DIAS report
Plant Production no. 92 ◆ May 2003
Review
Wheat grain composition and
implications for bread quality
J. Emanuelson, B. Wollenweber, J. R. Jørgensen, S. B. F. Andersen & C. R. Jensen
Ministry of Food, Agriculture and Fisheries
Danish Institute of Agricultural Sciences
DIAS report Plant Production no. 92 • May 2003
Wheat grain composition and implications for bread quality
J. Emanuelson
The Royal Veterinary and Agricultural University
Danish Veterinary and Agricultural Library
Dyrlægevej 10
DK-1870 Frederiksberg C
B. Wollenweber & J. R. Jørgensen
The Danish Institute of Agricultural Sciences
Research Centre Flakkebjerg
Department of Plant Biology
Flakkebjerg
DK-4200 Slagelse
S. B. F. Andersen
The Royal Veterinary and Agricultural University
Department of Agricultural Sciences
Plant Breeding and Crop Science
Thorvaldsensvej 40
DK-1870 Frederiksberg C
C. R. Jensen
The Royal Veterinary and Agricultural University
Department of Agricultural Sciences
Laboratory for Agrohydrology and Bioclimatology
Agrovej 10
DK-2630 Taastrup
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Print: DigiSource
ISSN 1397-9884
J. Emanuelson, B. Wollenweber, J.R. Jørgensen, S.B.F. Andersen,
C.R. Jensen
Review
Wheat grain composition and implications for bread quality
J. Emanuelson
The Royal Veterinary and Agricultural University,
Danish Veterinary and Agricultural Library.
Dyrlægevej 10, DK-1870 Frederiksberg C
B. Wollenweber & J.R. Jørgensen
The Danish Institute of Agricultural Sciences,
Department of Plant Biology,
Research Centre Flakkebjerg, DK-4200 Slagelse
S.B.F. Andersen
The Royal Veterinary and Agricultural University,
Department of Agricultural Sciences,
Plant Breeding and Crop Science,
Thorvaldsensvej 40, DK-1870 Frederiksberg C
C.R. Jensen
The Royal Veterinary and Agricultural University,
Department of Agricultural Sciences,
Laboratory for Agrohydrology and Bioclimatology,
Agrovej 10, DK-2630 Taastrup
Contents
Abstract………………………………………………………………………….…3
Origin of domesticated wheat…………………………………………………..6
Kernel composition……………………………………………………………....7
Kernel quality; Genetic variation
Composition of the endosperm………………………………………………..12
Carbohydrates; Vitamins and minerals; Lipids; Proteins
Composition of grain proteins……………………………………………………15
Albumins and globulins; Gluten proteins; Gliadins; Glutenins;
Protein structure; Quality scores; Chromosome structure
Bread-making quality…………………………………………………………...28
Kneading; Additives; Grain; Flour; Dough; Baking
References………………………………………………………………………...33
3
Abstract
The origin of domesticated wheat and prerequisites for its cultivation are specified. Bread
wheat genetic background is a hexaploid species with six groups of seven chromosomes in
each group i.e. 2n=42 chromosomes with the genomes A, B and D and the genome constitution of AABBDD. Common wheat includes varieties that may have spring or winter growth
habits, hard or soft endosperm, red or white bran colour. Strong flours from hard wheat are
used for bread, while weaker flours from soft wheat are mainly used in cakes, pastries, bisquits and certain noodles (FAO, 1990-1998). Spring wheat is grown in areas with maximum
rainfall in the spring and early summer, and maximum temperature in mid and late summer.
These conditions favours a production of rapidly maturing grain with endosperm of vitreous
texture and high protein content (varying from 8-15 %), traditionally suitable for breadmaking
(Kent and Evers, 1994; Posner, 2000). Winter wheat is grown in climates of relatively even
temperatures and rainfall.
Kernel composition, hardness and strength are dealt with in details. Wheat kernels are made
up of three major parts - the germ, the bran and the endosperm. Starch is the carbohydrate
present in the greatest amount in the endosperm and is enclosed in granules in the endosperm.
These granules are embedded in a protein matrix. The protein matrix found in hard wheats
tends to be tough and to hold starch granules tightly. Wheats are classified according to the
texture of the endosperm (vitreous or mealy) and the protein content and to the amount of αamylase. The genetic basis for the two major texture classes (hard and soft) is well established
as a single major gene whose allelic expression is associated with mutations in the protein
puroindoline a and b. In general high quality varieties produce good bread over a wide range
in protein content, and poor bread varieties produce poor bread quality even if the protein
content is high due to fertilization. Genetic variation in the gluten quality of wheat for breadmaking appears to derive directly from differences in the structure of the glutenin components
and several independent features of the glutenin structure appears to be involved.
There are four different types of proteins in a wheat kernel: albumins, globulins, gliadins and
glutenins. White flour contains mainly the endosperm storage proteins, the gluten proteins
consisting of a mixture of gliadins and glutenins. Albumins and globulins (the non-gluten proteins) are biologically active proteins and are responsible for starch breakdown and other enzymatic activity. The albumins are mainly enzymes - carbohydrases as α-amylases, βamylases or glucosidases, or proteolytic enzymes etc. High molecular weight (HMW) albumin subunits are mainly β-amylases. The globulins are divided into two groups, the 7S and
11S globulins. Both the 7S and 11S globulins are composed of multiple subunits. The 7S and
11S globulins are characterized by low contents of sulphur containing amino acids. The 7S
globulins are trimeric, comprising three subunits of MW 40,000-60,000 Da and resulting in a
protein of MW 150,000-190,000 Da. The subunits lack cysteine residues and are unable to
form disulphide bonds. The trimeric structure is only stabilised by non-covalent forces. An
11S globulin related protein, called triticin, is present in small amounts in the wheat en-
4
dosperm cells. The 11S globulins are hexamers comprising six subunits of MW 60,000 Da
and are polymeric storage proteins. The large globulin subunits contain lysine residues.
The gluten proteins (63-90% of total wheat protein) are traditionally classified into two
groups the gliadins and the glutenins. When kneading/mixing flour with water, gluten proteins
enable the formation of a cohesive visco-elastic dough that is capable of holding carbon dioxide and oven-rise, resulting in a fixed open foam structure of bread after baking.
The gliadins are monomeric proteins and mostly connected by intrachain polypeptide disulfide linkages. The α- (α- and β-gliadins) and γ-gliadins has disulphide bonds mainly formed
within the polypeptide chain taking part in formation of gluten polymers via interchain disulphide linkage are restricted, but their SS bonds are important in retaining the folding structure
that determines the nature of non-covalent bonding. The ω-gliadins is limited in their interactions in dough to non- covalent forces, primarily hydrogen bonds.
The glutenins consist of polymeric proteins characterized by interpolypeptide cross-linking by
disulphide bridges and are classified as (HMW subunits) prolamins and sulphur-rich (LMW
subunits) prolamins. The HMW subunit glutenins form the ’elastic backbone’ of gluten. The
HMW subunit glutenins comprise three separate domains, a central domain, which forms a
loose spiral structure (β-spiral), flanked by globular N- and C-terminal domains, which contain most or all of the cysteine residues that forms the interchain disulphide bonds. These disulphide-stabilised polymers are considered to form an elastic network, which interacts with
other gluten proteins, by disulphide bonds (HMW and LMW subunits) and non-covalent interactions, primarily by hydrogen bonds (gliadins).
The quality of gluten is genetically determined and varies between cultivars. The genotypic
variation in the contribution of glutenin to breadmaking quality is due to variation in the
number of specific HMW subunits.
Altogether over 50 individual components can be resolved by two-dimensional electrophoresis of total gluten proteins, and molecular and chemical analyses indicate, that most of these
are the products of single genes. The genes have been mapped using classical Mendelian
analysis as well as modern methods.
The Mixograph proved to be a powerful tool to investigate indices of bread making quality.
5
Origin of domesticated wheat
Domestication of wheat started more than 10.000 years ago with culture of einkorn wheat
(Triticum monococcum) (Heun et al., 1997), and the modern hexaploid bread wheat evolved
about 8.000 years ago. Wild wheats are commonly divided into two subgroups: goatgrasses in
the genus Aegilops L., and wild wheats in the genus Triticum L. All cultivated wheats are included in the genus Triticum. Wild species in Aegilops and Triticum occur in polyploid series,
based on a haploid number of n = x = 7 chromosomes. Diploid (2x), tetraploid (4x) and
hexaploid (6x) species of Aegilops occur naturally (24 wild species), whereas wild species of
Triticum only occur at the diploid and tetraploid levels. Cultivated wheats exist at the diploid,
tetraploid and hexaploid levels. The (about 25) species of the genus Triticum L. are divided
into three groups according to their chromosome number in diploid, tetraploid and hexaploid
species (Waines, 1994).
In agriculture today the following Triticum spp. are cultivated: common or bread wheat (T.
aestivum), durum (T. durum) and spelt (T. spelta). Common wheat is the species known as
wheat. Wheat is the most widely grown small-grain cereal of the Triticum species (more than
95%) and accounts for about 30% of the cereals crop production in the world (FAOSTAT database 1998). Wheat is the main species used for breadmaking around the world, and wheat
grain is also important as livestock feeds (Kent and Evers, 1994; Bushuk, 1998; Kelly and
George, 1998). Currently about 4,000 different wheat varieties are grown around the world
(Posner, 2000).
Triticum aestivum subspecies vulgare
Common or bread wheat - is a hexaploid species with six groups of seven chromosomes in
each group i.e. 2n=42 chromosomes with the genomes A, B and D and the genome constitution of AABBDD. The tetraploid species T. turgidium ssp. dicoccum was formed through hybridization of T. urartu (AA) with an unknown diploid B genome (possibly Aegilops speltoides). The dicoccum wheat was cultivated and later a hexaploid species was produced
spontaneous through hybridization with a weedy goatgrass T. tauschii (Aegilops squarrosa)
with the diploid D genome. The D genome greatly increased the stress tolerance (to drought,
heat or cold) (Waines, 1994). The AA, BB and DD genomes of wheat are closely related and
its 42 chromosomes have been classified into seven homoeologous groups, each composed of
three similar chromosomes (Vasil and Vasil, 1999).
