9

Chapter 2

REVIEW OF LITERATURE

2.1. PECTIN

2.1.1. Introduction

Pectin plays an important role in food processing as food additives and as a source

of dietary fiber. Pectin gels are very important in creating or modifying the texture of

jams, jellies, confectionary and in low fat dairy products. They are also used as

ingredients in the pharmaceuticals industry and lower the glucose response. In order to

understand their type and content, pectins are separated based on their solubility by

sequential extraction in water or buffer solutions, solutions of chelating agents, dilute

acids, or dilute sodium hydroxide or sodium carbonate. It is also considered a safe

additive with no limits on acceptable daily intake (FAO, 1969; Gnanasambandam &

Proctor, 1999). Factors affecting the functionality of pectins include composition, degree

of methylation, solubility, pH, temperature and presence of soluble solids. Good quality

of pectin based on the high degree of esterification and intrinsic viscosity with low acetyl

content. Degree of methylation is related to the rate of gel formation. High methoxyl

pectins gel in the presence of sugar gel but low methoxyl pectin gel in the presence of

calcium. Gel strength depends on the length of molecule. At very low molecular weight,

pectin is unable to form gels under conditions (Pagan et al., 1999). Although pectin

occurs commonly in most of the plant tissues, the number of sources that may be used for

the commercial manufacture of pectin is limited. Various sources of pectin include citrus

peels, dried apple pomace, sugar beets, sunflower heads, residues of mango, guava,

papaya, coffee and cocoa processing. Currently half of the commercial pectins used in the

food industry are extracted from citrus peels (Voragen et al., 1995). Citrus pectins are

light cream to light tan in color whereas apple pectins are often darker. Gnanasambandam

& Proctor (1999) isolated pectins from soy hull, a co-product of soyabean processing

using nitric acid as well as enzymes. Enzymatic pretreatment of soyhulls increased

content of alkali soluble pectins as well as X-ray diffraction intensities. Studies showed

10

that enzyme pretreatment improved the galacturonic acid content of pectins prepared

using conventional acid extraction process. Pectins are natural ionic polysaccharides with

many applications in food and pharmaceutical industry because of their ability to form

gels in the presence of divalent cations such as calcium (Braccini & Perez, 2001). Pectin

isolated from sugar beet pectin differs from other pectins in that it has higher branched

region in the form of acetyl content, which make sugar beet pectin unable to form gel.

Pectins also contain a small amount of protein around 2%, which make used as a

emulsion stabilizer also (Siew & Williams, 2008). Pectin is present not only in the

primary cell walls but also in the middle lamella between plant cells where it helps to

bind the cells together. The amount, structure and chemical composition of the pectin

differs between plants, within a plant over time and in different parts of a single plant.

During ripening, pectin is broken down by the enzymes pectinase and pectin esterase,

resulting in the process where the fruit becomes softer. This is because the middle lamella

that primarily consists of pectin breaks down and cells become separated from each other.

Pectin is thus also a natural part of human diet but does not contribute significantly to

nutrition. In human digestive system, pectin escapes the digestion in small intestine but is

acted upon by microbial growth of large intestines. Therefore, it also acts as soluble

dietary fiber. Pectin also reduces cholesterol absorption by increasing the viscosity in the

intestinal tract (Srivastva & Malviya, 2011). Pectin is a linear polysaccharide consisting

of a few hundred to one thousand saccharide units. The average molecular weight of

pectin varies from 50,000 to 150,000 (Whistler & Bemiller, 1997). It consists of D-

galacturonic acid units with very small amount of neutral sugars. The poly galacturonic

acid is partly esterified with methyl groups and the free acid groups may be partly or fully

neutralized with sodium, potassium or ammonium ions. (Monsoor et al., 2001).

Depending on the pectin source and the extraction mode, carboxyl groups are partially

esterified by methanol and in some cases, hydroxyl groups are partially acetylated.

Neutral sugars such as galactose, glucose, rhamnose, arabinose and xylose may also be

present bounded to the galacturonic acid as side chains and inserted into the main chain.

(Rolin & De Vries, 1990). Depending upon the degree of esterification, pectins are

divided in two categories: high-ester pectin with DE higher than 50% and low-ester

pectin, with DE lower than 50% (Thakur et al., 1997). In HMP, the gel is formed by

11

building a junction zone resulting from the cross-linking of homogalacturan through

hydrogen bond and the hydrophobic interaction between methoxyl groups. In low-ester

pectin, junction zones are formed by calcium cross-linking between free carboxyl groups

(Willats et al., 2006). Pectin is also used in fillings, sweets, as a stabilizer in fruit juices

and as a source of dietary fiber. In nature, around 80% of carboxyl groups of galacturonic

acids are esterified with methanol. This proportion is although reported to decrease more

or less during pectin extraction. The ratio of esterified to non-esterified galacturonic acid

determines the behavior of pectin in food applications (Srivasatva & Malviya, 2011).

2.1.2. Pectin Isolation

The extraction of pectin involves the hydrolysis of insoluble protopectin into

soluble pectins and then leaching them out of the fruit tissues. Several methods have been

reviewed for the hydrolysis. Extraction with hot water is the simplest and oldest method

for recovering pectic substances from plant tissues (Hermann (1919)). A wide range of

reagents could be used for the extraction of pectin. The most commonly used are mineral

acids such as sulphurous acid, sulphuric acid, hydrochloric acid, phosphoric acid and

nitric acid. HCl is most widely used because it is cheap and high yield of pectin obtained

(Sudhakar, 1991). Francis & Bell (1975) reviewed the commercial pectins and postulated

that its anhydrouronic acid content, methoxyl content, degree of esterification, jelly grade

and jelly units, mainly judges the quality of pectin. Anhydrouronic acid content indicates

the percentage of other organic material present while ash content represents the

inorganic impurities. In order to understand their type and content, pectins are separated

based on their solubility by sequential extraction in water or buffer solutions, solutions of

chelating agents, dilute acids or dilute alkalis. Enzymes are also used in pectin extraction

i.e. endo-polygalacturronase or combination of pectin esterase/endo-polygalacturonase

and endo-arabinase and endo-galactanase. Less degrative non-pectolyic enzymes might

be useful in preparation of pectin ingredients in their native state without altering the

properties (Gnanasambandam & Proctor, 1999). Sakai & Okushima (1980) also prepared

pectin from citrus peels using. In this method, a strain of Trichosporon penicilatum (a

protopectin degrading enzyme producer) was used. Microorganism was added in the

citrus peels and fermentation was allowed for a period of 15-20 hrs at 30 °C. 20-25 g

12

pectin / kg peels were obtained having high amount of neutral sugars. Prickly pear fruit

skin was used by Habibi et al. (2005) to isolate pectin by acid extraction followed by

fractionation. Fractionation was done to characterize the pectin structure using Anion

exchange chromatography as well as size exculsion chromatography. Results revealed

that alcohol soluble pectin 1 fraction was neutral having linear β- galactan. One of the

acid fractions alcohol soluble pectin 2 with galacturonic content consisted of repeating

disaccharide units. Alcohol soluble pectin 3 showed high galacturonic acid content

having alternate homogalacturonan blocks and rhamnogalacturonana blocks with same

amount of galactopyranosyluronic acid residues in each block. Researchers have also

extracted pectin from sunflower heads by alkali and optimize the extraction process using

response surface methodology. Most important factors found out to be temperature and

washing time of sunflower heads, pH, temperature, washing time and solvent: solid ratio

and their interactions significantly affected pigment removal. It has been observed that

slightly alkaline solution and lower solvent: solid ratio can be used to speed up the

pigment removal without affecting the pectin quality and yield (Shi et al., 1996).

