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    • 专利摘要:
    Food product for animals comprising Carvacrol, P-Cimenol and Aliine, and process for obtaining it. The present invention relates to an animal food product comprising carvacrol, p-cimenol and alliin. The present invention also relates to a process for obtaining the animal feed product comprising carvacrol, p-cimenol and alliin. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2730385A1
    申请号:ES201830456
    申请日:2018-05-09
    公开日:2019-11-11
    发明作者:Valls Jaime Borrell
    申请人:Biovet S A；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] Field of the Invention
    [0005]
    [0006] The present invention relates to the field of animal feeding. In particular, it refers to an animal food product comprising carvacrol, p-cimenol and alliin, and its process of obtaining.
    [0007]
    [0008] Background of the invention
    [0009]
    [0010] It is known that in the feeding of animals high amounts of undigested proteins reach the intestine, which, when not digested, cannot be absorbed and, on the other hand, are used by pathogenic bacteria, such as Clostridium, which do metabolize them. This causes Clostridium populations to increase, reducing the population of the intestine's own biota. Therefore, the intestine will function in an incorrect way, and even in some cases diarrhea can occur.
    [0011]
    [0012] The present invention provides a novel product with a content in its active ingredients that helps to improve the physiological state of the epithelial cells of the intestine and consequently, the digestion of food. The present inventors have obtained a series of products extracted from plant extracts whose combination has surprisingly been found to be optimal for the intestinal health of animals.
    [0013]
    [0014] The association of the three active principles of the present invention (carvacrol, p-cimenol and alliin) allows to obtain a final product with great genomic interaction capacity for the regeneration of enterocytes, great capacity for normalization of the physiological state of the immune cells of the intestine through an epigenetic mechanism by stimulation of the genes of IL-1, IL-12, Il-18 and CXC-chemokines and, ultimately, surprisingly improves the productivity of farms as the conversion rate of the animals (relationship between the weight gained by the animal and the food consumed) by improving their digestive well-being.
    [0015] Additionally, the present inventors have developed a process for obtaining the product of the present invention that allows obtaining a powder product, easy to store and administer, but which in turn contains the nutritional contents of a fresh product.
    [0016]
    [0017] Brief description of the drawings
    [0018]
    [0019] Figure 1 represents the food consumption (g) at different intervals of days for lots T1 to T4 according to example 5.1.
    [0020] Figure 2 represents the average daily gain (g) at different intervals of days for lots T1 to T4 according to example 5.1.
    [0021] Figure 3 represents the conversion rate (ratio between the weight gained by the animal and the food consumed) at different intervals of days for lots T1 to T4 according to example 5.1.
    [0022] Figure 4 represents lesions according to the type of Eimeria on day 21 for lots T1 to T4 according to example 5.1.
    [0023] Figure 5 represents lesions according to the type of Eimeria on day 35 for lots T1 to T4 according to example 5.1.
    [0024] Figure 6 represents the relative quantification of the genes transcribed by IL-1 for batches A to D according to example 5.4.
    [0025] Figure 7 represents the relative quantification of the genes transcribed by IL-12 for batches A to D according to example 5.4.
    [0026] Figure 8 represents the relative quantification of the genes transcribed by IL-18 for batches A to D according to example 5.4.
    [0027]
    [0028] Summary Description of the Invention
    [0029]
    [0030] A first objective of the invention is an animal food product comprising carvacrol, p-cimenol and alliin.
    [0031]
    [0032] A second objective of the invention relates to a process for obtaining the food product according to the first aspect of the invention.
    [0033] Description of the invention
    [0034]
    [0035] In a first aspect, the present invention relates to an animal food product comprising carvacrol, p-cimenol and alliin. Preferably, said animal refers to livestock production animals that include, without limitation, ruminants, pigs and birds.
    [0036]
    [0037] In a preferred embodiment, carvacrol is present in at least 3.4% by weight in the food product of the invention. Said carvacrol is preferably contained in Oríganum vulgare flour , more preferably it is contained in a weight percentage between 17 and 20% by weight.
    [0038]
    [0039] Carvacrol is also called cymophenol and has the following formula:
    [0040]
    [0041]
    [0042]
    [0043] carvacrol
    [0044]
    [0045] Carvacrol inhibits growth and eliminates a high number of pathogens that would produce harmful toxins in the intestine. Some of these pathogens are associated with gastroenteritis, hemorrhagic diarrhea, renal failure, among other disorders or diseases. Carvacol damages the integrity of the cell wall of these bacteria. It is particularly interesting to indicate that carvacrol has very little or little interaction with the probiotic flora of the intestine. At concentrations where it inhibits the growth of pathogenic bacteria between 97 and 100%, it only affects probiotic bacteria between 3 and 5%.
