foods

Article
Flour Functionality, Nutritional Composition, and In Vitro
Protein Digestibility of Wheat Cookies Enriched with
Decolourised Moringa oleifera Leaf Powder
Temitayo D. Agba 1 , Nurat O. Yahaya‑Akor 1 , Amarjit Kaur 1 , Moira Ledbetter 2 , James Templeman 2 ,
Jonathan D. Wilkin 2 , Bukola A. Onarinde 1 and Samson A. Oyeyinka 1,3, *

1 Centre of Excellence in Agri‑Food Technologies, National Centre for Food Manufacturing, University of
Lincoln, Holbeach PE12 7PT, UK; [email protected] (T.D.A.);
[email protected] (N.O.Y.‑A.); [email protected] (A.K.);
[email protected] (B.A.O.)
2 Division of Engineering and Food Science, School of Applied Sciences, Abertay University,
Dundee DD1 1HG, UK; [email protected] (M.L.); [email protected] (J.T.);
[email protected] (J.D.W.)
3 Centre for Innovative Food Research (CIFR), Department of Biotechnology and Food Technology, Faculty of
Science, University of Johannesburg, P.O. Box 17011, Johannesburg 2028, Gauteng, South Africa
* Correspondence: [email protected]

Abstract: This study investigated the potential of decolourised Moringa oleifera leaf powder
(D‑MOLP) in cookies to meet consumer demand for healthier food options, addressing the issue
of low acceptability due to its green colour. D‑MOLP and its non‑decolourised counterpart (ND‑
MOLP) were incorporated into wheat flour to produce cookies. The results showed that neither
decolourisation nor addition level (2.5 or 7.5%) significantly affected water activity or flour func‑
tionality, though slight differences in cookie colour were observed. The Moringa‑enriched cook‑
Citation: Agba, T.D.; Yahaya‑Akor, ies exhibited an improved spread ratio as well as higher protein, phenolic content, antioxidant ac‑
N.O.; Kaur, A.; Ledbetter, M.;
tivity, and in vitro protein digestibility compared to control cookies. The detected phenolic acids
Templeman, J.; Wilkin, J.D.;
included chlorogenic, ferulic, and fumaric acids, with the D‑MOLP cookies showing superior nu‑
Onarinde, B.A.; Oyeyinka, S.A. Flour
tritional properties, likely due to nutrient concentration and reduced antinutrients. Notably, glu‑
Functionality, Nutritional
tamic acid was the major amino acid in all the cookies, but only lysine significantly increased across
Composition, and In Vitro Protein
Digestibility of Wheat Cookies
the cookie types. This suggests D‑MOLP could be a promising alternative for food enrichment.
Enriched with Decolourised Moringa Future research should address the consumer acceptability, volatile components, and shelf‑life of
oleifera Leaf Powder. Foods 2024, 13, D‑MOLP‑enriched cookies.
1654. https://doi.org/10.3390/
foods13111654 Keywords: amino acid; bioactive; cookies; decolourisation; Moringa oleifera; wheat flour

Academic Editors: Laura Gazza and

Mahsa Majzoobi

Received: 24 April 2024 1. Introduction

Revised: 15 May 2024
The Moringa plant is a valuable source of macro‑ and micronutrients that could be
Accepted: 23 May 2024
used to address protein and energy malnutrition globally [1]. According to Asare et al. [2],
Published: 25 May 2024
all the different parts of the tree, including its flowers, leaves, and seeds are useful in folk
medicine for treating different ailments. Of the various parts of the Moringa plant, the
leaves are the most nutrient‑dense; for example, they are an excellent source of essential
Copyright: © 2024 by the authors.
amino acids and vitamins [3–5] and have been suggested as ingredients for food enrich‑
Licensee MDPI, Basel, Switzerland. ment and fortification [1]. Traditionally, the fresh leaves of Moringa are consumed as a
This article is an open access article snack and integrated into salads and vegetable soups [6,7]. Other promising ways of using
distributed under the terms and the leaves are through the food enrichment and fortification of commonly consumed foods
conditions of the Creative Commons like yoghurt [8], cake [9], bread [10], and biscuits or cookies [11–20].
Attribution (CC BY) license (https:// The utilisation of Moringa oleifera leaf powder (MOLP) in biscuit or cookie enrichment
creativecommons.org/licenses/by/ seems to be an attractive option, especially with the growing demand for healthier foods
4.0/). with better functionality. Shahzad et al. [21] noted that the demand for functional foods

Foods 2024, 13, 1654. https://doi.org/10.3390/foods13111654 https://www.mdpi.com/journal/foods

Foods 2024, 13, 1654 2 of 16

is increasing presumably due to their perceived health benefits such as the prevention of
noncommunicable diseases. Fapetu et al. [15], for example, showed that MOLP can be
incorporated into wheat flour for making functional cookies with antidiabetic properties
due to its inhibitory abilities against α‑amylase and α‑glucosidase enzymes. The Moringa
plant contains phytonutrients such as carotenoids, tocopherols, and ascorbic acid [22,23],
as well as bioactive compounds like quercetin and kaempferol glucoside with known phar‑
macological benefits [24]. This underscores the plant’s value as a versatile ingredient for
diverse industrial applications. Furthermore, the remarkable ability of the Moringa plant
to thrive in water‑stressed environments [25] suggests its potential as a sustainable food
source, aligning with Sustainable Development Goals (SDGs) 2 and 3. SDG‑2 aims to en‑
sure access to nutritious food, especially for vulnerable populations, while SDG‑3 focuses
on promoting health and well‑being across all age groups. The incorporation of Moringa,
particularly its nutrient‑rich leaves in foods, could contribute significantly to achieving
these goals while also supporting environmental sustainability. By reducing reliance on an‑
imal protein consumption, which is known to have a substantial carbon footprint, Moringa
offers a promising solution to address both food security and climate change concerns.
Previous research reported the use of varying levels (2–20%) of MOLP in cookies or
biscuit enrichment [11–20]. Fapetu et al. [15] reported that cookies enriched with MOLP
showed reduced thickness while the diameter and spread ratio increased. Furthermore,
the authors found a significant increase in bioactive compounds and antioxidant proper‑
ties in the MOLP‑enriched cookies. According to their report, a 2.5% level of addition of
MOLP was acceptable to consumers compared to other MOLP‑substituted cookies. Other
studies reported that MOLP can enhance the nutritional value of foods [26–28], improve
haemoglobin levels in an adolescent [29] and confers slowly digestible starch properties
and higher resistant starch content on cookies enriched with MOLP [30]. Although the use
of Moringa oleifera has been reported to improve the nutritional value of foods, neverthe‑
less, the appearance of enriched products is generally reported to be unacceptable [26–28].
The low acceptability of Moringa‑enriched foods has been associated with their green
colour, due to the chlorophyll content of the leaves which usually mask fortified or en‑
riched foods [1,26,27]. For example, some authors reported that MOLP‑enriched bread had
an herbal flavour and generally had low acceptability with increasing levels of MOLP [31].
Ntila et al. [32] also reported low acceptability for porridge enriched with MOLP at 2–3%
levels. To address this challenge and to further increase the utilisation of the leaves in
food application, one practical approach would be to decolourise the leaves before its use
in food enrichment.
Earlier researchers demonstrated the use of laboratory‑grade ethanol to decolourise
MOLP without significant changes in nutritional value [33,34]. The decolourisation of
MOLP l under optimised conditions of extraction time (10–30 min), solute‑to‑solvent ra‑
tio (1:20 to 1:5 w/v), and solvent concentration (50–95% v/v) using a Box–Behnken design
(Response Surface Methodology) reportedly reduced the chlorophyll content of the leaves
and increased protein, ash, and fibre contents [34]. Oyeyinka et al. [34] found that de‑
colourisation under optimised conditions of solute‑to‑solvent ratio (1:20), solvent concen‑
tration (95%), and extraction time of 30 min can remove high levels of chlorophyll with
minimal loss of certain bioactive compounds, such as phenolic compounds, into the ex‑
traction solvent. Thus, we hypothesize that the use of decolourised MOLP (D‑MOLP) in
cookie enrichment may produce a product with a better appearance, colour, and nutri‑
tional quality compared to cookies enriched with non‑decolourised MOLP (ND‑MOLP).
So far, there have been no studies utilising decolourised MOLP in cookies. Hence, this
study investigated the functionality of MOLP‑enriched wheat flour, in terms of its physi‑
cal properties, proximate composition, amino acid and phenolic acid content, antioxidant
properties, in vitro protein digestibility, and microbial quality of the resulting cookies.
Foods 2024, 13, 1654 3 of 16

