Variability in Presence, Abundance, and Timing of Bovine Milk- and Soy Beverage-Derived Peptides in Human Milk After Maternal Bovine Milk and Soy Beverage Consumption: A Randomized, Cross-over, Dietary Intervention Trial

BACKGROUND: Food-borne allergens in human milk (HM) may cause allergic responses in HM-fed infants, but variability of allergen transfer complicates recommendations for individuals nursing food-allergic infants. OBJECTIVE: We aimed to identify bovine- and soy-derived peptides in HM after maternal elimination and reintroduction of bovine milk (BM) and soy beverage (SB). METHODS: In this randomized, cross-over, dietary intervention trial, 38 lactating participants underwent 2 study phases, each including a 5-day diet elimination, 3-day diet intervention, and 2-day washout. Each diet intervention required daily consumption of increasing amounts of BM or SB (175, 295, and 415 mL). Peptidomics analysis was performed on a subset of HM samples (24 participants) collected after dietary elimination, and 2 and 4 h after BM/SB consumption (415 mL). Peptides were isolated via ethanol precipitation and C18 solid-phase extraction, analyzed by LC-MS/MS, and identified with Proteome Discoverer. RESULTS: We identified 121 bovine-derived peptides (associated with 6 proteins) in HM collected during the BM phase. From most to least abundant, these proteins were β-lactoglobulin, κ-casein, αs1-casein, β-casein, α-lactalbumin protein variant D, and glycosylation-dependent cell adhesion molecule 1. Generalized linear mixed models demonstrated differences in relative abundance for 14 peptides when comparing before, and 2 and 4 h after BM consumption. We identified 8 peptides of possible soy origin in HM collected during the SB phase, but they were not matched to parent proteins with adequate confidence. CONCLUSIONS: The relative abundance of some BM-derived peptides, while low overall, may differ in human milk collected after maternal BM dietary elimination compared to 2 and 4 h after BM consumption. Five days of dietary elimination may not be adequate for the elimination of BM-derived peptides or low levels of these non-human peptides may be present in HM from other sources. Soy-derived peptides

背景：人乳（human milk，HM）中的食源性过敏原可能会引发接受人乳喂养的婴儿产生过敏反应，但过敏原转运的差异性使得为食物过敏婴儿的哺乳者制定喂养建议变得更为复杂。
目的：本研究旨在明确在产妇剔除并重新引入牛乳（bovine milk，BM）与大豆饮品（soy beverage，SB）后，人乳中牛源与大豆源肽类的存在情况。
方法：本项随机交叉膳食干预试验共纳入38名泌乳受试者，分为2个研究阶段，每个阶段均包含5天饮食剔除期、3天饮食干预期以及2天洗脱期。每个干预期要求受试者每日递增剂量摄入牛乳或大豆饮品，剂量分别为175、295与415 mL。本研究对24名受试者的部分人乳样本进行了肽组学分析，样本采集时间分别为饮食剔除结束后，以及摄入415 mL牛乳/大豆饮品后2小时与4小时。肽类通过乙醇沉淀与C18固相萃取进行分离，经液相色谱-串联质谱（LC-MS/MS）分析后，采用Proteome Discoverer进行肽段鉴定。
结果：在牛乳干预阶段采集的人乳样本中，我们共鉴定出121条牛源肽段，对应6种蛋白质。按丰度从高到低排序，这些蛋白质分别为β-乳球蛋白（β-lactoglobulin）、κ-酪蛋白（κ-casein）、αs1-酪蛋白（αs1-casein）、β-酪蛋白（β-casein）、α-乳白蛋白蛋白变体D（α-lactalbumin protein variant D）以及糖基化依赖细胞黏附分子1（glycosylation-dependent cell adhesion molecule 1）。广义线性混合模型分析显示，在摄入牛乳前、摄入后2小时与4小时这三个时间点，14条肽段的相对丰度存在显著差异。在大豆饮品干预阶段采集的人乳样本中，我们鉴定出8条可能为大豆来源的肽段，但这些肽段无法以足够置信度匹配到其亲本蛋白质。
结论：尽管整体丰度较低，但部分牛源肽段的相对丰度在产妇剔除牛乳饮食后采集的人乳样本，与摄入牛乳后2小时、4小时采集的人乳样本中存在差异。5天的饮食剔除期可能不足以完全清除牛源肽段，或者这类非人生源肽段可能通过其他途径存在于人乳中。大豆源肽段