/REVIEW • © 1995 Nature Publishing Group http://www.nature.

com/naturebiotechnology

What's Brewing in Barley Biotechnology?

David McElroy* and Jake Jacobsen 1
University of California-Berkeley/USDA-ARS Plant Gene Expression Center, 800 Buchanan Street, Albany, CA 94710. 1CSIRO Division of Plant
Industry, GPO Box 1600, Canberra, ACT 2601, Australia, and Co-operative Research Centre for Plant Science, GPO Box 475, Canberra, ACT
2601 Australia. *Corresponding author (e-mail: mcelroy@nature.berkeley.edu).

The primary aim of barley (Hordeum vulgare) improvement for use in malting and brewing is to expand
the germplasm available to breeders. The development of a transformation system for barley, along with
an improved understanding of the biology underlying malting quality, has made it possible to consider
improving barley by making planned genetic changes. In this review, we discuss the opportunities for
biotechnology as they apply to the use of barley in malting and brewing.
t has often been said that biotechnology is ·as old as plasts5 and the first report of fertile transgenic plants regenerated
civilization itself. This cliche holds well for the malting from transformed protoplasts6 • Although the regeneration of
and brewing industries where the microbial infection of transgenic maize7 •8 and wheat9 plants from protoplasts was
soaked barley to produce an alcoholic beverage first accomplished, the regeneration of fertile transgenic plants from
occurred at least 5,000 years ago'. Malting and brewing cereal protoplasts was not a trivial extension of the technology
have since evolved into multi-million dollar industries. developed for rice' 0 • This situation also held for barley where
For example, in the USA over the period 1983-1992, the protoplasts derived from various tissues were shown to tran-
annual added-value on barley production (valued at $1 billion as siently express exogenous DNA' 1- 15 , but fertile transgenic barley
a raw commodity) was estimated to be $3 billion. Over this plants were never recovered.
period the US brewing industry generated $167 billion in total The use of microparticle bombardment' 6 of embryogenic
business activity, its 2.7 million employees earned $51 billion in tissue culture material, with subsequent selection and regenera-
wages, and taxes on malt beverages generated $13.6 billion in tion of transformed plants, overcame the problems encountered
federal, state and local revenues 2 • with the protoplast approach. Using this technology, fertile
The goals of barley improvement, as they apply to malting transgenic plants have been obtained from rice 17 · 18 , maize 09- 21 ,
and brewing, reflect the need to minimize agronomic problems oats22 , and wheat2 3•24 • Recently, a similar approach using imma-
in the growing of barley, to maximize yield, and to maintain ture zygotic embryos, young callus and microspore-derived
efficient, trouble-free, and cost-effective production of high embryos was used to generate large numbers of independently
quality malt and beer. Traditional plant breeding methods have . transformed, self-fertile barley plants25 •
made considerable progress toward these goals, and they will Although a routine transformation method· for barley has
remain at the center of barley crop improvement. However, the been developed, a number of significant challenges remain. The
traditional process of barley improvement is protracted, labor transformation technology needs to be extended to elite malting
intensive, and imprecise, relying on the selection of chance barley cultivars. In addition, efforts must be made to ensure
combinations of genes drawn from a finite gene pool. In some the heritability and fidelity of foreign gene expression under
cases, the biochemical processes and molecular biology under- diverse field conditions26 • Much work also remains to isolate
lying yield, disease resistance, and malting quality characteris- those regulatory sequences (promoters, etc) that will control the
tics are beginning to be understood. This, coupled to recent correct temporal (e.g. germination-specific) and/or spatial (e.g.
developments in barley transformation, has raised the possibility aleurone-specific, endosperm-specific) expression of intro-
of making planned genetic changes to modify these processes, duced malting quality traits in developing seeds'. Finally, anther
thus expanding the germplasm available to barley breeding culture (for double haploid production) and transformation
programs. technologies will need to be integrated in order to speed up
barley breeding programs, which currently take 10-15 years to
Barley Transformation complete.
Although it has been over ten years since the genetic engi- While much remains to be done in the area of barley tissue
neering of dicot plants was first accomplished, cereals have culture, the development of a routine method for barley transfor-
lagged behind. This is due in large part to the fact that they did mation will allow an evaluation of those traits that might be
not readily yield to the Agrobacterium-based transformation genetically manipulated to improve barley quality for malting
methods employed for dicots 3• Therefore, transgenic cereal and brewing.
technology initially focused on direct gene transfer to isolated,
regenerable protoplasts•. Rice led the way in cereal transforma- Barley Malting and Brewing
tion, with both the first report of plant regeneration from proto- In order to fully appreciate the industrial opportunities of

