Milk and Milk Product

Didik Setiyo W.
dsw (c) 2013 dept. of chemistry
Introduction
Milk normal secretion of the mammary
gland for the feeding of the young
mammals,
c.q.
lacteal secretion, practically free from
colostrums, obtained by the complete
milking with contain not less than 8.25%
milk solid-not-fat and not less than 3.25%
milk fat
Composition
Major component:
Lactose (occur naturally in milk)
Casein (occur naturally in milk)
Proteins (essential amino acids)
Milk fat (palatable mix. of glycerides of fatty acids)
Lactose and mineral salt------ true solution
Protein and casein phosphate ------ colloidal solt.
Fat ------ macroscopic dispersion of the milk plasma
Other constituents ----vitamin, enzyme, pigment, and
lactic acid
dsw (c) 2013 dept. of chemistry
Composition (cont.)
Cows milk in average gross composition: water 87%, fat 3.7%,
lactose 4.9 %, proteins 3.5%, ash 0.7 %
Water
87%
Solid
13%
27% Protein: casein
30% Fat: combined
with water is called
cream
37% lactose: milk
sugar
6% ash/minerals:
vitamins and
minerals including
calcium and
phosphorus
Protein
27%
Fat
30%
Lactose
37%
Ash/mineral
6%
Milk Solids
dsw (c) 2013 dept. of chemistry
Back
Variation in composition is due to breed diff., time of year, time of day,
individual diff., age of mammals, period of lactation, portion of any one
milking, feeding etc.
Breed difference: greatest magnitude effect
Time of year/day:
Milk in fall and early winter is richer than spring and summer
Morning milk is richer in fat than evening milk
After first few days after calving, it is known as colostrums and
differ materially from normal milk
Lactation period:
In the final lactation: there is some increase in fat and casein
Portion: first portion (fore milk) of milk drawn in milking process are
lower in fat than the last portion
Composition (cont.)
dsw (c) 2013 dept. of chemistry
Fat
Fat and associated lipids material as an emulsion
of small individual globule in an aqueous phase
Surrounding each milk fat globule is a number of
membrane composed of cholesterol, phospholipids
protein, and other surface-active molecules
The membrane prevent individual fat globules from
combining with others to form a larger globule
(emulsifying agent for the fat suspended in milk)
Fat globule membrane:
Inner layer: phospholipids-protein complex
Outer layer: enzymes originating in mammary
gland
Membrane agitation
1. disruption of the membrane (rancid flavor)
2. cause for globule to coalesce to form larger globule
and butter
Milk Fat: 12.5% glycerol, 85.5% fatty acid (by weight)
Glyceride tri- (98-99%), di- (0.05%), mono- (0.04%)
contains more than 16 diff. fatty acids combined with in a
number of diff. ways.
Fatty Acids (even number of carbon atom)
Milk Fat is the only natural food fat that contains butyric acid in its glyceride
structure
Butyric acid, C
6
, C
8
, C
10
saturated fatty acids give strong
characteristic odour
60-75% saturated fatty acids
25-35% unsaturated fatty acids
4% polyunsaturated fatty acids
Oleic acid and palmitic acid constitute 50% of the total fatty acid in
milk fat
Minor component: FFA, cholesterol, phospholipids, Fat-soluble vitamins
(A,D,E,K)
Fat (cont.)
Principal fatty acids found in milk
triglycerides
Molecular formula Chain length Melting point
Butyric CH
3
(CH
2
)
2COOH
C
4
8C
Caproic CH
3
(CH
2
)
4COOH
C
6
2C
Caprylic CH
2
(CH
2
)
6COOH
C
8
16C
Capric CH
3
(CH
2
)
8COOH
C
10
31.5C
Lauric CH
3
(CH
2
)
10COOH
C
12
44C
Myristic CH
3
(CH
2
)
12COOH
C
14
58C
Palmitic CH
3
(CH
2
)
14COOH
C
16
64C
Stearic CH
3
(CH
2
)
16COOH
C
18
70C
Arichidonic CH
3
(CH
2
)
18COOH
C
20

Oleic CH
3
(CH
2
)
7CH
=CH(CH
2
)
7COOH
C
18: 1
13C
Linoleic CH
3
(CH
2
)
4
(CH=CH.CH
2
)
2
(CH
2
)
6COOH
C
18: 2
5C
Linolenic CH
3
.CH
2
(CH=CH.CH
2
)
3
(CH
2
)
6COOH
C
18: 3

