I’m not a real hillbilly, but I play one on the internet…

My first “Will It Ferment” project is now in process.

See, I’m stuck with a somewhat unpredictable schedule and a need to travel frequently, cramped spaces to work in at the moment, and a streak of gustatory perversion that I just can’t help an urge to rebel against purists at the moment.

Quick bit of background on that latter statement: I’ve noticed that people seem to think there are only two-and-a-half kinds of “real” non-commercial fermented beverages. There’s beer (which is apparently defined as a strong tisane of hops, flavored by mixing it into fermented malt), there’s wine (which is always made of grapes of course), and out on the fringes of respectability is Mead (“Honey wine”) which seems to be slowly gaining some acceptance as a mildly exotic brew. The attitude is that anything else you might want to brew (say, a beer flavored and preserved with something besides hops, or a wine made out of anything but grapes) is probably some quaint “country” (i.e. hillbilly) thing for people who either live too far from civilization or are too poor to just buy a “real” beverage, or are too ignorant to know the difference. Either that, or it’s just some desperate attempt to make something to get drunk on. Might as well be making pruno.

The attitude kind of annoys me, so I’m trying to make a sort of “free person’s pruno”. I figure if the end result is as palatable as it could be, it’ll probably resemble “Zima”. Uh, no, I don’t expect it to be great – this is merely an experiment. My must has an Original Specific Gravity (“O.G.”) of approximately 1.054, about the same as most lagers start out. As I write this, my gallon of must is on the stove in a stainless steel pot. I’m going to try to heat it to boiling and let it boil for a few minutes in hopes of driving off much of the benzoic acid in it, so as not to inhibit the yeast fermentation.

To your right, you should see the ingredients used in this project. Yes, those two bottles in the background are there on purpose. I’m trying to make…Mountain Dew Wine.

Here’s the plan:

  • Last night, I took a couple of clean glass 1 Qt milk bottles, rinsed them with iodophor solution to sanitize, let them dry. Then, I put about 12 ounces of tap water (unchlorinated) into one and dumped the contents of both yeast packets you see in the picture into it to rehydrate.
  • The yeast packets were opened over a year ago – I don’t even recall what I did with the tiny amount of yeast I poured out. They’ve been sitting in the ‘fridge since then. What’s more, they had expired in December of 2006 to begin with.
  • The two yeasts (Red Star Montrachet and Red Star Flor Sherry) were both mixed into the same container in hopes that I could encourage a yeast orgy, giving me as much genetic diversity as possible from the two strains and maximizing the chance that I’d be able to get a culture which can grow in flat Mountain Dew and whatever benzoic acid (from Sodium Benzoate) might remain in it. I had read that V8 juice was actually an excellent medium for inducing yeast sporulation. Since sporulation occurs as a result of yeast cells mating, I made a huge leap of logic towards thinking the V8 might maximize my chances of getting some yeast mating going on.
  • After a few minutes to rehydrate, I dumped the 12-ounce can of V8 into the bottle and shook well. I also crushed up a children’s chewable vitamin (see bottle at lower-left) and a small portion (perhaps 1/5) of a capsule of the L-Arginine as a nitrogen source and added them as well. I then poured it back and forth between the two bottles a few times to aerate, then split the mixture between the two bottles, capped loosely, and went to bed.
  • As of this morning, fermentation was obviously occurring in the mixture, so sitting in my fridge open for over a year hadn’t killed off ALL of the cells. I opened the bottles and swirled to re-aerate, and added about a tablespoon of “corn sugar” (glucose a.k.a. dextrose) to wake the yeast cells back up. Since then, I’ve been pouring a bit of Mountain Dew right out of a third bottle into each of the yeast starter bottles every couple of hours. The amount of bubbling I see suggests to me that fermentation is still going on (and is not just from the carbonation of the Mountain Dew). Hopefully this will help the yeast culture acclimate a bit to the benzoic acid.
  • I dumped the other 2 2L bottles of Mountain Dew into a stainless steel 8-qt pot and heated to boiling, whisking with a steel whisk frequently to help get the dissolved gases to bubble out (hopefully along with some benzoic acid in the steam). At this moment, it’s up to 75C (about 165F) according to the thermometer I have stuck in it. It’s steaming a bit, and I can smell some of the citrusy aroma boiling off, unfortunately. I was afraid that’d happen. UPDATE: our ancient stove with one working burner seems to have trouble getting this much liquid above 200F without cranking it up all the way and risking the bottom getting too hot. Since there’s a substantial amount of hot water vapor coming off, I’m going to hope that’s carrying away some benzoic acid and just let it start cooling down. I’ll stir it vigorously and frequently with the whisk until the temperature drops to about 160F and then I’ll cover it for the night.
  • Meanwhile, before I go to bed, I’m going to recombine as much of the two yeast culture bottles as I can into one bottle, then rinse out the other. In that one, I’ll mix up about 12 ounces of tap water and enough corn sugar to reach a gravity of about 1.050-1.055. I’ll crush up another chewable vitamin and about half of the remaining arginine capsule into it, mix well, and warm it with a quick spin in the microwave (no exact measurements, just until it’s “obviously warm” to the touch after mixing). Then I’ll shake the V8 culture well to mix, and splash about a tablespoon’s worth into the new bottle.
  • In the morning, I’ll re-aerate the new culture and add a tablespoon of corn sugar to wake it back up, then once it’s going, I’ll start adding the now-cooled cooked flat Mountain Dew to it in small increments. Assuming it keeps going, I’ll dump in the remainder of the L-Arginine capsule, crush in one last chewable vitamin, and them combine the new culture with the rest of the cooked flat Mountain Dew in a nice plastic 2-gallon “water” container that I picked up (already rinsed with iodophor and dried). Cap it with a latex glove attached with a rubber band as described in the “Pruno” entry in Leon Kania’s “Alaskan Bootlegger’s Bible” (Click image for link) just because I thought it was the funniest airlock design I’d ever run into. Yes, I am easily amused.
  • If I’m lucky, there’ll be so much live and active yeast in the second culture at that point that the fermentation will finish quickly, because I’m driving to Texas the following day. Alternatively, I may be lucky and the remaining benzoic acid will slow the yeast down, so I can safely leave it fermenting (sitting in my sink in case of overflow while I’m gone) for the week that I’ll be gone.

