BACKGROUND OF THE INVENTION A number of research studies focused on the prevention of cardiovascular disease by n-3 polyunsaturated fatty acids (n-3 PUFA), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). n-3 PUFA have shown these acids favourably affect atherosclerosis, coronary heart disease, inflammatory disease, and perhaps even behavioural disorders.

Dietary n-3 PUFA act to prevent heart disease through a variety of actions.

They prevent arrythmias, are prostaglandin and leukotriene precursors, have anti-inflammatory properties, inhibit synthesis of cytokines and mitogens, stimulate endothelial-derived nitric oxide, are antithrombotic, have hypolipidemic properties and inhibit atherosclerosis (Connor; 2000; Am. J. Clin.

Nutr., 71: 171S-5S).

It has also been shown that diets, like Mediterranean diet, rich in monounsaturated fatty acids (MUFA) and poor in saturated fat have favourable effects on the cardiovascular risk profile (Feldman ; 1999; Am. J. Clin. Nutr., 70: 953-4).

In order to prevent cardiovascular diseases, an increased consumption of products enriched in n-3 polyunsaturated fatty acids and monounsaturated fatty acid would be desirable and beneficial for the population. However, the present way of life, the established food habits and other factors make optimum intake levels of n-3 polyunsaturated fatty acids and monounsaturated fatty acids quite difficult to achieve, since these nutrients are not present in most widely consumed products.

The invention described here provides a feasible and convenient way to increase the population consumption of n-3 polyunsaturated fatty acids, monounsaturated fatty acids and even vitamins which to a greater or lesser extent protect the cardiovascular system.

SUMMARY OF THE INVENTION The present invention provides an oil blend comprising n-3 polyunsaturated fatty acids (n-3 PUFA) and monounsaturated fatty acids (MUFA), nutritional products containing these oil blends and also the use of both the oil blends and the nutritional products in the prevention of cardiovascular diseases.

An aspect of the invention relates, thus, to an oil blend comprising n-3 polyunsaturated fatty acids (n-3 PUFA) and monounsaturated fatty acids (MUFA).

A second aspect of the invention relates to a process for preparing the oil blend, which comprises the following steps: a) preparing an oil rich in n-3 polyunsaturated fatty acids from plants and/or fish oil, b) preparing a mixture of vegetable oils rich in oleic acid, c) incorporating and mixing the oil obtained in step a) with the mixture obtained in step b) in the absence of oxygen and, optionally,

d) adding to the oil blend a vitamin selected from the group consisting of A, B6, B12, C, D, E or folic acid or a mixture thereof.

A third aspect of the invention relates to a nutritional product comprising the mentioned added oil blend.

Finally, a fourth aspect of the invention relates to the use of the nutritional product in the prevention of cardiovascular diseases and as diet supplement for infants, adolescents, elderly people, pregnant women, athletes and clinical nutrition formula.

DETAILED DESCRIPTION OF THE INVENTION As mentioned above, the first aspect of the invention relates to an oil blend comprising n-3 polyunsaturated fatty acids (n-3 PUFA) and monounsaturated fatty acids (MUFA). In this blend, the n-3 PUFA preferably include eicosapentaenoic acid or docosahexaenoic acid or a mixture thereof; whereas the MUFA preferably include oleic acid. A preferred blend composition comprises from 1 % to 85% in weight of eicosapentaenoic acid or docosahexaenoic acid or a mixture thereof and oleic acid. The relationship in weight between MUFA and PUFA is preferably 30 to 70/1, more preferably 50/1. Furthermore, the preferred weight relationship between eicosapentaenoic acid, docosahexaenoic acid and oleic acid is of 1/0.2-10/30-90, more preferred 1/1.2-2. 0/45-80, most preferred 1/1.6/50, 1/0. 65/ 40 or 1/1.6/27.

The oil blend also has other components as saturated fatty acid (less than 10% in weight) and n-6 polyunsaturated fatty acids (less than 20% in weight).

