Combination of nucleobases and brown algae for improving immunity

Abstract

The present invention relates to pharmaceutical and food compositions comprising nucleobases and / or sources of nucleobases and a mixture of brown algae, preferably, marine brown algae. The compositions preferably include Nucleoforce® and Algaemix®. Said compositions can be used in the treatment and / or prevention of bacterial infection as well as for improving immune function.

Description

[0001] Combined preparation for use in enhancing immunity

[0002] The present invention relates to pharmaceutical and food compositions comprising nucleobases and / or sources of nucleobases and a mixture of brown algae. Preferably, the compositions include Nucleoforce® and Algaemix®. Such compositions are useful in the treatment and / or prevention of a bacterial infection; they are also useful in the stimulation of immune function. Therefore, the present invention falls within the fields of biotechnology, pharmacy, and food, more particularly within compositions intended for the prevention and / or treatment of a bacterial infection and the enhancement of a subject's immunity.

[0003] STATE OF THE ART

[0004] The whiteleg shrimp, Litopenaeus vannamei, is one of the world's largest farmed tropical shrimp species, native to the Pacific Ocean. The shrimp is characterized by rapid growth, high tolerance to a wide range of water salinities (5–40 ppt), high intensification, low protein requirements, and other traits suitable for super-intensive aquaculture. However, environmental factors such as water temperature, salinity fluctuations, sharp and rapid changes in pH, low dissolved oxygen, and toxins such as ammonia, nitrite, hydrogen sulfide, and heavy metals affect shrimp growth and survival rates.

[0005] The global progress of shrimp farming is facing increasing environmental and pathological challenges, in addition to a low capacity to tolerate cold water in most shrimp-producing countries. The use of natural immunostimulants is recommended as a healthier and safer therapy than antibiotics and / or vaccines to control aquaculture pathogens. Increasing shrimp immunity is an important direction in current support policies to prevent shrimp diseases.

[0006] Nucleotides are low-molecular-weight chemical compounds that play a role in virtually all biochemical processes, including energy production (e.g., ATP), metabolism, and cell signaling (e.g., cAMP), providing the natural building block of DNA and RNA, and essential cofactors in the biosynthesis of proteins and enzymes (e.g., NAD+). They are naturally present in foods, albeit in limited quantities (<400 mg / 100 g). Tissues with high cell proliferation rates, such as the immune system or the intestine, are unable to meet their needs using de novo nucleotide synthesis alone, which is preferentially used to facilitate the salvage pathway for dietary nucleotides.

[0007] It has been reported that, in situations of immunological stress, dietary supplementation with nucleotide formulations is essential to maintain the humoral and cellular immune response (Gil., European Journal of Clinical Nutrition, 56 Suppl 3, S1 - S4. 2002). In particular, it has been shown that supplementation with exogenous nucleotides stimulates lymphoid cell proliferation and the lymphoproliferative response to alloantigens and mitogens. Furthermore, such supplementation contributes to the T cell response, increases the delayed cutaneous hypersensitivity response, increases graft rejection, reverses immunosuppression associated with malnutrition, increases resistance to certain infections, regulates the number of natural killer (NK) cells and macrophages and promotes immunoglobulin synthesis (Maldonado et al., Early Human Development, 65 Suppl., S69 - S74. 2001).

[0008] On the other hand, some studies have shown that dietary supplementation with certain polysaccharides from fungal, yeast, or bacterial extracts (especially α-glucans and β-glucans) has a positive effect on the immune response. β-glucans are a heterogeneous group of glucose polymers that have been studied in recent years for their biological effects in mammals. β-glucans have been described as activators of the immune system and modifiers of cellular response (Akramiene et al., Medicina (Kaunas), 43(8): 597–606, 2007).

[0009] In view of the above, there is a need in the state of the art for new approaches and strategies to improve various immunological indices of shrimp and the management of bacterial infections. DESCRIPTION OF THE INVENTION

[0010] By conducting tests aimed at improving the breeding performance of crustaceans, the inventors have observed that the combined administration of a nucleobase (or a source of nucleobases) and a mixture of brown algae, preferably marine brown algae, as a dietary supplement, improves the immunity of the subjects, favoring both the prevention and treatment of a bacterial infection.

