Chapter: 1

1.1) Introduction
• Pakistan council of scientific and industrial research (PCSIR) was
establishing in 1953 under societies act to promote the cause of
science and technology in country. PCSIR was established by the
scientist “Prof. Dr. Salim-uz-zaman Siddiqui” in 1953 for the
development of scientific and technical research and development
and to provide infrastructure to the industrial development.
• The 21- member Council is the policy making body of the PCSIR,
1 which is composed of Chairman , three member of the Governing
body , three Directors of PCSIR Laboratories, four representative
from ministries, four Directors of Industries , one from each
province and six representative of the industry.
 1.2) PCSIR Labs, Complex Lahore
• The Lahore Laboratories complex formally known as the West
Regional Laboratories started functioning with the staff in a
wing of the Punjab University, institute of chemistry in 1953.
• This setup was shifted in 1956 to the present parliament site for
which 68.5 acres of the land were earmarked by the Punjab
Government in 1955.
• The Laboratories are assisting the academic institutions by
providing facilities to their B.Sc. M.Sc., M.Phil. And PhD.
2
Students. Various centers and division are manned by high
trained researchers. Necessary additional facilities such as
workshops, libraries and pilot plants, are adequately available
meet the R&D support needs.
 1.3) Objectives of PCSIR
 The optimum utilization of indigenous raw material resources
for the development of industrial processes
 The development of technologies around local resources from
bench to pilot plant stages, the leasing them out for industrial
exploitation leading to import substitution and export
enhancement.
 The conduct R&D work on problems feed by the industrial
3 sector and maintains linkage through seminars, workshops,
publications, and provision of assistance to academic
institutions.
  The undertake cooperative research with local and foreign R&D
organization and commerce-industrial outfits on projects of
national interest.
 Human resources development through organized training course
and diffusive on job grooming of manpower for industry and
research center to broaden the science & technology in country.

4
 1.5) Main Activities
I. Research and Development work
II. Industrial linkage

1.6) Main group of the centre

 Cereal Technology Laboratory
 Food and Vegetables Proceeding Laboratory
 Dairy / Feed Nutrition Technology Laboratory
 Meat and Meat Products Laboratory
5  Food Additives & Contaminant Laboratory
 Microbiology Laboratory
 1.4) Food and Biotechnology Research Centre
• Food and Biotechnology Research Centre was Established in 1977
by the merger of two separate divisions namely, Biological
Evaluation and Fermentation and Food Technology & Nutrition.
• Among the major objective of this centre are the assistance in the
establishment / development of the Food and Biotechnology in the
country through value added output from low-price raw material ,
bio material, bio resource development and utilization , quality
6 assurance of finished products , resolution of industrial trouble-
shooting and attracting small and medium level Entrepreneurs to
establish the industry.
 1.7) Introduction to Dairy and Meat
Laboratory
• Milk testing and quality control is an essential component of any
milk processing industry.
• In FBRC dairy and nutrition feed lab all facilities are available for
adulteration, protein, fat, SNF, TSS, Ash, moisture and protein test
of pure milk, UHT milk ,Pasteurized milk, powder milk, Skim milk,
adult nutritional formulas, child nutritional formula ,ice cream,
7 butter, margarine and spread.
• For all kinds of other milk, milk product, meat and meat products
testing facilities are available in this lab and accredited methods of
testing are follow in this lab to check quality under ISO/IEC
17025:2005.
 Chapter 2
Lab Safety Rules
1. Conduct yourself in a responsible manner at all times in the
laboratory.
2. Follow all written and verbal instructions carefully.
Never work alone in the laboratory.
3. When first entering a science room, do not touch any equipment,
chemicals, or other materials in the laboratory area until you are
instructed to do so.
8
4. Do not eat food, drink beverages, or chew gum in the laboratory.
5. Always work in a well-ventilated area.
6. Observe good housekeeping practices.
 7. Dispose of all chemical waste properly.
8. Labels and equipment instructions must be read carefully before
use.
9. Keep hands away from face, eyes, mouth, and body while using
chemicals or lab equipment.
10. Know the locations and operating procedures of all safety
equipment including: first aid kit(s) and fire extinguisher.
11. Any time chemicals, heat, or glassware are used, students will wear
safety goggles.
9 12. A lab coat or smock should be worn during laboratory
experiments.
 Chapter: 3
Solution Preparations:
3.1) Normal Solution:
Normality of solution is the gram equivalent weight of a solute per liter
of solution.

