Term Infant Formulas Influencing Gut Microbiota: An Overview

Intestinal colonization of the neonate is highly dependent on the term of pregnancy, the mode of delivery, the type of feeding [breast feeding or formula feeding]. Postnatal immune maturation is dependent on the intestinal microbiome implementation and composition and type of feeding is a key issue in the human gut development, the diversity of microbiome, and the intestinal function. It is well established that exclusive breastfeeding for 6 months or more has several benefits with respect to formula feeding. The composition of the new generation of infant formulas aims in mimicking HM by reproducing its beneficial effects on intestinal microbiome and on the gut associated immune system (GAIS). Several approaches have been developed currently for designing new infant formulas by the addition of bioactive ingredients such as human milk oligosaccharides (HMOs), probiotics, prebiotics [fructo-oligosaccharides (FOSs) and galacto-oligosaccharides (GOSs)], or by obtaining the so-called post-biotics also known as milk fermentation products. The aim of this article is to guide the practitioner in the understanding of these different types of Microbiota Influencing Formulas by listing and summarizing the main concepts and characteristics of these different models of enriched IFs with bioactive ingredients.     

Human Milk Oligosaccharides and Lactose Differentially Affect Infant Gut Microbiota and Intestinal Barrier In Vitro

Background: The infant gut microbiota establishes during a critical window of opportunity when metabolic and immune functions are highly susceptible to environmental changes, such as diet. Human milk oligosaccharides (HMOs) for instance are suggested to be beneficial for infant health and gut microbiota. Infant formulas supplemented with the HMOs 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) reduce infant morbidity and medication use and promote beneficial bacteria in the infant gut ecosystem. To further improve infant formula and achieve closer proximity to human milk composition, more complex HMO mixtures could be added. However, we currently lack knowledge about their effects on infants’ gut ecosystems. Method: We assessed the effect of lactose, 2′-FL, 2′-FL + LNnT, and a mixture of six HMOs (HMO6: consisting of 2′-FL, LNnT, difucosyllactose, lacto-N-tetraose, 3′- and 6′-sialyllactose) on infant gut microbiota and intestinal barrier integrity using a combination of in vitro models to mimic the microbial ecosystem (baby M-SHIME^®) and the intestinal epithelium (Caco-2/HT29-MTX co-culture). Results: All the tested products had bifidogenic potential and increased SCFA levels; however, only the HMOs’ fermented media protected against inflammatory intestinal barrier disruption. 2′-FL/LNnT and HMO6 promoted the highest diversification of OTUs within the Bifidobactericeae family, whereas beneficial butyrate-producers were specifically enriched by HMO6. Conclusion: These results suggest that increased complexity in HMO mixture composition may benefit the infant gut ecosystem, promoting different bifidobacterial communities and protecting the gut barrier against pro-inflammatory imbalances.     

Building a Beneficial Microbiome from Birth

The microbiota has recently been recognized as a driver of health that affects the immune, nervous, and metabolic systems. This influence is partially exerted through the metabolites produced, which may be relevant for optimal infant development and health. The gut microbiota begins developing early in life, and this initial colonization is remarkably important because it may influence long-term microbiota composition and activity. Considering that the microbiome may play a key role in health and disease, maintaining a protective microbiota could be critical in preventing dysbiosis-related diseases such as allergies, autoimmunity disorders, and metabolic syndrome. Breast milk and milk glycans in particular are thought to play a major role in shaping the early-life microbiota and promoting its development, thus affecting health. This review describes some of the effects the microbiota has on the host and discusses the role microbial metabolites play in shaping newborn health and development. We describe the gut microbiota structure and function during early life and the factors that determine its composition and hypothesize about the effects of human milk oligosaccharides and other prebiotic fibers on the neonatal microbiota.     

人乳低聚糖对婴幼儿免疫的作用

人乳低聚糖是一类复合糖,由单糖通过糖苷键连接而成,其有助于婴幼儿免疫系统的发展。人乳低聚糖的一小部分被吸收至体循环,大部分达到结肠。人乳低聚糖除在肠腔和肠粘膜表面,可在全身多个部位发挥免疫功能。本文综述了在婴幼儿期人乳低聚糖与肠道菌群的相互作用,介导的免疫调节,对病原体和过敏反应的抑制。     

Human milk oligosaccharides: prebiotics and beyond

Human milk oligosaccharides (HMO) are complex glycans that are present at very high concentrations in human milk but not in infant formula. The significant energy expended by mothers to make these complex glycans suggests they must be important. How do maternal HMOs benefit the breast-fed infant? How are HMOs synthesized in the human mammary gland? How can we provide formula-fed infants with HMOs or HMO-like glycans? This article reviews current knowledge and open questions on the biosynthesis and functions of HMOs.     

