MICROPARTICLES COMPRISING A FAT SOLUBLE FRACTION COMPRISING DHA AND THEIR

PRODUCTION

The present invention relates to microparticles comprising a docosahexaenoic acid moiety having a low content of saturated fatty acid moieties and a low content of free surface fat. The invention further relates to uses of such microparticles, methods of their manufacture and products comprising the microparticles.

5 Compositions comprising significant amounts of highly unsaturated fatty acids have frequently been shown to possess valuable properties for nutrition and general health. Particularly, polyunsaturated fatty acids (PUFA) have been implicated in treatment or prevention of cardiovascular risks including a lower risk of thrombosis, reduced arteriosclerosis, antiarrhythmic effects, inhibition of cytokine release, reduction of blood viscosity

10 and reduction of arterial hypertension. In view of these effects, omega-3 polyunsaturated fatty acids have received special attention. They are long chain polyunsaturated fatty acids having multiple non-conjugated carbon-carbon double bonds with the first of their double bonds at the third carbon atom from their methyl terminus. Among these, docosahexaenoic acid (DHA, C22:6 n-3, cervonic acid) and (all-Z)-5,8,11 ,14,17- eicosapen-

15 taenoic acid (EPA, C20:5 n-3, timnodonic acid) are considered particularly important. Other common omega-3 fatty acids include, but are not limited to, octadecatrienoic acid (C18:3 n-3, alpha-linolenic acid), octadecatetraenoic acid (C18:4 n-3; stearidonic acid), eicosatetraenoic acid (C20:4 n-3) and docosapentaenoic acid (C22:5 n-3).

DHA is one of the predominant fatty acids in the structural phospholipids of the human brain and retina and accumulates rapidly in foetal and infant neural tissue during the last months of gestation and the first months of postnatal life. It is thus particularly recommended that pregnant, neonate and infant persons shall receive a required dose of omega-3 fatty acids and particularly of DHA.

Omega-3 fatty acids and particularly DHA and EPA are mainly contained in marine oils and particularly fish oils like tuna oil. Other sources of DHA and EPA include oils gener- ated from algae and fungi. However, fish oils and biotechnologically produced algal or fungal oils are generally not consumed voluntarily in their recommended amount. This is particularly true for fish oils, which frequently exhibit a repulsive odour due to the production of low molecular amines and oxidation products during storage of such oils.

It has thus frequently been tried to produce formulations comprising omega-3 fatty acids in a stabilized form. For example, WO 94/01001 discloses a microencapsulated oil or fat product, wherein at least one oil or fat is dispersed in a matrix material as particles or drops having an average diameter of less than or equal to 2 μm, the oil or fat containing at least 10 wt.-% of highly unsaturated fatty acids, preferably omega-3 and omega-6 fatty acids, the level of free fatty acids being below 5 wt.-% and preferably below 0.5 wt.-% and the matrix material consisting of caseinate and optionally at least one carbohydrate. According to this document, such microencapsulated product can be used in foods, such as infant formulas, health functional foods, dietetic foods and pharmaceuticals, wherein a high content or fat or oil containing at least one highly unsaturated fatty acid or a derivative thereof is desired. The microencapsulated product particularly addresses require- ments of a European Community Commission directive of 14 May 1991 regarding ingredients of infant formulas. Further nutritional compositions are described in EP 0 764 405 A2, WO 03/024237 A1 and EP 0 425 213 A2. The present invention intends to further these advantages.

It is thus generally desired to produce products containing a high amount of DHA, while the product is still in a stabilized form with low inclination of developing unpleasant odours during storage. However, there are constraints making the development of such products difficult: According to an approach as described in WO 94/01001 polyunsaturated fatty acids are combined with a carrier, i. e. a caseinate, to produce a powder. For such encapsulation products, the content of free surface fat is an important parameter. A content of free surface fat of more than 1 wt.-% is considered an impaired quality, as such product is prone to fast deterioration. Furthermore, such products develop stickiness and cannot readily be handled as a free flowing powder. Instead, special provisions for handling of such products have to be taken.

Another constraint is that such products require a high amount of carrier to prevent fatty acids from becoming free surface fat. An increase in carrier content, however, necessarily entails a decrease of total fat content, thus limiting the maximum total fat content. It is thus desired to avoid an increase in carrier content when trying to achieve a high total content of fat-soluble substances. Also, the relationship between total fat content, amount of carrier material and type of carrier material and total fat is not understood; it was thus not possible to optimize the selection and ratio of fat and carrier materials by routine experimentation. Due to lack of guidance by any established principles, the teachings of any of the above prior art documents could not be extended or combined with a reasonable expectation of success.

A generally preferred source of DHA in a natural oil is tuna oil. A typical refined tuna oil for human consumption, however, contains approximately 23-25 wt.-% of DHA moieties and approximately 28 wt.-% of saturated fatty acid moieties. Saturated fatty acids have been implicated in unwanted decreases of low density lipoprotein (LDL) levels, high cholesterol levels and unwanted high triglyceride levels in blood, which are all believed to increase the risk of coronary illnesses. It is thus desired to reduce the content of saturated fatty acids in DHA containing compositions while maintaining high DHA levels.

