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\begin{document}

\title{Effect of fermentation duration on the microstructure and selected
nutritional quality of acha ( Digitaria exilis) flour and acceptability
of cookies production}



\author[1]{Sunday Sobowale}%
\author[1]{Oluwole Omotoso}%
\author[2]{Joy Ikedichi Agbawodike}%
\affil[1]{Moshood Abiola Polytechnic}%
\affil[2]{New Mexico State University}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
This study investigated the effect of fermentation durations on the
\emph{acha} flour and resultant cookies. The \emph{acha} sample was
fermented at 24, 48 and 72 h, while the native \emph{acha} flour served
as the control sample. The functional, pasting, nutritional and
micro-structural properties of the fermented \emph{acha} flour and
sensory evaluation of the cookies were examined. The results indicate
significant differences (p\textless{}0.05) in the functional and pasting
properties of the flour and cookies samples. An increase in fermentation
duration enhances oil absorption capacity and dispersibility of the
samples. The protein and ash content of the \emph{acha} flour samples
increased. The mineral element showed significant difference
(p\textless{}0.05) in the calcium, sodium, and zinc content. The Fourier
transform infrared (FTIR) spectra increase the band intensities. The
peaks occurred in the range 3860 - 3650 cm \textsuperscript{-1}. The
X-ray diffraction (XRD) pattern revealed a slight increase in the
crystallinity of the fermented flour samples. The flour samples
exhibited an A-type XRD pattern while the cookies samples had V-type XRD
pattern. Scanning electron microscopy (SEM) revealed that fermentation
modified the microstructure of the flour, causing it to change from an
irregular compact granular to a regular loose structure. Cookies samples
produced from native and fermented \emph{acha} flour were acceptable by
the panelist. Therefore, cookies with improved nutritional and
acceptable sensory properties were produced from native and \emph{acha}
flour fermented between 24 and 72 h. This suggests that fermented
\emph{acha} flour holds promise for various food applications, including
complementary foods, and gel formulations with reduced syneresis.%
\end{abstract}%



\sloppy


\textbf{Effect of fermentation duration on the microstructure and
selected nutritional quality of \emph{acha} (\emph{Digitaria
exilis)}flour and acceptability of cookies production}

\textbf{Running title}

Influence of fermentation on quality of cookies

\textbf{Sunday Samuel Sobowale\textsuperscript{1,2}*, Benjamin Oluwole
Omotoso\textsuperscript{1} and Joy Ikedichi
Agbawodike\textsuperscript{3}}

\textsuperscript{1}Department of Food Technology, Moshood Abiola
Polytechnic, Abeokuta, Ogun State, Nigeria.

\textsuperscript{2}Department of Food Science and Technology, College of
Basic and Applied Science, Mountain Top University, Kilometre 12,
Lagos-Ibadan Expressway, Prayer City, Ogun State, Nigeria.

\textsuperscript{3}Department of Family and Consumer Science, New Mexico
State University, Las Cruces, NM, USA.

*Correspondence author: sobowale.sam@gmail.com; sssobowale@mtu.edu.ng

\textbf{Abstract}

This study investigated the effect of fermentation durations on
the\emph{acha} flour and resultant cookies. The \emph{acha} sample was
fermented at 24, 48 and 72 h, while the native \emph{acha} flour served
as the control sample. The functional, pasting, nutritional and
micro-structural properties of the fermented \emph{acha} flour and
sensory evaluation of the cookies were examined. The results indicate
significant differences (p\textless{}0.05) in the functional and pasting
properties of the flour and cookies samples. An increase in fermentation
duration enhances oil absorption capacity and dispersibility of the
samples. The protein and ash content of the \emph{acha} flour samples
increased. The mineral element showed significant difference
(p\textless{}0.05) in the calcium, sodium, and zinc content. The Fourier
transform infrared (FTIR) spectra increase the band intensities. The
peaks occurred in the range 3860 - 3650 cm\textsuperscript{-1}. The
X-ray diffraction (XRD) pattern revealed a slight increase in the
crystallinity of the fermented flour samples. The flour samples
exhibited an A-type XRD pattern while the cookies samples had V-type XRD
pattern. Scanning electron microscopy (SEM) revealed that fermentation
modified the microstructure of the flour, causing it to change from an
irregular compact granular to a regular loose structure. Cookies samples
produced from native and fermented \emph{acha} flour were acceptable by
the panelist. Therefore, cookies with improved nutritional and
acceptable sensory properties were produced from native and
\emph{acha}flour fermented between 24 and 72 h. This suggests that
fermented\emph{acha} flour holds promise for various food applications,
including complementary foods, and gel formulations with reduced
syneresis.

\textbf{KEYWORDS}

\emph{Acha} flour and cookies, fermentation, nutritional quality,
micro-structural properties

\textbf{1 INTRODUCTION}

The annual cereal crop ``\emph{acha} ''~(Digitaria exilis and Digitaria
iburua) is native to West Africa and is grown there for its edible
grains and straw. Known by many as hungry rice or folio, it is a member
of the graminae family (Sobowale \emph{et al} . 2023; Alamu,
2001).\emph{Acha} are generally eaten whole, possibly as a result of
their small shape (Ayo \emph{et al.,} 2018). When the whole~grain is
consumed, it serves as a great source of dietary fiber and has
additional nutriceuticals benefits that are helpful for those who are
health-conscious and for conditions like diabetes and obesity. According
to Karsada (2001), \emph{acha} ~posses nutritional qualities and like
other developing ancient grains, it is thought to represent the highest
quality of~minerals, vitamins, amino acids, and~fiber, which makes it
potential for use in the development of new products (Sobowale et al.
2021a).