Multifunctional Benefits of Prevalent HMOs: Implications for Infant Health

Breastfeeding is the best source of nutrition during infancy and is associated with a broad range of health benefits. However, there remains a significant and persistent need for innovations in infant formula that will allow infants to access a wider spectrum of benefits available to breastfed infants. The addition of human milk oligosaccharides (HMOs) to infant formulas represents the most significant innovation in infant nutrition in recent years. Although not a direct source of calories in milk, HMOs serve as potent prebiotics, versatile anti-infective agents, and key support for neurocognitive development. Continuing improvements in food science will facilitate production of a wide range of HMO structures in the years to come. In this review, we evaluate the relationship between HMO structure and functional benefits. We propose that infant formula fortification strategies should aim to recapitulate a broad range of benefits to support digestive health, immunity, and cognitive development associated with HMOs in breastmilk. We conclude that acetylated, fucosylated, and sialylated HMOs likely confer important health benefits through multiple complementary mechanisms of action.     

A Comparison of the In Vitro Effects of 2’Fucosyllactose and Lactose on the Composition and Activity of Gut Microbiota from Infants and Toddlers

Because of the recognized health benefits of breast milk, it is recommended as the sole nutrition source during the first 6 months of life. Among the bioactive components are human milk oligosaccharides (HMOs) that exert part of their activity via the gut microbiota. Here, we investigated the gut microbiota fermentation of HMO 2’fucosyllactose (2’-FL), using two in vitro models (48 h fecal incubations and the long-term mucosal simulator of the human intestinal microbial ecosystem [M-SHIME^®]) with fecal samples from 3-month-old breastfed (BF) infants as well as 2–3 year old toddlers. The short-term model allowed the screening of five donors for each group and provided supportive data for the M-SHIME^® study. A key finding was the strong and immediate increase in the relative abundance of Bifidobacteriaceae following 2’-FL fermentation by both the BF infant and toddler microbiota in the M-SHIME^®. At the metabolic level, while decreasing branched-chain fatty acids, 2’-FL strongly increased acetate production together with increases in the health-related propionate and butyrate whilst gas production only mildly increased. Notably, consistently lower gas production was observed with 2’-FL fermentation as compared to lactose, suggesting that reduced discomfort during the dynamic microbiome establishment in early life may be an advantage along with the bifidogenic effect observed.     

From Mum to Bum: An Observational Study Protocol to Follow Digestion of Human Milk Oligosaccharides and Glycoproteins from Mother to Preterm Infant

The nutritional requirements of preterm infants are challenging to meet in neonatal care, yet crucial for their growth, development and health. Aberrant maturation of the gastrointestinal tract and the microbiota could affect the digestion of human milk and its nutritional value considerably. Therefore, the main objective of the proposed research is to investigate how the intestinal microbiota of preterm and full-term infants differ in their ability to extract energy and nutrients from oligosaccharides and glycoproteins in human milk. This pilot study will be an observational, single-center study performed at the Neonatal Intensive Care Unit at Isala Women and Children’s Hospital (Zwolle, The Netherlands). A cohort of thirty mother–infant pairs (preterm ≤30 weeks of gestation, n = 15; full-term 37–42 weeks of gestation, n = 15) will be followed during the first six postnatal weeks with follow-up at three- and six-months postnatal age. We will collect human milk of all mothers, gastric aspirates of preterm infants and fecal samples of all infants. A combination of 16S rRNA amplicon sequencing, proteomics, peptidomics, carbohydrate analysis and calorimetric measurements will be performed. The role of the microbiota in infant growth and development is often overlooked yet offers opportunities to advance neonatal care. The ‘From Mum to Bum’ study is the first study in which the effect of a preterm gut microbiota composition on its metabolic capacity and subsequent infant growth and development is investigated. By collecting human milk of all mothers, gastric aspirates of preterm infants and fecal samples of all infants at each timepoint, we can follow digestion of human milk from the breast of the mother throughout the gastrointestinal tract of the infant, or ‘From Mum to Bum’.     

