Title of Invention

A PROCESS FOR PREPARING VEGETABLE OIL FRACTIONS RICH IN NON-TOCOLIC, HIGH -MELTING, UNSAPONIFIABLE MATTER

Abstract 1. A process for preparing a vegetable oil fraction rich in non-tocolic, high-melting, unsaponifiable matter which comprises the following steps: a) a vegetable oil, or a fraction thereof, having a slip melting point of not more than 30° C and a content of unsaponifiable matter of at least 0.5 % by weight is hydrogenated to fully saturate the fatty acids of the glycerides and to reach a slip melting point of at least 57 °C; b) to the hydrogenated oil is added from 1 to 75 % by weight of the unhydrogenated starting oil or another oil having a slip melting point of max. 30°C to act as a carrier and vehicle for the unsaponifiable matter; c) to the oil mixture from b) is added a solvent in a ratio from 1:2 to 1:20, and the mixture is heated to transparency; d) the mixture from c) is cooled in one or more steps to a final temperature in the range from -35 to +30°C, and the precipitated high-melting fraction(s) is (are) filtered off; e) the filtrate obtained from d) is desolventised, leaving a. fraction rich in unsaponifiable matter.
Full Text FORM 2
THE PATENTS ACT 1970
[39 OF 1970]
&
THE PATENTS (AMENDMENT) RULES 2006
COMPLETE SPECIFICATION
[See Section 10; rule 13]
"A PROCESS FOR PREPARING VEGETABLE OIL FRACTIONS RICH IN NON-TOCOLIC, HIGH-MELTING, UNSAPONIFIABLE
MATTER"
AARHUSKARLSHAMN DENMARK A/S, M.P. Brunns Gade 27, DK-8000 Arhus C, Denmark,
The following specification particularly describes the nature of the invention and the manner in which it is to be performed:-
GRANTED
2 APR 2007

A process for preparing vegetable oil fractions rich in
non-tocolic, high-melting, unsaponifiable matter
FRACTIONATION PROCESS
The present invention refers to a new process for the 5 fractionation of vegetable oils and fats which gives a fraction highly enriched in non-tocolic, high-melting, unsaponifiable matter. Basically the process does not generate trans-fatty acids. The oil fractions can be tai¬lored to specific applications as claimed.
10 BACKGROUND
Vegetable oils and fats are mainly triglycerides and other saponifiable matter such as mono and diglycerides and traces of free fatty acids. Besides these substances they have a varying content and composition of unsaponi-15 fiable constituents.
The unsaponifiable matter (or unsaponifiables) is the material which can be extracted by petroleum ether or other similar solvent after alkaline hydrolysis of a sample. The unsaponifiable constituents and the relative 20 composition are typical for the individual oil. A large number are biologically active components, and the following listing is not exhaustive, but it reflects their importance when it comes to documented functionality, as well as their main sources:
25 1. Tocols
The constituents are tocopherols and tocotrienols.
Tocopherols are present in practically all vegetable oils. Soybean, corn, sunflower and rapeseed oils are the main commercial sources. Tocotrienols are mainly present


in palm, rice bran, barley and wheat germ oil. Commercial quantities of tocotrienols are sourced from palm and rice bran oil.
Tocopherols are nature's major lipid-soluble antioxidant 5 and are often referred to as natural vitamin E (d-a-toco-pherol has the highest biopotency, and its activity is the standard against which all the others are compared). Vitamin E is an in vivo antioxidant protecting cell mem¬branes against the damaging effects of free radicals. The 10 application as a supplement and an additive in food and feed is well established. The vast data available also point to its importance in formulating functional c.smet-ics.
Tocotrienols are related to tocopherols and are consid-15 ered to be powerful antioxidants. Some studies suggest that the antioxidant potential is even greater than that of the tocopherols. Topical application of tocotrienols has also been found helpful in fighting oxidative damage to the skin while preserving the existing vitamin E in 20 skin cells.
2. Phytosterols
The major constituents are (3-sitosterol, campesterol and stigmasterol. Normally they account for more than 70% relative of the sterol fraction. Minor, normally occur-25 ring sterols are: stigmastenol, avenasterols and choles¬terol.
Sterols are present in almost all vegetable oils. Commer cial quantities are mainly sourced from soybean oil. Se¬lection criterions are availability, total content and 30 composition. As an example, rapeseed oil would be a good choice if brassicasterol was wanted as a constituent.

Topical application of a preparation that contains phy¬tosterols results in an increase in both moisture in the skin and lipid content on the skin. The phytosterols ad¬hering to the surface of the skin impart a hydrophobic surface, which is not reversed after exposure to water and soap. There are several studies reported in litera¬ture on the beneficial effect of sterols in restoring skin irritation which originates from external sources, i.e. UV-radiation, detergents, wet nappy exposure, etc.
Nutritional compositions containing phytosterols, mainly β-sitosterol and stannol esters, are reported to have a cholesterol-lowering action. The mechanism is not fully understood, but the plant sterols appear to inhibit the uptake of cholesterol from the gut. The effect is that both total and LDL cholesterol level in the blood are re¬duced.
Sterols are furthermore useful in the pharmaceutical in¬dustry for conversion into steroid derivatives.
3. Triterpene Alcohols (TTPA)
The major constituents are a- and β-amyrin, butyrospermol and lupeol. They are mainly present in the form of cinna-mic, acetic and fatty acid esters.
The main source is shea butter, but TTPAs are also pre¬sent in illipe, sal and shorea butter.
For centuries, shea butter has been used traditionally on the African continent for its outstanding properties in protecting and restoring the skin. This is especially at¬tributed to the unsaponifiable matter. A number of tests on human volunteers have proven a series of effects on various kinds of skin problems: cicatrising action in treatment of chapping, restoring dermatitis and erythema

from sunburn, etc. In animal tests the butyrospermol fraction is reported to have a cortisone-like action on oral and parenteral administration.
4. y-Oryzanol
5 y-Oryzanol is generally assigned to ferulic acid esters of steroid moieties. The main steroids are β-sitosterol, campesterol, cycloartenol and 24-methylcycloartenol.
y-Oryzanol is found in rice germ and rice bran oil. It is reported to have growth-accelerating effect on animals. 10 Topical application of an ointment which contains y-oryz-anol stimulates the blood flow in the peripheral veins. In tests on human volunteers it is reported to have a cholesterol-lowering effect on both total and LDL choles¬terol level.
15 5. Carotenoids
Carotenoids are mainly present in palm oil. The oil of Elaeis oleifera has the highest content of mixed natural carotenoids.
The major constituents are a- and p-carotene.
20 P-carotene is providing vitamin A activity. It is report¬ed to possess anti-cancer properties for certain types of cancer, but in some investigations ά-carotene is found to be more potent. It is also worth noting that other caro¬tenoids present in palm oil, phytolene and especially
25 lycopene, have been reported to possess even better anti¬cancer properties.
The mixed natural carotenoids can furthermore be used as a natural colouring agent in various applications.

Besides the classes of unsaponifiables listed above, there are many other types of interest where the sourcing has to be evaluated according to the relative presence and total concentration in the oil. This is illustrated 5 by the following examples: Olive oil has an interesting content of the triterpene polyunsaturated aliphatic hy¬drocarbon, squalene. In avocado oil approx. 50% of the unsaponifiable matter consists of alkylfuranes. Sesame seed oil contains sesamolin and sesamin.
10 All in all, this clearly demonstrates the need for indus¬trial processes tailored to enrich and standardise these valuable substances making it possible to direct and dose them in a proper manner for the application in question.
STATE OF THE ART
15 In general the concentration of unsaponifiable matter is low in the raw material. Consequently, industrial fatty residues, normally considered as waste, are in use as starting material. An example of this is the production of phytosterols (mainly sitosterols) based on wood-deriv-
20 ed by-products .
By the processing of vegetable oils and fats huge quanti¬ties of waste are generated i.e. soap stock by the alka¬line refining method and deodoriser distillate. Deodor¬iser distillate is of particular interest as it contains
25 10-30 % unsaponifiable matter of which approximately half is tocopherols and sterols. The separation and purifica¬tion of the tocopherols and sterols is a complex and ex¬pensive process involving physical and chemical methods. The commercial products available are based mainly on de
30 odoriser distillate from soybean oil.

