High-Temperature Pasteurization Used at Donor Breast Milk Banks Reduces Melatonin Levels in Breast Milk

Introduction

Breast milk contains melatonin (5-methoxy-N-acetyltryptamine), a hormone secreted by the pineal gland, with strong diurnal fluctuations associated with regulating the sleep–wake cycle of infants. ¹ Melatonin in the breast milk of healthy mothers has been found to clearly exhibit a 24-hour pattern,^1–4 where concentrations are barely detectable in daytime milk but present in evening and night milk.^5,6

During pregnancy, a fetus receives melatonin through rapid transplacental transmission in response to maternal circadian secretion, ⁷ which is important for embryonic growth and neurodevelopment. ⁸ When an infant is born, they do not yet produce their own melatonin. ¹ Instead, maternal melatonin from breast milk helps support the natural development of the infant's circadian biology.^9–11 In preterm infants this seems more important, with research showing a significant decrease in oxidative stress and cell death due to a reduction in inflammation after melatonin administration.^10,12,13 Significant improvement of sepsis and clinical outcomes have also been reported within 24, 48, and 72 hours after melatonin was administered to preterm infants with neonatal sepsis.^12,14,15 Melatonin is now considered a potential new tool for neuroprotection in preterm newborns, highlighting the important role that melatonin has on healthy infant development. ¹⁶

Donor breast milk banks play an important role in providing vital breast milk to infants. These donor banks exist because, under certain circumstances, the mother's own breast milk is often not available or is insufficient. These circumstances include a delayed onset of breastfeeding after a premature birth, the baby's inability to suckle, the separation of mother and child, or an illness of the mother. The donor breast milk banks collect, store, and distribute pasteurized breast milk.^17,18 Pasteurization ensures that bacteria and viruses that may be in the donated breast milk are destroyed. The pasteurization technique used in Australia is the Holder Pasteurization (HoP) process, which involves heating breast milk to +62.5°C for 30 minutes, or by any other heat treatment known or proven to result in an equivalent elimination of microorganisms.

The pasteurized donor breast milk is then slowly cooled from 62°C to 21°C within 2 hours and from 21°C to 5°C within the next 4 hours. ¹⁷ This is slightly different from other countries' guidelines, which either do not specify or state to rapidly cool the breast milk after pasteurization.^19,20 Research has shown that melatonin is stable in breast milk and can be frozen and thawed without affecting the concentration. ²¹ It is not known, however, if melatonin levels in breast milk are impacted by this pasteurization process. A similar study has looked at the effect of pasteurization on melatonin levels, ²² but to date no studies have examined the affect that pasteurization has on melatonin in breast milk using the Australian Donor Milk bank guidelines. ¹⁷ The aim of this project is to measure the level of melatonin in night breast milk before and after pasteurization.

Materials and Methods

A within-samples repeated measures study design was used for this project. This study was part of a larger study approved by Austin Health Human Research Ethics Committee (HREC/81999/Austin-2022). The collection of breast milk samples was performed by the participant, in their own home. Based on previous research protocols, ³ samples were collected between 10 pm and 5 am when the melatonin levels were likely highest. ¹

A total of 10 breast milk samples were collected from five mothers. Each sample was divided into two groups of 5 mLs (group A and group B). Group A remained unpasteurized and group B was pasteurized. Pasteurization was undertaken using the Australian guidelines for the HoP process. ¹⁷ The HoP process involved heating the breast milk samples to 62.5°C for 30 minutes and then cooling from 62.5°C to 21°C within 2 hours and from 21°C to 5°C within the next 4 hours. ¹⁷ A control tube of 5 mLs of breast milk was used during pasteurization, to ensure the temperature of the breast milk inside the tubes got to the required temperature. Melatonin levels in the breast milk samples were then analyzed using a commercially available precoated enzyme-linked immunosorbent assay (ELISA) Melatonin Elisa 96T (Serum/Plasma) tray from IBL. The results were read using CLARIOstarPlus (BMG Labtech) at 405 nm wavelength.

Statistical Analysis

Statistical analyses were performed using statistical software for social sciences (SPSS) statistical package (version. 29) for Windows (SPSS, Inc., Chicago, IL). The melatonin levels (pg/mLs) from the unpasteurized breast milk (group A) were compared with those of pasteurized breast milk (group B), using paired t-tests. A p-value <0.05 was considered statistically significant.

Results

The results showed a significant reduction on average of 23.6% after pasteurization (mean ± standard deviation = 51.92 pg/mL ± 19.54 versus 39.66 pg/mL ± 13.05, p = 0.01). Table 1 presents the breast milk sample collection times and melatonin levels before and after pasteurization, along with the difference between these.

