Were going to spend the next 45 minutes talking about hops. - - PDF document

We’re going to spend the next 45 minutes talking about hops. Specifically, we’re going to be talking about some exciting research that’s going on in the world of hops, and how you can incorporate that research into your homebrewing. This is an ambitious seminar: we are trying to cover a lot of ground, spanning 4 different areas of hop research, and we aim to keep it accessible for beginning and intermediate homebrewers while still making sure you advanced folks walk away learning something new. So with that said, strap on your seatbelts, and let’s get after it. 1

Who are we? Chris: My name is Chris Hotz and I began homebrewing in 2004. In 2012 I began taking my brewing more seriously by joining QUAFF homebrew club in San Diego and entering my beers into competitions. In 2013 was accepted into the first class of UC San Diego brewing certificate program and was privileged to learn from the likes of Chris White, Yuseff Cherney and Mitch Steele. In 2014 I began working as a Quality Analyst at Ballast Point Brewing before becoming an R&D brewer two year later in 2016 where I currently remain. While I can not claim as many homebrew awards as this man (Doug Brown), I have earned two NHC medals. Doug: And I haven’t won any medals at NHC, but I’m working on it, and hoping that changes tomorrow. I am the current Vice President of the QUAFF homebrew club in San Diego. I started homebrewing in the year 2000, and played around with the hobby on and off for several years with extract brewing before taking the all-grain plunge around 2012. In late 2013 I started to get more serious about beer: I joined a homebrew club, QUAFF, and in January of 2014 I enrolled in the Brewing Program at UC San Diego., which I completed in June of 2015. That experience was a real turning point for me, not only because it was in that program that I met Chris, but also because I got my first exposure to the world of brewing research… 2

Doug: Much of that research today comes from the Master Brewers Association of the Americas. The MBAA is a global -- no longer limited to just “the Americas” -- community committed to expanding and sharing our collective understanding of brewing &

• fermentation. Founded in 1887, by German Americans who brought their brewing

culture with them when they immigrated to the New World, today the MBAA is a 501(c)(3) non-profit organization with over 4,000 members from over 50 countries worldwide. Every year the MBAA holds its Master Brewers Conference, during which brewers, scientists, and other industry members present their findings and studies . I was fortunate to be able to attend the 2017 Master Brewers Conference, which was in Atlanta, and which takes us into our first topic for this seminar… 3

Auxiliary Bitter Compounds. Specifically, what are Auxiliary Bitter Compounds in hops, and how do they affect the quality of bitterness in beer? This is research coming out of Germany, and was presented at the Master Brewers Conference by Andreas Gahr, the Brewmaster of the Research Brewery at Hopfenveredlung St. Johann. I was really intrigued by his presentation, and Brewmaster Gahr graciously agreed to let me share his findings with y’all here today. 4

This wouldn’t be much of a scientific presentation without a bunch of three-letter acronyms being used incessantly. To make sure we’re on the same page, here are the main abbreviations I’ll be using. First, IAA, for Isomerized Alpha Acids. Most of us know isomerized alpha acids as being responsible for bitterness in beer. Hops contain alpha acids. When we buy hops, the packaging usually indicates the amount of alpha acids that are in the hops, as a percentage by weight. Those alpha acids aren’t very bitter, nor are they very soluble in water or beer. But when those alpha acids are heated, they undergo a chemical conversion – called isomerization – that turns them into isomerized alpha acids. Those isomerized alpha acids – or IAA – are very bitter, and they are relatively soluble in beer. This is one of the primary reasons we boil our wort, in order to isomerize the alpha acids in the hops to create Iso-Alpha Acid and add bitterness to the beer. 5

Next up are the Auxiliary Bitter Compounds, or ABC. These are the focus of this study, and we’ll get into some examples of them and their significance in a bit. For now, the important thing to know is that ABC refers to compounds contributing to the bitterness of beer other than the isomerized alpha acids, or IAA. 6

Our third acronym here is one we’re all probably familiar with: the IBU, for International Bittering Unit. And although this is a term that’s used all the time in the world of brewing, it’s a problematic word from a scientific perspective because it can refer to different things, and isn’t always used consistently. In theory, as it is often described, the IBU is defined such that 1 IBU is equal to 1 ppm,

• r 1 mg per Liter, of isomerized alpha acid. This is the definition of IBU that’s given in

many brewing texts, and it’s something that can be measured in a lab using high- performance liquid chromatography, or HPLC, which is a piece of sophisticated and relatively new laboratory technology that is capable of isolating isomerized alpha acids and measuring their concentration in a solution. But an HPLC is expensive, and only very small percentage of breweries utilize them. So most people measure bitterness in a lab using a piece of equipment called a spectrophotometer, which is much less expensive and uses older technology. A spectrophotometer works by targeting a sample with a specific wavelength of light and measuring how much light is absorbed. It turns out that isomerized alpha acids strongly absorb light with a wavelength of 275 nm. So by using a spectrophotometer set at 275 nm, you can get an idea how much Iso-Alpha Acid is in a beer, and thus measure IBU that way. 7

However, Iso-Alpha Acids aren’t the only thing that absorb 275 nm light, nor are they the only thing in beer that contributes to bitterness. As it turns out, there are a host

• f other things in beer that also absorb 275 nm light, and some of them contribute to

bitterness, too. There’s a non-exhaustive list on the slide in front of you, including:

• Alpha acids, meaning un-isomerized alpha acids: these are not very bitter, and they

absorb 275 nm light weakly, but they are sometimes found in beer and they do contribute somewhat to bitterness.