BIO/TECHNOLOGY VOL. 13 MARCH 1995 245

 MALTING • © 1995 Nature Publishing Group http://www.nature.com/naturebiotechnology

Steeping-initiation of cell metabolism and

induction of endosperm hydration
OPPORTUNITIES FOR BIOTECHNOLOGY
Modifying seed metabolism to ensure a rapid loss of dormancy prior to malting

Germination-growth phase following Developing genetic "switches" to ensure uniform, rapid, and complete germination in the malt
house
steeping to produce "malt"

Kilning-interruption of growth and metabo-

Ensuring retention of high levels of (1-3, 1-4)-13-glucanase, diastatic, protease, and other malt
lism, development of malt color and flavor, enzymes
and reduction of malt moisture

Malt Aging-slow diffusive balancing of ker-

nel moisture

BREWING
Mashing-conversion of malt components
Modifying seed starch structure and composition to increase starch deposition and maximize
and adjunct materials into a fermentable sub- starch hydrolysis
strate. The "mash" is carried through a se-
Increasing diastatic activity (a-amylase, 13-amylase, a-glucosidase, and limit dextrinase) to
ries of controlled temperature rises in order guarantee adequate endosperm conversion
to promote starch gelatinization and hydroly-
Developing novel market opportunities by modifying seed enzymology, e.g., introducing
sis of starch to sugars and proteins to amino glucoamylase genes for increased dextrin conversion and reduced beer calorie content
acids by malt enzymes

Lautering and Wort Boiling-settlement Reducing wort viscosity and improving beer filtration by lowering 13-glucan content through the
and filtration of the mash through its own in~ germination-specific over-expression of (thermostable) 13-glucanase genes
soluble components. The emerging "wort" is Utilization of antisense or co-expression technologies to lower the potential for the formation of
then boiled (with hops) to: extract hop com- "off" flavors from dimethyl sulphide and the products of lipoxygenase action
ponents; improve flavor; inactivate any re-
maining enzymes; and sterilize prior to fer-
mentation

Fermentation-consumption of fermentable Increasing glucose availability (and thus wort fermentability) by the genetic modification of limit
sugars by yeast resulting in the production dextrinase expression
of alcohol

Aging-completion of fermentation and fla- Developing proanthocyanidin-free barley to reduce haze-formation

vor maturation
FIGURE 1. Opportunities for biotechnology in barley malting and brewing.

