31

3. MATERIALS AND METHODS

3.1. Materials

3.1.1. Raw Materials

The following raw materials were procured from the Mysore local market for

processing of fibres and development of fibre rich products.

Ashgourd (Benincasa hispida), Radish (Raphanus sativus), Peas with pods (Pisum

sativum), Cardamom (Elettaria cardamomum), Carrot (Daucus carota), Onion (Allium

cepa), Potato (Solanum tuberosum).

3.1.2. Chemicals, reagents and kits

All the chemicals, organic solvents and acids used were of analytical grade.

Amyloglucosidase, Termamyl, Pancreatin, Protease (Sigma-Aldrich, USA), Pepsin (S.D.

Fine Chem Limited), Emulsifier [Polyoxyethene sorbitan monolaurate: Tween 20],

glucose oxidase-peroxidase Kit (Peridochrom Glucose, GOD-POD, Boehringer

Mannheim, Germany) and double distilled water were used in the analyses.

3.1.3. Other Ingredients

Semolina, maida, icing sugar, milk powder, custard powder, salt, ajwain

(Trachyspermum copticum), desi ghee, dalda, marvo fat, refined sunflower oil,

desiccated coconut, cashew nuts, tomato puree, chilli powder, jeera powder, coriander

seed powder, green gram dhal were procured from the local market.

32

3.2. Methods

3.2.1. Processing methods

Isolation of fibres: Ashgourd, bittergourd, ridgegourd, bottlegourd and radish were

washed thoroughly, peeled and cut into small pieces. These vegetables were then

blanched in boiling water for 2 min to eliminate surface microflora, cellular gases and

enzyme activity. Further, the pieces were subjected to juice extraction (3 times) using a

juice extractor (RayLons Metal Works, Bombay, India). The residue obtained was

dehydrated in a cabinet drier (Everflow Tray Drier, Chennai, India) at 60-70 oC to obtain

a moisture content of 5-7%. The vegetables such as cabbage, cauliflower and beans were

washed, cut, blanched (1.5 min in boiling water bath). Thereafter, these vegetables were

dehydrated as described above, to obtain a moisture content of 5-7%. Green peas were

procured from the market, washed thoroughly with fresh water to remove outer dust.

Thereafter the seeds were separated from the pods and peels were immediately dipped in

2% sodium carbonate solution for 30 min. The peels were washed with water to remove

the alkali. Then the peels were blanched in boiling water bath (containing 0.1%

magnesium oxide + 0.1% sodium hydrogen carbonate + 0.4% potassium metabisulphite)

for 3 min. Thereafter, the excess water was drained and peels were dried in preheated

cabinet drier at 65-70 oC, to get moisture content of 4-6%. Blanching provided the

products with good appearance in terms of colour, due to the inhibition of enzymes

responsible for enzymatic browning reactions. The dehydrated samples were ground,

33

sieved and packed as mentioned in the above procedure. Different cardamom varieties

i.e. white bold cardamom, white thin cardamom, green cardamom and black cardamom,

were procured from the local market and were decorticated to separate seed and peel.

The cardamom peels were dried in cabinet drier for 2 h at 55 oC to remove surface

moisture and facilitate grinding. All the dehydrated fibre samples were ground in a flour

mill and then passed through ISI standard sieves using a sieve shaker (Jayant Scientific,

Mumbai), equipped with 30 mesh, 60 mesh and 100 mesh sieves to get 250-410 µm,

140-230 µm and 40-110 µm particle size samples, respectively, to ensure the

homogeneity and uniform particle size. The various samples (500 g) were packed in PFP

[paper (42 GSM) / Al foil (0.02 mm) / polyethylene 60 µ] pouches.

Processing of vegetables and other ingredients: The vegetables were processed as per

the procedure described by Premavalli et al. (2008), wherein carrots were washed and cut

into cubes, blanched in boiling water (7 min), followed by dehydration at 65-70 oC to a

final moisture content of 3-4%. Green pea seeds were depoded, blanched in boiling water

containing magnesium oxide (0.1%), sodium metabisulphite (0.1%) and salt (2%) and

dehydrated (65-70 oC), in a cabinet drier, to a final moisture of 4-5%. Potatoes were

peeled, cut into cubes, blanched in boiling water containing salt and metabisulphite and

dehydrated to final moisture of 4-5%. The potato cubes were further deep fat fried in

dalda at 180 2 oC. Onion was peeled, cut to thin slices, dipped in 2% salt solution for

30 min and dehydrated to a final moisture content of 4-5%, thereafter the slices were

deep fat fried in dalda at 180 2 oC. All the vegetables were packed in tri-laminated PFP

pouches. Green gram dhal was dry roasted at 170 ± 5 oC for 20-25 min to obtain a golden

34

brown color and roasted flavor. Thereafter, it was cooled to room temperature, grinded in

flour mill and packed in tri-laminated PFP pouches. Sugar crystals and cardamom seeds

were powdered separately. Cardamom powder was sieved (60 mesh) and packed in tri-

laminated PFP pouches.

3.2.2 Development of fibre rich products

The development of fibre rich product was approached by the following two

methods for optimization of ingredients and finalizing the formulations. The methods

followed were:

Permutation-Combination method Response Surface Methodology

1. Sweet cookies

2. Savoury cookies

3. Vermicelli kheer

4. Savoury vermicelli

5. Roasted snack

6. Fried snack

A. Permutation-Combination method

These products were developed by optimizing the major ingredients, studying the

important physicochemical properties and by the acceptability of the product. The

ingredient composition, considering the functionality was chosen along with the

responses based on the type of the product. However, sensory evaluation was taken as a

constant response for all the developed products.

35

a. Sweet Cookies

Different levels (5-20%) of ashgourd fibre, pea peel fibre and cardamom peel

fibre were incorporated in sweet cookies. The cardamom peel fibre (5%) imparted

bitterness to the product, while ashgourd fibre (10%) gave a chalky aftertaste, thus was

not acceptable for incorporation in sweet cookies. The pea peel fibre was acceptable in

sweet cookies at 10% level and thus optimized.

Ingredients for preparation of 10 kg sweet cookies:

Maida : 4 kg

Pea peel fibre : 1 kg

Marvo fat : 2.8 kg

Iceing sugar : 2 kg

Cardamom powder : 20 g

Milk powder : 450 g

Custard powder : 200 g

Cashewnut : 200 g

Desiccated coconut : 200 g

The ingredients were blended and kneaded to form the dough which was then sheeted to

a thickness of 5 mm and round cookies were cut out using a circular mould. The cookies

were baked for 10-12 min at 204 ± 2 oC, cooled to room temperature (10 min), packed in

pouches of polypropylene (75 µ) (PP) and paper (42 GSM)/Al foil (20 μm)/polyethylene

36

(60 µ) laminated (PFP) pouches, each containing 15 cookies and stored at room

temperature (RT) i.e. 15-35 oC, 37

oC and 5

oC.

b. Savoury Cookies

For preparation of savoury cookies, different levels (5-20%) of ashgourd fibre,

radish fibre and pea peel fibre were incorporated during preparation of the cookies.

