50

CHAPTER 3

MATERIALS AND METHODS

In this chapter the discussion is focused on (1) amla pieces, (2) amla residue

incorporated bread and (3) proximate composition analysis. In the first heading

development of osmotically dehydrated amla pieces with spices development are

described stepwise. The first step is optimization of process variables for osmotic

dehydration process by response surface methodology; further the protocols for

parameters to be analyzed during different stages of processing are explained. In the

second heading process flow chart for amla residue preparation, amla residue

incorporated bread preparation by straight dough method are dealt in detail. Further

gluten test for bread and protocols to measure changes in physical properties of bread

during processing is briefed. In the third part the proximate composition and other

analysis protocols for the amla products, procedure for antioxidant activity

determination, colour analysis and sensory evaluation is discussed in detail for both

the products. The methodologies for microbial load count is also elaborated.

3.1 AMLA PIECES

The experiment “Process parameters optimization for the development of

spices incorporated amla (Emblica officinalis) pieces using response surface

methodology” was conducted with the objective of finding out the best treatment for

the preparation of dehydrated amla. The details of the materials and methods used in

carrying out this experiment are discussed. The experimental set up and the process

are given along with specific process physical parameters to be analyzed for amla

pieces during processing.

3.1.1 Procurement of the materials

Mature fruits of amla were procured from Tambaram, Chennai market.

Further cumin, fenugreek and fresh ginger were also procured from the same market.

51

Matured, disease free, uniform sized fruits of 3.5 cm to 3.7 cm diameter were

selected for the process. Fruits were then washed properly under running tap water so

that dirt that adhered on the surface of the fruits was properly cleaned. Cumin seeds

were ground using food processor and then sieved using hand siever. The fresh

ginger was washed thoroughly under running tap water and peeled manually. It was

then ground in laboratory mixer grinder. The fresh ginger extract was collected.

3.1.2 Process flow chart

The process flow chart for amla pieces with spices were shown in figure

3.1.1. The procured fruits were washed well with running water. The washed fruits

were wiped with clean cotton cloth to remove surface moisture, and then the fruits

are cut into pieces through their ribs.

Amla ↓

Pre-treatment ↓

Osmotic Dehydration ← Ingredients ↓

Draining ↓

Drying ↓

Packaging ↓

Storage

Figure 3.1.1 Process flowchart for preparation of amla pieces with spices

Salt (sodium chloride) is dissolved in distilled water at room temperature.

For different samples the salt concentration and the ingredients are varied. The

various types of ingredients selected for solution preparation are fenugreek powder,

cumin powder and fresh ginger concentrate. The salt concentration varies from 4, 8

to 12% as well as the spices of equal quantity was taken in three different

concentrations (3%, 6% and 9%). The ratio between amla pieces and solution was

52

taken as 3:1, 4:1 and 5:1. The process was optimized by response surface

methodology. The optimum time for OD was also fixed by determining dependant

variables such as weight loss, solid gain, vitamin C loss, acidity and over all

acceptability of amla pices after OD. The osmotically dehydrated amla pieces were

blotted with blotting paper to remove solution adhered on the surface of the amla

pieces after OD. The fruit pieces were spread as 0.35g/cm2 thickness in the trays and

were dried at 500C, 600C and 700C by tray drier. The drying time and temperature

were optimized based on their colour value and moisture ratio. Osmotic dehydrated

samples were also dried by Lyodel freeze dryer at – 500C ±20C under 10-3 vacuum

pressure after pre freezing them at 00C ±40C for 6 h. The drying time and drying

temperature were optimized, by subjecting the final product to analysis. The final

products were packed in low density polyethylene (LDPE) material with a thickness

of 0.05mm. The packages were heat-sealed and were kept in tight plastic container to

conduct storage studies at room temperature. The plastic container was avoided to

have direct contact with sun.

3.1.3 Experimental design

Response Surface Methodology (RSM) is a collection of mathematical and

statistical techniques that are useful for modeling and analysis of problems in which

the response is influenced by several variables. It is reported to be an efficient tool

for optimizing a process when the independent variables have the joint effect on the

responses [216]. It has been applied in optimizing food-processing operations by

several investigators [115,150,216-218]. Hence, response surface methodology was

used to design the experiments. The second order Box- Behnken design of four

variables and three levels each with three-center point combination was used [219].

This design was taken as it fulfilled most of the requirements needed for optimization

of the pretreatment (osmotic dehydration) process. In the above X1, X2, X3, X4 are

the coded independent osmotic process variables which are related to un-coded

variables using the following relation;

Xi = 2 (xi - �̅� Ri) / di (3.1.1)

53

Where xi is variable in actual units of the ith observation, �̅� Ri is the mean of the

highest and lowest variable value of xi and di is the difference between the highest

and lowest value of xi. The process variables and their levels in the form of coded

variables for three-factor three level response surface analyses are given in

Tables 3.1.1 (a) and 3.1.1 (b).

Table 3.1.1 (a) Level of process variables for the osmotic dehydration of amla

Independent variables Symbols

Levels

Symbols Coded Un-coded

Salt concentration (%) (P) X1

-1 4 (Min)

0 8

1 12 (Max)

Time (min) (t)

X2

-1 60 (Min)

0 75

1 90 (Max)

Solution to fruit ratio (STFR)

X3

-1 3 (Min)

0 4

1 5 (Max)

Spices concentration (%) (S)

X4

-1 3 (Min)

0 6

1 9 (Max)

The independent process variables were osmotic salt concentration, time,

solution to fruit ratio and spices concentration. The low level and high level in the

actual (un-coded) form were 4-12% (w/v), 60-90 min, 3-9% (w/v), 3-5 (v/w) for salt

concentration, process duration, spices concentration and solution to fruit ratio

respectively.