If I’m UNlucky, either it won’t ferment at all (and I can then use it to try to develop a Mountain Dew Tolerant strain of yeast), or it’ll ferment but taste utterly disgusting (in which case I can use it to try to obtain some Gluconobacter strains and make Mountain Dew Vinegar), or it’ll be “infected” and will already BE Mountain Dew Vinegar, which would also be pretty funny anyway. So, other than completely unforseen results, this experiment can’t be a TOTAL waste.

[Update 20080727: Preliminary results of this perverse project may be read In this more recent post…]

Boosting fermentation with science

All right then – I’ve got five pounds of honey, a pound of frozen cherries, packets of a couple of different dried yeasts, miscellaneous other potential additives, two 2-gallon polyethylene terphthalate fermentation containers with screw-top lids and spigots, several feet of aquarium airline tubing and connectors, silicone sealant, and miscellaneous kitchen gadgets (including a hydrometer). Now it’s time to discuss what I’m about to do and fish for comments and criticisms before I jump into it.

My goal here with this brewing experiment is a quick primary fermentation. And to compare the results from two different yeast strains, uh, TWO goals, quick fermentation, yeast strain comparison, and fermentation container design. THREE goals. Quick fermentation, comparing yeast strains, fermentation container design, and to try to keep the yeast cultures from dying off too quickly during the fermentation. FOUR. Four goals…

In this post, I’ll stick to talking about what I’m putting into the brew and how I hypothesize my additives and process with speed the fermentation along and help keep a large portion of the yeast viable during the primary fermentation.

Actually, the health of the yeast populations and the speed of fermentation are overlapping goals; more cells remaining alive and healthy means more cells simultaneously chewing up sugars and spitting out ethanol for me, resulting (hypothetically) in faster primary fermentation. In this experiment, I’m going to be focussing on nutrients and spices that are reported to benefit yeast activity. Here’s the process I am currently planning to follow, focussing primarily on the fermentation-boosting parts:

  • I’ll boil the 5 pounds of honey with enough tap-water to make about 2 gallons of must, adding the frozen cherries sometime after the boil gets underway.
  • Fermentation boost: we have water so hard that you have to wear a helmet to take a shower. (Joke stolen from my Environnmental Chemistry instructor, so you can blame Dr. Rosentreter for that one). It’s loaded with Mg2+ and Ca2+, which seem to be able to help the yeast to produce ethanol faster and survive higher ethanol concentrations better[1][2] as well as just being general nutrients[4].

