The polymeric chains

CHAPTER 1

INTRODUCTION

This chapter will have a general review on proteins which include its sources, usage and so on. The importance of cereal proteins and its consumptions in the world population is also discussed.

1.1 Introduction

Proteins are polymeric chains that are built from monomers called amino acids. All structural and functional properties of proteins derive from the chemical properties of the polypeptide chain. There are four levels of protein structural organization: primary (1), secondary (2), tertiary (3), and quaternary (4). Primary structure is defined as the linear sequence of amino acids in a polypeptide chain. The secondary structure refers to certain regular geometric figures of the chain. Tertiary structure results from long-range contacts within the chain. The quaternary structure is the organization of protein subunits, or two or more independent polypeptide chains. (Carey & Hanley)

Protein from animal sources (meat, fish, dairy products, egg white) is considered a "complete" protein because all ten essential amino acids are present in these proteins. Examples of essential amino acids are histidine and valine (Bruice, 2006). These amino acids are called essential amino acids because they cannot be synthesized by the body and have to be obtained from our daily dietary sources. An exception to this rule is collagen-derived gelatin which is lacking in tryptophan, which is also an essential amino acid.

Plant sources of protein (grains, legumes, nuts, and seeds) generally do not contain sufficient amounts of one or more of the essential amino acids. Thus protein synthesis can occur only to the extent that the limiting amino acids are available. These proteins are considered to have intermediate biological value or to be partially complete because, although consumed alone they do not meet the requirements for essential amino acids, they can be combined to provide amounts and proportions of essential amino acids equivalent to high biological proteins from animal sources. Plants that are entirely lacking in essential amino acids are considered incomplete proteins or sources of low biological value protein. These sources include most fruits and vegetables (Carey & Hanley).

One of the sources of plant proteins mentioned above are grains. Grains or cereals have been the most important crop for thousands of years. In most countries of the world the proportion of land devoted to cereals is the highest among cultivated plants. According to statistical data produced by Yada (2004), the world production of cereals was estimated at 1815 million tonne in the period of 1986 to 1990. The most marked increases were observed in the production of wheat, maize, rice, and barley. The majority of the cereals produced are used for human consumption. However, these cereals are also used as animal feed, industrial uses, and so on. The total protein consumption of the world population is estimated to be more than 100 million tonnes. Cereal proteins provide more than half of the total protein production of the world. The rise of cereal production in the world is shown in Table 1.1 below. If we consider that the production of milk and meat is also based on feed containing cereals, it is clear that in the provision of protein requirements the cereal proteins play the most important role.

1.1 Research Objective

The aim of this research is to review the protein extraction, purification and analysis method from grain proteins. The target protein chosen is prolamin and different types of prolamin from grains such as wheat, maize and rice are compared.

1.2 Research Scope

The scopes of this research are to review the extraction methods of prolamin from different source of grains and also to evaluate the prolamin purification and analysis method. Results from previous journals will be assessed and discussed to ascertain the best prolamin extraction and analysis method.

1.3 Benefit and Application of Research

This research was carried out to fully understand the grain protein extraction and analysis method. With this knowledge, it is possible to further explore the potential of grain proteins in a more beneficial ways to create a highly nutritional food for consumers. Moreover, the ability to achieve a highly efficient and productive method for prolamin extraction and analysis will also help in the engineering modification and gene expression of grain protein to produce crops that have better dietary value than the ones in the existing market.

Besides that, the extracted protein has potential to become advance animals supplement by directly consuming these extracted protein. This could be suitable for all types of animals such like ruminant, monogastric and even aquaculture.

1.4 Organization of Research

This report was arranged into a few chapters so that readers can understand the research made in detail. There are five main chapters in this report which include introduction, literature review, methodology, results and discussion and conclusion.

In the introductory chapter, brief description about proteins which includes the components, sources and usage of plant proteins are explained. Information regarding cereal crops in general is also included. Apart from that, the benefits and application of the research is also discussed.

The second chapter has a review on the literatures, researches, theories and data which had been published previously are reviewed. Different methods of plant protein extraction, purification and analysis are discussed in detail in this chapter.

In the methodology chapter, the materials, chemicals, equipments and experimental procedures used in the journals that were reviewed are described in detail. The equipments and apparatus used are also mentioned.

In the fourth chapter, the results obtained from the journals studied in the previous chapter are discussed. Data in the form of tables and graph obtained from the journals are also shown.

In the final chapter, the best methods for prolamin extraction, purification and analysis are determined based on the reviews on previous chapters. Future works that can be done from the application of these methods are also discussed.

CHAPTER 2

LITERATURE REVIEWS

This chapter explains in detail the types of protein, its characteristics and classes. Given that this research focuses mainly on grain protein, the different sources of grain protein together with its fractions and amino acid compositions are also discussed. An in-depth review on the protein extraction, purification and analysis methods from different journals are also made.

2.1 Basics of Protein

Essentially, the word protein came from the Greek word proteios which means primary or in other words, to take the first place. Proteins and peptides are polymers of amino acids linked together by amide bonds. An amino acid is the building blocks of a protein which has the structure of carboxylic acid with an amino group on the a-carbon. It consists of mainly carbon, hydrogen, oxygen, nitrogen, sulphur and phosphorus. The repeating units of amino acids are called amino acid residues (Bruice, 2006). The amino acids are almost always called by their common names. The table below shows the most common naturally occurring amino acids.

The building blocks of proteins consists of 20 most commonly occurring natural amino acids with the length range from 10 to 1000 amino acid residues. A protein with two amino acid residues is called dipeptides whereas protein with three amino acid residues is known as tripeptide. Protein with more than three amino acid residues is known as polypeptide.

