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Malolactic fermentation and acids

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Apimyces

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Hi,

I'm looking into experimenting further with malolactic bacteria (Oenococcus oeni, mostly). It's often reduced to "turns tart malic acid into a more mellow lactic acid", but these little buggers do so, so much more. More example, We tried them in mead, made with nothing but honey and pollen, and could easily tell apart the batch done with and without these bacteria, preferring the one with the bacteria. And yet... there was no malic acid in there...

What I'm looking at right now, though, is their metabolization of citric acid into acetic acid and diacetyl. I don't think citric acid is really considered "bad", but the latter two, I believe, are. I've been toying with the idea of using MLF when adding very tart fruits, but before I get started, I want to explore what may occur, to help me in the dosage and such.

The two fruit juices I'm looking to work with are cranberry and japanese quince. For the moment, let's stick with the former.

"The primary organic acids in cranberries are citric, malic, quinic, and L-ascorbic. Mean organic acid content of fresh and frozen berries varied slightly (2041mg/100g and 2035mg/100g, respectively) despite the large differences in individual acids. Malic was the primary acid in fresh berries at 64%. Citric and quinic made up 25% and 11%, respectively. In the frozen berry, citric and malic acids were evenly matched at 42% and 4 1 %, respectively. Quinic remained low at 17%"

Here, I'm thinking of using natural cranberry juice (not cocktails that include water and sugar). Now, if we run on the assumption that we dilute this in a mash, and bring it to an appropriate gravity and pH, we can assume the MLF will convert that malic acid (the main acid) into lactic acid, making it less tart and overpowering. But it will also convert all that citric acid into diacetyl and acetic acid... Yeast can metabolize diacetyl given time... but what about the acetic acid? Is it completely going to ruin the softening of the malic acid? If I put 1L of juice in a 5 gal brew, is it going to taste like vinegar in the end? Does anyone know if quinic acid is metabolized? And does anyone know if any strain can metabolize acetic into something more pleasant?

I was planning to use only Saccharomyces cerevisiae and Oenococcus oeni, but I'm open to adding more friends on the team if needed. My next two experiments with these targeted a sour english IPA and a sour american stout. For both I'll have three versions: one without juice and MLB, one with juice and no MLB, and one with both juice and MLB.

Thoughts, opinions, peer-reviewed articles? ;)
 

Vale71

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Keep in mind that beer itself contains organic acids, including citric.
Here is a study with some actual measurements:


https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1974.tb06797.x

Basically, you will be turning all that citric acid plus what you bring in with the fruit into diacetyl and acetic acid. :(
Yeast can reduce diacetyl, but if levels get really high that might not be enough, some form of Kräusening might be needed. As for acetic, I'm afraid you'll be stuck with it and then it's just a matter of whether you like the taste or not in the end.
 
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Apimyces

Apimyces

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Interesting, I was not familiar with the term "krausening", and yet, I was planning on doing something quite like that. My thoughts was to start the wort without juice or MLB, and, once the fermentation nears its end, boil the juice and add it to the wort along with the MLB. I was planning on giving these enough time to ferment before bottle-conditioning them (minimum 2 months, perhaps 6?). The reasoning for this, though, was to prevent the acidity from the juice from harming the primary fermentation. I know from my quince juice, that when I added yeast and MLB to it, nothing happened, because the pH was so insanely low (around 2 I think, that stuff is amazingly sour!), so I figured that, even diluted, it was best to wait for the yeast to be finishing up their job first.

"The flavour threshold of acetic acid in beer is approximately 90 mg / l. The precise figure depends on the pH value of the beer."

At 1L/20L of cranberry juice, I'm guesstimating (assuming a 1:1 citric:acetic conversion which is presumably untrue but simpler) an approximate net result of 255 mg/ l of acetic acid (am I calculating this wrong?), thus 2,8x the detection treshhold, but overall a 0,255% solution, which is still 1/19 strength of regular 5% vinegar. After all, the intended product is intended to be sour. Not trying to avoid sour, just trying to avoid bad/wrong/disgusting sour.

As for the article, citrate is not citric acid, but maybe it can be converted into it? In ales it gives 71-133 mg/l of citrate. Thus an addition of about a quarter to half of what the juice is giving, presuming citrate=citric acid, functionally. Plus, say, 13-44 (excluding the outlier 159) mg/l of acetate.

Ideally, I'm love to get some data on "well appreciated" sour beers, namely their acid compositions. How much acetic acid can a sour beer be and still be appreciated? On that subject, I found this, for example:

"
The volatile acidity for the lambic (gueuze) beer ranged from 3.97 g/L to 17.27 g/L, where Oude Artisan had the highest volatile acidity, while Boon had the lowest. Volatile acidity refers to the organic acids (such as acetic or butyric acids) that are more volatile or are more easily vaporized than non-volatile or fixed acids. Total acidity (g/L) for the lambic (gueuze) beer ranged from 2.62 to 7.83 g/L with Oude Boon exhibiting the lowest value and Oude Artisan having the highest value."

So say my cranberry MLF'd sour gets anywhere between 250-500 mg/l of acetic acid, that seems... completely within the normal and even lower range of commercial lambic beers? Thus, nothing to worry about?
 
