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Korean J. Food Sci. An. 37(6): 955~961 (2017)
https://doi.org/10.5851/kosfa.2017.37.6.955
pISSN 1225-8563
·
eISSN 2234-246X
ARTICLE
kosfaj.org 955
Defatting and Sonication Enhances
Protein Extraction from Edible Insects
Byoung Deug Choi, Nathan A. K. Wong, and Joong-Hyuck Auh*
Department of Food Science and Technology, Chung-Ang University, Anseong 17546, Korea
Abstract
Edible insects are attracting growing interest as a sustainable source of protein for addition to
processed meat and dairy products. The current study investigated the optimal method for
protein extraction from mealworm larvae (Tenebrio molitor), cricket adults (Gryllus bimacu-
latus), and silkworm pupae (Bombyx mori), for use in further applications. After defatting
with n-hexane for up to 48 h, sonication was applied for 1-20 min and the protein yield was
measured. All samples showed a total residual fat percentage below 1.36%, and a 35% to 94%
improvement in protein yield (%). In conclusion, defatting with n-hexane combined with son-
ication improves the protein yield from insect samples.
Keywords protein, mealworm, cricket, silkworm, sonication
Introduction
Edible insects are attracting increasing interest in the food industry because of
their potential to serve as an alternative protein source. Although insects have been
eaten in many cultures for centuries, Western societies are only recently adopting
such traditions. In many parts of the world, such as Africa, Asia, and Latin Amer-
ica, where insects are part of the diet, they are considered both a delicacy and a
nutritional requirement (van Huis et al., 2013). According to the World Health
Organization (Fishman et al., 2002), approximately 27% of children under 5 years
of age are experiencing undernutrition stemming from the lack of adequate protein
access. In these underdeveloped areas food security is prominent and entomoph-
agy plays a key role as a solution.
As a food source, insects are not only comparable in nutritional value to conven-
tional meat but also have many advantages in a variety of areas (van Huis et al.,
2013). In terms of the environment, insect rearing produces far fewer greenhouse
gases, requires clearing of less land, and requires less feed per unit of protein pro-
duced (12 times less than cattle), due to insects’ cold-blooded nature (van Huis et
al., 2013). Along with their rapid growth, efficiency, and availability (over 1900
known edible species), insects show promise for use in sustainable agriculture
(van Huis et al., 2013).
Although in Western societies insects are thought of as bothersome pests,
research has shown that they display high availability of protein, lipid, vitamin,
and mineral contents (Finke, 2002; Ghosh et al., 2017; Rumpold and Schlüter,
2013; Yi et al., 2013). All insects have a high abundance of amino acids (Ghosh et
Received November 27, 2017
Revised December 10, 2017
Accepted December 11, 2017
*Corresponding author
Joong-Hyuck Auh
Department of Food Science and
Technology, Chung-Ang University,
Ansung 17546, Korea
Tel: +82-31-670-3079
Fax: +82-31-675-4853
E-mail: jhauh@cau.ac.kr
Copyright © Korean Society for Food
Science of Animal Resources
This is an open access article distri-
buted under the terms of the Creat-
ive Commons Attribution Non-Com-
mercial License (http://creativecom-
mons.org/licences/by-nc/3.0) which
permits unrestricted non-commercial
use, distribution, and reproduction in
any medium, provided the original
work is properly cited.
December 2017 Volume 37 Issue 6
956 https://doi.org/10.5851/kosfa.2017.37.6.956
al., 2017; Melo et al., 2011; Yi et al., 2013; Zielińska et
al., 2015), and in-depth analysis of individual insects pro-
vides further conclusions as well (Hall et al., 2017; Long-
vah et al., 2011; Zhao et al., 2016). Based on these
studies, insects have been shown to contain a protein con-
tent ranging from 50-82% (dry weight). Compared to
conventional protein sources such as beef, pork, chicken,
and lamb, many insects, for example termites, grasshop-
pers, caterpillars, and houseflies, possess higher values
(Rumpold and Schlüter, 2013).
Because insects possess physical and sensory character-
istics that make them unattractive as food in Western cul-
ture, methods for extraction of protein for addition to
other food have been tested, to increase their acceptance
(Yi et al., 2013). Because insects show high lipid bio-
availability that can interfere with protein extraction, lipid
separation can be applied to enhance protein extraction.
