Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010
Program Plan BarleyFunFood (20082010)
Exploiting the barley endosperm as a resource for value added food and feed
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Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010
Overall program objectives (2008-2013)
 Look for diversity in carbohydrate composition of barley kernels
 Increase our understanding of polysaccharide biosynthesis in barley
 Produce barley materials with improved carbohydrate composition
 Increase our understanding of nutritional effects of cereal carbohydrates
Program outline (2008-2010)
BarleyFunFood is a thematic research program in which the Faculty of Natural Resources and
Agricultural Sciences, SLU is collaborating with the agro-industrial sector (Lantmännen Food
R&D and SW Seed, Svalöf Weibull AB) in order to increase our understanding of barley
biology and nutritional effects of cereal carbohydrates.
The program is funded by the faculty (50% funding) and the industry (50% funding). The
participating SLU departments contribute with additional resources such as saleries for
professors while participating companies have internal projects of importance for the program
but these are not included in the program.
The BarleyFunFood research team includes plant biologists, plant breeders, chemists, food
scientists, microbiologists and nutritionists working during the first three years within two
subprograms. The first subprogram Barley Biology includes 6 projects (collection and
cultivation of barley, screening of barley kernels with NIT and NIR, functional genomics in
barley, characterization of carbohydrates in barley kernels, development of NMR-based
methods for characterization of biological samples and metabolic fluxes, and carbohydrate
incorporation in developing barley endosperm). Since at this stage it is not possible to study
new genotypes from the program, the second subprogram Nutritional Effects of Cereal
Carbohydrates will start with available cereal fractions with known carbohydrate composition,
i.e. fractions rich in arabinoxylan, mixed-linkage -glucan, fructan and different types of
starch. This subprogram includes three subprojects (characterization of carbohydrates in
cereal fractions/cereal foods, cereal fibers and satiety, and prebiotic effects of different cereal
carbohydrates).
Background
1. The Cereal Endosperm
The cereal endosperm is our largest single primary food source, and thus among the most
economically important structures in biology. Development of the cereal seed is orchestrated by
the coordinated activities of a large number of genes that encode metabolic and regulatory
enzymes and other proteins. This results in a triploid endosperm, the embryo, pericarp and other
tissues of the mature grain. The endosperm consists of two tissues, the interior starch-filled
endosperm and the outer epidermal layer called the aleurone.
The unique value of the endosperm of cereal grains can be traced to the evolution of different
pathways for carbohydrate synthesis, such as starch, ß-glucans, arabinoxylans and fructans (Fig.
1). Fructans, which are mainly synthesized in vegetative tissues of some
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Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010
Fig. 1. Sucrose is uploaded to the barley
endosperm and serves as carbon source for
different metabolic pathways.
cereals, accumulate to significant amounts in mature rye and young barley grains. The
importance of the polysaccharide composition in the cereal grain for human and animal
consumption is multi-facetted:
 The type and composition of different carbohydrates is of increasing interest in both human
and animal nutrition. To a large extent this is due to the effects that different dietary
polysaccharides have on the intestinal microflora (Macfarlane and Macfarlane, 2007). There
is ample evidence of the positive effects of the colonic microflora on human health. The
microflora is dependent on a continuous supply of carbohydrates, the most important energy
substrate in the colon. Carbohydrates possessing the capacity to affect the colonic microflora
in a beneficial way and/or with the ability to form metabolically active compounds are called
prebiotic carbohydrates. (See section 9 below).
 The value-added properties of certain cereal carbohydrates on human health also include
immune-boosting, cholesterol-lowering, satiating and cancer-preventive effects.
 Cereal grain is an important feed ingredient for most intensively managed horses. However,
although cereals provide a valuable source of digestible energy, feeding low-glycemic cereal
grains to horses is always associated with some risk because of incomplete digestion of
resistant starch (RS), ß-glucan, arabinoxylan (AX) and fructan in the small intestine and
significant amounts of carbohydrates passing through to the cecum and colon (large intestine,
or hind gut). Rapid fermentation of carbohydrates in the hind gut leads to accumulation of
acidic end products and low pH. Just how the acid, build up in the hind gut, affects the horse
is not clear but there is no doubt that acid accumulation in the hind gut is the primary cause
of laminitis as well as many of the behavioural disorders commonly associated with feeding
grain to horses.
