CA2245114A1 - Non-chromatographic process for selective crystallization of vegetable steroids - Google Patents

Abstract

A process is provided for sequentially extracting lipids from unsaponifiable plant material. The process consists of dissolving the plant material in an aprotic solvent, then precipitating a first lipid, a phytosterol. A
second lipid, a tocol, is recovered from the supernatant by distillation to remove the aprotic solvent. The solvent can be reused to dissolve further plant material.

Description

CA 0224~114 1998-08-14 NON- HROMATOGRAPHIC PROCESS FOR SELECTIVE
CRYSTALLIZATION OF VEGETABLE STEROID
This invention relates tc extraction of lipids from plants, and specifically to extraction of sterols and tocols.
Backqround of the Invention Phytosterols are steroid ~olecules derived from plants. They are usecl as starting materials for synthetic steroid drugs and, in addition, have application in the manufacture of pharmaceuticals, nutriceu~ic~als, cosmetics and personal care products. Recent studies nave shown that phytosterol enriched ractions fron canola are particularly effective in ameliorating skin irr~tation caused by surfactants (I.oden, M. and Andersson, A-C. 1996. Brit. J.
Derm. 134:215--220). Being lipophi ic, phytosterols generally are extractable from plants along with other plant lipids such as glycerides and tocols (also known as tocopherols).
Plant tocols are forms o:- vitamin E which is well known as an anti-oxidant, preserva ive, free radical scavenger and dietary supplement. In mammal , vitamin E is a metabolic requirement for maintenance of skir quality and fertility and it has a well established commercial value. Natural plant tocols comprise a series of methyl~ted 4-hydroxyphenyl-alkadiene and ~omologous alkatrienes.
The -omposition of phytosterols and tocols differ from plant species to plant species. However, the physico-chemical properties cf these classes remain essentially the same across all plants.
In industrial refinin~ cf vegetable oil products, the phytosterol and tocol components are removed and CA 0224~114 1998-08-14 frequently are dlscarc1ed. In stanaard Nort,h American rapeseed and canola processing, these components are contained in what is referred tc as the "deodorizer c.istillat,e." This fraction is typically added to spent plant meal ~Thic~h is then disposed of as waste or occasionally finds applicat on as an ingredient in animal feed.
A common problem in commercial p~-oduction and utilization of phytost:erols and tocols has been the difficulty of obtaining reasonab:Ly pure class fractions of phytosterols (i.e. more or less tocol free) and tocol (_.e. more or less phytosterol f~ee) frorn gross plant mixtures.
U.S~ Patent 3,153,054 describes separation of drug-synthesis ste~ols frorn tocols in deodorizer sludges by tandem double liquid--liquid raction extra~tions using various polar and non-polar so~vent systems. U.S. patent, 3,335,154 describes esterificat:ion of glycerides as a preliminary step to the process of U.S. patent. 3,15~~,054. This process failed to produce either high yields or pure fractions of sterols.
U.S. Patent 4,610,100 describes a process of repeated crys~,allisat:ions of phytosterol m:Lxtures from petroleum ether, benzene or toluene which --esults in enrichment in stigmas_erol. Mother liquor~, produced a fraction enriched in sitosterol.
U.S. Patent 4,148,810 describes sterol isolations having relatively good yields which are produced by trans-esterification in met:hanol using calcium cihloride to obtain vitamin D3 substances. USSR Patent- 292,524 describes extraction of phytosterols in fair yield from salts of fatty CA 0224~114 1998-08-14 acids (i.e. na-ural soaps) by crys~allization from hot alcohol assisted by u:L~rasonic vibration.
Japanese patents 60/041694 and 92/007359 describe separation of sterols from deodorl7er distillates by mixed-solvent fractlonal crystallizatlon uslng centrifugal sedimentation and filtration. The solvent systems include alkanol-alkano.c acid mixtures and alkanol-ketone pairs. The process is specifically adapted fo~ small ,cale use and requires basket type centrifugal sedimenta_ion as an essential step in the process. The process ls not al~apted to large scale product'on.
Japanese patent 60/21569'3 describes separation of a brassicastero_-rich fraction by heating a rapeseed deodorizer distillate wit;h sodium or potassiurr~ hydroxide in methanol, ethanol, propanol or butanol. Japanese pa~ent 60/181098 describes ref ning sterol by reverse phase colurr~n chromatography The target product in both patents is iso-brassicasterol (3,5-cycloergost-22-en-6-ol).
The methods described in the prior art suffer from the disadvantage of being complex and difficult to adapt to a large manufact;ur~ng scale. Accord:ngly, there is a need for an inexpensive, reliable and simple method to obtain pure and colourless sterols and tocols in h:gh yield and on any desired scale.
