Edited by: Zorica D. Juranic, Institute of Oncology and Radiology of Serbia, Serbia
Reviewed by: Oscar J. Cordero, Universidade de Santiago de Compostela, Spain; Ivan Milos Stankovic, University of Belgrade, Serbia
This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology
†These authors have contributed equally to this work
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Donor human milk (DHM) is submitted to Holder pasteurization (HoP) to ensure its microbiological safety in human milk banks but this treatment affects some of its bioactive compounds. The objective of this work was to compare the effects of HoP and high temperature short time (HTST) treatments on some bioactive compounds found in DHM. A total of 24 DHM batches were processed in a continuous HTST system (70, 72, and 75°C for 5–25 s) and by HoP (62.5°C for 30 min). The concentrations of immunoglobulins (Igs) A, G, and M, transforming growth factor-beta 2 (TGF-β2), adiponectine, ghrelin, and leptin were measured using a multiplex system, whereas the concentration of epidermal growth factor (EGF) was determined by ELISA. In relation to Igs, IgG showed the highest preservation rates (87–101%) after HTST treatments, followed by IgA (54–88%) and IgM (25–73%). Ig retention after any of the HTST treatments was higher than after HoP (
Mother's own milk (MOM) is the gold standard for infant feeding in early life. It provides the macronutrients and micronutrients that fulfill the nutritional requirements of the newborn and, also, a myriad of bioactive compounds, such as immunoglobulins (Igs), cytokines, growth factors (GFs), and hormones (
DHM is usually pasteurized to ensure its microbiological safety and, currently, Holder pasteurization (HoP) is the most common method being applied in human milk banks (HMB). This method, which involves heating DHM at 62.5°C for at least 30 min, destroys high-risk viruses and non-spore-forming bacteria (
Alternative methods for DHM treatment are been investigated in order to ensure a proper microbial inactivation, while improving the preservation of its bioactive components (
Therefore, the objective of this work was to compare the effects exerted by the routine HoP treatment or HTST treatments performed in the cited system on the concentration of some of the main Igs, GFs and hormones present in donor milk. In addition, the effect of HTST pasteurization at different temperature/time pairs on these bioactive compounds was also evaluated.
DHM samples were obtained from the Regional Human Milk Bank “Aladina-MGU” located at the Hospital Universitario 12 de Octubre (Madrid, Spain). Milk collection was performed following a specific protocol for donor mothers approved by the Hospital 12 de Octubre Clinical Research Ethics Committee (ethical approval code: 12/325); informed consent was obtained from each donor in accordance with the Declaration of Helsinki. Milk was collected at home, frozen (−18°C) in a domestic refrigerator, and transported to the HMB in an insulated box provided with ice packs.
Milk samples collected from multiple donors were thawed in a shaking water bath (at 37°C) and mixed to compose a production batch (10 L; around 12 donors per batch). A total of 24 DHM production batches were used in this study. An aliquot (120 mL) of pooled raw milk from each production batch was used as control (raw milk). Another aliquot (120 mL) was transferred to a glass bottle (150 mL), and subjected to HoP (62.5°C for 30 min) and fast cooling at 4°C in shaking water baths (Jeio Tech BS-21, Lab Companion, Oxfordshire, UK) following the current HMB protocol. The rest of the production batch was HTST-processed at a fixed temperature (70, 72, or 75°C) and different times (5, 10, 15, 20, and 25 s) using the HTST equipment described by Escuder-Vieco et al. (
All aliquots of untreated (raw) or heat-processed (both HoP and HTST) DHM were stored frozen (−20°C) until analysis. To avoid interferences in immunoassays, the fatty layer was removed from samples by centrifugation at 14,000 × g for 10 min at 4°C.
Concentrations of Igs (IgA, IgG, and IgM), transforming growth factor-beta 2 (TGF-β2) and hormones (adiponectin, ghrelin, and leptin) were determined in duplicate using a Bioplex 200 system instrument (Bio-Rad, Hercules, CA, USA) and the Bio-Plex Pro Human Isotyping Assay, Bio-Plex Pro Human TGF-β Assay and Bio-Plex Pro Human Diabetes Assays kits (Bio-Rad). The concentration of EGF was measured using the RayBio® Human EGF ELISA kit (RayBiotech, Norcross, GA). Every assay was performed according to manufacturer's instructions. Standard curves were performed for each analyte. The analytes were assayed in, at least, three batches for each HTST treatment.
