Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.

Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study

Qualitative And Quantitative Analysis For

Adulteration And Nutritional Value Of Milk: A
Case Study
Sandip Sen1*, Himaja Donthula2,Bairam Ravindar3, Sirikonda Jala4,Chaitanya Mitta5, Swathi
Kalepu6

Abstract
Background: The present work aimed to determine the qualitative and quantitative analysis of the adulteration
and nutritional value of open and packed samples of milk obtained from the local market of Siddipet.
Methods: The different milk samples collected and samples were analyzed qualitatively and quantitatively. The
qualitative study includes an adulteration study and microbial analysis. The quantitative study was carried out by
a UV-visible spectrophotometer for casein and lactose. The fat analysis was carried out by a Gerber butyrometer.
Results: The result indicates water contents are present within acceptable limits as per the official standard. A
detailed adulteration screening was carried out on 50 samples. The samples contain common adulterants like
urea (0%), starch (0%), neutralizers (46%), detergent (26%), sugar (4%), glucose-dextrose (4%), sodium
chloride (32%), hydrogen peroxide (0%), mastitis (34%), formaldehyde (40%), maltodextrin (30%), nitrate- 227
nitrogen (0%). Detailed microbial analysis was carried out on 30 samples (DS1-DS30) namely standard plate
count, lactic acid bacterial count, resazurin test and direct microscopic count. The nutritional value of the best-
quality of milk samples was determined.
Conclusion: All the samples contained the required amount of casein, lactose, and fat as per the FSSAI standard.

Keywords: Milk, Adulteration tests, Standard plate count, Lactic acid bacterial count, Resazurin test and direct
microscopic count, Method development and validation, Lactose, Casein, Fat.
DOI Number: 10.14704/Nq.2022.20.17.Nq88031 Neuroquantology 2022; 20(17):227-237

INTRODUCTION milk processing was carried out under

Dairy production plays an important role in unhygienic conditions and ultimately affecting
supporting livelihoods and economies across the quality and nutritional value of milk2. In
India. As per a survey in 2018, India is the addition to diminishing the nutritional value of
largest milk producer with ~19% of the global a milk-based product, these practices can affect
market share. The milk processing industry in the health of consumers by causing allergies,
India is assumed to increase at a compound nausea, diarrhea, and other gastrointestinal
annual growth rate (CAGR) of ~14.8% between disorders; respiratory irritation; bleeding; kid-
the years 2018 - 2023 and will reach INR ney disorders3.
2,458.7 Bn in the year 20231. India's Export of Generally, the most common adulteration in
Dairy products was 51,421.85 MT to the world milk is water or whey along with added
for a worth of 186.71 USD Million during the nitrogen (e.g., ammonium salts, melamine,
year 2019-20. The demand for milk urea, and non-dairy proteins) to maintain the
consumption in India is directly proportional to protein level caused by the fraud4. Some other
its population. As of the year 2018, ~81.1% of added adulterants to adjust the visual aspects
*Corresponding Author:-Sandip Sen
Address:1*Department of Pharmaceutical Chemistry, School of Pharmacy, Guru Nanak Institutions Technical Campus,
Ibrahimpatnam, Hyderabad-501506.Telangana, India.Email: sandipsen2010@gmail.com
2,3,4Srikrupa Institute of Pharmaceutical Sciences, Velikatta, Siddipet, Telangana-502277
5,6BojjamNarasimhulu Pharmacy College For Women, Saidabad, Hyderabad, Telangana 500059

