analytical method for the analysis of cheese different methods are included some of the other detection method

1. General Analytical Method For Cheese
Including Adulterations
Subject :- FOOD ANALYSIS, MPH 104-T
Submitted to :- Dr. Mohammad Shahar Yar
Submitted by :- Shameer (Master of Pharmacy -Pharmaceutical Analysis)
Semester/year:- 1/1
Session :- 2020-2021

2. Contents:-
1. Introduction of cheese
2. Casein micelles
3. Preparation of the sample of Cheese for analysis
4. Steps involve in the Determination of Moisture in Cheese
5. Steps Involve In The Determination Of Fat In Cheese
6. Adulteration In Cheese

3. Cheese
 Cheese belong to the food category in dairy slots and
Being available in variety of flavors texture it find its way
to be one of the most known and widely used among
dairy products.
 Talking about Cheesemaking it's a controlled process in
which removing of water from milk is done, that causes
the concentration of milk protein, fat and other nutrients
present in it and there by increases its shelf life.

4. In broader term Cheesemaking involves
coagulating the casein protein.
Separating the milk into solid curds and
liquid whey(The liquid whey is drained away)
Formed curds are salted,
Given shape
Most important and sensitive step ripen in
a controlled environment.
*The selection of type of microorganism only detremine the
type,texture and flavor of cheese
*Microorganism are used in the entire processes of
cheesemaking

5. Casein micelles
 Studies propose that casein micelles consist of a complex of submicelles. The
calcium phosphate and hydrophobic interactions between submicelles
contribute to the stability of the micelle. Kappa casein around the outside
stabilize the micelles against aggregation.
 Key
A: submicelle
B: protruding chain
C: calcium phosphate
D: kappa casein
E: phosphate group

6. Preparation of the sample of Cheese
for analysis

7. Step-1
 In order to analyze cheese
sample, first step is
to remove the rind or
smear or moldy surface
layer of the cheese.
 With the piece of sample
in hand, subject it to Grind
or grate the sample by
means of an appropriate
device (if possible grind it
for second time)
 If the sample cannot be
ground or grated, mix it
thoroughly
by intensive stirring

8. Step-2
 Transfer the grated sample
to an air-tight container to
await analysis(as soon as
possible)
 Transfer the grated sample
to an air-tight container to
await analysis
 I order to prevent
condensation of moisture
on the inner surface of
the container. The storage
temperature should be
maintained below 10°C.

9. Steps involve in the
Determination of
Moisture in Cheese
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.
Apparatus + Requirements
Ø Flat-bottom dishes with lid Dishes of nickel,
aluminium or of other suitable metal (not
affected by boiling water) Dimentions
of the dishes :- 70 to 80 mm in diameter and
not more than 25 mm deep)
Ø Hot air oven
Ø Desiccator
Ø Sand particle size range 500 μ - 179
μ sieve.(prepration of the sand shall
be by digestion with concentrated HCl acid
, followed by thorough washing with
water. It shall then be dried and ignited till
it is dull red)

11. Procedure
Saturate the sand
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.
Weigh accurately 3 g of the prepared sample of Cheese
into a flat-bottomed dish (with a cover) previously dried,weighed containing about 20 g of prepared sand and a
stirring rod.
Heat the flat-bottomed metal dish
containing 20 g of prepared sand and a stirring rod, in
hot air oven for 1 h.
Allow it to cool in desiccator for 30-40 min

12. Repeat the process
Repeat the process of drying, cooling and weighing, until the successive weighing do not differ by more than 0.5 mg. Record the
weight.
Record the weight.
Heat the uncovered dish 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.
Cooling the sample
After 4 h replace the lid, transfer the covered dish to the
desiccator, allow it to cool to room temperature
weigh it accurately and quickly to the nearest 0.1 mg.
water-bath
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.

