Exclusively Breastfed Infant Microbiota Develops over Time and Is Associated with Human Milk Oligosaccharide Intakes

Temporal development of maternal and infant microbiomes during early life impacts short- and long-term infant health. This study aimed to characterize bacterial dynamics within maternal faecal, human milk (HM), infant oral, and infant faecal samples during the exclusive breastfeeding period and to document associations between human milk oligosaccharide (HMO) intakes and infant oral and faecal bacterial profiles. Maternal and infant samples (n = 10) were collected at 2–5, 30, 60, 90 and 120 days postpartum and the full-length 16S ribosomal RNA (rRNA) gene was sequenced. Nineteen HMOs were quantitated using high-performance liquid chromatography. Bacterial profiles were unique to each sample type and changed significantly over time, with a large degree of intra- and inter-individual variation in all sample types. Beta diversity was stable over time within infant faecal, maternal faecal and HM samples, however, the infant oral microbiota at day 2–5 significantly differed from all other time points (all p < 0.02). HMO concentrations and intakes significantly differed over time, and HMO intakes showed differential associations with taxa observed in infant oral and faecal samples. The direct clinical relevance of this, however, is unknown. Regardless, future studies should account for intakes of HMOs when modelling the impact of HM on infant growth, as it may have implications for infant microbiota development.     

Docosahexaenoic acid and arachidonic acid levels are correlated in human milk: Implications for new European infant formula regulations

From February 2022, all infant formula sold in the European Union must contain docosahexaenoic acid (DHA) at ~0.33%–1.14% of total fat with no minimum requirement for arachidonic acid (ARA). This work examines the association between DHA and ARA levels in human milk, the gold standard for infant feeding. Human milk (n = 470) was collected over 12-weeks postpartum from lactating mothers (n = 100) of infants born weighing <1250 g (NCT02137473). Fatty acids were analyzed by gas chromatography. ARA and DHA concentrations were associated in human milk (β = 0.47 [95% confidence interval 0.38–0.56] mol%), including transitional and mature milk, but not colostrum. This remained significant upon adjustment for percentages of other saturated, monounsaturated, n-3, or n-6 fatty acids, day of sample collection, or maternal characteristics (body mass index, ethnicity, education, and income). Infant formulas containing relatively high concentrations of DHA without ARA, as permitted by the new regulations, would not reflect the balance of these fatty acids in human milk.     

The Diverse Antimicrobial Activities of Human Milk Oligosaccharides against Group B Streptococcus

Streptococcus agalactiae or Group B Streptococcus (GBS) is a Gram-positive bacterial pathobiont that is the etiological cause of severe perinatal infections. GBS can colonize the vagina of pregnant patients and invade tissues causing ascending infections of the gravid reproductive tract that lead to adverse outcomes including preterm birth, neonatal sepsis, and maternal or fetal demise. Additionally, transmission of GBS during labor or breastfeeding can also cause invasive infections of neonates and infants. However, human milk has also been shown to have protective effects against infection; a characteristic that is likely derived from antimicrobial and immunomodulatory properties of molecules that comprise human milk. Recent evidence suggests that human milk oligosaccharides (HMOs), short-chain sugars that comprise 8–20 % of breast milk, have antimicrobial and anti-biofilm activity against GBS and other bacterial pathogens. Additionally, HMOs have been shown to potentiate the activity of antibiotics against GBS. This review presents the most recent published work that studies the interaction between HMOs and GBS.     

The Role of Human Milk Lipids and Lipid Metabolites in Protecting the Infant against Non-Communicable Disease

Non-communicable diseases continue to increase globally and have their origins early in life. Early life obesity tracks from childhood to adulthood, is associated with obesity, inflammation, and metabolic dysfunction, and predicts non-communicable disease risk in later life. There is mounting evidence that these factors are more prevalent in infants who are formula-fed compared to those who are breastfed. Human milk provides the infant with a complex formulation of lipids, many of which are not present in infant formula, or are present in markedly different concentrations, and the plasma lipidome of breastfed infants differs significantly from that of formula-fed infants. With this knowledge, and the knowledge that lipids have critical implications in human health, the lipid composition of human milk is a promising approach to understanding how breastfeeding protects against obesity, inflammation, and subsequent cardiovascular disease risk. Here we review bioactive human milk lipids and lipid metabolites that may play a protective role against obesity and inflammation in later life. We identify key knowledge gaps and highlight priorities for future research.     

