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Citation: Gharibzahedi, S.M.T.;
Altintas, Z. Ultrasound-Assisted
Alcoholic Extraction of Lesser
Mealworm Larvae Oil: Process
Optimization, Physicochemical
Characteristics, and Energy
Consumption. Antioxidants 2022,11,
1943. https://doi.org/10.3390/
antiox11101943
Academic Editors:
Soraya Rodriguez-Rojo and
Naiara Fernández
Received: 16 August 2022
Accepted: 23 September 2022
Published: 28 September 2022
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antioxidants
Article
Ultrasound-Assisted Alcoholic Extraction of Lesser Mealworm
Larvae Oil: Process Optimization, Physicochemical
Characteristics, and Energy Consumption
Seyed Mohammad Taghi Gharibzahedi 1,2 and Zeynep Altintas 1, 2, *
1Institute of Chemistry, Faculty of Natural Sciences and Maths, Technical University of Berlin, Straße des 17,
Juni 124, 10623 Berlin, Germany
2Institute of Materials Science, Faculty of Engineering, Kiel University, 24143 Kiel, Germany
*Correspondence: zeynep.altintas@tu-berlin.de or zeynep.altintas@tf.uni-kiel.de; Tel.: +49-30-314-23727
Abstract:
The ultrasound-assisted extraction (UAE) of oil from lesser mealworm (Alphitobius dia-
perinus L.) larvae powders (LMLPs) using ethanol/isopropanol as the superior solvent was opti-
mized. The evaluation of time (9.89–35.11 min), solvent-to-LMLPs (2.39–27.61 v/w), and temperature
(16.36–83.64
◦
C) showed that the highest extraction efficiency (EE, 88.08%) and
in vitro
antioxidant
activity (IVAA) of reducing power (0.651), and DPPH free-radical scavenging capacity (70.79%) were
achieved at 22.5 v/wsolvent-to-LMLPs and 70
◦
C for 22.64 min. Optimal ultrasound conditions
significantly improved the EE than n-hexane extraction (60.09%) by reducing the electric energy
consumption by ~18.5 times from 0.637 to 0.035 kWh/g. The oil diffusivity in ethanol-isopropanol
during the UAE (0.97
×
10
−9
m
2
/s) was much better than that of n-hexane (5.07
×
10
−11
m
2
/s).
The microstructural images confirmed the high efficiency of ethanol-isopropanol in the presence of
ultrasounds to remove oil flakes from the internal and external surfaces of LMLPs. The improved
IVAA was significantly associated with the total phenolic (4.306 mg GAE/g, r = 0.991) and carotenoid
(0.778 mg/g, r = 0.937) contents (p< 0.01). Although there was no significant difference in the fatty
acid profile between the two extracted oils, ethanol-isopropanol under sonication acceptablyimproved
oxidative stability with lower peroxides, conjugated dienes and trienes, and free fatty acids.
Keywords:
insect oil; ultrasound; extraction; diffusion; fatty acid; antioxidant; bioactive compounds
1. Introduction
In the last decade, a serious need for new natural sources and extraction processes for
edible oils has emerged due to the increased consumer demand. This potential integrated
strategy can contribute to overcoming the most important challenges facing the industry for
producing future edible oils with excellent quality and health-promoting properties [
1
,
2
].
Solvent extraction is usually performed to extract edible oils from a wide span of fruit and
vegetable seeds in the industry [
3
]. n-Hexane is the most common consumable solvent
to extract lipids due to its easy evaporation, low energy cost, and high selectivity to oils.
However, it causes ecological worries and human health problems, such as high flammabil-
ity and acute toxicity through inhalation, ingestion, and eye or skin contact [4]. Therefore,
its substitution with some efficient solvents is considered necessary for the sustainable
processing of edible oils. Recently, some emerging technologies have been utilized to
extract lipids, such as microwave heating, ultrasonication, high hydrostatic pressure, su-
percritical/subcritical, and enzyme-assisted extractions. These green technologies usually
run at lower extraction times with a minimum solvent usage compared to conventional
methods and increase the extraction yield of edible oils with a better oxidative quality [5].
Insect oil is one of the most promising and sustainable sources of edible oil supply.
Although the number of edible insects has reached 1900 species, the oil quality of a small
number of them, such as yellow mealworm (Tenebrio molitor), super worm (Zophobas morio),
Antioxidants 2022,11, 1943. https://doi.org/10.3390/antiox11101943 https://www.mdpi.com/journal/antioxidants
Antioxidants 2022,11, 1943 2 of 19
black soldier fly (Hermetia illucens), house cricket (Acheta domesticus), and Dubia cockroach
(Blaptica dubia), have been evaluated as substitutions for soybean, fish, and palm oils
in aquaculture and poultry [
6
,
7
]. Tzompa-Sosa et al. [
8
] recently examined the sensory
attributes of hummus and crackers made of deodorized yellow mealworm oil instead of
vegetable oils. The visual appearance of these food products did not change, but lower
overall acceptability and hardness compared to vegetable oil-based ones were reported.
However, a blend of rapeseed, peanut, and insect oils could improve the flavor features for
more consumer preference. Although lesser mealworm (Alphitobius diaperinus) is one of
the most important species of oil-supplying insects, not enough information on its lipid
potential in industrial applications is available. However, the young larvae powders of
this insect rich in linoleic, palmitic, and oleic acids have been recently formulated as a new
baking constituent to produce high-protein, mineral-dense snacks [9].
Due to the acoustic cavitation, the ultrasonication process can increase the mass
transfer and diffusion rate with higher solvent penetration into the solid matrix through
the disruption of cell structure [
10
]. Sound waves during ultrasonic treatment can be
propagated into the liquid medium and subsequently forms alternate cycles of compression
(high pressure) and rarefaction (low pressure). The generation of cavitation phenomenon
and very fine bubbles provides microturbulence and intense collision of particles in the
solvent, accelerating the internal and eddy diffusion [
11
]. This non-thermal process is
adaptable in oil industries because of its practical advantages, such as easy operation, fewer
costs, higher extraction efficiency (EE), lower oxidation rate, more maintenance of thermo-
sensitive bioactive compounds, and fewer negative impacts on the ecosystem [12,13].
To propose efficient ultrasound-assisted solvent extraction processes in terms of quan-
tity and quality improvements, it is necessary to develop a collection of mathematical and
empirical techniques for optimizing this process even in the presence of complex inter-
actions. Response surface methodology (RSM) is one of the most practical mathematical
techniques to optimize industrial processes by relying on appropriate experimental de-
signs [
14
]. RSM cannot only determine the interactions between processing factors, but also
reduces the number of experimental trials, development time, and overall cost [
15
]. The
successful application of RSM to optimize the UAE process to obtain edible oils from plant
tissues, such as hemp seeds [
16
], Moringa oleifera leaves [
17
], Moringa Peregrina seeds [
18
],
fresh aerial parts of Angelica Keiskei Koidzumi [
19
], and red mombin seeds (Spondias pur-
purea L.) [
20
], has been recently performed. In addition, Susanti et al. have recently reported
that using the UAE process under the optimal conditions determined by RSM is an efficient
method to substitute the conventional technique for the simultaneous extraction of oil and
phenolic compounds from red fruit (Pandanus conoideus) [21].
