need verification. Cell count: 1.0 x 10⁸ (medium100 million) cells/high aciditymL (10 billion cells in a 100 mL homebrew pouch)<ref name="wyeast_cellcounts"></ref>.

| [[SouthYeast LabsWyeast]] || Lactobacillus 2 4335 || Unknown ''Lactobcillus delbruekii'' || Homofermentatative Heterofermentative || || SourceThere are [https: Prickly pear fruit (South Carolina)//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 no longer offered by Wyeast. Best suits strong soursWhen asked about this product, the Wyeast customer support reported that the "4335" product was renamed to "5335" fifteen years ago, and lambic (high acidity)the "5335" and "4335" products are the same culture. It is unclear why "4335" was labeled as ''L. delbruekii'', but it is likely that it was originally misidentified <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2265479906813544/?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 disappearance of WY4335 ''L. delbruekii''. 09/04/2018.]</ref>.

''<blockquote>Lance Shaner: "Full disclosure: I own Omega Yeast Labs. Pitching at 120F is a bad idea with this blend. The bug doing most of the work in this blend is Lactobacillus plantarum. The best temp for plantarum is 80-90F95F <ref name="adi_oyl605">[https://www.facebook.com/groups/MilkTheFunk/permalink/1328002250561319/?comment_id=1329009390460605&reply_comment_id=1331204400241104&comment_tracking=%7B%22tn%22%3A%22R%22%7D Clarification on optimal OYL-605 temps by Adi hastings on MTF. 06/16/2016.]</ref>. It does not work [as well] over 100F105°F <ref name="adi_oyl605"></ref>. Also, we regularly make a 1 liter starter with the Lacto blend for faster souring. Simply pitch the contents of the pouch into 1 liter of sterile 1.040 wort and let sit for 24 hours at 70-80F 80°F before pitching (no need to stir). Adi Hastings mentioned the imperial stout we just kettle soured. We previously brewed a Berliner using the same method. At 17 hours, pH was at 3.42 and temp was 75F 75°F (original pitch temp was 85F85°F). At 40 hours, pH was 3.24, at which time we boiled. Lower pH in the Berliner compared to the stout at 17 hours likely has to do with different buffering capacities in different worts."</blockquote> This blend is very susceptible to hops. It is recommended to not use any hops when souring with this ''Lactobacillus''. If hops must be used (some commercial breweries have to use hops for legal reasons), a maximum of 2 IBU is recommended.

''A From their website: a great lactic acid bacterial strain that will add a pleasant tangy sourness. RVA 600 is a pure culture of Lactobacillus rhamnosus GG which is found in many commercial probiotic products which have been shown in clinical studies to have many beneficial effects. These are homofermentative (only produces lactic acid, no carbon dioxide or ethanol) and are hop-sensitive. For more pronounces souring add before you add your yeast. You can sour to taste then add a yeast strain to outcompete the bacteria. Again, hop sensitive so easy on them…or dry hop the heck out of it! You may also want to experiment with blending sour low hop beer with an ale strain beer. ''<ref>From RVA Yeast Lab's [http://www.rvayeastlabs.com/bacteria.html Website]</ref>.

''... the <blockquote>Malachy McKenna: "The amount of bacteria in our homebrew units should eliminate the use of a starter. Pitching on the warm side will speed up the souring but RVA 600, a pure culture of Lactobacillus rhamnosus GG, the first commercially available probiotic propagated for use in brewing, does just fine at room temp. We had originally developed RVA 600 as a mix but fell in love with the pure strain.''<ref>Discussion on Milk The Funk Facebook group with Malachy McKenna</ref>.</blockquote>

''"It is intended for secondary, so you only need to do a starter if you are doing a primary fermentation with it. DME would be the best substrate... Since its a Lacto species, you don't really want to aerate it. A slow stir-plate would be good, to keep it moving, but not much more than that."'' - Sarah Neel, White Labs, Sales and Customer Service (quoted with permission). Since WLP672 is meant for long term aging, it is not preferred to use for kettle souring/wort souring. See also [[100%25_Lactobacillus_Fermentation#History|100% ''Lactobacillus'' fermentation]].

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 without access . 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 won't want to add that much MRS media to their beer since MRS mediahas 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. MRS 09/22/2015.]</ref>. Therefore, growing ''Lactobacillus'' in a wort-based starter media is much 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 expensive 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 materials required 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 method general guideline (see below ). Note that the exact advisable 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 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, 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 DMEwort 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#External_ResourcesSamuel_Aeschlimann.27s_Starter_Procedures|External ResourcesSamuel 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 <ref name="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 additional ''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 guidesmedia. 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]]. =====Samuel Aeschlimann's Starter Procedures=====[[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.]]

Pitching ~0.5-1 liter per ~20 liters of wort (~0.75-1 gallon per barrel) of ''Lactobacillus'' starter is the general guideline. The exact advisable pitching rates of commercial cultures may differ from manufacture to manufacturer. <ref>[https://wwweurekabrewing.facebookwordpress.com/groups2015/MilkTheFunk05/permalink18/1077639885597558evaluate-starter-media-to-propagate-lactobacillus-sp/ MTF thread started by Brad Primozic. 5/29/2015.Samuel Aeschlimann from Eureka Brewing Blog]</ref>. Counting the exact number ran a set of cells can be difficult to achieve due experiments that found a DME based recipe for starter wort that produces a very high cell density similar to the small size that of bacteria cellsMRS media, so starter volumes are generally used instead when talking about ''pitching which provides optimal growth 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 Heit. 5/6/2015.]</ref>.

The recipe for this starter wort is: '''1.040 SG (10°P) Dried Malt Extract wort with 10% apple juice + 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 is relatively nonreactive with CO₂ compared to something like baking soda, so it won'Starter Procedures:t be consumed by exposure to air or due to CO₂ production by the ''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]], 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:

# Make Add 100 grams of DME to around 900 mL of water and heat pasteurize/boil as you would normally do for a 1 liter starter of '''. This should make 1.040 SG (10°P) Dried Malt Extract starter wort.# Cool the DME wort with 10% 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''' for growth results that are close to MRS media growth results (as per [https://eurekabrewing.wordpress.com/2015/05/18/evaluate-starter-media-to-propagate-lactobacillus-sp/ Samuel Aeschlimann from Eureka Brewing Blog]). 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.

# 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). # Pour all If a stir plate is not used, one indication that the starter is done will be when the top of the liquid starter begins to clear (turbidity is an indication that the culture is growing, and once the top portion of the starter into 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 wort/beerbecause of its buffering effect. The chalk will sediment within hours of being added to the starter, or if a stir plate is used, leaving a couple of hours after the sediment behindstir 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 will to sediment outagain. After the chalk sediments to the bottom of the flask, and is not desirable to pitch 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 <refname="bryan_lacto_starters">[http://suigenerisbrewing.blogspot.ca/2015/05/lacto-starters.html Heit, Bryan. "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 Heit of Sui Generis blog] for additional information on ''Lactobacillus'' starters.* For media for growing from stocks, see [[Laboratory_Techniques#Lactobacillus.2FPediococcus|Laboratory Techniques]].

