Acta Univ.

Sapientiae, Alimentaria, 16 (2023) 75–89

DOI: 10.2478/ausal-2023-0006

Mineral and anti-nutritional properties

of pearl millet and pumpkin leaf flour
as affected by fermentation
O. A. Ojo1,* A. Oshin1
e-mail: [email protected] e-mail: [email protected]

O. A. Obadina1,2,3 E. K. Oke1
e-mail: [email protected] e-mail: [email protected]
1
Department of Food Science and Technology, Federal University of Agriculture,
Abeokuta, Ogun State, Nigeria
2
Department of Biotechnology and Food Technology, University of Johannesburg,
Doornfontein Campus, South Africa
3
Department of Microbiology, Babcook University, Ilisan, Ogun State, Nigeria

Abstract. This study was aimed at determining the mineral and anti-
nutritional properties of naturally fermented millet and pumpkin leaf flour
blends. The millet grains were allowed to ferment spontaneously for 24 hrs
and 48 hrs and were processed into flour. Dried pumpkin leaves were blended
into flour and substituted using D-optimal mixture design, which resulted
in ten experimental runs. The mineral content and the anti-nutritional
properties of the flour blend formulation were analysed. Duncan’s multiple
range test was used to evaluate the mean at p < 0.05 with SPSS version 21.0.
Significant differences were observed in the mineral and anti-nutritional
composition of the fermented millet and pumpkin leaf flour blends at 24
hrs and 48 hrs of fermentation time respectively. Calcium, potassium, and
iron content increased significantly (p < 0.05) with increasing the amount
of pumpkin leaf flour in the flour blends. The values for tannins and total
phenolic composition ranged from 0.089 to 0.162% and from 0.075 to 0.120%
for 24 hrs and from 0.080 to 0.141% and from 0.060 to 0.120% for 48 hrs of
fermentation time respectively. Results showed that fermentation technique
could be used to enrich the nutritional and bioactive potential of millet.

Keywords and phrases: tannin, phytate, trypsin inhibitor, phenol, D-optimal

mixture
76 O. A. Ojo, A. Oshin, O. A. Obadina, E. K. Oke

1. Introduction
Millet is a seeded grass that is widely grown for human and animal nutrition all
over the world (Ranasalva & Visvanathan, 2014). It is an important crop in semi-arid
tropics of Africa and Asia, particularly in Nigeria, due to its ability to grow under
adverse weather conditions such as little rainfall, no fertilizer availability, or various
other scarcities; therefore, they are mostly recommended for farmers having difficult
circumstances (Soumya et al., 2016). The most common types of cultivated millet
are foxtail millet (Setaria itallica), finger millet (Eleusine coracona), pearl millet
(Pennisetum typhoideum), and proso millet (Panicum miliaceum) (Thilagavathi
et al., 2015). Pearl millet provides higher energy compared to other cereal grains
such as rice and wheat and is considered a significant source of nutrients such as
calcium, potassium, thiamine, niacin, and riboflavin (Shweta, 2015). Millet can be
used in producing food and beverage products such as papad, muruku, bread, hot
kolukattai, milk, malt beverage, and alcoholic beverage (Singh et al., 2021; Mahajan
et al., 2021). Due to the presence of tannins, polyphenolics, and phytic acid, the
bioavailability of these nutrients is low. They are typically considered anti-nutrients
and have been associated with inhibitory effect on protein and starch digestibility
and mineral bioavailability. Studies have shown that these phytochemicals can be
reduced substantially by food processing operations such as dehulling, malting,
fermentation, and heat treatment (Marston et al., 2016).
Fermentation is one of the oldest and widely used methods for processing millet,
especially in Africa and other developing countries where modern food preservation
methods are rarely available. It is a process involving the transformation of substrates
(millets) into new products through the action of certain microorganisms (lactic
acid bacteria and yeasts). This is regarded as an economical traditional processing
technique adopted to yield large amounts of products. During fermentation, enzymes
are activated through the action of microorganisms leading to changes in the pH; this
is in addition to other biochemical changes that occur during this process leading to
the modification of the substrate (Adebiyi et al., 2018; Srivastava et al., 2020). These
biochemical changes contribute to preservative properties, improved flavours, and
significant increase in nutritional properties (Obilana et al., 2014). Fermentation also
ensures the safety of the food by suppressing the growth and survival of undesirable
microflora. Fermentation has been reported to effectively improve the nutritional
quality of millet by increasing protein content, in-vitro protein digestibility (IVPD),
and mineral extractability (Ranasalva & Visvanathan, 2014; Adebiyi et al., 2018).
Other significant roles of fermentation in millet processing include the development
of a wide variety of flavours, aromas, and textures, detoxification, and a decrease in
cooking time (Jay et al., 2005; Adebiyi et al., 2018).
Other sources of nutrients, such as legumes and leaves, could be used to
supplement cereal flours in complementary porridge and bakery products to
Mineral and anti-nutritional properties of pearl millet and pumpkin leaf… 77