Relevant conflicts of interest/financial disclosures: The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study
and physicochemical properties of diluted milk,
such as sucrose, sodium chloride, surfactants,
and vegetable oils. To increase the milk shelf
life formaldehyde, hydrogen peroxide,
hypochlorite, dichromate, and salicylic acid is
added as an impurity5.
Around 68.7 percent of the milk and milk
products market in India is not as per the
standards laid down by the Food Safety and
Standards Authority of India (FSSAI). This not
only causes economic loss but is also a major
factor in health concerns6. The local population
does not only consume the milk from Siddipet
but also transports it to surrounding cities like
Karminnagar, Hyderabad, etc. Nevertheless,
handling, sale, and transportation processes
are practiced affecting the overall quality of
milk. Thus the present study is planned to
evaluate the adulterations and composition of
milk sold at Siddipet.
MATERIALS AND METHODS
Adulteration kit (Nice chemicals(P) Ltd. Mum-
bai, India), Lactose Amyl alcohol (Research lab
fine chem. Industries, Mumbai, India), Casein
(Merck), Concentrated sulphuric acid, Sodium
hydroxide, Chloroform, Methanol, Picric acid,
Phenol, Sodium disulfide and Trichloroacetic
acid (TCA) (SD Fine chemicals, Mumbai, India),
Resazurin solution (Sigma-Aldrich, Mumbai,
India),
Milk samples
The 50 unprocessed milk samples were
collected from dairy shops, milk collectors, and
local vendors at different sale points of Siddipet
(Telangana state, India, Location coordinates:
18.1°N 78.85°E) for the qualitative and
quantitative detection of the various
adulterants.
Experimental Section
Adulteration Tests
The qualitative analysis was performed for the
adulteration study by using the standard
procedure given by the Nice milk adulteration
test kit7,8.
Urea
5 ml of milk samples were mixed well with 5 ml
para dimethyl amino benzaldehyde reagent. If
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the solution turns distinct yellow indicates the
presence of urea
Starch
3 ml of milk samples in a test tube, fully boiled
Allow the milk to come to room temperature.
2–3 drops of a 1% iodine solution If the color
changes to blue, this indicates that the milk has
been contaminated with starch.
Neutralizer
5 ml of milk samples were placed in a test tube,
followed by 5 ml of alcohol and 2-3 drops of
rosolic acid. If the color of the milk turns
pinkish-red, it is assumed that the milk has
been contaminated with sodium
carbonate/sodium bicarbonate.
Detergent
With a test tube, place 5 ml of milk and mix in
1-2 drops of bromocresol purple solution. The
presence of detergent in milk is indicated by
the emergence of a violet hue.
Sugar 228
In a test tube, 10 ml of milk was mixed with 5
mL of hydrochloric acid and 0.1 g of resorcinol.
Then, shake the test tube and immerse it in a
boiling water bath for 5 minutes. The presence
of a red hue in milk suggests the presence of
added sugar.
Chloride
Add 2.0 ml of 0.1 N silver nitrate solutions to
5.0 ml of milk sample. Mix well, then add 0.5 of
a 10% potassium chromate solution and check
the color. The presence of dissolved chloride is
indicated by the emergence of a yellow tint.
Glucose and Dextrose
3 ml of milk was placed in a test tube. 3 ml of
Barford's reagent was added and carefully
mixed. Place the test tube in a boiling water
bath for 3 minutes, then immerse it in cold tap
water for 2 minutes. 1 ml of phosphomolybdic
acid was added and mixed. If the blue tint is
apparent, the milk sample contains glucose.
Acidity& heat stability
In a test tube, put 2ml of milk and 2ml of acidity
reagent. Mix the materials well, taking notice of
the color and the presence of any flakes or
clots. The presence of a flake or clot shows that
the milk passed the acidity test. Color lilac (up
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Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study
to 0.14%) (0.14% to 0.17%) faint red (0.17% to
0.20%) reddish-brown to brown (over 0.20%)
brownish yellow to yellow.
Hydrogen peroxide
5 ml of milk was placed in a test tube. Shake in
3 drops of paraphenylenediamine. The change
in the hue of the milk to blue indicates that it
has been tainted with hydrogen peroxide.
Mastitis
In a test tube, place 5ml of milk sample and 1ml
of mastitis reagent, and mix thoroughly. The
hue of regular milk is yellow. Milk from
diseased udders becomes green.
Formaldehyde
Fill a test tube with 10 ml of milk. Without
shaking, pour 5 mL of concentrated sulphuric
acid through the sides of the test tube. If a
violet or blue ring develops at the junction of