Perspective: Milk microRNAs as Important Players in Infant Physiology and Development

Evolutionary selective pressure on lactation has resulted in milk that provides far more than simply essential nutrients, delivering a complex repertoire of agents from hormones to intact cells. Human infants are born with low barrier integrity of their gut, which means that many of the complex biopolymer components of milk enter and circulate in lymph and blood, reaching organs throughout the body. Due to this state of gut maturation, all components of milk are potentially part of the crosstalk between mother and infants. This article highlights the functions of milk''s complex biopolymers, more specifically the potential role of microRNAs (miRNAs) contained in extracellular vesicles in human milk. miRNAs are key effectors in the regulation of many biological processes during early-age development, and consequently milk-sourced miRNAs must be considered to provide unique biological assets to the infant during breastfeeding. This article interprets the evidence of the potential action of human milk miRNAs on infant development, taking into account their abundance in milk based on the literature and current knowledge. Human milk miRNAs appear to influence lipid and glucose metabolism, gut maturation, neurogenesis, and immunity. We also show growing evidence that human milk miRNAs are epigenetic modulators that play a pivotal role in the regulation of tissue-specific gene expression throughout life. Furthermore, this article addresses the ongoing debate regarding the potential influence of human milk miRNAs on viral infection as a new research area. This article highlights that these bioactive molecules are now being incorporated into our overall understanding of nutrient needs for healthy infant development, preparing each individual infant to succeed as a healthy and protected adult throughout its life. In essence, miRNAs are a new language in the Rosetta stone of health that is mammalian lactation.     

Clinical Evaluation of 16-Week Supplementation with 5HMO-Mix in Healthy-Term Human Infants to Determine Tolerability,Safety, and Effect on Growth

Human milk oligosaccharides (HMOs) are complex sugars that occur naturally in human breast milk and provide many beneficial functions. Most formula products lack HMOs or contain only the most abundant HMO, 2′-fucosyllactose; however, benefits of HMOs come from multiple sugars. We therefore developed a mixture of five HMOs (5HMO-Mix) mimicking the natural concentrations of the top five HMOs (5.75 g/L total, comprising 52% 2′-fucosyllactose, 13% 3-fucosyllactose, 26% lacto-N-tetraose, 4% 3′-sialyllactose, and 5% 6′-sialyllactose) representing the groups of neutral, neutral-fucosylated, and sialylated HMOs. We conducted the first multicenter, randomized, controlled, parallel-group clinical study assessing the safety, tolerability, and effect on growth of formula containing the 5HMO-Mix in healthy infants. We enrolled 341 subjects aged ≤14 days; 225 were randomized into groups fed either with infant formula containing 5HMO-Mix (5HMO-Mix) or infant formula without HMOs (IF) for 4 months, with the others exclusively breastfed. There were no differences in weight, length, or head circumference gain between the two formula groups. The 5HMO-Mix was well tolerated, with 5HMO-Mix and breastfed infants producing softer stools at a higher stool frequency than the control formula group. Adverse events were equivalent in all groups. We conclude that the 5HMO-Mix at 5.75 g/L in infant formula is safe and well tolerated by healthy term infants during the first months of life.     

Acceleration of Small Intestine Development and Remodeling of the Microbiome Following Hyaluronan 35 kDa Treatment in Neonatal Mice

The beneficial effects of human milk suppressing the development of intestinal pathologies such as necrotizing enterocolitis in preterm infants are widely known. Human milk (HM) is rich in a multitude of bioactive factors that play major roles in promoting postnatal maturation, differentiation, and the development of the microbiome. Previous studies showed that HM is rich in hyaluronan (HA) especially in colostrum and early milk. This study aims to determine the role of HA 35 KDa, a HM HA mimic, on intestinal proliferation, differentiation, and the development of the intestinal microbiome. We show that oral HA 35 KDa supplementation for 7 days in mouse pups leads to increased villus length and crypt depth, and increased goblet and Paneth cells, compared to controls. We also show that HA 35 KDa leads to an increased predominance of Clostridiales Ruminococcaceae, Lactobacillales Lactobacillaceae, and Clostridiales Lachnospiraceae. In seeking the mechanisms involved in the changes, bulk RNA seq was performed on samples from the terminal ileum and identified upregulation in several genes essential for cellular growth, proliferation, and survival. Taken together, this study shows that HA 35 KDa supplemented to mouse pups promotes intestinal epithelial cell proliferation, as well as the development of Paneth cells and goblet cell subsets. HA 35 KDa also impacted the intestinal microbiota; the implications of these responses need to be determined.     

