Polverone
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Yeasts, amino acids, and fusel oils
I am interested in the production of fusel oils (alcohols higher than ethanol formed by yeast fermentation). I have discovered the following
information about them; please correct me if any of it has been "discovered" in error:
1) These higher alcohols are always present to some extent in raw fermentation products.
2) These higher alcohols are formed from amino acids.
3) Addition of ammonium salts reduces the extent to which yeast produce alcohols from amino acids.
4) (Unclear) the main portion of higher alcohol production may be while dissolved oxygen is still present and yeast are reproducing.
Most of the information I've seen online is about minimizing fusel oil formation in brewing. I have started to look through paper references too
but haven't found a lot in either place about *maximizing* production of fusel oils.
I'd like answers to all of the following questions, eventually:
1) What's an inexpensive source of relatively pure amino acids?
2) Failing number 1, can I simply add some protein-rich material like soy flour, egg white, or whey powder to the fermentation mixture and expect it
to be available to the yeast?
3) Failing number 2, can I reduce inexpensive (food) protein sources to their constituent amino acids with hydrochloric acid or enzymes, then use them
in fermentations (possibly after neutralizing acids or other processing)?
4) Assuming that I do have a pure amino acid or mixture of amino acids that I want to add to the fermentation mixture, how concentrated can I go? To
what extent will yeast act upon amino acids in addition to or instead of ordinary sugars? At least initially, I am more interested in achieving a high
fusel oil concentration than in using amino acids efficiently.
I'm excited by the idea of producing quantities of mixed "fusel oils" similar to those produced by ordinary brewing as well as specific
alcohols from purified amino acids.
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sparkgap
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Here's an interesting link: http://www.mbaa.com/TechQuarterly/Abstracts/1975/tq75ab03.ht... . I don't know, however, how to get the article save buying it or e-mailing
the authors.
Now, for two your questions:
1) Congeners are present in almost any alcohol produced via fermentation. I'd expect those higher alcohols to be present. That's why we get
hangovers.
2) I don't think so. If my memory serves me right, the higher alcohols come from the incomplete decomposition of whatever carbohydrates there are
in the medium.
3) (might be stupid) Clarification first. You want your amino acids optically pure or chemically pure? It depends on the amino acid. Glutamic acid,
for instance, can be gotten from MSG. Glycine can be made from complete digestion of animal gelatin. The other amino acids, I don't know.
4) I don't know about the second approach but the third approach seems viable. HCl might be OK, but why not just use meat tenderizer?
I don't know about the others. Haven't tried it myself save growing the yeast.
sparky
P.S. I'm assuming you'll only be using S. cerevisiae, no?
[Edited on 19-1-2005 by sparkgap]
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Polverone
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Many sources including the one you linked to consider amino acids an important part of fusel oil formation. For example, according to <A
HREF="http://www.chromspec.com/pdf/lit/Agilent%20app%20fusel%20fuels%2000043786.pdf">this report</A>, isoleucine and valine both
lead to 2-methyl-1-butanol, valine leads to 3-methyl-1-butanol, threonine to 1-propanol, and 2-phenylalanine to 2-phenylethanol. I'm glad that
your linked source reminded me that higher brewing temperatures lead to higher fusel oil formation; I'd read of that before but forgotten it.
I would like my amino acids to be optically pure as well as chemically pure. If I buy them from a health food store, for example, I expect that
they're optically pure (since they're produced via various organisms) but the capsules or powders may contain silica, magnesium stearate,
and other non-amino-acid components. The extra bits probably wouldn't interfere too much, but health supplement prices per unit are also a bit
high for my tastes unless you buy bulk powders, in which case the total price is a bit high. I suppose I was wondering if amino acids were used in
compounding animal food or any other application where they'd be optically pure and reasonably chemically pure yet cheaper than I'd find
them at lab suppliers or health food stores.
I suggested the use of HCl to break proteins into amino acids since it seems to be a common way of hydrolyzing proteins for analysis. The process
destroys tryptophan, though. Apparently NaOH can be used instead of HCl to preserve tryptophan, but it destroys other amino acids and racemizes those
that survive.
Yes, I plan on using good old <i>S. cerevisiae</i>.
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chemoleo
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Interesting that you mention ammonium salts preventing the formation of fusel oils, even in the presence of amino acids.
