3.1. Phytase Interventions
The studies reviewed took place in various locations, which featured different geographical and socio-economic contexts. In Europe, studies were conducted in Switzerland, specifically Zurich and other university settings [
13,
15,
17,
20], as well as in Denmark [
21] and Sweden [
23], providing insights into phytase’s effects in developed countries high-income settings with access to well-controlled research environments. In Africa, research was done in Gambia [
14], Burkina Faso [
16], and Benin [
18], which presented data from developing countries with a focus on improving micronutrient absorption among young children through dietary interventions. Additionally, a study in South Africa explored the impact of phytase on school-aged children’s micronutrient status, highlighting regional differences in dietary challenges and intervention strategies [
19]. The study conducted in Venezuela provided data on phytase’s impact on iron absorption from a controlled experimental setting, contributing insights from a Latin American context [
22]. These diverse locations underscore the broad applicability of phytase in enhancing micronutrient absorption across different populations and dietary environments.
The majority (82%, 9 out of 11) of the reviewed studies consistently reported that phytase supplementation enhanced micronutrient absorption, namely for zinc and iron [
14,
15,
16,
17,
18,
19,
20,
22,
23]. Notably, studies of Bach Kristensen et al., 2005 and Herter-Aeberli et al., 2020 reported no effect of phytase supplementation [
13,
21], while the remaining 9 studies strongly support the hypothesis of micronutrient bioavailability enhancement during phytase supplementation [
14,
15,
16,
17,
18,
19,
20,
22,
23]. For instance, two studies observed significant increases in fractional zinc absorption with phytase supplementation (P < 0.001 and P < 0.0001) respectively [
14,
16]. Similarly, another study reported that phytase nearly doubled iron absorption from FeSO4-fortified millet porridge (P < 0.001) [
18], and Brnić et al., 2014 found that consuming the active phytase enzyme together with a cereal meal increased zinc absorption to the same extent as when the meal was completely dephytinized before consumption [
17]. Studies that combined phytase with ascorbic acid showed synergistic effects in improving iron absorption. For example, the authors of one study demonstrated that adding phytase and ascorbic acid to meals significantly increased iron absorption from both FeSO4 and NaFeEDTA (P < 0.001) [
20], which is reflected in the fact that the combination of phytase and ascorbic acid led to greater iron absorption [
18]. Despite the overall positive impact of phytase, there were discrepancies in iron absorption results. Herter-Aeberli et al., 2020 found no significant increase in iron absorption from tef injera with added phytase compared to control meals (P > 0.05) [
13], contrasting with the significant improvements observed in other studies [
15,
18]. This inconsistency may arise from differences in food matrices, phytase types, and intervention durations, highlighting the need for context-specific adjustments in phytase application. Bach Kristensen et al., 2005 found that adding phytase to fiber-rich wheat bread did not prevent a decline in iron status, which contrasts with findings from other studies where phytase effectively improved iron absorption [
21].
The duration of interventions across the reviewed studies varied significantly, offering a diverse perspective on the effectiveness of phytase on micronutrient absorption. Short-term studies, focused on single-day interventions to assess immediate effects on zinc and iron absorption, revealed significant short-term increases in nutrient absorption [
14,
16,
18]. In contrast, other studies extended their observations over a few days, offering a slightly broader view of short-term effects [
13,
15,
23]. Longer-duration studies spanned several weeks to months, providing insights into the sustained impact of phytase on iron and zinc status, highlighting the potential for long-term nutritional benefits and the necessity to monitor for any prolonged consumption issues [
19,
21]. Also, two studies used crossover designs and controlled settings over several weeks to months, offering robust evidence by incorporating repeated measures and washout periods [
17,
22]. Finally, a study employed a two-day design for each segment to evaluate various combinations of enhancers, emphasizing the flexibility and adaptability of phytase interventions [
20].
The type of phytase used across studies was predominantly
Aspergillus niger, indicating a common preference for this microbial source due to its effectiveness in degrading phytic acid [
13,
14,
15,
16,
17,
18,
19,
20]. Also, in the study of Sandberg et al., 1996 the authors utilized
Aspergillus niger, consistent with the previous studies but with a notably high enzyme activity dose. Bach Kristensen et al., 2005 used a phytase produced from
Aspergillus Oryzae NOVO-L, showing some variation in phytase source but still within the
Aspergillus genus [
21]. Interestingly, Layrisse et al., 2000 diverged by using phytase from wheat, suggesting potential differences in enzymatic activity or stability [
22].
