The gut microbiome plays an important role in early life, protecting newborns from enteric pathogens, promoting immune system development and providing key functions to the infant host. Currently, there are limited data to broadly assess the status of the US healthy infant gut microbiome. To address this gap, we performed a multi-state metagenomic survey and found high levels of bacteria associated with enteric inflammation (e.g. Escherichia , Klebsiella), antibiotic resistance genes, and signatures of dysbiosis, independent of location, age, and diet. Bifidobacterium were less abundant than generally expected and the species identified, including B. breve, B. longum and B. bifidum, had limited genetic capacity to metabolize human milk oligosaccharides (HMOs), while B. infantis strains with a complete capacity for HMOs utilization were found to be exceptionally rare. Considering microbiome composition and functional capacity, this survey revealed a previously unappreciated dysbiosis that is widespread in the contemporary US infant gut microbiome.

Purpose: The neonatal period represents a unique stage of life when critical foundations of lifelong health are established. Throughout this period, the gut microbiome provides protection against infections and is crucial for the proper development of the immune system. Deviations from optimal microbiome compositions during the postnatal stage, conceptually referred to as dysbiosis, are associated with the absence of key infant-associated Bifidobacterium known to facilitate the utilization of human milk oligosaccharides (HMOs), increased enteric inflammation, and an increase in potentially pathogenic taxa. Although previous studies have revealed a marked dysbiosis …

Emerging research into the bioactivities of indigestible carbohydrates is illuminating the potential of various foods and food streams to serve as novel sources of health-promoting compounds. Oligosaccharides (OS) are widely present in milks and some plants. Our previous research demonstrated the presence of OS in brewed coffee and spent coffee grounds. Armed with this new knowledge, the next step toward improving the utilization of these valuable components involved investigating the effect of roasting on the formation and abundance of coffee OS. In the present study, we used advanced mass spectrometry to analyze a variety of coffee samples and demonstrated that a great structural diversity and increased abundance of OS is associated with higher roasting intensity. The present investigation also evaluated methods for OS extraction and fractionation. A preparative-scale chromatographic method, based on activated carbon, was developed to isolate enough amounts of OS from coffee to enable future confirmation of prebiotic and other in vitro activities.

Oligosaccharides are indigestible carbohydrates widely present in mammalian milk and in some plants. Milk oligosaccharides are associated with positive health outcomes; however, oligosaccharides in coffee have not been extensively studied. We investigated the oligosaccharides and their monomeric composition in dark roasted coffee beans, brewed coffee, and spent coffee grounds. Oligosaccharides with a degree of polymerization ranging from 3 to 15, and their constituent monosaccharides, were characterized and quantified. The oligosaccharides identified were mainly hexoses (potentially galacto-oligosaccharides and manno-oligosaccharides) containing a heterogeneous mixture of glucose, arabinose, xylose, and rhamnose. The diversity of oligosaccharides composition found in these coffee samples suggests that they could have selective prebiotic activity toward specific bacterial strains able to deconstruct the glycosidic bonds and utilize them as a carbon source.

