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The Protein Chemistry of Cereal Grains
The Protein Chemistry of Cereal Grains F Be´ke´s, FBFD PTY LTD, Beecroft, NSW, Australia CW Wrigley, The University of Queensland, St Lucia, QLD, Australia ã 2016 Elsevier Ltd. All rights reserved.
Topic Highlights
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The importance of dietary protein from cereal grains – rice, wheat, maize, and others. The purification of wheat gluten from dough – as an experiment in the kitchen and as a commercial product. The simple structure of proteins – a linear polymer of amino acids – produces the diversity of properties of enzymes, hormones, hair and horn, silk and sinew, and gluten together with the many other grain storage proteins. Folding and cross-linking of the amino acid polymer gives three-dimensional structure that is needed for many protein functions, such as enzymatic activity. Osborne’s solubility-based protein classes: water-soluble albumins, salt-soluble globulins, prolamins extractable with 70% aqueous ethanol, and the residual glutelins. Protein quantity is determined by various methods, especially by near-infrared spectroscopy, based on total nitrogen content. Protein quality relates to its nutritional value and its contribution to functional uses. The specific proteins are described for wheat, rye, triticale, barley, rice, maize, and sorghum.
Learning Objective To achieve an understanding of proteins as critical components of the grains of cereal species and their function for the plants and for our dietary needs.
Introduction Worldwide, plant proteins contribute more than half of our dietary supply of protein. Over 200 million tonnes of protein are harvested each year in the form of cereal grains, based on FAO statistics (http://faostat.fao.org). This protein harvest includes that derived from the three major cereal grains, wheat, rice, and maize, and also less widely grown species: barley, rye, triticale, and sorghum. In some developing countries, a single cereal species may be the major or sole source of protein in the diet. In many countries, cereal proteins enter the diet in various forms, including wheaten breads and pastries, noodles, fried rice dishes, rye crispbreads, cookies, biscuits, pearl barley, pasta, corn tortillas, breakfast cereal, oatmeal porridge, couscous, and many more foods. Most obviously, cereal grain proteins are eaten directly in these many processed forms. The second route is indirect, in the form of animal products, such as meat, milk, and eggs, following the feeding of cereal grains to animals. Whereas the protein part of the grain is important nutritionally, it also serves functional purposes, relevant to the
Reference Module in Food Sciences
processing of the grain. Of these, wheat and rye are unique because of the dough-forming properties of their storage proteins, known as ‘gluten’ when formed into dough.
The Discovery of Protein Structure Cereal Proteins in Prehistory Historically, the proteins of the cereal grains have played a significant part in the civilization of mankind, through their role in providing a reliable source of amino acids, the essential dietary compounds that are the building blocks of proteins in the body. Grains as a source of dietary proteins could be obtained via agriculture, permitting the establishment of a permanent dwelling place, providing an important change from the nomadic existence of the hunter-gatherer way of life. The establishment of permanent towns led, in turn, to specialization of occupation and to opportunities for mankind to pursue cultural pursuits and scientific inquiry. However, it is only in recent centuries that the chemistry of proteins has been elucidated.
Early Concepts The word ‘protein’ was proposed by the French scientist Berzelius, writing to the German scientist Mulder, with the appropriate meaning of ‘primary substance,’ as recently as 1838, long after the acceptance of the terms for specific types of proteins, such as ‘gluten,’ ‘zymom,’ and ‘fibrin.’ Early concepts of protein chemistry imagined it to have the complex formula C20H31N5O6, meaning that 20 molecules of carbon (C) were compounded with 31 molecules of hydrogen, five molecules of nitrogen, and six molecules of oxygen. The dogma of those early days also maintained (incorrectly) that this complex was absorbed (unchanged) directly by the intestine. It is now known that dietary proteins are hydrolyzed to the component amino acids for absorption into the bloodstream.
Wheat Gluten: The First Purified Protein One of the first proteins to be obtained in reasonably pure form was gluten, because it could be prepared so readily (by the washing of dough made from wheat flour). This important experiment, first reported by the Italian scientist Beccari in 1729, can be simply performed in the home kitchen. To do so, a dough is mixed by adding water, a little at a time, to wheat flour until a cohesive mass is formed. This dough is kneaded between the fingers under a gentle stream of water from the tap. If a glass is placed underneath, a milky-white stream of starch can be caught in the glass, while the dough in the fingers becomes smaller and tougher. The gluten ball held in the fingers is mainly protein, separated very simply from the starch that settles at the bottom of the glass. Strictly speaking, gluten is a protein–lipid–carbohydrate complex formed as a result of specific covalent and
http://dx.doi.org/10.1016/B978-0-08-100596-5.00101-3
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PROTEINS | The Protein Chemistry of Cereal Grains
noncovalent interactions from flour components during dough making as the components are hydrated and energy from mechanical input from the mixing process is provided. This gluten-washing procedure has since become very big business internationally, because the gluten thus isolated is needed for bolstering the protein content of a wide range of food products – bread, especially, and also breakfast cereals, meats, cheeses, snack foods, and texturized products – or consumed directly as ‘seitan.’ Nonfood applications include pet and aquaculture feeds, biodegradable plastics, films, coatings, adhesives, inks, cosmetics, and pharmaceuticals. Late in the nineteenth century, the American scientist Thomas Osborne studied the proteins of many edible grains, using a succession of solvents to extract diverse classes of proteins from the crushed grain. As a result, he proposed the generic names of ‘albumin’ (extracted with water), ‘globulin’ (extracted subsequently by salt solution), ‘prolamin’ (extracted by 70% aqueous ethanol), and ‘glutelin’ (extracted finally by a dilute acid). Subsequently, the prolamins of wheat have been understood to be single polypeptides, whereas wheat glutelins (known as glutenins) have been shown to be disulfide-linked polymers built up of glutenin subunits. The cover of one of Osborne’s famous publications appears in Figure 1. In many cereal grains, the metabolic proteins are albumins and globulins, many of these being the residual enzymes that have functioned in synthesizing the components of the maturing grain, plus others that are present in preparation for germination. On the other hand, the cereal storage proteins are often of the prolamin and glutelin classes; their water insolubility offers the advantage that they will be retained when the grain becomes waterlogged in the germination process. Osborne’s names for these major protein classes have since continued in use, but their definitions have been refined, and it has become clear that any one of these protein classes contains a large number of distinct but related proteins. The modern classification of cereal proteins is based on their genetics, also based on the chromosomal location of their coding genes. The term ‘prolamin’ in this nomenclature system covers all the polypeptides soluble in ethanol, independently whether they exist in the grain in monomeric or polymeric form. In the case of wheat (Figure 2), this prolamin group is subdivided into three groups as sulfur-rich, sulfur-poor, and high-molecular-weight (HMW) prolamins, representing the a-, b-, and g-gliadins plus the low-molecular-weight (LMW) glutenin subunits, the o-gliadins, and finally the HMW glutenin subunits, respectively, in the old classification system.
So What Is a Protein? A protein molecule is simply a linear polymer of amino acids. The structure of protein molecules also depends on the folding and cross-linking of the polymers, giving a three-dimensional structure that is especially important for functions such as enzymatic activity. It is no wonder that these early protein chemists wrestled with reaching this understanding, when we now see that this relatively simple chemical structure can produce the great diversity of properties that are found in proteins, such as enzymes and epidermis, hair and horn, feathers and flagella, silk and sinew, and even spider’s web.
Figure 1 The cover of one of Osborne’s early publications (1912) on the proteins of cereal grains.
The diversity depends on the selection and sequence of the amino acids plus the length of the protein (polypeptide) chain. Further diversity is provided by cross-linking between polypeptide chains and from linking to accompanying molecules (lipids, carbohydrates, or metal ions). The resulting protein molecules are large compared with many biological compounds, with molecular weights often in the range of 10 000–100 000 Da (1 Da being equivalent to the mass of a hydrogen atom). Some proteins are even larger, the largest glutenin proteins of wheat gluten having sizes extending up to tens of millions of Daltons. There are about 20 different amino acids, each of them having the combination of an acidic group (COOH) and a basic group (NH2) and each having a distinct side chain that provides the functional differences between them all. The amino acids are joined together by peptide bonds to form a polypeptide chain, by the following formula (for two amino acids, R1 and R2): R 1 COOH þ H2 N R 2 ! R 1 CO NH R2 þ H2 O The sequence and specificity of the amino acids are ‘spelled out’ exactly in the genetic code of the genes, thereby ensuring that the protein will be made in the same manner each time in the specific species and tissue. For this reason, the
PROTEINS | The Protein Chemistry of Cereal Grains
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Wheat proteins
Soluble proteins
Gluten proteins
Monomeric gliadin
Albumins
Globulins
ω gliadins
Polymeric glutenin
LMW
α gliadins β gliadins glutenin subunits γ gliadins
S-poor prolamins
S-rich prolamins
HMW prolamins
HMW glutenin subunits
HMW prolamins S-poor prolamins
HMW glutenin subunits
ω gliadin
γ LMW glutenin subunits
gliadin
β gliadin α gliadin
S-rich prolamins
Figure 2 Classification of the proteins of the wheat grain, showing the electrophoretic patterns for some of them.
determination of grain protein composition is a valuable means of establishing the identity of a variety within a grain species.
