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Techniques for the storage of human breast

milk: Implications for anti-microbial functions
and safety of stored milk

Article in European Journal of Pediatrics · December 2000

DOI: 10.1007/s004310000577 · Source: PubMed

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Eur J Pediatr (2000) 159: 793±797 Ó Springer-Verlag 2000

REVIEW

Michael Oladipo Ogundele

Techniques for the storage of human breast milk:

implications for anti-microbial functions
and safety of stored milk

Received: 23 February 2000 / Accepted: 2 May 2000

Abstract Storage of human breast milk by freezing or refrigeration of milk with and
without heating have been recommended. This can hardly be avoided because of the
social circumstances of most mothers who are regularly separated from their infants
because of work or schooling as well as the particular needs of some pre-term or sick
babies to be fed with expressed breast milk. The greatest fear that has hindered the
prospects of in-vitro storage of breast milk for any considerable period of time is the
possibility of bacterial contamination and growth of infectious pathogens in the stored
milk, thereby rendering them unsafe for human consumption. Bacteriological examina-
tion of refrigerated milks has proven their safety for human consumption for even up to
72 h. For a storage over longer periods up to 1 month, freezing at )20 °C could be
recommended, but the most preferred method, especially for longer storage would be
fresh freezing at )70 °C, if a€ordable or available. The expressed fears arising from
increased titratable acidity of such stored milk samples have been unfounded, since it has
been shown to be mainly attributable to levels of free fatty acids, rather than lactic acid,
which might have been produced by bacterial fermentation of milk sugars.
Conclusion Evidence shows that temporary storage of human milk under appropriate
conditions is not dangerous for babies and infants. This would further encourage the
practice of prolonged exclusive breastfeeding and allow the families to reap its multi-fold
bene®ts.

Key words Human milk á Infection á Anti-microbial factors á

Complement activation á Pasteurisation

other diseases, especially in developing countries [7, 12].

Introduction
Considering the fact that breast milk is the most optimal
source of nutrients and an unmatched source of indis-
The World Health Organisation and other international
pensable anti-microbial and other protective substances,
interest groups have recently mounted increasing cam-
the need to store breast milk for at least limited periods
paign e€orts aimed at enlightenment and education
of time is unavoidable in a neonatal unit taking care of
about the indispensable values of breast milk and
sick and pre-term newborn infants [3, 13, 14]. For pre-
breastfeeding, thereby advocating prolonged exclusive
term neonates, who are particularly susceptible to in-
and complementary breastfeeding [33].
fection and often require prolonged intensive care in the
There is conclusive evidence that breastfeeding pro-
hospital, maternal milk may still be inadequate for the
tects the infant against a wide range of infectious and