Microwaves are also used by some researchers to extract pectin from orange peels

(Kratchanova et al., 2004). Orange peels were exposed to microwaves for different

duration of 5-15 minutes with different power of 0.45 to 0.9 kW. Pectin was then

extracted using HCl. Microwave pretreatment of peels led to destructive changes in the

plant tissues. The changes resulted in an increase in the capillary porous characteristics

and the water absorption capacity of the plant material. Theses changes led to increased

pectin yield and improve its characteristics by improving the water absorption capacity of

peels. Mango peels were studied to evaluate the impact of different extraction conditions

on the yield and some biochemical characteristics of mango peels pectins in order to

access the feasibility of using mango peel as a source of pectin (Koubala et al., 2008).

Isolated pectin was then compared with lime pectin. Extraction conditions shown to have

a deep impact on the extraction yields and on the biochemical and macromolecular

characteristics of the extracted pectins. Ammonium oxalate led to high extraction yields

and proved to be an outstandingly interesting extractant. HCl also led to high extraction

yields but the extracted pectin was partly degraded. Water came out as a poor extractant

with respect to extraction yields. Whatever the extraction method used, the pectins

13

recovered were highly methylated. High methylated pectins from gels with high amounts

of sugar and acid. Many factors influence the conditions of gel formation and the gel

strength achieved but under equal conditions, gel strength increases with the molar mass

of pectin used (Voragen et al., 1995). Ammonium oxalate extracted pectins are thereby

most likely to exhibit good gelling properties i.e. high average molar mass, intrinsic

viscosity and a high degree of methylation. Extraction process was developed by

Turquois et al., (1999) to extract pectin from sugar beet pulp and potato pulp with high

gelling properties rather than yield. Rheological measurements were done to study the

effect of concentration of the extracted product, calcium content, sequestrant effect and

hydration temperature. Study included acidic and alkaline procedures for pectin

extraction. Extraction process developed maintains the structural integrity of the pectins

as much as possible. Theses procedures yielded products, which possessed both high

pectic substance content with low degree of esterification and a high gelling ability in the

presence of calcium. Sugar beet and potato pulps could be used as new sources of

pectins. Low quality apples were also used by Rascon-Chu et al., (2009) to extract pectin

and determine their gelling ability using acid extraction. Extracted pectin was then

characterized for composition and functional properties. Studies revealed that low quality

apple resulted in pectin with high galacturonic acid content, high intrinsic viscosity

allowing the formation of firm physical gels. Concentration of the pectins in a system

also affected the textural properties of gels. Hardness and adhesiveness found to be

increased with increase in pectin concentration. Therefore, the pectin recovered could be

used as a food additive to texturize or stabilize different food products. A new method

was developed by Cho et al., (2003) in which pectin was concentrated efficiently

decreasing the amount of ethanol need for the precipitation of pectin. Cross-flow

filtration was used to concentrate and purify isolated pectin. Cross microfilteration,

effectively concentrated pectin extracts which saved 75% of ethanol consumption

required for pectin precipitation. Undesirable impurities were also effectively removed.

2.1.3. Properties of Pectin

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Alkali salts of pectinic and pectic acids are usually water soluble in water. Dry

powdered pectin has tendency to hydrate very rapidly and form clumps on addition of

water. These clumps consist of semi dry packets of pectin contained in an envelope of

highly hydrated outer coating. Further solubilisation of such clumps is very slow. Dilute

pectin solutions are Newtonian but at moderate concentration, they exhibit non-

Newtonian, pseudo plastic behavior characteristics. Factors that increases gel strength

will increase the tendency to gel, decrease solubility and increase viscosity and vice-

versa. These properties of pectins are a function of their structure. Solutions of

monovalent salts of pectins exhibit stable viscosity because each polymer chain is

hydrated, extended and independent. Reduction in the pH reduce the ionization, the

polysaccharide chains no longer repel each other over their entire length and can

associate and form a gel. Pectins are mainly used as gelling agents but can also act as

thickener, water binder and stabilizer. Low methoxy pectins form thermo reversible gels

in the presence of calcium ions at low pH whereas high methoxyl pectin rapidly form

thermally irreversible gels in the presence of sufficient sugars such as sucrose and at low

pH. The lower the methoxyl content, slower is the gel set (Kohn, 1982). Effect of

chemical and physical modification on the thermal behavior of pectins was studied by

Einhorn-Stoll et al., (2009) to understand the physical state and state transitions resulting

from structural changes during preparation and processing of pectin materials. It has been

observed that degradation parameters varied with the degree of modification. All

chemically modified pectins were more sensitive to thermal degradation as compared to

their native materials. Transition temperatures as well as weight loss found to be

decreased with increased degree of modification. Very different behavior has been

observed in case of mechanically degraded pectins that showed starting of thermal

degradation earlier and ended later with decreased molecular weight. Pectin origin also

influenced the thermal degradation. Chemical changes in the cell wall as well as turgor

changes has been directed towards characterization of changes in pectic substances

during processing and storage operations, Pectins can be demethylated and

depolymerised by both enzymatic and non-enzymatic reactions. A simple model has been

proposed by Chang et al., (1993) to study the basic interaction between pectin molecules

and other cell-wall constituents effecting chemical and textural changes of vegetable

15

tissues during different cooking treatments. Krall & McFeeters (1998) studied the effect

of temperature, degree of methylation, pH and calcium on the hydrolysis of pectin. It has

been concluded that rate of pectin hydrolysis decreased at pH 3. Dynamic light scattering

study had been carried out by Kjoniksen et al., (2004) to study the temperature-induced

association and gelation of aqueous solutions of low methoxy pectin at different

temperatures and polymer concentrations. Results revealed that as the temperature

decreased formation of association complexes promoted and strengthened with increased

polymer concentration. Slow relaxation time for the highest polymer concentration rises

strongly with decreasing temperature suggested enhanced polymer chain association. At

low temperature of 10 °C, gelation occurs in the semi dilute regime and a transparent gel

is formed. Low temperature stabilized the association complexes and the gel net work

through intermolecular hydrogen bonds, which are broken-up at higher temperatures.

Panchev et al., 1989 proposed a model to describe the kinetics of apple pectin extraction

including the dissolution of pectin from protopectin and the degradation of dissolved

pectin. Minkov et al., (1996) also proposed a mathematical model to study the hydrolysis

of solid-phase protopectin to solid-phase pectin and the transformation of solid-phase

pectin into liquid-phase pectin. Cho & Hwang (2000) proposed mathematical model to

evaluate yield and intrinsic viscosity of pectin in acidic solubilisation of apple pomace.

