TECHNOLOGY OF CEREALS FG3.7 electon micrograph of one large starch granule and numerous small starch granules arge granule shows the equatorial groove. From A D. Evers, Starke, 1971, 23: 15 Reproduced with permission of the Editor of Die Starke. an experienced microscopist can identify the cereals are similar in shape to the smaller popula source, either from observation of an aqueous tion of Triticeae granules, but rice and oats have suspension at room temperature or with the some compound granules in which many granuli additional help of observed changes when the fit together to produce large ovoid wholes. Shapes suspension is heated, leading to gelatinization at of high-amylose granules are varied and may be a temperature characteristic of the species and related to their individual composition. The later type (snyder, 1984). The characteristic blue developers tend to be filamentous, some resembling staining reaction with iodine/potassium iodide strings of beads. Characteristics of starch granules solution does not occur with waxy granules, from cereals are shown in Table 3.4 which contain virtually no amylose, they stain Within the endosperm of a species small differ brownish red to yellow. It is characteristic ences in granule shape may arise as a result for amylose percentage to increase during of packing conditions. These can be seen in endosperm development, consequently staining grains as mealy and vitreous(or horny)regions reactions change during growth In mealy regions, packing is loose and Granules of cereals from the Triticeae tribe(see adopt what appears to be their natural form. In Ch. 2)are of two distinct types. The larger ones horny regions close packing causes granules to are biconvex while the smaller ones are nearly become multi-faceted as a result of mutual pres spherical (Fig. 3.7). Granules from most other sure. Small indentations can also arise from other
58 TECHNOLOGY OF CEREALS FIG 3.7 Scanning electon micrograph of one large starch granule and numerous small starch granules of wheat. The large granule shows the equatorial groove. From A.D. Evers, Stiirke, 1971, 23: 157. Copyright by Leica U.K., Reproduced with permission of the Editor of Die Stiirke. cereals are similar in shape to the smaller population of Triticeae granules, but rice and oats have some compound granules in which many granuli fit together to produce large ovoid wholes. Shapes of high-amylose granules are varied and may be related to their individual composition. The later developers tend to be filamentous, some resembling strings of beads. Characteristics of starch granules from cereals are shown in Table 3.4. Within the endosperm of a species small differences in granule shape may arise as a result of packing conditions. These can be seen in grains as mealy and vitreous (or horny) regions. In mealy regions, packing is loose and granules adopt what appears to be their natural form. In horny regions close packing causes granules to become multi-faceted as a result of mutual pressure. Small indentations can also arise from other an experienced microscopist can identify the source, either from observation of an aqueous suspension at room temperature or with the additional help of observed changes when the suspension is heated, leading to gelatinization at a temperature characteristic of the species and type (Snyder, 1984). The characteristic blue staining reaction with iodine/potassium iodide solution does not occur with waxy granules, which contain virtually 'no amylose, they stain brownish red to yellow. It is characteristic for amylose percentage to increase during endosperm development, consequently staining reactions change during growth. Granules of cereals from the Triticeae tribe (see Ch. 2) are of two distinct types. The larger ones are biconvex while the smaller ones are nearly spherical (Fig. 3.7). Granules from most other
CHEMICAL COMPONENTS FIG 3. 8 Scanning electron micrograph of maize starch granules of spherical and angular types. Some angular granules show indentations due to pressure from protein bodies endosperm constituents such as protein bodies. labelled precursors incorporated into growing granules(Badenhuizen, 1969). Such a system Pitting on the surface can be caused by enzymic of growth allows for the change in shape that hydrolysis and it is possible to find such granules occurs in starches of the Triticeae, by preferential in some cereal grains in which germination has deposition on some parts of the surface. As a begun or in which insect damage has occurred. result they change from tiny spheres to larger There is no evidence that these two physical lentil shaped granules(Evers, 1971) modifications to granule form change the chemical Some structures not evident in untreated granules can be revealed or exaggerated by treat As granules are transparent some manifesta- ment with