13. Calculation:-
𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 % 𝑏𝑦 𝑚𝑎𝑠𝑠= 𝑀1− 𝑀2𝑀1−𝑀 𝑥 100
Where,
 M = mass in g, of the empty dish with containing glass rod;
 M1 = Initial mass in g of the dish, lid, glass rod along with the material taken for
analysis;
 M2 = the final mass in g of the dish, lid, glass rod along with the material after
drying.
Sample Types % Moisture-A
(Manufacturere A)
% Moisture-B
(Manufacturere B)
% Moisture –
difference
Natural Mozzarella 47.68 47.80 0.12
Processed
American
38.96 38.97 0.01
Mascarpone 43.41 43.25 -0.16
Feta 55.87 56.11 0.24

14. Steps Involve In The Determination Of Fat In
Cheese

15. Procedure
Weigh accurately 1-2 g
of prepared sample in a
100 ml beaker.
Add 10 ml of conc.
hydrochloric acid.
Heat on a Bunsen
burner, stirring
continuously with a
glass rod, or on a boiling
water bath until all solid
particles are dissolved.
Cool to room
temperature
Add 10 ml of ethyl
alcohol first to the
beaker

16. later transfer the contents
to the Mojonnier fat
extraction flask or the
Rohrig
Transfer to the Mojonnier
fat extraction flask
later transfer the contents
to the Mojonnier fat
extraction flask or the
Rohrig tube Transfer to
the Mojonnier fat
extraction flask
Shake vigorously for one
minute. Add 25 ml of
petroleum ether and
repeat vigorous shaking
for one minute.
Centrifuge Mojonnier
flask at about 600 rpm. (If
Rohrig tube is used, let it
stand until upper liquid is
practically clear)
Wash the tip and the
stopper of the extraction
flask or tube with a
mixture of equal parts of
the two solvents .
and add the washings to
the weighing flask or tube
and
repeat extraction of liquid
remaining in the flask or
tube successively using
15 ml of each solvent.
Add, if necessary, water
to bring its level in tube to
original mark

17. Repeat extraction
using 15 ml of each
solvent.
Evaporate the solvent
completely on water
bath at a temperature
that does not cause
sputtering or bumping.
Dry the fat in oven at
102 ± 2°C to a
constant weight.
Weigh the cooled flask
ormetal dish or glass
bowl
Remove the fat
completely from the
container with warm
petroleum ether and
weigh as before

18. Calculation:-
 Fat, % (w/w) =100 (W1 − W2)W3
Where:-
 W1 = Weight in g of contents in the flask or metal dish or glass bowl
before removal of fat.
 W2 = Weight in g of contents in the flask or metal dish or glass bowl
after removal of fat and
 W3 = Weight in g of material taken for the test.

19. Adulteration In Cheese
 Adulteration of milk/cheese is done accidentally or to obtain illicit profit and
it is typically detrimental to quality
 Water is added to increase the volume of milk, which can increase the risk of
introducing contaminants and to increase the charge of pathogenic
microbes.
 Preservatives are added to increase the shelf-lives of milk and dairy
products, which improves profit.
 Cheaper bovine milk is often mixed with more expensive non-bovine milk
in cheese-making.

20.  Denatured whey proteins fraudulently added to cheese to increase
curd yield are not easily detected.
 Milkfat can be (in part) replaced by partially hydrogenated vegetable
oils.
 Mixtures of short chain fatty acids containing triacylglycerol (e.g.
coconut oil) with deposit fats can be passed off as milkfat.

21. Detection Of Coal-Tar Dyes
 5 ml of dil.H2SO4 or conc. HCl is added in a test tube containing 5ml of
melted sample to cheese and shaken well
 Pink color in case of H2SO4 or crimson color in case of HCl indicates the
presences of coal tar in cheese

22. Vanaspati Or Margarine
 Take 5ml of melted sample of cheese and add equal amount of Conc. HCl to it
.
 Add a pinch of sugar in to it subject it to shaken for 1 min and kept aside for
five minutes
 Appearance of crimson color indicates the presences of vanaspati or
margarine in it

23. Gelatin
 Take 10 ml of sample either melted or dissolve in suitable non-reactive
solvent
 Add 10 ml of acidic mercuric nitrate solution + add 20 ml of water
 Mix properly and filter
 If the filtrate is opaque it indicates the presences of gelatin in it
 Appearance of yellow precipitate after the addition of saturated
aqueous solution of picric acid in the filtrate confirms the presences of
gelatin

24. Urea
 Take 2 ml sample either melted or dissolve in suitable non-reactive solvent
 Add 2 ml of di methyl amino benzaldehyde solution (60ml of alcohol +40 ml
of distilled water +1.6 g di-mi=ethyl amino benzaldehyde + 10 ml HCl
 Place the test tube in hot water bath for few minutes
 Development of distinct yellow color indicates added urea in milk