\emph{Acha} 's low protein and fiber content enables it excellent baking
properties and the potential to considerably enhance~economic status,
wellness, and whole grain diets (Ayo and Kajo, 2016). One of the most
popular kinds of quick snacks is cookies (Farheena \emph{et al.,} 2015).
According to Olatidoye \emph{et al} . (2019b), cookies are nutritious
snacks~made from unappealing dough that is heated in an oven to create a
tasty final product. These are well-known~bakery products
and~ready-to-eat snacks with a long shelf life and~widespread
consumption and can serve as vehicles for important nutrient
(Ajibola\emph{et al.,} 2015).

Fermented foods are of great importance due to their ability to supply
and preserve large amounts of nutritious foods with a diverse range of
aroma, texture and flavor that enhance the human diet
(Olatidoye\emph{et} \emph{al.,} 2019a). In addition to preserving food,
fermentation process~produces~essential enzymes, omega-3 fatty acids,
and B-vitamins. \emph{Acha} grains contain abundant fermentable
carbohydrates that, when reaching the gut, exhibit greater probiotic
efficacy compared to wheat, increasing the number of lactobacilli and
bifida bacteria in the feces of humans~(Fayemi \emph{et al} ., 2022).

The potential of utilizing fermentation processes to
transform\emph{acha} flour into various products, including
confectionery, ready-to-eat food (RTE), weaning food, and breakfast
cereals, remains largely untapped (Vodouhe \emph{et al} ., 2012).
Despite \emph{acha}flour's availability in the market, its incorporation
into processed food products is currently underutilized. \emph{Acha}
stands out as one of the most nutritionally rich grains, containing
essential amino acids like methionine and cystine (Sobowale et. al.,
2021b). These amino acids play a crucial role in supplying sulfur and
other compounds necessary for normal metabolism and growth within the
body. The nutritional advantages of incorporating fermented \emph{acha}
into food products, particularly in the production of cookies, have not
been thoroughly explored. This oversight brings about a vital
examination into the potential benefits and effects of utilizing
fermented \emph{acha} flour into cookies production in alleviating the
prevailing food insecurity.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Materials and Equipments}

The \emph{Acha} grains were bought in Lagos State, Nigeria, at Mile 12
Market (7.5 \textsuperscript{o}N and 4.5 \textsuperscript{o}E).
Additional ingredients, including eggs, milk, margarine, sugar, and
baking powder, were procured from local Market in Abeokuta, Ogun State,
Nigeria.

\textbf{2.2 Sample preparation}

2.2.1 Preparation of native \emph{acha} flour

\emph{Acha} flour was prepared following Ayo \emph{et al.} (2007)
method. Dust and chaff~were taken out by winnowing the grains. Residual
dust and stones were washed away through sedimentation using large
bowls. The cleaned and de-stoned grains were subjected to drying in a
cabinet dryer at 45 \selectlanguage{ngerman}°C. After drying, the grains underwent milling using
an attrition milling machine, and the resulting flour was sieved to
achieve a particle size passing through a 50 \selectlanguage{greek}µ\selectlanguage{english}m mesh. An airtight
container was used to package the \emph{acha} flour.

2.2.2 Preparation of fermented \emph{acha} flour

Fermented \emph{acha} flour was prepared as described by Onweluzo and
Nwabugwu (2009). \emph{Acha} grains were soaked in distilled water and
left to ferment for 3 days. Following fermentation, the soaked water was
drained, and the seeds were dried in a cabinet dryer for 24 h at 40 \selectlanguage{ngerman}°C.
Subsequently, the dried grains underwent milling using an attrition
milling machine, and the resulting flour was sifted to achieve a
particle size passing through a 500 \selectlanguage{greek}µ\selectlanguage{english}m mesh.

2.2.3 Preparation of \emph{acha} cookies

About 100\% \emph{acha} -based cookies was prepared and baked as
described by Okpala \emph{et al.} (2011). The formulation comprised of
200 g of \emph{acha} flour (native flour, fermented four, malted flour)
2 g of salt, 2 g of baking powder, 62 g of egg, 80 g of margarine, 50 g
of sugar, 15.6 g of milk and 10 ml of vanilla flavor. Margarine and
sugar were blended to uniformity with an electric mixer operating at a
medium speed for 5 min. Eggs and milk were incorporated into the mixture
and mixed for duration of 30 min. Additional ingredients, including
flour, salt, vanilla flavor, and baking powder, were thoroughly blended
to make the dough. The dough was kneaded to achieve a consistent
thickness of 0.5 cm, and then circular cuts with a diameter of 5 cm were
made. The dough was placed in the oven and baked at 150 \selectlanguage{ngerman}°C for 30 min,
followed by a cooling.

\textbf{2.3} \textbf{Functional properties}

The water and oil absorption capacities was determined using the method
described by Adeyinka \emph{et al.,} (2016). The method explained
by~Adeleke and Odedeji (2010) was used to determine bulk density.
Swelling capacity was determined using the method described by
Sobowale\emph{et al} . (2021a).

\textbf{2.4} \textbf{Pasting Properties}

The Rapid Visco Analyzer (RVA) (Techmaster parten N103802 Australia) was
used to determine the pasting properties, as reported by Li \emph{et
al.} (2011). After weighing about 3.5 g of the samples into an empty,
dry canister, 25 ml of distilled water was poured into it. The solution
was well mixed, and the~canister was properly placed into the RVA as
specified. The slurry was heated to a temperature of~50 and
90\textsuperscript{o}C for 2 min. At a heating rate of 11.25 °C/min, the
pasting profile, including final viscosity, peak viscosity, setback,
breakdown, pasting temperature, and peak time, was obtained using
thermocline for windows software connected to a computer.

\textbf{2.5 Nutritional quality}

2.5.1 Proximate Composition

The proximate composition of \emph{acha} flour and cookies was
determined where protein was measured using the Kjeldahl method (N X
6.25), and moisture, ash, and fat were determined using methods 934.01,
996.06, and 923.03 of AOAC (2000).