SummarY of the Invention The ~nvention provides a process for sequentially extracting lip~ds from unsaponifiable plart material comprising extraction of a first l_pid in a first vessel by CA 0224~114 1998-08-14 (.) dissolvinc3 the un,aponiflable plarlt mG erial in an aprotic solvent to form a solution, (ilj cooling the solution until a precipitate of the fi:rst lipid is ~-ormed leaving a liquid supernatant, and (iii) recovering t-he prec.iFitate from the supernatant.
Optionally, the process comprlse, the additional step of extract::.ion a second lipid by (i) removal of the supernatant to a second vessel, (i.) distliling the supernatant to boil o:ff the aprotic solver~ leaving a residue of the second lipid and (iii) drying the re~sidue to remove any traces of the aprotic solvent.
In a prefer:red embodiment of the _nvention, the aprotic solvent used :is hexane. In a furt~ler preferred embodiment of the invention, the unsaponif:.able plant material used is derived from ~anola.
In a furthe:r embodiment of the invention, the hot aprotic solvent boiled off during ciistillation of the supernatant is reused to dissolve additional unsaponifiable plant material, thus producing a cycle which results in conservation of solvent and energy.
In a further embodiment of the invention, the process can be carried out simultaneously in several vessels, but synchronized at d:ifferent point.s in the process, in order to provide fo~ a cont:inuous process.
The process of the invent.ion prcvides the following advantages. First, it is uni.versally appl:Lcable in that it can be used wlt:h any lype of unsaponifiable plant material and, in particular, can be used wit.h deodo~-izer distillate CA 0224~114 1998-08-14 produced by refining of any vegeta}le 3'1 (eg.
rapeseed/canola, sunf:lower or soybean). Preferably, the distillate comprises ~ 12~ phytosterol. Second, the process is adapted to be compatible with st:andard operations and ec~uipment cur ently i31 use for processing and refining of ollseeds and other plant materials Third the process of the invention can be used on a small scale in the laboratory, on a pilot scale or on a large manufacturing scale. Fourth, the invention provides for improved yield, homogeneity and physical appearance of the sterol product.
Brief Description of I)rawinqs Figure 1 is a schematic cliagram :llustrating current methodology for refining vegetable ~il.
Figure 2 is a schematic cliagram ;llustrating the process of the invent:Lon.
Detailed Description The present invention provides a new and improved method for extracting phytosterols and tocols from mixtures of unsaponifiable plant material using aprotic solvents. The process can be applied to mixtures of unsaponifiable materials from any plant, for example, rapeseed/canola, sunflower, soybean. It lS particularly well adapted to be applied to deodorizer distillate, a byproduct of stanclard vegetable oil processing and refining.
The process is effected by dissolving a mixture of unsaponifiable plant material in an aprotic solvent that may be hot. In a preferred embodiment of the invention, the solvent used is hexane, commercial mixtures of isomeric CA 0224~114 1998-08-14 hexane, or anv of the hexane-like clkanes, such as pentanes, heptanes or octanes. Use of pentare may result in less efficient deposition of sterols due tc the smaller temperature range availab~e. Higher homologues of hexane function well but are more expensive and require mor-e energy and higher temperatures for solvent distillati~n and ~ecovery. Octanes including fuel-grade octanes give satisfactcry results.
However, hexane has the double advantage o- performing very well and of be;ng wel:L suited to the infrastructure of most existing oilseed processing facilities, wh:ich already use hexane for ex.raction of plant mea .
The amount of solvent used will affect both the yield and the purity of the phytosterol extract. A ratio of 1:7 (starting material:solvent) produces good yield and purity. This ratio i, convenient for small scale processing but can be impractical on a large scale wi h respect to the volume of hexane required. Lower volumes of solvent result in a less pure in t~al product as demonstrate~ by a yellow colour and an oily cons_sten~y (however, the puri~y of this initial product can be improved by simple recrystallization).
However, if recovery of a tocol is not a priority, sterol extraction can be performed at a ratio of starting material to solvent, preferably hexane, of ~:1 to 3:1. At these ratios, the extraction is preferably carried out at ambient temperature.
