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

NICU中的早产儿从他们自己的母亲或捐赠者那里喂养母乳，并接受各种处理做法，包括新鲜母乳、冷冻和解冻的母乳以及Holder巴氏杀菌捐赠者的母乳。冷冻-解冻循环和巴氏杀菌会改变牛奶蛋白水解酶和蛋白质的结构，而这些变化会影响乳多肽的释放。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。牛奶来源、加工和储存的不同可能会导致生物活性多肽的不同释放，从而导致婴儿胃肠道活动的不同。迫切需要评估牛奶处理方法对婴儿体内多肽释放及其生物活性的影响。我们的长期目标是确定早产儿的喂养方法，以促进最佳的体外发育和生长。这项建议的总体目标是确定常见的牛奶处理做法如何影响早产儿肠道内促进肠道健康的多肽的释放。我们的中心假设是，喂食这些牛奶的早产儿肠道内释放的乳肽的特性和数量将显著不同，因此将具有不同的抗菌、肠细胞和巨噬细胞免疫调节作用。本研究的具体目的是测定喂养新鲜母乳(MM)、冷冻和解冻母乳(FTMM)、巴氏杀菌母乳(HPMM)和巴氏杀菌供体乳(HPDM)的早产儿肠道内容物中多肽的抗菌和双歧活性，2)肠细胞免疫调节活性，3)巨噬细胞免疫调节活性。我们的方法是采集新生儿重症监护病房(NICU)中早产儿在喂养不同处理方式的牛奶后的肠道样本，从这些样本中提取多肽成分，在体外测试它们的抗菌、双歧以及肠细胞和巨噬细胞免疫调节活性，并通过质谱仪和数据库搜索技术鉴定这些多肽。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多肽的生物活性，并检查了处理人类牛奶的做法的影响。在该项目结束时，我们预计已经确定了1)早产儿肠道中释放的乳多肽表达与肠道健康相关的生物活性的程度；以及2)不同的牛奶处理方法如何影响多肽释放和生物活性。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，并可能确定有助于改善婴儿肠道内生物活性的牛奶补充策略。

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.