This application claims the benefit of European Patent Application EP20383060.9 filed on December 4, 2020.

Technical Field

The present invention relates to the field of animal feed additives. Particularly, it relates to a combination of a capsaicinoid, piperine and gingerol that can be added to animal feeds, and to an animal feed additive and an aquaculture feed comprising the combination. It also relates to the use of the mentioned combination for improving the quality of flesh of aquatic animals such as fish muscle.

Background Art

It is known that omega 3 long chain polyunsaturated fatty acids (n-3 LC-PUFAs) are highly beneficial to human health. This is related to the direct intake of a sufficient quantity of n-3 LC-PUFA together with a balanced dietary omega-3/omega-6 PLIFA ratio (n-3/n-6 ratio).

However, twenty-first-century Western society diets are generally characterized by a gross imbalance in the n-3 to n-6 PLIFA ratio. As such, for health reasons, an increase in the consumption of n-3 LC-PUFA is highly recommended. Consequently, fish and seafood, having a high concentration of n-3 LC-PUFA such as EPA (eicosapentaenoic acid, 20:5n-3) and DHA (docosahexaenoic acid, 22:6n-3) together with a high n-3 1-6 PUFA ratio, are believed to be a fundamental part of a healthy human diet.

Carnivorous fish species do not have a physiologically relevant ability to desaturate and elongate short-chain PUFAs such as LA (linoleic acid, 18:2n-6) and ALA (a-linolenic acid, 18:3n-3) to the biologically active and essential LC-PUFAs. Nevertheless, originally, farmed fish feed contained high levels of fish oil, supplying the required n-3 LC-PUFAs.

However, the dramatic increase in aquaculture production, accompanied by the increased demand for fish-based raw materials for feed production and the static or decreasing supply of fish raw materials, has led to the partial replacement of fish oil by terrestrial animal fats or vegetable oils in aquafeeds. This replacement affects the fatty acid profile of the fish, which will have a lower content of n-3 LC-PUFAs and lower n-341-6 PUFA ratio than those fed with a fish oil-based diet. Therefore, there is a need for finding sustainable alternatives for production of aquaculture feeds, while providing an improvement in the nutritional quality of the final product, such as of fish fillet, particularly of aquatic animals fed with aquafeeds having a relatively low proportion of fish oil.

Summary of Invention

The present inventors have found that the intake of a combination of a capsaicinoid, piperine and gingerol when added to an aquafeed positively affects the lipid metabolism in aquatic animals such as fish.

Surprisingly, it has been found that the mentioned combination provides un unexpected effect, since when added to an aquaculture feed (comprising fish oil) the levels of total n-3 LC-PUFAs such as EPA, DPA (docosapentaenoic acid, 22:5n-3) and DHA in total lipids (i.e. , in the total lipid contents) in the aquatic animal (such as in a fish, particularly in the muscle of the fish) fed with such feed are enhanced compared to the ones in an aquatic animal fed with a non-supplemented feed (i.e. the same feed but in the absence of the combination). Additionally, the combination also allows lowering the total n-6 PLIFA in the fish muscle, and therefore to increase the n-341-6 PLIFA ratio. This allows reducing the amount of fish oil in the aquaculture feed by partially substituting fish oil by terrestrial animal fats, vegetable oils or both of them. Thus, the supplementation of a fish oil-reduced aquaculture feed (i.e. an aquafeed containing a relatively low proportion of n-3 PLIFA and high proportion of n-6 PLIFA, or a relatively high proportion of the monounsaturated oleic acid, or a relatively high proportion of saturated fatty acids, or a combination of these features) with the mentioned combination allows bringing the balance between n-3 and n- 6 PLIFA in the aquatic animal closer to that found in fish fed with a non-reduced fish oil aquaculture feed (i.e. wherein fish oil has not been partially substituted by terrestrial animal fats or vegetable oils).

Thus, in a first aspect the present disclosure relates to an animal feed additive for aquatic animals, particularly fish such as marine fish, comprising: a combination comprising a capsaicinoid, piperine, gingerol, and a compound selected from cinnamaldehyde, a curcuminoid, and a mixture thereof; and at least one feed acceptable excipient.

Advantageously, by the use of this additive, not only the nutritional quality due to the improved polyunsaturated fatty acid profile of the final product, such as of the fillet of a fish, is enhanced, but also a decreased fat deposition is observed. Additionally, an improvement in feed conversion ratio (FCR) and weight gain are also observed, what is reflected in positive effects on the productive yield.

In a second aspect, the present disclosure relates to an aquaculture feed comprising an effective amount of the animal feed additive for aquatic animals as defined above.

The animal feed additive for aquatic animals of the present disclosure can be used for increasing the nutritional quality of aquatic animals, namely for improving polyunsaturated fatty acid profile and decreasing fat deposition, particularly of aquatic animals fed with fish oil-reduced aquafeeds (i.e., wherein fish oil usually contained in aquafeeds has been partially substituted by terrestrial animal fats or vegetable oils). A fish oil-reduced aquafeed is characterized by having a relatively low proportion of n-3 PLIFA and high proportions of n-6 PLIFA, or a relatively high proportion of the monounsaturated oleic acid, or a relatively high proportion of saturated fatty acids, or a combination of these features, compared with a non-reduced fish oil aquafeed.

