The invention also concerns plant stanol esters and their derivatives and salts in accordance with claim 28, plant sterol and stanol dicarboxylic acid derivatives in accordance with patent claim 34, amino acid derivatives in accordance with patent claim 35, citric acid derivatives in accordance with patent claims 36-37, and plant sterol and stanol 3R hydroxybutyric acid esters and their derivatives and salts of the derivatives in accordance with patent claim 39.

The invention also concerns a method in accordance with patent claim 41 for the manufacture of plant stanol derivatives.

Plant sterols or phytosterols refer to the sterols appearing in the plant kingdom which closely resemble cholesterol in terms of structure. They are, like cholesterol in mammals, a certain structural component of external and internal membranes and thus essential constituents for the living functions of cells. Isolated plant sterols often appear in poorly soluble, crystalline form. The phytosterols appearing in nature are intrinsically fat solutions, however. Chemically, natural sterols are C26... C30 alcohols, which have an aliphatic side chain in the C-17 position.

Cholesterol (5-cholestene-3-p-ol) and its hydrogenated form cholestanol ((3ß, 5a) cholestane-3-ol) are found mainly in humans and animals. The sterols found in the animais, plants and mushrooms of marine organisms and sea-weeds form a wide variety of oxidation, double bond, methyl group substitution and C-17 side-group structures. The a configuration of the C-5 position when a hydrogen atom is linked to it is common to the natural sterols. A small number of plant stanols are also found in plants, the isolation of which is not economically profitable. Using a catalyst,

phytosterols isolated in commercial applications can be hydrogenated into corresponding stanols.

Many phytosterols (independent of origin) are closely reminiscent of cholesterol in structure. The most well-known and the most studied are e. g. p-sitosterot (24p-ethyl- A5-cholestene-3p-ol), stigmasterol (24p-ethyl-22,23-dehydro-l\5-cholestene-3ß-ol), y- sitosterol (S24 (a)-ethyl-A5-cholestene-3ß-ol), campesterol, brassicasterol, cycloartenol, 24-methylenecycloartenol and cyclobranol. The sterol that is characteristic of yeast and mushrooms is ergosterol (p24-methyl-22, 23-dehydro-#5, A'-cholestadien-3p-ol=5,7,22-ergostatrien-3p-ol; pro-vitamin D). The anticholesteric nature of the two first-mentioned phytosterols is generally known. It is possible that other sterols suited for this purpose can be found in nature, in addition to the ß- sitosterol.

Plant sterols form part of our natural nourishment. The sources of plant sterols in our diet include plant oils and the margarines made from them, white phytosterols can also be found in grain products, soy beans and rice. The regular daily diet includes 0.2-0.3 g of plant sterols.

A certain physiologically important group of compounds is formed by relatives of the sterols, the cholic acids whose role in the food digestion organs is to act as a "biological soap", as an emulsifying agent of fat and as an absorption aid. Human bile contains several cholic acids conjugated with glycine and taurine (2- aminoethanesulphonic acid); glycine conjugates are those which mostly appear.

The effect preventing the cholesterol absorption of the p-sitosterot is assumed to be based on its ability to displace the cholesterol molecule in cholic acid/fat micelle.

(Ikeda et al. 1989, J. Nutr. Sci. Vitaminol. 35: 361-369). Many pieces of research into plant sterols have also shown that crystalline phytosterols do not dissolve very effectively in the micelle phase and therefore are not able to effectively prevent the absorption of cholesterol from the digestive tract. When water is present, solubility of phytosterols in plant oils is restricted to 2 % at room temperature and to 3 % at body temperature.

According to the observations of Heineman et al. 1991 (Eur. J. Clinic. Pharmacol. 40 Suppl. 1, p. 50-63), plant sterols inhibit cholesterol absorption only in fat-soluble form. A saturated form of p-sitosterot, p-sitostanol, inhibited cholesterol absorption in an infusion test substantially more effectively than the corresponding sterol (83 % vs 50 %). In general, the plant sterols are absorbed into the blood circulation poorly and the stanols not at all. Normally, the concentration of plant sterols in serum is 1/300'h part of the serum cholesterol level. In addition, according to the observations of Miettinen et al. (US patent 5 502 045), an increase in sitostanol concentration in nutrition also lowers the dosed ß-sitosterol and campesterol concentration of the blood serum.

According to the observations of Mattson et al. (1977, J. Nutr. 107: 1139-1146), it is probable that the molecules that really prevent cholesterol absorption are free phytosterol molecules which are hydrolytically released from their esters. This claim is supported by the fact that the cholesterol absorption lowering effect of the phytosterols does not depend on the length of the carbon chain of the fatty acid.

Molar quantities of the fatty acid esters such as acetate, decanoate and oleate are equally as effective. In addition, the dicarboxylic acid esters such as phytosterol hemisuccinate lower the cholesterol content of blood as effectively as the phytosterol monocarboxyl acid esters.

The previously known methods to bring the p-sitosterot to a aqueous solution are based on the particular compound's glycosides obtainable from nature, e. g. the ß- sitosteryl-p-D-glucoside. The carrier in the aqueous solution is, for example, lecithin, while the solvent may be isopropanol and the additive isopropylmyristate. In addition, the sterols'glycoside derivatives are bonded from the alcohol solution and/or emulsion to the starch (DE publication 2113215).

GB patent publication 938 937 describes a synthetic chemical, water-soluble phytosterol hemisuccinate-polyethyleneglycol condensate which has proved, in animal tests, to be effective in lowering blood cholesterol concentrations. The publication does not, however, state an opinion on the compound's hydrolytic

decomposition in the system, or whether this kind of polyester derivative is absorbed into the blood circulation or not. The compound is planned for use as a component of various juices, soups and drinks. It is clear, however, that the widespread use of the compound in food will be limited by the ethyleneglycol-based polyether component it contains and which is foreign to the human body.

Herting and Harris (1960, Fed. Proc. 19: 18) have reported on a water-soluble soy sterol-2-carbamate glutaric acid K+-salt which has been proved to lower blood cholesterol concentrations. Carbamates can be made by e. g. a reaction between chloroformates and primary amines. The carbamine acids are unstable and break up, releasing amine and carbon dioxide. The salts of carbamine acids are much more stable, however. As the chemical environment of food varies greatly, one can justifiably assume that there will be significant restrictions to the use of the compound. The publication does not indicate whether the presented compound is planned at all for use in foods.

A rise in the blood cholesterol levels is among the more important risk factors, along with smoking and higher blood pressure, in the spread of cardiac and vascular diseases. US patent publication 5502045 describes the production of p-sitostanoi fatty acid esters and their addition to food, especially as a part of edible fats in margarine, for example. The research carried out by Miettinen et al. 1995 (New Engl.

J. Med. 333: 1308-1312) observed a decrease in the total serum cholesterol when <BR> <BR> <BR> the daily supply of plant sterol of the test individuals had stabilised at the 2-3 g level.

The test used a saturated form of plant sterol, sitostanol, whose fat solubility had been increased by the esterification process. The tendency of the plant sterols to oxidate was reduced by the hydrogenation process.

A sitostane-3p-, 5a-, 6p-triol-3,6-diformiate (SU 574 450) classifie as a stanol derivative has also been made from p-sitosterot. The compound has been proved to moderately lower the triglyceride content of the blood serum of rats and effectively lower the triglyceride content of liver (54 % in guinea pigs). Tests measuring acute <BR> <BR> <BR> toxicity and performed on white male mice did not show toxicity at a 3.5 g dose per 1 kg of the animal's live weight. The ß-sitosterol used as a source material did not give

a positive response in this test. The compound is planned for use in the prevention or treatment of fatty liver. The compound is fat-soluble. It does not dissolve very well in alcohol and not at all in water.

In patent publication EP 0 430 078 concerning cholesterols and phytosterols, a large number of (alkylene-ester/ether/amide functional hydroxyphosphine) coline hydroxide salts were made, which can be taken in doses orally in the form of tablets, capsules, emulsions, suspensions and in soluble form in order to lower the cholesterol content of blood plasma. The patent publication mentions as a synthesis intermediate stage a group of sterol and stanol hydroxy acid derivatives, namely cholest-5-en-3p-yl- (S)-2-hydroxypropionate, stigmasta-5.22-diene-3-P-yl--3- hydroxypropionate, stigmastanol-3-p-glycolate, stigmastanol-3-ß-hydroxypropionate and stigmastanol-3-P-4-hydroxybutyrate (= y-hydroxybutyrate). The aforementioned hydroxy acid esters were not used or tested, however, as nutritional additives to lower the cholesterol in blood. According to the patent publication, the synthesis of the compound group requires very powerful and toxic reagents such as phosgene, oxalyl chloride, trityl chloride or phosphoryoxy chloride, so that it can justifiably be assumed that their use is restricted to pharmaceutical preparations. The publication expresses no opinion on the behavior of sterol derivatives in the digestive system.

P-sitosterols have been fused with H02C (CH2) nCO2H (n=0-8) di-acids at a 40-50% supply and the liquid crystallization of the resulting compounds has been examined (Mukhina et al. 1977. Leningr. Khim-Farm. Inst., Leningrad USSR 24. Obshch. Khim.

47 (6): 1429-1430). The liquid crystals have been conventional applications of sterols e. g. cholesterol esters.

Previously known are the 2- (2'-alkenyl) succinates made from cholesterols and phytosterols (EP 0554 897) and their use in cosmetics. The use of the C2 C6 hydroxy acid esters of phytosterols as components of hair shampoo compositions is known from patent publication JP 09194345. Phytosterols have also been esterified with lactic acid-oligomers and the resulting steroi esters have been used in formulations usable in e. g. hand creams and lip sticks (JP 58008098).

The use of phytosterols and their natural metabolites such as deoxycoline acid ascorbic, glutaric, tartaric and lactic acid esters for medical purposes other than the prevention or treatment of hypercholesterolemia is known from patent publication DE 19701264.

The p-sitosterot has also been esterified with 2,3-dihydroxy cinnamic acid and the antioxydative properties of the compound have been studied (Takagi, T. and lida, T.

