9

Chapter-2

REVIEW OF LITERATURE

2.1. Amaranthus leaves characteristics

The genus Amaranthus belongs to the family Amaranthaceae and includes more

than 60 species, of which three viz., A. hypochondriacus, A. cruentus and A. caudatus,

are the essential grain species (Stallknecht and Schulz-Schaeffer, 1993; Brenner et al.,

2000). Amaranth is a very versatile crop that is grown in a wide range of agro-climatic

conditions; it resists drought, heat, and pests, and adapts readily to new environments,

including some that are inhospitable to conventional cereal crops (Brenner et al., 2000;

Rana et al., 2007). It is one of the few multi-purpose crops, which can supply grains and

tasty leafy vegetables of high nutritional quality as a food and animal feed, and

additionally an ornamental plant, because of attractive inflorescence coloration (Breene,

1991; Mlakar et al., 2009). Several amaranth species are of paramount importance;

therefore, consumers can take advantage of them for different uses, such as flour from

seeds, salads from fresh leaves, inflorescences as source of natural red dye, or waste

products as animal foodstuff (Juan et al., 2007). Three amaranth species are mainly used

for seed production; these are Amaranthus cruentus L., A. caudatus L., and A.

hypochondriacus L. (Hernández and Herrerías, 1998); these species produce big

inflorescences full of seeds (Morales et al., 2009). A few studies have reported that A.

lividus leaves as well as flowers have antioxidant capacity (Ozsoy et al., 2009). Araceli

López-Mejía et al. (2014) evaluated the effect of extraction method and solvent type on

antioxidant capacity and total phenolic content of extracts from amaranth (Amaranthus

hypochondriacus L.) seeds or leaves. These authors extracted antioxidant compounds by

two methods: magnetic stirring or Soxhlet. For these two methods, methanol, ethanol, or

hexane, were tested as solvents. Antioxidant capacity was determined by inhibition of

DPPH· radical. Total phenolics were determined by the phenol Folin–Ciocalteu assay. A

significant effect of extraction method and solvent type on antioxidant capacity and total

phenolics was observed. The highest antioxidant capacities, up to 1070 equiv. of Trolox

10

(mg/100 g dry weight), and total phenolics, up to 619 equiv. of gallic acid (mg/100 g dry

weight), were determined in extracts from the Soxhlet extraction method; methanol was

observed as a better extraction solvent for amaranth seeds while ethanol was for leaves.

Amaranth leaves extracts was observed to exhibited more antioxidant capacity than those

from seeds. Antioxidant capacities of studied extracts are not only due to phenolics.

2.2 Grain Characteristics

Aamaranth seeds are small and lenticular in shape with each seed averaging 1.0-

1.5 mm in diameter and 1,000 seeds weighing 0.6-1.2 g (Jain and Hauptli, 1980;

Saunders and Becker, 1984). Amaranth grown for grain is pale-seeded, with seed colors

ranging from off-white to brown (Irving et al., 1981). Amaranth contains 15–22%

protein, 58–66% starch with a low gelatization temperature and granule size varying

between 1 and 3.5 lm, depending on variety (Tosi et al., 2001). Amaranth seeds contain

9–16% dietary fiber and 3.1–11.5% lipids (Pedersen et al., 1987). They are characterized

by high concentrations of calcium, phosphorus, iron, potassium, zinc, vitamins E and B

complex, and a low level of antinutritional factors (Tosi et al., 2001). Alvarez-Jubete et

al. (2010) reported the presence of polyphenols in A.caudatus seeds. Repo-Carrasco-

Valencia et al. (2010) observed flavonoids and betalains in amaranth seeds. Barba de la

Rosa et al. (2009) analyzed A. hypochondriacus seeds and they find out phenolics with

antioxidant properties. Pa´sko et al. (2009) reported the presence of phenolics and

anthocyanins in A. cruentus seeds. There are several ways for extracting compounds from

fresh vegetable foods; one of them is by using solvents such as water, ethanol, methanol,

ethyl acetate, or hexane, among others (Singh et al., 2002). Conventional solvents as

methanol and hexane were recognized for providing high extraction yields, but their uses

has raised safety, environmental, and health issues; while, ethanol was reported to be a

safe alternative (Velasco et al., 2007). The 18 different Amaranthus genotypes seeds were

analysed for their content of 11 polyphenols and the variations among genotype, species

and location (Steffensen et al., 2011). The flavonoid, rutin, exhibited large variations with

varying environmental conditions whereas the flavonoid, nicotiflorin, was affected less.

Amaranthus hypochondriacus displayed the most stable content of polyphenols with a

high end content of flavonoids. The variations between location/environmental condition

11

were primarily described by the variations in the content of p-coumaric acid and

protocatechuic acid in the seed samples.

Nutrients were concentrated in the seed-coat embryo fraction, reaching 2.3-2.6

times as much nitrogen, fat, fibre, and ash, 2.4-3.0 times as much thiamin, riboflavin, and

niacin and 1.4-2.5 times as much of several mineral elements as the original, intact seed.