Accordingly, another aspect of the present disclosure relates to the use of the combination, the animal feed additive for aquatic animals, or the aquaculture feed as defined above, for increasing the amount of omega 3 long chain polyunsaturated fatty acids in total lipids, or for increasing the ratio of omega 3/omega 6 polyunsaturated fatty acids in total lipids, or both of them, in an aquatic animal fed with a diet comprising the combination (or the animal feed additive), compared to the amount of omega 3 long chain polyunsaturated fatty acids in total lipids, the ratio of omega 3/omega 6 polyunsaturated fatty acids in total lipids, or both of them, in an aquatic animal fed with the same diet but in the absence of the combination (or the animal feed additive). This aspect can also be formulated as a method for increasing the amount of omega 3 long chain polyunsaturated fatty acids in total lipids of an aquatic animal, for increasing the ratio of omega 3/omega 6 polyunsaturated fatty acids in total lipids of an aquatic animal, or both of them, in an aquatic animal fed with a diet comprising the combination (or the animal feed additive), compared to the amount of omega 3 long chain polyunsaturated fatty acids in total lipids, the ratio of omega 3/omega 6 polyunsaturated fatty acids in total lipids, or both of them, in an aquatic animal fed with the same diet but in the absence of the combination (or the animal feed additive), the method comprising feeding the aquatic animal with a diet comprising an effective amount of the combination (or the animal feed additive) as defined herein above or below.

Brief Description of Drawings

Fig. 1 shows the total amount of lipids in fish fillet (mg/g dry weight (DW) of fillet). Bars with different superscripts differ significantly (P<0.05, assessed by one-way Analysis of Variance (ANO A) followed by Tukey's multiple comparisons test).

Fig. 2 shows the levels of individual PLIFA in fish fillet lipids (mg FA/g lipids), i.e. the amount (in mg) of each PLIFA in each g of lipids in the fillet. Within each graphic, columns with different superscript letters are significantly different (P<0.05, assessed by one-way Analysis of Variance (ANOVA) followed by Tukey's multiple comparisons test).

Fig. 3 shows the total levels of n-3 PLIFA and n-6 PLIFA in fish fillet lipids (mg/g lipids). The ratio between n-3/n-6 PLIFA is indicated in the secondary (right-hand) axis.

Detailed description of the invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions of terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

Capsicum oleoresin is an oily extract of the dried, ripened fruit of chili peppers of plants of the genus Capsicum. Capsaicinoids are considered the active ingredients of capsicum oleoresin. Thus, the oleoresin is used generally as a natural source of capsaicinoids. The amount of capsicinoids in the capsicum oleoresin can be from about 5.0 wt% to about 6.6 wt%, such as of 5.5 wt%.For the purpose of the present invention, the term "capsaicinoid" refers to a capsaicinoid or a salt thereof. Particularly, the capsaicinoid is selected from the group consisting of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin nonivamide, and mixtures thereof. More particularly, the capsaicinoid is capsaicin.

Black pepper oleoresin is an oily extract of dried berries of Piper nigrum, and it is used generally as a natural source of piperine. Piperine in black pepper oleoresin can be in an amount from 34 wt% to 42 wt%, such as of about 33 wt% or about 36 wt.%.

Ginger oleoresin is an oily extract of the ground-dried rhizomes of ginger (Zingiber officinale), and it is used generally as a natural source of gingerol. Gingerol in ginger oleoresin can be from about 4 wt% to about 12 wt%, such as of about 8 wt% or about 9 wt%.

Turmeric oleoresin is an oily extract of dried ripe rhizomes of turmeric (Curcuma longa L), and it is used generally as a natural source of curcuminoids. The amount of curcuminoids in turmeric oleoresin can be from about 28 wt% to about 38 wt%, such as of about 32 wt%. For the purpose of the present invention, the term "curcuminoid" refers to a curcuminoid or a salt thereof. Particularly, the curcuminoid is selected from the group consisting of curcumin, demethoxycurcumin and bisdemethoxycurcumin, and mixtures thereof. More particularly, the curcuminoid is curcumin.

Capsicum oleoresin, black pepper oleoresin, ginger oleoresin, turmeric oleoresin, and cinnamaldehyde are commercially available.

The term "animal feed additive", as used herein, refers to a composition suitable for or intended for being incorporated in an animal feed. In the present disclosure, the animal feed additive is for aquatic animals.

The term "aquatic animal" refers to crustaceans such as shrimps, prawns and crabs; fish such as breams, basses, salmon, trout, groupers, carps, tilapias, catfishes; molluscs such as clams, oysters, limpets and abalone; cephalopods such as octopus, squid and cuttlefishes; and other invertebrates such as cucumbers and sea urchins.

The term "animal feed" refers to a preparation or mixture suitable for or intended for intake by an animal. The terms "aquaculture feed” or "aquafeed" refer to an animal feed intended for intake by aquatic animals, that is a manufactured or artificial diet to supplement or to replace natural feed.