To the best of our knowledge, there are no studies found in the literature on the process
optimization of ultrasound-assisted extraction (UAE) to obtain lesser mealworm oil (LMO)
with the desired physicochemical. As a result, this study aimed (i) to examine the UAE
parameters (i.e., solvent-solid ratio, extraction time, and extraction temperature) to achieve
the maximum EE and antioxidant capacity of LMO, (ii) to compare the physicochemical
properties and fatty acid profile of the oils extracted by n-hexane and UAE under the
optimal conditions, and (iii) to calculate energy consumption and diffusion coefficients
between the two extraction techniques.
2. Materials and Methods
2.1. Materials, Chemicals, and Reagents
The insect powder of lesser mealworm (A. diaperinus L.) containing 59.6% protein,
28.7% fat, 3.7% fiber, 2.7% carbohydrate, and 0.9% salt was purchased from Snack-Insects
Co. (Witzeeze, Germany). The total content of protein, fat, and carbohydrate of LMLPs had
been determined by the Kjeldahl, Soxhlet, and Bradford (the rapid colorimetrics of phenol-
sulfuric acid with the standard of D-glucose) techniques, respectively. Ethanol, isopropanol,
n-hexane, diethyl ether, methanol, toluene, iso-octane, cyclohexane, anhydrous sodium
sulfate, sodium carbonate, iron (III) chloride, potassium hydroxide (KOH), potassium
Antioxidants 2022,11, 1943 3 of 19
ferricyanide, hydrochloric acid (HCl), trichloroacetic acid (TCA), gallic acid (GA), 1,1-
diphenyl-2-picrylhydrazyl (DPPH
·
), Folin–Ciocalteu’s reagent, phenolphthalein, phosphate
buffer tablets, and vitamin C were purchased from Sigma-Aldrich Chemical Co. (Darmstadt,
Germany). All other chemicals used were of analytical grade and obtained from either
Sigma-Aldrich or Merck Chemical Co. (Darmstadt, Germany).
2.2. Conventional Solvent Extraction
To determine the best organic solvent for the insect oil extraction, lesser mealworm
larvae powders (LMLPs) before defatting were dried at 150
◦
C for 5 min in a vacuum
oven (Salvis Lab VC-20 Vacucentre, Rotkreuz, Switzerland) and then sieved through
0.2 mm mesh. The solvent-assisted oil extraction by n-hexane, ethanol, isopropanol,
and their binary mixtures (1:1 v/v) was carried out in a solvent:solid ratio of 10:1 w/v
(10.0 g LMLPs), time of 2 h, and a stirring rate of 400 rpm after doing preliminarily ex-
perimental investigations. The extraction time differed depending on the boiling point
temperature for each organic solvent and azeotrope data for their binary mixtures. The
average extraction temperature for n-hexane, ethanol, isopropanol, n-hexane/ethanol, n-
hexane/isopropanol, and ethanol/isopropanol were considered to be 65
◦
C, 75
◦
C, 80
◦
C,
57
◦
C, and 78
◦
C, respectively. The power consumption for heating and stirring the magnetic
stirrer used for the conventional solvent extraction (CSE) was 550 and 8.5 W, respectively.
After finishing the extraction process, the liquid–solid mixtures were filtered by passing via
a Whatman filter paper using an aspirated Büchner funnel. The residue was re-extracted
twice, and the filtrates were combined from the three extraction stages. The solvent was
removed by a rotary evaporator (model Hei-VAP Value, Heidolph, Schwabach, Germany)
under a vacuum at 40
◦
C. The collected oil was passed via the layer of anhydrous sodium
sulfate placed over a filter paper in a funnel. The oil was lastly weighed and transferred
into 10 mL vials, gently flushed with nitrogen gas, capped, and stored at
−
18
◦
C until
further analysis.
2.3. Ultrasound-Assisted Extraction Process
The LMLPs (10.0 g) preheated in a vacuum oven (150
◦
C, 5 min) with a diameter of
0.2–0.5 mm were mixed with the superior solvent of ethanol-isopropanol (1:1 v/v) at ratios
of 2.39–27.61 v/win a 500 mL Erlenmeyer flask. The insect powder–solvent suspensions
were treated by ultrasound for 9.89–35.11 min at the frequency of 35 kHz and power equal
to 240 W within the ultrasonic bath (type RK 106, Bandelin electronic GmbH & Co. KG,
Berlin, Germany) with 75% filled by distilled water at 16.36–83.64
◦
C. The liquid level in the
Erlenmeyer was lower than that of the water bath. The defined levels of time, temperature,
and solvent-to-solid ratio were determined based on the experimental design of RSM
(Table 1). A digital thermometer was used to check the accuracy of water-bath temperature
while the circulation of water controlled the temperature increase in the water bath during
the experimental trials. For this, the temperature during extraction was continuously
adjusted and maintained at the desired level within ±1◦C by adding hot or cold water at
the appropriate level. The crude extracts’ filtration and concentration were also performed
per the CSE method.
2.4. Extraction Efficiency
Regarding the oil extraction performed from LMLPs with certain fat content, the EE
than extraction yield was a better index to show the solvent and UAE capability. The extrac-
tion yield can be defined as the ratio of recovered oil weight (
WRO
) to the weight of LMLPs
before extraction (
WLMLPs
, 10.0
g
). However, the EE indicates that the percentage of ex-
tracted oil concerning the quantity of oil presents in the LMLPs (
WOC
, 0.287
g oil/g LMLPs
),
which can be calculated by the following equation (Equation (1)) [22]:
EE (%) = WRO
(WLMLPs ×WOC)×100 (1)
Antioxidants 2022,11, 1943 4 of 19
Table 1. RSM-CCRD and experimental and predicted results for response variables.
