Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
  • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/12—Antidiarrhoeals
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/21—Streptococcus, lactococcus

Definitions

• the present invention belongs to the pharmaceutical area, particularly to the field of medicinal compositions for veterinary use.
• the invention relates to a microbial composition comprising probiotic microorganisms to reduce diarrhea and increase the vitality of neonatal cattle. DESCRIPTION OF THE STATE OF THE TECHNIQUE
• probiotics as living microorganisms that when administered in adequate doses, produce beneficial effects on the health of the recipient entity [1].
• a probiotic product is composed of microorganisms that survive and can be implanted in different organs of the digestive tract such as the stomach, small intestine or colon, with the aim of improving the functioning of the host's intestinal flora, helping to completely degrade food for subsequent absorption [2].
• probiotics in the intestinal flora can induce some proteins to undergo conformational changes and thereby activate intracellular biochemical mechanisms that favor the production of inflammation mediators, promoting cell differentiation or cell apoptosis and activating the immune response against any possible infection [3].
• Ruminant mammals have a very particular digestive morphology and physiology.
• the ability of ruminants to take advantage of fibrous carbohydrates from the diet is due to rumen, reticulum and omasum, which are the organs that precede the abomasum [4].
• the rumen is a fermentation chamber with anaerobic environment and variable pH that allows high retention of long particles of forage and stimulates rumination and body metabolism, maintaining an appropriate environment for the growth and reproduction of microorganisms.
• Ruminal microorganisms are favored by the absence of oxygen, the product of urea hydrolysis, a process that requires oxygen consumption by bacteria attached to the wall. These microorganisms have the ability to digest complex polysaccharides (e.g. cellulose, hemicellulose, pectin) to produce carbohydrates and also take advantage of non-protein nitrogen for the synthesis of amino acids and proteins [5].
• complex polysaccharides e.g. cellulose, hemicellulose, pectin
• the gastrointestinal tract of the calves is sterile and the microorganisms of the intestinal flora are only introduced from contact with their mothers.
• calves are separated from their mothers at birth and fed with milk substitutes, without allowing them to even feed on colostrum, which significantly alters the development of their intestinal flora. Consequently, in these production systems, the main cause of disease in calves up to three months of life is diarrhea.
• antibiotic agents can be used, which can generate an undesirable phenomenon of antimicrobial resistance, or probiotic products based on microorganisms can be used.
• probiotic products based on microorganisms
• Some commercial probiotic products for cattle such as Prokura®, Provita®, BioBoost® and Probios Calf® contain non-ruminal aerobic microorganisms, such as Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium bifidum, and Bacillus subtilis [7].
• US 3956482 describes a composition of ruminal microorganisms comprising Megasphaera elsdenii, Streptococcus boviss, Lactobacillus acidophilus, Bifidobacterium teenagers, Bacteroides ruminicola and Butyrivibrio fibrisolvens, which are adapted in a nutrient medium and administered to the animal during the first 24 hours and / or in the period between 80 and 140 days old.
• WO 2012147044 discloses a method for reducing methane production in ruminants which comprises administering a mixture of bacteria strains of the genus Propionibacterium and Lactobacillus, preferably Propionibacterium jensenii P63, Lactobacillus plantarum Lpll5 and Lactobacillus rhamnosus Lr32. Similarly, the document notes that the administration of these microorganisms can also stimulate the growth of the animal.
• the present invention relates to a microbial composition
• a microbial composition comprising at least one probiotic microorganism selected from the group consisting of Fibrobacter succinogenes, Ruminococcus flavefaciens, Streptococcus bovis and Butytrivibrio fibrisolvens, together with adjuvants and an acceptable vehicle.
• the composition of the invention exhibits adequate efficacy in reducing the incidence of diarrhea and promotes weight gain in newborn bovines.
• FIG 1. Corresponds to the results of the Log viability (CFU / ml) of the microbial composition of Example 2 stored at 4 ° C +/- 2 ° C for 6 months. Treatments with the same letter do not show significant differences according to Tukey's test (95%).
• FIG 2. Corresponds to results of the Log viability (CFU / ml) of the microbial composition of Example 2 stored at 18 ° C +/- 2 ° C for 6 months. Treatments with the same letter do not show significant differences according to Tukey's test (95%).
