IES20000995A2 - A process for the manufacture of sterol esters - Google Patents

Abstract

A process for the manufacture of a sterol ester product having hypocholesterolemic properties comprises purifying a sterol ester blend derived from a phytosterol mixture including B-sitosterol and campesterol to enrich the level of sitosterol and reduce the level of campesterol and other sterols and interesterification of the purified mixture to form a phytosteryl ester product enriched for sitosteryl ester at the expense of other sterols and steryl esters. The starting material is typically a soya derived mixture. The purification may be achieved by recrystallisation in a solvent such as ethanol or dichloromethane which favours the crystallisation of sitosterol. Alternatively, the purification can be achieved by reslurry in a like solvent using gradient temperature technology. Optionally the product may be further purified by reslurry in dry ethanol.

Description

The present invention relates to a process for the manufacture of sterol esters, and particularly to the manufacture of a plant derived fatty acid ester or mixture having hypocholesterolemic properties.

Jn humans, the link between elevated levels of serum cholesterol and risk of heart disease are ' 1 * well established. “'.f' $ -: 5 Tt has also been known since the early 1950’s that plant sterols are effective in lowering blood 'f·· serum cholesterol levels. The phytosterols are thought to reduce the absorption and re·Τ,.Λ 5 absorption of dietary cholesterol in the intestine. The most abundant plant sterols are βI sitosterol, campesterol and stigmasterol. It has been shown that sitosterol and its hydrogenated form are the main sterols of interest for lowering serum cholesterol.

’’ Phytosterols are perceived to have poor organoleptic characteristics and in particular lend a grainy mouth-feel to foods. Furthermore, sterols have low solubility in oils and fats.

Together, these factors have contributed to an under enrichment of phytosterols in food below levels which would supply humans with the perceived effective dose of 1.5g sterol/day, delivered primarily in oily preparations such as spreads, butter, cheese or cheese spreads, snack foods or the like.

The solubility of phytosterols in oil can be improved by esterification. For example, Hoffman. G., The chemistry and technology of edible oils and fats and their high fat products, Academic Press Limited, 1989, describes how the solubility and poor mouth feel of β-sitosterol is improved by esterification of the 3-position of the steroid nucleus with a fatty acid in an SN2 interesterification reaction catalysed by an alkoxide catalyst. The esterified product has a high fat solubility (30%-50%) and a palatable texture, even in its pure state.

IE000995 WO 98/01126 discloses a process for preparing a mixture of fatty acid esters from plant sources such as rice bran and sheanut by hydrolysing a sterol ester or mixture of sterol esters from the plant source to obtain a mixture comprising phenolic acids and/or fatty acids and free sterols, followed by esterifying the so-obtained free sterols with particular fatty acids. The phenolic acids and/or fatty acids may optionally be separated from the reaction mixture.

In another process disclosed in WO 98/38206, there is described the preparation of a mixture of stand esters, particularly plant-derived stand esters which can be incorporated into food products in order to lower blood serum cholesterol levels in humans. The stanol esters are prepared by hydrogenating a sterol blend at an elevated temperature, followed by interesterifying the intermediate stanol blend with a fatty acid methyl ester at elevated temperature, followed by purification of the resultant blend.

Plant sterols derived from soya are relatively high in sitosterol and campesterol content, both of which appear to be effective in reducing cholesterol levels. However, studies have shown that compesterol is relatively more absorbed in the gut than sitosterol.

Since the atherogenicity of absorbed campesterol is unknown it is desirable that levels of campesterol in sterol preparations are minimised. Therefore there is a need for a phytosterol mixture for incorporation into food products which has lower levels of campesterol, which has improved solubility in oil and which has good organoleptic characteristics.

Accordingly the present invention provides a process for the manufacture of a sterol ester blend derived from a phytosterol mixture including β-sitosterol and campesterol, the process comprising the steps of: purifying the phytosterol mixture to enrich the level of sitosterol and reduce the levels of campesterol and other sterols in the phytosterol mixture; and IE000995 interesterification of the purified phytosterol mixture to produce a phytosteiyl ester product with enriched levels of sitosteryl ester and reduced levels of other sterols and steryl esters.

