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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.

Collaboration among scientists has a major influence on scientific progress. Such collaboration often results from scientific meetings, where scientists gather to present and discuss their research and to meet potential collaborators. However, most scientific meetings have inherent biases, such as the availability of research funding or the selection bias of professional societies that make it difficult to study the effect of the meeting per se on scientific productivity. To evaluate the effects of scientific meetings on collaboration and progress independent of these biases, we conducted a study of the annual symposia held by the International Milk Genomics Consortium (IMGC) over a 12-year period. In our study, we conducted permutation testing to analyze the effectiveness of the IMGC in facilitating collaboration and productivity in a community of milk scientists who were meeting attendees relative to non-attendees. Using the number of co-authorships on published papers as a measure of collaboration, our analysis revealed that scientists who attended the symposium were associated with more collaboration than were scientists who did not attend. Furthermore, we evaluated the scientific progress of consortium attendees by analyzing publication rate and article impact. We found that IMGC attendees, in addition to being more collaborative, were also more productive and influential than were non-attendees who published in the same field. The results of our study suggest that the annual symposium encouraged interactions among disparate scientists and increased research productivity, exemplifying the positive effect of scientific meetings on both collaboration and progress., Competing Interests: DGL and JBG have received travel expenses for attendance to the annual IMGC meeting. Prior to her death, GP was the Executive Director of the California Dairy Research Foundation, a non-profit public research management organization. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors have no other competing interests.

Triclosan is frequently used for its antimicrobial properties and has been detected in human serum, urine, and breast milk. Animal and molecular studies have shown that triclosan exerts a wide range of adverse health effects at both high (ppm) and low (ppb) concentrations. Since triclosan is of growing concern to human and environmental health, there is a need to improve extraction procedures and to study additional effects from triclosan exposure. In this study, we have improved triclosan extraction from breast milk by using salt (MgSO 4 ) to reduce emulsion formation and increase water polarity and water (∼80%) to enhance the overall extraction efficiency (∼3.5 fold). This extraction method was applied to breast milk samples collected from donors who i) recorded their use of triclosan-containing personal care products and ii) provided matching infant stool samples. Of the participants who had detectable amounts of triclosan in their breast milk, nine (75%) of them reported daily use of triclosan-containing personal care products. Levels of triclosan in breast milk were compared to the donor's infant's fecal microbiome. We found that the bacterial diversity in the fecal microbiome of the infants exposed to breast milk with detectable triclosan levels differed compared to their peers exposed to milk containing non-detectable amounts. This finding implies that exogenous chemicals are impacting microbiome diversity., (Published by Elsevier Ltd.)

Children with autism spectrum disorders (ASD), characterized by a range of behavioral abnormalities and social deficits, display high incidence of gastrointestinal (GI) co-morbidities including chronic constipation and diarrhea. Research is now increasingly able to characterize the "fragile gut" in these children and understand the role that impairment of specific GI functions plays in the GI symptoms associated with ASD. This mechanistic understanding is extending to the interactions between diet and ASD, including food structure and protein digestive capacity in exacerbating autistic symptoms. Children with ASD and gut co-morbidities exhibit low digestive enzyme activity, impaired gut barrier integrity and the presence of antibodies specific for dietary proteins in the peripheral circulation. These findings support the hypothesis that entry of dietary peptides from the gut lumen into the vasculature are associated with an aberrant immune response. Furthermore, a subset of children with ASD exhibit high concentrations of metabolites originating from microbial activity on proteinaceous substrates. Taken together, the combination of specific protein intakes poor digestion, gut barrier integrity, microbiota composition and function all on a background of ASD represents a phenotypic pattern. A potential consequence of this pattern of conditions is that the fragile gut of some children with ASD is at risk for GI symptoms that may be amenable to improvement with specific dietary changes. There is growing evidence that shows an association between gut dysfunction and dysbiosis and ASD symptoms. It is therefore urgent to perform more experimental and clinical research on the "fragile gut" in children with ASD in order to move toward advancements in clinical practice. Identifying those factors that are of clinical value will provide an evidence-based path to individual management and targeted solutions; from real time sensing to the design of diets with personalized protein source/processing, all to improve GI function in children with ASD.

