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Preservation of Milk and Milk Products


for Analytical Purposes
a a a a
Neelam Upadhyay , Ankit Goyal , Anil Kumar , Darshan Lal Ghai &
a
Richa Singh
a
Dairy Chemistry Division, National Dairy Research Institute, Karnal,
Haryana, India
Accepted author version posted online: 18 Apr 2014.Published
online: 26 Jun 2014.

To cite this article: Neelam Upadhyay, Ankit Goyal, Anil Kumar, Darshan Lal Ghai & Richa Singh (2014)
Preservation of Milk and Milk Products for Analytical Purposes, Food Reviews International, 30:3,
203-224, DOI: 10.1080/87559129.2014.913292

To link to this article: http://dx.doi.org/10.1080/87559129.2014.913292

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Food Reviews International, 30:203–224, 2014
Copyright © Taylor & Francis Group, LLC
ISSN: 8755-9129 print / 1525-6103 online
DOI: 10.1080/87559129.2014.913292

Preservation of Milk and Milk Products


for Analytical Purposes

NEELAM UPADHYAY, ANKIT GOYAL, ANIL KUMAR,


DARSHAN LAL GHAI, AND RICHA SINGH
Dairy Chemistry Division, National Dairy Research Institute, Karnal, Haryana,
India
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Milk is a complete food containing high-quality protein, the only source of lactose,
and an excellent source of calcium. Thus, milk is prone to adulteration and to com-
bat this menace, in India, the government appoints food safety officers who carry out
chemical examination of milk and milk products. Formalin is the only legally permit-
ted preservative for milk and milk products, in India, meant for analytical purposes.
Scientific reports suggest that formalin interferes in maintaining the compositional pro-
file; since accuracy in analysis of milk components is very important, these changes
can be problematic. The other preservatives that have been described in the litera-
ture include mercuric chloride, potassium dichromate, hydrogen peroxide, bronopol,
and azidiol Most of these preservatives are bactericidal but either involve a health haz-
ard or environmental hazard or are unsuitable for long-term storage. Hence, there is a
need to uncover combinations of antibacterial agents and bacteriostatic antibiotics for
preserving milk and milk products until analyzed.

Keywords Chemical preservatives, Food safety officer, Formalin, Hydrogen peroxide,


Milk and milk products

Background and Introduction


Consumers need nutritious, wholesome, pure, and safe food. In recent years, consumers
have placed increased emphasis on food safety. Governments worldwide have regulated
foods with two general objectives: first, to ensure the safety and wholesomeness of the food
supply and, second, to prevent economic fraud or deception. These objectives encompass
such concepts as safety, purity, wholesomeness, and value.
Milk and milk products contribute a very significant proportion in our daily diet. Milk,
considered to be closest to the nature’s perfect food, is an excellent source of calcium, a
good source of minerals and high-quality proteins, the only source of lactose, and a source
of lipids, the most valuable component, which also forms the basis of milk pricing. India is
the largest milk producing country in the world, with 127 million tonnes(1) of milk produced
annually. Out of total milk production, ∼70% is utilized in the form of liquid milk and
various indigenous dairy products such as chhana, curd, khoa, paneer, and ghee (milk fat).
In India, milkmen are paid on the basis of milk fat, whereas in industry the basis is both,
milk fat and solids not fat (SNF) content. Thus, milk is more prone to adulteration by

Address correspondence to Neelam Upadhyay, Research Scholar, Dairy Chemistry Division,


National Dairy Research Institute, Karnal, Haryana 132001, India. E-mail: neelam_2912@yahoo.
co.in

203
204 Upadhyay et al.

the addition of vegetable oils, starch, sugar, and flour to adjust its composition illegally.
Addition of urea, detergents, and pond water in sour and spoiled milk is also in practice
to make it fit for processing and consumption. These practices lead to economic losses,
deterioration of the quality of end products, and a risk to consumers’ safety.(2)
To control such type of malpractices and ensure food safety and security, governments
appoint food safety officers who carry out the chemical examinations of milk and milk
products and other foods also. As milk and milk products are very perishable and deterio-
rate rapidly due to high moisture content, chemical preservatives are added to milk samples
so that the composition does not change until the analysis. Various studies have been con-
ducted that reported the effects of chemical preservatives on the composition of milk and
milk products preserved for analytical purposes,(3−10) but the major studies describing var-
ious preservatives and their limitations have not been previously described. The present
review deals with the various types of preservatives and their effects on milk components
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during preservation of milk and milk products meant for chemical analysis.
Chemical milk preservation has been practiced for a long time since milk fat test-
ing became a standard and routine exercise. Several substances and formulations have
been used in the past, but the search for the ideal milk sample preservative continues. The
search for preservatives depends upon the purpose for which it is required. Different types
of preservatives may be required in different situations. If the sample requires short-term
preservative, for example, for carrying the sample from the farm to the laboratory for anal-
ysis, a preservative providing small increase in shelf life will suffice. On the other hand,
in situations where samples are collected from the market by food authorities and sent
to a food laboratory for analysis and when disputes require the intervention of referral
laboratory, a preservative providing longer shelf life is required.
Accuracy of milk component analysis is very important to all dairy farmers and
the dairy industries. Many factors contribute to the variation of the parameters ana-
lyzed for payment purposes. The most important factor is the method used for sample
preservation.(11) Although some organic preservatives such as formaldehyde, toluene, or
hydrogen peroxide have been described in the literature,(12) the first chemical preservatives
used in developed countries were potassium dichromate, mercuric chloride, boric acid, or
combinations of these.(13) Some newer preservatives, such as bronopol or sodium azide,
have also been recommended.(14) Most of the chemical preservatives are bactericidal in
nature.
Coliform bacteria and staphylococci are more sensitive to the effect of sodium
azide than mesophilic bacteria, enterococci, and lactobacilli,(14) whereas enterococci are
more resistant to chloramphenicol than Salmonella sp., Esherichia. coli, Lysteria sp., and
Staphylococcus aureus.(15) Research dealing with combinations of antibacterial agents and
bacteriostatic antibiotics such as chloramphenicol and nitrofurazone brought out new mix-
tures that were good enough to preserve milk for 3 days without affecting the instrumental
analysis of milk by MilkoScan, BactoScan, and Fossomatic systems. One of these mixtures,
Azidiol (containing chloramphenicol and sodium azide), was most commonly used in all
milk-testing laboratories in Spain.(16)

Sampling Procedure for Chemical Examination


Food safety officers carry out periodic inspections of premises in which manufacture,
process, or business in milk or any milk product is carried out, with a view to ensure com-
pliance with the provisions of the Food Safety and Standards (FSS) Rules and Regulations,
2011.(17) A representative sample is collected so that the analysis will furnish reliable data
Preservation of Milk and Milk Products for Analysis 205