Using experimental data, yield and intrinsic viscosity models were determined as forms

of an Arrhenius-type equation and an exponential function of temperature. Comparative

study on functional properties of beet and citrus pectin was studied by Mesbahi et al.,

(2005). The highest yield of pectin was obtained at pH 1, temperature 90° C with

extraction periods of 4 hrs. Results indicated that the extracted beet pulp pectin could be

used in certain foods such as ketchup tomato sauce as a thickener or as an agent

increasing the viscosity. Beet pectin did not have good gelling properties in the food

products and could not produce a strong network to trap free water as much as citrus

pectin does. However, beet pectin showed higher hydrodynamic volume when dissolved

in water therefore can be used as a thickening agent. Degradation of pectins in alkaline

was studied by Renaurd & Thibault (1996) at mild alkaline pH and temperatures between

15-45 °C and kinetic constants of pectin saponification was reported by the liberation of

methanol. At higher pH a marked deviation was observed from the expected first order

16

kinetics with respect to methyl-esterified carboxyl groups. Deviation may be attributed to

the changes in pH that occurred during saponification. Physicals gels i.e. low methoxyl

pectins were evaluated for their rheological properties by Gigli et al., (2009). Influence of

ionic strength and temperature on the gelation of low methoxyl pectin was studied.

Studies revealed that gel transition is very sensitive to the ionic strength of the medium

while the viscoelastic properties of the gel structure were retained up to 60 °C. The

mechanical spectrum of low methoxyl pectin gel approaches the beginning of the

terminal region where the rheological behavior is mainly dominated by viscous flow, thus

indicating a liquid like character of a material and thus accounting for the large

proportion of solvent contained in the network. Effect of ultrasound waves on the acid

desertification of low methoxyl pectin was studied by Panchev et al., (1994). Treated

samples were then evaluated to study the effect of temperature, time and nitric acid on the

yield and quality of low esterified pectins. Ultrasonication of the treated material resulted

in pectin with decreased degree of esterification and higher yield. Optimum ultrasound

treatment proved to be within 24-30 minutes.

2.1.4. Pectin Modification

When a substantial portion of the methyl ester groups is removed by hydrolysis,

the modified pectin attains the ability to form uniform strong gels in the presence of

bivalent cations over a wide pH range. This property makes low-methoxyl pectin useful

for numerous applications in which ordinary pectin cannot be used (Graham & Shepherd,

1953). Modification of the pectins is done to increase their reactivity by partial hydrolysis

of the ester groups. Pectins can modified by saponification catalyzed by mineral acids,

bases, slats of weak acids, enzymes, and concentrated ammonium systems. The acid and

base hydrolysis is the simplest efficient procedures for modifying pectins. Modification

decreased the methoxyl content and increased the free carboxyl groups content of the

pectin after saponification. Chemical modification also increased the sorption capacity of

the pectins for heavy metals simulating the electrolyte composition of human body

(Kupchik et al., 2006). Low methoxyl pectin found its application in the making of jellied

fruit cocktails, low-solids gels, milk puddings, candy centers and coatings for various

food materials. Low methoxyl pectins are versatile materials, which make possible the

17

preparation of many new food products and the preparation of old food products in new

or easier ways (Graham & Shepherd, 1953). Pectins with higher degree of esterification

will gel at higher pH because they have fewer carboxylate anions at any given pH.

Dissolved pectins are decomposed spontaneously by de-esterification as well as by

depolymerisation. Rate of degradation depends on pH, water activity and temperature.

Maximum stability is found at pH 4. At low pH values and elevated temperatures,

degradation due to hydrolysis of glycosidic linkages is observed. Deesterification is also

favored by low pH. De-esterification of high methoxyl pectin slows the setting and

gradually adapts the characteristics of low methoxyl pectin. High temperature along with

high temperature favors elimination process that results in loss of viscosity and gelling

properties. At alkaline pH pectin is rapidly de-esterified and degraded even at room

temperature. Powdered high methoxyl pectin slowly lose their ability to form gels if

stored under humid or warm conditions while low methoxy pectin are more stable and

loss should not be significant after one year storage at room temperature (Srivastva &

Malviya, 2011). Khondkar et al., (2007) have studied rheological behaviors of starch and

pectin gels. Study was conducted to observe the influence of pectin on starch properties.

Starches were mixed wit hydrocolloids to improve their rheological properties (Descamp

et al., 1986). Cross linked of pectin and starch affected the rheological properties by

increasing the elasticity of starch gels. Cross linked starch-pectin mixtures (2:3 and 3:2)

showed quite high storage and loss moduli indicating that these gels have greater degree

of elasticity and very well structured.

2.1.5. Applications

Starch films and coatings have been used for various food and pharmaceutical

applications. Native and modified starches can be used for making edible films and

coatings. Films prepared from starches are isotropic, odorless, tasteless, non-toxic and

biodegradable. The starch films have low oxygen permeability, starch coatings are

nutritious, safe and economic and have been used in the storage and marketing of foods.

It has been observed by various researchers that starch films are similar to plastic films in

various properties like physical characteristics, chemical resistance and mechanical

properties. Maize starch was used to demonstrate a novel method, which enables the

18

preparation of starch films and coatings with good thickness control. 5% starch was

gelatinized in de-ionized water at 120 °C for 30 min with small addition of dispersant and

ethanol followed by ultrasonication to stabilize starch solution (Pareta & Edirisinghe,

2006). Pectin-starch blends are also used for making films. Theses films exhibit

extremely good modulus and tensile properties. Such films are biodegradable, recyclable

and may help satisfy increasing consumer and regulatory demands for materials with

these properties. To take the advantage of its polymeric properties, starch has been

suggested that starch must be gelatinized to disintegrate granules and overcome the

strong crystalline intermolecular forces before mixing with pectin. The disruption of the

starch granules and the resulting degree of solubilisation are highly dependent on the time

and temperature conditions to which the starch is exposed (Coffin et al., 1995). A

potential industrial use for the pectin/starch blends includes edible bags for soups and

noodle ingredients (Fishmann et al., 2000). To protect sensitive functional compounds

and deliver them safely to the intestine and colon, various edible coatings find attractive

approach for the food industry.

Pectin coating was also utilized to enhance spray-dry stability of pea protein-

stabilized oil-in-water emulsions by Gharsallaoui et al., (2009). Effect of drying on the

physical stability of oil-in-water emulsions containing pea protein-coated and pea

protein/pectin-coated droplets has been studied. It has been observed that pea protein-

pectin coating developed provide superior stability to oil droplets in terms of ageing and

pH changes, which may be due to increased steric repulsion by pectin that formed a less

charged protective layer around the protein interfacial film surrounding the oil droplet.

