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 del....

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,

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 Staphyloc.......

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

List of materials used in this article

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