''<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 ''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 spin [use a stir plate with an airlock]" <ref>[https://drive.google.com/open?id=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>.  For the influence on cell growth in mixed cultures, see [[Mixed_Cultures#Effects_of_Mixed_Cultures_on_Growth|Effects of Mixed Cultures on Growth]]. ====Effects of Oxygen==== 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 on 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=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 benchtop!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.  The important take away here is that oxygen doesn't significantly affect most ''Lactobacillus'' species. They do not care if oxygen is present in order to grow and produce energy for themselves and lactic acid for brewers. They also do not produce significant amounts of [[Butyric_Acid|butyric acid]] or [[Isovaleric_Acid|isovaleric acid]] in the presence of oxygen <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/10311154302500041182597671768445/?comment_id=10312283635720441182773928417486&offsetreply_comment_id=01183047521723460&total_commentscomment_tracking=24 %7B%22tn%22%3A%22R9%22%7D Conversation with Nick Impellitteri Dr. Bryan Heit of Sui Generis Blog on Milk The Funk Facebook groupMTF regarding butyric acid production by Lactobacillus. 311/523/2015.]</ref>. ~ Nick Impellitteri  Although it is most likely not relevant to beer brewing applications, there are a few exceptions to this in the scientific literature. For example, ''L. plantarum'', which is a facultatively heterofermentative species, is homolactic without the presence of oxygen and produces only lactic acid. After food sources have been exhausted and in the presence of oxygen, however, ''L. plantarum'' switches to heterolactic fermentation, and further converts lactic acid into acetic acid. In a lab setting the conversion of lactic acid to acetic acid only happened when glucose was no longer available (this may not be the case in wort where other limiting factors such as low pH can prevent ''L. plantarum'' from continuing their metabolic processes before all glucose is consumed, which is the case in kettle souring), and only during the stationary phase (after growth stopped). During the conversion of lactic acid to acetic acid, ''L. plantarum'' also produces hydrogen peroxide (H²0²), which is toxic to microorganisms and is thought to be a protection mechanism for ''L. plantarum'' <ref>[http://www.ncbi.nlm.nih.gov/pubmed/6480562 Physiological role of pyruvate oxidase in the aerobic metabolism of Lactobacillus plantarum. Sedewitz B, Schleifer KH, Götz F.1984.]</ref><ref name="Quatravaux_plantarum"></ref><ref name="microbewiki_plantarum">[https://microbewiki.kenyon.edu/index.php/Lactobacillus_plantarum_and_its_biological_implications Lactobacillus plantarum and its biological implications. Microbe Wiki. Retrieved 6/7/2015.]</ref><ref name="shaner_plantarum"></ref>. This same process has also been observed in one strain of ''L. brevis'' and under similar conditions of depleted nutrients and oxygen <ref>[http://aem.asm.org/content/early/2017/08/21/AEM.01659-17.abstract The oxygen-inducible conversion of lactate to acetate in heterofermentative Lactobacillus brevis ATCC367. Tingting Guo, Li Zhang, Yongping Xin, ZhenShang Xu, Huiying He, and Jian Kong. 2017.]</ref>. It is important to understand that these results were under lab settings and without any sort of brewing application considered in these studies, so they should not be assumed to be important to the brewing process. Omega Yeast Labs reports that no noticeable acetic acid is produced if the oxygen is not purged with their OYL-605 ''Lactobacillus'' blend which contains ''L. plantarum''; brewers should not aerate wort during souring but purging O2 is not required <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1222363364458542/?comment_id=1222371257791086&reply_comment_id=1225212894173589&comment_tracking=%7B%22tn%22%3A%22R%22%7D MTF conversation with Adi Hastings from Omega Yeast BayLabs on the effects of oxygen presence in OYL-605. 02/02/2016.]</ref>. This indicates that acetic acid and hydrogen peroxide production in typical brewing scenarios where little or no oxygen is present is probably not a concern. Oxygenating for yeast is recommended even if the ''Lactobacillus'' is not killed with a second boil, especially if the brewer is relying on one strain of brewers yeast to finish the fermentation. See also [[Mixed_Fermentation#Aeration|Aeration for Mixed Fermentation]] on general . In some cases, oxygen could benefit some species. For example, one strain of ''L. brevis'' has been shown to increase growth rates in the presence of oxygen <ref name="brevis_aeration"></ref>. Thomas Hübbe's master thesis showed that under lab growth media "VLB-S7-S", "NBB®-A", and MYPG + cyclohexmide, both strains of ''Lactobacillus cell '' tested (''L. brevis'' and ''L. parabrevis'') did not show growth in an aerobic chamber, but did grow in an anaerobic chamber, although this might be an effect of the growthmedia and not representative of brewing conditions <ref name="Hubbe">[https://www.facebook.com/groups/MilkTheFunk/permalink/1407620509266159/ Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016.]</ref>. See also:* [[Wort_Souring#Souring_in_the_Boiler_.28Kettle_Sour.29|Kettle souring]].

''"The following are comments by Bryan of [http://suigenerisbrewing.blogspot.com/ Sui Generis Brewing blog]: <blockquote>There is a fair bit of research into hop tolerance out there; its it's not a simple topic as a number of factors come into play to produce hop tolerance. To make things even more complicated, hop tolerance is an inducable inducible trait in many lacto ''Lactobacillus'' species - meaning that a seemingly susceptible strain can become resistant by culturing in ever-increasing doses, and a seemingly resistant strain can become susceptible after a generation or four in a hop-free media. I've been trying to generate a permanently high-alpha acid resistant lacto strain for a few months now. I've been culturing ''L. brevis'' in escalating IBU wort (starting at 10, currently at 25). Every 4th generation (1 generation = a subculture of a stationary-phase lacto culture, not as in # cell divisions) I pass it through 2 generations of an IBU-free media to try and select for strains which maintain this resistance. This seems to have worked up to ~18 IBU (update: 20-30 IBU), but past that point the resistance appears to remain inducible. I'm hoping a few more generations will provide me with a permanently tolerant strain. Update: I had made a strong (~80ibu) wort that I diluted with unhopped wort. I would grow the ' 'Lactobacillus'' for a few passages (1 passage = grow culture to completion, then dilute ~ 1:100 into fresh wort to start next passage) at a set the IBU level, then passage to a wort 3 or so IBU higher. I did 50-60 generations before I got to the high IBU levels (~3 months, 1-2 days per generation). I never did anything to determine how much of that resistance was inheritable <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1421792324515644/?comment_id=1422244054470471&reply_comment_id=1422263561135187&comment_tracking=%7B%22tn%22%3A%22R6%22%7D Conversation 3 with Bryan Heit of Sui Generis Blog on Milk The Funk regarding Lactobacillus hop tolerance. 09/30/2016.]</ref>. 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>[httphttps://suigenerisbrewingwww.blogspotfacebook.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 Brewing blogBlog 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. Lactobacilli usually don't have these MDT genes 'on', which is why a lot of strains won't do well with hops in the first batch of beer, but over time become more and more tolerant as they increase expression of the MDT's. The overall MDT expression level, in theory, determines the maximum resistance of the bacteria. In the case of my experiments, I'm looking for mutants whose MDT's are permanently stuck 'on' for the resistant strain and 'off' for the sensitive strain <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1143165635711649/?comment_id=1155715074456705&offset=0&total_comments=50&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Conversation 2 with Bryan of Sui Generis Blog on Milk The Funk regarding Lactobacillus hop tolerance. 09/28/2015.]</ref>.</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 Hop Toleranceestern (data reported and translated by Benedikt Koch on Milk THe Funk Facebook group). 1987.]</ref>.  Methner's data is shown below; graphs created by Benedikt Koch <ref name="Methner" />. Y axis = pH, X axis = days. <gallery>File:Methner 7IBU.JPG|'''7 IBU''' File:Methner 9IBU.JPG|'''9 IBU''' File:Methner 11IBU.JPG|'''11 IBU''' File:Methner 13IBU.JPG|'''13 IBU''' File:Methner 18IBU.JPG|'''18 IBU''' </gallery>::'''L70''': ''L. coryniformis''::'''L14, L18, L22, L29, L88, L92''': ''L. Brevis'' See also:* [[Hops#Antimicrobial_Properties|Hops Antimicrobial 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''.]

