Lipid Extraction of Black Soldier Fly Larva Using Aqueous Enzymatic and Soxhlet Method

. 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.


Introduction
Biodiesel is a kind of clean biofuel, which shows to be a promising alternative in the development of renewable energy. At present, the domestic biodiesel raw materials are agricultural crops, oil rich plants and oil-producing microorganisms, whose supply can hardly meet the market demand. It is urgent to find a sustainable supply of renewable biodiesel feedstock with low cost and high yield. In recent years, the use of food waste as feedstock to raise carrion insects such as BFSL to extract insect lipids as biodiesel feedstock emerges as a topic of great 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 BSFL is high and matches the quality of products from other animal and herbal sources. The study of L Zheng et al. showed that the biodiesel produced from the BSFL meets the EN14214 standard and is an ideal raw material for biodiesel [3].
Defatting BSFL is the primary processing step in the downstream valorization and multiple methods of lipid extraction from BSFL have been reported, such as Soxhlet extraction [4], mechanical pressing [5], supercritical fluids extraction [6] and solvent extraction [7,8], aqueous enzyme method [9]. Mechanical pressing is a traditional method but restricted in extracting insect fats because of potential protein denaturation at high pressure. The Soxhlet extraction method uses organic solvents to extract the oils and fats from the material and then rotary evaporation to separate crude oils and fats who have a varied boiling point. Long extraction time prohibited the application of Soxhlet extraction in industry. Currently, the aqueous enzyme method shows promising to scale up which use enzymes to separate oils and proteins, mainly through enzymatic degradation to increase the extraction rate of oils and fats. Despite enzyme cost and low recovery, the aqueous enzyme method has the advantages of mild treatment conditions, simple process, low energy consumption and high oil quality, as well as high purity and reusability of the protein [10]. A lack of published data on various lipid extraction process makes the comparison among technologies difficult. Therefore, the aim of this work is to compare the performance of aqueous enzymatic method and Soxhlet extraction method for the lipid extraction of BSFL as well as to evaluate their potentials for scalable biofuel production.

BSFL Pretreatment
BSFL were provided by Kailier LIMITED and stored at -20 °C in plastic bags until use. Frozen BSFL were grinded with laboratory grinder and immediately used for the different analysis/treatments of extraction. The enzymes employed for sample hydrolysis were alcalase enzyme (10000 U/g) and trypsase enzyme (250 U/mg), purchased from FengAn Zhejiang Biopharmaceutical LIMITED. Petroleum ether (boiling point 40~60°C) was of analytical grade (>98% purity). All the other chemicals were of analytical grade and purchased from Sigma-Aldrich.

Lipid Extraction by Aqueous Enzymatic Method
As shown in figure 1, 10 g of the crushed BSFL were added to 50ml distilled water, and the pH was adjusted at 8.5 after the temperature was raised to 50°C in a constant temperature magnetic stirrer. Enzymes were added at 800 U/g (dry insect biomass) and hydrolyzed 2h, then heated at 90 °C for 10 min to inactivate the enzymes. The hydrolyzed substrate was centrifuged at 4000 rpm at 4 °C for 15 min. Three fractions were obtained, from top to bottom: the lipid fraction I, the supernatant, and the pellet. The pellet was frozen at 18°C for 20 h followed by centrifugation to obtain lipid fraction II. Lipid mass was calculated by adding up lipid fraction I and fraction II.

Lipid Extraction by Soxhlet Extraction Method
10 g of the crushed BSFL were added to 120ml petroleum 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.

Crude fat and Lipid Quality of BSFL
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 Vegetable oils-Determination of iodine value. Acid value was referred to GB/T5530 Animal and vegetable fats and oils-Determination of acid value and acidity. Peroxide value was measured according to GB/T5538 Oils and fats-Determination of peroxide value and measurement of saponification value was in reference to GB/T5534 Animal and vegetable fats and oils-Determination of saponification value.

Crude Fat Composition
Fresh BSFL have a moisture content around 60%, to make them ready for lipid abstraction, the larvae were dried to about 6%, as shown in table 1. Dried larvae has around 29.6% of oil, a value comparable to 33.7% in reference [5]. Dried larvae with a lower moist content shows similar oil and protein profile as common vegetable oil seeds at 17~54.55% and 12.4~40% respectively. By de-oiling insect larvae, it provides a high potential of contribution to the supply for sustainable fat and protein alternatives to oilseeds.

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

Lipid Qualities
The quality of the lipids abstracted were also compared and summarized in Table 1. For reference, revelent data from a previous research [5] on the properties of BSFL lipid abstracted from cold screw pressing technique. As it can be seen from Table 2, the color of the oil extracted by the aqueous enzyme method is better than that of the petroleum ether extraction.
Due to the difference of sample's fatty acid composition, higher iodine numbers were found in this experiment implying a higher content of unsaturated fatty acids in our insect samples.
Corresponding to the chain length of fatty acids, the saponification value is a measure of average molecular weight of all fatty acids. The number of saponification of BSFL oil extracted with petroleum ether was less than that of the aqueous enzyme extraction, indicating that the oil may contain some unsaponifiable impurities that were raised with the oil by the petroleum ether during the extraction process. The quality of lipid from cold screw pressing is less refined with a value of 252 mg KOH·g -1 which is comparable with that of vegetable oils, such as coconut fat 250-262 mg KOH·g -1 .
Peroxide value is another indicator of lipid quality of importance. The peroxide value describe the oxidative statue of the fat. The peroxide value and acid value of BSFL extracted by the aqueous enzymatic method are smaller than those of petroleum ether extraction which means that the oxidation and rancidity of the oil in the process of BSFL oil extraction by the aqueous enzymatic method is small, and the nutrients in the oil can be better retained. However, peroxide value drop dramatically as low as 0.29 meqO 2 /kg in the work of Matthaus [5] because hydroperoxides were decomposed at high temperature during cold screw pressing process.

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 reported 5 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.