Table 1
The quantity of sample of food to be sent to the food analyst/director for analysis

Approximate quantity to be
Article of food supplied
Milk 500 mL
Sterilized milk/UHT milk 500 mL
Malai/Dahi 200 g
Yoghurt/Sweetened dahi 500 g
Chhana/Paneer/Khoya/Shrikhand 250 g
Cheese/Cheese spread 200 g
Evaporated milk/Condensed milk 200 g
Ice cream/Softy/Kulfi/Ice candy/Ice lolly 300 g
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Milk powder/Skimmed milk powder 250 g


Infant food/Weaning food 500 g
Malt food/Malted milk food 300 g
Butter/Butter oil/Ghee/Margarine/Cream/Bakery 200 g
shortening
Vanaspati, edible oils/Fats 400 g

representing the sample. A food safety officer takes a sample and divides it into four equal
parts in separate containers, which are marked and sealed. One sample portion is sent for
analysis to a food analyst, whereas two parts are held in safe custody in case a retest is
required, and the remaining portion is sent for analysis to an accredited laboratory. The
laboratory to which the sample is sent reports the results of the analysis to the registering
authority.
Under the Food Safety and Standards Authority (FSSA) of India, samples of milk
and milk products for analysis are taken in clean, dry bottles or jars or in other suitable
containers that can be tightly closed to prevent leakage, evaporation, or in the case of dry
substance, entrance of moisture. A food safety officer taking a sample of any food analy-
sis under the Act adds a preservative to the sample to maintain it for analysis. Whenever
any preservative is added to a sample, the identity and quantity of the preservative added
is clearly noted on the label affixed to the container. The quantity of sample of food to
be sent for analysis is specified in Table 1.(17) Thus, the food samples collected by the
food safety officer for analysis are required to be kept for a long time. The time varies
from 3 to 6 months and sometimes longer. Sometimes, the food samples, especially some
prepared foods, are found to be decomposed by bacteria or mold when they reach the
laboratory. Such samples are declared unfit for analysis and rejected. Therefore, for the
proper maintenance of the perishable food samples, use of a suitable preservative is a
necessity.(18)

Chemical Preservatives
“Preservative” for milk may be defined as any chemical compound or process that, when
applied to milk, prevents alterations caused by growth of microorganisms. The ideal means
of preserving milk samples for several days prior to compositional analysis is refrigera-
tion at as near the freezing point as possible without freezing the product. This minimizes
206 Upadhyay et al.

growth of bacteria and all the chemical and physical changes following such growth. Ice
crystal formation usually leads to disruption of physical structures in the milk, and makes
it difficult to withdraw representative portions. An ideal preservative assumes and assures
protection from mechanical fat globule disruption (churning), which results in free-fat for-
mation, as milk containing free fat is not suitable for accurate or reproducible subsample
withdrawal. Churning of fat in milk samples is prevented by minimal agitation. For samples
to be transported, minimal agitation is achieved by total filling of container, such as plastic
bag packaging, without air inclusion.
The minimum requirement for a suitable chemical preservative for milk sample is that
it must assure “testability of sample.” This means that the milk sample maintains its origi-
nal composition from the time of milking to the time of analysis and that the preservative
does not affect the outcome of testing. Other requirements for a satisfactory chemical
preservative are described as follows(19) :
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1. Broad-spectrum activity. A critical necessity is the pernicious ability of the preservative


to act against all types of microorganisms in milk. The broader the spectrum of the
preservative, the better is its utility.
2. Efficient minimum inhibitory levels. The preservative should be effective at low
concentrations in milk to minimize sample dilution, costs, and expedite handling
procedures.
3. High water solubility. Because the average milk sample has about 87% water, it is
imperative that the preservative has the capacity to function against the microorgan-
isms in the aqueous phase; high water solubility also assures easy miscibility without
excessive agitation.
4. Stability. The preservative must be stable under most storage conditions.
5. Presence of color. Unlike cosmetic preservatives, a desirable quality of a milk
preservative is that it imparts some color to the treated milk sample for identification
and safety concerns.
6. Compatibility. A good milk preservative should be just as effective in pooled milk
samples as in fresh samples from individual cows or other mammals and be suitable
for high fat as well as nonfat milk samples.
7. Shelf-life activity. The milk preservative should be effective in milk for a period of
6 months to 1 year (the expected time interval involved in litigations from the stage
of sampling till the stage of final chemical analysis through public health laboratory at
primary level and referral laboratory at the secondary level).
8. Toxicity and disposability. The preservative should be nonallergenic and exhibit no
demonstrable toxicity towards handlers or others coming in contact with it. Despite
its necessary biocidal property, it should not become an environmental hazard after
disposal.
9. Economy. Cost should be minimal and the preservative should be readily available.
10. Dispensing ability. Dispensing in solid form, as in tablets, would be preferable to
the liquid form, because of the inherent difficulty in handling liquids and the greater
accuracy in dispensing solids.
The most commonly used preservative in the case of samples of any milk, includ-
ing toned, separated, skimmed, and standardized milk, chhanna, cream, ice-candy, dahi,
khoa, or khoa-based and Paneer-based sweets, such as Kalakand and Burfi, is the liquid
commonly known as “formalin” (about 40% of formaldehyde in aqueous solution) in the
proportion of 0.1 mL (2 drops) for a 25-mL or 25-g sample. In case of samples of ice cream
and ice cream mix, the amount of preservative used is 0.6 mL for 100 mL or l00 g.(17) Under
Preservation of Milk and Milk Products for Analysis 207

IS 1479 part I,(20) if any sample is to be used for cryoscopic examination, mercuric chloride
is permissible as a preservative, provided the bottle is properly labeled as “POISON.”

Preservatives for Preserving Liquid Milk for Analytical Purposes

Mercuric Chloride (HgCl2 )


Mercuric chloride (HgC12 ), also known as corrosive sublimate, was approved in the United
States as a preservative of raw milk samples. It is poisonous and corrosive, but is an effective
preservative. It was often sold as a tableted mixture with other oxidizing agents, and usually
is available in two tablet sizes of about 0.5 and 1 g. About 46.7% of a tablet is HgC12 , the
remainder is inert filling and binding material such as sodium chloride, acacia, and mineral
oil.(21)
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By the end of the 1960s, the use of HgC12 as milk preservative was discontinued
due to the perception of environmental hazards related to mercury, which gets converted
to methyl mercury and consequently contaminates the micro-life, and ultimately harms
human health. Also, its relatively high toxicity (median lethal dose = 37 mg/kg, orally in
rats) makes HgC12 an occupational hazard. Furthermore, its corrosive nature contributes to
material wear and tear in laboratories performing instrumental analysis.(22)

Potassium Dichromate (K2 Cr2 O7 )


