Vitamin A deficiency (VAD) continues to be a significant health concern in developing countries affecting more than 190 million preschool-age children and 19.1 million pregnant women globally [1
]. Blindness, anemia, and infant mortality are all consequences of VAD that impact the quality of life in affected regions. Aggressive and transformative strategies are needed to overcome this nutritional challenge. A major factor in VAD is the lack of dietary vitamin A or provitamin A carotenoids (pVAC) in staple foods. Biofortification of grain-based staple foods including maize, sorghum, and rice with pVACs has been touted as a promising approach to address this issue directly for broader portions of affected populations. Biofortified maize (Zea mays L.
) genotypes have been developed through conventional breeding programs that produce yellow and orange kernels with high contents of β-carotene and other pVACs [2
]. Success in terms of biofortification of maize includes a target level of 15 μg/g of β-carotene, to provide an ~50% of the Estimated Average Requirement (EAR) for vitamin A in maize-eating regions [4
]. Included in this EAR estimates are that these levels should be retained through post-harvest and food processing [6
] and considers ultimate bioavailability in humans [8
With breeding efforts now generating elite genotypes with >20 μg/g of total pVACs, downstream processing and bioavailability are becoming critical factors to study. Losses during postharvest handling (drying, milling, and storage) and food processing can be significant as pVACs are sensitive to heat, oxygen, light, and acidic conditions [12
]. In African countries, maize and other cereals are commonly processed by steeping, in which natural fermentation is allowed to occur by soaking cereals in water spontaneously. Additionally, maize is utilized in several common preparations including bread, beverages, and porridges known such as ogi (Nigeria/West Africa), kenkey (Ghana), uji (Kenya), togwa (Tanzania), amahewu (South Africa), and mawé (Benin) [13
]. These spontaneously fermented products are dietary staples for adults and children alike [14
]. Most of these fermentations are spontaneous and rely on native lactic acid bacteria, sometimes accompanied by yeast fermentation [14
]. The fermentation process can improve nutrient quality, and density, and increase the bioavailability of certain nutrients in foods [15
]. For example, fermentation of grains has been associated with increased starch and protein digestibility [17
], as well as an increase in lysine and phenolics bioavailability [18
]. Organic acids produced during fermentation have been reported to enhance iron and zinc bioavailability through the formation of soluble ligands [20
]. Fermentation also lowers the pH which can favorably affect activity of endogenous phytase, resulting in lower phytic acid [21
]. These efforts combined suggest that fermentation of grains has the potential to reduce the risk of mineral deficiency among populations, especially in developing countries where unrefined cereals and pulses are highly consumed [22
As fermentation of maize for porridge production is commonly practiced throughout sub-Saharan Africa, it is critical to developing a better understanding of its impact on carotenoid stability, and potential bioavailability is needed. Carotenoids in starchy staple crops are believed to be minimally affected during fermentation. Thakkar et al. [23
] reported that β-carotene retention was 92% after three days fermentation of grated cassava, followed by 63% retention after roasting (165 °C, 10 min). A similar study on transgenic cassava roots carried out by Failla et al. [24
] reported that fermentation followed by roasting resulted in less than 40% retention of β-carotene. Recently, Aragón et al. [25
] working with an elite selection of biofortified cassava roots reported that spontaneous fermentation decreased levels of total carotenoid content (TCC) and β-carotene equivalent, with retentions ranging from 72% to 96% among ten genotypes.
While promising, less has been reported from biofortified maize grains. Li et al. [26
] studied the effect of spontaneous fermentation at room temperature (30 °C) in the dark for 48 h (solid-state fermentation) as part of the preparation of Ogi, a fermented maize porridge, made from high pVACs maize inbreeds. An initial 7% β-carotene loss was observed during 24 h soaking and milling, followed by an additional 10% loss during spontaneous fermentation, and a 7% loss during cooking.