The n-3 polyunsaturated fatty acids of the oil blend may be obtained from fish oil or from plants, especially from seaweed, or may be a mixture of oil obtained from fish, plants and seaweed. The oil blend also includes monounsaturated

fatty acids coming from the following vegetable oils : safflower, rapeseed, borage, high oleic sunflower, sunflower, palm, soy, corn and olive oil. The oil blends of the invention can also include vitamins selected from the group selected from vitamins A, B6, B, 2, C, D, E or folic acid. These vitamins may be present in a percentage to satisfy at least 15% of the daily Recommended Dietary Allowances (RDA), assuming a daily consumption of 25g of the oil blend described in the invention.

The oil blends of the invention can be employed in cardiovascular disease prevention. They can also be employed as food or diet supplement.

A second aspect of the invention relates to a method for preparing the oil blend mentioned above. This method comprises the following steps: a) preparing an oil rich in n-3 polyunsaturated fatty acids from plants and/or fish oil, which comprises a previous processing of degumming and refining of the oil followed by a reduction of the acidity to values less than 0. 1%. The refining process being an alkali type-refining using 25% excess of NaOH. The process continues with a bleaching process using Tonsil activated bleaching clay at 80-100°C for 30-60 minutes to remove peroxide compounds and obtaining a color reduction. Later, the oil is deodorized using a steam flow at 150- 200°C for 2-4 h and is finally stabilized with an added mixture of natural antioxidants, b) preparing a mixture of vegetable oils to obtain the desired composition in monounsaturated fatty acid (mainly oleic acid), c) incorporating and mixing the oil rich in n-3 polyunsaturated fatty acids obtained in step a) with the vegetable oils prepared in step b) in the absence of oxygen and, optionally,

d) adding to the oil blend a vitamin selected from the group consisting of A, B6, B, 2, C, D, E or folic acid or a mixture thereof.

A third aspect of the invention relates to a nutritional product to which the oil blend of the invention defined above has been added. By the expression "nutritional product"it is understood any food product. The nutritional products of the invention preferably comprise from 0.05% to 99% of the added oil blend of the invention. A more preferred range comprise from 0.05% to 30% of the added oil blend of the invention. Nutritional products containing this amount of added oil blend can be milk, dairy products, yoghurts, fermented milks, fruit and vegetable juices, biscuits, cakes, infant food and dehydrated food. Other more preferred range comprise from 10 % to 99% of the added oil blend of the invention. Nutritional products containing this amount of added oil can be margarine, butter, oils and spreads.

The addition of the oil blend to the nutritional products is effected by mixture and homogenisation according to the technological procedure of each product.

Another components such as vitamins and or minerals can be added to the nutritional products. Thus, vitamins A, B6, B, 2, C, D, E or folic acid or a mixture of one or more thereof can be added to nutritional products according to the present invention. The addition of these compounds can be effected either previously or after the heat treatment according to the specific technological procedure of each nutritional product.

Finally, a fourth aspect of the invention relates to the use of the nutritional product of the invention in the prevention of cardiovascular diseases as well as diet supplement for infants, adolescents, elderly people, pregnant women and athletes, and clinical nutrition.

The following examples are only presented to illustrate the object of the present invention.

Example 1 PREPARATION OF AN OIL BLEND A oil blend was prepared using the following ingredients: Ingredient Amount per Kg Olive oil 270 High oleic sunflower oil 630 g Stabilized fish oil 100 g Processing technology.- The preparation of the oil blend comprises: 1. Obtaining the oil rich in n-3 polyunsaturated fatty acid, which comprises a previous processing of degumming and refining of the oil followed by a reduction of the acidity to values less than 0. 1%. The refining process consists of an alkali-refining using 25% excess of NaOH. The process continues with a clay bleaching using Tonsil activated bleaching clay at 80-100°C for 30-60 min to remove peroxide compounds and obtaining a reduction of color. Later, the oil is deodorized by steam using 150-200°C steam for 2-4 h. Finally, the oil is stabilized with a mixture of natural antioxidants.

