MATERIALS

AND METHODS
 Materials and methods

4. Materials and Method:

The experiments relating to main quality and biochemical parameters,

proximate analysis and its evaluation were undertaken at the Department of
Bioscience, Sardar Patel University, Vallabh Vidyanagar.

4.1 Materials:

Promising and newly developed Indian and exotic germplasms of Brassica

oilseed species, namely Brassica juncea L., Brassica campestris L. (Yellow
and Brown sarson), Brassica napus L., Brassica carinaeta were received from
the Research Scientist, Oilseed Research Station, Sardarkrushinagar
Dantiwada Agricultural University, Sardar Krushinagar, Gujarat. All samples
are cleaned and stored in airtight bottles.

The sources of the chemicals and related reagents used in this study were
purchased from standard companies. The related standards for this study
were purchased from Sigma, USA.

4.2 Estimation of total oil content:

The total oil content was estimated using method given by Sadasivam (1992).
Principle: Oil from a known quantity of the seed extracted with petroleum
ether or hexane. It is then distilled off completely, dried, the oil weighed and
the percentage of oil is calculated.

57
 Materials and methods

Reagents and materials:

Petroleum ether (40-60°c)

Thimble (Whatman)
Absorbent cotton
Soxhlet apparatus

Procedure:

> 10 g clean dry seeds were finely grand in mortar pestle and put into
thimble.
> Cotton plug was placed at the top of thimble to evenly distribute the
solvent as it drops on the sample during extraction.
> The sample packet was placed in the butt tube of the Soxhlet
apparatus.
> Extract with petroleum ether (40-60°c) with the flew drop rate at150
drops/min for 6 to 8 hours.
> Allow to cool and dismantle the extraction flask.
> Extraction flask was kept on water bath to remove the odour of
petroleum ether.
> Flask was cooled at room temperature.
> The dirt and moisture outside of the flask was removed and weigh the
flask.

Calculation:

Oil in sample (%) = Weight of oil (al x 100

Weight of sample (g)

58
 Materials and methods

4.3 Physico chemical characteristics:

4.3.1 Estimation of acid value:

The acid value was determined using method given by Cox H.E. (1962).

Principle: The free fatty acid in an oils estimated by titreting against KOH in
the presence of phenolphthalein indicator. The acid value is the mg of KOH
required to neutralize the free fatly acids present in 1 mg sample.

Reagents:

1%phenolphthalin in 95% alcohol.

0.1 N KOH
Neutral solvent - 25ml ether + 25ml 95% alcohol. Add 1 ml 1%
Phenolphthalein and neutralize with N/10 alkali.

Procedure:

> 5 g oil was dissolved in 50 ml neutral solvent.

> Add few drops of phenolphthalein.
> The mixture was titrated against 0.1 N KOH with constant stirring.
> Shake the mixture constantly until a pink colour, which persists for
fifteen seconds, was obtained.

59
 Materials and methods

Calculation:

Acid value = Titer value x N of KOH x 56.1

(mg KOH/g) Weight of sample (g)

4.3.2 Estimation of saponification value:

The saponification value was determined by using method give by William
Horowitz (1975)

Principle: A known quality of oil is refluxed with an excess amount of

alcoholic KOH. After saponification, remaining KOH is estimated by titrating
against standard acid.

Reagents:

0.5N alcoholic KOH

Standard 0.5N HCI (Standardize against 0.5N Na2C03)
1% phenolphthalein in alcohol.

Procedure:

> Accurately weigh 2 g of oil was taken in the flask and add 25 ml
alcoholic KOH. Dissolved the oil completely.
> The flask was connected to air condenser and boil for 30 minutes on
boiling water bath.
> Cool the mixture and add 3 drops of phenolphthalein as an indicator.
> The mixture was titrated against std. 0.5N HCI until the pink colour
disappears.

60
 Materials and methods

> Similarly the blank was titrated in the absence of oil.

Calculation:

Saponification value of oil = (Blank - Titer) x 28.06

Weight of oil (g)

4.3.3 Estimation of Peroxide value:

The peroxide value was estimated by using method give by Cox H.E. (1962)

Principle: Peroxide value is a measure of the peroxides contained in the oil.

