Effect of pasteurization on selected immune components of donated human breast milk

Abstract

Objective:

Pasteurized, donated milk is increasingly provided to preterm infants in the absence of mother's own milk. The aim of this study was to determine the effect of pasteurization on the concentration of selected components in donated human breast milk.

Study Design:

Donated milk from 34 mothers was pooled into 17 distinct batches (4 mothers per batch). Aliquots of each batch were then Holder pasteurized (62.5 °C for 30 min). Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70 and IL-13 were measured in a multiplex enzyme-linked immunosorbent assay (ELISA). Granulocyte colony-stimulating factor (G-CSF), heparin-binding epidermal-like growth factor (HB-EGF) and hepatocyte growth factor (HGF) were measured by ELISA. Lipids were assessed by gas chromatography and gangliosides by the resorcinol-HCl reaction.

Result:

IFN-γ, TNF-α, IL-1β, IL-10 and HGF were significantly reduced by pasteurization (P<0.05). Gangliosides were not affected, but the proportion of medium-chain saturated fats was increased (P<0.05) with a trend towards a decreased proportion of oleic acid (P=0.057).

Conclusion:

Pasteurization significantly reduced the concentration of several immunoactive compounds present in breast milk, but did not have an impact on others.

Introduction

Mother's own milk (MOM) is the ideal source of nutrition for infants.1 In cases where MOM is not available for the infant, an alternative is donor milk (DM). DM is pasteurized to avoid potential transmission of infectious agents, typically by heating to 62.5 °C for 30 min (Holder pasteurization).2 Some studies on the impact of Holder pasteurization have been reported. For example, pasteurization completely inactivates all cellular components of milk, including T cells, B cells, macrophages, and neutrophils.3, 4, 5 Immunoglobulin A, and particularly immunoglobulin G are also significantly reduced, as are numerous other immunoactive components, such as lactoferrin,6, 7, 8 lysozyme7, 9 and erythropoietin.10 Some growth factors have been reported to be reduced by pasteurization, such as insulin-like growth factor-1 and insulin-like growth factor-2,11 whereas others like epidermal-like growth factor (EGF)11 and transforming growth factor-β12 appear capable of withstanding heat treatment. Likewise, the concentration of milk oligosaccharides, known to encourage a healthy microflora, is not altered by pasteurization.13 Despite the reduction or abolition of some bioactive components of milk, the ability of pasteurized breast milk to exert beneficial effects may not be lost, at least in vitro. Pasteurized milk has been reported to maintain much of its capacity to induce T-cell proliferation10 and still provides substantial inhibition on the growth of E. coli.14

As DM in hospitals is fed primarily to preterm infants who are at elevated risk of developing necrotizing enterocolitis (NEC), loss of immune factors through pasteurization may have significant implications, as many of the immunoactive components of milk have been hypothesized to be involved in the prevention of NEC. For example, a number of anti-inflammatory cytokines are known to be involved in suppressing intestinal inflammatory response, as well as growth factors, such as heparin-binding-EGF (HB-EGF), hepatocyte growth factor (HGF) and granulocyte colony-stimulating factor (G-CSF), all of which are important regulators of intestinal integrity.

In a series of experiments using a neonatal rat model of NEC, Feng et al.15, 16, 17 determined that treatment with HB-EGF resulted in reduced apoptosis, increased proliferation and migration, as well as maintenance of the epithelial barrier and mucosal integrity compared with control animals.

Human milk from mothers delivering at term contains significant concentrations of G-CSF,18 a cytokine involved in the regulation of neutrophil production by inhibiting apoptosis of granulocyte progenitors and supporting their proliferation and differentiation.19 A placebo-controlled pilot study of preterm infants demonstrated a significant reduction in clinical progression from stage I to stage II NEC when infants were provided enteral recombinant human G-CSF.20

HGF promotes cell growth in epithelial cells and is present in amniotic fluid and breast milk in sufficient amounts to profoundly affect gastrointestinal maturation, potentially contributing to the decreased rate of NEC in breastfed infants.21

Gangliosides and some long-chain polyunsaturated fatty acids are also known to play a role in preventing NEC.22, 23 To date, no investigations on the impact of pasteurization on these immunoactive compounds relevant to NEC have been reported.

The objective of this study was to determine the effect of DM pasteurization on a panel of cytokines (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70 and IL-13), growth factors G-CSF, HB-EGF and HGF, gangliosides, and relative fatty acid concentrations.

Methods

Collection and pasteurization of human milk

Frozen mature breast milk was donated by 34 healthy mothers (at least 1 month postpartum) whose children were former patients at The Hospital for Sick Children. All donors provided informed consent to use their breast milk in this study after all information that could link the milk with either the mother or the patient was removed from containers. Institutional approval to conduct this study was provided by the Quality and Risk Management Department at The Hospital for Sick Children.