Another requirement and thus object of the invention is to provide a particulate product having storage and transportation stability as well as mechanical strength and performance during further processing into tablets, extrudates and reconstituted emulsions or other edible end products. Storage and transport stability requires that a particulate product must not smell of old fish after 2 weeks storage in the dark at 25 ⁰ C, 50 % humid- ity, and also the anisidine value must not exceed 15 as determined according to European Pharmacopoeia 6.6 section 2.5.36 "Anisidine value". - A -

Another requirement is that the oil to be used in the manufacture of the particulate product should have a minimum content of 3 wt.-% of saturated fatty acids. Oils having a lower content of saturated fatty acids are presently intolerably expensive; the costs for producing a particulate product using an oil having less than 3 wt.-% of saturated fatty acids would be too high for incorporating the product into infant nutrition products.

SUMMARY OF THE INVENTION

The invention thus generally discloses a microparticle including a collection of such microparticles and a nutritional or pharmaceutical product comprising such microparticles. The microparticles comprise a fat soluble fraction and a matrix for forming the microparti- cle and embedding the fat soluble fraction. The fat soluble fraction comprises a docosa- hexaenoic acid moiety. The total amount of the fat soluble fraction is 31 - 44 wt.-% of the microparticle, the total amount of docosahexaenoic acid moieties is at least 1 1 wt.-% of the microparticle, the total amount of saturated fatty acid moieties is at most 12.5 wt.-% of the microparticle and at least 3 wt.-% of the fat soluble fraction, and the total amount of free surface fat is at the most 0.7 wt.-% of the microparticle.

The invention further provides a method of producing a microparticle. The method comprises the steps of

(a) providing a water phase comprising a matrix former, preferably a hydrocolloid matrix former and preferably also comprising a plasticizer,

(b) adding a concentrate and optionally a natural oil to the water phase to form the fat soluble fraction of an oil-in-water-emulsion,

(c) homogenising the emulsion, and

(d) spray drying the emulsion to achieve microparticles.

In this method, the steps and ingredients are chosen such that the total amount of the fat soluble fraction is 31 - 44 wt.-% of the microparticle, the total amount of docosahexaenoic acid moieties is at least 11 wt.-% of the microparticle, the total amount of saturated fatty acid moieties is at most 12.5 wt.-% of the microparticle, and the total amount of free surface fat is at the most 0.7 wt.-% of the microparticle. DETAILED DESCRIPTION OF THE INVENTION:

As used herein, the term ,,microparticle" denotes a particle comprising a matrix material and a fat soluble fraction. Microparticles according to the invention preferably have a mean diameter of 5 mm or smaller, particularly preferably 2 mm - 0.01 mm, more prefer- ably 1.5 mm - 0.2 mm and further preferably 0.6 - 0.1 mm measured by sieving in accordance with European Pharmacopoeia 6.6, section 2.9.38, using a Retch AS200 to agitate the sieves for 15 minutes.

The term "dispersion" as used herein denotes both an emulsion and a suspension. An emulsion as used herein denotes a liquid matrix comprising droplets of at least one further liquid phase. The term "suspension" is used herein denotes a liquid phase comprising solid particles dispersed therein.

The term "free surface fat" as used herein denotes such fatty substances as being readily extractable by an organic solvent and specifically petroleum ether under the conditions specified later herein. The free surface fat typically is located on the microparticle surface or in cavities within the microparticle but having contact to the particle surface through capillaries or cracks in the microparticle matrix.

In the context of the present invention, the term "fat solube" means hydrophobic organic compounds soluble in lipids.

All references to fatty acid moieties shall be understood to refer to both the fatty acid as such and to any substance having the respective fatty acid connected to the remainder of the substance via the fatty acid's carboxy terminus. Particularly, monoglycerides, diglyce- rides and triglycerides of fatty acids constitute the main ingredients of lipids, oils and fats.

Thus, for example the term "docosahexaenoic acid moiety" refers to docosahexaenoic acid as such, and also to that part of a substance, e.g. of a mono-, di- or triglyceride, which would be docosahexaenoic acid after hypothetical cleavage of the covalent bond at its carboxy terminus.

As used herein, the term "natural oil" refers to an oil derived from a living entity and containing at least one ester of an omega-3 fatty acid. A "fish oil" within the meaning of the present description thus is a natural oil derived from a fish or other marine organism, and is preferably refined for human consumption. Refining preferably includes de- acidification (remove free fatty acids), bleaching (reduce colour and heavy metal and pollutants (organic substances)), distillation and deodorization (steam injection during vacuum) and in some cases winterization (cooling down to precipitation of high melting fats/triglycerides). Fish oils tend to have a triglyceride content of at least 90 wt.-% of the oil. When referring to "natural oil" or "fish oil", tuna oils are generally particularly preferred due to their naturally high content of docosahexaenoic acid moieties. A typical refined tuna oil composition is:

Triglycerides: 95 - 99 wt.-%, Diglycerides: 2 - 5 wt.-%, Monoglycerides: 0 - 1 wt.-%, Free fatty acids: 0 - 1 wt.-%,

all weight percentages referring to the total oil. The content of saturated fatty acid moieties typically is at least 28 wt.-% of the tuna oil. The most prominent saturated fatty acid moieties are palmitic acid (19.5 wt.-%), stearic acid (5 wt.-%) and myristic acid (3.8 wt.- %), again referring to the total tuna oil.

As used herein, the term "concentrate" refers to a mixture obtainable or obtained from a natural oil and preferably a fish oil, by at least one step of concentrating desired fatty acids and reesterification of the concentrated fatty acids to yield a fat or an oil, or by deesterification of undesired fatty acids, preferably by enzymatic cleavage, and removal of such undesired fatty acids.

