The aim of this study is to measure the effect of a prebiotic (high dose/low dose) mixture at different doses within a wheat-based instant cereal, on fractional iron absorption (FIA), gut microbiota and inflammation after three weeks. FIA will be compared with and without three weeks of pre-feeding with two different doses of the prebiotic mixture.

Iron deficiency anemia (IDA) is prevalent in infants and preschool children in sub-Saharan Africa. Several studies have been conducted to mitigate IDA. Some of these studies had adverse effects on the gut microbiome and diarrhea due to the high iron dose administered in areas of high burden of malaria and other common childhood infections. As a potentially safer solution, we investigated the effect of lowering the iron dose in the iron supplements and including 7.5 g galacto-oligosaccharides (GOS). Findings from these studies demonstrated that in infants, consumption of iron and GOS improved iron status, improved the gut microbiota and reduced risk of illness. We recently demonstrated that habitual consumption of GOS for three weeks resulted in improved iron absorption that correlated with a decrease in colonic pH. It is not known whether other prebiotics or a combination will also result in increased iron absorption and what the effect of using half the dose of prebiotics would be. The composition of Human Milk Oligosaccharides (HMOs) in breast milk might be an important determinant of the gut microbiome composition and health of the breastfed infant by determining the composition of Bifidobacteria species that are the main commensal 'barrier' bacteria in the infant's gut microbiome. However, little is known about the specific HMOs composition of breast milk in African populations and the potential impact on the gut microbiota composition of the breastfed infants. We therefore aim to measure the effect of a prebiotic (high dose/low dose) mixture at different doses within a wheat-based instant cereal, on fractional iron absorption (FIA) from a wheat-based instant cereal containing no prebiotic. We will compare the FIA with and without three weeks of pre-feeding with two different doses of prebiotic mixture. In addition, we will investigate the effect of two different doses of this prebiotic (high dose/low dose) mixture on gut microbiota and inflammation after three weeks.

In south coast Kenya, we will enroll 195 infants and randomize them into three intervention groups. Daily for 3 weeks, infants will consume a newly formulated wheat-based instant cereal (3.6 mg iron with Ascorbic acid (AA)) at home. Group 1 will receive the cereal with 7.5 g prebiotic mixture; group 2 will receive the cereal with 3 g prebiotic mixture and group 3 will receive the cereal with no prebiotics. In a subset of 70 infants, we will administer four labelled test meals. Two test meals will be fed 2 weeks before beginning the intervention study to investigate the acute effect of prebiotics on FIA and the other two will be fed at the end of the 3 weeks intervention study to investigate the effect of chronic consumption of prebiotics on FIA. Breast milk samples from the mothers will be collected and analyzed for the composition of human milk oligosaccharides (HMOs). We hypothesize that: 1) Infants receiving the new wheat-based instant cereal containing prebiotic (high dose/low dose) mixture will have a higher iron absorption, and 2) higher fecal Bifidobacteria abundance than infants receiving the cereal without prebiotics; 3) in infants receiving the wheat-based instant cereal, the addition of 7.5 g of prebiotic (high dose/low dose) mixture will result in higher FIA, and 4) in greater ratio of Bifidobacteria to Enterobacteriaceae, lowest fecal calprotectin, intestinal fatty acid-binding protein (I-FABP) and lowest abundances of enteropathogens than the addition of 3 g of prebiotics; 5) the addition of 7.5 g of prebiotic (high dose/low dose) mixture will result in greater FIA when administered acutely or chronically and these two effects will be additive; 6) the wheat-based instant cereal will improve iron status, including hemoglobin (Hb), plasma ferritin (PF) and soluble transferrin receptor (sTfR), and inflammation status, including C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP), fecal calprotectin and I-FABP; 7) the wheat-based instant cereal will decrease fecal pH, thereby providing a less favorable growth environment for enteric pathogens; 8) Maternal secretor status will affect the infant gut microbiota, with infants of secretor mothers having higher abundances of Bifidobacterium and Bacteroides but lower abundances of enteropathogens; and 9) Effects of co-provision of prebiotic mixture on the infant gut microbiota and on iron absorption, will be stronger among infants of non-secretor mothers.

This study will generate crucial data for optimizing iron and prebiotic-composition of wheat-based instant cereal designed for complementary feeding during infancy. This cereal could potentially be part of continued research on interventions to reduce the burden of IDA using safer formulations not adversely affecting the infant's gut microbiome.

Inclusion Criteria:

• 6 to 11 months at baseline
• Anticipated residence in study area for the study duration
• In good health as assessed by professional staff at Msambweni County Referral hospital
• Caregiver is willing to participate and able to comply with study procedures
• Informed consent form has been read and signed by the caregiver (or has been read out to the caregiver)

Exclusion Criteria:

• Hemoglobin concentration less than 70 g/L
• Severe underweight (Z-score weight-for-age <-3)
• Severe wasting (Z-score weight-for-height <-3
• Chronic or acute illness or other conditions that in the opinion of the Principal Investigator (PI) or study pediatrician would jeopardize the safety or rights of a participant in the trial or would render the participant unable to comply with the protocol
• Participant taking part in other studies requiring the drawing of blood
• Regular intake (>2 days) of iron-containing mineral and vitamin supplements or fortified foods within the last 2 months
• Antibiotic treatment in the past 4 weeks prior to study enrollment