improve the nutritional and sensory qualities of such products (Chikondi et al.,
2018). The consumption of vegetables, such as pumpkin leaves, has a significant
impact on human health: they typically protect against chronic diseases and
contain large amounts of iron, folic acid, vitamin A and C. It is a drought-tolerant
plant; the young shoots and leaves are used in cooking soups, yam and vegetable
sauces and also for medicinal purposes (Mashiane et al., 2021). Deficiency of
iron (anaemia) is a major problem in underdeveloped and developing countries
(Llanos et al., 2016). The causes of anaemia can be classified into blood loss,
reduced production and increased destruction of red blood cells (Thilagavathi et
al., 2015). Debasmita and Binata (2017) reported that the major cause of anaemia
is the inadequate nutrient intake of iron, vitamins A, B12, C, folic acid, niacin
and pantothenic acid, which are also responsible for maintaining the level
of haemoglobin in the blood. To prevent the growing rate of anaemia, dietary
improvement, fortification, and supplementation are beneficial ways for the entire
population or for certain groups of people. The nutritional potentials of millet and
fluted pumpkin leaves as composite flour in food products would enhance food
security and improve the overall health status of consumers of such formulated
food products (Adeyeye, 2016). Many researchers (Onuoha et al., 2017; Srivastava
et al., 2020; Devi & Rajendran, 2021; Azeez et al., 2022) have worked on the
fermentation of pearl millet with other legumes, however, not in combination
with pumpkin leaves. Therefore, the objective of this work is to determine the
effect of fermentation on the minerals and anti-nutritional properties of flours from
fermented pearl millet and fluted pumpkin leaves.

2. Materials and methods

Millet grains and fluted pumpkin leaves were procured in 2022 at the Odo-Eran
market, Abeokuta Ogun State, Nigeria.

Fermented millet flour production

Fermented millet flour was produced according to the method described by

Adebayo-Oyetoro et al. (2017). The millet grains were cleaned and soaked in
potable water for 24 hrs. They were wet milled and sieved through a fine-mesh
sieve (muslin cloth). The millet slurry was allowed to ferment naturally in clean
plastic bucket for 24 hrs and 48 hrs. The fermented meal was then pressed using
a fine-mesh sieve to produce the fermented cake. The cake was dried using a
cabinet drier (LEEC Limited, Serial No 3114, United Kingdom) at 60oC for 10 hrs
and milled with a milling machine (Fritsch, D-55743, Idaroberstein-Germany) to
produce the fermented millet flour and was packaged in polyethylene bags.
78 O. A. Ojo, A. Oshin, O. A. Obadina, E. K. Oke

Fluted pumpkin leaf flour production

Fluted pumpkin leaf flour was produced using the modified method of Lawal et
al. (2021). Pumpkin leaves were washed thoroughly with tap water to remove dirt
and sorted to remove stalks. The green leaves were drained using a plastic sieve,
then sliced and dried at 60°C for 6 hrs using cabinet drier (LEEC Limited, Serial
No 3114, United Kingdom). Dried pumpkin leaves were milled using blender,
cooled and stored in high-density polyethylene bags.