Extracellular fungal glycolipid biosurfactants have attracted attention because productivities can be high, cheap substrates can be used, the molecules are secreted into the medium and the downstream processing is relatively simple. Three classes of extracellular fungal glycolipid biosurfactants have provided most of the scientific advances in this area, namely sophorolipids, mannosylerythritol lipids and cellobioselipids. Polyol lipids, a fourth class of extracellular fungal glycolipid biosurfactants, comprise two groups of molecules: liamocins produced by the yeast-like fungus Aureobasidium pullulans, and polyol esters of fatty acids, produced by some Rhodotorula yeast species. Both are amphiphilic, surface active molecules with potential for commercial development as surfactants for industrial and household applications. The current knowledge of polyol lipids highlights an emerging group of extracellular fungal glycolipid biosurfactants and provides a perspective of what next steps are needed to harness the benefits and applications of this novel group of molecules., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective:  A pilot study to determine lipoprotein classes and subclasses in premature infants and examine associations with nutritional intake, gestational age (GA), and morbidity., Study Design:  Plasma lipoprotein particle concentrations were analyzed in a cohort of 15 premature infants in the first 5 days of life and again at 2 weeks. Breast milk samples were analyzed for fatty acid content. Associations between lipoprotein particle subclasses and GA, breast milk intake, milk fatty acid intake, and chronic lung disease (CLD) were determined., Results:  At 2 weeks of age, more premature infants had higher concentrations of total very low-density lipoprotein and lower concentrations of total high-density lipoprotein (HDL) and large HDL particles (similar to profiles seen in adults and children with infectious disease, cardiometabolic disease, and diabetes). Lower total HDL, large HDL, and medium HDL and a higher small HDL:total HDL ratio at 2 weeks were each associated with CLD with GA a likely confounder. Intake of human milk C18 and C20 fatty acids was inversely correlated with plasma total LDL concentration at 2 weeks of age., Conclusion:  Dyslipidemia was common in extremely premature infants and was associated with CLD and with lower intake of specific long chain fatty acids., Competing Interests: None., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Scope: Body weight responds variably to the intake of dairy foods. Genetic variation may contribute to inter-individual variability in associations between body weight and dairy consumption., Methods and Results: A genome-wide interaction study to discover genetic variants that account for variation in BMI in the context of low-fat, high-fat and total dairy intake in cross-sectional analysis was conducted. Data from nine discovery studies (up to 25 513 European descent individuals) were meta-analyzed. Twenty-six genetic variants reached the selected significance threshold (p-interaction <10 -7) , and six independent variants (LINC01512-rs7751666, PALM2/AKAP2-rs914359, ACTA2-rs1388, PPP1R12A-rs7961195, LINC00333-rs9635058, AC098847.1-rs1791355) were evaluated meta-analytically for replication of interaction in up to 17 675 individuals. Variant rs9635058 (128 kb 3' of LINC00333) was replicated (p-interaction = 0.004). In the discovery cohorts, rs9635058 interacted with dairy (p-interaction = 7.36 × 10 -8) such that each serving of low-fat dairy was associated with 0.225 kg m -2 lower BMI per each additional copy of the effect allele (A). A second genetic variant (ACTA2-rs1388) approached interaction replication significance for low-fat dairy exposure., Conclusion: Body weight responses to dairy intake may be modified by genotype, in that greater dairy intake may protect a genetic subgroup from higher body weight., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein digestion is critical for infants. Dissimilarities between infants and adults in food intake and digestive physiology lead to distinct patterns of proteolysis between individuals. However, such differences are not well represented in many studies on protein digestion of baby foods. The complex biological structures of baby foods and the physiology of the infant digestive system are key factors affecting proteolysis during the first two years of life. Well-controlled in vitro studies have demonstrated that varying digestion conditions alter the specificity, rate, and extent of proteolysis of baby foods. Nonetheless, these models do not completely replicate in vivo proteolysis or the complex biogeography of the gastrointestinal tract. Animal and clinical studies have revealed the fate of dietary proteins along the digestive tract and the overall health impact on subjects. Building comprehensive and annotated datasets from human infants will require innovative and standardized measurement. Now, more systematic evaluations of digestion are emerging to advance the knowledge and its translation as food design for effective diet and health management in infants., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scientific, technological, and economic progress over the last 100 years all but eradicated problems of widespread food shortage and nutrient deficiency in developed nations. But now society is faced with a new set of nutrition problems related to energy imbalance and metabolic disease, which require new kinds of solutions. Recent developments in the area of new analytical tools enable us to systematically study large quantities of detailed and multidimensional metabolic and health data, providing the opportunity to address current nutrition problems through an approach called Precision Nutrition. This approach integrates different kinds of "big data" to expand our understanding of the complexity and diversity of human metabolism in response to diet. With these tools, we can more fully elucidate each individual's unique phenotype, or the current state of health, as determined by the interactions among biology, environment, and behavior. The tools of precision nutrition include genomics, metabolomics, microbiomics, phenotyping, high-throughput analytical chemistry techniques, longitudinal tracking with body sensors, informatics, data science, and sophisticated educational and behavioral interventions. These tools are enabling the development of more personalized and predictive dietary guidance and interventions that have the potential to transform how the public makes food choices and greatly improve population health.

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 long-term 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.

Anorexia nervosa (AN) is a complex psychiatric disorder with high morbidity and mortality rates. While many individuals make full recoveries, up to a third of patients develop a chronic, treatment-resistant form of the illness that leads to a premature death in 15-20% of those affected. There have been few advances in treatment, both in terms of psychological or pharmacologic treatment over the last 30 years. Food aversion is commonly cited by patients with AN as a barrier to normalizing eating and weight. Our group has a keen interest in examining factors that might allow this to be addressed, thus improving treatment outcomes through personalized dietary plans or nutritional supplementation related to underlying genetic status. We demonstrated that polyunsaturated fatty acids (PUFAs)-derived bioactive lipids (eicosanoids) are implicated in not only the risk of AN, but also with its comorbid psychopathology. Of interest, the differential postprandial omega 6-derived eicosanoid shift observed in AN highlights the possibility that the metabolism of PUFAs is an important mechanism underlying the profound food version, contributing to pathological food restriction in AN. A concise knowledge of the relationships among PUFAs, eicosanoids, and AN clinical course and psychopathology could be the key to developing personalized nutritional rehabilitative treatments for those suffering from AN. This paper provides a comprehensive overview of the literature on PUFAs in AN. We also selectively reviewed the clinical benefits PUFA treatments exert in other psychiatric diseases, on weight and appetite regulation, and for resolution of inflammation, all of which are relevant in the disease course and outcome of AN. We propose that personalized PUFA formulation be developed and tested as a novel adjunctive treatment for patients with AN. We hypothesize that with personalized PUFA formulation, food aversion and anxiety about eating will decrease while mood, dietary behavior, and weight restoration will improve in AN, leading to improvements in the overall treatment outcome., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Proteins are not equally digestible-their proteolytic susceptibility varies by their source and processing method. Incomplete digestion increases colonic microbial protein fermentation (putrefaction), which produces toxic metabolites that can induce inflammation in vitro and have been associated with inflammation in vivo. Individual humans differ in protein digestive capacity based on phenotypes, particularly disease states. To avoid putrefaction-induced intestinal inflammation, protein sources, and processing methods must be tailored to the consumer's digestive capacity. This review explores how food processing techniques alter protein digestibility and examines how physiological conditions alter digestive capacity. Possible solutions to improving digestive function or matching low digestive capacity with more digestible protein sources are explored. Beyond the ileal digestibility measurements of protein digestibility, less invasive, quicker and cheaper techniques for monitoring the extent of protein digestion and fermentation are needed to personalize protein nourishment. Biomarkers of protein digestive capacity and efficiency can be identified with the toolsets of peptidomics, metabolomics, microbial sequencing and multiplexed protein analysis of fecal and urine samples. By monitoring individual protein digestive function, the protein component of diets can be tailored via protein source and processing selection to match individual needs to minimize colonic putrefaction and, thus, optimize gut health.

Microbial oils have been analyzed as alternatives to petroleum. However, just a handful of microbes have been successfully adapted to produce chemicals that can compete with their petroleum counterparts. One of the reasons behind the low success rate is the overall economic inefficiency of valorizing a single product. This study presents a lab-scale analysis of two yeast species that simultaneously produce multiple high-value bioproducts: intracellular triacylglycerols (TG) and extracellular polyol esters of fatty acids (PEFA), two lipid classes with immediate applications in the biofuels and surfactant industries. At harvest, the yeast strain Rhodotorula aff. paludigena UCDFST 81-84 secreted 20.9 ± 0.2 g L -1 PEFA and produced 8.8 ± 1.0 g L -1 TG, while the yeast strain Rhodotorula babjevae UCDFST 04-877 secreted 11.2 ± 1.6 g L -1 PEFA and 18.5 ± 1.7 g L -1 TG. The overall glucose conversion was 0.24 and 0.22 g (total lipid) g (glucose) -1 , respectively. The results present a stable and scalable microbial growth platform yielding multiple co-products.

