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    • 专利摘要:
    Food product for animals comprising aloin, apigenin and caffeic acid, and a process for obtaining them. The present invention relates to a food product for animals comprising aloin, apigenin and caffeic acid. The present invention also relates to a process for obtaining the animal food product comprising aloin, apigenin and caffeic acid, comprising the steps of (a) obtaining an extract of aloin from the leaves of Aloe vera; (b) obtaining an extract of apigenin from the flower of Taraxacum officinale; (c) obtain an extract of caffeic acid from the total flower of Equinacea purpurea; and (d) mixing the extracts of steps (a), (b) and (c) to homogeneity. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2684782A1
    申请号:ES201700436
    申请日:2017-03-29
    公开日:2018-10-04
    发明作者:Jaime Borrell Valls
    申请人:Biovet S A；BIOVET SA；
    IPC主号:
    专利说明:

    DESCRIPTION

    Food product for animals comprising aloin, apigenin and caffeic acid, and process for obtaining it.
     5
    Field of research

    The present invention relates to the field of animal feeding. In particular, it refers to an animal food product comprising aloin, apigenin and caffeic acid, and its method of production. 10

    Background of the invention

    There are several factors in livestock and poultry production that lead to low performance of the animals' immune system and make it necessary to have a product in the market for this purpose.

    On the other hand, due to the prohibition of many antibiotics, the periods of suppression that they have or the concern about waste by consumers, it has been necessary to orient the supply to pronutrients that do not present any of the above problems, 20 understood as pronutrient a micro-ingredient included in the formulation of an animal feed in relatively small quantities with specific physiological and microbiological functions different from other nutrients.

    There are three factors that currently make the immune system of animals vulnerable and therefore it is convenient to administer immunostimulants in production animals:

    1. Young animals: the immaturity of their immune system makes them more predisposed to infections. 30

    In birds: The development of the immune system continues after the hatching of the egg. During the first seven days of life, a large part of the immunity is formed by maternal antibodies.
     35
    In piglets and ruminants, during the first weeks of life, the active immune system is not mature and the passive immunity acquired by colostrum decreases over time.

    Therefore, the administration of a general immunostimulant is necessary to prevent infections and strengthen the immune system of young animals. 40

    2. The genetic selection of breeds of poultry, pigs and ruminants to achieve a high growth rate or greater prolificacy causes a weakening of the immune system due to metabolic wear.
     Four. Five
     Fast-growing animals, after vaccination, take 21 days to develop a specific serological immunity. In many cases, young chickens (“broilers”) are slaughtered on day 32-35, for this reason in most cases it is considered that vaccination is useless because the animal is slaughtered before being completely immunized. fifty

     In animals of longer life, sometimes, to improve the results obtained with vaccines, it is necessary to use vaccines with very virulent strains, since they allow to achieve greater immunity.

    3. As a complement to antimicrobial therapy: In addition to drug treatment, immunostimulants help in the body's immune response against infection. Immunostimulants improve the animal's immune status, accelerate recovery and reduce the impact of diseases on productive parameters. Immunostimulants 5 also limit secondary and recurrent infections (especially in viral superinfections). They can also replace antimicrobial therapies when, due to the withdrawal period, pharmacological products cannot be administered. Highly virulent strains can cause side effects or even symptoms very similar to the disease. If an immunostimulant is administered the days prior to vaccination, a better immunity is achieved than only by using vaccination and there is no need to use highly virulent strains

    Therefore, in light of the importance of immunostimulation to production animals, together with the growing concern of consumers about residues in meat not to create resistance, the present inventors have developed a food product for animals to plant extract base that acts as immunostimulant.

    Summary Description of the Invention
     twenty
    A first objective of the invention is an animal food product comprising aloin, apigenin and caffeic acid.

    A second objective of the invention relates to a process for obtaining the food product according to the first aspect of the invention. 25

    Brief description of the drawings

    Figure 1: Western-Blot test.
     30
    Figure 2: Representation of the results of example 6.

    Description of the invention

    In a first aspect, the present invention relates to an animal food product comprising aloin, apigenin and caffeic acid. Preferably, said product is intended for livestock production animals that include, without limitation, ruminants, pigs and poultry.

