WO2020232519A1 - Nutritional compound formed by bacterial fermentation content for use as a supplement or additive for animal feed - Google Patents

Nutritional compound formed by bacterial fermentation content for use as a supplement or additive for animal feed Download PDF

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WO2020232519A1
WO2020232519A1 PCT/BR2020/050168 BR2020050168W WO2020232519A1 WO 2020232519 A1 WO2020232519 A1 WO 2020232519A1 BR 2020050168 W BR2020050168 W BR 2020050168W WO 2020232519 A1 WO2020232519 A1 WO 2020232519A1
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animal feed
additive
supplement
grains
content
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PCT/BR2020/050168
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French (fr)
Portuguese (pt)
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Bernardo DE LEÃO ROSENMANN
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De Leao Rosenmann Bernardo
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/33—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from molasses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention reports the production of a nutrient broth by means of fermentative processes using a genetically modified non-pathogenic bacterium. Genetic and metabolic engineering techniques were used to develop a strain of Corynebacterium glutamicum capable of producing aminolevulinic acid (5-ALA).
  • the nutritional broth obtained contains sugars, vitamins, amino acids, including 5-ALA, organic acids and nucleotides, and is added to the animal feed guaranteeing benefits, such as greater growth and better immunity of animals such as cattle, pigs, birds, fish and small animals animals like dogs and cats (pet food).
  • the fermentative processes are carried out from the use of different microorganisms, being bacteria or fungi, for the production of several biomolecules that are of commercial interest.
  • Microorganisms metabolize different substrates present in the reaction medium, generating primary metabolites, for example, amino acids, and secondary ones, such as antibiotics.
  • Fermentation processes are used in industries of various branches, such as food, cosmetic, pharmaceutical and livestock. On an industrial scale, fermentation technology is involved in the production of amino acids, vitamins, enzymes, organic acids, among other biomolecules.
  • 5-Aminolevulinic acid is an amino acid present in plants and animals. This amino acid can be found in some foods, such as red wine and tea (KR 101 180266). 5-ALA is a precursor to biomolecules such as chlorophyll and vitamin 12 (CN 101863703). The biosynthesis of 5-ALA generally occurs from succinyl-CoA and glycine by the action of the enzyme 5-aminolevulinic acid synthase (US 9333156).
  • 5-ALA already known for its wide application, being used in cancer treatments, acne, among other skin problems, as well as in plants to improve chlorophyll production and promote better plant growth (KR 20100083425, KR 101 180266, CN 101863703, KR 20090027269). When applied to livestock animal feed, chicken, among others, it increases immunity and promotes growth (KR 20100083425, CN 102870970, KR 20090027269).
  • Certain known and studied microorganisms are used in the industrial production of metabolites. These microorganisms are of great commercial interest because they are easy to manipulate, safer, have less contaminants and obtain greater production of biomolecules on a large scale. Thus, different genes are introduced in these microorganisms in order to obtain several metabolites of commercial interest that are difficult to be produced on a large scale by their origin organisms.
  • Corynebacterium glutamicum is a microorganism of commercial interest. As it is a non-pathogenic microorganism, it is used in industry for the production of L-glutamate and other amino acids, presenting a potential for the production of organic acids and biofuels.
  • C. glutamicum is applied in the biotechnology industry and has been studied for the production of 5-ALA, due to the fact that L-glutamate is a precursor to ALA and the microorganism is considered safe, presenting no risks to human health ( YU et al., 2015).
  • the amino acid 5-ALA can be converted into hemoglobin in the body and promote the development and maturation of red blood cells.
  • Hemoglobin is an important molecule in the transport of oxygen, responsible for dilating blood vessels, maintaining a stable pH in the body and promoting the healthy growth of the animal (CN 102870970).
  • Higher hemoglobin production directly interferes with oxygen and nutrient transport in cattle, for example, increases resistance to disease and activates metabolism (KR 100459918).
  • the amino acid 5-ALA has some benefits for animals such as cattle, pigs, chickens, as well as animals such as fish, dogs and cats. Among these benefits are promoting greater growth of animals, increasing immunity avoiding / reducing the use of antibiotics, improving physical size and metabolism and in the case of birds, increasing egg production (KR 20100083425, KR 20090027269, CN 102870970, KR 100459918).
  • the medium from fermentation with the bacterium C. glutamicum must be added to the animal feed, together with the 5-ALA produced in the process, which may contain other amino acids, sugars, nucleotides, organic acids and vitamins (CN 102870970, KR 100459918).
  • KR patent No. 100459918 reports the production of an animal food supplement (livestock) containing aminolevulinic acid.
  • the amino acid can come from fermentation, and it can be the fermented product, containing ALA, added to the feed.
  • the inventors do not describe the composition of this fermented medium or the microorganism used.
  • the present invention is differentiated by the fact that the fermented broth from the fermentation of genetically modified C. glutamicum is incorporated into the animal feed, the active ingredient being the broth containing sugars, amino acids, including the 5-ALA produced by fermentation, organic acids , vitamins and nucleotides.
  • 2012136786 depicts a feed additive for chickens and pigs containing 5-ALA and a fermented mixture of Sanguisorba officinalis and Rhus japonica by Aspergillus oryzae or Lactobacillus casei.
  • the amino acid was added to a fermented mixture, it does not originate from fermentation and the entire mixture is considered as the active ingredient.
  • Patent KR No 20100040165 also describes a mixture of a fermentate with the addition of 5-ALA. In that case, chitosan is fermented. The mixture of fermented chitosan and 5-ALA guarantees a better growth rate and immunity.
  • Patent application KR No 20090027269 exposes an animal feed composition to replace the use of antibiotics that presents different complex compounds such as corn, molasses and soy, and ALA.
  • the amino acid is added to make up animal feed.
  • ALA is responsible for increasing the animal's immunity, making it possible to give less antibiotics.
  • the present invention describes a composition to be added to the animal feed, the fermented broth containing the amino acid ALA, in which case the amino acid is produced by the fermentative process and not added to the medium.
  • Chinese patent application No. 1 02870970 describes a growth promoter for livestock animals.
  • the promoter is based on a mixture of amino acids, such as 5-ALA and lysine, in addition to coenzyme Q10, and a carrier is used, the zeolite powder.
  • the invention described here does not use a carrier for the fermented medium and the 5-ALA in addition to the rest of the broth composition comes from fermentation.
  • WO publication 2007141 1 1 1 reports a process for the production of an animal feed additive based on a fermented medium containing a high content of L-lysine. Fermentation takes place using corineform bacteria selected from mutations. The invention reports the possibility of the bacterial biomass being incorporated into the animal feed together with the fermented broth, since the bacterium is considered safe, such as C. glutamicum.
  • patent number US5840358A describes the use of a product obtained by fermentation, so that the process uses genetically modified E. coli or C. glutamicum for the production of L-lysine. The resulting final product consists of nutrient-rich biomass and fermented broth with a high concentration of L-lysine produced by microorganisms.
  • the invention described here reports the use of the genetically modified non-pathogenic bacteria C. glutamicum used in a fermentation process aimed at the production of 5-ALA.
  • the final product, to be used as animal feed, is characterized by a mixture of different components where only the fermentation broth or that together with the inactive bacterial biomass can be incorporated into the final formulation.
  • the present invention deals with the use of genetically modified strains of C. glutamicum in order to produce 5-aminolevulinic acid.
  • the differential proposed here involves the use of the fermented broth of the said strains composed of a mixture rich in nutrients, in addition to 5-aminolevulinic acid, used as an additive or supplement for animal feed such as cattle, goats, birds, pigs, fish and dogs and cats (pet food).
  • the steps involved in the manufacture of the final product are characterized by (a) a fermentative process using a C. glutamicum strain producing 5-ALA, (b) obtaining the fermented broth containing the biomass or separation of biomass and fermented broth by centrifugation or filtration, (c) reduction of pH to preserve the product and inactivation of biomass, (d) partial or total drying of the fermented broth with or without biomass and (d) preparation of the final product by mixing the fermented broth containing or not biomass with other components that make up animal feed.
  • the C. glutamicum strain ATCC 13032 used as a chassis for genetic modifications was used because it is a wild strain and its genome is completely sequenced (Ikeda, 2003).
  • the production of 5-ALA in C. glutamicum can occur by modulating gene expression by increasing the number of copies of the gene of interest to be inserted into the chromosome, using a regulated or constitutive promoter, in order to induce, increase or decrease expression of the desired gene, alteration of the initiation codon, or use of the gene in a plasmid that has stability when inserted into the strain of interest.
  • hemA of the bacterium Rhodobacter capsulatus optimized by means of a specialized algorithm for expression in C. glutamicum.
  • the gene codes for the enzyme 5-aminolevulinate synthase (ALAS), which produces 5-aminolevulinic acid from L-glycine and succinyl-CoA;
  • ALAS 5-aminolevulinate synthase
  • c) be A, serB and serC, from the strain itself, which results in the production of the enzymes phosphoglycerate synthase, phosphoserine aminotransferase and phosphoserine phosphatase, respectively;
  • araA, araB, araD and araE from E. coli, which code for the enzymes arabinose isomerase, ribulokinase, ribulose-5-phosphate-4-epimerase, and for the arabinose transporter, respectively f) glory, glpK and glpD of Corynebacterium diphteriae, which code for the glycerol carrier protein and for the enzymes glycerol kinase and glycerol-3-phosphodihydrogenase, respectively.
  • the resulting genetically modified strain can be used in fermentative processes that use different types of culture medium, and may contain different types of carbon and nutrients.