the two layers, formalin is present.
Maltodextrin
5ml milk in a glass beaker, boiled and chilled
The milk is then coagulated using a 10%
Trichloroacetic acid (TCA) solution. The filtrate
is obtained after filtering using Whatman filter
paper no.42. Mix 2ml of barium chloride
solution to the filtrate. The presence of
Maltodextrin in milk is indicated by the
emergence of a blue tint.
Nitrate nitrogen
2 g diphenylamine was weighed and dissolved
in sulphuric acid to make a final volume of 100
ml (reagent). Fill a test tube with 2 mL of milk.
Drain the milk from the tube after rinsing it
with it. Along the side of the test tube, place 2-3
drops of the reagent. Take note of the
developed color. When nitrates are present, a
deep blue hue is generated.
Standard Plate Count (SPC) & Lactic Acid
Bacterial Count (LABC)
Milk is serially diluted 10-fold using saline to
reduce the load of microbial growth and to
obtain well-isolated colonies followed by the
spread plate technique. After making dilutions,
plate out 0.1ml onto 10⁻⁴, 10⁻⁵ and 10⁻⁶on
nutrient agar which supports the growth of all
organisms. Incubate the plates for 48 hours at
32°C (90°F) 48hr. Observe and counts the
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colonies from all plates and interpret them by
comparing them to the standard9.
Ten-Minute Resazurin Test
The 5 ml of milk sample was transferred into a
tube containing 1 ml of the resazurin solution.
When the color falls between two disc
numbers, it is recorded because the half value,
for instance, a reading between 3 and 4
recorded as 3S. Tubes giving a reading between
1-3.5 streaky pink and pale pink - are recorded
as 0.510.
Direct Microscopic Count
The direct microscopic method was performed
by using a Trinocular microscope as per the
literature procedure (Yamoto TM-100) at X40
equipped with working station software (D-
View, V 1.0)11. Mix the sample of milk
thoroughly by shaking, if a single 0.01 ml
pipette is used, .rinse it thoroughly in sterile
water (25o to 35°C) between samples. Touch
the tip of the pipette to the center of a one-
square-centimeter area on a slide and expel the
entire volume of milk. After drying, submerge 229
the slides in clear xylene and air-dried. Fix the
film in ethyl alcohol for one minute either by
submerging the slide air-dried completely.
Stain the slides in alcoholic methylene blue
staining solution. Place one drop of immersion
oil on the film and examine it under the oil-
immersion objective. Count the single
organisms or isolated clumps on microscopic
fields. Calculate the average number of clumps
per field and multiply by the microscopic factor
(MF) to give the Direct Microscopic Count per
milliliter.
Method for estimation of casein in milk by
UV-Spectroscopy 12, 13
Milk samples: The sample of milk was
purchased from the local market.
Milk solutions: The milk sample was prepared
in the following concentration of 0.125 g/l for
the UV-219nm.
Extractor solution of chloroform and
methanol: A solution of chloroform-methanol
(2:1 v/v) was prepared and used for the
extraction of casein in milk samples.
Standard Solutions: Casein (Riedel) solutions
were prepared in NaOH 0.1 M and used as a
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Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study
standard in the following concentration, 0.125
g/l for the UV-219.
Assay method for estimation of casein in
milk:
To 5 ml of sample milk, 18.0 ml of the solution
of chloroform-methanol was added. The tubes
were sealed and shaken vigorously for 5 min.
The solutions were filtered using a quantitative
filter paper and the organic phase was
discarded. Then, 6.0 ml of chloroform and 6.0
ml of water were added to the solid samples
and they were shaken for 5 min and filtered.
The filtered solution is diluted with a working
solution up to 10ml and makes serial dilutions
by using the above solution 10-50μg /ml.
Prepare 10-50 μg /ml concentration in 6 spikes
and check the absorbance then interpolate the
values in the standard calibration curve.
Method for estimation of Lactose in milk by
UV-Spectroscopy14,15
All spectral measurements were made on
ELICO SL 244 double beam UV-VIS spectro-
photometer using 1.00 cm quartz curettes.
Digital weighing balance (SHIMADZU AUX 220)
used for weighing.
Milk samples: The sample of milk purchased
from the local market.
Milk solutions: The milk sample was prepared
in the following concentration 1000µg/ml and
make serial dilutions of 10-50µg/ml
Extractor solution by using Trichloroacetic
acid: 5ml of 24%TCA was added to 5ml of milk
and mixed thoroughly used for extraction of
lactose in milk samples.
Reagents: The following stock solutions were