2. Mixture of the vegetable oils to obtain the desired composition in monounsaturated fatty acid (mainly oleic acid).

3. Incorporation and mixture of the oil rich in n-3 polyunsaturated fatty acids with the mixture obtained in step 2 in absence of oxygen.

4. Adding to the oil blend a vitamin selected from the group consisting of A, B6, B, 2, C, D, E, folic acid or a mixture thereof.

Example 2 PREPARATION OF ENTERAL FORMULA An enteral formula was prepared using the following ingredients: Ingredient Amount per litre Skimmed milk 814, 30 Whey protein concentrate 11, 80 g Oil blend 32, 50 g Sacarose 27, 00 g Maltodextrine 71,00 g Soluble Fiber 10,00 g Vitamin A 1200 mg Vitamin D 7. 5 mcg Vitamin E 15 mg Vitamin K 120 mcg Vitamin C 90 mg Thiamin 2. 25 mg Riboflavin 2. 55 mg Pyridoxine 3 mg Vitamin B12 3 mcg Niacin 28. 5 mg Folic acid 30 mcg Panthotenic acid 4 mg Biotin 40 mcg Calcium 1200 mg Phosporus 970 mg Magnesium 47 mg Sodium 480 mg Potassium 1000 mg Chloride 700 mg Iron 43 mg Zinc 24. 5 mg lodine 150 mcg Manganese 100 mcg Selenium 10 mcg Mono-and diglycerides 1, 50 g Bisodium phosphate (Na2HP04 5H20) 0,60 g Carrageenan 4, 00 g Desmineralised Water 70, 00 g Arome 2, 75

Processing technology.- To an appropriately sized blend tank with agitation and heating all solid ingredients were mixed with the liquid milk and water. Then, the oil blend (obtained by the procedure described above) was admixed. The mixture was then heated at 60-70° C and emulsified through a single stage homogeniser at 6 to 7 MPa in absence of oxygen. After emulsification the mixture was heated to 140-150° C, 4-6 s, and was then passed through a two stages homogeniser (27-29 MPa and 3-4 MPa). Finally the mixture was packaged in the absence of oxygen.

Example 3 PREPARATION OF A FERMENTED MILK WITH THE OIL BLEND A fermented milk product was prepared using the following ingredients: Ingredient Amount per Kg Skimmed milk 890 g Oil blend 23. 2 g Sugar 70 g Milk protein 8 g Starter cultures 0. 1 g Lecitine 0. 5 g Flavours 100 mg Vitamin C 90 mg Vitamin B1 2. 1 mg Vitamin B2 2. 4 mg Vitamin B6 3 mg Vitamin B12 1. 5 mcg Vitamin A 1. 2 mg Vitamin D 7. 5 mcg Folic acid 300 mcg

Processing technology.- Fermented milk base was prepared by addition and further mix of all ingredients (except the oil blend) in skimmed milk. Then, the oil blend (obtained by the procedure described above) was admixed. The mixture was pasteurised and homogenized previously to fermentation process, which took place by inoculation of starter cultures and further incubation. Finally, the product was cooled and packaged.

Example 4 PREPARATION OF A LIGHT SPREADABLE A light spreadable was prepared using the following ingredients: Ingredient Amount per kg Butter 200 g Oil blend 208 g Starch 8 g Caseinate 6 g Emulsifier 2 g Maltodextrine 6 g Water 570 g

Processing technology.- Aqueous phase with water soluble ingredients was first prepared. Emulsifiers were melted and then were mixed with the oily phase. Aqueous phase then was incorporated into the oily phase by continuous addition at high temperature and further mix. The previous mix was pasteurised by using scrapped surface heat exchangers. The final solid product was obtained using a high speed rotor equipment with an external cooling system.

