DC 221

Department of Dairy Chemistry

College of Dairy Science and Technology
Guru Angad Dev Veterinary and Animal Sciences University
Ludhiana-141004
 A K Barui
Veena N
S K Mishra
H Panwar

2018

Department of Dairy Chemistry

College of Dairy Science and Technology
Guru Angad Dev Veterinary and Animal Sciences University
Ludhiana-141004
 TABLE OF CONTENTS

S. Experiment Date of Date of Remarks

No experiment submission
1. Determination of fat in cream
2. Determination of titratable acidity in cream
3. Determination of moisture content in butter
4. Determination of curd content in butter
5. Determination of fat content in butter
6. Determination of salt content in butter sample
7. Determination of moisture content in ghee
8. Determination of B.R. (Butyro-Refractometer) or
refractive index reading of ghee.
9. Determination of acidity in ghee.
10. Determination of soluble and insoluble volatile
acids (Reichert-Meissl, Polenske value and
Kirschner value) in ghee.
11. Determination of sucrose in condensed milk
12. Determination of moisture content in dried milk
13. Determination of bulk density in dried milk and
dried milk products
14. Determination of titratable acidity in dried milk
15. Determination of total ash on dry basis in dried
milk.
16. Determination of fat in dried milk by Rose-
Gottlieb method
17. Determination of total solids in ice-cream
18. Determination of fat in ice-cream (by Rose-
Gottlieb method)
19. Determination of moisture content in cheese
20. Determination of fat content in cheese
21. Determination of chloride content in cheese
22. Determination of total solids content in khoa
23. Determination of fat content in khoa: Gravime tr ic
method
24. Determination of moisture content in paneer
25. Determination of fat content in paneer by Rose-
Gottlieb method
 CHEMICAL ANALYSIS OF CREAM
Introduction
Cream is that portion of milk, rich in milk fat, which rises to the surface of milk on standing,
or is separated by skimming from milk by centrifugal force using a mechanical separator. Cream is
one of the most important products of milk, partly because of the ready sale which it enjoys in its
natural state, and partly because it is a raw material for butter, ghee and certain varieties of cheese.
Cream represents milk in which the Table 1: Cream types and typical minimum fat
normal proportion of fat has been contents
increased, mainly at the expense of the Product description Typical minimum fat
content % by weight
water removed during the process of
Clotted cream 55.0
separation or skimming. The quantity of Double cream 48.0
protein and lactose present in cream is Whipping cream 35.0
Whipped cream 35.0
approximately half that contained in an Creak or single cream 18.0
average cow’s milk, although the fat Half cream 12.0
Sterilized cream 23.0
percentage may vary considerably Sterilized half cream 12.0
according to the method of separation. Plastic cream 65.0
Source: Komorowski and Early (1992)
Cream is used as an ingredient in a wide
range of foods and beverages, and, in many cases, the type of cream used is determined by the fat
content. Table 1 lists a selection of cream types and their typical minimum fat contents. The
viscosity and thickness of cream vary according to fat content, as do functional properties such as
whipping ability. Cream is usually heat treated by pasteurization, sterilization or UHT processing.
• Clotted cream, a product traditionally manufactured in the southwestern counties of UK, has a
very high viscosity, golden creamy color and granular texture as a result of the way it is made,
and because it is often produced from the rich milk of Channel Island breeds of cow. It is not
pourable but spoonable and is used mostly as a spread on scones, in conjunction with fruit
preserve.
• Double cream is a thick pouring cream, though with homogenization and/or age thickening it
may require spooning for use. It is used principally as a dessert cream and may be whipped,
although the effective degree of aeration is less than achieved with whipping cream, and care
must be taken during whipping not to churn the cream to butter. In the United States, the product
may be described as Heavy Cream, although this may be of lower fat content (36% milk fat,
minimum).
• Whipping cream and whipped cream have a carefully controlled fat content, so as to maximize
volume during aeration. The best foam volume and stability is achieved with a fat content
between 38 and 40%, with the cream refrigerated at less than 5°C for 24 h before use. Over-
 whipping will churn the cream to butter. Whipping cream is used in both domestic and
industrial context for the production of toppings and fillings for baked goods.
• Cream or single cream is used in the domestic context mainly as a pouring cream for desserts,
although it may be used in coffee. In the industrial context it is used as an ingredient in the
manufacture of canned soups and sauces.
• Half cream is used predominantly in coffee, but it may also be used as a breakfast cream poured
over fruits and cereals. Half-and-half cream is manufactured in the United States from a mixture
of milk and cream, to a fat content of not less than 10.5% but less than 18%.
• Sterilized cream is usually sold in cans, with a shelf-life of up to 2 years. It is thick spoonable
cream with a distinct caramelized flavor, as a result of the in-can sterilization.
• UHT cream is manufactured as double,
Table 4.2 Chemical composition of cream
whipping, single and half cream packed in Constituents Percentage
cartons. Like sterilized cream it is often I II III
Fat 30.0 25.00 50.00
used as a standby product, although Water 64.0 68.20 45.45
portions for coffee are significant in the Protein 2.40 2.54 1.69
Lactose 3.50 3.71 2.47
catering and fast food markets. UHT cream Ash 0.40 0.56 0.37
is also produced in aerosols containers Total solid 36.0 31.80 54.55
Solids-not-fat 6.0 6.80 4.55
with a nitrous oxide propellant, for the
Source: Pagote, 2002
production of whipped cream toppings.
• Sterilization of cream extends its keeping quality considerably, besides preserving the quality
of the original cream. Production of sterilized cream is now an accepted practice for conserving
surplus milk fat to meet its shortage, and it is now being produced by a number of dairies in
India. Prior to the sterilization of cream either by in-can sterilization or by UHT method,
additives, i.e. stabilizing salts, are added. These salts have an effect on the pH value (buffer
salt) and complex Ca +2 (ion exchanger) resulting in a decreased aggregation of casein micelles
during the subsequent heat treatment and in hot coffee beverages. The salts most often used are
phosphates and citrates.
• Cultured or soured cream is a product made by fermenting 18% milk fat cream with cultures of
lactic acid producing bacteria. The product finds various applications as additives in rounding
off sauces or dressings. Normally, souring is achieved by inoculation with mesophilic lactic
acid bacteria. Acidified sour cream which is soured chemically (by glucono-δ-lactone, lactic
acid and others) is an exception. The most common mesophilic cultures comprise Lactococcus
lactis subsp. lactis, Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis var. diacetylactis and
Leuconostoc mesenteroides subsp. cremoris.
• Plastic cream is highly viscous than other types of cream. Its fat content is between 65 to 68%
and is prepared by (i) re-separating normal cream (30 to 40% fat) in a normal cream separator,
 or by (ii) separating milk in an especially designed ‘plastic cream separator’. Plastic cream is
usually used for the manufacture of butter oil.
Composition
The average chemical composition of cream with different fat content is given in the Table 2.
Compositional standards for cream in India
Food Safety and Standards specifications
According to FSS Rules (2011) cream including sterilized cream means the product of cow or
buffalo milk or a combination thereof. It shall be free from starch and other ingredients foreign to
milk. It may be of following three categories, namely;
• Low fat cream: containing milk fat not less than 25.0% by weight.
• Medium fat cream: containing milk fat not less than 40.0% by weight.
• High fat cream: containing milk fat not less than 60.0% by weight.
Note: Cream sold without any indication about milk fat content shall be treated as high fat
cream.
BIS specifications
BIS (BIS, 1968) has laid down specifications for only sterilized cream. Sterilized cream means
the product containing 20% or more of fat which has been homogenized, canned and sterilized. It
may contain sodium salts of phosphoric and polyphosphoric acid, citric acid, carbonic acid and
calcium chloride in quantities not
Table 3 BIS requirements for sterilized cream
exceeding 0.3%. Additions of thickening Characteristics Requirement
agents is prohibited. Sterilized cream shall Fat, % by weight, Min 20.0
Titratable acidity (as lactic 0.15
have a color ranging from white to yellow. acid), % by weight, Max
It shall be clean and free from any Incubation test To satisfy the
requirement of
objectionable discoloration and adulterants. the test
The sterilized cream shall have a clean, pleasant odour. It shall be free from objectionable off flavor.
The product shall also comply with the requirement specified in the Table 3.
Preparation of cream sample for chemical analysis
The preparation of cream sample depends upon its physical condition. If, at room temperature,
the cream is thin to pour easily, mix by repeated inversion of the container. If the cream is too thick,
stir gently, taking care that the top and the bottom layers get well mixed. It may not be possible to
mix by gentle stirring if the cream is very thick and the fat is partly separated, or if on stirring, the
cream becomes thick or fat separates. Under these circumstances, warm the cream sample to
temperature between 30 to 40°C in water-bath and, while cooling it to room temperature, shake the
container gently or stir the contents at intervals. Keep the container covered as much as possible to
avoid loss of moisture by evaporation. If the cream sample shows any abnormality, it should be
recorded. If satisfactory mixing cannot be achieved, the sample should not be tested. In the
laboratory, the exposure of cream sample to temperature below freezing point should be avoided.
Cream samples should be protected from light, heat, contaminating odors and be kept in a cool
place, at a storage temperature of 0 to 5°C.
Note: Sour cream should not be warmed but should be thoroughly stirred.
Determination of fat in cream; Gerber method: IS: 1124 (Part II) -1977
Apparatus
1. Butyrometers 0 to 70% scale. For specifications and calibration please refer Chapter_.
2. Wash bottle: Containing cold water (30 to 40°C) and another containing hot (about 70°C)
distilled water.
3. Stemless funnel.
4. Small scoop of suitable material (for e.g. aluminum, nickel or plastic). It is convenient if this
scoop has a counterpoise.
5. In addition to the above apparatus, use rest of the apparatus as required for the determination
of fat in milk by Gerber method.
Reagents
1. Gerber sulfuric acid: Sulfuric acid shall have a density of 1.807 to 1.812 g/ml at 27°C
corresponding to a concentration of sulfuric acid from 90 to 91% by mass.
2. Dilute Sulfuric acid: Prepared by mixing Gerber sulfuric and distilled water (1: 1).
3. Isoamyl Alcohol: The isoamyl alcohol shall have density of 0.803 to 0.805 g/ml at 27°C.
Procedure
1. Weigh 5.00  0.01 g of the cream sample into dried and tarred 50 ml beaker. Add in small
quantities freshly prepared dilute sulfuric acid (1:1) to the cream in the beaker.

2. Dissolve the cream and transfer the mixture carefully, with the help of a dry funnel, to the
cream butyrometer (0 to 70% scale). Rinse the beaker about six times with small quantities
of the dilute sulfuric acid to make sure that all cream has been transferred to the butyrometer.
Altogether 18 to 20 ml of the dilute acid should suffice to fill the butyrometer, leaving
sufficient space for the addition of isoamyl alcohol and mixing.

3. Add one ml of amyl alcohol by means of the 1 ml automatic measure. Do not wet the neck of
the butyrometer with isoamyl alcohol.

4. Close the neck of the butyrometer firmly with the lock stopper without disturbing the contents
using lock stopper key.

5. Shake the butyrometer carefully without inverting until the contents are thoroughly mixed,
the curd is dissolved, and no white particles or flakes are seen in the butyrometer. Then invert
the butyrometer a few times to mix the contents thoroughly.

6. Transfer the butyrometer quickly, with the bulb upside, into a water-bath maintained at a
temperature of 65  2°C and keep it for not less than 5 min. Take care to have the water level
in the bath above the top of the fat column in the butyrometer.

7. Take out the butyrometer from water-bath, wipe it with a cloth and transfer it to the centrifuge,
placing two butyrometers diametrically opposite so as to balance the rotating disc.
 8. Centrifuge at the maximum speed for 4 to 5 min. Bring the centrifuge to stop gradually.
Transfer the butyrometers, stopper downwards, into a water-bath maintained at a temperature
of 65  2°C and allow the butyrometer to stand in the water-bath for not less than 3 min and
not more than 10 min.

9. Before taking the reading, adjust the position of the fat column to bring the lower meniscus
of the fat column on to a main graduation mark. Record the fat percentage age reading to the
nearest 0.5% that is, half of the smallest scale division.

10. If after centrifugation there is not a sharp dividing line between the fat and the acid, or if the
acid layer in not clear, repeat the temperature adjustment and centrifuging before taking the
reading.

Notes:
1. Fat column should be clear. If a fluffy layer is observed, test should be rejected and more care
should be taken to dissolve the curd completely.
2. If the fat column is so dark as to make the reading difficult to read, the test should be rejected
and the density of sulfuric acid should be checked.
Determination of titratable acidity in cream
The titratable acidity of fresh cream should be consistent with the fat percentage of cream.
There exists an inverse relationship between percent fat and percent titratable acidity of cream. The
percent titratable acidity of freshly separated cream is always lower than that of milk, from which
it is separated, and can be calculated by the formula
Percent titratable acidity of cream:
= (% serum in cream⁄% serum in milk) × % titratable acidity of milk
= (100 – % fat in cream) ∕ (100 – % fat in milk) × % titratable acidity of milk
The determination of titratable acidity in cream has importance during the neutralization of sour
cream to be converted into butter. The purpose of neutralization of sour cream is to avoid excessive
fat loss in butter making that result from churning of highly acidic pasteurized cream. During
pasteurization of sour cream, the casein curdles, thereby entrapping fat globules; as a result, bulk
of the curd goes in butter milk, which causes high fat loss. The cream acidity should be reduced to
0.25 to 0.30% before churning in case of butter for early consumption, whereas cream acidity should
be reduced 0.06 to 0.08% before churning for butter to be stored for longer period. Salted acid
butter develops a fish flavor during commercial storage at -23 to -29°C (De, 1980). In the following
method (BIS, 1966), the cream sample is diluted with water, followed by titration with a standard
alkali using phenolphthalein as an indicator.
Apparatus
1. Burette: 10 ml capacity
2. White porcelain dishes: 60 ml capacity.
3. Stirring rods of glass, flattened at one end.
Reagents
1. Standard sodium hydroxide solution: 0.1 N (aq.).
2. Phenolphthalein indicator solution: Dissolve 1 g of phenolphthalein in 110 ml ethanol (95%;
v/v). Add 0.1 N sodium hydroxide solution until one drop gives a faint pink coloration. Dilute
with distilled water to 200 ml.
3. Rosaline acetate stock solution: Dissolve 0.12 g of Rosaline acetate in approximately 50 ml
of rectified spirit containing 0.5 ml of glacial acetic acid. Make up to 100 ml with rectified
spirit.
4. Bench solution: Dilute 1 ml of the stock solution to 500 ml with a mixture of rectified spirit
and distilled water in equal portions by volume.
Note: The stock solution and the bench solution should be stored in dark brown bottles
securely stoppered with rubber bungs.
Procedure
1. Weigh 10.0 g cream into each of two white porcelain basins of approximately 60 ml capacity.
2. Add 10 ml of water to both basins and stir to disperse the cream.
 3. Prepare from one dilution a color control by adding and stirring 2 ml dilute Rosaline acetate
solution. Stir 2 ml phenolphthalein solution into the other dilution and, while stirring
vigorously, titrate as rapidly as possible using sodium hydroxide solution, from a 10 ml
burette until the color matches the pink color of the control. The titration shall be preferably
done in north daylight or under illumination from a daylight lamp.
Calculation
Express the titratable acidity in terms of ‘lactic acid’, g/100 g of cream.
Titratable acidity (as lactic acid), % by weight = 9AN/M
Where
A = volume in ml of standard sodium hydroxide required for titration;
N = normality of the standard sodium hydroxide solution (0.1 N); and
M = weight in g of cream taken for the test.
 ANALYSIS OF BUTTER
Introduction
Butter is a fat rich product obtained by churning the cream that has been separated from cow,
buffalo or mixed milk. It is then worked, i.e. excess
Table 1: Average composition of butter
water is removed, and the mass is rendered
Constituents Percentage
homogenous usually with the addition of salts. Butter Butterfat 80.0
consists of unaltered fat globules and moisture Moisture 16.0
Salt 0.03 to 1.8
droplets embedded in a continuous phase of butterfat. Curd 1.0 to 2.0
The textural properties are largely dependent on the proportion of liquid to solid fat in the
continuous phase. The flavor of butter is due to diacetyl and to a lesser extent to butyric, acetic,
propionic, formic acids, acetaldehyde and acetoin. Basically, butter contains butterfat, water and
curd, the last consisting of caseins, lactose and mineral matter. The average composition of butter
is shown in the Table 5.1 below. The vitamin A content of butter is very variable being dependent
on various factors such as season, climate and feeding practices of the animal.
Many types of butter are manufactured in the market. These differ with the type of cream used
for the preparation of butter, and the different manufacturing processes. Unless specifically
mentioned, different kinds of butter may or may not have been salted. A brief description of several
kinds of butter is as follows
1. Pasteurized cream butter: Made usually from pasteurized sweet cream. Such butter usually
had mild flavor than that made from similar cream not subjected to pasteurization.
2. Ripened cream butter: Prepared from cream in which a delicate pleasant aroma has been
developed before churning by ripening i.e. cream is cultured with butter culture and is kept
at a desired temperature. Ripened cream butter has delicate flavor which is sometimes
referred to as real butter flavor.
3. Butter prepared from un-ripened cream: The flavor of such butter is usually mild.
4. Salted butter: Butter in which common salt (NaCl) has been added.
5. Sweet cream butter: Acidity of churned cream should not exceed 0.2% lactic acid.
6. Unsalted butter: Butter containing no salt.
7. Sour cream butter: Butter prepared from cream which has acidity more than 0.2% lactic acid.
8. Fresh butter: Butter prepared from fresh cream and which is not kept more than 3 weeks.
FSS Specifications
According to FSS Rules (2011) table creamery butter is the product obtained from cow or
buffalo milk or a combination thereof, or from cream or curd obtained from cow or buffalo milk or
a combination thereof, with or without the addition of common salt and annatto or carotene as
coloring matter. It shall be free from other animal fats, wax and mineral oils, vegetable oils and fats.
No preservatives except common salt and no coloring matter except annatto or carotene shall be
added. It shall contain not less than 80.0% by weight of milk fat, not more than 1.5% by weight of
curd and not more than 3.0% by weight of common salt. Diacetyl may be added as a flavoring agent
but, if so used, the total diacetyl content shall not exceed 4.0 ppm. Calcium hydroxide, sodium
bicarbonate, sodium carbonate, sodium polyphosphate (as linear phosphates) may be added for
regulating the hydrogen ion concentration in the finished product not exceeding 0.2% by weight of
butter as a whole.
According to FSS Rules (2011) desi (cooking) butter means the product obtained from cow or
buffalo milk or a combination thereof or curd obtained from cow or buffalo milk or a combination
thereof without the addition of any preservative including common salt, any added coloring matter
or any added flavoring agent. It shall be free from any other animal fats, wax and mineral oils,
vegetable oils and fats. It shall contain not less than 76.0% of milk fat by weight. Butter sold or
offered for sale without any indication is to whether it is table butter or desi butter, the standards of
quality prescribed for table butter shall apply.
BIS Specifications
As per BIS (IS 13690: 1992), Butter has been graded into two categories, i.e. Table Butter and
White Butter
1. Table Butter: Table butter means the product made from pasteurized cream obtained from
pasteurized cow or buffalo milk or a combination thereof with or without ripening with the
use of standard lactic culture, addition of common salt, annatto or carotene as coloring matter
and diacetyl as flavoring agent.
2. White Butter: White butter means the product made from pasteurized cow or buffalo milk
or a combination thereof or pasteurized cream of cow or buffalo or a combination thereof
without ripening and without the addition of common salt, any added coloring matter or any
added flavoring agent.
Pasteurized butter shall also comply with the requirements given in Table 1.
Preparation of butter sample for chemical analysis.
Warm the laboratory
Table 1: BIS Requirements for Pasteurized Butter
sample in the original unopened Characteristic Table White
Butter Butter
container, which should be
Milk fat, % by mass, Min 80.0 82.0
from one-half to two-thirds full, Moisture, % by mass, Max 16.0 16.0
to a temperature at which the Acidity (as lactic acid), % by mass, Max 0.15 0.06
Curd, % by mass, Max 1.0 1.5
sample will be soft enough to Common salt, % by mass, Max 2.5 -
facilitate thorough mixing to a Diacetyl, ppm, Max 4 -
Coliform count, per ml, Max 5 5
homogeneous state (free from Total yeast and mold count, per ml, Max 20 20
un-softened pieces) either by mechanical shaker or by hand. The temperature of mixing shall
normally not exceed 39°C. Allow the butter sample to attain ambient temperature with frequent
mixing. Open the sample container and stir (not longer than 10 s) with a suitable device, for example
a spoon or spatula, before weighing.
Determination of moisture content in butter
Apparatus
1. Hot air oven: Maintained at 100  1 °C.
2. Flat bottom moisture dish: Dishes of height at least 25 mm and at least 50 mm in diameter
and made of appropriate material (for example stainless steel, nickel or aluminum) not
affected by boiling water.
3. Glass rods with one end flattened and about 9 cm in length.
4. Desiccator with an efficient desiccant.
5. Boling water-bath with rings to take dishes of 50 mm diameter.
6. Clay pipe triangles.
Procedure
1. Clean the dish and the glass rod and dry them in the hot air oven maintained at 100  1°C for
at least 1 h.
2. Allow to cool to the room temperature in a desiccator and weigh the dish. Accurately weigh
(to the nearest 0.1 mg) into the dish 3 to 4 g of the prepared butter sample.
3. Place the dish on a boiling water-bath supported on a clay pipe triangle for at least 20 min,
stirring at frequent intervals until no moisture can be seen. Wipe the bottom of the dish and
transfer it to the oven maintained at 100  1°C and keep it for 90 min. Allow the dish to cool
in the desiccator and weigh to the nearest 0.1 mg.
4. Heat the dish again in an oven for 30 min. Repeat the process of heating, cooling and
weighing until the differences between two consecutive weights does not exceed 0.1 mg.
Record the lowest mass and preserve the residue for the determination of curd.
Note: As per IDF procedure (IDF, 1986), the weight of butter sample taken for moisture
determination is 5 to 10 g and drying temperature is 102 ± 2°C for 1 h.
Calculation
Moisture, % by mass = 100 (M1 - M2 )/ (M1 - M)
Where,
M1 = mass in g, of the dish with the material before heating to constant weight;
M2 = mass in g, of the dish with the material after heating to constant weight; and
M = mass in g, of the empty dry dish.
Determination of curd content in butter
Butter contains small amounts of milk proteins. The quantity varies with the method of
manufacture, and the number of washings given after churning is over. In order to ensure good
storage quality, butter should contain as little of the protein residue as possible. Normally protein
residue does not exceed 1.5%. It is estimated by making fat free the residue obtained after the
determination of moisture. The residue is dried and weighed.
Apparatus
1. Gooch crucible or sintered funnel - with filter flask and adapter.
2. Glass funnel with folded 12.5 cm Whatman filter paper Grade 1.
3. Flat bottom flask: 250 ml capacity
4. Desiccator with efficient desiccant.
5. Asbestos.
6. Hot air oven: Maintained at 100 ± 1°C.
7. Conical flask: 250 ml capacity.
8. Glass beads
Reagent
n-Hexane or, alternatively, light petroleum hydrocarbon solvent (petroleum spirit) with boiling
range between 40 to 60°C. The reagent shall not leave more than 1 mg of residue after evaporation
of 100 ml.
Procedure
1. Prepare an asbestos mat in a Gooch crucible or sintered funnel. Dry it in a hot air oven
maintained at 100  1°C, cool in the desiccator and weigh. Alternatively, dry, cool and weigh
ordinary glass funnel with folded 12.5 cm filter paper.
2. Melt the residue in the moisture dish and add 25 to 50 ml of petroleum solvent and mix
well.
3. Fit the crucible to the filter flask or place the funnel with filter paper on a filter stand.
4. Wet the asbestos mat or the filter paper with petroleum solvent and decant the fatty solution
from the dish into the asbestos or the filter paper, leaving the sediment in the dish. Macerate
the sediment twice with 20 to 25 ml of petroleum solvent and decant again the fatty solution
into the asbestos or the filter paper.
5. Filter the solution and collect the filtrate in a clean, dried, tared 250 ml flat bottom flask
containing 1 to 2 glass beads.
6. With the aid of a wash-bottle containing petroleum solvent, wash all the fat and sediment
from the dish into the crucible or the filter paper.
 7. Finally, wash the crucible or the filter paper until free from fat, collecting all the filtrate in
the flask. Preserve the filtrate for the determination of fat. Dry the crucible or filter paper in
the oven maintained at 100  1°C for at least 30 min.
8. Cool in the desiccator and weigh. Repeat drying, cooling and weighing until the loss of weight
between the consecutive weighing does not exceed 0.1 mg. Preserve the residue for the
determination of salt.
Calculation
Curd and salt, % by mass = 100 (M1 - M2 )/M
Where,
M1 = mass in g, of the filter paper with residue;
M2 = mass in g, of the filter paper alone; and
M = mass in g, of the sample.
Note: Curd, % by mass, is obtained by subtracting the value of salt, % by weight from the value
obtained as described later.
Determination of fat content in butter (Gerber method)
Apparatus
As required for the determination of fat in cream by Gerber method.
Reagent
As required for the determination of fat in cream by Gerber method.
Procedure
1. Weigh 2.5 g of butter sample in a 50 ml beaker and mix it with small portion of 1:1 sulfuric
acid. Transfer the contents into the butyrometer with the help of a small funnel. Rinse the
beaker about six times with small quantities of diluted sulfuric acid to make sure that all butter
has been transferred. Add between 15 to 20 ml of the mixture to the butyrometer depending
on its size.
2. Add 1 ml of isoamyl alcohol. Do not wet the neck of the butyrometer with iso-amyl alcohol.
Close the neck of the butyrometer firmly with the stopper without disturbing the contents
using lock stopper key.
3. Shake the butyrometer carefully without inverting it until the contents are thoroughly mixed,
the curd is dissolved, and no white particles or flakes are visible in the butyrometer. Then
invert the butyrometer a few times to mix the contents thoroughly. Transfer the butyrometer
quickly, with the bulb uppermost, into a water bath having a temperature of 65  2°C and
leave it there for not less than 5 min. Take care to have the water level in the bath above the
top of the fat column in the butyrometer.
4. Take out the butyrometer from water-bath, dry it with a cloth and transfer it to the centrifuge,
placing two butyrometers diametrically opposite so as to balance the rotating disc.
5. Centrifuge the butyrometers at the maximum speed for 4 to 5 min. Bring the centrifuge to
stop gradually. Transfer the butyrometers, stopper downwards, into a water-bath having a
temperature of 65  2°C and allow the butyrometer to stand in the water-bath for not less than
3 min and not more than 10 min.
6. Before taking the reading, adjust the position of the fat column to bring the lower meniscus
of the fat column on to a main graduation mark. Record the fat percentage.
Calculation
Fat percentage in butter sample = Butyrometer reading X 2
Determination of salt content in butter sample (Volhard's method)
In this method (BIS, 1966) salt present in the butter sample is extracted with hot water from the
dried fat-free residue obtained in moisture determination. The chlorides are precipitated by adding
excess of silver nitrate. The unused silver nitrate is titrated with potassium thiocyanate using ferric
ammonium sulphate indicator.
Reaction
Ag+ (excess) + Cl- AgCl (solid)
+ -
Ag + SCN AgSCN (solid)
Fe+3 + SCN- [FeSCN]+2 (Reddish brown)
Apparatus
1. Beakers: 100, 250 ml capacity.
2. Volumetric flask: 100 ml, 1 L capacity.
3. Conical flask: 250 ml capacity
4. Water-bath: Maintained at 60 to 70°C
Reagents
1. Standard silver nitrate solution: 0.05 N, standardized against standard sodium chloride.
Dissolve slightly more than theoretical quantity (8.7 g per 1 L of water) of silver nitrate
(equivalent weight 169.89) in halogen-free water and dilute to volume (1 L).
2. Nitric acid: sp. gr. 1.42 approx. 70 % (m/m).
3. Nitric acid: Approximately 5 N.
4. Ferric ammonium sulphate indicator solution: Dissolve 50 g of ferric ammonium sulphate
[Fe2 (SO4 )3 . (NH4 )2 SO4 .24H2 O] in 95 ml of water containing 5 ml of 5 N nitric acid.
5. Standard potassium thiocyanate (KCNS) solution (Approx. 0.05 N): Standardized against
standard silver nitrate. Weigh approx. 5.25 g KCNS and dissolve in 1 L water. Allow to stand
overnight and filter, if necessary, to get a clear solution, standardize by titration against 0.05
N AgNO3 and dilute with requisite volume of water to get exactly 0.05 N KCNS solution.
Procedure
1. Extract the salt from the residue of curd and salt by repeated washing of the Gooch crucible
or filter paper with hot water, or by placing the crucible or filter paper in a beaker of hot
water.
2. Collect the rinsing in a 100 ml volumetric flask passing the solution through a filter paper.
Allow to cool to room temperature and make up to volume.
3. Take 25 ml water extract into a 250 ml conical flask and add an excess (normally 25 to 30
ml) of 0.05 N silver nitrate solution.
4. Acidify with nitric acid; add 2 ml of the indicator solution and 1 ml nitrobenzene. Mix and
determine the excess of silver nitrate by titration with the potassium thiocyanate solution until
the appearance of an orange tint, which persist for 15 s.
 5. In the same manner determine the equivalent of 25 ml or the added amount of silver nitrate
as thiocyanate using the same volumes of reagents and water.
Calculation
NaCl, % by mass = 23.38 X N X (A-B)/M
Where,
N = normality of potassium thiocyanate solution (0.005 N);
A = volume in ml, of potassium thiocyanate in blank titration;
B = volume in ml, of potassium thiocyanate in the sample titration; and
M = mass in g, of the butter sample.
Determination of salt content in butter sample (Mohr's method)
In this method, the butter sample is melted in hot water, and the chlorides present in the mixture
are titrated with a solution of silver nitrate using potassium chromate as indicator.
Reaction
AgNO3 + NaCl AgCl + NaNO3
2AgNO3 + K2 CrO4 Ag2 CrO4 + 2KNO3
(Brick–red ppt)
Apparatus
1. Conical flask: 250 ml capacity.
2. Burette: 50 ml capacity, graduated to 0.1 ml.
3. Pipette: capable of delivering 2.0 ml
4. Measuring cylinder: 100 ml capacity, graduated.
Reagents
1. Standard silver nitrate solution (0.1 N): Standardized against standard sodium chloride.
Dissolve slightly more than theoretical quantity of silver nitrate (equivalent weight 169.89)
in halogen-free water and dilute to volume. Dissolve between 17 g and 19 g of silver nitrate
in 1 L of water which is practically free from carbon dioxide. Standardize the silver nitrate
solution against standard sodium chloride solution. Store the solution away from direct
sunlight.
2. Potassium chromate indicator (5%, w/v): Dissolve 50 g of potassium chromate (K 2 CrO4 ) in
1 L of water.
3. Calcium carbonate: Analytical Grade, free from chloride.
Procedure
1. Weigh accurately 5 g of butter sample into the 250 ml conical flask. Carefully add 100 ml of
boiling distilled water. Mix the contents of the conical flask. Allow to stand with occasional
swirling for 5 to 10 min.
2. After cooling to 50 to 55°C (titration temperature), add 2 ml of potassium chromate solution.
Mix by swirling. Add about 0.25 g of calcium carbonate and mix by swirling.
3. Titrate at 50 to 55°C with standard silver nitrate solution while swirling continuously, until
the brownish color persists for half a minute.
4. Carry out a blank test with all the reagents in the same quantity except the butter sample. The
maximum deviation between duplicate determinations should not exceed 0.02% of sodium
chloride.
Calculation
NaCl, % by mass = 5.85 N (V 1 -V2 )/M
Note: IDF (2000) has suggested the following formula for the calculation NaCl content in
butter.
NaCl, % by mass = 5.844 N (V 1 -V2 )/M
Where,
N = normality of silver nitrate solution (0.1N);
V1 = volume in ml, of silver nitrate used in the sample titration;
V2 = volume in ml, of silver nitrate used in the blank titration; and
M = mass in g, of the butter sample.
 ANALYSIS OF GHEE
Introduction
Ghee, also known as anhydrous milk fat is the most widely used fat rich product in Asia
particularly in Indian subcontinent. Ghee is the Indian name for the clarified butter fat and is one of
best cooking or frying medium. It is usually prepared from cow milk, buffalo milk or mixed milk
by the clarification of the milk fat at high temperature. In the Middle East, ghee is commonly made
form goat, sheep or camel milk and it is known as maslee or some variant of Arabic term called
samna. The IDF (1977) has defined ghee as a product exclusively obtained from milk, cream or
butter from various animal species by means of processes which results in almost total removal of
moisture and solids-not-fat and which gives the product a particular physical structure. Ghee is a
good source of fat soluble vitamins A, D, E and K. Cow ghee has excellent nutritional qualities
especially beneficial to persons convalescing after chronic illness. Ancient Sanskrit literature
describes ghee (ghrit) as the food fit for gods and a commodity of enormous value. It is used for
performing yagna in all the religious ceremonies.
In general, ghee is prepared by four methods, namely, desi, creamery butter, direct cream and pre-
stratification method. The essential steps involved in the preparation of ghee by these methods
involve three distinct operations:
- Concentration of milk fat
- Heat clarification of fat rich milk portion at 105 to 110°C, and
- Removal of residue from the pure heat clarified fat.
The concentration of fat in form of cream, makkhan or creamery butter helps in reducing the
load of ghee residue, fat loss in ghee residue and amount of water to be evaporated by heat
clarification. During the moisture removal process by heat, ghee acquires its characteristic flavor,
and solids-not-fat contents are converted to brown residue which facilitates removal of maximum
fat from it. In the third step ghee residues are separated by decantation, cloth or pressure filtration
or the centrifugal clarification techniques.
The direct cream method is reportedly most economical for preparing ghee and the product has
better keeping quality. In the pre-stratification method, advantages such as economy in fuel
consumption and production of ghee with low acidity and comparatively longer shelf life have been
claimed (Ganguly and Jain, 1972).
The quality of ghee depends on milk, cream, dahi or butter, methods of preparation, temperature
of clarification, storage conditions and the type of animal feed. These factors in turn will determine
the physico-chemical characteristics of ghee. The principal measurements of ghee quality are
peroxide value, acidity and flavor.
Chemical composition of ghee
Chemically, ghee is a complex lipid of glycerides (usually mixed), free fatty acids,
phospholipids, sterols, sterol esters, fat soluble vitamins, carbonyls, hydrocarbons, carotenoids
(ghee derived from cow milk), small amounts of charred casein and traces of calcium, phosphorus,
iron etc. Glycerides
constitute about 98% of the Table 1. Chemical composition of ghee
total material. Of the remaining Constituents Cow Buffalo
Fat (%) 99 to 99.5 99 to 99.5
constituents of about 2%, sterols,
Moisture (%) Less than 0.5 Less than 0.5
mostly cholesterol occurs to the Carotene (mg/g) 3.2 to 7.4 -
Vitamin A (I.U. / g) 19 to 34 17 to38
extent of about 0.5% (Ganguli
Cholesterol (mg/100 g) 302 to 362 209 to 312
and Jain, 1972). Tocopherol (mg/g) 26 to 48 18 to 31
Specifications for ghee in India Free fatty acid (FFA) (%) 2.8 2.8
Reference: Aneja et al., 2002
FSSAI specifications
Ghee means pure clarified fat derived solely from milk or curd or from desi (cooking) butter or
from cream to which no coloring matter or preservative has been added. The standards for the
quality of ghee produced in a state/union territory are given in the table 5.2.
Table 2 Standards of ghee under FSS rules
S. Name of the state & U.T.
40°C
Reading at
B.R.

