THE EFFECT OF INCORPORATING NUTRIENT-DENSE NATIVE AFRICAN PLANT MATERIALS ON THE BIOACCESSIBILITY OF PROVITAMIN A CAROTENOIDS FROM COMPOSITE CEREAL-BASED FOOD PRODUCTS

Vitamin A deficiency is the leading cause of childhood blindness affecting over 190 million preschool children around the world where the highest rates are found in Sub-Saharan Africa (1). The coexistence of this deficiency with shortfalls in iron and zinc has resulted in a shift in intervention strategies from single targeted approach to broader diet diversification. As a result, food-based strategies leveraging local nutrient-dense plants as natural fortificants have gained significant interest for their potential to simultaneously address multiple micronutrient, and in some instances macronutrient, deficiencies. However, the efficacy of such approach depends upon several factors including knowledge on the nutritional composition of native plant materials as well as strategies for their incorporation into staple consumer products. Additionally, there is lack of information on impact of concurrent introduction of mineral and provitamin A rich plants on the stability and bioavailability of each individual nutrients including changes in these factors over extended periods of exposure. This is a key point considering that many of these materials are reported to have potential inhibitors of carotenoid absorption (minerals, fiber and phenolics).

To address these research gaps, this dissertation focuses on three areas including 1) micronutrient, phytochemical and polysaccharide characterization of three commercially available native micronutrient dense African plant materials [Adansonia digitata (baobab), Moringa Oleifera (moringa) and Hibiscus Sabdariffa (hibiscus)] that have been targeted for use as natural iron fortificants; 2) determination of the impact of these materials on the bioaccessibility and intestinal uptake of provitamin A carotenoids from model composite cereal products and 3) assess the effect of longer term exposure to baobab and moringa on provitamin A carotenoid absorption and cellular differentiation biomarkers of human intestinal Caco-2 cells to better understand the potential impacts of extended exposure periods on long term micronutrient uptake.

Characterization of the plant fortificants focused on understanding both nutritive components and potential limiters of carotenoid bioavailability. Baobab, moringa and hibiscus all were found to contain key phytochemical and polysaccharide components that could be leveraged as nutritional and function ingredients. The relatively higher levels of lutein (57  4.6 g/g), zeaxanthin (11  0.1g/g) and -carotene (20  2 g/g) in moringa leaf powder support the notion that this plant material can be used as a source of provitamin A and non-provitamin A carotenoids. Phenolic analysis revealed the presence of substantial amounts of flavan-3-ols (1234  16 mg/100g) in baobab, anthocyanins (2001  56 mg/100g) in hibiscus, and flavonols (5352  139 mg/100g) in moringa leaf powder. Polysaccharide analysis demonstrated that the primary monosaccharide in baobab was found to be xyloglucan (47 %) which is in agreement with the tentative identification Xyloglucans (hemicellulosic polysaccharide) based on linkage analysis. Hibiscus was found to contain similar amounts of xylose (20%) and galactose (27%) supporting the presence of similar proportions of xyloglucans and pectic polysaccharides (type I, type II AG, RG I). The main monosaccharide in moringa was found to be galactose (36%) followed by glucose (23%) and linkage analysis revealed the presence of high proportions of pectic polysaccharides (type I, type II AG, RG I). These results provide insight into presence of potential enhancer or inhibitors of target micronutrient (provitamin A carotenoids or iron/zinc) bioavailability when used as functional and nutritional food ingredients.

Subsequently, the impact of mineral-rich baobab formulated at levels relevant for iron fortification on the bioaccessibility of provitamin A carotenoids (proVAC) from composite millet porridges containing dried carrot and mango was assessed using in vitro digestion. Proportions of millet flour and plant materials were dry blended to deliver ~25% of the RDA for vitamin A(VA) and iron(Fe) as follows: decorticated extruded millet (Senegalese Souna var.) (40-60%), dried proVA rich carrot and mango blend (30%), and dried Fe and ascorbic acid rich Adansonia digitata (baobab) (0-25%). While there were no significant differences in proVAC bioaccessibility from porridge formulations with 5 and 15% baobab (18.8+/-2.0 and 18.8±2.0% respectively) as compared to control containing no mineral-rich plant (23.8 +/- 1.2%), 25% baobab resulted in a significant decrease (p<0.05) in bioaccessibility of proVAC (13.3+/-1.6%). However, baobab inclusion did not impact intestinal uptake efficiency of provitamin A carotenoids by Caco-2 human intestinal cells

(3.3-3.6% -carotene and 3.7-4.5% for -carotene) across all formulation. These results suggest that any potential negative effects of baobab inclusion may be limited to food matrix interactions and digestion. This was confirmed in separate experiments that with experiments on baobab and carotenoid blends showing that digested baobab did not affect carotenoid absorption by Caco-2 cells. Overall these data support the notion that that modest inhibition of carotenoid bioaccessibility by baobab may not significantly limit carotenoid delivery from composite porridges. Furthermore, bioaccessible provitamin A content of a serving (200 g) of composite porridges can provide 27 - 48% of the RDA of vitamin A for children 1-3 years of age.

Finally, we evaluated the impact of long-term exposure to baobab and moringa digesta on Caco-2 cell differentiation biomarkers and provitamin A uptake to gain insight into how inclusion of these materials in to a daily diet may alter absorption and transport of nutrients or otherwise have potential negative effects on the intestine. Based on NMR analysis of intracellular metabolites in differentiating Caco-2 monolayers, significant alterations in specific osmotic pressure regulators, particularly glycerophosphocholine, taurine and myo-inositol were observed with repeated exposure to all treatment groups including the control (digested 0.9% saline solution). Changes in these metabolites levels have been linked with specific cellular function including protection against hyperosmotic stress and regulation of paracellular permeability of Caco-2 cells. Evaluation of carotenoid uptake comparing acute and acute on repeated exposure to treatment groups demonstrated that there was an overall significant reduction in carotenoid uptake with repeated exposure across all treatment groups including the control. Despite the reduction in carotenoid uptake, mRNA and protein levels of carotenoid transporters (CD-36, SR-B1 and FABP1) were not significantly altered with exposure through differentiation (except for SR-B1 protein levels). Decrease in SR-B1 levels may be due to bile acid accumulation from the digesta matrix which is known to regulate its own biosynthesis by a mechanism that involves the down-regulation SR-B1 expression to protect cells from cytotoxicity. Our results provide some insight into the impact of simulated gastrointestinal fluids alone on provitamin A uptake in this model system which are usually not taken into consideration in most Caco-2 cell studies. However, overall, these findings indicate that the introduction of baobab and moringa at levels relevant for delivery of meaningful levels of iron (15-23% RDA) should not have negative impacts on human intestinal function or carotenoid uptake over chronic use.

Taken together, our findings indicate that the three native Africa plant materials selected for investigation in these studies can be important sources of key micronutrients (iron, zinc and provitamin A carotenoids) and have potential as natural fortificants with application in staple foods such as cereal porridges. Incorporation of these plant materials, do not appear to negatively affect carotenoid bioavailability although there is a potential for their interaction during micellarization of carotenoids during normal digestion. While in vivo studies evaluating the bioavailability of provitamin A carotenoids from such composite formulations are required, these data support the further exploration of such natural fortification strategies in addressing micronutrient deficiencies in local African communities.