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Lactobacillus

4,661 bytes added, 12:09, 16 May 2017
updated WL672 and 677 based on WL data sheet
| [[Craft Cultures]] || CCYL512 || L. brevis || Heterofermentative || || Typically produces more lactic acid than Lactobacillus delbrueckii. Commercial pitches only.
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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">[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>.
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| [[Wyeast]] || 5335 || L. buchneri || Heterofermentative <ref name="mtf_wiki_shaner"></ref> || 1 liter starter for a 5 gallon batch of beer, 1.020 DME sterile wort, no stir plate, no O2, starter at 90°F if possible 5-7 days || Incubate at 90°F for 5-7 days for greater lactic acid production. Cell count: 1.0 x 10<sup>8</sup> (100 million) cells/mL (10 billion cells in a 100 mL homebrew pouch) <ref name="wyeast_cellcounts">[https://drive.google.com/folderview?id=0B8CshC9nxYHdZmE4MmoyLXA2WVk&usp=sharing Wyeast Specifications 2015 Retail Products. 2015.]</ref>.
Although 100% apple juice or 100% DME starters will "work" for ''Lactobacillus'' starters, they do not provide optimal growth conditions. [https://eurekabrewing.wordpress.com/2015/05/18/evaluate-starter-media-to-propagate-lactobacillus-sp/ Samuel Aeschlimann from Eureka Brewing Blog] ran a set of experiments that found a DME based recipe for starter wort that produces a very high cell density similar to that of MRS media, which provides optimal growth rates for ''Lactobacillus''.
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]] suggests 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:
# Add 100 grams of DME to around 900 mL of water and heat pasteurize/boil as you would normally do for a starter. This should make 1.040 SG (10°P) starter wort.
<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 ''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 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>.
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 estern (data reported and translated by Benedikt Rausch on Milk THe Funk Facebook group). 1987.]</ref>.
 
Methner's data is shown below; graphs created by Benedikt Rausch <ref name="Methner" />. Y axis = pH, X axis = days.
 
<gallery>
File:Methner 18IBU.JPG|'''7 IBU'''
File:Methner 18IBU.JPG|'''9 IBU'''
File:Methner 18IBU.JPG|'''11 IBU'''
File:Methner 18IBU.JPG|'''13 IBU'''
File:Methner 18IBU.JPG|'''18 IBU'''
</gallery>
::'''L70''': ''L. coryniformis''
::'''L14, L18, L22, L29, L88, L92''': ''L. Brevis''
See also:
| Enterococcus faecalis <ref name="fao"></ref> || ||
|}
 
===Sugar Utilization===
''Lactobacillus'' generally prefers glucose, fructose, and maltose, and does not ferment maltotriose. Some species may prefer certain types of sugars over others. For example ''L. plantarum'' ferments glucose first, and then fructose if it is available. ''L. reuteri'' ferments maltose first, while ''L. brevis'' feeds on maltose, glucose, and fructose. Disaccharides such as sucrose and maltose enter the cells through specific types of membrane transport proteins called permeases, and are broken down into monosaccharides through phosphorolysis before they enter the normal carbohydrate metabolic pathway <ref name="peyer_review"></ref>. Peak sugar consumption without competition from yeast is typically 48 hours, and very little alcohol or CO2 is produced (around 0.10-0.30% ABV, far less than the 0.5% required for non-alcoholic drinks). Consumption of sugars occurs mainly during the 48 hour growth period, but also occurs after growth has stopped. No more than 0.5-1°P worth of sugar is consumed by ''Lactobacillus''. Rather than high residual sugar concentration being the limiting factor on growth it is thought that low pH and other metabolic byproducts weaken and finally stop the growth of ''Lactobacillus'' <ref name="Peyer"></ref>. For a chart and in depth discussion on what types of sugars are fermentable by different species of ''Lactobacillus'', as well as charts on secondary metabolites, see [http://phdinbeer.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/ Matt Humbard's ''Physiology of Flavors in Beer – Lactobacillus Species'' blog article].
 