(Min)
Value
Meissl)
(Reichert
R.M.
(Max)
Oleic acid)
FFA % (as
% (Max)
Moisture
No.

1. Bihar, Chandigarh, Delhi, Punjab, 40 to 43 28 3 0.5

Haryana (Areas other than cotton tract
areas), West Bengal (Areas other than
cotton tract), Sikkim
2. Manipur, Meghalaya, Mizoram, 40 to 43 26 3 0.5
Arunachal Pradesh, Orissa, Nagaland,
Tripura, Assam, Goa, Kerala, Himachal
Pradesh, U.P., J & K, Maharashtra (Areas
other than Jodhpur Division), Haryana
(Cotton Tract Areas), Lakshadweep
3. Karnataka (Belgaum Distt.), Madhya 40 to 44 26 3 0.5
Pradesh (Areas other than cotton tract
areas), Pondicherry
4. Andhra Pradesh, Daman & Diu, Dadar &
Nagar Haveli, Karnataka (Areas other than 40 to 43 24 3 0.5
Belgaum Distt.)
5. Andaman & Nicobar Island, Tamil Nadu 41 to 44 24 3 0.5
6. Gujarat (Areas other than cotton tract) 40 to 43.5 24 3 0.5
7. Gujarat (Cotton tract areas), Madhya 41.5 to 45 21 3 0.5
Pradesh (Cotton tract areas), Maharashtra
(Cotton tract areas), Rajasthan (Jodhpur
sub division), West Bengal (Bishnupur sub
division)
- Baudouin test shall be negative.
- By cotton tract is meant the areas in the state where cotton seed is extensively fed to the cattle
and so notified by the state Govt. concerned.
 - Usually such cotton tract areas ghee has low RM value and high BR reading compared to
other areas.
- Ghee may contain BHA not more than 0.02 % as antioxidant.
Agmark specifications
The grading of ghee in India is in general done by Agmark Advisor to the Government of India
under provisions of Agricultural Produce (Grading and Marketing) Act of 1937. All ghee graded
by Agmark has to be pure and prepared from only cow or buffalo milk. Regional specifications
also exist in such grading due to variation in compositional properties of ghee influenced by the
type of animal feed.
As per Agmark grading, ghee is graded either as special or general separately for cow and
buffalo based on butyro-refractometer reading at 40°C, moisture, saponification value, Reichert-
Meissl value (RM value), Polenske value, and free fatty acids content.
Table 3 Agmark standards for ghee.
Parameter All India Regional
Winter Summer
Baudouin test Negative Negative Negative
Phytosterol acetate test Negative Negative Negative
Butyro-refractometer 40.0 to 43.0 41.5 to 44.0 42.5 to 45.0
reading at 40°C
Reichert-Meissl value* Not less than 28 Not less than 23 Not less than 21
Polenske value 1.0 to 2.0 0.5 to 1.2 0.5 to 1.0
Moisture content (%) Not more than 0.3 Not more than 0.3 Not more than 0.3
Free fatty acids (% oleic
acid)
a. Special grade Not more than 1.4 Not more than 1.4 Not more than 1.4
(Agmark Red Label)
b. General grade
(Agmark Green Not more than 2.5 Not more than 2.5 Not more than 2.5
Label)
*Ghee produced in cotton-tract area, the Reichert-Meissl value should not be less than 23.
BIS requirements (BIS, 1992) for butter oil (butter fat)

Table 4 BIS requirements (BIS, 1992) for butter oil (butter fat)
S. No. Characteristic Requirements
1 Milk fat, % by mass, Min 99.5
2 Moisture, % by mass, Max 0.5
3 Butyro-refractometer reading at 40°C 40.0 to 44.0
4 Reichert Meissl value, Min 21, 24, 26, 28 (see note)
24 (in case of imported butter
oil)
5 Free-fatty acid, % by mass (as oleic acid), Max 0.3
Peroxide value, milli-equivalent of oxygen per kg of 0.8
butter oil, Max
6 Coliform per g Absent
 Butter oil is prepared by melting butter at a temperature not exceeding 80°C, whereas ghee is
manufactured at 105 to 110°C. Butter oil has a bland flavor whereas ghee has a pleasing flavor.
Ghee has less moisture, contains more protein solids and differs in fatty acid and phospholipids as
compared to butter oil. Butter oil can be reconstituted with skim milk powder whereas ghee cannot
be.
Note: Reichert-Meissl values as applicable for different States/UT are:
RM State / UT
Value
21 Cotton tract areas in Gujarat, cotton tract areas in Madhya Pradesh, cotton tract areas in
Maharashtra, Jodhpur Division of Rajasthan and Bishnupur Sub-division of West
Bengal.
24 Andhra Pradesh, Andaman and Nicobar Islands, Dadra and Nagar Haveli, Daman and
Diu, areas other than cotton tract areas of Gujarat, areas other than Belgaum district of
Karnataka, Tamil Nadu.
26 Arunachal Pradesh, Assam, Goa, Cotton tract areas of Haryana, Himachal Pradesh,
Jammu and Kashmir, Belgaum district of Karnataka, Kerala, Lakshadweep, areas other
than cotton tract areas of Madhya Pradesh, areas other than cotton tract areas of
Maharashtra, Manipur, Meghalaya, Mizoram, Nagaland, Orissa, Pondicherry, areas
other than Jodhpur Division of Rajasthan, Tripura and Uttar Pradesh.
28 Bihar, Chandigarh, Delhi, areas other than cotton tract areas of Haryana, Punjab, areas
other than Bishnupur Sub-division of west Bengal and Sikkim.
Preparation of the test sample of ghee.
1. Mix the ghee sample in the container in which it is received until homogenous. Carry out this
operation in a cool place, away from direct sunlight, and complete it in shortest possible time.
In the event of any separation taking place in between, that is, mixing and commencement of
the chemical analysis for moisture determination, remix the sample. Use this for determining
of moisture.
2. After moisture determination, place the container or glass bottle containing ghee in a water-
bath at a temperature not higher than 50°C till completely melted. Filter through a dried,
fluted open-texture 15 cm Whatman filter paper Grade 4 with the help of a hot water funnel,
directly into receiving bottle. The filtered ghee should be bright and clear.
Determination of moisture content in ghee.
The moisture content of ghee is the loss in mass, expressed as a percentage by mass when the
product is heated in a hot air oven at 105 ± 1°C to constant mass
Apparatus
1. Flat-bottom moisture dishes with cover: Of stainless steel, nickel or aluminum having
approximately 50 mm diameter and 25 mm depth. The dishes shall have lids which fit well
and can readily be removed.
2. Hot air oven: Maintained at 105 ± 1°C.
3. Desiccator: Containing an efficient desiccant (e.g. freshly dried silica gel with a hygrometric
indicators).
Procedure
1. Uncover a dish and place the dish and its lid in a hot air oven at 105 ± 1°C for 1 h. Place the
lid on the dish, transfer the covered dish from the hot air oven to the desiccator. Allow it to
cool to room temperature and weigh it.
2. Transfer into the dish, about 10.0 g of the prepared sample of ghee and weigh quickly, with
the lid on the dish.
3. Transfer the uncovered dish containing the test portion of ghee sample to a hot air oven
maintained at a temperature of 105 ± 1°C, placing the lid along its side, for 1 h.
4. After 1 h, cover the dish with lid and immediately transfer it to a desiccator. Allow to cool to
room temperature for about 30 min and weigh to the nearest 0.1 mg. Return the dish,
uncovered, and the lid to the oven and heat again for 30 min.
5. Remove the dish to the desiccator, cool and weigh, as before. Repeat the operations of heating
(for 30 min), cooling and weighing until the difference in mass between two consecutive
weighing does not exceed 1 mg. Record the lowest mass.
6. Preserve the dried sample for the determination of insoluble impurities.
Calculation
Moisture and volatile matter content, % by mass = {100(M2 -M)/(M1 -M)}
Where
M = mass in g, of the empty dish, with lid;
M1 = mass in g, of the dish, lid, along with the ghee sample before drying; and
M2 = mass in g, of the dish, lid, along with the ghee sample after drying.
Express the results to the nearest 0.01% (m/m). The maximum deviation between duplicate
determinations shall not exceed 0.1%.
Determination of B.R. (butyro-refractometer) or refractive index reading of ghee.
Refractive index is the ratio of the velocity of light in vacuum to the velocity of light in the
sample medium; more generally, it is expressed as the ratio between the sine of angle of incidence
to the sine of the angle of refraction, when a ray of light of a definite known wavelength (usually
589.3 m the mean of the D-line of sodium) passes from air into ghee.
The refractive index of ghee may be read on an Abbe refractometer which gives the true
refractive index or on a butyro-refractometer, which reads on an arbitrary scale (B.R. reading) at
constant temperature. Since ghee is often solid or semi-solid at room temperature, the B.R. reading
is usually taken at 40°C, at which the ghee sample is clear and transparent (BIS, 1966).
The general values for B.R. reading of milk fat (40 to 43) and vegetable oils and fats (above
50) are so wide apart that this property can be employed as an indicator for milk fat adulteration
with vegetable oils and fats, except coconut oil (38 to 39) and palm oil (39 to 40). The B.R. reading
of animal body fats are in the range of 44 to 51 (Kumar et al., 2002). An increase in B.R. reading
in ghee is also caused by a decrease in the content of lower chain fatty acids, or by an increase either
in higher saturated or unsaturated fatty acids, particularly the latter (Rangappa and Achaya, 1974).
Apparatus
1. Precision Butyro-refractometer fitted with an accurate thermometer reading from 40 to 50°C.
Check the calibration of the instrument as per the manufacturer’s instructions.
2. Hot water circulating device to maintain the temperature of prism constant at 40  1°C.
3. Sodium lamp: Daylight can also be used if the refractometer has an achromatic compensator.
Reagent
Standard fluid for checking the accuracy of the instrument. Usually this fluid is provided by the
manufacturer for calibration of the refractometer.
Procedure
1. The sample should be rendered optically clear and free from water and suspended impurities.
This can be done by using following procedure.
2. After moisture determination, place the container or glass bottle containing ghee in a water-
bath at a temperature not higher than 50°C till completely melted. Filter through a dried,
fluted open-texture 15 cm Whatman filter paper Grade 4 with the help of a hot water funnel,
directly into receiving bottle. The filtered ghee should be bright and clear.
3. The correctness of the butyro-refractometer shall be tested before carrying out the test with
liquid of known refractive index (at 40°C). Open the double prism of the instrument and place
a few drops of the ghee sample on prism. Ghee shall completely fill the space between the
two prisms and shall show no air bubble. Close prisms firmly. Allow the instrument to stand
for few min before reading is taken so that temperature of ghee sample and instrument are
 same. The reading shall be taken after ghee has been kept in the prism for 2 to 5 min and after
it has been ensured that it has attained constant temperature (40°C) by taking two or more
readings. B.R. reading of ghee decreases with the rise in temperature and vice versa. A
correction is needed whenever; temperature of the butyro-refractometer is either below or
above 40°C.
4. Method of measurement is based upon observation of position of border line on the scale of
the instrument. Hold sector firmly and move backward or forward until field of vision is
divided into light and dark portion. Line dividing these portions may not be sharp but a band
of colors. The colors are eliminated by rotating screw head of compensator until sharp,
colorless line is obtained. Read the BR of the sample directly on the scale.
Notes
1. It should be born in mind that presence of free fatty acids considerably lowers the refractive
index.
2. In the homologues series of saturated fatty acids from butyric to stearic, the refractive index
rises steeply among the lower members, and flattens out at higher chain lengths. A double
bond elevates the refractive index: stearic acid has a lower refractive index than oleic, which
in turn has a lower value than linoleic.
3. For conversion of refractive index values into butyro-refractometer reading and vice versa
use the following formula (Rangappa and Achaya, 1974) or table (BIS, 1966) as follows:
Inter-conversion of Refractive index and Butyrometer Degrees
A. Refractometer index to Butyrometer degrees
Butyrometer reading in degrees = 42.0 + Factor (Observed refractive index - 1.4538)
The factor to be used varies with the range of refractive index, as shown below:
Observed refractive index range Factor
1.4500 - 1.4515 1400
1.4515 - 1.4530 1410
1.4530 - 1.4545 1420
1.4545 - 1.4560 1430
1.4560 - 1.4575 1440
B. Butyrometer degrees to Refractometer index
Refractometer index = 1.4538 + Factor (Observed BR - 42.0)
The factor to be used varies inversely with temperature
Observed BR reading range Factor
37.5 - 40.0 0.00072
40.0 - 42.5 0.00071
42.5 - 45.0 0.00070
45.0 - 47.5 0.00069
 4. The refractive index decreases with a rise and increases with a fall in temperature. If the
temperature is not exactly at 40°C, X is added to the observed reading for each degree above
or subtracted for each degree below 40°C pro rata, where
X for butyro-refractometer = 0.55
X for Abbe refractometer = 0.000365
Normally the temperature of observation shall not deviate by more than  2°C.
Table: Butyro-refractometer readings and indices of refraction
B.R. Refractive B.R. Refractive B.R. Refractive
Reading Index Reading Index Reading Index
35.0 1.4488 40.5 1.4527 46.0 1.4565
35.5 1.4491 41.0 1.4531 46.5 1.4569
36.0 1.4495 41.5 1.4534 47.0 1.4572
36.5 1.4499 42.0 1.4538 47.5 1.4576
37.0 1.4502 42.5 1.4541 48.0 1.4579
37.5 1.4506 43.0 1.4545 48.5 1.4583
38.0 1.4509 43.5 1.4548 49.0 1.4586
38.5 1.4513 44.0 1.4552 49.5 1.4590
39.0 1.4517 44.5 1.4555 50.0 1.4593
39.5 1.4520 45.0 1.4558
40.0 1.4524 45.5 1.4562

