University Place Wednesday Nite at the Lab  Mark Schmitt Brewing A Better Barley  January 23, 2013 UP825   cc >> Welcome, everyone, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the UW-Madison Biotechnology Center. I also work for UW Extension Cooperative Extension, and on behalf of those folks and our other sponsors, Wisconsin Public Television, the Wisconsin Alumni Association, and the UW-Madison Science Alliance, thanks for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. It's your chance to explore more about science here at UW-Madison. Tonight, I'm delighted to be able to introduce to you Mark Schmitt. He's with the US Department of Agriculture, ARS, which is the Agricultural Research Service. He's at a unit here on the UW-Madison campus called the Cereal Crops research unit. For many of us, it's fondly and formerly known as the Barley and Malt Lab, and he will be speaking to us tonight about his research on how you get the malt in the barley. 

Mark is from a Big 10 background. He was born at the University of Michigan. He was an undergraduate education from Ohio State, but he went to graduate school at UW-Madison. [LAUGHTER] Where he earned his PhD in horticulture and botany. He studied photorespiration and photosynthesis with Gerald Edwards. For the next 20 years, he conducted basic research at several companies both large, for example, DuPont, Monsanto, BASF, and small, EniChem Americas. Research projects of his have ranged from assimilate partitioning, to crop engineering, and fungicide mode-of-action. His work resulted in patents for inventions ranging from methods to protect crops from fungal pathogens, to identification of novel anticancer chemotherapeutic agents for pharmaceutical use. And in 2003, he joined the USDA Agricultural Research Service, Cereal Crops Research Unit here at Madison, Wisconsin, as a project leader for basic research into fundamental aspects of malting quality. They must have immediately started building the new building to keep him happy because I think that opened in 2007. 

Is that correct? >> That's the date. >> Yes. He has additional responsibilities for applied support for malting barley varietal development. One of the most interesting stories that I read in Jim Feldman's book on the buildings of the University of Wisconsin Madison was the origins of that building that comes out of the end of Prohibition, and the idea that the nation needed to catch up on a lost generation of expertise in malting and in barley cultivar development. That's why we have the Barley and Malt Lab here. Still the nice little yellow brick building on Campus Drive and the Cereal Crops Research Unit. Tonight we get to hear more about how you get the malt out of the barley or the malt into the barley. Please join me in welcoming Mark Schmitt to Wednesday Nite at the Lab. [APPLAUSE]  >> Thanks very much, Tom. I hope people can hear me. 

If you don't, wave your arms or make something otherwise observable, and I'll do my best to speak up. One other little piece of interesting, not quite trivia, but it may be something you're interested in is that our new building, the Cereal Crops Research Unit, as Tom said, was opened in 2007, but it may actually be the last single-story building built on the UW campus. It's actually a federal building, so it was built according to federal specifications. And I understand all UW buildings have to be at least three stories or taller. So, put that away in your useless trivia information file. [LAUGHTER] Bring it out as appropriate. So, what I want to talk about today is not necessarily just the research aspect of things, but give you a little bit of an introduction to how malting varieties are developed. Sort of an overview of the process, the hurdles that the malting barley breeders face, the support we get from stakeholders and the malting brewing industries, what we actually do in the malting quality lab to provide the breeders with malting quality information that they use to select the varieties, and then I'll give you a little bit of a flavor of some recent research we've been doing as well. So, basically, what I want to talk about today is what they do, and they I'm looking at in four different parts. So, as said, the malting barley breeders, the stakeholders associations, our QA team, as well as some laboratory research that we've been doing. 

A little bit about barley just in terms of basics. It's diploid grass, fairly simple genetics. It's just got seven chromosomes. Diploid is chromosome numbers 14. It's actually the fourth most abundant cereal worldwide, after maize, rice, and wheat. It's an old crop. People found specimens in archeological digs from long, long ago. It's a cool season crop that's widely adapted to a variety of different climatic conditions. It's fairly stress tolerant, drought tolerant. It actually does reasonably well on marginal lands. 

So it doesn't necessarily have to take the prime cropland. It's fairly widely grown. Until the 20th century, there were primarily land races that had sort of developed on their own over time. From after the time of Prohibition there has been more emphasis on actually breeding for malting characteristics. And, again, I'll talk about that a little bit later. It's a true breeding, self-fertile crop, but it's easily hybridized. It's used for food, human food, animal feed, and malting and brewing purposes. Brewing is probably what catches most people's interest. But worldwide, actually about 80% of the barley crop goes for animal feed. It's a little different in the United States, as I'll show in a slide later, but worldwide there are lots of other uses for barley other than malting. 

It's been around for, as I said, for quite a while It's been used in brewing because as it germinates, it produces fairly high quantities of enzymes that are important for converting starch and protein into forms that can be useful in brewing. And it also has hulls on the seed as well which are useful for some of the technical processing filtration in the brewing process. And, most recently, it has been approved for a heart-healthy nutrition claim. As oats, it has relatively high levels of soluble dietary fiber, beta glucans. >> Do you make flour out of it? >> Sure. You can buy barley flour in lots of different places. It has some flavor characteristics that aren't quite as palatable as I understand as wheat flour, but it certainly is useable for a variety of things. You can get barley muffins and whatnot. Anyway, I didn't bring it with me, but I was going to bring a couple visual aids in addition to a beer both, which it think Tom brought along. I was going to bring a bag of malted milk balls because barley malt is the malt part in the malted milk, and also, my favorite and every morning I face it, are the Grape-Nuts. 