Normality = Gram equivalent weight/ Liter of Solution

10 3.2) Molar Solution:

A molar solution is an aqueous solution that contains 1 mole (gram-
molecular weight) of solute in 1 liter of the solution

Donated by (M)
 M = No of moles of solute/ liter of solution

3.3) Percent Solution:

A relationship of a quantity of solute to quantity of solution multiply by
100, expressed in terms of mass of solute per mass of solution multiply
by 100.

Percent solutions can take the form of weight/volume % (wt/vol % or

w/v %)
11
3.4) PPM:
It is defined as number of parts of solute per million parts of solution

PPM=mg/kg
 3.5) PPb:
It is defined as the number of parts of solute per billion parts of
solution.

1 ppb = 0.001 mg/kg; 1 mg/kg = 1000 ppb

3.6) Molecular Weight:
Molecular Weight is the weight of all atoms in a given molecular
formula.
12
3.7) Concentrated solution:
A solution that contains high concentration of solute is called solution.
 3.8) Specific Gravity:
Specific gravity is defined as density of sample divided by density of
water. it is required for determination of solid not fat (SNF) and total
solids (TS) of milk sample.

3.9) pH:
Hydrogen ion concentration of a solution is called pH.

13 3.10) Acidity:
Acidity of milk means total acidity (natural + developed) or titratable.
 Chapter: 4
Proximate Analysis of Milk
4.1) Determination of Fat in dried milk sample

4.1.1) Principle:
The Gerber Sulphuric acid is used to dissolve the casein in milk
without charring the fat. The Iso-amylalcohal is used to lower down the
14 surface tension in the medium.
On centrifugation, The fat being lighter will be separated on top of the
solution.
 4.1.2) Apparatus:
 Butyrometer

 Test tube stand

 Cork

 Water bath

 Pipette

15 4.1.3 Instrument:
 Centrifuge
4.1.4) Reagents:
 200ml of Sulphuric acid H2SO4(92%v/v)
  Take 187ml of concentrated H2SO4 and make its volume up to
mark at 200ml with distilled water.
 Iso-amyl alcohol
4.1.5) Procedure:
1. Take 10ml of 92% H2SO4 solution in a Butyrometer.

2. Add 10.94ml milk sample gradually through the corners of the

butyrometer.

16 3. Add 1ml of Iso-amylalcohal gradually through the corners of the

butyrometer.

4. Add 2ml of distil water until it touches the column just below the
neck.
 5. Put tightly fitted cork at the neck.

6. Mix it well.

7. H2SO4 gives exothermic reaction and isoamylalcohal help to

separate milk fat .Protein and all other constituents are burned due the
presence of H2SO4 rather than fat.

8. Centrifuge at 1100rpm for 5 minutes.

9. After it tempered it to Boiling Water bath.

17
10.Note the fat percentage in milk sample.
 4.2) Determination of moisture in Powder milk
4.2.1) Principle:
The sample is dried to constant weight at 102 0C for 3hours in hot air
oven and the loss in the weight reported as moisture.

4.2.2) Apparatus:
 Petri dish

 Spatula

18  Tongue

 Desiccator
 4.2.3) Instruments:
• Weighing balance
• Hot air oven
4.2.4 Procedure:
1. Take petri dishes and put into hot air oven for 1 hour.

2. Place the empty dishes into desiccators and allow it to room

temperature.
19
3. Weight the empty petri dishes and note the reading.

4. Then place petri dishes onto weighing balance and weight 2.5g of
sample in case of dried milk sample and note the reading .
 5. Then place these dishes into Hot air oven at 102 0C for 3 hours.

6 .After that remove the dishes from oven and place into desiccator that
help to allow it to room temperature without outside moisture
absorbance.
7. Again weight the sample after drying onto weighing balance and note
the reading.

4.2.5 Calculation:
20 % Moisture = (Wt.of sample before drying-Wt.of sample after drying)/
wt. of sample x100
 4.3) Determination of Ash in milk
4.3.1) Principle:
This test is performed to check the level of minerals in milk sample. All
volatile compounds are evaporated and sample turn into ash at (550
0C) in muffle furnace.
4.3.2) Apparatus:
 Crucible
21
 Spatula

 Tongue

 Desiccator
 4.3.3) Instrument:
 Muffle furnace
 Weighing balance
 Burner

4.3.4) Procedure:
1. Take empty crucible and place into hot air oven for 1hour to dry it.

