Early Human Development 87 (2011) 577–580
Contents lists available at ScienceDirect
Early Human Development
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e a r l h u m d ev
Analysis of the influence of pasteurization, freezing/thawing, and offer processes on
human milk's macronutrient concentrations☆
Alan Araujo Vieira 1, Fernanda Valente Mendes Soares 1, Hellen Porto Pimenta 2,
Andrea Dunshee Abranches 2, Maria Elisabeth Lopes Moreira 1,⁎
Instituto Fernandes Figueira, Av. Rui Barbosa 716, Rio de Janeiro, RJ CEP 22540-020, Brazil
a r t i c l e
i n f o
Article history:
Received 12 March 2007
Received in revised form 24 April 2011
Accepted 27 April 2011
Keywords:
Human milk
Pasteurization
Infrared analyzer
a b s t r a c t
Introduction: The macronutrient concentrations of human milk could be influenced by the various processes
used in human milk bank.
Aims: To determine the effect of various process (Holder pasteurization, freezing and thawing and feeding
method) on the macronutrient concentration of human milk.
Methods: The samples of donated fresh human milk were studied before and after each process (Holder
pasteurization, freezing and thawing and feeding method) until their delivery to newborn infants. Fifty-seven
raw human milk samples were analyzed in the first step (pasteurization) and 228 in the offer step. Repeated
measurements of protein, fat and lactose amounts were made in samples of human milk using an Infrared
analyzer. The influence of repeated processes on the mean concentration of macronutrients in donor human
milk was analyzed by repeated measurements ANOVA, using R statistical package.
Results: The most variable macronutrient concentration in the analyzed samples was fat (reduction of 59%).
There was a significant reduction of fat and protein mean concentrations following pasteurization (5.5 and
3.9%, respectively). The speed at which the milk was thawed didn't cause a significant variation in the
macronutrients concentrations. However, the continuous infusion delivery significantly reduced the fat
concentration. When the influence of repeated processes was analyzed, the fat and protein concentrations
varied significantly (reduction of 56.6% and 10.1% respectively) (P b 0.05). Lactose didn't suffer significant
reductions in all steps.
Conclusion: The repeated processes that donor human milk is submitted before delivery to newborn infants
cause a reduction in the fat and protein concentration. The magnitude of this decrease is higher on the fat
concentration and it needs to be considered when this processed milk is used to feed preterm infants.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Human milk bank is the practice of collecting milk from lactating
mothers with the aim to feeding another woman's babies. Since 1909,
milk banks have been practiced throughout the world but their
popularity has been affected by several factors: development of infant
formula, emergency of HIV/AIDS and improvement in neonatal care
that enabled an increase survival of very low birth weight infant [1].
Currently, there is across the world, a growing enthusiasm about
☆ Acknowledge financial support: CNPq.
⁎ Corresponding author at: Rua Prudente de Moraes 368/808, Rio de Janeiro, RJ
CEP 22420-040, Brazil. Tel./fax: + 55 21 25541819.
E-mail addresses: [email protected] (A.A. Vieira), [email protected]
(F.V.M. Soares), [email protected] (H.P. Pimenta), bebeth@iff.fiocruz.br
(M.E.L. Moreira).
1
Participate in conception and design of the study, acquisition of data, analysis and
interpretation of data and drafting the article.
2
Participate in acquisition of data, analysis and interpretation of data and drafting
the article.
0378-3782/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.earlhumdev.2011.04.016
human milk banks and several countries has developed guidelines
and facilities to implement donors human milk banks [1–4].
Despite of human milk banks around the world have arisen,
evidences about the role of donor breast milk in current neonatal
practice remains to be established. Meta-analysis based on three
studies found a lower risk of NEC in infants receiving donor breast
milk compared with formula (combined RR 0.21, 95% CI 0.06 to 0.76)
but donor breast milk was associated with slower growth in the early
postnatal period [5].
The donor milk must be collected, processed and stored in way
that ensures its microbiological safety and nutritional quality [6]. The
nutritional quality control of human milk storage is complex, although
very necessary. The pasteurization is a mandatory step in order to
inactivate pathogenic microorganisms. For storage, freezing and
thawing is necessary. All these process could reduce macronutrients
concentrations and this donor milk from human milk bank could be
not enough to meet the specific needs of preterm infants [7,8].
Besides that, the breast milk varies widely in its composition:
throughout the lactation cycle, throughout the day, with maternal diet
578
A.A. Vieira et al. / Early Human Development 87 (2011) 577–580
and length of gestation. The energy content of milk is lower at the
beginning of a feed than at the end and in drip breast milk than in
expressed breast milk. Donors have also usually delivered at term or
have been lactating for some time, both of which result in lower
nutritional content [1,8].
