Upadhyay 2014
Upadhyay 2014
Upadhyay 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
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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
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.
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)
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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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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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.”
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)
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 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 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 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)
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.
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)
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
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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.
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.
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
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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References
1. National Dairy Development Board. Annual report 2011–2012. http://www.nddb.org/English/
AnnualReports/nddb-annual-report-2011-2012.pdf (accessed June 12, 2013).
2. Mabrook, M.F.; Petty, M.C. A novel technique for the detection of added water to full fat milk
using single frequency admittance measurements. Sensors Actuat. B Chem. 2003, 96, 215–218.
3. Harridans, K.B.; Narayanan, K.M. Effect of additional of formalin and storage on chemical
composition of paneer. J. Food Sci. Technol. 1976, 13, 155–156.
4. Gursel, A.; Gursoy, A.; Odabai, S.; Simer, A.; Ceylan, M.S.; Deveci, O. Effect of the LP-system
activation on distribution of the nitrogenous compounds in cow milk. GIDA J. Food. 2000, 25,
355–362.
5. Belewu, M. A.; Belewu, K.Y.; Nkwunonwo, C.C. Effect of biological and chemical
preservatives on the shelf life of West African soft cheese. Afr. J. Biotechnol. 2005, 4,
1076–1079.
6. Williams, P.; Garry, E.; Ouattara, G. The effect of Bronopol on the freezing point and
Downloaded by [Laurentian University] at 21:30 07 December 2014
impedance of milk samples. Advanced Instruments Inc., Institute Food Technology, 2007, 1–6.
http://www.aicompanies.com/documents/file/EffectBronopol_Brochure_WEB.pdf
7. Nigussie, H.; Seifu, E. Effect of the lactoperoxidase system and container smoking on the
microbial quality of goats’ milk during storage at an ambient temperature. J. Cell Anim. Biol.
2008, 2, 166–170.
8. Malinowski, E.; Smulski, S.; Gehrke, M.; Kłossowska, A.; Kaczmarowski, M. Effect of storage
conditions and preservation with Bronopol on somatic cell count with the DeLaval cell counter
in cow milk. Medycyna Weterynaryjna 2008, 64, 1299–1303.
9. Kamau, P.M.N.; Lamuka, P.O.; Wangoh, J. Effect of lactoperoxidase-thiocyanate-hydrogen
peroxide system and storage temperature on keeping quality of raw camel milk. Afr. J. Food
Agric. Nutri. Dev. 2010, 10, 4185–8201.
10. Boulares, M.; Mankai, M.; Hassouna, M. Effect of thiocyanate and hydrogen peroxide on the
keeping quality of ovine, bovine and caprine raw milk. Int. J. Dairy Technol. 2011, 64, 52–56.
11. Elizondo, J.; Aldunate, A.; Ezcurra, P.; Gallego, I.; Saigos, E.; Ulayar, E.; Izco, J.M. Efficiency
of the proportion of azidiol on preservation of ewe’s milk samples for analysis. Food Control
2007, 18, 185–190.
12. Mabon, R.M.; Brechany, E.Y. The measurement of urea in fresh and stored goats milk. Lab.
Pract. 1983, 32, 81. [Cited in Food Control. 2007, 18, 185–190.]
13. Grappin, R.; Jeunet, R. The Milko-Tester automatic for routine determination of fat in milk.
Lait 1970, 50, 233–256.
14. Benda, P. The effect of some preservatives on natural microflora in milk samples. Vet. Med.
(Prague) 1995, 40, 359–364.
15. Azanza, J.R.; Honorato, J. Tetraciclinas y fenicoles. In Farmacologıa Humana; Florez, J.,
Armijo, J.A., Mediavilla, A. (Eds.); Barcelona: Ediciones Cientıficas y Tecnicas: Barcelona,
1992; pp 1029–1037.
16. Rapp, M.; Muench, S. Newly developed liquid preservation agents for milk. Dtsch. Molkerei
Ztg. 1984, 105, 1264–1272.
