Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

Summary

The article describes a protocol for reconstituting pasteurized donor milk microbiota using the mother's own milk microbiota in practical, real-world settings. It demonstrates effective bacterial growth and microbiome modulation, supporting the feasible application of this procedure within the routine care of a maternity hospital and its associated human milk bank.

Abstract

Mother's own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot breastfeed, the preferred alternative is pasteurized donor human milk (PDM), which is routinely provided by human milk banks. PDM offers a superior range of nutritional and immunological elements compared to any commercially available formula. However, to ensure biosafety, PDM undergoes pasteurization, a process that inactivates commensal microbiota and reduces certain bioactive compounds. This study presents a protocol designed to restore the microbiota of PDM using MOM as a microbial source, adapting the approach to a real-world clinical setting.

The protocol was implemented in a clinical trial conducted at a maternity hospital and its associated human milk bank, with the aim of providing personalized donor milk to preterm infants whose mothers cannot produce sufficient milk. The methodology involves inoculating PDM with 10% of MOM, followed by incubation at 37 °C for 4 h. Microbiological analysis demonstrated successful bacterial growth in the inoculated milk (IM) post incubation, with the microbiota profile of the reconstituted milk (RM) closely resembling that of MOM, indicating effective microbiota restoration. These results suggest that the reconstitution protocol is feasible for implementation in neonatal care, with the potential to enhance the nutritional and immunological quality of PDM, thereby supporting the health and development of non-breastfed newborns.

Introduction

Breast milk is widely recognized as the best source of nutrition for newborns, providing not only essential macro and micronutrients but also a complex array of elements, including various metabolites and components of the immune system, such as antibodies, cytokines, and cells¹. Additionally, the beneficial properties in breast milk also arise from a community of commensal microorganisms. All these elements of breast milk play a crucial role in the development of the newborn¹. The community of microorganisms, known as the microbiota, is influenced by a combination of factors such as diet, maternal lifestyle, type of delivery, and gestational age, resulting in each milk having a microbiota with particular characteristics². The mechanisms by which the breast milk microbiota benefits neonatal development range from protecting the enteric surface through colonization by commensal bacteria to promoting immune system maturation through the production of metabolites that interact with immune cells in the intestine³. For this reason, the individualized profile of microorganisms in each mother's milk makes it a form of personalized medicine for newborns⁴.

The presence of a healthy microbiota can help protect the newborn against colonization by potentially pathogenic microorganisms by occupying the ecological niche and supporting nutrition, especially for premature infants, who are more vulnerable to these external impacts due to being in a hospital environment and having an immature immune system. Moreover, mothers who give birth prematurely often have difficulty producing milk in sufficient quantities for their newborns. Milk production is typically lower the younger the gestational age⁵. In these cases, the best option for feeding the newborn is pasteurized donor milk provided by Human Milk Banks (HMB)⁶.

Brazil has the largest and most extensive network of HMBs in the world⁷. This network collects, prepares, and distributes more than 160,000 L of pasteurized donor human milk (PDM) throughout the country at no cost to parents, through the national unified health system (Sistema Único de Saúde), managed by the Ministry of Health⁷. Pasteurization is essential to ensure the biosafety of the donated milk intended for newborns. This process is usually carried out using the Holder method, which involves immersing a container of raw donor breast milk in a water bath at 62.5 °C for 30 min^6,8. The resulting PDM is then stored under refrigeration until it is ready for consumption. However, pasteurization reduces the viability of the milk's commensal microbiota and decreases the bioavailability of various bioactive components. This is because the pasteurization process is standardized to prevent the transmission of pathogenic microorganisms to the newborn. In this context, in 2017, Cacho and colleagues described the possibility of reconstituting the commensal microbiota of a mother's own milk by inoculating it in various proportions into PDM⁹. Their findings demonstrated that using a 10% concentration of the mother's own milk (MOM) is the optimal concentration for maintaining the original composition of the MOM microbiome profile. This approach reduces the risk of overgrowth of specific bacteria, which, although typically commensal, could become harmful if disproportionately present in the reconstituted milk. Specifically, the study observed that a 1:10 dilution (10% MOM + 90% PDM), incubated for 4 h, resulted in a well-balanced bacterial load — approximately 60% of that found in undiluted MOM -- indicating a safer and more effective approach. In contrast, higher dilutions, such as 3:10, led to excessive bacterial growth, highlighting the critical need for controlled concentrations and conditions to ensure safety.

In this article, we demonstrate that this procedure of reconstituting the breast milk microbiota in PDM can be achieved in real-world situations within the routine care of a maternity hospital and its associated human milk bank.

Protocol

This protocol was performed as part of the procedures of a clinical trial conducted by our research group (Brazilian Registry of Clinical Trials, ReBEC RBR-729kr8x), and received Ethics Committee approval (process CAAE nº 41063520.4.0 000.0121) and informed consent were obtained from all participants prior to randomization and sample colletction.