54

Table 3.1.1 (b) Coded and Un-coded Process Variables

Coded Process Variables Un-Coded Process Variables X1 X2 X3 X4 C (%) t (min) STFR SC (%) -1 -1 0 0 4 60 4 6 1 -1 0 0 12 60 4 6 -1 1 0 0 4 90 4 6 1 1 0 0 12 90 4 6 0 0 -1 -1 8 75 3 3 0 0 1 -1 8 75 5 3 0 0 -1 1 8 75 3 9 0 0 1 1 8 75 5 9 -1 0 0 -3 4 75 4 3 1 0 0 -3 12 75 4 3 -1 0 0 1 4 75 4 9 1 0 0 1 12 75 4 9 0 -1 -1 0 8 60 3 6 0 1 -1 0 8 90 3 6 0 -1 1 0 8 60 5 6 0 1 1 0 8 90 5 6 -1 0 -1 0 4 75 3 6 1 0 -1 0 12 75 3 6 -1 0 1 0 4 75 5 6 1 0 1 0 12 75 5 6 0 -1 0 -1 8 60 4 3 0 1 0 -1 8 90 4 3 0 -1 0 1 8 60 4 9 0 1 0 1 8 90 4 9 0 -1 0 0 8 60 4 6 0 0 0 0 8 75 4 6 0 1 0 0 8 90 4 6

Where X1, X2, X3, X4 are coded process variables; C=salt concentration; t=time; STFR=solution to fruit ratio; SC=spices concentration.

3.1.4 Osmotic dehydration of amla

Fresh amla fruits bought from the market were sorted for uniform size, color

and free from physical damage; washed with fresh water then wiped with muslin

cloth. The initial moisture content of amla was 82% w.b. The brine solution of

different concentrations (4%, 8%, 12%) was prepared in a glass bottle to which

different concentrations (3%, 6%, 9%) of spices (fine powder of cumin, fenugreek

and fresh ginger extract) were added. All the spices were taken in equal ratio of

weight. The amla fruit was cut in to pieces along the ridges (2cm thick).This pieces

were added to the preset osmotic solution and the glass was covered with aluminum

foil. The ratio of amla pieces to osmotic solution was 3:1, 4:1 and 5:1. The glasses

55

were left undisturbed at room temperature (35°C±3°C) and osmotic dehydration

continues.

During experimentation, it was assumed that the amount of solid leaching out

of cut amla pieces during osmosis was negligible [220,221]. After the specified

process time the amla pieces were removed from the glass and the excess solution is

adhering to the amla pieces were wiped with a blotting paper. The change in weight

of amla pieces before and after osmotic dehydration was noted. After osmotic

dehydration a portion of pretreated amla (20-25g) were used to determine the dry

matter by oven method [222]. The remaining portion of the pretreated amla samples

were used to determine vitamin C loss (%), acidity (%) and over all acceptability.

The optimized pretreated amla sample was dried by vacuum freeze drier and tray

drier to 10% w.b moisture content. The dried samples were packed in LDPE bags

with thickness of 0.05mm and kept at room temperature (35°C±3°C) for storage

studies.

Water loss (WL) and solute gain (SG) during osmotic dehydration

The mass transfer parameters i.e. water loss (WL) and solute gains (SG)

reflecting as one of the quality attribute of amla were calculated by the following

equations 3.1.2 and 3.1.3.

% WL = 100)]()[(

0

00 xW

SSWW tt −+− (3.1.2)

% SG = 100xW

)SS(

0

0t − (3.1.3)

Where W0 is the initial weight of fruit taken for osmotic dehydration (g), Wt is the

weight of fruit after osmotic dehydration at any time t(g), S0 is the initial dry matter

of fruit(g), and St is the dry matter of fruit after osmotic dehydration. The other

quality parameters i.e. vitamin C content, acidity are determined by the procedures

explained below and over all acceptability were evaluated by the procedure

mentioned in the following subsections.

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3.1.5 Optimization of osmotic parameters

Response surface methodology was applied to the experimental data using a

commercial statistical package, Design-Expert version 8.0.5(Stat-ease Inc.,

Minneapolis, USA, Trial version). The same software was used for the generation of

response surface plots, contour plots and optimization of process variables. The

response surface and contour plots were generated for different interaction for any

two independent variables, while holding the value of other two variables as constant

(at the central value). Such three-dimensional surfaces could give accurate

geometrical representation and provide useful information about the behavior of the

system within the experimental design [223,224]. The optimization of the osmotic

dehydration process aimed at finding the levels of independent variables viz. salt

concentration, spices concentration, time and solution to fruit ratio, which could give

maximum possible water loss (WL), over all acceptability (OAA), solute gain (SG),

minimum percentage of acidity and vitamin-C loss (VCL).

3.1.6 Drying process

The amla pieces after OD were dried using cabinet tray dryer and vacuum-

freeze dryer. Both of the processes are discussed in the following paragraphs.