2.5.2 Mineral Content

The mineral composition of \emph{acha} flour and cookies was determined
according to AOAC, (2000) method. 10 ml~of 5 N concentrated
hydrochloride were used to digest 5 g~of the samples. The mixtures were
set on a water bath and evaporated nearly to dryness. The solution~was
allowed to cool, then sieved into a~standard flask of 100 ml
capacity~and diluted with distilled water to its full volume. Following
the acid digestion of the sample, the minerals were analyzed separately
with an atomic absorption spectrophotometer.

\textbf{2.6 Total phenolic content (TPC) and Total flavonoid content
(TFC)}

2.6.1 Total phenolic content (TPC)

Using Ainsworth and Gillepsie's (2007) Folin-Ciocalteu procedure, the
TPC of the flours and cookie extracts was evaluated. A solution of 10 \selectlanguage{greek}µ\selectlanguage{english}L
extract and 50 \selectlanguage{greek}µ\selectlanguage{english}L Folin-Ciocalteu phenol reagent (Sigma Aldrich,
Germany) was added to 500 \selectlanguage{greek}µ\selectlanguage{english}L of distilled water. After letting it stand
in the dark for three minutes, 200 \selectlanguage{greek}µ\selectlanguage{english}L of 20\% (g/v)
Na\textsubscript{2}CO\textsubscript{3} (Sigma Aldrich, Germany) was
added, followed by the addition of 245 \selectlanguage{greek}µ\selectlanguage{english}L of distilled water. The
mixture (300 \selectlanguage{greek}µ\selectlanguage{english}L) was precisely pipetted into a 96-well microplate,
covered with aluminum foil, and then incubated in darkness for 30 min.
The plate's absorbance was then measured using a micro plate reader
(iMark, Biorad, South Africa) at 750 nm. The standard utilized was
garlic acid (Sigma Aldrich, Germany), and the results were reported as
mg GAE/g sample.

2.6.2 Total flavonoid content (TFC)

The TFC was determined using the method proposed by Ar-Farsi and Lee
(2008), involving the mixing of 30 \selectlanguage{greek}µ\selectlanguage{english}L of the extract with 20 \selectlanguage{greek}µ\selectlanguage{english}L of 36 mM
NaNO\textsubscript{2}. About 20 \selectlanguage{greek}µ\selectlanguage{english}L of 94 mM AlCl\textsubscript{3} was
then added. A micro plate reader (iMark, Biorad, South Africa) was used
to measure the mixture's absorbance at 450 nm. Using catechin (Sigma
Aldrich, Germany) as~a reference, the results were reported as mg of
catechin equivalents per g of the sample (mg CE/g sample).

\textbf{2.7 Micro-structural properties of \emph{Acha} flour and
cookies}

2.7.1 Fourier transmission-infrared (FTIR) spectroscopy

Fourier transmission-infrared spectroscopy analysis was carried out
using the method of Adebiyi \emph{et al.} (2016); Animashaun and
Sobowale (2024). An FTIR spectrophotometer (Thermo Scientific Smart iTR,
(Attenuated Total Reflectance), Thermo Scientific Inc., USA) was used to
determine the samples' FTIR spectra. Before sample (0.5 g of
\emph{acha}flour and cookies) were mounted on the instrument, the
background spectra of the instrument were collected and at 16 runs per
scan, the records of the spectra were taken with characteristics peaks
in wave numbers ranging from 400 to 4000 cm\textsuperscript{-1} at.

2.7.2 X-ray diffraction (XRD)

The Adebiyi et al. (2016) method was used to conduct the X-ray
diffraction analysis. 5 g of samples were put into the XRD sample holder
and compressed with a stainless steel weight. The crystalline property
and X-ray diffraction of the samples were examined using an X-ray
diffractometer (Rigaku-ultima IV, Japan) fitted with a divergence slit.
The diffractometer was operated at 40 kV 40 mA with a scan speed of 1
0/min. The X-ray diffractogram's areas for the crystalline and amorphous
regions were used to calculate the relative crystallinity (RC).

2.7.3 Scanning electron microscopy (SEM)

The Adebiyi et al. (2016) method~was used to analyze the~scanning
electron microscopy. The \emph{acha} flour and cookies were placed on
aluminum stubs and sprayed with a carbon thin film using a carbon coated
Quorum Q150TE, Quorum Technologies, UK, to avoid the charge effect.
After coating, the samples were transported to an electron beam specimen
chamber in a scanning electron microscope (Vega 3 XMU, TESCAN Czech
Republic) and exposed to an electron beam while under vacuum.

\textbf{2.8 Sensory Analysis}

The sensory evaluation was determined using 50 panelists consisting of
students from food technology department, Moshood Abiola Polytechnic,
Abeokuta, Nigeria. A 9-point hedonic scale, with 1 denoting ''Like
extremely'' and 9~denoting ''dislike extremely,'' was used to code and
serve cookie samples to panelists on a white plastic plate. In between
the evaluations, the mouth was rinsed with table water. The coded
samples were to be assessed by the panelists for color,~crispiness,
aroma,~taste,~and general acceptability (Adeyanju \emph{et al.,} 2018).

\textbf{2.9 Statistical Analysis}

Using the statistical package for the social sciences (SPSS) software
version 20, one-way variance (ANOVA) was used to analyze all the data
obtained,~and Duncan's multiple range test was~used to separate the
means,~a significance level was established at P \textless{}~0.05
(Steel~and Torrie, 1980).