After the ban of HgCI2 , potassium dichromate (K2 Cr2 O7 ) was adopted as the preferred
milk sample preservative. It is relatively noncorrosive. Potassium dichromate appears as
bright orange-red due to the formation of the dichromate ion (Cr2 O7 −2 ). It is not hygro-
scopic or deliquescent (as compared with sodium dichromate). Pharmaceutically, it is used
primarily as an oxidizing agent. It was selected for Milko Tester, an electronic device that
rapidly determines fat content, use, and is also the accepted preservative for milk samples
in Germany where recommended amounts are 0.1–0.2%. The average weight of a tablet is
100 mg, containing 41 mg of the active ingredient. Consequently, 1 tablet in 100 mL milk
amounts to about 0.04% of K2 Cr2 O7 .
Concentrations of 0.08–0.80% K2 Cr2 O7 were effective in maintaining the fat content
of milk samples tested with the Milko Tester Automatic when stored at 21–27 ◦ C.(22) Fat
content depression greater than 0.1% occurred after the fifth day. This effect became more
pronounced as storage time increased. It could be due to changes in the physical structure
of the aging milk samples. Preservative concentration was thought to have no influence on
depression of fat results.(21)
Herd milk samples were tested with K2 Cr2 O7 in concentrations of 0.3–0.4% for up
to 20 days under refrigeration.(23) However, differences were observed between differ-
ent breeds. With Jersey samples, fat losses were demonstrated sooner than with individual
samples from Holstein-Friesian cows. Acidity developed in samples held at ambient tem-
perature in prolonged storage.(23) In a comparative study using K2 Cr2 O7 preservative,
similar results were obtained for estimation of fat content by Gerber method and the Milko
Tester Automatic-MK-III and it was observed that K2 Cr2 O7 had no adverse effect on the
analysis. However, formaldehyde, used in a related study, proved unsatisfactory.(24)
Problems associated with K2 Cr2 O7 are that the samples preserved with K2 Cr2 O7 even-
tually get sour and coagulate. This is accompanied by milk changing from yellow to
gray-green, an indication of reduction, most likely by reducing bacteria or by acidic con-
ditions, of the orange hexavalent chromium anion in dichromate (Cr2 O7 2− ) to the trivalent
208 Upadhyay et al.

chromium ion, which may appear as green Cr3+ or Cr(OH)4− in solution. Such milk cannot
be accurately analyzed for fat by the Milko Tester.
Although K2 Cr2 O7 has proven through laboratory and field tests to work well as a milk
sample preservative, it is occasionally pointed out as problematic. Its molecular structure
contains “chromium VI” (hexavalent chromium), which is a known irritant in some people
and causes rhinitis, nosebleed, and ulcerated nasal mucosa. It is also a potent sensitizer
for allergic dermatitis. Varying degrees of eczema have been reported in persons exposed
to “chromium VI.”(25) Since it is biocidal, problems in sewage systems will occur with
massive disposal.

Formaldehyde (HCHO)
Formaldehyde is commercially available as a 37–40% aqueous solution known as formalin.
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On storage, HCHO may form polymers, namely paraformaldehyde (CH2 O)n , which is
mostly in the form of trimers, known as meta-formaldehyde or trioxymethylene (CH2 O)3 .
These trimers show no reducing properties, since no free –CHO is available. In order to
inhibit the polymerization of formaldehyde, 10–15% methanol is added to the commer-
cially available formalin. Formalin, being a storage antiseptic and disinfectant, is used in
the preservation of various anatomical species and also foodstuffs. Being a known car-
cinogen, its use for edible purposes is prohibited; however, it has been permitted in India
for the preservation of milk and milk products meant for chemical analysis at a level of
0.1 mL/25 mL, i.e., 0.4% (v/v, v/w) of milk or milk products.(17,26) Formaldehyde acts
through alkylation of amino, carboxyl, or hydroxyl group, and probably damages nucleic
acids. It inactivates all microorganisms, including spores.
Use of formalin at such level has shown variable results regarding its ability to preserve
samples. Various reasons, such as use of substandard formalin, storage period, storage tem-
perature, and nature of the sample preserved, have been proposed to explain this variability.
The preservative has also been shown to affect various physicochemical properties as well
as the fat, protein, lactose, and total solid contents during storage. This has created a prob-
lem and brought industry in direct conflict with the regulatory agencies. Many research
workers have studied the compositional changes in milk samples as affected by formalin
preservation.(27) These are described below.

Effect of formalin on physicochemical properties of milk and milk products

Effect on acidity. Most researchers have reported that an immediate increase in acidity
of milk sample occurs on addition of formalin, which continues during subsequent storage.
This increase in acidity has been attributed to formalin-amino reactions. Dawood et al.(28)
found that addition of 0.1% formalin to milk increased the titratable acidity from 0.175%
to 0.190%. Fahmi et al.(29) found an increase of 0.05% in acidity of milk on addition of
5 ppm formalin. Similarly, the initial increase in acidity of milk, according to Bansal and
Singhal,(30) was proportional to the level of formalin added and was due to liberation of
H+ ions, but on continued storage beyond 5 months, the increase in acidity was attributed
to higher proteolytic activity in samples with the lower level of formalin studied. Likewise,
Karmakar and Ghatak(31) observed an increase in acidity in milk from 0.126% to 0.130%
with addition of 0.4% formalin, which continued to increase to 0.185% after 30 days.
Gupta and Gupta(32) studied the compositional change in cross-bred and local cow milk
as affected by 0.3% and 0.5% formalin preservative. Addition of formalin increased the
acidity, whereas casein percentage decreased in both types of milk. The extent of decrease
Preservation of Milk and Milk Products for Analysis 209

during storage was similar in samples containing 0.3% and 0.5% formalin. No signifi-
cant difference was recorded in lactose, total solids, fat, and specific gravity on addition
of formalin in milk, fresh as well as during storage up to 48 hours. It was concluded that
0.3% and 0.5% formalin was best for preservation of milk samples for chemical examina-
tion only. In contrast, Sandhu et al.(33) reported that addition of 0.4% and 0.6% formalin to
milk stored at room and refrigeration temperatures had no significant changes in acidity up
to 1 year of storage.

Effect on viscosity. An increase in viscosity of milk samples with different levels of


formalin has been reported by Bansal and Singhal,(30) who observed that after 2 months of
storage, the buffalo milk samples became so viscous that it was not possible to determine
their viscosity by Ostwald viscometer. Similarly, Bajaj and Rai(34) showed an increase in
viscosity of buffalo milk preserved with 0.4% formalin after 21 days at 30 ◦ C.
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Effect on cream layer formation. Sandhu et al.(33) reported that during storage of sam-
ples at 37 ◦ C in dark, a cream plug appeared within 24 hours. There were three distinct
layers in the samples, the top layer of cream plug, middle layer of liquid, and bottom layer
of semisolid material. However, on blending, these layers mixed readily and the resulting
samples were stable. These layers were more distinct in case of buffalo milk samples as
compared with the corresponding cow milk samples.

