Taste Testing and Other Research Progress

Yesterday, we finally were able to hold our tasting panel for the Sierra Nevada Stout Clone. There were two groups, BJCP testers and triangle testers. The BJCP group consisted of four volunteers who analyzed each of the four samples (3-step and single-step controls and Clarity-Ferm added beers) according to BJCP guidelines for category 13E, American Stout (the guidelines can be found here: http://www.bjcp.org/2008styles/style13.php#1e and the evaluation sheet here: http://www.bjcp.org/docs/SCP_BeerScoreSheet.pdf). The triangle testers were given three beers at a time, two of which were the same. Their task was to identify the beer that was different while performing some basic analysis. Many said this was very difficult to do, which was good news for us as it means that the Clarity-Ferm batches taste quite similar to the controls despite being gluten-free.

Cups set out and organized.

Beer poured. Just waiting for victims, err, volunteers.

Crackers, water, and evaluation sheets out.

Some of our gracious volunteers.

The rest of the wonderful volunteers.

The results of the BJCP tests were a little mixed. Some liked the Clarity-Ferm samples the most, while others liked the controls more. Some liked the 3-step and others liked the single-step. The overall consensus was positive, however.

A few days ago, we ran gels on the samples of beer we collected over the course of the summer. Oddly, they all have the same band at around 37 kD. After conducting some literature research, it seemed like the protein could be β-glucosidase. To test this assumption, we concentrated the beer samples through centrifugation and performed an assay using ONPG. Unfortunately, we did not see any activity. The protein could have been lost during purification or could be something completely different. We plan to run another gel in the near future to see if the protein is still present.

Microcentrifuge tubes waiting for samples.

This research certainly involves a lot of pipetting.

Heating the samples with Laemmli buffer.

Loading the gels.

Loaded gel.

Gels staining.

One of two gels. Notice the thick and consistent band at ~37 kD.

Over the past few days we've bottled our Oberon clone. At this point, it tastes pretty close to what we were hoping for, with little difference between the control and Clarity-Ferm samples. We'll perform another tasting panel in August to determine how it tastes after carbonation.

All our bottles from both the stout and Oberon clone.

After the last ELISA I performed, it seems as if the controls for the Oberon clone have well over 150 ppm gluten, the samples with 2 mL of Clarity-Ferm are bordering on gluten-free, and the samples with 4 mL of Clarity-Ferm are all completely gluten-free.

There likely won't be any updates for a while, but check back in August to see the results of our final taste test and detailed analysis of gluten content!

Research Update & Oberon Clone

Last Monday we started to brew our clone of Bell's Oberon, a wheat beer. We brewed the three-step on Monday and single-step on Tuesday. Both brews went off without a hitch. Instead of using dry yeast, we used a smack pack from Wyeast we had grown up over the weekend. This method seemed to be very effective as almost every carboy was fermenting violently over the first few days.

Sparging the three-step Oberon clone.

Normalizing the trub post-boil.

Came into lab on Tuesday morning to find that the three-step had started to ferment rather violently.

Three-step carboys pulled out with FermCapS ready to be added.

Three-step carboys after cleaning out the airlocks.

We ran another ELISA on Friday and determined that all of the samples with ClarityFerm added (both three-step and single-step) were quantifiably gluten-free. The controls were relatively low-gluten (all less than 40 ppm), but not low enough to be considered gluten-free. We're looking forward to them being completely carbonated to see how they compare to each other and to the commercially available Sierra Nevada Stout.

The ever-wonderful fish gelatin being added to solution.

Near the end of the week, we decided to take advantage of our new UV-Vis spectrophotometer and test the IBUs of the beer. IBUs (international bittering units) are a measure of the bitterness of the beer on a (theoretical) scale of 1-100. Technically, values above 100 are possible, but it's almost impossible to taste a difference after 100. A typical American Stout can vary from 35-75 IBU.

The process of determining IBU requires mixing a sample of 10 mL of beer with 1 mL of
3 M HCl. 50 µL of octanol and 20 mLof isooctane are added to this diluted beer and shaken for 15 minutes. We had to play around with centrifugation, as none of the instructions gave specifics. We determined that 10 minutes at 8000 g was sufficient. After centrifugation, there is an organic layer which contains the isohumulone (the molecule from hops largely responsible for the bitterness), a small emulsion layer, and an aqueous layer with most of the other molecules. After the spectrophotometer is blanked with a mixture of octanol and isooctane, the organic layer is read at 275 nm. A simple calculation is performed to determine IBUs.

Reagents and beer!

Tube post-centrifugation.

Initially, the values we were getting seemed very odd and incredibly high (well over 100 IBU), so we had to do some experimentation. After doing quite a few runs, I remembered reading that UV light can degrade the compounds we were analyzing. I decided to wrap the racks in masking tape, shielding the centrifuge tubes from light when they were sitting on the benchtop. We also added a step of washing the cuvette with water in addition to isopropanol. The combination of these two steps seemed to fix our problems, and values started to make sense.

Makeshift dark centrifuge tube rack.

Spectral analysis for determination of IBU.