=====Other Plant Type Tolerance=====Other types of plants can also inhibit the growth of ''Lactobacillus''. Nadia Marlen Aasen's Master's Thesis from Norwegian University of Life Sciences reported that ''Juniperus communis''Edit: I tried to add (but facebooks aversion to white space almost defeated mecommon juniper) that Itwigs have a significant impact on the growth of ''Lactobacillus''. The species tested were ''L. plantarum'', ''L. brevis'', and ''L. buchneri''ve been trying . Individual juniper infusions in water were created using twigs, needles, ripe berries, and unripe berries, and the infusions were measured in grams of plant material per mL of water. Juniper infusions are common in farmhouse brewing, and in Norway they are referred to generate a permanently highas ''einerlog''. Juniper twigs infused in water had the most impact on the growth of the species of lactic acid bacteria tested, with ''L. plantarum'' being partially inhibited at around 0.031-alpha acid resistant lacto strain for a few months now1 g/mL. I ''ve been culturing L. brevis in escalating IBU wort (starting '' was partially inhibited at 100.5-1.0 g/mL, currently at 25)and ''L. buchneri'' was inhibited by 0.125-1 g/mL. Every 4th generation Juniper needles had a very limited impact based on the concentrations that were tested (up to 1 generation = a subculture of g/mL). Unripe berries had a stationary-phase lacto culturevery slight impact, and ripe berries did not as in # cell divisions) I pass it through 2 generations of an IBU-free media to try and select for strains which maintain this resistancehave a significant impact at all on ''Lactobacillus'' growth. This seems No further research was done to have worked upto ~18 IBU, but past determine what the components within juniper twigs are that point the resistance appears to remain inducableinhibit LAB or what their concentrations were <ref name="Østlie">[https://nmbu.brage.unit.no/nmbu-xmlui/handle/11250/2681970?show=full Nadia Marlen Aasen. I'm hoping a few more generations will provide me Growth, metabolism and beer brewing with a permanently tolerant strainkveik. Master''s Thesis. Norwegian University of Life Sciences. 2020.]</ref>.

====Alcohol and Sugar Tolerance====''There are some other option; ILactobacillus''ve purified is generally tolerant of alcohol and high levels of sugar (but didn't keep - dohalthough growth is diminished once sugar content exceeds 20% due to osmosis stressing the cell wall) some pretty resistant . In the presence of high amounts of ethanol, the alcohol tolerant strains from grain by by making plates where you half-fill a platepack their cell walls with fatty acids, which slows the fluidity of the cell membrane. The alcohol tolerance of ''Lactobacillus'' is dependent on an angleboth strain and the growth substrate, with a high-IBU wortalcohol tolerance generally higher when glucose or starch are available. In one study that looked at 31 strains of ''Lactobacillus'', all strains grew effectively at 4% ABV. All of them still grew at 10% ABV, although some strains exhibited difficulty growing effectively at 10% ABV. Growth was diminished in general at 12% ABV (and then overlay the few strains that with a no-IBU wortwere alcohol intolerant stopped growing), but most still achieved some growth. This gives you a gradient plate Eight of the strains tested were still able to exhibit significant growth at 16% ABV, with low-IBUs and in general, most strains were able to exhibit at least small growth at 16% ABV (more so on starch and glucose versus cellobiose, lactose, or xylose). In general, the end where the hoppedspecies that were less tolerant of high amounts of ethanol (10-wort layer is thinnest and high IBUs 16%) where it is thickest: ''L. Some amylovorous'' (1 out of those 4 strains were resistant to over 30IBUwas particularly intolerant), ''L. hilgardii'' (1 strain still grew at 16% ABV, but less than the others), ''L. pentosus'' (1 strain still grew on starch medium, but being early not on glucose above 10% ABV), and ''L. casei'' (most strains grew in my yeast farming days I didnmost growth media, but generally less than other species). In general, the strains of 't bother keeping those'L.brevis''and ''L. plantarum'' were more tolerant of high ABV concentrations <ref>[https://link.springer.com/article/10.1007/BF01583633 Ethanol tolerance and carbohydrate metabolism in lactobacilli. R. Shane GoldM. M. MeagherR. HutkinsT. Conway. 1992.]</ref>.

