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Barley What are the basic barley types? Barley is classified primarily according to two of its characteristics—its growth habit (winter or spring) and the arrangement of the kernels on the mature barley head (two-rowed or six-rowed, Fig. 3.1). Winter barley seed is sown in the fall and harvested the following spring, whereas spring barley is planted in the spring and harvested during the following summer. While both two- and six-rowed barley can be of either the winter or spring type, nearly the entire U.S. crop is composed of spring barley. Research is being carried out at several locations into the development of winter barley varieties for the Americas.
Two-rowed barley The head of the barley plant is a spike with three florets at each node. In two-rowed barley, the central floret is fertile and the two lateral florets are sterile. Therefore, the mature head, when viewed along the axis of the spike, shows two rows of kernels on opposite sides of the spike. The main two-rowed growing states are Montana, Idaho, Wyoming, and Colorado. In Canada, the two-rowed and six-rowed barley production is spread across the provinces of Alberta, Saskatchewan, and Manitoba. The two-rowed malting barley is grown on both dryland and irrigated fields in the western United States. Partly because of the tworowed spike design, the kernels are more uniform, plump, brighter, higher in extract, and lower in protein than sixrowed barley. Six-rowed barley In six-rowed barley, all three florets at each node are fertile. When viewed along the axis of the spike, six rows of kernels are visible. The main six-rowed growing states are North Dakota, South Dakota, and Minnesota. Starch conversion enzymes such as alpha and beta amylase are generally produced in the aleurone layer of cells surrounding the endosperm. Since six-rowed barleys have smaller lateral kernels, the ratio of aleurone layer to endosperm is higher, so six-rowed malts have more enzymatic power than tworowed malts. The Midwest growing area is wetter, which means that six-rowed barleys will be more susceptible to disease and discoloration from molds and blights. In Europe, most of the malting barley is spring tworowed; however, some winter six-rowed malting barley is being grown.
How much does a bushel of barley weigh?
Fig. 3.1. Six-rowed barley head (left) and a two-rowed head (right).
A typical weight for a bushel of barley is 48 pounds. An old rule of thumb was that one bushel of barley, after malting and brewing, produced one barrel of beer (31 U.S. gallons). Although this is a good estimate for full-strength all-malt
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beers, less barley is required to produce beers containing high adjunct levels or light beers.
How can barley planting, growth, and harvest be characterized? Barley is grown across much of the United States and most of Canada. The crop is a member of the grass family as are other major world grain crops such as corn, oats, rice, rye, and wheat. The head or ear of barley can be six-rowed or two-rowed types as shown in Figure 3.1. Cultivars or varieties of each type exist that are suitable for malting and brewing Another division among various barleys is their habit of growth. Those with a spring growth habit are planted in the spring and harvested in the mid to late summer. Winter barleys are seeded in the fall and harvested in the early to mid summer. Winter barleys typically exhibit higher yield potentials in areas where both types can be grown. There are efforts to develop winter barleys in the western United States with excellent malting and brewing quality, but currently there are no winter malting varieties grown in North America. The growing of barley varies from region to region depending on climate, soil type, and growing season. Rather than trying to detail a set of specific farming practices for each region, it may be more instructive to discuss some of the production trends. Conservation tillage practices are replacing deep tillage to reduce soil erosion, conserve soil moisture, and lessen fuel consumption. A disadvantage of conservation tillage practices is that the increased plant residues left on the soil surface can lead to the spread of disease. Reduced yields, kernel plumpness, and excessive staining can result from the presence of many diseases. A fungal disease called Fusarium head blight (FHB) or scab has appeared periodically and, among other factors, pushed malting barley production west in North America. This disease is worse when following corn, wheat, or barley crops and when the previous crop’s residue is not worked well into the soil. At high levels of contamination diseased kernels can cause beer to gush. The practice of summer fallow, where no crop is planted the year prior to seeding a barley crop, is becoming less common. Summer fallow rotations were practiced in the most arid regions of the plains and prairies as a way to maximize soil moisture and yield of the eventual crop. This trend has led to increased acres available for planting in any given year but also makes crops more vulnerable to drought conditions that can reduce kernel plumpness and increase total protein content. Typically, barley is one of the earliest crops to be planted. It germinates at relatively low soil temperatures and is fairly tolerant of spring frosts. It does not tolerate
