Iron and zinc deficiencies are amongst the most common micronutrient deficiencies globally and are estimated to affect over 2 billion people [1
]. These deficiencies are associated with anemia (iron) [4
] and impaired immunity and development (zinc) [5
] and lead to major losses of human potential [6
]. A significant part of the population that is suffering from micronutrient deficiencies consume beans as part of their daily diet, especially in Latin America and Eastern Africa [8
]. Diets of rural and poor populations in these regions are mostly plant-based, in which legumes (and more specifically beans) are an essential component of daily diets [9
]. Common beans (Phaseolus vulgaris
L.) are an excellent source of not only iron and zinc but also proteins, dietary fiber, and vitamins [10
Biofortification, a nutrition-sensitive agricultural intervention, aims to improve the nutritional status of resource-poor populations through increasing the nutrient content of food crops, by developing more nutrient-rich crop varieties [11
]. HarvestPlus, a global interdisciplinary alliance of research and implementing agencies engaged in biofortification, use conventional breeding to improve the nutritional quality of staple crops without compromising other agronomic qualities (e.g. yield, drought resistance, etc.) [12
]. Iron beans are biofortified lines of beans with increased levels of iron and zinc that have been developed by HarvestPlus and have been released in 18 countries in Latin America and 26 countries in Africa [13
]. Micronutrient targets for breeding biofortified crops are established based on the food intake of target populations, nutrient losses during storage and processing, and bioavailability of the target nutrient to the human body [14
]. Current breeding targets for iron beans are 94 μg·g−1
compared to an average of 50 μg·g−1
as the baseline content of conventional varieties of beans [12
Studies conducted to date on the iron bioaccessibility and bioavailability from (iron biofortified) beans have been using Caco-2 cell models, in vitro digestion models [15
], poultry studies [16
], and human feeding trials [25
]. These studies show the influence of specific polyphenols on iron bio-accessibility and bioavailability depending on the type of bean. Furthermore, the positive effects of biofortified beans on iron status and other nutritional and functional indicators in humans are described. Mineral absorption from plant foods is generally low, which is mainly due to limited bioavailability of the iron and zinc to the body [29
]. In particular, anti-nutritional compounds hamper the potential nutritional impact of consuming plant foods and iron beans, specifically [30
]. Examples of such anti-nutritional compounds are phytic acid, polyphenols, lectins, and tannins.
Current research suggests that phytic acid is one of the major and significant inhibitors of mineral bioavailability from beans, next to polyphenols [8
]. Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate) and its salt phytate are known for their negative effect on iron absorption and can decrease iron status [8
]. Phytic acid is the main storage form of phosphorus and mineral storage in the bean seed and plant. It has been demonstrated that reductions in phytic acid levels in beans are not associated with reduced plant health or yields [30
]. Hence, it is possible to develop low phytic acid (lpa
) beans, with preferable agronomic traits.
For micronutrient biofortification strategies to successfully impact on human nutrition, sufficient levels of retention of target micronutrients after typical processing, storage, and cooking practices must be demonstrated [32
]. Also, mineral absorption of the biofortified crops should be similar or better than non-biofortified crops. However, absorption of iron and zinc in biofortified crops could be limited by its antinutrient content, such as phytic acid. In the case of beans, common processing techniques include soaking, boiling, and refrying. Micronutrients are lost in preparation methods due to chemical degradation (isomerization and oxidation) and physical loss, through the leaking of soluble solids into water or water loss [32
]. For instance, soaking has been shown to reduce phytic acid by solubilizing them in the soaking water, while on the other hand, it can also cause leaching of minerals [33
]. Micronutrient losses during food processing and cooking can be measured by determining True Retention (TR), where the changes in solids of food during processing and cooking are taken into account, to provide an accurate estimation of actual retention during the different processes [32
]. Retention studies that have tested conventional [34
] and biofortified beans [32
] have been published. However, the studies to date have not reported TR, which makes it difficult comparing results across different studies.
Low phytic acid mutant lines have been developed using a mutant allele of a gene that prevents the storage of phytic acid in the bean [30
]. Whereas research has been conducted to study retention in conventional bean varieties, no research on retention levels in these relatively new lpa
lines has been published. If these more freely available or weakly bound minerals are retained in beans while being processed, this could provide a route for further development of biofortified beans that combine high mineral and lpa
traits. Therefore, we aimed to assess the iron, zinc and total phytic acid levels of lpa
, biofortified and conventional beans and evaluated the iron, zinc and phytic acid retention when preparing common bean recipes using the different classes of bean varieties.