    In a preferred embodiment, the aloin is present in at least 2.8% by weight in the food product of the invention. Said aloin is preferably contained in an extract of the Aloe vera leaf, more preferably it is contained in a weight percentage between 7 and 11% by weight in said extract. Aloin is usually prepared by extracting aloe, a yellowish exudate that emanates from the front of the skin of aloe leaves. The latex, known as "aloe juice", subsequently dehydrates and turns into powder.

    Aloine is an anthraquinonic glycoside, which means that its anthraquinone skeleton has been modified by the addition of a sugar molecule. It acts by stimulating the immune system at the level of the nonspecific type, increasing the activity of macrophages and 50 neutrophils, and stimulating the elimination of microorganisms.



    In another preferred embodiment, apigenin is present in at least 1.25% by weight in the food product of the invention. Said apigenin is preferably contained in an extract of Taraxacum officinale, more preferably it is contained in a weight percentage between 3.5 and 5.5% by weight in said extract. The properties of apigenin are immunomodulatory and immunostimulant by increasing unspecific defenses. In vitro tests, the increase in phagocytic activity of macrophages has been demonstrated by 23%. Macrophages are cells of the defense system that quickly migrate to the site of infection to stop bacteria. 10



    In yet another preferred embodiment, caffeic acid is present in at least 3.3% by weight in the product of the invention. Said acid is preferably contained in an extract 15 of Echinacea purpurea, more preferably it is contained in a weight percentage between 5.5 and 8.5% by weight in said extract.



    Echinacea (Echinacea purpurea) is a plant native to North America that has traditionally been known for its immune system boosting properties. It increases the multiplication of white blood cells (T lymphocytes), and increases the capacity of phagocytosis (of white blood cells macrophages). It also improves the interaction of antibodies, produced by neutrophil white blood cells, making defenses more efficient.

    In a second aspect, the present invention relates to a process for obtaining an animal feed product comprising aloin, apigenin and caffeic acid, comprising the steps of:

    - (a) obtain an aloin extract from the leaves of Aloe vera.
     fifteen
    - (b) obtain an apigenin extract from the flowers of Taraxacum officinale; interpreting that "flower" or "flowers" means any part of the flower.

    - (c) obtain an extract of caffeic acid from the flower of Echinacea purpurea.
     twenty
    - (d) mix the extracts from steps (a), (b) and (c) until homogeneous.

    Preferably, step (a) comprises the following sub-stages:

    - (a1) clean, cut the tips and peel the Aloe vera leaves sufficient to obtain an ecological gel with a concentration between 0.5-1% by weight of aloin, preferably 0.7% by weight of aloin.

    - (a2) press and filter the gelatinous matter obtained in point (a1).
     30
    - (a3) clarify and sterilize gelatinous matter to eliminate bacteria and other substances of lower molecular weights.

    - (a4) concentrate the gelatinous matter by ultrafiltration to a concentration between 7 and 11% by weight of aloin in the extract, which is between 10 and 20 times higher than the concentration of the leaves.

    - (a5) lyophilize.

    In this way the aloin extract is obtained. 40

    Preferably, step (b) comprises the following sub-stages:

    - (b1) dry the fresh flowers, usually for 4 days.

    - (b2) suspend the flowers of Taraxacum in an enzymatic solution in emulsion. 5

    - (b3) acidify the previous suspension obtained in (b1), usually with 15% sulfuric acid by weight.

    - (b4) boil the suspension for 20 minutes under reduced pressure. 10

    - (b5) shake.

    - (b5) perform a methanolic extraction.
     fifteen
    - (b6) evaporate the methanol.

    In the sub-stage (b2), the enzymatic solution used is preferably composed of cellulase and xylanase in a volume ratio of 80% cellulase and 20% xylanase.
     twenty
    In this way the apigenin extract is obtained.

    Preferably, step (c) comprises the following sub-stages and does not require any hydrolysis stage:
     25
    - (c1) suspend the flower of Echinacea purpurea, prior to cleaning and disinfection, in 96% volume ethanol in a proportion in vegetable weight: alcohol of 1: 8 (for example, 1 kilo of vegetable in 8 kilos of ethanol), for 5 hours

    - (c2) filter to separate the solid part. 30

    - (c3) add an aqueous solution of 0.5-1.5 mM citric acid, preferably 1 mM.