J Am Oil Chem Soc 57 (10): 326-330 (1980).

Citric acid has previously been used to make fat-soluble glyceride derivatives, which observably have been effective antioxidants in plant oil and food which has a high fat content (Qiu et al. Zhogguo Youzhi. 1996,21,3,14-18). In addition, a synergetic antioxydative effect with tocopherol has been noticed in the case of citric acid, monoacyl glycerol citrate and ascorbic acid (Aoyma et al., Yakagaku, 1985.34 (1), 48- 52.

In previously known methods a phytosteroi or phytostanol derived from soy oil is dissolved in some edible medium (US 5244887), which is used as a suspension or dispersion in food products. Generally known dispersing agents such as lecithin are used as additives.

Previously known techniques also include methods, in which hydrophobic sterols and stanols and their fatty acid esters are dissolved or fixed in e. g. fat and phospholipid emulsions (DE publication 4038385).

According to the the prior art, a fat-soluble ß-sitosterol derivative is usually made from methyl esters by transesterification. Other generally known esterification methods are the reaction of sterols with fatty acid chlorides and anhydrides. Stanols, which are hydrogenated forms of plant sterols, can be used in the manufacture of esters.

The natural tendency of sterols to oxidize is reduced chemically by the hydrogenation process. In addition, the stereochemical structure of the molecule is

altered by the hydrogenation process. In catalytic hydrogenation, the main product is the trans-fusion of the A, B rings, i. e. the hydrogen in the C-5 position is a-orientated, so that the molecule is almost flat. A small amount of C-5 position p-orientated hydrogen (cis-fusion) is obtained as a byproduct, when the molecule has a curved shape. Trans-fusion is characteristic for natural hormones and cis-fusion for cholic acid.

According to the prior art, heterocyclic derivatives of ß-sitosterol are manufactured by making the hemisuccinate of the p-sitosterot react with SOC12 and thiols, amines and phenols. The antilipemic properties of the esters obtained have been investigated, but there are no reports of the hemisuccinate of P-sitosterol being soluble in ethanol, acetic acid or water, nor of the interaction of ethanol/fat solutions of ß-sitosterol hemisuccinate with water solutions (Arch. Pharm. (Weinheim. Ger.) (1990), 3232 (7), 401-4).

A drawback with these methods, however, is the minimal possibility to alter the solvency properties of the compound. p-sitostero ! and most of its known ester derivatives are either insoluble or very poorly soluble in solutions containing water.

Because of this, it is understandable that the extensive use of plant sterols has been limited at present to plant oil products.

Even though edible fat products that reduce cholesterol are available at present, it would be advantageous, if cholesterol lowering compounds could also be obtained from other foodstuffs. For example, alcoholic beverages are often consumed together with a fatty meal with a high cholesterol content. The unhealthy effects of the meal could be mitigated by consuming, with the meal, alcoholic beverages containing cholesterol reducing compounds. Another group of foodstuffs, to which the addition of cholesterol reducing compounds would be justified, are products with an acetic acid content. Sterol compounds to be added to alcoholic beverages or products containing acetic acid should be soluble in ethanol and/or acetic acid, but they should also be able to be dissolved or dispersed in ethanol and acetic acid solutions of such strengths that they can be consumed (in alcoholic beverages, alcohol less than 80% by volume, in acetic acid products, acetic acid less that 15%

by volume). It has not been previously proposed to add cholesterol reducing compounds to alcoholic beverages or products containing acetic acid. There are also no previous descriptions of natural water-based beverages that reduce cholesterol levels.

The results of the experiments showed that B-sitosterol dissolves very poorly in concentrated ethanol solution and concentrated acetic acid. Therefore, amounts of sterol compounds that would have a cholesterol lowering effect when consumed cannot be added to alcoholic beverages or products containing acetic acid.

According to this invention, this solubility problem was solved by synthesizing phytosterol and phytostanol esters, preferably B-sitosterol and B-sitostanol esters that dissolve and/or disperse in acetic acid, ethanol and/or water, from which a sterol or stanol ester water and/or acetic acid and/or ethanol solutions or dispersions can be prepared.

The invention is based on the fact that phytosterols or phytostanols, preferably B- sitosterols or B-sitostanols, can be chemically modified using a monomeric or oligomeric polar ester group. According to the invention, B-sitosterols or B-sitostanols can be made to react with aliphatic hydroxy acids, ketoacids, dicarboxylic acids or amino acids to form B-sitosterol esters or B-sitostanol esters or produce salts of these acids, amino acid ester amine salts, or the polyolesters of the aforementioned acids, poly-or oligoesters and amide and a-amino acid amide derivatives. The esters formed are soluble and/or dispersable in acetic acid, ethanol and water.

The compounds according to the invention have a highly lipophilic sterol or stanol structure and a highly polar hydrophilic structural unit.

More precisely, it is characteristic for the phytosterols and phytostanols according to the invention that the hydroxyl group in the 3 13-position in the sterol or stanol ring is esterified with a polar substituent, for example a dicarboxylic acid, hydroxy acid or a mixed ester or amino acid, or the oligomeric polyesters of the aforementioned acids or the dicarboxylic acids or hydroxy acids mixed esters, for example with an alcohol

component or a fatty acid component.

Characteristic of the invention is that phytosterol and phytostanol derivatives contain no structural units that are foreign to the human body. More specifically, the bond between a phytosterol or phytostanol and a substituent group is always an ester bond. In addition to the ester functional group, the side group may also possess an amine or amide, which equals an amino compound found in the body, such as an amino acid or taurine (2-aminoethanosulphonic acid), a structural unit of bile acid.

In one embodiment of the invention compounds according to the invention are added to various beverages, such as soft drinks, energy drinks or alcoholic beverages.

In another embodiment of the invention compounds according to the invention are added to products that typically contain acetic acid, such as spirit vinegar, malt vinegar or wine vinegar. The compounds according to the invention are added to these vinegar products, that are made by dilution of synthetically produced concentrated acetic acid or through fermentation.

More precisely, the compositions according to the invention are those beverage compositions which are defined in the characterizing part of patent claim 1,2 and 12 and the acetic acid compositions as defined in the characterizing part of claims 14 and 15.

Plant sterol or plant stanol esters according to the invention can be added to various strong alcoholic beverages such as bitters or liqueurs, or to weak alcoholic beverages such as low-alcohol carbonated beverages. Compounds according to the invention can also be added to beverages that do not contain alcool, such as soft drinks and energy drinks.

Plant sterol or plant stanol esters according to the invention can be added to food products containing acetic acid, for example mayonnaise, salads containing mayonnaise, salad dressings, mustards, ketchup, and vinegar preserves. Citric and/or tartaric acid, or their salts, are often added to the aforementioned food products. Phytosterol citrates or tartrates according to the invention can also be

used to achieve the same effect in these food products.

Plant sterol or plant stanol derivatives according to the invention can also be added as acetic acid solutions to low-calorie vegetarian foods and foods containing mushrooms, being highly beneficial with as small an amount as possible. The nutritional value of mushrooms, for example, as dry weight is almost the same as, say, a pea, and the fat content is only 1-2%, and which are mainly polyunsaturated fatty acids. On the other hand, close to 10-15% of the dry weight may be minerals.

Mushrooms are also high in fiber (Korhonen, M. 1995. Uusi Sienikirja. Otava). The mushroom's ability to absorb liquids can be taken advantage of when making food products containing phytosterols. For example, 5g of powdered mushrooms is able to absorb up to 100ml of phytosterol solution. The method is particularly well suited for plantation mushrooms and some wild, commercially gathered mushrooms, such as Boletus edulis (Cepe), Boletus pinophilus (Pine cepe), Suillus variegatus, Cantharellus tubaeformis, Cantharellus cibarius (Chanterelle). All the aforementioned mushrooms are excellent for drying and can also be used without being pre-treated. Many of the species mentioned are also ingredients in commercial soup mixes, canned food and salads.

Compounds according to the invention, e. g. citrate and tartrate, are examples of a group of organic acids which are known to not be easily absorbed. In large doses, these organic ions have been used as osmotic laxatives. (Tuomisto, J. and Paasonen, M. 1982. Pharmacology and Toxicology (in Finnish).

Kandidaattikustannus Oy. Helsinki. pp. 526-529).

A sterol or stanol derivative group according to the invention, is based on natural compounds, which are extensively used in food products, as safe techno chemicals and materials suitable for a biological environment. They can also be synthesized economically and with minimum side products. In this case, processes producing side products means, for example the acid chloride method, in which 1 mole of sterol reactant produces 1 mole of hydrochloric acid, and in the anhydride method the moles of reactant produces the same number of moles of carboxylic acid, or the reaction of a sterol with an enol ester (e. g. propene acetate) produces the same number of moles of acetone.

Chemicaily, plant sterol and stanol esters according to the invention can be classified as hydrolyzing esters. Even though we are not able to commit to any theory regarding the action mechanism of the compounds, we can justifiably assume that the compounds according to the invention act as substances that lower the serum cholesterol level, and that, in the body, the carboxylic acids act in accordance with their characteristics.

In the present invention, derivatives of the plant sterols and plant stanols have been produced that offer a calorie-free alternative to enjoy a product that lowers blood cholesterol in soft drinks, juices and products containing acetic acid.

The invention will now be explained in more detail with the aid of the following detailed description and with the reference to a number of working examples.

The aim of the present invention is to advantageously produce a hydrophilic derivative from B-sitosterols, preferably a B-sitosterol dicarboxylic acid semiester, a B- sitosterol hydroxy acid carboxylate, a B-sitosterol hydroxy acid hemiester, a B- sitosterol polyhydroxy acid mixed ester, a B-sitosterol hemiester polyol condensate and/or a B-sitosterol-a-amino acid ester or respective B-sitostanol derivative. The aim of the invention is particularly to produce a B-sitosterol ester or B-sitosterol oligoester or polyester, which has a carboxylic acid component that is a natural compound found in the body and/or in food products and/or is useable, preferably succinic acid, maleic acid, glutaric acid, ketoglutaric acid, malic acid, tartaric acid, citric acid, lactic acid, 3 (R)-hydroxybutyric acid or an amino acid derivable from proteins, or a derivative of these, or a respective B-sitostanol compound. The polyol component is preferably a gycerol ester, but it can just as well be ascorbic acid, for example. Polyol components can also be further modified by phosphorylation, which in this case means the ester of a phosphoric acid. The carboxyl group can be further modified with, for example taurine (2-aminoethane sulphonic acid), causing the molecule to gain a strong suiphonic acid functional group.

