Lactobacillus

Lactobacillus (often referred to as Lacto) is a genus of lactic acid bacteria (LAB) which produces acidity and sour flavors in the form of lactic acid (and sometimes acetic acid) found in lambics, Berliner Weiss, sour brown ales, and gueuze. Lacto can form a pellicle. See Pediococcus, Brettanomyces, Saccharomyces, and Mixed Cultures charts for other commercially available cultures. See the Sour Worting and Mixed Fermentation pages for brewing techniques with Lactobacillus. See the More Sour Worting Methods section for culturing Lactobacillus from grains, yogurt, or probiotics.

Commercial Lactobacillus Cultures

Culture Charts

Name	Mfg#	Taxonomy	CO2 Producer (Het/Hom)	Starter Note	Fermentation/Other Notes
Brewing Science Institute	L. delbrueckii	Lactobacillus delbrueckii			A Lactobacillus bacteria that produces a clean lactic sourness.
White Labs	WLP677	L. delbrueckii	Heterofermentative	no stir plate, room temp	Incubate at > 90°F and < 117°F for 5-7 days for greater lactic acid production.
White Labs	WLP672	L. brevis	Heterofermentative	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. Temperature range: 70-95°F; 80% attenuation (this may not reflect actual attenuation of wort in a real brewery; see reference ).
Wyeast	5335	L. buchneri		1L starter, 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.
Wyeast	5223-PC	L. brevis	Heterofermentative	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 2L starter of 1.020 dme) and set it on a stir plate at 95°F . The beer wort was not aerated, and the fermenter was flushed with CO2. These methods need verification.
Omega Yeast Labs	OYL-605	L. brevis, delbrueckii, and plantarum blend		1 liter starter at room temperature for 24-48 hours. no stir plate	Quick souring. Pitch into 65°F-90°F. Holding temperature is not required. No longer contains delbruekii .
GigaYeast	GB110	L. delbrueckii?	Heterofermentative	no stir plate, room temp	Incubate at 98°F for 48-72 hours; use low IBU wort. Sometimes referred to as GigaYeast's "Fast Acting Lacto". This strain is hop sensitive .
RVA Yeast Labs	RVA 600	L. rhamnosus GG	Homofermentative	No starter necessary	Homofermentative Lacto strain found in probiotics; sensitive to hops; does well at room temperature.
SouthYeast Labs	Lactobacillus 1	Unknown	Heterofermentative		Source: Spontaneously infected beer (South Carolina). Best suits Light sours, gose, farmhouse saison (medium/high acidity).
SouthYeast Labs	Lactobacillus 2	Unknown	Homofermentatative		Source: Prickly pear fruit (South Carolina). Best suits strong sours, and lambic (high acidity).

Manufacturer Tips

Omega Yeast Labs on OYL-605

The following is a statement by Lance Shaner, owner of Omega Yeast Labs:

Lance Shaner: Full disclosure: I own Omega Yeast Labs. Pitching at 120F is a bad idea with this blend. The bug doing most of the work in this blend is Lactobacillus plantarum. The best temp for plantarum is 80-90F. It does not work [as well] over 100F. Also, we regularly make a 1 liter starter with the Lacto blend for faster souring. Simply pitch the contents of the pouch into 1 liter of sterile 1.040 wort and let sit for 24 hours at 70-80F before pitching (no need to stir). Adi Hastings mentioned the imperial stout we just kettle soured. We previously brewed a Berliner using the same method. At 17 hours, pH was at 3.42 and temp was 75F (original pitch temp was 85F). At 40 hours, pH was 3.24, at which time we boiled. Lower pH in the Berliner compared to the stout at 17 hours likely has to do with different buffering capacities in different worts.

Wyeast on 5335

The following is an excerpt with Jess Caudill, Brewer/Microbiologist, at Wyeast Laboratories, Inc. concerning usage of Wyeast 5335 and making a Berliner Weissbier.

1. Use 5335.
2. If using our 5335, don’t use ANY hops. You can always blend in some IPA or hopped wort after souring takes place if you really need some bitterness or hop flavor/aroma in the beer.
3. From one 5335 pack, make a 1L starter with 1.020 DME sterile wort. No O2! Incubate at 90°F if possible for 5-7 days.
4. Brew your 5 gallons of wort. Again… no hops. Sterilize the wort. (No need for sour mashes). Cool to 90°F and add 1L 5335 starter. No O2. Try to maintain 90°F for 5-7 days depending on how sour you want the beer.
5. After 5-7 days, cool wort to around 68. Pitch with a low pH tolerant strain such as 1007 or 2124. No O2. Ferment for around 1-2 weeks… until you hit terminal.
6. Package beer. If bottle conditioning, use 4021 as a bottling strains. Very tolerant to low pH.

RVA Yeast Labs on RVA 600

A great lactic acid bacterial strain that will add a pleasant tangy sourness. RVA 600 is a pure culture of Lactobacillus rhamnosus GG which is found in many commercial probiotic products which have been shown in clinical studies to have many beneficial effects. These are homofermentative (only produces lactic acid, no carbon dioxide or ethanol) and are hop-sensitive. For more pronounces souring add before you add your yeast. You can sour to taste then add a yeast strain to outcompete the bacteria. Again, hop sensitive so easy on them…or dry hop the heck out of it! You may also want to experiment with blending sour low hop beer with an ale strain beer.

... the amount of bacteria in our homebrew units should eliminate the use of a starter. Pitching on the warm side will speed up the souring but RVA 600, a pure culture of Lactobacillus rhamnosus GG, the first commercially available probiotic propagated for use in brewing, does just fine at room temp. We had originally developed RVA 600 as a mix but fell in love with the pure strain.

