FPT 243 - Processing Technology of Legumes ana se s

Date:
E x e r c i s e : 2

Determination of proximate composition of selected pulses and oilseeds.

Title

Objective:

Determination of proximate composition viz. moisture, total ash, protein, crude

fbre, crude fat and total carbohydrates of selected pulses and oilseeds.

Oven,
Apparatus: Ov Muffle furnace and Sample
I. M o i s t u r e

Weigh previously dried and tared dish (W).

Weigh accurately about 5g of sample (W1) in a previously dried and tared dish.

Place the dish with its lid underneath in the oven maintained at 130 133°C for
2h.
The time should be reckoned from the moment the oven attains 130°C after the
dishes have been placed.

Remove the dish after after attaining constant weight, cool in the desiccators and
weigh (W2).
Observation table:

Pulses and W Wi W2
S.No.
Oilseeds (g) () (g
1.

2
Calculation: To calculate the moisture content of the sample following equation
can be used.

Moisture (%) Wi-W2

W1- W
x 100
Total solids (%) = 100-Moisture (%)

Results:
S. No. Moisture content (% wb) Total solids (%)|
Pulses and Oilseeds

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 FPT 243 - Processing Technologyof Legumes
and Oilseod.
II. Total ash
ceds
Weigh previously dried and tared dish (W).
Take fresh sample (W) for the determination, rather than lef
than left over
determination of moisture.
after
Ignite the fresh sample with the flame of a burner till charred.
Transfer muffle furnace maintained at 550
to a -

600°C and continue

in.
grey ash is obtained. ion till
Cool in desiccator and
a
weigh (W2).
Repeat
the process of
heating,
cooling and weighing at half hour
difference in weight in two consecutive intervalc till the
weighing is less than 1 mng.
Note the lowest
weight.
If ash still contains black particles add 2-3
Break the ash and drops of pre-heated water at 60.
is white or
evaporate dryness 100-110°C. Re-Ash at
to at .
slightly grey. 550°C. Until ach
ash
Observation table:

S. No. Pulses and W

Oilseeds Wi
(g) W2
1. (g)
(g)
2.

Calculation: To calculate the total ash

content of the
can be used. sample following equation
Total ash (%)) =
W
W-wX100 x 100

Results:

S. No. Pulses and Oilseeds

1. Total ash content
(%)
2.

College of Food Processing

Technology & Bio-Energy, AAU, Anand 10
 FPT 243 - Processing Technology ofLegumes and Oilseeds

IL. Protein (Kjeldahl method)

Principle:

Theprotein conte is determined from the organic ogen content by Kjeldahl method.
The various ni nitrogenous compounds are converted into ammonium sulphate by boiling
trated sulphuric
with concentrat
acid. The ammonium formed is decomposed with an
alkali (NaOOH
sulphate
H) and the ammonia liberated is absorbed in excess of standard solution of
id and then back titrated with standard alkali.
aci

Apparatus: Kjeldahl digestion flask (500 or 800

mL), Kjeldahl distillation apparatus
(same digestion flask fitted with rubber
stopper through which passes lower end
of efficient rubber bulb or trap to
prevent mechanical carryover of NaOH
during distillation or apparatus as shown
below), Conical flask (250 mL), Burette
(50 mL) and Sample

Reagents: Kjeldahl distillation apparatus

Concentrated sulphuric acid (Specifiic gravity
1.84)
in 1000 mL
45% Sodium hydroxide (Dissolve 450g of Sodium hydroxide pellets
water)
Standard sulphuric acid solution 0.1N
Standard sodium hydroxide solution 0.1
N

Methyl red indicator solution (Dissolve 0.5g methyl red in 100 mL of alcohol)

Procedure:
transfer to a 500 or 800 mL Kjeldahl flaskk
Weigh 1-2g of the sample and
about
flask.
to see that no portion
of the sample clings to the neck of the
taking care
mL of
15g of Potassium Sulphate and 40
Add 0.7g of Mercuric oxide, of
oxide is added to increase the rate
concentrated sulphuric acid (Mercuric environmental/safety
acid digestion. Because of
organic breakdown during sulphate can be used.
concerns over handling
and disposal of mercury, copper
into
view as mercury vapours might escape
This is important from safety point of Missouri catalyst tablets
the distillation process. Also
the environment during Sodium sulphate & 48.9%
tablets (Composition: 48.8%
known as Kjeldahl
sulphate) can also be
used.
Potassium sulphate &0.3% copper

Add two to three glass beads.

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College of Food
 Technology of Legun
umes and Oilseed
FPT 243-Processing

the stand in the digestion ou

P l a c e the flask in an inclined position

on
chamber and
digest.
flame until the
initial frothing ceases and th..
Heat the flask gently at low rotate the flask several xlu
boils moderate rate. During heating
steadily at a

hour or more until the colour of the dicn.

