J. Inst. Brew.. March-April. 1996, Vol. 102, pp. 97-102

MALTING AND BREWING SCIENCE: CHALLENGES AND OPPORTUNITIES'*

By A. W. MacGregor

(Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, Manitoba R3C3G8, Canada)

Received 15 September 1995

Molecular technologies have been developed for the transformation of barley. These technologies

complement current methods of barley breeding. In addition, they offer the potential of altering

specific components in barley that affect malting quality and of introducing foreign genes, control

ling desirable traits, into barley. Application of genetic engineering to improving malt quality factors

such as cell wall degradation, protein modification, starch hydrolysis and flavour stability, is

discussed. Limitations to the use of this technology for improving malt-related functional properties

of barley components such as cell walls and starch granules are also evaluated. Some possible

constraints to the utilization of genetic engineering for malt quality improvements are identified.

Key Words: Barley, genetic engineering, malt quality, cell walls,

proteins, starch, hydrolytic enzymes.

Introduction

Horace Brown was a scientist with very wide interests who

made significant contributions to a range of scientific disci

plines31. These included studies on the effects of rock form

ations on brewing water quality, the microbiology of spoilage

organisms in beer, the use of hops in brewing, the role of

nitrogen in malting and brewing and the physiology of barley

germination. It is this latter work, some of which is described

in a classic publication10 of 1890, that is associated mostclosely with Brown and that is still referred to today. The

publication contains a detailed description of the structure of

the barley kernel based on painstaking observations using

microscopy, carefully developed arguments to support the con

clusions drawn and thoughtful and insightful speculation.

The intent of this aspect of Brown's research was to build a

sound knowledge-base of the germination process for the

malting and brewing industries. This would not only provide

answers to problems encountered during processing of barley

and malt but would provide a spring board to improve

the efficiency of malting and brewing technologies, provide

impetus to the development of new technologies and improve

the quality of the final product. In other words, Brown was

intent on building a strong scientific foundation for the techno

logies (malting and brewing) with which he was working so

that advantage could be taken of advances in relevant scientific

knowledge as these become available. This is an approach that

is as relevant today as it was in Brown's time.

What are some of the challenges and opportunities facing

the malting and brewing industries today? Stable, if not

declining, beer and malt markets in many countries have led to

increased competition for market share and the developmentof "new" beer types such as dry and ice beer with associated

technological problems. As governments and industries curtail

spending there is less funding for research and there has been a

repositioning in research emphasis leading to a decrease in

longer term fundamental research and more emphasis on

shorter term "problem solving" research. An exploration of

the long term negative impact of this de-emphasis of funda

mental research is beyond the scope of this discussion. Along

with these changes are the increasing demands of consumers

•Based on Horace Brown Lecture presented at the Royal Society,

London, October 1993.

fPaper No. M201 of the Grain Research Laboratory, Canadian Grain

Commission. 1404—303 Main Street, Winnipeg, Manitoba R3C 3G8,Canada

for purity in food systems., i.e., a demand for "natural" foods

or foods made with minimum additives. This applies also to

the preparation of malt and beer and so extra demands will be

made on the quality of raw materials such as barley and malt.

Advantage must be taken, therefore, of new technologies that

have potential for improving the malting quality of barley.

Such a technology is plant genetic engineering3456-70 or the

manipulation, addition or deletion of quality-related genes in

barley. This offers the possibility of manipulating individual

components within the grain without changing other com

ponents. Equally important, however, is the potential for trans

ferring desirable genes from any organism into barley. This

opens up exciting possibilities for quality improvement of

malting barley.

Prerequisites for Genetic Manipulation

Several technological problems had to be overcome before

the new technique of genetic manipulation could be applied

successfully to cereal grains, such as barley35. For example,efficient methods had to be developed for the delivery of DNA

into single cells37, selection of the transformed cells, regeneration of fertile plants from the transformed cells, and

expression of the new or modified DNA in appropriate tissues

of the grain96. Most of these problems have been overcome sothat all the common cereals have been transformed with at

least some degree of success (Table I). Several groups have

reported the successful transformation of barley so the techno

logy is now available43"521771'00

From past experience with transformation of other crops it

is likely for economic reasons that most of the immediate

research effort on barley will concentrate on developing

cultivars with herbicide tolerance and increased resistance to

diseases22-69. Use of the technology to improve malting qualityshould be driven by the malting and brewing industries. They

have the monetary and personnel resources to control and

guide this approach to barley improvement.