[0011] In the examples of the present description, it is demonstrated, through the use of in vivo models, that the combined preparation defined in the claims synergistically improves the growth performance at the productive level (Example 1) and the capacity for tolerance to a bacterial infection (Example 2).

[0012] Thus, in a first aspect, the present invention relates to a combined preparation, hereinafter “combined preparation of the invention”, comprising: a) A first composition comprising a nucleobase and / or a source of nucleobases selected from the group consisting of nucleosides, nucleotides, RNA, DNA, and combinations thereof, and b) A second composition comprising a mixture of brown algae, wherein the concentration of the first composition relative to the second composition is between 100 and 600 ppm.

[0013] The term “combined preparation” refers to a preparation comprising more than one composition, in this case the first composition defined a) and the second composition defined b). In the combined preparation of the invention, both compositions do not need to be present in a union in order to be available, but can be separated so that their application can be separate, either simultaneously, sequentially, or mixed in a single solution. In this way, it does not necessarily result in a true combination, given the possible physical separation of both compositions. Preferably, the first and second compositions within the combined preparation are administered simultaneously. The combined preparation of the invention comprises a first composition a) comprising a nucleobase and / or a source of nucleobases selected from the group consisting of nucleosides, nucleotides, RNA and DNA.

[0014] As used herein, nucleobases are cyclic organic compounds that include two or more nitrogen atoms. They are a fundamental component of nucleosides, nucleotides, and nucleic acids. The most preferred nucleobases, which are part of the first composition of the combined preparation of the invention, are the purine bases (adenine and guanine) and pyrimidine bases (cytosine, thymine, and uracil).

[0015] The term “nucleobase source” refers to nucleosides, nucleotides, RNA (ribonucleic acid), DNA (deoxyribonucleic acid), or equivalent.

[0016] Examples of nucleosides include, but are not limited to, ribose nucleosides, such as adenosine, guanosine, uridine, and cytidine; and deoxyribose nucleosides, such as deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxyuridine.

[0017] Examples of nucleotides include, but are not limited to, phosphate esters of mononucleosides, such as adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), deoxythymidine monophosphate (dTMP), and deoxycytidine monophosphate (dCMP); and di- and trinucleoside phosphates, such as uridine diphosphate (UDP) and uridine triphosphate (UTP).

[0018] The source of nucleobases is preferably yeast, meat or similar products.

[0019] As understood by those skilled in the art, combinations of nucleobase sources are also contemplated within the first composition of the combined preparation of the invention. Thus, the first composition may simultaneously comprise two or more of said sources, such as nucleosides, nucleotides, RNA, and DNA. In another preferred embodiment of the combined preparation of the invention, alone or in combination with each and every one of the previous preferred embodiments, the first composition comprises uridine monophosphate, adenosine monophosphate, cytidine monophosphate, and disodium guanine-5-monophosphate.In another preferred embodiment of the combined preparation of the invention, alone or in combination with each and every one of the previous preferred embodiments, the first composition comprises between 8 and 9% uridine monophosphate, between 7 and 8% adenosine monophosphate, between 4 and 5% cytidine monophosphate and between 8 and 9% disodium guanine-5'-monophosphate (% by weight with respect to the total weight of the first composition). In all the intervals mentioned in the present description the extremes are included.

[0020] In another preferred embodiment of the combined preparation of the invention, alone or in combination with each and every one of the previous preferred embodiments, the first composition is Nucleoforce®. The Nucleoforce® composition is a solution whose main components are free nucleotides, such as, but not limited to, 8.3% uridine monophosphate, 7.45% adenosine monophosphate, 4.83% cytidine monophosphate and 8.27% disodium guanine-5'-monophosphate (% by weight with respect to the total weight of the first composition). Likewise, Nucleoforce® may also comprise active precursors. Nucleoforce® can be obtained from the extraction, purification and hydrolysis of RNA from Saccharomyces cerevisiae. This composition is marketed by Bioiberica SAU (www.bioibehca.com) and can be purchased by those skilled in the art through the usual sales channels.