Many Different Cereal Grain Proteins In the past century, we have come a long way from Osborne’s concept of four classes of proteins to realize that within each of his classes, there are a large number of proteins differing in charge and size; this great diversity and heterogeneity can readily be shown by fractionation methods such as gel electrophoresis. Each of the horizontal lines (‘bands’) in the electrophoretic patterns in Figure 3 represents a separate protein component (more than one in many cases). The combination of gel electrophoresis and gel isoelectric focussing as used in proteomics, provides a two-dimensional display of spots (Figure 4), each being a distinct protein (polypeptide, meaning polymer of amino acids). The diversity of grain protein composition is such that differences in composition can readily be demonstrated between closely related varieties of a grain species. This is evident in Figure 3 where each column of bands represents the prolamin proteins extracted from grain samples of specific varieties of wheat and of barley. Each of the two sets of varieties has a distinct pattern of bands, while all the wheat patterns differ from all the barley patterns. This type of analysis is thus useful for the identification of varieties in the practical situation of grain delivery when identity must be established to define quality type and royalty payment for distinct varieties.
Why Is Protein So Important in Grains? The protein component of the grain is critical to the grain’s role as a seed – the beginning of a new plant. The grain protein has the natural purpose to serve as a reserve of amino acids for the embryo (germ) to draw upon during germination, while the roots and shoots are growing until the leaves can support growth and development via photosynthesis. The protein component of the grain is important nutritionally as a source of amino acids, especially the essential amino acids that we cannot synthesize ourselves. The amino acids that are essential include lysine, methionine, leucine, isoleucine, and several more. On the other hand, grain protein is important for the grain processing industry, because it is often the most important part of the grain in determining processing quality and nutritional value. In grain composition, protein is next in abundance after starch and the fiber components (Figure 5). The protein content of cereal grains ranges from 8% to 15% of the grain weight (Table 1). Even within a grain species, the actual protein concentration may cover a range of values, depending on the genetic potential of a specific variety of that grain species, on the nutritional status of the plant, and on climatic and other environmental factors. Most of the protein is in the grain’s endosperm (the part that becomes white flour after milling the cereal grain), the endosperm being the largest part of the grain by mass. However, the proportion of protein may be higher in some of the non-endosperm tissues of the grain, especially in the germ (the embryo, which will become the new plant after germination).
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PROTEINS | The Protein Chemistry of Cereal Grains
Figure 3 Lab-on-a-chip analysis of the composition of prolamin proteins extracted from grains of wheat varieties (L to R: Halberd, Janz, Frame, and Yitpi) and of barley varieties (Excalibur, Sloop, Schooner, Flagship, Gairdner, and Baudin). Each protein extract was applied to a small lab chip for microfluidic capillary electrophoresis. Each analysis took about 50 s. The elution patterns were manipulated to simulate gel electrophoresis patterns.
kDa 130
3
pH
10
10 Figure 4 Proteome map of the polypeptides (reduced proteins) extracted from mature wheat grain (cv “Rosella”). Fractionation involved isoelectric focussing between pH 3 (left) and pH 10 followed by SDS gel electrophoresis in a gradient from 8% (top) to 18% polyacrylamide. After in-gel digestion with trypsin, 315 protein spots (as numbered) were excised and identified by MALDI-TOF and LC–MS/MS. Adapted from Mak, Y., Willows, R. D., Roberts, T. H., Wrigley, C. W., Peter Sharp, P., and Copeland, L., 2009. Germination of wheat: A functional proteomics analysis of the embryo. Cereal Chemistry 86 (3), 281–289.
The barley grain is taken as an example of this in Figure 6. Figure 7 shows the chemical composition of wheat grain, with respect to the distribution of protein (and other components) in its various anatomical parts.
Crude protein
Crude fat
Crude fiber
Crude ash
Starch
Cellulose
Figure 5 Chemical composition typical of a whole cereal grain.
The importance of protein content is seen in the valuation of grain shipments. The ‘single figure’ of the protein content of a grain sample is an important specification in the marketing of most cereal grains. In general, grain of higher protein content commands a higher value. For wheat, a higher protein content indicates better baking quality, and a higher protein content for feed grains provides a richer supply of essential amino acids (irrespective of the proportion of essential amino acids in their storage protein).
PROTEINS | The Protein Chemistry of Cereal Grains
However, malting barley is an exception to the rule that high protein content is desirable, because of the need to have a high starch content to provide a correspondingly high extraction of fermentable sugars in the malting process. Nevertheless, even for malting barley, there is a minimum level of protein (8%), to ensure that there is an adequate supply of the hydrolytic enzymes needed throughout the malting process.
Analysis of Protein Content Today, the routine determination of protein content of all grain species generally involves the use of near-infrared (NIR) spectroscopy, applied to whole grain, milled grain, or flour, based on correlations to total nitrogen analyses (Table 2). In the case of wheat only, protein content can also be determined by washing out the gluten protein and weighing its wet mass. Even though gluten protein is less than 80% of the total grain Table 1 Approximate protein content of the more common cereal species, as percentages of the ‘as is’ weight of whole grain, except for rice (milled grain) Protein content
Barley Maize (corn) Oats (as groats) Rice (milled) Rye Wheata
9–12 8–12 12–15 8–11 12–15 9–16
Durum wheat, a distinct species from common wheat, generally requires a higher protein range, say, 12–16%.
protein, this approach can be used if appropriate correlations are established between the level of washed gluten and ‘true’ grain protein content. Further possibilities include colorimetric methods (e.g., the biuret reaction) or the amide distillation procedure (Table 2). The standard (‘true’) method for protein determination involves the complete digestion of the grain or flour to its elements and determination of the proportion of the element nitrogen. This figure is multiplied by 6.25 to obtain the protein content or by a factor of 5.7 in the case of wheat. This procedure assumes that virtually all the nitrogen in the grain is in the form of protein. A lower multiplication factor is used for wheat because its protein contains a high proportion of the amino acid glutamine, giving its protein unusually high nitrogen content (Figure 8). The total nitrogen content is used as an absolute value in establishing correlations for the routine method of NIR spectroscopy to determine the protein content of whole grain or of milled products (Table 2).
Protein Quality
Cereal species
a
The ‘single figure’ of protein content, however, does not take into account the quality of the protein. This quality is indicated for nutritional purposes by analysis of the levels of essential amino acids after hydrolysis of the peptide bonds. For wheat, however, the quality of the protein also relates to the dough strength provided by the particular combination of gluten proteins present, determined partly by the genotype (the variety) and also by the growth conditions. For the animal feed industry, the amino acid composition is especially important, due to the need to ensure an adequate supply of essential amino acids. The protein of cereal grains is a
Crude protein Crude fat Crude fiber Crude ash Nitrogen-free extract Cellulose
Crude fiber
20
Crude fat
15
Crude protein
5
13.5
4
13.0 12.5
3
12.0
10 2 5
1
0
0 Whole
Pearled
Hulls
11.5 11.0 10.5 Whole
5
Pearled
Hulls
Figure 6 Chemical composition of the barley grain; distribution between the different parts.
Whole
Pearled
Hulls
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PROTEINS | The Protein Chemistry of Cereal Grains
12% 2% 60% 2% 6% 2% 2%
% of grain 0 Embryo
Scutellum
50 Pericarp
Protein Lipids Starch Red. sugars Pentosans Cellulose Ash
Aleurone
100 Endosperm
Protein Lipids Starch Red. sugars Pentosans Cellulose Ash Figure 7 Chemical composition of the wheat grain; distribution between anatomical parts. ‘Red. Sugars’ means reducing sugars, such as glucose. Table 2 Methods used to determine the protein content of cereal grain, especially wheat Process
Material assayed
Assay method
Grain Flour/meal
NIR NIR
Mill # Wash (for wheat only) # Extract
Gluten
Weight (wet or dry)
Protein in solution
Biuret or dye-binding reaction
# Hydrolyze
Amino acids
Gas or liquid chromatography
Amide N
Titrate ammonia released
Ammonia or nitrogen
Kjeldahl or Dumas method
# Alkaline digestion # Acid digestion
good source of most of the essential amino acids, with the exception of lysine and possibly tryptophan, which are lower in the cereal grains than in most animal proteins. The amino acid composition for wheat is shown in Figure 8, with respect to the major classes of wheat proteins. The amino acid compositions of whole grains of several cereals are compared in Table 3.
Comparison of the Proteins of Cereal Species The various cereal species differ in the composition of their grain proteins. Just as the various cereal species are related genetically, these genetic similarities and distinctions are reflected directly in the amino acid sequences of their storage proteins. Both the relevant gene sequences and the corresponding amino acid sequences also reflect the taxonomic
relationships among the cereal species, as is illustrated in Figure 9. To a large extent, these differences explain how the storage proteins of the respective cereal grains make their distinct contributions with respect to their food uses. The most obvious example is wheat, whose gluten-forming storage protein is unique among the cereals in suiting wheat flour for bread making. Furthermore, the proteins of the rice grain contribute to its eating quality. The storage proteins of the cereal endosperm have been given the specific names according to each cereal genus, as listed in Figure 9.