\textbf{3 RESULTS AND DISCUSSION}

The functional and pasting properties of the native and
fermented\emph{acha} flour and cookies samples were presented in Table
1. The loose and tapped bulk density of \emph{Acha} flour and cookie
samples was observed to decrease significantly (P\textless{}0.05) with
increased fermentation duration. The values ranged from 0.490 to 0.568
g/ml and 0.777 to 0.842 g/ml respectively for the flour sample, and
0.494 to 0.527g/ml and 0.710 to 0.867g/ml for the cookie samples. The
highest value was observed in native \emph{acha} flour and cookie
samples while least value was observed in sample fermented for 72 h. The
decrease observed in the loose and tapped bulk density might possibly
result from increased particle breakdown of the fibrous materials during
fermentation (Olapade \emph{et al.,} 2014). According to Nzigamasabo and
Hui (2006), pectinolytic and cellulotic enzymes are elaborated~by
fermenting microorganisms and aid in the breakdown of cell membranes.
The formation of carbon dioxide gas during fermentation, which causes
the bubbles to grow in size and increase in volume may also be the
reason of the decrease in bulk density of flour samples with longer
fermentation durations (Elmehdi \emph{et al.,} 2007). The cookie
sample's low loose bulk density suggests that less food would be
packaged in a constant volume, which ensures cheap packaging. The
ability of flour to absorb water and swell, improving food consistency,
is known as its water absorption capacity. In food systems, WAC is
desired to increase yield, consistency, and food body
(Osundahunsi\emph{et al.,} 2003). There was significant
(P\textless{}0.05) decrease in the water absorption capacity of the
flour (1.08 to 1.29g/g) and cookie samples (0.76 to 0.89g/g) with
increase in fermentation duration. Highest value was observed in native
\emph{acha} flour while least value was observed in sample fermented for
72 h. The alteration of macromolecules during fermentation may be the
cause of the samples' decreased~water absorption capacity (Oloyede
\emph{et al.} , 2016). The result is consistent with the pattern
observed by Ogodo \emph{et al} . (2018) in their research on lactic acid
bacteria-fermented soybean flour and Ntsamo \emph{et al.} (2020) on
fermentation on sorghum flour properties. Giami and Bekebain (1992) also
found that longer fermentation durations greatly reduced the microbial
activity of food products that have low water absorption capacities. As
a result, the product's shelf life would be increased. The oil
absorption capacity ranged between 0.72 -- 0.64 g/g for the flour
sample, and 0.65 to 0.77g/g for the cookie sample. With increased
fermentation duration, there was no significant (P\textgreater{}0.05)
increase in the samples' oil absorption capacity. \emph{Acha} flour
fermented for 72 h had the highest oil absorption capacity value while
native \emph{acha} flour had the least value. Intrinsic variables like
as protein structure, hydrophobicity or surface polarity may be
responsible for the increase in oil absorption capacity with increased
fermentation durations (Chandra and Samsher, 2013). The study of Oloyede
\emph{et al.} (2015) and Periago \emph{et al.} (1998) on pea flour and
moringa seed flour respectively, showed similar findings. Flour samples
with good oil absorption capacity are suitable for food preparations
that require oil mixing, such as bakery products, where oil is an
essential ingredient (Sobowale \emph{et al} ., 2021). The dispersibility
of the flour and cookie samples varies significantly (P\textless{}0.05)
as the values increased with increase in fermentation duration.
\emph{Acha} flour fermented for 72 h had the highest dispersibility
value while native\emph{acha} flour had the least value. Higher
dispersability value enables the~flour to~reconstitute~more readily in
water (Adebowale\emph{et al} . 2008). This indicates that the flour
samples' capacity to reconstitute effectively in water has been improved
by fermentation. This suggests that the cookies samples could digest
easily (Adebowale\emph{et al} ., 2008). The swelling capacity and
solubility index of the flour and cookie samples decreased with an
increase in fermentation period. This might be related to the granules
degree of association and binding force (Hoover, 2002). The values
obtained in this study for the functional properties of cookies conform
to the report of Razzaq\emph{et al.} (2012).

