Materials and Methods

MATERIALS AND METHODS

3.1 COLLECTION

Field trips were undertaken to different parts of the Indian coast over a period of
two years. Plants of Sargassum wightii were collected from different sites of
Mandapam, Tamil Nadu (9°17' N and 79°11' E) in the months of January, March,
May, July, September, November 2009 and in March, May, July, September,
November 2010. Plants of Gracilaria verrucosa were collected from Parikud of
Chilika Lake, Odisha (19°28'-19°54' N and 85°05'-85°38' E) during the months of
January, May, July 2009 and January, May, July 2010.

After collection, the algal samples were washed in seawater several times to
remove debris as well as epiphytes and dried in the sun for 3-4 days in the field. The
sun dried materials were brought to the laboratory in Delhi and further washed
thoroughly in tap water to remove the salt and sand particles. The wet thalli of
Sargassum were semidried with the help of blotting sheets. Main axis, young and old
blades were dissected out in sufficient quantity with the help of a fine scissor. These
dissected parts of Sargassum as well as cleaned thalli of Gracilaria were dried in
shade in the laboratory and subsequently cut into small pieces, sieved to attain a
particle size of 0.5-1.5 mm and stored in sealed plastic bags. Further analyses were
carried out from the whole seaweed, old and young blades, main axis as well as the
left over pulp.

3.2 EXTRACTION OF ALGINATE

Alginate was extracted from Sargassum wightii as per Zubia et al., (2008) using
formaldehyde alkali treatment method. 10 g oven dried sample was treated with 500
ml of 1% formaldehyde for 12h at room temperature (Fig. 3.1). Then the sample was
filtered through a muslin cloth followed by washing in tap water for 30 minutes. The
washed biomass was treated with 0.2N H2SO4 and kept on a shaker overnight and
again filtered through muslin cloth. Residue was washed in 50-100 ml of distilled
water, incubated with 500 ml of 1% Na2CO3 and kept at 25 °C for 12h and diluted
with distilled water to make the volume 1 L.

The whole solution was filtered through a double layered of muslin cloth and
the filtrate was treated with 50 ml of 0.1-0.2% NaCl and stirred. The resultant solution

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was gradually added to twice the volume of ethanol with continuous stirring with a
glass rod resulting in precipitation of sodium alginate. The precipitant washed in
absolute ethanol followed by acetone and dried at 40 °C for 12h. Remaining pulp was
collected and dried at 60 °C for further analysis.
METHOD OF ALGINATE EXTRACTION

Figure 3.1. (A) Bed of Sargassum wightii; (B) Field drying of Sargassum; (C) Cutting and drying; (D)
1% formaldehyde incubation; (E) Filtering and washing; (F) Shaking with 0.2N H2SO4; (G)
Filtration; (H) Washing to eliminate acid and salt; (I) Incubation with Na2CO3; (J) Dilution;
(K) Filtration; (L) Filtrate with 50 ml NaCl; (M) Sodium alginate precipitation in ethanol;
(N) Sodium alginate attached with glass rod; (O) Newly formed sodium alginate; (P)
Washing with absolute ethanol and acetone; (Q) Sodium alginate; (R) Residual pulp; (S)
Washing of pulp; (T) Dried sodium alginate and (U) Dried pulp

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3.3 EXTRACTION OF AGAR

Agar was extracted from Gracillaria verrucosa after Istini et al., (1994) using alkali
treatment method (Fig. 3.2).

METHOD OF AGAR EXTRACTION

A B C D

E F G H

I J K

L M N O
Figure 3.2. (A) Field drying of Gracilaria verrucosa; (B) Washing; (C) Cutting and drying; (D)
Weighing; (E) NaOH incubation; (F) Washing in tap water; (G) Neutralizing with
1.5% H2SO4; (H) Washing to eliminate acid and salts; (I) Boiling with distilled water;
(J) Filtering; (K) Remaining residue or pulp; (L) Dried pulp; (M) Thawing of freeze
agar; (N) Agar after thawing and (O) Dried Agar

50 g of clean oven dried sample was incubated in 1.0 L of 5.0% NaOH solution at
80 °C for 2h. After incubation the biomass was washed in continuous tap water and

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neutralized with 1.5% H2SO4 at room temperature for 2h and further washed in
continuous tap water overnight to eliminate the acid completely. The treated biomass was
boiled for 90 minutes in 1.0 L of distilled water and filtered through a double layered
muslin cloth. The filtrate was gelled up at room temperature and the residue (pulp) was
dried in oven at 60 °C for further analysis. The solidified agar was then cut into small
strips and kept for freezing at -20 °C for 24h. The frozen agar was thawed in tap water,
soaked in acetone and then dried at room temperature.

