Combined preparation of nuclebases and a mixture of triterpenic acid and polyphenols for use thereof in improving immunity

Abstract

The present invention relates to pharmaceutical and food compositions comprising nucleobases and / or sources of nucleobases and a mixture of triterpenic acid and polyphenols. Preferably, the compositions include Nucleoforce® and Aquolive®. Said compositions are useful in the treatment and / or prevention of bacterial infection; as well as being useful in improving immune function.

Description

[0001] COMBINED PREPARATION OF NUCLEOBASES AND A MIXTURE OF TRITERPENIC ACID AND

[0002] POLYPHENOLS FOR USE IN IMMUNE SUPPORT

[0003] The present invention relates to pharmaceutical and food compositions comprising nucleobases and / or sources of nucleobases, and a mixture of nucleobases.

[0004] 5 triterpene and polyphenols. Preferably, the compositions include Nucleoforce® and Aquolive®. Such compositions are useful in the treatment and / or prevention of a bacterial infection; they are also useful in stimulating 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.

[0005] STATE OF THE ART 5 The whiteleg shrimp, Litopenaeus vannamei, is one of the largest farmed tropical shrimp species in the world, 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 fluctuation, 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. 5 In cases of stress, the risk of opportunistic infections, especially those related to the respiratory and digestive systems, increases.A clear example of stress-related infections in early life comes from animal production. These infections are particularly prevalent in animals raised under commercial conditions.

[0006] The global advancement of shrimp farming faces increasing environmental and pathological challenges, in addition to a low capacity to tolerate cold water in most shrimp-producing countries. Therefore, increasing shrimp immunity is an important direction in current support policies5 to prevent the diseases that affect them. There is growing concern about the overuse of antibiotics to combat opportunistic infections due to the emergence of antimicrobial resistance. Thus, the use of natural immunostimulants is recommended as a healthier and safer therapy than the administration of antibiotics and / or vaccines to control pathogens in aquaculture.

[0007] Dietary supplement ingredients have been reported to improve immune function and reduce the risk of infectious diseases. Some studies suggest that supplementation with multiple immune-supporting micronutrients is beneficial, but more research is needed (Gombart et al., Nutrients, 6;12(1):236. 2020).

[0008] Thus, other isolated nutritional compounds exist, and the purported benefits of such compounds include, among numerous other effects, immune support, anti-inflammatory effects, anti-aging effects, cardiac support, and digestive support. Unfortunately, there is only a fairly small body of evidence to support some aspects of these purported benefits when these vitamins and other isolated nutritional compounds are ingested.

[0009] In view of the above, new approaches and strategies are needed for the management of bacterial infections, and in general, for improving a subject's immunity.

[0010] DESCRIPTION OF THE INVENTION

[0011] By conducting trials 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 terpenic acid and polyphenols, as a dietary supplement, improves the immunity of the subjects, favoring both the prevention and treatment of a bacterial infection.

[0012] 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).

[0013] 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 triterpenic acid and polyphenols, wherein the concentration of the first composition with respect to the second composition is between 250 and 750 ppm.

[0014] The term "combined preparation" refers to a preparation comprising more than one composition, in this case the first composition defined as a) and the second composition defined as b). In the combined preparation of the invention, both compositions do not need to be present in a union to be available, but can be separated so that they can be administered separately, 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.

[0015] 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.

[0016] 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 present invention, are the purine bases (adenine and guanine) and pyrimidine bases (cytosine, thymine, and uracil). The term “nucleobase source” refers to nucleosides, nucleotides, RNA (ribonucleic acid), DNA (deoxyribonucleic acid), or equivalent.

[0017] 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.

[0018] 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).

[0019] The source of nucleobases is preferably yeast, meat or similar products.

[0020] 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 a preferred embodiment of the combined preparation of the invention, the first composition comprises uridine monophosphate, adenosine monophosphate, cytidine monophosphate, and disodium guanine-5-monophosphate.

[0021] 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.

[0022] 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.

[0023] Nucleoforce® can be obtained from the extraction, purification, and hydrolysis of Saccharomyces cerevisiae RNA. This composition is marketed by Bioiberica SAU (www.bioibehca.com) and can be purchased by experts in the field through standard sales channels.

[0024] The components of the first composition 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 composition of the invention according to said nutritional needs.

[0025] The combined preparation of the invention comprises a second composition b) comprising a mixture of terpenic acid and polyphenols, wherein the concentration of the first composition with respect to the second composition is between 350 and 650 ppm.

