Background/Objectives: Human milk (HM) meets the nutritional needs of term-born infants. However, it remains unclear whether HM macronutrient concentrations and intakes differ by infant sex. We investigated sex- and country-specific differences in HM macronutrient and energy concentrations among exclusively breastfeeding Danish and Australian mothers, and sex-specific differences in 24 h HM intake and intakes of HM macronutrients, energy, and energy ratio in Australian infants. Methods: In this cross-sectional, multicenter study, 77 Danish and 84 Australian mothers donated HM samples between 2.5 and 5.5 months postpartum. Mid-infrared spectroscopy analyzed macronutrient concentration in the Danish samples. The creamatocrit method analyzed fat and biochemical assays analyzed lactose and protein in the Australian samples. Milk intake was measured using the test-weighing method. We used linear mixed-effect models to investigate sex- and country-specific differences. Results: There were no sex-specific differences in macronutrient and energy concentrations within either Danish or Australian cohorts. In all 161 HM samples, we found no sex-specific differences in lactose and protein concentrations. The 24 h median protein intake in Australian infants was 7.82 g in males and 7.26 g in females, (p = 0.274). The protein:energy ratio intake was 0.06 and 0.07 in male and females, respectively (p = 0.154). We also found no significant sex-specific differences in 24 h HM intake or intakes of fat, lactose, or energy. Conclusions: These findings suggest that males and females have similar macronutrient requirements during infancy. However, this needs to be confirmed in larger studies measuring 24 h milk intake.

Background/Objectives: Late-onset neonatal sepsis (LOS) remains a leading cause of morbidity and mortality in very low birth weight (VLBW) infants (<1500 g and/or gestational age <32 weeks), with limited preventive strategies. We evaluated whether early enteral bovine lactoferrin (bLf), given its antimicrobial, immunomodulatory, and antioxidant properties, reduces LOS and improves immunologic, antioxidant, and hematologic markers in these infants. Methods: In this randomized, double-blind, placebo-controlled trial, 103 VLBW infants received bLf (150 mg/kg/day; n = 50) or the placebo (n = 53) within 72 h of birth for four weeks or until discharge. Outcomes included culture-confirmed LOS, mortality, and major morbidities. Risk ratios (RRs) were calculated, adjusting for gestational age, human milk intake, and ventilatory support when ≥25 events occurred. Pre/post changes in cytokines, total antioxidant capacity (TAC), and hemoglobin (Hb) were analyzed for interaction effects (time x intervention). Results: bLf reduced LOS (adjusted RR 0.54; 95% CI 0.31-0.93; p = 0.028), without differences in other morbidities or mortality. bLf preserved MCP-1 levels, declining in the placebo group (interaction p = 0.022). Among LOS infants receiving bLf, IL-6 remained stable and MCP-1 increased, while both declined in other groups (interaction p = 0.007 for IL-6; p = 0.052 for MCP-1). Although TAC showed a non-significant interaction, the placebo group declined (p = 0.002), while bLf remained stable (p = 0.400) in the post hoc analysis. In non-transfused infants, bLf increased Hb by 0.9 g/dL vs. controls (p = 0.028). Conclusions: Early bLf supplementation safely reduces LOS in VLBW infants and may support immunologic, antioxidant, and hematologic stability.

Breast Milk Intake from 1 to 8.5 Months of Lactation in the Multisite Mothers, Infants and Lactation Quality (MILQ) Study