2. Materials and Methods

2.1. Materials
Baking items such as wheat flour (British Wheat Flour, Tesco, UK‑Moisture content:
8% and Protein content: 9.9%), margarine (Stork Baking Spread, Tesco), sugar (Silver Spoon
British granulated sugar), baking powder (Stockwell Eco, Tesco), salt (British cooking salt,
Tesco, UK), and eggs (Large free range egg) were all purchased from Tesco Superstore
Holbeach, Spalding, Lincolnshire, UK, except milk which was purchased from a local store
(Powdered Peak milk, Nigeria). Moringa powder (Naturale Bio) obtained from 100% dried
Moringa leaves (moisture content 7.80 ± 0.21%) was purchased from Veganok Company,
India, on Amazon, UK.

2.2. Preparation of Decolourised Moringa Leaf Powder

The Moringa leaves were decolourised using the optimised method of
Oyeyinka et al. [34]. In brief, the optimum conditions for decolourisation of MOLP reported
by these authors were a solute (MOLP)‑to‑solvent (ethanol) ratio of 1:20, extraction time
of 30 min, and a solvent concentration of 95%. The MOLP was dissolved in the solvent,
homogenized to dissolve properly, and the mixture was placed on an orbital shaker at
150 rpm for 30 min. Subsequently, the sample was centrifuged at 3200× g for 15 min using
a centrifuge (Eppendorf, Hamburg, Germany), and the supernatant was discarded. The
resulting slurry was dried at 50 ◦ C overnight and further air‑dried for 48 h to remove any
residual ethanol. The dried MOLP was then packed into a thick low‑density polyethylene
bag for storage and use.

2.3. Preparation of Cookies

For the preparation of cookies, a previously reported recipe was used with slight mod‑
ifications [35]. The control cookie recipe included wheat flour (400 g), salt (4 g), sugar
(200 g), milk (40 g), margarine (80 g), baking powder (8 g), and 1 egg, combined in se‑
quence. In the enriched cookies, the wheat flour was substituted with decolourised MOLP
(D‑MOLP) and non‑decolourised MOLP (ND‑MOLP) at different ratios of 2.5% and 7.5%.
This resulted in five types of cookies, including the 100% wheat flour control. The choice
of 2.5% and 7.5% substitution levels was based on a preliminary study, where differences
in cookie colour and appearance were not significant (p ≥ 0.05). Additionally, considering
that higher MOLP levels provide more nutrients, these levels were chosen for the study
design. The dry ingredients including wheat flour, baking powder, salt, and Moringa pow‑
der were sieved using a particle‑size sieve to guarantee consistent integration and remove
any foreign material. A food mixer (KitchenAid, Benton Harbor, MI, USA) was used to
cream butter and sugar for 5 min at low speed until they were light and fluffy. Next, the
egg and yolk were added, and the cream mixture was thoroughly mixed with the dry in‑
gredients, followed by milk for 5 min. Then, kneading was performed for another 5 min
to form dough that was not sticky and consistent, and it was refrigerated for 30 min. A
circular cookie cutter was used to cut the dough into circular shapes after it had been hand‑
flattened onto a flat ceramic surface to a thickness of about 0.5 cm using a wooden hand
roller. The cookies were baked for 20 min at 150 ◦ C in a pre‑heated oven. After baking, the
cookies were carefully removed from the baking pan using gloves, placed on a tray, and
covered with foil in an enclosed space.

2.4. Analyses on Flour Mixture with MOLP

2.4.1. Water and Oil Absorption Capacity
The ability of flour to absorb water or oil was assessed by the method described by
Oladunjoye et al. [35]. For water absorption capacity (WAC), one gram of flour sample
was dispersed in 10 mL of distilled water in a pre‑weighed centrifuge tube. The sample
was thoroughly vortexed and left to stand for 30 min at room temperature, after which the
mixture was centrifuged (Eppendorf, Hamburg, Germany) at 4000 rpm for 30 min. The
tube was reweighed after decanting the supernatant from it, and the WAC was expressed
Foods 2024, 13, 1654 4 of 16

as the gram of water bound per gram of flour. The same procedure was repeated for oil
absorption capacity (OAC), except that the water was replaced with sesame seed oil.

2.4.2. Bulk Density

The loose bulk density (LBD) and packed bulk density (PBD) of the flour samples
were determined using the modified method of Kaur et al. [36] described by previous re‑
searchers [35]. Briefly, a 100 cm3 graduated cylinder that had previously been tarred was
gently filled with the flour samples and weighed. The loose bulk density was estimated as
the sample weight‑to‑volume ratio (g/cm3 ). To calculate the packed bulk density, the cylin‑
der’s lid was gently tapped until no more changes in the sample level marked was observed.
The packed bulk density was estimated as the sample volume‑to‑weight ratio (g/cm3 ).

2.4.3. Foam Capacity and Foam Stability

The foam capacity (FC) and foam stability (FS) of the flour samples were measured
using the method reported by Oladunjoye et al. [35]. In a measuring cylinder, 2 g of the
flour sample was dispersed in 50 mL of distilled water at room temperature. The dispersed
solution was vigorously mixed and agitated to foam, and the volume was recorded after
30 s. The FC was calculated as a percentage rise in volume, whereas the FS was calculated
as a percentage difference in volume from the initial amount after 1 h of whipping.

