YIC: Temperature-dependent Flavor Determination

*This is part three of the Yeast from the Iron City (YIC) series. For sampling, media, and plating information check out Part 1, and for Isolation Part 2.

Now that we had isolated our very own yeast strain, it was time to pray to the many gods of beer and hope that it would result in something drinkable. We wanted to use a fairly simple recipe to make sure the yeast would be the star player in terms of flavor. To determine the temperature that resulted in the best flavor profile (see ester post), the wort was split into three 1 gallon batches and placed under 3 different temperature regimes (Cold 45 °F, room temp 68 °F, warm 72 °F). The recipe is in the style of a Belgian pale ale and is as follows:

Fermentables
5.5 lb Light DME
.25 lb Caramel 40
.25 lb Caramunich
Hops
0.5 oz Galena       30 min
0.5 oz Hallertauer 15 min
.25 oz Chinook      5 min
Yeast
The Fringe
Estimated
OG: 1.050
FG: 1.018
ABV: 4.2%
IBU: 40

Starter:
A starter was made from the isolated yeast by going from a 100 ml starter to a 1 gal starter. The large starter was then allowed to ferment and settle. The liquid was decanted off until only approximately 2 L remained. I then swirled the solution to bring the yeast back into solution and filled 6 sanitized White Lab Vials. The remainder was then pitched into a 500 ml starter for this recipe.

Brew Day Notes:
The day went on without a hitch. Heated 4 gallons of water to 155 °F and steeped the grain for 30 minutes. The grain bag was then removed and the DME was then whisked in. The wort was then brought to a boil and the hops were added as is shows in the recipe. The wort was then cooled with the immersion chiller to 70 °F. The batch was poured into a bottling bucket for easy measuring and topped up to 3 gallons with filtered water. The batch was split between 3-1 gal jugs and 160ml of stater was added to each batch. The jugs were left at room temperature (68 °F) until fermentation had begun (about 12 hours). They were then placed at their respective temperatures (Warm, Room, and Cold temperatures).

It took approximately 2 weeks both the warm and room temperature batches to complete fermentation. The cold batch took about 3.5 weeks total. The vessel needed to be removed from the fridge for fermentation to begin again and then was placed back into the fridge. After fermentation seemed to slow the vessel was left at room temperature to finish fully and to clean up diacetyl.

Measured 
Vol: 3 1-gal
OG: 1.051
FG: (Warm-1.018; Room-1.017; Cold-1.018)
ABV:4.3%

Tasting:
1-28-14:
Warm (72 °F): Pours copper with frothy white head. Aroma of clove, spices, slight fruit, slightly dry. Taste is slightly sweet up front with a bitter finish. Slightly tart. Very clean taste compared to the aroma. Light body.

Room (68 °F): Same appearance. Aroma of slight fruit otherwise clean. Taste is light grain, sweeter front than the warm with a slight bitter finish. No noticeable tartness. Light body.

Cold (45 °F): Same appearance. Aroma of light fruit, grain, Belgian spices, caramel, it is the sweetest of the three. Slight banana notes. bitter sweet finish. Light body.

Overall: This style and recipe was not exceptionally great, but we expected this one to be very mild in flavor because of the very simple light grain bill. The yeast strangely seems to be quite clean producing slight Belgian notes but otherwise no funk or noticeable off flavors. Amazingly this seems to be the case across the entire range of temperatures. The only real noticeable effect of the temperature was a longer fermentation (obviously) and the beer ended at a slightly higher FG and lead to a slightly sweeter beer overall at cool fermentation temperatures. This was quite surprising to us to have a first attempt wild yeast that both attenuated relatively well (64%) at all three temperatures and did not impart any crazy off flavors. It would be interesting to pitch this yeast into a more complex Belgian style where the grain bill is more complex, like a Dubbel or a Quad, especially to see how it handles the higher levels of alcohol.

Currently, the yeast is being worked up in the lab to determine what genus and hopefully species it is so that we can better characterize it. Updates will follow!

YIC: Isolation, and Making a Starter

*This is part two of the Yeast from the Iron City (YIC) series. For sampling, media, and plating information check out the first post, Yeast for the Iron City: Sampling and Plating

Figure 1. Mixed culture from several fruit. (picture looks hazy because of condensation on lid)

Now that we have a culture growing wild on the agar plates that looks something like figure 1, its time to move on to the next step, Isolation. One of the best isolation techniques to use is what's known as a 4-quadrant streak (Figure 2). This does take a bit of practice to get perfect, but is pretty easy and very effective at isolating colonies of microorganisms. The best tool to use is the inoculation loop. If you have a nichrome loop (non-disposable metal loop) you will need to run the wire and loop through a flame until it is red hot to sterilize it. Sterile plastic disposable loops are available, but you would need 4 per plate and who wants to buy something to throw it away after a single use?

Figure 2. 4- Quadrant Streak Technique. (Numbered as steps)

It may look like a 5 year old with the ability to color in the lines can do this,but as I said earlier, this technique takes a little practice to get good isolation. I'll give you the steps with a nichrome loop because that is what we used. If you are using plastic, one, I'm disappointed, and two, whenever the nichrome loop gets sterilized, toss yours in the garbage (or you can save them and autoclave them I suppose if they are autoclave-safe) and get out a new one. 