Formulation of fermented pearl millet and fluted pumpkin leaf flour

blends

Using D-optimal design, different blends of composite flour samples were

prepared by combining fermented pearl millet and fluted pumpkin leaf flour in
the following ratios: 93.75:6.25, 91.25:8.75, 90.00:10.00, 90.00:10.00, 95.00:5.00,
92.50:7.50, 92.50:7.50, 90.00:10.00, 95.00:5.00, and 100.00:0.00.

Mineral analysis of fermented pearl millet and fluted pumpkin leaf

flour blends

The mineral content of the samples was assessed using the procedure of AOAC
(2000). Calcium, iron, sodium, and potassium were measured using atomic absorption
spectrophotometer (Thermo scientific S Series Model GE 712354) after they have been
digested with perchloric-nitric acid mixture. Prior to digestion, 0.50 g of samples were
weighed into a 125-ml Erlenmeyer flask, over which 4 ml of concentrated perchloric-
nitric acid, 25 ml of concentrated nitric acid, and 2 ml of concentrated sulphuric acid
were added under a fume hood. The contents were mixed, heated gently in a digester
(Buchi Digestion unit K-424) with low to medium heat on a hot plate under perchloric
acid fume hood, and heating was continued for about 30 s until the appearance of
dense white fume. Then it was allowed to cool followed by addition of 50 ml distilled
water. The solution was allowed to cool and filtered completely with a wash bottle
into a Pyrex volumetric flask and then made up with distilled water. The solution
was then read on atomic absorption spectrophotometer.

Determination of tannins

The determination of tannin content was done using the method of Swain
(1979). Ground sample (0.2 g) was measured into a beaker (50 ml), 20 ml of 50%
methanol was added, covered, and placed in 80oC water bath for about 1 h and was
stirred to avoid clumping. Double-layered Whatman No. 1 filter paper was used
in the filtration of solution into a volumetric flask (100 ml), and 50% methanol
was used for rinsing. It was made up to the mark with distilled water and mixed;
Mineral and anti-nutritional properties of pearl millet and pumpkin leaf… 79

an extract of 1 ml was pipetted into a volumetric flask (50 ml); 20 ml of distilled

water and 10 ml of 17% Na2C03 and Folin–Denis reagent were thoroughly mixed.
This was made up with distilled water and allowed to stand for about 20 min until
a bluish-green colour was visible. Standard tannic acid solutions in the range of
0–10 (µg/L) were treated similarly to the 1 ml of the sample. The absorbance of
tannic acid standard solutions as well as the sample were read on the Spectronic
21D Spectrophotometer at a wavelength of λ = 760 nm.

Determination of trypsin inhibitor activity

Determining trypsin inhibitor activity was done by the method described by

Kakade et al. (1974). Flour sample of 1 g was extracted with 50 ml of 0.01 M of
NaOH for 1 h, and the pH of the resulting slurry was adjusted to between 9.4 and
9.6 with 1 M NaOH or 1 M HCI. At this stage, the slurry was shaken and left at 4°C
overnight; but occasionally it was more convenient to stir at ambient temperature
for 3 hrs or to macerate continuously (Ultra-Turrax) for 2 min. After extraction, the
suspension was shaken and diluted with water, so an amount of 1 ml produced
trypsin inhibition of between 40% and 60%. Trypsin solution of 2 ml was added
to the test tube; the tubes were placed in 37°C water bath and were allowed to
stay there for 10 min. Then 1 ml of 30% acetic acid was added and the contents
were thoroughly mixed and filtered with Whatman No. 3. The absorbance was
measured at λ = 410 nm against a blank reagent.