Common wheat includes varieties that may have spring or winter growth habits, hard or soft
endosperm, red or white bran colour. Strong flours from hard wheat are used for bread, while
weaker flours from soft wheat are mainly used in cakes, pastries, bisquits and certain noodles
(FAO, in the definition to the FAOSTAT database 1990-1998). One fifth of the world wheat
production is used in animal feeds (International Wheat Council, 1992).
6
Spring and winter wheat
Spring wheat is grown in areas with maximum rainfall in the spring and early summer, and
maximum temperature in mid and late summer. In areas where the winter season is too cold
or in areas with heavy snow covers wheat is cultivated as a spring crop (as in the northern part
of Scandinavia). These conditions favour a production of rapidly maturing grain with endosperm of vitreous texture and high protein content, traditionally suitable for breadmaking.
Spring wheat varieties have generally a better baking quality due to higher gluten content but
also a smaller yield compared to winter wheat varieties. Spring crop varieties can also be cultivated as a winter crop in areas with a warmer climate, winter precipitation and summer
drought. Spring wheat is either day-neutral or a long-day plant without vernalisation needs
(Kent and Evers, 1994; Kelly and George, 1998; Posner, 2000).
Winter wheat is grown in climates of relatively even temperatures and rainfall and it will be
grown in preference to spring wheat. In the fall the plant establish a root system before the
dormancy period starts and in spring there is no delay in waiting until the field becomes sufficiently dry to cultivate. The crop matures more slowly producing a higher yield with lower
protein content. Wheat is cultivated as a winter crop in many European countries. Winter
wheat is a long-day plant and requires vernalisation (Kent and Evers, 1994; Kelly and George,
1998).
Other Triticum species
Durum wheat (T. durum) - is used primarily as pasta but with an increasing importance of durum wheat in Italian bread types. Durum wheat is a tetraploid species and 2n=28 chromosomes with the genomes A and B. Nearly all of the remaining 5 % of the cultivated wheat is
durum. The yield of durum wheat is lower than that of bread wheat and it is grown in drier areas (Bushuk, 1998).
Spelt (T. spelta.) is grown on small scale and mostly in mountain regions in Europe, even
though it has obtained a new actuality in organic farming, while it demands less nutrients and
it is appreciated in the baking industry. Spelt is harvested before ripening because of a fragile
rachis (speltoid characteristics). Spelt is a hexaploid species with 2n=42 chromosomes with
the genomes A, B and D (Kelly and George, 1998; Vasil and Vasil, 1999).
Einkorn wheat (T. monococcum) – a diploid species with 2n=14 chromosomes with the A genome is only cultivated occasionally as animal feed (Vasil and Vasil, 1999).
Kernel composition
Wheat kernels are made up of three major parts - the germ, the bran and the endosperm. The
germ is an embryonic plant. It makes up about 2-4 percent of a wheat kernel by weight. The
germ consists of the embryo and the scutellum. The germ will grow into a new wheat plant if
the kernel is planted. The germ has high concentrations of non-gluten proteins, lipids, vitamins and minerals (ashes) (Posner, 2000). The bran (7-8 percent of kernel weight) is the outside covering. The bran consists of several cellulose layers. It is made up of tissues high in fi7
bre that protect the kernel, and tissues high in protein that digest starch. The vitamin, mineral
and lipid content are high in the bran. The volume of bran is smaller in large than in small
grains from samples of the same types of wheat. The bran and aleurone layer are separated together during milling (about 15 % of kernel weight) (Posner, 2000).
The endosperm (81-84 percent of kernel weight) is on the inside of the kernel. It is comprised
of starch and protein. The endosperm is the food source for the embryonic plant and becomes
when milled the material we commonly call flour. The maximum yield of pure flour is
roughly 82 percent - the amount of endosperm in a kernel. However, normal rates are generally lower than this (70-75 percent) because a complete separation of bran and endosperm are
not possible. The aleurone cell layer is a part of the endosperm and has a high soluble protein
and mineral content. The aleurone cell layer is separated with the bran, and constitutes of
about 5-8 % of the wheat kernel. Incomplete separation is evidenced as coloured specks
(pieces of bran) in white flour (Posner, 2000).
Kernel quality
Wheats are often classified according to the texture of the endosperm and the protein content
and to the amount of α-amylase.
Texture
The endosperm texture may be vitreous (steely, flinty, glassy, horny) or mealy (starchy,
chalky) (Dowell, 2000). The opacity of mealy kernels is an optical effect due to the presence
of minute vacuous or air-filled fissures between and perhaps within the endosperm cells. The
fissures form internal reflecting surfaces, preventing light transmission, and giving the endosperm a white appearance. Such fissures are absent from vitreous endosperm (Kent and
Evers, 1994; Haddad et al., 1999). The development of mealiness seems to be connected with
maturation. Immature grains of all wheat types are vitreous.
Vitreous grains are found in spring wheat varieties (with short growth period and quick ripening) and in wheat varieties growing at dry continental climates. Vitreousness can be induced
by nitrogen fertilizers and is positively correlated with high protein content. Mealy grains are
characteristic of varieties that grow slowly with a long maturation period. Mealiness are favoured by heavy rainfall, light sandy soils and dense planting and positively correlated with
high grain yielding capacity. The character is hereditary but is also affected by the growth
conditions (Kent and Evers, 1994).
Kernel hardness
Kernel hardness is strongly correlated to functionality of the grain during processing. It determines the type of milling process and the physical nature of the milled product. It is the
kernel hardness which generally determines the end-use (Pomeranz and Williams, 1990;
Bushuk, 1998). The milling times, milling energy requirements, and the amount of starch
damage produced in the milled flour are all influenced by grain hardness (Anjum and Walker,
1991; Pena, 1999). Hardness and softness are milling characteristics related to the way the
endosperm breaks down and related to the degree of adhesion between starch granules and the
8
surrounding protein. The ease of sifting is also affected by other parameters such as the moisture content. Unfortunately, wheat hardness has never been clearly defined, and published results are often expressed in arbitrary units that sometimes do not accurate reflect the actual
physical situation (Pujol et al., 2000).
Hard wheats
Hard wheats yield coarse, gritty flour, which is easily sifted in the milling processing. During
milling breakage occur along the cell walls and across this interface. Generally harder wheats
tend to be higher in protein content, although not in the entire range of kernel hardness
(Greenway, 1969; Anjum and Walker, 1991; Kent and Evers, 1994). Hard wheat kernels have
a continuous protein matrix that physically traps starch granules and causes hardness
(Stenvert and Kingswood, 1977). Varieties with hard textured endosperms will suffer much
more starch damage during milling than soft textured wheats, and their flours will consequently absorb more water. Since the water content of dough’s is optimised from each sample, this is likely to increase bread volume since a fixed weight of dry flour is used in official
bread quality testing for ranking on the National List (Payne et al., 1987a; Anjum and Walker,
1991).
Soft wheats
Soft wheats gives very fine flour consisting of irregular-shaped fragments of endosperm cells,
the particles adhere together and sifting of the flour is difficult. Intracellular spaces exist
around the starch granules of soft, but not of hard wheat, forming a discontinuity in the starchprotein matrix. This physical discontinuity provides a natural path for shearing forces during
kernel disruption, leading to softer material that is more easily reduced in particle size
(Stenvert and Kingswood, 1977; Glenn and Saunders, 1990). The breakage occurs between
the interface and through cell walls, the subaleurone endosperm cells tends to fragment into
some portion coming away while the rest remains attached to the bran. Grains of soft wheats
tend to have a weak, crumbly protein matrix. This matrix holds starch granules loosely and
tends to release whole granules when milled and the flour is composed primarily of whole
starch granules. Encapsulated starch absorbs less water than free starch. Soft wheats tend to
have lower water absorbing capacity than hard wheats and therefore generally result in
smaller bread volumes with a fixed weight of dry flour.
Flour yield
The flour yield is a simple measure on how much flour that can be obtained relative to the
weight of whole grains. The maximum yield of white flour in the milling of kernels is dependent of the endosperm content and is affected by the size and shape of the grain and the
thickness of the bran. The amount and quality of protein in flour will influence the amount of
water that it will hold. Ultimately, water makes up about 14% in flour, 45% in bread dough
and about 35% in the bread itself. The classification of wheat types as hard or soft, and as
strong or weak are shown in Table 1.
9
Table 1. Summary of grain quality characteristics (Hansen and Poll, 1999; Kent and
Evers, 1994).
Wheat species endosperm hardness grain protein content gluten properties/ baking strength
spelt
soft
low/medium
weak
winter wheat
soft/medium
low
weak
hard
medium
medium
spring wheat
hard
high
strong
durum wheat
extra hard
high
strong
α-amylase activity
The α-amylase activity is strongly influenced by the growth conditions either from frequent
rainy weather or if the crops lodges severely and the heads are in contact with the wet soil
(Kettlewell, 1996a). Four modes for accumulation of α-amylase activity, possibly leading to
low Hagberg falling number have been described in UK wheat varieties (Kettlewell and
Cashman, 1997; Lunn et al., 2001a; Lunn et al., 2001b). Three of the modes occur relatively
early in wheat grain development. The first early mode is termed retained pericarp α-amylase,
which has been identified as a possible cause of cyclic variations in pre-harvest Hagberg falling number of Scandinavian crops. It is regarded as the least important mode. The second
early mode is termed pre-maturity pericarp α-amylase and is due to deposition of α-AMY-1
isozymes in the endosperm cavity of the grain in particular genotypes in some environments.
The third (little recognised) early mode is termed pre-maturity sprouting and it involves germination early in development when the wheat grains are still of high moisture content, normally above 35%. The fourth (well recognised) mode, which is regarded as the most important phenomenon, is termed pre-harvest sprouting. Pre-harvest sprouting appears to form a
continuum of pre-maturity sprouting, but is discriminated by the definition of maturity as 35%
grain moisture.