Biofortification strategies to improve human nutrition require not only the development of biofortified varieties with high levels of micronutrients, but also of varieties that have lower levels of anti-nutritional compounds. Such anti-nutritional compounds can limit the bioavailability and uptake of micronutrients. In addition, for biofortified foods such as beans which are processed and cooked prior to consumption, it is essential that micronutrients are retained during the preparation of such foods in sufficient quantities to impact on human nutrition.
Here we demonstrate that the levels of iron and zinc found in the dry beans are comparable with those found in other studies [34
]. We also detect a positive trend between iron and zinc levels, which has been observed by others [45
]. Phytic acid levels found in conventional and biofortified beans in our study are also comparable to other studies, where phytic acid concentrations ranging from 4 to 26 mg·g−1
of beans have been reported [8
We found that the cooking times assessed using the Mattson cooker showed a large variation in the cooking times of the lpa
genotypes. Overall, the cooking time results should be interpreted with caution since storage time and temperature have been shown to influence cooking time. However, the cooking time was within the usual reported cooking times for beans [55
Our iron retention results are comparable to a study with non-soaked beans in Rwanda that showed a retention close to 100% after boiling the beans. In the Rwandan study, cooking broth was not discarded, which prevented iron loss through the broth [32
]. In contrast, in our study, the cooking broth was discarded, which led to a higher loss of iron. Carvalho et al (2012) found that iron retention for both soaked and non-soaked bean grains of six different common bean cultivars led to a loss of 13–19% of iron in non-soaked and soaked beans, which is similar to an average of 16% loss for both non-soaked and soaked beans in our study [34
]. Refrying increased the iron TR, most certainly due to adding cooking broth to prepare the refried beans. This broth contained the iron that leaked into the cooking broth during boiling. To our knowledge, no other studies have reported iron retention after refrying beans. Values > 100% for AR as reported in our study were also reported before by Ongol et al. [36
] and Ferreira et al. [35
]. The high AR of > 100% for iron retention can possibly be explained by the leakage of solubles in the water (10.1–20.5%).
Retention of zinc was studied by Carvalho et al. and showed that zinc levels in broth after boiling beans did not differ between soaked or non-soaked beans [34
]. Although we did not measure broth zinc concentrations, we did find a significant difference in zinc retention between soaked and non-soaked boiled beans; however, this difference was small. In addition, Carvalho et al. concluded that most zinc remained in the bean after boiling and was concentrated in the cooked bean [34
Refrying increased the zinc TR up to 100% for conventional non-soaked beans, this was most likely due to adding the cooking broth to prepare the refried beans. This broth contained the zinc that leaked into the cooking broth during boiling.
bean genotypes showed substantial losses of zinc into the boiling water, which is partly reconstituted during refrying, where differences in retention are much smaller between the lpa
and conventional group. No other studies have reported zinc retention after refrying beans. The higher affinity of zinc to phytic acid [57
], the relatively high zinc amount trapped in the pericarp rich in phytic acid after soaking and steaming rice [58
], and lower zinc retention in lpa
beans during boiling soaked beans suggest that during soaking and cooking, zinc from the cotyledon in non-lpa
beans possibly interacted with the phytic acid, preventing excessive zinc losses in the soaking and cooking water. However, phytic acid in lpa
beans was found in relatively low quantities, and the zinc from these beans may not have interacted much with the limited amounts of phytic acid remaining, causing larger zinc losses in the soaking and cooking water. This possibility should be investigated further, not only for zinc, but also for iron because most iron is also found in the cotyledon of the bean [59
] despite lpa
beans having a different retention pattern compared to zinc.
Phytic acid levels were significantly reduced (> 10%) by soaking in our study. Another study in different types of Canadian pulses showed only a slight increase in phytic acid after soaking a black bean variety (2.34%) and pinto bean (1.86%). A decrease in phytic acid was found for a dark red kidney bean and a navy bean variety (−0.54% and −1.03%, respectively) [53
]. A review of Haileslassie et al. compared 15 studies in which beans were soaked under various conditions. Results were ranging from no significant difference on phytic acid levels after soaking up to a 66% reduction in phytic acid after soaking in an autoclave [60
In a study of Shi et al, cooking various bean varieties resulted in very modest decreases in phytic acid. Compared to the raw values for different types of beans, the decreases were between −2.29% and −0.29% [53
]. This is very minimal in comparison with our study where phytic acid was reduced up to 50% after boiling. For the soaked samples, the soaking water was discarded and therefore higher losses of phytic acid were reported in comparison with the non-soaked beans when preparing boiled and refried beans. No other studies have published phytic acid retention after refrying beans.
