The impact of freeze-drying infant fecal samples on measures of their bacterial community profiles and milk-derived oligosaccharide content.

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

Growth and Morbidity of Gambian Infants are Influenced by Maternal Milk Oligosaccharides and Infant Gut Microbiota.

Human milk oligosaccharides (HMOs) play an important role in the health of an infant as substrate for beneficial gut bacteria. Little is known about the effects of HMO composition and its changes on the morbidity and growth outcomes of infants living in areas with high infection rates. Mother's HMO composition and infant gut microbiota from 33 Gambian mother/infant pairs at 4, 16, and 20 weeks postpartum were analyzed for relationships between HMOs, microbiota, and infant morbidity and growth. The data indicate that lacto-N-fucopentaose I was associated with decreased infant morbidity, and 3'-sialyllactose was found to be a good indicator of infant weight-for-age. Because HMOs, gut microbiota, and infant health are interrelated, the relationship between infant health and their microbiome were analyzed. While bifidobacteria were the dominant genus in the infant gut overall, Dialister and Prevotella were negatively correlated with morbidity, and Bacteroides was increased in infants with abnormal calprotectin. Mothers nursing in the wet season (July to October) produced significantly less oligosaccharides compared to those nursing in the dry season (November to June). These results suggest that specific types and structures of HMOs are sensitive to environmental conditions, protective of morbidity, predictive of growth, and correlated with specific microbiota

Lipid-Based Nutrient Supplements During Pregnancy and Lactation Did Not Affect Human Milk Oligosaccharides and Bioactive Proteins in a Randomized Trial.

BACKGROUND: Human milk oligosaccharides (HMOs) and bioactive proteins are beneficial to infant health. Recent evidence suggests that maternal nutrition may affect the amount of HMOs and proteins in breast milk; however, the effect of nutrient supplementation on HMOs and bioactive proteins has not yet been well studied. OBJECTIVE: We aimed to determine whether lipid-based nutrient supplements (LNSs) affect milk bioactive protein and HMO concentrations at 6 mo postpartum in women in rural Malawi. These are secondary outcomes of a previously published randomized controlled trial. METHODS: Women were randomly assigned to consume either an iron and folic acid capsule (IFA) daily from ≤20 wk gestation until delivery, followed by placebo daily from delivery to 6 mo postpartum, or a multiple micronutrient (MMN) capsule or LNS daily from ≤20 wk gestation to 6 mo postpartum. Breast milk concentrations of total HMOs, sialylated HMOs, fucosylated HMOs, lactoferrin, lactalbumin, lysozymes, antitrypsin, immunoglobulin A, and osteopontin were analyzed at 6 mo postpartum (n = 647). Between-group differences in concentrations and in proportions of women classified as having low concentrations were tested. RESULTS: HMO and bioactive protein concentrations did not differ between groups (P > 0.10 for all comparisons). At 6 mo postpartum, the proportions of women with low HMOs or bioactive proteins were not different between groups except for osteopontin. A lower proportion of women in the IFA group had low osteopontin compared with the LNS group after adjusting for covariates (OR: 0.5; 95% CI: 0.3, 0.9; P = 0.016). CONCLUSION: The study findings do not support the hypothesis that supplementation with an LNS or MMN capsule during pregnancy and postpartum would increase HMO or bioactive milk proteins at 6 mo postpartum among Malawian women. This trial was registered at clinicaltrials.gov as NCT01239693

Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation

BackgroundThe quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards.ObjectiveThe 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.MethodsThis 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.ResultsA 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).ConclusionsHMOs 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

Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation.

BackgroundThe quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards.ObjectiveThe 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.MethodsThis 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.ResultsA 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).ConclusionsHMOs 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

The impact of freeze-drying infant fecal samples on measures of their bacterial community profiles and milk-derived oligosaccharide content.

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

Digestion of Human Milk Oligosaccharides by Bifidobacterium breve in the Premature Infant.

ObjectiveThe aim of this study was to measure consumption and absorption of human milk oligosaccharides (HMOs) in a cohort of premature infants treated with probiotic Bifidobacterium breve.MethodsTwenty-nine premature infants (median gestational age 28 weeks, range 23-32 weeks) cared for in the neonatal intensive care unit of the King Edward and Princess Margaret Hospital in Perth, Australia, were treated with B breve at a dose of 1.66 billion organisms per day. Samples of feces, urine, and milk were obtained at initiation of the probiotic and again 3 weeks later. 16S ribosomal RNA from the feces was analyzed by next-generation sequencing. Quantitation of HMO content of the milk, urine, and feces was performed using nano-high-performance liquid chromatography-chip/time-of-flight mass spectrometry.ResultsThere was heterogeneity in colonization with bifidobacteria. "Responders" received milk with higher percentages of fucosylated HMOs and had higher percentages of bifidobacteria and lower percentages of Enterobacteriaceae in their feces than "nonresponders." Several individual HMOs in the milk were associated with changes in fecal bifidobacteria over time. Changes over time in milk, fecal, and urine HMOs suggested heterogeneity among HMO structures in consumption by microbes in the gut lumen and absorption from the intestine.ConclusionsColonization of the premature infant intestinal tract with probiotic B breve is influenced by prebiotic HMOs. B breve is a selective consumer of HMOs in the premature infant

Growth and Morbidity of Gambian Infants are Influenced by Maternal Milk Oligosaccharides and Infant Gut Microbiota.

Human milk oligosaccharides (HMOs) play an important role in the health of an infant as substrate for beneficial gut bacteria. Little is known about the effects of HMO composition and its changes on the morbidity and growth outcomes of infants living in areas with high infection rates. Mother's HMO composition and infant gut microbiota from 33 Gambian mother/infant pairs at 4, 16, and 20 weeks postpartum were analyzed for relationships between HMOs, microbiota, and infant morbidity and growth. The data indicate that lacto-N-fucopentaose I was associated with decreased infant morbidity, and 3'-sialyllactose was found to be a good indicator of infant weight-for-age. Because HMOs, gut microbiota, and infant health are interrelated, the relationship between infant health and their microbiome were analyzed. While bifidobacteria were the dominant genus in the infant gut overall, Dialister and Prevotella were negatively correlated with morbidity, and Bacteroides was increased in infants with abnormal calprotectin. Mothers nursing in the wet season (July to October) produced significantly less oligosaccharides compared to those nursing in the dry season (November to June). These results suggest that specific types and structures of HMOs are sensitive to environmental conditions, protective of morbidity, predictive of growth, and correlated with specific microbiota