2.4.4. Particle Size Distribution

The particle size distribution of the flour samples was determined using laser diffrac‑
tion analysis (Malvern Mastersizer Hydro 2000, Malvern Panalytical Ltd., Malvern, UK).
Briefly, the sample was dispersed in distilled water filled in the dispersion tank while a stir‑
rer was rotating at 2300 rpm to ensure the sample was representative due to its homogene‑
ity. Measurements were performed in triplicate for each sample. The mean particle sizes
at the 10th, 50th, and 90th percentiles of the particle size distribution curves were recorded.

2.4.5. Colour
The colour parameters (Lightness = L*, using the axis ranging between 0 for black
and 100 for white, redness (+a*), greenness (−a*), yellowness (+b*), and blueness (−b*)
were assessed using a bench‑top chromameter (Konica Minolta, Tokyo, Japan). The total
colour change (∆E) of the flour containing MOLP was compared with the wheat control
using the equation below [37].
√
∆E = 〖(∆L)〗^2 〖+(∆a)〗^2 〖+(∆b)〗^2

2.5. Analyses of Cookies with MOLP

2.5.1. Colour and Physical Dimensions
The colour parameters of the cookies were assessed as described in Section 2.4.5. For
physical dimension measurements, including diameter, thickness, and spread ratio, six
randomly chosen cookies from each sample were used as previously reported [38,39]. The
diameter of the cookies was determined by taking two measurements from each cookie
while rotating it 90 degrees using a ruler. The thickness was measured by taking three
readings from each cookie using digital vernier callipers with a 0.01 mm accuracy, and the
mean value for all six cookies were recorded. The results for thickness and diameter were
expressed in millimetres (mm). The spread ratio was determined by dividing the diameter
measurement by the thickness.

2.5.2. Texture Analysis

The breaking strength of the cookies was measured by following the triple beam snap
method (also called three‑point break) using Texture Analyzer (Stable Microsystems, Go‑
dalming, UK). A cookie sample was placed on two supporting beams placed 2.5 cm apart.
Another beam connected to a moving part was brought down to break the cookies at a
Foods 2024, 13, 1654 5 of 16

crosshead speed at 10 mm/min and load cell of 10 kg. Care was taken to see that the point
of contact was equivalent from both the supporting beams. The hardness (g/force) and
brittleness were recorded, and the average values were calculated.

2.5.3. Water Activity

To record the water activity (aw ), a calibrated water activity meter (AQUALAB 4TE,
Pullman, DC, USA) was used at room temperature according to a modified method of
Naknaen et al. [40]. Cookies previously milled into fine powder were put into the cup
(half‑filled) to prevent light from disrupting the light sensors in the meter. The readings
were taken after the device made a beep sound, and this took about 5 min or less for each
sample. The sample cup was cleaned after each reading.

2.5.4. Proximate Composition

Except for the moisture content, that was determined using a Halogen moisture anal‑
yser (PMB 202, Model), and the carbohydrate content, which was calculated using percent‑
age differences [100 − (ash + fat + fibre + moisture + protein)], all the other components—the
ash, protein (Micro‑Kjeldahl method) and fat content (Soxhlet extraction method)—were
determined using a standard method of AOAC [41].

2.5.5. Total Phenolic Content and Antioxidant Capacity

The Folin–Ciocalteu reagent was used to calculate the total phenol content [42]. Using
a spectrophotometer (UV–VIS spectrophotometer), absorption was measured at 765 nm
and compared to a standard calibration curve made using gallic acid (Sigma Aldrich, Lon‑
don, UK). The findings were represented as mg Gallic acid equivalents per gram of material
(mg GAE/g).
The antioxidant capacity of the cookies was analysed using a 2,2‑Diphenyl‑1‑
Picrylhydrazyl (DPPH) assay, following the method described by Maleke and Adebo [43].
Briefly, the extract (15 µL) was treated with 285 µL of DPPH solution (working solution
prepared by diluting 0.024 g of DPPH in 100 mL of methanol (MeOH) and incubating
in the dark for 20 min). This was allowed to incubate at 37 ◦ C for 15 min, and the ab‑
sorbance was read at a wavelength of 570 nm on a spectrophotometer (Bibby Scientific
Limited, Stone, UK).

2.5.6. Phenolic Acids Quantification

The phenolic acids present in the cookies were identified by a liquid chromatograph–
mass spectrometry/quadrupole time of flight (LC‑MS/QToF) technique. The cookies were
extracted using methanol and water in a ratio 60:40 (3 mL). The samples were diluted by
a factor of 5 and filtered (0.45 µm nylon) and transferred to a HPLC vial for analysis as
described by Ritvanen et al. [44]. The gradient was 0% B at 0.4 mL/min for 1 min, increasing
to 100% B over 10 min, maintaining for 2 min, returning to initial conditions over 0.1 min,
and re‑equilibrating for 6.9 min. The MS was equipped with an electrospray ionization
(ESI) source and was operated in negative ionization mode. A sample of 1 µL was injected
in the column. The MS source conditions were as follows: the capillary voltage was set at
3.0 kV, the gas temperature was set at 300 ◦ C, nitrogen was used as a drying gas at 8 L/min,
the nebulizer was set at 25 psi, and a sheath gas temperature of 300 ◦ C. The phenolic acids
were quantified by external standard calibrations. The retention time, linear calibration
range and R2 for each amino acid are reported in Table S1.

2.5.7. In Vitro Protein Digestibility

Protein digestibility was measured following the methods described by
Awobusuyi et al. [45]. The cookies were crushed into fine powder, and 200 mg was accu‑
rately weighed into a 50 mL centrifuge tube and labelled. Then, 35 mL of 0.1 M phosphate
buffer (pH of 2) and enzyme pepsin (1.5 mg/mL) were added to the tube containing the
sample. The sample solution was incubated in a shaking water bath at 3 ◦ C for 2 h to al‑
Foods 2024, 13, 1654 6 of 16

low for digestion by the enzyme. Thereafter, 2 M NaOH (2 mL) was added to terminate
the digestion, and the suspension was centrifuged at 4800 rpm at 4 ◦ C for 20 min. The
supernatant was discarded, and the residue was washed with 15 mL of a buffer solution
(0.1 M phosphate, pH 7). Centrifugation was repeated as described above, and the super‑
natant was discarded. The residue was washed on a Whatman’s No 3‑filter paper, and the
undigested protein residue in the filter paper was dried in an oven (Binder, FED 260 E3.1,
Tuttlingen, Germany) at 80 ◦ C for 2 h. The protein content of the initial and final sample
was determined using the Kjeldahl method, while the percentage protein digestibility was
calculated as shown below.

protein content(undigested cookies − residue after digestion)