Poor diets and micronutrient deficiencies during pregnancy and lactation are common in Indonesia, potentially affecting linear growth. National maternal programs focus predominantly on iron-folic acid (IFA) supplementation, but adherence is poor. We explored a strategy utilizing locally available micronutrient-rich foods in the form of micronutrient-enriched crackers (MEC) to improve neonatal and infant growth. The Sustainable Intervention of Supplementation to Improve Kid's (SISTIK) Growth Study was a double-blind, cluster randomized controlled trial conducted in 28 village clusters in Sumedang, Indonesia (Nov 2020-Jan 2023). Villages-unit of randomization-were stratified by size and assigned to receive either MEC or standard wheat crackers (SWC), along with nutrition and health education, morbidity control, and national IFA supplementation programs. Eligible pregnant women (19-35 years; 7-13 weeks gestation) were enrolled participated. The intervention lasted through five months postpartum. Individual mother-infant dyads were the unit of analysis. Primary outcomes were birth length (cm), attained length (cm), LAZ-score, and length velocity (cm/month) from birth to five months postpartum. Secondary outcomes included birth weight, morbidity, and maternal hemoglobin (Hb) (g/dL). A general linear model, including village as a random effect and intervention as a fixed effect was used. In each group 137 mother-infant dyads completed the study. Baseline characteristics were balanced. Intervention adherence was low (72-103 gr/week) compared to 525 gr/week (75 gr/day) recommended, with no significant intervention effect on growth outcomes, after adjusting for maternal height, sex, and human milk intake at 5 months. There were no differences between MEC and SWC groups in maternal Hb (mean ± SD) (12.0 ± 1.1 vs. 12.3 ± 1.2 g/dL) and anemia (11.9 vs 11.3%) at 35-36 weeks GA or morbidity. Compared with the same setting earlier at 5 mos pp, mean ± SD LAZ-score were less negative and stunting prevalence was lower in both groups. Overall, across the SISTIK study population, mean ± SD LAZ-score improved from -0.93 ± 0.85 to -0.51 ± 0.91, and stunting prevalence declined from 11 to 6%. Although this study did not show a treatment effect on the outcomes measured, future research should explore whether providing comprehensive nutrition and health support during pregnancy and post-partum could help reduce stunting in Indonesia. NCT04564222 (ClinicalTrials.gov); 25/09/2020.

The significance of human milk in an infant’s diet is well-established, yet accurately measuring human milk intake remains challenging. Current methods are either unsuitable for large-scale studies, such as the dose-to-mother stable isotope technique, or rely on set amounts of human milk, regardless of known variability in individual intake(1). There is a paucity of data on how much infants consume, particularly in later infancy (&gt;6 months) when complementary foods have been introduced. This research aimed to estimate human milk intakes and total infant milk intakes (including infant formula) in New Zealand infants aged 7-10 months, explore factors that predict these intakes, and develop and validate equations to predict human milk intake using simple measures. Human milk intake data were obtained using the dose-to-mother stable isotope technique in infants aged 7-10 months and their mothers as part of the First Foods New Zealand study (FFNZ)(2). Predictive equations were developed using questionnaire and anthropometric data (Model 1) and additional dietary data from diet recalls (Model 2)(3). The validity of existing methods to estimate human milk intake (NHANES and ALSPAC studies) was compared against the dose-to-mother results. FFNZ included 625 infants, with 157 mother-infant dyads providing complete data for determining human milk volume. Using the dose-to-mother data, the measured mean (SD) human milk intake was 785 (264) g/day. Older infants had lower human milk and total milk intakes, male infants consumed more total milk. The strongest predictors of human milk intake were infant age, infant body mass index, number of breastfeeds a day, infant formula consumption, and energy from complementary food intake. When the predictive equations were tested, mean (95% CI) differences in predicted versus measured human milk intake (mean, [SD]: 762 [257] mL/day) were 0.0 mL/day (-26, 26) for Model 1 and 0.5 mL/day (-21, 22) for Model 2. In contrast, the NHANES and ALSPAC methods underestimated intake by 197 mL/day (-233, -161) and 175 mL/day (-216, -134), respectively. The predictive equations are presented as the Human Milk Intake Level Calculations (HuMILC) tool, designed for use in large-scale studies to more accurately estimate human milk intakes of infants. The use of objective quantifiable assessment methods enhances our understanding of infant human milk intakes, improving our ability to accurately assess nutritional adequacy in infants.

Effect of kangaroo mother care in low birth weight infants on human milk intake: a randomized controlled trial.