The Proteins of Wheat, Rye, and Triticale Wheat and rye are closely related; triticale is a man-made hybrid between wheat and rye. The genetic control of the storage proteins of these cereals is detailed in the specific articles on wheat, rye, and triticale. Their protein compositions are thus similar to a limited extent, but the dough-forming quality of rye and triticale is much poorer than that of wheat. The popularity of rye-based foods is not so widespread as that of foods made from wheat. Rye is especially popular in Eastern European and Scandinavian countries. Baked products are commonly crispbread and varieties of bread, including pumpernickel. Whereas ‘true’ rye bread is made from 100% rye, it is common for rye bread to be made from a blend of rye and wheat flours, the contribution of the wheat gluten being needed to provide the baking quality that is lacking in the rye flour. The poorer dough-forming characteristics of rye gluten are largely due to the lower level of the glutelin-type protein, combined with the more hydrophilic (water-loving) properties of the rye secalins, compared with the corresponding classes of protein in wheat. The water-soluble proteins of rye contain the albumin and globulin classes of protein, similar to those in wheat, but for rye, there is generally a higher level of amylase enzymes than in wheat because of the greater susceptibility of rye to preharvest
PROTEINS | The Protein Chemistry of Cereal Grains
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Albumin
Globulin
Prolamin
Glutenin
Ile Leu Lys Met Cys Phe Tyr Thr Trp Val Arg His Ala Asp Glu Gly Pro Ser NH3 Figure 8 Amino acid composition of the Osborne fractions of wheat.
Table 3 The amino acid compositions of whole grains of several cereals, expressed as grams per 16 g of nitrogen Amino acid
Wheat
Rice
Corn
Barley
Oats
Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
3.6 4.6 4.9 2.5 29.9 3.9 2.3 3.3 6.7 2.9 1.5 4.5 9.9 4.6 2.9 1.1 3.0 4.4
6.0 8.3 10.3 1.1 20.6 5.0 2.5 3.8 8.2 3.8 2.3 5.2 4.7 5.4 3.9 1.2 3.5 5.5
7.5 4.2 6.3 1.6 18.9 3.7 2.7 3.7 12.5 2.7 1.9 4.9 8.9 5.0 3.6 0.6 3.8 4.8
4.0 4.7 5.7 2.3 23.6 3.9 2.1 3.6 6.7 3.5 1.7 5.1 10.9 4.0 3.3 1.5 3.1 5.0
4.5 6.3 7.7 2.7 20.9 4.7 2.1 3.8 7.3 3.7 1.7 5.0 5.2 4.7 3.3 1.3 3.0 5.1
sprouting and the consequent production of starch-degrading enzymes. The essential amino acid composition of rye is slightly better than that of wheat, because of the lower content of prolamin-type proteins in rye (Figure 9). The properties of triticale are intermediate between those of rye and wheat, reflecting the genetic origins of triticale.
The Proteins of the Barley Grain The protein concentration of cultivated barley ranges from 9% to 12%. For barley, the protein content is generally calculated as total nitrogen content multiplied by the factor of 6.25. The protein content of hull-less (naked) barley genotypes is
generally higher (even up to 18%) because of the absence of the outer hull layers (the glumes – the palea and lemma), which contain relatively little protein. The production of the enzymes of germination is an important part of a good malting barley, but paradoxically, high protein content (e.g., over 11%) is not necessarily desirable for the best malting quality. This is because the ultimate test of malting quality is the extent of ‘extract’ produced during the mashing process. The amount of extract determines how much beer can be produced from a given amount of malt. ‘Extract’ represents the amount of soluble fermentable sugars from the hydrolysis of starch, resulting from the combination of very active amylases and high starch content. For this reason, a low protein content (i.e., less than 8%) is also undesirable, as it may have the consequence of inadequate enzyme production. Consumption for human food is a traditional use of barley, but this now accounts for only 5% of total barley consumption worldwide. Feed use predominates, amounting to 75% of barley use. The amino acid composition of barley does not provide a consistently better balance nutritionally than other cereal grains, still being deficient in lysine, methionine, and threonine. Nevertheless, it is a popular source of protein and energy in feedstuffs, in combination with other sources of energy. High-lysine barley genotypes have been developed, analogous to those in maize, the first of these being the Hyproly line. The hordein polypeptides show considerable polymorphism, like the prolamins of wheat and rye. A large proportion of the barley prolamins are disulfide-linked (both intra- and interchain linkages) with naturally occurring polymers of high molecular weight. Unlike the glutenin polymers of wheat, these are not desirable for the processing of barley. Because they generate problems in the malting process and downstream, they are likely to produce haze in the final product (i.e., beer) if they have not been adequately hydrolyzed during processing. The hordein polypeptides are classified into groups (A, B, C, and D hordeins) according to their decreasing mobilities
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PROTEINS | The Protein Chemistry of Cereal Grains
Globulins
7S
Prolamins
11/12S
Maize, oats, rice, barley, rye, wheat
Wheat
Oats
7S globulin
T riticin
Globulin
Other prolamins
Rice
Maize
Glutelin
The prolamin superfamily
Sorghum
Maize
Rice
Kaffirin
Oats
Triticeae: barley, wheat, rye
Avenin 16 kDa 10 kDa 13 kDa
-Zein
-Zein
-Zein
-Zein HMW S-rich S-poor
Embryo, aleurone
Endosperm
Figure 9 Storage protein classes in cereals. Adapted from Shewry, P. R., Tatham, A. S., and Halford, N. G., 1999. The prolamins of the Triticeae. In: Shewry, P. R. and Casey, R. (Ed.), Seed Proteins. Kluwer Academic, The Netherlands, pp. 35–78. Table 4
Classes of barley proteins (hordeins) and corresponding loci
Hordein class
Loci
Gene copy number
Proteins present in the cv ‘Carlsberg’
MW (kDa)
Corresponding wheat prolamin
B C D g
Hor 2 Hor 1 Hor 3 Hor 5
20–30 20–30 1 Low
10 9 1 7
36–45 50–60 105 36–45
LMW glutenins o-Gliadins HMW glutenins g-Gliadins
during SDS gel electrophoresis. The C hordeins have analogy to the omega-gliadins of wheat, having a general absence of the sulfur-containing amino acids. They are coded by genes (locus Hor 1) on the short arm of barley chromosome 1H, as indicated in Table 4. The genes (Hor 2 and Hor 5) for the sulfur-rich B and gamma-hordeins are also coded on chromosome 1H. The D hordeins, which occur as disulfide-linked polymers, are analogous to the HMV subunits of wheat glutenin; like them, their genes (Hor 3) are located on the long arm of the corresponding barley chromosome (1H).
The Proteins of the Rice Grain The proteins of rice are a contrast to those of wheat and barley, being predominantly of the globulin class. As a protein source, milled rice contains the lowest amount of protein (about 5%) among the major cereals. Moreover, this protein is not easily digestible by humans and monogastric animals. However, compared with that of other cereal proteins, the overall amino acid composition of rice protein is significantly more balanced due to the relatively higher level of lysine content. This unusual amino acid composition results because rice is one of the few cultivated plants in which there are significant levels of globulins and prolamins. These are the two major classes of storage proteins in the seeds of higher plants. Unlike other cereals that accumulate prolamins as their primary
nitrogen reserve, the major storage proteins in rice are the glutelins, which are homologous at the primary sequence level to the 11S globulin proteins (Figure 9), a class that is the dominant form of nitrogen deposition in legumes. Rice prolamin proteins have a number of characteristics that are different from the prolamins present in most other cereals. The most important characteristic of the deposition of nitrogen in the rice kernel is that there are three kinds of protein bodies in the rice endosperm, namely, large spherical, small spherical, and crystalline protein bodies. Each of these is surrounded by a single continuous membrane. Although the spherical protein bodies form within vacuoles, the proteins are synthesized in the endoplasmic reticulum and in the Golgi apparatus and are then transported to the vacuoles via vesicles. Removal of the husk (hull) from rough (paddy) rice yields the kernel. This is composed of the pericarp, seed coat, aleurone, endosperm, and the germ and is known as ‘brown rice.’ It has a protein content of 9–10%, with a significantly higher nutritional value than the most commonly utilized rice product – white polished (milled) rice, whose protein content is 8%. The milling process for paddy rice results in 40–55% white milled rice, together with three major by-products, namely, husks (20%), bran (10%), and brokens (10–22%). These byproducts have protein contents of 3%, 17%, and 9%, respectively. The aleurone layer, the tissue with the highest level of proteins and nutritionally important minor components, is removed during the rice milling process.
PROTEINS | The Protein Chemistry of Cereal Grains
The subaleurone region of the rice grain plays an important part because of its nutritional value. It is a globulin-rich layer, being several cell layers thick. Its lysine content is much higher than that of the proteins located in the rice endosperm. Thus, rice should be milled as lightly as possible to retain as much as possible the subaleurone layer. The albumin fraction of rice is highly heterogeneous, containing biologically important components. It can be separated into four subfractions, based on the molecular size of its proteins. More than 50 individual polypeptides were observed in the albumin fraction, using isoelectric focussing. As for cereal grains in general, the albumins are mainly enzymes and enzyme inhibitors. Like the prolamins from other cereals, rice prolamins are readily soluble only in alcohol/water mixtures. However, the classical extraction procedure had not been useful in early studies. More recently, these estimates of prolamin content have been shown to be low. Using 55% propan-L-ol and reducing agents, higher yields of prolamins (20–25% of the total protein) have been obtained. Rice prolamins are 10–16 kDa in molecular weight. Thus, their molecular size range is significantly smaller than the prolamins of other cereals. They are highly variable between rice cultivars, based on their electrophoretic and isoelectric focussing fingerprints. According to the Osborne classification system, the least soluble proteins of the rice grain are oligomeric globulins representing up to 80% of the total protein fraction (Figure 9). They have been difficult to study because of their general insolubility in all solvents except dilute alkali, due to their high molecular weight and their heterogeneity. When analyzed by gel filtration chromatography, the purified globulin subunits were resolved into three subunits, linked by disulfide bonds, which varied in stoichiometry depending on the report and on the rice variety used. Despite their general insolubility, rice glutelins are homologous in structure to 11S globulins (Figure 9). The structural relationship is evident when comparing the N-terminal amino acid sequences and using immunologic methods. The extent of homology between the rice glutelin and legume 11S globulins is 30–35%. Structural similarity is evident in the primary sequences of homology to a motif common in wheat, rye, barley, and maize prolamins.
