AU1347200A - Fat based food products - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION PETTY PATENT TITLE OF INVENTION FAT BASED FOOD PRODUCTS Name and Address of Applicant: UNILEVER PLC of Unilever House, Blackfriars, London EC4P 4BQ, England The following statement is a full description of this invention, including the best method of performing it known to me:- F 7286au#5 (V) Fat based food products The present invention concerns a fat based food product comprising natural fat components which have a blood cholesterol lowering effect in amounts sufficient to obtain a blood cholesterol lowering effect if the food product is used according to the common needs of the consumer.

Such fat based food products are available oh the market for quite some time, and are to a large extend based on the use of fat which comprises a particular amount of "triacylglycerides rich in poly-unsaturated fatty acid (pufa) chains. In particular, the use of butter-like spreads comprising a significant amount of these 15 triacylglycerides having poly- unsaturated fatty acid chains (hereinafter called pufa rich triglycerides) is well known in the western world to reduce high blood cholesterol levels.

20 The variation in the mean plasma total cholesterol concentration among populations is highly correlated with the variation in the extent of atherosclerosis and in the incidence of Coronary Heart Disease (CHD), which is one of the major causes of death in the Western society.

Populations with a low cholesterol level (less than 180 mg/dl (4.7 mmol/L) are found to be less sensitive to atherosclerosis and coronary heart disease, whereas those with mean cholesterol levels above 220 mg/dl (5.7 mmol/L; hypercholesterolaemia) have increased rates of death due to CHD. Thus, a clear need for a method by which the cholesterol level can be lowered exists.

With the common products which comprise triglycerides rich in pufa, a particular amount of fat should be eaten on a F 7286au#5 (V) 2 daily bases to ensure a high enough intake of these triglycerides. A contribution of a minimal intake of pufa glycerides of 3 to 4 percent of the metabolizable energy is recommended.

However, if fat based food products are used in which part of the fat is dispensed with, for example by the use of thickeners and/or fat replacers, products with a low fat level such as low fat spreads comprising less than 40% fat would have a less blood cholesterol lowering effect than a 10 product comprising 80% fat, given that a similar amount of pufa rich triglycerides is present in the fat. This is even more the case for the products which were even further reduced in their fat content, such as low fat spreads comprising less than 20% fat.

For this type of products, a need for further improvement of the cholesterol lowering effect in fat based food products exists. Also for fat based food products which up till now did not comprise pufa rich triglycerides, the presence of components which have a cholesterol lowering effect is desired.

On the other hand, also the daily consumption of full fat food products per se is decreasing i.e. not solely by replacement of these products by low fat food products but also by a decreasing consumption of fat based food products as such. This is mainly due to the increasing awareness of the need to reduce the fat intake in general, wrongly based on the scientific consensus towards the necessity to reduce the consumption of triacylglycerides with saturated fatty acids in particular. Because of the reasons mentioned above, for cholesterol reducing PUFA-triglycerides containing fat based products a reasonable consumption is still preferred for the majority of the population in the majority of the effluent countries. Nevertheless, to F 7286au#5 (V) 3 compensate for the reduction in pufa through reduction of the consumption of triglycerides rich in pufa, also for those consumers that use high fat food products (e.g.

40-80% yellow fat spreads), which contain more than pufa rich triglycerides on fat, in a decreasing extend, the presence of components that have an additional cholesterol lowering effect is highly preferred.

Also for those consumers for which a significant reduction in plasma cholesterol is desired, i.e.

hypercholesterolaemia patients, the consumption of a high or low fat food product that contains more than 30% and preferably more than 45% pufa-triglycerides on fat and also natural components that have an additional cholesterol lowering effect would be most desirable.

As consumers these days have a clear preference for food products which are obtained from natural sources our **.invention deals with food products which contribute to the health of consumers by the use of ingredients obtained from natural vegetable, oil related sources and to include such particular natural ingredients in their context, i.e.

in food products comprising ingredients similar to the source of the particular ingredients. In the context of vegetable oil and fat based products, we have now found that some particular natural non-triglyceride minor oil components can be used in food products which are used on a daily basis, and which have a possible contribution to health in general, and in particular to the lowering of blood cholesterol level.

In European patent application no. EP 619 952 (Amano), a food additive is described, by which the level of cholesterol in food is lowered by treating the food with the food additive. Upon using the additive, (y-oryzanol F 7286au#5 (V) 4 being mentioned), a complex with the cholesterol in the food is formed. The food containing the additive can therefor not be applied to lower the blood cholesterol level in mamals.

In International Application no. WO 92/19640 (Raision Marganiini oy) a substance of 3-sitostanol fatty acid ester is described that can be used as such or added to food. It is described that an ordenary diet contains plant sterols 10 100-300 mg/day, and that these sterols are poorly absorbed from the intestines. In WO 92/19640, it is thus argued that the use of the plant sterols cannot be used for reducing the serum cholesterol levels in the human diet.