  • Two approximately -liter amounts of the must will be put into clean glass quart bottles and used to develop the initial yeast culture for pitching (each one for a different strain of yeast).
  • Fermentation Boost: Growing up a large population of yeast from the dried yeast packets before pitching will give me a faster start. In addition, the large headspace and the use of cloth rather than plastic or rubber covering of the top will allow oxygen to get into the starter culture, helping it to develop more quickly and in a more healthy fashion (i.e. a larger proportion of healthy, viable cells).

  • Nitrogen supplementation: Capsules of arginine picked up cheap at a certain big-box store will be added to the yeast starter.
  • Fermentation Boost: “Free Amino Nitrogen” is perhaps the most important bulk nutrient for yeast, and arginine seems to be the preferred amino acid source[3][4], presumably because it contains the most reduced nitrogen per molecule of the amino acids. I actually want to try to develop a process for using dry milk powder instead, but achieving sufficient hydrolysis of the milk proteins looks like it’s going to take some development on my part. For now I’ll “cheat” and use arginine instead.

  • Vitamin supplementation: A single well-crushed children’s “chewable vitamin” (“Flintstones” or generic equivalent) will be added to each starter culture as well.
  • Fermentation Boost: Pantothenic Acid (Vitamin B5), Inositol, trace minerals, and small amounts of additional potassium and phosphate to supply vital nutrients to the yeast culture.[4]

  • Fermentation-enhancing spices: I will be adding ground ginger and cinnamon (actually cassia) to the must near the end of the boil.
  • Fermentation Boost: In addition to providing what I think will be excellent complementary flavors to the final product, it appears that even fairly large amounts of these two spices – among others – provide a boost to fermentation rate[5] (via Shirley O. Corriher’s “Cookwise”[6]) of Saccharomyces cerevisiae cultures. If I’m doing the conversions appropriately, the peak fermentation boost for ginger works out to something like 3 tbsp of ground ginger per liter, or something like (very roughly) 10 tablespoons per gallon. I don’t plan to add quite so much, but a couple of tablespoons of each spice in the two-gallon batch ought to provide some nice flavor while still hopefully providing a boost to the fermentation rate as well.

“Cinnamon”: In the US, the rust-colored stuff labelled “Cinnamon” is not, actually, cinnamon. True cinnamon (Cinnamomum zeylanicum)is actually tan in color. What you get in the US when you buy a bottle of “Ground Cinnamon” actually comes from Cassia (Cinnamomum aromaticum), a closely related plant. Realistically, as far as I have been able to find out so far, there’s not likely to be a huge difference in the active components or flavor. While I haven’t yet gotten my hands on a copy of the old article from Cereal Chemistry[5] mentioned above, I’d give good odds that the “cinnamon” used in the study was also actually cassia anyway.

There’s one more thing that I hypothesize would help promote my goals that could be added: small amounts of oxygen[7] (say, less than 13% O2, or very roughly speaking, around half of the normal atmospheric concentration or less). However, I’m still trying to work out an easy way to achieve this automatically and am not yet ready to try it. Besides, this is already pretty poorly-designed for a “real” scientific experiment as it is, considering the number of variables that are really contained in this brewing process. Really, my hypothesis here boils down to a relatively vague “This mixture and process will allow me to finish the primary fermentation within a day or two of pitching”. If I ever have opportunity to do serious experimentation on this, it’ll require setting up a large number of separate fermentation reactions to assess the effects varying each individual set of hypothetically-fermentation-boosting additives. Hopefully one of these days things will settle down enough to let me try it.

