This invention relates to fat blends and to their uses. In particular, the invention relates to fat blends which can increase the levels of essential fatty acids in the body.

Docosahexaenoic acid (commonly known as DHA; 22:6(ω-3), a//-c/s-docosa- 4,7,10,13,16,19-hexa-enoic acid) is an omega-3 essential fatty acid. DHA is present in fish oils and is frequently taken as a nutritional supplement for its health benefits. DHA is the most abundant essential fatty acid in the brain and the retina and is believed to be beneficial in the treatment or prevention of many conditions and disorders including heart disease and disorders of the brain.

In the human body, DHA is provided by the diet or is synthesised from eicosapentaenoic acid (EPA). In turn, EPA is either derived directly from the diet or is synthesised from alpha-linolenic acid (ALA). ALA is another essential fatty acid and is present in relatively large amounts in oils such as linseed oil.

Attempts have been made to alter the levels of fatty acids, such as DHA, by supplementation of the diet with other fatty acids.

Eder et al, Journal of Animal Physiology and Animal Nutrition, 89, 2005, 45-54 describes the effect of linseed oil supplementation on the concentration of polyunsaturated fatty acids in liver phospholipids of rats fed diets containing oils rich in conjugated linoleic acid (CLA), sunflower oil or high-oleic sunflower oil. One of the diets tested contained ALA and CLA and oleic acid in respective amounts of 31 , 30 and 10 % by weight of the fatty acids present.

Attar-Bashi et al, Asia Pac J Clin Nutr, 2003, 12, suppl:S44 tested whether dietary CLA in combination with ALA could increase DHA levels compared to ALA alone and concludes that CLA does not increase DHA levels.

It has surprisingly been found, according to the invention, that certain compositions containing ALA and CLA can increase DHA levels. Moreover, the presence of oleic acid in the fat blend, in combination with the ALA and CLA, has been found to be important. WO 2007/096148 discloses the unrelated use of a conjugated fatty acid for enhancing immune system development. A diet used in the examples contains 8.6 % ALA, 11.2 % CLA and 19 % oleic acid, based on the weight of the fatty acids present.

WO 2005/063230 relates to the use of conjugated linolenic acids for another unrelated use, which is reducing body fat mass. All of the diets were relatively low in CLA content.

Bolukbasi et at, Journal of Animal and Veterinary Advances, (2007), 6(2), 262- 266, relates to the effects of CLA on broiler performance and describes a diet comprising 1.5 % olive oil and 1.2 % CLA.

There remains a need for compositions that can increase the level of DHA in a subject when they are consumed. There is also a need to provide an alternative to conventional sources of DHA, which is usually derived from fish or algae. Both algal DHA and DHA from fish tend to have problems of poor taste and DHA from fish is unacceptable to vegetarians.

Accordingly, the present invention provides fat blends comprising alpha-linolenic acid (ALA), conjugated linoleic acid (CLA) and oleic acid, wherein the fat blend comprises:

(i) greater than 3 % by weight alpha-linolenic acid (ALA); (ii) greater than 12 % by weight conjugated linoleic acid (CLA); and

(iii) greater than 20 % by weight oleic acid,

the percentages being based on the total weight of C16 to C20 fatty acids in the blend.

In another aspect, the invention provides the use of a fat blend comprising alpha- linolenic acid (ALA), conjugated linoleic acid (CLA) and greater than 15 % by weight oleic acid based on the total weight of C16 to C20 fatty acids in the blend, for increasing the levels of docosahexaenoic acid (DHA) in the blood of a subject by oral consumption. In yet another aspect, the invention provides a fat blend comprising alpha- linolenic acid (ALA), conjugated linoleic acid (CLA) and greater than 15 % by weight oleic acid based on the total weight of C16 to C20 fatty acids in the blend, for increasing the levels of docosahexaenoic acid (DHA) in the blood of a subject by oral consumption.

Also provided by the invention is a method for increasing the levels of docosahexaenoic acid (DHA) in the blood of a subject which comprises providing the subject with a fat blend comprising alpha-linolenic acid (ALA), conjugated linoleic acid (CLA) and greater than 15 % by weight oleic acid based on the total weight of C16 to C20 fatty acids in the blend, for oral consumption.