Vegetable oils are used as the starting material in a process that involves either saponification of the oil or conversion of the glycerides into fatty acid methyl es¬ters followed by extraction and purification of the unsa-5 ponifiable matter. The commercial products are tocophe-rol/tocotrienol mixtures and natural mixed carotenoids based on rice bran or palm oil.
The processes described so far are in use on an indus¬trial scale. The following processes are directly appli-10 cable to oils and fats and mainly related to sheanut oil as the starting material. Sheanut oil unsaponifiables have interesting, documented dermatological properties, and the oil has a beneficial high non-glyceride content of approx. 6 %.
15 In WO 96/03137 it is illustrated that a sheanut oil frac¬tion with 10 % unsaponifiable matter can be enriched to 50% by short-path distillation.
By applying supercritical CO2 extraction on sheanut oil it is experimentally demonstrated that a selective frac-20 tionation of the constituents is possible at temperatures of 40-80 °C and pressures of 100-400 bar (Turpin et al. 1990. Fat Sci. Technol. 92 (5), 179-184).
Another method for the production of lipid fractions en¬riched in unsaponifiables is by fractional crystallisa-25 tion from solvents .
It is experimentally demonstrated that tocopherol concen¬trates can be obtained from vegetable oils by almost com¬pletely removing the glycerides and sterols by fractional crystallisation from solvents at low temperatures. As an 30 example, partially hydrogenated cottonseed oil yielded a concentrate containing 32.1% tocopherols by crystallisa-

tion of the oil from 8 times its own weight of acetone at -74 °C. Acetone proved to be the most suitable solvent (Singleton and Bailey 1944. Oil & Soap 21, 224-226).
EP 069004 teaches how to prepare two fractions rich in unsaponifiable matter from shea butter. The fat is treated with a polar solvent of the ketone type in order to recover one fraction insoluble in the hot ketone sol¬vent (polyisoprenic hydrocarbons), The hot ketone solvent is cooled to a temperature below 0 °C to crystallise the hot soluble material, which is filtered off. The filtrate is desolventised giving a second fraction rich in unsapo¬nifiable matter (sterols and TTPA-esters). The preferred solvent is acetone. The two fractions are mixed at least in part or preferably in total to give a mixture claimed to contain 18-50% by weight of the unsaponifiable matter especially useful in dermatological compositions.
WO 99/63031 teaches how to fractionate a vegetable oil which gives one or more solid fractions suitable for con¬fectionery applications as well as a liquid fraction en¬riched in unsaponifiable matter. The oil to be fraction¬ated must have a slip melting point of 32-55 °C and, if necessary, the oil is partly hydrogenated to reach said melting interval. In the description page 9, line 2 from the bottom, it is stated that if the slip melting point of the oil is more than 55 °C, the amount of liquid to solubilise the desired components will be too small and they will be precipitating together with the solid triglycerides, and further, that the recovery of the small amount of the liquid fraction will also be more difficult than if a lower slip melting point is used. The fat is mixed with a solvent (acetone is preferred) and heated to transparency and cooled to precipitate a solid fraction. The filtrate is desolventised to give a liquid

fraction in which the unsaponifiable matter is enriched. The process is exemplified in a rapeseed fraction claimed to contain 1.5-5% unsaponifiable components, useful as an ingredient in cosmetic and pharmaceutical preparations 5 for providing moisturising, UV-protecting and anti¬inflammatory properties. The process is further exempli¬fied in a shea butter fraction claimed to contain 15-36% unsaponifiable components, useful as an ingredient in cosmetic and pharmaceutical preparations for providing 10 UV-protection and skin moisturising properties.
To summarise the following can be stated:
The use of waste material, especially the deodoriser dis¬tillate, has proven that it is possible to produce toco¬pherols and phytosterols of high purity. The process is 15 complex and expensive but driven by the high prices of tocopherols. The main problem is the sourcing of the starting material, as vegetable oil production is scat¬tered.
The extraction and purification via the methylester or 20 saponification route is expensive and leaves high amounts of cheap by-products i.e. fatty acids, glycerine and methyl esters. The commercial products have a content of 30-50% unsaponifiable matter.
The direct processing from oils and fats by short-path 25 distillation and supercritical fluid extraction is at present on an experimental stage.
The use of dry or solvent fractional crystallisation has limitations. If the concentration of unsaponifiable mat¬ter is low in the oil, the.resulting enrichment is also 30 low. Furthermore, if the starting material is liquid, it has to be partly hydrogenated before fractionation. The

trans-fatty acids formed during partial hydrogenation of
the unsaturated fatty acids in the glycerides may in¬crease the blood cholesterol level and the risk of coro¬nary heart disease if eaten. This makes the enriched 5 fractions unsuited for the fortification of food and as a nutritional supplement or a pharmaceutical or pharma-like product for oral administration.
OBJECT OF THE INVENTION
The invention aims at providing a process for preparing 10 oil fractions highly enriched in non-tocolic unsaponifi-able matter by which the enrichment of the higher melting unsaponifiables (e.g. phytosterols, triterpene alcohols and esters, y-oryzanol, carotenoids, etc.) and the compo¬sition of the glyceridic part of the oil fraction can be 15 tailored to the specific application. Basically the proc¬ess should not generate trans-fatty acids.
SUMMARY OF THE INVENTION
This is achieved by the process of the invention which comprises the following steps:
'.0 a) a vegetable oil, or a fraction thereof, having a
slip melting point of not more than 30 °C and a con¬tent of unsaponifiable matter of at least 0.5 % by weight is hydrogenated to fully saturate the fatty acids of the glycerides and to reach a slip melting
5 point of at least 57 °C; -
b) to the hydrogenated oil is added from 1 to 75 % by weight of the unhydrogenated starting oil or another oil having a slip melting point of max. 30 °C to act as a carrier and vehicle for the unsaponifiable mat- ter;

c) to the oil mixture from b) is added a solvent in a
ratio from 1:2 to 1:20, and the mixture is heated
transparency;
d) the mixture from c) is cooled in one or more steps
5 to a final temperature in the range from -35 to +3
°C, and the precipitated high-melting fraction(s) (are) filtered off;
e) the filtrate obtained from d) is desolventised,
leaving a fraction rich in unsaponifiable matter.
10 By the hydrogenation in step a) besides the unsaturated fatty acid moieties also some of the unsaponifiables cor taining double bonds are converted to the corresponding hydrogenated forms; especially in triterpene alcohols double bonds in side chains will be hydrogenated.
15 Often it is desirable to conduct step b) by adding from to 75 % by weight, preferably from 2 to 50 % by weight, and more preferably from 5 to 25 % by weight, of the un-hydrogenated starting oil as a carrier to the hydrogen¬ated oil from step a). This makes it possible to adjust
20 the content of unsaponifiable matter in the resulting en riched fraction and, at the same time, to keep the trans fatty acid content of the enriched fraction at maximum 2 %.
Another way of conducting step b) is by adding from 1 to 25 75 % by weight, preferably from 2 to 50 % by weight, and more preferably from 5 to 25 % by weight, of a triglyc¬eride oil different from the unhydrogenated starting oil as a carrier to the hydrogenated oil from step a), said triglyceride oil predominantly consisting of triglyc-30 erides of saturated and unsaturated C8-C22 fatty acids, and preferably being a vegetable oil predominantly con-

sisting of triglycerides of saturated and unsaturated C16-C22 fatty acids. Hereby, it is possible to select a carrier oil having specific beneficial effects in combi¬nation with the unsaponifiable matter for the intended 5 use.
Further, it may be desirable that the starting oil con¬taining the unsaponifiable matter is interesterified be¬fore the hydrogenation step and/or that the resulting oil mixture is interesterified before the fractionation steps 10 c) and d)
Normally, the solvent used in the fractionation steps c) and d) is of a non-polar or semi-polar type, e.g. select¬ed from the group consisting of hydrocarbons and linear and branched alkanols of 1-5 carbon atoms; and it is 15 preferably hexane or petroleum ether.
Suitably, the starting vegetable oil containing the unsa¬ponifiable matter originates from one of the following or their hybrids: rapeseed, canola, soybean, corn, maize-germ, sunflower, flax (low-linolenic linseed), mango, 20 avocado, olive, sesame, rice bran, wheat germ, oat and oat bran, palm, sal, shorea, illipe and shea, as well as any fraction or mixture thereof.
A starting oil of particular interest is a dekaritenised, lower-melting fraction of shea butter enriched in unsapo-25 nifiable matter, which is obtained as follows:
The crude shea butter is deacidified and subsequently partly dekaritenised by mixing it with a semi-polar solvent to precipitate the major part of the polyiso-prenic hydrocarbons (karitene) which are disposed of; 30 the solvent is distilled off and the oil is mixed with a suitable solvent e.g. hexane (ratio approx. 1:2 to

