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In addition to the starter information given in the [[Lactobacillus#Manufacturer_Tips|Manufacturer Tips]] above, this section includes general advice for ''Lactobacillus'' starters for homebrewers and brewers. For growing ''Lactobacillus'' in a lab environment, or from an initial grouping of cells from a plate/slant or smaller cell count, MRS media is the most efficient growth media. However, for full pitches of ''Lactobacillus'' in beer/wort, brewers probably don't want to add that much MRS media to their beer since MRS media has a distinct odor and smell that would not be desirable in beer <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1152135114814701/?comment_id=1152674674760745&offset=0&total_comments=9&comment_tracking=%7B%22tn%22%3A%22R0%22%7DConversation with Lance Shaner and Nick Impellitteri on Lacto starters on MTF. 09/22/2015.]</ref>. Therefore, growing ''Lactobacillus'' in a wort-based starter media is recommended for building full pitches of ''Lactobacillus''. See [[Lactobacillus#External_Resources|External Resources]] for additional starter guides.
Pitching ~0.5-1 liter per ~20 liters of wort (~0.75-1 gallon per barrel) of ''Lactobacillus'' starter is the general guideline. The exact advisable pitching rates of commercial cultures may differ from manufacture to manufacturer. <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1077639885597558/ MTF thread started by Brad Primozic. 5/29/2015.]</ref>. Counting cells and pitching an exact cell count is the best approach. However, counting cells of ''Lactobacillus'' under a microscope can be difficult to achieve due to the small size of bacteria cells, so starter volumes are generally used instead when talking about pitching rates for ''Lactobacillus'' <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1068323126529234/?comment_id=1068337639861116&offset=0&total_comments=47&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation on MTF with Bryan Heitof Sui Generis Blog. 5/6/2015.]</ref>. Matt Miller of Sour Beer Blog and Richard Preiss of Escarpment Yeast Labs advise that if ideal growth (1-2 billion cells/mL) can be achieved (for example by using [[Lactobacillus#Samuel_Aeschlimann.27s_Starter_Procedures|Samuel Aeschlimann's starter procedure]] or MRS media), then pitching as little as 100-125 mL of fresh ''Lactobacillus'' starter for 5 gallons of beer can achieve desirable acidity within 24 hours <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1180630378631841/?comment_id=1180733761954836&reply_comment_id=1180740128620866&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Richard Preiss on MTF regarding Lacto starters. 11/19/2015.]</ref><ref name="sbb2.0"></ref>. Moderately ideal growth in a starter results in around 500 million cells/mL, resulting in a 400 mL starter for 5 gallons, and low growth results in around 100 million cells/mL, resulting in a 2 liter starter for 5 gallons, according to Matt Miller (see [http://sourbeerblog.com/lactobacillus-2-0-advanced-techniques-for-fast-souring-beer/ Matt Miller's article] for more details) <ref name="sbb2.0"></ref>. However, it is difficult to verify how much growth has occurred with cheaper microscopes, so these rates may or may not be helpful to the brewer.
Another thing to consider is that achieving a pH of 4 as fast as possible is advisable for preventing off-flavors from other 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 Heit 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="heit_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, 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>.
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.
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 Heit of Sui Generis Blog about starters and agitation. 11/09/2015.]</ref>. Although ''Lactobacillus'' are aerotolerant and oxygen usually does not negatively affect their growth (except in the case of ''L. plantarum'', which has been shown to produce acetic acid when exposed to oxygen<ref name="microbewiki_plantarum"></ref>), it is also generally not needed (an exception to this may be ''L. brevis'', which has been shown to increase growth rates in the presence of oxygen <ref name="brevis_aeration"></ref>). Therefore, it is generally best practice to prevent aerating the starter with an airlock for ''Lactobacillus'' starters.
=====Samuel Aeschlimann's Starter Procedures=====
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 (MRS media 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 Heit of Sui Generis Blog regarding the use of chalk as a buffer in Lacto starters. 11/20/2015.]</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.
# The chalk is not desirable to pitch into the beer because of its buffering effect. The chalk will sediment within hours of being added to the starter, or if a stir plate is used, a couple of hours after the stir plate is turned off <ref>[https://en.wikipedia.org/wiki/Stokes%27_law Stokes' law. Wikipedia. retrieved 09/24/2015.]</ref><ref name="Sam_starter2"></ref>. The ''Lactobacillus'' should stay in suspension for at least a day or two after the starter is done, so swirling the starter isn't necessary, although it is certainly an option. If the starter is swirled, allow a couple of hours for the chalk to sediment out again. After the chalk sediments to the bottom of the flask, pour all of the liquid from the top of the starter into the wort/beer, and leave the chalk sediment behind. Avoid cold crashing the starter because it can have an adverse effect on the bacteria's health <ref name="heit_lacto_starters">[http://suigenerisbrewing.blogspot.ca/2015/05/lacto-starters.html Heit, Bryan. Lacto Starters. Sui Generis Blog. Retrieved 6/15/2015.]</ref><ref name="sam_starter"></ref>.