On the whole, proteins can be split into animal proteins and plant proteins. Animal protein is considered to be complete protein because it contains all the essential amino acids whereas plant protein is known as incomplete protein because not all essential amino acids are available in plant protein. In the table above, the essential amino acids are marked with asterisk (*).

Furthermore, proteins are the major structural components of all cells of the body and amino acids are the building blocks of protein. Proteins can function as enzymes, membrane-carriers and hormones. Proteins encompass many important chemicals including immunoglobulin and enzymes. In short, they form the foundation of muscles, skin, bone, hair, heart, teeth, blood and brain and the billions of biochemical activities going on in the body (Muhammad-Lawal & Balogun, 2007).

2.1.1 Classes of Protein

Proteins can be divided roughly into two classes. Fibrous proteins contain long chains of polypeptides that occur in bundles (Bruice, 2006). These proteins are insoluble in water. Examples of fibrous proteins in human body are structural proteins such as collagen, tendons and keratin. Another class of protein are globular protein which have somewhat spherical shapes and are soluble in water. Examples of globular proteins are enzymes.

2.1.2 Protein Structure

Protein molecules are described by several levels of structure. The primary structure describes the sequence of amino acids and also the location of all disulfide bridges in the chain. The secondary structure describes the repetitive conformations assumed by segments of the backbone chain of the protein (Bruice, 2006). The tertiary structure is formed by packing such structural elements into one or several compact globular units called domains. The final protein may contain several polypeptide chains arranged in a quaternary structure (Branden & Tooze, 1991).

2.1.3 Properties of Protein

The main properties of proteins that will be discussed here is the acid-base property as well as solubility of proteins. Proteins are greatly affected by pH since every amino acid has a carboxyl group and an amino group, and each group can exist in an acidic form or basic form depending on the pH of the solution in which the amino acid is dissolved (Bruice, 2006).

The typical amino acid building block for polypeptide synthesis has a central carbon atom that is attached to an amino (-NH2) group, a carboxyl (-COOH) group, a hydrogen atom, and a side chain (-R). At pH 7, the amino group is protonated (i.e., the addition of a proton) to form NH3+ and the carboxyl group is deprotonated to form -COO- so that the amino acid has the structure shown in figure 2.2. These amino acids are termed a-amino acids in accordance with a pre-IUPAC nomenclature system, in which the atoms in a hydrocarbon chain attached to a carboxyl (-COOH) group are designated by Greek letters. The carbon atom closest to the carboxyl group is designated a, the next , and so forth (Tropp, 2007).

It should also be noted that the isoelectric point (pI) of an amino acid is the pH at which it has no net charge. It is the point where the amount of positive charge in an amino acid exactly balances its negative charge (Bruice, 2006). An amino acid will be overall positively charged if the pH of the solution is less than the pI of the amino acid and vice versa.

As for solubility, proteins solubility depends on several factors mainly pH, concentration, and also temperature. Different classes of proteins may also exhibit different solubility as mentioned above in the case of fibrous and globular protein.

2.1.4 Protein Sources

Protein can be obtained mainly from animal and plant. The meat from cattle, goat, sheep, pig and poultry including the offal are the main sources of animal protein (Muhammad-Lawal & Balogun, 2007). The main differences between animal and plant protein is that animal products contain different ratios of amino acids and higher concentrations of protein than most plant products do (Muhammad-Lawal & Balogun, 2007).

Plant sources of protein (grains, legumes, nuts, and seeds) generally do not contain sufficient amounts of one or more of the essential amino acids. Thus protein synthesis can occur only to the extent that the limiting amino acids are available. These proteins are considered to have intermediate biological value or to be partially complete because, although consumed alone they do not meet the requirements for essential amino acids, they can be combined to provide amounts and proportions of essential amino acids equivalent to high biological proteins from animal sources. Since this research paper is focussed on grain protein, detailed explanation regarding the sources of grain protein is discussed in the next section.

In the industries, there is increasing demand for plant proteins because they are used as ingredients in formulated foods and also as fillers and substitutes for animal feeds. Soy protein is one of the most common sources for the production of animal feeds. As a result of its high demand, the price of soy protein has become very high and the usage of soy protein for Malaysian agricultural industries will bring less profit. Thus, there is need to find for an alternate source which is cheaper and also readily available. Hence, grain proteins can be seen as a good solution for this.

2.2 Grain Protein

Grain proteins are the most important source of plant protein and are largely consumed in and around the world. Sources of grain protein are mainly wheat, maize, barley and oat. These major grain protein sources are discussed below.

2.2.1 Wheat

Wheat, scientifically known as Triticum aestivum L., is considered as an important food source in the world food supply. Wheat are made into flour that consists predominantly of starch (about 70-80% dry weight), with lower amounts of protein (usually about 10-15% dry weight), lipids (1-2% dry weight) and other components such as non-starch polysaccharides (which correspond to cell wall fragments). Even with the low content of protein in wheat flour, it has great importance in determining the functional properties and the characteristics of wheat dough created.

Wheat is generally used for making commercial foods. These includes:-

  • Breads (leavened pan, steamed etc.)
  • Noodles and pastas
  • Baked products (cakes, cookies etc.)
  • Breakfast cereals

The varied usage of wheat highlights the importance of the quality for specific end uses. Thus, it is essential to improve wheat protein quality so that a higher nutritional value can be obtained from these products.

2.2.2 Maize

Maize is unique in that it cannot reproduce itself without the aid of humans -its seeds can't be released because they are tightly wrapped around the ear. Wild maize has never been found and domesticated maize was probably developed through hybridisation. It is one of the world's most important crops because it is used widely in food manufacture, in the production of industrial products and as animal feed. Maize are mainly used to make cornstarch, cornflour, corn meal, popcorn and so on.