Considering these findings, it is critical to include other elite biofortified maize genotypes as well as considering the potential impacts on product properties that may relate to carotenoid bioavailability and texture qualities [27
]. The texture of the final product is critical to the acceptability for consumers of biofortified fermented porridge. Textural qualities of Ogi depend on many factors, which include maize genotype, maize flour particle size, fermentation, and souring periods [28
]. Starch and its biochemical characteristics related to the ratio of amylose and amylopectin influence the viscosity and gelatinization properties of the starch, which impact the final texture and sensory attributes of typical maize-based foods [29
]. The characteristic of the starch granule, such as swelling, breakdown, and retrogradation, largely determine the overall pasting properties and stability of starchy foods [30
]. Establishing the impact of critical processes such as fermentation on the recovery of pVACs while simultaneously selecting rheological properties of the biofortified maize flour is critical to realizing the potential of these biofortified grains by facilitating their transfer to consumer foods. With this in mind, the objective of this study was to assess the impact of spontaneous fermentation on carotenoid stability and pasting properties of finished flours from five selected elite biofortified maize genotypes.
The primary purpose of this study was to evaluate the impact of steeping on carotenoid stability as a marker of the potential delivery of carotenoids through traditional African maize porridges. A secondary objective was to assess the changes in pasting properties on the resulting food product to characterize textural properties related to consumer acceptance of biofortified maize and potential impact on carotenoid stability. Five experimental maize (Zea mays L.
) genotypes (Table 1
) were chosen based on their unique carotenoid profiles and different kernel characteristics. Total carotenoid levels in these genotypes were generally consistent with previous results reported by our group, Ortiz et al. [6
Ogi, a fermented cereal porridge generated by steeping whole kernels and consumed by nearly 150 million West Africans [36
], was selected as a model to test the impact of steeping on carotenoid stability. Significant variability in the procedures used by native African communities to ferment cereals has been reported in the literature. Several authors reported differences in the starting materials [36
] (guinea corn, millet, sorghum white, and yellow corn), milling methods (whole kernels or degerminated flours), number of fermentation periods involved, number of times that the steeping water is replaced by freshwater, and number of thermal processes involved [28
]. Given this context, it was essential to begin to define fermentation parameters that may best preserve pVACs and allow for optimal textural characteristics aligned with consumer preferences. In general, ogi preparation involves an initial fermentation period called steeping with a duration between 24–120 h. The second fermentation period is souring with durations between 12–48 h. As this is one of the first studies to investigate the impact of fermentation on the stability of carotenoids from biofortified maize genotypes, the decision was made to simplify the process and ferment whole biofortified kernels with only one steeping period (with durations of 0, 24, 72, and 120 h) without changing the steeping water. Expecting this would serve to minimize losses due to leaching and therefore provide a baseline for future investigations. The pH of biofortified Ogi flours obtained in this study was found to have a higher pH (~4.97) than reported previously (3.7–4.06) for conventional Ogi flours after the steeping period [28
]. Genotypes 3 and 4 tended to produce products with a slightly higher acidity after the steeping period in comparison to the other genotypes. Ogi relies on spontaneous fermentation [46
], thus differences in the local lactic acid bacteria species populations in Africa and Indiana, USA may have contributed to different fermentation outcomes. This latter has been previously reported in native maize varieties (white vs. yellow) [47
] and may also be responsible, in part, for differences in the outcomes of these experiments compared to other fermentation experiments. In addition, maize kernels used in this experiment were stored for 2-years at −20 °C prior to steeping to induce spontaneous fermentation. Therefore, it is possible that these storage conditions might have altered the native microbiota in a way that could result in an incomplete fermentation.
Carotenoid stability was significantly impacted by the duration of the steeping period (p
< 0.001) across all biofortified maize genotypes. In general, the overall carotenoid retention was 92%, 82%, and 37% after 24, 72, and 120 hours, respectively. Li and others [26
] reported 10% β-carotene (95% CI 8.5–11.9) and 21.5% zeaxanthin (95% CI 14.6–28.4) losses on a wet maize flour that was allowed to spontaneously ferment at room temperature (30 °C) in the dark for 48 h (pH 4). Carotenoid retention during spontaneous fermentation has also been investigated on cassava. Aragón and others [25
] reported that fermentation of biofortified cassava roots significantly decreased levels of β-carotene equivalent with retentions ranging from 72% to 96% for β-carotene equivalent, with some cassava genotypes showing superior carotenoids retention during spontaneous fermentation. Thakkar and others [23
] found that fermentation of grated cassava for three days at room temperature in the preparation gari resulted in a minimal loss (8% loss) of the total β-carotene content. Carotenoid degradation can be induced by light, oxygen, extremes in pH, or a combination of all three [48
]. However, light exposure and pH might not be the main degradative factors, since maize fermentation was carried out at controlled temperature (27 °C) with no light exposure, and in general, carotenoids are reported to be stable to pH changes in foods over the range pH 2–7 [26
]. Even though dissolved oxygen in the steeping water decreases during ogi fermentation [45
], direct oxidation might be a factor contributing to the full range of carotenoid losses observed.