Optimal infant growth is reliant on the sufficient intake of human milk. Studies in animal models speculate that multiparous mothers produce a higher milk yield compared to primiparous mothers. We aimed to examine if there are relationships between consecutive lactations and infant demographics and both maternal 24 h milk production (MP) and infant milk intake (MI). Lactating mothers 1-6 months postpartum (n = 36; 25 fully breastfeeding (FBF), 11 supplementing with commercial milk formula (partly breastfeeding (PBF)) test-weighed their infants for 24 h during two consecutive lactations and provided demographic information. Twenty-four-hour MP by breast, infant MI (including mothers' own expressed milk and formula), breastfeeding and expressing frequencies were measured. The statistical analysis used linear mixed modelling accounting for infant birth weight (FBF) or time postpartum (PBF) and for the random effect of the participant. In the FBF group, there were no differences between lactations in terms of MP (p = 0.31) or the infant mother's own MI (p = 0.14). The birth weight was higher for consecutive lactation infants (p = 0.008). Infant sex was not associated with MP (p = 0.12) or the infant mother's own MI (p = 0.090). In the PBF group, the breastfeeding frequency (p = 0.042), MP (p = 0.025) and infant mother's own MI (p = 0.019) were higher in consecutive lactations whilst formula intake was lower (p = 0.004). This study suggests that in fully breastfeeding women, there is no significant effect of consecutive lactation or infant sex on MP or infant MI during established lactation.

ABSTRACT Background The oral microbiome of breastfed infants is distinct from that of formula-fed infants. However, breastfeeding characteristics, such as time spent breastfeeding (min/24 h), breastfeeding frequency (number of breastfeeds per day), and human milk intake (ml/day) vary significantly between breastfeeding dyads. Objectives Given that human milk and breastfeeding exposures likely influence early colonisation of the infant oral microbiome, this study aimed to elucidate the impact of breastfeeding characteristics on the development of the infant oral microbiome. Materials and methods Oral swabs (n = 55) were collected from infants at three months of age, alongside breastfeeding data collected over a 24-hour period. Bacterial DNA profiles were analysed using full-length 16S rRNA gene sequencing. Results Variations in breastfeeding characteristics contributed to differences in microbial community structure. Total breastfeeding duration (min/24 h) was positively associated with Bifidobacterium longum and Lactobacillus gasseri, while breastfeeding frequency was negatively associated with Veillonella sp. Additionally, human milk intake (ml/24 h) was negatively associated with Streptococcus parasanguinis. Conclusion These findings underscore the significant influence of early life feeding practices on oral microbial communities and emphasise the importance role of breastfeeding in shaping the oral microbiome during early life.

Human milk (HM) is the optimal source of nutrition for infants. Yet the suitability of HM macronutrient composition, paired with the challenge of regulating HM intake, may deserve some consideration for infants with inherited metabolic disorders (IMDs) requiring restrictive and controlled dietary management. Except for classic galactosemia, HM feeding is expected to be feasible, allowing infants to maintain metabolic stability, while growing and developing optimally. However, information about HM feeding in nonphenylketonuria (PKU) literature is scarce. In this systematic review, 52 studies were included, representing 861 infants (86% PKU) receiving HM after IMD diagnosis (mean duration 4-10 months depending on the IMD). For non-PKU IMDs (e.g., other amino acidopathies, urea cycle disorders, organic acidemias, fatty acid oxidation disorders), outcomes of HM feeding were available for few infants, except for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (n = 48). In PKU, HM feeding combined with phenylalanine-free formula, led to adequate metabolic control (25 studies), growth (15 studies), and neurodevelopment (10 studies). For other IMDs, more evidence is required, but the limited data suggest that HM feeding is possible, with attentive monitoring and disease-specific formula supplementation where applicable. In MCAD deficiency, ensuring adequate HM intake is essential, as symptoms were more frequently reported in exclusively breastfed infants. No IMD-specific articles were found on the relationship between HM feeding and many other outcomes of interest (e.g., immune status or comorbidity risk later in life). With the exception of galactosemia, HM feeding is expected to benefit infants with IMD. More data should be published for IMDs other than PKU.

