13,691
edits
Changes
added graphs for hop sensitivity data
| [[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>.
|-
| [[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 DME) 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 10<sup>8</sup> (100 million) cells/mL (10 billion cells in a 100 mL homebrew pouch) <ref name="wyeast_cellcounts"></ref>.
|-
| [[Omega Yeast Labs]] || OYL-605 || L. brevis, <span style="text-decoration: line-through;">delbrueckii</span>, and plantarum blend || Hetero/Hetero <ref name="mtf_wiki_shaner"></ref> || 1 liter starter for a 5 gallon batch of beer at room temperature for 24-48 hours. No stir plate unless kept anaerobic. || Quick souring. Pitch into 65°F-95°F <ref name="adi_oyl605"></ref>. Holding temperature is not required. No longer contains delbruekii <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1065268213501392/?comment_id=1065669443461269&offset=0&total_comments=18&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Raymond Wagner of Oso Brewing Co on Milk The Funk. 4/30/2015.]</ref>. Don't use any hops if possible. 2 IBU is a good target if hops must be used <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1092523807442499/?comment_id=1092571350771078&offset=0&total_comments=6&comment_tracking=%7B%22tn%22%3A%22R1%22%7D Conversation with Lance Shaner on MTF in regards to IBU tolerance of OYL-605. 6/15/2015.]</ref>. Contains ~150 billion cells per homebrew pitch <ref name="sbb2.0">[http://sourbeerblog.com/lactobacillus-2-0-advanced-techniques-for-fast-souring-beer/ Lactobacillus 2.0 – Advanced Techniques for Fast Souring Beer. Sour Beer Blog. Matt Miller. 11/18/2015. Retrieved 11/19/2015.]</ref>.
| [[SouthYeast Labs]] || Lactobacillus 2 || Unknown || Homofermentatative || || Source: Prickly pear fruit (South Carolina). Best suits strong sours, and lambic (high acidity).
|-
| [[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 || ||
|-
| [[Escarpment Laboratories]] || Lactobacillus Blend || L. brevis and L. plantarum || Heterofermentative || || This blend is designed to be usable at a wide range of temperatures, and is especially suited for kettle souring/sour worting. We recommend pre-acidifying wort to 4.5 with lactic acid, then pitching the Lactobacillus blend in a CO2-purged kettle or fermentor at 32-42°C.
| [[Escarpment Laboratories]] || Lactobacillus brevis || L. brevis || Heterofermentative || || This strain is moderately hop-tolerant, and as such it can also be used for long-term souring of <10IBU beers. It also performs well in kettle souring/sour worting where fast and clean lactic acidity is desired. We recommend pre-acidifying wort to 4.5 with lactic acid, then pitching the Lactobacillus blend in a CO2-purged kettle or fermentor at 35-45°C.
|-
| [[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>.
|}
====[[White Labs]] on WLP672====
"It is intended for secondary, so you only need to do a starter if you are doing a primary fermentation with it. DME would be the best substrate... Since its a Lacto species, you don't really want to aerate it. A slow stir-plate would be good, to keep it moving, but not much more than that." - Sarah Neel, White Labs, Sales and Customer Service (quoted with permission).
Since WLP672 is meant for long term aging, it is not preferred to use for kettle souring/wort souring. See also [[100%25_Lactobacillus_Fermentation#History|100% ''Lactobacillus'' fermentation]].
===General Advice===
Another thing to consider is that achieving a pH of 4 as fast as possible is advisable for preventing off-flavors from contaminating microbes <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1180630378631841/?comment_id=1181674265194119&reply_comment_id=1181715048523374&comment_tracking=%7B%22tn%22%3A%22R7%22%7D Conversation with Bryan of Sui Generis Blog on MTF regarding speed of acid production with Lacto. 11/20/2015.]</ref>. Larger pitch rates tend to achieve a lower pH faster <ref name="bryan_lacto_starters"></ref>. Therefore, unless using [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's starter procedure]], pitching 0.5-1 liters of starter for 5 gallons of wort is advisable in general. Even when using [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's starter procedure]], over-pitching ''Lactobacillus'' is not a concern (pitching an overly massive starter wort could produce undesirable flavors), so the same pitch rate should still be considered unless the brewer is confident that high growth rate has been achieved in the starter. Other factors that might affect the effectiveness of a volume based starter is the species/strain of the ''Lactobacillus'' being used, how much yeast contamination has occurred, and how old the ''Lactobacillus'' starter is. Some species/strains may require a larger volume of starter, as well as if yeast has contaminated the starter or wort (see [[Lactobacillus#100.25_Lactobacillus_Fermentation|100% Lactobacillus Fermentation]]). If a ''Lactobacillus'' culture is older than 1 month, then a fresh starter should be made. Keeping a separate Erlenmeyer flask for ''Lactobacillus'' starters can help to prevent yeast contamination <ref>Private correspondence with Richard Preiss to Dan Pixley. 11/20/2015.]</ref>, as well as using sterilization equipment such as an autoclave or pressure cooker.