Polyol esters of fatty acids (PEFA) are amphiphilic glycolipids produced by yeast that could play a role as natural, environmentally friendly biosurfactants. We recently reported discovery of a new PEFA-secreting yeast species, Rhodotorula babjevae, a basidiomycetous yeast to display this behavior, in addition to a few other Rhodotorula yeasts reported on the 1960s. Additional yeast species within the taxonomic order Sporidiobolales were screened for secreted glycolipid production. PEFA production equal or above 1 g L -1 were detected in 19 out of 65 strains of yeast screened, belonging to 6 out of 30 yeast species tested. Four of these species were not previously known to secrete glycolipids. These results significantly increase the number of yeast species known to secrete PEFA, holding promise for expanding knowledge of PEFA synthesis and secretion mechanisms, as well as setting the groundwork towards commercialization.

Background: Inflammation is associated with increased bone resorption; the role of inflammation in postprandial bone turnover has not been explored. Consumption of milk fat globule membrane (MFGM) reduces inflammation in animal models. This study aimed to measure postprandial changes in bone turnover after intake of high saturated fat test meals, with- and without the anti-inflammatory ingredient MFGM., Methods: Subjects ( n  = 36 adults) were obese (BMI 30-39.9 kg/m 2 ) or overweight (BMI 25-29.9 kg/m 2 ) with two traits of Metabolic Syndrome. Subjects consumed a different test meal on four occasions at random; blood draws were taken at baseline and 1, 3, and 6 h postprandial. Test meals included whipping cream (WC), WC + MFGM, palm oil (PO) and PO + MFGM. Biomarkers of bone turnover and inflammation were analyzed from all four time points., Results: Test meal (treatment) by time interactions were significant for bone resorption marker C-telopeptide of type 1 collagen (CTX) ( p  < 0.0001) and inflammatory marker interleukin 10 (IL-10) ( p  = 0.012). Significant differences in overall postprandial response among test meals were found for CTX and soluble intercellular adhesion molecule (sICAM), with the greatest overall postprandial suppression of CTX occurring in meals containing MFGM. However, test meal by MFGM interactions were non- significant for bone and inflammatory markers. Correlations between CTX and inflammatory markers were non-significant., Conclusion: This exploratory analysis advances the study of postprandial suppression of bone turnover by demonstrating differing effects of high SFA meals that contained MFGM; however MFGM alone did not directly moderate the difference in postprandial CTX response among test meals in this analysis. These observations may be useful for identifying foods and ingredients which maximize the suppression of bone resorption, and for generating hypotheses to test in future studies examining the role of inflammation in postprandial bone turnover., Trial Registration: Clinicaltrials.gov NCT01811329. Registered 11 March 2013.

Lactoferrin is a multifunctional glycoprotein found in the milk of most mammals. In addition to its well-known role of binding iron, lactoferrin carries many important biological functions, including the promotion of cell proliferation and differentiation, and as an anti-bacterial, anti-viral, and anti-parasitic protein. These functions differ among lactoferrin homologs in mammals. Although considerable attention has been given to the many functions of lactoferrin, its primary nutritional contribution is presumed to be related to its iron-binding characteristics, whereas the role of glycosylation has been neglected. Given the critical role of glycan binding in many biological processes, the glycan moieties in lactoferrin are likely to contribute significantly to the biological roles of lactoferrin. Despite the high amino acid sequence homology in different lactoferrins (up to 99%), each exhibits a unique glycosylation pattern that may be responsible for heterogeneity of the biological properties of lactoferrins. An important task for the production of biotherapeutics and medical foods containing bioactive glycoproteins is the assessment of the contributions of individual glycans to the observed bioactivities. This review examines how the study of lactoferrin glycosylation patterns can increase our understanding of lactoferrin functionality.

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.

Mechanistic research suggests a unique evolutionary relationship between complex milk oligosaccharides and cognate bifidobacteria enriched in breast-fed infants. Bovine milk oligosaccharides (BMO) were recently identified as structurally and functionally similar to human milk oligosaccharides. The present single-blind three-way crossover study is the first to determine the safety and tolerability of BMO consumption by healthy human participants ( n 12) and its effects on faecal microbiota and microbial metabolism. Participants consumed each supplement (placebo-control; low- and high-BMO doses) for eleven consecutive days, followed by a 2-week washout period prior to initiating the next supplement arm. Low and high BMO doses were consumed as 25 and 35 % of each individual's daily fibre intake, respectively. Safety and tolerability were measured using standardised questionnaires on gut and stomach discomfort and stool consistency. Faecal extracts were profiled for bacterial populations by next-generation sequencing (NGS) and bifidobacteria presence was confirmed using quantitative PCR. Urine was analysed for changes in microbial metabolism using nuclear magnetic resonance spectroscopy ( 1 H-NMR). Consumption of both the low and high BMO doses was well tolerated and did not change stool consistency from baseline. Multivariate analysis of the NGS results demonstrated no change in faecal microbiota phyla among the placebo-control and BMO supplement groups. In conclusion, BMO supplementation was well tolerated in healthy adults and has the potential to shift faecal microbiota toward beneficial strains as part of a synbiotic treatment with probiotic cultures that selectively metabolise oligosaccharides.

Milk proteins are a complex and diverse source of biological activities. Beyond their function, intact milk proteins also act as carriers of encrypted functional sequences that, when released as peptides, exert biological functions, including antimicrobial and immunomodulatory activity, which could contribute to the infant's competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself. A complex array of proteases produced in mother's milk has been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant's stomach, possibly even to a larger extent than the infant's own proteases. As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease-based peptide release system., (© 2017 Nestec Ltd., Vevey/S. Karger AG, Basel.)

Background: The quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards., Objective: The objective of our study was to rapidly measure the absolute concentrations of HMOs in milk using LC-mass spectrometry (MS) and to determine the phenotypic secretor status of the mothers., Methods: This quantitative method for measuring HMO concentration was developed by using ultraperformance LC multiple reaction monitoring MS. It was validated and applied to milk samples from Malawi (88 individuals; 88 samples from postnatal month 6) and the United States (Davis, California; 45 individuals, mean age: 32 y; 103 samples collected on postnatal days 10, 26, 71, or 120, repeated measures included). The concentrations of α(1,2)-fucosylated HMOs were used to determine the mothers' phenotypic secretor status with high sensitivity and specificity. We used Friedman's test and Wilcoxon's signed rank test to evaluate the change in HMO concentration during the course of lactation, and Student's t test was used to compare secretors and nonsecretors., Results: A decrease (P < 0.05) in HMO concentration was observed during the course of lactation for the US mothers, corresponding to 19.3 ± 2.9 g/L for milk collected on postnatal day 10, decreasing to 8.53 ± 1.18 g/L on day 120 (repeated measures; n = 14). On postnatal day 180, the total concentration of HMOs in Malawi milk samples from secretors (6.46 ± 1.74 mg/mL) was higher (P < 0.05) than that in samples from nonsecretors (5.25 ± 2.55 mg/mL ). The same trend was observed for fucosylated species; the concentration was higher in Malawi milk samples from secretors (4.91 ± 1.22 mg/mL) than from nonsecretors (3.42 ± 2.27 mg/mL) (P < 0.05)., Conclusions: HMOs significantly decrease during the course of lactation. Secretor milk contains higher concentrations of total and fucosylated HMOs than does nonsecretor milk. These HMO concentrations can be correlated to the health of breastfed infants in order to investigate the protective effects of milk components. The trials were registered at clinicaltrials.gov as NCT01817127 and NCT00524446., Competing Interests: 2 Author disclosures: G Xu, JCC Davis, E Goonatilleke, JT Smilowitz, JB German, and CB Lebrilla, no conflicts of interest., (© 2017 American Society for Nutrition.)