A typical concentrate according to the present invention will have a composition of:

Triglycerides: 50 - 65 wt.-%, Diglycerides: 30 - 50 wt.-%, Monoglycerides: 0 - 5 wt.-%, Free fatty acids: 0 - 1 wt.-%,

each time referring to the total concentrate. The content of saturated fatty acid moieties preferably is at most 5 wt.-% of the concentrate. Concentrates preferably have a content of less than 1 wt.-% of myristic acid moieties, less than 2 wt.-% of palmitic acid moieties, and less than 3 wt.-% of stearic acid moieties, again referring to the total concentrate. For the present invention, the term "embedding" denotes a thorough mixture of matrix material and fat soluble fraction in a substantially dry form, i.e. at a water content of the microparticles of less than 5 wt.-%. In an emulsion or after dissolving of the microparticles in water, the fat soluble fraction forms droplets of up to 1 μm average diameter, and said droplets preferably have an average diameter in water of up to 0.5 μm. The average diameter is measured with a Mastersizer 2000 (Malvern) laser diffractometer.

The microparticles of the present invention comprise a fat soluble fraction. Main ingredients of such fat soluble fraction preferably are natural oils, particularly tuna oil, and concentrates, as will become apparent hereinafter. The fat soluble fraction can further comprise other substances besides natural oils and concentrates as will be detailed later.

The total amount of the fat soluble fraction is 31 - 44 wt.-% of the microparticle, preferably 35 - 43 wt.-% of the microparticle. It has suprisingly been found that at this concentration, the desired minimum concentration of 11 wt.-% of docosahexaenoic acid moieties can be achieved, and still the particle maintains a low content of free surface fat.

The total amount of free surface fat is thus at most 0.7 wt.-% of the microparticle, preferably 0.4 - 0.01 wt.-% and most preferably 0.13 - 0.02 wt.-%. Without wanting to be bound by any particular theory, it is contemplated that the low amount of total free surface fat is decisive for providing a free flowing powder of microparticles of the present invention, as the microparticles do not significantly adhere to each other, and is also beneficial to ensure a good oxidation stability.

By the process of the invention it has also surprisingly become possible to increase the amount of docosahexaenoic acid moieties and the amount of fat soluble fraction without increasing the amount of matrix material, particularly of hydrocolloids. Conventional attempts to include a higher amount of fat soluble fraction where hampered by a need to achieve or maintain a high content of hydrocolloid and particularly of hydrocolloids to prevent formation of a high amount of free surface fat. Thus, the process for preparing the microparticles of the invention is cost-efficient.

Further, as the amount of free surface fat can be kept low according to the present invention, the fraction of docosahexaenoic acid moieties and other ingredients of the fat so- luble fraction directly exposed to air and thus to oxidation stress conditions can be kept low. Therefore, the microparticles are mechanically and chemically stable during storage, transport and further processing, e.g. into tablets, extrudates or food mixes.

In view of the total amount of saturated fatty acid moieties, the present invention allows to reduce this amount compared to a hitherto used tuna oil by 10 %. Thus, health concerns can be effectively addressed by the microparticles of the present invention and their respective method of manufacture. Considering the compositions of tuna oil and concentrate as given above, a typical reduction in total saturated fatty acid moieties of 10 % relative to the total fat soluble fraction of a typical tuna oil can be achieved.

Preferably, the total amount of saturated fatty acids is at most 22.7 wt.-%, even more preferably at most 20 wt.-% and most preferably at most 17 wt.-% of the fat soluble fraction, corresponding to a decrease of approximately 20%, 30% and 40% compared to a typical refined tuna oil. A minimum amount of saturated fatty acid moieties is 3 wt.-% of the fat soluble fraction.

Among the saturated fatty acid moieties, a reduction is particularly desired for myristic acid moieties, and/or palmitic acid moieties, and/or stearic acid moieties. Thus, according to the invention the microparticles preferably contain a total amount of myristic acid moieties of at most 3.5 wt.-%, preferably at most 3 wt.-%, even more preferably at most 2.7 wt.-% and most preferably at most 2.3 wt.-%, relative to the total fat soluble fraction. The microparticles of the present invention also preferably contain a total amount of palmitic acid moieties of at most 17.5 wt.-%, more preferably at most 15.6 wt.-%, even more preferably at most 13.7 wt.-% and most preferably at most 11.7 wt.-% of the total fat soluble fraction. And also preferably the microparticles of the present invention comprise a total amount of stearic acid moieties of at most 4.5 wt.-%, more preferably at most 4 wt.-%, even more preferably at most 3.5 wt.-% and most preferably at 3.1 wt.-%, relative to the total fat soluble fraction. Thus, compared to a conventional refined tuna oil a reduction in myristic, palmitic and/or stearic acid moieties of 10 %, more preferably of 20 %, even more preferably of 30 % and most preferably of 40 % can be achieved.

Likewise, the total amount of triglycerides preferably is at most 85.5 wt.-%, more preferably at most 76 wt.-%, even more preferably at most 66.5 wt.-% and most preferably at most 57 wt.-% of the fat soluble fraction. Again, this corresponds to a reduction compared to a conventional tuna oil of 10 %, more preferably of 20 %, even more preferably of 30 % and most preferably of 40 %. Preferably, the fat soluble fraction of the microparticles of the present invention comprise

- 0 - 5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties,

- 0 - 16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7 - 12 wt.-% of eicosapentaenoic acid moieties,

- 0 - 5 wt. -% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and

- 0 - 5 wt. -% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction.