Carotenoid losses during fermentation may also be attributed to carotenoid leaching in the discarded liquid during food processing. Bechoff et al. [49
] reported significant provitamin A carotenoids losses that were attributed to physical losses of pVACs and chemical losses (oxidation) during the processing of biofortified cassava. Nevertheless, one beneficial effect of fermentation is an improvement in extractability and bioavailability of minerals in cereal matrices [50
]. This latter is achieved primarily by the reduction of phytic acid, a powerful chelator agent for minerals. This fact might have an indirect role in the oxidation of carotenoids during fermentation, since fermentation may enhance mineral release from the food matrix and directly impact stability through their ability to stimulate oxidative processes and carotenoid oxidation through electron transfer reaction mechanism even at lower oxygen tensions [48
]. While not directly assessed in this experiment, it is plausible that the positive effect that fermentation has for mineral availability might also be responsible for a negative effect on carotenoid stability, particularly during subsequent processing.
Wet cooking of maize native and fermented whole flour further reduced total carotenoid content with retention ranging from 69% to 95% among genotypes, with average retention through the processing of 80% (Supplementary Table S2
). Kean et al. [52
] reported TCC retention of 52% in whole yellow dent cornmeal wet cooked with a 10% vegetable shortening and continuous stirring at 95 °C for 20 min. However, the cooking conditions used by Kean et al. [52
] might have enhanced carotenoid extraction into bulk lipid and susceptibility to oxidation over the extended cooking period (20 min). Lipkie et al. [32
] working with biofortified sorghum porridges made similarly to the present study reported retention of all-trans-β-carotene of ~77% (48%–100%) after 5 min wet cooking of sorghum slurries in boiling water stirred by hand. Carotenoid stability during wet cooking was found to be dependent on carotenoid structure (p
< 0.0001), with xanthophylls showing superior stability (82% retention) compared to carotenes (30% retention) during wet cooking (Figure 1
). A similar finding was reported in wheat flour [53
]. Carotenoids are known for properties as a chain-braking antioxidant in scavenging and quenching singlet oxygen. Mechanistic studies on the radical scavenging properties of carotenoids showed that the ability of carotenoids to scavenge radical cations increase with the extension of the chromophore and maximum overlap of the carbon-carbon double bond molecular orbital [54
]. Thus, the presence of additional hydroxyl groups on the carotenoid structure and the decreasing number of coplanar conjugated bonds decrease xanthophyll’s reactivities in radical scavenging reaction and, as such, would make them less susceptible to oxidative reactions [55
], which is consistent with our results (Figure 1
The pasting properties are reflected as changes in viscosity during heating of a suspension, and the changes in the starch physical and chemical structures [57
]. In our study, fermentation increased the viscosity for Ogi porridges, achieving the highest value after 120 h of steeping period among all genotypes evaluated. A similar phenomenon was observed by Osungbaro [40
] as well as by Adeyemi and Beckley [37
] in the natural fermentation of white maize grains (FARZ-27 and DMR-white varieties). They reported an increased viscosity for Ogi porridges of up to 4 days steeping or souring, after which further fermentation led to substantial viscosity reduction. On the other hand, it was noticed that C17 × DE3 genotype showed higher viscosity, HPV, and CPV values compared with genotype 5, Hi27 × CML328, and orange ISO genotypes. Genotype differences in the pasting properties might be related to differences in starch composition among genotypes. Notably, CI7 × DE3 (named C17 × DE exp by Sowa et al. [58
]) is a genotype of temperate origin with a dent type of kernel [59
]. All other genotypes are partially or entirely of subtropical origin and are of partial or full flint type of kernel. Relative to dent kernels, flinty kernels have a larger relative area of the vitreous or horny endosperm in proportion to the soft starchy endosperm. The vitreous endosperm is related to the compactness of the starch–protein matrix and amylose/amylopectin ratio [60