I presume it is because it competes with the transaminases, and various other enzymes involved in the amino acid metabolism. By the way, in
biochemistry we often grow bacteria in an NH4Cl solution (with glucose and other things), where the NH4Cl is the only nitrogen source present.
Regarding S.cerevisiae growing in a protein rich solution- I don't see why not. If you make a small batch culture with yeast, in glucose solution
(where it gets used to growing in solution), and then transfer this to a larger batch containing dissolved (sterilised) protein, it should be able to
grow on it. The yeast will secrete proteases and ingest the amino acids, or ingest the proteins altogether, to be proteolysed inside the cell. This
should save you the time of boiling it with acid, neutralisation etc. Would be interesting to see how well S.cerevisiae grows on protein nutrient
only, with no carbohydrates present. I imagine a lot of ammonium derivatives will be produced as well.
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sparkgap
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I just dug up and reread my microbiology book again. You're right; amino acids are precursors of the higher alcohols produced in fermentation.
Sorry for that.
Maybe you should stick with meat tenderizer for degrading the proteinaceous material you have on hand. I don't see why proteases wouldn't do
as well as HCl or NaOH without ruining the amino acids. You are right, the OH- will cause a lot of headaches for you as it will scramble the
chirality.
Buying the amino acids from a health food store isn't a very good idea. I presume you'll be paying more for the brand than for the amino
acids per se.
Then again, I have to concur with chemoleo. The yeasts have good enough proteases; they should be able to feed on a properly chosen source of protein.
I'd advice you to experiment.
As to chemoleo's comment on growing the yeast in protein medium, I can do no better than to quote from this post from another site:
"...Yeast are obligate aerobes, i.e. they need oxygen. They seem to thrive in places where there is an abundance of carbohydrates. An abundance
of bacterial action tends to make the whole mess go anaerobic (low oxygen levels), which would slow down yeast growth...
...This whole mess would probably decay more rapidly at a temperature closer to body temperature (37 ºC.) or perhaps slightly higher than it would at
room temperature (25 ºC.) Such an environment might favor the growth of bacteria rather than yeast, especially if the pile was rich in proteins
rather than carbohydrates. Agitation will also speed up the decay process... "
(from http://www.hypography.com/scienceforums/showthread.php?t=187)
In short, they need the carbohydrates, too. Don't be stingy.
sparky
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Polverone
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Hmmm. Yeast need oxygen? I thought they produced ethanol, at least, only in anaerobic conditions, though they thrive and multiply in aerobic ones.
I'm a bit confused about which conditions I want to maintain to maximize generation of alcohols from amino acids.
My first experimental little batch was prepared as follows: 150 g of sucrose were dissolved in 1 liter water in a clean 2-liter soda bottle. The raw
egg white from one small egg was added and distributed with agitation. 10 grams of dry, active bakers' yeast was added and the whole mix agitated
and loosely capped (so that CO2 can escape but the outside world stays out).
It has been fermenting for about 36 hours now. I realize that it may be a rather nutrient-poor mixture. Hopefully it will consume all the sugar
anyway. I kept the sugar concentration moderate since I knew the yeast wouldn't be in tip-top shape.
On some sites I've seen it said that high-temperature fermentations produce more congeners. In another paper I found, it said that conversion of
amino acids to alcohols via yeast fermentation is maximized at 21-23 C. I suppose both could be correct and at the higher temperature more carbonyl
compounds and esters are formed. I'm not really sure.
It appears that yeasts can use glutamic acid as a nitrogen source. This might be the perfect substance to experiment with since monosodium glutamate
is pure and inexpensive in bulk. It would be very nice if the yeasts can use proteins as-is, though. I will see.
I have used sucrose because it is very inexpensive and plenty of people seem to have no problems using it to feed yeast. I can get fructose and
glucose too, but sucrose is cheaper than fructose which is cheaper than glucose.
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sparkgap
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Cleared it up a bit. While I was trying to remember my microbial experiments, I seem to recall that I grew the yeast in a carbohydrate-rich medium
under aerobic conditions for about a day and a half before transferring them to a, um, medium more suitable for producing alcohol ( can't say
where I transferred them, or I'll get my ass whupped  ). Yes, alcohol only
comes out at anaerobic conditions. But that is precisely because they're oxygen-starved. Their metabolism can only do so much without oxygen.