Determination of total phenolic content

The method described by Turturică et al. (2016) was used in the determination
of total phenolic content. 1 g of sample was ground with pestle and mortar with
the addition of 10 ml 80% ethanol. The mixture was centrifuged at 10,000 rpm
for 20 min. The supernatant was kept, and the residue was further extracted with
80% ethanol, centrifuged, and exposed for evaporation to take place. The residue
was to dissolve in 5 ml of distilled water. The sample was measured into the test
tubes, and the volume of each was made up with distilled water to 3 ml. 0.5 ml of
Folin–Ciocâlteu reagent was added to the test tubes. After about 3 min, 2 ml sodium
carbonate (20%) solution was added to each tube. The contents of the test tubes
were mixed thoroughly, and the tubes were placed in a water bath for 1 min. It was
allowed to cool, and absorbance was read at λ = 650 nm against a blank reagent.

Determination of phytate

The method described by Maga (1982) was used in the determination of phytate.
2 g of sample was soaked in 20 ml of 0.2 M of HCl for 3 hrs and filtered. The filtrate
(0.5 ml) was mixed with 1 ml of 70% ferric-ammonium-sulphate solution in a test
80 O. A. Ojo, A. Oshin, O. A. Obadina, E. K. Oke

tube, boiled for 30 min in the water bath (100°C), cooled in ice, and centrifuged
at 3,000 rpm for 15 min. 1 ml of the supernatant was mixed thoroughly with 0.1
M of 2,2-pyridine solution (1.5 ml), and the absorbance was read at λ = 519 nm
using spectrophotometer.

Statistical analysis

Each analysis was carried out in triplicate. Mean values of the three replicates’
results were subjected to one-way analysis of variance (ANOVA) to determine
the significant difference, and the means were separated using Duncan’s multiple
range test at 95% confidence level (p < 0.05).

3. Results and discussions

The mean values for the mineral composition of fermented millet grains (FM)
and pumpkin leaf flour (PF) at 24 hrs and 48 hrs of fermentation time are shown
in figures 1–2. Significant (p < 0.05) difference was observed in the mineral
composition of 24 hrs and 48 hrs of fermentation time. The mean values of calcium
ranged from 49.49 mg/100 g to 206.39 mg/100 g and from 51.86 mg/100 g to 202.90
mg/100 g for 24 hrs and 48 hrs of fermentation time respectively. Calcium increased
Mineral and (p
significantly anti-nutritional
< 0.05) with properties of pearl in
increase millet and pumpkin
pumpkin leafflour
leaf flour…in the flour blends.
7

300

250
Mineral composition (mg/100g)

200
Calcium
150
Potassium
100 Sodium
Iron
50

0
0 5 5 6.25 7.5 7.5 8.75 10 10 10
Pumpkin flour (w/w%)

Figure 1. Mineral composition of 24 hrs fermented millet

Figure 1. Mineral composition of 24 hrs fermented millet and pumpkin leaf flour
and pumpkin leaf flour
350
n (mg/100g)

300

250
 0
0 5 5 6.25 7.5 7.5 8.75 10 10 10
Pumpkin flour (w/w%)
Mineral and anti-nutritional properties of pearl millet and pumpkin leaf… 81
Figure 1. Mineral composition of 24 hrs fermented millet and pumpkin leaf flour