The action of α-amylase decreases the size of the starch molecule and therefore reduces the
viscosity of a starch solution. High α-amylase activity causes more water to be released from
the surface of starch granules than normal, due to excessive breakdown of starch to maltose.
The α-amylase becomes active during dough handling and the early stages of baking.
Tests that measure viscosity, such as the Hagberg falling number test (Hagberg, 1960) and the
amylograph, are used to assess α-amylase activity to determine flour quality and early sprouting of wheat grains. Excessive α-amylase activity impairs wheat grain quality since enzymatic hydrolysis of starch during food manufacture can lead to processing problems and unsatisfactory end-products (Lunn et al., 2001b).
The α-amylase activity of flour is high if the germ has started to germinate. High α-amylase
activity in harvested grain can occur if dormancy breaks before harvest and if the grain is
moist enough to germinate. Hagberg Falling Number Value is a measure of sprouting resistance or of α-amylase enzyme activity and the analysis tells about germination of the germs.
If the starch has started to germinate then starch is being degraded. Excessively high levels of
α-amylase impair the quality of both the dough and the final bread because α-amylase cleaves
the starch molecules reducing the paste viscosity, and can lead to sticky dough’s. Some varie10
ties contain genes from rye which causes a decrease in bread-making quality, mainly (Payne,
1987). Unlike α-amylase, β-amylase is present in not field-sprouted grain, but it remains practically unchanged during germination (Rasper and Walker, 2000).
Genetic variation
The genetic basis for the two major texture classes (hard and soft) is well established as a single major gene whose allelic expression is associated with mutations in the protein puroindoline a and b (Baker, 1977; Anjum and Walker, 1991; Bettge and Morris, 2000). However
exceptional weather conditions such as excessive rainfall during the harvest period, N management, tillage system, pest infestations, and their interactions can influence grain hardness
strongly (Bushuk, 1998). Grain exposed to warm dry climates during the fruiting and filling
periods tends to be harder in texture and has a higher protein content (Bergman et al., 1998).
In general high quality varieties produce good bread over a wide range in protein content, and
poor bread varieties produce poor bread quality even if the protein content is high due to fertilization (Kent and Evers, 1994).
Kernel hardness (milling character) and strength (baking character) are inherited separately
and independently. The amount of free lipid present in flour is largely genetically determined
and only weakly correlated with applied nitrogen fertilizers, flour protein or season effects.
Hard grain cultivars have higher free lipid levels than the soft grain cultivars (Panozzo et al.,
1993).
There exist a negative correlation between grain yield and protein concentration. Characters
ranked in decreasing order of influence from cultivar (or increasing order of influence of the
growing environment): hardness, protein quality, α-amylase activity, test weight (the weight
of a fixed volume of grain), thousand kernels weight (TKW in gram per 1000 kernels), protein concentration, kernel size, dockage (Kettlewell, 1996a; Kettlewell, 1996b).
Among wheat cultivars used for breadmaking there are a linear relationship between flour
protein content and bread volume. A wide range of quality is possible giving differences in
the slope of the protein content – bread relationship (Bushuk, 1998).
In wheat cultivars of similar hardness, the genotypic variation in protein quality depends almost entirely on variations in the glutenin components of the flour. The amounts of acetic
acid insoluble glutenin are directly related to bread volume (Orth and Bushuk, 1972). Biochemical and genetic studies have identified a specific group of gluten proteins, the HMW
glutenin subunits to be the most important determinants of bread-making quality (Payne et al.,
1987b; Vasil and Vasil, 1999). Some varieties contain genes from rye, which causes a decrease in bread-making quality, mainly by increasing the dough stickiness (Payne et al.,
1987b).
Genetic variation in the gluten quality of wheat for breadmaking appears to derive mostly
from differences in the structure of the glutenin components. Several independent features of
the glutenin structure appears to be involved (Bushuk, 1998):
1. contents of glutenin relative to gliadin (not highly variable among genotypes),
2. average molecular weight and the molecular weight distribution of polymeric glutenin,
11
3. relative amount of HMW and LMW subunits,
4. allelic composition of HMW and LMW subunits, and
5. Presence or absence of some specific subunits (Payne quality scores).
In particular, the Payne quality scores based on electrophoresis (Payne et al., 1987b) have
been widely used for early selection in breeding programs for bread making quality. In addition to presence or absence of different HMW glutenin subunits also their relative amount has
been shown to affect dough characters and bread volume (Wieser and Zimmermann, 2000)
and a detailed molecular understanding of picture of the genetic structure of HMW glutenins
is being revealed (de Bustos et al., 2001) which will enable very efficient PCR based selection
for desirable alleles in the future. However, in addition to effects from HMW glutenin subunits also some LMW glutenin and some gliadin subunits affect quality (Branlard et al., 2001;
Rousset et al., 2001). Recent development in QTL mapping in wheat indicates considerable
genetic effect on bread making quality from QTLs unrelated to glutenins and gliadins (Blanco
et al., 1998; Perretant et al., 2000) which may be used in future breeding programs.
Figure 2 shows the factors affecting wheat dough rheological properties.
Composition of the endosperm
Endosperm is composed of three major components - starch, protein and other materials such
as water, oils and fibre. Starch makes up roughly 70 percent of the endosperm, protein 8 to 15
percent or more, water about 14%, oils and fibre the remaining portion.
The concentration of protein in grain is a balance between accumulation of protein and starch
(Schnyder, 1993), which originates mainly from photosynthesis during kernel growth and retranslocation from stem reserves. A change in the quantity of either protein or starch will impact on the protein% of the grain and consequently its apparent quality. Limited nitrogen conditions within the plants will lead to a competition between carbon (C) and nitrogen (N)
metabolism for energy- (ATP) and reduction- (NADPH) equivalents. Due to lack of available
amino-groups, less carbon-skeletons will be used for amino acid synthesis while carbohydrate
synthesis increases. On the other hand, since a large proportion of nitrogen is stored as a component of the photosynthetic system in the essential enzyme RUBISCO, extensive nitrogen
retranslocation will be associated with reduced photosynthetic activity (Gregory et al., 1981;
Al Khatib and Paulsen, 1999). Therefore less photosynthate will be available to contribute to
yield, but grain protein concentration will be high (Stoy and Bertholdsson, 1984; Giese and
Andersen, 1984). This relationship between nitrogen retranslocation and photosynthesis is a
major reason for the typical negative correlation between grain yield and grain protein concentration (Benzian and Lane, 1981; Morris and Paulsen, 1985; Simmonds, 1995).
Nitrogen is a major component of protein, and nitrogen supply to cereal crops is the principal
factor influencing grain protein concentration. Nitrogen fertilizer is one of the most-used tools
for altering grain yield and quality, based upon the knowledge that it can increase grain yield,
grain protein percentage, or both (El Negoumy et al., 1982; Kirkman et al., 1982). Addition of
N from a low base level increases yield, whereas that from a higher base level increases pro12
tein percentage. Much of the protein in the grain derives from nitrogen taken up during the
vegetative phase and is subsequently remobilized and translocated from senescing leaves and
other green plant parts.
Variation of N application strategies can have a beneficial effect for grain yield. In particular,
the timing of nitrogen supply has considerable influence on protein concentration (Bulman et
al., 1994). Early applications at seeding or stem-elongation can increase yield since they are
able to influence the size of the leaf canopy and thereby the supply of photosynthate for increasing both head number and grain number per head, i.e. the two components of the sink
organs (Rhodes and Mathers, 1974). The application of early-season nitrogen however may
not always be sufficient to maintain protein accumulation throughout the grain-filling period.
Late applications in early grain growth cannot increase the size of the leaf canopy because
tillering and leaf expansion have ceased, thus the sink size is frequently the limiting factor for
yield (Winkler and Schön, 1980). Applications of fertilizer at the beginning of kernel growth
have little or no effect on yield, but have the greatest effect on protein concentration. Any restriction to uptake of nitrogen after anthesis (e.g. low soil fertility, water deficits) will increase
the proportion of grain protein derived from retranslocation from green parts of the plant.
However, this is often accompanied by reductions in the proportion of several essential amino
acids in the grain protein including lysine, threonine, cysteine and methionine (Pomeranz et
al., 1977; Bulman et al., 1994).
Carbohydrates
Starch is the carbohydrate present in the greatest amount in the endosperm. Starch is a polymer of D-glucose most of the hexose sugar molecules being joined together by α-(1-4) bonds.
Starch is an association of two polysaccharides, the straight-chained amylose and the
branched-chain amylopectin (about 70-80% by weight of the whole granule starch), with side
chains attached to α-(1-6) bonds (Stone, 1996).
Starch is enclosed in granules in the endosperm. These granules are embedded in a protein
matrix. The protein matrix found in hard wheats tends to be tough and to hold starch granules
tightly. Some of these tightly held granules tend to be sheared or broken in the milling process
and release their starch contents, about 4-10 pct of the starch granules are damaged during the
milling process which leads to the release of sugars into the flour. Free starch can absorb large
amounts of water.
The granules are divided into different types due to their size: the A-type granules being lenticular and 15 to 40 µm in size and the B-type granules being spherical, small granules and 1
to 10 µm in size (Matz, 1991). Polysaccharides other than starch are found in cell walls, such
as pentosans (arabinoxylans and β-D-glucans). Cell walls of the lignified bran layers contain
appreciable amounts of cellulose (Matz, 1991). Wheat endosperm and flour contains only minor amounts of dietary fibre. Bran contain on the average about 43 % dietary fibre. The bran
that is removed during the various stages of the milling process is made up of fractions that
differ in size and endosperm content (Posner, 2000).
The bulk of the starch derives from photosynthesis during kernel growth, about 10% derives
from retranslocation from stem reserves (Blum, 1998). Relatively cool temperatures during
13
kernel growth promote the formation of carbohydrates, prolong the seed filling duration and
result in larger quantities of starch and consequently lower content of protein. Whereas relative high temperatures with lower soil moisture content after anthesis reduces the photosynthesis and shortenes the seed filling duration, resulting in lower grain yield of higher protein
content (Pomeranz, 1968). Genotypic and environmental factors affects the carbohydrate reserve accumulation and remobilisation for grain filling (Blum, 1998).