Effect of formalin on milk constituents

Effect on fat content. Low-fat values in the formalin-treated milk samples when ana-
lyzed by Gerber method have been observed by several workers. Sharma and Sarwar(35)
reported that fat contents of formaldehyde-treated milk samples could not be determined
accurately by original Gerber method. Certain modifications such as prolonging the heat-
ing of milk in butyrometer at 65 ◦ C and repeated centrifugations were suggested, which,
however, gave enormously high values. They recommended that formalin should not be
used for milk samples meant for chemical analysis by Gerber method. However, Karmakar
and Ghatak(31,36) have reported no change in Gerber fat values in the milk samples pre-
served for varying periods to 1 year at room temperature with 0.4% and 0.6% formalin.
To get accurate results of fat test by Gerber method, Raj and Singhal(37) have suggested the
use of increased percentage of H2 SO4 in Gerber acid to 95% instead of 90–91%. In another
study,(35) milk was treated with four enzymes, namely, pronase, trypsin, rennin, and nutrase,
to break down the protein to affect their dissolution under Gerber test. Reproducible results
were obtained with Gerber test using pronase enzyme, whereas no reliable results were
found in case of Rose-Gottlieb analysis.
Contradictory reports are available for estimation of fat by Rose-Gottlieb/Mojonnier
method. Bansal(38) reported that the levels of formalin did not significantly affect the fat
values determined by the Rose-Gottlieb method, although a steady decrease in fat values
was observed on storage of the preserved milk samples. Bajaj and Rai(39) showed that
there was no immediate impact of formalin addition on Mojonnier fat test, but there was
a constant decrease during storage. The decrease was due to hardening effect of formalin
on protein that entangles some of the fat, which is difficult to recover on treatment with
ammonia.
There are differences of opinion on the effect of formalin in milk samples on results
of Milko Tester. Several workers reported that formaldehyde did not alter the results of
fat determination by Milko Tester.(40,41) However, reports of some workers(38,39) indicated
210 Upadhyay et al.

the variability in the Milko Tester fat tests due to the presence of formalin as the milk
preservative.
Effect on protein content. It is an established fact that formaldehyde reacts with amino
groups of protein to form a harder formal-protein complex. Bector and Narayanan(42) and
Bansal(38) reported no change in protein either on addition of formalin or during storage
up to 6 months with Kjeldahl method, the variations were within the experimental error.
However, formalin caused an immediate decrease in protein content of cow and buffalo
milk by the dye-binding method. These values further declined up to 1 month and there-
after became more constant in both types of milk. The difference between the percentage
decrease in protein content of cow and buffalo milk samples was due to the difference in
dye-binding capacity (DBC) of different fractions of protein. The decrease in protein values
is due to blocking of active sites on protein by formalin. The active sites are responsible
for forming the protein-dye complex. A steady state after 1 month of storage was reached
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because the available active sites were saturated with formaldehyde and equilibrium was
achieved.(34,43)
Bajaj and Rai(34) reported that formaldehyde adversely affected the protein con-
tent determined by the Lowery method. The values further decreased on storage and
became constant after 1 month of storage. The decrease in protein was due to reaction
of formaldehyde with tyrosine. However, Kleczkowaski et al.(44) observed that accuracy of
Folin-Ciocalteu method was not affected when the milk was preserved with formaldehyde.
Effect on lactose content. Formaldehyde is known to have an effect on fat and protein
estimation. However, lactose content did not change, either on addition of formalin or dur-
ing subsequent storage for up to 6 months at 30 ± 2 ◦ C,(34) because of the fact that lactose
did not play any role towards increase in acidity in formalin-treated milk. The increase
in acidity of milk is merely due to formalin-amino reactions. Many workers(33,34,38,45)
reported no change in lactose by Lane-Eynon method in formalin-preserved samples during
storage up to 1 year.
The lactose contents of milk samples with added formalin measured by the picric acid
method showed an initial decrease followed by erratic results for lactose content. The possi-
ble reasons may be (a) the incomplete precipitation of protein, (b) the absorption of lactose
by the coagulated protein, or (c) extensive hydration of protein coagulum, which resulted
in concentration of lactose in the serum.(34)

Effect of formalin on microbiological quality of milk. Milk is prone to rapid microbial


spoilage because of its high nutrient (such as lactose in readily available form) content and
high water activity. Improper or delayed chilling or long-term storage may lead to increased
growth of bacteria in raw milk, causing spoilage of milk products.(46) Formalin interacts
with the amino groups of adenine, cytosine, and guanine in the nucleic acid component,
denaturing them and resulting in the inhibition of the growth of microorganisms.(47) Milk
samples preserved with 0.4% formalin for 6 months did not show any mold growth.(42)
Similarly, Sandhu et al.(33) preserved milk samples satisfactorily for 1 year at 37 ◦ C
with 0.4% or 0.6% formalin. Bansal and Singhal(30) showed that formalin-preserved cow
and buffalo milk samples were almost free of bacterial growth for 1 year. Karmakar and
Ghatak(31) stated that formalin at the level of 0.4% is a good preservative for milk at refrig-
eration temperature. Effect of formalin on methylene blue reduction time (MBRT) test has
been studied by Fahmi et al.(29) They reported that addition of 50 ppm formalin increases
the MBRT to 7 and 9 hours for buffalo and cow milk, respectively, whereas 100–1000 ppm
inhibited the reduction for more than 24 hours.
Preservation of Milk and Milk Products for Analysis 211

Hydrogen Peroxide (H2 O2 )


H2 O2 is a strong oxidizing, bleaching, and germicidal agent. The germicidal properties of
H2 O2 have been known since its discovery by the French chemist Thenard in 1818.(48) The
first experiments in preserving milk with H2 O2 were made as early as 1883.(48) Hydrogen
peroxide (H2 O2 ) is the traditional preservative to inhibit microbial proliferation and milk
spoilage. One milliliter of a 23% H2 O2 solution in 300 mL raw milk is capable of main-
taining decreased bacterial counts during 6 days storage at 8–10 ◦ C.(49) However, because
H2 O2 solutions are liquid and have relatively short-term effectiveness, this preservative is
not suitable for samples held long term.
The antimicrobial efficiency of H2 O2 is largely dependent upon the type of organ-
ism and the initial quality of the raw milk (i.e., the level of microbial contamination and
the somatic cell count). In general, gram-positive lactic acid bacteria are more resistant to
H2 O2 treatment than gram-negative bacteria.(50,51) Also, the antimicrobial action of H2 O2
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is determined by the concentration used, temperature, and duration of treatment. The quan-
tity of H2 O2 required for significant inhibition of bacterial growth is small and does not
make any appreciable change in the total volume of milk; hence, it is of no consequence
in terms of percentage of fat and proteins. Most of the H2 O2 added is decomposed by
the catalase-positive microorganisms. Its decomposition yields water and oxygen. In dilute
H2 O2 solutions, the oxidizing effect is reduced. Generally, the influence of H2 O2 on milk
is intensified by an increase of the temperature and prolongation of the period of treatment.
For use in preservation of milk, the H2 O2 solution must be of analytical quality and free
from metallic or other impurities.