 Carbohydrates compose about 70% of a dairy cow's dry matter intake and are the
predominant energy source to produce milk and support rumen function and microbial
growth. Since the energy content of available starch is higher than that of low-glycemic
polysaccharides, cereal grain containing high levels of non-fibrous starch and low levels of ßglucans and AX is preferable for maximum output in dairy farms. However, the fiber effect
of ß-glucans, AX and cellulose is required for proper rumination, and rations lacking fiber
generally result in low percentage of fat in the milk. Furthermore, a starch-rich diet
contributes to digestive disturbances. For example, whereas starch is a major source of
propionic acid from rumen fermentation, which may affect rumen pH and lead to acidosis, ßglucans are generally fermented to acetic acid, which has less affect on rumen pH.
 In poultry, as in other monogastic animals and humans, endogenous enzymes have a limited
ability to digest non-starch polysaccharides, and the poor performance of barley-fed chicken
is attributed to the relatively high proportion of ß-glucan in the barley grain. To control this
anti-nutritive effect, enzymes that hydrolyze non-starch polysaccharides are often added to
feeds and thus enhance the overall digestibility. Poultry producers also generally select barley
varieties low in ß-glucans in an attempt to control for these anti-nutritive properties.
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Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010

Barley ß-glucan polymers originating from the endosperm cell walls are one of the major
concerns in the brewing industry. The amount and molecular mass of ß-glucan in malt affect
brewhouse extract yield and wort and beer viscosities, as well as lautering, diatomaceous
earth, and membrane filtrations. Barley ß-glucan is also associated with beer hazes. While it
is possible to select barley varieties for malting on the basis of low levels of -glucan, there is
no clear relationship between ß-glucan content and malt quality. Although ß-glucan is the
major constituents of the endosperm cell walls, AX and other polysaccharides may also
contribute to the overall quality of malting barley. The coexistence of several biopolymers in
the cell walls, their spatial organization, and the nature of interactions (cross-linking) among
them might contribute to the mechanical strength, permeability, and solubility, and therefore
to enzymic susceptibility of cell walls during malting.
During the mashing process in brewing, hydrolytic enzymes are more active on the large
granules as compared to small granules, and therefore, A-type starch granules contribute
more to malting quality than B-type granules. Small starch granules may also cause problems
during malting as they adhere easily to proteins leading to clogging of filters. Thus there has
been a desire from the malting industries to produce malt from barley lines with a lower
content of small starch granules.
2. Starch
Starch is the predominant storage carbohydrate in plants and the second most abundant
biopolymer on earth, after cellulose. Starch is a mixture of amylose and amylopectin, both
glucose polymers. Amylose is a mostly linear polymer of 105-106 Da with 200-2000 -1,4 linked
glucose moieties with rare -1,6 branch points. Amylopectin, on the other hand, is highly -1,6
branched, with a complex structure of 107-108 Da and up to 3 × 106 glucose subunits, making it
the largest biological molecule in nature. In the plant, starch is deposited as starch granules in
chloroplasts of source organs such as leaves (transitory starch) or in amyloplasts of sink organs
such as seeds, tubers and roots (storage starch). The starch of mature barley endosperm is
composed of two types of granules; large lenticular (A-type; >10 m) and small round (B-type;
<10 m) granules. The large granules are initiated before 15 days-post-anthesis (dpa), whereas B
granules are formed after 15 dpa. In mature grain, only 10-15% of the granules are of the A-type,
but they account for most of the starch by weight (~90%). Large and small starch granules differ
slightly in their amylose content, the overall lipid composition, and the amount of proteins
attached to the surface.
The functional properties of starch, such as retrogradation, gelatinization, viscosity, swelling,
film formation, thermostability, crystallinity, texture, color, taste, and smell, determine its value
for food or non-food applications. These properties, in turn, are controlled by the
amylose/amylopectin ratio, the branching pattern of the amylopectin molecules, the chain lengths
of the amylose molecules and the amylopectin branches, the size and modality of the granules,
and starch-protein and starch-lipid interactions. The quality further depends on the exact
amylose/amylopectin ratio and the nature of the amylopectin molecules. Starch with a low
amylose/amylopectin ratio yields highly digestible, high-glycemic index foods. These highglycemic index starches have been associated with diet-related conditions such as type 2 diabetes
and insulin resistance. Modification of starch to increase the amylose to amylopectin ratios
would be greatly useful in improving the glycemic index and physiological responses to the
starchy foods that are prevalent in the diet in many developing countries. Improving the glycemic
indices of staple starchy food crops such as grains will improve their nutritional quality, and
subsequently have an effect on the prevalence of attendant disease conditions.
In addition to its exceptional value in food and feed, starch is used as a feedstock in a wide
array of applications for both bulk and commodity products in food and non-food industries. The
interest in starch as a renewable and CO2-neutral polymer and source for monomers to
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Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010
supplement and replace segments of the petro-chemical industry, is progressively increasing, as
are the prospects of tailor-production of starches in transgenic crops.