Peyer et al. (2017) examined the effects of the sugar content of brewer's wort on a strain of 'I'm hoping Lactobacillus amylovorous''. They found that the higher gravity of the wort increased the growth of the ''Lactobacillus'', and therefore also the lactic acid production. This increase was linear until the extract reached 16 Brix (1.065 SG), at which time the growth increase began to have slow down. The higher growth and more lactic acid production in higher gravity wort was probably due to increased nutrients as well as a blog post higher buffer capacity. This growth increase plateaued in wort that was 18-20 Brix, which was likely due to osmotic stress on that sometime in the next few monthscells <ref name="Peyer_2017">[http://www.asbcnet.org/publications/journal/vol/2017/Pages/ASBCJ-2017-3861-01.aspx Sour Brewing: Impact of Lactobacillus amylovorus FST2.11 on Technological and Quality Attributes of Acid Beers. Lorenzo C. Peyer, but life is nuts right now so I offer no guaranteeMartin Zarnkow, Fritz Jacob, David P. Schutter, Elke K. Arendt. 2017.]</ref>." ====Tolerance of Extreme Temperature====Most ''Lactobacillus'' species have a thermal death rate of ~151°F (66°C) after 5 minutes <ref>[https://wwwonlinelibrary.facebookwiley.com/groupsdoi/MilkTheFunkpdf/permalink10.1002/1002795743081973j.2050-0416.1957.tb02903.x SOME INVESTIGATIONSON LACTOBACILLUS INFECTION. By R.T.Dean,M.Sc.(Carllon and United Breweries,Ltd.,Melbourne,Australia). Received 11th October,1956.]</?comment_id=1003625646332316&offset=0&total_comments=16&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation ref>. Freezing without glycerol will kill most cells, but it is possible for a very small number of cold-resistant mutant cells to survive <ref>[http://fermentationnation.net/2015/11/episode-26-quality-assurance-w-jessica-davis-of-the-bruery/ Fermentation nation Podcast interview with Bryan Heit on Milk Jessica Davis, QA for The FunkBruery.]</ref> (~1:19:00 in). 01In very rare occasions, some strains have been identified as being extremely thermotolerant. For example, [https://patents.google.com/patent/US20180042256A1/en this patent] claims that a strain of ''Lactobacillus delbrueckii'' subsp. ''lactis'' can acidify milk within 2.5 hours when held at temperatures between 45-65°C (113-149°F).  In another example, [https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2672.1999.00607.x Jordan and Cogan (1999)] described the heat tolerance of three strains of ''Lactobacillus'' isolated from cheese (two strains of ''L. plantarum'' and one strain of ''L. paracasei''). When temperatures were finally able to kill the strains, they observed a non-linear death curve with either very little death for the first 15 minuets, or a tailing of the curve. For the ''L. paracasei'' strain, the culture was able to survive temperatures between 50 and 55°C for two hours. At 60°C, there was no significant cell death for 15 minutes, but after 15 minutes the culture began to slowly die, and then more quickly die after an hour of heat exposure. At 65°C, the ''L. paracasei'' strain had a fairly steep death curve for the first 10 minutes, and then a tailing curve showing a small number of cells surviving up to 25 minutes. Even at 72°C, which is typical for pasteurization in the milk industry, the death rate for this strain was slow enough to survive 15 second pasteurization methods. The two strains of ''L. plantarum'' were less heat tolerant, with death starting to occur at around 50-56°C <ref>[https://pubmed.ncbi.nlm.nih.gov/10499302/19Jordan KN, Cogan TM. Heat resistance of Lactobacillus spp. isolated from Cheddar cheese. Lett Appl Microbiol. 1999 Aug;29(2):136-40. doi: 10.1046/2015j.1365-2672.1999.00607.x. PMID: 10499302.]</ref>. Assuming the heat penetrates all surfaces, exposure to water heated to 72-80°C for 1 minute should be more than adequate for eliminating heat tolerant strains of ''Lactobacillus'', as well as any other beer contaminate species, from the brewing environment. See also:* [[Quality_Assurance#Pasteurization|Pasteurization]]* [[Barrel#Sanitizing|Barrel Sanitizing]]

Regarding dry 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'"Yes' 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, refrigerate themand 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>. In Sugar storage creates more acid as the ''Lactobacillus'' ferments it, so it may not be ideal unless the lab course I run we compare probiotics ''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 temp 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) 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>:# Cold crash the starter until the wort is clear.# Decant the liquid, and then refill it with water (distilled is recommended, but not required). # Cold crash again until the liquid falls clear.# Decant the liquid.# Store the resulting slurry at refrigeration temperatures. A slight drop in pH may still occur, but this technique should keep the storage solution well above 4.0 pH. 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 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 Michael. 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] for more information. ====Effects on Mixed Fermentation====''Lactobacillus'' can have an impact on ''S. cerevisiae'' metabolism and the flavor-contributing metabolites that it produces. A study by [https://onlinelibrary.wiley.com/doi/10.1002/jib.569 Dysvik et al. (2019)] compared beer pre-soured with ''L buchneri'' versus pre-soured with lactic acid versus lactic acid added post yeast fermentation and found that lactic acid by itself did not significantly change the amount of volatile flavor compounds produced by the yeast strain that was tested (Fermentis US-05). However, the beers soured with ''L. buchneri'' had significantly different volatile alcohols compared to the beers with just lactic acid added to them. Specifically, the beer that was pre-soured with ''L. buchneri'' ended up with less 2-methyl-1-butanol (alcohol, malty notes), 2-methyl-1-propanol (fruity/winey) and phenylethyl alcohol (rose/honey). The esters ethyl heptanoate and ethyl octanoate where highest when the ''L. buchneri'' pre-soured the wort but was left alive rather than boiled and killed. Acetic acid was also much higher in the beers soured with ''L. buchneri'' versus the beers with just lactic acid added, but the acetic acid was still below flavor threshold. Formic acid was around twice as much in the wort soured with ''L. buchneri'' versus wort with lactic acid or no lactic acid/bacteria, but the formic acid disappeared completely in all of the beers tested at bottling time and after maturation in the bottle. Although this study used a neutral ale yeast (US-05) and alcohols/esters across all samples were below flavor threshold (although combinations of different alcohols/esters under threshold can have a synergistic flavor impact), this supports anecdotal reports from brewers that adding lactic acid to beer to make a sour beer does not produce the same beer than when the souring is done with ''Lactobacillus''. It is interesting to note that both the beers with ''L buchneri'' and the beers with just lactic acid added had similarly significantly lower levels of pyruvic acid compared to the beer fermented with just yeast, as well as less haze, indicating that lactic acid alone inhibits the amount of pyruvic acid produced by the yeast as well as haze (perhaps because a lower pH reduces protein-polyphenol haze formation, or maybe the lower pH increased yeast flocculation) <ref name="Dysvik_2019">[https://onlinelibrary.wiley.com/doi/10.1002/jib.569 Pre‐fermentation with lactic acid bacteria in sour beer production. Anna Dysvik, Kristian Hovde Liland, Kristine S. Myhrer, Bjørge Westereng, Elling‐Olav Rukke, Gert de Rouck, Trude Wicklund. 2019. DOI: https://doi.org/10.1002/jib.569.]</ref>.  This study also compared two pre-soured beers with ''L buchneri'' where one was boiled and hopped after souring and the other was not boiled but instead blended with hoppy wort before fermentation (the viability of the ''L. buchneri'' was greatly reduced in the beer that was blended with hoppy wort, but not completely killed as was the case for the kettle soured beer). The beer that was blended with hoppy wort ended up having the most acetic acid (still below threshold), and the highest level of fruity tasting esters: ethyl heptanoate and ethyl octanoate, indicating that if the ''Lactobacillus'' is allowed to live then it can contribute to more complexity over time. There were no differences in any of the beers as far as ethanol production or CO₂ production, and terminal acid shock did not occur (probably because the beers were only 4% ABV and 3.6 pH as opposed to the 8.4% ABV and 3.17 pH of the beer tested in the fridge [[Saccharomyces#Fermentation_Under_Low_pH_Conditions|terminal acid shock study by Rogers et al.]]). It is important to note that the overall sensory differences reported in this study between the beers soured with ''L. buchneri'' and the beers soured with lactic acid were minor from a statistical analysis point of view <ref name="Dysvik_2019" />.  Souring wort with ''Lactobacillus'' can stall or slow a sequential yeast fermentation. This is likely due to low pH. The presence of lactic acid might change the way yeast ferments by allowing them to consume multiple types of sugars regardless of whether or not glucose is present, although it has been demonstrated that this alone is not the cause for stuck fermentations (see [[Lactic Acid]] for more information). Peyer et al. (2017) observed that growth of US- viable bacterial numbers dip off ~80x 05 was 82% at a pH of 3.51, and 53% at a pH of 3.17. Fermentation was delayed by 2-4 days (the lower the pH, the longer the start of fermentation was delayed). In a co-fermentation of ''Lactobacillus amylovorus'' and US-05, the initial growth of the ''L. amylovorus'' continued for 3 days while the US-05 was delayed. On day 7, the US-05 recovered and continued growth, and the growth of the ''Lactobacillus'' was slowed starting on day 5. This was due to the increase in ethanol from fermentation, lower pH, and the depletion of nutrients for the ''Lactobacillus''. It is also possible that the yeast benefited from the autolysis of the ''Lactobacillus'', which is speculated to have released nutrients that were made available to the yeast <ref name="Peyer_2017" />. Santeri Tenhovirta's master thesis agreed with this. Tenhovirta pitched several species of ''Lactobacillus'' for 48 hours, and then pitched Fermentis US-05. The control US-05 fermentation without any ''Lactobacillus'' started to ferment as expected after 20 hours, while the samples that were pre-acidified with ''Lactobacillus'' took around 2 days to begin yeast fermentation <ref name="Tenhovirta_masters">[https://helda.helsinki.fi/handle/10138/303018 The Effects of Lactic Acid Bacteria Species on Properties of Sour Beer. Santeri Tenhovirta; master thesis in Food Science from the University of Helsinki. 2019.]</ref>.  Ciosek et al. (2019) observed the opposite effect. A faster fermentation was achieved when pitching ''Lactobacillus brevis'' WLP672 (White Labs Inc, USA) for 1, 2, or 3 days before pitching Fermentis Safale US-05. However, the yeast reached a slightly higher final gravity after 7 days and the yeast growth was lower than when the yeast was pitched first or at the same time as the WLP672 ''L. brevis''. Interestingly, the lowest final gravity was achieved when the yeast and ''L. brevis'' were pitched at the same time. This indicates that some species of ''Lactobacillus'' can have a synergistic effect on yeast, while other species might be more antagonistic towards yeast, and that multiple stress factors such as a combination of both low pH and the presence of ethanol can be factors that prevent yeast from attenuating as well as it would have done if the pH wasn't lowered by the presence of lactic acid. Another surprising observation was made by Ciosek et al. (2019) for the samples that were fermented with US-05 first, and then after 1, 2, or 3 days the WLP672 ''L. brevis'' was added and allowed to ferment for 7 days total: these samples did not have a significant drop in pH from the ''L. brevis'', and remained at a pH of 4.0 or higher. This result indicates that this particular strain does not produce much lactic acid in nonthe presence of ethanol or there aren't enough simple sugars left after the yeast fermentation (at least in the short amount of time that they were tested). When the ''L. brevis'' was pitched first, it took 72 hours for the pH to get lower than 4, but this approach ended up with the lowest final pH after yeast fermentation was finished (~3.4 pH), and the co-pitch ended up at ~3.7 pH. The researchers declared that only when the ''L. brevis'' was allowed to ferment by itself for 2-refrigerated 3 days before the yeast was pitched did the finished beers have enough lactic acid content to be considered "sour beer" (~6 g/l of lactic acid for the samplesfermented with ''L.brevis'' first versus 1.8-2.8 g/l for all of the other samples), which is based on a definition from "American Sour Beers"by Michael Tonsmeire that states that sour beers are defined as having 3-6 g/l of lactic acid. However, no sensory data was presented in this study, nor were [[Titratable_Acidity|titratable acidity]] measurements taken, and Methner's 1987 thesis on Berliner Weisse reported 1-3 g/l of lactic acid in a survey of these German sour beers, so it is conceivable that all of the sample beers in this study had an adequate sour flavor <ref name="Ciosek_2019">[https://onlinelibrary.wiley.com/doi/pdf/10.1002/jib.590 Sour beer production: impact of pitching sequence of yeast and lactic acid bacteria. Aneta Ciosek, Iga Rusiecka, Aleksander Poreda. 2019. DOI: https://doi.org/10.1002/jib.590.]</ref><ref>[http://herr-rausch.de/MethnerBerliner.pdf Methner's thesis on Berliner Weisse, 1987 (German). Retrieve 11/15/2019.]</ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3052332944794899/?comment_id=3053207211374139 Benedicth Koch. Milk The Funk Facebook group thread on the lactic acid content of Berliner Weisse. 11/15/2019.]</ref>. Also found in the Peyer study was an increase in [https://en.wikipedia.org/wiki/Diacetyl diacetyl] and [https://en.wikipedia.org/wiki/Acetoin acetoin] in the beers that were co-fermented with ''L. amylovorus'' and US-05 versus the beers that were kettle soured or mash soured. Both of these compounds are responsible for the buttery taste in beer. Normally, after primary fermentation the yeast reduces diacetyl to acetoin, which is then converted to butanediol, however during a co-fermentation with ''Lactobacillus'', this conversion was inhibited in this study <ref name="Peyer_2017" />.