2. Then place into desiccator and allow it to room temperature.

22
3. Then place empty crucible onto weighing balance note the weight of
empty crucible.

4. Then place 1g of milk sample into crucible and note the reading.
 5. Then cherring of sample perform with the help of burner cherring
took place until flame is produce and cool of sample in the crucible
turned to cherry red color or black.

6. After cherring place the sample for 5 to 6 hours into a muffle

furnace.

7. Then the white color ash is appearing in the crucible.

8. Place it into desiccators to allow it to reduce its temperature.

23
9. Then weight the crucible with sample.

10. Note the reading.

 4.3.5) Calculation:
%Ash = (wt. after ashing - wt. empty crucible)/Wt. of sample x 100
4.4) Lactometer Reading (LR) test of milk

4.4.1) Principle:
This test gives the detection of total solids and density of the milk
sample.

4.4.2) Apparatus:
24
 Volumetric cylinder

 LR meter

 Beaker
 4.4.3) Procedure:
1. Take 100ml of milk sample in a beaker.

2. Note the temperature of the milk sample temperature of milk sample

should be 20 0C.

3. Pour milk sample into volumetric cylinder in such a way that milk
flows upon LR meter until LR meter dip thoroughly into milk
sample and milk touches the corner of the volumetric cylinder.
25
4. Allow it to stand for about 30seconds to stabilize it.

5. Then note the reading on LR meter.

 4.5) Butyrometer refractive index (BR value) test of milk
4.5.1) Principle:
Butyro-refractometer reading of milk fat is comparatively much less
than most of other fat/oil.
4.5.2) Apparatus:
• Spatula

• Centrifuge cup
26
• Beaker

• Burner
 4.5.3) Instruments:
 Butyro-refractometer

 Centrifuge

 Water bath

4.5.4) Procedure:
1. Take 100ml of milk sample in a beaker.
27
2. Fill the centrifuge cup with milk sample.

3. Now centrifuge the milk sample at 4000rpm for 14 minutes for the
separation of fat layer from milk.
 4. Remove the upper layer of fat from milk sample with the help of
spatula and place for 2 minutes on burner for clear fat separation.
5. Then allow it to cool for 2 to 3 minutes.
6. Maintains the temperature of Butyro-refrectrometer at 40 0C through
water flow in water bath.
7. Then cleans the slide of Butyro-refractometer with 2 drops of Iso-
amyl alcohol.
8. Then place 1 to 2 drops of separated fat sample onto slide of
28
Butyro-refractometer.
9. Note the reading.
 Precautions:
 Temperature of Butyro-refractometer at the time of reading should
be 40 0C.

4.6) Determination of Acidity in Milk sample

4.6.1) Apparatus:
 Pipette
 Erlenmeyer Flask
29
 Burette
 Beaker
 Petri plate
 4.6.2) Reagents:
1. 0.1N NaOH

2. 50ml of 96% Ethanol

3. 1% of Phenopthalein indicator
4.6.3) Procedure:
1. Prepare 13% solution of milk( weight 13g of powder milk sample
and dissolve into 40ml of distilled water then up to mark at 100ml).
30
2. Give stay time for 1hour.

3. Take 20ml of milk sample with the help of pipette into 200ml of
Erlenmeyer Flask and 10 ml of milk sample in case of liquid milk.
 4. Add 0.5ml of Phenolphthalein indicator and then titrate with 0.1N
NaOH until Faint Pink color appears.
5. Give it Stay time for 30 seconds.

4.6.4) Calculation:
%Titratable acidity = ( mlxVx90x100)/(Vx1000)
Ml = ml(0.1N)NaOH used
N = normality of( 0.1N)NaOH

31 V = ml milk solution
Interpretation:
Appearance of faint pink color in milk sample gives the detection of
acidity.
 4.7) Determination of pH

4.7.1) Apparatus:
 pH meter

 Beaker

4.7.2) Procedure:
1. Place 50ml of milk sample in 100ml of beaker.
32
2. Warm milk sample at 20 0C.