3. -Glucan
-Glucan is known to be a linear polysaccharide composed of 1,4-linked -D-glucopyranosyl
residues (about 70%) substituted at 3- or 4-position, and 1,3-linked -D-glucopyranosyl residues
(about 30%) substituted at 4-position. Most of the 1,4-linkages occur in groups of two
(cellotriosyl) or three (cellotetraosyl) separated by isolated 1,3-linkages. Longer blocks of 5-15
contiguous 1,4-linkages in small but significant proportions are also present. The distribution of
cellotriosyl and tetraosyl residues within the polymer has been suggested to be in a random
fashion but this has been debated over the years.
Mixed-linkage -glucan has been thoroughly studied owing to its importance in brewing and
feeding of mono-gastric animals. Their presence in barley seeds is restricted to the endosperm
cell walls, of which they contribute to about 75% of the dry matter. Both cultivar and growing
conditions are known to influence the content and solubility of the -glucan in barley and a dry
and hot climate favors high -glucan content and extract viscosity. In brewing, a low content of
-glucan in the malting barley reduces wort viscosity and improves filtration and haze formation
in the beer. Content and properties of the -glucan and -glucanase activity are therefore key
factors for good malting characteristics. A high content of -glucan in barley feed reduces feed
intake, feed conversion ratio and growth when fed to mono-gastric animals. This is explained by
a reduced ileal digestibility of energy, protein and other nutrients as well as the frequency of
sticky droppings. Addition of -glucanase to the feed significantly improved all these negative
factors for broiler production.
More recent attention has focused on the potential use of -glucan from barley and oats as a
functional ingredient in innovative foods (Brennan and Cleary, 2005; Åman, 2006). Several
companies are involved in the production of these types of functional ingredients or foods and
Sweden is one of the leading actors in this field. The health-related importance of -glucan, as
part of a balanced diet, has been known for decades and -glucan has been shown to have
glycemic, insulinemic and cholesterol lowering properties as well as prebiotic and immunemodulating effects. All these effects are most likely related to the content, structure, solubility
and molecular mass distribution of the -glucan in the food. Concurrently, research and
development have focused on the inclusion of -glucan rich fractions into both cereal- and dairybased food systems, illustrating their potential as ingredients to manipulate structure, texture and
health profile of a food.
4. Arabinoxylan
Arabinoxylans (AX) are the main complex sugars found in the hemicelluloses of plants and are
part of dietary fiber. In barley, AX constitutes ~70% of aleurone walls, ~20% of walls from the
starchy endosperm. The barley AX consists of 1500 - 5000 (1,4)--D-xylopyranosyl residues that
form a molecular backbone carrying single -L-arabinofuranosyl moities. AX is degraded in the
colon by intestinal bacteria possessing AX-degrading enzymes. Although the health effects of
arabinoxylans are well documented, the effects of the degradation products, the arabinoxylan
oligosaccharides (AXOS), are less studied.
5. Fructans
Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families.
Fructans have a general structure of a glucose linked to multiple fructose units. In plants up to
200 fructose units can be linked in a single fructan-molecule. These types are distinguished on
the basis of the glycosidic linkages by which the fructose residues are linked to each other. They
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Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010
can broadly be divided into three groups. In the first group there are the inulins, which are linear
fructans, where the fructose units are linked via a ß-2,1 bond. All fructans found in the
dicotyledons, as well as some monocotyledons are of this type. In the second group there are the
levans, which are also linear fructans, but in these the fructose units are linked via a ß-2,6 bond.
This type of fructan is found in a large part of the monocotyledons. In the third group there are
the fructans of the mixed type, which are also referred to as the graminan type. These fructans
have both ß-2,1 and ß-2,6 linkages between the fructose units, and also contain branches. These
fructans are found in grasses for example.
Fructans have a variety of applications. Small fructans have a sweet taste, whereas longer
fructan chains form emulsions with a fat-like texture and a neutral taste. The human digestive
tract does not contain enzymes able to degrade fructans; therefore, there is strong interest from
the food industry to use them as low-caloric food ingredients. Moreover, fructans have been
implicated in the prevention of osteoporosis by enhanced calcium uptake, a lower blood
cholesterol and triglyceride status, and prevention of colon cancer. In plants, fructans may have
functions other than carbon storage; they have been implicated in protecting plants against water
deficit caused by drought or low temperatures. Fructans are produced in the vacuole by the action
of specific enzymes (fructosyltransferases) that transfer fructose from sucrose to the growing
fructan chain.