Grape-Nuts are made with wheat and malted barley. And in fact, if you actually eat the Grape-Nuts themselves, it tastes very similar to what you would taste if you grabbed a handful of freshly made barley malt. It's really surprising. If you ever go take a tour of a malt plant, they'll often end that by letting you grab a little handful of the malt, pop it in your mouth, and people are just sort of surprised at the taste if they're familiar with what Grape-Nuts taste like. As I said, worldwide barley is used primarily for animal food. As I understand, it's a favorite food of camels in Saudi Arabia. It's fed, I guess, for livestock in a fairly large amount, Australia, for example, is a very large exporter of barley. In the US things are a little bit different proportioned. There's a relatively smaller use of barley for feed and other sort of purposes. About 40% of the overall domestic barley, with about 60% or so being used in beer. 

Barley was originally developed from Hordeum, wild Hordeum spontaneum. That was originally, I guess, found in the Fertile Crescent through Israel, Jordan, Lebanon, up around and into Iran. The domesticated barley modern varieties we use are derived primarily from the land races that come from the western portion of the Fertile Crescent. Portions from the east eventually moved their way through the Middle East into South Asia and up into China, and as they progressed here there was actually some, I guess, gene flow and the Himalayan land races actually have a slightly different proportion of some specific alleles or they're able to separate out the populations. And, again, while all of the land races are essentially natural derivatives, the Hordeum spontaneum that was domesticated into Hordeum vulgare is believed to actually come from Israel and Jordan. But barley and malt really are two different beasts. Malt is made from barley, but malt itself is a partially germinated product. And in a true botanical or biochemical sense, germination is the very early stages where the enzymes and various activities in the hydrated seed begin and the beginning of the root protrudes outside of the seedling. Germination in a malting sense actually takes place past that. So most malting will take place over a period of three to five days, sometimes six days depending on the particular variety and the conditions used. 

As I said, the important parts of malting are the activation of the enzymes in the seed and synthesis of the enzymes. So these are both enzymes that degrade carbohydrates as well as proteins in the cell walls as well. At the end of the germination process, the barley is stabilized by heating and drying. They will take it through a period of perhaps a day or so where temperatures start at about 45 or 50 degrees and ultimately ramp up with different time intervals going to 85 degrees or so for normal malting, and then can be taken higher if different characteristics are desired in the malt. By taking it to higher temperatures, you can get darker malts, different flavors. There's a variety of different characteristics you can use in generating the malt, largely by how it's heated and dry. One point I'll make at the end as well is the germination has been studied by the details, malting, not so much. There's been enormous amount of research over decades into the processes taking place when seeds germinate, but what actually happens in a malting environment is not quite the same and people haven't looked at it quite so carefully. And a little bit of the material I'll talk about at the end of the talk will address that, and I hope I can convince you that there's still reason to keep people employed to look at details of malting barley biochemistry. Malting varieties actually are specific and are chosen to be able to perform reproducibly in the malting conditions. 

Malts just have to be able to rely on specific response known that under a certain set of conditions, the product that they will get out of the process at the end is as they expected. The malting industry, malting and brewing industries have a very carefully developed set of specifications of different characteristics of both malt and the brewing products derived from it, wort and then ultimately the fermented beer. Again, I'll make the point that barley grain is not the final product for malting. The malt has to be able to germinate, produce the hydrolases and modify the reserves in the seeds. This is actually different than wheat, corn, soybeans, other crops. When those crops are harvested, the grain they get at the end is it. That's what the product is. Barley is a little bit different when it's going to be used to make into malt because when you harvest the barley grain out of the field at the end of the season, it still has to be able to be dried down and stored for a period of time and then be able to respond appropriately to generate the hydrolases, convert the seed reserves and so forth to be able to produce the product. So, if you produce something at the end of the season, which is essentially unviable or doesn't really grow well, then it really is no use as a malting variety. It has to be able to sort of go that step beyond the harvest in the field. 

So, storage conditions are important and the ability for the grain to be able to respond reproducibly after a period of time is also something that is important. One other thing I'll say as well is that there's a premium paid to the grower if the barley produced meets the malting specifications. If the grain doesn't meet the malting specifications, then basically it is sold as a feed grade barley which has a lower value. So, it's very much in the farmer's interest to be able to produce a crop which meets the malting specifications so that the farmer can get the malting premium from it. I have a great admiration for barley breeders. They face a lot of challenges, as do any sort of seed breeders. But it seems like the barley breeders have a number of challenges stacked at them that other breeders may not have. They face challenges in agronomy, disease resistance, especially in malting quality, and also there's some systemic constraints that come in as well that because barley is essentially a private sector crop, I'm sorry, a public sector, the barley breeders are mostly in universities or USDA facilities. There is a relatively small breeding effort by corporations. Basically, barley is a small enough crop that there really aren't the profits for it, and it's just not really feasible or economically conducive to be able to breed for malting barley. 