    [0046]
    [0047] In another preferred embodiment, p-cimenol is present at least 2.8% by weight in the food product of the invention. Said p-cimenol is preferably contained in an extract of Thymus vulgaris, more preferably it is contained in a weight percentage between 14 and 16% by weight.
    [0048] P-Cimenol is a compound whose functions as bactericide, fungicide and preservative are known and is present in many botanical varieties, including Thymus vulgaris.
    [0049]
    [0050]
    [0051]
    [0052]
    [0053] In vitro tests and field tests indicate that the cimenol ring is effective against bacteria, yeasts and fungal pathogens of the digestive system whose mechanism of action is as follows:
    [0054]
    [0055] Mechanism of action in bacteria
    [0056]
    [0057] Contact with the cimenol ring causes the immediate release of the cell contents into the bacteria, caused by the perforation of the bacterial membrane that leads to the destruction of the cell.
    [0058]
    [0059] At the intracellular level, the cimenol ring affects the biosynthetic pathways of ATP (an essential molecule for energy metabolism), cell pH and the balance of inorganic ions.
    [0060]
    [0061] Mechanism of action in yeasts and fungi
    [0062]
    [0063] On yeasts and fungi, adequate concentrations of cimenol ring manage to eliminate 100% of microorganisms in just 10 minutes of contact. Lysis of the cells and release of cellular content to the medium is observed. To the electron microscope, Yeasts and fungi have deformities and fractures in cell walls, especially those found in the proliferation phase.
    [0064] Additionally, the cimenol ring inhibits the biosynthesis of ergosterol, the most important sterol in the yeast cell membrane, contributing to the destruction of the cell.
    [0065]
    [0066] In yet another preferred embodiment, the alliin is present in at least 7.8% by weight in the product of the invention. Said alliin is contained in an extract of Allium sativum, more preferably it is contained in a weight percentage between 13 and 14% by weight.
    [0067]
    [0068]
    [0069]
    [0070]
    [0071] The underground bulb of Allium sativum (commonly garlic) contains numerous active components, among which sulfur compounds stand out. If the bulb is intact and fresh, the major compound is S-allyl-cysteine sulfoxide or Aliine. This is an unstable compound that is hydrolyzed by the action of the enzyme Alinase that is also present in the bulb. Allicin is obtained. Said allicin in turn is also a highly volatile compound. Alliin is present in the cytosol, which is also the substrate of the enzyme allinase found in separate vacuoles. When the garlic is crushed, the vacuoles containing the enzyme are broken and the reaction with the alliin takes place to form an intermediate product that condenses to give the allicin.
    [0072]
    [0073] In a second aspect, the present invention relates to a process for obtaining an animal feed product comprising carvacrol, p-cimenol and alliin, which comprises the steps of:
    [0074]
    [0075] (a) obtain an extract of carvacrol from the leaves of Origanum vulgare;
    [0076] (b) obtain an extract of p-cimenol from the leaves of Thymus vulgaris;
    [0077] (c) obtain an alliin extract from the bulb of Allium sativum;
    [0078] (d) mix the extracts from steps (a), (b) and (c) until homogeneous.
    [0079]
    [0080] Preferably, step (a) comprises the following sub-stages:
    [0081] (a1) grind and dry the Origanum vulgare leaves to obtain a powdered product; (a2) macerate the powder obtained in (a1) in ethanol, preferably, for 4 hours;
    [0082] (a3) solidify the oleoresin obtained in (a2) by absorption with silica gel and evaporate the solvent, preferably, maintaining the same temperature
    [0083] (a4) centrifuge, preferably, centrifuge at 6000 rpm for 30 minutes.
    [0084] (a5) discard aqueous phase.
    [0085]
    [0086] In this way carvacrol extract is obtained.
    [0087]
    [0088] Preferably, step (b) comprises the following sub-stages:
    [0089] (b1) cold press the leaves of Thymus vulgans;
    [0090] (b2) suspend in water the extract obtained in (b1);
    [0091] (b3) distill the oil from the leaf extract;
    [0092] (b4) extract the oil from the aqueous phase;
    [0093] (b5) extract with a base the phenolic content of the oil and solubilize it in water;
    [0094] (b6) extract the p-cimenol with ether;
    [0095] (b7) evaporate the ether.