Mineral composition (mg/100g) 350

300

250

200 Calcium

150 Potassium
Sodium
100
Iron
50

0
0 5 5 6.25 7.5 7.5 8.75 10 10 10
Pumpkin flour (w/w%)
Figure
Figure 2. 2. composition
Mineral Mineral composition of 48 hrs
of 48 hrs fermented fermented
millet millet
and pumpkin leaf flour
and pumpkin leaf flour
7
The regression coefficient of the flours at 24 hrs and 48 hrs of fermentation time
are shown in tables 1–2. Their linear and interaction effect was not significant
(p > 0.05) on the calcium content of fermented millet flour and fluted pumpkin
leaves for 24 hrs and 48 hrs. The coefficients of determination (R2) values were
0.51 and 0.70 for the flour blends of 24 hrs and 48 hrs of fermentation time
respectively.
Potassium content for the fermented millet and pumpkin leaf flour at 24 hrs
and 48 hrs ranged from 145.15 mg/100 g to 245.02 mg/100 g and from 200.92
mg/100 g to 316.11 mg/100 g respectively. Potassium increased significantly (p
< 0.05) with increase in pumpkin leaf flour in the flour blends. The least value
at both the 24 hrs and 48 hrs millet fermentation period was observed in 0%
PF formulation blends, while the maximum values were observed at 10% PF
formulation blends. In addition, the interaction effect of 24 hrs fermented millet
flour and fluted pumpkin leaf had significant effect (p < 0.05) on the potassium
content of the formulation blend. However, the linear effect of 48 hrs fermented
millet flour and fluted pumpkin leaves, as well as the interaction effect had no
significant effect (p > 0.05) on the potassium content of formulation blends.
The coefficients of determination (R2) values were 0.84 and 0.62 respectively
for the flour blends of 24 hrs and 48 hrs fermented time. This indicates that the
equation is a good fit in predicting the potassium content of the flour blend.
There was at first a decrease and then later an increase in the potassium content
of the formulation blends in both fermentation periods as the fluted pumpkin
leaf flour decreased.
82 O. A. Ojo, A. Oshin, O. A. Obadina, E. K. Oke

Table 1. Regression coefficient of mineral composition of fermented millet and

pumpkin leaf flour at 24 hrs
Parameter Calcium Potassium Sodium Iron
A 120.03 218.41 149.49 79.81
B 147.57 243.17 134.56 85.80
AB 51.84 -71.33* 31.03 4.70
F-value 0.53 15.42 1.82 14.20
R2 0.509 0.8371 0.5779 0.8256
Notes: * Significant at (p < 0.05); A: fermented millet flour, B: fluted pumpkin leaf flour,
AB: interaction effects of fermented millet and pumpkin leaf flour, R2: coefficient of
determination.

Table 2. Regression coefficient of mineral composition of fermented millet and

pumpkin leaf flour at 48 hrs
Parameter Calcium Potassium Sodium Iron
A 120.58 297.02 126.93 81.40
B 148.92 315.65 120.22 90.51
AB 48.96 -56.47 37.92 11.00
F-value 0.61 4.88 1.02 10.85
R2 0.701 0.620 0.541 0.783
Notes: * Significant at (p < 0.05); A: fermented millet flour, B: pumpkin leaf flour,
AB: interaction effects of fermented millet and pumpkin leaf flour, R2: coefficient of
determination.

The sodium content of fermented millet grains and pumpkin leaf flour ranged
from 132.13 mg/100 g to 173.98 mg/100 g and from 120.70 mg/100 g to 155.10
mg/100 g for 24 hrs and 48 hrs of fermentation time respectively. Sodium decreased
significantly (p < 0.05) with increase in pumpkin leaf flour in the blends. The
least values were observed in 8.75% PF formulation blends, while the maximum
values were observed in 6.25% PF formulation blends. The maximum values were
observed in 93.75% FM and 6.25% PF formulation blends, whereas the least values
were observed in 90% FM and 10% PF formulation blends. The main and the
interaction effect showed no significant (p > 0.05) effect on the sodium content of
formulation blend for 24 hrs and 48 hrs of fermentation time for millet flour and
fluted pumpkin leaves. The coefficients of determination were 0.58 and 0.54 for
the flour blends of 24 hrs and 48 hrs respectively.
The mean values of the iron content of the fermented millet grains and
pumpkin leaf flour blends ranged from 76.30 mg/100 g to 86.19 mg/100 g and
from 79.63 mg/100 g to 94.34 mg/100 g for 24 hrs and 48 hrs of fermentation
Mineral and anti-nutritional properties of pearl millet and pumpkin leaf… 83

