Clots on Boiling (COB) test of liquid milk Reference #

Used for: Practical implementation, for study Date:

and for reference.
Defination and explanation:
Acidity decreases the heat stability of milk. The clot-on- boiling test is used to determine
whether milk is suitable for processing, as it indicates whether milk is likely to coagulate during
processing (usually pasteurization). It is performed when milk is brought to the processing plant
— if the milk fails the test it is rejected.
The test measures the same characteristics as the alcohol test but is somewhat more lenient. It
has the advantage that no chemicals are needed. However, its disadvantage is that at high altitude
milk (and all liquids) boils at lower temperature and therefore the test is even more lenient.
Principle:
Acid developed in milk due to microbial activities during procuring, handling and transportation
of milk. This developed acid in milk make milk protein casein more heat sensitive so such milk
with more acidity (than natural milk) when boiled coagulate. On the other hand if milk sample
contains more whey proteins as globulin or albumins, such milk also show coagulation after
heating/ boiling as whey proteins are more heat sensitive than casein under natural acidic
condition of raw milk.
Scope and application:
This method is used to detect too advanced acidification in fresh milk that makes milk
completely unfit for purchase in field and accepting at reception (in field and at plant) and is
also used in factory to determine the heat stability of any kind of fluid milk for heat processing
in factory.
Standard: COB should be - ve for all kinds of fluid milk.
Reagents Required: NIL

Apparatus Required: 1. Test tube

2. Spirit lamp or Bunsen burner
3. Wooden test tube clamp
4. Pipette 10 mL or any available.
Procedure:
1. Take 4-5 ml sample in test tube with the help of a pipette.
2. Then heat the sample on Bunsen burner or spirit lamp to boiling point.
3. After boiling tilt the test tube gently and observe.
Observations: Presence of clot/ precipitate will indicate COB +ve.
Repeatability & Give same results for same kinds of samples.
Reproducibility:
Precautions:
1. Thoroughly mix the milk to get representative sample.
2. Glass ware used should be properly washed and cleaned.
Hygiene Requirements:
1. Wash and clean all glass ware, use detergent where required.
2. Place each and every chemical and glassware after use to their respective place.
3. Cover the each reagent bottle after using chemical/reagents.
4. Clean your work bench after performing analyses.
Reference:
 1. LYONS, J.; O'SHEA, M.J., 1950. Page 161 in Commercial methods of testing milk and
milk products, Cork Univ. Press, B.H. Blackwell Ltd., Oxford.UK.
2. Davis, J. G. 1951. Page 128 in Milk Testing: The Laboratory Control of Milk, Dairy
Industries Ltd., London, UK.

Alcohol Precipitate Test (APT) Reference #

Protein stability Test of liquid Milk
Used for: Practical implementation, for study Date:
and for reference.
Defination and explanation:
It is based on instability of the proteins when the levels of acid and/or rennet are increased and
acted upon by the alcohol. Also increased levels of albumen (colostrum milk) and salt
concentrates (mastitis) results in a positive test.
Principle: Protein of milk and alcohol compete for water available in milk. The higher the
concentration of alcohol used; greater will be it’s power to attract water. Consequently less
water will be available to keep the casein in suspension.
Scope and application:
This method is used to detect too advanced acidification in fresh milk in filed and at reception
both in field and at plant and is used to determine the heat stability of protein of fluid milk for
heat processing in factory.
Standards:
Raw milk should be - ve at least at 55 % Ethanol solution (V/V)
Reagents Required: Ethanol solution 68%, 65 % ,60% and 55 % (v/v)
Apparatus Required: 1. Pipette (10 mL)
2. Test tubes
3. Glass Petri dishes (where required)
Procedure:
1. Thoroughly mix the milk to get representative sample.
2. Take 2 ml milk sample with the help of pipette in a clean test tube.
3. Add 2 ml ethanol solution of required strength and mix, without shaking.
Observations: Presence of precipitate will be considered as APT +ve.
Repeatability & Give same results for same kinds of milk.
Reproducibility:
Precautions:
1. Strength of Ethanol used should be clearly marked on each type used.
2. Pipette and Petri dishes / test tubes should be clean no accumulation of milk residue in
it.
3. Light arrangement should be proper for proper visibility.
4. Make sure that none of the chemicals being used is expired.
Hygiene Requirements:
1. After performing the test clean and wash all the related apparatus and glassware.
2. Place all the chemicals in their specific place by properly labeling and covering.
3. Necessary instructions should be written about chemicals (hazardous) where required.
4. In case of any spillage or breakage, immediately clean the workbench carefully.
5. After working with solutions always wash the hands with soap.
Reference:
1. Roder, G., Grundzuge der Milchwirtschaft und des Molkereiwesens. Hamburg
u.Berlin,Verlag Paul Parey (1954) 680.
2. Sommer, Hugo H. 1938. Page 147 in Market Milk and Related Products. Madison.
Wisconsin.