Starch has been found to be potential alternative to commonly used coatings that escapes

digestion in small intestines. The combination of pectin with starch is a potential formula

for food grade coatings. Dimantov et al., (2004) mixed pectin with high amylose

cornstarch to prepare coatings. Coatings were then evaluated for their surface

characterization and dissolution properties. It has been observed as the amount of

cornstarch increased in the coating system the roughness of the film increased whereas

dissolution of the coatings decreased at stomach and intestine pH with increased amount

of cornstarch in the coating system. Pectin/starch blends were also plasticized with

glycerol using extrusion to make edible films by Fishman et al., (2000). Studies revealed

19

that plasticized pectin and pectin starch films have a large glass transition at about -50°C

indicating that these films are reasonably flexible at room temperature. Higher storage

modulus of these films makes them ideal for many thin film applications like edible bags

for soup, medical delivery systems etc.

Zsivanovits et al., (2004) have also studied mechanical properties of different types of

pectins. The material properties of pectin networks should show as strong dependence on

the concentrations, particularly hydration of the network. The hydration of the network is

influenced b the balance between the osmotic stress and cross-linking of the network,

which tends to restrict swelling and the affinity of the network for water, which drives

swelling. Mechanical behavior of pectins was examined at high concentrations relevant to

the behavior of pectin in the plant cell wall and as a film-forming agent. Mechanical

properties were examined as a function of counter ion type, concentration and extent of

hydration. Result revealed that swelling and stiffness of the films are strongly dependent

on pectin source and ionic environment. At a fixed osmotic stress, both Ca+

or Mg+

ions

reduce swelling and increase the stiffness of the films. Study has concluded that swelling

of high methoxyl pectin films at a constant osmotic stress is dependent on the source of

the pectin. Swelling is also dependent on counter ion type and concentration. The swollen

films behave as viscoelastic solids with a simple proportionality between polymer

concentration and tensile modulus.

2.2. KIDNEY BEAN

2.2.1. Grain characteristics

Legumes are processed and consumed in a variety of forms all over the world.

These methods are being widely used to improve nutritive value of legumes, primarily by

reducing the level of heat labile, non-nutritive compounds and by increasing the

bioavailability of nutritional components. Beans are an important legume crop consumed

throughout the world. Polyphenolic components present in beans act as antioxidants.

Lower glycemic index and the presence of alpha amylase inhibitors are proving

beneficial for diabetic patients as well as reduction of cholesterol level (Singh et al.,

2010). Legumes are commonly used as a source of protein and carbohydrate in human

diet in many countries of the world. Legumes are edible fruits and seeds of pod bearing

20

plants belonging to the family Leguminosae, containing about 750 genera and 16000-

19000 species (Allen & Allen, 1981). The grain legumes are ranked fifth in terms of

annual world grain production (171 million tons) (Ratnayake et al., 2001). India is the

largest producer and consumer of pulses in the world, accounting for 33% of the world

area and 22% of the world production of pulses (Singh et al., 2004). Legumes are sources

of complex carbohydrate, proteins and dietary fiber, having significant amounts of

vitamins and minerals and high energetic value (Tharanathan & Mahadevamma, 2003).

Protein content ranged between 17 to 40% in contrast to 7-13% of cereals, and being

equal to the protein content of meats (18-25%) (de Almeida Costa et al., 2006). Legumes,

including annual oilseeds, are high in protein, micronutrients, vitamins, minerals and

plant fibers. In addition, legumes are able to fix nitrogen from the air (through their

symbiotic association with the rhizobium bacteria), and they are adaptable to a variety of

cropping systems. Legumes are the major source of protein and constitute an important

supplement to the predominantly cereal-based diet of Asians (Singh, 1988). Cereals are

deficient in amino acid lysine, which is compensated for by the surplus in legumes, while

legumes are deficient in sulfur containing amino acids, which is compensated for by a

relative surplus in cereals (Thirumaran & Seralathan, 1988). Whole legume seeds were

recommended for consumption as the endosperm is main source of starch and protein and

seed coat is a good source of dietary fiber. Legume seed proteins mostly consist of salt-

soluble globulins, which are synthesized during seed development and hydrolyzed during

germination to provide nitrogen and carbon for developing seedlings (Chau et al., 1998).

The grains of food legumes are similar in structure but differ significantly from each

other in size, shape, color and thickness of the seed coat. Legume seeds have two major

parts; seed coat and the kernel (embryo and cotyledons). On an average, pulses (including

soybean) contain 11% seed coat, 2% embryo and 87% cotyledons. Legume proteins are

of two types – storage and structural – more versatile and useful in the Indian diets.

Storage proteins (70-80 percent) occur within the cells in discrete protein bodies. About

20-30 percent is the structural proteins responsible for cellular activities including

synthesis of structural and storage proteins. The cotyledons, account for 93 percent of

methionine and tryptophan of the whole seed, while the seed coat is the poorest in these

amino acids. The embryo is rich in methionine and tryptophan but it contributes only

21

about 2 percent of their total quantity in the seed (Kapoor & Gupta, 1977). Legume

proteins are deficient in methionine and trytophan. Dietary fibers are necessary to prevent

various diverticular and degenerative diseases. Recommended daily intake levels range

between 25-50 gm of fiber. Legumes are excellent sources of dietary fibers. It ranges

from as low as 6 percent in peanuts to as high as 25 percent in kidney beans and green

gram (Paul & Southgate, 1978; Kamath & Belavady, 1980). Low dietary fiber intake is

linked with increased incidence of cancer of the colon and rectum, diverticular disease,

coronary heart disease, diabetes and gallstone in affluent societies of the West (Burkitt &

Trowell, 1975). The hypocholeslterolemic effect is attributed to the dietary fiber fraction

of legumes (Cummings, 1978; Hellendoorn, 1979) because of its high content of pectins,

gums and galactants. Dietary fiber also absorbs bile salt. It is aided by saponins. Kidney

bean (Phaseolus vulgaris L.) is the most widely produced and consumed food legume in

Africa, India, Latin America and Mexico (FAO, 2002). This bean usually contains 20–

30% protein on a dry basis, and the protein has a good amino acid composition but is low

in sulphur-containing amino acids (notably methionine) and tryptophan (Gueguen &

Cerletti, 1994; Sathe, 2002). Dry beans have recovered prestige in the diets of developed

countries. This is due, in part, to health problems related to meat consumption, as well as

the discovery of the benefits of legumes in the diet and the protection they afford against

colon disease (Champ, 2001; Hangen & Bennink, 2003; Lee et al.,1992; Mathres, 2002).

