Tag: benzoic acid

Benzoic Acid Part 2: “Sour Stuff”

Okay, now that the boring review is over with…

Consider the cell. It doesn’t matter what kind of cell – bacterial, archael, fungal, animal, whatever. It’s still a tiny droplet of slightly salty water, thickened by a bunch of enzymes, other proteins, and various other substances floating around in the water. There’s also one other component that makes this a “cell” rather than soup: a bubble made of fatty material that the droplet is wrapped in, called the cell membrane. Depending on what kind of cell you’re thinking of, there may or may not be a “cell wall” made of some sort of rigid material, with the cell membrane inside of it. There may also be more than one membrane as is the case with the classic “Gram negative” style of bacterium, which has a second “outer” membrane wrapped around its cell wall. If it’s a eukaryotic cell, it’ll even have tiny little “organelles” inside itself wrapped in their own little membranes…but whatever. It’s the innermost one, inside of whatever cell wall may be there but wrapped around the cell’s guts, that we’re concerned with here.

Since stuff that will dissolve readily in water doesn’t tend to dissolve well into fats, and vice-versa, the cell membrane not only prevents stuff dissolved in the water inside the cell from leaking out, it also prevents stuff in the water outside from getting in. This lets a cell maintain itself at near neutral pH even if it happens to live in a very acidic environment, or an appropriate level of, say, sodium salts even if it lives in the Great Salt Lake.

This brings us back again to benzoic acid, which you should recall from the previous post alternates between a dissociated hydrogen-ion-and-benzoate-ion form and a combined, netural form in water. You may have noticed that foods preserved with benzoates tend to be sour, like fruit juices or soda. That’s because “sour” is the flavor of acid, and benzoic acid’s ability to be a preservative is only good in acidic environments

Useless Knowledge Break: the German word for acid is “Saurstoff”. Yes, that is pronounced like “sour stuff”, and no, that is not a coincidence.

An acidic environment means lots of extra hydrogen ions (“protons”) floating around. That also means that when a molecule of benzoic acid splits into a hydrogen ion and benzoate ion, it takes less time before another hydrogen ion comes by and the molecule can recombine again and therefore a bigger majority of the benzoate floating around at any moment is in the combined, somewhat fat-soluble neutral form. In that form, it can soak into a cell membrane if it encounters one.

If that molecule drifts through the membrane and gets to the inside of the cell, it may touch the less acidic watery environment there and dissociate into ions again and be unable to return through the membrane. The released hydrogen ions mean the inside of the cell becomes more acidic. As of today (20080806), the Wikipedia entry for Sodium Benzoate cites a single paper from the early 1980’s saying that when the inside of a yeast cell gets acidic enough, it prevents a specific step in the energy-generating process from working. This may be true, but there’s more to the story than this.

Obviously the membrane can’t totally seal the cell off from the outside, or the cell would be unable to excrete wastes or take in food molecules, so there are numerous specialized “transport” proteins that stick through the membrane to allow specific kinds of molecules in and out. Lots of biochemical reactions release hydrogen ions, so there are transport proteins that can shove hydrogen ions out of the cell and into the cell’s surroundings. The problem is that all substances naturally diffuse from areas of higher concentration to areas of lower concentration, so in an acidic environment the natural direction that hydrogen ions “want” to flow is into the more neutral cell. These transport proteins can shove the hydrogen ions in the opposite direction, but like pushing a boulder uphill it costs energy. This seems to be the primary reason that benzoic acid prevents bacteria and yeasts from growing – it makes them waste energy that they would be using for growth just to keep taking the hydrogen ions that the benzoic acid helps leak in through the cell membrane and shoving them back outside. The figure above is linked to a page at Helsinki university that discusses this type of preservative action in more detail.

Simple and elegant, and this seems to have been assumed to be the whole explanation for some time. But what happens to the benzoate ion when its hydrogen ion gets pumped away? Does it do anything?

Coming up next: Endocannibalism!

Why Benzoic Acid Works: Part 1 – “Some Boring Review Material”

It’s about time I got to the long-promised post about benzoic acid. The thing is, I don’t want to assume everybody reading this is well-versed in chemistry or anything, so after much thought I’m going to split this into three posts. This first one is a bit of chemistry review for some topics that are important to how benzoic acid acts as a preservative. People who are bored by this or know more about it than I do are welcome to either wait for the next post or leave corrections or questions in the comments as you see fit. (Brief note to people reading this from the RSS feed – I’ve noticed that the stylesheet information doesn’t transfer with the RSS, so you won’t see where the web page view would indicate that there is additional information available for some of the terms here. Try hovering over various words and phrases in this post, though, and the information should pop up if it’s there…or just pop in at the main site and post questions if you have any.)