The refractive index decreases with a rise and increases with a fall in temperature. If the
temperature is not exactly at 40°C, X is added to the observed reading for each degree above
or subtracted for each degree below 40°C pro rata, where
X for butyro-refractometer= 0.55
X for Abbe refractometer = 0.000365
Normally the temperature of observation shall not deviate by more than + 2°C.
Accuracy of the Method
The maximum difference between duplicate determinations shall not exceed 0.0002 units for
the refractive index and 0.1 for the butyro-refractometer reading.
Determination of acidity in ghee.
According to Agmark standards FFA level should not be more than 1.4% for Special Grade
(Red label) ghee and FFA should not be more than 2.5% for general grade (Green label) ghee and
according to FSS rules, ghee should not contain FFA more than 3%.
The acidity (free fatty acid content) of a fat is normally a measure of the extent to which
hydrolysis has liberated the fatty acids from there ester linkage with the parent glyceride molecule.
Partly for this reason, acidity of ghee is extensively quoted as a free fatty acid content percent (%
FFA). The FFA content of fresh ghee varies from 0.09 to 0.28% with an average of 0.16%. The
average FFA value of ghee increases to 0.28, 0.34, 0.39 and 0.44 during storage of ghee at 37°C for
1, 2, 3 and 4 months respectively (Kumar and Bector, 1985). The sensory quality of ghee
deteriorates with increase in FFA content.
Reaction
CH2 OCOR1 CH2 OH R1 COOH
| | +
CHOCOR2 + 3 H2 O CHOH + R2 COOH
| | +
CH2 OCOR3 CH2 OH R3 COOH

Triglyceride Glycerol Free Fatty Acids

RCOOH + NaOH RCOONa + H 2 O
Level of acidity shows the proneness of fat to oxidation. Highly acidic ghee shows faster
oxidation, and thus has poor keeping quality. With the increase in acidity, there is decrease in
consumer acceptability. The FFA present in ghee can be estimated by acid-base titration with alkali
(NaOH) using phenolphthalein as an indicator and the end point comes at around pH 8.3 (BIS,
1966).
Apparatus
1. Conical flasks: 250 ml capacity.
2. Burette: 50 ml, graduated to 0.1 ml.
Reagents
1. Ethanol or rectified spirit: 95 % (v/v), sp. gr. 0.816, neutral to phenolphthalein.
2. Sodium hydroxide or Potassium hydroxide: 0.1 N aqueous solution accurately standardized
against oxalic acid (AR grade) or potassium phthalate.
3. Phenolphthalein indicator: 1.0% solution in 95 % (v/v) ethanol or rectified spirit.
Procedure
1. Weigh 10 g of the ghee sample in a 250 ml conical flask. In another flask bring 50 ml of
ethanol to the boiling point and while still above 70°C, neutralize it to phenolphthalein (using
0.5 ml) with 0.1 N NaOH.
2. Add the neutralized alcohol to flask containing ghee sample and mix the contents of the flask.
 3. Bring the mixture to boil and while it is still hot, titrate with 0.1 N NaOH, shaking vigorously
during the titration. The end point of the titration is reached when the addition of single drop
produces a slight, but a definite color change persisting for at least 15 sec.
Calculation
The acidity of ghee can be expressed in different ways:
1. Acid value: The number of mg of KOH required to neutralize the free fatty acids present in
1 g of the ghee sample using following formula:
Acid value = 5.61T/M
Where
T = volume of 0.1 N alkali required for titration in ml;
M = mass in g, of sample taken.
2. Free fatty acids: The acidity of ghee is frequently expressed as the percentage of free fatty
acids in the sample, calculated as oleic acid, using following formula:
Free fatty acids = 2.82 T/M
Where
T = volume of 0.1 N alkali required for titration in ml;
M = mass in g, of ghee sample taken.
3. Degree of acidity: It is the total titratable acidity present in the ghee sample expressed as
percentage:
Degree of acidity = 100N/M
Where
N = ml of 1 N alkali used for titration;
M = mass in g, of ghee sample taken.
The maximum deviation between duplicate determination shall not exceed 0.2 degree of the
acidity or equivalent.
Derivation
1 ml of N NaOH = 1 ml of N oleic acid = 0.282 gm of oleic acid
1 ml of 0.1N NaOH = 0.0282 gm of oleic acid
Now, if T ml of 0.1N NaOH is used for W gram of the fat sample.
%FFA = 2.82 V/W
Determination of soluble and insoluble volatile acids (Reichert-Meissl, Polenske value and
Kirschner value) in ghee.
The Reichert-Meissl (R.M.) value is the number of ml of 0.1 N aqueous alkali solution required
to neutralize the water-soluble, steam volatile fatty acids distilled from 5 g of ghee under the precise
conditions specified in the method.
The Polenske value (P.V.) is the number of ml of 0.1 N aqueous alkali solution required to
neutralize the water-insoluble, steam volatile fatty acids distilled from 5 g of ghee under the precise
conditions specified in the method.
The Kirschener value is the number of ml of 0.1 N aqueous alkali solution required to neutralize
the water-soluble steam volatile fatty acids which form water-soluble silver salts distilled from 5 g
of ghee under the precise conditions specified in the method.
The R.M. value is substantially a measure of the lower fatty acids of ghee like butyric (C 4:0 ),
and caproic (C6:0 ). Butyric acid contributes about three fourth and caproic acid one-fourth to the
R.M. value, while the P.V. is made up of one-fourth caprylic (C8:0 ) and three fourth capric acid
(C10:0 ). Since the presence of lower fatty acids is peculiar to milk fat, the R.M. and P.V. are
important characteristics of ghee. In general, R.M. value and P.V. for cow and buffalo ghee is about
28 and 1.5 respectively. Of the common vegetable oils, only coconut and palm kernel contain steam
volatile acids, and both exhibit R.M. of 7 and P.V. of 13. In general, ghee is required to have a
R.M. value not less than 28. It is however of interest that ghee from milk of animals fed cotton
seeds has much lower R.M. value of about 20. Polenske value of cow ghee is higher (2 to 3) than
buffalo ghee (1 to 1.5). No significant seasonal variations have, however, been recorded for their
fat constants (Ganguly and Jain, 1972).
In the following method (BIS, 1966), ghee (5 g) is saponified using glycerol-potash diluted
with water and acidified, and thereafter steam distilled in a glass apparatus (Polenske distillat ion
apparatus) at a controlled rate. The condensed and cooled distillate is filtered; the water-soluble
acids which pass through are estimated by titration with alkali to give the R.M. value, while the
water-insoluble acids collected on the filter paper are dissolved out in alcohol and titrated to give
the P.V.
Reactions
CH2 OCOR1 CH2 OH+ R1 COONa
| |
CHOCOR2 + 3 NaOH CHOH + R2 COONa
| |
CH2 OCOR3 CH2 OH+ R3 COONa
Triglyceride Glycerol Fatty acids
RCOONa + H2 SO4 Na2 SO4 + RCOOH
RCOOH + NaOH RCOONa + H2 O
Fatty acids Sodium salt of fatty acid
Apparatus
1. Graduated Cylinders: 25 ml and 100 ml capacities.
2. Pipette: 50 ml capacity.
3. The assembly of the apparatus for the distillation is shown in figure 1 and details of the
constituent parts are given below:
Figure 1 Polenske distillation apparatus
Flat-bottom boiling flask (Polenske): The flask shall be made of heat-resistance glass and
shall conform to the following details:
Volume contained to bottom of neck 310 ± 10 ml
Length of neck 75 ± 5 mm
Internal diameter of neck 21 ± 1 mm
Overall height 160 ± 5 mm
Diameter of base 45 ± 5mm
Still-head: The still-head shall be made of glass tubing of wall thickness 1.25 ± 025 mm, and
shall conform to the shape shown in Figure 6.3, and with the following dimensions:

A 180 ± 5 mm
B 107.5 ± 2.5 mm
C 80 ± 5 mm
D 70 ±5 mm
E 20 ± 2 mm
F 4 ± 1 mm
G (external diameter of bulb) 37.5 ± 2.5 mm
Internal diameter of tubing 8.0 ± 0.5 mm
Acute angle between slopping
part of still-head and vertical 60 ± 2°
A rubber stopper fitted below the bulb of the longer arm of the still-head and used for
connecting it to the flask shall have its lower surface 10 mm above the center of the side hole
of the still-head.
Condenser: The condenser shall be made of glass and conform to the following dimensions:
Overall length 520 ± 5 mm
Length of water jacket 300 ± 5 mm
Length of widened part above water jacket 70 ± 10 mm
Wall thickness of widened part 1.25 ± 0.25 mm
Internal diameter of widened part 20 ± 1 mm
External diameter of inner tube 12 ± 0.5 mm
within water jacket
 Wall thickness of inner tube 1.0 ± 0.2 mm
Wall thickness of outer jacket 1.25 ± 0.25 mm
External diameter of water jacket 30 ± 2 mm
Receiver: The receiver shall be a flask with two graduation marks on the neck, one at 100 ml
and the other at 110 ml.
Asbestos-Board: An asbestos-board of 120 mm diameter and 6 mm in thickness, with a
circular hole of about 65 mm in diameter shall be used to support the flask over the burner.
During distillation the Polenske flask shall fit snugly into the hole in the board to prevent the
flame from impinging on the surface of the flask above the hole. A new asbestos-board may
conveniently be prepared by beveling the edge of the hole, soaking in water, molding the
edge with a flame, and drying.
Gas Burner: The burner should be sufficiently large to allow the distillation to be completed
in the time specified in the procedure.
The apparatus shall be supported on a retort stand.
Glass Funnel: Of approximate diameter 6 cm.
Reagents
1. Glycerol: 98% (m/m) conforming to AR grade.
2. Sodium hydroxide solution (50% (m/m)): Dissolve sodium hydroxide in an equal weight of
water and store the solution in a bottle protected from carbon dioxide. Use the clear portion
free of the solution from deposit.
3. Dilute sulfuric acid: Dilute 25 ml of concentrated sulfuric acid to 1:1 and its concentration is
adjusted with water until 40 ml of diluted sulfuric acid neutralize 2 ml of the 50% NaOH
solution.
4. Ethanol: 95 % (v/v), neutralized to phenolphthalein immediately before use, or neutralized
denatured spirit.
5. Glass Beads: Approximately 1.5 to 2.0 mm in diameter.
6. Phenolphthalein indicator: 0.5% solution in 95% (v/v) ethanol or rectified spirit.
7. NaOH solution: Approximately 0.1 N aqueous solution of NaOH of accurately determined
strength.
8. Barium hydroxide solution: 0.1 N.
9. Silver sulfate: Powdered AR grade.
10. Whatman filter paper: Grade 4.
Procedure
1. Weigh 5.00  0.01 g of ghee sample into a Polenske flask. Add 20 g of glycerol and 2 ml of
50% NaOH solution. Heat the flask over a direct flame using a Bunsen burner, with
continuous mixing, until ghee, including any drops adhering to the upper parts of the flask,
 is saponified, and the liquid becomes perfectly clear; avoid overheating during this
saponification. Cover the flask with a watch-glass.
2. Make a blank test without ghee but using the same quantities of reagents and following the
same procedure, again avoiding overheating; such overheating would be indicated by
darkening of the solution. Measure 93 ml of boiling distilled water, which has been
vigorously boiled for 15 min, into a 100 ml graduated cylinder.
3. When the soap is sufficiently cool to permit addition of the water without loss, but before the
soap has solidified, add water, draining the cylinder for 5 sec, and dissolve the soap. If the
solution is not clear (indicating incomplete saponification), or is darker than yellow
(indicating overheating), repeat the saponification with a fresh sample of ghee. Add two glass
beads, followed by 50 ml of the dilute sulfuric acid, and connect the flask at once with the
distillation apparatus.
4. Heat the flask without boiling its contents, until the insoluble acids are completely melted,
then increase the flame and distil 110 ml in between 19 and 21 min. Keep the water flowing
in the condenser at a sufficient speed to maintain the temperature of the issuing distillate
between 18 and 21°C.
5. When the distillate reaches the 110 ml mark, remove the flame and replace the 110-ml flask
by a cylinder of about 25 ml capacity, to collect draining. Close 110 ml flask with its stopper
and without mixing the contents; place it in water at 15°C for 10 min so as to immerse the
110 ml mark. Remove the flask from the water, dry from outside, and invert the flask carefully
avoiding wetting the stopper with insoluble acids. Mix the distillate by four or five double
inversions, without violent shaking.
6. Filter through a dry 9 cm open-texture filter paper (Whatman filter paper Grade 4) which fits
snugly into the funnel. Reject the first running’s and collect 100 ml in a dry volumetric flask;
cork the flask and retain the filtrate for titration.
7. Detach the still-head and wash the condenser with three successive 15 ml portions of cold
distilled water, passing each washing separately through the cylinder, the 110 ml flask, the
filter and the funnel, nearly filling the paper each time and draining each washing before
filtering the next. Discard the washings.
8. Dissolve the insoluble acids by three similar washings of the condenser, the cylinder, and the
filter, with 15 ml of neutralized ethanol, collecting the solution in the 110-ml flask and
draining the ethanol after each washing. Cork the flask and retain the solution for titration
(for Polenske value).
9. Reichert-Meissl or soluble volatile acid value: Pour 100 ml of the filtrate (obtained in step 6)
containing the soluble volatile fatty acids into a titration flask, add 0.1 ml of phenolphthalein
indicator and titrate with 0.1 N NaOH solution until the liquid becomes pink.
 10. If the Kirschner value is to be obtained, the titration flask shall be dried before use; note the
actual volume of barium hydroxide solution used; drain the 100 ml flask into the titration
flask, close with a cork and continue as given below (See step 12).
11. Polenske or insoluble volatile acid value: Titrate the alcoholic solution of the insoluble
volatile acids after addition of 0.25 ml of phenolphthalein indicator, with the 0.1 N barium or
sodium hydroxide solution until the solution becomes pink.
12. Kirschner value: Add 0.5 g of finely powdered silver sulfate to the neutralized solution
reserved in step 15. Allow the flask to stand in the dark for 1 h with occasional shaking and
filter the contents in the dark through a dry filter. Transfer 100 ml of the filtrate to a dry
Polenske flask, add 30 ml of cold distilled water, recently boiled for 15 min, 10 ml of the
dilute sulfuric acid and 0.1 g of the pumice powder or two glass beads. Connect the flask with
standard apparatus and repeat the process as described above, i.e. the distillation of 110 ml in
19 to 21 min, the mixing (but without the cooling for 10 min), and the filtration and the
titration of 100 ml of the filtrate with the barium hydroxide solution.
Calculassions
Reichert-Meissl Value = 1.10 (T1 – T2 )
Polenske value = T3 – T4
Kirschner value = {121(100+T1) (T5-T6)}/10000
Where
T 1 = volume in ml of 0.1 N barium or sodium hydroxide solution used for sample under step 9;
T 2 = volume in ml of 0.1 N barium or sodium hydroxide solution used for blank under step 9;
T 3 = volume in ml of 0.1 N barium or sodium hydroxide solution used for sample under step 11,
T 4 = volume in ml of 0.1 N barium or sodium hydroxide solution used for blank under step 11;
T 5 = volume in ml of 0.1 N barium hydroxide solution used for sample under step 12; and
T 6 = volume in ml of 0.1 N barium hydroxide solution used for blank under step 12.
Polenske values, and to a much slighter extent Reichert values, have been found to be low
when determined at low barometric pressures, such as may occur at high altitudes. The
following factors may be applied to values determined at a barometric pressure to convert
them to the values determined at normal pressure.
Correct Reichert Value = [{(observed value-10) log760}/log P] +10
Corrected Polenske Value = Observed value x {(760-45)/(P-45)}
Where
P = barometric pressure in mm of mercury at the place and time of determination.
Accuracy of the Method
Reichert-Meissl Value: The maximum deviation between duplicate determinations shall not
exceed 0.5 units.
 Polenske Value: The maximum deviations between duplicate determinations shall not exceed
0.3 units.
Kirschner Value: The maximum deviations between duplicate determinations shall not exceed
0.5 units.
 Evaporated and Condensed milk
Compositional standards for dried milk in India
FSSAI specifications
Condensed milk unsweetened (Evaporated milk) means the product obtained from cow or
buffalo milk or a combination thereof or from standardized milk, by the partial removal of water.
It may contain added calcium chloride, citric acid and sodium citrate, sodium salts of
orthophosphoric acid and polyphosphoric acid (as linear phosphate) not exceeding 0.3% by weight
of the finished product. Such additions need not be declared on the label. Condensed milk
unsweetened shall contain not less than 8.0% milk fat and not less than 26.0% milk solids.
If the product is subjected to Ultra High Temperature (UHT) treatment by heating it at a temperature
of not less than 140°C for a minimum period of 3 sec followed by aseptic packaging, it shall be
designated as UHT and labeled as specified under clause of sub-rule (B) or Rule 42.
Condensed milk sweetened means the product obtained from cow or buffalo milk or a
combination thereof or from standardized milk, by the partial removal of water and after addition
of cane sugar. It may contain added refined lactose, permitted flavor, calcium chloride, citric acid,
sodium citrate, sodium salts of orthophosphoric and polyphosphoric acid (as linear phosphate) not
exceeding 0.3% by weight of the finished product. Such additions need not be declared on the label.
Condensed milk sweetened shall contain not less than 9.0% milk fat and not less than 31.0%t total
milk solids and not less than 40.0% cane sugar. If the product is subjected to Ultra High
Temperature (UHT) treatment by heating it at a temperature of not less than 140°C for a minimum
period of 3 sec followed by aseptic packaging, it shall be designated as UHT and labeled as specified
under clause of sub-rule (B) or Rule 42.
Condensed skimmed milk unsweetened (Evaporated skimmed milk) means the product
obtained from cow or buffalo skimmed milk or a combination thereof by the partial removal of
water. It may contain added calcium chloride, citric acid and sodium citrate, sodium salts of
orthophosphoric acid and polyphosphoric acid (as linear phosphate) not exceeding 0.3% by weight
of the finished product. Such additions need not be declared on the label. Condensed skimmed milk
unsweetened shall contain not less than 20.0% total milk solids. The fat content shall not exceed
0.5% by weight. If the product is subjected to Ultra High Temperature (UHT) treatment by heating
it at a temperature of not less than 140°C for a minimum period of 3 sec followed by aseptic
packaging, it shall be designated as UHT and labeled as specified under clause of sub-rule (B) or
Rule 42.
Condensed skimmed milk sweetened means the product obtained from cow or buffalo skimmed
milk or a combination thereof or from standardized milk, by the partial removal of water and after
addition of cane sugar. It may contain added refined lactose, calcium chloride, citric acid, sodium
citrate, sodium salts of orthophosphoric and polyphosphoric acid (as linear phosphate) not
exceeding 0.3% by weight of the finished product. Such additions need not be declared on the label.
Condensed skimmed milk sweetened shall contain not less than 26.0% of total milk solids and not
less than 40.0% cane sugar. The fat content shall not exceed 0.5% by weight.
BIS specifications
BIS has specified the requirements for condensed milk and UHT condensed milk. Whereas for
evaporated milk, no specifications have been defined.
The products shall be manufactured from fresh, whole, standardized, reconstituted or recombined
milk obtained from cow milk or buffalo milk or a mixture thereof (milk solids suitably processed
may also be used), suitably standardized to give the final products as given the following tables.
The milk and / or milk solids used in the manufacture shall be free from non-permitted additives.
It contains added sugar.
The products may contain added refined lactose, permitted flavors, calcium chloride, citric acid,
sodium citrate, sodium salts of orthophosphoric acid and polyphosphoric acid. Such additions shall
not exceed 0.3% by mass of the finished product.
Table 1 Compositional specifications for condensed milk, partly skimmed and skimmed
condensed milk
S. No Characteristic Condensed Partly skimmed Skimmed
milk condensed milk condensed
milk
1. Total milk solids, % by mass, Min 31.0 28.0 26.0
2. Fat, % by mass Not less Above 3.0 & Not more
than 9.0 below 9.0 than 0.5
3. Sucrose, % by mass, Min 40.0 40.0 40.0
4 Titratable acidity (as lactic acid), % 0.35 0.35 0.35
by mass, Max
5. Bacterial count per g 500 500 500
6. E. coli per g Absent Absent Absent
7. Shigella and Salmonella per 25 g Absent Absent Absent
8. Coagulase positive Staphylococcus Absent Absent Absent
aureus per g
9. Yeast and mold count per g 10 10 10
10. Accelerated storage test To satisfy the requirements of the test

