Human milk oligosaccharide associated with the firmicutes-to-bacteroidetes ratio among stunted infants in Malang, Indonesia.

BACKGROUND: Human milk oligosaccharide (HMO) is a unique component of breastmilk. To date, no study has investigated the correlation between HMO and infant nutritional status particularly through the lens of gut microbiota. Therefore, our study aims to investigate the relationships between 2'-Fucosyllactose (2'-FL) in HMO and Firmicutes/Bacteroidetes (F/B) ratio among stunted infants. METHODS: A case-control study was conducted among 103 mother-infant pairs in Malang City, Indonesia. The quantification of 2'-FL HMO was assessed using High-Performance Liquid Chromatography (HPLC). The F/B ratio was analyzed with real-time poly-chain reaction (RT-PCR). For bivariate analysis, we employed the Spearman correlation and Mann‒Whitney tests, while for multivariate analysis, we utilized multiple linear regression. RESULTS: The findings showed that the stunted nutritional status was detected in 49 out of 103 infants. In this group, 40.81% of mothers of infants with a stunted nutritional status had a secretor-positive status, while all mothers of infants with appropriate nutritional status tested positive for the secretor status (100%). However, the association between maternal secretor status and infant nutritional status was not statistically significant (p>0.05). The average levels of 2'-FL HMO in breast milk were lower in the group with stunted infants compared to non-stunted infants (1.21 mg/L vs 1.40 mg/L). The regression analysis revealed a significant association of 2'-FL HMO levels with the presence of Bacteroidetes and value of the F/B ratio (p>0.05). CONCLUSIONS: The breast milk component 2'-FL HMO significantly influences the gut microbiota of stunted infants. Future research aimed at elucidating the mechanisms by which 2'-FL HMO modulates infant gut microbiota should consider not only concentration and specific bacterial taxa but also intake levels (Tab. 2, Fig. 1, Ref. 37). Text in PDF www.elis.sk Keywords: 2'-fucosyllactose, human milk, oligosaccharide, firmicutes, bacteroidetes, stunting, infant.

Recent advances on 2'-fucosyllactose: physiological properties, applications, and production approaches.

The trisaccharide, 2'-fucosyllactose (Fucα1-2Galß1-4Glc; 2'-FL), is the most abundant oligosaccharide in human milk. It has numerous significant biological properties including prebiotics, antibacterial, antiviral, and immunomodulating effects, and has been approved as "generally recognized as safe" (GRAS) by the Food and Drug Administration (FDA) and as a novel food (NF) by the European Food Safety Authority (EFSA). 2'-FL not only serves as a food ingredient added in infant formula, but also as a dietary supplement and medical food material in food bioprocesses. There is considerable commercial interest in 2'-FL for its irreplaceable nutritional applications. This review aims at systematically elaborating key functional properties of 2'-FL as well as its applications. In addition, several approaches for 2'-FL production are described in this review, including chemical, chemo-enzymatical, and cell factory approaches, and the pivotal research results also have been summarized. With the rapid development of metabolic engineering and synthetic biology strategies, using the engineered cell factory for 2'-FL large-scale production might be a promising approach. From an economic and safety point of view, microbial selection for cell factory engineering in 2'-FL bioprocess also should be taken into consideration.

Longitudinal Changes in Human Milk Oligosaccharides (HMOs) Over the Course of 24 Months of Lactation.