It has been established that the most assessed set of flour quality
attributes is its pasting properties, which accurately indicate the
quality of the flour (Offia-Olua, 2014). With an increase in the
duration of fermentation, there was a significant (P\textless{}0.05)
decrease in the peak viscosity of the flour samples. The
\emph{acha}flour sample that was fermented for 72 h had the lowest
value, while the native \emph{acha} flour had the highest value. The
similar phenomena~have~been reported for rice flour by Min et al.
(2004),~Yong and Tao (2008), Lu et al. (2005), and~soybean flour by
Olanipekun et al. (2009). The ability of the starch granules in the
flour sample to bind water is indicated by the decrease in peak
viscosity of the flour samples with an increase in fermentation
duration. As shown in Table 1, the peak viscosity of the cookie sample
increased significantly (P\textless{}0.05) with increase in fermentation
duration. High peak viscosity also indicates the fragility of the
swelled granules, which swell initially before disintegrating due to the
Rapid Visco Analyzer constant mixing (Adebowale \emph{et al} ., 2008;
Sanni \emph{et al} ., 2006). The trough viscosity values of the flour
sample decreased gradually with an increase in fermentation duration.
The decrease in trough viscosity with increase in fermentation duration
may be due to the swelling capacity of the starch granules in flour
samples (Ohizua\emph{et al.} , 2017). The flour samples generally had
high trough values, indicating higher cooked paste stability and a
higher ability to withstand shear at elevated temperatures (Farhat
\emph{et al.} , 1999). The trough viscosity values of the cookie samples
increase significantly (p\textless{}0.05) with increase in fermentation
duration. The breakdown viscosity values of the flour samples decreased
with an increase in the fermentation duration. The highest break down
value was obtained in native \emph{acha} flour while the least was
obtained in sample fermented for 72 h. A study by Chinma \emph{et al} .
(2010) also revealed that low breakdown values suggest that the starch
in question has cross-linking characteristics whereas high breakdown
values show that the swollen starch granules are relatively weak against
hot shearing. The lower breakdown viscosity values of flour samples
obtained in this study indicate high fermentation duration impact into
the flour sample ability to possess cross-linking properties and also
high stability against hot shearing. The breakdown values of the cookie
sample increased significantly (p\textless{}0.05) as fermentation
duration increased. Better palatability is indicated by a higher
breakdown viscosity (Tren \emph{et al} ., 2001). The final viscosity
values of the flour was observed to decrease significantly
(P\textless{}0.05) with increased fermentation duration. The simple
kinetic effect of cooling on viscosity and the re-association of starch
molecules in the samples may be the cause of the change in the final
viscosity with longer fermentation durations (Ikegwu \emph{et al} .,
2009). The relatively high value of final viscosity of the \emph{acha}
flour samples suggests the ability of the flour samples to form more
viscous pastes. The final viscosity values of the cookie samples
increased significantly (P\textless{}0.05) with increase in fermentation
duration. The final viscosity changes as fermentation time increases and
this was due to~high amylose content of the cookie samples and hydrogen
bonding during cooling. (Alais and Linden, 1986 cited by Iwe \emph{et
al} ., 2016). The retrogradation of the flour-based product during
cooling decreases with increasing setback value (James and Nwabueze,
2014). As the duration of fermentation increased, the setback viscosity
values of the flour samples decreased~significantly (P\textless{}0.05)
reduced. The setback viscosity value was highest in native \emph{acha}
flour while the least value was observed in sample fermented for 72 h.
This indicates that increase in fermentation duration has improved the
retrogradation tendency of the flour samples. When cooling paste made
from starch or a starch-based product, low setback suggests increased
resistance to retrogradation (Sanni et al., 2004), while~high setback is
linked to syneresis or weeping during freeze/thaw cycles (Adebowale et
al., 2005). The setback values of the cookie samples increased with
increased fermentation duration. It has been reported that setback
viscosity has a major impact on how easily a food product can be
digested when consumed. Higher setback values~are correlated with lower
digestibility (Shittu \emph{et al} ., 2001). The result indicates that
increased fermentation duration decreases the digestibility of the
cookies samples. The peak time of the flour samples increased
significantly (P\textless{}0.05) with increase in fermentation duration.
This shows that increased fermentation duration also increases the
cooking time of the \emph{acha} flour samples. The peak time values~are
comparable to those of Adebowale \emph{et al.} (2008) for instant
yam-breadfruit composite flour~and Chinma \emph{et al} . (2007)
for~germinating tiger-nut flour. The peak time increased with an
increase in fermentation duration. One of the properties that show the
minimum temperature needed to cook a sample, the energy costs involved,
and the stability of other components is the pasting temperature
(Iwe\emph{et al} ., 2016). The pasting temperature of the flour was
observed to increase with increased level of fermentation duration. A
greater pasting temperature is indicative of a larger ability to bind
water, a higher inclination to gelatinize, and a lesser swelling
property in starch-based flour because of the strong associative forces
that exist between the starch granules (Adebowale \emph{et al.,} 2008).
Same observation was reported by Danbaba \emph{et al.} (2012) for the
pasting properties of `ofada rice' varieties. Generally, the values
obtained for the pasting properties of flour samples are within those
recommended for commercial flour (Soni \emph{et al.,} 1993; Kemas
\emph{et al.,} 2012). The pasting temperature of the cookie samples
differs significantly (P \textless{} 0.05) as the values increased with
increase fermentation duration. This implies that fermentation duration
has increased the cooking temperature for cookies samples since pasting
temperature is an indication of minimum temperature required to cook a
sample (Iwe\emph{et al} ., 2016).

Table 2 shows the nutritional quality (proximate and mineral) of native
and fermented \emph{acha} flour and cookie samples. Significant
differences (p\textless{}0.05) were observed in fat, fibre and protein
content of the samples. The moisture content of the \emph{acha} flour
and cookies sample was observed to decrease with increase in
fermentation duration. The samples' decreased moisture content with
longer fermentation times might~likely be attributed to bacteria using
some moisture for metabolic processes (Igbabul \emph{et al.} , 2014).
According to the work of Hwabejire \emph{et al.} (2023), the moisture
content of spontaneously fermented \emph{acha} flour was observed to
increase within 48 h of fermentation time and decreased by 72 h. The
decrease in moisture content could be due to the breakdown of complex
carbohydrates into simpler sugars, which are then used by microorganisms
during fermentation. The moisture content of the cookie's samples was
observed to decrease generally after baking when compared to
the\emph{acha} flour samples. Food stability and value are indicated by
the amount of moisture in the food (Nonaka, 1997). The moisture content
values of the cookie samples found in this study fall within the World
Food Programme's (WFP) maximum moisture content for flour baked products
for effective storage, which is set at 10\%.

The protein content of the \emph{acha} flour samples increased with
increase in fermentation duration. It is possible that the increased
microbial mass during fermentation led to the extensive breakdown of
protein molecules into amino acids and other simple peptides, leading to
why the protein content of the flour samples was reported to increase
(Okechukwu \emph{et al.} , 2015). The result of protein content observed
corresponds to the observation of Michodjehoun-Mestres \emph{et al} .,
(2005) on increase in protein content during fermentation of sorghum,
and the work of Igbabul et al. (2022) on mahogany bean flour. The
protein content of the cookies was observed to decrease when compared
to\emph{acha} flour samples after baking and the values varies
significantly (P\textless{}0.05). The decrease in protein content
observed in cookies samples could be because of denaturation of protein
due to baking temperature (Ogundele \emph{et al.} , 2019).

The fat content of the \emph{acha} flour samples was observed to
decrease with increased fermentation duration. The breakdown of lipids
during fermentation may have led to the low fat level found
in\emph{acha} flour samples (SefaDedeh and Kofi-Agyir, 2002). The
reduced fat content contributes to an extended shelf life for
\emph{acha} flour by minimizing the likelihood of developing rancid
flavors. The fat content of the cookie samples increased after baking
when compared to the flour samples. The high fat content observed in
cookies samples than the flour samples could be because of baking fat
added as an ingredient during dough preparation and agreed with other
authors (Caponio \emph{et al.} , 2008). The ash content of the
\emph{acha} flour and cookie samples was observed to increase initially
at fermented period of 24 h, after which the ash content decreased with
increase in the fermentation duration. Highest value was observed in
sample fermented for 24 h, while the least value was observed in samples
fermented for 72 h shows to have no significant effect on the ash
content of the \emph{acha} flour samples, though numerical differences
was observed. The reduction in ash content following a 24 h fermentation
period may be attributed to the leaching of soluble mineral elements
into the medium or to the general activities of the fermenting
microorganisms, whose enzymatic activity broke down the food components
into forms that could be absorbed (Igbabul \emph{et al.} , 2014).