3. Dip the Electrode of pH meter in milk sample.

4. Record the Ph of milk sample.

 Inference:
Ph of milk is 6.4 to 6.68. A Ph of 7.0 or above indicates over-
neutralization.
4.8) Determination of COB

4.8.1) Principle:
The organoleptic tests for determining the quality of milk is not very
reliable. Milk with highly developed acidity clots on boiling.
33
4.8.2 Apparatus:
 Test tube

 Test tube stand

  Boiling water bath

 Burner

4.8.3) Procedure:
 Take 1ml of milk sample in test tube.
 Place the test tube in boiling watetr bath for 5minutes.
 Observe clotting on sides and bottom of test tube.

34
Inferences:
 Flakes or curd on sides of test tube indicate positive COB test and
should be rejected.
 No curd take place in case of normal milk sample.
 4.9) Determination of Protein in milk
4.9.2) Apparatus:

 Kjeldalh,s apparatus

 Distillation apparatus complete with heat source bulb and delivery

tube

 Pipette (5ml,10ml)

 Digital burette for titration

35
4.9.3) Reagents:

1. Sodium Hydroxide
2. Boiling Stones
 3. Methyl red indicator

4. Hydrochloric acid standard solution of 0.1N upto N/70

5. Sodium Hydroxide standard solution of 0.1N

6. 2% Boric acid

7. Digestion mixture(K2SO4,CuSO4,FeSO4)

4.9.4) Procedure:
36
Digestion:
1. Take 5ml of sample in kjeldhal’s bulb.

2. Add 3gm of digestion mixture.

 3. Add 20ml of concentrated H2SO4.

4. Heat it until frothing ceased and after that boiled until transparent
and clear.

5. Took the sample and made its volume upto 100ml in measuring
flask.

Distillation:
1. Take 5ml sample in distillation assembly and added 15ml NaOH.
37
2. Took 10ml boric acid and kept it at the bottom of condenser.

3. Started the distillation process and wait for the time when pink color
of boric acid changed to transparent.
 4. Then note the time of 2 minutes and after that remove the boric

acid.

5. Distilled the contents and NH3 was absorbed by boric acid.

Titration:
1. Titrate the distillate with N/70 HCl till pink color is obtained.

2. Note the reading.

38
4.9.5) Calculation:
% Total Protein =(Titrate used x 0.4) /wt.of sample x 6.38
 4.10) Determination of NCN and NPN
4.10.1) Apparatus:
 Kjeldalh,s apparatus

 Distillation apparatus complete with heat source, bulb and delivery

tube

 Pipette (5ml,10ml)

 Digital burette for titration

39
4.10.2) Procedure:
4.10.2.1) NCN (sample preparation):

 Take 10ml Milk sample

  Take 70ml Distilled water.
 Add 1ml acetic acid 10%.
 Add 1ml sodium acetate. (1 Molar)
Collect the filtrate and make volume upto100ml.

4.10.2.2) NPN (sample preparation) :

 Take 10ml Milk sample.

 Add 15% 40ml TCA.
40  Filter the precipitate.
 Collect the filtrate and make volupto50ml.
Digestion:

 Take 5ml of sample in kjeldalh,s bulb.

  Add 3gm of digestion mixture.
 Add 20ml of concentrated H2SO4.
 Heat it until frothing ceased and after that boiled until transparent
and clear.
 Took the sample and made its volume up to 100ml in measuring
flask.
Distillation:

41
 Take 5ml sample in distillation assembly and added 15ml NaOH.
 Took 10ml boric acid and kept it at the bottom of condenser.
 Started the distillation process and wait for the time when pink color
of boric acid changed to transparent.
  Then note the time of 2 minutes and after that remove the boric acid.
 Distilled the contents and NH3 was absorbed by boric acid.
Titration:

 Titrate the distillate with N/70 HCl till pink color is obtained.
 Note the reading.

4.10.3 Calculations:
Calculations for NCN: (factor 0.196 )
42
Calculations for NPN (factor 0.098)
 Chapter: 5
Determination of Adulterants in Milk
5.1) Detection of urea
5.1.1) Principle:
P_Dimethylaminobenzaldehyde form yellow color complex with urea.

5.1.2) Apparatus:
 Test tube
43
 Pipette
 Test tube stand
 Beaker
  Filter paper

 Volumetric flask

5.1.3) Reagents:
1. Tricholoroacetic acid (TCA) 24%

2. P-Dimethylaminobenzaldehyde( P-DMAB 1.6%, w/v)

5.1.4) Procedure:
1. Take 5ml of milk sample in a test tube.
44
2. Add 5ml of TCA solution and then filter.