prepared in distilled water: 1% phenol, 5%
sodium hydroxide, 1% picric acid and 1%
sodium disulfite (Na2S205).
Standard Solutions: Lactose solutions were
prepared in a working solution and used as the
standard in the following concentration, 1000
µg/ml for the visible-482.
Methods:
A 5ml of sample milk and 5ml of 24% TCA was
added and mixed thoroughly. The sample was
then filtered through Whatman no. 40 filter
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paper and 1 ml of the clear filtrate was diluted
to 10 ml with a working solution. The Spectro-
photometric methods of visible-482 nm were
used 16.
Method Validation for estimation of casein
and lactose in milk
The proposed methods have been extensively
validated as per ICH guidelines in terms of
specificity, linearity, accuracy, precision,
robustness, ruggedness, limits of detection
(LOD)and quantification (LOQ) 17.
Fat Analysis
Fat content in milk was determined by using
Gerber butyrometer as per the literature
procedure given for the Gerber method7.
Accurately 10 ml of sulphuric acid was added
to the milk sample and allowed the milk level
to fall so that the top of the meniscus is level
with the mark. Add 1 ml of Amyl alcohol, and
close with a stopper. Shake until homogeneous,
inverting it for the complete admixture of the
acid. Keep in a water bath for 5 min. at 65 ± 2°C 230
taking care to have casein particles if any
dissolve fully, and centrifuge for 4 min. at 1100
rpm. After removing the butyrometer tubes
and place them in a water bath for 5 min. at 65
± 2°C.
Statistical Analysis:
The statistical analysis was carried out through
computerized statistical packages i.e. graph pad
prism software (V.7.01, La Jolla, CA, USA).
RESULTS AND DISCUSSION
Though milk is produced throughout the year
addition of water is common practice to extend
the quantity of milk. In a study by Suryasnata
et. Al. 2016 at Hyderabad it was found that
there was a gradual deterioration in the quality
of milk18. In the present study majority of the
sample contain water as a common adulterant.
In a survey in 2015, it was found that 100% of
samples in Hyderabad were admixed with
water19. It not only reduces nutritional value
but also causes major health concerns.
However, if contaminated water is added is a
serious health concern to the consuming
community. Lactometer readings in Figure 1
shows 29 samples have a water content of
about 25 %, whereas 17 samples have 50% and
4 samples have 75% of water content.
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Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study
Figure 1: Percentage of water content present in different
milk samples
The presence of urea in milk overburdens the
kidney as they need to try and do more work to
throw out urea contents from the body. this
might cause kidney failure in some cases. It also
leads to swollen limbs and impaired vision20.
Besides, the areas are additionally harmful to
the guts and liver. that specific milk could also
be obtained from the cattle or buffalo, those
may consume the fodder fertilized with urea,
which can be excreted to exploit21. In the
present investigation, there was no urea was
found as adulteration.
Starch and sugar were added to extend the
thickness of the milk after the addition of
water22. Our study result marked that each one
of the examined milk samples was free from
starch. The absence of starch in examined milk
samples during this study had concordance
with the findings of the earlier study. Rideout
et al. (2008) mentioned that a higher amount of
starch may cause diarrhea due to the
consequences of undigested starch in the colon.
Its accumulation within the body may prove
very fatal for diabetic patients.
The samples also contain some nontoxic
adulterants like Sugar, Glucose, and
maltodextrin. Sucrose is added to the milk to
extend the carbohydrate content of the milk
and thus the density of milk is going to be
increased23. Samples DS 1 and DS 21 contained
sucrose as an adulterant. Glucose is added to
exploit to extend consistency and taste. Poor-
quality glucose is usually added to exploit.
The present study revealed that 46% of milk
sample was adulterated with neutralizer, and
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samples containing neutralizer as an adulterant
may produce severe toxicity. They like to act as
a slow poison for those affected by
hypertension and heart ailments. Sodium
hydroxide deprives the body of utilizing lysine,
an important amino alkanoic acid in milk,
which is required by growing babies. Such
artificial milk is dangerous for about is more
harmful to pregnant women24. Carbonates and