Table 2 Compositional specifications for different types of UHT condensed milk and UHT
condensed skimmed milk
S. No Characteristic UHT Condensed UHT Condensed
milk skimmed milk
1. Total milk solids, % by mass, Min 26.0 20.0
2. Fat, % by mass Not less than 8.0 Not more than
0.5
3. Sucrose, % by mass, Max 18.0 18.0
4 Titratable acidity (as lactic acid), % by mass, 0.35 0.35
Max
5. Titratable acidity (as acetic acid) after 0.37 0.37
incubation of packages for 7 days at 37°C, %
by mass, Max
6. Bacterial spores (Plate count) per 0.1 g, Max 10 10
7. Coliform Absent Absent
8. Coagulase positive Staphylococcus aureus Absent Absent
 The products shall have whitish to light brown color. The flavor of the products shall be pleasant
and clean. They shall be free from rancid, fruity, moldy, tallowy, cooked, sour, sandy and any other
objectional odour and taste. Sugar used in the manufacture of these products should of good quality
food grade. The products may be fortified with vitamins A, D and B groups. The vitamins shall be
of food grade. Levels of fortification shall be declared on the container. However, even with this
fortification, exclusive use of condensed milk for infant is not recommended. The material shall not
have more than 30% of the lactose crystals of size greater than 15 µm.
Analysis of evaporated and condensed milk
Preparation of the test sample
1. Samples of recently manufactured products in which no appreciable separation of
components may be expected: Open the container, transfer all material adhering to the lid
into the container and thoroughly mix by an up and down movement of a spoon in such a way
that the top layers and the contents of the lower corners are moved and mixed. When the
product is in a can, transfer the contents to a jar with a well-fitting lid. When the product is
in a collapsible tube, transfer as much as possible of the contents to a jar with a well-fitting
lid; then cut open the tube, scrape out all material adhering to the interior and transfer this
also to the jar. Mix the contents of the jar as described above.
2. Sample of the older products and sample in which separation of components may be expected:
Heat in a water bath at about 40°C until the sample has nearly reached this temperature, open
the container and proceed as described above (a). When the product is in a can or tube, transfer
the contents to a jar, scrape out all material adhering to the walls (in the case of a collapsible
tube, after cutting open the tube) and continue the mixing until the whole mass in
homogeneous, reducing the size of any large crystals by crushing them with a glass rod. Close
the jar with a well-fitting lid. Allow to cool.
Determination of sucrose in condensed milk
Lane-Eynon Method (Volumetric method); IS: 4079-1967
Apparatus
1. Volumetric flasks: 100, 250, 500 and 1000 ml capacities.
2. Burette: 50 ml, graduated to 0.1 ml.
3. Funnels.
4. Conical flasks: 250 ml capacities
5. Pipette: 1, 5 and 10 ml capacities.
6. Measuring cylinder: 25 and 50 ml capacities.
7. Whatman filter paper: Grade 1.
8. Watch glass.
9. Hot plate.
Reagents
1. Sodium Hydroxide solution (approximately 0.1N).
2. Stock solution of invert sugar: Weigh accurately 9.5 g of pure sucrose on a watch glass and
transfer it to a 1-liter volumetric flask with 100 ml of water. Add 5 ml of concentrated
hydrochloric acid. Allow this to stand for 3 days at 20 to 25°C and make up the final volume
to 1 L with distilled water. (This solution is stable for several months).
3. Standard solution of invert sugar: Neutralize a known aliquot of stock solution of invert sugar
(reagent 2) with dilute sodium hydroxide solution using litmus paper and dilute with distilled
water to a known volume so that more than 15 ml but less than 50 ml of it shall be required
to reduce all the copper in 10 ml of Fehling's solution taken for titration. Note the
concentration of invert sugar in this solution as mg per 100 ml. Prepare this solution fresh
every day.
Note: When 10 ml of Fehling’s solution is taken for titration, a standard invert sugar solution
containing 0.12 to 0.30 % (w/v) of invert sugar is used.
4. Methylene Blue indicator solution: Dissolve 0.2 g of methylene blue in water and dilute to
100 ml.
5. Fehling's Solution: This is prepared by mixing, immediately before use, equal volumes of the
following two solutions, known as Fehling's A and Fehling's B.
a. Fehling’s A: Dissolve 34.639 g copper sulphate (CuSO 4 .5H2 O) in water, add 0.5 ml of
concentrated sulfuric acid of sp. Gr. 1.84 and dilute to 500 ml in a volumetric flask with
water. Filter this solution through prepared asbestos.
b. Fehling’s B: Dissolve 173 g of Rochelle salt (potassium sodium tartrate -
KNaC4 H4 O6 .4H2 O) and 50 g of sodium hydroxide, AR grade, in water, dilute to 500 ml
in a volumetric flask and allow the solution to stand for two days. Filter this solution
through prepared asbestos.
 Standardization of Fehling's solution: Pour standard invert sugar solution (reagent 3) into
a 50 ml burette. Pipette 10 ml of Fehling’s solution into a 250 ml conical flask and run in
from the burette almost the whole of the standard invert sugar solution required to effect
reduction of all the copper, so that not more than 1 ml will be required later to complete
the titration. Heat the flask containing the mixture over a wire gauze. Gently boil the
contents of the flask for 2 min. At the end of this period, add, without interrupting the
boiling, 1ml of methylene blue indicator solution. While the contents of the flask continue
to boil, begin to add standard invert sugar solution (one or two drops at a time) from the
burette till blue color of the indicator just disappears. Titration should be completed
within 1 min so that contents of the flask boil altogether for 3 min without interruption.
Note the titer (that is, the total volume in ml of standard invert sugar solution used for the
reduction of all the copper in 10 ml of Fehling’s solution).
6. Zinc Acetate solution (2 N): Dissolve 21.9 g of zinc acetate dehydrate [Zn(C2 H3 O2 )2 .2H2O]
and 3 ml of glacial acetic acid in distilled water and make up the final volume to 100 ml with
water.
7. Potassium hexacyanoferrate (II) solution (1 N): Dissolve 10.6 g of potassium
hexacyanoferrate (II) trihydrate (K 4 Fe(CN)6 .3H2 O) in distilled water and make up the final
volume to 100 ml with water.
8. Concentrated Hydrochloric acid solution: sp. gr. 1.16.
9. Concentrated ammonia solution: sp. gr. 0.88.
10. Dilute ammonia solution: Dilute 10 ml of concentrated ammonia solution to 100 ml with
water.
11. Dilute acetic acid solution: Approximately equivalent to the dilute ammonia solution in
strength.
Procedure
A. Preparation of test portion and solutions for titration
1. Weigh accurately about 40 g of the well-mixed sample of condensed milk and transfer to a
100 ml beaker.
2. Add 50 ml of hot water (80 to 90°C) and mix well. Transfer the mixture to the 250 ml
volumetric flask, rinsing the beaker with successive quantities of water at 60°C, until the total
volume is between 120 to 150 ml.
3. Mix, and cool to room temperature. Add 5 ml of dilute ammonia solution. Mix again and then
allow to stand for 15 min.
4. Neutralize ammonia by adding an equivalent quantity of the dilute acetic acid solution, being
determined beforehand the exact number of milliliters by titration of ammonia solution with
bromothymol blue as indicator.
 5. Mix and add 12.5 ml of the zinc acetate solution followed by 12.5 ml of the potassium
hexacyanoferrate (II) solution. Mix again, and make up the final volume to 250 ml.
6. Stopper the flask and mix thoroughly by vigorous shaking. Allow to settle and filter. Mark
this as solution B1 .
7. Pipette 50 ml of solution B1 into a 100 ml volumetric flask, add 5 ml of concentrated HCl and
heat at 68°C for 5 min. Cool the solution and neutralize with NaOH solution. Make up the
final volume to 100 ml. Mark this as solution A 1 .
8. Dilute the solutions B1 and A1 so that the volume of solution required to react with 10 ml of
Fehling's solution is between 15 and 50 ml. Mark these diluted solutions as solution B2 and
solution A2 , respectively.
B. Titration
1. Incremental method of titration:
A. Pour the prepared solution (See Steps A-7 and A-8) into a 50 ml burette, pipette 10 ml
Fehling’s solution into a 250 ml conical flask and run in from the burette 15 ml of the
solution.
B. Without further dilution, heat the contents of the flask over a wire gauze and boil. (After
the liquid has been boiling for about 15 sec, it will be possible to judge if the copper is
almost all reduced by the bright red color imparted to the boiling liquid by the suspended
cuprous oxide).
C. When it is judged that nearly all the copper is reduced, add 1 ml of methylene blue
indicator solution, continue boiling the contents of the flask for 1 to 2 min from the
commencement of ebullition and then add the prepared solution in small quantities (1 ml
or less at a time), allowing the liquid to boil for about 10 secs between successive
additions, till blue color of the indicator just disappears.
D. In case there appears to be still much unreduced copper, after the mixture of Fehling’s
solution with 15 ml of the prepared solution has been boiling for a quarter of a min, add
the prepared solution from the burette in larger increments (more than 1 ml at a time,
according to judgment) and allow the mixture to boil for a quarter of min after each
addition.
E. Repeat the addition of the prepared solution at intervals of 15 sec until it is considered
unsafe to add a large increment of the prepared solution. At this stage, continue boiling
for an additional 1 to 2 min, add 1 ml of methylene blue indicator solution and complete
the titration by adding the prepared solution in small quantities (less than 1 ml at a time).
Note 1: It is advisable not to add the indicator until the neighborhood of the end point has
been reached because the indicator retains its full color until the end point is almost reached
and thus gives warning to the operator to go slowly.
 Note 2: When the operator has had a fair amount of experience with the method, a
sufficiently accurate result may often be obtained by a single estimation by the incremental
method of titration, but for the utmost degree of accuracy of which the method is capable a
second titration should be carried out by the standard method of titration.
2. Standard method of titration:
Pipette 10 ml of Fehling's solution into a 250 ml conical flask and run in from burette almost
the whole of the prepared solution B2 required to effect reduction of all the copper determined
as above (see Step B-1), so that, if possible, not more than 1 ml shall be required later to
complete the titration. Gently boil the contents of the flask for 2 min. At the end of this period,
add, without interrupting boiling, 1 ml of methylene blue indicator solution. While the
contents of the flask continue to boil, begin to add the prepared solution (one or two drops at
a time) from the burette till blue color of the indicator just disappears and the red or bright
orange color of precipitated Cu2 O appears. Titration should be completed within 1 min, so
that the contents of the flask boil altogether for 3 min without interruption.
Note 3: The indicator is so sensitive that it is possible to determine the end point within one
drop of the prepared solution in many cases. Complete decolorization of methylene blue is
usually indicated by the whole reaction liquid in which cuprous oxide is continuously churned
up becoming bright red or orange in color. In case of doubt, the flame may be removed from
the wire gauze for 1 or 2 sec and the flask held against a sheet of white paper. (A holder of
paper, suitably fixed round the neck of the flask, is very convenient for this purpose as it may
be left round the neck of the flask without the risk of over-balancing it). The top edge of the
liquid would appear bluish if the indicator is not completely decolorized. It is inadvisable to
interrupt the boiling as the indicator undergoes back oxidation rather rapidly when air is
allowed free access into the flask, but there is no danger of this as long as a continuous stream
of steam is issuing from the mouth of the flask.
Note 4: It should be observed that with both the incremental and the standard methods of
titration, the flask containing the reaction mixture is left on the wire gauze over the flame
throughout titration.
Note 5: In adding sugar solution to the reaction mixture the burette may be held in hand over
the flask. The burette may be fitted with a small outlet tube bent twice at right angles, so that
the body of the burette can be kept out of the steam while adding sugar solution. Burettes
with glass taps are unsuitable for this work as taps become heated by steam and are liable to
jam.
Repeat the titration as given in B-1 and B-2 using solution A2 .
Calculation
Sucrose, % by mass = 20W1 /W2 (2f2 / v2 -f1 / v1 )
Where
W1 = mass in mg of sucrose corresponding to 10 ml of Fehling's solution;
W2 = mass in g of the material taken for the determination;
f2 = dilution factor for solution A2 from A1 ;
v2 = volume in ml of solution A 1 corresponding to 10 ml of Fehling's solution;
f1 = dilution factor for solution B2 from B1 ;
v1 = volume in ml of solution B2 corresponding to 10 ml of Fehling's solution;
 Dried milk
Introduction
Milk is an extremely perishable item and shelf-life of raw milk is limited to 3 to 4 h depending
upon the temperature of storage. Although various heat treatments and packaging process are used
to extend its shelf-life for liquid consumption, but it has become obligatory to convert surplus milk
into milk powder and ghee. Conversion of milk or milk by products into dried form offers benefits
to both manufacturer and the users. The main objective of converting into milk powder is to enhance
its keeping quality without much loss in the nutritive value. Milk powder can be used either in the
preparation of reconstituted milk, a substitute for fluid milk or as an ingredient in the manufacture
of formulated foods. During the past few decades many technological methods for the drying of
food have been developed and some are particularly suitable for the production of milk powder.
The keeping quality of milk powder is directly correlated with the initial quality of raw milk,
processing variables, packaging and storage conditions. Under commercial conditions, raw milk of
varying chemical quality is available for the manufacture of dried milk. High acidic milk when
neutralized with alkali and converted into milk powder shows high lactate content which is one of
the important parameter to be analyzed before exporting the powders.
Compositional standards for dried milk in India
FSSAI specifications
Milk powder means the product prepared by spray drying of standardized milk obtained from
fresh cow milk or buffalo milk or a mixture thereof. It may contain calcium chloride, citric acid,
and sodium citrate, sodium salts of orthophosphoric acid and polyphosphoric acid (as linear
phosphate) not exceeding 0.3% by weight of the finished product and 0.01% of butylated
hydroxyanisole (BHA) by weight of the finished product. Such addition need not be declared on
the label. For improving dispersibility, it may contain lecithin to a maximum limit of 0.5% under
label declaration as per Rule 42 (ee). Milk powder shall contain no more than 4.0% moisture, not
less than 26.0% milk fat, not less than 96.0% total solids and not more than 7.3% total ash on dry
basis. The total acidity expressed as lactic acid shall not be more than 1.2%. The plate count shall
not exceed 40,000 per g. Coliform count and coagulase positive Staphylococcus aureus shall be
absent in 0.1 g of the powder. Salmonella and Shigella shall be absent in 25 g of powder. The
insolubility index shall not be more than 2.0 ml. The spray dried product shall be packed in nitrogen
or mixture of nitrogen and carbon dioxide in hermetically sealed containers.
Skimmed milk powder means the product obtained from skimmed milk of cow or buffalo milk
or a combination thereof by the removal of water. It may contain added calcium chloride, citric
acid, and sodium citrate, sodium salts of orthophosphoric acid and polyphosphoric acid (as linear
phosphate) not exceeding 0.3% by weight of the finished product. Such addition need not be
declared on the label. Skimmed milk powder shall not contain more than 1.5% milk fat and moisture
shall not exceed 5.0%. The total acidity expressed as lactic acid shall not exceed 1.5%. The plate
count shall not exceed 50,000 per g and coliform count shall be absent in 0.1 g of the powder. The
insolubility index (maximum) in case of roller dried is 15.0 ml and in spray dried is 1.5 ml. The
total solids shall not be less than 95.0% and total ash on dry basis shall not be more than 8.2%. The
process of drying shall be mentioned on the label.
Partly skimmed milk powder means the product obtained from partly skimmed cow or buffalo
milk or a combination thereof by the removal of water. It may contain added calcium chloride, citric
acid, and sodium citrate, sodium salts of orthophosphoric acid and polyphosphoric acid (as linear
phosphate) not exceeding 0.3% by weight of the finished product. Such addition need not be
declared on the label. Partly skimmed milk powder shall not contain more than 5.0% moisture and
fat content of the product shall be more than 1.5% and less than 26.0%. Butylated hydroxylanisole
(BHA) not exceeding 0.01% by weight of the finished product may be added. The exact fat content
shall be indicated on the label. The insolubility index (maximum) in case of roller dried is 15.0 ml
and in spray dried is 1.5 ml. The total solids shall not be less than 95.0% and total ash on dry basis
shall not be more than 8.2%. The total acidity expressed as lactic acid shall not be more than 1.5%.
The process of drying shall be mentioned on the label. The spray dried product120 shall be packed
in hermetically sealed containers.
BIS specifications
Table 1 Compositional specifications for different types of dried milks
S. No Characteristics Type of dried milk
Skim Milk Partly Skimmed milk
powder powder
IS: 13334 (Part II) - 1992
IS: 13334 (Part I) - 1992