Human milk intake has many benefits which could influence outcomes in biliary atresia (BA). However, the role of human milk in BA has not been examined. We hypothesized that human milk intake would be associated with improved outcomes in BA. We assessed the impact of any human milk (AHM) as compared to formula only (FO) intake before Kasai portoenterostomy (KP) on outcomes in 447 infants with BA using the PROBE database (NCT00061828) post hoc. The primary outcome was clearance of jaundice (COJ = total bilirubin (TB) < 2 mg/dL by 3 months post-KP). Secondary outcomes included 2-year survival with native liver (SNL), bilirubin levels, cholangitis, ascites, and growth. We assessed the fecal microbiome (n = 8) comparing AHM versus FO. At baseline, 211 infants received AHM and 215 received FO. 53.9% of AHM and 50.5% of FO achieved COJ (p = NS). SNL was insignificantly increased in AHM (odds ratio = 1.47, 95% confidence interval: 1.00-2.12, p = 0.053). TB decreased in AHM from 4 weeks to 3 months post-KP [4.8-4.0 mg/dL (p = 0.01)] unlike the FO group (4.9-4.9 mg/dL, p = 0.4). At 3 months post-KP, AHM infants had greater weight gain (1.88 ± 0.66 vs. 1.57 ± 0.73 kg, p < 0.001) and mid-upper arm circumference (12.9 ± 1.4 vs. 12.2 ± 1.7 cm, p < 0.001). Other secondary outcomes were not different. Microbiome differences were seen between AHM and FO. Human milk intake in infants with BA did not significantly improve COJ or SNL. However, growth parameters were improved, and TB 3 months post-KP was decreased. Thus, human milk intake should not be discouraged. Prospective studies with detailed assessment of human milk intake are needed.

SummaryNecrotizing enterocolitis (NEC) is a severe intestinal disease of very preterm infants with mother’s own milk (MOM) providing protection, but the contribution of the MOM microbiota to NEC risk has not been explored. Here, we analyze MOM of 110 preterm infants (48 NEC, 62 control) in a cross-sectional study. Breast milk contains viable bacteria, but there is no significant difference in MOM microbiota between NEC and controls. Integrative analysis between MOM microbiota, human milk oligosaccharides (HMOs), and the infant gut microbiota shows positive correlations only between Acinetobacter in the infant gut and Acinetobacter and Staphylococcus in MOM. This study suggests that NEC protection from MOM is not modulated through the MOM microbiota. Thus, “‘restoring” the MOM microbiota in donor human milk is unlikely to reduce NEC, and emphasis should instead focus on increasing fresh maternal human milk intake and researching different therapies for NEC prevention.

Human milk (HM) composition, including metabolic hormones and lipids, is influenced by various factors, including lactation stage and, potentially, infant sex, which may affect infant body composition (BC) development. We aimed to: (a) characterize the longitudinal concentration and intake profiles of HM leptin, adiponectin, insulin, and total lipids; (b) determine if their concentrations and intakes differ by infant sex; and (c) explore the intakes relationships with the development of infant BC. Milk samples (n = 501) were collected from 82 mother-infant dyads during the first 6 months postpartum. Infant 24 h HM intake was measured, and the average cumulative HM component intakes were calculated. The statistical analysis used linear mixed modeling. Intakes of HM leptin, adiponectin, insulin, and total lipids increased to 1 month postpartum and then remained stable. HM intake and total lipids intake but not hormone intakes were positively associated with infant BC (fat-free mass, fat-free mass index, fat mass, fat mass index, percentage fat mass, and fat mass to fat-free mass ratio). HM component concentrations and intakes did not differ by sex. These findings advance our understanding of the temporal nature of HM components, emphasizing the role of infant 24 h HM and total lipids intake in development of infant lean and adipose tissue.