In regards to liquid cultures and storage: ''Sensorily speaking, the kettle soured beer in the Peyer study tasted "purer"I'm not sure about the liquid cultures with fewer off- I freeze mine at flavors and a plum-80°C (lab freezer with 20% glycerol), which (stores) indefinitelylike aroma. As The sour mash and kettle sour beers had a rule refrigerated liquid cultures should last longer... but there is anecdotal evidence (for some species) of poorer survival of refrigerated versus room temp in liquid cultures. IMOlingering sour aftertaste, stable temps are likely more important for nonwhile the beer co-frozen stocks than hitting an fermented with 'ideal' temperatureL."amylovorus'' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1067614393266774/ Conversation with Bryan Heit on Milk The Funkand US-05 was described as having an astringent aftertaste. 05/04/2015.]This astringent aftertaste was speculated by the authors to be caused by LAB cell autolysis, which might have also contributed to a more complex flavor profile in the co-fermented beer <ref name="Peyer_2017" /ref> ~ Bryan Heit . Studies looking at how ''Lactobacillus'' might impact more characterful strains of [http://suigenerisbrewing''S.blogspot.com/ Sui Generis Brewing blog]. It is also recommended to store liquid cultures of Lacto with a few grams of a buffering chemical cerevisiae'', such as potassium phosphateBelgian strains, calcium sulfate (gypsum), or calcium hydroxide (pickling lime)have not been done yet. The exact amounts should be adjusted to reach a pH of about 4.0 for the entire solution (begin with 1 or 2 grams per liter, and adjust as needed) <ref> See also:* [https://www.facebook.com/groups/MilkTheFunk/permalink/10954493504832781739694606058746/?comment_id=1095492120479001&offset=0&total_comments=23&comment_tracking=%7B%22tn%22%3A%22R6%22%7D Conversation with Adi Hastings on MTFPoster for the referenced Peyer study. 6/20/2015]* [[Mixed_Fermentation#Staggered_Versus_Co-Pitching|Mixed Fermentation Staggered vs Co-pitching]].* [[Mixed_Fermentation#Souring_Without_Brettanomyces|Mixed Fermentation without ''Brettanomyces'']]</ref>.

All data provided by [httpshttp://matthumbard.wordpressphdinbeer.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].

