Data_Sheet_1_Frozen Mother’s Own Milk Can Be Used Effectively to Personalize Donor Human Milk.csv

Feeding preterm infants mother’s own milk (MOM) lowers rates of sepsis, decreases necrotizing enterocolitis, and shortens hospital stay. In the absence of freshly expressed MOM, frozen MOM (FMOM) is provided. When MOM is unavailable, preterm infants are often fed pasteurized donor human milk (DHM), rendering it devoid of beneficial bacteria. We have previously reported that when MOM is inoculated into DHM to restore the live microbiota [restored milk (RM)], a similar microbial diversity to MOM can be achieved. Yet, it is unknown if a similar diversity to MOM can be obtained when FMOM is inoculated into DHM. The goal of this study was to determine whether a similar microbial composition to MOM could be obtained when FMOM is used to personalize DHM. To this end, a fresh sample of MOM was obtained and divided into fresh and frozen fractions. MOM and FMOM were inoculated into DHM at different dilutions: MOM/FMOM 10% (RM/FRM10) and MOM/FMOM 30% (RM/FRM30) and incubated at 37°C. At different timepoints, culture-dependent and culture-independent techniques were performed. Similar microbiota expansion and alpha diversity were observed in MOM, RM10, and RM30 whether fresh or frozen milk was used as the inoculum. To evaluate if microbial expansion would result in an abnormal activation on the innate immune system, Caco-2 epithelial cells were exposed to RM/FRM to compare interleukin 8 levels with Caco-2 cells exposed to MOM or DHM. It was found that RM samples did not elicit a significant increase in IL-8 levels when compared to MOM or FMOM. These results suggest that FMOM can be used to inoculate DHM if fresh MOM is unavailable or limited in supply, allowing both fresh MOM and FMOM to be viable options in a microbial restoration strategy.母乳喂养早产儿自身母乳（Mother’s Own Milk, MOM）可降低败血症发生率、减少坏死性小肠结肠炎的发生，并缩短住院时长。在无法获取新鲜母乳的情况下，临床会使用冷冻母乳（Frozen MOM, FMOM）。当自身母乳完全不可得时，早产儿通常会接受巴氏杀菌捐赠人乳（Pasteurized Donor Human Milk, DHM）喂养，但这类乳剂缺乏有益菌群。本团队此前已有研究报道，将自身母乳接种至捐赠人乳中以恢复其活性微生物群（复原乳，Restored Milk, RM），可获得与母乳相似的微生物多样性。然而，若使用冷冻母乳接种至捐赠人乳中，是否也能获得同等水平的微生物多样性，目前仍不明确。本研究的目的在于探究：使用冷冻母乳对捐赠人乳进行微生物个性化修复后，能否获得与母乳相似的微生物组成。为此，我们采集了一份新鲜母乳样本，并将其分为新鲜和冷冻两部分。分别以新鲜母乳、冷冻母乳为接种源，按10%（RM10/FRM10）和30%（RM30/FRM30）的稀释比例接种至捐赠人乳中，随后在37℃条件下进行培养。在不同时间节点，我们分别采用依赖培养和非依赖培养技术对样本进行检测。结果显示，无论以新鲜母乳还是冷冻母乳作为接种源，母乳、RM10及RM30组均呈现出相似的微生物群扩增效果与α多样性水平。为评估微生物扩增是否会导致先天免疫系统异常激活，我们将Caco-2上皮细胞暴露于RM/FRM样本中，并与暴露于母乳或捐赠人乳的Caco-2细胞对比其白细胞介素8（IL-8）分泌水平。结果发现，与母乳或冷冻母乳组相比，RM样本并未引发IL-8水平的显著升高。上述结果表明，若新鲜母乳不可得或供应有限，可使用冷冻母乳作为接种源对捐赠人乳进行微生物修复，这使得新鲜母乳与冷冻母乳均可作为微生物修复策略中的可行方案。