hot temperatures during flowering and the grain fill period, especially in regions where nighttime temperatures do not cool down. Barley is a relatively low-input crop. The use of fertilizer, herbicides, and fungicides is typically minimal. Adding less nitrogen fertilizer satisfies the brewer’s desire for moderate to low grain protein but can reduce the overall yield of the crop. The need for herbicides is reduced or eliminated when barley gets off to an early start, shades the ground quickly, and competes well with weeds. The advantage barley has in this area could be challenged by other glyphosate-resistant crops since this class of herbicide has a broad spectrum and is relatively inexpensive. Fungicides are applied to barley, but usually only after growers have determined that the yield or quality losses in a specific field warrant treatment. A germinating barley seed will send up a main stem and several side shoots or tillers. Each main stem and tiller will develop a head. Two-rowed barleys tend to have more tillers per planted seed and six-rowed types are apt to have more seeds per head. The plumpness of two-rowed kernels tends to be greater than that of six-rowed barleys. Malting barley can be grown under irrigation or as a dryland (natural rainfall) crop. Nearly all irrigated malting barley is grown under contract with malting or brewing companies. The irrigated crop is generally grown in regions with very low rainfall. When sufficient irrigation water is available, these regions can produce high yields of good quality malting barley on a routine basis. Approximately one month after the barley heads emerge from the plant, the crop is mature and ready for harvest. In more arid regions, growers wait until the kernel moisture reaches 10–12%. The barley is then direct combined (Fig. 3.2) and trucked to on-farm storage, an elevator, or a processor. In more humid regions, barley is commonly cut into swaths for windrows (Fig. 3.3). Swathing promotes final drying of the grain and reduces any weeds that might cause significant problems during combining and possibly result in more foreign matter (or dockage) in the grain. Barley must be carefully handled during harvest, transportation, storage, and processing to avoid peeling or skinning of the grain. Losing portions of husk can lead to problems in malting and brewing. The husk protects the growing acrospire (shoot) and embryo during malting. If unprotected, an acrospire or whole embryo can be separated from the kernel, killing it and leaving an incompletely modified kernel. Skinned barley is also more prone to infection by fungi and bacteria during storage or malting. Malting barley with very low kernel moisture prior to harvest is more prone to skinning. Certain varieties also can exhibit more peeling problems. Historically, the United States has been self-sufficient in malting barley production. Attracted by premiums for malting barley, growers in marginal regions have often
Brewing Chemistry and Technology in the Americas
Fig. 3.2. Direct combining of barley. (USDA photo)
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The Canadian barley crop is large, with more acreage planted than in the United States. The varieties grown in Canada are somewhat different, with more acreage devoted to two-rowed types. Much of the Canadian crop is exported. The harvest of malting barley in Canada may be as large as 300 mbu. As in the United States, only the finest quality is selected for malting, so the amount of barley sent to malting is less than the total harvest of malting barley varieties. North American malting barley producers decide which varieties to grow prior to planting. Decisions are made based on how certain varieties have yielded and sold in their area in the past, what varieties are being contracted, and what signals the malting and brewing industries are giving. In the United States, a list of recommended barley varieties is compiled by the American Malting Barley Association, Inc. (AMBA). AMBA’s list and a couple of varieties not on this list can be found in Table 3.1. Descriptions of these varieties can be found in the publication titled Know Your Malting Barley Varieties. (An online version of this can be found at http://www.ambainc.org/pub/kymbv/2002_ KYMBV.htm.) In Canada, recommendations for planting are made by the Canadian Malting Barley Technical Centre (CMBTC). The varieties recommended for 2006–2007 are shown in Figure 3.4. (An online version of the list can be obtained at http://www.cmbtc.com.)