3. Take 2ml of filtrate in a test tube.

4. Add 2ml of P_DMAB solution.

 5. Observe the color.

Interpretation:
A characteristics yellow color in the filtrate from milk indicates the
presence of urea .

5.2) Detection of Cane Sugar

5.2.1) Principal:
Cane sugar reacts with Resorcinol in acid media and gives characteristics
45
red color.

5.2.3) Apparatus:
 Test tubes
  Test tubes holder

 Pipette

 Beaker

 Boiling water bath

5.2.4) Reagents:
0.5%Resorcinol solution
5.2.5) Procedure:
46
1. Take 1ml of milk sample with the help of pipette.

2. Add 1ml of Resorcinol solution and mix it well.

3. Place the tube in boiling water bath for 5 minutes.

 4. Withdraw the tube and observe the color.

Interpretation:
Appearance of deep red color indicates the presence of sucrose or
ketose sugar. The limit of detection of method is 0.1%.

5.3) Detection of Formalin

5.3.1) Principal:
H2SO4 containing traces of ferric chloride (FeCl3) react with
47 formaldehyde to gives characteristic violet color ring at the junction in
milk sample.

5.3.2) Apparatus:
 Test tubes
  Test tubes holder

 Pipette

 Volumetric cylinder

 Volumetric flask

 Weighing balance

 Petri dish

48 5.3.3) Reagents:
1. 3% of FeCl3 solution

2. 90% concentrated H2SO4

 5.3.4) Procedure:
1. Take 2ml of milk sample with the help of pipette in test tube.

2. Add 2ml of 90% H2SO4 containing traces of FeCl3 from the side

of test tube slowly.

3. Observe the appearance of purple color ring at the junction of test

tube.

49 Interpretation:
Formation of violet color ring at the junction of test tube indicates the
presence of formaldehyde in milk sample.
 5.4) Detection of Hydrogen peroxide Milk
5.4.1) Principle:
Vanadium pent oxide (V2O5) reacts with Hydrogen peroxide in the
milk sample to give characteristics brown color.

5.4.2) Apparatus:
 Test tubes

 Test tube holder

50
 Weighing balance

 Pipette

 Volumetric cylinder
 5.4.3) Reagents:
1. 1% solution of vanadium pent oxide (V2O5)

2. 6ml concentrated H2SO4

5.4.4) Procedure:
1. Take 10ml of milk sample in a test tube with the help of pipette.

2. Add 10 to 20 drops of V2O5 solution in test tube with the help of

51 pipette.

3. Mix it well.

4. Observe the color.

 Interpretation:
Appearance of characteristics brown color indicates the presence of
H2O2 in the milk sample.
5.5) Detection of Neutralizer in milk

5.5.1) Principle:
Rosalic acid reacts with alkali (NaOH, Na2CO3, and NaHCO3) to
gives raised red or pink color whereas pure milk show only brownish
color.
52
5.5.2) Apparatus:
 Test tubes

 Test tube holder

  Weighing balance

 Pipette

 Volumetric cylinder

 Volumetric flask

 Beaker

5.5.3) Reagents:
1. Ethyl alcohol Solution (60%v/v)
53
2. Rosalic acid Solution(0.05%w/v)

3. Na2CO3 Solution(1%)

4. NaHCO3 solution (1%)

 5.5.4) Procedure:
1. Take 2ml of milk sample with the help of test tube in the case of
negative sampling and 1ml of milk sample in case of positive
sampling.

2. Add 1ml of Na2CO3 or NaHCO3 solution in case of positive

sample of pure milk preparation but not in case of negative.

3. Add 2ml of Rosalic acid solution in sample.

54 4. Note the color changes.

Interpretation:
Appearance of pink or red color in positive milk sample gives the
indication of presence of alkali.
 5.6) Detection of Detergent
5.6.1) Principle:
Methylene blue indicator and chloroform react with detergent to gives
intense blue color at bottom layer.

5.6.2) Apparatus:
 Test tubes

 Test tube holder

55
 Weighing balance

 Pipette

 Volumetric cylinder
  Volumetric flask

 Beaker

5.6.3) Instrument:

 Centrifuge
5.6.4) Reagent:
1. Methylene blue indicator(0.05%)

2. Chloroform
56
5.6.5) Procedure:

1. Take 1ml of milk sample.

2. Add 1ml of Mathylene blue indicator and 2ml of chloroform.

 3. Centrifuge at 1100rpm for 5 minutes.

4. Observe the color.

Inference:
Intense blue color at the bottom layer indicates the presence of
detergent in milk sample.