Several different strategics arc available for manipulating

genes in cereals96. Insertion of desirable genes from other

TABLE I Transformation of Cereals

Cereal Year Transformed

Rice

Maize

Wheat

Oats

Bariey

1988 "■"■",990U.23J4

199297102I99290I99443-52-76-'00

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98 OPPORTUNITIES IN MALTING AND BREWING SCIENCE [J. Inst. Brew.

organisms into barley has the potential to exert the greatest

change in quality parameters. It is possible also to alter exist

ing genes to produce subtle but specific changes in a gene

product but little progress has been made to date, with this

approach. Yet another approach would be to alter the pro

moter part of genes that control hydrolytic enzymes21 so thatsynthesis of the enzymes would be switched on or activated

more rapidly during germination. This should have obvious

advantages in decreasing malting times. The use of antisense

technology to block the action of existing genes is a potentially

powerful aspect of genetic manipulation103. This technique hasbeen used with considerable success in a number of plants and

is being used currently to change the starch characteristics in

wheat".

Potential for Improving Malt Quality through

Genetic Engineering

Desired improvements in malt quality through changes in

barley characteristics have been discussed in several published

reports25-45-55. Many of these improvements could be achievedthrough conventional plant breeding but the specificity of

genetic manipulation and the access it offers to a range of gene

pools make it a powerful complement to breeding programs.

Some possible applications of the technology will now be

discussed.

Endosperm Cell Walls

These form a barrier to the movement of enzymes in the

endosperm during malting and cause severe technological

problems during brewing if not degraded adequately during

malting3. The major component of the walls is a mixed-linkageP-glucan that has been well characterized107. Despite that, littleis known about the biosynthesis of the polysaccharide in

barley. It is desirable to lower the levels of barley P-glucan but

the most effective way of accomplishing this at the present

time is through traditional breeding methods. The growing

environment also affects P-glucan levels in barley but this is

difficult to counteract6. Arabinoxylan, the minor componentof endosperm cell walls in barley, may also cause technological

problems because this polysaccharide, also, has the potential to

form viscous solutions58. The composition, structure, proper

ties and biosynthesis of arabinoxylans are not well understood.

Therefore, genetic manipulation of this minor but techno

logically important barley component is unlikely in the near

future.

Cell Wall Degrading Enzymes

Barley contains low levels of P-glucanase but a high pro

portion of the enzyme found in malt is synthesized in scutellar

and aleurone cells during germination1. It is important that

P-glucanase diffuses through the endosperm during malting

and degrades p-glucan throughout the endosperm3. Theenzyme has poor heat stability and may show limited activity

during mashing42. Therefore, to ensure minimal processingproblems, P-glucan degradation should be completed by the

end of malting. There are two major p-glucanases in malt and

they have been well characterized20-28. Their functional properties, including their detailed action on P-glucan, have been

documented, their sites of synthesis during barley germination

have been located and the genes controlling synthesis of the

enzymes have been identified.

It is not surprising that the p-glucanase system in malt has

been targeted for genetic manipulation21. One approach hasbeen to insert genes for a heat stable p-glucanase from Tricho-

derma reesei into barley52 but the effectiveness of manipulating

barley in this way has yet to be evaluated. Parameters that

confer heat stability on proteins are not well understood60 butwhen these have been identified, it may be possible to re-design

the appropriate barley gene to produce a more heat stable

P-glucanase. Research is underway to produce hybrid P-

glucanases that would combine desired heat stability with

specified hydrolytic activity8. Results of such research mayfind application in fermentation industries. P-Glucanases have

limited time to complete P-glucan hydrolysis during malting

and so their synthesis must be initiated rapidly during germin

ation. Research aimed at increasing the effectiveness of the

promoter region of the P-glucanase gene in barley would be

a worthwhile approach to increasing the effectiveness of P-

glucanase during malting21.Even low molecular weight products from P-glucan hydro

lysis can cause technological problems108. Complete hydrolysis

of these products to glucose would prevent such problems and

also provide extra fermentable extract to the brewer. Barley

does contain a P-glucosidase83 that hydrolyzes the tri- and

tetrasaccharides produced by p-glucanase*0 but the effective

ness of the enzyme during malting and mashing has not yet

been determined. The enzyme is encoded by a single gene and

the probable sequence of the gene has been reported. This is

another barley gene that could be manipulated to increase the

usefulness of an enzyme for malting and mashing purposes

and so lead to an improvement in the malting quality of the

barley.