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.     

Synthesis of the Human Milk Oligosaccharide Lacto‐N‐Tetraose in Metabolically Engineered,Plasmid‐Free E. coli

Human milk oligosaccharides (HMOs) constitute the third most abundant solid component of human milk. HMOs have been demonstrated to show positive effects on the infant’s well‐being. Despite numerous studies, more physiological analyses of single compounds are needed to fully elucidate these effects. Although being one of the most abundant core structures in human milk, the HMO lacto‐N‐tetraose (LNT) is not available at reasonable prices. In this study, we demonstrate the construction of the first E. coli strain capable of producing LNT in vivo. The strain was constructed by chromosomally integrating the genes lgtA and wbgO, encoding β‐1,3‐N‐acetylglucosaminyltransferase and β‐1,3‐galactosyltransferase. In shake‐flask cultivations, the strain yielded a total concentration of 219.1±3.5 mg L^?1 LNT (LNT in culture broth and the cell pellet). After recovery of LNT, structural analysis by NMR spectroscopy confirmed the molecule structure.     

Concentrations of calcium,magnesium, sodium and potassium in human milk and infant formulas

Concentrations of calcium, magnesium, sodium and potassium were determined in 55 samples of mature human milk from Canary women and 5 samples of powdered infant formula. According to the literature our data fell within the normal intervals described for each kind of milk. The mean concentration of Ca, Mg, Na y K of powdered infant formula was higher than those concentrations found in the human milks. Significant differences among the concentrations of Ca, Mg and Na for the milks of the considered mothers were observed. Only the Ca intakes for infants fed with human milk were lower than those requirements recommended by the Food and Nutrition Board (1989). However, the infants fed with powdered infant formula had an adequate intake of all the studied metals. A progressive decrease of the Na, K and Ca concentrations with the lactation stage was observed. Maternal age, parity and sex of the newborns did not affect the metal concentrations significantly.     

Maternal nutritional status and milk volume. Is there a cause-effect relationship?

Studies on human lactation were examined in order to gather some answers about questions concerning the effect of maternal food intake, size, fatness and economic status on milk production. Up to date, evidence in the literature is insufficient to permit definitive answers, but a general conclusion can be drawn: milk volume varies little among mothers with largely variable energy intakes, sizes and economic status. There is a great need for more controlled studies focusing on the relationship between maternal energy balance and milk output. Although many studies have separately addressed the nutritional changes in mothers throughout lactation (1-8) and milk consumption by infants (9-17), very few have correlated maternal nutritional conditions and the volume of milk consumed. This report will consider investigations published from 1975 and on, combining data on maternal nutritional status and milk production in the same individual. The rationale is that around 1975 more accurate and standardized methodology began to be used in related studies. Milk output is estimated by the summary of the differences of body weights of infants obtained before and after each milking episode during 24 hours. Before 1975 the balances used for such a purpose had very poor precision, and this interfered seriously on the inter and intra-personal variability of the measurements. Electronic scales made available after that year gave enough reliability to the procedure. This report is comprised of studies from birth to four months postpartum, when energy supplementation is less common, and quantitatively less important. Nutritional status of the mothers will be analyzed on the basis of four categorizing variables: social and economic status, anthropometry, food intake and body composition.     

Temporal Changes of Phospholipids Fatty Acids and Cholesterol in Breast Milk and Relationship with Diet

Phospholipids (PLs) and cholesterol in human milk (HM) are affected by lactation, and differential lipids are closely related to maternal diet. The contents of PLs and cholesterol in Chinese HM are quantified by gas chromatography and high performance liquid chromatography, respectively, and the relationship between differential lipids and the maternal diet is obtained by Pearson. The result shows that SFA, MUFA, and polyunsaturated fatty acid (PUFA) are not affected by lactation and geography for total fatty acids, but almost all sn‐2 fatty acids are influenced by geography and remain unchanged during lactation. Most SFAs show absolute sn‐2 selectivity and the majority of MUFAs and PUFAs are esterified at the sn‐1 position. Cholesterol (13.8–22.6 mg per 100 g milk) and 25‐hydroxycholesterol (0.45–1.01 mg per 100 g milk) increase significantly and remain constant during lactation, respectively, and they are affected by regions. In addition, the differential lipids (22:1n‐9, C9:0, trans‐PUFA, 22:4n‐6, etc.) of PLs are closely related to the maternal diet. PLs and cholesterol content differ from western research and infant formula, which will help to design an infant formula that is more suitable for Chinese babies in the future. Practical Application: Compared with PLs and cholesterol in western countries and infant formula, the specificity of Chinese HM can more accurately target the development of formulas suitable for the growth of Chinese infants. At the same time, according to the influence of the mother?s diet on the composition of HM, it is more reasonable to guide the diet of the mother.     