5.7) Detection of Starch in milk

57 5.7.1) Principle:
Iodine solution turns starch granules into intense blue to blackish color
by acting on amylase due to the formation of an unstable starch-iodo
compounds.
 5.7.3) Apparatus:
 Test tubes

 Test tube holder

 Weighing balance

 Pipette

 Volumetric flask

 Beaker
58
 Burner

 Petri dish

 Spetula
 5.7.4) Reagent:
1. Iodine Solution

5.7.5 ) Procedure:
1. Take about 4ml of milk sample in a test tube.

2. Bring it to boiling condition and allow the test tube to room

temperature.

3. Add 2-3 drops of Iodine solution to the test tube.

59
4. Observe the color.

Inference:
Development of blue color indicates the presence of starch.
 5.8) Detection of Salt (sodium chloride):
5.8.1) Principle:
Silver nitrate develops characteristics red color in milk sample
containing sodium chloride in the presence of potassium chromate.

5.8.3) Apparatus:
 Test tubes
 Test tube stand
60  Pipette

5.8.4) Reagents:
1. Potassium chromate Solution 10%(w/v)
2. Silver nitrate solution (0.1N)
 5.8.5) Procedure:
1. Take 2ml of milk sample in test tube.

2. Add 0.5ml of Potassium chromate in milk sample.

3. Then add 1ml of silver nitrate in milk sample.

4. Mix it thoroughly.

5. Observe the color changes.

61 Interpretation:
Appearance of red color precipitate indicates the presence of sodium
chloride (NaCl) in milk.
 Chapter: 6
Analysis of Cake Rusk
6.1) Determination of moisture in (cake Rusk)

6.1.1) Principle:
The sample is dried to constant weight at 102℃ for 3hours in hot air
oven and the loss in the weight reported as moisture.
62 6.1.2) Apparatus:
 Petri dish
 Spatula
 Desiccator
 6.1.3) Instruments:
 Weighing balance
 Hot air oven
6.1.4) Procedure:
1. Take petri dishes and put into hot air oven for 1 hour.
2. Place the empty dishes into desiccators and allow it to room
temperature.
3. Weight the empty Petri dishes and note the reading.
63
4. Then place Petri dishes onto weighing balance and weight 5g of
sample and note the reading.
5. Then place these dishes in hot air oven at 102℃ for 3 hours.
 6. After that remove the dishes from oven and place into desiccator.
7. Again weight the sample after drying onto weighing balance and
note the reading.
6.1.5) Calculation:
% Moisture = (wt. of sample before drying - wt. of sample after drying)/
wt. of sample x 100
6.2) Determination of Ash in (Cake Rusk)
6.2.1) Principle:
64 This test is performed to check the level of minerals in cake rusk sample.
All volatile compounds are evaporated and sample turn into ash at 550℃
for 5 hour in muffle furnace.
 6.2.2) Apparatus:
 Crucible
 Spatula
 Desiccator

6.2.3) Instruments:
 Muffle furnace
 Weighing balance
65
 Burner

6.2.4) Procedure:
1. Take empty crucible and place into hot air oven for 1hour to dry it.
 2. Then place into desiccator and allow it to room temperature.

3. Then place empty crucible onto weighing balance note the weight
of empty crucible.

4. Then place 1g of turmeric sample into crucible and note the reading.

5. Then charring of sample perform with the help of burner charring

took place until flame is produce and color of sample in the

crucible turned to cherry red color or black.

66
6. After charring place the sample for 5 to 6 hours into a muffle

furnace.

7. Then the white color ash is appear in the crucible.

 8. Place it into desiccators to allow it to reduce its temperature.

9. Then weight the crucible with sample.

10. Note the reading.

6.2.5) Calculation:
%Ash = (wt. after ashing - wt. empty crucible)/Wt. of sample x 100
6.3) Determination of Fat in cake rusk

67 6.3.1) Equipment:
 Analytical balance (at least 1 mg sensitivity).
 Electrical drying oven to be operated at 102ºC± 1ºC.
 Soxhlet extraction
 • Heat source
 Desiccator
 Glass rod

6.3.2) Reagents:
 Petroleum spirit boiling point 60-80ºC
 Cotton wool free of fat
 Acid washed sand

68 6.3.3) Procedure:
1. Rinse all glassware with petroleum spirit, drain, dry in an oven
at 102ºC for 30 min. and cool in a desiccator.
2. Place a piece of cotton wool in the bottom of a 100 mL beaker.
 Put a plug of cotton wool in the bottom of an extraction thimble and
stand the thimble in the beaker.