There are several ways people separate types of molecules into opposites. For example, ionic vs. covalent, polar vs. non-polar, or hydrophilic vs. hydrophobic. Although these three categories are each a little different from each other, they all relate to the same thing. As with all other chemistry, it all has to do with what the electrons are doing.

When atoms react with each other, they have a big fight over each other’s electrons. The reaction “finishes” (reaches equilibrium) when this custody battle is concluded. Each of the three categories above relate to how equitable the electron-sharing arrangment ends up being. Once the molecule’s atoms arrange themselves, if the custody of the electrons is distributed fairly evenly around the entire molecule, the molecule is considered “non-polar”. On the other hand, if the atoms at some corner of the molecule end up with more custody of the electrons than the other areas, the molecule ends up having an end that’s slightly more negatively charged (remember electrons are arbitrarily defined as being “negative”) than the others, and the molecule is “polar”. If you dissolve that polar molecule in water and the atoms remain together stubbornly clinging to the shared electrons, the molecule is considered “covalent” (“valence” refers to the area around atoms that electrons “orbit”), whereas if one or more of the atoms readily gains or gives up complete custody of one or more electrons and drifts away from the rest of the molecule, the molecule is considered “ionic”. (It amuses me to think of these latter two terms as “homoelectrical” and “heteroelectrical”. Yes, I am easily amused, why do you ask?) Plain old table salt is what you get when atoms of Sodium (“Na“) and Chlorine (“Cl”) get into one of these electron fights. If you were to look at a Periodic Table of Elements, take a look at the column way over on the left, with Sodium (Na) and Potassium (K) and so on. All of these have one electron that they just don’t really give a crap about. Way over on the other side of the table, one column over from the far right, you’ll see Fluorine (F), Chlorine(Cl) and so on. All of THOSE desperately want an extra electron (Chlorine is the third most electron-greedy – “electronegative” – atom, behind Fluorine and Oxygen). Stick Sodium Chloride in water, and Chlorine says “MINE!”, and Sodium says “Ah, whatever, who needs it?” and the gentle pull of the water molecules around them easily overcome the electric charge based attraction of the now positively charged sodium ion and the now negatively charged chlorine ion, and the two atoms drift apart.

This brings us to “hydrophobic” and “hydrophilic”. There’s a truism in chemistry that “like dissolves like”. Polar substances tend to dissolve well in other polar substances, and non-polar substances tend to dissolve well in other non-polar substances, but polar and non-polar substances don’t mix well at all. Water is a polar substance – it’s got an electron-greedy oxygen atom in between two comparatively electron-apathetic hydrogen atoms. What’s more, the two hydrogen atoms aren’t on exactly opposite sides of the oxygen atom. The “H-O-H” arrangement is actually bent (at just over 104°, if you care), so a water molecule ends up being slightly triangular, with one corner being a little bit negative (where the oxygen atom clings more to the electrons) and two corners with the hydrogens being a little bit positive. Any other molecule with a slightly-positive or slightly-negative part will find that part attracted to one side or the other of water molecules, and as a result will tend to be pulled out into the water as the molecules bounce around [i.e. it will dissolve]. On the opposite end of the scale, molecules with their electrons relatively evenly spread over them tend not to be soluble in water. Large molecules like fats are in this category, which is why fat floats on top of water rather than dissolving in it.

There are two other random facts that I need to wedge in here somewhere. First, the line between “covalent” and “ionic” is actually kind of arbitrary. Water is considered “covalent”, but a very small fraction of the times that two water molecules run into each other, they’ll hit just right so that the slightly-negative oxygen atom on one of them manages to attract one of the slightly-positive hydrogen atoms enough to make it leave an electron behind and jump over. When that happens, you end up briefly with a positively-charged “hydronium” ion (“H3O+“) and a negatively-charged “hydroxide” (OH–) ion. It doesn’t take too long for a “hydronium” to find a “hydroxide” again and rearrange back into two water molecules, but in pure water at “standard temperature and pressure” (defined as 25°C and one atmosphere of pressure) at any time there are about 620,000,000,000,000,000 hydroniums and hydroxides floating around in a liter of water – assuming I didn’t screw up my math there.

And, finally: a classical definition of an “acid” is something that “donates protons” (that is, hydrogen ions). In water, that means a molecule that provides extra available hydrogen atoms that water can pull off to form “hydronium” ions more often that water alone does.