M. O. Ogundele (&)1 Present address:

1
Department of Immunology, Department of Medical Information Science,
Georg-August University, University of Applied Sciences, Luxemburger Str. 10,
Kreuzbergring 57, 37075 GoÈttingen, 13353 Berlin, Germany
Germany e-mail: mogundel@yahoo.com
Tel.: +49-30-45490489, Fax: +49-30-45490489
794
®rst few days due to an inadequate suckling stimulation
by the weak neonate [22, 31]. The diculties involved in
transportation from the home by the mother who may
be living far away from the hospital, with an increased
risk of bacterial contamination associated with unhy-
gienic preparation and improper storage of arti®cial
formula feeds, are often indications for babies to be fed
with donor breast milk that is aseptically stored [3, 9, 18,
26, 31]. Some small-for-dates babies could also need to
be given additional milk, other babies may be excessively
hungry, while others may have mothers who are unwell
after delivery and unable to express breast milk e.g. after
caesarean section.
Increasing numbers of mothers, even in developing
countries, have to go out to work soon after delivery
[24, 26] and who still want to exclusively breastfeed their
children. Modern milk banks are often located in large
central hospitals, while milk donated from the smaller
hospitals and voluntary donors often needs to be tem-
porarily stored before being transported in special cool
boxes to the milk bank. Even after arriving at the milk
bank, performing microbiological and other control
tests often cause a delay in making the milk available for
use. The donated milk is often stored for varying peri-
ods before being ready for use [13]. The need for storage
is no longer a debatable subject, however the choice of
the most appropriate storage method is not often easy
to make. The greatest fear that has hindered the pros-
pects of in-vitro storage of breast milk for any consid-
erable period of time is the possibility of bacterial
contamination and growth of infectious pathogens in
the stored milk, thereby rendering it unsafe for human
consumption [34]. This short review aims to assist in
arriving at the right decision concerning the mode of
milk storage depending on the anticipated duration,
based on recent advances in our knowledge of milk
physiology.
Table 1 Major anti-microbial factors in human milk
E€ect against microbes
Secretory IgA Trans-luminar transport, neutralisation,
antigen binding, complement activation
IgG Neutralisation, activation of complement
IgM Neutralisation, activation of complement
Bi®dobacterium Bacteriostatic
growth factor
Lysozyme, Bacteriostatic, anti-in¯ammatory
lactoperoxidase
Complement Lysis, opsonisation, viral neutralisation
Lactose Increased calcium bio-availability
and Lactobacillus growth
Lactoferrin Bacteriostatic (bind iron)
Gangliosides, Receptor-like functions
glycoconjugates
Cytokines Immuno-modulation, anti-allergy
Lipid products Lysis
Milk cells Phagocytosis, immune-modulation,
antibody production
Antimicrobial factors in human milk
Table 1 shows a summary of the major anti-microbial
components of human milk and the e€ects of heating.
These include among others speci®c anti-viral antibod-
ies, antibacterial antibodies, non-speci®c IgG, IgA, IgM,
lactoferrin, GM1-like gangliosides, glycoconjugates, ol-
igosaccharides, products of milk lipids, di€erent cyto-
kines, lymphocytes, polymorphonuclear leucocytes,
macrophages, and complement proteins [10, 20, 21, 23].
Milk enzymes such as lactoperoxidase and lysozyme are
also known to have bacteriostatic e€ects. The clinical
signi®cance of these defence factors has been demon-
strated in several reports comparing the morbidity and
mortality of breast-fed and formula-fed infants [7, 12].
The availability or otherwise of these protective factors
to the infant depends largely on the method of milk
storage [8, 16, 27, 35±37].
Effects of different storage techniques
on the antibacterial components of human breast milk
A number of protective breast milk components may
be altered or irreversibly damaged by such processes as
freeze-thawing or heating. The major changes observed
in breast milk stored frozen at )20 °C for up to
1 month include progressive loss of up to 89% cell
viability and loss of bacteriostatic activity, whereas the
other immunoproteins IgA, IgG, IgM, lactoferrin,
lysozyme, C3 and C4 complement components, amino-
acids and fatty acids could be largely preserved or less
signi®cantly a€ected [4, 6, 29]. The proportion of viable
cells in those surviving is often reduced, with the
appearance of a large number of inactive ``ghost'' cells
[11, 16].
E€ect of heat Reference
After 30 min stable at 56 °C, some loss [4, 16, 21, 28, 29, 31]
at 62.5 °C; destroyed by sterilisation
After 30 min stable at 56 °C, some loss [4, 16, 28, 29, 31]
at 62.5 °C; destroyed by sterilisation
After 30 min stable at 56 °C, destroyed [4, 29, 31]
at 62.5 °C
Stable on sterilisation [4, 20]
Some loss at 56 °C for 30 min; destroyed [4, 20]
by sterilisation for 15 min
Destroyed after 30 min at 56 °C [23, 25]
Stable at 85 °C for 30 min [4, 20, 31]
66% destroyed after 30 min at 62.5 °C [4, 20, 31]
Stable on sterilisation [4, 29, 31]
Unknown [10]
Stable on sterilisation [15, 19, 36, 37]
Destroyed after 30 min at 62.5 °C [4, 6, 11, 16, 22, 29, 31]