2.2.3 Rice

Rice is considered as the staple food for over 60% of the world's population. Its annual production for food use is second in line to wheat. World rice production was 525 million metric tons in 1992 (FAO 1992). Apart from being the main food staple in most Asian countries, rice is also used in the productions of gels, puddings, ice creams, and baby formulas. This is because of the unique properties of rice which includes:-

  • Gluten-free
  • Hypo-allergenic
  • Hypocholesterolemic
  • Antioxidant
  • Antihypertensive
  • Anticancer

Being gluten-free is important because gluten can cause celiac disease that can damage the mucose of small intestines which can later cause malabsorption in gluten intolerant people. These added advantages over other wheat products comes with several drawbacks because protein denaturation during rice drying, milling, storage, and processing can lead to different functional characteristics in foods.

Proteins and starch are the two major components of rice, with approximately 8 and 80%, respectively (Marshall and Wordsworth 1994). Rice protein is valuable because it has unique hypoallergenic properties and ranks high in nutritive quality (rich in the essential amino acid lysine) among the cereal proteins (Bean and Nishita 1985). The hypoallergenic property and the high nutritive quality could make rice protein concentrate or isolate a competitive protein ingredient in the food ingredients market (Ju et al., 2001).

2.2.4 Barley

Barley was one of the first grains eaten by humans. Today, most of the world's supply is used to feed cattle and to make beer. It is still eaten in regions of the world where wheat cannot be grown such as Tibet, northern Germany, Finland, the Italian Alps, Israel, the Sahara and Ethiopia. The main food uses of barley include:-

  • Malted barley
  • Scotch barley
  • Barley flour

2.2.5 Oat

Oats grow well in moist, temperate to cool climates, and therefore thrive in conditions which wheat or barley would not tolerate. Popular in the British Isles, an early nineteenth century survey showed that the Welsh ate more oats than all other cereals combined and that in Scotland the ordinary people ate almost no other grain. Oats are mainly fed to livestock. However, an increasing amount of oats are being grown for human consumption (mainly in breakfast foods) due to consumer interest in the cholesterol-lowering benefits of oat bran. The main food uses of oat include:-

  • Oatmeal
  • Oat bran
  • Instant oat

2.3 Amino acid and Nutrient Composition of Grain Protein

The amino acid composition from different grain sources are shown in the table below. It should be noted that these data were obtained from cereal flours. The actual amino acid composition of unprocessed grain is higher than the ones shown below.

2.4 Grain Protein Fractions

In this research paper, studies will be done mainly on the grain proteins from wheat, maize and rice. The study of plant proteins, or more specifically cereal grain protein was started by T.B. Osborne, who was working at the Connecticut Agricultural Research Station from the year 1886 until 1928. He had classified cereal proteins into four groups, based on their sequential extraction in a series of solvents. According to his research, plant proteins are classified into four categories which include:-

  • Albumins which are water soluble protein. Example: enzymes
  • Globulins which are insoluble in water but soluble in dilute salt solution; these are the major proteins in leguminous seeds.
  • Prolamins which are soluble in aqueous alcohol and are present as major proteins in maize, wheat and barley.
  • Glutelins which are insoluble in all the above mentioned solvents but soluble in dilute acids or dilute alkalies. These are the most abundant proteins present in rice.

The three protein classes of globulins, prolamins, glutelins are nonenzymatic and have been called as storage proteins. These storage protein are synthesized during seed development and are stored for utilization at later stage for supply of nitrogen and sulphur during seed germination (Croy et al., 1984, Boulter and Croy, 1997).

2.4.1 Storage Protein

Seed storage proteins are the most abundant source of proteins in human and animal nutrition, and have being used in the development of new biodegradable materials for engineering and biological applications. However, the nutritional quality of these proteins is limited by deficiencies in the content of some essential amino acids, mainly lysine and tryptophan. The nutritional or technological quality of these proteins can be improved by genetic manipulation and a detailed knowledge of the protein structures is important for designing improved proteins with altered contents of specific amino acids.

This research paper will focus on the extraction, purification and analysis of storage protein from different grains namely wheat, maize and also rice. Prolamin is chosen as the specific target protein in this research because it is the most abundant protein that can be found in the grains mentioned above.

Research concerning prolamins is particularly difficult because of their heterogeneity and insolubility in most solvents. Studies on prolamins are hindered by the specific chemical constitution of these proteins, since they make up a complex of many polypeptides differing in electrical charge, molecular weight, sequence and isoelectric point. The identifcation of more or less allergenic fractions requires previous separation and purification of specific proteins forming the complex. Further details regarding the prolamin content in grain proteins are discussed below.

  • Wheat Prolamin
  • Wheat contains a high amount of gluten, which is identified as the water insoluble proteinaceous mass left after removal of bulk of starch and other components from the wheat dough. These glutens mainly consists of glutelin and prolamin protein fractions which have been named as glutenins and gliadins respectively. According to Shewry et al. (1989), gliadins are monomeric, soluble in 70% ethanol and constitute about 50% of the seed protein whereas glutenins are polymeric and require the presence of a reducing agent for breaking disulphide bonds during extraction.

    Gliadins have been classified into four groups as a- - ?- and ?-gliadins on the basis of their mobility. These have also been described on the basis of their amino acid composition as sulphur-rich prolamins (a- - and ?- gliadins) and sulphur-poor prolamins (?-gliadins) (Matta et al, 2009). With their molecular weights in the range of 30 to 45 kDa, a, and ? gliadins are poor in lysine, arginine and histidine, and hence are responsible for the poor nutritional quality of wheat. The ?-gliadins however, are resolved in the range of higher mol. wt. of 44 - 80 kDa (Charbonnier, 1974).