17. Food Safety and Standards Authority of India (FSSAI). Food Safety and Standards (Laboratory
and Sample Analysis) Regulations, 2011. http://fssai.gov.in/Portals/0/Pdf/Food%20Safety%
20and%20Standards%20(Laboratory%20and%20sampling%20analysis)%20regulation,%
202011%20(.pdf (accessed November 10, 2013).
18. Mukherjee, G.; Mathew, T.V. Effect of certain preservatives on food samples preserved for
analysis. J. Food Sci Technol. 1974, 11, 30–31.
19. Wilson, T.P. Evaluation of sodium omadine, bronopol, and Dowicil 200 as replacements
for potassium dichromate in milk sample preservation. M.S. thesis, The Pennsylvania State
University, State College, PA, 1983.
20. Indian Standards 1479 part I. Reaffirmed 2003. Ed. 1.4. Bureau of Indian Standards: New Delhi,
1960.
Preservation of Milk and Milk Products for Analysis 221
21. Kroger M. Instrumental milk fat determination. I. Effects of potassium dichromate concentra-
tion and sample storage tank on Milko-Tester results. J. Dairy Sci. 1971, 54, 735–737.
22. Kroger, M. Milk sample preservation. J. Dairy Sci. 1985, 68, 783–787.
23. Minzner, R.A.; Kroger. M. Physicochemical and bacteriological aspects of preserved milk
samples and their effect on fat percentage as determined with the Milko-Tester. J. Milk Food
Technol. 1974, 37, 123–128.
24. Olshevski, P.A.; Menshiko, N.G. Effects of some factors on accuracy fat estimation in milk.
Molochn. Prom. 1974, 8, 27–28. [Cited in Dairy Sci. Abstr. 1975, 37, 2172.]
25. Kaaber, K.; Veien, N.K. The significance of chromate ingestion in patients allergic to chromate.
Acta Derm. Venereal. (Stockh.) 1977, 57, 321–323. [Cited in MMWR Morb. Mort. Wkly. Rep.
1982, 31, 111–2, 118.]
26. Prevention of Food Adulteration Rules. Ministry of Health, Family Planning and Urban
Development, Government of India: New Delhi, 1955.
27. Boghra, V.R.; Borkhatriya, V.N. Physico-chemical properties and compositional profile of milk
samples as affected by formalin preservation—A review. Indian J. Dairy Sci. 2003, 56, 65–71.
Downloaded by [Laurentian University] at 21:30 07 December 2014
28. Dawood, A.E.; Naghmoush, M.R.; Nofel, A.A. The effect of certain additives on acidity and
formal number of milk. Ala. J. Agric. Res. 1974, 22, 73–77. [Cited in Dairy Sci. Abstr. 1976,
38, 8238.]
29. Fahmi, A.H.; Tawab, G.A.; Abou-El-Neba, A. Effect of added neutralizers and preservatives on
the Methylene Blue Reduction Test. Egypt J. Dairy Sci. 1982, 10, 15–21.
30. Bansal, A.; Singhal, O.P. Preservation of milk samples with formalin—Effect on acidity. Indian
J. Dairy Sci. 1991, 44, 573–576.
31. Karmakar, B.; Ghatak, P.K. Effect of chemical preservatives on different constituents of cow
milk during storage under refrigerated conditions. Cheiron, 1997, 26, 89–93.
32. Gupta, H.C.L.; Gupta, D. Compositional change in cross bred and local cow milk as affected
by formalin preservative. Pantnagar J. Res. 2010, 8, 219–221.
33. Sandhu, J.S.; Nusrath, N.; Narayanaswamy, M.; Kanpur, O.P. Study on the effect of formalin as
preservative on different constituents of raw milk samples during storage. J. Food Sci. Technol.
1984, 21, 424–425.