3.1.6.1 Tray drying

The optimized Amla sample was dried in a cabinet tray drier (Industrial and

laboratory tools corporation, HE.12D) shown in plate 3.1.1. The moisture ratio

(amount of moisture per unit area of dry material) and colour values (L*, a*, and b*)

were calculated to finalize the drying process. Amla samples were taken at every 60

min time interval and calculated the moisture loss for three different temperatures

(50°C, 60°C and 70°C). The dried samples were measured for its colour values to see

the effect of drying on the colour. The cabinet tray drier took 12-15h to dry the amla

pieces with the tray load of 0.35g / cm2 [64] till to get desired moisture content.

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Plate 3.1.1. Cabinet tray drier

3.1.6.2 Freeze drying

The optimized amla sample was freeze dried (Lyodel, 153-06-10; size R 410,

5kg). The osmotically dehydrated amla sample was pre-frozen in a deep freezer at

0- 4°C for 6h. The freeze drier (plate 3.1.2) was loaded with amla pieces and left for

freeze-drying for 13h at -50°C [220] and absolute pressure 1.0-3m bar. The final

moisture content of amla pieces were reduced to the maximum extent (10±1%).

Plate 3.1.2. Freeze drier

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3.1.7 Physical properties of amla pieces

The physical prosperities like volume, bulk density and surface area of the

amla pieces at different processing stages were determined as per the procedure

mentioned in the Sahay and Singh [225].

Volume (m3) = )/(

)(3mkgwaterofDensitykgwaterdisplacedofWeight (3.1.4)

Bulk density (kg/m3) = )(

)(3mamlaofVolumekgairinamlaofWeight (3.1.5)

Volume of the amla pieces were measured by water displacement method

using the equation 3.1.4. Bulk density of the amla pieces were measured by using the

equation (3.1.5) determining the weight of amla with respect to volume of it. Surface

area of the amla pieces were measured by counting number of square in the projected

surface of the amla pieces in the graph sheet.

3.1.8 Statistical analysis

Multiple regression models for parameters effecting amla pieces were

developed using SPSS 16 trial version. Paired sample test was conducted for surface

area, volume, density and specific gravity of the amla pieces for fresh amla, amla

after osmotic dehydration, after tray and freeze drying of the samples. The proximate

compositions of the samples were analyzed by paired sample test.

3.2 AMLA RESIDUE INCORPORATED BREAD

This experiment was conducted with the objective of preparing and

optimizing the amla residue incorporated bread. The details of the materials and

methods used to carry out this work consists of procurement of raw materials such as

amla, maida, yeast, salt sugar, milk powder, fat, food grade gluten etc. Amla residue

was separated from the de-seeded amla then dried and ground to obtain fine amla

residue powder. The amla residue incorporated bread was prepared by adding amla

residue along with other ingredients by straight dough method. The proximate

composition of the bread was determined by using appropriate methods.

59

Fruit

Washing

Cutting

Seed Removal

Cut pieces

Grinding

Juice extraction Juice

Dehydrated pulp

Drying

Grinding

Sieving

Amla residue

Figure 3.2.1. Preparation of amla residue

3.2.1 Collection of fruits

Amla fruits were procured from Tambaram, Chennai market. Matured,

disease free, uniform sized fruits of 3.5 to 3.7 cm diameter were selected for the

process. Fruits were then washed properly under running tap water so that dirt

adhered on the surface of the fruits was properly cleaned. The surface water was

removed by wiping with cotton cloth. Then the dried fruits were stored in LDPE

(0.05mm thickness) bags for further usage.

3.2.2 Collection of ingredients for bread preparation

All the necessary ingredients for bread preparation such as maida, dried

baker’s yeast, sugar, iodized salt, food grade gluten, edible vegetable fat, milk

powder etc. were collected from the local market and kept in air tight container in a

clean and dry place.

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3.2.3 Preparation of amla residue

The process flowchart for the preparation amla residue is shown in the figure

3.2.1. Fresh amla were brought from local market and washed thoroughly to remove

all the dirt. The seed was removed from the fruit and cut into pieces. Seed removal

and cutting was done manually. Then the small cut pieces were ground by food

processor and the juice was filtered using a muslin cloth. The dehydrated pulp was

dried with a tray drier (Hitech Equipments India, Chennai) at 45°C for 4-5h. Then

the dried sample were ground using food processor and sieved using hand siever with

400µ to get the fine amla residue.

3.2.4 Bread making process

The amla residue incorporated bread was prepared by straight dough method

by following process flowchart as shown in the Figure 3.2.2. In this method all

ingredients were mixed in one stage and kneaded until the gluten is well developed

then the dough was allowed to ferment for a predetermined time. This fermentation

period depends on the quantity and quality of the yeast, water, salt, strength of the

flour, temperature etc. In this process fermentation was brought about by flying

fermentation method. In general fermentation is brought about by yeast. When yeast

grows it consume sugar and gives off carbon dioxide. This has an effect upon the

material in which it is growing.

All the necessary ingredients such as flour 100g, sugar 4g, salt 2g, yeast 3g,

edible vegetable fat 5g, milk powder 3g and water were taken in the respective

containers. A small quantity of water was taken, little sugar and yeast was added and

mixed well. Then it was given rest for 5 to7 min. Small quantity of flour was added

and made it as a paste and allowed to raise for 10 to15min. This mixture is known as

“flying ferment”. Then it was added to the flour and mixed with all the other

ingredients. In case of residue incorporated bread with gluten (RBG), 3g of residue

was added to the flour with extra quantity of 4g gluten. In residue incorporated bread

without gluten (RBWG) only 3g of residue was added to the flour. All samples were

prepared separately in clean steel vessels. They were mixed well until a soft dough

ball forms. Kneaded, folded and pressed the dough continuously for about 6 to 10

min, then covered the bowl container with a piece of plastic wrap or aluminum foil.