Example 5 PREPARATION OF AN ENRICHED JUICE An enriched juice was prepared using the following ingredients: Ingredient Amount per kg Juice from concentrated 500 g Oil blend 4 9 Insoluble fiber 10 g Lecitine 0. 5 g Flavours 2 g Vitamin C 90 mg Vitamin B1 2. 1 mg Vitamin B2 2. 4 mg Vitamin B6 3 mg Vitamin B12 1. 5 mcg Vitamin A 1. 2 mg Vitamin D 7. 5 mcg Folic acid 300 mcg Processing technology.- The final product was prepared from a concentrated juice by addition of water and water soluble ingredients. Then, the oil blend of the invention was added and mixed and the resulting product was pasteurised and homogenized.

Finally, the product was cooled and packaged.

Example 6 PREPARATION OF AN U. H. T MILK BASED PRODUCT A milk based product was prepared using the following ingredients: Ingredient Amount per litre Skimmed milk 960 g Skimmed milk podwer 17g Oil blend 19 Bisodium phosphate 0. 5 g Tripotasium phosphate 0. 2 g Water 2 g Vitamin B6 3 mg Vitamin B12 3. 8 mcg Vitamin A 1200 mcg Vitamin D 7. 5 mcg Vitamin E 15 mg Folic acid 300 mcg

Processing technology.- All solid ingredients were mixed with the liquid milk and water. Then, the oil blend (obtained by the procedure described above) was admixed and homogenised in the absence of oxygen. The resulting dairy product was then subjected to U. H. T. treatment (150° C for 4 to 6 seconds) and finally packaged in the absence of oxygen.

Experimental design Ninety volunteers (45 men and 45 women, age: 42. 5 17.2, range: 20-65y) participated in the study. Volunteers were defined as healthy normolipidaemic subjects after medical history and physical examination. No subject was taking any medication known to influence lipid metabolism 1 month before the study and they were not suffering from any chronic or metabolic diseases. Women volunteers were not using oral contraceptives. Volunteers were instructed not to change their physical activity and their usual diet, but to avoid eating fish until the end of the study. Subjects drank 500 mL/day of semi skimmed milk from the beginning of the study (T) for four weeks (To). At time To, subjects were randomised in three different groups and replaced the semi skimmed milk with 1) a dairy product containing the fat mixture of the invention described in the example number 6, containing oleic acid and polyunsaturated fatty acids of

the n-3 series plus vitamins A, D, E, C, B6, B12 and folic acid (group OB); 2) a similar dairy product containing milk fat and polyunsaturated fatty acids of the n-3 series plus vitamins A, D, E, C, B6, B, 2 and folic acid (group co3) ; and 3) a dairy product containing oleic acid plus vitamins A, D, E, C, B6, B12 and folic acid (group O). After an overnight fast lasting 10h, a blood sample (30 mL) was taken at times T4 (beginning of the study), To (after consumption of the semi skimmed milk) and then, at 8 weeks after the supplementation with the nutritional product) (T8). Composition of the nutritional products used in the study is given in tables 1 and 2.

Table 1. Nutrient composition of the nutritional product used in the study Nutrient composition Grops O, #3 and OB Energy (kcal/100 ml) 52 Protein (g/100ml) 3. 5 Carbohydrate (g/100ml) 5.2 Folic acid (µg/100ml) 30 Vitamin A (µg/100ml) 120 Vitamin D (µg/100ml) 0.75 Vitamin E (mg/100ml) 1. 5 Vitamin B6 (mg/100m 0. 3 Vitamin B12 (µg/100ml) 0.38 Folic acid (µg/100ml) 30 Table 2. Fat composition of the nutritional product used in the study Olive oil group #3 group Oil Blend group Total fat (g/100 mL) 1.9 1,9 1, 9 oleic acid (g/100 mL) 1. 2 0. 4 1,2 EPA+DHA (mg/100 mL) undetectable 60 60

