Milk Composition and Properties Guide

Milk and Milk
Properties
Dr. Arti Nile

Def:
►It is the secretion of mammary
glands in human and animals
after labour.
►It is the only food for newborn for a long
period till weaning time.
►Before birth the fetus receives its nutrition
through the placenta.

►Milk secretion is stimulated by Prolactin.
►Milk flow is decreased by Estrogen and
Progesterone while increased by
Thyroxin. Also Squalene present in
HELBA increase milk flow.
►Initiation of lactation may result from
sudden removal of the placenta and
other factors.
►Oxytocin (posterior pituitary hormone)
responsible for milk ejection.

Milk is considered as a complete diet as:-
It contains all of the components necessary for
growth, maintenance of life and reproduction.
e.g:
Carbohydrates, Lipid, Protein, Some minerals and
Vitamins.
But it is deficient in:
Vitamin C, Vitamin D, Vitamin K, Iron, Copper.

1- Color:
White color → due to presence of:
► Fat globules in emulsion form.
► Protein in colloidal form.
► Ca. phosphate and Ca. Casinate.
* Yellowish (creamy) color → due to:
► Presence of Carotene and Xanthophyll pigments
specially in cow's milk and colostrums.

2- Reaction:
Fresh milk is amphoteric in reaction as it
contains acid and base.
Milk is an excellent buffer as it contain:
 Protein.
 Phosphate.
 Bicarbonate.
 Citrate.
** PH of fresh milk:
 6.6 – 6.8 Cow's milk.
 6.8 – 7.4 Human's milk

 Milk pH changed to alkaline in case of:
Mastitis
Late period of lactation.
If the milk left to stand for a long period
specially in warm temperature, lactose is
fermented to lactic acid by bacteria present
normally in milk as:
* Streptococcus Lactis.
* Lactobacillus Lactis.
Result in decrease in milk PH and milk Souring.

3- Specific Gravity:
It is the ratio between weight of a given
volume of milk compared with the same volume of
water at a specific temperature.
 It measure total solids of milk, and determine if
any constituent added or removed from milk..
 Measured by Lactometer.
 Normal specific gravity:
 1020 – 1030 Cow's milk.
 1030 – 1035 Human's milk.

 Fat is the only constituent in milk with specific
gravity lower than 1000, so, when fat present
in milk in high amount, specific gravity
decrease than normal values.
 When milk is skimmed (removal of fat
content), the specific gravity increased due to:
Removal of light constituents of milk.
 When water is added to milk (adulteration)
the specific gravity decreased due to dilution
of total solids.

4- Taste:
 Normal characteristic milky taste.
 Milk taste changed in case of :
 Souring: due to increased acidity.
 Boiling: due to certain biochemical changes
and evaporation of volatile fatty acids.
 Late stage of lactation: due to increase
chloride percent.
 Mastitis: inflammation of udder.
5- Odor:
Characteristic milky odor.

6- Freezing point of milk:
► It is the temperature at which the liquid milk
freeze or crystallize.
► The freezing point of cow or buffalo milk
ranges from (-0.53) to (-0.57) °C with average
(-0.55) °C
► Milk freezes at a temperature slightly lower
than that of water due to the soluble
constituents in milk such as lactose, minerals
which lower the freezing point.

►This test is important for detection of
the amount of water added to milk
►The addition of 9% water to milk will
raise the freezing point by 0.05 °C.
 i.e., if milk freezing point is -0.45 °C,
it means addition of 18% water.
-0.45 – (-0.55) = -0.45 + 0.55 = +0.10
0.05 → 9 %
0.10 → X
X = = 18 %
9x0.1
0.05

7- Boiling Point:
► Milk boiling point is 100.5 °C , more than
water due to presence of dissolving
substances
8- Cream Line Formation:
► When milk left at room temperature the
fat globules coalesce, clumped at surface
of the container and form cream line.

