WO2021226690A1 - Nutritional composition for dogs or cats, consisting of biomass from a genetically modified organism, expressing fibrillar proteins of the animal muscle, associated with other nutritional sources originating from agro-industrial waste, and method for obtaining same - Google Patents

Nutritional composition for dogs or cats, consisting of biomass from a genetically modified organism, expressing fibrillar proteins of the animal muscle, associated with other nutritional sources originating from agro-industrial waste, and method for obtaining same Download PDF

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WO2021226690A1
WO2021226690A1 PCT/BR2021/050191 BR2021050191W WO2021226690A1 WO 2021226690 A1 WO2021226690 A1 WO 2021226690A1 BR 2021050191 W BR2021050191 W BR 2021050191W WO 2021226690 A1 WO2021226690 A1 WO 2021226690A1
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proteins
yeast
biomass
animal
genetically modified
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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
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/02—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from meat
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/18—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from yeasts
    • 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
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • 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
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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

  • NUTRITIONAL COMPOSITION FOR DOGS OR CATS CONSISTING OF BIOMASS FROM A GENETICALLY MODIFIED ORGANISM, EXPRESSING FIBRILLARY PROTEINS OF ANIMAL MUSCLE, ASSOCIATED WITH OTHER NUTRITIONAL SOURCES FROM AGROINDUSTRIAL WASTE, AND
  • the present invention is dedicated to the dog and cat nutrition sector.
  • the ideal protein concentration range is 23 to 25 g/day in rations dry or 6.4 to 8 g/day in wet rations.
  • the consumption of meat for animal feed can be equivalent to significant portions of the total national production.
  • this figure reaches 31.52%.
  • 39.21% of meat production is destined for pet food, while in Italy this figure is 31.45% (LEENSTRA, VELLINGA &BESSEI; 2018).
  • EPP ecological paw print
  • microbial biomass which can be yeasts, filamentous fungi or bacteria, as a product with high protein content to be used as an integral part of pet food.
  • yeast cells including Candida utilis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae and Saccharomyces fragilis to be mixed with vegetable and animal protein sources, as fish protein concentrate, slaughterhouse rejects and bone meal.
  • yeast biomass as a component (from 5 to 50%), like many formulations, still it depends on animal parts to meet nutritional requirements in terms of protein.
  • flavoring additives to increase the attractiveness of the product, which is intended exclusively for human consumption, without prospecting for application as animal feed.
  • yeast in addition to approximately 50% of its composition being proteins, yeast can also be a source of vitamin B - involved in the nervous and circulatory systems - in addition to selenium - auxiliary antioxidant mineral in pancreatic and endocrine activity.
  • Another advantage of its admission as a compost for pet food is its high digestibility (between 60 and 80%), making the macro and micronutrients present in the cellular structure to be used by dogs and cats.
  • yeasts in rations are also carried out by the potential that these organisms have to increase the acceptability of the product by improving the palatability of formulations. This effect is mainly observed in dogs with the addition of yeast extract, in cases where there is a thermal pre-treatment of the mixture before it is finished (LIMA et al., 2016).
  • patent CN102008026B is cited, in which the proposed formulation suggests the use of yeast biomass or Bacillus genus bacteria as a source of microproteins.
  • animal parts such as organs and skin, flour or whole protein grains and animal and vegetable lipid compounds, in addition to vitamins and other minerals, microbial biomass represents an additional source of amino acids and polypeptides.
  • yeasts as a nutritional component in the feed, not just as an additive, the invention is still dependent on animal parts as sources of proteins and lipids, a fact that further supports the maintenance of high consumption of meat by dogs and cats.
  • patent CA2735659C is cited, in which the formulation of a pet food composed mainly of a vegetable matrix is described, reaching up to 70% of the total mass.
  • the method despite dispensing sources animals in its composition and insert pre and probiotic elements, yeast biomass appears only as a adjuvant.
  • Bacillus, Bacteroides, Bifidobacterium, Enterococcus, Lactobacillus, Leuconostoc, Saccharomyces, Candida or Streptococcus genus bacteria and yeasts are used exclusively for the formation of a food covering layer with probiotic properties, without taking advantage of the potential of these microorganisms as a protein source.
  • the patent follows a great trend of using microbial cells only as an additive, underusing a material with high protein concentration and a source of other macro and micronutrients.
  • Animal proteins destined for the pet market can come from organs or other co-products of the refrigeration industry, but the greatest use is in the form of pieces of muscle. These tissues are formed by fibrillar proteins responsible for allowing the formation of contraction and relaxation structures, in addition to being rich in essential amino acids for the maintenance of metabolic functions in dogs and cats.
  • Animal myofibrillar proteins such as a-actin, troponin and tropomyosin are sources of amino acids such as lysine and methionine, which are an integral part of the hormonal system, act as compounds for enzymatic structures, are involved in oxygen transport and mainly in muscle activity of animals (MALVA et al., 2018).
  • MALVA et al., 2018 the maintenance of a meat-based diet for pets is justified, reiterating the need to find new ways to produce these proteins without reinforcing the environmental impacts they generate in animal husbandry.
  • yeasts as they are eukaryotic beings, are capable of expressing heterologous mammalian proteins with the most diverse purposes.
  • One of the examples more concrete of this technology are the numerous strains of Saccharomyces cerevisiae that produce polypeptides from other organisms.
  • the wide dissemination of the application of the species for this purpose is based on its ability to withstand the conditions of industrial cultivation, in addition to admitting the genetic sequences inserted according to the manufacturing need (GOMES et al., 2018).
  • patent RU0002515913 proposes a method of transforming the VKPM Y-3928 strain of Saccharomyces cerevisiae into a vector with the GAL1 promoter aiming at the expression of recombinant human fusion proteins.
  • the invention shows the feasibility of expression of mammalian proteins by yeasts of this species.
  • the patent US8753866B2 is cited, in which the production of secreted proteins in microbial eukaryotic cells is described.
  • the technique concerns the synthesis in Saccharomyces cerevisiae of any secreted protein, as a therapeutic or industrial enzyme such as lipases, cellulases, or proteases.
  • the coding sequences of proteins synthesized by yeast can be of microbial origin, such as fungi of the genus Trichoderma or Aspergillus, or of insect or mammalian cells. This method, despite including the expression of mammalian proteins, follows the great trend of applying the yeast mutation technique for the synthesis of heterologous polypeptides, aiming only at the production of enzymes.
  • patent CN108203697A is cited, in which the genetic modification of a strain of Saccharomyces cerevisiae to express fish NK (natural killer) cell proteins is described to be administered in aquatic animal cultures and reduce the use of medicines in them.
  • the invention consists of extracting the genetic material from fish cells, replicating it by PCR with specific primers for the desired region, inserting the replicated fragments in Escherichia coli TOP10 with restriction enzymes to obtain a recombinant plasmid, selecting the transformed bacteria and inserting the plasmid in Saccharomyces cerevisiae cells.
  • the patent has a very well consolidated yeast transformation technology, using the Escherichia coli TOP10 bacterium as an intermediary for the replication of plasmids containing the gene of interest before inserting into S. cerevisiae. Despite this, it follows the line of many similar technologies using the same yeast, focusing on the application of the expressed product as a therapeutic resource in animals, not exploring the possibility of synthesizing proteins for food purposes, in addition to using yeast as a direct administration to the animal, without processing or purification steps for the product.
  • patent CN106399135A is cited, in which the cultivation of the C20140911 strain of Saccharomyces cerevisiae that holds a high productive potential of proteins is detailed. Biomass is grown in a liquid medium, and fermentation products are dispersed in the reaction environment.
  • the great advantage of this invention resides in the high synthesis of proteins and amino acids when compared to an unmutated strain.
  • the expression of peptides and polypeptides proposed by this invention follows a common and very well defined path in other similar technologies, since the focus is on the synthesis of applicable amino acids in pig feed.
  • the use of mixtures resulting from metabolism of yeasts analogous to this one in animal feed is already a widely used and described technique.
  • the patent US6737262B1 is cited, which is the method of obtaining an animal feed containing polypeptides expressed in a genetically modified organism.
  • the proposed technology is based on the modification of yeast for the expression of a series of essential amino acids for animal nutrition. Grains containing a plurality of amino acids are also associated with the mixture. produced by the microbial route.
  • the patent provides for the use of a genetically modified organism, which is a yeast cell, it limits the application of the technique to express amino acids. Furthermore, it does not foresee a mixture of biomass with the selected grains, failing to explore its existing nutritional properties, focusing only on its use as a peptide expression vector.
  • Yeasts of the species Saccharomyces cerevisiae have very well consolidated their application as a translation system for proteins for industrial use, which are usable therapeutically or as food or food supplements for animals and humans.
  • a recently explored potential of this microorganism is to express mammalian myostatin, a protein capable of modulating animal weight and muscle composition.
  • S. cerevisiae was able to be modified with an expression cassette containing foreign genes responsible for the synthesis of the peptide of interest, which was used as an oral vaccine in rats, showing efficacy as a heterologous protein in generating an immune response (ZHANG et al., 2012 ).
  • patent US10485259B2 is cited, in which a series of proteins included in the composition of eggs are expressed in yeast cells (Saccharomyces cerevisiae or Pichia pastoris).
  • yeast cells Sacharomyces cerevisiae or Pichia pastoris.
  • the invention demonstrates the possibility of constructing a vector that allows the translation of one or more sequences, demonstrating that a eukaryotic organism can play the role of simultaneously expressing more than one polypeptide of interest. Still, it does not explore the expression of mammalian fibrillar proteins in yeast.
  • Feeding dogs and cats depends on a series of components to meet their metabolic and growth needs. Since meat consumption is closely related to major environmental impacts by agriculture, the solution is the expression of such proteins through a microbiological route. On the other hand, there are still elements of animal feed that can be replaced by nutritional sources from leftovers or rejects from agro-industrial processes.
  • agro-industrial by-products for animal feed are those that are rich sources of energy compounds, such as rice bran or soybeans and husks, bagasse, leaves or seeds of fruits, vegetables and roots.
  • Cassava husks and cereal residues are included as protein sources, as well as flours and filter cakes from oilseed processing. Finally, they can be incorporated leftovers from cereal milling and oil refining as a source of crude fiber (AULA et al., 2012).
  • DDGS dried beans with soluble distillers
  • patent KR100733906B1 is cited, in which a method of natural nutrition for pets is proposed.
  • the invention consists of a mixture based on egg, fruit and vegetable flour, animal or vegetable lipids and another component that can be bread flour, powdered seaweed, powdered cheese or brown rice flour. Finally, leftover meat is added in powdered form to then be packaged and stored. Despite using nutritional sources of tailings, the invention is still dependent on animal products to supply the protein content required by the food of dogs and cats.
  • yeasts Based on the ability of yeasts to admit heterologous mammalian genes and with their cellular machinery to be able to express a range of animal proteins, in addition to being safe microorganisms with widely described industrial application, it is possible to create a capable mutant strain to express fibrillar proteins rich in essential amino acids for the food of dogs and cats without relying on any animal product.
  • Figure 1 shows a simplified flowchart of the process, encompassing the stages of cultivation of mutated yeast biomass, formulation with agro-industrial residues and eventual post-processing steps to obtain the product.
  • the present invention deals with the preparation of a protein compound associated with other nutritional sources and applicable in dog and cat food.
  • the technique comprises the steps described in Figure 1 of (a) creating a genetically modified yeast strain to express heterologous animal fibrillary proteins; cultivation of mutated biomass to obtain microbial biomass expressing such proteins in the reaction medium and obtaining protein concentrate containing biomass and expressed proteins; and (b) formulation with other essential nutritional compounds for dogs and cats from agro-industrial waste and destination for post-processing stages of the product.
  • Step (a) of creating a genetically modified yeast strain provides for the preferential use of Saccharomyces cerevisiae, which can be associated or relocated by variants of the same or another species such as the genus Pichia, preferably Pichia pastoris.
  • this invention describes the construction of a yeast strain capable of producing animal proteins in its cytosol, so that the heterologous expression of some of the main polypeptides involved in the formation of animal muscle fibers (such as actin, troponin and tropomyosin).
  • the chosen sequences referring to the coding regions of actin, troponin and tropomyosin are, respectively, those identified as SEQ ID NO: 1, 2 or 3, SEQ ID NO: 4, 5 or 6, and SEQ ID NO: 7, 8 or 9, representing the three proteins for species of bovine (Bos taurus), chicken (Gallus gallus) or swine (Sus scrofá) - whose original species were chosen for their application in food as a source of protein.
  • the proteins identified in SEQ ID NO: 10, 11 or 12, SEQ ID NO: 13, 14 or 15, and SEQ ID NO: 16, 17 or 18 can be expressed simultaneously in the same microorganism or individually so that , during the fermentation process, there is the option of co-cultivation of yeasts of the same species, but expressing different recombinant proteins. In view of the technique developed, the expression of other muscle proteins is made possible in order to obtain a product that is even richer in proteins.
  • yeast For the genetic modification of yeast, it is used as an instrument of gene insertion the native capacity of the yeast to carry out homologous recombination as a repair mechanism to break the double strand of DNA. Therefore, from a site-directed gene editing methodology, a break in the yeast chromosome must be induced and made available in the cytosol of this DNA fragments containing the gene to be inserted - with a promoter compatible with the host species - marked by regions of break site homology, which allow, therefore, homologous recombination with this heterologous fragment and, therefore, the insertion of the gene - in this case, of the chosen fibrillar protein - in the pre-determined site by the break of the yeast chromosome.
  • the chosen gene editing methodology must, therefore, be able to target the location of the break in the chromosome and, consequently, target the location of the gene insertion in yeast.
  • sequences SEQ ID NO: 1, 2 or 3, SEQ ID NO: 4, 5 or 6, and SEQ ID NO: 7, 8 or 9 can be arranged in the form of an operon, which is formed by a promoter free constitutive regulation, followed by each gene containing a ribosome binding sequence (RBS) compatible with yeast of the S. cerevisiae species, and the coding region of each gene.
  • RBS ribosome binding sequence
  • Another strategy predicts that genes can be expressed differently, each being controlled by a specific promoter compatible with the yeast used.
  • the genes can be integrated into the microorganism's chromosome, aiming at the stability of the recombinant DNA and guaranteeing that it will remain intact in the progenies arising from cell division in a reaction medium, or expressed by maintaining the plasmid in the selected yeast.
  • the modification of the strain of Saccharomyces cerevisiae had, therefore, the steps of construction of vectors containing, each one: a gene or more for the expression of one or more animal fibrillary proteins; a promoter compatible with the yeast strain chosen for expression preceding the coding region of the gene; the homology region of the yeast chromosome, marking the gene sequence and other essential elements for the functionalization of the vector according to the chosen methodology - such as regions of compatibilization with the chosen microorganism for the propagation of the plasmid before transformation into yeast.
  • the vectors - may have one or more genes for the different fibrillar proteins -, it is necessary to carry out the transformation and multiplication of the plasmids in a compatible organism, preferably using the bacterium Escherichia coli.
  • the plasmids used for cloning the actin, troponin and tropomyosin genes come from the methodology described by Jessop-Fabre et al, and can be associated or replaced by other plasmids with widespread use in the species mentioned herein that are also capable of carrying the information genetics to the yeast of interest and cause the proteins to be expressed.
  • the vectors containing the genes with the promoter to be inserted and the homology region that directs the insertion into the chromosome must be purified and linearized to remove all other elements before present in the vector in order to allow its multiplication by the bacterial system.
  • the S. cerevisiae cells are then subjected to growth in a medium containing yeast extract, peptone and dextrose at a temperature of 28 to 40°C, until reaching about 2x1 Cl ⁇ cells/mL, and then washed with water deionized.
  • Transformation of the linearized DNA fragments is then performed by incubating the yeast cells with lithium acetate for 20 to 40 minutes at 30 to 50°C together with the heterologous DNA fragments to be inserted and the vectors containing the guide elements corresponding to the region of predetermined homology for the functioning of the methodology described by Jessop-Fabre et al., (2016).
  • the cells are incubated for 2 to 3 hours in medium containing yeast extract, peptone and dextrose constituting the recovery phase, to then be plated on solid medium with the same composition plus agar and specific antibiotics (G418 and Noursetromycin).
  • Plating is a fundamental step for the proper selection of positive clones for the presence of plasmids essential for the functioning of the gene editing system described by the methodology of Jessop-Fabre et al, 2016. Confirmation of plasmid DNA integration in the chromosome takes place by selection by PCR reaction and sequencing using specific oligonucleotides for the integrated region.
  • the successful clones selected can individually express all the genes of the fibrillar proteins described herein or it is possible to select more than one clone where each one expresses one or more of the fibrillar proteins mentioned herein.
  • step (a) In the phase subsequent to step (a), after the selection of the desired clones, aiming at the cultivation of the mutated biomass to obtain the proteins in the reaction medium, a pre-inoculum is initially carried out with the clone or selected clones in the medium containing yeast extract, peptone and dextrose, which can be supplemented with other macro and micronutrients required by the microorganism.
  • the pre-inoculums are added to the reactor in which the production of yeast biomass expressing the fibrillar proteins should occur.
  • the elements of the culture medium are incorporated, composed of a carbon source, a nitrogen source and a micronutrient source - preferably, the carbon source being glucose and nitrogen, urea or free amino acids.
  • the fermentation process takes place at 28°C to 45°C for up to 144h under constant agitation at 120 to 450 rpm in liquid medium under controlled conditions in a bioreactor.