Protein digestibility = × 100
protein content of undigested cookies

2.5.8. Total Amino Acid Sample Preparation and Quantification by LC‑MS/QToF

Samples were extracted as described in Section 2.5.6 and were diluted by a factor of 5
and filtered (0.45 µm nylon) and transferred to a HPLC vial for analysis. The total amino
acid composition was determined by acid and alkaline hydrolysis, as described by Ritva‑
nen et al. [44] with some modifications. The extracted proteins (100 mg) were hydrolysed
in hydrochloric acid (6 M) or sodium hydroxide (5 M) at 105 ◦ C for 24 and 18 h, respec‑
tively. The sample was centrifuged (Eppendorf centrifuge 5415 R, Hamburg, Germany) at
10,200× g for 2 min. Aliquots of the solution were neutralized with sodium hydroxide (5 M)
and hydrochloric acid (6 M), respectively. The acid‑hydrolysed samples were diluted by a
factor of 50, and the alkali samples were diluted by a factor of 40. The samples were filtered
(0.45 µm nylon) and transferred to an HPLC vial for analysis. The samples were further di‑
luted as required. The amino acids in the extracted proteins were identified and quantified
by LC‑MS/QToF based on the method of Liu and Rochfort [46] with modifications. The sys‑
tem consisted of an Agilent 1290 Infinity II system including a pump, auto sampler, and
column oven, and an Agilent 6546 LC/Q‑ToF (Stockport, UK). The chromatographic sepa‑
ration was performed on a Synergi Hydro‑RP column (150 × 4.6 mm, 4 µm, Phenomenex)
fitted with a guard cartridge of the same stationary phase, maintained at 30 ◦ C. Mobile
phase A was water containing 0.1% formic acid, and mobile phase B was methanol con‑
taining 0.1% formic acid. The gradient was 0% B at 0.4 mL/min increasing to 100% B over
10 min, returning to the initial conditions over 0.1 min and re‑equilibrating for 6.9 min.
The MS was equipped with an electrospray ionization (ESI) source and was operated in a
positive ionization mode. A sample of 1 µL was injected in the column. The MS source con‑
ditions were as follows: the capillary voltage was set to 3.0 kV, the gas temperature was set
to 325 ◦ C, nitrogen was used as a drying gas at 10 L/min, the nebulizer was set at 35 psi, and
the sheath gas temperature was set to 325 ◦ C. The data were collected with MassHunter
Workstation LC‑MS data acquisition for 6200 TOF/6500 Version11.0.221.1 (Stockport, UK).
The amino acids were quantified by external standard calibrations. The retention time,
linear calibration range, and R2 for each amino acid are reported in Table S1.

2.6. Statistical Analysis

The samples were prepared in triplicate, and the analyses were performed in tripli‑
cate unless otherwise stated. The IBM Statistical Package for Social Science (SPSS) Ver‑
sion 27 was used to analyse the data using one‑way ANOVA, which was presented as
the mean ± SD (standard deviation) of three measurements. The mean separation was by
Fisher Least Significance Difference (p < 0.05).

3. Results and Discussion

3.1. Functional Properties and Particle Size of Flour
The functional properties including WAC, OAC, LBD, PBD, FC, and FS of the wheat
flour were not significantly affected (p ≥ 0.05) by the addition of either the decolourised
or non‑decolourised MOLP (Table 1). The particle sizes of the wheat flour and MOLP‑
Foods 2024, 13, 1654 7 of 16

enriched flours were also very similar (Table 1). Although the functional properties of the
wheat flour did not change significantly, there was a slight increase in its WAC and OAC,
likely due to the protein content in the MOLP. In this study, the decolourised MOLP (D‑
MOLP) had slightly higher protein content (32.13%) compared to the non‑decolourised
MOLP (ND‑MOLP) which had a value of 29.03%. Fapetu et al. [15] similarly reported a
non‑significant increase in OAC for wheat flour enriched with ND‑MOLP, even up to a
10% level of addition, but a decrease in the WAC. The variation in the WAC observed in
their study compared to our findings may be attributed to differences in the particle sizes
of the flours, as well as variations in starch granule structure and the availability of water
binding sites among the different flours used in the respective studies [26,47].

Table 1. Colour, moisture, water activity, functionality, and particle size of wheat–Moringa flour.

97.5% Wheat Flour + 92.5% Wheat Flour + 97.5% Wheat Flour + 92.5% Wheat Flour +
100% Wheat
Parameters 2.5% Decolourised 7.5% Decolourised 2.5% Non‑Decolourised 7.5% Non‑Decolourised
Flour
Moringa Moringa Moringa Moringa
L* 94.04 ± 0.12 a 89.56 ± 0.12 b 86.09 ± 0.58 c 87.78 ± 0.17 d 81.53 ± 0.15 e
a* 3.83 ± 0.03 a 2.99 ± 0.03 b 2.86 ± 0.06 c 1.38 ± 0.06 d −0.04 ± 0.03 e
b* 12.22 ± 0.13 e 13.84 ± 0.06 c 13.21 ± 0.26 d 14.62 ± 0.02 b 17.91 ± 0.13 a
∆E ‑ 4.84 ± 0.12 d 8.08 ± 0.60 b 7.14 ± 0.13 c 14.28 ± 0.18 a
aw 0.48 ± 0.00 a 0.46 ± 0.00 a 0.45 ± 0.01 a 0.46 ± 0.30 a 0.46 ± 0.01 a
WAC (g water/
0.93 ± 0.12 a 0.97 ± 0.14 a 1.17 ± 0.33 a 1.07 ± 0.41 a 1.16 ± 0.23 a
g flour)
OAC (g oil/g flour) 1.27± 0.40a 1.39 ± 0.05 a 1.41 ± 0.39 a 1.33 ± 0.00 a 1.37 ± 0.50 a
LBD (g/mL) 0.50 ± 0.01 ab 0.47 ± 0.00 b 0.48 ± 0.00 b 0.48 ± 0.00 ab 0.51 ± 0.01 a
PBD (g/mL) 0.67 ± 0.00 a 0.66 ± 0.00 a 0.66 ± 0.02 a 0.67 ± 0.03 a 0.69 ± 0.00 a
FC (%) 28.00 ± 0.00 a 26.00 ± 1.41 a 26.50 ± 0.70 a 26.50 ± 0.70 a 26.50 ± 0.70 a
FS (%) 24.00 ± 0.00 a 25.50 ± 0.70 a 24.50 ± 0.70 a 24.00 ± 0.00 a 24.50 ± 2.21 a
d 0.1 (µm) 14.10 ± 1.28 a 13.65 ± 0.72 a 15.09 ± 0.81 a 14.38 ± 0.73 a 15.17 ± 0.76 a
d 0.5 (µm) 79.67 ± 5.41 a 80.20 ± 3.47 a 84.82 ± 2.77 a 85.68 ± 2.46 a 83.88 ± 1.90 a
d 0.9 (µm) 213.25 ± 7.36 a 213.26 ± 3.37 a 223.08 ± 5.88 a 222.24 ± 1.75 a 218.83 ± 0.80 a
Values are reported as mean ± standard deviation. Mean values with different superscripts are significantly
different (p < 0.05). WAC: Water absorption capacity; OAC: Oil absorption capacity; LBD: Loose bulk density;
PBD: Packed bulk density; FC: Foaming capacity; FS: Foaming stability.