34. Bajaj, V.K.; Rai, T. Effect of formalin on comparative efficiency of protein and lactose
estimation. Indian J. Dairy Sci. 1993, 46, 21–25.
35. Sharma, R.; Sarwar. Determination of fat in formalin preserved milk samples. Indian J. Dairy
Biosci. 2000, 11, 138–141.
36. Karmakar, M.B.; Ghatak, P.K. Effect of chemical preservatives on chemical quality of buffalo
milk during storage. Indian J. Dairy Sci. 1995, 48, 559–561.
37. Raj, D.; Singhal, O.P. Stem graduated milk pipette for accurate fat estimation in milk by
Gerber method. In Brief Communications of the XXIII International Dairy Congress, Montreal,
October 8–12, 1990, Vol. I; International Dairy Federation: Brussels, Belgium, 1990; pp 225.
38. Bansal, A. Effect of formalin on different constituents of cow and buffalo milks with partic-
ular reference to Fat Globule Membrane (FGM). PhD dissertation, Kurukshetra University,
Kurukshetra, Haryana, India, 1989.
39. Bajaj, V.K.; Rai, T. Comparative efficiency of various analytical methods for fat and total solids
determined gravimetrically in formalin-preserved milk samples. Indian J. Anim. Sci. 1992, 62,
1096–1098.
40. Csok, J. Investigation of the applicability on Milko-Tester MK-II. Tejipari Kutat. Kozlem. 1973,
16, 3–10. [Cited in Dairy Sci. Abstr. 1975, 37, 7431.]
41. Patrattii, A.P; Aristova, V.P.; Agienko, K.S.; Otradnova, A.V.; Bykova, N.A. Introduction of
physical methods for monitoring fat content of milk deliveries. Molochnaya Promyshlesnnost
1984, 4, 23–38. [Cited in Dairy Sci. Abstr. 1985, 47, 5812.]
42. Bector, B.S.; Narayanan, K.M. Studies on the changes in composition of milk containing
HCHO during storage. Food Ind. J. 1973, 5, 5–6.
43. Tschorner, F. Milkchwissenschaft. 1965, 20, 234–240.
222 Upadhyay et al.
44. Kleczkowaski, K.; Jaisorwaski, H.; Kurowaski, H. A colorimetric method for the estimation of
protein in milk. Roczn. Nauk. Rol. 1961, 77, 923–930. [Cited in Dairy Sci. Abstr. 1962, 24,
1793.]
45. Grimbleby, F.H. The determination of lactose in milk. J. Dairy Res. 1956, 23, 229–237.
46. Bilkis, T.; Islam, M.; Sumy, M.C.; Mandal, M.; Ali, N.; Uddin, G.M. Rapid estimation of quality
of raw milk for its suitability for further processing in the dairy industries of Bangladesh. Int. J.
Dairy Sci. 2013, 8, 1–11.
47. Haselkorn, R; Doty, P. The reaction of formaldehyde with polynucleotides. J. Biol. Chem. 1961,
236, 2738–2745.
48. Rosell, M. Leperoxyde d’hydrogene moyen le plus pratique jusqua ce jour, pour la preservation
et degermisation du lait dans les pays tropicaux. Milchwissenschaft. 1954, 9, 180–184.
49. Luck, H. The use of hydrogen peroxide in milk and dairy products. In Milk Hygiene: Hygiene in
Milk Production, Processing and Distribution; World Health Organization Monograph Series
No. 48. World Health Organization: Geneva, 1962; pp 423–447.
50. Bjorck, L.; Rosen, C.G.; Marshall, V.; Reiter, B. Antibacterial activity of the lactoperoxidase
Downloaded by [Laurentian University] at 21:30 07 December 2014
system in milk against pseudomonads and other gram-negative bacteria. Appl. Microbiol. 1975,
30, 199–204.
51. Reiter, B.; Marshall, V.M.; Rosén, C.G. Nonspecific bactericidal activity of the
lactoperoxidases-thiocyanate-hydrogen peroxide system of milk against Escherichia coli and
some gram-negative pathogens. Infect. Immun. 1976, 13, 800–807.