:''Editor's note: the following section was reviewed for accuracy by MTF members [http://suigenerisbrewing.blogspot.com/ Bryan Heitof Sui Generis Blog], [http://phdinbeer.com/ Matt Humbard], [http://www.omegayeast.com/ Lance Shaner], and [http://www.escarpmentlabs.com/ Richard Preiss].''

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'' metabolism is described as the cell catabolizing one molecule of glucose to yield two molecules of pyruvate, which is then further reduced to two molecules of lactate (lactic acid). ''Homolactic'' fermentation only allows the fermentation of hexoses (glucose, mannose, etc.). ''Homolactic'' metabolism follows the ''Embden-Meyerhof-Parnas'' pathway <ref name="Effects on Food Properties"></ref>.

''Heterolactic'' metabolism is described as the cell catabolizing one molecule of glucose into one molecule of CO2CO₂, 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 ''pentose phosphateLactobacillus'' pathway, such as fructose or also called 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 CO₂ produced by this fermentation in brewer's wort was negligible. This is most likely due to the small amount of sugar consumed by ''phosphogluconateLactobacillus'' pathway <ref name="Effects on Food PropertiesCiosek_2019"></ref>. See [[Lactobacillus#100.25_Lactobacillus_Fermentation|100% ''Lactobacillus'' fermentation]] for more information.

There are three categories of ''Lactobacillus'' based on the type of fermentation they are capable of (''homolactic'', ''heterolactic'', or both). # '''Obligatory homofermentative''' Lactobacillus only perform ''homolactic'' fermentation, and thus only produce lactic acid <ref name="Effects on Food Properties"></ref>. # '''Obligatory heterofermentative''' Lactobacillus only perform ''heterolactic'' fermentation, and thus produce lactic acid, CO2, and ethanol (or sometimes acetic acid instead of ethanol) <ref name="Effects on Food Properties"></ref>. # '''Facultatively heterofermentative''' Lactobacillus generally are ''homolactic'' when there is an abundance of carbohydrates, but can also perform ''heterolactic'' fermentation when carbohydrates are not abundant <ref name="Effects on Food Properties"></ref>.

Other factors can determine if a # ''facultative heterofermentative'Obligatory homofermentative' species uses ''homolactic'' or ''heterolacticLactobacillus'' only perform homolactic fermentation, and thus only produce lactic acid <ref name="Effects on Food Properties"></ref>. For example, L. plantarum, which is a # '''facultatively Obligatory heterofermentative'' species, is ''homolactic'' without the presence of oxygen. In the presence of oxygen, however, it performs Lactobacillus''only perform heterolactic'' fermentation, and produces thus produce lactic acid, CO2, and ethanol (or sometimes acetic acid instead of ethanol) <refname="Effects on Food Properties">[https://microbewiki.kenyon.edu/index.php/Lactobacillus_plantarum_and_its_biological_implications Lactobacillus plantarum and its biological implications. Microbe Wiki. Retrieved 6/7/2015.]</ref>. # '''Facultatively heterofermentative''' ''Lactobacillus'' generally are homolactic when there is an abundance of carbohydrates, but can also perform heterolactic fermentation when carbohydrates are not abundant <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1083842231643990/?comment_idname=1084922688202611&offset=0&total_comments=28&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Lance Shaner about L. plantarum "Effects on MTF. 6/7/2015.]Food Properties"></ref>.

====100% Lactobacillus Fermentation==== The amount of CO2 produced is very small in ''Other factors can determine if a facultative heterofermentative'' speciesuses homolactic or heterolactic fermentation. Lance Shaner of Omega Yeast Labs noted that although For example, ''L. brevis is classified as plantarum''obligatory , which is a facultatively heterofermentative''species, is homolactic without the human eye cannot detect any CO2 production in the Omega Yeast Lactobacillus blend (OYL-605)presence of oxygen. Lance still needs to test this blend to see if In the presence of oxygen, however, it performs heterolactic fermentation, and produces any CO2 at allacetic acid <ref name="Quatravaux_plantarum"></ref><ref name="microbewiki_plantarum">[https://microbewiki.kenyon. It is clear though that any type of ''edu/index.php/Lactobacillus_plantarum_and_its_biological_implications Lactobacillus'', regardless of whether it is ''heterofermentative'' or ''homofermentative'', cannot produce a krausenplantarum and its biological implications. Krausens are sometimes seen with the use of commercially available ''Lactobacillus'' culturesMicrobe Wiki. If a krausen develops in wort when it is the only culture that is pitched, this is indicative of cross contamination of ''Saccharomyces'' or ''Brettanomyces'' in either the wort, or the ''Lactobacillus'' culture itself Retrieved 6/7/2015.]</ref><refname="shaner_plantarum">[https://www.facebook.com/groups/MilkTheFunk/permalink/1083842231643990/?comment_id=10846461248969341084922688202611&offset=0&total_comments=2628&comment_tracking=%7B%22tn%22%3A%22R822R%22%7D Discussion Conversation with Lance Shaner about L. plantarum on MTF. 6/7/2015.]</ref>. In addition to this, ''heterolactic'' fermentation by ''Lactobacillus'' can only produce 10-20% of the ethanol that Saccharomyces can produce <ref name="PhysioLacto">[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.

| 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. rhamnosus <ref>[http://cajgh.pitt.edu/ojs/index.php/cajgh/article/view/113 Complete Genome Sequence of the Probiotic Lactic Acid Bacterium Lactobacillus Rhamnosus. Samat Kozhakhmetov, Almagul Kushugulova, Adil Supiyev, Indira Tynybayeva, Ulykbek Kairov, Saule Saduakhasova, Gulnara Shakhabayeva, Kenzhebulat Bapishev, Talgat Nurgozhin, Zhaxybay Zhumadilov. 2013.]</ref> || L. pontis || L. bavaricus <ref name="fao"></ref>|- | 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> || |-| Streptococcus thermophilus <ref name="fao"></ref> || L. fermentatum <ref name="fao"></ref> |||- | Pediococcus acidilactici <ref name="fao"></ref> || L. sanfrancisco <ref name="fao"></ref> || |-| Pediococcus damnosus <ref name="fao"></ref> || Leuconostoc dextranicum <ref name="fao"></ref> |||-| Pediococcus pentocacus <ref name="fao"></ref> || Leuconostoc mesenteroides <ref name="fao"></ref> |||-| Enterococcus faecium <ref name="fao"></ref> || Leuconostoc paramesenteroides <ref name="fao"></ref> || |-| Enterococcus faecalis <ref name="fao"></ref> || ||

