FIELD OF INVENTION

The present invention relates to the field of pigments for aquaculture and in particular to astaxanthin formulated in a self-emulsifying system especially useful for the preparation of feed.

STATE OF THE ART

Pigmentation of farmed salmonids is a practice used to change the color of flesh, achieved by supplying feed, frequently adopted in rainbow trout farming. Since several years the pigmented trout, better known as salmon trout, represents an important share of the market, although in some important markets such as Germany and Denmark the main export continues to concern the non pigmented trout, commercially called "white trout".

The pigment retained in the muscle of farmed salmonids does not modify or make any changes to the organoleptic characteristics of the fish. The pigmentation consists only in a change of the muscle color, achieved by supplying feed, in order to reach the same flesh color of wild salmonids. Consumers consider flesh pigmentation to be an indicator of quality, both in the case of the rainbow trout and of farmed salmon.

The pink/red color of wild salmonid flesh is the result of their diet; the coloration grade varies in intensity depending on whether the diet is more or less rich in pigments, provided naturally by rich shellfish, algae, krill or small fishes, and depending on the physiological state of the fish. Salmonids, in fact, do not possess the ability to genetically synthesize pigments, but they are able to accumulate exogenous pigments in the muscle, in the skin and also in the gonads.

Pigments responsible for the coloration of salmonids muscles are called carotenoids; fish feed industries use these pigments for the coloration of farmed salmonids. Synthetic canthaxanthin and astaxanthin are the most commonly used carotenoids in aquaculture: astaxanthin is the predominant pigment found when analyzing wild salmonid muscle.

Pigmentation represents a significant contributor to the cost of wild salmon feed. In salmon breeding, pigmentation costs can represent up to 20% of total feeding expenses. The market is increasingly demanding with regard to muscle coloration, so that this feature has become a parameter for assessing the quality of aquaculture products.

The trout has a greater ability to fix the pigments in the flesh than salmon; this characteristic allows to adopt a different strategy for pigmentation, which can be reached in a shorter period of time and with lower pigment concentrations in the feed.

Despite the trout ability to retain the pigment well, the cost of pigmentation must still be carefully evaluated by farmers and food processors; it is in fact necessary to design proper strategies for the pigmentation process, adapted to meet sales strategies, in order to reach on schedule the flesh coloration intensity required by the market.

It is important to remember that the pigments, like other dietary ingredients, have well defined levels of digestibility: in practice, less than the full amount of pigment included in the feed will be digested by fish. Digestibility rates of carotenoids used in animal feed vary between 50% and 60%. Also, not all of the digested amount of pigment will deposit in the most common places of storage, i.e. muscles, gonads and skin; it is estimated that the amount held by the muscle can vary between 4 and 20% of total digested pigments.

Muscle pigmentation levels of farmed salmonids vary according to market requirements, fish species and commercial presentation.

For example, desired pigment concentrations in the muscle of a trout weighting 300 grams ranges between 4 and 6 ppm, while they reach about 20 ppm for a salt-water farmed trout weighting over two kilos. By knowing the amount of pigment that must be achieved in fish flesh, it is possible to draw a pigmentation strategy, setting the initial average weight of pigmentation, the type of feed to be used, the time necessary to achieve the desired coloration and type of pigment to be used. This process is carried out in order to minimize the costs of the pigmentation process. Astaxanthin is a very expensive and highly perishable carotenoid. Carotenoids are compounds prone to degradation and during production processes they can undergo structural changes due to oxidative and thermal alterations. Such structural changes can degrade these compounds and make them colorless molecules without their native properties. (Villota R. and Hawkes J. G. (1992). "Reaction kinetics in food systems" in Handbook of food engineering. Edited by D. Heldman and D. Lund. Marcell Dekker, Inc., New York).

Astaxanthin is a hydrophobic compound that is not easily absorbed by animals gastro-intestinal tract. It has already been highlighted that increased levels of lipids in the diet have a positive effect on astaxanthin retention rate in salmonids, and that this phenomenon is probably related to an increased digestibility of the pigment. The intestinal absorption of carotenoids involves several stages, which include: destruction of the food matrix, dispersion in a lipid emulsion, and solubilization in bile salt micelles before they are absorbed through the brush border of the enterocytes (Olsen et al., Aquaculture, 2005, 250, 804-812).

The objective of the present invention is therefore to provide astaxanthin in a formulation that minimizes degradation and maximizes the level of absorption in salmonids.