[0021] The combined preparation of the invention comprises a second composition b) comprising a mixture of brown algae, wherein the concentration of the first composition relative to the second composition is between 100 and 600 ppm.

[0022] In the present invention, "brown algae" refers to the photosynthetic organism eucalyptus, which belongs to the Protista kingdom (class Phaeophyceae). Its characteristic color is due to the presence of the carotenoid fucoxanthin in the chloroplasts. A large amount of alginic acid is produced in the cell wall of Phaeophyceae, accounting for up to 70% of the alga's weight. Brown algae are almost exclusively marine. Only a few species from eight genera grow in freshwater bodies.

[0023] Any brown algae may form part of composition b) of the combined preparation of the invention. Examples of brown algae include, but are not limited to, species of the genera Macrocystis and Ascophyllum, such as Macrocystis pyrifera, Macrocystis integrifolia, Ascophyllum nodosum, etc. In a preferred embodiment of the combined preparation of the invention, the brown algae of the second composition is a marine brown algae, which, in another even more preferred embodiment, the marine brown algae is Macrocystis integrifolia or Macrocystis pyrifera. Likewise, in a preferred embodiment of the combined preparation of the invention, the second composition may comprise one, two or more brown algae, more preferably, two or more marine brown algae, even more preferably, the at least two marine brown algae are Macrocystis integrifolia and Macrocystis pyrifera.Preferably, the algae make up at least 70%, 75%, 80%, 85% or 90% by weight of the total of the second composition.

[0024] The second composition of the combined preparation of the invention may comprise other components, such as carriers, diluents, or other compounds provided by the algae, such as nutrients, or one or more bioactive compounds. In the present invention, "bioactive compound" is understood to be a compound that influences the cellular and physiological activities of a subject, thus exerting a beneficial effect on its growth and health. From a chemical perspective, these compounds are of diverse origin and act through different mechanisms of action. Thus, there are polyphenol carotenoids, terpenes, lignans, organosulfur compounds, glucosilonates, saponins, marine polysaccharides, etc. However, in a preferred embodiment of the combined preparation of the invention, the second composition also comprises one, two, or three bioactive compounds selected from the group consisting of fucoidans, alginic acid, and p-glucans.In another even more preferred embodiment, the second composition b) comprises fucoidans and alginic acid; or fucoidans and p-glucans; or alginic acid and p-glucans. In another even more preferred embodiment, the second composition b) comprises fucoidans, alginic acid, and p-glucans.

[0025] In another preferred embodiment of the combined preparation of the invention, the second composition is Algaemix®. The Algaemix® composition is commercially available and can be purchased by those skilled in the art through the usual sales channels. The Algaemix® composition is a nutritional solution whose main component is a mixture of brown algae, preferably marine brown algae (mostly brown algae of the genus Macrocystis) that ensures an adequate content of bioactive components, prebiotics, polysulfated polysaccharides and other natural growth promoters (such as, but not limited to, protein, fiber, ash, fats, moisture, carbohydrates, calcium, iron, zinc, boron, magnesium, fucoidans, alginic acid, and p-glucans).

[0026] The components of the first and second compositions of the combined preparation of the invention may be present in any quantity / concentration, and said quantities may vary depending on the nutritional needs of the subject for whom the composition of the invention is intended. Those skilled in the art know how to adapt the quantities of the different components of the first and second compositions of the invention based on said nutritional needs.

[0027] In another preferred embodiment of the combined preparation of the invention, the first composition is the commercial product Nucleoforce® and the second composition is the commercial product Algaemix®.

[0028] The inventors of the present invention have analyzed different concentrations of the first and second compositions within the combined preparation until obtaining the optimal concentrations of each of them to exert a greater effect (greater than the effect that would be obtained by adding the effects of each of the two compositions used separately). Thus, in the combined preparation of the invention, the concentration of the first composition with respect to the second composition is between 100 and 600 ppm (extremes included). In the present invention, ppm or part per million (10 -6 ) to the number of units of a given substance (agent, etc.) that there are per million units of the set.