3.2. Water Activity and Colour of Flour

The water activity (aw ) of food ingredients and products is indicative of shelf stability
and susceptibility to microbial spoilage. It is the amount of available water in a food that
supports microbial growth and participates in chemical and enzymatic reactions. The ad‑
dition of MOLP generally reduced the aw values of wheat flour, but the reduction was not
significant (Table 1). However, MOLP addition to the wheat flour resulted in a significant
change in the colour of the flour samples (Table 1). The wheat flour containing D‑MOLP
had much higher lightness (L*) values (86.09–89.56) compared to the wheat flour contain‑
ing ND‑MOLP (81.53–87.78). The decolourisation step reduced the greenness of MOLP
through the removal of the chlorophyl in the leaves. This may explain the lower a* values
(greenness) recorded for the wheat flour containing ND‑MOLP. A previous study reported
significantly lower chlorophyll content (0.56 mg/g) for D‑MOLP compared to ND‑MOLP
(0.64 mg/g) [34]. Furthermore, the level of addition of MOLP significantly influenced the
colour of the flours. Higher levels of MOLP, whether decolourised or not, increased the
greenness of the wheat flour. This impacted the appearance and colour values of the cook‑
ies, as discussed in Section 3.4.

3.3. Geometrical Characteristics of the Cookies

The addition of MOLP generally decreased the thickness and diameter of the cookies
(Table 2). However, the decrease was only significant (p < 0.05) at a 7.5% level of addition.
The control wheat cookies recorded the highest thickness and diameter compared to all
the enriched cookies. On the other hand, the spread ratio of the cookies enriched with
ND‑MOLP was significantly higher than that of the control and those of samples enriched
with D‑MOLP. This may be due to the significant decrease in thickness of the cookies. The
Foods 2024, 13, 1654 8 of 16

spread ratio (7.91–10.05) of the enriched cookies in this study was higher than values previ‑
ously reported (4.05–7.04) for wheat flour enriched with ND‑MOLP [17,48]. A high spread
ratio value is desirable and is usually associated with better cookie quality [49,50]. Differ‑
ences may be observed in the spread ratio of cookies from composite flours. Watters [51]
reported a decrease in spread ratio value for cookies supplemented with non‑wheat flours
and associated the decrease to competition by the non‑wheat flours for limited free water
available in the cookie dough. The competition for water is thought to result in the forma‑
tion of aggregates which increases the number of hydrophilic sites in the flour [48]. During
dough formation, free water is partitioned between the hydrophilic sites, thereby increas‑
ing dough viscosity [52]. Thus, the higher spread ratio in the cookies with ND‑MOLP
suggests that there is less competition for free water by the added ND‑MOLP.

Table 2. Physical, chemical, and physicochemical properties of cookies from wheat–Moringa flour.

97.5% Wheat Flour + 92.5% Wheat Flour + 97.5% Wheat Flour + 92.5% Wheat Flour +
100% Wheat
Parameters 2.5% Decolourised 7.5% Decolourised 2.5% Non‑Decolourised 7.5% Non‑Decolourised
Flour
Moringa Moringa Moringa Moringa
L* 47.87 ± 0.17 a 49.41 ± 1.92 a 47.50 ± 0.69 a 48.68 ± 0.53 a 46.86 ± 0.13 a
a* 8.89 ± 0.50 a 9.38 ± 0.46 a 4.05 ± 0.23 b 2.21 ± 0.61 c 2.27 ± 0.11 c
b* 5.55 ± 0.49 c 11.63 ± 0.66 a 10.13 ± 0.45 b 11.46 ± 0.13 a 10.70 ± 0.09 ab
∆E ‑ 6.41 ± 1.12 b 6.70 ± 0.10 b 8.97 ± 0.41 a 8.45 ± 0.01 a
Hardness (N) 131.97 b ± 3.97 151.31 ab ± 10.52 151.06 ab ± 2.68 151.03 ab ± 18.64 153.56 a ± 7.7
Brittleness (mm) 1.39 ± 0.35 a 0.96 ± 0.05 b 1.11 ± 0.14 ab 0.94 ± 0.06 b 0.98 ± 0.15 b
Diameter (mm) 82.33 ± 2.94 a 80.08± 0.98 ab 78.67 ± 1.51 b 81.33 ± 3.27 ab 79.00 ± 2.00 b
Thickness (mm) 10.35 ± 0.87 a 10.12 ± 0.93 ab 9.56 ± 2.02 abc 8.09 ± 1.03 c 8.33 ± 1.99 bc
Spread ratio 7.96 ± 0.59 b 7.91 ± 0.75 b 8.23 ± 1.84 ab 10.05 ± 1.33 a 9.49 ± 2.64 ab
Moisture (%) 2.05 ± 0.28 bc 1.52 ± 0.11 c 2.42 ± 0.11 b 3.24 ± 0.37 a 2.12 ± 0.25 bc
Protein (%) 8.46 ± 0.03 b 8.77 ± 0.20 b 9.56 ± 0.05 a 8.67 ± 0.03 b 9.69 ± 0.16 a
Fat (%) 12.40 ± 0.08 a 12.86 ± 0.18 a 12.78 ± 0.04 a 12.38 ± 0.28 a 12.45 ± 0.36 a
Ash (%) 2.05 ± 0.22 b 2.66 ± 0.41 a 2.53 ± 0.08 ab 2.08 ± 0.05 b 2.36 ± 0.00 ab
Carbohydrate
74.23 ± 0.07 a 74.23 ± 0.73 a 72.35 ± 0.03 b 73.28 ± 0.63 ab 73.06 ± 0.76 ab
(%)
aw 0.44 ± 0.08 a
0.30 ± 0.06 b 0.32 ± 0.04 b 0.29 ± 0.04 b 0.28 ± 0.06 b
pH 7.08 ± 0.24 a 6.96 ± 0.03 a 6.97 ± 0.23 a 6.96 ± 0.03 a 6.79 ± 0.20 a
Total phenolic
content (mg 1.21 ± 0.03 d 2.49 ± 0.01 c 3.19 ± 0.02 b 2.54 ± 0.01 c 4.92 ± 0.39 a
GAE/g)
DPPH (%) 23.77 ± 1.65 b 87.70 ± 1.88 a 84.97 ± 5.64 a 86.24 ± 0.50 a 87.30 ± 0.45 a
Values are reported as mean ± standard deviation. Mean values with different superscripts are significantly
different (p < 0.05).