Trial
Independent Variables Response Variables
UAE Time (min, X1) Solvent/Solid (v/w, X2)UAE Temperature
(◦C, X3)EE (%, Y1) SCDPPH·(%, Y2) RP (Y3)
1 15 (−1, Factorial) 7.5 (−1, Factorial) 30 (−1, Factorial) 37.6 ±1.2 45.0 ±0.6 0.326 ±0.010
2 30 (+1, Factorial) 7.5 (−1, Factorial) 30 (−1, Factorial) 56.0 ±2.4 58.1 ±0.4 0.417 ±0.014
3 5 (−1, Factorial) 22.5 (+1, Factorial) 30 (−1, Factorial) 68.9 ±1.8 66.6 ±0.3 0.532 ±0.009
4 15 (−1), Factorial 22.5 (+1, Factorial) 30 (−1, Factorial) 79.5 ±1.0 69.3 ±1.0 0.666 ±0.001
5 30 (+1, Factorial) 7.5 (−1, Factorial) 70 (+1, Factorial) 63.0 ±1.0 56.6 ±0.3 0.432 ±0.004
6 5 (−1, Factorial) 7.5 (−1, Factorial) 70 (+1, Factorial) 78.1 ±2.0 64.5 ±1.1 0.509 ±0.012
7 15 (−1, Factorial) 22.5 (+1, Factorial) 70 (+1, Factorial) 84.7 ±2.2 69.3 ±0.7 0.575 ±0.007
8 30 (+1, Factorial) 22.5 (+1, Factorial) 70 (+1, Factorial) 92.3 ±1.3 60.4 ±0.5 0.532 ±0.007
9 9.89 (−α, Axial) 15 (0, Center) 50 (0, Center) 65.5 ±0.6 40.0 ±0.8 0.274 ±0.005
10 35.11 (+α, Axial) 15 (0, Center) 50 (0, Center) 85.4 ±0.7 58.1 ±1.7 0.434 ±0.006
11 22.5 (0, Center) 2.39 (−α, Axial) 50 (0, Center) 51.6 ±1.0 56.4 ±1.5 0.402 ±0.013
12 22.5 (0, Center) 27.61 (+α, Axial) 50 (0, Center) 90.4 ±1.2 78.4 ±1.1 0.796 ±0.001
13 22.5 (0, Center) 15 (0, Center) 16.36 (−α, Axial) 41.0 ±1.5 65.3 ±0.8 0.632 ±0.003
14 22.5 (0, Center) 15 (0, Center) 83.64 (+α, Axial) 80.2 ±1.2 73.9 ±0.8 0.686 ±0.013
15 22.5 (0, Center) 15 (0, Center) 50 (0, Center) 73.3 ±0.7 59.5 ±1.2 0.479 ±0.014
16 22.5 (0, Center) 15 (0, Center) 50 (0, Center) 76.9 ±0.4 60.3 ±0.6 0.488 ±0.009
17 22.5 (0, Center) 15 (0, Center) 50 (0, Center) 71.5 ±0.9 63.5 ±0.4 0.545 ±0.005
18 22.5 (0, Center) 15 (0, Center) 50 (0, Center) 75.4 ±1.3 62.3 ±0.5 0.492 ±0.004
19 22.5 (0, Center) 15 (0, Center) 50 (0, Center) 73.4 ±1.9 63.1 ±0.3 0.535 ±0.004
20 22.5 (0, Center) 15 (0, Center) 50 (0, Center) 69.9 ±1.0 61.3 ±0.8 0.529 ±0.002
2.5. DPPH·Scavenging Capacity Assay
The scavenging capacity of DPPH free-radical (SC
DPPH.
) of extracted LMOs was
measured according to the method described by Gharibzahedi et al. [
23
]. Briefly, 100 mg
of each oil sample in 1.0 mL toluene were vortexed with 3.9 mL of the DPPH
·
solution
(0.1 mM) in toluene for 30 s, kept in the dark for 1 h at ambient temperature, and finally,
their absorbance (A
s
) was read at 515 nm using a UV–vis spectrophotometer (Cary 60,
Agilent Technologies, Santa Clara, CA, USA). The same procedure was performed in
toluene instead of oil sample (A
0
) to determine the control absorbance. Vitamin C as a
positive control compound was used. The SC
DPPH·
was assessed as follows (Equation (2)):
SCDP PH (%) = 1−As
A0×100 (2)
2.6. Reducing Power Assay
The method of Ogbunugafor et al. [
24
] with small modifications was applied to
determine ferric ions’ reducing power (RP). 2.5 mL of each oil sample was initially mixed
with 2.5 mL of 1% potassium ferricyanide, incubated at 50
±
2
◦
C for 25 min, and then
cooled rapidly. An amount of 2.5 mL of 10% TCA solution to the mixture was added,
vortexed, and centrifuged at 3000 rpm for 10 min. In the next step, 2.5 mL of the supernatant
solution was mixed with 2.5 mL of Millipore water and 0.5 mL of 0.1% iron (III) chloride.
Then the absorbance was measured using a UV–vis spectrophotometer at 700 nm.
2.7. Single-Factor Exploratory Tests
Single-factor experiments (SFEs) were carried out to assess the effects of the following
parameters on EE, SC
DPPH·
, and RP: UAE time (5–60 min), UAE temperature (10–90
◦
C),
and solvent-to-LMLPs ratio (5:1–45:1 v/w). According to the single-factor experimental
data, the best range of response values in these three experiments were selected to input
into RSM’s central composite rotatable design (CCRD).
2.8. Experimental Design and Response Surface Optimization
An RSM-CCRD was employed using Design-Expert software (version 8.0., Statease
Inc., Minneapolis, MN, USA). Three operating variables involved in the UAE of LMO
including time (X
1
, 9.89–35.11 min), solvent-to-LMLPs ratio (X
2
, 2.39–27.61 v/w), and
Antioxidants 2022,11, 1943 5 of 19
temperature (X
3
, 16.36–83.64
◦
C) were optimized to achieve the highest EE, SC
DPPH·
, and
RP amounts. Table 1shows 20 experimental combinations of independent variables at
five levels in random order. The above-mentioned range for each studied parameter
was determined after performing preliminary trials. Second-order polynomial regression
equations were evaluated for the response functions. The generalized response surface
model to predict the optimal point is represented as follows (Equation (3)):
x=0+
3
∑
i=1
iXi+
3
∑
i=1
ii X2
i+
2
∑
i=1
3
∑
j=1+1
ij XiXj+ε(3)
where Yis the dependent variables,
0
is the model constant,
i
,
ii
, and
ij
are the model
coefficients, and
ε
is the error. They represent the linear, quadratic, and interaction effects
of the variables.
The significance of regression equations was statistically checked. As the significant
terms in the model were found by analysis of variance (ANOVA) for each response, in-
significant terms were removed in the final model. The quality of all fitted models was
assessed based on several statistical parameters such as the coefficient of determination
(R
2
), adjusted R
2
(R
2adj
), coefficient of variation (CV), and adequate precision (AP) [
25
].
Five extra tests were performed to verify the accuracy of the optimal points predicted by
the RSM package’s response optimizer. The validity of the fitted models was also confirmed
by comparing the actual and predicted data according to Student’s t-test using SPSS V.21
(SPSS Inc., Inc., Chicago, IL, USA) software at a significant level of 5%.
2.9. Evaluation of Total Phenolic Content
The total phenolic content (TPC) of oil samples was determined based on the mod-
ified method of Singleton and Rossi [
26
]. A calibration curve with the standard of GA
(10–100
µ
g/mL) in methanol was constructed for this test. 400
µ
L of GA in each concentra-
tion with 2 mL of dilution Folin–Ciocalteu’s reagent (1:10) was mixed, and then 1.6 mL of
7.5% sodium carbonate was added. The same experiment with oil samples instead of GA
was done to measure the TPC. After vortexing, the mixtures were incubated for 60 min in
the dark at room temperature, and their absorbance using a UV–visible spectrophotometer
was read at 765 nm. The mean TPC results were expressed as milligrams of GA equivalents
per gram of oil (GAE/g oil).
