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The Effect of Boiling on Milk Microbial Contents and Quality

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 Journal of American Science, 2011;7(2) http://www.americanscience.org

The Effect of Boiling on Milk Microbial Contents and Quality

Ahmed M.M. Metwally1, Nadia M.A.Dabiza*2, Wagih I.El-Kholy2 and Zeinab I.Sadek2
1
Dairy Technol.Dept. Faculty of Agric. Cairo University, Giza, Egypt
2
Dairy Science Department, National Research Center Dokki, Giza, Egypt
[email protected] *

Abstract: Though milk boiling is a widespread heat treatment in number of countries, the process was not
thoroughly studied. In this study, the effect of boiling buffaloes´ and cow's milk samples for different periods on
their microbiological contents, keeping quality and bacterial ecology contents and chemical changes were
determined. Lethality rate of 6.53, 6.77, 7.301 and 7.44 in buffaloes` and 6.76, 7.059, 7.012, 7.15 and 7.159 log10
cfu/ml in cows´ milk were obtained on boiling the samples for 0.5, 1,2 and 5min., respectively. Boiling milk for 0.5
and 1min decreased the bacterial count from 3.6×109 in cow's milk into 6.3×102 and 3.2 ×102 and from 7.8×109 in
buffaloes ‘milk into 2.26×103 and 1.3×103 cfu/ml, respectively. On cold storage, the microbial content of boiled
milk, not only did not increase but also declined on the first week. Boiling destroyed bacterial vegetative cell leaving
behind spores of the sporeformer which were dominated with B.cereus and Micrococcus leteus. Boiling affected
milk quality far less than the effect occurred in UHT milk as determined by O.D- value measurements.
[Ahmed M.M. Metwally, Nadia M.A.Dabiza, Wagih I.El-Kholy and Zeinab I. Sadek. The Effect of Boiling on
Milk Microbial Contents and Quality. Journal of American Science 2011;7(2):110-114]. (ISSN: 1545-1003).
http://www.americanscience.org.

Key words: Boiling period, Bacillus cereus, Buffaloes` and Cows` milk, UHT milk

1. Introduction: studied. Period of boiling varies between individuals,

In developed countries, only 2% of the milk some turns off the heat as milk boils & foams and
produced is consumed in its raw form, most of the others boil milk for 5 or 10 min., however at such
milk in underdeveloped countries is consumed raw. high temperature, period of heating has a decisive
In number of African countries as well as in Egypt effect on milk quality. For example, furosine
large percentage of milk is retailed directly to production in boiling milk was increased for different
consumers by farmers and small-scale traders period of heating from 110.4 to 116.7 and 128.9
including hawkers. Most of the dairy farmers are mg/100g proteins as the period increased from zero
small holders with 2 or 3 animals lacking the into 5 and 10min., respectively (Sun and Wang,
hygienic conditions of production and veterinarian 2009).
services; therefore their milk is not of best quality. In Therefore this research was carried out to
many of these countries, there is no enough determine the proper boiling period which results in
processing plants to coup up with the milk produced the required safety through a proper shelf life with
.Moreover, the industry processes mostly UHT milk, minimum quality deterioration. Moreover, chemical
which is expensive for most consumers, a matter that changes occurring in boiled milk and UHT-milk were
encourages them to consume retailed raw milk. compared using a UV- absorption method.
There fore they have to rely on boiling to
establish milk safety and a reasonable shelf life. Not 2. Materials and methods
only UHT milk is expensive but also the treatment Fresh buffaloes´ and cow's milk samples
produces number of undesirable changes and from the herd of the faculty of Agriculture, Cairo
products such as maillard reaction products and univ., Bacillus cereus NRRL, B.3711, from Northern
lactulose (Seiquer, et al.2010). Boiling is a common Regional research Laboratory, Ill.USA and Listeria
practice in many countries and WHO (World Health monocytogenes type 1 from Hungarian National
Organization) recommended milk boiling for African Collection of Medical Bacteria, OKI, Budapest,
countries and described the process in its training Hungary.
manual (Israel-Ballard and Chantry, 2010). Boiling Bacillus cereus and Listeria monocytogenes
milk is simple process and does not need temperature were activated in Brain- heart infusion broth (oxoid)
gauges or timing devices which limit the use of at 37°C for 24hr. After sufficient growth, the cultures
pasteurization. were diluted in saline to about 7×106 cfu/ml
However, period of boiling treatment, the measured by standard plate count (SPC).
effect of boiling milk on microbial contents, keeping
quality and chemical changes were not thoroughly