  • the fermentation process for the production of said mixture rich in nutrients containing different compounds, such as saccharides, nucleic acids, lipids and amino acids, including 5-aminolevulinic acid must contain: a) at least one carbon source, such as for example glucose, xylose, arabinose, among others, as long as the strain is able to metabolize these molecules;
  • At least one nitrogen source such as yeast extract, peptone or inorganic salts
  • At least one source of phosphorus such as monobasic sodium phosphate, dibasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, or another phosphorus inorganic salt that is assimilated by said microorganism;
  • inorganic salts derived from minerals or vitamins, such as sodium chloride, calcium carbonate, ammonium sulfate, among others, as long as they are assimilated by the said organism.
  • at least one organic acid such as citric acid, succinic acid, acetic acid, among others.
  • the process for producing the fermented broth should preferably be carried out in bioreactors with controlled conditions of temperature, pH, food, agitation and aeration. This process can also be carried out in flasks such as erlenmeyers or similar flasks, provided that the conditions for biomass growth are provided.
  • the growth of the microorganism must be carried out in the ambient temperature ranges at 37 ° C, preferably from 25 ° C to 34 ° C, more preferably from 28 ° C to 32 ° C.
  • the growth of the microorganism must be carried out in a pH range of 5.0 to 7.5, preferably from 5.5 to 7.0, more preferably from 6.0 to 6.5.
  • the fermentation process can the growth of the microorganism can be initiated with concentrations of carbon source varying from 5g / L to 60g / L, preferably from 20g / L to 50g / L, more preferably from 30g / L L to 40g / L.
  • feeding can be carried out constantly or in pulses, as long as the concentration of the carbon source is maintained from 2g / L to 8g / L, preferably from 4g / L to 6g / L, more preferably from 5g / L.
  • the growth of the microorganism must be carried out with an agitation range that varies from 150 to 250 rpm and aeration between 1 to 3 vvm, preferably agitation from 180 to 230 rpm and aeration between 1, 5 at 2 vvm.
  • the growth of the microorganism must start with an inoculum where the concentration of cells corresponds to ⁇ .Odoo of value between 1 to 10, preferably 5 to 15, more preferably [035]
  • the fermentation process can take place for 12 to 96 hours, preferably for 24 to 60 hours, more preferably for 36 to 48 hours according to the conditions previously mentioned.
  • the total or partial content of the fermenter can be subjected to two processes:
  • the separation can be carried out by filtration or ultrafiltration, preferably in a vacuum, using 0.45 micron porosity filters, preferably 0.22 ⁇ m porosity,
  • the acid to be used in this step can be preferably citric acid, but not limited to it, and ascorbic acid, acetic acid, lactic acid, malic acid, fumaric acid, gluconic acid, among others, can also be used.
  • the fermented broth, containing the inactivated biomass or not can be mixed with other components for the formulation of animal feed in liquid form, or can be subjected to partial or total drying, under vacuum or not, under temperatures of 40 to 80 ° C, preferably from 50 to 70 ° C, more preferably from 55 to 65 ° C, so that the residual humidity is 0.1 to 99.9%, preferably from 5 to 90%, more preferably from 10 to 80 %, more preferably 20 to 50%.
  • the content obtained can then be used as powder, granules or liquid and be mixed with other compounds for the formulation of animal feed.
  • the product resulting from the fermentation process described above can be mixed with other components in different proportions in order to generate an animal feed formulation.
  • These other molecules can be saccharides, amino acids or peptides, nucleic acids and other acids organic, lipids, micronutrients such as inorganic salts and enzymes or growth factors.
  • the final formulation of the feed to be made available for cattle, poultry, pigs, goats, fish and small animals such as dogs and cats may comprise, in addition to the product from the described fermentation process, the following compounds:
  • a) maize grains and their by-products such as silage, corn, bran, cornmeal, flour, dry distillers grains (Distiller Dried Grain), dry distillers grains with soluble (Distiller Dried Grain with Solubles) and any other by-products obtained from of any type of processing involving corn grains,
  • soybeans and their by-products such as silage, husk, bran, flour, textured soy protein, micronized soy protein, concentrated soy protein, water-soluble soy extract, defatted flakes, lecithins and any other by-product obtained from any type of processing involving soybeans,
  • wheat grains and their by-products such as silage, husk, fine bran, coarse bran, flour and any other by-product obtained from any type of processing involving wheat grains
  • sorghum grains and their by-products such as silage, husk, bran, flour, dry distillers grains, dry distillers grains with soluble, processed sorghum protein, and any other by-product obtained from any type of processing involving grains of sorghum,
  • rice grains and their by-products such as silage, husk, fat bran, stabilized bran, chirera, starch, flour, endosperm protein, and any other by-product obtained from any type of processing involving rice grains,
  • rye grains and their by-products such as silage, husk, flakes, bran, flour, and any other type of by-product obtained from any type of processing involving rye grains
  • barley grains and their by-products such as silage, bark, flakes, bran, flour, bagasse, and any other type of by-product obtained from any type of processing involving barley grains
  • molasses or molasses from vegetables such as sugar cane, corn, soybeans, rice, sorghum, among others, processed in different ways, such as powder, concentrated liquid containing different degrees of humidity, or any other type of by-product obtained from production process of molasses or molasses.
  • the formulation of the animal feed can follow different composition patterns using the components described above, so that a mixture of all or some of the aforementioned ingredients can make up the final feed.
  • the formulation of animal feed may also contain additional molecules such as thickeners, emulsifiers, acidulants, texturizers, flavors, flavors, stabilizers, among others, so that the final product has acceptable and sensory characteristics for each animal to be destined .
  • the feed can be produced in different formats, such as powder, pellets, fine granules, coarse granules, paste, among others, whether dry or moistened, so that it represents the best condition for availability for the animal to be destined.
  • a single colony of strain NB_CgALA05 present in a microbiological plate is inoculated in 15 ml of medium containing 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride. This pre-inoculum is incubated at 30 ° C for 16 hours with shaking at 180 rpm. The previous sample is added to 500mL of the same medium and incubated under the same conditions, in order to generate a greater amount of biomass. Finally, the previous inoculum is diluted in 1 L of the medium for the fermentation process in a 5 L bioreactor.
  • the culture medium for the fermentation process is initially composed of 40g / L of glucose, 10g / L of tryptone, 5g / L of yeast extract, 10g / L of sodium chloride and 10g / L of L-glycine.
  • the fermentation process is maintained with a minimum of 4g / L and a maximum of 40g / L of glucose available in the medium and the pH maintained between 6.0 and 6.5 by the addition of sodium hydroxide or another base. Fermentation takes place for up to 72 hours at 30 ° C with 300rpm agitation and constant addition of 2 v.v.m. of oxygen.
  • the total content is subjected to pH reduction by adding citric acid until the pH reaches a value close to 3.0. This process guarantees the complete inactivation of biomass. Afterwards, the product is subjected to drying until it contains between 10 and 15% moisture and then stored at 4-6 ° C.
  • the mixture for formulating animal feed is carried out by mechanically stirring the solid components in the form of powder or fine granules.
  • the ration described here consists of 0.1 -1% of the product from the process fermentative, 50-70% corn by-product from ethanol production (in this case dry distillers grains with soluble - DDGS), 10-17.5% hydrolyzed yeast, 10-30% soy bran , 5-10% of sugar cane molasses and 1.5-5% of components, such as thickeners, flavors, acidulants, among others.
  • the components are mixed in an industrial feed mixer with 480 rpm agitation for about 1 hour at room temperature. After the mixing process is complete, the feed is made available to chickens in the form of fine bran feed.
  • IKEDA M.
  • NAKAGAWA S.

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Abstract

The present invention describes the composition of an animal feed additive containing a nutrient-rich fermented broth obtained by means of a fermentation process using a genetically modified microorganism. The non-pathogenic bacterium Corynebacterium glutamicum, which is of great commercial interest, has been modified to produce aminolevulinic acid (5-ALA) more efficiently. The nutritional medium obtained is rich in biomolecules, such as sugars, organic acids, amino acids including 5-ALA, vitamins, and nucleotides, inter alia. In addition, the inactivated bacterial biomass can be added together with the fermented broth, acting as a source of nutrients without harming the animals. When added to animal feed, this broth promotes a series of benefits such as improved growth and immunity, reduces the use of antibiotics and supports the metabolism. Animals, such as pigs, poultry, cattle and fish, have a commercial value and any gain generates a financial return.

Description

COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OU ADITIVO PARA RAÇÃO ANIMAL BACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR ADDITIVE TO ANIMAL FEED
CAMPO DA INVENÇÃO FIELD OF THE INVENTION
[01] A presente invenção relata a produção de um caldo nutritivo por meio de processos fermentativos utilizando uma bactéria não patogênica geneticamente modificada. Técnicas da engenharia genética e metabólica foram empregadas para o desenvolvimento de uma cepa de Corynebacterium glutamicum capaz de produzir o ácido aminolevulínico (5-ALA). O caldo nutritivo obtido contem açúcares, vitaminas, aminoácidos, entre eles o 5-ALA, ácidos orgânicos e nucleotídeos, e é adicionado à ração animal garantindo benefícios, como maior crescimento e melhor imunidade de animais como gado, porcos, aves, peixes e pequenos animais como cães e gatos (pet food). [01] The present invention reports the production of a nutrient broth by means of fermentative processes using a genetically modified non-pathogenic bacterium. Genetic and metabolic engineering techniques were used to develop a strain of Corynebacterium glutamicum capable of producing aminolevulinic acid (5-ALA). The nutritional broth obtained contains sugars, vitamins, amino acids, including 5-ALA, organic acids and nucleotides, and is added to the animal feed guaranteeing benefits, such as greater growth and better immunity of animals such as cattle, pigs, birds, fish and small animals animals like dogs and cats (pet food).
