molecules

Article
Ultrasound-Assisted Extraction of Protein from Moringa oleifera
Seeds and Its Impact on Techno-Functional Properties
Khushar Fatima 1 , Muhammad Imran 1 , Muhammad Haseeb Ahmad 1 , Muhammad Kamran Khan 1, *,
Waseem Khalid 1,2 , Ammar AL-Farga 3 , Wafa S. Alansari 3 , Ghalia Shamlan 4 and Areej A. Eskandrani 5

1 Department of Food Science, Faculty of Life Sciences, Government College University,

Faisalabad 38000, Pakistan
2 University Institute of Food Science and Technology, The University of Lahore, Lahore 54000, Pakistan
3 Biochemistry Department, Faculty of Science, University of Jeddah, Jeddah 21577, Saudi Arabia
4 Department of Food Science and Nutrition, College of Food and agriculture Sciences, King Saud University,
Riyadh 11362, Saudi Arabia
5 Chemistry Department, Faculty of Science, Taibah University, Medina 30002, Saudi Arabia
* Correspondence: [email protected]

Abstract: Plant proteins can be an important alternative to animal proteins subject to minor modifi-
cation to address sustainability issues. The impact of ultrasound application on the yield, techno-
functional properties, and molecular characteristics of protein extracted from Moringa oleifera seeds
was studied. For this purpose, a central composite design (CCD) was applied to optimize ultrasound-
assisted extraction (UAE) parameters such as amplitude (25–75%), solute-to-solvent ratio (1:10–1:30),
and pH (9–13) for obtaining the maximum protein yield. At the optimized conditions of 75% am-
plitude, 1:20 solute-to-solvent ratio, and 11 pH, a protein yield of 39.12% was obtained in the UAE
process. Moreover, the best sonication time at optimized conditions was 20 min, which resulted
in about 150% more extraction yield in comparison to conventional extraction (CE). The techno-
functional properties, for instance, solubility, water (WHC)- and oil-holding capacity (OHC), and
Citation: Fatima, K.; Imran, M.; emulsifying and foaming properties of the protein obtained from UAE and CE were also compared.
Ahmad, M.H.; Khan, M.K.; Khalid, The functional properties revealed high solubility, good WHC and OHC, and improved emulsify-
W.; AL-Farga, A.; Alansari, W.S.; ing properties for protein obtained from UAE. Although protein from UAE provided higher foam
Shamlan, G.; Eskandrani, A.A.
formation, foaming stability was significantly lower.
Ultrasound-Assisted Extraction of
Protein from Moringa oleifera Seeds
Keywords: Moringa oleifera; seed; protein; ultrasound; functional properties
and Its Impact on Techno-Functional
Properties. Molecules 2023, 28, 2554.
https://doi.org/10.3390/
molecules28062554
1. Introduction
Academic Editor: Constantinos
Proteins impart some important techno-functional properties such as emulsification,
K. Zacharis
foaming, and/or gelling, owing to which they are considered to be one of the main com-
Received: 2 February 2023 ponents of food products [1]. Proteins with specific functionalities are either synthesized
Revised: 3 March 2023 chemically or extracted from animal and plant sources. Due to the intensive competition
Accepted: 9 March 2023 among industries and more specified demands from the consumers and to address sus-
Published: 11 March 2023 tainability issues, food industries always look for solutions to meet market challenges.
Currently, plant-derived proteins, particularly from agro-industrial waste, receive substan-
tial attention as a sustainable alternative to animal-based proteins due to the rising cost of
animal proteins and food security and sustainability issues [2].
Copyright: © 2023 by the authors.
Moringa oleifera (Lam.), which is a widely cultivated species in native parts of Asia
Licensee MDPI, Basel, Switzerland.
This article is an open access article
and Africa, belongs to the family Moringaceae [3]. M. oleifera, also known as Drumstick
distributed under the terms and
tree, is considered to be a nutritionally dense plant and is also referred to as a ‘miracle
conditions of the Creative Commons Tree’ because of its multi-purpose uses. Ease of cultivation makes it a cheap source of
Attribution (CC BY) license (https:// high-quality nutrients and ingredients in traditional herbal medicines [4]. The extract of
creativecommons.org/licenses/by/ its leaves is rich in important phytochemicals and has potential as an antioxidant [5], anti-
4.0/). microbial [6], anti-inflammatory [7], and anticancer agent [8]. Although seeds of M. oleifera

Molecules 2023, 28, 2554. https://doi.org/10.3390/molecules28062554 https://www.mdpi.com/journal/molecules

Molecules 2023, 28, 2554 2 of 17

are rich in protein (35–36%) and oil (38–39%) [9], they are still used mainly as feedstock [10]
and currently have no value-added ingredients or products. The present study revolves
around the extraction of M. oleifera seed protein (MOSP) to develop a high value-added
ingredient for food products.
The main challenge while extracting the proteins is to choose the appropriate extraction
technique. Traditionally, methods such as alkaline, organic solvent, salt, and enzymatic
extraction, which are used for protein extraction, are less efficient, require long extraction
time, give less protein yield, involve a high amount of solvents, and ultimately lead
to an environmental burden [11]. The application of novel and more accurate protein
extraction techniques can enhance the functional properties of foods. Ultrasonic-assisted
extraction is considered to be an efficient and environmentally friendly extraction technique
compared to conventional and other novel techniques because of its lower extraction time,
high extraction yield, low solvent consumption, and enhanced functional properties of
protein [12]. Sonication technique is associated with the phenomenon of acoustic cavitation,
during which the collapse of bubbles releases energy for enhanced mass transfer from and
to the interface; thus, this technique is classified as a sustainable and green technique [13].
The extraction efficiency of ultrasound is influenced by different parameters, such as
ultrasonic intensity, solute-to-solvent ratio, treatment time, temperature, etc., which need
to be optimized using appropriate combinations or statistical designs [14].
Conclusively, the study is mainly focused on the effect of techno-functional properties
such as protein solubility, water- and oil-holding capacity, and emulsifying and foaming
properties of M. oleifera seed protein extracted via the sonication technique. Moreover,
fluorescence and Fourier-transform infrared spectra will be taken to identify changes in
functional groups.