% Acidity Determination of liquid milks (Th°) Reference #

Used for: Practical implementation, for study Date:

and for reference.
Definition and explanation:
The acidity of fresh milk (natural acidity) is due to phosphates, casein and whey proteins, citrates
and carbon dioxide. The natural acidity of milk is 0.16 - 0.18%. Figures higher than this signifies
developed acidity due to the action of bacteria on milk sugar.
Bacteria that normally develop in raw milk produce more or less of lactic acid which contributes
towards the developed acidity in milk. The percentage of acid present in dairy products at any
time is a rough indication of the age of the milk and the manner in which it has been handled.
Fresh milk has an initial acidity due to its buffering capacity.
Total acidity of milk is sum of natural and developed acidity in milk and is analyzed as titratable
acidity.
Percent Titratable Acidity (%TA) is the percent acid in a sample based on titration data. More
specifically, it is usually defined in terms of the phenolphthalein end point (pH 8.3), which is
really an approximation of the true equivalence point (the point where all the acid is converted
to base). However, from the stand point of TA, the volume of base required to titrate to the end
point or equivalence point is what is of importance, and generally there is virtually no difference
between these volumes.
There are two fundamentally different methods of expressing acidity: (a) titratable acidity
expressed as percent lactic acid, and (b) hydrogen ion concentration or pH. The former measures
the total acidity but does not measure the strength of the acids. The pH indicates the strength of
the acid condition.
Principle: Acid-base Titration
Phenolphthalein is colorless in acidic media. The base added in the sample neutralizes the acid
present in sample and addition of one more extra drop base tends the whole media to become
basic and phenolphthalein present turns pink as it gives this color in basic media.
Scope and application:
This is a quantitative method used to determine the total titratable acidity of fresh milk in degree
thornic (Th°) at reception at plant, reception in area and in factory and also can be used to
determine the acidity of other fresh milk products in factory.
Standard: 0.08-0.14 for raw milk
1. 1.0 % Phenolphthalein indicator-
2. 0.1 N NaOH solution.
Reagents Required: 3. Distilled water
1. Weighing Balance( accurate to 0.1 g) or any available
2. Burette or acidimeter (25ml- graduated in 0.1 mL divisions)
3. Pipette 10mL or any available
Apparatus 4. Glass Beaker (100 ml or any other)
Required: 5. pH meter with electrode
6. Buffer solutions (pH 4.01 and 7.01)
Procedure:
1. Weigh 9 mL (approximately equal to 9 gm) sample in a 100ml beaker.
2. Add 0.5 mL (3-4 drops) of phenolphthalein indicator and swirl to mix.
3. Fill burette with 0.1 N NaOH up to 0 mL mark on top and allow it to drain until all air
bubbles have been removed.
4. Refill burette and drain until meniscus is at 0 mL mark.
5. Allow NaOH to drip into the beaker with continuous stirring titrate to a permanent pink
color the phenolphthalein end point is at pH 8.3.
6. Read level of NaOH in burette by standing at eye level with meniscus and read number
of milliliters of NaOH delivered.
Alternative Method:
1. weigh 9 g sample in a beaker
2. Use a pH meter and electrode. Stir sample with electrode while adding NaOH.
Stop addition of NaOH at pH 8.3. At pH 8.3 end point al the hydrogen is ionized.
Acidity is expressed as % Lactic acid.
1mL of 0.1 N NaOH = 0.0009 g of lactic acid
% acidity = mL of 0.1 N NaOH usedx0.0009x100
Weight of sample
Calculations: % acidity = mL of 0.1 N NaOH used x 0.1
Repeatability & + 0.01
Reproducibility:
Precautions:
1. Thoroughly mix the milk to get representative sample.
2. Solutions and indicator should be standardized.
3. Glassware being used should be clean and dry.
Hygiene requirements:
1. After performing the test clean and wash all the related apparatus and glassware.
2. Place all the chemicals by proper covering to their specific place after each test
3. Clean your work bench with clean cloth piece and alcohol (where necessary) after
working.
4. In case of any spillage or breakage, immediately clean the workbench carefully.
5. After working with solutions always wash the hands with soap.
Reference:
1. Standard Methods for the Examination of Dairy Products, 14th Edition, 1978, American
Public Health Association, Washington, D.C. Pages 355-357.
2. Atherton H.V. & JA. Newlander.1987. Page 250 in Chemistry and testing of dairy
products.AVI publishing Co.,Westport,U.S.A.