The dosage of phytase varied across the studies, reflecting different experimental designs and objectives. Studies of Herter-Aeberli et al., 2020 and Troesch et al., 2011 both used a dose of 380 FTU [
13,
19], whereas Zyba et al., 2019 used a higher dose of 588 x 2 FTUs [
14] and the study of Brnić et al., 2016 used a lower dose of 20.5 FTU [
16]. Notably, 3 studies had consistent doses, each using 190 FTU [
15,
17,
20]. Cercamondi et al., 2013 used a similar approach with 200 mg/portion, approximately 400 FTU [
18], while Bach Kristensen et al., 2005 administered a significantly higher dose of 2500 FTU/100 g [
21]. In contrast, Layrisse et al., 2000 used a more moderate dose of 304U/100g [
22], and Sandberg et al., 1996 applied an exceptionally high activity dose of 4 x 10^7 FTU/L [
23]. These variations highlight the lack of a standardized dosing protocol and suggest that further research is needed to determine optimal dosing for different populations and settings.
The test meals and supplements across the 11 studies demonstrated considerable diversity, reflecting the different research goals and contexts. However, there were notable correlations among studies that used similar or different approaches. Several studies utilized maize-based meals, either as porridge or in combination with other ingredients [
13,
15,
17,
20]. These studies consistently investigated the effect of phytase on iron or zinc absorption, demonstrating the relevance of maize as a common dietary staple and the potential benefits of phytase in enhancing nutrient absorption from maize-based foods. Others focused on millet-based porridges, exploring the impact of phytase on zinc and iron absorption in young children [
14,
16,
18]. These studies consistently found that the addition of phytase significantly improved micronutrient absorption, highlighting millet as another key staple where phytase can be beneficial. One study provided a sweetened maize porridge with a micronutrient powder containing iron and zinc [
19], while another focused on wheat bread with or without phytase [
21]. The differences in these test meals underscore the versatility of phytase across various food forms, although the outcomes varied, with Bach Kristensen et al., 2005 not showing a significant improvement in iron status despite phytase addition [
21]. Layrisse et al., 2000 used a basal breakfast with ferrous sulfate or ferrochel [
22], and Sandberg et al., 1996 tested white wheat rolls with wheat bran, comparing phytase-active and phytase-deactivated conditions [
23]. These studies, while employing different food bases, consistently found that the addition of phytase significantly enhanced iron absorption, reinforcing the effectiveness of phytase in improving iron bioavailability across different meal types.
Finally, the methodological quality and study designs of the 11 studies varied, encompassing randomized controlled trials (RCTs), crossover designs, and controlled experimental studies. Most studies were RCTs [
14,
16,
18,
19,
21,
23], providing robust evidence through randomization and control groups. Other studies employed crossover designs [
13,
15,
17,
20], allowing each participant to serve as their own control, thereby reducing inter-individual variability and increasing the reliability of the findings. Layrisse et al., 2000 utilized a controlled, but not randomised, experimental design to test iron absorption under various conditions, adding valuable comparative data [
22].
3.2. Dietary Phytic acid Interventions
The 17 included studies encompass a wide range of geographical and socio-economic contexts. Research was conducted in developed countries such as Switzerland [
26,
30], Sweden [
27,
35] and the USA [
29,
33], as well as in low-resource settings such as Guatemala [
32,
38] and Rwanda [
39]. Further, these studies also range in setting, from controlled laboratory environments [
34,
37] to real-world settings, such as community feeding programs [
38] and home-based interventions [
32]. Such geographical and socioeconomic diversity highlights the widespread applicability of the findings and underscores the global importance of understanding the impact of phytic acid on micronutrient absorption.
