Tag: fermentation

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 75°C (about 165°F) 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 200°F 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 160°F 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…]

I finally got to Bristol Brewing Company…

Signage in front of the Bristol Brewing CompanyAfter spotting the company’s mention in Jeff Sparrow’s “Wildbrews“, I’ve been wanting to visit Bristol Brewing Company, particularly since they may be the only such brewery using local yeasts and bacteria that they isolated themselves from their local environment.

Since we’re moving from Southeastern Idaho to Southeastern Texas, I’ve been going back and forth between the two location. It just so happens that along with New Belgium Brewing Company, Bristol Brewing is actually right along a convenient route between the two locations. (Let’s see if I can figure out how to work the Google Maps plugin):

How’s that look? How does it work? (Those of you reading this on the RSS feed: The interactive map only appears on the website, it would seem. Please check it out here.) I made the KML file myself…

Bristol Brewing is a cozy little brewpub, and the people there are encouragingly helpful. They were in the middle of bottling, so there were no tours. Also, there were not currently any of the skull-‘n-bones beers available. However, I did get an educational series of tastes of their current brews on tap (thank you to the employee I spent most of the time talking to whose name I’ve embarrassingly forgotten, but who I believe was Tad Davis judging from the photos on the web site). I also lucked out and their microbiologist, Ken Andrews, happened to be there. I asked about their native Colorado brewing flora. Turns out Bristol Brewing isn’t quite as bold as I originally thought. They’re still doing their primary fermentation with “normal” brewing yeasts. What they’ve done is inoculated some wine barrels with the locally-isolated yeasts and bacteria, and they use the barrels for a secondary fermentation and aging instead. Much safer if you have to worry about having a drinkable product at the end, and of course it makes me feel like more of a crazed rebel for wanting to isolate my own local bugs for the main fermentation. So, a great visit overall.

Incidentally, it seems they’ll be tapping a new Skull-‘n-Bones brew in a couple of weeks, at 5pm on Tuesday, May 27th (2008). It sounds like they’ll probably have some available for a few days before it all disappears, so I’m hoping my return to Texas can be timed such that I can swing by and at least get a taste.

I Hate You, Carl Zimmer!

Carl Zimmer wrote a book. Of course, that’s no reason to hate him, and I don’t hate him for that.

His book is all about Escherichia coli (“E.coli”). The friggin’ “Microsoft” of the biotech world. Accursed E. coli, hogging up all the print space and protocol development and sucking up electricity for -80°F freezers. I mean, come on people! You could be doing transformation of B. subtilis and related organisms instead, which form nice, sturdy endospores which you can dry out and keep in an any cool, dry place, no -80°F freezer needed! Or you could use something like Agrobacterium tumefaciens, and as a bonus be able to then transfer your nice transformed genes into plants, too! But NOOOOOooo….it’s always “E.coli, E.coli, E.coli.” DAMN YOU, E.COLI!

Of course, none of that is Carl Zimmer’s fault, either, so this is also no reason to hate him.

Now, if his book was lousy, that MIGHT be a reason to hate him, but as far as I can tell there’s no reason to think the book is lousy, so this is no reason to hate him either. In fact, that’s kind of the problem.

No, the reason I Hate Carl Zimmer is that he’s written a book about friggin’, stage-hogging E.coli…and I want it. (Well, a copy of it anyway.) It sounds like a very interesting book. I feel like a Republican who wants a copy of “The Audacity of Hope”. Or a Democrat who wants to plan a vacation to visit the George W. Bush Presidential Library. The cognitive dissonance torments me, and it’s all Carl Zimmer’s fault! CURSE YOU CARL ZIMMER!

Okay, got that out of my system. A review might follow eventually if I manage to get a copy of the book. Meanwhile, for a change of pace, anybody want to hear about my Asterisk setup? Or should I just get back to the fermentation stuff?

P.S. Here’s a bit of trivia for you: “Frig” is apparently an old-English word meaning “to wiggle”…

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 ½-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, Sá-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.

Fermentation: not just for alcohol

What does gluconic acid taste like, anyway?

Well, that was an interesting reminder. I’m tracking “fermentation” on Twitter, and caught a random reference to an interesting fermented beverage being made in Germany. The “reminder” I drew from this serendipitous reference was that “fermentation” doesn’t necessarily mean alcoholic fermentation.

“Fermentation” seems to be slightly tricky to define accurately. Most definitions seem to directly mention alcohol production from sugar, but this is only an example and not a definition. I’ve also seen the term used to mean simply “to grow a culture of microorganisms” (because the tank they are grown in can be referred to as a “fermentor”.)

Properly speaking, fermentation is what you get when you have microbes growing under conditions where the elelectrons that get sucked away from “food” molecules like sugars ends up on another, simpler carbon compound rather than something like oxygen, and therefore fermentation is implicitly anaerobic although that’s not the same as saying that fermentation cannot happen in the presence of oxygen (e.g. the Crabtree Effect, and of course fermentation of ethanol to vinegar requires oxygen). The end product is generally assumed to be organic acids (like acetic acid [vinegar]) or alcohols, and carbon dioxide. So, making beer and wine is fermentation. Making vinegar is fermentation. Making yogurt (lactic acid) is fermentation. Citric acid can be made by fermentation of glucose by Aspergillus molds, as can malic (apple) acid (see US Pat#3063910). You can make tartaric (grape) acid from glucose by fermentation as well (see US Pat#2314831).

I am familiar with the flavors of all of those products. One I’ve never directly tasted is gluconic acid, which is the main product of the fermentation process used to make “BIONADE®” (it seems to be written in all-caps everywhere).

According to their English-language page discussing their process – linked from the image at right, click to view – they are starting with malt, just as one would for beer, but instead of Saccharomyces yeasts, they are fermenting this wort-like liquid with “acid bacteria”. I’m going to hazard a guess that the bacterium in question is a strain of Gluconobacter oxydans or one of its close relatives. This group of bacteria is in the Acetobacteraceae family of bacteria which is involved in turning your wine into vinegar. It would appear that under the right conditions, the enzyme Glucose Oxidase (EC 1.1.3.4) produced by G.oxydans converts glucose to a compound which reacts with water to form gluconic acid. BIONADE® then adds flavor extracts and juices to the filtered fermentation product, carbonates it, and bottles it.

Not being familiar with the flavor of gluconic acid, I’m aching to get my hands on some of this stuff and try it.

For another example of a relatively non-alcoholic fermented beverage, see also Kombucha, which is essentially sweetened tea fermented by acetic-acid bacteria and non-Saccharomyces yeasts…which I also have yet to taste.

geostr:50.4600,10.2208:200804110105-06:geostr (at least if Google Maps interpretation of the address I could find at the moment is correct, and assuming the information I dug up and my interpretation of it is correct, this should be the approximate location of the brewery responsible for BIONADE® production.)