BACKGROUND: Human milk oligosaccharides (HMOs) are complex glycans that are highly abundant in human milk. While over 150 HMOs have been identified, it is unknown how individual HMOs change in concentration over 24 months of lactation. OBJECTIVES: To understand how HMO concentrations change over 24 months of lactation. METHODS: Breast milk samples were collected from participants in a longitudinal cohort study of Hispanic mother-infant pairs at 1, 6, 12, 18, and 24 months postpartum. Concentrations of 19 of the most abundant HMOs were measured using HPLC. Because the parent study is ongoing and not all participants have finished all time points yet, the sample sizes ranged per time point (n = 207 at 1 month; n = 109 at 6 months; n = 83 at 12 months; n = 59 at 18 months; and n = 28 at 24 months). Approximately 88% of participants were classified as HMO secretors-a genetic factor that affects concentrations of HMOs such as 2'fucosyllactose (2'FL) and lacto-N-fucopentaose I-while the remaining 12% were classified as nonsecretors. Mixed models were used to examine changes in HMO concentrations and relative abundances over the course of lactation. RESULTS: The majority of HMOs significantly decreased in concentration over the course of lactation. The exceptions were 2'FL, sialyl-lacto-N-tetraose b, and disialyl-lacto-N-tetraose, which did not change with time, and 3-fucosyllactose (3FL) and 3'-sialyllactose (3'SL), which significantly increased. The concentration of 3FL increased 10-fold, from 195 (IQR 138-415) µg/mL at 1 month to 1930 (1100-2630) µg/mL at 24 months, while 3'SL increased 2-fold, from 277 (198-377) µg/mL to 568 (448-708) µg/mL over the same time period. CONCLUSIONS: These results indicate that HMOs do not decrease in concentration uniformly across lactation. In particular, 3FL and 3'SL increased over the course of lactation in this cohort. Future studies are required to fully understand the functions of these HMOs.

Glycosynthase reaction meets the flow: Continuous synthesis of lacto-N-triose II by engineered ß-hexosaminidase immobilized on solid support.

The D746E variant of Bifidobacterium bifidum ß-N-acetyl-hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective ß-1,3-glycosylation of lactose from a d-glucosamine 1,2-oxazoline donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145-200 mM) at quantitative yield from the donor substrate. The wild-type ß-N-acetyl-hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d-glucosamine. By enabling short residence times (2 min) that are challenging for mixed-vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80-100 mM/min) from the slower side reactions (decomposition of donor substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.

Development of a quantitative assay for 2´-fucosyllactose via one-pot reaction with α1,2-fucosidase and l-fucose dehydrogenase.

2'-Fucosyllactose (2'-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2'-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2'-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and l-fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli. The recombinant enzymes were produced as 6 × His-tagged proteins and were purified to homogeneity using Ni2+ affinity chromatography. The purified α1,2-fucosidase and l-fucose dehydrogenase are monomers with molecular masses of 63 kDa and 36 kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2'-FL in a large number of samples simultaneously. This method can be used to quantify 2'-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2'-FL.

Infrared-assisted extraction followed by high performance liquid chromatography to determine angoroside C, cinnamic acid, and harpagoside content in Scrophularia ningpoensis.

BACKGROUND: Angoroside C, cinnamic acid, and harpagoside are bioactive constituents in Scrophularia ningpoensis. Currently, an infrared-assisted extraction (IRAE) method coupled with high-performance liquid chromatography with ultraviolet detection (HPLC-UV) for the analysis of bioactive constituents in this plant is lacking. METHODS: A method based on HPLC following IRAE has been developed for quantifying angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis. Four main factors, namely, extraction solvent, solid/liquid ratio, illumination time, and distance between the infrared lamp and the round-bottom flask, were optimized for extraction. Furthermore, conventional ultrasonic extraction (USE) and microwave-assisted extraction (MAE) were also investigated to validate the developed method. RESULTS: The optimal extraction conditions were as follows: ethanol concentration, 37.5%; solid/liquid ratio, 1:25; illumination time, 10 min; and distance between infrared lamp and round-bottom flask, 3 cm. The results of method validation demonstrated that the developed method meets the requirement of analysis. CONCLUSION: The results show that the IRAE-HPLC is a simple, accurate, and green analytical preparatory method for the potential extraction and quantification of angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis.

In vitro assessment of iron availability from commercial Young Child Formulae supplemented with prebiotics.