Fig. 3.3. Swathing of a barley field.
seeded malting varieties in the hopes that they will produce quality malting barley. This is particularly true in the states of Minnesota, North Dakota, and South Dakota, where adapted feed varieties do not always have sufficient yield advantages to warrant their planting. Maltsters and brewers select the very best of the crop for their needs and the bulk of the remainder is used as livestock feed. The development of shorter season corn and soybean varieties, changes in the U.S. farm program, and disease problems have lead to a decline in acreage in the last 10–15 years. Total barley production has fallen from an average of 406 million bushels (mbu) in 1988–1992 to an average of 286 mbu in 1998–2002. There are no official estimates of what percentage of production is malting barley, but a gross estimate can be made based on the area planted to malting types and average state yields. An estimate derived from these numbers from 1998 to 2002 gives an average production of malting barley of 157 mbu. It must be noted that quality problems (plumpness, staining, protein, etc.) would mean that considerably less than this would be available for malting and brewing. To offset these quality losses, significant amounts of barley have been imported from Canada in the past decade to satisfy U.S. malting needs.
What are the current barley disease and pest concerns and control measures? In barley, production losses due to diseases and pests occur each year. The extent to which abiotic and biotic stresses affect production varies greatly from one year to the next. Weather conditions during the growing season play an important role in both the incidence and severity of diseases as well as pests. In Plant Diseases Development and Management, a North Dakota State University Extension Service publication, Dr. Marcia McMullen and Dr. Art Lamey provide an excellent introduction to how biotic and abiotic stresses can affect production.
Table 3.1. Common Malting Barley Varieties in the United States Two-Rowed B1202 Conlon Garnet Harrington Merit Moravian 14 Moravian 37
Six-Rowed Drummond Excel Lacey Legacy Morex Robust Stander
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Fig. 3.4. Recommended malting barley varieties, 2006–2007. (Reproduced, with permission, from the Canadian Malting Barley Technical Centre)
Abiotic stresses Common abiotic stresses in barley production are heat, moisture (both deficient and excess), salinity, and nitrogen deficiency. Nutrient deficiencies, like a nitrogen deficiency, can generally be prevented or remedied with soil sampling and adequate fertilization. Options to limit the effects of the other abiotic stresses are limited. Early seeding is paramount to avoid heat stress. Infectious (biotic) stresses Biotic stresses can be subdivided into fungal, bacterial, and viral diseases and insect infestations. Fungal diseases are most common in barley and in order of economic importance are 1) FHB, 2) net and spot blotch, 3) kernel discoloration, 4) common root rot, 5) stem and stripe rust, and 6) powdery mildew. Barley yellow dwarf virus affects production occasionally as do Xanthomonas translucens, the cause of bacterial stripe and black chaff, and Pseudomonas syringae, the cause of bacterial leaf blight. Several cereal aphids, as well as grasshoppers, armyworms, and barley thrips, are occasional pests. No control options are available for the bacterial diseases or barley yellow dwarf virus. Control strategies using insecticides and fungicides are available for the insect pests and a number of the fungal pathogens. The impact of fungal diseases can generally be limited with an integrated approach of host resistance, fungicides, and cultural practices like crop rotation. Stem rust has not been a serious threat to barley production during the last several
decades. Powdery mildew is readily controlled with fungicides, should an outbreak occur, and plant breeders have selected cultivars that have less kernel discoloration. FHB has been very difficult to control and has a huge impact on production. FHB is caused by the fungus Fusarium graminearum. Since 1993, producers have experienced moderate to severe epidemics in the Upper Midwest region. One of the most insidious aspects about this disease is that F. graminearum can produce a mycotoxin called deoxynivalenol, which can contaminate the grain and ultimately the finished product (beer). Producers are concerned about this disease because it can reduce both yield and test weight. Maltsters are concerned about this disease because it can reduce germinative energy, malt extract, and kernel brightness. FHB-infected barley malt is of paramount concern to brewers. First, brewers do not want to use any malt that might be contaminated with mycotoxins because of possible carryover into the beer. Second, FHB-infected malt can lead to off-flavors in beer. Third, beer made from malt with FHB has a higher propensity to gush. Management of FHB in barley is difficult and requires an integrated approach. Although there are fungicides that have good activity against FHB, they have not always been consistent in reducing mycotoxins to levels that are acceptable to the brewing industry. Additionally, resistant cultivars are not currently available. Thus, this disease is best managed by reducing crop residue that harbors the fungal pathogen in the field, rotating barley with crops
Brewing Chemistry and Technology in the Americas that are not congenial hosts to the FHB pathogen, and applying fungicides when economically feasible.
