Changes

| [[Brewing Science Institute]] || L. delbrueckii || Lactobacillus delbrueckii || Homofermentative || || A Lactobacillus bacteria that produces a clean lactic sourness.

| [[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">[www.whitelabs.com/sites/default/files/R%26D%20Wild%20Yeast%20and%20Bacteria%20Experiments_2.pdf "R&D Wild Yeast and Bacteria Experiments". White Labs data sheet. Retrieved 05/16/2017.]</ref>

| [[White Labs]] || WLP672 || 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>.

| [[Wyeast]] || 5223-PC || L. brevis || Heterofermentative <ref name="mtf_wiki_shaner"></ref><ref name="nick"></ref> || no stir plate, room temp is fine || Heterofermentative (produces lactic acid, ethanol and CO2), more hop tolerant. Does well at room temperature. AVAILABLE ONLY FROM JULY THROUGH SEPTEMBER 2014 (Michael Dawson from Wyeast indicated that this culture may return at some point). Jamie Daly indicated on MTF that he got almost no sourness after 24 hours at 100°F (37.8°C). He lowered the temperature to 90°F-95°F (32.2°C-35°C) for 36 hours, and the pH of the wort went down to 3.29. Thus, Jamie recommends 90°F-95°F (32.2°C-35°C) for 60 hours for better souring; avoid warmer temperatures. He also aerated his starter of L. brevis (brevis 2L starter of 1.020 dmeDME) and set it on a stir plate at 95°F <ref name="brevis_aeration">[http://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(5): 1025–1030.]</ref>. The beer wort was not aerated, and the fermenter was flushed with CO2. These methods need verification. Cell count: 1.0 x 10⁸ (100 million) cells/mL (10 billion cells in a 100 mL homebrew pouch) <ref name="wyeast_cellcounts"></ref>.

| [[Inland Island Brewing & Consulting|Inland Island Yeast Laboratories]] || INISBC-991 || L. 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 || L. 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 || L. fermentum || Heterofermentative || ||

| [[The Yeast Bay]] || Lactobacillus Blend || L. plantarum, L. brevis, and an unidentified ''Lactobacillus'' species || Heterofermentative || || The Lactobacillus Blend includes three strains: Lactobacillus plantarum, Lactobacillus brevis and a strain of Lactobacillus isolated from a very unique brewer of American sour beers the returned a sequencing result of "uncultured Lactobacillus". Sure to please anyone with a knack for creating sour beers, it can quickly produce acidity across a wide range of temperatures. This blend can be used on its own for kettle souring prior to pitching yeast to create acidity quickly, or co-pitched with yeast to create sourness over time. It will produce a pronounced and rounded acidity that is the foundation of any complex sour beer. We recommend 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 very slow or no souring (testing is still ongoing to determine IBU at which lactic acid production is inhibited). Temperature: 70-100°F90°F. Cell count: 50-80 million cells/mL (1.75-2.8 billion cells for 35 mL homebrew vials) <ref name="WL_cellcounts"></ref><ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1280135442014667/?comment_id=1280341068660771&reply_comment_id=1280498695311675&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Nick Impellitteri on MTF regarding TYB Lactobacillus Blend cell counts. 04/08/2016.]</ref>. Recommended temperature range for fastest acid production for kettle souring is 85-90°F, although if kept in the 70's it should produce good acidification in 48-72 hours. A major drop off of in acid production is seen above 90°F <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1616265398401668/?comment_id=1617001948328013&comment_tracking=%7B%22tn%22%3A%22R%22%7D Impellitteri, Nick. Milk The Funk Facebook group. 03/17/2017.]</ref>.

<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 80-95F <refname="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 105°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 70-80°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 75°F (original pitch temp was 85°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>

Starter mediums that brewers have used include unhopped DME wort starters and apple juice starters. These tend to be adequate for many brewers. However, [https://eurekabrewing.wordpress.com/2015/05/18/evaluate-starter-media-to-propagate-lactobacillus-sp/ Samuel Aeschlimann from Eureka Brewing Blog] showed that using DME with a little bit of apple juice, chalk, and yeast nutrients provides close to optimal cell densities that match MRS media cell densities. See [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's Starter Procedures]]. Specific nutrients that will increase growth is thiamine (vitamin B1), or a combination of thiamine and riboflavin (vitamin B2). For example, it has been shown that thiamine is required in order for ''L. brevis'' to efficiently convert pyruvate into lactic acid and ethanol. The addition of these nutrients can help encourage growth <ref>[http://onlinelibrary.wiley.com/doi/10.1002/jib.385/full The influence of thiamine and riboflavin on various spoilage microorganisms commonly found in beer. Barry Hucker, Melinda Christophersen, Frank Vriesekoop. 2017.]</ref>.