52. Giolitti, G. The effect of high concentrations of hydrogen peroxide on the chemical composi-
tion of milk. Atti Soc. Ital. Sci. Vet. 1949, 3, 543–547. http://whqlibdoc.who.int/monograph/
WHO_MONO_48_(p423).pdf
53. Banerjee, N.L. Use of hydrogen peroxide as a milk preservative. Indian Med. Gaz. 1947, 82,
156–159.
54. Atamer, M. Butter Technology. Ankara Univ. Agric. Fac. Press No. 1313: Anakara, 1993; 1–89.
55. Luck, H. Effects of diluted hydrogen peroxide solutions on casein. Biochem. Z. 1956, 328,
216–227.
56. Luck, H.; Joubert, F.J. Influence of dilute hydrogen peroxide solution on whey proteins.
Biochem. Z. 1955, 327, 221–230.
57. Dahiya, S.; Speck, M.L. Hydrogen peroxide formation by lactobacilli and its effect on
Staphylococcus aureus. J. Dairy Sci. 1968, 51, 1568–1572.
58. Romani, B. Chim. E Industr. (Milano). 1947, 29, 143. http://whqlibdoc.who.int/monograph/
WHO_MONO_48_(p423).pdf
59. Negretti, F. Arch. Vet. Ital. 1956, 4, 266. http://whqlibdoc.who.int/monograph/
WHO_MONO_48_(p423).pdf
60. Rokhsana, F.; Das, U.K.; Yeasmin, R.; Nahar, A.; Parveen, S. Studies on the preservation of
Raw cow’s milk by chemical method. Bangladesh J. Sci. Ind. Res. 2007, 42, 317–326.
61. Anonymous. Use of sodium azide as a preservative of milk samples for fat and protein determi-
nation with the Milko-Scan. D. Milchwirtsch. 1977, 28 (27, Bell. Lebensmittel-Labor), XI–XII.
[Cited in Dairy Sci. Abstr. 1978, 40, 1053.]
62. Sanchez, A.; Sierra, D.; Luengo, C.; Corrales, J.C.; De La Fe, C.; Morales, C.T.; Contreras, A.;
Gonzalo, C. Evaluation of the MilkoScan FT 6000 milk analyzer for determining the freezing
point of goat’s milk under different analytical conditions. J. Dairy Sci. 2007, 90, 3153–3161.
63. Barcina, Y.; Zorraquino, M.A.; Pedauye, J.; Ros, G.; Rincon, F. Azidiol as a preservative for
milk samples. An. Vet. (Murcia) 1987, 3, 65–69.
64. Anonymous. Somatic cells in milk. Their significance and recommended methods for counting,
Document 114. FIL/IDF: Brussels, Belgium, 1979; pp 9–20.
65. Pintic-Pukec, N.; Barac, Z.; Dakic, A.; Strucic, D.; Blazek, D. Determination of the freezing
point in cow milk samples. Mljekarstvo 2011, 61, 336–340.
66. Ardo, Y. Bronopol as a preservative in milk samples. Milchwissenschaft 1979, 34, 14–16.
67. Brunt, K.D.; Higton, F.R. Preserving agent and method of use thereof. The Boots Company Plc
(GB2), assignee. U.S. Patent 4,636,476, 1987.
Preservation of Milk and Milk Products for Analysis 223
68. Ruttan, G.R.S. Milk sample preservative. U.S. Patent 5,196,344, Mar. 23, 1993.
69. Shepherd, J.A.; Waigh, R.D.; Gilbert, P. Antibacterial action of 2-bromo-2-nitropropane-1,3-
diol (Bronopol). Antimicrob. Agents Chemother. 1988, 32, 1693–1698.