Although lipid separation of foods has been explored
using a variety of materials for fat segregation, soybeans
have been best studied (Karki et al., 2010; L'Hocine et
al., 2006; Lou et al., 2010). Lipid extraction has been
shown to not only isolate lipids specifically but also to
improve certain functionalities and characteristics of pro-
teins (L'Hocine et al., 2006; Lee et al., 2016). For edible
insects, ethanol defatting has been applied to the larvae of
yellow mealworm Tenebrio molitor (Zhao et al., 2016)
for lipid determination, and hexane has been applied to a
variety of insects (Longvah et al., 2011; Purschke et al.,
2017; Yi et al., 2013) for comprehensive analysis. Altho-
ugh many alternatives to lipid extraction have been imp-
lemented (e.g. aqueous extraction, supercritical CO2), hex-
ane defatting yields 96% or greater oil removal on aver-
age (Ricochon and Muniglia, 2010).
Ultrasound assisted extraction (UAE) or sonication is
widely incorporated in food processing, as it provides an
alternative extraction method to other heat or pressure
treatments (microwave assisted, supercritical CO2, high-
pressure processing). Ultrasound technology has seen an
increase in utilization owing to its effectiveness and eco-
nomic advantages (Preece et al., 2017). Ultrasound tech-
nology has been applied for protein extraction from a
variety of foods including soy (Jambrak et al., 2009; Lee
et al., 2016; Lou et al., 2010; Preece et al., 2017), whey
protein concentrate (Chandrapala et al., 2011), and egg
whites (Arzeni et al., 2012). Although Tzompa-Sosa et al.
(2014) applied sonication technology to insects, no pro-
tein enhancement was noted; instead, it aided in lipid iso-
lation when used as a pretreatment to the Folch and aque-
ous extraction procedures. To date no reports have been
published on whether ultrasound technology is effective
for extraction of protein from insects.
Edible insects have been studied as ingredients for for-
mulation of meat and dairy products (Kim et al., 2016;
Omotoso, 2006; Rumpold and Schlüter, 2013; Wang et
al., 2017). Recently, Rumpold and Schlüter (2013) sug-
gested application of edible insects in animal feed and
human food products, with an emphasis on their use as a
texturizing agent based on their high emulsion capacity.
Kim et al. (2016) applied protein powders from edible
insects in sausages, which increased cooking yield and
hardness. Peptides extracted from silkworm pupae exhib-
ited a positive effect on the quality of yogurt but require
further research for application in dairy products (Wang
et al., 2017).
The present study aimed to establish the optimum con-
ditions for protein separation from three edible insects
using ultrasound, for application in the food industry.
Materials and Methods
Materials
n-Hexane was of chemical grade and purchased from
Honeywell Burdick & Jackson (Thomas Scientific, USA).
The bovine serum albumin powder was purchased from
Equitech-Bio, Inc. (USA).
Edible insects
Yellow mealworm larvae (Tenebrio molitor), field cric-
ket adults (Gryllus bimaculatus), and silkworm pupae
(Bombyx mori) were obtained from Insect Vision (Korea).
The T. molitor and G. bimaculatus samples were previ-
ously microwave dried and the B. mori samples were fro-
zen. The B. mori samples were freeze-dried following
receipt in our laboratory, and all insect samples were
immediately stored at -40°C until use. Prior to defatting,
insect samples were ground into coarse meal using a tra-
ditional pestle and mortar.
Hexane defatting for lipid removal
Lipids were extracted from the insect samples using n-
hexane as a solvent, at a solvent to sample ratio of 1:20.
Samples were stirred for 12 h and the hexane was remo-
ved by filtering, then replaced every 12 h for a total time
of 48 h. Samples were emptied onto aluminum foil and
left to dry overnight under a fume hood. Total lipid con-
tent was determined using the Soxhlet extraction method
Protein Extraction from Edible Insect
https://doi.org/10.5851/kosfa.2017.37.6.956 957
(AOAC, 2000).