The Proteins of Maize Maize (or corn), often classed in world trade as one of the ‘coarse’ grains, is an important source of protein in the diets of many people in countries such as Africa and South America. In addition, maize is used extensively as a feed grain and in a range of industrial processes. The process of corn starch manufacture produces the grain protein as a by-product. It is sometimes referred to as ‘corn gluten’ in the trade, but this name is inappropriate, because maize protein has no relationship to the gluten of wheat and is safe for consumption by those susceptible to celiac disease. Like the other cereal grains, maize protein is slightly deficient in lysine. This limits its utilization as an animal feed. This nutritional imbalance is largely due to the low lysine content of ‘zein,’ the major group of maize proteins (Figure 9). The
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development of the ‘opaque-2’ and ‘floury-2’ mutations has led to the development of maize genotypes with higher levels of lysine, due to a lower content of zein and more of the nutritionally superior albumin type of protein. Although these new maize types offer nutritional advantages, they carry a grain yield penalty. The zein protein of maize can be isolated by extraction of the crushed endosperm (the starchy part of the grain) with 70% aqueous ethanol, after breaking disulfide bonds. Maize zein has been further fractionated into subclasses of proteins, designated alpha-, beta-, gamma-, and delta-zeins. The genes for their synthesis have been mapped to maize chromosomes 6 and 7.
The Proteins of Sorghum The major storage proteins of sorghum, termed ‘kafirins’ (Figure 9), are fractionated by a procedure similar to that for the zeins of maize. Alpha-kafirin, composing about 80% of the overall kafirin content, can be further subdivided into two proteins with molecular weights of about 23 000 and 25 000 Da. Three beta-kafirins and one gamma-kafirin have been identified, all of them having molecular weights in the range of 16–28 kDa. Like maize, high-lysine mutants of sorghum have been developed.
Future Prospects Our current understanding of grain protein chemistry contrasts strongly with the rudimentary knowledge of the early twentieth century. That knowledge has progressed from the recognition of only a few distinct grain proteins to fractionating and characterizing thousands of proteins from all the tissues of the cereal grain – the endosperm, germ, scutellum, and bran. This knowledge permits more efficient selection for improved processing quality in the breeding of new varieties. The knowledge is also being applied to testing for protein quality after the grain is harvested, so that grains of specific quality can be segregated for appropriate marketing and processing. Knowledge at the chemical level is now complementing new genetic insights, so that breeders and molecular geneticists can employ conventional and novel methods to create quality types that have previously not been possible – varieties with new protein functionality. Thus, the future holds great promise for grains that will be processed more efficiently with nutritional advantages. For example, it has been possible to incorporate purified gluten proteins into rice dough, thus to monitor their effects on the mixing properties. The protein content of the cereal grains has always been important for marketing and processing. The generally desirable trait of high-protein concentration has often been inversely proportional to grain yield. Improvements in genetics and agronomic practice have tended to reduce grain protein levels, because high yield has been a common goal for farmers. The coming decades will bring a new factor that will further erode protein levels – the rising concentrations of carbon dioxide in the atmosphere. For whatever reason, it is inevitable that CO2 levels will continue to rise, although the rate of increase is difficult to predict. Many experiments have been
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PROTEINS | The Protein Chemistry of Cereal Grains
conducted to simulate effects of future levels of CO2. They have consistently indicated increased yields of grain and biomass, together with decreased levels of grain protein content. The higher atmospheric concentration of CO2 acts as a carbon fertilizer, with consequent increases in starch synthesis but little change in protein synthesis. There will thus be a need to rethink the role of protein in cereal grains if it will gradually become lower in concentration. At the manufacturing level, there is increasing interest in providing greater protein content for cereal-based snacks and breakfast foods. Cereal proteins are also undergoing texturization and extrusion with the aim of creating synthetic meat products.
Exercises for Revision
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How did the early chemists’ views on protein chemistry differ from our present knowledge? Why were they unaware of the great heterogeneity of grain proteins? What are the aspects of protein composition that explain the great differences between the various proteins in their properties? Prepare a table of differences and similarities between the major cereal species with respect to their protein compositions. What are the nutritional deficiencies of cereal grain proteins with respect to the essential amino acids? Describe the genetic achievements that have partially overcome these deficiencies.
Exercises for Readers to Explore the Topic Further
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•
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Examine the taxonomic relationships between the major cereal species, as provided in this encyclopedia. Compare the associations between the proteins of these grains in Figure 8. Look at the genus names to see the origins of the grain-specific protein names. Obtain the amino acid compositions of the grain proteins for the major cereals. What are the main differences? How would these differences account for functional and nutritional differences between these grain species? Contrast the differences in function for an enzyme (to catalyze a specific reaction) versus a storage protein (a source of amino acids for the new plant). Why would both types of protein be produced under the same strict genetic control? Compare the various methods of determining the amount of protein in a grain or flour sample (Table 2 and elsewhere). Which would you expect to be the most accurate?
See also: The Basics: Grain Crops, Overview (00001); Taxonomic Classification of Grain Species (00002); The Cereal Grains: An Overview of the Family of Cereal Grains Prominent in World Agriculture (00006); Proteins: Enzyme Activities (00102); Protein Synthesis and
Deposition (00106); Proteomics (00107); Wheat-Based Foods: Wheat grain utilization: Overview (00142); Wheat Processing: Wheat, Grain Proteins and Flour Quality (00164); Grain Marketing and Grading: Variety Identification of Cereal Grains (00175); Genetics of Grains: Maize, Quality Protein Maize (00223); Maize, Other Maize Mutants (00224).
Further Reading Bailey HC (1941) A translation of Beccari’s lecture “Concerning Grain” (1729). Cereal Chem. 18: 555. Bushuk W (ed.) (2001) Rye: Production, Chemistry and Technology, second ed. St. Paul, MN: American Association of Cereal Chemists. Dendy DAV (ed.) (1995) Sorghum and Millets: Chemistry and Technology St. Paul, MN: American Association of Cereal Chemists. Hartley H (1951) Origin of the word protein. Nature 168: 244. Henry RJ and Kettlewell PS (1996) Cereal Grain Quality. London: Chapman and Hall. Islam S, Ma W, Yan G, Be´ke´s F, and Appels R (2011) Modifying processing and health attributes of wheat bread through changes in composition, genetics and breeding. In: Cauvain SP (ed.) Bread making. Improving quality, second ed. Boston, New York: CRC Press. Juhasz A, Bekes F, and Wrigley CW (2014) Wheat proteins. In: Ustunol Z (ed.) Applied Food Protein Chemistry, Chapter 11, pp. 219–304. West Sussex, UK: Wiley-Blackwell, John Wiley & Sons. Juliano BO (ed.) (1985) Rice: Chemistry and Technology, second ed. St. Paul, MN: American Association of Cereal Chemists. Kent NL (1975) Technology of Cereals, with Special Reference to Wheat, second ed. Oxford, UK: Pergamon. Lasztity R (1999) Cereal Chemistry. Budapest, Hungary: Akade´miai Ko¨nyvkiado´. McGregor AW and Bhatty RS (eds.) (1993) Barley: Chemistry and Technology. St. Paul, MN: American Association of Cereal Chemists. Mertz ET (ed.) (1992) Quality Protein Maize. St. Paul, MN: AACC International. Pomeranz Y (ed.) (1988) Wheat: Chemistry and Technology, third ed. St. Paul, MN: American Association of Cereal Chemists. Seneweera S, Fernando N, Panozzo J, Tausz M, Norton RM, Fitzgerald GJ, Myers S, and Nicolas ME (2014) Intra-specific variation of wheat grain quality in response to elevated [CO2] at two sowing times under rain-fed and irrigation treatments. J. Cereal Sci. 59(2): 137–144. Shewry PR and Casey R (eds.). (1999) Seed Proteins. The Netherlands: Kluwer Academic. Shewry PR and Miflin BO (1985) Seed storage proteins. In: Pomeranz Y (ed.) Advances in Cereal Science and Technology vol. 7, pp. 1–83. St. Paul, MN: American Association of Cereal Chemists. Sikorski ZE (2001) The Chemical and Functional Properties of Food Proteins. Lancaster, PA: Technomic Publishing. Watson SA and Ramsted PE (eds.) (1987) Corn: Chemistry and Technology St. Paul, MN: American Association of Cereal Chemists. Webster FH (ed.) (1985) Oats: Chemistry and Technology. St. Paul, MN: American Association of Cereal Chemists. Wrigley CW (2012) Proteins – the basis of life. Teach. Sci. 58: 56–59. Wrigley CW and Batey IL (eds.) (2010) Cereal Grains: Assessing and Managing Quality Oxford, UK: Woodhead Publishing Ltd.
Relevant Websites http://www.aaccnet.org – American Association of Cereal Chemists. http://www.campden.co.uk – Campden & Chorleywood Food Research Association. http://www.icc.or.at – International Association for Cereal Science and Technology. http://www.usda.gov – United States Department of Agriculture. http://www.wheat.pw.usda.gov – Grain genes.