For physiological reasons, it is further preferred that the microparticles of the present invention comprise DHA and EPA moieties in a ratio of total weights of at least 4 (DHA):1(EPA) and further preferably up to 5.5:1 , thereby mimicking the ratio found in human milk.

Preferably, the fat soluble fraction of the microparticle of the present invention comprises

- a natural oil comprising docosahexaenoic acid moieties, preferably a marine oil, more preferably a fish oil and most preferably a tuna oil, and

- a concentrate comprising docosahexaenoic acid moieties,

wherein the total amount of docosahexaenoic acid moieties in the natural oil is less than the amount required to achieve a total amount of docosahexaenoic acid moieties of at least 11 wt.-% in the microparticle, and

wherein the total amount of docosahexaenoic acid moieties in the concentrate is suffi- cient to complement the amount of docosahexaenoic acid moieties in the natural oil to achieve a total amount of docosahexaenoic acid moieties of at least 1 1 wt.-% of the microparticle.

It has now been found that by reducing the amount of natural oil in a microparticle, the beneficial properties of such microparticles can actually be promoted. Thus, instead of raising the amount of natural oil in a microparticle to achieve a total content of docosa- hexaenoic acid moieties in the microparticle of at least 11 wt.-%, it has now been found that at least part of the natural oil should be replaced by a concentrate containing doco- sahexaenoic acid moieties. This way, the amount of saturated fatty acid moieties and the amount of triglycerides in a microparticle of the invention can be decreased without lowering the total content of docosahexaenoic acid moieties or increasing the amount of surface fat.

The concentrate preferably is a natural oil concentrate - preferably a tuna oil concentrate -, obtainable or obtained by a process comprising the steps:

(a) deesterification of fatty acid di- and/or triglycerides of a natural oil, preferably a marine oil, more preferably a fish oil and most preferably a tuna oil, wherein deesterification is by enzymatic cleavage or another process,

(b) isolation of selected fatty acids and removal of non-selected fatty acids, preferably by distillation,

(c) reesterification of the isolated fatty acids into monoglycerides, diglycerides and triglycerides.

Preferably, the total amount of docosahexaenoic acid moieties is at least 20 wt.-%, particularly preferably 21 - 30 wt.-% and even more preferably 23 - 25 wt.-% of the natural oil. Also preferably, the total amount of docosahexaenoic acid moieties is at least 42 wt.- %, particularly preferably 43 -80 wt.-% and more preferably 44 - 75 wt.-% of the concentrate.

Such compositions of natural oil and/or concentrate are particularly adapted to achieve the desired high DHA content of the microparticle while simultaneously achieving the desired low content of saturated fatty acid moieties and free surface fat. It is thus particu- larly preferred to combine them in a ratio of (natural oil:concentrate) 86:14 to 50:50 parts by weight. In view of the present description, the skilled person can select a ratio suitable for achieving a minimum content of DHA moieties of 11 wt.-% of the microparticle while maintaining the total amount of fat soluble fraction, free surface fat and content of saturated fatty acid moieties. Another preferred concentrate is obtainable or obtained by

a) deesterification of a natural oil, preferably a marine oil, more preferably a fish oil and most preferably a tuna oil, preferably by enzymatic cleavage, of fatty acid moieties other than DHA and/or EPA, and

b) removal of deesterified fatty acids.

In this case, the total amount of DHA moieties is at least 21 wt.%, preferably 22-39 wt.% and particularly preferably 24-29 wt.% of the total concentrate. Also, the total amount of EPA moieties in the concentrate is preferably 5-15 wt.% and more preferably 5-12 wt.%. A concentrate of natural oils wherein undesired fatty acid moieties have been deesterified and removed as described in steps a) and b) above is preferably used instead of natural oil when preparing the microparticles of the present invention and is thus preferably not mixed with a natural oil to produce the fat soluble fraction of the microparticles.

The microparticle of the present invention comprises a matrix for forming the micropar- ticle and embedding the fat soluble fraction therein. The matrix preferably comprises a hydrocolloid material and preferably also a plasticizer. Particularly preferred the hydrocol- loid material is selected from the group consisting of milk protein or milk protein hydroly- sates, whey protein, caseinates, gelatine, polysaccharides and mixtures of the aforementioned substances. Preferred polysaccharides are alginate, carrageenan, gum arabic, gum acacia, modified gum acacia, pectins, modified pectins and modified starch, prefera- bly sodium octenyl succinate modified starch. Particularly preferred matrix materials comprise caseinate, particularly sodium and/or potassium caseinate. It has been found that sodium or potassium caseinates and particularly potassium caseinate are particularly suitable for embedding the fat soluble fraction to maintain a desired low surface fat content.

Although caseinate may be used as the only matrix material, it is preferred to use a combination of caseinate and a plasticizer, preferably a carbohydrate and/or carbohydrate alcohol, as matrix material.