]. Vitreous endosperm has been associated with higher levels of α-zein protein and amylose levels relative to those of floury endosperm [61
]. Viscosity is also an indicator of starch hydration. Starch with more branched amylopectin absorbs and retains more water and achieve high peak viscosities [60
]. Therefore, the higher peak viscosity exhibited by the dent type CI7 × DE3 compared to flint type genotypes might be related to its starch and protein composition. Considering these results, genotype effects related to Ogi thickness is a critical factor related to the acceptability of biofortified orange maize given than African consumers prefer high viscosity porridges [42
shows a trend on the setback values among all genotypes evaluated. The setback values of whole maize porridges significantly increase (p
< 0.05) from 0 h to 72 h steeping time, followed by a significant decrease (p
< 0.05) from 72 h to 120 h. The setback value in the pasting viscosity curve is likely related to the aging trend and stability. Therefore, the aging trend of fermented whole maize porridge increased significantly with the steeping time compared to non-fermented whole maize flour.
Notwithstanding that natural fermentation of whole maize grain affects the pasting properties of the fermented Ogi by increasing the viscosity and setback with either fermentation or souring time [37
], the effect of natural fermentation on isolated starch from different plant sources was opposite reported [57
]. Natural fermentation on isolated starch decreased peak viscosity and final viscosity on waxy maize, wheat, and cassava starches [62
]. In addition, the setback of isolated starches decreased with respect to steeping time. A smaller setback value means better stability [65
]. The viscosity reduction observed in fermentation of isolated starches could be attributed to the hydrolysis of the starch main molecules, especially those of short-chain amylopectin, associated with both the action of microbial amylases and modest hydrolysis produced by the organic acids generated during fermentation [62
]. Structural alteration on the outer layers of the maize bran during spontaneous fermentation of whole maize kernels might have a role on the viscosity increase during the fermentation of maize grains. The outermost structure of maize kernels is a composite plant material consisting of thick-walled cells originating from the aleurone layer, testa, pericarp, and residual endosperm and is composed mainly of heteroxylans (approximately 67%) and cellulose (22%), but also content significant amounts, phenolic acids (approximately 4%, mainly ferulic and diferulic acid), and acetic acid [66
]. Alkali-soluble components of the maize bran also have functional properties as adhesives, thickeners, and stabilizers [67
]. As a matter of fact, the release components of the maize bran during alkaline cooking during tortilla preparation improved the viscosity, cohesiveness, and adhesiveness of the masa and tortillas [68
]. Therefore, it is possible that the increase in viscosity found as a consequence of the fermentation duration time might be caused by an alteration in the outer layer of the maize grain and/or starch complex formation between starch and components of the maize bran.
In summary, fermentation of biofortified maize grains resulted in a considerable reduction (63%) on total carotenoid content, with a generalized effect across all carotenoids forms after long steeping periods. Natural fermentation might also disrupt starch granules and the release of bran components, which translate into changes in the rheological properties of the end-product. Further studies are need to confirm this hypothesis. The extent to which these effects might enhance the solubility of minerals and soluble fibers, as previously reported, [50
] remains to be fully assessed. However, it appears that these factors would play a role in destabilizing carotenoids during fermentation. Although all carotenoid forms are susceptible to degradative reactions during thermal processing, consistent with previous reports, carotenoid susceptibility was dependent on the number of hydroxyl groups present in the structure with xanthophylls more stable to thermal processing than carotenes. These results suggest that fermented maize products made from steeping of biofortified maize genotypes can serve as a source of pVACs, but compatibility of steeping and thermal processing parameters must be further investigated to ensure that carotenoid stability and, eventually, bioavailability can be maximized in these staple foods.