Dairy Whitener##
IS: 14542 - 1998

IS: 12299 - 1998

IS: 14542- 1998
Standard Grade
IS: 1165-2002

Type II (sour)
Extra Grade#
Milk Powder

Type I

1 Moisture, % by mass, 4.0 4.0 3.5 4.0 4.0 4.0

Max
2. Total solids (milk 96.0 96.0 96.5 96.0 96.0 -
solids and added
salts), % by mass, Min
3 Milk solids (non-fat), - - - - 57.0*
% by mass, Min
4 Milk fat, % by mass 26.0 1.5 1.25 More than More than 20.0
(Max) (Max) (Max) 1.5 but less 1.5 but less (Min)
than 26.0 than 26.0
5 Insolubility index, ml, 2.0 Roller 0.5 Roller dried Roller dried 1.5
Max dried = = 15 =-
15 Spray dried Spray dried
Spray = 1.5 = 25
dried =
1.5
 6 Total ash (on dry 7.3 8.2 7.3 8.2 8.2 5.5
basis), % by mass,
Max
7 Acid insoluble ash, % - - - - - 0.1
by mass, Max
8 Titratable acidity (as 1.2 1.5 1.5 1.5 1.5 -
lactic acid), % by
mass, Max
9 Lactate content, mg/g, - - 1.5 - - -
Max
10 Scorched particles, - - 15.0 - - -
mg, Max Disc B
11 Total added sugar (as - - # - - 18.0
sucrose), % by mass,
Max
12 BHA, % by mass of 0.01 - # 0.01 0.01 0.01
the finished product,
Max
13 Lecithin, % by mass of 0.5 - # - - 0.5
the finished product,
Max
14 Added salts**, % by 0.3 0.3 # 0.3 0.3 along 0.3
mass of the finished with others
product, Max ***
15 Bacterial count per g, 40,000 50,000 40,000 50,000 50,000 40,000
Max
16 Coliform in 0.1 g Absent Absent Absent Absent Absent Absent
17 Staphylococcus Absent - Absent - - Absent
aureus (coagulase
positive) per 0.1g of
powder
18 Salmonella in 25 g Absent - Absent - - Absent
powder
19 Shigella in 25 g Absent - Absent - - -
powder
#The product shall be free from any extraneous matter and no additive is allowed.
##The product prepared by spray drying of cow milk, buffalo milk or a mixture thereof. The milk
may be modified by partial removal / substitution of milk solids (non-fat) with carbohydrates
such as sucrose, dextrose or dextrin and may contain permitted stabilizers and emulsifiers.
* Milk solids (non-fat) may be calculated by the formula: 100 - (moisture + fat + total added
sugar).
** Calcium chloride, citric acid, and sodium citrate, sodium salts of orthophosphoric acid and
polyphosphoric acid (as linear phosphate).
***Sodium bicarbonate as a neutralizer provided that the resultant product is labelled as “Unfit
for Direct Consumption”. Type II Partly skimmed milk powder (sour) primarily for use by
industry like bakery.
Analysis of dried milk
Preparation of the test sample
 Transfer the sample to a clean, dry container (provided with an air-tight lid) of a capacity about
twice the volume of the sample.
Close the container immediately and thoroughly mix the contents by repeatedly shaking and
inverting the container. During these operations, exposure of the sample to the atmosphere should
be avoided as far as possible, to minimize absorption of water.
Determination of moisture content in dried milk
The moisture content of milk powder is the loss in mass, expressed as a percentage by mass
when the product is heated in a hot air oven at 102 ± 2°C to constant mass. (IS: 11623:1986).
Apparatus
1. Flat-bottom moisture dishes with cover: Of stainless steel, nickel or aluminum having
approximately 50 mm diameter and 25 mm depth. The dishes shall have lids which fit well
and can readily be removed.
2. Hot air oven: Maintained at 102 ± 2°C.
3. Desiccator: Containing an efficient desiccant.
Procedure
1. Uncover a dish and place the dish and its lid in a hot air oven at 102 ± 2°C for 1 h. Place the
lid on the dish, transfer the covered dish from the hot air oven to the desiccator. Allow it to
cool to room temperature and weigh it.
2. Put approximately 1 g of the dried milk sample in the dish, cover the dish with the lid and
weigh the covered dish accurately and quickly.
3. Uncover the dish and put it with its lid in the hot air oven maintained at 102  2°C for 2 h.
4. Replace the lid, transfer the covered dish to the desiccator, allow it to cool to room
temperature (for approximately 30 - 45 min) and weigh it accurately and quickly.
5. Heat the uncovered dish and lid in the hot air oven at 102 ± 2°C for further 1 h, replace the
lid, allow the covered dish to cool to room temperature in the desiccator and weigh it. Repeat
the process of drying, cooling and weighing, until the successive weighing does not differ by
more than 0.5 mg. It is usually found that drying is complete after the first 2 h.
Calculation
100 (M1 - M2)
Moisture % by mass = M1 − M
Where
M = mass in g, of the empty dish;
M1 = initial mass in g, of the dish and lid with the material taken for analysis;
M2 = final mass in g, of the dish and lid with the material after drying.
The maximum deviation between duplicate determinations should not exceed 0.06% by mass
of moisture.
Determination of bulk density in dried milk and dried milk products
Bulk density of dried milks is economically, commercially and functionally an important
property. When shipping dried milk over a long distance, the producers are interested in high bulk
density to reduce the shipping volume and transportation cost. Also, high bulk density saves in
packaging material and storage capacity, on the other hand, a low bulk density, as influenced by
agglomeration, is also an important part of powder properties influencing the instant characteristics.
Milk powders prepared using
Table 2 Bulk density of skim milk powders produced by
nozzle atomization results in a various processes
higher bulk density than Drying process Bulk density (g cm-3 )
Spray-drying 0.50 – 0.60
centrifugal atomization. The Roller-drying 0.30 – 0.50
manufacturing procedure and Foam spray-drying 0.32
Spray-drying (commercial) 0.26
conditions greatly influence bulk Instant spray-drying (commercial) 0.59
density, principally by the effect on air content. Consequently, step to reduce the occluded air will
increase density. Minimizing the air content of the concentrate prior to drying would achieve this
aim. Increasing the total solids of the concentrate (a high concentration can be used with centrifugal
atomization), reducing the spray pressure or using a large orifice increases bulk density. Less
uniformity in particle size distribution results in closer packing and thus in a higher bulk density.
The bulk density of skim milk powders produced by various processes is presented in Table 8.2.
By adding certain non-dairying ingredients, e.g. sucrose or additives to milk before drying, the bulk
density of the dry products is affected.
Bulk density is the defined as the weight of a volume unit of powder and in practice it is
expressed in g/ml, g/100 ml or g/L. The reciprocal value of bulk density, is known as bulk volume,
is also occasionally used and is expressed in ml/100 g or ml/g. Very often this value is incorrectly
called ‘bulk density’. In the Systeme International (SI) units, the concepts of density as defined
below is expressed in kg/m3 . In commercial practice, however the densities of dried milk and dried
milk products are traditionally expressed in g/ml.
Poured Bulk Density is the quotient of the mass and volume of a powder after transferring it to
a specific cylinder. It is expressed in g/ml for dried milk and dried milk products. Loose Bulk
Density is the quotient of the mass and volume of a powder after 100 tapings under the condition
specified in the following procedure. Bulk Density is the quotient of the mass and volume of a
powder after 625 tapings under the conditions specified in the following procedure. In the following
method (IDF 134A: 1995) a test portion of dried milk is tapped in a measuring cylinder. After a
specified number of taps, the volume of the product is recorded, and bulk density is calculated.
Apparatus
1. Measuring cylinder: 250 ml capacity, graduated from 0 – 250 ml, of scale length 245 ± 4 mm,
of mass 190 ± 15g and capable of being fixed on the apparatus (Figure 1).
2. Bulk density apparatus: Having the following components (Figure 1).
 a. Screwing device: To fasten the measuring cylinder on the apparatus, of 450 ± 10 g mass.
b. Tapping device: That can lift up the screwing device and the measuring cylinder to a
height of 3 ± 0.1 mm, and capable of tapping at a frequency of 250 ± 15 per min.
c. Interval counting device: Capable of recording from 0 – 625 taps, fitted with an
automatic stop, capable of being regulated to stop after a previously defined number of
taps.
3. Spatula.
4. Beaker: 250 ml capacity.
5. Powder funnel with short tube in glass or antistatic material. Total height: 100 mm; length of
the tube: 30 mm; diameter of the tube: 20 mm; upper diameter of the funnel: 100 mm.
6. Brush.
Procedure
Before proceeding with the determination, ensure that the dried milk sample has been
maintained at laboratory temperature (20 – 25°C). Thoroughly mix the sample by repeatedly
rotating and inverting the container. The container should not be more than two-third full. In the
case of instant dried milk, the mixing shall be performed very gently to avoid reducing the particle
size of the sample.
1. Weigh into a 250 ml beaker, 100 ± 0.1 g of the milk powder. If 100 g of powder does not fit
into the measuring cylinder, reduce the mass to 50 ± 0.1 g.
2. Place the funnel on the measuring cylinder and transfer the powder to the cylinder using the
spatula. If necessary, use the brush for removing last powder into the measuring cylinder. In
order to make the reading easier, level off the surface with the spatula and record the volume
in ml (V0 ).
3. Fix the measuring cylinder in the bulk density apparatus and tap 100 times. Level off the
surface with the spatula and record the volume in ml (V 100 ).
4. Regulate the number of taps to 625 (the 100 taps of Step 3 included). After tapping, level off
the surface with the spatula and record the volume in ml (V 625 ).
Calculation
Calculate the result, expressed in g/ml, according to the appropriate formula given below:
1. Poured bulk density (ρ0 ) = M/V0
2. Loose bulk density (ρ100 ) = M/V100
3. Bulk density (ρ 625 ) = M/ V625
Where
M = mass in g, of the test portion
V0 , V100 and V625 = volumes in ml, after transfer, after 100 tapings and after 625 tapping’s
respectively. ρ = density in g/ml. Express the results to third decimal place.
Determination of titratable acidity in dried milk
Method for Skimmed milk powder and partly skimmed milk powder
The titratable acidity of dried milk is the number of ml of 0.1 N sodium hydroxide solution
required to neutralize, in the presence of phenolphthalein, a quantity of the reconstituted milk
corresponding to 10 g of solids-not-fat, until the appearance of a pink coloration (pH 8.40).
The principle of this method (IS 11766: 1986) involves preparation of reconstituted milk by addition
of water to a test portion of dried milk corresponding accurately to 5 g of solids-not-fat. Titration
with 0.1 N sodium hydroxide solution using phenolphthalein as indicator and cobalt (II) sulphate
as reference color solution. Multiplication of the number of ml used in the titration by the factor 2,
in order to obtain the number of ml in terms of 10 g of solids-not-fat. The amount of sodium
hydroxide solution required is a function of the amount of natural buffering substances present in
the product, and of developed or added acid or alkaline substances.
Apparatus
1. Burette: 25 ml capacity, graduated to 0.1 ml with an accuracy of ± 0.05 ml.
2. Porcelain basin or conical flask: 100 ml capacity.
3. Measuring cylinder: 50 ml capacity.
4. Pipette: 2 ml capacity.
Reagents
1. Water to be used shall be distilled or deionized water, free from carbon dioxide by boiling
for 10 min before use.
2. Standard Sodium Hydroxide solution: 0.1 ± 0.0002 N.
3. Reference color solution: Dissolve 3.0 g of cobalt (II) sulphate heptahydrate (CoSO 4 .7H2O)
in water and make up to 100 ml.
4. Phenolphthalein Indicator Solution: Dissolve 2 g of phenolphthalein in 75 ml of 95% (v/v)
ethanol and add 20 ml of water. Add NaOH solution (0.1 N) to this solution until one drop
gives a faint pink coloration. Dilute with water to 100 ml.
Procedure
1. Mix the dried milk sample thoroughly. Weigh (500/a) ± 0.01 g of the dried milk sample into
each of two conical flasks (or porcelain basin), ‘a’ being the solids-not-fat content of the
sample, expressed as a percentage to two decimal places.
Note: The solids-not-fat content of the sample may be calculated by subtracting the fat content
and the moisture content from 100.
2. Prepare reconstituted milk by adding 50 ml of water at about 20°C to each flask and agitate
vigorously. Allow to stand for about 20 min.
3. Add to one of the conical flask 2.0 ml of reference color solution to obtain a color standard
and mix by slight swirling. If a series of determinations on similar products is to be carried
out, this color standard may be used throughout. However, it should be discarded after 2 h.
 4. Add 2.0 ml of the phenolphthalein indicator solution to the second conical flask and mix by
slight swirling.
5. Titrate the contents of the second conical flask, while swirling, by adding standard sodium
hydroxide solution from the burette until a faint pink color similar to that of color standard
persists for about 5 sec. The titration should be completed within 45 sec.
6. Record the volume in ml, of standard NaOH solution used to the nearest 0.05 ml (say V ml).
Calculation
Titratable acidity = 2 X V
Where
V = volume in ml, of standard NaOH (0.1 N) used for titration
Express the results to one decimal place.
Method for Whole milk powder
Apparatus
1. White porcelain basin: 60 ml capacity, hemispherical.
2. Pipette: 1, 2 and 10 ml capacity.
3. Burette: 25 ml capacity, graduated to 0.1 ml.
4. Measuring cylinder: 25 ml capacity.
5. Stirring rods: Of glass, flattened at one end.
6. Electric hot plate.
Reagents
1. Standard Sodium Hydroxide solution (0.1N).
2. Phenolphthalein Indicator Solution: Dissolve 1 g of phenolphthalein in 100 ml of ethanol
(95%, v/v). Add NaOH solution (0.1 N) to this solution until one drop gives a faint pink
coloration. Dilute with distilled water to 200 ml.
3. Rosaline Acetate Solution (Stock solution): Dissolve 0.12 g of Rosaline acetate in
approximately 50 ml of ethanol (95%, v/v) containing 0.5 ml of glacial acetic acid. Make the
volume to 100 ml with ethanol (95%, v/v).
4. Rosaline Acetate Solution (Bench solution): Dilute 1 ml of the stock solution to 500 ml with
a mixture of ethanol (95%, v/v) and distilled water in equal proportions by volume.
Note: The stock solution and the bench solution should be stored in dark-brown bottles
securely with rubber bungs.
Procedure
1. Weigh accurately about 1.0 g of whole milk powder sample into each of the two porcelain
dishes. Add 10 ml of boiling water to each dish and stir with the flat end of a glass rod until
a perfectly smooth liquid is obtained. Cool to room temperature.
2. Use the contents of one dish as a blank by stirring in 2 ml of bench solution of Rosaline
acetate.
3. Add 1.0 ml of phenolphthalein indicator solution to the second dish and titrate the contents
against standard NaOH (0.1 N) solution adding drop by drop from the burette, stirring the
sample with glass rod until by comparison the color matches the pink tint of the solution in
the dish containing the Rosaline acetate solution. The time taken for complete titration shall
not exceed 20 seconds. Note down the volume of 0.1 N NaOH used in the titration. The
titration shall be preferably made in north light or under illumination from a day light lamp.
Calculation
Titratable acidity (as lactic acid) % by mass = 9 AN / M
Where
A = volume in ml, of standard NaOH (0.1N) required for titration;
N = normality of the standard NaOH solution (0.1N);
M = mass in g, of milk powder taken for the test.
Determination of total ash on dry basis in dried milk.
Apparatus
1. Platinum or silica crucible: About 70 mm diameter and 25 to 50 mm deep.
2. Muffle furnace: Capable of being controlled at 550 ± 20°C.
3. Desiccator: Containing an efficient desiccant.
4. Safety tongs having long arms.
5. Bunsen burner or electric hot plate.
Procedure
1. Weigh accurately about 3 g of the dried milk sample in the crucible, previously dried in a hot
air oven and weighed.
2. Heat the crucible gently on a burner or hot plate at first and then strongly in a muffle furnace
at 550  20°C till grey ash is obtained.
3. Cool the crucible in a desiccator and weigh it. Heat the crucible again at 550  20°C for 30
min. Cool the crucible in a desiccator and weigh.
4. Repeat this process of heating for 30 min, cooling and weighing until the difference between
two successive weighing is less than 1 mg.
5. Record the lowest mass.
Calculation
Total ash (on dry basis), % by mass = 100 (M2 - M) / (100 - M0 ) (M1 - M)
M2 = mass in g, of the crucible with ash;
M = mass in g, of the empty crucible;
M1 = mass in g, of the crucible with the material taken for the test; and
M0 = moisture, % by mass, calculated as per the method for dried milk.
Determination of fat in dried milk by Rose-Gottlieb method
The following procedure is based on the Rose-Gottlieb principle and is described here using
Mojonnier type extraction flask. The method can be carried out using fat extraction tube with siphon
or wash bottle fittings. In this method (IS 11721:1986), ethanol and ammonia are added to the
reconstituted dried milk sample. Addition of concentrated ammonia dissolves the fat globule
membranes and subsequent addition of ethanol facilitates the passage of the fat globules from the
aqueous phase to the solvents. The fat is then extracted into a mixture of diethyl ether and petroleum
ether. The amount of fat extracted is determined gravimetrically after removal of the solvents by
distillation or evaporation.
Apparatus
1. Centrifuge: Centrifuge in which the fat-extraction flasks or tubes can be spun at a rotational
frequency of 500 to 600 min-1 to produce a gravitational field of 80 to 90 g n at the outer end
of the flasks or tubes. (The use of the centrifuge is optional but recommended).
2. Distillation or evaporation apparatus: To enable the solvents and ethanol to be distilled from
the fat-collecting flasks or to be evaporated from beakers and dishes at a temperature not
exceeding 100°C.
3. Hot air oven: Maintained at 102 ± 2°C.
4. Water-bath: Maintained at 40 to 60°C.
5. Boiling water-bath or hot plate.
6. Mojonnier-type fat-extraction flasks: It is also possible to use fat-extraction tube (or flasks)
with siphon or wash-bottle fittings. The long inner limb of the fitting may have a hooked end
if desired. The flasks (or tubes) shall be provided with good quality bark corks or stoppers of
other material e.g. silicon rubber or polytetrafluoroethylene (PTFE) unaffected by the
reagents used. Bark corks shall be extracted with the diethyl ether, kept in water at 60°C or
more for at least 15 min, and shall then be allowed to cool in the water so that they are
saturated when used.
7. Rack: To hold the fat-extraction flasks or tubes.
8. Fat-collecting vessels: e.g. flat bottom flask, 125 to 250 ml capacity.
9. Measuring cylinder: 25 and 50 ml capacities.
10. Pipettes: Graduated, 5, 10 ml capacities.
11. Boiling chips or glass beads: Fat free.
12. Beakers: 100 and 250 ml capacities.
13. Tongs: Made of metal suitable for holding the fat collecting vessels.
Reagents
1. Ammonia solution: Approximately 25%, m/m (ρ20 910 g/L). If ammonia solution of this
concentration is not available, a more concentrated solution of known concentration may be
used.
 2. Ethanol: 95 to 96% (v/v).
3. Diethyl ether: Free from peroxides, containing no, or not more than 2 mg of antioxidants per
kg. For the preparation of peroxide free ether See Appendix 1.
4. Petroleum ether: Boiling range 40 to 60°C.
5. Mixed solvent: Freshly prepared by mixing equal volumes of the diethyl ether and light
petroleum ether.
6. Congo-red solution: Dissolve 1 g of Congo-red in water and dilute to 100 ml with water.
Note: The use of this solution, which allows the interface between the solvent and aqueous
layers to be seen more clearly, is optional. Other aqueous color solutions may be used
provided that they do not affect the result of the determination.
Procedure (Using the Mojonnier Fat-Extraction Tube)
1. Weigh to the nearest 1 mg, directly or by difference, into a fat extraction tube one of the
following test portions:
a. About 1 g of dried high- fat milk, of dried whole milk or of dried butter serum.
b. About 1.5 g of dried partly skimmed milk, of dried skimmed milk, of dried whey or of
dried butter milk.
2. The test portions shall be delivered as completely as possible into the lower (small) bulb of
the extraction tube. Add 10 ml of water at 65 ± 5°C so as to wash the test portion into the
small bulb of the tube and mix thoroughly until the product is completely dispersed. Cool
the Mojonnier tube in running water.
3. Add 2 ml of ammonia solution to the tube. Mix thoroughly with the dispersed test portion in
the small bulb of the tube. After the addition of ammonia, continue the determination without
delay.
4. Heat the flask at 65 ± 5°C in a water-bath for 15 to 20 min with occasional shaking and then
cool to laboratory temperature. Add 10 ml of ethanol and mix gently but thoroughly by
allowing the contents of the flask to flow backward and forward between the two bulbs; avoid
bringing the liquid too near to the neck of the flask. If desired, add two drops of Congo-red
solution.
5. Add 25 ml of the diethyl ether, close the flask with a cork saturated with water or with a
stopper wetted with water, and shake the flask vigorously, but not excessively (in order to
avoid the formation of persistent emulsions), for 1 min with the flask in a horizontal position
and the small bulb extending upwards, periodically allowing the liquid in the large bulb to
run into the small bulb. If necessary, cool the flask in running water, then carefully remove
the cork or stopper and rinse it and the neck of the flask with a little of mixed solvents so that
the rinsing run into the flask or the prepared fat-collecting vessel.
6. Add 25 ml of the petroleum ether. Close the flask with the rewetted cork or rewetted stopper
(by dipping in water) and shake the flask gently for 30 secs as described above.
7. Centrifuge the closed flask for 1 to 5 min at a rotational frequency of 500 to 600 min-1 . If a
centrifuge is not available, allow the closed flask to stand in the rack for at least 30 min until
the supernatant layer is clear and distinctly separated from the aqueous layer. If necessary,
cool the flask under running water.
8. Carefully remove the cork or stopper and rinse it and the inside of the neck of the flask with
a little of the mixed solvent so that the rinsing run into the flask or into the fat-collecting
vessel. If the interface is below the bottom of the stem of the flask, raise it slightly this level
by gently adding water down the side of the flask to facilitate the decantation of solvent (See
Figure 1 & 2).
9. Holding the extraction flask by the small bulb, carefully decant as much as possible of the
supernatant layer into the prepared fat-collecting vessel (fat-collecting vessel is prepared by
drying a vessel with a few glass beads in a hot air oven for 1 h, allow the vessel to cool to
room temperature for at least 1 h) containing a few glass beads avoiding decantation of the
aqueous layer. Rinse the outside of the neck of the Mojonnier flask with a little of the mixed
solvent, collecting the rinsing in the fat-collecting vessel and taking care that the mixed
solvent does not spread over the outside of the extraction flask.
10. Add 5 ml of ethanol to the contents of the extraction flask, using the ethanol to rinse the inside
of the neck of the flask and mix as described in Step 4. Carry out a second extraction by
repeating the operations as described in Step 5 to Step 9 inclusive but using only 15 ml of the
diethyl ether and 15 ml of the petroleum ether; use the ether to rinse the inside of the neck of
the Mojonnier flask. If necessary, raise the interface to slightly above the middle of the stem
of the extraction flask to enable the final decantation of solvent to be as complete as possible.
11. Carry out a third extraction without addition of ethanol by again repeating the operations
described in Step 5 to Step 9 inclusive but using only 15 ml of the diethyl ether and 15 ml of
the petroleum ether; use the ether to rinse the inside of the neck of the extraction flaks. If
necessary, raise the interface to slightly above the middle of the stem of the extraction flask
to enable the final decantation of solvent to be as complete as possible.
12. Remove the solvents (including ethanol) as completely as possible from the fat-collecting
vessel by distillation or by evaporation, rinsing the inside of the neck of the flask with a little
of the mixed solvent before commencing the distillation.
13. Heat the fat-collecting vessel (flask placed on its side to allow solvent vapor to escape) for 1
h in the hot air oven, maintained at 102 ± 2°C. Remove the fat-collecting vessel from the
oven, allow to cool (not in a desiccator, but protected from the dust) to room temperature (for
at least 1 h) and weigh to the nearest 0.1 mg. Do not wipe the vessel immediately before
weighing. Place the vessel on the balance using tongs (to avoid, in particular, temperature
variations).
 14. Repeat the operations of heating and weighing until the mass of the fat-collecting vessel
decreases by 0.5 mg or less or increases, between two successive weighing. Record the
minimum mass as the mass of the fat-collecting vessel and extracted matter.
15. Add 25 ml of the petroleum ether to the fat-collecting vessel in order to verify whether or not
the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the fat is
dissolved.
16. If the extracted matter is wholly soluble in the petroleum ether, take the mass of fat as the
difference between the final mass of the vessel containing the extracted matter and its initial
mass.
17. If the extracted matter is not wholly soluble in the petroleum ether, or in case of doubt and
always for regulatory purposes or in case of dispute, extract the fat completely from the vessel
by repeatedly washing with warm petroleum ether. Allow any trace of insoluble material to
settle and carefully decant the petroleum ether without removing any insoluble material.
Repeat this operation three more times, using the petroleum ether to rinse the inside of the
neck of the vessel.
18. Finally, rinse the outside of the top of the vessel with mixed solvents so that the solvent does
not spread over the outside of the vessel. Remove petroleum ether vapors from the vessel by
heating the vessel for 1 h in hot air oven, maintained at 102 ± 2°C, allow to cool and weigh
as described in Step 13 and Step 14.
19. Take the mass of fat as the difference between the mass determined in step 14 and this final
mass.
20. Carry out a blank test simultaneously with the determination, using the same procedure and
reagents but replacing the test portion of the sample (dried milk) by 2 ml of water.
Calculation
The fat content expressed as a percentage by mass
(M1 - M2 ) - (M3 - M4 )
= ---------------------------------- X 100
M0
Where
M0 = mass in g, of the test portion;
M1 = mass in g, of the fat-collecting vessel along with the extracted matter in step 14;
M2 = mass in g, of the prepared fat-collecting vessel, or, in the case of undissolved material,
of the fat-collecting vessel and insoluble residue determined as above (Step 18 to 19);
M3 = mass in g, of the fat-collecting vessel used in the blank test and any extracted matter
determined; and
M4 = mass in g, of the prepared fat-collecting vessel used in blank test, or, in the case of
undissolved material, of the fat-collecting vessel and insoluble residue determined as above
(Step 18 to 19).
Calculate result to the nearest 0.01 % (w/w).
Note: If the value of M3 -M4 is 0.5 mg or less, this can be neglected in the calculation provided
that the diethyl ether does not contain antioxidants.
 Ice-Cream
Introduction
Ice-cream is a frozen dairy food made by freezing a pasteurized and homogenized mix with
agitation to incorporate air and to ensure uniformity of consistency. The mix is composed of a
combination of milk products, sugar, dextrose, corn syrup in dry or liquid form, water and may
include eggs or egg products, harmless flavorings, and added stabilizers or emulsifiers. Ice-cream
or related products are generally classified as frozen desserts, which include ice-cream, frozen
custard, ice milk, Table:1 Approximate composition of commercial ice-cream
sherbets, water ice, Type of Milk fat MSNF Sugar Stabilizers & Total solids
ice-cream (%) (%) (%) emulsifiers (%)
frozen confections, Economy 10 10 to 11 13 to 15 0.30 to 0.50 35 to 37
and mellorine and ice-cream 12 9 to 10 13 to 15 0.25 to 0.50 35 to 37
Good 12 11 15 0.30 37 to 39
parevine type
average 14 8 to 9 13 to 16 0.20 to 0.40 37 to 39
products. Ice- ice-cream
Deluxe 16 7 to 8 13 to 16 0.20 to 0.40 40 to 41
cream must contain
ice-cream 18 6 to 7 13 to 16 0.25 40 to 41
not less than 10% 19 5 to 6 14 to 17 0.25 40 to 41
milk fat and 20% total milk solids except in the case of bulky flavors, the fat and total milk solids
must not be less than 8 and 16% respectively. It must contain no more than 0.5% stabilizers. The
approximate composition of different types of ice cream is given in the Table:1.
The physical structure of ice-cream is a complicated physicochemical system. Air cells are
dispersed in a continuous liquid phase with embedded ice crystals. The liquid phase also contains
solidified fat globules, milk proteins, insoluble salts, lactose crystals in some cases, stabilizers of
colloidal dimension, and sugars and soluble salts in solution. The finished product consists of liquid,
air, and solid, and constitutes a three-phase system.
Ice-cream can be classified on the basis of different types of ingredients which are as follows.
Plain ice-cream: An ice-cream in which the total amount of color and flavoring ingredients is
less than 5.0% of the volume of unfrozen ice-cream e.g. vanilla, coffee and caramel ice-cream.
Chocolate ice-cream: Ice-cream flavoured with cocoa or chocolate.
Fruit ice-cream: Ice-cream containing fruits with or with without additional fruit flavoring or
color. Fruit such as strawberry, apricot, or pineapple may be fresh, frozen, canned or preserved.
Nut ice-cream: Ice-cream containing nut meats, such as almonds, pistachio or walnuts, with or
without additional flavoring or color.
French custard ice-cream: One or more of the optional egg ingredient permitted are used in
such quantity that the total weight of egg yolk solids content is not less 1.4% of the weight of the
finished frozen custard or less than 1.12% for bulky flavoured products.
Ice-milk: A product containing 2-7% fat and 12-15% MSNF, sweetened, flavoured, and frozen
like ice-cream.
 Ice: Made of fruit juice, sugar and stabilizer, with or without additional fruit acid, color,
flavoring or water, and frozen to the consistency of ice-cream. Usually contains 28-30% sugar, 20-
25% over run, and no dairy products.
Fruit sherbet: Made of fruit juices, sugar, stabilizer and milk products. It is similar to ice except
that milk-whole, skim, condensed or powdered-or ice-cream mix is used in place of all or part of
the water in an ice.
Mellorine type product: Similar to ice-cream in which butterfat has been replaced by suitable
vegetable fat. Minimum fat standard ranges from 6-10%.
Soft ice-cream (softy): This product is sold in soft condition and is ready for consumption
shortly after it is drawn from the freezer without hardening.
Novelties: A novelty ice-cream or frozen confection is an especially shaped ice-cream whose
main appeal consists in its shape, size, color or convenience for eating.
Compositional standards for ice -cream in India
FSS specifications
Ice-cream, kulfi and chocolate ice-cream: Ice-cream, kulfi and chocolate ice-cream means the
frozen product obtained from cow or buffalo milk or combination thereof or from cream and/or
other milk products, with or without the addition of cane sugar, (dextrose, liquid glucose and dried
liquid glucose, maltodextrin) eggs, fruits, fruit juices, preserved fruits, nuts, chocolate, edible
flavors and permitted food colors. It may contain permitted stabilizers and emulsifiers not exceeding
0.5 % by weight. The mixture shall be suitably heated before freezing. The product shall contain
not less than 10.0% milk fat, 3.5% protein and 36.0% total solids. Starch may be added to a
maximum extent of 5.0% under a declaration on a label. The above standard for ice-cream shall
also apply to softy ice-cream. In case of ice-cream, where the chocolate or like covering portion
forms a separate layer, only the ice-cream portion shall conform to the standards of ice-cream.
Dried ice-cream mix: Dried ice-cream mix shall be the material prepared by spray or roller
drying of ice-cream mix. It shall contain milk solids, sucrose or corn syrup or refined sugar. It may
contain permitted colors and flavors. It may contain stabilizers and emulsifiers not exceeding 1.25%
by weight. The product shall contain not less than 27.0% milk fat and 9.5% protein and moisture
shall not be more than 4.0% by weight. The sucrose content shall not be more than 40.0%, by
weight. The process of drying shall be mentioned on the label. It shall be packed in hermetically
sealed clean sound container.
Milk ices or milk lollies: Milk ices or milk lollies means the frozen product obtained from
milk, skimmed milk or milk product with or without the addition of cane sugar, (dextrose, liquid
glucose and dried liquid glucose) eggs, fruits, fruit juices, nuts, chocolate, edible flavors and
permitted food colors. It may contain permitted stabilizers not exceeding 0.5 % of the product. The
mixture shall be suitably heat-treated before freezing. The product shall contain no more than 2.0%
milk fat, not less 3.5% proteins and not less than 20.0% total solids.
BIS specifications
Ice-cream, fruit ice-cream, nut ice-cream, chocolate ice-cream means the frozen food made
from heat treated mix made out of milk, cream and /or other milk products (derived from cow and
buffalo milk) and with or without sweetening ingredients, eggs, water, fruit, nuts, chocolate,
permitted stabilizers/emulsifiers, edible common salt, permissible flavoring and coloring matter.
The ice-cream shall comply with the requirements given in the Table. No fat other than milk
fat shall be present in the product with the exception of that derived from eggs, cocoa, and
emulsifiers (monoglycerides). Milk, cream, butter, evaporated milk, sweetened condensed milk,
dried milk, skim milk, evaporated skim milk, dried skim milk may be used in the ice-cream. Milk
and milk products shall be of good hygienic quality. Milk and cream should be fresh and sweet,
free from any off flavor, or other defects. Condensed evaporated and dried milk shall be free from
rancidity, gassiness and off flavors. Butter shall be fresh and free from rancidity and off flavor.
Properly prepared and matured fruit free from piths, seeds, skin and core, where necessary may be
used. The fruit may be fresh, frozen or canned. Currants, figs, dates, prunes or raisins in the dried
form may also be used. Canned fruit shall comply with the requirement of the Fruit Products Order
(FPO), 2011.
Proper laboratory BIS requirements for ice cream (IS: 2802-1964)
control is important Characteristic Plain Ice-cream Fruits, Nut and
Chocolate ice-cream
for efficient operation
Weight in grams, per liter, 525 540
and for maintaining Min
uniform quality of ice- Total solids, % by weight, 36.0 36.0
Min
cream. It consists of Milk fat, %, min 10.0 8.0
both chemical and Acidity % (as lactic acid), 0.25 -
Max
bacteriological Sucrose, % by weight, Max 15.0 15.0
analyses of the Total colony counts, per g, 250,000 250,000
not more than
ingredients used, of Coliform count/g, not more 100 100
the finished product, than
Phosphatase test of mix Negative Negative
and of other samples Emulsifiers and stabilizers 0.5 0.5
taken in an attempt to %, Max
Other Additives May contain edible flavors and permitted
remedy undesirable colors, edible common salts.
conditions.
CHEMICAL ANALYSIS OF ICE-CREAM
Preparation of -cream sample for chemical analysis
For samples taken in small packages, remove the packaging and place the sample in a clean,
dry container fitted with an airtight closure. For samples taken from bulk or from large packages,
keep them in their sampling containers.
 Plain Product: Allow the sample to soften at room temperature. It is not advisable to soften the
ice-cream sample by heating on water bath or over flame because melted fat tends to separate and
rises to the surface. After softening of ice-cream, mix it thoroughly by stirring with spoon or
eggbeater or by pouring back and forth between beakers.
Fruit nut and chocolate ice-cream containing insoluble particles: Use a mixer capable of
comminuting product to fine, uniform pulp. Use 100 - 200 g of ice-cream sample to fill the cup of
mixer full to about one-third. Melt the product at room temperature or in an incubator at 37°C in a
closed container. Transfer entire contents to the mixer cup and mix until insoluble particles are
finely divided (about 3-5 min for fruit ice-cream and up to 7 min for nut ice-cream). Alternatively,
the product may be grounded in porcelain or glass pestle and mortar.
Transfer the mixed ice-cream sample to a suitable container for convenience in weighing. After
weighing operation, return the remainder of the ice-cream sample to the refrigerator, preferably at
a temperature not exceeding -15°C.
Note: For determination of over-run in ice-cream, the entire ice-cream carton/cup should be
taken as such.
Determination of total solids in ice -cream
Apparatus
1. Flat bottom moisture dish: Dishes of height at least 25 mm and at least 75 mm in diameter
and made of appropriate material (for example stainless steel, nickel or aluminum) not
affected by boiling water, with lids, provided with short glass stirring rods having a widening
flat end.
2. Sand: Which passes through IS sieve 500 µ and is retained on IS sieve 180 µ. It shall be
prepared by digestion with concentrated HCl, followed by thorough washing with water till
free from chlorides. It shall then be dried and ignited to dull red heat. (Acid washed sand is
also commercially available).
3. Hot air oven: Capable of operating at 102 ± 1°C.
4. Water-bath: capable of being controlled at 45 ± 1°C
5. Boiling water-bath.
Procedure
1. Heat the metal dish containing about 20 g of the prepared sand and with a stirring rod, in an
oven at 102 ± 1°C for about 1 h.
2. Allow the dish with lid on to cool in an efficient desiccator for 30 to 40 min. Weigh accurately
3.0 g of the prepared ice-cream sample into the dish saturate the sand by careful addition of
few drops of water, and thoroughly mix the wet sand with the ice-cream by stirring with the
glass rod, smoothing out lumps and spreading the mixture over the bottom of the dish.
3. Place the dish on a boiling water-bath for 20 - 30 min, carefully stirring the mixture from
time to time, then wipe the bottom of the dish and transfer it along with the glass rod to the
well-ventilated oven at 102  1°C. The lid shall be placed beside the dish.
4. After 4 h, remove the dish; cover it with the lid and place in an efficient desiccator. Allow to
cool and weigh. Replace the dish in the oven for a further period of 1 h at 102  1°C, remove
to desiccator, cool and weigh again. Repeat the process of heating, cooling and weighing after
every 1 h till consecutive weighing agree to within 0.5 mg.
Calculation
100 (M2 - M)
Total solids % by mass = M1 − M
Where
M = mass in g, of the empty dish with lid containing glass rod;
M1 = initial mass in g, of the dish, lid, glass rod along with the material before drying;
M2 = final mass in g, of the dish, lid, glass rod along with the material after drying.
Note: As per IDF procedure, the drying temperature for moisture determination in ice-cream
samples is 102 ± 2°C for 2 h.
DETERMINATION OF FAT (BY ROSE-GOTTLIEB METHOD)
The following procedure is based on the Rose-Gottlieb principal and is described here using
Mojonnier type extraction flask. The method can be carried out using fat extraction tube with siphon
or wash bottle fittings (Fig 2 and 3).
In this method, ethanol and ammonia are added to the ice-cream sample. Addition of
concentrated ammonia dissolves the fat globule membranes and subsequent addition of ethanol
facilitates the passage of the fat globules from the aqueous phase to the solvents. The fat is then
extracted into a mixture of diethyl ether and petroleum ether. The amount of fat extracted is
determined gravimetrically after removal of the solvents.
Reagents
1. Concentrated Ammonia solution: Approximately 35%, w/w (sp. gr. 0.88).
2. Ethanol: 95 to 96 % (v/v).
3. Diethyl ether: (containing not more than 4% water. sp. gr. 0.713 to 0.716): Free from
peroxides containing no, or not more than 2 mg/kg of antioxidants. For the preparation of
peroxide free ether see Appendix 1
4. Light petroleum ether: Boiling range 40 to 60°C.
5. Mixed solvent: Freshly prepared by mixing equal volumes of the diethyl ether and light
petroleum ether.
6. Congo-red solution: Dissolve 1 g of Congo-red in water and dilute to 100 ml.
Note: The use of this solution, which allows the interface between the solvent and aqueous
layers to be seen more clearly, is optional. Other aqueous color solutions may be used
provided that they do not affect the result of the determination.
Apparatus
1. Centrifuge: Centrifuge in which the fat-extraction flasks or tubes can be spun at a rotational
frequency of 500 to 600 min-1 to produce a gravitational field of 80 to 90 g n at the outer end
of the flasks or tubes. (The use of the centrifuge is optional but recommended).
2. Distillation or evaporation apparatus: To enable the solvents and ethanol to be distilled from
the fat-collecting flasks or to be evaporated from beakers and dishes at a temperature not
exceeding 100°C.
3. Hot air oven: Maintained at 102 ± 2°C.
4. Water-bath: Maintained at 40 to 60°C.
5. Boiling water-bath or hot plate.
6. Mojonnier type fat-extraction flasks: The flasks (tubes) shall be provided with good quality
bark corks or stoppers of other material e.g. silicon rubber or polytetrafluoroethylene (PTFE)
unaffected by the reagents used. Bark corks shall be extracted with the diethyl ether, kept in
water at 60°C or more for at least 15 min, and shall then be allowing to cool in the water so
that they are saturated when used. It is also possible to use fat-extraction flask or tubes with
 siphon or wash-bottle fittings. The long inner limb of the fitting may have a hooked end if
desired.
7. Rack: To hold the fat-extraction flasks or tubes.
8. Fat-collecting vessels: e.g. flat bottom flask, 125 to 250 ml capacity.
9. Measuring cylinder: 25 and 50 ml capacities.
10. Pipettes: Graduated, 10 ml capacity.
11. Boiling chips or glass beads: Fat free.
12. Beakers: 100 and 250 ml capacities.
13. Tongs: Made of metal suitable for holding the fat collecting vessels.
Procedure (Using the Mojonnier Fat-Extraction Tube)
1. Weigh accurately 4 to 5 g (nearest to 1 mg) of the prepared sample of ice-cream in 50 ml
beaker. Transfer the sample to the fat extraction tube. Rinse the beaker with 5 to 6 ml water
and transfer the washings to the fat extraction tube. Dilute the content of the fat extraction
tube with water to total volume of 10 to 11 ml.
2. Add 2 ml of concentrated ammonia solution and mix thoroughly but without splashing the
contents to the upper part of the tube.
3. Heat in water bath for 20 min at 60°C with occasional shaking and then cool to room
temperature. Add 10 ml of ethanol and mix well by allowing the liquid to flow backwards
and forwards between the two bulbs; avoid bringing the liquid too near to the neck of the
flask. If desired, add two drops of Congo-red solution.
4. Add 25 ml of the diethyl ether, close the flask with a cork saturated with water or with a
stopper wetted with water, and shake the flask vigorously but not excessively (to avoid the
formation of persistent emulsions) for 1 min with the flask in a horizontal position and the
small bulb extending upwards, periodically allowing the liquid in the large bulb to run into
the small bulb.
5. Carefully remove the cork and add 25 ml of the light petroleum ether. Close the flask with
the rewetted cork (by dipping in water) and shake the flask gently for 30 sec.
6. Centrifuge the closed flask for 1 to 5 min at a 500 to 600 rpm. If a centrifuge is not available,
allow the closed flask to stand in the rack for at least 30 min until the supernatant layer is
clear and distinctly separated from the aqueous layer. If necessary, cool the flask in running
water.
7. Carefully remove the cork and rinse it and the inside of the neck of the flask with a little of
the mixed solvent so that the rinsing run into the flask or into the fat-collecting vessel. If the
interface is below the bottom of the stem of the flask, raise it slightly this level by gently
adding water down the side of the flask to facilitate the decantation of solvent (Fig 1 & 2).
8. Carefully decant as much as possible of the supernatant layer into the fat-collecting vessel
containing a few glass beads avoiding decantation of the aqueous layer. Rinse the outside of
 the neck of the Mojonnier flask with a little of the mixed solvent, collecting the rinsing in the
fat-collecting vessel and taking care that the mixed solvents does not spread over the outside
of the extraction flask.
9. Carry out a second extraction by repeating the operations described as above but using only
15 ml of the diethyl ether and 15 ml of the light petroleum ether; use the ether to rinse the
inside of the neck of the Mojonnier flask. If necessary, raise the interface to the middle of the
stem of the extraction flask to enable the final decantation of solvent to be as complete as
possible.
10. Carry out a third extraction by again repeating the steps as described above using only 15 ml
of the diethyl ether and 15 ml of the light petroleum ether. Repeat the decantation step as
above.
11. Remove the solvents as completely as possible from the fat-collecting vessel by distillat ion
or by evaporation, rinsing the inside of the neck of the flask with a little of the mixed solvent
before commencing the distillation.
12. Heat the fat-collecting vessel (flask placed on its side to allow solvent vapor to escape) for 1
h in the hot air oven, maintained at 102 ± 2°C. Remove the fat-collecting vessel from the
oven, allow to cool (not in a desiccator, but protected from the dust) to room temperature (for
at least 1 h) and weigh to the nearest 0.1 mg. Do not wipe the vessel immediately before
weighing. Place the vessel on the balance using tongs (to avoid in particular, temperature
variations).
13. Repeat the operations of heating and weighing until the weight of the fat-collecting vessel
decreases by 0.5 mg or less or increases, between two successive weighing. Record the
minimum weight as the weight of the fat-collecting vessel and extracted matter.
14. Add 25 ml of the light petroleum ether to the fat-collecting vessel in order to verify whether
or not the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the
fat is dissolved.
15. If the extracted matter is wholly soluble in the light petroleum ether, take the weight of the
fat as the difference between the final weight of the vessel containing the extracted matter
and its initial weight.
16. If the extracted matter is not wholly soluble in the light petroleum ether, or in case of doubt,
extract the fat completely from the vessel by repeatedly washing with warm light petroleum
ether. Allow any trace of insoluble material to settle and carefully decant the light petroleum
ether without removing any insoluble material. Repeat this operation three more times, using
the light petroleum ether to rinse the inside of the neck of the vessel.
17. Finally, rinse the outside of the top of the vessel with mixed solvents so that the solvent does
not spread over the outside of the vessel. Remove light petroleum ether vapors from the vessel
 by heating the vessel for 1 h in hot air oven, maintained at 102 ± 2°C, allow to cool and weigh
as described above.
18. Take the weight of fat as the difference between the weight determined in 13 and this final
weight.
19. Carry out a blank test simultaneously with the determination, using the same procedure and
reagents, but replacing the dissolved ice-cream sample by 10 ml water.