Incorporation of radish fibre in cookies, even at a lower percentage (5%) was not

acceptable by panelists, because of the high sulphur flavour imparted by radish, which

decreased the acceptability of the product. On the other hand, pea peel fibre incorporation

imparted a fibrous texture and dark colour to the product. Ashgourd fibre was quite well

acceptable to a level of 15% and was rated as good as control. The ashgourd fibre was

acceptable in savoury cookies at 15% level and thus optimized.

Ingredients for preparation of 10 kg savoury cookies:

Maida : 4.6 kg

Ashgourd fibre : 1.5 kg

Marvo fat : 2.6 kg

Milk powder : 500 g

Custard powder : 500 g

Salt : 150 g

Spices : 400 g

Desiccated coconut : 110 g

37

Fibre rich savoury cookies were prepared in a similar manner as that of sweet

cookies. The cookies were packed in pouches of polypropylene (75 µ) (PP) and paper (42

GSM)/Al foil (20 μm)/polyethylene (60 µ) laminated (PFP) pouches, each containing 15

cookies. The packed cookies were stored at room temperature (RT) i.e. 15-35 oC, 37

oC

and 5 oC.

c. Vermicelli Products

Fibre rich vermicelli were prepared by replacing semolina with different

percentage of ashgourd fibre, radish fibre, pea peel fibre and cardamom peel fibre,

ranging from 0-30%. Incorporation of ashgourd fibre more than 5%, gave a mashy look

to the product, where the taste of the product was acceptable but the texture was not up to

the mark. Vermicelli kheer with more than 5% pea peel fibre was not as well accepted as

it gave an uncooked flavour and fibrous feel during consumption. Addition of cardamom

fibre on the other hand was well accepted upto 10%. Radish fibre vermicelli was

acceptable at 25% fibre level with good texture of the product. Emulsifier, Polysorbate 20

(0.8%) was added during preparation for further improvement of texture. The ingredients,

semolina, fibre and emulsifier were mixed thoroughly and 35% hot water was slowly

added to the dry mix to avoid lumping. The mix was homogenized in pasta machine

(Model D-35, La Parmigiana) for 5 min and then extruded at ambient temperature using a

spiral die. The extruded samples were dried in a tray drier at 65 oC for about 3 h and

packed in PP (polypropylene, 75 µ) pouches and PFP (paper-foil-polyethylene) tri-

laminated pouch and stored at room temperature (15-35 oC), 37

oC and 5

oC.

38

Vermicelli kheer: For preparation of kheer, the following composition of ingredients was

weighed:

Cardamom peel fibre vermicelli : 20 g

Sugar : 40 g

Milk powder : 30 g

Cardamom seed powder : 1.2 g

Cardamom peel fibre vermicelli (20 g) was cooked in 100 ml boiling water for 2 min

stained and washed under running water. Weighed quantity of sugar, milk powder and

cardamom seed powder were added to boiling water (100 ml) and stirred continuously.

Cooked vermicelli was added to boiling mixture, stirred continuously and was cooked for

2.5 min and served hot.

Savoury Vermicelli: For preparation of savoury vegetable vermicelli, the following

composition of ingredients was weighed:

Radish fibre rich vermicelli : 30 g

Salt : 1.5 g

Jeera powder : 0.5 g

Coriander seed powder : 0.5 g

Chilli powder : 0.8 g

Tomato puree : 2 g

39

Dehydrated carrot : 3 g

Dehydrated and fried onion : 3 g

Dehydrated and fried peas : 3 g

Dehydrated and fried potato : 3 g

Radish fibre vermicelli (30 g) was roasted in 3 g ghee for 1 min, with continuous stirring.

Thereafter in 5 g hot ghee, the weighted spices were added and stirred. Water (1:5 - water

: vermicelli) was immediately added, followed by dehydrated vegetables and tomato

puree. Roasted vermicelli was then added and cooked for 5 min at low flame and served

hot.

B. Response Surface Methodology

The statistical software package design expert®

6.09, Stat-Ease Inc., Minneapolis, USA

(www.statease.com) was used to construct the experimental design to optimize the

ingredient levels in products as well as to analyze the data. A Central Composite

Rotatable Design (CCRD) was used without any blocking. The number points in the

design were obtained on basis of the number of independent factors (variables) decided

for the product. The parameters that influence the product quality and acceptability or

functionality were taken in responses. The products were developed based on the

statistical modeling and experimental designs (Wadikar et al., 2008). The factorial design

consisted of 4 factorial points, 6 axial points and 5 central points leading to 15 sets of

experiments. Each independent variable investigated in this experiment had five levels

40

which were -1.4142, -1, 0, +1 and +1.4142. A total of 15 level combinations (design

points) were generated for the three independent variables and the α-values in the design

outside the ranges were selected for rotatability of the design. The center point (the level

combination in which the value of each coded variable was 0) was repeated five times for

the three-variable design and was selected keeping the ingredients at levels expected to

yield, at least, satisfactory experimental results. The over all acceptability (OAA) and

texture were selected as the responses.

a. Roasted snack

This product was also optimized using RSM software, wherein ashgourd fibre,

green gram dhal and ghee were chosen as independent variables while over all

acceptability and total volatiles were the responses. Fifteen different combinations were

drawn. Green gram dhal (25-50 g) was roasted at 170 ± 5 oC for 20-25 min till light

brown color was formed with attractive roasted flavor. After cooling to room

temperature, the roasted dhal was milled into flour. Further, the green gram dhal and

ashgourd fibre (5-25 g) were mixed well in different proportions, and mixed with hot

molten ghee (15-30 g) in a steel vessel, on the flame. The mixture was stirred

continuously for 3 min. Thereafter, it was cooled and finally sugar (45 g) and cardamom

seed powder (4 g) were added and mixed well for uniformity and packed in PP

(polypropylene, 75 µ) pouches and PFP (paper foil polyethylene) tri-laminated pouch and

stored at room temperature (15-35 oC), 37

oC and 5

oC.

41

b. Fried Snack

The product was optimized using RSM software, wherein radish fibre was

incorporated in the ingredients to get a fibre enriched product. In case of this product, a 5-

level small central composite design (SCCD) was used with 3 independent variables

(ingredient factors) viz. fibre, maida and ghee and the codes of these variables were A, B

and C, respectively. The over all acceptability and texture were selected as the responses.