3.4. Physical Characteristics of the Cookies

The addition of MOLP to wheat flour does not seem to influence the lightness (L*)
values of the cookies (Table 2). However, the impact of the MOLP was significant (p < 0.05)
on the a‑values, especially for cookies enriched with ND‑MOLP. In addition, a higher level
of MOLP in the cookies resulted in a decrease in the a‑values. A shift to a lower a‑value
suggests a tendency towards greenness of the sample since a positive a‑value indicates red‑
ness and a negative a‑value suggests greenness of the sample. MOLP contains a high level
of chlorophyll which would normally mask the appearance and colour of foods enriched
with it. Thus, the much lower a‑value is impacted by the ND‑MOLP, and this was further
confirmed by the calculated total colour difference (∆E), which was highest for cookies en‑
riched with ND‑MOLP (Table 2). This suggests that the use of D‑MOLP can enhance the
colour of cookies, confirming the hypothesis stated above. The sensory characteristics of
the cookies, however, need further investigation since humans are the ultimate consumers.
In terms of hardness and brittleness, MOLP addition generally increased hardness but de‑
creased the brittleness of the cookies (Table 2). The hardness of cookies is influenced by
the development of a gluten network [53] and water–starch–protein interactions within
the ingredients used in the production of cookies [54]. The addition of MOLP to wheat
flour possibly increased the viscosity of the dough, thereby increasing its hardness after
baking. Previous studies also found an increase in cookie hardness following the addi‑
Foods 2024, 13, 1654 9 of 16

tion of MOLP to wheat flour using instrumental methods [17,55] and subjective sensory
analysis [56]. Another plausible reason for the increase in hardness is the higher level
of protein in the Moringa‑enriched cookies (Table 2) as explained later (Section 3.5). Pro‑
teins can absorb moisture, which can significantly affect the overall moisture balance in
the dough. When proteins absorb moisture, they reduce the amount of water available for
other ingredients, such as sugars and starches. This reduction in available moisture can
lead to a drier dough consistency. Consequently, the cookies may have a harder texture
after baking because the dough lacks the necessary moisture to maintain a soft and ten‑
der crumb. McWatters et al. [57] similarly attributed the harder texture of cookies with
added cowpea to the increased protein content and its interaction with the dough during
dough development.
The Moringa‑enriched cookies were very brittle compared to the control wheat cook‑
ies (Table 2). Brittle materials, even those of high strength, absorb relatively little energy
before fracture. Thus, while MOLP increased the hardness of the cookies, it negatively
impacted the brittleness. Optimisation may be required to obtain cookies with minimal
brittleness and moderate hardness.

3.5. Proximate Composition of the Cookies

The proximate composition data for the control cookies and the enriched samples
are presented in Table 2. Carbohydrate (72.35–74.23%), fat (approx. 13%), and protein
(8.46–9.69%) were the major components in the cookies. The ash (2.05–2.66%) and mois‑
ture content (1.52–3.24%) were generally low. The moisture content of the cookies, though
significantly different (p < 0.05), did not follow any trend with the level of addition or de‑
colourisation of the MOLP. All the cookie samples had low levels of moisture typical of
cookies, indicating reduced likelihood of microbial growth. The moisture values obtained
in this study are within the approved standard of ≤5% [58]. Moringa‑enriched cookies gen‑
erally showed higher protein content than the control wheat cookies. However, the level
of increase at 2.5% was not significant (p ≥ 0.05) compared to cookies enriched at 7.5%.
The cookies enriched with 7.5% D‑MOLP and ND‑MOLP both showed an approximately
14% increase in their protein content compared with the wheat cookies. Fapetu et al. [15]
also observed a significant increase in the protein content of cookies enriched with MOLP.
MOLP is a good source of protein with an appreciable amount of amino acid which is vital
for health [59]. Its inclusion in food products can enhance nutritional value, addressing
protein deficiencies and providing a sustainable, plant‑based protein alternative, benefi‑
cial for improving overall diet quality, especially in protein‑limited regions. Several other
authors have reportedly used the leaves in the enrichment of various foodstuffs including
cake [9], bread [10], and yoghurt [8].

3.6. Water Activity of the Cookies

All the Moringa‑enriched cookies showed significantly (p < 0.05) lower aw (0.28–0.32)
compared with the control sample (0.44). Meanwhile, there was no significant (p ≥ 0.05)
difference among the aw water values for the enriched cookies. This suggests that, with
or without decolourisation, the aw value of the cookies is not impacted. The aw values in
this study are within the range of values (0.31 to 0.50) reported for cookies enriched with
Strobilanthes crispus [60] and cookies enriched with the leaves of Mangifera indica (0.30 to
0.49) [61]. Food products with a high aw value have a high risk of bacterial proliferation,
destructive pathogens, and poor shelf‑life [62]. Thus, the enriched cookies may have a
longer shelf‑life than the control wheat cookies. Studies on the shelf stability of the cookies
may be required to validate this claim.

3.7. Total Phenolic, Antioxidant, and Phenolic Acid Content of the Cookies
The total phenolic content (TPC) of the cookies was significantly different among the
samples (Table 2). Both decolourisation and the level of addition of Moringa influenced the
TPC of the cookies. Generally, the TPC increased with increasing levels of Moringa addi‑
Foods 2024, 13, 1654 10 of 16

tion, which may be associated with the presence of phytochemicals, such as flavonoids and
some phenolic compounds in the leaf [63]. These compounds are well‑known to possess
antioxidant properties with health‑promoting benefits. Similarly, Moringa‑enriched cook‑
ies showed higher antioxidant properties than the control wheat cookies. However, the
antioxidant properties of the cookies as measured using the DPPH assay were not different
among the enriched cookies, though the cookies enriched with ND‑MOLP showed slightly
higher values than those containing D‑MOLP. Alves et al. [33] reported a decrease in the
DPPH scavenging activity of MOLP after decolourisation. As earlier noted, Moringa leaves
have phytochemicals with antioxidant properties. Sreelatha and Padma [64] reported vary‑
ing levels of phenolic compounds in tender and matured Moringa leaves. In this study,
six phenolic acids including fumaric, gallic, chlorogenic, syringic, p‑coumaric, and ferulic
acids were screened in the cookies using HPLC, but only three were detected in the cook‑
ies (Figure 1). Ferulic acid, which was the major phenolic acid (1263–1834 mg/100 g) in
Foods 2024, 13, 1654 the cookies, has been suggested to be important in improving cognitive functions11[65] of 16
as
well as possess prebiotic activity by selectively promoting the growth of Lactobacillus and
Parabacteroides in mice [66].