HISTÓRICO DA INVENÇÃO HISTORY OF THE INVENTION
[02] Os processos fermentativos são realizados a partir do uso de diferentes microrganismos, sendo bactérias ou fungos, para a produção de várias biomoléculas que apresentam um interesse comercial. Microrganismos metabolizam diferentes substratos presentes no meio reacional gerando metabólitos primários, por exemplo, aminoácidos, e secundários, como antibióticos. Os processos fermentativos, são empregados em indústrias de diversos ramos, como alimentício, cosmético, farmacêutico e pecuário. Em escala industrial a tecnologia de fermentação está envolvida na produção de aminoácidos, vitaminas, enzimas, ácidos orgânicos, entre outras biomoléculas. [02] The fermentative processes are carried out from the use of different microorganisms, being bacteria or fungi, for the production of several biomolecules that are of commercial interest. Microorganisms metabolize different substrates present in the reaction medium, generating primary metabolites, for example, amino acids, and secondary ones, such as antibiotics. Fermentation processes are used in industries of various branches, such as food, cosmetic, pharmaceutical and livestock. On an industrial scale, fermentation technology is involved in the production of amino acids, vitamins, enzymes, organic acids, among other biomolecules.
[03] Com o desenvolvimento e aprimoramento de novas ferramentas para a análise genômica foi possível um maior conhecimento dos microrganismos. Essas novas técnicas garantiram o emprego da engenharia genética e metabólica para a realização de mutações específicas, com isso, possibilitaram uma maior e melhor produção de biomoléculas de interesse comercial. [03] With the development and improvement of new tools for genomic analysis, a greater knowledge of microorganisms was possible. These new techniques ensured the use of genetic and metabolic engineering to carry out specific mutations, thereby enabling greater and better production of biomolecules of commercial interest.
[04] Com a utilização dos conhecimentos e ferramentas da engenharia genética é possível a realização de mutações em genes específicos, sendo evitadas alterações indesejadas no genoma microbiano que podem prejudicar a produção de metabólitos de interesse. As modificações genéticas são capazes de aumentar ou diminuir a expressão de uma determinada proteína, sendo possível a inserção e expressão de genes de diferentes organismos no genoma microbiano. [04] With the use of knowledge and tools of genetic engineering, it is possible to carry out mutations in specific genes, avoiding undesired changes in the microbial genome that may impair the production of metabolites of interest. Genetic modifications are able to increase or decrease the expression of a certain protein, making it possible to insert and express genes from different organisms in the microbial genome.
[05] O ácido 5-aminolevulínico (5-ALA) é um aminoácido presente em plantas e animais. Esse aminoácido pode ser encontrado em alguns alimentos, como vinho tinto e chá (KR 101 180266). O 5-ALA é precursor de biomoléculas como clorofila e vitamina 12 (CN 101863703). A biossíntese do 5-ALA, geralmente, ocorre a partir de succinil-CoA e glicina pela ação da enzima ácido 5- aminolevulínico sintase (US 9333156). [05] 5-Aminolevulinic acid (5-ALA) is an amino acid present in plants and animals. This amino acid can be found in some foods, such as red wine and tea (KR 101 180266). 5-ALA is a precursor to biomolecules such as chlorophyll and vitamin 12 (CN 101863703). The biosynthesis of 5-ALA generally occurs from succinyl-CoA and glycine by the action of the enzyme 5-aminolevulinic acid synthase (US 9333156).
[06] O método mais utilizado para a produção do 5-ALA é a síntese química, porém, esse processo apresenta desvantagens, como o custo mais elevado, baixa conversão e poluição ambiental. Os processos fermentativos surgem como uma alternativa para evitar a síntese química, nesse caso determinados microrganismos são modificados geneticamente para produzir o aminoácido de maneira mais eficiente e viável economicamente sem gerar danos ambientais (CN 10445081 2). [06] The most used method for the production of 5-ALA is chemical synthesis, however, this process has disadvantages, such as higher cost, low conversion and environmental pollution. Fermentative processes appear as an alternative to avoid chemical synthesis, in which case certain microorganisms are genetically modified to produce the amino acid in a more efficient and economically viable manner without generating environmental damage (CN 10445081 2).
[07] O 5-ALA já conhecido por sua vasta aplicação, sendo utilizado em tratamentos de câncer, acne, entre outros problemas de pele, como também em plantas para melhorar a produção de clorofila e promover um melhor crescimento das plantas (KR 20100083425, KR 101 180266, CN 101863703, KR 20090027269). Na aplicação em ração animal de gado, frango, dentre outros, aumenta a imunidade e promove o crescimento (KR 20100083425, CN 102870970, KR 20090027269). [08] Certos microrganismos conhecidos e estudados são empregados na produção industrial de metabólitos. Esses microrganismos apresentam um grande interesse comercial por serem fáceis de manipular, mais seguros, possuírem menos contaminantes e obterem maiores produções de biomoléculas em grande escala. Com isso, genes diferentes são introduzidos nesses microrganismos com o intuito de obter diversos metabólitos de interesse comercial que são difíceis de serem produzidos em grade escala pelos seus organismos de origem. [07] 5-ALA already known for its wide application, being used in cancer treatments, acne, among other skin problems, as well as in plants to improve chlorophyll production and promote better plant growth (KR 20100083425, KR 101 180266, CN 101863703, KR 20090027269). When applied to livestock animal feed, chicken, among others, it increases immunity and promotes growth (KR 20100083425, CN 102870970, KR 20090027269). [08] Certain known and studied microorganisms are used in the industrial production of metabolites. These microorganisms are of great commercial interest because they are easy to manipulate, safer, have less contaminants and obtain greater production of biomolecules on a large scale. Thus, different genes are introduced in these microorganisms in order to obtain several metabolites of commercial interest that are difficult to be produced on a large scale by their origin organisms.
[09] A bactéria Corynebacterium glutamicum é um microrganismo que apresenta interesse comercial. Por ser um microrganismo não patogênico é utilizado na indústria para a produção de L-glutamato e outros aminoácidos, apresentando um potencial para a produção de ácidos orgânicos e biocombustíveis. A C. glutamicum é aplicada na indústria biotecnológica e vem sendo estudada para a produção de 5-ALA, isso devido ao fato de que o L- glutamato é um precursor do ALA e o microrganismo é considerado seguro, não apresentando riscos à saúde humana (YU et al., 2015). [09] The bacterium Corynebacterium glutamicum is a microorganism of commercial interest. As it is a non-pathogenic microorganism, it is used in industry for the production of L-glutamate and other amino acids, presenting a potential for the production of organic acids and biofuels. C. glutamicum is applied in the biotechnology industry and has been studied for the production of 5-ALA, due to the fact that L-glutamate is a precursor to ALA and the microorganism is considered safe, presenting no risks to human health ( YU et al., 2015).
[010] O aminoácido 5-ALA pode ser convertido no organismo em hemoglobina e promover o desenvolvimento e maturação das hemácias. A hemoglobina é uma molécula importante no transporte de oxigénio, responsável por dilatar os vasos sanguíneos, manter o pH estável no organismo e promover o crescimento saudável do animal (CN 102870970). Uma maior produção da hemoglobina interfere diretamente no transporte de oxigénio e transporte de nutrientes, nos gados, por exemplo, aumenta a resistência a doenças e ativa o metabolismo (KR 100459918). [010] The amino acid 5-ALA can be converted into hemoglobin in the body and promote the development and maturation of red blood cells. Hemoglobin is an important molecule in the transport of oxygen, responsible for dilating blood vessels, maintaining a stable pH in the body and promoting the healthy growth of the animal (CN 102870970). Higher hemoglobin production directly interferes with oxygen and nutrient transport in cattle, for example, increases resistance to disease and activates metabolism (KR 100459918).
[01 1 ] Devido ao uso de antibiótico para prevenir e tratar doenças nos animais de importância comercial, eles desenvolveram resistência aos antibióticos e acumularam resíduos dessas drogas podendo representar risco para a saúde humana. Com isso e uma maior regulamentação na utilização de antibióticos na pecuária, novas alternativas são necessárias para reduzir o uso dessa substância (KR 20100083425, KR 20090027269, KR 100459918). [01 1] Due to the use of antibiotics to prevent and treat diseases in animals of commercial importance, they developed resistance to antibiotics and accumulated residues of these drugs, which may represent a risk to human health. With this and greater regulation in the use of antibiotics in the livestock, new alternatives are needed to reduce the use of this substance (KR 20100083425, KR 20090027269, KR 100459918).
[012] A União Europeia já se mobilizou quanto ao assunto e proibiu a adição de antibióticos, responsáveis por promover o crescimento, na ração animal, além disso, restringiu o uso dos mesmos para o tratamento de certas doenças (KR 20090027269). [012] The European Union has already mobilized on the subject and has banned the addition of antibiotics, responsible for promoting growth, in animal feed, in addition, has restricted the use of them for the treatment of certain diseases (KR 20090027269).
[013] O aminoácido 5-ALA apresenta alguns benefícios para animais como gado, porcos, frangos, além de animais como peixes, cães e gatos. Dentre esses benefícios estão promover um maior crescimento dos animais, aumentar a imunidade evitando/reduzindo o uso de antibióticos, melhorar o porte físico e o metabolismo e no caso das aves, aumentar a produção de ovos (KR 20100083425, KR 20090027269, CN 102870970, KR 100459918). [013] The amino acid 5-ALA has some benefits for animals such as cattle, pigs, chickens, as well as animals such as fish, dogs and cats. Among these benefits are promoting greater growth of animals, increasing immunity avoiding / reducing the use of antibiotics, improving physical size and metabolism and in the case of birds, increasing egg production (KR 20100083425, KR 20090027269, CN 102870970, KR 100459918).