SUMMARY OF THE INVENTION

In light of the above-mentioned considerations, a self-emulsifying system was designed and developed, whereby the active ingredient can be solubilized in an oil phase and then surrounded by surfactant molecules that facilitate the passage through the intestinal mucosa.

The subject-matter of the present invention is a composition, that is, a self- emulsifying system, comprising:

a) Astaxanthin 0.05-2.0%

b) Mono- and diglycerides of medium-chain fatty acids 20-25%

c) Saturated polyglycolized glycerides 20-25%

d) Surfactant 50-55%

e) Lipophilic antioxidant 0.5-2.0%

where % are % by weight referred to the total weight of the composition.

Subject of the invention is also a method for the preparation of the self-emulsifying system, with this method comprising:

heating components b), c) and d) of the composition at a temperature of 90-1 10 °C; adding astaxanthin under stirring up to complete dissolution;

cooling to room temperature and adding of component e). Surprisingly, it has been discovered that heating at a temperature of 100 degrees is enough to ensure pigment dissolution. In this way there are no leaks in the production process. Heating the oils and surfactant first, and adding the pigment only once they have reached the desired temperature, allows to minimize the degradation of the pigment due to temperature. Furthermore, the use of ethoxyquin as lipophilic antioxidant guarantees the self-emulsifying system to provide the pigment with stability for at least 5 months, in an oven at 40 degrees.

The innovative self-emulsifying system included in this invention can be easily and conveniently used to add astaxanthin in feed for livestock use. A further object of the invention is thus a feed for livestock use comprising the self-emulsifying system described above.

In in vivo trials of salmonids pigmentation, a feed administered according to this invention, that is, a feed including 7g/kg of astaxanthin (for absorption on the surface of feed pellets) added with the self-emulsifying system, proved to be at least as effective as available commercial feeds that include a higher quantity of astaxanthin (10g/kg). The invention's self-emulsifying system proves to be particularly useful to lower production costs and improving nutritional performance of feeds including astaxanthin.

Further advantages of this invention are explained in detail below.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the trend in plasma concentration profiles during 30 days of treatment with feed prepared according to this invention and two commercial feeds.

Fig. 2 shows the trend in astaxanthin concentrations in trout muscle during 30 days of treatment with feed prepared according to this invention and the two commercial feeds.

DETAILED DESCRIPTION OF THE INVENTION

Self-emulsifying systems are mixtures of oils and surfactants (sometimes containing also co-surfactant) that self-emulsify spontaneously in the gastro-intestinal tract under mild agitation, forming fine oil-in-water microemulsions. They thus display all the advantages of microemulsions, but they do not contain water within the formulation. Microemulsions are liquid nanosystems (NS), thermodynamically stable and transparent, which form spontaneously by mixing two immiscible liquids, typically oil and water, in the presence of a surfactant that stabilizes the interfacial film. They are characterized by a reduced droplets size in the dispersed phase (200 nm): this guarantees an increase of the surface area and therefore a greater absorption of the molecule contained within them. Unlike emulsions that require a large amount of energy to form, microemulsions form spontaneously by mild agitation: this is important when considering the cost of commercial production of the two types of systems (Lawrence et al., Adv. Drug Del. Rev., 2000, 45, 89-121 ). They can be of two types: oil-in-water (O/W) if the dispersed phase is oil or water-in-oil (W/O) if the dispersed phase is water.

The components of the invention's self-emulsifying system b) mono- and diglycerides of medium chain fatty acids and c) saturated polyglycolized glycerides are digestible oils, usually of vegetable origin, or medium-chain triglycerides and/or mixtures of glycerides. These oils are commonly used in the food and pharmaceutical industries.

Mono- and diglycerides of medium-chain fatty acids b) are selected from the group consisting of Caprylic/Capric Glycerides (Akoline MCMTM), Hydrogenated Vegetable Glycerides Citrate (Akoline GCTM), Sodium Stearoyl Lactylate (Akoline SLTM), Glyceryl Stearate Citrate (Akoline LCTM), Polyglyceryl-3 Stearate (Akoline PG7™), Polyglyceryl-3 Polyricinoleate (Akoline PGPR™) and Glyceryl Stearate (Akoline MD50™). The emulsifying b) is preferably Caprylic/Capric Glycerides (Akoline MCMTM) commercially available from Huwell Chemicals, Milan, Italy. Saturated polyglycolized glycerides c) are mixtures of mono-di-triglycerides and mono-diesters of PEG 400 with C8 and C10 fatty acids or caprylocaproyl polyoxylglycerides or PEG-8 Caprylic/Capric Glycerides, commercially known as Labrasol® and commercially available from Gattefosse (Sain-Priest, France).

