HULK MASH!

Modified From: http://beforeitsnews.com/motor-junkies/2013/04/hulk-smash-2463796.html

Who knows, the big green wrecking machine may also be an avid homebrewer when he hangs up his purple shorts and returns to being Bruce Banner the mild mannered scientist. Most everyone is familiar with the basic principles of how beer, and for that matter all alcoholic beverages are made; you take some variation of a sugar solution, add yeast and let the magic happen. In the case of beer and malt-liquor, you have a sugar solution made primary up of maltose; but where does this maltose come from exactly?

Well as the name implies, from malt! More specifically, malted barley, wheat, rye, or other cereals. The only trick is that this maltose is locked away inside the grain as large polysaccharide (sugar) chains that cannot be broken down by yeast alone. This is where the mashing process comes into play. Typically, when you get grain from your LHBS it has already been malted (basically controlled germination). It may have also been roasted or kilned, creating malts of various flavor and color. In simple terms, the malting process activates the production of certain enzymes in the seed to mobilize sugars it requires for growth into a new plant. This process is then halted by drying; stopping the enzymatic breakdown of the starch but preserving the enzymes.

 Figure 1. Crystaline structure of α-amylase (left) and β-amylase (right) (wikipedia.org)

The enzyme that we as brewers are mostly concerned with is amylase. In general, amylase catabolizes (the metabolic breakdown of complex molecules resulting in simpler molecules and energy) larger polysaccharide chains resulting in smaller chains, some of which can be used by yeast to make alcohol and CO₂. Amylase catalyzes the hydrolysis (chemical breakdown with the addition of water) of the polysaccharides amalose and amylopectin, the two molecules that make up starch into smaller oligosaccharides. There are two types of amylase, α-amylase and β-amylase that have several key differences (Figure 1). α-amylase cuts at random α-1,4-glycosidic bonds, making it relatively fast, cleaving amalose into both maltriose and maltose and amylopectin into glucose and "limit dextrin" at branch points (Figure 2). This results in the production of some longer unfermentable sugars that can be cleaved further given enough time and high enough α-amylase concentrations ⁽¹⁾.

Figure 2.  α-amylase (Hulk) action on a starch

β-amylase also works on α-1,4-glycosidic bonds, but only on the non-reducing end of the polysaccharide chain, making it slower, cleaving two glucoses off at a time resulting in a single maltose molecule⁽²⁾ (Figure 3).

Figure 3. β-amylase (Red Hulk) action on starch

Figure 4. Products of hydrolyzation (Left to Right) Glucose, Maltose, Maltotriose, Limit Dextrin (wikipedia.org)

The activity of these enzymes can be controlled by the temperature of the mash.  α-amylase has a temperature optimum from 149 °F to 158 °F ⁽³⁾ and β-amylase from 134 °F to 141 °F ⁽⁴⁾. By controlling the mash temperature you can push the optimal temperature to α-amylase or β-amylase which will result in more or less body respectively in the final product .

Only certain grains have still have enough amylase to convert the starches to fermentable sugars. Typically, this includes grains that have not been extensively roasted or kilned as these processes denature the amylase enzymes. This difference in amylase enzyme is measured in diastatic power. The greater the diastatic power the more amylase present and the greater ability to convert available starches. American 2-Row, American 6-Row, and Maris Otter are examples of "base malts" with relatively high diastatic power (140, 160, and 120 respectively) and are able to convert about an equal amount of malt with no diastatic power. Munich malts do still have diastatic power (72) but it is much lower than that of the base malts. Kilned malts like black malt have no diastatic power. Other partially processed adjuncts like flaked oats and wheat do have starches available for conversion but no diastatic power and must be mashed with base malts to extract the sugars from the grain.

If you're wanting to venture into brewing beers with a more complex grain bill than the typical extract recipe or just want to have more control of what exactly is going into your wort remember the words of the mighty Hulk, "HULK SMASH!".