1:4), heated to transparency, and cooled to a tempera¬ture at which the high-melting glycerides precipitate the precipitated fraction is filtered off and the fil¬trate desolventised leaving a lower-melting fraction 5 with a slip melting point of max. 30 °C suitable for further processing.
Generally, the vegetable oil fraction enriched in unsapo-nifiable matter obtained by the process of the invention may be subjected to further concentration by means of su-
10 percritic carbondioxide, molecular distillation, chroma¬tography or recrystallisation and/or to chemical modifi¬cation such as hydrogenation, ethoxylation, esterifica-tion or interesterification without leaving the inventive concept. Also, the physical state of the vegetable oil
15 fraction obtained may further be changed e.g. by emulsi-fication, admixing with a high-melting fat and spray cooling, encapsulation or incorporation in liposomes or nanosomes.
The present invention also concerns a vegetable oil frac-20 tion obtainable by the process according to the inven¬tion, said fraction being enriched in non-tocolic, high-melting, unsaponifiable matter by a factor of at least 3 and having a trans-fatty acid content of maximum 2 % by weight.
25 In such vegetable oil fraction the unsaponifiable matter is predominantly comprised of or derived from:
- carotenoids;
- sesamin and sesamolin;
- y-oryzanol;
30 - sterols, methyl sterols and dimethyl sterols;
- triterpene alcohols and their esters with cinnamic
acid, acetic acid and fatty acids.

A particular enriched fraction of shea butter obtainable by the process according to the invention has a content of at least 30 % by weight of non-tocolic, high-melting, unsaponifiable matter and is further characterised by 5 containing hydrogenated triterpene alcohols and/or their esters and by having a trans-fatty acid content of maxi¬mum 2 % by weight. Preferably, such shea butter fraction has a content of 40-90 % by weight of unsaponifiable mat¬ter. Further, it may be desirable that the preparation of 10 such shea butter fraction has involved at least one in-teresterification step before the fractionation steps c) and d).
The present invention further comprises the use of a vegetable oil fraction according to the invention for the 15 fortification of food products.
Thus, the invention comprises the use of a vegetable oil fraction according to the invention as a constituent of a dairy or dairy-like food product selected from the group consisting of milk, cream, ice cream, butter, cheese (in-20 Cluding soft, cream and processed cheeses), yoghurt and other fermented milk products.
It also comprises the use of a vegetable oil fraction ac¬cording to the invention as a constituent of a food prod¬uct selected from the group consisting of margarines, 25 butters and blends, spreads, mayonnaise, shortenings, dressings and salad oils at a concentration of 2-50% by weight.
Further, the invention comprises the use of a vegetable oil fraction according to the invention as an ingredient 30 in cosmetics, pharmaceuticals or pharma-like products for topical application or as an ingredient in nutritional

supplements, pharmaceuticals or pharma-like products for
oral administration.
In particular, the invention comprises the use of a vege¬table oil fraction according to the invention as an in-5 gredient in a nutritional supplement, a pharmaceutical or a pharma-like product for oral administration to humans and other mammals with the purpose of lowering the blood cholesterol level. Specifically, it may be used as an ad¬ditive to fat-containing food products or as an additive 10 to oils and speciality fats to be incorporated in food
products for humans and other mammals with the purpose of lowering the blood cholesterol level.
Finally, the invention comprises the use of a vegetable oil fraction according to the invention for the manufac-15 ture of a medicament for lowering the blood cholesterol level.
DETAILED DESCRIPTION OF THE INVENTION
The starting oil is obtained by means of standard crush¬ing, pressing and extraction techniques, optionally fol-
20 lowed by acid water treatment, deacidification and
bleaching steps normally used in the processing of vege¬table oils. If the starting oil has a slip melting point higher than 30 °C the oil is dry or solvent fractionated to obtain a lower melting fraction suitable as a starting
25 oil for the process. The starting oil can also be a mix¬ture of oils or fractions of oils obtained from different sources of raw materials. The prerequisite is that the starting oil has a slip melting point of not more than 30 °C and an unsaponifiable content of at least 0.5 % by
30 weight.

I. In a first aspect the present invention relates to a process for preparing an oil fraction rich in non-tocol-ic, high-melting, unsaponifiable matter which comprises the following steps:
5 a) the starting oil is hydrogenated to fully satu¬rate the fatty acids of the glycerides and to reach a slip melting point of at least 57 °C;
b) to the hydrogenated oil from a) is added a suit¬
able amount of the starting oil to act as a car¬
lo rier and vehicle for the unsaponifiable matter.
The concentration of the carrier oil in the oil mixture is 1-75 % by weight, preferably 2-50 % by weight, and more preferably 5-25 % by weight;
c) to the oil mixture from b) is added a solvent in
15 a ratio from 1:2 to 1:20, preferably from 1:3 to
1:15, and the mixture is heated to transparency;
d) the mixture from c) is cooled in one or more
steps to a final temperature in the range from -
35 to +30 °C, preferably from -20 to +20 °C, and
20 the precipitated high melting fraction(s) is
(are) filtered off;
e) the filtrate obtained from d) is desolventised,
leaving a fraction enriched in unsaponifiable
matter.'
25 Suitable conditions for the hydrogenation under a) are illustrated in the examples, but other variations are possible and within the ability of a person skilled in the art of processing vegetable oils.
The amount of starting oil to be added in step b) is de-30 pendent upon the original content of unsaponifiable mat-

ter in the starting oil and the type of unsaponifiable matter as well as the final concentration of unsaponifi¬able matter needed for the application in question. The concentration in the enriched fraction and its melting . 5 behaviour is regulated by varying the mixture ratio of carrier oil to the hydrogenated oil. This is further il-. lustrated in the examples,
The solvent used in c) is of a non-polar or semi-polar type such as hydrocarbons and linear and branched alka-10 nols of 1-5 carbon atoms. A preferred solvent in the process is hexane or petroleum ether.
In the fractionation step of d) one would expect the higher melting unsaponifiables to be entrapped and co-precipitated with the high melting glycerides, especially 15 if the amount of liquid carrier oil in the oil mixture is low. Quite unexpectedly the major part of the total mass of high-melting unsaponifiables is recovered.
The oil fraction prepared by this first aspect of the in¬vention is characterised by having a trans-fatty acid 20 content of' maximum 2 % by weight.
The enriched fraction can be further purified by deacidi-fication, bleaching and deodorisation in the normal man¬ner. During deodorisation a part of the tocopherols etc. are stripped off and, therefore, antioxidants and other 25 stabilisers may optionally be added to the product. If the product is liquid or semi-liquid, addition of a structuring fat or wax is useful in order to prevent sud¬den precipitation of supersaturated unsaponifiables in the product.
30 II. In a second aspect the present invention relates to a process as detailed under heading I, except that the car-

rier oil added to the fully hydrogenated starting oil in step b) is different from the starting oil.
The carrier oil used in this aspect is selected to incor¬porate beneficial effects in combination with the unsapo-5 nifiable matter for the intended use, e.g. nutritional aspects such as the degree and type of unsaturation of the fatty acid residues of the oil, and oxidative resis¬tance, etc. The selected carrier oil predominantly con¬sists of triglycerides of saturated and unsaturated C8-10 C22 fatty acids, and has a slip melting point of not more than 30 °C. Preferably the selected carrier oil is a vegetable oil predominantly consisting of triglycerides of saturated and unsaturated C16-C22 fatty acids.
If the selected carrier oil is obtained from unhydrogen-15 ated material, then the resulting enriched oil fraction will have a trans-fatty acid content of maximum 2 % by weight. If the carrier oil is the liquid fraction of a partly hydrogenated oil, the resulting enriched oil frac¬tion may have a trans-fatty acid content exceeding 2 % by 20 weight.
The magnitude of enrichment and the total content of unsaponifiable matter obtainable by the procedures under I and II are illustrated in the examples and summarised in the following table.