Like all other grains and seeds, amaranth seeds have three basic anatomical parts: a seed

coat (or bran) to protect the seed from the outside environment, an embryo (or germ)

which will grow into a new plant, and food storage tissue to nourish the growing embryo

(Hoseney, 1994). In amaranth the main food storage tissue is the perisperm, while in

grains (e.g. wheat) the main food storage tissue is the endosperm; both are composed of

starch granules embedded in a protein matrix. The seed coat is thin, while the germ is

relatively large (accounts for approximately 25 % of the seed weight) and forms a ring

that surrounds the perisperm (Belton and Taylor, 2002). Amaranth grains have higher

protein content (12–18%) than most of the other cereal grains, with a significantly higher

content of lysine, and acceptable levels of tryptophan and methionine, which are found in

low concentrations in cereals and leguminous grains of common usage (Mendoza and

Bressani, 1987; Teutonico and Knorr, 1985). Grain amaranth protein contains around 5%

lysine and 4.4% sulfur amino acids, which are the limiting amino acids in other grains

(Senft, 1980). Besides protein, the grains are a good source of dietary fibre and minerals,

such as magnesium, phosphorus, copper, and especially manganese. The Amaranth grain

oil contains significant amount (8%) of squalene (Sun et al., 1997), which has many

important direct and indirect beneficial effects on health, and has the potential of

replacing other squalene sources, e.g. whale or shark liver oil. Squalene, has been found

to act as an anticancer agent and has hypocholesterolemic effects (Das et al., 2003; Shin

et al., 2004). The importance of squalene as a food component has been attributed to its

ability to lower cholesterol levels by inhibiting its synthesis in the liver (Escudero et al.,

2006). The biomass of amaranth, both as green and dried, has high biological value, and

is used as source of protein, amino acid and dietary fibre for fattened pigs, rabbits, and

lambs (Andrasofszky et al., 1998; Zraly et al., 2004). The lipid fraction of the amaranth

grains is similar to other cereals consisting mainly of unsaturated fatty acids, with linoleic

acid being the predominant fatty acid. Amaranth oil fractions have also been reported to

12

have tocotrienols, which are known to effect cholesterol levels in the mammalian systems

(Lyon and Becker, 1987). The Amaranth grain can be an excellent choice for gluten free,

as well as casein free diets. In addition to the unique characteristics of the major

components of proteins, carbohydrates and lipids, the amaranth grains also contain high

levels of calcium, iron and sodium, compared to cereal grains (Becker et al., 1981). In

India, its grains are roasted like pop corn and mixed with honey, jaggery or molasses to

produce a candy type product known as laddoos (Singh and Singh, 2011). While in other

countries its popped grains are mixed with chocolate or puffed rice to make different

products. It is consumed in different forms in several other countries with the

consumption spreading to Europe and parts of North America. Amaranth flour is used as

thickener in gravies, soups and stews, custards etc and is blended with wheat flour in the

preparation of unleavended flat bread known as ‘chapattis’ in India and ‘tortillas’ in Latin

America (Singh and Singh, 2011).

2.3 Starch characteristics

2.3.1 Physicochemical properties

Starch consists of two structural isomers, an essentially linear polysaccharide

amylose (α-1, 4-anhydroglucopyranose) and a highly branched polysaccharide

amylopectin (including α-1,4 –linkage and α, 1,6-linkage) with ratio of amylose and

amylopectin ranges between 25-28% and 72-75% respectively (Manner,1989).The extent

of branching has been shown to increase with the molecular size of amylose (Greenwood

and Thomson., 1959). Amylopectin is the major component with an average molecular

weight of the order 107

to 109

(Aberle et al., 1994). Amorphous and semi crystalline radial

growth rings of 120-400 nm thickness emanating from the hilum are report that starch has

layered organization. The amorphous rings consists of amylose and amylopectin in a

disordered conformation, Whereas the semi crystalline is consist of the lamellar structure

of alternating crystalline and amorphous regions with a repeat distance of 9-11 nm

(Cameron and Donald., 1992). The crystalline regions of lamella is mainly made up by

double helices of amylopectin side chain packed laterally into a crystalline lattice, As the

amorphous region is consist of amylose and the amylopectin branching points.

Amylopectin cluster may contain amylose molecules that passed through crystalline and

13

amorphous layer. These amylose molecules are organized in a straightened conformation

in a crystalline region and in a disordered conformation in amorphous region (Matveev et

al., 1998). Four types of super molecules structure has different in macromolecules

organization as well as characteristics are shown in starches. These are crystalline and

amorphous lamellae (~4-6 nm), amylopectin cluster (~9 nm) semi crystalline and

amorphous rings (~120-400) as well as granule themselves (~0.5-100μm) (Bouleon et al.,

1998; Jenkins and Donald, 1995; Kozlov et al., 2007).Small-angle X-ray scattering and

neutron scattering have been shown to be useful for studying the arrangement of lamellar

structures in semi-crystalline starch granules (Waigh et al., 1996).

Villarreal et al. (2011) isolated starch from Amaranthus cruentus whole grain

(WG) and whole grain flour (WGF) using both the alkaline method (AM) and AM

combined with food degree protease digestion (AMP). The methods involved successive

soaking in NaOH solution (0.25 g/100 ml in AM and 0.05 g/100 ml in AMP), fibrous

fraction wet milling, enzymatic hydrolysis in AMP and multi-staged centrifugation.

Milling the amaranth grains in both methods increased significantly starch yield,

recovery, and purity when compared against WG and lowered soaking times as well.

Starch yield and recovery were 116.7% and 123.6% higher in WGF while protein, fiber,

and ash contents showed decreases of about 44.4%, 34.8%, and 30.4%, respectively. The

effect of the extracting methods was observed less notorious than that of the grain

milling. The authors suggested that both methods were suitable for extracting starch from

previously milled grains despite the fact that the AM showed significant operative

advantages. Amaranthus starch comprises two major types of biomacromolecules,

amylose and amylopectin. Amaranth starch displays an A-type X-ray pattern (Choi et al.,

2004; Hoover et al., 1998; Qian and Kuhn, 1999). Its amylose content is low, ranging

from 3–8% depending on different genotypes (Choi et al., 2004; Hoover et al., 1998;

Marcone, 2001; Qian and Kuhn, 1999; Uriyapongson and Rayas-Duarte, 1994).