weak acid or amylolytic enzymes. In tions of internal structure can be detected, even cereal starches a lamellate structure results if their significance cannot be fully appreciated. from removal of more susceptible layers and One such internal feature is the hilum exhibited persistence of more resistant layers. Layers may by granules of some species. It is a small air be spaced progressively more closely towards the space, considered to represent the point of initia- outside. The number of rings appears to coincide tion around which growth occurred(Hall and with the number of days for which a granule Sayre, 1969). This assumes that granules grow grows(Buttrose, 1962 ). Lamellae cannot be by deposition of new starch material on the outer revealed in granules from plants grown under surface of existing granules, and indeed this has. conditions of continuous illumination(Evers been demonstrated by detection of radioactively 1979
CHEMICAL COMPONENTS 59 FiG 3.8 Scanning electron micrograph of maize starch granules of spherical and angular types. Some angular granules show indentations due to pressure from protein bodies. endosperm constituents such as protein bodies. (Fig. 3.8). Pitting on the surface can be caused by enzymic hydrolysis and it is possible to find such granules in some cereal grains in which germination has begun or in which insect damage has occurred. There is no evidence that these two physical modifications to granule form change the chemical properties of the granules. As granules are transparent some manifestations of internal structure can be detected, even if their significance cannot be fully appreciated. One such internal feature is the hilum exhibited by granules of some species. It is a small airspace, considered to represent the point of initiation around which growth occurred (Hall and Sayre, 1969). This assumes that granules grow by deposition of new starch material on the outer surface of existing granules, and indeed this has . been demonstrated by detection of radioactively labelled precursors incorporated into growing granules (Badenhuizen, 1969). Such a system of growth allows for the change in shape that occurs in starches of the Triticeae, by preferential deposition on some parts of the surface. As a result they change from tiny spheres to larger lentil shaped granules (Evers, 1971). Some structures not evident in untreated granules can be revealed or exaggerated by treatment with weak acid or amylolytic enzymes. In cereal starches a lamellate structure results from removal of more susceptible layers and persistence of more resistant layers. Layers may be spaced progressively more closely towards the outside. The number of rings appears to coincide with the number of days for which a granule grows (Buttrose, 1962). Lamellae cannot be revealed in granules from plants grown under conditions of continuous illumination (Evers, 1979)
TECHNOLOGY OF CEREALS Size distributions less than half the total starch present. Some 70% The literature contains many tables of granule all the amylose bu size ranges and size distributions of granules from must also include much of the amylopectin. The different botanical sources While such tables evidence of biochemical studies and electron microscopy has pointed to the existence of struc useful guides they do not all accord in detail and tures with a periodicity of 5-10 nm,reflecting the some fail to indicate the nature of the distribution. alternating crystalline and amorphous zones of For example the bimodal distribution of the Triticeae is not always indicated although this is amylopectin an important characteristic by which the source of a starch may be recognized. In wheats the Granule surface and minor components proportional relationship between large biconvex The distribution of amylose and amylopectin and small spherical granules is fairly constant molecules in one starch granule was estimated by (approx 70% large granules w/w), and this is the same for rye and triticale French (1984): for one spherical granule 15 um G In barley there is a wider variation, in part due in diameter, with a mass of 2.65 x 10 g there to the existence of more mutant types( Goering would be about 2.5 x 10% molecules of amylose et al., 1973). Among 29 cultivars, small granules (D P=1000, 25% of total starch)and 7.4 x 10 counted for between 6% and 30%of the total molecules of amylopectin(D P.= 100,000,75% of starch). If the molecular chains are perpend- icular to the surface of the granule there would be about 14 x 10 molecular chains terminating Granule organization at the surface. Of these 3.5 x 10 would be amylose molecules and the rest would be Under crossed polarizers starch granules amylopectin chains exhibit birefringence in the form of a maltese Surface characteristics of granules are also cross. This indicates a high degree of order affected by the minor components of starches within the structure. The positive sign of the Bowler et al.