SouthYeast Labs on Lactobacillus 1 and 2

The L2 strain grows best at 86°F-100°F (30°C-37.7°C), and does not work well over 100°F. Keep IBU's low to none. L1 will likely be discontinued due to high amounts of acetic acid production .

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).

General Advice

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Nick Impellitteri from The Yeast Bay on general Lactobacillus cell growth:

"I typically grow it by itself anaerobically in MRS media. Seems to work very well and results in good growth. I've personally had the best success with MRS media and in an anaerobic environment, though I know some lactobacillus strains grow aerobically just fine. The problem with growing lactic acid bacteria is the acid they produce will eventually inhibit their own growth. MRS contains a buffer to help combat the drop in pH as a result of LAB metabolism, which keeps the pH around 6-6.5 (I think) for optimal growth. I usually grow them at 35 C, but sometimes incubator space is at a premium (like right now) and I just spin it on the benchtop!" .

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Bryan Heit of Sui Generis Brewing blog on Lactobacillus and Hop Tolerance:

"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 lacto 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.

Edit: I tried to add (but facebooks aversion to white space almost defeated me) that 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 option; 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.

I'm hoping to have a blog post on that sometime in the next few months, but life is nuts right now so I offer no guarantee."

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Bryan Heit of Sui Generis Brewing blog on storing dried Lactobacillus, such as probiotics or Dry Yeast for Sour Ales BlackManYeast:

"Yes, refrigerate them. In the lab course I run we compare probiotics stored at room temp versus in the fridge - viable bacterial numbers dip off ~80x faster in non-refrigerated samples."

And in regards to liquid cultures and storage:

"I'm not sure about the liquid cultures - I freeze mine at -80°C (lab freezer), which (stores) indefinitely. As a rule refrigerated liquid cultures should last longer... but there is anecdotal evidence (for some species) of poorer survival of refrigerated versus room temp in liquid cultures. IMO, stable temps are likely more important for non-frozen stocks than hitting an 'ideal' temperature."

Commercially available Lactobacillus strains and their pH change over time

All data provided by Matt Humbard

pH change at 86*f

pH change of Lactobacillus at 86° f. Data provided by: Matt Humbard

Time (hours)	Wyeast Lactobacillus buchneri	White Labs Lactobacillus brevis	White Labs Lactobacillus delbrueckii	Omega Lactobacillus plantarum
0	5.9	5.9	5.9	5.9
6	5.57	5.13	5.59	4.81
18	4.4	3.94	4.62	3.6
24	4.2	3.78	4.6	3.45
75.5	3.88	3.44	4.46	3.27
120	3.85	3.4	4.4	3.25

pH change at 98*f

pH change of Lactobacillus at 98° f. Data provided by: Matt Humbard

Time (hours)	Wyeast Lactobacillus buchneri	White Labs Lactobacillus brevis	White Labs Lactobacillus delbrueckii	Omega Lactobacillus plantarum
0	5.9	5.9	5.9	5.9
6	5.61	5.05	5.55	5.02
18	4.15	3.74	4.54	3.22
24	4.05	3.67	4.54	3.2
75.5	3.5	3.31	4.32	3.22
120	3.5	3.29	4.3	3.21

pH change at 102

pH change of Lactobacillus at 102° f. Data provided by: Matt Humbard

Time (hours)	Wyeast Lactobacillus buchneri	White Labs Lactobacillus brevis	White Labs Lactobacillus delbrueckii	Omega Lactobacillus plantarum
0	5.9	5.9	5.9	5.9
6	5.45	5.02	5.5	5.11
18	4.13	3.73	4.61	3.4
24	4.06	3.6	4.58	3.29
75.5	3.72	3.33	4.47	3.23
120	3.68	3.3	4.4	3.2

pH change at 108

pH change of Lactobacillus at 108° f. Data provided by: Matt Humbard

Time (hours)	Wyeast Lactobacillus buchneri	White Labs Lactobacillus brevis	White Labs Lactobacillus delbrueckii	Omega Lactobacillus plantarum
0	5.9	5.9	5.9	5.9
6	5.6	4.95	5.51	4.99
18	4.49	3.71	4.8	3.54
24	4.37	3.52	4.6	3.45
75.5	4.33	3.35	4.62	3.34
120	4.29	3.3	4.51	3.3

Metabolism

All metabolism by Lactobacillus, including growth, will require sugar to be consumed and lactate (lactic acid) to be produced. Two categories of metabolism exist, homofermentative and heterofermentative. Homofermentative metabolism is described as the cell catabolizing one molecule of glucose to yield two molecules of pyruvate, which is reduced to two molecules of lactate (lactic acid). Heterofermentative metabolism is described as the cell catabolizing one molecule of glucose into one molecule of CO2, one molecule of glyceraldehyde phosphate, and one molecule of acetyl phosphate. The molecule of glyceraldehyde phosphate is reduced to one molecule of lactate, and the acetyl phosphate is reduced to on molecule of ethanol. In summary, homofermentative LAB produces only lactic acid, while heterofermentative LAB produce lactic acid, CO2, and ethanol .

External Resources

• Physiology of Flavors in Beer – Lactobacillus Species - Matt Humbard's overview of different species of Lactobacillus physiology, discussion on homoefermentative vs heterofermentative physiology, which species can ferment different types of sugars, and secondary metabolites. Extensive data points included.

• Lactobacillus Starter Guide by Derek Springer. - Information about starters for both pure strains, as well as culturing from grains.

• 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.

• Lacto Starters, by Bryan Heit of Sui Generis blog - Step by step guide to making a starter for Lactobacillus.