Protein extraction using ultrasound
A Sonics® Vibra-Cell™ VCX 750 ultrasonic unit
(Sonics & Materials Inc., USA) was used, with a maxi-
mum power output of 2.5 kW. It was operated at 20 kHz
with a 75% AMP and pulsed every 3 s. Insects ground to
a coarse meal (12.5 g) were mixed with 200 mL of dis-
tilled water containing 9.46 mM ascorbic acid. The sus-
pension was then sonicated for 20 min and aliquots were
collected at 1, 2, 5, 10, 15, and 20 min. The procedure
was performed on ice and the sample was allowed to rest
between intervals of equal time. Samples were sieved
through a stainless-steel filter (pore size of 1 mm) and fil-
trates were collected and freeze-dried for further experi-
ments. To determine protein amount and calculate protein
yield, the samples were analyzed by the Dumas method
using an NDA 701 Dumas Nitrogen Analyzer (Velp Sci-
entifica, Italy). Protein yield (%) and total protein content
of each insect sample were calculated based on protein
quantity measured and reported as %N using the standard
conversion factor of 6.25. The amino acid composition of
the defatted insect samples was analyzed using an Agilent
1100 HPLC with an Eclipse AAA column (4.5 × 150
mm, 5 µm) from Agilent Technologies (USA).
Statistical analysis
All trials were performed in triplicate unless indicated
otherwise. Data were analyzed using SPSS (IBM, USA).
Significance of differences among means was determined
using analysis of variance (ANOVA) and Duncan’s mul-
tiple-range test, with a significance threshold of 5%.
Results
Total lipid content from hexane defatting
The lipid removal process was performed for all three
insect samples, using conventional hexane defatting for
24-48 h (Fig. 1). Subsequent analysis of fat content, using
the Soxhlet method, revealed crude residual fat values of
less than 2.0% for all samples. The lowest average fat
values, achieved after 48 h of defatting for mealworm,
cricket, and silkworm samples, were 0.69%, 1.07% and
0.34%, respectively. No significant differences were ob-
served after 36 h of defatting. Based on the values dis-
played in Fig. 1, the optimal time for defatting was 36 h.
Total protein content based on (%N) was highest in the
cricket samples, which contained 74% protein after defat-
ting. Silkworm pupae contained approximately 65%,
while the mealworm showed the lowest protein content at
just under 63% total protein.
Protein yield and amino acid composition
Application of ultrasound sonication to the three insect
samples was performed for various time periods, to a
maximum of 20 min. The experiment was performed in
triplicate and the averages are displayed in Fig. 2. Protein
yield percentage was calculated based on the total protein
amount of each insect. Although the yield showed a gen-
eral increasing trend with time for all samples, a signifi-
cant difference was observed between the silkworm pupae
and the other two insects. After 20 min of sonication, the
maximum protein yield of the silkworm pupae sample
was 94%, 76% greater than its yield without sonication
(18%, 0 min). After 5 min of treatment, the protein yield
increased exponentially to 89%. Sonication for 20 min
increased protein yield, but not significantly. Thus, for B.
mori 5 min appears to be the most efficient period of son-
ication.
Mealworm and cricket samples showed a lower protein
yield. Of the three insects, mealworm had the lowest pro-
tein yield at 35%; cricket had a yield of 37% (15 min).
Both insect samples showed an increase in protein yield
after 15 min of sonication, of 28% for mealworm and
34% for cricket. The highest values were obtained at 15
min for both the mealworm and cricket samples, and the
Fig. 1. Residual fat (%) in edible insects after hexane defat-
ting. Residual fat (%) was calculated using the total fat content
for each insect sample after 24-48 h n-hexane defatting. Each
value is expressed as the mean±S.D. (n=3); values marked with
different letters showed significant differences (p<0.05). Meal-
worm larvae, Tenebrio molitor larvae; cricket, Gryllus bimacula-
tus; silkworm pupae, Bombyx mori.
December 2017 Volume 37 Issue 6
958 https://doi.org/10.5851/kosfa.2017.37.6.956
slight decrease at the 20-min mark indicates that 15 min
of sonication was sufficient for both samples.
The amino acid composition of each edible insect was
determined (Fig. 3). The essential amino acids are the first
eight shown on the x-axis and the following represent the
non-essential amino acids. The three insects displayed sim-
ilar profiles, with highest levels of glutamine and lowest
levels of methionine. Levels of both proline and tyrosine,
however, were notably high for the mealworm sample.
