milk

1. Milk, And MilkAdulteration
• Group Member:
• SONIA NABI
• SIDRA AHMED SIDDIQUE
• BAKHAT RASA MATEEN
MPhil-FoodScience&Technology
UniversityOfKarachi

2. MILK
Milk is an emulsion or colloid of butterfat globules within a water-based
fluid that contains dissolved carbohydrates and protein aggregates with
minerals.
Milk is one of the most important constituents of human diet and is not only an excellent natural
source of carbohydrates, fats and proteins but also of vitamins, minerals and enzymes
Milk is known for its nutritious importance and its role in strengthening of bones, teeth, muscles
and lowering the risk of osteoporosis, osteoarthritis, ischemic heart disease, depression and
cancers

3. IMPORTANCE OF MILK
■ Calcium: Builds healthy bones and teeth;
maintains bone mass
■ Protein: Serves as a source of energy;
builds/repairs muscle tissue
■ Potassium: Helps maintain a healthy blood
pressure
■ Phosphorus: Helps strengthen bones and
generate energy

4. IMPORTANCE OF MILK
■ Vitamin D: Helps maintain bones
■ Vitamin B12: Maintains healthy red blood cells
and nerve tissue
■ Vitamin A: Maintains the immune system; helps
maintain normal vision and skin
■ Riboflavin (B2): Converts food into energy
■ Niacin: Metabolizes sugars and fatty acids

6. IMPORTANT
TERMINOLOGIES
ADULTERATION:
Making the article impure by the addition of some of
the legally prohibited substances into a more
valuable product or the subtraction into a more
valuable component.

7. ADULTERATION OF MILK:
With the increase demand of milk, the adulteration of
this commodity is on increasing day by day. The problem
of adulteration is more during summer and rainy
seasons, when milk production in comparatively much
less than the winter season.

8.  The driving force behind the cause of food adulteration is
the price gap between original and adulterated version.
 The more price gap means the commodity is more likely to
be adulterated.
 The more it is hard to analyze or distinguish the
commodity with its adulterated version the more
likelihood of it being adulterated
Psychology of Food Adulteration

9. Types of Adulteration
The commonly used adulterants are:
• Addition of water.
• Addition of separated milk.
• Addition of thickening agents when milk is watered, it becomes
deficient in density and viscosity which may be restored by
addition of substances like gelatin, cane sugar, starch, glucose.
• Partial skimming.
• Unusual adulterants, a few new types of adulterants like urea,
melamine, ammonium sulphate etc. have come up.

10. Prevalence of Milk Fraud in Chinese market and its
relationship with Fraud vulnerabilities in the Chain
• 1. Study aimed to survey the prevalence of ultra high temperature (UHT) processed milk samples suspected of being
adulterated in Chinese market.
• 2. For this 12 UHT milk samples from five different brands were purchased from local supermarket in Beijing in Nov 2018
as reference sample.
• ✓ Four label as premium quality (protein content>3.4%) from north of china.
• ✓Four of were normal quality also producing from north.
• ✓ Remaining four were of normal quality and produced in south.
• 3. So, accordingly 3 milk pools were prepared first one is mixture of premium quality (Pool A), second one was the mixture
of normal quality(Pool B) from north and the third one was mixture of normal quality(Pool C) from south of China. The
ratio of four milk samples in each pool was 1:1:1:1
• 4. Three milk pools of 100g, were prepared to which adulterant was added at several concentration.
• 5. Twelve reference sample and three milk pools were from major producers considered as control sample(ie. Non
adulterated) .These 15 samples comprised the training set.
• 6. Now 52 commercial UHT milk samples were purchased from local market in Beijing and e-commerce(across china)
during winter of 2018/2019 (test set). Same adulterants and same adulteration level applied.