Amaranthus starches was reported to have high crystallinity degree (around 39%),

associated to their waxy features (Villarreal et al. 2013 ). The amylose content of starch

granule varies with the botanical source of the starch and is affected by climatic

conditions and soil type during growth (Juliano et al., 1964; Morrison et al., 1984;

14

Morrison and Azudin, 1987). Starch properties depend on the physical and chemical

characteristics such as mean granule size, granule size distribution, amylose/amylopectin

ratio and mineral content (Madsen and Christensen, 1996; Singh et al., 2003). It is mainly

located in the perisperm, where it is present as very small granules embedded in a protein

matrix (Belton and Taylor, 2002). Depending on variety, the amount of starch varies from

48 % to 69 % (Resio et al., 2009), and the average starch granule diameter ranges from 1

to 3 μm (Wilhelm et al., 2002). Native starch granules are insoluble in cold water but

swell in warm water. When starch granules are heated in the presence of water, an order-

to disorder phase transition occurs. Swelling of starch granules exert a pressure on

neighboring crystallites and tends to distort them. Further heating leads to uncoiling or

dissociation of double helical regions and break-up of amylopectin crystallite structure.

Starch molecules have tendency to contract to obtain a random coil conformation

providing a constraint in direction of the chain against swelling. Further hydration

resulted in increased mobility permitting a redistribution of molecules and the smaller

linear amylose molecules diffuse out. Heating and hydration both weakened the granule

to the point where it can no longer hold the pressure developed inside the starch granule

and eventually a sol results. Collapse (disruption) of molecular orderliness within the

starch granule resulted in irreversible change in properties such as granular swelling,

crystallite melting, loss of birefringence, viscosity development and solubilisation (Flory,

1953). Leach et al. (1959) concluded that strong bonds resists the swelling of granules

whereas weak bonds undergo very rapid and unrestricted swelling and at relatively low

temperature. Swelling power and solubility provide evidence of the magnitude of

interaction between starch chains within the amorphous and crystalline domains. The

extent of this interaction is influenced by the amylose/amylopectin ratio, and by the

characteristics of amylose and amylopectin in terms of molecular weight/distribution,

degree and length of branching, and conformation (Hoover, 2001). The swelling power of

the starch is associated more with granule structure and chemical composition,

particularly amylose and lipid content. Presence of lipid results in the formation of

amylose-lipid complex, which are believed to restrict swelling and amylose leaching.

Once the amylose-lipid complexes dissolve, the rate of amylose leaching out of the

granules increases substantially.

15

2.3.2 Paste clarity

Paste clarity varies considerably with the starch source, the amylose/amylopectin

ratio, chemical or enzymatic modifications and addition of solutes. Swelling and brittleness

of the starch molecules affect the clarity of starch pastes (Craig et al., 1989). Solutes like

sucrose and glucose increased the starch paste clarity whereas lipids increased the opacity.

Salt reduce the transmittance as well as visual clarity of potato starch paste. Starch paste

clarity affected by the phosphorus. Phosphorus is present as phosphate monoester and

phospholipids in various starches. Phosphate monoester are covalently bound to the

amylopectin fraction of starch and known to increase starch paste clarity and viscosity,

while the presence of phospholipids results in opaque and lower viscosity pastes. When a

beam of light is reflected back and the starch appears white and opaque due to the surface

of the granule being larger than the wavelength of light. Separation of starch chains during

gelatinization decreases the reflecting ability of starch granules and thus increases the

percentage transmittance of a starch paste (Hoover et al., 1996).

2.3.3 Pasting properties

Ceaser (1932) and Ceaser and Moore (1935) using a consistometer first

recognized pasting characteristics of starch and starch containing products. The first

Brabender Viscoamylograph became available in 1930s. It has become a standard

equipment piece of equipment used by industry for characterization of starches and starch

containing products. Pasting characteristics were shown by Sandstedh and Abbott (1961)

to be significantly affected by starch concentration. Rapid Visco Analyzer (RVA) is an

instrument which measures the viscous properties of cooked starch and flour and co

relate with functionality of structural properties (Jane et al., 1999; Lindeboom, et al.,

2004) synergistic effect produced on the viscosity of the starch due to amylose molecular

size and amylopectin chain length distribution (Jane and Chen, 1992). A major advantage

of RVA when used to measure paste viscosities is the speed with which the procedure

may be carried out. A rapid cycle of heating and cooling is probably more appropriate in

mimicking processing methods than the longer heat- cool cycle used to assess pasting

properties. Pasting characteristics of all- purpose flour measured by RVA were found to

be similar to those measured by Brabender Viscoamylograph (Walker et al., 1988).

16

According to Hermansson and Svegmark (1996), during gelatinization, the starch

granules first swell like balloon, subsequently collapse and become folded and

simultaneously amylose leaches out from inside the granule, A three dimensional

network is formed by leached out amylose (Eliasson 1985a; Tester and Morrison, 1990).

The swelling behavior of starch is property of its amylopectin content and amylose acts

both as a dilutant and inhibitor of swelling (Tester and Morrison, 1990). Starch exhibits

unique viscosity behavior with the change of temperature, concentration and shear rate

(Nurul et al., 1999). Starches that are capable of swelling to a high degree are also less

resistant to breakdown on cooking and hence exhibit viscosity decreases significantly

after reaching the maximum value. The increase in viscosity during the cooling period

indicated the tendency of various constituents present in hot paste to associate or

retrograde as the temperature of the paste decreases. Viscosity of gelatinized starch

suspension may be attributed to the frictional dissipation of energy in the movement of

the swollen starch granules relative to one another (Miller et al., 1973). Cooked starch

behaved as non- Newtonian fluids due to secondary bonds between the hydrodynamic

units, either directly or through intermediate water molecules (Schutz., 1971).