The invention also provides the use of a fat blend comprising alpha-linolenic acid (ALA), conjugated linoleic acid (CLA) and greater than 15 % by weight oleic acid based on the total weight of C16 to C20 fatty acids in the blend, in the manufacture of a composition for treating or preventing one or more conditions selected from neurodegenerative diseases, depression, Alzheimer's disease, obesity and cardiovascular disease.

The invention also provides the use of a fat blend comprising alpha-linolenic acid (ALA), conjugated linoleic acid (CLA) and greater than 15 % by weight oleic acid based on the total weight of C16 to C20 fatty acids in the blend, in the manufacture of a composition for improving one or more of brain function, brain development, retinal function and retinal development.

Further provided by the invention is a foodstuff or nutritional supplement comprising a fat blend of the invention.

The invention is based on the finding that certain compositions can increase the level of DHA when orally consumed.

The compositions used in the invention are fat blends. The fat blends comprise alpha-linolenic acid (ALA), conjugated linoleic acid (CLA) and oleic acid.

The fat blends of the invention comprise:

(i) greater than 3 % , preferably greater than 5 %, more preferably from 7 to 65 %, such as 7 to 30 %, by weight alpha-linolenic acid (ALA); (ii) greater than 12 %, preferably greater than 15 %, more preferably from 16 to 77 %, even more preferably from 17 to 55 %, by weight conjugated linoleic acid (CLA); and

(iii) greater than 20 %, preferably from 25 to 82 %, more preferably from 25 to 45 %, by weight oleic acid.

All of the percentages are based on the total weight of C16 to C20 fatty acids in the blend.

The CLA, ALA and oleic acid may take different chemical forms in the fat blends of the invention. For example, the CLA, ALA and oleic acid may independently be in the form of free acids, salts or esters, or combinations thereof. Preferably, at least the ALA and the oleic acid are in the form of triglycerides, most preferably, all of the ALA, CLA and oleic acid are in the form of triglycerides. Suitable salts are non-toxic, pharmaceutically acceptable and/or acceptable for use in food products and/or pharmaceuticals and include, for example, salts with alkali metals and alkaline earth metals such as sodium, calcium and magnesium, preferably sodium. Suitable esters include, for example, mono-, di- and triglycerides and mixtures thereof, and C ₁ to C ₆ alkyl esters (where the alkyl group can be straight chain or branched), as well as esters formed with alcohols that are acceptable in food products or pharmaceutical products, such as are disclosed in EP-A-1167340, the contents of which are incorporated by reference herein.

The fat blends of the invention will typically contain C12 to C24 fatty acids other than ALA, CLA and oleic acid. The term "fatty acid", as used herein, refers to straight chain, saturated or unsaturated carboxylic acids having from 12 to 24 carbon atoms. The fatty acids may be present as free fatty acids, esters or salts, or combinations thereof, and will usually be in the form of glycerides, most preferably triglycerides.

Alpha-linolenic acid (ALA) is typically present in the fat blend in an amount of from 5 to 30% by weight, more preferably from 6 to 25 % by weight based on the total weight of C16 to C20 fatty acids in the blend. The ALA may be present as a free fatty acid, an ester or a salt, or a combination thereof, and will usually be in the form of a glyceride, most preferably the triglyceride. At least a part of the alpha-linolenic acid (ALA) in the blend is preferably provided by the presence in the fat blend of linseed (flaxseed) oil. Other sources of ALA include, for example, rapeseed (canola), soybeans and walnuts.

Oleic acid is preferably present in the fat blend in an amount of from 20 to 50 % by weight, more preferably from 25 to 40 % by weight based on the total weight of C16 to C20 fatty acids in the blend. The oleic acid may be present as a free fatty acid, an ester or a salt, or a combination thereof, and will usually be in the form of a glyceride, most preferably the triglyceride.

At least a part of the oleic acid in the blend is preferably provided by the presence in the fat blend of olive oil. Olive oil typically contains relatively high levels of oleic acid.

CLA is typically present in the fat blend in an amount of from 12 to 50 % by weight, more preferably from 16 to 45 % by weight based on the total weight of C16 to C20 fatty acids in the blend. The CLA may be present as a free fatty acid, an ester or a salt, or a combination thereof, and will usually be in the form of a glyceride, most preferably the triglyceride.