[0029] The first composition of the combined preparation of the invention may be at any concentration within the indicated range. However, in a preferred embodiment, the concentration of the first composition relative to the second is between 200 and 500 ppm (extremes included), which, in another even more preferred embodiment, is between 300 and 400 ppm (extremes included), which, in another even more preferred embodiment, is 334 ppm.

[0030] As understood by those skilled in the art, the combined preparation of the invention can be formulated in various ways depending on how it is to be administered. Therefore, the combined preparation of the invention can be in liquid form (solutions, emulsions, suspensions, syrups, etc.), solid form (capsules, tablets, powders, etc.), or semi-solid form (gels).

[0031] The combined preparation of the invention may form part of a food product or a pharmaceutical composition.

[0032] Therefore, another aspect of the present invention relates to a food product, hereinafter "food product of the invention," comprising the combined preparation of the invention. In the context of the present invention, the terms "food product" and "nutritional composition" are considered equivalent and may be used interchangeably throughout this description.

[0033] The food product of the invention may be for animal use / consumption, including a mammal and, therefore, humans, and refers to any product whose intake beneficially affects the subject, positively promoting its growth and improving the state of immunological health in the sense indicated in the present invention.

[0034] In a preferred embodiment of the food product of the invention, said food product is a food or a supplement (dietary or nutritional), an ingredient of a functional food, a medicinal food or a nutraceutical.

[0035] Examples of foods include, but are not limited to, vegetable products, meat products, snacks, beverages, cereals, bakery products, biscuits, dairy products, milks, fermented milks (such as yogurt or cheese), ice creams, butters, margarines, etc. In a preferred embodiment of the food product of the invention, the food is a feed, a beverage (e.g., smoothies, juices, sports nutrition drinks, etc.), a dairy product (e.g., milk, yogurt, cheese, kefir, etc.), a pastry product, a protein bar, cereals, a cereal bar, a gel, or a soup.

[0036] The term "supplement" refers, in the context of the present invention, to products or preparations intended to supplement or complement a subject's normal diet by providing nutrients or other substances with a physiological and / or nutritional effect. Examples of supplements include, but are not limited to, a dietary supplement, a nutritional supplement, a food supplement, etc.

[0037] The term "nutraceutical", in the context of the present invention, refers to products of natural origin with active biological properties, beneficial for health and with defined preventive and / or therapeutic capacity. Examples of nutraceuticals include, but are not limited to, folic acid, sterols, soluble fiber, phenolic compounds (flavonoids, anthocyanins, isoflavones, carotenoids, etc.), OMEGA-3 fatty acids (ALA (alpha-linolenic acid), EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid)).

[0038] By definition, "functional foods" refer to natural foods or foods to which a component has been added, or foods from which a component has been removed through technological or biological means; therefore, they are foods that, in addition to their nutritional content, contain ingredients that perform a specific activity in the physiological functions of the human body, promoting physical capacity and mental state. Examples of functional foods include, but are not limited to, foods that contain certain minerals, quantities of vitamins, fatty acids, fiber, and probiotics containing cultures of beneficial live microorganisms. They are usually presented in the form of foods for daily use enriched with certain nutrients or substances beneficial to health.

[0039] The main difference between a nutraceutical and a functional food is that a nutraceutical is composed of the biological part of a functional food, for example, one that reduces cholesterol (i.e., phytosterol).

[0040] The combined preparation of the invention may be part of a pharmaceutical composition. Thus, in another aspect, the present invention relates to a pharmaceutical composition, hereinafter "pharmaceutical composition of the invention," comprising the combined preparation of the invention.

[0041] The pharmaceutical composition of the invention can be administered to an animal, including a mammal and, therefore, to a human being, and refers to any composition whose administration beneficially affects the subject, positively promoting its growth and improving the state of immunological health in the sense indicated in the present invention.