With the help of software, fifteen different combinations were drawn and the products

were prepared. Maida (80-100 g), radish fibre (5-20 g) and ghee (15-30 g) were mixed

well in different proportions, followed by salt (2.8 g), chilly powder (2 g) and ajwain (4

g) as fixed variables. Maida was replaced by fibre to an extent of 20-25%. The dry

powder was thoroughly mixed, followed by melted ghee and hot water to make good

consistency, pliable dough. Small round balls were made from the dough, rolled and

flattened into circular shape (15 cm diameter) and cut into desirable shape. These pieces

were fried in refined sunflower oil heated upto 160 ± 5 oC for 3 min on medium flame to

get golden brown colour and the fried snacks were packed in PP (polypropylene, 75 µ)

pouches and PFP (paper foil polyethylene) tri-laminated pouch and stored at room

temperature (15-35 oC), 37

oC and 5

oC.

42

3.2.3. Analytical Evaluation

The samples were ground/ mixed well prior to analysis for uniformity and all

the samples were analyzed in triplicates.

3.2.3.1. Proximate Composition

Moisture: Moisture content in dietary fibre and other food samples were determined by

AOAC (1984) procedure. Five gram powdered sample was accurately weighed in tarred

aluminum dishes and heated in a hot air oven at 105 2 oC for 1 h. The dishes were

removed, cooled in desiccators containing fused calcium chloride to room temperature.

The dishes were weighed and again heated at 105 2 oC for 1 h, cooled and weighed.

This process was continued till the difference in weight was constant. The moisture

content was calculated from loss in weight of the sample on heating at 105 2 oC using

the equation below:

Moisture (%) = W2 - W1 x 100

W2 – W

Where W2 and W1 are weights of aluminum dishes along with the samples before

and after heating respectively and W is the empty aluminum dish weight. The results

were expressed in percentage.

43

Total Ash: Total ash content in the samples was estimated by AOAC (1984) procedure.

The clean silica crucibles were tarred by keeping them in a muffle furnace at 400 oC for

half an hour, followed by cooling in a desiccator. Weight of the empty crucible was noted

(W1). Thereafter, 5 g sample was weighed into the crucibles and weight was recorded

(W2). Then the crucible with the sample was heated over bunsen burner till the sample

got charred, later transferred to muffle furnace maintained at 650 10 oC incinerated for

5 h, cooled and weighed (W3). The results were expressed in percentage total ash as

follows:

Total ash (%) = W3- W1 x 100

W2- W1

Total Protein: Total protein was estimated by Kjeldhal method (Hawk 1965) using

Gerhardt nitrogen digestion (turbotherm) and distillation (Vapodest) auto apparatus, Bio-

incorporation, India. Sample (0.5 g) was weighed into Kjeldhal digestion tubes followed

by addition of concentrated sulphuric acid (10 ml) and digestion mixture (0.5 g). The

tubes were kept in digestion chamber for 3 h. The digested sample was distilled using

programmed distillation unit. The distilled samples were collected in a conical flask

containing boric acid and mixed indicator. The distilled samples were titrated against

standard 0.1 N hydrochloric acid and titer value was noted. The results were expressed in

percentage Nitrogen as follows:

%Nitrogen (g) = Titre value x Normality of HCl x 14 x Dilution x 100

Weight of sample x Volume x 1000(dilution)

44

Percent nitrogen obtained was multiplied by the factor 6.25 and converted to percentage

protein.

Total Fat: Fat content in terms of petroleum extractable fat was estimated by using

Soxhlet apparatus as described by AOAC (1984). The empty thimble (W1) was weighed

and 10 g sample was transferred into the thimble and weighed (W2) again. The difference

gives the weight of the sample. Weight of the empty flask was noted (W) and the

thimbles were placed in Soxhlet extractor and extracted with petroleum ether (40-60 oC)

for 14-16 h. The excess solvent in the flask was evaporated on a steam bath and the

residual fat was dried at 80 oC in hot air oven for 1 h, cooled in a desiccator, weight of the

flask (W3) was noted. The results were expressed in percentage fat as follows:

Fat (%) = W3- W x 100

W2- W1

Crude Fiber: Crude fibre was estimated using fibra plus instrument (FES 6, Pelican

Equipments, Chennai, India). Sample (1 g) was weighed into a glass crucible, weight

noted (W) and the crucible fixed into the digestion tubes. Thereafter, 1.25% sulphuric

acid (200 ml) was added and heated at 400 oC for 30 min. The crucibles were cooled and

acid solution was filtered off. The residue was washed thrice with hot distilled water. The

same procedure was repeated with 200 ml of 1.25% of sodium hydroxide. The crucibles

were removed and kept for drying in air oven at 100 oC for 1 h. Thereafter, the crucibles

45

were cooled in a desiccator. The cooled crucibles were weighed (W1) and kept for ashing

in muffle furnace at 550 5 oC for 4 h. After ashing the crucibles were cooled in a

desiccator and weighed (W2). The results were expressed in percentage crude fibre as

follows:

Crude Fiber (%) = W1- W2 x 100

W

Total Starch: Total starch was estimated according to Homs et al. (1986). Milled

sample (0.5 g) was suspended in 15 ml of distilled water in a conical flask. Termamyl

(100 mg) was added and flasks were placed in boiling water bath for 15 min, with mixing

every 5 min. The suspension was allowed to cool under continuous agitation. After

cooling the sample was transferred to a 25 ml volumetric flask. The flask was rinsed

carefully and volume made up with distilled water. One ml portion was then transferred

to a test tube. Amyloglucosidase and 2 ml 0.1 M sodium acetate buffer (pH 4.75) were

added and incubated at 60 oC for 30 min. The sample was then transferred to 50 ml

volumetric flask, made up to 50 ml with distilled water. To 1 ml of the solution, 1 ml of

water and 4 ml glucose reagent were added and incubated at 37 oC for 60 min and optical

density noted at 450 nm.

Standard: Standard solution of D-glucose containing 25, 50, 75 and 100 µg of glucose

were taken in tubes followed by addition of 2 ml distilled water incubated with glucose

reagent for 60 min and optical density noted at 450 nm. Total starch was expressed in

percentage starch as follows:

46

Total starch (%) = µg of glucose x dilution factor x 0.9 x 100

Sample weight

3.2.3.2. Dietary fibre profile

Total dietary fiber was estimated in fibre samples and processed products using

method of Asp et al. (1983). Sample (1 g) was taken in Erlenmeyer flask, 25 ml of 0.1 M

sodium phosphate buffer (pH 6.0) added to suspended uniformly. Termamyl (100 mg)

was added and incubated in a boiling water bath for 15 min, cooled and 20 ml of distilled

water added and pH adjusted to 1.5 with 4 N hydrochloric acid. Pepsin (100 mg) was

added and incubated at 40 oC with agitation for 60 min, cooled, 20 ml of water added and

pH adjusted to 6.8 with 4 N sodium hydroxide. Thereafter, 100 mg of pancreatin enzyme

was added and incubated at 40 oC with agitation for 60 min, cooled and pH was adjusted

to 4.5 with 4 N hydrochloric acid. The solution was filtered through the dry celite as the

filter aid.