  • the aeration of the medium is also monitored, maintained between 0.3 and 1.5 vvm, the pH from 4 to 8, and the production of foam, controlled, respectively, by the increase or reduction of the injection of filtered air, acid or base and defoamer.
  • the fermented broth can admit a solid-liquid separation step in order to retain the microbial biomass, in which case the unit operation is preferably carried out by a disc centrifuge or decanter or by a rotating drum-type filter.
  • the polypeptides of interest are intracellular, it is possible to add a cell lysis step to make them more bioavailable for animal consumption.
  • the lysis can occur before or after the filtration of the biomass, admitting the incorporation of the elements present in the fermented broth or opting for a resuspension of the separated cells in order to break them up.
  • the cell lysis of yeast is compatible with a series of physical-chemical, mechanical or enzymatic methods. Disruption steps can be chosen by means of high pressure homogenization, agitation in a ball mill or ultrasound, as long as the mechanical shocks or cavitation are controlled so that they do not degrade the proteins of interest. They are also viable and less costly options, such as osmotic shock, freezing and thawing or heating, since they do not depend on specific equipment and are based on temperature fluctuations or the concentration of salts in the intracellular environment. Furthermore, chemical methods are an alternative with the use of alkalis, acids, solvents or detergents capable of degrading the cell wall and releasing its internal content. Finally, similar to physicochemical methods in terms of practicality and exemption from additional equipment are enzymatic methods. Enzymes such as b-1,3-glucanases associated with a pH control for optimal enzymatic activity are effective in breaking down yeast cells.
  • a final product is obtained that can be liquid as an emulsion or pasty in order to be incorporated into the animal feed.
  • the advantage of using fermented broth is the maintenance of micro and macro nutrients excreted by the yeast during fermentation, which can enrich the nutritional composition of the final product.
  • the dispensability of costly downstream is presented as a technological advantage due to the economy in the process and the non-generation of waste during the production process.
  • the fermented broth containing yeast biomass expressing animal fibrillar proteins can be used directly without any downstream step for formulations with other elements nutritional products from agro-industrial residues;
  • the fermented broth containing yeast biomass expressing animal fibrillary proteins can be filtered or centrifuged in order to obtain the biomass with a lower liquid content to be used in the formulation with other nutritional elements from agro-industrial residues - thus dispensing the broth fermented and maintaining the biomass expressing fibrillar proteins;
  • the fermented broth containing yeast biomass or filtered or centrifuged yeast biomass can go through a cell lysis step to then be used in the formulation with other nutritional elements from agro-industrial residues.
  • the subsequent step concerns the formulation with other essential nutritional compounds for dogs and cats from agro-industrial waste. Based on an adequate composition for such animals and entirely from the production of genetically modified biomass expressing fibrillar proteins associated with nutritionally adequate agro-industrial residues, some compounds are defined as a source of proteins, carbohydrates, crude fibers, lipids, vitamins and minerals, having considering that the contents of these macronutrients in the residues can be variable as they depend closely on their state of processing and conservation.
  • the protein elements will come from the fermentation and lysis of the mutated Saccharomyces cerevisiae strain synthesizing animal fibrillary polypeptides.
  • Bran such as rapeseed, cottonseed, peas, soybeans or other legumes can also be added because they are rich in proteins and rejects from grain processing.
  • FDA Food and Drug Administration
  • AAFCO Association of American Feed Control Officials
  • the protein contents in dog food must not be less than 18% for the corporal maintenance of an adult animal, and a minimum of 22% to ensure conditions for growth and reproduction. For cats, this content for the same purposes is 26 and 30%.
  • Crude fiber can come from distillery grains, citrus pulp, bran such as peanuts, legume husks or grains, oil extraction filter cakes, fruit and vegetable husks, among others.
  • bran such as peanuts, legume husks or grains, oil extraction filter cakes, fruit and vegetable husks, among others.
  • the fibers still play an important role in the formulation by incorporating the feed mixture.
  • Sources of carbohydrates can be residues rich in sugars such as molasses, processed cereals such as barley, rice and wheat; and from fruit pulp or from remaining parts of tubers and other roots.
  • the variation in the rate of carbohydrates in the ration will depend on which objective you want to supply. So-called low-carbohydrate rations may contain lower levels than those intended for growth, with high energy demand or destined for the initial stages of development of pets.
  • Lipids in turn, can be added in the form of oils from seeds or legumes, present in lecithin, or from other sources, such as stone filter cake.
  • Crude Fiber rice, wheat, corn, barley, rye, oats
  • bagasse cakes of 1.2 0.9 filtration
  • Essential amino acids, vitamins, macro and microminerals that may not be sufficient when coming from the ingredients proposed in the feed formulation, can be added in other ways, so that the nutritional and metabolic needs of pets are met.
  • agro-industrial residues may be added to fulfill functions of sensory additives or to change the physicochemical conditions of the mixture.
  • Possible tailings used as feed additives are dreche, DDGS (distilled dried grains) or peanut bran to increase palatability, glycerin as a moisture retainer in dry or extruded feeds, lecithin as a thickening agent, and molasses as a binder.
  • Pet foods are available in commercial variations of form and moisture. Solid rations contain 6 to 10% moisture, canned 60 to 90% and semi-solid 25 to 35%. Following the formulation stage, the mixture obtained goes to the product post-processing stage, which may include extrusion, pelletizing, coating, drying, among others. The final form of the product will depend on the desired palatability and nutrient concentrations, the animal's ease of digestion, the lifetime for which it is intended, whether or not additives or preservatives are inserted, the desired shelf life and the cost of the feed. finished.
  • EXAMPLE 1 Creation of cloning vectors
  • the process of transforming the strain begins s288c of Saccharomyces cerevisiae with the construction of vectors containing the genes of animal fibrillary proteins.
  • an assembly containing the gene responsible for the expression of the proteins of interest is necessary, accompanied by a promoter compatible with the yeast cellular machinery in the upstream of the coding strand, and flanked by sequences coincident with the region of homology chosen on the basis of knowledge of the structure of the yeast chromosomes.
  • the constructed vector consisted of the gene of SEQ ID NO: 1, preceded by the natural yeast promoter identified in SEQ ID NO: 19 and delimited by regions of homology compatible with the of SEQ ID NO: 22.
  • a second vector was elaborated containing the necessary elements for the expression of the protein of SEQ ID NO: 13, as the gene of SEQ ID NO: 4, preceded by the same promoter of SEQ ID NO. : 19 for its greater compatibility with the transcription and translation machinery of the Saccharomyces cerevisiae s288c.
  • the constructed vector contained sequences capable of being linked with this region in the repair process described in Example 2.
  • heterologous protein of SEQ ID NO: 16 by Saccharomyces cerevisiae s288c the third vector constructed consisted of the promoter of SEQ ID NO: 19, followed by the gene of SEQ ID NO: 7.
  • the vector was finished with the presence at the ends of the fragment of sequences capable of being reconciled with the chosen region.
  • EXAMPLE 2 Cloning of selected sequences in Saccharomyces cerevisiae and linearization in the chromosome
  • the methodology proposed by Jessop-Fabre et al. (2016) consists of carrying out a site-directed mutation and therefore predicts that, for the correct insertion of genes of interest in the desired regions of homology to occur, the technique relies on an endonuclease to make the cuts in the yeast chromosomes and a guide RNA (gRNA) for each chosen region of homology, causing the endonuclease to be directed to the exact region of homology in which the inserts are to be incorporated.
  • gRNA guide RNA
  • plasmids containing the gene for translation into gRNA and a plasmid containing the sequence for the synthesis of the endonuclease used to open the strands of the chromosomes are inserted.
  • Saccharomyces cerevisiae s288c was initially cultivated in peptone yeast extract medium dextrose with 20g/L dextrose for approximately 48 hours at 30 °C until reaching I CL cells/mL - quantified in a counting chamber - and subsequently washed with sterile deionized water.
  • EXAMPLE 3 Cultivation of genetically modified yeast to obtain protein concentrate containing biomass expressing animal fibrillar proteins
  • yeast extract medium peptone dextrose with 20g/L liquid dextrose is performed in order to reach the concentration in the bioreactor for the production of 5 g/L of biomass.
  • the inoculum is cultured for 72 hours at 37 °C with constant stirring at 120 rpm until reaching a cell concentration of 1x10 7 CFU.mL'.
  • the process continues with the step of cultivation of the mutated biomass in a bioreactor.
  • a liquid medium containing glucose and free amino acids at concentrations of 20 and 8 g/L, respectively.
  • the previously cultivated inoculum is added, reaching an initial concentration of 5 g/L of biomass in the bioreactor.
  • the fermentation process for biomass production propagates for 72 hours at 35 Q C with pH 5, maintained at a constant rotation of 320 rpm. Aeration was maintained at 0.5 vvm and increased to 1.2 vvm after 12 hours. At the end of fermentation, a final biomass concentration of 30 g/L was obtained.
  • EXAMPLE 4 Formulation of a protein-rich compound for dog food
  • a suitable composition may contain 70% yeast expressing fibrillar proteins, associated with 10% palm kernel flour as a protein source, plus 10% sugarcane molasses capable of offering reducing sugars, 15% oil of soy to supply the demand for lipids, and 2% of dry distillery grains providing the fibers and being admitted as an agent to increase palatability.
  • a suitable composition can contain 80% of yeast expressing fibrillar proteins, plus 10% of remaining parts of potato capable of offering reducing sugars, 10% of soybean oil to supply the lipid demand, and 1% of rejects from corn milling giving the fibers.
  • composition results in a nutritious compound with dry base element rates of 38% crude protein, 11.4% fat, 3.9% crude fiber and 94% digestible energy.
  • Such formulation suits the macronutrient demands of cats and also admits the addition of additives or supplements of vitamins, minerals and amino acids, or serves as an ingredient for other formulations intended for this group of animals.
  • the composition resulting from the mutated yeast biomass plus agroindustrial residues as a nutritional source follows the mixing step for an extruder with the desired grain millimeter, operating between 60 to 100 Q C, eliminating pathogens such as Enterococcus spp. or Salmonella spp. without causing damage to the ingredients of the formulation.
  • the formulated and shaped feed grains are dried by heated air in convection operating from 100 to 130 Q C, obtaining a final product with humidity between 6 and 10%.
  • the resulting composition of the mutated yeast biomass plus agro-industrial residues as a nutritional source proceeds from the mixing step to one for an extruder operating at 60 to 100 Q C, eliminating pathogens such as Enterococcus spp. or Salmonella spp. without causing damage to the ingredients of the formulation.
  • the extruded dough with 20-30% moisture is cut into pieces of the size of interest and mixed with a jelly or paste to achieve the desired final moisture of 60-90% before being packaged or canned.
  • SEQ ID NO: 5 Coding sequence for the tropomyosin alpha 1 chain of Gallus gallus
  • SEQ ID NO: 6 Coding sequence for the tropomyosin alpha 1 chain of Sus scrofa
  • SEQ ID NO: 7 Coding sequence for the troponin T1 chain of Bos taurus
  • SEQ ID NO: 8 Coding sequence for troponin T1 chain from Gallus gallus
  • SEQ ID NO: 12 Sus scrofa alpha 1 actin amino acid sequence
  • SEQ ID NO: 13 Bos taurus alpha 1 tropomyosin amino acid sequence
  • SEQ ID NO: 14 Gallus gallus tropomyosin alpha 1 amino acid sequence
  • SEQ ID NO: 15 Sus scrofa tropo
  • SEQ ID NO: 20 Saccharomyces cerevisiae s288c X chromosome homology region located between base pairs 194944 and 195980
  • SEQ ID NO: 21 Saccharomyces cerevisiae s288c chromosome XI homology region located between base pairs 93378 and 94567
  • SEQ ID NO: 22 Saccharomyces cerevisiae s288c chromosome XI homology region located between base pairs 91575 and 92913
  • BEITZ D.C.
  • BAUER J.E.
  • BEHNKE K.C.
  • DZANIS D.A.
  • FAHEY G.C.
  • HILL HILL
  • JESSOP-FABRE M.M.; JOClNAS, T; STOVICEK, V.; DAI, Z.; JENSEN M.K.; KEASLING, J.D.; BORODINA, I. EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnology Journal, Vol. 11. 2016.
  • LEENSTRA F.
  • VELLINGA TV
  • BESSEI W. Environmental footprint of meat consumption of cats and dogs. Lohmann Information. 33, Vol. 52(1). June/2018.
  • FIGLIOLA L;
  • RISOLIA L.W.
  • SABCHUCK T.T.
  • MURAKAMI F.Y.
  • FÉLIX AP
  • MAJORKA A.
  • DE OLIVEIRA S.G. Effects of adding dried distillers with soluble grains (DDGS) to dog diets supplemented with xylanase and protease. Brazilian Journal of Animal Science, Vol. 48. 2019.
  • ZHANG T.; SUN. L; XIN, Y.; BAD. L. ZHANG, Y.; Wang, X.; XU, K.; REN, C.; ZHANG, C.; CHEN, Z.; YANG, H.; ZHANG, Z.

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Abstract

The present invention relates to a nutritional composition for feeding dogs or cats, comprising an alternative protein composition to animal protein, consisting of the biomass from an organism genetically modified to express fibrillar proteins of the animal muscle, associated with agro-industrial waste as other nutritional sources, and to the method for obtaining this composition. The method comprises the steps of: (a) creating a strain of Saccharomyces cerevisiae or Pichia pastoris genetically modified to heterologously express fibrillar proteins of the animal muscle; cultivating the mutated biomass to obtain the microbial biomass expressing such proteins in the reaction medium, and obtaining the protein concentrate containing the biomass and expressed proteins; and (b) formulation with other nutritional compounds essential for dogs and cats, originating from agro-industrial waste, and sending to steps for post-processing of the product.

Description

COMPOSIÇÃO NUTRICIONAL PARA CÃES OU GATOS, CONSTITUÍDA DE BIOMASSA DE UM ORGANISMO GENETICAMENTE MODIFICADO, EXPRESSANDO PROTEÍNAS FIBRILARES DO MÚSCULO ANIMAL, ASSOCIADA A OUTRAS FONTES NUTRICIONAIS PROVENIENTES DE RESÍDUOS AGROINDUSTRIAIS, E NUTRITIONAL COMPOSITION FOR DOGS OR CATS, CONSISTING OF BIOMASS FROM A GENETICALLY MODIFIED ORGANISM, EXPRESSING FIBRILLARY PROTEINS OF ANIMAL MUSCLE, ASSOCIATED WITH OTHER NUTRITIONAL SOURCES FROM AGROINDUSTRIAL WASTE, AND
PROCESSO DE OBTENÇÃO GETTING PROCESS
Campo da invenção field of invention
[001] A presente invenção dedica-se ao setor de nutrição de cães e gatos. [001] The present invention is dedicated to the dog and cat nutrition sector.
Fundamentos da invenção Fundamentals of Invention
[002] A domesticação de animais acompanha a história humana, uma vez que cães eram uma ferramenta para garantir a segurança de residências ou auxiliar em caças, e gatos apresentavam um papel importante no controle de roedores. A alimentação destes ocorria de acordo com a nutrição humana e na base das dietas, entre outras fontes de nutrientes, encontrava-se a carne. [002] The domestication of animals follows human history, as dogs were a tool to ensure the security of homes or assist in hunting, and cats played an important role in rodent control. The feeding of these occurred according to human nutrition and on the basis of diets, among other sources of nutrients, was meat.
[003] Atualmente, com a tendência à industrialização e com cães e gatos assumindo um papel majoritário como animais de estimação, instaura-se um mercado que demanda rações fabricadas e de alta qualidade. Ainda assim, permanece como fonte proteica predominante a carne bovina, de frango, suína, de peixes ou oriunda de outros animais também consumíveis por humanos. [003] Currently, with the trend towards industrialization and with dogs and cats assuming a major role as pets, a market is established that demands high quality manufactured feed. Even so, the predominant protein source remains beef, chicken, pork, fish or meat from other animals that are also consumable by humans.
[004] As demandas de fontes energéticas para cães e gatos se diferenciam de acordo com as necessidades nutricionais de cada grupo. De acordo com a Escola de Medicina Veterinária da Universidade de Cornell, gatos são animais que dependem da inserção de carne em suas dietas devido aos aminoácidos essenciais nela presentes (principalmente carnitina e taurina). Já cães assumem uma alimentação mais flexível em termo de proteínas, podendo assimilar outras fontes como vegetais, cereais e leguminosas. Apesar de adotarem uma dieta maleável, para cães a proteína animal ainda é considerada um meio completo de obtenção dos aminoácidos essenciais não produzidos pelo animal (BEITZ et al., 2006). [004] The demands of energy sources for dogs and cats differ according to the nutritional needs of each group. According to the Cornell University School of Veterinary Medicine, cats are animals that depend on meat being added to their diets because of the essential amino acids in it (mostly carnitine and taurine). Dogs, on the other hand, assume a more flexible diet in terms of proteins, being able to assimilate other sources such as vegetables, cereals and legumes. Despite adopting a malleable diet, for dogs animal protein is still considered a complete means of obtaining essential amino acids not produced by the animal (BEITZ et al., 2006).