11. Prevalence of Milk Fraud in Chinese market and its
relationship with Fraud vulnerabilities in the Chain
• 7. A total of 24 adulterant were used categorized into five group named protein rich adulterant, Nitrogen based
adulterant, Carbohydrates based adulterant, preservative and water. Both single and combined adulteration test were
conducted.
• 8. In Combined adulteration 40g water added in 100g of milk pool sample then one of adulterants from either protein,
CHO, nitrogen based added to diluted milk pool to increase apparent protein content with 40% w/w, same with other two.
• 9. Ultimately a total of 288 single adulterated sample and 51 combined adulterated samples prepared These 339
adulterated sample were considered as adulterant test set.
• 10. Milk scan FT120 equipment used to measure milk composition. It is based on FTIR technology and reporta series of
milk compositional parameter namely protein, fat, lactose, total solid(TS), solid non fat(SNF) content, density and freezing
point depression (FPD).
• 11. All measured compositional features of premium milk pool (pool A) were higher than the normal milk pool from both
North and South (pool B & C).
• 12. The protein content of control sample (3.4%- 3.7%) w/w was higher than that of raw milk collected from Chinese
Holstein cattle which is 2.9-3.3% w/w.
• 13. Fat and lactose content of Chinese sample is 4.0% and 5.1% respectively. While in Dutch sample it is low about 3.8%
and 4.7% respectively.

12. Milk
Adulteration and
Its Toxicological
Effects

13. ADULTERANTS
IN MILK
■ Cane sugar
■ Starch
■ Urea
■ Sodium chloride
■ Neutralizers
■ Quaternary ammonium compounds
■ Anionic detergents
■ Skimmed milk powder
■ Formalin
■ Hydrogen peroxide
■ Boric acid and borates

14. FORMALIN
• Formalin is added in milk to preserve it for longer
period.
• Many cases of biological mutation and reproduction
complication have been noted.
• On toxicity it causes tumors, formalin causes CNS
disorders and irritates nose, throat and lungs.
• When taken in high dose it causes cancer and may lead
to coma.

15. UREA
• Urea is generally added in the preparation of synthetic milk to raise the SNF value Protein content.
• Potential symptoms are burning sensation in throat and chest
• Cough, dyspnea, asthma (one case); redness, in eyes and skin, headache; nausea, vomiting, lung
damage, inflammation.
• Affected organs are respiratory system, skin, eyes.

16. Ammonium
Sulphate
 The presence of ammonium sulphate increases the
lactometer reading.
 The product has low toxicity.
 On ingestion small quantities are unlikely to cause toxic
effect and large quantities give rise to gastro-intestinal
disorders.

17. Hydrogen Peroxide
• To enhance the shelf life of milk for consumption without microbial
contamination.
• To minimize the processing cost by heating the milk.
• For the purpose to activate Lacto peroxidase system, which destroy the bacterial
population by its antibacterial effect.
• Lacto peroxidase (LP) system is naturally present in raw milk that prevents the
bacterial multiplication due to its bacteriostatic effect. By activating the LP-system,
shelf life of milk can be increased for 8 to 12 hours.

18. Side Effects of Hydrogen Peroxide
• Eye exposure to dilute hydrogen peroxide may cause extreme irritation and pain but is unlikely to cause
permanent damage.
• Dilute hydrogen peroxide has been reported to cause serious gastrointestinal side effects.
• An overuse of H2O2 can cause damage to DNA cells, and prevent them from replicating, hence leading to
premature aging.
• The use of hydrogen peroxide can cause your skin to dry out or rather drains away the moisture content in skin,
which is required to keep your skin looking young and supple.
• Vitiligo is a disease that is marked by the loss of skin pigment, which affects a lot of people. Studies show that it
is due to the high levels of hydrogen peroxide in the blood and epidermis.
• It can cause irritation in gums and teeth, since H2O2 is a highly reactive compound.

19. Sugar
Table sugar like sucrose is added to the
milk to increase the carbohydrate content
of the milk and thus the density of milk
will be increase.
The milk can now be adulterated with
water.
It will not be detected during the
lactometer test.
Sugar will react with the resorcinol to give
a red colored precipitate, indicating the
presence of Table sugar in milk.

20. SIDE EFFECTS OF SUGAR
• Sugar can suppress your immune system.
• Sugar can produce a significant rise in triglycerides.
• Sugar reduces high-density lipoproteins (HDL).
• Sugar can increase the levels of glucose in the blood much higher than complex carbohydrates in
a glucose tolerance test can.

21. Salt
Addition of salt in milk is
mainly restored with the
aim of increasing the
corrected lactometer
reading.
Excess Salt
Consumption Has
Been Linked To:
 Exercise – induced
asthma.
 Heartburn.
 Osteoporosis. It
can cause
hypertension.
 Uneven heart rate
 Extreme thirst
 Gastric cancer is
associated with
high level of
sodium.
 Hypertension

22. Vegetable
Fat
• It is separated to make the cream and sold at high
prices.
• People separate the cream from the milk and add
vegetable fat into the milk and then sell it after
homogenization.