Lactometer Reading for SNF calculation

and specific gravity determination of Reference #
liquid milk.
Used for: Practical implementation, for
Issue date:
study and for reference.
Definition and explanation:
Addition of water to milk can be a big problem where we have unfaithful farm workers, milk
transporters and greedy milk hawkers. Any buyer of milk should therefore assure
himself/herself that the milk he/she purchases is wholesome and has not been adulterated. Milk
has a specific gravity. When its adulterated with water or other materials are added or both
misdeeds are committed, the density of milk change from its normal value to abnormal. The
lactometer test is designed to detect the change in density of such adulterated milk. Carried out
together with the Gerber butterfat test, it enables the milk processor to calculate the milk total
solids (% TS ) and solids non fat (SNF).
Specific gravity is the relation between the mass of a given volume of any substance and that
of an equal volume of water at the same temperature.
Since 1 ml of water at 4°C weighs 1 g, the mass of any material expressed in g/ml and its specific
gravity (both at 4°C) will have the same numerical value. The specific gravity of milk averages
1.032, i.e. at 4°C 1 ml of milk weighs 1.032 g.
Since the mass of a given volume of water at a given temperature is known, the volume of a
given mass, or the mass of a given volume of milk can be calculated from its specific gravity.
For example, one litre of water at 4°C has a mass of 1 kg, and since the average specific gravity
of milk is 1.032, one litre of average milk will have a mass of 1.032 kg.
Principal:
The function of the Lactometer/hydrometer is based on Archimedes principle that a solid
suspended in a liquid will be buoyed up by a force equal to the weight of the liquid displaced.
Thus, the lower the density of the substance, the lower the lactometer/hydrometer will sink.
In light liquids like kerosene, gasoline, and alcohol, the hydrometer must sink deeper to displace
its weight of liquid than in heavy liquids like brine, milk, and acids. In fact, it is usual to have
two separate instruments, one for heavy liquids, on which the mark 1.000 for water is near the
top, and one for light liquids, on which the mark 1.000 is near the bottom of the stem.
Scope and Application:
This method is rapid and only approximate and is convenient to use in conjugation with gerber
and babcock methods for fat. This most is suitable to determine the SNF contents and specific
gravity (by using formulae) of fresh milk at reception (in field and at plant) can also be used to
determine SNF and specific gravity of fresh milk and other liquid fresh milk products in factory
but it is preferable to determine total solid contents of liquid milks by drying in factory.
Reagents Required: Nil
Glass Ware & Equipment 1. Calibrated thermometer (0 – 110° C) or any available.
Required: 2. Calibrated Lactomter (14 – 42) Quenne or any
available.
3. A Plastic Cylinder (250 ml) at least 4 mm greater in
diameter than the bulb of lactometer.
4. Ice Box
5. Hot Water Bath
6. SS Beaker
Procedure:
1. Take about 500 ml milk sample in SS Beaker. Heat the sample at 40-45° C for about
five minutes and then cool down to 20° C.
2. Rinse the cylinder with some milk sample and drain it.
3. Rinse the Lactometer with the milk sample.
 4. Now fill the cylinder with the milk sample completely.
5. Place the Lactometer over the cylinder and leave it gently.
6. Add some more milk in cylinder to remove the foam.
7. Wait for about 20 – 30 seconds till the Lactometer become stable, and then note the
reading (LR).
Calculations: SNF=(LR/4)+0.72+(0.22 X Fat % of sample)
Specific gravity= LR/1000+1
Repeatability & Reproducibility: ±0.5
Precautions:
1. Prior to test make sure that all glassware and equipments are ok and neat & clean.
2. Lactometer must not touch the walls of cylinder.
3. Foam must be removed over the sample.
4. Temperature of milk sample must be maintained at 20° C.
Hygiene requirements:
After performing the test clean and wash all the related apparatus and glassware.