The CLA may comprise one isomer or a mixture of two or more different isomers including: cis, cis; cis, trans; trans, cis; and trans, trans isomers. Preferred isomers are the transiO, cis12 and cis9, trans 11 isomers, including these isomers in relatively pure form, as well as mixtures with each other and/or mixtures with other isomers. More preferably, the CLA comprises transiO, cis12 and cis9, trans11 isomers and the weight ratio of transiO, cis12 isomer to cis9, trans11 isomer, or vice versa, is at least 1.2:1, such as 1.3:1 , even more preferably at least 1.5:1 , e.g., in the range 1.5:1 to 100:1 or 1.5:1 to 10:1 , such as a 60:40 or 80:20 mixture of the transiO, cis12: cis9, trans11 isomers. Particularly preferred are compositions comprising the cis9, trans11 isomer as the major isomer component i.e., present in an amount of at least 55 %, preferably at least 60 %, more preferably at least 70 %, even more preferably at least 75 %, most preferably at least 80 %, such as at least 90 % or even 100 % by weight based on the total amount of CLA.

CLA can be produced in conventional ways. For example, CLA can be produced by known methods, such as that described in EP-A-902082, the contents of which are incorporated herein by reference. CLA enriched in one or more isomers is disclosed in WO 97/18320, the contents of which are also incorporated herein by reference.

The preferred fat blends of the invention comprise linseed oil, olive oil and a source of conjugated linoleic acid (CLA). In one aspect, the invention contemplates a fat blend comprising linseed oil, olive oil and CLA.

One particularly preferred fat blend of the invention comprises:

(i) from 15 to 25 % CLA; (ii) from 10 to 20 % ALA; and (iii) from 20 to 50 % oleic acid; all percentages being based on the total weight of C16 to C20 fatty acids in the blend.

Another preferred fat blend of the invention comprises:

(i) from 30 to 50 % CLA; (ii) from 4 to 15 % ALA; and

(iii) from 20 to 40 % oleic acid; all percentages being based on the total weight of C16 to C20 fatty acids in the blend.

It will be appreciated that the sum of the percentages for any given composition cannot exceed 100 % but that it may be less than 100 % where, for example, the composition contains other C16 to C20 fatty acids.

The fat blends of the invention are suitable for human consumption and are preferably adapted for human consumption. The fat blends can be consumed as such but are preferably formulated into food products or nutritional supplements.

The fat blends of the invention (and food products or nutritional supplements comprising them) are preferably consumed at a different time from a meal, for example, at least 30 minutes, more preferably at least one hour, before or after a meal. Suitable food products that may comprise the fat blends of the invention include, for example: margarines; low fat spreads; very low fat spreads; bicontinuous spreads; water continuous spreads; confectionery products, such as chocolates, coatings or fillings; ice creams; ice cream coatings; ice cream inclusions; dressings; mayonnaises; sauces; bakery fats; shortenings; cheese; meal replacement products; health bars; muesli bars; drinks; dairy products; low carbohydrate products; low calorie products; soups; cereals; and milk shakes.

Other examples of product forms for the fat blends of the invention are nutritional supplements (which term includes food supplements), such as in the form of a soft gel or a hard capsule preferably comprising an encapsulating material selected from the group consisting of gelatin, glycerol, starch, modified starch, starch derivatives such as glucose, sucrose, lactose and fructose. The encapsulating material may optionally contain cross-linking or polymerizing agents, stabilizers, antioxidants, light absorbing agents for protecting light- sensitive fills, preservatives and the like. Preferably, the unit dosage of the fat blend in the food supplements is from 1 mg to 1000 mg (more preferably from 100 mg to 750 mg). The amount of the fat blend that is used in a unit dosage form is preferably from 100 mg to 2000 mg, for example 250 mg to 1500 mg (e.g., 750 mg), for example for taking four times a day.

The compositions of the invention may contain other additives that are well known in the art of food and pharmaceutical products including, but not limited to, flavouring ingredients, colouring agents, sweeteners and emulsifiers.

According to the invention, certain fat blends are useful for increasing the levels of docosahexaenoic acid (DHA) in the blood of a subject by oral consumption. The fat blends comprise ALA, CLA and greater than 15 %, more preferably greater than 20 %, such as greater than 30% or greater than 40 %, by weight oleic acid based on the total weight of C16 to C20 fatty acids in the blend. Preferably, the ratio of ALA/CLA is from 0.3 to 0.6. Typically, the fat blends will be fat blends of the invention as defined above.