Insoluble dietary Fiber (IDF): The residue was washed with 2 x 10 ml of 95% alcohol

and acetone, dried at 105 ± 2 oC to constant weight and weight of the crucibles were

noted (D1) and incinerated at 550 ± 100 oC for 5 h. After cooling weight of the crucibles

were noted (I1).

Soluble dietary fiber (SDF): The volume of the filtrate was made upto to 100 ml and

400 ml of warm 95% alcohol added, kept for 1 h to precipitate, then filtered through the

dried and weighed crucible (D2) and washed with 2 x 10 ml of alcohol and acetone and

47

dried at 105 ± 2 oC over night. Incinerated at 550 ± 100

oC for 5 h, cooled and the

weights of the crucibles were noted (I2).

Total dietary fiber (TDF): It was calculated as sum of IDF and SDF. The results were

expressed in percentage dietary fibre as follows:

Blank (B): Insoluble and soluble blanks were also run without sample following the same

procedure.

IDF (%) = D1 – I1 – B × 100

W

SDF (%) = D2 – I2 – B × 100

W

TDF (%) = IDF + SDF

where: W = weight of the sample

Resistant Starch: Resistant starch was estimated as per the modified method of Goni et

al. (1996) method. Sample (0.1 g) was weighed into a centrifuge tube and 10 ml of

potassium chloride-hydrogen chloride (KCl-HCl) buffer (pH 1.5) was added to it. The

contents in the tubes were mixed well after addition of 0.2 ml of the pepsin solution

48

(1g/10 ml) and incubated at 40 oC for 60 min with constant shaking. After cooling the

tubes to room temperature 9 ml of 0.1 M tris-maleate buffer (pH 6.9) and 1 ml of

termamyl solution (40 mg/ml tris-maleate buffer) were added and incubated for 16 h at

37 oC. The solution was centrifuged (3000 rpm, 15 min) and supernatant was discarded.

The residue was washed with 10 ml of distilled water, centrifuged and supernatant was

discarded. To the residue 3 ml of distilled water and 3 ml of 4 M potassium hydroxide

were added and kept at 37 oC for 30 min. After 30 min 5.5 ml of 2 M hydrochloric acid

and 3 ml of 0.4 M sodium acetate buffer (pH 4.75) along with amyloglucosidase were

added and incubated at 60 oC for 60 min. The solution was centrifuged, the supernatant

collected and volume made upto 100 ml with distilled water. Thereafter, 0.5 ml of each

sample, standard and water were pipetted into test tube containing glucose oxidase-

peroxidase reagent. The optical density was read at 505 nm after 30 min incubation at 37

oC. A standard curve was plotted using standard glucose.

3.2.3.3. Physicochemical properties of Dietary Fibre

Particle Size Distribution: The fibre samples (100 g) were submitted to granulometry

determination (Ancona et al., 2004) in a sieve shaker (equipped with 18, 30, 60,100 and

150 mesh sieves). Each sample was placed in the top sieve, with the largest mesh and

shaken for 20 min. The retained material on each sieve was weighed carefully and

expressed as a percent of the original sample weight.

49

Water Holding Capacity: Water Holding Capacity was determined using the method

described by Sowbhagya et al. (2007). Sample (1 g) was accurately weighed into a

graduate test tube and 30 ml of distilled water added. The content was allowed to hydrate

for 18 h at ambient temperatures and then filtered through Whatman No.1 filter paper.

The hydrated residue weight was recorded and it was dried at 105 ± 2 oC to get a constant

weight. The results were expressed as gm of water held/ gm of dry sample.

Water Holding Capacity (g/g) = Residue hydrated weight – Residue dry weight

Residue dry weight

Water Binding Capacity: Water Binding Capacity was determined as outlined by

Sowbhagya et al. (2007). Sample (1 g) was accurately weighed into a graduate test tube

and 30 ml of distilled water added. The content was allowed to hydrate for 18 h at

ambient temperature, centrifuged (4000 rpm, 20 min) and the suspended solution was

removed. A portion of wet sample was removed, weighed and dried at 100 oC to get

constant weight. The results were expressed as gm of water bound/ gm of sample.

Water Binding Capacity (g/g) = Residue hydrated weight – Residue dry weight (after centrifugation)

Residue dry weight

Oil Binding Capacity: Oil Binding Capacity was determined as outlined by Sangnark

and Noomhorm (2004a) and was expressed as g of oil held per gram of fibre. Sample (1

g) was weighed into a pre weighed centrifuge tube (W1) and the weight noted (W2), to

which 25 ml of the groundnut oil was added and stirred. The content was allowed to

50

hydrate for 18 h at ambient temperature, centrifuged (4000 rpm, 20 min) and the

supernatant was decanted and the tubes were held in slant position to drain excess oil.

The weights of the tubes were noted (W3). The difference in the weight of the centrifuged

tubes with dry sample and after oil binding, gives the amount of fat absorbed. The results

were expressed as gm of water bound/ gm of sample.

Oil Binding Capacity (g/g) = Initial wt of sample – Final wt of sample after oil uptake

Swelling Capacity: Swelling Capacity was measured as described by Sowbhagya et al.

(2007). Sample (1 g) was placed in a graduated test tube and the volume noted (V1).

Thereafter, 30 ml distilled water was added and the contents allowed to hydrate for 18 h.

The final volume attained by the sample was measured (V2). The result was expressed as

ml of swollen sample per g of dry initial sample.

Swelling Capacity (ml/g) = Initial volume - Final volume

Particle Density: Particle Density was determined as described by Sangnark and

Noomhorm (2004a), through displacement in petroleum ether (40-60

oC). Petroleum ether

(40-60 oC), was transferred into a pre-weighed (W1) volumetric flask. The weight was

noted (W2). Repeated the similar procedure after transferring 2 g sample and noted the

weight (W3).

Particle Density (g/cm3) = Density of petroleum ether × Weight of sample

Weight of petroleum ether displaced

51

Cation Exchange Capacity: Cation Exchange Capacity was measured according to the

method described by Gorecka et al. (2000), by converting the fibre sample to acidic form

by treatment with excess of 2 M hydrochloric acid for 48 h. After filtration, 100 ml of 5%

sodium chloride was added to 0.5 g of wet residue in an Erlenmeyer flask. The flask was

shaken during incubation at 37 oC for 2 h and the solution was titrated against sodium

hydroxide. The result was expressed as Meq/g dry initial sample.