Reference:
1. FAO Manual of Food Quality Control, 14/8, page 12-1986-determination of total solid
(Rapid method) milk.
2. Sommer, Hugo H. 1938. Page 179 in Market Milk and Related Products. Madison.
Wisconsin.

Fat Determination of liquid milk (Gerber Reference #

method)
Used for: Practical implementation, for study Date:
and for reference.
Definition and explanation:
11 ml pipette (which delivered about 10.90 ml milk) was used in the original Gerber method.
Volume used in some countries are as follow:
1. India, 10.75 ml
2. The Netherlands, 10.66 ml
3. Hungary, 10.8 ml
4. UK., 10.94 ml
5. USA., 11 ml
New stopper of natural rubber absorb fat to some extant. Results are in gm fat/100 gm of milk.
Principle:
Everything in milk except the fat dissolves in sulphuric acid. The fat floats to the top. The
centrifuge ensures complete separation with no bubbles in the fat, iso-amylalcohol ensures the
complete separation of fat, and the fat content can be measured using the graduations on the
butyrometer.
Scope and application:
Gerber method is quick, quantitative method to determine the fat contents of fresh milk at
reception at plant and in factory can be used for liquid milks fat determination. This method is
not suitable for formalin preserved samples. This method can also be used in field by using
manual centrifugal machine but in this case result may vary.
Standard: Raw milk: 3.5-8.5 %
 1. Isoamyl alcohol (sp. Gravity 0.081 ± 0.01)
Reagents Required: 2. Sulfuric acid (d=1.816 ± 0.003 g/ml, 90 – 91% purity with clear
color).
1. Calibrated Butyrometer ( 8 % )
2. Calibrated Pipette (10.94mL)
3. Key Stopper
Apparatus 4. Auto measure 10 mL with stand or pipette 10 mL.
Required: 5. Auto measure 1 mL with stand or pipette 1 mL.
6. Water bath
7. Centrifuge machine (1100 rpm, 65°C temperature)
8. Calibrated Thermometer
Procedure:
1. Take about 500 – 800 ml of milk sample in a SS Beaker. Heat the sample at 40-45°
C and then cool down to 20 o C by continuously agitating.
2. Take 10 ml sulphuric acid in butyrometer with the help of Auto-measure.
3. Mix the milk sample well and rinse the pipette with the milk sample.
4. Now fill the pipette with sample above the mark.
5. Pour the samples from the pipette so that the upper meniscus of the sample reaches
to the mark.
6. Now pour the filled pipette into the Butyrometer gently making an angle of 45° with
butyrometer neck in 30-40 seconds.
7. Add 1 ml iso-amyl alcohol with the help of Auto-measure or pipette.
8. Insert the stopper into Butyrometer with the help of Stopper Key.
9. Mix the contents well by inverting butyrometer 3- 4 times and further shaking if
required.
10. Centrifuge the Butyrometer (by placing in such a way that graduated column remains
upward) in Gerber Machine for 3 min. for raw and 10 minutes for UHT milk at 65°C
and 1100 rpm.
11. Remove the Butyrometer from the Gerber Machine and immediately read the lower
meniscus of fat column in butyrometer.
12. Fat %age of sample directly correspond to the reading in butyrometer column.
Appearance of the Test
1. The colour of the fat column should be straw yellow.