    - (c4) treat with ethyl p-hydroxybenzoate: 1: 5 emulsifier (weight ratio).
     35
    - (c5) homogenize between 30-35 minutes.

    - (c7) remove the solvent by steam entrainment.

    In this way the extract of caffeic acid is obtained. 40

    Here, it should be understood that any of the described embodiments of the present invention may be combined with each other, unless specifically indicated otherwise.
     Four. Five
    The present invention is further illustrated by reference to the following examples without representing a limitation of the scope of the present invention.

    Examples
     fifty
    Example 1. Obtaining Aloin from Aloe Vera

    1. Blade washing: in stainless steel or plastic tubs, in manual or mechanized operation, by using recirculation pumps of the washing solution composed of water and a surfactant such as sodium laurel sulfate or laurel ether sulfate sodium.

    2. Blade rinsing: in stainless steel or plastic tubs, in manual or mechanized operation, by using recirculation pumps of the rinse solution composed of water and a bactericidal agent, such as iodine, hydrogen peroxide or other convenient.

    3. Cutting of the spiny edges or edges: manual operation by means of blades attached to the debarking table.

    4. Automatic debarking: using adjustable height blades and roller driving on the debarking table.
     fifteen
    5. Pressing: cold press to eliminate the fibers and pulp for the production of juice. It is necessary to disperse the glass by using a pulper that does not cut or shred excess fibers to facilitate subsequent removal. Smoothie is not recommended.
     twenty
    6. Filtration: by using a centrifugal filter at room temperature to remove plant fibers.

    7. Clarification of the mucilage by using a high speed disc clarifying centrifuge that offers better results and can even eliminate large bacteria. 25

    8. Sterilization is performed at room temperature by using membranes with porosity of up to 0.5 µm. Bacteria have sizes larger than 1 µm.

    9. Cold purification is achieved by ultrafiltration through porosity membranes 30 to retain substances from a molecular weight of 400 Da. This method manages to increase the concentration of aloin between 10 and 20 times compared to that found in the untreated leaf.

    10. For the final drying, the lyophilization process is used, which involves the freezing of the product and the subsequent sublimation of the ice formed, without going through the liquid phase of the water. It is, without a doubt, the best drying method when you want to preserve the original properties of the product. It allows to obtain an easily reconstitutable powder. A yellowish powder with greenish reflections is obtained.
     40
    11. A standardization of the extract is carried out to verify its purity and richness.

    Example 2. Obtaining apigenin from the flowers of Taraxacum officinale
     Four. Five
    1. To obtain apigenin, dried flowers were dried for four days. From every 4 kilos of fresh matter, 1 kg of dry matter will be obtained.

    2. To obtain a higher concentration of active ingredient, the dry matter was introduced into a balloon with enzymatic solution with water, sufficient to achieve the suspension of the sample. 50 This is usually a ratio of 1 kilo of dried Taraxacum in 7 liters of water. The smallest possible manipulation of the sample is sought in order not to degrade the active ingredients.

    3. Acidify with 15% sulfuric acid.
    4. Boil for 20 min.

    5. To improve the yield of the extract, it is reduced under reduced pressure to a butyrous consistency.
     5
    6. The residue is resuspended in methanol and reduced pressure is reapplied until the extract is brought to dryness.

    7. A standardization of the extract is carried out to verify its purity and richness. 10

    Example 3. Obtaining caffeic acid from the flowers of Echinacea purpurea

    1. To obtain caffeic acid, a cleaning and disinfection of the flower is carried out by means of the use of recirculation pumps of the washing solution composed of water and a sodium laurel sulfate or sodium laurel ether sulfate surfactant.

    2. Rinse: in stainless steel or plastic tubs, in manual or mechanized operation, by using recirculation pumps of the rinse solution composed of water and a bactericidal agent such as iodine, hydrogen peroxide or other convenient. twenty

    3. Suspend the plant in 96% ethanol by volume in a proportion by plant weight: alcohol of 1: 8 (for example, 1 kilo of vegetable in 8 kilos of ethanol) for 5 hours.