The recipe for this starter wort is: '''1.040 SG (10°P) Dried Malt Extract wort with 10% apple juice + 20 grams of chalk (CaCO3) per liter + yeast nutrients'''. Regarding the use of chalk, it is the preferred buffer because it does not react with CO2 (unlike baking soda), so it won't be consumed by exposure to air due to CO2 production by the Lacto. It also has a pKa (maximum buffering capacity) of around 4.6, which is ideal for ''Lactobacillus'' growth. The fact that it easily precipitates out also makes it ideal to use as a buffer <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1180630378631841/?comment_id=1181674265194119&reply_comment_id=1181743348520544&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Bryan of Sui Generis Blog regarding the use of chalk as a buffer in Lacto starters. 11/20/2015.]</ref>. Jeff Mello from [[Bootleg Biology]] and Nick Impellitteri from [[The Yeast Bay]] suggest that using the smaller amount of 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>. To create a 1 liter starter for 20 liters of wort, follow these directions:

# Reference the above [[Lactobacillus#Culture_Charts|Culture Charts]] for how long the starter should be incubated for before pitching. If a stir plate is not used, one indication that the starter is done will be when the top of the starter begins to clear (turbidity is an indication that the culture is growing, and once the top portion of the starter starts to clear then that is a sign that growth has stopped) <ref name="Sam_starter2">[https://www.facebook.com/groups/MilkTheFunk/permalink/1131778916850321/?comment_id=1131806746847538&offset=0&total_comments=6&comment_tracking=%7B%22tn%22%3A%22R2%22%7D Conversation with Sam Aeschlimann of Eureka Brewing Blog on MTF. 08/20/2015.]</ref>.

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>.  Cell growth can also be influenced by the presence of other microorganisms, such as ''Saccharomyces'' and ''Brettanomyces''. One study by Hübbe showed that ''L. brevis'' and ''L. parabrevis'' grew to the normal high cell counts when grown individually and without competition. When co-fermented with ''Brettanomyces'', the cell count of ''L. brevis'' was halved, and the growth rate of ''L. parabrevis'' was greatly diminished to about 15-20% (the pitching rate of ''Brettanomyces'' was also tested, and seemed to not have an effect on the ''Lactobacillus'' growth). When co-fermented with both ''S. cerevisiae'' and ''Brettanomyces'', the ''Lactobacillus'' growth was greatly diminished to about 2-13% of what the normal cell growth was without competition. This appears to correspond with anecdotal reports from brewers that some ''Lactobacillus'' species/strains do not compete well with yeast, especially ''S. cerevisiae'' <ref name="Hubbe"></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), 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>. 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>. ''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 <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>.

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 ''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 upto up to ~18 IBU(update: 20-30 IBU), but past that point the resistance appears to remain inducableinducible. 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 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>[https://www.facebook.com/groups/MilkTheFunk/permalink/1002795743081973/?comment_id=1003625646332316&offset=0&total_comments=16&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation 1 with Bryan of Sui Generis Blog on Milk The Funk regarding Lactobacillus hop tolerance. 01/19/2015.]</ref>.

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>.

Lance Shaner's experiment on testing [[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 [[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>.

Recent studies on lactic acid fermented malt beverages shows that ''Lactobacillus'' produces only about 0.1% ABV, producing "non-alcoholic" fermented malt beverages <ref name="Dongmo" /><ref name="Peyer" />. Elde 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):