15 It has now been found that a lowering of blood cholesterol level is obtainable by the regular consumption of fat based food products which comprise at least one compound of the group consisting of tocotrienol, phytosterol and oryzanol, S. and mixtures thereof, in an amount of at least 0.2 wt% for tocotrienol, 0.25, preferably 0.5 wt%, and further preferred at least 1 wt% for phytosterol and at least 0.25 wt%, preferably at least 0.5 wt% and further preferred at least 1 wt% for oryzanol or their relative amounts if mixtures of one or more of these components are used, the weight percentages being based on the total weight of the fat based food product.

A preference exists for a fat based food product which comprises a combination of these compounds in amounts. In this embodiment, the fat based food product comprises at least 0.1 wt% tocotrienol, 0.25 wt% oryzanol and 0.25 wt% phytosterol. The use of at least one of phytosterol and oryzanol is preferred, these components showing an effect on blood cholesterol even stronger than tocotrienol.

F 7286au#5 (V) Thus, in another and preferred embodiment of the invention, it has now been found that a significant lowering of blood cholesterol level is obtainable by the regular consumption of fat based food products which comprise at least one compound of the group consisting of phytosterol and oryzanol, and mixtures thereof, in an amount of at least 0.25, preferably 0.5 wt%, and further preferred at least 1 wt% for phytosterol and at least 0.25 wt%, preferably at least 0.5 wt% and further preferred at least 1 wt% for 10 oryzanol or their relative amounts if mixtures of one or more of these components are used, the weight percentages being based on the total weight of the fat based food product.

For economical reasons, amounts between 0.5 to 20 wt%, preferably 0.5-10 wt% and further preferred 1-10 wt% for phytosterol, and between 0.25 and 20 wt%, preferably 20 wt%, more preferred 0.5-10 wt%, whereas sometimes an amount between 0.25 and 6 wt% for oryzanol will be sufficient for these ingredients to obtain a cholesterol lowering effect. Of course, higher amounts can be applied if so desired.

Preferably, the fat based food product of the present invention comprises at least one compound of the group consisting of phytosterol and oryzanol, in an amount of 2-8 wt% for phytosterol, and 0.5-8 wt%, with amounts of wt% to be found effective in some specific cases for oryzanol or their relative amounts if mixtures of one or more of these components are used, the weight percentages being based on the total weight of the fat based food product. In an more preferred embodiment the fat based food product comprises at least 4 wt% phytosterol or at least 1.2 wt% oryzanol or a mixture thereof in their relative weights. It is preferred in some cases to use more than the F 7286au#5 (V) 6 relative weights.

Tocotrienol, phytosterol, and oryzanol are hereinafter also referred to as 'healthy components'.

The fat based food products of the present invention comprise preferably more than one compound of the group consisting of tocotrienol, phytosterol, and oryzanol. In this case, the amount of the single components can be less than those indicated hereinabove, as long as care is taken that for the compound, a relative same weight amount of the other compounds is used. This amount can be indicated as the minimum effective dose (MED). For phytosterol, the minimum effective dose is 0.5 wt%, for oryzanol 0.5 wt%, although 15 sometimes 1 and 1 wt% respectively can be regarded as such.

If a lower amount of one or more of these components is used, e.g. 60% of the MED of one of the components is used, a total of 40% of the sum of the MED of the other **components should be present as well.

The amounts are amounts normally being sufficient to obtain statistically significant effects after use of these products in amounts similar to the amounts of the prior art fat based products, and for a prolonged period of time.

These amounts are based on common daily intake of the fat based food products. If more or less of such products are being used according to the particular use in a particular area or habits, the amounts can be adapted accordingly.

In particular, the invention concers a fat based food product comprising natural fat components which have a blood cholesterol lowering effect in amounts sufficient to obtain a blood cholesterol lowering effect, wherein the fat based food product comprises at least one compound of the group F 7286au#5 (V) 7 consisting of tocopherol and polyphenol, and the fat comprises at least 4 wt% phytosterol, the weight percentages being based on the total weight of the fat based food product. By the regular consumption of the now found fat based food products a positive contribution to health in general, and in particular to the lowering of the blood cholesterol level can be found.

e 10 Tocotrienol, phytosterol, and oryzanol are compounds which are known per se. Japanese patent laid open number 04/320,645 describes the use of unsaponified substances of rice bran oil in rice bran oil, and mentions that the oil exhibited a very good physiological action. No 15 substantiation of this is given.

*oo 9 In the present application, by the use of these components not only a cholesterol lowering effect, but also some antioxidative working can be found.

:9 In a specific embodiment of the present invention, also at :least one of tocopherol and polyphenol is present. These components are known as such, and have been described in different publications. For example, in "Tocopherole Antioxidantien der Natur; by Pongracz et al.;Fat Sci.