6. Barley
Barley (Hordeum vulgare L.) is a true diploid closely related to wheat and rye; it is one of the
most important crop species in the world. It is the most cultivated crop in Sweden, Norway,
Denmark and Finland. Globally, it is the fourth-most cultivated cereal crop (after wheat, maize
and rice) and is grown (2002) on 54 Mha with an annual yield of 132 Mt
(www.fao.org/WAICENT/), and 1.9 Mha and 9 Mt respectively for the Nordic countries. Barley
is ideally suited not only for practical improvements but also as a model system for wheat, rye,
oats, and related forage grasses because of the large and dedicated international effort at finding
genes (ESTs) and mapping them in this crop, the available diagnostic mutants, and the analytical
methods that have been developed. Barley grain is largely used as animal feed and malt and, to a
lesser extent, as food. It is an excellent energy source because the grain consists of 80%
carbohydrates, mostly starch; in many countries in Africa and Asia barley is an important part of
the diet. The soluble fiber of barley makes it a good “functional food” and for this reason it has
been rediscovered in Europe. The plant is remarkably plastic in its adaptation to altitude, latitude,
soil moisture and salinity, and temperature. Its wide geographic distribution has led to a vast
array of genetic variability in the germplasm, stored in collections such as the Nordic Gene Bank
(Alnarp), and the Risø and Carlsberg barley collections, most of which remains to be
characterized and exploited. A large collection of barley mutants affecting the endosperm
phenotype is available and constitutes an enormous potential resource, hitherto almost
unexploited, for crop improvement given appropriate molecular markers to prevent gene drag in
the crosses. Many of these mutants have unknown effects on the metabolome (all pools of
metabolites in a cell), which includes carbohydrate and storage protein biopolymers.
7. Carbon Allocation in the Developing
Cereal Endosperm
Carbohydrate metabolism in the cereal
endosperm is a complex issue.
Metabolic reactions involving hexoses
take place in the cytosol and in the
amyloplast of these cells (Fig. 2). With
the exception of the nonoxidative
Fig. 2. Some metabolic fates of imported sucrose in the barley
endosperm. -GluSy, -glucan synthase; DAG, diacyl
glycerol; HxK, hexokinase; Inv, sucrose invertase; PC,
phosphatidyl choline; SuSy, sucrose synthase; SSE, starch
synthesis enzymes; TAG, triacyl glycerol.
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Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010
branch of the pentose phosphate pathway, which is localized in the cereal amyloplast, both
compartments are characterized by an almost redundant set of enzymes catalyzing both anabolic
and catabolic reactions. The metabolite pools of the cytosol and the amyloplast are efficiently
connected by transporters for triose phosphate, hexose phosphate, pentose phosphates, and ADPGlc, which enable metabolite flux and maintain the phosphorus balance in the different
compartments of the cell.
Whereas source-sink communication controls the quantity of sucrose that enters the
endosperm, an interplay between source-sink interaction and regulatory switches inside the
endosperm cells govern the fate of the imported sucrose. The incoming sucrose in the barley
endosperm is the carbon source for a variety of metabolic pathways (Figs. 1, 2); mainly starch or
-glucan synthesis, or glycolysis. Other end products are fructans and other polysaccharides. In
barley, the route to storage fat (oil bodies) is minute compared to starch synthesis. However in
another cereal, oats, significant amounts of oil are produced in the endosperm. How the carbon
flux along these different routes is determined and regulated is not clear. Non-destructive NMRbased metabolic flux analysis has the power to resolve carbon flux into starch and other pools
and sinks within a complex metabolic context.
8. Metabolic Flux Analysis
In recent years, in vivo NMR/MS metabolic flux analysis (MFA) has become a major tool in
metabolic engineering of microorganisms, and it is now emerging as a powerful strategy also in
plant metabolic engineering (Krishnan et al., 2005; Schwender et al., 2004). In concert with
transcriptome and proteome analyses, MFA allows a systems biology approach to plant
metabolic engineering. The aim of MFA is a detailed quantification of all metabolic fluxes in the
central metabolism of an organism. The result is a flux map that shows the distribution of
anabolic and catabolic fluxes over the metabolic network. Based on such flux maps possible
targets for genetic modifications can be identified, the result of an already performed genetic
manipulation can be judged, or conclusions about the cellular energy metabolism can be drawn.
MFA with the stable “magnetic” 13C isotope is based on carbon labeling experiments. In such an
experiment a specifically 13C labeled substrate like e.g. [1-13C]-glucose or [U-13C12]-sucrose is
used as a tracer and fed to the biological system. The labeled carbon atoms are then distributed
all over the metabolic landscape and the isotopic enrichment in the intracellular metabolite pools
can subsequently be measured nondestructively by NMR. The resulting data provide a large
amount of information that is evaluated using advanced mathematical isotopomer models and,
assuming metabolic and isotopic steady-state during the experiments, the metabolic fluxes in a
given metabolic network can be calculated.