Attempts to alter intestinal dysbiosis via administration of probiotics have consistently shown that colonization with the administered microbes is transient. This study sought to determine whether provision of an initial course of Bifidobacterium longum subsp. infantis (B. infantis) would lead to persistent colonization of the probiotic organism in breastfed infants. Mothers intending to breastfeed were recruited and provided with lactation support. One group of mothers fed B. infantis EVC001 to their infants from day 7 to day 28 of life (n = 34), and the second group did not administer any probiotic (n = 32). Fecal samples were collected during the first 60 postnatal days in both groups. Fecal samples were assessed by 16S rRNA gene sequencing, quantitative PCR, mass spectrometry, and endotoxin measurement. B. infantis-fed infants had significantly higher populations of fecal Bifidobacteriaceae, in particular B. infantis, while EVC001 was fed, and this difference persisted more than 30 days after EVC001 supplementation ceased. Fecal milk oligosaccharides were significantly lower in B. infantis EVC001-fed infants, demonstrating higher consumption of human milk oligosaccharides by B. infantis EVC001. Concentrations of acetate and lactate were significantly higher and fecal pH was significantly lower in infants fed EVC001, demonstrating alterations in intestinal fermentation. Infants colonized by Bifidobacteriaceae at high levels had 4-fold-lower fecal endotoxin levels, consistent with observed lower levels of Gram-negative Proteobacteria and Bacteroidetes. IMPORTANCE The gut microbiome in early life plays an important role for longterm health and is shaped in large part by diet. Probiotics may contribute to improvements in health, but they have not been shown to alter the community composition of the gut microbiome. Here, we found that breastfed infants could be stably colonized at high levels by provision of B. infantis EVC001, with significant changes to the overall microbiome composition persisting more than a month later, whether the infants were born vaginally or by caesarean section. This observation is consistent with previous studies demonstrating the capacity of this subspecies to utilize human milk glycans as a nutrient and underscores the importance of pairing a probiotic organism with a specific substrate. Colonization by B. infantis EVC001 resulted in significant changes to fecal microbiome composition and was associated with improvements in fecal biochemistry. The combination of human milk and an infant-associated Bifidobacterium sp. shows, for the first time, that durable changes to the human gut microbiome are possible and are associated with improved gut function.

The development of probiotics as therapies to cure or prevent disease lags far behind that of other investigational medications. Rigorously designed phase I clinical trials are nearly non-existent in the field of probiotic research, which is a contributing factor to this disparity. As a consequence, how to appropriately dose probiotics to study their efficacy is unknown. Herein we propose a novel phase I ascending dose trial of Bifidobacterium longum subsp. infantis (B. infantis) to identify the dose required to produce predominant gut colonisation in healthy breastfed infants at 6 weeks of age.

Breast milk is a multifunctional biofluid that provides nutrients along with highly diverse non-nutritive bioactive components such as antibodies, glycans, bacteria, and immunomodulatory proteins. Research over the past decade has confirmed the essential role of breast milk bioactives in the establishment a healthy intestinal microbiota within the infant. The intestinal microbiota of an exclusively breastfed baby is dominated by several species of Bifidobacteria - the most influential member of which is Bifidobacterium longum subspecies infantis (B. infantis) - and is referred to as the milk-oriented microbiome (MOM). MOM is associated with reduced risk of infection in infancy as well as a reduced risk of certain chronic illnesses in adulthood. Establishment and persistence of MOM is dependent on the selective digestion of complex sugar structures in breast milk that are otherwise indigestible to the infant by B. infantis and its relatives. This review focuses primarily on the influence of breast milk glycans and glycosylated proteins on the development of the intestinal microbiome, and how maternal phenotype may influence the development of MOM providing a framework to understand how variation in diet shapes a protective intestinal microbiome.

Metabolomics provides a valuable strategy for describing and annotating the structures, compositions, and functions of mammalian milk. Detailed analyses of the complex components of milk have revealed an unexpected diversity of glycans consisting of oligosaccharides, glycoproteins, and glycolipids, all of which help shape the intestinal environment and in particular the intestinal microbiome of breastfed babies. Using complete and partial ensembles of glycan mixtures, the holistic principles of metabolomics analytics were leveraged for microbial screening studies. The complex glycans of human milk proved to be highly selective in their ability to support the growth of only a very rare group of enteric bacteria. These studies led to the conclusion that a signature achievement of breast milk is the development of a unique milk-oriented intestinal microbiota that results from a functional overlap of stereospecific glycan biosynthesis in maternal mammary epithelia with equally stereospecific glycosidase enzymes encoded within the genome of the commensal bacteria. Clinical evidence in support of that hypothesis has now been generated by the simultaneous administration and quantitation of the entire repertoire of glycans in the milk going in and the feces coming out of human infants. These platforms of systems biology combining separation technologies coupled to highly accurate and sensitive mass spectrometry with exhaustive library development and computational tools provide a model for success in understanding biological processes. Metabolomics is now extending that understanding of the infant microbiota and its phenotype to the role of complex glycans in the microbiota of all ages. The relentless selective pressure on the process of lactation within the mammary epithelial cell over millennia of evolution has been to nourish, protect, and support the survival of the mother infant pair. The principles that have emerged to nourish infants provide a guiding model for diet and health of all humans. The tools of metabolomics are proving successful in revealing the mechanisms behind milk’s “genius.”

