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

INSECT FATS FOR COSMETICS 191 starting volumes of fat. Fifty g of fat was heated to 90°C and 15 ml of a 50 mM citric acid solution was added and incubated for 30 min (90°C) while stirring. The solution was centrifuged (5 min, 2,000 × g) and the oil phase was washed with 10 ml warm water fol- lowed by centrifugation (5 min, 2,000 × g) and retaining of the oil phase. The wash step was repeated once. The degummed fat was heated to 95°C while stirring and 10% NaOH was added to neutralize the FFAs for 45 min. The mixture was centrifuged for 10 min (2,000 × g) and the solution was washed twice with 6 ml demi water. Bleaching of the fats was done by adding 1% of Fuller’s earth and incubating at 95°C for 30 min. The fat fraction was removed by centrifugation (5 min, 2,000 × g). Finally the fats were deodorized by removal of the remaining solvents, aldehydes, ke- tones, and alcohols using a vacuum steam distillation, run for 3 h. ACID VALUE The acid value was determined by titration with phenolphthalein as an indicator. 0.25 g fat was dissolved in 50 ml ethanol and was titrated with a standardized NaOH solution. The acid value was subsequently calculated according to the formula [based on (20)]: FW 100 1.99 acidvalue q q q V Cq mq1,000 Here V is the volume of NaOH added to reach the infl ection point, C is the effective concentration of NaOH, and m is the mass of fat. FW is the approximate average molecu- lar weight of the fatty acid profi le of the insect (e.g., ±235 for BSF and ±275 for the cricket and the locust). 1.99 is a conversion factor. FATTY ACID PROFILE DETERMINATION WITH GC–MS The fatty acid composition was determined by GC coupled with MS using an Agilent 78020A. The fatty acids were fi rst converted to fatty acid methyl esters to make them volatile. 1.5 ml 0.5 M methanolic NaOH was added to 25 mg fat and the mixture was heated to 100°C for 5 min. The mixture was then cooled to room temperature, 2.4 ml of the cata- lyst BF3 was added, and the mixture was incubated at 100°C for 30 min. The mixture was cooled to room temperature, 1 ml isooctane was added, and the mixture was vor- texed. Five milliliter of a sodium chloride solution was added, the mixture was vortexed and subsequently centrifuged at 2,700 × g. The supernatant was collected and the pellet was washed twice with isooctane, followed by centrifugation and collection of the super- natant. The supernatant was dehydrated using Na2SO4, and after fi ltering, the solution was brought to 5 ml with isooctane. From this mixture, three dilutions (each containing an internal standard methyl heptadecanoate) in hexane were made for analysis by GC–MS. Quantifi cation was performed by comparing the chromatogram to standard curves de- rived for each fatty acid that was to be detected.