Topic Highlights
• • •
• • • • •
The importance of dietary protein from cereal grains – rice, wheat, maize, and others. The purification of wheat gluten from dough – as an experiment in the kitchen and as a commercial product. The simple structure of proteins – a linear polymer of amino acids – produces the diversity of properties of enzymes, hormones, hair and horn, silk and sinew, and gluten together with the many other grain storage proteins. Folding and cross-linking of the amino acid polymer gives three-dimensional structure that is needed for many protein functions, such as enzymatic activity. Osborne’s solubility-based protein classes: water-soluble albumins, salt-soluble globulins, prolamins extractable with 70% aqueous ethanol, and the residual glutelins. Protein quantity is determined by various methods, especially by near-infrared spectroscopy, based on total nitrogen content. Protein quality relates to its nutritional value and its contribution to functional uses. The specific proteins are described for wheat, rye, triticale, barley, rice, maize, and sorghum.
Learning Objective To achieve an understanding of proteins as critical components of the grains of cereal species and their function for the plants and for our dietary needs.
Introduction Worldwide, plant proteins contribute more than half of our dietary supply of protein. Over 200 million tonnes of protein are harvested each year in the form of cereal grains, based on FAO statistics (http://faostat.fao.org). This protein harvest includes that derived from the three major cereal grains, wheat, rice, and maize, and also less widely grown species: barley, rye, triticale, and sorghum. In some developing countries, a single cereal species may be the major or sole source of protein in the diet. In many countries, cereal proteins enter the diet in various forms, including wheaten breads and pastries, noodles, fried rice dishes, rye crispbreads, cookies, biscuits, pearl barley, pasta, corn tortillas, breakfast cereal, oatmeal porridge, couscous, and many more foods. Most obviously, cereal grain proteins are eaten directly in these many processed forms. The second route is indirect, in the form of animal products, such as meat, milk, and eggs, following the feeding of cereal grains to animals. Whereas the protein part of the grain is important nutritionally, it also serves functional purposes, relevant to the
Reference Module in Food Sciences
processing of the grain. Of these, wheat and rye are unique because of the dough-forming properties of their storage proteins, known as ‘gluten’ when formed into dough.
The Discovery of Protein Structure Cereal Proteins in Prehistory Historically, the proteins of the cereal grains have played a significant part in the civilization of mankind, through their role in providing a reliable source of amino acids, the essential dietary compounds that are the building blocks of proteins in the body. Grains as a source of dietary proteins could be obtained via agriculture, permitting the establishment of a permanent dwelling place, providing an important change from the nomadic existence of the hunter-gatherer way of life. The establishment of permanent towns led, in turn, to specialization of occupation and to opportunities for mankind to pursue cultural pursuits and scientific inquiry. However, it is only in recent centuries that the chemistry of proteins has been elucidated.
Early Concepts The word ‘protein’ was proposed by the French scientist Berzelius, writing to the German scientist Mulder, with the appropriate meaning of ‘primary substance,’ as recently as 1838, long after the acceptance of the terms for specific types of proteins, such as ‘gluten,’ ‘zymom,’ and ‘fibrin.’ Early concepts of protein chemistry imagined it to have the complex formula C20H31N5O6, meaning that 20 molecules of carbon (C) were compounded with 31 molecules of hydrogen, five molecules of nitrogen, and six molecules of oxygen. The dogma of those early days also maintained (incorrectly) that this complex was absorbed (unchanged) directly by the intestine. It is now known that dietary proteins are hydrolyzed to the component amino acids for absorption into the bloodstream.
Wheat Gluten: The First Purified Protein One of the first proteins to be obtained in reasonably pure form was gluten, because it could be prepared so readily (by the washing of dough made from wheat flour). This important experiment, first reported by the Italian scientist Beccari in 1729, can be simply performed in the home kitchen. To do so, a dough is mixed by adding water, a little at a time, to wheat flour until a cohesive mass is formed. This dough is kneaded between the fingers under a gentle stream of water from the tap. If a glass is placed underneath, a milky-white stream of starch can be caught in the glass, while the dough in the fingers becomes smaller and tougher. The gluten ball held in the fingers is mainly protein, separated very simply from the starch that settles at the bottom of the glass. Strictly speaking, gluten is a protein–lipid–carbohydrate complex formed as a result of specific covalent and
http://dx.doi.org/10.1016/B978-0-08-100596-5.00101-3
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PROTEINS | The Protein Chemistry of Cereal Grains
noncovalent interactions from flour components during dough making as the components are hydrated and energy from mechanical input from the mixing process is provided. This gluten-washing procedure has since become very big business internationally, because the gluten thus isolated is needed for bolstering the protein content of a wide range of food products – bread, especially, and also breakfast cereals, meats, cheeses, snack foods, and texturized products – or consumed directly as ‘seitan.’ Nonfood applications include pet and aquaculture feeds, biodegradable plastics, films, coatings, adhesives, inks, cosmetics, and pharmaceuticals. Late in the nineteenth century, the American scientist Thomas Osborne studied the proteins of many edible grains, using a succession of solvents to extract diverse classes of proteins from the crushed grain. As a result, he proposed the generic names of ‘albumin’ (extracted with water), ‘globulin’ (extracted subsequently by salt solution), ‘prolamin’ (extracted by 70% aqueous ethanol), and ‘glutelin’ (extracted finally by a dilute acid). Subsequently, the prolamins of wheat have been understood to be single polypeptides, whereas wheat glutelins (known as glutenins) have been shown to be disulfide-linked polymers built up of glutenin subunits. The cover of one of Osborne’s famous publications appears in Figure 1. In many cereal grains, the metabolic proteins are albumins and globulins, many of these being the residual enzymes that have functioned in synthesizing the components of the maturing grain, plus others that are present in preparation for germination. On the other hand, the cereal storage proteins are often of the prolamin and glutelin classes; their water insolubility offers the advantage that they will be retained when the grain becomes waterlogged in the germination process. Osborne’s names for these major protein classes have since continued in use, but their definitions have been refined, and it has become clear that any one of these protein classes contains a large number of distinct but related proteins. The modern classification of cereal proteins is based on their genetics, also based on the chromosomal location of their coding genes. The term ‘prolamin’ in this nomenclature system covers all the polypeptides soluble in ethanol, independently whether they exist in the grain in monomeric or polymeric form. In the case of wheat (Figure 2), this prolamin group is subdivided into three groups as sulfur-rich, sulfur-poor, and high-molecular-weight (HMW) prolamins, representing the a-, b-, and g-gliadins plus the low-molecular-weight (LMW) glutenin subunits, the o-gliadins, and finally the HMW glutenin subunits, respectively, in the old classification system.
So What Is a Protein? A protein molecule is simply a linear polymer of amino acids. The structure of protein molecules also depends on the folding and cross-linking of the polymers, giving a three-dimensional structure that is especially important for functions such as enzymatic activity. It is no wonder that these early protein chemists wrestled with reaching this understanding, when we now see that this relatively simple chemical structure can produce the great diversity of properties that are found in proteins, such as enzymes and epidermis, hair and horn, feathers and flagella, silk and sinew, and even spider’s web.
Figure 1 The cover of one of Osborne’s early publications (1912) on the proteins of cereal grains.
The diversity depends on the selection and sequence of the amino acids plus the length of the protein (polypeptide) chain. Further diversity is provided by cross-linking between polypeptide chains and from linking to accompanying molecules (lipids, carbohydrates, or metal ions). The resulting protein molecules are large compared with many biological compounds, with molecular weights often in the range of 10 000–100 000 Da (1 Da being equivalent to the mass of a hydrogen atom). Some proteins are even larger, the largest glutenin proteins of wheat gluten having sizes extending up to tens of millions of Daltons. There are about 20 different amino acids, each of them having the combination of an acidic group (COOH) and a basic group (NH2) and each having a distinct side chain that provides the functional differences between them all. The amino acids are joined together by peptide bonds to form a polypeptide chain, by the following formula (for two amino acids, R1 and R2): R 1 COOH þ H2 N R 2 ! R 1 CO NH R2 þ H2 O The sequence and specificity of the amino acids are ‘spelled out’ exactly in the genetic code of the genes, thereby ensuring that the protein will be made in the same manner each time in the specific species and tissue. For this reason, the
PROTEINS | The Protein Chemistry of Cereal Grains
3
Wheat proteins
Soluble proteins
Gluten proteins
Monomeric gliadin
Albumins
Globulins
ω gliadins
Polymeric glutenin
LMW
α gliadins β gliadins glutenin subunits γ gliadins
S-poor prolamins
S-rich prolamins
HMW prolamins
HMW glutenin subunits
HMW prolamins S-poor prolamins
HMW glutenin subunits
ω gliadin
γ LMW glutenin subunits
gliadin
β gliadin α gliadin
S-rich prolamins
Figure 2 Classification of the proteins of the wheat grain, showing the electrophoretic patterns for some of them.
determination of grain protein composition is a valuable means of establishing the identity of a variety within a grain species.