    The glutenin polymers held together by disulphide linkages may occur as aggregates of very high mol. wt. upto 20,000 kDa the largest in the plant kingdom (Matta et al, 2009). It is mentioned that due to their similarities such as solubility in alcohol, higher proline and glutamine content and structural homology, glutenins and gliadins both have been considered as prolamins (Shewry et al., 1981).

  • Maize Prolamin
  • The most abundant proteins in maize which are represented by prolamins constitute 60% of the endosperm protein (Nelson, 1966) and have been named as zeins. Known as one of the extensively studied cereal proteins, zeins are subdivided into four types a, , ? and d fractions. According to Matta et al (2009), the a-zeins contribute about 75 of total zeins and are constituted by polypeptides of mol. wt. 19 kDa and 22 kDa. The -zeins consist of polypeptides of mol. wt. 14 kDa and 16 kDa, and account for 10 - 15% of total zeins whereas ?-zein and d zein are represented by polypeptides of mol. wt. 27 kDa and 10 kDa respectively.

    The low content of lysine and tryptophan in all the zein fractions makes the maize proteins inferior in nutritional quality. The a-zeins, due to one or two cysteine residues per molecule are present either as monomers or oligomers, while the , ? and d zeins have higher levels of cysteine and /or methionine and form alcohol insoluble polymers that can be extracted only under reducing conditions. In this way, due to very low cysteine and methionine, a-zeins have lower nutritional value as compared to , ? and d zeins.

  • Rice Prolamin
  • Unlike the alcohol-soluble prolamins dominating in grains of most of the cereals, glutelins represent the major protein fraction in rice. According to Matta et al (2009), glutelins constitutes 80% of the total seed protein. In another extraction protocol, Krishnan and White (1995) reported a lower proportion of 53% for glutelins. These are formed by polypeptide pairs of mol.wt. 57 kDa, each consisting of a large acidic (37 - 39 kDa) and a small basic (22 - 23 kDa) subunit (Yamagata et al., 1982).

    With respect to the molecular weights of subunit pairs and their subunits, rice glutelins show similarity with the legumin-like proteins of pea and soybean; these proteins have also been considered homologous due to similarity in their biosynthesis and amino acid sequences (Matta et al, 2009). The alcohol soluble prolamins which are present in PB-I type of protein bodies, account for approx. 35% of rice protein.

2.5 Protein Extraction

In general, there are several methods that can be used for the extraction of protein mainly the saline treatment and also the alkaline treatment. In order to achieve good extraction results, the protein extraction procedure must meet three major criteria which include permitting the solubilization of all proteins, being compatible with the subsequent purification and detection procedures and finally there should not be any involuntary modifications of the protein structure.

In the case of grain proteins, several different methods have been developed to extract grain protein. Generally, grain proteins are extracted by the conventional method suggested by Osborne (1924). By taking a look at rice protein extraction in the research done by Ju et al (2001), rice flour is first defatted for protein extraction. Rice protein consists of four fractions with different solvent solubility: albumin (water-soluble), globulin (salt-soluble), glutelin (alkali-soluble), and prolamin (alcohol-soluble). Based on this, the storage protein fractions can be sequentially extracted by water, salt, ethanol, and alkali buffer (Luthe 1984). Extraction conditions must be optimized for high protein recovery: 70% n-propanol was recommended for prolamin extraction by Sugimoto et al (1986) after they had tested various concentrations. The simplified diagram of the Osborne extraction method is shown below.

Another prolamin extraction method includes the addition of reducing agent in aqueous alcoholic solvent. Landry and Moureaux (1970) incorporated the use of reducing agents into their fractionation of maize zeins. The Landry-Moureaux method divides prolamins into two major classes: those extractable in aqueous alcohol alone and those extractable in aqueous alcohol plus a reducing agent. These classes have been termed zein or zein-I, and zein-like or zein-II (also called G1 or alcohol-soluble glutelins). According to Hamaker et al (1995), another minor fraction, termed prolamin-like (G2 or salt-soluble glutelins), contains a prolamin-like protein that may be the y-zein. This fraction is obtained from the alcohol-extracted pellet using a solution containing salt and reducing agent at pH 10. Total prolamin content has frequently been estimated as that obtained by adding the two classes (zein and zein-like) together or by extraction with aqueous alcohol plus reducing agent.

2.6 Protein Content Measurement

There are several methods that can be used for protein content measurement mainly Kjeldahl method, Dumas method, Biuret method, Infrared Spectroscopy and also Lowry method. In the Dumas method for nitrogen analysis, the sample is combusted in a carbon dioxide atmosphere in contact with cupric oxide. The latter converts carbon and hydrogen to carbon dioxide and water vapor respectively, and these are swept into a chamber containing sodium hydroxide, where they are absorbed. The volume of nitrogen gas remaining is then measured. This procedure is particularly useful for azo, nitroso, nitro, and other compounds which are more or less refractory to Kjeldahl analysis (Malnutrition, 1963).

In this research, Kjeldahl method will be reviewed for protein content measurement. The general steps of Kjeldahl method include digestion, neutralization and finally titration (Nielsen, 2003). It is based on the principles of nitrogen being one of the five major elements found in protein.

In this procedure, proteins and other organic food components in a protein containing sample are digested or oxidized with sulphuric acid in the presence of catalysts. As the organic material is oxidized the carbon it contains is converted to carbon dioxide and the hydrogen is converted into water.

The nitrogen, from the amine groups found in the peptide bonds of the polypeptide chains, is converted to ammonium ion, which dissolves in the oxidizing solution, and can later be converted to ammonia gas. The Kjeldahl method of nitrogen analysis is the worldwide standard for calculating the protein content in a wide variety of materials ranging from human and animal food, fertilizer, waste water and fossil fules.