Out of the 17 studies, eight studies specifically examined the absorption of iron. More specifically, four of these studies confirmed that phytic acid inhibits iron absorption [
26,
27,
39,
40]. For example, high phytic acid content was consistently associated with reduced fractional iron absorption [
39,
40]. However, other studies, such as Lind et al., 2003 and Hoppe et al., 2018, showed less pronounced effects or mixed results [
35,
37]. Also, six other studies examined zinc absorption. Half of these studies confirmed that phytic acid inhibits zinc absorption [
26,
31,
32]. For example, diets rich in phytic acid were associated with lower fractional zinc absorption [
36]. However, a few studies found no significant difference in zinc absorption with low phytic acid maize or beans when compared to controls [
38,
39]. Furthermore, 3 other studies examined calcium absorption. Most of these studies showed that high phytic acid content results in reduced calcium absorption [
31,
33]. For example, calcium absorption was significantly greater from low phytic acid maize tortillas when compared to control ones [
33]. Out of the 17 studies, only one study examined the effect of phytic acid on manganese absorption, stating that high phytic acid content inhibits its absorption [
26]. Also, a single study assessed the absorption of copper and found no significant impact of phytic acid on copper absorption [
30]. These contradictory findings imply that while phytic acid generally affects micronutrient absorption, its effect may be influenced by the dietary context and food matrix.
The duration of the studies’ interventions differed considerably with intervention periods ranging from one day [
26,
34] to six months [
37]. The studies of Heany et al., 1991 and Bohn et al., 2004 involved single-day interventions, focusing on the acute effects of phytic acid on micronutrient absorption [
26,
34]. On the contrary, more longitudinal studies, such as those conducted by Lind et al., 2003 and Hoppe et al., 2018, were conducted over several weeks to months, investigating the effect of phytic acid over greater periods [
35,
37]. For example, Lind et al., 2003 conducted an intervention period of 6 months, while Hoppe et al., 2018 ran interventions for 12 weeks. This diversity reflects different research objectives, from acute to chronic effects, offering a comprehensive insight into the effects of phytic acid on various time scales.
Across all 17 studies, interventions were primarily concerned with controlling the phytic acid content of the test meals. In most cases, this was achieved either by using low phytic acid, bean, maize or cereal variants [
37,
38,
40] or by adding phytic acid to the meals to test its effects [
27,
31,
34]. A few studies used dehydrated foods to evaluate changes in nutrient absorption [
27,
30]. These interventions aimed to isolate the effect of phytic acid by comparing low and high phytic acid foods or by adding measured levels of phytic acid to test meals, thus providing evidence of how phytic acid affects micronutrient bioavailability.
There was a wide variety of test meals and diets used in the interventions. Examples include, bread made from different types of flour [
26], bread prepared with different phytic acid content [
27], and maize tortillas with different levels of phytic acid [
33]. While some studies used complete meals that included different types of foods, such as beans, maize, and cereals [
39,
40], other studies focused on specific ingredients, such as iron-fortified infant formulas or cereals with reduced phytic acid [
35,
37]. This diversity of test meals underlines the adaptability of the research designs to different dietary contexts and provides a broad scope for assessing the effects of phytic acid in different food matrices.
The methodological quality and study design of the 17 studies differed substantially. Almost half of the studies (9 out of 17) used randomized controlled trials (RCTs) [
27,
31,
32,
34,
35,
37,
38,
39], which are perceived to be robust for assessing causal effects. However, other studies used less stringent designs, such as community-based interventions [
38] or non-randomized trials [
34]. Designs also differed in terms of blinding and control measures, with some using double blinding [
35,
37], while others did not specify blinding methods. Such variability in study design both reflects the different technical and organizational challenges faced by the researchers in diverse settings and adds to a well-rounded understanding of the impact of phytic acid on micronutrient absorption.
3.3. Dephytinisation Interventions
The 14 included studies cover a broad spectrum of geographical and socio-economic settings. Study settings spanned multiple continents, including Europe (France, Switzerland) [
43,
45], Africa (Malawi, Benin, Rwanda) [
46,
53,
54], Asia (Korea, Singapore) [
49,
52], and North America (USA) [
42]. These studies involved populations with varying socio-economic backgrounds, from research universities and urban health centers to rural communities with limited access to advanced healthcare. For instance, Manary et al., 2000 and Petry et al., 2014 focused on communities in Malawi and Rwanda, where high levels of dietary phytic acid are prevalent due to staple food sources [
53,
54]. In contrast, studies like Davidsson et al., 1997 and Zhang et al., 2007 were conducted in more developed settings, such as France and Sweden, with controlled dietary conditions and higher resources for research infrastructure [
43,
47]. This diversity highlights the global relevance of studying food dephytinisation’s impact on micronutrient absorption and the varying challenges faced by different populations.