70. Amores, J.; Sanchez, A.; Martin, A.G.; Corrales, J.C.; Contreras, A.; De la Fe, A. Viability
of Mycoplasma agalactiae and Mycoplasma mycoides ssp. capri in goat milk samples stored
under different conditions. Vet. Microbiol. 2010, 145, 347–350.
71. Anonymous. Encyclopedia of Chemical Technology, 3rd ed., Vol. 7; Grayson, M., Ed.; John
Wiley & Sons: New York, 1979.
72. Smyth, J.C. A novel air sampling and analytical method for determination of airborne bronopol.
PhD thesis/dissertation, University of South Florida Graduate School, FL, USA, 2006. http://
scholarcommons.usf.edu/etd/2708 (accessed November 11, 2013).
73. OSP Microcheck Inc. Bronopol—Environmental Safety, 4; OSP Microcheck Inc.: Calgary,
Alberta, Canada, 2006. http://www.ospmicrocheck.com
74. Maibach, H.I. Dermal sensitization potential of 2-bromo-2-nitro-propane diol (Bronopol).
Contact Dermatitis 1977, 3, 99. [Cited in Boots Co., Ltd. Tech. Bull. Data No. 3, 1979.]
Downloaded by [Laurentian University] at 21:30 07 December 2014
75. Frosch, P.J.; White, I.R.; Rycroft, R.J.G.; Lahti, A.; Burrows, D.; Camarasa, J.G.; Ducombs,
G.; Wilkinson, J.G. Contact allergy to bronopol. Contact Dermatitis 1990, 22, 24–26.
76. Wilson, C.L.; Powell, S.M. An unusual case of allergic contact dermatitis in a veterinary
surgeon. Contact Dermatitis 1990, 23, 42–43.
77. Rudzki, E.; Rebandel, P.; Grzywa, Z. Occupational dermatitis from cosmetic creams. Contact
Dermatitis 1993, 29, 210–219.
78. Bertrand, J.A. Influence of shipping container, preservative, and breed on analysis of milk
components of shipped samples. J. Dairy Sci. 1996, 79, 1–148.
79. Barbano, D.M.; Woiciechowski, K.L.; Lynch, J.M. Effect of preservatives on the accuracy of
mid-infrared milk component testing. J. Dairy Sci. 2010, 93, 6000–6011.
80. Sanchez, A.; Slerra, D.; Luengo, C.; Corrales, J.C.; Morales, C.T.; Contreras, A.; Gonzalo, C.
Influence of storage and preservation on Fossomatic cell count and composition of goat milk.
J. Dairy Sci. 2005, 88, 3095–3100.
81. Gonzalo, C.; Martınez, J.R.; Carriedo, J.A.; San Primitivo, F. Fossomatic cell-counting on ewe
milk: Comparison with direct microscopy and study of variation factors. J. Dairy Sci. 2003, 86,
138–145.
82. Vermunt, A.E.M.; Loeffen, G.J.M.; Van der Voet, H.; Naber, M.A.A.M. Development of ref-
erence samples for calibration and quality control of somatic cell count using a Fossomatic
instrument. Neth. Milk Dairy J. 1995, 49, 111–123.
83. Lee, K.L.; Dayton, K.P.; Kroll, G.; McGilliard, M.L. Effects of preservative, storage time, and
storage temperature on milkfat percent, protein percent and somatic cell count determination.
J. Dairy Sci. 1986, 69 (Suppl. 1), 211. [Abstract]
84. Butler, G.; Stergiadis, S. Suitability of bronopol preservative treated milk for fatty acid
determination. J. Dairy Res. 2011, 78, 220.
85. Kaylegian, K.E.; Lynch, J.M.; Fleming, J.R.; Barbano, D.M. Lipolysis and proteolysis of mod-
ified and producer milks used for calibration of mid-infrared milk analyzers. J. Dairy Sci. 2007,
90, 602–615.
