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Short communication: Effect of refrigerated storage on the pH and bacterial content of pasteurized human donor milk

  • Author Footnotes
    2 SAMID Network (Spanish Collaborative Maternal and Child Health Research Network); RD12/0026/007.
    S. Vázquez-Román
    Correspondence
    Corresponding author
    Footnotes
    2 SAMID Network (Spanish Collaborative Maternal and Child Health Research Network); RD12/0026/007.
    Affiliations
    Neonatology Department, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain

    Pediatrics Department, Universidad Complutense, 28040 Madrid, Spain
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  • Author Footnotes
    2 SAMID Network (Spanish Collaborative Maternal and Child Health Research Network); RD12/0026/007.
    D. Escuder-Vieco
    Footnotes
    2 SAMID Network (Spanish Collaborative Maternal and Child Health Research Network); RD12/0026/007.
    Affiliations
    Neonatology Department, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain

    Pediatrics Department, Universidad Complutense, 28040 Madrid, Spain
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  • M.D. Martín-Pelegrina
    Affiliations
    Neonatology Department, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain

    Pediatrics Department, Universidad Complutense, 28040 Madrid, Spain
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  • B. Muñoz-Amat
    Affiliations
    Neonatology Department, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain

    Pediatrics Department, Universidad Complutense, 28040 Madrid, Spain
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  • L. Fernández-Álvarez
    Affiliations
    Pediatrics Department, Universidad Complutense, 28040 Madrid, Spain

    Nutrition and Bromatology Department, Universidad Complutense, 28040 Madrid, Spain
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  • P. Brañas-García
    Affiliations
    Microbiology Department, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain
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  • D. Lora-Pablos
    Affiliations
    Clinical Research Unit, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain

    CIBER de Epidemiología y Salud Pública (CIBERESP), Madrid, Spain
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  • J. Beceiro-Mosquera
    Affiliations
    Neonatology Department, Hospital Universitario Príncipe de Asturias, 28805 Alcalá de Henares, Spain
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  • Author Footnotes
    2 SAMID Network (Spanish Collaborative Maternal and Child Health Research Network); RD12/0026/007.
    C.R. Pallás-Alonso
    Footnotes
    2 SAMID Network (Spanish Collaborative Maternal and Child Health Research Network); RD12/0026/007.
    Affiliations
    Neonatology Department, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain

    Pediatrics Department, Universidad Complutense, 28040 Madrid, Spain
    Search for articles by this author
  • Author Footnotes
    2 SAMID Network (Spanish Collaborative Maternal and Child Health Research Network); RD12/0026/007.
Open ArchivePublished:October 03, 2018DOI:https://doi.org/10.3168/jds.2018-14984

      ABSTRACT

      Once pasteurized donor milk is thawed for its administration to a preterm or sick neonate, and until it is administered, it is kept refrigerated at 4 to 6°C for 24 h. After this time, unconsumed milk is discarded. This time has not been extended, primarily because of the concern of bacterial contamination. The aim of this study was to determine the changes in pH and bacterial count when pasteurized donor milk was kept under refrigeration for a prolonged period (14 d). In this prospective study, 30 samples of pasteurized donor milk from 18 donors were analyzed. Milk was handled following the regular operating protocols established in the neonatal unit and was kept refrigerated after thawing. pH measurements and bacteriology (on blood agar and MacConkey agar plates) were performed on each sample at time 0 (immediately after thawing) and then every day for 14 d. Changes in pH of samples over time were evaluated with linear mixed-effects regression models. A slow but gradual increase in milk pH was observed starting from the first day [mean (±SD) pH of 7.30 (±0.18) at time 0 and 7.69 (±0.2) on d 14]. No bacterial growth was observed in any of the samples throughout the complete trial except in one sample, in which Bacillus flexus was isolated. In conclusion, pasteurized human donor milk maintains its microbiological quality when properly handled and refrigerated (4–6°C). The slight and continuous increase in milk pH after the first day could be due to changes in the solubility of calcium and phosphate during refrigerated storage.