Plant stanol esters and their derivatives are particularly preferred compounds

according to this invention.

Preferred plant sterol or plant stanol ester derivatives, particularly in products containing acetic acid, are the sulphonates, taurine amide and glycine amide, and carboxyl amides. Citrate derivatives are also possible.

Taurine derivatives are particularly preferred compounds for preparation of acetic acid solutions (acetic acid in the ratio 5-15 volume-%) which contain high sterol or stanol ester concentrations.

Mixed esters that are soluble in alcool, glycerol esters, and compounds that disperse in ethanol are preferred compounds for alcoholic beverages. The lower the concentration of alcohol in the beverage the more important is its solubility in water, and therefore particularly preferable compounds are ester salts according to this invention.

A phytosterol and phytostanol hydroxy acid ester can also be an oligomeric polyester, that is defined by the more general definition polyhydroxy alkanoate. in this case, poly (L-lactic acid) and the ester of 3R hydroxy butyric acid, 13-hydroxy butyrate.

Phytosterol and phytostanol lactic acid esters (-mono-L-lactate and-poly [L-lactic acid]) according to the invention are compounds that are soluble in ethanol and fats, which thus have a potentially wide range of application.

B-sitosterol and B-sitostanol-oligo-/poly-L-lactic acid ester is a viscose liquid between 50-100°C, which can be dispersed in water with common methods, if necessary. Regular emulsifiers used in food products, such as sugar lipids and lecithin, can be used as additives, and proteins, modified starches, hydrolyzed carboxy methyl cellulose are some examples of substances that can be used as protective colloids.

Compounds according to the invention are soluble and/or dispersable in water, ethanol and/or acetic acid and also in fats, meaning that compounds according to the

invention can be dissolved/dispersed in water and/or acetic acid phase and dissolved in the fatty component of the product.

Compounds according to the invention can be e. g. in an approx. 6% fat-containing cream liqueur dissolved both in the water/alcohol solution and in the fatty component of the product.

Hydroxy acid esters of phytosterols, such as citrate and tartrate, can be modified by e. g. mixed esterification, when the COOH-group is esterified using either an aliphatic alcohol or polyol.

Hemiesters of phytosterol and phytostanol can be modified by mixed esterification with aliphatic alcohol or polyol.

The most preferred polyol component is glycerol, though it can also be ascorbic acid.

According to preferred embodiments of the present invention, phytosterols and phytostanols are esterified with citric acid, succinic acid, glutaric acid, lactic acid or 3R-hydroxy butyric acid.

According to this invention, phytosterol and phytostanol dicarboxylic acid derivatives such as potassium, sodium and ammonium salts of steryl dicarboxylic acid hemiesters, steryl dicarboxylic acid carboxy amides, glycine and taurine derivatives of steryl dicarboxy acid hemiesters, and their salts, are particularly preferred compounds for adding to various alcoholic beverages or vinegar products.

Dicarboxyiic acid derivatives of plant sterol or plant stanol have the formula: S-02CRC02Y (I) in which S = a plant sterol or plant stanol R = C2... C6 carbon chain; Y = C2Hs or-CH2CH (OH) CH20H or = K+, Na+ or NH4+

or S and R as above and Y=-C(C02Y,) (CH2Co2Y2) (CH2Co2Y3), when Y1=Y2=Y3=H or -C2H5 or K+, Na+ or NH4+ or the formula: S-O2CRCONH2(II) in which S and R are the same as above or the formula: S-O2CRCONHCH2CO2Y(III) in which S and R are the same as above and Y=Hor=K+, Na+orNH4+ or the formula: S-O2CRCONHCH2CH2SO3Y (IV) in which S and R are the same as above and Y = H or = K+, Na+ or NH4+ According to this invention, steryl or stanyl dicarboxylic acid glycerol mixed esters are preferred phytosterol or phytostanol dicarboxylic acid and polyol mixed esters.

S-O2CRCO2CH2CH(OH)CH2OH(V) in which S and R are the same as above.

Preferred compounds according to this invention are amino acid esters of phytosterols or phytostanols, which have the formula:

S = a plant sterol or plant stanol R= H or a naturally occurring structure in L-amino acids or the amine group is in quaternary form, meaning that the anion is a chloride, sulfate, sulfite, phosphate, phosphite or acetate.

According to this invention, particularly citric acid esters of phytosterol and phytostanol, their derivatives and salts are preferred compounds for adding to various alcoholic beverages or products containing acetic acid, and have the formula: S = plant sterol or plant stanol R1=HorK+,Na+orNH4+andR3=HR2 or R, = R2 = H and R3 = C2... C22 carboxylic acid residue or R, = R2 =-C2Hs and R3=H or R, = R2 =-C2Hs and R3 = C2.. C22 carboxylic acid residue or R, = Rz =-CH2CHOHCH20H and R3 =H or R, = R2 =-CH2CHOHCH2OH and R3 = OCCH3

Therefore, preferred citric acid esters include for example steryl dipotassium, disodium and diammonium citrate, steryl acyl citrate, steryl diethyl citrate (R, = R2 = C2H5), steryl acyt diethyl citrate (R, = R2 =-C2H5 and R3 = C2.. C22 carboxylic acid), steryl citrate glycerol ester (R3 = H, R, = R2 = -CH2CHOHCH2OH), steryl acetyl citrate glycerol ester (R3 = OCCH3) and the steryl poly (citric acid) with the formula: S = plant sterol or plant stanol R = H or-C2H5 or K+, Na+ or NH4+ or -CH2CHONCH2OH 2-10n= According to this invention, particularly preferred amino nitrogen-containing citric acid derivatives are steryl citrate dicarboxyamide, steryl citrate diglycine amide and steryl citrate ditaurine amide, with the following structures:

S = plant sterol or plant stanol According to the present invention, dicarboxylic acid and hydroxy acid esters of phytosterol and phytostanol are classed as hydrolyzing esters, whose properties can be assumed to differ from known and commercial stanol esters. Factors known from general organic chemistry affect the speed of hydrolysis of different esters. Steric and electronic properties are the most important. A substituent in the ester's side chain that draws electrons, for example, increases the speed of hydrolysis. On the other hand, a side chain substituent group that donates electrons, such as an alkyl group, decreases the speed of hydroiysis. Thus, poly (hydroxy succinate) is hydrolyzed faster than poly (2-S-hydroxy propionate) for example.

Esterification of citric acid and phytosterol or phytostanol in different conditions produces different quantities of di-B sitosteryl or di-B-sitostanyl citrate. Due to its poor solubility ( « 0.5% in 96.4% ethanol), the compound can be easily separated from the steryl or stanyl monocitrate produced in the same reaction system. Disteryl and distanyl citrates can be partially hydrolyzed in a water solution or a water-ethanol solution with excess of alkali (theor. 1 mol: 2 mol disterol citrate: alkali) to a respective sterol monocitrate salt and free sterol, which will not dissolve in the water phase.

If required, the water solubility of the plant sterol citrates according to the invention can be improved by preparing salts from citrates, and ethanoi solubility can be

improved by preparing ethyi citrates.

Citric acid esters and their derivatives are already widely used in food products.

Citric acid is used in various drinks to regulate acidity, the product's scent and taste can be emphasized with the aid of citric acid, preservation time can be improved and, as mentioned earlier, citric acid has properties which prevent oxidation. Citric acid esters of phytosterol, and their derivatives are particularly preferred in products containing ethanol and acetic acid, because the fresh citric acid taste suits many drinks and vinegar products.

Preferred compounds according to the invention tlso include esters formed by phytosterol or phytostanol with 3 R-butyric acid or lactic acid. These are soluble in ethanol, acetic acid and also fats, which increases their potential uses.

Alternatively, liquid B-sitosterol or B-sitostanol esters (preferably lactic acid esters) can be dispersed in water (preferably to form a 10-40% emulsion) and then be added in the required concentration to the food product being prepared.

A"water and/or ethanol solution"refers to a clear, water-ethanol solution which contains dissolved B-sitosterol or B-sitostanol esters. Such a solution contains approximately 60-96.4 volume-% of ethanol. A concentrated ethanol solution preferably contains 0.5-30g/l of B-sitosterol or B-sitostanol esters.

"Plant sterol and/or stanol"refers to sitosterol, stigmasterol, campesterol, brassicasterol, cycloartenol, 24-methylene cycloartenol and cyclobranol and/or their hydrated forms, and mixtures, and which have a serum cholesterol lowering effect.

Even though the terms B-sitosterol or B-sitostanol are used in conjunction with this invention, it also includes other plant sterols and/or stanols which have a cholesterol lowering effect.

A"water and/or ethanol dispersion"refers to a water or water-ethanol dispersion in which B-sitosterol or B-sitostanol esters are dispersed. When B-sitosterol or B- sitostanol esters containing concentrated, over 60 vol-% ethanol, solution is diluted

with water, the esters begin to form dispersions in the water-ethanol solution, forming a milky emulsion. It is preferable to have 0.1-20g of B-sitosterol or ! 3-sitostanol esters per liter of dispersion.

A"water and/or acetic acid solution"refers to a clear, 0-15 weight-%, preferably 4-10 weight-% acetic acid solution with dissolved B-sitosterol or B-sitostanol esters. In such a solution, it is preferred to have 0.1-20g/l of B-sitosterol or B-sitostanol esters in solution.

A"water and/or acetic acid dispersion"refers to a water or water-acetic acid dispersion in which B-sitosterol or B-sitostanol esters are dispersed. In the water and/or acetic acid dispersion, the B-sitosterol or B-sitostanol ester concentration can preferably be up to 100g/l.

B-sitosterol or B-sitostanol esters can be dissolved and/or dispersed in water or in a weak 0-15 weight-%, preferably a 4-10 weight-% acetic acid solution.