A small number of strains of ''Lactobacillus'' can also break down polysaccharides and starches. They are referred to as "amylolytic LAB". They generally belong to the species ''Lb. manihotivorans'', ''L. fermentum'', ''L. amylovorus'', ''L. amylophilus'', ''L. plantarum'' or ''L. amylolyticus''. This seems to be associated with a gene called "amyA", which encodes for extracellular alpha-amylase activity, as well as alpha-glucosidase, neopullulanase, amylopectin phosphorylase, and maltose phosphorylase. This activity is limited by high amounts of glucose, maltose, or sucrose <ref name="peyer_review"></ref>. Some species can also produce beta-glucosidase capable of breaking down monoglycosides (see [[Glycosides]]), 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>.
 
====100% Lactobacillus Fermentation====
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.
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):
<youtube>9a-ZpF2LDm8</youtube>
* See also [[100% Lactobacillus Fermentation]].
===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 maltose, and does not ferment maltotriose. Some ethanol/acetate (acetic acid) in heterofermentative species may prefer certain types of sugars over others. For example ''L. plantarum'' ferments glucose first, and then fructose if Acid production is at it is available. ''L. reuteri'' ferments maltose first, while ''L. brevis'' feeds on maltose, glucose, and fructose. Disaccharides such as sucrose and maltose enter s highest during the cells through specific types of membrane transport proteins called permeasesexponential growth phase, and are broken down but continues into monosaccharides through phosphorolysis before they enter the normal carbohydrate metabolic pathway <ref name="peyer_review"></ref>stationary and decline phases. Peak sugar consumption without competition from yeast is typically 48 hours, and very little alcohol or CO2 is produced (around 0.10-0.30Typically just under 50% ABV, far less than of the 0.5% required for nonlactic acid produced is L-alcoholic drinkslactic acid (more nutritionally relevant). Consumption of sugars occurs mainly during while the 48 hour growth period, but also occurs after growth has stopped. No more than 0.5slight majority is D-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'' lactic acid <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 The amount of strains of ''Lactobacillus'' can also break down polysaccharides lactic and starches. They are referred acetic acids produced varies from species to as "amylolytic LAB"species. They generally belong to For example, the species referenced study showed that ''LbL. manihotivoransplantarum'', produces more than twice the amount of lactic acid than ''L. fermentumbrevis'', and ''L. amylovorusreuteri'', produced slightly more lactic acid than ''L. amylophilusbrevis'', . ''L. plantarumreuteri'' or produced around twice as much acetic acid than ''L. amylolyticusbrevis''. 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]]), 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>.  Many strains of ''LactobacillusL. plantarum'' 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 produced very little acetic acid). The enzymatic breakdown small amount of tannins provides a food source for the acetic acid produced by ''LactobacillusL. plantarum''. In the cited in this studywas explained by oxygen exposure during sampling, a strain of while the obligate heterofermentative species (''L. plantarumreuteri'' 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''L. Tannase has been brevis'') produced acetic acid 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 direct result of Tannase Produced by Lactic Acid Bacteria, Lactobacillus plantarum H78. Mari Matsuda, Yayoi Hirose, and Makoto Kanauchi. 2016.]their heterolactic fermentation </ref><ref>[http://www.beveragedaily.com/R-D/New-enzyme-aims-to-take-the-haze-out-of-iced-tea name="http://www.beveragedaily.com/R-D/New-enzyme-aims-to-take-the-haze-out-of-iced-teaPeyer". 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>.