PURPOSE: Iron is essential for development and growth in young children; unfortunately, iron deficiency (ID) is a significant public health problem in this population. Young Child Formulae (YCF), milk-derived products fortified with iron and ascorbic acid (AA, an enhancer of iron absorption) may be good sources of iron to help prevent ID. Furthermore, some YCF are supplemented with prebiotics, non-digestible carbohydrates suggested to enhance iron bioavailability. The aim of our study was to evaluate iron bioavailability of YCF relative to prebiotic and AA concentrations. We hypothesised that YCF with the highest levels of prebiotics and AA would have the most bioavailable iron. METHODS: We used the in vitro digestion/Caco-2 cell model to measure iron bioavailability from 4 commercially available YCF with approximately equal amounts of iron, but varying amounts of: AA and the prebiotics fructo- and galacto-oligosaccharides. Caco-2 cell ferritin formation was used as a surrogate marker for iron bioavailability. RESULTS: The YCF with the highest concentration of prebiotics and AA had the highest iron bioavailability; conversely, the YCF with the lowest concentration of prebiotics and AA had the lowest. After the addition of exogenous prebiotics, so that all tested YCF had equivalent amounts, there was no longer a significant difference between YCF iron bioavailability. CONCLUSION: Our results suggest that ascorbic acid and prebiotics in YCF improve iron bioavailability. Ensuring that iron is delivered in a bioavailable form would improve the nutritional benefits of YCF in relation to ID/IDA amongst young children; therefore, further exploration of our findings in vivo is warranted.

Determination of 2'-Fucosyllactose and Lacto-N-neotetraose in Infant Formula.

Human milk oligosaccharides (HMO) are the third most abundant solid component of human milk. It is likely that they are responsible for at least some of the benefits experienced by breast-fed infants. Until recently HMO were absent from infant formula, but 2'-fucosyllactose (2'-FL) and lacto-N-neoteraose (LNnT) have recently become available as ingredients. The development of formula containing these HMO and the quality control of such formula require suitable methods for the accurate determination of the HMO. We developed two different approaches for analysis of 2'-FL and LNnT in formula; high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD). In lab trials using blank formula spiked with the two oligosaccharides, both approaches worked well with recoveries of 94⁻111% (HPAEC-PAD) and 94⁻104% (HILIC-FLD) and RSD (iR) of 2.1⁻7.9% (HPAEC-PAD) and 2.0⁻7.4% (HILIC-FLD). However, when applied to products produced in a pilot plant, the HPAEC-PAD approach sometimes delivered results below those expected from the addition rate of the ingredients. We hypothesize that the oligosaccharides interact with the formula matrix during the production process and, during sample preparation for HPAEC-PAD those interactions have not been broken. The conditions required for labeling the HMO for detection by the FLD apparently disrupt those interactions, and result in improved recoveries. It is likely that both analytical approaches are appropriate if a suitable extraction process is used to recover the HMO.

FUT2-dependent breast milk oligosaccharides and allergy at 2 and 5 years of age in infants with high hereditary allergy risk.

PURPOSE: Manifestation of allergic disease depends on genetic predisposition, diet and commensal microbiota. Genetic polymorphism of mothers determines their breast milk glycan composition. One major determinant is the fucosyltransferase 2 (FUT2, secretor gene) that was shown to be linked to commensal microbiota establishment. We studied whether FUT2-dependent breast milk oligosaccharides are associated with allergic disease in breast-fed infants later in life. METHODS: We analyzed FUT2-dependent oligosaccharides in breast milk samples of mothers (n = 266) from the placebo group of a randomized placebo-controlled trial of prebiotics and probiotics as preventive against allergic disease in infants with high allergy risk (trial registry number: NCT00298337). Using logistic regression models, we studied associations between FUT2-dependent breast milk oligosaccharides and incidence of allergic disease at 2 and 5 years of age. RESULTS: At 2 years, but not at 5 years of age, we observed a presumed lower incidence (p < 0.1) for IgE-associated eczema manifestation in C-section-born infants who were fed breast milk containing FUT2-dependent oligosaccharides. By logistic regression, we observed a similar relation (p < 0.1) between presence of FUT2-dependent breast milk oligosaccharides and IgE-associated disease and IgE-associated eczema in C-section-born infants only. When testing with the levels of breast milk oligosaccharide 2'-fucosyllactose as proxy for FUT2 activity, we observed significant (p < 0.05) associations in the C-section-born infants with 'any allergic disease,' IgE-associated disease, eczema and IgE-associated eczema. CONCLUSION: The data indicate that infants born by C-section and having a high hereditary risk for allergies might have a lower risk to manifest IgE-associated eczema at 2 years, but not 5 years of age, when fed breast milk with FUT2-dependent milk oligosaccharides. Further studies with larger cohorts and especially randomized controlled intervention trials are required to build on these preliminary observations.

Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3'-, 4-, and 6'-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo.

BACKGROUND: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known. OBJECTIVE: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS. METHODS: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1ß (IL-1ß) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo. RESULTS: The 3 galactosyllactoses (3'-GL, 4-GL, and 6'-GL) expressed in colostrum rapidly declined over early lactation (P < 0.05). In H4 cells, HMOS attenuated TNF-α- and IL-1ß-induced expression of IL-8, MIP-3α, and MCP-1 to 48-51% and pathogen-induced IL-8 and MCP-1 to 26-30% of positive controls (P < 0.001). GOS reduced TNF-α- and IL-1ß-induced inflammatory responses to 25-26% and pathogen-induced IL-8 and MCP-1 to 36-39% of positive controls (P < 0.001). GOS and HMOS mitigated nuclear translocation of nuclear transcription factor κB (NF-κB) p65. HMOS quenched the inflammatory response to Salmonella infection by immature human intestinal tissue ex vivo to 26% and by GOS to 50% of infected controls (P < 0.01). CONCLUSION: Galactosyllactose attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. Thus, galactosyllactoses are strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation.

Cow's milk-based beverage consumption in 1- to 4-year-olds and allergic manifestations: an RCT.

BACKGROUND: Nutrients such as docosahexaenoic acid (DHA), prebiotics and ß-glucan have been associated with reduced incidence of respiratory illnesses and allergic manifestations (AM). Our objective was to assess if consumption of a cow's milk-based beverage with these and other nutrients supports respiratory, gastrointestinal, and skin health in otherwise well-nourished, healthy children. METHODS: In this double-blind, randomized, controlled trial, healthy children (1-4 years of age) from two daycare centers in Brazil were fed three servings/day of a cow's milk-based beverage (CMBB; n = 125) containing DHA, the prebiotics polydextrose (PDX) and galactooligosaccharides (GOS), ß-glucan, and other key nutrients, or a control cow's milk-based beverage (control; n = 131) for up to 28 weeks. Occurrence of respiratory infections, diarrheal disease and AM was assessed by study pediatricians and the number of episodes were analyzed with the Cochran-Mantel-Haenszel test and the Andersen-Gill model. RESULTS: The CMBB group had fewer episodes of AM, which included allergic rhinitis or conjunctivitis, wheezing, allergic cough, eczema and urticaria, compared to the control group (p = 0.021). The hazard ratio for increased number of episodes of AM was lower in the CMBB group compared to control (HR, 0.64; 95 % CI 0.47-0.89; p = 0.007). There was no difference in the incidence of respiratory infections and diarrheal disease between groups. CONCLUSION: A cow's milk-based beverage containing DHA, PDX/GOS, and yeast ß-glucan, and supplemented with micronutrients, including zinc, vitamin A and iron, when consumed 3 times/day for 28 weeks by healthy 1- to 4-year-old children was associated with fewer episodes of allergic manifestations in the skin and the respiratory tract. TRIAL REGISTRATION: registration number: NCT01431469.

Oligosaccharide composition of breast milk influences survival of uninfected children born to HIV-infected mothers in Lusaka, Zambia.