Alternatively, B-sitosterol or B-sitostanol esters can be dissolved and/or dispersed in a concentrated 100 weight-% acetic acid solution that is diluted with water to a 4-10 weight-% acetic acid solution. Such a solution contains approximately 0.1-100 9/l, preferably 1-70 g/I of B-sitosterol or B-sitostanol esters. Depending on its use, a 4 -10 weight-% acetic acid solution for nutritional use may contain 0.5-30 g/i of B- sitosterol or B-sitostanol esters. An acetic acid solution or dispersion for industrial use may contain 70-100 g/I of B-sitosterol or B-sitostanol esters, for example.

By a"dicarboxylic acid"here is meant an organic acid that has two carboxyl groups (-COOH). Suitable dicarboxylic acids in this invention are, for example, glutaric acid and its derivatives, and succinic acid. Maleic acid is an example of an unsaturated dicarboxylic acid.

In an esterification reaction, the-OH group of the carboxylic acid is released and forms water, while the alcool, in this case B-sitosterol or B-sitostanol, reacts with the carboxylic acid residue to form an ester.

By a"hemiester"here is meant that only one of the dicarboxylic acid's-COOH groups has reacted with the alcool.

By a"ketoacid"here is meant an organic acid that contains a keto-group in the hydrocarbon structure of the acid. Ketoglutaric acid is an example of a ketoacid.

By a"hydroxy acid"here is meant an organic acid that contains a hydroxyl group in the hydrocarbon structure of the acid. In this invention, suitable hydroxy acids include malic acid, tartaric acid, citric acid, lactic acid, and 3 (R)-hydroxy butyric acid.

By an"amino acid ester of B-sitosterol"is meant, for example B-sitosterol glycinate (=B-sitosterol amino acetic acid ester). The amino group may, of course, have originated from any amino acid, poly (amino acid) or peptide.

By the"salts of the hydroxy acid, ketoacid, dicarboxylic acid or amino acid esters of B-sitosterol or B-sitostanol"is meant that at least one-COOH group is in the form -COO-M+ (M = Na+, K+ or NH4+'.

By the"amine salts of the amino acid esters of B-sitosterol or B-sitostanol"is meant that the amino group is e. g. in the form-NH3+X-.

By an"amino acid ester of B-sitosterol"is meant, for example, B-sitosterol glycine (=B-sitosterol amino acetic acid ester). The amino group can, of course, originate from any amino acid, poly (amino acid), or peptide.

By an"a-amino acid amide of B-sitosterol hemiesters"is meant, for example, N- (B- sitosteryi succinyl)-glycine. The free-COOH group of the amino acid component can, of course, originate from any amino acid, poly (amino acid), or peptide.

A"B-sitosterol oligo/polyester"refers to the formation of a B-sitosterol ester from a diester or oligoester, which was formed from numerous hydroxy acids or dicarboxylic acids or amino acids. One example of such an ester is the B-sitosterol succinate (acyl glycerol) ester.

Rsc-TISED SHEET (RULE 91)

"Polyol"refers to a compound containing at least two hydroxyl groups. Glycerol is an example of a polyol.

By"poly/oligoesters of B-sitosterol or B-sitostanol"it is meant that B-sitosterol has been esterified with, for example lactic acid which has at least two L-lactic acid units.

"Phytosterol/stanol polyhydroxy alkanoates"refers to the following, common chemical structures: sterol 0 R 11 1 Or-o--C--n stanol or sterol) O 11 or'-0- (-C \ 0-)-. CH3 CH3 stanol R=C,.. C6 alkyl n=1-10 By"alcoholic beverages"here it refers to beverages that contain ethanol. They include beverages prepared from both fermented or distilled ethanol. The ethanol concentration can vary between > 0 vol-% to 80 vol-%.

Beverages other than alcoholic beverages refers to aqueous beverages that do not contain ethanol. Examples of these include carbonated and non-carbonated soft drinks, berry and fruit juices, nectars, and so-called sports drinks. It is particularly preferable to add compounds according to the invention to citrus fruit-based beverages.

According to this invention, the composition of the beverage can contain water and 0 -80 volume-% ethanol. Following quantities of the other components can also be in the beverage: >0-3 weight % of the hydroxy acid, dicarboxylic acid or amino acid ester of B- sitosterol or B-sitostanol.

>0-40 weight % of sugar or artificial sweetener of the respective sweetness, for example aspartame or acesulfamine K >0-15 weight % of aromatic substances (various extracts, distillates), and >0-1 weight % of colors.

Of the components listed above, it is assumed that the beverage will at least have water as a liquid medium, the hydroxy acid, dicarboxylic acid or amino acid ester of B-sitosterol or B-sitostanol, and preferably also aromatic substances.

The amount of hydroxy acid or dicarboxylic acid or amino acid ester of B-sitosterol or B-sitostanol can be dosed into the beverage according to the estimated daily consumption of the beverage per person. For example, a soft drink or alcoholic beverage that has an ethanol content of approximately 2-7 vol-% can have quite a low amount of sterol ester or stanol ester, say 0.1-3.0 g/I, compared to a stronger alcoholic beverage of 20-40 vol-%, which would have a daily consumption of 2 restaurant drinks at most, and have a sterol ester or stanol ester content of 30 g/l at most.

"Acetic acid composition"refers to products containing acetic acid that can be used for nutritional purposes. These acetic acid preparations can be made by either fermentation or dilution of a synthetic, concentrated acetic acid. The acetic acid concentration can vary from > 0 weight-% to 15 weight-%, preferably 4-10 weight- %.

According to this invention, the acetic acid composition can contain 1-10 weight-% of hydroxy acid, ketoacid, dicarboxylic acid or amino acid esters of ß-sitosterol or ß- sitostanol, the salts of said acids, or the amine salts of the amino acid esters of said

acids, or the polyol esters of said acids, poly/oligoesters or amide or a-amino acid amide derivatives.

Food products containing acetic acid refers to such food products that contain an acetic acid component. Food products of this type include mayonnaise, salads containing mayonnaise, salad dressings, mustards, ketchup, and canned vinegar products.

A B-sitosterol or B-sitostanol ester according to the invention can be added, for example, to preservative liquid, which also contains water, 4-10 weight-% acetic acid, sugar, and spices. Composition of such a preservative may contain, for example: 4-10 weight-% acetic acid 0.01-3 weight-% citrate derivatives, tartrate derivatives, or hemiester amides 40-80 volume-% sugar (e. g. aqueous starch sugar): dry constituents 70% 1-2 weight-% mixed spices (mustard seeds, bay teaves) The hydroxy acid or carboxylic acid esters of B-sitosterol or B-sitostanol can be dosed into an acetic acid composition or a food product containing acetic acid according to how much it is estimated that a person will consume of this vinegar preparation or nutritional substance containing acetic acid. In a vinegar preparation, for example, that has an acetic acid content of approximately 4-10 vol-%, and it is estimated that a person will consume a maximum of 2-3 dl daily, the amount of sterol ester or stanol ester may be 0.5-20 g/l. If the vinegar preparation is such that only a few tablespoons of it is used per person, the sterol ester or stanol ester content can be 70-100 g/l, for example. It is aimed that the daily consumption per person will be 2 -3 g of the sterol or stanol ester according to the invention.

A concentrate for industrial use of the compounds according to the invention can be prepared for adding to food products. The concentrate can be prepared for example by using a commoniy known drying method, such as lyofilization or evaporation, from the salt of the appropriate sterol or stanol ester.

The invention is based on the surprising observation that the regular chemistry based on the direct esterification and/or transesterification of alcohol component used in the formation of polyesters (e. g. alkydes) can be used in the preparation of B- sitosterol or B-sitostanol esters that have a hydroxyl, carboxyl, amino, amide or ester functional group.

One distinctive characteristic of the method according to the invention is that B- sitosterol or B-sitostanol are esterified in a one-step process with a dicarboxylic acid, ketoacid, hydroxy acid or amino acid, meaning that there will be at least one carboxylate, keto, hydroxyl, or amino group, or a combination of the aforementioned functional groups in the ester's side chain. Also distinctive is that the sterol/stanol esters either dissolve or disperse in polar solvents, preferably into water, ethanol and/or acetic acid, and in this form they can then be used as components in various beverages, such as soft drinks or alcoholic beverages, food products containing acetic acid, or products containing vinegar or that sterol/stanol esters are also soluble in fats or fat emulsions, in which form they can be used as a structural component of food products prepared from fats or fat emulsions.

Hemiesters and polycarboxylic acid esters of B-sitosterol react in the same way as carboxylic acids, e. g. with ammonia and amines to form respective amides. Thus, any scientifically known method, such as the dehydration of carboxylammonium or amine salts, ester aminolysis, carboxylic acid chloride methods etc. can be used in the preparation of amide-functional sterol or stanol derivatives.

One very simple example of an amide derivative of B-sitosterol is B-sitosterol succinate carboxy amide.

It is known that in natural compounds polar groups greatly affect the water and alcohol solubility of the compound. One such group is the steroid-structure bile acids. For example, at 15°C, 0.33 gel of N-cholyl glycine dissolves in water. At the same temperature, 274 g/l of a respective Na salt will dissolve in water. One example of a synthetic, water-soluble sterol derivative is cholesterol PEG-900 (polyethylene glycol 900 mono (cholesteryl) ether sebacate). It is also commonly

known that tartaric acid-based polyesters can be water soluble.

B-sitosterol can also be esterified with an amino acid derivable from a protein, after which an amine salt is prepared with an acid, for example sulfate, sulfite, phosphate, phosphite etc. Of the organic acids, sulphonic acid, acetic acid etc. can be used for this. a-aminoacid derivatives with amide functional groups can also be made from ß- sitosterol derivatives, and their salts, preferably Na+, K+ or NH4+ can be used to increase solubility and/or dispersibility in water and/or acetic acid.