Effect of H2 O2 on milk sugar and butterfat. Giolitti(52) found no changes for lactose, fat,
total nitrogen, and pH after the addition of 0.04% by weight of H2 O2 to milk. According
to other experiments, the lactose content of peroxide-treated milk was somewhat lower
than that of untreated sample.(53) The sugar content of untreated milk was 5.01%, but after
treatment with 0.01%, 0.02%, 0.04%, and 0.08% by weight of H2 O2 at 30 ◦ C for 16 hours,
the values were 5.01%, 4.95%, 4.95%, and 4.60%, respectively.

Effect of H2 O2 on casein. H2 O2 in higher concentration oxidizes proteins, resulting


in formation of aldehydes, ketones, and acids, which are known sources of increased
acidity.(54) Dilute peroxide solutions do not show this effect. H2 O2 treatment of pure casein
solution (0.1–0.4% by weight) increases the proportion of nitrogen not precipitated by
trichloroacetic acid and reduces the viscosity of the solution.(55) This may be why H2 O2
treatment (0.04% by weight) of milk increases the albumin content and decreases the casein
content.(52)

Effect of H2 O2 on whey proteins. Beta-lactoglobulin (β-Lg) shows similar changes to


those noted with casein. The storage of skim milk to which has been added 0.4% by weight
of H2 O2 for 7 days at 4 ◦ C resulted in a complete breakdown of β-Lg into a component of
lower molecular weight. At lower peroxide concentration, the effect was reduced. Solutions
of pure crystalline β-Lg appeared to be more resistant to dilute H2 O2 solution than the
natural β-Lg in milk.(56)

Effect of H2 O2 on shelf life. Dahiya and Speck(57) observed an inverse relationship


between acid production and H2 O2 concentration, due, perhaps, to an inhibition of
the activity of lactic acid bacteria. Romani(58) tried preservation of milk with different
212 Upadhyay et al.

concentrations of H2 O2 and reported that milk can be kept at 5 ◦ C for 24–35, 32–40, and
up to 100–110 days by the addition of 0.4%, 0.8%, and 1.2% by weight of H2 O2 , respec-
tively. Similar results have been reported by Negretti(59) who found that milk could be
preserved up to 39 days at 28 ◦ C with 1% H2 O2 . Cheese whey was preserved for more
than 10 days by the addition of 0.02% by weight of H2 O2 soon after separation.(49) Milk
samples preserved with 0.05% H2 O2 at refrigeration temperature, and analyzed for protein
(by Kjeldahl method), fat (by precipitating casein by ethanol and extracting fat by diethyl
and petroleum ether), and lactose (by copper reduction method) showed that the chemical
parameters remained almost the same for a period of 20 days.(60)

Toxic aspect of H2 O2 in dairy. The effect of H2 O2 treatment on preservation of the con-


stituents of milk is less marked than that of other accepted processes applied in the dairy
industry. The milk does not lose in nutrition value, apart from a small decrease of some
vitamins and a considerable loss of ascorbic acid.(49)
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Sodium Azide
Sodium azide is a colorless, odorless, crystalline solid (salt-like) or solution. It is solu-
ble in water or liquid ammonia, and slightly soluble in alcohols. Coliform bacteria and
Staphylococci are sensitive to sodium azide. It is effective in stabilizing milk samples used
for measuring fat and protein content.
In one study, the use of sodium azide for the preservation of raw milk samples led
to low results for fat and protein as determined by the Foss MilkoScan. The reductions
averaged 0.036% for fat and 0.020% for protein for additions of 1 tablet/30 mL milk with
correspondingly greater reductions for higher additions. The origin of such discrepancies
was traced to the use of NaCl (200 mg/tablet) as a filler. Equivalent amounts of sucrose
showed the same effect. The researchers suggested that this problem could be corrected
by calibrating the instrument against a milk of known composition containing the same
amount of azide in tablet form as the preserved sample.(61) According to these studies,
sodium azide was very effective in stabilizing milk samples used for fat and protein content
through various methods. Sanchez et al.(62) found that an increased level of sodium azide
caused freezing point depression of milk samples.
Caution: The major problem associated with sodium azide is that it is capable of react-
ing with metals in waste system pipes within laboratory buildings and in sewage systems,
producing spontaneous explosions.(22) It is unlikely that a chemical of such explosive nature
would be looked upon with favor by milk-testing laboratories.

Azidiol
Azidiol is commonly used in all milk-testing laboratories in Spain. It has two components:
sodium azide and chloramphenicol. Coliform bacteria and Staphylococci are sensitive to
sodium azide, whereas Salmonella sp., E. coli, Listeria sp., and S. aureus are more sensitive
to chloramphenicol (citation).
Barcina et al.(63) added 0.1 mL azidiol (containing 12 mg sodium azide and 0.5 mg
chloranphenicol/100 mL milk) as a preservative to 30 mL of milk in two sets, one of which
was kept at 4 ◦ C and the other at 20 ◦ C (Fig. 1). Milk samples were analyzed for fat, pro-
tein, and lactose by the infrared method by using a MilkoScan.(64) All the samples were
preserved with azidiol at 4 ◦ C during the storage time for analysis of fat, lactose, and pro-
tein. Samples stored more than a week at 20 ◦ C showed significant alteration (P < 0.01) in
fat and lactose components of milk compared with the control with no azidiol (first day
Preservation of Milk and Milk Products for Analysis 213

Figure 1. Changes of lactose content in milk stored with azidiol at 4 ◦ C (•) and 20 ◦ C () and
without preservative at 4 ◦ C () and 20 ◦ C ().(63)
© Anales de Veterinaria. Reproduced with kind permission of Anales de Veterinaria. Permission to
Downloaded by [Laurentian University] at 21:30 07 December 2014

reuse must be obtained from the rightsholder.

Figure 2. Changes of protein content in milk stored with azidiol at 4 ◦ C (•) and 20 ◦ C (◦) and without
preservative at 4 ◦ C () and 20 ◦ C ().(63)
© Anales de Veterinaria. Reproduced with kind permission of Anales de Veterinaria. Permission to
reuse must be obtained from the rightsholder.

of analysis). However, the protein component was not modified when milk samples were
stored during 15 days with azidiol at 20 ◦ C prior to analysis (Fig. 2). Samples for chemi-
cal analysis with azidiol could be preserved for more than 15 days at 4 ◦ C and at least for
a week at room temperature (about 20 ◦ C). Pintic-pukec et al.(65) measured the freezing
point of milk samples preserved with azidiol and observed that the average freezing points
of azidiol-preserved samples were significantly (P < 0.05) lower as compared with that of
control samples.