      Key words

      Short Communication

      Pasteurized donor milk is the first option for feeding premature or ill newborns when their mother's milk is not available (
      • WHO (World Health Organization)
      Infant and Young Child Nutrition Global Strategy on Infant And Young Child Feeding Report. 55 World Health Assembly. Accessed mon/day/year.
      ;
      • PATH (Program for Appropriate Technology in Health)
      Strengthening human milk banking: A global implementation framework. Version 1.
      ). Although donor milk comes from healthy women, it is pasteurized to prevent the transmission of infectious microorganisms to an especially vulnerable population, such as preterm babies or infants admitted to the neonatal intensive care unit. The most common practice in human milk banks is to pasteurize donor milk according to the Holder method (62.5°C, 30 min;
      • PATH (Program for Appropriate Technology in Health)
      Strengthening human milk banking: A global implementation framework. Version 1.
      ). Holder pasteurization denatures viral proteins (
      • Orloff S.L.
      • Wallinford J.C.
      • Mcdougal J.S.
      Inactivation of human inmunodeficiency virus type 1 in human milk: Effects of intrinsic factors in human milk and of pasteurization.
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      • de Oliveira P.R.
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      • de Andrade Gomes S.
      • Heck A.R.
      • de Castro Figueiredo J.F.
      • Mussi-Pinhata N.M.
      Hepatitis B viral markers in banked human milk before and after Holder pasteurization.
      ;
      • Landers S.
      • Updergrove K.
      Bacteriological screening of donor human milk before and after Holder pasteurization.
      ;
      • Donalisio M.
      • Cagno V.
      • Vallino M.
      • Moro G.E.
      • Arslanoglu S.
      • Tonetto P.
      • Bertino E.
      • Lembo D.J.
      Inactivation of high-risk human papillomaviruses by Holder pasteurization: Implications for donor human milk banking.
      ) and destroys all non-spore-forming viable bacteria in milk, although some strains of Bacillus spp. or other sporulated species can remain viable. In addition, some of the milk's beneficial properties are lost during pasteurization, such as the cellular components that confer immunologic properties (B cells, T cells, macrophages, and neutrophils); and immunologic proteins such as IgA, IgG, lactoferrin, lysozyme, and erythropoietin, are significantly reduced. Other components, such as oligosaccharides, remain intact (
      • Akinbi H.
      • Meinzen-Derr J.
      • Auer C.
      • Ma Y.
      • Pullum D.
      • Kusano R.
      • Reszka K.J.
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      Alterations in the host defense properties of human milk following prolonged storage or pasteurization.
      ;
      • Ewaschuk J.B.
      • Unger S.
      • Harwey S.
      • O'Connor D.L.
      • Field C.J.
      Effect of pasteurization on immune components of milk: implications for feeding preterm infants.
      ;
      • Gómez de Segura A.
      • Escuder D.
      • Montilla A.
      • Bustos G.
      • Pallás C.
      • Fernández L.
      • Corzo N.
      • Rodríguez J.M.
      Heating-induced bacteriological and biochemical modifications in human donor milk after Holder pasteurisation.
      ;
      • Chang J.C.
      • Chen C.H.
      • Fang L.J.
      • Tsai C.R.
      • Chang Y.C.
      • Wang T.M.
      Influence of prolonged storage process, pasteurization, and heat treatment on biologically-active human milk proteins.
      ).
      Unpasteurized mother's milk has bactericidal properties that confer protection against neonatal infections as well as against contamination during milk handling. This bactericidal activity remains unchanged during the first 72 h of refrigerated storage but is lost subsequently (
      • Silvestre D.
      • López M.C.
      • March L.
      • Plaza A.
      • Martínez-Costa C.B.
      Bactericidal activity of human milk: Stability during storage.
      ;
      • Miranda M.
      • Gormaz M.
      • Romero F.J.
      • Silvestre D.
      Estabilidad de la capacidad antioxidante y pH en leche humana refrigerada durante 72 horas: Estudio longitudinal.
      ). Holder pasteurization significantly reduces (50–70%) the bactericidal effect, but if the pasteurized donor milk is kept refrigerated, the residual bactericidal capacity remains stable for up to 72 h (
      • Silvestre D.
      • López M.C.
      • March L.