Technol.; 97. Jahrgang Nr. 3, pp. 90-104" a study of the use of tocopherols as antixoxidants in, inter alia, food is described.

Applicants have now found that the use of these healthy minor oil components results in a blood cholesterol lowering stronger than obtainable by the use of any pufa rich triglyceride fat blend alone.

F 7286au#5 (V) 8 As a further improvement of fat based food products comprising one of these components, it was found that the use of a mixture of these healthy ingredients shows an effect on health, in particular blood cholesterol lowering, which is much stronger than the effect which can be expected on the basis of the studies published regarding the effect of cholesterol lowering per weight amount of intake of each of these components alone. Moreover, this synergy was not found for blood cholesterol lowering alone, 10 but also on other health aspects.

In particular, combinations of oryzanol, phytosterols and *9e* pufa rich glycerides show a very beneficial effect which could not be expected on the basis of the health effect of these compounds alone.

*9The oil related components used in the fat based foods of *9*9 the present invention can be found as minor components in most plants from which oil is recovered. As conventional oil milling aims at producing triacylglycerols with a minimum amount of unsaponifiable matter, only limited amounts or none of the non-triglyceride healthy components *ObS at all are found in the oil presently available. The components to be used in the products of the present invention are found in the waste product streams which results upon the refining processes for obtaining commonly, classically refined oils.

As only very minor amounts of the suitable components are found in the waste streams of the usual oil refinings, e.g. sunflower oil, soybean oil, rapeseed oil, maizgerm oil and the like, using the waste of commonly used seed material will require enormous amounts of the waste material to be further process. However, relative significant amounts of one or more of these components can be found in materials not always used (yet) on a large scale as an fat or oil source. In particular, the F 7286au#5 (V) 9 components very suitable for the present invention can be found in, for example, rice bran (in particular oryzanol, phytosterols, and also tocopherols), wheat and maize germ (in particular phytosterols and tocotrienols), oat and oat bran in particular phytosterols and tocotrienols), sesame seed (in particular phytosterols), soybean, sheanuts (phytosterol) and palm oil (tocotrienols). The healthy minor oil components can also found, although in amounts lower than the sources mentioned before, in, for example, sunflower, rape, olive, line, peanuts, cotton, and safflower.

Oryzanol consist of a mixture of ferulic acid esters of unsaturated triterpene alcohols and is also referred to as 15 gamma-oryzanol. In this invention only the term oryzanol is used. Within the group of tocopherols and tocotrienols one can distinguish alpha, beta, gamma and delta tocopherols and tocotrienols depending on the number and position of the methyl substituents on the chromane ring of the molecule. In this invention the terms tocopherol and tocotrienol is used to cover this whole family of molecules. For a further description and schematic drawing of the oryzanol and tocopherols and tocotrienols meant in this description, reference is made to "Separation of Vitamin E and gamma-Oryzanols from Rice Bran by Normal- Phase Chromatography", M. Diack and M. Saska, JAOCS Vol.

71, no. 11, pp. 1211. Phytosterols, also known as plant sterols or vegetable sterols can be classified in three groups, 4-desmethylsterols, 4-monomethylsterols and 4,4'-dimethylsterols. In oils they mainly exists as free sterols and sterol esters of fatty acids although sterol glucosides and acylated sterol glucosides are also pres.ent.

There are three major phytosterols namely beta-sitosterol, stigmasterol and campesterol Schematic drawings of the components meant are as given in "Influence of Processing F 7286au#5 (V) on Sterols of Edible Vegetable Oils", S.P. Kochhar; Prog.

Lipid Res. 22: pp. 161-188. In this invention the term phytosterol is used to cover the whole group of free phytosterols, phytosterol fatty acid esters and (acylated) phytosterol glucosides. The term polyphenols (or phenolics) can be defined chemically as a substance which possesses an aromatic ring bearing one or more hydroxy substituents, including functional derivatives. Reference is made to "Phenolic Compounds in Food", Chi-Tang Ho; Phenolic 10 Compounds in Food and Their Effects on Health II, Am.

Chemical Soc., 1992 In this invention the term polyphenols refers to all plant phenolic molecules derived from a plant source with an antioxidant activity and not covered by the terms oryzanol, tocotrienol and tocopherol, e.g: simple 15 phenols and phenolic acids, hydroxycinnamic acid derivatives (e.g coumaric and ferulic acid) and flavonoids.

Tocopherol and polyphenol can also be obtained from most of these natural vegetable, oil related sources. These components can additionally be present where reference is made to 'healthy components'. Very small amounts may be present in the phytosterol composition, but usually, amounts are less than 500 ppm based on total fat level. In this invention, amounts of over 800 ppm, or even over 1000 ppm, based on total fat level, can suitably be present.

