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Lactobacillus

2,300 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>.
<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:
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>

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