Preferred plasticizers are lactose, maltose, saccharose, glucose, glucose syrup, fructose, lactose, invert sugar, sorbitol, manitol, trehalose, targatose, pullulan, raftilose (oligofruc- tose), dextrin, maltodextrin, glycerin and mixtures thereof, such as saccharose, trehalose, pullulan, dextrin and raftilose and mixtures thereof. Particularly preferred plasticizers are glucose syrup, maltodextrin, saccharose, maltose and lactose, most preferably maltodex- trin and/or saccharose. It has been found that a weight ratio of plasticizer : caseinate (particularly potassium caseinate) of 8.5:1 to 4.5:1 provides an increased mechanical stability of the microparticles for the presently selected total amount the fat soluble fraction and its composition according to the present invention when compared to a matrix consisting of caseinate or saccharose as such. Further, said weight ratio minimizes the proportion of free surface fat of the microparticles.

The weight ratio between the fat soluble fraction and the hydrocolloid, preferably casei- nate and most preferably K-caseinate, is preferably at least 4.5:1 , particularly preferably at least 6:1 and more preferably at least 7:1. These weight ratios are particularly adapted to achieve a sufficiently stable microparticle under mechanical and oxidation (exposure to ambient air) stress as well as a good storage stability combined with a low content of free surface fat.

The microparticle of the present invention preferably is a spray dried microparticle. For spray drying, preferably an oil-in-water-emulsion of the fat soluble fraction, the matrix material and water is homogenised and atomised into a spray drying tower to evaporate water of the emulsion. Most preferably, the gas phase in the spray drying tower contains a starch or modified starch and/or further anticaking agent to finely cover the drying microparticles.

A preferred microparticle of the present invention will thus comprise 20-30 wt.-% starch, relative to the total microparticle, 4-10 wt.-% further substances, preferably including or consisting of sodium ascorbate, relative to the amount of fat soluble fraction, 17-36 wt.-% sucrose, relative to the total microparticle, 10-50 wt.-% of hydrocolloid, relative to the amount of fat soluble fraction, a total of 31-44 wt.-% of fat soluble fraction, relative to the total microparticle, and up to 5 wt.-% of water, relative to the total microparticle.

The free surface fat content of the microparticle of the present invention at most 0.7 %, particularly preferably at most 0.4 %, even more preferably at most 0.15 % and most preferably at most 0.13 %. It has further been found that a minimum amount of free surface fat of 0.01 % yields an acceptable, free flowing powder of microparticles of the present invention. The fat soluble fraction of the microparticles of the present invention can comprise further substances and moieties besides substances having a docosahexaenoic acid moiety, i.e. docosahexaenoic acid as such and/or preferably mono-, di- and/or triglycerides comprising at least one docosahexaenoic acid moiety. Preferred further contents of the fat so- luble fraction are provitamins and vitamins, particularly vitamin A and esters thereof, vitamin E and esters thereof, preferably vitamin E-acetate, vitamin D, preferably vitamin D2 and/or vitamin D3, vitamin K, preferably vitamin K1 , further monounsatu rated fatty acids and polyunsaturated fatty acids besides docosahexaenoic acid, preferably conjugated linolenic acid (CLA), carotenoids, preferably beta-caroten, lutein, lycopene, beta- cryptoxanthin, astaxanthin, cantaxanthin, citranaxanthin and zeaxanthin, curcumin and benzoquinones, preferably coenzyme Q10 (ubidecarenone). Further preferred are fat soluble antioxidants, particularly ascorbyl palmitate. Thus, the microparticles of the present invention comprise a fat soluble fraction preferably consisting of or preferably essentially consisting of the aforementioned fatty acid moieties, particularly DHA and preferably also EPA, and one or more of the substances mentioned before in this paragraph.

The microparticles of the present invention may contain further substances which are not necessarily part of the fat soluble fraction. Particularly preferred, the microparticles may contain

- antioxidants, preferably T-butyl hydroxyl toluene (BHT), T-butyl hydroxyl anisole (BHA), ascorbic acid, sodium ascorbate, citric acid, sodium citrate EDTA and its salts, tocopherols, preferably natural tocopherol and particularly preferably gamma- tocopherol, tert.-butylhydroquinone (TBHQ), ethoxyquine, propyl gallate, herb extracts, preferably rosemary and/or oregano extract;

- anticaking agents, preferably tricalciumphosphate and silicate, particularly preferably silicon dioxide and sodium aluminium silicate, tricalciumphosphate being most preferred;

- emulsifiers and surfactants, particularly preferably ascorbyl palmitate, sucrose esters, mono- and diglycerides of fatty acids and derivatives thereof, and lecithin.

Thus, the matrix of the microparticles of the present invention preferably consists of or preferably consists essentially of the matrix material as described above, preferably including one or more hydrocolloids and further preferably including one or more plasti- cizers, and preferably one or more emulsifiers and/or surfactants.

The present invention further relates to end products comprising the microparticles of the present invention. A preferred end product is a food, a food supplement, a beverage, a pharmaceutical or veterinary product, a feed or feed supplement and a personal care product. Particularly preferred end products are nutritional, nutrition supplementary and pharmaceutical end products. Among these, infant nutrition compositions including neonate nutrition products are particularly preferred. The skilled person understands that the microparticles of the present invention are not included in such end product in an amount insufficient for achieving a physiological effect. Instead, the skilled person will work with synthetical propensity, i.e. building up rather than tearing down, to achieve the benefits available via the microparticles of the present invention. For example, he will include enough microparticles to achieve a physiologically sensible intake of DHA moieties by a consumer, particularly preferred an infant or neonate, while maintaining a low intake of saturated fatty acid moieties. The microparticles of the present invention may be dissolved in the end product and no longer be present in microparticulate form. By including microparticles of the present invention in an end product of the above type, the beneficial effects particularly for infants and neonates referred to above can be achieved.