    [0096]
    [0097] In this way the p-cimenol extract is obtained.
    [0098]
    [0099] Preferably, step (c) comprises the following sub-stages and does not require any hydrolysis stage:
    [0100] (c1) suspend Allium sativum teeth in vegetable oils, preferably sunflower oil, under negative pressure and grind;
    [0101] (c2) filter to separate the skins
    [0102] (c3) let stand and decant approximately half the volume of the oil phase; (c4) add an aqueous solution of 0.5-1.5 mM citric acid, preferably 1 mM;
    [0103] (c5) treat with ethyl p-hydroxybenzoate in a 1: 5 ratio and an emulsifier;
    [0104] (c6) homogenize between 30-35 minutes;
    [0105] (c7) use a bentonite to absorb the liquid phase and obtain the powder alliin extract.
    [0106]
    [0107] Through this step (c) an extract of Allium sativum is obtained that preserves intact the content of alliin and allinase so that the biochemical process of conversion of alliin in its derivatives occurs when the product comes into contact with saliva, whose conditions are the ideal ones for the activation of the enzyme for the hydrolysis of alinases, so that the benefits of the pronutrients contained in Allium sativum are maximum, since their concentration would be equivalent to ingesting it in crude oil without losses. This allows to improve the offer, with wide application in the animal food industry based on substances authorized as food additives.
    [0108]
    [0109] The present invention is further illustrated by reference to the following examples without representing a limitation of the scope of the present invention.
    [0110]
    [0111] EXAMPLES
    [0112]
    [0113] Example 1. Obtaining carvacrol from Origanum vulgare
    [0114]
    [0115] First, a de-stemming of the oregano plant ( Origanum vulgare) was carried out to separate the leaves from the woody parts thereof. Origanum vulgare leaves were milled and dried to facilitate subsequent ethanolic extraction. This extract could be considered ecological because it does not have any type of chemical treatment.
    [0116]
    [0117] 100 kilos of dry powder of Origanum vulgare leaves were macerated with 96% ethanol in a 1: 3 w / v ratio for 4 hours. A product was obtained with the consistency of an oleoresin that is necessary to adsorb with silica gel and process in the rotary evaporator at 6,000 rpm for 30 minutes.
    [0118]
    [0119] 100 kilos of powdered product have a yield of 12 kg of extract rich in carvacrol (17-20%). A standardization of the extract was carried out to verify its purity and richness.
    [0120]
    [0121] Example 2. Obtaining p-cimenol from the leaves of Thymus vulgaris
    [0122]
    [0123] In order to obtain organic p-cimenol, cold pressing of dried and ground thyme leaves was carried out. The cake obtained contains approximately 4% p-cimenol.
    [0124]
    [0125] To obtain a higher concentration of active ingredient, the previous cake was placed in a balloon with enough water to achieve the suspension of the sample. This is usually a ratio of 1 kilo of dried thyme in 3 liters of water. The smallest possible manipulation of the sample is sought in order not to degrade the essential oils that are the main source of the compound to be purified. Therefore, for the extraction of essential oil Thyme was used with steam distillation distillation. Due to the volatility and insolubility of the oil in water, the product will be expected to present two phases.
    [0126]
    [0127] Subsequently, an extraction was made to separate the pure essential oil with a separating funnel. The essential oil was separated in a covered container and protected from light to avoid light decomposition. With the aqueous part, a new extraction with ether was carried out in order to finally separate the excess oil that is solubilized in this phase. Traces of water with sodium sulfate as a desiccant were extracted from the ether phase. To reduce the amount of components to be separated by chromatography, an acid-base extraction was performed.
    [0128]
    [0129] To obtain the phenols, among which p-cimenol, which are the most acidic compounds in the essential oil, an acid-base extraction was performed. First, the oils obtained together with distilled water were placed in the separating funnel. Next, 50 ml of 1N potassium hydroxide was added making the phenols, being the most acidic compounds present in the oil, soluble in the aqueous phase.