In addition to providing complete postnatal nutrition, breast milk is a complex biofluid that delivers bioactive components for the growth and development of the intestinal and immune systems. Lactation is a unique opportunity to understand the role of diet in shaping the intestinal environment including the infant microbiome. Of considerable interest is the diversity and abundance of milk glycans that are energetically costly for the mammary gland to produce yet indigestible by infants. Milk glycans comprise free oligosaccharides, glycoproteins, glycopeptides, and glycolipids. Emerging technological advances are enabling more comprehensive, sensitive, and rapid analyses of these different classes of milk glycans. Understanding the impact of inter- and intraindividual glycan diversity on function is an important step toward interventions aimed at improving health and preventing disease. This review discusses the state of technology for glycan analysis and how specific structure-function knowledge is enhancing our understanding of early nutrition in the neonate.

Human milk oligosaccharides (HMOs), as an abundant and bioactive component of breast milk, work in many ways to promote the health of breast fed infants. The expression of HMOs has been shown to vary in accordance with Lewis blood type and secretor status, as women of different blood types differ in the expression of α1,2 fucosyltransferase (FUT2) and α1,3/4 fucosyltransferase (FUT3). In this study, HMOs were extracted from the milk of 60 women from The Gambia, Africa with various Lewis and secretor blood types. The HMOs were profiled using high resolution HPLC-Chip/TOF mass spectrometry. Notably, the amounts of fucosylation varied significantly between Le(a+b-) nonsecretors, Le(a-b+) and Le(a-b-) secretors, and Le(a-b-) nonsecretors. With higher frequency of expression of the recessive Lewis negative and nonsecretor phenotypes in West African populations, the HMO profiles of several milks from women of these phenotypes were examined, demonstrating decreased amounts of total oligosaccharide abundance and lower relative amounts of fucosylation. Also in this study, four specific fucosylated structures (2'FL, LNFP I, LDFT, and LNDFH I) were determined to be specific and sensitive glycan markers for rapidly determining secretor status without the need for serological testing.

Milk fat globules (MFGs) are accepted primarily as triacylglycerol delivery systems. The identification of nanometer-sized lipid-protein particles termed "lactosomes" that do not contain triacylglycerol raises the question of their possible functions. MFGs were isolated by slow centrifugation, and lactosomes were isolated by ultracentrifugation at a density equivalent to plasma high-density lipoproteins (HDL) (d > 1.063 g/mL) from human milk obtained from six volunteers at different lactation stages. Isolated lactosomes were analyzed and compared with MFGs for their size distribution, lipidome, proteome, and functional activity. Lactosomes from early milk, day 8, were found to be similar in size as those from mature milk >28 days, averaging ∼ 25 nm in diameter. In total, 97 nonredundant proteins were identified in the MFG and lactosome fractions, 46 of which were unique to the MFG fraction and 29 of which were unique to the lactosome fraction. The proteins identified in the lactosome and MFG fractions were enriched with proteins identified with immunomodulatory pathways. Unlike MFGs and GM1-laden reconstituted HDL that served as a positive control, lactosomal binding capacity to cholera toxin was weak. Lipidomic analyses found that lactosomes were devoid of triacylglycerol and gangliosides, unlike MFGs, but rich in a variety of phospholipid species. The data found differences in structure, composition, and function between lactosomes and MFG, suggesting that these two particles are derived from different biosynthetic and/or secretory pathways. The results reveal a bioactive lipid-protein, nanometer-length scale particle that is secreted into milk not to supply energy to the infant but to play unique, protective, and regulatory roles.

