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

新生儿重症监护室的早产儿由自己的母亲或捐赠者喂养母乳，并接触各种处理方法，包括新鲜母乳，冷冻和解冻母乳以及霍尔德巴氏消毒供体母乳。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，这些变化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的活性，包括抗菌、肠细胞和巨噬细胞免疫调节。不同的奶源、加工和储存方式可能产生不同的生物活性肽释放，因此婴儿胃肠道中的活性也不同。有一个关键的需要，以评估乳汁处理的做法对释放多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喂养的做法，早产儿促进最佳的体外发育和生长。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，喂养这些牛奶的早产儿肠道内释放的乳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1)抗菌和双歧杆菌活性，2)肠细胞免疫调节活性，3)巨噬细胞免疫调节活性的肽在早产儿肠道内容物新鲜母乳（MM），冷冻和解冻母乳（FTMM）， Holder-巴氏灭菌母乳（HPMM）和Holder-巴氏灭菌供体乳（HPDM）。我们的方法是在新生儿重症监护病房（NICU）的早产儿中收集不同处理方式的母乳喂养后的肠道样本，从这些样本中提取肽成分，在体外测试其抗菌、双歧性和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。这项研究具有创新性，因为它确定了体内肠道消化过程中释放的肽的生物活性，并检查了母乳处理实践的影响。在这个项目结束时，我们期望已经确定1)早产儿肠道中释放的乳肽表达肠道健康生物活性的程度；2)不同的牛奶处理方式对肽释放和生物活性的影响。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，从而提高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.