Normal
Chemical composition
of milk

Milk is formed of:
1) Water: form 87%.
2) Solids: form 13%.
A- Organic constituents of milk.
1. Protein.
2. Lipid.
3. Carbohydrate.
B- Inorganic constituents of milk:
1. Minerals.
2. Vitamins.

A- Organic Constituents of Milk

1- Protein:
► Milk protein less in human than in cow's milk.
► All milk protein synthesized in the mammary gland.
► Milk protein of high coefficient digestibility (85 –
95%)
X 100
Amount of N2 absorbed
Total N2 in the protein

Characterized by:
1- Protein of high biological value as:
►It contains all essential amino acids.
►Easily digested.
►Easily absorbed.
►Easily metabolized.
2- Contain moderate amount of non essential
amino acids to decrease stress on body cells.
3- Essential to keep positive nitrogen balance
(nitrogen intake more than nitrogen output).
5- Essential to maintain growth of newborn.

6. Total amount of protein differ according to
species and affect rate of growth of young:
e.g:
► Young rabbit reach double weight in 5 days as
milk protein 10 gm/dl.
► Young calf reaches double weight in 50 days as
milk protein 3.7 gm/dl.
► Young child reach double weight in 5 - 8
months as milk protein 1.5 gm/dl.

Types of Milk Proteins are:
1. Casein.
2. Lactalbumin.
3. Lactglobulin.
4. Milk enzymes.

1) Casein
►It is the main and most dominant milk
protein.
►It represents 25% in human's milk and 83% in
cow's milk.
►It is a compound protein (Phospho-protein) of
high biological value.
►The high phosphate content of casein allows
it to associate with calcium and form calcium
phosphate salts.
 So, at normal PH of fresh milk (6.6 PH)
casein present as insoluble Ca. caseinate
phosphate complex.

►Casein contains 0.7% phosphorous and 0.7%
sulpher, but it is Phospho-protein Not Sulpho-
protein. Why??
1) Phosphorous:
 Attached to (OH) group of serine to form
phospho-serine.
 Attached to (OH) group of therionine to
form phospho-therionine.
2) Sulpher:
 Found in methionine (But masked by the
methyl group) So, act as Lipotropic factor
that prevent accumulation of fats on liver.

►Casein is suspended in milk in colloidal
suspension complex of casein micelles that are
dispersed in the water phase of milk.
►Casein micelles are spherical in shape and
much smaller in diameter than fat globules,
and consists of a network of casein sub-
micelles, each one micelle contains 400-500
submicelles, bonded by calcium phosphate
bridges.

►Casein is deficient in cystiene and cystin so give
negative result with sulpher test.
►Casein : (Lactalbumin - Lactglobulin) ratio is 1:3
which facilitate its digestion.
►According to molecular weight and electrophoresis
process, casein is separated in to 3 fractions, each
one of them has its own amino acid composition :
 α Casein → 75%.
 β Casein → 22%.
 γ Casein → 3%.
1
=
Casein
3
Lactalbumin + Lactglobulin

►Casein is the only milk protein that not
coagulated on boiling.
►The net charge of milk protein is negative
charge, so protein molecules remain
separated from each others due to repulsion
forces.
 Isoelectric point of casein: It is the point of
absence of net electrical charge on protein
molecules (So, protein PPT and not affected
by the electric field).

Milk Souring (Milk Curdling – Zabadi):
►The principle of coagulation, or curd formation, at
acidic pH is the basis for Yoghurt (Zabadi) formation.
How?
► Fermentation of lactose to lactic acid by:
 Lactic acid producing bacteria present normally in milk.
 Addition of acid (acetic acid).
 Addition of starter culture (yoghurt).
Lactose + lactic acid bacteria Lactic acid ↓ pH
Worm temp.
Fermentation