[0042] In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0043] The term "excipient" refers to a substance that aids in the absorption of any of the components of the composition, i.e., the compositions comprised in the combined preparation of the invention, or stabilizes said components and / or aids in the preparation of the pharmaceutical composition by giving it consistency or by providing flavors that make it more pleasant. Thus, excipients could have the function of keeping the components together (e.g., starches, sugars, or cellulose), of sweetening, of providing a coloring, of protecting the active ingredient (e.g., to isolate it from air and / or moisture), of filling a tablet, capsule, or any other form of presentation, of disintegrating to facilitate the dissolution of the components, etc., without excluding other types of excipients not mentioned in this paragraph.Therefore, the term "excipient" is defined as that substance which, included in the galenic forms, is added to the active ingredients or their combinations to enable their preparation and stability, modify their organoleptic properties or determine the physical-chemical properties of the pharmaceutical composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of the compounds of the pharmaceutical composition, that is, it must be compatible with the compositions included in the combined preparation of the invention.

[0044] The "galenic form" or "pharmaceutical form" is the arrangement in which the active ingredients and excipients are arranged to constitute a composition or a drug. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered.

[0045] The "vehicle" or "carrier" is preferably an inert substance, and like the excipient, its purpose is to facilitate the incorporation of other compounds, allow for improved dosage and administration, and / or provide consistency and shape to the pharmaceutical composition. Therefore, the vehicle is a substance used in the drug to dilute any of the components of the pharmaceutical composition of the present invention to a given volume or weight; or, even without diluting said components, is capable of allowing for improved dosage and administration and / or providing consistency and shape to the drug. When the presentation form is liquid, the pharmaceutically acceptable vehicle is the diluent.

[0046] Furthermore, the excipient and vehicle must be pharmacologically acceptable, meaning that the excipient and vehicle must be permitted and evaluated so as not to cause harm to the subject to whom they are administered. In each case, the form of presentation of the pharmaceutical composition will be adapted to the type of administration used. Therefore, the composition can be presented in the form of solutions or any other clinically permitted form of administration and in a therapeutically effective amount. The pharmaceutical composition of the invention can be formulated in solid (tablets, powders, microgranules (pellets), tablets, pills, capsules, granules, patches, etc.), semi-solid (gels), or liquid (solutions, suspensions, emulsions, syrups, drops, drinkable vials, etc.) forms. In a preferred embodiment of the pharmaceutical composition of the invention, said composition is formulated for oral administration.The form adapted for oral administration refers to a physical state that can allow oral administration.

[0047] As the examples below show, the combined preparation of the invention is useful for increasing the growth performance of the subjects. Furthermore, in a simulated in vivo bacterial infection situation (cultivation under laboratory conditions), an unexpected increase in the survival rate of the subjects occurs, which represents a positive impact on the subjects' tolerance capacity in an infectious situation, demonstrating a synergistic effect associated with the combined use of the compositions comprised in the combined preparation of the invention. In summary, the combined preparation of the invention has a positive and synergistic effect on bacterial growth and infections.

[0048] Thus, in another aspect, the invention relates to the combined preparation, food product, or pharmaceutical composition of the invention for use as a medicament, or alternatively, to the use of the combined preparation, food product, or pharmaceutical composition of the invention for the manufacture of a medicament. The term "medicament" refers to a substance that is used for the prevention, alleviation, treatment, or cure of diseases, syndromes, or clinical conditions in animals, including humans. In the context of the present invention, it refers to the combined preparation, food product, or pharmaceutical composition in a therapeutically effective amount.

[0049] As used in this description, the term "therapeutically effective amount" refers to the amount of the combined preparation, food product, or pharmaceutical composition of the invention that produces the desired effect. The dosage to obtain a therapeutically effective amount depends on a variety of factors, for example, the age, weight, sex, or tolerance of the subject.

[0050] The medicament of the invention can be used either alone or in combination with other medicaments or compositions to promote or enhance immunity in a subject, and to treat and / or prevent a bacterial infection in a subject. The medicament of the present invention can be used together with other active ingredients or therapies as a combination therapy. The other active ingredients can be part of the same composition or can be provided by a separate composition, being administered simultaneously or sequentially.