2.3.4 Thermal properties

The gelatinization of the native starch is required in almost all- culinary and

industrial uses of starch (Blanshard, 1987). Gelatinizatiotion leads to change in

organization of the granule as a function of temperature and water content. The

crystalline order in starch is often underlying factor influencing its functional properties.

Atwell et al. (1988) reported that collapse of crystalline order within the starch granule

manifests itself as irreversible changes in properties such as granule swelling, pasting,

loss of birefringence and starch solubility. Many techniques like Differential scanning

calorimeter (DSC), X-ray scattering, light scattering, optical microscopy, thermomechanical

analysis (TMA) and NMR spectroscopy have been employed to study these events in an

attempts to understand the precise structural changes underlying gelatinization (Jenkins

and Donald, 1998). DSC has been widely used to study the thermal behavior of starches,

including gelatinization, glass transition temperature and crystallization. Stevens and

Elton (1971) first reported the application of DSC to measure the heat of gelatinization of

17

starch. DSC has been widely used to study the thermal behavior of starches as it helped to

understand phase transition in starch upon heating in the presence of water (Ghaisi et al.,

1982). DSC is a technique whereby the difference in energy input into a substance and a

reference material is measured as a function of temperature while both materials are

subjected to programmed heating or cooling. When a thermal transition occurs, the

energy absorbed by bin the transition, a recording of this balancing energy yields a direct

calorimetric measurement of the energy transition which is then recorded as a peak. The

area under the peak is directly proportional to the enthalpic change (ΔH) and its direction

indicates whether the thermal event is endothermic or exothermic (Karim et al., 2000).

Thermal properties predict the qualities suitable for industrial use. Temperature and the

water content leads to a change in the organization of the granule during gelatinization.

Gelatinization caused a collapse of crystalline order within the starch granules, which

resulted in irreversible changes in properties like swelling, solubility, loss of

birefringence. The point of initial gelatinization and the range over which it occurs are

governed by starch concentration, method of observation, granule type and heterogeneity

within the granule population under observation (Atwell, 1988). Gelatinization occurs

initially in the amorphous regions as opposed to the crystalline regions of the granule,

because hydrogen bonding is weakened in these areas. Amylopectin plays a major role in

starch granule crystallinity; the presence of amylose lowers the melting points of

crystalline regions and the energy for starting gelatinization (Flipse et al., 1996).

Recrystallization of amylopectin branch chains has been reported to occur in less ordered

manner in stored starch gels as it is present in native starches. This explain the

observation of amylopectin retrogradation endotherms at a temperature range below that

for gelatinization (Ward et al.,1994). The variation in thermal properties of starches after

gelatinization and during refrigerated storage may be attributed to the variation in

amylose to amylopectin ratio, size and shape of the granules and presence and absence of

lipids.

2.3.5 Morphology

Starch is laid down in the form of granules that function as an energy reserve. The

granules vary in size and shape with botanical origin. There have been many studies of

18

starch granule structure and these have used a wide variety of techniques. Microscopy

(predominantly optical and scanning electron microscopy) is mainly used for looking at

the whole granule. An examination of these granules under optical or electron

microscopy reveals pronounced concentric rings (French, 1984). The combined repeat

distance of crystalline and amorphous lamellae accounts for the peak observed in small

angle X – ray and neutron scattering experiments (Sterling, 1962; Blanshard et al., 1984

and Oostergetel and Van Bruggen, 1989). Morphological characteristics of starches from

different plant sources vary with the genotype and cultural practices. The variation in size

and shape of starch granules may be due to the biological origin. Granule size reported to

be influence the pasting properties of starch (Aoo and Jane, 2007; Shinde et al., 2003).

The molecular architecture of amylopectin and its molecular arrangement within the

granule is related with granule size (Geera et al., 2006; Jane., 2006; Raeker et al., 1998).

X – ray diffraction has been used to study the crystalline change and to characterize the

transition of crystal structure during starch gelatinization. Starch granules are categorized

based on packing of parallel stranded double helices in the granule, into three types- A, B

and C type. A- type granules are found in cereals like maize, rice, B-type granules are

found in tubers like potato and C- type granules which is a mixture of A and B are found

in legumes. A having close packing and B having loose packing with more amount of

inter- helical water (Cooke and Gidley, 1992).

2.3.6 Dynamic Rheology

Dynamic rheometer allows the continuou

) is measure of the energy stored in the material and recovered from it per

cycle while the loss modulus (G’’) is measure of the energy dissipated or lost per cycle of

sinusoidal deformation (Ferry, 1980). The ratio of the energy lost to the energy stored for

each cycle can be defined by tan δ, which is an another parameter indicating the physical

behavior of a system. The initial increase in G’ could be attributed to the degree of

granular swelling to fill the entire available volume of the system (Eliasson, 1986) and

intergranule contact might from a three- dimensional network of the swollen granules

(Evans and Haisman, 1979; Wong and Lelievre, 1981). With further increase in

19

temperature resulted in decreased G’ indicated the disruption of gel structure during

prolonged heating (Tsai et al., 1997). The of gel structure may be attributed to the melting

of crystalline region remaining in the swollen granules, which deforms and loosen the

particles (Eliasson, 1986).

2.4 Protein isolates characteristics

2.4.1 Functional properties

Proteins are commonly employed as food ingredients on the basis of their

importance in the human diet. The best proteins belong to animal sources since they meet

human nutritional requirements and because they have a suitable functionality. However,

the high cost of animal proteins makes vegetable proteins the main dietary component for

most of the world’s population (Scilingo al., 2002). Amaranth is a dicotyledoneous plant

with a well balanced protein content, and has been proposed as a new alternative source

of high quality protein (Castellani et al., 2000). Amaranth proteins contain acceptable

levels of essential amino acids, particularly lysine, tryptophan, and methionine, which are

found in low concentrations in cereals and leguminous grains of common usage

(Mendoza and Bressani, 1987; Teuto´nico and Knorr, 1985). Among seed proteins, those

of soybean and amaranth stand out because of their high nutritional quality. Their amino

acid compositions are close to the human diet requirements and are complementary

(Bressani, 1994; Liu, 2000). The most promising source is plant proteins that could

substitute either partly or completely animal proteins in human nutrition (Rodríguez-

Patino et al., 2007; Tavano et al., 2008). The main proteins in the grains are represented

by albumins and globulins and to a lesser extent by glutelins (Duarte-Correa et al., 1986).