The poor weight gain observed in preterm infants who are fed by
pooled human milk and the low fat and energy content of stored
human milk offered to preterm infants caught attention [9,10]. There
is a consensus about the special needs and the importance of
delivering the needed nutrients to feed very low birth weight infants
in order to promote adequate growth and development [11].
The use of banked donor term milk from Human Milk Banking is
still controversy, non-evidence based and need to research-driven
standardization of practice. The objective of this study is to determine
the effect of pasteurization, freezing, thawing and feeding method on
fat, lactose, and protein concentrations in human milk.
Raw human milk
80ml
Analyze
6ml
Pasteurization
(LTLT)
Pasteurized human
milk
analyse = 57
Pasteurized human
milk
analyse
n = 57
Quickly-thawed
human milk
n = 114
Freezing at -20°C
Slowly-thawed
human milk
2. Methods
After formal consent, experimental study samples with eighty
milliliters of raw human milk were donated by nursing women. The
samples were collected by hand expression or by the use of a breast
pump (manual or electric), stored in glass flasks and managed
throughout all the habitual processes of pasteurization, freezing,
thawing (both quick and slow) and being offered to preterm infants
(gavage and continuous infusion). At each stage, 6 ml of human milk
was reserved from the samples to be analyzed by the infrared technique
[12]. Time between expression and pasteurization was at most 30 min.
Fifty-seven raw human milk samples, each one with 80 ml, were
analyzed before and after the first step (pasteurization). After dividing
each original sample into two aliquots, all 114 samples were frozen for
24 h. Later, the 114 samples were thawed and analyzed and again
divided into two aliquots, resulting in 228 samples that were analyzed
after the 2 feeding methods. At each step, 6 ml of human milk was
forwarded to infrared analyses (Fig. 1).
The pasteurization technique followed the worldwide guidelines
for human milk banks (Long Term/Low Temperature technique—LTLT
−62.5 °C for 30 min, followed by rapid cooling) [3]. After pasteurization, the samples were divided into two aliquots with 40 ml each
and frozen at −20 °C.
After 24 h, the thawing of the samples was performed in two
ways — slow thaw: immersion of the samples in water bath at 40 °C
for 10 min; and quick thaw: microwave oven for a period of 45 s, with
caution taken not to let the milk boil and remove the hazard of
product contamination due to water entering through the bottle.
The feeding method was performed by the two main ways of
feeding preterm infants that cannot suck — gavage and continuous
infusion. In the gavage process, the human milk sample (8 ml) was
offered using a 10 ml syringe and a nasogastric tube number 4, with
the milk pushed by gravitational power. In the continuous infusion
process, a 10 ml syringe and a nasogastric tube number 4 were also
used, but a 40 cm perfusion tube was added between the syringe and
the nasogastric tube. The established time for the continuous infusion
was 1 h and the amount of sample that remained in the perfusion and
nasogastric tubes was measured. The syringe pump was put in a
horizontal position. After the delivery of the milk, 6 ml of the sample
was taken to the infrared analyzer for macronutrient measurements.
To perform the analyses of the samples we use the infrared
analyzer (Milko-scan Minor™), [12] previously calibrated for human
milk measurements using Kjeldahl method for protein, Chloramine-T
for lactose and Gerber for fat.
We also analyzed 10 samples without procedures as controls and
perform 4 repeated measurements for protein, fat and lactose in each
sample using infrared analyzer.
The mean macronutrient contents of the human milk samples
were compared throughout each step of the process by Friedman and
n = 57
n =114
Analyze
6ml
Analyze
6ml
n = 57
Gavage
offer
n = 57
n = 57
n = 57
Continuous
infusion offer
Continuous
infusion offer
Gavage
offer
Analyze – 6ml of each offer process
Fig. 1. Description of the study design.
t tests. The mean macronutrient contents of human milk samples after
the thawing and offer processes were compared by ANOVA, using the
statistical package SPSS9.0. The influence of repeated processes on the
mean concentration of macronutrients in human milk was analyzed
by repeated measurements ANOVA, using R statistical package.
Sample calculation considered a difference between the means of
0.5 mg%, an alpha error of 5%, and a beta error of 20%. This study was
approved by the Ethics Committee of Fernandes Figueira Institute.
3. Results
There was a significant difference between the mean concentrations of fat and protein throughout the processes studied, a reduction
of 5.5% and 3.9%, respectively. There was no significant difference on
lactose concentration through the processes studied (Table 1).
The most variable macronutrient concentration in the analyzed
samples was fat (0.96–8.26 mg%).
