Food Chemistry 286 (2019) 651–658

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Promoting the use of locally produced crops in making cereal-legume-based T

composite flours: An assessment of nutrient, antinutrient, mineral molar
ratios, and aflatoxin content
Patchimaporn Udomkuna, , Chanin Tirawattanawanichb, John Ilukorc, Piyanut Sridonpaid,
⁎

Emmanuel Njukwea, Pélagie Nimbonae, Bernard Vanlauwef

a
International Institute of Tropical Agriculture (IITA), Bujumbura, Burundi
b
Department of Physiology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand
c
Development Data Group – Survey Unit, World Bank, Kampala, Uganda
d
Institute of Nutrition, Mahidol University, Nakhon Pathom, Thailand
e
National Center for Food Technology, Bujumbura, Burundi
f
IITA, Nairobi, Kenya

ARTICLE INFO ABSTRACT

Keywords: Cassava, rice, and banana flours were used individually to replace wheat flour in cereal-legume-based composite
Functional properties flours. The proximate composition, mineral content, antinutritional effect, mineral molar ratios, and aflatoxin
Mineral availability level were investigated. Replacing wheat flour with rice flour significantly (P < 0.05) improved protein, fat,
Antinutrient potassium, and phosphorus content in samples. The molar ratios of phytate or oxalate to minerals (calcium and
Composite flour
zinc) in all composite flours were lower than the reported critical values, except phytate to iron. However, all
Indigenous crops
samples, except full replacement by rice flour, might not provide adequate zinc bioavailability when the effect of
calcium and phytate on zinc absorption was collectively considered. Although all composite flours were con-
taminated with aflatoxins, only the control composed of wheat flour did not meet the EU regulatory threshold
(4.0 µg/kg) for total aflatoxins. The findings showed that nutritional properties and aflatoxin content of com-
posite flours can be improved by replacement with local crops.

1. Introduction the addition of wheat flour (Shittu, Raji, & Sanni, 2007; Renzetti, Dal
Bello, & Arendt, 2008).
Cereal–legume-based flour is a basic staple food for many African One of the major problems of plant-based flours is the presence of
populations and is commonly consumed as porridge for breakfast, as an antinutrients such as phytate, oxalate, and tannin which limit their
ingredient for fufu, or used as a child-weaning food. Most cereals, such utilization (Kathirvel & Kumudha, 2011). Although, some health ben-
as maize, wheat, and sorghum, are a source of calories and complex efits from tannin and phytate have been shown, such as reductions in
carbohydrates but have a low protein and lysine content. On the other blood glucose and insulin response to starchy foods, and/or in plasma
hand, legumes like soybean and other beans are relatively richer in cholesterol and triglycerides (Thompson, 1993), and oxalate is rather a
proteins and dietary fiber, as well as a significant number of vitamins pro-oxidant with reports of toxicity due to a build-up of free radicals
and minerals (Tharanathan and Mahadevamma, 2003). Today, many (Kumar, Sinha, Makkar, & Becker, 2010), these antinutrients can ad-
countries in Sub-Saharan Africa (SSA), except Sudan and Ethiopia, have versely interfere with the absorption of minerals, such as calcium, zinc,
become completely dependent on imported wheat resulting in a debt and iron (Kruger et al., 2013) and prevent efficient utilization of pro-
burden (Eleazu et al., 2014). Another challenge associated with wheat teins (Gibson, Bailey, Gibbs, & Ferguson, 2010). The processing method
consumption is the development of celiac disease (gluten-sensitive en- either by soaking and/or heating have been found to reduce those anti-
teropathy) (Elijah, 2014). To counteract such problems, the use of nutritional factors (Patterson, Curran, & Der, 2017), however it could
composite flour consisting of several flours obtained from local crops still pose a significant health impact especially in cases where the ori-
has been emphasized in many low-income countries, with or without ginal levels of the anti-nutritional factors are high (Gibson, Perlas, &

⁎
Corresponding author.
E-mail address: [email protected] (P. Udomkun).

https://doi.org/10.1016/j.foodchem.2019.02.055
Received 30 October 2018; Received in revised form 2 February 2019; Accepted 5 February 2019
Available online 20 February 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
P. Udomkun, et al. Food Chemistry 286 (2019) 651–658