2.10. Determination of Total Carotenoid Content
The method described by Naebi et al. [
27
] was used to assess the total carotenoid
content (TCC). For this experiment, 7.5 g of each oil sample in a 25 mL volumetric flask
was brought up to the volume with cyclohexane, and then their absorbance was measured
by a UV–visible spectrophotometer at 470 nm. The TCC (mg/kg) was calculated based on
the following formula (Equation (4)):
TCC (mg/kg)=A470 ×106
2000 ×100 ×L(4)
where
A470
is the absorbance amount at
λ
= 470 nm, and Lis the thickness of the spec-
trophotometer cell (1 cm).
2.11. Analysis of Fatty Acids Profile
The AOAC procedure was used to prepare the fatty acid methyl ester (FAME) [
28
]. At
first, LMO (50 mg) dissolved in 4.0 mL of methanolic HCl (0.5 M) was highly vortexed and
incubated for 4 h at 50
±
2
◦
C. Then, the mixture was immediately cooled down to room
temperature and the FAME was purified with 10 mL of n-hexane. The layer of anhydrous
sodium sulfate was used to dry the clear upper layer containing FAME. Identification
and quantification of the extracted FAME profile were carried out using gas chromatog-
raphy (GC, Agilent 6890, Agilent Technologies, Wilmington, DE, USA) equipped with a
Antioxidants 2022,11, 1943 6 of 19
Chrome-pack BPX5 capillary column (30 m
×
0.25 mm
×
0.25
µ
m) and an ionization mass
detector (Agilent 5973N, USA). The flow rate of carrier gas (helium) and the split ratio were
1.0 mL/min and 100.0, respectively. The oven temperature program included: initially
set at 60
◦
C (isothermal for 10 min) and gradually increased from 70 to 140
◦
C with a
10
◦
C/min rate. After holding for 10 min at 140
◦
C, it was increased to 250
◦
C with a rate of
7
◦
C/min, and lastly, isothermally kept at 280
◦
C for 10 min. In addition, the mass detector
was set under the following conditions: capillary direct interface temperature of 240
◦
C,
ionization energy of 70 eV, scanning interval of 0.5 s, and mass range of 40–1000 m/z[
29
].
The comparison of retention indices of fatty acids with their authentic samples and the mass
spectral data available in the library (Wiley-VCH 2001 data software, Weinheim, Germany)
contributed to identifying and determining each fatty acid.
2.12. Assessment of Physicochemical Properties
The apparent viscosity, specific gravity, and refractive index were assessed by Brook-
field rotational viscometer (DV-II+PRO model, Brookfield Engineering Labs., Inc., Middle-
boro, Brookfield, MA, USA), pycnometer, and Abbe refractometer (Carl Zeiss, model G,
Jena, Switzerland), respectively. The browning index (BI) was measured by determining
the spectrophotometric absorbance of the diluted mixture of oil with n-hexane (1:20, w/v)
at 420 nm [
30
]. The photometric color index (PCI) of the oil in the visible spectrum (
λ
= 460,
550, 620, and 670 nm) was estimated by the following formula (Equation (5)) [31]:
PCI = 1.29 (A460) + 69.7 (A550 ) + 41.2 (A620)−56.4 (A670 ) (5)
The acid (AV, mg KOH/g), saponification (SV, mg KOH/g), iodine value (IV), peroxide
(PV, meq O
2
/kg), and p-anisidine (p-AnV) values of the extracted oils were respectively
determined based on the AOCS standard methods of Cd 3a-63, Cd 3-25, Cd 1d-92, Cd 8-53,
and Cd 18-90 [
32
]. The totox value (TxV) is calculated by the formula of 2PV + p-AnV
to indicate an oil’s overall oxidation state. The conjugated diene (K
232
) and triene (K
270
)
levels based on the standard procedure of ISO 3656:2011 were measured by determining the
absorbance of 1% (w/v) oil solution in cyclohexane at 232 and 270 nm, respectively [
33
]. The
oxidative stability of LMOs was evaluated using the Rancimat (Rancimat 679, Metrohm,
Herisau, Switzerland) at an airflow rate of 20 L/h and a temperature of the heating block
of 120
◦
C [
34
]. The procedure of Wang et al. [
35
] with small modifications was applied
to evaluate the content of free fatty acid (FFA). After dissolving LMO (2.0 g) in ethanol-
diethyl ether (50 mL, 1:2 v/v), the mixture was stirred for 30 min at room temperature and
titrated against 0.05 M KOH using the phenolphthalein indicator. The FFA content was
then calculated using the following formula (Equation (6)):
FFA(mg/kg)=(V×C×56.11)
m(6)
where Vis the volume of KOH exhausted by LMOs in mL, Cis the concentration of KOH
(0.05 M), and mis the mass of the oil sample in g (2.0).
2.13. Estimation of Diffusion Coefficients
Fick’s second law (FSL) can be used to describe the oil extraction in CSE and UAE
processes (Equation (7)) [4]:
∂C
∂t=D∂2C
∂X2(7)
where C,t,X, and Dare the solute concentration, the time (s), the particle thickness (m),
and the diffusion coefficient (m2/s), respectively.
There were three assumptions, including (i) taking into account insect powders as
small spheres, (ii) neglecting the resistance to the external mass transfer due to the vigorous
Antioxidants 2022,11, 1943 7 of 19
stirring, and (iii) the free-solute solvent at the beginning of the process. The FSL solution
for a stirred solution in a limited volume can be done by Equation (8) [36]:
Mt
M∞=1−
∞
∑
n=1
6α(α+1)ex p−Dq2
nt
α2
9+9α+q2
n+α2(8)
where
Mt
and
M∞
are the total oil amount in LMLPs at the time tand after an infinite time
of diffusion, respectively.
α
is the mass ratio of the solvent and LMLPs. Furthermore,
qn
is
non-zero positive roots of Equation (9):
tanqn=3qn
3+αq2
n
(9)
2.14. Calculation of Electric Energy Consumption
The electric energy consumption (EEC) of UAE and CSE processes per gram LMO can
be calculated as follows (Equation (10)) [37]:
EEC(kW.h/g)=P×t
m(10)
where P,t, and mare the power consumption (kW), the extraction time (h), and the mass of
obtained oil (g), respectively.
2.15. Scanning Electron Microscopy
The LMLPs before and after defatting by the CSE and UAE methods were vacuum-
dried at 75
◦
C for 24 h, coated with a thin layer of gold employing a desktop sputtering
system, and visualized using the field emission-scanning electron microscopy (FE-SEM,
Zeiss Gemini DSM 982, Carl Zeiss Ltd, Oberkochen, Germany) at a 30
µ
m scale bar under
1000×magnification and an accelerating voltage of 6 kV.
2.16. Statistical Analysis
The data were a mean of three experimental replications and subjected to ANOVA us-
ing SPSS V.21 software. Significant differences were assessed by Duncan’s test
(p< 0.05). Pearson’s coefficient performed the correlation analysis between antioxidant
activity (RP/SCDPPH) and bioactive compounds (TPC/TCC).