The crude fibre content of the \emph{acha} flour samples and cookie
sample values decreased with increase in fermentation duration. Highest
value was observed in native \emph{acha} flour/cookie while the least
value was observed in samples fermented for 72 h. Due to the actions of
microorganisms known for the bioconversion of carbohydrates and
lignocelluloses, the fibrous tissues of the \emph{acha} seeds may have
softened during fermentation, which could account for the decrease in
crude fiber content of the \emph{acha} flour samples with longer
fermentation periods (Igbabul \emph{et al} ., 2014). This result
correlates with that of Forsido \emph{et al.,} (2020) who observed a
decrease in the crude fibre content of cereal-based complementary foods,
as fermentation duration increased. The fibre content of the cookies
varies significantly (P\textless{}0.05) and was not appreciably affected
by baking. All the cookies' fiber content, however, fell below the
FAO/WHO (1994) suggested range of no more than 5 g of dietary fiber per
100 g of dry matter, which is in line with~Okpala \emph{et al.} (2013).
The flour samples' comparatively high carbohydrate content indicates
that they may be an energy source. The result observed in this study
correlated with those observed by Igbabul \emph{et al} ., (2014) for
fermented cocoyam flour.

The mineral composition revealed that calcium, sodium and zinc content
of the \emph{acha} flour and cookie samples varies significantly
(P\textless{}0.05). The calcium, sodium and zinc content of the samples
increased initially, but fermentation duration above 24 h cause a
decrease in the mineral content of the samples with increase in
fermentation duration. Highest value for calcium, sodium and zinc was
observed in sample fermented for 24 h while the least value was observed
in sample fermented for 72 h. The decrease in the mineral content of
the\emph{acha} flour samples with increased fermentation duration might
be attributed to leaching of the mineral elements in the fermenting
water during prolong fermentation (Assohoun \emph{et al.,} 2013).
However, there was no significant (P\textless{}0.05) difference among
the samples in terms of magnesium, copper and phosphorus. This shows
that fermentation duration has no significant effects on the magnesium,
copper and phosphorus content of the flour samples, although numerical
differences were observed. The magnesium, copper and phosphorus content
increased initially, with fermentation duration above 24 h, the mineral
parameters decreased with increased fermentation duration.
Assohoun\emph{et al.} (2013) also reported that possible microbial
utilization and conversion could be responsible for the reduction in the
levels of minerals during prolonged fermentation. Similar results were
found in Samitya \emph{et al,} (2021) on plant-based foods. The mineral
content of the cookie samples was lower when compared to the flour
samples. The baking process was observed to slight decrease the mineral
content of the cookie samples except for the sodium and copper content
which was observed to increase after the baking process. This implies
that both Na and Cu are very heat resistant minerals, and that baking
may have made them slightly more extractable. This observation can~be
attributed to the Maillard reaction that occurred during baking.
Delgado-Andrade et al. (2011) stated that the Maillard reaction can
result in disintegration or creation of compounds that may have an
impact on the availability and solubility of minerals. Despite the trend
observed in the mineral content, the cookies samples still contain
appreciable quantities of mineral elements that can supply the
recommended daily allowance of the mineral elements as stated above. The
same results for extruded \emph{acha} -soybean blends and fermented
popcorn-African locust bean blends were obtained by Anuonye et al.
(2010) and Ijarotimi and Keshinro (2013). The phenolic and flavonoid
compounds of the fermented flour and cookies samples were shown in
Tables 3. The values of phenolic and flavonoid compounds increased
significantly (p\textless{}0.05) with increased fermentation duration.
According to research, consuming phenolic compounds may have a
significant impact on health, since it~regulates~metabolism, chronic
disease, weight~and cell proliferation (Cory \emph{et al} ., 2018).