So, I'll come back to those again a little bit later. The agronomic challenges are usually the same sorts of things that most breeders would face for yield. If you don't yield well then there's not going to be enough crop to sell to recover cost and make a profit. Specifics for height, heading date, lodging, so forth, are fairly important to be able to meet a window of sort of local condition suitability. Something that's become fairly important recently as an opportunity is to develop malting barley that's winter hardy. In Europe, probably about half of the barley crop that's grown from malting barley is grown as a winter barley and about half is grown as a spring barley. Winter barley is planted in the fall. It germinates and establishes itself, grows to a small height, and then over-winters. When conditions become favorable again in the spring, it resumes growth and as such, gets sort of a head start, can complete the crop cycle sooner or is harvested earlier.  Generally, it has better water use because water is available during the wintertime, and when the crop is ready to start growing, generally the winter barley eat a little bit more. 

And also, the timing of the harvest is useful. In some places it may be possible to get a double-cropping in where you grow winter barley followed by another crop. In the US, there haven't been a lot of winter barleys released. There have been a few, mostly from the western part of the US. Winter hardiness hasn't been great. Although, they do survive and they can yield a crop if the winters aren't too bad. But the winter hardiness of a malting barley is something that's an active breeding program. The barley breeder at the University of Minnesota, Kevin Smith, has a program working specifically to develop winter malting barley for the Midwest, Minnesota and Wisconsin as well. So, this is an area of ongoing interest. Disease resistance is, of course, another concern the breeders have to face. 

There are any number of historic diseases that the barley needs to be able to withstand. About 15 years or so ago the barley growers became faced with a problem with the Fusarium head blight or scab. This was a fungal disease that causes yield loss, and the fungus produces a fungal toxin called vomitoxin which, as you might guess, isn't something you'd really want to have in a crop that you would eat or use for brewing. So that really took the heart out of a lot of the Midwest malting barley growth. Nowadays, most of the malting barley production has moved west into western North Dakota and Montana and Idaho, and so forth. But, again, the Midwestern breeders have been making progress bringing in some resistance from some Chinese barleys to the locally adapted varieties. I think there have been two or three different varieties released and accepted as a malting variety in the last five years or so which have shown 20% to maybe 40% reduction in Fusarium infestation. A more current problem is a stem rust that's referred to as Ug99 or by the plant pathology wonks is TTKSK. Ug99 actually refers to the where the disease was first identified in Uganda in 1999. So, it was first found here, in central Africa I guess. 

It's a problem because it's a race of the rust to which none of the current varieties of either wheat or barley have any significant resistance. There's resistance available but the resistance is largely in unidentified varieties which are, not unidentified but unadapted varieties which don't perform particularly well in the regions where the barley is grown. So, the breeders are actively working, trying to get the resistance into current varieties. But there is some concern globally about the spread of the disease. The diagram here shows that over the last couple years it's actually spread up into the Middle East, and there is some real concern that it has the possibility of spreading further into west Asia and up into China. This is a very heavily, very intensively cultivated region for wheat, particularly, but also barley as well. And if the disease spreads that far and causes some significant epidemics in those grain-growing regions, there can be some really horrific yield losses and problems for the people who depend on those crops. I guess I'll just say at this point that this is something that people are aware of and are actively working on. Resistance has been identified, and again, they're working to get it into the varieties that are adapted to local culture. Malting quality, this is the part that's more near and dear to my heart. 

Malting quality is kind of a fuzzy thing. A maltster or a brewer would say he knows malting quality when he sees it, and the malting and brewing industries can develop a chart listing a variety of values for the different measurements that are made. But most of the characteristics that are made are from fairly poorly defined targets. For example, one of the most common malting quality, one of the most important malting quality characteristics that's measured is referred to as malt extract which basically is the amount of soluble material that can extracted from the ground up barley as it's given a hot water soak for a period of time. Now, that actually consists largely of sugar, mostly from starch degradation, but there are amino acids and peptides and cell wall fragments and a whole bunch of other different sorts of things in there. It's essentially just by simply physically measuring the density or refractive index or osmotic potential of the wort that's produced as the barley is given a hot water extract. So, it's something that's important because the malt extract directly translates to dollars in the brewer's bank account because the extract is what the yeast ferments to produce alcohol which the brewer sells but really isn't a very well-defined term so it's not something that a molecular biologist could go in and say I'm going to find the extract gene and we'll work on that or find some discrete defined target. Also, another problem that comes up is that the malting quality phenotype, this collection of traits, characteristics, is something that's easily disturbed. So, as a breeder is trying to bring in resistance to scab or stem rust or one of the other diseases, it's fairly easy to lose the package of malting characteristics. It's a fairly difficult thing to try to bring that suite of characteristics back together while retaining the trait that is being introgressed. 