The World Health Organization (WHO) recommends exclusive breastfeeding for the first 6 months of life followed by complementary foods and sustained breastfeeding for at least 2 years, underscoring its pivotal role in reducing infant mortality and preventing various illnesses. This perspective delves into the intricate relationship between breastfeeding practices, early life antibiotic exposure, and infant gut microbiome development, highlighting their profound influence on child health outcomes. Antibiotics are extensively prescribed during pregnancy and childhood, disrupting the microbiome, and are related to increased risks of allergies, obesity, and neurodevelopmental disorders. Breastfeeding is a significant determinant of a healthier gut microbiome, characterized by higher levels of beneficial bacteria such as Bifidobacterium and lower levels of potential pathogens. Despite widespread recognition of the benefits of breastfeeding, gaps persist in healthcare practices and support mechanisms, exacerbating challenges faced by breastfeeding families. This highlights the pressing need for comprehensive research encompassing breastfeeding behaviors, human milk intake, and their impact on infant health outcomes. Additionally, promoting awareness among healthcare providers and families regarding the detrimental effects of unnecessary formula supplementation could facilitate informed decision-making and bolster exclusive breastfeeding rates. Moreover, donor human milk (DHM) is a promising alternative to formula, potentially mitigating disruptions to the infant gut microbiome after antibiotic exposure. Overall, prioritizing breastfeeding support interventions and bridging research gaps are essential steps towards improving child health outcomes on a global scale.

Processing speed is suboptimal among preterm-born children which is of concern as it is a foundational skill supporting higher-level cognitive functions. The study objective was to evaluate associations between early-life nutrition and processing speed in childhood. Macronutrient and human milk (mother's own, donor) intakes from 137 children born preterm with very low birth weight enrolled in a nutrition feeding trial were included. Processing speed was evaluated at age 5 using the Wechsler Preschool and Primary Scale of Intelligence-fourth edition Processing Speed Index. Associations between early-life nutrition and processing speed were explored through linear regression. Children had a mean (standard deviation [SD]) birth gestational age of 28.1 (2.5) weeks, weight of 1036 (260) g and 52% were male. The mean (SD) assessment age was 5.7 (0.2) years. Sex-dependent relationships were identified between first postnatal month protein, lipid and energy intakes and processing speed at5 years. For females, lower protein (per 0.1 g/kg/d: -0.88, 95% confidence interval [CI]: -1.53, -0.23; p = 0.01) and energy (per 10 kcal/kg/d: -2.38, 95% CI: -4.70, -0.05; p = 0.03) intakes were related to higher processing speed scores. Mother's milk provision was positively associated (per 10% increase: 0.80, 95% CI: 0.22, 1.37; p = 0.01) and donor milk was negatively associated (per 10% increase: -1.15, 95% CI: -2.22, -0.08; p = 0.04) with processing speed scores; no sex differences were observed. First postnatal month nutrition was related to processing speed at age 5 in children born preterm with very low birth weight. Early-life nutrition that supports processing speed may be leveraged to improve later cognitive outcomes for this vulnerable population.

Background: Exclusive breastfeeding (EBF) of infants during the first 6 months of their life is strongly recommended. Video‐based communication is an innovative method that could promote EBF. The present study is aimed at assessing the effectiveness of video‐based intervention in promoting breastfeeding practices from birth to 6 months postpartum. This was evaluated through maternal reports, as well as EBF of infants at 3 and 5 months of age, using the deuterium oxide dose‐to‐mother (DTM) technique.Methods: In a cluster randomized community trial, 16 communities were assigned to receive a video‐based behavior change communication (Video‐Health) or standard health care messages (Control). Pregnant women in their first trimester (12 ± 2 weeks) were enrolled and followed up, together with their infants, until 6 months postpartum. Data on breastfeeding practices were collected by questionnaire, monthly, in all the participants (n = 508). Human milk intake (HMI) and nonmilk oral intake (NMOI) were measured in a subsample of 60 mother–infant pairs at 3 and 5 months postpartum using DTM. Mixed models and logistic regression were used to examine the difference in continuous and discrete breastmilk practices between the intervention and the control arms, respectively.Results: The majority of mothers initiated breastfeeding early (92.1%), 82.4% reported colostrum feeding, and 17.5% provided prelacteal feeding. No significant differences were found between Video‐Health and Control arms (p &gt; 0.05). The intervention significantly improved reported EBF rates at 4 and 5 months postpartum (p &lt; 0.05). DTM results showed that the proportion of women practicing EBF was 50%–67% less than reported at 3 months for both arms and at 5 months in the intervention arm. The intervention did not significantly affect measured EBF at 3 and 5 months postpartum but did improve HMI at 3 months. Non significant differences in NMOI were observed at 3 months, but at 5 months, there was a significant difference between the study arms.Conclusions: Video‐based behavior change communication did not result in significant improvements in reported breastfeeding practices, except for a higher reported adherence to EBF beyond 3 months. Additionally, the intervention had no effect on EBF as measured by the DTM. Early introduction of non‐breastmilk foods and liquids persisted, despite self‐reported EBF extending up to 6 months postpartum.Trial Registration: ClinicalTrials.gov identifier: NCT04414527