Kidney beans have numerous health benefits, e.g., they reduce heart and renal disease

risks, lower glycemic index for persons with diabetes, increase satiation, and prevent

cancer. Furthermore, kidney beans are regarded as an important source of protein and

minerals for livestock feed production, as well as potential raw materials for processing

into human food (Shimelis & Rakshit, 2007). Cookability has been defined as 'the

conditions by which seeds achieve a degree of tenderness during cooking, acceptable to

the consumer'. In most countries, legumes are commonly prepared for traditional

consumption by soaking for varying periods and then boiling. A characteristic property of

nearly all dried legumes is the long cooking time of 3-4 h required to attain the required

degree of softness and palatability ('doneness'). In high altitude regions, such as the

highland plateau of Africa, cooking time is even further increased. Traditional processing

methods and pretreatments designed to reduce cooking time include soaking in water for

22

periods of up to 24 h. Water absorption is an important determinant of the rate of

hydration and of cooking properties. Water absorption is to some extent determined by

heredity, but it is also influenced by environmental factors, such as agronomic and

storage conditions. Starch and protein are the major components involved in the

hydration process. Agbo (1982) demonstrated significant differences in processed food

quality between two dry bean strains of the same genotype that differed only in a single

gene for seed-coat color. Initial moisture content, seed-coat thickness, texture and

permeability, and storage temperature have been shown to affect water uptake in cowpea

(Sefa-Dedeh & Stanley 1979, Moscoso et al., 1984) and dry kidney bean. Phytic acid

content and Cookability found to be correlated. Hard texture of grains is due to calcium-

pectic complexes formed. Phytic acid chelates the calcium and reduce the formation of

complex thus results in texture softening. (Kohn 1968, Kumar et al.,1978, Mattson et al.,

1950).

Long storage periods under tropical conditions result in hard to cook (HTC). This

phenomenon has been reported in several species of legumes including cowpea and red

kidney bean (Jackson & Varriano-Marsten 1981, Sefa-Dedeh et al., 1979). HTC results

from deterioration during storage and reduced water absorption (hard shell) and

cookability of cotyledons (Sclerema), accompanied by deleterious changes in texture and

flavor. Mejia (1979) reported a significant correlation between an increase in tannin

content and hardness, attributable to temperature- and humidity-dependent changes in

condensed tannins, and continued development of tannin from low-molecular mass non

tannin material. The loss of cookability of dry kidney bean in storage has been related to

the reduction in phytic acid phosphorus, and changes in the ratio of monovalent to

divalent cations in soaked bean. The reduction in phytic acid and monovalent cations

results in lower solubilisation of pectic substances through chelation and ion exchange

during cooking (Moscoso et al., 1984). HTC is overcome by the use of salt solution for

soaking the legumes. Salt alters the configuration and conformation of native proteins,

thus increasing their solubility, reducing steric hindrance, and exposing more peptide

bonds to hydrolysis. Salts also break the hydrogen bonds between protein and condensed

tannins. Salt reduces the calcium- and magnesium-mediated interactions between phytic

acid and protein and between minerals and pectin, altering the microstructure of black

23

bean, making them more porous and permitting easier penetration of heat and water

(Sievwright & Shipe 1986). Soaking dry bean in food-grade salt reduced HTC reported

by Rockland & Metzler (1967).

Digestibility of legumes is limited by few antinutritional factors like (ANFs) like

trypsin inhibitor and others. These are chemical substances, which, although non-toxic

generate adverse physiological responses and interfere with the utilization of nutrients.

ANFs are protease inhibitors, lectins, goitrogens, antivitamins and phytates, saponins,

oestrogens, flatulence factors, allergens and lysinoalanine (Liener, 1981). Some other

ANFs are cyanogens, favism factors, lathyrism factors, amylase inhibitors, tannins,

aflatoxins and amines. Although only a few legumes may contain all these ANFs, many

contain a few of them. Most of the ANFs are heat-labile and since humans only consume

legumes after cooking, it would not constitute any major health hazard. Heat stable

compounds such as polyphenols and phytates are, however, not easily removed by simple

soaking and heating. These could be reduced by germination and/or fermentation.

Legumes are rich source of polyphenolic compounds. Till recently, some of these (e.g.

tannins), were considered as anti-nutrients due to their adverse effects on protein

digestibility. However, nowadays, there is considerable interest in the antioxidant activity

of these compounds and in their potential health benefits, especially in the prevention of

cancer and cardiovascular disease (Menon, 2000). Dark colored legumes like red kidney

beans, black beans, black gram and soybean have higher amount of these polyphenolic

compounds. Cooking improved the antinutrients, protein and starch digestibility of food

legumes (Rehman & Shah, 2005). Cooking reduce the tannin and phytic acid content.

Maximum improvement in protein and starch digestibility was observed by cooking

legumes at 121 °C for 10 minutes.

2.2.2. Flour

Physicochemical and functional properties of flours prepared from common beans

and green mung beans were studied by Dzudie & Hardy (1996). Common beans flours

showed significantly better water and oil absorption capacity than green mung bean flour.

Mung bean flour showed higher bulk density, emulsion capacity whereas flour from

common bean showed more stability. Studies revealed the samples are rich in protein,

24

potassium, phosphorus and calcium with good functional properties. Therefore, flours

form these legumes can be used as a protein supplement in human diets. Rheology of the

Bengal gram flour was also studied by Bhattacharya et al., (1992). Effect of

concentration, particle size, consistency index and apparent viscosity were investigated.

Bengal flour suspension was pseudo plastic and exhibited yield stress. Particle size and

concentration of flour strongly influenced the rheological behavior of the suspension.

Chemical composition of raw or soaked beans played an important role in indicating the

texture of beans after cooking (Pujola et al., 2007). Amount of protein and amylose

present in raw beans provide a good indication of these substances in cooked beans.

Magnesium content in the raw seeds showed a strong correlation with that found in

cooked seed coat. Varietal differences also found to play role in having greater tendency

to lose starch during processing. Functionality has been defined as any property of a food

ingredient having great impact on its utilization, except its nutritional quality, functional

properties affect the processing applications, food quality directly and indirectly and

ultimately their acceptance in food and food formulations (Mahajan & Dua., 2002).

Functional properties of 10 legume flours have been investigated by Sosulski et al.,

(1976). Functional properties of the flours are due to protein content, complex

carbohydrates, pectins and mucilage. Protein composition as well as non-protein

components may contribute substantially to the emulsification properties of protein-

containing products like legume flour McWatters, (1983). Flours from different black

gram cultivars were investigated for functional, thermal and pasting properties and were

correlated with each other (Kaur & Singh, 2007). Flours shown to have low breakdown

viscosity indicating their resistance to break during cooking or thermostability in other

words.

2.3. Starch

Starch is the most abundant storage reserve carbohydrate in plants. Starch is a

versatile and useful polymer not only because of the ease with which its physicochemical

properties can be altered through chemical or enzymatic modification and/or physical

treatment (James et al., 2003). Most starches are composed of a mixture of two molecular

25

entities (polysaccharides), a linear fraction, amylose, and a highly branched fraction,

amylopectin. Amylose contributed to 15 and 25% for most starches. The ratio of amylose

and amylopectin in starch varies from one starch to another. The two polysaccharides are

homoglucans with only two types of chain linkages, an α -(1 → 4) of the main chain and

an α -(1 → 6) of the branch chains. Hydrophilic properties of the polymer are imparted

by the abundance of hydroxyl groups along the amylose molecules, giving it an affinity

for moisture. Because of their linear nature, mobility, and the presence of many hydroxyl

groups along the polymer chains, amylose molecules have a tendency to orient

themselves in a parallel fashion and approach each other closely enough to permit

hydrogen bonding between adjacent chains. As a result, the affinity of the polymer for

water is reduced and the solution becomes opaque. Amylopectin is a highly branched

polysaccharide. The structure consists of α – D -glucopyranose residues linked mainly by