• Humulinones: Chris will be talking to you about humulinones more in a bit, but for

now, understand that they are also bitter, and they are also picked up by a spectrophotometer at 275 nm.

• Also beta acids, some beta acid-derived compounds like hulupones, certain

polyphenols, etc. Put all this together and hopefully some of the confusion around IBU is evident. If we think of IBU as referring only to Iso-Alpha Acid concentration, we’re missing the impact of all these other compounds that can contribute to bitterness. On the flip side, if we think of IBU as simply a measure of everything picked up by a spectrophotometer at 275 nm, we’re going to get a result that is only approximate, based heavily on how well these various compounds absorb 275 nm wavelength light, and one that for many beers that deviates from what we might expect based on any

• f the bitterness formulas we’re familiar with like Tinseth, Rager, Garetez, etc.

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So there’s really no perfect way to quantify or measure bitterness. To make the best

• f this imperfect situation, going forward in this talk I’m going to use IBU to refer to

bitterness as it would be measured by a spectrophotometer, capturing both Iso-Alpha Acids and Auxiliary Bitter Compounds. In other words, let’s think of IBU as being equal to the sum of IAA plus ABC. Using the term IBU in this way, the ratio of a beer’s IBU to Iso-Alpha Acid concentration becomes a useful proxy for identifying how much of a beer’s bitterness is derived from Auxiliary Bitter Compounds. Since IBU includes all of a beer’s IAA plus its ABC, a beer that derives all of its bitterness from Iso-Alpha Acid will have an IBU to IAA ratio of exactly 1. As the percentage of a beer’s bitterness coming from Auxiliary Bitter Compounds increases, the ratio of IBU to IAA will also increase, since adding more Auxiliary Bitter Compounds to a beer increases IBU (the numerator) while leaving the IAA (the denominator) constant. And now that I’ve set all this up, the question is: so what? Why do we care how much of a beer’s bitterness is derived from Auxiliary Bitter Compounds…? 9

Here’s why, and this is the key takeaway of the research that Brewmaster Gahr presented to the MBAA. According to repeated experiments, the higher the percentage of a beer’s bitterness that’s derived from Auxiliary Bitter Compounds, the more harmonious or pleasing that bitterness is perceived to be by the people who drink the beer. By this, an improved harmony or quality of bitterness refers to reduced harshness and reduced lingering character of bitterness, so as to produce a more balanced and pleasant overall impression. Before you are the results of two such studies. On the left, you see a study of the quality of bitterness in 16 single-hop beers. The x-axis shows the ratio of IBU to IAA, and the y-axis indicates the harmony or quality of bitterness as rated by a sensory

• panel. As you can see, there’s a steady, statistically significant relationship here,

indicating that increasing IBU to IAA ratio is positively correlated with an increase in the harmony and quality of bitterness. The graph on the right aggregates data from 17 beers across three other similar

• studies. Again, we see a statistically significant positive correlation between IBU to

IAA ratio and quality of bitterness. So, to really drive the point of all this: as the amount of Auxiliary Bitter Compounds increases relative to the amount of Iso-Alpha Acid in a beer, the bitterness in that beer becomes more harmonious and pleasing to human drinkers. 10

Now it sounds like we’re on to something that could actually be useful in making better beer. How do we put this information into practice? One idea is to bitter our beers with hops that have a higher ratio of beta acids to alpha acids. Recall that both beta acids and some beta-derived compounds were among the list of compounds that qualify as Auxiliary Bitter Compounds, while alpha acids are what get isomerized into Iso-Alpha Acid. By using hops that have more beta acids per unit of alpha acid, we’re thus going to increase the ratio of IBU to IAA. The above graph illustrates this point empirically. This is from another experiment in Brewmaster Gahr’s study, and it shows 16 different single hop beers that were hopped using hops with different ratios of beta acids to alpha acids. The x-axis shows the ratio of beta to alpha, and the y-axis shows the ratio of IBU to IAA. As the ratio of beta to alpha increases, so does the ratio of IBU to IAA, showing a positive and linear correlation. 11

Another strategy to increase the IBU to IAA ratio in our beers is to use less simply high-alpha hop for bittering. High-alpha hops are traditionally referred to as “bittering hops,” since less of them is needed to achieve a certain target bitterness and their use is thus more efficient in the brewhouse. But if our goal is to increase the IBU to IAA ratio, we’re not necessarily so concerned with brewhouse efficiency, and using less of these high-alpha hops for bittering will help decrease the IAA denominator of that ratio, and thus increase the ratio overall. 12