Holz, 2006). Shi, Arntfield, and Nickerson (2018) reported that the weighed and mixed for 10 min with a double cone solids blender which
soaking and cooking processes were found to be more effective in re- was locally fabricated in Burundi. All composite flour samples were
ducing lectins and oxalate in common beans and soybean, except phytic stored in airtight containers prior to biochemical analyses.
acid. The optimal exploitation of ingested nutrients is therefore ham-
pered, rendering to decreasing their nutritive value (Fekadu, Beyene, & 2.2. Biochemical analyses
Desse, 2013; Gemede, Haki, Beyene, Woldegiorgis, & Rakshit, 2015).
Low mineral availability in staple foods can lead to malnutrition and to 2.2.1. Proximate analysis
physiological pathology, such as osteoporosis, impairment of child Moisture content was determined using the method of AOAC
growth, and anemia (Akhter, Saeed, Irfan, & Malik, 2012). In addition, (1990). The sample was dried at 105 °C for 16 h in a draft of air (model
cereals and legumes are susceptible to aflatoxin contamination during UF55, Memmert Oven, Germany). The loss in weight was recorded as
production and storage which causes serious health complications in the moisture content. Protein content was investigated by the Kjeldahl
human and animals, such as stunting in children, liver damage, and method. A conversion factor of 6.25 was used to convert from total
cancer (Ismail et al., 2018). nitrogen to percentage crude protein. Ash content was determined by
To achieve efficient utilization and promotion of cereal–legume- the method of AOAC (1990) that involved burning off moisture and all
based composite flours among the poor, especially in rural areas, a organic constituents at 600 °C in a VULCAN™ furnace (model 3-1750,
study of their functional properties and aflatoxin contamination is ne- Cole-Parmer, Illinoi, USA). The weight of the residue after incineration
cessary. Therefore, the objective of this study aims at evaluating the was recorded as the ash content. Fat content of the samples was also
nutrient, antinutrient, mineral molar ratios, and aflatoxin contents in determined by the method of AOAC (1990), using the Soxhlet extrac-
cereal-legume-based composite flours. tion technique (model FOSS Soxtec™ extraction, Sweden). The crude
fiber content was determined using fiber extraction equipment (model
FOSS Fibertec™ 2010, Sweden). The percentage of carbohydrate con-
2. Materials and methods
tent was calculated by subtraction of the percentages of moisture, crude
protein, ash, fat, and crude fiber from 100. The caloric value (kcal/
2.1. Composite flour preparation
100 g) of each loaf was calculated using Atwater’s conversion factors,
based on the caloric coefficients corresponding to the contents of pro-
Cereal-legume-based flours from a mixture of maize, wheat, sor-
tein (4 kcal/g), carbohydrate (4 kcal/g), and fat (9 kcal/g). All mea-
ghum, soybean, and sesame flours were prepared as the control,
surements were carried out in triplicate.
whereas flours obtained from local crops, including cassava, rice, and
banana flours, were used to partly and fully replace wheat flour in the
2.2.2. Determination of total sugar content
formula. All grain and seed samples were procured from local markets
Total sugar content was determined by the method of Dubois, Gilles,
in Bujumbura, Burundi, except wheat flour which was obtained “as it
Hamilton, Rebers, and Smith (1956). About 0.02 g of the sample was
is”. Maize, sorghum, soybean, and sesame samples were separately
weighed into a centrifuge tube; 1 mL ethanol, 2 mL distilled water, and
cleaned, sorted, sun-dried for 1–2 days, and roasted at 150 °C for
10 mL hot ethanol were added. The mixture was vortexed and cen-
20 min. The roasted maize and soybean seeds were dehulled and al-
trifuged at 2000 rpm for 10 min. The supernatant was decanted and
lowed to cool at room temperature; rice seeds were sorted and roasted
used for determining sugar content. Phenol sulfuric acid reagent was
at 150 °C for 5 min. Fresh cassava was peeled and cleaned, subsequently
used for color development, and glucose standards were used for esti-
soaked in water for 12–16 h, then grated and pressed to remove water.
mation of sugar. The absorbance was read with a spectrophotometer
The pressed mash obtained was sun-dried for 3–4 days. Fresh green and
(model Genesys G10S, USA) at 490 nm.
firm banana was also peeled, cleaned, and then cut into small pieces
before being sun-dried for 2 days. All samples were dried/roasted to a
2.2.3. Mineral analysis
moisture content of 7.5 ± 0.5 g water/100 g, ground to obtain a fine
Potassium (K) in composite flour samples was determined using a
powder, and passed through a 750 µm mesh sieve.
Flame photometer (model 410, Sherwood Scientific Ltd., UK); analysis
The composition and ratio of the control (CON) was selected, based
of phosphorus (P) was performed using the UV spectrophotometer
on a commercial product. The ratio of maize, sorghum, sesame, and
(model 7305, Jenway, UK). Contents of calcium (Ca), iron (Fe), mag-
soybean flours were fixed in all treatments (Table 1); the composition of
nesium (Mg), and zinc (Zn) were analyzed using the Atomic Absorption
wheat flour was partly (P) and fully (F) replaced by cassava (C), rice
Spectrophotometer (AAS) (model 205, Buck Scientific, USA). All mea-
(R), and banana (B) flours. In each treatment, all flours were accurately
surements were carried out in triplicate.
Table 1
Seven different treatments of composite flours. 2.2.4. Determination of antinutrients
The method of Latta and Eskin (1980) was modified to determine
Composition (g/100 g) CON1 P-C F-C P-R F-R P-B F-B
the phytate content. Phytate was extracted from 1 g of composite flour
Maize flour 30 30 30 30 30 30 30 with 20 mL of 2.4% (v/v) hydrochloric acid (HCl) by shaking at room
Wheat flour 25 10 – 10 – 10 – temperature for 2 h, followed by high speed centrifugation of the sus-
Sorghum flour 15 15 15 15 15 15 15 pension for 15 min. The supernatant decanted and filtered through
Sesame flour 10 10 10 10 10 10 10 Whatman No. 1 filter paper. A 3 mL aliquot of filtrate was diluted to
Soybean flour 20 20 20 20 20 20 20
Cassava flour – 15 25 – – – –
18 mL with distilled water and the diluted sample was passed through a
Rice flour – – – 15 25 – – 200–400 mesh AG1-X8 chloride anion exchange resin (Bio-Rad La-
Banana flour – – – – – 15 25 boratories GmbH, München, Germany). Inorganic phosphorus was
Total 100 100 100 100 100 100 100 eluted with 0.07 M sodium chloride (NaCl) followed by elution of
1 phytate with 0.7 M NaCl. Phytate was determined colormetrically based
CON = control with 25 g wheat flour /100 g composite flour.
on pink color of Wade reagent, which is formed upon the reaction of the
P-C = partial substitution of wheat flour with 15 g/100 g of cassava flour.
F-C = full substitution of wheat flour with 25 g/100 g of cassava flour. ferric ion and sulfosalicyclic acid. One milliliter of Wade reagent
P-R = partial substitution of wheat flour with 15 g/100 g of rice flour. (0.03% solution of FeCl3⋅6H2O containing 0.3% (v/v) sulfosalicyclic
F-R = full substitution of wheat flour with 25 g/100 g of rice flour. acid in water) was added to 3 mL of the clear supernatant sample and
P-B = partial substitution of wheat flour with 15 g/100 g of banana flour. then centrifuged at 2000 rpm for 15 min. The absorbance was measured
F-B = full substitution of wheat flour with 25 g/100 g of banana flour. by a spectrophotometer (model Genesys G10S, USA) at 500 nm. The