2. The ends of the fat column should be clearly and sharply defined.
3. The fat column should be free from specks and sediment.
4. The water just below the fat column should be perfectly clear.
5. The fat should be within the graduation.
Problems in test results
Curdy tests:
 Too lightly coloured or curdy fat column can be due to:
 Temperature at milk or acid or both too low.
 Acid too weak.
 Insufficient acid.
 Milk and acid not mixed thoroughly.
Charred tests:
 Darkened fat column containing black speck at the base is due to:
  Temperature of milk-acid mixture too high.
 Acid too strong.
 Milk and acid mixed too slowly.
 Too much acid used.
 Acid dropped through the milk.
Calculations: Nil.
Repeatability & ± 0.05
Reproducibility:
Precautions:
1. Use gloves & goggles while handling acid.
2. Prior to test make sure that all glassware and equipments are calibrated and neat & clean
no fat accumulation in any of glassware.
3. During filling of butyrometer take care that no acid falls on the outside of the
butyrometer.
4. Always mix the contents in butyrometer very carefully better to cover the butyrometer
with piece of cloth.
5. Make sure that stopper is properly fixed in butyrometer.
6. Read the fat% as early as possible.
7. While starting centrifuge machine make sure that it is properly balanced.
8. Stir the sample gently and continuously while heating and cooling.
9. Always read the meniscus accurately.
10. Use calibrated Gerber Machine, Thermometer & pipette.
11. In some Butyrometers water needs to be added or Stopper needs to be adjusted to
maintain the level of Fat Column.
12. Note the reading immediately because esterification of iso-amyl alcohol starts if remains
at 65°C for more time and it results in high fat reading.
13. After reading, always remove the stopper & contents from the butyrometer under tap
water to avoid any suffocation from acid alcohol vapors, while the butyrometer is in
straight position to avoid any kind of intact with its contents.
14. In case of any breakage, make its entry in breakage record.
15. When any butyrometer is broken inside the machine, during its cleaning always use
gloves or cloth so that to prevent any injury caused by glass pieces.
16. Water bath should be handled carefully during working, always take care that hands
don’t touch the electric connections.
17. Always turn off the electric supply of the Gerber machine, water bath etc. before
cleaning.
Hygiene requirements:
1. After performing the test clean and wash all the related apparatus and glassware.
2. Place all the beakers and thermometers used to their specific place after
washing/cleaning.
3. Clean your work bench with clean cloth piece and alcohol (where necessary) after
working.
4. In case of any spillage or breakage, immediately clean the workbench carefully.
5. After working with solutions always wash the hands with soap.
Reference:
1. Davis, J. G. 1951. Page 59 in Milk Testing: The Laboratory Control of Milk, Dairy
Industries Ltd., London, UK.
2. FAO Manual of Food Quality Control, 14/8, page 8-1986-determination of milk fat by
Gerber method.