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.

中文翻译：

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储存、温度和提取试剂盒对母乳微生物群中检测到的系统发育组成的影响

母乳被认为是新生儿的最佳喂养方式，并且是发育中的婴儿胃肠道的细菌来源。然而，与所有低生物量样品一样，需要对样品收集、储存和提取方法等变量进行标准化，以消除研究中微生物组成的差异。本研究的目的是调查与新鲜样品相比，不同的储存方法、温度、防腐剂和提取试剂盒如何影响母乳微生物组。母乳样品通过六种不同的方法进行处理：新鲜（方法 1）、-80°C 冷冻（方法 2）、用 RNAlater 处理并储存在 4°C 或 -80°C（方法 3 和 4）、以及处理在室温下用牛奶保存液保存（方法 5 和 6）。^{方法 1-5 使用 PowerFood TM}微生物 DNA 分离试剂盒 (Mobio)提取，方法 6 使用牛奶 DNA 保存和分离试剂盒 (Norgen BioTek) 提取。在属水平上，方法 1-5 共有最丰富的属。与新鲜样品相比，-80°C 冷冻样品的显着变化最小，而使用牛奶保存和分离试剂盒处理和提取的样品变化最显着。多样性分析表明微生物群组成的变化与所使用的方法和提取试剂盒有关。这项研究强调，当无法从新鲜牛奶样品中提取时，-80°C 冷冻是保持牛奶微生物组完整性的下一个最佳选择。此外，我们的结果表明，提取试剂盒的选择对牛奶中检测到的微生物群具有深远的影响。