Update me on feeding yeast with egg white. If that succeeds, you may not have to bother with acquiring MSG.
sparky
[Edited on 21-1-2005 by sparkgap]
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chemoleo
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Eggwhite is actually not such a bad idea, as it contains lysozyme - an enzyme that that kills bacteria by 'lysing' them open, so eggwhite
straight from an egg washed with ethanol (sterilising it) should yield sterile protein.
Lysozyme is harmless for yeast though - which is what you want.
Otherwise, you could add antibiotics, which won't affect yeast. I am just mentioning this becuase i'd be always paranoid about sterility, I
wouldn't want bacteria messing in the fermentation, or other fungi (which are presumably even more of a problem).
Sterilising protiens by heat would be difficult as they tend to denature, which would produce flocculant precipitates.
As to the need for carbohydrates or not, in a protein rich nutrient solution, best is probably just to do the experiment and see which grows better.
By the way, I remember beer-brewing experiences where we just covered the brew with heated paraffin-based oil (food grade). This kept it nice and
sterile.
By the way, Polverone, how do you intend to determine the amount of fusel oils made from each experiment, as opposed to the ethanol produced? Or the
composition of the fusel oils?
[Edited on 21-1-2005 by chemoleo]
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sparkgap
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I most certainly agree with the lysozyme. Eucaryotes do well with lysozyme; procaryotes (well, most) tend to just dissolve in oblivion after being
exposed.
Side note: tears have lysozyme.
Acquiring the antibiotics won't be cheap. If you plan to, though, buy streptomycin from someone who supplies agricultural chemicals. The brand
name, IIRC, is "Agristrep".
The quantitative test I can think off the top of my head as of this moment is to use KCr2O7. You could replace this with KMnO4 if you're
concerned about disposing of chromium, but the oxidation is slower.
sparky
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Polverone
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Very unfortunately, I don't have the space for much glassware, and this means no distillation  . I plan on trying to saturate the liquid with salt and then perform a solvent extraction. The higher alcohols should
be more hydrophobic and should also remain behind last when the solvent is evaporated.
Some of the alcohols are said to have characteristic odors of their own; I can also try making esters of the mixture and then seeing how it smells.
This is crude, I know. Some of the materials I extract will not be alcohols at all. I will deal with it as it comes. The first question is "how
much not-ethanol can I make and extract from a given fermentation?" My liter of egg + sugar media has what appear to be oily droplets floating on
the surface right now. I hope that these are interesting fermentation byproducts. I could probably satisfy most of my curiosity about all this by
spending enough time with the library, but I like to make and watch things as well as read about things.
My first project was going to be the production of glycerol via bisulfite/sulfite addition to fermentation, but I didn't have enough nutrient
material around to run it the way I really wanted to. I will be getting those materials and trying that. I'm actually more interested in the
acetaldehyde/bisulfite adduct formed than in the glycerol.
I find it very interesting that bisulfite will capture acetaldehyde under the relatively diluted conditions of fermentation. The adducts formed with
acetone and benzaldehyde, at least, do not appear/stay insoluble in dilute aqueous media.
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Organikum
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You might try nutrient salts in special MgSO4, (super)phosphate and ammoniumsulfate (nitrogen source).
Urea is a fine nitrogen source too.
For ph-control citrate buffers are easy and work fine. Or some hydrogenphosphates - works too.
Thiamine might help as sodium pyruvate, but this all depends....
...depends on so many factors that I cannot give true advice now and here.
Using large amounts of yeast from start diminishes problems with infections, H2O2 (dil.) is useful for sterilizing igredients, the yeast can stand
quite some of this - bacteria not.
Of course you have to decide if you want to do this anareobic or aerobic. Or somehow inbetween/cycled perhaps? 
Sorry I am have no time to look deeper into this now.
/ORG
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sparkgap
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Interesting links I have come across:
http://aem.asm.org/cgi/content/full/66/10/4187
http://www.wynboer.co.za/recentarticles/1200ethyl.php3
(apparently, urea for yeast is a bad idea, unless you want urethane)
http://byo.com/feature/1162.html
(egg white and beer-making)
Now for my own input:
The "most" in my last post is most certainly correct. Gram-positive bacteria die; Gram-negative bacteria scoff lysozyme. That is why
Salmonella infection is a risk in handling raw egg white, since poultry tend to harbor these nasty microbes.
Using large amounts of yeast, as Organikum suggested, puts you at a risk of diminishing returns (crowding and nutrient depletion come to mind). Best
is to start small and let them reproduce for a while in aerobic conditions before transferring them to an anaerobic medium.