[0051] In another aspect, the invention relates to the combined preparation, food product, or pharmaceutical composition of the invention, for use in enhancing immunity in a subject, or alternatively, to the use of the combined preparation, food product, or pharmaceutical composition of the invention for the manufacture of a medicament for enhancing immunity in a subject. Similarly, the present invention relates to a method for enhancing immunity in a subject, comprising administering the combined preparation, food product, or pharmaceutical composition of the invention to said subject.

[0052] In the context of the present invention, the expression “enhancing the immunity of a subject” refers to promoting, increasing, stimulating, increasing or inducing the immune system in a subject, that is, promoting, increasing, stimulating, increasing or inducing the set of biological elements and processes inside the subject (lymphocytes, leukocytes, cytokines, etc.) that allow it to maintain homeostasis or internal balance against external aggressions, whether of a biological nature (pathogenic agents) or physical-chemical (such as pollutants or radiation) and internal (for example, cancer cells).

[0053] In another aspect, the invention relates to the combined preparation, food product, or pharmaceutical composition of the invention, for use in the treatment and / or prevention of a bacterial infection in a subject, or alternatively, the combined preparation, food product, or pharmaceutical composition of the invention for the manufacture of a medicament for the treatment and / or prevention of a bacterial infection in a subject. Similarly, the present invention relates to a method for the treatment and / or prevention of a bacterial infection in a subject, comprising administering the combined preparation, food product, or pharmaceutical composition of the invention to said subject.

[0054] The term “treatment” or “treating” as understood in the present invention refers to combating the effects caused as a consequence of the disease or pathological condition of interest in a subject, which includes:

[0055] (i) inhibit the disease or pathological condition, that is, stop its development;

[0056] (i) alleviate the disease or pathological condition, that is, cause the regression of the disease or pathological condition or its symptomatology;

[0057] (iii) stabilize the disease or pathological condition.

[0058] The term “prevention” or “preventing” an infection as understood in the present invention, consists of avoiding the onset of the disease or clinical condition, that is, preventing the disease or pathological condition from occurring in a subject, in particular, when said subject has a predisposition to the pathological condition, but has not yet been diagnosed as having it.

[0059] In a preferred embodiment of the invention, the bacterial infection is caused by Vibrio parahaemolyticus (also known as V. parahaemolyticus). The genus Vibrio, belonging to the family Vibrionaceae, has 48 species, of which at least 12 are recognized as human pathogens. Of these, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the most important pathogenic vibrios, both in terms of their disease-causing capacity and the magnitude of the overall disease burden. V. parahaemolyticus is a gram-negative, slightly curved, facultative aerobic, halophilic, oxidase-positive, glucose- but not sucrose-containing rod, and variable urease-containing rod. It requires selective media for its growth. This organism is frequently isolated from a variety of raw seafood products, particularly shellfish.

[0060] The term “subject” refers to a human or a non-human animal, preferably a mammal or an arthropod. The subject to which the present invention relates may be, for example, but not limited to, a ruminant such as a sheep, goat or cattle, dromedary, camel, llama, kangaroo, pig; poultry such as a turkey, duck, quail, goose, pigeon, chicken, hen or rooster, horse; companion animals such as a dog or cat; as well as arthropods such as crustaceans or myriapods.

[0061] Therefore, in a preferred embodiment of the combined preparation, food product, or pharmaceutical composition for therapeutic use according to the present invention, the subject is a mammal or an arthropod. In a more preferred embodiment, the mammal is a primate, preferably a human. In an even more preferred embodiment, the arthropod is a crustacean, preferably a carid, more preferably a shrimp, a prawn, a shrimp, or a squid.