Plasma isolation y plasma biochemical parameters Blood was withdrawn in EDTA-containing vacutainers and plasma was obtained by centrifugation at 1 000 x g for 10 min at 4°C. Triacylglycerols, (TG), total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL- C), plasma total antioxidant capacity (CAP), TBARS measured as malondialdehyde (MDA), intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and vitamin E were measured in plasma at the beginning of the study (T4), after one month of consumption of semi skimmed milk (To) and after eight weeks of consumption of the nutritional product assigned to each group (T8). TG, LDL-C and HDL-C were measured with commercially available kits obtained from Biosystems (Spain). For plasma lipid peroxidation, fresh plasma (50 tl) were incubated with 100 mM of the free radical generator AAPH (Wako Ltd, Japan), at 37°C for 2 h, and MDA was determined as described elsewhere. Total antioxidant capacity was measured using Trolox as standard with a commercially available kit (Randox, UK).

VCAM-1 and ICAM-1 were measured by ELISA using the a commercial kit purchased from cytoscreen (Biosource international, USA). Plasma vitamin E concentration was measured by HPLC with uv detection.

Table 3. Plasma concentrations of Triacylglycerols, (TG), total cholesterol (TC), LDL cholesterol (LDL-C), HDL-cholesterol (HDL-C), plasma total antioxidant capacity (CAP), Malondialdehyde (MDA), vitamin E, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), were measured at the beginning of the study (T), after one month of consumption of semi skimmed milk (To) and after eight weeks of consumption of the dairy product supplemented oleic acid (group O), omega 3 (group cl) 3), or with the oil blend of the invention (group OB),. Parameter T4 To T8 group O T8 Group co 3 T8 group OB TG (mmol/L) 1. 330. 04a 1.35~0.04a 1. 360. 05 a 1. 300. 06 a 1. 110. 04 b TC (mmol/L) 4. 830. 10 a 4. 880. 09 a 4. 550. 12 b 4. 890. 12 a 4. 130. 09c LDL-C (mmol/L) 2. 820. 12 a 2. 830. 11a 2.55~0.12b 2. 71~0.11a 2. 280. 11' HDL-C (mmol/L) 1. 510. 06 a 1. 520. 07a 1. 550. 03 a 1. 650. 03 b 1. 750. 02 C Vitamin E (mg/L) 39. 72. 1 a 36. 81. 5 a 39. 72. 4 a 38. 01. 7 a 39. 21. 0 a CAP (pM Trolox) 20. 50. 1 a 19. 40. 2 a 20. 50. 2a 19.9~0.2a 20.5~0.2a MDA (pmol/L) 17. 70. 8 a 16. 70. 8 a 17. 20. 8 a 17. 00. 7 a 16. 80. 7 a ICAM-1 (pg/L) 8. 810. 27 a 8. 800. 37 a 8. 600. 37 a 8. 380. 34 a 7. 910. 34 VCAM-1 (pg/L) 11. 30. 25 a 11. 20. 35a 11 0+0 3 a 10. 140. 2 b 9. 560. 19 C

Average~SEM. a,b,c, Values with different superscript letters are significantly different, P < 0.05.

Plasma levels of triacylglycerols, total cholesterol and LDL-cholesterol in the oil blend group significantly decreased by 17%, 15% and 20%, respectively, whereas HDL cholesterol levels increased by 15% compared to plasma levels at the beginning the study (T-4). In contrast, TG levels in group O remained unchanged, total cholesterol and LDL cholesterol values only decreased by 6% and 10%, respectively, and a minor increase of 3% in HDL cholesterol levels was observed. With respect to group m 3, plasma levels of TG remained unchanged compared to values obtained at T 4 LDL cholesterol only decreased by 4% and HDL cholesterol levels increased by approximately 9%, whereas total cholesterol levels remained unchanged throughout the study for this group.

Plasma vitamin E, total antioxidant capacity and plasma oxidability levels (measured as MDA) remained unchanged throughout the study which suggests the addition of PUFAs, being more prone to in vitro oxidation, does not affect plasma oxidability.