Another strategy to increase IBU to IAA ratio, and probably the most relevant to contemporary brewing practices, is to utilize shorter hop boiling times and/or increased late hop additions. Most Auxiliary Bitter Compounds are sufficiently extracted during short boiling times, and some of them in fact disappear with longer boiling times. A short boiling time is thus adequate to extract much if not all of our Auxiliary Bitter Compounds, while also producing fewer Iso-Alpha Acids due to reduced isomerization. This is similar, of course, to the impact that shorter hop boiling times have on hop aroma/flavor relative to bitterness, as well. Like most Auxiliary Bitter Compounds, the volatile hop oils that are responsible for flavor and aroma are sufficiently extracted during short boil times, and can also quickly volatize out if boiled too long. You can think of ABC in much the same way: if you want your hop additions to produce a higher ratio of IBU to IAA, consider adding hops later to the process, as you would with so-called “flavor” or “aroma” hop additions. 13

Taking this idea one step farther, a final strategy for increasing IBU to IAA ratio is dry

• hopping. Though not all Auxiliary Bitter Compounds are transferred by dry hopping,

some of them are. These include alpha acids and humulinones, as Chris will explain later on. Meanwhile, there are no Iso-Alpha Acids created during dry hopping, since there is insufficient heat to produce isomerization. So adding dry hop increases your ABC – and thus your top-line IBU – with no increase to IAA, boosting the overall IBU to IAA ratio. Taking this to the extreme: in one experiment, beers brewed with no hot-side hopping at all were exclusively dry hopped at a rate of 500 grams per HL, which is about 2/3 of an ounce per gallon, or 4 ounces per 6 gallons. Those beers achieved measured bitterness values up to 28 IBU with IAA concentrations of 1 ppm or less. I’m not sure where even that 1 ppm of IAA would be coming from in those cases, but that would still produce an IBU to IAA ratio of around 28, which is way off the charts compared against the ratios in the beers we’ve been discussing. 14

Chris: We are about to get super geeky about a specific auxiliary bitterness compound, humulinone, bitterness units and their contribution to perceived bitterness. This is research comes from Hopsteiner, and was presented at the 2015 Master Brewers Conference and as a webinar with the American Society of Brewing Chemists.

• Dr. John Paul Maye was kind enough to let me share his findings with you today.

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What are humulinones? Humulinones are organic compounds found in hops, similar in molecular structure to iso-alpha acids Where iso-alpha acids are formed from the isomerization of alpha acids, humulinones are formed from oxidative reactions on alpha acids. The exact mechanism of how humulinones form in hops is unknown. It appears there is something in the leaf material of the hop that facilitates the oxidation and isomerization of α-acids to form

• humulinone. This reaction appears to be limiting, and this is why the concentration of

humulinones increases a few days following hop pelleting and then stops. Exposing hop pellets or hop powder to air caused little to no increase in the overall humulinone concentration. Therefore, by the time you purchase hops as a homebrewer, the humulinone concentration in the pellets has stabilized. 16

Polarity: Humulinones are more polar than iso-alpha acids and therefore should be more beer soluble than iso-alpha acids. In fact, Hopsteiner analyzed 30 commercial IPA’s by HPLC and found the humulinone concentration in those beers to range from 3ppm to 33ppm. This upper range is significant, as we will soon see. Bitterness: sensory experiments indicate that humans perceive humulinones to be about 66%, or two-thirds, as bitter as iso-alpha acids. Again, this becomes significant when looking at the commercial beers tested by Hopsteiner with elevated concentration of humulinones of 33ppm, as that would be similar to getting as much as 24 ibu’s from iso-alpha acids. Concentration in pellets: This is thought to be because when hops are baled, about 10– 20% of the lupulin glands are broken, whereas when they are pelletized nearly 100% of the glands are broken. But again, in both pelletized and whole leaf hops, the concentration of humulinones in hops stops and does not increase with aging. Concentration varies by hop variety: I should mention here that hop varieties with higher hop storage index (HSI) show higher concentration of humulinones. HSI is a spectrophotometric measurement that provides an idea as to how fast a hop varietal

• ages. The higher the HSI in hops or hop pellets generally speaking the higher the

humulinone concentration that can be expected from those hops and this relationship is variety dependent. In other words, not all hops with an HSI of 0.3 will 17

have similar humulinone concentration. 17

Dry hop conditions and the kind of hops used:

• Centennial hop pellets assayed 0.35 w/w% Humulinone the day of dry hopping.
• Hop pellets were simply dumped on top of the beer.
• Beer Type: Low IBU (8.6 ppm or IBU) and High IBU (48 ppm or IBU)
• Temperature of dry hopping, 16C (60F)
• Contact Time: 5 Days