===Sugar Utilization and Secondary Metabolites===''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 ''LactobaccilusLactobacillus'', 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====  Lance Shaner's experiment on testing [[Lactobacillus_Fermentation|100% Lactobacillus Fermentation]] showed that '''pure cultures''' of WLP677, WLP672, Wyeast 5335, Wyeast 5223-PC, and the ''L. plantarum'' from Omega Yeast OYL-605, could not fully attenuate a 1.037 SG wort. The most attenuative ''Lactobacillus'' culture, WLP677, was only able to attenuate down to 1.03255 SG. It is likely that all species and strains of ''Lactobacillus'' available to brewers cannot fully attenuate wort. In addition, this study showed at most a 0.29% ABV in 100% ''Lactobacillus'' fermentations (attributed to WLP677). See [[Lactobacillus_Fermentation|100% Lactobacillus Fermentation]] for more information. If a higher attenuation is achieved, cross contamination of yeast is most likely the cause. Thomas Hübbe's masters thesis also supports that ''Lactobacillus'' attenuates less than 10% of the sugars in wort <ref name="Hubbe"></ref>. The amount of CO2 produced is very small in heterofermentative species. Lance Shaner of Omega Yeast Labs noted that although ''L. brevis'' is classified as obligatory heterofermentative, the human eye cannot detect any CO2 production in the Omega Yeast Lactobacillus blend (OYL-605). Lance still needs to test this blend to see if it produces any CO2 at all. There have been reliable reports of pure ''Lactobacillus brevis'' cultures 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&notif_t=group_comment_reply&notif_id=1468974761019794# Conversation with Richard Preiss on MTF regarding pure Lactobacillus fermentation. 07/19/2016.]</ref>. It is clear though that any type of ''Lactobacillus'', regardless of whether it is heterofermentative or homofermentative, cannot produce a krausen. Krausens are sometimes seen even with the use of commercially available ''Lactobacillus'' cultures and good sanitation techniques. If a krausen develops in wort when it is the only culture that is pitched, this is indicative of cross-contamination of ''Saccharomyces'' or ''Brettanomyces'' in either the wort or the ''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>. In addition to this, heterolactic fermentation by ''Lactobacillus'' can only produce 10-20% of the ethanol that Saccharomyces can produce <ref name="PhysioLacto">[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.  Recent studies on lactic acid fermented malt beverages also support that ''Lactobacillus'' produces only about 0.1% ABV, producing "non-alcoholic" fermented malt beverages <ref name="Dongmo" /><ref name="Peyer" /><ref name="Tenhovirta_masters" />. Elke Arendt, a brewing scientist that specializes in ''Lactobacillus'' presented her work at the Belgian Brewing Conference 2015. In it she explained that LAB will only ferment 0.5°P of wort regardless of the gravity of that wort. When asked at the end of the presentation why ''Lactobacillus'' only ferments ~0.5°P (note that Shaner's experiment shows ''Lactobacillus'' fermenting ~1°P, although this may be due to a margin of error since Shaner only performed this experiment once), considering that ''Lactobacillus'' ferments maltose and there is plenty of maltose in wort, Arendt responded that she believes that the bacteria reaches max cell density in the wort with relatively little sugar requirements (~16 mins in and ~25 mins in): <youtube width="300" height="200">9a-ZpF2LDm8</youtube> An in-house experiment by Bell's Brewery which was presented at the MBAA Conference 2017 by Timothy Lozen reported slightly higher amoutns of ABV from a few species of ''Lactobacillus''. Out of 7 different species of ''Lactobacillus'' that were tested, ''L. bucherni'' (White Labs) produced the most alcohol at 0.64% ABV. ''L. rossiae'' (White Labs) and ''L. brevis'' (Bell's Brewery) produced around 0.4% ABV. ''L. delbruekii'' subsp. ''bulgaricus'' (ATCC #11842) produced around 0.5% ABV. The other strains, which were ''L. delbruekii'' subsp. ''lactis'' (ATCC #12315), ''L. casei'' (White Labs), and ''L. plantarum'' (Goodbelly) produced 0.1% or lower ABV <ref name="lozen_2017" />. * 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>. 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 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 Bell's Brewery, ''L. casei'' (White Labs), ''L. buchneri'' (White Labs), ''L. delbruekii'' subsp. ''bulgarius'' (ATCC #11842), ''L. plantarum'' (Goodbelly), and ''L. rossiae'' (White Labs). The wort soured with ''L. bucherni'' was the most preferred with a score of 5.9, followed closely by ''L. casei'' which scored 5.3, ''L. brevis'' wich scored 4.9, ''L. plantarum'' which scored 4.6, and ''L. rossiae'' which scored 4.3. The ''L. delbruekii'' subsp. ''bulgarius'' scored lowest on preference with a score of 1.6, and was characterized as "cheesy/pukey and funky". ''L. casei'' was characterized as the most sour, while ''L. buchneri'' was characterized as the most citrusy and ''L. plantarum'' was characterized as the most fruity. The different strains also produced a range of titratable acidity and diacetyl with ''L. casei'' producing the most and ''L. delbruekii'' subsp. ''bulgarius'' the lowest titratable acidity and the most diacetyl. ''L buchneri'' produced the most alcohol at 0.64% ABV <ref name="lozen_2017">[https://www.mbaa.com/meetings/archive/2017/proceedings/Pages/92.aspx Timothy Lozen. "A comparison of selected lactic acid bacteria for use in the production of sour wort and beer." Presentation by Bell's Brewery for the 2017 Master Brewers Conference. 2017.]</ref>. See also [https://www.masterbrewerspodcast.com/085 MBAA podcast #85 with Tim Lozen].  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/S0924224415300625 Lactic Acid Bacteria as Sensory Biomodulators for Fermented Cereal-Based Beverages. Lorenzo C. Peyer , Emanuele Zannini , Elke K. Arendt. 2016.]</ref><ref name="Tenhovirta_masters" />. Acetaldehyde produced from ''L. plantarum'' helps to produce pyranoanthocyanins that stabilize wine's 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, Siyu Lic, Hongfei Zhaoa, Pan Gua, Yuqi Chena, Bolin Zhanga, Baoqing Zhu. 2018. https://doi.org/10.1016/j.foodres.2018.03.032]</ref>. Some strains may also produce fusel alcohols and other off-flavors. For example the referenced study found an accumulation of the fusel alcohol n-Porponal in the sample of ''L. reuteri'', and a small decrease of isovaleric acid coupled with a small increase of [https://en.wikipedia.org/wiki/Hexanoic_acid hexanoic acid] by ''L. brevis'', ''L. plantarum'', and ''L. reuteri'' (only 0.25-0.32 mg/L was found, and the flavor threshold of hexanoic acid is 5.4 mg/L <ref>[http://www.leffingwell.com/odorthre.htm Leffingwell & Associates website. Odor Thresholds. Retrieved 12/30/2015.]</ref>) <ref name="Peyer"></ref>. Heterofermentative species can also produce [[Tetrahydropyridine|tetrahydropyridines (THP)]], which is the cause of "mousy" off-flavors <ref name="Costello">[http://pubs.acs.org/doi/abs/10.1021/jf020341r Mousy Off-Flavor of Wine:  Precursors and Biosynthesis of the Causative N-Heterocycles 2-Ethyltetrahydropyridine, 2-Acetyltetrahydropyridine, and 2-Acetyl-1-pyrroline by Lactobacillus hilgardii DSM 20176. Peter J. Costello and Paul A. Henschke. 2002.]</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"></ref>. A few species, especially most strains of ''L. fermentum'', and some strains of ''L. delbrueckii'' subsp. ''bulgaricus'', can produce ropiness in the form of exopolysaccharides, similar to [[Pediococcus]] <ref name="peyer_review"></ref>.  [http://www.sciencedirect.com/science/article/pii/S0308814617302911#t0005 Dongmo et al. (2017)] found 56 volatile flavor compounds, including various esters, alcohols, ketones, aldehydes, acids, ethers compounds, sulfur compounds, heterocyclic compounds, phenols (including guaiacol and 4-vinylguaiacol), terpenes, lactones, and several unidentified compounds. Key compounds produced by ''Lactobacillus'' include acetaldehyde (thought to be a major flavor contributor to kettle soured beers <ref name="Peyer_2017" />), β-Damascenone, furaneol, phenylacetic acid, 2-phenylethanol, 4-vinylguaiacol, sotolon, methional, vanillin, acetic acid, nor-furaneol, guaiacol and ethyl 2-methylbutanoate. Acetaldehyde was the most impactful aroma compound found followed by propan-1-ol and γ-dodecalactone. Acetaldehyde was generally produced in much higher amounts (~23-64 µg/L) by the select strains of ''L. plantarum'', while ''L. amylolyticus'' and ''L. brevis'' produced only 1.5-3 µg/L. In fact, the levels of all of these compounds differed significantly based on the species and strain. The selected strains of ''L. brevis'' were associated as having worse aromas that were dominated by methional (cooked potatoes), acetic acid (vinegar), and nor-furaneol (caramel-like). The ''L. plantarum'' strains selected were identified as producing more positive aromas from compounds such as β-damascenone (apple/fruit juice), furaneol (strawberry), 2-phenylethanol (rose/caramel) and ethyl 2-methylbutanoate (citrus). Small but significant amounts of linalool and geraniol were also found, which are normally terpenes found in [[Hops|hops]]. Vanillan is formed from ferulic acid by some ''Lactobacillus'' species as well as ''Oenococcus oeni'' <ref name="Dongmo">[http://www.sciencedirect.com/science/article/pii/S0308814617302911 Key volatile aroma compounds of lactic acid fermented malt-based beverages – impact of lactic acid bacteria strains. Sorelle Nsogning Dongmo, Bertram Sacher, Hubert Kollmannsberger, Thomas Becker. 2017. doi:http://dx.doi.org/10.1016/j.foodchem.2017.02.091.]</ref>. Some strains of ''Lactobacillus'' can break down chlorogenic acids, which are esters found in plants, into their phenolic derivatives. For example, some strains of ''L. fermentum'', ''L. helveticus'', and ''L. reuteri'' were found to hydrolize chlorogenic acids into caffeic acid, which can then be converted into other phenols such as 4-vinylcatechol and 4-ethylcatechol by ''[[Brettanomyces#Phenol_Production|Brettanomyces]]'' <ref>[https://www.sciencedirect.com/science/article/pii/S0963996918303363 Site-specific hydrolysis of chlorogenic acids by selected Lactobacillus species. Elsa Anaheim Aguirre Santos, Andreas Schieber, Fabian Weber. 2018. DOI: https://doi.org/10.1016/j.foodres.2018.04.052.]</ref>.  Some strains of ''L. plantarum'', ''L. brevis'', and ''Pediococcus pentosaceus'' can reduce hydroxycinnamic acids such as p-coumaric, caffeic, and ferulic acids, into their vinyl phenol derivatives, similar to POF+ ''Saccharomyces'' and ''Brettanomyces''. Furthermore, some strains of ''L. plantarum'' have been found to further reduce the vinyl phenols into ethyl phenols (the same phenolic compounds produced by ''Brettanomyces'') by excreting a protein enzyme called ''VprA''. So far ''L. plantarum'' is the only species of lactic acid yeast that has been identified as being capable of creating ethyl phenols from vinyl phenols, and only certain strains have the genetic capability to do this <ref>[http://aem.asm.org/content/early/2018/06/20/AEM.01064-18.abstract Ethylphenols formation by Lactobacillus plantarum: Identification of the enzyme involved in the reduction of vinylphenols. Laura Santamaría, Inés Reverón, Félix López de Felipe, Blanca de las Rivas and Rosario Muñoz. 2018. doi: 10.1128/AEM.01064-18.]</ref><ref>[https://www.ncbi.nlm.nih.gov/pubmed/25261518 Hydroxycinnamic acids used as external acceptors of electrons: an energetic advantage for strictly heterofermentative lactic acid bacteria. Filannino P, Gobbetti M, De Angelis M, Di Cagno R. 2014. DOI: 10.1128/AEM.02413-14.]</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.