barley genetic engineering, we will give an overview of the reduce malt moisture for subsequent storage. These objectives
processes involved in malting and brewing27 • These are sum- are achieved by exposing the malt to hot dry air in a malt kiln.
marised in Figure 1. The kilning process is regulated in order to reduce kernel mois-
Malting. The aims of malting are: the hydration and solubili- ture to 3-5 % while at the same time ensuring the survival of the
zation of seed endosperm cell wall materials; the moderate hydrolytic enzymes that will be required in the brewing process.
solubilization of protein matrices; the production of malting It is during kilning that much of the color and flavor of the malt is
enzymes, e.g. a-amylase, ,6-glucanase; and the limited enzy- developed. Malt Aging. Aging allows for the slow diffusive
matic breakdown of endosperm starch granules. The malting balancing of moisture within the kernel prior to milling.
process itself involves germination, growth interruption, and Brewing. Mashing. During mashing malt components (and
malt aging. Steeping. Barley grain is water-steeped (1-2 days, any adjunct material) are converted into a fermentable substrate
10-20°C) to remove CO2 , replace 0 2 , and dissipate heat. Steep- that includes sugars, amino acids, nucleic acids, vitamins and
ing induces cell elongation and respiration in the embryo, stimu- minerals . Adjunct is any starch-rich material that is used as !l
lates embryo secretory activity, activates enzymes., and initiates source of fermentable sugars , e.g. com or rice kernel frag-
endosperm hydration. During the steeping process kernel mois- ments . The "mash" (malt in warm water) is carried through a
ture content increases from 10-15% to 42-46%, with grain series of controlled temperature rises and holds (40-75°C) in
volume increasing by approximately 25 % . Botanically defined order to gelatinize and solubilize the seed starch and to solubi-
germination is initiated at approximately 30% moisture content. lize carbohydrate-degrading enzymes. The mashing process
Germination. Germination is the controlled growth phase (3-6 provides time for a-amylase to liquefy the starch and for pro-
days) after steeping and before kiln drying. The germinated teolytic enzymes to release free amino &cids that will serve as
grains are referred to as " green malt". Germination is con- the sole nitrogen source for yeast during subsequent fermenta-
trolled using cooled and humidified air to remove heat and tion. The mash is raised to 70°C for the production of ferment-
improve gas exchange. Kilning. The objects of kilning are to able sugars by a-amylase activity and is finally raised to a
arrest seedling growth, interrupt internal modification, and temperature of75°C to complete the breakdown of starch deriv-