3. Results and Discussion
3.1. Selection of the Organic Solvent
Figure 1compares the EE of LMOs extracted by the pure organic solvents and their
binary mixtures. The results showed the use of two binary mixtures of n-hexane/ethanol
(78.18%) and ethanol/isopropanol (74.03%) than the other solvents (47.21–63.85%) signifi-
cantly had more capability to extract oil from LMLPs (p< 0.05). As there was no significant
difference in the EE between ethanol/isopropanol and n-hexane/ethanol, the alcoholic
mixture was chosen for the UAE due to the removal of n-hexane. In general, the solvent
polarity can be determined based on its dielectric constant. Accordingly, polar solvents
like ethanol (24.55 C
2
/N
·
M
2
, 25
◦
C) and isopropanol (19.92 C
2
/N
·
M
2
, 25
◦
C) compared
to the nonpolar solvent such as n-hexane (1.88 C
2
/(N
·
M) are more able to extract polar
constituents from natural substances, resulting in a higher EE [
38
,
39
]. In other words,
the difference in oil extraction yields obtained from pure organic solvents (i.e., n-hexane,
ethanol, and isopropanol) and their binary mixtures can be related to the co-extraction of
any polar compounds, lipidic or not, enhancing the mass of extracted oil. Espinosa-Pardo
et al. realized that ethanol compared to hexane, had a better ability to increase the extraction
yield of corn germ oil due to the extraction of other polar compounds from the lipid ma-
trix [
40
]. Accordingly, higher EE of n-hexane/ethanol may be contributed to the separation
Antioxidants 2022,11, 1943 8 of 19
of phospholipids by the polar phase (ethanol) and more nonpolar lipids like triglycerides
by the non-polar phase (n-hexane) [
38
,
41
]. Recently, a combination of these two solvents
(hexane/ethanol; 1:4 v/v) has resulted in the highest efficiency in extracting oil from shrimp
by-products [
41
]. The high ability of polar solvents to increase EE of LMOs explains that
the alcoholic mixture contained more polar lipids such as mono-glycerides (MAG), di-
glycerides (DAG), phospholipids, and lipoproteins) than n-hexane or its combination with
each of the alcohols.
3.2. Single-Factor Experiments
The results of SFEs showed that the best ranges of UAE time, UAE temperature,
and solvent-to-LMLPs ratio to achieve the maximum EE, SC
DPPH·
, and RP amounts were
10–35 min
, 30–70
◦
C, and 5:1–30:1 v/w, respectively (Figure 2a–c). As shown in Figure 2a,
the EE rate fluctuated between 72.87 and 85.67% for the insect oil in the time range of
10–35 min
. The maximum SC
DPPH·
(58.06–64.87%) and RP (0.623–0.722) amounts were also
found in the time duration of 10–35 min. However, an increase in the UAE time from 35 to 60
min led to a significant reduction in the EE and antioxidant activity (Figure 2a). An increase
in the UAE time increases the effective disruption of the cell walls and leads to a better
mass transfer of intracellular products into the solvent. However, a long UAE time can
reduce the permeability of solvent into the cell walls because of over-suspended impurities.
Figure 1.
The EE comparison of oils extracted from LMLPs by pure and binary mixed organic
solvents. Means with different superscript letters (a–c) in columns indicate the significant statistical
difference (p< 0.05).
It was found that the longer exposure of lipid substances in the extract can degrade
them during the oxidation processes and decrease their antioxidant ability [42]. Figure 2b
illustrates the effect of UAE temperature on the EE and antioxidant activity of A. diaperinus
oil. A significant increase was observed in the EE (74.48–84.56%), SC
DPPH·
(51.11–64.12%),
and RP (0.628–0.721) amounts when the UAE temperature increased from 30 to 70
◦
C.
However, these quantitative and qualitative properties meaningfully dropped at tempera-
tures above 70
◦
C and less than 30
◦
C (Figure 2b). Increased UAE temperature probably
improves the solubility and viscosity of A. diaperinus larvae lipids and bioactive compounds
in ethanol/isopropanol.
Nevertheless, the yield reduction of EE at higher temperatures may be attributed to
the intensity reduction of the cavitation phenomenon. Under this condition, the cohesive
force developed by the count of cavitation bubbles can decrease the tensile strength of the
liquid. Accelerated evaporation at high temperatures also can facilitate the formation of
free radicals to oxidize available lipids in the extract [
43
,
44
]. The solvent-to-LMLPs ratio
effect on the EE and antioxidant activity of the extracted insect oil is exhibited in Figure 2c.
An increase in this ratio from 5 to 30 v/wincreased the EE and antioxidant activity. This
fact can be owing to the mass transfer improvement of oil and bio-functional constituents
Antioxidants 2022,11, 1943 9 of 19
at a larger concentration difference between the liquid (ethanol/isopropanol) to solids. No
significant change in the EE and antioxidative responses in the solvent volume of more
than 30 v/wwas detected as the mass transfer of oil globules and bioactive compounds
(e.g., carotenoids and phenolics) are more confined to the solid interior. A similar result
was also reported by Zhang et al. [
45
] and Zhang et al. [
46
] for the UAE of edible oils from
flaxseeds and autoclaved almond powders, respectively.
Figure 2.
The effect of UAE time (
a
), extracted at 240 W, 65
◦
C, and 20:1 (v/w) solvent-to-LMLPs),
UAE temperature (
b
), extracted at 240 W, 20 min, and 20:1 (v/w) solvent-to-LMLPs), and solvent-
to-LMLPs (
c
), extracted at 240 W, 20 min, and 60
◦
C using ethanol/isopropanol on the EE, SC
DPPH·
,
and RP.
3.3. Fitting the Mathematical Models
Highly significant second-order polynomial models (p< 0.0001) with an insignificant
lack-of-fit were satisfactorily fitted to predict the independent variables based on the
multiple linear regression analysis of the experimental data (Table 2). The obtained models
Antioxidants 2022,11, 1943 10 of 19
for the EE (Y
1
), SC
DPPH.·
(Y
2
), and RP (Y
3
) in terms of the experimental (uncoded, Table 1)
data are given as follows (Equations (11)–(13)):
Y1= 73.37 + 6.28X1+ 11.40X2+ 10.37X3−1.97X1X2−2.31X2X3−4.25X32. (11)
Y2= 61.72 + 3.31X1+ 5.74X2+ 1.93X3−3.39X1X2−2.09X1X3−3.02X2X3−4.63X12+ 1.84X22+ 2.63X32(12)
Y3= 0.51 + 0.039X1+ 0.094X2−0.036X2X3−0.064X12+ 0.044X32. (13)
Table 2.
ANOVA table for the experimental variables of each response variable and corresponding
coefficients for the predictive models.