Vitamins and minerals
Vitamins and minerals are found in high concentration in germ and bran. The grain is considered as a significant source of the vitamins thiamin, niacin, riboflavin and tocopherol (Fujino
et al., 1996).
The ash content does not affect the baking quality of the flour and it relates basically to the
level of bran in the flour. The ratio of bran to endosperm may be higher in small kernels
(Posner, 2000). Minerals represent about 1.6 percent of the grain and decreases to much lower
levels on milling to about 0.4 percent (Fujino et al., 1996).
Lipids
Lipid content of wheat grains range from 2-4 %. Lipids are found in all tissues with triglycerides being more abundant in the aleurone and embryo and present in low levels in the endosperm while phospholipids and glycolipids are present at higher concentrations in the endosperm. Lipids represent about one percent of the starch granules (Fujino et al., 1996). The
embryo contains about one third of its weight as oil. The endosperm is lowest in oil and the
outer layers have an intermediate content (Posner, 2000).
Free lipids constitute about 1-2% of flour weight. The amount of free lipids (hexane extractable lipids) and especially the glycolipids (polar fraction) are highly correlated with bread
volume (Bekes et al., 1991; Matz, 1991). The increase is affected by small additions; large
amounts have no additional improving effect. The lipids seem to satisfy certain functional
properties. Once those requirements are met, no additional benefit can be derived by adding
more lipids (Matz, 1991).
Proteins
Protein is the other major part of the endosperm. Protein makes up roughly 8 to 15 percent of
a wheat kernel. Factors favouring protein accumulation like dry, sunny weather, nitrogen fertilizers and spring-sown varieties will increase grain protein and vice versa. Much of the protein in the grain derives from nitrogen taken up during the vegetative phase and subsequently
remobilized and translocated from senescent leaves and tillers (Stone and Savin, 1999). The
protein content is strongly influenced by the environment and by fertilizer application,
whereas the composition of the storage proteins is determined mainly by the genotype. The
composition of grain proteins varies with climate, genotype, nitrogen fertilizers and methods
of measurement (Stone and Nicolas, 1996; Stone and Savin, 1999).
There exist a curvilinear increase in protein content in response to nitrogen fertilizers. Breadmaking quality increases with the N supply, and reaches a peak at an N supply level above
14
that needed to achieve maximum yield (Borghi, 1999). After this peak the protein quality decreases with increasing protein content because the extra N accumulated in the grain is represented by gliadins or nonprotein nitrogen, which depress the breadmaking quality (Borghi,
1999).
The content of the essential amino acid lysine in wheat is lower than in other cereals. Increase
in protein content of the grain results in increase in lysine per unit weight of grain. As the
grain protein level is increased, the level of lysine decreased. There exist a negative curvilinear correlation between the lysine content (% of protein) and the protein content, as the grain
protein level increases most pronounced between 10-15 % range of protein (Aykroyd and
Doughty, 1970; Bajaj, 1990).
Composition of grain proteins
There are four different types of proteins in a wheat kernel: albumins, globulins, gliadins and
glutenins. White flour contains mainly the endosperm storage proteins, the gluten proteins
consisting of a mixture of gliadins and glutenins.
The systematics of cereal proteins was classified into groups on the basis of their solubility by
Osborne (1924) as shown in Table 2.
Table 2. Solubility of wheat proteins
Proteins
Albumins
Non-gluten proteins
Gluten proteins
Soluble in
Location in
water
embryo (metabolic proteins) and
endosperm cells (cytoplasmic
proteins)
embryo and aleurone layer (storage
proteins) and endosperm cells
(cytoplasmic proteins)
Globulins dilute salt solutions (O.5 M NaCl)
Gliadins 70-80% ethanol
Glutenins dilute acids or alkalis solutions
(0.05 M Acetic acid)
endosperm (storage proteins)
Fractionation on a solubility basis, followed by Kjeldahl nitrogen analysis of each protein
fraction made an accurate method to determine the grain protein composition, and this simple
method of separation and classification forms still the basis of modern cereal chemistry. Modern methods have enabled a subdivision of the wheat proteins into different classes by polymerisation with inorganic oxidising agents and separated by electrophoresis. In Table 3 the protein composition of a typical wheat grain is shown with references to researchers from
different countries.
15
Table 3. Protein composition of typical wheat grain.
I, Australia (Stone and Savin, 1999), II, USA (Shukla, 1975); III, Canada (Orth et al., 1972);
IV, Germany (Wieser et al. 1980);V, FAO (Aykroyd and Doughty, 1970).
Proteins
Non-gluten
I
II
III
IV
V
Albumins
22%
3-5%
8-9%
15%
Globulins
15%
10%
4-5%
7%
Gliadins
40%
69%
35-40%
33%
45%
Glutenins
23%
16%
35-50%
46%
40%
15%
Gluten
The protein fractions varied between varieties as well. In flours of 26 wheat varieties (grown
at one location in Canada) the protein fractions varied between 3.5 and 10.5 % in albumin cotent, 3.5 and 6 % in globulin content, 30 to 42 % in gliadin content and between 38 and 55 %
in glutenin content. The glutenin content was separated into glutenin content between 6 and
27 % and in residue protein content between 15 and 37%. Some of the proteins (between 3 to
13%) were lost during dialysis of the salt extracts (Orth and Bushuk, 1972).
The supply of nitrogen influences the balance of protein fractions, which accumulate in the
grain. Both the rate and duration of synthesis differ for each of the major protein fractions. In
addition, the deposition of the different classes of proteins is highly asynchronous as both the
balance and the amount of the different protein fractions varies throughout grain growth
(Gupta et al., 1996; Stone and Nicolas, 1996). The metabolic proteins (albumin and globulin)
are the first to accumulate in appreciable amounts. They make up approximately 90% of the
total grain protein in the first ten days of grain growth. Even though they are deposited in the
grain for most of the grain-filling period, the proportion of albumin and globulin in total grain
protein declines during grain growth, and make up 20-30% at maturity. This decline occurs
because the synthesis of storage protein (70-80% of the mature protein mass) occurs somewhat later in grain filling (Lásztity, 1996). Gliadins are the first storage proteins to accumulate
in readily measurable amounts (10% of the total protein) five to ten days after anthesis. Glutenins/prolamins are frequently the last of the proteins to appear in the grain, and may not be
present in significant quantities until 20 days after anthesis, reaching a maximum of 30 to
40% in the grain at harvest (Stone and Nicolas, 1996; Lásztity, 1996). This asynchronicity of
fractional protein deposition means that causes of disturbance in grain growth are also likely
to affect grain protein composition, via disruption during a certain phase of grain growth (and
so protein deposition) or through a general truncation of grain filling. The total protein concentration increases with nitrogen (Byers et al., 1978; Eppendorfer and Eggum, 1994; Simmonds, 1995). For wheat, barley and maize the prolamin protein fractions increase most in response to increasing supply of nitrogen.
However, while the proportion of non-essential amino acids (glutamate, glutamine and asparagine) in the glutelin/prolamin fraction increases, the accompanying decrease in essential
16
amino acids will result in a lower nutritional quality. Thus, as the amino acid that is available
in least supply (lysine, threonine and methionine) in relation to the animals requirement determines the extent of protein synthesis and thus the protein-value of the diet, the availability
and balance between (non-) essential amino acids in feed ingredients is now becoming a
greater concern as feed often has to be supplemented by alternative protein sources.
Increased knowledge of the physiology of the fractional protein deposition would allow the
manipulation through agronomic means. Therefore there is increasing interest in the repercussions of the reduced nitrogen fertilizer quotas for the nutritional value in cereals. Practices
such as specifically timed fertilizer application and reducing environmental stress events at
given stages of grain growth may be used to intelligently alter grain protein composition and
consequently grain quality.
Albumins and globulins
Albumins and globulins (the non-gluten proteins) are water and salt-water soluble proteins,
respectively. They are biologically active proteins and are responsible for starch breakdown
and other enzymatic activity. While albumins are metabolic proteins, the globulins are storage
proteins located in protein bodies in cells of the embryo and aleurone layer. A major fraction
of albumins and globulins are enzymes (i.e. amylases and proteases). During germination and
growth of the seedling the enzymes make nutrients and energy available by their hydrolytic
and proteolytic capacity (Stone and Savin, 1999).
In comparison with the gluten proteins, albumins and globulins has a better spectrum of essential amino acids (higher amount of ionic and S-containing amino acids i.e. Lysine, Arginine and Aspartic acid). This quality is nullified when considering on the overall grain composition because of the high content of gluten proteins (Shukla, 1975).
The albumins and globulins play only a minor role in the protein interactions, which are required in the formation of cohesive gluten. They are cytoplasmic proteins of the endosperm
cells and may be involved in some of the protein-protein interactions relating to the gluten
proteins but they are considered to be less significant as that of gliadins and glutenins
(Wrigley et al., 1998).
Most of these proteins are primarily located in the embryo, which are excluded from flour
during the milling process (Stone and Savin, 1999), but albumins and globulins are found to
be present in all three mill fractions – bran, embryo and endosperm (Shukla, 1975).
Albumins
Albumins can be divided into 6 different classes with molecular weight (MW) between
17,000 to 28,000 Da. The six classes are different in electrophoretic properties regarding
charge and ion mobility. Many albumins are known to be glycoproteins.The albumins are
mainly enzymes - carbohydrases as α-amylases, β-amylases or glucosidases, or proteolytic
enzymes etc. (Matz, 1991) High molecular weight (HMW) albumin subunits are mainly βamylases (Gupta et al., 1996).
17
Globulins
The globulins are divided into two groups, the 7S and 11S globulins. Both the 7S and 11S
globulins are composed of multiple subunits. The 7S and 11S globulins are characterized by
low contents of sulphur containing amino acids (Shewry, 2000).
