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	FORUM ON BEER, HOMEBREWING, AND RELATED ISSUES
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Contents:
  Re: more esters, and also acids/pt1. ("-S")
  Re: more esters, and also acids/pt2. ("-S")
  searching HBD ("Peter A. Ensminger")
  esters, acids, and practical ideas for fermenting beer (Matt)
  La Binchoise ("Chad Stevens")
  RE: health-beneficial properties of hops (jmcdonald)


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Date: Wed, 14 Sep 2005 04:37:56 -0400
From: "-S" <-s at adelphia.net>
Subject: Re: more esters, and also acids/pt1.

Uh Oh  8klimit bit me.
 ...
Matt asks for more on esters ...

> Among other things, Steve explains how "stuck ferments" (shortage of
> some growth factor other than sugar causes yeast to stop growing) are
> likely to lead to increased acetate esters in practice.

Not just acetate esters, esters generally.

I referred to the class of acylCoA, not just the specific acetylCoA
molecule.  acetylCoA is the rather hairy CoenzymeA molecule attached to the
part of the acetic acid molecule minus a hydroxyl group (-OH)
{ http://en.wikipedia.org/wiki/Acetic_acid ,
http://en.wikipedia.org/wiki/Image:Acetyl_Coenzyme_A_1.jpg }.

AcylCoA is similar but it can have ANY acyl group attached in place of the
acetyl group.  An acyl group is just an organic ion with a terminal carboxyl
group { terminal carbon attached to a carbonyl oxygen (=0) }.   These are
formed from carboxylic acids, like acetic.  The other large class of
carboxylic acids in yeast are fatty acids which also form fatty acylCoA
molecules and then fattty esters.

- -- 

So here is the ester story again in in shorthand.
a/    R1.COOH + ATP + CoASH -> R1.COSCoA  +ADP + PPi { via acytlCoA
synthetase }
b/    R1.COSCoA + R2.OH -> R1.COO.R2 + CoASH  { via R2 specific alcohol
transferase }

Step a/:  R1.COOH is the carboxylic acid, and could be acetic or a fatty
acid or something else.   ATP is merely supplies energy to this step and
results in the AMP and PPi residue.  CoASH is a shorthand for the
unencumbered CoenzymeA molecule.  That notation may be clear if you realize
that the "working end" of CoA is a sulfur 'S' atom and in the free state has
a thiol -SH terminus.  The resulting product, ( R1.COSCoA ), is the acylCoA
where "R1.CO" is the residue from the original carboxylic acid with the
terminal carbonyl, and the rest is the CoA ion in place of the missing
hydroxyl.

Step b/: The acylCoA molecule (R1.COSCoA) interacts with an alcohol (R2.OH)
via an alcohol transferase enzyme(AAT) and the result is the ester
(R1.COO.R2) and the freed CoASH.  Note that ALL esters have this general
form ( http://en.wikipedia.org/wiki/Ester ).

So this is a general mechanism for esters in beer, not just the acetate
esters.
- --

Most of the acetic acid for for the formation of acetate esters during
fermentation comes from pyruvate to acetaldehyde converted via acetaldehyde
dehydrogenase into acetic acid.  Many books will state that the acetylCoA
arises directly from the action of pyruvate dehydrogenase enzyme on pyruvate
(bypassing step a/) however this dehydrogenase enzyme is absent during
fermentation ["Brewing Yeast & Fermentation", Boulton&Quain].  AcetylCoA may
come from catabolizing amino acids, or beta-oxidation of fatty acids, but
that's a minor source of acetylCoA in normal fermentation.

- --
more to follow ...

 -S



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Date: Wed, 14 Sep 2005 04:42:23 -0400
From: "-S" <-s at adelphia.net>
Subject: Re: more esters, and also acids/pt2.

continued ...

> In the interesting theory reported by John Palmer, an increase in total
> growth leads to the production of more AAT enzymes, thus to more esters
> produced "after the growth phase."

Weak tea.   There is no evidence ever presented, and it is certainly not
adequate to explain experimental results.   I suspect that the big "growth
cessation" ester term is controlled by the yeast final biomass (not growth)
and that the "background" ester term is probably too complex to parse out
easily but is dependent on either the acylCoA level or the AATase level and
early on the alcohol level  - which ever happens to be rate limiting at the
moment..

The paper "A flavor model for beer fermentation", by Gee & Ramirez(JIB,
v160, pp321-329) attempts to numerically model many factors (sugar uptake,
yeast mass, fusels, esters, alcohols, certain VDKs) and they they perform
some test ferments and attempt to derive the coefficients for their model.
The model assumed that the ester ethyl-acetate would be proportional to
sugar uptake and therefore similar to their model for biomass growth.  Some
other esters not dependent on acetylCoA were modelled as proportional to
growth.  The model matched many experimentally measured parameters
wonderfully, but the fusel model was a bit off and the ester model wasn't
great at all.    It was clear that *all* ester production was very low
during the first 50 hours of the lager fermentation and many quite low
through 75 hours, but the model predicted an early ramp-up in line with
yeast growth.  That didn't happen, so the model that the "background" esters
rate is proportional to growth is fundamentally broken.

