HOMEBREW Digest #4847 Wed 14 September 2005

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  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 Return to table of contents
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 Return to table of contents
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/ Return to table of contents
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 Return to table of contents
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 Return to table of contents
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 Return to table of contents
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