2. Results and Discussion

2.1. Optimization of Ultrasonic-Assisted Extraction (UAE) of Protein
2.1.1. Fitting the Proposed Model
Response surface methodology (RSM) is a useful statistical technique for evaluating
the influence of different factors and for designing experiments such as the one in this
study [15]. An empirical model can be designed to evaluate the optimized conditions using
the required response. In the present study, the effect of ultrasonication on the extraction
efficiency of M. oleifera seed protein (MOSP) was evaluated to observe the maximum
recovery of the protein using RSM. For this, a central composite design (CCD) was applied
using three independent variables: amplitude (%) (A), solute-to-solvent ratio (g/mL) (B),
and pH (C) (Table 1) with 17 runs with 3 central points. Usually, if one ultimately misses
any runs, the accuracy of the remaining runs in the Box–Behnken Design (BBD) becomes
critical to the dependability of the model, so a CCD is preferred.
The MOSP yield obtained ranged between 30 and 39%, depending on the combination
of the trial. Experimental run 16 showed the lowest yield with an amplitude of 25%, a
solute-to-solvent ratio of 1:10, and a pH of 9, while experimental run 10 showed the highest
yield with an amplitude of 75%, a solute-to-solvent ratio of 1:20, and a pH of 11. The
difference between the measured values and the predicted values is minimal, which shows
the rationality of the measured values. Following quadratic polynomial regression, an
equation of amplitude (A), solute-to-solvent ratio (B), and pH (C) was developed to obtain
the response model of extraction yield:

Y1 = 36.59 + 3.08 A + 0.4920 B + 0.1120 C + 0.0050 AB + 0.0050 AC − 0.0250

BC − 0.3726 A2 + 0.1124 B2 − 1.71 C2

The results obtained were further processed for analysis of variance (ANOVA) with
a 95% confidence level in order to check the significance and suitability of the response
model (Table 2).
Molecules 2023, 28, 2554 3 of 17

Table 1. Central composite design representing the experimental trials along with M. oleifera seed
protein (MOSP) yield.

Independent Variables Independent Variables Response

(Coded Values) (Actual Values) (MOSP Yield (%))
Run A: B: Solute-to- A: B: Solute-to-
Amplitude Solvent Ratio C: pH Amplitude Solvent Ratio C: pH Measured Predicted
(%) (g/mL) (%) (g/mL)
1 0 0 −1 50 1:20 9 34.76 34.77
2 1 −1 1 75 1:10 13 37.34 37.32
3 −1 0 0 25 1:20 11 33.15 33.13
4 −1 1 1 25 1:30 13 32.10 32.11
5 (c.p.) 0 0 0 50 1:20 11 36.62 36.59
6 (c.p.) 0 0 0 50 1:20 11 36.52 36.59
7 1 −1 −1 75 1:10 9 37.06 37.07
8 −1 −1 1 25 1:10 13 31.17 31.18
9 0 −1 0 50 1:10 11 36.22 36.20
10 1 0 0 75 1:20 11 39.30 39.29
11 1 1 1 75 1:30 13 38.28 38.29
12 (c.p.) 0 0 0 50 1:20 11 36.57 36.59
13 0 0 1 50 1:20 13 35.02 34.99
14 −1 1 −1 25 1:30 9 31.94 31.93
15 0 1 0 50 1:30 11 37.20 37.19
16 −1 −1 −1 25 1:10 9 30.92 30.91
17 1 1 −1 75 1:30 9 38.11 38.10
c.p. = central point.

Table 2. Analysis of variance (ANOVA) for quadratic model.

Source Sum of Squares df Mean Square F-Value p-Value

Model 111.81 9 12.42 11,418.21 <0.0001 **
A: Amplitude 94.93 1 94.93 87,243.93 <0.0001 **
B:
Solute-to-solvent 2.42 1 2.42 2224.75 <0.0001 **
ratio
C: pH 0.1254 1 0.1254 115.29 <0.0001 **
AB 0.0002 1 0.0002 0.1838 0.6810 ns
AC 0.0002 1 0.0002 0.1838 0.6810 ns
BC 0.0050 1 0.0050 4.60 0.0693 ns
A2 0.3720 1 0.3720 341.87 <0.0001 **
B2 0.0338 1 0.0338 31.11 0.0008 **
C2 7.81 1 7.81 7180.25 <0.0001 **
Residual 0.0076 7 0.0011
Lack of Fit 0.0026 5 0.0005 0.2093 0.9308 ns
Pure Error 0.0050 2 0.0025
Cor Total 111.82 16
R2 0.9999
R2 adjusted 0.9998
** Significant at 0.01 level; ns = non-significant; df = degree of freedom.

The results indicate that the p-value of the overall model is less than 0.0001, which
is highly significant, and it indicates the sustainability of the fitted model. Additionally,
the F-value documented the significance of the response model. Moreover, the value for
determination coefficient (R2 ) was 0.9999, and the adjusted value (R2 adjusted) for the
extracted yield was 0.9998, which shows that the model was correctly interpreted using
the measured data. Generally, R2 values higher than 0.75 are considered to be superlative
for a good fitted model [16]. Furthermore, the values of the coefficient estimate report
the expected change in the response factor value when all remaining factors are held at
a medium level (Table 3). Coefficients with positive values represent a linear increase in
 tracted yield was 0.9998, which shows that the model was correctly interpreted using the
measured data. Generally, R2 values higher than 0.75 are considered to be superlative for
a good fitted model [16]. Furthermore, the values of the coefficient estimate report the
expected change in the response factor value when all remaining factors are held at a
medium level (Table 3). Coefficients with positive values represent a linear increase in
Molecules 2023, 28, 2554 4 of 17
the response factor, and those with negative values document the linear decrease in the
dependent factor. The accuracy of the model was checked via the correlation coefficient.

the response
Table factor,
3. Coefficient and those
estimation with negative
in terms values document the linear decrease in the
of coded factors.
dependent factor. The accuracy of the model was checked via the correlation coefficient.
Coefficient
Factor df Standard Error 95% CI Low 95% CI High VIF
3. Coefficient estimation in terms of coded factors.
TableEstimate
Intercept 36.59 1 0.0141 36.55 36.62
Factor Coefficient Estimate df Standard Error 95% CI Low 95% CI High VIF
A: Amplitude 3.08 1 0.0104 3.06 3.11 1.0000
Intercept 36.59 1 0.0141 36.55 36.62
B: Solute to solvent ratio 0.4920 1 0.0104 0.4673 0.5167 1.0000
A: Amplitude 3.08 1 0.0104 3.06 3.11 1.0000
C:
B: Solute to pH 0.1120 1 0.0104 0.0873 0.1367 1.0000
0.4920 1 0.0104 0.4673 0.5167 1.0000
solvent ratioAB 0.0050 1 0.0117 −0.0226 0.0326 1.0000
C: pH AC 0.1120 0.0050 1 1 0.0104
0.0117 0.0873 −0.0226 0.1367 0.0326 1.0000
1.0000
AB BC 0.0050 −0.0250 1 1 0.0117
0.0117 −0.0226 −0.0526 0.0326 0.0026 1.0000
1.0000
AC 0.0050 1 0.0117 −0.0226 0.0326 1.0000
BC A 2
−0.0250 −0.3726 1 1 0.0202
0.0117 −0.0526 −0.4203 0.0026−0.3250 1.54
1.0000
A2 B2 −0.3726 0.1124 1 1 0.0202
0.0202 −0.4203 0.0647 −0.32500.1600 1.541.54
B2 C2 0.1124 −1.71 1 1 0.0202
0.0202 0.0647 −1.76 0.1600 −1.66 1.541.54
C2 −1.71 1
df = degree of freedom; 0.0202
CI = confidence interval; − 1.76
VIF −1.66factor.
= variance inflation 1.54
df = degree of freedom; CI = confidence interval; VIF = variance inflation factor.
2.1.2. Single-Factor Analysis for Protein Yield
2.1.2.According
Single-Factor to Analysis
the results for obtained,
Protein Yieldit was observed that the amplitude (A), so-
Accordingratio
lute-to-solvent to the(B),
results
and obtained, it was observed
pH (C) independent that
factors theaamplitude
had significant(A), solute-to-
effect on the
solvent ratio (B), and pH (C) independent factors had a significant
yield of MOSP. The variation in the response factor (yield, %) due to independent effect on the yield of
varia-
MOSP. Theform
bles in the variation in thelevels
of coded response factor
is also (yield,in
presented %)Figure
due to1.independent
The effect ofvariables in the
the individual
form of coded levels is also presented in Figure 1. The effect of the
variable was noted while keeping the other two at their medium values. The single-factor individual variable
was notedgraph
response while shows
keepingthat thethe
other two at their
amplitude medium
variable hasvalues.
a directThe single-factor
effect response
on the increase in
graph shows that the amplitude variable has a direct effect on the increase
yield. On the other hand, the pH imparts an increasing trend for the response yield until in yield. On
the
the other
central hand,
point,thewhile
pH imparts an increasing
a decrease in pH leads trend
to afor
lowthe response
yield yield until
of response factortheafter
central
the
point, while a decrease in pH leads to a low yield of response
central point. Moreover, the independent solute-to-solvent ratio factor has shown a factor after the central
point. Moreover,impact
non-significant the independent solute-to-solvent
on the response factor before ratio
andfactor
afterhas
the shown
centralapoint.
non-significant
impact on the response factor before and after the central point.
40