Lipids in infant formulas: Current and future innovations

Lipids in infant formulas are designed to mimic the composition of human milk and/or approximate the performance of the breastfed infant. Human milk lipids exhibit a complex, highly variable nature, varying by population, dietary intake, time of day, length of feeding and others. Continued understanding and characterization of human milk lipids has led to infant formula innovations aimed at a better comparison to human milk and/or improved dietary lipid utilization in the formula‐fed infant. The underlying aim of such innovation is the generation of outcomes as comparable to human milk feeding as possible. The present paper briefly reviews the current understanding of human milk lipid composition, with a focus on how this knowledge drives current and future innovations in the area of infant formula lipids.     

Anti-biofilm Activity of Human Milk Oligosaccharides in Clinical Strains of Streptococcus agalactiae with Diverse Capsular and Sequence Types

Group B Streptococcus (GBS) is an encapsulated Gram-positive bacterial pathogen that causes severe perinatal infections. Human milk oligosaccharides (HMOs) are short-chain sugars that have recently been shown to possess antimicrobial and anti-biofilm activity against a variety of bacterial pathogens, including GBS. We have expanded these studies to demonstrate that HMOs can inhibit and dismantle biofilm in both invasive and colonizing strains of GBS. A cohort of 30 diverse strains of GBS were analyzed for susceptibility to HMO-dependent biofilm inhibition or destruction. HMOs were significantly effective at inhibiting biofilm in capsular-type- and sequence-type-specific fashion, with significant efficacy in CpsIb, CpsII, CpsIII, CpsV, and CpsVI strains as well as ST-1, ST-12, ST-19, and ST-23 strains. Interestingly, CpsIa as well as ST-7 and ST-17 were not susceptible to the anti-biofilm activity of HMOs, underscoring the strain-specific effects of these important antimicrobial molecules against the perinatal pathogen Streptococcus agalactiae.     

Effect of 9,12-Octadecadiynoic Acid on Neurobehavioral Development in Caenorhabditis elegans

Human breast milk lipids have major beneficial effects: they promote infant early brain development, growth and health. To identify the relationship between human breast milk lipids and infant neurodevelopment, multivariate analyses that combined lipidomics and psychological Bayley-III scales evaluation were utilized. We identified that 9,12-octadecadiynoic acid has a significantly positive correlation with infant adaptive behavioral development, which is a crucial neurodevelopment to manage risk from environmental stress. To further clarify the biological function of 9,12-octadecadiynoic acid in regulating neurodevelopment, Caenorhabditis elegans (C. elegans) was used as a model to investigate the effect of 9,12-octadecadiynoic acid on neurobehavioral development. Supplementation with 9,12-octadecadiynoic acid from the L1 to L4 stage in larvae affected locomotive behaviors and foraging ability that were not socially interactive, implying that 9,12-octadecadiynoic acid is involved in regulating the serotonergic neuronal ability. We found that supplementary 0.1 μM 9,12-octadecadiynoic acid accelerated the locomotive ability and foraging ability via increasing the expression of serotonin transporter mod-1. Antioxidant defense genes, sod-1, sod-3 and cyp-35A2 are involved in 9,12-octadecadiynoic acid-induced motor neuronal activity. Nevertheless, supplementary 9,12-octadecadiynoic acid at concentrations above 1 μM significantly attenuated locomotive behaviors, foraging ability, serotonin synthesis, serotonin-related gene expressions and stress-related gene expression, resulting in the decreased longevity of worms in the experiment. In conclusion, our study demonstrates the biological function of 9,12-octadecadiynoic acid in governing adaptive behavioral development.     