The storage of human milk at +4 °C for 48 h after
expression could also result in a signi®cant loss of cel-
lular viability but does not denature milk proteins. The
loss of milk macrophages and neutrophils occur pre-
sumably via cell adhesion to the milk container or
cytolysis. The milk lymphocyte concentration, however,
is not signi®cantly a€ected by storage [4, 27]. The
antibacterial activity of the remaining milk cells is,
however, largely retained after storage at +4° C for
24±48 h [22].
After standard heat sterilisation, IgA and lactoferrin
were undetectable in milk samples [28]. Others have
found this to signi®cantly alter the levels of major anti-
microbial components of milk causing up to 47% loss of
IgA, more than 88% loss of IgG, 41%±47% loss of
lactoferrin and alpha-1-antitrypsin, and some proteins
are also denatured [4, 16, 31]. Holder pasteurisation
(62.5 °C, 30 min) could cause a less signi®cant loss of
IgA but destroyed the small content of IgM. Lysozyme
and lactoferrin seem to be stable to this treatment.
Lysozyme was however progressively destroyed with
increasing temperature, to near 100% at 100 °C [8].
Pasteurisation also leads to near total loss of milk cells
[4]. Inactivation of milk bile-stimulated lipase by pas-
teurisation is also capable of a signi®cant decreases in
the absorption of milk lipids by about 33% corre-
sponding to about 16% of the consumed energy [36, 37].
Microbiological quality of breast milk
under different storage conditions
Freshly collected breast milk is rarely sterile and nor-
mally contains bacteria originating from the maternal
skin and nipple duct micro-¯ora, but it also sometimes
contain potential pathogens [1, 6, 26]. However, the
ability of these organisms to infect the suckling infant is
uncertain and probably minimal [4]. Nevertheless, heavy
bacterial contamination at the time of milk collection
would be undesirable and should be avoided [26]. Some
of the milk pathogens could produce lipases, proteases
and decarboxylases which may cause damage to anti-
microbial proteins or convert free amino-acids into toxic
amines [4]. Other factors associated with increased
bacterial contamination of expressed breast milk include
undue delay of the initiation of milk expression in the
post-partum period [2].
The most convenient and a€ordable means of milk
storage at home or small clinics for short periods is by
refrigeration. This process has a number of advantages
over freezing. It prevents thawing and prolonged re-
warming that may accompany freezing. Many bacteri-
ological studies have concentrated on examining the
changes occurring in breast milk stored by refrigeration
or left at room temperature [11, 26]. From studies con-
ducted under tropical conditions, unheated breast milk
could be stored for at least 8 h at room temperature and
up to 24 h at +4 °C in the refrigerator before bacterial
multiplication occurs beyond unacceptable levels [25].
795
Similar studies under temperate climatic conditions have
shown that breast milk stored for up to 48±72 h in the
refrigerator is bacteriologically acceptable [4, 6, 13].
Most milk samples show a signi®cant drop in the level of
bacterial colony counts during refrigeration. This is in
sharp contrast to cow's milk formula which shows sig-
ni®cant increase in the bacterial counts during 6 h of
storage at both room temperature and under refrigera-
tion and no evidence of bacteriostatic activity at any
period [1, 11, 13, 26]. A similar decrease in bacterial
colony count has also been demonstrated in human milk
after 5±21 days freezing at )20 °C followed by thawing
[6, 11].
Bacteriostatic activity of human milk against arti®-
cially inoculated bacteria of di€erent strains has been
shown to be highest in fresh refrigerated or fresh frozen
(at )70 °C) and thawed milk. Bacteriostatic activity of
frozen milk (at )20 °C) tends to progressively deterio-
rate after 1 month of storage. This activity is however
lost after pasteurisation [11, 29]. The possibility of ac-
cumulation of undesirable bacterial products such as
enterotoxins, bacterial enzymes and reactive amines is
very unlikely even at refrigerator temperatures because
most bacteria are metabolically inactivated at this tem-
perature and the other antibacterial systems in the milk
act to prevent any signi®cant bacterial growth and
activity [4, 17].
Storage enhances anti-microbial functions
of human milk
The bacteria-adhesive role of high molecular weight
mucus components of the milk fat globule membrane
has been implicated in preventing the colonisation and
infection of epithelial mucosa by certain meningitis-
causing bacterial strains in neonates [32]. Recent studies
have shown that the ability of colostrum samples stored
by refrigeration (at +4 °C in the absence of anticoag-
ulants) to bind to pathogenic bacteria in-vitro was
greatly enhanced within the ®rst 3 days of storage, in
comparison to freshly collected milk or that frozen at
)20 °C or )70 °C. The initial increase in the levels of
sequestered bacteria was followed by a progressive fall,
gradually approaching the level cultured from the nor-
mal saline control after 3 weeks (personal observations).
Similar observations were also made for other transi-
tional and mature whole-milk samples. This sequestra-
tion activity was lost by de-fattening of the milk samples
by centrifugation. This enhancement of bacteria
sequestration has been proposed to be attributed to the
e€ects of accumulated fragments of complement
activation in the stored milk.
Refrigeration and deep freezing of milk have also
been associated with enhanced bacteriostatic activities
and reduced contaminant bacterial load [11, 29]. Fur-
thermore, freezing of milk for at least 1 day is known to
destroy cytomegalovirus shed into the milk from
infected mothers [5].
796
Free fatty acids produced during prolonged storage
of breast milk by the e€ect of milk lipases have been
shown to have very potent cytolytic e€ects on normal
human blood cells [30], and on pathogenic organisms,
including killing of intestinal parasites, Giardia lamblia
and Entamoeba histolytica, predominantly gram-positive
bacteria and yeast, and causing a membrane disruption
of enveloped viruses in cultured cells. The levels of free
fatty acids progressively increase in the stored milk in a
temperature-dependent manner, but is highest if it is
initially cooled by refrigeration at +4 °C [15]. The
possible activation and involvement of complement
components in contributing to this enhanced in-vitro
anti-microbial activity have been suggested [25]. The
increased titratable acidity of such stored milk samples
has been shown to be mainly attributable to levels of free
fatty acids rather than lactic acid, which is feared to have
been produced by bacterial fermentation of milk sugars
[19].
Conclusion
The increasing advocate for prolonged exclusive and
complementary breastfeeding by mothers who may be
working or schooling, as well as the particular needs of
some pre-term babies to be fed with expressed breast
milk, would require more mothers to express milk
during the periods of their separation and keep them
safe for their babies consumption. Transport to and
from milk banks and processing of milk also often
necessitate temporary or prolonged storage of milk.
Freezing or refrigeration of milk with and without
heating has been recommended [4]. The potential ability
of rapid freeze-thawing to disrupt milk fat globule
membranes and the progressive deterioration in the
bacteriostatic capacity of frozen milk, would suggest
that refrigeration would be preferable for short-term
storage. For storage over periods longer than 3 days up
to 1 month, freezing at )20 °C could be recommended
and fresh freezing at )70 °C for longer-term storage.
Fears arising from increased acidity of stored milk and
about the possible accumulation of undesirable bacte-
rial products such as enterotoxins, bacterial enzymes
and reactive amines have been allayed. On the contrary,
certain defensive functions of stored milk are potentially
enhanced.
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