The studies collectively demonstrate that phytase effectively dephytinises foods, enhancing micronutrient absorption, albeit the results are mixed. For iron absorption, half of the studies (7 out of 14) consistently show that dephytinisation increases iron bioavailability [
43,
45,
46,
47,
51,
54]. Notably, Petry et al., 2014 found that removing up to 95% of phytic acid significantly improved iron absorption from biofortified beans [
54], while Hurrell et al., 2003 observed increased iron absorption from cereal porridges with reduced phytic acid content [
45]. On the other hand, Davidsson et al., 1997, 1995 reported that, while dephytinisation generally improved iron bioavailability, the effect was not always pronounced, and the presence of ascorbic acid further enhanced absorption in some cases [
43,
44]. For zinc absorption, five studies investigated the impact of phytase treatment [
49,
50,
52,
53,
54]. More than half of these studies found that phytase treatment improved zinc absorption [
52,
53,
54], with Manary et al., 2000 showing greater fractional absorption in children on a diet with low phytic acid content [
53]. However, Petry et al., 2010 and Couzy et al., 1998 reported mixed results indicating, that while phytase treatment enhanced the absorption, the impact varied depending on the presence of polyphenols and the baseline phytic acid levels [
49,
50]. For manganese absorption, only one study specifically addressed this, showing that dephytinisation significantly increased manganese absorption, doubling the fractional absorption compared to non-dephytinised soy formula [
44]. This highlights the potential of phytase to enhance the bioavailability of multiple micronutrients, although the effects can vary based on the specific nutrient and context of the diet.
The types, doses, and durations of phytase interventions varied significantly across the studies. Most studies used
Aspergillus niger phytase, but dosages ranged widely. The doses of phytase used in the studies ranged from as low as 40 FTU/L [
51] to as high as 5000 FTU/g [
52,
53]. For instance, Manary et al., 2000 used 5000 FTU/g of phytase for a short duration of 3-7 days [
53], while Davidsson et al., 1995 did not specify the dose but utilized a 1-day intervention [
44]. Kim et al., 2007 administered 5000 FTU/g of
Aspergillus niger phytase over 9 days for high and low phytic acid diets [
52]. Some studies, like Hurrell et al., 2003, also did not specify doses, but ensured effective phytic acid degradation [
45]. The intervention durations overall, varied from 1 day [
43,
44,
46,
52] to 42 days [
53], reflecting different study designs and objectives. This variation underscores the need for standardization in phytase treatment research to better compare outcomes.
The test meals or diets across the studies were diverse, and tailored to the specific dietary patterns and research aims of each study. Davidsson et al., 1997 used iron-fortified cereals with native and dephytinised phytic acid content [
43], while Petry et al., 2014 employed beans with varying phytic acid levels [
54]. Manary et al., 2000 provided corn-plus-soy porridge, with and without phytase, to assess zinc absorption [
53]. Zhang et al., 2007 tested oat-based beverages supplemented with different iron compounds and phytase [
47]. Other studies included soy formulas [
51], fonio porridges [
46], and roller-dried cereal porridges [
45]. This variety reflects the adaptation of interventions to local dietary habits and nutrient challenges, highlighting the importance of context-specific solutions.
The methodological quality and study designs varied considerably across the 14 studies. Most studies (8 out of 14) employed randomized controlled trials [
43,
46,
47,
49,
51,
52,
53,
54], which are robust methods for assessing the effects of dietary interventions. For instance, Petry et al., 2014 and Kim et al., 2007, and Davidsson et al., 1994 used RCT and crossover designs to evaluate the impact of phytase on iron and zinc absorption [
51,
52,
54]. Other studies used interventional designs with a focus on community-based approaches [
48,
53]. Despite this, variations in study quality were evident, with some studies lacking detailed information on phytase dosages [
43,
44,
45,
46,
47,
50]. Overall, while the diversity in study designs enriches the understanding of the dephytinisation impact, it also emphasizes the need for consistent reporting and methodological rigor to enhance the comparability and generalizability of findings.