So, what often happens is the brewer ends up, or I'm sorry, the breeder ends up breeding within a fairly narrow background. I'll show some pedigree information later on which shows in the Minnesota program that they have the same varieties essentially contributing back into the pedigree at different stages through the varietal development. So, what that can mean is that you end up losing some diversity and some potential for making further selections to somehow enhance the characteristics that you're trying to develop. There are ways around that, but it's something that's a bit of a problem. Also, I didn't mention it here but there's an additional problem where a number of traits are fairly tightly linked and they can't be readily separated So, while you might be trying to increase extract and decrease beta glucan, it may be that for various genetic reasons, those characteristics just segregate together apart from where they might be on the chromosomes, and you might think they would segregate independently, but the disequilibrium may actually prevent those traits from being separated. The malting quality measurements are actually critical for helping the breeders choose the right sorts of varieties to advance, but they're a bit problematic in a number of ways because they usually take place late on in the development cycle for a couple of reasons. One of which is the traditional seed requirements call for about 250 grams or so of grain to be able to malt and perform the malting quality analyses. It's a bulk analysis. You can't really get specific on that. And also, it's relatively slow, expensive, and of somewhat limited capacity. 

Generally, people refer to the malting quality measurements as being the limiting factor to the variety development. I'll show a little bit of something we've developed recently which may help speed things up a bit. Although, there are a number of approaches that are being brought to bear. As I mentioned, the malting quality metrics, the suite of traits that breeders look at, this is a table from the American Malting Barley Association website where they give the breeders' targets. So these are the values that they suggest are sort of optimal for commercial malts. So breeders breeding two-row barley would try to make sure that the kernels are always more than 90% plump. They have beta glucans below 100 parts per million. For six-row barley, they'll accept them up to 120 parts per million. Soluble protein sort of in an intermediate range. Extracts above 80%, essentially, for either two-row or the six-row and specified enzyme activities for diastatic power which is kind of a combined measurement of all the potential carbohydrate activity in the malt or alpha amylase is one specific enzyme activity. 

There's the mouse. Alphas amylase as well as another measurement of that. There's a bit of a dichotomy or developing split within the brewing world. Most of the American light lagers generally have been going for a fairly, well, essentially they use adjunct for the brewing to add some additional starch. So, some breweries may add rice to add additional starch to provide more alcohol. Other brewers may use corn grits to provide the additional alcohol. Craft brewers tend to use more strictly malt, so it's an all-malt brew so that they don't use any adjunct, any additional cereals. And so, the brewers doing the craft brews with all-malts tend to need relatively lower enzyme activities, the diastatic power and alpha amylase, because they're adding a larger fraction of the malt, where brewers that add, essentially, other grains to the fraction of barley malt in the mash need to have each barley kernel having relatively more enzyme activity. So, there's some tendency to be interested in developing two different types of, or two different flavors if you will, of barley varieties. One for an adjunct brew and one for an all-malt brew. 

The barley breeder at University of Minnesota, Kevin Smith, several years ago released a new malting variety Rasmusson. It was actually named after his predecessor, Don Rasmusson. This is a timeline that he and Al Buddy, who worked in the Malt Quality Lab with me, a timeline they developed showing the actual length of time that it took to develop Rasmusson. This was, I guess, a fairly normal timeline. So, the initial cross of the first parents of Lacey and one of the Minnesota breed lines, M95, took place in a greenhouse in the fall of 1996. Kevin made some increases in a winter greenhouse, ran it out to increase in the field, and then a couple additional selfings through to the summer of 1998 when they had the first selection for agronomic traits and malting quality. This was the first time that we actually got the barley at the CCRU to be able to take a look at its malting quality. And then through a number of other years they had additional field trial malting quality analysis. It was included in a Mississippi Valley regional malting barley nursery for several years. It looks like five years as it's shown here. 

This nursery is where the breeders contribute their best lines which are grown together with the number of check varieties so that the agronomic performance, malting quality characteristics, disease resistance, can all be compared amongst the sort of standards and the upcoming varieties. There are also a number of pilot and plant scale trials through AMBA, the American Malting Barley Association. This is where the stakeholders group actually gets their first chance to look at the grain, both as it malts and then as they brew it later on in their own hands. So, the pilot scale tests are where a kilogram goes out to a number of different collaborators. They malt it up and then run it through the malting quality analyses and see how it stacks up with what's been grown other places. Subsequently, they go to a plant scale testing if the pilot scale testing has been satisfactory. The pilot scale, as I said, uses about a kilogram of grain. The plant scale testing uses, according to one person, about 7300 bushels, which is about 10 to the eighth grams of barley. So I think a technical term is it's called a whacking great load of barley. [LAUGHTER] So, when they get up to the plant scale testing, it's actually fairly serious. 