The three-step control was around 31-32 IBU and the ClarityFerm sample was around 33-34 IBU. The single-step control is about 36 IBU, and the ClarityFerm sample is approximately 38 IBU. The increase in IBU from the controls to ClarityFerm samples could be due to the fact that ClarityFerm breaks down proteins which would normally bind with some of the isohumulone and settle out during fermentation. The decreased amounts of these proteins means that less isohumulone would settle out and thus more would be in solution, increasing bitterness. The difference between the three-step and single-step is potentially due to the increased boil time of the three-step (we had to boil off more in the three-step in order to get equal volumes).

We're going to open a few bottles of the stout this week to see how carbonation is coming along and hope to hold the official taste-test next week. We also plan to bottle the Oberon clone sometime next week. Once all of that is done, we'll be able to run an IBU analysis on the Oberon clone to see how close it is to what is expected.

A Long Overdue Update

My apologies for taking so long to post anything new. Between medical school applications and research, the past few weeks have been pretty busy. I'll try to summarize what we've been up to below, mostly in the form of pictures.

Week 2:
Dr. Hamilton wanted to run gels to compare the proteins in the different beer samples. Before doing so, I ran a Bradford assay to make sure I didn't overload the gels.

Preparation of cuvettes for Bradford.

Spec and cuvettes ready for analysis.

Starting polymerization on 10% SDS gels

Gels running

Gels just before destain

Gel #1

Gel #2

Unexpectedly, the Bradford assay returned protein concentrations relatively equal across all samples, and the gels showed the same bands with the same intensities throughout, the largest of which was just over ~35 kD.


One of the required reagents for the RIDASCREEN ELISA is a solution of ethanol and fish gelatin. This reagent is a pain to make as the ethanol can cause the fish gelatin to crash out of solution. The gelatin is also about twice the viscosity of honey. Below is a picture of me trying to vortex the mixture to get everything into solution.

Fish gelatin and ethanol!

All 12 gallons. Notice how the 1 step (on the right) have a lot more trub than the 3 step.

Full RIDASCREEN Plate:
Below are pictures of the RIDASCREEN ELISA performed on a full plate (verses the semi-full plate shown before)

Prepping of samples. 1 mL of beer + 9 mL of fish gelatin/ethanol solution.

Supernatant and a diluent post centrifugation and ready to add.

Standards and samples added along with the enzyme conjugate.

Incubating on the rocker.

Samples post-incubation.

Plate after third wash with 250 µL wash buffer.

Plate after addition of 100 µL of chromagen to each well.

Plate after incubating with chromagen for 10 minutes.

After addition of 100 µL stop reagent to each well. Read in the plate reader immediately.

My best friend for the ELISA. It allows me to pipette the same amount multiple times without refilling or changing the tip. It speeds up the process of having to pipette into all 96 wells.

After our third assay (through Day 10 of fermentation), we determined that all of the samples with Clarity Ferm added were almost completely gluten free, all reading less than 4 ppm (<20 ppm can be sold as being gluten free). We will continue to take samples and analyze them after bottling to make sure that the beer truly is gluten free.

Week 3:
Trip to Ann Arbor
Dr. Hamilton and I needed to make a trip to Ann Arbor to pick up some brewing supplies at Adventures in Home Brewing. Since we were already there, we decided to stop at Arbor Brewing for lunch. The beer was definitely worth the detour.

Flight of everything Arbor Brewing had on tap.

Dr. Hamilton enjoying some of the flight.

Bottling!

Dr. Hamilton taking a minute to help scrub some bottles. It took a lot longer than we thought to get all the bottles we would need clean and ready to go. Thankfully, we started the day before bottling.

About half of the bottles we needed. Thanks to Dr. VanZant for giving us extra bottles!

After struggling to get the bottles upside down to dry, I decided to turn the cooler on its side to stack the bottles. To my surprise, it worked perfectly. The bottles were left upside down overnight to drain excess PBW.

In the midst of bottling, a new UV-Vis spectrophotometer came! I felt like a kid on Christmas morning helping set it up.

Prepped and ready to start bottling.

Sanitizing a bottle before adding the beer.

 

Another view of the sanitizing.

 

Racking from the carboy to bottling bucket with priming sugar added (18 g sugar/gallon of beer if you were wondering).

  

Dr. Hamilton just finished bottling in this picture.

For some reason, the capper was super cool to me.

All of our bottles after two mornings of bottling.

I get really defensive of my beer, er, research.

What kind of people would we be if we wasted the extra? Even before carbonation, it tastes pretty good.

Milling:
Since the efficiency of the stout we brewed was a little low, we decided to mill the grains for our Oberon clone a bit more than the company we bought them from online had. We'll be brewing this on Monday and Tuesday.

Milling the grain for the Oberon clone.

Power tools are absolutely fantastic. Dr. Hamilton also knows how to capture me at my finest.

Check back for updates periodically! After a few weeks we'll be able to test the stout for taste and final gluten content. I already have a list of people willing to help, for science of course.

Bonus picture of my housemate Wyatt celebrating that his research on MRSA seems to be working