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The plastic wrap is preferred as one can see the dough as it rises. Then dough was

allowed to rise for about 45 to 60min. After the dough has risen, it was removed

from container/bowl and again kneaded, by folding then kneading for another 4 to 6

min.

Baking pan was prepared by slightly greasing it to prevent the final product

from sticking to the pan after baking. Dough was cut to size and placed in baking

pan. Dough was allowed to rise to twice of its size in the pan before putting pan into

oven. The oven was preheated to 200 to 210ºC. Then baking pan was placed with

dough in oven and allowed to bake for about 25 to 30 min. Pan was removed from

oven and bread was allowed to cool. After cooling the bread was sliced into pieces.

Preparation of ingredients

Preparation of flying ferment

Dough mixing

Bulk fermentation

Knock Back

Dividing

Intermediate proofing

Moulding and final proofing

Backing

Cooling

Slicing and wrapping

Figure 3.2.2 Flowchart of bread making

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3.2.5 Determination of Gluten content

Accurately about 25g of the flour was weighed into a porcelain bowl; 15 ml

of the water was added to the material and kneaded into dough. Care should be taken

that as far as possible all the materials were taken into the dough. Dough was kept in

a beaker, covered with water and allowed to stand for 1h. Then the dough was

removed and kneaded under a stream of tap water, holding it with a piece of nylon

cloth, care should be taken to retain gluten while the starch and bran were run away.

The presence of starch was confirmed by Iodine test. Washing was continued till

starch removed completely from the dough. The wet gluten was placed on a

previously dried, weighed Wattman’s filter paper, as much as possible into a thin

layer and placed in hot air oven maintained at 100±2 ºC. Dried for 6h and cooled in a

desiccator and weighed. This process was repeated until the weight difference is not

less than 2mg. The final weight was noted as wet gluten content.

3.2.6 Analysis of the Dough Development

The dough was prepared like a semi sphere. The height and diameter was

measured by placing and marking it in a butter paper before and after the dough rise.

All the three types of the sample RBG, RBWG and CB were analyzed and measured

for their respective diameter and height before and after the dough development.

Triplicates were taken for observation. The rise in the dough volume was calculated

by using the following formula (3.2.3).

Volume of the Semi sphere = 23

ΠR3 (3.2.3)

Where R is the radius of the hemisphere.

3.2.7 Texture Profile Analysis

Crumb and crust texture for CB, RBG and RBWG were determined by a

texture analyzer TA-XT.Plus (Stable Micro Systems) provided with the software

‘‘Texture Expert’’, and equipped with a 75 mm diameter aluminium cylindrical

probe. Slices of 2cm thickness were compressed to 50% of their original height in a

‘‘Texture Profile Analysis’’ double compression test (TPA), at 5mm/s speed test,

with a 7s delay between first and second compression. Primary parameters (hardness

63

(gram-force, gf), cohesiveness, springiness and resilience) and secondary mechanical

characteristics (gumminess (gf) and chewiness (gf)) were calculated from the TPA

graphic.

3.2.8 Statistical analysis

Multiple regression models for parameters effecting the amla residue

incorporated breads was developed using SPSS 16 trail version. Experiments were

conducted in triplicates and the results were expressed as the mean of those values ±

standard deviation. Student’s t –test of mean of two populations were carried out in

between the different types of breads (Control Bread (CB), RBG, RBWG, Multi

Grain Bread (MGB) and Whole Wheat Bread (WWB)) to find out significance

difference between over all acceptability at p<0.05 level of the sensory evaluation.

Student’s paired t-test was also carried out in between the changes in nutrients

contents during the storage period of the three types of sample (CB, RBG and

RBWG) and the results were analyzed to find out the significant difference in

changes at p<0.05 level. Statistical analysis was performed by using of Minitab

Version 14 statistical software.

3.3 ANALYSIS OF AMLA PRODUCTS

This part deal with the protocol for the physico chemical analysis of amla,

amla pieces, amla residue and amla residue incorporated breads along with sensory

evaluation method by 9 point Hedonic scale rating, protocol for antioxidant activity

determination and number of bacteria, yeast and mould count determination during

stability study of the product.

3.3.1 Moisture content

Moisture content was determined according to A.O.A.C [226] method. In this

the prepared samples were accurately weighed in a previously weighed moisture cup

and dried in a hot air oven at 70°C. After drying, the sample was cooled in

desiccators and reweighed. Moisture cups were again kept in an oven and repeated

until the consecutive weighing does not vary. Percentage moisture content was

determined by the following formula (3.3.1).

64

Moisture content (%) =(𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 – 𝐹𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡) 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡

× 100 (3.3.1)

3.3.2 Titrable Acidity

10g sample was crushed in mortar and pistol with 20ml distilled water, placed

aside for some times and shook well and filtered. Known volume of aliquot was

taken and was titrated with 0.1N NaOH using phenolphthalein as indicator. The

endpoint was denoted by the appearance of pink colour. The titration was repeated

thrice and the average value was recorded. Percentage titrable acidity was determined

by the following formula (3.3.2).

