A question about bacterial/yeast cultures

[bernardsmith]

I am experimenting using rejuvelac (a lactic bacteria-rich drink made from sprouted grains (in this case winter wheat berries) to sour mead. Has anyone assayed rejuvelac and can say what bacteria and yeast strains are likely to be present? The local honey I am using is raw and may contain enough yeast to fully ferment the must but it would be very useful for me to know if a) there may be Brux or Brett in the rejuvelac and what kinds of LAB are typically found present in wheat berries. Thanks.

 

[RPh_Guy]

Sounds like rejuvelac is made by steeping some semi-malted grain at room temp. In brewing this would be called a wild starter using grain, similar to a sour mash.
I'm not sure what kind of conversion you'd get so early in the malting process. Probably at least some sugar, along with nutrients. Have you tested gravity at various points in the process? Just curious.

Rejuvelac could have almost any microbe in it.
I'd guess at minimum it would contain wild Lactobacillus, mold, and probably some wild Sacc, with possibly other fungi, yeast, and bacteria.

There's nothing special about the microbes growing on wheat. You could just go grab any plant from outside and dunk/steep it in a sugar + nutrient solution to get a similarly random mix of microbes. Then you can proudly tell people the microbes are locally sourced!

Have you been successful souring mead with lactic acid bacteria? I'll be pleasantly surprised if my "lambic mead" actually becomes sour to my taste.

If you haven't explored MTF, here's a whole bunch of info on the topic.
http://www.milkthefunk.com/wiki/Alternative_Bacteria_Sources
http://www.milkthefunk.com/wiki/Wild_Yeast_Isolation
http://www.milkthefunk.com/wiki/Spontaneous_Fermentation

 

[bernardsmith]

Thanks RPh Guy, but I think your basic assumption is inaccurate. The grains are raw and as part of the process you sprout them. Takes about 1-3 days to grow the "tails" (rootlets). Once sprouted you pour a volume of water over the sprouts and allow the sprouted grains to sit under this water - at room temperature - for about 3 days. The liquid tastes tart and quite fruity - citrus-like, the pH is about 3.70 and there is a great deal of effervescence. Haven't measured the gravity. Not sure what those readings would provide. The bacteria are converting sugars into acids. But I have added no sugar to the water. What does change - and changes very significantly is the pH - from about 7 - 3.71 in 3 days.
Additionally, the grains themselves now taste sweet so something is converting the complex carbohydrates into shorter sugars: I often make wheat berry salads with the same grains and these are magnitudes sweeter - but that conversion may be through the sprouting itself.

I am familiar with MTF. They seem (largely) to avoid indigenous souring: not enough control over the final product, I think. I've searched their discussions about Rejuvelac and the use of sweet whey (from hard cheese making - so culture -rich, and not acid-rich in the way that softer cheeses are often made today with vinegar or other acids, rather than the use of cultures to break down the lactose into lactic acid ) but there is nothing very substantive about the flora and fauna found in these substances.

My current experiments with rejuvelac involve raw honey without the addition of any yeast and I cannot be sure whether the honey is fermenting because the indigenous yeast likes the environment that the rejuvelac creates when mixed with honey OR there is enough yeast in the rejuvelac to ferment the honey OR whether there is enough viable yeast when both honey and rejuvelac work together. The obvious next step (I think) is to create a must with no indigenous yeast apart from what might be in the rejuvelac and see how the gravity drops. BUT.. the gravity would likely drop if the bacteria were converting sugars into acids without making ethanol. Is there any simple test for the presence of ethanol other than boiling a sample and burning the vapor to see what color flame it produces?

 

[RPh_Guy]

I think your basic assumption is inaccurate. The grains are raw and as part of the process you sprout them.

You are malting the wheat yourself

the grains themselves now taste sweet so something is converting the complex carbohydrates into shorter sugars: I often make wheat berry salads with the same grains and these are magnitudes sweeter - but that conversion may be through the sprouting itself.

Unmalted wheat contains starch (and protein and maybe some dextrins), not fermentable sugars, and no "diastatic power".
You couldn't make rejuvelac using raw wheat flour, right?

Upon germination, the wheat produces enzymes (alpha and beta amylase) that can rapidly convert the starches into sugars. The amount of enzymes is called the diastatic power and the process of activating the enzymes is called malting.