► Leading to decrease milk pH to 4.6 (Isoelectric
point of casein) leads to PPT of casein as:
1) By souring and lactic acid production: some
protein molecules gain the hydrogen ion with its
(+Ve) charge.
2) Calcium and phosphate are dissolved and
separated from casein.
3) The (+Ve) charge protein attract to the (-Ve) charge
protein at 4.6 pH, leading to PPT of casein.
4) Part of lactic acid combines of with Ca. of casein to
form Ca. lactate and free casein which PPT with fat
and fat soluble vitamins.
Ca. Caseinate + lactic acid Casein (pure) + calcium lactate
PPT ↓ (soluble)

Whey of milk curd:
It is the residual clear fluid which formed after removal
of casein and contain:
►Water.
►Lactic acid.
►Lactalbumin & lactglobulin (whey protein)
►Minerals.
►Water soluble vitamins.
The greenish yellow color of whey is due to
riboflavin (vitamin B2)

Milk Clotting (Cheese Manufacturing)
During digestion (in new born):
►Rennin enzyme acts on casein, converting it to
soluble Paracaseinate which is bounded to Ca and
form insoluble Ca. paracaseinate (milk clot).
►Rennin enzyme:
 Derived from 4th stomach of calves and used
externally in cheese manufacture.
 It is secreted inactive as pro-rennin.
 Its optimum PH 4.
 It is absent in adult stomach.
►Formation of milk clot: prevents rapid passage of
milk from stomach to intestine so give the sense of
fullness to newborn.

Whey of milk clot:
Residual clear fluid after removal of casein which
contain:
►Water.
►Lactose.
►Lactalbumin & lactglobulin (whey
protein)
►Minerals except Ca.
►Water soluble vitamins.

B- Lactalbumin:
► Represent 87% of whey protein.
► PPT by full saturation with ammonium
sulphate.
► Rich in cystein and cystin so give +ve result
with sulpher test.
► Simple protein.
► Soluble protein.
► Easily digested.

►Consists of two fractions:
 α Lactalbumin 32% of whey protein.
 β lactglobulin 55% of whey protein.
N.B:
β lactglobulin called globulin because it need
small amount of NaCl to be dissolved but it is
not PPT by half saturation as albumin.

3- Lactglobulin:
► Represent 13% of whey protein.
► PPT by half saturation with ammonium
sulphate solution.
► Rich in cystein and cystin so give +ve result
with sulpher test.
► Simple protein.
► Soluble protein.

►Consists of two fractions:
 True globulin (Euglobulin) 50%.
 False globulin (Pseudoglobulin) 50%.
►They carry antibodies causing immunity so
called immunoglobulins.
►They present in higher concentration in
colostrum.

4- Milk enzymes:
1. Catalase.
2. Peroxidase.
3. Xanthin oxidase.
4. Alkaline phophatase.
5. Amylase.
6. Lipase.
7. Aldehyde oxidase.

1- Catalase:
► Carried by fat globules.
► Act on hydrogen peroxide (H2O2) producing
water and oxygen.
► Increased in case of mastitis.

2- Peroxidase:
► Carried by lactalbumin.
► Act on hydrogen peroxide (H2O2).
► It is heat stable enzyme (resist destruction by heat).
► It is destroyed by milk sterilization.
Milk sterilization:
Heating of milk to 116◦C for 15 minutes which destroy
all harmful and non harmful microorganisms.
► Milk sterilization leads to alteration in milk taste and
destruction of vitamin C and vitamin B2.
N.B:
Absence of peroxidase enzyme indicates milk
sterilization.

3- Xanthin oxidase:
► Act on hypoxanthin and xanthin to produce
uric acid.

4- Alkaline phophatase:
► Catalyze hydrolysis of phosphate ester.
► Destroyed by the same temperature that destroys the
harmful bacteria.
e.g:
Tubercle bacilli.
► Absence of Alkaline phophatase enzyme indicates
milk pasteurization.
► Milk pasteurization:
Heating of milk to 60◦C for 30 minutes or 70◦C for 15
minutes followed by sudden cooling which destroy
the harmful microorganisms only.