Surfactants d) used are of non-ionic type because when used in oral formulations they have a lower critical micelle concentration (CMC) compared to non-ionic surfactants and are less sensitive to the pH (Lawrence et al., Adv. Drug Del . Rev., 2000, 45, 89-121 ; Shafiq et al., Eur. J. Pharm. Biopharm., 2007, 66, 227-243). These non-ionic surfactant are preferably selected among Span (sorbitan esters), Tween (polysorbates), esters of polyethylene glycols and sucrose esters.

The preferred surfactant is Tween 80.

The lipophilic antioxidant e) is preferably Ethoxyquin or similar.

The self-emulsifying system of the invention, once prepared, can be included in the feed preparation during the phase of greasing after extrusion and drying, according to the usual manufacturing process known as state of the art. In this way the feed production process does not change except for the fact that the pigment is not mixed as powders in the first phase of the process but is instead added at the end during the greasing phase.

Adsorption tests of the self-emulsifying system on feed surface and subsequent in vivo testing were performed employing the most critical type of fish feed, that is, fish feed with high protein content and with potentially very high lipid levels (22-30% crude fat). Using a type of feed with such high lipid levels, it was possible to verify the feasibility of the manufacturing process of feed under the extreme technological conditions.

The graphs below present in vivo performances on fish of the feed prepared with the self-emulsifying system of the invention, compared to performance of two commercial feeds.

The present invention will be better understood in light of the following examples. EXPERIMENTAL PART

• mixture of mono-di-triglycerides and mono-diesters of PEG 400 with C8 and C10 fatty acids (Labrasol®, Gattefosse Sas, Milan), mixture of mono-diglycerides of C8 and C10 fatty acids (Akoline MCM®, Huwell Chemicals Sri, Milan), polysorbate oleic acid (Tween® 80, Sygma Aldrich, Milan), polysorbate monolaurate (Tween® 20, Sygma Aldrich, Milan).

EXAMPLE 1 - Preparation of a self-emulsifying system containing astaxanthin. Components: (expressed as weight % of the total weight of the composition) b) Akoline MCM: 22%

c) Labrasol: 22%

d) Tween 80: 54,44% a) Astaxanthin: 0.063%

e) Ethoxyquin: 1 .5%

To prepare the self-emulsifying system with astaxanthin, the mixture components b) , c) and d) were heated in a silicone bath at a temperature of 100 °C. Once the temperature was reached, 0.063% of astaxanthin was dispersed in the self- emulsifying system under magnetic-induced agitation for 15' to facilitate dissolution of the pigment. During this dissolution step, a vacuum-argon line was also used, in order to prevent oxidation. After the self-emulsifying system cooled off, a lipophilic antioxidant, ethoxyquin, was added to the mixture at a concentration of 1 .5% (as allowed by the European legislation in force).

EXAMPLE 2 - Stability studies performed on self-emulsifying system of Example 1 To assess the stability of the self-emulsifying system with astaxanthin, samples of the formulation were stored in ovens at 25 °C, 30 °C and 40 °C. The concentration of pigment was assessed with triple sampling at 0, 10, 30 and 150 days by means of HPLC analysis (HPLC method reported below). It was found that the pigment concentration did not vary significantly, demonstrating the stability of the system within the timeframes and at the temperatures considered. Therefore, ethoxyquin plays its role effectively by reducing pigment degradation over time in the formulation.

Astaxanthin concentrations in the formulation maintained at 25, 30 and 40 °C after different storage times.

EXAMPLE 3. Preparation of astaxanthin added feed.

The feed was prepared by simple adsorption of the self-emulsifying system of example 1 on commercial not-medicated feed. Adsorption of the formulation on pellets surface took place after extrusion and drying, during greasing phase. 1 1 .8% of self-emulsifying system containing astaxanthin according to example 1 was loaded on the feed so as to obtain a feed containing on average 7g/kg of astaxanthin. The size of droplets in the dispersed phase released from the feed after this process resulted to be compatible with the self-emulsifying system, whereby the micro-emulsifying properties were maintained after loading onto the solid carrier. Commercial not-medicated feed used for this example preparation is a fish feed containing: fish meal, wheat gluten, fish oil (5%), wheat starch, mineral and vitamin supplements, soy lecithin. It is commercially available in non-greased form (feed for Excel 3P, to grease, produced by Hendrix SpA, Verona, Italy). EXAMPLE 4 - Comparative in vivo trial performed on fish after administration of feed prepared according to Example 3 and commercial feeds.