The expression "an effective amount", as used herein, refers to any amount sufficient to achieve a desired and/or required effect, i.e. to increase the amount of omega 3 long chain polyunsaturated fatty acids in total lipids, increase the ratio of omega 3/omega 6 long chain polyunsaturated fatty acids, or both of them, in an aquatic animal with regard to the amount and/or ratio of the mentioned fatty acids without the addition of the combination of the present disclosure.

The term "feed conversion ratio (FCR)" refers to a measure of an animal's efficiency in converting feed mass input into increases of the desired output. In animals raised for meat (such shrimp and fish) the output is the body mass gained by the animal. FCR is calculated as feed intake divided by weight gain, over a specified period. Improvement in FCR means reduction of the FCR value.

The term "specific growth rate (SGR)" refers to the daily increase in body weight (in %). The term "percentage by weight" or "wt.%" refers to the percentage by weight of the ingredient per weight of the overall composition, unless otherwise stated.

As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the”, also include the plural of the noun.

As mentioned above, the animal feed additive for aquatic animals of the present disclosure comprises a combination comprising a capsaicinoid, piperine, gingerol, and a compound selected from cinnamaldehyde, a curcuminoid, and a mixture thereof; and at least one feed acceptable excipient.

In an embodiment:

- the capsaicinoid is in an amount from 0.1 wt.% to 1.0 wt.%, or from 0.2 wt.% to 0.7 wt.%,

- piperine is in an amount from 0.025 wt.% to 0.25 wt.%, or from 0.1 wt.% to 0.2 wt.%,

- gingerol is in an amount from 0.002 wt.% to 0.05 wt.%, or from 0.01 wt.% to 0.04 wt.%, and either

- cinnamaldehyde is in an amount from 0.5 wt.% to 3.0 wt.%, or from 0.75 wt.% to 2.5 wt.%, such as 2.0 wt.%, and

- the curcuminoid is in an amount from 0 wt.% to 3.0 wt.%, or from 0.5 wt.% to 3.0 wt.%, or from 1.0 wt.% to 2.5 wt.%, such as 1.5 wt.%, or, alternatively

- cinnamaldehyde is in an amount from 0 wt.% to 3.0 wt.%, or from 0.5 wt.% to 3.0 wt.%, or from 0.75 wt.% to 2.5 wt.%, such as 2.0 wt.%, and

- the curcuminoid is in an amount from 0.5 wt.% to 3.0 wt.%, or from 1.0 wt.% to 2.5 wt.%, such as 1.5 wt.%, wherein weight percentages are relative to the total weight of animal feed additive.

In another embodiment, optionally in combination with one or more features of the particular embodiments defined above, the capsaicinoid is selected from the group consisting of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin, nonivamide, and mixtures thereof, and the curcuminoid is selected from the group consisting of curcumin, demethoxycurcumin and bisdemethoxycurcumin, and mixtures thereof.

In another embodiment, optionally in combination with one or more features of the particular embodiments defined above, the capsaicinoid is capsaicin. In another embodiment, optionally in combination with one or more features of the particular embodiments defined above, the curcuminoid is curcumin.

In an embodiment, optionally in combination with one or more features of the particular embodiments defined above, cinnamaldehyde is in an amount from 0.5 wt.% to 3.0 wt.%, and the curcuminoid is in an amount of 0 wt.%.

In another embodiment, optionally in combination with one or more features of the particular embodiments defined above, curcuminoid is in an amount from 0.5 wt.% to 3.0 wt.%, and the cinnamaldehyde is in an amount of 0 wt.%.

In another embodiment, optionally in combination with one or more features of the particular embodiments defined above, the animal feed additive for aquatic animals comprises a capsaicinoid, piperine, gingerol, and cinnamaldehyde, wherein the amount of capsaicinoid is from 0.25 wt.% to 1.0 wt.%, or from 0.35 wt.% to 0.85 wt.%, such as of 0.62 wt.%; the amount of piperine is from 0.025 wt.% to 0.25 wt.%, or from 0.04 wt.% to 0.2 wt.%, such as 0.13 wt.% or 0.14 wt.%; and the amount of gingerol is from 0.002 wt.% to 0.04 wt.%, or from 0.005 wt.% to 0.03 wt.%, such as of 0.02 wt.%; and the amount of cinnamaldehyde is from 0.5 wt.% to 3.0 wt.%, or from 0.75 wt.% to 2.5 wt.%, such as of 2.0 wt-%; relative to the total weight of animal feed additive and, particularly, the amount of curcuminoid is 0 wt%.

In another particular embodiment, optionally in combination with one or more features of the particular embodiments defined above, the animal feed additive for aquatic animals comprises a capsaicinoid, piperine, gingerol, and a curcuminoid, wherein the amount of capsaicinoid is from 0.1 wt.% to 0.5 wt.%, or from 0.15 wt.% to 0.4 wt.%, such as of 0.26 wt.%; the amount of piperine is from 0.04 wt.% to 0.2 wt.%, or from 0.06 wt.% to 0.16 wt.%, such as of 0.11 wt.%; the amount of gingerol is from 0.01 wt.% to 0.05 wt.%, or from 0.015 wt.% to 0.04 wt.%, such as of 0.03 wt.%; and the amount of curcuminoid is from 0.5 wt.% to 3.0 wt.%, or from 1.0 wt.% to 2.5 wt.%, such as of 1.5 wt.%; relative to the total weight of animal feed additive and, particularly, the amount of cinnamaldehyde is 0 wt%.