25. Detergent
 Take 2-5 ml each of a suspect and sample and pure sample/standard in
separate test tubes
 Add 7.5 ml of ethanol to each to the precipitate the protein filter it via
whatman No.1 filter paper
 Collect 2 ml of filteratae from each in the separate test tubes and add 2
ml of methylene blue solution (25mg/100ml water) to each and mix well
 Add 4 ml of chloroform to each and mix well
 Allow the layer of the choloroform to separate
 Compare the color of methylene blue in the choloroform layer extracted
form a suspect sample with that from a pure milk sample
 Appearance of more intense color of chloroform layer in the suspect
sample compare with that in the pure milk sample indicate s the
prescences of detergent.

26. Ammonium compound
 Take 1ml of sample either melted or dissolve in suitable non-reactive solvent in
a test tube add 5ml of 2% sodium hydroxide solution .5ml of 5% phenol
solution heat for 20 sec in boiling water bath
 A blush color immediately forms which turns deep blue in case of sample
containg HCl in ammonium sulphate
 The color remains stable for 12 hours the devlopment of pink color shows the
sample is free from ammonium sulphate

27. Boric Acid /Borax
 Five ml of sample either melted or dissolve in suitable non-reactive
solvent is taken in a test tube add 1 ml of concenterated HCl and mixed
well to of a turmeric paper is dipped into the acidified milk it is dipped in a
watch glass at 100c or over a small flame
 If the turmeric paper turns to red it indicates the prescences of borax or
boric acid
 Conformation can be maded by adding a drop of ammonia solution on
turmeric paper and if the red color changes to green it shows the
prescences of boric

28. Countinuing...
 Traditionally, milk adulterations have been monitored with chemometric
methods.
 More recent approaches, by now considered classic strategies:-include
Elisa
DNA analysis,
Electrophoretic
and chromatographic methods.

29. Method Of Detection Of Melamine In Milk
And Milk product
 A liquid chromatography triple quadrupole tandem mass spectrometry (LC-
MS/MS) method for residues of melamine consists of an initial extraction
with 2.5% aqueous formic acid, followed by a series of filtration,
centrifugation, and dilution steps. The method is used for detection of
both Melamine and Cyanuric Acid using HILIC LC Column.
 Melamine is detected in positive ion mode and cyanuric acid in negative ion
mode. The extracts are analyzed by LC-MS/MS. Analyte concentrations
are calculated using external standard calibration with a standard curve
prepared in a pre-fortified control matrix which has been carried through
the extraction procedure.

30. Determination Of Starch Content In Adulterated Fresh
Cheese Using Hyperspectral Imaging
 The aim of this study was to measure the starch content in adulterated fresh
cheese using hyperspectral imaging
 technique. Adulterated fresh cheese was prepared using concentrations of
starch of 0.055–12.705 mg g−1
 (0.0055–1.2705%); subsequently, hyperspectral imaging in the range of 200–
1000 nm, distributed in 101 bands
 were acquired. The modeling of starch content was performed by the method
of partial least squares regression(PLSR)
 A correlation coefficient (R2) of 0.9915 and a Root Mean Square Error of
cross-validation (RMSECV) of
 0.3979 was obtained. With five latent variables, a correlation coefficient of
validation (R2) of 0.8321 and a
 RMSEP of 1.3515 was obtained for a reduced model.

31. Detection Of Adulteration In Italian Mozzarella
Cheese Using Mitochondrial DNA Templates As
Biomarkers
 Considering the importance of monitoring adulterations of genuine cheeses in
the dairy industry, a polymerase chain reaction–based method was developed to
detect bovine-
 specific mitochondrial DNA sequence in Italian water buffalo Mozzarella
cheese.
 DNA was isolated from cheese matrix and governing liquid by organic
extractions and kit purifications.
 Amplifications of a 134-bp fragment were performed with a bovine–specific
 set of primers designed on the sequence alignment of bovine and buffalo
mitochondrial cytochrome oxidase subunit I. The specificity of the primers was
tested using DNA from the blood of two species (water buffalo and bovine),
which are present together in adulterated Italian Mozzarella cheese.
 This method reliably detected a content of 0.5 % of bovine milk, making it
suitable for routine fraud monitoring.