Supplemental Conjugated Linoleic Acid Consumption Does Not Influence Milk Macronutrient Contents in all Healthy Lactating Women

The term “conjugated linoleic acid” (CLA) refers to a group of positional and geometric isomers that are derived from linoleic acid and are found primarily in meat and milk products from ruminant animals. Due to the array of putative benefits associated with various forms of CLA, there has been recent interest in supplementing human diets with these fatty acids especially when weight loss is desired. However, in many animal models, CLA has been shown to decrease milk fat production. There is some concern, therefore, that maternal CLA supplementation during lactation might inadvertently decrease nutrient supply to the nursing infant. However, there is only limited research on the effect of CLA consumption on milk fat content in women. Based on previously published work from our laboratory, we hypothesized that CLA supplementation would reduce the milk fat percentage in lactating women in a dose-dependent manner. Breastfeeding women (n = 12) were assigned randomly to treatments of 4 g/day safflower oil (SFO), 2 g/day CLA plus 2 g/day SFO, or 4 g/day CLA in a double blind, 3 × 3 Latin square design. Conjugated linoleic acid supplements contained approximately equal amounts of cis9,trans11–18:2 and trans10,cis12–18:2; the two most common isoforms of CLA. Milk was collected by complete breast expression on the last day (day 5) of each intervention period and analyzed for macronutrient and fatty acid composition. On day 4 of each intervention period, infant milk consumption was estimated by 24 h weighing of the infant. Washout periods were 9 days in length. We observed a dose-dependent increase in the concentrations of cis9,trans11–18:2 and trans10,cis12–18:2 in the milk fat. However, we detected neither a change in overall macronutrient composition nor infant milk consumption. These data do not support those obtained from animal models or our previous human work suggesting that consumption of CLA mixtures necessarily reduces milk fat. It is possible that either (1) the interpretation of our previously published data should be reevaluated, and/or (2) there are important intra- and inter-species differences in this regard.     

Peptides from the Intestinal Tract of Breast Milk-Fed Infants Have Antimicrobial and Bifidogenic Activity

For bioactive milk peptides to be relevant to infant health, they must be released by gastrointestinal proteolysis and resist further proteolysis until they reach their site of activity. The intestinal tract is the likeliest site for most bioactivities, but it is currently unknown whether bioactive milk peptides are present therein. The purpose of the present study was to identify antimicrobial and bifidogenic peptides in the infant intestinal tract. Milk peptides were extracted from infant intestinal samples, and the activities of the bulk peptide extracts were determined by measuring growth of Escherichia coli, Staphylococcus aureus, and Bifidobacterium longum spp. infantis after incubation with serial dilutions. The peptide profiles of active and inactive samples were determined by peptidomics analysis and compared to identify candidate peptides for bioactivity testing. We extracted peptides from 29 intestinal samples collected from 16 infants. Five samples had antimicrobial activity against S. aureus and six samples had bifidogenic activity for B. infantis. We narrowed down a list of 6645 milk peptides to 11 candidate peptides for synthesis, of which 6 fully inhibited E. coli and S. aureus growth at concentrations of 2500 and 3000 µg/mL. This study provides evidence for the potential bioactivity of milk peptides in the infant intestinal tract.     

Preparation of Human Milk Fat Substitutes from Lard by Lipase‐Catalyzed Interesterification Based on Triacylglycerol profiles

Human milk fat substitutes (HMFSs) with triacylglycerol profiles highly similar to those of human milk fat (HMF) were prepared from lard by physical blending followed by enzymatic interesterification. Based on the fatty acid profiles of HMF, different vegetable and single‐cell oils were selected and added to the lard. Blend ratios were calculated based on established physical blending models. The blended oils were then enzymatically interesterified using a 1,3‐regiospecific lipase, Lipozyme RM IM (RML from Rhizomucor miehei immobilized on Duolite ES562; Novozymes A/S, Bagsværd, Denmark), to approximate HMF triacylglycerol (TAG) profiles, particularly with respect to the distribution of palmitic acid in the sn?2 position. The optimized blending ratios were determined to be: lard:sunflower oil:canola oil:palm kernel oil:palm oil:algal oil:microbial oil = 1.00:0.10:0.50:0.13:0.12:0.02:0.02. The optimized reaction conditions were determined to be: enzyme load of 11 wt%, temperature of 60 °C, water content of 3.5 wt%, and reaction time of 3 hours. The resulting product was evaluated for total and sn?2 fatty acids, polyunsaturated fatty acids, and TAG composition. A high degree of similarity was obtained, indicating the great potential of the product as a fat alternative for use in infant formulas.     