(1 → 4)-linkages (as in amylose) but with a greater proportion of nonrandom α - (1 → 6)-

linkages, which gives a highly branched structure. Amylopectin is one of the largest

biological molecules and its molecular weight ranges from 106 to 10

9 g × mol

–1,

depending on botanical origin of the starch, fractionation of starch, and method used to

determine the molecular weight. Amylopectin has limited mobility in solution because of

the branched nature and eliminate the possibility of significant levels of inter chain

hydrogen bonding. On average, amylopectin has one branch point every 20 to 25

residues. The branch points are not randomly located. Linear branched chains with DP

~15 in amylopectin are the crystalline regions present in the granules. Another unique

feature of amylopectin is the presence of covalently linked phosphate monoesters. They

occur largely in starch from tuberous species, especially potato starch. Phosphate

monoesters increased the electrostatic repulsion between which results in the change of

gelatinization and pasting properties of starch (Qiang, 2005). Functional properties of

starch depend on the ratio of amylose: amylopectin which further depend on the botanical

origin from which the starch extracted (Swinkles., 1985). Starch is used as ingredient and

provides texture to several foods. Thus, it can be used as thickeners, stabilizers, binders,

adhesives, tonics, coagulants, gelling and forming agents, emulsion and foam stabilizers

and water retention agents (Freidman, 1995). The reduced bioavailability of pulse

starches has been attributed to the presence of intact tissue/cell structure, high levels of

26

amylose, high content of viscous soluble dietary fiber component, the presence of a large

number of antinutrients and strong interactions between amylose chains, which could

influence the rate and extent of pulse starch digestibility related to slow and moderate

postprandial glucose and insulin responses (Hoover & Sosulski, 1985; Edward, 1993:

Hoover & Zhou, 2003). Swelling factor, amylose leaching, gelatinization temperature,

gelatinization enthalpy, relative crystallinity and chain length distribution of amylopectin

affected the digestibility of the pulse starches (Chung et al., 2008).

2.3.1. Physicochemical properties

Physicochemical properties like swelling power, solubility, and transmittance

were reported to be significantly correlated with the average granule size of the starches

from various sources. (Zhou et al., 1998). 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. The property of starch depends on the physical and chemical

characteristics such as granule size distribution, amylose/amylopectin ratio and mineral

content. Swelling power has been reported to be influenced by strongly bonded micellar

network (Gujska, 1994). Amylose content known to be affected by climatic conditions,

botanical sources and soil type during growth (Julaino et al., 1964; Morrison et al., 1984).

27

Amylose plays a very important role in restricting the initial swelling because swelling

proceeds more rapidly after amylose has been exuded. Granules become more susceptible

to shear disintegration as they swell and release soluble material as they disintegrate.

Legumes have been characterized by a high amylose content of 25-65% (Hoover &

Sosulski, 1991). El-faki et al., 1983 and Lineback & Ke (1975) reported amylose content

of chickpea to be ranged between 30-32% and 28-33%, respectively. Hoover & Sosulski

(1991) reported that swelling power and solubility also get affected by temperature,

which might be due to melting of the crystallites. The swelling power of 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.

Pulse starch contains varying amount of phosphate monoester derivatives, which

result in increased paste clarity and viscosity (Jane et al., 1996). The starch granules of

pulse have greater stability against mechanical shear than those of the fragile swollen

wheat starches because of the hot paste viscosity that does not show any breakdown point

in legumes (Iyer & Singh, 1997). Clarity of starch is one of the important attributes in

starch applications. Swelling and brittleness of the starch molecules affect the clarity of

starch pastes. 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 monoesters 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.

Seperation 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).

28

2.3.2. Thermal properties

Whenever a material undergoes a change in physical state (e.g. melting) or

transforms from one crystalline form to another or whenever it reacts chemically, heat is

either absorbed (endothermic) or liberated (exothermic). Various techniques are used to

understand the changes occur during gelatinization like Differential Scanning

Calorimetry (DSC), X-ray scattering (Jenkins & Donald., 1998). DSC has been widely

used to study thermal behavior of starches as it helped to understand phase transition in

starch upon heating in 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 thermal transitions occurs, the energy absorbed bin the

transition, a recording of this balancing energy yields a direct calorimetric measurement

of the energy transitions which is then recorded as a peak. The area under peak is directly

proportional to the enthalpic change (Karim et al., 2000). Thermal properties predict the

qualities suitable for industrial use. Temperature and water content leads to a change in

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 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 crystalline regions of the granules due to weak hydrogen bonds in amorphous

regions. Amylopectin played a major role in starch granule crystallinity; the presence of

amylose lowers the melting point of crystalline regions and the energy to start

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 starch gels. This explains the behavior of amylopectin retrogradation endotherms

at a temperature below that for gelatinization (Ward et al., 1994). Transition temperatures

are influenced by the molecular architecture of the crystalline region corresponding to the

distribution of amylopectin short chains (DP 6-11) and not by the proportions of

crystalline regions corresponding to the amylose/amylopectin ratio (Noda et al., 1998).

29

Transition temperatures were positively related to long chains amylopectin. Tp and Tc

showed significant positive correlation with peak viscosity, breakdown, setback and final

viscosity. Transition temperature of starches was dependent on the proportion of granules

and amylopectin and more on the former. Starches with higher proportion of the longer

side chain amylopectin fraction showed higher transition temperature, which is consistent

with their greater crystallinity as indicated by higher gelatinization enthalpy (Singh et al.,

2011). Cooke & Gidley (1992) reported that ΔHgel reflects the loss of double helical order

rather than the loss of crystallinity whereas according to Tester & Morrison (1990a.

1990b) ΔHgel reflects the overall crystallinity of amylopectin. Legumes have higher

transitions temperatures, which make them suitable for application where high processing

temperatures are used to assure its thickening effects since use of starches with low

gelatinization temperatures in canned products resulted in formation of

pyrodextrinization and reactions with other components of the system to certain extent

(Betancur et al., 2001).

2.3.3. Retrogradation properties

Retrogradation is a term used for the behavior of gelatinized starch on cooling and

storage. It is of great importance as it affects quality, acceptability and shelf-life of starch

containing foods (Biliaderis, 1991). Starch retrogradation has been used to describe

changes in physical behavior following gelatinization. Retrogradation is the reassociation

of starch molecules forming an ordered structure such as double helices during storage. In

an initial step, two chains may associate. Ultimately, under favorable conditions, a

crystalline order appears and physical phase separation occurs (Atwell, 1988).