===Sugar Utilization and Primary/Secondary Metabolites=======Primary Metabolites====Lactic acid is the primary metabolite for ''Lactobacillus'' generally prefers glucose, fructose, as well as CO2 and maltoseethanol/acetate (acetic acid) in heterofermentative species. Acid production is at it's highest during the exponential growth phase, but continues into the stationary and does not ferment maltotriosedecline 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>. Some The amount of lactic and acetic acids produced varies from species to species may prefer certain types of sugars over others. For example , the referenced study showed that ''L. plantarum'' ferments glucose firstproduces more than twice the amount of lactic acid than ''L. brevis'', and then fructose if it is available. ''L. reuteri'' ferments maltose first, while produced slightly more lactic acid than ''L. brevis'' feeds on maltose, glucose, and fructose. Disaccharides such ''L. reuteri'' produced around twice as sucrose and maltose enter the cells through specific types of membrane transport proteins called permeasesmuch acetic acid than ''L. brevis'', and are broken down into monosaccharides through phosphorolysis before they enter the normal carbohydrate metabolic pathway <ref name="peyer_review"></ref>''L. Peak sugar consumption without competition from yeast is typically 48 hours, and plantarum'' produced very little alcohol or CO2 is produced (around 0.10-0.30% ABV, far less than the 0.5% required for non-alcoholic drinks)acetic acid. Consumption The small amount of sugars occurs mainly acetic acid produced by ''L. plantarum'' in this study was explained by oxygen exposure during sampling, while 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 obligate heterofermentative species (''LactobacillusL. reuteri''. 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 ''LactobacillusL. brevis'' ) produced acetic acid as a direct result of their heterolactic fermentation <ref name="Peyer"></ref>. For a chart and in depth discussion on what types of sugars are fermentable by different species of ''Lactobacillus'', as well as charts on secondary metabolites, see [http://phdinbeer.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/ Matt Humbard's ''Physiology of Flavors in Beer – Lactobacillus Species'' blog article].

A small number of strains ====Secondary Metabolites====Both primary and secondary metabolites play a large role in the flavor and aroma profile of wort fermented with ''Lactobacillus'' can also break down polysaccharides and starches. They Secondary metabolites are compounds that are referred not directly related to as "amylolytic LAB"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. They generally belong to the species ''Lb. manihotivorans''Demain AL, ''LFang A. fermentum'', ''L2000. amylovorus'', ''L]</ref>. amylophilus'' These secondary metabolites are produced by the pathways mentioned above, ''Land 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. plantarum'' or ''L 12/29/2015. amylolyticus''</ref>. This seems Thus, different species and strains can produce a wide variety of flavors and aromas (compare this to be associated with a gene called "amyA", food grade lactic acid in which encodes for extracellular alpha-amylase activity, as well as alpha-glucosidase, neopullulanase, amylopectin phosphorylase, and maltose phosphorylasenone of these secondary metabolites exist). This activity is limited by high amounts These secondary metabolite are the result of glucose, maltose, or sucrose carbohydrate fermentation and amino acid metabolism <ref name="peyer_review"></ref>. Major secondary metabolites that

Lactic acid is the primary metabolite for An example from one study showed that ''L. plantarum'' produced significantly more diacetyl, acetoin (yogurt-like flavor), and acetaldehyde than ''L. reuteri'' and ''LactobacillusL. brevis''. These three compounds were associated with dairy-related notes of "buttery", "lactic", as well as CO2 and ethanol"yogurt" flavors identified during sensory testing <ref name="Peyer"></acetate (acetic ref>. Some LAB can release these compounds through the catabolism of citric acid) , which is found in heterofermentative specieswort. Acid Ester production is at it's highest generally insignificant, although significant ester formation has been found during the exponential growth phasemalolactic fermentation in red wines, but continues into the stationary and decline phases. Typically just under 50% of the lactic acid ethyl acetate has been found to be produced is L-lactic acid (more nutritionally relevant) while the slight majority is D-lactic acid in malt based beverages <ref name="Peyerpeyer_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>. The amount of lactic Some strains may also produce fusel alcohols and acetic acids produced varies from species to speciesother off-flavors. For example, the referenced study showed that found an accumulation of the fusel alcohol n-Porponal in the sample of ''L. plantarumreuteri'' produces more than twice the amount , and a small decrease of isovaleric acid coupled with a small increase of lactic [https://en.wikipedia.org/wiki/Hexanoic_acid hexanoic acid than ] by ''L. brevis'', and ''L. reuteriplantarum'' produced slightly more lactic acid than , and ''L. brevisreuteri''(only 0. ''L25-0. reuteri'' produced around twice as much acetic acid than ''32 mg/L. brevis''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. plantarum'' produced very little acetic acid]</ref>) <ref name="Peyer"></ref>. The small amount 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 acetic acid produced the Causative N-Heterocycles 2-Ethyltetrahydropyridine, 2-Acetyltetrahydropyridine, and 2-Acetyl-1-pyrroline by ''LLactobacillus hilgardii DSM 20176. Peter J. Costello and Paul A. Henschke. 2002.]</ref>. plantarum'' in this study was explained by oxygen exposure during sampling 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, while the obligate heterofermentative and valine to form fruity flavors <ref name="peyer_review"></ref>. A few species (, especially most strains of ''L. reuterifermentum'' , and some strains of ''L. brevisdelbrueckii subsp. bulgaricus'') produced acetic acid as a direct result , can produce ropiness in the form of their heterolactic fermentation exopolysaccharides, similar to [[Pediococcus]] <ref name="Peyerpeyer_review"></ref>.