BACKGROUND: Human milk oligosaccharides (HMOs) have multiple immunomodulatory functions that influence child health. OBJECTIVE: In this study we investigated whether HMO composition influences survival to 2 y of age in HIV-infected and HIV-exposed, uninfected (HEU) children during and after breastfeeding. METHODS: In the context of an early weaning trial in 958 HIV-infected women in Lusaka, Zambia, we conducted a nested case-cohort analysis of mortality to 2 y of age among 103 HIV-infected and 143 HEU children. Breast-milk samples collected at 1 mo postpartum were analyzed for HMO content. Samples were selected to include mothers of all HIV-infected children detected by 6 wk of age, of whom 63 died at <2 y of age; mothers of all HEU children who died at <2 y of age (n = 66); and a random sample of 77 HEU survivors. Associations before and after weaning in HIV-infected and HEU infants separately were investigated by using Cox models. RESULTS: Among HEU children, higher maternal breast-milk concentrations of 2-linked fucosylated HMOs [2'-fucosyllactose and lacto-N-fucopentaose (LNFP) I] (HR: 0.33; 95% CI: 0.14, 0.74) as well as non-2-linked fucosylated HMOs (3-fucosyllactose and LNFP II/III; HR: 0.28; 95% CI: 0.13, 0.67) were significantly associated with reduced mortality during, but not after, breastfeeding after adjustment for confounders. Breastfeeding was protective against mortality only in HEU children with high concentrations of fucosylated HMOs. Among HIV-infected children, no consistent associations between HMOs and mortality were observed, but breastfeeding was protective against mortality. CONCLUSIONS: The oligosaccharide composition of breast milk may explain some of the benefits of breastfeeding in HEU children. HIV infection may modulate some of the consequences of HMOs on child survival.

Influence of processing procedure on the quality of Radix Scrophulariae: a quantitative evaluation of the main compounds obtained by accelerated solvent extraction and high-performance liquid chromatography.

An improved high-performance liquid chromatography with diode array detection combined with accelerated solvent extraction method was used to simultaneously determine six compounds in crude and processed Radix Scrophulariae samples. Accelerated solvent extraction parameters such as extraction solvent, temperature, number of cycles, and analysis procedure were systematically optimized. The results indicated that compared with crude Radix Scrophulariae samples, the processed samples had lower contents of harpagide and harpagoside but higher contents of catalpol, acteoside, angoroside C, and cinnamic acid. The established method was sufficiently rapid and reliable for the global quality evaluation of crude and processed herbal medicines.

[Assessment Method of Remnant α-1, 3-galactosyle Epitopes in Animal Tissue-derived Biomaterials].

The aim of this study was to establish an assessment method for determining α-Gal (α-1, 3-galactosyle) epitopes contained in animal tissue or animal tissue-derived biological materials with ELISA inhibition assay. Firstly, a 96 well plate was coated with Gal α-1, 3-Gal/bovine serum albumin (BSA) as a solid phase antigen and meanwhile, the anti-α-Gal M86 was used to react with α-Gal antigens which contained in the test materials. Then, the residual antibodies (M86) in the supernatant of M86-Gal reaction mixture were measured using ELISA inhibition assay by the α-Gal coating plate. The inhibition curve of the ELISA inhibition assay, the R2 = 0.999, was well established. Checking using both α-Gal positive materials (rat liver tissues) and α-Gal negative materials (human placenta tissues) showed a good sensitivity and specificity. Based on the presently established method, the α-Gal expression profile of rat tissues, decellular animal tissue-derived biological materials and porcine dermal before and after decellular treatment were determined. The M86 ELISA inhibition assay method, which can quantitatively determine the α-Gal antigens contained in animal tissues or animal tissue-derived biomaterials, was refined. This M86 specific antibody based-ELISA inhibition assay established in the present study has good sensitivity and specificity, and could be a useful method for determining remnant α-1, 3Gal antigens in animal tissue-derived biomaterials.

Select human milk oligosaccharides directly modulate peripheral blood mononuclear cells isolated from 10-d-old pigs.