Retrogradation is important in industrial use of starch, as it can be a desired end-point in

certain applications but it also causes instability in starch pastes. Starch retrogradation is

influenced by the botanical source (e.g., cereal starch vs. tuber starch) and the fine

structure of amylopectin (e.g., chain length and distribution), molecular size and size

distribution of starch affect the rate of retrogradation. Amylose is responsible for short

term (Goodfellow & Wilson, 1990) whereas amylopectin for long-term rheological and

structural changes of starch gels (Gudmundsson, 1994). Moisture content in the starch gel

and storage temperature can affect the rate and extent of starch retrogradation. Certain

30

polar lipids, surfactants, and sugars can retard or reduce the extent of retrogradation. At

lower water contents, water acts as a plasticizer, which will affect the Tg (glass transition

temperature) of a partially crystalline polymer (Bizot, 1997). Therefore, the amount of

water will affect the glass transition of starch-based foods, hence the properties,

processing, and stability of many starch-based food products (Slade & Levine., 1987). On

ageing starch molecules in pastes, gels and baked foods begin to associate resulting in

precipitation, gelation and changes in consistency and opacity followed by gradual

increase in rigidity and phase separation between polymer and solvent (D’Appolonia &

Morad., 1981; Kulp & Ponte., 1981). Retrogradation is also desirable in some cases

where hardening and reduced stickiness is required (Collona et al., 1992). Amylopectin is

responsible for retrogradation of starch (Eliasson, 1985). Stability of these crystallites is

very less than amylose due to limited dimensions of the chain (Miler et al., 1985).

Amylose content found to be influenced the retrogradation (Baik et al., 1997; Fan &

Marks., 1998). Amylopectin and other intermediate materials also play an important role

in starch retrogradation during refrigerated storage. The intermediate materials with

longer chains than amylopectin may also form longer double helices during

retrogradation (Yamin et al., 1999). Starch retrogradation is influenced by the botanical

source (e.g., cereal starch vs. tuber starch) and the fine structure of amylopectin (e.g.,

chain length and distribution).

2.3.4. Morphology

Morphological features depends upon the size, shape and size distribution of

granules of starch from different botanical sources. Granule size reported to be influence

the pasting properties of starch (Ao & Jane, 2007; Shinde et al., 2003). Geera et al., 2006

reported that granule size is related to the molecular architecture of amylopectin and its

molecular arrangement with the starch granule. Morphology may be depended on the

biochemistry of the chloroplast or amyloplast as well as the physiology of the plant

(Badahuizen, 1969). In nature, starch exists in the form of granules, which can differ in

size and shape. The origin of starch granules can be inferred from their size, shape, and,

the hilum position (the original growing point of granule). X-ray diffraction has been

used to study the crystallinity change and to characterize the transition of crystal structure

31

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 & B are found in legumes. A having close packing

and B having loose packing with more amount of inter-helical water (Cooke & Gidley.,

1992). Legume seed starch granules are bean-like with a central elongated or starred

hilum. The size of starch granules vary from 2 to 100 μm in diameter. The tightly packed

A type structure would be expected to be more stable. A type starch has a higher melting

temperature and hence is more stable than the B type (Gidley, 1987). C-type starches

showed unique characteristics differ from A- and B- type granules starches since legume

starches have higher amylose content which lowers the melting point of crystallites and

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

2.3.5. Pasting properties

When starch is cooked, the flow behavior of a granule slurry changes markedly as

the suspension becomes a dispersion of swollen granules, partially disintegrated granules,

then molecularly dispersed granules. The cooked product is called a starch paste. In

general, a starch paste can be described as a two-phase system composed of a dispersed

phase of swollen granules and a continuous phase of leached amylose. It can be regarded

as a polymer composite in which swollen granules are embedded in and reinforce a

continuous matrix of entangled amylose molecules (Ring, 1985). If the amylose phase is

continuous, aggregation with linear segments of amylopectin on cooling will result in the

formation of a strong gel. Consistometer was used by Ceaser (1932) and Ceaser & Moore

(1935) to study the pasting characteristics and starch containing products. Brabender

viscoamylograph and viscoamylograph was used to study pasting properties. Sandstedh

& Abbott (1961) reported that starch concentration affected the pasting properties while

Mazurs et al., (1957) develop graphical presentation of amylograph data to compare

properties, which are independent of starch concentration. Rapid Visco Analyzer (RVA)

was then introduced as an alternative to Brabender viscoamylograph to measure pasting

characteristics. RVA has the advantages of using a small sample size, short testing time,

and the ability to modify testing conditions. Starches have been classified into four types

32

based on their gelatinized paste viscosity profiles. Type I is high swelling starches, which

are characterized by a high peak viscosity followed by rapid thinning during cooking

(potato, tapioca, waxy cereal). Type II is moderately swelling starch, which shows a

lower peak viscosity, and much less thinning during cooking (normal cereal starches).

Type III consists of restricted swelling starches, which show a relatively less pronounced

peak viscosity and exhibit high viscosity that remains constant or increases during

cooking (legume starches). Type IV is highly restricted starch, which does not swell

sufficiently to give a viscous solution (high amylose maize starch) Schoch & Maywald

(1968). Starches that are capable of swelling to a high degree are also less resistant to

breakdown on cooking and hence exhibit viscosity decrease significantly after reaching

the maximum value. Increase in viscosity during cooling indicated the tendency of

various constituents present in hot paste to reassociate or retrograde as the temperature of

the paste decrease. Viscosity of the 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).

2.3.6. Rheological properties

Starch rheology is to study the stress-deformation relationships of starch in

aqueous systems. The rheological properties of starch are important to both food and

industrial processing applications. During processing, starch dispersions will be subjected

to combined high heating and shearing that affect their rheological change as well as the

final characteristics of the product. Starch gelatinization, especially granular swelling,

changes the rheological properties of starch. The subsequent retrogradation will further

modify the rheological properties of starch. Rheology is directly related to the

microstructure of starch. The rheological properties of starch are influenced by many

factors such as the amylose/amylopectin ratio, minor components, the chain length of

amylose and amylopectin molecules, the concentration of starch, shear and strain, and

temperature (Qiang, 2005). Dynamic rheometery allows the continuous assessment of

dynamic moduli at various temperatures (Ferry, 1980). There is a great opportunity to

33

utilize various strains or deformation forces to obtain a more complete view of a

material’s physical properties. Very low strain allow measurements without disturbing or

destroying the inherent gel structure, which is of great value in describing the time and

temperature dependent changes in starch gels during aging ( Karim et al., 2000). The

storage modulus (Gɂ) is a measure of the energy stored in the material and recovered for it

per cycle while (Gʺ) measure the energy dissipated or lose per cycle of sinusoidal

deformation (Ferry,1980). Initial increase in Gɂ could be attributed to the degree of

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

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

(Evans & Haisman, 1979; Wong & Lelievre, 1981), further increase in temperature

resulted in decreased Gɂ indicated the disruption of gel structure during prolonged heating

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

crystalline region remaining in the swollen granules, which deforms and loosens the

particles (Eliasson, 1986). Another parameter, which maybe useful in indicating the

physical behaviour of a system is the loss tangent (tan δ). It is the ration of the energy lost

to the energy stored for each cycle of the deformation, i.e. tan δ= Gʺ/ Gɂ. It is a useful

indicator of the relative contributions of the viscous (Gʺ) and elastic (Gɂ) components to

the viscoelastic properties of a material.