It's something where they are literally malting at a malt plant scale to see how the barley functions in a real commercial operation. And then if things seem to go reasonably well there, one of the brewers will actually take that lot of malt and brew with it and run it through their taste panels and see how it works. If it works in the process, if it gives right flavor, if it seems to be a reasonable player, if it's handled well. So, if it gets satisfactory in two years of plant scale testing, then AMBA may recommend it as an official recommended malting variety. It's an official recommendation in the sense that it's a statement that the malting and brewing industries see potential commercial use in this variety. So it'd give growers some confidence that they can actually plant the material and grow it, and if it makes malting specifications, there likely will be somebody who wants to buy it. Anybody can grow any variety of barley, but if it isn't a variety that's known, if you don't know how it's going to perform and nobody's interested in buying it, it gets a feed grade classification. It doesn't get a malting premium. Anyway, through the full process here, it took, I guess, 13 or 14 years from the time of the first cross of the parents to when it was actually a recommended malting variety and when there was enough seed available that it could start being distributed somewhat. So, it's not a fast and easy process. 

It takes a while to be able to work through the whole cycle, to be able to grow the line out, see how it performs, make sure it performs adequately, generate enough information that people can trust it. In a summary that Kevin put together he said, basically, from the crossing event, back in 1996 I think it was, there were four greenhouse harvests, two winter harvests, winter harvests usually are where they're grown in Arizona or New Zealand or someplace over the winter where they can get a crop in. Obviously, it does better than trying to grow something in the winter in Minnesota. As well as harvest from 10 summer crops. A total of 143 station years. So, that means, a station year is grown at one site in one year. So, if you had a year where you tested out in eight different locations, that would be eight station years. So, over the number of years that it took to develop the variety, Kevin was able to get data from 143 station years. One thing I'll point out here as well is that nine years of the maltings came to us for a quality analysis, and we analyzed it in one variety or another or in one location of another about 70 times. So I got to know Rasmusson reasonably well. 

It got two out of two in the pilot scale testing program and two out of two in the plant scale testing, so it was recommended for inclusion on the list of the recommended malting varieties. Sure. >> What was the purpose of Rasmusson? Why did they decide to create this? What were they... >> There's always a need for new varieties that have improved agronomic characteristics, disease resistance, malting quality. There are older varieties around one of the ones here. Robust, it was registered in '83. It was still very heavily grown for the next 20 years, but eventually it fell out of favor. There were other new varieties that came that had better malting characteristics, higher extract, more reproducible low beta glucan. Any of a variety of different reasons that you would sort to bring out a new variety to replace deficiencies in the existing varieties. I guess I'm not sure how else to... 

>> Is there something very specific about this that they said we want this, we're breeding this for these particular characteristics? Were they very specific what their goals were or was it a very general set of goals? >> For Rasmusson or earlier, it's basically just bigger, faster, better, shinier, improved, a suite of things. Subsequent varieties have been actually released which show reductions the Fusarium head blight, reductions in the mycotoxin production. So those are ones that were developed to have, were sort of adequate and all the other characteristics but also had a reduced susceptibility to the disease pressure. Rasmusson was sort of the last of the ones that came through the normal development cycle without being specifically selected for tolerance and scab resistance. Well, I guess the point I'll just make from this, again, is that you can see the early parents, Morex, which was a long-time standard and has sort of now become the laboratory standard. It's sort of the Arabidopsis of the malting barley world. Appears in the pedigree in a number of different places. Morex and Manker yielded Robust, that gave us Excel, Stander, Lacey, released in 2000, ultimately down to Rasmusson. So, as I mentioned, there can be problems or there's concern about a loss in diversity and variability in the breeding lines. This is all Minnesota breeding program, and so they're all somewhat interrelated, but that really is necessary because of the relative ease with which the suite of malting characteristics can be lost. 

And it's fairly easy for a variety to quickly sort of go to hell if you bring in traits that are rather distant. And then, the last thing I'll mention is sort of the systemic constraints on the breeders. Malting barley, as I said, comes from a few public breeding programs and the university and USDA. The competing crops, so corn, soybeans, wheat, canola, are all bred from private companies that have deep pockets and throw a lot of money at developing new varieties. A lot of the new varieties of the other crops actually are displacing barley so that the maize and other crops can be grown further north in regions where barley was the primary crop. So, there's a bit of an issue maintaining enough of a barley acreage and barley crop around to be able to get the reliable supply of malting barley. At present, the genetically modified grain really isn't a viable option. Brewers really don't want it unless they're forced to take it because wheat and other crops which are sort of competing head to head for acreage with barley are genetically modified or soon will be. It may be a situation where barley will be forced to go that route, but currently there's more of an emphasis on research into the molecular biological basis of malting quality and not using the tools for selecting GM barley. I'll mention just briefly, the American Malting Barley Association, AMBA, they're a stakeholder group, very strong supporters of both the industry and out unit as well. 