As mentioned above, usually capsaicinoids are commercialized in the form of capsicum oleoresin, piperine in the form of black pepper oleoresin, gingerol in the form of ginger oleoresin, and curcuminoids in the form of turmeric oleoresin.

Thus, in another embodiment, optionally in combination with one or more features of the particular embodiments defined above, in the animal feed additive for aquatic animals of the present invention, the capsaicinoid is provided in the form of capsicum oleoresin; piperine is provided in the form of black pepper oleoresin; gingerol is provided in the form of ginger oleoresin, and the curcuminoid, if present, is provided in the form of turmeric oleoresin.

The amount of each one of the oleoresins in the animal feed additive will depend on its content of the corresponding active ingredient. In particular, knowing the content of the active ingredient in the corresponding commercial oleoresin, an expert will know the amount of each oleoresin to be added in the feed additive in order to obtain the desired percentage by weight of each active ingredient.

Thus, this embodiment can also be formulated as an animal feed additive for aquatic animals as defined above comprising: a combination comprising

- capsicum oleoresin as a source of the capsaicinoid, particularly, wherein the oleoresin is in an amount which provides an amount of capsaicinoid as defined above;

- black pepper oleoresin as a source of piperine, particularly, wherein the oleoresin is in an amount which provides an amount of piperine as defined above;

- ginger oleoresin as a source of gingerol, particularly, wherein the oleoresin is in an amount which provides an amount of gingerol as defined above; and

- cinnamaldehyde, particularly, in an amount as defined above; or, alternatively, turmeric oleoresin as a source of the curcuminoid, particularly, wherein the oleoresin is in an amount which provides an amount of curcuminoid as defined above; and at least one feed acceptable excipient.

Thus, in a particular embodiment, the amount of capsicum oleoresin is from about 1.8 wt.% to about 18 wt.%, the amount of black pepper oleoresin is from about 0.07 wt.% to about 0.7 wt.%, the amount of ginger oleoresin is from about 0.02 wt.% to about 0.6 wt.%, and the amount of turmeric oleoresin is from about 1.5 wt.% to about 9.4% wt.%.

The animal feed additive for aquatic animals of the present disclosure can be incorporated in an animal feed composition in order to obtain an aquaculture feed comprising a capsaicinoid, piperine, gingerol, and a compound selected from cinnamaldehyde, a curcuminoid, and a mixture thereof, as additional components.

Thus, the aquaculture feed of the present disclosure comprises a) an animal feed composition for aquatic animals, particularly an aquaculture feed containing fish oil such as in an amount from 1 wt.% to 25 wt.% relative to the total weight of aquaculture feed, and, more particularly, an aquaculture feed containing fish oil wherein fish oil has been partially substituted by terrestrial animal fats or vegetable oils; and b) an effective amount of the animal feed additive for aquatic animals as defined herein above and below.

In an embodiment of the aquaculture feed of the present disclosure, optionally in combination with one or more features of the particular embodiments defined above, the amount of the animal feed additive for aquatic animal in the aquaculture feed is from 0.025 wt.% to 0.30 wt.%, or from 0.05 wt.% to 0.25 wt.%, or from 0.10 wt.% to 0.20 wt.%, or from 0.10 wt.% to 0.15 wt.%, relative to the total weight of aquaculture feed. In a particular embodiment, the amount of the animal feed additive for aquatic animal is of 0.15 wt.% relative to the total weight of aquaculture feed.

The recommended dietary formulations vary in every kind of aquatic animal such as in every kind of fish. Accordingly, in an embodiment, optionally in combination with one or more features of the particular embodiments defined above, the aquaculture feed of the present disclosure comprises an amount of added oils from 3 wt.% to 35 wt.%, such as 30 wt.% relative to the total weight of aquaculture feed, wherein the added oils comprise fish oil, and animal fats, vegetable oils, or both of them. As an example, the amount of fish oil in the aquaculture feed can be from 1 wt.% to 25 wt.% relative to the total weight of aquaculture feed.

The amount of added oils depends on the cultured aquatic animal. As an example, the amount of added oils in standard seabream or seabass aquaculture feeds is from 10 wt.% to 16 wt.%, wherein the amount of fish oil can be, for instance, from 3 wt.% to 12 wt.%; and the amount of added oils in standard salmonid aquaculture feeds is from 17 wt.% to 35 wt.%, such as 25 wt.%, wherein the amount of fish oil can be, for instance, from 4 wt.% to 35 wt.%, or from 10 wt.% to 25 wt.%.

The combination or animal feed additive for aquatic animals of the present disclosure is particularly effective when incorporated in aquafeeds wherein a proportion of the fish oil contained therein has been substituted by terrestrial animal fats or vegetable oils.

Thus, in an embodiment, optionally in combination with one or more features of the particular embodiments defined above, the aquaculture feed comprises fish oil and terrestrial animal fats or vegetable oils, or both of them. Terrestrial animal fats or vegetable oils contain reduced levels of n-3 PLIFA and increased levels of n-6 PLIFA, or a relatively high proportion of the monounsaturated oleic acid, or a relatively high proportion of saturated fatty acids, or a combination of these features.