    [0130]
    [0131] Once the aqueous phase was isolated from the rest, it was taken to a new separatory funnel for acidification with 15 ml 2N hydrochloric acid and 100 ml of ether was added. In this way, only phenols in the etheric phase were obtained. All the ether phases were combined and filtered with anhydrous sodium sulfate as a desiccant to remove traces of water. Subsequently, the compound to be heated in a water bath was taken to evaporate the ether and thus obtain the isolated phenols. As with the protease, this oil is absorbed with a bentonite, which allows the product to be in a fine powder with 14-16% p-cimenol. A standardization of the extract was carried out to verify its purity and richness.
    [0132]
    [0133] Example 3. Obtaining alliin from the bulbs of Allium sativum
    [0134]
    [0135] To obtain organic alliin, without chemical treatments, the extraction was carried out in sunflower vegetable oil, which, by avoiding contact with air, minimizes the hydrolysis of the compound by the action of the allinase. The whole garlic cloves were put in the oil and grinding was carried out in an environment with negative pressure to avoid the presence of oxygen to the maximum. Finished this was filtered to separate the skins. The product contains 5% alliin.
    [0136] To increase the concentration of alliin from this emulsion, an concentration of alliin was performed. For this, it was allowed to stand for 2 hours and the oil part was decanted reducing its volume in half. A 1 mM aqueous citric acid solution was added and the remaining emulsion was treated with ethyl p-hydroxybenzoate in a ratio of 1: 5 and an emulsifier and homogenized for 30 minutes. In this way, an Allium sativum extract with a richness of alliin of 13-14% was obtained.
    [0137]
    [0138] Example 4. Obtaining the food product of the invention.
    [0139]
    [0140] Once all the active ingredients have been purified, after quantitative analysis to check the concentrations, the preparation of the final product was carried out.
    [0141]
    [0142] This preparation took place by simple mixing of the extracts to obtain the product as a liquid.
    [0143]
    [0144] For the preparation of the powdered food, 75 kilos of Allium sativum flour , 25 kilos of Oríganum vulgare flour and 25 kilos of Thymus vulgaris flour were placed . They were mixed for 6 minutes, at which time the homogeneity of the product can be ensured. At this time, a grinding of the product was carried out to guarantee an appropriate particle size for use in industrial facilities, which will be the end users of the product. The complementary food obtained by the incorporation of natural ingredients from aromatic plants is ready to be consumed with a minimum guaranteed composition of 3.4% carvacrol, 2.8% cimenol and 7.8% alliin. It was packaged properly.
    [0145]
    [0146] Example 5. Efficacy test
    [0147]
    [0148] There are a number of protozoa, coccidia, which infest the epithelial tissues of the intestines causing serious economic losses. Currently, coccidia infections are treated by administering chemical coccidiostats or by vaccination.
    [0149]
    [0150] To demonstrate the efficacy of the product of the invention against coccidia, several tests have been performed, which compare the use of the product of the invention with the methods currently used.
    [0151] Example 5.1 The first one was carried out in Scotland and the efficacy of the product of the invention was tested against various coccoidia species called Eimeria, specifically E. acervulina, E. maxima, E. tenella, E. necatrix in broilers
    [0152]
    [0153] Its design is schematized as follows:
    [0154]
    [0155] ANIMALS: 960 broilers in 4 treatment groups distributed in 32 sheds of 30 animals (8 sheds per treatment). Animals of 2 weeks of age were infected experimentally with pathogenic oocysts of: Eimeria tenella, E. acervulina, E. maxima and E. necatrix
    [0156]
    [0157] EVALUATED PARAMETERS:
    [0158] - Food consumption (see figure 1)
    [0159] - Average daily gain (see figure 2)
    [0160] - Conversion rate (see figure 3)
    [0161] - Severity of intestinal lesions on days 21 and 35 (days 7 and 14 post infection) (see Figures 4 and 5)
    [0162] - Oocysts per gram (day 20, 22 and 28).
    [0163] - The severity of intestinal lesions was evaluated on a scale from 0 to 4.
    [0164]
    [0165] TREATMENTS:
    [0166] - Treatment 1 (T1): Not infested and untreated (NINT)
    [0167] - Treatment 2 (T2): Infested untreated (INT)
    [0168] - Treatment 3 (T3): Product of the invention at 0.5 kg / T from day 0 until sacrifice (INV0.5).
    [0169] - Treatment 4 (T4): Product of the invention at 1 kg / T from day 0 until sacrifice (INV1).
