Jornal de Pediatria - Vol. 76, Nº5, 2000 357
0021-7557/00/76-05/357
Jornal de Pediatria
Copyright
© 2000 by Sociedade Brasileira de Pediatria
ORIGINAL ARTICLE
Effect of home boiling and refrigeration
on bacterial load of pasteurized milk
Tania B. Morais1, Dirce M. Sigulem2
Abstract
Objectives: to assess the efficacy of in-home boiling of pasteurized milk in reducing the bacterial load
and the ability of the refrigeration in preserving the milk after boiling.
Methods: thirty samples of pasteurized milk bought in São Paulo, Brazil, were submitted to in-home
boiling procedure at the laboratory. Portions of samples were taken before and after boiling, and after 2,
4, 6 and 24 hours under refrigeration for microbiological analyses. Methods used were mesophilic bacteria
count, and coliforms and Escherichia coli (E.coli) enumeration.
Results: no sample presented mesophilic bacteria count above the Brazilian standard for pasteurized
milk. E.coli was not recovered from any sample. Ten samples (33%) had coliform bacteria; of these, 3
samples (10%) were above the standard. Mesophilic bacteria count after boiling was significantly lower
than before boiling. After 24 hours under refrigeration, mesophilic bacteria count was significantly higher
than after boiling. No significant differences were found between the intervals of 2, 4, 6 and 24 hours under
refrigeration. Samples before boiling presented significantly higher coliform bacteria. No coliform bacteria
were recovered at any time after boiling.
Conclusions: in-home boiling of milk reduced bacterial load, while refrigeration kept bacteria under
low counts.
J Pediatr (Rio J) 2000; 76(5): 357-60: milk, contamination.
Introduction
The introduction of pasteurized milk, in the beginning
of this century, was a remarkable advance both in terms of
technology and public health. Reports at the time describe
an impressive reduction in infant mortality in Europe and in
the United States related to pasteurization.1,2 However,
even nowadays, both in Brazil and in developed countries,
shortcomings in the processing of milk have allowed the
contamination of pasteurized milk by pathogenic
microorganisms,3-6 such as saprophytic bacteria, capable
of multiplying in low temperatures. These bacteria may
cause milk - which is very susceptible, due to its
physicochemical and nutritional properties - to deteriorate.
In Brazil, the microbiological quality of pasteurized
milk has been unsatisfactory due to deficiencies in the
production, processing, and commercialization of this
product, as one shown in Table 1.3,7-15 Among the
commercially available milks, type C has more flexible
legal microbiological standards than other types of milk.
1. Ph.D., Sciences. Laboratory of Food Chemistry and Microbiology.
2. Ph.D.; Associate Professor, Department of Pediatrics.
Universidade Federal de São Paulo, Escola Paulista de Medicina.
357
Effect of home boiling and refrigeration... - Morais TB et alii
358 Jornal de Pediatria - Vol. 76, Nº5, 2000
Since it is the least expensive type of pasteurized milk, it is
also the most used one in children of low-income families.16
Although the safety offered by the use of pasteurized
milk is considerable in populational terms, compendia on
pediatrics17,18 have been careful to recommend home boiling
of the milk given to children; in our population, both high
and low socioeconomic-level populations have widely
adopted this measure.19 However, no experimental work
demonstrating the efficacy of this procedure in decreasing
bacterial load was found.
This gap motivated the performance of the present
study, whose objectives were to assess the efficacy of
boiling in reducing the bacterial load of pasteurized milk,
and the ability of refrigeration in preserving this product.