Filt~ation of precipitated sterols (which do not dissolve appreciably in this amount of hexane without heating) is easy, and the crude sterol product can be brought to CA 0224~114 1998-08-14 acceptable purity (al~ays above 85~ and usLIally above 90%) by simple hexane-washing in the filte~ bed. ~lternately, the crude product may be washed with ccld hexane in the bed and then recrystallized from hexane or other sc~lvents, such as heptane, methanol, ethanol, propancl, acet~ne or ethyl acetate, after remova: from the bec.. This provldes sterol content in the range of 94-98%. Or-, the crude sterol may be removed from the filter bed, washec.~ thoroughly with cold hexanes, and ther re-i--iltered.
The extract:Lon of sterol from distillates can be performed at ambient t:emperatures ~20-25~C', provided that sterol hydrat~cn is used. Crude precipi.a~:ed sterols can then be filtered o_f or centrifuged out direc~ within 30-60 minutes of the blending-in of water for hyciration (discussed below). No heating o; any component is required, so that effectively the hot stage of the thermal cvcle is eliminated in this option. There is no recrystallizat:ion effect, but nonetheless the recovered sterols may be of- high enough purity for certain purposes. Given a filter-bed wash of the crude product, sterol contents above 85% ~an be realized consistently at tonne scale.
Best yields are obtained, up to 8C% in some instances, by retaining a cold cyc e. In the just-mentioned "no-heat" ext~action option, the crude mixture is cooled to as low as 2~C bef-ore fillration to recover sterols. The filtrate can be recycled back into subsequent extractions as in the originally conceived process, or preferably hexane can be stripped off and recycled for that purpose. This latter CA 0224~114 1998-08-14 option is useflll where it is desir~d to reccver the non-sterol components of the distillate, in particula- tocopherols with Vitamin E activity.
Cool ng to temperatures ~ower than 2~ can be disadvantageous because free fatty aclds can begin to co-precipitate with the ,terols. Canola seed Gil is very high (up to 90%) in oleic acid (melting point 1:3CC), so in that application the co-precipitate is mostly c:eic acid. Cooling to about 5~C s prefe:rred and prov des a reasonable balance between yield and pur:ity. The cooling staqe can be eliminated altogether in the interests of faster (and therefore cheaper) processing, or for other reasons.
In summary, the process may be carried out using "hot-stage" extraction as originally descr:bed, with or without cooling of the precipitatecl crude product mixture to below ambient temperature before filt~ation. The extraction step may also be carr:ied out at am~ient-temperature with or without pre-flltratioIl cooling. A variely of hexane:distillate ratios may be used. The invention allows for these options without affecting the option for recovery and recycling of hexane used.
It lS preferable that step a~ of the process of the invention is carried out in the presence of- water at 1 to 5%
by weight of the unsaponifiable plant mate~ial, more preferably 2 to 4% by weight. In practice, about 3% water by weight is routinely added. The water leve may be adjusted in the plant material pr:lor to extraction or water may be added to the extraction vessel at the time of processing. The ~ 76451-7 CA 0224jll4 1998-08-14 latter is preierred for reason of easier handling. In either case, vigorous blending is important in achieving an even and rapid distribution of the water through the mass of material.
Added water hydrates the sterol molecules quite rapidly (within 10-15 minutes), and these hydrated forms have reduced solubility in lipophilic media and therefore precipitate readily in the overall distillate matrix or in hexane. In fact, the fully hydrated sterols will barely dissolve in 10-12 volumes of boiling hexane. Hydration increases the relative hexane-solu'cility difference between sterols and other components of the distil]ate, making their separation easier. It also decreases in aosolute terms the solubility of sterols in the other main components of the distillate (i.e., oleic and related fatty acids).
The hydration reaction is moderately rapid, requires no heating, and does not liberate significant amounts of heat.
In the current procedure, 100 partC distillate (of low or ~zero" water content) and a quantity of hexane are stirred rapidly while Z-4 part:s of water are added. After a few minutes, the hexane solution/suspen.sion, which is initially clear or somewhat turbid, becomes opaque, and copious sterol particles appear. Aft:er 15-30 minutes the mixture resolves into precipitated sterol solids anc hexane--soluble supernatant liquid. These phases can be conveniently separated by filtration, by centriiugation, or by decant:ation in cases where a high hexane-d,stillate ratio allows this. Filtration is preferable in that ln situ washing of the sterols with cold hexane can convenient.y be carried out.