2.7 Protein Purification and Analysis

As for protein purification, there are several methods used which include salt precipitation and also isoelectric precipitation method. Another method for protein purification is using chromatography which will be discussed further in this section. The main reason for conducting protein purification is to carry out functional and structural studies on the protein. The purified protein is also used for industrial and pharmaceutical applications such as in the production of insulin, vaccines, and enzymes. Apart from that, purification of proteins enables the identification of new proteins which are yet to be recognized.

2.7.1 Salt Precipitation

The solubility of proteins is strongly dependent on the salt concentration (ionic strength) of the medium. Proteins are usually poorly soluble in pure water. Their solubility increases as the ionic strength increases, because more and more of the well-hydrated inorganic ions (blue circles) are bound to the proteins surface, preventing aggregation of the molecules (salting in). At very high ionic strengths, the salt withdraws the hydrate water from the proteins and thus leads to aggregation and precipitation of the molecules (salting out) (Koolman & Roehm, 2005).

Thus, adding salt such as (NH4)2SO4 at high concentration makes it possible to precipitate the proteins. The ability of salts to effect salting out of proteins depends on their solubility and on the ionic strength of their concentrated solutions. Salts of di- and trivalent anions, such as sulphate or phosphate, are more efficient than those of monovalent anions, such as chlorides. It is not surprising, therefore, that the salts that have been used most commonly are those that combine high solubility with multivalency, i.e., the phosphates of sodium and potassium and the sulphates, particularly those of ammonia and sodium. Of these, the phosphates, being comparatively expensive, are normally restricted to small-scale concentration and fractionation, whereas sodium sulphate, because of its relatively low solubility can generally only be used effectively at temperatures of 20C and above. The remaining salt, ammonium sulphate, is cheap and very soluble and has found widespread use in the concentration of culture filtrates (Norris, 1970).

Unlike the phosphates, however, it is not a buffer, and on prolonged storage solutions of the salt may become acid because of loss of ammonia. This can be corrected by the addition of a small amount of sodium bicarbonate to the solution (Norris, 1970).

Solubility in (NH4)2SO4 decreases at increasing temperature, as expected for a hydrophobic effect; this is used in crystallizing proteins, bringing them to an (NH4)2SO4concentration where they are just soluble at 4 and letting them warm up to room temp. so that they will precipitate slowly, though usually in very small crystals, too small for X-ray crystallography.

2.7.2 Isoelectric Precipitation

The principle of isoelectric precipitation lies on the solubility of protein. The solubility in a protein is a result of polar interactions with the aqueous solvent, ionic interactions with the salts present, and, to some extent, repulsive electrostatic forces between like-charged molecules or small (soluble) aggregates of molecules. Small aggregates cancel out strong attraction charges. In the ionic strength range from zero to physiological, some proteins form precipitates because the repulsive forces are insufficient. For example, a high surface hydrophobicity means low interaction with the solvent and fewer charged groups to interact with salts. An overall charge near zero minimizes electrostatic repulsion, and may, close to the isoclectric point, cause hydrophobic forces to attract molecules to each other. This is called isoelectric precipitation.

Most proteins have a minimum solubility around their isoelectric point, which in the case of globulins is low enough to result in precipitation. In a protein mixture, the situation is greatly complicated by heterogeneous interactions; different proteins with similar properties aggregate to form the isoelectric precipitate. In many cases isoelectric precipitates can be formed in a tissue extract by lowering the pH to between 6.0 and 5.0 (Scopes, 1982).

2.7.3 Chromatography

Another method of protein purification is called column chromatography. This method separates proteins in a mixture by repeated partitioning between a mobile aqueous solution and an immobile solid matrix. The solution containing the protein mixture is percolated through a column containing the immobile solid matrix consisting of thousands of tiny beads. As the solution passes through the column, proteins interact with the immobile matrix and are retarded. If the column is long enough, it can separate proteins that have different migration rates. Proteins released from the column can be detected by an ultraviolet monitor and then collected in tubes by a fraction collector.

Besides that, protein separation can be improved by increasing the solid matrixs surface area through the use of a longer column or finer beads. This modified procedure known as high-performance liquid chromatography (HPLC) is now widely used in protein purification (Tropp, 2007). Several different kinds of chromatography methods are gel filtration, size exclusion chromatography (SEC), ion-exchange chromatography and so on.

Gel filtration or molecular exclusion chromatography separates protein molecules by size. The technique should be used when sample volumes have been minimised. Since buffer composition does not directly affect resolution buffer conditions can be varied to suit the sample type or the requirements for the next purification, analysis or storage step (Amersham Biosciences, 2003). This method depends upon special beads that permit small proteins to penetrate into their interior while excluding large proteins from this region.

A gel filtration column has two different water compartments: the internal compartment consists of the aqueous solution inside the beads and the external compartment consists of the aqueous solution outside the beads. Small protein molecules have access to both compartments, whereas large protein molecules only have access to the external compartment. Therefore, the large proteins appear in earlier fractions than do the small proteins. A protein mixture is introduced at the upper end of the column and elution is carried out by passing a buffer solution through the column. Large protein molecules (red) are unable to penetrate the particles, and therefore pass through the column quickly. Medium-sized (green and small particle (blue) are delayed for longer or shorter period. The proteins can be collected separately from the effluent (eluate) (Koolman & Roehm, 2005). The figure below shows the schematic view of gel filtration process.