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 Journal of American Science, 2011;7(2) http://www.americanscience.org

Thermal resistance of Bacillus cereus and Listeria colony shape and color, microscopic examination,
monocytogenes: reaction to gram stain, catalase test, and production of
The test tube method of Donnelly and acid from glucose, mannitol and zylose.
Briggs (1987) was used to determine heat resistance
of both microorganisms. One tenth of ml of 24 old 3. Results and Discussion:
culture (after the come up period) was inoculated into The effect of heating milk at different
10 ml of sterilized cow's milk in screw- capped tubes temperatures on its microbial content was
and were heated in a thermostatically oil bath. Milk determined, Fig (1).Maximum lethality values of
was heated at 100ºC for 0.5, 1, 5 and 10 min, and 5.793, 6.09 and 6. 60 log10
then samples were cooled in ice bath. Aerobic plate cfu/ml for buffaloes´ milk and 5.85, 6.18 and 6.959
counts using plate count agar (oxoid) at 37°C for log10 cfu/ml for cow's milk were obtained in milk
48h.were used to determine the survivals. Rates for heated at 80, 90 and 100°C for 15s, respectively. The
thermal inactivation of each bacterium were destruction effect was temperature dependent. The
determined graphically by plotting the log10 cfu/ml of 80ºC resembles ultra-pasteurization treatment.
surviving cell population versus heating time. A line Lethality values of above 6 log10 cfu/ml
was drawn through the date points and D-values were which obtained by boiling treatment, was greater than
obtained from the slope of the best lit line (El- the minimum microbial inactivation of 5 log10
Shenawy et al, 1989). required for pasteurization. Boiling milk for 15s
reduced microbial count from 2 or 3 × 109 cfu/ml into
Effect of milk different heat treatments on bacterial few hundreds about 900 cfu/ml. The 15s of heating
contents: resembles the boiling flow-over end point used by
A half liter milk samples in glass beakers regular consumer at home.
were heated at 80°C, 90ºC and 100ºC for 15 second
in thermostatically controlled oil bath followed by 10
rapid cooling in ice-bath to 5ºC. In another 9
Mean ABC(log cfu/ml )
-1

experiment buffaloes´ and cow's samples (0ne liter) 8

Buffaloes`
were boiled for different periods and followed by 7 Cows` milk
rapid cooling. Samples were tested microbiologically 6
for viable bacteria using aerobic plate count in 5
nutrient agar (oxoid) (37°C for 48hr) after the 4
standard methods for the examination of dairy 3
products. Lethality was calculated as the difference 2
between the log of colony counts of the untreated 1
(No.) and treated samples (N1) ( log10 No- log10 N1) 0
(Roig-Saguès et al, 2009). untreated milk 80ºC 90 ºC 100ºC

Chemically, the samples were tested using a Heat treatment

UV- method for evaluation of heat treatment of milk
(Sun and Wang, 2009). Fig (1): Change in aerobic bacteria counts (ABC)
of cow's & uffaloes` milk heated at different
Boiled milk keeping quality: temperatures for 15 second.
Cow's and buffaloes´ milk of one liter
samples were boiled for different periods (1.0, 2.0., Table (1) shows the effect of increasing the
3.0 and 5.0 min) and after cooling, the samples were period of boiling into 0.5, 1, 2, 5 and 10min.on milk
kept in a household refrigerator (~ 7-8°C) for 10 microbial contents. The lethality rate increased by
days. Total bacterial contents of the samples were increasing boiling period to reach 6.76, 7.059, 7.012,
determined after 5, 7 and 10days of cold storage 7.15 and 7.59 in cow's milk and 6.53, 6.77, 7.301,
using plate count agar (oxoid) at 6.76 and 7.44 log10 cfu/ml in buffaloes´ milk at 0.5, 1,
37ºC for 48h. 2, 5 and 10min., respectively. Boiling for 0.5, 1.0 and
2min reduced total bacterial count from 7.8×109 to
Bacterial ecology of boiled milk: 2.26× 103, 1.3 × 103, 3.9×102 in buffaloes´ milk and
Buffaloes´ milk was boiled at 100ºC for 10 from 3.6× 109 to 6.3× 102 and 3.2× 102 to 3.5× 102 in
min and after cooling, milk was plated on nutrient cows` milk. Increasing the holding period beyond the
agar at 37ºC for 48hr. Colonies present were visually 0.5 min progressively reduced the numbers, but
examined and colonies representative of each insignificantly. Lethality values of greater than 67
distinct morphology were counted, isolated and were obtained by boiling milk for 1 and 2 min for
identified. Identification was carried out by observing buffaloes´ and cow's milk. Boiling periods beyond