3. Accurately weigh 5 g of sample into the thimble. Add 1 - 1.5 g of

sand and mix the sand and sample with a glass rod. Wipe the glass
rod with a piece of cotton wool and place cotton wool in the top of
the thimble. Dry the sample in an oven at 102ºC for 5 hour.
4. Allow the sample to cool in a desiccator.
5. Take the piece of cotton wool from the bottom of the beaker and
place it in the top of the thimble.
69
6. Insert the thimble in a Soxhlet liquid/solid extractor.

7. Accurately weigh a clean, dry 150 mL round bottom flash and put

about 90 mL of petroleum spirit into the flask.

 8. Assemble the extraction unit over either an electric heating mantle.

9. Heat the solvent in the flask until it boils. Adjust the heat source so

solvent drips from the condenser into the sample chamber at the

rate of about 6 drops per second.

10. Continue the extraction for 6 hours. piece of cotton wool from the

bottom of the beaker and place it in the top of the thimble.

11. Remove the extraction unit from the heat source and detach the
70
extractor and condenser.

12. Place the flask in an oven at 102ºC.

13. Cool the flask in a desiccator and weigh the flask and contents.
 6.3.4) Calculations:
Weight of sample = S % Crude fat = (W2 – W1) x 100 S

6.4) Determination of Protein in (Cake Rusk):

6.4.1) Principle:
This method depends upon the oxidation of organic matter with
sulphuric acid in the presence of catalyst and the simultaneously
formation of ammonium salts and amines from the nitrogen in the
71
turmeric sample.

6.4.2) Apparatus:
 Kjeldal apparatus
  Distillation apparatus complete with heat source, bulb and delivery
tube
 Pipette (5ml,10ml)
 Digital burette for titration
6.4.3) Reagents:
1. NaOH
2. Boiling Stones( 8 to 10 meshes).

3. Methyl red indicator

72
4. Hydrochloric acid standard solution of 0.1N upto N/70

5. Sodium Hydroxide standard solution of 0.1N

6. 2% Boric acid
 7. Digestion mixture (k2So4,CuSO4,FeSO4)
6.4.4) Procedure:

 Digestion:
1. Take 5ml of sample in kjeldal bulb.

2. Add 3gm of digestion mixture.

3. Add 20ml of concentrated H2SO4.

4. Heat it until frothing ceased and after that boiled until transparent
73 and clear.

5. Took the sample and made its volume upto 100ml in measuring

flask.
  Distillation:
1. Take 5ml sample in distillation assembly and added 15ml NaOH.

2. Took 10ml boric acid and kept it at the bottom of condenser.

3. Started the distillation process and wait for the time when pink
color of boric acid changed to transparent.

4. Then note the time of 2 minutes and after that remove the boric
acid.
74
5. Distilled the contents and NH3 was absorbed by boric acid.

 Titration:
1. Titrate the distillate with N/70 HCl till pink color is obtained.
 6.4.5) Calculation:
% Total Protein =(Titrate used x 0.4) /wt. of sample x 6.38

Chapter: 7
Conclusion:

It was great experience to work in PCSIR . During this period I gained

hands on practical experience in various aspects of food science.

75 I learned how different test are performed like protein, fat, moisture,

crude fiber, crude fat, crude protein, ash and I also perform different

adulteration test of milk like urea, formalin, cane sugar etc.

 Chapter: 8
References:
1. Sullivan DM, Carpenter DE (eds) (1993) Methods of analysis for nutritional

labeling. AOAC International, Arlington, VA

2. Manual of Method of Analysis of Foods, Ministry of Health and Family Welfare,

Government of India, (New Delhi, 2015)

76 3. ISO 8968 /IDF 20-1;20- , “ N ) Kj ” G & Horwitz.1988: Determination of

Protein Content by Kjeldalh,s Method. Journal of AOAC,vol.71, nr.5:893-897