The peroxides present are determined by titration against thiosulphate in the
presence of Kl. Starch is used as an indicator.

Reagents:

CH3COOH: CHCI3 (3:1) v/v

Saturated potassium iodide (Kl) - Dissolve excess amount of Kl in fresh
boiled water. Excess solid should remain. Kept in the dark.
Standard 0.01 N Na2S203 5H20 (Standardize against 0.01 N K2Cr207)
1 % starch - 1 gm starch dissolved in 10 ml water. Pour the suspension into
90 ml of boiling water for 2 minutes.

Procedure:

> 5 gm of oil was dissolved in 30 ml CH3COOH: CHCI3

> Mix and dissolve the oil completely.

61
 Materials and methods

> 0.5 ml of saturated Kl was added in to above mixture. Mix well and
allowed stand for 1 minute.
> In the mixture add 430 ml water and 3-4 drops of starch and mix.
> The mixture was titrated against std. 0.01 N Na2S203.5H20 until the
blue colour just disappears.
> The blank was titrated similarly in the absence of oil.

Calculation:
Peroxide value = Ax N x 1000
Weight of oil (g)
Where, A = Test - blank
N = Normality of Na2S203

4.3.4 Estimation of Iodine value:

The iodine value was determined by using method of AOAC (1975)

Principle: The oil contains both saturated and unsaturated fatty acids.
Halogens add across the double bonds of unsaturated fatty acids to form
addition compounds. Iodine monochioride is allowed to react with fat in the
dark. Iodine gets incorporated into the fatty acid chain wherever the double
bond exist. The amount of iodine consumed is then determined by titrting
amount of iodine (after adding Kl) with standard Sodiumthiosulphate and
comparing with a blank in which the oil is omitted. Hence, the measure of
iodine absorbed by oil gives the degree of unsaturation.

62
 Materials and methods

Reagents:

Hanus iodine solution: Dissolve 8 gm iodine in 500ml glacial acetic acid and heat
to got dissolved. Cool and add 1.5ml of bromine.
15% Potassium iodide (Kl) in water.
Standard 0.1 N Na2S203.5H20 (standardize against 0.1N K2Cr207)
1% starch
Chloroform AR

Procedure:

> 2 g of oil was dissolved in to 20 ml chloroform and dissolved it completely.

> 25ml Hanus iodine solution was added in to mixture. Mix well and kept
the mixture in dark for 30 minutes.
> 20ml Kl was added in the mixture and mix well.
> The mixture was titrated against standard 0.1 N Na2S203.5H20 using
starch as an indicator with constant stirring to extract the iodine from
the chloroform layer.
> The blank was treated similarly without oil.

Calculation:

Iodine value = Ax Nx 0.1269x100 g l2 /100g of oil

Weight of oil (g)
Where,
A = Blank - test (ml of Na2s203)
N = Normality of Sodiumthiosulphate

63
 Materials and methods

4.3.5 Determination of Unsaponifiable matter:

The unsaponifiable matter was determined by method given by manual
methods of Test and Analysis for Food (Oils & fats), Directorate General of
Health Services, Ministry of Health & Family Welfare, Govt, of India, New
Delhi.

Principle: The oil is completely saponified with alcoholic potassium hydroxide

solution and extracted with petroleum ether. The petroleum ether extract is
washed with aqueous alcohol and then again with water. The washed ether
extract is evaporated and the residue weighed. Unsaponifiable matter is this
residue minus the fatty acid present in it, which is determined by titration with
sodium hydroxide solution in alcoholic medium.

Reagents:

Alcoholic Potassium hydroxide solution - Dissolved 70g KOH in 90ml water

and add equal amount of ethyl alcohol. Make up to 1 liter. Allow the mixture to
stand overnight, decant the clear liquid.
95% ethyl alcohol
1% phenolphthalein
Petroleum ether
Aqueous alcohol
0.02N standard sodium hydroxide (NaOH) solution
Acetone AR

64
 Materials and methods

Procedure:

> 5 gm of oil was dissolved in 50 ml of alcoholic KOH.

> The mixture was boiled gently but steadily under reflux condenser for 1
hour or until the saponification was to be completed.
> The condenser was washed with about 10 ml of ethyl alcohol.