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P. Udomkun, et al. Food Chemistry 286 (2019) 651–658

phytate content was calculated using a standard phytic acid curve and (mins) were aflatoxin-B1 7.62; aflatoxin-G1 4.01; aflatoxin-B2 5.21; and
results were expressed as mg/100 g. aflatoxin-G2 2.53. To assess the reliability of the method, standards for
Tannin content was determined by weighing 0.5 g of a composite aflatoxin-B1, aflatoxin-G1, aflatoxin-B2, and aflatoxin-G2 were spiked
flour sample and then adding 5 mL of 1% (v/v) of HCl in methanol. The into blank flour samples at three concentration levels: 2.25, 3.75, and
sample was allowed to stand at room temperature for 15 min before 5.25 mg/kg. The selection of these concentration levels was based on
vortex mixing and centrifugation at 3000 rpm for 10 min. The super- the level of background contamination of collected samples. On
natant of 2.5 mL was transferred to a 10 mL flask containing 7.5 mL of average, 95.5% of aflatoxin-B1 was recovered; 101.2% of aflatoxin-G1;
water, then 0.5 mL of Folin-Denis regent and 1 mL of sodium carbonate 98.5% of aflatoxin-B2; and 99.7% of aflatoxin-G2.
were added. The final volume was adjusted to 10 mL with water, and
the absorbance was determined after 30 min of incubation at room 2.3. Statistical analyses
temperature using a UV/Vis spectrophotometer (model Genesys G10S,
USA) at 760 nm. The content of tannin was calculated from the stan- Statistical analysis was performed using the general linear model.
dard curve of tannic acid solution. Least significant difference (LSD) was used to estimate the significant
Total oxalate content was assayed using the method of Savage, differences among the means for each treatment at 5% probability level
Vanhanen, Mason, and Rose (2000) with a slight modification. Total using the SAS program (Ver. 9.4, SAS Inst., Cary, NC, USA).
oxalates were extracted from 0.5 g of composite flour samples with
50 mL of 0.2 M HCl and then incubated in a water bath at 80 °C for 3. Results and discussion
15 min. The extracts were allowed to cool to room temperature before
being transferred to a 100 mL volumetric flask; the final volume was 3.1. Nutritional composition
adjusted with water. Subsequently, the extracted samples were cen-
trifuged at 2800 rpm for 15 min. The supernatant was filtered through a The result showed that the moisture content of all composite flours
0.45 µm cellulose acetate filter (Sartorious, Göttingen, Germany) prior was in the range of 7.2–7.7 g water/100 g, with no statistically sig-
to analysis with High-Performance Liquid Chromatography (HPLC). nificant difference between the flours (Table 2). A significant increase
The HPLC system used consisted of a binary pump (model ProStar 210, (P < 0.05) of protein (17.5–18.5 g/100 g) and fat content
Varian, USA), auto-sampler (model Triathlon, Spark, Holland) with a (10.1–11.0 g/100 g) was found in partial (P) and full (F) replacement
UV detector set at 210 nm (model Waters 2487 Dual λ, absorbance with rice flours (P-R and F-R) when compared with the corresponding
detector, Waters Corporation, USA). Separation was conducted in a values of other samples. The higher level of protein observed in re-
Supelco reversed phase column (4.6 mm I.D. × 250 mm length). The placement with rice could be an advantage to those who suffer from
mobile phase was 0.25 g/100 mL dehydrogenate phosphate and protein deficiency, assisting them to regain loss of functions, including
0.0025 mol/L tetrabutyl ammonium hydrogen sulphate buffered at pH growth and maintenance of enzymes, hormones, neurotransmitters, and
2.0 with ortho-phosphoric acid. The column temperature was set at body tissues. An increase of dietary fat would be able to compensate for
room temperature and the total running time was 20 min at a flow rate reduced carbohydrate-derived energy and facilitate the absorption of
of 1 mL/min. The injection volume was 5 µl. Identification of oxalic fat-soluble vitamins as well as increasing palatability by absorbing and
acid was carried out by comparing the HPLC retention time of unknown retaining flavors. However, it is a concern that cereal-legume-based
peaks to the chromatogram with added standard. foods are known to be susceptible not only to deterioration by microbial
growth, especially mycotoxins but also prone to lipid oxidation (Temba,
2.2.5. Determination of molar ratio of antinutrients to minerals Njobeh, & Kayitesi, 2017). Thus, more attention needs to be paid to the
The predicted bioavailability of dietary minerals was determined storage conditions for these composite flours.
based on the molar ratio (Gemede et al., 2015). The molar ratio of The content of crude fiber (2.8–2.9 g/100 g) and ash (2.9–3.0 g/
phytate and oxalate to calcium, iron, or zinc was obtained after dividing 100 g) was significantly higher (P < 0.05) in both partial and full
the mole of the antinutrient with the mole of the mineral. substitution by banana flours (P-B and F-B). Total carbohydrate content
accounted for more than 60 g/100 g in control (CON), partial replace-
2.2.6. Aflatoxin analysis ment with cassava (P-C), full replacement with cassava (F-C), P-B, and
Composite flour samples were extracted according to the procedure F-B samples. The carbohydrate content was significantly lower
described by ISO (2003) method number 16,050, with minor mod- (P < 0.05) in P-R and F-R. Even though the lower carbohydrate con-
ifications. Briefly, 12.5 g of sample was weighed into a 100 mL amber tent is statistically significant, it is less of a concern as the total car-
glass conical flask, and 3 g of sodium chloride was added, followed by bohydrate content is still high. There was no significant difference in
50 mL of methanol and purified water (30 mL: 20 mL). The mixture was total sugar content (2.4–2.9 g/100 g) and gross energy value
shaken (model HS 501 D Shaker, IKA, Germany) at 200 rpm for 1 h. The (396.1–402.2 kcal/100 g) among all composite flours, suggesting that
suspension was allowed to settle before being filtered through they can serve as a good source of energy. Additionally, the energy
Whatman No. 1 paper. Fifty milliliters of the filtrate were collected into values obtained from composite flours were in accordance with the
a conical flask, then 10 mL of the filtrate was placed in a beaker and recommendations of WHO/FAO (2003) which specify energy-supplying
30 mL of phosphate buffer solution was added. The pH value was then micronutrients, for example, protein (10–15% of total energy), total fat
adjusted to 7.4 using 0.1 mol/L sodium hydroxide (NaOH). The sample (15–30% of total energy), and total carbohydrate (55–75% of total
was passed through an immune-affinity column (AflaStar TM R-IAC, energy), could prevent chronic non-deficiency diseases.
Romer Labs Inc., Missouri, USA), followed by a 20 mL rinse with ultra- Minerals are essential in metabolism and homeostasis of the human
pure water. Total aflatoxins were eluted with 1.5 mL of water-free body. Deficiency of minerals can lead to symptoms of common dis-
methanol (HPLC grade). orders and diseases (Gharibzahedi & Jafari, 2017). In this study, sig-
A HPLC system (Agilent 1260, Agilent Technologies Inc., USA) nificantly higher (P < 0.05) amounts of potassium (600.8–1182.1 mg/
consisting of a binary pump, auto sampler, fluorescence detector, re- 100 g) and phosphorus (215.2–257.8 mg/100 g) were observed in all
verse phase column C18 (Hypersil ODS, 4.6 × 100 mm, 5 µm), and samples, particularly in F-R and F-B, when compared with CON
column heating chamber was used for the separation and quantification (Table 2). The contents of calcium (1580.3–1623.6 mg/100 g) and iron
of aflatoxins. The mobile phase consisted of an isocratic mixture of (13.9–14.5 mg/100 g) were highest (P < 0.05) in P-C and F-C samples.
water/acetonitrile (75 mL: 25 mL) and flow rate was set at 1.0 mL/min. Partial and full replacement of wheat flour with banana flour (P-B and
The fluorescence detector was set at an excitation wavelength of F-B) led to a significant reduction (P < 0.05) of iron content. Among
360 nm and an emission wavelength of 423 nm. The retention times composite flours, the lowest amount of magnesium (191.1–210.4 mg/