5- Amylase:
► Carried by lactalbumin.
► Catalyze hydrolysis of α 1, 4 glycosidic
linkage of starch and glycogen.
► Increased in case of mastitis.

6- Lipase:
► Carried by casein.
► Catalyze hydrolysis of primary ester linkage in
triacylglycerol.
► Act on milk fat producing free fatty acids and
undesirable taste (Rancidity).

7- Aldehyde oxidase (Schardinger's enzyme):
► Its absence indicates milk boiling.
► Schardinger's test:
Milk + Methyl blue + Formaldehyde
 Blue color appeared in case of boiled milk.
 Colorless in case of unboiled milk.

N.B:
Beside milk proteins milk also contains:
A) Lactoferrin binding protein:
 It contains iron which is bounded to a glycoprotein.
 It facilitates iron transport and storage.
 Found in high concentration in human colostrums
and milk.
B) Vitamin B12 binding protein:
 It carries vitamin B12.
Lactoferrin and vitamin B12 binding protein
deprive pathogenic intestinal bacteria from iron
and vitamin B12 so they have bacteriostatic
action.

2- Lipid:
► Human's and cow's milk contain the same
amount 3.5 gm/dl but buffalo's milk is a little
higher 7 gm/dl.
► Easily separated on standing.
► Responsible for white color of milk
► It consists mainly of triacylglycerol
distributed as coarse emulsion which contains
oleic, myristic, palmitic and stearic fatty acids.

Also contain small amounts of:
►phospholipids 0.1%.
 Milk phospholipids are lecithin, cephalin,
sphingomyelin (9:5:1).
 Phospholipids in cow's milk twice that of
human milk.
►Cholesterol 0.01%.
Cow's milk contains higher proportion (mainly free
form) than human milk mainly (ester form)
► Milk contain fat soluble substance:
e.g:

►Fat soluble vitamins (vitamin A) which is
higher in human milk than cow's milk.
► Milk fat is formed mainly in lactating mammary gland
from:
 Active acetate.
 Glycerol: derived from glucose
(dihydroxyaceton phosphate).
► Milk fat may be derived from blood triacylglycerol but
to a limited extent.

Human's milk
Cow's milk
Contain no short chain fatty
acids.
Contain short chain fatty
acids 10%.
Contain 10% essential
(polyunsaturated) fatty acids
specially linoleic fatty acids.
Contain 0.05% essential
(polyunsaturated) fatty
acids.
Saturated fatty acids 48%.
Saturated fatty acids 58%.
Unsaturated fatty acids
52%.
Unsaturated fatty acids
42%.

3- Carbohydrates:
► Lactose (milk sugar) is the only carbohydrate
of milk.
► It is a reducing disaccharide consists of glucose
and galactose.
► Human's milk contains 7% lactose while cow's
milk contains 5% lactose.
► Lactose may be excreted in urine during last
third of pregnancy physiologically so it should
be differentiated from glucose by osazon test.

Importance of lactose:
1. It is less sweet than sucrose so allow the baby
to take large amount of milk without causing
nausea.
2. It is non fermentable carbohydrate so it doesn't
produce CO2 in GIT and the baby doesn't
suffer from abdominal colic or distention.
3. Lactose help growth of lactic acid producing
bacteria so help in absorption of Ca, P, Fe, Cu
which prefer acidic medium for their
absorption.
4. Lactose inhibits growth of putrefactive bacteria
which cause abdominal distention by
increasing the acidity of the intestine.

Lactose synthesis:
Lactose is synthesized in mammary gland from α
D glucose and β D galactose.
 Galactose converted to galactose -1-
phosphate by galactokinase enzyme.
 Galactose -1- phosphate and UDP glucose are
converted to UDP galactose and glucose -1-
phosphate by uridyle tranferase enzyme.
 UDP galactose bounded to glucose by lactose
synthase enzyme forming lactose.