But in fairness to Organikum, his advice on buffers sounds good. Not too much salts though, or plasmolysis might set in.
Just my two cents worth...
sparky
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Organikum
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| Quote: | | Best is to start small and let them reproduce for a while in aerobic conditions before transferring them to an anaerobic medium.
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This is doubtless true but requires rigorous sterile conditions when you want reproducible results.
Starting with the amount needed for the anaerobic fermentation avoids this.
(addition of ethanol to the fermentation broth suppresses further ethanol production and often favors other compounds - it depends if the compounds
wanted are coupled directly to ethanol production or not. This also prevents infections)
Nutrient depletion is avoided by:
- addition of glucose
- addition of thiamine
- addition of sodium pyruvate
Addition of pyruvate alone is an almost unfailable means to revive the metabolism of a yeast which has ceased fermenting - as long the yeast isnt
already completely dead of course
Growing the yeast oneself allows to condition the yeast for special purposes, to acid-harden it or to make it resistant against elevated temperatures
for example, whats advantagesous, but I wouldnt start this way as it adds to many variables.
/ORG
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JohnWW
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"Fusel oil" alcohols are propyl, isopropyl, butyl, isobutyl, pentyl (amyl) isopentyl, hexyl, and isohexyl alcohols, with straight carbon
chains; some esters of the corresponding carboxylic acids also occur. They are toxic in large quantities. In distillation of spirits, their content in
alcoholic drinks is reduced; but it is not reduced in alcoholic drinks drunk as fermented. Fusel oil is concentrated in alcoholic liquor in which the
water is partly removed by freezing instead of distillation.
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sparkgap
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How are the fusel oils coming?
How are the little buggers doing producing your coveted fusel oils? Did the egg whites work?
sparky (~_~)
P.S. How do you get pyruvate OTC?
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Polverone
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fermentation progress
Though my interests are spurred by unconventional applications of yeasts, I decided that I needed to get a better feel for ordinary fermentations
before moving on to modified schemes. So right now I'm trying to come up with a good nutrient mix and other conditions (acidity) to allow
baker's yeast to ferment relatively high concentrations of sugar to alcohol. I've seen many nutrients suggested in various places. Many
sites suggest buying prepackaged nutrient mixes or using various organic materials like tomato paste. Of course I'm interested in using mixtures
of pure chemicals for the inorganic nutrients. Most sites also assume that you're fermenting plant products (from grapes, barley, etc.) and that
doesn't apply here. Sites on home distilling have given me what seems to be pretty good advice for preparing sugar-based wash.
I think I've achieved decent results on my most recent run, which used NaHCO3, KHCO3, NH4NO3, H3PO4, MgSO4, CaCl2, and a multivitamin pill to
provide nutrients. This may be overkill since I tried adding a bit of everything that different sites suggested. In the future I will probably use
urea instead of NH4NO3 since I learned that baker's yeast cannot make use of nitrate. Of course during the fusel oil production experiments I
will withhold nitrogen sources other than amino acids to encourage fusel production.
According to a number of references, yeasts cannot directly use proteins, only free amino acids and small peptides. My fermentation with egg white
seemed to complete okay. It developed a bit of an H2S odor. I had insufficient salt on hand to saturate the fermentation liquid before extraction, so
I eventually tossed it out without attempting an extraction. I need to go to the big grocery store and stock up on sugar, salt, and dry active yeast
from the bulk section.
Calcium pyruvate is sold at health food stores, or pyruvic acid can be prepared by heating tartaric acid with KHSO4.
I rinse out my fermentation containers with chlorine bleach and water before starting a new fermentation. Is this a good way to sterilize?
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chemoleo
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| Quote: | | According to a number of references, yeasts cannot directly use proteins, only free amino acids and small peptides. |
Now that is interesting (to me anyway). Could you please quote these references? It seems surprising to me, that an organism as versatile as S.
cerevisiae, does not produce extracellular proteases that allow its survival on proteins (eggwhite). With this I should assume that S.
cerevisiae only survives on free a.a's, meaning that its growth is dependent on other organisms that degrade proteins (i.e. a symbiotic
relationship)? That'd be a surprise indeed.
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sparkgap
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Quote:
...an organism as versatile as S. cerevisiae, does not produce extracellular proteases that allow its survival on proteins...
That, or it has really lame extracellular proteases.