Infant formulas lack the complex mixture of oligosaccharides found in human milk. These human milk oligosaccharides (HMO) may be pivotal to the development of the neonatal immune system. Few comprehensive analyses of the effects of HMO on immune cells from neonates have been undertaken. Herein, the direct effects of HMO on immune cells were analysed ex vivo. Peripheral blood mononuclear cells (PBMC) isolated from 10-d-old sow-reared (SR) or colostrum-deprived formula-fed (FF) pigs were stimulated for 72 h with single HMO, mixtures of single HMO or a complex mixture of HMO isolated from human milk (iHMO). T-cell phenotype, cytokine production and proliferation were measured by flow cytometry, immunoassay and [³H]thymidine incorporation, respectively. Stimulation with HMO had direct effects on PBMC. For instance, cells stimulated with iHMO produced more IL-10 than unstimulated cells, and cells stimulated with fucosylated HMO tended to proliferate less than unstimulated cells. Additionally, co-stimulation with HMO mixtures or single HMO altered PBMC responses to phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) stimulation. Compared with PBMC stimulated with PHA alone, cells co-stimulated with iHMO and PHA proliferated more and had fewer detectable CD4⁺CD8⁺ T cells. Compared with PBMC stimulated by LPS alone, cells co-stimulated with a mixture of sialylated HMO and LPS proliferated more and tended to have fewer detectable CD4⁺ T cells. Differences in the baseline responses of PBMC isolated from the SR or FF pigs were observed. In summary, HMO directly affected PBMC populations and functions. Additionally, ex vivo measurements of PBMC phenotype, cytokine production and proliferation were influenced by the neonate's diet.

Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix: Application to 2'-fucosyllactose (2'-FL) in breast milk.

Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass-to-charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility-mass spectrometry (DI-IM-MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM2) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se- (non-secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2'-fucosyllactose (2'-FL) and lacto-N-fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI-IM-MS was also investigated. The standard addition method allowed the determination of the 2'-FL despite the presence of other oligosaccharides, including 3-fucosyllactose (3-FL) isomer in breast milk. This proof-of-concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures.

Maternal Diet Associated with Oligosaccharide Abundances in Human Milk from Latina Mothers.

Growing evidence indicates that human milk oligosaccharides (HMOs) are important bioactive compounds that enhance health and developmental outcomes in breastfed babies. Maternal dietary intake likely contributes to variation in HMO composition, but studies identifying diet-HMO relationships are few and inconsistent. This study aimed to investigate how the maternal intake of macronutrients and micronutrients-specifically proteins, fats, vitamins, and minerals-associated with HMOs at 1 month (n = 210), 6 months (n = 131), and 12 months postpartum (n = 84). Several associations between maternal dietary factors and HMO profiles were identified utilizing partial correlation analysis. For example, maternal free sugar (rho = -0.02, p < 0.01), added sugar (rho = -0.22, p < 0.01), and sugary sweetened beverage (rho = -0.22, p < 0.01) intake were negatively correlated with the most abundant HMO, 2'-fucosyllactose (2'-FL), at 1 month, suggesting that higher sugar consumption was associated with reduced levels of 2'-FL. Further, vitamins D, C, K, and the minerals zinc and potassium were positively correlated with 2'-FL at 1 month (pAll < 0.05). For the longitudinal analysis, a mixed-effects linear regression model revealed significant associations between maternal vitamin intake and HMO profiles over time. For example, for each unit increase in niacin intake, there was a 31.355 nmol/mL increase in 2'-FL concentration (p = 0.03). Overall, the results provide additional evidence supporting a role for maternal nutrition in shaping HMO profiles, which may inform future intervention strategies with the potential of improving infant growth and development through optimal HMO levels in mothers' milk.

The human milk metabolome reveals diverse oligosaccharide profiles.

Breast milk delivers nutrition and protection to the developing infant. There has been considerable research on the high-molecular-weight milk components; however, low-molecular-weight metabolites have received less attention. To determine the effect of maternal phenotype and diet on the human milk metabolome, milk collected at day 90 postpartum from 52 healthy women was analyzed by using proton nuclear magnetic resonance spectroscopy. Sixty-five milk metabolites were quantified (mono-, di-, and oligosaccharides; amino acids and derivatives; energy metabolites; fatty acids and associated metabolites; vitamins, nucleotides, and derivatives; and others). The biological variation, represented as the percentage CV of each metabolite, varied widely (4-120%), with several metabolites having low variation (<20%), including lactose, urea, glutamate, myo-inositol, and creatinine. Principal components analysis identified 2 clear groups of participants who were differentiable on the basis of milk oligosaccharide concentration and who were classified as secretors or nonsecretors of fucosyltransferase 2 (FUT2) gene products according to the concentration of 2'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose I. Exploration of the interrelations between the milk sugars by using Spearman rank correlations revealed significant positive and negative associations, including positive correlations between fucose and products of the FUT2 gene and negative correlations between fucose and products of the fucosyltransferase 3 (FUT3) gene. The total concentration of milk oligosaccharides was conserved among participants (%CV = 18%), suggesting tight regulation of total oligosaccharide production; however, concentrations of specific oligosaccharides varied widely between participants (%CV = 30.4-84.3%). The variability in certain milk metabolites suggests possible roles in infant or infant gut microbial development. This trial was registered at clinicaltrials.gov as NCT01817127.