Structural and functional aspects of prebiotics used in infant nutrition

Breast-feeding is associated with several benefits. Among them, the balanced postnatal development of the immune system is 1 of the key functions of breast-feeding. Although this effect is of multifactorial origin, it is widely accepted that the entire intestinal microbiota of breast-fed infants represents an important stimulating factor of the postnatal development of the immune system. The effect of breast-feeding on the intestinal microbiota can not be attributed to a single compound, but there is accumulating evidence that human milk oligosaccharides play a crucial role. Because there is a broad consensus that the intestinal microbiota plays an important physiological role for the host, many attempts have been made to influence the intestinal flora by dietary interventions. This article summarizes results of intervention studies in which nonmilk oligosaccharides have been used to mimic the prebiotic effect of breast-feeding. A second focus has been related to the question of whether the prebiotic activity has beneficial effects on the postnatal development of the immune system. The data clearly demonstrate that prebiotics of nonmilk origin can mimic the prebiotic effect of breast-feeding, and this has positive consequences for the postnatal development of the immune system.     

The Gut‒Breast Axis: Programming Health for Life

The gut is a pivotal organ in health and disease. The events that take place in the gut during early life contribute to the programming, shaping and tuning of distant organs, having lifelong consequences. In this context, the maternal gut plays a quintessence in programming the mammary gland to face the nutritional, microbiological, immunological, and neuroendocrine requirements of the growing infant. Subsequently, human colostrum and milk provides the infant with an impressive array of nutrients and bioactive components, including microbes, immune cells, and stem cells. Therefore, the axis linking the maternal gut, the breast, and the infant gut seems crucial for a correct infant growth and development. The aim of this article is not to perform a systematic review of the human milk components but to provide an insight of their extremely complex interactions, which render human milk a unique functional food and explain why this biological fluid still truly remains as a scientific enigma.     

Human Milk Oligosaccharide Profiles and Associations with Maternal Nutritional Factors: A Scoping Review

Human milk oligosaccharides (HMOs) are complex unconjugated glycans associated with positive infant health outcomes. This study has examined current knowledge of the effect of maternal diet and nutritional status on the composition of HMOs in breast milk. Using the PRISMA-ScR guidelines, a comprehensive, systematic literature search was conducted using Scopus, Web of Science, Global Health (CABI), and MEDLINE. Titles and abstracts were screened independently by two reviewers against predefined inclusion and exclusion criteria. Fourteen studies met the inclusion criteria and reported on maternal dietary intake (n = 3), maternal body composition indices (n = 9), and dietary supplementation interventions (n = 2). In total, data from 1388 lactating mothers (4011 milk samples) were included. Design methodologies varied substantially across studies, particularly for milk sample collection, HMO analysis, dietary and body composition assessment. Overall, this review has identified potential associations between maternal dietary intake and nutritional status and the HMO composition of human milk, though an abundance and sufficiency of evidence is lacking. Standardised procedures for human milk sample collection and HMO analysis, along with robust and validated nutrition assessment techniques, should be employed to further investigate the impact of maternal nutritional factors on HMO composition.     

Variations in the Composition of Human Milk Oligosaccharides Correlates with Effects on Both the Intestinal Epithelial Barrier and Host Inflammation: A Pilot Study

Background: Human milk oligosaccharides are complex, non-digestible carbohydrates that directly interact with intestinal epithelial cells to alter barrier function and host inflammation. Oligosaccharide composition varies widely between individual mothers, but it is unclear if this inter-individual variation has any impact on intestinal epithelial barrier function and gut inflammation. Methods: Human milk oligosaccharides were extracted from the mature human milk of four individual donors. Using an in vitro model of intestinal injury, the effects of the oligosaccharides on the intestinal epithelial barrier and select innate and adaptive immune functions were assessed. Results: Individual oligosaccharide compositions shared comparable effects on increasing transepithelial electrical resistance and reducing the macromolecular permeability of polarized (Caco-2Bbe1) monolayers but exerted distinct effects on the localization of the intercellular tight junction protein zona occludins-1 in response to injury induced by a human enteric bacterial pathogen Escherichia coli, serotype O157:H7. Immunoblots showed the differential effects of oligosaccharide compositions in reducing host chemokine interleukin 8 expression and inhibiting of p38 MAP kinase activation. Conclusions: These results provide evidence of both shared and distinct effects on the host intestinal epithelial function that are attributable to inter-individual differences in the composition of human milk oligosaccharides.     