Proteins
Principles protein: casein, lactalbumin (0.1-
0.4%), lactoglobulin (0.4%)
Casein: as a colloidal calcium caseinate
calcium phosphate complex and forms about
3% of the whole milk
Other component: small amount (2%) of
heat-stable milk serum protein
dsw (c) 2013 dept. of chemistry
Milk protein fractions
dsw (c) 2013 dept. of chemistry
Casein
Casein is defined as the fraction of skim milk
that is precipitated by acidifying to pH 4.6-4.7,
leaving the milk serum protein in solution
Three electrophoretic components designated
as , , and -casein in order of decreasing
mobility
complex of -casein-casein which is
hydrolyzed from casein via the action of rennin
to give para--casein
para--casein is insoluble in the presence of
calcium ion, but precipitate, instead, as a clot or
curd
Basis of cheese manufacture
Milk of ruminant (buffalo, cow, ewe, goat, etc.)
contain at least 2.5% casein give cheese-
type-curd with rennet (milk of others, not)
Rennin: enzyme
produced by
cells
encapsulating
digestion track
to coalesce
milk. This
enzyme effects
soluble-milk
protein which
convert to
insoluble
ensuring milk to
take more time
before
hydrolysis by
protein-
digesting
enzyme
Whey proteins
After the casein proteins have been precipitated, the whey
proteins remain in solution.
Whey soluble protein retained in the supernatant
include lactalbumin and lactoglobulin
unaffected by diluted acid and rennin (casein
affected)
both are coagulated by heat (depend on pH,
T, t, and salt concentration)
Component: -lactalbumin, lactoglobulin, protease-
peptone, blood serum, (serum albumin,
immunoglobulin)
Other proteins
After removal of casein, albumin, globulin from skim milk
filtrate contains a fraction that may be precipitated by
saturation with ammonium sulfate
Fraction protease-peptone fraction that consist of smaller
molecules size than true protein

Nonprotein Nitrogen
Includes:
Amino acid, vitamin, uric acid, ammonia, creatin, creatinin
End product of nitrogen metabolism and in synthesis of milk in
the udder
Lactose(milk sugar)
Composed of D-glucose and D-galactose
Content: 4-5% (the more lactose contains, the sweeter its
taste), 1/3 as sweet as sucrose)
Reducing Fehlings solution
Soluble in hot acetic acid, insoluble in alcohol and ether
Fermented by bacteria lactic acid (souring of milk), but
odor is due to other volatile product (during fermentation)
Hydrolyzed by lactase (in digestive track) into
monosaccharide as well as -galactosidase (from bacteria)
Over lactose intake (exceeds the amount that it can
hydrolyse) symptoms of lactose intolerance (flatulence,
cramps, diarrhea)

Ash (mineral matter)
Average ash content: 0.7% include
Free acid radicals (citrate, carbonate, etc.)
Sulfur, phosphorus
Calcium, magnesium
Potassium and sodium as its chloride, phosphates and
citrate
Composition:
Major constituents: chloride and oxide of K, Ca, P
Trace elements: Zn, Cu, Fe, Flourine, Iodine, Al, Ba, B, Co,
Li, Sr, Ti, Si, Sn, and Mo (some are of enzyme constituents,
Fe in peroxidase, Mo in xanthine oxidase
Many of these elements are attached to proteins of fat-
globule membrane
dsw (c) 2008 dept. of chemistry
Vitamins
Fat-soluble vitamins: A, D, E, K
Ascorbic acid
Thiamin, riboflavin, niacin, pantotenic acid, pyridoxine, folic
acid, and cyanocobalamin
Water-soluble vitamins
(CH=CH-CH=CH)
2
CH2OH
CH
3
CH
3
H
3
C
Vitamin A:
UsesMaintains skin and mucous membrane of
oral cavity, and digestive, respiratory, reproductive,
and urinary track. Critical for vision
Vitamins (cont..)
Vitamins (cont..)
Vitamins (cont..)
Enzyme
Lypase catalyze the hydrolysis of lipids
Phosphatase hydrolyze ester of
phosphoric acid
Lactoperoxidase, catalase, amilase,
protease, xanthine oxidase

dsw (c) 2013 dept. of chemistry
Grade of milk
Grade
Grade A raw milk
Grade A pasteurized milk
Certified milk
Classes
Class 1: fluid purpose (highest price milk)
Class 2:any other purpose