Starter mediums that brewers have used include unhopped 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>.
Although more experiments and probably needed, agitation is believed to be an important factor for both yeast and bacteria in general. Gentle stirring on a stir plate or orbital shaker, or frequent gentle manual agitation leads to faster growth and a higher number of organisms. Agitation keeps the microbes in solution. It also maximizes the microbes' access to nutrients and disperses waste evenly. In a non-agitated starter, the microbes are limited to the diffusion rate of nutrients, leading to a slower and more stressful growth <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1174865305875015&reply_comment_id=1176092372418975&total_comments=1&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Bryan of Sui Generis Blog about starters and agitation. 11/09/2015.]</ref>. If agitation is not possible for whatever reason, a successful starter can be made without agitation. Sam Aeschlimann reported good success with ''Lactobacillus'' starters that are not agitated <ref name="Sam_starter2"></ref>.
Although 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:
* [[Hops#Antimicrobial_Properties|Hops antimicrobial properties]].
* [http://www.garshol.priv.no/blog/337.html How hops prevent infection, by Lars Garshol].
* [https://www.youtube.com/watch?v=J2g5P7ZlGn4 Per Buer's Video Demonstration of how dry hopping inhibits ''Lactobacillus''.]
* [https://www.ratebeer.com/forums/lab-and-hops_289071.htm "CLevar" on Ratebeer.com data point on ''Lactobacillus'' being inhibited by hops, but not as much by iso-alpha acid hop extract.]
====Storage====
* See [http://basementbrewlab.com/lab/lab-media/sla/ Rogosa SL Agar (Lactobacillus); Basement Brew Lab, by Dustin Metzger] for more information.
====Effects on Mixed Fermentation====
The presence of ''Lactobacillus'' can stall or slow yeast fermentation. This is likely a combination of low pH as well as the ability of lactic acid to change the way yeast ferments. Normally, yeast will ferment glucose first before any other sugars that are also available. The presence of lactic acid appears to change the way yeast ferments by allowing them to consume multiple types of sugars regardless of whether or not glucose is preset. See [[Lactic Acid]] for more information.
== Commercially available Lactobacillus strains and their pH change over time ==
| 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¬if_t=group_comment_reply¬if_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 Acid production is at it''L. plantarum'' ferments glucose firsts highest during the exponential growth phase, but continues into the stationary and then fructose if it is availabledecline phases. ''Typically just under 50% of the lactic acid produced is L. reuteri'' ferments maltose first, -lactic acid (more nutritionally relevant) 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 slight majority is D-lactic acid <ref name="peyer_reviewPeyer"></ref>. Peak sugar consumption without competition from yeast is typically 48 hours, The amount of lactic and very little alcohol or CO2 is acetic acids produced (around 0varies from species to species.10-0.30% ABV For example, 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 referenced study showed that ''LactobacillusL. plantarum''. Rather produces more 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 twice the growth amount of lactic acid than ''LactobacillusL. brevis'' <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://phdinbeerL.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/ Matt Humbardreuteri's 'produced slightly more lactic acid than 'Physiology of Flavors in Beer – Lactobacillus Species'' blog article]L. A small number of strains of brevis''Lactobacillus'' can also break down polysaccharides and starches. They are referred to as "amylolytic LAB". They generally belong to the species ''LbL. manihotivoransreuteri'', produced around twice as much acetic acid than ''L. fermentumbrevis'', and ''L. amylovorusplantarum'', produced very little acetic acid. The small amount of acetic acid produced by ''L. amylophilusplantarum''in this study was explained by oxygen exposure during sampling, while the obligate heterofermentative species (''L. plantarumreuteri'' or and ''L. amylolyticusbrevis''. This seems to be associated with ) produced acetic acid as 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 direct result of glucose, maltose, or sucrose their heterolactic fermentation <ref name="peyer_reviewPeyer"></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.
===External Resources===
* [https://www.microbes.info/resource-topic/lactobacillus-eubacteria-microorganisms-general-microbiology Microbes.info links to species databases and microbiology resources.]
* [https://matthumbard.wordpress.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/ Physiology of Flavors in Beer – Lactobacillus Species] - Matt Humbard's overview of different species of Lactobacillus physiology, discussion on homofermentative vs heterofermentative physiology, which species can ferment different types of sugars, and secondary metabolites. Extensive data points included.
* [http://phdinbeer.com/2015/08/05/beer-microbiology-lactobacillus-ph-expeirment/ Beer Microbiology – Lactobacillus pH experiment] - Matt Humbard's experiment to determine final pH of individual ''Lactobacillus'' strains used in the graphs on this page.