86. Sierra, D.; Sanchez, A.; Contreras, A.; Luengo, C.; Corrales, J. C.; De la Fe, C.; Guirao,
I.; Morales, C.T.; Gonzalo, C. Effect of storage and preservation on total bacterial counts
determined by automated flow cytometry in bulk tank goat milk. J. Dairy Sci. 2009, 92,
4841–4845.
87. Armandola, P. Preservation of raw milk samples. Minerva Diet 1967, 7, 256–257. [Cited in
Dairy Sci. Abstr. 1969, 31, 727.]
88. Armandola, P. Preservation of milk samples for analytical purposes. Ind. Latte. 1969, 5, 33–38.
[Cited in Dairy Sci. Abstr. 1969, 31, 3144.]
89. Kvapilik, J.; Suchanek, B. Effect of different methods of sampling preservation on results of
milk analysis. Zivecisna Vyroba 1974, 19, 31–38. [Cited in Dairy Sci. Abstr. 1975, 37, 1393.]
224 Upadhyay et al.
90. Wolfschoon, A.F. Tests on the use of the Milko-Tester MK-III for the determination of milkfat.
Rev. Inst. Lat. Candido Tostes 1978, 33 (196), 11–12. [Cited in Dairy Sci. Abstr. 1979, 41, 487.]
91. Jandal, J.M.; Rai, T. Effect of storage temperatures on compositional changes in milk samples
added with preservatives. Indian J. Anim. Sci. 1988, 58, 1245–1247.
92. Seskena, R.; Janevica, L. Influence of chemical preservatives on the quality and composition
indices of raw milk samples. Acta Univ. Latviensis 2007, 723, 171–180.
93. Gupta, R.K. Quality of raw milk in India. In Advances in Processing and Preservation of Milk;
Lecture Compendium; National Dairy Research Institute: Karnal, 1999 [Cited in J. Food Sci.
Technol. 2014, 51, 821–831.]
94. Kumar, S.; Rai, D.C.; Niranjan, K.; Bhat, Z.F. Paneer—An Indian soft cheese variant: A review.
J. Food Sci. Technol. 2014, 51, 821–831. doi:10.1007/s13197-011-0567-x.
95. Ramaiah, B.K.; Narayanan, K.M. Effect of addition of formalin and storage on chemical
composition of ice cream. Indian J. Dairy Sci. 1976, 29, 135–137.
96. Dinakar, P.; Sharma, U.P. Efficiency of formalin in preservation of khoa samples for analytical
purposes. Asian J. Dairy Sci. 1989, 8, 65–70.
Downloaded by [Laurentian University] at 21:30 07 December 2014
97. Joseph, J.K.; Akinyosoye, F.A. Comparative studies on red sorghum extracts and other
chemicals as preservatives for West African soft cheese. Int. Dairy J. 1997, 7, 193–198.
98. Bertrand, J.A. Influence of shipping container, preservative and breed on analysis of milk
components of shipped samples. J. Dairy Sci. 1996, 79, 145–148.
99. Environmental Health and Safety. Potassium dichromate. 2008. http://www.jtbaker.com/msds/
englishhtml/P5719.htm (last accessed September 13, 2010). [Cited in J. Dairy Sci. 2010, 93,
6000–6011.]
100. Chang, S.; Lamm, S.H. Human health effects of sodium azide exposure: A literature review and
analysis. Int. J. Toxicol. 2003, 22, 175–186.
101. Andersen, I.; Molhave, L. Controlled human studies with formaldehyde. In Formaldehyde
toxicity, Gibson J.E., Ed.; Hemisphere Publishing Corporation: Washington, DC, 1983; pp
154–165.
102. Ardoe, Y. Bronopol as a preservative in milk sample for the determination of cell content using
Fossomatic. Milchwissenschaft 1982, 37, 139–142.
103. Dunham, J.R.; Kroger, M. Milk preservatives. Dairy herd improvement. Kansas, 1985; 2 p.
http://eeb.lu.lv/EEB/2007/Seskena.pdf (accessed October 24, 2013).