The deuterium dose-to-mother (DTM) method measures the human milk intake of breastfed children. Recently, the use of this method has been expanded to classify babies objectively as exclusively breast fed (EBF) or not (non-EBF) based on quantification of non-milk oral water intake (NMOI). However, the calculation of NMOI estimates involves atmospheric temperature and humidity. To evaluate the effects of atmospheric temperature and humidity on NMOI calculation and the classification of exclusive breastfeeding. The effect of indoor temperature and relative humidity on NMOI and the estimated prevalence of non-EBF were examined in two existing data sets of DTM in children by varying temperature in the range of 15 to 35°C and relative humidity in the range of 20 to 80% representing the maximum span of indoor conditions expected. Population-level estimates of NMOI distributions were derived using the rstan package for R v2.21.2. The NMOI decreased at a rate of -1.15 g/day per °C increase and at a rate of -1.01 g/day per percent increase in relative humidity; this was due to variations in non-oral water intake from the atmosphere, a component of the calculation of NMOI, which is dependent on temperature and humidity. For the various locations considered, the mean calculated NMOI varied between 24.6 and 53.3 g/day using the same input data. In the mixed-fed sample of babies, the prevalence of non-EBF based on the earlier defined NMOI cut-off of 86.6 g/day was reduced by 19% when relative humidity was increased by 60%. Atmospheric conditions are essential factors in the computation of NMOI, used in the objective classification of babies as exclusively breast fed or not, and should be considered when the DTM method is used to classify exclusive breastfeeding.

This study aimed to assess the agreement in EBF between maternal recall and the dose-to-mother (DTM) technique. Indonesia, Malaysia, Mongolia, Pakistan, Sri Lanka, Thailand, and Vietnam participated in the study. A total of 207 and 118 mother-infant pairs were assessed at 3 and 6 months of child's age. Using a standardized questionnaire, mothers were asked to recall child feeding during the previous 24 h, at 3 and 6 months. Those recalled to be EBF proceeded to be assessed using DTM technique. Non-milk oral intake (NMOI) cutoff of 86.6 g/d was used to classify EBF. According to DTM, 66% of infants were EBF at 3 months, while only 22% were EBF at 6 months. At 3 months, the overall % agreement between maternal recall and DTM method was 68%, kappa 0.06 (95% CI: 0.07-0.20), and at 6 months, the % agreement was only 21%, kappa -0.031 (95% CI -0.168 to 0.107). Human milk intakes were similar at 3 months and 6 months when expressed as g/d, but decreased when expressed as g/kg/d, with a large variation within and between countries; Pakistan being the lowest. This study showed there were declining levels of EBF from 3 to 6 months in the participating countries from Asia and the agreement between maternal recall and DTM technique to classify EBF was low. To ensure that the DTM technique can be more widely used in evaluating breastfeeding promotion programs, consensus on the appropriate NMOI cutoff and simplification of the DTM protocol is necessary.

