Effect of storage, temperature, and extraction kit on the phylogenetic composition detected in the human milk microbiota

Human milk is considered the optimum feeding regime for newborns and is a source of bacteria for the developing infant gastrointestinal tract. However, as with all low biomass samples, standardization across variabilities such as sample collection, storage, and extraction methods is needed to eliminate discrepancies in microbial composition across studies. The aim of this study was to investigate how different storage methods, temperatures, preservatives, and extraction kits influence the human milk microbiome, compared to fresh samples. Breast milk samples were processed via six different methods: fresh (Method 1), frozen at −80°C (Method 2), treated with RNAlater and stored at 4°C or −80°C (Methods 3 and 4), and treated with Milk Preservation Solution at room temperature (Methods 5 and 6). Methods 1‐5 were extracted using PowerFood(TM) Microbial DNA Isolation kit (Mobio), and Method 6 was extracted using Milk DNA Preservation and Isolation kit (Norgen BioTek). At genus level, the most abundant genera were shared across Methods 1‐5. Samples frozen at −80°C had fewest significant changes while samples treated and extracted using Milk Preservation and Isolation kit had the most significant changes when compared to fresh samples. Diversity analysis indicated that variation in microbiota composition was related to the method and extraction kit used. This study highlighted that, when extraction from fresh milk samples is not an option, freezing at −80°C is the next best option to preserve the integrity of the milk microbiome. Furthermore, our results demonstrate that choice of extraction kit had a profound impact on the microbiota populations detected in milk.