The experiments were carried out on rainbow trout. One hundred and twenty rainbow trout (average weight 350 g) were randomly divided in three raceway tanks (40 fish/tank) and allowed to adapt for one week. Fish were starved for 4 days prior to the administration of feed. This was a parallel design study in which each of the three tanks was used to test one of the feeds. Medicated feeds were supplied, at the rate of 1 % biomass to fish, directly in the tanks for 30 days, as repeated once daily administrations.

Group I was fed with feed medicated with Novu®, group II was fed with feed medicated with Carophyll Pink® and group III was fed with feed loaded with the self- emulsifying system.

Commercial feed used as comparison are produced by mixing, before the extrusion process, feed components in powder form with respectively 10% of Carophyll pink® and 10% of Novu® (pigments). As a result of this operation, both commercial feed contained 10 g/kg of astaxanthin.

The palatability and nutritional capacity of the three different feeds were compared and resulted very similar, as can be seen in three tables below. Weight gain after administration of Pink ® loaded feed

Weight gain after administration of Novu® loaded feed Weight gain after administration of invention feed

At each of the scheduled times: 0 (before administration), 1 0, 20 and 30 days ten toruts were randomly selected from each tank and anesthetize with tricaine methanesulphonate. Animal were then weighted for evaluating the weight gain and plasma samples (3 ml) taken from caudal vein. Ten plasma samples per treatment group and time point were pooled and stored at 20 C until analysis.

After blood sample, the same animals were sacrificed and a sample of fillet was taken from each. The single samples of blood and muscle were treated according to the methods of extraction named in the following paragraphs, and then analyzed by HPLC.

Extraction of the plasma samples

200 microliters of plasma were added to 200 microliters of ethanol and 1 ml of ethyl acetate. The mixture thus obtained was then vortexed for 30s and then centrifugated at 9400g for 4 minutes. The supernatant was then transferred into a glass test tube of 10 ml. The sediment remained in an Eppendorf vial and was re-extracted two consecutive times with a further 1 ml of ethyl acetate and then 1 ml of hexane. The three supernatants, brought together in the test tube of 10 ml, were then evaporated to dryness in a centrifugal concentrator. Dry residues were redissolved in 200 μΙ of mobile phase, vortexed, centrifuged for 10 minutes at 14000 rpm and injected into HPLC at a rate of 100 μΙ.

Extraction of muscle samples

Astaxanthin concentration was analyzed in each fish muscle. Samples of each fillet have been cut, chopped and carefully weighed. To samples of 7.5 g were added 7.5 ml of water and 7.5 ml of methanol. The mixture thus obtained was homogenized with a mixer for 30 seconds. Subsequently 21 ml of chloroform were added and the samples were further homogenized for another 30 seconds. Then, the muscle samples were centrifuged (15 min, 2500 rpm). At the end of the centrifuge, 2 ml of chloroform (lower phase) containing astaxanthin, were collected with a pipette and transferred into test tube, and then evaporated to dryness in a centrifugal concentrator. Before analysis, dried samples were redissolved in 1 ml of mobile phase, filtered through syringe filters from 45 μΙ and injected in HPLC.

HPLC method

The analysis was performed with a liquid chromatograph Agilent (Palo Alto, CA) equipped with DAD detector and assembled with a quaternary LC 1 100 series pump, an under vacuum microdegasser, an auto-sampler. The chromatographic separation was obtained, at room temperature, using a reverse phase column Symmetry C18 (3,5μιη, 150 X 4.6 mm) of Waters (Milford, MA). As mobile phase was used: Acetonitrile 70, 50 Acetone and Methanol 5. The analysis was performed at a flow rate of 1 ml/min with the UV detector set at 480 nm, with UV detection. The injection volume was 100 μΙ/ml in DCL while run time last 16 minutes.

Analysis of the muscle samples by colorimetric method

Colour values were measured using a portable spectrophotometer Minolta CM 2600 d (Ramsey, NJ, USA) with a 8 mm aperture, using Standard llluminant D65 light source and 10 viewing angle geometry. The values recorded included L ^* , a ^* , b ^* , Chroma ^* and Hue ^* scores, and was taken on 5 points per fillet (per animal).

Here below are reported the graph and tables with the results obtained in the plasma of the fish. Astaxanthin concentrations in trout plasma (in bold final concentrations detected).