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.

The infant intestinal microbiota is often colonized by two subspecies of Bifidobacterium longum: subsp. infantis (B. infantis) and subsp. longum (B. longum). Competitive growth of B. infantis in the neonate intestine has been linked to the utilization of human milk oligosaccharides (HMO). However, little is known how B. longum consumes HMO. In this study, infant-borne B. longum strains exhibited varying HMO growth phenotypes. While all strains efficiently utilized lacto-N-tetraose, certain strains additionally metabolized fucosylated HMO. B. longum SC596 grew vigorously on HMO, and glycoprofiling revealed a preference for consumption of fucosylated HMO. Transcriptomes of SC596 during early-stage growth on HMO were more similar to growth on fucosyllactose, transiting later to a pattern similar to growth on neutral HMO. B. longum SC596 contains a novel gene cluster devoted to the utilization of fucosylated HMO, including genes for import of fucosylated molecules, fucose metabolism and two α-fucosidases. This cluster showed a modular induction during early growth on HMO and fucosyllactose. This work clarifies the genomic and physiological variation of infant-borne B. longum to HMO consumption, which resembles B. infantis. The capability to preferentially consume fucosylated HMO suggests a competitive advantage for these unique B. longum strains in the breast-fed infant gut., Competing Interests: J.B.G., C.B.L. and D.A.M., are co-founders of Evolve Biosystems, Inc., a company focused on diet-based manipulation of the gut microbiota.

Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population. In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan. Marker gene sequencing of the fecal samples revealed bifidobacteria as the dominant inhabitants of the infant gastrointestinal tracts. A glycosidase from Bifidobacterium longum subsp. longum was then expressed to digest HMOs in vitro, which showed that the digested oligosaccharides in feces corresponded to the action of glycosidases on HMOs. Similar expression of endoglycosidases also showed that N-glycans were released by bacterial enzymes. Although bifidobacteria may dominate the gut, it is possible that specific minority species are also responsible for the major products observed in feces. Nonetheless, the enzymatic activity correlated well with the known glycosidases in the respective bacteria, suggesting a direct relationship between microbial abundances and catabolic activity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The ability to distinguish pathogens from self-antigens is one of the most important functions of the immune system. However, this simple self versus non-self assignment belies the complexity of the immune response to threats. Immune responses vary widely and appropriately according to a spectrum of threats and only recently have the mechanisms for controlling this highly textured process emerged. A primary mechanism by which this controlled decision-making process is achieved is via Toll-like receptor (TLR) signaling and the subsequent activation of the immune response coincident with the presence of pathogenic organisms or antigens, including lipid mediators. While immune activation is important, the appropriate regulation of such responses is also critical. Recent findings indicate a parallel pathway by which responses to both viral and bacterial infections is controlled via the secretion of soluble TLR2 (sTLR2). sTLR2 is able to bind a wide range of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). sTLR2 has been detected in many bodily fluids and is thus ubiquitous in sites of pathogen appearance. Interestingly, growing evidence suggests that sTLR2 functions to sequester PAMPs and DAMPs to avoid immune activation via detection of cellular-expressed TLRs. This immune regulatory function would serve to reduce the expression of the molecules required for cellular entry, and the recruitment of target cells following infection with bacteria and viruses. This review provides an overview of sTLR2 and the research regarding the mechanisms of its immune regulatory properties. Furthermore, the role of this molecule in regulating immune activation in the context of HIV infection via sTLR2 in breast milk provides actionable insights into therapeutic targets across a variety of infectious and inflammatory states.

Background: 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., Methods/design: This is a parallel-group, placebo-controlled, randomised, double-blind ascending dose phase I clinical trial of dietary supplementation with B. infantis in healthy breastfed infants. The objective is to determine the pharmacologically effective dose (ED) of B. infantis required to produce predominant (>50 %) gut colonisation in breastfed infants at 6 weeks of age. Successively enrolled infant groups will be randomised to receive two doses of either B. infantis or placebo on days 7 and 14 of life. Stool samples will be used to characterise the gut microbiota at increasing doses of B. infantis., Discussion: Probiotic supplementation has shown promising results for the treatment of a variety of ailments, but evidence-based dosing regimes are currently lacking. The ultimate goal of this trial is to establish a recommended starting dose of B. infantis for further efficacy-testing phase II trials designed to evaluate B. infantis for the prevention of atopic dermatitis and food allergies in at-risk children., Trial Registration: Clinicaltrials.gov # NCT02286999 , date of trial registration 23 October 2014.

Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.

California's rural agricultural communities face an increased burden of obesity and metabolic disease. The present objective is to define the social and environmental influences to child obesity and physical activity within Mexican-origin communities in California's Central Valley. A range of data (anthropometric, socioeconomic, demographic, cultural and environmental) were collected on more than 650 children enrolled in Niños Sanos, Familia Sana. Physical activity data were gathered from a subsample of children 4-7 years of age (n = 148) via accelerometer. Cross sectional analyses explored the relationship between BMI and physical activity and the influence of numerous social and environmental variables. In this sample 45 % of children were determined to be overweight or obese. Boys had a higher daily average moderate-to-vigorous physical activity than girls (p = 0.008). Chi square analyses showed weight status was associated with activity level in girls (p = 0.03) but not boys. Multivariate regression revealed several social and environmental indicators influenced BMI and physical activity (p = 0.004). In this population of school-age children of Mexican-origin, girls may benefit more from targeted efforts to increase MVPA. Family and community support systems may also boost child participation in physical activities.