[005] Tomando como base o consumo diário intermediário de animais de cães e gatos de médio porte, a faixa de concentração ideal de proteínas é de 23 a 25 g/dia em rações secas ou 6,4 a 8 g/dia em rações úmidas. Considerando as porções de ração recomendadas e o número de animais para cada 1000 habitantes, o consumo de carne por rações animais pode equivaler a parcelas significativas do total de produção nacional. Em países com elevado número de cães e gatos, como os Estados Unidos, este valor chega a 31 ,52%. Em outros países, como os do Reino Unido, 39,21% da produção de carne é destinada para rações de animais de estimação, já na Itália esse valor é de 31 ,45% (LEENSTRA, VELLINGA & BESSEI; 2018). [005] Based on the intermediate daily consumption of medium-sized dogs and cats, the ideal protein concentration range is 23 to 25 g/day in rations dry or 6.4 to 8 g/day in wet rations. Considering the recommended feed portions and the number of animals for every 1000 inhabitants, the consumption of meat for animal feed can be equivalent to significant portions of the total national production. In countries with a high number of dogs and cats, such as the United States, this figure reaches 31.52%. In other countries, such as the United Kingdom, 39.21% of meat production is destined for pet food, while in Italy this figure is 31.45% (LEENSTRA, VELLINGA &BESSEI; 2018).
[006] Associado ao vasto consumo de carne animal está o impacto ambiental causado principalmente pelas culturas de aves, suínos e bovinos. A emissão de gases causadores do efeito estufa, degradação de áreas nativas para pastagem, e consumo de água representam uma ameaça para o meio ambiente. Em uma analogia à pegada de carbono - análise das consequências geradas pelo consumo humano de diversos produtos - estudos são feitos para determinar a “ecological paw print” (EPP) provocada pelo perfil de alimentação animal. [006] Associated with the vast consumption of animal meat is the environmental impact caused mainly by the cultures of poultry, swine and cattle. The emission of greenhouse gases, degradation of native areas for pasture, and water consumption represent a threat to the environment. In an analogy to the carbon footprint - analysis of the consequences generated by human consumption of various products - studies are carried out to determine the “ecological paw print” (EPP) caused by the profile of animal feed.
[007] No Japão, por exemplo, a população de cães e gatos domésticos tem o potencial consumidor de 3,6 a 15,6% de toda a comida produzida no país em um ano, podendo chegar à emissão de 2,5 a 10,7 milhões de gases do efeito estufa nesse mesmo período (SU & MARTENS, 2018). [007] In Japan, for example, the population of domestic dogs and cats has a potential consumer of 3.6 to 15.6% of all food produced in the country in one year, reaching an emission of 2.5 to 10 ,7 million greenhouse gases in the same period (SU & MARTENS, 2018).
[008] Assim, há uma crescente tendência para o desenvolvimento de produtos baseados em fontes proteicas alternativas, visando tanto o bem-estar de cães e gatos quanto a mitigação do impacto ambiental causado pelo perfil de consumo de proteínas animais. Uma possibilidade é o uso de biomassa microbiana, podendo ser de leveduras, fungos filamentosos ou bactérias, como produto de alto teor proteico para ser usado como parte integrante de rações para animais de estimação. [008] Thus, there is a growing trend towards the development of products based on alternative protein sources, aiming at both the welfare of dogs and cats and the mitigation of the environmental impact caused by the consumption profile of animal proteins. One possibility is the use of microbial biomass, which can be yeasts, filamentous fungi or bacteria, as a product with high protein content to be used as an integral part of pet food.
[009] Como referência, cita-se a patente US3939284A, na qual é proposto um método de obtenção de células de levedura, entre elas Candida utilis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae e Saccharomyces fragilis para serem misturadas a fontes de proteínas vegetais e animais, como concentrado proteico de peixe, rejeitos de abatedouros e farinha de ossos. Apesar de apresentar a associação de biomassa de levedura como componente (de 5 a 50%), como muitas formulações, ainda depende de partes animais para suprir as necessidade nutricionais em termos de proteína. Além disso, conta com aditivos flavorizantes para aumentar a atratividade do produto, o qual é destinado exclusivamente à alimentação humana, sem prospectar a aplicação como ração animal. [009] As a reference, the patent US3939284A is cited, in which a method of obtaining yeast cells is proposed, including Candida utilis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae and Saccharomyces fragilis to be mixed with vegetable and animal protein sources, as fish protein concentrate, slaughterhouse rejects and bone meal. Despite presenting the association of yeast biomass as a component (from 5 to 50%), like many formulations, still it depends on animal parts to meet nutritional requirements in terms of protein. In addition, it has flavoring additives to increase the attractiveness of the product, which is intended exclusively for human consumption, without prospecting for application as animal feed.
[0010] Dentre as inúmeras possibilidades de microrganismos GRAS (geralmente reconhecido como seguro) para serem usados como biomassa proteica em alimentação, um destaque é feito para leveduras, como a Saccharomyces cerevisiae. [0010] Among the numerous possibilities of GRAS microorganisms (generally recognized as safe) to be used as protein biomass in food, a highlight is made for yeasts, such as Saccharomyces cerevisiae.
[0011] Além de aproximadamente 50% de sua composição ser de proteínas, leveduras também podem ser fonte de vitamina B - envolvida nos sistemas nervoso e circulatório - além de selênio - mineral antioxidante auxiliar na atividade pancreática e endócrina. Outra vantagem de sua admissão como um composto de rações de animais de estimação é sua alta digestibilidade (entre 60 e 80%), fazendo com que os macro e micronutrientes presentes na estrutura celular sejam aproveitados por cães e gatos. [0011] In addition to approximately 50% of its composition being proteins, yeast can also be a source of vitamin B - involved in the nervous and circulatory systems - in addition to selenium - auxiliary antioxidant mineral in pancreatic and endocrine activity. Another advantage of its admission as a compost for pet food is its high digestibility (between 60 and 80%), making the macro and micronutrients present in the cellular structure to be used by dogs and cats.
[0012] A adição de leveduras em rações também é realizada pelo potencial que estes organismos têm de aumentar a aceitabilidade do produto por melhorarem a palatabilidade de formulações. Esse efeito é observado principalmente em cães com a adição de extrato de levedura, nos casos em que há um pré-tratamento térmico da mistura antes de finalizada (LIMA et al., 2016). [0012] The addition of yeasts in rations is also carried out by the potential that these organisms have to increase the acceptability of the product by improving the palatability of formulations. This effect is mainly observed in dogs with the addition of yeast extract, in cases where there is a thermal pre-treatment of the mixture before it is finished (LIMA et al., 2016).
[0013] Como referência, cita-se a patente CN102008026B, na qual a formulação proposta sugere a utilização de biomassa de leveduras ou bactérias do gênero Bacillus como uma fonte de microproteínas. Associada a partes animais como órgãos e peles, farinha ou grãos proteicos inteiros e compostos lipídicos animais e vegetais, além de vitaminas e outros minerais, a biomassa microbiana representa uma fonte adicional de aminoácidos e polipeptídeos. Apesar de inserir leveduras como um componente nutricional na ração, não somente como um aditivo, a invenção ainda é dependente de partes animais como fontes de proteínas e lipídeos, fato que ainda corrobora para a manutenção do consumo elevado de carne por cães e gatos. [0013] As a reference, patent CN102008026B is cited, in which the proposed formulation suggests the use of yeast biomass or Bacillus genus bacteria as a source of microproteins. Associated with animal parts such as organs and skin, flour or whole protein grains and animal and vegetable lipid compounds, in addition to vitamins and other minerals, microbial biomass represents an additional source of amino acids and polypeptides. Despite inserting yeasts as a nutritional component in the feed, not just as an additive, the invention is still dependent on animal parts as sources of proteins and lipids, a fact that further supports the maintenance of high consumption of meat by dogs and cats.
[0014] Como referência cita-se a patente CA2735659C, na qual é descrita a formulação de uma ração para animais de estimação composta majoritariamente por uma matriz vegetal, chegando a até 70% da massa total. O método, apesar de dispensar fontes animais em sua composição e inserir elementos pré e probióticos, a biomassa de levedura aparece apenas como um coadjuvante. São utilizadas bactérias e leveduras dos gêneros Bacillus, Bacteroides, Bifidobacterium, Enterococcus, Lactobacillus, Leuconostoc, Saccharomyces, Candida ou Streptococcus exclusivamente para a formação de uma camada de cobertura da ração com propriedades probióticas, sem aproveitar o potencial destes microrganismos como fonte proteica. A patente segue uma grande tendência de utilização de células microbianas apenas como um aditivo, subutilizando um material com alta concentração proteica e fonte de outros macro e micronutrientes. [0014] As a reference, patent CA2735659C is cited, in which the formulation of a pet food composed mainly of a vegetable matrix is described, reaching up to 70% of the total mass. The method, despite dispensing sources animals in its composition and insert pre and probiotic elements, yeast biomass appears only as a adjuvant. Bacillus, Bacteroides, Bifidobacterium, Enterococcus, Lactobacillus, Leuconostoc, Saccharomyces, Candida or Streptococcus genus bacteria and yeasts are used exclusively for the formation of a food covering layer with probiotic properties, without taking advantage of the potential of these microorganisms as a protein source. The patent follows a great trend of using microbial cells only as an additive, underusing a material with high protein concentration and a source of other macro and micronutrients.
[0015] A tendência de alimentação de cães e gatos alternativa à carne deu lugar a um nicho de opções de rações à base de proteínas vegetais. Apesar de uma alternativa ambientalmente mais amigável, não supre a totalidade de necessidades nutricionais. Fontes proteicas provenientes de grãos e cereais apresentam uma deficiência em aminoácidos essenciais que animais de estimação necessitam e não conseguem produzir por conta própria (DONADELLI, JONES & BEYER, 2019). [0015] The trend of feeding dogs and cats as an alternative to meat has given way to a niche of vegetable protein-based feed options. Despite being a more environmentally friendly alternative, it does not meet all nutritional needs. Protein sources from grains and cereals present a deficiency in essential amino acids that pets need and cannot produce on their own (DONADELLI, JONES & BEYER, 2019).
[0016] As proteínas animais destinadas ao mercado pet podem ser provenientes de órgãos ou outros co-produtos da indústria frigorífica, mas a maior utilização se dá em forma de peças de músculos. Estes tecidos são formados por proteínas fibrilares responsáveis por permitir a formação de estruturas de contração e relaxamento, além de serem ricas em aminoácidos essenciais para a manutenção de funções metabólicas em cães e gatos. [0016] Animal proteins destined for the pet market can come from organs or other co-products of the refrigeration industry, but the greatest use is in the form of pieces of muscle. These tissues are formed by fibrillar proteins responsible for allowing the formation of contraction and relaxation structures, in addition to being rich in essential amino acids for the maintenance of metabolic functions in dogs and cats.
[0017] Proteínas animais miofibrilares como a-actina, troponina e tropomiosina são fontes de aminoácidos como lisina e metionina, os quais são parte integrante do sistema hormonal, atuam como compostos para estruturas enzimáticas, estão envolvidos no transporte de oxigénio e principalmente na atividade muscular de animais (MALVA etal., 2018). Assim, justifica-se a manutenção de uma dieta a base de carne para animais de estimação, reiterando a necessidade de descobrir novas formas de produzir estas proteínas sem reforçar as criações de animais os impactos ambientais por elas gerados. [0017] Animal myofibrillar proteins such as a-actin, troponin and tropomyosin are sources of amino acids such as lysine and methionine, which are an integral part of the hormonal system, act as compounds for enzymatic structures, are involved in oxygen transport and mainly in muscle activity of animals (MALVA et al., 2018). Thus, the maintenance of a meat-based diet for pets is justified, reiterating the need to find new ways to produce these proteins without reinforcing the environmental impacts they generate in animal husbandry.
[0018] Sabe-se que leveduras, por serem seres eucarióticos, são capazes de expressar proteínas heterólogas de mamíferos com os mais diversos propósitos. Um dos exemplos mais concretos desta tecnologia são as inúmeras cepas de Saccharomyces cerevisiae que produzem polipetídeos de outros organismos. A grande disseminação da aplicação da espécie para esta finalidade está pautada em sua capacidade de suportar as condições de cultivo industrial, além de admitir as sequências genéticas inseridas conforme a necessidade de fabricação (GOMES et al., 2018). [0018] It is known that yeasts, as they are eukaryotic beings, are capable of expressing heterologous mammalian proteins with the most diverse purposes. One of the examples more concrete of this technology are the numerous strains of Saccharomyces cerevisiae that produce polypeptides from other organisms. The wide dissemination of the application of the species for this purpose is based on its ability to withstand the conditions of industrial cultivation, in addition to admitting the genetic sequences inserted according to the manufacturing need (GOMES et al., 2018).
[0019] Como referência cita-se a patente RU0002515913, na qual propõe-se um método de transformação da cepa VKPM Y-3928 de Saccharomyces cerevisiae em um vetor com o promotor GAL1 visando a expressão de proteínas de fusão humanas recombinantes. A invenção mostra a viabilidade da expressão de proteínas de mamíferos por leveduras desta espécie. [0019] Reference is made to patent RU0002515913, which proposes a method of transforming the VKPM Y-3928 strain of Saccharomyces cerevisiae into a vector with the GAL1 promoter aiming at the expression of recombinant human fusion proteins. The invention shows the feasibility of expression of mammalian proteins by yeasts of this species.
[0020] Como referência cita-se a patente US8753866B2, na qual é descrita a produção de proteínas secretadas em células eucarióticas microbianas. A técnica refere-se à síntese em Saccharomyces cerevisiae de qualquer proteína secretada, como uma enzima terapêutica ou industrial tais como lipases, celulases, ou proteases. A sequências codificantes das proteínas sintetizadas pela levedura podem ser de origem microbiana, como fungos do gênero Trichoderma ou Aspergillus, ou de células de insetos ou mamíferos. Este método, apesar de incluir a expressão de proteínas de mamíferos, segue a grande tendência de aplicação da técnica de mutação de leveduras para síntese de polipeptídeos heterólogos, visando somente a produção de enzimas. [0020] As reference, the patent US8753866B2 is cited, in which the production of secreted proteins in microbial eukaryotic cells is described. The technique concerns the synthesis in Saccharomyces cerevisiae of any secreted protein, as a therapeutic or industrial enzyme such as lipases, cellulases, or proteases. The coding sequences of proteins synthesized by yeast can be of microbial origin, such as fungi of the genus Trichoderma or Aspergillus, or of insect or mammalian cells. This method, despite including the expression of mammalian proteins, follows the great trend of applying the yeast mutation technique for the synthesis of heterologous polypeptides, aiming only at the production of enzymes.
[0021] Como referência cita-se a patente CN108203697A, na qual é descrita a modificação genética de uma cepa de Saccharomyces cerevisiae para expressar proteínas de células NK {natural killer) de peixes para serem administradas em culturas animais aquáticas e reduzir o uso de medicamentos nas mesmas. A invenção consiste em extrair o material genético de células de peixe, replicá-lo por PCR com primers específicos para a região desejada, inserir os fragmentos replicados em Escherichia coli TOP10 com enzimas de restrição para obter um plasmídeo recombinante, selecionar as bactérias transformadas e inserir o plasmídeo em células de Saccharomyces cerevisiae. A patente conta com uma tecnologia de transformação de leveduras muito bem consolidada, utilizando a bactéria Escherichia coli TOP10 como intermediária para replicação dos plasmídeos contendo o gene de interesse antes de inserir na S. cerevisiae. Apesar disso, segue a linha de muitas tecnologias semelhantes utilizando a mesma levedura, focando a aplicação do produto expressado como um recurso terapêutico em animais, não explorando a possibilidade de sintetizar proteínas para fins alimentares, além de destinar a levedura como administração direta ao animal, sem etapas de processamento ou purificação do produto. [0021] As a reference, patent CN108203697A is cited, in which the genetic modification of a strain of Saccharomyces cerevisiae to express fish NK (natural killer) cell proteins is described to be administered in aquatic animal cultures and reduce the use of medicines in them. The invention consists of extracting the genetic material from fish cells, replicating it by PCR with specific primers for the desired region, inserting the replicated fragments in Escherichia coli TOP10 with restriction enzymes to obtain a recombinant plasmid, selecting the transformed bacteria and inserting the plasmid in Saccharomyces cerevisiae cells. The patent has a very well consolidated yeast transformation technology, using the Escherichia coli TOP10 bacterium as an intermediary for the replication of plasmids containing the gene of interest before inserting into S. cerevisiae. Despite this, it follows the line of many similar technologies using the same yeast, focusing on the application of the expressed product as a therapeutic resource in animals, not exploring the possibility of synthesizing proteins for food purposes, in addition to using yeast as a direct administration to the animal, without processing or purification steps for the product.
[0022] Como referência cita-se a patente MX2015017899A, a qual descreve o uso do gene da quimosina bovina otimizado com uma sequência sintética para a produção de proteína recombinante, expressando em Pichia pastoris. A invenção parte de uma sequência de aminoácidos com alto nível de similaridade à codificada por bois, mas com mudanças pontuais para que seja assimilada pela levedura usada como hospedeira. Com o código genético aprimorado para a síntese de quimosina bovina, ocorre a inserção do plasmídeo pPICZa encarregado de transformar as células de Pichia pastoris. Esta tecnologia mostra a aceitabilidade que espécies de leveduras têm com genes de mamíferos, mas como tantos outros processos similares foca na expressão de uma enzima, não como um vetor de tradução de outras proteínas com função de suprimento proteico. [0022] As reference is cited the patent MX2015017899A, which describes the use of the bovine chymosin gene optimized with a synthetic sequence for the production of recombinant protein, expressing in Pichia pastoris. The invention starts with an amino acid sequence with a high level of similarity to that encoded by oxen, but with specific changes so that it is assimilated by the yeast used as a host. With the improved genetic code for the synthesis of bovine chymosin, the pPICZa plasmid responsible for transforming Pichia pastoris cells is inserted. This technology shows the acceptability that yeast species have with mammalian genes, but like so many other similar processes it focuses on the expression of an enzyme, not as a translation vector of other proteins with a protein supply function.