The phytosterol starting mixture is preferably a soya derived mixture and typically contains as major components the sterols β-sitosterol, campesterol, stigmasterol and brassicasterol.

In one arrangement the phytosterol mixture is purified by recrystallization in a solvent which favours the crystallization of the sitosterol while retaining the campesterol, stigmasterol and brassicasterol in solution. Typical solvents which are suitable for this purpose are ethanol or dichloromethane.

In a second arrangement the phytosterol mixture is purified by reslurry in a chosen solvent using gradient temperature technology. Suitable solvents include ethanol or dichloromethane. Gradient temperature technology is a purification process which involves filtering a hot resluny mixture of phytosterol in the chosen solvent.

Preferably, the purified phytosterol mixture is dried to remove water from the mixture, since it has been found that removal of the water improves the yield of subsequent reactions.

In one arrangement, the phytosterol mixture is dried by vacuum drying.

In a second arrangement the phytosterol mixture is dried by prolonged heating at a temperature in the range of 130-150°C in a drying oven. Additionally, a dry nitrogen blanket is continually bled through the drying oven to remove condensing water.

Preferably, an alkoxide catalyst is used for the interesterification reaction. Alkoxide catalysts are particularly useful in nucleophilic substitution interesterification reactions.

IE000995 Sodium ethoxide is the catalyst preferably used in the interesterification reaction since it is pharmaceutically acceptable and is also easily quenched and removed at the end of the reaction.

In a preferred embodiment of the invention, the phytosteryl ester product may be reslurried in dry ethanol in order to remove excess methyl ester starting material and to increase further the concentration of sitosteryl ester.

It is important that all reagents used, including the catalyst, are anhydrous or substantially so, with water levels of 0.01% or less.

The ester starting material for the interesterification step, which should likewise be dry, may preferably comprise a fatty acid methyl ester derived from rapeseed oil with a fatty acid composition predominated by Cl8 fatty acids. Conveniently, the Cl8 acids comprise between about 79% and 84% of the total fatty acids.

Soya derived starting sterol esters mixture is preferred. Such a phytosterol mixture typically has the composition outlined in Table 1 Table 1 Soya Phytosterol Mixture β-Sitosterol Campesterol Stigmasterol 50% - 60% 28% - 38% % -15% Brassicasterol 2% - 8% Such a mixture is commercially available from EISAI as a ‘sitosterol’ mixture. The first step is to purify the phytosterol mixture by recrystallization or by reslurry using gradient temperature IE000995 technology in a solvent such as ethanol or dichloromethane. The purified mixture is enriched so that the relative level of β-sitosterol increases and of campesterol decreases.

The mixture is then dried before the interesterification step. It is important that all reagents are practically anhydrous, that is, having water levels below 0.01% to improve the yield of the interesterification reaction.

This can be achieved by vacuum drying or by prolonged heating at a temperature in the range of 130 -150°C in a reaction vessel such as a drying oven. A dry nitrogen blanket which is continually bled can be effective in removing condensate from the reaction vessel. Alternatively, the reaction vessel can be heated under vacuum.

The dried mixture is then interesterified using a catalyst and a fatty acid ester. The catalyst for this type of interesterification reaction is well documented, but for pharmacological reasons sodium ethoxide is preferred. The catalyst is added reslurried in a quantity of dried ester starting material.

As mentioned above, the ester starting material is preferably a fatty acid methyl ester based on rapeseed oil with a fatty acid composition of mostly (79-84%) Cl 8 acids. The hydroxy substituent at position 3 of the steroid nucleus of the β-sitosterol present is esterified with a fatty acid during the interesterification reaction. This reaction is particularly promoted by the presence of an alkoxide catalyst. The esterified β-sitosterol thus prepared has a high fat solubility (30-50%) and an improved palatable texture, even in its pure state.

