Stealth viable lactic acid bacteria growth detection within 4 hours
A major issue for brewmasters during brewing process is to be able to identify any abnormal lactic acid bacteria growth very quickly without false positives.
A very dangerous & critical situation is the stealh growth of lactic acid bacteria ruining your wort or beer.
Why do we speak of stealth growth? Hop resistant lactic acid bacteria are very difficult to grow on standard agar media used for lactic acid bacteria (1).
Why? because these bacteria are used to the harsh unfriendly beer environment, and suddenly change their environment for a very rich growth media. It will be exactly as if a starving person, would be suddently overfed in a feast, that person may die because his/her organism is not used to take so much food.
Are there available rapid methods alternatives enabling immediate corrective action?
Not really. PCR method are costly, time consuming, and require a minimum of 48 hours incubation in selective media to just give the presence/absence of a specific strain. Besides PCR does not reveal of your lactic acid bacteria is dead or alive. Dead DNA binds easily on particles, dead bacteria maintain their DNA inside their cell. If a lactic acid bacteria is not growing during incubation time with a PCR kit using also a selective media, you won’t notice it.
Lactobacilus lindneri a known as a major beer spoiler which “goes under the radar” as many are either fastidious to cultivable or are viable but not culturable on standard plate count media, but grows very well in beer.
When lactic acid bacteria reach critical levels of 10 exp5 /mL of beer it may trigger irreversible enzymatic beer spoilage activity. For example as few as 20,000 pediococci per ml in fermenting beer have been reported (2) to produce a diacetyl concentration of 0.36 mg/liter.
Is classic Atp Bioluminescence detection the solution?
Other methods as Atp bioluminescence methods have been used. Atp bioluminescence is based on the principle that only living cells produce a molecule ATP or Adenosin Triphosphate. Once this ATP is released from viable cells, when in contact with a substrate-enzyme Luciferin-luciferase (used by fireflies to produce light), it produce a bioluminescent reaction.
Higher the amount of ATP higher the bioluminescence. There are however two issues to solve for rapid lactic acid bacteria beer analysis.
- Free ATP released from dead barley or yeast cells, are very resistant, it can resist up to more than 500°Celsius, so it is no wonder that you find it in high amounts in beer released from dead yeast cells or barley or wheat plant cells. If you do not get read of it, it will cause false positives…
- Yeast ATP have huge amount of intracellular ATP, if your ATP bioluminescence method does not discriminate bacteria ATP from yeast ATP you have a problem. ATP bioluminescence will come essentially from your abundant yeast cells in beer, and you will not be able to discriminate bacteria growth then. And that is the case of all ATP biolumiscence detection kits available on the market.
ATP bioluminescence can only be used, if this method is able to eliminate free ATP & yeast ATP, so as to analyze specifically bacteria intracellular ATP. Actually no Atp kits available on the market are able to discriminate bacteria ATP from yeast ATP, even when using with specific bouillon incubation. For example if MRS broth is added with cycloheximide (Brettanomyces as others yeasts are cycloheximide resistant, even used as a ingredient in Brettanomyces agar media).
French technology with US patent solves these issues following 3 steps.
Issues solved in 3 steps:
- Step 1: selective release of yeast ATP & elimination with free ATP
innovative reagents selectively destroy yeast ATP from lactic acid bacteria targeting a specific target common to yeasts and not present in lactic acid bacteria
- Step 2: lactic acid bacteria are lysed by conventionnal cell lysis reagents, releasing lactic acid bacteria ATP
- Step 3: lactic acid bacteria ATP released mixed with luciferin-luciferase produce a bioluminescence increasing with the population of lactic acid bacteria in the sample.
Scientific bibliography
- Suzuki.K et al. 2012. 125th anniversary review: microbial instability of beer caused by spoilage bacteria. Journal of the Institute of Brewing, 117, 131-155
- Kissel, T. L., and P. E. Dakin. 1956. Pediococcus: culture method for their detection and a report on their growth characteristics,p. 44-53. In A. Beech (ed.), Proceedings of the 69th Convention of the Master Brewers Association of the Americas.