Note: IDF procedure recommend addition of 5 ml of ethyl alcohol before second extraction
during the procedure.

Calculations
The fat content expressed as a percentage by mass

(M1 - M2 ) - (M3 - M4 )
= ---------------------------------- X 100
M0
Where
M0 = mass, in g of the ice-cream sample test portion.
M1 = mass, in g of the fat-collecting vessel along with the extracted matter in step 13.
M2 = mass, in g of the prepared and fat-collecting vessel or in the case of undissolved material,
of the fat-collecting vessel and insoluble residue determined as above.
M3 = mass, in g of the fat-collecting vessel used in the blank test and any
extracted matter determined.
M4 = mass, in g of the prepared fat-collecting vessel used in blank test or in the case of
undissolved material, of the fat-collecting vessel and insoluble residue determined as above.
Calculate result to the nearest 0.01 % (m/m).
 Cheese
Introduction
Cheese, a concentrated dairy food Table: 1. Some major types of cheese that can be
prepared from milk, is defined as the fresh recognized on the basis of moisture content and/or
method of maturation
or matured product obtained by draining the Description Popular Country
whey (the moisture or serum of the original examples
Extra-hard cheese Grana Italy
milk) after coagulation of casein, milk’s
(grating) Padano Italy
major protein. Casein is coagulated by acid Parmigiano
(Parmesan) France
produced by selected microorganisms
Passelan
and/or by coagulating enzymes resulting in Hard cheeses Cantal France
Cheddar England
curd formation. Milk may also be acidified
Leicester England
by added food grade acidulants in the Manchego Spain
Semi-hard cheeses Caerphilly Wales
manufacture of certain varieties of cheese
Port du Salut France
such as cottage cheese. Cheeses with Edam Holland
There are at least 1000 named cheese “eyes” Emmental Switzerland
Guryere Switzerland
varieties, most of which have very limited Maribo Denmark
production. The principal families are Cheeses internally Danablu Denmark
ripened with molds Gorgonzola Italy
Cheddar, Dutch, Swiss and Pasta Filata Roquefort France
(Mozzarella) which together accounts for Stilton England
Cheeses surface- Brie France
about 80% of total cheese production. ripened with molds Camembert France
Manufacture of cheese curd is essentially a Cheeses surface- Bel Paese Italy
ripened with Klosterkase Germany
concentration process in which the milk fat bacteria Limburger Belgium
and casein are concentrated about tenfold Tilsit Germany
Un-ripened soft Cottage USA
while the whey proteins, lactose and soluble cheeses cheese USA
salts are removed in the whey. The acid- Cream France
cheese
coagulated and acid/heat-coagulated Fromage
cheeses are normally consumed fresh, but frais
Italian-style Cotronese Italy
the vast majority of rennet coagulated cheeses (pasta Mozzarella Italy
cheeses are ripened (matured) for a period filata) Provolone Italy
White-brined Feta Greece
ranging from three weeks to more than two cheeses
years during which various biochemical, microbiological, physical and chemical changes occur,
resulting in characteristic flavor, aroma and texture.
Processed cheese
Processed cheese is manufactured by blending shredded natural cheeses of different types and
degrees of maturity with emulsifying agents, water, flavors and colors and by heating the blend
under a partial vacuum with constant agitation until a homogeneous mass is obtained (which is
usually packed while hot). Processed cheeses are characterized on the basis of composition, water
content and consistency; according to these criteria three main groups may be distinguished,
processed cheese block, processed cheese foods and processed cheese spread.
Proper selection of natural cheese is of utmost importance for the successful production of
processed cheese. In some countries, processed cheeses manufactured from only one variety of
cheese of different degrees of maturity are very popular. e.g. processed cheddar cheese in U.K. and
Australia; Cheddar, Gruyere and Mozzarella in the USA and Canada; Emmental in Western Europe.
The most important criteria for cheese selection are: type, flavor, maturity, consistency, texture and
pH. Natural cheese with microbial defects should not be selected for processing: spore forming,
gas producing, and pathogenic bacteria are particularly hazardous.

Table: 2 Proximate composition of selected varieties of cheeses

Cheese variety Moisture Fat Protein Salt Ash pH
(%) (%) (%) (%) (%)
Blue 42.0 29.0 21.0 4.5 6.0 6.5
Brick 40.0 30.0 22.5 1.9 4.4 6.4
Camembert 52.5 23.0 18.5 2.5 3.8 6.9
Cheddar 37.0 32.0 25.0 1.5 3.70 5.2-5.4
Cottage (uncreamed) 79.5 0.3 15.0 0.80 1.0 4.8-5.0
Cottage (Creamed) 79.2 4.3 13.2 0.80 1.0 4.8-5.0
Cream cheese 50.0 33.5 10.0 0.75 1.3 4.6
Edam 43.0 24.0 26.1 2.0 3.0 5.7
Feta 47.0 25.0 19.0 4.0 5.0 4.3
Gouda 41.0 28.5 26.5 2.0 3.0 5.8
Gruyere 33.5 30.0 30.0 1.1 4.1 5.7
Limberger 45.0 28.0 22.0 2.0 4.8 6.8
Mozzarella 50.30 24.80 20.31 1.72 2.8 5.2-5.6
Parmesan 31.0 25.0 36.0 2.6 5.4 5.4
Provolone 42.5 27.0 25.0 3.0 4.0 5.4
Quarg (low fat) 79.0 0.2 15.0 0.70 1.0 4.5
Ricotta 72.0 10.0 12.5 1.2 3.6 5.9
Roquefort 40.0 31.0 21.5 3.5 6.0 6.4
Stilton 49.53 24.0 18.87 2.93 2.10 5.52
Swiss/Emmental 35.5 30.5 27.5 1.2 3.5 5.6
Heat processed cheese 40.0 30.0 23.2 1.5-2.0 4.9 5.6-5.7
(Cheddar)
Heat processed cheese 40.0 26.9 26.4 1.5-2.0 5.1 5.6-5.7
(Swiss)

Compositional standards for Cheese in India

FSS specifications
Hard Cheese: Hard Cheese means the product obtained by draining after coagulation of milk
with a harmless milk coagulation agent under the influence of harmless bacterial cultures. It shall
not contain any ingredient not found in milk, except coagulating agent, sodium chloride, calcium
chloride (anhydrous salt) not exceeding 0.02% by weight, annatto or carotene color and may contain
emulsifiers and/or stabilizers, namely citric acid, sodium citrate or sodium salts of orthophosphoric
acid and polyphosphoric acid (as linear phosphate), not exceeding 0.2% by weight. Wax used for
covering the outer surface shall not contain anything harmful to health. In case the wax is colored
only permitted food color shall be used. Hard cheese shall contain no more than 43.0% moisture
and not less than 42% milk fat of the dry matter. Hard cheese may contain up to 3000 ppm sorbic
acid or its sodium, potassium or calcium salts calculated as sorbic acid, and/or 12.5 ppm nisin either
singly or in combination. Natamycin may be used for surface treatment only; subject to the
following conditions, namely (i) maximum level of application shall not exceed 2 mg/dm3 of cheese
surface, (ii) the penetration depth shall not exceed 2 mm and (iii) the maximum residue level in the
finished product shall not exceed 1 mg/dm3 .
Processed cheese: Processed cheese means the product obtained by heating one or more types
of hard cheese with permitted emulsifiers and/or stabilizers, namely citric acid, sodium citrate,
sodium salts of orthophosphoric acid and phosphoric acid (as linear phosphates) with or without
added condiments and acidifying agents, namely vinegar, lactic acid, acetic acid, citric acid and
phosphoric acid. Processed cheese may contain no more than 4.0% of anhydrous permitted
emulsifiers and/or stabilizers provided that the content of anhydrous inorganic agent shall in no
case exceed 3.0 % of the finished product. It shall not contain more than 47.0% moisture. Processed
cheese chiplets (packed sliced cheese) when sold in a package other than tin, shall not contain more
than 50% moisture. The milk fat content shall not be less than 40% of the dry matter. Processed
cheese may contain up to 3000 ppm sorbic acid or its sodium, potassium or calcium salts (calculated
as sorbic acid) and/or 12.5 ppm nisin either singly or in combination. It may contain calcium
chloride (anhydrous) not exceeding 0.02% by weight.
Processed cheese spread: Processed cheese spread means a product obtained by comminuting
and mixing one or more types of cheeses into a homogeneous mass with the aid of heat. It may or
may not contain butter, cream, butter oil, milk, milk powder, cheese whey, butter milk or one or
more of these or any of these from which part of water has been removed. It may also contain
permitted emulsifying and stabilizing agents. It may also contain one or more of the
sodium/potassium salts of citric acid, phosphoric acid, tartaric acid, lactic acid in such quantities
that mass of the solids of such emulsifying agents is not more than 4% of the mass of the processed
cheese spread. It may contain sequestering and buffering agents, namely, lactic acid, acetic acid,
citric acid and phosphoric acid. It may contain vegetable coloring matter such as annatto, carotene,
permitted flavoring agents and milk coagulating enzymes with or without purified calcium chloride
(anhydrous salt) not exceeding 0.02% and sodium citrate not exceeding 2.0% may be added. It may
contain natural sweetening agents, namely, sugar, dextrose, cane sugar, corn syrup, honey, corn
syrup solids, maltose, malt syrup and hydrolyzed lactose in a quantity necessary for seasoning and
spices and condiments. It may contain sodium chloride not exceeding 3.0% by weight. Processed
cheese spread may contain up to 3000 ppm sorbic acid or its sodium, potassium or calcium salts
(calculated as sorbic acid) and/or 12.5 ppm nisin. It shall not contain more than 60% moisture and
milk fat content (on dry basis) shall not be less than 40% by weight.
BIS specifications
Table 3 BIS specifications for hard cheese, processed cheese, processed cheese spread
and soft cheese
Characteristic Cheese type
Hard Processed Processed Soft cheese Soft cheese
spread milk skim milk
Moisture, % by mass 43.0 47.0 60.0 48.0-70.0 48.0-70.0
(Max) (Max) (Max)
Milk fat (on dry 42.0 40.0 (Min) 40.0 (Min) 50.0 (Min) 13.0 (Max)
basis), % by mass (Min)

Salt (added NaCl), % 3.0 3.0 (Max) 3.0 (Max) Optional Optional
by mass (Max)