Association of Maternal Microbiota and Diet in Cord Blood Cytokine and Immunoglobulin Profiles

Mothers confer natural passive immunization to their infants through the transplacental pathway during the gestation period. The objective of the present study was to establish at birth the maternal and cord plasma concentration and relationship of immunoglobulins (Igs), cytokines (CKs), and adipokines. In addition, the impact of the maternal microbiota and diet was explored. The plasma profile of these components was different between mothers and babies, with the levels of many CKs, IgM, IgG2a, IgE, IgA, and leptin significantly higher in mothers than in the cord sample. Moreover, the total Igs, all IgG subtypes, IgE, and the Th1/Th2 ratio positively correlated in the mother–infant pair. Maternal dietary components such as monounsaturated fatty acids-polyunsaturated fatty acids and fiber were positively associated with some immune factors such as IgA in cord samples. The microbiota composition clustering also influenced the plasma profile of some factors (i.e., many CKs, some Ig, and adiponectin). In conclusion, we have established the concentration of these immunomodulatory factors in the maternal–neonatal pair at birth, some positive associations, and the influence of maternal diet and the microbiota composition, suggesting that the immune status during pregnancy, in terms of CKs and Igs levels, can influence the immune status of the infant at birth.     

Characterization and Oxidative Stability of Human Milk Fat Substitutes Enzymatically Produced from Palm Stearin

Production of human milk fat substitutes (HMFSs) from three types of palm stearin with palmitic acid (PA) of 91.3, 70.3 and 62.6 %, respectively, was scaled up to a kilogram scale. The physiochemical properties of these products including fatty acid profiles, triacylglycerol compositions, tocopherol contents, oxidative stability and melting and crystallization profiles were compared with those of HMFSs from lard, butterfat and tripalmitin and fats from infant formulas. Based on their chemical compositions, HMFSs from palm stearin with PA contents of 70.3 and 62.6 % produced by enzymatic acidolysis were found to have the highest degree of similarity to human milk fat, which indicated that these HMFSs were the most suitable for use in infant formulas. However, HMFSs from palm stearin with PA content of 91.3 % had the highest tocopherol contents. By investigation of the primary and secondary oxidation products during accelerated oxidation, the oxidative stability of HMFSs was found to be positively correlated to the contents of tocopherols, and the volatile oxidation compounds with the highest relative contents in HMFSs were aldehydes analyzed by solid-phase microextraction-GC–MS. All HMFSs had final melting points lower than body temperature.     

Lipids and human milk

To support the growth and development of the breast‐fed infant, human milk provides the dietary essential fatty acids, linoleic acid (LA; 18:2n‐6), α‐linolenic acid (ALA, 18:3n‐3), as well as longer‐chain polyunsaturated fatty acids including arachidonic acid (20:4n‐6) and docosahexanoic (DHA 22:6n‐3). The linoleic acid, alpha‐linolenic acid, DHA and arachidonic acid concentration of pasteurized and unpasteurized human milk remains stable during the first month of storage at –20°C and –80°C. However after the first month, a slow decrease in concentration progresses until the end of 6 months of storage at both temperatures. The levels of n‐6 and n‐3 fatty acids, particularly linoleic acid, alpha‐linolenic acid and DHA, in human milk vary widely within and among different populations, and are readily changed by maternal dietary intake of the respective fatty acid. The present paper reviews recent understanding from key researchers of maternal diet and human milk fat composition and form our work the effect of milk fat composition on storage conditions. It is important to understand that maternal diet can affect human milk fat composition and subsequently infant development and growth.     

Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.     

Characterization and Oxidative Stability of Structured Lipids: Infant Milk Fat Analog

Structured lipids (SLs) for infant milk formulation were produced by enzymatic interesterification of tripalmitin with vegetable oil blends and fish oil. The SLs were characterized by fatty acid content and structure, melting profiles, oxidative stability index, free fatty acid (FFA) concentration, and tocopherol content. Oxidative stability was studied using accelerated methods by quantifying FFA, peroxides (peroxide value) and aldehydes (p-anisidine value) production. Total oxidation (TOTOX value) was calculated as 2 × (peroxide value) + (p-anisidine value). The structured lipids after purification by distillation and addition of antioxidants had melting profiles, oxidative stability index, and initial FFA concentration that were similar to that of the starting oil blends, while the fatty acid composition and structure of the SLs were similar to that of human milk fat. Oxidative stability of the SLs was improved with tocopherol addition as antioxidants and was comparable to that of the vegetable oils and oil blends.