[0026] “T-terpenic acid” is a pharmacologically highly effective organic compound derived from isoprene, which is primarily of plant origin and is widely distributed in nature. T-terpenic acids predominantly occur as pentacyclic compounds. Examples of t-terpenic acids include, but are not limited to, corosolic, oleanolic, maslinic, asiatic, madecassic, betulinic, glycyrrhetinic, ursolic, and boswellic acids. In the context of the present invention, the second composition of the combined preparation of the invention may comprise a t-terpenic acid and / or a derivative of a t-terpenic acid. Examples of compounds derived from t-terpenic acids are widely known in the state of the art.

[0027] In the present invention, "polyphenols" are understood to be compounds with a molecular structure characterized by the presence of one or more phenolic rings. They originate primarily in plants, which synthesize them in large quantities as a product of their secondary metabolism. Some are essential for plant physiological functions. Others participate in defense functions against stress situations and various stimuli (water, light, etc.). They are also a group of substances with high antioxidant capacity and positive effects on your health.

[0028] Examples of polyphenols include, but are not limited to, phenolic acids, stilbenes, lignans, phenolic alcohols, hydroxytyrosol, oleuropein, and flavonoids.

[0029] Both triterpenic acid and polyphenols are active compounds obtainable from plants. In the present invention, "active substance" or "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 carotenoids, polyphenols, terpenes, lignans, organosulfur compounds, glucosilonates, saponins, marine polysaccharides, etc. In another preferred embodiment of the combined preparation of the invention, alone or in combination with all or each of the previous preferred embodiments, the triterpenic acid and the polyphenols of the second composition are obtained from a plant extract, preferably from the Olea genus, more preferably Olea europaea.Preferably, the mixture of triterpenic acid and polyphenols represents at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% by weight with respect to the total of the second composition.

[0030] The second composition of the combined preparation of the invention may comprise fulvic acids. "Fulvic acids" are a mixture of weak aliphatic and aromatic organic acids that dissolve in water at all pH levels (acidic, neutral, and alkaline), which is produced during the decomposition of organic matter. It can be found in soil, marine sediments, wastewater, and compost. In another preferred embodiment of the combined preparation of the invention, alone or in combination with all or each of the previous preferred embodiments, the second composition further comprises fulvic acids. In another preferred embodiment of the combined preparation of the invention, the second composition is Aquolive®. The Aquolive® composition is commercially available and can be purchased by those skilled in the art through standard sales channels.

[0031] 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 Aquolive®.

[0032] 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 250 and 750 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.

[0033] 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 350 and 650 ppm (extremes included), which, in another even more preferred embodiment, is between 450 and 550 ppm (extremes included), which, in another even more preferred embodiment, is 500 ppm.

[0034] 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).

[0035] The combined preparation of the invention may be part of a food product or a pharmaceutical composition. 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.

[0036] 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.

[0037] 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.

[0038] Examples of foods include, but are not limited to, vegetable products, meat products, snacks, beverages, cereals, bakery products, cookies, dairy products, milks, fermented milks (such as yogurt or cheese), ice creams, butters, margarines, etc. In another 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.

[0039] 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.

[0040] The term "nutraceutical" refers, in the context of the present invention, 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)).

[0041] 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 containing 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 specific nutrients or substances beneficial to health.

[0042] 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).

[0043] 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.

[0044] 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.

[0045] In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 forms (tablets, powders, microgranules (pellets), tablets, pills, capsules, granules, patches, etc.), semi-solid forms (gels), or liquid forms (solutions, suspensions, emulsions, syrups, drops, drinkable vials, etc.). In another 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.

[0050] 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.

[0051] Therefore, in another aspect, the invention relates to the combined preparation, the food product or the pharmaceutical composition of the invention, for use as a medicament or, alternatively, to the use of the combined preparation of the invention, the food product of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament.

[0052] The term "medicament" refers to a substance 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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 physicochemical (such as pollutants or radiation) and internal (for example, cancer cells).

[0057] 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.

[0058] 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:

[0059] (i) inhibit the disease or pathological condition, that is, stop its development;

[0060] (i) alleviate the disease or pathological condition, that is, cause the regression of the disease or pathological condition or its symptomatology;

[0061] (iii) stabilize the disease or pathological condition.

[0062] 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.

[0063] 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 Vibrionaceae family, 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 global disease burden. V. parahaemolyticus is a gram-negative, slightly curved, facultative aerobic, halophilic, oxidase-positive, glucose- but not sucrose-containing rod, with a variable urease activity. It requires selective media for its growth, with a 1% NaCl concentration, and is widely distributed in marine environments. This organism is frequently isolated from a variety of raw seafood products, particularly shellfish.