39

38
Protein Yield (%)

37

36

35

Amplitude (%)
34
Ratio
pH
33
-1.5 -1 -0.5 0 0.5 1 1.5
Coded Levels

Figure 1. Linear effect of studied parameters on the MOSP

MOSP yield.
yield.

2.1.3. Effect of
2.1.3. Effect of Mutual
Mutual Interactions
Interactions on
on Protein
Protein Yield
Yield
The interactions that occurred between amplitude
The interactions that occurred between amplitude and and solute-to-solvent ratio (AB),
solute-to-solvent ratio (AB),
amplitude and pH (AC), and solute-to-solvent ratio and pH (BC) had a non-significant
amplitude and pH (AC), and solute-to-solvent ratio and pH (BC) had a non-significant
effect on protein yield (p > 0.05). The interaction between independent variables is also
shown in the form of 3D response surface plots (Figure 2). Through the study of the
obtained results, the maximum yield obtained by the mutual interaction of amplitude
and solute-to-solvent ratio (Figure 2a) was observed. It was observed from the results
that when interaction between amplitude and pH occurred with the average value of the
solute-to-solvent ratio, the yield percentage slightly decreased at the maximum amplitude
(Figure 2b). On the other hand, there was a decrease in the yield percentage as the solute-
to-solvent ratio and pH were considered to be interacting variables by taking the average
fixed amplitude (Figure 2c). These plots showed that the selected factor levels were logical
 tained results, the maximum yield obtained by the mutual interaction of amplitude and
solute-to-solvent ratio (Figure 2a) was observed. It was observed from the results that
when interaction between amplitude and pH occurred with the average value of the so-
lute-to-solvent ratio, the yield percentage slightly decreased at the maximum amplitude
(Figure 2b). On the other hand, there was a decrease in the yield percentage as the so-
Molecules 2023, 28, 2554 5 of 17
lute-to-solvent ratio and pH were considered to be interacting variables by taking the
average fixed amplitude (Figure 2c). These plots showed that the selected factor levels
were logical enough and had a positive influence on extraction yield. According to the
enough
figures, and had a positive
the interactions influence
between on extraction
the variables yield. According
significantly to extraction
affected the the figures, the
yield,
interactions between
which was also the variables
interpreted throughsignificantly
ANOVA (Tableaffected
2). the extraction yield, which was
also interpreted through ANOVA (Table 2).

40 40

38
38
Protein Yield (%)

Protein Yield (%)

36
36
34

34
32

32 30
30 13
25 80 12 80
20 60 11 60
Ra pH
ti o 15 40 ( %) 10 40 ( %)
e e
litud litud
10 20 Am p 9 20 Am p

(a) (b)

38

37
Protein Yield (%)

36

35

34
13
12 30
11 25
pH 20
10
15 o
9 Ra ti
10

(c)
Figure 2. Response surface plots representing the effect of mutual interactions of studied parame-
Figure 2. Response surface plots representing the effect of mutual interactions of studied parameters
ters on the protein yield. Interaction between amplitude & ratio (a), amplitude & pH (b) and ratio &
on
pHthe
(c) protein yield. Interaction
while keeping between
third parameter amplitude
at central value.& ratio (a), amplitude & pH (b) and ratio &
pH (c) while keeping third parameter at central value.
2.1.4. Optimization
2.1.4. Optimization andand Validation
Validation
The predicted
The predictedextraction
extractionyield
yieldatat optimized
optimized conditions
conditions waswas noted
noted fromfrom the experi-
the experimen-
mental design. According to CCD and contour plots, the best extraction
tal design. According to CCD and contour plots, the best extraction yield (39.90%) yield (39.90%)
was
was predicted at the optimized conditions of an amplitude higher than
predicted at the optimized conditions of an amplitude higher than 75%, a solute-to-solvent 75%, a so-
lute-to-solvent
ratio ratio
of 1:22, and a pH ofof
1:22, andwhich
11.37, a pH were
of 11.37, whichoff
rounded were rounded
to 75%, 1:20, off
andto11,
75%, 1:20, and
respectively,
11, the
for respectively, for the ease ofValidation
ease of experimentation. experimentation. Validation
of the statistical of the
model andstatistical modelequa-
the regression and
the regression equation was confirmed by repeating the experimental run
tion was confirmed by repeating the experimental run at defined optimized conditions. Theat defined op-
timized conditions. The measured value of the extraction yield (39.12%)
measured value of the extraction yield (39.12%) at optimized conditions (amplitude 75%, at optimized
conditions (amplitude
solute-to-solvent ratio of75%,
1:20,solute-to-solvent ratio the
and pH 11) confirmed of 1:20, andofpH
validity the11) confirmed
model. the va-
The obtained
values were quite close to the predicted ones. Overall, the results showed that the model
for the extraction of MOSP through ultrasonication was fit and acceptable. Furthermore,
a comparison of protein yield was made between UAE and conventional extraction (CE).
While keeping the other parameters at optimized levels, the sonication treatment resulted
in a 150% increase in the protein yield in comparison to CE (Figure 3).
Molecules
Molecules2023, 28,28,
2023, x FOR
2554PEER REVIEW 7 of6 17
of 17