Discussion
Conventional hexane defatting was carried out for 24-
48 h on mealworm, cricket, and silkworm samples. Our
results showed average residual crude fat values of 0.75%
for mealworm, 1.17% for cricket, and 0.47% for silk-
worm pupae. Our proximate composition analysis data
for crude fat prior to defatting (33.46% for mealworm;
19.77% for cricket; 30.87% for silkworm pupae: data not
shown) was comparable to the findings of others (Finke,
2002; Ghosh et al., 2017; Hall et al., 2017; Yi et al., 2013).
In particular, our results corroborated those of Ghosh et
al. (2017), who assayed 5 species of insects currently eaten
in South Korea, including two of the three contained in
our current study (T. molitor larvae, G. bimaculatus
adults). A direct comparison of the yellow mealworm
(Tenebrio molitor) sample was performed by Purschke et
al. (2017) for extraction of oils with conventional hexane
vs. supercritical CO2 (SCO2). Total defatting percentages
(%) were 96.56% for n-hexane and 92.57% for SCO2
under conditions of 325 bars, 55°C, and 75 min. Although
the SCO2 method produced a more rapid result, the energy
output also needs to be considered, and the two methods
yielded products with similar physio-chemical properties
and fat composition (Purschke et al., 2017). We also app-
lied SCO2 for defatting of mealworms, but only 30% of
lipids were removed at 50°C (data not shown), which was
not sufficient pretreatment for protein separation. Hexane
defatting of mealworm showed equally high defatting per-
centages, but with a higher hexane to sample ratio. These
Fig. 2. Effect of sonication time on protein extraction from
edible insects. Protein yield (%) calculated using total amount
of protein in each insect sample after 1-20 min sonication treat-
ment. Each value is expressed as the mean±S.D. (n=3); values
marked with different letters showed significant differences
(p<0.05). Mealworm larvae, Tenebr io m olito r larvae; cricket, Gryl-
lus bimaculatus; silkworm pupae, Bombyx mori.
Fig. 3. Amino acid profiles of proteins extracted from edible insects. Contents are expressed as percentages for each amino acid
after extraction (mg/g protein). Mealworm larvae, Tenebrio molitor larvae; cricket, Gryllus bimaculatus; silkworm pupae, Bombyx mori.
Protein Extraction from Edible Insect
https://doi.org/10.5851/kosfa.2017.37.6.956 959
findings coupled with our results showcase hexane’s effi-
ciency to reduce the lipid content of insect samples.
Few studies have been published using insect samples,
and soybean protein may serve as an alternate point of
comparison. Comparable proximate analyses of soy pro-
tein including essential amino acid profiles (Yi et al.,
2013) provide a point of reference for assessment. A study
was conducted on the defatting of soybeans to compare
different defatting solvents such as ethanol and methanol
to hexane, due to reports of its effect on functionality and
availability, as well as tighter emission restrictions and
safety concerns (L'Hocine et al., 2006). Residual fat per-
centages in 100 g samples with a 1:3 solvent ratio were
1.4% for hexane, 3.8% for ethanol, and 14.5% for metha-
nol.
The protein content detected on a dry matter basis after
defatting of each insect ranged from 62% to 74%, with
the cricket samples displaying the highest values (results
not shown). Research on proximate nutritional content
showed similar values for all insects (Ghosh et al., 2017;
Longvah et al., 2011; Purschke et al., 2017; Rumpold and
Schlüter, 2013; Yi et al., 2013; Zhao et al., 2016; Ziel-
ińska et al., 2015). On a dry weight basis, the protein con-
tent of Tenebrio molitor larvae ranged from 51% to 58%
for non-defatted samples, while Yi et al. (2016) obtained
a protein content value of 76.5% for n-hexane defatted
mealworm samples after 6 h of treatment. Protein content
of Bombyx mori pupae ranged from 48% to 58%, for non-
defatted samples based on dry weight. A non-defatted
protein percentage of 58.32% calculated by Ghosh et al.
(2017) for Gryllus bimaculatus is comparable to our res-
ult. To date, values of protein content specifically for de-
fatted B. mori pupae and G. bimaculatus have yet to be
reported.