Mike Davis, who's the president of AMBA, and Dave Peterson, in the audience, both were main movers in getting out new building built. I think it was a $12 million earmark. Maybe a bad word not necessarily all the time. But that's what got our building built in 2007 along with other money to run the operation, as well as supporting the other aspects of barley research from Minnesota to Oregon. They really are focused on trying to maintain an adequate supply of high quality malting barley for the industry, and to do that they are also are fairly strong supporters of basic malting barley research. Recently, AMBA has been picking up more and more members, and that's a good thing. They have a number of full members, full dues paying as well as a number of associate members that are sort of taking a taste from the well. But it's the dues from these brewing and malting organizations which actually allow AMBA to provide the support that allows us to do some of our malting quality work and supports a number of other research programs developing malting barley. As I mentioned, barley is being out-competed in many ways by a number of other crops so that over the last, what is it, I guess 20 years, 25 years or so, there's been a fair reduction in the US acreage of barley that's grown. It's stabilized a little bit recently, but it still is of concern. 

As I mentioned, we're in what really is not only a new building but a terrific building. It's a very attractive building, and even more importantly, it's extremely functional. It's a very well designed building and very good to work in. We currently are in the Biotech Center, which is here on campus. Cereal Crops Research Unit is way over on the far end here with just the Forest Products Complex and then the hospital complex is being at the far end of the west end of campus. This is just a schematic diagram of the research building where we have research labs for six principal investigators along the, I guess it's the east wall, east side of the building. >> East wall. >> Yeah. And then on the west side is where they have the suite of the malting quality labs. For the three labs for the malting quality are basically the malting lab, a sort of seed prep lab, and the malting quality analysis or the instrumental lab. 

You can see there's actually a fair fraction of the space in our facility that's devoted to the malting quality support program. Now, to talk about malting quality on campus, you tend to think of small things. A little pot here, maybe a little microtube, but in real commercial production, a malting quality bin is a big thing. This is a photograph down one of the bins, or just a linear malting bin, at a cargo plant that was in Jefferson Junction. This plant actually is a little bit smaller than many. This one has about 6,000 bushels capacity in each bin. So the bed of malt here is perhaps five or six feet deep, and I'm sure, maybe 30 or 40, 50 yards long and 10 yards wide. It's a substantial quantity of grain. As I said, it comes out to about a million grams of barley. To try to analyze how the malt is going to do in a commercial malting facility, we've developed some methods over the years, people well before I got there, to go through the malting process so the three parts of it, the steeping, germination, and kilning, at smaller scales that are able to generate a malt product which is actually representative of what they get in a commercial pale malt malting run. 

So we have some seed tanks that are built. These have individual boxes which are able to drop into the water underneath so we and adjust individually the steeping time or how long the grain is hydrated. Some fatter grain takes longer to hydrate. Skinnier grain takes shorter time to hydrate. The whole process is designed to come out of steeping at 45% to 47% moisture, which is what really is optimal for the germination process which takes place in the next devices where. We move the grain into these round cans that are put in on a turntable. So it rotates, I think it's five minutes every hour or about that. The grain is contained inside the chamber here. It's maintained near saturating humidity in the darkness for about five days, after which it's moved into the kilns. The kilns blow heated air through the bottom of the containers with the grain in it out the top. 

It can be recirculated and program the temperature to dry the grain out according to a desired profile. We also have a couple instruments called the Joe White Micromalters, which basically is a big steel box from Australia that does everything all in one. When it works, it's great. [LAUGHTER] And when it works, it's great. [LAUGHTER] This is, as I said, an all in one thing, and it can be a very good thing. We use it for some specialty projects, which I'll come back to a little bit later. And then we also have the analytical lab where people use the mash baths to be able to make the hot water extractions of the ground malt and then separate it out by filtration and then analyze it on a variety of instruments. There's a whole range of different techniques that are done as part of the standard malting accounting profile. Over the years, we tried to increase the capacity and be able to provide more samples more quickly to our collaborators. So this is a view of about, well, from the early '90s through about 2010 with individual measurements here and then sort of five-year average blocks showing we've been able to increase the overall capacity of the lab from a little over 4,000 to about 5500 samples per year. 

Recent years we've been up over 6,000 samples, so the capacity is increasing. Although, it's still a bit limited. All the readers indicate they'd like to have more samples. If they send a sample, it arrives in Madison on Tuesday, they want the answer on Thursday, which usually is not feasible. So, again, comparing the quantities, a commercial malting bin has about a million grams in it. Our smaller scale malting boxes for the steeping and mashing have about 140-gram capacity. So, it's actually six orders of magnitude less than commercial bins. So, actually, the term micromalting is realistic. And then when it's mashed, it's about a 25-gram quantity that's used to generate the extract. I thought for a little bit about what to call it. 

I didn't know if we should call the smaller scale stuff we'd be talking about extreme malting, or I thought maybe next generation malting might be a little bit presumptuous, self-promoter that I am. But, basically, what we've done is modified the conditions so instead of using the relatively large quantity of grain that is the standard amount, we're able to reduce it by about two orders of magnitude, so we only use about two grams of the grain. Contain that inside, literally, an inch-and-a-half diameter stainless steel tea ball which goes into, is bulk up with a number of other individual samples in the Joe White run through the cycle. And we can actually generate some reasonable malt with that. Taking things down to the other end when we go to do the mashing as well. We're able to reliably mash about 180 milligrams of tissue, so that's about the equivalent of five seeds which, thinking about it, it's remarkable we're able to get reasonable numbers from that. I'll just give you a quick run through here since I see the clock is moving on and my talk is running on. But to try to malt smaller quantities, we realize that there are a number of issues with trying to malt these smaller quantities individually. When you get to a small quantity like this, surface area to volume issues become problematic, moisture, gas flow, other things become an issue. So, the small quantities of grain don't really behave as they would in a big bulk of the grain. 