50

MOSP yield (%)

40
30
20
10
0
0 10 20 30
Extraction time (min)

Figure 3. MOSP yield (%) during ultrasound-assisted extraction () and conventional extraction (N).
Figure 3. MOSP yield (%) during ultrasound-assisted extraction ( ) and conventional extraction
(▲). For a commercial point of view, the yield of protein is highly important, particularly
for the efficient utilization of agro-waste and to achieve the goal of sustainable development.
2.2. Functional Properties
Conventional extraction of MOSP
techniques for protein other than ultrasonication are either time-
2.2.1. Solubility
consuming or less productive. Therefore, UAE was applied for the extraction of protein
from M. oleifera
Solubility is seeds.
one ofAs theit was
mostnoticed during
important this study,properties
functional various studies have also
of protein. It isshown
the
an increase in the protein yield with the increase in the power/amplitude
thermodynamic property which can be elaborated as the ‘protein concentration in a sat- of the sound
waves,
urated for instance,
solution’, and itwampee
must beseed protein [11],
at equilibrium lupin
with theseed
solidprotein
phase at [17], Eurycoma
optimal longifolia
conditions.
root protein [18], and Dolichos lablab L. bean protein [19]. It is known
Solubility of proteins can be altered by any extrinsic and intrinsic factors [26]. At neutral that ultrasonication
pH,works on the principal
the solubility of MOSP of cavitation,
extracted and as aconventional
via the result of the cavitational
method waseffect, 5.56 a± mechanical
0.13%. A
significant increase was observed in the solubility of MOSP extracted withbarrier,
force is generated which helps in the transport of protein across the cell either by
the ultrasonic
increasing the flow of solvent on both sides or, sometimes, by
method, which was 29.82 ± 0.21% (Table 4). Ultrasonication of MOSP resulted in the en- rupturing the cell barrier [20].
Although of
hancement ultrasonic
protein amplitude
solubility. remained
This may promising
have happenedfor a high
becauseMOSP of yield, the solute-to-
the reduction in
solvent ratio presented a minor effect on the protein yield. The lower solute-to-solvent
particle size of the MOSP alteration in the molecular structure and changes in the con-
ratio led to the lower difference in protein concentration inside and outside the cell matrix,
formation [27], thus exposing more hydrophilic groups in the medium for increased
thus reducing the protein yield [11]. On the other hand, the higher solute-to-solvent ratio
solubility. Similar results were observed on the solubility of whey protein [28] and pea
resulted in less ultrasonic energy density per unit volume and ultimately, lower protein
protein [29].
yield was observed. Although there was a minimal effect of the solute-to-solvent ratio on
the MOSP
Table yield,properties
4. Functional the optimum of M. value
oleiferaof 1:20
seed g/mL(MOSP)
protein was found to be best forextraction
from conventional the maximum(CE)
andMOSP yield. The solute-to-solvent
ultrasonic-assisted extraction (UAE).ratio often varies depending on the protein source, the
structure of the protein, the extraction medium, and others [21]. The pH value significantly
Functional Properties
affected the protein yield, MOSP (CE)protein yield increased
as the MOSPwith (UAE) the increase in pH value up
Solubilityto(%)
11. This might be attributed5.56 ± 0.13 to the breakage of H-bonds29.82 ± in 0.21the cell matrix and, thus, an
increase in the protein0.86
WHC (g/g) yield [22]. Any further increase
± 0.009 1.02in± pH
0.006 resulted in the degradation
of
OHC (g/g) protein, causing reduced solubility,
0.91 ± 0.015 and consequently,
1.91 ± 0.013yield of MOSP decreased.
the
Similar
Emulsion capacity results were obtained during the extraction of proteins from wampee seed [11] and
Dolichos lablab L. [19]. 58.39 ± 1.68 75.93 ± 1.19
(mg/mL)
Foaming capacityAt (%)optimized conditions,13.21 ± 0.27different ultrasonic times 24.23(0–30
± 0.64min) were observed for the
maximum MOSP yield, and a higher yield was found at 20 min (Figure 3). With further
WHC = water-holding capacity; OHC = oil-holding capacity.
increases in time, a slight decrease was observed. The 30 min treatment was counterpro-
ductive
The high by dropping
amplitude thelevel
protein yield. Thisescalates
of ultrasound might bethe due to the solubility
protein structuralby degradation
altering
of protein, which generally develops aggregates by folding
the conformation and structure of protein; this results in the inside aperture of hydro- to resist the extreme condi-
tions, and ultimately, proteins do not solubilize and possibly
philic ends of amino acids toward water [30]. A larger area of protein was covered up finish with the centrifugation
residue [23]. In the case of conventional extraction, the yield of MOSP remained less than
with water, as the molecular weight of the treated protein was reduced due to the high
that of UAE at each time period, which further confirms the effectiveness of the sonication
ultrasonic amplitude [31]. The temperature rise due to ultrasonication also had a signifi-
treatment. In most of the studies based on the sonication treatment for the extraction
cant effect on the improvement of protein solubility, as protein solubility rose with the
of protein from different plant sources, the best extraction time ranged between 15 and
rise of temperature, as reported in the studies for soy protein [32]. Enhancement in pro-
20 min [21,23,24]. The time may increase depending on the ultrasound intensity and equip-
tein solubility could also be due to the alteration in the three-dimensional structure of
ment; for instance, this ultrasound-assisted extraction time may reach more than 60 min if
globular protein, which resulted in a higher number of charged groups established with
an ultrasonic cleaner was used rather than an ultrasonic probe [25].
high electrical conductivity, unlike the CE sample. Under those conditions, as more water
interacted with proteins, and electrostatic forces increased, the inter-linkage between
water and protein improved, thus increasing the protein solubility.
Molecules 2023, 28, 2554 7 of 17

2.2. Functional Properties of MOSP

2.2.1. Solubility
Solubility is one of the most important functional properties of protein. It is the ther-
modynamic property which can be elaborated as the ‘protein concentration in a saturated
solution’, and it must be at equilibrium with the solid phase at optimal conditions. Solubil-
ity of proteins can be altered by any extrinsic and intrinsic factors [26]. At neutral pH, the
solubility of MOSP extracted via the conventional method was 5.56 ± 0.13%. A significant
increase was observed in the solubility of MOSP extracted with the ultrasonic method,
which was 29.82 ± 0.21% (Table 4). Ultrasonication of MOSP resulted in the enhancement
of protein solubility. This may have happened because of the reduction in particle size of
the MOSP alteration in the molecular structure and changes in the conformation [27], thus
exposing more hydrophilic groups in the medium for increased solubility. Similar results
were observed on the solubility of whey protein [28] and pea protein [29].

Table 4. Functional properties of M. oleifera seed protein (MOSP) from conventional extraction (CE)
and ultrasonic-assisted extraction (UAE).