Our analysis quantified the total amount of phytic acid in the samples, including other dephosphorylated forms of myoinositol with less phosphate groups (IP-3/4/5
). These other compounds (especially the lower phosphorylated forms) do not necessarily inhibit mineral absorption to the same extent and therefore could lead to an overestimation of their actual effect. In vitro studies using Caco-2 cell lines demonstrated the inhibiting effect of phytic acid for different degrees of phosphorylation (IP-3/4/5/6
) for both Fe and Zn [61
were reported to not have an effect on zinc absorption in an animal study [62
]. In a series of five human studies using extrinsic labelling, it was found that only inositol phosphates lower than IP3
had no effect on iron bioavailability [63
]. Future research could further identify the type of phytic acid present in the different types of beans, as this might be another angle of explaining the differences in retention and eventually the effect on the bioavailability of minerals to the human body [50
The molar ratios of phytic acid to iron found in this study are comparable to other studies where lpa
beans were consumed by different groups of women to compare the iron bioavailability from different types of bean seeds [31
]. Studies have shown that lpa
beans have a higher iron bioavailability caused by the low concentration of phytic acid compared to conventional beans [31
]. No data was found on the zinc bioavailability from lpa
A multiple meal isotope bean study showed that both biofortified and lpa
beans provided more bioavailable iron in comparison with conventional beans, however, there was no difference in fractional iron absorption [46
]. In another single meal study, a 50–60% higher fractional absorption was found for lpa
beans compared to conventional beans. In addition, it was reported that studies based on single meals often exaggerate the inhibiting effect of phytic acid on absorption of both iron and zinc [50
]. One study used dephytinized beans (95% phytic acid reduction) and compared these to conventional and biofortified varieties for the fractional iron absorption in a multiple meal study [64
]. Results showed a fractional absorption of iron of 13.2, 9.2 and 7.1% for respectively dephytinized, biofortified and conventional beans. When these results are extrapolated to the findings from our study, one portion of boiled beans could contribute for 14, 16 and 23% for respectively conventional, biofortified and lpa
beans (taken as 95% dephytinized beans) of the physiological requirements of iron in an adult woman. Hence, the indications are that the lpa
trait is promising and of public health relevance, especially in settings with a high iron deficiency prevalence, a high phytic acid diet, and a high consumption of beans.
In addition, the phytic acid content of the whole diet has shown to be of influence, particularly on the zinc bioavailability from beans. Absorption from diets with a phytic acid to zinc ratio of 12–15 compared to a ratio of 5 was approximately 50% less [65
]. For iron, an increase in bioavailability influenced by phytic acid ratios is only found at very low ratios of 0.4–1.0 [66
]. Hence, when lpa
beans are used to replace conventional beans and would be added to an already low-phytic acid diet this could potentially increase the absorption of both iron and zinc significantly. Further research is needed to test to what extent low phytic acid-mineral ratios in beans can lead to a higher bioavailability of iron and zinc, when part of a whole diet.
The use of extrinsic labelling in determining the iron absorption and bioavailability has shown to not always be consistent when compared with intrinsic labelled foods, therefore, interpretation of these studies should be taken with caution [67
]. Future studies should, where possible, be carried out with the use of intrinsically labelled foods to prevent these unwanted effects, or set up using in vitro digestion/Caco-2 cell models coupled with a poultry model that has also shown to be in strong agreement with human studies and a reliable tool for screening varieties [16
The cotyledons contain 75–80% of iron, this location could potentially be the cause of the discrepancy between intrinsic and extrinsic labelling. The cotyledon cell walls represent a barrier for iron absorption from the bean, however, breaking these cell walls did not show an increase in the bioavailable fraction of iron. This suggests that the intracellular matrix of the bean potentially inhibits the exchange of iron with the cell transport mechanism [68
The present study focused on total phytic acid content of beans and its possible effect on the bioavailability of iron and zinc. However, we recognize that polyphenols are an additional class of anti-nutritionals that need to be considered in high-Fe bean biofortification efforts and also with reference to the lpa
trait. It has been shown in a series of in vitro digestion/Caco-2 cell models and/or coupled with poultry model studies that specific polyphenols in especially black beans inhibit iron uptake and that breeding for more iron in black beans does not lead to more bioavailable iron due to higher levels of polyphenolic compounds [19
]. The overall inhibitory effect of polyphenols is combinatorial, whereby some polyphenols (catechin, 3,4-dihydroxybenzoic acid, kaempferol, and kaempferol 3-glucoside) promote iron uptake while others (myricetin, myricetin 3-glucoside, quercetin, and quercetin 3-glucoside) inhibit iron uptake.
As the lpa
trait could be combined with different types and colors of beans, an optimal combination could be sought that has not only high mineral availability, but also good acceptability by consumers. One possible combination could be the yellow Manteca bean, which has shown to be fast-cooking and has a high iron bioavailability [17