bicarbonates are added to exploit, which may
disrupt the hormone signaling that regulates
development and reproduction. Carbonates in
milk produce gastrointestinal problems
including peptic ulcer, diarrhea, colon ulcer and
electrolyte disturbance25. The samples DS5,
DS12, DS14, DS16, DS17, DS18, DS19, DS20,
DS23, DS24, DS26, DS29, DS30, DS31, DS34,
DS35, DS36, DS 37, DS 40, DS 41, DS 46, DS 50
contain neutralizer as an adulterant.
Detergents are added to emulsify and dissolve
the oil in the water giving the frothy solution,
the characteristic white color of milk26. As per
this study, 26% of milk samples were shown 231
positive for detergents. The octylphenol and
nonylphenol of detergents cause carcinoma
and also decrease sperm production from testi-
cles27. The ICMR (1993) in one of its reports,
states detergents cause food poisoning and
gastrointestinal complications. Sample no DS 5,
DS 14, DS 16, DS 17, DS 23, DS 24, DS 28, DS 35,
DS 38, DS 46, DS 48, DS 49, and DS 50 contain
detergent as an adulterant.
Chlorine is added to compensate for the density
of the diluted milk after the addition of water.
Chlorinated can cause clogging in arteries and
develop heart problems. The addition of
formalin as an adulterant in milk causes
vomiting, diarrhea, and abdominal pain28, 29. It
also results in a decrease in body temperature,
shallow respiration, weak irregular pulse and
unconscious. It also causes blindness by
damaging the optical nervous and it's
carcinogenic in nature and peroxide causes
gastritis, and inflammation of the intestines
and results in bloody diarrhea. The result
shows samples DS 3, DS 5, DS 6, DS 7, DS 8, DS
10, DS 11, DS 13, DS 16, DS 23, DS 33, DS 34, DS
44 DS,46 DS47, DS 48 contained chlorine as an
adulterant.
Drinking milk having mastitis, won't harm you.
The milk from the affected breast could also be
a bit saltier than normal, but it's safe to drink
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Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study
but considered an adulterant. Sample no DS 3,
DS 4, DS 5, DS 6, DS 7, DS 8, DS 14, DS 16, DS 37,
DS 38, DS 44, DS 45, DS 46, DS 47, DS 48, DS 49,
DS 50 contained mastitis as an adulterant.
Generally, formaldehyde is used as a milk
preservative, when utilized in the proportion of
1 part formaldehyde in 10,000, milk remained
fresh without curdling for seven days. But long-
run exposure may cause liver toxicity. The
samples like DS 1, DS 2, DS 5, DS 11, DS 12, DS
13, DS 17, DS 23, DS 24, DS 26, DS 27, DS 28, DS
29, DS 30, DS 35, DS 36, DS 37, DS 39, DS 41
contained formaldehyde as an adulterant.
Maltodextrin can be utilized in dairy foods or
soya milk. This product improves the nutrition
function, the standard and flavor are often
enhanced and it also can reduce the value of
the products30. Samples DS 1, DS 2, DS 3, DS 7,
DS 8, DS 9, DS 11, DS 12, DS 13, DS 42, DS 43,
DS 44 DS, DS 48, DS 49, and DS 50 contained
maltodextrin as an adulterant.
Figure2 contain common adulterant like Urea
(0%), Starch (0%), hydrogen peroxide (0%),
Nitrate nitrogen (0%), Neutralizers (46%),
Detergent (26%), Sugar (4%), Glucose-
dextrose (4%), common salt (32%), Mastitis
(34%), Formaldehyde (40%), and Maltodextrin
(30%).
50
% of Adulteration
indicate an increased risk of pathogenic micro-
organisms and are not suitable for
consumption. The presence of formaldehyde
and detergents in milk (packet samples) shows
less growth of microorganisms because these
reagents will inhibit the growth of
microorganisms. The poor handling and
unhygienic transportation of milk (open
samples) showed the high growth of micro-
organisms32, 33.
The basic principle behind the resazurin
reduction test is the disappearance of the pink
color of the resazurin solution due to bacterial
metabolite. Due to the presence of microbes,
the amount of oxygen is reduced in the milk.
The amount of oxygen is inversely proportional
to bacterial count34. So if the microbial count is
more the disappearance of color will be faster.
The resazurin test usually measures the
metabolic reaction that takes place at the cell
surface of bacteria.
Detailed microbial analysis was applied on 30
samples (DS1-DS30) namely Standard plate
count, lactic acid bacterial count, Resazurin 232
test, and direct microscopic count Table 1. The
standard plate count accustomed determines
the entire number of bacteria present in a
specified amount of milk, usually a milliliter.
The coliform plate count is widely used to
determine the entire number of coliforms
present in one mL of milk sample.
The result represents in, showing excellent
results (DS4, DS6, DS19, DS29, DS30), nine