In principle, the amino acid esters of ß-sitosterol or p-sitostanot can be made using any generally known peptide-synthetic method, for example, the activated esters (p- nitrophenyl, hyrdoxy succinic imide, pentachloro phenyl esters) of amino acids together with an N-protection (for example, BOC = tert-butyl oxycarbonyl and benzyl oxycarbonyl). It is preferred to use benzyl protection, because as a powerful protective group it permits, for example, transesterification when making sterol esters. Benzyl protection is particularly preferred for a secondary amino group. L- proline and 4-hydroxy-L-proline are examples of such groups. In addition, benzyl protection can be removed by hydrogenation.

Amino acid esters according to the invention include p-sitosterof a-amino carboxylates and the a-amino acid amides of p-sitostero ! hemiesters.

According to one preferred embodiment of the invention, the ß-sitosterois are hydrogenated using generally known methods into corresponding stanols and the esterification reactions are carried out only after this, using the stanols.

Compounds according to the invention are manufactured by combining the reactants in the desired molar ratios (batch process) and removing water (preferably azeotropically or by molecular sieving), alcool, or other volatile condensation products from the reaction mixture by distillation, giving a high yield of sterol or stanol esters that are soluble in acetic acid, ethanol and water.

According to instructions in literature, (Fieser & Fieser, 1967. Reagents for Organic Synthesis, Vol. 1. p. 799) sterols can be hydrogenated, at a high yield and in moderate reaction conditions, to form stanols (0.02% PtO2 of the mass of the sterol.

EtOAc, H2/103kPas, 40-500C, 1/2 h, acid co-catalyst). The reaction products are 88% sitostane-3p-ol, 3.4% sitostane-3a-ol and 0.9% sitostane. In addition to this, 1.6% sitostane-3p-acetate is obtained from the exchange reaction.

A large number of heterogenic and homogenic hydrogenation catalysts and reagents are known in organic chemistry (e. g. March, J. Advanced Organic Chemistry, Reactions, Mechanism and Structure, 4 edition. Chapters 5-9 and in the notes to them), by means of which the double bond of a sterol can be saturated without converting the functional group of the 3p-substituent, which can be, for example OH, COOH, CONHR or COOR.

The esterification of a ß-sitosterol or p-sitostano ! is carried out at a high temperature 110-280C, either by a direct esterification method, in which water is removed azeotropically from the reaction mixture, or by transesterification, when some volatile condensation product is distille from the reaction mixture. Hemiesters (for example, succinate and glutarate) are more economically manufactured directly from the corresponding cyclic anhydride at a temperature of 90-200oC.

In esterification based on the removal of water, it is technically possible to use numerous azeotrope systems, advantageously hydrocarbons, such as cyclohexane, toluene or technical xylene. The task of the aforementioned solvent in the reaction mixture is not only water removal, but also the control of the temperature of the reaction mixture. More precisely, the temperature of the reaction mixture is controlled by adjusting the ratio of the weights of the solvent and the reactants to 15-50% of the mass of the reactants.

Thus, when esterifying at a temperature of less than 1400C, it is preferred to use cyclohexane or toluene as the solvent and xylene at higher temperatures.

Technically, it is also preferred to allow water, which is possibly in the reactants, to

leave as an azeotrope, before arriving at the reaction temperature; thus a separate drying stage for the reactants may be unnecessary.

In direct esterification, it is also preferred to use a strong acid as the catalyst, which need not be removed from the reaction mixture. Esterification with cyclic anhydrides can also be carried out thermally without a catalyst. Esterification can be carried out with cyclic esters, such as lactons, glycolides and lactides, using the conventional methods known to the literature of chemistry.

The catalyst used in transesterification can be equally well strong acids (TsOH (= paratoluene sulphonic acid), H2SO4, H3PO4, etc.), alkalis (NaOH, KOH, NaOEt, t- BuOK (= potassium tert-butylate) etc.) or the generally used weak Lewis acids. From the point of view of the intended application of the present invention, it is not, however, preferred for metals to appear in the reaction mixture, except for Na, K, Ca, Mg. Generally, it is not preferred from the point of view of the intended application for halogenides to appear in the reaction mixture.

In direct esterification, it is also preferable to use some strong acid that need not be removed from the reaction mixture. Esterification with cyclic anhydrides can also be carried out thermally, without a catalyst, for example, using lactons together with either an acid or a base catalyst or using a lactide.

The hydrophilic properties and ethanol and/or acetic acid solubility of the sterol derivatives described above is an extremely useful property. Stable dispersions can be made simply by diluting strong ethanol solutions of sterol esters with water.

Strong ethanol solutions may preferably contain 0.5-30 g/l of the esters according to the invention. Stable water/acetic acid solutions/dispersions can be made simply, either by diluting strong acetic acid solutions of the sterol esters with water or by dissolving and/or dispersing in water/acetic acid solutions, the acetic acid content of which is 0-15% by weight.

When making aqueous solutions and/or dispersions, the ethanol can, if necessary, be removed by vacuum evaporation. The solution or dispersion can be assisted by well-known emulsifiers used in food products, such as fatty acid derivatives of

sugars or sugar alcools, phospholipids, or lecithin. In most cases, no emulsifier is required, as the P-sitosterol or p-sitostano ! hemiesters are emulsified as such with the sodium, ammonia and/or amine salts of an alcohol solution containing water.

In principle, there is no obstacle to fermenting the dispersions in a ratio of 10-15% to the ethanol and/or to their chemicai oxidation to form their respective acetic acid solutions. As this is an oxidative reaction system, it is preferrable that the compounds according to the invention are stanol derivatives.

Commonly known emulsifiers used in food products, such as fatty acid derivatives of sugars or sugar alcools, phospholipids, or lecithin, can be used as additives for dissolving and dispersing. In most cases, no emulsifier is required, as ß-sitosterol or ß-sitostanol hemiesters are emulsified as such into water-based acetic acid solution as their sodium, ammonium, and/or amine salts.

In the preparation of various dispersions, it is sometimes preferred to select a sterol or stanol esterification method and starting materials so that the functional group of the sterol or stanol side chain is a neutral hydroxy carboxylic acid ester. In such cases, the preferred starting materials are the ethyl esters of hydroxy acids, which are transesterified with a sterol or the hemiester of the sterol reacts with some polyol, preferably glycerol.

Sterol and stanol esters can be purified with cold precipitation from an organic solvent. Therefore, according to the present invention, the sterol is esterified directly with a carboxylic acid using a water removal system based on azeotropic distillation and/or molecular sieves. If required, as a continuation process sterol can be converted to its respective stanol by hydragenation in ethanol. Sterol and stanol esters are dispersed in a water-based alcohol solution by diluting their alcohol mother liquor, which is preferably a strong alcohol solution, for example a 96.4% ethanol solution.

According to one preferred embodiment, phytosterol/stanol oligomeric lactates are manufactured from compounds according to the invention by non-catalytic

esterification at a temperature of 155-120C, starting from a 90% L-lactic acid water-based solution and phytosterols or phytostanols, with toluene acting to form a water azeotrope. Under such weak reaction conditions, it is justified to assume that the risk of racemization of the lactic acid is very small.

According to one preferred embodiment of the invention, p-sitosterots are esterified and sterol esters are hydrogenated to form stanols. Because, for example sterol lactates are highly soluble in alcool, a sterol ester can be hydrogenated to directly form its respective stanol ester. This achieves the significant advantage that hydrogenation can be carried out at room temperature. As the solubility of phytosterol in alcohol at 20OC is < 1 %, phytosterols cannot be hydrogenated at similar conditions to form stanols. Sterols must be hydrogenated to form stanols in ethanol at 65-770C.

Sterol esters that are soluble in acetic acid can be hydrogenated in concentrated acetic acid to form stanols. It is practical for the hydrogenation to be carried out directly in an ethanol or acetic acid solution, which will be used later in the manufacture of beverages or vinegar products.

The following examples illustrate the invention. They do not, however, limit the patent's scope of protection.

Example 1 The starting material for compounds according to the invention was a plant sterol mixture (Weinstein Nutritional Products), which was composed of 45-55% P- sitosterol, 20-30% campesterol and 15-25% stigmasterol. The lactic acid used in the formation of the lactate was Purac heat-stable L-lactic acid, with a lactic acid content of 88%. The citric acid was a commercial citric acid monohydrate (food product quality). In the preparation of the derivatives, the azeotropic solvent used was technical-grade toluene, and the solvent used in the hydrogenation of sterol was ethanol (Primalco Oy 96.5 volume-% ethanol). The hydrogenation catalyst was 5%

Pd/C (Aldrich).

Example 2 Hydrogenation of ßsitosterol 5.0 kg of ethanol (96.4 volume-%) and 0.2 kg of p-sitostero ! was mixed in a 10-liter reactor. The reactor was flushed thoroughly with nitrogen twice, after which 1-3.5 g of hydrogenation catalyst (Pd/C) suspended in ethanol was added to the reactor.

After this, a vacuum was created in the reactor twice. Hydrogen was fed into the reactor while the temperature was increased slowly to 54-57 oC. When it appeared that hydrogen was no longer being consumed (the pressure did not decrease), the hydrogen pressure was released, the reactor was flushed with nitrogen, and the reaction mixture was filtered while hot to remove the catalyst.

Once the reaction mixture had cooled, stanol began to crystallize from the solution.

The solvent was evaporated to approximately half the original volume, and the remaining stanol was crystallized by cooling the reaction mixture at 10 oC. The product was air-dried at room temperature. Individual hydrogenation tests showed process technical differences, which have been recorded in Table 1.

Hydrogenation was carried out in a 10-liter BUCH pilot reactor. The vacuum- nitrogen flushing cycles were carried out before the reaction. In the hydrogenation experiments, hot water and/or steam heating were used instead of thermal oil. H2 flow measurement was used.