The 7S globulins probably serve a role as seed storage proteins and are placed in embryonic
and aleurone tissues, and their hydrolysis during germination may provide an immediate resource of nitrogen and carbon to the young seedling. The 7S globulins are similar to other
seed storage proteins in containing relatively high amounts of glutamate, glutamine and arginine. The 7S globulins are trimeric, comprising three subunits of MW 40,000-60,000 Da and
resulting in a protein of MW 150,000-190,000 Da. The subunits lack cysteine residues and are
unable to form disulphide bonds. The trimeric structure is only stabilised by non-covalent
forces (Shewry, 2000).
The 7S globulins are highly regulated and co-ordinated via gene expression patterns during
seed development; they have a positive regulation of expression by ABA. The identification
of recombinant DNA clones corresponding to 7S globulins in both wheat and barley was the
result of screening for genes which were inducible by the phytohormone ABA (Kriz, 1999).
An 11S globulin related protein, called triticin, is present in small amounts in the wheat endosperm cells. The 11S globulins are hexamers comprising six subunits of MW 60,000 Da
and are polymeric storage proteins. The large globulin subunits contain lysine residues
(Shewry, 1996).
Proteolysis of the subunits occurs to give products of small MW ~20,000 Da (basic) and
large subunits MW 40,000 Da (acidic) associated by an interchain disulphide bond. These
products are referred to as ’subunit pairs’. The six subunits again associates by non-covalent
forces, giving an protein of MW 320-450,000 Da (Shewry, 2000).
Gluten proteins
The gluten proteins are traditionally classified into two groups the gliadins and the glutenins.
Previous the gliadins were grouped as prolamins and the glutelins as glutenins. Comparison of
the amino acid sequences led to an alternative classification. All of the gluten proteins are
now defined as prolamins because they are soluble, either in the native state or after reduction
of interchain disulphide bonds, in alcohol-water mixtures and because of their high contents
of proline and glutamine (Shewry et al., 1986; Madgwick et al., 1992). The prolamins has low
contents of the amino acids lysine, threonine and tryptophan (Shewry, 2000).
Gluten comprises about 63-90% of the total wheat grain protein as shown in Table 3.
In the milling process the endosperm cells are separated to give white flour. In the dough
processing the proteins are brought together on wetting and mixing to form a continuos network in the dough. The gluten component of flour can be separated from starch by washing.
The gluten network has viscoelastic properties, which allows the dough to be processed into a
lot of different end-use products including breads. This viscoelastic properties of the gluten
network, are due to interactions among the gliadins and glutenins and the other components of
the gluten i.e. lipids, small amounts of carbohydrates and soluble proteins (Pomeranz, 1968).
18
When kneading/mixing flour with water, gluten proteins enable the formation of a cohesive
visco-elastic dough that is capable of holding gas (carbon dioxide released by fermentation or
from chemical reactions) and oven-rise, resulting in a fixed open foam structure (a light, porous crumb structure) of bread after baking. The precise balance between viscosity (extensibility) and elasticity (dough strength) is important and highly elastic doughs are required for
breadmaking. The extensibility depends mainly of the monomeric gliadin content whereas the
dough strength depends of the glutenin polymers.
Gliadins
The gliadins are monomeric proteins and mostly connected by intrachain polypeptide disulfide linkages. Gliadins are a series of heterogenous proteins with similar low molecular
weight about MW 35,000 Da. The gliadin protein molecules are made of small, stable, folded
structures supported by disulfide bonds, whose solution properties probably are dictated by
their amino acid compositions (Shukla, 1975).
The gliadins are thought to be arranged in a fibrillar form at pH greater than 5 and as partially
unfolded monomers at lower pH. The microfibrillar aggregation is enhanced when the ionic
strength is increased, and hydrophobic interactions are probably also involved in stabilising
the structure.
The gliadins are divided into three groups: α-(α- and β-), γ-, and ω-gliadins, based on their
amino acid sequences and electrophoretic mobility at low pH, but more than 30 components
can be separated by two-dimensional electrophoretic procedures. A single variety contains
about 45 gliadin components (Wrigley and Shepherd, 1973; Payne, 1987).
α- and γ-gliadins
The α- (α- and β-gliadins) and γ-gliadins has disulphide bonds mainly formed within the
polypeptide chain (intrachain disulphide bonds). The amino acid composition of the α- and γgliadins are similar to the whole gliadin fraction. The α- and γ-gliadins are classified into the
group of S-rich prolamins (Wrigley et al., 1998; Shewry, 2000). The two types of proteins
have similar structures, with most components consisting of between 250 and 300 residues.
The sequences can be divided into two clear domains: the N-termini domain with repeated
peptides rich in proline and glutamine residues and the C-terminal (non-repetitive domain).
All γ-gliadins contain eight cysteine residues located in the C-terminal, and six of the cysteine
residues are also present in the α-gliadins. The disulphide bonds formed by the cysteine residues are important in stabilising the protein structure (Shewry and Tatham, 1997).
The α- and γ-gliadins ability to take part in formation of gluten polymers via interchain disulphide linkage are restricted, but their SS bonds are important in retaining the folding structure
that determines the nature of non-covalent bonding (Wrigley et al., 1998; Shewry, 2000). The
strong non-covalent protein-protein interactions (mainly hydrogen bonds and hydrophobic interactions) of the gliadins are mainly responsible for the viscosity and extensibility of gluten
(Madgwick et al., 1992; Shewry, 2000).
19
A high content of gliadins in the grain gives a poor nutritional quality of the flour, because the
gliadins are a very poor source of lysine, tryptophan, and sulphur containing amino acids
(Shukla, 1975).
ω-gliadins
The ω-gliadins is different in amino acid composition. The ω-gliadins contains no disulphide
bonds, and therefore virtually no sulphur-containing amino acids (little or no cysteine or methionine) and only small amounts of basic amino acids. They are classified as the S-poor
prolamins (as shown in figure 1). The ω-gliadins is limited in their interactions in dough to
non-covalent forces, primarily hydrogen bonds (Shewry, 2000).
Glutenins
The glutenins consist of polymeric proteins characterized by interpolypeptide cross-linking by
disulphide bridges (interchain disulphide bonds) (Shukla, 1975). Glutenins can be separated
into individual polypeptide subunits by reduction of interchain SS bonds and are then classified into high molecular weight (HMW) glutenin subunits and low molecular weight (LMW)
glutenin subunits on the basis of their molecular weight by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE).
The glutenins are classified as HMW prolamins (HMW glutenin subunits) and sulphur-rich
prolamins (LMW glutenin subunits). The groups of prolamins are shown in figure 1. The
prolamins are divided into three groups: the HMW prolamins (HMW glutenin subunits), the
sulphur-rich prolamins (LMW glutenin subunits, γ-gliadins and α-gliadins) and the sulphurpoor prolamins (ω-gliadins) on the basis of amino acid composition and molecular weight
(Miflin et al., 1983; Madgwick et al., 1992; Shewry, 1996; Shewry, 2000).
The glutenin subunits are assembled into high molecular weight polymers with MW above 1
x 106 Da and possibly exceeding 1 x 107 Da. The interchain disulphide bonds needs to be reduced before the subunits are soluble in alcohol-water mixtures. The term ‘subunit’ refers to
the single chain polypeptides obtained after reduction of the disulphide bonds of glutenin. The
glutenins, which contain both intrachain and interchain disulphide bonds, exist as polymers
stabilised by the interchain disulphide bonds (Shewry, 2000).
Glutenins are large, heterogenous molecules built up from about 19 different subunits that are
linked by disulphide bonds. In flour particles polymeric glutenin comprising three, four or
five high molecular weight (HMW) subunits and about 15 low molecular weight (LMW) subunits is pre-packed in protein bodies. More than 30 different HMW subunits have been identified in the world wheat collection. The number of different LMW subunits present in genotypes of common wheat has not yet been determined (Bushuk, 1998). The allelic variation are
about 500 million or above although there are only nine major gene loci in bread wheat that
contain prolamin genes, see Table 6.
All hexaploid bread wheat cultivars contain six HMW subunit genes, but only three, four or
five of these genes are expressed. The protein produced from each expressed gene accounts in
average for about 2% of the total seed protein, and the combined proportion of HMW subunits varies from about 6 to 10% of the total (Shewry et al., 1998). There are clear correla20
tion's between the numbers of expressed genes, the amount of HMW subunit protein, and of
HMW elastic glutenin polymers and breadmaking performance (Shewry and Tatham, 2000;
Shewry, 2000).
The interchain disulphide bonds needs to be reduced before the subunits are soluble in alcohol-water mixtures. The polypeptide subunits of glutenin can be separated by sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Other techniques are amount of
acetic acid insoluble glutenin and a SDS gel protein test (Bushuk, 1998) or a size-exclusion
high performance liquid chromatography (SE-HPLC) of dough protein extracted with sonication into SDS containing buffer to indicate the molecular weight distribution (Wrigley et al.,
1998). The reduced subunits can in turn be separated into HMW and LMW groups on the basis of molecular weight.
The composition of glutenin subunits into two distinct, unequal groups the predominant low
molecular weight (LMW) subunits and the high molecular weight (HMW) subunits (Stone
and Savin, 1999) are shown in Table 4, where the HMW subunits comprise about 8.5 % of total gluten proteins.
Table 4. Glutenin composition (Stone and Savin, 1999).
Solubility
LMW subunits HMW subunits
SDS-soluble glutenin (8%)
70%
30%
SDS-insoluble glutenin (15%)
60%
40%
The HMW glutenin subunits
The HMW subunits contain significantly less proline and had MW from 67,500 to 87,700 Da
(A type) (Shewry et al., 1992) or about a molecular weight MW of 70,000 Da (Payne, 1987).
Analysis of genes encoding nine different HMW subunits (including allelic and homoallelic
forms derived from A, B and D genomes) demonstrates that the proteins have similar structures. All consist of three domains, with short N-terminal (about 80-100 residues) and Cterminal (42 residues) regions flanking a central repetitive domain that varies in length from
480 to 700 residues (Shewry et al., 1998).