In other words, the wheat berries are seeds. The plant stores its energy in the seed as starch so it's not available to microorganisms. When the seed has water to start growing, it's cellular mechanisms activate the enzymes and it converts the starch into sugar so it can use it as energy.

Certain molds can convert starches to sugars but this is a much longer process. Traditional rice wine for example relies on mold (and gelatinization).

The liquid tastes tart and quite fruity - citrus-like, the pH is about 3.70 and there is a great deal of effervescence.

As the sugars are produced by the newly malted wheat the wild yeast and bacteria begin fermentation.
Lactic Acid Bacteria (LAB) produce lactic acid accounting for the pH drop, specifically Lactobacillus spp. and possibly Pediococcus spp. Yeast produce alcohol and CO2. Kloeckera spp. and Saccharomyces cerevisiae are likely the dominant yeasts at this point, giving off the fruity flavors and aroma. Possibly some other Saccharomyces spp. (e.g. S. bayanus) or Brettanomyces spp. may be present (Brett grows much more slowly and is less common).
Many other yeasts, molds, bacteria, and fungi are likely present but do not contribute to fermentation (besides possibly Acetobacter). Because they don't have an impact, I suppose scientists don't really care what other microbes are actually there at the start. As I mentioned before, almost anything could be present and there are almost limitless types of microbes. Fermentation (lol autocorrect tried to put Frankenstein) should kill most of these other microbes. The rapid pH drop and alcohol hopefully inhibits the dangerous microbes (Clostridium spp., Enterobacteriaceae, mold).

Isolating and identifying each microbe isn't the easiest thing (some are difficult to grow on plates), and costs money (isolating, propagating/incubating, staining, PCR, electrophoresis, etc.).

Haven't measured the gravity. Not sure what those readings would provide.

I was just curious how fast the sugar conversion occurred relative to how fast it was being fermented. Doesn't matter.

MTF. They seem (largely) to avoid indigenous souring: not enough control over the final product, I think.

Absolutely! Wild fermentation is unpredictable and unreproducible. Wild yeasts can produce some really foul flavors.
However it's not that uncommon. As seen in the links I posted above they do explain how to wrangle natural microbes.

The obvious next step (I think) is to create a must with no indigenous yeast apart from what might be in the rejuvelac and see how the gravity drops. BUT.. the gravity would likely drop if the bacteria were converting sugars into acids without making ethanol.

LAB do not drop gravity more than 1-2 points. Their metabolism stops when the pH reaches the floor, around 3.0-3.4 depending on species & strain. It would be pretty unusual to have a plant with no yeast on it, so you'd almost certainly have some. The surface of a plant is much less hostile to yeast than honey!

Is there any simple test for the presence of ethanol other than boiling a sample and burning the vapor to see what color flame it produces?

Yes but you will know by observing CO2 production that alcohol is being made.
They also sell test strips that can determine alcohol presence. They are marketed for mothers testing breast milk.

Actually measuring ABV is more difficult but can be done.

It is possible to measure alcohol percentage at home.
In The New Cider Maker's Handbook, Claude describes the process and calculations in detail.

Basically you boil a sample of the product to remove the alcohol, then add back distilled water. You can then accurately calculate the alcohol by comparing the difference in density before and after.
You need a high resolution hydrometer.

He explains that vinometer and hydrometer/refractometer methods are probably not sufficiently accurate.

Hope I've cleared up some things!

 

[bernardsmith]

You have confirmed many of my hypotheses but they are still only hypotheses as I don't know what the actual flora in my must are. The other point is one I really don't understand. You state that I am malting the grains. How? I am not applying any heat warmer than room temperature so there is no Maillard reaction and no caramelization of any sugars. These grains are simply soaked in volumes of water for several days. They are still quite raw. But I suspect and someone with far more knowledge about brewing and Egyptology than I can chuckle that the first beers ever made were simply made not from bread (creating malted sugars) that was soaked in water - either deliberately (to make a soup?) or accidentally but from piles of grain that accidentally sprouted and were then left to drown in water. Because isn't malting a) for flavor and b) to to halt the growth of the seed which would otherwise reduce the amount of sugar available to the brewer. Drowning the seed in water seems to have the same effect in halting the seed's development. Of course I may be dead wrong, .