In another particular embodiment of the aquaculture feed of the present disclosure, optionally in combination with one or more features of the particular embodiments defined above, up to a 90 wt%, such as from 5 wt.% to 90 wt. %, or from 15 wt.% to 75 wt. %, or from 25 wt.% to 60 wt.%, of the total amount of fish oil has been replaced by the same weight amount of terrestrial animal fats, vegetable oils, or both of them.

Particularly, fats substituting fish oil are incorporated in the form of terrestrial animal fats, more particularly mammal's fat such as tallow and lard (e.g. rendered beef and/or swine fat).

As mentioned above, the aquatic animals can be crustaceans, fish, molluscs, cephalopods, and other invertebrates such as cucumbers and sea urchins. Particularly, the aquatic animals are fish, more particularly marine fish.

The animal feed additive for aquatic animals and the aquaculture feed of the present disclosure can be prepared by conventional methods known to those skilled in the art, such as the ones described in the examples. Aquaculture feed is most commonly produced in the form of pellets, manufactured by pelletisation or extrusion.

As an example, a process for the preparation of an aquaculture feed as defined herein above and below can comprise the steps of manufacturing the animal feed additive comprising a capsaicinoid, piperine and gingerol and, optionally, cinnamaldehyde, or a curcuminoid, or a mixture thereof, and, then, mixing the animal feed additive with an animal feed composition for aquatic animals.

As an example, a process for manufacturing the animal feed additive comprises preparing an active mixture by mixing the ingredients comprising a capsaicinoid, piperine, gingerol, and an ingredient comprising a compound selected from cinnamaldehyde, a curcuminoid, or a mixture thereof, and then further mixing with other feed acceptable excipients, comprising at least one carrier, one antioxidant, one emulsifier and a fat matrix material, to finally atomize the whole composition, for instance in a spray cooling atomizer, to obtain the animal feed additive in a fat coated encapsulation form.

Particularly, the active mixture can be obtained by mixing a capsicum oleoresin as a source of the capsaicinoid, black pepper oleoresin as a source of piperine; ginger oleoresin as a source of gingerol; and a component selected from cinnamaldehyde, turmeric oleoresin as a source of curcuminoid, or a mixture thereof.

Furthermore, an animal feed additive obtainable by the process as defined above also forms part of the invention. All the embodiments of the process of the invention contemplate all the combinations providing all the embodiments of the animal feed additive of the invention and combinations thereof.

In an embodiment, optionally in combination with one or more features of the particular embodiments defined above, the animal feed additive for aquatic animals is in a fat coated encapsulation form.

Examples of feed acceptable ingredients or additives include vegetable fats and oils such as hydrogenated palm fat or soybean oil; antioxidants such as BHT, emulsifying agents such as lecithins or fatty acids esterified with glycerol and binders such as colloidal silica.

As another example, ingredients of a feed composition for aquatic animals can be mixed for instance in a mixer, then the mixture can be mixed with the feed additive in the form of a powder, and subsequently extruded or pressed to form a pellet. Alternatively, the feed composition can be transformed into pellets by extrusion or pressing, and then a feed additive according to the present disclosure comprising water or an edible oil such as a vegetable oil can be coated onto the pellets, particularly in the amounts defined herein above, in order to obtain the final animal feed.

Animal feed compositions wherein the combination of the animal feed additive of the present disclosure can be added are commercially available.

As an example, an animal feed composition for aquatic animals can comprise one or more components selected from proteins including fish meal and vegetable meals, carbohydrates including starch, lipids including fish oil, terrestrial animal fats and vegetable oils, vitamins, amino acids and other allowed feed additives and feed materials.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”.

The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

Commercially available capsicum oleoresin containing a 5.5 wt% of capsaicinoids, black pepper oleoresin containing a 33 wt.% piperine, ginger oleoresin containing an 8 wt.% of gingerol, and turmeric oleoresin containing a 32 wt.% of curcuminoids, and cinnamaldehyde were used.

Diets containing either feed additive A or B shown in Table 1 were tested.

Table 1

The amounts of capsaicinoids contained in the capsicum oleoresin, of piperine contained in the black pepper oleoresin, of gingerol contained in the ginger oleoresin, and of curcuminoids contained in the turmeric oleoresin are shown in Table 2 below.

Table 2 wt.% are with respect to the total amount of additive. *Mean values appearing in the raw material specifications. Six experimental diets were tested. In Table 3 the content of fish oil, mammal's fat, feed additive A, and feed additive B on the tested diets are shown, wherein diets AO.5, A1.0, A1.5 and B1.0 are based on the NC formulation supplemented with the tested feed additives A and B in the specified amounts.