Subject Example 1 Rapeseed Example 2 Rapeseed Example 3 Shea Butter Example 3 Shea Butter
Unsaponifiable content in starting oil, in % 0.9 0.9 12.5 12.5
Carrier oil content in the mixture, in% 2 5 10 20
Yield of the enri¬ched fraction, in% 5.9 8.2 21.0 29.0
Unsaponifiable content in enriched fraction, in% 14.5 10.9 53.0 38.1
Enrichment factor 16.1 12.1 4.2 3.0
Recovery of un¬saponifiable matter, in% 94.4 98.4 89.0 88.4
III. In a third aspect the invention relates to a process as detailed under headings I or II, wherein the starting oil containing the unsaponifiable matter is interesteri-5 fied before the hydrogenation step a) and/or the oil mix¬ture from step b) is interesterified before the frac¬tionation steps.
The interesterification of the starting oil increases the oil solubility of a part of the unsaponifiables, and the 10 interesterification of the oil mixture randomises the fatty acid content and thus incorporates unsaturated fatty acids in the glyceridic part of the resulting en¬riched oil fraction.
The interesterification is performed in the presence of a 15 suitable catalyst, e.g. methoxide, ethoxide, or a mixture of glycerol and caustic lye, as illustrated in the exam¬ples, but other variations are possible and within the ability of a person skilled in the art of processing vegetable oils.
20 As mentioned in the beginning, any vegetable oil or frac¬tion thereof is suited to be processed according to the

invention provided that it has a slip melting point of not more than 30 °C and an unsaponifiable content of at least 0.5 % by weight. Preferred starting oils for the process originate from the following or their hybrids: rapeseed and canola (Brassica napus, campestris etc.), soybean (Glycine max), corn (maize) germ (Zea mays), sun¬flower (Helianthus annuus), flax (Lineum usitatissimus), mango (Mangifera indica), avocado (Persea americana), olive (Olea europea) , sesame (Sesamum indicum), rice-bran (Oryza sativa), wheat germ (Triticum aestivum), oat and oat bran (Avena sativa), palm (Elaeis guineensis, oleif-era etc.), sal (Shorea robusta), illipe (Madhuca spp.), shorea (Shorea stenoptera), sheanut (Butyrospermum parkii) or fractions or mixtures thereof.
Sheanut oil (shea butter) is of particular interest due the nature of the unsaponifiable constituents and a total content of approx. 6% thereof in the shea butter. As the slip melting' point of shea butter is normally higher than 30 °C, a liquid fraction has to be used as the starting oil. The liquid fraction of shea butter is a result of the production of the high melting fraction known in the industry under the INN-designation "Shea Stearine". The preferred liquid fraction has a further benefit as it has a content of 8% unsaponifiables or higher.
A more preferred fraction is obtained by the fractiona¬tion of a partly dekaritenised shea butter. This fraction, is especially suitable, as the polyisoprenic hydrocarbons (karitene) are.unwanted in edible products. Furthermore, as the karitene is highly unsaturated it thereby gives rise to problems associated with oxidation. The following is an illustration of the typical relative composition of the main types of unsaponifiables in shea butter and the liquid fractions:

Types of unsaponifjable Shea butter Liquid fraction of shea butter Liquid fraction
of dekaritenised
shea butter
TTPAs and esters 65% 70% 85%
Phytosterols and others 6% 6% 8%
Karitene 29% 24% 7%
On the basis of the liquid fractions of shea butter the invented process makes it possible to manufacture en¬riched fractions with an unsaponifiable content in the range 30 - 90% by the fractionation of oil mixtures con¬taining up to 50% by weight of the unhydrogenated shea butter fraction, that acts as the carrier oil for the unsaponifiable matter. Fractionation of an oil mixture containing up to 75 % by weight of the unhydrogenated shea butter fraction will result in an enriched fraction with an unsaponifiable content of 20 % by weight which corresponds to an enrichment factor of at least 3 vs. the normal content in shea butter.
POSSIBLE USES OF THE ENRICHED FRACTIONS OBTAINED BY THE PROCESS
Some of the obvious applications are already illustrated in the section BACKGROUND above.
Due to the high concentrations of unsaponifiables obtain¬able by the process of the invention the enriched frac¬tions can be used as a starting material for further mechanical and/or chemical processing.
Some new and special applications are related to frac¬tions made on the basis of sheanut oil.

It is known from published studies (Weststrate and Meijer 1998. Eur. J. Clin, Nutr. 52(5):334-343/ Sierksma, West¬strate and Meijer 1999. Brit. J. Nutr. 82:273-282; Vissers, Zock, Meijer and Katan 2000. Am. J. Clin. Nutr.
5 72:1510-1515) that the naturally occurring unsaponifiable matter in sheanut oil is not suited for lowering choles¬terol levels in serum. Surprisingly it has been found that shea butter fractions made according to the process of the present invention are able to reduce the total and
LDL cholesterol level in the human blood. The results are detailed in Example 9.
As a result of this, a fraction of shea butter manufac¬tured according to the invention can be used in food products and nutritional supplements, and due to the high concentrations obtainable by the process it can be used as an active ingredient in capsules and tablets for oral administration for the purposes illustrated in Example 9.
The invention will be better understood with reference to the following examples that are illustrative and should not be taken as limiting the scope of the present inven¬tion as described in the claims.
EXAMPLES
Methods of analysis and definitions
Total unsaponifiable matter ( Total unsaponifiable matter (> 15%), is determined as 100% minus the total glyceridic content. The glyceridic content is calculated on the basis of the actual content of fatty acids in the sample analysed by absolute quan¬titative GC according to IUPAC 2.301 and 2.304.

Karitene is determined by methylating the sample with 0.5n KOH in methanol. The remanence is washed with hot methanol, dissolved in chloroform and the content deter¬mined gravimetrically,
5 TTPA, hydrogenated TTPA and esters thereof are analysed by GC according to a modified IUPAC 2.323 using choles-teryl palmitate as internal standard.
Slip melting point is determined according to AOCS Cc 3-25.
10 SFC (solid fat content) is analysed by puls-NMR according to IUPAC 2.150.
Fatty acids are analysed by GC according to IUPAC 2.301 and 2.304.
Total content of tocopherols is determined by HPLC with 15 0.6% pentanol in hexane as mobile phase and with fluores¬cence detection (extinction 291 nm, emission 330 nm) vs. a reference sample.
Acid value is determined according to IUPAC 2.2 01.
Iodine value is determined according to IUPAC 2.205.
20 Enrichment factor is calculated as the fraction: conten-t of unsaponifiables in the enriched fraction divided by the content in the starting oil.
"bdl" denotes: "below detection limit".
"Transparent" means that the liquid is allowing light to 25 pass through so that objects behind can be clearly seen, i.e. more clear than translucent.