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Humulinone concentration can be measured using an HPLC, which the Hopsteiner team did on each of these beers after the experiment. Two points to understand from this chart: 1) As expected, the higher the dose rate of the dry hops, the higher the concentration of humulinones in the resulting beer. In fact, humulinones increased at a roughly linear rate. This is a result of the high solubility of humulinones in beer, which is a consequence of the high polarity we discussed

• earlier. It also suggests that at least up to 2 lbs per barrel, we haven’t reached any

sort of saturation point in the beer, since the humulinone concentration keeps increasing at about the same rate. 2) Perhaps less expectedly, the iso-alpha acid concentration in the beers is decreasing as the dry hop rate increases. This is especially true in the high IBU beer, where the concentration of iso-alpha acids drops almost in half, from 48 ppm without dry hops to 30 ppm with 2 lbs per barrel of dry hop. This suggests that while the addition of dry hops is increasing the concentration of humulinones, it is also stripping iso-alpha acids from the beer. The higher impact

• f this on the high IBU beer is likely because iso-alpha acids are not especially

polar, so when they are found in higher concentration they are more easily stripped from solution. 19

Dry hop conditions and the kind of hops used:

• Cascade hop pellets assayed 5.7% alpha acids, 5.5% Beta Acids and 0.23w/w%

Humulinone the day of dry hopping.

• Dose Rate: 0, 1, 2, 3, 4, and 6 lbs/barrel
• Hop pellets were simply dumped on top of the beer.
• Beer Type: 42 ppm Iso-Alpha Acid Beer by HPLC (High IBU)
• Temperature of dry hopping, 16C (60F)
• Contact Time: 3 Days

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• Even up to 6 lbs per barrel of dry hop, humulinone concentration seems to be

increasing at an almost linear rate.

• Alpha acid concentration also increases, though not as sharply as it does for the

first 1-2 pounds of dry hop. Remember from the ABC portion of this talk by Doug, alpha acids also contribute some bitterness to beer, although they are much less bitter (~10%) than iso-alpha acids.

• Iso-Alpha Acid concentration consistently decreases as more dry hops are used,

but the rate of iso-alpha acid stripping seems to slow down as dry hop rate increases, suggesting there may be a limit to this effect. 21

So, tying all this together, based on the results of these studies the researchers at Hopsteiner came up with a concept called calculated bitterness. Remember from before that humulinones are about 66% as bitter as iso-alpha acids and alpha acids 10% as bitter. In trying to calculate the perceived bitterness of a beer, we therefore need to account for the bitterness concentration of the humulinones in the beer. But we also need to account for the role that dry hopping plays in stripping Iso-Alpha Acids from the beer, especially in high IBU beers. What you see before you is the graph that Hopsteiner came up with to account for the anticipated change in perceived bitterness – which they call calculated bitterness – based on the rate of dry hopping in beer. Notice that when dry hopping at 1 lb per barrel, the calculated bitterness actually

• decreases. This is because the increase in bitterness from adding humulinones to the

beer is not enough to off-set the decrease in Iso-Alpha acid concentration caused by the dry hopping. But at some point after this, the Iso-Alpha Acid stripping seems to taper off, while the humulinone concentration continues to increase at about the same rate. Under these conditions, dry hopping at over 2 pounds per barrel, the calculated bitterness increases to above that of the original beer. This is significant when considering that many NEIPA are dry hopped at these higher levels…in essence, you can be increasing your calculated bitterness when dry hopping above 3lbs per bbl. 22

So most of us have had beers that listed their IBU’s at 90+ units, but felt their perceived bitterness was lower than that listed. This is because the IBU’s listed for the beers were measured most likely using the spectro IBU method. Because this method was developed to measure anything that is picked up at 275nm, dry hopped beers that contain humulinone and alpha acids and polyphenols will give IBU test results that don’t correlate with perceived bitterness. That is, the perceived bitterness may be significantly lower than the IBU test result would suggest. Perhaps this is a reason why the IBU test should be used more for quality control to analyze consistency between batches, but lot listed, or taken for granted, by consumers. 23

The impact of humulinones is particularly relevant to the production of dry hopped

• beers. Accordingly, there are a number of ways homebrewers can incorporate this

knowledge into their recipes in order to more precisely shape the perceived bitterness of their beers: 1. Consider humulinone impact along with IBU in predicting the perceived bitterness of dry hopped beers. 2. HSI selection: humulinone concentration is variety dependent, and that hop varieties with higher HSI generally have higher humulinone

• concentrations. So if you want more humulinone concentration in

beer, select hop varieties with higher HSI. But if you want lower humulinone concentration – such as if you are hoping to add a lot of dry hops without increasing the bitterness – select hop varieties with lower HSI. 3. I state this here because it has been observed that beers with high levels of BU’s contributed by humulinones display a smooth, non- lingering bitterness, in relationship to beers with similar BU’s contributed by mostly iso-alpha acids.. This would make sense as humulinones are more polar than iso-alpha acids and therefore would not stick to the tongue as long as iso-alpha acids. 24