Functional Properties MOSP (CE) MOSP (UAE)

Solubility (%) 5.56 ± 0.13 29.82 ± 0.21
WHC (g/g) 0.86 ± 0.009 1.02 ± 0.006
OHC (g/g) 0.91 ± 0.015 1.91 ± 0.013
Emulsion capacity (mg/mL) 58.39 ± 1.68 75.93 ± 1.19
Foaming capacity (%) 13.21 ± 0.27 24.23 ± 0.64
WHC = water-holding capacity; OHC = oil-holding capacity.

The high amplitude level of ultrasound escalates the protein solubility by altering the
conformation and structure of protein; this results in the inside aperture of hydrophilic
ends of amino acids toward water [30]. A larger area of protein was covered up with water,
as the molecular weight of the treated protein was reduced due to the high ultrasonic
amplitude [31]. The temperature rise due to ultrasonication also had a significant effect on
the improvement of protein solubility, as protein solubility rose with the rise of temperature,
as reported in the studies for soy protein [32]. Enhancement in protein solubility could
also be due to the alteration in the three-dimensional structure of globular protein, which
resulted in a higher number of charged groups established with high electrical conductivity,
unlike the CE sample. Under those conditions, as more water interacted with proteins, and
electrostatic forces increased, the inter-linkage between water and protein improved, thus
increasing the protein solubility.

2.2.2. Water (WHC)- and Oil-Holding Capacity (OHC)

WHC and OHC are important functional properties of protein used for the moderation
of the texture and viscosity of the food product and also for the reduction in the processes
of dehydration during food storage [33]. The ability of proteins to retain or hold water in
their three-dimensional structure is known as the WHC of protein. Proteins with a high
WHC during application in a food product can dehydrate the other ingredients present in
food, and thus, the product becomes less sensitive to storage humidity [34].
The WHC of MOSP extracted with CE was 0.86 ± 0.009 g/g, and that of the UAE
sample was 1.02 ± 0.006 g/g (Table 4). The increase in water-holding capacity after
ultrasonication was probably due to the spongy structure generated by peptide chains and
due to the ionized polarity groups formed after ultrasound treatment [35]. Because of these
groups, loose structures were formed and produced more space for water storage; thus,
this condition resulted in a higher WHC. Similar results were obtained after the application
of ultrasonication on the beef Longissmus lumborum [36].
Oil-holding capacity (OHC) is the ability of proteins to trap oil or fat within their
non-polar chains. The oil-holding capacity of the MOSP sample extracted with CE was
0.91 ± 0.015 g/g (Table 4). According to the results obtained, the OHC of the MOSP ex-
Molecules 2023, 28, 2554 8 of 17

tracted with UAE was higher than that of the CE one. The ultrasonically treated MOSP
sample had a significantly higher OHC (1.91 ± 0.013 g/g). Ultrasonication increased the
surface exposure of hydrophobic groups, due to which a strong linkage formed with triglyc-
eride molecules, resulting in the improved OHC (Boukhari, Doumandji et al. 2018). The
same trends were observed in related studies on whey protein isolates [16] and tamarind
seeds protein isolates [37].
The improved WHC and OHC are considered helpful for enhancing the shelf life
of processed foods given the fats and water reduced from the surface [38]. Their higher
values also improve the mouth feel of the product. Overall, it was observed that ultrasonic
treatment had a significant impact on water- and oil-holding capacities.

2.2.3. Emulsion Capacity and Emulsion Stability

The emulsifying property of protein is its ability to form an emulsion and to maintain
the stability of the newly formed emulsion [39]. It is an important parameter in the pro-
duction of various fabricated foods. The emulsion capacity of MOSP extracted with CE
was 58.39 ± 1.68 mg/mL (Table 4). Next, the ultrasonic treatment emulsion capacity of
MOSP significantly increased to 75.93 ± 1.19 mg/mL. Prepared emulsions were kept at
room temperature to calculate the emulsion stability. A slight improvement was observed
in the emulsion stability of the MOSP extracted with UAE (W = 4%) as compared to that of
the CE one (W = 6%); this improvement might be associated with the higher hydrophobic
levels and increased droplet size of oil in the water emulsion, which was established via ul-
trasonication [40]. In addition, it has been reported that the increased solubility of proteins
resulted in a maximal emulsifying capacity. Furthermore, the increased emulsifying capac-
ity of MOSP might be related to the alternation in aggregation, solubility, and secondary
structure [41]. A recent study which was conducted to study the effects of ultrasonication
on animal and vegetable proteins showed similar development in the emulsion properties
of protein [42,43].

2.2.4. Foaming Capacity and Foaming Stability

The total inter-facial area formed by the whipping of protein is called the foaming
capacity (FC) of the protein [44]. The stability of foam is calculated as the total time
required to lose the volume of the foam. It is an important functional property of protein
that helps in the production of formulated foods. The foaming capacity of MOSP extracted
with CE was 13.21 ± 0.27% (Table 4). The foaming capacity of MOSP was improved to
24.23 ± 0.64% after ultrasonic treatment. The observed enhancement in foaming capacity
might be associated with the effect of ultrasonic homogenization, which improved the
foaming power [45]. This homogenization evenly distributed the particles of protein, which
increased the foaming ability of the MOSP sample extracted with UAE. Corresponding
results were obtained in another study on whey protein [46]. The increased exposure of
hydrophobic groups aided in the dispersion of protein molecules toward air-water interface
and their surface assimilation on it. A similar trend in the foaming capacity of protein was
also shown in an earlier study conducted to study the effects of high-intensity ultrasonic
treatment on food proteins [47]. However, the trend was opposite in the case of foaming
stability (FS). There was no significant improvement observed in MOSP extracted with UAE
when compared to the CE one. The reason for this might be the ultrasonic cavitation which
increased MOSP solubility, and the foaming stability dropped because of the decrease in
surface activity [27]. Furthermore, the breakdown of larger peptide units into smaller ones
also causes the reduction in foaming stability. It was reported earlier in a few studies that
ultrasonication may have had no significant effect on foaming stability, as observed in
de-hulled yellow mustard protein [48] and pea protein [49] isolates.
 stability dropped because of the decrease in surface activity [27]. Furthermore, the
breakdown of larger peptide units into smaller ones also causes the reduction in foaming
stability. It was reported earlier in a few studies that ultrasonication may have had no
significant effect on foaming stability, as observed in de-hulled yellow mustard protein
Molecules 2023, 28, 2554 [48] and pea protein [49] isolates. 9 of 17