Meals high in SFA, particularly palmitate, are associated with postprandial inflammation and insulin resistance. Milk fat globule membrane (MFGM) has anti-inflammatory properties that may attenuate the negative effects of SFA-rich meals. Our objective was to examine the postprandial metabolic and inflammatory response to a high-fat meal composed of palm oil (PO) compared with PO with an added dairy fraction rich in MFGM (PO+MFGM) in overweight and obese men and women (n 36) in a randomised, double-blinded, cross-over trial. Participants consumed two isoenergetic high-fat meals composed of a smoothie enriched with PO with v. without a cream-derived complex milk lipid fraction ( dairy fraction rich in MFGM) separated by a washout of 1-2 weeks. Serum cytokines, adhesion molecules, cortisol and markers of inflammation were measured at fasting, and at 1, 3 and 6 h postprandially. Glucose, insulin and lipid profiles were analysed in plasma. Consumption of the PO + MFGM v. PO meal resulted in lower total cholesterol (P = 0·021), LDL-cholesterol (P = 0·046), soluble intracellular adhesion molecule (P = 0·005) and insulin (P = 0·005) incremental AUC, and increased IL-10 (P = 0·013). Individuals with high baseline C-reactive protein (CRP) concentrations (≥3 mg/l, n 17) had higher (P = 0·030) insulin at 1 h after the PO meal than individuals with CRP concentrations <3 mg/l (n 19). The addition of MFGM attenuated this difference between CRP groups. The addition of a dairy fraction rich in MFGM attenuated the negative effects of a high-SFA meal by reducing postprandial cholesterol, inflammatory markers and insulin response in overweight and obese individuals, particularly in those with elevated CRP.

Dietary recommendations suggest decreased consumption of SFA to minimise CVD risk; however, not all foods rich in SFA are equivalent. To evaluate the effects of SFA in a dairy food matrix, as Cheddar cheese, v. SFA from a vegan-alternative test meal on postprandial inflammatory markers, a randomised controlled cross-over trial was conducted in twenty overweight or obese adults with metabolic abnormalities. Individuals consumed two isoenergetic high-fat mixed meals separated by a 1- to 2-week washout period. Serum was collected at baseline, and at 1, 3 and 6 h postprandially and analysed for inflammatory markers (IL-6, IL-8, IL-10, IL-17, IL-18, TNFα, monocyte chemotactic protein-1 (MCP-1)), acute-phase proteins C-reactive protein (CRP) and serum amyloid-A (SAA), cellular adhesion molecules and blood lipids, glucose and insulin. Following both high-fat test meals, postprandial TAG concentrations rose steadily (P < 0·05) without a decrease by 6 h. The incremental AUC (iAUC) for CRP was significantly lower (P < 0·05) in response to the cheese compared with the vegan-alternative test meal. A treatment effect was not observed for any other inflammatory markers; however, for both test meals, multiple markers significantly changed from baseline over the 6 h postprandial period (IL-6, IL-8, IL-18, TNFα, MCP-1, SAA). Saturated fat in the form of a cheese matrix reduced the iAUC for CRP compared with a vegan-alternative test meal during the postprandial 6 h period. The study is registered at clinicaltrials.gov under NCT01803633.

Infant fecal samples are commonly studied to investigate the impacts of breastfeeding on the development of the microbiota and subsequent health effects. Comparisons of infants living in different geographic regions and environmental contexts are needed to aid our understanding of evolutionarily-selected milk adaptations. However, the preservation of fecal samples from individuals in remote locales until they can be processed can be a challenge. Freeze-drying (lyophilization) offers a cost-effective way to preserve some biological samples for transport and analysis at a later date. Currently, it is unknown what, if any, biases are introduced into various analyses by the freeze-drying process. Here, we investigated how freeze-drying affected analysis of two relevant and intertwined aspects of infant fecal samples, marker gene amplicon sequencing of the bacterial community and the fecal oligosaccharide profile (undigested human milk oligosaccharides). No differences were discovered between the fecal oligosaccharide profiles of wet and freeze-dried samples. The marker gene sequencing data showed an increase in proportional representation of Bacteriodes and a decrease in detection of bifidobacteria and members of class Bacilli after freeze-drying. This sample treatment bias may possibly be related to the cell morphology of these different taxa (Gram status). However, these effects did not overwhelm the natural variation among individuals, as the community data still strongly grouped by subject and not by freeze-drying status. We also found that compensating for sample concentration during freeze-drying, while not necessary, was also not detrimental. Freeze-drying may therefore be an acceptable method of sample preservation and mass reduction for some studies of microbial ecology and milk glycan analysis.

Introduction: There is a high degree of inter-individual variability among people in response to intervention with omega-3 fatty acids (FA), which may partly explain conflicting results on the effectiveness of omega-3 FA for the treatment and prevention of chronic inflammatory diseases. In this study we sought to evaluate whether part of this inter-individual variability in response is related to the regulation of key oxylipin metabolic genes in circulating peripheral blood mononuclear cells (PBMCs)., Methods: Plasma FA and oxylipin profiles from 12 healthy individuals were compared to PBMC gene expression profiles following six weeks of supplementation with fish oil, which delivered 1.9 g/d eicosapentaenoic acid (EPA) and 1.5 g/d docosahexaenoic acid (DHA). Fold changes in gene expression were measured by a quantitative polymerase chain reaction (qPCR)., Results: Healthy individuals supplemented with omega-3 FA had differential responses in prostaglandin-endoperoxide synthase 1 (PTGS1), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), and interleukin 8 (IL-8) gene expression in isolated PBMCs. In those individuals for whom plasma arachidonic acid (ARA) in the phosphatidylethanolamine (PE) lipid class decreased in response to omega-3 intervention, there was a corresponding decrease in gene expression for PTGS1 and ALOX12. Several oxylipin product/FA precursor ratios (e.g. prostaglandin E2 (PGE2)/ARA for PTGS1 and 12-hydroxyeicosatetraenoic acid (12-HETE)/ARA for ALOX12) were also associated with fold change in gene expression, suggesting an association between enzyme activity and gene expression. The fold-change in PTGS1 gene expression was highly positively correlated with ALOX12 gene expression but not with PTGS2, whereas IL-8 and PTGS2 were positively correlated., Conclusions: The regulation of important oxylipin metabolic genes in PBMCs varied with the extent of change in ARA concentrations in the case of PTGS1 and ALOX12 regulation. PBMC gene expression changes in response to omega-3 supplementation varied among healthy individuals, and were associated with changes in plasma FA and oxylipin composition to different degrees in different individuals., Trial Registration: clinicaltrials.gov NCT01838239.