2.4. Modification of starch

Modification of starch was carried out to overcome the shortcomings of native

starches such as insolubility in cold water, loss of viscosity, and thickening power after

cooking. In addition, retrogradation occurs after loss of ordered structure on starch

gelatinization, which results in syneresis or water separation in starchy food systems.

However, these shortcomings of native starch could be overcome, for example, by

introducing small amounts of ionic or hydrophobic groups onto the molecules. The

modifications alter the properties of starch, including solution viscosity, association

behavior, and shelf life stability in final products. Another purpose of starch modification

is to stabilize starch granules during processing and make starch suitable for many food

and industrial applications. Starch can be physically modified to improve water solubility

and to change particle size. The physical modification methods involve the treatment of

34

native starch granules under different temperature/moisture combinations, pressure,

shear, and irradiation. It also includes mechanical attrition to alter the physical size of

starch granules. Starch is widely modified by chemical methods. The most common

chemical modification processes are acid treatment, cross-linking, oxidation, and

substitution, including esterification and etherification. Chemical modification can be

carried out on three starch states:

• In suspension, where the starch is dispersed in water, the chemical reaction is carried

out in water medium until desired properties are achieved. The suspension is then

filtered, washed, and air-dried.

• In a paste, where the starch is gelatinized with chemicals in a small amount of water, the

paste is stirred, and when the reaction is completed, the starch is air-dried.

• In the solid state, where dry starch is moisturized with chemicals in a water solution, air

dried, and finally reacted at a high temperature (i.e., ≥ 100˚C).

The most common chemical modification includes oxidation, esterification, and

etherification. The chemical modification of starch results in enhanced molecular stability

against mechanical shearing, acidic, and high temperature hydrolysis; obtaining desired

viscosity; increasing interaction with ion, electronegative, or electropositive substances;

and reducing the retrogradation rate of unmodified starch.

Another type of starch modification that helps improve the application of starch is

crosslinking. Researchers have used different type of crosslinking agents so far like

phosphorus oxychloride, sodium tripolyphosphate, epichlorohydrin (Hoover & Sosulski,

1985, Rutenberg & Solarke, 1984; Woo & Seib, 1997; Wuzurberg, 1986) to improve the

mechanical properties as well as water stability of starch products (Kunaik &

Marchessault, 1972; Seker & Hanna, 2006). Starch with a low level of cross-linking

shows a higher peak viscosity than that of native starch and reduced viscosity breakdown.

The chemically bonded cross-links may maintain granule integrity to keep the swollen

granules intact, hence, prevents loss of viscosity and provides resistance to mechanical

shear. Increasing the level of cross-linking eventually will reduce granule swelling and

decrease viscosity. At high cross-linking levels, the cross-links completely prevent the

granule from swelling and the starch cannot be gelatinized in boiling water even under

autoclave conditions. Cross-linking of legume starches has been shown to decrease

35

amylose-leaching, water binding capacity, α-amylase digestibility, granular swelling but

also increased thermal stability and setback viscosity (Hoover et al., 2010).

Cross-linked starches are used in salad dressings to provide thickening with stable

viscosity at low pH and high shear during the homogenization process. Cross-linked

starches with a slow gelatinization rate are used in canned foods where retort sterilization

is applied; such starches provide low initial viscosity, high heat transfer, and rapid

temperature increase, which are particularly suitable for quick sterilization (Rutenberg &

Solarke, 1984). Cross-linked starches have been applied in soups, gravies, sauces, baby

foods, fruit filling, pudding, and deep fried foods (Wuzurberg, 1986).

2.5. Pectin-Starch Blends

Starch films are similar to plastic films in various properties like physical

characteristics, chemical resistance and mechanical properties. Maize starch was used to

demonstrate a novel method, which enables the preparation of starch films and coatings

with good thickness control. 5% starch was gelatinized in de-ionized water at 120 °C for

30 min with small addition of dispersant and ethanol followed by ultrasonication to

stabilize starch solution (Pareta & Edirisinghe, 2006). Pectin-starch blends are also used

for making films. Theses films exhibit extremely good modulus and tensile properties.

Such films are biodegradable, recyclable and may help satisfy increasing consumer and

regulatory demands for materials with these properties. To take the advantage of its

polymeric properties, starch has been suggested that starch must be gelatinized to

disintegrate granules and overcome the strong crystalline intermolecular forces before

mixing with pectin. The disruption of the starch granules and the resulting degree of

solubilization are highly dependent on the time and temperature conditions to which the

starch is exposed (Coffin et al., 1995). A potential industrial use for the pectin/starch

blends includes edible bags for soups and noodle ingredients (Fishmann et al., 2000). To

protect sensitive functional compounds and deliver them safely to the intestine and colon,

various edible coatings find attractive approach for the food industry.

Pectin coating was also utilized to enhance spray-dry stability of pea protein-stabilized

oil-in-water emulsions by Gharsallaoui et al., (2010). Effect of drying on the physical

stability of oil-in-water emulsions containing pea protein-coated and pea protein/pectin-

36

coated droplets has been studied. It has been observed that pea protein-pectin coating

developed provide superior stability to oil droplets in terms of ageing and pH changes,

which may be due to increased steric repulsion by pectin that formed a less charged

protective layer around the protein interfacial film surrounding the oil droplet. Starch has

been found to be potential alternative to commonly used coatings that escapes digestion

in small intestines. The combination of pectin with starch is a potential formula for food

grade coatings. Pectin was mixed with high amylose cornstarch to prepare coatings by

Dimantov et al., (2004). Coatings were then evaluated for their surface characterization

and dissolution properties. It has been observed as the amount of cornstarch increased in

the coating system the roughness of the film increased whereas dissolution of the

coatings decreased at stomach and intestine pH with increased amount of corn starch in

the coating system. Pectin/starch blends were also plasticized with glycerol using

extrusion to make edible films by Fishman et al., (2000). Studies revealed that plasticized

pectin and pectin starch films have a large glass transition at about -50°C indicating that

these films are reasonably flexible at room temperature. Higher storage modulus of these

films makes them ideal for many thin film applications like edible bags for soup, medical

delivery systems etc. Mechanical properties of different types of pectins have also been

studied by Zsivanovits et al., (2004). The material properties of pectin networks should

show as strong dependence on the concentrations, particularly hydration of the network.

The hydration of the network is influenced b the balance between the osmotic stress and

cross linking of the network, which tends to restrict swelling and the affinity of the

network for water, which drives swelling. Mechanical behavior of pectins was examined

at high concentrations relevant to the behavior of pectin in the plant cell wall and as a

film-forming agent. Mechanical properties were examined as a function of counter ion

type, concentration and extent of hydration. Result revealed that swelling and stiffness of

the films are strongly dependent on pectin source and ionic environment. At a fixed

osmotic stress, both Ca+

and Mg+ ions reduce swelling and increase the stiffness of the

films. Study has concluded that swelling of high methoxyl pectin films at a constant

osmotic stress is dependent on the source of the pectin. Swelling is also dependent on

counter ion type and concentration. The swollen films behave as viscoelastic solids with a

simple proportionality between polymer concentration and tensile modulus.