2.3. Structural Study of MOSP

2.3.1. 2.3.
FT-IR AnalysisStudy of MOSP
Structural
The
2.3.1.FT-IR
FT-IRspectrum
Analysis of MOSP can be described in three importance wavelength
bands (Figure 4).
The FT-IR Generally,
spectrumthe FT-IR can
of MOSP spectrum is based
be described on the
in three different amide
importance zonesbands
wavelength
[50]. The amide-I zone representing C=O bonds is stretched over
(Figure 4). Generally, the FT-IR spectrum is based on the different amide zones the wavelength range
[50]. The
from amide-I
1700 to 1600 cm −1; the amide-II zone is associated with N–H bonds which are spread
zone representing C=O bonds is stretched over the wavelength range from 1700 to
over the
16001575
cm−to 1 ; 1480 cm−1 wavelength
the amide-II range; and
zone is associated withtheN–Hamide-III
bonds zone whichrepresenting
are spread overthe N–the 1575
H bending and C–N
− 1 is stretched over range of 1400–1200 cm −1 . Among
to 1480 cm wavelength range; and the amide-III zone representing the N–H bending these bands, the
amide-I
andisC–N
the most responded
is stretched overarea to of
range any1400–1200
chemical cm change in the secondary
−1 . Among these bands, structure of
the amide-I is
protein. The FT-IR spectra of MOSP isolated via conventional extraction
the most responded area to any chemical change in the secondary structure of protein. and ultrason-
ic-assisted extraction
The FT-IR spectra are ofshown
MOSP in Figurevia
isolated 4. conventional
It can be observed that and
extraction sonication signifi-
ultrasonic-assisted
cantlyextraction
affects theare amide-I region based on C=O stretching, while only a
shown in Figure 4. It can be observed that sonication significantly change in the in-affects
tensity
theofamide-I
peaks was observed
region for C=O
based on otherstretching,
regions. Awhile
similar trend
only was observed
a change on quinoa
in the intensity of peaks
seed protein isolatesfor
was observed [51] and regions.
other Moringa oleifera seed
A similar protein
trend wasisolates
observed [52]ontreated
quinoawith
seedson-protein
ication.
isolates [51] and Moringa oleifera seed protein isolates [52] treated with sonication.
E:\FT-IR\Moringa Protein Untreated 4.0 03/03/2023 15:15:20
90
80
Transmittance [%]
70
60
50
40

3500 3000 2500 2000 1500 1000

Molecules 2023, 28, x FOR PEER REVIEW Wavenumber cm-1
10 of 17
Page 1 of 1

(a) CE
E:\FT-IR\Moringa Protein Treated.0 03/03/2023 15:22:04
90
80
Transmittance [%]
70
60
50

3500 3000 2500 2000 1500 1000

Wavenumber cm-1
Page 1 of 1

(b) UAE
Figure Figure
4. FT-IR
4. spectra of M. oleifera
FT-IR spectra seed protein
of M. oleifera (MOSP)
seed protein isolate isolate
(MOSP) from conventional extraction
from conventional (a)
extraction (a)
and ultrasonic-assisted extraction (b).
and ultrasonic-assisted extraction (b).

2.3.2. Intrinsic Fluorescence Patterns

The fluorescence spectrum is basically a representation of amino acid residues such
as tryptophan, tyrosine, and phenylalanine present in a protein, which are, in principal,
detrimental to the tertiary structure of protein. Most of the studies related to tertiary
structure using intrinsic fluorescence are based on the changes in tryptophan intensity
 50
3500 3000 2500 2000 1500 1000
Wavenumber cm-1
Page 1 of 1
Molecules 2023, 28, 2554 10 of 17
(b) UAE
Figure 4. FT-IR spectra of M. oleifera seed protein (MOSP) isolate from conventional extraction (a)
and ultrasonic-assisted extraction (b).
2.3.2. Intrinsic Fluorescence Patterns
2.3.2. Intrinsic Fluorescence Patterns spectrum is basically a representation of amino acid residues such
The fluorescence
The fluorescence
as tryptophan, spectrum is basically
tyrosine, andaphenylalanine
representation ofpresent
amino acid in aresidues
protein,such
which are, in principal,
as tryptophan, tyrosine, and phenylalanine present in a protein, which are, in principal,
detrimental to the tertiary structure of protein. Most of the studies related to tertiary
detrimental to the tertiary structure of protein. Most of the studies related to tertiary
structure using intrinsic fluorescence are based on the changes in tryptophan intensity [52].
structure using intrinsic fluorescence are based on the changes in tryptophan intensity
Usually,
[52]. Usually, max of tryptophan
λmax ofλtryptophan < 330the
< 330 nm shows nmoccurrence
shows the occurrence
of the amino acid ofinthe
theamino acid in the non-
polar environment, while
non-polar environment, while λmax > 330 nm λ > 330 nm shows its presence in
maxshows its presence in the polar environment the polar environment due to
conformational
due to conformational changes
changes in thein the tertiary
tertiary structure
structure of proteinof [51].
protein
The [51]. Theofpresence of tryptophan,
presence
tryptophan, tyrosine,and
tyrosine, andphenylalanine
phenylalanine residues
residues in
in MOSP
MOSPisisevident
evidentfrom fromprevious
previous studies [53,54]. The
studies [53,54].
fluorescence spectra of tryptophan, tyrosine, and phenylalanineres-
The fluorescence spectra of tryptophan, tyrosine, and phenylalanine residues for the protein
idues for the protein obtained from conventional extraction and ultrasound-assisted ex-
obtained from conventional extraction and ultrasound-assisted extraction are shown in
traction are shown in Figure 5. It can be observed that λmax for tryptophan, phenylalanine,
Figure
and tyrosine in the5.case
It can be observed
of MOSP from CE wasthatobserved
λmax forattryptophan,
380 nm, 380 nm, phenylalanine,
and 390 nm. and tyrosine in the
case of MOSP from CE was observed at 380 nm, 380
After MOSP was obtained by using UAE, no shift in λmax was found except for phenyl- nm, and 390 nm. After MOSP was
obtained
alanine, which shiftedbytousing UAE,
360 nm. no shift
Moreover, in λmax in
an increase wasthefound exceptintensity
fluorescence for phenylalanine,
was which shifted
to of360
found in all thenm.
studiedMoreover, an increase
amino residues. in the
The spectra fluorescence
of tyrosine give someintensity was found in all of the
noise, but
they couldstudied
be helpful for observing
amino residues.theThe
considerable changes
spectra of in the
tyrosine chemical
give somestructure of they could be helpful
noise, but
MOSP. for observing the considerable changes in the chemical structure of MOSP.