[0062] In the present invention, "arthropods" are understood to be invertebrates equipped with an external skeleton (exoskeleton), a body divided into segments, and articulated appendages, among other characteristics. Arthropods constitute the most numerous and diverse phylum within the 29 phyla that make up the Animal Kingdom. Depending on the number of legs, arthropods are divided into: insects (three pairs of legs), arachnids (four pairs of legs), crustaceans (five pairs of legs), and myriapods (with more than 10 legs).

[0063] DESCRIPTION OF THE FIGURES

[0064] Figure 1. Food intake (g) after 8 weeks in each of the production trial groups. Different superscript letters indicate statistically significant differences (p<0.05). NF: Nucleoforce®; AL: Algaemix®.

[0065] Figure 2. Evolution of the survival rate (%) in the different study groups over time. Different superscript letters indicate statistically significant differences (p<0.05). NF: Nucleoforce®; AL: Algaemix®; PC: Positive Control; NC: Negative Control.

[0066] EXAMPLES

[0067] The invention will then be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

[0068] Combined preparation

[0069] The components of the combined preparation of the invention used in the examples of the present patent application are shown in Tables 1 and 2.

[0070] Algae comprise 80% by weight of the total composition.

[0071] Example 1. Study of the effect of the combined preparation of the invention on the productivity of whiteleg shrimp (Litopenaeus vannameí) under recirculating aquaculture system (RAS) conditions.

[0072] A total of 1500 specific pathogen-free whiteleg shrimp (Litopenaeus vannamei) from a shrimp farm in Vietnam were acclimatized for 2 days and then sorted into 6 treatment groups (250 shrimp per group; 5 replicates (400L tanks) per treatment with 50 shrimp / tank) (see Table 3).

[0073] During the 8 weeks of the trial, several common production parameters were evaluated:

[0074] - Variations in weight and biomass

[0075] - Food intake

[0076] - feed conversion ratio

[0077] Tables 4 and 5 show the feed intake results after 8 weeks of treatment in the different groups. Similarly, Figure 1 shows how the NF500 and AL500 groups significantly improved (p<0.05) their feed intake compared to the Control group. On the other hand, while the 1:1 combination (NF500AL500 group) did not achieve a significant increase (p>0.05) compared to the Control group, the 1:2 combination (NF334AL666 group) did achieve a statistically significant increase (p<0.05) compared to the Control group. Furthermore, the NF334AL666 group also showed a significantly higher feed intake (p<0.05) than the NF500 and AL500 groups.This demonstrates a synergy at the production level between the components Nucleoforce® and Algaemix® when combined in a 1:2 ratio (NF334AL666 group), as well as the superiority of this combination in a 1:2 ratio (NF334AL666 group) compared to the 1:1 ratio (NF500AL500 group). Example 2. Study of the effect of the combined preparation of the invention on the survival of whiteleg shrimp (Litopenaeus vannamei) when challenged with Vibrio parahaemolyticus under laboratory conditions. A total of 840 whiteleg shrimp from a shrimp farm in Vietnam were used to evaluate the subjects' tolerance to bacterial infection by V. parahaemolyticus.

[0078] Once at the facility, shrimp were acclimatized connected to an open flow system for 2 days and subsequently sorted into 7 treatment groups (120 shrimp per group; 4 replicates (120L tanks) per treatment with 30 shrimp / tank) (see Table 6). Water temperature was maintained between 24°C and 30°C. Each tank was equipped with an aeration system with two airstones to maintain oxygen levels above 6 ppm during the study period, a biofilter bucket, and a plastic lid to reduce the risk of cross-contamination. The light regime (photoperiod) was 12:12 h (Light:Dark). The maximum stocking density was 200 shrimp / m3.

[0079] The feeding regimen was small and frequent; once every 4 hours for a 24-hour period (i.e., 6 times a day). The shrimp were fed until satiated.

[0080] The bacterial challenge was carried out as follows: a V. parahaemolyticus isolate was collected from shrimp affected by acute hepatopancreatic necrosis (AHPN) at a shrimp farm in Vietnam. The shrimp were challenged by immersion. Bacterial suspensions were added to the tanks to achieve the desired density, which was measured by optical density absorbance (OD600 nm), to achieve a lethal dose of 90% mortality within 10 days of challenge. Shrimp were followed for 10 days after challenge to quantify and compare the survival rate between groups.

