E3S Web of Conferences 406, 03013 (2023) https://doi.org/10.

1051/e3sconf/202340603013
ICEMEE 2023

Lipid Extraction of Black Soldier Fly Larva Using Aqueous

Enzymatic and Soxhlet Method
Lijun Wu1, 2, 3, Zhijun Li4, Jianhang Li1,5, Haifeng Fang1, Xiaopeng Qiu1,* , Pengrui Fu1 and Tong Zhu1
1 Power China Huadong Engineering Corporation Limited, Hangzhou 311122, Zhejiang, China
2 College of Environmental and Resource Science, Zhejiang University, Hangzhou 310029, Zhejiang, China
3 Huadong Eco-Environmental Engineering Research Institute of Zhejiang Province, Hangzhou 311122, Zhejiang, China
4 Hangzhou Linjiang Environmental Energy Co.LTD, Hangzhou 310003, Zhejiang, China
5 Yangtze River Delta (Jiaxing) Ecological Development Co., Ltd., Jiaxing 314000, Zhejiang, China

Abstract. This study aimed to explore the efficacy of lipid extraction methods on black solider fly larvae
(BSFL) which is a potential sustainable source of biodiesel. The raw fat content of BSFL in exam was 29.6%
on dry matter basis, which is comparable to other studies. Both aqueous enzymatic method and Soxhlet
method are effective in BSFL lipid extraction at levels around 80%. The lipid quality from aqueous enzymatic
method is superior in addition to other merits such as mild, easy and fast. Further research is recommended
to scale up its potential as bioenergy alternatives.

1 Introduction through enzymatic degradation to increase the extraction

rate of oils and fats. Despite enzyme cost and low recovery,
Biodiesel is a kind of clean biofuel, which shows to be a the aqueous enzyme method has the advantages of mild
promising alternative in the development of renewable treatment conditions, simple process, low energy
energy. At present, the domestic biodiesel raw materials consumption and high oil quality, as well as high purity
are agricultural crops, oil rich plants and oil-producing and reusability of the protein[10]. A lack of published data
microorganisms, whose supply can hardly meet the market on various lipid extraction process makes the comparison
demand. It is urgent to find a sustainable supply of among technologies difficult. Therefore, the aim of this
renewable biodiesel feedstock with low cost and high work is to compare the performance of aqueous enzymatic
yield. In recent years, the use of food waste as feedstock method and Soxhlet extraction method for the lipid
to raise carrion insects such as BFSL to extract insect extraction of BSFL as well as to evaluate their potentials
lipids as biodiesel feedstock emerges as a topic of great for scalable biofuel production.
interests. Raised on organic waste, BSFL is reported as a
qualified raw material for biodiesel production containing
35~40% lipids on average [1, 2]. The quality of lipids from 2 Material and Methods
BSFL is high and matches the quality of products from
other animal and herbal sources. The study of L Zheng et 2.1 BSFL Pretreatment
al. showed that the biodiesel produced from the BSFL
meets the EN14214 standard and is an ideal raw material BSFL were provided by Kailier LIMITED and stored at -
for biodiesel[3]. 20 °C in plastic bags until use. Frozen BSFL were grinded
Defatting BSFL is the primary processing step in the with laboratory grinder and immediately used for the
downstream valorization and multiple methods of lipid different analysis/treatments of extraction. The enzymes
extraction from BSFL have been reported, such as Soxhlet employed for sample hydrolysis were alcalase enzyme
extraction [4], mechanical pressing [5], supercritical fluids (10000 U/g) and trypsase enzyme (250 U/mg), purchased
extraction [6] and solvent extraction[7,8], aqueous from FengAn Zhejiang Biopharmaceutical LIMITED.
enzyme method[9]. Mechanical pressing is a traditional Petroleum ether (boiling point 40~60°C) was of analytical
method but restricted in extracting insect fats because of grade (>98% purity). All the other chemicals were of
potential protein denaturation at high pressure. The analytical grade and purchased from Sigma-Aldrich.
Soxhlet extraction method uses organic solvents to extract
the oils and fats from the material and then rotary
2.2 Lipid Extraction by Aqueous Enzymatic
evaporation to separate crude oils and fats who have a
Method
varied boiling point. Long extraction time prohibited the
application of Soxhlet extraction in industry. Currently, As shown in figure 1, 10 g of the crushed BSFL were
the aqueous enzyme method shows promising to scale up added to 50ml distilled water, and the pH was adjusted at
which use enzymes to separate oils and proteins, mainly 8.5 after the temperature was raised to 50°C in a constant
* Corresponding author: [email protected]