Example 1. Fraction of rapeseed oil enriched in unsaponifiable content
A semi-refined low-erucic rapeseed oil with an iodine value of 115.7 is fully hydrogenated to an iodine value 5 of 1.3 and a slip melting point of 68.0 °C. Rapeseed oil is added to the hydrogenated oil to a content of 2% by weight. The oil mixture is prepared by adding 20 g of rapeseed oil to 980 g of melted hydrogenated oil to reach a total weight of 1000 g.
10 In a jacketed vessel equipped with a stirrer 10 litre of hexane is added to the oil mixture and the suspension is heated to transparency. From a temperature of approx. 50 °C the mixture is cooled at a rate of approx. 1 °C/min to reach a final temperature of 5 °C. The precipitated high-
15 melting fraction is filtered off and washed with hexane. The high-melting fraction has an iodine value of 0.5 and a slip melting point of 68.7 °C.
The filtrate is desolventised leaving an enriched frac¬tion in a yield of 58.5 g corresponding to 5.9 % by 20 weight of the oil mixture.

25


Parameter Startina oil Enriched fraction
Total unsaponifiables, % 0.9 14.5
Total tocopherols, ppm 698 5593
Total trans-fatty acids, % bdl 1.0
Enrichment factor calc. on
total unsaponifiables 16.1
Enrichment factor calc. on
total tocopherols 8.0

Based on the analytical figures enrichment factors have 30 been calculated. This clearly demonstrates that by apply¬ing this new process of the invention on an oil with a low content of unsaponifiable matter it is possible to

produce a highly enriched fraction that at the same time has a low trans-fatty acid content making it suitable for food and non-food applications.
The enrichment factor for total unsaponifiables is much 5 higher than for the low melting tocopherols.
Furthermore, the invented process is recovering the major part of the total unsaponifiable matter. The total mass in the oil mixture is 9 g (0.9 % of 1000 g), and the yielded fraction'contains 8.5 g (14.5 % of 58.5 g) corre-10 sponding to a recovery of 94.4 % of the total unsaponifi¬able matter.
Example 2. Oil enriched with unsaponifiable matter origi¬nating from rapeseed
The procedure in Example 1 was repeated, except that the 15 carrier oil used was a speciality oil with a high oxida¬tive resistance. This oil is commercially available under the trade name "Cremeol PS-6" (Cremeol is a trademark of Aarhus Olie). The oil is produced by multiple dry-frac¬tionation of partly hydrogenated rapeseed, sunflower or 20 soybean oil.
The oil is characterised by the following typical values:
Saponification value 186-195
Iodine value 82-92
Slip melting point, °C 6
25 Solid fat content at 20 °C, % 0
Rancimat value at 120 °C, h 40
Predicted shelf life at 20 °C, years 5
Fatty acids bv GC:
Palmitic, C16:0, % 4
30 Stearic, C18:0, % 4
Total C18:1,% 84
Linoleic, C18:2, % 5
Total trans-fatty acids, % 29

The carrier oil "Cremeol PS-6" was added to the fully hy-drogenated rapeseed oil in an amount of 5% of the total oil mixture. The fractionation of the oil mixture was performed as described in Example 1.
5 The results are described in the following table.

10
15


Parameter Starting. Oil Enriched Fraction
Total unsaponifiables, % 0.9 10.9
Total Tocopherols, ppm 698 5322
Total trans-fatty acids, % bdl 22.8
Enrichment factor calc.
on total unsaponifiables 12.1
Enrichment factor calc.
on total tocopherols 7.6
Yield of fraction in g 82
Recovered amount of
unsaponifiables in g 8.9
Recovery in % of total matter 98.9

The enriched fraction appeared to have a consistency similar to petroleum jelly. The slip melting point was 20 44.9 °C.
This clearly demonstrates that it is possible to enrich a suitable carrier oil with the unsaponifiable matter in question by applying this new process of the invention. Like in Example 1 the enrichement factor for total 25 unsaponifiables is much higher than for the low melting tocopherols.
As can be seen, the process does not generate trans-fatty acids. The content of trans-fatty acids in the fraction is inherent to the preferred carrier oil.
30 Due to the high oxidative resistance provided by the car¬rier oil the produced fraction is ideally suited as an

ingredient in cosmetics, toiletries and pharma products for dermal application.
Example 3. Fraction of sheanut oil enriched in unsaponi-fiable matter
5 The starting oil in this example is an industrially pro¬duced, semi-refined and partly dekaritenised fraction of shea butter obtained by the following processing steps commonly used in the vegetable oil industry for the pro¬duction of food ingredients:
10 1. The crude oil is obtained by means of the stan¬dard crushing, pressing and extraction tech¬niques.
2. The free fatty acids are removed in an alkaline
deacidification process followed by a bleaching
15 step to reduce the colour.
3. The semi-refined oil is dekaritenised by mixing
it with a semi-polar solvent by which the main
part of the karitene precipitates, and is subse¬
quently disposed of. The solvent is distilled off
20 leaving a partly dekaritenised shea butter.
4. In the following fractionation process the fat is
mixed with hexane and heated to transparency fol¬
lowed by a cooling designed to crystallise the
high-melting fraction known under the INN-
25 designation "Shea Stearine".
5. The Shea Stearine is filtered off and the fil¬
trate desolventised leaving a low-melting, partly
dekaritenised shea butter fraction. After post
refining in the form of an alkaline deacidifica-

tion and a subsequent treatment with bleaching earth the fraction is named PR 589.
The shea butter fraction PR 589 is characterised by the .following typical values vs. shea butter:
5 Parameter Shea fraction PR 589 Refined Shea
Butter
Slip melting point, °C 20 32
Acid value 0.1 0.3
Iodine value 74 62
10 Total unsaponifiables, % 12.5 6
Polyisoprenic hydrocarbons, % 0.8 2
The shea butter fraction was fully hydrogenated to a slip melting point of 68 °C by the following process:
To 2500 g of the fraction was added 0.5% catalyst ("Gir-15 dler G-53", Sud-Chemie AG) and the mixture was heated to 180 °C. Hydrogen was applied at 5 ato for 160 minutes. The catalyst was filtered off, and the hydrogenated oil was denickled in the normal way. To the hydrogenated oil was added 10% by weight of shea butter fraction PR 589 to 20 produce an oil mixture ready for fractionation. In the
same manner another oil mixture was made by adding 20% of shea butter fraction PR 589 to the hydrogenated oil.
In jacketed vessels equipped with stirrer and containing 1000 g each of the two oil mixtures hexane was added in a
25 ratio oil to hexane of 1:10 w/v and the mixture was
heated to transparency. From a temperature of 36 °C the mixture was cooled at a rate of approx. 1 °c/min to reach a final temperature of 0 °C. The precipitated high-melt¬ing fractions are filtered off and the filtrate desol-.
30 ventised leaving two fractions rich in unsaponifiable matter. The two fractions were obtained in a yield of 21.0% and 29.0%, respectively.

10

The total mass of unsaponifiables in the oil mixture is 125g (12.5% of 1000 g). As can be seen in the following table the major part is recovered by the process.
Parameter Shea fraction Fraction I Fraction
PR-589 (10%PR-589) (20%PR-589)
Total unsaponifiables, % 12.5 53.0 38.1
Total trans-fatty acids, % bdl 0.7 0.4
Enrichment factor calc. on
total unsaponifiables 4.2 3.0
Yield of fraction in g 210 290
Recovered amount of
unsaponifiables in g 111.3 110.5
Recovery in % of total matter 89.0 88.4