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Apimyces

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http://www.craftbrewersconference.com/wp-content/uploads/2015_presentations/W1320_Kara_Taylor.pdf

Also, on trials over sourness of beers, in addition to the objectively quantifiable concentration of acids (in mg / l), their perception is also a story in its own. Carbonation levels, alcohol levels, and fruit/yeast/other flavors might impact perception of acidity. Also, surprisingly not mentioned there, but fundamental: sweetness level. The higher the FG, the less sour the final product will taste. As for these other variables, I'm assuming they all work the same way: more equals less sour. More alcohol, more carbonation, and a less neutral yeast might make the end product taste less sour.

Now, by this point, one may wonder "why are you trying to make a sour that's no so sour by adding things that are very sour to then transform the into things that are less sour and then finally make that sour result taste even less sour by masking it with other things".

And that'd be a pretty good question. Why not, I guess? Haha. I've had some unexpected good results from MLB in non-standard usage in the past, I guess I'm just really interested in exploring the full potential of Oenococcus oeni past simply treating it as some "malic to lactic enzyme" many people seem to dismiss it as. I'm hoping for a more complex flavor and mouthfeel with the addition of both a sour fruit juice and MLB to any given style than one would obtain with either or neither, as well as a more vibrant color.
 

RPh_Guy

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Most of us folks interested in sours enjoy the flavors added by Brettanomyces. Something to consider.

I've never intentionally used O. oeni because I prefer drinks more sour. Could you elaborate more on the flavor contribution?
 
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Apimyces

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Most of us folks interested in sours enjoy the flavors added by Brettanomyces. Something to consider.

I've never intentionally used O. oeni because I prefer drinks more sour. Could you elaborate more on the flavor contribution?
Me and my wife are rather traditional, when it comes to beer, I guess is a way to put it. My wife even more so than I. We enjoy brews with more punch and flavor than your typical Budweiser/Coors Light/Molson Ex/Labatt Blue/etc. But the tendency of many microbreweries to try to go to extremes... does not please our sensitive palettes. :p

I can appreciate light brett touches, and mild sourness, but if my wife is to begin appreciating those flavors herself, she'll need mild introductions to them too, because so far she hasn't really liked what she has tasted, much. ;)

So that's a bit what I'm aiming for here, a mild sourness, that is unquestionably perceptible, but... soft-ish. I know Bretts are common to many sours, but those characteristic contributions were not things I was looking for at the moment. Also, Brett can turn both ethanol and sugars into acetic acid... whereas I'm trying to minimize the presence of actetic acid.

Cranberry is also largely a proxy for quince, in these trials. I have hundreds of quince plants, which I've somewhat abandoned due to not having much to do with them, but they are very flavorful and aromatic, despite being extremely sour. Pure quince juice is about: pH of 2.6, 4g/100ml malic acid, 1,1g/100ml quinic acid, and 9mg/100ml succinic acid. Description:

"The chaenomeles juice was very acidic, with a pH of 2.5–2.8, and the titratable acidity was at most 4.2% calculated as anhydrous citric acid. The content of vitamin C and phenols was in general high, 45–109 mg ascorbic acid and 210–592 mg phenol per 100 ml juice, respectively. Three main organic acids were detected in the juice: malic acid, quinic acid and succinic acid."

Maybe it's a poor proxy, given quince has apparently no citric acid and limited to insane levels of malic acid (and about twice the acids of cranberry), but cranberry juice can be bought in-store, and I only have so much quince juice on hand. Still, I'll be doing trials with both.

As for the contributions of O. oeni on my mead, it's been too long ago, and we didn't really take good notes on that, other than both of us definately preffered the version that had MLB in it to the one that did not, though both were given a positive appreciation (and came from the same must).
 

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Brett can turn both ethanol and sugars into acetic acid... whereas I'm trying to minimize the presence of actetic acid.
Acetic acid formation from ethanol is a strictly aerobic process.
Keep oxygen out = no acetic acid.

I'm not sure about acetic acid from citric acid.
 

S-Met

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turns tart malic acid into a more mellow lactic acid", but these little buggers do so, so much more.
I occasionally use O. Oeni in ciders, but MLC can occur naturally as well given time.
Next question is for my knowledge: Are you certain of it's affects on the other acids? Do you have any papers or studies to support the claim, or only personal anecdotes? I don't discount that it affects other acids, rather that I am unfamiliar with its process outside of MLC.

@Vale71 thank you for the link, I glossed it, but will read it more in-depth soon. I appreciate studies like this.

why are you trying to make a sour that's no so sour by adding things that are very sour to then transform the into things that are less sour and then finally make that sour result taste even less sour by masking it with other things".
Funny, but I never questioned this. My assumption was that either you liked to play or you were specifically looking to accentuate the features you like or suppress ones you do not.
 
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Apimyces

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I occasionally use O. Oeni in ciders, but MLC can occur naturally as well given time.
Next question is for my knowledge: Are you certain of it's affects on the other acids? Do you have any papers or studies to support the claim, or only personal anecdotes? I don't discount that it affects other acids, rather that I am unfamiliar with its process outside of MLC.