There is an inadequate understanding of the daily variations in hormones and macronutrients in human milk (HM), and sample collection protocols vary considerably from study to study. To investigate changes in these milk components across 24 h, 22 lactating women collected small milk samples before and after each breastfeed or expression from each breast. Test weighing was used to determine the volume of HM consumed in each feed. The concentrations of leptin, adiponectin, insulin, fat, and glucose were measured, and the intakes were calculated. A linear mixed model was fitted to assess within-feed and circadian variation in HM feed volume and concentration, and intakes of several components. The average infant intake of HM was 879 g/24 h. Significantly higher pre-feed concentrations were found for adiponectin and glucose and lower post-feed concentrations were found for insulin and fat. Significant circadian rhythms were displayed for leptin, adiponectin, insulin, glucose (both concentration and intake), fat concentration, and milk volume. These findings demonstrate the necessity for setting up standardised and rigorous sampling procedures that consider both within-feed and circadian variations in HM components to gain a more precise understanding of the impacts of these components on infant health, growth and development.

Human milk (HM) offers important nutritional benefits. However, except for phenylketonuria (PKU), there are little data on optimal levels of consumption of HM and a special formula free of disease-related amino acids (SF-AA) in infants with inborn errors of metabolism of amino acids and proteins (IEM-AA-P). We designed a spreadsheet to calculate the amounts of SF-AA and HM required to cover amino acid, protein, and energy needs in patients with the nine main IEM-AA-P in infants aged under 6 months. Upon entering the infant's weight and the essential amino acid or intact protein requirements for the specific IEM, the spreadsheet calculates the corresponding required volume of HM based on the amino acid concentration in HM. Next, the theoretical daily fluid intake (typical range, 120-200 mL/kg/day) is entered, and the estimated daily fluid intake is calculated. The required daily volume of SF-AA is calculated as the difference between the total fluid intake value and the calculated volume of HM. The spreadsheet allows for the introduction of a range of requirements based on the patient's metabolic status, and includes the option to calculate the required volume of expressed HM, which may be necessary in certain conditions such as MMA/PA and UCD. In cases in which breastfeeding on demand is feasible, the spreadsheet determines the daily amount of SF-AA divided over 6-8 feeds, assuming that SF-AA is administered first, followed by HM as needed. Intake data calculated by the spreadsheet should be evaluated in conjunction with data from clinical and nutritional analyses, which provide a comprehensive understanding of the patient's nutritional status and help guide individualized dietary management for the specific IEM.

BackgroundThe combination of cultivation studies with molecular analysis approaches allows characterization of the complex human gut microbiota in depth. In vitro cultivation studies of infants living in rural sub-Saharan Africa are scarce. In this study, a batch cultivation protocol for Kenyan infant fecal microbiota was validated.MethodsFresh fecal samples were collected from 10 infants living in a rural area of Kenya. Samples were transported under protective conditions and subsequently prepared for inoculation within less than 30 h for batch cultivation. A diet-adapted cultivation medium was used that mimicked the daily intake of human milk and maize porridge in Kenyan infants during weaning. 16 S rRNA gene amplicon sequencing and HPLC analyses were performed to assess the composition and metabolic activity, respectively, of the fecal microbiota after 24 h of batch cultivation.ResultsHigh abundance of Bifidobacterium (53.4 ± 11.1%) and high proportions of acetate (56 ± 11% of total metabolites) and lactate (24 ± 22% of total metabolites) were detected in the Kenyan infant fecal microbiota. After cultivation started at an initial pH 7.6, the fraction of top bacterial genera (≥ 1% abundant) shared between fermentation and fecal samples was high at 97 ± 5%. However, Escherichia-Shigella, Clostridium sensu stricto 1, Bacteroides and Enterococcus were enriched concomitant with decreased Bifidobacterium abundance. Decreasing the initial pH to 6.9 lead to higher abundance of Bifidobacterium after incubation and increased the compositional similarity of fermentation and fecal samples. Despite similar total metabolite production of all fecal microbiota after cultivation, inter-individual differences in metabolite profiles were apparent.ConclusionsProtected transport and batch cultivation in host and diet adapted conditions allowed regrowth of the top abundant genera and reproduction of the metabolic activity of fresh Kenyan infant fecal microbiota. The validated batch cultivation protocol can be used to study the composition and functional potential of Kenyan infant fecal microbiota in vitro.