Materials and methods
Thirty cartons of 1-liter type C pasteurized milk were
bought in São Paulo (10 cartons of three different brands of
milk). In the laboratory, the total content of each carton was
poured into a covered, sterilized milk pot, and submitted to
boiling. Boiling was defined as the procedure of fireheating until there was ebullition, characterized by the
Table 1 -
Percentage of samples in disagreement with legal
standards according to the type of milk in different
Brazilian regions
Location
Type of
milk (n)
Samples in
disagreement (%)
São Paulo, SP, 19893
A (103)
B (162)
C (165)
71.0
36.0
21.0
C (36)
14.0
Campina Grande, PB, 19957
Belo Horizonte, MG,
19958
Salvador, BA, 19959
Ribeirão Preto, SP, 199610
Natal, RN, 199811
Maringá, PR,
199812
C (1795)
25.0
– (31)
33.0
whole (141)
B (188)
C (368)
25.5
26.0
11.0
C (71)
53.5
C (50)
77.0
Bauru, SP, 199813
A (21)
B (30)
C (29)
71.0
50.0
52.0
Belém, PA, 199914
C (18)
72.0
B (25)
40.0
Uberlândia, MG,
“rise” of the milk on the recipient walls up to the superior
border. At this point, the milk pot was immediately removed
from the fire and placed inside a domestic refrigerator at 7
ºC. The temperature/time ratio (measured with a certified
thermometer and a chronometer) reached by the boiling
procedure was approximately 95 °C/3 seconds.
We took aliquots from each sample in six occasions for
laboratory analysis: a) immediately before boiling (BB); b)
immediately after boiling (AB); c) after 2 hours of
refrigeration (R2); d) after 4 hours of refrigeration (R4); e)
after 6 hours of refrigeration (R6); f) after 24 hours of
refrigeration (R24). The samples were submitted to two
laboratory analyses: standard count of facultative aerobic
mesophilic bacteria plaques, and determination of the most
probable number of total coliforms and Escherichia coli (E.
coli).20
The results obtained were compared to the maximum
microbiological standards established by the Brazilian
Ministry of Health for type C milk (Edict 451, 1997
(mesophils: 3 x 105/ml, coliforms: 10/ml, and fecal coliforms:
2/ml).
Sigma Stat for Windows was used for statistical analyses.
Considering the mesophilic bacteria count results, we
analyzed the differences between the brands, as well as the
differences between BB, AB, R2, R4, R6, and R24. In order
to determine the differences between brands, we applied the
Kruskal-Wallis analysis-of-variance-by-ranks test,
complemented by a multiple comparison test when the
difference was significant. For that, we used the results
obtained before boiling in the 10 samples of each brand. In
order to assess the count differences according to
refrigeration time, we used the Friedman test, complemented
by a multiple comparison test whenever the difference was
significant, grouping the results of the 30 samples without
considering individual brands. The efficacy of boiling was
determined through the MacNemar significance test, using
the presence/absence of coliforms in the 30 analyzed
samples, both before and after the boiling procedure. Values
of P<0.05 were considered statistically significant.
199915
Results
Table 2 presents the distribution of frequencies of
mesophilic bacteria counts in the three brands analyzed.
Median counts for brand C were significantly higher than
for brands A and B. Brands A and B did not present
statistically significant differences.
No samples (of any of the analyzed brands) presented
mesophilic bacteria count above the legal standards. Ten
samples (33%) presented coliforms - two samples of brand
A and one of brand C (10%) presented coliforms above the
allowed standards. Brand B did not have any sample with
coliforms above the standards; however, it was the brand
that presented the higher number of positive samples for
Effect of home boiling and refrigeration... - Morais TB et alii
Table 2 -
Nº de
bacteria/ml
Distribution of the frequency of facultative mesophilic
bacteria counts in three brands of type C pasteurized
milk
Brand A
n=10
Brand B
n=10
Brand C
n=10
0 |– 103
0 (0%)
0 (0%)
0 (0%)
103 |–
104
5 (50%)
7 (70%)
1 (10%)
104 |–| 105
5 (50%)
3 (30%)
9 (90%)
Median
13,700
8,250
24,400
P25-P75
5,300–21,800
4,800–10,500
20,700–43,000
coliforms (five samples). E. coli was not detected in any
sample.