Extensive hydrolysis of arabinoxylans is also desirable

during malting. Limited information is available on the malt

enzymes required for such hydrolysis7-86 and so it is prematureat this time to envisage using genetic engineering technology to

increase the levels of these enzymes in malt although this

would be beneficial for malt quality.

Non-Enzymic Barley Proteins

The influence of barley proteins on malt and beer quality is

complex because of the large number of proteins present

in barley, the diversity of their functional properties and

the changes that they undergo during malting53-71-88-104. Someeffects of these interactions are listed in Table II.

Hordeins are a complex group of proteins80 that must be

modified extensively during malting or they will cause numer

ous processing problems during brewing. They are composed

of three main groups (B, C, D), each of which is heterogenous

but group D contains only one major component. Attempts to

evaluate the effects of each of these groups on malting quality

have had limited success although there is some indication that

a low D- to B-hordein ratio would be desirable71. Progress has

been made on identifying the hordcin genes98 but practical

utilization of this knowledge must wait for more detailed

information on the functionality of individual hordein pro

teins. It would then be feasible to alter proportions of different

proteins present in barley81 without increasing the total protein

content, which normally would not be desirable. The hordein

proteins also form a matrix in the barley endosperm that

embeds the starch granules. This matrix must be extensively

hydrolyzed during malting to release the starch granules so

that they can be solubilized effectively during mashing.

Gel-forming proteins have been identified in barley63-85-89.

These proteins reduce malt extract and cause filtration prob

lems and so have been the subject of several studies. The gels

consist of mixtures of high and low molecular weight proteins,

including B- and D-hordeins, that are joined together through

intra- and inter-molecular disulphide bonds63. Some components of the gel that is formed during mashing appear to be

TABLE II Protein Interactions During

Malting and Brewing

Viscosity-associated proteins

Head retention

Beer stability

Wort fermcntability

Protein matrix and starch granules

Protein type/quantity

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Vol. 102, 1996] OPPORTUNITIES IN MALTING AND BREWING SCIENCE 99

derived from barley proteins that have been partially hydro-

lyzed by proteolysis during malting85. Again, insufficient in

formation is available on proteins that are essential for gel

formation and so it is premature to develop a strategy for

limiting their levels in barley.

Proteins are also actively involved in the formation and

retention of beer foam110 along with other components derived

from malt and hops. Recent results suggest that hydrophobic

proteins derived from hordeins are major components of beer

foam2-36. Other proteins such as protein Z44 and a trypsin/a-amylase inhibitor36 have also been identified in beer foam. Itis the hydrophobic nature of a range of polypeptides rather

than a few specific proteins that appear to be important

constituents of beer foam. It is difficult with current know

ledge to increase specifically the head retention potential of

barley and malt because the property does not reside in

specific proteins.

Proteins and polyphenols have been implicated in the forma

tion of hazes in beer during storage61. This problem has been

controlled by judicious use of proteases to degrade the pro

teins and selective absorbents to remove proanthocyanidins,

the active polyphenol component of haze16-51. Identification of

the proanthocyanidins99 in barley that are responsible, alongwith proteins and other beer components, for haze formation

has led to the development of proanthocyanidin-free barley

that does reduce the haze-forming potential of the resulting

malt". Little information is available on the barley proteins,either intact or modified by proteolysis during malting, that

participate in haze formation. It is possible that some protein

cross-linking is involved in haze formation and so some of the

same proteins may be responsible for foam stability and haze

formation. Until more information is available on the identity

of haze-related proteins, little can be done to lower their levels

in barley either through genetic manipulation or through more

traditional breeding methods.

Proteolytic Enzymes

During germination barley proteins, mainly insoluble, stor

age proteins in the endosperm, must be converted into soluble

proteins, peptides and amino acids to supply nutrients to the

developing embryo. From a technological standpoint, the same

changes are important during malting because they lead to

destruction of the protein matrix, release of starch granules,

and to formation of amino acids in the malt that are required

for yeast nutrition during brewing. These changes are brought

about by a complex array of proteolytic enzymes15-54'74'"3.