Titrable Acidity (%) = 𝑇𝑖𝑡𝑟𝑒 × 0.1 × 0.064 × 100𝑊𝑡.𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛

(3.3.2)

3.3.3 Ascorbic Acid

Reagents and their preparations

Dye solution

52mg 2,6-dichlorophenol-indophenol was dissolved in 150ml distilled water

containing 42mg sodium bicarbonate. It was diluted with distilled water to 200ml.

The solution was stored on brown bottle in a refrigerator at about 30ºC, standardized

everyday and fresh solution was prepared every week.

Standard ascorbic acid solution

100mg L-ascorbic acid was dissolved in a small volume of 4% oxalic acid

solution and the volume was made up to 100ml with the same solution. 10ml of this

stock solution was diluted to 100ml with 4% oxalic acid (0.1mg ascorbic acid per

ml).

Preparation of sample and titration

5ml working standard was pipette out and diluted with 10ml of 4% oxalic

acid. It was then titrated against the dye (V1 ml) end point was the appearance of

pink colour that persists for few minutes. The amount of dye consumed was the

amount of ascorbic acid present. 1ml sample was extracted with 4% oxalic acid and

made up to 100ml and centrifuged. 5ml supernatant was pipette out; 10 ml of 4%

65

oxalic acid was added and titrated against the dye (V2 ml). Ascorbic acid content was

determined by the following formula (3.3.3).

Ascorbic acid (mg/100gm) = 0.5 ×𝑉2 × 100 ×100𝑉1× 5 × 𝑤𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛

(3.3.3)

3.3.4 Total carbohydrate content

Total sugar content of whole amla, amla residue, amla pieces with spices and

bread samples were determined by colorimetric method using a standard curve of

Glucose.

Preparation of anthrone reagent

200mg anthrone was dissolved in 100ml of ice-cold 95% H2SO4. Fresh

solution was prepared before use.

Preparation of standard graph

100mg D-glucose was dissolved in 100ml water. 10ml stock was diluted to

100ml with distilled water. 1ml of working standard contains 1mg glucose. 0 to 1ml

working standard was pipetted into the test tubes and final volume of the test tube

was made up to 1ml by distilled water. 4ml anthrone reagent was added mixed well

and kept in boiling water bath for 8min. After boiling, the absorbance was measured

at 630nm in a spectrophotometer using an experiment blank (4ml anthrone reagent +

1ml distilled water) for zero setting. The absorbance was plotted against the known

concentration of glucose on a graph paper.

Preparation of sample

10g sample was crushed and put into a test tube containing boiling water and

hydrolysed keeping it in a boiling water bath for 3h with 5ml of 2.5N HCl and cooled

to room temperature. Then it was neutralised with solid Na2Co3. After the

effervescence ceases the volume was made up to 100ml and centrifuged. From the

supernatant 1 ml aliquots were taken in to the test tube to which 4 ml of anthrone

reagent was added and kept in boiling water bath for 8min. After cooling absorbance

was recorded at 630nm. From the standard graph the amount of carbohydrate present

in the sample was calculated.

66

3.3.5 Protein content

Protein content of whole amla, amle residue, amla pieces with spices and

bread samples were measured by Lowry’s method [227].

Preparation of reagents

(A) 50ml of 2% sodium carbonate was mixed with 50ml of 0.1N NaOH

solution (0.4g in 100ml distilled water).

(B) 10ml of 1.56% copper sulphate solution was mixed with 10ml of 2.37%

sodium potassium tartarate solution. Analytical reagent C was prepared

by mixing 2ml of (B) with 100ml of (A).

Preparation of standard graph

50mg bovine serum albumin was dissolved in 50ml distilled water. 10ml of

stock was diluted to 50ml with distilled water. Now 1ml of working standard

contains 0.2mg of bovine serum albumin. 0 to 1ml of working standard was pipetted

into the test tubes and final volume of the test tube was made up to 1ml by distilled

water. 5ml reagent C was added, mixed well and incubated at room temperature for

10min. Then 0.2ml of Folin ciocalteau was added and the reaction mixture and kept

in dark for 30min. After 30min, the absorbance was measured at 660nm in a

spectrophotometer using an experiment blank (5ml reagent C+ 0.2ml Folin

ciocalteau + 1 ml distilled water) for zero setting. The absorbance was plotted against

the known concentration of bovine serum albumin on a graph paper.

Preparation of sample

A known weight of sample was taken and extracted with Phosphate buffer of

pH 7. Then the extract is centrifuged at 10,000rpm for 15-20min. The supernatant

was collected. 0.2ml of supernatant was taken in triplicate and the total volume was

made up to 1ml in a test tube. To the solution 5ml of reagent C was added and

incubated at room temperature for 10min. After 10min 0.5ml of Folin ciocalteu was

added and incubated in dark for 30min. After 30min absorbance was recorded at

67

660nm. From the standard graph the amount of protein present in the sample was

calculated.

3.3.6 Total Phenol content

Preparation of standard graph

50mg catechol was dissolved in 50ml distilled water. 10ml of stock was

diluted to 50ml with distilled water. Now 1ml of working standard contains 0.2mg

catechol . 0 to 3ml of working standard was pipetted into the test tubes; final volume

of the test tube was made up to 3ml by distilled water. 0.5ml reagent Folin ciocalteu

was added, mixed well and incubates at room temperature for 2min. Then 20%

Na2CO3 was added and the reaction mixture is kept in boiling water bath for exactly

1min. After 1min, the absorbance was measured at 650nm in a spectrophotometer

using an experiment blank (3ml distilled water + 0.5ml Folin ciocalteau + 2ml

Na2CO3) for zero setting. The absorbance was plotted against the known

concentration of catechol on a graph paper.