Cation Exchange Capacity (meq/g) = A-B

where, A: ml sodium hydroxide for titration of sample

B: ml sodium hydroxide for titration of blank

Note: 1 ml 0.1 M sodium hydroxide = 0.1 Meq

Free radical scavenging capacity (DPPH activity): Antioxidant activity was measured

using the stable radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) as described by Amin &

Mukhrizah (2006) and Dasgupta & Bratati, (2004). Sample (2 g) was suspended in 20 ml

methanolic solution (water: methanol, 1:2). The suspension was shaken thoroughly using

orbital shaker at 130 rpm for 2 h, at room temperature. It was then filtered to a pre-

weighed flask using Whatman No. 1 filter paper. The methanolic extract was evaporated

using flash evaporator at 50 oC. The flask was cooled, weighed and rinsed with 2 × 3 ml

methanolic water. This extract (0.2ml) was added to 6 ml 0.004% methanolic DPPH

52

solution. Control sample was run simultaneously (Ao). The sample was kept in dark for

30 min and absorbance was determined at 517 nm, against methanolic water.

Antioxidant activity (%) = Absorbance of blank – Absorbance of sample × 100

Absorbance of blank

Reducing power: The reducing power of the samples was determined according to the

method of Amin and Mukhrizah (2006). Sample extract (50-500 µl) prepared in case of

DPPH activity was mixed with 2.5 ml of citric-phosphate buffer (0.2 M, pH 6.6) and 2.5

ml of 10 gL-1

potassium ferricyanide. The mixture was then incubated at 50 oC for 20

min. Thereafter, 2.5 ml of 100 mlL-1

trichloroacetic acid was added to the mixture and

centrifuged at 5000 rpm for 10 min. To the upper layer of solution (2.5 ml), 2.5 ml

distilled water and 0.5 ml of 1g L-1

ferric chloride was added. The absorbance was

measured at 700 nm.

Total Phenols: Total phenol estimation in fibre samples was carried out using Folin-

Ciocalteu reagent (FCR), according to the method described by Thimmaiah (2006a).

Sample (0.5 g) was weighed and ground with 10 times of 80% ethanol in a pestle-mortar.

The homogenate was centrifuged at 10,000 rpm for 20 min and the supernatant was

decanted and kept. The procedure was repeated again with 5 volumes of ethanol and the

supernatant was decanted, both the supernatant were pooled and evaporated using flash

evaporator at 40 oC. The residue was dissolved in 5 ml distilled water and 0.2-2 ml

aliquots were pipetted into the test-tubes. The final volume was made up to 3 ml,

53

followed by 0.5 ml FCR. After 3 min, 2 ml 7% sodium carbonate was added and the

mixture was placed in a boiling water bath for 2 min. The tubes were cooled and

absorbance was measured at 650 nm, against blank. A standard curve was plotted using

standard catachol graph

Total Volatiles: The total volatiles were estimated in roasted product, as described by

Cronin (1982). Powdered sample (10 g) was transferred into distillation flask, 100 ml

water was added, mixed well and was steam distilled, till 360 ml distillate was collected.

The aqueous layer was extracted with diethyl ether (3-4 times). A pinch of anhydrous

sodium sulphate was added to the combined diethyl ether extract, in a dry conical flask.

The ether layer was carefully transferred to a pre-weighed conical flask (W1). This step

was repeated 3 times to transfer maximum amount of ether in the pre-weighed flask. The

ether was evaporated at a low temperature (40 oC) using a rotary evaporator and the flask

was cooled in a desiccator and weighed (W2). The results were expressed in percentage.

Total Volatiles (%) = W2 – W1 × 100

Weight of sample

3.2.3.4. In vitro Studies

To know the behavior of these fibres at different particle size (30, 60 and 100

mesh), in human digestive system, an in vitro study was conducted. For modeling

different conditions existing in particular part of human digestive tract, the methods were

54

modified and environmental parameters (pH) were adjusted in oral cavity: pH 6.6,

passage time 7 min; in stomach pH 1.8 for 135 min; and in duodenum pH 8.7 for 60 min.

The water binding capacity was measured by transferring of fixed amount of fibre

into three plastic centrifuge tubes and 30 ml of the buffers with pH 6.6, pH 1.8 and pH

8.7, were added in individual tubes which were kept in the water bath at 37 oC and

shaken for 7 min, 135 min and 60 min, respectively. The soaked fibre was then

centrifuged at 3000 g for 15 min and supernatant was carefully removed. The tubes were

kept in a slant position for 30 min to drain excess of water and weighed. A portion of wet

sample was removed, weighed and dried to constant weight at 100± 2 oC. The results

were expressed as g water bound per g sample

The swelling capacity was measured by transferring of 1 g fibre in three glass

cylinder and initial volume was noted. Thereafter, 30 ml of the buffers maintained at pH

6.6, pH 1.8 and pH 8.7, were added and the samples were allowed to swell for 7 min, 135

min and 60 min, respectively. The final volumes were noted. The results were expressed

as ml of swollen sample per g of dry initial sample.

The cation exchange capacity was measured by converting the fibre sample to acidic

form by treatment with excess of 2 M hydrochloric acid for 48 h. After filtration, 100 ml

of 5% sodium chloride in buffers (pH 6.6, pH 1.8 and pH 8.7) was added to 0.5 g of wet

residue. The flasks were shaken during incubation at 37 oC for 7 min, 135 min and 60

55

min and the solutions were titrated with sodium hydroxide/hydrochloric acid. The results

were expressed as Meq per gram sample.

Glucose Diffusion Assay: For the glucose diffusion assay, fibre samples were prepared

by removing glucose and other sugars from the prepared samples. The water-insoluble

solids (WIS) were prepared according to the method of Chau et al. (2003), WIS was

separated from the prepared fibre samples, by homogenizing the fibre sample in distilled

water (fibre : water ratio of 1 : 20, w/v) using a high-speed vortex for 2 min. After

filtration, WIS was thoroughly washed with 70% ethanol thrice and dried at 45 oC. On

the basis of the method of Ou, et al. (2001), wherein 0.5 g of fibre sample was well mixed

in 25 ml of glucose solution (100 mM L-1

) and dialyzed against 200 ml distilled water

(pH 7.0) in an thermostatic orbital shaker cum incubator at 37 oC for 24 h, using a

dialysis membrane with a cutoff molecular weight of 12,000. The beaker was given a

gentle shaking motion (to simulate intestinal peristalsis) for 3 h, followed by 3 h without

shaking. This continued for initial 12 h incubation. For the remaining 12 h the incubation

was done without shaking. The glucose content in the dialysate was determined

spectrophotometrically at regular intervals of 3 h for the first half of total incubation

period using a glucose oxidase-peroxidase method (Peridochrom Glucose, GOD-POD,

Boehringer Mannheim, Germany). At the end of 24 h, the glucose content in the dialysate

was again measured. A control test was carried out without addition of fibre.

Determination of Glucose Adsorption Capacity (GAC): According to the method

described by Ou, et al. (2001), the glucose-adsorption capacity (Mm g

-1) was determined

56

by mixing 1 gm of fibre sample with 100 ml of glucose solution (50 Mm L-1

) and

incubated at 37 oC for 6 h. After centrifugation at 4000 g for 20 min, the final glucose

content in the supernatant was measured using the glucose assay kit (Peridochrom

Glucose, GOD-POD, Boehringer Mannheim, Germany), to estimate the amount of

glucose adsorbed by the fibre sample.