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 Journal of American Science, 2011;7(2) http://www.americanscience.org

2min did not show any increase in the lethality rate. 2min period.
Therefore, boiling milk need not to exceed than more

Table (1) Total bacterial count in for milk boiled for different period.
Period of boiling, min1. Type of milk2
Cow's Buffaloes´
Bacterial counts, × 109 cfu/ml
Raw milk (untreated) 3.6±26.46 7.8±31.22
Bacterial counts, × 103 cfu/ml
0.5 0.63±0.03 2.26±0.26
1.0 0.32±0.05 1.3±0.13
2.0 0.35±0.07 0.39±0.04
5.0 0.25±0.06 0.28±0.03
10.0 0.25±0.07 0.29±0.04
1- LSD(0.05) = 2.5 2- LSD (0.05) = 2

Microbial lethality at boiling temperature This means that B.cereus as spore formers would
was higher in cow's milk than in buffaloes´ milk. This tolerate the boiling treatment; however, storage cold
is may be due to buffaloes´ milk contains more fat temperature would prevent its germination and
and T.S than cow's milk, this high T.S reduces heat growth.
rate of exchange and the high fat content protects the
microorganisms against heat. This is expected to
increase bacterial heat resistance in buffaloes´ than
cow's milk. Some workers (Nasr, 2008) found that a
temperature of 75°C for 25 seconds was required to
kill L.monocytogenes in buffaloes´ milk while the
72ºC/15s was enough for cows` milk. But still boiling
for both milks could be for 1 or 2 min at the most.
Therefore, regular boiling practices at home
regardless the period used reduces the microbial load
into a level considered to be safe for human
consumption, particularly
that all pathogens are also destroyed. The bacterial
counts were reduced by boiling to levels significantly
lower than the 2o.ooo cfu/ml limit required for grade
(A) pasteurized milk in pasteurized milk ordnance
(Ranieri, et al., 2009).
To determine the safety of boiled milk for human
consumption, the survival of Listeria monocytogenes
and Bacillus cereus, two pathogens usually found in
milk, through boiling was studied. The D-value of
both microorganisms at 100ºC was determined. L.
momocytogenes (7.5×106 cfu/ml) was completely
destroyed at 100ºC for all periods starting from the
first instant of boiling, therefore, its D- value could
not be determined. This means that non- spore former
pathogens pose no danger in boiled milk. The D-
value of B.cereus was determined in both milks to be
7.5min & 10.4 min for cow's and buffaloes` milk, Fig (2): D-value of Bacillus cereus determined in
respectively (Fig 2). These results are in the boiled milk.
agreement with that obtained by El-kholy, 1993 who
reported that D-value of L. momocytogenes was 1.4 Actually boiling destroys all vegetative cells
sec. at 70ºC. So, 100ºC is more than enough for leaving behind spores of sporeformers. This was
destroying the pathogen. found when the ecology of boiled milk was studied;
results are presented in Table (2). Of the 15 gram-
positive spore forming bacteria isolated in the present

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 Journal of American Science, 2011;7(2) http://www.americanscience.org

study from boiled milk for 10min, 5 isolates were leteus, 7 isolates were M. varians and one was
Bacillus cereus and the rest were characterized to be M.roseus.
of the genus Micrococcus. Five isolates were M.

Table (2) Bacterial isolates obtained from milk boiled for 10min.
Number of Isolates
Bacterial isolates
isolates (%)
Bacillus cereus 5 38.4
Micrococcus leteus 5 38.4
Micrococcus varians 2 15.3
Micrococcus roseus 1 7.6
Non –of these isolates were psycrotolerant samples boiled for 1, 2 and 5min were stored for 10
endospore since after 10 days of cold storage the days in household refrigerator to determine the
counts insignificantly increased as in Table(3) which keeping quality.
presents the keeping quality of boiled milk. Milk