Table 3

In Table 3:

- PC - Positive control diet mimicking a commercial diet for gilthead seabream containing fishmeal, fish protein hydrolysate, feather meal hydrolysate, porcine blood meal, poultry meal, corn gluten meal, soybean meal and rapeseed meal as major protein sources, and fish oil as the main fat source;

- NC - Negative control diet, having the same composition as PC diet but for 45% of the fish oil having been replaced by a blend of terrestrial animal fats, designated as mammal’s fat, and particularly rich in palmitic acid (16:0, 23.8%), stearic acid (18:0, 14.4%), oleic acid (18:1n-9, 39.5%) and linoleic acid (18:2n-6, 9.4%) fatty acids;

- AO.5 diet - NC diet + Feed additive A at 0.5 g/kg (0.05 wt.%);

- A1.0 diet - NC + Feed additive A at 1.0 g/kg (0.1 wt.%);

- A1.5 diet - NC + Feed additive A at 1.5 g/kg (0.15 wt.%); and

- B1.0 diet - NC + Feed additive B at 1.0 g/kg (0.1 wt.%).

Moreover, PC and NC formulations were supplemented with specific vitamins, crystalline amino acids and inorganic phosphate to avoid any nutritional deficiencies, and both of them targeted a composition of 46% crude protein, 16% crude fat, 20.5 MJ/kg gross energy.

Diets were produced by extrusion. Powder ingredients and feed additives A or B were mixed with the NC formulation in a double-helix mixer (model 500L, TGC Extrusion, France) and grounded (below 400 pm) in a micropulverizer hammer mill (model SH1, Hosokawa-Alpine, Germany). Diets (pellet size: 4.0 mm) were manufactured with a twin- screw extruder (model BC45, Clextral, France) with a screw diameter of 55.5 mm. Extruded pellets were dried in a vibrating fluid bed dryer (model DR100, TGC Extrusion, France). After cooling, oils were added post-extrusion by vacuum coating (model PG- 10VCLAB, Dinnissen, The Netherlands).

The composition of the experimental diets is shown in Table 4.

Table 4 - Fish feed composition

Fatty acids with <0.25% are not represented. Values are average ± standard deviation (n=2). For fatty acids a single analysis was done.

As can be seen from the data in Table 4, replacement of 45% of fish oil by mammal’s fat caused an important change in the fatty acid composition of the tested fish feeds. Main effects were an increase in the level of saturated fatty acids (SFA), associated with rises in palmitic (16:0) and stearic (18:0) acids; an increase in the level of monounsaturated fatty acids (MLIFA), caused by contents in oleic acid (18:1n-9); and a substantial modification of the polyunsaturated fatty acid profile (PLIFA) of the n-3 and n-6 series, resulting in an important decrease in the n-3/n-6 PLIFA ratio.

In terms of C18 PLIFA’s, the partial fish oil substitution caused a rise in the percentage of linoleic acid (LA, 18:2n-6) and a reduction in alpha-linolenic acid (ALA, 18:3n-3) in total lipids. However, all of the long-chain PUFA (LC-PUFA, C>20) of both the n-6 and n-3 series were decreased in total lipids of the fish oil-replaced diet - this included arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3).

EXAMPLE 1

The effect of supplemental feed additives A and B above on the growth performance of gilthead seabream fed diets with a high level of a rendered animal fat was assessed as follows.

1. Growth performance trial

1.1. Test animals

Experimental species under testing was gilthead seabream (Sparus aurata). Fish were stocked at the research facilities for more than 6 months during which they were fed a standard commercial feed.

1.2. Rearing conditions

At the start of the experiment, triplicate groups of 40 gilthead seabream with a mean initial body weight (IBW) of 84.7 ± 3.8 g were allotted to 18 circular tanks (volume: 1000 L). Tanks were located outdoors, supplied with open-flow aerated seawater (water-flow rate: 5.0 L/min; dissolved oxygen > 6.9 mg/L) and subjected to natural photoperiod conditions during the Winter-Spring period (January till May). Water temperature fluctuated between 11.3 and 19.6°C (average 16.2 ± 1.9°C) and mean water salinity was 35.1 psu.

1.3. Feeding and samplings

Each replicate tank was fed one of the six diets during 112 days. Fish were fed to apparent satiety, by hand, twice a day (10.00 and 16.00h) during week days and once a day during weekends (10.00h), with utmost care to avoid feed losses. Distributed feed was quantified throughout the trial. Anesthetized fish were group weighed at the start of the trial, at days 29, 59, 92 and day 112.

2. Analytical methods

Experimental diets were grinded prior to analysis. The chemical composition analysis of diets was made using the following procedures: dry matter after drying at 105°C for 24 h; ash by combustion at 550°C for 12 h; crude protein (Nx6.25) by a flash combustion technique followed by a gas chromatographic separation and thermal conductivity detection (LEGO FP428); fat by dichloromethane extraction (Soxhlet); gross energy in an adiabatic bomb calorimeter (I KA).

The fatty acid analysis of mammals fat and experimental diets was performed by a standard gas-chromatography method such as explained in Example 2 below.

3. Criteria for evaluating growth and nutrient utilization

IBW (g): Initial mean body weight.

FBW (g): Final mean body weight.

Specific growth rate, SGR (%/day): (Ln FBW- Ln IBW) x 100/days.

Feed conversion ratio, FCR: crude feed intake I weight gain.

Feed intake, Fl (%BW/day): (crude feed intake I (IBW+FBW) / 2 / days) x 100. Protein efficiency ratio, PER: wet weight gain I crude protein intake.

4. Statistical analysis

Data are presented as mean of three replicates ± standard deviation. Data were subjected to a one-way analysis of variance. Prior to ANOVA, values expressed as % were subjected to arcsine square root transformation. Statistical significance was tested at 0.05 probability level. All statistical tests were performed using the IBM SPSS V21 software.