Samples were thawed overnight in a refrigerator, warmed in a water bath (37 °C) and gently inverted to produce a homogeneous mixture. The warmed milk from 4 different women was pooled to produce 17 batches of 120 ml each (each donor contributed to 2 pools). Each pooled batch was mixed by gentle inversion and 1 ml aliquots of the unpasteurized milk were collected and frozen at −80 °C for later analysis. Remaining milk was processed in a Breast Milk Pasteurizer (T30/USA, Sterifeed, Medicare Colgate, Devon, UK), which involved submerging bottles into a preheated water bath (63.2 °C) followed by a cool water bath (<9 °C). A temperature probe was positioned in a centrally placed non-sample bottle to ensure milk samples were maintained at 62.5 °C for 30 min. Aliquots of 1 ml of the pasteurized DM were collected and stored at −80 °C for later analysis.

Cytokine analysis

A TH1/TH2 human 10-plex kit (for IL-2, IL-4, IL-5, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IL-1β and IL-13) was purchased from Mesoscale Discovery (MSD; Gaithersburg, MD, USA). All reagents were provided with the kit. Each 96-well plate had 10 carbon electrodes in the bottom of each well, each precoated with the 10 antibodies of interest. The standards were reconstituted in the assay diluent provided. Assay diluent (25 μl) was added to all the wells as a blocking agent, and incubated for 30 min at room temperature on an orbital shaker. Calibration and standardization were accomplished with standards supplied by the manufacturer, and linear responses were obtained from 2.4 to 10 000 pg ml−1. The lower limit of detection for the analytes ranged from 0.39 to 2.3 pg ml−1, and percent recovery from spikes in various biological samples ranged from 81 to 128%. Samples (aqueous portion of milk), standards and controls were added in duplicate at 25 μl per well. The plate was incubated for 2 h at room temperature on an orbital shaker. Wells were washed three times using 200 μl phosphate-buffered saline+0.05% Tween 20. Detection antibody was added as per manufacturer's instructions at 25 μl per well, and incubated for 1 h at room temperature on an orbital shaker. At the end of the incubation, the plate was washed three times. MSD Read Buffer (150 μl) was added to each well and the MSD plates were measured on the MSD Sector Imager 2400 plate reader. The raw data were measured as electrochemiluminescence signal (light) detected by photodetectors and analyzed using the Discovery Workbench 3.0 software (MSD). A 4-parameter logistic fit curve was generated for each analyte using the standards, and the concentration of each sample calculated.

Enzyme-linked immunosorbent assay (ELISA)

Kits for the analysis of human G-CSF, HB-EGF and HGF were purchased from R&D Systems (Minneapolis, MN, USA). Assays were optimized before analyses, and were carried out according to manufacturer's directions using only the aqueous portion of the milk. Standards were supplied in the kits. All samples were analyzed in duplicate with coefficient of variation <10%. All plates were read according to the manufacturer's specifications in a microplate reader (SpectraMax 190; Molecular Device, Sunnyvale, CA, USA). The HGF ELISA exhibited no cross-reactivity to EGF or insulin-like growth factor-1, the HB-EGF exhibited no cross-reactivity to amphiregulin, betacellulin, epiregulin, neuroregulin-1α or transforming growth factor-α, and the G-CSF assay exhibited no cross-reactivity to recombinant G-CSF-R.

Fatty acid analysis

Total breast milk lipids were extracted using a modified Folch protocol, chloroform:methanol (2:1)/CaCl2 (5:1) v/v.24 A 15:0 internal triglyceride standard was added (50 μg Nu-Chek Prep, Elysian, MN, USA) to identify the fatty acids and determine their relative concentration. Fatty acid methyl esters were prepared from the extracted lipids by heating (110 °C) with methanolic KOH for 1 h, and then heating an additional hour with BF3 and hexane.25 Samples were allowed to cool and water was used to separate the lipid layers. The hexane layer was removed and dried down by nitrogen gas. The total lipid extracted was resuspended in 500 μl fresh hexane and injected into a gas chromatograph (Agilent model # 7890A GC System, Agilent Technologies, Mississauga, ON, Canada). The instrument's operating conditions, method and column (CP-Sil 88 fused-silica capillary column: 100 m × 0.25 mm i.d. × 0.2 μm; Varian, Mississauga, ON, Canada) were established in a previously published method.26 Fatty acids were identified according to commercial standard 463 (Nu-Chek Prep, Elysian, MN, USA) and expressed as a relative percent (% w/w). All solvents and gases used were of analytic and ultra pure grade and were purchased by Fisher Scientific and Praxair in Edmonton, AB, Canada.