Galactosemia:
►It is a condition occurs due to deficiency of
galactose -1- phosphate uridyle transferase.
►Characterized by accumulation of galactose and
galactose -1- phosphate in blood.

Lactose synthase enzyme:
Consists of two subunits:
1. Catalytic subunit:
 Can't synthesize lactose.
 It catalyze the attachment of galactose to protein to
form glycoprotein.
2. Modifier subunit:
 Alter the specificity of the catalytic subunite which
make galactose bounded to glucose to form lactose.
► During pregnancy little modifier subunite is formed in
the mammary gland so no lactose synthesized But at
birth large amount of modifier is formed helping the
synthesis of lactose in mammary gland.

Importance of lactose:
1. It is less sweet than sucrose so allow the baby
to take large amount of milk without causing
nausea.
2. It is non fermentable carbohydrate so it doesn't
produce CO2 in GIT and the baby doesn't
suffer from abdominal colic or distention.
3. Lactose help growth of lactic acid producing
which prefer acidic medium for their
absorption.

4. Lactose inhibits growth of putrefactive
bacteria which cause abdominal
disturbance by increasing the acidity of
the intestine.
5. On hydrolysis it produce glucose and
galactose:
 Glucose act as source of energy.
 Galactose used for synthesis of
galactolipids so help in development of
nerve tissues.

N.B:
Lactose level in milk tend to vary directly with the
weight of the adult brain:
Man has the largest brain in proportion to the
body weight of all animals this may be related
to the galactolipids of the brain which contains
galactose.
6. Lactose has a laxative effect as some of
glucose is fermented by the intestinal
bacteria producing CO2 which stimulate
the intestinal movement.

N.B:
If the milk is taken by the adult in large
amount result in diarrhea due to decrease
in lactase enzyme so lactose is hydrolyzed
by intestinal bacteria to glucose and
galactose.
 Glucose is fermented producing CO2 and
abdominal distention.
 Non fermentable galactose and
unhydrolyzed lactose increase osmotic
pressure of the intestine leads to diarrhea.

B-Inorganic constituents of
milk

1- Minerals:
► Human milk contain less mineral elements
(0.4%) than cow's milk (0.8%).
► Milk rich in Ca and P which are present in
their proper ratio for absorption (2:1) in human
milk while in cow's milk (1:2) which is not
suitable for their maximum absorption .
► Ca and P are essential for:
1. Growth of bone and teeth.
2. Stability of casein.

►Milk is deficient in Fe and Cu which are
supplied by their storage in liver during prenatal
life (this store is sufficient till weaning time).
N.B:
Milk is deficient in Iron but it is more in human
milk than cow's milk Thus anaemia in breast
feeding is less common.
►Milk contain adequate amount of Na, K, Mg.
►Human milk contains Na:K (1:2) which is
suitable for the optimal growth of newborn.

2- Vitamins:
► Milk is deficient in:
► Vitamin C.
► Vitamin D.
► Vitamin K.
► Milk contain adequate amount of vitamin B complex
which are sufficient for first week of life
e.g:
► Pantothenic acid.
► Riboflavin (gives the whey the greenish tint in
sunlight).

N.B:
►Vitamin C must be supplied to the growing baby
in the form of fruit juices to withstand infection.
►Fortified vitamin D milk is used in order to
supply the baby with vitamin D requirement
which is added from cod liver oil.
►Exposure to sunlight in the early morning or
before sunset help in formation of active vitamin
D from cholesterol.

Milk is an adequate diet for
children
Although milk is deficient in Fe, Cu, vitamin C,
vitamin D, and vitamin K it is complete natural
food for the following reasons:

1. Easily digested absorbed and metabolized.
2. Contain all the nutrients required for the
newborn at early stage of life.
3. Balanced ratio between carbohydrates, lipid
and protein.
4. Milk protein:
Protein of high biological value as:
 It contains all essential amino acids.
 Easily digested.
 Easily absorbed.
 Easily metabolized.