---

1. Greenwood, C.T., MacGregor, D.Sc., and MacGregor, A.W. 1965. The isolation of α-amylase from barley and malted barley, and a study of the properties and action-patterns of the enzymes. J. Inst. Brew. 71:405-417
2. Enevoldsen, B.S., Bathgate, G.N. 1969. Structural analysis of wort dextrins by means of β-amylase and the debranching enzyme, pullulanase.J. Inst. Brew. 75:433-443
3. Bertoft, E., Andtfolk, C., and Kulp S.E. 1984. Effect of pH, temperature, and calcium ions on barley malt α-amylase isoenzymes. J.Inst. Brew. 90:298-302
4. Eglinton, J.K., Langridge, P., Evans, D.E. 1998. Thermostability variation in alleles of barley beta-amylase. J Cereal Science 23(3): 301-309

'Tis the Season

The holiday season is upon us and it is that time of year when most breweries release their obligatory Winter/Seasonal Ale. While Winter Ale is not a recognized style, a few styles of beer and ingredients lend themselves to producing beers hardy enough for winter. I generally think of barley wines, malty beers, spiced ales, and old ales with high alcohol content. Recently I’ve run across a couple of winter ales that don’t really fit into the winter ale category. They weren’t necessarily bad but definitely not what I expected or didn’t satisfy my winter cravings. Below is a sampling of those beers.

Smuttynose Winter Ale, Portsmouth, NH

Amber/brown colored with a medium tan head. Aroma of roasted malts, caramel, and chocolate. Light body with some caramel notes but lacking on malt characteristics. I was a little disappointed.

Verdict: Not Naughty nor Nice

Big Sky Powder Hound, Missoula, MT

Copper colored with large white head. Aroma of floral/earthy hops and malt. Medium body with floral/earthy hop bitterness upfront and a nice malt backbone. Seems like an IPA guised as a Winter Ale.

Verdict: Not bad for an IPA but deserves a visit from Krampus

  

                
Harpoon Winter Warmer, Boston, MA

Amber/brown colored with small tan head. Aroma of cinnamon and nutmeg with some malt sweetness. Light body with overpowering cinnamon upfront which overpowers anything else. The aroma was the best thing about this.

Verdict: Like a Christmas Wreath, best thing about it is its aroma

Goose Island Sixth Day, Chicago, IL

Brown colored with small tan head. Light aroma of hops and roasted malt. Medium body with earthy hop bitterness upfront, some caramel and chocolate notes, and a warm alcohol finish.

Verdict: On Santa’s Nice List

Victory Winter Cheer, Downington, PA

Golden colored with large white head and nice lacing. Aroma of wheat, banana, and citrus. Light body with citrus hops upfront, fruit notes, and some wheat body. Another summer beer masquerading as a Winter Ale.

Verdict: Shouldn’t be surprised with coal in the stocking

Sam Adams Winter Lager, Boston, MA

Dark amber colored with small tan head. Aroma of caramel and citrus. Light body with malt sweetness upfront and notes of cinnamon and caramel.

Verdict: On Santa’s Nice List

Great Divide Hibernation Ale, Denver, CO

Brown colored with small tan head. Aroma of roasted malts and coffee. Medium body with malt body and coffee notes upfront some hop bitterness and a dry finish.

Verdict: A gift from St. Nick himself

I am not one to say beers should follow the exact guidelines of a style or that brewers shouldn’t experiment with new ingredients and techniques but when I get a Winter Ale I have some expectations. I expect not to get an IPA guised or repackaged as a Winter Ale and I expect to get something that’s warming (high in alcohol) or at least malty. Just simply slapping a winter label on a beer does not make it a Winter Ale. Have a happy holidays and prost!

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.

A Tale of Two Yeasts

It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, and where there was one, now there are many, yeast that is. No this is not some Dickens’ novel but the real life tale of brewer’s yeast. Most homebrewers are aware that there are generally two types of yeast used to make beer, ale yeast (Saccharomyces cerevisiae) and lager yeast (Saccharomyces pastorianus aka S. carlsbergensis), but things are rarely as simple as they seem.