Preparation of sample

A known weight of sample was ground in a mortar and pestle along with 10-

time volume of 80% ethanol, the homogenate sample was centrifuged at 10,000rpm

for 20min. Supernatant was saved and the residue was re-extracted with five times

the volume of 80% ethanol and centrifuged. The supernatant was evaporated until

dry; the residue was dissolved in 5ml of distilled water, 0.2 to 2ml of different

aliquots was pipette out into test tubes. In each test tube the volume was made up to

3ml with water, 0.5ml of Folin ciocalteau reagent, after 2min, 2ml of Na2Co3 was

added in each test tube. The test tubes were placed in boiling water for exactly one

minute and cooled. The absorbance was measured at 650nm. From the standard

graph amount of total phenol content of the sample was calculated against the

standard graph.

3.3.7 Tannin content

Tannin content of samples was determined by colorimetric method [228]

using a standard curve of tannic acid.

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Preparation of standard curve

Standard tannic acid (1ml=0.1mg of tannic acid) solution of 0 to 10ml was

pipetted into 100ml volumetric flask containing 75ml of distilled water. 5ml of Folin-

Denis reagent and 10ml of saturated Na2CO3 solution were added into each of the

volumetric flask and the volume was made up to 100ml with distilled water. It was

mixed well and kept for 30min. After filtration, the absorbance was measured at

750nm in a spectrophotometer using an experiment blank (5ml Folin-Denis reagent +

10ml Na2CO3+ 85ml distilled water) for zero setting. The absorbance was plotted

against the known concentration of tannic acid on a graph paper.

Preparation of sample and determination of Tannin

A known weight of sample was mixed with 50 - 75ml of distilled water in a

100ml volumetric flask. 5ml Folin-Denis reagent and 10ml Na2CO3 solutions were

added and volume was made up to 100ml with distilled water, mixed well by shaking

and kept for 30min. After filtration absorbance was measured as in the case of

standard curve. The values were matched with the standard curve and percentage

tannin content of the sample was determined using the following formula (3.3.4).

Tannin content (%) = mg of tannic acid × dilution ×100 ml of sample taken for estimation × weight of sample × 1000

(3.3.4)

3.3.8 Ash Content

10-50g sample was weighed in a dish. Weight of empty dish was noted. The

dish was put in a sand bath and then placed in a hot bath. It was heated gently to a

point of ignition and allowed to burn spontaneously. Carbon residue obtained from

this were kept in a crucible and placed in muffle furnace at a temperature of 550ºC

for 4-5h till the black color disappear and to get a constant reading. Then it was

allowed to cool and weight was taken to calculate the percentage ash contents using

the following formula (3.3.5).

Percentage ash content = (𝑚2−𝑚)𝑚1

× 100 (3.3.5)

where m1 - Weight of the sample

m2 - Weight of the crucible + ash

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m - Weight of the empty crucible

3.3.9 Dietary fibre

This assay AOAC, International determines the total dietary fibre content of

samples using a combination of enzymatic and gravimetric methods. Samples of

dried, fat-free foods are gelatinized with heat stable α-amylase and then

enzymatically digested with protease and amyloglucosidase to remove the protein

and starch present in the sample. Ethanol is added to precipitate the soluble dietary

fibre. The residue is then filtered and washed with ethanol and acetone. After drying,

the residue is weighed. Half of the samples were analyzed for protein and the others

were ashed. Total dietary fibre is the weight of the residue less the weight of the

protein and ash.

Moisture and fat free samples were analyzed for their TDF contents by

enzymatic and gravimetric method of the Association of Official Analytical

Chemists [222], using TDF-100 kit obtained from Sigma Aldrich chemical company,

USA.

SDF, IDF and TDF were determined by an enzymatic-gravimetric method

(AOAC method 32.1.17 (45)). Duplicate test samples were sequentially treated for

starch gelatinization and enzymatic starch and protein digestion in three incubation

steps: heat stable α-amylase (or termamyl) (1500-3000 units mg- l protein; Sigma

Chemical Co.) at 100°C, 30min, pH 6.0; amyloglucosidase (5000-8000 units ml-~;

Sigma Chemical Co.) at 60°C, 30min, pH 4.0-4.6; and protease (7-15 units mg -j

protein; Sigma Chemical Co.), pH 7.5. The enzyme digestate was then filtered using

acid washed celite on a Fibertec system E1023 filtration unit (Tecator, Sweden). For

IDF and SDF, after the enzyme digestate is filtered, the residue left is the IDF and

the filtrate is the SDF. For SDF, filtrate is precipitated with 95% ethanol before

filtering. TDF, IDF and SDF residue values are all corrected for undigested protein,

ash and blank. Crude protein was determined as nitrogen x 6.25; nitrogen was

measured by a micro-Kjeldahl method (Kjeltec, Model 1035 analyser). Ash was

determined by incinerating at 600°C in a muffle furnace for 6h.

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Reagents preparation

Distilled or deionized water was used to prepare solutions as given in the

steps.

1. 78% Ethanol: 207ml of water was placed into a one liter volumetric flask and

then diluted to volume 1l with 95% ethanol.