Analysis of Fluid Milk Product
Sampling:
Collecting milk in one container from one or more container
Mixing (with no shaking) just stir
Storage in a nonadsorbing, airtight container; cold (but not
freezing)
Addition a suitable preservative may allowed
Tablet of HgCl
2
-K
2
Cr
2
O
7
not exceeding 1 g;
formal dehyde 36% (0.1 % per 30 mL milk);
if phosphatase test is to be made, chloroform is the only
permisible preservative)
Bring sample to about 20
0
C, mix by pouring sample into a
receptacle and back again several times
Weigh or measure the test portion
dsw (c) 2013 dept. of chemistry
Weighing Method
Principles:
Transferring a weighed quantity of milk into a flat
dish (porcelain, aluminum) containing a layer of
sand (to increase the drying surface)
Drying the dish and content to a constant weight
Cooling it in desiccator
The increasing in weight correspond to the total
solid in the sample
Analysis of (cont.): Total Solids
Dried solid
should be
practically
white. Any
appreciable
browning
indicates that
some of lactose
has been
caramelized
(error in result)
dsw (c) 2013 dept. of chemistry
Procedure:
Add 10-15 g of white sand to flat dish
Heat the dish at 100
0
C in drying oven until a
constant weigh
Weigh 5 g of prepared milk into the dish
Dry on a steam bath for 15 minutes, then in air
oven at 98
0
-100
0
C for 3 hr.
Cool in a desiccator and weigh rapidly
Report the percentage of residue as total solid
Analysis of (cont.): Total Solids
dsw (c) 2013 dept. of chemistry
composition
Lactometer Method
Constituents of normal milk are present in a fairly constant
ratio total solid can be calculated with a fair degree of
accuracy if sp.gr. and fat percentage are known
Procedure:
Bring sample to 16
0
, determine the sp gr of the milk with a
Quevenne lactometer
Total Solid (TS) = L + 1.2f + 0.14
with L = Lactometer reading
f = percentage of fat in the milk (by the method laterin
det. of crude fat bellow)
Analysis of (cont.): Total Solids
dsw (c) 2013 dept. of chemistry
Determination of Crude Fat
Volumetrically:
Babcock
method as a
rapid method for
general dairy
testing


Gravimetrically:
Mojonnier
method
BABCOCK Method
Principle:
Milk H
2
SO
4
(concentrated)

Protein denaturated hydrolyzed

Fat to coalesce processes follows
(centrifuging, water adding)

dsw (c) 2013 dept. of chemistry
dsw (c) 2013 dept. of chemistry
Babcock Method
Sample of well-mixed milk in a standard test milk bottle
Hold the bottle at an angle then pour H
2
SO
4
slowly, in small portion,
while rotating the bottle
Mix the acid and milk until all traces of curd have disappeared. The
mixture become hot and turns uniformly dark colored
Transfer the bottle to a centrifuge, counter balance with bottle
containing water then centrifuge for 5 min.
Remove the bottle, add hot distilled water until the bulb of the bottle is
filled
Centrifuge for 2 min., again. add hot water until the fat column is
forced up to the neck, centrifuge 1 min., at 55-60
0
C
Place the bottle into the water bath (immerse to the top of the fat
column)
After equilibrium, remove bottle from the bath, dry and measure the
height of the column (read directly by weight of the fat in the milk)
Determination of Crude Fat
Babcock Method:

Well-mix milk------------Mixing -----------centrifuging

----hot water adding ------------- centrifuging ---hot water

again -----------centrifuging -----------------measurement
Determination of Crude Fat
Test milk bottle
H
2
SO
4