Ultrasound is widely used for extraction of substances
such as herbal oils, protein, and bioactive components in
plant material (flavones, polyphenolics) (Vilkhu et al.,
2008). For insects, sonication methods have not been
reported, although electric blending (Hall et al., 2017; Yi
et al., 2013) and extraction with NaOH (Zhao et al., 2016)
or HCL (Longvah et al., 2011) have been reported. Thus,
in our experiment the extraction of protein through soni-
cation was performed on three edible insects up to a dura-
tion of 20 min. Peak protein yield was 94% for silkworm
pupae, 37% for cricket, and 35% for mealworm. Sonica-
tion for 1 min, to a maximum of 15 or 20 min, produced
an increase in protein yield for the aforementioned insects
of 76%, 34%, and 28% respectively. Due to the lack of
research on ultrasound-assisted extraction of protein from
insects, the results cannot be thoroughly discussed in det-
ail. The differences in protein yield percentage between
silkworm pupae and the other two insects may be attri-
buted to size differences (surface area of crude samples)
and the greater prominence of chitin in the exoskeletons
of both mealworms and crickets. Our experimental proce-
dure might have caused some sample loss, leading to
underestimation of protein percentage, especially for sam-
ples with a higher chitin content. After sonication, the
suspensions contained suspended particles of the insect
being tested, and the sieved residues were discarded to
reduce the insoluble fraction. The silkworm pupae sample
was the most soluble of the three, which might explain
the results obtained. A mechanical method to more finely
homogenize the samples prior to sonication and testing of
the insoluble fractions caught in the sieve should be
investigated to improve accuracy. Using soy protein as an
alternative reference point, ultrasound technology applied
to defatted soy protein flakes showed not only a signifi-
cant reduction in particle size, from 1000 µm to 90 µm,
but also a 10.5% increase in overall protein release (Karki
et al., 2010). After 2 min of sonication, the protein yield
increased by 14% and 18% in mealworm and cricket
samples, comparable to the results published by Karki et
al. (2010).
The amino acid profiles of the selected edible insect
samples are summarized in Fig. 3. No significant changes
in amino acid profiles were observed after sonication. In
comparison to previous reports (Ghosh et al., 2017; Long-
vah et al., 2011; Rumpold and Schlüter, 2013; Yi et al.,
2016; Zielińska et al., 2015), the selected insects showed
a high-quality amino acid profile that meets typical amino
acid requirements for human nutrition. The amino acid
profiles of insects are usually quite similar, but both pro-
line and tyrosine values were much higher in mealworms.
Since tyrosine is biologically related to phenylalanine,
their contents are commonly reported together (Rumpold
and Schlüter, 2013; Yi et al., 2016) but others have rep-
orted a much lower tyrosine content in T. molitor larvae
(Zielińska et al., 2015). Initial levels of proline in T. moli-
tor, prior to sonication treatment (7.21%), were similar to
levels reported by Yi et al. (2013), at 6.6%, but higher
than the 4.34% reported by Zielińska et al. (2015). An
extraction method that did not use a defatting step dis-
played lower protein values than the method reported
here (Ghosh et al., 2017) but the trends were comparable.
Thus, our results generally corroborate previously pub-
December 2017 Volume 37 Issue 6
960 https://doi.org/10.5851/kosfa.2017.37.6.956
lished results.
Conclusion
Hexane defatting of whole insect samples yielded 1.36%
or less residual fat content, and a protein content of 62-
74%. Protein extraction using sonication increased pro-
tein yield percentages for all insects. Although the inc-
rease in protein yield was lower for mealworm and cric-
ket samples than for silkworm pupae (94%), this may have
resulted from characteristics of the insect specimens them-
selves or their life stage. Hexane defatting significantly
decreased total fat percentage and ultrasound-assisted ext-
raction increased protein yield percentage. These findings
will further the exploration of edible insect protein as an
ingredient in meat and dairy products.
Acknowledgements
This research was supported by Korea Institute of Plan-
ning and Evaluation for Technology in Food, Agriculture,
Forestry (IPET) through the High Value-added Food Tech-
nology Development Program and by Chung-Ang Univ-
ersity Excellent Student Scholarship.
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