Fig.1 shows plasma profiles obtained after 30 days of repeated administration of different types of feeds: two commercial feeds and one feed prepared according to the invention.

As can be seen from the graph and the values in the table, the Pink® formulation is less bioavailable, having lower AUC, and is characterized by poor absorption in the first 20 days of treatment and a high concentration of pigment and comparable to the other two feed only after 30 days. This feed is characterized by a slow absorption and would probably require more than one month of treatment.

The Novu® feed shows AUC comparable to the invention feed but its maximum plasma concentration occurs after 20 days of treatment. The invention feed has peak plasma already at 10 days that remained almost unchanged throughout the treatment. The bioavailability is comparable as it was said to that of the Novu® formulation. Considering that the formulation of the invention has a lower dose (30% less) of the pigment, these results are even more remarkable.

In order to verify the effectiveness of treatment with the pigment is, however, more important, than the plasma concentrations, determine the astaxanthin deposition in the muscle.

Astaxanthin concentrations in trout flesh (in bold final concentrations detected). Day PINK S.D. INVENTION S.D. NOVU S.D.

(Mg/ml) (Mg/ml) (Mg/ml) (Mg/ml) (Mg/ml) (Mg/ml)

0 0 0 0 0 0 0

10° 0 0 0 0 0 0

20° 0 0 0,1 13605 0,1666868 0,03805671 0,091771

30° 0,43071 0,306749 0,4326 0,3444829 0,58407873 0,458937

Values of astaxanthin concentrations in the muscle reported in the table and in Fig. 2 show that, after 1 0 days of administration of all types of feeds, no pigment can be detected in the samples. The Pink® sample in agreement with previous plasma data, settles more slowly into the muscle and the pigment is detected only after 30 days of treatment. These results indicate that it requires a more prolonged treatment. Novu® feed, always in accordance with the plasma data, settles slowly but reaches major title. Feed of the invention provides a final titre of 0.43 mg/ml; the latter, however, already at 20 days has an appreciable concentration. Also in consideration of the reduced loading of astaxanthin in this feed (7g/kg compared to 1 0 mg/kg) these results are particularly encouraging.

Fig. 2 shows astaxanthin concentrations in trout flesh obtained after 30 days of repeated administration of different types of feeds: two commercial feeds and one feed prepared according to the invention.

Evaluation using colorimeter (Minolta CM 2600 d, Ramsey, NJ, USA)

To the data obtained by HPLC analysis it followed an analysis of the degree of salmon color of the threads by colorimeter. The trout treated fillets were analyzed by 20 and 30 days, similarly to what was done with HPLC. As indicated in the experimental part, the recorded values (colorimetric space L * a * b *) were L * (brightness), a * (redness index), b * (yellow index), more Croma (C, chroma) and Tint (h, hue), in 5 different points for each thread and are reported in the table below (table 1 1 ). The value a * generically reveals the best correlation with the levels of carotenoids present. Also the fat content in the muscle is correlated with parameters L *, a * and b *; so fillets that contain the same levels as astaxanthin but the highest concentrations in fat nevertheless possess high values of the parameter a ^* , and then the red coloration but also high values of h (hue) and then of a more yellow coloring. Results analysis with colorimeter after 20 and 30 days.

By a ^* values thus obtained there is a perfect correlation between the red indexes and pigment concentrations analyzed in muscle. In particular, Pink® feed after 20 days shows a red low index and reaches levels comparable to the other two only after 30 days of treatment. Novu® has a slower pace and a max 30 days after always in perfect agreement with muscle data. Very interesting, however, the values measured after 20 days of treatment with the feed of the invention feed which proves very effective in the short term, ensuring a significant pigmentation of the flesh. Its final red index is comparable to the other two feed, while being dosed to 70% with respect to these. As evident from these results, astaxanthin delivered inside the invention is absorbed more rapidly and hence utilized more effectively. This difference is probably due to the fact that astaxanthin was dissolved within the self-emulsifying system and adsorbed on the surface of the extruded feed and so it was readily available to be absorbed. When delivered inside commercial feed instead, the pigment is supplied in the form of microspheres which are mixed with feed components and fish oil that works as a binder solution. For this reason, the pigment before being absorbed must be released from fish oil and solubilize in the gastro-intestinal fluids after digestion lipid/emulsification process. In the self-emulsifying system such steps are bypassed because the pigment is already available for absorption, given that the SES system gives rise spontaneously to a microemulsion in contact with gastrointestinal fluids.