[014] Para garantir benefícios melhores e mais rápidos, o meio proveniente da fermentação com a bactéria C. glutamicum deve ser adicionado à ração animal, juntamente com o 5-ALA produzido no processo, que podem conter ainda outros aminoácidos, açúcares, nucleotídeos, ácidos orgânicos e vitaminas (CN 102870970, KR 100459918). [014] To ensure better and faster benefits, the medium from fermentation with the bacterium C. glutamicum must be added to the animal feed, together with the 5-ALA produced in the process, which may contain other amino acids, sugars, nucleotides, organic acids and vitamins (CN 102870970, KR 100459918).
ESTADO DA ARTE STATE OF ART
[015] A patente KR No 100459918 relata a produção de um suplemento alimentar para animal (pecuária) contendo o ácido aminolevulínico. Como é citado na patente, o aminoácido pode ser proveniente de uma fermentação, podendo ser o produto fermentado, contendo o ALA, adicionado à ração. Os inventores não descrevem a composição desse meio fermentado e nem o microrganismo utilizado. A presente invenção se diferencia pelo fato de que o caldo fermentado proveniente da fermentação de C. glutamicum modificada geneticamente é incorporado à ração animal, sendo o ingrediente ativo o caldo contendo açúcares, aminoácidos, entre eles o 5-ALA produzido pela fermentação, ácidos orgânicos, vitaminas e nucleotídeos. [016] A patente sul-coreana No 2012136786 retrata um aditivo alimentar para frangos e porcos contendo o 5-ALA e uma mistura fermentada de Sanguisorba officinalis e Rhus japonica por Aspergillus oryzae ou Lactobacillus casei. Nesse caso o aminoácido foi adicionado a uma mistura fermentada, ele não tem origem da fermentação e toda a mistura é considerada como o ingrediente ativo. A patente KR No 20100040165 descreve também uma mistura de um fermentado com a adição do 5-ALA. Nesse caso a quitosana é fermentada. A mistura da quitosana fermentada e do 5-ALA garante uma melhor taxa de crescimento e imunidade. [015] KR patent No. 100459918 reports the production of an animal food supplement (livestock) containing aminolevulinic acid. As mentioned in the patent, the amino acid can come from fermentation, and it can be the fermented product, containing ALA, added to the feed. The inventors do not describe the composition of this fermented medium or the microorganism used. The present invention is differentiated by the fact that the fermented broth from the fermentation of genetically modified C. glutamicum is incorporated into the animal feed, the active ingredient being the broth containing sugars, amino acids, including the 5-ALA produced by fermentation, organic acids , vitamins and nucleotides. [016] South Korean patent No. 2012136786 depicts a feed additive for chickens and pigs containing 5-ALA and a fermented mixture of Sanguisorba officinalis and Rhus japonica by Aspergillus oryzae or Lactobacillus casei. In this case the amino acid was added to a fermented mixture, it does not originate from fermentation and the entire mixture is considered as the active ingredient. Patent KR No 20100040165 also describes a mixture of a fermentate with the addition of 5-ALA. In that case, chitosan is fermented. The mixture of fermented chitosan and 5-ALA guarantees a better growth rate and immunity.
[017] O pedido de patente KR No 20090027269 expõe uma composição de ração animal para substituir o uso de antibióticos que apresenta diferentes compostos complexo como milho, melaço e soja, e o ALA. O aminoácido é adicionado para compor a ração animal. O ALA é responsável por aumentar a imunidade do animal, sendo possível uma administração menor de antibióticos. A presente invenção descreve uma composição a ser adicionada à ração animal, o caldo fermentado contendo o aminoácido ALA, no caso o aminoácido é produzido pelo processo fermentativo e não adicionado ao meio. [017] Patent application KR No 20090027269 exposes an animal feed composition to replace the use of antibiotics that presents different complex compounds such as corn, molasses and soy, and ALA. The amino acid is added to make up animal feed. ALA is responsible for increasing the animal's immunity, making it possible to give less antibiotics. The present invention describes a composition to be added to the animal feed, the fermented broth containing the amino acid ALA, in which case the amino acid is produced by the fermentative process and not added to the medium.
[018] O pedido de patente chinesa No 1 02870970 descreve um promotor de crescimento para animais da pecuária. O promotor é baseado na mistura de aminoácidos, como o 5-ALA e a lisina, além da coenzima Q10, e um carreador é utilizado, o pó de zeolite. A invenção aqui descrita não utiliza um carreador para o meio fermentado e o 5-ALA além do resto da composição do caldo é proveniente da fermentação. [018] Chinese patent application No. 1 02870970 describes a growth promoter for livestock animals. The promoter is based on a mixture of amino acids, such as 5-ALA and lysine, in addition to coenzyme Q10, and a carrier is used, the zeolite powder. The invention described here does not use a carrier for the fermented medium and the 5-ALA in addition to the rest of the broth composition comes from fermentation.
[019] A publicação WO No 2007141 1 1 1 relata um processo para a produção de um aditivo de ração animal baseado em um meio fermentado contendo um alto teor de L-lisina. A fermentação ocorre utilizando bactérias corineforme selecionadas de mutações. A invenção relata a possibilidade da biomassa bacteriana ser incorporada a ração animal juntamente com o caldo fermentado, uma vez que a bactéria é considerada segura, como a C. glutamicum. De maneira semelhante, a patente de número US5840358A descreve o uso de um produto obtido por via fermentativa, de modo que o processo emprega E. coli ou C. glutamicum modificados geneticamente para a produção de L-lisina. O produto final resultante é constituído da biomassa rica em nutrientes e do caldo fermentado com alta concentração de L-lisina produzida pelos microrganismos. [019] WO publication 2007141 1 1 1 reports a process for the production of an animal feed additive based on a fermented medium containing a high content of L-lysine. Fermentation takes place using corineform bacteria selected from mutations. The invention reports the possibility of the bacterial biomass being incorporated into the animal feed together with the fermented broth, since the bacterium is considered safe, such as C. glutamicum. Similarly, patent number US5840358A describes the use of a product obtained by fermentation, so that the process uses genetically modified E. coli or C. glutamicum for the production of L-lysine. The resulting final product consists of nutrient-rich biomass and fermented broth with a high concentration of L-lysine produced by microorganisms.
[020] A invenção aqui descrita relata o uso da bactéria não patogênica C. glutamicum geneticamente modificada empregada em um processo fermentativo visando a produção do 5-ALA. O produto final, a ser utilizado como ração para animais, é caracterizado por uma mistura de diferentes componentes onde apenas o caldo fermentativo ou esse em conjunto com a biomassa bacteriana inativa podem ser incorporados na formulação final. [020] The invention described here reports the use of the genetically modified non-pathogenic bacteria C. glutamicum used in a fermentation process aimed at the production of 5-ALA. The final product, to be used as animal feed, is characterized by a mixture of different components where only the fermentation broth or that together with the inactive bacterial biomass can be incorporated into the final formulation.
DESCRIÇÃO DA INVENÇÃO DESCRIPTION OF THE INVENTION
[021 ] A presente invenção trata do uso de estirpes de C. glutamicum geneticamente modificadas a fim de produzir o ácido 5-aminolevulínico. O diferencial aqui proposto envolve o uso do caldo fermentado das referidas estirpes composto por uma mistura rica em nutrientes, além do ácido 5- aminolevulínico, utilizado como aditivo ou suplemento para ração de animais como bovinos, caprinos, aves, suínos, peixes e cães e gatos (pet food). [021] The present invention deals with the use of genetically modified strains of C. glutamicum in order to produce 5-aminolevulinic acid. The differential proposed here involves the use of the fermented broth of the said strains composed of a mixture rich in nutrients, in addition to 5-aminolevulinic acid, used as an additive or supplement for animal feed such as cattle, goats, birds, pigs, fish and dogs and cats (pet food).
[022] Para que a presente invenção seja sustentável, as etapas envolvidas para a fabricação do produto final são caracterizadas por (a) processo fermentativo utilizando uma estirpe de C. glutamicum produtora de 5-ALA, (b) obtenção do caldo fermentado contendo a biomassa ou separação da biomassa e caldo fermentado por centrifugação ou filtração, (c) redução do pH para preservação do produto e inativação de biomasa, (d) secagem parcial ou total do caldo fermentado com ou sem biomassa e (d) preparo do produto final por meio da mistura do caldo fermentado contendo ou não biomassa com outros componentes que compõem ração animal. [023] A estirpe de C. glutamicum ATCC 13032 empregada como chassi para as modificações genéticas foi utilizada por ser uma estirpe selvagem e possuir o seu genoma completamente sequenciado (Ikeda, 2003). [022] For the present invention to be sustainable, the steps involved in the manufacture of the final product are characterized by (a) a fermentative process using a C. glutamicum strain producing 5-ALA, (b) obtaining the fermented broth containing the biomass or separation of biomass and fermented broth by centrifugation or filtration, (c) reduction of pH to preserve the product and inactivation of biomass, (d) partial or total drying of the fermented broth with or without biomass and (d) preparation of the final product by mixing the fermented broth containing or not biomass with other components that make up animal feed. [023] The C. glutamicum strain ATCC 13032 used as a chassis for genetic modifications was used because it is a wild strain and its genome is completely sequenced (Ikeda, 2003).
[024] A produção do 5-ALA na C. glutamicum pode ocorrer pela modulação da expressão gênica por meio do aumento do número de cópias do gene de interesse a ser inserido no cromossomo, uso de promotor regulado ou constitutivo, a fim de induzir, aumentar ou diminuir a expressão do gene desejado, alteração do códon de iniciação, ou uso do gene em um plasmídeo que possua estabilidade quando inserido na estirpe de interesse. [024] The production of 5-ALA in C. glutamicum can occur by modulating gene expression by increasing the number of copies of the gene of interest to be inserted into the chromosome, using a regulated or constitutive promoter, in order to induce, increase or decrease expression of the desired gene, alteration of the initiation codon, or use of the gene in a plasmid that has stability when inserted into the strain of interest.