3.2.3.5. Physical Properties of fibre rich products

Physical Properties of cookies: Physical parameters i.e. diameter, thickness, weight,

volume, density were estimated according to the methods described by Leelavathi & Rao

(1993) and Gujaral et al. (2003). Four cookies were laid edge-to-edge and diameter (mm)

was measured using a scale. Similarly six cookies were stacked on top of each other and

thickness (mm) was measured using a scale. Cookies were rearranged and measurements

were made. Spread was determined by calculation i.e. diameter divided by thickness

(Gujaral et al., 2003). Weight of the cookies was measured using weighing balance.

Volume of the cookies was determined by water displacement method wherein water in

measuring cylinder was filled to a known level (W1) and four cookies were submerged at

a time in that measuring cylinder. The water displaced was noted (W2). Volume of

cookies (ml) was calculated as (W2-W1). Density of cookies was calculated by dividing

weight (g) by volume (ml) (Leelavathi and Rao, 1993).

57

3.2.3.6. Storage Analysis of fibre rich products

Storage studies were conducted for all products at room temperature (RT), 37

oC and 5

oC for 6 to 8 months. The parameters studied throughout the storage period were

Free fatty acids, Peroxide value, Thiobarbituric acid value, Induction time, Browning

index, Texture profile and over all acceptability (OAA) of the product.

a. Free Fatty Acids: Free Fatty acids (FFA) in all the samples were estimated by

titrometric method (AOCS 1973). Sample (5 g) was taken in a stoppered conical flask, 50

ml of chloroform added, shaken for 1 h and filtrated. Filtrate (10 ml) was taken in a pre-

weighed conical flask (W1) and solvent was evaporated completely in boiling water bath.

Residual fat (W2) was noted, dissolved in 50 ml of neutralized benzene-alcohol mixture

(1:1) and titrated against standardized alcoholic potassium hydroxide solution using

phenolphthalein as indicator to a pale pink end point. The FFA content was calculated as

follows an expressed as % oleic acid.

Free Fatty Acids (%oleic acid) = Titre value x Normality of alkali x 28.2 × 100

Weight of oil sample

b. Thiobarbituric Acid (TBA) Value: Thiobarbituric acid (TBA) value was

determined by the method of Tarledgis et al. (1960). Sample (10 g) was weighed, treated

with 7.5 ml of 3 N hydrochloric acid and 75 ml distilled water. The content was steam

distilled at a constant rate of 5 ml per min and 50 ml of the distillate was collected in 10

min. To 25 ml of aliquot of distillate 2 ml TBA solution (0.67% in glacial acetic acid)

58

was added and the contents were heated in a boiling water bath for 35 min. The color was

measured at 540 nm against blank, which was prepared by taking 20 ml distilled water.

TBA value was calculated using the formula given below:

TBA value (mg malonaldehyde/kg) = 3.2 x Absorbance

0.15 x Weight of sample

c. Peroxide Value: Peroxide value was determined by iodometric titration (AOCS

1973). Sample (5 g) was weighed into a 250 ml iodine flask and 50 ml of chloroform

added. The mixture was shaken for 1 h in mechanical shaker and filtered through

Whatman No. 1 filter paper. 20 ml of filtrate was transferred to another iodine flask and

30 ml acetic acid was added followed by 1 ml saturated potassium iodide solution. It is

kept in dark for half an hour. Then the flask was removed and 50 ml distilled water was

added. The contents of flask were titrated against sodium thiosulphate solution (0.02 N)

using starch (1%) as indicator to colorless end point.

Peroxide value(meq O2 /Kg fat) = Titre value Normality of sodium thio sulphate 1000

Weight of fat*

*Note: In case of samples, with less than 1g fat content, the PV values are expressed on sample

basis.

d. Browning Index: Browning intensity was determined as described by Sharma et

al. (1992), by measuring the optical density of the alcoholic extract at 420 nm. Sample (4

59

g) was added to 100 ml 70% ethanol and shaken in mechanical shaker for 2 h. The

content of the flask was filtered through Whatman No. 1 and optical density of the filtrate

was measured at 450 nm.

3.2.3.7. Estimation of major Fibre components

Neutral Detergent Fibre, acid detergent fibre, cellulose, hemicellulose and

lignin contents (%) in these plant fibres were estimated according to the methods

described by Goering and Van Soest (1975). The cell wall or neutral detergent fibre

comprises the hemicellulose, cellulose and lignin components of fibre, while acid

detergent fibre measures the lignin and cellulose content generally known as

‘Lignocellulose’. Thus, the hemicellulose content was calculated as the difference

between neutral detergent fibre and acid detergent fibre, while cellulose was calculated as

the difference between acid detergent fibre and lignin content.

Neutral Detergent Fibre (NDF): Sample (0.5 -1.0 g) (W1) was weighed in a 600 ml

Berzelius beaker and 100 ml of NDF solution was added. The NDF solution was

prepared by dissolving 30 g sodium lauryl sulphate, 18.61 g disodium dihydrogen

ethelene diamine tetra dehydrate, 6.81 g sodium borate decahydrate, 4.56 g disodium

hydrogen phosphate, 10 ml triethylene glycol in one liter distilled water. The sample in

NDF solution was heated to boil for 10 min. Then the suspension was further boiled for

60 min at a low flame. Further, the content was filtered through a pre-weighed ash-less

filter paper (W2), under vacuum. The residue was rinsed with hot water several times,

then with acetone and dried at 100 oC in a hot air oven for 24 h. The filter paper was

60

cooled in a desiccator and weighed (W3). The filter paper containing residue was further

kept for ashing in a pre-weighted crucible (W4), at 500 oC for 4 h. The crucible was

cooled and weighed (W5). The results were expressed in percentage NDF as follows:

Neutral Detergent Fibre (%) = [(W3-W2) – (W5 –W4)] × 100

W1

Where, Sample weight: W1; Weight of filter paper: W2; Weight of residue with

filter paper: W3 ; Weight of crucible: W4; Weight of crucible with ash: W5

Acid Detergent Fibre (ADF): Sample 0.5 -1.0 g (W1) was weighed in a beaker and 100

ml ADF solution was added. The ADF solution was prepared by adding 27.84 ml

sulphuric acid to a 1000 ml capacity volumetric flask and the volume was made up with

distilled water. Cetyl trimethyl ammonium bromide (20 g) was added and thoroughly

mixed. The sample along with ADF solution was heated to boil for 10 min. Then the

suspension was further boiled for 60 min at a low flame. Further, the content was filtered

through crucible, under vacuum and the residue was rinsed with hot water several times,

then with acetone and dried at 100 oC in a hot air oven for 24 h. The crucible containing

residue was cooled in a desiccator and weighed (W2). Further it was kept for ashing in the

muffle furnace at 500 oC for 4 h. The crucible after ashing was cooled and weighed (W3).