The HMW glutenin subunits are classified as HMW prolamins in figure 1. Especially the
HMW glutenin subunits are responsible for the elasticity of dough (Payne, 1987; Payne et al.,
1987b; Madgwick et al., 1992; Shewry et al., 1992).
21
Gluten proteins
insoluble in alcoholwater mixtures
70% (v/v) ethanol
small amounts
Glutenins
(polymers)
HMW-SU
interchain SS bonds
HMW
prolamins
soluble in alcohol-water mixtures
70% (v/v) ethanol
Gliadins
(monomers)
γ-gliadins
LMW -SU
intrachain SS bonds
S-rich
prolamins
α-gliadins
ω-gliadins
no SS bonds
S-poor
prolamins
Figure 1. Summary of the classification and nomenclature of wheat gluten proteins.
HMW glutenin subunits are classified as HMW prolamins and forms interchain disulphide bonds. LMW glutenin subunits, γ-gliadins and α-gliadins are classified as S-rich
prolamins and forms intrachain disulphide bonds. The ω-gliadins are classified as Spoor prolamins and forms no disulphide bonds (Madgwick et al., 1992;Shewry, 1996).
The LMW glutenin subunits
The LMW glutenin subunits resembles the monomeric α-type gliadins in amino acid composition and has similar MW ranging from 30,000 to 40,000 Da (C type) and from 40,000 to
50,000 Da (B type) divided on the basis of mobility on SDS-PAGE. Direct disulphide bond
determination demonstrates that the cysteine residues, located in conserved positions (within
the amino acid sequence and sequence context) present in the S-rich prolamins (α-gliadins, γtype gliadins and LMW subunits) form intrachain disulphide bonds while additional cysteines
residues present only in the LMW subunits form interchain bonds with cysteines in HMW
subunits and other LMW subunits (Shewry and Tatham, 1997). The LMW subunits are interacting with the HMW subunit polymers by covalent disulphide bonds (Shewry, 2000).
Protein structure
The structural studies indicate that the HMW subunit glutenins form the ’elastic backbone’ of
gluten. The HMW subunit glutenins comprise three separate domains, a central domain,
which forms a loose spiral structure (β-spiral), flanked by globular N- and C-terminal domains, which contain most or all of the cysteine residues that forms the interchain disulphide
22
bonds. These disulphide-stabilised polymers are considered to form an elastic network, which
interacts with other gluten proteins, by disulphide bonds (HMW and LMW subunits) and noncovalent interactions, primarily by hydrogen bonds (gliadins) (Shewry, 2000).
Molecular and biophysical studies have revealed details of the HMW subunit structure that
may relate to their role in viscoelastic polymers. These are the number and distribution of cysteine residues (which are the potential cross-linking sites), and the properties of the β-spiral
structure formed by repetitive sequences of the central part of the proteins. It is likely that
many water molecules in gluten are replaced by interchain hydrogen bonds with other proteins, both gliadins and glutenins. The formation of such interchain hydrogen bonds could occur as the grain dehydrates during maturation and/or be favoured during dough formation and
processing (Shewry et al., 1998).
Disulphide bonds play a key role in determining the structure and properties of wheat gluten
proteins. Comparison of the sequences of monomeric gliadins and polymeric glutenin subunits allows the identification of conserved and variant cysteine residues. Direct disulphide
bond determination demonstrates that the conserved cysteine residues present in S-rich
prolamins (α-type gliadins, γ-type gliadins and LMW subunits) form intrachain disulphide
bonds while additional cysteines residues present only in the LMW subunits form interchain
bonds with cysteines in HMW subunits and other LMW subunits. Conserved and variant cysteine residues are also present in the HMW subunits but their patterns of disulphide bond
formation are less well-understood(Shewry and Tatham, 1997).
The monomeric gliadins with intrachain or no disulphide bonds, interact via non-covalent interactions (hydrogen bonding, van der Waals’, electrostatic and hydrophobic interactions) and
contributes to viscosity. The polymeric glutenins, in addition to the non-covalent interactions,
interact via both intra- and interchain molecular disulphide bonds and contribute to elasticity.
The ratio of monomeric gliadins to polymeric glutenin determines the balance between dough
viscosity and elasticity and affects gluten protein quality (Shewry and Tatham, 1997).
Monomers and polymers
The grain proteins can be divided into polymeric or monomeric types, based on their aggregating properties (i.e. intermolecular disulphide linkage formation). Gliadins and typical albumins and globulins constitutes the monomeric proteins (Gupta et al., 1996; Stone and
Savin, 1999).
There are four main types of polypeptides (subunits) that have been identified as assembling
into disulphide bond mediated polymers in the aggregated protein fraction in wheat seed.
These are HMW glutenin subunits and LMW glutenin subunits (the glutenin subunits make
up about 85%), HMW albumin subunits (mostly beta-amylases) and globulin subunits
(triticins) (MacRitchie, 1992; Gupta et al., 1995; Gupta et al., 1996). Glutenin contributes significantly to the viscoelastic properties of doughs.
Deposition in the seed of HMW glutenin subunits, LMW glutenin subunits and HMW albumin subunits (β-amylases) began as early as 7 days after anthesis and progressed until maturity. Disulphide-linked aggregation (polymerization) of these subunits also began at 7 days after anthesis. However significant changes in size distribution of the polymers occurred only
23
during the late stages of seed development which coincided with a rapid increase in the
amounts of the glutenin subunits (Gupta et al., 1996).
The amount of unextractable polymeric protein in total polymeric protein was very strongly
correlated with dough strength properties and accounted for the variation in these parameters
to a much greater degree than did the percent total polymeric protein in protein or in flour
(Gupta et al., 1993). The unextractable polymeric protein had a significantly greater HMW to
LMW subunit ratio than did the extractable polymeric protein, thus confirming that the molecular size distribution shifts to greater molecular size as this ratio increases. When both
HMW and LMW glutenin subunits were present together, they interacted in a positive way
with each other in polymer formation and thus provided greater amounts of large polymers
than expected from their individual contribution (Gupta et al., 1995).
In some cultivar lines, the proportion of HMW to LMW glutenin subunit content can account
for large percentage of the differences in dough properties. The reduced dough properties
(lower resistance and extensibility, may be due to a reduction in the proportion of total glutenin in their protein and especially to the reduction of HMW glutenin subunits (Singh et al.,
1991).
LMW glutenin subunits (Glu-3 loci) affected the quantity and/or size distribution of the
polymers due to differences in the amounts and/or probably the quality of subunits produced.
LMW glutenin subunits rather than gliadins were responsible for positive effects of Glu3/Gli-1 genetic linkages. The positive effects of these loci on dough strength is due to variation in the polymeric proteins (glutenins) rather than in the monomeric proteins (gliadins or
albumins/globulins) (Gupta and MacRitchie, 1994). The effect of Glu-1 loci on dough/gluten
strength and molecular size distribution of the polymeric protein was far greater than that of
the Glu-3 loci, and this is attributed to more than the difference in the size of these subunits
(Gupta et al., 1995). The chromosome structure of wheat is shown in Table 6.
Wheat genetic lines lacking HMW and/or similarly lacking LMW glutenin subunits from one,
two or three Glu-1 or Glu-3 loci respectively, were used to study the effects of these polypeptides on glutenin polymer formation and dough/gluten properties. Dough and gluten properties were significant correlated with both total and more strongly with the proportions of unextractable polymers. Loss of all the Glu-1 subunits, on an equal weight basis, reduced the
amounts of the larger polymers to a much greater extent than the loss of all the Glu-3 subunits, reflecting more than the different molecular size in these subunits. Both Glu-1 and Glu3 subunits form large polymers on their own. When both the HMW and the LMW subunits
were present together, the amounts of large polymers were much greater than the sum of the
amounts when only one group was present. This suggests a positive interaction between these
two groups of subunits with respect to polymer formation (Gupta et al., 1995).
The dough strength or gluten elasticity of wheat flours depends largely of the polymeric protein present and especially of the amount of HMW glutenin polymers and the unextractable
polymeric protein. A significant linear correlation was obtained between the relative quantity
of unextractable polymeric protein (in total polymeric protein or in total protein) and the
dough strength (breadmaking quality) over a range of genotypes and environments (including
flour protein levels) (Gupta et al., 1993; Gupta et al., 1995).
24
The HMW glutenin polymers have long been known to be responsible for gluten and dough
elasticity, with gliadins contributing mainly to the extensibility. The balance of glutenin and
gliadin varies with genotype and environment, with insufficient elasticity being the most
common cause of poor breadmaking quality. Variation in the amount of and composition of
the HMW glutenin subunits is associated with differences in breadmaking performance
(Payne, 1987; Payne et al., 1987b; Gupta et al., 1991; Bushuk, 1998; Shewry, 2000).
Quality scores
The quality of gluten is genetically determined and varies between cultivars. The genotypic
variation in the contribution of glutenin to breadmaking quality is due to variation in the
number of specific HMW subunits. Payne et al. assigned quality scores by ranking the presence of certain glutenin subunits with high bread making quality (Payne, 1987). Some of the
glutenin subunits (based on electrophoresis - the SDS-sedimentation value) were given quality scores, and for each wheat cultivar the total quality score was summarized from all the
present individual or pairs of glutenin subunits at the list (Glu-1 quality score). The quality
scores were adjusted if a rye translocated chromosome was present, see Table 5 (Payne et al.,
1987b). The possible maximum score is 10 and the minimum is 3 in the 84 British wheat varieties. 67 percent of the total variation in bread making quality was contributed to Glu-1
quality score (rye adjusted score) in the British varieties (Payne et al., 1987b).