 

[RPh_Guy]

https://en.m.wikipedia.org/wiki/Malt

Malting activates the amylase enzymes needed for converting starch to sugar. Normally the germination is halted by drying, no heat required.

Kilning, which is adding heat, is the process to add flavor. That's how we get all different kinds of flavor from barley/wheat/rye/spelt malt, but it all begins with a similar process that you're doing.
Simply kilning grains without first allowing them to germinate will not produce "malt" and the resulting cooked grain will not yield fermentable sugars.

I don't know what the actual flora in my must are.

And you cannot, other than observing the effects.
Observe that you have fermentation, ergo yeast, and measurable acidity, ergo LAB.
Unless you want to start isolating and identifying the organisms yourself or send a sample to a lab you'll need to come to terms with not knowing what's in there

HTH, cheers.

 

[bernardsmith]

But by what criteria do I even know that "fermentation" is taking place. I see bubbles and I see a reduction in gravity but I am unable to determine if what I am seeing/tasting is ethanol or some other compound. I have not pitched any yeast so any fermentation is being done by indigenous yeast but how does one know that fermentation is occurring and not simply LAB eating sugar and producing acids... How does one determine the presence of ethanol using chemical or other methods?

 

[RPh_Guy]

Humans have been producing alcoholic beverages for thousands of years simply by observing the signs of fermentation, long before they had any concept of microbiology or pitching yeast.
Yeast grow all over the stuff that ferments, and basically anything growing outside.

There's no other explanation for the bubbling and gravity drop besides yeast fermentation that produces CO2 and ethanol.

Above I already mentioned several scientific ways for determining ethanol presence/content. If you don't like those, you can always try the does-it-get-me-buzzed test!

 

[bernardsmith]

Ha ha ha... A good test - but I wonder how breathalyzers work. Perhaps there is a device that is sold to the public. Will check that and see but in the meantime I took measurements from two experimental batches.
Batch 1 started 6/14/18 with rejuvelac and one pound local raw and 1 lb sage honey. No added yeast. Today the SG is 1.010 and the pH is 3.54. Batch 2 begun 7/1/18 with 3 lbs of local honey and 2.5 oz of inchet (gesho) no added yeast and today the gravity is 1.052 and the pH is 3.51.
Not a chemist but if so much sugar has been removed but the pH has dropped .20 or thereabouts then the likely explanation is that yeast is involved... Though perhaps a better indictaion might be to measure the TA rather than pH - pH is telling me WHAT acid is present (its strength) TA would tell me HOW MUCH acid is present.. The taste by the way of the t'ej is really good and that of Batch 1 is pleasantly sour... but will need some more sweetness to balance.
Oh... and may the Fourth be with you.

 

[RPh_Guy]

Happy 4th!

how does one know that fermentation is occurring and not simply LAB eating sugar and producing acids

Sorry, I forgot to answer this more thoroughly.

Generally speaking LAB don't wont drop gravity more than 1-2 points, so you definitely have yeast fermentation.
http://www.milkthefunk.com/wiki/100%_Lactobacillus_Fermentation

The reason behind this is that LAB growth/metabolism becomes inhibited once the pH drops (they are self limiting).
Think of it like the alcohol tolerance of yeast. They can only drop gravity a certain number of points before the alcohol kills them. It just happens that LAB can only consume a much smaller amount of sugar before becoming inhibited relative to yeast.

I've kettle soured with Lacto several times. No visible CO2 production and no gravity drop.

You've made yogurt and kefir, so you've also seen lacto-only fermentation first hand and know they don't produce CO2 or krausen like yeast (milk is full of protein so you'd definitely get a krausen if lacto could make one).

Our GI tract is full of Lacto. When we eat sugar we don't get huge gas production.

If you want to get into chemistry, measuring TA can tell you the same thing. Count the carbon atoms in the lactic acid and then you can calculate exactly how much sugar they consumed. Once you calculate the amount of sugar you can determine how much they dropped gravity. Carbon utilization by LAB metabolism goes almost entirely toward lactic acid production.

You absolutely have yeast fermentations (and consequently alcohol); there's no doubt.