Lactic acid is ====Secondary Metabolites====Both primary and secondary metabolites play a large role in the primary metabolite for flavor and aroma profile of wort fermented with ''Lactobacillus'', as well as CO2 and ethanol/acetate (acetic acid) in heterofermentative species. Acid production is at it's highest during Secondary metabolites are compounds that are not directly related to the exponential growth phaseof an organism, 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 often assist with survival <ref name="Peyer"><[http://ref>www.ncbi.nlm.nih. gov/pubmed/11036689 The amount natural functions of lactic and acetic acids produced varies from species to speciessecondary metabolites. For exampleDemain AL, the referenced study showed that ''LFang A. 2000.]</ref>. plantarum'' produces more than twice These secondary metabolites are produced by the amount of lactic acid than ''L. brevis''pathways mentioned above, and ''Ldifferent strains probably regulate the enzymes involved in various pathways differently and produce different secondary metabolites <ref>Private correspondence with Richard Preiss from Dan Pixley. reuteri'' produced slightly more lactic acid than ''L 12/29/2015. brevis''</ref>. ''L. reuteri'' produced around twice as much acetic acid than ''L. brevis''Thus, different species and ''L. plantarum'' produced very little acetic strains can produce a wide variety of flavors and aromas (compare this to food grade lactic acidin which none of these secondary metabolites exist). The small amount of acetic acid produced by ''L. plantarum'' in this study was explained by oxygen exposure during sampling, while These secondary metabolite are the obligate heterofermentative species (''L. reuteri'' and ''L. brevis'') produced acetic acid as a direct result of their heterolactic carbohydrate fermentation and amino acid metabolism <ref name="Peyerpeyer_review"></ref>. Major secondary metabolites that
Both primary An example from one study showed that ''L. plantarum'' produced significantly more diacetyl, acetoin (yogurt-like flavor), and secondary metabolites play a large role in the flavor acetaldehyde than ''L. reuteri'' and aroma profile of wort fermented with ''LactobacillusL. brevis''. Secondary metabolites are These three compounds that are not directly were associated with dairy-related to growth 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 an organismcitric acid, which is found in wort. Ester production is generally insignificant, although significant ester formation has been found during malolactic fermentation in red wines, but often assist with survival and ethyl acetate has been found to be produced in malt based beverages <refname="peyer_review">[http://www.ncbisciencedirect.nlm.nih.govcom/science/article/pubmedpii/11036689 The natural functions of secondary metabolitesS0924224415300625 Lactic Acid Bacteria as Sensory Biomodulators for Fermented Cereal-Based Beverages. Demain ALLorenzo C. Peyer , Emanuele Zannini , Fang AElke K. 2000Arendt. 2016.]</ref>. These secondary metabolites are produced 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 the pathways mentioned above''L. brevis'', ''L. plantarum'', and ''L. reuteri'' (only 0.25-0.32 mg/L was found, and different strains probably regulate the enzymes involved in various pathways differently and produce different secondary metabolites flavor threshold of hexanoic acid is 5.4 mg/L <ref>Private correspondence with Richard Preiss from Dan Pixley[http://www.leffingwell.com/odorthre.htm Leffingwell & Associates website. Odor Thresholds. Retrieved 12/2930/2015.]</ref>) <ref name="Peyer"></ref>. Thus, different Heterofermentative species and strains can also produce a wide variety [[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 aromas their associated non-fusel alcohols (compare this 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 food grade lactic acid in which none form fruity flavors <ref name="peyer_review"></ref>. A few species, especially most strains of ''L. fermentum'', and some strains of these secondary metabolites exist)''L. delbrueckii subsp. These secondary metabolite are bulgaricus'', can produce ropiness in the result form of carbohydrate fermentation and amino acid metabolism exopolysaccharides, similar to [[Pediococcus]] <ref name="peyer_review"></ref>.