Table 5. Quality scores (Glu-1 quality scores) assigned to individual or pairs of HMW
glutenin allelic subunits. For each wheat cultivar a total quality score was calculated by
a summation of all the present individual or pairs of glutenin subunits at the list. Some
points are subtracted dependent of the quality score if the chromosome 1R from rye is
present (i.e.3 points if the quality score are above 6, 2 points from 6 and 5, and 1 point if
the quality score were 4 or below). E.g. in the variety Alexandria the glutenin subunits
1Ax1, 1Bx7+1By9 and 1Dx5+1Dy10 are present given a total quality score of 9 (Payne et
al., 1987c).
Score
4
3
3
2
2
1
1
Chromosome
1A
1
2*
null
-
1B
17+18
7+8
7+9
7
6+8
1D
5+10
2+12
3+12
4+12
-
The grain proteins of 84 English grown wheat varieties were analysed by SDS-PAGE to determine the subunit composition and the Payne quality score was determined for each variety.
25
In the British varieties the most common occurring subunits were those associated with poor
breadmaking quality. The insufficient elasticity, which causes poor dough strength, has long
been recognised as the main deficiency in wheats grown in Great Britain (Payne, 1987). In 67
Canadian cultivars the proteins were fractionated by SDS-PAGE to determine their high MW
glutenin subunit composition. The Payne quality scores (Glu-1 quality scores) accounted for
59-69% of the variation in bread-making quality (Lukow et al., 1989).
Ng and Bushuk (1988) used the HMW subunit composition to develop an equation for predicting the unit bread volume (bread volume per 1% protein content) of a bread wheat cultivar. All cultivars having a good score in breadmaking quality based on SDS-Sedimentation
test assigned to single pairs of HMW glutenin subunits contains the glutenin subunit composition Glu-1D (5+10). All breeding lines in Canada are now selected to contain Glu-1D (5+10)
(Bushuk, 1998).
Based on an Australian experiment the alleles from 74 recombinant inbred lines were analysed, and Gupta et al. (1994; 1994)determined the relative effects of HMW and LMW glutenin subunits on bread-making quality. The effects on dough resistance of the individual
LMW and HMW glutenin subunit alleles from different loci are largely cumulative, accounting for 80% of the variation observed; interactions between these loci accounted for other
10% of the variation in dough resistance. It suggests that the alleles with positive (superior)
additive effects can be accumulated to improve wheat quality. Therefore the dough strength
can be improved further without increasing the grain protein levels (via additional nitrogen
fertilization) and also without reducing the grain yield (Gupta et al., 1994).
A guide to wheat breeders who wish to develop varieties with improved bread-making quality
is therefore to cross genotypes that have complementary good subunits coded by different loci
and to select progeny with a high Glu-1 quality score (Payne et al., 1987b). Biochemical basis
for the association between glutenin alleles and dough strength lies in the effects of these alleles on the amounts and size distributions of native glutenin (polymeric protein) because of
differences in quantity and /or types (polymerising behaviour) of the subunits produced
(Gupta and MacRitchie, 1994).
The 5+10 biotype (with HMW glutenin allelic subunits 5+10 in the Glu-D1d allele) accumulated larger polymers more quickly than the 2+12 biotype (with allelic subunits 2+12 in the
Glu-D1a allele). The difference becoming apparent at 28 days after anthesis which could be
attributed to variation in the accumulation rate of HMW glutenin subunits (Gupta et al.,
1996).
It is probably due to better ability of the HMW subunits (of the 5+ 10 biotype) to polymerise
into very large macromolecules, which are associated with a greater dough strength (this leads
to longer mixing time and can be shown as a longer time to peak development in the
Mixograph). Therefore dough properties might be modified primarily by factors that alter
chain length for glutenin polymers (Wrigley et al., 1998).
26
Chromosome structure
-B1>Glu-B3>Glu-A3>Glu-D3=Glu-A1 with respect to maximum dough resistance, Rmax.
In contrast to Rmax only 25% of the variability in dough extensibility (Ext) could be accounted for by the glutenin alleles. Only Glu-D1 and Glu-D3 loci showed significant effects
on this parameter. Altogether over 50 individual components can be resolved by twodimensional electrophoresis of total gluten proteins, and molecular and chemical analyses indicate, that most of these are the products of single genes. The genes have been mapped using
classical Mendelian analysis as well as modern methods (Madgwick et al., 1992). The chromosome structure of wheat is shown in Table 6.
Table 6. Chromosome structure of wheat
Chromosome number
Arm
1
1A
loci (genes)
Genome
1B
1D
Proteins
Glu-A1, Glu-B1, Glu-D1 glutenin HMW-SU long
Glu-A3, Glu-B3, Glu-D3 glutenin LMW-SU short
Gli-A1, Gli-B1, Gli-D1
gliadins *
short
long
2
3
4
5
short
6
short
1B or 1D/1Rrye
2A
3A
4A
5A
6A
2B
3B
4B
5B
6B
2D
3D
4D
5D
Ha-gene on 5D
6D
grain protein content QTL
Vrn1 gene
drought-induced ABA accumulation gene
LEA-gene?
Gli-A2, Gli-B2, Gli-D2
gliadins**
CM proteins
friabilin
or gene (osmoregulation)
Per-A4 gene
CM proteins
*all of ω, most γ gliadins. ** all of α, some γ gliadins. Modified from Friedli (1996), Payne (1987) and Wrigley
et al. (1998).
7
7A
7B
7D
The HMW subunits are controlled by three gene loci located on the long arm of chromosomes
1A, 1B and 1D and identified as Glu-1A, Glu-1B and Glu-1D (Payne, 1987; Wrigley et al.,
1998). The different glutenin subunit loci could be ranked as Glu-D1>Glu.
The effects of individual Glu-3 (Gli-1) alleles (the LMW glutenin subunits) are largely additive to those of Glu-1 alleles (the HMW glutenin subunits)(Gupta et al., 1994). Wheats giving
flour of the strong type have been found to carry gliadin blocks 1A3 (or 1A4), 1B1, 1D1 (or
1D5), 6A3, 6B1 and 6D1 (or 6D2), which are markers of high gluten quality, frost hardiness
and drought resistance.
Some varieties contain the short arm of chromosome 1R from rye combined with the long
arm of chromosome 1B or 1D (1BL/1RS or 1DL/1RS)(Singh et al., 1991). Only one of the
loci (Glu-3/Gli-1 loci) is replaced by the rye chromosome and causes a decrease in bread-
27
making quality, mainly by increasing the dough stickiness (Payne et al., 1987a). The positions
of the grain nitrogen content quantitative trait loci (QTL) on barley chromosomes 5HS and
5HL were comparable to QTL on wheat chromosomes 5A and 5D that affect grain protein
content (Bezant et al., 1997).
Chromosome 5A was likely to carry a gene or genes having major effect on drought-induced
ABA accumulation. Maximum likelihood position for the ABA QTL is about 4 cM from
xpsr426 towards xpsr575 in wheat. The vernalization responsive allele of Vrn1 in wheat is associated with lower ABA accumulation, higher leaf weight, lower leaf dry weight %, higher
tiller number and more root mass per tiller (Quarrie et al., 1997).
Hardness/softness of the wheat endosperm is genetically controlled of a gene (Ha) located on
the 5D chromosome. The gene controlling formation of the molecular weight 15 kDa protein,
friabilin, is also situated on the 5D chromosome, and it is either identical with the Ha gene or
is located quite close to it (Greenblatt et al., 1995; Preston, 1998). Friabilin is strongly present
in soft-endosperm wheats that fragments easily and at random, but is only weakly present, or
entirely absent from, hard wheats that fragments only with difficulty.
The osmoregulation gene (or) has a position at approximately 60 cM from the centromere on
the short arm of chromosome 7A, close to a peroxidase gene (Per-A4) conditioning differences in a peroxidase isozyme which is only active in the endosperm. Peroxidase has been
shown, in vitro, to enhance cross linkages of several soluble proteins. Backcross lines, which
had been bred for an osmoregulation gene to improve the drought tolerance, had significantly
negative effect on breadmaking quality (shorter dough development times and significantly
lower maximum resistances to extension than recurrent parents). A probable explanation of
the dough strength effect lay in a difference in peroxidase activity due to linkage between the
endosperm peroxidase, Per-A4, locus, and the osmoregulation, or, locus. On average, lines
with high osmoregulation (lower dough strength) had lower peroxidase activities than recurrent parents (higher dough strength). This effect, however, depended on protein content and
genotype (Morgan, 1999).
Bread-making quality
Baking quality is largely determined by the composition and content of proteins in the grain.
There exists a positive correlation between protein content and bread volume (Finney and
Barmore, 1948; Tipples and Kilborn, 1974). Water absorption is related to dough mixing
quality and increases with protein concentration. In fact, the difference in baking quality of
flour becomes most clear in the baking test. In bread wheat varieties a wide range of quality is
possible. There exist a linear relationship between bread volume and flour protein content and
the slope is a measure of the performance of a cultivar. The performance of a cultivar at a
specific protein level is measured as the highest bread quality such as the bread volume in the
baking test (Bushuk, 1998). Dough properties might be modified primarily by factors as influences the length of glutenin polymers. In addition non-covalent interactions between the
chains can be seen to determine the slippage of these chains against each other, thus also affecting dough properties (Wrigley et al., 1998).
28
The bread-making quality of flours are further determined by amounts and types of HMW
glutenin subunits present in the dough (Payne, 1987; Payne et al., 1987b; Gupta and MacRitchie, 1994; Gupta et al., 1995; Gupta et al., 1996; Vasil and Vasil, 1999).
Bread volume was directly related to the proportion of the acetic acid-insoluble glutenin and
inversely related to the proportion of the acetic acid-soluble glutenin in samples of 26 cultivars of diverse baking quality grown in four locations (Orth and Bushuk, 1972).