Apart from gel filtration, reversed-phase chromatography (RPC) can also be used for protein purification. RPC separates proteins and peptides with differing hydrophobicity based on their reversible interaction with the hydrophobic surface of a chromatographic medium. Samples bind as they are loaded onto a column. Conditions are then altered so that the bound substances are eluted differentially. Due to the nature of the reversed phase matrices, the binding is usually very strong and requires the use of organic solvents and other additives (ion pairing agents) for elution. Elution is usually performed by increases in organic solvent concentration, most commonly acetonitrile (Amersham, 2003). However, RPC is not recommended for protein purification if recovery of activity and return to a correct tertiary structure are required, since many proteins are denatured in the presence of organic solvents.

Another method of chromatography used is hydrophobic interaction chromatography (HIC). HIC separates proteins with differences in hydrophobicity. The separation is based on the reversible interaction between a protein and the hydrophobic surface of a chromatographic medium. This interaction is enhanced by high ionic strength buffer which makes HIC an ideal next step for purification of proteins which have been precipitated with ammonium sulphate or eluted in high salt during IEX chromatography. Samples in high ionic strength solution (e.g. 1.5 M NH2SO4) bind as they are loaded onto a column. Conditions are then altered so that the bound substances are eluted differentially. Elution is usually performed by decreases in salt concentration. Most commonly, samples are eluted with a decreasing gradient of ammonium sulphate, as shown in Figure 2.5. Target proteins, which are concentrated during binding, are collected in a purified, concentrated form.

In the case of grain protein, researches done by Huebner et al (1990) have mentioned about rice protein analysis by gel electrophoresis, two-dimensional electrophoresis and electrofocusing, gel filtration chromatography, and reversed-phase (RP-) high-performance liquid chromatography (HPLC). Of these methods, two-dimensional electrophoresis best resolves proteins and differentiates genotypes but is difficult and slow (Huebner et al, 1990).

RP-HPLC effectively separates alcohol-soluble prolamins of many cereals, permitting varietal identification. In rice, however, such analyses appear less useful, because prolamins are a minor fraction. According to Huebner et al (1990), there are close similarity among chromatograms of varieties analyzed by HPLC. An alternative extraction procedure was devised by Hussain et al (1989), permitting RP-HPLC differentiation of many varieties; in this work, proteins from all classes may be present.

2.8 Summary

In conclusion, the extraction and purification of plant protein, especially grain protein has become increasingly important for both food and also non-food industries. Thus, this research aims at utilizing this opportunity to come out with a better method in the protein extraction, purification and analysis process of prolamin. In the following chapter, detailed methodology of the extraction, purification and analysis of grain prolamin from wheat, maize and rice will be discussed.

CHAPTER 3

METHODOLOGY

3.1 Protein Extraction

As mentioned in the previous chapter, several steps of extraction process are needed for the extraction of prolamin from different grain sources. It is also necessary to determine whether the extraction method chosen is suitable for further purification and analysis of protein.

3.1.1 Osborne Method

In a research done by Ju, Hettiarachchy, & Rath (2001), extraction method based on the studies of Osborne (1924) were used. In this method, proteins are extracted according to its solubility in different types of solvents. The extraction method is discussed in detail below.

The sample preparation was done by using 100g of rice flour which was defatted with 400 ml of hexane. The defatted rice flour was dried under a hood at ambient temperature for 24 h. This was followed by several steps of protein extraction to separate the different types of protein from the rice flour. The first step was the extraction of albumin whereby the flour was shaken with 400 ml of distilled water at 20 ?C for 4 h and centrifuged at 3000 x g for 30 min. Next, globulin extraction is done by treating the flour with 400 mL of 5% NaCl at 20 ?C for 4 h and centrifuging at 3000 x g for 30 min. The third step was the extraction of glutelin by using 300 mL of 0.02 M NaOH to get a of pH 11.0, at 20 ?C for 30 min. Finally, prolamin extraction is done with 300 mL of 70% ethanol at 20 ?C for 4 h (Sugimoto and others 1986). Each extraction was repeated two times in order to remove all the protein of each fraction.

In this method, the albumin, globulin, and glutelin that were obtained were precipitated from their supernatants by adjusting the pH to their isoelectric points (Ips). The determination of Ips for each supernatant were done by testing a portion from each supernatant to a pH ranging from 3.0 to 10.0 and determining the turbidity (optical density at 320 nm) with a spectrophotometer. The pH that gave the maximum turbidity was taken as the Ip for each supernatant.

According to Tecson et al (1971), the prolamin was precipitated by adding acetone to the supernatant. The precipitated proteins of albumin, globulin, glutelin, and prolamin were washed twice with distilled water, adjusted to pH 7.0, freeze-dried, and then stored at 4 ?C.

This comprehensive step-by-step extraction method suggested by Osborne (1924) might not be necessary for wheat and maize prolamin because the prolamin content in these grains are higher when compared to rice. Thus, a simpler extraction method with the usage of fewer reagents can be done for wheat and maize prolamin extraction.

3.1.2 Landry-Moureaux method

The Landry-Moureaux extraction method which utilizes the usage of reducing agent was demonstrated in the research done by M. R. Bugs, et al. (2004) on maize prolamin whereby Zein and pennisetin were extracted using a simplified protocol which is a modification of a previously described method in Forato et al. (2000). In this research, endosperm meal from the maize cultivar was extracted with a threefold (w/v) excess of 70% ethanol containing 25 mM Tris-HCl at pH 9 for 6 h at 4 ?C under mild agitation in the absence of salt. After a brief centrifugation, the supernatant was applied at room temperature to a Superdex 75 HR 26/100 (Amersham Biosciences) column equilibrated and eluted with 25 mM Tris-HCl, (pH 9) containing 70% ethanol.