Contrarily to most common grains, the proteins in amaranth are mainly composed of

globulins and albumins, and contain very little or no storage prolamin proteins, which are

the main storage proteins in cereals, and also the toxic proteins in celiac disease

(Drzewiecki et al., 2003; Gorinstein et al., 2002). Several studies suggest that grain

amaranth derivatives represent interesting ingredients for food formulations and

promissory materials for the development of edible and/or biodegradable films (Colla et

al., 2006; Elizondo et al., 2009; Tapia-Blácido et al., 2005). Structural characteristics of

these proteins influence their functional properties. Protein isolates represent an

20

interesting ingredient for such food formulations to which they must contribute not only

with good nutritional properties, but also with suitable functional properties (Ventureira

et al., 2010). Isoelectric precipitation can also produce certain anti-nutritional factors

(lysinoalanine) and the reduction of protein nutritional quality due to the loss of some

essential amino acids through the beta-elimination reaction (Sarwar et al., 1999). Another

possible adverse effect is the retention and concentration of natural antinutritional

components like phytic acid (Rahma et al., 2000). Avanza et al., (2005) showed that

amaranth proteins were able to form self-supporting gels that could be applied in

different gel-like foods. Ventureira et al., (2012) and Bolontrade et al. (2013) studied

emulsifying and foaming properties of amaranth protein isolates and showed that

improvement in these properties could be achieved by altering pH. Tomoskozi et al.,

(2008) studied the functional properties of amaranth protein fractions and protein isolates

and found that emulsifying and foaming properties were relatively poor in comparison

with casein and soy protein isolates.

The foaming properties of proteins are related both to the processing procedure

and to the protein composition (Kiosseoglou et al., 1999).The most important factor for

the foaming capacity of a protein solution is the velocity at which the protein can reduce

the interfacial tension while a larger surface is being created due to stirring or bubbling

(Graham and Phillips 1976).In order to form foams the protein must reach the interface

quickly to accommodate and change its conformation (Damodaran, 1994); therefore, the

protein must be in soluble form (Walstra, 1989; Yalçin and Çelik, 2007). Flexible protein

molecules showing an important surface hydrophobicity present a higher foaming

capacity, whereas foam stabilitydepends on the protein capacity to develop

intermolecular bonds forming a viscoelastic film (Damodaran, 1997; Utsumi, et al.,

1997). Proteins are good foam stabilizers when their molecules canform a viscoelastic

film in the interface through intermolecular interactions (Damodaran, 1997). Amaranth

proteins have turnedmore soluble with better foaming properties upon

enzymatichydrolysis (Condes, et al., 2009; Scilingo, et al., 2002).

Aceituno-Medina et al. (2013) developed amaranth protein isolate ultrathin

structures using the electrospinning technique. The effects of pH, type of solvent and

21

surfactant addition on the spinnability, morphology and molecular organization of the

obtained structures were also reported. Regarding the effect of pH on API

electrospinning, capsule morphologies were only obtained at extreme pH values (i.e. pH

2 and pH 12), which allowed the solubilisation of the proteins, and the process was

favoured when the solutions were previously heated to induce protein denaturation.

Fibre-like morphologies were only obtained when the solvent used for electrospinning

was hexafluoro-2-propanol, as this organic solvent promotes the formation of random

coil structures and, thus, an increase in the biopolymer entanglements.

Rheological properties such as viscoelasticity and texture are closely related with

microstructure of the matrix gel. The gels of fine-stranded matrix (Foegeding et al.,

1995), are harder and retain more water than those of more open matrices (particulate

gels). The contributions of covalent and noncovalent bonds to gel texture and

viscoelasticity are different in nature. Disulfide bonds usually play an important role in

increasing gel matrix hardness whereas hydrogen and hydrophobic interactions used to be

responsible for keeping network structure (Puppo and An˜o´n, 1998; Zheng et al., 1993).

2.4.3 Fourier transform infrared (FTIR)

FTIR spectroscopy is a well established tool in the determination of protein

secondary structure and can provide valuable information in the development process of

ideal formulations and process parameters (Barth et al., (2002)). FTIR is necessary to

compare the properties of soluble protein fractions (Gorinstein et al., 2005). Gorinstein, et

al, (1996) studied the susceptibility of amaranth globulins (A-G) to chymotrypsin using

FTIR gave a quantitative estimation of protein denaturation in solid state and conclude

that there is disappearance of the α-helix. FTIR spectrum of chymotrypsin and its amide

bands containing information on secondary structure. FTIR is an infrared spectroscopic

technique used to analyze the secondary structure of proteins and peptides based on the

infrared bands in the amide I region (1700–1600 cm−1

) (Surewicz and Mantsch, 1988).

The peptide bonds of the protein give rise to three major signals within the FTIR

absorbance spectrum, referred to as the amide I, II and III bands. The amide I band (1700

cm-1

- 1600 cm-1

) is used by most authors for the determination of secondary structure as

it is built up by few molecule vibrations with plenty of information available in literature

22

(Susi et al. 1985; Dong et al. 1995; Carpenter et al. 1998). Characteristic peaks for the

amaranth protein isolate were identified at around 1634 cm-1

and 1533 cm-1

, which

correspond to the amide I and II regions respectively (Aceituno-Medina et al. 2013). The

absorption peak atw1634 cm-1

can be attributed to the stretching of the C=O (Amide I)

while the peak at 1533 cm-1

is due to stretching of C-N and bending of N-H (Amide II).