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 growth of an organism, but often assist with survival <ref>[http://www.ncbisciencedirect.nlm.nih.govcom/science/article/pubmedpii/11036689 The natural functions of secondary metabolitesS0308814617302911#t0005 Dongmo et al. Demain AL(2017)] found 56 volatile flavor compounds, including various esters, alcohols, ketones, aldehydes, acids, ethers compounds, sulfur compounds, heterocyclic compounds, phenols, terpenes, lactones, Fang A. 2000.]</ref>and several unidentified compounds. These secondary metabolites are Key compounds produced by the pathways mentioned above''Lactobacillus'' include acetaldehyde, β-Damascenone, furaneol, phenylacetic acid, 2-phenylethanol, 4-vinylguaiacol, sotolon, methional, vanillin, acetic acid, nor-furaneol, guaiacol and different strains probably regulate ethyl 2-methylbutanoate. Acetaldehyde was the enzymes involved in various pathways differently most impactful aroma compound found followed by propan-1-ol and produce different secondary metabolites <ref>Private correspondence with Richard Preiss from Dan Pixleyγ-dodecalactone. 12Acetaldehyde was generally produced in much higher amounts (~23-64 µg/29/2015L) by the select strains of ''L. plantarum'', while ''L. amylolyticus'' and ''L. brevis'' produced only 1.<5-3 µg/ref>L. ThusIn fact, different the levels of all of these compounds differed significantly based on the species and strain. The selected strains can produce a wide variety of flavors and ''L. brevis'' were associated as having worse aromas that were dominated by methional (compare this to food grade lactic cooked potatoes), acetic acid in which none of these secondary metabolites exist(vinegar), and nor-furaneol (caramel-like). These secondary metabolite are the result 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 carbohydrate fermentation linalool and amino geraniol were also found, which are normally terpenes found in [[Hops|hops]]. Vanillan is formed from ferulic acid metabolism by some ''Lactobacillus'' species as well as ''Oenococcus oeni'' <ref name="Peyer_reviewDongmo">[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>.

An example from one 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 showed that compared volatile acids produced by a probiotic strain of ''L. plantarum'' produced significantly more diacetyl, acetoin (yogurt-like flavorNCIMB 8826)when fermented in oats, barley, malted barley, and acetaldehyde than ''Lwheat. reuteri'' In oats, there was slight increase in oleic acid and linoleic acid and ''L. brevis''a decrease when fermented in wheat, barley, or malted barley. These three In malted barley, there were small increases in flavor active compounds were associated with dairy-related notes of such as furfural ("butteryalmond"flavor), 2-ethoxyethyl acetate and isoamyl alcohol, but little to none detected when fermented in oats, "lactic"wheat, and "yogurt" flavors identified during sensory testing <ref name="Peyer"></ref>or unmalted barley. Some LAB can release these compounds through the catabolism of citric Acetic acid, which is found production was higher in barley and malted barley than it was in wortoats and wheat. Ester production is generally insignificantMany other organic acids in the oats, wheat, barley, although significant ester formation has been found and malted barley were supposedly taken up by the ''L. plantarum'' during malolactic fermentation in red wines. In barley, and ethyl acetate has been found to be produced there were trace amounts of new acids created that were not already in malt based beverages the barley itself <ref name="peyer_review">[http://www.sciencedirect.com/science/article/pii/S0924224415300625 Lactic Acid Bacteria as Sensory Biomodulators for Fermented CerealS0308814609004373 Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-Based Beverages. Lorenzo C. Peyer based substrates Ivan Salmeron, Pablo Fuciños, Emanuele Zannini Dimitris Charalampopoulos, Elke KSeverino S. ArendtPandiella. 20162009.]</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 species of ''L. reuteriLactobacillus'', and a small decrease of isovaleric acid coupled with a small increase of [https://en.wikipedia.org/wiki/Hexanoic_acid hexanoic acid] by including ''L. brevislactis'', and ''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 diacetyl (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 can be reduced to acetoin and Paul A. Henschke. 2002.]</ref>. Aldehydes (2-methyl-1-propanal, 2-methyl-1-butanal, 3-methyl-1-butanalbutanediol) and their associated non-fusel alcohols (2-methyl-1-propanolas an intermediate metabolite from consuming sugar, 2-methyl-1-butanolcitrate, 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 speciesHowever, especially most strains of ''L. fermentum''citrate levels are rather low in malted barley (but higher in sorghum), and some strains of ''L. delrueckii subsp. bulgaricus'', can produce ropiness diacetyl production has been observed to be very low in the form of exopolysaccharides, similar to [[Pediococcus]] barley and oat based worts <ref name="peyer_review"></ref>.