5. Milk lipid:
Milk contain essential fatty acids
e.g:
 Linoleic acid.
 Linolenic acid.
6. Milk sugar (lactose):
A. It is less sweet than sucrose so allow the baby to
take large amount of milk without causing nausea.
B. It is non fermentable carbohydrate so it doesn't
produce CO2 in GIT and the baby doesn't suffer
from abdominal colic or distention.
C. Lactose help growth of lactic acid producing
which prefer acidic medium for their absorption.

D. Lactose inhibits growth of putrefactive bacteria
which cause abdominal distention.
E. On hydrolysis it produce glucose and galactose.
F. Lactose has a laxative effect.
7. Provide Ca and P in proper ratio for absorption (2:1)
which are essential for:
1. Growth of bone and teeth.
2. Stability of casein.
8. Good source of vitamins.
9. Contain antibodies specially in colostrums which
provide immunity to the newborn.
10. Cheep in price and of good caloric value.

N.B:
►Certain foreign substance in the blood
plasma may enter the milk as:
►Volatile oils of certain foods (onions –
garlic).
►Drugs (sulphonamids – salicylate –
morphin – alcohol).
►Inorganic elements (iorn – iodin – lead –
mercury).

Factors affecting milk composition

1. Species:
►Milk protein:
Rabbit's milk 10% while cow' milk 3.7%.
►Milk fat:
Cow's milk 3.5% while buffaloe's milk 7%.
2. Age:
►Old age decrease fat contents of milk.
3. Seasonal variation:
►In winter increase in fat, protein and mineral
contents of milk.

4. Nutritional variation:
► Overfeeding has no effect on milk composition.
► Underfeeding decrease in milk volume and protein
and lactose contents of milk while fat contents of
milk increased.
5. Infection of mammary gland:
► Mastitis decrease fat, lactose and casein contents of
milk while whey protein and chloride ions
increased.

6. Stage of lactation:
► Colostrum secreted during 1st week of
lactation.
► Intermediate milk secreted during 1st month of
lactation except 1st week.
► Mature milk secreted during 1st year of
lactation except 1st month.
► Late milk secreted after 1st year of lactation.
 Watery in consistancy.
 Contains less protein, lipid, lactose and
vitamins.
 Contains more minerals than mature milk
(less sweet to help weaning).

N.B:
Witch's milk:
Fluid secreted by mammary gland of
infants during first few days of life due to
the effect of placental hormones on
mammary gland of infants.

A- Colostrum:
► Yellowish fluid secreted by mammary gland during
1st week of lactation.
► Volume of colostrum in human (150 – 300 ml/day).
► Specific gravity (1.040 – 1.060)
Colostrum is very important for survival of new born due
to:
1. High level of protein (20%):
 40 – 55% globulin specially γ globulin
(immunoglobulin) which provide immunity for
newborn up to 6 months .
 Globulin is a coagulable protein by heat.

 Absorbed without digestion due to its low
molecular weight.
 Globulin contain prolin which enter in Hb
synthesis.
2. Low fat (3 g%) and carbohydrate (4 g%) content.
► Contains higher content of cholesterol and
lecithin.
3. High mineral content.
► Iron found in higher concentration than mature
milk.
4. High fat soluble vitamins (E, D, A, K) and water
soluble vitamins (riboflavin, nicotinic acid).

5. Trypsin inhibitor:
 Inhibit action of trypsin enzyme so:
immunoglobulins absorbed as such.
6. Yellowish color of colostrum is due to high
level of β carotene which is present 50 – 100
times as much as in mature milk.
► Colostrum contains large granular bodies
(colostrum corpuscles):
Alveolar cells of mammary gland and
leukocytes baded with fat.

Function:
1. Has a laxative effect so help excretion of
meconium.
2. Contain high level of immunoglobulins which
provide immunity for newborn.
3. Easily digested, absorbed and metabolized.
4. Has high nutritive value (rich in protein,
minerals and vitamins).