• the microbial compositions of the invention comprise at least one probiotic microorganism as an active ingredient, adjuvants and an acceptable carrier.
• Probiotic microorganisms according to the present invention may be, among others, facultative anaerobic bacteria or strict anaerobic bacteria. The definition, characteristics and properties of each of them can be found in detail in the text Manual of Determinative Bacteriology [9] which is incorporated in its entirety as a reference.
• the compositions comprise, as an active ingredient, anaerobic microorganisms selected from the group consisting of Fibrobacter succinogenes, Ruminococcus flavefaciens, Streptococcus bovis and Butytrivibrio fibrisolven, which can be quantified, serving as measurement units, the concentration and their viability.
• concentration of each of said microorganisms of the active ingredient of the present invention is between 1x10 3 and 1x1011 CFU / ml, more preferably between 10 x 6 and 10 10 CFU / ml, and even more preferably, 10 9 CFU / ml.
• the active ingredient may be contained in water, in a solvent, in a mixture of solvents, in a liquid culture medium, in a lyophilisate, in an aqueous suspension or in a concentrated paste, either in equal or different amounts of each of probiotic microorganisms.
• the compositions of the invention include, in addition to the active ingredient, different adjuvants with specific functions to give shape and characteristics to the final presentation (eg form emulsions, regulate pH, improve stability and increase shelf life).
• the concentration of the active ingredient in the compositions of the invention is preferably between 0.1% and 99.9% (w / w), more preferably between 20.0% and 60.0% (w / w) and even more preferably , at 40.0% (w / w).
• Coadjuvants include all those known in the technical field, including water, organic solvents, mineral oils, vegetable oils such as soybean oil, corn oil, cane oil, olive oil, coconut oil , wheat germ oil and mixtures thereof, polysorbates, polyols, polymers, lipids, saponifiable lipids, support substances (eg kaolin, talc, bentonites, silicates), diluents, emulsifying agents, viscous agents, surfactants, pH regulators, stabilizers and dyes.
• water organic solvents
• mineral oils such as soybean oil, corn oil, cane oil, olive oil, coconut oil , wheat germ oil and mixtures thereof
• polysorbates eg kaolin, talc, bentonites, silicates
• diluents emulsifying agents, viscous agents, surfactants, pH regulators, stabilizers and dyes.
• the concentration of the adjuvants in the compositions of the invention is preferably between 0.01% and 99.99% (w / w) and more preferably, between 0.1% and 60 , 0% (p / p).
• Emulsifying agents include, but are not limited to polysorbates, sorbitan esters, noniphenol, sodium lauryl sulfate and mixtures thereof.
• Viscose agents include, but are not limited to polymers, gums, hydrocolloids, finely divided solids, waxes and mixtures thereof.
• PH regulating agents include but are not limited to carbonates, phosphates, citrates and borates.
• acceptable vehicle for purposes of the present invention can be defined as a mixture of substances (eg solvents, solutions, emulsions and suspensions) capable of containing the active ingredient and / or adjuvants, without affecting its ability to Perform the desired function.
• substances eg solvents, solutions, emulsions and suspensions
• compositions of the invention may be in the form of powders, soluble granules, dispersible granules, dispersible tablets, suspension or emulsion.
• soluble granulate is intended to include granules for application after dissolution of the active ingredient in water as a solution, optionally containing insoluble formulation aids.
• dispenser granulate refers to granules for application in suspension form, after disintegration and dispersion in water or other aqueous solvent.
• the term "dispersible tablet” refers to a formulation in the form of tablets to be used individually to form a suspension of the active ingredient after its disintegration in water.
• the term “suspension” refers to liquids containing the active ingredient and adjuvants stably suspended, either to be applied directly or diluted in water.
• the term “emulsion” is intended to include heterodispersed systems with different degrees of viscosity, which give rise to liquid or semi-solid systems, which can be encapsulated or not, and used to generate solid pharmaceutical forms.
• an emulsion type composition can be prepared by mixing an aqueous phase containing the active ingredient, with an oil phase containing emulsifying agents. Once the two phases have been mixed, the emulsion is gasified with C (3 ⁇ 4 and the pH regulators and stabilizing agents are added.
• the microbial composition of the invention is in the form of an emulsion, it has as an active ingredient anaerobic probiotic microorganisms and adjuvants such as emulsifiers, polymers and pH regulators that improve the viability, efficacy and shelf life of the product.