[0023] Como referência cita-se a patente CN106399135A, na qual é detalhada o cultivo da cepa C20140911 de Saccharomyces cerevisiae que detém um alto potencial produtivo de proteínas. A biomassa é cultivada em meio líquido, e os produtos de fermentação ficam dispersos no ambiente reacional. A grande vantagem desta invenção reside na alta síntese de proteínas e aminoácidos quando comparada com uma cepa não mutada. Apesar de demonstrar uma eficácia competitiva, a expressão de peptídeos e polipeptídeos proposta por esta invenção segue um rumo comum e muito bem definido em outras tecnologias semelhantes, uma vez que o enfoque é na síntese de aminoácidos aplicáveis em ração de porcos. O uso de misturas resultantes de metabolismo de leveduras análogos à esta em alimentação de animais já é uma técnica amplamente difundida e descrita. [0023] As a reference, patent CN106399135A is cited, in which the cultivation of the C20140911 strain of Saccharomyces cerevisiae that holds a high productive potential of proteins is detailed. Biomass is grown in a liquid medium, and fermentation products are dispersed in the reaction environment. The great advantage of this invention resides in the high synthesis of proteins and amino acids when compared to an unmutated strain. Despite demonstrating a competitive efficacy, the expression of peptides and polypeptides proposed by this invention follows a common and very well defined path in other similar technologies, since the focus is on the synthesis of applicable amino acids in pig feed. The use of mixtures resulting from metabolism of yeasts analogous to this one in animal feed is already a widely used and described technique.
[0024] Como referência cita-se a patente US6737262B1 , a qual se trata do método de obtenção de uma ração animal contendo polipeptídeos expressos em um organismo geneticamente modificado. A tecnologia proposta é baseada na modificação de levedura para a expressão de uma série de aminoácidos essenciais à nutrição animal. Também são associados grãos detentores de uma pluralidade de aminoácidos à mistura produzida por via microbiana. Apesar da patente prever o uso de um organismo geneticamente modificado, sendo este uma célula de levedura, limita a aplicação da técnica para expressar aminoácidos. Além disso, não prevê uma mistura da biomassa com os grãos selecionados, deixando de explorar suas propriedades nutricionais existentes, focando apenas em seu uso como um vetor de expressão de peptídeos. [0024] As a reference, the patent US6737262B1 is cited, which is the method of obtaining an animal feed containing polypeptides expressed in a genetically modified organism. The proposed technology is based on the modification of yeast for the expression of a series of essential amino acids for animal nutrition. Grains containing a plurality of amino acids are also associated with the mixture. produced by the microbial route. Although the patent provides for the use of a genetically modified organism, which is a yeast cell, it limits the application of the technique to express amino acids. Furthermore, it does not foresee a mixture of biomass with the selected grains, failing to explore its existing nutritional properties, focusing only on its use as a peptide expression vector.
[0025] Leveduras da espécie Saccharomyces cerevisiae têm muito bem consolidada sua aplicação como um sistema de tradução para proteínas de emprego industrial, as quais são utilizáveis de forma terapêutica ou como alimento ou suplementos alimentares animais e humanos. Um potencial recentemente explorado deste microrganismo é o de expressar miostatina de mamíferos, uma proteína capaz de modular peso e composição muscular de animais. A S. cerevisiae foi capaz de ser modificada com um cassete de expressão contendo genes estrangeiros responsáveis pela síntese do peptídeo de interesse, que foi utilizada como vacina oral em ratos, apresentando eficácia como proteína heteróloga ao gerar resposta imune (ZHANG et ai., 2012). [0025] Yeasts of the species Saccharomyces cerevisiae have very well consolidated their application as a translation system for proteins for industrial use, which are usable therapeutically or as food or food supplements for animals and humans. A recently explored potential of this microorganism is to express mammalian myostatin, a protein capable of modulating animal weight and muscle composition. S. cerevisiae was able to be modified with an expression cassette containing foreign genes responsible for the synthesis of the peptide of interest, which was used as an oral vaccine in rats, showing efficacy as a heterologous protein in generating an immune response (ZHANG et al., 2012 ).
[0026] Células de Saccharomyces cerevisiae e Pichia pastoris tiveram seu potencial analisado para a expressão de a-tropomiosiona, uma proteína muscular fibrilar, analisando sua similaridade com a produzida por mamíferos. O estudo mostra que a estabilidade, compatibilidade de sequência de aminoácidos, bem como a funcionalidade da proteína dependem do número de cópias do plasmídeo, força do promotor e composição do meio reacional (ALEGRIA et ai., 2003). Apesar de demonstrar a eficácia na expressão de uma proteína fibrilar de mamíferos, a aplicação da mesma reserva-se a estudos comparativos, não explora a possibilidade de associar esta com outras proteínas similares, tampouco destina seu emprego à alimentação animal, mostrando que esta é uma rota não traçada. [0026] Cells of Saccharomyces cerevisiae and Pichia pastoris had their potential analyzed for the expression of a-tropomyosone, a fibrillar muscle protein, analyzing its similarity with that produced by mammals. The study shows that stability, amino acid sequence compatibility, as well as protein functionality depend on plasmid copy number, promoter strength and composition of the reaction medium (ALEGRIA et al., 2003). Despite demonstrating the effectiveness in the expression of a mammalian fibrillar protein, its application is reserved for comparative studies, it does not explore the possibility of associating it with other similar proteins, nor does it allocate its use to animal feed, showing that this is a undrawn route.
[0027] Como referência cita-se a patente US10485259B2, na qual são expressas em células de levedura ( Saccharomyces cerevisiae ou Pichia pastoris) uma série de proteínas compreendidas na composição de ovos. A invenção demonstra a possibilidade de construir um vetor que permita a tradução de uma ou mais sequências, demonstrando que um organismo eucariótico pode desempenhar o papel de expressar simultaneamente mais de um polipeptídeo de interesse. Ainda assim, não explora a expressão de proteínas fibrilares de mamíferos em leveduras. [0028] A alimentação de cães e gatos depende de uma série de componentes para suprir suas necessidade metabólicas e de crescimento. Uma vez que o consumo de carne está inti mamente relacionado com grandes impactos ambientais pela agropecuária, encontra- se como solução a expressão de tais proteínas por uma via microbiológica. Em contrapartida, ainda existem elementos da ração animal que podem ser substituídos por fontes nutricionais oriundas de sobras ou rejeitos dos processos agroindustriais. [0027] As reference, patent US10485259B2 is cited, in which a series of proteins included in the composition of eggs are expressed in yeast cells (Saccharomyces cerevisiae or Pichia pastoris). The invention demonstrates the possibility of constructing a vector that allows the translation of one or more sequences, demonstrating that a eukaryotic organism can play the role of simultaneously expressing more than one polypeptide of interest. Still, it does not explore the expression of mammalian fibrillar proteins in yeast. [0028] Feeding dogs and cats depends on a series of components to meet their metabolic and growth needs. Since meat consumption is closely related to major environmental impacts by agriculture, the solution is the expression of such proteins through a microbiological route. On the other hand, there are still elements of animal feed that can be replaced by nutritional sources from leftovers or rejects from agro-industrial processes.
[0029] De acordo com a FDA, agência federal do departamento de saúde americano, está previsto que os alimentos destinados à nutrição animal devem seguir os mesmos parâmetros que para humanos, sendo ingredientes seguros para ingestão, produzidos em condições sanitárias, livres de substâncias prejudiciais e devidamente identificados em rótulos. Portanto, partes remanescentes de processamento de grãos, subprodutos de atividades agroindustriais, restos de frutas e verduras em devida condição de armazenamento e conservação, ou biomassa de outros processos fermentativos podem ser algumas das opções empregadas na formulação de rações para cães e gatos. [0029] According to the FDA, federal agency of the American Department of Health, it is expected that foods intended for animal nutrition must follow the same parameters as for humans, being safe ingredients for ingestion, produced under sanitary conditions, free of harmful substances and properly identified on labels. Therefore, remaining parts from grain processing, by-products from agro-industrial activities, leftovers of fruits and vegetables in proper storage and conservation conditions, or biomass from other fermentation processes may be some of the options used in the formulation of feed for dogs and cats.
[0030] Dentre as maiores vantagens do uso destes materiais estão a constituição dos resíduos ser quase completamente orgânica, seu preço ser reduzido quando comparado com os mesmos insumos in natura, serem boas fontes de fibra bruta e terem compatibilidade com o organismo dos animais. [0030] Among the greatest advantages of using these materials are the constitution of the residues being almost completely organic, its price being reduced when compared to the same raw materials, being good sources of crude fiber and having compatibility with the organism of animals.
[0031] Sabendo que resíduos de cultivo rural e da cadeia produtiva alimentar correspondem à porção majoritária da produção agrícola anual, podendo chegar a até 30%, e que grande parte destes refugos ainda contêm nutrientes disponíveis e admitidos por animais de estimação, viabiliza-se um composto contendo elementos desta origem associados à uma biomassa reconhecida como segura expressando proteínas fibrilares animais. [0031] Knowing that residues from rural cultivation and the food production chain correspond to the majority portion of the annual agricultural production, which can reach up to 30%, and that a large part of these residues still contain nutrients available and admitted by pets, it becomes viable a compound containing elements of this origin associated with a biomass recognized as safe expressing animal fibrillar proteins.
[0032] Dentre os subprodutos agroindustriais mais usuais para a alimentação animal estão aqueles que são fontes ricas em compostos energéticos, como farelo de arroz ou soja e cascas, bagaços, folhas ou sementes de frutas, verduras e raízes. Estão incluídos como fontes proteicas as cascas de mandioca e resíduos de cereais, bem como farinhas e tortas de filtração do processamento de oleaginosas. Por fim, podem ser incorporados sobras da moagem de cereais e do refino de óleos como origem de fibra bruta (AULA et al., 2012). [0032] Among the most common agro-industrial by-products for animal feed are those that are rich sources of energy compounds, such as rice bran or soybeans and husks, bagasse, leaves or seeds of fruits, vegetables and roots. Cassava husks and cereal residues are included as protein sources, as well as flours and filter cakes from oilseed processing. Finally, they can be incorporated leftovers from cereal milling and oil refining as a source of crude fiber (AULA et al., 2012).
[0033] A seleção de resíduos a serem utilizados na formulação de rações para cães e gatos dependem de características nutricionais como proteína e fibra bruta, além da taxa de digestibilidade de matéria orgânica. Ademais de reduzir o custo de insumos e mitigar o impacto ambiental ao reaproveitar rejeitos, uma composição alimentar com esta base ainda tem o potencial de trazer benefícios substanciais aos animais de estimação. [0033] The selection of residues to be used in the formulation of feed for dogs and cats depend on nutritional characteristics such as protein and crude fiber, in addition to the rate of digestibility of organic matter. In addition to reducing the cost of inputs and mitigating the environmental impact by reusing tailings, a food composition on this basis still has the potential to bring substantial benefits to pets.
[0034] Um exemplo de aplicação é o de grãos secos de destiladores com solúveis (DDGS) em rações de cães e gatos. Por ser um resíduo rico em fibras insolúveis seu uso em monogástricos geralmente é feito em menores quantidades. Porém, o DDGS ainda possui maiores concentrações de proteínas, gorduras vitaminas e minerais se comparado ao milho antes de processado. É um composto facilmente incorporado em misturas para refeições animais, não afeta o processo industrial e tem potencial de aumentar a palatabilidade de alimentação de pets (RISOLIA et al., 2019). [0034] An example of application is that of dried beans with soluble distillers (DDGS) in dog and cat food. As it is a residue rich in insoluble fibers, its use in monogastrics is usually done in smaller amounts. However, DDGS still has higher concentrations of proteins, fats, vitamins and minerals compared to corn before processing. It is a compound easily incorporated into mixtures for animal meals, does not affect the industrial process and has the potential to increase the palatability of pet food (RISOLIA et al., 2019).
[0035] Como referência cita-se a patente KR100733906B1 , na qual é proposto um método de nutrição natural para pets. A invenção consiste em uma mistura à base de ovo, farinhas de frutas e vegetais, lipídeos de origem animal ou vegetal e outro componente que pode ser farinha de pão, algas em pó, queijo em pó ou farinha de arroz integral. Por fim, são adicionados restos de carne em forma de pó para então ser empacotado e armazenado. Apesar de utilizar fontes nutricionais de rejeitos, a invenção ainda é dependente de produtos de origem animal para suprir o teor de proteínas demandada pela alimentação de cães e gatos. [0035] As a reference, patent KR100733906B1 is cited, in which a method of natural nutrition for pets is proposed. The invention consists of a mixture based on egg, fruit and vegetable flour, animal or vegetable lipids and another component that can be bread flour, powdered seaweed, powdered cheese or brown rice flour. Finally, leftover meat is added in powdered form to then be packaged and stored. Despite using nutritional sources of tailings, the invention is still dependent on animal products to supply the protein content required by the food of dogs and cats.
[0036] Como referência cita-se a patente US6338866B, em que é produzida uma ração para pets a partir dos resíduos de algas e fungos contendo ácidos graxos. A tecnologia prevê a reutilização de biomassa gerada como subproduto em processos produtivos de ácidos graxos, misturando-a com ingredientes para gerar uma formulação com aroma suave aceitável por pets e humanos. Esta invenção demonstra a possibilidade do reuso de biomassa proveniente de outros processos com aceitabilidade pelos animais de estimação, mas parte unicamente desta fonte nutricional. Não associando-a com outros produtos pode ocorrer uma deficiência de nutrientes essenciais. [0037] Diante do anteriormente exposto, sabe-se que é expressivo o consumo alimentar de cães e gatos em nível mundial, mas que ainda depende fortemente de uma das maiores indústrias poluidoras, a da carne animal. Sabendo que, juntamente com as inferências em ecossistemas causadas pela criação de gados e granjas, um dos grandes problemas da agroindústria é a geração de resíduos não aproveitados. [0036] As a reference, the patent US6338866B is cited, in which a pet food is produced from the residues of algae and fungi containing fatty acids. The technology foresees the reuse of biomass generated as a by-product in fatty acid production processes, mixing it with ingredients to generate a formulation with a mild aroma acceptable to pets and humans. This invention demonstrates the possibility of reusing biomass from other processes that are acceptable to pets, but it is based solely on this nutritional source. Not associating it with other products can lead to a deficiency of essential nutrients. [0037] Given the above, it is known that the food consumption of dogs and cats is expressive worldwide, but that it still depends heavily on one of the largest polluting industries, that of animal meat. Knowing that, together with the inferences in ecosystems caused by the creation of cattle and farms, one of the great problems of agribusiness is the generation of unused residues.
[0038] Partindo da capacidade de leveduras admitirem genes heterólogos de mamíferos e com sua maquinaria celular serem capazes de expressar uma gama de proteínas animais, além de que são microrganismos seguros com aplicação industrial amplamente descrita, viabiliza-se a criação de uma cepa mutante capaz de expressar proteínas fibrilares ricas em aminoácidos essenciais para a alimentação de cães e gatos sem depender de nenhum produto de origem animal. [0038] Based on the ability of yeasts to admit heterologous mammalian genes and with their cellular machinery to be able to express a range of animal proteins, in addition to being safe microorganisms with widely described industrial application, it is possible to create a capable mutant strain to express fibrillar proteins rich in essential amino acids for the food of dogs and cats without relying on any animal product.
[0039] Associando esta biomassa mutada a outras fontes biodisponíveis em rejeitos agroindustriais, cria-se uma formulação com aceitabilidade, altamente nutritiva, bioquimicamente segura e que é capaz de surgir de uma via proteica alternativa à carne e de subprodutos ou restos transformados em componentes alimentares compatíveis com as necessidade de animais domésticos. [0039] Associating this mutated biomass with other bioavailable sources in agro-industrial waste, a formulation with acceptability, highly nutritious, biochemically safe and capable of arising from an alternative protein pathway to meat and by-products or waste transformed into food components is created compatible with the needs of domestic animals.
Breve descrição dos desenhos Brief description of the drawings
A Figura 1 apresenta um fluxograma simplificado do processo, englobando as etapas de cultivo da biomassa de levedura mutada, de formulação com resíduos agroindustriais e de eventuais etapas de pós processamento para obtenção do produto. Figure 1 shows a simplified flowchart of the process, encompassing the stages of cultivation of mutated yeast biomass, formulation with agro-industrial residues and eventual post-processing steps to obtain the product.
Descrição da invenção Description of the invention
[0040] A presente invenção trata-se da elaboração de um composto proteico associado a outras fontes nutricionais e aplicável na ração de cães e gatos. A técnica compreende as etapas descritas na Figura 1 de (a) criação de uma cepa de levedura geneticamente modificada para expressar proteínas fibrilares animais heterólogas; cultivo da biomassa mutada para obtenção da biomassa microbiana expressando tais proteínas no meio reacional e obtenção do concentrado proteico contendo biomassa e proteínas expressadas; e (b) formulação com outros compostos nutritivos essenciais para cães e gatos provenientes de resíduos agroindustriais e destinação para etapas de pós- processamento do produto. [0040] The present invention deals with the preparation of a protein compound associated with other nutritional sources and applicable in dog and cat food. The technique comprises the steps described in Figure 1 of (a) creating a genetically modified yeast strain to express heterologous animal fibrillary proteins; cultivation of mutated biomass to obtain microbial biomass expressing such proteins in the reaction medium and obtaining protein concentrate containing biomass and expressed proteins; and (b) formulation with other essential nutritional compounds for dogs and cats from agro-industrial waste and destination for post-processing stages of the product.