Doug: Changing gears here a bit, our next study relates to an always popular subject: diacetyl. This is from research presented by Devin Tani, of White Labs, also at the 2017 Master Brewer’s Conference in Atlanta. 25

Diacetyl: I suspect most of you are familiar with it—that rich, slick, buttery compound that most of us very rarely want in our beer. Diacetyl is a natural byproduct of brewer’s yeast fermentation, but healthy yeast will usually reabsorb enough of it during the maturation phase so that it’s imperceivable in beer. 26

When we’re talking about controlling diacetyl, there are all sorts of helpful tips available to us homebrewers. Do a diacetyl rest, take a look at our fermentation profile and yeast handling, consider yeast strain selection, make sure we’ve got good cleaning and sanitation, etc. … But in recent years, especially as folks have started to really push the boundaries as far as hop usage is concerned, craft brewers have identified another thing we now need to keep our eyes on when we’re talking about diacetyl control: dry hopping. 27

White Labs and others have been looking at this since at least 2013, when Kara Taylor – also of White Labs – presented a paper entitled “Relationships between Diacetyl and Dry Hopped Beers” at the 2013 Master Brewers Conference. For this latest study, Devin Tani went looking to try to understand why dry hopping seems to produce diacetyl in some beers but not others. So he went around and collected samples of dry hopped beers from different breweries around San Diego

• County. He collected those samples before dry hopping, and then daily during the

dry hop process. He then chemically analyzed those samples to get a better sense of what was going on within. What you see on the graph in front of you is a plot of time, on the x-axis, against the diacetyl concentration, on the y-axis. That horizontal red line at 100 ppb represents the generally accepted taste threshold for diacetyl, of 100 parts per billion. There are at least two key things here I want you to notice: first, almost all of the beers experienced some sort of an increase in diacetyl at some point during the dry hop

• process. And second, in three of those beers – the beers numbered 11, 12, and 13 –

this dry hop increase was significant enough to push the beer over threshold at some point. 28

Coinciding with this increase in dry hop, Devin noticed something else: a decrease in specific gravity. This graph zooms in on those three beers – numbers 11, 12, and 13 – that experienced the biggest increases in diacetyl, and plots both the diacetyl concentration and the specific gravity as a function of time. The diacetyl for each beer is represented by the blue, red, and green bars, which you can see have all risen above the 100 ppb threshold by Day 4. Meanwhile, the corresponding lines of the same colors start to decrease around Day 1 or 2, indicating that the specific gravity of the beers decreased after the dry hops were introduced. Considering that all of these beers were presumably done fermenting at the time the dry hops were added, this gravity drop was a bit unexpected. 29

Zooming in even further, Devin observed that not only did the gravities for these beers decrease, but so did their dextrin concentrations. Remember that dextrins are those long chain sugars, which we get from things like crystal malt, that aren’t fermentable by brewers yeast. By the end of fermentation, when most of the other sugars have been consumed by the yeast, the dextrins remain and provide body and sweetness to the beer. I want to emphasize at this point that it’s not scientifically clear exactly why the dry hopping would correspond with a decrease in the dextrin concentration in these

• beers. One theory, though, is that some enzymes present in the dry hops act on

these dextrins in the beer in a way that makes them fermentable, which in turn gets the suspended yeast going again with a small amount of fermentation. This re- activated fermentation could explain why the specific gravity of the beers decreases during dry hopping. It could also explain where the diacetyl is coming from, since that re-activated fermentation would result in the yeast producing some additional diacetyl during the dry hop. What’s worse, the yeast at that point may no longer be sufficiently active to really clean up this newly-produced diacetyl, like it normally would in the initial fermentation. This isn’t proven, but it’s one of the leading theories that’s starting to emerge from this area of research. 30

As brewers and homebrewers, this can feel a little frustrating. If the pros, and if science, don’t even completely understand why dry hopping sometimes leads to increased diacetyl, how can we be expected to control it in our beers?! Well, not fully understanding this stuff is part of the fun, right? And there are some practices we can incorporate to avoid diacetyl in our dry hopped beers, regardless. One of those is to monitor gravity through the dry hop process. At a minimum, take samples right before dry hopping, and then at the end of the dry hop process, and

• bserve whether there’s been a gravity drop in the beer. The lack of a gravity drop

would probably signal that you’re in the clear. A decrease in gravity, on the other hand, would signal that some additional fermentation probably went on during dry hopping, so you have the potential for some diacetyl above the taste threshold. 31

Next up, especially if you observed a gravity drop after dry hopping, is a forced diacetyl test. This is good practice in general, but it’s especially good practice now given what we know about dry hopping and diacetyl. Best of all, it’s a simple test that all of us can do at home with no fancy equipment. Start by going over to your beer at the end of the dry hop process and collect two samples of from the fermenter. Keep one at room temperature, and heat the other to about 140 F for 30 mins. Do not microwave. A water bath works great, and a sous vide is perfect for this, though you can also use a stove top and a thermometer. After that 30 minutes, cool the heated sample so that both are room temperature, and do a side by side comparison. If you smell butter in the sample that was heated, you’re going to end up with perceivable diacetyl in your beer. If you don’t, you’re probably in the clear. 32