The procedure for reconstituting the milk microbiota is done in three steps:

1. Obtaining mother's raw milk

NOTE: The procedure for obtaining milk can be found elsewhere^10,11.

1. Before the procedure, obtain informed consent from the mother according to the ethical committee and be sure to explain everything that will be done to the mother, addressing any doubts, concerns, fears, and anxieties about the procedure to keep the mother relaxed.
2. Have the mother express milk using an electric breast pump into a sterilized container under the supervision of the nurse in charge of the unit, following the established local hygiene standards of each maternity ward.
3. After collection, label the container holding the milk with the mother's information, date, and time of collection. Keep the mother's own milk (MOM) at a temperature of -4 °C or lower for up to 12 h.

2. Defrosting of pasteurized donor's milk (PDM)

NOTE: The HMBs have their own stock of PDM, frozen at a temperature below -4 °C for a maximum period of up to 6 months, and the procedure is well described in the literature elsewhere^12,13,14.

1. Thaw raw milk or PDM stored in sterilized glass containers with plastic lids in a water bath at 40 °C, ensuring temperature maintenance and a shorter processing time.
   NOTE: Do not thaw PDM at room temperature or at refrigeration temperature (inside the refrigerator), as thawing times in these cases are extended, which promotes microbial exposure and growth.
2. Remove human milk bottles from the water bath before the milk in the bottle reaches a temperature above 5 °C.

3. Inoculation of PDM with MOM for microbiome reconstitution

NOTE: The standard volumetric ratio for the process of inoculating PDM with MOM is 90%: 10% (v/v). The steps of the procedure are as follows:

1. Instruct the technician to wear a lab coat, mask, cap, and gloves.
2. Clean the work surface with a surface cleaning solution following local institutional protocol.
3. Arrange the materials to be used and prepare the equipment. Preheat the incubator (water bath) with water to 37 °C.
4. Plan the final volume of inoculated milk to be used and select an appropriately sized container. Place the container with fresh raw MOM and the PDM in liquid form on the work surface.
5. Collect an aliquot of up to 1 mL of the MOM using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot in a DNase/RNase-free cryotube at -80 °C.
6. Transfer the remaining volume, equivalent to 10% of the final desired volume of milk, using a sterile volumetric instrument (e.g. syringe, pipette) to a food-safe container to be immersed in a water bath at 37 °C.
7. Collect a 1 mL aliquot of the PDM using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot in a DNase/RNase-free cryotube at -80 °C.
8. Transfer an amount of PDM equivalent to 90% of the final desired volume of milk using sterile volumetric instrument (e.g., syringe, pipette) to the container with the MOM from the previous step, now forming the inoculated mother's milk (IM).
9. Seal the container with a lid that prevents any external liquid from contaminating the inside of the bottle and immerse it in the water bath at 37 °C for 4 h to allow the microorganisms inoculated in the milk to multiply.
10. After 4 h, remove the container with the fermented IM from the water bath, now called reconstituted milk (RM).
11. Reserve an aliquot of up to 1 mL using sterile equipment for subsequent 16S sequencing and bacterial count. Store the aliquot store in a DNase/RNase-free cryotube at -80 °C.
12. Transfer the remaining volume to a sanitized container for feeding newborns.

4. Validation of the method by bacterial culturing on plates

NOTE: All procedures must be performed under sterile conditions to prevent contamination and ensure the accuracy of the dilutions. Bacterial growth can be estimated using methods described elsewhere^9,15.

1. Prepare serial dilutions of each milk sample (PDM, MOM, IM, RM) in sterile saline at 10^-1, 10^-2, 10^-3, and 10^-4 concentrations. Mix each dilution thoroughly to ensure uniform bacterial distribution.
2. Pippete and spread 100 µL of each diluted sample onto Man-Rogosa-Sharpe (MRS) and Mannitol Salt Agar (MSA) plates in triplicates, using a sterile Drigalski loop or glass spreader. Make sure that the volume is uniformly spread on the agar plate.
3. Incubate MRS agar plates in an anaerobic jar containing to create a low-oxygen environment for lactic acid bacteria growth.
4. Incubate Mannitol Salt Agar (MSA) plates aerobically to support the growth of Staphylococcus species.
5. Incubate the plates at 37 ± 2 °C for 48-72 h.
6. Select the dilution with a countable number of bacterial colonies (25-250 colonies) on each type of agar
7. Count visible colonies on each of the three replicate plates for the selected dilution.
8. Calculate the average colony count from triplicate plates for the selected dilution in both MRS and MSA media.
9. Determine the number of colony-forming units (CFU) per mL for each sample using the following formula and convert to a base-10 logarithmic scale (log10) to facilitate interpretation. Proceed with statistical analysis of the results.
   Number of CFU/mL = Number of colonies × total dilution factor/volume plated in mL

5. Milk microbiome analysis

NOTE: The milk microbiome analysis can be performed according to the methods and pipeline described elsewhere¹⁶. A screenshot of the R script used in this analysis is presented in Supplemental Figure S1.