The inter-assay coefficients of variation were below manufacturers' instructions for all the immune markers, and the lower limit of quantification (LLOQ) for every analyte in human milk were: 0.21 μg/L for IgA, 0.78 μg/L for IgM, 2.19 μg/L for IgG, 1.57 ng/L for TGF-β2, 0.03 ng/L for EGF, 23.10 ng/L for adiponectin, 7.40 ng/L for ghrelin, and 11.45 ng/L for leptin.
The variation of
Normality of data distribution was tested through Saphiro–Wilks tests. Results are displayed as the median and interquartile range (IQR). The effect of heat treatments on Igs, GFs, and hormones concentration was expressed as the retention rate in relation to raw DHM samples. Kruskal–Wallis test was used to compare the effect of HTST treatments and HoP on the concentration of these bioactive compounds. Pairwise
Significance was set at
All the Igs, GFs, and hormones analyzed in this work were detected in all the batches of raw DHM and showed a wide range of concentrations (Table
Concentration of immunoglobulins, growth factors, and hormones in raw DHM batches.
IgA (mg/L) | 18 | 473.32 | 381.51–694.43 | 109.88–905.02 | 43.21 |
IgG (mg/L) | 17 | 17.99 | 13.59–46.84 | 7.80–75.95 | 69.98 |
IgM (mg/L) | 20 | 18.79 | 9.22–47.46 | 5.06–87.26 | 85.33 |
EGF (μg/L) | 6 | 3.51 | 3.24–3.99 | 3.14–4.01 | 10.27 |
TGF-β2 (μg/L) | 21 | 1.84 | 1.20–2.45 | 446.36–4592.45 | 50.76 |
Adiponectin (μg/L) | 24 | 7.74 | 6.41–9.27 | 3.74–17.6 | 36.68 |
Ghrelin (ng/L) | 15 | 28.24 | 16.25–43.93 | 10.55–57.84 | 49.97 |
Leptin (ng/L) | 20 | 116.97 | 79.05–184.50 | 68.4–348.19 | 53.14 |
The percentages of retained Ig concentrations in DHM after the different HTST treatments and HoP are shown in Figure
Percentage of IgA, IgG, and IgM retention in DHM before and after HTST treatment (at 70, 72, and 75°C for 5, 10, 15, 20, 25 s) or Holder pasteurization (62.5°C, 30 min). Different letters indicate a significant difference among retention rates.
Regarding GFs, the retained TGF-β2 concentration oscillated between 78 and 107% for HTST-treated DHM and 54–92% after HoP (
Percentage of transforming growth factor-beta 2 (TGF-β2) and epidermal growth factor (EFG) retention in DHM before and after HTST treatment (at 70, 72, and 75°C for 5, 10, 15, 20, 25 s) or Holder pasteurization (62.5°C, 30 min).
No differences were observed between HoP and the HTST treatments in relation to the concentrations of adiponectin and ghrelin in DHM (
Percentage of adiponectin, ghrelin, and leptin retention in DHM before and after HTST treatment (at 70, 72, and 75°C for 5, 10, 15, 20, 25 s) or Holder pasteurization (62.5°C, 30 min). Different letters indicate a significant difference among retention rates.
IgA and IgG retention values showed no differences among the HTST treatments carried out at different temperatures and times, as it was confirmed by a two-way ANOVA test with temperature and time as factors (Supplementary Table
70 | −0.0077 | 130 | 0.842 |
72 | −0.0113 | 88 | 0.867 |
75 | −0.0203 | 49 | 0.821 |
The impact of temperature and time during HTST treatments on the retention rates of GFs and hormones was also analyzed using two-way ANOVA tests (Supplementary Table
In this work, the effect of HMB-operative HTST treatment on the concentration of some milk bioactive factors, including Igs, GFs, and hormones, was assessed and compared to that exerted by the traditional HoP method. Globally, HTST treatments were associated to a significantly higher preservation of, at least, some of the biologically active compounds tested in this study. The effect of this HTST system on the microbiological quality of DHM had been evaluated previously (
Human milk provides a high number of bioactive factors with immunological, anti-inflammatory and anti-infectious properties (
Secretory IgA (SIgA) is the predominant Ig class found in human milk and its role in the prevention of respiratory and gastrointestinal infectious diseases in breast-fed infants is well documented (
IgMs are also relevant for neonatal health playing an important role in the opsonization of Gram-negative pathogens (
The levels of Igs in human milk are highly variable depending, among other factors, on the stage of lactation, decreasing rapidly in the first 4 weeks post-partum (
Igs are thermolabile compounds but the advantage of pasteurization at high temperatures over HoP for the preservation of IgA shown in this study has been widely acknowledged (
A varying degree of Ig preservation was observed among the samples analyzed in this study when they were submitted to the same heat treatment. This fact may be related to the different Ig concentrations that each sample had before treatment since protein denaturation is a concentration-dependent process (
Breastfeeding has been shown to prevent NEC and neonatal nosocomial infections in preterm infants in neonatal intensive care units (NICUs) worldwide (
Human milk is rich in GFs, including TGF-β2 and EGF. Presence of high concentrations of TGF-β2 is a common feature of human milk under physiological conditions (
