Assessment of Iron Bioavailability in Whole Wheat Bread by Addition of Phytase-Producing Bifidobacteria Juan Mario Sanz-Penella, † Jose ́ Moise ́ s Laparra, ‡ Yolanda Sanz, ‡ and Monika Haros* ,† † Cereal Group and ‡ Microbial Ecophysiology and Nutrition Group, Institute of Agrochemistry and Food Technology (IATA-CSIC), Avenida Agustín Escardino 7, Parque Científico, 46980 Paterna-Valencia, Spain ABSTRACT: In this study, the influence of phytase-producing Bifidobacterium strains during the breadmaking process (direct or indirect) on final bread Fe dialyzability and ferritin formation in Caco-2 cell as a measure of cell Fe uptake was assessed. The addition of bifidobacteria significantly reduced the InsP 6 + InsP 5 concentrations compared to control samples. Fe-dialyzable contents for samples with bifidobacteria were increased 2.3-5.6-fold, and dialyzability was improved by 2.6-8.6% compared to controls. However, this was not reflected in an increase of Fe uptake by Caco-2 cells as was predicted by the phytate/Fe molar ratios. The results demonstrated the usefulness of phytase-producing bifidobacteria to reduce phytate during the breadmaking process and to increase Fe accessibility, although the effects appeared to be still insufficient to improve Fe bioavailability in Caco- 2 cells. Further refinement of the use of phytase-producing bifidobacterial strains and/or breadmaking technological processes is deserved for improving Fe uptake. KEYWORDS: whole wheat bread, sourdough, Bifidobacterium, phytate-degrading enzyme, Fe dialyzability, Fe uptake, Caco-2 cells ■ INTRODUCTION For many years, public health concerns have focused on Fe deficiency because this is a health problem in most countries of the world. Fe deficiency results in the depletion of body Fe stores and is believed to affect 20-50% of the world’s population. This problem is even more pronounced in populations consuming monotonous plant-based diets with little meat, where most of the dietary Fe is in nonheme form. Nonheme Fe is found mainly in plant foods such as cereals, legumes, fruits, and vegetables, and its absorption is often <10%. 1 In addition, the absorption of this kind of Fe can be affected by many dietary components that are able to act as enhancers or inhibitors. 1 Bread is a staple food in many countries and is therefore of global importance in international nutrition. Nevertheless, the presence of phytic acid or phytate (myo-inositol hexaphosphate, InsP 6 ) in whole grains, which have strong chelating properties, interferes with mineral absorption by forming insoluble complexes with nutritionally important minerals such as Fe, Zn, and Ca. 2 Food fortification, such as fortified cereal flours, is the most practical and best long-term strategy to prevent Fe deficiency. However, Fe addition often causes unacceptable sensory changes in food vehicles and has low bioavailability. 3 Many studies have indicated that hydrolysis of phytate is a way to overcome its negative effect on mineral absorption, 4,5 so that substantial decreases of phytic acid in cereal products could improve Fe availability to the consumers. Cereal grains contain endogenous phytase, an enzyme capable of hydrolyzing phytate to free inorganic phosphate and lower inositol phosphate esters, thus decreasing or eliminating the antinutritional effect of phytates. 4 However, in cereal grain products phytates remain at high concentrations due to inefficient enzymatic degradation. 6 To overcome this limitation, the addition of exogenous enzymes, mainly from fungal origin, to increase phytase activity has been the best strategy to reduce the phytate content of cereals. The effect of phytase from Aspergillus niger in InsP 6 degradation during the breadmaking process was studied by Tü rk and Sandberg 7 and later by Haros et al. 6 Its use to reduce phytate content in bread was an effective method for increasing Fe absorption in humans. 5 The addition of sourdough for breadmaking has also been used for InsP 6 degradation by activation of cereal endogenous phytase due to the decrease of pH. 8 Some studies have improved Fe absorption in humans by reducing phytate via the addition of fungal phytase or promoting the endogenous phytase activity into food processing. 9-11 Nevertheless, human studies are often impractical because they are costly, lengthy, and complex. The Caco-2 cell line has been used extensively as an in vitro method to assess Fe bioavailability. A strong correlation has been found between the published human absorption data and Fe uptake by the Caco-2 cells, indicating the usefulness of this method in assessing human Fe absorption. 12 The study of the phytate effects on Fe bioavailability in whole grain products by the Caco-2 cell line has been well documented. 13-15 Some years ago it was reported that strains of the Bifidobacterium genus have phytase activity, 16,17 suggesting their possible utility in the production of bakery products with low InsP 6 levels. 18,19 The use of fungal phytases is currently approved in animal feed, but so far has not been certified for use in foods intended for human consumption. Thus, the use of bifidobacteria, which are GRAS/QPS (generally regarded as safe/qualified presumption of safety) microorganisms, could be a strategy particularly suitable to reduce the phytate content in whole grain products for human consumption. Received: December 8, 2011 Revised: February 6, 2012 Accepted: February 28, 2012 Published: February 28, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 3190 dx.doi.org/10.1021/jf205048r | J. Agric. Food Chem. 2012, 60, 3190-3195