The type of grain that Aging has a large impact on the aromas and flavors produced by ''Lactobacillus'' fermentation over time and 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 typically influenced by a probiotic strain temperature of ''L. plantarum'' (NCIMB 8826) when fermented in oatsthe environment, barley, malted barleyoxygen exposure, and wheatthe byproducts of fermentation. In oatsGenerally, there was slight increase in oleic acid fermentation has a positive effect on preserving some aroma and linoleic acid and a decrease when fermented in wheat, barley, or malted barleyflavor compounds. In malted barley, there were small increases in flavor active Other compounds such as furfural ("almond" flavor)may change, 2-ethoxyethyl acetate causing aroma 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 wheatflavor changes. Many other organic acids in the oatsFor example, wheat, barley, and malted barley were supposedly taken up by the one study characterized wort freshly fermented with ''L. plantarum'' during fermentationas "butter" and honey", and when aged as "yogurt" and "sour". 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 some species of ''Lactobacillus''same study, including ''L. lactisreuteri'' was characterized as "sour" when fresh, and "honey" and "pungent" when aged. ''L. plantarumbrevis'', produce diacetyl (which can be reduced to acetoin and 2,3-butanediol) was characterized as an intermediate metabolite from consuming sugar, citrate"soy sauce" when fresh, and amino acids. However, citrate levels are rather low in malted barley (but higher in sorghum), "yeasty" and diacetyl production has been observed to be very low in barley and oat based worts "cider" when aged <ref name="Peyer_reviewPeyer"></ref>.

Aging has a large impact on the aromas and flavors produced by Many strains of ''Lactobacillus'' fermentation over time and other lactic acid bacteria can produce tannase, which is typically influenced by temperature an enzyme that breaks down a certain class of the environment, oxygen exposuretannins called "hydrolizable tannins" (for example, and the byproducts of fermentationtannic acid). Generally, fermentation has The enzymatic breakdown of tannins provides 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 food source for the ''L. plantarumLactobacillus'' as "butter" and honey", and when aged as "yogurt" and "sour". In the same cited study, a strain of ''L. reuteriplantarum'' was characterized selected out of 47 other tannase producing LAB as "sour" when freshbeing 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 "honey" 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 "pungenthttp://www.beveragedaily.com/R-D/New-enzyme-aims-to-take-the-haze-out-of-iced-tea" when aged. Beveragedaily.com. Guy Montague-James. 04/04/2011. Retrieved 011/09/2016.]</ref>. Some ''L. brevisLactobacillus'' was characterized as "soy sauce" when fresh, and "yeasty" and "cider" when aged strains could therefore have a positive effect on beer clarity by breaking down some haze forming tannins <ref name>[https://www.facebook.com/groups/MilkTheFunk/permalink/1464383586923184/?comment_id=1465361093492100&reply_comment_id=1465496463478563&comment_tracking="Peyer">%7B%22tn%22%3A%22R3%22%7D Review of this entry by Mike Lentz via MTF. 11/10/2016.]</ref>.