The healthy components can be added as single, pure components, as a mixture of such single, pure components, as components present in a concentrate obtained from specific processing of the natural source, as components obtained by mixing such concentrates obtained from different sources) and the like.

The mixtures of the healthy oil components can be prepared by mixing single, pure components (bought at specialized F 7286au#5 (V) 11 suppliers) or be obtained by treating the oils and oil containing raw materials in such a way that the minor components remain therein. By specific processing, concentrates comprising a high amount of healthy minor oil components can be obtained. These are applied by preference if the 'naturalness' of a product is considered a particular issue. The concentration of the healthy minor oil components should be such that, if the concentrate is applied, the amounts indicated before are present in the fat based food product. Consequently, this means that the concentration should be high if it is desired to make a product in which the fat is replaced by non-fat ingredients, and a large amount of the other ingredients in the concentrate are fatty components.

*0.Fat based food products are food products (partially) based on fat and regarded by the user, in particular the consumer, as 'fatty type of products'. Examples are yellow fat spreads (containing vegetable fat and/or animal fat such as butterfat), dressings, coffeecreamer, shortenings, cooking and frying oils, fillings and toppings, and the o0eo like. These products in most cases comprise a particular amount of fat. In some cases, however, products are still regarded as 'fatty type of products', despite a replacement of part or even all the fat by fat replacers. Fat based food products in which the fat is partially or completely replaced by fat replacers are also covered by the term fat based food products of this invention.

Fat products consisting of fat only are also considered as fat based food products in this specification. These are not only used as cooking and frying oils, but also sometimes in the industry in the preparation of food products, e.g. in baking.

The food products as such are common products in the F 7286au#5 (V) 12 western world, and are used by consumers on a daily basis in amounts different for each individual.

The invention is in particular very suitable for yellow fat spreads, dressings, cheese, shortenings and cooking and frying oils, and more in particular for yellow fat spreads which can comprise 0 (zero) to 90 wt% fat (usually 5-80 Dressings can comprise 0 to 85 wt% faty (usually 5-80 shortenings, cooking and frying oil more than fat. Suitable yellow fat spreads can be margarines 10 comprising 80 wt% of fat, 70 wt% or 60 wt% of fat, or so called halvarines comprising 50 wt%, 40 wt% of fat, or low fat spreads which often comprise between 20 wt% and 25 wt% of S..fat.

In a highly preferred embodiment, the food product is a yellow fat spread comprising 0 to 60 wt% fat, preferably 0 to 40 wt% fat, even more preferred 0 to 25 wt% fat, the product comprising at least one of phytosterol and oryzanol and mixtures thereof, in an amount of at least 0.5 wt%, preferably at least 1 wt% and more preferred at least 2 wt% for phytosterol, and at least 0.5 wt%, and preferably at least 1 wt% for oryzanol or their relative amounts if mixtures of one or more of these components are used, the weight percentages being based on the total weight of the fat based food product.

Yellow fat spreads comprising less than 40 wt% fat and at least 3 wt% phytosterol and 1.2 wt% oryzanol can be regarded as very beneficial in that these not only have a very low fat content, but also show an antioxidative effect and a significant lowering of the blood cholesterol working if applied similar to a common butterlike spread on a regular, daily basis. This applies even more for products with less than 25 wt% fat.

F 7286au#5 (V) 13 The use of the minor healthy components in cheese shows an additional beneficial effect, which is that the cholesterol in cheese is adsorbed by the body in a far lesser amount if phytosterol is present in the cheese than without. Hence, whereas cheese consumption usually adds cholesterol to the body, with the use of healthy minor oil components, in particular phytosterol, a strongly reduced absorbtion and a blood cholesterol lowering effect will be found. This effect can also be found for food products other than *cheese comprising cholesterol or consumption of cholesterol containing food products in combination with the products as described in the invention. The amount of minor healthy 15 components to be added on the preparation of the cheese is higher than the amount of cholesterol on a molar weight basis, so that the cheese product obtained still contains these minor healthy components in a free and active form.

The fat that is applied in these fat based food products can be any fat, such as dairy fat and/or vegetable fat.

However, if fat is present, for health reasons the use of one or more vegetable fat sources is preferred. In particular, the use of liquid fats is preferred. These can be hydrogenated, interesterified, and the like. The fat can be one single fat or a blend. The fat or one of the fats applied can also beneficially be obtained from the same source as the healthy components are obtained from. E.g., rice bran oil can be obtained and mixed with one or more of the healthy components mentioned in this description which also are obtained from rice bran, or a mixture of wheat germ oil and rice bran oil can be used with one or more health components obtained from wheat germ and/or rice bran.