Source DF EE (%, Y1)1SCDPPH·(%, Y2)1RP (Y3)1
C SS p-Value C SS p-Value C SS p-Value
Model 29 73.37 4152.90 <0.0001 61.72 1357.42 <0.0001 0.51 0.26 <0.0001
X11 6.28 539.28 <0.0001 3.31 149.35 0.0010 0.039 0.02 0.0076
X21 11.40 1773.81 <0.0001 5.74 449.44 <0.0001 0.094 0.12 <0.0001
X31 10.37 1469.62 <0.0001 1.93 50.78 0.0230 - 0.01 0.2409 ns
X121 - 14.29 0.1377 ns −4.63 308.65 <0.0001 −0.064 0.06 0.0002
X221 - 4.63 0.3800 ns 1.84 49.00 0.0249 - 0.007 0.0748 ns
X321−4.25 260.41 <0.0001 2.63 99.72 0.0037 0.044 0.028 0.0031
X1X21−1.97 31.17 0.0385 −3.39 91.89 0.0048 - 0.002 0.5403 ns
X1X31 - 5.53 0.3393 ns −2.09 35.07 0.0499 - 0.0007 0.1469 ns
X2X31−2.31 42.74 0.0191 −3.02 73.14 0.0092 −0.036 0.004 0.0386
Residual 10 54.91 70.55 0.018
LoF 15 22.88 0.6395 ns 58.13 0.0578 ns 0.014 0.0911 ns
Pure
error 5 32.03 12.42 0.004
Total 19 4207.81 1427.97 0.28
R20.9870 0.9506 0.9348
R2adj 0.9752 0.9061 0.8760
CV 3.31 4.31 8.35
AP 33.86 18.36 15.41
1
DF—Degree of Freedom, C—Coefficient, SS—Sum of squares, ns—non-significant, LoF—Lack-of-fit.
2
X
1
—UAE
time, X2—solvent-to-LMLPs ratio, and X3—UAE temperature.
The quadratic models (p< 0.0001) for EE, SC
DPPH.
, and RP responses were fitted with
high R
2
(0.9348–0.9870), R
2adj
(0.8760–0.9752), and AP (15.41–33.86) accompanied by low
CV (3.31–8.35) values, signifying the adequacy of fitted models and experiments’ high
precision and reliability. Overall, a high R
2adj
proves that non-significant terms have not
been included in the model. In addition, the AP measures the signal-to-noise ratio, while a
ratio greater than 4.0 would be desirable [
47
]. Table 1shows that the polynomial regression
models sufficiently cover the experimental range of each response variable.
3.4. Effects of Independent Variables on the EE
Table 2illustrates that, among the three independent variables, solvent-to-LMLPs ratio
exerted the maximum significance on the EE (p< 0.0001; SS = 1773.81) followed by UAE
temperature (p< 0.0001; SS = 1469.62) and UAE time (p< 0.0001, SS = 539.28). The only
quadratic effect of UAE temperature was highly significant among the quadratic terms.
The mutual interaction between solvent/solid ratio and UAE time and solvent/solid ratio
and UAE temperature was found to be significant (p< 0.05, Table 2). Figure 3a,b show
that an increase in all the independent variables in the studied range resulted in increased
EE. The individual optimum condition showed that maximum EE (92.42%) was predicted
to be obtained in the UAE under 29.88 min time, 22.50 (v/w) solvent/solid ratio, and
64.42 ◦C temperature.
Antioxidants 2022,11, 1943 11 of 19
Figure 3.
Response surface plots of significant mutual interactions on the EE (
a
,
b
), SC
DPPH
(
c
–
e
), and
RP (f) of LMO extracted by UAE using ethanol/isopropanol.
The cavitation phenomenon can be increased with an increase in the applied sonication
time. More ultrasonic waves at prolonged times generated high-shear gradients by causing
microstreaming to form collapsing bubbles in the vicinity of the cell membrane. As a result,
more disruption of cellular membranes increased the contact surface between oil flakes
and alcoholic solvents to accelerate the solvent penetration into insect substances for the oil
liberation from cells into the solvent with an improvement in the mass transfer rate [
48
].
The increased mass transfer at higher temperatures may be attributed to the reduction
Antioxidants 2022,11, 1943 12 of 19
of viscosity and density of solvent mixtures. In addition, it was possible to develop a
cohesive force to decrease the tensile strength of the liquid by increasing the count of
microbubbles under cavitation with low vapor pressure, reducing the solvent viscosity [
44
].
The reduced EE at extremely high UAE temperatures may be related to the isomerization
and degradation of polyunsaturated fatty acids and other oily constituents [
49
]. Increasing
the EE with an increase in the solvent/solid ratio was based on the principle of mass
transfer because the concentration gradient between solids and liquids is considered a
driving force for mass transfer [50,51].
3.5. Effects of Independent Variables on the Antioxidant Activity
Table 2shows that the linear, quadratic, and interaction effects of all the independent
variables on the SC
DPPH·
were significant. However, the RP was significantly affected by
the linear effects of UAE time (p< 0.01) and solid/solvent ratio (p< 0.0001). In addition,
the quadratic effects of UAE time and temperature were significant on the RP. Only the
significant cross term on the RP was found between the solid/solvent ratio and UAE
temperature (Table 2). Interestingly, the most significant effect on the SC
DPPH
and RP
belonged to the main effect of the solid/solvent ratio. Figure 3c–f illustrate that the SC
DPPH
and RP were significantly boosted by increasing the UAE temperature and solvent-to-
LMLPs ratio. However, RP was reduced at higher extraction times. From the individual
optimization data, a combination of 24.87 min time, 22.50 (v/w) solvent/solid ratio, and
31.5
◦
C temperature was predicted to achieve the highest antioxidant activity (SC
DPPH·
(73.24%) and RP (0.705)) of LMO extracted by the UAE.
Free radicals can be formed when hydrogen atoms are lost from double bonds in
the molecular structure of unsaturated fatty acids. The inhibition of free radicals with
some natural antioxidants such as phenolics, carotenoid pigments, and tocopherols can
spontaneously retard or prevent the initiation of the chain reaction and, subsequently,
lipid oxidation. Therefore, more protection of these bioactive compounds during the UAE
process can improve the antioxidant activity of LMO because the donation of hydrogen
atoms or the transfer of electrons to free radicals can contribute to the production of stable
radicals by interfering with the propagation reaction [
52
]. Samaram et al. [
53
] also found
that oil-soluble antioxidants from papaya seeds were more recovered in warm media and
additional movements. Increasing the antioxidant activity at higher temperatures may be
attributed to the higher solubility of bioactive compounds such as carotenoids and phe-
nolics [
54
]. An increase in the content of phenols of grape seeds with increasing the UAE
temperature from 33 to 67
◦
C was earlier reported [
55
]. High solvent ratios probably inten-
sify the capacity of organic solvents to transfer antioxidant ingredients with an increment
in the diffusion rate by reducing the viscosity [
52
,
53
]. Reducing the antioxidant capacity
at longer extraction times can be due to the degradation of unstable minor compounds
such as phenols due to the collapse of cavitation bubbles and the generation of short-lived
localized hot spots with extremely high local temperature and pressure [56].
3.6. Optimal Conditions and Verification of the Models
The overall optimum region with the highest EE (88.08%), SC
DPPH
(70.79%), and
RP (0.651) of LMO was obtained in the UAE under 22.64 min time, 70
◦
C temperature,
and the solvent-to-LMLPs ratio of 22.5 v/wusing the RSM package’s response optimizer.