(1985) reviewed developments in birefringence suggests that molecules are organized work on these although they point out that this in a radial direction(French, 1984). Amylomaize is an under-researched area. Non-starch materials starch exhibits only weak birefringence of an present in commercial starch granules can arise unusual type(Gallant and Bouchet, 1986) from two sources. They may be inherent com Starch granules exhibit X-ray patterns, indicat- ponents of the granules in their natural condition ing a degree of crystallinity. Cereal starches or they may arise as deposits of material solubilized give an a pattern, tuber, stem and amylomaize or dispersed during the process by which the starches give a B pattern and bean and root starch is separated arches a c pattern the c pattern is considered The main non-starch components of starch to be a mixture of A and B. It is accepted that granules are protein and lipid amounts vary with the crystallinity is due to the amylopectin as it is starch type: in maize 0. 35% of protein (n X 6.25) shown by waxy granules. Furthermore, amylose is present on average Slightly more is present can be leached from normal granules without in wheat starch(0.4%). The most significant ffecting the X-ray pattern. The a and B patterns proteins in terms of their recognized effects or are thought to indicate crystals formed by double starch behaviour are amylolytic enzymes bound helices in amylopectin. The double helices occur to the surface Even traces of alpha-amylase can in the outer chains of amylopectin molecules, have drastic effects on pasting properties through where they form regions or clusters. The crystal- hydrolyzing starch polymers at temperatures up line parts of starch granules are responsible for to the enzymes inactivation temperatures many of the physical characteristics of the granules' SDS PAGE(sodium dodecyl sulphate, poly- structure and behaviour. Nevertheless they involve acrylamide gel elecrophoresis) showed surface
60 TECHNOLOGY OF CEREALS Size distributions less than half the total starch present. Some 70% is amorphous; this comprises all the amylose but must also include much of the amylopectin. The evidence of biochemical studies and electron microscopy has pointed to the existence of structures with a periodicity of 5-10 nm, reflecting the alternating crystalline and amorphous zones of amylopectin. Granule surface and minor components The distribution of amylose and amylopectin molecules in one starch granule was estimated by French (1984): for one spherical granule 15 pm in diameter, with a mass of 2.65 x lO-9 g there would be about 2.5 x lo9 molecules of amylose (D.P = 1000, 25% of total starch) and 7.4 x lo7 molecules of amylopectin (D.P. = 100,000, 75% of starch). If the molecular chains are perpendicular to the surface of the granule there would be about 14 x 10' molecular chains terminating at the surface. Of these, 3.5 x 10' would be amylose molecules and the rest would be Surface characteristics of granules are also affected by the minor components of starches. Bowler et al. (1985) reviewed developments in work on these although they point out that this is an under-researched area. Non-starch materials present in commercial starch granules can arise from two sources. They may be inherent components of the granules in their natural condition or they may arise as deposits of material solubilized or dispersed during the process by which the starch is separated. The main non-starch components of starch granules are protein and lipid. Amounts vary with starch type: in maize 0.35% of protein (N x 6.25) is present on average. Slightly more is present in wheat starch (0.4%). The most significant proteins in terms of their recognized effects on starch behaviour are amylolytic enzymes bound to the surface. Even traces of alpha-amylase can have drastic effects on pasting properties through hydrolyzing starch polymers at temperatures up to the enzymes' inactivation temperatures. SDS PAGE (sodium dodecyl sulphate, polyacrylamide gel elecrophoresis) showed surface The literature contains many tables of granule size ranges and size distributions of granules from different botanical sources. While such tables are useful guides they do not all accord in detail and some fail to indicate the nature of the distribution. For example the bimodal distribution of the Triticeae is not always indicated although this is an important characteristic by which the source of a starch may be recognized. In wheats the proportional relationship between large biconvex and small spherical granules is fairly constant (approx 70% large granules w/w), and this is the same for rye and triticale. In barley there is a wider variation, in part due to the existence of more mutant types (Goering et al., 1973). Among 29 cultivars, small granules accounted for between 6% and 30% of the total starch mass. Granule organization exhibit birefringence in the form of a maltese cross. This indicates a high degree of order within the structure. The positive sign of the birefringence suggests that molecules are organized in a radial direction (French, 1984). Amylomaize starch exhibits only weak birefringence of an unusual type (Gallant and Bouchet, 1986). Starch granules exhibit X-ray patterns, indicating a degree of crystallinity. Cereal starches give an A pattern, tuber, stem and amylomaize starches give a B pattern and bean and root starches a C pattern. The C pattern is considered to be a mixture of A and B. It is accepted that the crystallinity is due to the amylopectin as it is shown by waxy granules. Furthermore, amylose can be leached from normal granules without affecting the X-ray pattern. The A and B patterns are thought to indicate crystals formed by double helices in amylopectin. The double helices occur in the outer chains of amylopectin molecules, where they form regions or clusters. The crystalline parts of starch granules are responsible for many of the physical characteristics of the granules' structure and behaviour. Nevertheless they involve Under crossed polarizers starch granules amylopectin chains
CHEMICAL COMPONENTS proteins of wheat starch to have molecular masses of water available during cooking. Digestibilit of under 50 k while integral proteins were over in the intestines of single-stomached animals is 50 K. Altogether ten polypeptides have been also increased by gelatinization separated between 5 K and 149 K. The major 59 K polypeptide is probably the enzyme respon- Gelatinization sible for amylose synthesis. It has been shown to be concentrated in concentric shells within This is a phenomenon manifested as several granules. Two other polypeptides of 77 K and changes in properties, including granule swelling 86 K are likely to be involved in amylopectin and progressive loss of organized structure synthesis. Perhaps the most interesting of the(detected as loss of birefringence and crystallinity), surface proteins is that in the 15 K band. increased permeability to water and dissolved This has been found in greater concentration or bstances (including dyes), increased leaching starches from cereals with soft endosperm than of starch components, increased viscosity of the on those from cereals with hard endosperm. The aqueous suspension and increased susceptibility protein has been called friabilin,, because of its to enzymic digestic association with a friable endosperm(cf Ch. 4) At room temperature starch granules are not (Greenwell and Schofield, 1989) totally impermeable to water, in fact water uptake Phosphorus is another important minor con- can be detected microscopically by a small increase stituent of cereal starches. It occurs as a com- in diameter. The swelling is reversible and the onent of lysophospholipids. They consist of 70% wetting and drying can be cycled repeatedly lysophosphatidyl choline, 20% lysophosphatidyl without permanent change. If the temperature of ethanolamine and 10% lysophosphatidyl glycerol. a suspension of starch in excess water is raised The proportion of lysophospholipids to free fatty progressively, a condition is reached, around acids varies with species: in wheat, rye, triticale 60C, at whic ible swelling begins, and and barley over 90%occurs as lysophospholipids, continues with increasing temperature. The in rice and oats 70% and in millets and sorghum change is endothermic and can be quantified by 55%. In maize 60% occurs as free fatty acids thermal analysis techniques Removal of lipids from cereal starches reduces starch are: wheat 19.7, maize 18.0, waxy majay (Morrison, 1985) Typical heats of gelatinization in J per g of he temperatures of gelatinization-related changes 19.7 and high amylose maize 31.79(Maurice et and increases peak viscosity of pastes. In other al. 1983). Swelling involves increased uptake of words they become more like the lipid-free potato water and can thus lead to increased viscosity by reducing the mobile phase surrounding the gran ules; accompanying leaching of starch polymers Technological importance of starch into this phase can further increase viscosity. The swelling behaviour of starch heated in water is Much of the considerable importance of starch often followed using a continuous automatic in foods depends upon its nutritional properties; viscometer, such as the Brabender Amylograph it is a major source of energy for humans and for ( Shuey and Tipples, 1980). Upon heating a slurry domestic herbivorous and omnivorous animals. of 7-10% starch w/w in water at a constant rate In the human diet it is usually consumed in a of 10-5C per min, starch eventually gelatinizes cooked form wherein it confers attractive textural and begins to thicken the mixture The tempera qualities to recipe formulations. These can vary ture at which a rise in consistency is shown is from those of gravies and sauces, custards and called the pasting temperature. The curve then pie fillings to pasta, breads, cakes and biscuits generally rises to a peak, called the peak viscosity (cookies). Much of the variation in texture depends When the temperature reaches 95C, that tem upon the degree of gelatinization, which in turn perature is maintained for 10-30 min and stirring depends upon the temperature, and the amount is continued to determine the shear stability of