23. Starch
• Starch is one such component that is added to
adulterate milk.
• Addition of carbohydrate to milk increases its solid
content.
• There by reducing the amount of fat present in the
milk.

24. Soap
• Soap is added to milk to increase the foaming of milk and thus to have thick milk.
• Addition of such chemicals will cause health problem especially related to
stomach and kidneys.

25. Neutralizers
• Added neutralizers (such as NaOH, KOH, carbonates, bicarbonates, ammonia, various
alkalis, etc.) in milk or cream.
• These prohibited substances may be added to fresh or badly preserved milk with the
tricky purpose to correct its pH and acidity values to optimal ones, and so pretend the
milk is freshly milked or perfectly preserved.

26. Quaternary Ammonium Compounds
Purpose:
■ They are used in cleaning agents as a disinfectant.
■ They are used in milk to increase shelf life where bacteria are growing rapidly.
Harmful effects of QAC:
■ Contact dermatitis
■ Triggering of asthma symptoms in people who already have asthma or new onset
of asthma in people with no prior asthma

27. Boric Acid
Purpose
■ Like formalin, boric acid and sodium borates are also common preservatives.
■ They are available in the form of either boric acid (H3BO3) or its salt as sodium
borate(Na3BO3) as commercial chemicals.
Harmful effects of Boric Acid
■ Boric acid is very toxic when taken internally.
■ When boric acid enters the body, it can
cause nausea, vomiting, diarrhea, dermatitis, kidney damage, acute failure of the
circulatory system, and even death.

28. Melamine
• Melamine is added into milk to increase the protein count at less cost.
• Melamine or 2,4,6-triamino-1,3,5-triazine (C3H6N6) is a nitrogen-rich compound which
has been used for producing plastics, adhesives, laminates, paints, permanent-press
fabrics, textile finishes, paper coatings and fertilizer mixtures.
Toxicity
• Causes renal and urinary problems and even infant death when it reacts with cyanuric
acid inside the body

29. https://www.researchgate.net/profile/Masood-
Akhtar/publication/267566928_Adulteration_and_Microbiological_Quality_of_Milk_A_Review/links/5582487608ae12bde6e4b
7b3/Adulteration-and-Microbiological-Quality-of-Milk-A-Review.pdf

32. Quantitative & Qualitative test for
determination of adulteration in milk

33. Methods of Detecting Adulterants in Milk
In general, these classified into detection methods
Qualitative detection methods.
Quantitative detection methods.
Quantitative detection methods are complex and diverse.This
includes biotechnological and electrical methods.
Qualitative detection methods are color based chemical reactions
(biochemical). Qualitative detections are advantageous because
these are simple, rapid and very easy to perform

34. Quantitative ( BIOTECHNOLOGICAL) Tests for
Determination of Adulteration in Milk

35. QUANTITATIVE DETECTION METHODS
The use of modern biotechnology should prove to be rapid,
sensitive and accurate methods for detection and analysis of
adulterants. Type of quantitative detection techniques depend
on the nature of adulterants in milk.

36. 1. LC (Liquid Chromatography) and ELISA
(Enzyme Linked Immunosorbent Assay)
They are the most common techniques used to detect
foreign protein.
In this, MIR (Medium Infra Red) spectroscopy performed better to
detect the adulterants such as tap water, whey, hydrogen peroxide,
synthetic urea and urine. Like urea, synthetic urine is also used in milk
to increase the nitrogen content.

37. 2. PCR (Polymerase Chain Reaction) and PAGE
(Polyacrylamide Gel Electrophoresis)
These methods are usually used to detect milk from different species as
adulterants.
This method identifies molecular masses to differentiate between proteins in
the milk of cow and goat.
Cow milk adulteration in caprine milk has been quantified by HPLC/ESI-MS
(High Performance Liquid Chromatography/Electrospray Ionization- Mass
Spectroscopy)

38. 3. On-line liquid chromatography-gas chromatography (LC-GC)
It has been applied to the detection of vegetable oils in
milk fat using β-sitosterol.

39. 4. Quantitatively melamine detection has
been possible using SERS (Surface
Enhanced Raman Spectroscopy)
SERS is a portable sensor based to detect melamine instantly).
In this, SB-ATR FTIR (Single Bounce Attenuated Total Reflectance - Fourier transform infrared
spectroscopy) has been used to quantify melamine in both liquid and powder milk.
This portable screening system based on Laser Raman spectroscopy has been developed to
quantify melamine
Using Raman band, melamine in dried milk powder has been immediately detected without
extracting melamine from milk (Okazaki et al., 2009).