Table 1 Test no. sterol (kg) EtOH (kg) Pd/C10% Pd (g) reaction conditions 1 0.2 3.9 3.0 H2: 1-2 bar, 430C, 4h, 4xH2 charge < 2 bar 2 0.2 4.7 3.0 H2: 1-2 bar, 770C, 4h, 4xH2 charge < 2 bar

3 0.2 4.7 3.0 H2: 1-1.5 bar, 770C, 3h, continuous H2 feed 4 0.2 4.7 1.5 same as previous Composition of the products of hydrogenation: 1.77% stanols, remainder sterols, no stigmastane/campestane 2. 78% stanols, no sterols, remainder stigmastane/campestane 3. 84% stanols, no sterols, remainder stigmastane/campestane 4. 93% stanols, no sterols, remainder stigmastane/campestane Example 3 Preparation of p-sitostero) hydrogen succinate The following substances were allowed to react: 9.0 g p-sitosterot 2.15 g succinic acid anhydride 1.7 g pyridine 20 mi toluene The mixture was heated intermittently for 5 h. The toluene was evaporated using a <BR> <BR> <BR> <BR> vacuum. 2.5 mi of 1 N HCI dissolved in 50 mi ethanol (96.4 volume-%) was added.<BR> <BR> <BR> <BR> <BR> <BR> <P>The white precipitate formed was filtered, filter-washed with a 2: 3 water-ethanol solution, and finally with distille water. The product was dried by gentle heating.

Example 4 ß-sitosterol citrate The following substances were allowed to react: 50.0 g ß-sitosterol

66.0 g citric acid monohydrate 100.0 ml xylene + an acid catalyst, such as paratoluene sulphonic acid, H2SO4 or H3PO4 The mixture was isothermally heated for 6 hours in nitrogen atmosphere at 1450C, or until the xylene circulating in the water separator clarified in the final stage of esterification. The cooled reaction mixture was a viscous reddish liquid. The xylene was evaporated by vacuum evaporation. The waxy reaction product was dissolved in ethanol to a concentration of 5-30 weight-%. The reaction product was precipitated as a yellowish wax using cold precipitation at a temperature of +/-0- 400C.

Example 5 P-sitosterol citrate; heterogeneous reactions Sterol, citric acid and toluene were mixed in a flask. The temperature of the reaction mixture was increased to boiling point, and the water collecte with a water separation device, to monitor the progress of the reaction. A catalytic amount of phosphoric acid was added to the reaction mixture. In the early phase, the reaction mixture was heterogeneous, but as the reaction progressed it changed into a one- phase mixture. After the reaction, the toluene was evaporated from the mixture using a rotavapor, and the product was dried in a vacuum oven at 40-600C. Table 2 shows, for the various experiments, the reaction conditions, the molar ratios of the starting materials, and the quantity of reaction water formed.

Table 2 Experiment Starting materials (g) molar ratio reaction conditions reaction water, ml (in mols) 1 100.0 sterol 1/3 12 h: 1100C, 3 h: 135-1490C 9.0 ml (0.5 mol) 149 citric acid 2 100.0 sterol 1/3 32 h: 110oC, 3 h: 140-1500C 20.0 ml (1.1 mol) 149 citric acid 3 100.0 sterol 1/4 11h30 min: 110-1200C 11.0 ml (0.6 mol)

184 citric acid 4 140 citric acid (btank) 40 h: 110OC 10-1.5 ml Example 6 P-sitosterol citrate from citrate condensation <BR> <BR> <BR> <BR> <BR> 185 g of anhydrous citric acid and 300 mi toluene were mixed in a 1000 cm3 flask, and 8 ml of water was azeotropically distille from the reaction mixture during the overall reaction time of 36 hours. The amount of water collecte during this time represents 45% of the total number of moles of citric acid. Next, 100 g of p-sitosterot (which represents 0.24 mol) was added, and the water distillation was continued until 8-1 Om ! more had been collecte, after which the reaction was finished. The raw product was treated as described in the previous example. Using this method, more alcohol-soluble citrates were obtained than in experiments 1,2 and 3 of the previous example.

Example 7 Di- (p-sitosteryt) citrate; heterogeneous reactions Di-p-sitosteryl citrate is formed in a heterogeneous reaction between citric acid and P-sitosterol, at the same reaction conditions as described in the previous section.

The compound is separated from the sterol monocitrate on the basis of solubility, using ethanol extraction. The method is based on the fact that the solubility of sterol monocitrate in ethyl alcohol is ten times that of di-p-sitosterol citrate.

Example 8 Preparation of sterolacetylcitrate in a homogenous reaction mixture

Crystalline citric acid dihydrate (100 g, 15.7% hydrate water), 100% acetic acid (400 g) and acetic anhydride (156.5 g) was mixed in a 1 liter volumetric flask, and heated intermittently for 4 h at 1200C. Next, 50 g of p-sitosterot was added to the reaction mixture. The solution was heated intermittently for 4-5h at 120OC and allowed to cool at room temperature. The white precipitate was separated by filtration. The filtrate was diluted with 5 liters of water, and a yellowish precipitate was formed. The precipitate was filtered and washed in the filter with water. The product was dissolved in ethanol. The light colored precipitate (1.3 g) was filtered. The actual reaction product (yield 16 g) was obtained as waxy crystals after the ethanol was evaporated. The advantage of the preparation method described in this example is that the product can be produced in acetic acid without using other solvents.

Example 9 Citric acid ester sterol condensate Ethyl-p-sitosteryl citrate The following substances were allowed to react: 50.0 g p-sitosterot 66.0 g triethyl citrate + catalyst such as paratoluene sulphonic acid, H2SO4, H3PO4, NaOH, KOH. NaOEt, t- BuOK or Lewis acids Ethanol was distille from the reaction mixture using nitrogen gas at 160-2500C for 3 -6 h. The cooled reaction mixture was a viscous reddish liquid. The sterol ester was purified by cooling the 5-15 weight-% ethanol mother liquor of the reaction product to a temperature of +/-0--400C, when a light-colored product was obtained as a waxy precipitate.

Example 10 Citric acid reaction with ß-sitosteryl, non-catalyzed I

25.0 g p-sitosterot, 50.0 g citric acid monohydrate and 150 mi toluene were heated intermittently for 6 h at 110-112oC. At this point, approximately 5 ml of water was collecte in the water collector.

The surplus, unreacted citric acid was removed from the cooled reaction mixture by filtration, the citric acid was washed in the filter with toluene, and the filtrates were combined. The toluene was removed by distillation at 60-80OC at a reduced pressure in a vacuum (29 mmHg). The yield was approximately 30 g.

Example 11 Citric acid reaction with p-sitosteryl, non-catalyzed 11 50.0 g p-sitosterot, 100.0 g citric acid and 200 ml cyclohexane were heated intermittently using a water separator for a minimum of 20 hours. The hydrate water (approximately 8.5 mi) was separated at the start of the heating. After this, water formed very slowly (theoretically 2.2 ml).

Heating was continued until the small droplets of water disappeared from the cyclohexane returning from the condenser, or at least until 1.5 ml of condensation water had been separated.

Initially, the cyclohexane was removed by distillation at normal pressure, and then at a reduced pressure (approximately 30 mmHg). Any remaining solvent residues in the solidified reaction mixture were evaporated in a vacuum (0.3 mmHg).

The unreacted citric acid was removed by extraction with water at room temperature.

The light-colored, yellowish, poorly soluble precipitate was filtered and dried. The yield was 60.0 g. The raw product was purified by dissolving it in 1000 mi 96.4 volume-% ethanol at room temperature. Any unreacted p-sitosterot was separated from the ethanol solution by filtration.

Useable product was obtained from the uncatalyzed ß-sitosterol reaction with citric acid, showing the usefulness and development potential of this preparation method.

Example 12 Acid catalyzed citric acid reaction with p-sitosteroi The following substances were allowed to react: 50.0 g p-sitosteroi<BR> 33.0 g citric acid monohydrate<BR> 54.0 g cyclohexane 2.9 mi of hydrate water was distille from the reaction mixture at a temperature of 80 -820C. At this point, the cyclohexane began to circulate clear in the water trap. The esterification stage was started by adding 200-300 mg of orthophosphoric acid.

Water distillation was continued for 8 hours at the original temperature, during which time 2.5 ml more water collected (theoretically 50.0 g/414 gmol-'x 18.0 mol-'= 2.2 ml H20). The reaction mixture, which was fluid during heating, changed into a viscous mass.

Using this method, at least 44% of the sitosterol would have reacted to form alcool- soluble citrates.

Example 13 P-sitosterol citrate salts While stirring, drops of 25% ammonia solution were added to the raw product (p- sitosterol citrate from example 12), which was to a 96.4% volume-% ethanol solution containing 6-10 weight-% of citrate, when a white, cheese-like precipitate began to form immediately. The precipitate was collecte on the filter using a partial vacuum and was flushed with cold ethanol. Next, the precipitated sterol citrate ammonium salt was dried in a vacuum using gentle heat. The dry product was a white powder.

The composition of the primary elements was 60.6% C and 8.1 % H, with an approximate molecular formula of C41H64013, i. e. a sterol substituted citrate dimer.

The composition of the primary elements of the ammonium salt was 62.05% C, 9.04% H, 3.63% N, which is represented by the molecular formulaC4, H68013N2, i. e. a sterol substituted citrate dimer ammonium salt.

Potassium salt is prepared similarly from the p-sitosteroi citrate in example 6, by adding 0.5 N KOH.

Example 14 <BR> <BR> <BR> <BR> <BR> <BR> A mixture containing 5.0 g of di-betasitosteryl citrate suspended in 300 g of water<BR> <BR> <BR> <BR> <BR> <BR> and 0.4 g of KOH was heated at 90-100OC for 5 h, after which the white precipitate was allowed to settle. Titration of the clear liquid with a 0.5 N HCI solution and phenolphthalein indicator to neutral showed that 86% of the KOH had reacted with the sterol. 0.9 g of dissolved material, was observed in the water solution and precipitated when the water solution was made strongly acidic with the HCI solution.

The reaction showed that partial hydrolysis of the disterol citrate produces a water soluble sterol monocitrate potassium salt, from which the corresponding carboxylic acid is released by a strong acid.

Example 15 P-sitosterol tartaric acid condensate Initially, the following substances were reacted: 50.0 g ß-sitosterol 36.1 g (+) tartaric acid 57.0 g xylene solvent 300 pI H3PO4 catalyst

Water was azeotropically distille from the reaction mixture at 1460C for 12-13 h.