CA 0224~114 1998-08-14 The result~ng solution is cooled, whereupon most of the component pAytosterols are deposited as precipitated solids on the walls of the vessel. Deposition of phytosterols can be effected more rapidly and thoroughly if the solution is chilled or seeded with phytosterol crystalc. The deposited phytosterols can be filtered off or, alterrately, the supernatant can be decanted or drained o f to another vessel, leaving the phytosterols behind. Phytoste ols can then be recovered by scraping the sides of ~he vessel. The crystalline phytosterols can be washed wit~l cold solvent and/or recrystallized from hexane cr other organic solvents.
Once the phytosterols ar~ precipitated out of solution, the supernat:ant solution contains principally a mixture of alpha and gamma tocols, both of which are recognized as common iorms of vitamin ~. ~'hese tocols can be freed from the solvent by simple distillat:on, followed by a further period under :Low vacuum to remove the last traces of solvent. The resulting residue comprises a fraction rich in tocols and reduced in phytosterols.
The warm solvent vapour producec by distillation can be directed back to the first vessel to dissolve the next batch of deodorizer distillate thus creating a cycle of cooling, phytosterol ~eposition and dra n-off. In this way, solvent can be recycled and energy can be ~onserved.
The composition of the phytosterol and tocol fractions obta ned will vary according to the compositions found in the particular plant source used. Nevertheless, use of the aprotic solvent and the mild separation conditions (no CA 0224~114 1998-08-14 acids, alkaliec, or high temperatures) c~f the invention produces a phytostero:L fraction of a higher purity level that can be produced by processes known in the art. The process of the invention is capable of producing phytosterol fractions with >98% purlty. Fu:-thermore, the process of the invention produces phytosterol :Eractions hav ng a light colour.
The sterol content of "crude" product from the full hot and cold cycle described is typically above.90% and typical yields over many trials have varied from 40-70% of theoretical, with the average beina near 60%. From hydrated, ambient-tempe-ature extractions, crude product has been obtained in yields of 60-80%, with sterol purities above 85%.
Indications to date suggest that the proce,s works progressively better at larger sca'e. Trial processing of 150 kg and 1000 kg confinn this point.
Crude sterols from the process carl be recrystallized to very high purlty by means already well i~nown in the art.
Methanol and ethanol may be used as recrystallizing solvents with good success, hol~ever, hexane is the preferred solvent.
Heptane may a]so be used. Air-dried final manufactured product consistently ,~ssays under ~0 ppm hexane, thus passing "food grade" requirements. A sing:Le recry,tallization of "85%
grade" materia' using 10-12 volumes of hex~ne gives recoveries of 80-90% and a sterol content of about 94-98%. The profile of the three major sterol molecules in canola product, brassicasterol, sitosterol and campesterol, is not particularly affected by process variations or by recrystallizing from hexane. Recrystalliz~tion from alcohols CA 0224~114 1998-08-14 can effect procressive enrichments of some components.
In a further embodiment of the invention, several vessels for extractin~ phytosterols can be operated in parallel, with each vessel synchror.ized at a different stage of the heating, cooling or draw-off cycle and with each being attached to a corresponding vessel for ext~-acting tocols.
This parallel, synchronized system allows f-or continuous operations and also provides back-up capacity for each step of the overall prccess.
The process of the invention can be used independently as a stand alone manufacturing process or it can be integrated into a larger process of vegetable oil extraction. The invention is well adapted to be compatible with existing oilseed refinery operations, especially where the aprotic solvent u.c,ed is hexane.
The method of the invention can be used on a small scale in the laboratory (1-500g starting material), on a pilot scale (500g-200kg starting material) or on a large manufacturing s-ale (200-4000kg).
Example 1 Deodorizer distillate (2.4g) fro~ canola was dissolved in 20ml of hexane heated to apprcximately 60~C. The solution was refrigerated for 48 hours ther~ _iltered, washed with a little cold hexane and freed of solvent by air evaporation for 12 hours. Phytosterols were deposited on the sides and the floor of the containe:r vessel as a white crystalline sol-id.
The cold solution was dra:ined or decanted into a CA 0224~114 1998-08-14 second vessel where it was reheated unti~ Ost of the solvent hexane was boiled off. The resulting resi~nue was subjected to a low vacuum tc remove the last traces of hexane.
Analysis of the resulting phytosterol fraction lndicated that it had 97~ purity. The phytosterol was white and had a scarcely det:ectable odour. Yiell was approximately 5G-60% of the theoretical phytosterol content of the starting material. The isolated phytostercl fraction had the following composition:
beta-sitosterol 47%
brassicasterol 29%
campesterol 22%
stigmasterol 1%
Example 2 The phytosterol extraction from Example 1 was repeated on a larger scale. 54.3g ~f deodorizer distillate was dissolved n 380 rnl of hot hexane, coc:ed and refrigerated for 48 hours. The resulting phytosterol precipitate was f ltered and dried. The extracted phytosterol demonslrated a white colour.
ExamPle 3 200g of deodorizer disti late from canola, from a different lot than used in Example 1 or 2, with 18~
phytosterol content was dissolved :n 1200g of hexane at about 50-60~C. The solution was then cooled at about 2~C for 16 hours. The so_ution was then filtered under vacuum, and washed with a small arnount (200g) of hexane to give 2.8g of white crystal'lne phytosterol material; about 8% of CA 0224~114 1998-08-14 theoretical yield.
ExamPle 4 97g of deodorizer distillate from sanola (from the same batch used in Example 3 was dissolved in 146g of hexane at room temperature. To this mixture was added 3g of water and a precipitate was observed immediately. The mixture was then cooled for about 12 hours, filtered, and the resulting crystalline material washed with 290g of cc,ld hexane. The residual solvent was t:hen removed giving l g of phytosterol material of 89~ purity; 65% of the theoretical yield.
ExamPle 5 A p_lot scale trial was performeci wherein llOOkg of deodorizer distillate from canola was dissolved in 1650kg of hexane at roorr temperature. To this mixture was added 22kg of water with stirring. A precipitate as observed within 5 minutes. The resulting mixture was then f ltered and washed with about 450kg of cold hexane. I'he residual solvent was removed giving 93kg of phytosterol materia] of 86% purity. A
small sample of this rnaterial washed with an additional amount of hexane assayed a~ :,95% purity.
All material from Examples 3, 4 and 5 had the following sterol profile:
beta-sitosterol46%
brassicasterol24%
campesterol 29%
stigmasterol 1%.