      • Plaza A.
      • Martínez-Costa C.B.
      Bactericidal activity of human milk: Stability during storage.
      ;
      • Bertino E.
      • Giribaldi M.
      • Baro C.
      • Giancotti V.
      • Piazzi M.
      • Peila C.
      • Tonetto P.
      • Arslanoglu S.
      • Moro G.E.
      • Cavallarin L.
      • Gastaldi D.J.
      Effect of prolonged refrigeration on the lipid profile, lipase activity and oxidative status of human milk.
      ).
      After pasteurization, donor milk is typically stored frozen. Afterward, once it is thawed and, until it is administered to the recipient, it is kept refrigerated at 4 to 6°C for a variable period of time, usually 24 to 48 h (
      • PATH (Program for Appropriate Technology in Health)
      Strengthening human milk banking: A global implementation framework. Version 1.
      ). This period is not extended due to the concern of bacterial contamination (
      • Frischknecht K.
      • Wälchli C.
      • Annen V.
      • Fuhrer T.
      • Gianoli P.
      • Stocker M.
      Recommandations pour l'organisation et le fonctionnement d'une banque de lait en Suisse.
      ) and reductions in nutritive properties and biological activity (
      • Meng T.
      • Perrin M.T.
      • Allen J.C.
      • Osborne J.
      • Jones F.
      • Fogleman A.D.
      Storage of unfed and leftover pasteurized human milk.
      ).
      Given that pasteurized donor milk is a scarce resource, studies have been conducted to determine whether this refrigeration time can be extended (
      • Slutzah M.
      • Codipilly C.N.
      • Potak D.
      • Clark R.M.
      • Schandler R.J.
      Refrigerator storage of expressed human milk in the neonatal intensive care unit.
      ;
      • Vickers A.M.
      • Starks-Solis S.
      • Hill D.R.
      • Newburg D.S.
      Pasteurized donor human milk maintains microbiological purity for 4 days at 4°C.
      ;
      • Meng T.
      • Perrin M.T.
      • Allen J.C.
      • Osborne J.
      • Jones F.
      • Fogleman A.D.
      Storage of unfed and leftover pasteurized human milk.
      ). These studies have measured the pH or bacterial growth in refrigerated pasteurized human donor milk for varying periods. However, none of these studies has performed a quality assessment of pasteurized donor milk during refrigerated storage by both pH and bacterial count measurement in the same samples of milk for more than 7 d. Therefore, the aim of the current study was to determine the changes in pH and bacterial counts in pasteurized donor milk stored refrigerated up to 14 d. Our initial hypothesis was that pasteurized human milk acidifies when refrigerated, although more slowly than unpasteurized milk.
      A prospective study was planned with the aim of determining the changes in pH and bacterial growth in pasteurized donor milk stored at 4 to 6°C. The study was performed at the Neo 12 Human Milk Bank, at University Hospital 12 de Octubre (Madrid, Spain). It was reviewed and approved by the Ethics Committee for Clinical Investigation (Comité Etico de Investigación Clínica) of University Hospital 12 de Octubre. Written informed consent was obtained from each donor during their first interview at the milk bank.
      Milk samples were 30 aliquots (120 mL each) of milk pasteurized in the Human Milk Bank following its regular procedures (no special pasteurizations were performed for the study). Milk was donated by healthy women; donor screening included a serological analysis for hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and syphilis, and the women completed a questionnaire on life habits. Donors were instructed on hygiene measures for milk extraction (such as using cap and facemask, hand washing, and breast pump sterilization before expression). Immediately after expression, milk was frozen at home for a maximum of 15 d, and then transported frozen to the milk bank where it was kept at a controlled temperature (−20°C) for a maximum of 41 d. To pasteurize the milk, it was thawed in a shaking water bath at 37°C (Jeio Tech BS-21; Lab Companion, Seoul, Korea) until a central block of ice (approximately 50% of the container) was present, and then transferred to the fridge to finish thawing at 4°C. Batches of milk from the same donor were created. For each batch, nutrient composition (fat, protein, and lactose contents) was measured using a MilkoScan FT 2 (Foss Iberia S.A., Barcelona, Spain). Holder pasteurization was then performed in a shaking water bath (Jeio Tech BS-21; Lab Companion) by heating milk samples (120 mL) at 62.5°C for 30 min, and then rapidly cooling to 4°C. After pasteurization, a microbiological analysis of the milk was performed. In the event of any bacterial growth, the entire batch was discarded, unless <500 cfu/mL of Bacillus cereus had grown. Properly pasteurized samples were stored frozen at −20°C.