Cheers

 

[bernardsmith]

so this is indeed, what I was hypothesizing - that at most LAB can only produce 1-3 % alcohol but that does not mean that LAB while under the 2% ceiling cannot eat sugar and transform the sugar into lactic acid. And THAT is my concern. Why does Michael Tonsmeire (The Mad Fermentationist) suggest that brewers pasteurize their wort after a few days of sitting on LAB if not to stop the LAB competing with the yeast for the available sugars.

If I suspect the LAB is NOT producing alcohol but I do suspect that the LAB is producing lactic acid THEN the drop in gravity may be due to the production of acids. Lactic acid is lactic acid and its pH is fixed, isn't it? One cc of Lactic acid will have the same pH as 50 barrels, no? But the TA of 1 cc will be far less than the TA of 1 liter , no?
Moreover, since we do NOT know what the precise microbial content of rejuvelac is and what yeasts there may be I have no idea what action THOSE yeasts might be performing and indeed how they may work with the S cerevisiae in the honey.

 

[RPh_Guy]

at most LAB can only produce 1-3 % alcohol but that does not mean that LAB while under the 2% ceiling cannot eat sugar and transform the sugar into lactic acid.

Not quite right....
LAB produce lactic acid, not ethanol (under 0.5% ABV, generally speaking), and not CO2. This causes minimal or no gravity drop.

Yeast produce ethanol and CO2. They cause a large gravity drop.

Both yeast and bacteria contribute flavor compounds. Both drop pH to different extents (lactic acid vs carbonic acid)

Why does Michael Tonsmeire (The Mad Fermentationist) suggest that brewers pasteurize their wort after a few days of sitting on LAB if not to stop the LAB competing with the yeast for the available sugars.

This is called a kettle sour. Boiling post-souring prevents large amounts of Lacto from potentially contaminating our equipment. A secondary benefit is being able to target a particular pH and then stop the souring when it is reached.

LAB do not compete with yeast, besides the acid slowing down yeast fermentation a little bit.

Lactic acid is lactic acid and its pH is fixed, isn't it? One cc of Lactic acid will have the same pH as 50 barrels, no? But the TA of 1 cc will be far less than the TA of 1 liter , no?

No.
pH is a direct measure of the concentration of hydrogen ions.
TA is a measure of the amount of a particular acid and it's salts.

So, as lactic acid is produced, TA will increase linearly.
pH is much more complicated and beyond the scope of me explaining it here. For starters the scale is logarithmic. Bottom line: in the pH range of our beverages as lactic acid is produced the pH will drop. A lactic acid solution can reach pH 2.4 at 0.1N concentration. Most compounds in solution contribute to pH to some degree.

Moreover, since we do NOT know what the precise microbial content of rejuvelac is and what yeasts there may be I have no idea what action THOSE yeasts might be performing and indeed how they may work with the S cerevisiae in the honey.

It's really not that mysterious.
Since you have both yeast and LAB the major fermentation products are ethanol, CO2, and lactic acid.
Acetic acid should be minimal depending on various factors.
Other compounds are produced in only tiny amounts as byproducts (esters, glycerol, diacetyl, sulfite, etc).

Cheers

 

[bernardsmith]

Aha! So here's my ignorance showing. If SG is really nothing other than a measure of the (relative) density of a solution and if the density of the solution is 1.xyz because of the amount of sugar dissolved in solution then I would have thought that anything that reduces the amount of sugar and changes the solution to something less dense would result in a less dense liquid, but you are saying despite the fact that LAB change sugar to lactic acid the density of the liquid does not change... But being a skeptic from Scotland I have to test that assumption. I will make a solution of rejuvelac and add to that a known quantity of lactose. Yeast cannot, I think ferment lactose but the lactose will first add to the density (gravity) of the liquid and then by your account (unless I am misunderstand your assertion) the gravity should remain more or less the same despite the fact that LAB will convert lactose to lactic acid. Unless, of course lactic acid dissolved in water is more dense than lactose dissolved in water....

 

[RPh_Guy]

Sounds like you're getting it!

Two things to keep in mind:
1. LAB consume only a small amount of the sugar (probably less than 4% by my calculation).
2. The lactic acid produced stays in solution and is more dense than water, so unlike ethanol which is far less dense than sugar, a lactic acid solution replacing a small portion of the sugar is less prone to causing a decrease in density.

