INSECT FATS FOR COSMETICS 189 applications and transport and associated costs. Therefore, identifying other suitable sources such as insect fats may prove useful. In this paper, three insect species, the BSF, the house cricket and the locust, were evalu- ated for their potential use in cosmetic applications. Fats extracted from these insects are evaluated in a hand cream formulation as a proof of principle to demonstrate the potential of using insects, cultivated on organic waste streams, in personal health-care products. MATERIALS AND METHODS INSECTS In general, insects are considered as farm animals and are to be treated as such according to the Belgian legislation (17). However, detailed guidance regarding insect welfare and euthanasia are lacking and little is known about the humane treatment and other ethical aspects in insects. Black soldier fl ies were bred at the Thomas More campus Geel in a greenhouse at an average day temperature of 30°C and relative humidity of 50–90%. The breeding process is similar to the process described in Sheppard et al.(18). Female fl ies deposit their eggs in cardboard structures. Eggs were harvested and placed shielded from light until they hatched. For this study, the larvae were cultivated on chicken feed. When they reached the pre-pupae stage, the pre-pupae migrated out of the chicken feed and were harvested. The pre-pupae were stored at -20°C until needed for fat extraction. Locusts and crickets were bought frozen from a local insect breeder (Desmedt Insects, Tessenderlo, Belgium). CHEMICALS AND FATS All chemicals, citric acid, sodium hydroxide, hexane, chloroform, propanol, diethyl ether, methanol acetic acid, phenolphthalein, Fuller’s earth, ethyl acetate, petroleum ether (40–65°C), NaCl, BF3, 2,2,4-trimethyl heptane, Na2SO4 used for the extraction, degum- ming, thin-layer chromatography (TLC), and gas chromatography–mass spectrometry (GC–MS) procedures were bought from Sigma-Aldrich (St. Louis, MO) and VWR Chemicals (Radnor, PA). Macadamia nut oil and a commercial vegetal oil mixture (a mixture of macadamia nut oil, cotton seed oil, and olive derived squalene) were purchased from IMCD Benelux BV (West-Knollendam, The Netherlands) and Clariant (Puget-sur-Argens CEDEX, France), respectively. EXTRACTION OF INSECT FATS Fats were extracted from all three insect species. The insects were dried, typically over- night at 65°C, and subsequently ground and sieved (maze 2.36 mm). A Soxhlet procedure using petroleum ether was applied to extract fats from small (15 g) insect samples. For preparation of larger volumes, the fats were extracted at room tem- perature in hexane. About 3 l of hexane was added to 1.5 kg of grinded insect material.

JOURNAL OF COSMETIC SCIENCE 190 The solution was incubated while stirring for 1 h and was then fi ltered using an air press fi lter, separating the hexane/fat mixture from the remaining solid fraction (consisting mainly of chitin and proteins). The petroleum ether (Soxhlet extraction) or hexane (large scale extraction) was subsequently removed (and recycled) by evaporation in a rotavapor device and the yield of raw fat was determined. FRACTIONATION OF BSF FATS BSF fat was fractionated according to a modifi cation of the method published by Kaluzny et al. (19). A 500-mg aminopropyl solid-phase extraction column (Chromabond NH2, Macherey-Nagel column 1) was washed twice with 2 ml hexane. A 10 mg sample of raw BSF fat was dissolved in 0.5 ml CHCl3 and applied to the solid-phase extraction column (SPE) column, and the CHCl3 was discarded. Four milliliters of CHCl3:2-propanol (2:1) was applied on the column and the elute was collected in Tube 1. Subsequently, 4 ml of 2% acetic acid in diethyl ether was applied on column 1 and the elute, which contains the free fatty acid (FFA) fraction, was captured in a separate tube. Column 1 was then eluted with 4 ml methanol and the elute, which contains the phospholipids, was collected in a separate tube. SPE column 1 was then discarded. The eluent in Tube 1 was then blow-dried with N2 gas and redissolved in 0.4 ml hexane. A second aminopropyl SPE column was prepared by washing it twice with 2 ml hexane and the redissolved eluent (Tube 1) was applied on the column. Four milliliters of hexane was applied. The eluent, which contains cholesteryl es- ters, was captured in a collection tube. Six milliliters of a 1% diethylether, 10% dichloro- methane in hexane solution was applied and the eluent, which contains triglycerides and cholesterol, was collected. The SPE column was then treated with 6 ml 5% ethyl acetate in hexane and the eluent (containing cholesterol) was collected. Diglycerides were collected by eluting the column with 4 ml 15% in hexane. Monoglycerides were eluted by applying 4 ml CHCl3:methanol (2:1). All collected fractions were dried under N2 gas and subsequently dissolved in 200 μl CHCl3 for visualization using TLC. TLC TLC was run on a 20 × 20 cm silica gel plate (ALUGRAM SIL G/UV254 Macherey-Nagel, Duren, Germany). Samples were loaded as individual spots separated by 1 and 1.5 cm from the bottom of the plate. Samples were loaded using an individual spot for the fat samples (obtained by dissolving 50 μl fat in 0.5 ml CHCl3) and applying three spots for the samples that were obtained by fractionation of the BSF fats. One hundred and two milliliters of running buffer (80 ml hexane:20 ml diethylether:2 ml acetic acid) was poured in a glass container and the loaded TLC plate was put in the tank and run until the front reached the top of the plate. The TLC plate was then put in a glass tank for I2-vapor visualization. REFINING OF INSECT FATS Raw fats still contain contaminants, such as phospholipids and FFAs, which limit in the shelf life. These contaminants should be removed by a degumming process. The proce- dure described here starts from 50 g of fats, but adapted volumes can be taken for other