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(https://creativecommons.org/licenses/by/4.0/).
E3S Web of Conferences 406, 03013 (2023) https://doi.org/10.1051/e3sconf/202340603013
ICEMEE 2023

temperature magnetic stirrer. Enzymes were added at 800 at 90 °C for 10 min to inactivate the enzymes.
U/g (dry insect biomass) and hydrolyzed 2h, then heated

Pellet Centrifugation
Alkali Enzymes

Grinded Larvae Magnetic stirring Solvent

Centrifugation Lipid Fraction II
powder with heater

Lipid Fraction I

Fig.1. Experimental process of aqueous enzymatic method

The hydrolyzed substrate was centrifuged at 4000 rpm

at 4 °C for 15 min. Three fractions were obtained, from
2.3 Lipid Extraction by Soxhlet Extraction
top to bottom: the lipid fraction I, the supernatant, and the
Method
pellet. The pellet was frozen at 18°C for 20 h followed by
centrifugation to obtain lipid fraction II. Lipid mass was 10 g of the crushed BSFL were added to 120ml petroleum
calculated by adding up lipid fraction I and fraction II. ether in a Soxhlet apparatus. The sample material was
extracted for 8 h at 50℃ water bath as shown in figure 2.
Then the solvent was removed by a rotary vacuum dryer
and the yield was calculated gravimetrically.

Fig.2.The experiment layout of soxhlet extraction

2.4 Crude fat and Lipid Quality of BSFL

3 Results and Discussion
Value of crude fat content was referred to GB/T5512
Inspect of grain and oilseeds-Determination of crude fat
content in grain. Iodine value was referred to GB/T5532 3.1 Crude Fat Composition
Vegetable oils-Determination of iodine value. Acid value Fresh BSFL have a moisture content around 60%, to make
was referred to GB/T5530 Animal and vegetable fats and them ready for lipid abstraction, the larvae were dried to
oils-Determination of acid value and acidity. Peroxide about 6%, as shown in table 1. Dried larvae has around
value was measured according to GB/T5538 Oils and fats- 29.6% of oil, a value comparable to 33.7% in reference [5].
Determination of peroxide value and measurement of Dried larvae with a lower moist content shows similar oil
saponification value was in reference to GB/T5534 and protein profile as common vegetable oil seeds at
Animal and vegetable fats and oils-Determination of 17~54.55% and 12.4~40% respectively. By de-oiling
saponification value. insect larvae, it provides a high potential of contribution
to the supply for sustainable fat and protein alternatives to
oilseeds.
Table 1. Composition of common oilseeds and BSFL
Source Oil % Moisture % Protein% Reference
Dried larvae 29.6 6 / /
Dried larvae 33.7 5.6 28 [5]
Cottonseed 23 8 23 [11]
Flaxseed 34 9 23 [11]
Rapeseed 39 9 22 [11]
Soybean 17-20 13 30-40 [11]
Sunflower seed 29.3-54.5 3-6 12.4-19.7 [12]

2
E3S Web of Conferences 406, 03013 (2023) https://doi.org/10.1051/e3sconf/202340603013
ICEMEE 2023

value with cold screw pressing method is given in the

3.2 Lipid Yields and Extraction Efficiency work of Matthaus[5].
The experimental setting of two lipid extraction
The lipid yields（Y）was calculated by the following
methods were as described in material and methods where
formula: 50℃ is mostly maintained due to the oil melting point of
Y 100% (1) about 30 ℃. In contrast, the press head was heated to 90℃
to allow the drainage of the oil in cold screw pressing
Where Wl is the weight of extracted lipid; Wp is
method[5].
weight of dried biomass powders. The lipid extraction
As it can be seen clearly in figure 3 that both aqueous
efficiency (η) was calculated using following expression:
enzymatic method and Soxhlet method show improved
η 100% (2) performance in extraction efficiency, 78.4% and 82.6%
Where P is the percentage of crude fat content in BSFL, respectively. Despite higher drainage temperature, the
which is determined to be 29.6% in the experiment. The P extraction efficiency of cold screw pressing is the lowest
at 72.7%.