[025] Tanto por meio da inserção de plasmídeos, por eletroporação ou choque- térmico, quanto por meio da inserção dos cassetes gênicos no cromossomo da estirpe chassi, por recombinação homóloga como relatado por Jiang e colaboradores, 2017, para que a estirpe seja capaz de produzir o 5-ALA de maneira viável é necessário que os seguintes genes sejam expressos: [025] Both through the insertion of plasmids, by electroporation or thermal shock, and through the insertion of the gene cassettes in the chromosome of the chassis strain, by homologous recombination as reported by Jiang et al., 2017, so that the strain is capable In order to produce 5-ALA in a viable way, the following genes must be expressed:
a) hemA da bactéria Rhodobacter capsulatus, otimizado por meio de algoritmo especializado para expressão em C. glutamicum. O gene codifica para a enzima 5-aminolevulinato sintase (ALAS), a qual produz ácido 5- aminolevulínico a partir de L-glicina e succinil-CoA; a) hemA of the bacterium Rhodobacter capsulatus, optimized by means of a specialized algorithm for expression in C. glutamicum. The gene codes for the enzyme 5-aminolevulinate synthase (ALAS), which produces 5-aminolevulinic acid from L-glycine and succinyl-CoA;
b) glyA, da própria estirpe, que resulta na produção da enzima serina hidroximetiltransferase, levando à produção de glicina a partir do aminoácido L- serina e tetrahidrofolato; b) glyA, from the strain itself, which results in the production of the enzyme serine hydroxymethyltransferase, leading to the production of glycine from the amino acid L-serine and tetrahydrofolate;
c) ser A, serB e serC, da própria estirpe, que resulta na produção das enzimas fosfoglicerato sintase, fosfoserina aminotransferase e fosfoserina fosfatase, respectivamente; c) be A, serB and serC, from the strain itself, which results in the production of the enzymes phosphoglycerate synthase, phosphoserine aminotransferase and phosphoserine phosphatase, respectively;
d) xylA e xylB de Escherichia coli, que resulta na produção das enzimas xilose isomerase e xiluloquinase, respectivamente d) Escherichia coli xylA and xylB, which results in the production of the enzymes xylose isomerase and xylulokinase, respectively
e) araA, araB, araD e araE de E. coli, que codificam para as enzimas arabinose isomerase, ribuloquinase, ribulose-5-fosfato-4-epimerase, e para o transportador de arabinose, respectivamente f) glpF, glpK e glpD de Corynebacterium diphteriae, que codificam para a proteína transportadora de glicerol e para as enzimas glicerol quinase e glicerol- 3-fosfodihidrogenase, respectivamente. e) araA, araB, araD and araE from E. coli, which code for the enzymes arabinose isomerase, ribulokinase, ribulose-5-phosphate-4-epimerase, and for the arabinose transporter, respectively f) glory, glpK and glpD of Corynebacterium diphteriae, which code for the glycerol carrier protein and for the enzymes glycerol kinase and glycerol-3-phosphodihydrogenase, respectively.
[026] Muitos processos fermentativos em escala industrial visando a produção de biomoléculas de interesse comercial fazem uso de organismos geneticamente modificados. No caso da presente invenção, o objetivo é obter um produto o qual possui em sua constituição o caldo fermentado que contenha ou não a biomassa, de modo que no caso da presença de biomassa, essa deve ser devidamente inativada de modo a não conter nenhum traço do organismo vivo. [026] Many fermentation processes on an industrial scale aimed at the production of biomolecules of commercial interest make use of genetically modified organisms. In the case of the present invention, the objective is to obtain a product which has in its constitution the fermented broth that contains biomass or not, so that in the case of the presence of biomass, it must be deactivated in such a way that it does not contain any trace of the living organism.
[027] No caso da presente invenção, a estirpe resultante modificada geneticamente pode ser empregada em processos fermentativos que utilizam diferentes tipos de meio de cultivo, podendo conter diferentes tipos de carbono e nutrientes. [027] In the case of the present invention, the resulting genetically modified strain can be used in fermentative processes that use different types of culture medium, and may contain different types of carbon and nutrients.
[028] O processo fermentativo para a produção da referida mistura rica em nutrientes contendo diferentes compostos, como sacarídeos, ácidos nucleicos, lipídeos e aminoácidos, dentre eles o ácido 5-aminolevulínico, deve conter: a) pelo menos uma fonte de carbono, como por exemplo glucose, xilose, arabinose, entre outras, desde que a estirpe seja capaz de metabolizar essas moléculas; [028] The fermentation process for the production of said mixture rich in nutrients containing different compounds, such as saccharides, nucleic acids, lipids and amino acids, including 5-aminolevulinic acid, must contain: a) at least one carbon source, such as for example glucose, xylose, arabinose, among others, as long as the strain is able to metabolize these molecules;
b) pelo menos uma fonte de nitrogénio, como por exemplo extrato de levedura, peptona ou sais inorgânicos; b) at least one nitrogen source, such as yeast extract, peptone or inorganic salts;
c) pelo menos uma fonte de fósforo, como fosfato de sódio monobásico, fosfato de sódio dibásico, fosfato de potássio monobásico, fosfato de potássio dibásico, ou outro sal inorgânico composto por fósforo que seja assimilado pelo referido microrganismo; c) at least one source of phosphorus, such as monobasic sodium phosphate, dibasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, or another phosphorus inorganic salt that is assimilated by said microorganism;
d) sais inorgânicos derivados de minerais ou vitaminas, como cloreto de sódio, carbonato de cálcio, sulfato de amónio, entre outros, desde que sejam assimilados pelo referido organismo. e) pelo menos um ácido orgânico como ácido cítrico, ácido succnínico, ácido acético, entre outros. d) inorganic salts derived from minerals or vitamins, such as sodium chloride, calcium carbonate, ammonium sulfate, among others, as long as they are assimilated by the said organism. e) at least one organic acid such as citric acid, succinic acid, acetic acid, among others.
[029] O processo para produção do caldo fermentado deve ser realizado preferencialmente em bioreatores com condições controladas de temperatura, pH, alimentação, agitação e aeração. O referido processo também pode ser realizado em frascos como erlenmeyers ou frascos semelhantes, desde que as condições para crescimento da biomassa sejam fornecidas. [029] The process for producing the fermented broth should preferably be carried out in bioreactors with controlled conditions of temperature, pH, food, agitation and aeration. This process can also be carried out in flasks such as erlenmeyers or similar flasks, provided that the conditions for biomass growth are provided.
[030] Em relação a temperatura, o crescimento do microrganismo deve ser realizado nas faixas de temperatura ambiente a 37°C, preferencialmente de 25°C a 34°C, mais preferencialmente de 28°C a 32°C. [030] In relation to temperature, the growth of the microorganism must be carried out in the ambient temperature ranges at 37 ° C, preferably from 25 ° C to 34 ° C, more preferably from 28 ° C to 32 ° C.
[031 ] Em relação ao pH, o crescimento do microrganismo deve ser realizado em faixa de pH de 5,0 a 7,5, preferencialmente de 5,5 a 7,0, mais preferencialmente de 6,0 a 6,5. [031] Regarding pH, the growth of the microorganism must be carried out in a pH range of 5.0 to 7.5, preferably from 5.5 to 7.0, more preferably from 6.0 to 6.5.
[032] Em relação a alimentação, o processo fermentativo pode o crescimento do microrganismo pode ser iniciado com concentrações de fonte de carbono variando de 5g/L a 60g/L, preferencialmente de 20g/L a 50g/L, mais preferencialmente de 30g/L a 40g/L. Ainda, a alimentação pode ser realizada de forma constante ou em pulsos, desde que a concentração da fonte de carbono seja mantida de 2g/L a 8g/L, preferencialmente de 4g/L a 6g/L, mais preferencialmente de 5g/L. [032] In relation to feeding, the fermentation process can the growth of the microorganism can be initiated with concentrations of carbon source varying from 5g / L to 60g / L, preferably from 20g / L to 50g / L, more preferably from 30g / L L to 40g / L. Furthermore, feeding can be carried out constantly or in pulses, as long as the concentration of the carbon source is maintained from 2g / L to 8g / L, preferably from 4g / L to 6g / L, more preferably from 5g / L.
[033] Em relação a agitação e aeração, o crescimento do microrganismo deve ser realizado com uma faixa de agitação que varia de 150 a 250 rpm e aeração entre 1 a 3 vvm, preferencialmente agitação de 180 a 230 rpm e aeração entre 1 ,5 a 2 vvm. [033] In relation to agitation and aeration, the growth of the microorganism must be carried out with an agitation range that varies from 150 to 250 rpm and aeration between 1 to 3 vvm, preferably agitation from 180 to 230 rpm and aeration between 1, 5 at 2 vvm.
[034] Em relação a biomassa, o crescimento do microrganismo deve ser iniciado com um inoculo onde a concentração de células seja correspondente a ϋ.Odoo de valor entre 1 a 10, preferencialmente 5 a 15, mais preferencialmente [035] O processo fermentativo pode se dar por 12 a 96 horas, preferencialmente por 24 a 60 horas, mais preferencialmente por 36 a 48 horas de acordo com as condições previamente mencionadas. [034] In relation to biomass, the growth of the microorganism must start with an inoculum where the concentration of cells corresponds to ϋ.Odoo of value between 1 to 10, preferably 5 to 15, more preferably [035] The fermentation process can take place for 12 to 96 hours, preferably for 24 to 60 hours, more preferably for 36 to 48 hours according to the conditions previously mentioned.