      For the study, 30 aliquots of 120 mL were randomly chosen from the milk available at the Human Milk Bank; samples came from 18 donors. Each one of the samples was obtained from an individual donor but some donors provided more than one sample. For each sample, the following data were recorded: maternal age and nationality, type of milk, nutritional composition, days from expression to pasteurization, and days from expression to the beginning of the study.
      The samples were partially thawed in a shaking water bath at 37°C until a central block of ice (approximately 50% of the container) was present, and transferred to a refrigerator at 4 to 6°C. Each 120-mL sample was divided into two 60-mL aliquots under the laminar flow hood, using 120-mL sterile glass containers with plastic stoppers. From this time forward, the samples were handled under the same conditions as commonly used in the neonatal unit to prepare feedings (nonsterile cap, facemask, gloves, and surfaces cleaned with alcohol), and in the same room, which was exclusively dedicated to human milk handling. Milk was kept in the neonatal unit refrigerator (5–6°C), not in the milk bank. The refrigerator used to store human milk in the neonatal unit does not have continuous temperature monitoring, because it is usually used to store milk for short periods. The temperature was set at 5°C and a complete count of the number of times the refrigerator was opened was performed for 72 h.
      Term milk was considered milk from donors whose children were born ≥37 wk of gestational age, and preterm milk was milk from mothers of children born <37 wk of gestational age. Colostrum was milk expressed during the first 7 d after birth, intermediate milk was that expressed from d 8 to 21, and mature milk was that expressed from 22 d postpartum.
      Initially (time 0) and then daily for the next 14 d, a 5.5-mL sample of refrigerated pasteurized donor milk was extracted from the 60-mL aliquot. Each aliquot was opened to extract samples a maximum of 7 times, simulating the maximum number of times containers are opened to prepare feedings for hospitalized infants.
      We measured pH and performed bacterial counts to evaluate the quality of the pasteurized milk samples during refrigeration. The pH of each sample was measured twice using a calibrated pH meter (BASIC 20, Crison Instruments, Barcelona, Spain). Bacterial counts were determined by spreading 10 μL of milk onto blood agar and 10 μL onto MacConkey agar plates (Biomerieux, Lyon, France), which were incubated at 37°C in an aerobic environment for 48 h. These culture media were chosen because blood agar is an enriched medium that allows the growth of almost any type of bacteria, whereas MacConkey medium is specific for gram-negative and lactose-fermenting bacteria. If any bacterial growth was detected, colonies were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in collaboration with the Department of Microbiology at University Hospital 12 de Octubre.
      The study was performed in 3 phases, with 10 samples analyzed in each phase. The first phase was conducted with 10 samples, and we analyzed pH and bacterial count in order to determine the sample size. As the sample size needed was 20 samples (assuming a mean difference of 0.10, SD of 0.25, a correlation of 0.80, and a 95% confidence level), the second phase was conducted with another 10 samples. The third phase was planned to confirm the pH results, and microbiological analyses were not performed because bacterial growth was not detected in previous phases.
      Characteristics of the patients and samples were described using means and standard deviations or absolute and relative frequencies. The normality of the data distribution was tested with the Shapiro–Wilk test. Changes in the pH of the samples over time were evaluated with linear mixed-effects regression models (
      • Hedeker D.
      • Gibbons R.D.
      Mixed-effects regression models for continuous outcomes.
      ). The data were analyzed using Stata 10 software (StataCorp, College Station, TX), and significant differences were defined as P < 0.05.