[0041] A etapa (a) de criação de uma cepa de levedura geneticamente modificada prevê a utilização preferencial de Saccharomyces cerevisiae, podendo ser associada ou realocada por variantes da mesma ou de outra espécie como do gênero Pichia, preferencialmente Pichia pastoris. Visando a obtenção de um microorganismo capaz de expressar as proteínas fibrilares animais, esta invenção descreve a construção de uma cepa de levedura capaz de produzir em seu citosol proteínas animais, de modo que é realizada a expressão heteróloga de alguns dos principais polipeptídeos envolvidos na formação de fibras musculares animais (como actina, troponina e tropomiosina). [0041] Step (a) of creating a genetically modified yeast strain provides for the preferential use of Saccharomyces cerevisiae, which can be associated or relocated by variants of the same or another species such as the genus Pichia, preferably Pichia pastoris. Aiming at obtaining a microorganism capable of expressing animal fibrillar proteins, this invention describes the construction of a yeast strain capable of producing animal proteins in its cytosol, so that the heterologous expression of some of the main polypeptides involved in the formation of animal muscle fibers (such as actin, troponin and tropomyosin).
[0042] As sequências elegidas referentes às regiões codificantes de actina, troponina e tropomiosina são, respectivamente, as identificadas como SEQ ID NO: 1 , 2 ou 3, SEQ ID NO: 4, 5 ou 6, e SEQ ID NO: 7, 8 ou 9, representando as três proteínas para espécies de bovinos ( Bos taurus), frangos ( Gallus gallus) ou suínos ( Sus scrofá) - cujas espécies de origem foram escolhidas pela sua aplicação na alimentação como fonte de proteína. As proteínas identificadas nas SEQ ID NO: 10, 11 ou 12, SEQ ID NO: 13, 14 ou 15, e SEQ ID NO: 16, 17 ou 18 podem ser expressas de forma simultânea no mesmo microorganismo ou de forma individual de modo que, durante o processo fermentativo, há a opção de um co-cultivo de leveduras da mesma espécie, porém expressando diferentes proteínas recombinantes. Diante da técnica desenvolvida, possibilita-se a expressão de outras proteínas musculares a fim de obter um produto ainda mais rico em proteínas. The chosen sequences referring to the coding regions of actin, troponin and tropomyosin are, respectively, those identified as SEQ ID NO: 1, 2 or 3, SEQ ID NO: 4, 5 or 6, and SEQ ID NO: 7, 8 or 9, representing the three proteins for species of bovine (Bos taurus), chicken (Gallus gallus) or swine (Sus scrofá) - whose original species were chosen for their application in food as a source of protein. The proteins identified in SEQ ID NO: 10, 11 or 12, SEQ ID NO: 13, 14 or 15, and SEQ ID NO: 16, 17 or 18 can be expressed simultaneously in the same microorganism or individually so that , during the fermentation process, there is the option of co-cultivation of yeasts of the same species, but expressing different recombinant proteins. In view of the technique developed, the expression of other muscle proteins is made possible in order to obtain a product that is even richer in proteins.
[0043] Para a modificação genética da levedura, é utilizado como instrumento da inserção gênica a capacidade nativa da levedura de realizar a recombinação homóloga como um mecanismo de reparo a quebra da fita dupla de DNA. Sendo assim, a partir de uma metodologia de edição gênica sítio dirigida, deve-se induzir uma quebra no cromossomo da levedura e disponibilizar no citosol desta fragmentos de DNA contendo o gene a ser inserido - com um promotor compatível a espécie hospedeira - balizado pelas regiões de homologia do local de quebra, que permitem, portanto, a recombinação homóloga com esse fragmento heterólogo e, por conseguinte, a inserção do gene - no caso, da proteína fibrilar escolhida - no local pré-determinado pela quebra do cromossomo da levedura. A metodologia de edição gênica escolhida deve, portanto, ser capaz de direcionar o local da quebra no cromossomo e, por consequência, direcionar o local da inserção gênica na levedura. [0043] For the genetic modification of yeast, it is used as an instrument of gene insertion the native capacity of the yeast to carry out homologous recombination as a repair mechanism to break the double strand of DNA. Therefore, from a site-directed gene editing methodology, a break in the yeast chromosome must be induced and made available in the cytosol of this DNA fragments containing the gene to be inserted - with a promoter compatible with the host species - marked by regions of break site homology, which allow, therefore, homologous recombination with this heterologous fragment and, therefore, the insertion of the gene - in this case, of the chosen fibrillar protein - in the pre-determined site by the break of the yeast chromosome. The chosen gene editing methodology must, therefore, be able to target the location of the break in the chromosome and, consequently, target the location of the gene insertion in yeast.
[0044] Além da inserção gênica no cromossomo da levedura, é possível também expressar as proteínas fibrilares por meio de plasmídeos contendo promotores e origens de replicação compatíveis com a espécie escolhida. Sendo os genes para as três proteínas fibrilares expressas em um único plasmídeo em forma de operon ou comparti mentalizadas a partir do uso de três plasmídeos individuais e suas combinações. Os plasmídeos contendo apenas um gene para uma proteína podem também ser inseridos em três diferentes clones, permitindo o co-cultivo das diferentes leveduras expressando cada uma alguma proteína fibrilar das aqui já citadas anteriormente. [0044] In addition to the gene insertion in the yeast chromosome, it is also possible to express fibrillar proteins through plasmids containing promoters and replication origins compatible with the chosen species. The genes for the three fibrillar proteins are expressed in a single plasmid in the form of an operon or compartmentalized using three individual plasmids and their combinations. Plasmids containing only one gene for a protein can also be inserted into three different clones, allowing the co-cultivation of different yeasts, each expressing some fibrillar protein of those mentioned above.
[0045] As sequências SEQ ID NO: 1 , 2 ou 3, SEQ ID NO: 4, 5 ou 6, e SEQ ID NO: 7, 8 ou 9 podem ser organizadas em forma de operon, o qual é formado por um promotor constitutivo livre de regulação, seguido de cada gene contendo uma sequência de ligação ao ribossomo (RBS) compatível com leveduras da espécie S. cerevisiae, e a região codificante de cada gene. Outra estratégia prevê que os genes podem ser expressos de forma distinta, sendo cada um controlado por um promotor específico compatível com as leveduras utilizadas. Em quaisquer das situações, os genes podem ser integrados no cromossomo do microrganismo, visando a estabilidade do DNA recombinante e a garantia de que este permanecerá íntegro nas progénies oriundas da divisão celular em meio reacional, ou expressos pela manutenção do plasmídeo na levedura selecionada. [0045] The sequences SEQ ID NO: 1, 2 or 3, SEQ ID NO: 4, 5 or 6, and SEQ ID NO: 7, 8 or 9 can be arranged in the form of an operon, which is formed by a promoter free constitutive regulation, followed by each gene containing a ribosome binding sequence (RBS) compatible with yeast of the S. cerevisiae species, and the coding region of each gene. Another strategy predicts that genes can be expressed differently, each being controlled by a specific promoter compatible with the yeast used. In either situation, the genes can be integrated into the microorganism's chromosome, aiming at the stability of the recombinant DNA and guaranteeing that it will remain intact in the progenies arising from cell division in a reaction medium, or expressed by maintaining the plasmid in the selected yeast.
[0046] Para a integração dos genes no cromossomo, foi utilizada a metodologia de inserção gênica sítio dirigida no cromossomo de Saccharomyces cerevisiae descrita por Jessop-Fabre et ai. (2016), na qual os elementos particulares dessa invenção - genes codificantes para proteínas fibrilares animais associados a um promotor compatível a espécie de levedura - foram inseridos em regiões específicas do cromossomo fazendo proveito da recombinação homóloga nativa e dos instrumentos disponíveis na metodologia selecionada. [0047] A modificação da cepa de Saccharomyces cerevisiae contou, portanto, com as etapas de construção dos vetores contendo, cada um: um gene ou mais para a expressão de uma ou mais proteínas fibrilares animais; um promotor compatível com a cepa de levedura escolhida para expressão antecedendo a região codificante do gene; a região de homologia do cromossomo da levedura balizando a sequência gênica e outros elementos essenciais para a funcionalização do vetor de acordo com a metodologia escolhida - como regiões de compatibilização com o microrganismo escolhido para a propagação do plasmídeo antes da transformação em levedura. Após a construção dos vetores - podendo eles possuir um ou mais genes para as diferentes proteínas fibrilares -, é preciso realizar a transformação e multiplicação dos plasmídeos em algum organismo compatível, sendo utilizado preferencialmente a bactéria Escherichia coli. Os plasmídeos utilizados para a clonagem dos genes da actina, troponina e tropomiosina são provenientes da metodologia descrita por Jessop-Fabre et ai, podendo ser associados ou substituídos por outros plasmídeos com uso já difundido na espécie aqui citada que também são capazes de carregar a informação genética até a levedura de interesse e fazer com que as proteínas sejam expressas. [0046] For the integration of genes into the chromosome, the site-directed gene insertion methodology in the chromosome of Saccharomyces cerevisiae described by Jessop-Fabre et al. (2016), in which the particular elements of this invention - genes encoding animal fibrillary proteins associated with a promoter compatible with the yeast species - were inserted into specific regions of the chromosome, taking advantage of native homologous recombination and the tools available in the selected methodology. [0047] The modification of the strain of Saccharomyces cerevisiae had, therefore, the steps of construction of vectors containing, each one: a gene or more for the expression of one or more animal fibrillary proteins; a promoter compatible with the yeast strain chosen for expression preceding the coding region of the gene; the homology region of the yeast chromosome, marking the gene sequence and other essential elements for the functionalization of the vector according to the chosen methodology - such as regions of compatibilization with the chosen microorganism for the propagation of the plasmid before transformation into yeast. After the construction of the vectors - they may have one or more genes for the different fibrillar proteins -, it is necessary to carry out the transformation and multiplication of the plasmids in a compatible organism, preferably using the bacterium Escherichia coli. The plasmids used for cloning the actin, troponin and tropomyosin genes come from the methodology described by Jessop-Fabre et al, and can be associated or replaced by other plasmids with widespread use in the species mentioned herein that are also capable of carrying the information genetics to the yeast of interest and cause the proteins to be expressed.
[0048] Após a propagação dos vetores preferencialmente em E. coli, os vetores contendo os genes com o promotor a serem inseridos e a região de homologia que direciona a inserção no cromossomo devem ser purificados e linearizados para a remoção de todos os outros elementos antes presentes no vetor a fim de permitir a sua multiplicação pelo sistema bacteriano. As células de S. cerevisiae são, então, submetidas a crescimento em meio contendo extrato de levedura, peptona e dextrose a uma temperatura de 28 a 40°C, até atingir cerca de 2x1 Cl· células/mL, para então serem lavadas com água deionizada. A transformação dos fragmentos de DNA linearizados é realizada, então, pela incubação das células de levedura com acetato de lítio de 20 a 40 minutos entre 30 e 50 °C juntamente com os fragmentos de DNA heterólogos a serem inseridos e os vetores contendo os elementos guia correspondentes a região de homologia pré- determinada para o funcionamento da metodologia descrita por Jessop-Fabre et al., (2016). Antes da inserção dos fragmentos gênicos, contudo, para o funcionamento da metodologia escolhida é preciso transformar na levedura um plasmídeo que permita a expressão da nuclease essencial ao funcionamento da edição gênica sítio dirigida aqui selecionada para executar a inserção dos genes no cromossomo da levedura. [0049] Posteriormente, as células são incubadas por de 2 a 3 horas em meio contendo extrato de levedura, peptona e dextrose constituindo a fase de recuperação, para então serem plaqueadas em meio sólido com a mesma composição acrescida de ágar e antibióticos específicos (G418 e Noursetromicina). O plaqueamento é uma etapa fundamental para a seleção adequada de clones positivos para a presença dos plasmídeos essenciais para o funcionamento do sistema de edição gênica descrita pela metodologia de Jessop-Fabre et ai, 2016. A confirmação da integração do DNA plasmidial no cromossomo se dá por seleção por reação de PCR e sequenciamento utilizando oligonucleotídeos específicos para a região integrada. Os clones bem sucedidos selecionados podem, individualmente, expressar todos os genes das proteínas fibrilares aqui descritas ou pode-se selecionar mais de um clone onde cada um expressa uma ou mais das proteínas fibrilares aqui citadas. [0048] After propagating the vectors preferably in E. coli, the vectors containing the genes with the promoter to be inserted and the homology region that directs the insertion into the chromosome must be purified and linearized to remove all other elements before present in the vector in order to allow its multiplication by the bacterial system. The S. cerevisiae cells are then subjected to growth in a medium containing yeast extract, peptone and dextrose at a temperature of 28 to 40°C, until reaching about 2x1 Cl· cells/mL, and then washed with water deionized. Transformation of the linearized DNA fragments is then performed by incubating the yeast cells with lithium acetate for 20 to 40 minutes at 30 to 50°C together with the heterologous DNA fragments to be inserted and the vectors containing the guide elements corresponding to the region of predetermined homology for the functioning of the methodology described by Jessop-Fabre et al., (2016). Before inserting the gene fragments, however, for the chosen methodology to work, it is necessary to transform in yeast a plasmid that allows the expression of the nuclease essential for the functioning of the site-directed gene editing selected here to carry out the insertion of genes into the yeast chromosome. [0049] Subsequently, the cells are incubated for 2 to 3 hours in medium containing yeast extract, peptone and dextrose constituting the recovery phase, to then be plated on solid medium with the same composition plus agar and specific antibiotics (G418 and Noursetromycin). Plating is a fundamental step for the proper selection of positive clones for the presence of plasmids essential for the functioning of the gene editing system described by the methodology of Jessop-Fabre et al, 2016. Confirmation of plasmid DNA integration in the chromosome takes place by selection by PCR reaction and sequencing using specific oligonucleotides for the integrated region. The successful clones selected can individually express all the genes of the fibrillar proteins described herein or it is possible to select more than one clone where each one expresses one or more of the fibrillar proteins mentioned herein.
[0050] Na fase subsequente à etapa (a), após a seleção dos clones desejados, objetivando o cultivo da biomassa mutada para obtenção das proteínas no meio reacional, é inicialmente realizado um pré-inóculo com o clone ou com os clones selecionados em meio contendo extrato de levedura, peptona e dextrose, podendo ser suplementado com outros macro e micronutrientes demandados pelo microrganismo. A etapa para incremento do número de células que serão inoculadas em reator ocorre de 24 a 72 horas com temperatura de 25 a 45 °C com agitação de 90 a 230 rpm até atingir de 1 ,0±0,5x10= a 1 ,0±0,5x10^ UFC/mL. Se houver a seleção de mais de um clone, pode- se realizar o co-cultivo ou o cultivo individual de cada OGM. [0050] In the phase subsequent to step (a), after the selection of the desired clones, aiming at the cultivation of the mutated biomass to obtain the proteins in the reaction medium, a pre-inoculum is initially carried out with the clone or selected clones in the medium containing yeast extract, peptone and dextrose, which can be supplemented with other macro and micronutrients required by the microorganism. The step to increase the number of cells that will be inoculated in a reactor takes place from 24 to 72 hours at a temperature of 25 to 45 °C with stirring from 90 to 230 rpm until reaching 1.0±0.5x10= to 1.0± 0.5x10^ CFU/ml. If more than one clone is selected, co-cultivation or individual cultivation of each GMO can be carried out.
[0051] Em seguida, os pré-inóculos são adicionados ao reator no qual deverá ocorrer a produção da biomassa da levedura expressando as proteínas fibrilares. Junto ao inoculo - adicionado na proporção 15 a 30% (v/v) - são incorporados os elementos do meio de cultivo compostos por uma fonte de carbono, uma fonte de nitrogénio e uma fonte de micronutrientes - preferencialmente, sendo a fonte de carbono glicose e a de nitrogénio, uréia ou aminoácidos livres. O processo fermentativo ocorre de 28°C a 45°C por até 144h sob agitação constante de 120 a 450 rpm em meio líquido sob condições controladas em biorreator. São monitorados também a aeração do meio, mantida entre 0,3 e 1 ,5 vvm, o pH de 4 a 8, e a produção de espuma, controlados, respectivamente, pelo aumento ou redução da injeção de ar filtrado, de ácido ou base e de antiespumante. [0052] Após o processo fermentativo, está compreendida a etapa de obtenção do concentrado proteico contendo a biomassa de levedura juntamente com as proteínas animais fibrilares sintetizadas pelo metabolismo celular. O caldo fermentado pode admitir uma etapa de separação sólido-líquido com o intuito de reter a biomassa microbiana, sendo nesse caso a operação unitária preferivelmente realizada por uma centrífuga de disco ou decanter ou por um filtro do tipo tambor rotativo. Como os polipeptídeos de interesse são intracelulares, é possível adicionar uma etapa de lise celular para torná- los mais biodisponíveis ao consumo animal. A lise pode ocorrer antes ou após a filtração da biomassa, admitindo incorporação dos elementos presentes no caldo fermentado ou optando por uma ressuspensão das células separadas para então rompê-las. [0051] Then, the pre-inoculums are added to the reactor in which the production of yeast biomass expressing the fibrillar proteins should occur. Along with the inoculum - added in a proportion of 15 to 30% (v/v) - the elements of the culture medium are incorporated, composed of a carbon source, a nitrogen source and a micronutrient source - preferably, the carbon source being glucose and nitrogen, urea or free amino acids. The fermentation process takes place at 28°C to 45°C for up to 144h under constant agitation at 120 to 450 rpm in liquid medium under controlled conditions in a bioreactor. The aeration of the medium is also monitored, maintained between 0.3 and 1.5 vvm, the pH from 4 to 8, and the production of foam, controlled, respectively, by the increase or reduction of the injection of filtered air, acid or base and defoamer. [0052] After the fermentation process, the stage of obtaining the protein concentrate containing the yeast biomass along with the fibrillar animal proteins synthesized by cell metabolism is understood. The fermented broth can admit a solid-liquid separation step in order to retain the microbial biomass, in which case the unit operation is preferably carried out by a disc centrifuge or decanter or by a rotating drum-type filter. As the polypeptides of interest are intracellular, it is possible to add a cell lysis step to make them more bioavailable for animal consumption. The lysis can occur before or after the filtration of the biomass, admitting the incorporation of the elements present in the fermented broth or opting for a resuspension of the separated cells in order to break them up.