246 BIO/TECHNOLOGY VOL. 13 MARCH 1995

 • © 1995 Nature Publishing Group http://www.nature.com/naturebiotechnology

atives to short-chain oligosaccharides. Mashing can involve the

addition of microbial enzymes (e.g. amyloglucosidase) to con-
vert all starch to fermentable sugars, as occurs in the production
of some low calorie beers. Lautering and Wort Boiling. The
sis is likely to be a function of the level of (1-3,1-4)-,B-glucanase
produced by the aleurone, and how much of the enzyme survives
high temperature kilning and mashing. Doubling the amount of
,B-glucanase activity is likely to ensure sufficient ,B-glucan
mash is allowed ·to settle and is then filtered through its own hydrolysis and this could be achieved either by increasing the
insoluble components (mainly husk and aleurone) in a process amount of enzyme synthesized or by changing the heat stability
called lautering. After the addition of hops, the emerging filtrate of ,B-glucanase33 ·34 • The former goal might be realized by intro-
is boiled to: sterilize prior to fermentation; improve color, flavor ducing genes with enhanced expression characteristics, a strat-
and odor, extract hop components; inactivate remaining egy which may be subject to co-suppression effects26 • The latter
enzymes; reduce volume; and coagulate polymers for their goal could be accomplished by using native barley genes engi-
subsequent removal. The fermenqible extract that emerges from neered for greater heat-stability34 or by introducing heat-stable
this process is called the "wort". Fermentation. During fer- ,B-glucanase genes from fungi33 or bacteria35 . In selecting micro-
mentation (3-9 days, 8-18°C) fermentable sugars are consumed bial genes for use, it is important to take into account not only
by the yeast resulting in the production of alcohol. Aging. heat-stability but also other characteristics such as pH optima,
During aging (also called lagering) fermentable sugar consump- substrate specificity and hydrolysis pattern.
tion is completed and flavors are allowed .to mature. The aged Proanthocyanidins. Non-biological haze formation in wort
product may then be filtered and pasteurized, or cold filtered . and beer is caused mainly by the formation of colloidal precipi-
tates between the polyhydroxylated proanthocyanidins (PA) and
Opportunities for Biotechnology in Malting protein36 and most (80 %) of the PA originates from the barley.
Barley Production, Malting and Brewing Barley PAs are polymers of (+)-catechin and ( +)-gallocatechin,
The definition of malting quality provides a useful frame- products of the flavonoid pathway that accumulate in the mater-
work within which to examine the prospects for biotechnology nally derived seed coat (testa) of developing barley grains.
in malting and brewing. Figure 1 gives an overview of some These PA polymers remain in the testa after completion of the
opportunities for applying genetic engineering to the improve- malting process and are extracted into the wort during mash-
ment of barley quality for malting and brewing. Although these ing36. Approaches to preventing haze formation include the
opportunities may not rank equally in importance throughout the addition of hydrolytic enzymes or chemicals to break down the
world, the general themes from country to country are similar. protein or cause protein precipitation and/or the reduction of PA
Potential targets for genetic engineering studies include: manip- in wort by selective adsorption. These processes are both unde-
ulating malting enzymes; altering endosperm protein content, sirable and expensive. Because most of _the PA in beer comes
which is inversely correlated with malting quality28•29 ; improv- from barley, one approach to alleviating the problem has been to
ing yield, disease and pest resistance; controlling preharvest genetically block the production of PA in the grain37. A number
sprouting; modifying dormancy; and producing rapidly germi- of barley mutants have been identified in which synthesis of
nating cultivars. Our knowledge of the biology underlying many anthocyanin or PA (or both) are impaired. The first mutations
of these processes is comparatively poor and we have chosen to obtained acted early in the PA biosynthetic pathway, and many
focus on certain goals for scientific and commercial reasons, downstream compounds, including anthocyanins as well as PA,
even if, in many cases, this rationale is based on correlative data. were not produced. However, although haze stability and beer
Dormancy and germination. Dormancy should be ade- quality were good, the mutations were associated with reduced
quate for preventing pre-harvest sprouting and it should decay yield and increased disease susceptibility38 • More recently,
quickly after harvest. Dormancy is affected by environmental mutations · affecting steps later in the PA biosynthetic pathway
factors (for example temperature and day length during grain have been identified, and in such lines only PA production is
formation) and consequently the extent of dormancy is variable. impaired. These new lines are currently being used to breed PA-
Dormancy can be broken by heating the grain for several days free malting barleys, and the first cultivar, Carninant, has been
but this is costly. The development of a way in which dormancy recommended for commercial use in Denmark. In the future, it
would be broken rapidly by using a " molecular switch" induced may be possible to control PA production in transgenic barley
during steeping may be useful. Uniform and complete breakage using anti-sense or co-suppression technology.
of dormancy would give the potential for rapid and uniform Off-flavors. Off-flavors in beer can be caused by excessive
germination. amounts of compounds like dimethyl sulphide (DMS) and trans-
Grain composition. ,B-Glucan, (1-3,1-4)-,B-Glucanase and 2-nonenal which are produced during brewing. DMS is formed
Wort Viscosity: ,B-Glucan is the major constituent of the cell primarily from S-methylmethionine (SMM) at high tempera-
walls of the starchy endosperm of barley. During malting the ,B- tures, such as occurs during kilning and wort boilint9 •40 • It may
glucan polymer is broken down by ( 1-3 ,1-4)-,B-glucanase synthe- be possible to regulate the amount of SMM production in germi-
sized in and secreted from the aleurone cells30. The cell wall nating grain using anti-sense or co-suppression technology.
,B-glucan molecules are very large (molecular weights from SMM is an essential component of metabolism (for example in
200,000- 300,0003'), water soluble, and produce viscous worts pectin formation), however, and large reductions in it synthesis
if not sufficiently reduced in size by hydrolysis. This causes slow may be harmful to barley growth and development. The lipoxy-
filtration of wort and beer, as well as the possibility of glucan genase (WX) pathway and the enzymes responsible for trans-
haze (precipitate) developing in the finished product. Selection 2-nonenal synthesis in barley have been explored''-43. Hydro-
for low ,B-glucan barleys is a common breeding aim, but clearly peroxidation of linolenic and linoleic acids by lipoxygenase
this can only proceed to a limited extent since glucan is an gives rise to 9-hydroperoxide, which is further metabolized to
essential cell wall component of the starchy endosperm. There is give aldehydes and alcohols, that confer off-flavors to beer.
also genetic variability in the size of the ,B-glucan molecules in Lipoxygenase is present in embryos in two forms. WX I is
barley32 . Although selection for small molecular weight ,B-glu- stable in the dry grain and survives kilning to cause off-flavors
can seems worth considering, reliable methods for screening during brewing. WX 2 is not stable. It would be worthwhile to
large numbers of samples for this trait have not been developed. use anti-sense or co-suppression to reduce WXl activity to see
Alternatively, there are several possibilities for increasing ,B- if this would lessen the off-flavor problem in beer production.
glucan hydrolysis using genetic engineering. ,B-Glucan hydroly- Enzyme content. Diastatic activity. A major process dur-