According to another research by Erasmus & Taylor (2003), the best solvent used in maize prolamin (zein) extraction is 60% tertiary butyl alcohol and 0.05% dithiothreitol which acts as a reducing agent. This method is suitable for academic studies but is not compatible for food industries. However, zein is also manufactured by a patented process using aqueous isopropyl alcohol or aqueous ethanol (Reiners, Wall, & and Inglett, 1973). In this process, maize gluten is extracted at 60C with 88% aqueous isopropyl alcohol containing 0.25% sodium hydroxide and the extract separated centrifugally. The clarified extract is then chilled to -15C causing the zein to precipitate. The supernatant is decanted and the lower layer, containing 30% zein, is dried on a vacuum drum drier or a flash dryer. A purer product is made by re-dissolving and re-precipitating the zein.

Another extraction method described by Wallace et al (1990) was done on the base of separation of maize protein according to its zein and nonzein fractions. This method is simpler than the one suggested by Osborne (1924) because multistep separation procedures are not needed. In this method, maize kernel proteins is separated into zein (subclassified as the a-, -, ?-, and d-zeins) and nonzein fractions. This method is based on extraction of virtually all the kernel proteins in borate buffer (pH 10) with a detergent (1% SDS) and reducing agent (2% 2-ME), followed by addition of alcohol to 70% total volume. This last step precipitates nonzein proteins, while zeins remain in solution. Under this procedure, clear separation of maize endosperm proteins can be obtained.

As for wheat prolamin (gliadins), Bietz et al. (1984) studied the extraction conditions most appropriate for extracting wheat gliadins (prolamins) for RP-HPLC analysis. Their best procedure involved extraction with 70% ethanol (1-2 mL per single ground kernel or 50 mg flour) for 30 min or less, with constant or intermittent agitation. This method is similar to Wallace et al (1990) whereby the defatting of samples and pre-extraction of other protein fractions are unnecessary.

3.2 Protein Purification and Analysis

As mentioned in the previous chapter, there are many methods that can be applied to analyse extracted protein content. Currently, the best method available is by using Reverse-Phase HPLC (RP-HPLC). Nevertheless, the different methods for prolamin analysis will be discussed below.

3.2.1 High Performance Liquid Chromatography (HPLC)

In a research done Lookhart & Bean (1995), analysis on wheat prolamin (gliadin) was done by using Hewlett-Packard 1090 liquid chromatograph equipped with a Vydac C18 reversed-phase column. In this method, Gliadins were eluted by a multistep linear gradient at 65C, starting at 25% CH3CN and 75% water each containing 0.1% trifluoroacetic acid (TFA). The CH3CN concentration increased to 30% at 5 min, to 40% at 55 min, to 100% at 56 min, and then returned to the initial condition (25% CH3CN) at 58 min. The total runtime from injection to injection was 68 min, including a 10-min reequilibration step between runs.

Gliadins were separated by HPLC from three separate extractions. Each peak was manually collected three to five times for each extract. Collected fractions of each peak were combined and lyophilized overnight and then redissolved in 100 l of 70% ethanol. A Hewlett-Packard 1040A diode-array detector was also used to detect the eluted components at 210 nm (0.500 absorbance units full scale). A data point was stored every 640 msec on a Hewlett-Packard (9000-300) computer for subsequent integration, replotting, and comparison. All samples were analyzed within four days of extraction.

3.2.2 High Performance Capillary Electrophoresis (HPCE)

Another method that can be used for protein analysis is by using HPCE. In the same research done by Lookhart & Bean (1995), Beckman P/ACE 2100 was used for HPCE analysis. The HPCE buffer was 0.1M (pH 2.5) phosphate buffer with a polymeric additive, hydroxypropylmethylcellulose (HPMC). HPCE injection times varied for samples due to the relative amounts of proteins and the viscosities of the extractant. Pressure injection times for the gliadins were 4 sec (0.25 nl). In this analysis, proteins were detected by UV absorbance at 200 nm.

3.2.3 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE)

According to a research done by Shyur et al (1994) on rice prolamin, the crude prolamins were dissolved in an equilibrat ion buffer (0.5% sodium dodecylsulfate (SDS), 50 mM Tris-HC1 (pH 8.5), and 50 mM -mercaptoethanol) and centrifuged at 10,000 x g for 30 mm. The supernatant liquid was applied to a Sephadex G- 150 column (26 x 96 cm) previously equilibrated with the equilibration buffer, and eluted with the same buffer solution. The flow rate was 9.2 ml per h and the effluent was collected in 5 ml fractions. An aliquot of each fraction was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Prolamins were found in the first peak eluted from the Sephadex G-150 column. After electrophoresis the gel was sliced into 0.75mm sections. Each slice was put into an eppendorf tube and a buffer consisting of 6 M urea and 50 mM TrisH C1 (pH 8.9) was added. Prolamins were extracted from the gel slices by vigorously shaking the mixture overnight at room temperature. By using this method, recovery of prolamins from the gel was estimated to be round 90%.

Another research was done on SDS-PAGE analysis of maize prolamin done by Hamaker et al (1995) by using protein extracted with the method suggested by Wallace et al (1990). SDS-PAGE was performed on a vertical gel electrophoresis system. The separating gel was a linear 1015% (w/v) polyacrylamide gradient containing 5M urea with a 4% (w/v) stacking gel. Lyophilized samples were dissolved in SDS-PAGE sample solvent (2% SDS, 1% 2-ME, O.066M Tris at pH 6.8, with 10% glycerol and bromophenol blue), heated in a boiling water bath for 3 mm, and placed in the sample wells. Electrophoresis was performed at 70 V for 18 hr. Proteins were stained with 0.25% Coomassie Brilliant Blue R 250 in 46% methanol and 8% acetic acid and destained in 20% ethanol and 10% acetic acid.