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 BeveragesS0308814617302911#t0005 Dongmo et al. Lorenzo C. Peyer (2017)] found 56 volatile flavor compounds, Emanuele Zannini including various esters, Elke K. Arendt. 2016.]</ref>. Some strains may also produce fusel alcohols , ketones, aldehydes, acids, ethers compounds, sulfur compounds, heterocyclic compounds, phenols, terpenes, lactones, and other off-flavorsseveral unidentified compounds. For example the referenced study found an accumulation of the fusel alcohol n-Porponal in the sample of Key compounds produced by ''L. reuteriLactobacillus''include acetaldehyde, β-Damascenone, furaneol, phenylacetic acid, 2-phenylethanol, 4-vinylguaiacol, sotolon, methional, vanillin, acetic acid, nor-furaneol, guaiacol and a small decrease of isovaleric acid coupled with a small increase of [https://enethyl 2-methylbutanoate.wikipedia Acetaldehyde was the most impactful aroma compound found followed by propan-1-ol and γ-dodecalactone.org Acetaldehyde was generally produced in much higher amounts (~23-64 µg/wiki/Hexanoic_acid hexanoic acid] L) by the select strains of ''L. brevisplantarum'', while ''L. plantarumamylolyticus'', and ''L. reuteribrevis'' (produced only 01.255-0.32 mg3 µg/L was found. In fact, and the flavor threshold levels of all of hexanoic acid is 5these compounds differed significantly based on the species and strain.4 mg/ The selected strains of ''L <ref>[http://www.leffingwell.com/odorthre.htm Leffingwell & Associates websitebrevis'' were associated as having worse aromas that were dominated by methional (cooked potatoes), acetic acid (vinegar), and nor-furaneol (caramel-like). Odor ThresholdsThe ''L. Retrieved 12plantarum'' strains selected were identified as producing more positive aromas from compounds such as β-damascenone (apple/30fruit juice), furaneol (strawberry), 2-phenylethanol (rose/2015.]</ref>caramel) and ethyl 2-methylbutanoate (citrus) <ref name="Peyer"></ref>. Heterofermentative species can Small but significant amounts of linalool and geraniol were also produce found, which are normally terpenes found in [[TetrahydropyridineHops|tetrahydropyridines (THP)hops]], which . Vanillan is the cause of "mousy" off-flavors formed from ferulic acid by some ''Lactobacillus'' species as well as ''Oenococcus oeni'' <ref name="CostelloDongmo">[http://pubswww.acssciencedirect.orgcom/doiscience/absarticle/10.1021pii/jf020341r Mousy Off-Flavor S0308814617302911 Key volatile aroma compounds of Wine:  Precursors and Biosynthesis lactic acid fermented malt based beverages – impact of the Causative N-Heterocycles 2-Ethyltetrahydropyridinelactic acid bacteria strains. Sorelle Nsogning Dongmo, Bertram Sacher, 2-AcetyltetrahydropyridineHubert Kollmannsberger, and 2-Acetyl-1-pyrroline by Lactobacillus hilgardii DSM 20176Thomas Becker. Peter J2017. Costello and Paul A doi:http://dx. Henschkedoi. 2002org/10.]<1016/ref>j. 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>foodchem. A few species, especially most strains of ''L2017. fermentum'', and some strains of ''L02. delrueckii subsp091. bulgaricus'', can produce ropiness in the form of exopolysaccharides, similar to [[Pediococcus]] <ref name="peyer_review"></ref>.
The type of grain that the ''Lactobacillus'' is fermented in may also play a role in the types and amounts of secondary metabolites that are produced. One study compared volatile acids produced by a probiotic strain of ''L. plantarum'' (NCIMB 8826) when fermented in oats, barley, malted barley, and wheat. In oats, there was slight increase in oleic acid and linoleic acid and a decrease when fermented in wheat, barley, or malted barley. In malted barley, there were small increases in flavor active compounds such as furfural ("almond" flavor), 2-ethoxyethyl acetate and isoamyl alcohol, but little to none detected when fermented in oats, wheat, or unmalted barley. Acetic acid production was higher in barley and malted barley than it was in oats and wheat. Many other organic acids in the oats, wheat, barley, and malted barley were supposedly taken up by the ''L. plantarum'' during fermentation. In barley, there were trace amounts of new acids created that were not already in the barley itself <ref>[http://www.sciencedirect.com/science/article/pii/S0308814609004373 Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-based substrates Ivan Salmeron, Pablo Fuciños, Dimitris Charalampopoulos, Severino S. Pandiella. 2009.]</ref>. Some species of ''Lactobacillus'', including ''L. lactis'' and ''L. plantarum'', produce diacetyl (which can be reduced to acetoin and 2,3-butanediol) as an intermediate metabolite from consuming sugar, citrate, and amino acids. However, citrate levels are rather low in malted barley (but higher in sorghum), and diacetyl production has been observed to be very low in barley and oat based worts <ref name="peyer_review"></ref>.
Aging has a large impact on the aromas and flavors produced by ''Lactobacillus'' fermentation over time and is typically influenced by 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.

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