40

30 samples are showing good results (DS3, DS5,

DS7, DS8, DS11, DS20, DS21, DS27, DS28), five
20
samples showing fair results (DS12, DS14,
10 DS15, DS22, DS24) and 11 samples showing
0 poor results (DS1, DS2, DS9, DS10, DS13, DS16,
DS17, DS18, DS23, DS25, DS26). lactic acid
al a

et ch

ex r
se Su t

ro e
St rs

N alt eh s
at de e
tr n
ge h se

rm a e

en
n

M ald iti
a

pe id
tr re

itr o yd
Fo M xid

ni ri
e

ge

ro C tro
-d g
D ar

bacteria (LAB) are among the foremost

og
n lor

st
iz

e xt
eu U

er

significant groups of probiotic organisms,

N

co
lu

yd

commonly utilized in fermented dairy

G

Adul terant
Figure 2: Percentage of adulterants in different milk
samples
The standard plate count and lactic acid
bacterial count (SPC and LABC) are used to
confirming the presence of bacteria under
aerobic conditions at 30 and 35°C. The bacteria
are generally obtained from different stressed
conditions during handling, storage and
processing31. The higher value values of SPC
products. Among other benefits, these
microorganisms can enhance lactose digestion,
stimulate the system, and prevent and treat
diarrhea.
The samples DS3, DS6, and DS19 showed
excellent results, and 12 samples are showing
good results (DS1, DS2, DS4, DS5, DS7, DS11,
DS12, DS20, DS21, DS24, DS27, DS30), eleven
samples are showing fair results (DS8, DS10,
DS13, DS14, DS15). Resazurin reduction test
results for the samples DS1, DS4, DS7, DS8,

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Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study
DS12, DS15, DS20, DS 22, DS24, DS25, DS27 shown pale pink color with doubtful results,
and DS28 showed pink color with satisfactory eight samples were shown streaky pink color
quality, ten samples (DS3, DS5, DS6, DS10, with unsatisfactory results (DS2, DS9, DS11,
DS13, DS14, DS17, DS19, and DS30) were DS16, DS18, DS21, DS23, DS29).