F 7286au#5 (V) 14 The use of fat compositions comprising a considerable amount of pufa rich triglycerides in addition to the use of the healthy components is in particular considered highly beneficial. For example, oils of sunflower, safflower, rapeseed, linseed, linola and/or soybean can be used in a preferred embodiment. Also the fat compositions mentioned in Netherlands patent documents no. NL 143115, NL 178559, NL 155436, NL: 149687, NL 155177, European patent documents EP 41303, EP 209176, EP 249282, and EP 470658 are highly suitable.

Wheat and maize germ, and soybean oil, are in particular good sources for the components used in the fat based food products of the current invention.

If a fat blend is used, it is preferred that it comprises at least 30 wt%, and more preferred at least 45 wt% of poly-unsaturated fatty acids, based on the total weight amount of the fat in the fat based food product. So, a strong effect on the cholesterol lowering effect is obtained if use is made of a mixture of healthy minor oil components such as tocotrienol, phytosterol, and S. oryzanol, or extracts comprising these components, or these components in combination with the antioxidants polyphenols and tocopherols, or extracts comprising all of these these components in a food product in which a fat blend comprising at least 30 wt.% of pufa rich triglycerides is used.

sunflower oil can be obtained and mixed with one or more of the healthy components mentioned in this description which are obtained from rice bran, wheat germ or a mixture of wheat germ oil and rice bran oil.

In another embodiment of the invention, it has been found that using specific technologies to incorporate the components in the food product significantly increased F 7286au#5 (V) their physiological beneficial activity. In addition thereto, the solubility of the healty components in the food product will increase, and, as a result thereof, the maximum concentration can be increased without altering the product characteristics and quality.

The technigues which can be applied beneficially are incorporation into the fat by the use of micro-emulsions, liposomes, incorporation into micro-particles or adhering to micro-particles. According to another method, which is less preferred, one or more of the healthy components that :-.-.might show a low solubility is esterified as desired with one or more fatty acids to increase their solubility. In the intestine these esters will be hydrolysed resulting in is the free fatty acids and active minor oil component.

Products so prepered are still considered, for the purpose of this invention, to contain the active minor oil component, as these are hydrolysed in the intestine.

The invention further envisages the use of an oil concentrate comprising more than 4 wt% of one or more of tocopherol oryzanol and phytosterol for the preparation of a fat based food product as described above. In another preferred embodiment, the invention concerns the use of an oil concentrate comprising more than 4 wt% of one or more of tocopherol oryzanol and phytosterol for the preparation of a yellow fat spread as described above.

F 7286au#5 (V) 16 Example I Male Syrian hamsters, aged 10-11 weeks, were fed semi-purified diets ad libitum, containing 30% of energy as fat; 23 en% as proteins; 47 en% as carbohydrates; 0.01% cholesterol; and either 0.38% oryzanol as obtained from a commercial source (Tsuno Rice Fine Chemical Ltd, Japan) or 0.34% oryzanol as oil concentrate (using an enriched 4.5% oryzanol containing mixture 1:12) of rice 10 bran oil and palm oil or nothing (control). The ratio of polyunsaturated (PUFA) to monounsaturated (MUFA) to saturated (SAFA) fatty acids in all the diets was 7.2:12:10.8 The semipurified diets were fed for 6 weeks, after a run-in period on the control diet for 2 15 weeks. The experimental groups comprised 12-14 hamsters/group. The hamsters were housed individually in Makrolon cages type II, with a layer of sawdust as bedding.

At the end of the experiment blood samples were collected S. and (heparin) plasma total cholesterol was determined using a commercially available enzymic method (CHOD-PAP).

Throughout the experiment body weights and feed intake did not differ between the experimental groups. No clinical signs were observed in any group. The average total plasma cholesterol in the control group was 3.83 mM while the total plasma cholesterol in the group fed with additional pure oryzanol and oryzanol as oil concentrate was on average lowered by 0.57 and 0.71 mM respectively.

Example II Male Syrian hamsters, aged 10-11 weeks, were fed semi-purified diets ad libitum, containing 30% of energy as fat; 23 en% as proteins; 47 en% as carbohydrates; 0.01% cholesterol; and either a 100 ppm tocotrienols F 7286au#5 (V) 17 mixture (composition alpha-tocopherol 8tocopherol gamma tocopherol delta-tocopherol alpha-tocotrienol gamma-tocotrienol (30.9), delta-tocotrienol or nothing (control). The ratio of polyunsaturated (PUFA) to monounsaturated (MUFA) to saturated (SAFA) fatty acids in the diet was either 7.2:12:10.8 (high PUFA) or 3:12:15 (high SAFA). The semipurified diets were fed for 6 weeks, after a run-in period on the control diet for 2 weeks. The experimental 10 groups comprised 12-14 hamsters/group. The hamsters were housed individually in Makrolon cages type II, with a layer of sawdust as bedding. At the end of the experiment blood samples were collected and (heparin) plasma total cholesterol was determined using a commercially available 15 enzymic method (CHOD-PAP).