* All types of ''Lactobacillus'' produce different levels of ''secondary metabolites'' (compounds that are not required for the organism to live <ref>[http://en.wikipedia.org/wiki/Secondary_metabolite Secondary Metabolite. Wikipedia. Retrieved 6/9/2015.]</ref>) in addition to the primary metabolites discussed above. These include acetaldehyde, diacetyl, fusel alcohols, and many more compounds (see [http://phdinbeer.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/ Matt's article] for more details). * Despite some beliefs by brewers, ''Lactobacillus'' has not been shown to produce significant levels [[Butyric Acid|butyric acid]] (see the [[Butyric Acid|butyric acid]] page for more information) in the presence of oxygen or otherwise in brewer's wort. Some species might be able to produce small amounts of [[Isovaleric_Acid|Isovaleric Acid]] in the presence of ''Streptococcus thermophilus'' or another alpha-keto acid producing microorganism (see the [[Isovaleric Acid]] page for details). * Although ''Lactobacillus'' are not not inhibited by oxygen, they are generally not considered to be organisms that produce oxygen-dependent metabolites. Some heterofermentative species produce acetic acid, butyric acid (not much in wort, but more so in fermented milk products), and propanoic acid, but they always do so through an anaerobic pathway. The presence of oxygen does not affect metabolism in general <ref name="heit_oxygen_LAB">[https://www.facebook.com/groups/MilkTheFunk/permalink/1182597671768445/?qa_ref=qd&comment_id=1182773928417486&reply_comment_id=1183047521723460&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Bryan of Sui Generis Blog on the effects of oxygen in LAB on MTF. 11/23/2015.]</ref>.  * The major off-flavor that some ''Lactobacillus'' strains might produce is acetaldehyde <ref name="heit_oxygen_LAB"></ref>. ''Saccharomyces'' will help to metabolize the acetaldehyde produced by ''Lactobacillus''. ===Foam Degradation===[[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 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>. ''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 [[Wort 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]]

* [[Sour WortingWort Souring]]

* [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 homoefermentative 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://suigenerisbrewing.blogspot.ca/2015/05/lacto-starters.html Lacto Starters, by Bryan Heit of Sui Generis blog] - Step by step guide to making a starter for Lactobacillus.* [http://www.garshol.priv.no/blog/335.html Yeast terminology, part 2: bacteria, by Lars Garshol.]* [https://www.youtube.com/watch?v=9a-ZpF2LDm8 Elke Arendt, presentation at Belgian Brewing Conference 2015 on scientific findings in LAB in malting and brewing.]* [http://sourbeerblog.com/lactobacillus-2-0-advanced-techniques-for-fast-souring-beer/ Lactobacillus 2.0 – Advanced Techniques for Fast Souring Beer. Sour Beer Blog.]