The domestication of barley in Sumeria 6,000 years ago likely led to the predecessor to modern day beer and the beginnings of yeast domestication. During the Middle Ages in Europe, ale-type beers (likely brewed with Saccharomyces cerevisiae) were beginning to be produced with lager-brewing arising in 15th century Bavaria⁽¹⁾. The first yeast strain (CBS1171) wasn’t isolated until 1883 by Emile Christian Hansen and was designated as the neo type (specimen a species is named for) of Saccharomyces cerevisiae. This isolation led to the first pure cultures of yeast strains used for commercial beer production in the late 1800’s⁽²⁾. It was generally recognized that ale and lager yeasts were different but it would take another 100 years to identify that difference (Figure 1).

S. cerevisiae 

S. pastorianus 

S. bayanus

Figure 1. See why yeast species identification is difficult. All three species are part of the Saccharomyces sensu stricto group.

In 1985, the Saccharomyces group known as Saccharomyces sensu stricto were split utilizing DNA analyses into four distinct species: ale style yeast, S. cerevisiae (neo type strain CBS1171NT); wine and cider yeast, S. bayanus (CBS380T); S. paradoxus (CBS432NT); and lager yeast, S. pastorianus (CBS1538NT)⁽³⁾. These experiments also revealed that S. pastorianus (lager yeast) was a hybrid species with S. cerevisiae and another species ⁽⁴⁾, but more on that coming up. Since 1985, this group has been divided into even more species. Many of these species are differentiated by only a few nucleotide base pairs at certain chromosomal loci known for sugar (maltose) and sulfite metabolism. Some species are allotetraploid hybrids (a duplication of each chromosome from the parents) or sterile haploids and diploids incapable of sexual reproduction, but yeast reproduction can be saved for another post.

As you can see, yeast genetics is complicated and contentious. Lager yeast (S. pastorianus) unlike ale yeasts, have never been isolated from the wild and depends on humans for its propagation ⁽⁴⁾. In 2011, scientists identified a cryotolerant species of yeast (S. eubayanus) in Patagonia that is likely the missing parent of lager yeast (Figure 2). These yeasts were isolated from beech trees and their associated fungi (Cyttaria sp.) in the Southern Hemisphere. These beech trees are equivalent to their northern cousins, oak trees ⁽⁵⁾. Saccharomyces spp. are often associated with oak trees in the Northern Hemisphere and are thought to be the origins of brewers and baker’s yeast in Europe. How South American yeast made their way to Europe is still under some debate. Some believe the early trade routes may have brought back exotic wood and the yeast hitched a ride to European breweries. Others believe that fruit flies stowed away on shipping vessels may have carried S. eubayanus in their guts until finding their way to Europe ⁽⁵⁾. We may never know how S. eubayanus found their way to Bavarian breweries but they have helped to create one of the most popular and commonly used yeast in the brewer’s arsenal. Prost!

Figure 2. Cyttaria from Chile growing in a tree branch where S. eubayanus was isolate   

---

1. Corran HS (1975) A History of Brewing (David and Charles, London, UK).
2. Nguyen H-V, Legras J-L, Neuve´ glise C, Gaillardin C (2011) Deciphering the Hybridization History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T. PLoS ONE 6(10): e25821. doi:10.1371/journal.pone.0025821
3. Vaughan-Martini A, Kurtzman CP (1985) Deoxyribonucleic Acid Relatedness among Species of the Genus Saccharomyces Sensu Stricto. International Journal of Systematic Bacteriology 35: 508–511.
4. Dunn B, Sherlock G (2008) Reconstruction of the genome origins and evolution of the hybrid lager yeast Saccharomyces pastorianus. Genome Res 18:1610–1623.
5. Libkind et al. (2011). Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast. PNAS 108:14539-14544.