3.4 DETERMINATION OF MOISTURE CONTENT

Samples measuring 0.5-1.5 mm in size were kept in an oven at 60 °C for 24h for
complete drying. After cooling at room temperature (20 °C), 1 g of sample was placed
in a preweighed crucible. The crucible was placed again in oven at 105 °C for 24h for
complete drying. Then crucible was kept in a desiccator and reweighed after cooling
down. Moisture content of each sample was calculated.

3.5 DETERMINATION OF ASH CONTENT

Samples measuring 0.5-1.5 mm in size were kept in an oven at 105 °C for 24h
for complete drying. Then samples were kept in a desiccator for cooling. After
cooling, 1 g of dried sample was kept in a preweighed crucible and the crucible was
transferred in a muffle furnace at 575 °C for 24h. Crucible was cooled by keeping in a
desiccator and reweighed after cooling down. Ash content of sample was calculated
as:

Weight of ash
% Ash content = x 100
Weight of sample

3.6 CARBOHYDRATE EXTRACTION

3.6.1 Sample preparation for carbohydrate analysis

3.6.1.1 Acidic extraction

0.3 g of sample was treated with 3 ml of 72% H2SO4 (v/v) in a 25 ml beaker,

stirred on a magnetic stirrer at 25 °C for 30 minutes and transferred in a 500 ml

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conical flask. Distilled water was added to maintain a final acid concentration of
2.5%. The reaction flask was then hydrolyzed in an autoclave for 30 min at 121 °C.
After cooling samples were distributed in two flasks. Sample of first flask was
neutralized with NaOH for determination of reducing sugar by DNSA method (Miller,
1959) and Glucose by GOD-POD kit. Sample of other flask was neutralized with
sodium carbonate for determination of total carbohydrates by Phenol–Sulphuric acid
method (Dubois et al., 1956).

3.6.1.1 Alcoholic extraction

0.1 g of sample was treated with 10 ml of 70% ethanol at 80 °C in a water bath for
2h. After cooling down it was filtered and the residue was retreated with 70% ethanol at
80 °C in water bath for complete extraction of soluble sugars. Both the filtrates were
mixed thoroughly and volume was made upto 100 ml with distilled water. This solution
was used for the estimation of total soluble carbohydrate and stored glucose estimation.

Note: Both samples were kept at 4 °C, until used and before carbohydrates
estimation both types of extracts were centrifuged at 10,000 rpm to remove any
type of turbidity.

3.6.2 Estimation of total carbohydrate

Phenol–Sulfuric acid method was used to estimate total carbohydrate (Dubois et al.,
1956). For determination of total carbohydrates in acidic or alcoholic extract, 0.1 or 0.2
ml of sample was pipetted out in test tube and volume was made upto 1 ml. A blank was
settled with distilled water and standard curve was prepared using standard glucose
solution ranging 10 to 100 µg /ml (Fig.3.3). All test tubes were shaken vigorously after
adding 5 ml of 96% H2SO4 (v/v) and 1 ml phenol. The test tubes were then incubated in
water bath for 20 min at 30 °C to develop orange-yellow colour, after cooling at room
temperature, optical density (O.D.) was measured at 490 nm. Total carbohydrates, total
soluble carbohydrates and total insoluble carbohydrates were calculated.

Total carbohydrate = Total carbohydrate estimated in acidic extracts

Total soluble carbohydrate = Total carbohydrate estimated in alcoholic extracts
Total insoluble carbohydrate = Total carbohydrate – Total soluble carbohydrate

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Materials and Methods

1.2
y = 0.0095x + 0.0448
1 R² = 0.9912
Absorption at 490 nm

0.8

0.6

0.4

0.2

0
0 20 40 60 80 100 120
Glucose concentration (µg/ml)

Figure 3.3. Standard curve for total carbohydrate estimation

3.6.3 Estimation of reducing and non-reducing sugar

Dinitrosalicylic acid (DNSA) method was used for determining reducing sugars
(Miller, 1959). According to this method, the reducing sugars reduce dinitrosalicyclic
acid to produce a reddish orange coloured complex, which can be measured by
spectrophotometer at 540 nm.