Elimination of Residual Lacto-N-triose II for Lacto-N-tetraose Biosynthesis in Engineered Escherichia coli.

Lacto-N-tetraose (LNT) is an important neutral human milk oligosaccharide (HMO) and acts as a significant core structure for complex HMO biosynthesis. We previously achieved high-yield LNT biosynthesis (57.5 g/L) using fed-batch fermentation; however, residual lacto-N-triose II (LNTri II) was also found (21.58 g/L). Here, we re-engineered an efficient LNT-producing Escherichia coli with low LNTri II accumulation using genetically stable LNTri II-producing strains with a genomic insertion of lgtA (encoding ß1,3-N-acetylglucosaminyltransferase). Comparable and low titers of LNT (3.73-4.61 g/L) and LNTri II (0.33-0.63 g/L), respectively, were obtained by introducing ß1,3-galactosyltransferase. To reduce residual LNTri II, the E. coli transporter gene setA was disrupted, obviously reducing the accumulation of LNTri II and LNT. Next, the gene encoding ß-N-acetylhexosaminidase (BbhI) was introduced into LNT-producing strains or E. coli BL21(DE3) for single- or mixed-strain cultivation, respectively. Finally, LNT was obtained (30.13 g/L) in a cocultivation system of mixed engineered strains without undesired LNTri II.

2'-Fucosyllactose (2'-FL) changes infants gut microbiota composition and their metabolism in a host-free human colonic model.

BACKGROUND: Breast milk is critical for neonates, providing the necessary energy, nutrients, and bioactive compounds for growth and development. Research indicated that human milk oligosaccharides (HMOs) have been shown to shape a beneficial gut microbiota, as well as their metabolism (e.g. short-chain fatty acids). 2'-Fucosyllactose (2'-FL) is one major HMO that composed of 30% of total HMOs. OBJECTIVES: This study aimed to understand the impact of 2'-FL on the composition and metabolism of infant gut microbiota. METHODS: Our study utilized an in-vitro human colonic model (HCM) to investigate the host-free interactions between 2'-FL and infant gut microbiota. To simulate the infant gut microbiota, we inoculated the HCM system with eight representative bacterial species from infant gut microbiota. The effects of 2'-FL on the gut microbial composition and their metabolism were determined through real-time quantitative PCR and liquid-chromatography mass spectrometry (LC/MS). The obtained data were analyzed using Compound Discoverer 3.1 and MetaboAnalyst 4.0. RESULTS: Our study findings suggest that the intervention of 2'-FL in HCM resulted in a significant change in the abundance of representative bacterial species. PCR analysis showed a consistent increase in the abundance of Parabacteroides. distasonis in all three colon sections. Furthermore, analysis of free fatty acids revealed a significant increase in their levels in the ascending, transverse, and descending colons, except for caproic acid, which was significantly reduced to a non-detectable level. The identification of significant extracellular polar metabolites, such as glutathione and serotonin, enabled us to distinguish between the metabolomes before and after 2'-FL intervention. Moreover, correlation analysis revealed a significant association between the altered microbes and microbial metabolites. CONCLUSIONS: In summary, our study demonstrated the impact of 2'-FL intervention on the defined composition of infant gut microbiota and their metabolic pathways in an in vitro setting. Our findings provide valuable insights for future follow-up investigations into the role of 2'-FL in regulating the growth and development of infant gut microbiota in vivo.