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P. Udomkun, et al. Food Chemistry 286 (2019) 651–658

Table 2
Proximate and mineral concentrations of composite flours.
Quality2 CON1 P-C F-C P-R F-R P-B F-B

Proximate (g/100 g)
Moisture 7.4a (1.0) 7.2a (1.2) 7.5a (0.8) 7.7a (0.5) 7.5a (0.8) 7.6a (1.0) 7.7a (0.9)
Protein 15.2b (1.2) 15.5b (0.9) 15.8b (1.3) 17.5a (1.4) 18.5a (1.1) 12.1c (1.2) 13.8c (1.5)
Fat 8.8b (0.6) 7.7b (1.1) 7.9b (0.8) 10.1a (1.0) 11.0a (0.8) 8.1b (0.8) 8.3b (1.1)
Crude fiber 1.8b (0.2) 2.1b (0.3) 2.2b (0.2) 2.2b (0.1) 2.3b (0.3) 2.8a (0.2) 2.9a (0.2)
Ash 1.9b (0.1) 2.2b (0.3) 2.2b (0.1) 2.2b (0.0) 2.4b (0.2) 2.9a (0.1) 3.0a (0.2)
Carbohydrate 64.9a (0.8) 65.3a (1.2) 64.4a (1.1) 60.3b (1.0) 58.3b (1.1) 66.5a (0.9) 64.3a (1.2)
Total sugar 2.6a (0.0) 2.4a (0.1) 2.5a (0.1) 2.8a (0.2) 2.9a (0.1) 2.5a (0.2) 2.7a (0.1)
Gross energy (kcal/100 g) 399.7a (1.3) 398.8a (1.3) 396.8a (1.5) 402.1a (1.8) 402.2a (1.0) 396.3a (1.5) 396.1a (1.2)