Throughout the experiment body weights and feed intake did not differ between the experimental groups. No clinical signs were observed in any group, although it was clear that some effect was found which could not be explained without assuming that the tocotrienol did have some kind of effect on the blood cholesterol in the plasma. The average total plasma cholesterol in the control groups on high SAFA and high PUFA diets was 4.25 and 3.95 mM respectively while the average total plasma cholesterol in the groups fed with 100 ppm tocotrienol mixture was 4.29 and 4.31 mM for the high SAFA and high PUFA diet respectively, i.e. no cholesterol lowering effect additional to PUFA was observed.

Example III Male Syrian hamsters, aged 10-11 weeks, were fed semi-purified diets ad libitum, containing 30% of energy F 7286au#5 (V) 18 as fat; 23 en% as proteins; 47 en% as carbohydrates; 0.01% cholesterol; and either oryzanol (using an enriched 4.5% oryzanol containing preparation dissolved in a mixture 1:12) of rice bran oil and palm oil (prepared as above)) alone, or in combination with 100 ppm tocotrienols or nothing (control). The ratio of polyunsaturated (PUFA) to monounsaturated (MUFA) to saturated (SAFA) fatty acids in all the diets was either 7.2:12:10.8 (high PUFA) or 3:12:15 (high SAFA). The 10 semipurified diets were fed for 6 weeks, after a run-in period on the control diet for ~2 weeks. The experimental groups comprised 12-14 hamsters/group. The hamsters were housed individually in Makrolon cages type II, with a layer of sawdust as bedding. At the end of the experiment blood 15 samples were collected and (heparin) plasma total cholesterol was determined using a commercially available enzymic method (CHOD-PAP).

Throughout the experiment body weights and feed intake did not differ between the experimental groups. No clinical signs were observed in any group. The average total plasma cholesterol in the high SAFA control groups was 4.25 mM. A decrease on average total plasma cholesterol in the high PUFA control group of 0.30 mM was observed. Addition of oryzanol as oil concentrate decreased the average total plasma cholesterol with 0.60 and 0.47 mM for the high SAFA and high PUFA diets respectively. Additional tocotrienols decreased the average total plasma cholesterol in the high PUFA group further with an additional 0.11 mM. No additional effect of tocotrienols was found in the high SAFA group.

The combination of high PUFA and oryzanol as oil concentrate in the diet lead thus to a total decrease of 0.77 mM compared to the high SAFA diet. With additional F 7286au#5 (V) 19 tocotrienols a total decreasing effect on average plasma cholesterol of 0.88 mM could be found.

Example IV Male Syrian hamsters, aged 10-11 weeks, were fed semi-purified diets ad libitum, containing 30% of energy as fat; 23 en% as proteins; 47 en% as carbohydrates; .0.01% cholesterol; and either 0.4% phytosterol 10 preparation (ex Kaukas Oy Finland) alone or in combination with 100 ppm tocotrienols or nothing (control). The ratio of polyunsaturated (PUFA) to monounsaturated (MUFA) to saturated (SAFA) fatty acids in all the diets was either 7.2:12:10.8 (high PUFA) or 3:12:15 (high SAFA). The 15 semipurified diets were fed for 6 weeks, after a run-in period on the control diet for 2 weeks. The experimental groups comprised 12-14 hamsters/group. The hamsters were S..housed individually in Makrolon cages type II, with a layer of sawdust as bedding. At the end of the experiment blood samples were collected and (heparin) plasma total cholesterol was determined using a commercially available enzymic method (CHOD-PAP).

Throughout the experiment body weights and feed intake did not differ between the experimental groups. No clinical signs were observed in any group. The average total plasma cholesterol in the high SAFA control groups was 4.25 mM. A decrease on average total plasma cholesterol in the high PUFA control group of 0.30 mM was observed. Addition of phytosterols decreased the average total plasma cholesterol with 0.94 and 0.95 mM for the high SAFA and high PUFA diets respectively. With additional tocotrienols, no additional decreased on the average total plasma cholesterol could be found.

F 7286au#5 (V) Example V Preparation of a spread 44 parts refined sunflower oil (65% PUFA as linoleic acid) was enriched with oryzanol obtained from rice bran oil and mixed with 18 parts of normal refined sunflower oil and 8 parts of a refined interesterified mixture of 50 parts fully hardened palm oil and 50 parts fully hardened palm .kernel oil. To 70 parts of this fatblend, 0.1 part soybean 10 lecithin, 0.1 part monoglyceride and 0.1 part of B-carotene solution are added.

To 29 parts water, 0.3 part whey protein powder, a small amount of flavour, and citric acid to obtain a pH of 4.8 **15 are added.