Thus, the invention provides a method for increasing the levels of DHA in the blood of a subject which comprises providing the subject with a fat blend of the invention for oral consumption. The fat blend is typically simply provided to the subject for oral consumption, either in the form of the fat blend itself, a food product or a nutritional supplement. Preferably, the subject is in need of increasing the level of DHA in the blood. The subject is preferably a mammal, most preferably a human.

The fat blends of the invention may be used to increase the level of DHA in vitro. This may have a number of benefits. For example, the fat blends may be useful in treating or preventing one or more conditions selected from neurodegenerative diseases, depression, Alzheimer's disease, obesity and cardiovascular disease. Alternatively or additionally, the fat blends may be useful for improving one or more of brain function, brain development, retinal function and retinal development.

The invention may involve the use of oleic acid to increase the effect of ALA and CLA in increasing the level of DHA in vitro, for example in fat blends according to the invention.

The following non-limiting examples illustrate the invention and do not limit its scope in any way. In the examples and throughout this specification, all percentages, parts and ratios are by weight unless indicated otherwise.

Examples

Example 1

Rats were fed different levels of ALA ₁ CLA and oleic acid. The dietary groups are presented in Table 1.

TABLE 1

% of olive oil, CLA and linseed oil (LO) in the dietary groups

Clarinol™ G80 is CLA in the form of a triglyceride available from Lipid Nutrition BV, Wormerveer, The Netherlands.

Plasma from the rats was analysed. The results are set out in Table 2.

Dietary Unseesd oil

Group OA ClA (LO) LO/CLA DHA ALA CLA mean SD mean SD mean SD

1 2 0,5 3,5 7,0 170,65 24,35 315,64 69,43 20,53 3,97

2 2 1,0 3,0 3,0 223,68 35,84 300,13 74,29 45,51 11,07

3 2 1,5 2,5 1,7 255,53 28,11 271,35 30,86 89,91 20,77

4 2 2,0 2,0 1,0 276,88 42,69 256,14 69,94 133,89 40,64

5 2 2,5 1,5 0,6 312,95 50,97 197,30 63,63 245,25 28,72

6 2 3,0 1,0 0,3 318,57 35,49 170,32 34,34 280,26 28,38

TABLE 2

Plasma levels (nmoles/ml of plasma) of DHA, ALA and CLA.

Figure 1 shows the linear regression of DHA plasma concentration in function to linseed oil (LO)/CLA ratio in the diet. Figure 2 shows the logarithmic regression of alpha linolenic acid (ALA) and CLA plasma concentrations in function to linseed oil/CLA ratio in the diet.

The data show that unexpectedly the highest yield in DHA was obtained with a ratio of ALA/CLA between 0.3-0.6 (see Table 2) and increased linearly in relation to ALA/CLA ratio (Figure 1). Plasma levels of ALA and CLA were proportional, even though not linearly, to the amount present in the diet (Figure 2).

Example 2

The following is an example of a yoghurt according to the invention.

Formulation (%):

Ingredient Dosage [%] Dosage white mass overall [%

Semi-skimmed milk 1.5% fat 90.920 72.736

Sugar 4.000 3.2

Skimmed milk powder 1.900 1.52

Fat blend 1.875 1.5

Whey protein powder (30% protein) 1.300 1.04

Direct starter culture 0.005 0.004

Sugar syrup 42% (w/w) aseptic 13.808

Strawberry puree °Brix aseptic 6.208

The fat blend contains 42 % Clarinol™ G80, 25 % linseed oil and 33 % olive oil and comprises:

(i) 33 % CLA; (ii) 13 % ALA; and (iii) 34 % oleic acid; the percentages being based on the total weight of C16 to C20 fatty acids in the blend.

A 10% pre-emulsion of the fat blend in milk is made by slowly mixing in the fat blend to milk of 60 ⁰ C under high-shear mixing. The mixture is homogenised dual-stage at 200/50 bar and the resulting emulsion is cooled to 4 ⁰ C. Dry blend sugar, milk powder and whey protein is mixed with the rest of the milk. Then the pre-emulsion is added. The milk is heated to 60 ⁰ C, homogenized dual-stage at 160/40 bar and heated 2 minutes at 95 ⁰ C. The milk is cooled to a fermentation temperature of 32 ⁰ C. Starter culture is added to the milk at 32 ⁰ C and milk is fermented till pH 4.3-4.5. The resulting yoghurt is cooled to about 20 "C ₁ stirred and sugar syrup and fruit are added to the yoghurt.