Five runs of additional confirmation tests under the optimal conditions showed that the
corresponding experimental values for EE, SC
DPPH·
, and RP were 89.4
±
1.8%,
71.3 ±0.8%
,
and
0.663 ±0.016
, respectively. There was no significant difference found between the
experimental and predicted values. Therefore, the second-order polynomial models pre-
sented in this study were efficient in optimizing the operating parameters involved in the
UAE of LMOs using the alcoholic mixture of ethanol/isopropanol.
Antioxidants 2022,11, 1943 13 of 19
3.7. Comparison of Ultrasound and n-Hexane Extraction Methods
3.7.1. Process Efficiency
Results showed the EE of LMO obtained under the optimal UAE using ethanol/isoprop
anol (89.41%) was much better than that of CSE with n-hexane (60.09%) (p< 0.05). The
calculated diffusion coefficients confirmed that LMO during ultrasonication was extracted
with a more velocity (0.97
×
10
−9
m
2
/s) compared to the CSE (5.07
×
10
−11
m
2
/s) (Table 3).
IN addition, the SEM images visualized in Figure 4show that the UAE and CSE drastically
changed the microstructure of untreated LMLPs with a smooth and intact surface. The
presence of solvents during stirring and ultrasonication led to the breakdown of cell walls
and surface perforations with different intensities and extensions. In the CSE, the sample
microstructure was partially damaged, whilst the microstructure by acoustic waves due
to the cavitational effect was destroyed, with many irregular pores (Figure 4). Thus, the
mixture of ethanol/isopropanol could easier penetrate the sample structure to extract lipids
with a higher mass transfer rate.
A comparison in the EEC reveals that the diffusion, disruption, and leaching out of
lipids in UAE (0.035 kW.h/g) needed much lower energy to be implemented than the CSE
(0.647 kW.h/g) (p< 0.01, Table 3). Accordingly, the EEC of CSE was 18.48 times more
than that of the UAE. This result was consistent with the findings of Ideris et al. [
57
], who
suggested the UAE method saves both time and energy for oil extraction from Canarium
odontophyllum kernels. The EEC reduction is related to the decrease in consumed power.
The applied power of the UAE (240 W) was much less than that of the conventional
method (558.5 W), based on Equation (10), the EEC reduction can be justified by decreasing
the extraction time in the UAE process. Ultrasonic waves produce vibrations for the
development of voids to rapidly transfer energy to solid particles immersed in the extraction.
Moreover, cavitation bubbles at a short time grow closer to the solid surface and collapse
at a higher amplitude forcing the cell wall to rupture, further accelerating the transfer of
desired compounds trapped inside into the solvent medium [10,58,59].
3.7.2. Bioactive Compounds and Antioxidant Activity
The SC
DPPH
and RP amounts of lipids extracted by the CSE were 60.10% and 0.517,
respectively, which were significantly lower than those of lipids extracted by the UAE
(Table 3). The TPC and TCC for lipids extracted by CSE were 3.652 mg GAE/g and 0.645
mg/g, respectively, whereas the corresponding values for UAE were 4.306 mg GAE/g
and 0.778 mg/g, respectively (p< 0.05). The SC
DPPH·
of lipids extracted by CSE and
UAE showed a significantly positive association with TPC (r = 0.968–0.991) and TCC
(r = 0.908–0.937)
. Furthermore, a strong correlation was identified between the RP and TPC
in both CSE (r = 0.856, p< 0.01) and UAE (r = 0.921, p< 0.01). However, no significant
correlation was found between RP and TCC of extracted lipids.
Although there is no report on the type of carotenoid compounds present in oils
extracted from lesser mealworm larvae, the presence of retinol, lutein, zeaxanthin,
β
-
cryptoxanthin,
α
-carotene, cis-
β
-carotene, and trans-
β
-carotene in locust (Locusta migrato-
ria), melon bug (Aspongopus viduatus), and black soldier fly (H. illucens) larvae oils were
evidenced [
60
]. Moreover, Nino et al. showed that the main compounds identified in the
phenolic profile of edible house cricket (A. domesticus) extracts were 4-hydroxybenzoic,
p-coumaric, ferulic, and syringic acids. Other phenolic compounds with strong free-radical
scavenging ability were quinic, gallic, chlorogenic, caffeic, sinapic, and 2-hydroxybenzoic
acids [
61
]. More presence of carotenoids and phenolics in lipids extracted by the optimal
UAE process can be a result of the stronger deterioration of plant tissues through the for-
mation of cavitational bubbles in liquids with a lower surface [
55
–
57
]. Shorter times in the
UAE also can maintain bioactive compounds against chemical alterations like hydrolysis,
isomerization, and oxidation [12].
Antioxidants 2022,11, 1943 14 of 19
Table 3.
The process efficiencies, bioactive compounds, antioxidant activities, fatty acids composition,
and physicochemical properties of LMOs extracted by CSE and UAE methods.
Property
Extraction Method 1,2
CSE with n-Hexane UAE with
Ethanol/Isopropanol
Process efficiency, energy, and
diffusion coefficient
Extraction efficiency (%) 60.09 ±1.32 b89.41 ±1.87 a
Diffusion coefficient (D, ×10−9m2/s) 5.07 ×10−11 b 0.97 ±0.05 ×10−9 a
Electric energy consumption (EEC, kW.h/g) 0.647 ±0.009 a0.035 ±0.003 b
Bioactive compounds and
antioxidant activity
Total carotenoid content (TCC, mg/g) 0.645 ±0.044 b0.778 ±0.032 a
Total phenolic content (TPC, mg GAE/g) 3.652 ±0.015 b4.306 ±0.029 a
DPPH scavenging capacity (SCDPPH·,%) 60.10 ±2.32 b71.31 ±0.84 a
Reducing power (RP) 0.517 ±0.012 b0.663 ±0.016 a
Physicochemical properties
Browning index (BI) 0.316 ±0.009 a0.299 ±0.05 a
Photometric color index (PCI) 14.98 ±0.09 a15.22 ±0.11 a
Specific gravity 0.9005 ±0.0003 a0.9003 ±0.0003 a
Refractive index 1.452 ±0.003 a1.450 ±0.002 a
Apparent visocisty (cP) 300.78 ±5.62 a300.29 ±7.01 a
Acid value (AV, mg KOH/g) 1.78 ±0.09 a1.67 ±0.10 b
Saponification value (SV, mg KOH/g) 221.05 ±1.35 a220.35 ±0.19 a
Peroxide value (PV, meq O2/kg) 0.303 ±0.009 a0.269 ±0.011 b
p-Anisidine value (AnV) 0.201 ±0.04 a0.193 ±0.02 b
Totox value (TxV) 0.807 ±0.005 a0.731 ±0.005 b
Iodine value (IV, g iodine/100 g) 88.70 ±0.71 a83.52 ±0.34 b
Conjugated diene (K232) 1.64 ±0.07 a1.55 ±0.03 b
Conjugated triene (K270) 0.126 ±0.005 a0.109 ±0.004 b
Induction time (IT, h) 17.22 ±0.61 b19.56 ±0.40 a
Free fatty acid (FFA, mg/kg) 0.34 ±0.04 a0.24 ±0.03 b
Fatty acids profile
Caproic acid (C6:0) 0.15 ±0.02 a0.15 ±0.01 a
Lauric acid (C12:0) 0.32 ±0.02 a0.29 ±0.01 a
Myristic acid (C14:0) 0.91 ±0.03 a0.93 ±0.07 a
Palmitic acid (C16:0) 28.65 ±0.26 a26.32 ±0.18 a
Palmitoleic acid (C16:1) 0.30 ±0.02 a0.28 ±0.00 a
Stearic acid (C18:0) 7.68 ±0.06 a7.56 ±0.38 a
Oleic acid (C18:1∆9c) 28.01 ±0.76 a29.54 ±0.25 a
Linoleic acid (C18:2n-6) 30.18 ±0.44 a31.52 ±0.63 a
α-Linolenic acid (C18:3n-3) 2.05 ±0.11 a2.01 ±0.05 a
Arachidic acid (C20:0) 0.31 ±0.05 a0.30 ±0.02 a
Gondoic acid (C20:1∆11) 0.38 ±0.03 a0.34 ±0.01 a
1
CSE and UAE are convention solvent extraction (with n-hexane, 120 min time, 65
◦
C temperature, solvent/solid
ratio of 10.0:1 v/w) and ultrasound-assisted extraction (with ethanol/isopropanol under the optimal conditions
(22.64 min time, 70.0
◦
C temperature, solvent/solid ratio of 22.5:1 v/w);
2
Differences in treatment means in each
row with the same statistical letter (a,b) are statistically non-significant.