1. Centrifuge 500 µL of the sample for 15 s at 180 × g to sediment cells and particulates.
2. Collect 350 µL of the supernatant and use it for DNA extraction.
3. Quantify the extracted DNA using a spectrophotometer.
4. Amplify the V3 and V4 regions of the 16S ribosomal RNA using 20 ng of DNA and primers 357F/805R (see Table of Materials)¹⁷.
5. Use magnetic beads to purify the resulting amplicons and quantify barcoded amplicons using a DNA fluorimeter.
6. Sequence the amplicons in paired-end 250 bp mode.
7. Process the resulting sequences by performing quality trimming and adapter removal¹⁸. Use paired-end mode (PE) with Phred33 scoring and set a minimum read length of 200 bp to ensure high-quality reads.
8. Import paired-end sequence data into QIIME 2¹⁹ using a manifest file format and the command: qiime tools import.
9. Process the imported sequences with DADA2 for denoising, dereplication, and chimera removal by using the command: qiime dada2 denoise-paired. This step generates high-quality representative sequences and a feature table.
10. Filter features and sequences to retain only those with sufficient representation using the commands: qiime feature-table filter-features and qiime feature-table filter-seqs.
11. Prepare a reference database by extracting regions amplified by the primers (V3-V4) using: qiime feature-classifier extract-reads.
12. Use the VSEARCH algorithm for taxonomic classification with the Silva reference database at a 97% identity threshold by executing the command:
    qiime feature-classifier classify-consensus-vsearch
13. Integrate taxonomic assignments with the feature table using BIOM tools: biom add-metadata.
14. Perform statistical analyses as described elsewhere^20,21.

Results

Bacterial growth analysis by plate culturing
The microbiological tests conducted to validate the method of inoculating PDM with the MOM, as proposed by Cacho et al.⁹, demonstrated that, based on the CFU/mL count, there were no significant differences between the inoculated milk samples before and after the 4 h incubation. However, differences in the number of colonies were observed at these two time points, indicating bacterial growth during incubation on MSA for Staphyl...

Discussion

Here we present a protocol to be applied in maternity hospitals associated with a human milk bank, aiming to provide PDM with the reconstitution of the milk microbiota from a specific mother who, for various reasons, cannot provide sufficient milk to her newborn. The protocol is simple and essentially based on two steps. The first involves the application of procedures routinely performed in human milk banks for the proper collection and subsequent pasteurization of donor milk, as well as the collection of the mother's o...

Materials

Name	Company	Catalog Number	Comments
Electric breast milk pump	Horigen	XN-2219M2	Soft pump dual plus
Pyrex media bottles	Corning	CLS1395100	Glass containers with plastic lids
1000 µL pipette Labmate pro	Corning HTL SA	5666	Variable volume pipettor
Cryogenic vial	Corning	CLS431417	DNase/RNase-free 2 mL vials
15 mL centrifuge tubes	Corning	CLS431470	DNase/RNase-free 15 mL tubes
Water bath	EcoSonics	Q3.0/40A
Man–Rogosa–Sharpe (MRS)	Difco	288210	Lactic acid bacteria growth media
Mannitol Salt Agar (MSA)	Himedia	MH118	Staphylococcus  growth media
Anaerobic Jar	Permution		Create a low-oxygen environment for lactic acid bacteria growth
Extracta  Kit – DNA e RNA	Loccus	MPTA-PV16-B Y	DNA extraction Kit
Nanodrop	Promega	E6150	Quantus DNA
GoTaqG2	Promega	M7841	PCR amplication System
Quantus Fluorometer	Promega	E5150	DNA / RNA quantitation kit
MiSeq System	Illumina Inc.	M-GL-00006 v4.0	Sequencing equipment
MiSeq Reagent Kit v2 (300-cycles)	Illumina Inc.	MS-102-2002	Sequencing kits and reagents
Software name
Trimmomatic	http://www.usadellab.org/cms/index.php?page=trimmomatic		Read trimming tool for Illumina NGS data
QIIME 2	https://docs.qiime2.org/2024.10/		Microbiome analysis package
Primer name	Primer sequence
16S_357F	TCGTCGGCAGCGTCAGA TGTGTATAAGAGACAGC CTACGGGNGGCWGCAG
16S_805R	GTCTCGTGGGCTCGGAG ATGTGTATAAGAGACAGG ACTACHVGGGTATCTAATC