The results were expressed in percentage ADF as follows:

Acid Detergent Fibre (%) = (W2–W3) × 100

W1

61

where, Sample weight: W1, Weight of crucible with residue : W2, Weight of crucible with

ash: W3

Lignin Content: The acid detergent fibre residue (without ashing) was further treated

with 72% sulphuric acid in a pre weighed crucible, with continuous stirring with a glass

rod to get a smooth paste. The crucible was refilled for 3 h to maintain the acid level in

the crucible. Then the acid was filtered through vacuum and the contents were washed

several times with distilled water, until acid free. The crucible was dried in a hot air oven

at 100 oC for 24 h, cooled in desiccator and weighed (W1). The crucible was further kept

for ashing in the muffle furnace at 500 oC for 4 h, cooled and weighed (W2). The results

were expressed in percentage lignin as follows:

Lignin (%) = W1 - W2 × 100

W

Pectin content: Pectin content was estimated according to the procedure described by

Thimmaiah (2006b). The fibre sample from the plant material was saponified with alkali

and precipitated as calcium pectate, which was washed until free from chloride, dried and

weighed. To 50 g sample 300 ml of 0.01 N hydrochloric acid was added and boiled for

30 min. It was filtered under suction, the residue was washed with hot water, while the

filtrate was collected. To the residue in a flask, 100 ml 0.05 N hydrochloric acid was

added and the content was boiled for 20 min. The contents were filtered as before, the

residue was washed with hot water, while the filtrate was collected. To the residue, 100

62

ml of 0.3 N hydrochloric acid was added again and the content was boiled for 10 min.

The contents were filtered as before, the residue was washed with hot water, while the

filtrate was collected. The filtrates were combined, cooled and made up to a volume of

500 ml. Further, 100 – 200 ml aliquots were pipetted into two 1000 ml beaker. 250 ml of

water was added and the acid was neutralized with 1 N sodium hydroxide using

phenolphatheline indicator. Excess of 10 ml of 1 N sodium hydroxide was added with

constant stirring and the content was allowed to stand overnight. Further, 50 ml of 1 N

acetic acid was added with continuous stirring, followed by 25 ml of 1N calcium chloride

solution (after 5 min). The content was allowed to stand for one hour, boiled for 2 min

and then filtered through Whatman No. 4 filter paper. The precipitate was thoroughly

washed with hot distilled water, until free from chlorides (test using silver nitrate). The

filter paper containing calcium pectate was transferred to a weighed dish, dried overnight

at 100 oC, cooled in desiccator and weighed. The results were expressed as percentage

calcium pectate as follows:

Calcium Pectate (%) = Wt of calium pectate 500 100

Volume of filtrate taken Wt of sample

3.2.3.8. Isolation of Major dietary Fibre components

Pectin isolate: The pectin isolation was carried out as described by Kertsz (1951). The

sample (10 ± 0.1 g) was suspended in an inorganic acid (hydrochloric acid) in the ratio

63

1:15 and the suspension (2 pH) was heated at 100 oC for 60 min (maintain the level of

water). The content was cooled and filtered through Whatman No. 3 filter paper. The

filtrate was collected and residue was again treated with hydrochloric acid. The above

procedure was repeated 3-4 times and the filtrate was pooled. The pH was checked and

the extract was precipitated with warm ethanol (1:2) and kept for 18 h. The precipitate

was filtered and washed with 75% ethanol, 85% and finally by 95% pure ethanol and

dried at a low temperature (40 oC). The isolate was cooled, packed, sealed and kept in a

dry place. The experiment was repeated 10 times to collect the cellulose isolate, which

was mixed well and used for studying the physical cum functional properties of the

isolate.

Cellulose isolate: The cellulose isolation was carried out as described by Togrul and

Arslan (2003). To fibre sample (8 ± 0.1 g) 350 ml 0.1 M sodium phosphate buffer (pH

7.5) was added and stirred well. Proteolysis was performed by adding protease (35 mg).

The content was incubated overnight at 37 oC (add few crystals of thymol to prevent

fermentation). To residue 700 ml 0.25% ammonium oxalate (w/v) was added, mixed and

pH 3.5 was maintained. The mixture was further shaken at 75 oC in water-bath for 60

min. Sodium hydroxide (10%) solution was added (1 g residue/100 ml extract) to the

fibre residue. The mixture was shaken at 35 oC in water bath for 22 h. The content was

filtered and the residue was washed thoroughly with distilled water to remove base. The

hemicellulose free fibre was mixed with 100 ml distilled water, 5 ml 10% acetic acid and

2 g sodium chloride. The mixture was shaken at 75 oC in water-bath for 60 min and

filtered. The isolate was washed with distilled water and 95% ethanol, dried at a low

64

temperature (45 oC), cooled, weighed, packed, sealed and kept in a dry place. The

experiment was repeated 10 times to collect the cellulose isolate, which was mixed well

and used for studying the physical cum functional properties of the isolate.

Hemicellulose isolate: The hemicellulose isolation was carried out as described by

Doner and Hicks (1997). Fibre sample (3 g) and aqueous 30% hydrogen peroxide (2.5

ml) were added to distilled water (75 ml) in them flask and the flask was kept in orbital

shaker for 1 h. The pH was adjusted to 11.5 by addition of 2 N sodium hydroxide (

9ml). The contents were shaken in orbital shaker for 1 h and pH during extraction was

maintained at 11.5 by drop wise addition of 50% sodium hydroxide. Then centrifuged at

18,000 rpm for 15 min and the residue was separated. Further, the supernatant was

adjusted to pH 4 with 4 N hydrochloric acid ( 2ml). The hemicellulose fraction was

allowed to precipitate overnight and then collected after centrifugation at 10,000 rpm for

15 min. The supernatant liquid was removed by decantation. The hemicellulose

precipitate was washed with isopropanol, collected by filtration and dried in vacuum at

45 oC. The experiment was repeated 10 times to collect the cellulose isolate, which was

mixed well and used for studying the physical cum functional properties of the isolate.

3.2.4. Organoleptic Evaluation

The optimized dietary fibre based functional foods were evaluated in relation to

the sensory preference using 9-point hedonic scale with anchor points, 1 (dislike very

much) and 9 (like very much). A semi-trained panel of 10 judges evaluated the samples

65

which were randomly presented initially and periodically for organoleptic evaluation on a

nine point hedonic scale. In this nine point hedonic scale, ‘6’ was considered as the cutoff

point for a stable and well accepted product. All panelists were between the age of 25 to

50 years. The order of presentation of samples was randomized and different 2-digit

number codes were used for the sample sets. The coded samples were served at room

temperature (25 oC) on a white disposable plastic plates and taste-neutral water was

provided for rinsing. The sensory evaluation was carried out for color, flavor, taste

texture and overall acceptability (OAA) of the product. The order of presentation

between the coded samples was varied from one storage time to another. The results were

presented as mean of 10 evaluations.