> The mixture was kept for cooling and transferred it in to a separating
funnel.

> The transfer was completed by washing the flask first with ethyl alcohol
and then with cold water.
> 50 ml of water was added in to the separating funnel followed by an
addition of 50 ml of petroleum ether.
> Insert the stopper and shake vigorously for at least one minute and
allow settling until both the layers clear.

> The lower layer was transferred and repeat the ether extraction for at
least six times more using 50 ml of petroleum ether for each extraction.
> The ether extracts were collected in separating funnel and it was
washed for three times by 25ml aqueous alcohol.

> Again the ether layer was washed with 20 ml water until the wash
water no longer turns pink on edition of a few drops of phenolphthalein
solution. Do not remove any ether layer.
> The ether layer was transferred to a flask containing a few pieces of
pumice stone.
> The mixture was kept on a water bath for the evaporation. The last
traces of ether were removed by placing the flask in air oven at 80°c for
about one hour.
> The last traces of moisture were removed by the addition of few ml of
acetone and dry at 50°c for 30 minutes.
 Materials and methods

Biuret reaction of the protein with the alkaline cupric tartrate solution. The
intensity of the blue colour is measured calorimetrically at 660 nm. ^his
intensity of the colour depends on the amount of these aromatic amino acids

present and will thus vary for different proteins.

Reagents:

Solution A: 2% Na2C03 (Anhydrous) in 0.1N NaOH

Solution B: 0.5% CuS04 5H20 in 1% Na-K- tartarte
Solution C: (Alkaline copper solution): Mix 50 ml of solution A with 1 ml of
solution B just prior use.
Solution D: Folin Ciocalteu Reagent regent (FCR, Commercial)
Stock standard: 50 mg of Bovine Serum Albumin (Fraction V) in 50 ml distilled
H20 in std. Flask.
Working standard: Dilute 10 ml of stock standard in 50 ml with distilled H2O in

std. Flask. One ml of this solution contains 200ng protein.

Procedure:

> 0.2, 0.4, 0.6. 0.8 and 1.0 ml of the working standards were taken in to
test tubes.
> 0.1 ml and 0.2 ml of the sample extract were taken into other test
tubes.
> The volume was made up to 1 ml with distilled H20 in all tubes.
> A tube with 1 ml of distilled H20 was served as a blank.
> 5 ml of solution C was added in all tubes mix well and incuba:e at
room temperature for 10 minutes.

67
 Materials and methods

> Add 0.5 ml FCR (solution D), was added in to mixture, mix well and
immediately incubate at room temperature in dark for 30 min.
> The absorbance was red at 660 nm against the blank.

> Plot a standard graph. The amount of protein was calculated in the
sample and expresses the results as mg/g or mg/100mg sampel or

percentage.

Protein (g%) =

^Std. Conc.(pgP r r
rSample OD~\ rvolume (ml)~\
X X
P° 1X 100
x
1

Aliquot (ml) 1
X Sample Wt (g)
Std. Conc.(pg) 1000 100
V J V J L J L J L J

68
 Materials and methods

4.5 Determination of Fatty acids profile:

The determination fatty acid profile and preparation of FAMEs were performed
by method given by Sadasivam (1992).

Rapeseed mustard oil contains lowest amount of saturated fatty acids,

essential fatty acids, oleic acid and erucic acid. It has very unique fatty acid
profile, which is determined by Gas chromatography.

Principle: Fatty acids are made volatile by converts them in to methyl esters.
The conversion of fatty acids in methyl ester is carried out directly by trans
esterification i.e. from glycerol ester to methyl esters. Esterification can also
done after saponification. The esters are identified and quantified by injecting
into GC- FID and comparing with a set of standard esters.

Reagents:

Petroleum ether (40-60°c) AR - Merck Co.

Methanol AR - Merck Co.
Potassium hydroxide AR - Merck Co.

Materials:
Borosil stopper glass test tube
100 nl glass syringe
Apparatus:
Gas chromatograph (Shimadzu GC- 15A) with FID.