Table 1: Results of Standard Plate Count, Lactic acid-producing bacterial count and ten minutes of Resazurin test
Stand plate Lactic acid Stand plate Lactic acid
Sample Resazurin test Sample Resazurin test
bacterial bacterial bacterial bacterial
code Color code Color
count (Cfu) count (Cfu) count (Cfu) count (Cfu)
DS 1 80×107# 1×106* Pink +++ DS 16 5×107 # 3×106 Streaky pink++
DS 2 120×107# 2×106* Streaky pink ++ DS 17 Overgrowth # 2×106 Pale pink+
DS 3 2×105* 1×105** Pale pink+ DS 18 Overgrowth # 3×106 Streaky pink++
DS 4 1×105** 2×105* Pink+++ DS 19 1×105** 1×105** Pale pink
DS 5 2×105 * 2×105* Pale pink+ DS 20 2×106 * 2×106 * Pink+
DS 6 1×105 ** 1×105** Pale pink+ DS 21 1×106 * 2×106 * Streaky pink++
DS 7 2×105 * 2×105* Pink+++ DS 22 4×107 † 2×107† Pink+++
DS 8 1×106 * 1×106† Pink+++ DS 23 Over growth # 5×107# Streaky pink++
DS 9 Over growth # 3×107# Streaky pink++ DS 24 3×103 † 2×103 * Pink +++
DS 10 Overgrowth # 3×107† Pale Pink+ DS 25 Overgrowth # 4×107# Pink +++
DS 11 1×106 * 1×106 * Streaky pink++ DS 26 Overgrowth # 3×107† Pale pink+
DS 12 1×107 1×106* Pink+++ DS 27 2×105 * 2×105 * Pink +++
DS 13 Overgrowth # 2×107† Pale pink+ DS 28 2×106 * 1×106† Pink +++
DS 14 3×107† 1×107† Pale pink+ DS 29 1×105 ** 4×105# Streaky pink ++
DS 15 1×107† 1×107† Pink+++ DS 30 1×105** 1×105 * Pale pink+
Control - - Control - -

# poor, *good, ** very good † fair ,+: Doubtful, ++: Unsatisfactory, +++ : Satisfactory
233
The direct microscopic count study was
administered on four samples namely DS2,
DS4, DS8, and DS13. The result presented in
Figure3indicated the presence of many cocci
and rods in clumps and patches in sample DS2
indicating improper cleaning of utensils. The
effect of the dusty and dirty environment was
found in sample DS4 by the presence of
excessive numbers of rod-shaped bacteria. The
effect of storage is often predicted by the
The bacterial microscopic count was found for sample DS
presence of a huge number of cocci in pairs or 4 was found to be less than 5 lakhs clumps count per ml
short chains in sample DS8. Mastitis infection and can be considered as good quality milk
was found in sample DS13 because of the
presence of a huge number of leucocyte cells.

The bacterial microscopic count was found for sample DS

The bacterial microscopic count was found for sample DS 8 was found to be more than 5 lakhs to 40 lakhs clumps
2 was found to be less than 5 lakhs clumps count per ml count per ml and can be considered as a fair quality milk
and can be considered as good quality of milk

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Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study

The bacterial microscopic count was found for sample DS

13 was found to be more than 5 lakhs to 40 lakhs clumps
count per ml and can be considered as a fair quality milk
Figure 5: Standard calibration curve of casein
Figure 3: The Results of Direct Microscopic Count
Similarly, the lactose content is determined by
Therefore, from the above results, it is often colorimetry at 482 nm (Figure 6) using 1 %
concluded that packed samples have better phenol, 5% NaOH, 1% acid,1 % sodium
quality to open samples concerning bisulfate (Table 2 and Figure 7). The
adulteration screening and microbial analysis. extraction was carried out by using 24 %
Whereas open samples are better compared to trichloroacetic acid. the tactic showed linearity
packed samples in respect of added chemical at 10-50 μg/Ml, Coefficient of correlation (r2)
adulterants. Thus, it had been further decided 0.999 and a couple of RSD values but 2%. Total
to work out the nutritional value of the fat content was decided by using Gerber's
simplest quality milk samples. So the butyrometer reported previously7.
234
determination of nutritional value (casein,
lactose and fat) were determined for DS3, DS6,
DS7, DS11, DS21, and DS27.
Before the estimation of casein and lactose
content in milk, the method was developed and
validated as per ICH guidelines. The casein
content was decided at 219 nm(Figure-4)
using 0.1M NaOH. The extraction was carried
out by using chloroform and methanol 2:1
(v/v). the tactic showed linearity at 10-50
μg/Ml, Coefficient of correlation (r2) 0.999 and
2 RSD value but 0.5 % (Table 2and Figure 5).
The method developed was used for the Fig 6: λmax of lactose at 482nm by UV-spectroscopy
estimation of Casein content in milk samples(
(DS3, DS6, DS7, DS11, DS21, DS27). The
samples contained 97-100 % of casein. 0.25
Y= 0.003x+0.002
R2 =0.999
0.20
Absorbance (nm)