Figure 1 illustrates the significant decrease in the number
of mesophilic bacteria after boiling, and the slow increase
in the number of these bacteria during the refrigeration
period.
The multiple comparison test showed that the median
mesophilic bacteria counts immediately after boiling (AB)
were significantly lower than the median before boiling
(BB). The median after 24 hours of refrigeration (R24) was
significantly higher than the median immediately after
boiling (AB). A significant increase was not observed for
median of counts when intervals R2, R4, R6, and R24 were
compared.
In the 10 samples that were positive for coliforms, these
microorganisms were not observed after boiling. The
McNemar test showed that the presence of coliforms in the
samples was significantly higher before boiling (P<0.0001).
Jornal de Pediatria - Vol. 76, Nº5, 2000 359
Discussion
Mesophilic bacteria, coliforms, and E. coli are
microorganisms also knwon as indicators; they are useful in
the determination of the microbiological quality of foods,
since they present ecological characteristics that are similar
to those of pathogenic microorganisms. However, laboratory
analysis of indicator microorganisms is faster, easier, safer,
and less expensive. The presence of indicators at certain
levels suggests that the foods have been exposed to conditions
that would also allow the presence of pathogenic
microorganisms. Therefore, they reveal the potential risk
for the presence of such agents. Indicators are also used to
verify if the treatments meant to ensure the innocuity of a
nutritive product were effective.
Although widely used, home boiling had not been tested
yet in terms of its efficacy in decreasing or eliminating the
bacterial load of milk. The results we obtained showed that
this is a highly efficient procedure - immediately after
boiling, there was a reduction of almost 200 times in the
mesophilic bacteria count, and the apparent elimination of
pasteurization-surviving coliforms. Regarding refrigeration,
we observed that the median of mesophilic counts was
significantly higher 24 hours after refrigeration when
compared to the results obtained immediately after boiling.
This is an expected result, since refrigeration does not
prevent bacterial multiplication, although it occurs very
slowly. The number of mesophilic bacteria found (465/ml),
however, is insufficient to cause any organoleptic alteration,
which usually occurs when counts reach more than 1,000,000
bacteria per ml. Concerning coliforms, if there were any
cells that survived boiling, refrigeration was enough to keep
them in non-detectable numbers.
Milk pasteurization is doubtlessly a technological
process that brought great benefits to public health. However,
due to processing defects, the presence of pathogenic
microorganisms in this product has been reported after
pasteurization.5,6 In Brazil, a study carried out in Fortaleza,4
CE, demonstrated the presence of Staphylococcus aureus
and Salmonella sp in 10% of the 20 analyzed samples.
The present study showed that there was an increase in
the quality of type C pasteurized milk commercialized in
São Paulo. In a study performed in 1989,3 21% of the
samples did not meet legal standards; in the present work,
this percentage was 10%. The quality of the milk
commercialized in São Paulo, however, is superior to that
of the milk commercialized in other regions of the state and
of the country. As it can be seen in Table 2, a high
percentage of the samples did not meet legal standards. This
situation calls for immediate action on the part of health
authorities.
Figure 1 - Medians of facultative aerobic mesophilic bacteria
counts (BB, AB, R2, R4, R6, and R24)
Taking into consideration the quality of pasteurized
milk commercialized in our country, the recommendation
to boil the milk given to children should be followed, since
this procedure, which is simple and accessible, along with
refrigeration, has proven efficient to increase the safety of
360 Jornal de Pediatria - Vol. 76, Nº5, 2000
pasteurized milk and in to preserve the organoleptic qualities
of the product.
Acknowledgements
To Janice T. Silva for the laboratory support.
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Correspondence
Dra. Tania B. Morais
Rua Araioses, 75
CEP 05442-010 São Paulo, SP, Brazil
Phone: + 55 11 262.6567 / 576.4525
Fax: + 55 11 571.1160