Minerals (mg/100 g)
Potassium (K) 582.2f (2.5) 600.8e (2.2) 650.3d (3.1) 959.2b (2.5) 1182.1a (2.4) 878.9c (2.0) 1008.5b (1.9)
Phosphorus (P) 193.5d (1.9) 241.9b (2.2) 252.2a (1.2) 240.2b (3.0) 257.8a (2.6) 215.2c (2.5) 248.7ab (2.7)
Calcium (Ca) 817.8c (5.3) 1580.3a (4.8) 1623.6a (3.2) 778.0 cd (2.7) 719.2d (4.1) 858.7bc (2.3) 958.4b (3.0)
Iron (Fe) 10.4c (0.1) 13.9b (0.2) 14.5a (0.3) 9.8c (0.1) 10.6c (0.1) 5.8d (0.0) 4.1d (0.2)
Magnesium (Mg) 224.3bc (3.4) 210.4c (2.4) 191.1d (2.0) 219.8c (2.8) 230.7b (2.5) 225.6bc (3.2) 244.0a (1.1)
Zinc (Zn) 5.2c (0.3) 4.1d (0.3) 3.4e (0.2) 6.2b (0.2) 8.3a (0.1) 5.2c (0.1) 4.8d (0.1)

1
CON = control with 25 g wheat flour /100 g composite flour.
P-C = partial substitution of wheat flour with 15 g/100 g of cassava flour.
F-C = full substitution of wheat flour with 25 g/100 g of cassava flour.
P-R = partial substitution of wheat flour with 15 g/100 g of rice flour.
F-R = full substitution of wheat flour with 25 g/100 g of rice flour.
P-B = partial substitution of wheat flour with 15 g/100 g of banana flour.
F-B = full substitution of wheat flour with 25 g/100 g of banana flour.
2
All values are expressed as mean (standard deviation) (N = 3).
a-f
, values within a row with different superscript letters are significantly different (P < 0.05).

100 g) was found in P-C and F-C samples, whereas P-R and F-R con- during the fermentation process. The low content of phytate in cassava
tained the highest amount of zinc (6.2–8.3 mg/100 g). flour might therefore be a result of the fermentation process as Marfo,
Simpson, Idowu, and Oke (1990) observed a substantial reduction of
phytate in cassava after 72 h of fermentation.
3.2. Antinutrient composition Nissar, Ahad, Naik, and Hussain (2017) stated that phytate content
should be lowered as much as possible, ideally ≤ 25 mg/100 g or 3% of
Phytic acid, known as myo-inositol hexakisphosphate (IP6), has the phytate-containing food should be consumed to minimize the mi-
been found to be effective in chelating metal ions. These health benefits cronutrient losses. The Reference Daily Intake (RDI) value of phytate
of phytic acid have been identified as having anti-carcinogenic and varied by country; for example, 180 mg RDI/ day has been specified for
antioxidant properties (Campos-Vega, Loarca-Piña, & Oomah, 2010). Sweden, whereas UK and USA accept 631–746 mg RDI/ day (Nissar
On the other hand, phytic acid can form very stable, insoluble com- et al., 2017). As phytate is heat stable, heat treatment is not able to
plexes with minerals such as calcium, zinc, and iron (Bohn, Meyer, & degrade it substantially owing to the formation of either insoluble
Rasmussen, 2008) as well as the chelation of amino acids, thereby de- complexes between phytate and other macro- and micronutrients, such
creasing mineral and amino acid bioavailability (Kruger et al., 2013). In as phytate–protein and phytate–protein–mineral complexes, or the
this study, the replacement of rice flour in P-R and F-R samples showed penta- and tetra-phosphate hydrolyzed products of inositol hexapho-
the significantly highest (P < 0.05) content of phytic acid sphate (Siddhuraju & Becker, 2001). However, some thermal degrada-
(198.4–211.6 mg/100 g) when compared to other flours, while the tion could have occurred during pressurized steam cooking as presented
content was significantly lower in composite flour with cassava flour as by Wu, Ashton, Simic, Fang, and Johnson (2018) in sorghum flaked
a replacement (Table 3). This is in an agreement with a finding of breakfast cereals.
Svanberg, Lorri, and Sandberg (1993) who report that phytases, which Tannins are water-soluble polyphenols found in plant-based foods
are special phosphatase enzymes, can catalyze the hydrolysis of phytate (Sehrawat, Sharma, & Sultana, 2006). Though tannins have many
in crops into lower inositol phosphates and inorganic phosphorus

Table 3
Antinutritional factor contents of composite flours.
Antinutrient (mg/100 g)2 CON1 P-C F-C P-R F-R P-B F-B

c d e a b c
Phytate 170.8 (2.3) 155.5 (4.2) 142.4 (3.1) 211.6 (2.8) 198.4 (2.2) 168.2 (3.1) 153.7d (4.5)
Tannin 161.2c (2.3) 122.3e (3.1) 95.6f (2.0) 142.5d (4.2) 120.8e (1.8) 222.5b (3.7) 250.8a (4.1)
Oxalate 32.2a (1.9) 29.9a (2.1) 27.4a (2.4) 31.4a (1.5) 29.9a (2.2) 29.2a (1.2) 27.1a (1.7)

1
CON = control with 25 g wheat flour /100 g composite flour.
P-C = partial substitution of wheat flour with 15 g/100 g of cassava flour.
F-C = full substitution of wheat flour with 25 g/100 g of cassava flour.
P-R = partial substitution of wheat flour with 15 g/100 g of rice flour.
F-R = full substitution of wheat flour with 25 g/100 g of rice flour.
P-B = partial substitution of wheat flour with 15 g/100 g of banana flour.
F-B = full substitution of wheat flour with 25 g/100 g of banana flour.
2
All values are expressed as mean (standard deviation) (N = 3).
a-f
, values within a row with different superscript letters are significantly different (P < 0.05).