0.15

0.10

0.05

0.00
0 10 20 30 40 50 60
Concetration (g/ml)

Figure 7: Standard calibration curve of lactose

Figure 4: λmax of casein at 219nm by UV-spectroscopy

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Neuroquantology | November 2022 | Volume 20 | Issue 17 | Page 227-237 | Doi: 10.14704/Nq.2022.20.17.Nq88031
Sandip Sen, D Himaja, Bairam Ravindar, Sirikonda Jala, Chaitanya Mitta, Swathi kalepu, Qualitative And Quantitative Analysis For Adulteration
And Nutritional Value Of Milk: A Case Study
Table 2:Summary of Validation Parameters for the Proposed Method for estimation of casein and lactose
Parameters Results of casein Results of lactose
Absorption maximum (λmax) 219nm 482nm
Beer’s Law limit (μg/ml) 10-50 μg/ml 10-50 μg/ml
Slope 0.009463 0.003379
Intercept 0.4169 0.008370
Coefficient of correlation 0.999 0.999
Accuracy (%recovery) 99% 99%
Repeatability 0.174 & 0.183% 1.29 & 1.41%
Reproducibility 0.016 & 0.212% 1.41 & 1.85%
Precision (% RSD)
Intermediate
0.174&0.279% 1.22 & 1.41%
precision
Robustness (% RSD) 1.015 & 1.53% 1.22& 2.0%
Ruggedness(% RSD) 0.174& 0.279% 1.22& 1.41 %
Limit Of Detection (LOD) 0.178 μg/ml 0.234 μg/ml
Limit Of Quantification (LOQ) 0.452 μg/ml 0.542 μg/ml

The method was used for the estimation of
casein, lactose and fat content in milk samples
DS3, DS6, DS7, DS11, DS21, and DS27 (Table 4).
The samples contained 62-100 % of lactose and
97-100 % of casein and 5-8 % of total fat.
Table 3: Estimation of casein, lactose and fat in milk
The reason behind this might be due to the
presence of added water in the milk already
confirmed by the lactometer reading. So from
the above discussion, we can conclude that
milk was collected from different sources of
Siddipet but was not authentic as per the
standard of FASSI.
CONCLUSION
From the above discussion, it is often
concluded that the sample obtained from the
local market contained extraneous water. But
differing types of adulterants are found both in
packed and opened samples namely
neutralizers (46%), detergent (26%), sugar
(4%), glucose-dextrose (4%), chloride ion
(32%), Mastitis (34%), Formaldehyde (40%),
Maltodextrin (30%). The packed samples
contain these adulterants like neutralizers,
detergents, sugar, chloride ion and glucose-
dextrose, formaldehyde. Where few open
samples contained this adulterant. The most
often found adulterant in the open sample was
mastitis. But microbial analysis indicated the
presence of more bacteria in the non-
pasteurized open sample. the precise reason is
235
unknown but could also be because of
improper storage, utility and condition even
animal hygiene could also be liable for this.
Though no such samples were recognized that
are freed from adulterants but based on the
comparative analysis (presence of detergent,
neutralizer, microbial analysis) the nutritional
values were determined for DS3, DS6, DS7,
DS11, DS21 and DS27. All the samples
contained the required amount of casein,
lactose, and fat as per the FASSI standard. So
we will conclude nutritional value is not a
matter of great concern but human hygiene and
safety.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGMENTS
The authors are also thankful to the Srikrupa
institute of pharmaceutical sciences, Siddipet
and School of Pharmacy, Guru Nanak
Institutions Technical Campus, Ibrahimpatnam.
for providing the necessary facilities for the
research work.
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