So, I thought that we could perhaps try something I've seen work in solid phase peptide synthesis where you have beads contained within a mesh bag that are then pooled with a number of other treatments in one common treatment to add on additional peptide in the synthesis cycle, and then the bags are taken out and handled separately after that. So, what we've done is placed a number of the tea balls on a bed of the grain covered over with another layer of the grain so that, essentially, the tea balls are surrounded by a little under a kilogram of just a carrier malt, and run it through the full malting cycle. My mouse, there we are. So, after the steeping period, you can see the grain has actually swollen. It comes up much higher on the malting in the bins. After 24 hours we found that the moistures were drying out a little bit so we give them a quick little dunk and then continue on with the rest of the cycle through the kilning process. And, son of a gun, the stuff actually works. The numbers, well, roughly we can increase the sample throughput by about threefold. So we can either look at that as processing three times the number of samples or getting the results back to the people submitting the samples three times faster. There's really no effective increase in the staffing requirements. 

That's what I've been told, I should say. Although, we can actually get by and use fewer people in the lab to be able to get the same sort of throughput. So, the glass is half full or half empty, I guess. And as I said, the results were comparable. We're able to use some laboratory devices. This is actually a 96-well plate bead beater. It essentially takes containers that are in the footprint of a 96-well marker plate. Containers can have a number of grains of the barley plus a couple stainless steel balls, and they extract some medium in it. You put it in there and shake the dickens out of it for a couple minutes. And it actually does a remarkably good job at grinding the grain, the malt, and dispersing it, centrifuge down the solid bits, take the liquid off the top, put it into a little laboratory thermal mixer. 

It's a little shaking incubator that's about yay big. Just a little bench top size. Very convenient. You can run it through the standard mashed temperature cycle that's prescribed as the one that's mostly commonly used for quality analysis. And a lot of data points, and, again, time is fleeting, so what I'll just direct you to is the fact that on the two axes here of each of the graphs, four on this page and then the four on the next one, we compare the results from a full scale malting and malt extract mashing analysis with what we get from the tea ball malting in the small scale analysis. So, you can see in each of the cases for the malt extract, for the carbohydrate enzymes, alpha amylase, and the overall starch capacity of diastatic power, we get really quite nice correlation coefficients. The numbers trend along the same line very well. Beta glucan is another story. That's always been the problem in the lab. The numbers are fairly variable. 

That is something, I think, is not so much a technique, but I think it's a facet of the biology of the beast. Beta glucan numbers are just variable. If you look at the nitrogen parameters as well, the soluble protein, the ratio of soluble protein to total malt protein, overall malt protein and, essentially, the amino acids, the free amino nitrogen, again we get nice trend lines. The goal of this is actually to be able to take the malt and separate the real poor performers from the good performers. We're not really trying to do a fine separation of the best from the next best, or one that's 5% better than another. What a breeder's goal really is is to get rid of the crap. So you get the bad stuff, the stuff that's never going to make it to a commercial malting variety, out of the way, out of your program so you can focus on the good stuff. And the numbers we get from the small scale methodology is certainly adequate to do that. In fact, one of our collaborators, the barley breeder from Montana State University, Tom Blake, had included in a grant proposal his view of what it takes, really, to do malting quality analysis. And he called it quality analysis in three feet of bench space. 

So, basically we have the bead beater, a NanoDrop spectrometer, fluorometer, the thermomixer, an osmometer to measure the solutes in the wort, and then the centrifuge to be able to separate things out. And doing that you can basically get the most important bits of information for malting quality analysis at the first pass level. So to be able to separate out the good from the bad. So, compare the three feet of bench space here with the half of the building that we needed for the full scale analysis. So, we're actually quite heartened when Tom sent this to us indicating an interest in collaborating saying that people really are believing that there is some potential in that. And the last little bit that I'll try to squeeze in before the clock strikes eight is a comment I got, it was actually at a table dinner. It was at the Garden Expo a year ago. We were sitting around the table. I was talking with a food scientist who shall remain unnamed. And his comment was, when I was telling him what I was working on, but don't we already know everything we need to know about that? And I said, well, sort of yes and no. 

We know a lot about germination, less so about malting. Malting really is a process that involves a lot of fairly carefully controlled parameters. Things that really not, well they're engineered but they're really empirically determined. So there's not necessarily a good fundamental equation which says you need to do X or Y or Z to be able to do it, but a good maltster has the art to be able to say we need to do this to be able to get the crop to produce things in a certain way. And, actually, if you look at it, the straight germinated barley, these are three grains of a robust malting variety that was germinated just in a plastic box in an incubator in the laboratory, as compared to three grains that came from a micromalting of the same seed lot. You can see that in the micromalt you've got a few fairly small rootlets that compare dramatically to the fairly long shoot, and quite extensive roots on the incubator germinated barley. Well, I guess somebody said that's obvious enough that a principal investigator could tell the difference between those two treatments. If we actually use some of our small methodology to do malting quality analysis on these few individual brains that have been kilned and had the shoots and rootlets removed, you can see that the actual carbohydrate related parameters are fairly comparable between the malt house, the million-gram malting, between the 150-gram malting, and between our two-gram malting. But interestingly, the numbers relating to the protein mobilization are quite different. So, the numbers agree fairly well between the micromalting and the commercial malting, but to actually malt protein is quite low. 