While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first 10 days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.

With the progressive aging of the world's population, immunosenescence is rapidly becoming a clinical concern as it accounts for a higher incidence of severe infections and poor response to vaccines. To identify nutritional approaches that may counteract immunosenescence is of obvious importance in clinical practice. Dairy products in general and whey proteins in particular share the capacity to stimulate the immune system within the digestive tract while the antibody response to Streptococcus pneumoniae vaccine is a good marker of the immune function. We performed a controlled, randomized, double‐blind pilot study to determine if an eight‐week supplementation with whey protein (or soy protein used as control) could enhance the serum response to pneumococcal vaccine in healthy senior citizens. Out of 127 volunteers, 17 subjects were eligible and completed the study receiving the vaccine after four weeks of supplementation. Antibody levels were measured at baseline and the end of the study against 14 pneumococcal types and a detailed nutritional questionnaire was administered to all subjects. Subjects receiving whey protein manifested a serum response higher compared to the control soy supplementation against 12/14 bacterial types. In particular, whey led to a higher frequency of response to all four more virulent types (4, 9, 14, and 23). Calorie and protein intake data suggest a better nutritional status in the whey group. Whey protein supplementation is a promising supplement to stimulate the immune response to vaccine in senior citizens and possibly to counteract immunosenescence while larger studies are warranted.

ABSTRACT Galacto-oligosaccharides (GOS) are versatile food ingredients that possess prebiotic properties. However, at present there is a lack of precise analytical methods to demonstrate specific GOS consumption by bifidobacteria. To better understand the role of GOS as prebiotics, purified GOS (pGOS) without disaccharides and monosaccharides was prepared and used in bacterial fermentation experiments. Growth curves showed that all bifidobacteria assayed utilized and grew on pGOS preparations. We used a novel mass spectrometry approach involving matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) to determine the composition of oligosaccharides in GOS syrup preparations. MALDI-FTICR analysis of spent fermentation media demonstrated that there was preferential consumption of selected pGOS species by different bifidobacteria. The approach described here demonstrates that MALDI-FTICR is a rapid-throughput tool for comprehensive profiling of oligosaccharides in GOS mixtures. In addition, the selective consumption of certain GOS species by different bifidobacteria suggests a means for targeting prebiotics to enrich select bifidobacterial species.

SummaryHuman milk contains approximately 200 complex oligosaccharides believed to stimulate the growth and establishment of a protective microbiota in the infant gut. The lack of scalable analytical techniques has hindered the measurement of bacterial metabolism of these and other complex prebiotic oligosaccharides. An in vitro, multi‐strain, assay capable of measuring kinetics of bacterial growth and detailed oligosaccharide consumption analysis by FTICR‐MS was developed and tested simultaneously on 12 bifidobacterial strains. For quantitative consumption, deuterated and reduced human milk oligosaccharide (HMO) standards were used. A custom software suite developed in house called Glycolyzer was used to process the large amounts of oligosaccharide mass spectra automatically with 13C corrections based on de‐isotoping protocols. High growth on HMOs was characteristic of Bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains tested, B. longum bv. longum, B. adolescentis, B. breve and B. bifidum, showed low or only moderate growth ability. Total oligosaccharide consumption ranged from a high of 87% for B. infantis JCM 7009 to only 12% for B. adolescentis ATCC 15703. A detailed analysis of consumption glycoprofiles indicated strain‐specific capabilities towards differential metabolism of milk oligosaccharides. This method overcomes previous limitations in the quantitative, multi‐strain analysis of bacterial metabolism of HMOs and represents a novel approach towards understanding bacterial consumption of complex prebiotic oligosaccharides.