What are monoclonal antibodies and ELISA tests, and how is this technology useful in monitoring FHB? Agricultural scientists have used antibodies for immunodiagnostic analyses since the 1920s. They have gained acceptance by the scientific community because they are specific, sensitive, and versatile for qualifying or quantifying organisms or substances in aqueous solutions. They have distinct advantages over standard chemical analytical techniques, bioassays, and molecular techniques because they are faster, simpler to use, less expensive, and environmentally benign. Immunogens are chemical compounds that are foreign to vertebrate animals and, when used in a vaccine, induce biosynthesis of antibodies. Not all compounds elicit an immune response, but those that do are usually proteins, and to a lesser extent, lipids and complex carbohydrates. For the purpose of this discussion, proteins will be considered as the model from which antibody technology can be described with special reference to detection of F. graminearum.
Polyclonal and monoclonal antibodies Antibodies are produced by immune systems of vertebrate animals as part of their defense strategy against invasion by disease-causing organisms. They are the culmination of a complex series of reactions involving numerous cell and tissue types that have elaborate signaling systems designed to turn on or turn off various components of immune function within the body. While this process is complex, a rudimentary knowledge of the immune system is needed to appreciate exactly what a monoclonal antibody is. When the body is invaded by a foreign organism (such as a bacterium, protein, or fungus) the body immediately recognizes the organism as a threat and begins processing proteins on the surface of the invading organism. Some of the surface proteins have vital roles, such as enzyme production, tissue recognition, or tissue attachment. Thus, to render the organism inactive, the surface proteins must somehow be inactivated by the immune system. To do so, specialized “macrophage” cells in the host animal sequester surface proteins from the invading organism, internalize the proteins, and process the proteins into oligopeptide units via proteases and peptidases. The macrophage cell has the ability to move the oligopeptides back to the surface of the macrophage cell, with the oligopeptides emanating from the cell surface much like pinheads from a pincushion. Thus, all the protein subunits are presented to the macrophage cell surface. A second set of cells, called B cells, receives signals to produce antibodies to the surface protein subunits from
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the invading organism, rendering the organism inactive. Bone marrow and splenic tissues in the body produce B cells. However, any given B cell does not produce a full complement of antibodies to all oligopeptides at the macrophage surface. Rather, individual B cells produce antibodies to a single oligopeptide subunit of a protein. Numerous B cells are thus required to produce antibodies to the numerous oligopeptides from an individual protein that has been processed by the macrophage cell. But, each B cell is capable of replicating itself in a clonal fashion so that all subsequent generations of B cells from any individual parent B cell will produce only the antibodies produced by the parent B cell. There are nearly infinite sets of clonal B cells producing antibodies to numerous fractions of any number of surface proteins from organisms that invade the body. The term “polyclonal” antibody is used to describe this type of immune reaction because numerous (poly) B cells are cloning themselves and generating antibodies to the protein subunits from the invading organisms (or vaccine). Monoclonal antibodies, on the other hand, are antibodies produced by a single B cell line. A distinguishing feature about monoclonal antibodies is that they can be produced under controlled laboratory conditions. The B cells cannot be cultured in vitro, so they are fused with a myeloma cell to produce a hybrid cell. This hybrid cell has the characteristics of the myeloma cell but retains the ability to produce the antibodies of the B cell to which the myeloma cell was fused. These cells require specialized media and environmental conditions to be cultured. To find cell lines that produce useful antibodies, an animal (usually a mouse) is vaccinated with a protein, and the spleen is harvested and homogenized after a suitable immune reaction has been obtained. The splenic cells are then fused with myeloma cells. The resulting hybrid cell lines can then be screened for their ability to produce useful antibodies. Those cell lines producing desirable antibodies are saved and the remaining cell lines terminated and discarded. An individual cell line will produce only one antibody to a single oligopeptide from a particular protein of interest. Once the cell line producing the desired antibody is found, it will produce an infinite amount of antibody so long as the cell line is viable. The quality of the antibody product is also consistent within the cell line. The hybrid cell lines can be put into cryogenic storage, retrieved, and re-cultured for future use, virtually guaranteeing an endless supply of antibody product for diagnostic use. The production of monoclonal antibodies is depicted in Figure 3.5.