Figure 1.1.Phenolic
Figure Phenolic acids
acids of wheat–Moringa
of wheat–Moringa cookies.
cookies. A: 100%A: 100%
wheat wheat flour;
flour; B: B:wheat
97.5% 97.5%flour
wheat+
flour + 2.5% decolourised Moringa; C: 92.5% wheat flour + 7.5% decolourised Moringa;
2.5% decolourised Moringa; C: 92.5% wheat flour + 7.5% decolourised Moringa; D: 97.5% wheat D: 97.5%
wheat+ 2.5%
flour flour +non-decolourised
2.5% non‑decolourised
Moringa;Moringa;
E: 92.5%E:wheat
92.5%flour
wheat flour
+ 7.5% + 7.5% non‑decolourised
non-decolourised Moringa.
Moringa.
Error Error bars
bars indicate indicate
standard standard
deviation (Ndeviation (N =letters
= 3). Different 3). Different letters mean
mean significantly significantly
different dif‑
(p < 0.05).
ferent (p < 0.05).
3.8. In Vitro Protein Digestibility of the Cookies
Chlorogenic acid was not found in the control cookies but was present in the Moringa‑
The addition of MOLP significantly (p < 0.05) influenced the in vitro protein digesti-
enriched cookies, indicating that the MOLP contributed to the chlorogenic acid in the cook‑
bility (IVPD) of the cookies as illustrated in Figure 2. The cookies containing D-MOLP or
ies. Previous studies found that MOLP is a good source of several phenolic acids including
ND-MOLP both exhibited significantly (p < 0.05) higher protein digestibility (58.82–
chlorogenic acid [67–70]. These phenolic compounds are recognised for their health ben‑
76.43%) compared to the control wheat cookies (52.54%). This improvement aligns with
efits, including anticancer properties, prevention and mitigation of oxidative stress, and
previous studies that reported enhanced protein digestibility of Moringa-enriched cookies
reduction in cellular damage caused by free radicals [43]. The chlorogenic acid content
[12,30,71,72]. The variation in protein digestibility within different foods can be attributed
was higher in the cookies enriched with 7.5% D‑MOLP (1672 mg/100 g) and the cook‑
to inherent differences in food proteins and the presence of antinutrients, which affect the
ies enriched with 7.5% ND‑MOLP compared with those containing 2.5% levels (481 and
bioavailability of amino acids. In this study, the cookies containing ND-MOLP showed
518 mg/100 g, respectively). However, the fumaric acid content of the cookies decreased
significantly
with increasing(p <levels
0.05) lower
of MOLPIVPD butthan
thethose enriched
decrease withsignificant.
was not D-MOLP. This difference
The control is
cook‑
likely due to the decolourisation process, which presumably reduced the levels
ies showed the highest level of fumaric acid (671 mg/100 g) compared with the enriched of antinu-
trients
cookiesin(551–631
the Moringa
mg/100powder.
g). Although the specific antinutrient levels in these cookies
were not determined, previous research indicated that decolourisation of MOLP can re-
duce antinutrients
3.8. In Vitro Proteinsuch as tannins
Digestibility andCookies
of the phytates by approx. 60% [34]. Tannins and phytates
are known to decrease
The addition of MOLPthe bioavailability
significantly (p of foodinfluenced
< 0.05) nutrients the
by forming complexes
in vitro protein with
digestibil‑
proteins
ity (IVPD) of the cookies as illustrated in Figure 2. The cookies containing D‑MOLP of
and minerals, respectively. The IVPD results suggest that the decolourisation or
MOLP could be a beneficial step in enhancing the nutritional quality of Moringa-enriched
food products.
 Foods 2024, 13, 1654 11 of 16

ND‑MOLP both exhibited significantly (p < 0.05) higher protein digestibility (58.82–76.43%)
compared to the control wheat cookies (52.54%). This improvement aligns with previous
studies that reported enhanced protein digestibility of Moringa‑enriched
cookies [12,30,71,72]. The variation in protein digestibility within different foods can be
attributed to inherent differences in food proteins and the presence of antinutrients, which
affect the bioavailability of amino acids. In this study, the cookies containing ND‑MOLP
showed significantly (p < 0.05) lower IVPD than those enriched with D‑MOLP. This dif‑
ference is likely due to the decolourisation process, which presumably reduced the levels
of antinutrients in the Moringa powder. Although the specific antinutrient levels in these
cookies were not determined, previous research indicated that decolourisation of MOLP
can reduce antinutrients such as tannins and phytates by approx. 60% [34]. Tannins and
phytates are known to decrease the bioavailability of food nutrients by forming complexes
Foods 2024, 13, 1654 with proteins and minerals, respectively. The IVPD results suggest that the decolourisa‑
12 of 16
tion of MOLP could be a beneficial step in enhancing the nutritional quality of Moringa‑
enriched food products.

Figure 2. Protein digestibility of wheat–Moringa cookies. A: 100% wheat flour; B: 97.5% wheat
Figure 2. Protein digestibility of wheat–Moringa cookies. A: 100% wheat flour; B: 97.5% wheat flour
+ flour
2.5% +decolourised
2.5% decolourised Moringa;
Moringa; C: wheat
C: 92.5% 92.5% flour
wheat+ 7.5%
flour decolourised
+ 7.5% decolourised
Moringa;Moringa;
D: 97.5%D:wheat
97.5%
wheat flour + 2.5% non‑decolourised Moringa; E: 92.5% wheat flour + 7.5% non‑decolourised
flour + 2.5% non-decolourised Moringa; E: 92.5% wheat flour + 7.5% non-decolourised Moringa.
Moringa.
Error Error bars
bars indicate indicate
standard standard
deviation (N =deviation (N =letters
3). Different 3). Different letters mean
mean significantly significantly
different dif‑
(p < 0.05).
ferent (p < 0.05).
3.9. Amino Acid Profile of the Cookies
3.9. Amino Acid Profile of the Cookies
Moringa addition mainly improved the non-essential amino acid profile of the cook-
Moringa addition mainly improved the non‑essential amino acid profile of the cook‑
ies, though lysine and threonine, which are essential amino acids, were also higher in
ies, though lysine and threonine, which are essential amino acids, were also higher in
Moringa-enriched cookies (Table 3). Glutamic acid was the major amino acid found in all
Moringa‑enriched cookies (Table 3). Glutamic acid was the major amino acid found in all
the cookies. Previous studies similarly reported glutamic acid as the most abundant amino
the cookies. Previous studies similarly reported glutamic acid as the most abundant amino
acid in Moringa-enriched cookies [73,74]. Among the essential amino acids, leucine con-
acid in Moringa‑enriched cookies [73,74]. Among the essential amino acids, leucine con‑
tent (456.47–493.86 mg/100 g) was the highest, while histidine was the lowest (9.98–22.48
tent (456.47–493.86 mg/100 g) was the highest, while histidine was the lowest
mg/100 g). Of all the amino acids, only lysine significantly (p < 0.05) increased in all the
(9.98–22.48 mg/100 g). Of all the amino acids, only lysine significantly (p < 0.05) increased
cookies.
in all theFurthermore, regardlessregardless
cookies. Furthermore, of Moringa of leaf type leaf
Moringa (ND-MOLP or D-MOLP),
type (ND‑MOLP higher
or D‑MOLP),
levels of Moringa leaves showed higher amino acid composition. However,
higher levels of Moringa leaves showed higher amino acid composition. However, the the Moringa
cookies
Moringa containing D-MOLP had
cookies containing slightly
D‑MOLP hadlower amino
slightly acidamino
lower content compared
acid with the
content compared
sample enriched with ND-MOLP, though the difference was not
with the sample enriched with ND‑MOLP, though the difference was not significantsignificant (p ≥ 0.05).
Oyeyinka et al. [34] reported a non-significant increase in histidine, serine,
(p ≥ 0.05). Oyeyinka et al. [34] reported a non‑significant increase in histidine, serine,glycine, glu-
tamic acid, threonine, alanine, proline lysine, tyrosine, methionine, and
glycine, glutamic acid, threonine, alanine, proline lysine, tyrosine, methionine, and isoleucine when
MOLP was when
isoleucine decolourised.
MOLP was Thedecolourised.
improvementThe in non-essential
improvementamino acids is lessamino
in non‑essential beneficial
acids
since
is lessthe body can
beneficial synthesise
since the bodythis category ofthis
can synthesise amino acids.
category of However,
amino acids. considering
However, the
con‑
other benefits
sidering derivable
the other from
benefits the cookies,
derivable such
from the as the significant
cookies, such as the improvement in lysine,
significant improvement
better bioactive compounds, and improved digestibility as shown in previous sections,
the use of D-MOLP in food enrichment may be a welcome development in the food man-
ufacturing sector.

