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Lactobacillus

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[[File:Omega-lacto-microscope.jpg|thumb|Omega Yeast Labs OYL-605 Lactobacillus Blend; photo by [https://www.facebook.com/groups/MilkTheFunk/permalink/1096077917087088/ Stephen Little].]]
'''Lactobacillus''' (often referred to by brewers as "Lacto") is a genus of Gram-positive, rod-shaped lactic acid bacteria (LAB) which produces acidity and sour flavors in the form of lactic acid and [[Lactobacillus#Sugar_Utilization_and_Secondary_Metabolites|secondary metabolites]] found in lambics, Berliner Weiss, sour brown ales, and gueuze. All ''Lactobacillus'' species are facultative anaerobes, which means they grow anaerobically but can also grow in the presence of oxygen and use oxygen to some degree <ref name="todar_lactics4"></ref>. There are more than 100 species, many of which are found in the human gastrointestinal track <ref name="todar_lactics4">[http://textbookofbacteriology.net/lactics_4.html ''Lactic Acid Bacteria''. Todar's Online Texbook of Bacteriology. Kenneth Todar, PhD. Pg. 4. Retrieved 07/28/2015.]</ref><ref name="Todar_nutgro4">[http://textbookofbacteriology.net/nutgro_4.html ''Nutrition and Growth of Bacteria''. Todar's Online Texbook of Bacteriology. Kenneth Todar, PhD. Retrieved 07/28/2015.]</ref>. In addition to beer, some species of ''Lactobacillus'' are also used to ferment yogurt, cheese, sauerkraut, pickles, wine, cider, kimchi, cocoa, and kefir <ref>[https://en.wikipedia.org/wiki/Lactobacillus ''Lactobacillus''. Wikipedia. Retrieved 07/28/2015.]</ref>. ''Lactobacillus'' can form a [[pellicle]] (need reference). See ''[[Pediococcus]]'', ''[[Brettanomyces]]'', ''[[Saccharomyces]]'', and [[Mixed Cultures]] charts for other commercially available cultures. See the [[Sour Worting]] 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.
==Commercial Lactobacillus Cultures==
This strain produces a nice, clean lactic acidity (down to ~pH 3.16-3.18) in unhopped wort within 36 hours at a temperature of ~72-77 F. The higher the temperature (up to 90 F is what we've tested), the faster the acid production. This is a great strain for kettle souring, as it grows rather quickly and produces acidity fast with no detectable off flavors. We are about to begin some trials in hopped wort to test out the acidification in the presence of hop compounds, though we presume there is some level of adaptation to hop compounds given the environment in the beer from which we cultured the strain. Temperature: 70-90 ºF.
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| [[White Labs]] || WLP677 || L. delbrueckii (potentially misidentified) || Heterofermentative <ref name="mtf_wiki_shaner">[http://www.milkthefunk.com/wiki/100%25_Lactobacillus_Fermentation Milk The Funk Wiki. 100% Lactobacillus Fermentation Test by Lance Shaner.]</ref><ref name="tmf_cultures">[http://www.themadfermentationist.com/p/commercial-cultures.html ''Commercial Brettanomyces, Lactobacillus, and Pediococcus Descriptions''. The Mad Fermentationist Blog. Michael Tonsmeire. Retrieved 3/4/2015.]</ref> || no stir plate, room temp ||Incubate at > 90°F and < 117°F for 5-7 days for greater lactic acid production. Cell count: 50-80 million cells/mL (1.75-2.8 billion cells in a 35 mL homebrew vial) <ref name="WL_cellcounts">Private correspondence with White Labs Customer Service and Dan Pixley. 10/29/2015.</ref>. Not a good strain for kettle souring, but can produce a "soft" acidity over a longer period of time <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1212455192116026/?comment_id=1212475888780623&reply_comment_id=1212476575447221&comment_tracking=%7B%22tn%22%3A%22R3%22%7D Conversation with Andrew Addkison on MTF. 01/12/2016.]</ref>. White Labs claims that it is tolerant to up to 20 IBU, although growth starts to become inhibited at 15 IBU <ref name="WL_datasheet" /><ref>[http://www.themadfermentationist.com/p/commercial-cultures.html "Commercial Brettanomyces, Lactobacillus, and Pediococcus Descriptions; Commercial Yeast Laboratories." The Mad Fermentationist blog. Michael Tonsmeire. Retrieved 12/12/2016.]</ref>. Generally heat tolerant, but sours faster between 100-110°F <ref name="WL_datasheet">[http://www.whitelabs.com/sites/default/files/R%26D%20Wild%20Yeast%20and%20Bacteria%20Experiments_2.pdf "R&D Wild Yeast and Bacteria Experiments". White Labs data sheet. Retrieved 05/16/2017.]</ref>
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| [[White Labs]] || WLP672 || L. brevis || Heterofermentative <ref name="mtf_wiki_shaner"></ref><ref name="nick">[https://www.facebook.com/groups/MilkTheFunk/permalink/1029638267064387/?comment_id=1030638553631025&offset=0&total_comments=24 Conversation with Nick Impellitteri from The Yeast Bay on the MTF Facebook Group. 3/4/2015.]</ref> || No stir plate, room temp|| Produced by [[The Yeast Bay]]. More hop tolerant than other Lacto strains, however TYB advises to use wort with less than 10 IBU. White Labs data sheet shows that growth is inhibited to 82% at 5 IBU, and 60% at 10 IBU <ref name="WL_datasheet" />. Temperature range: 70-95°F (greatly inhibited at 110°F) <ref name="WL_datasheet" />; 80% attenuation (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>). <ref>[http://www.theyeastbay.com/wild-yeast-and-bacteria-products/wlp672-lactobacillus-brevis The Yeast Bay website. Retrieved 3/2/2015.]</ref> Cell count: 50-80 million cells/mL (1.75-2.8 billion cells for 35 mL homebrew vials) <ref name="WL_cellcounts"></ref>.
The important take away here is that oxygen doesn't significantly affect ''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/1182597671768445/?qa_ref=qd&comment_id=1182773928417486&reply_comment_id=1183242405037305&comment_tracking=%7B%22tn%22%3A%22R7%22%7D Conversation with Bryan of Sui Generis Blog on MTF regarding butyric acid production by Lactobacillus. 11/23/2015.]</ref>.
There are, however, 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 (H<sup>2</sup>0<sup>2</sup>), 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>. 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'', and brewers should not aerate wort during sour 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 Labs 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. ''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'' 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 growth media and not representative of brewing conditions <ref name="Hubbe">[https://www.facebook.com/groups/MilkTheFunk/1407620505932826/ Effect of mixed cultures on microbiological development in Berliner Weisse (master thesis). Thomas Hübbe. 2016.]</ref>.
====Hop Tolerance====
An example from one 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>. 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>.
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>.
===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 tend to are toxins target other species that are closely related to the toxin-producing species (although there are some exceptions to this in the literature)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 may still 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 the this [https://www.facebook.com/BootlegBiology/photos/a.148869931970401.1073741829.124634287727299/465185997005458/?type=1&theater Bootleg Biology notes for [[Pediococcus#Commercial_Pediococcus_Cultures|"Sour Weapon"]Facebook post]).
==See Also==

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