Holder pasteurization fosters richer gut microbiomes in preterm infants, closely mimicking the benefits of mothers’ own milk, while Retort processing appears to diminish key protective bacteria.
Impact of donor human milk pasteurization methods on the gut microbiome of preterm infants. Image Credit: RMC42 / Shutterstock
In a recent study published in the journal Pediatric Research, researchers at Vanderbilt University Medical Center in the USA investigated the impacts of two typical donor human milk (DHM) treatments, Holder and Retort, on infant gut microbiomes. The study leveraged a cohort of pre-term infants (n = 150) fed either Holder- or Retort-pasteurized donor human milk (DHM) and prospectively collected weekly stools but analyzed one stool per infant to evaluate the impacts of the two donor human milks (DHMs) on gut microbiota.
Study findings highlight the importance of heat duration and temperature in maintaining milk quality, revealing that infants receiving Holder-pasteurized milk exhibited significantly higher gut microbial diversity and a greater abundance of beneficial bacteria. Notably, Holder-pasteurization resulted in a microbiome that more closely resembled that of infants fed their own mother's milk.
Background
The first few months of life are a crucial period of infant growth and development. Human (mother’s) milk is the established gold standard for infant nutrition, providing the optimal balance of nutrients and bioactive factors to support healthy growth and immune development during this critical period. It contains a unique blend of nutrients, antimicrobial proteins, antibodies, human milk oligosaccharides, and other immune-protective compounds that promote gut maturation, shape the developing microbiome, and reduce the risk of infections, inflammatory conditions such as Necrotizing enterocolitis (NEC), and other serious neonatal complications.
Early life nutrition is particularly relevant to pre-term infants in the Neonatal Intensive Care Unit (NICU). When a mother's own milk (MOM) is insufficient, pasteurized donor human milk (DHM) is the recommended alternative. However, unlike breastfed MOM, DHM must be treated to eliminate harmful pathogens and ensure its safety to immunocompromised infants.
The two most common methods of DHM treatment are Holder pasteurization (heating milk to 63°C for 30 minutes) and Retort pasteurization (heating milk to 121°C for 5 minutes at ~15 PSI). While both are effective, the harsher Retort method is hypothesized to degrade more of the milk's beneficial bioactive components.
Unfortunately, the impacts of these treatments on infant gut microbiota, a crucial determinant of long-term physiological and developmental well-being, are unknown.
About the study
The present study aims to address this knowledge gap and inform pediatricians and parents by directly comparing the impacts of the two pasteurization methods on the gut microbiota of a large cohort of pre-term infants at the Vanderbilt University Medical Center.
Notably, the NICU at Vanderbilt had used Retort-pasteurized DHM from December 2019 to March 2022 due to pandemic-related supply shortages, but then switched back to their standard Holder-pasteurized DHM, unintentionally generating the ideal sample group for the present investigation.
The study analyzed one stool sample per infant collected once they were on exclusive full-volume DHM for at least a week, from 150 pre-term infants born at less than 34 weeks' gestation. The infants (study participants) were divided into three groups based on their exclusive diet: 1. Retort-pasteurized DHM (n = 80 infants), 2. Holder-pasteurized DHM (n = 54 infants), or 3. Their own mother's milk (n = 16 infants). Antibiotic exposure was much lower in the MOM group (18.8%) than in either the DHM group (~80%), and the Retort group had a higher C-section delivery rate (82.5% vs. 64.8% in the Holder group).
Whole-metagenome sequencing (WMS) was used to identify and characterize all the microorganisms present in the stool samples, thereby assessing the diversity and composition of the gut microbiome and elucidating its functional capabilities.
Study findings
WMS analyses revealed significant differences in the gut microbiomes of infants depending on the type of milk they received, particularly in terms of microbial diversity. Infants fed Holder-pasteurized DHM demonstrated significantly higher alpha-diversity, a key measure of a healthy, rich ecosystem, than their Retort-pasteurized DHM-fed counterparts (Chao-1 index, p = 0.007). Shannon diversity did not differ significantly, and beta-diversity analyses revealed that diet and delivery mode had a significant influence on microbiome composition.
Notably, observed diversity differences were driven by the abundance of specific beneficial anaerobic bacteria known to support gut health, including Bacteroides thetaiotaomicron, Clostridium species, and Bifidobacterium species. In contrast, these beneficial microbes were less abundant in the Retort group. Functional profiling revealed enrichment of genes involved in carbohydrate metabolism and sugar transport systems, including ABC transporters and phosphotransferase systems, in infants fed with Holder's formula.
Comparisons of data from infants fed MOM revealed that the microbiome of the Holder-fed infants was much more similar to that of infants fed their mother's own milk, suggesting the gentler pasteurization method better preserves the components of human milk that help cultivate a healthy gut.
However, despite these profound microbial differences, the study found no statistically significant variations in short-term clinical outcomes, including NEC, bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH), mortality, or length of hospital stay between the two DHM groups. However, retinopathy of prematurity (ROP) was lower in the MOM group than in either the DHM group or the control group.
Conclusions
The article provides the first direct evidence demonstrating that the method used to pasteurize donor human milk has a measurable and significant impact on the gut microbiome of pre-term infants. It highlights that the gentler Holder pasteurization process results in a more diverse and beneficial microbial community, closely mimicking the effects of the gold-standard mother's own milk.
The authors note that the single-timepoint design and limited sample size may have reduced power to detect clinical outcome differences, and that group differences in delivery mode and antibiotic exposure could have influenced the results. Nevertheless, these pre-term infant microbiome findings represent a crucial step forward in safeguarding this nutritionally vulnerable subpopulation.
Journal reference:
- Ocampo-Chih, C., Hendricks, H., Weitkamp, S., Gowda, N. S., Singh, H., Banerjee, R., Rajagopala, S. V., & Weitkamp, J.-H. (2025). Impact of donor human milk pasteurization methods on the gut microbiome of preterm infants. Pediatric Research. DOI: 10.1038/s41390-025-04386-2. https://www.nature.com/articles/s41390-025-04386-2