Some of these are present in barley, others are synthesized

during malting and are active in the endosperm, while some

may be active only in the aleurone29. There is some controversyabout the activity of these enzymes during mashing41 because

they arc heat labile and so may be inactivated rapidly. There

fore, it is important that effective protein degradation is com

pleted by the end of malting. Over 40 different proteolytic

activities have been identified in malt"3 but only a few pro

teases have been characterized in detail. Proteases, like other

enzymes, have bond specificities and some of these have been

determined through elegant and very necessary studies33 but

much remains to be done in this area. With current knowledge

it is not possible to contemplate changing the protease com

plement of malt in the near future. An additional complication

is the presence of endogenous protease inhibitors in barley32.The extent to which they may modulate protein modification

during malting has yet to be determined.

Barley Starch

Rapid degradation of starch to fermentable carbohydrates

during mashing depends on the starch being fully gelatinized18before any of the starch-degrading enzymes are inactivated.

Starch granules in barley, therefore, should have the lowest

gelatinization temperatures possible. Factors affecting the gela-tinization temperature of barley starch granules include

growing environment95, granule size48 and, possibly, starchstructure"2. Little can be done to offset the environmental

effect. Barley cultivars containing a high proportion of large

granules would be preferred for malting because small granules

have higher gelatinization temperatures48 and do have the

potential to cause problems during brewing. Although signi

ficant progress has been made in identifying the enzymes

responsible for starch synthesis in cereals5172-73'87, controllingand changing the proportion of large granules does not yet

appear to be feasible. There is some evidence that reducing the

length of the outer chains in amylopectin may reduce gelatiniz

ation temperatures of the starch"2, but, again, the biochemical

pathways controlling this parameter are not yet understood.

High amylose and waxy barley starches tend to have higher

gelatinization temperatures than do normal, large granules46

and so they do not offer any advantage to the brewer. The

starch content of potatoes has been increased by the insertion

of a starch synthesis gene92 and a similar effect could be

achieved in barley.

Starch-degrading Enzymes

It is unlikely that a-amylase levels are quality limiting in

barley malts66. Dextrin profiles in wort and beer4' suggest that

a-amylolysis of starch is usually complete by the end of mash

ing. The two major a-amylase components in malt have similar

activities and action patterns on solubilized starch51 so there is

no incentive to change the proportion of these components in

malt. ct-Amylase gene families in malt have been identified and

characterized65 and so could be manipulated if required. Thereis a potential inhibitor of malt a-amylase 2 in barley64105106.

Since the inhibitor does not appear to play a role in malting or

mashing there is no obvious advantage to lowering its level in

barley.

P-Amylase plays a crucial role during mashing because it is

responsible for the degradation of starch and products of

a-amylase hydrolysis of starch to maltose, the most abundant

fermentable carbohydrate in wort94. The two amylases working

together are more effective in degrading starch than are the

two enzymes acting independently, p-amylase is synthesized

during barley development but is rendered fully active during

malting26-91. The enzyme is heat labile and a significant proportion of the activity is lost during kilning4-9. Enzyme activity

is also lost rapidly during mashing84 when the temperature

approaches 65°C. It is important, therefore, that sufficiently

high levels of p-amylase are present during mashing to hydro-

lyze the starch completely in the temperature range 58-60°C

(starch gelatinization temperature) to 65°C (temperature of

rapid inactivation of P-amylase). The presence of linear malto-

dextrins such as maltotetraose or maltohexaose in wort or

beer indicates incomplete hydrolysis of starch dextrins by (3-

amylase47. Obviously, it would be beneficial to increase levelsof p-amylase in barley and/or increase the temperature stabi

lity of the enzyme. Genes coding for P-amylase have been

detected391" so it would be possible to increase barley P-amylase levels. The parameters that control the heat stability of

enzymes are poorly understood but some success has been

achieved in improving the heat stability of barley p-amylase68.

This opens up the possibility of improving the thermal stabilities of other barley and malt enzymes.

The technological significance of a-glucosidase is not clear.It is important during malting for the conversion of maltose to

glucose93, a sugar that can be assimilated and metabolized bythe active embryo. However, brewing yeasts rapidly metabolize

maltose and so do not require the prior hydrolysis of maltose

to glucose. a-Glucosidase may increase the effectiveness of

P-amylase during mashing by removing maltose, a possible

competitive inhibitor of P-amylase75. There is no obvious

reason at the present time to alter a-glucosidase levels in malt.