Antioxidants 2022,11, 1943 15 of 19
Figure 4.
The SEM images of untreated LMLPs (
a
) and substances defatted by CSE (
b
) and UAE
under optimal conditions (c).
3.7.3. Fatty Acid Composition and Physicochemical Properties
The GC-MS analysis showed that the linoleic (C18:2n-6, 30.18–31.52%), oleic (C18:1
∆
9c,
28.01–29.54%), palmitic (C16:0, 26.32–28.65%), stearic (C18:0, 7.56–7.68%), and
α
-linolenic
(C18:3n-3, 2.01–2.05%) acids were the most dominant fatty acids in the LMO composition.
Myristic (C14:0), gondoic (C20:1
∆
11), arachidic (C20:0), palmitoleic (C16:1), lauric (C12:0),
and caproic (C6:0) acids in very small levels were also found. There was no significant
difference in the fatty acid profile of lipids extracted by CSE and UAE methods (Table 3).
Oleic, linoleic, and palmitic acids were also the major fatty acids in lipids extracted from
yellow mealworm, buffalo mealworm, house cricket, and Dubia cockroach [
62
]. Similar
findings were reported by Roncolini et al. [
9
], who found that the main fatty acids of A.
domesticus were C18:2 (33.66%), C18:1 (28.97%), C16:0 (24.98%), and C18:0 (7.23%). Jalili
et al. [
63
] also showed that the fatty acid profile was hardly affected by UAE as there was no
significant difference in the fatty acid composition of canola seed oils extracted by soxhlet
and UAE. Moreover, significant differences in none of the physical properties of LMO, such
as apparent viscosity, specific gravity, refractive index, BI, and PCI between the two oils,
were detected. Except for the SV, lipids extracted by the UAE showed lower AV, PV, p-AnV,
TxV, K
232
, K
270
, and FFA levels compared to CSE (p< 0.05, Table 3). Hence, more thermal
stability was determined for LMOs extracted by UAE under optimal conditions than CSE.
The IT evaluated using the Rancimat for LMOs extracted by CSE and UAE was 17.22
and 19.56 h, respectively (p< 0.05). The AV of oil indicates its deterioration degree because
it measures the number of free carboxylic acid groups [
64
]. Lower AV of LMO extracted by
UAE shows that this oil had better quality due to a shorter time and a higher solvent/solid
ratio [
65
]. Longer times in the CSE process probably escalated the decomposition and
oxidation of triacylglycerols, resulting in a rise in the FFA content and other oxidative
parameters [
64
,
65
]. Better oxidative properties along with the high similarity of fatty acids
profile showed that the UAE with ethanol/isopropanol can be an excellent alternative for
the CSE of edible oil from A. domesticus using n-hexane.
4. Conclusions
The current work evaluated the combined potential of optimal ultrasonication and
selected organic solvent to improve the extraction yield and quality of LMO. The initial
assessment of LMOs extracted by pure and binary mixed organic solvents showed that the
use of ethanol/isopropanol resulted in a good EE. Second-order polynomial models con-
structed in RSM-CCRD revealed sufficient reliability in predicting the EE and antioxidant
activity of extracted LMOs during the UAE. The UAE time of 22.64 min, UAE temperature
of 70
◦
C, and the solvent-to-LMLPs ratio of 22.5 v/wwere selected and applied for effective
extraction. Satisfactory EE, SC
DPPH
, and RP of LMO were achieved using the optimal
conditions. The antioxidant potential of LMOs strongly depended on bioactive compounds
such as carotenoids and phenolics. A comparison between CSE (with n-hexane) and UAE
(with ethanol/isopropanol) methods revealed that the optimal ultrasonication process
significantly produced LMO with a higher EE and bioactivity under the reduced EEC.
Antioxidants 2022,11, 1943 16 of 19
Ultrasonication caused an extensive structural rupture of LMLPs and improved the quick
transfer of oil from external and internal parts into ethanol/isopropanol as an effective sub-
stituent of n-hexane. Therefore, the diffusivity of oil in the selected solvent was remarkably
increased by acoustic waves. The ultrasound extracted LMOs with more thermal stability
without any significant effect on the main dominant fatty acids. More in-depth studies
are in progress on the kinetic behavior of LMO-UAE. However, the optimized ultrasonic
approach is satisfactory for obtaining high-quality oils from other insects with at least simi-
lar cell structures to lesser mealworms. Since consuming less solvent for the UAE makes
it economically reasonable and feasible for industrial use, more effort should be directed
to reducing the volume of solvent used in the extraction process. It is recommended that
other bioactive minor compounds (e.g., tocopherols) be analyzed, in addition to the profile
of carotenoids and phenolic compounds, and the content and type of polar lipids present
in LMOs extracted by different solvents and techniques.
Author Contributions:
Conceptualization, S.M.T.G.; investigation, S.M.T.G.: writing—original draft
preparation, S.M.T.G.; writing—review and editing, Z.A.; supervision, Z.A.; project administration:
Z.A. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was supported by the Alexander von Humboldt Foundation, German Research
Foundation (DFG, Grant number: 428780268), and Aventis Foundation (Grant number: 80304368).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors acknowledge the support of the Alexander von Humboldt Founda-
tion for S.M.T.G. via the Georg Forster Research Fellowship.
Conflicts of Interest: The authors declare no conflict of interest.
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