time respectively. Iron content increased significantly (p < 0.05) with increase
in pumpkin leaf flour in the flour blends. The linear and interactive effect of 24
hrs and 48 hrs of fermentation time for millet flour and fluted pumpkin leaves
does not show a significant level (p > 0.05) regarding the iron content of the
formulation blends. The coefficients of determination (R2) values were 0.83 and
0.78, respectively, for the flour blends of 24 hrs and 48 hrs of fermentation time
for millet flour and fluted pumpkin leaves. This indicates that the equation is a
good fit in predicting the iron content of the flour blend. There was a decrease
in the iron content of the formulation blends in both fermentation periods as the
fluted pumpkin leaf flour decreased.
The mean values for the anti-nutritional composition of the formulated blends
of 24 hrs and 48 hrs fermentation time for millet grains and pumpkin leaf flour
are shown in tables 3–4. Tannin content ranged from 0.089 to 0.162% and from
0.080 to 0.141% respectively.

Table 3. Anti-nutritional composition of fermented millet and

pumpkin leaf flour at 24 hrs
PF Tannin Trypsin Total phenolic Phytate
(w/w%) (%) inhibitor (%) (%) (%)

6.25 0.135 ± 0.00b 0.024 ± 0.00a 0.120 ± 0.00k 0.004 ± 0.01bc

8.75 0.134 ± 0.00 h
0.040 ± 0.00a
0.106 ± 0.01 g
0.004 ± 0.01bc
10.00 0.141 ± 0.11lm 0.027 ± 0.00a 0.111 ± 0.00i 0.004 ± 0.01bc
10.00 0.142 ± 0.00mn 0.026 ± 0.00a 0.112 ± 0.00ij 0.004 ± 0.01bc
5.00 0.138 ± 0.00i 0.024 ± 0.00a 0.107 ± 0.00gh 0.004 ± 0.01bc
7.50 0.161 ± 0.00o 0.029 ± 0.01a 0.119 ± 0.01k 0.004 ± 0.00bc
7.50 0.162 ± 0.00 o
0.029 ± 0.01a
0.119 ± 0.01 k
0.006 ± 0.01bc
10.00 0.143 ± 0.00n 0.029 ± 0.00a 0.113 ± 0.00j 0.006 ± 0.01e
5.00 0.139 ± 0.00ij 0.025 ± 0.00a 0.108 ± 0.00bc 0.005 ± 0.00de
0.00 0.089 ± 0.00a 0.063 ± 0.00a 0.075 ± 0.00a 0.001 ± 0.00a
Notes: Mean values with different superscripts within the same column are significantly
different (p < 0.05); PF: pumpkin flour.

As shown in Table 5, the linear effect of 24 hrs fermentation time for millet
and fluted pumpkin leaves, as well as the interaction had no significant (p > 0.05)
effect on the tannin content of the blend formulation. However, the interaction
of 48 hrs fermentation time for millet flour and fluted pumpkin leaves had a
significant (p < 0.05) effect on the tannin content of formulation blend, as shown
in Table 6.
84 O. A. Ojo, A. Oshin, O. A. Obadina, E. K. Oke