Polyclonal vs. monoclonal antibodies for diagnostic purposes Antibodies, both monoclonal and polyclonal, are very useful for qualitative and quantitative diagnostic procedures. Polyclonal antibodies can be harvested by collecting blood and separating the serum. The serum containing
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Fig. 3.5. Development of monoclonal antibodies. A mouse is vaccinated with a protein antigen (a) and the spleen harvested (b) once an immune response is detected. The spleen is homogenized (c); myeloma cells (d) are fused with spleen cells (e); the fused cells are cloned and cell lines screened for antibodies (f).
antibodies can be used directly for immunochemical assays. Polyclonal antibodies are generally elicited in laboratory animals such as rabbits or small farm animals. They have very high affinity to their target compounds and can be configured to provide a more rapid analysis (from seconds to minutes) than monoclonal antibodies. However, the use of polyclonal antibodies carries other limitations associated with the cost of maintaining blood donor animals and the need to revaccinate whenever serum is to be harvested. Furthermore, immune responses vary whenever booster injections are administered, so serum needs to be recharacterized prior to use. The limited quantity of serum that can be obtained from small animals limits their use to narrow applications where small quantities of antibodies are needed. Polyclonal antibodies may also cross-react with proteins if the protein subunits of the target are similar to subunits of other (spurious) proteins. Monoclonal antibodies, on the other hand, are easily prepared by precipitation from the growth media for the cell lines and suspended in standard solutions whereby the antibodies are stable for years. It is easy to obtain additional antibody since they are produced by cell lines and are amenable to broad-scale applications where numerous analyses are needed. Since cell lines produce the antibodies, there is no donor animal or need for revaccination to harvest additional antibody, and acquisition of additional antibody is more cost effective than for polyclonal antibodies. A major benefit of monoclonal antibodies is their specificity. Because they are specific to only one oligopeptide of the target protein, they are much less likely to cross-react with spurious proteins. Analytical procedures using monoclonal antibodies generally require more time (minutes to hours) than those of polyclonal antibodies, an obvious limitation to their utility.
Analytical formats for antibodies Two immunochemical analytical formats are relevant for diagnostics of F. graminearum in barley. Both formats re-
quire a solid support phase, but it is the type of support phase that differentiates the format. Immunoblotting is conducted on a white opaque solid support membrane while enzyme-linked immunosorbent assay (ELISA) is conducted on a transparent solid support membrane. Both require extraction, transfer, and adherence of the target protein to the solid support (Fig. 3.6). The support with the protein is then exposed to antibodies that have chromogens conjugated to them. The antibodies attach to the target proteins, excess antibody is washed from the support, and a chromophore is added to give a color reaction. Immunoblots are generally used for qualitative analyses but are adaptable to crude quantitative analyses as well. The barley samples tested by immunoblot in Figure 3.7 contained varying amounts of F. graminearum, and thus gave different intensities of color reaction. Immunoblot formats called dipstick analyses (similar to a home pregnancy test) can easily be developed for field and grain elevator assessment of Fusarium in barley. The immunoblot gives an assessment of whether the fungus is present and can be used to identify grain lots that could be problematic during malting. The gas or liq-
Fig. 3.6. Immunoanalysis. Target and other proteins from a sample attach to a solid support; the monoclonal antibody (mAb) is specific to the target. A chromogenic agent is conjugated to the antibody and develops color when the chromophore is added.
Fig. 3.7. Immunoblot. The top third shows seeds with an average of 2.2 µg of Fusarium per g of seed; the middle shows seeds that were not infected with Fusarium (and therefore no color reaction); and the bottom third shows seed with an average of 12.5 µg of Fusarium per g of seed.