      Overall, 77.8% (14/18) of the donors were Spanish. Median age at the time of the study was 37 yr (34–37 yr). In total, 77.8% of the samples were mature, term milk and 22.2% were mature, preterm milk. In Table 1, the composition of the milk analyzed before pasteurization is shown. The mean number of days from expression to pasteurization was 26.4 ± 9.6 d and from expression to the first day of the study was 48.9 ± 18 d. The neonatal unit's refrigerator was opened an average of 58 times per day.
      Table 1Donor milk composition (mean of all milks combined ± SD unless otherwise noted) analyzed before pasteurization
      CompositionValue
      Protein, g/dL1.80 ± 0.10
      Lactose, g/dL (median and IQR
      IQR = interquartile range.
      7.55 (7.31–7.94)
      Fat, g/dL2.90 ± 1.08
      Energy, kcal/dL63.5 ± 8.3
      1 IQR = interquartile range.
      We observed a slight increase in pH over time (Table 2). The mean pH of the milk samples at room temperature (23 ± 2.5°C) was 7.30 (±0.18) at time 0 and 7.69 (±0.21) at d 14 (Figure 1), with a statistically significant difference (P = 0.000). The pH increased significantly from d 0 to 1 (P = 0.000), and then remained stable until d 4, when the difference was again significant (P = 0.02).
      Table 2pH values during the study period (n = 30)
      DayMean pHSDMinimumMaximumP-value
      07.310.186.817.55
      17.390.196.837.660.00
      27.390.226.847.790.57
      37.400.236.807.680.44
      47.450.226.877.770.02
      57.560.197.097.910.00
      67.580.197.067.770.16
      77.600.187.227.870.49
      87.610.186.967.830.36
      97.590.246.907.920.22
      107.650.246.907.860.00
      117.650.207.007.920.91
      127.650.217.097.960.88
      137.660.197.117.930.52
      147.690.217.117.990.00
      Figure thumbnail gr1
      Figure 1Mean pH values at time zero and at d 14 (P = 0.00). The central rectangle represents the SD and the line and the cross inside the rectangle show the median and the mean, respectively; the whiskers indicate the maximum and minimum values; and the black dots outside the rectangles are suspected outliers (>1.5 interquartile range). Color version available online.
      We detected no relationships between the days from expression to pasteurization or the days from expression to the first day of the study and the difference in pH over time, the composition of the milk, or the sociodemographic characteristics (mean age and country of origin) of the donors.
      We observed bacterial growth in only 1 of the 20 samples analyzed. In the 14 cultures performed on this sample, Bacillus flexus was identified (100 cfu/mL). We did not detect any bacterial growth in the remaining samples.
      The pH of pasteurized human milk increased slightly when stored refrigerated at 4 to 6°C for 14 d, and no bacterial contamination was observed. Our initial hypothesis was that pasteurized human milk would acidify when refrigerated, although more slowly than unpasteurized milk. As we have shown, the results found were the opposite of our initial hypothesis.
      Milk acidity is a quality indicator (
      • Novak F.R.
      • Cordeiro D.R.
      The correlation between aerobic mesophilic microorganism counts and Dornic acidity in expressed human milk.
      ;
      • Vázquez-Román S.
      • García-Lara N.R.
      • Escuder-Vieco D.
      • Chaves-Sánchez F.
      • de la Cruz-Bertolo J.
      • Pallás-Alonso C.R.
      Determination of Dornic acidity as a method to select donor milk in a milk bank.
      ). Milk can acidify by lipolysis (transformation of triglycerides in free fatty acids) or by the production of lactic acid by lactose-fermenting bacteria. Acidic milk is of poorer quality because it is more osmolar and has poorer bioavailability of phosphorus and calcium (
      • Erickson T.
      • Gill G.
      • Chan G.M.
      The effects of acidification on human's milk cellular and nutritional content.
      ). Milk acidity can be measured using 2 methods: Dornic acidity and pH. When milk acidifies, the pH decreases but the degree of Dornic acidity increases (
      • Escuder-Vieco D.
      • Vázquez-Román S.
      • Sánchez-Pallás J.
      • Ureta-Velasco N.
      • Mosqueda-Peña R.
      • Pallás-Alonso C.R.
      Determination of acidity in donor milk.
      ). Milk with Dornic acidity >7 (pH 6.57) is discarded because it is considered to be of poor quality (
      • Clark R.M.
      • Hundrieser K.H.
      • Ross S.
      • Brown P.B.
      Effect of temperature and length of storage on serum-stimulated and serum independent lipolytic activities in human milk.
      ;