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P. Udomkun, et al. Food Chemistry 286 (2019) 651–658

health-promoting properties such as antioxidant, anti-carcinogenic, in a reduction of calcium absorption (Brogren & Savage, 2003). The
anti-inflammatory, anti-arteriosclerotic, and anti-microbial activities molar ratio of [oxalate]: [calcium] has been reported as hazardous
(Kumar et al., 2010; Mazni, Ho, Azizul, & Nurdin, 2016), their anti- when it is > 2.0 (Israr, Frazier, & Gordon, 2013); the inhibitory effect of
nutritional properties have been reported, including reduction of pro- phytate on iron absorption is dose-dependent (Gibson et al., 2010) and
tein digestibility, leading to poor absorption of iron (Chung, Wong, Wei, can occur at a very low phytate concentration. Although Hurrell et al.
Huang, & Lin, 1998). The result in this study showed that the re- (1992) indicated that a strong inhibitory effect of phytate on iron exists
placement of banana flour in P-B and F-B samples significantly in- even when the [phytate]: [iron] molar ratio is as low as 0.2, the critical
creased (P < 0.05) tannin content (310.4–332.1 mg/100 g) in compo- value of the [phytate]: [iron] molar ratio of < 1.0 is accepted as phy-
site flours when compared with CON; the content was significantly tate begins to lose its inhibitory effect on iron absorption at this level
lower in flours in which rice (120.8–142.5 mg/100 g) and cassava (Hurrell, 2004). Zinc absorption is also a dose-dependent response
(95.6–122.3 mg/100 g) were the replacements (Table 3). Fekadu et al. (Nävert & Sandström, 1985) and poor availability of zinc is encountered
(2013) indicated that the anti-nutritional effect of tannins depends on when the molar ratio of [phytate]: [zinc] is > 15 (Sandberg, Anderson,
chemical structure and dosage and the acceptable total daily intake of Carlesson, & Sandström, 1987). In a study by Hunt and Beiseigel
tannic acid for a man is 560 mg/100 g. Therefore, the content of tannin (2009), the amount of zinc absorbed from a 1-day diet was reduced by
in composite flours might not cause a toxic effect on ingestion. 25% (1 mg) when the [phytate]: [zinc] molar ratio was increased from
Oxalate is a metabolic end-product of ascorbate, glyxylate, and 4 to 15. Ellis et al. (1987) recommended a ≤10 [phytate]: [zinc] molar
glycine metabolism in humans. Oxalate content tends to be highest in ratio for adequate zinc bioavailability. It could be seen that the phytate
the leafy part of vegetables rather than in the seeds and stems (Savage, or oxalate to mineral (calcium and zinc) molar ratios of all composite
2000). There are two major forms of oxalates, soluble and insoluble. flours were lower than the reported critical values, while phytate to
Soluble oxalates are mostly bound to sodium, calcium, and ammonium iron was significantly higher. Therefore, a significant adverse effect of
ions, while insoluble oxalates are bound to calcium, magnesium, and the composite flours, if any was indicated on the absorption of iron was
iron ions (Savage et al., 2000). Insoluble oxalates are less absorbed in indicated.
the digestive tract and are excreted in the feces (Massey, 2003). High Interestingly, the effect of phytate on the bioavailability of minerals
intake of soluble oxalate can cause calcium oxalate crystallization and generally depends not only on the phytate content in the staple food but
the formation of kidney stones (nephrolithiasis) in the urinary tract also on the interaction between phytate and minerals (Ma, Li, Zhai,
(Morozumi & Ogawa, 2000). Kok, & Yang, 2007). Some studies have reported that high dietary
There was no significant difference in oxalate content calcium impairs zinc absorption in the presence of a high intake of
(27.1–32.2 mg/100 g) among composite flours; however, a reduction phytate (Gibson et al., 2010; Gemede et al., 2015). Therefore, the molar
was found at full replacement by cassava, rice, and banana flours ratio of [phytate][calcium]/[zinc] might be better used as an indicator
(Table 3). By the presence of oxalate content in composite flours, pa- of zinc bioavailability than the molar ratio of [phytate]: [zinc] alone
tients with kidney stone should be carefully considered as they are (Obah & Amusan, 2009). The molar ratio of [phytate][calcium]/[zinc]
advised to limit total oxalate intake to not more than 50–60 mg/day of < 0.5 mol/kg has been positively associated with zinc absorption
(Massey, Palmer, & Horner, 2001). However, the adverse effect of (Adetuyi, Osagie, & Adekunle, 2011). From the [phytate][calcium]/
consuming food that are high oxalate-containing has been shown to be [zinc] molar ratio of cereal-legume based composite flours, a sig-
considerably reduced by forming insoluble oxalate with calcium-rich nificantly higher amount (P < 0.05) of [phytate][calcium]/[zinc] was
foods (Faudon & Savage, 2014). Also, processing methods such as found in composite flour where cassava flour had been the replacement,
soaking and cooking may cause considerable skin rupture and facilitate while only full replacement with rice flour (F-R) had a value less than
the leakage of soluble oxalate, resulting in low available oxalate for the critical level.
absorption in the digestive tract (Savage et al., 2000). To lower the inhibitory effect of phytate on minerals in plant-based
complementary foods, some methods such as food preparation and
3.3. Molar ratios and predicted mineral bioavailability processing as well as fortification with micronutrients including zinc
have been suggested (Gibson & Holz, 2001; Gibson et al., 2006). A
As the concentrations of phytate and oxalate were high in studied study by Hurrell, Reddy, Juillerat, and Cook (2003) exhibited that
cereal–legume-based composite flours, it is important to consider their complete phytate degradation can improve the absorption of iron in
effects on the bioavailability of critical minerals, calcium, iron, and cereal porridge when being prepared with water but not with milk. This
zinc. The molar ratios of antinutrients to minerals are important for finding indicates that it is difficult to predict the fraction of mineral
predicting the potential mineral bioavailability, with lower molar ratios absorption since it can be varied by several factors such as the com-
indicative of higher mineral bioavailability. From the molar ratio of the position and preparation methods of ingested food, and age and nu-
antinutritional factor to minerals in composite flours, it could be seen tritional status of the target group (Mazariegos et al., 2006; Gibson
that the [phytate]: [calcium] molar ratio ranged from 0.005 to 0.017; et al., 2010).
[phytate]: [iron], 0.833–3.181; [phytate]: [zinc], 2.354–4.125; [ox-
alate]: [calcium], 0.017–0.042; and [phytate][calcium]/[zinc], 3.4. Aflatoxin contamination
0.423–1.674 mol/kg (Table 4). In addition, the molar ratio of [phytate]:
[calcium] was significantly lower (P < 0.05) in P-C and F-C samples, All composite flours in this study were contaminated with aflatoxins
while the molar ratio of [phytate]: [iron] was significantly affected and levels ranged from 2.6 to 8.8 µg/kg (Table 5). Among the samples,
(P < 0.05) by the replacement with banana flour. A significant in- a high concentration of total aflatoxins was significantly detected
crease (P < 0.05) of [phytate]: [zinc] molar ratios was observed in P-C (P < 0.05) in CON. High levels of aflatoxin-B1 were found in all
and F-C samples when compared with CON, while F-R showed the composite flours, followed by aflatoxin-G1, and aflatoxin-B2. However,
lowest values. The molar ratio of [oxalate]: [calcium] was significantly aflatoxin-G1 was not detected in P-B and F-B samples, whereas all
decreased (P < 0.05) by the replacement with cassava and banana samples were not contaminated by aflatoxin-G2. Although all samples
flours. met the proposed East Africa regulatory threshold of 10 µg/kg
Generally, the suggested critical value of the [phytate]: [calcium] (Udomkun et al., 2017), CON, P-C, P-R, and P-B did not meet the EU
molar ratio is < 0.17 (Umeta, West, & Fufa, 2005). However, Morris regulatory threshold for total aflatoxins of 4.0 µg/kg (EC, 2007,2010).
and Ellis (1985) mentioned that a [phytate]: [calcium] molar ratio > In general, aflatoxin contamination in agricultural commodities can
0.24 will diminish calcium absorption. In addition, phytate was found be compounded by a wide range of pre- and post-harvest factors such as
to increase available soluble oxalate, which can bind calcium, resulting high temperature and drought conditions (Paterson & Lima, 2010;