40. 5. The researchers used Illumina's Solexa
sequencing technology
This method is used to screen for microRNAs in healthy cow milk.
The researchers believe that these microRNAs can serve as biomarkers for reflecting
the quality of milk and dairy products, including milk powder.
Manipulated milk would not be able to replicate the expression levels of these
microRNAs in a standard test
However, mostly such types of detection methods are not accessible at all level of
farmer, milk collection points, or at the spot of dairy cooperatives.

41. Analytical methods to carry out the
control of adulteration
It is based on chromatographic techniques such as;
1. Reversed phase high performance liquid chromatography (HPLC)
2. Gas chromatography coupled to mass spectrometry (GC-MS)
3. Mid-infrared microspectroscopy
4. Nuclear magnetic resonance (NMR)
5. Matrix assisted laser desorption/ionization time of flight (MALDI-TOF)
6. Raman spectroscopy

42. QUALITATIVE (BIOCHEMICAL)DETECTION METHODS
For urea detection
Urea forms a yellow complex with DMAB in a low acidic solution at room temperature.
Presence of hydrogen peroxide
Take 1 ml of milk sample in a test tube. add 1 ml of the potassium iodide-starch reagent and mix well. observe the
color of the solution in the tube. blue colour will be developed in the presence of h2o2, whereas pure milk sample
remain white in color.
FOR PRESENCE OF FORMALIN
Formalin reacts with Sulphuric acid and ferric chloride to give a purple colored ring at the junction of the milk layers.
FOR DETECTION OF HYPOCHLORITE
The chloride test allows to identify any added salt water in the milk, a sophistication that the normal cryoscopic
examination does not detect. This analysis allows to quantify the concentration of salt in products such as cheese,
vegetable purees and sauces, and liquid of processing and storage of dairy products.
FOR DETECTION OF MELAMINE
Formerly in 2008, a national standard method issued by the Chinese authorities (AQSIQ and SAC 2008a) identified
HPLC as the technique for rapid determination of melamine in raw milk. The standard showed a detection limit of
0.05 mg/kg and applied to raw milk and milk-based products without additives.

43. FOR DETECTION OF STARTCH
Starch in milk uses iodine solution, addition of which turns the milk solution to
blue black color due to the formation of starch –Iodo complex, in the presence of starch
FOR DETECTION OF CANE SUGAR
Resorcinol produces red color solution with sucrose in acidic media.
Detection of skim milk powder in milk
As per the law, use of skimmed milk powder (SMP) is not allowed for adjustment of SNF in case of sale of
cow/buffalo or mixed milk. The method is based on the fact that the coagulum obtained from reconstituted
skim milk powder by addition of acetic acid, gives intense blue colour on boiling with phosphomolybdic
acid due to certain reducing groups present in the proteins of milk powder which are able to cause
reduction of molybdenum blue resulting in formation of blue colour.
Detection of vegetable oil in milk
Fat is extracted from milk and its refractive index is determined and compared with that of the pure milk
fat.

44. Big order of Supreme Court on
packaged milk of all brands
Chief Justice of Pakistan Justice Mian Saqib Nisar, on January 13, 2018, ordered the authorities to recall
packaged milk of all brands from the markets of Karachi for testing purpose. Hearing a suo motu case
at the Karachi Registry regarding sale of substandard packaged milk, he ordered the relevant
authorities to conduct laboratory tests of packaged milk.

45. Effectual Gold Nanoprobe Sensor for screening Cow Milk Adulteration in goat
milk-Comparision with PCRArticle in journal agriculture science April
2021Published by Canadian center of science and education
• 1. It is rapid (less than 20 min), reliable and cheap method (4euros/reaction).
• 2. Gold nanoparticles detect target DNA sequences and for it's detection it doesn't need any
instrument or modification chemistry.
• 3.PCR (polymerase chain reaction) reacting with bacterial DNA provides direct and rapid
detection of milk adulteration.
• 4. Procedure is very simple and relies on color changes of 20nm ,if pink color remained means the
result is positive,if color changes into purple means the result is negative and adulteration is
present.
• 5. After visual indications,it is confirmed by absorption spectroscopy which eliminates any sort of
color blindness error.