Background: Human milk oligosaccharides (HMOs) shape the intestinal microbiota in term infants. In premature infants, alterations in the intestinal microbiota (dysbiosis) are associated with risk of necrotizing enterocolitis (NEC) and sepsis, and the influence of HMOs on the microbiota is unclear., Methods: Milk, urine, and stool specimens from 14 mother-premature infant dyads were investigated by mass spectrometry for HMO composition. The stools were analyzed by next-generation sequencing to complement a previous analysis., Results: Percentages of fucosylated and sialylated HMOs were highly variable between individuals but similar in urine, feces, and milk within dyads. Differences in urine and fecal HMO composition suggest variability in absorption. Secretor status of the mother correlated with the urine and fecal content of specific HMO structures. Trends toward higher levels of Proteobacteria and lower levels of Firmicutes were noted in premature infants of nonsecretor mothers. Specific HMO structures in the milk, urine, and feces were associated with alterations in fecal Proteobacteria and Firmicutes., Conclusion: HMOs may influence the intestinal microbiota in premature infants. Specific HMOs, for example those associated with secretor mothers, may have a protective effect by decreasing pathogens associated with sepsis and NEC, while other HMOs may increase dysbiosis in this population.

Unlabelled: Many symbiotic gut bacteria possess the ability to degrade multiple polysaccharides, thereby providing nutritional advantages to their hosts. Like microorganisms adapted to other complex nutrient environments, gut symbionts give different metabolic priorities to substrates present in mixtures. We investigated the responses of Bacteroides thetaiotaomicron, a common human intestinal bacterium that metabolizes more than a dozen different polysaccharides, including the O-linked glycans that are abundant in secreted mucin. Experiments in which mucin glycans were presented simultaneously with other carbohydrates show that degradation of these host carbohydrates is consistently repressed in the presence of alternative substrates, even by B. thetaiotaomicron previously acclimated to growth in pure mucin glycans. Experiments with media containing systematically varied carbohydrate cues and genetic mutants reveal that transcriptional repression of genes involved in mucin glycan metabolism is imposed by simple sugars and, in one example that was tested, is mediated through a small intergenic region in a transcript-autonomous fashion. Repression of mucin glycan-responsive gene clusters in two other human gut bacteria, Bacteroides massiliensis and Bacteroides fragilis, exhibited variable and sometimes reciprocal responses compared to those of B. thetaiotaomicron, revealing that these symbionts vary in their preference for mucin glycans and that these differences occur at the level of controlling individual gene clusters. Our results reveal that sensing and metabolic triaging of glycans are complex processes that vary among species, underscoring the idea that these phenomena are likely to be hidden drivers of microbiota community dynamics and may dictate which microorganisms preferentially commit to various niches in a constantly changing nutritional environment., Importance: Human intestinal microorganisms impact many aspects of health and disease, including digestion and the propensity to develop disorders such as inflammation and colon cancer. Complex carbohydrates are a major component of the intestinal habitat, and numerous species have evolved and refined strategies to compete for these coveted nutrients. Our findings reveal that individual bacteria exhibit different preferences for carbohydrates emanating from host diet and mucosal secretions and that some of these prioritization strategies are opposite to one another. Thus, we reveal new aspects of how individual bacteria, some with otherwise similar metabolic potential, partition to "preferred niches" in the complex gut ecosystem, which has important and immediate implications for understanding and predicting the behavioral dynamics of this community., (Copyright © 2015 Pudlo et al.)

Breast milk enhances the predominance of Bifidobacterium species in the infant gut, probably due to its large concentration of human milk oligosaccharides (HMO). Here we screened infant-gut isolates of Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum using individual HMO, and compared the global transcriptomes of representative isolates on major HMO by RNA-seq. While B. infantis displayed homogeneous HMO-utilization patterns, B. bifidum were more diverse and some strains did not use fucosyllactose (FL) or sialyllactose (SL). Transcriptomes of B. bifidum SC555 and B. infantis ATCC 15697 showed that utilization of pooled HMO is similar to neutral HMO, while transcriptomes for growth on FL were more similar to lactose than HMO in B. bifidum. Genes linked to HMO-utilization were upregulated by neutral HMO and SL, but not by FL in both species. In contrast, FL induced the expression of alternative gene clusters in B. infantis. Results also suggest that B. bifidum SC555 does not utilize fucose or sialic acid from HMO. Surprisingly, expression of orthologous genes differed between both bifidobacteria even when grown on identical substrates. This study highlights two major strategies found in Bifidobacterium species to process HMO, and presents detailed information on the close relationship between HMO and infant-gut bifidobacteria.

Background: Human milk is the gold standard of nutrition for infants, providing both protective and essential nutrients. Although much is known about milk from mothers giving birth to term infants, less is known about milk from mothers giving birth to premature infants. In addition, little is known about the composition and diversity of small molecules in these milks and how they change over the first month of lactation., Objective: The objective was to understand how milk metabolites vary over the first month of lactation in mothers giving birth to term and preterm infants., Methods: (1)H nuclear magnetic resonance (NMR) metabolomics was used to characterize metabolites that were present in micromolar to molar concentrations in colostrum (day 0-5 postpartum), transition milk (day 14), and mature milk (day 28) from mothers who delivered term (n = 15) and preterm (n = 13) infants. Principal components analysis, linear mixed-effects models (LMMs), and linear models (LMs) were used to explore the relation between infant maturity and the postpartum day of collection of milk samples., Results: By using a standard NMR metabolite library, 69 metabolites were identified in the milks, including 15 sugars, 23 amino acids and derivatives, 11 energy-related metabolites, 10 fatty acid-associated metabolites, 3 nucleotides and derivatives, 2 vitamins, and 5 bacteria-associated metabolites. Many metabolite concentrations followed a similar progression over time in both term and preterm milks, with more biological variation in metabolite concentrations in preterm milk. However, although lacto-N-neotetraose (LMM, P = 4.0 × 10(-5)) and lysine (LM, P = 1.5 × 10(-4)) significantly decreased in concentration in term milk over time, they did not significantly change in preterm milk., Conclusion: Overall, the metabolic profile of human milk is dynamic throughout the first month of lactation, with more variability in preterm than in term milk and subtle differences in some metabolite concentrations. This trial was registered at clinicaltrials.gov as NCT01841268., (© 2015 American Society for Nutrition.)

Introduction: In California's agricultural Central Valley, the rate of childhood obesity is higher than the national average. Adequate physical activity contributes to obesity prevention and its assessment is useful to evaluate the impact of interventions., Methods: Niños Sanos, Familia Sana (Healthy Children, Healthy Family [NSFS]) uses community-based participatory research to implement an intervention program to reduce childhood obesity among people of Mexican origin in the Central Valley. Anthropometric measurements were conducted on more than 650 children enrolled in NSFS. Physical activity data from a subgroup of children aged 4 to 7 years (n = 134) were collected via a wearable accelerometer., Results: Children were classified on the basis of age and sex-adjusted body mass index as healthy weight (57.7%); overweight (19.3%), or obese (23%). Logistic regression showed that moderate to vigorous physical activity (MVPA) was associated with a child's likelihood of having a healthy BMI (odds ratio: 1.03; 95% CI, 1.01-1.05; P = .017)., Conclusion: NSFS's community-based participatory approach resulted in successful use of a commercial electronic device to measure physical activity quantity and quality in this hard-to-reach population. Promotion of adequate daily MVPA is an appropriate and necessary component of NSFS's childhood obesity prevention strategy.