      • Hedeker D.
      • Gibbons R.D.
      Mixed-effects regression models for continuous outcomes.
      ).
      Human milk has 2 types of lipases (
      • Clark R.M.
      • Hundrieser K.H.
      • Ross S.
      • Brown P.B.
      Effect of temperature and length of storage on serum-stimulated and serum independent lipolytic activities in human milk.
      ;
      • Bertino E.
      • Giribaldi M.
      • Baro C.
      • Giancotti V.
      • Piazzi M.
      • Peila C.
      • Tonetto P.
      • Arslanoglu S.
      • Moro G.E.
      • Cavallarin L.
      • Gastaldi D.J.
      Effect of prolonged refrigeration on the lipid profile, lipase activity and oxidative status of human milk.
      ): bile salt–dependent lipase, which seems to be active in the newborn, helping the immature pancreas to digest and absorb fat, and lipoprotein lipase, which is active in the lactating mammary gland (where it is involved in the uptake of circulating fatty acids) but not in the infant. In unpasteurized milk, the lipases remain active when refrigerated or frozen (
      • Penn A.H.
      • Altshuer A.E.
      • Small J.W.
      • Taylor S.F.
      • Dobkins K.R.
      • Schimd-Schönbein G.W.
      Effect of digestion and storage of human milk on free fatty acid concentration and cytotoxicity.
      ). Previous studies have shown that if unpasteurized human milk is kept refrigerated, acidification of the milk will occur because of 2 factors: lipolysis by the lipases (and thus the release of fatty acids into the medium) and the production of lactic acid by lactose-fermenting bacteria (
      • Rona M.S.
      • Novak F.R.
      • Portilho M.
      • Pelissari F.M.
      • Tozzo Martins A.B.
      • Matioli G.
      Effect of storage time and temperature on the acidity, calcium, protein and lipid content of milk from human milk banks.
      ;
      • Slutzah M.
      • Codipilly C.N.
      • Potak D.
      • Clark R.M.
      • Schandler R.J.
      Refrigerator storage of expressed human milk in the neonatal intensive care unit.
      ;
      • Ghoshal B.
      • Lahiri S.
      • Kar K.
      • Sarkar N.
      Changes in biochemical contents of expressed breast milk on refrigerator storage.
      ;
      • Ahrabi A.F.
      • Handa D.
      • Codipilly C.N.
      • Shah S.
      • Williams J.E.
      • McGuire M.A.
      • Potak D.
      • Aharon G.G.
      • Schanler R.J.
      Effects of extended freezer storage on the integrity of human milk.
      ). However, when milk is pasteurized, lipases are destroyed and the bacteria present in the milk are eliminated. Thus, in the current study these 2 processes cannot be implicated (
      • Ghoshal B.
      • Lahiri S.
      • Kar K.
      • Sarkar N.
      Changes in biochemical contents of expressed breast milk on refrigerator storage.
      ;
      • Penn A.H.
      • Altshuer A.E.
      • Small J.W.
      • Taylor S.F.
      • Dobkins K.R.
      • Schimd-Schönbein G.W.
      Effect of digestion and storage of human milk on free fatty acid concentration and cytotoxicity.
      ;
      • Ahrabi A.F.
      • Handa D.
      • Codipilly C.N.
      • Shah S.
      • Williams J.E.
      • McGuire M.A.
      • Potak D.
      • Aharon G.G.
      • Schanler R.J.
      Effects of extended freezer storage on the integrity of human milk.
      ;
      • Baro C.
      • Giribaldi M.
      • Arslanoglu S.
      • Giuffrida M.G.
      • Dellavalle G.
      • Conti A.
      • Tonetto P.
      • Biasini A.
      • Coscia A.
      • Fabris C.
      • Moro G.E.
      • Cavallarin L.
      • Bertino E.
      Effect of two pasteurization methods on the protein content of human milk.
      ); therefore, the change in milk pH likely due to other factors.
      A study published in 2015 (
      • Vickers A.M.
      • Starks-Solis S.
      • Hill D.R.
      • Newburg D.S.
      Pasteurized donor human milk maintains microbiological purity for 4 days at 4°C.
      ) analyzed 42 samples of pasteurized donor milk. The samples were thawed and kept refrigerated for 9 d. None of the cultures conducted grew any microbes. As part of a larger study on unpasteurized milk (35 samples of unpasteurized milk),
      • Slutzah M.
      • Codipilly C.N.
      • Potak D.
      • Clark R.M.
      • Schandler R.J.
      Refrigerator storage of expressed human milk in the neonatal intensive care unit.
      analyzed 5 samples of pasteurized milk that were kept refrigerated for 96 h; they observed that neither the pH nor the concentration of fatty acids changed, and there was no bacterial growth. In a previous study conducted in our unit (