    [0170]
    [0171] The results obtained for each of the parameters evaluated are the following: aa: I consume ea men ogv ase am na gura
    [0172] Lot 0 - 14 days 14 - 21 days 21 - 28 days 28 - 35 days 35 - 42 days 0 - 42 days T1 488.08 722.92 1114.83 1417.33 b 1725.88 5469.04 T2 481.38 693 , 83 1076.04 1389.12 to 1709.25 5349.63 T3 487.12 727.69 1097.77 1395.86 ab 1726.62 5435.07 T4 483.42 728.42 1113.79 1419.00 b 1728 , 50 5473.12
    [0173] Table 2: Average daily gain (g) (see also Figure 2)
    [0174]
    [0175] Lot 0 - 14 days 14 - 21 days 21 - 28 days 28 - 35 days 35 - 42 days 0 - 42 days T1 417.04 471.96 685.50 b 779.00 bc 873.88 3228.38 T2 411.25 396.38 656.12 to 765.25 to 869.75 3098.75 Q3
    [0176] T4
    [0177]
    [0178] Table 3: Conversion rate (see also figure 3)
    [0179]
    [0180] Lot 0 - 14 days 14 - 21 days 21 - 28 days 28 - 35 days 35 - 42 days 0 - 42 days
    [0181]
    [0182]
    [0183]
    [0184]
    [0185] a, b, c: statistical coefficients that indicate the significant differences between the different groups
    [0186]
    [0187] CONCLUSIONS:
    [0188]
    [0189] 1. Despite the infestation, the batches with the product of the invention obtain the same daily gain (0.5 kg / Tm) or even higher (1kg / Tm) than the NINT batch. The average daily gain is better than that of the INT group.
    [0190]
    [0191] 2. Despite the infestation, conversion rates similar to the control group (NINT) are obtained with the product of the invention . The conversion rate is higher than in lot INT.3
    [0192]
    [0193] 3. The INT lot shows a decrease in feed consumption during the 3rd and 4th week of life of the animals. This is because the infestation has produced a disease and that, as a consequence, the final weight and the conversion rate of this batch is worse than in the batch with the product of the invention.
    [0194]
    [0195] 4. The productive parameters are maintained with the product of the invention despite having more serious injuries than in the control lot (NINT). This is because the product does not prevent cell infestation, but promotes the elimination of coccidia.
    [0196]
    [0197] Example 5.2 . This second test was carried out by comparison of the product of the present invention against chemical coccidiostats in industrial poultry farming in Romania.
    [0198]
    [0199] Test design:
    [0200]
    [0201] TYPE OF STUDY: On own farms, at random
    [0202] ANIMALS: Broilers, Cobb, 0 to 42 days old.
    [0203] The study was carried out in three different farms: A, B and C
    [0204] EVALUATED PARAMETERS:
    [0205] - Presence of coccidia
    [0206] - Daily weight gain
    [0207] - Average weight
    [0208] - Mortality
    [0209]
    [0210] • FARM A (TREATMENT WITH PRODUCT of the invention 0.250 kg / T from day 0 until sacrifice.)
    [0211] - 4 buildings
    [0212] - BUILDING 1: 19320 animals.
    [0213] - BUILDING 2: 19350 animals.
    [0214] - Other unused buildings
    [0215]
    [0216] • FARM B (TREATMENT WITH CHEMICAL COCCIDIOSTATICS: Diclazuril 200g / T, 0 at 20 days, Lasallócido 600 g / T, 21 at 35 days. Suppression period, 35 at 42 days)
    [0217] - 10 buildings
    [0218] - BUILDING 3: 16950 animals.
    [0219] - BUILDING 4: 16950 animals.
    [0220] - Other unused buildings
    [0221]
    [0222] • FARM C (TREATMENT WITH PRODUCT of the invention 0.500 kg / T from day 0 until slaughter)
    [0223] - 10 buildings
    [0224] - BUILDING 5: 16800 animals.
    [0225] - BUILDING 6: 16300 animals.
    [0226] - Other unused buildings
    [0227]
    [0228] RESULTS:
    [0229] 1.- PRESENCE OF COCCIDES:
    [0230] - FARM A: Coccidia appear on day 19 in building 1 and on day 25 in building 2.
    [0231] - FARM B: Coccidia appear on day 24 in building 3 and on day 25 in building 4.