Table 3. Amino acid composition of cookies from wheat–Moringa flour.

Foods 2024, 13, 1654 12 of 16

in lysine, better bioactive compounds, and improved digestibility as shown in previous sec‑
tions, the use of D‑MOLP in food enrichment may be a welcome development in the food
manufacturing sector.

Table 3. Amino acid composition of cookies from wheat–Moringa flour.

97.5% Wheat Flour + 92.5% Wheat Flour + 97.5% Wheat Flour + 92.5% Wheat Flour +
100% Wheat
Parameters 2.5% Decolourised 7.5% Decolourised 2.5% Non‑Decolourised 7.5% Non‑Decolourised
Flour
Moringa Moringa Moringa Moringa
Lysine 25.80 ± 0.33 c 29.22 ± 0.63 b 55.11 ± 2.19 a 28.49 ± 1.07 b 52.85 ± 1.19 a
Histidine 11.83 ± 0.40 b 11.61 ± 0.23 b 22.48 ± 0.69 a 9.98 ± 0.50 c 22.11 ± 0.46 a
Threonine 70.78 ± 1.28 b 71.43 ± 0.38 b 103.88 ± 4.70 a 71.59 ± 0.90 b 103.21 ± 2.50 a
Valine 268.45 ± 16.07 a 258.23 ± 3.99 a 266.17 ± 21.12 a 239.44 ± 22.62 a 257.46 ± 11.17 a
Methionine 96.54 ± 7.12 a 87.69 ± 23.78 a 110.92 ± 5.91 a 92.79 ± 1.27 a 104.32 ± 14.86 a
Isoleucine 220.19 ± 7.56 a 210.43 ± 1.71 ab 213.86 ± 20.21 ab 198.41 ± 11.34 b 213.51 ± 0.66 ab
Leucine 493.86 ± 12.28 a 474.39 ± 10.21 ab 490.56 ± 20.32 a 456.47 ± 18.21 b 478.07 ± 14.49 ab
Phenylalanine 298.17 ± 14.40 a 276.01 ± 14.26 a 293.28 ± 17.15 a 273.47 ± 30.62 a 277.53 ± 12.38 a
Arginine 47.04 ± 1.71 b 50.98 ± 1.60 b 142.11 ± 3.52 a 43.02 ± 4.86 b 143.67 ± 8.11 a
Serine 20.46 ± 1.17 b 20.17 ± 0.87 b 32.14 ± 1.31 a 23.27 ± 5.26 b 33.42 ± 0.62 a
Aspartic acid 72.61 ± 2.82 c 75.08 ± 1.64 c 102.15 ± 1.58 a 86.48 ± 9.11 b 100.65 ± 3.78 a
Alanine 59.88 ± 2.51 b 63.12 ± 0.63 b 94.52 ± 4.18 a 63.11 ± 1.11 b 92.02 ± 2.28 a
Glycine 8.90 ± 0.23 b 8.89 ± 0.69 b 12.29 ± 0.35 a 9.86 ± 1.15 b 11.61 ± 0.42 a
4‑Hydroxyproline 0.90 ± 0.41 b 1.12 ± 0.50 b 4.48 ± 0.53 a 2.67 ± 2.06 ab 3.35 ± 0.29 a
Glutamic acid 2333.02 ± 18.28 a 2268.73 ± 31.06 ab 2213.10 ± 83.15 ab 2111.42 ± 100.90 b 2210.92 ± 39.24 ab
Tyrosine 170.39 ±0.74 bc 181.87 ±3.21 a 164.38 ± 4.12 cd 159.20 ± 5.23 d 176.59 ± 10.06 ab
Proline 820.93 ± 68.43 a 759.43 ± 23.42 ab 770.75 ± 41.67 a 664.38 ± 71.68 b 755.69 ± 29.76 ab
Values are reported as mean ± standard deviation. Mean values with different superscripts are significantly
different (p < 0.05).

4. Conclusions
The functional, nutritional, and in vitro protein digestibility properties of wheat–
Moringa cookies have been reported in this study. The addition of Moringa flour at 2.5 and
7.5% did not change the functional properties of the wheat flour. However, the decolouri‑
sation and level of addition of Moringa leaves increase the spread ratio, protein content,
antioxidant potential, and in vitro protein digestibility of cookies made from the enriched
flours. Although glutamic acid was the major amino acid in the cookies, lysine was the only
amino acid that increased significantly in the enriched cookies compared to the control. All
the cookies except the control wheat cookies are good sources of chlorogenic, ferulic, and
fumaric acids. Decolourisation did not negatively affect the bioactive compounds in the
cookies including their antioxidant properties as measured by the DPPH assay. This study
addresses the major challenge, of greenish discolouration imparted to foods by Moringa
leaves and provides some insight into the application of decolourised Moringa leaves in
cookie production. This research marks the first report on using decolourised Moringa
oleifera leaf powder in cookie formulation. Our findings not only highlight the potential
of enhancing access to nutritious food (aligned with SDG‑2) but also demonstrate the fea‑
sibility of delivering a nutrient‑dense staple suitable for all age groups, thereby promot‑
ing health and well‑being (SDG‑3). This is particularly promising given the widespread
consumption of cookies across various age demographics. The decolourisation of MOLP
presents a promising processing strategy applicable to food products with low consumer
acceptability. Future research should focus on assessing sensory characteristics, consumer
acceptability, volatile components, and the shelf‑life of the cookies. Additionally, explor‑
ing environmentally friendly methods like enzymatic decolourisation is recommended for
further investigation.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/foods13111654/s1, Table S1, Retention time, linear calibration
range, and R2 for amino and phenolic acids.
Foods 2024, 13, 1654 13 of 16

Author Contributions: Conceptualization, S.A.O. and B.A.O.; methodology, S.A.O., B.A.O., M.L.,
J.T. and J.D.W.; validation, S.A.O., J.D.W. and B.A.O.; formal analysis, S.A.O., T.D.A., A.K., N.O.Y.‑
A. and B.A.O.; investigation, A.K., T.D.A., N.O.Y.‑A., S.A.O., M.L., J.T. and J.D.W.; resources, S.A.O.
and B.A.O.; data curation, A.K., T.D.A., N.O.Y.‑A., M.L. and J.T.; writing—original draft prepara‑
tion, S.A.O., A.K., T.D.A. and N.O.Y.‑A.; writing—review and editing, S.A.O., M.L., J.T. and J.D.W.;
supervision, S.A.O. and B.A.O.; project administration, S.A.O., B.A.O., M.L., J.T. and J.D.W.; fund‑
ing acquisition, S.A.O. and B.A.O. All authors have read and agreed to the published version of
the manuscript.
Funding: The authors wish to thank the College of Science, University of Lincoln, for the Pump mini
grant awarded to Dr Samson Oyeyinka and Dr Bukola Onarinde.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Materials, further inquiries can be directed to the corresponding author.
Conflicts of Interest: The authors declare no conflicts of interest.

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