      • Tobío-Gimeno A.
      • Escuder Vieco D.
      • Flores-Antón B.
      • Vázquez-Román S.
      • Pallás-Alonso C.R.
      Changes in pasteurized donor human milk during refrigeration.
      ), we analyzed the pH of 30 samples of unfrozen pasteurized donor milk kept in a refrigerator, and the pH remained unchanged for 4 d. A more recently published study analyzed bacterial growth in refrigerated pasteurized milk and found no bacterial growth in 7 d (
      • Meng T.
      • Perrin M.T.
      • Allen J.C.
      • Osborne J.
      • Jones F.
      • Fogleman A.D.
      Storage of unfed and leftover pasteurized human milk.
      ).
      In this study, we found the opposite result to what we had expected: we observed a slight increase in milk pH during refrigerated storage of pasteurized donor milk. This change was not related to bacterial growth, given that postprocessing contamination was not evident; thus, some milk component could be involved in this pH shift. Human milk has a complex mineral composition that plays a crucial role in sustaining normal growth and development of the newborn, but this composition also has an important influence on many milk properties. Certain milk salts are soluble, whereas others, such as calcium phosphate, exist partly in soluble form and partly in the colloidal phase associated with casein micelles. Changes in temperature modify the equilibrium between the soluble and colloidal phases. Moderate heating, such as Holder pasteurization, decreases the solubility of calcium phosphate due to the formation of colloidal calcium phosphate, which is accompanied by a concomitant decrease in milk pH. Subsequent cooling and keeping the milk at low temperature restores the original equilibrium by increasing both the solubility of calcium phosphate and the milk pH (
      • Gaucheron F.
      The minerals of milk.
      ;
      • Lucey J.A.
      • Horne D.S.
      Milk salts: Technological significance.
      ).
      Bacterial growth was detected in only one sample: Bacillus flexus grew in all aliquots of the same sample (from d 1 to 14), with no increase in the number of colonies over refrigeration time, and never exceeding the acceptance limit of the milk bank. Bacillus spp. are sporulated microbes whose spores are highly resistant to heat and disinfectants. They can therefore remain as surface contaminants in laboratories and are difficult to eradicate. Occasional contamination of pasteurized milk with Bacillus spp. is a common problem in virtually all human milk banks and in the food industry (
      • Hanson M.L.
      • Wendorf W.L.
      • Houk K.B.
      Effect of heat treatment of milk's activation of Bacillus spores.
      ;
      • Landers S.
      • Updergrove K.
      Bacteriological screening of donor human milk before and after Holder pasteurization.
      ;
      • Gómez de Segura A.
      • Escuder D.
      • Montilla A.
      • Bustos G.
      • Pallás C.
      • Fernández L.
      • Corzo N.
      • Rodríguez J.M.
      Heating-induced bacteriological and biochemical modifications in human donor milk after Holder pasteurisation.
      ).
      In this study, alkalization of human milk was observed. Further studies are required that focus on understanding the processes that pasteurized milk undergoes when refrigerated and the potential consequences of these processes on the absorption of macronutrients and micronutrients and the activity of elements with biological value. We have found no previous references on the alkalization of human milk.
      The main limitation of the study was that refrigerator temperature (and its variation) was not monitored during the 14 d of study, given that we planned the study with the intention of imitating the conditions under which human milk is stored in neonatal units.
      In conclusion, it appears that pasteurized human donor milk, if kept refrigerated and handled with appropriate hygiene measures, does not become contaminated. Based on these results, we believe that, from a microbiological point of view, the validity period of refrigerated donor milk could be safely extended to 48 h, given that it is a scarce resource. The alkalization process that human milk undergoes needs further study to determine its consequences on milk quality.

      ACKNOWLEDGMENTS

      This study was financed by the Carlos III Spanish Health Research Funding, grant number PI 12/02128, 28029, Madrid, Spain.

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