If anybody sees anything stupid (or just interesting) up there, please say something…

[1] Dombek KM, Ingram LO: “Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation.”; Appl Environ Microbiol. 1986 Nov;52(5):975-81.
[2] Nabais RC, S-Correia I, Viegas CA, Novais JM: “Influence of Calcium Ion on Ethanol Tolerance of Saccharomyces bayanus and Alcoholic Fermentation by Yeasts.”; Appl Environ Microbiol. 1988 Oct;54(10):2439-2446.
[3] Carter BL, Halvorson HO: “Periodic changes in rate of amino acid uptake during yeast cell cycle.”; J Cell Biol. 1973 Aug;58(2):401-9.
[4] Fugelsang KC, Edwards CG: “Wine Microbiology – Practical Applications and Procedures (2nd Ed.)”; 2007; Springer Science+Business Media LLC; pp 15-18
[5] Wright WJ, Bice CW, Fogelberg JM: “The Effect of Spices on Yeast Fermentation.”; Cereal Chemistry. 1954 Mar;Vol.31,100-112
[6] Corriher, SO: “Cookwise”; 1997; HarperCollins Publishers, inc; New York; pp 69-70
[7] Nagodawithana TW, Castellano C, Steinkraus KH: “Effect of dissolved oxygen, temperature, initial cell count, and sugar concentration on the viability of Saccharomyces cerevisiae in rapid fermentations.”; Appl Microbiol. 1974 Sep;28(3):383-91.

The care and feeding of Saccharomyces

Let me pause now for a moment to review what I’ve learned so far:

  • Yeast are filthy little jerks
  • No, seriously. I’ve previously reviewed their promiscuous sex lives,
    their sexually-transmitted diseases, and their toiletry habits. Somehow, though I still want to do more brewing, so let’s continue.

    Bag of 'Parodina Yeast Chow'.  I am not affiliated with Purina Mills corporation!  This image is PARODY!

  • Yeast need to be fed particular sugars
  • The three major elements needed by pretty much every living thing for “food” are Carbon, Nitrogen (as reduced “amino” nitrogen), and phosphorus (as oxidized phosphate) (Reduced sulfur is also needed in small amounts for proteins). Glucose (“dextrose” or “corn sugar”), fructose, or sucrose (“table sugar”, each molecule of which is made of a molecule of glucose attached to a molecule of fructose) are all used as carbon sources by Saccharomyces yeasts. Possibly also Galactose under certain conditions[1]. Saccharomyces yeasts don’t appear to be able to use lactose (“milk sugar”, each molecule of which is made of a molecule of glucose and a molecule of galactose), so some recipes include lactose in order to ensure there is some residual “sugar” in the mix at the end, for flavor and “body”.

  • Yeast need reduced nitrogen (amino nitrogen or ammonia…or urea)
  • Aside from sugars, this seems to be possibly the most important yeast nutrient. The most
    “natural” source of this nutrient would seem to be amino acids or very short peptides (2-5 amino acids long). Apparently urea (carbamide) also makes a good yeast nutrient, but:

  • You don’t want TOO much nitrogen available to the yeast, or there’ll be excess urea dumped back into the brew
  • This could combine with the ethanol to make “ethyl carbamate”, which is considered
    a probable carcinogen, at least if it’s present at a high enough level. Obviously if you use urea as a
    yeast nutrient, that’s only going to increase the possibility of a problem.

  • Saccharomyces yeasts are effectively incapable of using proteins for nutrition.
  • Proteins can be a source of amino nitrogen (and carbon and sulfur), but like all real microbes, yeast cells cannot just “eat” chunks of protein. They have to be broken down into very small chains of amino acids or even as individual amino acid molecules before the yeast can suck them up and use them. Saccharomyces yeasts do not appear to normally excrete protein-digesting enzymes, so by themselves they cannot make any use of protein for nutrition[3].

  • Yeast need oxygen
  • Oxygen is necessary for making certain components of the cell membrane, in addition to it’s more obvious role in respiration. Without a way to replace used up membrane components, the yeast stop reproducing and eventually fall apart and die. There seems to be some suggestion that to a certain extent one can substitute some raw membrane material for oxygen here (either as “yeast hulls” or possibly even certain of the natural waxes on some fruits).

  • If you give yeast oxygen, though, they consume the sugars entirely instead of making alcohol…
  • …or do they? Between the “Crabtree effect” (when there are high concentrations of glucose, alcohol production continues even in the presence of oxygen) and indications in scientific papers[2], it seems SMALL amounts of oxygen may not be a problem, and might very well be beneficial.