The microparticles of the present invention are typically not produced or sold one single particle a time. Thus, whenever the present description refers to a "microparticle" of the present invention, it is to be understood that also a multiplicity of microparticles is meant. A preferred quantity of microparticles of the present invention is at least 5 kg of microparticles, more preferably at least 20 kg of microparticles and most preferably at least 25 kg of microparticles. A preferred production batch quantity is thus preferably at least 5, 20 or 25 kg, but more preferably is at least 100 kg, more preferably at least 500 kg and most preferably at least 1000 kg.

The invention further relates to a method of producing a microparticle according to the present invention. In the method as given above a natural oil and a concentrate is added to a water phase to form the fat soluble fraction of an oil-in-water-emulsion. Preferable compositions and amounts of natural oil, concentrate and further ingredients of the fat soluble fraction have been indicated above. Homogenization is preferably performed using a high shear system, further preferably a rotor-stator system, and most preferably a high pressure homogenizer. The method of the present invention allows to produce the microparticles of the present invention reproducibly and in the form of a free flowing powder at an economically sensible large scale.

The invention will hereinafter be further described by reference to preferred examples. The examples shall be understood as not to limit the scope of the claims or the scope of disclosure of the present description.

Example 1 : Test method for determination of free surface fat

Principle: The microparticles are dispersed in petroleum ether, whereby the amount of fat which is not embedded dissolves and is determined by means of weight analysis. Free surface fat content is expressed as the extracted amount of fat in relation to the weighed amount of product, as described below.

Method: Weigh 10.00 g product into a 250 ml Erlenmeyer flask.

Add 50.0 ml petroleum ether to the flask and shake for a few seconds.

Decant the petroleum ether into a counterbalanced 100 ml Erlenmeyer flask through a Whatman No. 4 paper filter, which has been moistened with petroleum ether.

Repeat the procedure with another 50 ml petroleum ether and again with 2 x 10 ml petroleum ether.

Evaporate the entire amount of petroleum ether under nitrogen at max. 4O ⁰ C and place the flask in an incubator at 105 ⁰ C for one hour and allow to cool in a desiccator.

Weigh the flask in grams (4 decimals).

Calculate the content of free surface fat content (%) in the samples by the following equation: i- J- Is ample ITi ₁ = Weight of flask (g)

ITi ₂ = Weight of flask with free fat (g)

msampie = Weighed sample (g)

Example 2: General manufacturing method

In a first step caseinate (preferably potassium caseinate) and saccharose are dissolved in water, preferably at a temperature of 50-70 ⁰ C. The solution is then degassed by reduction of air pressure, and followingly preferably kept under an atmosphere of nitrogen. Sodium ascorbate is added to the solution before or after degassing.

Fish oil, preferably tuna oil, concentrate, tocopherol, a fat soluble antioxidant mixture comprising ascorbyl palmitate, lecithin and further mono- and diglycerides of fatty acids are mixed and are added to the degassed solution under vigorous agitation at 50-70 ⁰ C to form a pre-emulsion. The pre-emulsion is then homogenised by high pressure homogeni- sation to yield an emulsion, but can also be emulsified in a high shear mixer, e.g. a rotor stator mixer. The emulsion is spray dried in hot air comprising starch and tricalcium phosphate.

The powder thus obtained (including the powders of examples 3a and 3b) is particularly suitable for preparing an infant or neonate formula to achieve the beneficial physiological effects referred to at the beginning of this description.

Microparticles of the present invention are obtained.

Example 3: Preferred methods of manufacture

In a general method according to example 2, the following contents were used:

Example 3a: DHA oil: caseinate ratio 8:1

288 potassium caseinate, 1346 g sucrose and 138 g sodium ascorbate were dissolved in 1500 ml water at 65 ⁰ C under agitation. 2300 g DHA oil (1978 g natural tuna oil and 322 g concentrate) was mixed with mixed tocopherol to 3000 ppm and 13.04 g fat soluble antioxidant (comprising ascorbyl palmitate, lecithin and further mono- and diglycerides of fatty acids), heated to 65 ⁰ C and added to the aqueous solution under agitation. The DHA oil was made of a mixture of tuna oil and concentrate and had a content of docosahexae- noic acid moieties of 282 mg/g of oil and a content of eicosapentaenoic acid moieties of 66 mg/g of oil. A pre-emulsion was thus obtained. The pre-emulsion was homogenised in a rotor/stator system and diluted followed by one pass at 550 bar through a high pressure homogeniser. After homogenisation, a sprayable emulsion (oil-in-water-emulsion) is obtained.

The sprayable emulsion was atomized in a spray drying tower with added maize starch and tricalcium phosphate as powdering agents.

The resulting dry powder of microparticles had the following characteristics:

Total amount of fat soluble fraction: 42.4 wt.-% of microparticles.

Total amount of docosahexaenoic acid moieties: 11.9 wt.-% of the microparticles.

Total content of eisocapentaenoic acid moieties: 2.8 wt.-% of microparticles.

Total amount of free surface fat: 0.13 %.

Total amount of myristic, palmitic and stearic acid moieties: 10.6 wt.-% of the microparticles.

Total content of triglycerides: 90.8 wt.-% of the fat soluble fraction.

The DHA oil was obtained by mixing tuna oil and concentrate in a ratio of 86 (tuna oil): 14 (concentrate). The microparticles were obtained as a free flowing powder.