Bronopol
Bronopol (C3 H6 BrNO4 ; 2-bromo-2-nitropropane-1,3-diol) is a preservative extensively
used in the cosmetics industries in Europe and USA and has been briefly studied as a
milk preservative.(66–70) Bronopol is a formaldehyde-releasing preservative particularly
because of its high activity against gram-negative bacteria,(71) especially Pseudomonase
aeruginosa. It is stable for a minimum of 1 year under normal storage conditions and
no photodecomposition is reported.(72) However, it decomposes more rapidly at elevated
214 Upadhyay et al.

temperatures and alkalinity to generate decomposition products that include formaldehyde,


nitromethane, nitrous acid, glycolic acid, hydrogen bromide, oxides of nitrogen, formic
acid, and methanol.(73) At lower concentrations (0.01–0.02%), bronopol does not cause irri-
tation or skin allergic reactions in humans(74) ; at higher concentrations, it has been shown
to produce skin irritation(75) and allergic dermatitis on dermal exposure.(76,77)
In dairy laboratories, use of bronopol is widespread, replacing other preservation
strategies.(78) A lot of studies have been done on the compositional effects of milk samples
preserved with bronopol. It has several advantages over the use of other preservatives; for
example, it is noncorrosive, nontoxic, stable at normal storage conditions, and does not pro-
duce any color when added in milk, unlike mercuric chloride and potassium dichromate.(79)
Bronopol received considerable attention when it was used in its traditional and well-
established antibacterial concentration of 0.02% (by weight), as it performs efficiently as a
sample preservative and does not interfere with the normal testing procedures.
Ruttan(68) developed bronopol-based antimicrobial composition for the preservation
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of milk samples that had 2-bromo-2-nitropropane-1,3-diol (6–12 mg) and natamycin


(0.3–0.6 mg) in effective amounts to prevent spoilage of the milk sample (50 mL) for a
period of at least 5–10 days. It was found that when natamycin was used in combination
with 2-bromo-2-nitropropane-1,3-diol, the milk samples did not require refrigeration and
often worked as well or better than potassium dichromate as preservative. Natamycin is
a commercially available yeast and mold inhibitor currently used to prevent their growth
in refrigerated food products, such as cheese. More specifically, it is a polyene antifungal,
antibiotic produced by Streptomyces natalensis and by S. chattanoogensis. At a weight ratio
of 10–40:1 of 2-bromo-2-nitropropane-1,3-diol to natamycin, it was optimal for preserving
milk samples. The natamycin remains on the top of the milk sample and does not mix with
the milk. It is therefore ideally placed to control the growth of yeast and mold, which com-
mences growth at the milk surface. Bronopol, on the other hand, mixes with the milk and
acts as an antibacterial agent. The control of the growth of yeast and mold at the surface
helps to eliminate bacteria in milk and thus enhances the action of bronopol.(68)
Sanchez et al.(80) evaluated the influence of bronopol (0.04 g/100 mL) and storage
temperatures (4 and −20 ◦ C) on somatic cell count (SCC) and composition of goat milk
and reported no significant difference in total proteins, lactose content, and total solids
up to 42 days in milk samples stored at 4 ◦ C. For the bronopol-preserved milk samples
at 4 ◦ C, relative decreases in SCC from storage times of 24 hours to 10, 25, or 42 days
post collection were 5%, 6.9%, and 15%, respectively. However, bronopol-preserved goat
milk samples stored at −20 ◦ C showed no variations in SCC during the study. In bronopol-
preserved sheep milk stored at refrigeration temperature, SCC values were reduced by 2.8%
from 3 hours to 9 days post collection.(81) Cow milk samples preserved with bronopol and
stored for longer periods at 4 ◦ C showed a decrease in the SCC of 10% and 16% at 6 and
10 weeks post collection, respectively.(82)
Higher milk fat (4.43% vs. 4.37%) and protein (3.7% vs. 3.67%) contents were
reported in cow milk preserved with bronopol compared with that of potassium
dichromate–preserved samples(78) and unpreserved samples.(83) Sanchez et al.(80) in
contrast, observed no significant differences in fat or protein content in case of bronopol-
preserved goat milk samples and concluded that goat milk samples preserved with
bronopol could be stored for 25 and 105 days at refrigeration and frozen temperature,
respectively.
Ardo(66) compared bronopol with methylene blue and sodium dichromate–preserved
milks and concluded that milk samples preserved with bronopol should not be analyzed by
ether extraction (i.e., Rose-Gottlieb); but in connection with instrumental methods for fat
Preservation of Milk and Milk Products for Analysis 215

and protein, preservation with bronopol is a very good alternative to sodium dichromate.
Currently, gas-liquid chromatography (GLC), high-performance liquid chromatography
(HPLC), mid-infrared analyzers, and flow cytometry are widely used analytical techniques
in establishing purity of milk samples, both qualitatively as well as quantitatively.(78,83,84)
Butler and Stergiadis(84) studied the suitability of bronopol-treated milk for fatty acid deter-
mination. They reported that milk treated with bronopol could reliably be used to evaluate
fatty acid composition in most cases; however, it might influence the concentration of a few
long-chain fatty acids present in relatively low concentrations.
Barbano et al.(79) studied the effect of bronopol (0.02%) on the accuracy of cow milk
components by mid-infrared testing and reported a significant difference in all compo-
nents, with the largest effect on protein content. Conversely, no change in fat and protein
readings was detected over 4-week periods, but a significant change (i.e., 0.02–0.03%) in
lactose reading occurred.(85) Sanchez et al.(62) used five different preservation strategies—
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no preservative, preservation with azidiol (0.006 or 0.018 g of sodium azide/100 mL), and
preservation with bronopol (0.020 or 0.040 g/100 mL)—in analyzing freezing point of
milk samples by MilkoScan. They concluded that the regression coefficient was maximum
(0.894) for the analytical conditions when the milk samples were preserved with bronopol
at 0.020 g/100 mL.
Sierra et al.(86) showed that bronopol-preserved milk samples stored at 4 and 10 ◦ C for
24 hours had lower log IBC (individual bacterial count)/mL values (6.343 ± 0.0199 and
6.312 ± 0.0199, respectively) compared with the azidiol-preserved samples at time 0. They
recommended that bronopol should not be used in goat milk samples when total bacte-
rial count (TBC) determination was done using the automated flow cytometry method.
One of the drawbacks of using bronopol is that at consistently high temperatures, the
product becomes unstable; it must be refrigerated to work properly. The use of 2-bromo-2-
nitropropane-1,3-diol therefore has not proven to be entirely successful and there is still a
need for an improved antimicrobial agent for preserving milk sample.