  • Yeast need vitamins and minerals
  • B1 (“Thiamine”) is commonly mentioned, though apparently the need for it varies from strain to strain. Also potentially important are Pantothenic Acid (B5), Niacin (Nicotinic Acid, Vitamin B3), Biotin, Inositol, as well as Potassium, Magnesium, and trace amounts of calcium and a few other minerals[4].

  • Unhealthy yeasts are more prone to make (EEK!) Off-Flavors and Off-Odors (EEK again!)
  • For one thing, it seems to be a general rule that you don’t want your brew sitting on the corpses of dead yeast (the “lees” of wine, or “trub” of beer), because that is a potential source of (insert dramatic music and crash of thunder here)Off-Flavors and Off-Odors. Yeast dying and falling apart is also a major source of urea being dumped into the brew, too. Some strains of yeast under certain conditions, such as insufficient pantothenic acid, may be prone to producing nasty-smelling sulfides as well.

So, in most cases what we want to do when brewing is keep our yeast as alive and happy as possible, and get them to hurry up and finish our primary fermentation before they start dying off. Coming up: My (as yet untested) plot for accomplishing this – without specialized scientific equipment or materials.

[1] Wilkinson JF: “The pathway of adaptive fermentation of galactose by yeast” Biochem J. 1949; 44(4): 460467
[2] Nagodawithana TW, Castellano C, Steinkraus KH: “Effect of dissolved oxygen, temperature, initial cell count, and sugar concentration on the viability of Saccharomyces cerevisiae in rapid fermentations.” Appl Microbiol. 1974 Sep;28(3):383-91.
[3] Bilinski CA, Russell I, Stewart GG: “Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects.” Appl Environ Microbiol. 1987 Mar;53(3):495-499.
[4] Fugelsang KG, Edwards CG: “Wine Microbiology: Practical Applications and Procedures” 2007; Springer Science+Business Media LLC, New York; pg 17

More on the shocking life of yeasts

(Brief Update: Hello Ontario! Did I attract the attention of a Toronto homebrewing club or something? Anyway – welcome!)

I am amazed at how much depravity I uncover as I explore the mystery that is
Saccharomyces cerevisiae.

I’ve previously discussed how the filthy little beasts have drunken orgies and exchange sexually transmitted diseases with each other. Now I find out the inebriated little jerks are peeing in my beer, possibly to try to give me cancer!

No, seriously. Given enough “Free Amino Nitrogen”, for example in the form of the pirate’s favorite amino acid, like tiny little single-celled bladders, the yeast will start excreting extra nitrogen in the form of Urea all over whatever they’re growing in.

Of course, the whole time they’ve also been excreting ethanol. It turns out, under certain conditions urea (more formally known as “carbamide” nowadays) and ethanol will combine like drunken evil “Wonder Twins” to form Ethyl Carbamate.

Front Cover of the bookI ran into this as I was reading through my shiny new Wine Microbiology book, which has two pages on this yeast pee byproduct. An article linked to from fark.com recently reminded me of it and prompted this post.

To be honest, this seems a lot like the acrylamide media circus (compare the two links…) that popped up back in 2002. In both of these cases, we’re talking about a substance that occurs as a natural result of the preparation process rather than some new industrial chemical, and in both cases the processes in question have been around probably since prehistory. And in both cases, the real situation seems to boil down to something like “pay attention to your preparation technique, and if you try to live entirely on a diet of overcooked French fries and dessert wines, you might be at an increased risk for cancer.” QED. Or perhaps DUH.

Other than not trying to live on a French McDonald’s diet, there are some things you can do when you brew to limit ethyl carbamate formation. Put very simply: don’t overfertilize your grapes because that can directly lead to unnecessarily high levels of nitrogen available in your wine, and don’t leave your bottles of brew in hot conditions for long, because ethyl carbamate forms faster in hot conditions.

There, problem solved. A more detailed “ethyl carbamate preventative action manual” may be found here. Meanwhile, I’m pretty sure our favorite drunken little micro-hedonists are too busy partying and making our wines and beers to be plotting our cancerous dooms.