Example 3b: Alternative DHA: Caseinate ratio of 8:1 238 g potassium caseinate, 1816 g sucrose and 114 g sodium ascorbate were dissolved in 1300 ml water at 65 ⁰ C under agitation. 1908 g of DHA oil (1240 g natural tuna oil and 668 g concentrate) was mixed with mixed tocopherol to 3000 ppm and 10.8 g fat soluble antioxidant (comprising ascorbyl palmitate, lecithin and further mono- and diglycerides of fatty acids), containing 350 mg/g docosahexaenoic acid moieties and 65 mg/g eicosapen- taenoic acid moieties, heated to 65 ⁰ C and added to the aqueous solution under vigorous agitation. A pre-emulsion was obtained.

The pre-emulsion was diluted, homogenized (two passes at 500 bar) and dried as described for example 3a. A free flowing powder of microparticles was obtained. This dried powder had the following characteristics:

Total amount of fat soluble fraction: 35.3 wt.-% of the microparticles.

Total content of docosahexaenoic acid moieties: 12.2 wt.-% of the microparticles.

Total content of eisocapentaenoic acid moieties: 2.3 wt.-% of microparticles.

Free surface fat: 0.03 %.

Total content of myristic, palmitic and stearic acid moieties: 7.0 % of the fat soluble fraction.

Total triglyceride content: 84.5 % of the fat soluble fraction.

The DHA oil was obtained by mixing tuna oil and concentrate in a ratio of 65 (tuna oil):35 (concentrate).

Example 4: End product

A preferred infant nutrition dry formula comprising the microparticles of the invention (Examples 3 and 5) and further adding arachidonic acid has the composition:

Example 5: Manufacturing method using enzymatically treated tuna oil

Example 3a was reproduced. However, the "DHA oil" was replaced by the identical quantitiy of a concentrate, said concentrate being an enzymatically treated tuna oil.

The invention is hereinafter further explained by way of some preferred embodiments, which achieve the advatages described above.

Embodiment 1 : Microparticle comprising a fat soluble fraction and a matrix for forming the microparticle and embedding the fat soluble fraction, wherein the fat soluble fraction comprises a docosahexaenoic acid (C22:6 omega-3,

DHA) moiety, characterized in that the total amount of the fat soluble fraction is 35-43 wt-% of the microparticle, and the total amount of docosahexaenoic acid moieties is at least 11 wt% of the microparticle, - the total amount of saturated fatty acid moieties is at most 12.5 wt% of the micro- particle, and at least 3 wt.-% of the fat soluble fraction, and the total amount of free surface fat is at most 0.7 % of the microparticle, preferably 0.01-0.4 wt. % an most preferably 0.02-0.13 wt.%.

Embodiment 2: A microparticle as described in Embodiment 1 , wherein the total amount of saturated fatty acids in the fat soluble fraction of the microparticle is at most 22.7 wt.- %, preferably at most 20 wt.-% and most preferably 3-17 wt.-% of the fat soluble fraction.

Embodiment 3: A microparticle as described in Embodiment 2, having

a) a total amount of myristic acid moieties of at most 3.5 wt.-%, preferably at most 3 wt.- %, even more preferably at most 2.7 wt.-% and most preferably at most 2.3 wt.-% of the total fat soluble fraction, and further having a total amount of

0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or 0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or

0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or - 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction; and/or

b) a total amount of palmitic acid moieties of at most 17.5 wt.-%, more preferably at most 15.6 wt.-%, even more preferably at most 13.7 wt.-% and most preferably at most 11.7 wt.-% of the total fat soluble fraction, and further having a total amount of - 0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or

0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or 0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction; and/or c) a total amount of stearic acid moieties of at most 4.5 wt.-%, more preferably at most 4 wt.-%, even more preferably at most 3.5 wt.-% and most preferably at 3.1 wt.-% of the total fat soluble fraction, and further having a total amount of

0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or - 0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or 0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction.

Embodiment 4: A microparticle having

a fat soluble fraction and a matrix for forming the microparticle and embedding the fat soluble fraction, wherein the fat soluble fraction comprises a docosahexaenoic acid (C22:6 omega-3, DHA) moiety, characterized in that the total amount of the fat soluble fraction is 31-44 wt.-% of the microparticle, and the total amount of docosahexaenoic acid moieties is at least 11 wt% of the microparticle, preferably 11-44 wt.-% and even more preferably 12-44 wt.-% of the microparticle, - the total amount of saturated fatty acid moieties is 3 to 9.99 wt.-% of the microparticle, and the total amount of free surface fat is at most 0.7 % of the microparticle, preferably 0.01-0.4 wt. % an most preferably 0.02-0.13 wt.%.

Embodiment 5: A microparticle of Embodiment 4, wherein the total amount of saturated fatty acid moieties is at most 8.8 wt.-%, preferably at most 7.48 wt.-% and more prefara- bly 0.93-7.31 wt.-%, all weight percentages relative to the total microparticle.