Example 3

Soft gel capsules are produced by rotary die processing. The material for the outside shell of the capsules, the gel, and the fill are formulated separately. Once the gel mass and the fill mass are ready, the gel is spread into thin film to form two gelatin ribbons which are then rolled over two separate dies which determine the size and the shape of the capsules. As the gelatin films adapt to the dies, a fat blend of the invention comprising alpha-linolenic acid, conjugated linoleic acid and oleic acid is carefully dosed to a level of 500mg, 750mg or "lOOOmg oil per capsule and injected between the two gelatin ribbons which are sealed immediately afterwards by applying heat and pressure. Capsules fall from the machine and are then dried under a stream of hot air.

Example 4

This example shows the effect of oleic acid on CLAJLO mixture in DHA formation. Total fat was balanced by adding coconut oil (CO) (diets 1 and 2) or increasing CLA and linseed oil (LO) keeping the same CLA/LO ratio.

Materials and Methods

Animals and diets

30 male Wistar rats (Harlan, Milan, Italy), with an average initial weight of 150- 170 g, were divided into 5 groups, and fed for 3 weeks 5 different diets. The diets were based on the AIN-93G formulation, with substitution of soybean oil with a blend of oils (olive oil, linseed oil, CLA and coconut oil, see Table 3 for ratios) which allowed the 5 diets to be equal for total fatty acids. All experiments were performed in accordance with the guidelines and protocols approved by the European Union (EU Council 86/609; D.L 27.01.1992, No. 116) and by the

Animal Research Ethics Committee of the University of Cagliari. Measurement of plasma fatty acid profile

Total lipids were extracted from plasma using the Folch method. Aliquots were mildly saponified in order to obtain free fatty acids for HPLC analysis. Separation of fatty acids was carried out with a Hewlett-Packard 1100 HPLC system (Hewlett-Packard, Palo Alto, California, USA) equipped with a diode array detector. A C-18 lnertsil 5 ODS-2 Chrompack column (Chrompack International BV, Middleburg, The Netherlands), 5 μm particle size, 15O x 4.6 mm, was used with a mobile phase of CH ₃ CN/H ₂ O/CH ₃ COOH (70/30/0.12, v/v/v) at a flow rate of 1.5 ml/min. Unsaturated fatty acids were detected at 200 nm. Spectra (195-315 nm) of the eluate were obtained every 1.28 s and were electronically stored. These spectra were taken to confirm the identification of the HPLC peaks.

Results

The data show that the highest yield in DHA was obtained with 2% of olive oil (Table 4). Lower levels of plasma DHA were detected when either olive oil was replaced with coconut oil or higher levels of CLA and linseed oil (Figures 3 and 4).

Figure 3 shows linear regression of DHA plasma concentration in function to olive oil in the diet when olive oil was replaced with coconut oil.

Figure 4 shows linear regression of DHA plasma concentration in function to olive oil in the diet when olive oil was replaced by increasing CLA and linseed oil (LO) keeping the same CLA/LO ratio.

Tables and Figures

^* comparative

TABLE 3. % of olive oil (OA), CLA, linseed oil (LO) and coconut oil (CO) in the dietary groups of Example 4

Dietary % of oils Plasma levels (nmoles/ml)

CO CLA LO OO DHA ALA CLA

229.12 193.97 285.44

2 2.5 1.5 0

58.52 44.00 65.63

268.30 181.37 265.79

1 2.5 1.5 1

61.04 42.91 41.94

356.85 164.20 246.83

0 2.5 1.5 2

51.62 32.07 30.48

282.55 176.33 311.49

0 3.2 1.9 1

38.71 26.39 13.74

257.28 207.22 349.58

0 3.8 2.3 0

20.21 41.33 56.10

TABLE 4. Plasma levels (nmoles/ml of plasma) of DHA, ALA and CLA in rats fed a mixture of olive oil (00), CLA, linseed oil (LO) and coconut oil (CO) for 3 weeks