    [0232] - FARM C: No coccidia appeared
    [0233]
    [0234] Table 4.- DAILY GROWTH (g / day):
    [0235] Week 1 2 3 4 5 6
    [0236] Technological standard 17.6 38.0 59.0 79.1 88.6 87.0
    [0237]
    [0238] FARM A
    [0239]
    [0240]
    [0241] FARM B
    [0242]
    [0243]
    [0244] FARM C
    [0245]
    [0246]
    [0247] Table 5.- WEIGHT AVERAGE (g):
    [0248]
    [0249] Week 1 2 3 4 5 6
    [0250] Technological standard 164 430 843 1397 2017 2626
    [0251]
    [0252] FARM A
    [0253] B2 145 402 810 1170 1474 1950
    [0254] B3 150 406 797 1300 1705 2273 FARM B
    [0255] B4 152 400 805 1305 1720 2300
    [0256] B5 151 427 897 1402 1937 2504 FARM C
    [0257]
    [0258]
    [0259] Table 6. MORTALI IDAD:
    [0260] Acumu Week 1 2 3 4 5 6
    [0261] Lada B1 0.65 0.38 0.30 1.19 1.55 0.45 4.52 FARM A
    [0262] B2 1.7 0.30 0.68 2.26 0.99 0.55 6.56 B3 0.81 0.48 0.24 0.87 1.38 0.49 4.27 FARM B
    [0263] B4 0.68 0.38 0.33 1.16 1.17 0.58 4.3 B5 0.41 0.30 0.36 0.51 0.62 1.62 3.8 FARM C
    [0264] B6 0.74 0.33 0.27 0.50 0.53 1.19 3.56
    [0265]
    [0266] CONCLUSIONS:
    [0267] 1. The best results were obtained on farm C with the product of the invention at doses of 0.5 kg / T.
    [0268] 2. In farm C with the product of the invention at doses of 0.5 kg / T it is the only one where coccidiosis is not observed.
    [0269] 3. In farm C with the product of the invention at a dose of 0.5 kg / T, better weight and lower mortality are achieved.
    [0270] 4. The product of the invention is effective as an optimizer of the intestinal mucosa with better results than chemical coccidiostats.
    [0271]
    [0272] If we compare the results of the farm with farm B (chemical coccidiostats), we can conclude that with the product of the invention, a production of 231 tons more meat is achieved for every 1,000,000 broilers.
    [0273]
    [0274] Example 5.3 . This third test was carried out by comparison of the product against vaccinations against coccidiosis in industrial poultry farming in El Salvador.
    [0275] Test design:
    [0276] - Farm of 83,000 chicks, from 1 to 16 weeks
    [0277] - Product of the invention against vaccination.
    [0278] - Lot distribution:
    [0279] Building 2B Building 3B
    [0280] Product of the invention / without vaccination Only vaccination
    [0281] 16,561 animals 15,952 animals
    [0282] Average weight: 36 g Average weight: 36 g 5 * 10
    [0283]
    [0284] Table 7
    [0285]
    [0286]
    [0287]
    [0288]
    [0289] CONCLUSIONS:
    [0290] • The administration of the product of the invention achieves, compared to vaccination:
    [0291] S Lower mortality (%), 2.53% vs. 3.20
    [0292] S Greater uniformity, 90% versus 85%, meaning uniformity the deviation of the weight of the animal with respect to the average weight of the group, ie the homogeneity of the weight within a group)
    [0293] S Similar weight and conversion rate
    [0294] Example 5.4. This fourth test includes the technical report of the project for the analysis of the molecular level responses of an intestinal optimizer.
    [0295]
    [0296] 5.4.1. Obtaining blood samples
    [0297] First, several blood samples were obtained from healthy adult birds, making three independent groups.
    [0298]
    [0299] 5.4.2. Obtaining a fraction of PMNs
    [0300]
    [0301] Once the heparinized blood samples were obtained, the cell fraction corresponding to the polymormonuclear cells (PMNs) was isolated by applying a ficoll gradient. For this, 12 ml of diluted blood 1: 1 in SBS (Salts Balanced Solution) on 9 ml of Ficoll-Paque PLUS was added without mixing both phases. After centrifuging at 400gs for 40 min at 20 ° C, the PMN phase was transferred to a new tube with a pasteur pipette and 3 washes were performed with 3V of SBS (100gs, 10 min, 20 ° C). PMNs from various extractions were resuspended in the appropriate volume of DMEM medium supplemented with fetal bovine serum and added in 12-well cell culture plates to obtain densities of approximately 105-106 cells / well.
