A3.

MATERIALS AND METHODS

3.1. Sources of materials:-
Fenugreek seeds were procured from local market of Allahabad city; they were
washed and dried under fans, grind to powder form and stored at room temperature. All the
chemicals were used for chemical analysis in extracts.

3.2. Processing of samples:-
The seeds were processed in the terms of dry heating at 40-50C under tray drier. The
sample was prepared for analysis of changes in proximate and antioxidant composition from
fresh material.
Soaked:-The clean seeds (50g) were soaked in distilled water at the ratio of 1:5 (w/v)at room
temperature for 12 hrs. The water was intermittently changed every 6 hrs. After 12 hrs ,the
excess water was discarded and seeds were dried in hot air oven at 40
0
C for 6 hrs.
Germinated:- Fenugreek seeds (50g) were soaked overnight in water at the ratio of 1:5 (w/v).
The excess water was drained and seeds were kept in the dark for germination (tied in muslin
cloth) at 27
0
2
0
C temperature for 24 hrs. The germinated seeds were dried in an oven at
40
0
C for 6 hrs.
Roasting:- Fenugreek seeds (50g) were roasted in an open pan at 1305
0
C for 7 min. It was
continuously stirred with ladle for proper and uniform roasting until it became slight brown
and left a peculiar aroma.
Pressure cooked:- Fenugreek seeds (50g) were cooked in pressure cooker for 20 min. Water
taken in a ratio of 1:5 (w/v). Water was discarded and the cooked sample were dried at 50
0
C.
3.3. Proximate Composition:-
The seeds were analysed for moisture, protein, fat, carbohydrates, ash and
crude fibre.
3.3.1. Moisture content:-
Moisture content of the sample was determined by AOAC (1984) procedure. The
sample (5g) was weighed accurately and transferred to a clean, dried and weighed glass dish.
The contents were dried in a hot air oven at 70
o
C until a constant weight is achieved. After
Initial weight of sample - Weight of dried sample
Moisture (%) = 100
Initial weight of sample

cooling the loss in weight was taken as moisture content and expressed in terms of
percentage.

3.3.2. Protein
Protein estimation was done by Micro Kjeldahl method (AOAC, 1984). Two grams
of the sample was transferred to a digestion flask followed by addition of 3g of digestion
mixture (K
2
SO
4
: CuSO
4
: SeO
2
: 100:20:1 ratio) and 40 ml of concentrated sulphuric acid. The
contents were then digested till a bluish green transparent liquid was obtained. The contents
were then quantitatively transferred to a 100 ml volumetric flask and volume was made up to
100 ml using glass distilled water. A 10 ml aliquot of digested mixture was distilled with
excess of 30 per cent NaOH and the liberated ammonia was collected in 30 ml of 2 per cent
boric acid solution containing 2-3 drops of mixed indicator (10 ml of 0.1 per cent
bromocresol green + 2ml of 0.1 per cent methyl red indicator in 95 per cent ethyl alcohol).
The entrapped ammonia was titrated against 0.01N HCl. A blank was also simultaneously
digested and distilled. Nitrogen content in the sample was calculated as follows:


N= Normality of hydrochloric acid (0.01 N)
% Protein = % Nitrogen x 6.25
3.3.3. Fat:-
Fat content of the samples was determined by Soxhlet Extraction Method (AOAC,
1984). Two grams of the dried sample was transferred to a thimble. The thimble was placed
in the Soxhlet apparatus and fat was extracted for 2-2 hrs with petroleum ether (B.P. 60-
14 x (Sample titre-Blank titre) x N x volume made up of digest
% Nitrogen = 100
1000 x Weight of the sample (g) x Aliquot of digest taken for distillation (ml)

80
o
C). Thereafter, the ether was evaporated and the extracted fat was weighed. Fat percentage
was calculated by the following expression:


Weight of ether extract= weight of extract with beaker-weight of empty beaker



3.3.4. Carbohydrate
The carbohydrate content in the samples was calculated by difference. The sum of
moisture, protein, fat and ash content was subtracted from hundred and the result was
designated as per cent carbohydrate content.
3.3.5. Ash
Ash content in the samples was determined according to AOAC (1984) method. Two
grams of sample was weighed in to a silica dish, charred and ignited in muffle furnace at
550
o
C until carbon free ash was obtained. The dish was cooled in dessicator and weighed.
The ash content in the sample was calculated by following formula:-

Weight of residue= weight of residue with silica dish- weight of empty silica dish
3.3.6. Minerals Composition:-
The seeds were analysed for calcium, phosphorous and iron.
3.3.7. Crude fibre
Crude fibre content of the samples was determined according to AOAC (1984)
procedure. Defatted sample was boiled in 1.25 per cent sulphuric acid for exactly 30 minutes,
Weight of the ether extract
Fat (%) = 100
Weight of the sample

Weight of the residue
Ash (%) = 100
Weight of the sample

filtered through Whatman No.54 filter paper and washed with hot distilled water. The residue
was then boiled in 1.25 per cent sodium hydroxide solution for exactly 30 minutes, filtered
through Whatman No. 54 filter paper and washed with hot distilled water. The filter paper
with the residue was dried in oven at 105
o
C for 3-4 hour till constant weight was obtained.
The difference in weight of filter paper with residue minus filter paper was reported as crude
fibre content of the sample and it was expressed as per cent crude fibre using the following
formula:

3.4. Preparation of aqueous decoction:-
The seeds were washed thoroughly with sterile double distilled water to make these
leaves completely free from any possible contamination. Aqueous decoction of seeds was
prepared by boiling 20 grams of seeds in 100 ml sterile distilled water over moderate flame
for 20 minutes. The aqueous extract was cooled, filtered through Whatman No. 1 filter paper
and then kept in sterile screw capped glass vials at 4C. The aqueous extracts were used to
determine antimicrobial activity of leaves. These crude extracts were reconfirmed as free of
any contamination by plating method (APHA, 1992). These crude extracts were diluted with
sterile double distilled water (as negative control) to obtain required concentrations before
experiments.
3.5. Preparation of solvent extract:-
20 g sample of raw and processed fenugreek seeds were soaked in ethanol(80%) for
48 hours at 24C with stirring (Liu and Nakano, 1996). The extracts were centrifuged and
filtered through Whatman No. 1 filter paper and evaporated using vacuum rotary evaporator
or water bath to near dryness and stored in glass vials in dark at 4C and extracts were used to
determine the antioxidants. These crude extracts were diluted with 10% dimethyl sulphoxide
(weight of residue + filter paper) - (weight of the filter paper)
% Crude fibre = 100
Weight of the sample

(DMSO- which is to be used as negative control) to obtain required concentrations before

experiments (for antimicrobial activity).
3.6. Antioxidant Analysis:-
3.6.1. Estimation of DPPH free radical scavenging method
The free radical scavenging activity of the fenugreek seeds extracts was measured by
measuring the decrease in absorbance of ethanolic DPPH solution at 517 nm in the presence
of the extract (Krings and Berger, 2001). The initial concentration of DPPH was 0.1 mM and
the readind was taken after allowing the solution to stand for 30 min. In cases where the
absorbance decreased too much before the 30 minutes period. The sample was appropriately
diluted. The antioxidant activity was expressed as:-




3.6.2. Ferric reducing power of plasma:-
The ferric reducing power of the seeds extracts was determined by using potassium
ferricyanide- ferric chloride method (Oyaizu, 1986). Different dilutions of extracts amounting
to 1 ml were added to 2.5 ml 0.2 M phosphate buffer (pH=6.6) and 2.5 ml potassium
ferricyanide (1%). The mixtures were incubated at 50C for 20 minutes, after which 2.5 ml
trichloroacetic acid (10%) was added. 2.5 ml of the mixture was taken and mixed with 2.5 ml
water and 0.5 ml 1% ferric chloride. The absorbance at 700 nm was measured after allowing
the solution to stand for 30 minutes.
3.6.3. Total Phenolic Content:-
Total phenolic content was determinbed by adopting Folin-Ciocalteu method
(Velioglu et al., 1998). Basically, 0.2 ml of extracts was added with 1.5 ml of Folin-Ciocalteu
reagent and mixture was allowed to stand at room temperature for 5 minutes. Then 1.5 ml of
sodium carbonate solution (6%) was added into the mixture. Absorbance was measured using
spectrophotometer at 725 nm after 1 hours standing at room temperature. Results were
expressed as gallic acid equivalent in mg/100 g dry weight (DW).



3.6.4. Total Tannin Content:-
The tannin content was determined by the method of Ranganna, 2005. Powdered
sample (0.5 g) was boiled with water (75ml) for 30 minutes and centrifuged at 2000 rpm for
20 minutes and the supernatant was collected. Folin Denis reagent and sodium carbonate is
added to the sample extract, solution is diluted tom100ml with water proper shaking and
absorbance is taken at 700 nm after 30 minutes.
3.7. Stastical Analysis:-
All the data were analysed stats by using technique of analysis of various method
Snede or Cochar linear correlation coefficient was calculated using SISS120 software.
3.8. Antibacterial Activity:-
3.8.1. Test Organisms:-
Four pathogenic bacterial strains were selected for the study to assess antimicrobial
activities of herb seeds against those strains. The strains were Escherichia coli, Styphlococcus
aureus, Salmonella enterica, Shigella flexeneri were obtained from Centre of Food
Technology, Institute of Professional Studies, University of Allahabad, Allahabad, India. All
bacterial cultures were maintained on tryptic soy agar (HiMedia) and subcultured regularly.
Standard inoculums was prepared by sub-culturing 4-5 freshly grown isolated colonies of
each strain in Tryptic soy broth (TSB) and incubated at 37C for 24 hours. Inocula were
standardized with sterile TSB to give final cell load

CFU/ml.


3.8.2. Well Agar Diffusion Method:-
The selected strains of bacteria were inoculated into 10 ml of sterile TSB, and
incubated at 37C for 16-18 hours. Using a sterile cotton swab, the TSB cultures were
swabbed on the surface of sterile TSA plates. Agar wells were prepared with the help of
sterilized cork borer or sterilized micropipette tip with 10 mm diameter. Using a
micropipette, 100 l of different concentrations of seeds extracts were added along with 10%
DMSO as negative and streptomycin as positive controls to different wells in the plate. The
plates were incubated in an upright position at 37C for 24 hours. The diameter of inhibition
zones was measured in mm and the results were recorded (Indu et al.,2006).
3.8.3. Determination of MICs:-
Minimum Inhibitory Concentrations (MICs) were determined by broth dilution
method in culture tubes (Jorgensen et al., 1999). Various concentrations (40, 30, 20, 10, 5,
2.5 mg dry weight/ ml) of extracts were added to broth immediately after inoculating with
fresh 0.2 ml cultures of the stain, keeping final volume at 5 ml. The cultures were incubated
on a rotary shaking incubator or normal incubator at 37C for 48 hours. The lowest
concentrations of the leaves extracts showing no visible growth was considered as the MIC.

Supervisor Student
Dr. Pinki Saini


A



Niharika Dubey
M.sc in Food TECHNOLOGY
Semester IV