The two fractions have a remarkably high content of unsa-15 ponifiable matter making them suited for a number of food and non-food applications. Especially in Fraction I the concentration has reached a level that makes it possible to use it in e.g. gelatine capsules for oral administra¬tion.
20 Example 4. Industrial scale test-production of a fraction of shea butter enriched in unsaponifiable matter
In order to verify the applicability of the process on an industrial scale a fraction similar to Fraction I in Ex¬ample 3 was produced as described in the following:
25 Shea butter fraction PR 589 was hydrogenated as described in Example 3. To 30 metric ton of the hydrogenated oil was added 4.5 metric ton of shea butter fraction PR 589.
To the oil mixture containing 13% carrier oil was added hexane in the ratio 1:10 v/v, and the mixture was heated 30 to transparency and fractionated by cooling the mixture to reach a final temperature of 0 °C. The precipitated high-melting fraction was filtered off and the filtrate

desolventised leaving a fraction rich in unsaponifiable matter in a yield of 23.5 % based on the weight of the oil mixture.
The recovered fraction was passed through a 50 \xra filter and further purified by deodorisation at 220 °C and 2 mbar. After cooling to 100 °C 25 ppm citric acid and 500 ppm natural mixed tocopherols were added to the product to improve the oxidative resistance.
The final product has the following characteristics:

10
15

Appearance
Slip melting point, °C
Solid fat content at 20 °C, %
Solid fat content at 30 °C, %
Solid fat content at 40 °C, %
Rancimat value, 120 °C, h
Acid value
Iodine value
Total unsaponifiable matter, %

Yellowish, homogeneous paste 28.6 10.3
7.1
3.8 31.7
0.84 80.8 45.7

Fatty acid composition of the glyceridic part in %

20
25
30

C12:0 C14.0 C16:0 C18:0 C18:1 cis C18:1 trans C 18:2 cis C 18:2 trans C18:3cis C20:0 C20:1 cis C22:0 Others

0.7
0A
8.0
33.3
45.9
1.8
7.0
0.1
0.3
1.3
0.3
0.2
0.7



35

The high-melting fraction was of a very good quality which makes it suited as a raw material for the produc¬tion of food ingredients.

This example demonstrates the applicability of the in¬vented process on an industrial scale.
Example 5. Interesterlfied fraction of shea butter en¬riched in unsaponifiable matter
In shea butter the major part of the unsaponifiable mat¬ter is a- and β-amyrin, butyrospermol and lupeol, present in the form of cinnamic and acetic acid esters and to a lesser extent as fatty acid esters and the free alcohols. A minor part is the phytosterols i.e. ά-spinasterol, stigmasterol, etc.
In order to increase the oil solubility of the unsaponi-fiables, the starting oil is interesterified before hy-drogenat:' in for the purpose of increasing the presence of fatty acid ester types. Shea butter fraction PR 589 is interesterified at a temperature of 120 °c with 0.1% so¬dium methoxide as a catalyst. The resulting interesteri¬fied product is named shea butter fraction PO 135. The relative composition and the total content of the unsapo¬nifiable matter is given below:

20
25


Constituents PR 589 P0 135
Total unsaponifiable content 12.5% 12.0%
comprising:
TTPA-cinnamic esters 50% 48%
TTPA-acetlc esters 22% 21%
TTPA-fatty acid esters TTPA and Phytosterols Karitene 4%
17%
7% 14% 9% 8%

The shea butter fraction PO 135 is fully hydrogenated ac¬cording to the procedure described in Example 3. An oil 30 mixture is prepared by mixing the fully hydrogenated and the unhydrogenated shea butter fraction PO 135 in the ra¬tio 1:1 by weight.

In order to randomise the glycerides the oil mixture is interesterified at a temperature of 120 °C for 15 minutes with 0.1% sodium methoxide as a catalyst. After treatment with citric acid and bleaching earth the resulting prod-5 uct is named shea butter fraction PO 136.
Shea butter fraction PO 136 is fractionated in two steps by mixing it with hexane in the ratio 1:4 (w/v) and heat¬ing the mixture to transparency and cooling it to 0 °C to precipitate the first high-melting fraction and filtering 10 it off. The filtrate is distilled until the oil to hexane ratio is 1:4 (w/v). The second high-melting fraction is disposed of after cooling to -15 °C. The filtrate is de-solventised leaving a fraction named PO 136EE.
Shea butter fraction PO 136EE has a total unsaponifiable 15 content of 34.0%. The product has the following composi¬tion in which "TTPA" comprises hydrogenated moieties:
Total glycerides 66.0%
TTPA-acetic acid esters 6.2%
TTPA-fatty acid esters 7.5%
20 TTPA-cinnamic acid esters 15.7%
Karitene 2.0%
Other unsaponifiable matter 2.6%
Fatty acid composition of the glyceridic part:

C14:0 0.1%
C16:0 4.0%
C16:1 cis 0.1%
C18:0 - 29.6%
C18:1 cis 54.4%
C18:1 trans 0.6%
C18:2 cis 9.4%
C18:2 trans 0.0%
C18:3 cis 0.3%
C20:0 0.8%
C20:1 cis 0.5%
C22.0 0.1%
C22:1 cis 0.1%


Shea butter fraction PO 136EE is tailored to be used for the nutritional fortification of any food product. Fur¬thermore, it is especially suited to be used as an ingre¬dient in fat-containing nutritional products for the pur-5, pose of lowering the blood cholesterol content.
If a higher degree of enrichment is needed for the in¬tended use, this can be achieved by the fractionation of an oil mixture containing less unhydrogenated shea butter fraction PO 135 cf. Example 3.
10 Example 6. Use of an oil, enriched with unsaponifiable matter originating from rapeseed, in a cosmetic formula¬tion
From literature {British Journal of Dermatology 134, 215-220 (1996)) it is known that unsaponifiables from rape-15 seed have a beneficial effect in restoring the barrier function on surfactant-irritated skin.
In the following formulation the oil fraction enriched in rapeseed unsaponifiables from Example 2, where 5% "Cre-meol PS-6" is added as carrier oil, is incorporated in a 20 hand cream intended to be used after manual cleaning.

CTFA/INCI Name % w/w
Vegetable Oil (and) Rapeseed (Brassica Campestris) Oil Unsaponifiables 6.00
Glyceryl Dioleate 6.00
Glyceryl Stearate SE 4.50
Octyl Stearate 2.00
Hydrogenated Vegetable Oil 1.00
Dimethicone 0.50
Water ad 100.00
Glycerin 4.00
q.s.

Ingredient
"Cremeol PS-6" enriched in rape-seed unsaponi¬fiables
"Cremeol FR-36"
"Tegin SE"
(Th Goldschmidt)
10
"Cetiol 868" (Cognis)
"Cremeol HF-52"
"Abil 100"
(Th Goldschmidt)
15 B;
Water, deionized
Glycerin, 99.5%
C: Perfume and
preservatives
20
The product is produced by heating A and B to 75-80 °C while stirring and combining the two phases. The mixture is homogenised at 70 °C. Cool slowly to 30 °C while stir¬ring. Add C and homogenise at 30 °C. The final viscosity of the emulsion is obtained after 2-3 days at room tem¬perature.
25 Example 7. Use of a fraction of sheanut oil, enriched in unsaponifiable matter, in cosmetic and pharma-like oint¬ments
The use of fraction II in Example 3 in two waterless for¬mulations intended to protect and restore the skin is il-
30 lustrated in the following way:

Inaredient CTFA/INCI Name % w/w
Shea fraction II Shea Butter (Butyro-spermum Parkii) Extract 2.00
"Cremeol VP" Veg. Oil (and) Hydrogenated Veg. Oil (and) Candelilla (Euphorbia Cerifera) Wax 73.40
"Cremeol HF-52" Hyd'-lenated Veg. Oil 13.00
Beeswax Bee 7.80
Carnauba wax Carr a (Copernicia 3.80
10
Cerifera) Wax
Fragrance q.s.
All the ingredients are heated to 70-80 °C and mixed. The mixture is filled into moulds or 5 ml tubes at 65-70 °C 15 and cooled.
The formulation was tested at various concentrations of shea fraction II by five female panelists skilled as daily users of lipbalm. A dosage of 2-3% was preferred in emolliency and incorporating an increase of the lasting 20 effect by one to two hours. At higher concentrations the product was too sticky and declined as being a cosmetic product.
II. Pharma-like Ointment
Ingredient % w/w
15 Shea fraction II 20
White Petrolatum, NF19 72
White Wax, NF19 3
Isopropyl palmitate 5
All the ingredients are heated to 70-80 °C and mixed. The 2o mixture is cooled while stirring. When the product starts to congeal it is filled into tubes. The ointment has a


slip melting point of 47.6 °C and passed a storage test at 40 °C.
Due to the restoring, cicatrising and bacteriostatic properties of the sheanut oil unsaponifiable matter the 5 product is intended for use on extremely dry, sensitive skins, wounds and various scars.
Example 8. Use of a fraction of sheanut oil, enriched in unsaponifiable matter, as an active ingredient in a soft gelatine capsule for oral administration
10 Due to the high content of unsaponifiable matter in frac¬tion I from Example 3 it is suited for encapsulation.
A mixture was made according to the following formula¬tion:
Ingredient % w/w
15 Shea fraction I 80%
"Shoguwar41 NG/NF" 20%
(NF 19, hydrogen, veg. oil - Type II)
"Shoguwar 41 NG/NF" is a soybean oil hydrogenated to a slip melting point of 41 °C. At 20 °C SFC is approx. 75% 20 and at 37 °C SFC is approx. 20%. This makes it a good processing agent and carrier that will keep the active material suspended and uniformly distributed.
The two ingredients are dosed, melted and mixed at 45 °C and fed to the filling machine to produce soft gelatine 25 capsules of 2 50 rag content.
A daily dose of 750 mg (250 mg three times daily) is equivalent to ca. 300 mg unsaponifiables.

Example 9. A clinical study of the efficacy of an inter-esterified fraction of sheanut oil enriched in non-tocol-ic, high-melting unsaponifiable matter on plasma lipids and lipoproteins in healthy subjects with normal to bor¬derline high plasma cholesterol

Setting:

Aalborg Sygehus, Hobrovej 18, 9000 Aal-borg, Denmark.



10

Project manager: Erik Berg Schmidt MD, D.M.Sci., Depart¬ment of Medicine, Hjorring/Bronderslev Hospital, Hjorring, Denmark.



Project team:

Inge Aardestrup, Department of Clinical Biochemistry, Aalborg Hospital, Aalborg, Denmark;



15

Jens Mellerup, R&D Department of Aarhus Oliefabrik A/S, Aarhus, Denmark;

Jeppe Hagstrup Christensen, Department of Nephrology, Aalborg Hospital, Aal¬borg, Denmark.
Background
10 Plasma total cholesterol and low-density lipoprotein
(LDL) cholesterol levels are strongly related to the risk of coronary heart disease (CHD), the major cause of pre¬mature death in Western societies. Thus, a reduction in total cholesterol of 1 % is associated with a 2 - 3 % de-
5 crease in the risk of CHD. A reduction of plasma choles¬terol levels is therefore of paramount importance both for the individual patients and for the whole community. Intake of plant sterols can lower the plasma cholesterol concentrations in humans. However, little is known about

The effects of the unsaponifiable constituents of sheanut .1, mainly the triterpene alcohols.
objective
study the effect of a sheanut oil fraction, prepared .cording to Example 5, on plasma lipids and lipoproteins healthy subjects with normal to borderline high plasma olesterol.
sign and Treatment
e hundred and five healthy volunteers (54 men of age .8 + 13.4 y, range 20 - 64 y, and 51 women of age 4 0.7 12.6 y, range 23 - 60 y) were randomly assigned to 2 satment groups in a double-blind study design. The sub-jets were randomized into treatment blocks of 16 persons order to secure an equal distribution of men and women the two groups. The Active group consumed 30 g/d of a anut oil spread, and the Control group consumed 30 g/d a sunflower oil spread. A dietary questionnaire was led out, and the subjects were asked to maintain their itual diet during the whole study period. The subjects asumed one unit of spread 3 times a day and usually on lice of bread, at breakfast, lunch, and supper. The ts were given for 6 consecutive weeks.
R&D Department, Aarhus Oliefabrik A/S, Denmark, spe-lly prepared the experimental spreads. They were ked in blinded foil envelopes each holding 10 g and eled with a four-digit code. The fat phase of both sads contained liquid oil and hard stock. The hard ck and the sunflower oil used in the two spreads came n the same batches. The liquid sheanut oil used, con-ling 33.2 % by weight of non-glyceridic constituents,
modified by fractionation, hydrogenation, and inter-

esterification according to the procedure described in Example 5.
The sheanut oil spread contained 10 % non-glycerides, i.e. triterpene acetic acid esters, triterpene cinnamic 5 acid esters, triterpene fatty acid esters, free triter¬pene alcohols, free sterols, and kariten (a naturally oc¬curring polyisoprenic hydrocarbon in sheanuts). The sun¬flower oil spread was similar to the range of well-known sunflower oil products with a long history of heart 10 health promotion.
Both spreads showed normal appearance and had almost identical structure. Compositions are shown in Table 1.



Results
The preparations were well tolerated, and all of the one hundred and five volunteers who started also completed the trial.,Fifty-three subjects were allocated to the Ac-5 tive group, and 52 subjects participated in the Control group. Baseline characteristics of the study population are given in Table 2. Pre-experimentally no significant differences in the plasma cholesterol levels existed be¬tween the Active group and the Control group.
TABLE 2
Characteristics of the volunteers before inclusion,
Men (n=54) Women (n=51)
mean± s.d. range mean± s.d, range
Age(y) 38.8± 13.4 20-64 40.7 ± 12.6 23-60
Total-cholesterol (mmoI/L) 5.04± 1.22 2.80 - 8.30 5.15± 1.01 3.45 - 7.85 HDL-cholesterol (mmol/L) 1.31 ± 0.25 0.80 - 2.00 1.63± 0.32 l 0.80 - 2.40
LDL-cholesterol (mmoI/L) 3.18± 1.14 2 1.35 - 6.40 3,00± 0.96 1.60 - 5.75
1 Significantly different from men: P 10 There were significant reductions in plasma total- and LDL-cholesterol of 5 and 8 %, respectively, after treat¬ment in the Active group.
The mean plasma linoleic acid concentration increased significantly by 6 % after the sheanut diet and signifi-
15 cantly by 10 % after the sunflower diet. The mean serum stearic acid concentration increased significantly by 4 % after the sheanut diet and non-significantly by 1 % after the sunflower diet. These results are in the directions expected from the fatty acid contents of the diets and
20 validate the participants' compliance to the dietary in¬structions.
The effect of the diets on plasma lipids and lipoproteins are given in Table 3 and the safety data are given in Ta¬ble 4.





Conclusion
In summary the study shows that consumption of a sheanut oil spread containing unsaponifiable material, which is a mixture of triterpene alcohols such as butyrospermol, ά-5 amyrin, β-amyrin, lupeol/ and germanicol, is effective and safe in lowering plasma total- and LDL-cholesterol levels in healthy adults. The observed reduction in LDL-choleste¬rol in the present study is of clinical relevance, and in the order of magnitude of what is commonly seen in subjects 10 given dietary advice for hypercholesterolemia. We therefore conclude that intake of this type of sheanut oil can make a contribution to the prevention of CHD.