Finally, be open to process adjustments. Take notes on what you do during dry hopping, and if you observe diacetyl in some of your dry hopped beers, go back and check your notes to see if you can identify any process control points that correspond to the presence of diacetyl. If you do, try changing those things up in future batches to see if it eliminates the diacetyl. To give a personal anecdote, this recently came into play for me with my own

• brewing. On the recommendation of Mitch Steele and others, I typically dry hopped

my beers at about 66 degrees Fahrenheit, and this was working fine for me. But I was frustrated by my inability to effectively harvest yeast from my fermenters before dry hopping at this temperature, and once I dry hopped the yeast was so mixed in with hop material that I didn’t want to use it again. So I talked with Chris about this and how they manage it at Ballast Point, and he explained that they usually drop their tanks to about 50 Fahrenheit after fermentation, which crashes the yeast enough that they can then harvest it before dry hopping. I tried this at home with the next two batches, and did have more success harvesting yeast, but also ended up with both beers containing diacetyl well above threshold. I don’t know why this happened, especially since the same method was working fine for Ballast Point, but it did. So, until I better understand what’s going on here, I did like Pavlov’s dog, and I simply stopped dropping my beer to 50 Fahrenheit before dry hopping. And what do you know, since I went back to my prior process, the last few batches all turned out fine. The moral of all of this isn’t to avoid dry hopping at 50 Fahrenheit, since it may work 33

for you like it works for Ballast, and surely others. The moral is to pay attention to what you’re doing, and be open-minded to process adjustments if you end up with results you don’t want. 33

And on that vein, aside from being occasionally frustrating, this study of dry hopping and diacetyl has opened up a number of frontiers for brewers to explore as we aim to control it. As homebrewers, we can try to better understand these things by changing process variables on our own systems. Does it matter how long we dry hop? Allowing the more time may leave the yeast with more time to uptake any diacetyl that was produced during dry hopping. But extending the contact time on the hops could also change the flavor and aroma, in particular by increasing grassiness. Is there a sweet spot here? How about hop age, variety, or form? Do those matter? Is this phenomenon isolated to pellet hops, or can Cryohops and other formats also trigger diacetyl production during dry hopping? Does yeast strain matter? Does temperature? Scientists like Devin are working to better understand these things. But there’s nothing stopping us homebrewers from experimenting with them at home, either. 34

Along these lines, short of brewing up a bunch of dry hopped beers trying to control for all these variables, what could we learn from a forced fermentation trial? Take a finished dry hopped beer, measure its specific gravity, and then pitch it with some fresh yeast. If the gravity drops, what does that tell us? Perhaps that the dry hopping somehow changed the sugar composition of the beer such that there was some new fermentable sugar for that fresh yeast to go to work on. 35

Or what about dry hopping before the beer is finished fermenting in the first place? Whatever might be going on here as far as dry hops and dextrins and fermentable sugars… if we cause that to happen while the yeast is still actively fermenting, might the yeast be active enough to do a proper diacetyl cleanup at the end of fermentation, as it would if we never dry hopped at all? And if this works, might we have identified a means of dry hopping that’s relatively safe from diacetyl production? 36

The folks at White Labs looked at this, and the results are before you. They brewed up four 15-bbl batches, each of which was fermented with their California Ale Yeast strain, and then dry hopped on Day 4 of the primary fermentation, when the specific gravity was still at around 1.020 to 1.024. After another 6 days on the hops, those beers all finished at about 1.012. And before you is a look at the diacetyl concentrations as a function of time. All 4 of them started out above the 100 ppm threshold, as we would expect for a beer that’s still fermenting and hasn’t had a proper diacetyl rest yet. The diacetyl concentration then spiked within a day of dry hopping, which we might also expect given what we’ve just learned about diacetyl and dry hopping. But then fast forward 5 days, and all 4 beers ended up very safely below threshold, with very low diacetyl concentrations around 20-25 ppb that aren’t likely to be perceived by anyone. Sounds promising to me. I might be a bit concerned about losing aroma, with hop volatiles potentially getting vented out of the beer while it emits CO2 during

• fermentation. But also, as Chris is about to explain, there may be more going on here

than just diacetyl reduction, too… 37

Chris:

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• What is biotransformation?
• A transformation of terpenoids into new terpenoids due to interaction between hop oils

and active yeast.