Using the Wilcoxon test, it was demonstrated that the process
responsible for the significant difference between the mean of the fat
and of the protein was pasteurization (p b 0.001).
There were no significant differences in fat, protein and lactose
concentrations between the slow and quick thaw (Table 2). However,
the difference of the fat means between the gavage and continuous
Table 1
Comparison of mean fat, protein and lactose concentrations (mg%) in human milk
through the studied processes.
Raw
Mean ± Sd
Pasteurized
Median Mean ± Sd
Fat
2.17±1.46 1.72
Protein 1.03±0.39 0.95
Lactose 6.36 ± 0.51 6.49
a
Friedman test.
Thawed
Median Mean ± Sd
2.05 ± 1.46 1.67
0.99 ± 0.42 0.92
6.28 ± 0.54 6.48
Median Pa
2.00 ± 1.45 1.60
0.97 ± 0.41 0.89
6.34 ± 0.55 6.48
b 0.001
b 0.001
0.427
A.A. Vieira et al. / Early Human Development 87 (2011) 577–580
Table 2
Comparison of mean fat, protein and lactose concentrations (mg%) in human milk
between the thawing processes.
Slow thaw
Fat
Protein
Lactose
a
Quick thaw
Mean ± Sd
Median
Mean ± Sd
Median
z
Pa
2.00 ± 1.42
0.95 ± 0.41
6.35 ± 0.54
1.61
0.88
6.47
2.00 ± 1.48
0.99 ± 0.42
6.33 ± 0.57
1.60
0.92
6.48
− 0.056
− 0.823
− 0.18
0.956
0.410
0.860
Mann–Whitney test.
infusion was significant, with the continuous infusion being responsible for the lesser fat mean concentration (53.6%). There was no
difference in protein and lactose between the two offer processes
(Table 3).
Analyzing the longitudinal variation of macronutrient mean
concentrations between each group studied, that is, the groups of
slow thaw with the human milk offered by gavage or by continuous
infusion and the groups of quick thaw with the human milk offered by
gavage or by continuous infusion, there were significant differences in
fat and protein concentrations, mainly in the groups where the human
milk was offered by continuous infusion (a reduction of 58.9% and
10.1%, respectively). There were no differences in mean concentration
of lactose among the groups studied (Table 4).
When we analyzed the raw milk without process (10 controls
samples), we didn't observe significant differences between feeding
method. The coefficient of variation between controls samples in four
repeated measurements for protein, fat and lactose was less than 1%
for fat and 0.7% for protein and lactose.
4. Discussion
Nowadays, human milk banks are experiencing a resurgence of
interest and resources [13]. However, the poor weight gain observed
in preterm infants who were fed by pooled donor human milk calls
into question the adequacy of using this milk for feeding preterm
infants [1,8]. Even with this inadequacy, human milk is still
recommended for very low birth weight (VLBW) infants because of
the benefits reaped in stimulating the defense mechanisms of the
body and the unique profile of its fat content [14].
The donor human milk necessarily needs to be processes before
being administered to newborn infants. Throughout these processes,
significant energy losses are detected, primarily from the fat content
which is the main caloric-energetic source of human milk [14]. This
fact was confirmed in this study, where the mean fat concentration
decreased up to 1.2 mg% (58.9%) throughout the studied processes
(pasteurization, freezing, thawing and offer). Previous study did not
show differences in fat content between raw human milk and
pasteurized milk but we find less fat content after pasteurization
(3%). Another difference between these 2 studies is the initial fat
content in raw donor milk [15].
Fat is the most variable changeable constituent of human milk,
with different concentrations in mature milk, hind milk, manually
expressed milk and milk collected in the afternoon, not to mention the
inter-individual variations [16].
Table 3
Comparison of mean fat, protein and lactose concentration (mg%) in human milk
between the offer processes.
Gavage
Fat
Protein
Lactose
a
Continuous infusion
Mean ± sd
Median
Mean ± sd
Median
t
Pa
1.89 ± 1.21
0.95 ± 0.39
6.34 ± 0.56
1.58
0.89
6.50
0.95 ± 0.99
0.90 ± 0.40
6.33 ± 0.55
0.65
0.83
6.55
− 7.11
− 1.19
− 0.58
b 0.001
0.410
0.559
Mann–Whitney test.
579
Table 4
Comparison of human milk macronutrient concentrations (mg%) throughout the
studied treatment and offer processes — analysis of associated effects on slowly-thawed
and quickly-thawed human milk (ANOVA for repeated measurements).