Ganglioside analysis

The ganglioside assay was based on modified Folch extraction where chloroform/methanol (2:1) is used to partition the gangliosides into an aqueous upper layer.24 Calcium chloride and centrifugation were used to facilitate layer separation. Gangliosides were isolated from the upper layer and a portion was applied to a C18 Sep-Pak column (Waters, Milford, MA, USA).27 The isolated ganglioside fraction was then dried down and assayed by the resorcinol-HCl reaction.28

Statistical analysis

The results are presented as a percentage increase or decrease of analyte post pasteurization (mean±s.e.m.), compared with raw milk. Statistically significant differences were assessed by comparing pre- and post-pasteurization values using a two-tailed paired t-test, with P<0.05 considered significant.

Results

Cytokine panel

IFN-γ, TNF-α, IL-1β and IL-10 concentrations were all significantly reduced by pasteurization at 62.5 °C for 30 min (P<0.05, Figure 1). Unexpectedly, IL-8 concentrations were significantly increased by pasteurization. IL-2, IL-4, IL-5, IL-12p70 and IL-13 were detectable in all samples, but were not significantly impacted by Holder pasteurization.

Figure 1
figure1

Cytokine concentrations in pasteurized donor milk (DM). Samples of DM (n=17) were Holder pasteurized (62.5 °C, 30 min), and aliquots analyzed in a 10-panel multiplex cytokine assay. Bars are mean±coefficient of variation. *P<0.05.

Growth factors

Of all the compounds measured, HGF was the most heat labile and was significantly reduced in pasteurized DM samples compared with raw milk samples (P<0.05, Figure 2). HB-EGF was reduced by pasteurization, but did not reach statistical significance (P=0.056). G-CSF was undetectable in all milk samples measured, both pasteurized and raw.

Figure 2
figure2

Donor milk (DM) concentrations of heparin-binding epidermal-like growth factor, hepatocyte growth factor and granulocyte colony-stimulating factor upon Holder pasteurization. Samples of DM (n=17) were pasteurized (62.5 °C, 30 min), and aliquots analyzed via enzyme-linked immunosorbent assay. Bars are mean±s.d. *P<0.05.

Fatty acids

Fatty acid analysis revealed slight changes in relative percent composition. Medium-chain saturated fats (C12:0, C14:0), were relatively increased by the pasteurization process; this was partially balanced by a trend toward a decrease in the proportion of oleic acid (C18:1n9, P=0.06) (Table 1). Long-chain polyunsaturates were not affected by pasteurization.

Table 1 Change in breastmilk fatty acids upon Holder pasteurization

Gangliosides

Pasteurization did not impact ganglioside concentrations (Figure 3).

Figure 3
figure3

Concentrations of gangliosides upon pasteurization. Samples of donor milk (n=17) were Holder pasteurized (62.5 °C, 30 min), and aliquots analyzed by the resorcinol-HCl reaction. Bars are mean±s.d.

Discussion

This study investigated the impact of pasteurization on a number of bioactive components of breast milk that are potentially associated with the prevention of NEC in preterm infants. IFN-γ, TNF-α, IL-1β, IL-10, HGF and monounsaturated fatty acids were reduced by pasteurization, whereas IL-2, IL-4, IL-5, IL-12p70, IL-13, HB-EGF (P=0.056), gangliosides and most fatty acids, including the essential fatty acids, were not.

Mothers of preterm infants often are unable to provide sufficient milk. In a study by Schanler et al.,29 only 27% of mothers were able to provide sufficient milk for their preterm infants. When adequate amounts of MOM are not available for preterm infants, two alternatives exist: formula milk, typically of bovine origin, or DM. A recent Cochrane meta-analysis by Quigley et al. investigating the effect of formula milk compared with DM on the growth and development of preterm or low birth weight infants concluded that although formula feeding resulted in higher rates of growth in the short term compared with DM, it also resulted in a statistically significant increase in the risk of NEC (relative risk 2.5, CI 1.2, 5.1).30 Sullivan et al.31 recently conducted a trial of extremely preterm infants who were randomized to three groups. Groups 1 and 2 received pasteurized donor human milk-based fortifier (Holder pasteurization) when the enteral intake was 100 and 40 ml kg−1 per day, respectively, and both groups received pasteurized donor human milk (high-temperature short time) when no MOM was available. Group 3 received bovine milk-based fortifier when the enteral intake was 100 ml kg−1 per day and preterm formula when no MOM was available. Rates of NEC and NEC requiring surgery were significantly decreased (50 and 90%, respectively) in infants receiving an exclusively human-based diet compared with those receiving any bovine-based milk. The reductions were so marked that this was concluded to be the most significant intervention on the prevention of NEC reported to date.32 This research, taken together, suggests that although MOM is the gold standard, DM is the best alternative when an adequate supply of MOM is unavailable.