Also read "Lipid metabolism and the regulation of volatile ester synthesis
in s.cerevisiae", Thurston et al, (JIB v88, pp90-94).  This paper presents
examples with graphics of measured growth *rates* and ester *rates* and it
is clear as a bell that while this ale yeast fermentation hits an
exponention plateau of about 12% biomass growth per hour from hours 8-21,
then drops off rapidly specifically due to oxygen/sterol limitation.
Meanwhile the ester levels chug along at a backgroung rate, then have a big
spike centered around hours 21-22 and drops off rapidly.   You can't see
this data and still believe all these mommilies and malarcky about growth
and esters.   There is a growth related background rate certainly but there
is also a clear early period with positive growth and no esters. Then there
is also a huge but brief ester production rate when growth is halted by a
non-carbolite growth limiting factor.   This growth cessation ester
production periods is probably common when O2 and aminos and sugars all
compete as the final lmit to growth in normal Hbrewing.


> 1.  I think pitching rate affects total growth ONLY because of the
> small amount of energy required for (non-growth-related) background
> maintenance of the cells.

Right.  Your yeast have to eat-up so much available sugar energy and
assuming we keep the yeast happy this is primarily used for and wort
fermentables is almost proportional to total biomass growth.

> But if
> there is a pool of Acetyl CoA laying around after growth stops, and AAT
> enzymes were produced in proportion to growth, then depending on
> reaction and denaturing rates, etc, small amounts if increased growth
> could lead to disproportionately large increases of acetate esters.

It's not a static pool.  acetylCoA will continue to be produced after growth
stops if sugars->pyruvate->acetaldehyde->acetic->acetlyCoA path was still
available (or alternatives).  The rate of production will fall, as the other
uses for fatty acids stop.  We see this all the time in stuck fermentations.
There is a still a little CO2 produced as fermentation proceeds to create
maintenance energy levels.  This means
sugar->pyruvate->acetaldehyde->ethanol is working.  There is no reason to
think that the acetaldehyde dehydrogenase isn't still active for producing
acetic->acetaldehyde too.

More important, I don't buy that AATase is produced in proportion to growth,
only a modest amount is needed till growth ceases and organisms are frugal.

The production of esters from carboxylic acids and alcohols is energetically
expensive, so there is a huge question looming - why do yeast bother ?   Why
do they work to get rid of carboxylic acids ?  One theory is that the yeast
do this to get rid of medium length fatty (carboxylic) acids.  Long fatty
acids are made two carbons at a time from acetylCoA *only*(almost).
Normally this lengthening stops around 16 to 18 carbons in length, and these
long free fatty acids are joined, three at a time to glycerol and this
combined molecule is an acyl-tri-glycerides (fat) which makes up the cell
membranes.  The very short enoic acids (acetic and propanoic for example)
aren't a big problem.  The headache is that medium length FA (C8 - C14) are
toxic, so when growth stops these unfinished long FAs require hazmat
treatment.  They are converted to esters to detoxify them.  AAT activity is
probably only critical to yeast survival AFTER they have toxic FAs and have
no means to lengthen them by the growth enabled mechanism.  I'd wager that
AAT level rises rapidly as growth ceases.

Unless ethanol and fusels are more toxic than the literature indicates,
there is no reason yeast should ever waste ATP and acetylCoA by converting
these to esters.  It's possibly just a side-effect of the short-FA removal
scheme above.

> 2. IS there a pool of Acetyl CoA lying around after growth stops?

It's not statically, "lying around" (see above).  All organisms have a vast
number of "feedback loops" implementented as enzymes and genetic expressions
of enzymes.   When growth stops and the major use of acetylCoA to make fatty
acids ceases, then the acetylCoA level will rise until some feedback
mechanism causes the acetylCoA creation rate to slow and match the new lower
consumption rate.   Of course there may be other feedback mechanisms
"upstream" which stop the production with growth, but that doesn't seem to
be the case.

> 3. Do fatty acids ever leave the cell walls and if so what is their
> fate?

Cell *MEMBRANE* (the wall is just a skeleton) has long
chain FAs formed into di- & tri-glycerides.  These can be mobilized and
oxidized for energy (beta-oxidation, fat burning), but this effectively
never happens in the fermenter.  The amount of mid & long FAs in beer is
measured in parts per billion.  Short carboxcylic acids (hexanoic C6 and
below) may reach a few ppm in beer, but don't come from the membrane.

> 4. More practically--can I increase the iso-amyl acetate in my beer,
> without increasing (and in fact reducing) isoamyl alcohol levels, by
> simply adding acetic acid to the wort?  I assume not, because how would
> it get into the yeast cells and how would it get transformed to
> Acetyl-CoA... but who knows?