Analysis of cheese and processed cheese products

Preparation of the test sample.
1. Prior to analysis, remove the rind or smear or moldy surface layer of the cheese, in such a
way as to provide a sample representative of the cheese as it is usually consumed.
2. Grind or grate the sample by means of an appropriate device; mix the ground or grated mass
quickly, and if possible grind or grate a second time, and again mix thoroughly. If the sample
cannot be ground or grated, mix it thoroughly by intensive stirring and kneading.
3. Transfer the test sample to an air-tight container to await analysis, which should be carried as
soon as possible after grinding. If delay is unavoidable, take all precautions to ensure proper
preservation of the sample, and to prevent condensation of moisture on the inside surface of
the container. The storage should be 10 to 12°C.
Determination of moisture content.
The moisture content of cheese is the loss in mass, expressed as a percentage by mass when the
product is heated in an air oven at 102 ± 2°C to constant mass (IS:2785:1979).
Apparatus
1. Flat-bottom dishes with lid: Dishes of nickel, aluminum or of other suitable metal not affected
by boiling water, 70 to 80 mm in diameter and not more than 25 mm deep, provided with
short glass stirring rod having a widened flat end. The dishes shall have lids which fit well
and can readily be removed.
2. Hot air oven: Maintained at 102 ± 1°C.
3. Desiccator: Containing an efficient desiccant.
4. Sand: Which passes through 500 μ sieve and is retained by 180 μ sieve. It shall be prepared
by digestion with concentrated HCl, followed by thorough washing with water. It shall then
be dried and ignited till it is dull red.
Procedure
1. Heat the flat-bottomed metal dish containing 20 g of prepared sand and a stirring rod, in hot
air oven for about 1 h. Allow to cool in an efficient desiccator for 30-40 min. Weigh
accurately 3 g of the prepared sample of cheese into a flat-bottomed dish (with a cover)
previously dried and weighed containing about 20 g of prepared sand and a stirring rod.
2. Saturate the sand by careful addition of a few drops of distilled water, and thoroughly mix
the wet sand with the cheese sample by stirring with the glass rod, smoothing out lumps and
spreading the mixture over the bottom of the dish.
3. Place the dish on a boiling water bath for 20 to 30 min, then wipe the bottom of the dish.
Transfer the dish containing the material, along with glass rod after uncovering in an oven
maintained at 102  1°C for about 4 h.
4. After 4 h replace the lid, transfer the covered dish to the desiccator, allow it to cool to room
temperature and weigh it accurately and quickly to the nearest 0.1 mg.
5. Heat the uncovered dish containing glass rod and lid in the oven at 102 ± 1°C for further 1 h,
replace the lid, allow the covered dish to cool to room temperature in the desiccator and weigh
it. Repeat the process of drying, cooling and weighing, until the successive weighing does
not differ by more than 0.5 mg. Record the weight.
Calculation
100 (M1 - M2)
Moisture % by mass = M1 − M
Where
M = mass in g, of the empty dish with lid containing glass rod;
M1 = initial mass in g, of the dish, lid, glass rod along with the material before drying;
M2 = final mass in g, of the dish, lid, glass rod along with the material after drying.
Express the results to the nearest 0.01% (m/m).
Note: The maximum deviation between duplicate determinations should not exceed 0.15%
of the ash.
Determination of fat content (Gerber method)
Apparatus
1. Cheese butyrometer: 0 - 40% scale having stoppered funnel having holes.
2. Gerber Centrifuge: Same as for milk fat testing.
3. Water bath: Same as for milk fat testing.
Reagents
1. Gerber sulfuric acid: Sulfuric acid shall have a density of 1.807 to 1.812 g/ml at 27°C
corresponding to a concentration of sulfuric acid from 90 to 91% by weight.
2. Iso-amyl Alcohol: The iso-amyl alcohol shall have density of 0.803-0.805 g/ml at 27°C.
Procedure
1. Weigh 3 ± 0.01 g of the prepared cheese sample into the stoppered funnel.
2. Transfer 10 ml of sulfuric acid into the butyrometer by means of automatic measure. Take
care not to wet the neck of the butyrometer with sulfuric acid.
3. Add gently from the wash-bottle sufficient warm water (30 to 40°C) to form a layer about 6
mm deep on top of the acid, allowing the water to flow down the side of the bulb.
4. Insert the neck of the funnel containing 3 g of cheese into the neck of the butyrometer.
Withdraw the stopper from the neck of the funnel and transfer all the cheese to the
butyrometer with the aid of the glass rod or spatula.
5. Transfer 1 ml of iso-amyl alcohol into the butyrometer with the help of automatic measure.
6. Add warm water (30 to 40°C) from the wash bottle until the butyrometer is filled to about 5
mm below the shoulder. Close the neck of the butyrometer firmly with the stopper. Shake the
butyrometer carefully without inverting it until all the contents are thoroughly mixed, the curd
is dissolved and no white particles are seen in the liquid. Then invert the butyrometer a few
times to mix the contents thoroughly.
7. Transfer the butyrometer quickly, with bulb upside, into a water-bath having a temperature
of 65 ± 2°C and keep it there for not less than 3 min and not more than 10 min.
8. Take out the butyrometer from the water-bath, wipe it with a cloth and transfer it to the
centrifuge. Centrifuge it for 5 min. Bring the centrifuge to stop gradually. Transfer the
butyrometer, stopper downwards, into a water bath having a temperature of 65±2°C and allow
the butyrometers to stand in the water bath at least 3 min and not more than 10 min
9. Note the reading of the scale of the fat column in the butyrometer after adjusting it with key
to the nearest 0.3 %, i.e., one-third of the smallest scale division. Repeat centrifugation, if no
sharp demarcation between the fat column and the acid is observed.
Note: For cheese containing more than 40% of fat, 1.5 g of cheese should be taken for the
test and the butyrometer reading multiplied by 2.
Gravimetric method
In the following procedure (BIS, 1979), a test portion is digested with hydrochloric acid
followed by addition of ethanol. The acid-ethanolic solution is subsequently extracted with diethyl
ether and light petroleum ether followed by removal of solvents by distillation or evaporation and
determination of mass of the substances extracted, which are soluble in light petroleum. This is
usually known as Schmid-Bondzynski-Ratziaff (SBR) principle.
Reagents
1. Hydrochloric acid solution: ρ20 approximately 1.125 g/ml. Dilute 675 ml of concentrated
hydrochloric acid , ρ20 1.18 g/ml, to 1000 ml with water.
2. Ethanol or ethanol denatured by methanol: at least 94% (w/v).
3. Congo red solution: Dissolve 1 g of Congo red in water and dilute to 100 ml
4. Diethyl ether: free from peroxides containing no, or not more than 2 g/kg of antioxidants.
5. Light Petroleum: having any boiling range between 30 and 60°C.
6. Mixed solvents: prepared shortly before use by mixing equal volumes of diethyl ether and
the light petroleum.
Apparatus:
1. Centrifuge: in which the fat-extraction flasks or tubes can be spun at a 500 to 600 rpm to
produce a gravitational field of 80 to 90 g, at the outer end of the flasks or tubes (the use of
the centrifuge is optional but recommended).
2. Distillation or evaporation apparatus: to enable the solvents and ethanol to be distilled from
the fat-collecting flasks or to be evaporated from beakers and dishes at a temperature not
exceeding 100°C.
3. Hot air oven: Maintained at 102 ± 2°C.
4. Boiling water bath or hot plate.
5. Mojonnier type fat-extraction tube: the flask shall be provided with good quality bark corks
or stoppers of other material for e.g. silicon rubber or polytetrafluoroethylene (PTFE)
unaffected by the reagents used.
6. Fat-collecting vessels: e.g. flat bottom flask of capacity 125 to 250 ml.
7. Measuring cylinder: 5 and 25 ml capacities.
8. Pipettes: Graduated, 10 ml capacity.
9. Glass beads.
Procedure:
1. Mix the test sample and weigh immediately to the nearest 1 mg, directly or by difference,
into a fat-extraction flask, or into 100 ml beaker or flask, 1.0 to 1.5 g of the test sample. Add
10 ml of HCl solution.
2. Boil gently, with shaking the content of the vessel either over a flame or in a boiling water-
bath, until all solid particles are entirely dissolved. Cool the tube in running water.
3. Add 10 ml of the ethanol and mix gently, but thoroughly by allowing the contents of the flask
to flow backward and forward between the two bulbs; avoid bringing the liquid too near to
the neck of the flask.
4. Add 25 ml of the diethyl ether, close the flask with a cork saturated with water or with a
stopper wetted with water, and shake the flask vigorously but not excessively (to avoid the
formation of persistent emulsions) for 1 min with the flask in a horizontal position and the
small bulb extending upwards, periodically allowing the liquid in the large bulb to run into
the small bulb. If necessary, cool the flask in running water, then carefully remove the cork
or stopper and rinse it and the neck of the flask with a little of mixed solvents so that the
rinsing run into the flask or the prepared fat-collecting vessel.
5. Add 25 ml of the light petroleum ether. Close the flask with the rewetted cork (by dipping in
water) and shake the flask gently for 30 sec.
6. Centrifuge the closed flask for 1 to 5 min at a speed of 500 to 600 rpm. If a centrifuge is not
available, allow the closed flask to stand in the rack for at least 30 min until the supernatant
layer is clear and distinctly separated from the aqueous layer. If necessary, cool the flask
under running water.
7. Carefully remove the cork and rinse it and the inside of the neck of the flask with a little of
the mixed solvent so that the rinsing run into the flask or into the fat-collecting vessel. If the
interface is below the bottom of the stem of the flask, raise it slightly this level by gently
adding water down the side of the flask to facilitate the decantation of solvent (Fig 1 & 2).
8. Carefully decant as much as possible of the supernatant layer into the fat-collecting vessel
containing a few glass beads avoiding decantation of the aqueous layer. Rinse the outside of
the neck of the Mojonnier flask with a little of the mixed solvent, collecting the rinsing in the
fat-collecting vessel and taking care that the mixed solvents does not spread over the outside
of the extraction flask.
9. Carry out a second extraction by repeating the operations described as above but using only
15 ml of the diethyl ether and 15 ml of the light petroleum ether; use the ether to rinse the
inside of the neck of the Mojonnier flask. If necessary, raise the interface to the middle of the
stem of the extraction flask to enable the final decantation of solvent to be as complete as
possible.
10. Carry out a third extraction by again repeating the steps as described above using only 15 ml
of the diethyl ether and 15 ml of the light petroleum ether. Repeat the decantation step as
above.
11. Remove the solvents as completely as possible from the fat-collecting vessel by distillat ion
or by evaporation, rinsing the inside of the neck of the flask with a little of the mixed solvent
before commencing the distillation.
 12. Heat the fat-collecting vessel (flask placed on its side to allow solvent vapor to escape) for 1
h in the hot air oven, maintained at 102 ± 2°C. Remove the fat-collecting vessel from the
oven, allow to cool (not in a desiccator, but protected from the dust) to room temperature (for
at least 1 h) and weigh to the nearest 0.1 mg. Do not wipe the vessel immediately before
weighing. Place the vessel on the balance using tongs (to avoid in particular, temperature
variations).
13. Repeat the operations of heating and weighing until the weight of the fat-collecting vessel
decreases by 0.5 mg or less or increases, between two successive weighing. Record the
minimum weight as the weight of the fat-collecting vessel and extracted matter.
14. Add 25 ml of the light petroleum ether to the fat-collecting vessel in order to verify whether
or not the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the
fat is dissolved.
15. If the extracted matter is soluble in the light petroleum ether, take the weight of the fat as the
difference between the final weight of the vessel containing the extracted matter and its initial
weight.
16. If the extracted matter is not soluble in the light petroleum ether, or in case of doubt, extract
the fat completely from the vessel by repeatedly washing with warm light petroleum ether.
Allow any trace of insoluble material to settle and carefully decant the light petroleum ether
without removing any insoluble material. Repeat this operation three more times, using the
light petroleum ether to rinse the inside of the neck of the vessel.
17. Finally, rinse the outside of the top of the vessel with mixed solvents so that the solvent does
not spread over the outside of the vessel. Remove light petroleum ether vapors from the vessel
by heating the vessel for 1 h in hot air oven, maintained at 102 ± 2°C, allow to cool and weigh
as described above.
18. Take the weight of fat as the difference between the weight determined in 14 and this final
weight.
19. Carry out a blank test simultaneously with the determination, using the same procedure and
reagents but replacing dissolved cheese sample by water.
Note: IDF procedure recommend addition of 5 ml of ethyl alcohol before second extraction
during the procedure.
Calculations
The fat content expressed as a percentage by mass

(M1 - M2 ) - (M3 - M4 )
= ---------------------------------- X 100
M0
Where
M0 = mass in g, of the cheese sample;
M1 = mass in g, of the fat-collecting vessel along with the extracted matter;
M2 = mass in g, of the empty fat-collecting vessel or in the case of undissolved material, of
the fat-collecting vessel and insoluble residue determined as above.
M3 = mass in g, of the fat-collecting vessel used in the blank test and any extracted matter
determined.
M4 = is the weight in g of the empty fat-collecting vessel used in blank test or in the case of
undissolved material, of the fat-collecting vessel and insoluble residue determined as above.
Calculate result to the nearest 0.01 % (w/w).
Note: If the value of W3 -W4 is 0.5 mg or less, this can be neglected in the calculation provided
that the diethyl ether does not contain antioxidants.
Determination of chloride content
All cheeses are salted, either by mixing dry salt with the drained curd (confined largely to
English varieties), rubbing dry salt on the surface of the pressed cheese (e.g. Romano or Blue
cheeses), or by immersion of the pressed cheeses in brine (most varieties). Salt concentration varies
from 0.7% (2% salt-in-moisture) in Emmental to 7-8% (15% salt-in-moisture) in Domiati. Salt
(sodium chloride) is added to cheese to improve flavor and to control ripening. NaCl influences
cheese ripening principally through its effect on water activity but it probably has some more
specific effects also which appear to be only partly due to water activity. Among the principal
effects of salt are:
- It is the principal factor affecting the water activity of young cheeses and has a major effect
on the growth and survival of bacteria and the activity of enzymes in cheese, and hence affects
and controls the biochemistry of cheese ripening.
- Salting promotes syneresis and hence reduces the moisture content of cheese.
- It has positive effect on flavor.
Cheese contributes to dietary sodium, high levels of which have undesirable nutritional
consequences, e.g. hypertension and osteoporosis. There is interest in many western countries in
the production of low-Na cheese and the most common approach at present is to replace some or
all of the NaCl by KCl. However, apart from cost, this practice affects the flavor of cheese since
the flavor of KCl is distinctly different from that of NaCl and a bitter flavor (not due to abnormal
proteolysis) is detectable in cheese containing > 1% KCl.
Chemical titration method
In this method (BIS, 1979), the cheese sample is digested using nitric acid in the presence of a
known amount of standard silver nitrate to give a clear solution. All chloride anions present are thus
precipitated as silver chloride. Ammonium iron (III) sulphate indicator is added followed by the
titration of excess silver nitrate with standard potassium thiocyanate solution to a faint reddish-
brown endpoint persisting for at least 15 sec (Fox, encyclopedia).
Reaction
Ag+ (excess) + Cl- AgCl (solid)
Ag+ + SCN- AgSCN (solid)
Fe+3 + SCN- [FeSCN]+2 (Reddish brown)
Reagents
1. Concentrated nitric acid: Sp. gr. 1.42 and chloride free.
2. Iron alum (ammonium ferric sulphate) solution: Cold saturated solution in 10% nitric acid.
3. Potassium chromate solution: 5% in water.
4. Standard silver nitrate solution: Prepare a 0.05 N solution by dissolving 8.5 g of solid silver
nitrate in 1 L of distilled water. Standardize the silver nitrate solution against a solution of
sodium chloride of known strength by the following manner:
 a. Preparation of 0.05 N sodium chloride: Place on watch glass about 5 g of sodium chloride
and dry in an oven at 250 to 350°C for 1-2 h. Cool in a desiccator. Weigh 2.9227 g of the
dried salt in a dish. Transfer the salt without loss to a small beaker. Dissolve the salt in
distilled water and transfer the solution to a 1 L volumetric flask, washing the beaker
repeatedly with distilled water and adding the washings to the content of the flask. Make
up the final volume with water.
b. Standardization of silver nitrate solution: Pour the silver nitrate solution into a burette
and pipette out 25 ml of the 0.05 N sodium chloride into the Erlenmeyer flask. Add 1 ml
of potassium chromate solution, place the flask on a white tile, add the silver nitrate
solution in a steady slow stream, rotating flask continuously. The red color of silver
chromate rapidly disappears at first, but as the concentration of sodium chloride is
progressively weakened, the red color will become more persistent until the addition of
one drop of silver nitrate causes the turbid yellow liquid to move towards a reddish color,
which persists after briskly shaking the flask. Repeat the titration until concordant results
are obtained. Now Calculate from this the normality of the silver nitrate and accordingly
adjust the strength of silver nitrate solution to exactly 0.05 N.
5. Standard Potassium Thiocyanate solution: Prepare 0.05 N solution by dissolving about 5.5 g
of potassium thiocyanate in 1 L water. Place the thiocyanate solution in a burette and, pipette
25 ml of the silver nitrate into an Erlenmeyer flask. Add about 5 ml of nitric acid solution
(one volume of pure acid diluted with 2 volumes of distilled water), followed by 1 ml of the
iron alum indicator. Add the thiocyanate solution with constant brisk rotation of the flask
until the reddish-brown color of ferric thiocyanate becomes more persistent. Proceed more
slowly, adding the thiocyanate drop by drop until one drop produces a faint brown color
which persists on shaking the flask. Repeat the titration until concordant results are obtained.
From this, calculate the normality of the thiocyanate solution and accordingly adjust the
strength of potassium thiocyanate solution to exactly 0.05 N.
Procedure
1. Weigh about 2 g of the prepared sample of cheese to an accuracy of 1 mg in a 300 ml
Erlenmeyer flask. Add 10 ml of distilled water and 25 ml of 0.05 N standard silver nitrate
solution.
2. Warm the contents of the flask at 75 to 80°C, to facilitate the dispersion of the cheese on
vigorous swirling.
3. Add 10 ml of concentrated nitric acid and boil the contents of the flask gently until the curd
is digested. This operation takes from 7 to 10 min. The finishing of the stage may be detected
when the silver chloride is granular, the liquid is of clear lemon-yellow color and the fat layer
is free from solid material.
 Note: The procedure can be modified by adding about 10 ml of saturated potassium
permanganate solution and 25 ml of concentrated nitric acid and then boiling the contents of
the flask gently until the curd is digested. At the end of the procedure as given above, add
about 0.25 g of urea to remove any nitrous acid, and 6 ml of acetone to improve the end point.
4. Add 2 ml of the iron alum solution and add about 50 ml of distilled water.
5. Determine the excess silver nitrate by titration with 0.05 N potassium thiocyanate solution
until the first appearance of an orange tint persists for 15 sec. Determine in the same manner
the equivalent of 25 ml of silver nitrate solution as thiocyanate, using the same volumes of
reagent and water.
Calculation
The salt content of the cheese is calculated by using the following formula:
0.292 (V1 -V2 )
Salt, % by mass = ------------------------
M
Where
V1 = volume in ml of standard potassium thiocyanate equivalent to 25 ml of silver nitrate;
V2 = volume in ml, of thiocyanate used in the titration of excess silver nitrate; and
M = mass in g, of cheese taken for the test
 Khoa
Khoa, an important Indian milk product, is prepared by continuous boiling of milk until desired
concentration (65 to 72% total solids) and texture is attained. During the manufacture of khoa from
milk, heat-coagulation of milk proteins, especially the whey proteins occurs, and characteristic
cooked flavor appears in the product. Vigorous stirring of the hot mass exerts an appreciable
homogenization action, so that when the pat is formed, all the fat globules are entrained in the mass.
The water is also dispersed in the form of fine droplets. Khoa form an important base material for
variety of popular
Type of khoa and their preferred end uses
sweets like burfi, Type of khoa Total solids (%) Fat (%) End uses
Dhap 56 to 63 20 to 23 Gulab jamuns, pantua.
peda, gulab jamuns,
Pindi 67 to 69 21 to 26 Burfi, peda.
kalakand etc. The Danedar 60 to 65 22 to 25 Kalakand, milk cake.
quality of khoa depends on the type of milk used and the method of manufacture. In general, khoa
made from buffalo milk is uniform whitish in color with a tinge of brown, a slightly oily or granular
texture and a rich nutty flavor which is associated with mildly cooked and sweet taste. Buffalo milk
with a higher total solids content also gives a higher yield of khoa than cow milk with a lower total
solids content. For commercial trade, three main types of khoa are recognized, i.e. dhap, pindi and
danedar, each type having preferred end uses as mentioned in the Table.
Composition
Chemical
Chemical composition of khoa
composition of khoa Type of milk Total solids Fat Protein Lactose Ash
depends on the initial (%) (%) (%) (%) (%)
Buffalo milk 78.40 30.50 17.70 23.90 5.90
composition of milk, Cow milk 80.70 25.20 15.80 33.50 4.10
degree of Mixed milk 79.80 29.00 16.70 30.10 5.20
Market sample 71.60 24.60 19.00 25.20 3.60
concentration of milk
solids and the losses or gains in handling. An average chemical composition of khoa is as follows.
The shelf life of khoa is 2 to 4 days under ambient conditions and three weeks under refrigerated
conditions.
Compositional standards for khoa in India:
FSSAI specifications
By whatever variety of names, it is sold such as Pindi, Danedar, Dhap, Mawa or kava means
the product obtained from cow or buffalo or goat or sheep milk or milk solids or a combination
thereof by rapid drying. The milk fat content shall not be less than 30.0% on dry weight basis of the
finished product. It may contain citric acid not more than 0.1% by weight. It shall be free from
added starch, added sugar and added coloring matter.
BIS specifications
As per BIS, Khoa as presently being marketed is designated as Pindi, Danedar and Dhap.
 For the preparation of khoa, only fresh, sweet, clean milk, free from colostrum and in every
way fit for human consumption shall be used. Milk shall be free from adulterants, preservatives and
any
matter foreign to
Chemical and microbiological requirements for khoa as per BIS
milk. The fat Characteristic Pindi Danedar Dhap
percentage of the milk Total solids, % by mass, Min 65.0 60.0 55.0
Milk fat, % by mass, (on dry basis), Min 37 37 37
shall be such that the Total ash, % by mass (on dry basis), Max 6.0 6.0 6.0
final product Titratable acidity (as lactic acid), % by 0.8 0.9 0.6
mass, Max
conforms to the Coliform count, per g, Max 90 90 90
requirements given in Yeast and mold count, per g, Max 50 50 50
the above table.

Sampling of khoa
In drawing, preparing and handling khoa samples, the following instructions shall be observed.
1. Samples shall be taken in a place protected against dust, soot, heat, dampness and
contamination.
2. The sample, the material being sampled, the sampling instrument and the containers for
samples shall be protected from adventitious contamination.
The sampling of khoa shall be carried out in accordance with the sampling of cheese.
Scale of sampling: In a single consignment all the containers of one type of material drawn
from a single batch of manufacture shall constitute a lot. Sample shall be tested from each lot
separately for ascertaining the conformity of the material to the requirements of the prescribed
standards.
Sampling of bulk units
Total number of units in the lot (N) Number of units to be selected (n)
1 1
2 to 8 2
9 to 25 3
26 to 50 4
51 to 100 5
Over 100 8

If the product is supplied in retain containers, the number of units to be sampled shall be
according to following Table
Sampling of retail units
Total number of units in the lot (N) Number of units to be selected (n)
Up to 25 3
26 to 100 5
101 to 500 8
501 to 1000 10
 1001 to 5000 13
Over 5000 20

These containers shall be selected at random from the lot. To ensure the randomness of
selection, a random number table as agreed to between the purchaser and the supplier shall be used.
In case such a table is not available, the following procedure shall be adopted.