3.2.5. Instrumental Analysis

Image Analyser (IA): The size of the dietary fibre particles was measured using image

analyser. Dietary Fibre particles of different mesh sizes were subjected to IA using Image

Analyzer (Olympus Micro Image Analyser, Lite Version 4.0, USA). Samples were

placed on clean transparent slides and it was attempted to put as many particles as

possible in a single layer in the visible field of the camera. The fibre particle images were

captured with the image analysis software. Data capture, achieved by a high resolution 3-

CCD camera and a Colour frame grabber was coupled to the Pentium personal computer

with the help of supported software. The mean diameter of the fibre particles was

tabulated.

66

Scanning electron microscopy (SEM): The microstructure of dietary fibre particles

were seen using SEM. The samples for SEM of dietary fibres were prepared by fixing

dietary fibre powder on the double-sided conductive adhesive tape and pasting the same

on a copper stub. A portion of these samples was hydrated for 24 h in excess of water.

Excess water was drained and the wet samples were dried overnight under the lamp. The

samples (hydrated and non-hydrated) were coated with gold (20 ) in a sputter coating

unit (EMS 550) for 8 min and then examined using a FEI Quanta-400 electron

microscope, at an accelerating voltage of 10 kV. The selected regions depicting distinct

morphological features of fibre samples were photographed.

Texture Profile: The texture of the cookies was expressed as breaking strength or

hardness (Newtons, N) and fracture point (N) were measured by the ‘3-point bend jig’

(three-point break test) (Part No. 01/2756) using a ‘Lloyd TA Plus Texture Analyzer

(Anutekh Lloyd Instruments Ltd., Hampshire, UK). The distance between the two

identical beams was 30 mm. Another identical beam was brought down from above at a

cross head speed of 60 mm/min with a load cell of 100 N to contact the cookies. The

downward movement was continued till the cookie breaks. Clearance and depression

were set at 3 mm and 15 mm, respectively. Force required to break cookie individually

was noted and the average of triplicate values was calculated.

Differential Scanning Calorimetry (DSC): The effect of different fibres on thermal

properties was studied using DSC 2010, TA instruments, New castle, USA, with temperature

programmer controller. Sample (100 0.01 mg) was mixed with water to make a paste

67

(1:3). From this, 10 mg of sample was accurately weighed into aluminum pans and

sealed. The pans were kept for equilibration in isothermal conditions for 30 min and

thereafter heated from 25 to 200 oC at the rate of 5

oC/min. Empty pan was used as the

reference. Thermal transition and its peak temperature and enthalpy change were

analyzed using software (Thermal Analysis ver 2000) linked with the equipment. The

area under the peak was taken as enthalpy change (∆H). Onset temperature (To), peak

temperature (Tp) and conclusion temperature (Tc) were automatically calculated. The

experiment was performed in triplicate.

Brabender Micro-Visco-Amylograph: Pasting Characteristics were determined using a

Brabender micro visco-amylograph (230V, 50/60 Hz), Duisburg, Germany equipped with

a 250 cmg measuring cartridge and variable speed. For the rheological measurements,

sample (15 ± 0.1g) on 14% moisture basis was mixed in distilled water (110 ml) at room

temperature and added into the heating bowl. The suspension was heated and cooled with

constant stirring at a speed of 250 rpm. The suspension was heated at the rate of 3 oC

min-1

with rising temperature in the range 30-92 oC, constant temperature 92

oC (5 min)

and cooling in the range of 92-55 oC. Pasting temperature, peak viscosity and set back

viscosity were read from the viscograms.

Rancimat: During storage analysis of fibre rich products, the induction time of the

products was determined using Rancimat equipment (Metrohm Rancimat 743), which

generally measures the oxidative stability of the fatty samples. The oil was extracted with

chloroform (1:3) using wrist action mechanical shaker (Culture Instruments, Banglore,

68

India) for 2 h. Thereafter, the sample was filtered using Whatman No.1 filter paper and

the chloroform was evaporated using flash evaporator (Superfit 02295) at 40 oC. The

extracted oil sample (3 g) was taken in a test tube and exposed to stream of air at 130 oC.

The volatile oxidation products were automatically transferred to the measuring vessel

through the air stream and absorbed in the measuring solution (double distilled water).

The conductivity of this measuring solution was recorded continuously to obtain an

oxidation curve whose point of inflection was taken as induction time. This induction

time provides a good characteristic value for the oxidative stability of the oil (Majumdar

et al., 2007).

Gel Filtration Chromatography (GPC): Apparent molecular weight (relative to

molecular weight markers) distribution of the pectin sample was determined by gel

permeation chromatography (GPC), which was performed on the Sepacryl S-200 HR

column matrix (cross-linked copolymer of allyl dextran and N, N - Methylene

bisacrylamide; column dimension 26 mm ×600mm) by elution with 75mM sodium

phosphate buffer (pH 7) at 25 cm/hr (2.2 ml/min). Blue dextran (200 kDa), bovine serum

albumin (66kDa) and carbonic anhydrase (29kDa) (Sigma-Aldrich, USA), were used as

molecular weight markers. individual standard marker were weighed (5 ± 0.05 mg)

separately and were dissolved into 2 ml distilled water and applied on the column to get

retention volume for individual sample. Each standard marker (5 mg each) i.e. blue

dextran, bovine serum albumin and carbonic anhydrase, were weighed and dissolved in 2

ml distilled water to get separation of individual peaks. Prior to the UV measurements,

the fractions were passed through a membrane filter of pore size 0.45 m. (Berth et al.,

69

1990). The amounts of eluents from the column were monitored using UV absorbance

(Shimadzu 2550 UV spectrophotometer) at 280 nm. The retention volume and area were

measured. Similarly, mixture of markers along with pectin isolate (from ashgourd fibre)

were loaded in the column and fractions eluted along with phosphate buffer (pH 7) were

monitored using UV absorbance at 280 nm. Likewise, commercial pectin was also

compared with standard markers at 280 nm. To validate the separating efficiency and method,

the isolated pectin along with commercial pectin were dissolved, filtered and loaded in

the column and the fractions eluted along with phosphate buffer (pH 7) were measured at

280 nm. Based on the retention volume (ml) and integrated area, the average molecular

weight of the pectin isolate was determined.

3.2.6. Statistical Analysis

All the analytical data in triplicate were analysed for mean and standard

deviation (SD). Data were further statistically evaluated by one-way analysis of variance

using Statistical Program for Social Science (SPSS) (version 10). Significance was

defined as p ≤ 0.05.