Although some countries successfully practice the feeding of raw DM to hospitalized infants,33, 34 the Human Milk Banking Association of North American mandates Holder pasteurization of all donated milk to remove pathogens such as Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and cytomegalovirus.2 Numerous studies have shown reductions in some of the other bioactive components present in human milk upon pasteurization (reviewed in ref. 35).

The reduction observed in cytokines may not have significant biological consequences, as both pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β) and an anti-inflammatory cytokine (IL-10) were reduced. It is possible that reducing pro-inflammatory cytokines in DM could exert beneficial effects in neonates at high risk for NEC, although in the context of the extensive number of cellular, immunologic and microbicidal compounds that are altered by pasteurization, this requires further research. Furthermore, this study was not an exhaustive analysis of all cytokines or their binding proteins, and therefore the clinical implications are unclear. An assessment of the effect of pasteurization on the capacity of breast milk to impact inflammation in vitro would help address this question.

One unexpected effect of pasteurization we observed was the significant increase in IL-8 concentration. This could be due to the pasteurization causing the release of a bound protein, rendering the IL-8 more readily able to bind anti-IL-8 antibodies in the ELISA process. The biological significance of this is difficult to predict, but presumably would not be overly impactful as the cleavage of a binding protein would likely occur in the stomach, regardless.

HB-EGF stimulates cell growth and differentiation of neutrophils and is upregulated in response to tissue damage, hypoxia, stress, and during wound healing and regeneration.15, 16, 17 HB-EGF is found in biologically significant quantities in breast milk and amniotic fluid. In a series of experiments using a neonatal rat NEC model, Feng et al.15, 16, 17 found that treatment with HB-EGF resulted in reduced apoptosis, increased proliferation and migration, as well as maintenance of the epithelial barrier and mucosal integrity compared with control animals. The reduction of HB-EGF in pasteurized milk may have negative implications on the intestinal integrity of preterm infants, compared with MOM.

Postnatally, human milk from mothers delivering at term contains significant concentrations of G-CSF,18 a hematopoietic cytokine involved in the regulation of neutrophil production by inhibiting apoptosis of granulocyte progenitors and supporting their proliferation and differentiation.19 Unexpectedly, none of the samples studied contained detectable concentrations of G-CSF, despite accurately quantifiable positive controls. The absence of G-CSF in human milk has not been reported as all investigations have been in early postnatal milk or colostrum and not mature milk, as in the current study. This too may have implications for the development of NEC, as in a hypoxia-induced rat NEC model, mucosal injury was prevented by administration of G-CSF when compared with control rats.36

Srivastava et al.21 suggest that the decreased risk of developing NEC among breastfed infants may be in part due to the high concentrations of HGF present in amniotic fluid and colostrum/early milk. HGF is a mitogen for multiple subsets of epithelial cells, including gastrointestinal epithelial cells.37 We observed a substantial reduction in HGF concentrations in pasteurized versus raw DM.

Long-chain polyunsaturated fatty acids and gangliosides have both been shown to have positive effects on the prevention of NEC.23, 38 In this study, pasteurization did not impact either long-chain polyunsaturated fatty acids or gangliosides. Some fatty acid alterations were observed, with pasteurization causing a reduction in relative percentage of oleic acid, and a relative increase in medium-chain fatty acids. The physiological implications of these changes are likewise difficult to predict, but the preservation of the long-chain polyunsaturated fatty acids is likely a favorable outcome. Results obtained in this study are similar to a previous report, where little change was observed in fatty acid composition upon pasteurization.39

In conclusion, we have shown that several immune factors, including cytokines and growth factors are significantly diminished upon pasteurization, whereas others are not impacted by the high temperature processing. The biological implications of these findings require further investigation. As human milk banking is rapidly increasing in North America, it is critical to understand the impact of pasteurization on human milk. This will allow for the development of new and innovative techniques for handling milk that will have the least impact on the biologic activity of human milk.

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Acknowledgements

The authors are grateful to Robert Polakowski and Paige Sorochan for technical assistance with ganglioside and fatty acid analysis, respectively.

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Correspondence to C J Field.

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Ewaschuk, J., Unger, S., O'Connor, D. et al. Effect of pasteurization on selected immune components of donated human breast milk. J Perinatol 31, 593–598 (2011). https://doi.org/10.1038/jp.2010.209

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Keywords

  • donor milk
  • NICU
  • necrotizing enterocolitis
  • preterm infants
  • formula milk

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