After cooling, the reaction mixture was a light-colored half-crystalline mass from which the xylene was removed using vacuum evaporation. After removal of the xylene, the product was kept in vacuum at 80-90oC in order that all xylene residues would be removed. The solvent-free product was a light gray powder with a composition of approximately 15% sterol substituted oligotartrates and the remainder p-sitosterot tartrate dimers.

Example 16 p-sitosterol succinate carboxy amide Initially, the following substances were reacted in nitrogen atmosphere: 10.0 g p-sitosterot hydrogen succinate prepared according to example 3 3.0 ml thionyl chloride 50.0 mi anhydrous cyclohexane During the reaction, the temperature was kept at approximately 20OC using a water bath. At first, the mixture was stirred slowly and the p-sitosterot succinate began to dissolve. Simultaneously, SO2 and HCI gases were released from the reaction mixture as bubbles, which left with the N2 stream. The speed of stirring was gradually increased as the sterol succinate dissolution progressed. In other words, the speed of the reaction was kept constant. After the formation of the solution, the temperature was increased very slowly to 40-450C and kept constant while stirring vigorously for half an hour, to remove any gaseous substances remaining in the solution.

Next, a 20 cm3 sample was taken with a pipette and transferred to the microdistillation apparatus. The unreacted thionyl chloride was removed from the reaction mixture by distillation in an N2 stream. Distillation was continued until the volume of the remaining liquid was approximately 10 cm3. The concentrated solution was quickly mixed with excess, cold 25% ammonia solution. Fast mixing was

continued for half an hour. The surplus ammonia and solvent were evaporated in a vacuum. The precipitate was separated from the water solution by filtration and washed with water, after which the product was dried in a vacuum exsiccator.

The nitrogen content of the product was found to be 1.95%, on the basis of which it was calculated that 72% of the theoretical yield of the desired amide had been obtained.

Using the wave lengths 1660-1680 and 1733cm-', carbonyl absorption in the FTIR spectrophotometer indicated that the product was the desired amide.

Example 17 Analyses of the primary elements Starting materials % C % H P-sitosterol (technical) 82.163 11.946 C29H50O 414.17g/mol 83.9 12.15 (calculated value) P-sitosterol products composition of primary elements closest molecular formula % C % H % N Hydrogen succinate (e. g. 3) 76.59 10.63 C33H5404 Citrate (e. g. 4) 71.57 9.38 C35H5607 Citrate dimer 67.00 8.52 C41H68O, 3 Diethyl citrate (e. g. 9) 79.10 10.78 C66HlO8o7 Citrate/citr. dimer (e. g. 10) 68.50 9.08 Citrate dimer (e. g. 12) 60.61 8.07 non-purified raw product Citrate dimer (NH4) 2 62.05 9.04 3.63 C4, H68013N2 (e. g. 13)

Oligo tartrate 53.965 7.22-(C4H405) n-steryl (e. g. 15) Tartrate dimer 63.96 8.23 C37H580 1 The starting material for the compounds according to the invention was a plant sterol mixture (Weinstein Nutritional Products), composed of 45-55% ß-sitosterol, 20-30% campesterol and 15-25% stigmasterol.

Example 18 FTIR spectrums of the products The esterification of p-sitosteroi (marked *) with various hydroxy acids was determined by FTIR spectroscopically.

Characteristic absorptions cm-' Example 3* hydrogen succinate 1178: ester stretching, 1464: CH3 1712: Carboxy carbonyl, 1732: ester carbonyl, 2860-3000: sterol C-H, 3440: (wide) H-bond carboxy-OH (instead OH stretch 1058 is missing from the spectrum).

Example 4* citrate deriv. Strong absorption from the ester at 1194- 1197; 1463: CH3, strong band of fusion 1733- 1737 suits ester carbonyl, absorption model ( (typical) for C-H hydrogen sterol ring system visible 2860-3000; Area 3000-3500 wide, absorption band-most likely citrate-OH and carboxyl-OH groups: can sometimes distinguish absorption 3450 (wide).

Example 9 di-*ethyl citrate Absorption from ester at 1193-1199 and 1219- 1222,1463: CH3,2 strong ester carbonyl bands 1725-1733 and 1776, a characteristic C-H absorption band for sterols 2860-3000; hydrogen- bonded OH absorption 3431.

Example 13 *citrate dimer (NH2) 4 1725: ester carbonyl, 2859: sterol C-H, 2945: sterol C-H, 3224 and 3403 wide bands.

Example 15 *tartrate oligomers 1084 suitable for OH stretch, 1203-1225 wide absorption band most likely related to ester bond, absorption 1442; 1464 suitable for the methyl groups of the sterol structure; strong absorption in the carbonyl area 1743; very wide, strong absorption 2500-3500, concentrated at 3433, originates from hydrogen-bonded OH and carboxyl groups. In the range 2869-3000 partially covered CH absorption whose model [2869,2936] also indicates that this is a substituted sterol in question.

References Technical P-sitosterol 1058: OH stretch, 1464 CH3: 2860-3000 [2868, 2937 more characteristic] Sterol C-H, 3390: h hydrogen-bonded OH.

P-sitosterol acetate 1058cm-'OH stretch; missing 1731 ester carbonyl 2668,2937 sterol C-H 3390 hydrogen-bonded OH; missing

Acetyl triethyl citrate 1736 cm-'ester carbonyls, strong 2981 medium, C-H Example 19 P-sitosterol lactate The following substances were allowed to react in toluene solution: 25.0 g p-sitosterot 30.0 g 90% (S)-2-hydroxy propionic acid (L-lactic acid) water solution 100.0 mi toluene A minimum of 7 mi of water was azeotropically distilled from the reaction mixture over 7-8 hours; as the conversion approached theoretical, upon cooling no lactic acid or plant sterols were crystallized from the reaction mixture. The cooled reaction mixture was a yellowish, viscose liquid. The toluene was removed from the reaction mixture in a vacuum (29 mmHg) at a temperature of 80-90OC. At a temperature of 60-100OC, the reaction product was a light-colored, yellowish, viscose liquid that was soluble in lipids and ethanol. As a whole, the material obtained was useable and contained very little or no free plant sterols and/or lactic acid.

Example 20 P-sitosterol lactate Preparation with Fischer apparatus <BR> <BR> 200 g p-sitosterot, 250 g lactic acid, and 500 mi toluene were dissolved in a 5 I flask.

The mixture was refluxed at 110-115 oC at the same time as water (free water + reaction water) was collecte using water separation apparatus, in order to monitor the progress of the reaction. Reaction time was 11.5 h. At this point, 68 mi of water had been collecte (free water: 33.8 mi + reaction water 34.2 ml), representing a

77.5% conversion in relation to the starting material. The toluene was evaporated from the reaction mixture using a rotavapor, and the product was dried in a vacuum oven at 40-600C. The esterification experiment was repeated 5 times.

Example 21 P-sitostanol derivatives Stanol lactate The stanol lactate was produced using the same method as the p-sitosterot lactate except that the starting material was ß-sitostanol Example 22 P-sitostanol derivatives Stanol lactate; the hydrogenation of sterol lactate 0.2 kg p-sitosteroi lactate was dissolved in 5 kg ethanol (96.4 volume-%, Primalco Oy, Nurmijarvi) and the same hydrogenation process was carried out as in the production of ß-sitostanol. A filter was used to separate the hydrogenation catalyst from the reaction mixture. The ethanol solution was concentrated by evaporation and the separated raw material was decanted. The raw product was again dissolved into a 10% solution of ethanol and precipitated by adding excess water. The precipitate could be filtered when the water-ethanol ratio was 1: 2. The resulting product was air-dried and then vacuum-dried at 20oC.

Example 23 The production of p-sitosterot dicarboxylic acid derivatives P-sitosterol succinate glycerol ester The p-sitosterol hemisuccinate was reacted with glycerol while toluene was used as

an azeotrope forming agent, as in example 19.

Ethyl p-sitosteryl succinate By transesterification at 180-200OC in the presence of a H+ catalyst, the p-sitostero ! and the diethyl succinate are in a molar ratio of 1: 2. The excess of diethyl succinate was distille at 100-1500C and 0.3 mmHg.

Example 24 The ß-sitosterol succinyl chloride prepared according to example 16 was reacted with triethyl citrate in toluene with a molar ratio of 1: 1. The acting HCI acceptor in the reaction mixture is pyridine (1 mol pyridine per 1 mol p-sitosterot succinate chloride).

Example 25 The production of a P-sitosterol basic dispersion The following were combined: 25.0 g 19.0 weight-% diethyl-p-sitosteryl citrate (refined) in 96.4 volume-% ethanol 85.0 g ethanol (96.4 volume-%) 80.0 g water These were mixed, producing a light-colored yellowish, very stable dispersion which can be used in the production of alcoholic beverages.

Example 26 A liqueur-like alcoholic beverage was produced as follows: 15 g ß-sitosterol citrate (from example 4) was dissolved into 285 mi 96.4 volume-% alcool. Water was added to bring the alcohol content of the solution to 35 volume- %. 6 mi coffee aroma and 4 ml cocoa aroma were added to the solution. 143 g liquid starch sugar FFS (Neson Oy, Jokioinen, Finland) at 70% dry matter content

was added, stirring continuously. Water was added so that with compression the volume was 1000 ml. The beverage's alcohol content was 28 volume-%.

Example 27 A bitters-type alcoholic beverage was produced from ethyl-ß-sitosterol citrate as follows: <BR> <BR> <BR> <BR> <BR> 5 g ethyl-p-sitosteryl citrate (from example 9) was dissolved into 385 ml 96.4 volume- % alcool. Water was added until the mixture's alcohol content was 40 volume-%.

24 ml bitter aroma and 100 g liquid starch sugar at 70% dry matter content FFS (Neson Oy, Jokioinen, Finland) were added gradually, stirring constantly. Water was added so that with compression the volume was 1000 ml. The finished beverage had an alcohol content of 28 volume-%.

Example 28 A non-alcoholic beverage can be produced from a p-sitosterot citrate dispersion as follows: > 0-3 weight fraction p-sitostero ! citrate, > 0-40 weight fraction sugar, > 0-15 weight fraction flavors > 0-1 weight fraction colors and water.