WE CLAIM:
1. A process for preparing a vegetable oil fraction rich in non-tocolic,
high-melting, unsaponifiable matter which comprises the following steps:
a) a vegetable oil, or a fraction thereof, having a slip melting point of not
more than 30° C and a content of unsaponifiable matter of at least 0.5 %
by weight is hydrogenated to fully saturate the fatty acids of the
glycerides and to reach a slip melting point of at least 57 °C;
b) to the hydrogenated oil is added from 1 to 75 % by weight of the unhydrogenated starting oil or another oil having a slip melting point of max. 30°C to act as a carrier and vehicle for the unsaponifiable matter;
c) to the oil mixture from b) is added a solvent in a ratio from 1:2 to 1:20, and the mixture is heated to transparency;
d) the mixture from c) is cooled in one or more steps to a final temperature in the range from -35 to +30°C, and the precipitated high-melting fraction(s) is (are) filtered off;
e) the filtrate obtained from d) is desolventised, leaving a. fraction rich in
unsaponifiable matter.
2. A process as claimed in claim 1, wherein from 2 to 50 % by weight, and preferably from 5 to 25 % by weight of carrier is added to the hydrogenated oil in step b).
3. A process as claimed in claim 1 or 2, wherein unhydrogenated starting oil is used as the carrier in step b).
4. A process as claimed in claim 1 or 2, wherein a triglyceride oil different from the unhydrogenated starting oil is used as the carrier in

step b) , said triglyceride oil predominantly consisting of triglycerides of saturated and unsaturated C8-C22 fatty acids, and preferably being a vegetable oil predominantly consisting of triglycerides of saturated and unsaturated C16-C22 fatty acids.
5. A process as claimed in any one of the preceding claims, wherein the starting oil containing the unsaponifiable matter is interesterified before the hydrogenation step in order to increase the oil solubility of a part of the unsaponifiables.
6. A process as claimed in any one of the preceding claims, wherein the oil mixture from step b) is interesterified before the fractionation steps c) and d) in order to randomise the fatty acid content and incorporate unsaturated fatty acid in the glyceridic part of the resulting enriched oil fraction.
7. A process as claimed in claim 5 or 6, wherein the interesterification is performed for at least 5 minutes in the presence of a suitable catalyst.
8. A process as claimed in claim 7, wherein the catalyst is selected from the group consisting of methoxide, ethoxide, and a mixture of glycerol and caustic lye.
9. A process as claimed in any one of the preceding claims, wherein the solvent used in the fractionation steps c) and d) is of a non-polar or semi-polar type.
10. A process as claimed in claim 9, wherein the solvent is selected from the group consisting of hydrocarbons and linear and branched alkanols of 1-5 carbon atoms, and is preferably hexane or petroleum ether.

11. A process as claimed in any one of the preceding claims, wherein the starting vegetable oil containing the unsaponifiable matter originates from one of the following or their hybrids: rapeseed, canola, soybean, corn, maize-germ, sunflower, flax (low-linolenic linseed), mango, avocado, olive, sesame, rice bran, wheat germ, oat and oat bran, palm, sal, shorea, illipe and shea, as well as any fraction or mixture thereof.
12. A process as claimed in any one of the preceding claims, wherein the starting vegetable oil is a dekaritenised, lower-melting fraction of shea butter enriched in unsaponifiable matter, which is obtained as follows:
The crude shea butter is deacidified and subsequently partly dekaritenised by mixing it with a semi-polar solvent to precipitate the major part of the polyisoprenic hydrocarbons (karitene) which are disposed of; the solvent is distilled off and the oil is mixed with a suitable solvent e.g. hexane (ratio approx. 1:2 to 1:4), heated to transparency, and cooled to a temperature at which the high-melting glycerides precipitate; the precipitated fraction is filtered off and the filtrate desolventised leaving a lower-melting fraction with a slip melting point of max. 30 °C suitable for further processing.
13. A process as claimed in any one of the preceding claims, wherein the vegetable oil fraction obtained is further subjected to concentration by means of supercritical fluid extraction, molecular distillation, chromatography or recrystallisation.
14. A process as claimed in any one of the preceding claims, wherein the vegetable oil fraction obtained is further subjected to chemical modification such as hydrogenation, ethoxylation, propoxylation, esterification or interesterification.

15. A process as claimed in any one of the preceding claims, wherein the physical state of the vegetable oil fraction obtained is further changed e.g. by emulsification, incorporation in liposomes or nanosomes, admixing with a high melting fat and spray cooling, or microencapsulation.
16. A vegetable oil fraction obtainable by the process as claimed in any one of claims 4 to 8, wherein the carrier oil added to the fully hydrogenated starting oil in step b) is different from the starting oil, or wherein the starting oil containing the unsaponifiable matter is interesterified before the hydrogenation step a) and/or the oil mixture from step b) is interesterified before the fractionation steps, said fraction being enriched in non-tocolic, high-melting, unsaponifiable matter by a factor of at least 3 and having a trans-fatty acid content of maximum 2% by weight.
17. A vegetable oil fraction as claimed in claim 16, wherein the unsaponifiable matter is comprised of or derived from:
carotenoids; sesamin and sesamolin; y-oryzanol;
sterols, methyl sterols and dimethyl sterols;
triterpene alcohols and their esters with cinnamic acid, acetic acid and fatty acids.
18. A vegetable oil fraction obtained by the process as claimed in claim 4, said fraction being enriched in non tocolic, high-melting, unsaponifiable matter by a factor of at least 3 and having a trans-fatty acid content inherent to the preferred carrier oil.
19. A vegetable oil fraction as claimed in claim 18, wherein the trans-fatty acid content is of maximum 2% by weight.

20. A shea butter fraction obtainable by the process as claimed in any one of claims 1-15, having a content of at least 30 % by weight of non-tocolic, high-melting, unsaponifiable matter and being further characterised by containing hydrogenated triterpene alcohols and/or their esters and by having a trans-fatty acid content of maximum 2 % by weight.
21. A shea butter fraction as claimed in claim 20 having a content of 40 - 90 % by weight of said unsaponifiable matter.
22. A shea butter fraction as claimed claim 20 or 21, obtained by the process as claimed in any one of claims 5 - 15, i.e. involving at least one interesterification step before the fractionation steps c) and d).
Dated this June 06, 2003.



Documents:

585-mumnp-2003-assignent(29-06-2005).pdf

585-mumnp-2003-cancelled pages(12-09-2007).pdf

585-mumnp-2003-claims(granted)-(12-09-2007).doc

585-mumnp-2003-claims(granted)-(12-09-2007).pdf

585-mumnp-2003-correspondece(14-09-2007).pdf

585-mumnp-2003-correspondece(ipo)-(17-08-2007).pdf

585-mumnp-2003-form 1(02-04-2007).pdf

585-mumnp-2003-form 1(06-06-2003).pdf

585-mumnp-2003-form 13(23-03-2006).pdf

585-mumnp-2003-form 13(29-10-2007).pdf

585-mumnp-2003-form 13(31-03-2004).pdf

585-mumnp-2003-form 18(01-12-2005).pdf

585-mumnp-2003-form 2(granted)-(12-09-2007).doc

585-mumnp-2003-form 2(granted)-(12-09-2007).pdf

585-mumnp-2003-form 3(06-06-2003).pdf

585-mumnp-2003-form 3(10-07-2007).pdf

585-mumnp-2003-form 3(16-09-2003).pdf

585-mumnp-2003-form 5(02-04-2007).pdf

585-mumnp-2003-form 5(06-06-2003).pdf

585-mumnp-2003-form 6(29-06-2005).pdf

585-mumnp-2003-form-pct-ipea-409(12-09-2007).pdf

585-mumnp-2003-form-pct-isa-210(12-09-2007).pdf

585-mumnp-2003-power of authority(02-04-2007).pdf

585-mumnp-2003-power of authority(05-01-2005).pdf

585-mumnp-2003-power of authority(08-07-2003).pdf


Patent Number 211425
Indian Patent Application Number 585/MUMNP/2003
PG Journal Number 45/2007
Publication Date 09-Nov-2007
Grant Date 29-Oct-2007
Date of Filing 06-Jun-2003
Name of Patentee AARHUSKARLSHAMN DENMARK A/S
Applicant Address M.P. BRUNNS GADE 27, DK-8000 ARHUS C, DENMARK.
Inventors:
# Inventor's Name Inventor's Address
1 JENS MELLERUP OSTERMARKSVEJ 58, DK-8381 TILST, DENMARK
2 MOGENS BACH PROVSTEVAEN 9, DK-8464 GALTEN, DENMARK.
3 JORGEN VALENTIN ENKELUND LYKKENSHOJ 50, DK-8220 BRABRAND, DENMARK.
PCT International Classification Number C11B 7/00
PCT International Application Number PCT/DK01/00849
PCT International Filing date 2001-12-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/260,591 2001-01-09 Denmark
2 PA 2000 01917 2000-12-21 Denmark