The Fourier transmission infrared (FTIR) spectra of fermented\emph{acha}
flour and cookies are shown in Table 4. Fourier Transmission-Infrared
(FTIR) spectroscopy is a technique for studying constituent functional
groups in an analyte (Adebiyi \emph{et al.,}2016). The infrared (IR)
spectra of the native and fermented \emph{acha}flours and cookies
samples showed similar peaks with variations in intensity. The FTIR
spectra of the flour and baked cookie samples reveal the changes brought
about by the fermentation process. The peaks were between 3860 - 3650
cm\textsuperscript{-1} (Figure 1). The high peak range observed could be
because of O--H bond stretching (Sun \emph{et al.,} 2014). The O--H
absorption peak of native \emph{acha} flour changed from 3650
cm\textsuperscript{-1} to 3670, 3830 and 3800 cm\textsuperscript{-1} for
24 h, 48 h and 72 h fermentation period, respectively. These changes
could be attributed to better lipophilic and hydrophilic properties of
fermented flour samples (Animashaun and Sobowale, 2024). A similar
pattern was also noted in the cookie samples, where the O-H peak varied
for the 24, 48 and 72 h fermentation periods, respectively, from 3610
cm\textsuperscript{-1} to 3800, and 3830 to 3860 cm\textsuperscript{-1}.
The degree of inter and intra-molecular bond formation is shown by the
fluctuation in the O-H peak (Sun \emph{et al.} , 2014). However, the
increase in the width of the fermented samples spectrum in both flour
and cookies could be as a result of characteristic presence of alcohol,
produced during fermentation (Adebiyi \emph{et al.} , 2016). Tightly
bound water in the form of moisture was the cause of the asymmetric
stretching of the C--H band in the region of 3030 -- 2980
cm\textsuperscript{-1} and the vibration peak at 1490 -- 1470
cm\textsuperscript{-1} observed in the spectrum (Shi\emph{et al.,}
2012). The carbonyl stretches peaks in the native and fermented
\emph{acha} flour samples ranged~between 1760 and 1770
cm\textsuperscript{-1}. However, the carbonyl peak values observed in
the cookie samples were at higher intensities in the absorption range of
1780 --1770 cm\textsuperscript{-1}. It was observed in Figure 1 that
fermentation process reduces the carbonyl peaks which might be due to a
decrease in the total lipids present in the samples as a result of
fermentation (Correia \emph{et al.} , 2005; 2008). The additional fat
added during dough preparation may have contributed to the greater
carbonyl peaks seen in the cookie samples. However, baking depolymerized
the fat, resulting in a strong carbonyl peak in every cookie sample. C-O
bond stretching and aliphatic C-N stretching are responsible for the
peaks at 1180 -- 1181 cm\textsuperscript{-1}, whereas the amide I band
formed in the 1550--1570 cm\textsuperscript{-1} range. The intensity of
the bands increased from 1550 cm\textsuperscript{-1} in the
native\emph{acha} flour samples to 1560 and 1560 to 1570
cm\textsuperscript{-1} in the fermented flour samples after 24, 48 and
72 h, respectively. This indicates alterations in the samples
crystallinity. There may have been a buildup of proteins in the
processed samples as indicated by the fermented samples' comparatively
greater amide I peak intensities (Correia \emph{et al.,} 2005).
Increases~in the amide peak intensities were also visible in the cookie
samples' spectra. This might be due to the embedded proteins \selectlanguage{greek}β-\selectlanguage{english}sheet
structures (Duodu \emph{et al.,} 2001). This study~is~comparable to
those published by Animashaun and Sobowale (2024) and Correia et al.
(2008) on the IR spectra for maize and sorghum respectively. Figure 2
shows the X-ray diffraction (XRD) pattern for native and
fermented\emph{acha} flour and its resultant cookie. The variations in
the crystallinity of the flours and cookies affected by processing are
examined using X-Ray Diffraction (XRD) patterns. Peak intensity relates
to the number of semi-crystalline structures that are arranged and/or
the variation in electron density between lamellae that are crystalline
and those that are amorphous. (Hamley, 2013), while the sharp peaks
correlated to crystalline region, the diffused peaks amounted to the
amorphous region of the flour and cookies samples (Hamley, 2013;
Adebiyi\emph{et al.,} 2016). The diffraction patterns for the native and
fermented \emph{acha} flour samples shows that all the flour samples
exhibited an A-type XRD pattern (Sun \emph{et al.,} 2014).
Native\emph{acha} had a diffraction pattern with major reflections at 2\selectlanguage{greek}θ
\selectlanguage{english}of 16.2\textsuperscript{o}, 17.9\textsuperscript{o},
19.0\textsuperscript{o}, and a diffused peak at 22.8\textsuperscript{o}.
Flour samples fermented for 24, 48 and 72 h had similar peaks with
stronger diffraction intensities with peaks occurring at 2\selectlanguage{greek}θ \selectlanguage{english}of
15.3\textsuperscript{o}, 17.4\textsuperscript{o},
20.2\textsuperscript{o} and 22.9\textsuperscript{o},
15.5\textsuperscript{o}, 17.5\textsuperscript{o},
19.4\textsuperscript{o} and 23.2\textsuperscript{o}, and
15.3\textsuperscript{o}, 18.0\textsuperscript{o},
19.4\textsuperscript{o} and 23.1\textsuperscript{o} respectively. The
fermented flour blends' XRD patterns showed variation, which might be
attributed to hydrolysis that occurred during the fermentation process
(Lu \emph{et al.,} 2005). Other cereal flours exhibit the
observed~A-type structure (Amadou \emph{et al.} , 2014; Adebiyi \emph{et
al.,} 2016). The X-ray diffraction plots show that the crystallinity in
cookies samples has little variation when compared to the flour samples.
The diffused V-type peaks seen in all samples are commonly
found~in~cereals that have been processed (Dharmaraj \emph{et
al.,}2014). This explains why all of the cookie samples showed increased
thermal~degradation and the disintegration of the crystalline region~of
the flour samples to an amorphous zone. The disruption of crystalline
region is due to heat processing resulting to a partial or complete
reorganization in the crystalline alignment (Sun \emph{et al.} , 2014).
Similar result was reported by Adebiyi \emph{et al.,} (2016) and
Dharmaraj \emph{et al} ., (2014) for malted and fermented pearl millet
biscuit and during heat treatments resulted in a decrease in
crystallinity of \emph{acha} flour and cookies, respectively.