[036] Ao término da fermentação, o conteúdo total ou parcial do fermentador pode ser submetido a dois processos: [036] At the end of the fermentation, the total or partial content of the fermenter can be subjected to two processes:
a) separação do caldo fermentado e biomassa por um processo de centrifugação em centrífuga industrial a velocidades de 1000 a 14000 rpm, preferencialmente de 2000 a 8000 rpm, mais preferencialmente de 3000 a 4000 rpm. Ainda, a separação pode ser realizada por filtração ou ultrafiltração, preferencialmente a vácuo, utilizando filtros de 0,45 micrometros de porosidade, preferencialmente de 0,22 um de porosidade, a) separation of the fermented juice and biomass by a centrifugation process in an industrial centrifuge at speeds of 1000 to 14000 rpm, preferably from 2000 to 8000 rpm, more preferably from 3000 to 4000 rpm. Furthermore, the separation can be carried out by filtration or ultrafiltration, preferably in a vacuum, using 0.45 micron porosity filters, preferably 0.22 µm porosity,
b) inativação da biomassa por meio da adição de qualquer reagente ácido de modo que o pH da solução seja reduzido para o valor de 3,0 a 3,5. O ácido a ser empregado nessa etapa pode ser preferencialmente o ácido cítrico, mas não limitado a esse, sendo que podem ser utilizados também ácido ascórbico, ácido acético, ácido lático, ácido málico, ácido fumárico, ácido glucônico, entre outros. b) inactivation of the biomass through the addition of any acid reagent so that the pH of the solution is reduced to the value of 3.0 to 3.5. The acid to be used in this step can be preferably citric acid, but not limited to it, and ascorbic acid, acetic acid, lactic acid, malic acid, fumaric acid, gluconic acid, among others, can also be used.
[037] O caldo fermentado, contendo a biomassa inativada ou não, pode ser misturado a outros componentes para formulação de ração animal na forma líquida, ou pode ser submetido a secagem parcial ou total, a vácuo ou não, sob temperaturas de 40 a 80°C, preferencialmente de 50 a 70°C, mais preferencialmente de 55 a 65°C, de modo que a umidade residual seja de 0,1 a 99,9%, preferencialmente de 5 a 90%, mais preferencialmente de 10 a 80%, mais preferencialmente de 20 a 50%. O conteúdo obtido pode então ser utilizado como pó, grânulos ou líquido e ser misturados a outros compostos visando a formulação de ração para animais. [037] The fermented broth, containing the inactivated biomass or not, can be mixed with other components for the formulation of animal feed in liquid form, or can be subjected to partial or total drying, under vacuum or not, under temperatures of 40 to 80 ° C, preferably from 50 to 70 ° C, more preferably from 55 to 65 ° C, so that the residual humidity is 0.1 to 99.9%, preferably from 5 to 90%, more preferably from 10 to 80 %, more preferably 20 to 50%. The content obtained can then be used as powder, granules or liquid and be mixed with other compounds for the formulation of animal feed.
[038] O produto resultante do processo fermentativo descrito anteriormente pode ser misturado a outros componentes em diferentes proporções a fim de gerar uma formulação de ração animal. Esses outros moléculas podem ser sacarídeos, aminoácidos ou peptídeos, ácidos nucleicos e outros ácidos orgânicos, lipídeos, micronutrientes como sais inorgânicos e ainda enzimas ou fatores de crescimento. [038] The product resulting from the fermentation process described above can be mixed with other components in different proportions in order to generate an animal feed formulation. These other molecules can be saccharides, amino acids or peptides, nucleic acids and other acids organic, lipids, micronutrients such as inorganic salts and enzymes or growth factors.
[039] A formulação final da ração a ser disponibilizada para bovinos, aves, suínos, caprinos, peixes e animais de pequeno porte como cães e gatos, pode- ser constituída, além do produto oriundo do processo fermentativo descrito, os seguintes compostos: [039] The final formulation of the feed to be made available for cattle, poultry, pigs, goats, fish and small animals such as dogs and cats, may comprise, in addition to the product from the described fermentation process, the following compounds:
a) grãos de milho e seus subprodutos, como silagem, quirera, farelo, fubá, farinha, grãos secos de destiladores (Distiller Dried Grain), grãos secos de destiladores com solúveis (Distiller Dried Grain with Solubles) e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de milho, a) maize grains and their by-products, such as silage, corn, bran, cornmeal, flour, dry distillers grains (Distiller Dried Grain), dry distillers grains with soluble (Distiller Dried Grain with Solubles) and any other by-products obtained from of any type of processing involving corn grains,
b) grãos de soja e seus subprodutos, como silagem, casca, farelo, farinha, proteína de soja texturizada, proteína de soja micronizada, proteína de soja concentrada, extrato hidrosolúvel de soja, flocos desengordurados, lecitinas e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de soja, b) soybeans and their by-products, such as silage, husk, bran, flour, textured soy protein, micronized soy protein, concentrated soy protein, water-soluble soy extract, defatted flakes, lecithins and any other by-product obtained from any type of processing involving soybeans,
c) grãos de trigo e seus subprodutos, como silagem, casca, farelo fino, farelo grosso, farinha e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de trigo, c) wheat grains and their by-products, such as silage, husk, fine bran, coarse bran, flour and any other by-product obtained from any type of processing involving wheat grains,
d) grãos de sorgo e seus subprodutos, como silagem, casca, farelo, farinha, grãos secos de destiladores, grãos secos de destiladores com solúveis, proteína de sorgo processada, e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de sorgo, d) sorghum grains and their by-products, such as silage, husk, bran, flour, dry distillers grains, dry distillers grains with soluble, processed sorghum protein, and any other by-product obtained from any type of processing involving grains of sorghum,
e) grãos de arroz e seus subprodutos, como silagem, casca, farelo gordo, farelo estabilizado, quirera, amido, farinha, proteína do endosperma, e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de arroz, e) rice grains and their by-products, such as silage, husk, fat bran, stabilized bran, chirera, starch, flour, endosperm protein, and any other by-product obtained from any type of processing involving rice grains,
f) grãos de centeio e seus subprodutos, como silagem, casca, flocos, farelo, farinha, e qualquer outro tipo de subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de centeio, g) grãos de cevada e seus subprodutos, como silagem, casca, flocos, farelo, farinha, bagaço, e qualquer outro tipo de subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de cevada, f) rye grains and their by-products, such as silage, husk, flakes, bran, flour, and any other type of by-product obtained from any type of processing involving rye grains, g) barley grains and their by-products, such as silage, bark, flakes, bran, flour, bagasse, and any other type of by-product obtained from any type of processing involving barley grains,
h) bagaços de vegetais como cana de açúcar, laranja, maçã, uva, caju, entre outros, processados de diferentes maneiras, como em pó, farelo, farinha, pellets, grânulos ou pedaços, ou qualquer outro tipo de subproduto obtido a partir de qualquer tipo de processamento envolvendo bagaços em geral, h) vegetable pomace such as sugar cane, orange, apple, grape, cashew, among others, processed in different ways, such as powder, bran, flour, pellets, granules or pieces, or any other type of by-product obtained from any type of processing involving bagasse in general,
i) melaços ou melados de vegetais como cana de açúcar, milho, soja, arroz, sorgo, entre outros, processados de diferentes maneiras, como em pó, líquido concentrado contendo diferentes graus de umidade, ou qualquer outro tipo de subproduto obtido a partir do processo de produção de melaços ou melados. i) molasses or molasses from vegetables such as sugar cane, corn, soybeans, rice, sorghum, among others, processed in different ways, such as powder, concentrated liquid containing different degrees of humidity, or any other type of by-product obtained from production process of molasses or molasses.
[040] A formulação da ração animal pode seguir diferentes padrões de composição utilizando os componentes descritos acima, de modo que uma mistura de todos ou de alguns dos referidos ingredientes pode compor a ração final. [040] The formulation of the animal feed can follow different composition patterns using the components described above, so that a mixture of all or some of the aforementioned ingredients can make up the final feed.
[041 ] A formulação da ração animal pode ainda conter moléculas adicionais como espessantes, emulsificantes, acidulantes, texturizantes, flavorizantes, aromatizantes, estabilizantes, entre outros, de modo que torne o produto final com características bromatológicas e sensoriais aceitáveis para cada animal a ser destinado. [041] The formulation of animal feed may also contain additional molecules such as thickeners, emulsifiers, acidulants, texturizers, flavors, flavors, stabilizers, among others, so that the final product has acceptable and sensory characteristics for each animal to be destined .
[042] Após a mistura dos componentes, a ração pode ser produzida em diferentes formatos, como pó, pellets, grânulos finos, grânulos grossos, pasta, entre outros, sejam estes secos ou umedecidos, de modo que represente a melhor condição para disponibilização para o animal a ser destinado. [042] After mixing the components, the feed can be produced in different formats, such as powder, pellets, fine granules, coarse granules, paste, among others, whether dry or moistened, so that it represents the best condition for availability for the animal to be destined.
[043] O exemplo descrito na presente invenção trata apenas de uma representação do processo fermentativo e posterior formulação de um aditivo ou suplemento para ração animal a fim de ilustrar uma das várias possibilidades de uso da mistura nutritiva, de modo que não limite as aplicações possíveis do produto aqui descrito a apenas este caso. [043] The example described in the present invention deals only with a representation of the fermentative process and subsequent formulation of an additive or supplement for animal feed in order to illustrate one of the several possibilities of use of the nutritious mixture, so that it does not limit the possible applications of the product described here to just this case.