Fig.3. Lipid extraction efficiency comparison of three methods

and summarized in Table 1. For reference, revelent data

3.3 Lipid Qualities from a previous research [5] on the properties of BSFL
lipid abstracted from cold screw pressing technique.
The quality of the lipids abstracted were also compared
Table 2. Properties of BSFL lipid from different abstraction methods
Properties Aqueous Soxhlet Screw pressing
enzymatic method [5]
method
Color(manually rated) Yellow/clear Tawny/clear Yellow
Iodine value(g·kg-1) 1058.4 1046.5 780
Saponification value(mgKOH·g-1) 195.42 157.03 252
Peroxide value (meq O2·kg-1) 3.871 6.272 0.29

As it can be seen from Table 2, the color of the oil may contain some unsaponifiable impurities that were
extracted by the aqueous enzyme method is better than raised with the oil by the petroleum ether during the
that of the petroleum ether extraction. extraction process. The quality of lipid from cold screw
Due to the difference of sample’s fatty acid pressing is less refined with a value of 252 mg KOH·g-1
composition, higher iodine numbers were found in this which is comparable with that of vegetable oils, such as
experiment implying a higher content of unsaturated fatty coconut fat 250-262 mg KOH·g-1.
acids in our insect samples. Peroxide value is another indicator of lipid quality of
Corresponding to the chain length of fatty acids, the importance. The peroxide value describe the oxidative
saponification value is a measure of average molecular statue of the fat. The peroxide value and acid value of
weight of all fatty acids. The number of saponification of BSFL extracted by the aqueous enzymatic method are
BSFL oil extracted with petroleum ether was less than that smaller than those of petroleum ether extraction which
of the aqueous enzyme extraction, indicating that the oil means that the oxidation and rancidity of the oil in the

3
E3S Web of Conferences 406, 03013 (2023) https://doi.org/10.1051/e3sconf/202340603013
ICEMEE 2023

process of BSFL oil extraction by the aqueous enzymatic Energy,147,584-593 (2020)

method is small, and the nutrients in the oil can be better 11. M. Bockisch, Handbuch der Lebensmittel-
retained. However, peroxide value drop dramatically as Technologie Nahrungsfette und öle. Verlag Eugen
low as 0.29 meqO2/kg in the work of Matthaus[5] because Ulmer, Stuttgart (1993)
hydroperoxides were decomposed at high temperature
12. K. J. Kaffka, K. H. Norris, J. Peredi, A. Balogh, Acta
during cold screw pressing process.
Alimentaria,11, 258 (1982)

4 Conclusions
The raw fat content evaluated in BSFL in this experiment
was 29.6% on dry matter basis. Both aqueous enzymatic
method and Soxhlet method are efficient in lipid fraction
at levels around 80%. The preliminary data collected in
this paper demonstrated that the differences between two
methods are mostly in lipid qualities. The quality of BSFL
oil extracted by aqueous enzymatic method is better than
that of Soxhlet method because the degree of oxidation
and rancidity of the oil is negligible which is beneficial for
lipid retain. Data from cold screw pressing as reported5
was used as reference. Due to higher operation temperate
with cold screw pressing process, nutrients are susceptible
to decomposing while abstraction efficacy is not improved
otherwise.
Overall, both aqueous enzymatic method and Soxhlet
method are prone to meet the standard expectations of the
industry and the use of insect oil as raw materials for
biodiesel production shows to be an attractive alternative
as bioenergy in the future.

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