Amide I band has been widely used to study protein folding, unfolding and aggregation

with infrared spectroscopy due to its sensitivity to secondary structure of proteins. The

amide II band (1600 cm-1

-1500 cm-1

) is mostly employed for evaluating the accessibility

of the protein backbone by H-D exchange (Haris et al. 1990; Wu, 2001), while the amide

III band (1330 cm-1

-1220 cm-1

) is only seldom utilized for structural evaluation due to its

weak intensity and complex composition (Costantino et al. 1995). The amide I band

(1700–1600cm-1

) is often used in literature as it is based on only few molecule vibrations

and sufficient information for cross-referencing is available (Carpenter et al., 1998). The

spectrum of the amaranth protein isolates powder (Aceituno-Medina et al. 2013) showed

bands at 1634 (strong) and 1692 cm-1

(weak), which are characteristic of β-sheet

structures. After electrospinning of proteins, the amide I band considerably broadened

indicated a greater conformational freedom of the protein chains (Barth, 2007). Kaiden et

al. (1987) applied IR spectroscopy to the study of the secondary structure of proteins and

found that heat or denaturation treatment resulted in the alterations of amide I, II, and III

bands. One wave number or wavelength in FTIR includes the large majority of the

information correlated with secondary structure content and no more than 3 significant

independent wave numbers/wave lengths could be found for any of the spectroscopic data

(Goormaghtigh et al., 2009).

2.4.4 Sodium dodecyl sulphate (SDS) gel electrophoresis (PAGE)

Sodium dodecyl sulphate (SDS) gel electrophoresis (PAGE) is a low cost,

reproducible and rapid method for quantifying, comparing and characterizing proteins.

The structural characteristics of proteins have been investigating using SDS-PAGE. This

method separates proteins based primarily on their molecular weight (Laemmlli, 1970).

SDS binds to hydrophobic portion of protein, disrupting its folded structure and allows it

to exist stably in solution in extended conformation. As a result, the length of SDS-

23

protein complex is propotional to its molecular weight. Slab gels have become more

widely used tube gels, since many samples can be run on the same gel. It the most

common method of electrophoresis in practice suitable for distinguishing closely related

rice varieties as well as the other investigated plants, was used (Edwards et al., 2004),

Gorinstein et al. (2005) found in SDS analysis that The low resolution of bands in the

region of low molecular weight (<20 kDa) and interpret that SDS–PAGE profiles in this

region are weak and not differentiate for proteins of different varieties of amaranth. The

identification of bands corresponding to the main protein fractions of amaranth in

Amaranth deffated flour protein profile is difficult because of the overlap of common

bands between the different fractions as reported by several authors. Even if the different

fractions were separated, the resolution was not improved (Martí-nez and Añón 1996).

Using SDS-PAGE electrophoresis,20 to 22 bands of rice, 29 of sorghum, 36 bands of

soybean seeds, 35 of buckwheat and from 28 to 39 bands of Amaranth species,

respectively, were detected (Gorinstein et al., 2005). The amaranth protein isolate was

reported to be consisted of a mixture of different proteins with molecular weights ranging

from around 10 to 83 kDa (Aceituno-Medina et al., 2013). Under denaturing conditions

(SDS-PAGE) the native isolate exhibited a pattern typical of an amaranth isolate

(Martínez et al., 1997) composed of high molecular weight soluble aggregates that did

not enter the gel, and polypeptides corresponding to the 7S fraction, to the 11S fraction,

and to the globulin Protein fraction naturally present in amaranth seeds (Castellani et al.,

2000; Marcone, 1999; Marcone et al., 1994).

2.5 Flour film characteristics

The agricultural biopolymers can be used for the development of edible and

biodegradable films. These films can provide an opportunity to increase their

applications, add value addition and contribute to decrease the environmental pollution

by substituting nondegradable synthetic plastic in food and pharmaceutical applications.

These biopolymers have been used to prepare edible films and coatings that can be used

in food protection and preservation. Their utility depends upon their capacity to act as an

adjunct for improving overall food quality, extending shelf-life, and possibly improving

cost–benefit of packaging materials (Guilbert, 1986; Kester and Fennema, 1986; Petersen

24

et al., 1999). The incorporation of plasticizers such as glycerol is necessary to reduce

polymer intermolecular forces, increasing the mobility of the polymeric chains, and

improving the mechanical characteristics of the film, such as the film extensibility

(Krochta and Sothornvit, 2001; Mali, 2002). Polysaccharides and proteins have good gas,

aroma, and lipid barriers and can also adhere to fruits or vegetable cut surfaces. However,

they are inefficient against water transfer because of its hydrophilic characteristics. Lipids

impart high water barrier properties and form brittle films causing anaerobic conditions at

higher storage temperatures and not sticking to hydrophilic cut surfaces (Peroval et al.,

2002 and Kester et al., 1988). The amaranth flour from Amaranthus caudatus has been

reported to have good film forming ability, thereby yielding films with excellent barrier

properties with respect to water vapor, moderate solubility, and high flexibility (Tapia-

Blácido et al., 2005). These properties were contributed from the balance between the

concentration of biopolymers and lipids and their natural interaction in the flour, which

prevents phase separation (Tapia-Blácido et al., 2007). The increase in non-biodegradable

waste material and the difficulty in recycling most of the available synthetic packaging

have been stressing to conduct research toward the development of new biodegradable

materials that were suitable for packaging (Davis and Song, 2006; Marsh and Bugusu,