Def:
It is a process by which cow's milk is made to be
as near as human's milk.
Aim:
 Protein of human's milk (casein) form 25% of
milk protein while casein in cow's milk form 5/6
of milk protein so it form dense clot in newborn
stomach which can't be digested leading to
vomition.
 Lactose in human's milk higher than that in
cow's milk (7:5).

Steps:
1. Pasteurization of milk:
 Heating of milk to 60 ◦C for 30 minutes followed by
sudden cooling then the pasteurized milk left in
cool place for 4 hours to allow separation of cream.
2. Separate the cream and the residual milk called
Skimmed milk.
3. Skimmed milk is divided to two halves.
 To one half add:
 Separated cream.
 Equal amount of water.
 Complete lactose to 7%.
 Iron, vitamin C, vitamin D may be added.
4. Mix well then sterilize the milk:
 Heating of milk to 116 ◦C for 15 minutes.

N.B:
1. The humanized milk still differs from human milk in
containing less iron and copper.
2. The protein in diluted cow's milk is usually still higher
than that of human milk so:
Bottle fed babies store greater quantities of
nitrogen than do breast fed babies. This
nitrogen retention result in a muscle mass
25% greater in bottle fed babies.
 Cow's milk digested more slowly than human milk.
 The faeces of bottle fed babies are more abundant
and of a harder consistency than those of breast fed
babies.

3. High mineral content of cow's milk may be a
load to cardiovascular system due to high Na+
content.
4. High phosphate content of cow's milk may
produce hyperphosphatemia and hypocalcemia
with convulsions in the infants.

Advantages of human milk (Breast Feeding):
1. Psychological effect on both child and
mother.
2. Breast milk is supplied in suitable
temperature.
3. Sterile and not liable to be contaminated.
4. Cheaper than animal milk.
5. Not liable to adulteration.
6. Lactation may act as a contraceptive
measure.
7. Lactation minimize the risk of breast tumors.
8. Lactation help involution of the uterus.

Principle:
Lactose in milk is estimated in the clear filtrate
after precipitating proteins. Tungestic acid, and
Trichloroacetic acid can be used to precipitate
milk proteins.

Instrument:
1. Estimated pipette.
2. Volumetric flask.
3. Funnel.
4. Beaker.
5. Burette.
6. Burette holder.
7. Porcelain dish.

Procedures:
a) PPT of casein:
►In a clean dry volumetric flask add:
 5ml of milk
 5ml of 10% sodium tungstate solution
 5ml of 2/3 N sulphuric acid drop by
drop.
►Shake gently after each addition .
►Complete to the 100-ml mark by distilled
water.
►Left for sometime (1/4 – 1/2 hr) until the
precipitate of casein settles down.

b) Filteration:
►Filter into a dry clean beaker.
N.B:
The filtrate must be clear, if not refilter again.

c) Reduction:
► When about half the volume is filtered, wash the burette
with little of the filtrate.
► Then fill the burette with the filtrate.
► In a porcelain dish place:
 10 ml of Fehling’s solution.
 20 ml of distilled water.
 Heat the Fehling’s solution until it gently boils and
► Begin the reduction adding the filtrate slowly and
regularly in order to keep Fehling’s solution boiling all
the time.
► Continue the reduction until the last trace of blue color
is just discharged.

►Using an external indicator near the end point
(Potassium Ferrocyanide solution acidified with
Glacial Acetic acid) to ensure the complete
reduction of Fehling's solution:
The end point is reached when no brown color or
precipitate is formed on the addition of one drop of
the solution to the external indicator. 10-ml Fehling’s
solution are reduced by 0.0678-gram lactose.
If brown color or PPT appeared it indicates:
 Incomplete reduction of fehling’s solution.
 Complete reduction till the yellow color of the
indicator doesn’t change.

d) Calculation:
► R = number of mls of the filterate used for complete
reduction of fehling’s solution.
► 10 ml Fehling’s solution are completely reduced by 0.0678 gm
lactose.
► Total dilution of milk (T.D):
► Lactose in milk (g%):
100
20 =
5
0.0678 x 20 x 100
Lactose in milk =
R

Estimation of glucose or lactose in
solution:

Principle of the test:
The quantitative determination of glucose or lactose in
gm/dl in the solution depends upon their reducing
property to Fehling’s solution.
Instruments used in the estimation:
► Pipette 5 and 10-ml capacity.
► Conical flask of 100 cc capacity.
► Burette 50 ml capacity
► Burette holder.
► Porcelain dish.
► Beaker.