0.8
0.7 (a) CE UAE
Fluorescence Instensity

0.6
0.5
0.4
0.3
0.2
0.1
0
Molecules 2023, 28, x FOR PEER REVIEW 11 of 17
200 250 300 350 400 450 500 550 600
Wavelength (nm)

4
CE
3.5 (b)
Fluorescence Intensity

UAE
3
2.5
2
1.5
1
0.5
0
200 250 300 350 400 450 500 550 600
Wavelength (nm)

0.12
CE
Fluorescence Intensity

0.1 (c)
UAE
0.08
0.06
0.04
0.02
0
200 250 300 350 400 450 500 550 600
Wavelength (nm)

Figure 5. Fluorescence spectra of tryptophan

Figure 5. Fluorescence spectra(a),
ofphenylalanine (b), and
tryptophan (a), tyrosine (c) residues
phenylalanine fortyrosine
(b), and the (c) residues for the
MOSP from CE and UAE.
MOSP from CE and UAE.
The increase in the intensity of amino residues is related to the oxidation of protein
by hydroxyl radicals produced during the sonication treatment [20]. A similar trend was
observed in the case of lupin protein [21] and pea protein [17]; however, the observation
was in conflict with the results of quinoa seed protein isolates [51] and walnut protein
isolates [55], which experienced a decrease in the intensity from the application of ultra-
 Molecules 2023, 28, 2554 11 of 17

The increase in the intensity of amino residues is related to the oxidation of protein
by hydroxyl radicals produced during the sonication treatment [20]. A similar trend was
observed in the case of lupin protein [21] and pea protein [17]; however, the observation
was in conflict with the results of quinoa seed protein isolates [51] and walnut protein iso-
lates [55], which experienced a decrease in the intensity from the application of ultrasound.
These discrepancies are related mainly to the production of different amounts of hydroxyl
radicals due to the variation in the intensity of ultrasound treatment, the duration of the
treatment, and the concentration and types of substrates of treated proteins. Unfortunately,
no study was found to provide correlating results for phenylalanine and tyrosine. Never-
theless, this increase in the intensity could be attributed to the response of different amino
acid residues to the variety of stresses to the protein due to the structure of the amino acid
residues [56].

3. Material and Methods

3.1. Raw Materials and Chemicals
M. oleifera seeds (Figure 6) were procured from Ayub Agriculture Research Institute,
Faisalabad, Pakistan. Dried M. oleifera seeds were milled in a commercial grinder and then
passed through a 100-mesh sieve to obtain fine seed powder. Peanut and corn oils were
purchased from a local market. Coomassie brilliant blue G-250 and bovine serum albumin
Molecules 2023, 28, x FOR PEER REVIEW
(BSA) of analytical grade were acquired from Sigma-Aldrich. The rest of the chemicals, such 12 of 1
as sodium hydroxide, hydrogen chloride, acetone, methanol, and others, were obtained
from Duksan, Korea.

Figure6.6.Moringa
Figure Moringaplant, pods,
plant, andand
pods, seeds.
seeds.
3.2. Ultrasonic-Assisted Extraction (UAE) of Seed Protein
3.2. Ultrasonic-Assisted Extraction (UAE) of Seed Protein
The UAE method of [18] was adopted with slight modification. Briefly, for each
The UAE
extraction, samplesmethod of [18] solute-to-solvent
with varying was adopted with slight
ratios (1:10,modification.
1:20, and 1:30)Briefly, for each ex
at different
traction,
pH samples
levels (9, with
11, and 13) varying
were solute-to-solvent
sonicated using a 13 mm proberatios[57]
(1:10, 1:20,
for 15 min and 1:30) at differen
at a frequency
pH
of 20levels
kHz and (9, a11,
netand 13) power
output were sonicated using
of 750 W but with a variable
13 mm probe [57] for
amplitudes (25, 15
50,min
and at a fre
75%)
quencyusing
ofsonication
20 kHz and apparatus VCX750power
a net output (Sonicsof&750
Materials,
W butInc. withNewtown,
variableCT, USA). In (25, 50
amplitudes
total,
and 17 runsusing
75%) were performed,
sonicationasapparatus
per response surface (Sonics
VCX750 statistical&design (see Table
Materials, Inc.1).Newtown,
After CT
optimization
USA). In total, 17 runs were performed, as per response surface statisticalwas
of amplitude, solute-to-solvent ratio, and pH, the best extraction time design (se
determined by performing trials for 0, 5, 10, 15, 20, 25, and 30 min at optimized conditions.
Table 1). After optimization of amplitude, solute-to-solvent ratio, and pH, the best ex
The conventional extraction (CE) of protein at optimized conditions but without amplitude
traction
was time was for
also performed determined by performing
the comparison trials
and validation forsonication
of the 0, 5, 10, 15, 20, 25, and 30 min a
application.
optimized conditions. The conventional extraction (CE) of protein at optimized condi
tions but without amplitude was also performed for the comparison and validation of th
sonication application.

3.3. Protein Quantification by Bradford Method

The amount of protein was determined by using the Bradford method [58]. Con
Molecules 2023, 28, 2554 12 of 17

3.3. Protein Quantification by Bradford Method

The amount of protein was determined by using the Bradford method [58]. Concisely,
after sonication/conventional extraction, each sample was centrifuged (Thermo Scientific
Heraeus Megafuge 8R-Germany) at 4 ◦ C at 7508× g for 20 min, and the supernatant was
collected and diluted. Then, 1 mL from each diluted solution was mixed with 5 mL of
Coomassie brilliant blue G-250 solution, and the mixture was kept for 2 min at room
temperature. The absorbance of each mixture was taken at 525 nm by a UV-Vis Double-
Beam spectrophotometer (Specord-200 Plus, Analytik Jena, Germany). Bovine serum
albumin (0–100 ppm) was used to prepare a standard curve for quantifying the protein
contents of the extracts. Based on the protein content of the extract, the protein extraction
yield (g/100 g sample) was calculated using following formula:

Extraction yield (%) = (Protein weight/powder weight) × 100 (1)

3.4. Isolation of Seed Protein

Protein from the supernatant obtained during UAE/CE under optimized conditions
was isolated by following acid–base methodology [59]. Briefly, the pH of the supernatant
was adjusted to 3, which is the isoelectric point for seed protein, and the solution was
stored at 4 ◦ C for 6 h. Precipitates were then collected on filter paper from each sample
by washing them twice with distilled water. These precipitates were then dispersed in
deionized water, followed by pH adjustment to 7. Finally, solutions were freeze-dried for
48 h to obtain M. oleifera seed protein (MOSP).

3.5. Functional Properties of M. oleifera Seed Protein (MOSP)

3.5.1. Solubility
The solubility of the protein samples was estimated by using the method described
by [33]. For this, 10 mg of MOSP was dispersed in 8 mL of deionized water, and the pH was
adjusted to 2–10 (if necessary) with either 1.0 M NaOH or HCl. The protein solution was
then stirred at room temperature for 30 min. The volume of the solution was then adjusted
to 10 mL by adding the respective pH solutions. These solutions were then centrifuged for
20 min at 7508× g. After the determination of the protein content in the supernatant via the
Bradford method, protein solubility was calculated via the following formula:

Solubility (%) = (Protein content in supernatant/Total protein in sample) × 100 (2)

3.5.2. Water- and Oil-holding Capacity

The method presented by Yılmaz and Hüriyet [60] and Saha and Deka [61] was
adopted to determine the water (WHC)- and oil-holding capacity (OHC) of samples. The
determination of the WHC/OHC of the samples was performed by mixing 0.3 g from each
sample with 5 mL of deionized water or peanut oil in centrifuge tubes. The mixtures were
vigorously vortexed and left for 30 min at room temperature. The mixture solutions were
centrifuged at 3003× g for 15 min. The calculation of WHC and OHC for each sample was
performed according to the following formula:

WHC or OHC (g/g) = (W2 − W1)/W0 (3)

where W0 = weight of dry sample in grams, W1 = weight of dry sample and tube,
W2 = weight of sediments and tube.