Discussion

This study found that HoP significantly reduces melatonin levels in breast milk. This finding contradicts the findings of a recent related study by Chrustek et al., ²² in 2022, who found no significant differences in melatonin levels pre- and postpasteurization. In addition, they reported slightly elevated melatonin after pasteurization. There are, however, key differences between this study and that conducted by Chrustek et al., ²² which may account for the varying findings. First, in the study of Chrustek et al., ²² breast milk samples were rapidly cooled after pasteurization, which contrasts to the slow cooling recommendations outlined by the Australian HoP guidelines that were applied in this study. ¹⁷ Furthermore, Chrustek et al. ²² did not report the time that the breast milk samples analyzed in their study were collected.

As melatonin fluctuates over a 24-hour period, it is important to collect milk at night when it is high in melatonin levels. The melatonin level range reported in the samples collected in by Chrustek et al. ²² before pasteurization was only 0.65–26.24 pg/mL, which does not meet the recommended range reported in the IBL international ELISA kit manual. In addition, the control tests in the kit ranged from 5.2–13.7 to 56.8–96.8 pg/mL. Having a sample with 0.65 pg/mL of melatonin prepasteurization does not leave much room for reduction in melatonin levels after pasteurization. Our study specifically used night-time breast milk samples, with a melatonin range between 25 and 76 pg/mL. Further research is needed to determine if the differences between the two studies are due to the rapid versus slow cooling stage of the pasteurization process.

Currently there are no policies at donor breast milk banks that consider the timing of breast milk expression and the presence of circadian timed hormones such as melatonin. As indicated by previous research, there might be consequences to feeding infants breast milk collected at different times of the day. ²³ Research by Booker et al. ²³ found that infants of mothers who expressed breast milk at a different time of the day to when the infant consumed the breast milk took on average 15 minutes longer to get to sleep at night than other feeding types. ¹ Also, these infants seemed to have slept longer during the day. A potential explanation was that these infants might be consuming daytime breast milk at night, which is rich in cortisol at night, keeping them up and/or consuming melatonin-rich night milk during the day. Therefore, these infants were consuming breast milk with the wrong circadian hormonal timing, possibly explaining the differences in their sleep.

Melatonin levels at night should be between 28.5 and 78.2 pg/mL. ²⁴ The presence of high levels of melatonin still after pasteurization in this study (average 40 pg/mL) means that donor breast milk banks might need to still consider the circadian timing in breast milk when feeding infants. It is hoped, however, that this article highlights the importance of melatonin and the benefits to the infant, so that the handling, storage, and provisions regarding the circadian changes in breast milk composition over a 24-hour period are considered. For example, labeling and keeping breast milk samples separated by day and night expression should be considered to ensure infants, especially preterm infants, are receiving donor breast milk that aligns the appropriate hormone levels for the time of day or night that it is consumed.

It was essential to determine the impact pasteurization has on melatonin because infants who receive donor breast milk might not be receiving the necessary protection and circadian timing benefits.^15,16,25 It is hoped that the outcome from this study will stimulate conversation and consideration regarding pasteurization techniques and the effect it might have on important hormones in donor breast milk, such as melatonin.

There are other processes of pasteurization that might not destroy hormones in breast milk including high-temperature short-time pasteurization, which is a thermal process that involves milk being forced between plates or pipes that are heated on the outside by hot water at a temperature of 72°C, ¹⁹ High Pressure Processing, which is a nonthermal process involving technology that inactivates microorganisms by applying a high hydrostatic or Ultraviolet-C, which uses short-wavelength ultraviolet radiation to destroy the nucleic acids of an organism. ¹⁹ It is recommended that future research examines the impact on melatonin levels from other processes of pasteurization.

This study followed the Australian Operational guidelines for donor breast milk banks, which may limit the generalizability of the findings to other countries. In addition, there was a variation of both the concentration and the amount of melatonin reduced after the HoP process. Further investigation is required to determine why this is the case. For instance, research on cow's milk has shown that different levels of fat content or vitamins may affect pasteurization nutrition.^26,27

Conclusion

The potential impact from the findings of this project is considerable. This study showed that there was a significant reduction in melatonin levels after pasteurization. Considering the growing evidence of research showing that melatonin is involved in an infant's development, especially if born premature, it is important to consider the consequences of the HoP technique on important hormones found in breast milk. It is hoped that outcomes from this study will help generate awareness of the impact of high-temperature pasteurization of breast milk hormones such as melatonin. More investigation is needed into the variance of melatonin reduction between samples as well as different pasteurization techniques.