And now, at last, we reach the subject of the preservative known as “benzoic acid”. If you read the ingredients lists of the food and drink you buy, you’ll probably never actually see “benzoic acid” on the label. Instead, you’ll see “sodium benzoate” or “potassium benzoate”. If you remember, sodium and potassium don’t really care about one of their electrons, so when you dump “sodium benzoate” in water, the sodium goes floating off to play with the water, leaving behind a negatively-charged benzoate ion with its electron. The extra electron hangs out around the part of the benzoate ion where the electron-greedy oxygen atoms are, making the molecule quite polar. Along comes a new “hydronium” ion, carrying a hydrogen that decides it misses its electron after all, and it jumps over to take over partial custody of the electron that the sodium left behind. In short, you’re going from Sodium + Benzoate + Hydronium + Hydroxide to Sodium + Hydroxide +…Benzoic Acid. (Plus a molecule of water, which is traditionally left out of these kinds of equations, which used to be the “hydronium”.) With the hydrogen attached and sharing the electron, benzoic acid no longer has so much of a charge imbalance and is a lot less polar. Being an acid, Benzoic Acid can also give that hydrogen ion back up again to a molecule of water – exactly the reverse of the reaction that formed it.

That’s the punchline to this: in water, a molecule of benzoic acid might at any one time be without it’s hydrogen and therefore charged/polar and hydrophilic, or it might have the attached hydrogen and be uncharged, relatively non-polar, and be comparatively hydrophobic…or “fat-soluble”.

Next post: So what?

(Tap Tap Tap) Is this thing on?…

Move is underway. Here’s a quick update / test.

Cornelia and Monk(eymutt), the Laser Dogs

On the way home (to Texas, that is, with a load of furniture from House v1.0 in Idaho) we adopted a co-dog. Now Cornelia isn’t the only official dog in the house.

Yeah, I know, pretty frivolous stuff. What do you want – this is mainly a test post. You guys ARE seeing this post, right? (Please comment and let me know). I’ve finally found and jumped through the hoops necessary to migrate the bigroom.org website over to the new host. It’s also running on the old server in House v1.0 on the DSL line as well, but this post isn’t getting put on it. If I’ve done everything correctly, hosting for bigroom.org should now be handled by Eskimo North. Just a few other minor tweaks and I’ll be set to return to House v1.0 and shut down the old server so I can move it down here to Texas.

I’ve gone from about 4400ft elevation down to about 250ft, but the plastic bottles of Mountain Dew® Wine seem to have positive pressure, so it would appear there is still some live yeast left in there and the priming sugar is slowly doing its job. I’m pretty sure the benzoic acid is what’s slowing down the activity so drastically, but it hasn’t killed it off yet.

I’ve also got a couple of entries in mind already for the next The Giants’ Shoulders blog carnival, and hopefully some more crazed brewing project news to offer sometime. And, of course, the promised post on why benzoic acid works. Stay tuned.

Mountain Dew® Wine: Disappointment strikes!

<whine excuse=”obligatory”>Have I ever mentioned what a huge hassle it is to relocate from one abode to another 1600 miles away?…</whine>

I’m finally back at House v1.0 where I can check on the progress of my Mountain Dew® Wine. It appears to have managed to ferment, in spite of the severe dose of preservatives in the stuff designed to prevent that from happening. It went somewhat slowly, but it’s gone from an original gravity of around 1.054 down to about 1.011 or so, suggesting about, say, 5-6% alcohol in the final product, which has faded to a pale, cloudy yellow color. Hopefully the cloudiness is from still-living yeast, which has now demonstrated that it is reasonably benzoic-acid-and-caffeine tolerant.

I fear I must report that the result is a crushing disappointment to me. It’s not very good. Worse yet, it’s not very bad, either. I was hoping that if it wasn’t surprisingly tasty that it would at least be shockingly awful in some interesting way so I’d have something entertaining to say about it here.

Actually, the adjective that comes to mind is “inoffensive”. If you would like to whip up a quick simulation of what I’ve got here, you might be able to do it like this: Take some citrus-flavored sparkling mineral water. Dilute it about half with (uncarbonated) distilled water. Then mix about 7 volumes of that with one volume of vodka. What I’ve got here is slightly sparkling, with a barely noticeable citrus flavor and little or no remaining sweetness. It’s a little surprising to me just how much of the flavor of Mountain Dew® apparently comes from its sweetness. Perhaps next time I try this (if there ever IS a “next time”…) I’ll have to mix regular and “diet” Mountain Dew® – with some bonus sugar to make up the difference, of course.

I’m not completely done here. I’m still going to dispense it into cleaned bottles with a little bit of sugar to prime it for full carbonation. Sanitized plastic soda-bottles of course – none of that snobby glass stuff for this here experimental drinkin’ substance! It may be high-class for “pruno”, but it’s sure as heck not Champagne™. (Besides, I want to evaluate reusing plastic bottles anyway – it’d be a lot easier to tell when there’s too much pressure and to let some of the pressure off if there is.) Plus, I need to take some of the still-live yeast and keep it alive. No point in developing a benzoic-acid-tolerant yeast strain and not keeping it!

