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.

JOURNAL OF COSMETIC SCIENCE 192 FOURIER TRANSFORM INFRARED SPECTROSCOPY Infrared spectra of the fats were acquired on a Brüker ALPHA spectrometer (Brussels, Belgium) with a platinum ATR single refl ection diamond module. Each spectrum was the result of averaging 24 scans taken at a resolution of 4 cm-1. All spectra were measured at 21°C. CHALLENGE TEST The effi cacy of microbial preservation was tested according to the method described in the European Pharmacopoeia version 7.0 (21). The inoculum consisted of Pseudo- monas aeruginosa, Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus brasiliensis. VISCOSITY The viscosities of the oils were determined using 125 ml of the oils in 250 ml beakers at 25°C using a Brookfi eld (Elscolab, Kruibeke, Belgium) type RVDV-II + program- mable viscometer using spindle 3 at 60 rpm. Several other oils were measured as a reference. These were isopropyl myristate (BASF, Ludwigshafen, Germany), cyclo- pentasiloxane (Dow Corning Europe, Barry, UK), octyl stearate (BASF), C12–C15 alkyl benzoate (Innospec, Littleton, CO), decyl oleate (BASF), C8/C10 triglyceride (BASF), octyldodecanol (BASF), and dimethicone (BRB International, Ittervoort, The Netherlands). SPREADABILITY The spreadability of the oils was measured following the protocol by Dietz (22). Gelatin fi lms made from lime bone were kindly donated by Rousselot (Gent, Belgium). Five mil- liliter of oil was pipetted onto the fi lms and after 5 min the drop diameter was measured using a caliper. The reported values are an average of 4 or 8 measurements. FORMULATING OF HAND CREAMS The insect fats were formulated as 1%, 2%, 4%, 5%, and 10% fractions in a hand cream. The composition of the formulation and the origin of the used materials are given in Table I. The typical formulation contains 5% fat, and the variation in fat con- tent was compensated by adding more or less water in phase 2. The formulations with insect fats were compared with formulations containing 5% mink or macadamia nut oil. Macadamia nut oil was purchased from IMCD Benelux BV. The origin of the mink oil is proprietary information. The natural antioxidant sunfl ower seed oil extract of rosemary leaf (INCI Helianthus Annuus Seed Oil Rosmarinus Offi cinalis Leaf Extract) was obtained from Gattefossé.