A variety of proteases release hundreds of endogenous peptide fragments from intact bovine milk proteins. Mass spectrometry-based peptidomics allows for high throughput sequence assignment of a large number of these peptides. Mastitis is known to result in increased protease activity in the mammary gland. Therefore, we hypothesized that subclinically mastitic milks would contain higher concentrations of released peptides. In this work, milks were sampled from three cows and, for each, one healthy and one subclinically mastitic teat were sampled for milk. Peptides were analyzed by nano-liquid chromatography quadrupole time of flight tandem mass spectrometry and identified with database searching. In total, 682 peptides were identified. The total number of released peptides increased 146% from healthy to subclinically mastitic milks ( p < 0.05), and the total abundance of released peptides also increased significantly ( p < 0.05). Bioinformatic analysis of enzyme cleavage revealed increases in activity of cathepsin D and elastase ( p < 0.05) with subclinical mastitis.

Background: Individuals with inactive alleles of the fucosyltransferase 2 gene (FUT2; termed the 'secretor' gene) are common in many populations. Some members of the genus Bifidobacterium, common infant gut commensals, are known to consume 2'-fucosylated glycans found in the breast milk of secretor mothers. We investigated the effects of maternal secretor status on the developing infant microbiota with a special emphasis on bifidobacterial species abundance., Results: On average, bifidobacteria were established earlier and more often in infants fed by secretor mothers than in infants fed by non-secretor mothers. In secretor-fed infants, the relative abundance of the Bifidobacterium longum group was most strongly correlated with high percentages of the order Bifidobacteriales. Conversely, in non-secretor-fed infants, Bifidobacterium breve was positively correlated with Bifidobacteriales, while the B. longum group was negatively correlated. A higher percentage of bifidobacteria isolated from secretor-fed infants consumed 2'-fucosyllactose. Infant feces with high levels of bifidobacteria had lower milk oligosaccharide levels in the feces and higher amounts of lactate. Furthermore, feces containing different bifidobacterial species possessed differing amounts of oligosaccharides, suggesting differential consumption in situ., Conclusions: Infants fed by non-secretor mothers are delayed in the establishment of a bifidobacteria-laden microbiota. This delay may be due to difficulties in the infant acquiring a species of bifidobacteria able to consume the specific milk oligosaccharides delivered by the mother. This work provides mechanistic insight into how milk glycans enrich specific beneficial bacterial populations in infants and reveals clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants.

Secretory immunoglobulin A (sIgA) is a major glycoprotein in milk and plays a key role in mediating immune protection of the gut mucosa. Although it is a highly glycosylated protein, its site-specific glycosylation and associated glycan micro-heterogeneity have still not been fully elucidated. In this study, the site-specific glycosylation of sIgA isolated from human colostrum (n = 3) was analyzed using a combination of LC-MS and LC-MS/MS and in-house software (Glycopeptide Finder). The majority of the glycans found are biantennary structures with one or more acidic Neu5Ac residues; however, a large fraction belonged to truncated complex structures with terminal GlcNAc. Multiple glycosites were identified with nearly 30 glycan compositions located at seven sites on the secretory component, six compositions at a single site on the J chain, and 16 compositions at five sites on the IgA heavy (H) chain. Site-specific heterogeneity and relative quantitation of each composition and the extent of occupation at each site were determined using nonspecific proteases. Additionally, 54 O-linked glycan compositions located at the IgA1 hinge region (HR) were identified by comparison against a theoretical O-glycopeptide library. This represents the most comprehensive report to date detailing the complexity of glycan micro-heterogeneity with relative quantitation of glycoforms for each glycosylation site on milk sIgA. This strategy further provides a general method for determining site-specific glycosylation in large protein complexes.

Background: Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland, leading to differences in protein expression and enzymatic activity, thereby resulting in an altered peptide profile., Objective: This study examined differences in peptides present between milk from women delivering at term and women delivering prematurely., Methods: Nano-LC tandem mass spectrometry was employed to identify naturally occurring peptides and compare their abundances between term and preterm human milk samples at multiple time points over lactation. Term milk samples were collected from 8 mothers and preterm milk was collected from 14 mothers. The 28 preterm and 32 term human milk samples were divided into 4 groups based on day of collection (<14, 14-28, 29-41, and 42-58 d)., Results: Preterm milk peptide counts, ion abundance, and concentration were significantly higher in preterm milk than term milk. Bioinformatic analysis of the cleavage sites for peptides identified suggested that plasmin was more active in preterm milk than term milk and that cytosol aminopeptidase and carboxypeptidase B2 likely contribute to extensive milk protein breakdown. Many identified milk peptides in both term and preterm milk overlapped with known functional peptides, including antihypertensive, antimicrobial, and immunomodulatory peptides., Conclusion: The high protein degradation by endogenous proteases in preterm milk might attenuate problems because of the preterm infant's immature digestive system. This trial was registered at clinicaltrials.gov as NCT01817127., (© 2015 American Society for Nutrition.)

Peptidomics is an emerging field branching from proteomics that targets endogenously produced protein fragments. Endogenous peptides are often functional within the body-and can be both beneficial and detrimental. This review covers the use of peptidomics in understanding digestion, and identifying functional peptides and biomarkers. Various techniques for peptide and glycopeptide extraction, both at analytical and preparative scales, and available options for peptide detection with MS are discussed. Current algorithms for peptide sequence determination, and both analytical and computational techniques for quantification are compared. Techniques for statistical analysis, sequence mapping, enzyme prediction, and peptide function, and structure prediction are explored., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human milk oligosaccharides (HMOs) play a key role in shaping and maintaining a healthy infant gut microbiota. This article demonstrates the potential of combining recent advances in glycomics and genomics to correlate abundances of fecal microbes and fecal HMOs. Serial fecal specimens from two healthy breast-fed infants were analyzed by bacterial DNA sequencing to characterize the microbiota and by mass spectrometry to determine abundances of specific HMOs that passed through the intestinal tract without being consumed by the luminal bacteria. In both infants, the fecal bacterial population shifted from non-HMO-consuming microbes to HMO-consuming bacteria during the first few weeks of life. An initial rise in fecal HMOs corresponded with bacterial populations composed primarily of non-HMO-consuming Enterobacteriaceae and Staphylococcaeae. This was followed by decreases in fecal HMOs as the proportion of HMO-consuming Bacteroidaceae and Bifidobacteriaceae increased. Analysis of HMO structures with isomer differentiation revealed that HMO consumption is highly structure-specific, with unique isomers being consumed and others passing through the gut unaltered. These results represent a proof-of-concept and are consistent with the highly selective, prebiotic effect of HMOs in shaping the gut microbiota in the first weeks of life. The analysis of selective fecal bacterial substrates as a measure of alterations in the gut microbiota may be a potential marker of dysbiosis.