[0053] A lise celular de leveduras é compatível com uma série de métodos físico- químicos, mecânicos ou enzimáticos. Podem ser optadas etapas de rompimento por homogeneização a alta pressão, agitação em moinho de bolas ou ultrassom, desde que controlados os choques mecânicos ou cavitação para que não degradem as proteínas de interesse. Também são opções viáveis e menos custosas como choque osmótico, congelamento e descongelamento ou aquecimento, uma vez que não dependem de equipamentos específicos e têm como base a oscilação de temperatura ou concentração de sais no meio intracelular. Ademais, métodos químicos são uma alternativa com o uso de álcalis, ácidos, solventes ou detergentes capazes de degradar a parede celular e liberar seu conteúdo interno. Por fim, análogo aos métodos físico-químicos quanto à praticidade e isenção de equipamentos adicionais, estão os métodos enzimáticos. Enzimas como b-1 ,3-glucanases associadas a um controle de pH para atividade enzimática ótima mostram-se efetivas para a ruptura de células de levedura. [0053] The cell lysis of yeast is compatible with a series of physical-chemical, mechanical or enzymatic methods. Disruption steps can be chosen by means of high pressure homogenization, agitation in a ball mill or ultrasound, as long as the mechanical shocks or cavitation are controlled so that they do not degrade the proteins of interest. They are also viable and less costly options, such as osmotic shock, freezing and thawing or heating, since they do not depend on specific equipment and are based on temperature fluctuations or the concentration of salts in the intracellular environment. Furthermore, chemical methods are an alternative with the use of alkalis, acids, solvents or detergents capable of degrading the cell wall and releasing its internal content. Finally, similar to physicochemical methods in terms of practicality and exemption from additional equipment are enzymatic methods. Enzymes such as b-1,3-glucanases associated with a pH control for optimal enzymatic activity are effective in breaking down yeast cells.
[0054] Após a separação ou não da biomassa do caldo fermentado e a ruptura celular ou não, é obtido um produto final que pode ser líquido como uma emulsão ou pastoso a fim de ser incorporado na ração de animais. A vantagem do uso do caldo fermentado é a manutenção dos micro e macro nutrientes excretados pela levedura durante a fermentação que podem enriquecer a composição nutricional do produto final. Além disso, a dispensabilidade de um downstream custoso se apresenta como uma vantagem tecnológica pela economia no processo e pela não geração de resíduos durante o processo produtivo. [0055] Sendo assim, há três opções possíveis de processamento para o produto obtido nesta etapa: (i) o caldo fermentado contendo a biomassa de levedura expressando as proteínas fibrilares animais pode ser usado diretamente sem nenhuma etapa de downstream para a formulações com outros elementos nutricionais provenientes de resíduos agroindustriais; (ii) o caldo fermentado contendo a biomassa de levedura expressando as proteínas fibrilares animais pode ser filtrado ou centrifugado objetivando- se obter a biomassa com um conteúdo líquido menor para ser usada na formulação com outros elementos nutricionais provenientes de resíduos agroindustriais - dispensando, portanto, o caldo fermentado e mantendo a biomassa expressando as proteínas fibrilares; e (iii) o caldo fermentado contendo a biomassa de levedura ou a biomassa de levedura filtrada ou centrifugada pode passar por uma etapa de lise celular para então ser utilizada na formulação com outros elementos nutricionais provenientes de resíduos agroindustriais. [0054] After the separation or not of the biomass from the fermented broth and cell rupture or not, a final product is obtained that can be liquid as an emulsion or pasty in order to be incorporated into the animal feed. The advantage of using fermented broth is the maintenance of micro and macro nutrients excreted by the yeast during fermentation, which can enrich the nutritional composition of the final product. In addition, the dispensability of costly downstream is presented as a technological advantage due to the economy in the process and the non-generation of waste during the production process. [0055] Therefore, there are three possible processing options for the product obtained in this step: (i) the fermented broth containing yeast biomass expressing animal fibrillar proteins can be used directly without any downstream step for formulations with other elements nutritional products from agro-industrial residues; (ii) the fermented broth containing yeast biomass expressing animal fibrillary proteins can be filtered or centrifuged in order to obtain the biomass with a lower liquid content to be used in the formulation with other nutritional elements from agro-industrial residues - thus dispensing the broth fermented and maintaining the biomass expressing fibrillar proteins; and (iii) the fermented broth containing yeast biomass or filtered or centrifuged yeast biomass can go through a cell lysis step to then be used in the formulation with other nutritional elements from agro-industrial residues.
[0056] A etapa subsequente diz respeito à formulação com outros compostos nutritivos essenciais para cães e gatos provenientes de resíduos agroindustriais. Tomando como base uma composição adequada para tais animais e integralmente proveniente da produção de biomassa geneticamente modificada expressando proteínas fibrilares associada à resíduos agroindustriais nutricionalmente adequados, definem-se alguns compostos como fonte de proteínas, carboidratos, fibras brutas, lipídeos, vitaminas e minerais, tendo em vista que os teores destes macronutrientes nos resíduos podem ser variáveis pois dependem intimamente do seu estado de processamento e conservação. [0056] The subsequent step concerns the formulation with other essential nutritional compounds for dogs and cats from agro-industrial waste. Based on an adequate composition for such animals and entirely from the production of genetically modified biomass expressing fibrillar proteins associated with nutritionally adequate agro-industrial residues, some compounds are defined as a source of proteins, carbohydrates, crude fibers, lipids, vitamins and minerals, having considering that the contents of these macronutrients in the residues can be variable as they depend closely on their state of processing and conservation.
[0057] Os elementos proteicos, e também fonte de aminoácidos essenciais, serão provenientes da fermentação e lise da cepa de Saccharomyces cerevisiae mutada sintetizando os polipeptídeos fibrilares animais. Ainda podem ser acrescidos farelos como o de colza, semente de algodão, ervilha, soja ou outras leguminosas por serem ricos em proteínas e rejeitos provenientes do processamento de grãos. Seguindo as determinações da FDA (Food and Drug Administration) pautadas pela AAFCO (Association of American Feed Control Officials), os teores de proteína em rações para cães não devem ser menores que 18% para manutenção corporal de um animal adulto, e um mínimo de 22% para garantir condições de crescimento e reprodução. Para gatos, esse teor para as mesmas finalidades são de 26 e 30%. [0058] A fibra bruta pode ser proveniente de grãos de destilaria, polpa cítrica, farelos como o de amendoim, cascas de leguminosas ou grãos, tortas de filtração de extração de óleos, cascas de frutas e verduras, entre outros. Além de auxiliarem no funcionamento gastrointestinal e serem parte integrante da formação do bolo fecal quanto mais solúveis, as fibras ainda cumprem um papel importante na formulação ao encorparem a mistura da ração. [0057] The protein elements, and also a source of essential amino acids, will come from the fermentation and lysis of the mutated Saccharomyces cerevisiae strain synthesizing animal fibrillary polypeptides. Bran such as rapeseed, cottonseed, peas, soybeans or other legumes can also be added because they are rich in proteins and rejects from grain processing. Following the determinations of the FDA (Food and Drug Administration) guided by the AAFCO (Association of American Feed Control Officials), the protein contents in dog food must not be less than 18% for the corporal maintenance of an adult animal, and a minimum of 22% to ensure conditions for growth and reproduction. For cats, this content for the same purposes is 26 and 30%. [0058] Crude fiber can come from distillery grains, citrus pulp, bran such as peanuts, legume husks or grains, oil extraction filter cakes, fruit and vegetable husks, among others. In addition to aiding in the gastrointestinal functioning and being an integral part of the formation of the fecal cake, the more soluble it is, the fibers still play an important role in the formulation by incorporating the feed mixture.
[0059] Fontes de carboidratos, por sua vez, podem ser de resíduos ricos em açúcares como o melaço, de cereais processados tais como cevada, arroz e trigo; e de polpa de frutas ou de partes remanescentes de tubérculos e outras raízes. A variação da taxa de carboidratos na ração dependerá de qual objetivo deseja suprir. Rações ditas de baixo carboidrato poderão conter menores níveis do que aquelas direcionadas à crescimento, de alta demanda energética ou destinada a fases iniciais de desenvolvimento dos animais de estimação. Lipídeos, por sua vez, podem ser acrescidos em forma de óleos de sementes ou leguminosas, presentes na lecitina, ou de outras origens, como da torta de filtração de caroços. [0059] Sources of carbohydrates, in turn, can be residues rich in sugars such as molasses, processed cereals such as barley, rice and wheat; and from fruit pulp or from remaining parts of tubers and other roots. The variation in the rate of carbohydrates in the ration will depend on which objective you want to supply. So-called low-carbohydrate rations may contain lower levels than those intended for growth, with high energy demand or destined for the initial stages of development of pets. Lipids, in turn, can be added in the form of oils from seeds or legumes, present in lecithin, or from other sources, such as stone filter cake.
[0060] A variação de teores dos principais macronutrientes em dietas de cães e gatos variam de acordo com o estágio de desenvolvimento do animal, bem como a finalidade da dieta, podendo ser de alta taxa de carboidrato, proteína ou gordura. Tomando como base uma dieta com enfoque em manutenção corporal de um animal adulto, os valores de fontes energéticas pode variar como disposto na Tabela 1 abaixo. Assim, conhecendo a composição de resíduos agroindustriais é possível modular uma formulação de ração que se adeqúe para o fim de alimentação animal. [0060] The variation in the contents of the main macronutrients in dog and cat diets vary according to the animal's stage of development, as well as the purpose of the diet, which may be high in carbohydrates, protein or fat. Based on a diet focused on body maintenance of an adult animal, the values of energy sources can vary as shown in Table 1 below. Thus, knowing the composition of agro-industrial residues, it is possible to modulate a feed formulation that is suitable for animal feeding purposes.
TABELA 1 - Possíveis resíduos como fonte nutricional em rações pet TABLE 1 - Possible residues as a nutritional source in pet food
Função % Requerida* Function % Required *
Exemplos de Resíduos Waste Examples
Nutricional Cães Gatos Nutritional Dogs Cats
33 a 34 a Melaço, partes remanescentes de tubérculos e outras raízes, polpa33 to 34 a Molasses, remaining parts of tubers and other roots, pulp
Carboidratos 58 52 cítrica Carbohydrates 58 52 citrus
16 a 24 a Resíduos de leguminosas, cereais maltados da indústria cervejeira,16 to 24 a Legume residues, malted cereals from the brewing industry,
Proteínas proteins
34 44 biomassa microbiana restante de bioprocessos 34 44 remaining microbial biomass from bioprocesses
12 a 12 to
Lipídeos 9 a 21 Lecitina, torta de filtração de oleaginosas ou caroços 28 Farelos, cascas e outros rejeitos de moagem de grãos e cereaisLipids 9 to 21 Lecithin, oilseed filter cake or lumps 28 Bran, husks and other waste from grain and cereal milling
0,7 a 0,7 a 0.7 to 0.7 to
Fibra Bruta (arroz, trigo, milho, cevada, centeio, aveia), bagaços e tortas de 1 ,2 0,9 filtração Crude Fiber (rice, wheat, corn, barley, rye, oats), bagasse and cakes of 1.2 0.9 filtration
*dieta de manutenção corporal para animais adultos (> 1 ano) (Adaptado de HALLA et ai, 2018). * body maintenance diet for adult animals (> 1 year) (Adapted from HALLA et al, 2018).
[0061] Aminoácidos essenciais, vitaminas, macro e microminerais que eventualmente não forem suficientes quando provenientes dos ingredientes propostos na formulação da ração, podem ser acrescidos de outras formas, de modo que sejam supridas as necessidades nutricionais e metabólicas de animais de estimação. [0061] Essential amino acids, vitamins, macro and microminerals that may not be sufficient when coming from the ingredients proposed in the feed formulation, can be added in other ways, so that the nutritional and metabolic needs of pets are met.
[0062] Além de serem ingredientes do composto proposto como ração para cães e gatos, os resíduos agroindustriais poderão ser adicionados de forma cumprir funções de aditivos sensoriais ou para alterar as condições físico-químicas da mistura. São considerados possíveis rejeitos utilizados como aditivos de ração o dreche, DDGS (grãos secos por destilação) ou farelo de amendoim para aumentar a palatabilidade, glicerina como um retentor de umidade em rações secas ou extrusadas, lecitina como um agente espessante, e melaço como um aglutinante. [0062] In addition to being ingredients of the proposed compound as feed for dogs and cats, agro-industrial residues may be added to fulfill functions of sensory additives or to change the physicochemical conditions of the mixture. Possible tailings used as feed additives are dreche, DDGS (distilled dried grains) or peanut bran to increase palatability, glycerin as a moisture retainer in dry or extruded feeds, lecithin as a thickening agent, and molasses as a binder.
[0063] Alimentos para animais de estimação são disponibilizados em variações comerciais de forma e umidade. Rações sólidas contém de 6 a 10% de umidade, enlatadas de 60 a 90% e semi-sólidas de 25 a 35%. Seguindo a etapa de formulação, a mistura obtida segue para a fase de pós processamento do produto, que pode incluir operações de extrusão, peletização, coating, secagem, entre outros. A forma final do produto dependerá da palatabilidade e concentrações de nutrientes almejadas, da facilidade de digestão pelo animal, do tempo de vida para a qual é destinada, da inserção ou não de aditivos ou preservantes, do tempo de prateleira desejado e do custo da ração finalizada. [0063] Pet foods are available in commercial variations of form and moisture. Solid rations contain 6 to 10% moisture, canned 60 to 90% and semi-solid 25 to 35%. Following the formulation stage, the mixture obtained goes to the product post-processing stage, which may include extrusion, pelletizing, coating, drying, among others. The final form of the product will depend on the desired palatability and nutrient concentrations, the animal's ease of digestion, the lifetime for which it is intended, whether or not additives or preservatives are inserted, the desired shelf life and the cost of the feed. finished.
Exemplos de concretizações da invenção Examples of embodiments of the invention
[0064] EXEMPLO 1 : Criação dos vetores de clonagem [0064] EXAMPLE 1: Creation of cloning vectors
[0065] Utilizando a metodologia de recombinação homóloga nativa de leveduras proposta por Jessop-Fabre et al. (2016), inicia-se o processo de transformação da cepa s288c de Saccharomyces cerevisiae com a construção de vetores contendo os genes de proteínas fibrilares animais. Para a construção de tais vetores, se faz necessária a montagem contendo o gene responsável pela expressão das proteínas de interesse, acompanhado de um promotor compatível com a maquinaria celular da levedura no upstream da fita codificante, e flanqueado por sequências coincidentes com a região de homologia alvo escolhidas com base no conhecimento da estrutura dos cromossomos da levedura. [0065] Using the yeast native homologous recombination methodology proposed by Jessop-Fabre et al. (2016), the process of transforming the strain begins s288c of Saccharomyces cerevisiae with the construction of vectors containing the genes of animal fibrillary proteins. For the construction of such vectors, an assembly containing the gene responsible for the expression of the proteins of interest is necessary, accompanied by a promoter compatible with the yeast cellular machinery in the upstream of the coding strand, and flanked by sequences coincident with the region of homology chosen on the basis of knowledge of the structure of the yeast chromosomes.
[0066] Para a expressão da proteína de SEQ ID NO: 10 o vetor construído constou com o gene de SEQ ID NO: 1 , antecedido pelo promotor natural de leveduras identificado na SEQ ID NO: 19 e delimitado por regiões de homologia compatíveis com a de SEQ ID NO: 22. De forma análoga, um segundo vetor foi elaborado contendo os elementos necessários para a expressão da proteína de SEQ ID NO: 13, como o gene de SEQ ID NO: 4, precedido pelo mesmo promotor de SEQ ID NO: 19 por sua maior compatibilidade com a maquinaria de transcrição e tradução da Saccharomyces cerevisiae s288c. Para a região de homologia na qual o gene foi inserido, foi escolhida a de SEQ ID NO: 21 , portanto no vetor construído constavam sequências capazes de serem ligadas com esta região no processo de reparo descrito no Exemplo 2. Por fim, para a expressão heteróloga da proteína de SEQ ID NO: 16 pela Saccharomyces cerevisiae s288c o terceiro vetor construído constou com o promotor de SEQ ID NO: 19, seguido do gene de SEQ ID NO: 7. Como a região de homologia ao cromossomo da levedura selecionada para a inserção deste gene de interesse foi a de SEQ ID NO: 20, o vetor foi finalizado com a presença nas extremidades do fragmento de sequências capazes de serem conciliadas com a região escolhida. [0066] For the expression of the protein of SEQ ID NO: 10, the constructed vector consisted of the gene of SEQ ID NO: 1, preceded by the natural yeast promoter identified in SEQ ID NO: 19 and delimited by regions of homology compatible with the of SEQ ID NO: 22. Similarly, a second vector was elaborated containing the necessary elements for the expression of the protein of SEQ ID NO: 13, as the gene of SEQ ID NO: 4, preceded by the same promoter of SEQ ID NO. : 19 for its greater compatibility with the transcription and translation machinery of the Saccharomyces cerevisiae s288c. For the region of homology in which the gene was inserted, SEQ ID NO: 21 was chosen, therefore, the constructed vector contained sequences capable of being linked with this region in the repair process described in Example 2. Finally, for expression heterologous protein of SEQ ID NO: 16 by Saccharomyces cerevisiae s288c the third vector constructed consisted of the promoter of SEQ ID NO: 19, followed by the gene of SEQ ID NO: 7. As the region of homology to the selected yeast chromosome for the insertion of this gene of interest was that of SEQ ID NO: 20, the vector was finished with the presence at the ends of the fragment of sequences capable of being reconciled with the chosen region.