I *suspect* that the acetic acid might make it into the yeast cells, but I
doubt it would increase acetylCoA level levels in a significant way.   Might
be fun to try.    An easier route to produce high ester levels has already
been outlined here.   Brew like a newbie minus the lack of sanitation
control.  Just be real nasty to your yeast.  Underpitch, under-oxygenate,
ferment a little too warm, don't use any nutrient, bump up the SG a few
points.  Of course you must start with a yeast which can produce the
right AAT or nothing will work.

 -S



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Date: Wed, 14 Sep 2005 11:11:30 -0400
From: "Peter A. Ensminger" <ensmingr at twcny.rr.com>
Subject: searching HBD

Try this:

1) Go to HBD homepage: http://hbd.org/
2) Click "Search" (under "Site Features" in left column)
3) Type in your search term(s)

Voila!

Cheerio!
Peter A. Ensminger
Syracuse, NY
http://hbd.org/ensmingr/



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Date: Wed, 14 Sep 2005 10:11:21 -0700 (PDT)
From: Matt <baumssl27 at yahoo.com>
Subject: esters, acids, and practical ideas for fermenting beer

Fredrik and Steve provide a lot of thoughts on my previous questions. 
For people like me who haven't had much exposure to the biochemistry
(and want it), the wikipedia.com pages that Steve included links for
are great.  Now if only those JIB papers were available online as well.

Anyway, I understand very well that in cases where some growth factor
other than sugar causes yeast growth to slow, we get a big spike in
ester production.  I am tempted to call this a "stuck ferment ester
spike," and then ignore it because even when I want lots of esters I
plan to avoid sticking my ferments.  BUT... maybe I'm going too far,
because maybe an ester spike happens even in healthy, sugar-limited
ferments as well.  (Also, perhaps there is no such thing as a
completely sugar-limited ferment in practice.)  This was really why I
asked whether there are still pools of Acetyl-CoA available after
growth stops--but I forgot to specify that I am most interested in the
case where growth stops *because the sugar runs out*.  Any thoughts on
that?  If ester production is (almost) always dominated by a late
spike/hump then I'll think a lot less about the "background"
production.

Also: Steve says "Most of the acetic acid for the formation of acetate
esters during fermentation comes from pyruvate to acetaldehyde
converted via acetaldehyde dehydrogenase into acetic acid."  An
acetaldehyde molecule has one less oxygen than an acetic acid molecule.
 Where does the oxygen come from to make this reaction happen?

A Practical Idea:
Let's say, first of all, that we pitch an adequate amount of healthy
yeast into a sufficiently nutritious/aerated wort, so that the yeast
are still happy and healthy when the sugar runs out.  Let's also say
that we want to minimize diacetyl and fusel levels.  Finally let's say
that we can easily raise the ferment temp to at least the low 70s
whenever we want, just by leaving our carboy at room temperature and
letting the yeast do its thing.  How do we use the ferment temp to
control esters?

If we want to minimize esters we can keep the temperature as low as
possible for the yeast strain we're using.  Great.

The point I want to make is that it *seems to me* that we should PITCH
THE YEAST INTO VERY COOL WORT EVEN IF WE WANT LOTS OF ESTERS.  By "very
cool" I mean the low end of our yeast strain's temp range.  If esters
are not really produced early on, then it costs us nothing in terms of
esters if we keep the initial ferment very cool to minimize diacetyl
and fusel production.  This may seem obvious, but I for one have in the
past believed things like "The Belgians ferment at 80F," and found out
later that breweries like Westvleteren and Unibroue pitch much cooler
and only later let the temp rise to startling heights.  I've also read
things like "The Belgians ferment at 80, but that didn't work for me so
I ferment Belgian styles at 64," and wondered whether such a strategy
really gives enough esters.  The concept of a single-temp ferment seems
convenient but flawed.  (Yes, I know I am probably not the first to say
this.)  

Ringwood yeast seems like another possible application.

Matt




		


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Date: Wed, 14 Sep 2005 11:39:27 -0700
From: "Chad Stevens" <zuvaruvi at cox.net>
Subject: La Binchoise

Anyone know the spice/fruit in La Binchoise Reserve Speciale?

Thanks,

Chad


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Date: Wed, 14 Sep 2005 15:57:03 -0700
From: <jmcdonald at library.caltech.edu>
Subject: RE: health-beneficial properties of hops

 

> Moreover, health-beneficial properties due to particular compounds  
> present in hops confer to beer a unique position in the  
> appreciation of (low-)alcoholic drinks.

Anyone know more about this assertion of "health-beneficial  
properties" of hops?

- ---------
I can't cite anything off the top of my head since I'm not a biologist, 
but if you go to PubMed http://www.ncbi.nlm.nih.gov/entrez/query.fcgiand

and search on 'Humulus' you can find many articles about the biology 
and chemistry of hops.  Searching other academic research 
databases will uncover many more articles documenting studies on 
the health benefits of compounds found in hops.  Use BIOSIS, Medline, 
or Web of Knowledge if you have access through a local university or 
public library.

I would guess that they did not cite specific studies since they are 
already incorporated in the scientific knowledge of the discipline that 
was their intended audience.  

John





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