2007). Various natural biodegradable polymers such as proteins, polysaccharides etc have

potential application in the production of environmentally-friendly packaging (Chandra

and Rustgi, 1998; Krochta and De Mulder-Johnston, 1997). The amaranth flour was

recently used as raw material for the production of edible films and coatings, still on a

laboratory scale (Colla et al., 2006; Tapia-Blácido et al., 2007; 2005a; 2011). Amaranth

flour films were obtained in a casting process using glycerol as plasticizer and it was

determined that glycerol content, pH, and process temperature were significant factors

affecting mechanical and barrier properties. The biofilms were characterized by a

yellowish color, moderate opacity, high flexibility, and low tensile strength; however,

they had less oxygen and water permeability than other polysaccharide and protein films

(Tapia-Bl´acido et al., 2005a). Amaranth flour films were reported to have interesting

mechanical and water vapor barrier characteristics that were later attributed to the

interactions formed between their polymers (starch and proteins) and lipids, to the

distribution of these interactions within the film matrix and to the natural concentrations

25

of each component in the film (Tapia-Blácido et al., 2007). The potential of edible films

made from a variety of materials to control water transfer and improve food quality and

shelf life were reported to be quite interesting (Krochta et al., 1997 and Koelsch, 1994).

The production of biodegradable and edible films from carbohydrates and proteins adds

value to low-cost raw materials and can play a significant role in food preservation

(Averous et al., 2001; Krochta and Miller, 1997). Protein films are made of raw materials

of high molecular weight such as gelatin, myofibrilar proteins, gluten, whey protein

among others. These films are characterized by good mechanical properties, although

they are usually quite permeable to water and gases (McHugh and Krochta, 1994).

Starches from different origins, such as potato, corn, wheat, rice, and cassava, both

natural and modified, have been utilized, mainly in the manufacture of edible films

(Lourdin et al., 1995; Ollett et al., 1991; Vicentini et al., 2002). The matrix of starch-

based film is normally formed during the drying of a gelatinized dispersion, as hydrogen

bonds form between hydroxyl groups (Lourdin et al., 1995). The incorporation of

plasticizers is necessary to reduce polymer intermolecular forces, increasing the mobility

of the polymeric chains, and improving the mechanical characteristics of the film, such as

the film extensibility (Krochta and Sothornvit, 2001; Mali, 2002). Glycerol is a

hydrophilic plasticizer, and when added at the correct level with respect to the

biopolymer content, can interfere with chain to chain hydrogen bonding and the water

solubility of the biopolymer (protein/starch mixtures), a process generally used to

improve the mechanical properties of edible films (Sobral et al., 2001; Sothornvit and

Krotcha, 2001). Edible plasticized films are thin, flexible materials made from

biopolymers and capable of forming a continuous matrix by adding food grade

plasticizers. Zein-glycerol films were reported to have good tensile strength of 27 MPa

(Singh et al., 2009).

2.6 Extrusion cooking

Extrusion cooking is used worldwide for the production of expanded snack foods,

modified starch, ready to- eat cereal foods, pet foods and porridge (Frame, 1994; Harper,

1981; Smith and Singh, 1996). Extrusion cooking is a low cost, high temperature short

time (HTST) process, used worldwide for processing of a number of food products

26

(Frame, 1994) including snacks, ready to eat (RTE) cereals, confectioneries and crisp

breads (Suknark et al., 1997). In this process, the food materials are mixed, wetted,

melted and cooked before being forced through a die to obtain products of definite shape,

size and porosity (Rahman et al., 2002). During extrusion, gelatinization of starch,

denaturation of protein, modification of lipid, development of the Maillard reaction and

inactivation of enzymes, microbes and many antinutritional factors (Bhattacharya and

Prakash,1994; Rufián-Henares et al., 2006a) all take place simultaneously. Extrusion

variables, composition of feed material, particle size distribution and additives,

significantly affect extrusion parameters and product properties (Ryu et al., 1993; Singh

et al., 1998; Singh et al., 1999; Singh and Smith, 1997). Cereal grains are generally used

as major raw materials in extruded snack foods due to their good expansion

characteristics. Physical characteristics of an extruded snack product such as expansion,

hardness and density are important parameters in terms of the consumer acceptability of

the final product, as well as its functional properties (Launay and Lisch, 1983; Jamora et

al., 2002; Tahnoven et al., 1998; Wagner, 1989). Corn meal is a major ingredient for

extruded foods, such as ready-to-eat breakfast cereals and snacks. The effect of various

process variables on extrusion behaviour of corn grits have been extensively studied

(Fletcher et al., 1985; Hsieh et al., 1990; Singh et al., 1998). De Muelenaere and

Buzzared (1969) reported the extruded degermed corn grits and whole corn meal and

found that degermed corn grit had much greater expansion than whole corn. Zhang and

Hoseney (1998) reported the extrusion behavior of corn meal with poor and good

expansion properties. Extrusion cooking is a versatile and very efficient technology,

widely used in grain processing. There is a trend in the food industry to develop

convenience products, such as puffed snack foods and breakfast cereals, of high

nutritional value. Interest in amaranth grain has increased in recent years because of its

nutritional components, particularly its high protein and lysine content (Bressani et al.,

1994.). Extrusion cooking increases the availability of proteins or nutrients in the

amaranth grain, and the available lysine remains the same as in the raw material

(Mendoza and Bresani, 1987). Extrusion cooking process for A. cruentus and A. caudatus

resulted in the protein nutritional quality that was comparable to casein (Mendoza et al.,

1987). Extruded amaranth grain exhibited better nutritional value than raw amaranth and

27

the product required no additional cooking prior to consumption (Bressani et al., 1992).