Continue heating for about an
IS pale
blue.
If black specs are present after 30 minutes of digestion, wrap the
vessel
aluminiunm foil and keep for 2-3 minutes. By doing this black specs would with
down from the walls in the digestion mixture. If the specs are still present
mo
remove
the vessel from heat and allow to cool for 10 mins. Do not modify the
intensity in the whole process. Alternatively, few drops of water may also he
down across the side of the flask. pour
Cool the digest and add slowly 200 mL of water.

Cool, add piece of granulated Zinc or anti bump granules and carefully nou
a

down the side

of the flask sufficient Sodium Hydroxide solution (450g/L) to pour
the contents
strongly alkaline (about 110 mL) before mixing the acid and 1ake
layer. alkaline
Connect the flask to a distillation apparatus
and condenser. incorporating an efficient flash head
.To the condenser fit
delivery tube which dips just below the
a

pipetted volume of standard acid contained surface of the

in a conical
(Precaution: The receiving solution must remain flask receiver.
ammonia). below 45°C to prevent loss of
Mix the contents of
the digestion flask and boil until
the receiver. 150 mL have distilled
into
Add 5 drops of methyl red
indicator and titrate with
Hydroxide solution. standardized 0.1 N Sodium
Carry out a blank titration
Safety and GLP Aspects: simultaneously. 1 mL of 0.1 NHSO4=
0.0014g N
.Perform digestion l distillation steps
Wear under well
gloves and ventilated fume hood.
sodium hydroxide. safety glasses when
handling concentrated
Ensure neutralization sulphuric acid a
of the Acid, before
Ensure check of disposal.
e.g. Before each tightness of
apparatus distillation, distillation
check apparatus and
is
perfectly clean and free that the rubber performance of
tnura
analysis remove it and rinse bung of the
the distillation
with water. from traces of salt. fter eachdistilia
After each seric
series of

College of Food
Processing Technology& Ria
 T 243-Procexsing1echnotoyyof Legumesand Oilseeds
Add hydrogen per0x ide (30,
w/wi afier the addition of sulphuric acid to prevent
Cxpioson%, T his
wOuld prevent
and foaming propcrties. foaming, caused by products with high 1at
cono
Calculatlon: Thc 1ollowing cquation can hc used to calculate protcin content r tne
sample;
Calculate proteln as = Nx 6.25

Protein on dry wt. basis, % Proteln Content x 100

100-Molsturecontent
Ideally the protein content of food stuff is calculated by multiplying its total nitrogen
contcnt by a factor 6.25, This factor is used whenever the nature of the protein is
unknown or when the product to be analysed is a mixture of different proteins with
different factors. However use of different Nitrogen conversion factors for different
matrices may lead to better accuracy of results.

Few cxamples:

6.38- for Milk and milk products (cheese/ cascinate/whey and derivatives/ yoghurt/
lactose/ ice-cream).

6.25-for Infant formula (hydrolysed / partially hydrolysed/ follow-on)

6.25-for Infant cereals with milk (complete cereals, to be reconstituted with water)

5.8- for Infant cereals without milk (standard cereals, to be reconstituted with milk

Results:

S. No. Pulses and Oilseeds Protein content (%)

2
IV. Crude fiber

Reagents:
Rcagents/chemicals used may be of AR grade.

Dilute Sulphuric acid 1.25% (w/v) accurately prepared

Sodium Hydroxide solution- 1.25% (w/v) accurately prepared
volume
Ethyl alcohol95% by

College of Food Processing Technology & Bio-Energy, AAU, Anand 13

 FPT 243- Processing Technology of Legumes
mes and Oilseeds

Petroleum ether
Procedure:
Weigh accurately about 2.5-3g sample
ether.
traction apparatus
and transfer to an extraction

(Soxhlet extractor) and extract with petroleum

Air dry the extracted sample and transfer to a dry 1 L conical flask.

(>10%), then treat it with

percentage of fat in the product high
is a
If
acetone and petroleum benzene. Excess of fat, if not removed on initial ixture
initial defattin
may affect the end result.
Transfer the whole of the boiling acid to the flask containing the defatted
and immediately connect the flask with a water cooled reflux
flux condenser material
condenser aand
so that the contents of the flask begin to boil within 1 minute. heat
Since there is a risk of foaming and bumping, add a few drops of octanol
after
addition of sulphuric acid, to prevent foaming and boiling/glass beads in the filask
to prevent bumping.
Rotate the flask frequently taking care to keep the material from remaining on
the
sides of the flask and out of contact with the acid.

Continue boiling for exactly 30 minutes.