9. Impact on the Intestinal Microflora: Effects of Dietary Carbohydrate Composition in the Barley
Endosperm
Bacterial communities (microbiota) of considerable biodiversity populate the gastrointestinal tract
of animals, including humans, and influence the physiology, biochemistry, and immunology of
the animal host. Of the several hundred species of bacteria that colonize the large intestine,
bifidobacteria are generally considered to be health promoting and beneficial. Predominance of
bifidobacteria is now recognized as being essential for the prevention of diseases and maintaining
good health. Accordingly, considerable research is being directed at promoting the growth of
bifidobacteria in the large intestine. One main strategy being employed is the use of selective
carbohydrate substrates for the growth of indigenous bifidobacteria, the "prebiotic" approach. To
be effective, these carbohydrates must reach the colon undigested and unabsorbed in the upper
gastrointestinal tract and be selectively utilized by the resident bifidobacteria (Fig. 3). RS, ßglucans, AX and fructans are examples of such carbohydrates.
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Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010
Starches are important as energy sources for humans and also for their interactions with the
gut microflora throughout the digestive tract. In the large bowel, starches that have escaped small
intestinal digestion, i.e. RS, together with proteins, other undigested carbohydrates and
endogenous secretions are fermented by the resident microflora. The resulting short chain fatty
acids contribute substantially to the normal physiological functions of the viscera. Specific types
of resistant starch (e.g. the chemically modified starches used in the food industry) may be used
to manipulate the gut bacteria and their products (including short chain fatty acids) so as to
optimise health. In the upper gut, these starches may assist in the transport of probiotic organisms
thus promoting the immune response and suppressing potential pathogens.
Once in the large intestine, RS is extensively fermented by the microflora to short chain fatty
acids (SCFA), primarily acetate, propionate and butyrate. The production of SCFA helps lower
Fig. 3. Polysaccharides that escape digestion in the small intestine are subject to degradation by benign
bacteria in the large intestine. Fermentation products from this process, in particular butyric acid, are
important for colonic cell growth and differentiation. Other beneficial effects of these low-GI carbohydrates
are also listed
the pH of the gut and reduce levels of toxic ammonia in the gut and blood. Studies in both
humans and rats inoculated with human microflora have shown that fermentation of RS produces
significantly higher levels of butyrate in relation to acetate or propionate. Butyrate is readily
metabolized by the cells lining the colon, which derive about 60% to 70% of their energy from
bacterial fermentation products, such as butyrate. Butyrate is therefore an important regulator of
colonic cell growth and differentiation. This may explain why the incidence of colon cancer is
inversely related to the intake of starch, in particular RS, and that diets high in RS appear to
provide protection against colon cancer. Notably, dietary intake of RS is two- to fourfold lower in
the US, Europe and Australia compared to that of populations consuming high-starch diets, such
as in India and China. Of special interest is the fact that different indigestible carbohydrates
produce different patterns of SCFA during fermentation in the colon, and it is thus possible to
control the formation of SCFA through diet.
Apart from the known effects of RS and fructans on the microbial activity in the intestinal
tract, information on the relationship between different starches and fructans and the microflora is
relatively sparse and substantial opportunities exist both for basic research and food product
development. Furthemore, ß-glucans and AX have been neglected in prebiotic research, even
though they are major components of grains such as barley and oats and are therefore common in
the food of farm animals and humans.
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Tema-forskningsprogram
BarleyFunFood; Barley Functional Food
Program Plan 2008-2010
Steering Committee and Project Management Group
A steering committee with principal investigators or their deputies from the partners and the vice
dean of the NL faculty has been established. The elected chairman of the steering committee calls
for meetings. A project management group (PMG) with all principal investigators or their deputy
and the project leaders has also been established. The program leader calls for meetings.
References
Brennan SB, Cleary LJ (2005) J Cereal Sci, 42:1-13.
Duncan SH, Scott KP, Ramsey AG, Harmsen HJM, Welling GW, Stewart CS, Flint HJ (2003) Appl Environ
Microbiol, 69:1136-1142.
Krishnan P, Kruger NJ, Ratcliffe RG (2005) J Exp Bot, 56:255-265.
Macfarlane GT, Macfarlane S (2007) Curr Opinon Biotechnol, 18:156-162.
Schwender J, Ohlrogge J, Shachar-Hill Y (2004) Curr Opin Plant Biol, 7:309–317.
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