Proteomics of human milk has been used to identify the comprehensive cargo of proteins involved in immune and cellular function. Very little is known about the effects of gestational diabetes mellitus (GDM) on lactation and breast milk components. The objective of the current study was to examine the effect of GDM on the expression of proteins in the whey fraction of human colostrum. Colostrum was collected from women who were diagnosed with (n = 6) or without (n = 12) GDM at weeks 24-28 in pregnancy. Colostral whey was analyzed for protein abundances using high-resolution, high-mass accuracy liquid chromatography tandem mass spectrometry. A total of 601 proteins were identified, of which 260 were quantified using label free spectral counting. Orthogonal partial least-squares discriminant analysis identified 27 proteins that best predict GDM. The power law global error model corrected for multiple testing was used to confirm that 10 of the 27 proteins were also statistically significantly different between women with versus without GDM. The identified changes in protein expression suggest that diabetes mellitus during pregnancy has consequences on human colostral proteins involved in immunity and nutrition.

Oligosaccharides are abundant in human milk. Production of these highly diverse structures requires significant energy expenditure by the mother and yet these human milk oligosaccharides offer no direct nutritive value to her infant. A primary function of human milk oligosaccharides is to shape the infant's intestinal microbiota with life-long consequences. Bifidobacterium longum subspecies infantis (B. infantis) is unique among gut bacteria in its prodigious capacity to digest and consume any human milk oligosaccharide structure, the result of a large repertoire of bacterial genes encoding an array of glycosidases and oligosaccharide transporters not found in other bacterial species. In vitro, B. infantis grows better than other bacterial strains in the presence of human milk oligosaccharides, displays anti-inflammatory activity in premature intestinal cells, and decreases intestinal permeability. In premature infants, B. infantis given in combination with human milk increases B. infantis and decreases Enterobacteriaceae in the feces. Probiotics containing B. infantis decrease the risk of necrotizing enterocolitis in premature infants. Colonization with B. infantis is also associated with increased vaccine responses. Probiotic organisms have historically been selected based on ease of production and stability. The advantages of B. infantis, selected through coevolution with human milk glycans, present an opportunity for focused manipulation of the infant intestinal microbiota.

The present study describes the physical, antioxidant, and antimicrobial properties of biodegradable films prepared by incorporating different concentrations (1, 2, and 3% v/v) of Zataria multiflora Boiss (avishan-e shirazi) essential oil (ZEO) into carboxymethyl cellulose (CMC) film. The films' tensile strength, elongation at break, water-vapor permeability, optical characteristics, microstructure, and antimicrobial and antioxidant properties were investigated. The results indicated that the film containing 1% ZEO had the highest tensile strength and elongation at break. The control film showed the lowest water-vapor permeability. The resulting optical data showed that the control films were transparent in appearance; transparency was significantly reduced by an increase in ZEO concentration. Solubility in water decreased with increased ZEO. Films with ZEO, especially at higher concentrations, were more effective against all tested bacteria than the control film. Those films incorporating essential oil revealed antioxidant properties as well; this effect was greatly improved when the proportion of ZEO was increased. The results indicated that the antioxidant and antibacterial activity of ZEO is retained when it is used in CMC film. These properties with some good physical characteristics suggest applications for ZEO-incorporated film in a wide range of food products., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Little is known about the digestive process in infants. In particular, the chronological activity of enzymes across the course of digestion in the infant remains largely unknown. To create a temporal picture of how milk proteins are digested, enzyme activity was compared between intact human milk samples from three mothers and the gastric samples from each of their 4-12 day postpartum infants, 2 h after breast milk ingestion. The activities of 7 distinct enzymes are predicted in the infant stomach based on their observed cleavage pattern in peptidomics data. We found that the same patterns of cleavage were evident in both intact human milk and gastric milk samples, demonstrating that the enzyme activities that begin in milk persist in the infant stomach. However, the extent of enzyme activity is found to vary greatly between the intact milk and gastric samples. Overall, we observe that milk-specific proteins are cleaved at higher levels in the stomach compared to human milk. Notably, the enzymes we predict here only explain 78% of the cleavages uniquely observed in the gastric samples, highlighting that further investigation of the specific enzyme activities associated with digestion in infants is warranted.

An extensive mass spectrometry analysis of the human milk peptidome has revealed almost 700 endogenous peptides from 30 different proteins. Two in-house computational tools were created and used to visualize and interpret the data through both alignment of the peptide quasi-molecular ion intensities and estimation of the differential enzyme participation. These results reveal that the endogenous proteolytic activity in the mammary gland is remarkably specific and well conserved. Certain proteins-not necessarily the most abundant ones-are digested by the proteases present in milk, yielding endogenous peptides from selected regions. Our results strongly suggest that factors such as the presence of specific proteases, the position and concentration of cleavage sites, and, more important, the intrinsic disorder of segments of the protein drive this proteolytic specificity in the mammary gland. As a consequence of this selective hydrolysis, proteins that typically need to be cleaved at specific positions in order to exert their activity are properly digested, and bioactive peptides encoded in certain protein sequences are released. Proteins that must remain intact in order to maintain their activity in the mammary gland or in the neonatal gastrointestinal tract are unaffected by the hydrolytic environment present in milk. These results provide insight into the intrinsic structural mechanisms that facilitate the selectivity of the endogenous milk protease activity and might be useful to those studying the peptidomes of other biofluids., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures.

Production of biodiesel from edible plant oils is quickly expanding worldwide to fill a need for renewable, environmentally-friendly liquid transportation fuels. Due to concerns over use of edible commodities for fuels, production of biodiesel from non-edible oils including microbial oils is being developed. Microalgae biodiesel is approaching commercial viability, but has some inherent limitations such as requirements for sunlight. While yeast oils have been studied for decades, recent years have seen significant developments including discovery of new oleaginous yeast species and strains, greater understanding of the metabolic pathways that determine oleaginicity, optimization of cultivation processes for conversion of various types of waste plant biomass to oil using oleaginous yeasts, and development of strains with enhanced oil production. This review examines aspects of oleaginous yeasts not covered in depth in other recent reviews. Topics include the history of oleaginous yeast research, especially advances in the early 20th century; the phylogenetic diversity of oleaginous species, beyond the few species commonly studied; and physiological characteristics that should be considered when choosing yeast species and strains to be utilized for conversion of a given type of plant biomass to oleochemicals. Standardized terms are proposed for units that describe yeast cell mass and lipid production., (Copyright © 2014. Published by Elsevier Inc.)