But Polverone mentioned that the eggwhite fermented and developed H2S odor. Assuming only yeast was there (You did it aseptically, no?) that means it
was able to cut a few peptide bonds (cysteine included).
.... S. cerevisiae only survives on free a.a's, meaning that its growth is dependent on other organisms that degrade proteins...
Now I'm curious. Just where did man get his wild yeast from back in the olden days?
Chemoleo: symbiotic, or more precisely, commensalistic.
To answer a question:
...rinse out my fermentation containers with chlorine bleach and water before starting a new fermentation. Is this a good way to sterilize?
Just rinse? You should have let the bleach stay for two minutes tops. Then you rinse. Thrice. Just to make sure.
sparky (^_^)
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unionised
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Just a thought, would yeast ferment aspartame to anything interesting?
I guess methanol is going to be one product, any guesses for anything else?
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chemoleo
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Yes, if you look at the structure, you'd get aspartic acid, methanol and phenylalanine (ie. by proteolysis, or in the testube by boiling with acid).
This would then be reduced to phenylethanol, the aspartic acid I don't know.
However, a single sugar replacement tablet probably only contains a few mg of the aspartame (being 160 x sweeter than sugar), so you'd have to
use quite a few tablets to get decent amounts of phenylethanol. I like the idea though 
Never Stop to Begin, and Never Begin to Stop...
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unionised
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I had a look at the sweetners in the shops, you can get glycine, leucine and aspartame at reasonable prices. (None of them pure, they're all
mixtures). Leucine should give methylbutanol if I've got the chemistry right. Just what you need for a fusel oil.
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Esplosivo
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This is the result of my first run using 1.5Lts of medium and a sachet of Baker's yeast, Saccharomyces cerevisae. I did not use any
amino acids in the mixture on purpose, so to compare results of further fermentations with what I obtained during this run. As a nutrient medium I
used a quasi saturated sucrose solution to which I added the following: MgSO4, CaCl2, KH2PO4, NaCl, KCl, NH4NO3 and a very small quantity of
salts containing Cu2+, Fe2+ and ammonium molybdate. This was left to ferment in an anoxic environment for approximately 4-5 weeks. After a crude
fractional distillation of the ferment I obtained the 'white'ish coloured (transparent) liquid smelling strongly of 'crude wine'.
A crappy picture is attached. I was thinking about an emulsion of some sort, such as for example ethanol emulsifying larger-chain alcohols such as
butanol. I also found it interesting that after settling a small amount of colourless layer formed above the solution (not visible in the attached
picture because the quality of the picture is absolutely not a good one), probably showing the presence of long-chain alcohols which are rather
insoluble.
I am currently running a 2.5Lt batch with nearly the same mixture so that I can obtain more of the product and carry out a proper fractionation (when
my ordered distillation set finally arrives).
Strange is the fact that I used absolutely no amino acid (and was quite careful there wasn't any). I will extract casein from milk in the near
future and hydrolyse it with hydrochloric acid, which can later be neutralized with sodium hydroxide. Some extra NaCl would probably cause no
difference in the fermentation process. Following the distillation of that product I will post other results. The process seems promising.
Edit1: Forgot to mention that I tried salting-out but without any success. If that is an emulsion is seems difficult to break. I will try adding some
chloroform or any other immiscible solvent so to see if the white colour is reduced, but not soon unfortunately.
[Edited on 20-4-2005 by Esplosivo]
Theory guides, experiment decides.
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Esplosivo
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Just made a quick test to verify wether the white distillate was an emulsion or not. I saturated a 40mL sample with NaCl (an excess, most was still
undissolved). I then added approx 5-10mL of ethyl ethanoate (this is why I saturated the water with sodium chloride, since the ester is appreciably
soluble in water). On agitating the mixture the whit colour vanished. After settling the two layers separated slightly again, but they are still
separating in the fridge. There therefore was a 'considerable' quantity of longer-chain alcohols without the use of any amino acid.
Edit1: Two layers seem to have settled. The lower conc. salt soln. is of about 4mL while the rest (which I am assuming is alcohols and the ethyl
ethanoate I used) amount for up to >45mL. I'm quite astonished really, thats quite a bit of alcohol content after a simple fractional
distillation from which I obtained nearly 300mL from 1.5Lts of ferment.
[Edited on 22-4-2005 by Esplosivo]
Theory guides, experiment decides.
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