See [http://suigenerisbrewing.blogspot.ca/2015/05/lacto-starters.html ''Lacto Starters'', by Bryan Heit of Sui Generis blog] for additional information on ''Lactobacillus'' starters.
====Cell Growth====
Most ''Lactobacillus'' species have a thermal death rate of ~145°F (63°C). Freezing without glycerol will kill most cells, but it is possible for a very small number of cold-resistant mutant cells to survive <ref>[http://fermentationnation.net/2015/11/episode-26-quality-assurance-w-jessica-davis-of-the-bruery/ Fermentation nation Podcast interview with Jessica Davis, QA for The Bruery.</ref> (~1:19:00 in).
All [https://en.wikipedia.org/wiki/Prokaryote prokaryotes], which includes all bacteria in general, are categorized based on the levels of oxygen in their environment in which they can grow and how they utilize oxygen if at all <ref name="Todar_nutgro4"></ref>. ''Lactobacillus'' species are usually considered to be "facultative anaerobes" (or "facultative aerobes") <ref name="todar_lactics4"></ref>. Facultative anaerobes make energy from oxygen if it is present via the [https://en.wikipedia.org/wiki/Oxidative_phosphorylation oxidative phosphorylation pathway], but otherwise engage in anaerobic fermentation <ref>[http://www.ncbi.nlm.nih.gov/books/NBK21208/ Biochemistry. 5th edition. Berg JM, Tymoczko JL, Stryer L. 2002. Chapter 18.]</ref><ref>[http://inst.bact.wisc.edu/inst/index.php?module=book&type=user&func=displayarticle&aid=111 Virtual Microbiology Textbook. Department of Bacteriology, University of Wisconsin-Madison. Retrieved 12/02/2015.]</ref>. Lactobacilli, however, are unique in that they blur the line between facultative anaerobes and another class of prokaryotes known as "aerotolerant anaerobes" (anaerobes that do not use oxygen to generate energy, but can grow in the presence of oxygen). They can utilize oxygen, but not through the oxidative phosphorylation pathway. They use an alternative pathway instead. This pathway uses flavine-containing oxidases and peroxidases to carry out the oxidation of NADH2 using O2. Lactobicalli then are a somewhat special case of facultative anaerobes <ref name="bergey">Bergey's Manual of Systematic Bacteriology, 2nd edition. pg 471</ref><ref>Correspondence with Bryan Heit of Sui Generis Blog from Dan Pixley. 12/01/2015.</ref>. The important take away here is that ''Lactobacillus'' do not care if oxygen is present in order to grow and produce energy for themselves and lactic acid for brewers.
====Hop Tolerance====
:''Editor's note: the following are comments by Bryan Heit of [http://suigenerisbrewing.blogspot.com/ Sui Generis Brewing blog]''
There is a fair bit of research into hop tolerance out there; its 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 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 ~18 IBU, but past that point the resistance appears to remain inducable. I'm hoping a few more generations will provide me with a permanently tolerant strain.
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 Heit 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 Heit of Sui Generis Blog on Milk The Funk regarding Lactobacillus hop tolerance. 09/28/2015.]</ref>.
See also:
====Storage====
For dried ''Lactobacillus'', such as probiotics or [[Dry Yeast for Sour Ales BlackManYeast]] products, [http://suigenerisbrewing.blogspot.com/ Bryan Heitof Sui Generis Blog's] lab studies have shown that they can lose viability ~80 times faster at room temperature than when stored at refrigeration temperatures. Therefore, it is recommended to store dried ''Lactobacillus'' at refrigeration temperatures.
Liquid cultures become stressed by two factors: storage in an acidic environment, and storage without sugar <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1154970097864536/?comment_id=1156656834362529&offset=0&total_comments=12&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Bryan Heit of Sui Generis Blog on stress factors in storing Lacto. 09/28/2015.]</ref>. Sugar storage creates more acid as the ''Lactobacillus'' ferments it, so it may not be ideal unless the ''Lactobacillus'' is continually fed. Ideally, liquid cultures of ''Lactobacillus'' should be stored frozen with 20% glycerol, or refrigerated as slants with water or mineral oil. Also, there is anecdotal evidence that certain species may survive better at room temperature. Bryan hypothesizes that stable temperatures may be more important than storing at an "ideal" temperature <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1067614393266774/ Conversation with Bryan Heit of Sui Generis Blog on Milk The Funk. 05/04/2015.]</ref>. For instructions on how to make slants at home capable of storing any microbe for potentially 2+ years, [http://suigenerisbrewing.blogspot.com/2015/11/easy-home-yeast-banking-and-video.html see Bryan Heit's video on Sui Generis Brewing (requires a pressure cooker)].