Claims (13)

1. A process for extracting lipids from unsaponifiable plant material comprising (a) extraction of a first lipid in a first vessel by (i) dissolving the unsaponifiable plant material in an aprotic solvent to form a solution, (ii) cooling the solution until a precipitate of the first lipid is formed leaving a liquid supernatant and (iii) recovering the precipitate from the supernatant.

2. The process according to claim 1 or claim 2, comprising an additional step (b) extraction of a second lipid by (i) removal of the supernatant to a second vessel, (ii) distilling the supernatant to boil off the aprotic solvent, leaving a residue of the second lipid and (iii) drying the residue to remove any traces of the aprotic solvent.

3. The process according to claim 1 or 2, wherein the first lipid is a sterol.

4. The process according to claim 2, wherein the second lipid is a tocol.

5. The process according to any one of claims 1 to 4, wherein the aprotic solvent is an alkane selected from the group consisting of pentane, isopentane, hexane, isohexane, heptane and octane.

6. The process according to claim 5, wherein the aprotic solvent is hexane.

7. The process according to any one of claims 1 to 6, wherein step (a)(i) is carried out at ambient temperature.

8. The process according to any one of claims 1 to 7, wherein the plant material is derived from any one of rapeseed, canola, corn, sunflower, palm and soybean.

9. The process according to claim 8, wherein the plant material is derived from canola.

10. The process according to any one of claims 1 to 9, wherein water is present in the first vessel in an amount of about 1 to 5% by weight of the unsaponifiable plant material.

11. The process according to claim 10, wherein the amount of water is 2 to 4% by weight of unsaponifiable plant material.

12. The process according to any one of claims 2 to 11 comprising an additional step (c) reusing the boiled off aprotic solvent from step (b) to dissolve unsaponifiable plant material.

13. A continuous process for sequentially extracting lipids from unsaponifiable plant material comprising carrying out the process of any one of claims 1 to 12 in more than one vessel simultaneously and synchronizing said processes such that at any point in time, the process in each vessel is at a different step.