    [0302]
    [0303] 5.4.3. Obtaining sporozoites from Eimería
    [0304]
    [0305] In collaboration with a parasitology laboratory, inocula of approximately 105 / ml of sporozoites of the species Eimería acervulina, Eimería maxima, Eimeria praecox and Eimeria tenella were obtained.
    [0306]
    [0307] 5.4.4. Treatments
    [0308] The following in vitro treatments were performed :
    [0309] 1 Negative control: PMEM medium DMEM (37 ° C 5% CO23 hours) - 3 samples 2- Eimeria treatment: PMN + Eimeria sporozoites 10: 1 approximately in DMEM medium (37 ° C 5% CO 2 3 hours) - 3 samples
    [0310] 3- Treatment with product of the invention: PMN product of the invention at a 1: 10,000 dilution in DMEM medium (37 ° C 5% CO 2 3 hours) - 3 samples
    [0311] 4- Treatment with the product of the invention Eimeria : PMN product of the invention (37 ° C 5% CO 2 1 hours) Eimeria 10: 1 sporozoites approx. in DMEM medium (37 ° C 5% CO 2 2 hours) - 3 samples
    [0312]
    [0313] 5.4.5 Obtaining RNA, cDNA and qPCR from key genes in responses against intracellular pathogens such as Eimeria
    [0314]
    [0315] The RNA was extracted from the PMNs after the treatments and the cDNA was synthesized.
    [0316]
    [0317] Each cDNA sample was used as a template for a quantitative PCR in order to analyze the transcriptional variation of the following genes:
    [0318]
    [0319] - ILs 1, 12, 18: relevant pro-inflammatory interleukins in infection processes by intracellular organisms
    [0320] - GADPH: gene housekeeping
    [0321]
    [0322] The results were analyzed by normalizing the difference in cycles with respect to the housekeeping gene (GAPDH), using the DcT calculation.
    [0323]
    [0324] Results for IL-1 (see figure 6), IL-12 (see figure 7), IL-18 (see figure 8)
    [0325]
    [0326] A: CONTROL
    [0327] B: Product of the invention 1: 10,000
    [0328] C: Eimeria 1:10
    [0329] D: Product of the invention + Eimeria
    [0330]
    [0331] IL-1:
    [0332] - Promotes the secretion of chemokines and cytokines by fibroblasts, macrophages and epithelial cells.
    [0333] - Attracts other inflammatory cells, such as macrophages, heterophiles and lymphocytes.
    [0334] - Amplifies the immune response.
    [0335]
    [0336] IL-12:
    [0337] - Triggers the Th1 immune response (T lymphocytes), which also plays an important role in the fight against coccidiosis (adaptive immunity).
    [0338] - Activates NK cells (cytotoxicity) and macrophages (phagocytosis).
    [0339] Increase the duration of the cellular immune response.
    [0340]
    [0341] IL-18:
    [0342] - Induces the activation of NK cells and cytotoxic T lymphocytes.
    [0343] - Stimulates the production of interferon gamma (macrophage activation and Th1 immune response).
    [0344] - Increases the effect of IL-12.
    [0345]
    [0346] CONCLUSIONS:
    [0347] The immuno-stimulation properties shown by the product of the invention may be due, among other effects, to the induction of pro-inflammatory interleukins IL-1, IL-12 and IL-18 at the transcriptional level. These molecules are essential to fight infections by intestinal parasites such as Eimería:
    [0348] - IL-1 synthesizes chemokines and cytokines to attract inflammatory cells
    [0349] - IL-12 and IL-18 promote the activation of the cellular immune response
    [0350] There is a greater expression of IL-1, IL-12 and IL-18 when the product of the invention is administered (as compared to the control).
    [0351] There is a greater expression of IL-1 and IL-12 when Eimeria infection occurs after exposure to the product of the invention.
    权利要求:
    Claims (17)
    [1]
    1. - Animal food product comprising carvacrol, p-cimenol and alliin.
    [2]
    2. - Food product according to claim 1, wherein carvacrol is present in at least 3.4% by weight in said product.
    [3]
    3. - Food product according to any of the preceding claims, wherein the p-cimenol is present in at least 2.8% by weight in said product.
    [4]
    4. - Food product according to any of the preceding claims, wherein the alliin is present in at least 7.8% by weight in said product.
    [5]
    5. - Food product according to any of the preceding claims, wherein the carvacrol is contained in Origanum vulgare flour .
    [6]
    6. - Food product according to claim 5, wherein carvacrol is present in the flour of Origanum vulgare in a weight percentage between 17 and 20% by weight.