• What are terpenoids?
• Organic compounds produced by a variety of plants
• The aromas vary widely: floral, fruity, minty and peppery
• Some terpenoids include: geraniol, linalool, beta-citronellol

39

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Biotransformation of hop aroma terpenoids by ale and lager yeast By Andrew J. King and J. Richard Dickson 1. Geraniol (floral, rose, sweet) and linalool (floral, orange) can both be transformed to B-Citronellol (citrusy, fruity) 2. Beta-myrcene, beta-caryophyllene or alpha-humulene (organic hydrocarbon), showed no signs of transformation Biotransformations of hop-derived aroma compounds by Saccharomyces cerevisiae upon fermentation By T. Praet, F. Van Opstaele, B. Jaskula-Goiris, G. Aerts, and L. De Cooman 1. Overview of scientific research thus far done on the research of biotransformation. 2. Emphasized the need for more research 2015 CBC Seminar on Understanding How to Control Flavor and Aroma Consistency in Dry Hopped Beer 41

Objective: Build on the knowledge contributed by prior studies to examine biotransformation of hop oils specifically while in the presence of the WLP001 California Ale Yeast (the "Chico Strain") strain. The Chico Strain is the most-used ale yeast strain among professional and amateur brewer's, but is generally believed to contribute only low levels of biotransformation in comparison to other yeast strains. Mosaic was used in this single hopped beer as it does contain 0.4 to 0.8% linalool and 0.5 to 0.9% geraniol, two such compounds that have been observed as contributing to biotransformation when in the presence of ale yeast. Recipe: Malt – 2-Row Hops – Mosaic Yeast – WLP001 Cali Calculated IBU – 30 bu* 42

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Here you see a spider graph. Spider graphs helps us analyze the strength or intensity

• f a specific aroma or flavor attribute. As points appear closer to the center of the

spider graph, the weaker the intensity of that particular attribute. This spider graph charts the aroma intensity of attributes characteristic of Mosaic hops. While each of the three beers demonstrate fairly similar attributes across beers, the intensity of these attributes differ from each beer as they were dry hopped at different points. Too Soon: For the most part, attributes consistent of Mosaic hops, had less intensity (besides apple/pear interestingly) in the beer dry hopped on day two of fermentation. During high krausen, fermentation is quite vigorous and there is a lot of CO2

• production. This could be pulling volatile aromas from the product resulting is less

aromatic intensity. This beer was most similar to the next beer, Just Right. Just Right: Had the highest intensity of aromatics characteristic of citrus/tropical, stone fruit, berry, melon and floral. Less desired aromatics such as grassy, apple, spicy has low intensity relative to the other two beers. Having been dry hopped at the end of fermentation, after capping the tank to hold in volatile aroma attributes, helping to keep desired aromatics. Too Late: This beer had the highest intensity of attributes I would consider least desirable, such as grassy, onion/garlic, resinous. This was dry hopped at 32F post filter, no yeast present. Perhaps the lack of yeast (biotransformation) and the cold 44

dry hop temperature contributed. 44

Similar attribute intensity’s as aroma. 45

I asked participants to rate the three beers from least preferred to most preferred amongst the three. Just Right stood out as the most preferred. While 0% of the 22 participants rated it their least favorite amongst the three beers. Too Late was by far the least preferred. Most participants notes the garlic/onion character, along with a vegetal attribute and resinous mouthfeel. While this beer was the only beer of the three to be filtered (pre-dry hop), it did use fining agents and regiment as the other two beers. I find it interesting the even though this beer was roused, the positive attributes still lacked the intensity of the other two beers. Why is that? Lack of yeast to interact with the hop oils? Or is the cold temperature playing a bigger role to the hop attributes extracted? 46

Biotransformation is real. It is very complicated. There is a lot of room for more experimentation and study of the subject by real scientist. By no means does my intro experiment answer any questions, necessarily. However, I find in this day and age with NEIPA’s as a hot topic, this has rejuvenated discussions regarding dry hop techniques and timing. 47

My goal with this experiment was to get real, in house, data regarding a yeast strain we use often in our beers along with a hop varietal that is popular among brewers, and to investigate what aroma attributes are impacted by these different dry hopping methods. Was biotransformation playing a part in these results…not sure  But that is something I hope to investigate more in future experiments. 48

Biotransformation will be dependent upon hop varietal. Hops containing terpenoids, such as geraniol and linalool, have demonstrated the effects of biotransformation. However, this observation is yeast strain specific. Some strains may demonstrate greater change of terpenoids to new terpenoids than other strains. Who knows, perhaps this is information that the cultivating community may some day be able to provide to brewers when they purchase yeast. 49

Temperature: At what temperature should one dry hop their beer? This will be dependent on a lot of variables, such as desired attributes one is trying to extract from their hops, equipment, efficiency, speed and ease, to name a few. Timing: When is the optimal time to dry hop? How does this effect intensity of aroma and flavor attributes? How does it effect throughput? Is it safe to dry hop during high krausen without the proper equipment (geyser!)? Yeast Strain: As mentions earlier… Hop Varietal: As mentioned earlier. Combination: What combination between yeast and hops produces the desired results? I highly suggest listening to a recent BeerSmith podcast where Brad Smith interviews Randy Mosher and another one with Stan Hieronymus. It is free, and they provide some interesting hop combinations that can lead to biotransformation of new compounds that each hop on their own, or used along other hops, would not produce certain attributes. 50