CHEMICAL COMPONENTS 61 proteins of wheat starch to have molecular masses of water available during cooking. Digestibility of under 50 K while integral proteins were over in the intestines of single-stomached animals is 50 K. Altogether ten polypeptides have been also increased by gelatinization. separated between 5 K and 149 K. The major Gelatinization 59 K polypeptide is probably the enzyme responsible for amylose synthesis. It has been shown to be concentrated in concentric shells within This is a phenomenon manifested as several granuies. Two other polypeptides of 77 K and changes in properties, including granule swelling 86 K are likely to be involved in amylopectin and progressive loss of organized structure synthesis. Perhaps the most interesting of the (detected as loss of birefringence and crystallinity), surface proteins is that in the 15 K band. increased permeability to water and dissolved This has been found in greater concentration on substances (including dyes), increased leaching starches from cereals with soft endosperm than of starch components, increased viscosity of the on those from cereals with hard endosperm. The aqueous suspension and increased susceptibility protein has been called 'friabilin', because of its to enzymic digestion. association with a friable endosperm (cf Ch. 4) At room temperature starch granules are not (Greenwell and Schofield, 1989). totally impermeable to water, in fact water uptake Phosphorus is another important minor con- can be detected microscopically by a small increase stituent of cereal starches. It occurs as a com- in diameter. The swelling is reversible and the ponent of lysophospholipids. They consist of 70% wetting and drying can be cycled repeatedly lysophosphatidyl choline, 20% lysophosphatidyl without permanent change. If the temperature of ethanolamine and 10% lysophosphatidyl glycerol. a suspension of starch in excess water is raised The proportion of lysophospholipids to free fatty progressively, a condition is reached, around acids varies with species: in wheat, rye, triticale 60"C, at which irreversible swelling begins, and and barley over 90% occurs as lysophospholipids, continues with increasing temperature. The in rice and oats 70% and in millets and sorghum change is endothermic and can be quantified by 55%. In maize 60% occurs as free fatty acids thermal analysis techniques. (Morrison, 1985). Typical heats of gelatinization in J per g of dry Removal of lipids from cereal starches reduces starch are: wheat 19.7, maize 18.0, waxy maize the temperatures of gelatinization-related changes 19.7 and high amylose maize 31.79 (Maurice et and increases peak viscosity of pastes. In other al., 1983). Swelling involves increased uptake of words they become more like the lipid-free potato water and can thus lead to increased viscosity by starch. reducing the mobile phase surrounding the granules; accompanying leaching of starch polymers into this phase can further increase viscosity. The swelling behaviour of starch heated in water is Technological importance of starch Much of the considerable importance of starch often followed using a continuous automatic in foods depends upon its nutritional properties; viscometer, such as the Brabender Amylograph it is a major source of energy for humans and for (Shuey and Tipples, 1980). Upon heating a slurry domestic herbivorous and omnivorous animals. of 7-10% starch w/w in water at a constant rate In the human diet it is usually consumed in a of 1°-5"C per min, starch eventually gelatinizes cooked form wherein it confers attractive textural and begins to thicken the mixture. The temperaqualities to recipe formulations. These can vary ture at which a rise in consistency is shown is from those of gravies and sauces, custards and called the pasting temperature. The curve then pie fillings to pasta, breads, cakes and biscuits generally rises to a peak, called the peak viscosity. (cookies). Much of the variation in texture depends When the temperature reaches 95"C, that temupon the degree of gelatinization, which in turn perature is maintained for 10-30 min and stirring depends upon the temperature, and the amount is continued to determine the shear stability of