I'm not sure about the actual density numbers and too lazy to figure them out but I know my 88% lactic acid solution is 1.21g/mL (whereas water is ~1g/mL).
These facts help explain why SG will only drop 0-2 points or so from LAB fermentation.

I've tested SG before and after Lacto-only fermentation (souring) in wort and had no change in density.

You can easily test this yourself the same way. Mix up a sterilized (heated) sugar solution with some nutrients by any means, and then add a pure Lacto culture (you can use yogurt). Keep covered air tight. Measure SG and either pH or TA initially and after a 2-5 days. Better yet, just make a kettle sour beer since you seem to like sour drinks

Your honey/rejuvelac thing could accurately be called a wild sour braggot.

 

[bernardsmith]

So these data are really interesting. My one quibble is with the use of yogurt. Yogurt is a thermophilic culture (high temperature) so adding yogurt at room temperature may not in fact be the way to make the best use of the LAB in yogurt. I have been using whatever cultures are to be found in and on the grain berries - at room temperature.
That said, as a purist I would argue that the use of raw grains in a mead to make a braggot is stretching the term braggot to breaking point if not beyond that. A braggot is made with beer and not simply with grains. I say that as someone who has made wine from rice and wine from wheat. The fermentation was from the sugars I added and not from those grains (the flavors were from the grains),
and I would argue that those wines were as close to sake (in the first instance) as my fruit sorbets are close to gelato and in the second case the wheat wine was as close to hefeweizen as my seitan is to kobe steak (and both my sorbets and my seitans are - excellent, even though I say so myself) so I will leave braggots as those meads made with malted grains. I don't intend to argue with BCJP rules or judges.

 

[RPh_Guy]

Generally speaking, all LAB are thermophilic. They are well adapted for living inside animals and grow best at body temperature.
Even L. plantarum that is widely touted for quickly souring at room temp in fact grows best at our body temp.

You ARE malting the grain.
Rejuvelac is an unconventional way to make [sour] beer, but it IS a yeast fermented product made with malted grain sugar.
With rejuvelac the malting process is combined with the mash and overlapped with fermentation. Really not the best way to make beer but I guess it works.

The inventor apparently made a LOT of false claims about her food products and their effects.

But hey, call it whatever you want, no point in arguing over the name
Cheers

 

[bernardsmith]

Sorry but my skepticism is showing: I make kefir and kefir has both meso and thermophilic bacteria. I can make Chevre cheese using the meso bacteria from the kefir by ripening the milk at about 76F, and "Romano" using the same culture to ripen the milk at about 131 F (cooking the milk at 131 F would kill all the meso but would encourage the thermophilic to produce lactic acids at this higher temperature resulting in very different flavor profiles of the cheese - (the different textures are due to the ways whey is removed) and hence my assumption that the LAB I am producing from the grains are not a single culture but may contain multitudes (to misquote Walt Whitman).

 

[RPh_Guy]

I suggested you could use yogurt as a source of Lacto simply because it probably doesn't contain yeast (at least not yeast that can ferment).
You can allow it (or any other source of pure Lacto) to sour your experiment (or beer) at any temp appropriate for that culture.

Lacto species typically don't like the cold, which makes them thermophilic in a broad sense (they prefer warmer than room temperature >70F). Apologies if that was confusing since you're used to hearing it in a more specific context.
Edit: "Thermophilic" does have a specific definition when taking about bacteria, and that's not what I meant!

 

[bernardsmith]

Cheese makers are familiar with certain LAB as mesophilic and others as thermophilic and confusing these two can result in cheeses that fail to taste the way one might expect - Compare styles of beer...All LAB may be "thermophilic" in the eyes of brewers but in the hands of cheese makers LAB are not the same. Working with LAB at the wrong temperature may result in cheeses that might as well have been cultured with ... dead or inactive bacteria.

 

[RPh_Guy]

I am no cheese maker. Sorry for the confusion about that.

Hope your batches of whatever you want to call them turn out well!

 

[bernardsmith]

The batches made with rejuvelac are very tasty; those made with sweet whey are more mixed (A tej I made with whey is garbage but the one I am making with rejuvelac is very drinkable and this with only indigenous yeast)