Embodiment 6: A microparticle of Embodiment 4, wherein

a) the total amount of myristic acid moieties is at most 3.5 wt.-%, preferably at most 3 wt.- %, even more preferably at most 2.7 wt.-% and most preferably at most 2.3 wt.-% of the total fat soluble fraction, and further having a total amount of

0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or 0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or 0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction;

b) the total amount of palmitic acid moieties is at most 17.5 wt.-%, more preferably at most 15.6 wt.-%, even more preferably at most 13.7 wt.-% and most preferably at most 11.7 wt.-% of the total fat soluble fraction, and further having a total amount of

0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or - 0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or 0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction; and/or

c) the total amount of stearic acid moieties is at most 4.5 wt.-%, more preferably at most 4 wt.-%, even more preferably at most 3.5 wt.-% and most preferably at 3.1 wt.-% of the total fat soluble fraction, and further having a total amount of

0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or 0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or 0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction.

Embodiment 7: A microparticle according to Embodiment 1 , 2, 3, 4, 5 or 6, having a mean particle diameter of 2 mm - 0.01 mm, more preferably 1.5 mm - 0.2 mm and further preferably 0.6 - 0.1 mm.

Embodiment 8: A microparticle according to any of Embodiments 1 , 2, 3, 4, 5, 6 or 7, which, when dissolved in still water at 25 ⁰ C and 1013 hPa, forms oil phase droplets having an average diameter of at most 1 μm, preferably at most 0.5 μm.

Embodiment 9: A microparticle comprising or consisting of 20-30 wt.-% starch, relative to the total microparticle, preferably 22-28 wt.-%, 4-10 wt.-%, preferably 4.5-9 wt.-%, of further substances, preferably including or consisting of sodium ascorbate, relative to the amount of fat soluble fraction,

17-36 wt.-%, preferably 21-31 wt.-%, of sucrose, relative to the total microparticle, - 10-50 wt.-%, preferably 12-25 wt.-%, of hydrocolloid, preferably including or consisting of sodium caseinate, relative to the amount of fat soluble fraction, a total of 31-44 wt.-%, of fat soluble fraction, relative to the total microparticle, and up to 5 wt.-% of water, relative to the total microparticle,

wherein

- the total amount of docosahexaenoic acid moieties is at least 11 wt% of the microparticle, and the total amount of saturated fatty acid moieties is at most 12.5 wt% of the microparticle and at least 3 wt.-% of the fat soluble fraction, and the total amount of free surface fat is at most 0.7 wt.-% of the microparticle.

Embodiment 9: A microparticle as described in Embodiment 8, having

a) a total amount of myristic acid moieties of at most 3.5 wt.-%, preferably at most 3 wt.-

%, even more preferably at most 2.7 wt.-% and most preferably at most 2.3 wt.-% of the total fat soluble fraction, and further having a total amount of

0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or - 0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or 0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction; and/or

b) a total amount of palmitic acid moieties of at most 17.5 wt.-%, more preferably at most 15.6 wt.-%, even more preferably at most 13.7 wt.-% and most preferably at most 11.7 wt.-% of the total fat soluble fraction, and further having a total amount of 0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or 0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or 0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction; and/or

c) a total amount of stearic acid moieties of at most 4.5 wt.-%, more preferably at most 4 wt.-%, even more preferably at most 3.5 wt.-% and most preferably at 3.1 wt.-% of the total fat soluble fraction, and further having a total amount of

0-5 wt.-% of arachidonic acid (C20:4 omega-6, AA) moieties, and/or 0-16 wt.-% of eicosapentaenoic acid (C20:5 omega-3, EPA) moieties, preferably at least 5 wt.-% and most preferably 7-12 wt.-% of eicosapentaenoic acid moieties, and/or

0-5 wt.-% of alpha-linolenic acid (C18:3 omega-3, alpha-LNA) moieties, and/or - 0-5 wt.-% of gamma-linolenic acid (C18:3 omega-6, gamma-LNA) moieties, all weight percentages relative to the total fat soluble fraction.

Preferred manufacturing process 1 : A process for manufacturing any of Embodiments 1 , 2, 3, 4, 5, 6, 7, 8 or 9, comprising the steps of

a) providing a water phase comprising a matrix former,

b1 ) obtaining a natural oil, preferably a marine oil and most preferably a tuna oil, having a total amount of DHA moieties of at least 20 wt.-%, preferably 23-25 wt.-%, the weight percentages being relative to the total oil,

b2) before, during or after step b1 ) obtaining a concentrate having a total amount of DHA moieties of at least 42 wt.-%, preferably 43-80 wt.-% and more preferably 44-75 wt.-%, the weight percentages beng relative to the total concentrate,

b3) mixing the oils of steps b1 ) and b2) in a ratio of (a:b) 86:14 to 50:50,

b4) adding the mixture to the water phase to form the fat soluble fraction of a resulting oil- in-water emulsion,

c) homogenizing the emulsion, and

d) spray drying the emulsion to obtain the microparticles. Preferred manufacturing process 2: A process for manufacturing any of Embodiments 1 , 2, 3, 4, 5, 6, 7, 8 or 9, comprising the steps of

a) providing a water phase comprising a matrix former,

b1 ) obtaining a natural oil, preferably a marine oil and most preferably a tuna oil, having a total amount of DHA moieties of at least 20 wt.-%, preferably 23-25 wt.-%, the weight percentages being relative to the total oil,

b2) deesterification of fatty acid moieties other than DHA and optionally EPA from the oil of step b1 ),

b3) removal of free fatty acids from the deesterified oil of step b2) to obtain a concentrate

b4) adding the concentrate to the water phase to form the fat soluble fraction of a resulting oil- in-water emulsion,

c) homogenizing the emulsion, and

d) spray drying the emulsion to obtain the microparticles.