    4. Filter to discard the solid part, since caffeic acid is already in solution in the liquid part.

    5. Press the solid parts, to obtain better extraction performance.

    6. Add an aqueous solution of 0.5-1.5 mM citric acid, preferably 1 mM. 30

    7. Treat with ethyl p-hydroxybenzoate: 1: 5 emulsifier (weight ratio).

    8. Homogenize mechanically for 30-35 minutes.
     35
    9. Bring the extract to dryness by removing the solvent by steam entrainment.

    10. A standardization of the extract is carried out to verify its purity and richness.
     40
    Example 4. Obtaining the food product of the invention

    Once all the active ingredients have been purified, after quantitative analysis to check the concentrations, the preparation of the final product was carried out.
     Four. Five
    This preparation takes place by simple mixing of the extracts to obtain the product in powder form.

    In a reactor, 75 kilos of Echinacea purpurea flour, 50 kilos of Aloe Vera flour and 50 kilos of Taraxacum officinale flour were introduced. They were mixed for 6 minutes, at which time the homogeneity of the product can be ensured. At this time, the product was milled to ensure proper particle size for use in industrial facilities, which will be the end users of the product. The food obtained by incorporating natural ingredients from aromatic plants, is ready to be
    consumed with a minimum guaranteed composition of 2.8% by weight of aloin, 1.25% by weight of apigenin and 3.3% by weight of caffeic acid.

    Example 5. Genomic interaction test of the product subject of the invention
     5
    The objective is to evaluate the RNA-messenger transduction rate in vitro as a measure of ribosomial activity that is stimulated by the presence of active pronutrients (aloin, apigenin and caffeic acid) of the food product. The indicator is puromycin. The detection is based on the use of an antipuromycin antibody and the development with a secondary antibody labeled with peroxidase. 10

    Cultures of intestinal cell tissues were performed that are fed with basic nutrients to which an indicator, an inhibitor of protein production and the pronutrients to be analyzed are added. Four different spots ((1) white from a basic cell culture, (2) with indicator, (3) with indicator and food product 1: 1000, (4) with 15 indicator and food product 1: 10,000) were prepared and prepared. deposited on an electrophoresis plate. Once developed they were stained. Staining of the plates was performed by Western Blot method. At greater intensity, greater presence of proteins from ribosomal activity.

    As can be seen in Figure 1, bands 3 and 4 have a greater amount of protein 20 synthesized by intestinal cells, which confirms that the presence of active pronutrients from the food product stimulates the transduction of messenger RNA, from the nucleus, in ribosomes and consequently more specific protein of the intestinal cell. A higher protein specifies more cellular activity.
     25
    Example 6. Test of antibody production in birds with the product of the invention

    The objective of this in vivo test is to determine the efficacy of the product object of the present invention by its effect on the immune response against three viral diseases of birds such as: Gumboro, Newcastle and infectious bronchitis; and its efficacy on general antibacterial resistance.

    The test consisted of dividing the chickens into 8 different batches (19,000 chickens per batch).

    - The product (2 lots) was administered to 38,000 chickens, while the rest (114,000 chickens) 35 were not given any immunity enhancer.

    - The product was administered at a dose of 500 ml / 1000 L in drinking water for 7 days (from 7 to 14) and another 7 days (from 28 to 35).
     40
    - On day 40, blood sample analysis of a significant number of birds was performed to evaluate the antibody titer against Newcastle, Gumboro and Bronchitis.

    The results obtained can be seen in Figure 2.
     Four. Five
    Therefore, it can be concluded that the birds to which the product of the invention was administered improved the number of antibodies for each of the diseases for which they were vaccinated, thereby improving the treatment result, ultimately resulting in a improvement of mortality data.
    fifty
    权利要求:
    Claims (16)
    [1]

    1. Animal food product comprising aloin, apigenin and caffeic acid.

    [2]
    2. Food product according to claim 1, wherein said animals are animals of livestock production.