Preparation of DNSA reagent (Ghose, 1987):

Distilled Water : 1416 ml

3, 5-Dinitrosalicylic acid : 10.6 g

NaOH : 19.8 g

Above chemicals were dissolved in a complete dark bottle to protect from direct
sunlight, to which 306 g of Rochelle salts (Na-K tartarate), 7.6 ml of phenol (melt at
50 °C) and 8.3 g of Na2SO3 were also added.

Note: In DNSA method colour develops only under alkaline condition. Thus acidic
samples were neutralized with NaOH.

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1 ml of DNSA reagent was added to 1 ml of algal sample in a test tube and

incubated in water bath at 100°C for 10 min. After cooling at room temperature, 10
ml of distilled water was added to it and O.D. was recorded at 540 nm. Standard curve
was prepared using standard glucose solutions ranging from 100 to 1000 µg/ml
(Fig. 3.4).

0.9
y = 0.7085x + 0.1097
0.8
R² = 0.992
Absorption at 490 nm

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1 1.2
Glucose concentration (mg/ml)

Figure 3.4. Standard curve for total reducing carbohydrate estimation

3.6.4 Estimation of glucose

Glucose was estimated in acidic as well as in alcoholic extract by GOD-POD

methods (Table 3.1). According to this method, glucose is oxidized by glucose
oxidase (GOD) to gluconic acid and hydrogen peroxide. Hydrogen peroxide formed
in this reaction oxidatively couples with 4-aminoantipyrine and phenol in the presence
of peroxidase (POD) to produce re-quinonimine dye. This dye has maximum
absorption at 505 nm. The intensity of color complex is directly proportional to the
concentration of glucose in the sample. The reaction scheme is as below:

β-D-Glucose + O2 + H2O = Gluconic acid + H2O2

H2O2 + 4 – aminoantipyrine + phenol = Red dye + H2O

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Table. 3.1. GOD-POD analysis

Standard Blank Test
Glucose Standard 10 µl - -
Working solution 1000 µl 1000 µl 1000 µl
Sample - - 10 µl

Algal samples were incubated at 37 °C for 15 min and then read against water
blank at 505 nm. Glucose concentration in samples was estimated by using the
following equation.

3.6.5 Estimation of cellulose and hemicellulose

Cellulose and hemicellulose content were estimated as per Ververis et al.,

(2007). Concentration of glucose (C1) by GOD-POD method and concentration of
reducing sugar (C2) by DNSA method (Miller, 1959) in acid extracted algal samples
were used in following equations.

Determination of cellulose:

% (w/w) cellulose = (0.9/0.96) x (V/M) x α x 100

Where 0.9 is the coefficient that results from the molecular weight ratio of the
polymer and monomer hexose. The saccharification yield was taken as 0.96, C1 as the
glucose concentration (g/L), V is the total volume of sugar solution (L), M is the dry
weight of the algal biomass sample (g) and α is the dilution factor of the sample.

Determination of hemicellulose:

% (w/w) hemicellulose = (0.88/0.93) x (C1 - C2) x (V/M) x α X 100

Where 0.88 is the coefficient that results from the molecular weight ratio of the
polymer and the monomer pentose, 0.93 is the saccharification yield of xylene to xylose,
C2 is the concentration of reducing sugars (g/L) determined from the DNS method, C1 is
the glucose concentration (g/L) from above, V is the total volume of sugar solution (L),
M is the dry weight of the algal biomass sample (g) and α is the dilution factor of the
sample.

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3.6.6 Analysis of other soluble sugars

Concentration of sugars from different parts of seaweeds and pulp were

analyzed using High Performance Liquid Chromatography (HPLC). Different parts of
thalli of S. wightii and their pulp were analyzed for mannitol, fructose, sucrose and
maltose whereas G. verrucosa and its pulp were analyzed for galactose, fructose,
sucrose and maltose. Both qualitative and quantitative analyses were carried out.