Treatment and offer processes
Lactose
Fat
Protein
Raw
Post-pasteurization
Slow-thawed-gavage offered
Slow-thawed-continuous
infusion offered
Quickly-thawed-gavage offered
Quickly-thawed-continuous
infusion offered
F
P
6.31 ± 0.51
6.28 ± 0.54
6.35 ± 0.57
6.38 ± 0.56
2.17 ± 1.46
2.05 ± 1.46
1.91 ± 1.21
0.89 ± 0.99
1.03 ± 0.39
0.99 ± 0.42
0.96 ± 0.41
0.90 ± 0.39
6.32 ± 0.54
6.36 ± 0.56
1.88 ± 1.22
1.00 ± 0.99
0.94 ± 0.38
0.89 ± 0.41
0.58
0.629
36.71
b 0.001
1.92
0.046
Besides that, the repeated process of freezing and thawing can
alter the fat globule structure in human milk. This fact, associated with
the use of plastic devices to deliver human milk to preterm infants,
mainly when this delivery process takes a long time (one hour, for
example), might be an explanation for the intense decline of fat
content in the samples that were delivered by continuous infusion —
the rupture of fat globule membranes results in coalescence and
facilitates adherence to flask walls and to utensils used to feed the
newborn infants (plastic equipment and syringes) [16–18]. Consequently, if the milk is infused by a gastric tube using syringe pumps
held vertically and milk is not homogenized before feeding, losses can
reach 34% of the fat content [18]. Large fat losses occurred, continuous
feeding giving rise to significantly greater losses than bolus feeding.
More fat loss occurred with low flow rates and there were also
significant protein losses [19].
It is important to remember that the pasteurization process
inactivates the bile salt-stimulated lipase, which is greatly responsible
for the digestion and absorption of fat in newborn infants, decreasing
the maximum utilization of the delivered human milk [20].
In the studied samples there was a difference in the mean protein
concentrations, mainly between the raw and post-pasteurization
samples (reduction of 3.9%). Pasteurization causes the loss of variable
amounts of IgA, IgM, IgG, lactoferrin, certain vitamins, and other milk
components; it might not affect the nutritional value of the proteins,
but it interferes in their bioactive function [21].
The mean loss of protein in the studied samples was 0.12 mg%
(13.6%), an amount that is of clinical importance. A plausible
explanation for the declining protein concentration found in the
continuous infusion offer samples could be the absorption of the
proteins into the fat globule surface when the milk fat globules were
disrupted during heat treatment. So, as the fat remained adhered to
the plastic surfaces of the delivery devices, a small amount of protein
was also absorbed by the fat globule membranes [22–24].
The small carbohydrate variation in human milk contents found in
this study is in accordance with available literature [25,26]. There
were no significant differences in mean concentrations of human milk
lactose among the processes studied (a reduction of 0.5%).
Concern over the low rate of weight gain of newborn infants fed
exclusively with human milk often leads to the use of artificial
formula with no prior evaluation of the quality of the human milk
being offered. Unformulated banked donor milk alone, does not have
sufficient macronutrient content or energy density to sustain a verylow-birth-weight preterm infant. The control of human milk
macronutrient variations could direct the attention to target supplementation of breast milk for VLBW infants based on spot sample
measurements. Through continuous monitoring of the macronutrient
content in human bank milk, it is possible to develop milk with
sufficient protein and energy content to cover the needs of preterm
infants, mainly the very low birth weight ones [1]. Fortification could
make up for these shortcomings, perhaps making formulated banked
580
A.A. Vieira et al. / Early Human Development 87 (2011) 577–580
donor milk a better choice for preterm infants than bovine-based
formulas when mother's milk is unavailable [27–29].
Determine the nutritional content of donor breast milk in Human
Milk Banking could be important to supply individualized fortification
to further improve the nutritional management of very-low-birthweight infants.
References
[1] Leaf A, Winterson R. Breast-milk banking: evidence of benefit. Pediatr Child Health
2009;19:395–9.
[2] National Network Human Milk Banks from Health Minister- Brazil . Available in
http://www.redeblh.fiocruz.br. Access in: 25/03/2011.
[3] Updegrove K. Human milk banking in the United States. Newborn Infant Nursing
Rev 2005;5:27–33.
[4] Hartmann BT, Pang WW, Keil AD, Hartmann PE, Simmer K. Best practice guidelines
for the operation of a donor human milk bank in an Australian NICU. Early Hum
Dev 2007;83:667–73.
[5] Boyd CA, Quigley MA, Brocklehurst P. Donor breast milk versus infant formula for
preterm infants: systematic review and meta-analysis. Arch Dis Child Fetal
Neonatal Ed 2007;92:169–75.