• the microbial composition of the invention is a water-oil emulsion (W / O), where anaerobic microorganisms are in the aqueous phase of the emulsion (internal phase), coated by the oil phase (phase external) that provides protection against oxygen from the external environment.
• the aqueous phase of the emulsion is a suitable culture medium containing the microorganisms, while the oil phase of the emulsion can be formed, among others, by vegetable oils, polysorbates and saponifiable lipids that favor the formation of the emulsion W / OR.
• suitable culture medium refers to any culture medium that contains the sources of nutrients and trace elements necessary for the growth of anaerobic microorganisms.
• the suitable culture medium comprises glucose, Yeast extract, an indicator of anaerobiosis, sodium bicarbonate, cysteine-HC1, volatile fatty acids, KHPO4, KH2PO4, ammonium sulfate, NaCl, MgS0 4 and CaCi2 at concentrations between 0.0001 and 100.0 g / L of each one of them.
• EXAMPLE 1 Obtaining strains of Butyrivibrio f ⁇ brisolvens (B9), Streptococcus bovis (CP, Ruminococcus flavefaciens (Rf) and Fibrobacter succinosenes (Fs) of the active ingredient of the Microbial Composition) and from a wild herbivore.
• Butyrivibrio f ⁇ brisolvens (B9) was isolated from a bovine of the Holstein-Friesand breed
• Streptococcus bovis (C2) was isolated from a bovine from the Cauca Valley region of the valley fed race
• Ruminococcus flavefaciens ( Rf) was isolated from a bovine of the Lucerne and Fibrobacter succinogenes (Fs) breed and was isolated from the blind of a Chigüiro in the Casanare region (Colombia) .
• the strains were reactivated in a culture medium rich in cellobiose-glucose and incuba
• the strains are stored in the Germplasm Bank of Microorganisms with Interest in Animal Nutrition of CORPOICA (BGMINA).
• a composition in the form of W / O emulsion was prepared with a mixture of Butyrivibrio f ⁇ brisolvens (B9), Streptococcus bovis (C2), Ruminococcus flavefaciens (Rf) and Fibrobacter succinogenes (Fs), as active ingredient.
• B9 Butyrivibrio f ⁇ brisolvens
• C2 Streptococcus bovis
• Rf Ruminococcus flavefaciens
• Fs Fibrobacter succinogenes
• this oil phase was mixed with the aqueous phase (culture medium of each bacterium in a 1: 1 ratio) with the aid of a Dynamic® homogenizer at the maximum agitation level for 5 minutes.
• the emulsion formed was also gasified with C (3 ⁇ 4. Table 1 shows the concentrations of each component.
• Example 2 To a microbial composition obtained according to Example 2, its stability was determined under storage conditions. The samples were stored at a temperature of 4 ° C +/- 2 ° C (TI) and 18 ° C +/- 2 ° C (T2), for 6 months. To carry out the test, 12 ml of the microbial composition was packed in a high density polypropylene dosing syringe, which corresponded to the experimental unit of each treatment. The stability study had a completely randomized experimental design with repeated measurements over time and all measurements were performed in triplicate. The results of the stability study were subjected to an analysis of variance and then to comparisons of means using the Tukey test (95%).
• Table 3 shows the values pH obtained in the three samples evaluated at each temperature, which are close to 7.0. [10].
• FIG 1 The feasibility results obtained for each of the treatments are illustrated in FIG 1 and in FIG 2.
• FIG 1 the results of the treatments stored at 4 ° C are observed, where it is evidenced that after 6 months of storage there was a significant reduction in viability with respect to zero time.
• the concentration of microorganisms is not less than 1x10 ( Figure 1).
• Group 1 Administration of fresh microbial composition.
• Group 2 Administration of microbial composition stored for 6 months.
• Control Group No microbial composition was administered. In a randomized complete design with a 3x2 factorial, 2 variables were analyzed: incidence of diarrhea and body weight gain. Each calf in groups 1 and 2 received 12 doses of 10 mL / day orally of a microbial composition according to Example 3. The microbial composition was administered for 10 consecutive days, starting on the day of birth (DI), while The next two doses were given at 15 and 30 days (DI 5 and D30).
• the weight gain of the calves was determined by monthly weighing with electronic scale, starting from the DI until three months of age.
• the presence of diarrhea was determined by direct observation in each animal and the frequency was recorded.
• the microbial composition tested showed that it reduces the incidence of diarrhea and increases body weight gain in the animals evaluated. The results are shown in Table 4.

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Cited By (3)

Citations (3)

• 2015
  • 2015-08-07 WO PCT/IB2015/056033 patent/WO2017025772A1/es not_active Ceased
  • 2015-08-07 BR BR112018002582-8A patent/BR112018002582B1/pt active IP Right Grant
  • 2015-08-07 US US15/751,083 patent/US20180228181A1/en not_active Abandoned
• 2018
  • 2018-02-07 CO CONC2018/0001271A patent/CO2018001271A2/es unknown

Patent Citations (3)

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