Analytical conditions for HPLC system

The equipment used was of Waters 600 controller liquid chromatograph

with an RI detector, model 410.

Column : 25 x 3.2 mm, 10 μm ODS bonded silica gel column

Mobile phase : acetonitrile: HPLC grade water (80:20)

Flow rate : 1.5 ml/min

Column temperature : 34 oC

Retention time : 15 min

Detection : Shimadzu spectrophotometer (Refractive Index Detector)

(Emission = 445 nm and Extraction = 360 nm)

3.6.6.1 Extraction and estimation of sugars by HPLC

Ethanolic extracts were used for HPLC analysis. Ethanol was evaporated from
sample by keeping sample in an oven at 60 °C and homogenized in acetonitrile:
HPLC grade water in the ratio 80:20. Whatman filter paper No. 42 and Millipore
syringe driven filter unit having 0.22 µm membrane filter was used to filter the
samples. Samples were sonicated and column was cleaned with acetonitrile: HPLC
water in the ratio 80:20, prior to injection. To quantify sugar content standards of
various concentrations were run. To avoid any moisture, sugar standards were oven
dried overnight at 60oC, dissolved in acetonitrile: water and filtered through a 0.22
µm membrane filter before injecting and a calibration curve was prepared (Fig. 3.5).

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All chemicals used in analysis were of HPLC grade. The samples (20 µL) were
injected into C-18 column using a syringe in Shimadzu HPLC system (SCL 4A).

Figure 3.5. HPLC calibration curve for various sugar analysis including (A) Mannitol and (B) Galactose

3.6.7 Estimation of dietary fibers

Major forms of dietary fibers were calculated in seaweeds and remaining pulp
following American Association of Cereal Chemist (AACC) (Donnelly, 2003).

Soluble dietary fiber = Agar/Alginate content of seaweeds

Insoluble dietary fiber = Cellulose content + Hemicellulose content + Lignin content
Total dietary fiber = Soluble dietary fiber + insoluble dietary fiber

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3.7 ESTIMATION OF PROTEIN

One gram of dried algal sample was dissolved in 100 ml of 10% NaCl, stirred
for 15 minutes and then filtered through Whatman filter paper No.1. Filtrates were
used as crude extract of protein and stored at 4 °C until use. Total protein was
estimated by Bradford’s (1976) method. The method is based on the fact that there is
a change in coloration of solution containing Coomassie Brillant Blue G-250 (CBB)
alone and along with protein. To prepare 0.01% (w/v) of protein reagent 100mg of
CBB was dissolved in 50 ml of 95% ethanol and 100 ml of 85% (w/v) phosphoric
acid. Final solution diluted upto 1 L with distilled water. Final concentrations in the
reagent were 0.01% (w/v) CBB, 4.75% (w/v) ethanol and 8.5% (w/v) phosphoric
acid. 0.01, 0.02….0.1 ml of sample were pipetted out and volume were made upto 0.1
ml. A blank was also settled with 0.1 ml of water. All the test tubes were shaken well
after adding 5 ml of protein reagent. Absorbance of this sample was noted at 595 nm
against blank. Standard curve was prepared using standard Bovine Serum Albumin
(BSA) solutions ranging from 10 to 100 µg/ml.

0.9
0.8 y = 0.008x + 0.0408
Absorbance at 595 nm

0.7 R² = 0.9889
0.6
0.5
0.4
0.3
0.2
0.1
0
0 20 40 60 80 100 120
BSA concentration (µg/ml)

Figure 3.6. Standard curve for total protein estimation

3.8 ESTIMATION OF TOTAL LIPID

Total lipid was estimated as per Bligh and Dyer method (1959). For the
determination of total lipid content, 10 g of algal sample homogenized in chloroform:
methanol (10:20 v/v) mixture for two min. Then solution was homogenized again in