Beer contains significant levels of branched starch dextrins"

indicating that the starch debranching enzyme, limit dextrinase,

is largely ineffective during mashing. This is due, in large

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100 OPPORTUNITIES IN MALTING AND BREWING SCIENCE [J. Inst. Brew.

measure, to the presence of limit dextrinase inhibitors in the

malt49. These low molecular weight proteins are present in

barley30 and, although their levels are reduced during malting,sufficient inhibitor remains in the malt to inhibit a high pro

portion of the malt limit dextrinase. The heat stability of limit

dextrinase is similar to that of (J-amylase84 and so the enzyme

should be reasonably effective during mashing in the absence

of the inhibitors. It would be beneficial to develop barleys with

low levels of inhibitor, using antisense technology, and in

creased potential to produce limit dextrinase during malting by

increasing the gene dosage for the enzyme. Neither of these

approaches is feasible at the moment because the genes coding

for limit dextrinase and the inhibitors have not been identified

and characterized. Increased limit dextrinase activity during

mashing would have to be controlled carefully so as not to

remove all branched dextrins because they contribute to mouth

feel and body in the final beer79. Genetic manipulation of the

limit dextrinase system in malt does have the potential to

increase markedly the fertnentability of worts.

Flavour Stability

Numerous components contribute to the flavour of beer and

several that produce undesirable off-flavours have been identi

fied59'67. Only one example will be discussed here to illustratehow genetic manipulation could be utilized to improve flavour

stability in beer. The undesirable "cardboard" flavour some

times found in beer has been traced to trans-2-nonenal18. This

aldehyde originates, via a series of reactions, from the un-

saturated fatty acids linoleic and linolenic that are produced

from barley lipids by the action of lipase38. A crucial step inthis series of reactions is the oxidation of these acids (mainly

linoleic) by the enzyme lipoxygenase (LOX) in the presence of

oxygen to form hydroperoxides. Very low levels of trans-2-

nonenal are detectable in beer (0.1 ug/l)59 and it is difficult to

exclude oxygen from all phases of brewing to the extent

necessary to prevent formation of such low levels of this

material.

There arc two LOX enzymes in malt; LOX-1 is derived

from barley and LOX-2 is formed during malting109. Current

evidence indicates that LOX-1 is mainly responsible for the

formation of trans-2-nonenal during brewing17. It should bepossible to locate the gene coding for this enzyme in barley

and block its action through antisense technology. The effect

of such a manipulation on barley germination would have to

be monitored in detail. Another, more desirable, approach

would be to reduce the levels of linoleic acid (the main source

of trans-2-nonenal) in barley using antisense technology to

block the gcne(s) controlling synthesis of the enzme(s) respon

sible for linoleic acid formation. Alternatively, barley could be

induced to synthesize another fatty acid with less harmful

functional properties. Either approach would lower the poten

tial for trans-2-nonenal formation. Judicious selection of kiln

ing programs27 can also be used to tackle the problem by

inactivating LOX-1.

There are constraining factors to the successful exploitation

of plant genetic engineering and they require serious con

sideration. Some of these are listed in Table III. The growing

environment has a strong influence on the quantity and func

tionality of grain components and may over-ride any poten

tial, genetic, improvements. Extensive evaluation must be

carried out after any genetic manipulation to ensure that only

the desired change has occurred and that the change has not

affected normal functioning of the grain or plant. Current

knowledge of plant biochemistry and physiology is in

complete! Some quality factors may be difficult to manipulate

because they are controlled by multiple genes, e.g., extract is a

composite of several factors. A major constraint to the utiliz

ation of transformed organisms is consumer acceptance62101.This was pointed out several years ago and is still a serious

problem in some countries. It does require ongoing consumer

education about the safety of engineered plants. Progress in

TABLE m Constraining Factors on

Genetic Manipulation

Effect on plant/grain

Specificity of desired change

Environment

Multiple genes/quality factor

Consumer resistance

this area will be made now that genetically engineered food

stuffs are available30.Changes to plants through genetic engineering are now a

reality. The malting and brewing industries must take advan

tage of this powerful, new technology. Brewers should decide

what types of malt they will require in the next 5-10 years,

keeping in mind that public pressure will reduce the use of

additives in food production so malt quality will become even

more important. Maltsters, in turn, must take the initiative in

identifying more clearly desired quality parameters in barley

and malt so that appropriate genes can be identified, located,

characterized and altered appropriately. A collaborative effort

involving many disciplines will be required if the full potential

of genetic engineering is to be exploited for the improvement

of barley malting quality.

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