2. Phosphate Buffer, 0.08M, pH 6.0: 1.4g of Na2HPO4 and 8.4g of NaH2PO4,

anhydrous was dissolved in approximately 700ml of water and then diluted to

one litre with water. pH was checked and adjusted if necessary with either

NaOH or H3PO4. Then it was stored in tightly capped container at room

temperature.

3. Sodium Hydroxide Solution, 0.275N: 275ml of 1.0N NaOH solution was diluted

to one litre with water in a volumetric flask. Then it was stored in a tightly

capped container at room temperature.

4. Hydrochloric Acid Solution, 0.325M: 325ml of 1.0M HCl solution was diluted to

one litre with water in a volumetric flask. It was stored in a tightly capped

container at room temperature.

Procedure for TDF Analysis

The crucibles were washed thoroughly. Heated for one h at 525° C and

cooled. It was soaked and rinsed in water and then air dried 0.5g of celite was added

to each crucible and dried at 130°C to constant weight. The dried crucibles with

celite were cooled in the desiccators and weighed to nearest 0.1mg. This is recorded

as celite plus crucible weight or W1. Blanks were run along with samples through the

entire procedure to measure any contributions to residue from reagents. Samples and

blanks to be tested for dietary fibre content were run in duplicate so that duplicate

protein and ash values were available for improved accuracy.

1. Four 1g samples were weighed out of the sample into tall form beakers. (Sample

weights should not differ by more than 20mg).

2. 50ml of pH 6.0 phosphate buffer was added to each beaker.

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3. 0.1ml α-Amylase was added to each beaker and mixed well.

4. Each beaker was covered with aluminum foil and placed in a boiling water bath.

Beakers were gently agitated at 5min intervals. Then incubated for 15min after

the internal temperature of the beakers reaches 95ºC.

5. Solutions were allowed to cool to room temperature.

6. The pH of the solutions was adjusted to 7.5 ±0.2 by adding 10ml of 0.275N

NaOH to each beaker. pH was checked and adjusted if necessary with either

NaOH or HCl.

7. Immediately before use, a 50mg/ml solution of Protease was made in phosphate

buffer. Pipetted out 0.1ml (5mg Protease) into each beaker.

8. Each beaker was covered with aluminum foil and placed in 60ºC water bath.

With continuous agitation, incubated for 30min after the internal temperature of

the beakers reached to 60ºC.

9. Solutions were allowed to cool to room temperature.

10. The pH of the solutions was adjusted to between pH 4.0 and 4.6 by adding 10ml

of 0.325M HCl to each beaker. pH was checked, adjusted if necessary with either

NaOH or HCl.

11. 0.1ml of Amyloglucosidase was added to each beaker.

12. Each beaker was covered with aluminum foil and placed in 60ºC water bath.

With continuous agitation, incubated for 30min after the internal temperature of

the beakers reached to 60ºC.

13. Four volumes of 95% ethanol were added to each beaker.

14. Solutions were set overnight at room temperature to allow complete precipitation.

15. Filtration: The beds of Celite in each crucible were wetted and redistributed using

78% ethanol. Gentle suction was applied to draw Celite onto frit as an even mat.

Gentle suction was maintained and quantitatively transferred the precipitate and

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suspension from each beaker to its respective crucible. The residue was washed

with three 20ml portions of 78% ethanol, two 10ml portions of 95% ethanol, and

two 10ml portions of acetone.

16. Crucibles containing residues were dried overnight in a 105ºC air oven.

17. All crucibles were cooled in a desiccator, weighed to nearest 0.1mg, and recorded

this weight as “Residue + Celite + Crucible Weight" or W2.

18. The residues from two samples were analyzed and two blanks for protein.

19. The residue in the crucibles was ashed from two samples and two blanks for 5 h

at 525ºC. Cooled in desiccator, weighed to nearest 0.1mg and recorded this

weight as "Ash + Celite + Crucible Weight" or W3.

Calculations:

Residue Weight = W2-W1

Ash Weight = W3-W1

B = R BLANK - PBLANK - A BLANK

% TDF = [R sample – P sample – A sample − B)SW

× 100 (3.3.6)

Where:

TDF = Total Dietary Fibre

R = Average Residue Weight (mg)

P = Average Protein Weight (mg)

A = Average Ash Weight (mg)

SW = Average Sample Weight (mg)

3.3.10 Antioxidant activity

Direct extraction of the raw material was done with methanol following

Eloff’s method [229] for the purpose of preliminary screening. In this method,

coarsely ground material was extracted with the organic solvent methanol in the ratio

1:10 (w/v), under shaking condition. The extract was filtered using Whatman No.1

filter paper and decanted into pre-weighed glass vials. The process was repeated

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thrice with the same material using fresh solvent. The extracts were pooled and

concentrated in a rotary evaporator at 40°C and stored for further use.

Direct aqueous extraction

Aqueous extract was prepared by following the method proposed by

Polaquini [230]. The raw material was extracted in distilled water (1:10 w/v) for 5h,

under shaking condition (120rpm), in shade. After shaking the material was filtered

in common filter paper and in Whatman No.1, lyophilised and stored in sterile flasks.

One gram of lyophilised material was dissolved in 100ml distilled water (0.01g/ml)

for the preparation of the aqueous extract.