Many Different Cereal Grain Proteins In the past century, we have come a long way from Osborne’s concept of four classes of proteins to realize that within each of his classes, there are a large number of proteins differing in charge and size; this great diversity and heterogeneity can readily be shown by fractionation methods such as gel electrophoresis. Each of the horizontal lines (‘bands’) in the electrophoretic patterns in Figure 3 represents a separate protein component (more than one in many cases). The combination of gel electrophoresis and gel isoelectric focussing as used in proteomics, provides a two-dimensional display of spots (Figure 4), each being a distinct protein (polypeptide, meaning polymer of amino acids). The diversity of grain protein composition is such that differences in composition can readily be demonstrated between closely related varieties of a grain species. This is evident in Figure 3 where each column of bands represents the prolamin proteins extracted from grain samples of specific varieties of wheat and of barley. Each of the two sets of varieties has a distinct pattern of bands, while all the wheat patterns differ from all the barley patterns. This type of analysis is thus useful for the identification of varieties in the practical situation of grain delivery when identity must be established to define quality type and royalty payment for distinct varieties.
Why Is Protein So Important in Grains? The protein component of the grain is critical to the grain’s role as a seed – the beginning of a new plant. The grain protein has the natural purpose to serve as a reserve of amino acids for the embryo (germ) to draw upon during germination, while the roots and shoots are growing until the leaves can support growth and development via photosynthesis. The protein component of the grain is important nutritionally as a source of amino acids, especially the essential amino acids that we cannot synthesize ourselves. The amino acids that are essential include lysine, methionine, leucine, isoleucine, and several more. On the other hand, grain protein is important for the grain processing industry, because it is often the most important part of the grain in determining processing quality and nutritional value. In grain composition, protein is next in abundance after starch and the fiber components (Figure 5). The protein content of cereal grains ranges from 8% to 15% of the grain weight (Table 1). Even within a grain species, the actual protein concentration may cover a range of values, depending on the genetic potential of a specific variety of that grain species, on the nutritional status of the plant, and on climatic and other environmental factors. Most of the protein is in the grain’s endosperm (the part that becomes white flour after milling the cereal grain), the endosperm being the largest part of the grain by mass. However, the proportion of protein may be higher in some of the non-endosperm tissues of the grain, especially in the germ (the embryo, which will become the new plant after germination).
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PROTEINS | The Protein Chemistry of Cereal Grains
Figure 3 Lab-on-a-chip analysis of the composition of prolamin proteins extracted from grains of wheat varieties (L to R: Halberd, Janz, Frame, and Yitpi) and of barley varieties (Excalibur, Sloop, Schooner, Flagship, Gairdner, and Baudin). Each protein extract was applied to a small lab chip for microfluidic capillary electrophoresis. Each analysis took about 50 s. The elution patterns were manipulated to simulate gel electrophoresis patterns.
kDa 130
3
pH
10
10 Figure 4 Proteome map of the polypeptides (reduced proteins) extracted from mature wheat grain (cv “Rosella”). Fractionation involved isoelectric focussing between pH 3 (left) and pH 10 followed by SDS gel electrophoresis in a gradient from 8% (top) to 18% polyacrylamide. After in-gel digestion with trypsin, 315 protein spots (as numbered) were excised and identified by MALDI-TOF and LC–MS/MS. Adapted from Mak, Y., Willows, R. D., Roberts, T. H., Wrigley, C. W., Peter Sharp, P., and Copeland, L., 2009. Germination of wheat: A functional proteomics analysis of the embryo. Cereal Chemistry 86 (3), 281–289.
The barley grain is taken as an example of this in Figure 6. Figure 7 shows the chemical composition of wheat grain, with respect to the distribution of protein (and other components) in its various anatomical parts.
Crude protein
Crude fat
Crude fiber
Crude ash
Starch
Cellulose
Figure 5 Chemical composition typical of a whole cereal grain.
The importance of protein content is seen in the valuation of grain shipments. The ‘single figure’ of the protein content of a grain sample is an important specification in the marketing of most cereal grains. In general, grain of higher protein content commands a higher value. For wheat, a higher protein content indicates better baking quality, and a higher protein content for feed grains provides a richer supply of essential amino acids (irrespective of the proportion of essential amino acids in their storage protein).
PROTEINS | The Protein Chemistry of Cereal Grains
However, malting barley is an exception to the rule that high protein content is desirable, because of the need to have a high starch content to provide a correspondingly high extraction of fermentable sugars in the malting process. Nevertheless, even for malting barley, there is a minimum level of protein (8%), to ensure that there is an adequate supply of the hydrolytic enzymes needed throughout the malting process.
Analysis of Protein Content Today, the routine determination of protein content of all grain species generally involves the use of near-infrared (NIR) spectroscopy, applied to whole grain, milled grain, or flour, based on correlations to total nitrogen analyses (Table 2). In the case of wheat only, protein content can also be determined by washing out the gluten protein and weighing its wet mass. Even though gluten protein is less than 80% of the total grain Table 1 Approximate protein content of the more common cereal species, as percentages of the ‘as is’ weight of whole grain, except for rice (milled grain) Protein content
Barley Maize (corn) Oats (as groats) Rice (milled) Rye Wheata
9–12 8–12 12–15 8–11 12–15 9–16
Durum wheat, a distinct species from common wheat, generally requires a higher protein range, say, 12–16%.
protein, this approach can be used if appropriate correlations are established between the level of washed gluten and ‘true’ grain protein content. Further possibilities include colorimetric methods (e.g., the biuret reaction) or the amide distillation procedure (Table 2). The standard (‘true’) method for protein determination involves the complete digestion of the grain or flour to its elements and determination of the proportion of the element nitrogen. This figure is multiplied by 6.25 to obtain the protein content or by a factor of 5.7 in the case of wheat. This procedure assumes that virtually all the nitrogen in the grain is in the form of protein. A lower multiplication factor is used for wheat because its protein contains a high proportion of the amino acid glutamine, giving its protein unusually high nitrogen content (Figure 8). The total nitrogen content is used as an absolute value in establishing correlations for the routine method of NIR spectroscopy to determine the protein content of whole grain or of milled products (Table 2).
Protein Quality
Cereal species
a
The ‘single figure’ of protein content, however, does not take into account the quality of the protein. This quality is indicated for nutritional purposes by analysis of the levels of essential amino acids after hydrolysis of the peptide bonds. For wheat, however, the quality of the protein also relates to the dough strength provided by the particular combination of gluten proteins present, determined partly by the genotype (the variety) and also by the growth conditions. For the animal feed industry, the amino acid composition is especially important, due to the need to ensure an adequate supply of essential amino acids. The protein of cereal grains is a
Crude protein Crude fat Crude fiber Crude ash Nitrogen-free extract Cellulose
Crude fiber
20
Crude fat
15
Crude protein
5
13.5
4
13.0 12.5
3
12.0
10 2 5
1
0
0 Whole
Pearled
Hulls
11.5 11.0 10.5 Whole
5
Pearled
Hulls
Figure 6 Chemical composition of the barley grain; distribution between the different parts.
Whole
Pearled
Hulls
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PROTEINS | The Protein Chemistry of Cereal Grains
12% 2% 60% 2% 6% 2% 2%
% of grain 0 Embryo
Scutellum
50 Pericarp
Protein Lipids Starch Red. sugars Pentosans Cellulose Ash
Aleurone
100 Endosperm
Protein Lipids Starch Red. sugars Pentosans Cellulose Ash Figure 7 Chemical composition of the wheat grain; distribution between anatomical parts. ‘Red. Sugars’ means reducing sugars, such as glucose. Table 2 Methods used to determine the protein content of cereal grain, especially wheat Process
Material assayed
Assay method
Grain Flour/meal
NIR NIR
Mill # Wash (for wheat only) # Extract
Gluten
Weight (wet or dry)
Protein in solution
Biuret or dye-binding reaction
# Hydrolyze
Amino acids
Gas or liquid chromatography
Amide N
Titrate ammonia released
Ammonia or nitrogen
Kjeldahl or Dumas method
# Alkaline digestion # Acid digestion
good source of most of the essential amino acids, with the exception of lysine and possibly tryptophan, which are lower in the cereal grains than in most animal proteins. The amino acid composition for wheat is shown in Figure 8, with respect to the major classes of wheat proteins. The amino acid compositions of whole grains of several cereals are compared in Table 3.
Comparison of the Proteins of Cereal Species The various cereal species differ in the composition of their grain proteins. Just as the various cereal species are related genetically, these genetic similarities and distinctions are reflected directly in the amino acid sequences of their storage proteins. Both the relevant gene sequences and the corresponding amino acid sequences also reflect the taxonomic
relationships among the cereal species, as is illustrated in Figure 9. To a large extent, these differences explain how the storage proteins of the respective cereal grains make their distinct contributions with respect to their food uses. The most obvious example is wheat, whose gluten-forming storage protein is unique among the cereals in suiting wheat flour for bread making. Furthermore, the proteins of the rice grain contribute to its eating quality. The storage proteins of the cereal endosperm have been given the specific names according to each cereal genus, as listed in Figure 9.