    [7]
    7. - Food product according to any of the preceding claims, wherein the p-cimenol is contained in an extract of Thymus vulgaris.
    [8]
    8. - Food product according to claim 7, wherein p-cimenol is present in said extract in a weight percentage between 14 and 16% by weight.
    [9]
    9. - Food product according to any of the preceding claims, wherein the alliin is contained in an extract of Allium sativum.
    [10]
    10. - Food product according to claim 9, wherein the alliin is present in said extract in a weight percentage between 13 and 14% by weight.
    [11]
    11. - Procedure for obtaining an animal food product comprising carvacrol, p-cimenol and alliin, comprising the steps of:
    (a) obtain an extract of carvacrol from the leaves of Origanum vulgare;
    (b) obtain an extract of p-cimenol from the leaves of Thymus vulgaris;
    (c) obtain an alliin extract from the bulb of Allium sativum;
    (d) mix the extracts from steps (a), (b) and (c) until homogeneous.
    [12]
    12. - Method according to claim 11, wherein step (a) comprises the following sub-stages:
    (a1) grind and dry the leaves of Oríganum vulgare to obtain a powder product; (a2) macerate the powder obtained in (a1) in ethanol;
    (a3) solidify the oleoresin obtained in (a2) by absorption with silica gel and evaporate the solvent;
    (a4) centrifuge.
    (a5) discard the aqueous phase.
    [13]
    13. - Method according to claim 12, wherein in step (a4) it is centrifuged at 6000 rpm for 30 minutes.
    [14]
    14. - Method according to any of claims 11 to 13, wherein step (b) comprises the following sub-stages:
    (b1) cold press the leaves of Thymus vulgaris;
    (b2) suspend in water the extract obtained in (b1);
    (b3) distill the oil from the leaf extract;
    (b4) extract the oil from the aqueous phase;
    (b5) extract with a base the phenolic content of the oil and solubilize it in water;
    (b6) extract the p-cimenol with ether;
    (b7) evaporate the ether.
    [15]
    15. - Method according to any of claims 11-14, wherein step (c) comprises the following sub-stages:
    (c1) suspend Allium sativum teeth in vegetable oils under negative pressure and grind;
    (c2) filter to separate the skins
    (c3) let stand and decant approximately half the volume of the oil phase; (c4) add an aqueous solution of 0.5-1.5 mM citric acid;
    (c5) treat with ethyl p-hydroxybenzoate in a 1: 5 ratio and an emulsifier;
    (c6) homogenize between 30-35 minutes;
    (c7) use a bentonite to absorb the liquid phase and obtain the powder extract.
    [16]
    16. - Method according to claim 15, wherein in step (c1) the vegetable oil is sunflower oil.
    [17]
    17. - Process according to claim 15 or 16, wherein in step (c4) the aqueous citric acid solution has a concentration of 1 mM.
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    同族专利:
    公开号 | 公开日
    ES2730385B2|2020-05-11|
    CO2018011577A1|2020-05-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2008049437A1|2006-10-26|2008-05-02|Biofiber-Damino A/S|Composition comprising lignin and antidi arrheal component|
    WO2009019255A2|2007-08-03|2009-02-12|Vetagro S.R.L.|Synergetic composition comprising flavouring substances and organic acids and use thereof|
    WO2017004161A1|2015-07-02|2017-01-05|Novus International Inc.|Antimicrobial compositions and uses thereof|
    US20170173059A1|2015-12-17|2017-06-22|Nicholas Magrone, JR.|Novel Combination of Naturally Occurring Compounds to Assist With Suspected Mycobacterial Infections Related to Autoimmune Conditions|
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    ES201830456A|ES2730385B2|2018-05-09|2018-05-09|FOOD PRODUCT FOR ANIMALS THAT INCLUDES CARVACROL, P-CIMENOL AND ALIINA, AND PROCEDURE FOR THEIR OBTAINING|ES201830456A| ES2730385B2|2018-05-09|2018-05-09|FOOD PRODUCT FOR ANIMALS THAT INCLUDES CARVACROL, P-CIMENOL AND ALIINA, AND PROCEDURE FOR THEIR OBTAINING|
    CONC2018/0011577A| CO2018011577A1|2018-05-09|2018-10-26|Alquernat zycox food product for animals comprising carvacrol, p-cimenol and alliin, and procedure for obtaining them|
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