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P. Udomkun, et al. Food Chemistry 286 (2019) 651–658

Table 4
Molar ratios of antinutritional factor to mineral of composite flours.
Molar ratio2 CON1 P-C F-C P-R F-R P-B F-B

b c c a a b
Phytate: Calcium 0.013 (0.003) 0.006 (0.002) 0.005 (0.002) 0.016 (0.004) 0.017 (0.003) 0.012 (0.002) 0.010b (0.001)
Phytate: Iron 1.393e (0.011) 0.949f (0.008) 0.833f (0.005) 1.832c (0.012) 1.588d (0.014) 2.460b (0.010) 3.181a (0.018)
Phytate: Zinc 3.235c (0.038) 3.735b (0.031) 4.125a (0.028) 3.361c (0.057) 2.354e (0.021) 3.185d (0.046) 3.153d (0.032)
Oxalate: Calcium 0.039a (0.003) 0.019d (0.002) 0.017d (0.003) 0.040a (0.002) 0.042a (0.001) 0.034b (0.005) 0.028c (0.002)
Phytate × Calcium: Zinc3 0.661d (0.011) 1.476b (0.009) 1.674a (0.018) 0.654d (0.022) 0.423e (0.018) 0.684d (0.019) 0.756c (0.017)

1
CON = control with 25 g wheat flour /100 g composite flour.
P-C = partial substitution of wheat flour with 15 g/100 g of cassava flour.
F-C = full substitution of wheat flour with 25 g/100 g of cassava flour.
P-R = partial substitution of wheat flour with 15 g/100 g of rice flour.
F-R = full substitution of wheat flour with 25 g/100 g of rice flour.
P-B = partial substitution of wheat flour with 15 g/100 g of banana flour.
F-B = full substitution of wheat flour with 25 g/100 g of banana flour.
2
All values are expressed as mean (standard deviation) (N = 3).
3
The unit of [Phytate][Calcium]/[Zinc] is mol/kg.
a-g
, values within a row with different superscript letters are significantly different (P < 0.05).