[0064] 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 refers 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 mynapods.

[0065] 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.

[0066] 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 mynapods (with more than 10 legs).

[0067] DESCRIPTION OF THE FIGURES

[0068] Figure 1. Feed conversion ratio (FCR) after 8 weeks in each study group. Different superscript letters indicate statistically significant differences (p<0.05). NF: Nucleoforce®; OA: Aquolive®.

[0069] 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®; AO: Aquolive®; PC: Positive Control; NC: Negative Control.

[0070] EXAMPLES

[0071] The invention will now be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention. Combined preparation

[0072] 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.

[0073] Example 1. Study of the effect of the combined preparation of the invention on the productivity of whiteleg shrimp (Litopenaeus vannamei) under recirculating aquaculture system (RAS) conditions.

[0074] A total of 1250 specific pathogen-free whiteleg shrimp (Litopenaeus vannamei) from a shrimp farm in Vietnam were acclimatized for 2 days and subsequently sorted into 6 treatment groups (250 shrimp per group; 5 replicates (400L tanks) per treatment) (see Table 3).

[0075] Each tank was equipped with an aeration system to maintain oxygen levels above 6 ppm throughout the study period. The maximum density was 200 shrimp / m². 3 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 to satiation. The water temperature was maintained between 28 and 32°C.

[0076] During the 8 weeks of the trial, several common production parameters were evaluated:

[0077] - Variations in weight and biomass

[0078] - Food intake

[0079] - feed conversion ratio (FCR)

[0080] Tables 4 and 5 show the feed intake results after 8 weeks of treatment in the different groups. Similarly, Figure 1 shows how, despite achieving a numerically better AO500 (a lower AO500 implies greater efficiency), AO500 does not achieve a statistically significant difference (p>0.05) compared to the control group. In the NF500 group, a significantly better AO500 (p<0.05) was observed compared to the control group. The most notable result is a very marked improvement in AO500 in the NF500A0500 group, which achieved a statistically significant improvement (p<0.05) compared to both the control and NF1000, NF500, and AO500. This demonstrates a productive synergy between the Nucleoforce® and Aquolive® components when combined in a 1:1 ratio.

[0081] Example 2. Study of the effect of the combined preparation of the invention on the survival of the whiteleg shrimp (Litopenaeus vannamei) against a challenge with Vibrio parahaemolyticus under laboratory conditions.

[0082] A total of 720 whiteleg shrimp affected by acute hepatopancreatic necrosis (AHPN) from a shrimp farm in Vietnam were used to evaluate the tolerance of the subjects to bacterial infection by V. parahaemolyticus. Once at the facility, the shrimp were acclimated connected to an open flow system for 2 days and subsequently classified into 6 treatment groups (120 shrimp per group; 4 replicates (90 L tanks) per treatment with 30 shrimp / tank) (see Table 6).

[0083] 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.

[0084] 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.

[0085] The bacterial challenge was carried out as follows: a V. parahaemolyticus isolate was collected from shrimp affected by acute hepatopancreatic necrosis (AHPN) from a shrimp farm in Vietnam. The shrimp were challenged by immersion. Bacterial suspensions were added to the tanks to achieve the desired density, 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.

[0086] Figure 2 shows the results of the evolution of the survival rate throughout the study 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 group (without challenge and diet without supplementation), which validates the model used and the bacterial challenge carried out. On the other hand, although the NF500, AO500 and NF1000 groups showed higher survival rates 240 hours post-challenge than the Positive Control group, this superiority did not reach the level of statistical significance (p > 0.05) in any of these cases, as can be seen in the superscript letters.In contrast, the NF500AC500 group combination achieved a significantly higher survival rate than the Positive Control group, with a statistically significant difference (p<0.05) compared to both the Positive Control and NF500 groups. This demonstrates a synergy between the Nucleoforce® and Aquolive® components against a bacterial challenge when combined in a 1:1 (500:500) ratio.

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 triterpenic acid and polyphenols, wherein the concentration of the first composition relative to the second composition is between 250 and 750 ppm.

2. Combined preparation according to claim 1, wherein the triterpenic acid and the polyphenols of the second composition are obtained from a plant extract, preferably, of the genus Olea, more preferably, Olea europaea.

3. Combined preparation according to claim 1 or 2, wherein the second composition further comprises fulvic acids.

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 350 and 650 ppm.

5. Combined preparation according to claim 4, wherein the concentration of the first composition with respect to the second is between 450 and 550 ppm.

6. Combined preparation according to claim 5, wherein the concentration of the first composition with respect to the second is 500 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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