Milk Exosomes Transfer Oligosaccharides into Macrophages to Modulate Immunity and Attenuate Adherent-Invasive E. coli (AIEC) Infection

Exosomes are abundance in human body fluids like urine, milk and blood. They act a critical role in extracellular and intracellular communication, intracellular trafficking and physiological regulation. Multiple immune-modulatory components, such as proteins, RNAs and carbohydrates (glycoproteins), have been found in human milk exosomes, which play immune-regulatory functions. However, little is known about oligosaccharides in milk exosomes, the “free sugars”, which act critical roles in the development of infant’s immature mucosal immune system. In this study, the profile of milk exosomes encapsulated human milk oligosaccharides (HMOs) was calibrated with characteristic oligosaccharides in colostrum and mature milk, respectively. The exosomes containing human milk oligosaccharides were uptaken by macrophages, which were responsible for the establishment of intestinal immunity. Furthermore, mice pretreated with exosome encapsulated HMOs were protected from AIEC infection and had significantly less LPS-induced inflammation and intestinal damage. Exosome encapsulated milk oligosaccharides are regarded to provide a natural manner for milk oligosaccharides to accomplish their critical functions in modifying newborn innate immunity. The understanding of the interaction between a mother’s breastfeeding and the development of an infant’s mucosal immune system would be advantageous. The transport of milk oligosaccharides to its target via exosome-like particles appears to be promising.     

Changes in HMO Concentrations throughout Lactation: Influencing Factors,Health Effects and Opportunities

Human milk oligosaccharides (HMOs) are important functional biomolecules in human breast milk. Understanding the factors influencing differences in HMO composition and changes in their concentration over lactation can help to design feeding strategies that are well-adapted to infant’s needs. This review summarises the total and individual concentration of HMOs from data published from 1999 to 2019. Studies show that the HMO concentrations are highest in colostrum (average 9–22 g/L), followed by slightly lower concentrations in transitional milk (average 8–19 g/L), with a gradual decline in mature milk as lactation progresses, from 6–15 g/L in breast milk collected within one month of birth, to 4–6 g/L after 6 months. Significant differences in HMO composition have been described between countries. Different HMOs were shown to be predominant over the course of lactation, e.g., 3-fucosyllactose increased over lactation, whereas 2′-fucosyllactose decreased. Recent clinical studies on infant formula supplemented with 2′-fucosyllactose in combination with other oligosaccharides showed its limited beneficial effect on infant health.     

Cytotoxic Lactalbumin-Oleic Acid Complexes in the Human Milk Diet of Preterm Infants

Frozen storage is necessary to preserve expressed human milk for critically ill and very preterm infants. Milk pasteurization is essential for donor milk given to this special population. Due to these storage and processing conditions, subtle changes occur in milk nutrients. These changes may have clinical implications. Potentially, bioactive complexes of unknown significance could be found in human milk given to preterm infants. One such complex, a cytotoxic α-lactalbumin-oleic acid complex named “HAMLET,” (Human Alpha-Lactalbumin Made Lethal to Tumor cells) is a folding variant of alpha-lactalbumin that is bound to oleic acid. This complex, isolated from human milk casein, has specific toxicity to both carcinogenic cell lines and immature non-transformed cells. Both HAMLET and free oleic acid trigger similar apoptotic mechanisms in tissue and stimulate inflammation via the NF-κB and MAPK p38 signaling pathways. This protein-lipid complex could potentially trigger various inflammatory pathways with unknown consequences, especially in immature intestinal tissues. The very preterm population is dependent on human milk as a medicinal and broadly bioactive nutriment. Therefore, HAMLET’s possible presence and bioactive role in milk should be addressed in neonatal research. Through a pediatric lens, HAMLET’s discovery, formation and bioactive benefits will be reviewed.     

Breast Milk: A Source of Functional Compounds with Potential Application in Nutrition and Therapy

Breast milk is an unbeatable food that covers all the nutritional requirements of an infant in its different stages of growth up to six months after birth. In addition, breastfeeding benefits both maternal and child health. Increasing knowledge has been acquired regarding the composition of breast milk. Epidemiological studies and epigenetics allow us to understand the possible lifelong effects of breastfeeding. In this review we have compiled some of the components with clear functional activity that are present in human milk and the processes through which they promote infant development and maturation as well as modulate immunity. Milk fat globule membrane, proteins, oligosaccharides, growth factors, milk exosomes, or microorganisms are functional components to use in infant formulas, any other food products, nutritional supplements, nutraceuticals, or even for the development of new clinical therapies. The clinical evaluation of these compounds and their commercial exploitation are limited by the difficulty of isolating and producing them on an adequate scale. In this work we focus on the compounds produced using milk components from other species such as bovine, transgenic cattle capable of expressing components of human breast milk or microbial culture engineering.