Sodium Omadine
Chemically, sodium omadine, a fungicide, is sodium-2-pyridinethiol-1-oxide. At 0.04%, it
is as effective as potassium dichromate in preserving raw milk held at 24 ◦ C up to 1 week
for fat and protein testing.(19) During the second week of storage, results were found to
be less reliable. At 32 ◦ C, the protein readings in both sodium omadine and potassium
dichromate–treated samples were less stable during the first week and even less consistent
thereafter.

Comparative Studies of Different Preservatives in Milk


The reports discussed above show the effect of individual preservatives on milk preserved
for analytical purpose. As can be observed from the reports, none of the preservatives is
suitable in all cases. Some preservatives are suitable either for a particular milk compo-
nent (such as proteins, fats, lactose) or for a specific test (e.g., Kjeldahl test for protein,
Gerber test for fats). Although formalin is the only preservative that showed the maximum
suitability in most analytical tests, there are still a few limitations as reported by several
researchers. By considering all these factors, various workers have performed compara-
tive studies to examine the effect of preservatives singly and in combination. Some of the
comparative studies are discussed below.
216 Upadhyay et al.

One study(87) compared the effect of the preservatives (a) formalin with 4%
paraformaldehyde, (b) 0.25% K2 Cr2 07 and 0.25% paraformaldehyde added to 250 mL
milk, and (c) 0.5% HgCl2 on density, fat content (by Gerber method), and acidity of raw
milk samples. The study showed that the three treatments were suitable for milk analysis,
although a slight increase in acidity was observed in all three treatments.
An analysis of several preservatives showed that K2 Cr2 07 (0.1%) and HgCl2 (0.1%)
were the best chemicals for stabilizing measurements of density and percent fat in analytical
milk samples for up to 4 months. However, with chloroform (2.0%), formaldehyde (0.04%),
and trioxymethylene (0.4%) as preservatives, changes occurred in the above-mentioned
parameters after 2 months of storage.(88)
Another study compared several preservatives in fresh milk samples, including 40%
formaldehyde solution at 0.1–0.3 mL/100 mL milk, saturated K2 Cr2 O7 solution at the same
concentration, Cumasin (a nontoxic commercial preservative) at 2 tablets/100 mL milk,
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and 6.4% solution of HgCl2 in ethanol. Analyzing the samples in 10-fold replication for fat
(by Milko Tester), protein (Pro-Milk method), and total solids (by Total Milk Solids Tester)
revealed that K2 Cr2 O7 at 0.3 mL/100 mL milk was generally suitable for all measurements.
Formaldehyde was unsatisfactory for fat and protein determinations but good for total solids
(TS) at 0.3 mL/100 mL milk. The HgCl2 treatment was reasonably satisfactory for protein
evaluation, but was considered unsuitable for Pro-Milk testing because of its relative tox-
icity and corrosive nature when in contact with metal parts of the testing apparatus.(89)
In a similar study using the Milko Tester MK-III and preserving fresh milk samples for
3 days at ambient temperature (20–23 ◦ C) with K2 Cr2 O7 , HgCl2 , or formaldehyde, results
indicated that over the longest periods of storage only the dichromate-preserved samples
yielded satisfactory results.(90)
Jandal and Rai(91) compared three preservatives, formalin (0.4%), HgCl2 (0.4% and
0.6%), and 1:1 HgCl2 and K2 Cr2 O7 (0.4% and 0.6%), for effects on color, clot-on-boiling,
acidity, fat, solids-not-fat (SNF), and loss of protein and whey proteins. During storage at
30 ± 1 ◦ C and 5–8 ◦ C for 90 days, they observed a small variation of 0.03–0.06 units in fat
content of milk samples preserved with various preservatives at refrigeration temperature,
except in the case of 1:1 HgCl2 and K2 Cr2 O7 , where an appreciable decrease in fat content
was observed. However, this decrease was greater at 30 ± 1 ◦ C (0.28–0.32 units). There was
no significant effect of the two storage temperatures on SNF content. Thus, they concluded
that all the preservatives except the mixture of HgCl2 and K2 Cr2 O7 can be used for the
determination of fat (modified Gerber method) and SNF content.
Seskena and Jankevica(92) conducted an experiment to assess the feasibility of sodium
azide, hydrogen peroxide, bronopol, azidiol, boric acid, and potassium sorbate as milk
preservatives and their influence on the quality and composition indices of raw milk sam-
ples. It was reported that 0.02% sodium azide, 0.4% azidiol, 0.04% bronopol, and 0.5%
potassium sorbate had no effect on fat and protein contents at 4 and 20 ◦ C, although a sig-
nificant decrease in fat content with hydrogen peroxide at 20 ◦ C without affecting protein
content was observed. The decrease in fat and protein was up to 0.31% and 0.74%, respec-
tively, in the presence of boric acid as preservative due to reaction between boric acid and
–CH groups of lipid molecules and –NH group of protein.
Barbano et al.(79) determined the effect of K2 Cr2 O7 and bronopol on the accuracy
of fat, protein, and lactose content determination in milk by mid-infrared (mid-IR) milk
analysis and reported that K2 Cr2 O7 had little or no effect on mid-IR test results. All
bronopol-based preservative approaches in the study differed in mid-IR test results com-
pared with K2 Cr2 O7 -preserved and unpreserved milks, with the largest effect on protein
content.
Preservation of Milk and Milk Products for Analysis 217