[0067] Para a obtenção em maiores quantidades dos plasmídeos construídos foi realizada a clonagem dos três vetores em três diferentes colónias de Escherichia coli TOP10 cultivada em meio LB suplementado com 100 pg/mL de ampicilina para seleção dos transformantes, uma vez que todos os vetores criados também continham gene para resistência ao antibiótico como forma de marcador de seleção. Após o cultivo em célula bacteriana, os plasmídeos foram extraídos e purificados para a etapa seguinte da metodologia proposta. [0068] Sabendo que os cromossomos da levedura são lineares, e que a expressão das proteínas fibrilares de interesse só se dá se devidamente incorporadas à esta estrutura, foi necessário um corte nos plasmídeos construídos após a replicação e purificação. Com a enzima de restrição Notl, os plasmídeos foram linearizados de forma a transformarem- se em fragmentos contendo o promotor, o gene de interesse e que esta estrutura estivesse delimitada em ambas as extremidades pelas regiões de homologia previamente selecionadas e descritas. [0067] To obtain larger quantities of the constructed plasmids, the cloning of the three vectors in three different colonies of Escherichia coli TOP10 cultivated in LB medium supplemented with 100 pg/ml of ampicillin for selection of transformants was performed, since all vectors created also contained the gene for antibiotic resistance as a form of selection marker. After cultivation in bacterial cells, plasmids were extracted and purified for the next step of the proposed methodology. [0068] Knowing that the yeast chromosomes are linear, and that the expression of fibrillar proteins of interest only occurs if properly incorporated into this structure, it was necessary to cut the plasmids constructed after replication and purification. With the restriction enzyme NotI, the plasmids were linearized so as to become fragments containing the promoter, the gene of interest and that this structure was delimited at both ends by the regions of homology previously selected and described.
[0069] EXEMPLO 2: Clonagem das sequências selecionadas em Saccharomyces cerevisiae e linearização no cromossomo [0069] EXAMPLE 2: Cloning of selected sequences in Saccharomyces cerevisiae and linearization in the chromosome
[0070] A metodologia proposta por Jessop-Fabre et al. (2016) consiste em realizar uma mutação sítio dirigida portanto prevê que, para que ocorra a inserção correta dos genes de interesse nas regiões de homologia desejadas, a técnica depende de uma endonuclease para realizar os cortes nos cromossomos da levedura e de um RNA guia (gRNA) para cada região de homologia escolhida, fazendo com que a endonuclease seja direcionada para a exata região de homologia em que se deseja incorporar os insertos. Portanto, juntamente com as sequências previamente linearizadas são inseridos os plasmídeos contendo o gene para a tradução em gRNA e um plasmídeo contendo a sequência para síntese da endonuclease usada para abrir as fitas dos cromossomos. [0070] The methodology proposed by Jessop-Fabre et al. (2016) consists of carrying out a site-directed mutation and therefore predicts that, for the correct insertion of genes of interest in the desired regions of homology to occur, the technique relies on an endonuclease to make the cuts in the yeast chromosomes and a guide RNA ( gRNA) for each chosen region of homology, causing the endonuclease to be directed to the exact region of homology in which the inserts are to be incorporated. Therefore, together with the previously linearized sequences, plasmids containing the gene for translation into gRNA and a plasmid containing the sequence for the synthesis of the endonuclease used to open the strands of the chromosomes are inserted.
[0071] Para a inserção destes plasmídeos também clonados em colónias distintas de E. coli TOP 10, replicados e purificados, juntamente com os fragmentos dos genes de interesse previamente construídos e linearizados, a Saccharomyces cerevisiae s288c foi inicialmente cultivada em meio extrato de levedura peptona dextrose com 20g/L dextrose por aproximadamente 48 horas a 30 °C até atingir I CL células/mL - quantificada em câmara de contagem - e posteriormente lavada com água deionizada estéril. [0071] For the insertion of these plasmids also cloned in distinct colonies of E. coli TOP 10, replicated and purified, together with the previously constructed and linearized fragments of the genes of interest, Saccharomyces cerevisiae s288c was initially cultivated in peptone yeast extract medium dextrose with 20g/L dextrose for approximately 48 hours at 30 °C until reaching I CL cells/mL - quantified in a counting chamber - and subsequently washed with sterile deionized water.
[0072] Em seguida foi submetida a acetato de lítio por 20 minutos a 40 °C juntamente com os elementos a serem inseridos nas células. A proporção usada na transformação foi de 1 pg de fragmentos linearizados de DNA com 1 pg do plasmídeo de gRNA auxiliares. As células foram, então, incubadas por 2 horas em meio extrato de levedura peptona dextrose com 20g/L dextrose para recuperação. Por fim, a seleção dos clones foi realizada pelo plaqueamento em meio sólido com o mesmo meio acrescido de ágar e antibióticos específicos (G418 e Noursetromicina), sendo a confirmação dos genes inseridos e de suas localizações no cromossomo da levedura realizada por PCR e identificação de banda em gel de eletroforese. [0072] Then it was subjected to lithium acetate for 20 minutes at 40 °C along with the elements to be inserted into the cells. The ratio used in the transformation was 1 pg of linearized DNA fragments with 1 pg of helper gRNA plasmid. The cells were then incubated for 2 hours in peptone dextrose yeast extract medium with 20g/L dextrose for recovery. Finally, the selection of clones was performed by plating on solid medium with the same medium plus agar and specific antibiotics (G418 and Noursetromycin), with confirmation of the inserted genes and their locations in the yeast chromosome performed by PCR and band identification in electrophoresis gel.
[0073] A determinação da inserção dos genes por PCR foi realizada para confirmar a viabilidade da cepa devidamente mutada com os genes de SEQ ID NO: 1 , 4 e 7, competente para a expressão das respectivas sequências de aminoácidos identificadas como SEQ ID NO: 10, 13 e 16. [0073] The determination of the insertion of genes by PCR was performed to confirm the viability of the properly mutated strain with the genes of SEQ ID NO: 1, 4 and 7, competent for the expression of the respective amino acid sequences identified as SEQ ID NO: 10, 13 and 16.
[0074] EXEMPLO 3: Cultivo da levedura geneticamente modificada para obtenção do concentrado proteico contendo a biomassa expressando as proteínas fibrilares animais [0074] EXAMPLE 3: Cultivation of genetically modified yeast to obtain protein concentrate containing biomass expressing animal fibrillar proteins
[0075] Após a seleção do clone de Saccharomyces cerevisiae s288c expressando constitutivamente os genes para as proteínas fibrilares animais de interesse, realiza-se um pré-inóculo em meio extrato de levedura peptona dextrose com 20g/L dextrose líquido a fim de atingir a concentração inicial no bioreator de produção de 5 g/L de biomassa. O inoculo é cultivado por 72 horas a 37 °C com agitação constante de 120 rpm até atingir uma concentração de células de 1x107 UFC.mL'. [0075] After selection of the Saccharomyces cerevisiae s288c clone constitutively expressing the genes for the animal fibrillary proteins of interest, a pre-inoculum in yeast extract medium peptone dextrose with 20g/L liquid dextrose is performed in order to reach the concentration in the bioreactor for the production of 5 g/L of biomass. The inoculum is cultured for 72 hours at 37 °C with constant stirring at 120 rpm until reaching a cell concentration of 1x10 7 CFU.mL'.
[0076] Após o cultivo do pré-inóculo de forma a acrescer a quantidade de células viáveis para o crescimento da levedura expressando as proteínas fibrilares animais de interesse, o processo tem continuidade com a etapa de cultivo da biomassa mutada em biorreator. Em meio líquido contendo glicose e aminoácidos livres em concentrações de 20 e 8 g/L, respectivamente, é acrescido o inoculo previamente cultivado atingindo uma concentração inicial de 5 g/L de biomassa no biorreator. O processo fermentativo para produção da biomassa, então, se propaga por 72 horas a 35 QC com pH 5, mantido à rotação constante de 320 rpm. A aeração foi mantida em 0,5 vvm e acrescida para 1 ,2 vvm após 12 horas. Ao final da fermentação, obteve-se uma concentração final de biomassa de 30 g/L. [0076] After the cultivation of the pre-inoculum in order to increase the amount of viable cells for yeast growth expressing the animal fibrillar proteins of interest, the process continues with the step of cultivation of the mutated biomass in a bioreactor. In a liquid medium containing glucose and free amino acids at concentrations of 20 and 8 g/L, respectively, the previously cultivated inoculum is added, reaching an initial concentration of 5 g/L of biomass in the bioreactor. The fermentation process for biomass production, then, propagates for 72 hours at 35 Q C with pH 5, maintained at a constant rotation of 320 rpm. Aeration was maintained at 0.5 vvm and increased to 1.2 vvm after 12 hours. At the end of fermentation, a final biomass concentration of 30 g/L was obtained.
[0077] Para obter o concentrado proteico derivado da biomassa de levedura expressando proteínas fibrilares, optou-se por não realizar nenhuma etapa de separação do caldo fermentado. Utilizou-se, portanto, a biomassa juntamente com o seu caldo fermentado a fim de manter os compostos presentes no caldo fermentado para a formulação do composto proteico final, tendo como vantagem a diminuição de uma operação unitária potencialmente custosa ao processo e manutenção dos inúmeros micronutrientes presentes no caldo fermentado pela levedura durante o crescimento da biomassa. [0077] To obtain the protein concentrate derived from yeast biomass expressing fibrillar proteins, it was decided not to carry out any step of separation of the fermented broth. Therefore, the biomass was used together with its fermented broth in order to keep the compounds present in the fermented broth for the formulation of the final protein compound, with the advantage of reducing a unit operation potentially costly to the process and maintenance of the numerous micronutrients present in the yeast fermented broth during biomass growth.
[0078] Para aumentar, contudo, a biodisponibilidade das proteínas fibrilares expressas pela levedura, optou-se por realizar a lise celular da biomassa, uma vez que as proteínas animais não são excretadas. Para a lise celular, o método utilizado foi o enzimático pela adição das seguintes enzimas no caldo fermentado e pela manutenção adequada das condições exigidas pela reação: b-1 ,3 Glucanases e quitinases em pH 6, 5 a 35 °C. Optou-se por não utilizar nenhum composto químico na etapa de lise a fim de não deixar nenhum resíduo potencialmente tóxico no concentrado proteico derivado da levedura, agora lisado, que será posteriormente utilizado na formulação com outras fontes nutricionais derivadas de resíduos agroindustriais para utilização na ração de cães e gatos. [0078] To increase, however, the bioavailability of fibrillar proteins expressed by yeast, it was decided to carry out cell lysis of the biomass, since animal proteins are not excreted. For cell lysis, the method used was enzymatic, by adding the following enzymes to the fermented broth and by maintaining adequate conditions required by the reaction: b-1,3 Glucanases and chitinases at pH 6.5 at 35 °C. It was decided not to use any chemical compound in the lysis step in order not to leave any potentially toxic residues in the protein concentrate derived from yeast, now lysed, which will later be used in the formulation with other nutritional sources derived from agro-industrial residues for use in the feed of dogs and cats.
[0079] EXEMPLO 4: Formulação de um composto rico em proteínas destinado a ração de cães [0079] EXAMPLE 4: Formulation of a protein-rich compound for dog food
[0080] Para uma formulação à base de resíduos agroindustriais associados com a biomassa de Saccharomyces cerevisiae mutada, visando a nutrição de cães e admitindo sua utilização como um ingrediente para outras rações, uma composição adequada depende de fontes de macronutrientes admissíveis por estes animais. A base de uma composição adequada pode conter 70% de levedura expressando proteínas fibrilares, associada à 10% de farinha de palmiste como fonte proteica, acrescida de 10% de melaço de cana-de-açúcar capaz de oferecer açúcares redutores, 15% de óleo de soja para suprir a demanda de lipídeos, e 2% de grãos secos de destilaria conferindo as fibras e sendo admitido como agente de aumento de palatabilidade. [0080] For a formulation based on agro-industrial residues associated with mutated Saccharomyces cerevisiae biomass, aiming at the nutrition of dogs and admitting its use as an ingredient for other feeds, an adequate composition depends on acceptable macronutrient sources for these animals. The base of a suitable composition may contain 70% yeast expressing fibrillar proteins, associated with 10% palm kernel flour as a protein source, plus 10% sugarcane molasses capable of offering reducing sugars, 15% oil of soy to supply the demand for lipids, and 2% of dry distillery grains providing the fibers and being admitted as an agent to increase palatability.
[0081] Essa composição resulta em um composto nutritivo com taxas de elementos em base seca de 36% de proteína bruta, 16% de gorduras, 3,7% de fibras brutas e energia digestível de 90%. Tal formulação adequa-se às demandas de macronutrientes por cães e ainda admite acréscimo de aditivos ou suplementos de vitaminas, minerais e aminoácidos, ou serve como um ingrediente para outras formulações destinadas a este grupo de animais. [0082] EXEMPLO 5: Formulação de um composto rico em proteínas destinado a ração de gatos [0081] This composition results in a nutritious compound with dry base element rates of 36% crude protein, 16% fat, 3.7% crude fiber and 90% digestible energy. Such formulation suits the macronutrient demands of dogs and also admits the addition of additives or supplements of vitamins, minerals and amino acids, or serves as an ingredient for other formulations intended for this group of animals. [0082] EXAMPLE 5: Formulation of a protein-rich compound for cat food
[0083] Para uma formulação à base de resíduos agroindustriais associados com a biomassa de Saccharomyces cerevisiae mutada, visando a nutrição de gatos e admitindo sua utilização como um ingrediente para outras rações, uma composição adequada depende de fontes de macronutrientes admissíveis por estes animais. A base de uma composição adequada pode conter 80% de levedura expressando proteínas fibrilares, acrescida de 10% de partes remanescentes de batata capaz de oferecer açúcares redutores, 10% de óleo de soja para suprir a demanda de lipídeos, e 1% de rejeitos da moagem de milho conferindo as fibras. [0083] For a formulation based on agro-industrial residues associated with mutated Saccharomyces cerevisiae biomass, aiming at the nutrition of cats and admitting its use as an ingredient for other feeds, an adequate composition depends on acceptable macronutrient sources for these animals. The base of a suitable composition can contain 80% of yeast expressing fibrillar proteins, plus 10% of remaining parts of potato capable of offering reducing sugars, 10% of soybean oil to supply the lipid demand, and 1% of rejects from corn milling giving the fibers.
[0084] Essa composição resulta em um composto nutritivo com taxas de elementos em base seca de 38% de proteína bruta, 11 ,4% de gorduras, 3,9% de fibras brutas e energia digestível de 94%. Tal formulação adequa-se às demandas de macronutrientes por gatos e ainda admite acréscimo de aditivos ou suplementos de vitaminas, minerais e aminoácidos, ou serve como um ingrediente para outras formulações destinadas a este grupo de animais. [0084] This composition results in a nutritious compound with dry base element rates of 38% crude protein, 11.4% fat, 3.9% crude fiber and 94% digestible energy. Such formulation suits the macronutrient demands of cats and also admits the addition of additives or supplements of vitamins, minerals and amino acids, or serves as an ingredient for other formulations intended for this group of animals.
[0085] EXEMPLO 6: Ração seca [0085] EXAMPLE 6: Dry feed
[0086] Visando a obtenção de uma ração seca com granulometria adequada, passível de ser compactada e com teor de umidade entre 6 e 10%, a composição resultante da biomassa de levedura mutada acrescida de resíduos agroindustriais como fonte nutricional segue da etapa de mistura para uma extrusora com a milimetragem de grãos desejada, operando entre 60 a 100 QC eliminando patógenos como Enterococcus spp. ou Salmonella spp. sem causar danos aos ingredientes da formulação. Com a mistura contendo de 20 a 30% após a extrusão, os grãos de ração formulada e modelada são secos por ar aquecido em convecção operando de 100 a 130 QC obtendo um produto final com umidade entre 6 e 10%. [0086] Aiming at obtaining a dry feed with adequate granulometry, capable of being compacted and with a moisture content between 6 and 10%, the composition resulting from the mutated yeast biomass plus agroindustrial residues as a nutritional source follows the mixing step for an extruder with the desired grain millimeter, operating between 60 to 100 Q C, eliminating pathogens such as Enterococcus spp. or Salmonella spp. without causing damage to the ingredients of the formulation. With the mixture containing from 20 to 30% after extrusion, the formulated and shaped feed grains are dried by heated air in convection operating from 100 to 130 Q C, obtaining a final product with humidity between 6 and 10%.