5. Results

Data on growth performance, feed conversion and protein efficiency ratio of seabream fed for 112 days with the various experimental diets is reported in Table 5 below. Table 5

Values are means ± standard deviation (n=3)

Different superscripts within a row denote a statistical difference (P<0.05)

After 112 days of experimental feeding, the overall growth performance of fish fed the PC diet (mimicking a commercial diet with fish oil) was considered as satisfactory and within the normal range for gilthead seabream reared at a water temperature profile fluctuating between 11.3 and 19.6°C (average 16.2 ± 1.9°C). During the first 15 days of the trial, the average water temperature was 13°C (from 11.3 to 14.9 °C), which strongly conditioned feed intake and growth of fish.

In the best performing treatment (PC), fish showed a 1.9-fold increase of their initial body weight. Feed conversion ratio (FCR) among the treatments varied between 1.24 and 1.43, suggesting overall adequate feeding practices.

Conclusions from the experimental data were:

- In comparison to the positive control (PC) with 100% fish oil, the negative control (NC) with 45% of fish oil replaced by mammal’s fat led to a significant reduction of FBW, SGR and PER and an increase of FCR. However, this negative effect of mammal’s fat could not be directly associated to a lower feed intake.

- Supplementation of the NC diet with additive A at 1.5 g/kg (A1.5 diet) showed a final body weight similar to PC. Moreover, when expressed as specific growth rate, we observe that additives supplementation in A1.0, A1.5 and B1.0 diets resulted on a growth rate similar to that found in the PC treatment.

- In comparison to the NC treatment, additive supplementation adopted in A0.5, A1.0, A1.5 and B1.0 diets led to a reduction of FCR, which was similar to that found in the PC treatment. Inversely, additive supplementation adopted in A0.5, A1.0, A1.5 and B1.0 diets led to an increase of protein efficiency ratio (PER) to levels similar to that found in the PC treatment and higher than that observed in the NC treatment.

EXAMPLE 2

The effect of supplemental feed additives A and B above on the lipid and fatty acid analysis of fillet samples of gilthead seabream at the end of the growth trial, i.e., after 112 days (see Example 1, section 1.3. Feeding and samplings) was assessed.

The skin was removed from the two whole fillets taken per fish (from 3 fish per triplicate tank, n=9 per treatment) and then fillets from each individual fish were minced together and divided into two sub-samples: one was used to determine the moisture content (dry weight) and the second was used fresh for lipid analysis. Total lipids were extracted from samples by homogenisation in chloroform/methanol (2:1 , v/v) containing 0.01 % butylated hydroxytoluene (BHT) and quantified gravimetrically after evaporation of the solvent under a stream of nitrogen, followed by vacuum desiccation overnight. Total lipids were stored in chloroform/methanol (2:1 , 10 mg ml’ ¹ ) at -20°C until final analysis.

Fatty acid methyl esters (FAME) were prepared from total lipid by acid-catalysed transesterification using 2 ml of 1 % H2SO4 in methanol plus 1 ml toluene, and FAME were then extracted and purified. FAME were separated and quantified by gas-liquid chromatography (Fisons GC8600, Carlo Erba, Milan, Italy) using a 30 m x 0.32 mm capillary column (CP wax 52CB; Chrompak, London, U.K) utilizing on-column injection at 50°C and flame ionization detection at 250°C. Hydrogen was used as carrier gas (2.0 ml min ^-1 constant flow rate), injection was on-column and temperature programming was from 50°C to 180°C at 40°C min ^-1 and then to 225°C at 2°C min ^-1 . Individual fatty acids were identified by comparison with well characterised fish oil and well known standards (Supelco) and quantified by means of the response factor to the internal standard, 21 :0 fatty acid, added prior to transmethylation.

The results are shown in Table 6 below.

Table 6. Fillet lipid and fatty acid (FA) composition

As can be seen from Table 6, the partial substitution of fish oil by mammalian fat caused a significant increase in fat deposition in fish fillet (mg of lipids per g dry weight of fillet), which was countered by feed additives A and B (see also Fig. 1).

The fish’s fillet fatty acid profile was significantly affected by the tested feed additives A (diets A1.0 and A1.5) and B in a manner that does not reflect the diet’s fatty acid composition (Table 6). Some unexpected results were observed in SFA levels (little affected by either fish feed or additive supplementation), but the main effects concerned the n-3 and n-6 PLIFA profile.

Both additives clearly modified the PLIFA profile of lipids in fish fillet when added to the negative control (NC) feed. Overall, the additives enabled offsetting the effects of mammal’s fat inclusion and approximating the fillet lipid profile to that of fish fed the positive control (PC) feed containing 100% fish oil. In particular, partial replacement of fish oil by mammal’s fat in the NC feed of the present invention caused a significant increase in the levels of C18 PLIFA, both LA and ALA, in total lipids and a significant decrease in LC-PUFA, including ARA, EPA, DPA and DHA, in total lipids compared to the PC treatment. However, the supplementation of the NC with feed additives A and B partially reverted these effects by decreasing the levels of LA and ALA in total lipids, and increasing the levels of ARA, EPA, DPA and DHA in total lipids of the fillet of fish fed these diets. Furthermore, a clear dose-response was observed for feed additive A, with more marked effects at the higher dose. The results are shown in Table 6 (see also Fig. 2).