DPPH (2, 2-diphenyl-1-picrylhydrazyl) method

The radical scavenging activity of the methanol extracts was determined by

using DPPH assay according to Chang [231]. According to this method, varying

concentrations of the extract (2µg to 20µg in DMSO) was added to 2.96ml of DPPH

(0.1mM in ethanol) and the mixture was incubated for 20min at room temperature

under dark condition. After incubation, the decrease in the absorption of the DPPH

solution due to addition of the extract was measured at 517nm. Ascorbic acid

(10mg/ml DMSO) was used as standard.

3.3.11 Sensory evaluation

Sensory evaluation for amla pieces and amla residue incorporated bread was

studied by nine- point Hedonic scale rating. Over all acceptability of the amla pieces

after OD was calculated for further discussion, whereas for the organoleptic studies

such as color, texture, taste, flavor, bread development and over all acceptability of

bread sample along with market samples were studied. The dietary fibre rich market

bread of Brand name MODERN, manufactured by Hindustan Unilever Ltd, was

purchased from MORE super market. The samples were given to 21 untrained

panelists. The evaluation was done on a nine point hedonic scale as given below.

Like extremely : 9

Like very much : 8

Like moderately : 7

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Like slightly : 6

Neither like nor dislike : 5

Dislike slightly : 4

Dislike moderately : 3

Dislike very much : 2

Dislike extremely : 1

3.3.12 Colour Analysis

The cieLab co-ordinates (L*, a*, b*) of the freeze dried amla pieces with

spices (FD), tray dried amla pieces with spices (TD), amla residue incorporated

bread with gluten (RBG), Amla residue incorporated bread without gluten (RBWG),

control bread (CB), whole wheat bread (WWB) and multi grain bread (MGB) were

directly read in a glass cuvette with a spectrophotocolorimetre MiniScan MS/Y-2500

(HunterLab, Reston, VA, USA), calibrated with a white tile (L*=94.0, a*= -1.1,

b*=0.6), at 60ᵒC with a D-65 illuminant source. The parameters determined were

L* [L* = 0(black) and L* = 100 (white)], a* (-a* = greenness and +a* = redness)

and b* (-b* = blueness and + b* = yellowness).

3.3.13 Stability studies

Osmo- tray dried and osmo- freeze dried amla pieces with spices were packed

into 0.05mm thickness LDPE pack and stored at room temperature (35°C ±3°C) for

60 days. The products stability was studied by conducting biochemical parameters

and microbial load at 20 days interval. The bread samples such as CB, RBG and

RBWG were packed in the same packing material and kept at refrigerated

temperature (12°C±3°C) for 6 days. The products stability was studied by conducting

biochemical parameters and microbial load at every alternate day.

3.3.14 Microbial studies

Culture of bacteria in agar plates

A sterile Petri dish containing nutrient agar was used to culture bacteria or

fungi is called agar plate. Culture in agar plate is usually done on solid medium. At

times semisolid medium containing 2% agar is also used to study bacterial motility.

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Pour plate method for evaluation of Bacteria

The pour plate technique was used for culture and enumeration of bacteria.

Conditions vary depending upon the types of bacteria being enumerated.

1. Agar was melted and kept at 44-46°C.

2. Serial dilutions were prepared using 0.1% peptone water. Following incubation,

one of the dilutions will be yielding growth of 30-300 colonies in the agar plate.

3. 1ml of the sample or dilution was transferred to a sterile, empty Petri dish

containing approximately 15ml of molten agar medium.

4. The sample and agar were mixed thoroughly by rotating the plate several times,

clockwise, then counterclockwise.

5. When the media was solidified, the plates were inverted and incubated.

6. After incubation colonies were counted using a Quebec counter and hand tally.

7. Otherwise in a simpler method, one ml of bacterial suspension was taken and

added it into culture tube containing molten agar. Mixed well and poured it into a

Petri plate. Allowed it to solidify and incubated at 24-26°C for 48h.

Yeast and Mold Count

As Amla is an acidic fruit and its processing products also content acidity up

to some extent, so there is a chance of growth of yeasts and molds during storage

period. For evaluation of the fungal colonies Potato Dextrose Agar (PDA) was used.

The sample was prepared and microbial analysis was carried on by pour plate

method under LAF. The plates were kept in incubator at 28ºC for 72h and the

colonies were counted by colony counter.

Coliform Count

During processing of amla pieces and amla residue incorporated bread, there

are possibilities of contamination due to unhygienic condition. There is also chances

of presence of Coliforms such as E. coli, salmonella etc. in the product. For

76

evaluation of the Coliform colonies Violet Red Bile Agar (VRBA) was used. The

sample was prepared and microbial analysis was carried on by pour plate method

under LAF. The plates were kept in incubator at 37ºC for 24h and the colonies were

counted by colony counter.

Colony counting

A colony counter is an instrument used to count colonies of bacteria or other

microorganisms growing on an agar plate. Early counters were merely lighted

surfaces on which the plate was placed, with the colonies marked off with a felt-

tipped pen on the outer surface of the plate while the operator kept the count

manually. More recent counters attempt to count the colonies electronically, by

identifying individual areas of dark and light according to automatic or user-set

thresholds, and counting the resulting contrasting spots. The maximum number of

colonies which may be effectively counted on a single plate is somewhere between

100 and 1,000 depends on the size of the colony and the type of organism.

Standard protocols were used for processing of amla pieces with spices and

amla residue incorporated breads. The analysis was done as per international

standard methods. All the measurements are done in triplicates and mean ± standard

deviation was taken for interpretation.