The maximum resistance (Rmax) did not always correlate well with the percentage of polymeric protein in total protein. Rmax always showed high correlation with the percent of nonextractable protein. Not all of the polymeric protein contributes to dough strength but only the
fraction of the largest molecular size,estimated to be about 250,000 kDa. A good correlation
was obtained between the unextractable protein and the HMW/LMW glutenin subunit. The
5+10 biotype had a greater Rmax than the 2+12 biotype. The presence of 5+10 subunits was
associated with a molecular weight distribution shifted to higher molecular weights than at the
presence of 2+12 subunits. The Ext parameter was reduced if the size of the polymeric protein
became too large, and the biotype 5+10, which had a larger molecular size of the glutenin
polymer had the lowest Ext (MacRitchie, 1998). Pechanek et al. (Pechanek et al., 1997) suggests that the ratio of HMW gluteins, especially the χ-type subunits, to total protein content
could be the best early detectable parameter with the best predictive value for breadmaking
quality. Several genes of LMW glutenin subunits were isolated from T.boeticum and used for
expression of proteins in bacterial cells. The dough incorporated with the LMW glutenin subunits showed significantly increased mixing time using a 2-gram mixograph. This indicated
that LMW glutenin subunits contribute positively to expanding the gluten network of the
dough and confirm the importance of cysteine residues involved in forming intermolecular disulphide bonds (Lee et al., 1998).
The width and height of the Mixogram was related to the proportion of unaggregated proteins
before the mixing peak and to polymeric proteins after the dough consistency reached a
maximum. The Mixograph proved to be a powerful tool to investigate indices of bread making quality.
Gupta et al. (1993) and Martinant et al. (1998) has shown that a greater amount of unextractable polymeric proteins would be expected to have a greater dough resistance (elasticity)
and a longer mixing requirement than those with a smaller proportion. Dough consistency, as
measured by the height of the Mixogram, was correlated with grain hardness, as expressed by
the starch damage content and the proportion (%) of flour particles larger than 37.8 mm
(Gupta et al., 1993; Martinant et al., 1998). The ability of the HMW subunits to polymerise
into very large macromolecules is associated with a greater dough strength (and this leads to
longer mixing time and can be shown as a longer time to peak development in the Mixograph)
(Wrigley et al., 1998).
29
Kneading
The amino acid cysteine is very important in the structural properties of the gluten proteins,
because free sulfhydryl groups (SH-groups) in the reduced state can react with another cysteine molecule and via an oxidation form a disulphide bond (SS-bonds) and the amino acid
cystine is formed (Shukla, 1975).
Cysteine with its SH-group has great importance for the gluten strength, because two cysteine
molecules can form a sulphur bridge in the form of cystine, which makes a stronger structure
than the free SH-groups. The cysteine molecules can be in the same peptide chain and form a
string or it can be placed between different peptide chains and combine them to a greater
molecule. Disulphide-bonds can move within a molecule and gives the protein a flexible
structure, its secondary structure. The tertiary structures of proteins are made of ion bonds and
hydrogen bonds (acid-base).
During the kneading process in dough making, the S-bridges can move around in the gluten
molecule. A lot of free S-groups are formed when dough with weak gluten strength is
kneaded, whereas many S-bridges are formed in dough with tight gluten strength when
kneaded (Zhao et al., 1999a; Zhao et al., 1999b; Luo et al., 2000). In a study of variation in
the breadmaking quality and rheological properties of UK wheat in relation to sulphur nutrition under field conditions it was found that grain S status (S concentration and N:S ratio) was
more influential on bread volume than grain protein concentration (Zhao et al., 1999b).
Additives
Oxidising agents can be very effective in improving the handling characteristics of dough.
These substances exert their effect on the mechanical properties of dough by causing the formation in additional cross-bonds between the gluten molecules (Matz, 1991). If the wheat
flour is too weak (as in Denmark or UK) different additives like ascorbic acid are added to
improve flour quality. The additive acts as an oxidant to increase the number of SS-bonds
through oxidation of the free SH-groups thus increasing the cross-linking of protein polymers
in the gluten matrix (Kent and Evers, 1994; Hansen and Poll, 1997; Marx et al., 2000). Furthermore, addition of peroxidase and addition of enzymes as thioredoxins is able to catalyse
the reduction of specific disulphide bonds, and thereby to increase dough strength (Marx et
al., 2000).
The addition of enzymes such as the NADP/thioredoxin system to a poor quality of soft white
flour resulted in increased dough strength by reducing the intrachain disulphides to provide
free SH-groups. Thus allowing the free SH-groups to interact with the sidechains of other protein subunits for the formation of interchain bonds (Marx et al., 2000).
Grain
Gluten proteins are initially deposited in discrete protein bodies that later subsequently fuse to
form a continuous protein matrix surrounding the starch granules in the starchy endosperm of
developing grain (Shewry, 2000). Wheat gluten proteins are folded and assembled within the
lumen of the endoplasmic reticulum of the developing endosperm cells, where disulphide
bond formation and exchange may be catalysed by the enzyme protein disulphide isomerase.
30
This enzyme catalyses the thiol/disulphide exchange. Similarly, reduction of disulphide bonds
to facilitate mobilisation during germination, may be catalysed by thioredoxin (Shewry and
Tatham, 1997). It is likely that many of the water molecules in gluten are replaced by interchain hydrogen bonds with other proteins, both gliadins and glutenins. The formation of such
interchain hydrogen bonds could occur as the grain dehydrates during maturation and/or be
favoured in dough formation and processing (Shewry et al., 1998). Gaines et al
(1997)observed in a study of the influence of kernel size and shrivelling on soft wheat milling
and baking quality, that the milling and baking properties of smaller kernels were equivalent
to those of larger kernels.
Flour
White flour is derived from the starchy endosperm of the wheat grain and therefore contains
mainly starch and prolamin storage proteins (i.e. gliadins and glutenins). The structure of
wheat gluten is not understood in detail, it is generally considered that the glutenins form an
elastic network stabilised by interchain disulfide bonds, while the gliadins interact with this
via non-covalent forces (principally hydrogen bonds and hydrophobic interactions) and contribute mainly to viscosity (Shewry, 1996). The HMW glutenin subunits can react with other
HMW or LMW subunits via interchain SS-bonds. The HMW glutenin subunits are important
in the formation of viscoelastic doughs owing to their molecular structure and ability to form
polymers stabilised by disulphide bonds (Shewry and Tatham, 1997).
The sulfhydryl-disulphide status changes during the processing of wheat grain as a result of
the reduction of protein disulphide bonds. The intramolecular to intermolecular bond exchanges increases in the wheat flour proteins during conditioning of the grain and maturation
of the flour resulting in dough of greater strength (Gobin et al., 1996). The potential for the
formation of S-S linkages is a determinant of dough strength. The cystine content has a positive correlation with dough strength (Pomeranz, 1968). Flour from S deficient wheat produces
dough with decreased extensibility and increased resistance to stretching, which results in reduced bread volume (Scherer, 2001).
Dough
The water-insoluble proteins of wheat (gliadins and glutenins), together with lipids, small
amounts of carbohydrates and some soluble proteins, interacts to form a component called
gluten (Pomeranz, 1968). When flour is wetted, mixed and kneaded into dough it consists of
starch, gluten and water. The gluten complex is formed into a proteineous network with viscoelastic properties. The gliadins and glutenins form the gluten complex and the elasticity/strength and extensibility/viscosity of the dough, are related to the types and quantity of
the present glutenins and gliadins respectively (Wieser et al., 1994; Vasil and Vasil, 1999).
Gluten is the three-dimensional matrix of bread dough and is directly responsible for its gas
retaining capability (the dough volume), the oven-spring during baking and the characteristics
of the leaved structure of baked products. Washing out most of the starch granules and other
dough solubles can isolate the gluten fraction.
31
On initiation of mixing, the hydrated proteins begin to alter their relationship to each other.
During mixing, it is known that considerable amount of water becomes bound. Mixing of hydrated proteins disrupts their original association in breaking of many intermolecular secondary bonds and resulting in the development of a cohesive gluten matrix (Shewry, 1996).
Oxidation occurs when flour is heated. The most important covalent cross-links in gluten are
the disulphide bridges. For kinetic reasons heating will accelerate the formation of these S-S
bridges. Once covalent coupling by S-S bonds has occurred, physical aggregation is difficult
to reverse.
The surface of the starch granule is assumed to be hydrophilic. This infers that gluten of poor
quality, which has a greater tendency to interact with starch, is more hydrophilic than is the
good quality gluten. A similar assumption can be reached from the solubility data; the good
quality gluten is more difficult to solubilise. Hydrophobic interactions in proteins of good
quality flours are stronger (or greater in number) than those in proteins of poor quality flours
(He and Hoseney, 1991).
During dough mixing, lipids become embedded into the protein matrix and are essential in the
viscoelastic properties of the protein network (Panozzo et al., 1993). The lipids almost certainly acts as plasticizers and contributes to dough viscoelasticity by interacting with gluten
(Shewry, 1996). Hard grained cultivars had higher levels of free lipid than the soft grained
wheats. Dough properties an baking performance are strongly dependent on both genotype
and environment (Peterson et al., 1992; Johansson and Svensson, 1998; Johansson and Svensson, 1999). Both flour protein and free lipid had a significant effect on bread volume, the effect of free lipid remaining significant after adjustment for protein. A condition necessary for
high bread volume is adequate protein and free lipid level (Panozzo et al., 1993).
Baking
The changes of the dough during baking are very complex. During the baking process the
sugars (mono- and disaccharides) released from damaged starch during milling are degraded
by yeast to carbon dioxide an important factor in developing the dough volume. In the dough
most of the water is bound to proteins. In a physical sense, dough changes from foam to a
sponge structure. The dough expands during heating in the oven (oven-rise), but this expansion is inhibited by crust formation. Water vapour condenses initially during baking on the
crust, and finally evaporates together with gasses (ethanol, CO2). Inside the dough water is
transported from the outer side to the inner side. Heat is transported by direct or indirect heat
to the loaf. In the crust and inside the loaf, starch gelatinises and proteins denature. After heating most of the water is probably bound to damaged starch. Although it is likely that gluten
still binds water after heating, there have been great changes in the water distribution during
heating. This affects the reaction kinetics of the denaturation of protein and gelatinisation of
remaining starch granules (Weegels and Hamer, 1998).
32
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