The molecular basis by which human breast milk supports the development of a protective intestinal microbiome in infants is unknown. Human milk oligosaccharides (HMOs) are quantitatively the third largest and most diverse component of breast milk. HMOs are believed to provide protection against pathogens and prebiotic enrichment of beneficial commensals such as bifidobacteria. Bifidobacterium infantis ATCC 15697, an isolate from the infant gut, preferentially consumes four milk oligosaccharides representing nearly 70% of all HMOs present in milk. The purpose of this study was to understand the role of HMOs in establishing and promoting a Bifidobacteria‐rich gut microflora in infants. When B. infantis is pre‐grown on HMOs versus lactose, there is a two‐fold increase in its binding ability to Caco2 cells. To further understand this interaction we have measured the global changes in gene expression of Caco2 cells in the presence of HMOs and B. infantis. This approach will serve as model for understanding how nutrients in a diet (HMOs) influence the interactions between gut microbiota and epithelial cells to effect human health. Research support provided by the California Dairy Research Foundation and UC Discovery Grants.

Human milk contains a high concentration of diverse soluble oligosaccharides, carbohydrate polymers formed from a small number of monosaccharides. Novel methods combining liquid chromatography with high resolution mass spectrometry have identified approximately 200 unique oligosaccharides structures varying from 3 to 22 sugars. The increasing complexity of oligosaccharides follows the general pattern of mammalian evolution though the concentration and diversity of these structures in homo sapiens are strikingly. There is also diversity among human mothers in oligosaccharides. Milks from randomly selected mothers contain as few as 23 and as many as 130 different oligosaccharides. The functional implications of this diversity are not known. Despite the role of milk to serve as a sole nutrient source for mammalian infants, the oligosaccharides in milk are not digestible by human infants. This apparent paradox raises questions about the functions of these oligosaccharides and how their diverse molecular structures affect their functions. The nutritional function most attributed to milk oligosaccharides is to serve as prebiotics - a form of indigestible carbohydrate that is selectively fermented by desirable gut microflora. This function was tested by purifying human milk oligosaccharides and providing these as the sole carbon source to various intestinal bacteria. Indeed, the selectively of providing the complex mixture of oligosaccharides pooled from human milk samples is remarkable. Among a variety of Bifidobacteria tested only Bifidobacteria longum biovar infantis was able to grow extensively on human milk oligosaccharides as sole carbon source. The genomic sequence of this strain revealed approximately 700 genes that are unique to infantis, including a variety of co-regulated glycosidases, relative to other Bifidobacteria, implying a co-evolution of human milk oligosaccharides and the genetic capability of select intestinal bacteria to utilize them. The goal of ongoing research is to assign specific functions to the combined oligosaccharide-bacteria-host interactions that emerged from this evolutionary pressure.

Human milk fat globules (HMFGs) are considered to constitute a triglyceride-rich source of fat and energy. However, milk contains lipid particles at different sizes ranging from tens of micrometers to less than 1 microm. In particular, the physical, chemical, and biological properties of submicron sized particles are poorly described. Individual HMFGs were analyzed using laser trapping confocal Raman spectroscopy, and their chemical signature was obtained and compared to 1, 5, and 10 microm globules. Significant differences in both lipid composition and relative lipid content were found between the classes of particles with different diameters. A strong Raman peak at 1742 cm(-1) corresponding to the triacylglycerol core was detected in the 5 and 10 microm diameter globules, whereas in the smaller HMFGs no detectable peak was found. In addition, the submicron particles produced Raman signals consistent with large quantities of unsaturated fatty acids. Moreover, cis and trans isomers of unsaturated fatty acids were found to be unequally distributed between large and small milk fat globules. Interestingly, trans unsaturated fatty acids were found only in 1 and 5 microm globules although more prominent in the 5 microm diameter range. This is the first evidence for size related differential lipid composition of various diameter classes of HMFGs. The results suggest that the milk fat globule size distribution determines milk lipid composition. In addition, large portions of the HMFGs are secreted into milk conspicuously not for fat delivery. Thus, small HMFGs may offer novel metabolic and nutritional functions.