A practical option for brewers without a pressure cooker is to store the liquid culture with a few grams of a buffering chemical such as calcium carbonate (chalk), potassium phosphate, calcium sulfate (gypsum), or calcium hydroxide (pickling lime). The exact amounts should be adjusted to reach a pH of about 4.0-6.0 for the entire solution (begin with 1 or 2 grams per liter, and adjust as needed) <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1095449350483278/?comment_id=1095492120479001&offset=0&total_comments=23&comment_tracking=%7B%22tn%22%3A%22R6%22%7D Conversation with Adi Hastings on MTF. 6/20/2015.]</ref>.
==Metabolism==
:''Editor's note: the following section was reviewed for accuracy by MTF members [http://suigenerisbrewing.blogspot.com/ Bryan Heitof Sui Generis Blog], [http://phdinbeer.com/ Matt Humbard], [http://www.omegayeast.com/ Lance Shaner], and [http://www.escarpmentlabs.com/ Richard Preiss].''
===Types of Metabolism===
[[File:Homolactic heterolactic.JPG|thumb|upright=2.5|Homolactic and Heterolactic pathways <ref name="Effects on Food Properties">[https://books.google.com/books?id=6SLB1j0rX78C&pg=PA76&lpg=PA76&dq=what+is+obligatory+facultatively+heterofermentative&source=bl&ots=6E4DB8Px_X&sig=PXZTNO4fB3ifCF_qAl5_I4euKmk&hl=en&sa=X&ei=Z-10VZqvFIWyggTInIHYBw&ved=0CCsQ6AEwAw#v=onepage&q=what%20is%20obligatory%20facultatively%20heterofermentative&f=false Fermentation: Effects on Food Properties. Bhavbhuti M. Mehta, Afaf Kamal-Eldin, Robert Z. Iwanski. CRC Press, Apr 12, 2012. Pg 76,77.]</ref>]]
* Despite some beliefs by brewers, ''Lactobacillus'' has not been shown to produce significant levels [[Butyric Acid|butyric acid]] (see the [[Butyric Acid|butyric acid]] page for more information) in the presence of oxygen or otherwise in brewer's wort. Some species might be able to produce small amounts of [[Isovaleric_Acid|Isovaleric Acid]] in the presence of ''Streptococcus thermophilus'' or another alpha-keto acid producing microorganism (see the [[Isovaleric Acid]] page for details).
* Although ''Lactobacillus'' are not not inhibited by oxygen, they are generally not considered to be organisms that produce oxygen-dependent metabolites. Some heterofermentative species produce acetic acid, butyric acid (not much in wort, but more so in fermented milk products), and propanoic acid, but they always do so through an anaerobic pathway. The presence of oxygen does not affect metabolism in general <ref name="heit_oxygen_LAB">[https://www.facebook.com/groups/MilkTheFunk/permalink/1182597671768445/?qa_ref=qd&comment_id=1182773928417486&reply_comment_id=1183047521723460&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Bryan Heit of Sui Generis Blog on the effects of oxygen in LAB on MTF. 11/23/2015.]</ref>.
* The major off-flavor that some ''Lactobacillus'' strains might produce is acetaldehyde <ref name="heit_oxygen_LAB"></ref>. ''Saccharomyces'' will help to metabolize the acetaldehyde produced by ''Lactobacillus''.
* [http://www.fivebladesbrewing.com/lactobacillus-starter-guide/ Lactobacillus Starter Guide by Derek Springer.] - Information about starters for both pure strains, as well as culturing from grains.
* [https://eurekabrewing.wordpress.com/2015/05/18/evaluate-starter-media-to-propagate-lactobacillus-sp/ Evaluate starter media to propagate Lactobacillus sp., Eureka Brewing Blog, by Samuel Aeschlimann.] - This experiment showed that growing Lactobacillus in '''10°P DME, 10% apple juice + CaCO3 (20 g L-1) + yeast nutrients''' lead to the best growth results, and close to expensive MRS media growth results.
* [http://suigenerisbrewing.blogspot.ca/2015/05/lacto-starters.html Lacto Starters, by Bryan Heit of Sui Generis blog] - Step by step guide to making a starter for Lactobacillus.
* [http://www.garshol.priv.no/blog/335.html Yeast terminology, part 2: bacteria, by Lars Garshol.]
* [https://www.youtube.com/watch?v=9a-ZpF2LDm8 Elke Arendt, presentation at Belgian Brewing Conference 2015 on scientific findings in LAB in malting and brewing.]