@Vale71 thank you for the link, I glossed it, but will read it more in-depth soon. I appreciate studies like this.


Funny, but I never questioned this. My assumption was that either you liked to play or you were specifically looking to accentuate the features you like or suppress ones you do not.
O. oeni will metabolize at least malic and citric acid. If may or may not have any impacts on other acids. However, this is a microorganism, that isn't limited to just transforming acids. Just as yeast don't just turn sugar into ethanol, O. oeni does much more than convert acids. Its behavior, though, is poorly documented. Trials with this bacteria mostly focus on malic acid, lactic acid, citric acid, acetic acid, and diacetyl. However, I do remember one study that quoted this species as a beneficial gut probiotic. I don't remember the details, I read it a while back, but still. If your question regarding O. oeni and acids related to my mead, though, I'm not certain that the impact had anything to do with acids. Though... maybe? Honey does contain traces of acids, as does pollen. And I do think that the pollen was more... digested... than the honey, if we can put it that way. The addition of pollen was about 40g/L I think, it was inspired off an article that tried pollen in meads as a natural way to reach YAN targets, since honey musts are extremely nutrient-poor. In this regard, I only have limited anecdotal evidence on the contributions of O. oeni for anything else than converting malic to lactic, and that experience itself is kind of old and my memory not great on the details. But the flavor contributions, I believe, were not due to acid changes, at least not mostly due to acid changes, but largely due to the rest of the metabolic processes of the bacteria, which can also, by the way, as our yeast can, turn sugars into ethanol. Do these bacteria produce specific esters, phenols, by metabolizing various molecules in the must differently than yeast would? Probably. Is there sound peer-reviewed articles on the subject? I'm not finding much.

But that's kind of what's prompting my curiosity here. I tried something a while back, a bit randomly, and know I was happy with the results. It's largely uncharted territory, as far as I can tell. Exploration is part of the pleasure! And indeed, I guess I like to play, though I'm also rather bookish, so I like to understand my play as much as possible.

I'm not telling everyone to start adding Oenococcus oeni to everything, not yet at least. But I will keep everyone posted of my results. I'm hoping to finish drafting my recipes by next week, to order the ingredients, and then get brewing (again) soon enough. 6x 1 gallon batch trials to begin with.

This answers some of my questions. Thank you
I think it answers many of my own questions as well, though I'll have to research further a few of the passages I'm not sure to understand. For example, it seems to say that turning citric acid to acetic is an aerobic process... but the formula does not include pure O2, so I'm under the impression it gets the oxygen from the citric acid itself. Lots of detailed data in there for sure, though, will take a few readings to assimilate no doubt.
 
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Apimyces

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https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-64-0222?journalCode=ujbc20

Well, this seems to suggest that malt naturally contains malic acid.

I started my base wort yesterday, will be splitting into experimental fermenters by the end of the week. With/without juice and with/without Oenococcus oeni will be trialed. I look forward to evaluating the results! Will keep everyone updated on the impacts of MLB on beer.

I just hope O. oeni tolerates hops well enough. I've got an estimated 35 IBU wort and will be adding roughly 13g/gallon of hops for dry hopping later. Can't really find any info on this subject because of the paywalls.
 

Vale71

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I just hope O. oeni tolerates hops well enough. I've got an estimated 35 IBU wort and will be adding roughly 13g/gallon of hops for dry hopping later. Can't really find any info on this subject because of the paywalls.
O. oeni is listed as being gram-positive so it should be sensitive to alpha-acids to some extent. Unfortunately I don't have information on how sensitive it actually is, sorry.
 
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Apimyces

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Interesting... and confusing. Lots of accronyms and codes I'm not familiar with in there. It doesn't seem to say anything about O. oeni and hops, though. So while it's true to affirm that it presents no evidence of hop resistance in O. oeni, I'm not sure we could go so far as to say that it presents evidence of a lack of hop resistance in O. oeni. If I'm getting this right, it might just be that none of the studies they looked up actually tested this variable with this species.

O. oeni is known to be a pretty tough organism, though. Can handle pretty adverse conditions.

Perhaps an IPA wasn't the best style to test MLB on... oh well, we'll see. Anyways all these antibiotic effects are always dose-dependant, I'll dry-hop after giving the MLB a few weeks to do their thing.
 
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Results are coming in. Did 6 variants of a base beer, half of which got oenococcus oeni, a third got 5% apple juice, a third got 5% quince juice.

There was a good difference in flavor between the plain beer and the one with MLB. In this case, tasters preferred the one without MLB.

In the beers with apple juice, the apple character was very subtle, barely noticeable. The MLB did not do as big of a difference, iirc (to be reconfirmed, though, I think those were too fresh when compared).

In the beers with quince juice, the MLB did a huge difference. The only without MLB was a moderately sour beer, much to my liking. My wife did not like, as she dislikes sours. The one with MLB was much, much softer, and my wife actually enjoyed that one. I enjoyed both equally, though quite different in mouthfeel. To be noted that japanese quince is extremely rich in malic acid, which is very sharp tasting.
 
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