Example 1: gms of the Soya phytosterol mixture of Table 1 was slurried in 500 mis dried ethanol at 50°C for three hours. The mixture was filtered while hot and the filter cake washed with 50 mis dry cold ethanol.

IE000995 The product was dried under vacuum at 104°C using a vacuum oven and used in the interesterification step.

Example 2: gms ofthe soya phytosterol mixture of Table 1 was slurried in 70mls of dichloromethane at 20°C for two hours with stirring. The mixture was filtered while hot, blown dry and washed with 100 mis of ethanol. The product was dried at 104°C in a vacuum oven.

Example 3: 7.6 gms of purified Soya phytosterol from Examples 1 or 2 were combined with 9.5 gms of the fatty acid methyl ester (supplied by Oleotec GMBH Hinter den Hoefen 11, D-51147 Cologne, Germany) in a dried reaction vessel and heated at 130°C for 2-3 hours with stirring to effect solution and dehydration. The mixture was cooled to 110°C and 28 mgms of sodium ethoxide slurried in 100 mgms fatty acid ester (dried) was added with vigorous stirring. A mild effervescence (evolution of methanol) was noted and the reaction was allowed to proceed for 2 hours.

The reaction mixture was then cooled to 95°C and 100 mgms distilled H2O was added to quench the catalyst.

The reaction product was washed with 3 x 25 mis H2O. The excess methyl ester was removed by vacuum distillation, this step being optional.

Example 4: gms of the dried reaction mixture from Example 3 was reslurried in 20ml dry ethyl alcohol for 2 hours at ambient temperature. The reaction product was collected by centrifugation.

The reaction product of Examples 3 and 4 was a brown, neutral tasting wax which was stabilised by the addition of 150 ppm d-delta rich tocopherol to prevent oxidation.

IE000995 The campesterol level in the final product ranges between about 7% and 11%. This final product can be incorporated into a range of fat-containing products for human consumption, including but not limited to spreads, butter, cheese and cheese spreads, snack foods and confectionery and in concentrations aimed at providing an adequate daily intake of the hypocholesterolemic product. It is considered that a daily intake of at least 1.5g of sitosterol per day is effective in reducing cholesterol levels.

It will of course be understood that the invention is not limited to the specific details described herein, which are given by way of example only, and that various modifications and alterations are possible within the scope of the invention as defined by the appended claims.

Claims (5)

CLAIMS:

1. A process for the manufacture of a sterol ester blend derived from a phytosterol mixture including β-sitosterol and campesterol, the process comprising the steps of: purifying the phytosterol mixture to enrich the level of sitosterol and reduce the levels of campesterol and other sterols in the phytosterol mixture; and interesterification of the purified phytosterol mixture to produce a phytosteryl ester product with enriched levels of sitosteryl ester and reduced levels of other sterols and steryl esters.

2. A process according to claim 1 in which the phytosterol mixture comprises a soya derived mixture and typically contains as major components the sterols β-sitosterol, campesterol, stigmasterol and brassicasterol and the mixture is purified by recrystallization in a solvent which favours the crystallization of the sitosterol while retaining the campesterol, stigmasterol and brassicasterol in solution or by reslurry in a solvent using gradient temperature technology, suitable solvents including ethanol or dichloromethane.

3. A process according to claim 2 in which the purified phytosterol mixture is dried to remove water from the mixture by vacuum drying or by prolonged heating at a temperature in the range of 130-150°C in a drying oven with a dry nitrogen blanket being continually bled through the drying oven to remove condensing water, and in which an alkoxide catalyst such as sodium ethoxide or equivalent is used for the interesterification reaction.

4. A process according to any of claims 1 to 3 in which the phytosteryl ester product is reslurried in dry ethanol in order to remove excess methyl ester starting material and to increase further the concentration of sitosteryl ester. IE000995

5. A process for the manufacture of a sterol ester blend derived from a phytosterol mixture including β-sitosterol and campesterol substantial as herein described with reference to the Examples.