Starting from any unit, count them as 1, 2, 3, …….., etc. up to r and so on in one order, where r is
equal to the integral part of N/n, N being the total number of units in the lot and n is the number of
units to be selected. Every rth unit thus counted shall be withdrawn to give required number of units
in the sample.
Chemical analysis of Khoa
Preparation of test sample of khoa
1. Grind or grate the laboratory sample of khoa by means of an appropriate device; mix the
ground or grated mass quickly, and if possible grind or grate a second time, and again mix
thoroughly. If the sample cannot be ground or grated, mix it thoroughly by intensive stirring
and kneading.
2. Transfer the test sample to an air-tight container to await analysis, which should be carried as
soon as possible after grinding. If delay is unavoidable, take all precautions to ensure proper
preservation of the sample and to prevent condensation of moisture on the inside surface of
the container. The storage temperature should be below 10°C.
Determination of total solids content in khoa
Apparatus
1. Flat-bottomed dishes: Made of aluminum, nickel or stainless steel, 50 to 75 mm diameter,
20 to 25 mm depth, and provided with well-fitting readily removable lids.
2. Boiling water-bath.
3. Water-bath: Maintained at 30 to 40°C.
4. Hot air oven: Maintained at 102  2°C.
5. Desiccator with efficient desiccant (for example freshly dried silica gel with a hygroscopic
indicator).
6. Short glass stirring rods: Flattened at one end and fitting in to the dish.
7. Quartz sand or sea sand: Which passes through 500 microns IS sieve and is retained on 180
microns IS sieve [IS: 460 (Part I) – 1978], and which passes the suitability test size as follows.
a. Place 20 g of sand in a dish containing a stirring rod. Heat the open dish and sand, stirring
rod and lid in a hot air oven controlled at 102 ± 1°C, for at least 2 h or preferably
overnight. Fit the lid, allow the dish to cool in a desiccator to room temperature and weigh
to the nearest 0.1 mg.
b. Moisten the sand with 5 ml of water, mix the sand and water using the stirring rod and
heat the dish and sand, stirring rod and lid in a hot air oven, controlled at 102 ± 1°C for
at least 4 h. Fit the lid, allow the dish to cool in the desiccator to room temperature and
weigh again to the nearest 0.1 mg. The difference between the two weighing shall not
exceed 0.5 mg.
c. If the requirement given above is not met, the sand may be made suitable for the
determination as follows.
d. Leave the sand immersed in 25% (m/m) hydrochloric acid solution for 3 days. Stir
occasionally, decant off the supernatant liquid as far as possible. Then wash the sand with
water until the acid reaction has disappeared. Calcine the sand at 550°C for at least 4 h
using a muffle furnace. Repeat the test for the suitability of the sand as described above.
Procedure
1. Heat a dish containing about 25 g of the sand with its lid alongside and a stirring rod on top
of the lid, in a hot air oven at 102° ± 1°C for about 1 h.
2. Place the lid (with the stirring rod on the top) on the dish, immediately transfer the dish to the
desiccator, allow to cool for at least 45 min, and weigh the dish with lid and rod to the nearest
0.1 mg. Tilt the sand to one side of the prepared dish, place on the clear space about 2.0 g of
the prepared test sample of khoa, replace the lid with the stirring rod on top and weigh the
dish to the nearest 0.1 mg.
3. Add 5 ml of distilled water to the test portion in the dish and mix with the stirring rod.
Thoroughly mix together the diluted test portion and the sand and spread the mixture evenly
 over the bottom of the dish. Leave the stirring end of the rod in the mixture with the other
end resting on the rim of the dish.
4. Heat the dish on a boiling water-bath, with as much as possible of the bottom of the dish
exposed to steam, for 30 min, stirring the mixture frequently in the early stages of drying so
that the mixture is well aerated and becomes crumbly. Lay the stirring rod flat inside the dish,
dry the bottom of the dish and heat the dish, with its lid alongside, in a hot air over maintained
at 102 ± 1°C for 4 h.
5. Place the lid on the dish, allow the dish to cool in the desiccator and weigh to the nearest 0.1
mg. Repeat the process of drying (heating the dish for 1 h), cooling and weighing, until the
difference in mass between two consecutive weighing does not exceed 0.5 mg. Record the
lowest mass.
Calculation
100 (M2 - M)
Total solids content, % by mass = M1 − M
Where
M = mass in g, of the dish, lid and stirring rod;
M1 = mass in g, of the dish, lid, stirring rod and test portion of khoa sample; and
M2 = mass in g, of the dish, lid, stirring rod and dried test portion.
Determination of fat content in khoa: Gravimetric method
In the following procedure (BIS, 1979), a test portion of khoa sample is digested with
hydrochloric acid followed by addition of ethanol. The acid-ethanolic solution is subsequently
extracted with diethyl ether and light petroleum ether followed by removal of solvents by distillat ion
or evaporation and determination of mass of the substances extracted, which are soluble in
petroleum ether. This is usually known as Schmid-Bondzynski-Ratziaff (SBR) principle.
Reagents
1. Hydrochloric acid solution: ρ20 approximately 1.125 g/ml. Dilute 675 ml of concentrated
hydrochloric acid, ρ20 1.18 g/ml, to 1000 ml with water.
2. Ethanol: 95 to 96% (v/v).
3. Diethyl ether: Free from peroxides, containing no, or not more than 2 mg of antioxidants per
kg. For the preparation of peroxide free ether See Appendix 1.
4. Petroleum ether: Boiling range 40 to 60°C.
5. Mixed solvent: Freshly prepared by mixing equal volumes of the diethyl ether and light
petroleum ether.
6. Congo-red solution: Dissolve 1 g of Congo-red in water and dilute to 100 ml with water.
Note: The use of this solution, which allows the interface between the solvent and aqueous
layers to be seen more clearly, is optional. Other aqueous color solutions may be used
provided that they do not affect the result of the determination.
Apparatus
1. Centrifuge: Centrifuge in which the fat-extraction flasks or tubes can be spun at a rotational
frequency of 500 to 600 min-1 to produce a gravitational field of 80 to 90 g n at the outer end
of the flasks or tubes. (The use of the centrifuge is optional but recommended).
2. Distillation or evaporation apparatus: To enable the solvents and ethanol to be distilled from
the fat-collecting flasks or to be evaporated from beakers and dishes at a temperature not
exceeding 100°C.
3. Hot air oven: Maintained at 102 ± 2°C.
4. Water-bath: Maintained at 40 to 60°C.
5. Boiling water-bath or hot plate.
6. Mojonnier-type fat-extraction flasks: It is also possible to use fat-extraction tube (or flasks)
with siphon or wash-bottle fittings. The long inner limb of the fitting may have a hooked end
if desired. The flasks (or tubes) shall be provided with good quality bark corks or stoppers of
other material e.g. silicon rubber or polytetrafluoroethylene (PTFE) unaffected by the
reagents used. Bark corks shall be extracted with the diethyl ether, kept in water at 60°C or
more for at least 15 min, and shall then be allowed to cool in the water so that they are
saturated when used.
7. Rack: To hold the fat-extraction flasks or tubes.
 8. Fat-collecting vessels: e.g. flat bottom flask, 125 to 250 ml capacity.
9. Measuring cylinder: 25 and 50 ml capacities, graduated.
10. Pipettes: Graduated, 5, 10 ml capacities.
11. Boiling chips or glass beads: Fat free.
12. Beakers: 100 and 250 ml capacities.
13. Tongs: Made of metal suitable for holding the fat collecting vessels.
Procedure
1. Mix the test sample of khoa and weigh immediately to the nearest 1 mg, directly or by
difference, into a fat-extraction flask, or into 100 ml beaker or flask, 1.0 to 1.5 g of the test
sample. Add 10 ml of hydrochloric acid solution.
2. Boil gently, with shaking the content of the vessel either over a flame or in a boiling water-
bath, until all solid particles are entirely dissolved. Cool the tube in running water.
3. Add 10 ml of the ethanol and mix gently, but thoroughly by allowing the contents of the flask
to flow backward and forward between the two bulbs; avoid bringing the liquid too near to
the neck of the flask.
4. Add 25 ml of the diethyl ether, close the flask with a cork saturated with water or with a
stopper wetted with water, and shake the flask vigorously but not excessively (in order to
avoid the formation of persistent emulsions) for 1 min with the flask in a horizontal position
and the small bulb extending upwards, periodically allowing the liquid in the large bulb to
run into the small bulb. If necessary, cool the flask in running water, then carefully remove
the cork or stopper and rinse it and the neck of the flask with a little of mixed solvents so that
the rinsing run into the flask or the prepared fat-collecting vessel.
5. Add 25 ml of the petroleum ether. Close the flask with the rewetted cork (by dipping in water)
and shake the flask gently for 30 sec.
6. Centrifuge the closed flask for 1 to 5 min at a speed of 500 to 600 rpm. If a centrifuge is not
available, allow the closed flask to stand in the rack for at least 30 min until the supernatant
layer is clear and distinctly separated from the aqueous layer. If necessary, cool the flask
under running water.
7. Carefully remove the cork or stopper and rinse it and the inside of the neck of the flask with
a little of the mixed solvent so that the rinsing run into the flask or into the fat-collecting
vessel. If the interface is below the bottom of the stem of the flask, raise it slightly this level
by gently adding water down the side of the flask to facilitate the decantation of solvent (Fig
1 & 2).
8. Holding the extraction flask by the small bulb, carefully decant as much as possible of the
supernatant layer into the fat-collecting vessel containing a few glass beads avoiding
decantation of the aqueous layer. Rinse the outside of the neck of the Mojonnier flask with a
 little of the mixed solvent, collecting the rinsing in the fat-collecting vessel and taking care
that the mixed solvents does not spread over the outside of the extraction flask.
9. Carry out a second extraction by repeating the operations as described in Step 4 to Step 8
inclusive but using only 15 ml of the diethyl ether and 15 ml of the light petroleum ether; use
the ether to rinse the inside of the neck of the Mojonnier flask. If necessary, raise the interface
to slightly above the middle of the stem of the extraction flask to enable the final decantation
of solvent to be as complete as possible.
10. Carry out a third extraction by again repeating the steps as described in Step 4 to Step 8
inclusive but using only 15 ml of the diethyl ether and 15 ml of the light petroleum ether; use
the ether to rinse the inside of the neck of the extraction flaks. If necessary, raise the interface
to slightly above the middle of the stem of the extraction flask to enable the final decantation
of solvent to be as complete as possible.
11. Remove the solvents (including ethanol) as completely as possible from the fat-collecting
vessel by distillation or by evaporation, rinsing the inside of the neck of the flask with a little
of the mixed solvent before commencing the distillation.
12. Heat the fat-collecting vessel (flask placed on its side to allow solvent vapor to escape) for 1
h in the hot air oven, maintained at 102 ± 2°C. Remove the fat-collecting vessel from the
oven, allow to cool (not in a desiccator, but protected from the dust) to room temperature (for
at least 1 h) and weigh to the nearest 0.1 mg. Do not wipe the vessel immediately before
weighing. Place the vessel on the balance using tongs (to avoid, in particular, temperature
variations).
13. Repeat the operations of heating and weighing until the mass of the fat-collecting vessel
decreases by 0.5 mg or less or increases, between two successive weighing. Record the
minimum mass as the mass of the fat-collecting vessel and extracted matter.
14. Add 25 ml of the petroleum ether to the fat-collecting vessel in order to verify whether or not
the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the fat is
dissolved.
15. If the extracted matter is soluble in the light petroleum ether, take the mass of fat as the
difference between the final mass of the vessel containing the extracted matter and its initial
mass.
16. If the extracted matter is not soluble in the light petroleum ether, or in case of doubt and
always for regulatory purposes or in case of dispute, extract the fat completely from the vessel
by repeatedly washing with warm petroleum ether. Allow any trace of insoluble material to
settle and carefully decant the petroleum ether without removing any insoluble material.
Repeat this operation three more times, using the petroleum ether to rinse the inside of the
neck of the vessel.
 17. Finally, rinse the outside of the top of the vessel with mixed solvents so that the solvent does
not spread over the outside of the vessel. Remove petroleum ether vapors from the vessel by
heating the vessel for 1 h in hot air oven, maintained at 102 ± 2°C, allow to cool and weigh
as described in Step 12 and Step 13.
18. Take the mass of fat as the difference between the mass determined in step 13 and this final
mass.
19. Carry out a blank test simultaneously with the determination, using the same procedure and
reagents but replacing the test portion of the khoa sample by 2 ml of water.
20. Carry out a blank test simultaneously with the determination, using the same procedure and
reagents but replacing dissolved khoa sample by water.
Calculation
The fat content expressed as a percentage by mass
(M1 - M2 ) - (M3 - M4 )
= ---------------------------------- X 100
M0
Where
M0 = mass in g, of the test portion;
M1 = mass in g, of the fat-collecting vessel along with the extracted matter in step 13;
M2 = mass in g, of the prepared fat-collecting vessel, or, in the case of undissolved material,
of the fat-collecting vessel and insoluble residue determined as above (Step 17 to 18);
M3 = mass in g, of the fat-collecting vessel used in the blank test and any extracted matter
determined.
M4 = mass in g, of the prepared fat-collecting vessel used in blank test, or, in the case of
undissolved material, of the fat-collecting vessel and insoluble residue determined as above
(Step 17 to 18).
Calculate result to the nearest 0.01 % (w/w).
Note: If the value of M3 -M4 is 0.5 mg or less, this can be neglected in the calculation provided
that the diethyl ether does not contain antioxidants.
 Paneer
Paneer is an indigenous milk product prepared by combined action of acid coagulation and heat
treatment of milk and subsequent drainage of whey. The phenomenon of coagulation involves the
formation of large structural aggregates of proteins in which milk fat and other colloidal and soluble
solids are entrained with the whey. The coagulum so obtained is poured over a muslin cloth to
separate the coagulum from the whey. The coagulum is then pressed to facilitate the formation of
paneer blocks of suitable sizes, followed by their immersion in chilled water to impart them a
distinctive texture. The cooling result in re-absorption of some water and this treatment presumably
imparts the characteristic springy and rubbery texture to paneer. Paneer is best made from buffalo
milk (standardized to 6% fat). Cow milk yields an inferior product in terms of body and texture.
The responsible factor for this remarkable difference in paneer quality is due to higher level of
casein minerals, particularly calcium and phosphorus, tends to produce hard and rubbery body while
cow milk tends to produce and soft and mellow characteristics. Buffalo milk paneer retains higher
fat, proteins and ash contents and less moisture and lactose when compared with the paneer from
cow milk. The optimum pH of coagulation is 5.3 for buffalo milk while for cow milk pH is 5.0.
Paneer is extensively used as an ingredient for cooking with vegetables in northern India.
Composition
The keeping quality Table: Chemical composition of paneer
of paneer made from
Constituent (%) Buffalo milk Cow milk Market samples
fresh, sweet milk is 5 to 6 (5.8% fat) (4% fat)
Moisture 50.72 52.40 51.91
days at 5 to 10°C, while
Fat 27.13 23.40 26.59
that made from acidic Protein 17.99 20.30 17.65
milk have a storage life Lactose 2.29 - 2.05
Ash 1.87 - 1.78
of 3 to 4 days at the same
temperature.
Compositional standards for paneer in India
BIS specifications
Paneer shall be clear and free from dirt, surface discoloration, insects and rodents contamination
and from adulterants. Table: Chemical and microbiological requirements for Paneer as per
It shall not have any BIS
S. No Characteristic Paneer
free moisture. Paneer 1. Moisture, % by mass, Max 60.0
shall have a pleasant 2. Milk fat, % by mass, (on dry basis), Min 50.0
odour and 3. Titratable acidity (as lactic acid), % by mass, Max 0.50
4. Bacterial count, per g, Max 500,000
characteristic milk 5. Coliform count, per g, Max 90
acidic flavor. It shall 6. Yeast and mold count, per g, Max 250
have a closely knit smooth texture, firm, cohesive and spongy body. Before coagulation, milk shall
be boiled or heated to a sufficiently high temperature for such time that it will result in the complete
destruction of pathogenic microorganisms.
The coagulants such as lactic acid, citric acid and their sodium and potassium salts shall be of
food grade and free from toxic substances. The sour whey shall be heat-treated to render it
microbiological safe.
In Paneer, either sorbic acid and its sodium and potassium salts or propionic acid and its sodium,
potassium and calcium salts or other permitted preservatives may be added up to the extent of 2000
mg/kg. No extraneous coloring matter shall be added to paneer.
FSS specifications
Paneer means the product obtained from the cow or buffalo milk or a combination thereof by
precipitation with sour milk, lactic acid or citric acid. It shall not contain more than 70.0% moisture
and the milk fat content shall not be less than 50.0% of the dry matter. Milk solids may also be used
in the preparation of this product.
In paneer, either sorbic acid and its sodium, potassium and calcium salts or propionic acid and
its sodium and potassium salts may be added up to the extent of 2000 mg/kg.
Sampling of paneer
In drawing, preparing and handling khoa samples, the following instructions shall be observed.
1. Samples shall be taken in a place protected against dust, soot, heat, dampness and
contamination.
2. The sample, the material being sampled, the sampling instrument and the containers for
samples shall be protected from adventitious contamination.
The sampling of paneer shall be carried out in accordance with the sampling of cheese. Scale
of sampling: In a single consignment all the containers of one type of material drawn from a single
batch of manufacture shall constitute a lot. Sample shall be tested from each lot separately for
ascertaining the conformity of the material to the requirements of the prescribed standards.
Sampling of bulk units
Total number of units in the lot (N) Number of units to be selected (n)
1 1
2 to 8 2
9 to 25 3
26 to 50 4
51 to 100 5
Over 100 8

If the product is supplied in retain containers, the number of units to be sampled shall be
according to following Table
Sampling of retail units
Total number of units in the lot (N) Number of units to be selected (n)
Up to 25 3
 26 to 100 5
101 to 500 8
501 to 1000 10
1001 to 5000 13
Over 5000 20
These containers shall be selected at random from the lot. To ensure the randomness of
selection, a random number table as agreed to between the purchaser and the supplier shall be used.
In case such a table is not available, the following procedure shall be adopted.
Starting from any unit, count them as 1, 2, 3, …….., etc. up to r and so on in one order, where
r is equal to the integral part of N/n, N being the total number of units in the lot and n is the number
of units to be selected. Every rth unit thus counted shall be withdrawn to give required number of
units in the sample.
Chemical analysis of Paneer
Preparation of the test sample of paneer
Grate the paneer sample quickly through a suitable grater. Mix the grated sample thoroughly.
Transfer the grated sample to an air-tight container to await analysis, which should be carried as
soon as possible after grinding. If delay is unavoidable, take all precautions to ensure proper
preservation of the sample, and to prevent condensation of moisture on the inside surface of the
container. The storage temperature should be below 10°C.
Determination of moisture content in paneer
The moisture content of paneer is the loss in mass, expressed as a percentage by mass when the
product is heated in an air oven at 102 ± 2°C to constant mass (IS 10484: 1983).
Apparatus
1. Flat-bottomed dishes: Made of aluminum, nickel or stainless steel, 70 to 80 mm diameter,
20 to 25 mm depth, and provided with well-fitting readily removable lids.
2. Hot air oven: Maintained at 102° + 1°C.
3. Desiccator with efficient desiccant (e.g. freshly dried silica gel with a hygrometric indicators).
4. Short glass stirring rods: Flattened at one end and fitting in to the dish.
Procedure
1. Heat a dish containing glass rod, with its lids alongside in the oven maintained at 102 ± 2°C
for at least 1 h. Place the lid on the dish and immediately transfer to the desiccator. Allow to
cool to room temperature (at least 30 min) and weigh it to the nearest 0.1 mg.
2. Transfer into the dish containing glass rod, about 2.0 g of the prepared sample of paneer and
weigh quickly, with the lid on the dish. Mix the paneer sample uniformly with 4 ml of hot
distilled water using flatted end of the glass rod. Wash off the paneer particles adhering to the
glass rod pouring additional 1 ml of hot distilled water and leave the glass rod in the dish.
3. Place the dish without lid on the vigorously boiling water-bath in such a way that the bottom
of the dish is maximally exposed to and directly heated by the steam. Leave the dish on the
boiling water-bath for 30 min.
4. Transfer the uncovered dish containing the dried material to a hot air oven maintained at a
temperature of 102 ± 1°C, placing the lid along its side, for 4 h.
5. After 4 h, cover the dish with lid and immediately transfer it to a desiccator. Allow to cool to
room temperature for about 30 min and weigh to the nearest 0.1 mg.
6. Repeat the process of heating in the hot air oven (for 1 h) followed by cooling and weighing
until the difference between two consecutive weighing is less than 1 mg. Record the lowest
mass.
Calculation
100 (M1 - M2)
Moisture % by mass = M1 − M
M = mass in g, of the empty dish containing glass rod, with lid;
M1 = mass in g, of the dish, lid, glass rod along with the material before drying; and
M2 = mass in g, of the dish, lid, glass rod along with the material after drying.
Express the results to the nearest 0.01% (m/m).
Determination of fat content in paneer by Rose-Gottlieb method
The following procedure is based on the Rose-Gottlieb principle and is described here using
Mojonnier type extraction flask. The method can be carried out using fat extraction tube with siphon
or wash bottle fittings (See Annexure B). In this method, ethanol and ammonia are added to the test
portion of paneer sample. Addition of concentrated ammonia dissolves the fat globule membranes
and subsequent addition of ethanol facilitates the passage of the fat globules from the aqueous phase
to the solvents. The fat is then extracted into a mixture of diethyl ether and petroleum ether. The
amount of fat extracted is determined gravimetrically after removal of the solvents by distillat ion
or evaporation.
Apparatus
1. Centrifuge: Centrifuge in which the fat-extraction flasks or tubes can be spun at a rotational
frequency of 500 to 600 min-1 to produce a gravitational field of 80 to 90 g n at the outer end
of the flasks or tubes. (The use of the centrifuge is optional but recommended).
2. Distillation or evaporation apparatus: To enable the solvents and ethanol to be distilled from
the fat-collecting flasks or to be evaporated from beakers and dishes at a temperature not
exceeding 100°C.
3. Hot air oven: Maintained at 102 ± 2°C.
4. Water-bath: Maintained at 40 to 60°C.
5. Boiling water-bath or hot plate.
6. Mojonnier-type fat-extraction flasks: It is also possible to use fat-extraction tube (or flasks)
with siphon or wash-bottle fittings. The long inner limb of the fitting may have a hooked end
if desired. The flasks (or tubes) shall be provided with good quality bark corks or stoppers of
other material e.g. silicon rubber or polytetrafluoroethylene (PTFE) unaffected by the
reagents used. Bark corks shall be extracted with the diethyl ether, kept in water at 60°C or
more for at least 15 min, and shall then be allowed to cool in the water so that they are
saturated when used.
7. Rack: To hold the fat-extraction flasks or tubes.
8. Fat-collecting vessels: e.g. flat bottom flask, 125 to 250 ml capacity.
9. Measuring cylinder: 25 and 50 ml capacities.
10. Pipettes: Graduated, 5,10 ml capacities.
11. Boiling chips or glass beads: Fat free.
12. Beakers: 100 and 250 ml capacities.
13. Tongs: Made of metal suitable for holding the fat collecting vessels.
Reagents
1. Ammonia solution: Approximately 25%, m/m (ρ20 910 g/L). If ammonia solution of this
concentration is not available, a more concentrated solution of known concentration may be
used.
 2. Ethanol: 95 to 96% (v/v).
3. Diethyl ether: Free from peroxides, containing no, or not more than 2 mg of antioxidants per
kg. For the preparation of peroxide free ether See Appendix 1.
4. Petroleum ether: Boiling range 40 to 60°C.
5. Mixed solvent: Freshly prepared by mixing equal volumes of the diethyl ether and light
petroleum ether.
6. Congo-red solution: Dissolve 1 g of Congo-red in water and dilute to 100 ml with water.
Note: The use of this solution, which allows the interface between the solvent and aqueous
layers to be seen more clearly, is optional. Other aqueous color solutions may be used
provided that they do not affect the result of the determination.
Procedure (Using the Mojonnier Fat-Extraction Flask)
1. Weigh to the nearest 1 mg, about 1 g of paneer sample into a clean and dry 50 ml beaker.
2. Add 8 ml of hot distilled water and make a smooth paste with a glass rod.
3. Add 3 ml of ammonia solution. Warm the contents of the beaker on a boiling water-bath.
Swirl gently to disperse the mixture till the paneer is dissolved completely. Cool the contents
of the beaker.
4. Using 10 ml of ethanol, transfer the contents of the beaker completely to the Mojonnier fat
extraction flask. Mix gently but thoroughly by allowing the contents of the flask to flow
backward and forward between the two bulbs; avoid bringing the liquid too near to the neck
of the flask. If desired, add two drops of Congo-red solution.
5. Add 25 ml of the diethyl ether, close the flask with a cork saturated with water or with a
stopper wetted with water, and shake the flask vigorously, but not excessively (in order to
avoid the formation of persistent emulsions), for 1 min with the flask in a horizontal position
and the small bulb extending upwards, periodically allowing the liquid in the large bulb to
run into the small bulb. If necessary, cool the flask in running water, then carefully remove
the cork or stopper and rinse it and the neck of the flask with a little of mixed solvents so that
the rinsing run into the flask or the prepared fat-collecting vessel.
6. Add 25 ml of the petroleum ether. Close the flask with the rewetted cork or rewetted stopper
(by dipping in water) and shake the flask gently for 30 secs as described above.
7. Centrifuge the closed flask for 1 to 5 min at a rotational frequency of 500 to 600 min-1 . If a
centrifuge is not available, allow the closed flask to stand in the rack for at least 30 min until
the supernatant layer is clear and distinctly separated from the aqueous layer. If necessary,
cool the flask under running water.
8. Carefully remove the cork or stopper and rinse it and the inside of the neck of the flask with
a little of the mixed solvent so that the rinsing run into the flask or into the fat-collecting
vessel. If the interface is below the bottom of the stem of the flask, raise it slightly this level
 by gently adding water down the side of the flask to facilitate the decantation of solvent (See
Figure 1 & 2).
9. Holding the extraction flask by the small bulb, carefully decant as much as possible of the
supernatant layer into the prepared fat-collecting vessel (fat-collecting vessel is prepared by
drying a vessel with a few glass beads in an hot air oven for 1 h, allow the vessel to cool to
room temperature for at least 1 h) containing a few glass beads avoiding decantation of the
aqueous layer. Rinse the outside of the neck of the Mojonnier flask with a little of the mixed
solvent, collecting the rinsing in the fat-collecting vessel and taking care that the mixed
solvent does not spread over the outside of the extraction flask.
10. Add 5 ml of ethanol to the contents of the extraction flask, using the ethanol to rinse the inside
of the neck of the flask and mix as described in Step 4. Carry out a second extraction by
repeating the operations as described in Step 5 to Step 9 inclusive but using only 15 ml of the
diethyl ether and 15 ml of the petroleum ether; use the ether to rinse the inside of the neck of
the Mojonnier flask. If necessary, raise the interface to slightly above the middle of the stem
of the extraction flask to enable the final decantation of solvent to be as complete as possible.
11. Carry out a third extraction without addition of ethanol by again repeating the operations
described in Step 5 to Step 9 inclusive but using only 15 ml of the diethyl ether and 15 ml of
the petroleum ether; use the ether to rinse the inside of the neck of the extraction flaks. If
necessary, raise the interface to slightly above the middle of the stem of the extraction flask
to enable the final decantation of solvent to be as complete as possible.
12. Remove the solvents (including ethanol) as completely as possible from the fat-collecting
vessel by distillation or by evaporation, rinsing the inside of the neck of the flask with a little
of the mixed solvent before commencing the distillation.
13. Heat the fat-collecting vessel (flask placed on its side to allow solvent vapor to escape) for 1
h in the hot air oven, maintained at 102 ± 2°C. Remove the fat-collecting vessel from the
oven, allow to cool (not in a desiccator, but protected from the dust) to room temperature (for
at least 1 h) and weigh to the nearest 0.1 mg. Do not wipe the vessel immediately before
weighing. Place the vessel on the balance using tongs (to avoid, in particular, temperature
variations).
14. Repeat the operations of heating and weighing until the mass of the fat-collecting vessel
decreases by 0.5 mg or less or increases, between two successive weighing. Record the
minimum mass as the mass of the fat-collecting vessel and extracted matter.
15. Add 25 ml of the petroleum ether to the fat-collecting vessel in order to verify whether or not
the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the fat is
dissolved.
16. If the extracted matter is soluble in the petroleum ether, take the mass of fat as the difference
between the final mass of the vessel containing the extracted matter and its initial mass.
 17. If the extracted matter is not soluble in the petroleum ether, or in case of doubt and always
for regulatory purposes or in case of dispute, extract the fat completely from the vessel by
repeatedly washing with warm petroleum ether. Allow any trace of insoluble material to settle
and carefully decant the petroleum ether without removing any insoluble material. Repeat
this operation three more times, using the petroleum ether to rinse the inside of the neck of
the vessel.
18. Finally, rinse the outside of the top of the vessel with mixed solvents so that the solvent does
not spread over the outside of the vessel. Remove petroleum ether vapors from the vessel by
heating the vessel for 1 h in hot air oven, maintained at 102 ± 2°C, allow to cool and weigh
as described in Step 13 and Step 14.
19. Take the mass of fat as the difference between the mass determined in step 14 and this final
mass.
20. Carry out a blank test simultaneously with the determination, using the same procedure and
reagents but replacing the test portion of the paneer sample by 1 ml of distilled water.
Calculation
The fat content expressed as a percentage by mass
(M1 - M2 ) - (M3 - M4 )
= ----------------------------- X 100
M0
Where
M0 = mass in g, of the test portion;
M1 = mass in g, of the fat-collecting vessel along with the extracted matter in step 14;
M2 = mass in g, of the prepared fat-collecting vessel, or, in the case of undissolved material,
of the fat-collecting vessel and insoluble residue determined as above (Step 18 to 19);
M3 = mass in g, of the fat-collecting vessel used in the blank test and any extracted matter
determined.
M4 = mass in g, of the prepared fat-collecting vessel used in blank test, or, in the case of
undissolved material, of the fat-collecting vessel and insoluble residue determined as above
(Step 18 to 19).
Calculate result to the nearest 0.01 % (m/m).
Note: If the value of M3 -M4 is 0.5 mg or less, this can be neglected in the calculation provided
that the diethyl ether does not contain antioxidants.