Studies on Preservation of Milk Products


Traditional milk products play a very important role in social, economic, and nutritional
well-being. The importance of dairy products has been recognized since Vedic times, since
they are considered to be a complete food.(93) In India, about 50% of the total milk is used
to prepare dairy products such as curd, butter, paneer, cheese, and ice cream.(94) These
products are perishable on account of high water activity and readily available nutrients.
Their preservation is also of considerable interest.
Mukherjee and Mathew(18) found that spraying formalin on cheese, chhanna, and khoa
(at the concentration of 0.4%) allowed the samples to be stored successfully for more than
10 months. Harridans and Narayanan(3) reported that paneer could be preserved well in
sterilized and stoppered glass bottles at 30 ◦ C for 6 days with the addition of formalin
(0.4% v/w). However, after 21 days of storage, the samples became moldy and there were
decreases in fat and total nitrogen content and increases in water-soluble nitrogen, tyrosine,
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and acidity of sample. Ramaiah and Narayanan(95) observed that addition of formalin at a
level of 0.4% preserved samples of plain ice cream (at 25–30 ◦ C) for 2 months, although
they observed an increase in acidity of formalin-preserved ice cream samples after 2 months
of storage at 25–30 ◦ C. Further storage caused appreciable decreases in total solid, fat, and
sucrose contents. Therefore, it was recommended that ice cream samples preserved with
0.4% formalin should be analyzed within a period of 2 months to check its compositional
standards.
A study on preservation of khoa at 35 ± 1 ◦ C with different levels of formalin (at
0.05, 0.10, 0.20, 0.25, and 0.30 mL per 25g of sample) was carried out by Dinakar and
Sharma.(96) There was minimal effect on fat ranging from 0.19% to 0.05% decrease,
whereas there was no effect on total protein. The water-soluble protein content increased
more at 0.05 mL of formalin per 25 g of sample. Lactose content remained steady for
2 months, followed by a decline, which was greater at formalin levels of 0.05 mL per 25 g
of sample. A dose of 0.2 mL of formalin per 25 g of khoa was found to be most effec-
tive in controlling the development of acidity from hydrolysis of lactose, volatile acidity,
and water-soluble protein. No significant effects were observed on moisture, fat, and total
protein content; however, vitamin A and vitamin C levels decreased. They suggested that
0.2 mL of formalin per 25 g of sample was more effective in preserving khoa samples
meant for analysis.
Belewu et al.(5) studied the effects of ginger extracts, propionic acid, and sodium ben-
zoate on shelf life of cheese samples at ambient temperature. It was observed that 0.8%
propionic acid and 0.8% sodium benzoate extended the shelf life of the samples until the
ninth day of storage, whereas the control sample spoiled on the second day. Titratable acid-
ity increased from 0.20% to 0.27% in cheese samples treated with ginger extracts. The
extended shelf life of samples treated with ginger extract could be due to its antioxidant
properties. Similarly, the use of 0.8% propionic acid and 0.8% sodium benzoate resulted in
the preservation of cheese for 8 days.(97)

Conclusions
The suitability of different preservatives for different milk components and
physicochemical properties is described in the review (Table 2). There are four types of
results for each method and each type of preservative mentioned in Table 2: (1) suitable
in all cases; (2) suitable in some references and not in others, the differences being that it
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Table 2
Preservative suitability for different milk components/properties
Parameters Preservatives
(milk
components/ Mercuric Potassium Formaldehyde/ Hydrogen Sodium Sodium
properties) chloride dichromate formalin peroxide azide Azidiol Bronopol omadine
Proteins NR  (76)  (38, 42, 44) (49, 57) (58, 89) (60, 89)  (59, 77, 83, X (19)
X (32, 34) 89)
X (75)
Lipids  (88)  (21, 76)  (31, 32, 36, (49, 57) (58, 89) (89)  (59, 83, 89) X (19)
40, 41) X (60) X (75)
X (35, 38, 39,

218
88)
Lactose NR  (76)  (32, 33, 34, (49, 57) (58) X (60)  (59, 77) X (19)
38, 45) X (50) X (83)
Acidity NR X (23)  (33) (49) (58) NR NR NR
X (28, 29, 30,
31)
Volume NR NR  (32) NR (58) NR NR NR
Specific NR NR  (32) NR NR NR NR NR
gravity
Viscosity NR NR X (30, 34) NR NR NR NR NR
Note. The numbers in parentheses indicate the references. NR = not reported;  = no significant change reported by authors for a particular parameter
against a corresponding preservative; X = different results (increase or decrease) reported by authors for a particular parameter against a corresponding
preservative.
Preservation of Milk and Milk Products for Analysis 219

is suitable in one type of analysis (e.g., protein by Kjeldahl method) but not for protein
methods using dye-binding; (3) there is conflict in the literature about the suitability for
the same type of analysis; and (4) not suitable in all cases. It was observed that most
studies reported using formalin and bronopol, with a very few reports on preservation with
mercuric chloride, potassium dichromate, and sodium omadine. Limited applications of
mercuric chloride can be attributed to its corrosive and highly toxic nature. Although potas-
sium dichromate, in most of the studies, did not affect the milk composition significantly,
its use is limited on account of being a known skin irritant, causing allergic dermatitis, and
is a biocide, which can cause sewage problems and elevated chromium levels in drinking
water.(22) Dichromate, its degradation product, is also toxic (carcinogenic and oxidizer) to
humans and is considered to be a corrosive poison.(98,99) Similarly, sodium azide is also
reported to cause no or minimal changes in milk composition (Table 2), but is harmful
and shows toxic effects to human health.(22,100) The most commonly reported health effect
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from sodium azide exposure is hypotension, which is almost independent of route of


exposure.(100) Fatal doses occur with exposures of ≥700 mg (10 mg/kg).
Formalin is the most effective and widely used preservative in preservation of milk
and some dairy products meant for analytical purposes and is the only legally permitted
preservative in India, according to FSSA rules and regulations. Although it is considered to
be the most effective preservative for milk and milk product samples stored at ambient tem-
perature, the efficiency of formalin in maintaining the composition profile of milk and milk
products is questionable based on widely conflicting scientific research reports. Several
enquiries about the effect of formalin on various physicochemical properties, constituents,
modus operandi of the sample analysis by regulatory agencies, and quality of formalin used
are of great concern in the present context of quality awareness among the manufacturers
and consumers with global competition and stringent food laws.
The use of formalin has shown contradictory results regarding the preservability of
samples, which may be due to the level of formalin used, use of substandard formalin,
storage period, storage temperature, and the nature of sample preserved, among other poten-
tial reasons. This has created a problem for the industry being in direct conflict with the
regulatory agencies. Formalin is also shown to have negative impacts on human health.
The most common symptoms include irritation of the eyes, skin, nose, and throat, along
with increased tearing, which occurs at air concentrations of about 0.4–3 parts per million
(ppm).(101) On the contrary, bronopol was reported to have no harmful effect vis-à-vis skin
and allergic reactions at lower concentration (0.01–0.02%), which is generally the dose
added for preservation purposes.(102,103) It has been used as a preservative in the pharma-
ceutical and cosmetic industry for a long time, probably due to its stability for more than
a year at room temperature.(72) Different companies provide bronopol in safe and cost-
effective forms such as liquid and solid (tablet). When added to milk, pure bronopol, unlike
mercuric chloride and potassium dichromate, produces no change in color.(79) However,
there are some contradictory reports on the effect of bronopol on the analysis of milk
components.
In view of the above, there is a need to revisit the issues related to preservation of sam-
ples of milk and milk products meant for analytical purposes. It requires reinvestigation on
the use of formalin and other preservatives used for this purpose. There is also a need to
look for alternate preservatives. The selected preservative should be antibacterial, antifun-
gal, and environmentally friendly. A combination of suitable chemical substances could be
a novel approach for efficient preservative action.
220 Upadhyay et al.

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composition of paneer. J. Food Sci. Technol. 1976, 13, 155–156.
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