Remove the flask and filter through fine linen (about 18 threads to a cm) held ina
fannel and wash with boiling water until the washings are no longer acid to litmus
(Crucible filter may be used in filtration steps as accidental tearing of linen may
lead to safety concerns and also accuracy of results may be better with use of
crucibles, Porosity 2 filter crucible, 50 mL volume- can be used).
Filter aids can be added for better filtration and recovery of the analyte [filter aid
Celite (R) 545].

Bring to boil some quantity of Sodium hydroxide solution.

Wash the residue on the linen into the flask with 200 mL of boiling Sodium
hydroxide solution. Immediately connect the flask to the reflux condenser and boil
for exactly 30 minutes.

Remove the flask and immediately filter through the filtering cloth.
Thoroughly wash the residue with
boiling water and transfer to a Gooch crucio
prepared with a thin compact layer of
ignited asbestos.
Wash the residue thoroughly first with hot water and then with about 15
ethyl alcohol.
m

Dry the Gooch crucible and contents at 105+2°C in air until

stant

weight is achieved.
an oven co
Cool and weigh.

College of Food Processing Technology & 14

Bio-Energy, AAU, Anana

 FPT 243-ProcessingTechnologyof Legumes and Oilseeds
ncinerate the contents of the Gooch crucible in a muffle furnace until all
carbonaceous matter is burnt.
Cool the Gooch crucible containing ash in a desiccator and weigh (Dry the
crucible with its residues in an oven at 130°C for 2
hours).
Performance characteristics to be defined in the current method as proposed below:
Limit of detection of approximately in the
0.2g/100g crude fibre product.
Repeatability limit of 0.3g/100g when the crude fibre content is less than
10g/100g product and 3% of the average when the crude fibre content is equal to
or greater than 10g/100g product.
Precautions:

I t is recommended to use fume-hoods for handling Sulphuric acid, Sodium

hydroxide etc.
Ensure neutralization of the acid/base used prior to disposal.
Dilute the concentrated sulphuric acid first.
Against use of asbestos it is recommended to use filter aid Celite (R) 545, 22140
Fluka.
Observation table:

S. N o . P u l s e s and W W W2
Oilseeds (g
1.

Calculation: Calculate crude fibre on dry wt. basis by giving correction for the moisture
content.

Crude fiber (%) = W2 x 100

W

Where,
W = wt in g of Gooch crucible and contents before ashing

W2 =wt in g of Gooch crucible containing asbestos and ash

W =wt in g of the dried material taken for the test

Results:

College of Food Processing Technology & Bio-Energy, AAU, Anand 15

 FPT243-Processing Technology of Legumes and Oilseeds
S. No. Pulses and Oilseeds Crude flbre content (%)

1.

2.

V. Crude fat (Soxhlet extraction method)

Total fat refers to the sum of triglycerides, phospholipids, wax ester, sterols and minor
amount of non-fatty materials. Crude fat includes free fat whereas total fat includes free
and bounded fat.

Apparatus: Soxhlet assembly (Condenser, Extraction tube, Flat bottom flask and Heating
mantle), Thimble and Sample

Reagents:
Diethyl ether, peroxide free
Petroleum ether, boiling range 40-60°C

Procedure:

Weigh about 10g (W1) of the material in a thimble.

.Plug it with cotton and place in the extraction tube.

Add 50 mL petroleum ether and 50 mL diethyl ether in the flat bottom flask (W)
and attach it with the extraction tube and condenser.

P u t assembly on heating mantle and start cold water circulation in the condenser.

Start the heating of the solvent and continue the extraction process for about 4-16h
depending on the fat content of the sample.
After completion of extraction remove the round bottom flask, evaporate solvents
in on the hot
plate and take weight (W2) of the flask containing fat.
Observation table:

Pulses and
S. No.
Oilseeds
W
W W2
() (g) (g
1.

2.
Calculation: Crude fat can be calculated
using following formula;

Crude fat (%) =

* W
x 100

College of Food Processing Technology &

Bio-Energy, AAU, Anand 16
 FPT 243-Processing Technology ofLegumes and Ollseeds
Results:

S. No. Pulses and Ollseeds Crude fat content (%)

1.

VI. Total carbohydrates (Difference method)

100 obtain
Add moisture, ash, protein, fibre and fat content and deduct the value from
to

total carbohydrates content by difference.

Observation table:

Crude fat
Pulses and Moisture Total ash Protein Crude fibre
S. No. (%) (%) (%) (%%)
Oilseeds (%)
1.
2.

be calculated using following formula.

Calculation: Calculate crude fat can

+ Fibre+Fat)
Total carbohydrate (%) =
100- (Moisture + Ash+ Protein

Results:
Pulses and Oilseeds
Total carbohydrate content (%)|
S. No.
1.

2.

Conclusion:

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