With regard to the adhesion molecules measured in the study (ICAM-1 and VCAM1), only the OB group showed a significant decrease in plasma levels of both. In atherosclerosis, elevated levels of soluble VCAM-1 and ICAM-1 have been reported. In a further study within the"Physician's Health Study", soluble ICAM-1 at entry appeared to be a predictor for long term cardiovascular events.

LDL isolation and biochemical parameters measured in LDL.

For LDL isolation, 10 ml of fresh plasma were transfered to ultracentrifuge tubes and its density was adjusted to 1.30 g/ml with solid KBr. Tubes were then filled up dropwise with a 0.15 M NaCI solution and centrifuged at 242 000 x g for 2.5 h at 4°C in a VTi50 rotor. LDL particles typically sedimented at the density range of 1.006-1. 063 g/ml. LDL fractions were pooled and dialysed in the dark for 24 h against three changes of 2 L each of 0. 01M phosphate buffered saline (PBS) 0.15 M NaCI, pH 7.4, and then frozen at-80°C until needed.

For lag time measurements, 50 jug of dialysed LDL in 1 ml PBS were incubated with'0 'M CUS04 for several hours at 37°C. The formation of conjugated dienes was monitored continuosly by measuring the increase in absorbance at 234 nm every 10 min. LDL lipid peroxidation was determined with the ferrous oxidation/xylenol orange reagent before and after AAPH induction. Vitamin E was measured as described above using 1 mg of LDL protein.

Table 4. Antioxidant parameters of LDL particles isolated from volunteers of the study. Parameter T 4 To T8 Group O T8 Grou T8 group W3 OB Vitamin E22. 31. 1 a 20. 6~0. 9 a 21. 51. 0a 24.2~1.5a 24. 21. 5 a (mg/g LDL protein) ElM LDL20. 1 1. 1 a 20. 0~1. 0 a 18. 70. 9 ab 19. 70. 5 a 17. 70. 6 b hydroperoxid es/mg protein Lag time 175. 3+6. 5 a 180. 46. 5 a 181. 25. 7 a 183. 24. 6 a 198. 25. 6 b (min)

AverageiSEM. a bS Values with different superscript letters are significantly different, P < 0.05. T-4, values at the beginning of the study; To, values after one month of consumption of semi skimmed milk ; T8 values after eight weeks of consumption of the dairy product supplemented with oleic acid (group O), omega 3 (group c3 3) or the oil blend (group OB).

LDL vitamin E values did not change during the study but 1) LDL basal hydroperoxides were significantly reduced, and 2) lag time was significantly increased by about 13% in the OB group. The lag time parameter indirectly indicates the ability of LDL lipoproteins to withstand oxidation, as it measures the time the lipoproteins take to reach the oxidation log phase upon incubation with an oxidant (copper). The results obtained suggest the administration of the nutritional product containing the oil blend significantly protects LDL particles from deleterious oxidations when compared to the other groups.

The effects on the biomarkers mentioned above observed for the group of volunteers that consumed the oil blend-supplemented dairy product was therefore much more prominent than the one estimated for each of the groups O and omega 3, and moreover, even more prominent than the two taken together. This results indicate there is a synergic effect in the components of the oil mixture described in the invention.

High cholesterol/triacylglycerides levels, low HDL cholesterol levels and specially oxidised LDLs are among risk factors associated with atherosclerosis and coronary heart disease. We have studied the effect that a nutritional product containing an oil blend has on biomarkers for cardiovascular events, and compared it with similar nutritional products enriched in oleic acid or omega 3, whose composition has been described in Tables1 and 2. Taking all the parameters measured into account, we conclude the components of the oil blend mixture described in the invention show a synergic effect, and the regular administration of the oil blend protects the cardiovascular system which, to a greater or lesser extent, prevents the occurrence of cardiovascular events and therefore may well be considered as a health promoting nutritional product.