The Proteins of Wheat, Rye, and Triticale Wheat and rye are closely related; triticale is a man-made hybrid between wheat and rye. The genetic control of the storage proteins of these cereals is detailed in the specific articles on wheat, rye, and triticale. Their protein compositions are thus similar to a limited extent, but the dough-forming quality of rye and triticale is much poorer than that of wheat. The popularity of rye-based foods is not so widespread as that of foods made from wheat. Rye is especially popular in Eastern European and Scandinavian countries. Baked products are commonly crispbread and varieties of bread, including pumpernickel. Whereas ‘true’ rye bread is made from 100% rye, it is common for rye bread to be made from a blend of rye and wheat flours, the contribution of the wheat gluten being needed to provide the baking quality that is lacking in the rye flour. The poorer dough-forming characteristics of rye gluten are largely due to the lower level of the glutelin-type protein, combined with the more hydrophilic (water-loving) properties of the rye secalins, compared with the corresponding classes of protein in wheat. The water-soluble proteins of rye contain the albumin and globulin classes of protein, similar to those in wheat, but for rye, there is generally a higher level of amylase enzymes than in wheat because of the greater susceptibility of rye to preharvest
PROTEINS | The Protein Chemistry of Cereal Grains
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Albumin
Globulin
Prolamin
Glutenin
Ile Leu Lys Met Cys Phe Tyr Thr Trp Val Arg His Ala Asp Glu Gly Pro Ser NH3 Figure 8 Amino acid composition of the Osborne fractions of wheat.
Table 3 The amino acid compositions of whole grains of several cereals, expressed as grams per 16 g of nitrogen Amino acid
Wheat
Rice
Corn
Barley
Oats
Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
3.6 4.6 4.9 2.5 29.9 3.9 2.3 3.3 6.7 2.9 1.5 4.5 9.9 4.6 2.9 1.1 3.0 4.4
6.0 8.3 10.3 1.1 20.6 5.0 2.5 3.8 8.2 3.8 2.3 5.2 4.7 5.4 3.9 1.2 3.5 5.5
7.5 4.2 6.3 1.6 18.9 3.7 2.7 3.7 12.5 2.7 1.9 4.9 8.9 5.0 3.6 0.6 3.8 4.8
4.0 4.7 5.7 2.3 23.6 3.9 2.1 3.6 6.7 3.5 1.7 5.1 10.9 4.0 3.3 1.5 3.1 5.0
4.5 6.3 7.7 2.7 20.9 4.7 2.1 3.8 7.3 3.7 1.7 5.0 5.2 4.7 3.3 1.3 3.0 5.1
sprouting and the consequent production of starch-degrading enzymes. The essential amino acid composition of rye is slightly better than that of wheat, because of the lower content of prolamin-type proteins in rye (Figure 9). The properties of triticale are intermediate between those of rye and wheat, reflecting the genetic origins of triticale.
The Proteins of the Barley Grain The protein concentration of cultivated barley ranges from 9% to 12%. For barley, the protein content is generally calculated as total nitrogen content multiplied by the factor of 6.25. The protein content of hull-less (naked) barley genotypes is
generally higher (even up to 18%) because of the absence of the outer hull layers (the glumes – the palea and lemma), which contain relatively little protein. The production of the enzymes of germination is an important part of a good malting barley, but paradoxically, high protein content (e.g., over 11%) is not necessarily desirable for the best malting quality. This is because the ultimate test of malting quality is the extent of ‘extract’ produced during the mashing process. The amount of extract determines how much beer can be produced from a given amount of malt. ‘Extract’ represents the amount of soluble fermentable sugars from the hydrolysis of starch, resulting from the combination of very active amylases and high starch content. For this reason, a low protein content (i.e., less than 8%) is also undesirable, as it may have the consequence of inadequate enzyme production. Consumption for human food is a traditional use of barley, but this now accounts for only 5% of total barley consumption worldwide. Feed use predominates, amounting to 75% of barley use. The amino acid composition of barley does not provide a consistently better balance nutritionally than other cereal grains, still being deficient in lysine, methionine, and threonine. Nevertheless, it is a popular source of protein and energy in feedstuffs, in combination with other sources of energy. High-lysine barley genotypes have been developed, analogous to those in maize, the first of these being the Hyproly line. The hordein polypeptides show considerable polymorphism, like the prolamins of wheat and rye. A large proportion of the barley prolamins are disulfide-linked (both intra- and interchain linkages) with naturally occurring polymers of high molecular weight. Unlike the glutenin polymers of wheat, these are not desirable for the processing of barley. Because they generate problems in the malting process and downstream, they are likely to produce haze in the final product (i.e., beer) if they have not been adequately hydrolyzed during processing. The hordein polypeptides are classified into groups (A, B, C, and D hordeins) according to their decreasing mobilities
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PROTEINS | The Protein Chemistry of Cereal Grains
Globulins
7S
Prolamins
11/12S
Maize, oats, rice, barley, rye, wheat
Wheat
Oats
7S globulin
T riticin
Globulin
Other prolamins
Rice
Maize
Glutelin
The prolamin superfamily
Sorghum
Maize
Rice
Kaffirin
Oats
Triticeae: barley, wheat, rye
Avenin 16 kDa 10 kDa 13 kDa
-Zein
-Zein
-Zein
-Zein HMW S-rich S-poor
Embryo, aleurone
Endosperm
Figure 9 Storage protein classes in cereals. Adapted from Shewry, P. R., Tatham, A. S., and Halford, N. G., 1999. The prolamins of the Triticeae. In: Shewry, P. R. and Casey, R. (Ed.), Seed Proteins. Kluwer Academic, The Netherlands, pp. 35–78. Table 4
Classes of barley proteins (hordeins) and corresponding loci
Hordein class
Loci
Gene copy number
Proteins present in the cv ‘Carlsberg’
MW (kDa)
Corresponding wheat prolamin
B C D g
Hor 2 Hor 1 Hor 3 Hor 5
20–30 20–30 1 Low
10 9 1 7
36–45 50–60 105 36–45
LMW glutenins o-Gliadins HMW glutenins g-Gliadins
during SDS gel electrophoresis. The C hordeins have analogy to the omega-gliadins of wheat, having a general absence of the sulfur-containing amino acids. They are coded by genes (locus Hor 1) on the short arm of barley chromosome 1H, as indicated in Table 4. The genes (Hor 2 and Hor 5) for the sulfur-rich B and gamma-hordeins are also coded on chromosome 1H. The D hordeins, which occur as disulfide-linked polymers, are analogous to the HMV subunits of wheat glutenin; like them, their genes (Hor 3) are located on the long arm of the corresponding barley chromosome (1H).
The Proteins of the Rice Grain The proteins of rice are a contrast to those of wheat and barley, being predominantly of the globulin class. As a protein source, milled rice contains the lowest amount of protein (about 5%) among the major cereals. Moreover, this protein is not easily digestible by humans and monogastric animals. However, compared with that of other cereal proteins, the overall amino acid composition of rice protein is significantly more balanced due to the relatively higher level of lysine content. This unusual amino acid composition results because rice is one of the few cultivated plants in which there are significant levels of globulins and prolamins. These are the two major classes of storage proteins in the seeds of higher plants. Unlike other cereals that accumulate prolamins as their primary
nitrogen reserve, the major storage proteins in rice are the glutelins, which are homologous at the primary sequence level to the 11S globulin proteins (Figure 9), a class that is the dominant form of nitrogen deposition in legumes. Rice prolamin proteins have a number of characteristics that are different from the prolamins present in most other cereals. The most important characteristic of the deposition of nitrogen in the rice kernel is that there are three kinds of protein bodies in the rice endosperm, namely, large spherical, small spherical, and crystalline protein bodies. Each of these is surrounded by a single continuous membrane. Although the spherical protein bodies form within vacuoles, the proteins are synthesized in the endoplasmic reticulum and in the Golgi apparatus and are then transported to the vacuoles via vesicles. Removal of the husk (hull) from rough (paddy) rice yields the kernel. This is composed of the pericarp, seed coat, aleurone, endosperm, and the germ and is known as ‘brown rice.’ It has a protein content of 9–10%, with a significantly higher nutritional value than the most commonly utilized rice product – white polished (milled) rice, whose protein content is 8%. The milling process for paddy rice results in 40–55% white milled rice, together with three major by-products, namely, husks (20%), bran (10%), and brokens (10–22%). These byproducts have protein contents of 3%, 17%, and 9%, respectively. The aleurone layer, the tissue with the highest level of proteins and nutritionally important minor components, is removed during the rice milling process.
PROTEINS | The Protein Chemistry of Cereal Grains
The subaleurone region of the rice grain plays an important part because of its nutritional value. It is a globulin-rich layer, being several cell layers thick. Its lysine content is much higher than that of the proteins located in the rice endosperm. Thus, rice should be milled as lightly as possible to retain as much as possible the subaleurone layer. The albumin fraction of rice is highly heterogeneous, containing biologically important components. It can be separated into four subfractions, based on the molecular size of its proteins. More than 50 individual polypeptides were observed in the albumin fraction, using isoelectric focussing. As for cereal grains in general, the albumins are mainly enzymes and enzyme inhibitors. Like the prolamins from other cereals, rice prolamins are readily soluble only in alcohol/water mixtures. However, the classical extraction procedure had not been useful in early studies. More recently, these estimates of prolamin content have been shown to be low. Using 55% propan-L-ol and reducing agents, higher yields of prolamins (20–25% of the total protein) have been obtained. Rice prolamins are 10–16 kDa in molecular weight. Thus, their molecular size range is significantly smaller than the prolamins of other cereals. They are highly variable between rice cultivars, based on their electrophoretic and isoelectric focussing fingerprints. According to the Osborne classification system, the least soluble proteins of the rice grain are oligomeric globulins representing up to 80% of the total protein fraction (Figure 9). They have been difficult to study because of their general insolubility in all solvents except dilute alkali, due to their high molecular weight and their heterogeneity. When analyzed by gel filtration chromatography, the purified globulin subunits were resolved into three subunits, linked by disulfide bonds, which varied in stoichiometry depending on the report and on the rice variety used. Despite their general insolubility, rice glutelins are homologous in structure to 11S globulins (Figure 9). The structural relationship is evident when comparing the N-terminal amino acid sequences and using immunologic methods. The extent of homology between the rice glutelin and legume 11S globulins is 30–35%. Structural similarity is evident in the primary sequences of homology to a motif common in wheat, rye, barley, and maize prolamins.