Table (3): Boiled milk keeping quality

Cold Cows’ Milk Buffaloes’ Milk
Storage2, Boiling3 Period (min).
days 1 2 5 1 2 5
× 104cfu/ml
0 2.3±0.05 1.4±0.23 1.3±0.21 2.4±0.21 2.8±0.21 0.9±0.2
5 2.9±0.09 1.6±0.21 1.2±0.2 2.22±0.26 3.6±0.44 0.20±0.10
7 0.65±0.02 0.65±0.02 0.6±0.18 0.8±0.09 0.2±0.03 0.2±0.02
10 1±0.26 1.55±0.03 0.75±0.04 0.33±0.03 0.2±0.20 2.2±0.02
1- LSD (0.05) (overall) = 2 2- LSD (0.05) = 1.2 3-LSD (0.05) = 2

There was insignificant count decrease after to the above reference, furosine formation at 100ºC is
the fifth day of storage. This was followed by a a straight line relationship with 0.D- values. This
significant decrease in the 7th day and the counts means that boiling is a more delicate treatment than
insignificantly changed on the 10th day of cold all kinds of UHT treatment by forming far less
storage. The counts were of a range between 2×103 amount of furosine.
and 7×103 cfu/ml on seventh day of storage, which A better flavor and causing less nutrition
means that boiling milk has a good keeping quality deterioration than UHT. The formation of furosine
under refrigeration. Actually, the samples remained reduces the nutritive value of milk by decreasing
in good condition after 20 days of storage. It was protein availability and may behave as chelating
found that spore formers are the major spoilage agents for metal cations affecting their
bacteria of heat treated milk .The bacterial ecology of bioavailability. Over heating such as in bottle
high temperature short time pasteurized milk in the sterilization which sometimes is used in UHT
US for example was found to be gram positive production may result in decreasing of food intake
endospore-forming bacteria (i.e Bacillus and (Seiquer, et al. 2010). It was found that UHT milk
Paenibacillus). During cold storage the predominant produce more furosine than boiling, boiling for 5min
spoilage genera shifted from Bacillus spp to produced 116.7 while commercial UHT contained
Paeinbacillus sp, some of these strains were 142 mg furosine/100g protein (Sun and Wang, 2009).
psycrotolerant endospores and their growth caused Also, UHT-milk was found to contain 0.181% free
milk spoilage, (Ranieri and Boor,2009). fatty acids, 0.453 mEq of O2/kg of fat peroxides and
To compare the effect of heat treatment on thiobarbituric acid (TBA) values of 0.019 as
milk quality a UV- method was use to discriminate compared to 0.118% Free fatty acids, 0.296 mEq of
between boiling & UHT treatments and the results O2/kg of fat peroxides and 0.018 TBA in 5min boiled
are in Fig (3). Boiling up to 5 min developed less milk (Meshref and Al-Rowaily,2008)
than half of 0.D- values of other heat treatments. The In conclusion, milk boiling for periods less
extended shelf life treatments was of intermediate than 2min whether boiling was carried out in an oil
0.D–value between boiling & UHT-milk. These 0.D- bath or on direct flame provides the consumer the
values correlate with furosine contents and according required safety which lasts for a reasonable shelf life

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 Journal of American Science, 2011;7(2) http://www.americanscience.org

under at cold temperature life. The method though is storage of milk and some dairy products and lipid
simple and inexpensive for regular consumers oxidation. Pakistan J of nutrition 7(1)118-125.
preserves more of milk nutritive value and flavor 6. Nasr,N.F.(2008). Advanced studies on
compared to UHT treatments which is develops old pasteurization of milk and juices: A Ph -Thesis,
or stale flavor. However, on boiling continuous Department of Agr.Microbiology, Faculty of
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to be sure that the formed foam is exposed to boiling 7. Ranieri, M.L.and Boor, K.J.(2009). Short
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0.7
4833–4840.
0.6
8. Ranieri, M.L., Huck, J. R.; Sonnen, M.; Barbano,
0.5 D. M. and Boor, K. J. (2009). High temperature,
0.D-value

0.4 short time pasteurization temperatures inversely

0.3 affect bacterial numbers during refrigerated
0.2
storage of pasteurized fluid milk J. Dairy Sci. 92:
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0.1
9. Roig-Sagués, A.X.; Velázquez, R.M.; Legre-
0 Agramont, P.M.; López-Pedemonte, T.J.Brinze-
a b c d e f g Zambrano,W.J.;Guamis-López,B. and Hernandez,
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c: Boiling for 2min d: boiling for 5min severe heat treatment of milk on calcium
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Corresponding author estimate the heat treatment of milk and milk-like
Nadia M.A.Dabiza systems. International J. of Dairy Technology.
Dairy Science Department, National Research Center 62(3)321-324.
Dokki, Giza, Egypt
[email protected] 12/12/2010
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