[6] Moltó-Puigmartí C, Permanyer M, Castellote AI, López-Sabater MC. Effects of
pasteurisation and high-pressure processing on vitamin C, tocopherols and fatty
acids in mature milk. Food Chem 2011;124:697–702.
[7] Tully DB, Jones F, Tully MR. Donor milk: what's in it and what's not. J Hum Lact
2001;17:152–5.
[8] Modi N. Donor breast milk banking. BMJ 2006;333:1133–4.
[9] Gianini NM, Vieira AA, Moreira ME. Evaluation of the nutritional status at
40 weeks corrected gestational age in a cohort of very low birth weight infants. J
Pediatr (Rio J) 2005;81:34–40.
[10] Vieira AA, Moreira MEL, Rocha AD, Pimenta HP, Lucena SL. Assessment of the
energy content of human milk administered to very low birth weight infants. J
Pediatr (Rio J) 2004;80:490–4.
[11] Shanler RJ. The use of human milk for premature infants. Clin Perinatol 2001;48:
206–19.
[12] Michaelsen KF, Pedersen SB, Skafte L, Jaeger P, Peitersen B. Infrared analysis for
determining macronutrients in human milk. J Pediatr Gastroenterol Nutr 1988;7:
229–35.
[13] Tully MR, Lockhart-Borman L, Updegrove K. Stories of success: the use of donor
milk is increasing in North America. J Hum Lact 2004;20:75–7.
[14] Shanler RJ. Suitability of human milk for the low-birthweight infant. Clin Perinatol
1985;22:207–22.
[15] Fidler N, Sauerwald TU, Demmelmair H, Koletzko B. Fat content and fatty acid
composition of fresh, pasteurized, or sterilized human milk. Adv Exp Med Biol
2001;501:485–95.
[16] Thomas MR, Chan GM, Book LS. Comparison of macronutrient concentration of
preterm human milk between two milk expression techniques and two
techniques for quantization of energy. J Pediatr Gastroenterol Nutr 1986;5:
597–601.
[17] Metha NR, Hamosh M, Bitman J, Wood DL. Adherence of medium-chain fatty acids
to tube feeding of human milk during gavage feeding. J Pediatr 1998;112:474–6.
[18] Thomaz ACP, Gonçalves AL, Martinez FE. Effects of human milk homogenization
on fat absorption in very low birth weight infants. Nutr Res (Los Angel) 1999;19:
483–92.
[19] Stocks RJ, Davies DP, Allen F, Sewell D. Loss of breast milk nutrients during tube
feeding. Arch Dis Child 1985;60:164–6.
[20] DeBelle RC, Vaupshas BS, Vitullo BB, Haber LR, Shafer E, Mackie GG, et al. Intestinal
absorption of salts: immature development in the neonate. J Pediatr 1979;94:
472–6.
[21] Henderson TR, Fay TN, Hamosh M. Effect of pasteurization on long chain
polyunsaturated fatty acid level and enzyme activities of human milk. J Pediatr
1998;132:876–8.
[22] Yo A, Singh H, Taylor MW, Anema SG. Interactions of fat globule surface proteins
during concentration of whole milk in a pilot-scale multiple-effect evaporator. J
Dairy Res 2004;71:471–9.
[23] Weber A, Loui A, Jochum F, Bührer C, Obladen M. Breast milk from mothers of very
low birthweight infants: variability in fat and protein content. Acta Paediatr
2001;90:772–5.
[24] Prentice A, Prentice AM, Whitehead RG. Breast-milk fat concentrations of rural
African women 1. Short term variations within individuals. Br J Nutr 1981;45:
483–94.
[25] Polberger S, Lönnerdal B. Simple and rapid macronutrient analysis of human milk
for individualized fortification: basis for improved nutritional management of
very-low-birth-weight infants? J Pediatr Gastroenterol Nutr 1993;17:283–90.
[26] Michaelsen KF, Skafte L, Badsberg JH, Jørgensen M. Variation in macronutrients in
human bank milk: influencing factors and implications for human milk banking. J
Pediatr Gastroenterol Nutr 1990;11:229–39.
[27] Wojcik KY, Rechtman DJ, Lee ML, Montoya A, Medo ET. Macronutrient analysis of a
nationwide sample of donor breast milk. Am Diet Assoc 2009;109:137–40.
[28] Morales Y, Schanler RJ. Human milk and clinical outcomes in VLBW infants: how
compelling is the evidence of benefit ? Semin Perinatol 2007;31:83–8.
[29] Cooke RJ. Adjustable fortification of human milk fed to preterm infants. J Perinatol
2006;26:591–2.