First identify a colony you suspect to be yeast that is mostly free of other colonies touching it. Typically, the yeast colonies I have seen will be opaque white/ off white with clean edges (Figure 3). The stuff you don't want to touch is either fuzzy (fungi), has a very slimy shiny appearance (typically bacteria) or is a color other than white/ off-white (Figure 1). Sterilize your loop in the method described earlier. Then wait about 10-15 seconds to let it cool (don't "shake it like a Polaroid picture" as they say to cool it faster, you will increase the chances of picking up microbes from the air). Take about half of the individual colony making sure not to touch any other colonies and streak onto the plate as in Figure 1-1. Sterilize your loop and let it cool. Make streak as in step two of figure 1. The key is to only cross over your initial streak a few times. Repeat these steps two more times with successively less crossing into the previous streak. 

Figure 3. Isolated Yeast Colonies

Let this stand at room temperature until you start to see growth. Theoretically, you will have diluted out the original streak to single individuals that will then form a single isolated colony in either the 3rd or 4th quadrant (Figure 4A). If you do Success! You can now take a sample (from a single colony) and look at it under magnification to tell whether or not you actually have yeast (Figure 4B).

Figure 4. A) Example of 4-Quadrant Isolation. B) Wild Yeast X400

At this point you can begin to build up a starter to use pitch. To avoid possible contamination, it is best to start with a relatively small starter and work up to the size you want to pitch with rather than just going straight from plate to a .5 L starter. This greater yeast to starter ratio will allow the yeast to out compete most other microbes that may have been introduced into the starter. Nothing left to do now but pitch and hope that you've caught a good one! The Saga continues in the next YIC post: Temperature-dependent Flavor Determination.

Yeast from the Iron City (YIC): Sampling and Plating

A friend and I decided  a while back to return to the roots of fermentation and try to go wrangle us up some home grown bugs. Well, really they grow on fruits. Ever notice the white powder on grape skins that you can wipe off to reveal a shiny skin underneath? What you just brushed off, in addition to the epicuticular wax of the fruit, were thousands of microorganisms, some of which were wild yeast (Figure 1). Grape skins aren't the only thing you'll find yeast on. Most fruits especially those that we think of as sweet are teeming with microorganisms. The skins of these fruits are acting like natural nutrient agar plates, growing microorganisms on any sugars they can get their hands on. It's pretty much harmless if you happen to eat it, unless you ingest so much that you create a miniature brewery in your gut. Don't believe me? Check out this story on NPR: Auto-Brewery Syndrome. Luckily for you and I, this has only been observed in a single case; so I don't think there is much to worry about.

Figure 1. Grapes with Epicuticular Wax (white powder)

Anyway, we set out to swab different fruits and bring back samples for culture with the following methods. Most of the stuff we used to take samples you can get from a drug store, the supplies for culturing it may require a click over to Amazon, or maybe your LHBS will have some of what you need.

Sample Collection

We used cotton swabs to swab the outsides of several types of fruit. The swabs were then placed into zip top bags or, if you have them pill pouches, (they are almost exactly the right size for a single cotton swab). We did not take the time to sterilize anything at this point because during the culturing step you should be able to form isolated colonies the different microorganisms present on a 4 quadrant plate. Culture media was made up from a recipe found on BKyeasts Blog. Essentially the materials needed are: something to boil your media in (we used a erlenmeyer flask inside a pot), petri-dishes (either sterile plastic or reusable glass) (Figure 2), agar (an extract from sea weed), malt extract (I've found that DME is easier to measure and store when using small quantities), yeast nutrient, and of course filtered tap water or DI water if you have it. You may also want to add in a hop pellet for its anti-microbial power to prevent some growth. Theoretically, the hop acids will help weed out some of the organisms that you definitely don't want. You can read more about that on a previous post IPA: Myth or Microbiology.

Figure 2. Glass Petri-Dishes (Left), 500ml Erlenmeyer Flask (Right)

Why go through the trouble of making agar media instead of making up a batch of dilute wort? One, you will be able to see the organisms you have much more easily on a plate. More importantly, culturing on agar media is the only way you will be able to isolate the yeast you want from all of the other nasty microorganisms that will just ruin your beer, but that is for a later post.

After boiling the malt agar to melt and sterilize it we let it cool to just above the temperature at which it starts to set up. This prevents a large amount of condensation from forming on the lids of the Petri-dish that could drip down onto the media and smear the microorganism colonies around. After letting the plates cool down for a day or so, you are ready for the streaking (of the microorganisms)! Before you begin the process of streaking your culture, you need to make sure you follow aseptic technique. A fancy term meaning keep everything as clean as possible. A diluted bleach solution or other antimicrobial cleaner should be used to clean any surfaces that you may come in contact with, and you should make sure to wash your hands very well. Gloves may be a good idea but are not absolutely necessary (we didn't use them and things turned out fine).

Figure 3. Swab Pattern for Microorganism Plating

For the initial transfer of the samples to the culture media we divided plates up into quarters, labeled them, and brushed the tips of each cotton swab on each quarter of a plate to save space (Figure 3). For now let the plates incubate at room temperature. You should start to be able to see some growth of various shapes, color, and texture. It took a little under a week for our samples to show much growth (Figure 4). Be watchful, after the initial growth started they seemed to grow exponentially leading me to place them in the fridge to slow them down until we could work with them again.

Figure 4. Plated Microorganisms After One Week of Incubation at Room Temperature.

There is pretty much a 100% chance that the samples swabbed will contain more than one type of microorganism (known as a mixed culture), so you will need to use a technique to isolate single colonies of microorganisms so you can pick and choose what you want to take to the next step. I'll write a brief overview of this isolation process and how we did it in a following post but for now, happy yeast hunting!