    [3]
    3. Food product according to claim 1 or 2, wherein the aloin is present in at least 2.8% by weight in said product.
     10
    [4]
    4. Food product according to any of the preceding claims, wherein the apigenin is present in at least 1.25% by weight in said product.

    [5]
    5. Food product according to any of the preceding claims, wherein caffeic acid is present in at least 3.3% by weight in said product. fifteen

    [6]
    6. Food product according to any of the preceding claims, wherein the aloin is contained in an extract of the Aloe vera leaf.

    [7]
    7. Food product according to claim 6, wherein the aloin is present in said Aloe vera leaf extract in a weight percentage between 7 and 11% by weight.

    [8]
    8. Food product according to any of the preceding claims, wherein the apigenin is contained in an extract of Taraxacum officinale.
     25
    [9]
    9. Food product according to claim 8, wherein the apigenin is present in said extract in a weight percentage between 3.5 and 5.5% by weight.

    [10]
    10. Food product according to any of the preceding claims, wherein the caffeic acid is contained in an extract of Echinacea purpurea. 30

    [11]
    11. Food product according to claim 10, wherein the caffeic acid is present in said extract in a weight percentage between 5.5 and 8.5% by weight.

    [12]
    12. Method for obtaining an animal food product comprising aloin, apigenin and caffeic acid, comprising the steps of:
    - (a) obtain an aloin extract from the leaves of Aloe vera.
    - (b) obtain an apigenin extract from the Taraxacum officinale flower. 40
    - (c) obtain an extract of caffeic acid from the total of the flower of Equinacea purpurea.
    - (d) mix the extracts from steps (a), (b) and (c) until homogeneous.
     Four. Five
    [13]
    13. The method according to claim 12, wherein step (a) comprises the following sub-stages:
    - (a1) clean, cut the tips and peel the Aloe vera leaves sufficient to obtain an ecological gel with a concentration between 0.5-1% by weight of aloin. fifty
    - (a2) press and filter the gelatinous matter obtained in the sub-stage (a1).
    - (a3) clarify and sterilize the gelatinous matter.
    - (a4) concentrate the gelatinous matter by ultrafiltration to a concentration between 7 and 11% by weight of aloin in the extract.
    - (a5) lyophilize.
     5
    [14]
    14. The method according to any of claims 12 or 13, wherein step (b) comprises the following sub-stages:
    - (b1) dry the fresh flowers.
     10
    - (b2) suspend Taraxacum flowers in an emulsion enzyme solution.
    - (b3) acidify the suspension obtained in the sub-stage (b1).
    - (b4) boil under reduced pressure. fifteen
    - (b5) shake.
    - (b6) perform a methanolic extraction.
     twenty
    - (b5) evaporate the methanol.

    [15]
    15. A method according to claim 14, wherein the enzymatic solution used in the sub-stage (b2) is composed of cellulase and xylanase in a volume ratio of 80% cellulase and 20% xylanase. 25

    [16]
    16. The method according to any of claims 12 to 15, wherein step (c) comprises the following sub-stages:
    - (c1) suspend the flower of Echinacea purpurea, after cleaning and disinfection, in ethanol 30 to 96% by volume in a proportion in vegetable weight: alcohol of 1: 8 for 5 hours.
    - (c2) filter to separate the solid part.
    - (c3) add an aqueous solution of 0.5-1.5 mM citric acid. 35
    - (c4) treat with ethyl p-hydroxybenzoate: emulsifier in a 1: 5 weight ratio.
    - (c5) homogenize between 30-35 minutes.
     40
    - (c6) remove the solvent.
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    同族专利:
    公开号 | 公开日
    ES2684782B1|2019-04-24|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2197632T3|1999-04-28|2004-01-01|Paxa N.V.|ALOINE PREPARATION PROCEDURE BY EXTRACTION.|
    CN1586268A|2004-08-06|2005-03-02|孙智斌|Composite fodder with aloe|
    CN103641807A|2013-12-25|2014-03-19|南通大学|Extraction method of apigenin, pharmaceutical composition for treating diabetes and application thereof|
    CN103965149A|2014-04-30|2014-08-06|桂林军供生化技术开发有限公司|Aloin extraction method|
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