Vargas-L´opez et al. (1991) investigated the extrusion cooking of amaranth under

alkaline conditions and concluded that the processed amaranth flour may be used for

tortilla preparation. It is difficult to directly produce expanded products by extrusion

cooking of amaranth grain alone due to their high fat content. Fat provides a powerful

lubricant effect in extrusion cooking and reduces product expan sion. Fat also modifies

the eating qualities of the extruded product (Guy, 1994). Ilo et al. (1999) reported that

additional starch must be used in extrusion cooking of amaranth to improve extrudability

and product properties. Therefore, extrusion cooking of amaranth in combination with

nutritionally complementary cereal grains such as rice is of even greater interest, because

it can be used to produce nutritionally balanced products in the well-accepted form of a

puffed extrudate. Snack foods with good acceptance and high nutritive value have been

developed by extrusion cooking of the defatted flour obtained from milling the

grain(Cha´vez-Ja´uregui et al., 2000). The extrusion cooking process is based on starch

gelatinization and protein denaturation using high pressure and high temperature (Areˆas,

1996). As well as its high acceptability (Cha´vez-Ja´uregui et al., 2000), such amaranth

snack foods also present characteristics such as cholesterol-lowering effects in

hypercholesterolemic rabbits (Plate and Areˆas, 2002), protein of high biologic value, and

high bioavailability of calcium, zinc, and magnesium (Ferreira, 1999). For patients

suffering from celiac disease gluten-free products are required and extrusion cooking is a

suitable process for producing gluten-free expanded snack foods since, unlike bread,

starch is the main component providing the desirable expanded structure in extruded

snack foods (Acs et al., 1996 and Chartrand et al., 1997). Direct-puffed snacks made by

extrusion process are classified as a second-generation snack. They are usually low in

bulk density and are often marketed as high-fiber, low-calorie, high-protein and

nutritional product (Liu et al., 2000). Sensory attributes of extrudates depend on

processing conditions and raw material composition. The interactions are so complex that

differentiation among the influences of individual variables on the changes in final

product characteristics is impossible (Meuser et at., 1984). The acceptance of snacks is

critical because of the specific quality attributes that attract people. The various sensory

attributes and of snack foods are appearance, texture, taste, colour and flavour. Such

28

products possess a classical brittle failure mechanism as a consequence of their cellularity

and lack of structural resiliency. They are often described as crunchy because of a

complex failure mechanism that involves the repetitive deformation and fracturing of the

cell structure (Barrett et al., 1994). The success or failure of a new extruded snack food

product is directly related to the sensory attributes and texture is the mostimportant

attribute (Anton and Luciano, 2007). Extruded amaranth grain products have specific

aroma and can be used as snack food, supplement in breakfast cereals, or as raw material

for further processing (Breene, 1991). Sanches– Marroquim et al., (1986) investigated

extruded blends of Amaranth with wheat and oats flour. The optimal combination of

these components, to their opinion is 5050׃, or 6040׃, respectively. Ramos Diaz et al.

(2013) showed that it was possible to prepare expanded gluten-free corn-based extrudates

containing amaranth, quinoa and kañiwa flour (20% of solids). The SEI was the highest

in extrudates containing amaranth and the lowest in pure corn extrudates. A study of

extrudates containing various contents of amaranth, quinoa or kañiwa may answer

questions on the interaction of biocomponents during extrusion and their effect on SEI

and hardness. They suggested that their investigation was a step forward in the

understanding and developing of novel gluten-free snack products.

2.7 Popping characteristics

Amaranth seeds can be popped by heat. Popping is a very attractive technology

for processing amaranth seeds, because of the easy way of processing and the pleasant

flavor of the end product (Bressani et al., 1992). The popping process on a hot plate has

been used for centuries and is the oldest way of popping amaranth grains (Lara and

Ruales, 2002). The dry seeds can be popped if they are exposed to temperature close to

200°C (Fuente and Tovar, 1995). Heat causes evaporization of the water contained in the

starch matrix. The steam produced fills the pores of the starch granules, increasing the

temperature and pressure. In this phenomenon, the starch granules swell and rupture the

pericarp, leading to the expansion of starch granules. The endosperm is transformed into

a bubbly matrix, which solidifies through the evaporation of water, yielding a spongy

structure (Schwartzberg et al., 1995; Lara and Ruales 2002). Amaranth seeds are

29

traditionally consumed after puffing. The seeds are spherical in shape with an average

diameter of about 1 mm, and are beige in color. Amaranth is also consumed by roasting

the flour or by grinding the seeds and extruding the flour. Candies and snacks made out

of puffed seeds are nutritious and widely consumed in Asia and South America (Singhal

and Kulkarni, 1988). Conventionally amaranth seeds are puffed over a hot skillet or using

fluidized bed heaters (Tovar et al., 1994) both of which are batch processes. Expanded

amaranth, obtained from the puffing operation, is commonly used as ready-to-eat

breakfast foods or as ingredients in snack formulation (Hozova et al., 1997). The puffing

operation of cereals consists on the sudden application of heat at atmospheric pressure so

that the water is vaporized inside the grain, reaching very high internal pressures (Song

and Eckhoff, 1994). The external tissue was broken and the grain was expanded, forming

the endosperm foam attached to fragments of pericarp and embryo (Hoseney et al., 1983;

Reeve and Walker, 1969). The most important quality parameter of the popped product

was reported as expansion volume which was influenced by the compositional

characteristics of the raw grain (Chen and Yeh, 2001) and the processing conditions

(Chandrasekhar and Chattopadhyay, 1990). Toasted and popped amaranth seeds are

reported to have a distinctive flavor, which is described as nutty, slightly toasty and sweet

(Pszczola, 1998). Popped amaranth seeds are mixed with milk and/or honey or syrup in

order to make confections. Review of literature indicated that a limited work on

functional properties of leaves and seeds of different amaranthus lines has been

conducted.