Brewing Chemistry and Technology in the Americas uid chromatographic test for deoxynivalenol assesses the presence of the toxin but not the organism that produces the toxin. Unfortunately the organism can be present without toxins currently being present, thus yielding a false sense of security. The immunoblot analysis is likely to be superior as a forensic Fusarium analysis. The ELISA format can also be used for quantitative and qualitative analyses. The ELISA format uses a plastic plate with multiple reaction wells as a reaction vessel in which up to 96 assays can be performed at once (Fig. 3.8). Quantitative assays are conducted with standards of known concentrations in reaction wells of the ELISA plate. These wells develop color signals of varying intensity that are detected with a modified spectrophotometer called a “microplate reader.” Regression analysis is conducted to establish a color signal to protein quantity relationship, and the color reactions in the remaining wells (samples) are quantified accordingly. Cooperation with barley researchers at North Dakota State University, the University of Minnesota, and Busch Ag Resources has permitted comparison of visual scoring for disease, the ELISA analysis for F. graminearum, and the gas chromatography/electron capture (GC/EC) analysis for deoxynivalenol in controlled field plots. The experiment consisted of a set of barley varieties and breeding lines that were uniformly tested in replicated trials and evaluated for disease at four field locations. Data were collected and statistical analyses and coefficients of variation compared to determine which test provided the most consistent results. The mean coefficients of variation were 51.2, 24.3, and 53.5 for disease scoring, ELISA, and GC/EC deoxynivalenol analysis, respectively. Thus, the ELISA system was a superior disease diagnostic method. One distinct advantage of immunochemistry over gas or liquid chromatography techniques is the simplicity of
Fig. 3.8. ELISA microplate. Color intensity of the reactions in the microtiter plate can be used to accurately measure quantity of the disease (yellow indicates no Fusarium, orange indicates moderate infection, while red indicates heavy infection of Fusarium).
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sample preparation. Testing protocols use whole grain extracted by shaking in aqueous solutions. There are no organic solvents used in the process, either for extraction or in analysis. So, there are no flammable liquids to contend with and no hazardous waste. The seed remains viable after extraction (a trait of great significance to plant breeders). The simplicity of the system makes it amenable to use where large volumes of samples need to be processed. In one experiment, 1,000 samples were shipped to a laboratory via overnight express, cataloged, processed, analyzed, and returned via overnight express to the client, all within five working days. In summary, immunochemical methods use the immune system to create antibodies that are highly specific to a target organism or chemical compound. The antibody developed to F. graminearum provides a more accurate assessment of disease than methods currently accepted as the norm. Extraction and preparation of the samples are simple, and the analysis is adaptable to field conditions.
How do the environmental factors of irrigation, fertilizer, soil, and growing conditions affect barley? Growth stages Before we can understand the effects of different production practices and environmental stresses on the performance and quality of barley, we need to know about the different barley growth stages and yield components. Different scales are available for describing barley growth, including the Feekes, Haun, and Zadoks growth stage scales. In simple terms, barley growth can be divided into four main stages. The first is the seedling stage, from seedling emergence until jointing. The initiation of jointing can be determined by feeling the lowest leaf node on the plant above the ground to see if it is solid. If the node is solid, the growing point of the plant (i.e., the developing spike) is above the ground, and the plant is susceptible to different stresses that may affect yield and grain quality. The next major growth stage is the boot stage. This occurs when the spike of the plant is preparing to emerge from the flag leaf sheath. At the late boot stage, self-pollination of the florets (immature kernels) is occurring, and the leaf sheath of the plant splits open because of the growing spike. The third main growth stage is heading. At this stage the spike of the plant is emerging from the leaf sheath and fertilization is nearly complete. The final growth stage is grain fill. This stage can be further subdivided into the milk, dough, and ripe stages. The milk and dough stages can be identified by squeezing the developing kernel between your thumb and finger. In the milk stage the endosperm of the kernel is mostly liquid; thus, the liquid exuded from the kernel will be milky in color. During this stage nutrients are being translocated to the developing kernels. During the dough stage the kernel is beginning to dry down; thus, when the