[0087] EXEMPLO 7: Ração úmida [0087] EXAMPLE 7: Wet feed
[0088] Visando a obtenção de uma ração úmida com 60 a 90% de água em sua composição e destinada à comercialização em latas ou sachês, a composição resultante da biomassa de levedura mutada acrescida de resíduos agroindustriais como fonte nutricional segue da etapa de mistura para uma para uma extrusora operando entre 60 a 100 QC eliminando patógenos como Enterococcus spp. ou Salmonella spp. sem causar danos aos ingredientes da formulação. A massa extrusada com 20 a 30% de umidade é cortada em pedaços com o tamanho de interesse e misturada com uma geleia ou pasta para atingir a umidade final desejada de 60 a 90% antes de ser empacotada ou enlatada. [0088] Aiming at obtaining a moist feed with 60 to 90% water in its composition and intended for sale in cans or sachets, the resulting composition of the mutated yeast biomass plus agro-industrial residues as a nutritional source proceeds from the mixing step to one for an extruder operating at 60 to 100 Q C, eliminating pathogens such as Enterococcus spp. or Salmonella spp. without causing damage to the ingredients of the formulation. The extruded dough with 20-30% moisture is cut into pieces of the size of interest and mixed with a jelly or paste to achieve the desired final moisture of 60-90% before being packaged or canned.
Definições Definitions
SEQ ID NO: 1 - Sequência codificante para a cadeia de actina alfa 1 de Bos taurusSEQ ID NO: 1 - Coding sequence for Bos taurus alpha 1 actin chain
SEQ ID NO: 2 - Sequência codificante para a cadeia de actina alfa 1 de Gallus gallusSEQ ID NO: 2 - Coding sequence for the Gallus gallus alpha 1 actin chain
SEQ ID NO: 3 - Sequência codificante para a cadeia de actina alfa 1 de Sus scrofaSEQ ID NO: 3 - Coding sequence for the Sus scrofa alpha 1 actin chain
SEQ ID NO: 4 - Sequência codificante para cadeia da tropomiosina alfa 1 de Bos taurusSEQ ID NO: 4 - Coding sequence for the alpha 1 tropomyosin chain of Bos taurus
SEQ ID NO: 5 - Sequência codificante para cadeia da tropomiosina alfa 1 de Gallus gallus SEQ ID NO: 6 - Sequência codificante para cadeia da tropomiosina alfa 1 de Sus scrofa SEQ ID NO: 7 - Sequência codificante para cadeia da troponina T1 de Bos taurus SEQ ID NO: 8 - Sequência codificante para cadeia da troponina T1 de Gallus gallus SEQ ID NO: 9 - Sequência codificante para cadeia da troponina T1 de Sus scrofa SEQ ID NO: 10 - Sequência de aminoácidos de actina alfa 1 de Bos taurus SEQ ID NO: 11 - Sequência de aminoácidos de actina alfa 1 de Gallus gallus SEQ ID NO: 12 - Sequência de aminoácidos de actina alfa 1 de Sus scrofa SEQ ID NO: 13 - Sequência de aminoácidos de tropomiosina alfa 1 de Bos taurus SEQ ID NO: 14 - Sequência de aminoácidos de tropomiosina alfa 1 de Gallus gallus SEQ ID NO: 15 - Sequência de aminoácidos de tropomiosina alfa 1 de Sus scrofa SEQ ID NO: 16 - Sequência de aminoácidos de troponina T 1 de Bos taurus SEQ ID NO: 17 - Sequência de aminoácidos de troponina T1 de Gallus gallus SEQ ID NO: 5 - Coding sequence for the tropomyosin alpha 1 chain of Gallus gallus SEQ ID NO: 6 - Coding sequence for the tropomyosin alpha 1 chain of Sus scrofa SEQ ID NO: 7 - Coding sequence for the troponin T1 chain of Bos taurus SEQ ID NO: 8 - Coding sequence for troponin T1 chain from Gallus gallus SEQ ID NO: 9 - Coding sequence for troponin T1 chain from Sus scrofa SEQ ID NO: 10 - Amino acid sequence of Bos taurus alpha 1 actin SEQ ID NO: 11 - Gallus gallus alpha 1 actin amino acid sequence SEQ ID NO: 12 - Sus scrofa alpha 1 actin amino acid sequence SEQ ID NO: 13 - Bos taurus alpha 1 tropomyosin amino acid sequence SEQ ID NO: 14 - Gallus gallus tropomyosin alpha 1 amino acid sequence SEQ ID NO: 15 - Sus scrofa tropomyosin alpha 1 amino acid sequence SEQ ID NO: 16 - Bos taurus troponin T 1 amino acid sequence SEQ ID NO: 17 - Gallus gallus troponin T1 amino acid sequence
SEQ ID NO: 18 - Sequência de aminoácidos de troponina T1 de Sus scrofa SEQ ID NO: 18 - Sus scrofa troponin T1 amino acid sequence
SEQ ID NO: 19 - Sequência do promotor TEF1 (fator de alongamento da tradução) nativo de Saccharomyces cerevisiae s288c SEQ ID NO: 19 - Sequence of the native TEF1 (translational elongation factor) promoter from Saccharomyces cerevisiae s288c
SEQ ID NO: 20 - Região de homologia do cromossomo X de Saccharomyces cerevisiae s288c localizada entre os pares de base 194944 e 195980 SEQ ID NO: 20 - Saccharomyces cerevisiae s288c X chromosome homology region located between base pairs 194944 and 195980
SEQ ID NO: 21 - Região de homologia do cromossomo XI de Saccharomyces cerevisiae s288c localizada entre os pares de base 93378 e 94567 SEQ ID NO: 21 - Saccharomyces cerevisiae s288c chromosome XI homology region located between base pairs 93378 and 94567
SEQ ID NO: 22 - Região de homologia do cromossomo XI de Saccharomyces cerevisiae s288c localizada entre os pares de base 91575 e 92913 SEQ ID NO: 22 - Saccharomyces cerevisiae s288c chromosome XI homology region located between base pairs 91575 and 92913
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Claims

REIVINDICAÇÕES
1. COMPOSTO PROTEICO CONSTITUÍDO PELA BIOMASSA DE UM ORGANISMO GENETICAMENTE MODIFICADO EXPRESSANDO PROTEÍNAS FIBRILARES ASSOCIADO A OUTRAS FONTES NUTRICIONAIS PROVENIENTES DE RESÍDUOS AGROINDUSTRIAIS caracterizado por (a) uma biomassa de levedura geneticamente modificada expressando proteínas fibrilares constitutivas da musculatura animal e (b) resíduos agroindustriais como fonte de outros elementos nutricionais essenciais para a ração de cães e gatos. 1. PROTEIN COMPOUND CONSTITUTED BY THE BIOMASS OF A GENETICALLY MODIFIED ORGANISM EXPRESSING FIBRILLARY PROTEINS ASSOCIATED WITH OTHER NUTRITIONAL SOURCES FROM AGROINDUSTRIAL RESIDUES characterized by (a) a genetically modified yeast biomass as an agroindustrial animal source expressing fibrillar proteins of other essential nutritional elements for the ration of dogs and cats.
2. COMPOSTO PROTEICO, de acordo com a reivindicação 1, caracterizado por a cepa de levedura geneticamente modificada ser dos gêneros Saccharomyces ou Pichia, preferencialmente Saccharomyces cerevisiae e Pichia pastoris. 2. PROTEIN COMPOUND according to claim 1, characterized in that the genetically modified yeast strain is of the genera Saccharomyces or Pichia, preferably Saccharomyces cerevisiae and Pichia pastoris.
3. COMPOSTO PROTEICO, de acordo com a reivindicação 1 , caracterizado por as proteínas fibrilares animais expressadas compreenderem actina, troponina e tropomiosina. 3. PROTEIN COMPOUND according to claim 1, characterized in that the expressed animal fibrillary proteins comprise actin, troponin and tropomyosin.
4. COMPOSTO PROTEICO, de acordo com a reivindicação 1, caracterizado por a mutação da levedura envolver a inserção de genes para a expressão de proteínas fibrilares de espécies utilizadas em alimentação humana ou animal em sua forma in natura, englobando bovinos {Bos taurus), aves ( Gallus gallus) e suínos ( Sus scrofa ). 4. PROTEIN COMPOUND according to claim 1, characterized in that the yeast mutation involves the insertion of genes for the expression of fibrillar proteins of species used in human or animal food in its in natura form, including cattle (Bos taurus), poultry ( Gallus gallus ) and swine ( Sus scrofa ).
5. COMPOSTO PROTEICO, de acordo com a reivindicação 1 , caracterizado por os resíduos agroindustriais incorporados na formulação serem integrados em quantidades suficientes para suprirem a demanda de macronutrientes de cães e gatos. 5. PROTEIN COMPOUND, according to claim 1, characterized in that the agro-industrial residues incorporated in the formulation are integrated in sufficient quantities to meet the demand for macronutrients from dogs and cats.
6. COMPOSTO PROTEICO CONSTITUÍDO PELA BIOMASSA DE UM ORGANISMO GENETICAMENTE MODIFICADO EXPRESSANDO PROTEÍNAS FIBRILARES ASSOCIADO A OUTRAS FONTES NUTRICIONAIS PROVENIENTES DE RESÍDUOS AGROINDUSTRIAIS caracterizado por a criação e cultivo de uma levedura - dos gêneros Saccharomyces ou Pichia, preferencialmente Saccharomyces cerevisiae e Pichia pastoris - geneticamente modificada para a expressão de proteínas fibrilares animais utilizada como parte integrante de uma formulação para rações de cães e gatos. 6. PROTEIN COMPOUND CONSTITUTED BY THE BIOMASS OF A GENETICALLY MODIFIED ORGANISM EXPRESSING PROTEINS FIBRILLARY ASSOCIATED WITH OTHER NUTRITIONAL SOURCES FROM AGROINDUSTRIAL WASTE characterized by the creation and cultivation of a yeast - of the genera Saccharomyces or Pichia, preferably Saccharomyces cerevisiae and Pichia pastoris - genetically modified for the expression of animal fibrillary proteins used as an integral part of a formulation dog and cat rations.
7. COMPOSTO PROTEICO, de acordo com a reivindicação 6, caracterizado por a alteração na levedura geneticamente modificada ser a inserção de genes para a expressão de proteínas fibrilares constitutivas da musculatura animal compreendidas pelas proteínas actina, troponina e tropomiosina, cujas sequências codificantes são compreendidas pelas sequências SEQ ID NO: 1 a 9 e as sequências de aminoácidos pelas SEQ ID NO: 10 a 18. 7. PROTEIN COMPOUND, according to claim 6, characterized in that the alteration in genetically modified yeast is the insertion of genes for the expression of fibrillar proteins constituting the animal musculature comprised by the proteins actin, troponin and tropomyosin, whose coding sequences are comprised by sequences of SEQ ID NO: 1 to 9 and the amino acid sequences of SEQ ID NO: 10 to 18.
8. COMPOSTO PROTEICO, de acordo com a reivindicação 6, caracterizado por a modificação na levedura para a expressão heteróloga das proteínas fibrilares animais compreender a associação dos genes com um promotor compatível com a levedura hospedeira e a expressão compreender o uso de plasmídeos inseridos na levedura ou a inserção dos genes em regiões específicas do cromossomo da levedura a partir de uma metodologia de edição gênica que utilize do mecanismo nativo de recombinação homóloga da levedura. 8. PROTEIN COMPOUND according to claim 6, characterized in that the modification in yeast for the heterologous expression of animal fibrillary proteins comprises the association of genes with a promoter compatible with the host yeast and the expression comprises the use of plasmids inserted in the yeast or the insertion of genes into specific regions of the yeast chromosome using a gene editing methodology that uses the yeast's native mechanism of homologous recombination.
9. COMPOSTO PROTEICO CONSTITUÍDO PELA BIOMASSA DE UM ORGANISMO GENETICAMENTE MODIFICADO EXPRESSANDO PROTEÍNAS FIBRILARES ASSOCIADO A OUTRAS FONTES NUTRICIONAIS PROVENIENTES DE RESÍDUOS AGROINDUSTRIAIS caracterizado por as etapas de (a) criação de uma cepa de levedura geneticamente modificada para viabilizar a expressão heteróloga de proteínas fibrilares animais e seu cultivo para obtenção da biomassa microbiana expressando tais proteínas no meio reacional resultando em um composto proteico; e (b) formulação com outros compostos nutritivos essenciais provenientes de resíduos agroindustriais com etapas de pós-processamento do produto destinado para alimentação de cães e gatos. 9. PROTEIN COMPOUND CONSTITUTED BY THE BIOMASS OF A GENETICALLY MODIFIED ORGANISM EXPRESSING FIBRILLARY PROTEINS ASSOCIATED WITH OTHER NUTRITIONAL SOURCES FROM AGROINDUSTRIAL RESIDUES characterized by the steps of (a) creation of a strain of genetically modified abrylar yeast animals and proteins its cultivation to obtain microbial biomass expressing such proteins in the reaction medium resulting in a protein compound; and (b) formulation with other essential nutritional compounds from agro-industrial residues with post-processing steps of the product intended for feeding dogs and cats.
10. COMPOSTO PROTEICO, de acordo com a reivindicação 9, caracterizado por a biomassa de levedura geneticamente modificada ser dos gêneros Saccharomyces ou Pichia, preferencialmente Saccharomyces cerevisiae e Pichia pastoris, para a expressão de proteínas fibrilares animais como fonte alternativa de proteína em rações de cães e gatos. 10. PROTEIN COMPOUND according to claim 9, characterized in that the genetically modified yeast biomass is of the genera Saccharomyces or Pichia, preferably Saccharomyces cerevisiae and Pichia pastoris, for the expression of animal fibrillar proteins as an alternative source of protein in dog food and cats.
11. COMPOSTO PROTEICO, de acordo com a reivindicação 9, caracterizado por a biomassa da levedura ser modificada para expressar proteínas fibrilares animais constitutivas da musculatura animal compreendidas pelas proteínas actina, troponina e tropomiosina, cujas sequências codificantes são compreendidas pelas sequências SEQ ID NO: 1 a 9 e as sequências de aminoácidos pelas SEQ ID NO: 10 a 18. 11. PROTEIN COMPOUND, according to claim 9, characterized in that the yeast biomass is modified to express animal fibrillar proteins constituting the animal musculature comprised by the proteins actin, troponin and tropomyosin, whose coding sequences are comprised by the sequences SEQ ID NO: 1 to 9 and the amino acid sequences of SEQ ID NO: 10 to 18.
12. COMPOSTO PROTEICO, de acordo com a reivindicação 9, caracterizado por a etapa de formulação da biomassa de levedura geneticamente modificada com resíduos agroindustriais como fonte de nutrientes essenciais na ração de cães e gatos compreender bagaços, cascas, farelos, tortas de filtração, biomassas microbianas ou vegetais passíveis de reutilização, óleos ou outros resíduos que não contenham traços de origem animal. 12. PROTEIN COMPOUND according to claim 9, characterized in that the step of formulating genetically modified yeast biomass with agro-industrial residues as a source of essential nutrients in dog and cat food comprises bagasse, bark, bran, filter cakes, biomass reusable microbial or vegetable substances, oils or other residues that do not contain traces of animal origin.
13. COMPOSTO PROTEICO, de acordo com a reivindicação 9, caracterizado por a aplicação da biomassa de levedura modificada na formulação para rações de cães e gatos ser compreendida por seu uso associada ou não com o caldo fermentado, englobando etapas de lise celular, de separação sólido-líquido ou de purificação das proteínas expressadas de maneira prévia à formulação. 13. PROTEIN COMPOUND, according to claim 9, characterized in that the application of modified yeast biomass in the formulation for dog and cat food is understood by its use associated or not with the fermented broth, encompassing stages of cell lysis, separation solid-liquid or purification of proteins expressed prior to formulation.
14. COMPOSTO PROTEICO, de acordo com a reivindicação 9, caracterizado por a formulação ter prevista na composição resíduos agroindustriais desempenhando funções nutricionais como fonte de macronutrientes como proteínas, carboidratos, fibras alimentares e lipídeos, micronutrientes, aminoácidos e vitaminas. 14. PROTEIN COMPOUND, according to claim 9, characterized in that the formulation has foreseen in the composition agro-industrial residues performing nutritional functions as a source of macronutrients such as proteins, carbohydrates, dietary fiber and lipids, micronutrients, amino acids and vitamins.
15. COMPOSTO PROTEICO, de acordo com a reivindicação 9, caracterizado por permitir a suplementação com outros aditivos que não contenham traços de origem animal - como aminoácidos essenciais, minerais, vitaminas e espessantes - com fim de aprimorar as condições físico-químicas do produto final e permitir etapas adicionais de pós-processamento do produto - como secagem, extrusão e outros - de acordo com a necessidade das características do produto final almejadas. 15. PROTEIN COMPOUND, according to claim 9, characterized in that it allows supplementation with other additives that do not contain traces of animal origin - such as essential amino acids, minerals, vitamins and thickeners - in order to improve the physical-chemical conditions of the final product and allow for additional post-processing steps of the product - such as drying, extrusion and others - according to the need for the desired characteristics of the final product.
PCT/BR2021/050191 2020-05-13 2021-05-07 Nutritional composition for dogs or cats, consisting of biomass from a genetically modified organism, expressing fibrillar proteins of the animal muscle, associated with other nutritional sources originating from agro-industrial waste, and method for obtaining same WO2021226690A1 (en)

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