The scanning electron microscope (SEM) image for native and
fermented\emph{acha} flour and corresponding cookies samples is
presented in Figure 3. The internal structure of food samples~is
commonly~evaluated and characterized using scanning electron microscopy
(SEM). SEM has been utilized by numerous authors to characterize the
morphology of starch granules, explain the endosperm's structural
arrangement, and examine how distinct treatments affect the granules
(Lauro et al., 2000; McPherson and Jane, 2000; Perera et al., 2001;
Naito et al., 2004; Wilson and Betchel, 2004). The scanning electron
microscope (SEM) images of native and fermented \emph{acha} flour
samples were compared to observe the morphological changes in them. It
was observed according to flour micrographs that the granules of native
\emph{acha} flour have a non-uniform, smaller, lenticular or disk-like
shape, which is spherical or ellipsoidal in shape. Regular and smoother
structural networks were observed with shape and structure almost the
same to native \emph{acha}flour in the fermented samples. The granules
had smaller pores~and seemed to be round or polygonal in shape. This
suggests that fermentation process had an effect in forming a regular
shape in their corresponding samples. The effect of osmotic stress
during the fermentation process is responsible for the fermented
samples' increased porosity, disorientation, and less~condensation as
seen in the SEM images (van der Weerd \emph{et al.,} 2001). The
spherical or ellipsoidal shape of the granules is like the shape of
granules in wheat and corn starches reported by Horstmann \emph{et al.}
(2017). The scanning electron microscopy images of the cookie samples
give a more varied arrangement of the granules when compared to the
flour samples. The baking process has been shown to disrupt the granular
structure as all the cookies samples had larger granular sizes with a
``honeycomb like'' structure. Similar observations were reported by
Dhamaraj \emph{et al.}(2014) and Adebiyi \emph{et al.} (2016) for high
temperature processed finger millet and pear millet respectively. The
high structural deformation of the samples may be because heat applied
during baking causes a higher diffusion rate. High-temperature levels,
while baking led to intensified water evaporation and melting of starch
granules (Izli and Polat, 2019). In general, the baking procedure
stabilized the colloidal batter system, enabled the formation of an
enveloping~crust, and altered the structural nature~of the cookie
samples. This situation may undermine the starch-protein matrix and
hence give rise to structural damage and finally rupture the compact
structure of the samples. The micrograph of native and fermented
\emph{acha} cookies was in close conformation with the findings of
Vega-G\selectlanguage{ngerman}álvez \emph{et al} . (2015) for high temperature processed
products structure.

The sensory attributes of cookies produced from native and
fermented\emph{acha} flour is presented in Figure 4. There were
significant (P\textless{}0.05) difference among all the samples produced
from native and fermented \emph{acha} flour. In terms of appearance and
crispness, the mean score shows decrease with an increase in the
fermentation duration. Highest mean score was observed in cookie
produced from native\emph{acha} flour, while the least mean score was
observed in sample produced from \emph{acha} flour fermented for 72 h.
Perhaps, decrease in the mean score for appearance and crispness with
increased fermentation duration could be attributed to dark colouration
developed because of mallard reaction during baking (Hussain \emph{et
al.,} 2006) and increase in mechanical force required require breaking
the samples due to increase in hardness of the samples facilitated by
decreased moisture content of the flour samples, respectively (Guinard
and Mazzucchelli, 1996; Luyten \emph{et al} ., 2004). Also, it was
observed that there is decrease in taste and aroma with increased
fermentation duration. The panelist rated the cookie produced from
\emph{acha} flour fermented for 24 h and sample produced from native
\emph{acha} flour the most preferred in terms of taste and aroma,
respectively. There was significant difference (P\textless{}0.05) among
samples in terms of color. The panelists preference for the colour of
the cookies decreased with increase in fermentation duration. The
decrease was as a result of increase fermentation duration which might
be attributed to more rapid mallard reaction between protein and
carbohydrate which produced cookies with darker colour which may not be
appealing to the consumer. Cookies sample produced from native
\emph{acha} flour was more preferred in terms of colour. The textural
properties of the samples decreased with increased fermentation
duration. This was in line with the report of Luyten \emph{et al} .
(2004). The panelists overall acceptability for the cookies samples
decreased with increase in fermentation duration. Generally, cookies
produced from native \emph{acha} flour were rated most acceptable while
fermented \emph{acha} flour for 72 h was rated the least (Figure 5). The
result obtained for sensory attributes in this study was similar with
the work of Okpala \emph{et al.} (2013) for cookies samples made from
blends of germinated pigeon pea, fermented sorghum, and cocoyam flour.

\textbf{CONCLUSIONS}

This study provides valuable insights into the impact of fermentation
duration on the functional, pasting, nutritional composition, phenolic,
flavonoid microstructure, and sensory attribute of \emph{acha} flour and
cookies. The results indicate that there were significant differences
(p\textless{}0.05) in the functional and pasting properties of the flour
and cookies samples. An increase in fermentation duration was observed
to enhance the oil absorption capacity and dispersibility of both flour
and cookies samples. Overall, the values obtained for the pasting and
functional properties of the flour samples fall within the recommended
range for commercial flour. Regarding the proximate composition, as the
fermentation duration increased, the protein and ash content of
the\emph{acha} flour samples increased. The mineral elements such as
calcium, magnesium, sodium, zinc, copper, and phosphorus initially
increased with fermentation up to 24 hours. Cookies with improved
nutritional composition and acceptable sensory attributes could be
produced from both native and \emph{acha} flour fermented for durations
between 24 and 72 h. This suggests that fermented \emph{acha} flour
holds promise for various food applications, including preparations,
complementary foods, and gel formulations with reduced syneresis.

\textbf{AUTHORSHIP CONTRIBUTIONS}

\textbf{Sunday Samuel Sobowale:} Conceptualization, project
administration, methodology and writing of original draft, reviewing and
editing of manuscript. \textbf{Benjamin Oluwole Omotoso:} Writing of
original draft, Proof reading and correcting the final draft of the
manuscript. \textbf{Joy Ikedichi Agbawodike:} Project administration,
methodology, Proof reading and correcting the final draft of the
manuscript.

\textbf{ACKNOWLEDGEMENTS}

The author(s) acknowledged the Tertiary Education Funds (TETFUND),
Nigeria for providing the research grant and the Management of Moshood
Abiola Polytechnic for given us the opportunity to access the TETFUND
grant for this research work.

\textbf{FUNDING INFORMATION}

This research was funded by Tertiary Education Funds (TETFUND), Nigeria.

\textbf{DECLARATION OF CONFLICTING INTERESTS}

The author(s) declared no conflicts of interest exist in the submitted
manuscript, and all authors approve it for publication.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{ORCID}

S.S. Sobowale ID https://orcid.org/0000-0002--1051-1133

J.I. Agbawodike ID https://orcid.org/0000-0002-0707-3419

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