[0081] Figure 2 shows the results of the evolution of the survival rate throughout the trial in the different groups. The Positive Control group (with challenge and diet without supplementation) ended 240 hours post-challenge with a statistically lower survival rate (p < 0.05) than the Negative control (without challenge and diet without supplementation), which validates the model used and the bacterial challenge that was carried out. On the other hand, although the NF500, AL500, NF1000 and NF500AL500 groups showed higher survival rates 240 hours post-challenge than the Positive Control group, this superiority did not reach the level of statistical significance in any of these cases (p > 0.05), as can be seen in the superscript letters.In contrast, the NF334AL666 combination achieved a much higher survival rate than the Positive Control group, and than the NF500 and AL500 groups separately, and also achieved a statistically significant difference (p<0.05), both compared to the Positive Control group and to the NF500, AL500, NF1000 and NF500AL500 groups. Thus, a synergy against a bacterial challenge between the Nucleoforce® and Algaemix® components is demonstrated when combined in a 1:2 ratio (NF334AL666 group).

Claims

CLAIMS 1. A combined preparation comprising: a) A first composition comprising a nucleobase and / or a source of nucleobases selected from the group consisting of nucleosides, nucleotides, RNA, DNA, and combinations thereof, and b) A second composition comprising a mixture of brown algae, wherein the concentration of the first composition relative to the second composition is between 100 and 600 ppm.

2. Combined preparation according to claim 1, wherein the brown algae of the second composition is a marine brown algae.

3. Combined preparation according to claim 1 or 2, wherein the second composition further comprises one, two or three bioactive compounds selected from the group consisting of fucoidans, alginic acid and p-glucans.

4. Combined preparation according to any one of claims 1 to 3, wherein the concentration of the first composition with respect to the second is between 200 and 500 ppm.

5. Combined preparation according to claim 4, wherein the concentration of the first composition with respect to the second is between 300 and 400 ppm.

6. Combined preparation according to claim 5, wherein the concentration of the first composition with respect to the second is 334 ppm.

7. A food product comprising a combined preparation according to any one of claims 1 to 6.

8. Food product according to claim 7, wherein the food product is a food, a supplement, an ingredient of a functional food, a medicinal food or a nutraceutical.

9. Food product according to claim 8, wherein the food is a feed, a beverage, a dairy product, a confectionery product, a protein bar, cereal, a cereal bar, a gel or a soup.

10. A pharmaceutical composition comprising the combined preparation according to any one of claims 1 to 6.

11. Pharmaceutical composition according to claim 10, further comprising a pharmaceutically acceptable carrier and / or excipient.

12. Pharmaceutical composition according to claim 10 or 11, wherein said composition is formulated for oral administration.

13. Combined preparation according to any one of claims 1 to 6, food product according to any one of claims 7 to 9, or pharmaceutical composition according to any one of claims 10 to 12, for use as a medicament.

14. Combined preparation according to any one of claims 1 to 6, food product according to any one of claims 7 to 9, or pharmaceutical composition according to any one of claims 10 to 12, for use in enhancing immunity in a subject.

15. Combined preparation according to any one of claims 1 to 6, food product according to any one of claims 7 to 9, or pharmaceutical composition according to any one of claims 10 to 12, for use in the treatment and / or prevention of a bacterial infection in a subject.

16. Combined preparation, food product or pharmaceutical composition for use according to claim 15, wherein the bacterial infection is caused by Vibrio parahaemolyticus.

17. Combined preparation, food product or pharmaceutical composition for use according to any one of claims 14 to 16, wherein the subject is a mammal or an arthropod.

18. Combined preparation, food product or pharmaceutical composition for use according to claim 17, wherein the mammal is a primate, preferably a human.

19. Combined preparation, food product or pharmaceutical composition for use according to claim 18, wherein the arthropod is a crustacean, preferably a carid, more preferably a shrimp, a prawn, a shrimp or a squill.

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