Reagents and solutions required:
► Glucose or lactose, solution under estimation.
► Fehling quantitative reagent.
► Potassium ferrocyanide acidified with glacial acetic
acid (external indicator).
Significance of the test:
► This test considered the bases of tests used for the
determination of blood glucose.
► Used for estimation of lactose in milk, for detection of
adulteration of milk by the addition of water.

procedures:
(A) Rough estimation:
► Fill the burette with glucose or lactose solution after its
careful rinsing with water.
► Put in the porcelain dish
10-ml of Fehling quantitative reagent.
20 ml of distilled.
Heat until the Fehling boils in the dish.
► Begin the reduction by slow addition of solution in
order to keep the Fehling boiling all the time.
► Continue reduction till the last trace of the blue color of
the Fehling’s reagent turn to red brown PPT of Cu2O.
► Record the rough reading R (number of mls of glucose
or lactose descended from the burette).

►If the (R) rough reading is below 5ml make
dilution to the glucose or the lactose solution
under the estimation.
►If the (R) rough reading is above 5ml
calculate the percentage of glucose or the lactose
solution under the estimation.

(B) Preparation of diluted sugar solution:
► Mutiply the (R) by 4.
► The result is equaled to 5, 10 or 20.
► In 100 ml volumetric flask add:
? ml of the glucose or lactose sugar solution.
Completed with distilled water to 100 ml mark
and well mixed.
► In this case the sugar solution is diluted 5, 10 or 20
times.

(C) Accurate estimation:
► Wash the burette with distilled water and rinse it with
little of diluted sugar,
► fill it again with the diluted sugar.
► In the porcelain dish add:
10 ml of Fehling’s quantitative solution.
 20 ml of distilled water.
Heat till gentile boiling.
► Begin the reduction by slow addition of the diluted
sugar solution drop by drop in order to keep the
Fehling’s solution boiling all the time.
► Continue reduction till the last trace of the blue color
disappeared and changed to red brown PPT of Cu2O

► Using an external indicator near the end point
(Potassium Ferrocyanide solution acidified with Glacial
Acetic acid) to ensure the complete reduction of
Fehling's solution:
If no brown color or PPT is formed on the addition
of one drop of the solution to the external indicator it
indicates:
 complete reduction of Fehling's solution.
If brown color or PPT appeared it indicates:
 Incomplete reduction of Fehling's solution.
 Complete reduction till the yellow color of the
indicator doesn’t change.
► Record the number of mls used for reduction of 10 ml
Fehling’s quantitative solution and give it symbol (V)
ml.

D) Calculation:
► 10 ml Fehling’s solution are completely reduced by
0.0678 gm lactose.
g/dl
0.0678 x 100
Lactose in solution =
R
g/dl
0.0678 x T.D x 100
Lactose in solution =
V

► 10 ml Fehling’s solution are completely reduced by
0.05 gm glucose.
g/dl
0.05 x 100
Glucose in solution =
R
g/dl
0.05 x T.D x 100
Glucose in solution =
V

N.B.:
► Fehling’s solution should boil gently all the
time.
► Never stir by glass rod.
► The rate of addition of sugar solution from the
burette should be equal to the rate of
evaporation of Fehling in the dish.
► Do not use the external indicator as long as
there is blue coloration seen by naked eye.
► The external indicator should be present in
white, clean porcelain slab.

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