Dark colored
Until bulb filled
Fat forced up
Cooling in bath
Mojonnier Method
Fat is released from other milk constituent by treatment with
ammonium hydroxide and ethanol then dissolved in a mixture
of ether and PE
Ether layer (containing fat) is decanted into a weighed dish,
the ether is evaporated then the dish again weighed
Different in weight is the amount of the fat extracted
Procedure:
Weighing approx. 10 g of well-mixed milk into a Mojonnier
extraction flask and adding 1.25 mL of concentrated
ammonium hydroxide. Add 10 mL of 95% ethanol, stopper
the flask and holding it in horizontally with a finger on the
stopper, shake thoroughly. Add 25 mL of ether and shake for
1 min., add 25 mL PE and again shake for 1 min., centrifuge
the flask or let stand until upper layer is clear. Remove the
stopper then decant off as much as possible of the ether layer
into an aluminum dish.
Repeat the extraction of the liquid remaining by adding
successively 5 mL ethanol, 15 mL ethyl ether and 15 mL of
PE, shaking after each addition as before. Let stand, then
decant the layer. Evaporate the ether layer and dry in an oven
at 135
)
C for 5 min., Cool in desiccator then weigh.
Calculate the percentage of the fat obtained
Determination of Crude Fat
Determination of Ash
Organic matter is oxidized by combustion
in a muffle furnace
Remaining residueashis weighed and
percentage of ash calculated
Ashing should be done at temperature of 500
0
C in order to prevent
volatilization of the chloride (sodium and potassium)
Procedure:
Heat a crucible at 500
0
C for 1 hr, cool and weigh
Pipette 10 mL of well-mixed milk into crucible and weigh.
Evaporate to dryness on a steam bath.
Transfer the crucible and dried sample to a muffle furnace until
the ash is carbon free (white ash)
Cool in desiccator, weigh and calculate the percentage of ash
Determination of Total Acidity
The acidity of fresh milk (apparent acidity)
is due to the presence of casein, phosphates, albumin, CO
2
, and
citrates
Any increase in acidity is due to the fermentation of lactose to lactic
acid by bacteria present (real acidity).
When lactic acid is sufficient produced, proteins coagulate (milk is
termed sour)
Procedure:
Weigh 20 g (about 20 mL) of well-mixed milk into beaker placed on a
white background), dilute with an equal volume of CO
2
-free water
(recently boiled and cooled), add pp indicator, titrate the milk solt. with .
0.1 N NaOH
Report the acidity as percentage of lactic acid by weigh (1 mL NaOH is
equivalent to 0.090 (cek! Benar apa salah) g lactic acid
OH
O
OH
dsw (c) 2013 dept. of chemistry
dsw (c) 2013 dept. of chemistry
Determination of Total Protein
Kjeldahl method
Casein and lactalbuminthe major protein
constituent in milk, are contained approx. 15.7% nitrogen
converting factor to protein = 6.38 (=100/15.7)
Procedur:
Transfer accurately a weighed sample of appox. 5 g of well-mixed
milk into an 800-mL Kjeldahl flask. Add the various kjeldahl
reagent, digest, distill and titrate the distillate (as previous
discussion)
Conduct a back determination
Calculate the percentage of protein present in the milk:

% protein = sample titration (mL) - blank titration (mL) x
N
solution
x 0.14 (meq of N) x 6.38
KMnO
4

Determination of Lactose (milk sugar)
Polarimetric determination: based on optic activity character of the
lactose
Munson-Walker method: based on reducing properties (Fehlings A and
B to produce Cu
2
O)
Iodine-Thiosulfate method: based on oxidation of Cu
2
O with nitric acid
to give soluble cupric salt
Permanganometric: also based on oxidation of Cu
2
O with ferric sulfate
and H
2
SO
4
, titration of the ferrous with standard potassium
permanganate

Cu
+
+ Fe
3+
Cu
2+
+ Fe
2+

Folin-Wu Colorimetric method:
Milk sample is freed from suspended component, then heated with an
alkaline copper solt. In a Folin-Wu sugar tube to prevent reoxidation.
Cu
2
O is oxidized by phosphomolybdic acid and bleu color is measured
in a colorimeter. Concentration of lactose is det. by comparison with a
sugar solution of known concentration treated in the same manner
Residual Phosphatase Test
The test is commonly used for detection of
improperly pasteurized milk
Fact: alkaline phosphatase, an enzyme present
naturally in raw milk, is progressively inactivated at
the temperature employed in pasteurization
Basis:
hydrolysis of disodium phenyl phosphate by the enzyme
Phenolate then is quanititated by any colorimetric reaction
Any addition of unpasteurized milk to pasteurized milk can
be detected
Determination of Minerals
Greatest interest: Calcium, iron
Other cations: sodium, potassium
AAS as an also sensitive method in detecting small amount of
contaminant (Pb Hg, etc.)
Method
Preliminary:
Removing organic matter (dry ashing or wet digestion for non-
Hg analysis)
Dissolving the residue in dilute acid
Measurement:
Aspiration to the flame
Determining absorption at spesific (a resonance line)
Preparation:
Stock solution of a salt of the metal to be determined in acid
Preparation a series of standard solution
dsw (c) 2013 dept. of chemistry
Chemical Analysis of Calcium
(Volumetrically)
Calcium is precipitated at pH 4 as the oxalate
The oxalate dissolve in acid liberating oxalic acid
Oxalic acid then is titrated with potassium permananganate
with bromocresol green as an indicator (until pink colour)
Equation:
2 KMnO
4
+ 5 CaC
2
O
4
+ 8 H
2
SO
4
2 MnSO
4
+ K
2
SO
4

+ 5 CaSO
4
+ 10 CO
2
+ 8 H
2
O
Calculation:
Jumlah calcium (mg) = Vol. KMnO
4
(mL) x N
KMnO4
x meq Ca


Follow the procedure!
Basis: formation of coloured complex of ferrous ion and
2,2-dipyridyl
Chemical Analysis of Iron
(UV-Vis Spectrometrically)
sample
protein solution
acid
heating
pH adjustment
2,2-dipyridyl
Coloured
solt.
Measuring A at 522 nm
Comparing with standard
data