CHAPTER 4

RESULTS AND DISCUSSION

In the previous chapter, different methods for extraction, purification and analysis of prolamin from wheat, maize and rice grains were reviewed. The results obtained from the studies on these methods are further discussed below.

4.1 Protein Extraction

In the research done by Ju, Hettiarachchy, & Rath (2001), the results obtained from sequential extraction method of rice prolamin are shown in the table below. Protein contents extracted were determined by the Kjeldahl method (AACC 1983) and the nitrogen content was multiplied by 5.95 according to Juliano (1994). The protein extraction efficiency was calculated as a percentage of the summed amount of proteins in the four supernatants to the total protein of defatted rice flour. Recovered efficiency (%) of each protein was calculated as a percentage of the amount of precipitated freeze-dried protein from supernatants to the total protein in the supernatants. These results are shown in the table below.

4.2 Protein Purification and Analysis

The results obtained from the different prolamin purification and analysis methods are discussed in the following sections.

4.2.1 High Performance Liquid Chromatography (HPLC)

From the research by Lookhart & Bean (1995) mentioned in the previous chapter, results of the analysis on wheat prolamin (gliadin) which was extracted with 70% ethanol without using the Osborne method are shown below. From the figure below, the two lines A and B represents new and old chromatography column respectively. This was done in the research to compare if there were any loss of resolution from the old column. The highest peaks for both lines were observed between 52 to 58 min for ?-gliadin.

4.2.2 High Performance Capillary Electrophoresis (HPCE)

From the figure above, the HPCE pattern for GLI- 1 exhibited major peaks between 4 and 8 min. Minor peaks also occurred from 1 to 3 min; The GLI-1 pattern had its largest peak at 6 min and showed good resolution of all major peaks.

In comparison between the two analysis methods used in Lookhart & Bean (1995), HPCE is faster than the HPLC method (Table 4.1). However, HPCE is more expensive than other electrophoretic methods, but it is much safer because it requires very small amounts of organic solvents and buffers and also toxic acrylamides usage is unnecessary.

4.2.3 Gel Filtration

In the research of M. R. Bugs et al., (2004), gel filtration was used to purify and analyze maize protein. Figure 4.3 shows the results of the gel filtration of seed extract with Superdex 75 of total protein measured with the absorbance at 280 nm. In Figure 4.3A, a smaller protein peak at an elution volume of 135 mL (peak a) is assigned to pennisetin, and the second peak at an elution volume of 170 mL (peak b) is the major constituent of the extract, which has a predominant yellow color and contains a low molecular weight protein. Figure 4.3B shows a smaller protein peak at an elution volume of 135 mL (peak a), which is assigned to the zein; as observed with pennisetin, the second peak at an elution volume of 170 mL (peak b) has a predominant yellow color.

4.2.4 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE)

From the figure above, the 15.5 and 14.2 kDa polypeptides of rice prolamins were isolated by preparative 14% SDS-PAGE. Equal amounts of these prolamins were analyzed by 520% SDS-PAGE. Protein bands were detected by silver staining. The amino acid composition obtained from the purified rice prolamin, 15.5 kDa and 14.2 kDa polypeptides is shown in the table below. It can be observed that these three prolamin fractions had similar amino acid compositions with high contents of glutamic acid, alanine, glycine, and arginine and also low levels of lysine and histidine.

Meanwhile, the SDS-PAGE analysis of maize prolamin (zein) from Wallace et al., (1990) extraction method done by Hammaker et al. (1995) is also shown below. From the results, it can be seen from the banding pattern that the zein fractions (lane 1) are free from any contaminants by other protein fractions. This also shows that the Wallace et al., (1990) extraction method is simpler and more effective method compared to the conventional method used by Osborne (1924).

CHAPTER 5

CONCLUSION AND FUTURE PROSPECTS

As a conclusion, this research had shown a variety of methods for prolamin extraction, purification and analysis. For prolamin extraction, the most common methods used are modifications of Osborne method and Landry-Moureaux method. As for prolamin purification and analysis, various chromatographic methods can be used such as High Performance Liquid Chromatography (HPLC), High Performance Capillary Electrophoresis (HPCE), Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE) and so on. Several journals were reviewed to establish the right method for these procedures.

In the prolamin extraction process, the Osborne method proved to be demanding because of the need to follow several steps before obtaining prolamin at the final stage. Although this method has promising results, there are concerns over the extensive treatment of the raw material with reagents that may cause denaturation of protein content. An easier method would be the ones suggested by Wallace et al (1990) which only seperates protein into prolamin and non-prolamin content. This method uses minimal amount of solvents thus ensuring that the proteins are not denatured during the extraction process. Nevertheless, this method was only tested on maize prolamin but it is assumed that it can also be applied to wheat and rice prolamins as well.

As for the purification and analysis process, there are no specific data pertaining on the efficiency of each method used. Thus, conclusion on the best purification and analysis method cannot really be determined in this research. Through the writers understanding from the journals reviewed, all the methods described, be it HPLC, HPCE or SDS-PAGE can be used for prolamin purification and analysis.

For future works, this research paper can be used as a foundation for experimental work on prolamin extraction and analysis. With all the required materials and equipments available, the experiments can be easily done at laboratory scale. The current recovery efficiency of 90% from the extraction process can also be optimized to acquire a better efficiency from these experimental works.

From the groundwork done in this research, it is believed that the potentials of grain protein extraction can and will be utilized for additional development of genetically modified crops to attain a more nutritionally valued cereal grains. It is essential to further exploit these techniques so that more information regarding the storage protein genes expression can be gained to improve the quality of cereal crops in the future.

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