On a related note, an article was mentioned on fark.com, saying that back in 1955, a scientist “proved” that it is not normally possible to get drunk on beer. Of course, he seems to have been referring to dilute mass-market bladderwash and his reasoning was that a typical human stomach cannot contain enough 3.7%-alcohol beer for a typical human to achieve a dangerous blood-alcohol level.

As usual, the articles (see here and here) go for the “a scientist says this” part but never bother to say WHERE the scientist says it – usually a real scientific publication.

A quick search of pubmed turns up a likely candidate:

Greenberg LA: “The definition of an intoxicating beverage.” Q J Stud Alcohol. 1955 Jun;16(2):316-25 (link goes to the pubmed entry, which has little more information that this).

I do believe it is a moral imperative that I get a copy of this article somewhere so that I may reference it later. Is there anyone out there reading this who might be able to get a copy of this paper somewhere for me? Please?…

Very brief post…

Sorry about having another brief bout of blogstipation. We’ve finally managed to close on a house and now we’re probationary Texans (y’all). I’ve been spending the last week+ driving back to SE Idaho (by way of Best Friends, since we now have room to hire a second dog to hopefully keep Cornelia the Laser Dog company. I’ve got to lug 3 cats (and one goldfish in a small fish tank) a distance of 1600 miles or so starting in about 6-8 hours. Wish me (good) luck.

After the weekend, I should have some time to get back to the real posts. The Mountain Dew Wine was virtually unfermented when I returned, even after sitting there 10 days, but since I’ve gotten back it’s started going. Still slowly – the bubbler spits out 3-5 bubbles ever 40 seconds or so – but it’s going. It’ll be interesting to see what I end up with. I wanted to do 2-3 posts on the effects of benzoic acid on yeasts (that’s the preservative in Mountain Dew®), and I would swear I had one or two others in mind. Oh, yes, and an update on getting a Magellan GPS replacement that can actually be used – they seem to have located a slightly lower-end model eXplorist that they can send me. I sent them back their “walled garden”-based Triton 500, so the replacement unit ought to show up next week, I think. Their service has been pretty good, at least.

“A small modification of Koch’s plating method.”

Only two more days for the Classic Papers Challenge, so if I’m going to get any more up, I’d better get my butt in gear.

Here’s a nice easy one:

Petri, R. J.:”Eine kleine Modification des Koch’schen Plattenverfahrens.” Centralblatt für Bacteriologie und Parasitenkunde; 1887; Vol. 1, pages 279-280.

The American Society for Microbiology has a translation available online. It’s only about a page-and-a-half of relatively large type – check it out.

There’s a trick we microbiologists use for growing bacteria. You make a solid (but wet) surface that contains whatever nutrients the microbe (bacteria, archaea, yeasts, mold spores…) you’re interested in need, and then you spread a diluted mixture of the microbe on it. The idea is that since the surface is solid the microbes can’t move around too much, and at any spot where a single cell starts initially, a whole pile of that cell and it’s genetically-identical (non-sexually-produced) clone-children will form until it gets big enough to see without a microscope. This cell-pile is called a “colony”, and you can poke or rub it with a sterile object, then stick the object into a sterile nutrient source. The end result is you have a “pure” culture of microbes that are effectively genetically identical. The solid material could be a lot of things – I’ve seen references to using slices of potato – though these days agar-agar gel mixed with nutrients is the preferred substance.

Koch (that is, Robert Koch of “Koch’s Postulates” fame, not Ed Koch the former mayor of New York City) used gelatin (so, hey, here’s another thing you can do with your expired Jell-O&reg;). He apparently used to have a stack of shallow bowls, and had to use a special pouring device to carefully dump the gelatin into each stacked bowl in turn, then cover the works with a bell jar in order to keep stuff from falling into them from the air and contaminating them.

This was kind of a pain to work with, so some clever guy named Julius came up with a modification of this method in 1887, using pairs of shallow dishes, one slightly larger than the other so that it could be turned upside down to use as a lid. Then, you don’t necessarily need the bell jar, and you don’t need to stack them so they’re easier to pour.

Julius Robert Petri’s idea was so useful that we still use it today. Oh, yeah, and they named the dish-and-lid combination after him.

How’s that for a “classic” paper?

Meanwhile, my “Mountain Dew® Wine” project is turning out to be substantially more educational and fascinating than I’d hoped. There seems to be a decent amount of information available on how benzoic acid affects yeasts. I intend to turn that into a post later, but first I’ll try to find at least one more old paper to post before tomorrow is over…

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…]