6 parts of the fat phase composition and 30 parts of the S..aqueous phase composition were mixed and kept at 60 degrees C. The mixture was then passed through a Votator line with 2 scraped surface heat exchangers (A-units) and 1 stirred crystallizer (C-unit) in AAC-sequence operating at 800, 800 and 100 rpm respectively. The product leaving the C-unit had a temperature of 11 degrees C. It was filled into tubs and stored at 5 degrees C. A good and stable, high PUFA, high fat-continuous spread enriched with 2% oryzanol was obtained.

Example VI Preparation of a spread 34 parts refined sunflower oil (65% PUFA as linoleic acid) was enriched with oryzanol obtained from rice bran oil and mixed with 6 parts of a refined interesterified mixture of 50 parts fully hardened palm oil and 50 parts fully F 7286au#5 (V) 21 hardened palm kernel oil. To 39 parts of this fatblend, 0.1 part soybean lecithin, 0.1 part monoglyceride and a small amount of B-carotene solution are added.

To 57 parts water, 2 parts gelatine, 0.3 part whey protein powder, a small amount of flavour, preservative and citric acid to obtain a pH of 4.7 were added.

parts of the fat phase composition and 60 parts of the 10 aqueous phase composition were mixed and kept at 60 degrees C. The mixture was then passed through a Votator line with 2 scraped surface heat exchangers (A-units) and 2 stirred crystallizers (C-unit), in ACAC-sequence operating at 500, 1000, 600 and 100 rpm respectively. The product leaving the last C-unit had a temperature of 10 degrees C. It was filled into tubs and stored at 5 degrees C. A good and stable, high PUFA, low fat-continuous spread enriched with oryzanol was obtained.

S

Example VII 0:00 Preparation of a dressing 33% fat 9e parts of water is mixed with 11 parts of various flavour components, preservatives, thickeners and emulsifiers. The mixture is thouroughly mixed in a stainless steel stirred vessel. To this aquous mixture 33 parts of sunflower oil PUFA as linoleic acid) enriched with oryzanol obtained from rice bran oil as described in Example 1 and a 1% tocotrienol mixture is added, thourouhly mixed for an additional 15 min, to obtain a pre-emulsion. The preemulsion is brought into a colloid mill (Prestomill and processed at a split-size between level 15 and 20 and a throughput between level 4 and 6. A good and stable water continuous dressing enriched with 1.5% oryzanol and 1% F 7286au#5 (V) 22 tocotrienol mixure is obtained.

Example VIII A 100 volunteers were choosen for this study. All reported to be not on a slimming, medically prescribed, vegaterian or vegan diet, reported not to receive medical treatment or having a current disease or history of metabolic diseased, chronic gastrointestinal disorder, cardiovascular disease,high blood pressure or high blood cholesterol. The did not use medicines except analgesics or oral contraceptives. Volunteers had at entry plasma total cholesterol levels below 8 mmol/L and normal plasma activities of L-(-glutamyltransferase, alanine amino- Stransferase and aspartate aminotransferase. White bloods 15 cell, red blood cell and platelet counts, haemoglobin concentration and haematocrit were all within the normal range. The volunteers were requested to retain their s normal lifestyle and dietary pattern.

Experimental design The study had a randomized double-blind placebo controlled balanced incomplete Latin square design with five treatments and four periods. Subjects received in four consecutive periods of 24 or 25 days (3.5 weeks) 30 g/d of a margarine in a coded tub for consumption at lunch and dinner. The spread was meant to replace the spreads habitually used by the volunteers. Three types of margarines were investigated.The statistician allocated each person to one of the treatments for each period such that the design was balanced.

Fasting blood was sampled after 2.5 and 3.5 weeks. Body weight was measured after 3.5 weeks. In each period, dietary intake was assessed with a food frequency method.

The study was designed to have a power of 90% with a confidence of 95% to assess differences in total F 7286au#5 (V) 23 cholesterol levels between treatments of 3-5% based on an expected plasma total cholesterol concentration of 5 mmol/L and an expected within-person variation in plasma total cholesterol level of 0.35 mmol/L (Personal communication, P Zock, 1996, Wageningen Agricultural University).

The test margarines were specially prepared spreads (VandenBergh Foods, Purfleet, UK, and Unilever Research Laboratory, Vlaardingen, the Netherlands), packed in blinded tubs and labeled with a colour code for each period of 3.5 weeks. The margarines were fortified with phytosterol (-ester) concentrates derived from either .predominantly soybean oil distillates (Henkel Corporation, LaGrange, USA) or rice bran oil (Tsuno, Wakayama, Japan).

15 The soybean oil sterols were esterified with fatty acids from sunflowerseed oil to an esterification degree of (Unilever Reasearch Laboratory, Vlaardingen, The Netherlands). The ricebran oil sterols contained only sterols esterified to ferulic acid (oryzanol). The sterol cocentrates were refined and used in spread production together with other edible oils and fats (sunflowerseed oil, rapeseed oil and hardstock) in order to achieve a product as close as possible in fatty acid composition as the nonfortified control. Final free phytosterol concentration was between 5.5 11% in the different porducts. As control article, Flora (VandenBergh Foods, Crawley, UK), a spread not fortified with sterols was used.