Also noteworthy, feed additives A and B affected the balance between n-3 and n-6 in the lipids of the fish fillet a positive way, from both the fish’s and consumer’s health point of view. Concretely, fillet lipids of fish fed the additive-supplemented feeds had enhanced levels of total n-3 PUFA and lowered total n-6 PUFA, and therefore a higher n-3/n-6 PUFA ratio, compared to fillets of fish fed the NC feed. As observed with the individual PUFA, the supplementation of a fish-oil reduced fish feed with additives A and B allowed bringing the balance between n-3 and n-6 PUFA in the fish fillet lipids closer to that found in the PC treatment (see Table 6, Fig. 3).

EXAMPLE 3

A similar assay was carried out but with the following additives:

Table 2 wt.% are with respect to the total amount of additive. *Mean values appearing in the raw material specifications.

The results were similar to the ones obtained in Examples 1 and 2.

For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

Clause 1. A combination comprising a capsaicinoid, piperine, and gingerol, and optionally a compound selected from cinnamaldehyde, a curcuminoid, and a mixture thereof.

Clause 2. The combination according to clause 1 , wherein the capsaicinoid is selected from the group consisting of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin, nonivamide, and mixtures thereof, and wherein the curcuminoid is selected from the group consisting of curcumin, demethoxycurcumin and bisdemethoxycurcumin, and mixtures thereof.

Clause 3. The combination according to clauses 1 or 2, wherein the capsaicinoid is capsaicin.

Clause 4. An animal feed additive for aquatic animals comprising the combination as defined in any one of clauses 1 to 3 and at least one feed acceptable excipient.

Clause 5. The animal feed additive for aquatic animals according to clause 4, comprising:

- from 0.1 wt.% to 1.0 wt.% of a capsaicinoid,

- from 0.025 wt.% to 0.25 wt.% of piperine,

- from 0.002 wt.% to 0.05 wt.% of gingerol,

- from 0 wt.% to 3.0 wt.% of cinnamaldehyde, and

- from 0 wt.% to 3.0 wt.% of a curcuminoid, wherein weight percentages are relative to the total weight of animal feed additive.

Clause 6. The animal feed additive for aquatic animals according to clause 5 comprising a capsaicinoid, piperine, gingerol, and cinnamaldehyde, wherein the amount of capsaicinoid is from 0.25 wt.% to 1.0 wt.%, the amount of piperine is from 0.025 wt.% to 0.25 wt.%, the amount of gingerol is from 0.002 wt.% to 0.04 wt.%, and the amount of cinnamaldehyde is from 0.5 wt.% to 3.0 wt.%, relative to the total weight of animal feed additive.

Clause 7. The animal feed additive for aquatic animals according to clause 5 comprising a capsaicinoid, piperine, gingerol, and a curcuminoid, wherein the amount of capsaicinoid is from 0.1 wt.% to 0.5 wt.%, the amount of piperine is from 0.04 wt.% to 0.2 wt.%, the amount of gingerol is from 0.01 wt.% to 0.05 wt.%, and the amount of curcuminoid is from 0.5 wt.% to 3.0 wt.%, relative to the total weight of animal feed additive.

Clause 8. The animal feed additive for aquatic animals according to any one of clauses 4 to 7, which is in a fat coated encapsulation form.

Clause 9. An aquaculture feed comprising a) an animal feed composition for aquatic animals comprising fish oil and b) an effective amount of the animal feed additive for aquatic animals as defined in clauses 4 to 8.

Clause 10. The aquaculture feed according to clause 9, wherein the amount of the animal feed additive for aquatic animal is from 0.025 wt.% to 0.30 wt.% such as of 0.15 wt.% relative to the total weight of aquaculture feed.

Clause 11. The aquaculture feed according to clauses 9 or 10, comprising an amount of added oils from 3 wt.% to 30 wt.%, wherein the added oils comprise fish oil, and animal fats, vegetable oils, or both of them.

Clause 12. The aquaculture feed according to clause 11 , wherein up to a 90 wt.%, such as from 5 wt.% to 90 wt. %, or from 15 wt.% to 75 wt.%, or from 25 wt.% to 60 wt.%, of the total amount of fish oil has been replaced by the same weight amount of terrestrial animal fats, vegetable oils, or both of them.

Clause 13. The animal feed additive for aquatic animals as defined in clauses 4 to 8 or the aquaculture feed of clauses 9 to 12, wherein the aquatic animals are fish, particularly marine fish.

Clause 14. Use of the combination as defined in clauses 1 to 3, the animal feed additive for aquatic animals as defined in clauses 4 to 8 or the aquaculture feed as defined in clauses 9 to 12 for increasing the amount of omega 3 long chain polyunsaturated fatty acids in total lipids of aquatic animals, increasing the ratio of omega 3/omega 6 polyunsaturated fatty acids in lipids of aquatic animals, or both of them.

Clause 15. The use according to clause 12, wherein the aquatic animals are fish, particularly marine fish.