Effects of human milk handling practices on peptide release and bioactivity in the preterm infant intestine

NICU中的早产儿由他们自己的母亲或捐赠者喂养母乳，并暴露于各种处理实践，包括新鲜母乳、冷冻和解冻母乳以及保持器巴氏消毒的捐赠者母乳。冻融循环和巴氏灭菌可以改变牛奶蛋白酶和蛋白质的结构，这些改变可以影响牛奶肽的释放。存在于婴儿肠道中的乳肽具有一系列活性，包括抗微生物、肠细胞和巨噬细胞免疫调节。奶源、加工和储存的差异可能产生生物活性肽的差异释放，从而产生婴儿胃肠道中的差异活性。有一个关键的需要，以评估牛奶处理的做法对释放的肽在婴儿和他们的生物活性的影响。我们的长期目标是确定早产儿的喂养方法，以促进最佳的子宫外发育和生长。本提案的总体目标是确定常见的牛奶处理方法如何影响早产儿肠道内肠道健康促进肽的释放。我们的中心假设是，这些牛奶喂养的早产儿肠道内释放的牛奶肽的身份和数量将显着不同，因此将具有不同的抗菌，肠细胞和巨噬细胞免疫调节作用。我们的具体目的是确定在喂养新鲜母乳（MM）、冷冻和解冻母乳（FTMM）、保持器巴氏灭菌母乳（HPMM）和保持器巴氏灭菌供体奶（HPDM）的早产儿的肠内容物中肽的1）抗微生物和生物生成活性、2）肠细胞免疫调节活性和3）巨噬细胞免疫调节活性。我们的方法将是从新生儿重症监护室（NICU）内喂养暴露于不同处理方法的牛奶后的早产儿中收集肠道样本，从这些样本中提取肽组分，体外测试其抗菌、生物合成、肠细胞和巨噬细胞免疫调节活性，并通过质谱和数据库搜索技术鉴定肽。这项研究是创新的，因为它确定了在体内肠道消化过程中释放的肽的生物活性，并检查了人乳处理实践的影响。在该项目结束时，我们预计已经确定1）早产儿肠道中释放的乳肽表达肠道健康相关生物活性的程度;以及2）不同的牛奶处理实践如何影响肽释放和生物活性。这项研究的积极影响是，它将有助于确定早产儿的最佳牛奶处理方法，以及可能的牛奶补充策略，从而改善婴儿肠道内的生物活性。

Preterm infants in the NICU are fed human milk from their own mothers or donors, and exposed to a variety of handling practices, including fresh mother’s milk, frozen and thawed mother’s milk and Holder pasteurized donor milk. Freeze-thaw cycles and pasteurization can alter the structure of milk proteases and proteins, and these alterations can affect the release of milk peptides. Milk peptides present in the infant intestine have an array of activities including antimicrobial, and enterocyte- and macrophage-immunomodulatory. Differences in milk sources, processing, and storage may yield differential release of bioactive peptides, and therefore differential activities in the gastrointestinal tract of infants. There is a critical need to evaluate effects of milk handling practices on release of peptides within the infant and their bioactivities. Our long-term goal is to determine feeding practices for preterm infants that promote optimal ex utero development and growth. The overall objective of this proposal is to identify how common milk handling practices affect the release of gut health-promoting peptides within the preterm infant intestine. Our central hypothesis is that the identity and amounts of milk peptides released within the intestine of preterm infants fed with these milks will be markedly different and therefore will have different profiles of antimicrobial, and enterocyte- and macrophage-immunomodulatory actions. Our specific aims are to determine the 1) antimicrobial and bifidogenic activities, 2) enterocyte-immunomodulatory activity, and 3) macrophage- immunomodulatory activity of peptides in the intestinal contents of preterm infants fed fresh mother’s milk (MM), frozen and thawed mother’s milk (FTMM), Holder-pasteurized mother’s milk (HPMM) and Holder- pasteurized donor milk (HPDM). Our approach will be to collect intestinal samples from preterm neonates within the neonatal intensive care unit (NICU) after feeding milk exposed to different handling practices, extract the peptide component from these samples, test their antibacterial, bifidogenic and enterocyte- and macrophage-immunomodulatory activity in vitro, and identify the peptides via mass spectrometry and database searching techniques. This research is innovative because it identifies the bioactivities of peptides released during in vivo intestinal digestion and examines the effect of human milk handling practices. At the conclusion of this project, we expect to have determined 1) the extent to which milk peptides released in the preterm infant intestine express gut health-related bioactivity; and 2) how different milk handling practices affect peptide release and bioactivity. The positive impact of this research is that it will help determine optimal milk handling practices for preterm infants, and possibly supplementation strategies for milk that will lead to improved bioactivity within the infant gut.