The soluble protein, or the amount of solubles in the liquid extraction are high. And there's a fairly high fraction of the soluble to the total, indicating that there was a large conversion of the protein reserves. Which actually makes sense if you realize that the roots actually are quite rich in nitrogen. So the germinating grain puts a lot of protein into growing the roots to be able to get the seed established to get water, nutrients and so forth. But that nitrogen has to come from some place, and so when the grain puts its effort into growing roots, it mobilizes the nitrogen from the malt, surprisingly, so we have lower nitrogen there. So, what does that really mean in terms of what we can identify for targets? This is some results from a gene chip experiment. Briefly, we extracted RNA from barley that was in the commercial malting and the micromalting and in the growth chamber growing, or the little plastic box germinated plants and compared the profiles of the various transcripts we got. So, we measured from the dry seed out of steep on days one through four or five, depending on the experiment. We look at what the overall abundance of the 22,000 probes that were on the chip were. And if you compare just how things change from the dry seed to out of steep in the commercial malting, you can see the most of the change happens during that first transition as the grain is hydrated and then it goes through germination. 

The number of differences in transcript abundance from one day to the next decrease with time down to a somewhat smaller amount. If you actually compare the commercial malt versus the micromalt for probes that are actually annotated to be related to various proteinases, we can see there are really not too many differences between the commercial malt and the micromalt. The incubator actually shows relatively larger differences that actually increase with time. That's just talking about the overall numbers of probes that go up, down, or sideways. If you sort through all that and put together some filters that separate out treatments that increase or decrease by a factor of two or four or some other characteristics, you can go and identify a number of particular probes which may be interesting. The proteinase EPB is one that was of the first proteinases that was identified in germinating barley and has been long implicated in germination and malting. It actually has been shown to digest hortein, which is the primary storage protein in barley. If you look at a number of the different probes, they all sort of seem to follow the same sort of trend, either increasing with time, which is a typical hydrolytic enzyme response through germination. Some of the probes don't really change too much over time. However, interestingly, there was one that was one specific probe, EPB1. 

This is given this HB26011RAT. This one, the three different treatments, so the commercial malt house, the micromalting, and the laboratory germination were all sort of similar through day one, but beyond that, the treatments diverged so that the treatment that had different nitrogen mobilization parameters measured by the malting quality also showed relatively higher activity of this one probe for the endoproteinase. We found similar sort of differentials for several other endoproteinases here where, again, it was the lab-germinated treatment that diverged, either increasing and diverging from it or, in one case, decreasing, diverging from the expression levels in the other probes. We found similar things for one of the -- proteinases. These type of proteinases have been implicated in malting quality in some other expression profiling studies using the barley one chip. And then, similarly, some serine carboxypeptidases, just another mechanistic class of proteinases, have been implicated statistically with some malting quality. Again, I'll show that there was one of the contigs which actually showed an increase, continued to increase over the course of germination in the lab-germinated but had a different time profile in the commercial and the micromalting. So, again, that shows there's something different going on. So, again, just quickly, we have a couple different expression of different transcripts for some proteinases that seem to be different between the laboratory germinated grain that has the different phenotype and different malting quality characteristics and the commercial and the micromalting treatments. So, at this stage, we think that there's something interesting going on here, but most of these other transcripts, the peptidases, the -- proteinase, and the A494, have largely been identified through studies in the shoots from senescence or some stress treatment. 

Really very little is known about how they actually function in the germinating grain. So what the actual roles of the different proteinases are in the mobilization of the seed reserves is what we'll pay somebody salary in the future most probably. [LAUGHTER] So, just to give you a quick rundown of some of the people. Mike Davis and Scott Heisel in the American Malting Barley Association have really been strong and key supporters of the malting barley research and the variety development. The malting barley breeders we work with closely are Kevin Smith in Minnesota, Rich Horsley in North Dakota, Tom Blake in Montana, Don Overt and his replacement in Aberdeen, Idaho, for the ARS, Steven Ullrich, who retired recently, he was replaced by Kevin Murphy at Washington State, and Pat Hayes at Oregon State who's a great collaborator. We've got a bunch of people, some in the audience here. Al Buddy, who retired about a year or so ago and had, up until that time, done a real bang up job at making the malting quality analysis operation just rock solid, reliable, and really the heart of the barley variety development. And also a couple people, Lori -- and Leslie Zalapa work with me in the research lab and have done some of the small scale malting and the research work. And with that, I thank you for your attention. [APPLAUSE]