Globally, TGF-β2 concentrations were higher at lower HTST temperatures and diminished as temperature and/or time of treatment increased, which is in agreement with previous findings (
EGF retention rates did not change significantly during heat treatments. This fact may be related to its disulfide bonding patterns that determine a high conformational stability of the tertiary structure (
The presence of hormones and GFs in human milk has been linked to the function of the mammary gland as an extra-uterine extension or replacer of the placenta to regulate the activities of various tissues and organs of the infant until the maturation and full function of his own endocrine system (
Adiponectin, having an appetite-stimulatory effect and, also, regulating energy metabolism, displays the highest concentration in human milk among appetite hormones, and its concentration is reduced over the course of lactation (
Ghrelin is involved in the short-term regulation of feeding and in the long-term regulation of weight and energy metabolism (
Leptin, which displays an anorexigenic effect, influence both short- and long-term regulation of energy balance and food intake. This hormone can be transferred from blood to milk before being secreted by epithelial cells in milk fat globules during lactation (
Leptin was highly affected by heat treatment and, therefore, it could not be detected in any DHM sample after HoP. Its retention percentages after HTST treatments were the lowest among the hormones studied in this work. In contrast, the concentrations of ghrelin and adiponectin remained unchanged after any of the heat treatments. The increase of these hormones after both HoP and HTST treatments may be attributable to adiponectin dissociation from trimeric to monomeric forms (
The analytical method employed in this study is based on a standard sandwich enzyme immunoassay approach, which is the most popular technique to determine the concentrations of immunological compounds in biological fluids. The multiplexing assay enables the simultaneous analysis of different compounds from a small amount of sample. However, this methodology does not discriminate between biologically active and inactive molecules carrying the antigenic determinant. Therefore, bioassays must be performed in future works to test the biological activity of the analyzed compounds after the heat treatments. These complementary bioassays are expensive and time-consuming but may help to define more precisely the role of these molecules in clinical practice. In addition, there are a high number of maternal and infant factors that influence the levels of bioactive compounds in human milk and their functions in the infant gut. These factors pinpoint the difficulty in relating unequivocally the variation of one compound to a specific infant outcome (
A wide variation in the concentration of several bioactive compounds (IgA, IgG, IgM, EGF, TGF-β2, adiponectin, ghrelin, and leptin) was found in raw DHM. HoP (62.5°C, 30 min) had an important impact on the concentrations of most of the bioactive compounds studied, in particular on those of IgA, IgM, IgG, and leptin, as indicated by their low remaining levels or their lack of detection after the treatment. In contrast, HTST treatment of DHM resulted in higher preservation of these compounds. IgG was the most thermostable Ig, followed by IgA, which concentration showed a reduction of ~30% independently of the temperature-time combination applied. IgM was the most susceptible Ig to the processing conditions, although about 50% of its concentration (was preserved after heating at 72°C for 10–15 s, conditions that allow to achieve the microbiological safety objectives currently established in HMBs. Leptin was also sensitive to the thermal treatment but, again, a 50% of the initial content remained after any of the HTST treatments applied in this work. The DHM concentrations of TGF-β2, EGF, adiponectin, and ghrelin were not affected by any heat treatment applied in this study. As a conclusion, milk quality after HTST pasteurization using the continuous system developed by Escuder-Vieco et al. (
DE-V participated in the design of the study, acquisition of the samples, carried out the pasteurization processes, performed the immunological assays, analyzed and interpreted the data, and drafted the manuscript. IE-M participated in the design of the study, carried out the analyses, and participated in the data analysis. JR designed the study and provided critical revisions of the manuscript for important intellectual content. CP-A and LF participated in the design of the study, funding acquisition, analysis of the data, and provided a critical revision of the manuscript. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
Donor human milk
Human milk bank
Holder pasteurization
High temperature short time
Neonatal intensive care unit
Mother's own milk
Immunoglobulin
Growth factor
Necrotizing enterocolitis
Secretory immunoglobulin A
Transforming growth factor-beta 2
Epidermal growth factor.