BIO/TECHNOLOGY VOL. 13 MARCH 1995 247

 • © 1995 Nature Publishing Group http://www.nature.com/naturebiotechnology

ing malting and mashing is the conversion of starch to ferment-

able sugars, mainly glucose and maltose. This is accomplished
by a group of enzymes consisting of a-amylase, /3-amylase, a-
glucosidase and limit dextrinase. These enzymes are known
genotypes with improved malting characteristics. The full real-
ization of this technology faces many obstacles. Since barley
represents a minor cereal crop in the agricultural economy of
most developed countries, there has been a relative lack of
collectively as "diastase" and their performance is known as public research investment into the study of its basic biology and
"diastatic activity". "Extract" is a major quality parameter agronomy. Only with a fuller understanding of the biology
representing the proportion of the grain solubilized in the wort. underlying quality traits, however, will the malting and brewing
Because starch constitutes about 75 % of the grain dry weight, industries be better able to identify worthwhile targets for
this conversion process makes a major contribution to the total genetic manipulation. While the malting and brewing industries
amount of extract. For rapid starch hydrolysis to occur, starch have leveraged their funding for barley improvement by supple-
granules must first be gelatinized. Although a-amylase was menting public investment in conventional breeding programs
thought to be solely responsible for this liquefaction process, there has been (with a few notable exceptions) little industrial
there is now evidence that a-glucosidase may also be . investment in barley biotechnology. Concerns about the public
involved44 .45 • Increasing either the amounts of these enzymes acceptance of consuming alcoholic beverages produced using
and/or their thermostability may beneficially speed up starch genetically engineered plants have been a factor in moving
granule hydrolysis. Increasing diastatic activity might also forward with these technologies. However, the lead times in
improve the efficient utilization of adjunct materials, producing agricultural biotechnology are long, and for those with the
additional fermentable sugar and additional alcohol. Efforts are foresight to take on the challenge, there are a significant number
presently underway to clone the · various diastase genes from of exciting opportunities brewing in barley biotechnology.
barley with the aim of using transformation technology to
increase their expression levels. Introducing foreign diastatic Acknowledgments
genes with increased heat stability to compensate for their loss in The authors wish to thank Moira Anderson , Peggy Lemamc, Kenneth
kilning and mashing may also be worthwhile. ,8-Amylase is MacKeller, Suzin McElroy, Odd-Arne Olsen, Robert Schuurink, David
Thomas, and Berry Treak for critical reading of our manuscript. D.Jvf. is
especially heat labile, and it might be advantageous to express supported by a Research Grant from the US National Barley Genome
heat-stable forms of this enzyme, either in the starchy endo- Mapping Program and by The Coors Brewing Company.
sperm or in aleurone29 • Limit dextrinase is a debranching
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manipulation. In: Biosynthesis and Manipulation of Plant Products. D. Grierson by the editors; suggestions and proposals
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48. Visser, G. F. and Jacobsen, E. 1993. Towards modifying plants for altered starch are welcome.
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50. Hammond, J. R. M. and Bamforth, C. W. 1993. Progress in the development of
fices,Bio/Technology, 65 Bleecker Street,
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