3.5.3. Emulsion Capacity and Emulsion Stability

The emulsion capacity (EC) and emulsion stability (ES) of samples were determined
according to the method of Jiang, et al. [62]. The EC of the samples was determined by
dissolving 1 g from each sample with 50 mL of 0.1 NaOH in a 250 mL beaker. Then, 50 mL of
corn oil was added, and the mixture was homogenized (FSH-2A Homogenizer, Changzhou,
China) at 10,000 rpm for 2 min to form an emulsion. The emulsion was then transferred
Molecules 2023, 28, 2554 13 of 17

into a 100 mL measuring cylinder. The EC was determined by calculating the difference
between the initial volume (Vi) of oil and the released volume (Vr) of oil against the weight
of the sample (W), which were taken as shown in the following formula:

EC = (Vi − Vr)/W (4)

The emulsion stability (ES) was observed after 48 h at room temperature by determin-
ing the amount of separated water from oil.

W (%) = Vol. of separated water (mL)/Original amount of water (mL) × 100 (5)

W is the percentage of water separated.

3.5.4. Foaming Capacity and Stability

The method adopted by Phongthai, et al. [63] for the determination of foaming capacity
(FC) and foaming stability (FS) was used for the purpose. To measure FC and FS, 50 mL
of 0.1% (w/v) from each protein solution was taken in a 150 mL beaker and homogenized
(FSH-2A Homogenizer, China) for 1 min at 24,000 rpm. The total volume was measured at
0 and 10 min. FC and FS were calculated according to the following formulae:

FC (%) = (V1 − V0)/V0 × 100 (6)

FS (%) = (V2 − V0)/V1 − V0 × 100 (7)

where V0 is the volume of the protein solution before homogenization; V1 is the volume of
the protein solution after homogenization at 0 min; V2 is the volume of the protein solution
after homogenization at 10 min.

3.6. Fourier-Transform Infrared (FT-IR) Spectroscopy

FT-IR is considered to be one of the best techniques for the determination of the
secondary structure of proteins [51]. In this study, spectra to assess the composition
of a protein’s secondary structure was acquired by an infrared spectrometer (Alpha II
FT-IR, Bruker, Billerica, MA, USA) equipped with an attenuated total reflection (ATR)
accessory without a temperature controller. A small amount of the samples was evenly
placed on the ZnSe ATR crystal to obtain the spectrum in the transmission mode ranging
from 4000 cm−1 to 400 cm−1 . The spectra obtained were processed using the latest OPUS
software version 7.0.

3.7. Fluorescence Spectroscopy

Fluorescence spectroscopy has undergone rapid development due to its enormous
technical advances, accuracy, and enhanced methods for analysis based on fluorophore
compounds. The fluorescence spectra of protein extracted by using ultrasound and conven-
tional alkaline extraction were taken using a FluoroMax4 Spectrofluorometer (HORIBA,
Piscataway, NJ, USA) equipped with a xenon lamp. The method described by Mir et al. in
2019 was followed with little modification to acquire spectra. Spectra were taken at the
fixed excitation wavelength of 220 nm for tyrosine, 260 nm for phenylalanine, and 280 nm
for tryptophan, while the emission range was between 300 and 550 nm with a slit width of
1 nm. Three spectra were taken for each amino acid, and the average spectrum is presented
in the results.

3.8. Experimental Design and Statistical Analysis

Based on the principle of central composite design (CCD), amplitude % (A), solute-
to-solvent ratio (B), and pH (C) as the studied parameters for the maximum extraction
of protein were optimized (Table 1) via response surface methodology (RSM) by using
MATLAB (2007a). An analysis of variance (ANOVA) with a 95% confidence level was then
carried out for each response variable in order to test the model significance and suitability.
 3.8. Experimental Design and Statistical Analysis
Based on the principle of central composite design (CCD), amplitude % (A), so-
lute-to-solvent ratio (B), and pH (C) as the studied parameters for the maximum extrac-
tion of protein were optimized (Table 1) via response surface methodology (RSM) by
using MATLAB (2007a). An analysis of variance (ANOVA) with a 95% confidence level
Molecules 2023, 28, 2554 14 of 17
was then carried out for each response variable in order to test the model significance and
suitability.

where Y denotes the predicted value of the response variable; β0 denotes the intercepts;
where Y denotes the predicted value of the response variable; β0 denotes the intercepts; and
and βi, βii, and βij are the linear, second order, and interaction regression coefficients pre-
βi , βii , and βij are the linear, second order, and interaction regression coefficients predictable
dictable by the model, respectively. Xi and Xj are the values of studied or independent
by the model, respectively. Xi and Xj are the values of studied or independent variables.
variables.
4. Conclusions
This research revealed that ultrasonic-assisted extraction is significantly better and had
beneficial effects in obtaining higher protein yields. During extraction, ultrasound power
and pH were among the parameters which significantly affected the protein yield. This
treatment altered the secondary and tertiary structure of MOSP, which were evident from FT-
IR and fluorescence spectroscopy, respectively. Ultrasonication did not yield any degrading
effects on MOSP in terms of functional properties. The functional properties of MOSP such
as solubility, water- and oil-holding capacity, emulsion stability, and foaming capacity and
stability improved in the beginning and then reduced as the amplitude of sonication further
increased. These results would give an understanding of the mechanism and interrelation
of structural changes with functional properties observed after the ultrasonic-assisted
extraction of MOSP and its further application in the development of advanced sustainable
food products.

Author Contributions: Conceptualization, K.F. and M.K.K.; methodology, K.F. and M.H.A.; software
and validation, M.I., W.K. and A.A.-F.; formal analysis, K.F. and M.H.A.; data curation, K.F. and
M.H.A.; writing—original draft preparation, K.F. and W.S.A.; writing—review and editing, G.S.,
A.A.E. and M.K.K.; supervision, M.K.K. All authors have read and agreed to the published version of
the manuscript.
Funding: The authors received no financial support for the research, authorship, and/or publication
of this article. The research was performed as part of the employment of the authors.
Institutional Review Board Statement: Not applicable
Informed Consent Statement: Not applicable
Data Availability Statement: Not applicable
Acknowledgments: The authors acknowledge Waqas Ahmed from the University of Veterinary and
Animal Sciences, Lahore, Pakistan, for access to FT-IR spectroscopy equipment and Amna Sahar from
University of Agriculture, Faisalabad, Pakistan, for access to fluorescence spectroscopy equipment.
Conflicts of Interest: The authors declare no conflict of interest.

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