Total fat content, fatty acid composition, free sterols, carotenoids and tocopherol content of margarines were measured. For determiation of the fat content samples orf margarine were mixed with celite, colled to -20 degrees C and freeze-dried at 10 degrees C. Fat was extracted with dicholromethane, after evaporation, the leftover was weighed. The triglyceride content was calculated by subtracting the total sterol/sterolester content from the F 7286au#5 (V) 24 total fat content. The fatty acid composition was analysed by methanolysis of the fatty acids in a sample of margarine extracted with a methanolic NaOH solution BF 3 as catalyst.

Analysis of sterol types was done by saponifying the margaine with KOH and extracting the unsaponifiable part into cyclohexane.

The homogeneity of sterols and their content in the margarines was analysed prior to the start by analysing and comparing samples taken atthree different times during the i 10 spread production process. Coefficients of variation in the content of the major sterols for these sampels were generally less than This indicates a good homogeneity ee. of the sterols in the margaines. The analysed margarine composition is given in Table 1.

Margarines were provided in lots of 30 tubs per period; volunteers were requested to store them in the refrigerator until use. At the end of each period remaining tubs returned to the dieticians.

*s F 7286au#5 (V) Table 1 FLORA soybean ricebran Total fat as glycerides (g/100 g) 70 I3 72 Water (g/100 g) 30 16 22 Major fatty acids (g/100 g) Lauric (C12:0) 1.6 1.2 1.1 Myristic (C14:0) 0.7 0.6 Palmitic (C16:0) 9.3 8.7 8.2 Stearic (C18:0) 2.7 3.0 Oleic (C18:1 cis) 20.2 24.6 24.7 Lineleic (C18:2, 9c, 12c) 32.1 38.4 37.2 Linolenic (C18:3, 9c, 12c, 15c) 1.5 1.8 1.6 Total sterols (g/100 g) 0.36 10.82 5.58 Major sterols (g/100 g) Campesterol 0.06 2.7 0.78 Campestanol 0.13 0.07 Stigmasterol 0.01 1.89 0.07 Sitosterol 0.16 5.03 0.50 Sitostanol 0.17 0.37 Cycloartenol 0.01 0.02 1.57 24 meth cycloartenol 0.01 2.07 Vitamin E (mg/10Og) 39 68 Alpha and beta carotene(mg/100 g) 0.47 0.53 0.44 Results The study started as intended with 100 adult volunteers, males and 50 females, with a mean age of 45 V 12.8 years and a body mass index of 24.2 V 2.16 kg/m 2 Baseline total- LDL- and HDL-cholesterol levels were 5.35 V 1.06, 3.54 V 0.97 and 1.26 V 0.35 mmol/L, respectively. During the study no changes were reported in the type of diet consumed. Average spread consumption was around 30 g/day.

The very large majority of margarines were used as intended at lunch and dinner, overall for each product in more than 99.2 of all meal occurrences.

Table 2 shows blood lipid data based on 2.5 and sampling results, including the minimal significant difference with 95% confidence.

In the analysis, the mean of the 2.5 and 3.5 weeks results is used for HDL- cholesterol, triglycerides and LDL cholesterol were used.

In table 3, the treatment effects are shown for tertiles based on the study entry values for total- or LDLcholesterol concentrations. The soybean spread F 7286au#5 (V) 26 significantly lowered total- and LDL-cholesterol concentrations by about 0.37-0.44 mmol/L compared to Florawithout an effect on HDL-cholesterol level.

Consequently the total- or LDL cholesterol to HDLcholesterol ratio was significantly reduced by the soybean sterol product. Total glycerol levels were not affected by the treatment. There was no significant difference between males and females in treatment effect.

Claims (3)

1. Fat based food product wherein the fat used in the product is a fat comprising at least 30 wt%, and prefearbly 45 wt% of pufa-rich triglycerides, calculated on the total weight of the fat present in the product, and wherein the fat comprises at least one compount of the group consisting of tocotrienol, phytosterol, and oryzanol in an amount of at 10 least 0.2 wt.% for tocotrienol, at least 0.25 wt% for phytosterol, and at least 0.25 wt.% for oryzanol, the weight percentages being based on the total weight of the fat based food product.

2. Fat based food product according to claim 1, wherein a mixture of phytosterol and oryzanol is present

3. Fat based food product according to claim 1 or 2, wherein at least one of tocopherol and polyphenol, is incorporated 20 into the fat. DATED Signed for and on behalf on UNILEVER PLC by Unilever Australia Limited AYELA THILO