(i) DD dissolved in different solvents (3 : 1, w/v), like methanol and acetone (ii) Crystallization at −20°C, 24 h, mixture of acetone and methanol (4 : 1) (iii) Centrifugation, filtration, washing solvent: DD = 3 : 1 (v/v)
93% recovery of squalene from DD (2 washes) in filtrate; 79.5% recovery of total sterols in the cake
(i) Extraction of oil from flour in a Soxtec System HT6 with petroleum ether (40–60°C) (ii) Remove the unsaponifiables with a mixture of EtOH 95% : KOH 50% (30 : 5; v/v) (iii) Unsaponifiables extraction 5x petroleum ether; washing, drying, filtration, solvent removing (iv) Isolation of squalene from ultrafiltrate by column chromatography on a silica gel (24 g, 70–230 mesh, Sigma Co.) column
=>squalene content increased from 4.2% in crude oil to 43.3% in the unsaponifiables (i) 94% purity squalene (ii) 90% recovery
Extraction and isolation of minor components following the steps (a) Esterification of the oil with an alcohol (b) Collection of the esters phase from glycerol (c) Distillation of the ester phase (d) Dilution of the concentrate in a nonpolar solvent ( 0.2–50 barr) (e) Adsorption of the concentrate on an adsorbent, (f) Extraction of the minor components using a mixture of solvents (g) Desorption of the minor components by a mixture of solvents
(a) MeOH (c) Short path distillation at < 200°C, mmTorr => concentrate with 5% carotene, 0.5% tocols, 0.7% squalene, 0.7% sterols (d) Hexane (e) Normal-phase silica gel packed column (12 cm × 4 cm i.d.); feed/adsorbent = 23 g/kg (f) Hexane : isopropanol = 99.5 : 0.5, < 1 barr (g) First fraction collected is squalene Squalene recovery = 92%
Process comprising four steps: saponification, cracking and esterification of the fatty acids, and extraction by super and/or subcritical gases into a high pressure extraction column with independently temperable column regions
Animal source, like shark liver oil or an extract thereof
Method comprising steps (a) Purification distillation at (b) Denaturing distillation at , and are temperature to cause squalene boiling; < 140°C and ≥ 200°C (c) saponification of the composition (shark liver oil or extract thereof) prior to or after distillation stages
(a) [70°C, 100°C] [0.5 μmHg, 5 μmHg] composition with >95% squalene (b) [200°C, 300°C] [0.5 mmHg, 5 mmHg] composition with at least 99.9% squalene
Process for the simultaneous extraction of squalene, sterols, vitamin E () Esterification of FFA () Transesterification of the combined FA with the same short alcohol () Three successive distillations; third distillate contains squalene
() Alcohol C1–C3: fatty acids in a molar ratio >5 Acid catalyst : distillate = 1 : 10 (w/w) < 95°C () Alcohol C1–C3, basic catalysis, < 100°C () First distillation on a column with 20 theoretical plates mbarr–10 mbarr; = 160°–180°C Second distillation on a column with 10 theoretical plates, mbarr–30 mbarr, = 220–225°C Third distillation on a column with 10 theoretical plates; 1 mbarr–10 mbarr, 220–260°C
(i) Supercritical CO2 extraction with or without cosolvent EtOH (2% or 5%) (ii) Extract fractionation by gradual decrease of pressure yields (at ambient conditions) to the highest concentration of squalene
(i) At 55 MPa and 5% cosolvent: 0.289 g squalene/100 g seeds (ii) Up to 17.9 g SQ/100 g oil (iii) Tocopherols were also extracted in the same process
Extraction of phytosterols, squalene, vitamin E, following the steps (a) Conversion of crude palm oil in palm oil methyl esters (b) 3 short path distillations of PO methyl esters (c) Saponification of phytonutrients obtained in stage (b) (d) Crystallization of phytosterols (e) Solvent partitioning of vitamin E and squalene
(a) MeOH/EtOH with NaOH/KOH; (b) I short path distillation at : 70°C–120°C and : 1.33 Pa–6.67 Pa II short path distillation at : 130°C–200°C and < 1.133 Pa III short path distillation at < 120°C and < 1.133 Pa (c) 10% NaOH/KOH (d) unsaponifiable fraction obtained in step (c) mixed with hydrocarbon solvent/C1–C4 alcohol/H2O = 25/1/1/, heat at 65–85°C, cool slow at 25–30°C (e) mixing filtrate from (d) with heptane/hexane/i-octane and C1–C4 alcohol (5 : 3); squalene extraction from hydrocarbon layer and vitamin E extraction from alcohol layer; original CPO contains 250–730 ppm squalene, recovery by this process up to 97%
Isothermal countercurrent column, without reflux, at 343 K, : 150–230 bar, solvent : feed ratio = 13 : 1; A product with up to 90% (wt) squalene was obtained
() Esterification in supercritical MeOH () Extraction of squalene with SC-CO2 at 52.05°C, bar, extraction time 180 min A raffinate with 75% squalene was obtained
(i) Saponification of FFAs with glycerol and acid catalyst (Zn) (ii) Glycerol esterified product is subject to SC-CO2 extraction: °C, bar, 30 Kg CO2/Kg sample, countercurrent column (3 m × 30 mm, i.d.) with Sulzer stainless steel rings as packing material; extract with 83.7% squalene; extraction yield = 83.7% (iii) Column with temperature gradient SC-CO2extraction 50 : 40 : 30 → 90% squalene in extract; extraction yield = 91.1% (iv) Pilot plant scale experiments
(i) 50 mL extractor (320 mm × 13 mm, i.d.) with glass beads packing; (ii) SC-CO2 extraction at 3 mL/min for 90 min, at 200 bar and 50°C (iii) 438.16 ppm squalene
(i) Short path distillation unit (KDL4 Model, UIC Inc., Joliet, IL) (ii) degumming of crude oil by heating at 90°C, adding 2% (w/w) H2O, stirring, resting for 24 h, centrifugation at 18,900 g for 20 min; (iii) Alkali-refining of the degummed oil with 20% (w/w) caustic soda (iv) Best result for fractionation by short path distillation at 180°C and 3 mtorr: (i) squalene concentration in distillate increased sevenfold, with 76% recovery
Mutant yeast obtained by genetic engineering techniques
Supercritical CO2 solvent extraction
(i) Two-stage cultivation system for yeast: aerobic for biomass growth, anaerobic for squalene production (ii) Disruption of cells in a glass bed mill; (iii) Separation of squalene in two steps (a) Solvent extraction from lysate with CHCl3 : MeOH = 2 : 1 (v/v) (b) Lyophilization, followed by SC-CO2 extraction → 95% purity squalene
(i) Cells after anaerobic fermentation subject to lyophilization; (ii) SFE at = 60°C, = 250–255 bar, CO2 flow rate: 0.2 L/min (iii) Yield: 430.52 µg squalene/g dry weight cells