Table 4. Anti-nutritional composition of fermented millet and

pumpkin leaf flour at 48 hrs
PF Tannin Trypsin Total phenolic Phytate
(w/w%) (%) inhibitor (%) (%) (%)
6.25 0.119 ± 0.00f 0.021 ± 0.00a 0.093 ± 0.00e 0.003 ± 0.01a
8.75 0.127 ± 0.00g 0.032 ± 0.15a 0.103 ± 0.00f 0.003 ± 0.01a
10.00 0.139 ± 0.01 ij
0.027 ± 0.00 a
0.076 ± 0.00 bc
0.003 ± 0.01a
10.00 0.140 ± 0.01jk 0.028 ± 0.00a 0.077 ± 0.00cd 0.004 ± 0.00bc
5.00 0.109 ± 0.00c 0.021 ± 0.01a 0.106 ± 0.00g 0.003 ± 0.01a
7.50 0.111 ± 0.01d 0.021 ± 0.01a 0.119 ± 0.00k 0.003 ± 0.01a
7.50 0.113 ± 0.01e 0.022 ± 0.01a 0.120 ± 0.00k 0.003 ± 0.01ab
10.00 0.141 ± 0.00 kl
0.029 ± 0.00 a
0.076 ± 0.00 d
0.005 ± 0.00de
5.00 0.110 ± 0.00c 0.022 ± 0.01a 0.107 ± 0.00gh 0.004 ± 0.00bc
0.00 0.080 ± 0.00a 0.014 ± 0.00a 0.060 ± 0.00a 0.003 ± 0.01a
Notes: Mean values with different superscripts within the same column are significantly
different (p < 0.05); PF: pumpkin flour.

Table 5. Regression coefficient of anti-nutritional composition of fermented

millet and pumpkin leaf flour at 24 hrs
Parameter Tannin Trypsin Total phenolic Phytate
inhibitor
A 0.14 8.2985 × 10-3 0.11 4.440 × 10-3
B 0.14 0.049 0.11 4.612 × 10-3
AB 0.049 0.20 0.030 2.019 × 10-4
F-value 1.35 0.50 2.05 0.020
R2 0.4102 0.7438 0.60 0.76
Notes: * Significant at p < 0.05); A: fermented millet flour, B: pumpkin leaf flour, AB:
interaction effects of fermented millet and pumpkin leaf flour, R2: coefficient of determination.

The coefficients of determination (R2) values were 0.41 and 0.93, respectively, for
the flour blends of 24 hrs and 48 hrs fermentation time for millet flour and fluted
pumpkin leaves. This is an indication that the equation is a poor fit in predicting
the tannin content of the flour blend of 24 hrs fermentation time for millet flour
and fluted pumpkin leaves, but it can accurately predict the tannin content of the
flour blends of 48 hrs fermentation time for millet flour and fluted pumpkin leaves.
Tannin intake has been implicated to cause depletion in the digestive enzymes
which are responsible for the secretion and production of endogenous protein,
Mineral and anti-nutritional properties of pearl millet and pumpkin leaf… 85

the malfunctioning of the digestive tract, and the toxic effect resulting from the
metabolites (Jan et al., 2022).

Table 6. Regression coefficient of anti-nutritional composition of fermented

millet and pumpkin leaf flour at 48 hrs
Parameter Tannin Trypsin Total phenolic Phytate
inhibitor
A 0.11 0.014 0.10 3.514 × 10-3
B 0.14 0.039 0.077 3.971 × 10-3
AB -0.039* 0.084 0.091* -3.326 × 10-3
F-value 40.34 0.48 10.87 1.92
R2 0.9308 0.84 0.7838 0.5906
Notes: * Significant at p < 0.05); A: fermented millet flour, B: pumpkin leaves flour,
AB: interaction effects of fermented millet and pumpkin leaves flour, R2: coefficient of
determination.