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Doug: Chris and I were pretty intrigued the potential for these Auxiliary Bitter Compounds to improve the quality of bitterness in our beers. So we set out to reproduce Brewmaster Gahr’s results at home. We chose an American Blonde as the base beer and mashed and lautered about 14 gallons of a single all-grain wort. We subdivided that wort 4 ways, feeding about 3.25 gallons for each batch into Chris’s Picobrew Zymatic. Using the Zymatic, we then did 4 separate boils that were identical except for the hopping regiment. We took the 4 resulting worts and fermented them in 4 separate but identical fermenters, all in the same chest freezer, and each was pitched with an identical mass of a single yeast

• slurry. After fermentation, each got the identical treatment of fining and force-

pressure carbonation. Ultimately, what we were trying to produce were 4 beers that would be as identical as possible in every way except for the hopping. 52

. . . And we came pretty close to that. You can see on the slide in front of you the 4 different hopping regiments we used. Only a 60-minute CTZ addition for Beer #1, only a 60-minute Saaz addition for Beer #2, a regiment of 60-minute CTZ and then 20- and 0-minute Saaz additions for Beer #3, and a schedule of 60-, 20-, and 0-minute Saaz additions for Beer #4. I sized these additions using the Tinseth formula, aiming for identical concentrations of Iso-Alpha Acid in each of the 4 beers. What was different, then, would be the amount of Auxiliary Bitter Compound in each beer. Because CTZ is a high-alpha hop, we expected Beer #1 with only a 60-minute addition

• f CTZ to have very low auxiliary bitter compounds, for an IBU to IAA ratio of about 1.

Saaz is a low-alpha hop with a higher ratio of beta-to-alpha, so Beer #2 should have had a somewhat higher IBU to IAA ratio. Returning to CTZ for a 60-minute addition while also adding some late additions to Beer #3 should have increased the IBU to IAA ratio even further. And then by using all Saaz, because it had those same late additions plus Saaz for the 60-minute addition, Beer #4 should have had the highest IBU to IAA ratio of all. In other words, the intent here was to hold the Iso-Alpha Acid concentration constant for each of the 4 beers, while increasing the IBU to IAA ratio for each beer going from #1 to #4. Based on the research just presented, the quality

• f bitterness should have also increased for each beer from #1 to #4.

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To assess that, we convened a tasting panel. What you see before you is an exact copy of the survey form we gave tasters, along with five samples, which were poured in identical cups and labeled A through E. To help identify if tasters could actually perceive a difference between the beers, 2 of the 5 samples were identical. I’ll now read to you, verbatim, the written instructions we provided: “You have been given five cups of beer, labeled A through E. Two of these cups contain the same beer. The others were all brewed from the same wort, and fermented under identical conditions using the same yeast, but differ from one another in the way they were hopped. Instructions:

• 1. Identify the two cups containing the identical beer, and mark the two “Same?”

boxes on this sheet that correspond to those two cups.

• 2. For each of the five cups of beer, please rate the quality of the beer’s bitterness
• n a scale of 0 to 10, with 0 representing the lowest quality (least pleasing) and

10 representing the highest quality (most pleasing). Please note that the intensity

• f bitterness is not necessarily the same as the quality of bitterness. Think of the

quality of bitterness as a measure of how pleasing or harmonious the bitterness is, relative to other beers you have experienced in your life. Do not feel obligated to use the entire scale.

• 3. Using the lines below, please list any descriptors that you believe characterize the

bitterness of that sample.” 54

And here are the results, which I’m presenting for the sake of intellectual honesty more than anything else. Two things jump out immediately here: 1) The beers turned out extremely similar to one another, which made the taste test very

• difficult. Only 6 out of 42 people were able to correctly identify the identical beers they were
• given. Given that there was a 1 in 10 chance of randomly guessing a correct answer, this

indicates our panel was better – but not much better – than a random number generator would have been. Of the folks who did correctly identify the same beers, multiple of them indicated that they did so based on aroma, rather than bitterness. So there’s a lot of reason to question whether our panel was able to reliably perceive bitterness differences between these beers. 2) And yet, when we average the sensory data, a clear – albeit subtle – pattern does emerge. The “Average Quality” column in the table before you represents the average quality of bitterness for each beer across the 42 tasters. You can see that the quality of bitterness more or less steadily decreased from Beer #1 to Beer #4, which is exactly the opposite of what our null hypothesis was. The “Screen Quality” column represents the same data, except filtered to only include the assessments of those tasters who correctly identified the identical samples. And across that data, we see the same pattern, even a little bit more exaggerated. So, we failed to reproduce the results. One reason for this may been that I failed to actually hold the Iso-Alpha Acid concentration steady across the four beers We didn’t have access to an HPLC to measure IAA concentration before this presentation, but are working on that. Also, you’ll see that the IBU concentrations – which we were able to measure using a spectrophotometer – did not steadily increase across the 4 beers, as expected. In short, this was a first effort, and these beers may well have not hit their targets, so I caution against

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reading much into this data.

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