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10ml of pure chloroform for an additional 1 min. Finally 10 ml of water was added in
mixture and homogenized for two min. Immediately, after homogenization final
solution was filtered using vacuum filter through Whatman filter paper No. 1. Then
total solution was transferred in a graduated cylinder. Filter paper was washed and
blended with 10 ml of chloroform, refilter and transfer to the cylinder. Total solution
was allowed to set for 5-10 min for phase separation. Total volume of lower layer of
chloroform was noted as ‘x’ ml. Then upper layer of methanol-water was removed
with small volume of the chloroform layer to ensure complete removal of the upper
layer. Volume of remaining chloroform layer was noted as ‘y’ ml. Then it was
transferred in a preweighed conical flask (‘a’ g). Chloroform was evaporated in a
water bath at 40-50°C under a stream of nitrogen gas. Residue was dried and cooled
over phosphoric anhydride in a vacuum desiccator. Then weight of flask was noted as
‘b’ g. 5 ml of chloroform was dissolved in the residue and evaporated again. This
process was repeated 3 times and final weight of flask was noted as ‘c’ g. Total lipid
content was calculated as:

Weight of lipids (g): (b-a) - (c-a) = ‘d’ g

100

3.9 ESTIMATION OF TOTAL PHENOLIC COMPOUNDS

Total phenolic compounds were estimated as per Malick and Singh (1980). For
the determination of total phenolics content 1 g of algal sample was homogenized
with 10 ml of 80% ethanol. The solution was centrifuged at 10,000 rpm for 20
minutes at 4°C. Supernatant was collected and residue was re-extracted with 80%
ethanol for 3 times. All the supernatants were mixed in a test tube and evaporated to
dryness in water bath at 100°C. To the residue 5 ml of distilled water was added and
mixed well. In a series of test tubes supernatant was pipetted out from 0.2-2 ml. Final
volume was made to 3 ml in each test tube with distilled water and then 0.5 ml of
Folin-Ciocalteu’s reagent (FCR) was also added. After 3 min, 2 ml of 20% sodium

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carbonate was added and immediately all the test tubes were kept in water bath at
100°C for exactly one minute. Contents were mixed well and the O.D. of blue colour
was noted at 650 nm. FCR reagent was used as blank. Standard curve was prepared
with pyro-catechol 10-100 µg/ml.

3.10 ESTIMATION OF LIGNIN

Total acid soluble lignin in seaweeds was estimated as per Ververis et al.,
(2007). 0.7 g sample was boiled with 5 ml of 72% (w/w) H2SO4 solution for 4.5h in
order to hydrolyze the cellulose, hemicellulose and other organic compounds. The
suspension was filtered through crucible No. 3 and the solid residue was dried at 105
°C for 24h and weighed (W1). The residue was then transferred to a pre-weighed dry
porcelain crucible, heated at 600 °C for 5h, cooled at room temperature and then
weighed (W2). Acid soluble lignin was then calculated by using following formula:

Total acid soluble lignin = Weight of crucible at 105 °C (W1) – Weight of crucible at 600 °C (W2)

3.11 ESTIMATION OF ELEMENTS AND MINERALS

Mineral elements including macro, micro and metals were estimated by using
Allen’s (1989) method. 0.2 g oven dried sample was taken in a Kjeldahl tube. 1 ml of
60% perchloric acid, 5 ml of conc. HNO3 and 0.5 ml of H2SO4 was added in Kjeldahl
tube. The samples were gently shaken and kept in digester until solution become
clear. After cooling, the volume of the mixture was adjusted to 10 ml with distilled
water and boiled for few minutes. The contents were filtered through Whatman filter
paper No. 44 and volume was made 50 ml. Atomic absorption spectrophotometer was
used for estimation of elements and minerals. The calculations were done by using the
following formula.

X (µg/g)

Where:
X = Element
C = Concentration
D.F. = Dilution Factor

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Materials and Methods

3.12 ESTIMATION OF CARBON, HYDROGEN, NITROGEN AND SULFUR

Carbon, hydrogen, nitrogen and sulfur were estimated by CHNSO analyzer.

Instrument used in present investigation was “Elementar Analysensysteme Gmbh
Vario EL V 3.00”.

3.13 FT-IR MEASUREMENT

Sargassum wightii, Gracilaria verrucosa and their pulp alongwith agar and
alginate (standard as well as extracted) were analysed by FT-IR. The solid samples
were mixed with 100 mg of dried potassium bromide (KBr) and compressed to
prepare as a salt disc. The disc was then read spectrophotometrically. The frequencies
of different components present in each sample were analyzed. The same procedure
was followed for the standards.