Bandyopadhyay et al., 2016; Kamika, Ngbolua, & Tekere, 2016), poor polished rice. Although there is no study on aflatoxin contamination in
farm practices (Hell, Mutegi, & Fandohan, 2010; Kang’ethe et al., banana flour, the result might be related to the presence of polyphenols
2017), and a lack of drying facilities and inappropriate storage practices in banana. Several polyphenols, such as gallic acid, catechins, epica-
(Matumba et al., 2017). Interestingly, the highest aflatoxin con- techins, anthocynins, and other flavonoid derivatives, are reported to
tamination in this study was found in all samples composed of wheat be essentially found in banana (Bennett et al., 2010; Pereira &
flour. This might be attributed to poor storage of wheat flour in the Maraschin, 2015). Scaglioni, Souza, Schmidt, and Badiale-Furlong
markets as well as poor governmental regulations and legislations on (2014) suggested that the polyphenol compounds have an ability to
aflatoxin contamination in the country. inhibit α-amylase enzyme—an important enzyme for microbial
In addition, the low level of aflatoxin contamination in composite growth—from the fungal species Aspergillus oryzae.
flour with full replacement by cassava flour could be explained by the
fact that the lactic acid bacteria and/or some microorganisms, such as
Saccharomyces cerevisiae strains obtained from the fermentation process 4. Conclusions
during the 12–16 h of soaking in cassava flour production, have the
ability to bind or degrade aflatoxins (Shetty, Hald, & Jespersen, 2007). This study provides data on nutrients, antinutrients, molar ratios of
Moreover, Rodriguez, Buschmann, Iglesias, and Beeching (2000) men- the anti-nutritional factor to minerals (mineral availability), and afla-
tioned that scopoletin—a thermostable coumarin phytoalexin with toxin content in cereal-legume-based composite flours with partial and
fungicidal properties—may also contribute to reducing aflatoxin-B1 in full replacement of wheat flour by local crops such as cassava, rice, or
cassava. In the case of rice flour, Pitt, Taniwaki, and Cole (2013) re- banana flour. Although the modified composite flours were observed to
ported that rice cultivated with an irrigation system leads to low levels be rich in some macro- and micro-nutrients and low in aflatoxin con-
of aflatoxin in the final product but the contamination becomes more tamination, the level of antinutrients should be addressed as it was
probable when rice seeds are collected wet and then dried. Likewise, found to influence the bioavailability of minerals, especially in diets
the milling process during polished rice production has been shown to that are high in phytate and calcium but low in zinc as well as iron.
decrease the level of aflatoxin in rice (Katsurayama et al., 2018). Sales At the household level, food preparation (soaking, germination, or
and Yoshizawa (2005) showed that the level of aflatoxins in brown rice fermentation) and processing methods (boiling or roasting) can be used
was reduced by 78% after being processed to polished rice, while a to reduce some antinutrients, while fortification with micronutrients,
study by Trucksess, Abbas, Weaver, and Shier (2011) indicated that including minerals, at appropriate levels can be an alternative option
aflatoxin was reduced from 158 µg/kg in brown rice to 56 µg/kg in for the commercial level. In conclusion, these composite flours are re-
commended as a practical and sustainable approach for improving

Table 5
Aflatoxin concentration in composite flours.
Aflatoxin (µg/kg)2 CON1 P-C F-C P-R F-R P-B F-B

Aflatoxin-B1 5.6a (0.2) 4.4b (0.1) 1.5c (0.0) 3.4b (0.1) 2.2c (0.1) 4.3b (0.2) 2.6bc (0.1)
Aflatoxin-G1 3.1a (0.1) 0.2b (0.0) 0.8b (0.0) 0.7b (0.1) 0.5b (0.0) ND ND
Aflatoxin-B2 0.1a (0.0) 0.1a (0.0) 0.3a (0.0) 0.1a (0.0) 0.2a (0.0) 0.5a (0.0) 0.2a (0.0)
Aflatoxin-G2 ND3 ND ND ND ND ND ND
Total aflatoxins 8.8a (0.2) 4.7b (0.1) 2.6c (0.0) 4.2b (0.1) 2.9c (0.0) 4.8b (0.1) 2.8c (0.1)

1
CON = control with 25 g wheat flour /100 g composite flour.
P-C = partial substitution of wheat flour with 15 g/100 g of cassava flour.
F-C = full substitution of wheat flour with 25 g/100 g of cassava flour.
P-R = partial substitution of wheat flour with 15 g/100 g of rice flour.
F-R = full substitution of wheat flour with 25 g/100 g of rice flour.
P-B = partial substitution of wheat flour with 15 g/100 g of banana flour.
F-B = full substitution of wheat flour with 25 g/100 g of banana flour.
2
All values are expressed as mean (standard deviation) (N = 3).
3
ND = non-detected.
a-d
, values within a row with different superscript letters are significantly different (P < 0.05).

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P. Udomkun, et al. Food Chemistry 286 (2019) 651–658

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Project (PJ-002057)” which is funded by the International Fund for Gibson, R. S., & Holz, C. (2001). Dietary diversification/modification strategies to en-
hance micronutrients content and bioavailability of diets in developing countries.
Agricultural Development (IFAD) and the European Commission (EC). British Journal of Nutrition, 85, S159–S166.
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Conflict of interest
maize for Sub-Saharan Africa. 10th International working conference on stored product
protection, Julius-Kühn-Archiv p. 425.
The authors have no conflict of interests. Hunt, J. R., & Beiseigel, J. M. (2009). Dietary calcium does not exacerbate phytate in-
hibition of zinc absorption by women from conventional diets. The American Journal
of Clinical Nutrition, 89, 839–843.
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(1992). Soy protein, phytate, and iron absorption in humans. The American Journal of
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