[044] Exemplo 1 : [044] Example 1:
[045] Preparo de uma mistura nutritiva por processo fermentativo utilizando uma estirpe de C. glutamicum produtora de ácido 5-aminolevulínico a ser incorporada a ração animal como aditivo ou suplemento: [045] Preparation of a nutritive mixture by fermentation process using a strain of C. glutamicum producing 5-aminolevulinic acid to be incorporated into animal feed as an additive or supplement:
[046] Uma única colónia da estirpe NB_CgALA05 presente em placa microbiológica é inoculada em 1 5mL de meio contendo 10g/L de triptona, 5g/L de extrato de levedura, 10g/L de cloreto de sódio. Esse pré-inóculo é incubado a 30°C por 16 horas sob agitação de 180rpm. A amostra anterior é adicionada à em 500mL do mesmo meio e incubada sob as mesmas condições, a fim de gerar maior quantidade de biomassa. Por fim, o inoculo anterior é diluído em 1 L do meio para o processo fermentativo em bioreator de 5L. O meio de cultura para o processo fermentativo é composto inicialmente de 40g/L de glucose, 10g/L de triptona, 5g/L de extrato de levedura, 10g/L de cloreto de sódio e 10g/L de L- glicina. [046] A single colony of strain NB_CgALA05 present in a microbiological plate is inoculated in 15 ml of medium containing 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride. This pre-inoculum is incubated at 30 ° C for 16 hours with shaking at 180 rpm. The previous sample is added to 500mL of the same medium and incubated under the same conditions, in order to generate a greater amount of biomass. Finally, the previous inoculum is diluted in 1 L of the medium for the fermentation process in a 5 L bioreactor. The culture medium for the fermentation process is initially composed of 40g / L of glucose, 10g / L of tryptone, 5g / L of yeast extract, 10g / L of sodium chloride and 10g / L of L-glycine.
[047] O processo fermentativo é mantido com um mínimo de 4g/L e máximo de 40g/L de glicose disponível no meio e o pH mantido entre 6,0 e 6,5 por meio da adição de hidróxido de sódio ou outra base. A fermentação ocorre por até 72 horas a 30°C sob agitação de 300rpm e adição constante de 2 v.v.m. de oxigénio. [047] The fermentation process is maintained with a minimum of 4g / L and a maximum of 40g / L of glucose available in the medium and the pH maintained between 6.0 and 6.5 by the addition of sodium hydroxide or another base. Fermentation takes place for up to 72 hours at 30 ° C with 300rpm agitation and constant addition of 2 v.v.m. of oxygen.
[048] Após o tempo de fermentação o conteúdo total é submetidos a redução de pH por adição de ácido cítrico até o pH atingir valor próximo a 3,0. Esse processo garante a completa inativação da biomassa. Após, o produto é submetido a secagem até conter entre 10 a 15% de umidade e então armazenado de 4-6°C. [048] After the fermentation time, the total content is subjected to pH reduction by adding citric acid until the pH reaches a value close to 3.0. This process guarantees the complete inactivation of biomass. Afterwards, the product is subjected to drying until it contains between 10 and 15% moisture and then stored at 4-6 ° C.
[049] A mistura para formulação da ração animal é realizada por meio da agitação mecânica dos componentes sólidos na forma de pó ou grânulos finos. A ração aqui descrita é constituída de 0,1 -1 % do produto oriundo do processo fermentativo, 50-70% de co-produto a base de milho oriundo da produção de etanol (neste caso grãos secos de destiladores com solúveis - DDGS), 10-17,5% de levedura hidrolisada, 10-30% de farelo de soja, 5-10% de melaço de cana de açúcar e 1 ,5-3% de componentes, como espessantes, flavorizantes, acidulantes, entre outros. [049] The mixture for formulating animal feed is carried out by mechanically stirring the solid components in the form of powder or fine granules. The ration described here consists of 0.1 -1% of the product from the process fermentative, 50-70% corn by-product from ethanol production (in this case dry distillers grains with soluble - DDGS), 10-17.5% hydrolyzed yeast, 10-30% soy bran , 5-10% of sugar cane molasses and 1.5-5% of components, such as thickeners, flavors, acidulants, among others.
[050] A mistura dos componentes se dá em misturador de ração industrial sob agitação de 480 rpm por cerca de 1 hora a temperatura ambiente. Após completo o processo de mistura, a ração é disponibilizada para frangos na forma de ração farelada fina. [050] The components are mixed in an industrial feed mixer with 480 rpm agitation for about 1 hour at room temperature. After the mixing process is complete, the feed is made available to chickens in the form of fine bran feed.
REFERÊNCIAS REFERENCES
IKEDA, M., NAKAGAWA, S. The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Applied Microbiology and Biotechnology. 62(2-3):99-109 (2003). IKEDA, M., NAKAGAWA, S. The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Applied Microbiology and Biotechnology. 62 (2-3): 99-109 (2003).
JIANG, Y. et al. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nature Communications. 8:15179 (2017). JIANG, Y. et al. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nature Communications. 8: 15179 (2017).
YU, X. et al. Engineering Corynebacterium glutamicum to produce 5- aminolevulinic acid from glucose. Microbial Cell Factories, 14:183 (2015). YU, X. et al. Engineering Corynebacterium glutamicum to produce 5-aminolevulinic acid from glucose. Microbial Cell Factories, 14: 183 (2015).
Patentes referenciadas: KR 101 180266, CN 101863703, US 9333156, CN 104450812, KR 20100083425, KR 20090027269, CN 102870970, KR Referenced patents: KR 101 180266, CN 101863703, US 9333156, CN 104450812, KR 20100083425, KR 20090027269, CN 102870970, KR
100459918, US5840358A. 100459918, US5840358A.

Claims

REIVINDICAÇÕES
1 . COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE1 . NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, caracterizado por um aditivo ou suplemento para ração animal, principalmente bovinos, aves, suínos, caprinos, peixes e pequenos animais como gato e cães (pet food), sendo o princípio ativo um composto fermentado contendo moléculas como carboidratos, lipídeos, aminoácidos, dentre eles o ácido 5-aminolevulínico, e ácidos nucleicos. ANIMAL FEED ADDITIVE, characterized by an additive or supplement for animal feed, mainly cattle, birds, pigs, goats, fish and small animals such as cats and dogs (pet food), the active ingredient being a fermented compound containing molecules such as carbohydrates, lipids , amino acids, including 5-aminolevulinic acid, and nucleic acids.
2. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE2. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com a reivindicação 1 , caracterizado por um caldo fermentado nutritivo oriundo de um processo fermentativo empregando uma estirpe de Corynebacterium glutamicum geneticamente modificada capaz de produzir aminoácidos, dentre eles, o ácido 5-aminolevulínico. ANIMAL FEED ADDITIVE, according to claim 1, characterized by a nutritious fermented broth from a fermentative process employing a genetically modified Corynebacterium glutamicum strain capable of producing amino acids, among them, 5-aminolevulinic acid.
3. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE3. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 e 2, caracterizado por um caldo fermentado composto por uma ou mais moléculas de carboidratos, como glicose, xilose e arabinose e fontes complexas de carbono, lipídeos, como fosfolipídeos, esfingolipídeos, e glicolipídeos, aminoácidos, como lisina, metionina, treonina, valina, triptofano e ácido 5-aminolevulínico, e ácidos nucleicos, como ácido deoxiribonucleico, ácido ribonucleico e nucleotídeos. ANIMAL FEED ADDITIVE, according to claims 1 and 2, characterized by a fermented broth composed of one or more carbohydrate molecules, such as glucose, xylose and arabinose and complex sources of carbon, lipids, such as phospholipids, sphingolipids, and glycolipids, amino acids, such as lysine, methionine, threonine, valine, tryptophan and 5-aminolevulinic acid, and nucleic acids, such as deoxyribonucleic acid, ribonucleic acid and nucleotides.
4. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE4. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 3, caracterizado por um composto rico em nutrientes derivado de um processo fermentativo onde a fração solúvel é separada da biomassa por centrifugação, filtração ou ultrafiltração e utilizada como aditivo ou suplemento na mistura de ração para animais. ANIMAL FEED ADDITIVE, according to claims 1 to 3, characterized by a nutrient-rich compound derived from a process fermentative where the soluble fraction is separated from biomass by centrifugation, filtration or ultrafiltration and used as an additive or supplement in the animal feed mixture.
5. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE5. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 4, caracterizado por um composto rico em nutrientes derivado de um processo fermentativo onde a biomassa é inativada por redução de pH, aumento de temperatura, pressão osmótica, pressão mecânica ou ultrassom, e utilizada, em conjunto com a fração solúvel, como aditivo ou suplemento na mistura de ração para animais. ANIMAL FEED ADDITIVE, according to claims 1 to 4, characterized by a nutrient-rich compound derived from a fermentative process where the biomass is inactivated by pH reduction, temperature increase, osmotic pressure, mechanical pressure or ultrasound, and used , together with the soluble fraction, as an additive or supplement in the animal feed mixture.
6. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE6. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 5, caracterizado por um composto rico em nutrientes derivado de um processo fermentativo onde a biomassa é separada por centrifugação, filtração ou ultrafiltração, inativada por redução de pH, aumento de temperatura, pressão osmótica, pressão mecânica ou por ultrassom, e utilizada como aditivo ou suplemento na mistura de ração para animais. ANIMAL FEED ADDITIVE, according to claims 1 to 5, characterized by a nutrient-rich compound derived from a fermentation process where the biomass is separated by centrifugation, filtration or ultrafiltration, inactivated by pH reduction, temperature increase, osmotic pressure , mechanical pressure or by ultrasound, and used as an additive or supplement in the mix of animal feed.
7. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE7. NUTRITIVE COMPOUND FORMED BY CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 3, caracterizado por um composto rico em nutrientes derivado de um processo fermentativo que pode ser utilizado como aditivo ou suplemento na mistura de ração para animais na forma de pó, pasta, líquido concentrado ou líquido não concentrado. ANIMAL FEED ADDITIVE according to claims 1 to 3, characterized by a nutrient-rich compound derived from a fermentation process that can be used as an additive or supplement in the mixture of animal feed in the form of powder, paste, concentrated liquid or non-concentrated liquid.
8. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE8. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de grãos de milho e seus subprodutos, como silagem, quirera, farelo, fubá, farinha, grãos secos de destiladores (Distiller Dried Grain), grãos secos de destiladores com solúveis (Distiller Dried Grain with Solubles), e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de milho, podendo estes serem misturados ou não a materiais fibrosos derivados de modificações químicas ou mecânicas do DDG ou DDGS. ANIMAL FEED ADDITIVE, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 to 99.9% of corn grains and their by-products, such as silage, corn, bran, cornmeal , flour, dry distillers grains (Distiller Dried Grain), dry distillers grains with soluble (Distiller Dried Grain with Solubles), and any other by-product obtained from any type of processing involving corn kernels, which can be mixed or not mixed fibrous materials derived from chemical or mechanical modifications of the DDG or DDGS.
9. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE9. NUTRITIVE COMPOUND FORMED BY CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OU ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de co- produto de a base de grãos de soja e seus subprodutos, como silagem, casca, farelo, farinha, proteína de soja texturizada, proteína de soja micronizada, proteína de soja concentrada, extrato hidrosolúvel de soja, flocos desengordurados, lecitinas e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de soja. BACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR ADDITIVE FOR ANIMAL FEED, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 99.9% co-product of soybean based products and by-products, such as silage, husk, bran, flour, textured soy protein, micronized soy protein, concentrated soy protein, water soluble soy extract, flakes defatted, lecithins and any other by-product obtained from any type of processing involving soybeans.
10. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE10. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OU ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de co- produto de a base de grãos de trigo e seus subprodutos, como silagem, casca, farelo fino, farelo grosso, farinha e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de trigo. BACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR ADDITIVE FOR ANIMAL FEED, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 a 99.9% co-product of wheat grain and its by-products, such as silage, husk, fine bran, coarse bran, flour and any other by-product obtained from any type of processing involving wheat grains.
1 1 . COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OU ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de co- produto de a base de grãos de sorgo e seus subprodutos, como silagem, casca, farelo, farinha, grãos secos de destiladores, grãos secos de destiladores com solúveis, proteína de sorgo processada, e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de sorgo. 1 1. NUTRITIVE COMPOUND FORMED BY BACTERIAL FERMENTATION CONTENT FOR USE AS A SUPPLEMENT OR ANIMAL FEED ADDITIVE, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 to 99.9% of co - product of sorghum grains and their by-products, such as silage, husk, bran, flour, dry distillers grains, dry distillers grains with soluble, processed sorghum protein, and any other by-product obtained from any type of processing involving sorghum grains.
12. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OU ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de co- produto de a base de grãos de arroz e seus subprodutos, como silagem, casca, farelo gordo, farelo estabilizado, quirera, amido, farinha, proteína do endosperma, e qualquer outro subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de arroz. 12. NUTRITIVE COMPOUND FORMED BY BACTERIAL FERMENTATION CONTENT FOR USE AS AN ADDITIVE OR ADDITIVE TO ANIMAL FEED, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 to 99.9% of co-product based on rice grains and their by-products, such as silage, husk, fat bran, stabilized bran, chirera, starch, flour, endosperm protein, and any other by-product obtained from any type of processing involving rice grains.
13. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE13. NUTRITIVE COMPOUND FORMED BY CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de co- produto de a base de grãos de centeio e seus subprodutos, como silagem, casca, flocos, farelo, farinha, e qualquer outro tipo de subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de centeio. ANIMAL FEED ADDITIVE, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 to 99.9% of co - product made from rye grains and their by-products, such as silage, husk, flakes, bran, flour, and any other type of by-product obtained from any type of processing involving rye grains.
14. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE14. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de co- produto de a base de grãos de cevada e seus subprodutos, como silagem, casca, flocos, farelo, farinha, bagaço, e qualquer outro tipo de subproduto obtido a partir de qualquer tipo de processamento envolvendo grãos de cevada. ANIMAL FEED ADDITIVE, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 to 99.9% of co-product based on barley grains and their by-products, as silage, bark, flakes, bran, flour, bagasse, and any other type of by-product obtained from any type of processing involving barley grains.
15. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OU ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de bagaços de vegetais como cana de açúcar, laranja, maçã, uva, caju, entre outros, processados de diferentes maneiras, como em pó, farelo, farinha, pellets, grânulos ou pedaços, ou qualquer outro tipo de subproduto obtido a partir de qualquer tipo de processamento envolvendo bagaços em geral. 15. NUTRITIVE COMPOUND FORMED BY BACTERIAL FERMENTATION CONTENT FOR USE AS AN ADDITIVE OR ADDITIVE TO ANIMAL FEED, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 to 99.9% of vegetable pomace such as sugar cane, orange, apple, grape, cashew, among others, processed in different ways, such as powder, bran, flour, pellets, granules or pieces, or any other type of by-product obtained from any type of processing involving bagasse in general.
16. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE16. NUTRITIVE COMPOUND FORMED BY CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 7, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, e 50 a 99,9% de melaços ou melados de vegetais como cana de açúcar, milho, soja, arroz, sorgo, entre outros, processados de diferentes maneiras, como em pó, líquido concentrado contendo diferentes graus de umidade, ou qualquer outro tipo de subproduto obtido a partir do processo de produção de melaços ou melados. ANIMAL FEED ADDITIVE, according to claims 1 to 7, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, and 50 to 99.9% of molasses or vegetable molasses such as sugar cane, corn, soy, rice, sorghum, among others, processed in different ways, such as powder, concentrated liquid containing different degrees of moisture, or any other type of by-product obtained from the production process molasses or molasses.
17. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE17. NUTRITIVE COMPOUND FORMED BY THE CONTENT OF
FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OUBACTERIAL FERMENTATION FOR USE AS A SUPPLEMENT OR
ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 16, caracterizado por uma composição para ração animal contendo de 0,1 a 50% do caldo fermentado com ou sem biomassa, inativada ou não, 0,1 % a 99,9% de grãos de milho e/ou seus subprodutos, 0,1 % a 99,9% de grãos de soja e/ou seus subprodutos, 0,1 % a 99,9% de grãos de trigo e/ou seus subprodutos, 0,1 % a 99,9% de grãos de sorgo e/ou seus subprodutos, 0,1 % a 99,9% de grãos de arroz e/ou seus subprodutos, 0,1 % a 99,9% de grãos de centeio e/ou seus subprodutos, 0,1 % a 99,9% de grãos de cevada e/ou seus subprodutos, 0,1 % a 99,9% de bagaços de vegetais e/ou seus subprodutos, 0,1 % a 99,9% de melaços ou melados e/ou seus subprodutos, de modo que pelo menos dois dos compostos supracitados sejam misturados com o caldo fermentado com ou sem biomassa, inativada ou não, nas proporções supracitadas. ANIMAL FEED ADDITIVE, according to claims 1 to 16, characterized by a composition for animal feed containing from 0.1 to 50% of the fermented broth with or without biomass, inactivated or not, 0.1% to 99.9% of corn grains and / or their by-products, 0.1% to 99.9% of soy beans and / or their by-products, 0 , 1% to 99.9% of wheat grains and / or their by-products, 0.1% to 99.9% of sorghum grains and / or their by-products, 0.1% to 99.9% of rice grains and / or its by-products, 0.1% to 99.9% of rye grains and / or its by-products, 0.1% to 99.9% of barley grains and / or their by-products, 0.1% to 99 , 9% vegetable pomace and / or its by-products, 0.1% to 99.9% molasses or molasses and / or its by-products, so that at least two of the aforementioned compounds are mixed with the fermented broth with or without biomass, inactivated or not, in the aforementioned proportions.
18. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OU ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 17, caracterizado por uma composição para ração animal à qual podem ser adicionadas moléculas como espessantes, emulsificantes, acidulantes, texturizantes, flavorizantes, aromatizantes, estabilizantes, entre outras, de modo que torne o produto final com características bromatológicas e sensoriais aceitáveis para cada animal a ser destinado. 18. NUTRITIVE COMPOUND FORMED BY BACTERIAL FERMENTATION CONTENT FOR USE AS AN ADDITIVE OR ADDITIVE TO ANIMAL FEED, according to claims 1 to 17, characterized by a composition for animal feed to which molecules such as thickeners, emulsifiers, acidulants, texturizers can be added , flavoring, flavoring, stabilizers, among others, so that the final product has acceptable chemical and sensory characteristics for each animal to be destined.
19. COMPOSTO NUTRITIVO FORMADO PELO CONTEÚDO DE FERMENTAÇÃO BACTERIANA PARA USO COMO SUPLEMENTO OU ADITIVO PARA RAÇÃO ANIMAL, de acordo com as reivindicações 1 a 18, caracterizado por uma composição para ração animal que pode ser produzida em diferentes formatos, como pó, pellets, grânulos finos, grânulos grossos, pasta, entre outros, sejam estes secos ou umedecidos. 19. NUTRITIVE COMPOUND FORMED BY BACTERIAL FERMENTATION CONTENT FOR USE AS AN ADDITIVE OR ADDITIVE TO ANIMAL FEED, according to claims 1 to 18, characterized by a composition for animal feed that can be produced in different formats, such as powder, pellets, granules fine, coarse granules, paste, among others, whether dry or moistened.
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