Besides water, the beverage contains sterol or stanol esters, and preferably also some flavors. If the beverage is to be carbonated, 5 g/l carbon dioxide is added.

Example 29 Dissolving ß-sitosterol into concentrated acetic acid

Initially, the following were combined: 1.49 g (cotd precipitated) ethyl-ß-sitosterol citrate from example 9 80 mi 100% acetic acid The sterol citrate and glacial acetic acid mixture was slowly diluted with water to 200 mi and simultaneously stirred rapidly with a magnetic stirrer.

100 mg Tween-80 assisting material was added and the mixture was rapidly diluted with water to its final volume of 850 mi.

The first stage of dilution appears critical. The emulsifier must be added at a diluting ratio of 70: 20-30 AcOH: H20.

The end result was a stable emulsion, with a sterol citrate content of 0.1-0.15 weight-% and acetic acid content of 10 volume-% Example 30 Acetic acid dispersions Initially, the following were combined: <BR> <BR> 8.0 g solid ß-sitosterol citrate from example 12<BR> 200.0 g Rajamaki Spirit Vinegar (Primalco Oy, Nurmijarvi) The mixture was mechanically shaken for 7 hours.

The ß-sitosterol citrate content in the mixture was 3.85 weight-% The light yellow emulsion that formed during shaking was centrifuged for 15 min at 2000 rpm. The dispersion above the sedimented material was decanted. The finished dispersion had a concentration of 2.75 weight-%, representing 71.5% of the theoretical yield.

Example 31 Acetic acid dispersions 6.80 g ß-sitosterol citrate from example 12 90.7 g Rajamaki Spirit Vinegar (Primalco Oy, Nurmijarvi), containing 10 weight-% acetic acid.

The mixture was allowed to stand without stirring for 12 hours. Determination of the amount of soluble matter from the solution showed that 0.74% of the sterol citrate had dissolved. The mixture was stirred for 6 hours, after which it was stirred rapidly with an Ultra Turrax homogenizer for at least 2 min.

The concentration of the final dispersion was found to be 7 weight-%. The dispersion was stable for at least three days, the cup viscosity was 30 s and the Brookfield viscosity n 124 cp at 230C.

Example 32 Nutritious acetic acid which contains P-sitosterol French salad dressing Recipe: 2 tbsp. p-sitostero ! citrate acetic acid dispersion from example 31 2 tbsp. wine vinegar (white wine vinegar, Primalco Oy, Nurmijarvi) 12 tbsp. cooking oil 2 spice measures salt 1/2 spice measure white pepper 2 tsp. mustard 4 spice measures castor sugar 1 clove of garlic, crushed Total approx. 150 g.

The ingredients were dosed into the bottle and shaken vigorously. Next the mixture was mixed in a food processor for 30 s, mainly to homogenize the garlic. A reference sample was made with the same method except that the vinegar was made up of 2 tablespoonfuls of spirit vinegar and 2 tbsp. wine vinegar.

There was 0.9 weight-% p-sitosterot citrate in the salad dressing.

According to taste, both dressings were quite successful and it was difficult to distinguish one from the other. There were differences in the structure of the specimens. The reference sample was"more oily,"i. e. oil drops were separated.

The dressing which contained sterol was more feculent. Oil became separated in both samples. At least in the beginning the separation was more rapid in the reference sample. The samples were allowed to stand at room temperature for one hour, after which they were refrigerated.

Example 33 <BR> <BR> <BR> <BR> <BR> 35.0 g p-sitosterot citrate acetic acid dispersion from example 31 and 60.0 g cooking<BR> <BR> <BR> <BR> oil (5 weight-% p-sitostanot lactate, 0.5 g satt, 11.5 g sugar and 3.0 g spices, white pepper, ground garlic or mustard) were dosed. The mixture contained 7.45 weight-% sterol derivative and had 55 weight-% fat content.

It was noted that a salad dressing containing acetic acid could be produced from the compounds in question, whose plant sterol or stanol content is comparable to a common plant stanol containing margarine.

Example 34 Nutritious acetic acid which contains ß-sitosterol Mayonnaise Recipe (Liimatainen, A. 1995. (In Finnish) The best home cooking. Mayonnaise in a

food processor, p. 123. WSOY). The following ingredients were mixed (at room temperature): 1 egg 1/2 tsp. salt 2 tsp. mustard Mix at full speed for 30 s (Moulinex Masterchef 65 Electronics) 1 tbsp. p-sitosterot citrate vinegar dispersion from example 31 Mix at full speed for 15 s (Moulinex Masterchef 65 Electronics) 2 dl cooking oil The cooking oil was added while mixing.

A reference sample was made using the same method, except that the acetic acid used was PIRKKA acetic acid (Primalco Oy, Nurmijarvi).

Example 35 Nutritious acetic acid which contains P-sitosterol Mushroom salad Recipe: 1 dl crushed onion (1 medium-sized onion) 2 dl fresh boiled crushed milk caps Onions and mushrooms are mixed.

3 tbsp. mayonnaise containing ß-sitosteroi citrate (from example 34) was mixed into 1 dl onion-mushroom mixture above.

A reference sample was prepared using the same method, except that the mayonnaise from example 33 was used. The mushroom salad containing ß- sitosterol citrate had the same taste as the reference sample.

Example 36 Nutritious acetic acid which contains p-sitostero ! Tomato ketchup Recipe (Kotivalmistus. Ed. Hannele Hietala. (In Finnish) Delicious microwaving p.

355. Valitut Palat-Reader's Digest A3. ISBW 951-8933-43x) 900 g ripe tomatoes cut in quarters 300 g jam sugar (Suomen Sokeri Oy, Kantvik) 2.5 dl vinegar dispersion 1 tsp. ground mustard seeds <BR> <BR> <BR> /2 tsp. ground cinnamon<BR> <BR> <BR> <BR> /4 tsp. ground cloves<BR> <BR> <BR> /2 spice measure Cayenne pepper 1 tbsp. corn starch (Maizena; CPC Food A/S) +2 tbsp. cotd water The vinegar dispersion was made as follows: 0.5 d ! p-sitosterot citrate vinegar dispersion from example 31 was diluted with water to 0.83 dl and then made up to 2.5 di with Rajamaki's Apple Wine Vinegar (Primalco Oy, Nurmijarvi) The tomatoes, sugar, acetic acid and spices were mixed in a pot and heated, stirring every now and then. During the heating the mixture thickened. While hot, the mixture was purée with a food processor (in 60 s pulses), and immediately sieved to separate the seeds. The sieved sauce was combined with the water-corn starch mixture, and the ketchup was boiled, stirring every now and then. At this stage the ketchup thickened. The ketchup was poured into boiled, warm 2.5 dl glass jars, leaving 5 cm free space between the product and the lid. The jars were refrigerated.

There was at least 0.24 weight-% p-sitostero ! citrate in the product.

A reference sample was prepared using the same method, except that the acetic acid product used was Rajamäki Apple Vinegar.

The ketchup containing p-sitosterot citrate was as good and its thickness even better compared to the reference sample.

Example 37 A preservative solution containing p-sitosterot and acetic acid 50.0 ml Rajamäki Spirit Vinegar (Primalco Oy, Nurmijarvi) 0.2-20 g/l citrate derivatives, tartrate derivatives or hemiester amides 60.0 ml liquid starch sugar (FFS, ka 70%, Neson Oy, Jokioinen) 0.15 g spice mix (mustard seed, bay leaf) Example 38 Mushroom salad containing p-sitosterol <BR> <BR> Preserving solution 3.0 g ß-sitosteroi derivative<BR> 1000 g Rajamaki Spirit Vinegar (Primalco Oy, Nurmijarvi)<BR> 1200 g starch liquid sugar (FFS, c. 70%, Neson Oy, Jokioinen) 15 g spice mix<BR> <BR> Combine 10 g dried mushrooms (Cantharellus tubaeformis) (-2dl)<BR> 20 g fresh carrot (thinly sliced) 1 pepper ring (7 g) cubed 1 clove garlic (2 g) cubed 1 dl previously prepared preserving solution, The solution was allowed to absorb for 0.5 h, and then slowly heated to boiling. The solution is then poured immediately into a boiled, glass jar which is still hot, and the

jar sealed.

Evaluation of results: The solution was absorbed very well, taste and aroma were excellent. It is worth mentioning that the mushrooms regain their natural shape.

Example 39 Various types of mayonnaise containing p-s ! tostero) Products were made in a food processor according to the following recipe: 1 egg 2 tsp. mustard 1 tsp. salt Homogenize at full speed for 60 s Add 1 tbsp. Rajamäki Honey-Apple Vinegar (Primalco Oy, Nurmijarvi) Homogenize at full speed for 15 s Add 2 dl cooking oil while mixing at full speed The p-sitosterot esters were added to the products white dissolved in cooking oil.

The reference sample was a product made according to the basic recipe. The mixing times for all samples were exactly the same.

Mayonnaise P-sitosterol derivative % fat salt added/not added No. 1 * hemisuccinate 2.5 same as reference- ample No. 2 * hemisuccinate 2.5 not added No. 3 * citrate 2.5 not added Evaluation of results: No. 1 more viscous than reference-sample; stronger flavor, but not unpleasant No. 2 same viscosity as previous; no complaints about taste No. 3 excellent structure, very dense, stays intact, no complaints about taste.

Example 40 A cream liqueur containing P-sitosterol <BR> <BR> 2.0 g p-sitostero ! hemisuccinate was dissolved into 550 g cream liquor concentrate (Creamy Creation, DMV, Division of Campina, Melkunie, the Netherlands) containing 12% fat, while mixing at 40-650C, so that thus the product's fatty portion contains 3% p-sitosteroi ester.

115 mi 96.4% alcohol was added and mixed well <BR> 160 g starch fluid sugar FFS (Neson Oy, Jokioinen), with a 70% dry matter content, was added and mixed. Finally, 250 ml water was added, stirring continuously. If desired, chocolate aroma, for example, can be added to the beverage.

This yields a cream liqueur with an alcohol content of 18 vol.-%.