69
 Materials and methods

Condition of the apparatus:

❖ Injector: Maintain the injectors of the vaporizing type at least at 210°C

❖ Oven temperature: 210°c,

❖ Column: 2.1m stainless steal column packed by 15% DEGS (Dietylase
glycol succinate) with 2mm x 3mm ID and WAW 80/100 mesh.
(Maximum temperature- 200°c.)
❖ Column temperature: 195°c.
❖ Detector. Flame ionization detector (FID)
❖ Detector temperature: 220°c.
❖ Carrier gas : Nitrogen
❖ Other gases : 0% Air and Hydrogen

Procedure:

> The three valves of hydrogen gas, nitrogen gas and air were opened
and adjusted the flow rate at 3kg/cm2, 6kg/cm2 and 6kg/cm2

respectively.
> The instrument was started and the temperature of column (oven),

injector block and detector block were adjusted at 195°c, 210°c and
220°c. respectively.
> The conditioning instrument was kept for 30 minutes.

> 1 |il petroleum ether was injected before injecting the sample or
standard for washing.

> Thenl ^l sample (FAME) was injected.

70
 Materials and methods

Preparation of FAME:

> 8 to 10 drops of oil was taken in clean glass stopper test tube.

> In the test tube 10 ml petroleum ether (40-60°c) was added.

> In the mixture 0.5 ml 4% methanolic KOH was added.
> The mixture was gently shaked for 30 times.
> The mixture was kept steady for 20 minutes.

> 1 fj.l was injected from the upper layer (FAME) in to GC-FID.

4.6 Determination of Glucosinoiate content:

The Glucosinoiate was determined by ISO (1992) HPLC method.

Brassica oil seed species (Rapeseed - mustard) are important animal and

human food source, mainly because of its high quality of oil and high protein
seed meal. However, the high glucosinoiate content in meal make it
unacceptable for animal consumption. There is a need to eliminate
antinutritive glucosinoiate present in the meal. Determination of individual or
total glucosinoiate content in Rapeseed - mustard meal is carried out by
advance HPLC method.

The suggested protocol to determine (quantity) individual or total

glucosinoiate was faster (reduction of extraction number and desulfatation
time). It was very useful for the analysis a high number of samples. Rate on
other hand analysis costs were reduced (manpower, delay of desalfatation,
costs of HPLC grade solvent). This was more precise and advance method.

71
 Materials and methods

Reagents and materials:

Rapeseed mustard meal

Methanol (HPLC grade)
Acetonitrile (HPLC grade)
Water (HPLC grade)
Sinigrin (Internal std., Sigma)
Helix pomatia sulfafase (Type H-1,Sigma)
DEAE Sephadex A- 25 (Sigma)
0.2 micro m filters (Biored Co.)
Biored ion exchange columns
Micropipettes and tips
HPLC system (Agilent 1100) with VWD and data recorder.
50 micro liter syringe.
Epperndofs

Procedure:

> 200mg of rapeseed meal was homogenized with 10ml of 75°C

methanol / water (70/30) and with an internal standard (Sinigrin) in
mortar pestle.
> The mixture was permanently agitated with a magnetic stirrer for 10
minutes.
> Then centrifuged the mixture for 15 minutes at 5000 rpm.
> 1-5 ml of centrifuged extraction of samples was put on the top of an ion
exchange column containing Dry DEAE sephadex A- 25 (25mg) and
then adds 10 micro liters of Helix pomatia sulfatase.

72
 Materials and methods

> The prepared ion exchange column was kept for minimum 11 hours. It
was necessary for the complete desulfatation in operating condition.
> The desulfo glucosinoiates were eluted and released with 4 x 0.5 ml
ultra pure water (Milli Q form mille pore).
> All the fractions were collected in high quality sterile vials for the
analysis.
> Desulfo- glucoinolates were separated by HPLC using an intertsil
30DS-3 column (100 x 3 mm, 3 micro m) with water / acetonitrile
gradient (from 2% to 25% in 35 min).
> Variable wavelength detector (VWD) was used for analysis at 229 nm.
> The data were recorded for the results and for the calculation.

4.7 Statistical analysis:

Arithmetic Mean and Standard Error were calculated for data the results were
statistically evaluated using One - way analysis of variance (ANOVA) using
SPSS (10.0 version). Individual differences between means were determined
with the Duncan post-hoc test.
A P<0.05 was considered as statistically significant.

73