The Proteins of Maize Maize (or corn), often classed in world trade as one of the ‘coarse’ grains, is an important source of protein in the diets of many people in countries such as Africa and South America. In addition, maize is used extensively as a feed grain and in a range of industrial processes. The process of corn starch manufacture produces the grain protein as a by-product. It is sometimes referred to as ‘corn gluten’ in the trade, but this name is inappropriate, because maize protein has no relationship to the gluten of wheat and is safe for consumption by those susceptible to celiac disease. Like the other cereal grains, maize protein is slightly deficient in lysine. This limits its utilization as an animal feed. This nutritional imbalance is largely due to the low lysine content of ‘zein,’ the major group of maize proteins (Figure 9). The
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development of the ‘opaque-2’ and ‘floury-2’ mutations has led to the development of maize genotypes with higher levels of lysine, due to a lower content of zein and more of the nutritionally superior albumin type of protein. Although these new maize types offer nutritional advantages, they carry a grain yield penalty. The zein protein of maize can be isolated by extraction of the crushed endosperm (the starchy part of the grain) with 70% aqueous ethanol, after breaking disulfide bonds. Maize zein has been further fractionated into subclasses of proteins, designated alpha-, beta-, gamma-, and delta-zeins. The genes for their synthesis have been mapped to maize chromosomes 6 and 7.
The Proteins of Sorghum The major storage proteins of sorghum, termed ‘kafirins’ (Figure 9), are fractionated by a procedure similar to that for the zeins of maize. Alpha-kafirin, composing about 80% of the overall kafirin content, can be further subdivided into two proteins with molecular weights of about 23 000 and 25 000 Da. Three beta-kafirins and one gamma-kafirin have been identified, all of them having molecular weights in the range of 16–28 kDa. Like maize, high-lysine mutants of sorghum have been developed.
Future Prospects Our current understanding of grain protein chemistry contrasts strongly with the rudimentary knowledge of the early twentieth century. That knowledge has progressed from the recognition of only a few distinct grain proteins to fractionating and characterizing thousands of proteins from all the tissues of the cereal grain – the endosperm, germ, scutellum, and bran. This knowledge permits more efficient selection for improved processing quality in the breeding of new varieties. The knowledge is also being applied to testing for protein quality after the grain is harvested, so that grains of specific quality can be segregated for appropriate marketing and processing. Knowledge at the chemical level is now complementing new genetic insights, so that breeders and molecular geneticists can employ conventional and novel methods to create quality types that have previously not been possible – varieties with new protein functionality. Thus, the future holds great promise for grains that will be processed more efficiently with nutritional advantages. For example, it has been possible to incorporate purified gluten proteins into rice dough, thus to monitor their effects on the mixing properties. The protein content of the cereal grains has always been important for marketing and processing. The generally desirable trait of high-protein concentration has often been inversely proportional to grain yield. Improvements in genetics and agronomic practice have tended to reduce grain protein levels, because high yield has been a common goal for farmers. The coming decades will bring a new factor that will further erode protein levels – the rising concentrations of carbon dioxide in the atmosphere. For whatever reason, it is inevitable that CO2 levels will continue to rise, although the rate of increase is difficult to predict. Many experiments have been
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PROTEINS | The Protein Chemistry of Cereal Grains
conducted to simulate effects of future levels of CO2. They have consistently indicated increased yields of grain and biomass, together with decreased levels of grain protein content. The higher atmospheric concentration of CO2 acts as a carbon fertilizer, with consequent increases in starch synthesis but little change in protein synthesis. There will thus be a need to rethink the role of protein in cereal grains if it will gradually become lower in concentration. At the manufacturing level, there is increasing interest in providing greater protein content for cereal-based snacks and breakfast foods. Cereal proteins are also undergoing texturization and extrusion with the aim of creating synthetic meat products.
Exercises for Revision
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How did the early chemists’ views on protein chemistry differ from our present knowledge? Why were they unaware of the great heterogeneity of grain proteins? What are the aspects of protein composition that explain the great differences between the various proteins in their properties? Prepare a table of differences and similarities between the major cereal species with respect to their protein compositions. What are the nutritional deficiencies of cereal grain proteins with respect to the essential amino acids? Describe the genetic achievements that have partially overcome these deficiencies.
Exercises for Readers to Explore the Topic Further
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Examine the taxonomic relationships between the major cereal species, as provided in this encyclopedia. Compare the associations between the proteins of these grains in Figure 8. Look at the genus names to see the origins of the grain-specific protein names. Obtain the amino acid compositions of the grain proteins for the major cereals. What are the main differences? How would these differences account for functional and nutritional differences between these grain species? Contrast the differences in function for an enzyme (to catalyze a specific reaction) versus a storage protein (a source of amino acids for the new plant). Why would both types of protein be produced under the same strict genetic control? Compare the various methods of determining the amount of protein in a grain or flour sample (Table 2 and elsewhere). Which would you expect to be the most accurate?
See also: The Basics: Grain Crops, Overview (00001); Taxonomic Classification of Grain Species (00002); The Cereal Grains: An Overview of the Family of Cereal Grains Prominent in World Agriculture (00006); Proteins: Enzyme Activities (00102); Protein Synthesis and
Deposition (00106); Proteomics (00107); Wheat-Based Foods: Wheat grain utilization: Overview (00142); Wheat Processing: Wheat, Grain Proteins and Flour Quality (00164); Grain Marketing and Grading: Variety Identification of Cereal Grains (00175); Genetics of Grains: Maize, Quality Protein Maize (00223); Maize, Other Maize Mutants (00224).
Further Reading Bailey HC (1941) A translation of Beccari’s lecture “Concerning Grain” (1729). Cereal Chem. 18: 555. Bushuk W (ed.) (2001) Rye: Production, Chemistry and Technology, second ed. St. Paul, MN: American Association of Cereal Chemists. Dendy DAV (ed.) (1995) Sorghum and Millets: Chemistry and Technology St. Paul, MN: American Association of Cereal Chemists. Hartley H (1951) Origin of the word protein. Nature 168: 244. Henry RJ and Kettlewell PS (1996) Cereal Grain Quality. London: Chapman and Hall. Islam S, Ma W, Yan G, Be´ke´s F, and Appels R (2011) Modifying processing and health attributes of wheat bread through changes in composition, genetics and breeding. In: Cauvain SP (ed.) Bread making. Improving quality, second ed. Boston, New York: CRC Press. Juhasz A, Bekes F, and Wrigley CW (2014) Wheat proteins. In: Ustunol Z (ed.) Applied Food Protein Chemistry, Chapter 11, pp. 219–304. West Sussex, UK: Wiley-Blackwell, John Wiley & Sons. Juliano BO (ed.) (1985) Rice: Chemistry and Technology, second ed. St. Paul, MN: American Association of Cereal Chemists. Kent NL (1975) Technology of Cereals, with Special Reference to Wheat, second ed. Oxford, UK: Pergamon. Lasztity R (1999) Cereal Chemistry. Budapest, Hungary: Akade´miai Ko¨nyvkiado´. McGregor AW and Bhatty RS (eds.) (1993) Barley: Chemistry and Technology. St. Paul, MN: American Association of Cereal Chemists. Mertz ET (ed.) (1992) Quality Protein Maize. St. Paul, MN: AACC International. Pomeranz Y (ed.) (1988) Wheat: Chemistry and Technology, third ed. St. Paul, MN: American Association of Cereal Chemists. Seneweera S, Fernando N, Panozzo J, Tausz M, Norton RM, Fitzgerald GJ, Myers S, and Nicolas ME (2014) Intra-specific variation of wheat grain quality in response to elevated [CO2] at two sowing times under rain-fed and irrigation treatments. J. Cereal Sci. 59(2): 137–144. Shewry PR and Casey R (eds.). (1999) Seed Proteins. The Netherlands: Kluwer Academic. Shewry PR and Miflin BO (1985) Seed storage proteins. In: Pomeranz Y (ed.) Advances in Cereal Science and Technology vol. 7, pp. 1–83. St. Paul, MN: American Association of Cereal Chemists. Sikorski ZE (2001) The Chemical and Functional Properties of Food Proteins. Lancaster, PA: Technomic Publishing. Watson SA and Ramsted PE (eds.) (1987) Corn: Chemistry and Technology St. Paul, MN: American Association of Cereal Chemists. Webster FH (ed.) (1985) Oats: Chemistry and Technology. St. Paul, MN: American Association of Cereal Chemists. Wrigley CW (2012) Proteins – the basis of life. Teach. Sci. 58: 56–59. Wrigley CW and Batey IL (eds.) (2010) Cereal Grains: Assessing and Managing Quality Oxford, UK: Woodhead Publishing Ltd.
Relevant Websites http://www.aaccnet.org – American Association of Cereal Chemists. http://www.campden.co.uk – Campden & Chorleywood Food Research Association. http://www.icc.or.at – International Association for Cereal Science and Technology. http://www.usda.gov – United States Department of Agriculture. http://www.wheat.pw.usda.gov – Grain genes.