The value of the trypsin inhibitor for the formulated blends of 24 hrs fermentation
time for millet and pumpkin leaf flour ranged from 0.024% to 0.063% resp. On the
other hand, the mean values of trypsin inhibitor for the formulated blends of 48 hrs
fermentation time for millet and pumpkin leaf flour ranged from 0.014% to 0.032%,
resp., as shown in Table 6. The main effect of the 24 hrs and 48 hrs fermentation
time for millet and fluted pumpkin leaf flour, as well as the interaction, had no
significant (p > 0.05) effect on the trypsin inhibitor of flour blends. In addition,
the R2 values were 0.74 and 0.84 for both the flour blends at 24 hrs and 48 hrs
respectively. This indicates that the equation is a good fit in predicting the trypsin
inhibitor content of the flour blend. There was first an increase and then later a
decrease in the trypsin inhibitor content of the formulation blends at 24 hrs and
48 hrs as the fermented millet flour increased. However, the fact that the trypsin
inhibitors are heat-labile suggests that they can be destroyed through processing
such as grinding and cooking (Venter & van Eyssen, 2001; Adane et al., 2013).
The values of the total phenolic content for the formulated blends at 24 hrs
fermentation time for millet grains and pumpkin leaf flour ranged from 0.075
to 0.120%. The least values were observed in 100% FM and 0% PF formulation
blends, while the maximum values were observed in 93.75% FM and 6.25%
PF formulation blends. The mean values of the total phenolic content for the
formulated blends of 48 hrs fermentation time for millet grains and pumpkin leaf
flour ranged from 0.060 to 0.120%. The maximum values were observed in 92.50%
FM and 7.50% PF formulation blends, whereas the least values were observed in
100% FM and 0% PF formulation blends. The linear effect of 24 hrs fermentation
time for millet flour and fluted pumpkin leaves, as well as the interaction, had
86 O. A. Ojo, A. Oshin, O. A. Obadina, E. K. Oke

no significant (p > 0.05) effect on the total phenolic content of the formulation
blend. However, the interaction of 48 hrs fermentation time for millet flour and
fluted pumpkin leaves had a significant (p < 0.05) effect on the total phenolic
content of flour blends. The coefficients of determination (R2) values were 0.60
and 0.78 for the flour blends of 24 hrs and 48 hrs fermentation time for millet and
fluted pumpkin leaf flour respectively. The total phenolic content was observed to
increase gradually upon the substitution levels of fermented millet and thereafter
decrease at higher substitution levels notwithstanding the varying fermentation
periods. During fermentation, the factors’ conditions (temperature, time, and pH),
the microorganism species present as well as the grain type all have significant
effect on phenolic compounds (Jan et al., 2022).
The values for the phytate content of the flour blends of 24 hrs fermentation
time for millet grains and pumpkin leaf flour ranged from 0.004 to 0.006%.
However, the mean values for the phytate content of the formulated blends of
48 hrs fermentation time for millet grains and pumpkin leaf flour ranged from
0.003% to 0.005%. The linear effect of 24 hrs and 48 hrs fermentation time
for millet flour and fluted pumpkin leaves, as well as the interaction, had no
significant (p > 0.05) effect on the phytate content of formulation blend. Also,
the coefficients of determination (R2) values were 0.76 and 0.59, respectively,
for the flour blends of 24 hrs and 48 hrs fermentation time for millet flour and
fluted pumpkin leaves. There was a slight decrease in the phytate content of
the formulation blends at 24 hrs as fermented millet flour increased and fluted
pumpkin leaf flour decreased. However, the phytate content of the formulation
blends at 48 hrs fermentation time for millet flour increased as the fluted pumpkin
leaf flour decreased – it was observed to initially decrease and then later increase
at higher levels of fluted pumpkin leaves.

Conclusions
The study shows the effect of fermentation time on minerals and anti-nutritional
factors of flour blends from pearl millet and pumpkin leaves. The calcium,
potassium, sodium, and iron content of the flour blends from fermented millet and
pumpkin leaf flour increased significantly at 24 hrs and 48 hrs fermentation time,
while tannin, total phenolic, and phytate decreased significantly at both 24 hrs and
48 hrs fermentation time. Calcium, potassium, and iron were maximized, while
sodium tannin, trypsin inhibitor, total phenolic, and phytate were set to “none”.
However, the optimized solutions from the D-optimal design were fermented
millet flour of 90.81% and pumpkin leaf flour of 9.19% at 24 hrs and fermented
millet flour of 90.52% and pumpkin leaf flour of 9.48% at 48 hrs fermentation
time respectively.
Mineral and anti-nutritional properties of pearl millet and pumpkin leaf… 87

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