3.13.1 Analytical conditions for FT-IR

Instrument – Spectrum BX FTIR (Perkin Elmer)
Source – Nicrome wire coated with allay
Detector – LiTaO3 (Lithium Tantalate)
Resolution – 64T01 cm-1
Beamspiltter – KBr
Power supply – 100 to 120 V or 220 to 240 V

3.14 ENZYMATIC HYDROLYSIS OF ALGAL PULP

Saccharification, fermentation and ethanol estimations were carried out as per

Gupta et al., (2009). Commercial cellulase (6.0 FPU/mg) from Trichoderma reesei
(ATCC 26921) and β-glucosidase (250 U/ml) from Aspergillus niger (Novozyme
188) were obtained from Sigma (St. Louis, MO, USA). The samples were suspended
in 0.05M citrate phosphate buffer (pH 5.0) at 50 ºC with solid content of 10% (w/v)
and soaked in rotatory incubator shaker (Innova 4400, New Brunswick Scientific, NJ,
USA) for 2h. The suspension was further supplemented with cellulase (20 FPU/g dry
substrate) and β-glucosidase (60 U/g dry substrate). Enzymatic hydrolysis was
performed at 50 ºC at 150 rpm. A dose of 0.005% (w/v) sodium azide was introduced

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to avoid any microbial contamination and 1.0% (v/v) Tween-80 was added to
facilitate the enzymatic action. The samples were collected every 6h and subsequently
analyzed for the glucose released in the reaction mixture. Glucose was estimated by
DNSA method.

3.15 FERMENTATION

For the fermentation of the seaweeds and pulp to ethanol commercial strain of
Saccharomyces cerevisiae was maintained in medium containing (g/L): glucose 30.0,
yeast extract 3.0, peptone 5.0, agar 20.0 at pH 6.0±0.2 and temperature 30 ºC. The
inoculum was multiplied by growing the cells at 30 ºC for 24h in the culture medium
containing (g/L) glucose 30.0, yeast extract 3.0, peptone 5.0, (NH4)2HPO4 0.25 at pH
6.0±0.2. The cells were grown to an optical density (OD600) of 0.6.

The fermentation of enzymatic hydrolysates was carried out separately in 250

ml conical flasks with working volume of 50 ml. The enzymatic hydrolysate
supplemented with 3g/L yeast extract and 0.25 g/L (NH4)2HPO4 was inoculated with
S. cerevisiae (6.0% v/v) at pH 6.0±0.2. Samples were withdrawn at 6h intervals and
centrifuged at 10,000 x g for 15 minutes at 4°C. The cell free supernatant was
evaluated for ethanol and residual sugar concentration.

3.16 ETHANOL ESTIMATION

Ethanol was estimated by gas chromatography (GC) (Perkin Elmer, Clarus 500)
with an elite-wax (cross bond-polyethylene glycol) column (30.0 m × 0.25 mm) at 85
°C and flame ionization detector (FID) at 200 °C. The ethanol standards were
prepared using commercial grade ethanol (Merck, Darmstadt, Germany). Nitrogen
with a flow rate of 0.5 ml/min was used as carrier gas. Total reducing sugars were
estimated by the DNSA method and the total phenolics released were determined by
the Folin–Ciocalteu’s reagent method (Malick and Singh, 1980) using vanillin as
standard.

The optical density (A600 nm) of culture filtrate was measured using a double
beam spectrophotometer (Specord 200). Dry biomass of yeast cells was measured
after drying the yeast pellets at 70 °C till constant weight.

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3.17 STATISTICAL ANALYSIS

Physical, biochemical and nutrient characteristics of S. wightii, G. verrucosa and

their pulp samples were analysed statistically. Significant changes from mean value
were tested by chi square (χ2) test. Relationships between different parameters were
analyzed by calculating square root of R2 value. R2 value was obtained from trendline
option by MS Excel. Significant changes among seasons and various parts were
analysed by two-way analysis of variance (ANOVA), followed by a one-way
ANOVA. Mean and standard deviation (SD) were obtained by descriptive statistics.

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